HK1120077B - Neutrokine-alpha and neutrokine-alpha splice variant - Google Patents

Description

Neutrokine-alpha and Neutrokine-alpha splice variants

The application is a divisional application of an invention patent application with the application date of 2000-2-22, the application number of 00806541.1 (international application number of PCT/US00/04336) and the invention name of Neutrokine-alpha and Neutrokine-alpha splice variant.

The present invention relates to a novel cytokine which has been designated Neutrokine-alpha. In addition, an apparent splice variant of Neutrokine- α, designated Neutrokine- α SV, has been identified. In particular embodiments, the invention provides nucleic acid molecules encoding Neutrokine-alpha and Neutrokine-alpha SV polypeptides. In additional embodiments, Neutrokine-alpha and Neutrokine-alpha SV polypeptides are also provided, as well as vectors, host cells, and recombinant methods for producing the same.

Prior Art

Human tumor necrosis factors (TNF-. alpha.) and (TNF-. beta., or lymphotoxins) are relevant members of a large class of polypeptide mediators, including interferons, interleukins, and growth factors, collectively referred to as cytokines (Beutler, B. and Cerami, A. Immunological analysis Res. 7: 625-. Sequence analysis of cytokine receptors has elucidated several subfamilies of membrane proteins: (1) the Ig superfamily, (2) hematopoietins (cytokine receptor superfamily), and (3) tumor necrosis factor (TNF/Nerve Growth Factor (NGF) receptor superfamily (see TNF superfamily, in Gruss and Down, blood 85 (12): 3378-.

Tumor necrosis factors (a mixture of TNF- α and TNF- β) were originally discovered due to their anti-tumor activity, however they are now recognized as pleiotropic cytokines with a variety of biological activities, including having the ability to apoptosis some transformed cell lines, mediate activation and proliferation of cells, and also play important roles in immune regulation and inflammation.

Known members of the TNF ligand superfamily include TNF- α, TNF- β (lymphotoxin- α), LT- β, OX40L, Fas ligand, CD30L, CD27L, CD40L, and 4-IBBL. The ligands of the TNF ligand superfamily are acidic, having approximately 20% (in the range of 12% to 36%) sequence-homologous TNF-like molecules in the extracellular domain and predominantly present in membrane-bound form with the biologically active form of a trimer/multimer complex. Soluble forms of the TNF ligand superfamily are currently identified only as TNF, β -LT, and Fas ligands (see Gruss, H and Dower, S.k., blood 85 (12): 3378-.

Tumor necrosis factor-alpha (TNF-alpha, also known as cachectin; hereinafter "TNF") was originally secreted by monocytes and macrophages in response to exotoxins or other stimulators as a homotrimer of soluble 17 KD protein subunits (Smith, R.A. et al, J. Biochem. 262: 6951-6954(1987)). A membrane-bound 26 KD precursor form of TNF has also been described (kriegIer, M. et al, cells 53: 45-53 (1988)).

The accumulated findings indicate that TNF is a regulatory cytokine with pleiotropic biological activity. These activities include: inhibition of lipoprotein lipase synthesis ("cachectin" activity (Beutler, B. et al, Nature 316: 552 (1985)), activation of polymorphonuclear leukocytes (Klebanoff, S.J. et al, J. Immunol 136: 4220 (1986); Perussia, B. et al, J. Immunol 138: 765(1987)), inhibition of cell growth or stimulation of cell growth (Vilcek, J. et al, J. exp. Med.163: 1986; Sugarman, B.J. et al, science 230: 943 (1985); Lachman. L.B. et al, J. Exp. Med.2913 (1987)), cytotoxic effects on certain transformed cells (Laehman, L.B. et al, e.g.; Darzynekiwicz, Z. et al, cancer research 44: 83(1984) (Kohase, M. et al, Sa. cell 45: 659, Sarty. J. et al, Nature's. 1986: Wolr.3532, Wolr. 1986; Nature Vol. J. 1986: 2; Nature No. (1986: 547.), (1986; Gen), J. m, et al, j.exp.med.162: 2163 (1985); and immunomodulatory activities, including activation of T cells (Yokota, S. et al, J. Immunol 140: 531 (1988)), activation of B cells (Kehrl. J. H. et al, J. exp. Med. 160: 786(1987)), activation of monocytes (Philip, R. et al, Nature 323: 86 (1986)), activation of thymocytes (Ranges, G.E. et al, J.exp. Med. 167: 1472(1988)), and stimulation of the expression of Major Histocompatibility Complex (MHC) class I and class II molecules on the cell surface (Couins, T. et al, Proc. Natl. Acad. Sci 83: 446 (1986); Pujol-Borrel, R. et al, Nature 326: 304 (1987)).

It is noted that TNF has pro-inflammatory effects, leading to tissue damage, such as inducing pro-coagulation on vascular endothelial cells (Pober, J.S. et al, J.Immunol 136: 1680 (1986)), increasing neutrophil and lymphocyte adhesion (Pober, J.S. et al, J.Exp.Med.166; 1390 (1987)), and stimulating the release of platelet-activating factor from macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al, J.Exp.Med.166; 1390 (1987)), recent findings relating to TNF in a number of infections (Cerami. A et al, modern immunology 9: 28(1988)), immune dysregulation, tumorigenic pathologies, such as fluid cachexia associated with some malignancies (Oliff, A et al, cell 50: Med (1987)), and a decrease in the metabolism of autoimmune pathologies of TNF in autoimmune and graft versus host pathologies (Piguet al, P.F. et al, Exp.166: 1280: 1287) and a major problem of cancer metabolism in patients with autoimmune pathologies of TNF infection and graft versus host pathologies, generally associated with anorexia. The result of continued wasting is the known "cachexia" (Kern, K.A., et al, J.Parent.enter.Nutr.12: 286-. Thus cachexia is associated with a significant morbidity and with most cancer mortality, and many studies have speculated that TNF is an important mediator of cachexia in cancer infectious pathologies and in other catabolic states.

TNF is thought to play a role in the pathophysiological consequences of gram-negative sepsis and endotoxic Shock (Michie, H.R. et al, Br.J.Surg.76: 670-671 (1989); Debets, J.M.H. et al, Second Vienna Shock Forum, P463-466 (1989); Simpson, S.Q. et al Crit.Care.Clin 5: 27-47 (1989); including fever, physical discomfort, anorexia and cachexia endotoxin is a potential monocyte/macrophage activator, stimulating TNF (Kornblath, S.K. et al, immunology 137: 2585-2591(1986)) and the production and secretion of other cytokines TNF mimics the biological activity of many Med endotoxins. TNF is thought to be the major mediator of the clinical manifestations of endotoxin-related diseases, TNF and other monocyte-derived cytokines mediating the metabolic and neurohormonal responses to endotoxin (Miche, J.318. J.318: 1986), acute diseases with influenza-like symptoms are produced, including fever, tachycardia, increased metabolic rate and stress hormone release (Revhauy, A. et al, Arch. Surg.123: 162-170 (1988)). Increased circulating TNF levels have also been found in patients with gram-negative sepsis (Waage, A. et al, Lancetl: 355-357 (1987)); hammerle, A.F et al, Second Vienna Shock Forum p.715-718 (1989); detts, j.m.h. et al crit.core.med.17: 489-497 (1989); colandra t, et al, j.infec.dis.161: 982-987(1990)).

Passive immunotherapy aimed at neutralizing TNF levels has a satisfactory effect on gram-negative sepsis and endotoxemia, based on the increased TNF production and elevated TNF levels in these pathological states as described above. Cerami et al (EPO patent publication 0212489, 3/4 1987) disclose antibodies directed against "modulators" (later found to be identical to TNF) identified as cachectin. Such antibodies are useful in diagnostic immunoassays and in the treatment of shock in bacterial infections. Rubrn et al (EPO patent publication 0218868, 22.4.1987) disclose monoclonal antibodies to human TNF, hybridomas secreting such antibodies, methods for producing such antibodies, and the use of such antibodies in immunoassays for TNF. Yone et al (Epo patent publication No. 0288088, 26/10 1988) disclose anti-TNF antibodies, including mAbs, and their role in the immunoassay diagnosis of pathologies, particularly Kawasaki's pathology and cellular infections. Patients with Kawasaki pathology (pediatric acute febrile mucocutaneous lymph node syndrome; Kawasaki, T., Auergg 16: 178 (1967); Kawasaki, T., Shonica (Pediatrics) 26: 935 (1985)) have increased levels of TNF in their body fluids, which is associated with pathological progression (Yone et al, supra).

Other researchers describe mAbs specific for recombinant human TNF that have neutralizing activity in vitro (Liang, C-M. et al, review of Biochemical biophysical research 137: 847-854 (1986); Megger, A. et al, hybridoma 6: 305-311 (1987); Hirai, M. et al, J. Immunol. methods 96: 57-62 (1987); Mouer, A. et al, cytokine 2: 162-169 (1990)). Some of these mAbs are useful for epitope mapping and development of enzyme immunoassays for human TNF (Fendly et al, supra; Hirai et al, supra; Mouer et al, supra), as well as facilitating purification of recombinant TNF (Bringman et al, supra). however, these studies do not provide a basis for the production of TNF neutralizing antibodies that are diagnostic or therapeutic in vivo due to immunogenicity, lack of specificity, and/or drug adaptability.

Neutralizing antisera or mAbs to TNF have been shown to eliminate adverse physiological changes and prevent death after experimental lethal studies of endotoxemia and bacteremia in mammals other than men. This effect has been demonstrated, for example, in rodent lethality assays and in primate pathology model systems (Mathison, J.C. et al, J.Clin 81: 1925-.

It should be noted that trials of treatment with anti-TNF mabs in humans have been limited, but have shown satisfactory therapeutic results, for example in the treatment of arthritis and sepsis see, for example, Elliott, m.j. et al, Baillieres clin. 633-52 (1995); feldmann m. et al, ann.n.y.acad.sci.usa 766: 272-8 (1995); poll, t, et al, shock 3: 1-12 (1995); wheny et al, Crit.Care.Med.21: s436-40 (1993); tyacey, k.j. et al, crit.care.med.21: s415-22 (1993).

Mammalian development is dependent on cellular proliferation and differentiation, as well as the occurrence of apoptosis through apoptosis l.walker et al, Methods achiev. exp.pathol.13: 18(1988). Apoptosis plays a key role in the destruction of immune thymocytes that recognize self-antigens. Aberrations in this normal elimination process can cause autoimmune disease (Gamma et al, modern immunology 12: 193(1991)).

Itoh et al (cell 66: 233(1991)) describe a cell surface antigen, Fas/CD95, which mediates apoptosis and is involved in the deletion of T cell clones. Fas is expressed in activated T cells, B cells, neutrophils, and in the thymus, liver, heart and lung and ovaries of adult mice in addition to activated T cells, B cells and neutrophils. Apoptosis is induced in the assay of cross-linking monoclonal Ab to Fas (Yonehare et al, J.Exp.169: 1747 (1989); Trauth et al, science 245: 301(1989)), and an example of binding monoclonal Ab to Fas to stimulate T cells under certain conditions (Alderson et al, J.Exp.178: 2231 (1993)).

The Fas antigen is a 45 Kd cell surface protein of associated MW. The Fas gene of human and mouse has been cloned by Watanabe-Fukunaga et al (J. Immunol 148: 1274(1992)) and Itoh et al (cell 66: 233 (1991)). The proteins encoded by these genes are transmembrane proteins with structural homology to the nerve growth factor/tumor necrosis factor receptor superfamily, which includes two TNF receptors, the low affinity nerve growth factor receptor and CD40, CD27, CD30 and OX 40.

Fas ligand has recently been described (Suda et al, cell 75: 1169 (1993)). The amino acid sequence shows that Fas ligand is a type II transmembrane protein belonging to the TNF family. Thus, FAS ligand polypeptides comprise 3 major domains: a short intracellular domain at the amino terminus, a longer extracellular domain at the carboxy terminus, and a hydrophilic transmembrane domain connecting these two domains. Fas ligand is expressed in splenocytes and thymocytes, consistent with T cell-mediated cytotoxicity. Purified Fas ligand has a MW of 40 Kd.

The interaction between Fas/Fas ligand has been demonstrated in recent years to be required for apoptosis following T cell activation (Ju et al, Nature 373: 444 (1995); Brunner et al, Nature 373: 441 (1995). activation of T cells induces two proteins on the cell surface. The subsequent interaction between the ligand and the receptor leads to apoptosis. This supports the possible regulation of apoptosis induced by the interaction between Fas/Fas ligand during normal immune response.

Thus, there is a need to provide cytokines similar to TNF involved in pathological conditions. Such novel cytokines can be used to produce novel antibodies or other antagonists that bind these TNF-like cytokines for the diagnosis and treatment of diseases associated with TNF-like cytokines.

Summary of The Invention

In accordance with one embodiment of the present invention, there is provided a novel extracellular domain of a Neutrokine-alpha polypeptide, and a novel extracellular domain of a Neutrokine-alpha SV polypeptide, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

In accordance with another embodiment of the present invention, isolated nucleic acid molecules, including mRNA, DNA, cDNA, genomic DNA, and biologically active and diagnostically or therapeutically useful fragments and derivatives thereof, are provided that encode human Neutrokine-alpha or Neutrokine-alpha SV.

The isolated nucleic acid molecules provided by the present invention comprise or consist of a polynucleotide encoding a cytokine and its apparent splice variants that are structurally similar to TNF and related cytokines and have similar biological effects and activities. This cytokine is designated Neutrokine-alpha and the invention encompasses Neutrokine-alpha polypeptides having at least a portion of the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO: 2) or having at least a portion of the amino acid sequence encoded by the cDNA clone deposited at the ATCC at 22/10.1996 with accession number 97768 (HNEDU 15). The nucleotide sequence determined by sequencing the deposited Neutrokine-alpha clone, shown in FIGS. 1A and 1B (SEQ ID NO: 1), contains an open reading frame encoding an entire polypeptide of 285 amino acid residues, the 285 amino acids including the N-terminal methionine, a deduced endodomain of about 46 amino acid residues, a deduced transmembrane domain of about 26 amino acid residues, a deduced ectodomain of about 213 amino acids, and the deduced molecular weight of the entire protein is about 31 kDa. Like other type II transmembrane proteins, the soluble form of Neutrokine-alpha includes all or part of the extracellular domain cleaved from the transmembrane domain, as well as polypeptides comprising the entire Neutrokine-alpha polypeptide deleted of the transmembrane domain, i.e., the extracellular domain linked to the intracellular domain.

The apparent splice variant of Neutrokine- α is referred to as Neutrokine- α SV and the invention encompasses Neutrokine- α SV comprising or consisting of at least a portion of the amino acid sequence shown in FIGS. 5A and 5B (SEQ ID NO: 19) or encoded by the amino acid sequence encoded by the cDNA clone HDPMC52 deposited at the ATCC at accession number 203518 at 12/10 of 1998. The nucleotide sequence determined by sequencing the deposited Neutrokine-alpha SV clone is shown in FIGS. 5A and 5B (SEQ ID NO: 18), which contains an open reading frame encoding a complete polypeptide of 266 amino acids, including the N-terminal methionine, a putative intracellular domain of about 46 amino acids, a putative transmembrane domain of about 26 amino acids, a putative extracellular domain of about 194 amino acids, and the entire protein was deduced to have a molecular weight of about 29 kDa. Like other type II transmembrane proteins, the soluble form of Neutrokine- α SV includes all or a portion of the extracellular domain cleaved from the transmembrane domain, and a polypeptide comprising the entire Neutrokine- α SV polypeptide deleted of the transmembrane domain, i.e., the extracellular domain linked to the intracellular domain.

Thus one embodiment of the invention provides an isolated nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a full length Neutrokine-alpha polypeptide having the sequence shown in figures 1A and 1B (SEQ ID NO: 2) or encoded by a cDNA clone contained in the deposit with ATCC accession No. 97768; (b) a nucleotide sequence encoding a putative extracellular domain of a Neutrokine-alpha polypeptide having the amino acid sequence from position 73 to 285 as shown in figures 1A and 1B (SEQ ID NO: 2), or encoded by a cDNA clone contained in the deposit with ATCC accession No. 97768; (c) a nucleotide sequence encoding a fragment of (b) a polypeptide having Neutrokine-alpha functional (i.e., biological) activity; (d) a nucleotide sequence encoding a polypeptide comprising the Neutrokine-alpha endodomain (presumed to be about 1-46 contiguous amino acid residues of FIGS. 1A and 1B (SEQ ID NO: 2)), or encoded by a cDNA clone contained in the deposit with ATCC accession No. 97768; (e) a nucleotide sequence encoding a polypeptide comprising the Neutrokine-alpha transmembrane domain (presumably contiguous amino acid residues about 47-72 of FIGS. 1A and 1B (SEQ ID NO: 2)), or encoded by a cDNA clone contained in the deposit with ATCC accession No. 97768; (f) a nucleotide sequence encoding a soluble Neutrokine-alpha polypeptide having an extracellular domain and an intracellular domain but lacking a transmembrane domain; and (g) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e) or (f) above.

Other embodiments of the invention include isolated nucleic acid molecules comprising or consisting of a polynucleotide having a nucleotide sequence at least 80%, 85% or 90% identical, preferably at least 95%, 96%, 97%, 98% or 99% identical to any of the nucleotide sequences in (a), (b), (c), (d), (e) (f) or (g) above, or which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e) (f) or (g) above. The polynucleotide to which it hybridizes does not hybridize under stringent conditions to a polynucleotide having a nucleotide sequence consisting of only a residues or only T residues.

Another embodiment of the invention provides an isolated nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence selected from the group consisting of; (a) a nucleotide sequence encoding a full length Neutrokine- α SV polypeptide having the complete amino acid sequence shown in fig. 5A and 5B (seq id NO: 19) or having the cDNA contained in the deposit deposited at 10.12/1998 as ATCC accession No. 203518, clonally encoded; (b) a nucleotide sequence encoding a putative extracellular domain of a Neutrokine- α SV polypeptide having the amino acid sequence at positions 73-266 shown in figures 1A and 1B (SEQ ID NO: 2) or encoded by a cDNA clone contained in the deposit with ATCC deposit No. 203518, deposited at 10.12.1998; (c) a polynucleotide sequence encoding a polypeptide comprising the intracellular domain of Neutrokine- α SV (putative about 1-46 consecutive amino acid residues of figures 5A and 5B (SEQ ID NO: 19)) encoded by the intracellular domain or cDNA clone contained in the deposit of ATCC deposit No. 203518 deposited at 10.12.1998; (d) a polynucleotide sequence encoding a polypeptide comprising the Neutrokine- α SV transmembrane domain (putatively contiguous amino acid residues about positions 47-72 of FIGS. 5A and 5B (SEQ ID NO: 19)), the transmembrane domain or a cDNA clone contained in the deposit deposited at 10.12/1998 as ATCC deposit No. 203518; (e) a nucleotide sequence encoding a soluble Neutrokine- α SV polypeptide having an extracellular domain and an intracellular domain but no transmembrane domain; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e) above.

Other embodiments of the invention include isolated nucleic acid molecules comprising or consisting of a polynucleotide having a nucleotide sequence at least 80%, 85% or 90% identical, preferably at least 95%, 96%, 97%, 98% or 99% identical to any of the nucleotide sequences in (a), (b), (c), (d), (e) or (f) above, or which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e) or (f) above. The polynucleotide to which it hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only a residues or only T residues.

In one embodiment, the apparent splice variant of Neutrokine- α comprises or consists of at least a portion of the amino acid sequence encoded by the sequence Gly142-Leu266 amino acids set forth in FIGS. 5A and 5B (SEQ ID NO: 19), or by the cDNA clone HDPMC52 deposited at ATCC at accession number 203518 at 12/10 of 1998.

In other embodiments, the nucleic acid molecule of the invention comprises or consists of a polynucleotide encoding an amino acid sequence of an epitope-bearing portion of Neutrokine-alpha or Neutrokine-alpha SV having the amino acid sequence in (a), (b), (c), (d), (e) (f) or (g) above. Another nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising or consisting of a polynucleotide encoding an amino acid sequence of a Neutrokine-alpha or Neutrokine-alpha SV polypeptide having an amino acid sequence comprising at least 1 but not more than 50, preferably not more than 40, more preferably not more than 30, most preferably not more than 20 amino acid additions, substitutions and/or deletions. Of course, it is particularly preferred that the amino acid sequence of the polynucleotide encoding the amino acid sequence of a Neutrokine-alpha or Neutrokine-alpha SV polypeptide contains no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1-100, 1-50, 1-25, 1-20, 1-15, 1-10 or 1-5 amino acid additions, substitutions and/or deletions, preferably conservative substitutions.

The invention also relates to recombinant vectors, including the isolated nucleic acid molecules of the invention, and to host cells containing the recombinant vectors, as well as methods of producing such vectors and host cells, and methods of using them to produce Neutrokine-alpha polypeptides by recombinant techniques.

According to another embodiment of the present invention, there is provided a method for producing such a polypeptide by recombinant techniques comprising culturing a recombinant prokaryotic and/or eukaryotic host cell containing a Neutrokine-alpha or Neutrokine-alpha SV nucleic acid sequence according to the present invention under conditions promoting expression of said polypeptide, followed by recovery of said polypeptide.

The invention also provides an isolated Neutrokine-alpha polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence having the complete amino acid sequence shown in FIGS. 1A and 1B (i.e., positions 1-285 of SEQ ID NO: 2), or the full length Neutrokine-alpha polypeptide encoded by the cDNA plasmid contained in the deposit with ATCC accession No. 97768; (b) has the sequence shown in SEQ ID NO: 2 except the complete amino acid sequence shown in the sequence table 2, namely the amino acid sequence of the full-length Neutrokine-alpha polypeptide of N-terminal methionine (namely the amino acid sequence from 2 to 285 of SEQ ID NO: 2); (c) a fragment of (b) a polypeptide having Neutrokine-alpha functional activity (i.e., biological activity); (d) the amino acid sequence of a putative extracellular domain of a Neutrokine-alpha polypeptide having the amino acid sequence shown in figures 1A and 1B (SEQ ID NO: 2) at positions 73-285 or encoded by a cDNA clone contained in the deposit with ATCC accession No. 97768; (e) a nucleotide sequence encoding a Neutrokine-alpha polypeptide, which has an amino acid sequence at position 134-285 as shown in FIGS. 1A and 1B (SEQ ID NO: 2); (f) the amino acid sequence of the intracellular domain of the Neutrokine-alpha polypeptide (putative about 1-46 contiguous amino acids of FIGS. 1A and 1B (SEQ ID NO: 2)) encoded by either the cDNA plasmid contained in the deposit with ATCC accession No. 97768; (g) the amino acid sequence of the Neutrokine-alpha transmembrane domain (putative about 47-72 contiguous amino acid residues in FIGS. 1A and 1B (SEQ ID NO: 2)) encoded by the cDNA plasmid contained in the deposit with ATCC accession No. 97768; (h) an amino acid sequence of a soluble Neutrokine-alpha polypeptide having an extracellular domain and an intracellular domain but no transmembrane domain, wherein the domains are as defined above; and (i), (a), (b), (c), (d), (e), (f), (g) or (h) a fragment of the polypeptide. The polypeptide of the present invention also includes a polypeptide having an amino acid sequence which is at least 80%, preferably at least 85% or 90%, more preferably at least 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequence (a), (b), (c), (d), (e), (f), (g), (h) or (i), and a polypeptide having an amino acid sequence which is at least 80%, 85%, or 90%, more preferably at least 95% similar to the above amino acid sequence. A further embodiment of the invention relates to a polypeptide comprising or consisting of the amino acid sequence of an epitope-bearing portion of a Neutrokine-alpha polypeptide having the amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), (h) or (i) above. Neutrokine-alpha polypeptides of the invention include a portion of such polypeptides having at least 4, at least 5, at least 6, at least 7, at least 8, preferably at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, more preferably at least about 30-50 amino acids, although any length up to an epitope-bearing polypeptide comprising the entire amino acid sequence of the polypeptide of the invention is also encompassed by the invention.

A particularly preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 80%, 85%, 90% identical, preferably at least 95%, 96%, 97%, 98%, 99%, or 100% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2). A preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 90% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 as shown in FIGS. 1A and 1B (SEQ ID NO: 2). A more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 95% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 as shown in FIGS. 1A and 1B (SEQ ID NO: 2). A more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which has at least 96% identity to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 as shown in FIGS. 1A and 1B (SEQ ID NO: 2).

In addition, a more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 97% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence 134-285 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2). In addition, a more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 98% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence 134-285 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2). In addition, a more preferred embodiment relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence 134-285 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2).

The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotides and nucleic acid molecules are also encompassed by the present invention.

The present invention further provides an isolated Neutrokine-alpha SV polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of a full-length Neutrokine-alpha SV polypeptide having the complete amino acid sequence shown in FIGS. 5A and 5B (i.e., amino acids 1-266 of SEQ ID NO: 19), or encoded by the cDNA clone deposited at the ATCC at deposit No.203518 on 10.12.1998; (b) an amino acid sequence of a full-length Neutrokine- α SV polypeptide having the amino acid sequence of SEQ ID NO: 19 (i.e., amino acids 2-266 of SEQ ID NO: 19) except for the N-terminal methionine; (c) the amino acid sequence of the putative extracellular domain of a Neutrokine- α SV polypeptide having the amino acid sequence at positions 73-266 shown in figures 5A and 5B (SEQ ID NO: 19) or encoded by the cDNA clone deposited at the ATCC with deposit No.203518 at day 10, 12 months 1998; (d) the amino acid sequence of the intracellular domain of the Neutrokine- α SV polypeptide, which is putatively the consecutive amino acid residues at positions about 1-46 as shown in FIGS. 5A and 5B (SEQ ID NO: 19), or encoded by the cDNA clone deposited at ATCC No.203518 at 12/10 of 1998; (e) the amino acid sequence of the transmembrane domain of Neutrokine- α SV, which is putatively the contiguous amino acid residues at positions 47-72 as shown in FIGS. 5A and 5B (SEQ ID NO: 19), or encoded by the cDNA clone deposited at ATCC No.203518 at 12/10 of 1998; (f) an amino acid sequence of a soluble Neutrokine-alpha SV polypeptide having an extracellular domain and an intracellular domain but no transmembrane domain, wherein each domain is as defined above; and (g) a fragment of the polypeptide of (a), (b), (c), (d), (e), or (f) above. The polypeptides of the invention also include polypeptides having an amino acid sequence which is at least 80%, preferably at least 85% or 90%, more preferably at least 95%, 96%, 97%, 98% or 99% identical to the sequence described in (a), (b), (c), (d), (e), (f) or (g) above, as well as polypeptides having an amino acid sequence which is at least 80%, 85% or 90% similar, preferably at least 95% similar to the above-mentioned sequence. Another embodiment of the present invention relates to a polypeptide comprising or consisting of the amino acid sequence of an epitope-bearing portion of a Neutrokine- α SV polypeptide having the amino acid sequence as described in (a), (b), (c), (d), (e), (f) or (g) above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Neutrokine-alpha SV polypeptide of the invention, including a portion of such polypeptide having at least 4, at least 5, at least 6, at least 7, at least 8, preferably at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, more preferably at least 30-50 amino acids, of course any length up to an epitope-bearing polypeptide comprising the entire amino acid sequence of the polypeptide of the invention are also encompassed by the invention.

Some non-limiting embodiments of the invention relate to a polypeptide having an amino acid sequence of an epitope-bearing portion of a Neutrokine-alpha or Neutrokine-alpha SV polypeptide having an amino acid sequence as described in (a), (b), (c), (d), (e), (f), (g), (h) or (i) above. In other embodiments, the invention provides isolated antibodies that specifically (i.e., uniquely) bind a Neutrokine-alpha or Neutrokine-alpha SV polypeptide having the amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), (h) or (i) above.

The invention also provides methods of isolating antibodies that specifically (i.e., uniquely) bind to Neutrokine-alpha or Neutrokine-alpha SV polypeptides having the amino acid sequences described above. Such antibodies are useful for diagnosis or therapy as described below.

The invention also provides pharmaceutical compositions comprising soluble Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, in particular human Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, and/or anti-Neutrokine-alpha antibodies and/or anti-Neutrokine-alpha SV antibodies, which may be used, for example, in the treatment, prevention, prognosis and/or diagnosis of tumors and tumor metastases, bacterial infections, viral and other parasitic infections, immunodeficiency, inflammation, lymphatic disease, autoimmune disease, graft-versus-host disease, stimulation of peripheral tolerance, disruption of some transformed cell lines, mediation of cell activation, survival and proliferation, to mediate immunomodulatory and inflammatory responses, and to enhance or inhibit immune responses.

In some embodiments, soluble Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or agonists thereof of the invention are administered to treat, prevent, prognose and/or diagnose immunodeficiency disorders (e.g., X-linked Severe Combined Immunodeficiency (SCID), autosomal SCID, adenosine deaminase deficiency (ADA deficiency), X-linked hypo-globulin (XLA), Bruton's disease, congenital hypo-globulin, X-linked pediatric hypo-globulin, acquired hypo-globulin, adult-onset hypo-globulin, late-onset hypo-globulin, abnormal hypo-globulin, blood-propanglobulin, pediatric-transient hypo-globulin, non-specific hypo-globulin, blood-propan-globulin hypo-globulin, Common Variable Immunodeficiency (CVID) (acquired), Wiskott-Aldrich syndrome (WAS), X-linked high IgM immunodeficiency, non-X-linked high IgM immunodeficiency, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated levels of IgS, thymoma immunodeficiency, Ig heavy chain deletion, K chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive hypogammaglobulinemia (Swiss type), reticulocyte dysplasia, neonatal neutropenia, severe congenital leukopenia, thymic lymphoid dysplasia-dysplasia or dysplastic immunodeficiency, dyskinetic telangiectasia, brachytic dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezolf combined IgS immunodeficiency syndrome, purine nucleoside phosphorylase (PNP deficiency), MHC class II deficiency (Bare lymphocyto syndrome), and severe combined immunodeficiency), or diseases associated with immunodeficiency.

In a specific embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or polynucleotides of the present invention, or agonists thereof, are administered to treat, prevent, prognose and/or diagnose common variable immunodeficiency.

In a specific embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or polynucleotides of the present invention, or agonists thereof, are administered to treat, prevent, prognose and/or diagnose X-linked hypogammaglobulinemia.

In another specific embodiment, a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or an agonist thereof, is administered to treat, prevent, prognose and/or diagnose Severe Combined Immunodeficiency (SCID).

In another specific embodiment, a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or an agonist thereof, is administered for the treatment, prevention, prognosis and/or diagnosis of Wiskott-Aldrich syndrome.

In another specific embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or polynucleotides of the present invention, or agonists thereof, are administered to treat, prevent, prognose and/or diagnose an Ig deficiency in X-linked high IgM levels.

In another embodiment, a Neutrokine- α antagonist and/or a Neutrokine- α SV antagonist (e.g., an anti-Neutrokine- α antibody) is administered for the treatment, prevention, prognosis and/or diagnosis of an autoimmune disease (e.g., rheumatoid arthritis, systemic lupus erythematosus, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, antiphospholipid (antiphospholipid) syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), multiple sclerosis, neuritis, uveitis eye disease, purpura (e.g., Henloch-Scoenlein purpura), Reiter's disease, Stiff-Man syndrome, autoimmune pneumonia, Guillain-Barre syndrome, insulin-dependent diabetes mellitus, and autoimmune ophthalmia, autoimmune thyroid disease, hypothyroidism (i.e.g., Hashimoto's thyroid disease), goodpasture's syndrome, pemphigus, receptor autoimmunity such as (a) Grave's disease, (b) Myastenia Grave, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, anti-collagen antibodies schleroderma, connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, primary Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigus, Siogren's syndrome, diabetes, and adrenergic drug resistance (including that of asthma or cystic fibrosis), chronic active hepatitis, primary cholecystoliosis, other endocrine gland disorders, vitiligo, vasculitis, post-MI, cardiotomy syndrome, rubella, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, grandilagmatous, degenerative, and atrophic) or autoimmune disease. In a specific preferred embodiment, rheumatoid arthritis is treated, prevented, prognosed and/or diagnosed with an anti-Neutrokine-alpha antibody and/or an anti-Neutrokine-alpha SV antibody and/or other antagonist of the invention. In another specific preferred embodiment, systemic lupus erythematosus is treated, prevented, prognosed and/or diagnosed with an anti-Neutrokine-alpha antibody and/or an anti-Neutrokine-alpha SV antibody and/or other antagonist. In another preferred embodiment, the treatment, prevention, prognosis and/or diagnosis of idiopathic thrombocytopenic purpura is with an anti-Neutrokine-alpha antibody and/or an anti-Neutrokine-alpha SV antibody and/or other antagonist. In another preferred embodiment, IgA nephropathy is treated, prevented, prognosed and/or diagnosed with an anti-Neutrokine-alpha antibody and/or an anti-Neutrokine-alpha SV antibody and/or other antagonists of the invention. In a preferred embodiment, autoimmune diseases, and disorders and/or pathologies associated with the above diseases and disorders are treated, prevented, prognosed and/or diagnosed with anti-Neutrokine-alpha antibodies and/or anti-Neutrokine-alpha SV antibodies.

The invention further provides a composition comprising a Neutrokine-alpha or Neutrokine-alpha SV polynucleotide, a Neutrokine-alpha or Neutrokine-alpha SV polypeptide, and/or an anti-Neutrokine-alpha or anti-Neutrokine-alpha SV antibody, administered to a cell in vitro, an ex vivo cell, a cell in vivo, or a multicellular organism. In a preferred embodiment, the compositions of the invention comprise a Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide to express a Neutrokine-alpha polypeptide and/or a Neutrokine-alpha SV polypeptide in a host to treat a disease. In a more preferred embodiment, the compositions of the invention comprise a Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide to express a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide in a host to treat an immunodeficiency disorder or a disorder associated with immunodeficiency. It is particularly preferred that the expression be in a patient for the treatment of a dysfunction associated with aberrant endogenous activity of Neutrokine-alpha or Neutrokine-alpha SV genes, (e.g., by increasing B cell number or increasing B cell life to enhance expression of aberrant B cell function).

The invention also provides a screening method for identifying compounds that enhance or inhibit a cellular response induced by Neutrokine-alpha and/or Neutrokine-alpha SV comprising contacting a cell expressing Neutrokine-alpha and/or Neutrokine-alpha SV with a candidate compound, assaying the cellular response, and comparing the cellular response to a standard cellular response, the standard response being assayed in the absence of the candidate compound; whereby a high cellular response and a standard response indicate that the compound is an agonist and a lower cellular response than the standard response indicates that the compound is an antagonist.

In another embodiment, a method of identifying Neutrokine-alpha and/or Neutrokine-alpha SV receptors is provided, as well as methods of using such receptor screens to assay agonists and antagonists. The assay involves determining the effect of a candidate compound on binding of Neutrokine-alpha and/or Neutrokine-alpha SV to the receptor for Neutrokine-alpha and/or Neutrokine-alpha SV. In particular, the method comprises contacting Neutrokine-alpha and/or Neutrokine-alpha SV receptor with a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide of the invention and a candidate compound, and determining whether binding of the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide to the Neutrokine-alpha and/or Neutrokine-alpha SV receptor is increased or decreased due to the presence of the candidate compound. Antagonists may be useful in the prevention of septic shock, inflammation, cerebral malaria, HIV virus activation, graft/host rejection, bone resorption, rheumatoid arthritis, cachexia (wasting or dystrophia), immune system dysfunction, lymphoma, and autoimmune dysfunction (e.g., rheumatoid arthritis and systemic lupus erythematosus).

The present inventors also revealed that Neutrokine-alpha is expressed not only in monocyte cell lines but also in kidney, lung, peripheral blood cells, bone marrow, T-cell lymphoma, B-cell lymphoma, activated T-cells, bone cancer, smooth muscle, macrophages, and umbilical cord blood tissues. The inventors further revealed that Neutrokine- α SV appears to be highly expressed only in primitive dendritic cells. Some disorders of these tissues and cells, such as tumors and tumor metastases, bacterial, viral and other parasitic infections, immunodeficiency (e.g., chronic variable immunodeficiency), septic shock, inflammation, brain abuse, HIV virus activation, graft/host rejection, bone resorption, rheumatoid arthritis, autoimmune diseases (e.g., rheumatoid arthritis, and systemic lupus erythematosus), and cachexia (wasting or malnutrition). It is believed that significantly higher or lower levels of Neutrokine-alpha and/or Neutrokine-alpha SV gene expression may be detected in certain tissues (e.g., bone marrow) or body fluids (e.g., serum, plasma, urine, infusions or cerebrospinal fluid) taken from individuals having such disorders, and that the "standard" Neutrokine-alpha or Neutrokine-alpha SV gene expression level is the expression level in tissues or body fluids taken from individuals without such disorders. Accordingly, the present invention provides a diagnostic method for use during diagnosis of a disease, comprising (a) analysing the level of Neutrokine-alpha and/or Neutrokine-alpha SV gene expression in cells or body fluids of an individual; (b) comparing the expression level with the standard Neutrokine-alpha and/or Neutrokine-alpha SV gene expression level, and comparing the result with the standard level higher or lower indicates the presence of the disease.

Other embodiments of the present invention relate to a method of treating an individual in need of increasing or maintaining a level of Neutrokine-alpha and/or Neutrokine-alpha SV activity in vivo comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or agonist thereof of the present invention.

Another embodiment of the present invention is directed to a method of treating an individual in need of reducing the level of Neutrokine-alpha and/or Neutrokine-alpha SV activity in vivo comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of Neutrokine-alpha and/or Neutrokine-alpha SV. Preferred antagonists for use in the present invention are antibodies specific for Neutrokine-alpha and/or antibodies specific for Neutrokine-alpha SV.

Brief Description of Drawings

The following drawings illustrate embodiments of the invention without limiting the scope of the claims.

FIGS. 1A and 1B show the nucleotide (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of Neutrokine-alpha. Amino acids 1-46 represent the putative intracellular domain, amino acids 47-72 represent the putative transmembrane domain (underlined double line), and amino acids 73-285 represent the putative extracellular domain (remaining sequence). Potential asparagine-linked glycosylation sites are indicated in FIGS. 1A and 1B by the bold asparagine symbol (N) in the Neutrokine-alpha amino acid sequence, with the bold # on the first nucleotide of the asparagine residue in the nucleotide sequence encoding Neutrokine-alpha. Potential N-linked glycosylation sequences are found at the following positions in the Neutrokine-alpha amino acid sequence: N124-Q127(N-124, S-125, S-126, Q-127) and N242-C245(N-242, N-243, S-244, C-245).

The regions of high identity between Neutrokine-alpha, Neutrokine-alpha SV, TNF-alpha, TNF-beta, LT-beta, and the closely related Fas ligand (FIG. 2 shows an alignment of these sequences) are underlined in FIGS. 1A and 1B. These regions are not limiting and are labeled in FIGS. 1A and 1B as Conserved Domains (CD) -I, CD-II, CD-III, CD-IV, CD-V, CD-VI, CD-VII, CD-VIII, CD-IX, CD-X, and CD-XI.

FIGS. 2A and 2B show the regions of identity between the amino acid sequences of Neutrokine-alpha (SEQ ID NO: 2) and Neutrokine-alpha SV (SEQ ID NO: 19), and TNF-alpha (TNF-alpha in FIGS. 2A and 2B, GenBank No. Z15026; (SEQ ID NO: 3), TNF-beta (TNF-beta in FIGS. 2A and 2B, GenBank No. Z15026; SEQ ID NO: 4), lymphotoxin-beta (LT-beta in FIGS. 2A and 2B, GenBank No. L11016; SEQ ID NO: 5), and FAS ligand (FASL, GenBank No. U1182I in FIGS. 2A and 2B, SEQ ID NO: 6), which are established by the "MegAlign" program, which is part of a computer program called "DAN STAR" sequence.

FIG. 3 shows Neutrokine-alpha amino acid sequence analysis. Using the default parameters of said computer program for the comparison of SEQ ID NO: 2, showing an alpha region, a beta region, a turn region and a coil region; hydrophilic and hydrophobic; an amphiphilic region; a flexible region; antigen index and surface probability. In the "antigenic index-Jameson-Wolf" diagram, the position of the highly antigenic region of Neutrokine-alpha, i.e.the region from which the epitope-bearing peptide of the invention can be obtained, is indicated. The antigenic polypeptide comprises SEQ ID NO: 2 from about Phe115 to Leu147, Ile150 to Tyr163, Ser171 to Phe194, Glu223 to Tyr246, Ser271 to Phe 278.

The data in fig. 3 is also shown in table form in table 1. Columns are labeled with "residues", "positions", and roman numerals I-XIV. The column designations refer to the following features of the amino acid sequences shown in FIG. 3 and Table 1: "residue" refers to SEQ ID NO: 2 and the amino acid residues of figures 1A and 1B; "position" refers to SEQ ID NO: 2 and the positions of the corresponding residues in FIGS. 1A and 1B; i: alpha region-Garnier-Robson; II: alpha region-Chou-Fasman; IH: beta region-Garnier-Robson; IV: beta region-Chou-Fasman; v: corner region-Garnier-Robson; VI: corner region-Chou-Fasman; VII: frizzled-Garnier-Robson; VIII: hydrophilicity plots-Kyte-Doolittle; IX: hydrophobicity plot-Hopp-Woods; x: the alpha amphipathic region-Eisenberg; XI: the beta amphipathic region-Eisenberg; XII: flexible region-Karplus-Schulz; XIII: antigen index-Jameson-Wolf; and XIV: surface probability map-Emini.

FIGS. 4A, 4B and 4C show the human cDNA clones related to the present invention designated HSOAD55(SEQ ID NO: 7), HSLAH84(SEQ ID NO: 8) and HLTBM08(SEQ ID NO: 9) in sequence alignment with the Neutrokine-alpha nucleotide sequence determined from the human cDNA clone deposited at ATCC No. 97768.

FIGS. 5A and 5B show the nucleotide sequence (SEQ ID NO: 18) and the deduced amino acid sequence (SEQ ID NO: 19) of the Neutrokine- α SV protein. Amino acids 1-46 represent the putative intracellular domain, amino acids 47-72 represent the putative transmembrane domain (underlined double line), and amino acids 73-266 represent the putative extracellular domain (remainder of the sequence). Potential asparagine-linked glycosylation sites are marked in the Neutrokine- α SV amino acid sequence with the bold asparagine symbol (N) in fig. 5A and 5B, and are marked with the bold # above the first nucleotide encoding an asparagine residue in the Neutrokine- α SV nucleotide sequence. Potential N-linked glycosylation sequences are found in the Neutrokine- α SV amino acid sequence at the following positions: n124 to Q127(N124, S125, S126, Q127), and N223 to C226(N223, N224, S225, C226). The antigenic polypeptide comprises SEQ ID NO: 19 amino acid sequence of about Pro 32-Leu 47, Glu 116-Ser 143, Phe153-Tyr173, Pro218-Tyr227, Ala232-Gln241, Ile244-Ala249, and Ser252-Val 257.

The regions of high identity between Neutrokine-alpha, Neutrokine-alpha SV, TNF-alpha, TNF-beta, LT-beta, and the closely related Fas ligand (an alignment of these sequences is shown in FIG. 2) are underlined in FIGS. 1A and 1B. These conserved regions (of Neutrokine- α and Neutrokine- α SV) are labeled in FIGS. 5A and 5B as Conserved Domains (CD) -I, CD-II, CD-III, CD-V, CD-VI, CD-VII, CD-VIII, CD-IX, CD-X, and CD-XI. Neutrokine- α SV does not contain the CD-IV sequence described in the FIG. 1A and FIG. 1B schematic illustrations.

Alignment of the Neutrokine-alpha polypeptide sequence (SEQ ID NO: 2) with APRIL, TNF-alpha, LT-alpha is shown in FIG. 7A. In fig. 7A, the β -sheet region is shown, as described below in the figure description of fig. 7A.

Figure 6 shows a Neutrokine- α SV amino acid sequence analysis using default parameters of the computer program to infer the amino acid sequence of SEQ ID NO: 19, an α region, a β region, a turn region, and a coil region; hydrophilic and hydrophobic; an amphiphilic region; a flexible region; antigen index and surface probability. The location of the highly antigenic region of Neutrokine-alpha protein, i.e.the region from which the epitope-bearing peptide of the invention can be obtained, is shown in the "antigenic index-Jameson-Wolf" diagram. Antigenic polypeptides include, but are not limited to, polypeptides comprising SEQ ID NO: 19 of the amino acid sequence of seq id no: about Pro32-Leu47, about Glu116-Ser143, about Phe153-Tyr173, about Pro218-Tyr227, about Ser252-Thr258, about Ala232-Gln241, about Ile244-Ala249, about Ser252-Val 257.

The data shown in fig. 6 may be represented in tabular form similar to the data shown in table 1. Such a table representing the exact data shown in fig. 6 can be generated using the DNA STAR computer sequence analysis package's megalign component, setting up default parameters. This is the same procedure used to generate fig. 3 and 6.

FIG. 7A: the amino acid sequence of Neutrokine-alpha, and the alignment of its putative ligand-binding domain with that of the ligand-binding domain of APRIL, TNF-alpha, LT-alpha (specifically, amino acid residues 115-250 of the human APRIL polypeptide (SEQ ID NO: 20; ATCC accession No. AF046888), amino acid residues 88-233 of TNF-alpha (SEQ ID NO: 3; GenBank No. Z15026), and amino acid residues 62-205 of LT-alpha (also known as TNF-beta, SEQ ID NO: 4); GenBank No. Z15026). The putative transmembrane region of Neutrokine-alpha is shown, and the cleavage site of Neutrokine-alpha is in italics. The underlined sequences (A-H) represent the putative beta-sheet regions.

FIG. 7B: neutrokine-alpha mRNA expression. Northern blot analysis was performed on poly (A) + Northern blots (Clonetech) obtained from a range of human tissue types and selected cancer cell lines using Neutrokine-alpha open reading frame as probe. A2.6 Kb mRNA of Neutrokine-alpha was detected at high levels in placenta, heart, lung, fetal liver, thymus and pancreas. This 2.6Kb Neutrokine-alpha mRNA was also detected in HL-60 and K562 cell lines.

Fig. 8A and 8B: the expression of Neutrokine-alpha is increased after activation of human monocytes by IFN-gamma. FIG. 8A: flow cytometric analysis of Neutrokine-alpha protein expression on monocytes cultured in vitro. Purified cells were cultured for 3 days with or without IFN-. gamma. (100U/ml). Cells were then stained with Neutrokine-alpha specific mAb (2E5) (solid line) or isotype matched control (IgGl) (dotted line). Comparable results were obtained with monocytes purified from three different donors in three separate experiments. FIG. 8B: neutrokine-alpha specific TagMan primers were prepared and used to determine the relative expression level of Neutrokine-alpha mRNA in unstimulated and IFN-. gamma.treated monocytes (100U/ml). The nucleotide sequence of the TagMan primer is as follows: (a) and (3) probe: 5'-CCA CCA GCT CCA GGA GAA GGC AAC TC-3' (SEQ ID NO: 24); (b) 5' amplification primer: 5'-ACC GCG GGA CTG AAA ATC T-3' (SEQ ID NO: 25); and (c) a 3' amplification primer: 5'-CAC GCT TAT TTC TGC TGTTCT GA-3' (SEQ ID NO: 26).

Fig. 9A and 9B: neutrokine-alpha is a potential B lymphocyte stimulator. FIG. 9A: the biological activity of Neutrokine-alpha was determined in a standard B lymphocyte co-stimulation assay using Staphylococcus aureus cowan 1(SAC) as a guide. SAC alone produces a background count of 1427 + -316. Values reported are the mean ± deviation of the results obtained in triplicate wells. Similar results were obtained with recombinant Neutrokine-alpha purified from stable CHO transfectants and transiently transfected HEK293T cells. FIG. 9B: neutrokine-alpha and its combination with anti-IgM were used to stimulate the proliferation of tonsil B-cells. Bioanalysis was performed as described for analysis with SAC, except that each well was pre-coated with goat anti-human IgM antibody at a concentration of 10 μ g/ml PBS.

Fig. 10A and 10B: expression of the Neutrokine-alpha receptor in normal human peripheral blood mononuclear cells and tumor cell lines. FIG. 10A: human peripheral blood nucleated cells were obtained from normal volunteers and separated by density gradient centrifugation. Cells were stained with biotinylated Neutrokine- α followed by staining with PE-conjugated streptavidin and mAbs specific for CD3, CD20, CD14, CD56 and CD66b conjugated with FITC or PerCP. Cells were analyzed on a Becton Dickinson FACScan using CellQuest software. Data represent one of 4 independent experiments. FIG. 10B: neutrokine-alpha is combined with the tissue cell line U-937 and the myeloma cell line IM-9.

11A, 11B, and 11C: the in vivo effects of Neutrokine-alpha administered in BALB/cAn NCR mice. FIG. 11A: formalin-fixed spleens were embedded with paraffin and 5 micron sections were stained with hematoxylin and eosin (upper panel). The lower panel is a section taken from the same animal, which was stained with anti-CD 45R (B220) mAb and developed with horseradish peroxidase-conjugated rabbit anti-mouse Ig (mouse adsorbed) and the substrate diaminobenzidine tetrahydrate (DAB). Slides were counterstained with Mayer's hematoxylin. CD45R (B220) expressing cells were brown in color. FIG. 11B: flow cytometric analysis of normal (left panel) and Neutrokine-alpha treated (right panel) stained with PE-CD45R (B220) and FITC-ThB (LybD). FIG. 11C: serum levels of IgM, IgG and IgA in normal and Neutrokine-alpha treated mice.

Detailed Description

The present invention provides an isolated nucleic acid molecule comprising a polynucleotide encoding a Neutrokine-alpha polypeptide having the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO: 2), as determined by sequencing a cDNA clone. The nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1) was obtained by sequencing the HNEDU15 clone deposited at the American Type Culture Collection (ATCC) at 22.10.1996, accession No.97768, deposited at 20110-. The deposited clone was contained in pBluescriptSK (-) plasmid (Stratagene, La Jolla, Calif.).

The invention also provides an isolated nucleic acid molecule comprising a polynucleotide encoding a Neutrokine-alpha SV polypeptide having the amino acid sequence shown in FIGS. 5A and 5B (SEQ ID NO: 19), as determined by sequencing a cDNA clone. The nucleotide sequence shown in FIGS. 5A and 5B (SEQ ID NO: 18) was obtained by sequencing the HDPMC52 clone deposited at the American Type Culture Collection (ATCC) at 12/10 1998 with accession number ATCC No. 203518. The deposited clones were contained in pBluescript SK (-) plasmid (Stratagene, La Jolla, Calif.).

The Neutrokine-alpha and Neutrokine-alpha SV polypeptides of the present invention are sequence homologous to the translated products of human mRNAs for TNF-alpha, TNF-beta, LT-beta, Fas ligand, APRIL and LT-alpha (see FIGS. 2A, 2B and 7A). As indicated above, TNF- α is considered to be an important cytokine that plays a role in cytotoxicity, necrosis, apoptosis, co-stimulation, proliferation, lymphangiogenesis, immunoglobulin class switching, differentiation, antiviral activity, and regulation of adhesion molecules and other cytokines and growth factors.

Nucleic acid molecules

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein are determined using an automated DNA sequencer (e.g., Model 373, Applied Brosystems, Inc, Foster City, Calif.) and all amino acid sequences of polypeptides encoded by DNA molecules determined herein are deduced by translation of the DNA sequences determined above. Thus, it is known in the art that as with any DNA sequence determined by such automated methods, there may be some error in any nucleotide sequence determined herein. Nucleotide sequences determined by automated methods are typically at least about 90%, more typically at least about 95% to 99.9% identical to the true nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more accurately determined by other methods, including artificial DNA sequencing methods well known in the art. It is also known in the art that a single insertion or deletion in a defined nucleotide sequence will result in a frame shift in nucleotide translation compared to the actual sequence, whereby from the point of such an insertion or deletion, the deduced amino acid sequence encoded by the defined nucleotide sequence will be completely different from the actual amino acid sequence encoded by the sequenced DNA molecule.

A "nucleotide sequence" of a nucleic acid molecule or polynucleotide is a deoxyribonucleotide sequence in the case of a DNA molecule or polynucleotide and the corresponding ribonucleotide sequence (A, G, C and U) in the case of an RNA molecule or polynucleotide, wherein each thymidine (T) deoxyribonucleotide in a specific deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U).

Using the information provided herein, such as the nucleotide sequences in FIGS. 1A and 1B, the nucleic acid molecules of the present invention encoding Neutrokine-alpha polypeptides can be obtained using standard cloning and screening methods, such as methods for cloning eDNA using mRNA as a starting material. As shown herein, the nucleic acid molecule (SEQ ID NO: 1) depicted in FIGS. 1A and 1B was found in a cDNA library derived from neutrophils. Expressed sequence tags corresponding to a portion of the Neutrokine-alpha cDNA have also been found in kidney, lung, peripheral blood leukocytes, bone marrow, T-cell lymphomas, B-cell lymphomas, activated T-cells, gastric cancer, smooth muscle, macrophages, and cord blood tissue. In addition, using the nucleotide information provided in FIGS. 5A and 5B, nucleic acid molecules of the present invention encoding Neutrokine- α SV polypeptides can be obtained using standard cloning and screening methods, such as those used to clone cDNA using mRNA as a starting material. As shown in the present invention, the nucleic acid molecules shown in FIGS. 5A and 5B (SEQ ID NO: 18) were found in a cDNA library derived from original dendritic cells.

Neutrokine-alpha plasmid HNEDU15, ATCC accession No. 97768, contains an open reading frame encoding a protein of about 285 amino acid residues, a putative intracellular domain of about 46 amino acids (amino acid residues about 1-46 of FIGS. 1A and 1B (SEQ ID NO: 2)), a putative transmembrane domain of about 26 amino acids (underlined amino acid residues about 47-72 of FIGS. 1A and 1B (SEQ ID NO: 2)), and a putative extracellular domain of about 213 amino acids (amino acid residues about 73-285 of FIGS. 1A and 1B (SEQ ID NO: 2)); the deduced molecular weight was about 31 KDa. The Neutrokine-alpha polypeptide shown in FIGS. 1A and 1B (SEQ ID NO: 2) has about 20% similarity and about 10% identity to human TNF-alpha, which is GenBank accession No. 339764.

Neutrokine- α SV plasmid HDPMC52, ATCC accession number 203518, contains an open reading frame encoding a protein of about 266 amino acid residues, a putative intracellular domain of about 46 amino acids (about amino acid residues 1-46 of FIGS. 5A and 5B (SEQ ID NO: 19)), a putative transmembrane domain of about 26 amino acids (about underlined amino acid residues 47-72 of FIGS. 5A and 5B (SEQ ID NO: 19)), and a putative extracellular domain of about 194 amino acids (about amino acid residues 73-266 of FIGS. 5A and 5B (SEQ ID NO: 19)); the deduced molecular weight was approximately 29 KDa.

The Neutrokine- α SV polypeptide shown in FIGS. 5A and 5B (SEQ ID NO: 19) is about 33.9% similar and about 22.0% identical to human TNF- α, having accession number 339764 in GenBank.

The skilled artisan will appreciate that the authentic intact Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides encoded by the deposited cdnas may sometimes be somewhat shorter, containing about 285 and 266 amino acids, respectively, due to possible errors in sequencing as described above. In particular, the defined Neutrokine-alpha and Neutrokine-alpha SV coding sequences contain a common second methionine codon that serves as the initiation codon for open reading frame translation at nucleotides 64-66 as shown in FIGS. 1A and 1B (SEQ ID NO: 18). More generally, the true open reading frame can be anywhere within + -20 amino acids, especially + -10 amino acids, of the codon deduced from the first or second methionine at the N-terminus shown in FIGS. 1A and 1B (SEQ ID NO: 1) and FIGS. 5A and 5B (SEQ ID NO: 18). It will be further appreciated that the polypeptide domains described herein are determined by computer analysis and, thus, the exact location of the ectodomains, endodomains and transmembrane domains of Neutrokine-alpha and Neutrokine-alpha SV polypeptides may vary depending on the analytical criteria used to identify the various domains. For example, the exact location of the extracellular domains of Neutrokine- α and Neutrokine- α SV in FIGS. 1A and 1B (SEQ ID NO: 2) and FIGS. 5A and 5B (SEQ ID NO: 19) may vary slightly depending on the criteria used to define the domain (e.g., the location may be "shifted" by about 1-20 residues, particularly about 1-5 residues). In this case, the terminal end of the transmembrane domain and the starting point of the extracellular domain are presumed on the basis of the identification of the hydrophobic amino acid sequences in the positions shown above, as shown in FIGS. 3 and 6 and Table 1. As described below, the present invention further provides polypeptides having various residues deleted from the N-terminus and/or C-terminus of an intact polypeptide, including polypeptides lacking one or more of the N-terminal amino acids of the extracellular domains described herein, which constitute the extracellular domains of Neutrokine-alpha and Neutrokine-alpha SV polypeptides in soluble form.

The nucleic acid molecules and polynucleotides of the invention may be in the form of RNA, such as mRNA, or in the form of DNA, including cDNA and genomic DNA obtained by cloning or synthetically produced. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or may be the non-coding strand, also known as the antisense strand.

An "isolated" nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that is isolated from its natural environment. For example, a recombinant DNA molecule contained in a vector of the invention is considered to be isolated. Isolated DNA molecules additionally include, for example, recombinant DNA molecules maintained in heterologous host cells, or DNA molecules purified (partially or substantially purified) in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the invention. However, the nucleic acid contained in a clone, which is a member of a library (e.g., a genomic or cDNA library) that has not been isolated from other members of the library (e.g., in the form of a homologous solution containing this clone and other library members), or a chromosome (e.g., a karyotype "chromosome smear") isolated or removed from a cell or cell lysate, is not isolated in the present invention. As further described herein, an isolated nucleic acid molecule according to the invention may be naturally occurring, recombinantly or synthetically produced.

The isolated nucleic acid molecules of the present invention include DNA molecules which comprise or consist of an Open Reading Frame (ORF) having the start codon at position 147-149 of the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1). In addition, isolated nucleic acid molecules of the present invention include DNA molecules comprising or consisting of sequences that differ substantially from the sequences described above, but which, due to the degeneracy of the genetic code, still encode a Neutrokine-alpha protein. Of course, the genetic code is well known in the art. Thus, degenerate variants as described above can routinely be generated by those skilled in the art. In another embodiment, the isolated nucleic acid molecule provided herein comprises or consists of a sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence encoded by the cDNA contained in the plasmid with ATCC accession No. 97768. Preferably, the nucleic acid molecule comprises or consists of a mature or soluble polypeptide sequence encoding an extracellular domain of a polypeptide encoded by a cDNA in the plasmid of ATCC deposit No. 97768 or a polypeptide.

The isolated nucleic acid molecules of the present invention also include DNA molecules comprising an Open Reading Frame (ORF) having the start codon at positions 1-3 of the nucleotide sequence shown in FIGS. 5A and 5B (SEQ ID NO: 18). In addition, isolated nucleic acid molecules of the present invention include DNA molecules comprising or consisting of sequences that differ substantially from the sequences set forth above, but which, due to the degeneracy of the genetic code, still encode a Neutrokine- α SV polypeptide. Of course, the genetic code is well known in the art. Thus, degenerate variants as described above can be routinely produced by those skilled in the art. In another embodiment, the isolated nucleic acid molecule provided herein comprises or consists of a sequence encoding a Neutrokine- α SV polypeptide having the amino acids encoded by the cDNA contained in the plasmid with ATCC accession No. 203518. Preferably, the nucleic acid molecule comprises or consists of a sequence encoding an extracellular domain of a polypeptide or a mature soluble polypeptide sequence encoded by the cDNA contained in the plasmid deposited under ATCC accession number 203518.

The present invention further provides a nucleic acid molecule comprising or consisting of the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1), or the nucleotide sequence of Neutrokine-alpha cDNA contained in the plasmid of ATCC No.97768, or a nucleotide sequence having a sequence complementary to one of the above sequences. In addition, the present invention provides an isolated nucleic acid molecule comprising or consisting of the nucleotide sequence shown in FIGS. 5A and 5B (SEQ ID NO: 18), or the nucleotide sequence of Neutrokine- α SV cDNA contained in the plasmid of ATCC No.203518, or a nucleotide sequence having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, have utility including, but not limited to, as probes for gene mapping by in situ hybridization to chromosomes, and for detecting the expression of Neutrokine-alpha and Neutrokine-alpha SV in human tissue, for example, by Northern or Western blot analysis.

In one embodiment, the polynucleotide of the invention comprises or consists of SEQ ID NO: 22. SEQ ID NO: 22 were constructed from several overlapping mouse EST sequences (AI 182472, AA 422749, AA 25047 and AI 122485) obtained from GenBank. Comparison of the EST sequences yields SEQ ID NO: 22, and a Neutrokine-alpha-like polynucleotide sequence. From SEQ ID NO: 22 is as shown in SEQ ID NO: shown at 23. SEQ ID NO: 22 and SEQ ID NO: 23 are also encompassed by the present invention.

In another embodiment, the polynucleotide of the invention comprises or consists of SEQ ID NO: 27, and/or a sequence encoding SEQ ID NO: 28, fragments, variants and derivatives thereof. Such polynucleotides are also encompassed by the present invention. For example, some embodiments of the invention relate to polynucleotides comprising or consisting of a sequence that encodes a polypeptide sequence that differs from the sequence set forth in SEQ ID NO: 28 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 68-219. From SEQ ID NO: 27 as set forth in SEQ ID NO: shown at 28. Comprises or consists of SEQ ID NO: 28 and the amino acid sequence of SEQ ID NO: 28, are also encompassed by the present invention. For example, some embodiments of the invention relate to polypeptides comprising or consisting of a polypeptide that differs from SEQ ID NO: 28, amino acids 68-219, having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity. Has the sequence shown in SEQ ID NO: 27 is obtained from cynomolgus monkey (i.e. Macaea irus) PBMC by RT-PCR using two degenerate primers. Briefly, total RNA was prepared from cynomolgus monkey PBMCs by using Trizol (purchased from Life Technologies, Inc, Rockville, MD) according to the manufacturer's instructions. Then, single-stranded cDNA was synthesized from cynomolgous monkey PBMC preparations using oligo-dT primers using standard methods. Neutrokine-alpha specific primers were designed based on conserved regions between murine and human Neutrokine-alpha molecules (SEQ ID NOS: 22 and 1, respectively). The following two degenerate oligonucleotide primers were then combined with cDNA template by PCR to generate a cyanologous monkey Neutrokine-alpha nucleic acid molecule. 5' primer: 5 ' -TAC CAG ITG GCI GCC ITG CAA G-3 ' (SEQ ID NO: 35) and 3 ' primers: 5 '-GTI ACA GCA GTT TIA IIG CAC C-3' (SEQ ID NO: 36). In the degenerate primer sequences (SEQ ID NOS: 35 and 36), "I" represents deoxyinosine or dideoxyinosine.

In another embodiment, the polynucleotide of the invention comprises or consists of SEQ ID NO: 29, and/or a sequence encoding SEQ ID NO: 30, fragments, variants and derivatives thereof. Such polynucleotides are also encompassed within the scope of the present invention. For example, some embodiments of the invention relate to polynucleotides comprising or consisting of a polynucleotide encoding a polypeptide that differs from SEQ id no: 30, or a polypeptide sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to amino acids 68-219 of the sequence shown in seq id No. 30. From SEQ ID NO: 29 is as shown in SEQ ID NO: shown at 30. Comprises or consists of SEQ id no: 30, and the amino acid sequence shown in SEQ ID NO: 29 and SEQ ID NO: 30 are also encompassed by the present invention. For example, some embodiments of the invention relate to polypeptides comprising or consisting of a polypeptide that differs from SEQ ID NO: 30, amino acids 68-219, or a polypeptide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical. Has the sequence shown in SEQ ID NO: 29 is obtained from cynomolgus PBMCs by RT-PCR using two degenerate primers. Briefly, total RNA was prepared from cynomolgus PBMCs by using Trizol (purchased from Life Technologies, Inc, Rockviue, MD) according to the manufacturer's instructions. Single stranded cDNA was then synthesized from cynomolgus PBMC preparations using oligo dT primers using standard methods. Neutrokine-alpha specific primers were designed based on conserved regions between murine and human Neutrokine-alpha molecules (SEQ ID NOS: 22 and 1, respectively). Macaque Neutrokine-alpha nucleic acid molecules were then generated by PCR using cDNA templates in combination with the following two degenerate oligonucleotide primers. 5' primer: 5 ' -TAC CAG ITG GCI GCC ITG CAA G-3 ' (SEQ ID NO: 35) and 3 ' primer: 5 '-GTI ACA GCA GTT TIA IIG CAC C-3' (SEQ ID NO: 36). In the sequence of the degenerate primer (SEQ ID NOS: 35 and 36), "I" represents deoxyinosine or dideoxyinosine.

The invention also provides a nucleic acid molecule having a sequence identical to SEQ ID NO: 1 and SEQ ID NO: 18, which has been determined from the following related cDNA clones: HSOAD 55(SEQ ID NO: 7), HSLAH84(SEQ ID NO: 8), and HLTBM08(SEQ ID NO: 9).

The invention further relates to nucleic acid molecules encoding a portion of the nucleotide sequences described herein, as well as fragments of the isolated nucleic acid molecules. In one embodiment, the present invention provides a polypeptide having an amino acid sequence representing SEQ ID NO: 1, consisting of the nucleotide sequence of a portion of SEQ ID NO: 1, 1-1001 nucleotides. In another embodiment, the present invention provides a polypeptide having an amino acid sequence represented by SEQ ID NO: 18, the portion consisting of the nucleotide sequence of SEQ ID NO: 18 from nucleotide 1 to nucleotide 798.

The invention further relates to fragments of the nucleic acid molecules (i.e., polynucleotides) described herein. A nucleic acid molecule fragment having, for example, a nucleotide sequence of at least 15, preferably at least 20 or 25, more preferably at least 30, most preferably at least 40, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 450, or 500 nucleotides in length, such as: the nucleotide sequence of the cDNA contained in the plasmid of ATCC deposit No. 97768, the nucleotide sequence encoding the polypeptide sequence encoded by the cDNA contained in the plasmid of ATCC deposit No. 97768, the amino acid sequence of SEQ ID NO: 1, encoding the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence of the cDNA contained in the plasmid of ATCC deposit No. 203518, the nucleotide sequence of the polypeptide sequence of SEQ ID NO: 18, encoding the nucleotide sequence of SEQ id no: 20, or the complementary strand thereof. These fragments have many uses, including but not limited to diagnostic probes and primers as described herein. Of course, larger fragments, such as fragments of length 501-1500 nucleotides, which correspond to most, if not all, of the following nucleotide sequences are also useful according to the invention: the nucleotide sequence of the cDNA contained in the plasmid of ATCC deposit No. 97768, the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of the cDNA contained in the plasmid of ATCC deposit No. 203518, and the nucleotide sequence of SEQ ID NO: 18. Preferred nucleic acid fragments of the invention include nucleic acid molecules encoding a polypeptide comprising or consisting of an epitope-bearing portion of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide as shown in FIGS. 1A and 1B (SEQ ID NO: 2) and FIGS. 5A and 5B (SEQ ID NO: 19), respectively, and as described in detail below. Polypeptides encoded by these polynucleotide fragments are also encompassed by the present invention.

A nucleic acid molecule fragment having, for example, a nucleotide sequence of at least 15, preferably at least 20 or 25, more preferably at least 30, most preferably at least 40, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 450 or 500 nucleotides in length, such as: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 27, SEQ ID NO: 29, encoding the nucleotide sequence of SEQ ID NO: 23, encoding the polypeptide sequence of SEQ ID NO: 28, encoding the polypeptide sequence of SEQ ID NO: 30 or the complementary strand thereof. These fragments have many uses, including but not limited to use as diagnostic probes and primers as described herein. Of course, larger fragments such as those of length 501-1500 nucleotides are also useful according to the invention, which correspond to most, if not all, of the following nucleotide sequences: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 27, SEQ ID NO: 29, encoding the nucleotide sequence of SEQ ID NO: 23, encoding the polypeptide sequence of SEQ ID NO: 28, encoding the polypeptide sequence of SEQ ID NO: 30, or the complementary strand thereof. Polypeptides encoded by these polynucleotide fragments are also encompassed by the present invention.

Representative fragments of Neutrokine-alpha polynucleotide fragments of the invention include, for example: comprises or consists of SEQ ID NO: 1 or the complementary strand thereof or the cDNA of ATCC accession No. 97768, wherein the cDNA comprises a fragment consisting of about nucleotide sequences at positions 1-50, 51-100, 101-146, 147-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 600-650, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050 and/or 1051-1082. The word "about" includes the specifically noted ranges as well as ranges of a few more or less (5, 4, 3, 2, or 1) nucleotides at either or both ends than this range.

Representative fragments of Neutrokine- α SV polynucleotide fragments of the present invention include, for example: comprises or consists of SEQ ID NO: 18 or the complementary strand thereof, or about 1-50, 51-100, 101-146, 147-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, and/or 851-900 of the cDNA of ATCC accession number 203518. The term "about" as used herein includes the specified range, as well as ranges of a few nucleotides more or less (5, 4, 3, 2, or 1) at either or both ends of the range.

In certain preferred embodiments, the polynucleotide of the invention comprises or consists of SEQ id no: 1, 571-627, 580-627, 590-627, 600-627, 610-627, 571-620, 580-620, 590-620, 600-620, 571-610, 580-610, 590-610, 571-600, 580-600 and/or 571-590.

In certain other preferred embodiments, the polynucleotide of the invention comprises or consists of seq id NO: 18 consisting of the following nucleotide residues: 1-879, 25-879, 50-879, 75-879, 100-879, 125-879, 150-879, 175-879, 200-879, 225-879, 250-879, 275-879, 300-879, 325-879, 350-879, 375-879, 400-879, 425-879, 450-879, 475-879, 500-879, 525-879, 550-879, 575-879, 600-879, 625-879, 650-879, 675-879, 700-879, 725-879, 750-879, 775-879, 800-879, 825-879, 850-879,1-850, 25-850, 50-850, 75-850, 100-850, 125-850, 150-850, 175-850, 200-850, 225-850, 250-850, 275-850, 300-850, 325-850, 350-850, 375-850, 400-850, 425-850, 450-850, 475-850, 500-850, 525-850, 550-850, 575-850, 600-850, 625-850, 650-850, 675-850, 700-850, 725-850, 750-850, 775-850, 800-850, 825-850,1-825, 25-825, 50-825, 75-825, 100-825, 125-825, 150-825, 175-825, 200-825, 225-825, 250-825, 275-825, 300-825, 325-825, 350-825, 375-825, 400-825, 425-825, 450-825, 475-825, 00-825, 525-825, 550-825, 575-825, 600-825, 625-825, 650-825, 675-825, 700-825, 725-825, 750-825, 775-825, 800-825,1-800, 25-800, 50-800, 75-800, 100-800, 125-800, 150-800, 175-800, 200-800, 225-800, 250-800, 275-800, 300-800, 325-800, 350-800, 375-800, 400-800, 425-800, 450-800, 475-800, 500-800, 525-800, 550-800, 575-800, 600-800, 625-800, 650-800, 675-800, 700-800, 725-800, 750-800, 775-800,1-775, 25-775, 50-775, 75-775, 100-775, 125-775, 150-775, 175-775, 200-775, 225-775, 250-775, 275-775, 300-775, 325-775, 350-775, 375-775, 400-775, 425-775, 450-775, 475-775, 500-775, 525-775, 550-775, 575-775, 600-775, 625-775, 650-775, 675-775, 700-775, 725-775, 750-775,1-750, 25-750, 50-750, 75-750, 100-750, 125-750, 150-750, 175-750, 200-750, 225-750, 250-750, 275-750, 300-750, 325-750, 350-750, 375-750, 400-750, 425-750, 450-750, 475-750, 500-750, 525-750, 550-750, 575-750, 600-750, 625-750, 650-750, 675-750, 700-750, 725-750,1-725, 25-725, 50-725, 75-725, 100-725, 125-725, 150-725, 175-725, 200-725, 225-725, 250-725, 275-725, 300-725, 325-725, 350-725, 375-725, 400-725, 425-725, 450-725, 475-725, 500725, 525-725, 550-725, 575-725, 600-725, 625-725, 650-725, 675-725, 700-725,1-700, 25-700, 50-700, 75-700, 100-700, 125-700, 150-700, 175-700, 200-700, 225-700, 250-700, 275-700, 300-700, 325-700, 350-700, 375-700, 400-700, 425-700, 450-700, 475-700, 500-700, 525-700, 550-700, 575-700, 600-700, 625-700, 650-700, 675-700,1-675, 25-675, 50-675, 75-675, 100-675, 125-675, 150-675, 175-675, 200-675, 225-675, 250-675, 275-675, 300-675, 325-675, 350-675, 375-675, 400-675, 425-675, 450-675, 475-675, 500-675, 525-675, 550-675, 575-675, 600-675, 625-675, 650-675,1-650, 25-650, 50-650, 75-650, 100-650, 125-650, 150-650, 175-650, 200-650, 225-650, 250-650, 275-650, 300-650, 325-650, 350-650, 375-650, 400-650, 425-650, 450-650, 475-650, 500-650, 525-650, 550-650, 575-650, 600-650, 625-650,1-625, 25-625, 50-625, 75-625, 100-625, 125-625, 150-625, 175-625, 200-625, 225-625, 250-625, 275-625, 300-625,325-625, 350-625, 375-625, 400-625, 425-625, 450-625, 475-625, 500-625, 525-625, 550-625, 575-625, 600-625,1-600, 25-600, 50-600, 75-600, 100-600, 125-600, 150-600, 175-600, 200-600, 225-600, 250-600, 275-600, 300-600, 325-600, 350-600, 375-600, 400-600, 425-600, 450-600, 475-600, 500-600, 525-600, 550-600, 575-600,1-575, 25-575, 50-575, 75-575, 100-575, 125-575, 150-575, 175-575, 200-575, 225-575, 250-575, 275-575, 300-575, 325-575, 350-575, 375-575, 400-575, 425-575, 450-575, 475-575, 500-575, 525-575, 550-575,1-550, 25-550, 50-550, 75-550, 100-550, 125-550, 150-550, 175-550, 200-550, 225-550, 250-550, 275-550, 300-550, 325-550, 350-550, 375-550, 400-550, 425-550, 450-550, 475-550, 500-550, 525-550,1-525, 25-525, 50-525, 75-525, 100-525, 125-525, 150-525, 175-525, 200-525, 225-525, 250-525, 275-525, 300-525, 325-525, 350-525, 375-525, 400-525, 425-525, 450-525, 475-525, 500-525,1-500, 25-500, 50-500, 75-500, 100-500, 125-500, 150-500, 175-500, 200-500, 225-500, 250-500, 275-500, 300-500, 325-500, 350-500, 375-500, 400-500, 425-500, 450-500, 475-500,1-475, 25-475, 50-475, 75-475, 100-475, 125-475, 150-475, 175-475, 200-475, 225-475, 250-475, 275-475, 300-475, 325-475, 350-475, 375-475, 400-475, 425-475, 450-475,1-450, 25-450, 50-450, 75-450, 100-450, 125-450, 150-450, 175-450, 200-450, 225-450, 250-450, 275-450, 300-450, 325-450, 350-450, 375-450, 400-450, 425-450,1-425, 25-425, 50-425, 75-425, 100-425, 125-425, 150-425, 175-425, 200-425, 225-425, 250-425, 275-425, 300-425, 325-425, 350-425, 375-425, 400-425,1-400, 25-400, 50-400, 75-400, 100-400, 125-400, 150-400, 175-400, 200-400, 225-400, 250-400, 275-400, 300-400, 325-400, 350-400, 375-400,1-375, 25-375, 50-375, 75-375, 100-375, 125-375, 150-375, 175-375, 200-375, 225-375, 250-375, 275-375, 300-375, 325-375, 350-375,1-350, 25-350, 50-350, 75-350, 100-350, 125-350, 150-350, 175-350, 200-350, 225-350, 250-350, 275-350, 300-350, 325-350,1-325, 25-325, 50-325, 75-325, 100-325, 125-325, 150-325, 175-325, 200-325, 225-325, 250-325, 275-325, 300-325,1-300, 25-300, 50-300, 75-300, 100-300, 125-300, 150-300, 175-300, 200-300, 225-300, 250-300, 275-300,1-275, 25-275, 50-275, 75-275, 100-275, 125-275, 150-275, 175-275, 200-275, 225-275, 250-275,1-250, 25-250, 50-250, 75-250, 100-250, 125-250, 150-250, 175-250, 200-250, 225-250,1-225, 25-225, 50-225, 75-225, 100-225, 125-225, 150-225, 175-225, 200-225,1-200, 25-200, 50-200, 75-200, 100-200, 125-200, 150-200, 175-200,1-175, 25-175, 50-175, 75-175, 100-175, 125-175, 150-175, 1-150, 25-150, 50-150, 75-150, 100-150, 125-150, 1-125, 25-125, 50-125, 75-125, 100-125, 1-100, 25-100, 50-100, 75-100, 1-75, 25-75, 50-75, 1-50, 25-50, and/or 1-25

In some further preferred embodiments, the polynucleotide of the invention comprises or consists of seq id NO: 1 consisting of the following nucleotide residues: 400-627, 425-627, 450-627, 475-627, 500-627, 525-627, 550-627, 575-627, 600-627, 400-600, 425-600, 450-600, 475-600, 500-600, 525-600, 550-600, 575-600, 400-575, 425-575, 450-575, 475-575, 500-575, 525-575, 550-575, 400-550, 425-550, 450-550, 475-550, 500-550, 525-550, 400-500, 425-500, 450-500, 475-500, 400-475, 425-475, 450-475, 400-450, 425-450, 571-800, 600-800, 625-800, 650-800, 675-800, 700-800, 725-800, 750-800, 775-800, 571-775, 600-775, 625-775, 650-775, 675-775, 700-775, 725-775, 750-775, 571-750, 600-750, 625-750, 650-750, 675-750, 700-750, 725-750, 571-725, 600-725, 725-725, 650-725, 625-725, 675-625, 700-700, 625-700, 675-700, 571-675-700, 675-700, 600-675-700, 650-700, 675-700, 571-675, 675-675, 625, 650-675, 650-600-650, 625, 600-650-625, 600-775

In other preferred embodiments, the polynucleotide of the invention comprises or consists of seq id NO: 1 consisting of the following nucleotide residues: 147-500, 147-450, 147-400, 147-350, 200-500, 200-450, 200-400, 200-350, 250-500, 250-450, 250-400, 250-350, 300-500, 300-450, 300-400, 300-350, 350-750, 350-700, 350-650, 350-600, 350-550, 400-750, 400-700, 400-650, 400-600, 400-550, 425-750, 425-700, 425-650, 425-600, 425-550, 450-1020, 450-1001, 450-950, 450-900, 450-850, 450-800, 450-775, 500-1001, 500-950, 500-900, 500-850, 500-800, 500-SA 775, 550-SA 1001, 550-SA 950, 550-SA 900, 550-SA 850, 550-SA 800, 550-SA 775, 600-SA 1001, 600-SA 950, 600-SA 900, 600-SA 850, 600-SA 800, 600-SA 775, 650-SA 1001, 650-SA 950, 650-SA 900, 650-SA 850, 650-SA 775, 700-SA 1001, 700-SA 950, 700-SA 900, 700-SA 850, 700-SA 800, 700-SA 775, 825-SA 1082, 850-SA 1082, 875-SA 1082, 900-SA 1082, 925-SA 1082, 950-SA 1082, 975-SA 1082, 1000-SA 1082, 1081082, and/or 1050-SA 1082

Preferably, a polynucleotide fragment of the invention encodes a polypeptide that exhibits Neutrokine-alpha and/or Neutrokine-alpha SV functional activity. A polypeptide having "functional activity" is a polypeptide that exhibits one or more known functional activities associated with a full-length and/or secreted Neutrokine-alpha polypeptide and/or Neutrokine-alpha SV polypeptide. Such functional activities include, but are not limited to, biological activity (e.g., ability to stimulate B cell proliferation, survival differentiation and/or activation), antigenicity [ ability to bind (or compete for binding to) an anti-Neutrokine-alpha and/or anti-Neutrokine-alpha SV antibody(s) ], immunogenicity (ability to produce antibodies that bind Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides), ability to form multimers with Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention, and ability to bind to receptors or ligands for Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides.

In other specific embodiments, the polypeptides encoded by the polynucleotide fragments of the invention comprise or consist of a putative intracellular domain of Neutrokine-alpha (amino acids 1-46 of SEQ ID NO: 2), a putative transmembrane domain (amino acids 47-72 of SEQ ID NO: 2), a putative extracellular domain (amino acids 73-284 of SEQ ID NO: 2), a putative TNF conserved domain (amino acids 191-284 of SEQ ID NO: 2). In another embodiment, a polynucleotide fragment of the invention comprises or consists of any combination of 1, 2, 3 or all 4 of the above domains. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another specific embodiment, the polypeptide encoded by a polynucleotide fragment of the invention comprises or consists of a putative intracellular domain of Neutrokine- α SV (amino acids 1-46 of SEQ ID NO: 19), a putative transmembrane domain (amino acids 47-72 of SEQ ID NO: 19), a putative extracellular domain (amino acids 73-266 of SEQ ID NO: 19), or a putative TNF conserved domain (amino acids 172-265 of SEQ ID NO: 19). In further embodiments, the polypeptide encoded by a polynucleotide fragment of the invention comprises or consists of any combination of 1, 2, 3 or all 4 of the above domains. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide fragment of the invention comprises or consists of a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 2, Met 1-Lys113, Leu114-Thr141, Ile142-Lus160, Gly161-Gln198, Val199-Ala248 and Gly250-Leu 285. In addition, polynucleotides encoding any combination of 2, 3, 4, 5 or more of these amino acid sequences are also encompassed by the present invention. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide fragment of the invention comprises or consists of a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 19, Met 1-Lys113, Leu114-Thr141, Gly142-Gln179, Val180-Ala229, Gly230-Leu266 residues. In addition, polynucleotides encoding any combination of 2, 3, 4, 5 or more of these amino acid sequences are also encompassed by the present invention. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide fragment of the invention comprises or consists of a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 23, Met 1-Lys106, Leu107-Thr134, Glu135-Asn165, Ile167-Lys184, Gly185-Gln224, Val225-Ala272 and Gly273-Leu309 residues. In addition, polynucleotides encoding any combination of 2, 3, 4, 5 or more of these amino acid sequences are also encompassed by the present invention. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide fragment of the invention comprises or consists of a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 28 of Tyr 1-Lys47, Leu48-Thr75, Ile76-Lys94, Gly95-Gln132, Val133-Ala182 and Gly183-Ala 219. In addition, polynucleotides encoding any combination of 2, 3, 4, 5 or more of these amino acid sequences are also encompassed by the present invention. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide fragment of the invention comprises or consists of a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 30, Tyr 1-Lys47, Leu48-Thr75, Ile76-Lys94, Gly95-Gln132, Val133-Ala182 and Gly183-Ala 219. In addition, polynucleotides encoding any combination of 2, 3, 4, 5 or more of these amino acid sequences are also encompassed by the present invention. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In another embodiment, the polynucleotide of the invention comprises or consists of SEQ ID NO: 21, and (b) the sequence composition shown in the figure. SEQ ID NO: 21 encodes a polypeptide having an initial methionine residue linked to the amino acid sequence of SEQ ID NO: 2, and the composition of Ala134-Leu285 residues of the Neutrokine-alpha polypeptide sequence shown in the figure 2. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In certain other preferred embodiments, the polynucleotide of the invention comprises or consists of seq id NO: 21 consisting of the following nucleotide residues: 1-459, 15-459, 30-459, 45-459, 60-459, 75-459, 90-459, 105-459, 120-459, 135-459, 150-459, 165-459, 180-459, 195-459, 210-459, 225-459, 240-459, 255-459, 270-459, 285-459, 300-459, 315-459, 330-459, 345-459, 360-459, 375-459, 390-459, 405-459, 420-459, 435-459, 450-459,1-450, 15-450, 30-450, 45-450, 60-450, 75-450, 90-450, 105-450, 120-450, 135-450, 150-450, 165-450, 180-450, 195-450, 210-450, 225-450, 240-450, 255-450, 270-450, 285-450, 300-450, 315-450, 330-450, 345-450, 360-450, 375-450, 390-450, 405-450, 420-450, 435-450,1-435, 15-435, 30-435, 45-435, 60-435, 75-435, 90-435, 105-435, 120-435, 135-435, 150-435, 165-435, 180-435, 195-435, 210-435, 225-435, 240-435, 255-435, 270-435, 285-435, 300-435, 315-435, 330-435, 345-435, 360-435, 375-435, 390-435, 405-435, 420-435,1-420, 15-420, 30-420, 45-420, 60-420, 75-420, 90-420, 105-420, 120-420, 135-420, 150-420, 165-420, 180-420, 195-420, 210-420, 225-420, 240-420, 255-420, 270-420, 285-420, 300-420, 315-420, 330-420, 345-420, 360-420, 375-420, 390-420, 405-420,1-405, 15-405, 30-405, 45-405, 60-405, 75-405, 90-405, 105-405, 120-405, 135-405, 150-405, 165-405, 180-405, 195-405, 210-405, 225-405, 240-405, 255-405, 270-405, 285-405, 300-405, 315-405, 330-405, 345-405, 360-405, 375-405, 390-405,1-390, 15-390, 30-390, 45-390, 60-390, 75-390, 90-390, 105-390, 120-390, 135-390, 150-390, 165-390, 180-390, 195-390, 210-390, 225-390, 240-390, 255-390, 270-390, 285-390, 300-390, 315-390, 330-390, 345-390, 360-390, 375-390,1-375, 15-375, 30-375, 45-375, 60-375, 75-375, 90-375, 105-375, 120-375, 135-375, 150-375, 165-375, 180-375, 195-375, 210-375, 225-375, 240-375, 255-375, 270-375, 285-375, 300-375, 315-375, 330-375, 345-375, 360-375,1-360, 15-360, 30-360, 45-360, 60-360, 75-360, 90-360, 105-360, 120-360, 135-360, 150-360, 165-360, 180-360, 195-360, 210-360, 225-360, 240-360, 255-360, 270-360, 285-360, 300-360, 315-360, 330-360, 345-360,1-345, 15-345, 30-345, 45-345, 60-345, 75-345, 90-345, 105-345, 120-345, 135-345, 150-345, 165-345, 180-345, 195-345, 210-345, 225-345, 240-345, 255-345, 270-345, 285-345, 300-345, 315-345, 330-345,1-330, 15-330, 30-330, 45-330, 60-330, 75-330, 90-330, 105-330, 120-330, 135-330, 150-330, 165-330, 180-330, 195-330, 210-330, 225-330, 240-330, 255-330, 270-330, 285-330, 300-330, 315-330,1-315, 15-315, 30-315, 45-315, 60-315, 75-315, 90-315, 105-315, 120-315, 135-315, 150-315, 165-315, 180-315, 195-315, 210-315, 225-315, 240-315, 255-315, 270-315, 285-315, 300-315,1-300, 15-300, 30-300, 45-300, 60-300, 75-300, 90-300, 105-300, 120-300, 135-300, 150-300, 165-300, 180-300, 195-300, 210-300, 225-300, 240-300, 255-300, 270-300, 285-300,1-285, 15-285, 30-285, 45-285, 60-285, 75-285, 90-285, 105-285, 120-285, 135-285, 150-285, 165-285, 180-285, 195-285, 210-285, 225-285, 240-285, 255-285, 270-285,1-270, 15-270, 30-270, 45-270, 60-270, 75-270, 90-270, 105-270, 120-270, 135-270, 150-270, 165-270, 180-270, 195-270, 210-270, 225-270, 240-270, 255-270,1-255, 15-255, 30-255, 45-255, 60-255, 75-255, 90-255, 105-255, 120-255, 135-255, 150-255, 165-255, 180-255, 195-255, 210-255, 225-255, 240-255,1-240, 15-240, 30-240, 45-240, 60-240, 75-240, 90-240, 105-240, 120-240, 135-240, 150-240, 165-240, 180-240, 195-240, 210-240, 225-240,1-225, 15-225, 30-225, 45-225, 60-225, 75-225, 90-225, 105-225, 120-225, 135-225, 150-225, 165-225, 180-225, 195-225, 210-225,1-210, 15-210, 30-210, 45-210, 60-210, 75-210, 90-210, 105-210, 120-210, 135-210, 150-210, 165-210, 180-210, 195-210,1-195, 15-195, 30-195, 45-195, 60-195, 75-195, 90-195, 105-195, 120-195, 135-195, 150-195, 165-195, 180-195,1-180, 15-180, 30-180, 45-180, 60-180, 75-180, 90-180, 105-180, 120-180, 135-180, 150-180, 165-180, 1-165, 15-165, 30-165, 45-165, 60-165, 75-165, 90-165, 105-165, 120-165, 135-165, 150-165,1-150, 15-150, 30-150, 45-150, 60-150, 75-150, 90-150, 105-150, 120-150, 135-150,1-135, 15-135, 30-135, 45-135, 60-135, 75-135, 90-135, 105-135, 120-135,1-120, 15-120, 30-120, 45-120, 60-120, 75-120, 90-120, 105-120,1-105, 15-105, 30-105, 45-105, 60-105, 75-105, 90-105, 1-90, 15-90, 30-90, 45-90, 60-90, 75-90, 1-75, 15-75, 30-75, 45-75, 60-75, 1-60, 15-60, 30-60, 45-60, 1-45, 15-45, 30-45, 1-30, and/or 15-30. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

Thus, a specific embodiment of the invention relates to a polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of beta sheet region A, A ', B, B', C, D, E, F, G or H as shown in FIG. 7A and in example 6. Other embodiments of the invention relate to polynucleotides encoding Neutrokine-alpha polypeptides comprising or consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or any combination of all 10 of the beta-sheet regions a-H shown in figure 7A and example 6. A further preferred embodiment of the invention relates to a polypeptide comprising or consisting of the Neutrokine-alpha amino acid sequence of the beta-sheet region A, A ', B, B', C, D, E, F, G or H as shown in FIG. 7A and in example 6. A further embodiment of the invention relates to a Neutrokine-alpha polypeptide comprising or consisting of any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9 or all 10 beta-sheet regions a-H as shown in figure 7A and example 6.

In certain other preferred embodiments, the polynucleotide of the invention comprises or consists of seq id NO: 21, 34-57, 118-123, 133-141, 151-159, 175-216, 232-255, 280-315, 328-357, 370-393 and/or 430-456 nucleotide residues. These polynucleotides and polypeptides correspond to the putative beta-sheet region shown in figure 7A. In certain embodiments, the polynucleotide of the invention comprises or consists of a polynucleotide that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the above beta-sheet regions. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotide sequences are also encompassed by the present invention. In another embodiment, the invention provides an isolated nucleic acid molecule comprising a polynucleotide that hybridizes under stringent hybridization conditions to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the beta sheet polynucleotides of the invention described above. "stringent conditions" as used herein are as described above.

In a further preferred embodiment, the polynucleotide of the invention comprises or consists of SEQ id no: 1, 576-599, 660-665, 675-683, 693-701, 717-758, 744-803, 822-857, 870-899, 912-935 and/or 972-998 nucleotide residues. Polypeptides encoded by these polynucleotide fragments are also encompassed by the present invention. These polynucleotide and polypeptide fragments correspond to the putative beta-sheet region shown in FIG. 7A.

In a further preferred embodiment, the polynucleotide of the invention comprises or consists of SEQ id no: 18, 457-, 472-, 480-, 490-, 498-, 514-, 571-, 600-, 619-, 654-, 667-, 696-, 699-, 732-, and/or 769-, 795-nucleotide residues. Polypeptides encoded by these polynucleotide fragments are also encompassed by the present invention. These polynucleotide and polypeptide fragments correspond to the putative beta-sheet region shown in FIG. 7A.

In a further preferred embodiment, the polynucleotide of the invention comprises or consists of SEQ id no: 22, 124-, 139-, 147-, 157-, 165-, 181-, 222-, 238-, 267-, 286-, 321-, 334-, 363-, 376-, 399-and/or 436-462. Polypeptides encoded by these polynucleotide fragments are also encompassed by the present invention. These polynucleotide and polypeptide fragments correspond to the putative beta-sheet region shown in FIG. 7A. Polypeptides comprising or consisting of an amino acid sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or any combination of all of these regions are also encompassed by the present invention.

The relative positions of several intron/exon boundaries of murine Neutrokine-alpha (SEQ ID NO: 22 and SEQ ID NO: 23) were determined based on sequence analysis of murine genomic DNA. The second exon (previously referred to as "exon 2") at the 5' end of the clone of the murine Neutrokine-alpha genome, is encoded by the sequence of SEQ ID NO: 23, and the sequence Tyr187-Gln 222. The apparent third exon at the 5' end of the clone of the murine Neutrokine-alpha genome (previously referred to as "exon 3") comprises the amino acid sequence of SEQ ID NO: 23, Val223-Gly273 of the sequence shown in the specification.

Thus, in one embodiment, the invention provides a polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 23 Tyr187-Gln222 amino acid sequence. The invention also relates to a nucleic acid molecule comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a murine Neutrokine-alpha polypeptide as described above. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention.

In another embodiment, the invention provides a polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 23, Val223-Gly273 residues. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a murine Neutrokine-alpha polypeptide as described above. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention.

In addition, the relative positions of the corresponding intron/exon boundaries in human Neutrokine-alpha (SEQ ID NO: 1 and SEQ ID NO: 2) were determined based on sequence alignment of murine and human Neutrokine-alpha polypeptides. The apparent second exon at the 5' end of human Neutrokine-alpha (also referred to as "exon 2" in advance) is encoded by SEQ ID NO: 2, and Tyr163-Gln 198. The apparent third exon at the 5' end of human Neutrokine-alpha (also referred to as "exon 3" in advance) is encoded by SEQ id no: 2, Val199-Gly 249.

Thus, in one embodiment, the invention provides a nucleic acid encoding a polypeptide comprising or consisting of SEQ id no: 2 Tyr163-Gln198 amino acid sequence. The present invention also relates to a nucleic acid molecule comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide as described above. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention.

In another embodiment, the invention provides a polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and a Val199-Gly249 amino acid sequence. The present invention also relates to a nucleic acid molecule comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide as described above. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and/or polynucleotide sequences are also encompassed by the present invention.

The functional activity of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, as well as fragments, variant derivatives, and analogs thereof, may be analyzed by various methods described herein and well known in the art.

For example, in one embodiment, various immunoassays known in the art may be used for their ability to bind or compete with full-length Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides for binding to anti-Neutrokine-alpha and/or anti-Neutrokine-alpha SV antibodies, or to bind Neutrokine-alpha receptor and/or Neutrokine-alpha SV receptor on B cells, including but not limited to competitive and non-competitive assays using the following methods: radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoradiometric assay, gel diffusion precipitation, immunodiffusion assay, in situ immunoassay (labeled with, for example, colloidal gold, an enzyme, or a radioisotope), Westem blot, precipitation, agglutination (e.g., gel agglutination, hemagglutination), complement fixation, immunofluorescence, protein A, and immunoelectrophoresis, among others. In one embodiment, antibody binding is detected by detecting a label on the original antibody. In another embodiment, the primary antibody is detected by detecting the secondary antibody and binding of the reagent to the primary antibody. In yet another embodiment, the secondary antibody is labeled. Numerous methods of detecting binding in immunoassays are known in the art and are within the scope of the present invention.

In another embodiment, when identifying Neutrokine-alpha and/or Neutrokine-alpha SV ligands, or evaluating the ability of a fragment, variant or derivative of the polypeptide of the invention to multimerize, binding may be analyzed, for example, by methods well known in the art such as reduced or non-reduced gel chromatography, protein affinity chromatography, and affinity blotting. See Phizicky, E.et al, 1995, microbiological research 59: 94-123. In another embodiment, the physiological relevance of Neutrokine-alpha and/or Neutrokine-alpha SV binding to its substrate is analyzed.

In addition, the assays described herein (e.g., see examples 6 and 7), as well as other assays known in the art, can be routinely used to determine the ability of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides and fragments, variant derivatives and analogs thereof to stimulate Neutrokine-alpha and/or Neutrokine-alpha SV-related biological activity (e.g., to stimulate or inhibit (in the case of Neutrokine-alpha and/or Neutrokine-alpha SV antagonists) B cell proliferation, differentiation and/or activation and/or prolong B cell survival in vitro or in vivo).

Other methods are well known to those skilled in the art and are also encompassed by the present invention.

In additional embodiments, the polynucleotides of the invention encode polypeptides having the functional characteristics of Neutrokine-alpha and Neutrokine-alpha SV. Preferred embodiments of the invention for this purpose include fragments comprising or consisting of the following regions of Neutrokine-alpha and Neutrokine-alpha SV polypeptides: an alpha helix and alpha helix forming region ("alpha region"), a beta sheet and beta sheet forming region ("beta region"), a turn and turn forming region ("turn region"), a coil and coil forming region ("coil region"), a hydrophilic region, a hydrophobic region, an alpha amphipathic region, a beta amphipathic region, a flexible region, a surface forming region and a high antigen index region.

It is believed that one or more of the beta-sheet regions of Neutrokine-alpha shown in FIG. 7A are important for dimerization and interaction between Neutrokine-alpha and its ligand.

Some preferred regions for this purpose are shown in fig. 3 (table 1). The data shown in fig. 3 and table 1 only represent when the default parameters of the DNA STAR computer program were analyzed for SEQ id no: 2 in different forms with the same result obtained with the amino acid sequence of 2.

The preferred regions described above and shown in FIG. 3 and Table 1 include, but are not limited to, the aforementioned types of regions identified by analysis of the amino acid sequences shown in FIGS. 1A and 1B. As shown in FIG. 3 and Table 1, such preferred regions include the Garnier-Robson α region, the β region, the turn region, and the coil region, the Chou-Fasman α region, the β region and the coil region, the Kyte-Doolittle hydrophilic region and the hydrophobic region, the Eisenberg α and β amphiphilic regions, the Karplus-Schulz flexible region, the Emini surface forming region, and the Jameson-Wolf high antigen index region. Highly preferred polynucleotides are those that encode polypeptides consisting of regions of Neutrokine-alpha and/or Neutrokine-alpha SV that combine some of the above (e.g., 1, 2, 3, or 4) features.

In addition, the data in columns VIII, IX, XIII and XIV of Table 1 can be routinely used to determine regions of Neutrokine-alpha that are highly potentially antigenic (column VIII of Table 1 represents hydrophilicity according to Kyte-DooLittle; column IX of Table 1 represents hydrophobicity according to Hopp-Woods; column XIII of Table 1 represents antigenic index according to Jameson-Wolf; column XIV of Table 1 represents surface probability according to Emini). Regions of high antigenicity are determined from the data shown in VIII, IX, XIII, and/or XIV by selecting values that represent regions of the polypeptide that are susceptible to exposure to the polypeptide surface in the context in which antigen recognition can occur at the onset of the immune response. The data shown in FIG. 6 are also represented in a similar tabular format, by simply examining the amino acid sequence shown in FIG. 6(SEQ ID NO: 19) using the DNA STAR pattern and program with default parameters. As described above, the amino acid sequence shown in FIG. 6 may also be used to determine regions of Neutrokine-alpha that are highly potentially antigenic, and may be represented graphically (e.g., in FIG. 6) or in the form of a table (e.g., in Table 1).

TABLE 1

TABLE 1 (continuation)

TABLE 1 (continuation)

TABLE 1 (continuation)

TABLE 1 (continuation)

Other preferred nucleic acid fragments of the invention include nucleic acid molecules comprising or consisting of a sequence encoding one or more epitope-bearing portions of Neutrokine-alpha. In particular, such nucleic acid fragments of the invention include nucleic acid molecules comprising or consisting of a sequence encoding a polypeptide selected from the group consisting of: SEQ ID NO: 2 amino acid sequence of about Phe115-Leu147, Ile150-Tyr163, Ser170-Phe194, Glu223-Tyr246, Ser271-Phe 278. "about" as used herein refers to the specified range as well as ranges that have more or less than 5, 4, 3, 2, or 1 amino acid residue at one or both of the amino and carboxy termini. Polypeptides encoded by these nucleic acid molecules are also encompassed by the present invention. Polypeptide fragments carrying antigenic epitopes of Neutrokine-alpha can be readily determined by one skilled in the art using the Jameson-Wolf antigen index analysis described above, as shown in FIG. 3. Methods for determining the epitope-bearing portion of other such Neutrokine-alpha are described in detail below.

Other preferred nucleic acid fragments of the invention include nucleic acid molecules comprising or consisting of a sequence encoding one or more epitope-bearing portions of Neutrokine- α SV. In particular, such nucleic acid fragments of the invention include nucleic acid molecules comprising or consisting of a sequence encoding a polypeptide selected from the group consisting of: SEQ ID NO: 19 amino acid sequence of about Pro32-Leu47, Glu116-Ser143, Phe153-Tyr173, Pro218-Tyr227, Ser252-Thr258, Ala232-Gln241, Ile244-Ala249, Ser252-Val 257. As used herein, "about" refers to a specific range, as well as ranges that have more or less than 5, 4, 3, 2, or 1 amino acid residue at one or both of the amino terminus and the carboxy terminus. Fragments of a polypeptide bearing an antigenic epitope of Neutrokine-alpha can be assayed using the Jameson-Wolf antigen index described above and can be readily determined by one skilled in the art. Methods for determining the epitope-bearing portion of other such Neutrokine- α SVs are described in detail below.

In specific embodiments, the polynucleotide of the invention is less than 100,000kb, 50,000kb, 10,000kb, 1,000kb, 500kb, 400kb, 350kb, 300kb, 250kb, 200kb, 175kb, 150kb, 125kb, 100kb, 75kb, 50kb, 40kb, 30kb, 25kb, 20kb, 15kb, 10kb, 7.5kb, or 5kb in length.

In another embodiment, a polynucleotide of the invention comprises at least 15, at least 30, at least 50, at least 100 or at least 250, at least 500, at least 1000 contiguous nucleotides of a Neutrokine-alpha coding sequence, but consists of less than or equal to 1000kb, 500kb, 250kb, 200kb, 150kb, 100kb, 75kb, 50kb, 30kb, 25kb, 20kb, 15kb, 10kb or 5kb of genomic DNA flanking 5 'or 3' coding nucleotides as shown in FIGS. 1A and 1B (SEQ ID NO: 1) or FIGS. 5A and 5B (SEQ ID NO: 18). In another embodiment, a polynucleotide of the invention comprises at least 15, at least 30, at least 50, at least 100 or at least 250, at least 500, or at least 1000 contiguous nucleotides of a Neutrokine-alpha coding sequence, but does not comprise all or part of any Neutrokine-alpha intron. In another embodiment, the nucleic acid comprising the Neutrokine-alpha coding sequence does not contain coding sequences for genes flanking the genome (i.e., the 5 'or 3' sequences of the Neutrokine-alpha gene in the genome). In other embodiments, the polynucleotide of the invention does not contain more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2 or 1 coding sequence of the genes flanking the genome.

In another embodiment, the invention provides an isolated nucleic acid molecule comprising a polynucleotide that hybridizes under stringent conditions to a portion of a polynucleotide in a nucleic acid molecule of the invention as described above, for example: the sequence of the cDNA clone deposited under ATCC accession number 203518, complementary to the coding and/or non-coding sequences shown in FIGS. 1A and 1B (SEQ ID NO: 1), complementary to the coding and/or non-coding sequences shown in FIGS. 5A and 5B (SEQ ID NO: 18), complementary to the sequence shown in SEQ ID NO: 21, (i.e., transcribed, untranslated) sequence complementary to the coding and/or non-coding sequence shown in SEQ ID NO: 22, complementary to the sequence of the coding and/or non-coding sequence shown in SEQ ID NO: 27, complementary to the coding and/or non-coding sequence of SEQ id no: 29, or a fragment of such a sequence (e.g., an open reading frame or fragment thereof). "stringent hybridization conditions" refers to incubation at 42 ℃ overnight followed by washing the filters with 0.1XSSC at about 65 ℃ in a solution containing: 50% formamide, 5XSSC (750mM NaCl, 75mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 XDenhard's solution, 10% dextran sulfate, and 20. mu.g/ml denatured sheared salmon sperm DNA

A polynucleotide that hybridizes to "a portion" of a polynucleotide refers to a polynucleotide (DNA or RNA) that hybridizes to any integer number of nucleotides in length from at least 15, preferably at least 20, more preferably at least 30, and most preferably at least 30-70 (e.g., 40, 50, or 60), particularly preferably 30-70 or 80-150, and even more preferably to a full-length reference polynucleotide. The uses of these polynucleotides include, but are not limited to, use as diagnostic probes and primers, as described in detail below. A portion of a polynucleotide "at least about 20 nucleotides in length" is intended to include, for example, the specified range. Greater than or less than 5, 4, 3, 2, 1, or 0 amino acids of one or both ends of the nucleotide sequence of the reference polynucleotide (e.g., either or both of the two deposited cDNAs, the complementary strand of the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1), the complementary strand of the nucleotide sequence shown in FIGS. 5A and 5B (SEQ ID NO: 18), the complementary strand of the nucleotide sequence shown in SEQ ID NO: 21, the complementary strand of the nucleotide sequence shown in SEQ ID NO: 22, the complementary strand of the nucleotide sequence shown in SEQ ID NO: 27, and/or the complementary strand of the nucleotide sequence shown in SEQ ID NO: 29). Of course, a polynucleotide that hybridizes only to the sequence of a poly (A) sequence (e.g., the 3 ' terminal poly (A) sequence segment of the Neutrokine- α cDNA shown in FIGS. 1A and 1B (SEQ ID NO: 1), the 3 ' terminal poly (A) sequence segment of the Neutrokine- α SV cDNA shown in FIGS. 5A and 5B (SEQ ID NO: 18), or the 3 ' terminal poly (A) sequence segment of the Neutrokine- α SV cDNA shown in SEQ ID NO: 22), or a complementary sequence of T (or U) residues is not included in the polynucleotides of the present invention for hybridizing to a portion of a nucleic acid of the present invention, as such a polynucleotide will hybridize to any nucleic acid molecule (e.g., virtually any double-stranded cDNA clone generated using oligo dT as a primer) that contains a poly (A) sequence or a complementary sequence of an oligo dT residue.

As indicated above, nucleic acid molecules of the invention encoding Neutrokine-alpha or Neutrokine-alpha SV polypeptides may include, but are not limited to: a polynucleotide which itself encodes the amino acid sequence of the extracellular domain of the corresponding polypeptide; the coding sequence for the extracellular domain of the corresponding polypeptide and additional sequences, such as sequences encoding the intracellular domain and transmembrane domain sequences, or a preprotein, proprotein or preproprotein sequence; the coding sequence for the extracellular domain of the corresponding polypeptide with or without the aforementioned additional coding sequences.

The nucleic acids of the invention also encode the above-described protein sequences with additional non-coding sequences, including, for example, but not limited to, introns and non-coding 5 'and 3' sequences, such as transcribed, untranslated sequences that function in transcription, mRNA processing including splicing and polyadenylation signals, e.g., chromosome binding and mRNA stability; additional coding sequences that encode additional amino acids, such as those that provide additional functionality.

Thus, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide. In some preferred embodiments of the invention, the marker amino acid sequence is a 6-histidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc, 9259 Eton, Averue, Chatsworth, CA, 91311), many of which are commercially available. Gentz et al, proceedings of the American academy of sciences 86: 821-824(1989), 6-histidine allows for routine purification of the fusion protein. The "HA" tag is another peptide used for purification, which corresponds to an epitope derived from the influenza hemagglutinin protein, as set forth by Wilson et al, cell 37: 767(1984). Other such fusion proteins include the Neutrokine-alpha or Neutrokine-alpha SV polypeptide fused to Fc at the N-or C-terminus, as described below.

The present invention also relates to variants of the nucleic acid molecules of the invention, which encode a nucleic acid sequence of SEQ ID NO: 2 or a portion, analog or derivative of a Neutrokine-alpha or Neutrokine-alpha SV polypeptide. Variants may be naturally occurring, such as natural allelic variants. An "allelic" variant refers to one of several variations of a gene occupying a given locus on a chromosome of an organism. See Gene II, Lewis, B.ed, John Wiley & Sons, New York (1985). Non-naturally occurring variants can be generated by mutagenesis methods known in the art, including, but not limited to, oligonucleotide-mediated mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis (see, e.g., Carter et al, nucleic acids Res. 13: 4331 (1986); and Zoller et al, nucleic acids Res. 10: 6487 (1982)), cassette mutagenesis (see, e.g., Wells et al, Gene 34: 315(1985)), and restriction-selection mutagenesis (see, e.g., Wells et al, Philos. Trans. R. Soc. London SerA 317: 415 (1986)).

Such variants include those produced by nucleotide substitution, deletion or addition. The substitution, deletion or addition may comprise one or more amino acids. Variants may be changes in the coding region, the non-coding region or both. Changes in the coding region may result in conservative or non-conservative amino acid substitutions, deletions or additions. Particularly preferred among these are silent substitutions, additions and deletions which do not alter the properties and activity of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or parts thereof. Also particularly preferred are conservative amino acid substitutions.

Other embodiments of the invention relate to isolated nucleic acid molecules comprising a polynucleotide encoding an amino acid sequence of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide having an amino acid sequence containing at least 1 but not more than 50, preferably not more than 40, more preferably not more than 30, especially preferably not more than 20, 10-20, 5-10, 1-5, 3-5, or 1-3 conservative amino acid substitutions. Of course, preferred sequences are, in order, polynucleotides encoding Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides that contain an amino acid sequence of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative amino acid substitution.

Other embodiments include isolated nucleic acid molecules comprising or consisting of a polynucleotide having a nucleotide sequence at least 80%, 85% or 90% identical, preferably at least 95%, 96%, 97%, 98%, or 99% identical to a polynucleotide selected from the group consisting of: (a) a nucleotide sequence encoding a Neutrokine-alpha polypeptide having the complete amino acid sequence shown in FIGS. 1A and 1B (i.e., positions 1-285 of SEQ ID NO: 2); (b) a nucleotide sequence encoding a Neutrokine-alpha polypeptide having the complete amino acid sequence except the N-terminal methionine shown in FIGS. 1A and 1B (i.e., amino acids 2-285 of SEQ ID NO: 2); (c) a fragment of (b) a polypeptide having Neutrokine-alpha functional activity (e.g., antigenic or biological activity); (d) a nucleotide sequence encoding the putative extracellular domain of a Neutrokine-alpha polypeptide having the amino acid sequence at positions 73-285 of FIGS. 1A and 1B (SEQ ID NO: 2); (e) a nucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 of FIGS. 1A and 1B (SEQ ID NO: 2); (f) a nucleotide sequence encoding a Neutrokine-alpha polypeptide having the complete amino acid sequence encoded by the cDNA clone deposited under ATCC accession No. 97768; (g) a nucleotide sequence encoding an extracellular domain of a Neutrokine-alpha polypeptide having the amino acid sequence encoded by the cDNA deposited under ATCC accession No. 97768; and (h) a nucleotide sequence complementary to any of the nucleotide sequences of (a), (b), (c), (d), (e), (f), (g) or (h) above. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotides and nucleic acid molecules are also encompassed by the present invention.

A particularly preferred embodiment of the invention relates to nucleic acid molecules comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 80%, 85%, 90% identical, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 of FIGS. 1A and 1B (SEQ ID NO: 2). A preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 90% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 of FIGS. 1A and 1B (SEQ ID NO: 2). A more preferred embodiment of the invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 95% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 of FIGS. 1A and 1B (SEQ ID NO: 2). A more preferred embodiment of the invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 96% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 of FIGS. 1A and 1B (SEQ ID NO: 2).

In addition, a more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 97% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 of FIGS. 1A and 1B (SEQ ID NO: 2). A more preferred embodiment of the invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 98% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 of FIGS. 1A and 1B (SEQ ID NO: 2). A more preferred embodiment of the invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 of FIGS. 1A and 1B (SEQ ID NO: 2).

Another embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide encoding an amino acid sequence of a Neutrokine-alpha SV polypeptide (a fragment of a Neutrokine-alpha SV polypeptide as described herein) containing at least 1 but not more than 50, preferably not more than 40, more preferably not more than 30, and most preferably not more than 20 conservative amino acid substitutions. Of course, particularly preferred polynucleotides are those encoding the amino acid sequence of a Neutrokine-alpha polypeptide having an amino acid sequence with no more than 7-10, 5-10, 3-7, 3-5, 2-5, 1-5, 1-3, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative amino acid substitution.

Other embodiments include isolated nucleic acid molecules comprising or consisting of a polynucleotide having a nucleotide sequence at least 80%, 85%, 90% identical, preferably at least 95%, 96%, 97%, 98%, or 99% identical to a polynucleotide selected from the group consisting of: (a) a nucleotide sequence encoding a Neutrokine- α SV polypeptide having the complete amino acid sequence shown in fig. 5A and 5B (i.e., positions 1-266 of SEQ id no: 19); (b) a nucleotide sequence encoding a Neutrokine-alpha SV polypeptide having the complete amino acid sequence except the N-terminal methionine shown in FIGS. 5A and 5B (i.e., positions 2-266 of SEQ ID NO: 19); (c) a nucleotide sequence encoding the putative extracellular domain of a Neutrokine- α SV polypeptide having the amino acid sequence at positions 73-285 of positions 5A and 5B (SEQ ID NO: 19); (d) a nucleotide sequence encoding a Neutrokine- α SV polypeptide having the complete amino acid sequence encoded by the cDNA clone deposited under ATCC accession No. 203518; (e) a nucleotide sequence encoding an extracellular domain of a Neutrokine- α SV polypeptide having the amino acid sequence encoded by the cDNA clone deposited under ATCC accession No. 203518; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d) or (e) above.

In addition, the polynucleotides encompassed by the present invention comprise or consist of a sequence that is at least 90% or 95% identical to any portion of at least about 10, 20, 25 or 30 contiguous nucleotides, preferably at least about 40 or 50 contiguous amino acids of the nucleotide sequence 1-1082 in FIGS. 1A and 1B (SEQ ID NO: 1), preferably excluding the nucleotide sequences identified from the 4 cDNA clones described above and the nucleotide sequences at positions 797, 1082, 810, 1082 and 346, 542. The present invention also includes a polynucleotide comprising or consisting of a sequence that is at least 90% or 95% identical to any portion of about 10, 20, 25 or 30 contiguous nucleotides, preferably at least about 40 or 50 nucleotides, of the sequence shown in FIGS. 5A and 5B (SEQ ID NO: 18), preferably excluding the nucleotide sequence determined from the 4 cDNA clones described above. The invention also includes a polynucleotide comprising or consisting of a sequence at least 90% or 95% identical to seq id NO: 21, preferably at least about 40 or 50 contiguous nucleotides, preferably excluding the nucleotide sequence determined from the 4 cDNA clones described above. The invention also includes a polynucleotide comprising or consisting of a sequence at least 90% or 95% identical to SEQ ID NO: 22, preferably at least about 40 or 50 contiguous nucleotides, preferably excluding the nucleotide sequence determined from the 4 cDNA clones described above. The invention also includes a polynucleotide comprising or consisting of a sequence at least 90% or 95% identical to SEQ ID NO: 27, preferably does not comprise the nucleotide sequence determined from the 4 cDNA clones described above, and preferably consists of a sequence of any portion of at least about 10, 20, 25 or 30 contiguous nucleotides, preferably at least about 40 or 50 contiguous nucleotides. The invention also includes a polynucleotide comprising or consisting of a sequence at least 90% or 95% identical to SEQ ID NO: 29, and preferably does not include the nucleotide sequence determined from the 4 cDNA clones described above, and preferably consists of a sequence of any portion of at least about 10, 20, 25 or 30 contiguous nucleotides, and preferably at least about 40 or 50 contiguous nucleotides. The term "about" as used herein includes the specified range, as well as more or less (i.e., 5, 4, 3, 2 or 1) amino acids at one or both ends.

A polynucleotide having a nucleotide sequence that is, for example, at least 95% "identical" to a reference nucleotide sequence encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include more than 5 mismatches per 100 nucleotides of the reference sequence encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference sequence, approximately 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or nucleotides accounting for 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. Such mutations in the reference sequence may occur at the 5 'or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, interspersed between individual nucleotides or one or more contiguous groups in the reference sequence. The reference (reference) sequence may be the entire nucleotide sequence encoding Neutrokine-alpha and/or Neutrokine-alpha SV as shown in FIGS. 1A and 1B (SEQ ID NO: 1) and FIGS. 5A and 5B (SEQ ID NO: 18), respectively, or any Neutrokine-alpha such as SEQ ID NO: 21, 22, 27, 28, or a fragment of any of the Neutrokine-alpha or Neutrokine-alpha SV polynucleotides described herein.

Indeed, whether any particular nucleic acid molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide sequence shown in fig. 1A and 1B, or the nucleotide sequence shown in fig. 5A and 5B, or the nucleotide sequence of a deposited cDNA clone, or any Neutrokine-alpha polynucleotide such as SEQ ID NO: the Neutrokine-alpha polynucleotide or fragment thereof, as set forth in 21, 22, 27 or 28, can be routinely determined using known computer programs (Wisconsin sequence analysis Package, Version 8 for Unix, genetics computer group, university research park, 575 Seience Drive, Madison, Wis 53711). Bestfit found the best segment of homology between the two sequences using the local homology alignment program of Smith and Waterman (applied math Advance 2: 482-. When using Bestfit or any other sequence comparison program to determine whether a particular sequence is, for example, 95% identical to a reference sequence of the invention, the parameters will, of course, be set such that the percent identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of 5% over the total number of nucleotides in the reference sequence are allowed.

In a specific embodiment, the identity between the reference (reference) sequence (the sequence of the invention) and the target sequence, also referred to as global alignment, is determined using the FAS TDB computer program based on alignment of Brutlag and co-workers (Comp. App. biosci 6: 237-. In a sequence alignment, both the reference and target sequences are DNA sequences. RNA sequences can be aligned by converting U to T. The results of the overall alignment are expressed as percent identity. Preferred parameters for FASTDB sequence alignment of DNA sequences to calculate percent identity are: matrix equals to unity, k-tuple equals to 4, Mismatch Penalty equals to 1, Joining Penalty equals to 30, Randomization Group Length equals to 0, Cutoff Score equals to 1, Gap Penalty equals to 5, Gap Size Penalty equals to 0.05, Window Size equals to 500 or the target nucleotide sequence Length, whichever is shorter. According to this embodiment, if the target sequence is shorter than the reference sequence due to a 5 'or 3' deletion rather than due to an intrinsic deletion, manual adjustment of the result is made because the FASTDB program does not take into account the 5 'and 3' truncations of the target sequence when calculating the percent identity. For target sequences that are truncated at the 5 'and 3' ends relative to the reference sequence, the percent identity is adjusted by calculating the percentage of unmatched/aligned bases of the 5 'and 3' reference sequences of the target sequence to the total bases of the reference sequence. The determination of whether a nucleotide is a match/alignment is determined by the result of a FASTDB sequence alignment. This percentage is then subtracted from the percentage of identity calculated by the FASTDB program described above using the specified parameters to obtain the final percentage of identity. The result of this adjustment is used for the purpose of this embodiment. Only bases outside the 5 'and 3' bases of the target sequence, which are not matched/aligned to the reference sequence, as shown by FASTDB sequence alignment, need to be calculated to manually adjust the percent identity results. For example, a 90 base target sequence is compared to a 100 base reference sequence to determine percent identity. Deletions occur at the 5 'taste end of the target sequence, and thus FASTDB sequence alignment does not show matches/alignments of the first 10 bases at the 5' end. The 10 unpaired bases represent 10% of the sequence (number of unmatched bases at the 5 'and 3' ends/total base of the reference sequence) and therefore these 10% should be subtracted from the percent identity results calculated by the FASTDB program. If the remaining 90 bases are sufficiently matched, the final percent identity is 90%. In another embodiment, a 90 base target sequence is compared to a 100 base reference sequence. This deletion is an intrinsic deletion and thus bases are provided at the 5 'and 3' ends of the target sequence that do not match/align with the reference sequence. In this case, the percent identity calculated from FASTDB does not need to be adjusted manually. Similarly, manual adjustment is only required if the 5 'and 3' bases of the target sequence do not match/align with the reference sequence. This embodiment does not require additional manual adjustment.

The present invention relates to nucleic acid molecules that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences (i.e., polynucleotides) disclosed herein, e.g., the sequences shown in FIGS. 1A and 1B (SEQ ID NO: 1) or the nucleotide sequences of the deposited cDNAs, whether they encode polypeptides having Neutrokine-alpha and/or Neutrokine-alpha SV functional activity (e.g., biological activity). In addition, the present invention also relates to nucleic acid molecules that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences shown in FIGS. 5A and 5B (SEQ ID NO: 18) or the nucleic acid sequences of the deposited cDNAs, whether or not they encode a polypeptide having Neutrokine- α SV activity. In addition, the invention also relates to a polypeptide having the sequence shown in SEQ ID NO: 21, 22, 27 or 28, whether or not they encode a polypeptide having Neutrokine-alpha activity. This is because even if a particular nucleic acid molecule does not encode a polypeptide having Neutrokine-alpha and/or Neutrokine-alpha SV activity, the skilled person will know how to use this nucleic acid molecule, for example as a hybridization probe or a Polymerase Chain Reaction (PCR) primer. Uses of the nucleic acid molecules of the invention that do not encode polypeptides having Neutrokine-alpha and/or Neutrokine-alpha SV activity include: (1) isolating Neutrokine-alpha and/or Neutrokine-alpha SV gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal smears to provide accurate chromosomal location of the Neutrokine-alpha and/or Neutrokine-alpha SV genes, as described by Verma et al, handbook of human chromosome basic methods, Pergamon Press, New York (1988); and Northern blot analysis to detect the expression of Neutrokine-alpha and/or Neutrokine-alpha SVmRNT in specific tissues.

However, preferred nucleic acid molecules are those that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences disclosed herein (e.g., the nucleotide sequences shown in FIGS. 1A and 1B (SEQ ID NO: 1), or to the nucleic acid sequence of the deposited cDNA, or fragments thereof) and which in fact encode a polypeptide having functional activity (e.g., biological activity) of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide. Also preferred are nucleic acid molecules which have at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the nucleic acid sequences shown in FIGS. 5A and 5B (SEQ ID NO: 18), or the nucleic acid sequences of the deposited cDNAs, and which in fact encode polypeptides having functional activity (e.g., biological activity) of the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides. It is also preferred to have a sequence identical to SEQ ID NO: 21, 22, 27, or 28, and which in fact encodes a polypeptide having functional activity (e.g., biological activity) of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide.

"polypeptide having functional activity (e.g., biological activity) of a Neutrokine-alpha polypeptide" and "polypeptide having functional activity (e.g., biological activity) of a Neutrokine-alpha SV polypeptide" refer to polypeptides which exhibit substantially similar, but not necessarily identical, activity as the activity of the extracellular domain or full-length polypeptide of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide of the invention, as determined in a particular functional assay (e.g., immunological or biological assay). For example, the functional activity of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide may be determined by the ability of the polypeptide sequence to form multimers (e.g., homodimers and homotrimers) with the entire Neutrokine-alpha and/or Neutrokine-alpha SV or the extracellular domain of Neutrokine-alpha and/or Neutrokine-alpha SV, and the ability to bind ligands of the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide. Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide functional activity may also be measured by determining the ability of a polypeptide of the invention to induce proliferation, differentiation or activation of lymphocytes (e.g., B cells) and/or prolong B cell survival. These functional assays can be routinely performed using the methods described herein (see, e.g., example 6) and other methods known in the art. In addition, the Neutrokine-alpha or Neutrokine-alpha SV polypeptide of the present invention modulates cell proliferation, cytotoxicity, cell survival and cell death. The effect of a protein on certain cells can be determined by in vitro cell proliferation, cytotoxicity, cell survival and cell death assays using reagents for detecting cell replication and/or death that are well known in the art and are generally commercially available. For example, many such assays for TNF-related protein activity are found in various references. Briefly, such assays, for example, involve collecting human or animal (e.g., murine) cells and mixing them with (1) a supernatant of transfected host cells containing Neutrokine-alpha protein (or candidate polypeptide), or (2) a supernatant of control untransfected host cells, and after a certain period of incubation, determining the effect on cell number or viability. Such cell proliferation and/or survival modulating activity as may be determined in such assays is useful in the treatment of tumors, tumor metastases, infections, autoimmune diseases, inflammation and other immune related diseases.

Neutrokine-alpha regulates cell proliferation and differentiation in a dose-dependent manner in the above assay. Thus, preferred "polypeptides having functional activity (e.g., biological activity) of a Neutrokine-alpha polypeptide" include polypeptides that also exhibit any of a number of cell modulating (particularly immunomodulatory) activities in the above assays in a dose-dependent manner. Although the degree of dose-dependent activity need not be equivalent to that of the Neutrokine-alpha polypeptide, it is preferred that "a polypeptide having functional activity of a Neutrokine-alpha polypeptide" exhibits substantially similar dose-dependence in a given activity as compared to the Neutrokine-alpha polypeptide (i.e., the candidate polypeptide exhibits a higher activity, or no less than 25 times, preferably no less than 10 times, the activity as compared to the reference Neutrokine-alpha polypeptide).

In certain preferred embodiments, "a polypeptide having functional activity (e.g., biological activity) of a Neutrokine-alpha polypeptide" and "a polypeptide having functional activity (e.g., biological activity) of a Neutrokine-alpha SV polypeptide" include polypeptides that also exhibit modulating (particularly immunomodulatory) activity of any of the same B cells (or other cells) as described in figures 8A, 8B, 9A, 9B, 11, 10, 12A and 12B and example 6.

Like other members of the TNF family, Neutrokine-alpha appears to act on blood cells including, for example, monocytes, lymphocytes (e.g., B cells) and neutrophils. Therefore, Neutrokine- α is active in inducing proliferation, differentiation and migration of these cells. This activity is useful for immune enhancement or suppression, bone marrow protection, stem cell migration, control of acute and chronic inflammation, and treatment of leukemia. Methods for determining such activity are known in the art. See, e.g., Peters et al, modern immunology 17: 273 (1996); young et al, journal of experimental methods 182: 1111 (1995); caux et al, Nature 390: 258 (1992); and Santiago-Schwarz et al, biological Experimental Process development 378: 7 (1995).

Of course, due to the degeneracy of the genetic code, one of skill in the art will immediately recognize that a large number of nucleic acid molecules having a nucleic acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to that contained in the cDNA clone contained in the deposit with ATCC accession number 97768, or the nucleic acid sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1), or fragments thereof, will encode a polypeptide "having functional activity (e.g., biological activity)". One of skill in the art will also immediately recognize that a number of nucleic acid molecules encoding a "polypeptide having functional activity (e.g., biological activity) of a Neutrokine-alpha SV polypeptide" have a sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence contained in the cDNA clone deposited under ATCC accession No. 203518, or the nucleic acid sequence shown in fig. 5A and 5B (SEQ ID NO: 18). In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled worker even without the above comparative analysis. It is further recognized in the art that such nucleic acid molecules, which are not degenerate variants, have some that also encode polypeptides having Neutrokine-alpha and/or Neutrokine-alpha SV activity. This is because it is well recognized by those skilled in the art that amino acid substitutions have little or no significant effect on protein function (e.g., replacement of one aliphatic amino acid with another aliphatic amino acid) as described in detail below.

Similarly, a nucleic acid encoding a polypeptide comprising all or part of SEQ ID NO: 2, appears to be a diagnostically and therapeutically valuable polynucleotide for detecting and/or altering the expression of Neutrokine-alpha or Neutrokine-alpha SV polynucleotide. In addition, spanning SEQ ID NO: the polynucleotide shown in 19 as the binding portion of the amino acid residues T141 and G142 of the Neutrokine-alpha SV polypeptide, between which the amino acid sequence V142-K160 of Neutrokine-alpha is apparently inserted, also appears to have diagnostic and therapeutic effects. This T141/G142 exhibits a higher probability of hybridizing to a Neutrokine- α SV polynucleotide than to a Neutrokine- α polynucleotide across the polynucleotide. Some non-limiting, non-exclusive examples of such Neutrokine- α SV encoded by the polynucleotides of the present invention comprise or consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: G121-E163 of 19; E122-E163; G123-E163; N124-E163; S125-E163; S126-E163; Q127-E163; N128-E163; S129-E163; R130-E163; N131-E163; K132-E163; R133-E163; A134-E163; V135-E163; Q136-E163; G137-E163; P138-E163; E139-E163; E140-E163; T141-E163; G142-E163; S143-E163; Y144-E163; T145-E163; F146-E163; U147-E163; P148-E163; W149-E163; L150-E163; L151-E163; S152-E163; F153-E163; K154-E163; R155-E163; G156-E163; S157-E163; A158-E163; L159-E163; E160-E163; E161-E163; K162-E163; G121-K162; G121-E161; G121-E160; G121-L159; G121-A158; G121-S157; G121-G156; G121-R155; G121-K154; G121-F153; G121-S152; G121-L151; G121-L150; G121-W149; G121-P148; G121-V147; G121-F146; G121-T145; G121-Y144; G121-S143; G121-G142; G121-T141; G121-E140; G121-E139; G121-P138; G121-G137; G121-Q136; G121-V135; G121-A134; G121-R133; G121-K132; G121-N131; G121-R130; G121-S129; G121-N128; G121-Q127; G121-S126; G121-S125; G121-N124; G121-G123; and G121-E122.

Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

Vectors and host cells

The invention also relates to a vector comprising the isolated DNA molecule of the invention, a host cell genetically engineered or otherwise engineered with the recombinant vector to produce the polypeptide of the invention, and a method for producing Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides by recombinant or synthetic means.

In one embodiment, the polynucleotide of the invention is linked to a vector (e.g., a cloning or expression vector). The vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally occurs only in the complementing host cell. The polynucleotide may be linked to a vector containing a selectable marker for propagation in a host. Introduction of the vector construct into a host cell can be carried out by techniques known in the art, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic liposome mediated transfection, electroporation, transduction, infection, or other methods. These methods are described in many standard laboratory manuals, such as Davis et al, molecular biology based methods (1986).

In general, recombinant expression vectors include an origin of replication and a selectable marker that allows transformation of host cells, such as the ampicillin resistance gene of E.coli and the s.cerevisiae TRP1 gene, and a promoter derived from a highly expressed gene that directs transcription of downstream structural sequences. Such promoters may be derived from an operon encoding a glycolytic enzyme such as 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heat shock protein. The heterologous structural sequences are assembled in the appropriate state with translation initiation and termination sequences, preferably leader sequences which directly secrete the translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence may encode a fusion protein comprising an N-terminal identifying peptide having the desired characteristics, e.g., stably or simply purifying the expressed recombinant product.

In one embodiment, the DNA of the invention is combined with suitable heterologous regulatory elements (e.g., promoters or enhancers), such as the lambda-phage PL promoter, E.coli Lac, Trp, PhoA and tac promoters, SV40 early and late promoters, and promoters of retroviral LTRs, among others. Other suitable promoters are also known in the art.

As mentioned above, the expression vector preferably comprises at least one selectable marker. Such markers include the dihydrofolate reductase, G418 or neomycin resistance genes in eukaryotic cell cultures and the tetracycline, kanamycin or ampicillin resistance genes in E.coli and other bacterial cultures. Suitable hosts include, for example, but are not limited to, bacterial cells such as E.coli, Streptomyces, Salmonella typhimurium; fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf 9 cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; and plant cells. Suitable media and culture conditions for the above-described host cells are known in the art.

The host cell may be a higher eukaryotic cell, such as a mammalian cell (e.g., a human-derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell may be a prokaryotic cell, such as a bacterial cell. Host strains can be selected that modulate the expression of the inserted gene sequences, or that modify and process the gene product, in a particular manner as desired. Expression from certain promoters may be increased in the presence of certain inducers; thus, expression of the genetically engineered polypeptide can be controlled. In addition, different host cells have the characteristics and specific mechanisms of translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be selected to enhance the desired modification and processing of the exogenously expressed protein. The selection of appropriate vectors and promoters for expression in a host cell is well known in the art, and the necessary methods for expression vector construction, introduction of the vector into a host, and expression in a host are also known to those skilled in the art.

Efficient expression vectors for use in bacteria are constructed by inserting a structural DNA sequence encoding the desired protein, together with appropriate translation initiation and termination signals, into an operable reading phase having a functional promoter. The vector comprises one or more phenotypic selectable markers and an origin of replication to maintain the vector and, if desired, provide for amplification within the host. Suitable prokaryotic hosts for transformation include E.coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although other species may alternatively be used. Expression vectors for use in cells typically, but not by way of limitation, may comprise a selectable marker and a bacterial origin of replication derived from a commercially available plasmid comprising the genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Such commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1(Promega Biotec, Madison, Wis., USA). These pBR322 "backbone" parts are combined with appropriate promoters and expressed structural sequences. Preferred vectors for bacteria include pHE4-5 (ATCCNO.209311; and variants thereof), pQE70, pQE60, and pQE-9, available from QIAGEN; pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16A, pNH18A, pNH46A, available from Stratagene; ptrc 99a, pKK223-3, pKK233-3, pDR540, pRIT5 from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZ α, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC 3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, Calif.). Preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG, available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL available from Pharmaeia.

Other suitable vectors are also known to those skilled in the art.

After transformation and growth of the appropriate host strain to the appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time. Cells were harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract was retained for further purification.

Microbial cells for protein expression can be disrupted by any conventional method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, which methods are well known to those skilled in the art.

In one such embodiment, Pichia pastoris is used in eukaryotic cell systems for the expression of Neutrokine-alpha protein. Pichia pastoris is a methylotrophic yeast that metabolizes methanol as its sole carbon source. One of the major steps in the methanol metabolic pathway is methanol and O₂Oxidized to formaldehyde. This reaction is catalyzed by alcohol oxidases. Pichia pastoris must produce high levels of alcohol oxidase for metabolism of methanol as its sole carbon source, since alcohol oxidase is associated with O₂Of (2)The harmony is low. Therefore, the promoter region of one or both of the alcohol oxidase genes (AOX 1) is highly active in growth media in which methanol is the main carbon source. In the presence of methanol, the alcohol oxidase produced from the AOX 1 gene comprises more than about 30% of the total soluble protein in pichia pastoris. See Ellis, s.b. et al, cell molecular biology 5: 1111-21 (1985); koutz, p.j. et al, yeast 5: 167-77 (1989); tschopp, j.f., et al, nucleic acids study 15: 3859-76(1987). Thus, heterologous coding sequences such as Neutrokine-alpha or Neutrokine-alpha SV polypeptides of the invention are expressed at unusually high levels in Pichia grown in the presence of methanol under the transcriptional regulation of all or part of the AOX 1 regulatory sequence.

In one embodiment, the plasmid vector pPIC9K is used to express DNA encoding a Neutrokine-alpha or Neutrokine-alpha SV polypeptide in a Pichia system essentially as described in the "Pichia scheme: methods in molecular biology ", edited by d.r.higgins and j.cregg, Humana press, Totowa, NJ, 1998. This expression vector expresses and secretes the Neutrokine-alpha or Neutrokine-alpha SV protein of the present invention by means of a strong AOX 1 promoter linked to a pichia pastoris alkaline Phosphatase (PHO) secretion signal peptide (i.e., leader sequence) located upstream of the multiple cloning site.

Those skilled in the art will recognize that many other yeast vectors may be used in place of pPIC9K, such as pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZ α, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC 3.5K, and PAO815, so long as the proposed expression construct provides the appropriate localization signals for transcription, translation, secretion (if necessary), and the like, including the desired in-frame AUG.

In one embodiment, high level expression of a heterologous coding sequence, such as a Neutrokine-alpha or Neutrokine-alpha SV polynucleotide of the invention, can be obtained by cloning a heterologous polynucleotide of the invention into an expression vector, such as pGAPZ or pGAPZ alpha, and culturing the yeast in the absence of fermentation.

The DNA encoding the polypeptide of the present invention is improved by transcription in higher eukaryotes and insertion of an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually 10-300bp in length, that act on a promoter to increase its transcription. For example, the SV40 enhancer comprising 100-270 bp on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer may be included.

Various mammalian cell culture systems can also be used to express recombinant proteins. Mammalian expression lines include, for example, the COS-7 cell line including monkey kidney fibroblasts described by Gluzman (cell 23: 175(1981)), and other cell lines capable of expressing compatible vectors, such as the C127, 3T3, CHO, Hela and BHK cell lines. Mammalian expression vectors contain an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcription termination sequences, and 5' flanking untranscribed sequences. DNA sequences derived from the SV40 spliceosome, as well as polyadenylation sites, may also be used to provide the desired untranscribed genetic element.

In a specific embodiment, a construct expressing a portion of the extracellular domain of Neutrokine-alpha (e.g., amino acid residues Ala 134-Leu 285) is preferred. Those skilled in the art will be able to use the sequences respectively represented by SEQ ID NOs: 1 and SEQ ID NO: 2 or SEQ ID NO: 18 and SEQ ID NO: 19 to yield such an expression construct.

In another embodiment, a construct expressing the entire putative extracellular domain of Neutrokine- α (i.e., Gln73-Leu285 amino acid residues) is preferred. Those skilled in the art will be able to use the sequences respectively represented by SEQ ID NOs: 1 and SEQ ID NO: 2 or SEQ ID NO: 18 and SEQ ID NO: 19 to yield such an expression construct.

In addition to host cells containing the vector constructs, the invention also encompasses primary, secondary, and immortalized vertebrate, especially mammalian, host cells that have been engineered to delete or replace endogenous genetic material (e.g., a Neutrokine-alpha coding sequence) and/or include genetic material (e.g., a heterologous polynucleotide sequence) that is associated with, activates, alters, and/or amplifies a Neutrokine-alpha polynucleotide of the invention. For example, methods known in the art can be used to operably bind a heterologous control region (e.g., promoter and/or enhancer) to an endogenous Neutrokine-alpha polynucleotide sequence by homologous combination (see, e.g., U.S. Pat. No.5641670 issued 24/6/1997; International publication No. WO 96/29411 published 26/9/1996; International publication No. WO 94/12650 published 4/8/1994; Koller et al, Proc. Natl. Acad. Sci. USA 86: 8932-.

The aforementioned host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, cell-free translation systems may also be used to produce polypeptides of the invention using RNA derived from DNA constructs of the invention.

The polypeptides of the invention may be expressed or synthesized in modified forms, such as fusion proteins (including polypeptides joined by peptide bonds to heterologous protein sequences (of different proteins)), and may include not only secretion signals, but also additional heterologous functional regions. Such fusion proteins can be produced by ligating the polynucleotides of the invention and the desired nucleic acid sequence encoding the desired amino acid sequence to each other in appropriate reading frame and expressing the fusion protein product by methods known in the art. Alternatively, such fusion proteins can be produced by protein synthesis methods, such as using a peptide synthesizer. Thus, for example, additional amino acid regions, particularly polar amino acids, can be added to the N-terminus of the polypeptide to improve stability and persistence during purification, or during subsequent retention and storage. Likewise, peptide components may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide components to polypeptides to produce secretion or excretion, or to improve stability and facilitate purification, etc., is a well known and commonly used method in the art.

In one embodiment, a polynucleotide encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide of the invention may be fused to a pelB pectate lyase signal sequence to improve the expression and purification efficacy of such a polypeptide in gram-negative bacteria. See U.S. patent nos. 5576195 and 5846818, which are incorporated by reference in their entirety.

A preferred fusion protein comprises a heterologous region of an immunoglobulin that is used to stabilize and purify the protein. For example, EP-A-0464533 (Canadian counterpart 2045869) discloses fusion proteins comprising portions of the constant region of an immunoglobulin molecule and other human proteins or portions thereof. In many cases, the Fc part of the fusion protein is very advantageous for use in therapy and diagnosis, thus improving, for example, the pharmacokinetic properties (EP-A0232262). On the other hand, it is desirable to be able to remove this Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described above. This is the case when the Fc portion is a barrier in therapy and diagnosis, for example when the fusion protein is used as an antigen for immunization. In drug development, for example, human proteins such as hIL-5 have been fused to Fc portions for high throughput screening assays to identify antagonists of hIL-5. See d.bennett et al, journal of molecular recognition 8: 52-58(1995) and K.Johanson et al, J. Biochem 270: 9459-9471(1995).

Polypeptides of the present invention include naturally purified products, chemically synthesized products, and products produced by recombinant means from prokaryotic or eukaryotic hosts, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending on the host used in the recombinant production method, the polypeptide of the invention may be glycosylated or non-glycosylated. In addition, the polypeptides of the invention may also include initially modified methionine residues resulting from host-mediated processing.

Polypeptides of the invention may be chemically synthesized using techniques known in the art (e.g., Creighton, 1983, principles of protein Structure and molecular biology, W.H.Freeman & Co., N.y., and Hunkapiller, M., et al, 1984, Nature 310: 105-111). For example, peptides corresponding to fragments of an intact Neutrokine-alpha or Neutrokine-alpha SV polypeptide of the invention may be synthesized using a peptide synthesizer. In addition, non-canonical amino acids or chemical amino acid analogs can be introduced as substitutions or additions to the Neutrokine-alpha or Neutrokine-alpha SV polynucleotide sequence, if desired. Non-classical amino acids include, but are not limited to, the D isomer of a common amino acid, 2, 4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, n-citrulline, cystine, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer (designer) amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and general analogs of amino acids. In addition, the amino acid may be D (dextrorotatory) or L (levorotatory).

The present invention encompasses Neutrokine-alpha or Neutrokine-alpha SV polypeptides that are specifically modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to antibody molecules or other cellular ligands, and the like. Any chemical modification can be carried out by known methods, including, but not limited to, by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄Chemical cleavage, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, and the like.

Other post-translational modifications contemplated by the invention include, for example, N-linked or O-linked carbohydrate chains, processing of the N or C terminus, attachment of chemical components to the amino acid backbone, chemical modification of N-linked or O-linked carbohydrate chains, and addition or deletion of the N-terminal methionine residue due to expression in a prokaryotic host cell. The polypeptide may also be modified with a detectable label, such as an enzymatic label, a fluorescein label, an isotope or an affinity label that can detect and isolate the protein. In addition, the polypeptides of the invention may be modified by iodination.

In one embodiment, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention may also be labeled with biotin. In another related embodiment, biotinylated Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention, for example, may be used as imaging agents or tools for identifying one or more Neutrokine-alpha and/or Neutrokine-alpha SV receptors or other co-receptor or co-ligand molecules.

The present invention also provides chemically modified derivatives of Neutrokine-alpha or Neutrokine-alpha SV which have additional advantages such as increased solubility, stability and in vivo or in vitro circulation time of the polypeptide, or reduced immunogenicity (see U.S. patent No. 4179337). The chemical component used for derivatization may be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. The polypeptide may be modified at random positions within the molecule, or at predetermined positions within the molecule, and may include 1, 2, 3, or more chemical components attached.

The polymer may be of any molecular weight and may be branched or unbranched. In the case of polyethylene glycol, the preferred molecular weight is between about 1-100kDa (the term "about" means that some molecules are heavier or lighter than the standard molecular weight in the preparation of polyethylene glycol) for ease of use and manufacture. Other sizes of polyethylene glycol may be used depending on the desired therapeutic criteria (e.g., duration of desired sustained release, effect on biological activity, ease of use, degree or loss of antigenicity, and known effect of polyethylene glycol on a therapeutic protein or analog). For example, the average molecular weight of the polyethylene glycol can be about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, 20000, 25000, 35000, 40000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000, or 100,000 KDa.

As indicated above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. patent nos. 5643575; morpurgo et al, Biochemical Biotechnology applications 56: 59-72 (1996); vorobjev et al, nucleotide 18: 2745-2750 (1999); and Caliceti et al, bioconjugate chemistry 10: 638-.

The effect of polyethylene glycol molecules (or other chemical components) on functional protein domains or antigenic domains should be considered when attaching them to proteins. A number of attachment methods are known in the art, such as EPO 401384 (PEG coupled to G-CSF), also found in Malik et al, exp. 1028 (1035) (pegylation of GM-CSF with tresyl chloride) described in (1992). For example, polyethylene glycol can be covalently bound to an amino acid residue through a reactive group such as a free amino or carboxyl group. Reactive groups are those groups to which the activated polyethylene glycol molecule can bind. Amino acid residues having a free amino group may, for example, include lysine residues and the N-terminal amino acid residue; the amino acid residue having a free carboxyl group may include aspartic acid residue, glutamic acid residue and C-terminal amino acid residue. Thiol groups may also be used as reactive groups for attaching polyethylene glycol molecules. For therapeutic purposes it is preferred to attach at the amino group, e.g. at the N-terminus or at the lysine group.

As indicated above, polyethylene glycol can be attached to proteins by linkage to any amino acid. For example, polyethylene glycol can be attached to a protein by covalent bonds to lysine, histamine, aspartic acid, glutamic acid, or cysteine. One or more reactive chemistries may be used to attach the polyethylene glycol to a specific amino acid residue of the protein (e.g., lysine, histamine, aspartic acid, glutamic acid, or cysteine), or to attach more than one amino acid residue of the protein (e.g., lysine, histamine, aspartic acid, glutamic acid, or cysteine, and combinations thereof)

One may particularly desire a protein that is chemically modified at the N-terminus. Taking polyethylene glycol as an example, various polyethylene glycol molecules (by molecular weight, branching, etc.) can be selected, and the ratio of the polyethylene glycol molecules to protein (or peptide) molecules in the reaction mixture can be selected to perform a PEGylation reaction type, so as to obtain the selected N-terminal PEGylated protein. The method of obtaining an N-terminally PEGylated preparation (i.e., separating this fraction from other mono-PEGylated fractions if desired) may be by purifying the N-terminally PEGylated substance from the population of PEGylated protein molecules. Chemically modified selected proteins modified at the N-terminus can be achieved by reductive alkylation reactions that exploit the different reactivities of different types of original amino groups (versus N-terminal lysines) suitable for derivatization in a particular protein. Under appropriate reaction conditions, selective derivatization of the protein at the N-terminus with a polymer-containing carbonyl group is achieved.

As indicated above, PEGylation of the proteins of the invention can be achieved by a number of methods. For example, polyethylene glycol can be attached to a protein directly or through an intervening linker. Linker systems for attaching polyethylene glycol to proteins are found in Delgado et al, crit.rev.thera.drug carriers, 9: 249-304 (1992); francis et al, Intern.J. Hematol.68: 1-18 (1998); U.S. patent nos. 4002531; U.S. patent nos. 5349052; WO 95/06058; and WO 98/32466. The above documents are incorporated by reference in their entirety.

A system for attaching polyethylene glycol directly to amino acid residues of proteins without intervening linkers utilizes tresylated MPEG, which uses 2, 2, 2-trifluoroethanesulfonyl chloride (ClSO)₂CH₂CF₃) Modified monomethoxypolyethylene glycol (MPEG). Polyethylene glycol is directly attached to the amine group of proteins based on their reaction with tresylated MPEG. Thus, the present invention includes protein-polyethylene glycol conjugates produced by reacting a protein of the present invention with a polyethylene glycol molecule having a 2, 2, 2-trifluoroethanesulfonyl group.

Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. patent No.5612460 (incorporated by reference in its entirety) discloses a urethane linker that links polyethylene glycol to a protein. In which polyethylene glycol is a protein-polyethylene glycol conjugate attached to a protein through a linker, can also be produced by reacting a protein with a compound such as MPEG-succinimidyl succinate, MPEG activated with 1, 1' -carbonyldiimidazole, MPEG-2, 4, 5-trichlorophenyl carbonate, MPEG-p-nitrophenyl carbonate, and various MPEG-succinate derivatives. Additional polyethylene glycol derivatives and reaction chemistries for attaching polyethanols to proteins are described in WO98/32466, which is incorporated by reference in its entirety. PEGylated protein products produced using the chemical reaction processes described herein are included in the present invention.

The number of polyethylene glycol moieties attached to each protein of the invention (i.e., the degree of substitution) may also vary. For example, pegylated proteins of the invention may be linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules on average. Similarly, the average degree of substitution is, for example, 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol components per protein molecule. Methods for determining the degree of substitution are described in Delgado et al, crit.rev.thera.drug Carrier sys.9: 249-304 (1992).

Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides may be recovered and purified by known methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") is preferred for purification.

Neutrokine-alpha polypeptide

Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the present invention may be monomeric or multimeric (i.e., dimeric, trimeric, tetrameric, and higher multimers). Thus, the present invention relates to monomers and multimers of the Neutrokine-alpha and Neutrokine-alpha SV polypeptides of the invention, their preparation and compositions (preferably pharmaceutical compositions) comprising them. In specific embodiments, the polypeptide of the invention is a monomer, dimer, trimer or tetramer. In another embodiment, the multimer of the invention is at least a dimer, trimer or tetramer.

The multimers encompassed by the present invention can be a homo-polymer or a hetero-polymer. The term "homologous polymer" as used herein refers to multimers that contain only Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides (including fragments, variants and fusion proteins of Neutrokine-alpha and/or Neutrokine-alpha SV) of the invention. These homologous polymers may contain Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides having the same or different amino acid sequences. In a specific embodiment, the homologous polymers of the invention are multimers that contain only Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides having the same amino acid sequence. In another specific embodiment, the homologous polymers of the invention are multimers comprising Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide having the same or different amino acid sequence), or a homotrimer (e.g., containing a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide having the same or different amino acid sequence). In a preferred embodiment, the multimer of the invention is a homotrimer. In another embodiment, the homomultimer of the invention is at least a homodimer, a homotrimer or a homotetramer.

The term "heteropolymer" as used herein refers to a multimer that contains a heterologous polypeptide (i.e., a polypeptide of a different protein) in addition to a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, heterotrimer, or heterotetramer. In further embodiments, the heteromultimer of the invention is at least a heterodimer, a heterotrimer, or a heterotetramer. In another embodiment, not exclusive, the heteromultimer of the invention comprises a CD40 ligand polypeptide sequence, or a biologically active fragment or variant thereof.

The polymers of the invention may be hydrophobic, hydrophilic, ionic and/or covalently associated and/or may be indirectly linked, for example by liposome formation. Thus in one embodiment, a multimer of the invention, such as a homodimer, or a homotrimer, is formed when a polypeptide of the invention is contacted with another polypeptide in solution. In another embodiment, a heteromultimer of the invention, such as a heterotrimer or heterotetramer, is formed when a polypeptide of the invention is contacted in solution with an antibody to a polypeptide of the invention, including an antibody to a heterologous polypeptide sequence in a fusion protein of the invention. In other embodiments, the multimer of the invention is formed by a covalent relationship with and/or a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide of the invention. Such a covalent relationship may comprise one or more amino acid residues comprised in the polypeptide sequence (the polypeptide sequence being the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 19 or the sequence of the polypeptide encoded by the deposited cDNA clone related to the present invention). In one instance, covalent association is a crosslink between interacting cysteine residues located in the polypeptide sequence in the native (i.e., naturally occurring) polypeptide. In another case, the covalent association is the result of a chemical or recombinant operation. Alternatively, such a covalent relationship may comprise one or more amino acid residues comprised by a heterologous polypeptide sequence in a Neutrokine-alpha and/or Neutrokine-alpha SV fusion protein. In one embodiment, the covalent association is a covalent association between heterologous sequences comprised in a fusion protein of the invention (see, e.g., U.S. patent No. 5478925). In a specific embodiment, the covalent association is a covalent association between heterologous sequences contained in a Neutrokine- α -Fc and/or Neutrokine- α SV-Fc fusion protein of the invention. In another specific embodiment, the covalent association of the fusion proteins of the invention is a covalent association between heterologous polypeptide sequences from another TNF family ligand/receptor member, which are capable of forming covalently associated multimers, such as osetoprotegerin (see international publication No. wo98/49305, the contents of which are incorporated herein by reference in their entirety). In another specific embodiment, the covalent association of the fusion protein of the invention is a covalent association between heterologous polypeptide sequences from CD40L or a soluble fragment thereof. In another embodiment, two or more Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention are linked by a synthetic linker (e.g., a peptide, carbohydrate or soluble polymer linker). Such peptide linkers are described, for example, in U.S. Pat. No.5073627 (incorporated by reference). Proteins comprising a plurality of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides separated by peptide linkers may be produced using conventional recombinant DNA methods.

Another method of making multimers of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention comprises using Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper or isoleucine zipper domain is a polypeptide that facilitates multimerization of proteins in which the zipper structure is found. Leucine zippers were originally identified in several DNA binding proteins (Landshulz et al, science 240: 1759(1988)) and have been found in many different proteins. Known leucine or isoleucine zippers are naturally occurring dimeric or trimerized peptides and derivatives thereof. Leucine zippers of Neutrokine-alpha and/or Neutrokine-alpha SV proteins suitable for producing soluble multimers are described, for example, by the incorporated-by-reference PCT application wo 94/10308. Recombinant fusion proteins comprising soluble Neutrokine-alpha and/or Neutrokine-alpha SV fused to a peptide that is dimeric or trimerized in solution are expressed in suitable host cells and the resulting soluble multimerized Neutrokine-alpha and/or Neutrokine-alpha SV are recovered from the culture supernatant by methods known in the art.

Some members of the TNF family of proteins are believed to be in trimeric form (Beutler and Huffel, science 264: 667, 1994; Banner et al, cell 73: 431, 1993). Accordingly, trimerized Neutrokine-alpha and/or Neutrokine-alpha SV has enhanced biological activity. Preferred leucine zipper components are those that preferentially form trimers. Other peptides derived from naturally occurring trimeric proteins may be used to prepare trimerized Neutrokine-alpha and/or Neutrokine-alpha SV, as described by Hoppe et al (FEBS letters 344: 191, (1994) and U.S. patent application Serial No. 08/446922, disclosing a leucine zipper derived from lung Surfactant Protein D (SPD).

In another embodiment, the protein of the invention is produced by including in Flag-Neutrokine-alpha or FlagFlag in Neutrokine-alpha SV fusion proteinPolypeptide sequences are linked by interactions between the sequences. In another embodiment, the protein of the invention is produced by including in Flag-Neutrokine-alpha or FlagHeterologous polypeptide sequence of-Neutrokine-alpha SV fusion protein and anti-FlagThe antibodies are associated by interactions between them.

Multimers of the invention can be produced using chemical methods known in the art. For example, polypeptides to be included in multimers of the invention can be chemically cross-linked using linker molecules and linker molecule length optimization methods known in the art (see U.S. Pat. No.5478925, incorporated by reference). In addition, the multimers of the invention can be generated by methods known in the art by forming one or more intramolecular crosslinks between cysteine residues located in the sequence of the polypeptides to be included in the multimer. (see U.S. Pat. No.5478925, incorporated by reference). In addition, the polypeptides of the invention can be routinely modified by the addition of cysteine or biotin at the C-or N-terminus of the polypeptide, and multimers of polypeptides containing one or more of these modifications can be generated using methods known in the art (see U.S. Pat. No.5478925, incorporated herein by reference). In addition, liposomes containing the polypeptide components to be included in the multimers of the invention can be produced by methods known in the art (see U.S. Pat. No.5478925, incorporated by reference).

Alternatively, multimers of the invention can be produced using genetic engineering methods known in the art. In one embodiment, the polypeptides contained in the multimers of the invention are recombinantly produced using the fusion protein methods described herein or other methods known in the art (see U.S. Pat. No.5478925, incorporated by reference). In a specific embodiment, a polynucleotide encoding a homodimer of the invention is synthesized by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then from the beginning C-terminus to the N-terminus in the reverse direction (deletion of the leader sequence) to a polynucleotide encoding the translation product of the polypeptide (see U.S. Pat. No.5478925, incorporated herein by reference). In another embodiment, recombinant polypeptides of the invention are produced using recombinant methods described herein or other methods known in the art, which polypeptides contain a transmembrane domain and can be incorporated into liposomes by membrane reconstitution methods (see U.S. Pat. No.5478925, incorporated by reference).

In one embodiment, the present invention provides an isolated Neutrokine-alpha polypeptide having the amino acid sequence encoded by the cDNA clone having ATCC accession number 97768, or having the amino acid sequence depicted in FIGS. 1A and 1B (SEQ ID NO: 2), or an isolated polypeptide comprising a portion (i.e., a fragment) of a polypeptide as described above. In another embodiment, the present invention provides an isolated Neutrokine- α SV polypeptide having the amino acid sequence encoded by the cDNA clone deposited under ATCC accession number 203518, or having the amino acid sequence depicted in FIGS. 5A and 5B (SEQ ID NO: 19), or a polypeptide comprising a portion or fragment of such a polypeptide.

The polypeptide fragments of the present invention include a polypeptide comprising or consisting of the amino acid sequence shown in (SEQ ID NO: 2), the amino acid sequence encoded by the cDNA in the plasmid deposited under ATCC accession number 97768, or the amino acid sequence encoded by a nucleic acid that hybridizes (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone or to the complementary strand of the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1).

In addition, polypeptide fragments of the present invention include a polypeptide comprising or consisting of the amino acid sequence set forth in (SEQ ID NO: 19), the amino acid sequence encoded by the cDNA contained in the plasmid deposited under ATCC accession number 203518, or the amino acid sequence encoded by a nucleic acid that hybridizes (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone or to the complementary strand of the nucleotide sequence set forth in FIGS. 5A and 5B (SEQ ID NO: 18).

In addition, the polypeptide fragments of the present invention include a polypeptide comprising or consisting of a polypeptide having an amino acid sequence substantially identical to seq id NO: 21 (e.g., under stringent hybridization conditions) to the complementary strand of the nucleotide sequence set forth in seq id no.

The polypeptide fragments of the present invention also include a polypeptide comprising or consisting of a sequence identical to SEQ id no: 23, or an amino acid sequence corresponding to SEQ ID NO: 22 (e.g., under stringent hybridization conditions) to the complementary strand of the nucleotide sequence set forth in seq id no.

In addition, the polypeptide fragments of the present invention include a polypeptide comprising or consisting of SEQ id no: 28, or an amino acid sequence corresponding to SEQ ID NO: 27 (e.g., under stringent hybridization conditions) to the complementary strand of the nucleotide sequence set forth in seq id no.

In addition, the polypeptide fragments of the present invention include a polypeptide comprising or consisting of SEQ id no: 30, or an amino acid sequence substantially identical to SEQ ID NO: 29 (e.g., under stringent hybridization conditions) to the complement of the nucleotide sequence set forth in seq id no.

The polypeptide fragments of the present invention include a polypeptide comprising or consisting of SEQ id no: 2, or an amino acid sequence encoded by a cDNA contained in the deposited clone, or a nucleic acid that hybridizes (e.g., under stringent hybridization conditions) to a nucleotide sequence contained in the deposited clone or shown in fig. 1A and 1B (SEQ ID NO: 1), or the complementary strand thereof. Protein fragments may be "free-existing", or comprised in a larger polypeptide, the fragment forming part of the polypeptide or a region, preferably as a single contiguous region. Polypeptide fragments of the invention typically include, for example, fragments comprising or consisting of SEQ ID NO: 2, 1-50, 51-100, 101-150, 151-200, 201-250, and/or 251-285 amino acid residues. In addition, a polypeptide fragment can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, or 200 amino acids in length.

In specific embodiments, the polypeptide fragment of the invention comprises or consists of amino acid residues 1-46, 31-44, 47-72, 73-285, 73-83, 94-102, 148-152, 166-181, 185-209, 210-221, 226-237, 244-249, 253-265 and/or 277-284 of the sequence shown in FIGS. 1A and 1B (SEQ ID NO: 2). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

One skilled in the art will appreciate that mutations directed to a region of a Neutrokine-alpha polypeptide of the invention encompassing an insert of 19 amino acids not found in the Neutrokine-alpha SV polypeptide sequence (i.e., Val 142-Lys 160 amino acid residues of the sequence shown in figures 1A and 1B and SEQ ID NO: 2) may affect the observed biological activity of the Neutrokine-alpha polypeptide. More particularly, such residues of the targetable mutagenic Neutrokine-alpha polypeptide sequence include, without limitation, SEQ ID NO: 2 the following amino acid residues of a Neutrokine-alpha polypeptide represented by: v142; t143; q144; d145; c146; l147; q148; l149; i150; a151; d152; s153; e154; t155; p156; t157; i158; q159 and K160. Encoded in SEQ ID NO: 2, a polynucleotide having one or more mutations in the V142-K160 region of Neutrokine-alpha polypeptide is desired. Polypeptides encoded by these polynucleotides are also encompassed within the scope of the invention.

A polypeptide fragment may be "free-standing", or contained within a larger polypeptide, the fragment forming a portion or region of the polypeptide, preferably as a single contiguous region. Polypeptide fragments of the invention typically include, for example, fragments comprising or consisting of SEQ ID NO: 2, about amino acid residues 1-15, 16-30, 31-46, 47-55, 56-72, 73-104, 105-. Polypeptide fragments of the invention additionally include, for example, fragments comprising or consisting of SEQ ID NO: 19, about amino acid residues 1-143, 1-150, 47-143, 47-150, 73-143, 73-150, 100-150, 140-145, 142-148, 140-150, 140-200, 140-225 and 140-266 of the amino acid sequence shown in the specification. In addition, a polypeptide fragment may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, or 200 amino acids in length. By "about" herein is meant a specified range, as well as such ranges of more or less, several, 5, 4, 3, 2, 1 amino acid residues at the amino terminus or the carboxy terminus or both. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention

Other preferred embodiments encompass polypeptide fragments comprising or consisting of the structure: a putative intracellular domain of Neutrokine-alpha (amino acid residues 1-46 of SEQ ID NO: 2), a putative transmembrane domain of Neutrokine-alpha (amino acid residues 47-72 of SEQ ID NO: 2), a putative extracellular domain of Neutrokine-alpha (amino acid residues 73-285 of SEQ ID NO: 2), a putative TNF conserved domain of Neutrokine-alpha (amino acid residues 191-284 of SEQ ID NO: 2), and a polypeptide comprising or consisting of a putative intracellular domain of Neutrokine-alpha fused to a putative extracellular domain (i.e., amino acid residues 1-46 of SEQ ID NO: 2 fused to amino acid residues 73-285). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Further preferred embodiments encompass polypeptide fragments comprising or consisting of the structure: a putative intracellular domain of Neutrokine- α SV (amino acid residues 1-46 of SEQ ID NO: 19), a putative transmembrane domain of Neutrokine- α SV (amino acid residues 47-72 of SEQ ID NO: 19), a putative extracellular domain of Neutrokine- α SV (amino acid residues 73-266 of SEQ ID NO: 19), a putative TNF conserved domain of Neutrokine- α SV (amino acid residues 172-265 of SEQ ID NO: 19), and a polypeptide comprising or consisting of a fusion of the putative intracellular domain of α SV-Neutrokine with the extracellular domain (amino acid residues 1-46 of SEQ ID NO: 19 fused with amino acid residues 73-266). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Other embodiments of the invention encompass polypeptide fragments comprising or consisting of the putative beta sheet region identified in figure 7A. These polypeptide fragments of the invention comprise or consist of SEQ id no: 2, Gln144-Ala151, Phe172-Lys173, Ala177-Glu179, Asn183-Ile185, Gly191-Lys204, His210-Val219, Leu226-Pro237, Asn242-Ala251, Gly256-Ile263 and/or Val276-Leu 284. In some further embodiments, the polypeptide fragments of the invention further comprise or consist of seq id NO: 19 Phe153-Lys154, Ala158-Glu160, Asn164-Ile166, Gly172-Lys185, His191-Val200, Leu207-Pro218, Asn223-Ala232, Gly237-Ile244, and/or Val257-Leu256 amino acid residues, and consisting of SEQ ID NO: 23, Phe42-Lys43, Ala47-Glu49, Asn53-Ile55, Gly61-Pro74, His80-Val89, Leu96-Pro107, Asn112-Ala121, Gly126-Ile133 and/or Asp146-Leu 154. In other embodiments, the polypeptide fragments of the invention further comprise or comprise SEQ ID NO: 28, Gln78-Ala85, Phe106-Lys107, Ala111-Glu113, Asn117-Ile119, Gly125-Lys138, His144-Val153, Leu160-Pro171, Asn176-Ala185, Gly190-Ile197, and/or Val210-Leu218, and consists of the amino acid residues at positions SEQ ID NO: 30, Gln78-Ala85, Phe106-Lys107, Ala111-Glu113, Asn117-Ile119, Gly125-Lys138, His144-Val153, Leu160-Pro171, Asn176-Ala185, Gly190-Ile197, and/or Val210-Leu 218. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Some polypeptides of the invention, for example, but not limited to, comprise or consist of a combination of amino acid sequences of the invention, for example, a polypeptide comprising SEQ ID NO: 2 [ Met1-Lys113] and [ Leu114-Thr141] and [ Ile142-Lys160] and [ Gly161-Gln198] and [ Val199-Ala248] and [ Gly250-Leu285 ]; SEQ ID NO: 2 [ Met1-Lys113] and [ Ile142-Lys160] and [ Gly161-Gln198] and [ Val199-Ala248] and [ Gly250-Leu285 ]; or SEQ ID NO: 2 [ Met1-Lys113] and [ Leu114-Thr141] and [ Ile142-Lys160] and [ Gly161-Gln198] and [ Val199-Ala248] and [ Gly250-Leu285 ]. Or SEQ ID NO: 2 [ Met1-Lys113] and [ Leu114-Thr141] and [ Ile142-Lys160] and [ Gly161-Gln198] and [ Gly250-Leu285 ]. Other combinations may include polypeptide fragments other than those described above (e.g., fusions of [ Leu114-Thr141] and [ Val199-Ala248] and [ Gly250-Leu285] and [ Ile142-Lys160] of SEQ ID NO: 2). Other combinations may also include heterologous polypeptide fragments as described herein and/or other polypeptides or polypeptide fragments of the invention (e.g., [ Met1-Lys113] and [ Leu114-Thr141] and [ Ile142-Lys160] and [ Gly161-Gln198] and [ Gly250-Leu285] fusions to FLAG tags of SEQ ID NO: 2). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In addition, the polypeptides of the invention include or consist of, for example, without limitation, combinations of amino acid sequences, such as those including SEQ ID NO: 19 [ Met1-Lys113] and [ Leu114-Thr141] and [ Gly142-Gln179] and [ Val180-Ala229] and [ Gly230-Leu266 ]; SEQ ID NO: 19 [ Met1-Lys113] and [ Gly142-Gln179] and [ Val180-Ala229] and [ Gly230-Leu266 ]; or SEQ ID NO: 19 [ Met1-Lys113] and [ Leu114-Thr141] and [ Gly142-Gln179] and [ Gly230-Leu266 ]. Other combinations may include polypeptide fragments other than those described above (e.g., fusions of [ Leu114-Thr141] and [ Val180-Ala229] and [ Gly230-Leu266] and [ Gly142-Gln179] of SEQ ID NO: 19). Other combinations may also include heterologous polypeptide fragments as described herein and/or other polypeptides or polypeptide fragments of the invention (fusions of [ Met1-Lys113] and [ Leu114-Thr141] and [ Gly142-Gln179] and [ Gly230-Leu266] to the FLAG tag of SEQ ID NO: 19). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

The polypeptides of the invention additionally comprise or consist of, for example, without limitation, combinations of amino acid sequences, including, for example, SEQ ID NO: 23 [ Met1-Lys106] and [ Leu107-Thr134] and [ Ile167-Lys184] and [ Gly185-Gln224] and [ Val225-Ala272] and Gly273-Leu309 ]; SEQ ID NO: 23 [ Met1-Lys106] and [ Glu135-Asn165] and [ Ile167-Lys184] and [ Gly185-Gln224] and [ Val225-Ala272] and [ Gly273-Leu309], or SEQ ID NO: 23 [ Met1-Lys106] and [ Leu107-Thr134] and [ Glu135-Asn165] and [ Ile167-Lys184] and [ Gly185-Gln224] and [ Gly273-Leu309 ]. Other combinations may include polypeptide fragments other than those described above (e.g., fusions of [ Met1-Lys106] and [ Gly185-Gln224] and [ Ile167-Lys184] and [ Val225-Ala272] and [ Leu107-Thr134] and [ Gly273-Leu309] of SEQ ID NO: 23). Other combinations may also include fusions of the heterologous polypeptide fragments described herein and/or other polypeptides or polypeptide fragments of the invention (e.g., [ Met1-Lys106] and [ Glu135-Asn165] and [ Ile167-Lys184] and [ Gly185-Gln224] and [ Val225-Ala272] and [ Gly273-Leu309] of SEQ ID NO: 23 with a FLAG marker). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

The polypeptides of the invention additionally comprise or consist of, for example, without limitation, combinations of amino acid sequences, including, for example, SEQ ID NO: 28 [ Tyr1-Lys47] and [ Leu48-Thr75] and [ Ile76-Lys94] and [ Gly95-Gln132] and [ Val133-Ala182] and [ Gly183-Ala219 ]; SEQ ID NO: 28 [ Tyr1-Lys47] and [ Leu48-Thr75] and [ Ile76-Lys94] and [ Val133-Ala182 ]; or SEQ ID NO: 28 [ Tyr1-Lys47] and [ Leu48-Thr75] and [ Ile76-Lys94] and [ Val133-Ala182] and [ Gly183-Ala219 ]. Other combinations may include polypeptide fragments other than those described above (e.g., fusions of [ Tyr1-Lys47] and [ Gly183-Ala219] and [ Val133-Ala182] and [ Leu48-Thr75] of SEQ ID NO: 28). Other combinations may also include fusions of the heterologous polypeptide fragments described herein and/or other polypeptides or polypeptide fragments of the invention (e.g., [ Leu48-Thr75] and [ Ile76-Lys94] and [ Gly95-Gln132] and [ Val133-Ala182] of SEQ ID NO: 28 with Fc receptor markers). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

The polypeptides of the invention additionally comprise or consist of, for example, without limitation, combinations of amino acid sequences, including, for example, SEQ ID NO: 30 [ Tyr1-Lys47] and [ Leu48-Thr75] and [ Ile76-Lys94] and [ Gly95-Gln132] and [ Val133-Ala182] and [ Gly183-Ala219 ]; SEQ ID NO: 30 [ Tyr1-Lys47] and [ Leu48-Thr75] and [ Ile76-Lys94] and [ Val133-Ala182 ]; or SEQ ID NO: 30 [ Tyr1-Lys47] and [ Ile76-Lys94] and [ Val133-Ala182] and [ Gly183-Ala219 ]. Other combinations may include polypeptide fragments other than those described above (e.g., fusions of [ Tyr1-Lys47] and [ Gly183-Ala219] and [ Val133-Ala182] and [ Leu48-Thr75] of SEQ ID NO: 30). Other combinations may also include heterologous polypeptide fragments as described herein and/or other polypeptides or polypeptide fragments of the invention (e.g., fusions of [ Leu48-Thr75] and [ Ile76-Lys94] and [ Gly95-Gln132] and [ Val133-Ala182] of SEQ ID NO: 30 with Fc receptor markers). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Other embodiments of the invention encompass fragments of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides comprising or consisting of functional regions of the polypeptides of the invention, such as the alpha, beta, turn and coil regions of Garnier-Robaon, the alpha, beta and coil regions of Chou-Fasman, the Kyte-Doolittle hydrophilic and hydrophobic regions, the alpha and beta amphipathic regions of Eisenberg, the Karplus-Schulz flexible region, the Emini surface forming region and the Jameson-Wolf high antigen index region, as shown in FIGS. 3, 6 and table 1. In a preferred embodiment, a polypeptide fragment of the invention is antigenic. The data shown in columns VIII, IX, XIII and XIV in Table 1 can be used to routinely determine regions of Neutrokine-alpha that are highly potentially antigenic. Highly antigenic regions are determined by selecting values from the data shown in columns VIII, IX, XIII and/or IV that represent regions of the polypeptide that are readily exposed on the surface of the polypeptide under circumstances in which antigen recognition may occur during the initiation of an immune response. Particularly preferred fragments of the invention are those which comprise regions of Neutrokine-alpha and/or Neutrokine-alpha SV combined with structural features such as those described above (e.g. 1, 2, 3 or 4). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another embodiment, the invention provides a polypeptide comprising or consisting of an epitope-bearing portion of a polypeptide of the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The epitope portion of a polypeptide is an immunogenic or antigenic epitope of a polypeptide of the invention. An "immunogenic epitope" refers to a portion of a protein that elicits an antibody response when the entire protein is an immunogen. On the other hand, "antigenic epitope" refers to a region of a protein molecule to which an antibody can bind. The number of immunogenic epitopes of a protein is generally less than the number of antigenic epitopes. See, for example, Geysen et al, Proc. Natl. Acad. Sci. USA 81: 3998-4002(1983).

To select polypeptides bearing antigenic epitopes (i.e., regions containing protein molecules to which antibodies can bind), it is well known in the art that relatively short synthetic peptides that mimic portions of the protein sequence, typically are capable of eliciting antisera that react with portions of the mimicking protein. See, for example, Sutcliffe, j.g., Shinnick, t.m., Green, n, and Learrer, R.A (1983) "antibodies reactive with predetermined sites on proteins", science 219: 660 — 666. Peptides capable of eliciting protein-reactive sera are typically represented in the primary sequence of a protein, can be characterized by some simple chemical rules, and are defined as being neither immunodominant regions of the intact protein (i.e., immunogenic epitopes), nor amino-or carboxy-terminal. The antigenic epitope-bearing peptides or polypeptides of the invention are therefore useful for the production of antibodies, including monoclonal antibodies that specifically bind to the polypeptides of the invention. See, e.g., Wilson et al, cell 37: 767 778(1984), p 777.

The antigenic epitope-bearing peptide or polypeptide of the invention preferably contains a sequence of at least 4, at least 5, at least 6, at least 7, preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, more preferably between about 15 and about 30 amino acids comprised within the amino acid sequence of the polypeptide of the invention. Preferred polypeptides comprising an immunogenic or antigenic epitope are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues in length. Other preferred antigenic epitopes include the antigenic epitopes disclosed herein and portions thereof.

Antigenic polypeptides or peptides that may be used to produce antibodies specific for Neutrokine-alpha and/or Neutrokine-alpha SV include, by way of example and without limitation: a polypeptide comprising or consisting of the amino acid residues at about Phe115-Leu147 as shown in FIGS. 1A and 1B (SEQ ID NO: 2); a polypeptide comprising or consisting of the amino acid residues at about Ile150-Tyr163 as shown in FIGS. 1A and 1B (SEQ ID NO: 2); a polypeptide comprising or consisting of amino acid residues at about Ser171-Phe194 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2); a polypeptide comprising or consisting of amino acid residues Glu223-Tyr246 as shown in FIGS. 1A and 1B (SEQ ID NO: 2); a polypeptide comprising or consisting of amino acid residues at about Ser271-Phe278 as shown in FIGS. 1A and 1B (SEQ ID NO: 2); . By "about" herein is meant a specified range, as well as more or less, several, 5, 4, 3, 2, or 1 amino acid at the amino terminus or the carboxy terminus or both within that range. These polypeptide fragments have been determined to carry antigenic epitopes of Neutrokine-alpha polypeptides by Jameson-Wolf antigen index analysis, as shown in FIG. 3 and Table 1.

Antigenic polypeptides or peptides that may be used to produce antibodies specific for Neutrokine-alpha and/or Neutrokine-alpha SV include, by way of example and without limitation: a polypeptide comprising or consisting of the amino acid residues at positions about Pro32-Leu47 as shown in FIGS. 5A and 5B (SEQ ID NO: 19); a polypeptide comprising or consisting of the amino acid residues at about Glu116-Ser143 shown in FIGS. 5A and 5B (SEQ ID NO: 19); a polypeptide comprising or consisting of the amino acid residues at about Phe153-Tyr173 as depicted in figures 5A and 5B (SEQ ID NO: 19); a polypeptide comprising or consisting of the amino acid residues at about Pro218-Tyr227 shown in FIGS. 5A and 5B (SEQ ID NO: 19); a polypeptide comprising or consisting of the amino acid residues at about Ala232-Gln241 of FIGS. 5A and 5B (SEQ ID NO: 19); a polypeptide comprising or consisting of amino acid residues about Ile244-Ala249 as shown in FIGS. 5A and 5B (SEQ ID NO: 19); a polypeptide comprising or consisting of the amino acid residues at about Ser252-Val257 as shown in FIGS. 5A and 5B (SEQ ID NO: 19). By "about" herein is meant a specified range, as well as more or less, several, 5, 4, 3, 2 or 1 amino acid residue at the amino terminus or the carboxy terminus or both within that range. Polynucleotides encoding these polypeptides are also encompassed by the present invention. These polypeptide fragments have been identified by Jameson-Wolf antigen index analysis as carrying antigenic epitopes of Neutrokine- α SV polypeptides, as shown in figure 6 and in tabular data generated by the Protean component of the DNA STAR computer program.

The epitope-bearing peptides and polypeptides of the invention can be produced by any conventional method. See, for example, Houghten, R.A (1985) general procedure for rapid solid phase synthesis of large quantities of peptides: specificity of antigen-antibody interaction at the individual amino acid level, proceedings of the american academy of sciences 82: 5131-; the method of "Simultaneous Multi-peptide Synthesis (SMPS)" by Houghten et al (1986) is described in U.S. Pat. No. 4631211.

Uses of the epitope-bearing peptides and polypeptides of the invention include, but are not limited to, induction of antibodies according to methods well known in the art. See, e.g., Sutcliffe et al, supra; wilson et al, supra; chow, m, et al, proceedings of the american academy of sciences 82: 910-914; and bitte, f.j. et al, gene virus journal 66: 2347-2354(1985).. The peptides of the invention bearing immunogenic epitopes, i.e. the part of the protein that elicits an antibody response when the entire protein is an immunogen, are identified according to methods known in the art. See, for example, Geysen et al, supra. In addition, Geysen, U.S. Pat. No.5194392, describes a method for detecting or determining the sequence of a monomer (amino acid or other compound) that is a topological equivalent of an epitope (i.e., mimotope) that is complementary to a particular paratope (antigen binding site) of a corresponding antibody. Geysen (1989) describes a method for detecting or determining the sequence of a monomer which is the topological equivalent of a ligand complementary to the ligand binding site of a corresponding particular receptor. Similarly, U.S. Pat. No.5480971 to Houghten, R.A. et al (1996) on mixtures of peralkylated oligopeptides discloses linear C1-C7-alkyl-peralkylated oligopeptides and libraries of such peptides, and methods of using such oligopeptide libraries to determine the sequence of peralkylated oligopeptides that preferentially bind to the corresponding receptor molecule. Thus, non-peptide analogs of the epitope-bearing peptides of the invention can also be routinely produced by these methods.

The present invention encompasses a polypeptide comprising or consisting of an epitope of: has the sequence shown in SEQ ID NO: 2, or a polypeptide encoded by a polynucleotide sequence contained in a clone contained in the deposit with ATCC deposit No. 97768, or a polypeptide encoded by a polynucleotide sequence substantially identical to seq id NO: 1 or the complement of the cDNA sequence contained in ATCC deposit No. 97768, under such hybridization conditions. The invention also encompasses polynucleotide sequences comprising or consisting of a sequence encoding an epitope of a polypeptide of the invention (as represented by SEQ ID NO: 1), a polynucleotide sequence encoding the complementary strand of a polynucleotide sequence of an epitope of the invention, and a polynucleotide sequence that hybridizes (under the hybridization conditions) to the complementary strand.

The invention also encompasses a polypeptide comprising or having the amino acid sequence of SEQ ID NO: 19, or the polypeptide sequence encoded by the polynucleotide sequence contained in ATCC deposit No. 203518, or the polypeptide sequence encoded by a polynucleotide sequence identical to the polynucleotide sequence shown in SEQ ID NO: 18 or the cDNA sequence contained in ATCC deposit No. 203518 under the hybridization conditions described herein. The invention also encompasses a polynucleotide sequence comprising or consisting of an epitope encoding a polypeptide of the invention (sequence shown in SEQ ID NO: 18), a polynucleotide sequence encoding the complementary strand of the polynucleotide sequence of the epitope of the invention, and a polynucleotide sequence that hybridizes to the complementary strand (e.g., under the hybridization conditions described).

The term "epitope" as used herein refers to a portion of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, more preferably a human. In a preferred embodiment, the invention encompasses a polypeptide comprising an epitope and a polynucleotide encoding such a polypeptide. An "immunogenic epitope" refers to a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, such as by the methods for producing antibodies as described above (see, e.g., Geysen et al, Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983)). The term "antigenic epitope" refers to a portion of a protein to which an antibody immunospecifically binds its antigen, as determined by any method well known in the art, e.g., by the immunoassay described. Immunospecific binding does not include non-specific binding, but does not necessarily include cross-reactivity with other antigens. The antigenic epitope need not be immunogenic.

Fragments which are epitopes can be produced by any conventional method (see, for example, Houghten, proceedings of the national academy of sciences USA 82: 5131-5135(1985), further, described in U.S. Pat. No. 4631211)

In the present invention, an antigenic epitope preferably comprises a sequence of at least 4, at least 5, 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, most preferably about 15 to 30 amino acids. Preferably, the polypeptide comprising an immunogenic or antigenic epitope is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100 amino acid residues in length. Other preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of 2, 3, 4, 5 or more of these antigenic epitopes. Antigenic epitopes can be used as target molecules in immunoassays (e.g., as described by Wilson et al, cell 37: 767-778 (1984); Sutcliffe et al, science 219: 660-666 (1983)).

Similarly, immunogenic epitopes can be used to induce antibodies, for example, according to methods well known in the art (see, e.g., Sutcliffe et al, supra; Wilson et al, supra; Chow et al, Proc. Natl. Acad. Sci. USA 82: 910-. Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as combinations of 2, 3, 4, 5 or more of these immunogenic epitopes. A polypeptide comprising one or more immunogenic epitopes may be present with a carrier protein such as albumin to elicit an antibody response of an animal type (e.g. rabbit or mouse), or the polypeptide may be carrier free if it is sufficiently long (at least about 25 amino acids). However, immunogenic epitopes comprising as few as 8-10 amino acids have been shown to be effective in generating antibodies that bind linear epitopes in at least denatured polypeptides. (as in Western blotting).

The epitope-bearing polypeptides of the invention can be used to induce antibodies according to methods well known in the art, including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al, supra; wilson et al, supra; and Bittle et al, journal of genoviruses 66: 2347-2354(1985). If in vivo immunization is used, the animal can be inoculated with free peptide; however, anti-peptide antibody titers can be enhanced by coupling the peptides to a macromolecular carrier such as Keyhole Limpet Hemocyanin (KLH) or tetanus toxoid. For example, peptides containing cysteine residues may be coupled to a carrier using a linker such as m-maleimidobenzoate-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to a carrier using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with free peptide or peptide coupled to a carrier, for example by intraperitoneal and/or intradermal injection of an emulsion containing about 100mg of the peptide or carrier protein and Freud's adjuvant or any other adjuvant known to stimulate an immune response. Some booster injections, for example, every approximately two weeks, are required to provide effective titers of anti-peptide antibodies, which can be detected, for example, by ELISA assays using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in the serum of an immunized animal can be increased by selecting anti-peptide antibodies, for example, by adsorbing the peptide to a solid support and eluting the selected antibodies according to methods well known in the art.

Those skilled in the art will appreciate and as noted above that polypeptides of the invention comprising immunogenic or antigenic epitopes can be fused to other polypeptide sequences. For example, a polypeptide of the invention can be fused to a constant region of an immunoglobulin (IgA, IgE, IgG, IgM), or a portion thereof (CH1, CH2, CH3, or any combination thereof and a portion thereof) to produce a chimeric polypeptide. Such fusion proteins are easy to purify and have an increased half-life in vivo. Chimeric proteins have been shown to consist of the first two domains of the human CD4 polypeptide and various domains of the constant region of mammalian heavy or light chains. See, e.g., Ep 394827; tyaunecker et al, Nature, 331: 84-86 (1988). Enhanced transport of antigens across epithelial barriers to the immune system has been shown to conjugate antigens (such as insulin) to FcRn binding partners such as IgG or Fc fragments (see, e.g., PCT publications WO 96/22024 and WO 99/04813). IgG fusion proteins having disulfide-linked dimeric structures due to disulfide bonds of IgG moieties have also been found to haveMore efficient ability to bind and neutralize other molecules. See, e.g., Fountoulakis et al, journal of biochemistry 270: 3958-3964(1995). The nucleic acids encoding the epitopes described above can also be recombined with the corresponding genes as epitope tags, such as the Hemagglutinin (HA) tag or the flag tag, to facilitate detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al facilitates the purification of undenatured fusion proteins expressed in human cell lines (Janknecht et al, Proc. Natl. Acad. Sci. USA 88: 89072-897). In this system, the corresponding gene was subcloned into a vaccinia recombinant plasmid such that the open reading frame of the gene was translationally fused to an amino-terminal marker consisting of 6 histidine residues. The label serves as the matrix binding domain of the fusion protein. Applying an extract from cells infected with recombinant vaccinia virus to Ni ²⁺Nitriloacetic acid agarose chromatography column, and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides and epitope-bearing fragments thereof of the present invention are fused to a heterologous antigen (e.g., a polypeptide, carbohydrate, phospholipid or nucleic acid). In specific embodiments, the heterologous antigen is an immunogen.

In a more specific embodiment, the heterologous antigen is the gpl20 protein of HIV or a fragment thereof. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides and epitope-bearing fragments thereof of the present invention are fused to the polypeptide sequence of another TNF ligand family member (or a biologically active fragment or variant thereof). In a specific embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention are fused to a CD40L polypeptide sequence. In a preferred embodiment, the CD40L polypeptide sequence is soluble.

Methods of gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling (collectively "DNA shuffling") can be used to modulate the activity of Neutrokine-alpha and/or Neutrokine-alpha SV, thereby efficiently producing agonists and antagonists of Neutrokine-alpha and/or Neutrokine-alpha SV. See U.S. patent nos. 5605793, 5811238, 5830721, 5834252, and 5837458, and Patten, p.a. et al, general opinion 8 biotechnology: 724-33 (1997); harayama, s. biotechnological trend 16 (2): 76-82 (1998); hansson, l.o, et al, journal of molecular biology 287: 265-76 (1999); and Lorenzo, m.m. and blasto, r. biotechnology 24 (2): 308-13(1998), which are incorporated herein by reference. In one embodiment, alterations to Neutrokine-alpha and or Neutrokine-alpha SV polynucleotides and corresponding polypeptides may be made by DNA shuffling. DNA shuffling involves assembly of two or more DNA segments into the desired Neutrokine-alpha and/or Neutrokine-alpha SV molecule by homology or site-specific recombination. In another embodiment, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and corresponding polypeptides may be altered by random mutagenesis by error-prone polymerase chain reaction, random insertion of nucleotides, or other methods prior to recombination. In another embodiment, one or more components, motifs, segments, portions, domains, fragments, etc. of Neutrokine-alpha and/or Neutrokine-alpha SV may be recombined with one or more components, motifs, segments, portions, domains, fragments, etc. of one or more heterologous molecules. In a preferred embodiment, the heterologous molecules are, for example, TNF- α, lymphotoxin- α (LT- α, also known as TNF- β), LT- β (found in the heterotrimeric LT- α 2- β complex), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF- γ (International publication No. WO 96/14328), AIM-I (International publication WO 97/33899), AIM-II (International publication WO 97/34911), APRIL (J.Experil.methods 188 (6): 1185-1190), endokine- α (International publication WO 98/07880), OPG, OX40, and Nerve Growth Factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International publication WO 96/34095), DR 39 3 (International publication WO 97/33904), DR4 (International publication WO 98/32856), TR5 (International publication WO 98/30693), TR6 (International publication WO 98/30694), TR7 (International publication WO 98/41629), TRANK, TR9 International publication WO 98/56892), TR10 International publication WO 98/54202), 312C2 (International publication WO 98/06842), TR12, CDA and V-FLIP. In other embodiments, the heterologous molecule is any member of the TNF family.

In a preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention (including biologically active fragments or variants thereof) are fused to a soluble CD40L polypeptide or a biologically active fragment or variant thereof.

Protein engineering may be performed to improve or alter the characteristics of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides. Recombinant DNA methods known to those skilled in the art can be used to generate novel mutant proteins or "muteins" that include one or more amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides may exhibit, for example, enhanced activity or improved stability. In addition, they can be purified in high yields and with better stability than the corresponding native polypeptide, at least under certain purification and storage conditions. For example, for many proteins, including the extracellular domain of a secreted protein in mature form, it is known in the art that one or more amino acids may be deleted at the N-terminus or C-terminus without substantial loss of biological function. For example, Ron et al, J. Biochem, 268: 2984-2988(1993) report on modified KGF proteins which have heparin-binding activity even if 3, 8 or 27 amino-terminal amino acids are lost.

Since the proteins of the present invention are members of the TNF polypeptide family, the deletion of the N-terminal amino acids up to Gly (G) residue 191 of FIGS. 1A and 1B (SEQ ID NO: 2) still retains some biological activity such as the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation and/or activation, and cytotoxicity to appropriate target cells. Polypeptides having deletions of further N-termini, including Gly (G) residues, are not expected to retain biological activity, since this residue in TNF-related polypeptides is known to be the start of a conserved domain required for biological activity. However, even if one or more functions of the protein are lost by deletion of one or more amino acids from the N-terminus of the protein, other functional activities may be retained. Thus, the ability of a shortened protein to induce and/or bind antibodies that recognize the intact protein, or the extracellular domain thereof, may be retained when a small portion of the residues of the intact protein, or the extracellular domain thereof, are removed from the N-terminus. Particular polypeptides lacking the N-terminal residue of the intact protein retain this immunological activity and can be determined by conventional methods described herein and by other methods known in the art.

Thus, the present invention further provides a polypeptide having one or more residues deleted from the amino terminus up to the 191 glycine residue (amino terminal Gly191) of the amino acid sequence of Neutrokine-alpha shown in FIGS. 1A and 1B (SEQ ID NO: 2), and a polynucleotide encoding such a polypeptide. In particular, the polypeptides provided by the invention comprise or consist of SEQ id no: n of 2 ¹An amino acid sequence of residue 285, wherein n¹Is SEQ ID NO: 2, an integer from amino acid positions 2 to 190 of the amino acid sequence set forth in seq id No. 2. Polynucleotides encoding these polypeptides are also encompassed by the present invention. More particularly, the present invention provides polynucleotides encoding polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 2-285, 3-285, 4-285, 5-285, 6-285, 7-285, 8-285, 9-285, 10-285, 11-285, 12-285, 13-285, 14-285, 15-285, 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, 59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285, 68-285, 69-285, 70-285, 71-285, 72-285, 73-285, 74-285, 75-285, 76-285, 77-285, 78-285, 79-285, 80-285, 81-285, 82-285, 83-285, 84-285, 85-285, 86-285, 87-285, 88-285, 89-285, 90-285, 91-285, 92-285, 93-285, 94-285, 95-285, 96-285, 97-285, 98-285, 99-285, 100-285, 101-285, 102-285, 103-285, 104-285, 105-285, 106-285, 107-285, 108-285, 109-285, 110-285, 111-285, 112-285, 113-285, 114-285, 115-285, 116-285, 117-285, 118-285, 119-285, 120-285, 121-285, 122-285, 123-285, 124-285, 125-285, 126-285, 127-285, 128-285, 129-285, 130-285, 131-285, 132-285, 133-285, 134-285, 135-285,1 36-285, 137-285, 138-285, 139-285, 140-285, 141-285, 142-285, 143-285, 144-285, 145-285, 146-285, 147-285, 148-285, 149-285, 150-285, 151-285, 152-285, 153-285, 154-285, 155-285, 156-285, 157-285, 158-285, 159-285, 160-285, 161-285, 162-285, 163-285, 164-285, 165-285, 166-285, 167-285, 168-285, 169-285, 170-285, 171-285, 172-285, 173-285, 174-285, 175-285, 176-285, 177-285, 178-285, 179-285, 180-285, 181-285, 182-285, 183-285, 184-285, 185-285, 186-285, 187-285, 188-285, 189-285 and 190-285. Polypeptides encoded by these polynucleotides are also encompassed by the present invention. The invention also relates to a nucleic acid molecule comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Also encompassed by the invention are polypeptides encoded by such nucleic acids and/or polynucleotides, which are polypeptides comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above-described amino acid sequence, and polynucleotides encoding such polypeptides.

In addition, since the putative ectodomain of the Neutrokine-alpha polypeptide of the present invention may itself elicit biological activity, deletion of the N-terminal and C-terminal amino acid residues of the putative ectodomain of the polypeptide (spanning amino acids Gln73-Leu285 of SEQ ID NO: 2) may still retain some biological activity, such as ligand binding, stimulation of lymphocyte proliferation, differentiation and/or activation, and modulation of cell replication or modulation of target cell activity. However, even if the deletion of one or more amino acids at the N-terminus of the putative extracellular domain of a Neutrokine-alpha polypeptide results in the loss of one or more biological functions of this polypeptide, other functional activities may be retained. Thus, the ability of a shortened polypeptide to induce and/or bind an antibody that recognizes the intact polypeptide or an extracellular domain thereof may be retained when a small portion of the residues of the intact or mature polypeptide or an extracellular domain thereof are removed from the N-terminus. Whether a particular polypeptide lacking the N-terminal residue of the intact polypeptide retains such immunological activity can be determined by conventional methods described herein as well as by other methods known in the art.

Accordingly, the present invention also provides a polypeptide having a sequence deleted from SEQ ID NO: 2 up to one or more residues of glycine residue 280 from the amino terminus of the Neutrokine-alpha amino acid sequence shown in figure 2. In particular, the invention provides polypeptides comprising or consisting of SEQ ID NO: n of 2 ²-285 amino acid sequence, wherein n²Is SEQ ID NO: 2, position 73 is the first residue position at the N-terminus of the putative extracellular domain of a Neutrokine-alpha polypeptide (SEQ ID NO: 2). Polynucleotides encoding these polypeptides are also encompassed by the present invention. More particularly, in certain embodiments, the invention provides polynucleotides encoding polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 2Q-73 to L-285; g-74 to L-285; d-75 to L-285; l-76 to L-285; a-77 to L-285; s-78 to L-285; l-79 to L-285; r-80 to L-285; a-81 to L-285; e-82 to L-285; l-83 to L-285; q-84 to L-285; g-85 to L-285; h-86 to L-283; h-87 to L-285; a-88 to L-285; e-89 to L-285; k-90 to L-285; l-91 to L-285; p-92 to L-285; a93 to L-285; g-94 to L-285; a-95 to L-285; g-96 to L-285; a-97 to L-285; p-98 to L-285; k-99 to L-285; a-100 to L-285; g-101 to L-285; l-102 to L-285; e-103 to L-235; e-104 to L-285; a-105 to L-285; p-106 to L-285; a-107 to L-285; v-108 to L-285; t-109 to L-285; a-110 to L-285; g-111 to L-285; l-112 to L-285; k-113 to L-285; 1-114 to L-285; f-115 to L-285; EI16 to L-285; p-117 to L-285; p-118 to L-285; a-119 to L-285; p-120 to L-285; g-121 to L-235; e-122 to L-285; g-123 to L-285; n-124 to L-285; s-125 to L-285; s-126 to L-285; q-127 to L-285; n-128 to L-285; s-129 to L-285; r-130 to L-285; n-131 to L-285; k-132 to L-285; r-133 to L-285; a-134 to L-285; v-135 to L-285; q-136 to L-285; g-137 to L-285; p-138 to L-285; e-139 to L-285; e-140 to L-285; t-141 to L-285; v-142 to L-285; t-143 to L-285; q-144 to L-285; d-145 to L-285; c-146 to L-285; l-147 to L-285; q-148 to L-285; l-149 to L-285; i-150 to L-285; a-151 to L-280; d-152 to L-285; s-153 to L-285; e-154 to L-285; t-155 to L-285; p-156 to L-285; t-157 to L-285; 1-158 to L-285; q-159 to L-285; k-160 to L-285; g-161 to L-285; s-162 to L-285; y-163 to L-285; t-164 to L-285; f-165 to L-285; v-166 to L-285; p-167 to L-285; w-168 to L-285; l-169 to L-285; l-170 to L-285; s-171 to L-285; f-172 to L-285; k-173 to L-285; r-174 to L-285; g-175 to L-285; s-176 to L-285; a-177 to L-235; l-178 to L-285; e-179 to L-285; e-180 to L-285; k-181 to L-285; e-182 to L-285; n-183 to L-285; k-184 to L-285; i-185 to L-285; l-186 to L-285; v-187 to L-285; k-188 to L-285; e-189 to L-285; t-190 to L-285; g-191 to L-285; y-192 to L-285; f-193 to L-285; f-194 to L-280; i-195 to L-285; y196 to L-285; g-197 to L-285; q-198 to L-285; v-199 to L-285; l-200 to L-285; y-201 to L-285; t-202 to L-285; d-203 to L-285; k-204 to L-285; t-205 to L-285; y-206 to L-285; a-207 to L-285; m-203 to L-285; g-209 to L-285; h-210 to L-285; l-211 to L-285; i-212 to L-285; q-213 to L-285; r-214 to L-285; k-215 to L-285; k-216 to L-285; v-217 to L-285; h-218 to L-285; v-219 to L-285; f-220 to L-285; g-221 to L-285; d-222 to L-285; e-223 to L-285; l-224 to L-285; s-225 to L-285; l226 to L-285; v-227 to L-285; t-228 to L-285; l-229 to L-285; f-230 to L-285; r-231 to L-285; c-232 to L-285; i-233 to L-285; q-234 to L-235; n-235 to L-285; m-236 to L-285; p-237 to L-285; e-238 to L-285; t-239 to L-285; l-240 to L-285; p-241 to L-285; n-242 to L-285; n-243 to L-285; s-244 to L-285; c-245 to L-285; y-246 to L-285; s-247 to L-285; a-248 to L-285; g-249 to L-285; 1-250 to L-285; a-251 to L-285; k-252 to L-285; l-253 to L-285; e-254 to L-285; e-255 to L-285G-256 to L-285; d-257 to L-285; e-258 to L-285; l-259 to L-285; q-260 to L-285; l-261 to L-285; a-262 to L-285; i-263 to L-285; p-264 to L-285; r-265 to L-285; e-266 to L-285; n-267 to L-285; a-268 to L-285; q-269 to L-285; i-270 to L-285; s-271 to L-285; l-272 to L -285; d-273 to L-285; g-274 to L-285; d-275 to L-285; v-276 to L-285; t-277 to L-285; f-278 to L-285; f-279 to L-285; and G-280 to L-285. Polypeptides encoded by these polynucleotides are also encompassed by the present invention. The present invention also relates to a nucleic acid molecule comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide as described above. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by such nucleic acids and/or polynucleotides which comprise or consist of an amino acid sequence which is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences are also encompassed by the present invention, as are polynucleotides encoding such polypeptides.

A particularly preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 95%, 96%, 97%, 98%, 99% or 100% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2). A preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 90% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2). A more preferred embodiment of the invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 95% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence 134-285 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2). A more preferred embodiment of the invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 96% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence 134-285 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2).

In addition, a more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 97% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2). In addition, a more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 98% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2). In addition, a more preferred embodiment of the present invention relates to a nucleic acid molecule comprising or consisting of a polynucleotide having a nucleotide sequence which is at least 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha polypeptide having the amino acid sequence at position 134-285 as depicted in FIGS. 1A and 1B (SEQ ID NO: 2).

In specific embodiments, a polypeptide consisting of one of the following Neutrokine-alpha and/or N-terminal deleted polypeptide fragments of Neutrokine-alpha SV is preferred: SEQ ID NO: 2, Ala71-Leu285 amino acid residues, Ala81-Leu285 amino acid residues, Leu112-Leu285 amino acid residues, Ala134-Leu285 amino acid residues, Leu147-Leu285 amino acid residues, and Gly161-Leu285 amino acid residues, and polynucleotides encoding these polypeptides are also encompassed by the invention.

Similarly, many biologically functional C-terminal deletion muteins are known. For example, interferon gamma improves 10 fold activity by deleting 8-10 amino acid residues from the carboxy terminus of its protein (Dobeli et al, J. Biotech 7: 199-216(1988)). since the protein of the invention is a member of the TNF polypeptide family, deletion of the C-terminal amino acid up to the leucine residue at position 284, it is desirable to retain most of the biological activity, e.g., ligand binding, ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation and/or activation, ability to modulate cell replication. Polypeptides that are deleted up to about 10 additional C-termini or residues (i.e., up to glycine residue 274) may also retain some activity, such as receptor binding activity, although such polypeptides lose a portion of their extension to SEQ ID NO: 2, the conserved TNF domain of Leu 284. However, even if deletion of one or more amino acids from the C-terminus of a protein results in loss of one or more biological functions of the protein, other functions may be retained. Thus, when a small number of residues of the intact or mature protein are removed from the C-terminus, the ability of the shortened protein to induce and/or bind antibodies that recognize the intact or mature protein remains. Whether a particular polypeptide lacking the C-terminal residue of an intact protein retains such immunological activity can be determined by conventional methods described herein and by other methods known in the art.

Thus, the present invention further provides a polypeptide having one or more residues deleted from the carboxy terminus of the amino acid sequence depicted in FIGS. 1A and 1B (SEQ ID NO: 2) up to glycine 274 (Gly274), and polynucleotides encoding the same. In particular, the invention provides a polypeptide comprising or consisting of SEQ ID NO: 2 amino acid sequence 1-m¹A polypeptide consisting of the amino acid sequence of residue(s) wherein m¹Is SEQ ID NO: 2 at amino acid residues 274-284. Polynucleotides encoding these polypeptides are also encompassed by the present invention. More particularly, the present invention provides polynucleotides encoding polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, residues 1-274, 1-275, 1-276, 1-277, 1-278, 1-279, 1-280, 1-281, 1-282, 1-283, and 1-284. Polypeptides encoded by these polynucleotides are also encompassed by the present invention. The invention also relates to a nucleic acid molecule comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by such nucleic acid and/or polynucleotide sequences which comprise or consist of at least 80%, 85%, 90%, 92%, 95% amino acid sequences as described above are also encompassed by the present invention Amino acid sequences that are 96%, 97%, 98% or 99% identical, and polynucleotides encoding these polypeptides are also encompassed by the invention.

The invention also provides a polypeptide comprising or consisting of one or more amino acids deleted from the amino terminus and the carboxy terminus, which can be described as having the amino acid sequence of SEQ ID NO: n of 2¹-m¹Residue of which n¹And m¹Are integers as described above. The present invention also includes a nucleotide sequence encoding a polypeptide comprising or consisting of a portion of the complete Neutrokine-alpha amino acid sequence encoded by the deposited cDNA clone deposited under ATCC accession No. 97768, wherein the portion does not include amino acids 1-190 of the amino terminus, or amino acids 1-11 of the C-terminus, of the complete amino acid sequence (or any combination of these N-and C-terminal deletions). Polynucleotides encoding all of the above-described deletion polypeptides are also encompassed by the present invention.

Similarly, the amino acid sequence from the C-terminal residue of the Neutrokine-alpha putative extracellular domain up to seq id NO: 2, can retain some biological activity, such as ligand binding, stimulation of lymphocyte (e.g., B cell) proliferation, differentiation and/or activation, and modulation of cell replication or modulation of target cell activity. Having a further C-terminal deletion comprising SEQ ID NO: 2, Leu79 is not expected to retain biological activity.

However, even if the deletion of one or more amino acids at the C-terminus of the polypeptide results in the loss of one or more biological functions of the polypeptide, other biological activities may be retained. Thus, the ability of a shortened polypeptide to induce and/or bind an antibody that recognizes an intact, mature polypeptide or its extracellular domain may be retained when a small number of residues of the intact, mature polypeptide or its extracellular domain are removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residue of the putative extracellular domain retains such immunological activity can be determined by conventional methods described herein as well as by other methods known in the art.

Accordingly, the art also provides polypeptides having a sequence deleted from SEQ ID NOs: 2 until the carboxyl terminal of the amino acid sequence of the putative extracellular domain of the Neutrokine-alpha polypeptideOne or more residues of leucine 79. In particular, the invention provides a polypeptide comprising or consisting of SEQ ID NO: 2, 73-m of the amino acid sequence shown in²A polypeptide consisting of an amino acid sequence consisting of residues, wherein m²Is SEQ ID NO: 2, and residue 78 is the first residue at the C-terminus of the putative extracellular domain of Neutrokine-alpha polypeptide (SEQ ID NO: 2). Polypeptides encoded by these polynucleotides are also encompassed by the present invention. More particularly, in certain embodiments the invention provides polynucleotides encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 2Q-73 to Leu-285; q-73 to L-284; q-73 to K-283; q-73 to L-282; q-73 to A-281; q-73 to G-280; q-73 to F-279; q-73 to F-278; q-73 to T-277; q-73 to V-276; q-73 to D-275; q-73 to G-274; q-73 to D-273; q-73 to L-272; q-73 to S-271; q-73 to I-270; q-73 to Q-269; q-73 to A-268; q-73 to N-267; q-73 to E-266; q-73 to R-265; q-73 to P-264; q-73 to I-263; q-73 to A-262; q-73 to L-261; q-73 to Q-260; q-73 to L-259; q-73 to E-258; q-73 to D-257; q-73 to G-256; q-73 to E-255; q-73 to E-254; q-73 to L-253; q-73 to K-252; q-73 to A-251; q-73 to I-250; q-73 to G-249; q-73 to A-248; q-73 to 8-247; q-73 to Y-246; q-73 to C-245; q-73 to S-244; q-73 to N-243; q-73 to N-242; q-73 to P-241; q-73 to L-240; q-73 to T-239; q-73 to E-238; q-73 to P-237; q-73 to M-236; q-73 to N-235; q-73 to Q-234; q-73 to I-233; q-73 to C-232; q-73 to R-231; q-73 to F-230; q-73 to L-229; q-73 to T-228; q-73 to V-227; q-73 to L-226; q-73 to S-225; q-73 to L-224; q-73 to E-223; q-73 to D-222; q-73 to G-221; q-73 to F-220; q-73 to V-219; q-73 to H-218; q-73 to V-217; q-73 to K-216; q-73 to K-215; q-73 to R-214; q-73 to Q-213; q-73 to I-212; q-73 to L-211; q73 to H-210; q-73 to G-209; q-73 to M-208; q-73 to A-207; q-73 to Y-206; q-73 to T-205; q-73 to K-204; q-73 to D-203; q-73 to T-202; q-73 to Y-201; q-73 to L-200; q-73 to V-199; q-73 to Q-198; q-73 to G-197; q-73 to Y-196; q-73 to I-195; q-73 to F-194; q-73 to F-193; q-73 to Y-192; q-73 to G-191; q-73 to T-190; q-7 3 to E-189; q-73 to K-188; q-73 to V-187; q-73 to L186; q-73 to I-185; q-73 to K-184; q-73 to N-183; q-73 to E-182; q-73 to K-181; q-73 to E-180; q-73 to E-179; q-73 to L-178; q-73 to A-177; q-73 to S-176; q-73 to G-175; q-73 to R-174; q-73 to K-173; q-73 to F-172; q-73 to S-171; q-73 to L-170; q-73 to L-169; q-73 to W-168; q-73 to P-167; q-73 to V-166; q-73 to F-165; q-73 to T-164; q-73 to Y-163; q-73 to S-162; q-73 to G-161; q-73 to K-160; q-73 to Q-159; q-73 to I-158; q-73 to T-157; q-73 to P-156; q-73 to T-155; q-73 to E-154; q-73 to S-153; q-73 to D-152; q-73 to A-151; q-73 to 1-150; q-73 to L-149; q-73 to Q-148; q-73 to L-147; q-73 to C-146; q-73 to D-145; q-73 to Q-144; q-73 to T-143; q-73 to V-142; q-73 to T-141; q-73 to E-140; q-73 to E-139; q-73 to P-138; q-73 to G-137; q-73 to Q-136; q-73 to V-135; q-73 to A-134; q-73 to R-133; q-73 to K-132; q-73 to N-131; q-73 to R-130; q-73 to S-129; q-73 to N-128; q-73 to Q-127; q-73 to S-126; q-73 to S-125; q-73 to N-124; q-73 to G-123; q-73 to E-122; q-73 to G-121; q-73 to P-120; q-73 to A-119; q-73 to P-118; q-73 to P-117; q-73 to E-116; q-73 to F-115; q-73 to I-114; q-73 to K-113; q-73 to L-112; q-73 to G-111; q-73 to A-110; q-73 to T-109; q-73 to V-108; q-73 to A-107; q-73 to P-106; q-73 to A-105; q-73 to E-104; q-73 to E-103; q-73 to L-102; q-73 to G-101; q-73 to A-100; q-73 to K-99; q-73 to P-98; q-73 to A-97; q-73 to G-96; q-73 to A-95; q-73 to G-94; q-73 to A-93; q-73 to P-92; q-73 to L-91; q-73 to K-90; q-73 to E-89; q-73 to A-88; q-73 to H-S7; q73 to H-86; q-73 to G-85; q-73 to Q-84; q-73 to L-83; q-73 to E-82; q-73 to A-81; q-73 to R-80; q-73 to L-79. Polypeptides encoded by these polynucleotides are also encompassed by the present invention. The invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence which is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide as defined above, and said polynucleotide sequence fused to a heterologous polynucleotide sequence . Polypeptides encoded by such nucleic acid and/or polynucleotide sequences are also encompassed by the present invention, as are polypeptides comprising or consisting of amino acid sequences that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above-described amino acid sequences, and polynucleotides encoding such polypeptides.

The invention also provides polypeptides having one or more amino acids deleted from the amino terminus and the carboxy terminus of the putative extracellular domain of Neutrokine-alpha, which can be described as having the amino acid sequence of SEQ ID NO: n of 2²-m²Residue of which n²And m²Are integers as described above.

In another embodiment, a nucleotide sequence encodes a polypeptide consisting of a portion of the ectodomain of a Neutrokine-alpha amino acid sequence encoded by the cDNA plasmid of No.97768 in the ATCC, said portion of the ectodomain not including amino acids 1-206 of the amino terminus, or amino acids 1-206 of the carboxy terminus, or any combination of the amino-terminal and carboxy-terminal deletions of the amino-and carboxy-terminal amino acid sequence of the ectodomain of Neutrokine-alpha amino acid sequence.

As noted above, even if deletion of one or more amino acids from the N-terminus of a polypeptide results in loss of one or more functional activities (i.e., biological activities) of the polypeptide, other functions or biological activities may be retained. Thus, the ability of a shortened Neutrokine-alpha mutein to induce and/or bind antibodies that recognize full-length or mature forms of a polypeptide or its extracellular domain may be retained when a small number of residues of the complete or mature polypeptide or its extracellular domain are removed from the N-terminus. Whether a particular polypeptide lacking the N-terminal residue of the intact polypeptide retains such immunological activity can be determined by the methods described herein and by methods known in the art. It is not impossible that the Neutrokine-alpha mutein lacking a large number of N-terminal amino acid residues still retains some functional (e.g., biological or immunological) activity. In fact, peptides consisting of as few as 6 Neutrokine-alpha amino acid residues will also typically elicit an immune response.

Accordingly, the present invention provides a polypeptide having a deletion from SEQ id no, and a polynucleotide encoding such a polypeptideID NO: 2 up to one or more residues of glycine residue 280 from the amino terminus of the putative full length amino acid sequence of Neutrokine-alpha. In particular the invention provides a polypeptide comprising SEQ ID NO: 2, sequence n³A polypeptide of amino acid sequence consisting of residues 285, wherein n³Is SEQ ID NO: 2 from 1 to 280 amino acid residues.

More particularly, the present invention provides polynucleotides encoding polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 2D-2 to L-285; d-3 to L-285; s-4 to L-285; t-5 to L-285; e-6 to L-285; r-7 to L-285; e-8 to L-285; q-9 to L-285; s-10 to L-285; r-11 to L-285; l-12 to L-285; t-13 to L-285; s-14 to L-285; c-15 to L-285; l-16 to L-285; k-17 to L-285; k-18 to L-285; r-19 to L-285; e-20 to L-285; e-21 to L-285; m-22 to L-285; k23 to L-285; l-24 to L-285; k-25 to L-285; e-26 to L-285; c-27 to L-285; v-28 to L-285; s-29 to L-285; i-30 to L-285; l-31 to L-285; p-32 to L-285; r-33 to L-285; k-34 to L-285; e-35 to L-285; s-36 to L-285; p-37 to L-285; s-38 to L-285; v-39 to L-285; r-40 to L-285; s-41 to L-285; s-42 to L-285; k43 to L-285; d-44 to L-285; g-45 to L-285; k-46 to L-285; l-47 to L-285; l-48 to L-285; a-49 to L-285; a-50 to L-285; t-51 to L-285; l-52 to L-285; l-53 to L-285; l-54 to L-285; a-55 to L-285; l-56 to L-285; l-57 to L-285; s-58 to L-285; c-59 to L-285; c-60 to L-283; l-61 to L-285; t-62 to L-285; v-63 to L-285; v-64 to L-285; s-65 to L-285; f-66 to L-285; y-67 to L-285; q-68 to L-285; v-69 to L-285; a-70 to L-285; a-71 to L-285; l-72 to L-285; q-73 to L-285; g-74 to L-285; d-75 to L-285; l-76 to L-285; a-77 to L-285; s-78 to L-285; l-79 to L-285; r-80 to L-285; a-81 to L-285; e-82 to L-285; l-83 to L-285; q-84 to L-285; g-85 to L-285; h-86 to L-285; h-87 to L-285; a-88 to L-285; e-89 to L-285; k-90 to L-285; l-91 to L-285; p-92 to L-285; a-93 to L-285; g-94 to L-285; a-95 to L-285; g-96 to L285; a-97 to L-285; p-98 to L-285; k-99 to L-285; a-100 to L-285; g-101 to L-285; l-102 to L-285; e-103 to L-285; e-104 to L-285; a-105 to L-285; p-106 to L-285; a-107 to L-285; v-108 to L-285; t-109 to L-285; a-110 to L-285; g-111 to L-285; l-112 to L-285; k-113 to L-285; i-114 to L-285; f-115 to L-285; e-116 to L-285; p-117 to L-285; p-118 to L-285; a-119 to L-285; p-120 to L-285; g-121 to L-285; e-122 to L-285; g-123 to L-285; n-124 to L-285; s-125 to L-285; s-126 to L-285; q-127 to L-285; n-128 to L-285; s-129 to L-285; r130 to L-285; n-131 to L-285; k-132 to L-285; r-133 to L-285; a-134 to L-285; v-135 to L-285; q-136 to L-285; g-137 to L-285; p-138 to L-285; e-139 to L-285; e-140 to L-285; t-141 to L-285; v-142 to L-285; t-143 to L-285; q-144 to L-285; d-145 to L-285; c-146 to L-285; l-147 to L-285; q-148 to L-285; l-149 to L-285; i-150 to L-285; a-151 to L-285; d-152 to L-285; s-153 to L-285; e-154 to L-285; t-155 to L-285; p-156 to L-285; t-157 to L-283; i-158 to L-285; q-159 to L-285; k-160 to L-285; g-161 to L-285; s-162 to L-285; y-163 to L-285; t-164 to L-285; f-165 to L-285; v-166 to L-285; p-167 to L-285; w-168 to L-285; l-169 to L-285; l-170 to L-285; s-171 to L-285; f-172 to L-285; k-173 to L-285; r-174 to L-285; g-175 to L-285; s-176 to L-285; a-177 to L-285; l-178 to L-285; e-179 to L-285; e-180 to L-285; k-181 to L-285; e-182 to L-285; n-183 to L-285; k-184 to L-285; i-185 to L-285; l-186 to L-285; v-187 to L-285; k-188 to L-285; e-189 to L-285; t-190 to L-285; g-191 to L-285; y-192 to L-285; f-193 to L-285; f-194 to L-285; i-195 to L-285; y-196 to L-285; g-197 to L-285; q-198 to L-285; v-199 to L-285; l-200 to L-285; y-201 to L-285; t-202 to L-285; d-203 to L-285; k-204 to L-285; t-205 to L-285; y-206 to L-285; a-207 to L-285; m-208 to L-285; g-209 to L-285; h-210 to L-285; l-211 to L-285; i-212 to L-285; q-213 to L-285; r-214 to L-285; k-215 to L-285; k-216 to L-285; v-217 to L-285; h-218 to L-285; v-219 to L-285; f-220 to L-285; g-221 to L-285; d-222 to L-285; e-223 to L-285; l-224 to L-285; s-225 to L-285; l-226 to L-285; v-227 to L-285; t-228 to L-285; l-229 to L-285; f-230 to L-285; r-231 to L-285; c-232 to L-285; i-233 to L-285; q-234 to L-285; n-235 to L-285; m-236 to L-285; p-237 to L-285; e-238 to L-285; t-239 to L-285; l-240 to L-285; p-241 to L-285; n-242 to L-285; n-243 to L-285; s-244 to L-285; c-245 to L-285; y-246 to L-285; s-247 to L-285; a-248 to L-285; g-249 to L-285; i-250 to L-285; a-251 to L-285; k-252 to L-285; l-253 to L-285; e-254 to L-285; e-255 to L-285; g-256 to L-285; d-257 to L-285; e-258 to L-285; l-259 to L-285; q-260 to L-285; l-261 to L-285; a-262 to L-285; 1-263 to L-285; p-264 to L-285; r-265 to L-285; e-266 to L-285; n-267 to L-285; a-268 to L-285; q-269 to L-285; i-270 to L-285; s-271 to L-285; b-272 to L-285; d-273 to L-285; g-274 to L-285; d-275 to L-285; v-276 to L-285; t-277 to L-285; f-278 to L-285; f-279 to L-285; and G-280 to L-285. The invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide as described above, and to such polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by such nucleic acid and/or polynucleotide sequences comprising amino acid sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences are also encompassed by the present invention, as are polynucleotides encoding such polypeptides.

As described above, even if one or more amino acids are deleted from the C-terminus of the protein, resulting in the loss of one or more functional activities (e.g., biological activities) of the protein, other functional activities may be retained. Thus, the ability of the shortened Neutrokine-alpha mutein to induce and/or bind antibodies recognizing the intact or mature polypeptide or extracellular domain may still be retained when a small number of residues of the intact or mature polypeptide or extracellular domain are removed from the C-terminus. Whether a particular polypeptide lacks the C-terminus of the intact polypeptide retains such immunological activity can be determined by the methods described herein and by other methods known in the art. It is not impossible that Neutrokine-alpha muteins lacking a large number of C-terminal amino acid residues still retain some functional (e.g., biological or immunological) activity. In fact, peptides consisting of as few as 6 Neutrokine-alpha amino acid residues will also typically elicit an immune response.

Accordingly, the present invention provides a polypeptide having a deletion from SEQ ID NO: 2 to the carboxy terminus up to one or more residues of glutamic acid at position 6, and polynucleotides encoding such polypeptides. In particular, the invention provides a polypeptide comprising seq id NO: 1-m of 2³A polypeptide of amino acid sequence consisting of residues, wherein m ³Is SEQ ID NO: 2, from 6 to 284 amino acid residues.

More particularly, the present invention provides polynucleotides encoding polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 2M-1 to L-284; m-1 to K-283; m-1 to L-282; m-1 to A-281; m-1 to G-280; m-1 to F-279; m-1 to F-278; m-1 to T-277; m-1 to V-276; m-1 to D-275; m-1 to G-274; m-1 to D-273; m-1 to L-272; m-1 to S-271; m-1 to I-270; m-1 to Q-269; m-1 to A-268; m-1 to N-267; m-1 to E-266; m-1 to R-265; m-1 to P-264; m-1 to I-263; m-1 to A-262; m-1 to L-261; m-1 to Q-260; m-1 to L-259; m-1 to E-258; m-1 to D-257; m-1 to G-256; m-1 to E-255; m-1 to E-254; m-1 to L-253; m-1 to K-252; m-1 to A-251; m-1 to I-250; m-1 to G-249; m-1 to A-248; m-1 to S-247; m-1 to Y-246; m-1 to C-245; m-1 to S-244; m-1 to N-243; m-1 to N-242; m-1 to P-241; m-1 to L-240; m-1 to T-239; m-1 to E-238; m-1 to P-237; m-1 to M-236; m-1 to N-235; m-1 to Q-234; m-1 to I-233; m-1 to C-232; m-1 to R-231; m-1 to F-230; m-1 to L-229; m-1 to T-228; m-1 to V-227; m-1 to L-226; m-1 to S-225; m-1 to L-224; m-1 to E-223; m-1 to D-222; m-1 to G-221; m-1 to F-220; m-1 to V-219; m-1 to H-118; m-1 to V-117; m-1 to K-216; m-1 to K-115; m-1 to R-114; m-1 to Q-113; m-1 to I-112; m-1 to L-211; m-1 to H-210; m-1 to G-209; m-1 to M-208; m-1 to A-207; m-1 to Y-206; m-1 to T-205; m-1 to K-204; m-1 to D-203; m-1 to T-202; m-1 to Y-201; m-1 to L-200; m-1 to V-199; m-1 to Q-198; m-1 to G-197; m-1 to Y-196; m-1 to 1-195; m-1 to F-194; m-1 to F-193; m-1 to Y-192; m-1 to G-191; m-1 to T-190; m-1 to E-189; m-1 to K-188; m-1 to V-187; m-1 to L-186; m-1 to I-185; m-1 to K-184; m-1 to N-183; m-1 to E-132; m-1 to K-181; m-1 to E-180; m-1 to E-179; m-1 to L-178; m-1 to A-177; m-1 to S-176; m-1 to G-175; m-1 to R-174; m-1 to K-173; m-1 to F-172; m-1 to S-171; m-1 to L-170; m-1 to L-169; m-1 to W-168; m-1 to P-167; m-1 to V-166; m-1 to F-165; m-1 to T-164; m-1 to Y-163; m-1 to S-162; m-1 to G-161; m-1 to K-160; m-1 to Q-159; m-1 to I-158; m-1 to T-157; m-1 to P-156; m-1 to T-155; m-1 to E-154; m-1 to S-153; m-1 to D-152; m-1 to A-151; m-1 to I-150; m-1 to L-149; m-1 to Q-148; m-1 to L-147; m-1 to C-146; m-1 to D-145; m-1 to Q-144; m-1 to T-143; m-1 to V-142; m-1 to T-141; m-1 to E-140; m-1 to E-139; m-1 to P-138; m-1 to G-137; m-1 to Q-136; m-1 to V-135; m-1 to A-134; m-1 to R-133; m-1 to K-132; m-1 to N-131; m-1 to R-130; m-1 to S-129; m-1 to N-128; m-1 to Q-127; m-1 to S-126; m-1 to S-125; m-1 to N-124; m-1 to G-123; m-1 to E-122; m-1 to G-121; m-1 to P-120; m-1 to A-119; m-1 to P118; m-1 to P-117; m-1 to E-116; m-1 to F-115; m-1 to I-114; m-1 to K-113; m-1 to L-112; m-1 to G-111; m-1 to A-110; m-1 to T-109; m-1 to V-108; m-1 to A-107; m-1 to P-106; m-1 to A-105; m-1 to E-104; m-1 to E-103; m-1 to L-102; m-1 to G-101; m-1 to A-100; m-1 to K-99; m-1 to P-98; m-1 to A-97; m-1 to G-96; m-1 to A-95; m-1 to G-94; m-1 to A-93; m-1 to P-92; m-1 to L-91; m-1 to K-90; m-1 to E-89; m-1 to A-88; m-1 to H-87; m-1 to H-86; m-1 to G-35; m-1 to Q-84; m-1 to L-83; m-1 to E-82; m-1 to A-81; m-1 to R-80; m-1 to L-79; m-1 to S-78; m-1 to A-77; m-1 to L-76; m-1 to D-75; m-1 to G-74; m-1 to Q-73; m-1 to L-72; m-1 to A-71; m-1 to A-70; m-1 to V-69; m-1 to Q-68; m-1 to Y-67; m-1 to F-66; m-1 to S-65; m-1 to V-64; m-1 to V-63; m-1 to T-62; m-1 to L-61; m-1 to C-60; m-1 to C-59; m-1 to S-58; m-1 to L-57; m-1 to L-56; m-1 to A-55; m-1 to L-54; m-1 to L-53; m-1 to L-52; m-1 to T-51; m-1 to A-50; m-1 to A-49; m-1 to L-48; m-1 to L-47; m-1 to K-46; m-1 to G-45; m-1 to D-44; m-1 to K-43; m-1 to S-42; m-1 to S-41; m-1 to R-40; m-1 to V-39; m-1 to S-38; m-1 to P-37; m-1 to S-36; m-1 to E-35; m-1 to K-34; m-1 to R-33; m-1 to P-32; m-1 to L-31; m-1 to 1-30; m-1 to S-29; m-1 to V-28; m-1 to C-27; m-1 to E-26; m-1 to K-25; m-1 to L-24; m-1 to K-23; m-1 to M-22; m-1 to E-21; m-1 to E-20; m-1 to R-19; m-1 to K-18; m-1 to K-17; m-1 to L-16; m-1 to C-15; m-1 to S-14; m-1 to T-13; m-1 to L-12; m-1 to R-11; m-1 to S-10; m-1 to Q-9; m-1 to E-8; m-1 to R-7; and M-1 to E-6. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide as described above. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by such nucleic acid and/or polynucleotide sequences comprising amino acid sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences are also encompassed by the present invention, as are polynucleotides encoding such polypeptides.

The invention also provides polypeptides having one or more amino acids deleted from the amino terminus and the carboxy terminus of a Neutrokine-alpha polypeptide, which can be described as having the amino acid sequence of SEQ ID NO: n of 2³-m³A position residue wherein n³And m³Are integers as described above.

In addition, since the putative extracellular domain of a Neutrokine- α SV polypeptide of the present invention may itself elicit functional activity (e.g., biological activity), the amino acid sequence set forth in SEQ ID NO: 19, the putative extracellular domain N and C-terminal amino acid deletions of the polypeptide at Gln73-Leu266 may still retain some functional activity, such as ligand binding, stimulation of lymphocyte (e.g., B cell) proliferation, differentiation and/or activation, modulation of cell replication, modulation of target cell activity and/or immune activity. However, even if one or more amino acids are deleted from the N-terminus of the putative extracellular domain of the Neutrokine- α SV polypeptide resulting in the loss of one or more functional activities, other functional activities may be retained. Thus, the ability of a shortened polypeptide to induce and/or bind an antibody that recognizes an intact or mature polypeptide or an extracellular domain thereof may be retained when a small number of residues of the mature or intact polypeptide or extracellular domain are removed from the N-terminus. Whether a particular polypeptide lacking the N-terminal residue of the intact polypeptide retains such immunological activity can be determined by conventional methods described herein and by other methods known in the art.

Accordingly, the present invention also provides a polypeptide having one or more residues deleted from the amino terminus of the Neutrokine- α SV amino acid sequence up to the glycine residue at position 261, and polynucleotides encoding the same. In particular the invention provides a polypeptide comprising SEQ ID NO: 19 n of⁴A polypeptide of the amino acid sequence of-266, wherein n⁴Is SEQ ID NO: 19 amino acid sequence 73-261, and position 261 is the first residue position at the N-terminus of the putative extracellular domain of a Neutrokine- α SV polypeptide (as shown in SEQ ID NO: 19).

More particularly, in certain embodiments, the present invention provides polynucleotides encoding a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: q-73 to L-266 of 19; g-74 to L-266; d-75 to L-266; l-76 to L-266; a-77 to L-266; s-78 to L-266; l79 to L-266; r-80 to L-266; a-81 to L-266; e-82 to L-266; l-83 to L-266; q-84 to L-266, G-85 to L-266; h-86 to L-266; h-87 to L-266; a-88 to L-266; e-89 to L-266; k-90 to L-266; l-91 to L-266; p-92 to L-266; a-93 to L-266; g-94 to L-266; a-95 to L-266; g-96 to L-266; a-97 to L-266; p-98 to L-266; k-99 to L-266; a-100 to L-266; g-101 to L-266; l-102 to L-266; e-103 to L-266; e-104 to L-266; a-105 to L-266; p-106 to L-266; a-107 to L-266; v-108 to L-266; t-109 to L-266; a-110 to L-266; g-111 to L-266; l-112 to L-266; k-113 to L-266; i-114 to L-266; f-115 to L-266; e-116 to L-266; p-117 to L-266; p-118 to L-266; a119 to L-266; p-120 to L-266; g-121 to L-266; e-122 to L-266; g-123 to L-266; n-124 to L-266; s-125 to L-266; s-126 to L-266; q-127 to L-266; n-128 to L-266; s-129 to L-266; r130 to L-266; n-131 to L-266; k-132 to L-266; r-133 to L-266; a-134 to L-266; v-135 to L-266; q-136 to L-266; g-137 to L-266; p-138 to L-266; e-139 to L-266; e-140 to L-266; t-141 to L-266; g-142 to L-266; s-143 to L266; y-144 to L-266; t-145 to L-266; f-146 to L-266; v-147 to L-266; p-148 to L-266; w-149 to L-266; l-150 to L-266; l-151 to L-266; s-152 to L-266; f-153 to L-266; k-154 to L-266; r-155 to L-266; g-156 to L-266; s-157 to L-266; a-158 to L-266; l-159 to L-266; e-160 to L-266; e-161 to L-266; k-162 to L-266; e-163 to L-266; n-164 to L-266; k-165 to L-266; i-166 to L-266; l-167 to L-266; v-168 to L-266; k-169 to L-266; e-170 to L-266; t-171 to L-266; g-172 to L-266; y-173 to L-266; f-174 to L-266; f-175 to L-266; i-176 to L-266; y-177 to L-266; g-178 to L-266; q-179 to L-266; v-180 to L-266; l-181 to L-266; y-182 to L-266; t-183 to L-266; d-184 to L-266; k-185 to L-266; t-186 to L-266; y-187 to L-266; a-188 to L-266; m-189 to L-266; g-190 to L-266; h-191 to L-266; l-192 to L-266; i-193 to L-266; q-194 to L-266; r-195 to L-266; k-196 to L-266; k-197 to L-266; v-198 to L-266; h-199 to L-266; v-200 to L-266; f-201 to L-266; g-202 to L-266; d-203 to L-266; e-204 to L-266; l-205 to L-266; s-206 to L-266; l-207 to L-266; v-203 to L-266; t-209 to L-266; l-210 to L-266; f-211 to L-266; r-212 to L-266; c-213 to L-266; i-214 to L-266; q-215 to L-266; n-216 to L-266; m-217 to L-266; p-218 to L-266; e-219 to L-266; t-220 to L-266; l-221 to L-266; p-222 to L-266; n-223 to L-266; n-224 to L-266; s-225 to L-266; c-226 to L-266; y-227 to L-266; s-228 to L-266; a-229 to L-266; g-230 to L-266; i-231 to L-266; a-232 to L-266; k-233 to L-266; l-234 to L-266; e-235 to L-266; e-236 to L-266; g-237 to L-266; d-238 to L-266; e-239 to L-266; l-240 to L-266; q-241 to L-266; l-242 to L-266; a-243 to L-266; i-244 to L-266; p-245 to L-266; r246 to L-266; e-247 to L-266; n-248 to L-266; a-249 to L-266; q-250 to L-266; i-251 to L-266; s-252 to L-266; l-233 to L-266; d-254 to L-266; g-255 to L-266; d-256 to L-266; v-257 to L-266; t-258 to L-266; f-259 to L-266; f-260 to L-266; and G-261 to L-266. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide sequence encoding the aforementioned Neutrokine- α SV polypeptide. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by such nucleic acid and/or polynucleotide sequences comprising amino acid sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences are also encompassed by the present invention, as are polynucleotides encoding such polypeptides.

Similarly, the C-terminal amino acid of the putative extracellular domain of Neutrokine- α SV up to SEQ ID NO: the deletion of leucine 79 may retain some functional activity, such as ligand binding, stimulation of lymphocyte (e.g., B cell) proliferation, differentiation and/or activation, modulation of cell replication, modulation of target cell activity and/or immunogenicity. Having the further SEQ ID NO: the C-terminal deletion of 19, including the Leu79 deletion, is not expected to retain biological activity.

However, even if one or more amino acids are deleted from the C-terminus of the polypeptide, resulting in loss of one or more functional activities (e.g., biological activities) of the polypeptide, other functional activities may be retained. Thus, the ability of a shortened polypeptide to induce and/or bind an antibody that recognizes an intact polypeptide, a mature polypeptide or an extracellular domain thereof may be retained when a small number of residues of the mature, intact polypeptide or the extracellular domain are removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residue of the putative extracellular domain retains such immunological activity can be determined by the routine methods described herein and by other methods known in the art.

Accordingly, the present invention further provides a polypeptide having a sequence deleted from SEQ ID NO: 19 to the carboxy terminus of one or more residues of the amino acid sequence of the putative extracellular domain of Neutrokine- α SV up to leucine at position 79. In particular, the invention provides a tool Having the sequence of SEQ ID NO: 19 of an amino acid sequence shown in 73-m⁴A polypeptide of amino acid sequence consisting of residue(s) wherein m⁴Is SEQ ID NO: 19 at one integer position from the 79 to 266 amino acid residue positions of the amino acid sequence shown in figure 19.

More particularly, in certain embodiments, the invention provides polynucleotides encoding polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: q-73 to K-264 of 19; q-73 to L-263; q-73 to A-262; q-73 to G-261; q-73 to F-260; q-73 to F-259; q-73 to T-258; q-73 to V-257; q-73 to D-256; q-73 to G-255; q-73 to D-254; q-73 to L-253; q-73 to S-252; q-73 to I-251; q-73 to Q-250; q-73 to A-249; q-73 to N-248; q-73 to E-247; q-73 to R-246; q-73 to P-240; q-73 to I-244; q-73 to A-243; q-73 to L-242; q-73 to Q-241; q-73 to L-240; q73 to E-239; q-73 to D-238; q-73 to G-237; q-73 to E-236; q-73 to E-235; q-73 to L-234; q-73 to K-233; q-73 to A-232; q-73 to I-231; q-73 to G-230; q-73 to A-229; q-73 to S-228; q-73 to Y-227; q-73 to C-226; q-73 to S-225; q-73 to N-224; q-73 to N-223; q-73 to P-222; q-73 to L-221; q-73 to T-220; q-73 to E-219; q-73 to P-218; q-73 to M-217; q-73 to N-216; q-73 to Q-215; q-73 to I-214; q-73 to C-213; q-73 to R-212; q-73 to F-211; q-73 to L-210; q-73 to T-209; q-73 to V-208; q-73 to L-207; q-73 to S-206; q-73 to L-205; q-73 to E-204; q-73 to D-203; q-73 to G-202; q-73 to F-201; q-73 to V-200; q-73 to H-199; q-73 to V-198; q-73 to K-197; q-73 to K-196; q-73 to R-195; q-73 to Q-194; q-73 to I-193; q-73 to L-192; q-73 to H-191; q-73 to G-190; q-73 to Q-73; q-73 to A-188; q-73 to Y-187; q-73 to T-186; q-73 to K-185; q-73 to D-184; q-73 to T-183; q-73 to Y-182; q-73 to L-181; q-73 to V-180; q-73 to Q-179; q-73 to G-178; q-73 to Y-177; q-73 to I-176; q-73 to F-175; q-73 to F-174; q-73 to Y-173; q-73 to G-172; q-73 to T-171; q-73 to E-170; q-73 to K-169; q-73 to V-168; q-73 to L-167; q-73 to I-166; q-73 to K-165; q-73 to N-164; q-73 to E-163; q-73 to K-162; q-73 to E-161; q-73 to E-160; q-73 to L-159; q-73 to A-158; q-73 to S-157; q-73 to G-156; q-73 to R-155; q-73 to K-154; q-73 to F-153; q-73 to S-152; q-73 to L-151; q-73 to L-150; q-73 to W-149; q-73 to P-148; q-73 to V-147; q-73 to F-146; q-73 to T-145; q-73 to Y-144; q-73 to S-143; q-73 to G-142; q-73 to T-141; q-73 to E-140; q-73 to E-139; q-73 to P-138; q-73 to G-137; q-73 to Q-136; q-73 to V-135; q-73 to A-134; q-73 to R-133; q-73 to K-132; q-73 to N-131; q-73 to R-130; q-73 to S-129; q-73 to N-128; q-73 to Q-127; q-73 to S-126; q-73 to S-125; q-73 to N-124; q-73 to G-123; q-73 to E-122; q-73 to G-121; q-73 to P-120; q-73 to A-119; q-73 to P-118; q-73 to P-117; q-73 to E-116; q-73 to F-115; q-73 to I-114; q-73 to K-113; q-73 to L-112; q-73 to G-111; q-73 to A-110; q-73 to T-109; q-73 to V-108; q-73 to A-107; q-73 to P-106; q-73 to A-105; q-73 to E-104; q-73 to E-103; q-73 to L-102; q-73 to G-101; q-73 to A-100; q-73 to K-99; q-73 to P-98; q-73 to A-97; q-73 to G-96; q-73 to A-95; q-73 to G-94; q-73 to A-93; q-73 to P-92; q-73 to L-91; q-73 to K-90; q-73 to E-89; q-73 to A-88; q-73 to H-87; q-73 to H-86; q-73 to G-85; q-73 to Q-84; q-73 to L-83; q-73 to E-82; q-73 to A-81; q-73 to R-80; q-73 to L-79; and Q-73 to S-78. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide sequence encoding the aforementioned Neutrokine- α SV polypeptide. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by such nucleic acid and/or polynucleotide sequences comprising amino acid sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences are also encompassed by the present invention, as are polynucleotides encoding such polypeptides.

The invention also provides polypeptides having one or more amino acids deleted from the amino terminus and the carboxy terminus of the putative extracellular domain of Neutrokine-alpha SV, which may be described as having the amino acid sequence of SEQ id no: 19 n of⁴-m⁴A position residue wherein n⁴And m⁴Are integers as described above.

In another embodiment, the nucleotide sequence encodes a polypeptide consisting of a portion of the extracellular domain of the Neutrokine- α SV amino acid sequence encoded by the deposited cDNA clone deposited under ATCC accession No. 203518, said portion of the extracellular domain not comprising amino acids 1-260 of the amino terminus of the extracellular domain, or amino acids 1-187 of the carboxy terminus of the extracellular domain, or any combination of the amino-terminal and amino-terminal deletions of the extracellular domain.

As noted above, even if the N-terminal deletion of one or more amino acids in a polypeptide results in the loss of one or more functional activities (e.g., biological activities) of the polypeptide, other functional activities may be retained. Thus, the ability of the shortened Neutrokine-alpha SV muteins to induce and/or bind to antibodies that recognize a full-length or mature polypeptide or its extracellular domain remains when a small number of residues of the full-length or mature polypeptide or its extracellular domain are removed from the N-terminus. Whether a particular polypeptide lacking the N-terminal residue of the intact polypeptide retains such immunological activity can be determined by conventional methods described herein and by other methods known in the art. It is not impossible that the Neutrokine- α SV mutein lacking a large number of N-terminal amino acid residues still retains functional (e.g., immunogenic) activity. In fact, peptides consisting of as few as 6 Neutrokine- α SV amino acid residues may also generally elicit an immune response.

Accordingly, the present invention provides a polypeptide having a sequence deleted from SEQ ID NO: 19 to one or more residues from the amino terminus up to glycine at position 261 of the putative full length amino acid sequence of Neutrokine- α SV. In particular the invention provides a polypeptide comprising SEQ ID NO: 19 of the sequence shown in⁵An amino acid sequence consisting of residues 266, wherein m⁵Is SEQ ID NO: 19 in an integer number of amino acid residues 1 to 261 of the amino acid sequence shown in seq id no.

More particularly, the present invention provides polynucleotides encoding polypeptides selected from the group consisting of amino acid sequences of seq id nos: SEQ ID NO: 19D-2 to L-266; d-3 to L-266; s-4 to L-266; t-5 to L-266; e-6 to L-266; r-7 to L-266; e-8 to L-266; q-9 to L-266; s-10 to L-266; r-11 to L-266; l-12 to L-266; t-13 to L-266; s-14 to L-266; c-15 to L-266; l-16 to L-266; k-17 to L-266; k-18 to L-266; r-19 to L-266; e-20 to L-266; e-21 to L-266; m-22 to L-266; k-23 to L-266; l-24 to L-266; k-25 to L-266; e-26 to L-266, C-27 to L-266; v-28 to L-266; s-29 to L-266; i-30 to L-266; l-31 to L-266; p-32 to L-266; r-33 to L-266; k-34 to L-266; e-35 to L-266; s-36 to L-266; p-37 to L-266; s-38 to L-266; v-39 to L-266; r-40 to L-266; s-41 to L-266; s-42 to L-266; k-43 to L-266; d-44 to L-266; g-45 to L-266; k-46 to L-266; l-47 to L-266; l-48 to L-266; a-49 to L-266; a-50 to L-266; t-51 to L-266; l-52 to L-266; l-53 to L-266; l-54 to L-266; a-55 to L-266; l-56 to L-266; l-57 to L-266; s-58 to L-266; c-59 to L-266; c-60 to L-266; l-61 to L-266; t-62 to L-266; v-63 to L-266; v-64 to L-266; s-65 to L-266; f-66 to L-266; y-67 to L-266; q-68 to L-266; v-69 to L-266; a-70 to L-266; a-71 to L-266; l-72 to L-266; q-73 to L-266; g-74 to L-266; d-75 to L-266; l-76 to L-266; a-77 to L-266; s-78 to L-266; l-79 to L-266; r-80 to L-266; a-81 to L-266; e-82 to L-266; l-83 to L-266; q-84 to L-266; g-85 to L-266; h-36 to L-266; h-87 to L-266; a-88 to L-266; e-89 to L-266; k-90 to L-266; l-91 to L-266; p-92 to L-266; a-93 to L-266; g-94 to L-266; a-95 to L-266; g-96 to L-266; a-97 to L-266; p-98 to L-266; k-99 to L-266; a-100 to L-266; g-101 to L-266; l-102 to L-266; e-103 to L-266; e-104 to L-266; a-105 to L-266; p-106 to L-266; a-107 to L-266; v-108 to L-266; t-109 to L-266; a-110 to L-266; g-111 to L-266; l-112 to L-266; k-113 to L-266; i-114 to L-266; f-115 to L-266; e-116 to L-266; p-117 to L-266; p-118 to L-266; a-119 to L-266; p-120 to L-266; g-121 to L-266; e-122 to L-266; g-123 to L-266; n-124 to L-266; s-125 to L-266; s-126 to L-266; q-127 to L-266; n-128 to L-266; s-129 to L-266; r-130 to L-266; n-131 to L-266; k-132 to L-266; r-133 to L-266; a-134 to L-266; v-135 to L-266; q-136 to L-266; g-137 to L-266; p-138 to L-266; e-139 to L-266; e-140 to L-266; t-141 to L-266; g-142 to L-266; s-143 to L-266; y-144 to L-266; t-145 to L-266; f-146 to L-266; v-147 to L-266; p-148 to L-266; w-149 to L-266; l-150 to L-266; l-151 to L-266; s-152 to L-266; f-153 to L-266; k-154 to L-266; r-155 to L-266; g-156 to L-266; s-157 to L-266; a-158 to L-266; l-159 to L-266; e-160 to L-266; e-161 to L-266; k-162 to L-266; e-163 to L-266; n-164 to L-266; k-165 to L-266; i-166 to L-266; l-167 to L-266; v-168 to L-266; k-169 to L-266; e-170 to L-266; t-171 to L-266; g-172 to L-266; y-173 to L-266; f-174 to L-266; f-175 to L-266; i-176 to L-266; y-177 to L-266; g-178 to L-266; q-179 to L-266; v-180 to L-266; l-181 to L-266; y-182 to L-266; t-183 to L-266; d-184 to L-266; k-185 to L-266; t-186 to L-266; y-187 to L-266; a-188 to L-266; m-189 to L-266; g-190 to L-266; h-191 to L-266; l-192 to L-266; i-193 to L-266; q-194 to L-266; r-195 to L-266; k-196 to L-266; k-197 to L-266; v-198 to L-266; h-199 to L-266; v-200 to L-266; f-201 to L-266; g-202 to L-266; d-203 to L-266; e-204 to L-266; l-205 to L-266; s-206 to L-266; l-207 to L-266; v-208 to L-266; t-209 to L-266; l-210 to L-266; f-211 to L-266; r-212 to L-266; c-213 to L-266; 1-214 to L-266; q-215 to L-266; n-216 to L-266; m-217 to L-266; p-218 to L-266; e-219 to L-266; t-220 to L-266; l-221 to L-266; p-222 to L-266; n-223 to L-266; n-224 to L-266; s-225 to L-266; c-226 to L-266; y-227 to L-266; s-223 to L-266; a-229 to L-266; g-230 to L-266; i-231 to L-266; a-232 to L-266; k-233 to L-266; l-234 to L-266; e-235 to L-266; e-236 to L-266; g-237 to L-266; d-238 to L-266; e-239 to L-266; l-240 to L-266; q-241 to L-266; l-242 to L-266; a-243 to L-266; i-244 to L-266; p-245 to L-266; r246 to L-266; e-247 to L-266; n-248 to L-266; a-249 to L-266; q-250 to L-266; i-251 to L-266; s-252 to L-266; u-253 to L-266; d-254 to L-266; g-255 to L-266; d-256 to L-266; v-257 to L-266; t-258 to L-266; f-259 to L-266; f-260 to L-266; and G-261 to L-266. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide as described above. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by such nucleic acid and/or polynucleotide sequences having amino acid sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences are also encompassed by the present invention, as are polynucleotides encoding such polypeptides.

As noted above, even if one or more amino acids are deleted from the C-terminus of the protein, resulting in loss of one or more functional activities (e.g., biological activities) of the protein, other functional activities may be retained. Thus, the ability of a shortened Neutrokine- α SV mutein to induce and/or bind antibodies that recognize an intact or mature polypeptide or its extracellular domain may be retained when a small number of residues of the intact or mature polypeptide or extracellular domain are removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residue of the intact polypeptide retains such immunological activity can be determined by conventional methods described herein and by other methods known in the art. It is not impossible that the Neutrokine- α SV mutein lacking a large number of C-terminal amino acid residues still retains some functional (e.g., immunogenic) activity. In fact, peptides consisting of as few as 6 Neutrokine- α SV amino acid residues will also typically elicit an immune response.

Accordingly, the present invention provides a polypeptide having a sequence deleted from SEQ ID NO: 19 to the carboxy terminus of the Neutrokine- α SV amino acid sequence at one or more residues up to the glutamic acid at position 6. In particular the invention provides a polypeptide comprising SEQ ID NO: 1-m of 19⁵A polypeptide of amino acid sequence of residues, wherein m ⁵Is SEQ ID NO: 19, and an integer number of amino acid residues from 6 to 265 of the amino acid sequence shown in SEQ ID NO.

More particularly, the present invention provides polynucleotides encoding polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: m-1 to G-265 of 19; m-1 to G-264; l-263; m-1 to A-262; m-1 to G-261; m-1 to F-260; m-1 to F-259; m-1 to T-258; m-1 to V-257; m-1 to D-256; m-1 to G-255; m-1 to D-254; m-1 to L-253; m-1 to S-252; m-1 to I-251; m-1 to Q-250; m-1 to A-249; m-1 to N-248; m-1 to E-247; m-1 to R-246; m-1 to R-245; m-1 to I-244; m-1 to A-243; m-1 to L-242; m-1 to Q-241; m-1 to L-240; m-1 to E-239; m-1 to D-238; m-1 to G-237; m-1 to E-236; m-1 to E-235, 5M-1 to L-234; m-1 to K-233; m-1 to A-232; m-1 to I-231; m-1 to G-230; m-1 to A-229; m-1 to S-228; m-1 to Y-227; m-1 to C-226; m-1 to S-225; m-1 to N-224; m-1 to N-223; m-1 to P-222; m-1 to L-221; m-1 to T-220; m-1 to E-219; m-1 to P-218; m-1 to M-217; m-1 to N-216; m-1 to Q-115; m-1 to I-214; m-1 to C-213; m-1 to R-112; m-1 to F-211; m-1 to L-210; m-1 to T-209; m-1 to V-208; m-1 to L-207; m-10 to S-206; m-1 to L-205; m-1 to E-204; m-1 to D-203; m-1 to G-202; m-1 to F-201; m-1 to V-200; m-1 to H-199; m-1 to V-198; m-1 to K-197; m-1 to K-196; m-1 to R-195; m-1 to Q-194; m-1 to I-193; m-1 to L-192; m-1 to H-191; m-1 to G-190; m-1 to M-189; m-1 to A-188; m-1 to Y-187; m-1 to T-186; m-1 to K-185; m-1 to D-184; m-1 to T-183; m-1 to Y-182; m-1 to L-181; m-1 to V-180; m-1 to Q-179; m-1 to G-178; m-1 to Y-177; m-1 to I-176; m-1 to F-175; m-1 to F-174; m-1 to Y-173; m-1 to G-172; m-1 to T-171; m-1 to E-170; m-1 to K-169; m-1 to V-168; m-1 to L-167; m-1 to 1-166; m-1 to K-165; m-1 to N-164; m-1 to E-163; m-1 to K-162; m-1 to E-161; m-1 to E-160; m-1 to L-159; m-1 to A-158; m-1 to S-157; m-1 to G-156; m-1 to R-155; m-1 to K-154; m-1 to F-153; m-1 to S-152; m-1 to L-151; m-1 to L-150; m-1 to W-149; m-1 to P-148; m-1 to V-147; m-1 to F-146; m-1 to T-145; m-1 to Y-144; m-1 to S-143; m-1 to G-142; m-1 to T-141; m-1 to E-140; m-1 to E-139; m-1 to P-138; m-1 to G-137; m-1 to Q-136; m-1 to V-135; m-1 to A-134; m-1 to R-133; m-1 to K-132; m-1 to N-131; m-1 to R-130; m-1 to S-129; m-1 to N-128; m-1 to Q-127; m-1 to S-126; m-1 to S-125; m-1 to N-124; m-1 to G-123; m-1 to E-122; m-1 to G-121; m-11 to P-120; m-1 to A-119; m-1 to P-118; m-1 to P-117; m-1 to E-116; m-1 to F-110; m-1 to I-114; m-1 to K-113; m-1 to L-112; m-1 to G-111; m-1 to A-110; m-1 to T-109; m-1 to V-108; m-1 to A-107; m-1 to P-106; m-1 to A-105; m-1 to E-104; m-1 to E-103; m-1 to L-102; m-1 to G-101; m-1 to A-100; m-1 to K-99; m-1 to P-98; m-1 to A-97; m-1 to G-96; m-1 to A-95; m-1 to G-94; m-1 to A-93; m-1 to P-92; m-1 to L-91; m-1 to K-90; m-1 to E-89; m-1 to A-88; m-1 to H-87; m-1 to H-86; m-1 to G-85; m-1 to Q-84; m-1 to L-83; m-1 to E-82; m-1 to A-81; m-1 to R-80; m-1 to L-79; m-1 to S-78; m-1 to A-77; m-1 to L-76; m-1 to D-75; m-1 to G-74; m-1 to Q-73; m-1 to L-72; m-1 to A-71; m-1 to A-70; m-1 to V-69; m-1 to Q-68; m-1 to Y-67; m-1 to F-66; m-1 to S-65; m-1 to V-64; m-1 to V-63; m-1 to T-62; m-1 to L-61; m-1 to C-60; m-1 to C-59; m-1 to S-58; m-1 to L-57; m-1 to L-56; m-1 to A-55; m-1 to L-54; m-1 to L-53; m-1 to L-52; m-1 to T-51; m-1 to A-50; m-1 to A-49; m-1 to L-48; m-1 to L-47; m-1 to K-46; m-1 to G-45; m-1 to D-44; m-1 to K-43; m-1 to S-42; m-1 to S-41; m-1 to R-40; m-1 to V-39; m-1 to S-38; m-1 to P-37; m-1 to S-36; m-1 to E-35; m-1 to K-34; m-1 to R-33; m-1 to P-32; m-1 to L-31; m-1 to I-30; m-1 to S-29; m-1 to V-28; m-1 to C-27; m-1 to E-26; m-1 to K-25; m-1 to L-24; m-1 to K-23; m-1 to M-22; m-1 to E-21; m-1 to E-20; m-1 to R-19; m-1 to K-18; m-1 to K-17; m-1 to L-16; m-1 to C-15; m-1 to S-14; m-1 to T-13; m-1 to L-12; m-1 to R-11; m-1 to S-10; m-1 to Q-9; m-1 to E-8; m-1 to R-7; and M-1 to E-6. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a Neutrokine- α SV polypeptide as described above. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by such nucleic acid and/or polynucleotide sequences comprising amino acid sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences are also encompassed by the present invention, as are polynucleotides encoding such polypeptides.

The present invention also provides polypeptides having one or more amino acids deleted from the amino terminus and the carboxy terminus of a Neutrokine-alpha SV polypeptide, which can be described as having the amino acid sequence of SEQ ID NO: 19 n of⁵-m⁵Residue of which n⁵And m⁵Are integers as described above. In further embodiments, the present invention provides a polypeptide comprising SEQ ID NO: 134-m of 2⁶A polypeptide of amino acid sequence at residue(s) wherein m⁶Is SEQ ID NO: 2, an integer of amino acid residues 140-285 as shown in sequence No. 2. For example, the invention provides polynucleotides encoding polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 2 a-134 to Leu-285; a-134 to L-284; a-134 to K-283; a-134 to L-282; a-134 to A-281; a-134 to G-280; a-134 to F-279; a-134 to F-278; a-134 to T-277; a-134 to V-276; a-134 to D-275; a-134 to G-274; a-134 to D-273; a-134 to L-272; a-134 to S-271; a-134 to I-270; a-134 to Q-269; a-134 to A-268; a-134 to N-267; a-134 to E-266; a-134 to R-265; a-134 to P-264; a-134 to I-263; a-134 to A-262; a-134 to L-261; a-134 to Q-260; a-134 to L-239; a-134 to E-258; a-134 to D-257; a-134 to G-256; a-134 to E-255; a-134 to E-254; a-134 to L-253; a-134 to K-252; a-134 to A-251; a-134 to I-250; a-134 to G-249; a-134 to A-248; a-134 to S-247; a-134 to Y-246; a-134 to C-245; a-134 to S-244; a-134 to N-243; a-134 to N-242; a-134 to P-241; a-134 to L-240; a-134 to T-239; a-134 to E-238; a-134 to P-237; a-134 to M-236; a-134 to N-235; a-134 to Q-234; a-134 to I-233; a-134 to C-232; a-134 to R-231; a-134 to F-230; a-134 to L-229; a-134 to T-228; a-134 to V-227; a-134 to L-226; a-134 to S-225; a-134 to L-224; a-134 to E-223; a-134 to D-222; a-134 to G-221; a-134 to F-220; a-134 to V-219; a-134 to H-218; a-134 to V-217; a-134 to K-216; a-134 to K-215; a-134 to R-214; a-134 to Q-213; a-134 to I-212; a-134 to L-211; a-134 to H-210; a-134 to G-209; a-134 to M-208; a-134 to A-207; a-134 to Y-206; a-134 to T-205; a-134 to K-204; a-134 to D-203; a-134 to T-202 (ii) a A-134 to Y-201; a-134 to L-200; a-134 to V-199; a-134 to Q-198; a-134 to G-197; a-134 to Y-196; a-134 to I-195; a-134 to F-194; a-134 to F-193; a-134 to Y-192; a-134 to G-191; a-134 to T-190; a-134 to E-189; a-134 to K-188; a-134 to V-187; a-134 to L-186; a-134 to I-185; a-134 to K-184; a-134 to N-183; a-134 to E-182; a-134 to K-181; a-134 to E-180; a-134 to E-179; a-134 to L-178; a-134 to A-177; a-134 to S-176; a-134 to G-175; a-134 to R-174; a-134 to K-173; a-134 to F-172; a-134 to S-171; a-134 to L-170; a-134 to L-169; a-134 to W-168; a-134 to P-167; a-134 to V-166; a-134 to F-165; a-134 to T-164; a-134 to Y-163; a-134 to S-162; a-134 to G-161; a-134 to K-160; a-134 to Q-159; a-134 to I-158; a-134 to T-157; a-134 to P-156; a-134 to T-155; a-134 to E-154; a-134 to S-153; a-134 to D-152; a-134 to A-151; a-134 to I-150; a-134 to L-149; a-134 to Q-148; a-134 to L-147; a-134 to C-146; a-134 to D-145; a-134 to Q-144; a-134 to T-143; a-134 to V-142; a-134 to T-141; and a-134 to E-140. The present invention also relates to nucleic acid molecules comprising or consisting of a polynucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide as described above. The present invention also encompasses the above polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by such nucleic acid and/or polynucleotide sequences having amino acid sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences are also encompassed by the present invention, as are polynucleotides encoding such polypeptides.

Other preferred polypeptide fragments of the invention comprise or consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: 2M-1 to C-15; d-2 to L-16; d-3 to K-17; s-4 to K-18; t-5 to R-19; e-6 to E-20; r-7 to E-21; e-8 to M-22; q-9 to K-23; s-10 to L-24; r-11 to K-25; l-12 to E-26; t-13 to C-27; s-14 to V-28; c-15 to S-29; l-16 to I-30; k-17 to L-31; k-18 to P-32; r-19 to R-33; e-20 to K-34; e-21 to E-35; m-22 to S-36; k-23 to P-37; l-24 to S-38; k-25 to V-39; e-26 to R-40; c-27 to S-41; v-28 to S-42; s-29 to K-43; i-30 to D-44; l-31 to G-45; p-32 to K-46; r-33 to L-47; k-34 to L-48; e-35 to A-49; s-36 to A-50; p-37 to T-51; s-38 to L-52; v-39 to L-53; r-40 to L-54; s-41 to A-55; s-42 to L-56; k-43 to L-57; d-44 to S-58; g-45 to C-59; k-46 to C-60; l-47 to L-61; l-48 to T-62; a-49 to V-63; a-50 to V-64; t-51 to S-65; l-52 to F-66; l-53 to Y-67; l-54 to Q-68; a-55 to V-69; l-56 to A-70; l-57 to A-71; s-58 to L-72; c-59 to Q-73; c-60 to G-74; l-61 to D-75; t-62 to L-76; v-63 to A-77; v-64 to S-78; s-65 to L-79; f-66 to R-80; y-67 to A-81; q-68 to E-82; v-69 to L-83; a-70 to Q-84; a-71 to G-85; l-72 to H-86; q-73 to H-87; g-74 to A-88; d-75 to E-89; l-76 to K-90; a-77 to L-91; s-78 to P-92; l-79 to A-93; r-80 to G-94; a-81 to A-95; e-82 to G-96; l-83 to A-97; q-84 to P-98; g-85 to K-99; h-86 to A-100; h-87 to G-101; a-83 to L-102; e-89 to E-103; k-90 to E-104; l-91 to A-105; p-92 to P-106; a-93 to A-107; g-94 to V-108; a-95 to T-109; g-96 to A-110; a-97 to G-111; p-98 to L-112; k-99 to K-113; a-100 to I-114; g-101 to F-115; l-102 to E-116; e-103 to P-I17; e-104 to P-118; a-105 to A-119; p-106 to P-120; a-107 to G-121; v-103 to E-122; t-109 to G-123; a-110 to N-124; g-111 to S-125; l-112 to S-126; k-113 to Q-127; i-114 to N-128; f-110 to S-129; e-116 to R-130; p-117 to N-131; p-118 to K-132; a-119 to R-133; p-120 to A-134; g-121 to V-135; e-122 to Q-136; g-123 to G-137; n-124 to P-138; s-125 to E-139; s-126 to E-140; q-127 to T-141; n-123 to V-142; s-129 to T-143; r-130 to Q-144; n-131 to D-145; k-132 to C-146; r-133 to L-147; a-134 to Q-148; v-135 to L-149; q-136 to I-150; g-137 to A-151; p-138 to D-152; e-139 to S-153; e-140 to E-154; t-141 to T-155; v-142 to P-156; t-143 to T-157; q-144 to I-158; d-145 to Q-159; c-146 to K-160; l-147 to G-161; q-148 to S-162; l-149 to Y-163; i-150 to T-164; a-151 to F-165; d-152 to V-166; s-153 to P-167; e-154 to W-168; t-155 to L-169; p-156 to L-170; t-157 to S-171; i-158 to F-172; q-159 to K-173; k-160 to R-174; g-161 to G-175; s-162 to S-176; y-163 to A-177; t-164 to L-178; f-165 to E-179; v-166 to E-180; p-167 to K-181; w-168 to E-182; l-169 to N-183; l-170 to K-184; s-171 to iI-185; f-172 to L-186; k-173 to V-187; r-174 to K-188; g-175 to E-189; s-176 to T-190; a-177 to G-191; l-178 to Y-192; e-179 to F-193; e-180 to F-194; k-181 to I-195; e-182 to Y-196; n-183 to G-197; k-184 to Q-198; i-185 to V-199; l-186 to L-200; v-187 to Y-201; k-188 to T-202; e-189 to D-203; t-190 to K-204; g-191 to T-205; y-192 to Y-206; f-193 to A-207; f-194 to M-208; i-195 to G-209; y-196 to H-210; g-197 to L-211; q-198 to I-212; v-199 to Q-213; l-200 to R-214; y-201 to K-215; t-202 to K-216; d-203 to V-217; k-204 to H-218; t-205 to V-219; y-206 to F-220; a-207 to G-221; m-208 to D-222; g-209 to E-223; h-210 to L-224; l-211 to S-225; i-212 to L-226; q-213 to V-227; r-214 to T-228; k-215 to L-229; k-216 to F-230; v-217 to R-231; h-218 to C-232; v-219 to I-233; f-220 to Q-234; g-221 to N-235; d-222 to M-236; e-223 to P-237; l-224 to E-238; s-225 to T-239; l-226 to L-240; v-227 to P-241; t-228 to N-242; l-229 to N-243; f-230 to S-244; r-231 to C-245; c-232 to Y-246; i-233 to S-247; q-234 to A-248; n-235 to G-249; m-236 to 1-250; p-237 to A-251; e-238 to K-252; t-239 to L-253; l-240 to E-254; p-241 to E-255; n-242 to G-256; n-243 to D-257; s-244 to E-258; c-245 to L-259; y-246 to Q-260; s-247 to L-261; a-248 to A-262; g249 to I-263; i-250 to P-264; a-251 to R-265; k-252 to E-266; l-253 to N-267; e-254 to A-268; e-255 to Q-269; g-256 to I-270; d-257 to S-271; e-258 to L-272; l-259 to D-273; q-260 to G-274; l-261 to D-275; a-262 to V-276; i-263 to T-277; p-264 to F-278; r-265 to F-279; e-266 to G-280; n-267 to A-281; a-268 to L-282; q-269 to K-283; i-270 to L-284; and S-271 to L-285. Preferably, such polypeptide fragments possess one or more functional activities (e.g., biological activity, antigenicity, and immunogenicity) of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide of the present invention and may be used, for example, in the production or screening of antibodies, as described below. The invention also relates to amino acid sequences comprising or consisting of at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to the above amino acid sequences. The present invention also encompasses the above amino acid sequences fused to heterologous amino acid sequences. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Other preferred polypeptide fragments of the invention comprise or consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: 19M-1 to C-15; d-2 to L-16; d-3 to K-17; s-4 to K-18; t-5 to R-19; e-6 to E-20; r-7 to E-21; e-8 to M-22; q-9 to K-23; s-10 to L-24; r-11 to K-25; l-12 to E-26; t-13 to C-27; s-14 to V-28; c-15 to S-29; l-16 to I-30; k-17 to L-31; k-18 to P-32; r-19 to R-33; e-20 to K-34; e-21 to E-35; m-22 to S-36; k-23 to P-37; l-24 to S-38; k-25 to V-39; e-26 to R-40; c-27 to S-41; v-28 to S-42; s-29 to K-43; i-30 to D-44; l-31 to G-45; p-32 to K-46; r-33 to L-47; k-34 to L-48; e-35 to A-49; s-36 to A-50; p-37 to T-51; s-38 to L-52; v-39 to L-53; r-40 to L-54; s-41 to A-55; s-42 to L-56; k-43 to L-57; d-44 to S-58; g-43 to C-59; k-46 to C-60; l-47 to L-61; l-48 to T-62; a-49 to V-63; a-50 to V-64; t-51 to S-65; l-52 to F-66; l-53 to Y-67; l-54 to Q-68; a-55 to V-69; l-56 to A-70; l-57 to A-71; s-58 to L-72; c-59 to Q-73; c-60 to G-74; l-61 to D-73; t-62 to L-76; v-63 to A-77; v-64 to S-78; s-65 to L-79; f-66 to R-80; y-67 to A-81; q-68 to E-82; v-69 to L-83; a-70 to Q-84; a-71 to G-85; l-72 to H-86; q-73 to H-87; g-74 to A-88; d-75 to E-89; l-76 to K-90; a-77 to L-91; s-78 to P-92; l-79 to A-93; r-80 to G-94; a-81 to A-95; e-82 to G-96; l-83 to A-97; q-84 to P-98; g-85 to K-99; h-86 to A-100; h-87 to G-101; a-88 to L-102; e-89 to E-103; k-90 to E-104; l-91 to A-105; p-92 to P-106; a-93 to A-107; g-94 to V-108; a-95 to T-109; g-96 to A-110; a-97 to G-111; p-98 to L-112; k-99 to K-113; a-100 to I-114; g-101 to F-115; l-102 to E-116; e-103 to P-117; e-104 to P-118; a-105 to A-119; p-106 to P-120; a-107 to G-121; v-108 to E-122; t-109 to G-123; a-110 to N-124; g-111 to S-125; l-112 to S-126; k-113 to Q-127; i-114 to N-128; f-115 to S-129; e-116 to R-130; p-117 to N-131; p-118 to K-132; a-119 to R-133; p-120 to A-134; g-121 to V-135; e-122 to Q-136; g-123 to G-137; n-124 to P-138; s-125 to E-139; s-126 to E-140; q-127 to T-141; n-128 to G-142; s-129 to S-143; r-130 to Y-144; n-131 to T-145; k-132 to F-146; r-133 to V-147; a-134 to P-148; v-135 to W-149; q-136 to L-150; g-137 to L-151; p-138 to S-152; e-139 to F-153; e-140 to K-154; t-141 to R-155; g-142 to G-156; s-143 to S-157; y-144 to A-158; t-145 to L-159; f-146 to E-160; v-147 to E-161; p-148 to K-162; w-149 to E-163; l-150 to N-164; l-151 to K-165; s-152 to I-166; f-153 to L-167; k-154 to V-168; r-155 to K-169; g-156 to E-170; s-157 to T-171; a-158 to G-172; l-159 to Y-173; e-160 to F-174; e-161 to F-175; k-162 to I-176; e-163 to Y-177; n-164 to G-178; k-165 to Q-179; i-166 to V-180; l-167 to L-181; v-168 to Y-182; k-169 to T-183; e-170 to D-184; t-171 to K-185; g-172 to T-186; y-173 to Y-187; f-174 to A-188; f-175 to M-189; i-176 to G-190; y-177 to H-191; g-178 to L-192; q-179 to I-193; v-180 to Q-194; l-181 to R-195; y-182 to K-196; t-183 to K-197; d-184 to V-198; k-185 to H-199; t-186 to V-200; y-187 to F-201; a-188 to G-202; m-189 to D-203G-190 to E-204; h-191 to L-205; l-192 to S-206; i-193 to L-207; q-194 to V-208; r-195 to T-209; k-196 to L-210; k-197 to F-211; v-198 to R-212; h-199 to C-213; v-200 to I-214; f-201 to Q-213; g-202 to N-216; d-203 to M-217; e-204 to P-218; l-205 to E-219; s-206 to T-220; l-207 to L-221; v-208 to P-222; t-209 to N-223; l-210 to N-224; f-211 to S-225; r-212 to C-226; c-213 to Y-227; i-214 to S-228; q-215 to A-229; n-216 to G-230; m-217 to I-231; p-218 to A-232; e-219 to K-233; t-220 to L-234; l-221 to E-235; p-222 to E-236; n-223 to G-237; n-224 to D-238; s-225 to E-239; c-226 to L-240; y-227 to Q-241; s-228 to L-242; a-229 to A-243; g-230 to I-244; i-231 to P-245; a-232 to R-246; k-233 to E-247; l-234 to N-24S; e-235 to A-249; e-236 to Q-250; g-237 to I-251; d-238 to S-252; e-239 to L-253; l-240 to D-254; q-241 to G-250; l-242 to D-256; a-243 to V-257; i-244 to T-258; p-245 to F-259; r-246 to F-260; e-247 to G-261; n-248 to A-262; a-249 to L-263; q-250 to K-264; i-251 to L-265; and S-252 to L-266. Preferably, such polypeptide fragments possess one or more functional activities (e.g., biological activity, antigenicity, and immunogenicity) of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide of the present invention and may be used, for example, in the production or screening of antibodies, as described below. The invention also relates to polypeptides comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences. The present invention also encompasses the above amino acid sequences fused to heterologous amino acid sequences. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Other preferred polypeptide fragments of the invention comprise or consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: 38M-1 to F-15; d-2 to C-16; e-3 to S-17; s-4 to E-18; a-5 to K-19; k-6 to G-20; t7 to E-21; l-8 to D-22; p-9 to M-23; p-10 to K-24; p-11 to V-25; c-12 to G-26; l-13 to Y-27; c-14 to D-28; f-15 to P-29; c-16 to I-30; s-17 to T-31; e-18 to P-32; k-19 to Q-33; g-20 to K-34; e-21 to E-35; d-22 to E-36; m-23 to G-37; k-24 to A-38; v-25 to W-39; g-26 to F-40; y-27 to G-41; d-28 to I-42; p-29 to C-43; i-30 to R-44; t-31 to D-45; p-32 to G-46; q-33 to R-47; k-34 to L-48; e-35 to L-49; e-36 to A-50; g-37 to A-51; a-38 to T-52; w-39 to L-53; f-40 to L-54; g-41 to L-55; i-42 to A-56; c-43 to L-57; r-44 to L-58; d-45 to S-59; g-46 to S-60; r-47 to S-61; l-48 to F-62; l-49 to T-63; a-50 to A-64; a-51 to M-65; t-52 to S-66; l-53 to L-67; l-54 to Y-68; l-55 to Q-69; a-56 to L-70; l-57 to A-71; l-58 to A-72; s-59 to L-73; s-60 to Q-74; s-61 to A-73; f-62 to D-76; t-63 to L-77; a-64 to M-78; m-65 to N-79; s-66 to L-80; l-67 to R-81; y-68 to M-82; q-69 to E-83; l-70 to L-84; a-71 to Q-85; a-72 to S-86; l-73 to Y-87; q-74 to R-88; a-75 to G-89; d-76 to S-90; l-77 to A-91; m-78 to T-92; n-79 to P-93; l-80 to A-94; r-81 to A-95; m-82 to A-96; e-83 to G-97; l-84 to A-98; q-85 to P-99; s-86 to E-100; y-87 to L-101; r-88 to T-102; g-89 to A-103; s-90 to G-104; a-91 to V-105; t-92 to K-106; p-93 to L-107; a-94 to L-108; a-93 to T-109; a-96 to P-110; g-97 to A-111; a-98 to A-112; p-99 to P-113; e-100 to R-114; l-101 to P-115; t-102 to H-116; a-103 to N-117; g-104 to S-118; v-105 to S-119; k-106 to R-120; l-107 to G-121; l-108 to H-122; t-109 to R-123; p-110 to N-124; a-111 to 125; a-112 to R-126; p-113 to A-127; r-114 to F-128; p-115 to Q-129; h-116 to G-130; n-117 to P-131; s-118 to E-132; s-119 to E-133; r-120 to T-134; g-121 to E-135; h-122 to Q-136; r-123 to D-137; n-124 to V-138; r-125 to D-139; r-126 to L-140; a-127 to S-141; f-128 to A-142; q-129 to P-143; g-130 to P-144; p-131 to A-145; e-132 to P-146; e-133 to C-147; t-134 to L-148; e-135 to P-149; q-136 to G-150; d-137 to C-151; v-138 to R-152; d-139 to H-153; l-140 to S-154; s-141 to Q-105; a-142 to H-156; p-143 to D-157; p-144 to D-158; a-145 to N-159; p-146 to G-160; c-147 to M-161; l-148 to N-162; p-149 to L-163; g-150 to R-164; c-151 to I-165; r-152 to I-166; h-153 to I-167; s-154 to Q-168; q-155 to D-169; h-156 to C-170; d-157 to L-171; d-158 to Q-172; n-159 to L-173; g-160 to I-174; m-161 to 175; n-162 to D-176; l-163 to S-177; r-164 to D-178; n-165 to T-179; i-166 to P-180; i-167 to A-181; q-163 to L-182; d-169 to E-183; c-170 to E-184; l-171 to K-185; q-172 to E-186; l-173 to N-187; i-174 to K-188; a-175 to I-189; d-176 to V-191; s-177 to V-191; d-178 to R-192; t-179 to Q-193; p-180 to T-194; a-181 to G-195; l-182 to Y-196; e-183 to F-197; e-184 to F-198; k-185 to I-199; e-186 to Y-200; n-187 to S-201; k-188 to Q-202; i-189 to V-203; v-190 to L-204; v-191 to Y-205; r-192 to T-206; q-193 to D-207; t-194 to P-203; g-195 to I-209; y-196 to F-210; f-197 to A-211; f-198 to M-212; i-199 to G-213; y-200 to H-214; s-201 to V-215; q-202 to I-216; v-203 to Q-217; l-204 to R-218; y-205 to K-219; t-206 to K-220; d-207 to V-221; p-208 to H-222; i-209 to V-223; f-210 to F-224; a-211 to G-225; m-212 to D-226; g-213 to E-227; h-214 to L-228; v-215 to S-229; i-216 to L-230; q-217 to V-231; r-218 to T-232; k-219 to L-233; k-220 to F-234; v-221 to R-235; h-222 to C-236; v-223 to 1-237; f-224 to Q-238; g-225 to N-239; d-226 to M-240; e-227 to P-241; l-228 to K-242; s-229 to T-243; l-230 to L-244; v-231 to P-245; t-232 to N-246; l-233 to N-247; f-234 to S-248; r-235 to C-249; c-236 to Y-250; i-237 to S-250; q-238 to A-252; n-239 to G-253; m-240 to I-254; p-241 to A-253; k-242 to R-256; t-243 to L-257; l-244 to E-253; p-245 to E-259; n-246 to G-260; n-247 to D-261; s-248 to E-262; c-249 to I-263; y-250 to Q-264; s-251 to L-265; a-252 to A-266; g-253 to I-267; i-234 to P-268; a-255 to R-269; r-256 to E-270; l-257 to N-271; e-258 to A-272; e-259 to Q-273; g-260 to I-274; d-261 to S-275; e-262 to R-276; i-263 to N-277; q-264 to G-278; l-265 to D-279; a-266 to D-280; i-267 to T-281; p-268 to F-282; r-269 to F-283; e-270 to G-284; n-271 to A-285; a-272 to L-286; q-273 to K-287; i-274 to L-288; and S-275 to L-289. Preferably, such polypeptide fragments possess one or more of the activities of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention and may be used, for example, in the production or screening of antibodies, as described below. The invention also relates to polypeptides comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the above amino acid sequences. The present invention also encompasses the above amino acid sequences fused to heterologous amino acid sequences. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Those skilled in the art will recognize that the amino acid sequences of some Neutrokine-alpha and Neutrokine-alpha SV polypeptides may be altered without significantly affecting the structure or function of the polypeptide. If such sequence differences are desired, it should be remembered that there are regions critical to determining the activity of the polypeptide.

Thus, the present invention also provides variants of a Neutrokine-alpha polypeptide which exhibit the functional activity (e.g. biological activity) of a Neutrokine-alpha polypeptide or which comprise a region of a Neutrokine-alpha polypeptide, such as a polypeptide fragment as described herein. The invention also includes variants of Neutrokine-alpha SV polypeptides that exhibit functional activity (e.g., biological activity) of the Neutrokine-alpha SV polypeptide, or that include regions of the Neutrokine-alpha SV polypeptide, such as polypeptide fragments described herein. Such mutants include insertions, deletions, transitions, repeats and pattern substitutions selected according to general rules known in the art and therefore have a minor effect on activity. For example, guidance on how to generate phenotypically silent amino acid substitutions is found in Bowie, j.u. et al, "information definitions in protein sequences that tolerate amino acid substitutions", science 247: 1306 (1990) in which the authors state that there are two main approaches to investigating the resistance of amino acid sequences to changes. The first method relies on an evolutionary process in which mutations are accepted or rejected by natural selection. The second approach is to use genetic engineering to make amino acid changes at specific positions of a cloned gene and to select or screen to identify sequences that retain functionality.

As described by the authors, these studies have demonstrated that proteins are surprisingly tolerant of amino acid substitutions. The authors also indicate that amino acid changes are allowed at some positions of the protein. For example, most cryptic amino acid residues require nonpolar side chains, while surface side chains are generally conserved. Other such phenotypically silent substitutions are found in Bowie, j.u., et al, supra, and in the references indicated herein. Obvious conservative substitutions are substitutions between the aliphatic amino acids Ala, Val, Leu and Ile; the exchange between the hydroxyl residues Ser and Thr, the exchange between the acidic residues Asp and Glu, the substitution between the amide residues Asn and Gln, the exchange between the basic residues Lys and Arg, and the substitution between the aromatic residues Phe, Tyr.

Thus, a fragment, derivative or analog of the polypeptide shown in FIGS. 1A and 1B (SEQ ID NO: 2) or the polypeptide encoded by the deposited cDNA plasmid may be one of the following: (i) wherein one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and such substituted amino acid residue may or may not be encoded by the genetic code; (ii) wherein one or more amino acid residues comprises a substituent; or (iii) wherein the extracellular domain of the polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol); or (iv) wherein additional amino acids are fused to the polypeptide extracellular domain, such as an IgG FC fusion region peptide or leader or secretory sequence or a sequence used to purify the polypeptide extracellular domain or a proprotein sequence. Such fragments, derivatives and analogs are considered to be within the scope of the present invention by those skilled in the art in light of the teachings herein.

Alternatively, the fragment, derivative or analog of the polypeptide shown in FIGS. 5A and 5B (SEQ ID NO: 19) or encoded by the deposited cDNA plasmid may be one of: (i) wherein one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and such substituted amino acid residue may or may not be encoded by the genetic code, or (ii) wherein one or more amino acid residues comprise a substituent, or (iii) wherein the extracellular domain of the polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol), or (iv) wherein other amino acids are fused to the extracellular domain of the polypeptide, such as soluble biologically active fragments of other TNF ligand family members (e.g., CD40 ligands), IgGFC fusion region peptides or leader or secretory sequences, or sequences useful for purifying the extracellular domain of the polypeptide or proprotein sequences. Such fragments, derivatives and analogs are considered to be within the scope of the present invention by those skilled in the art in light of the teachings herein.

Thus, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the present invention may comprise one or more amino acid substitutions, deletions or additions, whether naturally mutated or artificially manipulated. As noted above, the changes are preferably minor, such as conservative amino acid substitutions that do not substantially affect protein folding or activity (see Table 2).

TABLE 2 conservative amino acid substitutions

Aromatic compounds	Phenylalanine tryptophan tyrosine
		Hydrophobicity	Leucine isoleucine valine
Polarity	Glutamine asparagine
		Basic property	Arginine lysine histidine
Acidity	Aspartic acid glutamic acid
		Small molecules	Alanine serine threonine methionine glycine

In one embodiment of the invention, the polypeptide comprises or consists of an amino acid sequence of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide having an amino acid sequence containing at least 1 but not more than 50, preferably not more than 40, more preferably not more than 30, and even more preferably not more than 20 conservative amino acid substitutions. Most preferred of course are amino acid substitutions which contain at least 1 but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitution.

For example, site-directed mutagenesis at the amino acid level of Neutrokine-alpha may be performed by replacing a particular amino acid with a conservative substituent. SEQ ID NO: 2, preferred conservative substitution mutations in the Neutrokine-alpha amino acid sequence set forth in fig. 2 include: substitution of M1 with A, G, I, L, S, T or V; replacement of M1 with A, G, I, L, T, or V; replacement of D2 with E; replacement of D3 with E; replacing S4 with A, G, I, L, T, M or V; replacement of T5 with A, G, I, L, S, M or V; replacement of E6 with D; replacement of R7 with H, or K; replacement of E8 with D; replacing Q9 with N; replacing S10 with A, G, I, L, T, or V; replacement of R11 with H, or K; substitution of L12 with A, G, I, S, T, M or V; replacement of T13 with A, G, I, L, S, M or V; replacing S14 with A, G, I, L, T, M or V; substitution of L16 with A, G, I, S, T, M or V; replacement of K17 with H, or R; replacement of K18 with H, or R; replacement of R19 with H, or K; replacement of E20 with D; replacement of E21 with D; substitution of M22 with a, G, I, L, S, T; replacement of K23 with H, or R; substitution of L24 with A, G, I, S, T, M or V; replacement of K25 with H, or R; replacement of E26 with D; substitution of V28 with A, G, I, L, S, T or M; replacing S29 with A, G, I, L, T, M or V; replacement of I30 with A, G, L, S, T, M or V; substitution of L31 with A, G, I, S, T, M or V; replacement of R33 with H, or K; (ii) a Replacement of K34 with H, or R; replacement of E35 with D; replacing S36 with A, G, I, L, T, M or V; replacing S38 with A, G, I, L, T, M or V; substitution of V39 with A, G, I, L, S, T or M; replacement of R40 with H, or K; replacing S41 with A, G, I, L, T, M or V; replacing S42 with A, G, I, L, T, M or V; replacement of K43 with H, or R; replacement of D44 with E; substitution of G45 with A, I, L, S, T, M or V; replacement of K46 with H, or R; substitution of L47 with A, G, I, S, T, M or V; substitution of L48 with A, G, I, S, T, M or V; replacement of A49 with G, I, L, S, T, M or V; replacement of A50 with G, I, L, S, T, M or V; replacement of T51 with A, G, I, L, S, M or V; substitution of L52 with A, G, I, S, T, M, or V; substitution of L53 with A, G, I, S, T, M or V; substitution of L54 with A, G, I, S, T, M or V; replacement of a55 with G, I, L S, T, M or V; substitution of L56 with A, G, I, S, T, M or V; substitution of L57 with A, G, I, S, T, M or V; replacing S58 with A, G, I, L, T, or V; substitution of L61 with A, G, I, L, S, T, M or V; replacement of T62 with A, G, I, L, S, M or V; substitution of V63 with A, G, I, L, S, T, or M; substitution of V64 with A, G, I, L, S, T, or M; replacing S65 with A, G, I, L, T, M, or V; replacement of F66 with W, or Y; replacement of Y67 with F, or W; replacing Q68 with N; substitution of V69 with A, G, I, L, S, T, or M; replacement of a70 with G, I, L S, T, M or V; replacement of a71 with G, I, L S, T, M or V; substitution of L72 with A, G, I, S, T, M or V; replacing Q73 with N; substitution of G74 with A, I, L, S, T, M or V; replacement of D75 with E; substitution of L76 with A, G, I, S, T, M or V; replacement of A77 with G, I, L, S, T, M or V; replacing S78 with A, G, I, L, T, M or V; substitution of L79 with A, G, I, S, T, M or V; replacement of R80 with H, or K; replacement of A81 with G, I, L, S, T, M or V; replacement of E82 with D; substitution of L83 with A, G, I, S, T, M or V; replacing Q84 with N; substitution of G85 with A, I, L, S, T, M or V; replacement of H86 with K or R; replacement of H87 with K or R; replacement of a88 with G, I, L S, T, M or V; replacement of E89 with D; replacement of K90 with H or R; substitution of L91 with A, G, I, S, T, M or V; replacement of a93 with G, I, L S, T, M or V; substitution of G94 with A, I, L, S, T, M or V; replacement of a95 with G, I, L S, T, M or V; substitution of G96 with A, I, L, S, T, M or V; replacement of a97 with G, I, L S, T, M or V; replacement of K99 with H or R; replacing a100 with G, I, L S, T, M or V; replacing G101 with A, I, L, S, T, M or V; replacement of L102 with A, G, I, S, T, M or V; replacing E103 with D; replacing E104 with D; replacing a105 with G, I, L S, T, M or V; replacement of a107 with G, I, L S, T, M or V; replacing V108 with A, G, I, L, S, T or M; replacement of T109 with A, G, I, L, S, M or V; replacing A110 with G, I, L S, T, M or V; replacement of G111 with A, I, L, S, T, M or V; replacement of L112 with A, G, I, S, T, M or V; replacement of K113 with H or R; replacement of I114 with A, G, L, S, T, M or V; replacing F115 with W or Y; replacement of E116 with D; replacement of a119 with G, I, L S, T, M or V; substitution of G121 with A, I, L, S, T, M or V; replacing E122 with D; replacing G123 with A, I, L, S, T, M or V; replacing N124 with Q; replacing S125 with a, G, I, L, T, M or V; replacing S126 with A, G, I, L, T, M or V; replacing Q127 with N; replacing N128 with Q; replacing S129 with A, G, I, L, T, M or V; replacement of R130 with H, or K; replacing N131 with Q; replacement of K132 with H or R; replacement of R133 with H, or K; replacement of a134 with G, I, L S, T, M or V; replacing V135 with A, G, I, L, S, T or M; replacing Q136 with N; replacement of G137 with A, I, L, S, T, M or V; replacement of E139 with D; replacing E140 with D; replacement of T141 with A, G, I, L, S, M or V; replacing V142 with A, G, I, L, S, T or M; replacement of T143 with A, G, I, L, S, M or V; replacing Q144 with N; replacing D145 with E; replacing L147 with A, G, I, S, T, M or V; replacing Q148 with N; substitution of L149 with A, G, I, S, T, M or V; replacement of I150 with A, G, L, S, T, M or V; replacement of A151 with G, I, L S, T, M or V; replacing D152 with E; replacing S153 with A, G, I, L, T, M or V; replacement of E154 with D; replacement of T155 with A, G, I, L, S, M or V; replacement of T157 with A, G, I, L, S, M or V; replacement of I158 with A, G, L, S, T, M or V; replacement of Q159 with N; replacement of K160 with H or R; substitution of G161 with A, I, L, S, T, M or V; replacing S162 with a, G, I, L, T, M or V; replacement of Y163 with F or W; replacing T164 with A, G, I, L, S, M or V; replacement of F165 with W or Y; replacement of V166 with A, G, I, L, S, T or M; replacing W168 with F or Y; substitution of L169 with A, G, I, S, T, M or V; replacing L170 with A, G, I, S, T, M or V; replacing S171 with A, G, I, L, T, M or V; replacing F172 with W or Y; replacement of K173 with H or R; replacement of R174 with H or K; substitution of G175 with A, I, L, S, T, M or V; replacing S176 with a, G, I, L, T, M or V; replacing A177 with G, I, L S, T, M or V; replacement of L178 with A, G, I, S, T, M or V; replacing E179 with D; replacing E180 with D; replacement of K181 with H or R; replacing E182 with D; replacement of N183 with Q; replacement of K184 with H or R; substitution of I185 with A, G, L, S, T, M or V; replacing L186 with A, G, I, S, T, M or V; replacing V187 with A, G, I, L, S, T or M; replacement of K188 with H or R; replacing E189 with D; replacing T190 with A, G, I, L, S, M or V; substitution of G191 with A, I, L, S, T, M or V; replacement of Y192 with F or W; replacement of F193 with W or Y; replacement of F194 with W or Y; replacement of I195 with A, G, L, S, T, M or V; replacement of Y196 with F or W; substitution of G197 with A, I, L, S, T, M or V; replacing Q198 with N; substitution of V199 with A, G, I, L, S, T or M; replacing L200 with A, G, I, S, T, M or V; replacing Y201 with F or W; replacing T202 with A, G, I, L, S, M or V; replacing D203 with E; replacement of K204 with H or R; replacement of T205 with A, G, I, L, S, M or V; replacing Y206 with F or W; replacing A207 with G, I, L S, T, M or V; replacing M208 with A, G, I, L, S, T or V; replacing G209 with A, I, L, S, T, M or V; replacement of H210 with K or R; (ii) replacement of L211 with A, G, I, S, T, M or V; replacement of I212 with A, G, L, S, T, M or V; replacing Q213 with N; replacement of R214 with H or K; replacement of K215 with H or R; replacement of K216 with H or R; replacing V217 with A, G, I, L, S, T or M; replacement of H218 with K or R; replacing V219 with A, G, I, L, S, T or M; replacing F220 with W or y; replacing G221 with A, I, L, S, T, M or V; replacing D222 with E; replacing E223 with D; substitution of L224 with A, G, I, S, T, M or V; replacing S225 with A, G, I, L, T, M or V; replacing L226 with A, G, I, S, T, M or V; replacing V227 with A, G, I, L, S, T or M; replacing T228 with A, G, I, L, S, M or V; substitution of L229 with A, G, I, S, T, M or V; replacing F230 with W or Y; replacement of R231 with H or K; replacement of I233 with A, G, L, S, T, M or V; replacing Q234 with N; replacing N235 with Q; replacing M236 with A, G, I, L, S, T or V; replacing E238 with D; replacing T239 with A, G, I, L, S, M or V; replacement of L240 with A, G, I, S, T, M or V; replacing N242 with Q; replacing N243 with Q; replacing S244 with a, G, I, L, T, M or V; replacement of Y246 with F or W; replacing S247 with a, G, I, L, T, M or V; replacing a248 with G, I, L S, T, M or V; substitution of G249 with A, I, L, S, T, M or V; replacing I250 with A, G, L, S, T, M or V; replacing a251 with G, I, L S, T, M or V; replacement of K252 with H or R; (ii) a Replacing L253 with A, G, I, S, T, M or V; replacing E254 with D; replacing E255 with D; replacing G256 with A, I, L, S, T, M or V; replacement of D257 with E; replacing E258 with D; substitution of L259 with A, G, I, S, T, M or V; replacing Q260 with N; replacing L261 with A, G, I, S, T, M or V; replacement of a262 with G, I, L S, T, M or V; substitution of I263 with A, G, L, S, T, M or V; replacement of R265 with H or K; replacement of E266 with D; replacement of N267 with Q; substitution of G, I, L, S, T, M or V for A268; replacement of Q269 with N; replacing I270 with A, G, L, S, T, M or V; replacing S271 with a, G, I, L, T, M or V; replacing L272 with A, G, I, S, T, M or V; replacing D273 with E; substitution of G274 with A, I, L, S, T, M or V; replacing D275 with E; replacing V276 with A, G, I, L, S, T or M; replacement of T277 with A, G, I, L, S, M or V; replacing F278 with W or Y; replacement of F279 with W or Y; replacing G280 with A, I, L, S, T, M or V; substitution of a281 with G, I, L S, T, M or V; replacement of L282 with A, G, I, S, T, M or V; replacement of K283 with H or R; replacement of L284 with A, G, I, S, T, M or V; and/or L285 is replaced with A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-alpha proteins of the invention may be routinely screened for Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties (e.g., enhanced or reduced stability and/or solubility). Preferably, the resulting proteins of the invention have increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha SV functional activity. More preferably, the Neutrokine-alpha and/or Neutrokine-alpha SV protein obtained by the invention has more than one increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical property.

In another embodiment, site-directed mutagenesis at the Neutrokine- α SV amino acid level can be performed by substituting a particular amino acid with a conservative substituent. SEQ ID NO: preferred conservative substitution mutations of the Neutrokine- α SV amino acid sequence as set forth in 19 include: replacement of M1 with A, G, I, L, S, T or V; replacement of D2 with E; replacement of D3 with E; replacing S4 with A, G, I, L, T, M or V; replacement of T5 with A, G, I, L, S, M or V; replacement of E6 with D; replacement of R7 with H or K; replacement of E8 with D; replacing Q9 with N; replacing S10 with A, G, I, L, T, M or V; replacement of R11 with H or K; substitution of L12 with A, G, I, S, T, M or V; replacement of T13 with A, G, I, L, S, M or V; replacing S14 with A, G, I, L, T, M or V; substitution of L16 with A, G, I, S, T, M or V; replacement of K17 with H or R; replacement of K18 with H or R; replacement of R19 with H or K; replacement of E20 with D; replacement of E21 with D; replacement of M22 with A, G, I, L, S, T or V; replacement of K23 with H or R; substitution of L24 with A, G, I, S, T, M or V; replacement of K25 with H or R; replacement of E26 with D; substitution of V28 with A, G, I, L, S, T or M; replacing S29 with A, G, I, L, T, M or V; replacement of I30 with A, G, L, S, T, M or V; substitution of L31 with A, G, I, S, T, M or V; replacement of R33 with H or K; replacement of K34 with H or R; replacement of E35 with D; replacing S36 with A, G, I, L, T, M or V; replacing S38 with A, G, I, L, T, M or V; substitution of V39 with A, G, I, L, S, T or M; replacement of R40 with H or K; replacing S41 with A, G, I, L, T, M or V; replacing S42 with A, G, I, L, T, M or V; replacement of K43 with H or R; replacement of D44 with E; substitution of G45 with A, I, L, S, T, M or V; replacement of K46 with H or R; substitution of L47 with A, G, I, S, T, M or V; substitution of L48 with A, G, I, S, T, M or V; replacement of A49 with G, I, L, S, T, M or V; replacement of A50 with G, I, L, S, T, M or V; replacement of T51 with A, G, I, L, S, M or V; substitution of L52 with A, G, I, S, T, M or V; substitution of L53 with A, G, I, S, T, M or V; substitution of L54 with A, G, I, S, T, M or V; replacement of A55 with G, I, L, S, T, M or V; substitution of L56 with A, G, I, S, T, M or V; substitution of L57 with A, G, I, S, T, M or V; replacing S58 with A, G, I, L, T, M or V; substitution of L61 with A, G, I, S, T, M or V; replacement of T62 with A, G, I, L, S, M or V; substitution of V63 with A, G, I, L, S, T or M; substitution of V64 with A, G, I, L, S, T or M; replacing S65 with A, G, I, L, T, M or V; replacement of F66 with W or Y; replacement of Y67 with F or W; replacing Q68 with N; substitution of V69 with A, G, I, L, S, T or M; replacement of A70 with G, I, L, S, T, M or V; replacement of A71 with G, I, L, S, T, M or V; substitution of L72 with A, G, I, S, T, M or V; replacing Q73 with N; substitution of G74 with A, I, L, S, T, M or V; replacement of D75 with E; substitution of L76 with A, G, I, S, T, M or V; replacement of A77 with G, I, L, S, T, M or V; replacing S78 with A, G, I, L, T, M or V; substitution of L79 with A, G, I, S, T, M or V; replacement of R80 with H or K; replacement of A81 with G, I, L, S, T, M or V; replacement of E82 with D; substitution of L83 with A, G, I, S, T, M or V; replacing Q84 with N; substitution of G85 with A, I, L, S, T, M or V; replacement of H86 with K or R; replacement of H87 with K or R; replacement of A88 with G, I, L, S, T, M or V; replacement of E89 with D; replacement of K90 with H or R; substitution of L91 with A, G, I, S, T, M or V; replacement of A93 with G, I, L, S, T, M or V; substitution of G94 with A, I, L, S, T, M or V; replacement of A95 with G, I, L, S, T, M or V; substitution of G96 with A, I, L, S, T, M or V; replacement of A97 with G, I, L, S, T, M or V; replacement of K99 with H or R; replacing A100 with G, I, L, S, T, M or V; replacing G101 with A, I, L, S, T, M or V; replacement of L102 with A, G, I, S, T, M or V; replacing E103 with D; replacing E104 with D; replacing A105 with G, I, L, S, T, M or V; replacing A107 with G, I, L, S, T, M or V; replacing V108 with A, G, I, L, S, T or M; replacement of T109 with A, G, I, L, S, M or V; replacing A110 with G, I, L, S, T, M or V; replacement of G111 with A, I, L, S, T, M or V; replacement of L112 with A, G, I, S, T, M or V; replacement of K113 with H or R; using A, G, L; s, T, M or V replaces I114; replacing F115 with W or Y; replacement of E116 with D; replacing A119 with G, I, L, S, T, M or V; substitution of G121 with A, I, L, S, T, M or V; replacing E122 with D; replacing G123 with A, I, L, S, T, M or V; replacing N124 with Q; replacing S125 with a, G, I, L, T, M or V; replacing S126 with A, G, I, L, T, M or V; replacing Q127 with N; replacing N128 with Q; replacing S129 with A, G, I, L, T, M or V; replacement of R130 with H or K; replacing N131 with Q; replacement of K132 with H or R; replacement of R133 with H or K; replacing A134 with G, I, L, S, T, M or V; replacing V135 with A, G, I, L, S, T or M; replacing Q136 with N; replacement of G137 with A, I, L, S, T, M or V; replacement of E139 with D; replacing E140 with D; replacement of T141 with A, G, I, L, S, M or V; replacing G142 with A, I, L, S, T, M or V; replacing S143 with A, G, I, L, T, M or V; replacement of Y144 with F or W; replacement of T145 with A, G, I, L, S, M or V; replacing F146 with W or Y; replacing V147 with A, G, I, L, S, T or M; substitution of W149 with F or Y; replacement of L150 with A, G, I, S, T, M or V; replacement of L151 with A, G, I, S, T, M or V; replacing S152 with a, G, I, L, T, M or V; replacement of F153 with W or Y; replacement of K154 with H or R; replacement of R155 with H or K; substitution of G156 with A, I, L, S, T, M or V; replacing S157 with a, G, I, L, T, M or V; replacing A158 with G, I, L, S, T, M or V; substitution of L159 with A, G, I, S, T, M or V; replacing E160 with D; replacing E161 with D; replacement of K162 with H or R; replacement of E163 with D; replacing N164 with Q; replacement of K165 with H or R; using A, G, L; s, T, M or V replaces I166; substitution of L167 with A, G, I, S, T, M or V; substitution of V168 with A, G, I, L, S, T or M; replacement of K169 with H or R; replacing E170 with D; replacement of T171 with A, G, I, L, S, M or V; replacing G172 with A, I, L, S, T, M or V; replacement of Y173 with F or W; replacement of F174 with W or Y; replacing F175 with W or Y; using A, G, L; s, T, M or V replaces I176; replacing Y177 with F or W; replacement of G178 with A, I, L, S, T, M or V; replacing Q179 with N; replacing V180 with A, G, I, L, S, T or M; replacement of L181 with A, G, I, S, T, M or V; replacement of Y182 with F or W; replacement of T183 with A, G, I, L, S, M or V; d184 was replaced with E; replacement of K185 with H or R; replacing T186 with A, G, I, L, S, M or V; replacing Y187 with F or W; substitution of G, I, L, S, T, M or V for A188; replacement of M189 with a, G, I, L, S, T or V; replacing G190 with A, I, L, S, T, M or V; replacement of H191 with K or R; replacing L192 with A, G, I, S, T, M or V; using A, G, L; s, T, M or V replaces I193; replacement of Q194 with N; replacement of R195 with H or K; replacement of K196 with H or R; replacement of K197 with H or R; replacement of V198 with A, G, I, L, S, T or M; replacement of H199 with K or R; replacing V200 with A, G, I, L, S, T or M; replacing F201 with W or Y; replacing G202 with A, I, L, S, T, M or V; replacing D203 with E; replacing E204 with D; replacement of L205 with A, G, I, S, T, M or V; (ii) a Replacing S206 with A, G, I, L, T, M or V; substitution of L207 with A, G, I, S, T, M or V; replacing V208 with A, G, I, L, S, T or M; replacement of T209 with A, G, I, L, S, M or V; replacing L210 with A, G, I, S, T, M or V; replacing F211 with W or Y; replacement of R212 with H or K; using A, G, L; s, T, M or V substitution I214; replacing Q215 with N; replacing N216 with Q; replacing M217 with A, G, I, L, S, T or V; replacing E219 with D; replacing T220 with A, G, I, L, S, M or V; replacing L221 with A, G, I, S, T, M or V; replacing N223 with Q; replacing N224 with Q; replacing S225 with A, G, I, L, T, M or V; replacement of Y227 with F or W; replacing S228 with A, G, I, L, T, M or V; substitution of G, I, L, S, T, M or V for A229; replacing G230 with A, I, L, S, T, M or V; using A, G, L; s, T, M or V replaces I231; replacing A232 with G, I, L, S, T, M or V; replacement of K233 with H or R; substitution of L234 with A, G, I, S, T, M or V; replacing E235 with D; replacing E236 with D; substitution of G237 with A, I, L, S, T, M or V; d238 was replaced with E; replacing E239 with D; replacement of L240 with A, G, I, S, T, M or V; replacing Q241 with N; replacing L242 with A, G, I, S, T, M or V; replacing A243 with G, I, L, S, T, M or V; using A, G, L; s, T, M or V replaces I244; replacement of R246 with H or K; replacing E247 with D; replacing N248 with Q; replacing A249 with G, I, L, S, T, M or V; replacing Q250 with N; using A, G, L; s, T, M or V replaces I251; replacing S252 with a, G, I, L, T, M or V; replacing L253 with A, G, I, S, T, M or V; replacing D254 with E; replacing G255 with A, I, L, S, T, M or V; replacing D256 with E; replacement of V257 with A, G, I, L, S, T or M; replacing T258 with A, G, I, L, S, M or V; replacement of F259 with W or Y; replacing F260 with W or Y; replacing G261 with A, I, L, S, T, M or V; replacing A262 with G, I, L, S, T, M or V; substitution of L263 with A, G, I, S, T, M or V; replacement of K264 with H or R; replacing L265 with A, G, I, S, T, M or V; and/or L266 is replaced with A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-alpha proteins of the invention may be routinely screened for Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties (e.g., enhanced or reduced stability and/or solubility). Preferably, the resulting proteins of the invention have increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha SV functional activity. More preferably, the resulting Neutrokine-alpha and/or Neutrokine-alpha SV protein of the invention has one or more increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties.

In another embodiment, site-directed mutagenesis at the Neutrokine-alpha amino acid level may be performed by replacing a particular amino acid with a conservative substituent. SEQ ID NO: preferred conservative substitution mutations of the Neutrokine-alpha amino acid sequence as depicted in fig. 23 include: replacement of R1 with H or K; substitution of V2 with A, G, I, L, S, T or M; substitution of V3 with A, G, I, L, S, T or M; replacement of D4 with E; substitution of L5 with A, G, I, S, T, M or V; replacing S6 with A, G, I, L, T, M or V; replacement of A7 with G, I, L, S, T, M or V; replacement of A10 with G, I, L, S, T, M or V; substitution of L13 with A, G, I, S, T, M or V; substitution of G15 with A, I, L, S, T, M or V; replacement of R17 with H or K; replacement of H18 with K or R; replacing S19 with A, G, I, L, T, M or V; replacing Q20 with N; replacement of H21 with K or R; replacement of D22 with E; replacement of D23 with E; replacement of N24 with Q; substitution of G25 with A, I, L, S, T, M or V; replacement of M26 with A, G, I, L, S, T or V; replacement of N27 with Q; substitution of L28 with A, G, I, S, T, M or V; replacement of R29 with H or K; replacement of N30 with Q; replacement of R31 with H or K; replacement of T32 with A, G, I, L, S, M or V; replacement of Y33 with F or W; replacement of T34 with A, G, I, L, S, M or V; replacement of F35 with W or Y; substitution of V36 with A, G, I, L, S, T or M; replacement of W38 with F or Y; substitution of L39 with A, G, I, S, T, M or V; substitution of L40 with A, G, I, S, T, M or V; replacing S41 with A, G, I, L, T, M or V; replacement of F42 with W or Y; replacement of K43 with H or R; replacement of R44 with H or K; substitution of G45 with A, I, L, S, T, M or V; replacement of N46 with Q; replacement of A47 with G, I, L, S, T, M or V; substitution of L48 with A, G, I, S, T, M or V; (ii) a Replacement of E49 with D; replacement of E50 with D; replacement of K51 with H or R; replacement of E52 with D; replacement of N53 with Q; replacement of K54 with H or R; using A, G, L; s, T, M or V substitution I55; substitution of V56 with A, G, I, L, S, T or M; substitution of V57 with A, G, I, L, S, T or M; replacement of R58 with H or K; replacing Q59 with N; replacement of T60 with A, G, I, L, S, M or V; substitution of G61 with A, I, L, S, T, M or V; replacement of Y62 with F or W; replacement of F63 with W or Y; replacement of F64 with W or Y; using A, G, L; s, T, M or V substitution I65; replacement of Y66 with F or W; replacing S67 with A, G, I, L, T, M or V; replacing Q68 with N; substitution of V69 with A, G, I, L, S, T or M; substitution of L70 with A, G, I, S, T, M or V; (ii) a Replacement of Y71 with F or W; replacement of T72 with A, G, I, L, S, M or V; replacement of D73 with E; using A, G, L; s, T, M or V substitution I75; replacement of F76 with W or Y; replacement of A77 with G, I, L, S, T, M or V; replacement of M78 with A, G, I, L, S, T or V; substitution of G79 with A, I, L, S, T, M or V; replacement of H80 with K or R; substitution of V81 with A, G, I, L, S, T or M; using A, G, L; s, T, M or V substitution I82; replacing Q83 with N; replacement of R84 with H or K; replacement of K85 with H or R; replacement of K86 with H or R; substitution of V87 with A, G, I, L, S, T or M; replacement of H88 with K or R; substitution of V89 with A, G, I, L, S, T or M; replacement of F90 with W or Y; substitution of G91 with A, I, L, S, T, M or V; replacement of D92 with E; replacement of E93 with D; substitution of L94 with A, G, I, S, T, M or V; replacing S95 with A, G, I, L, T, M or V; substitution of L96 with A, G, I, S, T, M or V; substitution of V97 with A, G, I, L, S, T or M; replacement of T98 with A, G, I, L, S, M or V; substitution of L99 with A, G, I, S, T, M or V; replacing F100 with W or Y; replacement of R101 with H or K; using A, G, L; s, T, M or V replaces I103; replacing Q104 with N; replacing N105 with Q; replacement of M106 with A, G, I, L, S, T or V; replacement of K108 with H or R; replacement of T109 with A, G, I, L, S, M or V; replacing L110 with A, G, I, S, T, M or V; replacing N112 with Q; replacing N113 with Q; replacing S114 with A, G, I, L, T, M or V; replacement of Y116 with F or W; replacing S117 with a, G, I, L, T, M or V; replacing A118 with G, I, L, S, T, M or V; replacement of G119 with A, I, L, S, T, M or V; using A, G, L; s, T, M or V replaces I120; replacing A121 with G, I, L, S, T, M or V; replacement of R122 with H or K; (ii) replacement of L123 with A, G, I, S, T, M or V; replacing E124 with D; replacing E125 with D; substitution of G126 with A, I, L, S, T, M or V; replacement of D127 with E; replacing E128 with D; using A, G, L; s, T, M or V replaces I129; replacing Q130 with N; replacement of L131 with A, G, I, S, T, M or V; replacing A132 with G, I, L, S, T, M or V; using A, G, L; s, T, M or V replaces I133; replacement of R135 with H or K; replacing E136 with D; replacement of N137 with Q; replacing A138 with G, I, L, S, T, M or V; replacement of Q139 with N; using A, G, L; s, T, M or V replaces I140; replacing S141 with a, G, I, L, T, M or V; replacement of R142 with H or K; replacement of N143 with Q; substitution of G144 with A, I, L, S, T, M or V; replacing D145 with E; replacing D146 with E; replacing T147 with A, G, I, L, S, M or V; replacement of F148 with W or Y; replacement of F149 with W or Y; replacement of G150 with A, I, L, S, T, M or V; replacing A151 with G, I, L, S, T, M or V; replacing L152 with A, G, I, S, T, M or V; replacement of K153 with H or R; substitution of L154 with A, G, I, S, T, M or V; and/or L155 is replaced with A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-alpha proteins of the invention may be routinely screened for Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties (e.g., enhanced or reduced stability and/or solubility). Preferably, the resulting proteins of the invention have increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha SV functional activity. More preferably, the resulting Neutrokine-alpha and/or Neutrokine-alpha SV protein of the invention has one or more increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties.

In another embodiment, site-directed mutagenesis at the Neutrokine-alpha amino acid level may be performed by replacing a particular amino acid with a conservative substituent. SEQ ID NO: 38, include: replacement of M1 with A, G, I, L, S, T or V; replacement of D2 with E; replacement of D3 with E; replacing S4 with A, G, I, L, T, M or V; replacement of A5 with G, I, L, S, T, M or V; replacement of K6 with H or R; replacement of T7 with A, G, I, L, S, M or V; substitution of L8 with A, G, I, S, T, M or V; substitution of L13 with A, G, I, S, T, M or V; replacement of F15 with W or Y; replacing S17 with A, G, I, L, T, M or V; replacement of E18 with D; replacement of K19 with H or R; substitution of G20 with A, I, L, S, T, M or V; replacement of E21 with D; replacement of D22 with E; replacement of M23 with A, G, I, L, S, T or V; replacement of K24 with H or R; substitution of V25 with A, G, I, L, S, T or M; substitution of G26 with A, I, L, S, T, M or V; replacement of Y27 with F or W; replacement of D28 with E; using A, G, L; s, T, M or V substitution I30; replacement of T31 with A, G, I, L, S, M or V; replacing Q33 with N; replacement of K34 with H or R; replacement of E35 with D; replacement of E36 with D; substitution of G37 with A, I, L, S, T, M or V; replacement of A38 with G, I, L, S, T, M or V; replacement of W39 with F or Y; replacement of F40 with W or Y; substitution of G41 with A, I, L, S, T, M or V; using A, G, L; s, T, M or V substitution I42; replacement of R44 with H or K; replacement of D45 with E; substitution of G46 with A, I, L, S, T, M or V; replacement of R47 with H or K; substitution of L48 with A, G, I, S, T, M or V; substitution of L49 with A, G, I, S, T, M or V; replacement of A50 with G, I, L, S, T, M or V; replacement of A51 with G, I, L, S, T, M or V; replacement of T52 with A, G, I, L, S, M or V; substitution of L53 with A, G, I, S, T, M or V; substitution of L54 with A, G, I, S, T, M or V; substitution of L55 with A, G, I, S, T, M or V; replacement of A56 with G, I, L, S, T, M or V; substitution of L57 with A, G, I, S, T, M or V; substitution of L58 with A, G, I, S, T, M or V; replacing S59 with A, G, I, L, T, M or V; replacing S60 with A, G, I, L, T, M or V; replacing S61 with A, G, I, L, T, M or V; replacement of F62 with W or Y; replacement of T63 with A, G, I, L, S, M or V; replacement of A64 with G, I, L, S, T, M or V; replacement of M65 with A, G, I, L, S, T or V; replacing S66 with A, G, I, L, T, M or V; substitution of L67 with A, G, I, S, T, M or V; replacement of Y68 with F or W; replacing Q69 with N; substitution of L70 with A, G, I, S, T, M or V; replacement of A71 with G, I, L, S, T, M or V; replacement of A72 with G, I, L, S, T, M or V; substitution of L73 with A, G, I, S, T, M or V; replacing Q74 with N; replacement of A75 with G, I, L, S, T, M or V; replacement of D76 with E; substitution of L77 with A, G, I, S, T, M or V; replacement of M78 with A, G, I, L, S, T or V; replacement of N79 with Q; substitution of L80 with A, G, I, S, T, M or V; replacement of R81 with H or K; replacement of M82 with A, G, I, L, S, T or V; replacement of E83 with D; substitution of L84 with A, G, I, S, T, M or V; replacing Q85 with N; replacing S86 with A, G, I, L, T, M or V; replacement of Y87 with F or W; replacement of R88 with H or K; substitution of G89 with A, I, L, S, T, M or V; replacing S90 with A, G, I, L, T, M or V; replacement of A91 with G, I, L, S, T, M or V; replacement of T92 with A, G, I, L, S, M or V; replacement of A94 with G, I, L, S, T, M or V; replacement of A95 with G, I, L, S, T, M or V; replacement of A96 with G, I, L, S, T, M or V; substitution of G97 with A, I, L, S, T, M or V; replacement of A98 with G, I, L, S, T, M or V; replacing E100 with D; replacing L101 with A, G, I, S, T, M or V; replacement of T102 with A, G, I, L, S, M or V; replacing A103 with G, I, L, S, T, M or V; replacing G104 with A, I, L, S, T, M or V; replacing V105 with A, G, I, L, S, T or M; replacement of K106 with H or R; replacement of L107 with A, G, I, S, T, M or V; replacing L108 with A, G, I, S, T, M or V; replacement of T109 with A, G, I, L, S, M or V; replacing A111 with G, I, L, S, T, M or V; replacing A112 with G, I, L, S, T, M or V; replacement of R114 with H or K; replacement of H116 with K or R; replacing N117 with Q; replacing S118 with A, G, I, L, T, M or V; replacing S119 with A, G, I, L, T, M or V; replacement of R120 with H or K; substitution of G121 with A, I, L, S, T, M or V; replacement of H122 with K or R; replacement of R123 with H or K; replacing N124 with Q; replacement of R125 with H or K; replacement of R126 with H or K; replacing A127 with G, I, L, S, T, M or V; replacement of F128 with W or Y; substitution of Q129 with N; replacing G130 with A, I, L, S, T, M or V; replacing E132 with D; replacing E133 with D; replacement of T134 with A, G, I, L, S, M or V; replacing E135 with D; replacing Q136 with N; replacement of D137 with E; replacement of V138 with A, G, I, L, S, T or M; replacement of E139 with D; replacing L140 with A, G, I, S, T, M or V; replacing S141 with a, G, I, L, T, M or V; replacing A142 with G, I, L, S, T, M or V; replacing A145 with G, I, L, S, T, M or V; substitution of L148 with A, G, I, S, T, M or V; replacement of G150 with A, I, L, S, T, M or V; replacement of R152 with H or K; replacement of H153 with K or R; replacing S154 with a, G, I, L, T, M or V; replacing Q155 with N; replacement of H156 with K or R; replacing D157 with E; replacing D158 with E; replacement of N159 with Q; replacing G160 with A, I, L, S, T, M or V; replacement of M161 with A, G, I, L, S, T or V; replacing N162 with Q; substitution of L163 with A, G, I, S, T, M or V; replacement of R164 with H or K; replacing N165 with Q; using A, G, L; s, T, M or V replaces I166; using A, G, L; s, T, M or V replaces I167; replacing Q168 with N; replacement of D169 with E; substitution of L171 with A, G, I, S, T, M or V; replacing Q172 with N; replacement of L173 with A, G, I, S, T, M or V; using A, G, L; s, T, M or V replaces I174; (ii) a Replacing A175 with G, I, L, S, T, M or V; replacing D176 with E; replacing S177 with a, G, I, L, T, M or V; replacing D178 with E; replacing T179 with A, G, I, L, S, M or V; replacing A181 with G, I, L, S, T, M or V; replacing L182 with A, G, I, S, T, M or V; replacement of E183 with D; replacing E184 with D; replacement of K185 with H or R; replacing E186 with D; replacing N187 with Q; replacement of K188 with H or R; replacing I189 with A, G, L, S, T, M or V; replacing V190 with A, G, I, L, S, T or M; replacement of V191 with A, G, I, L, S, T or M; replacement of R192 with H or K; replacing Q193 with N; replacement of T194 with A, G, I, L, S, M or V; substitution of G195 with A, I, L, S, T, M or V; replacement of Y196 with F or W; replacement of F197 with W or Y; replacement of F198 with W or Y; using A, G, L; s, T, M or V replaces I199; replacing Y200 with F or W; replacing S201 with A, G, I, L, T, M or V; replacing Q202 with N; replacing V203 with A, G, I, L, S, T or M; replacing L204 with A, G, I, S, T, M or V; replacement of Y205 with F or W; replacing T206 with A, G, I, L, S, M or V; replacing D207 with E; using A, G, L; s, T, M or V replaces I209; replacing F210 with W or Y; replacing A211 with G, I, L, S, T, M or V; replacement of M212 with a, G, I, L, S, T or V; replacement of G213 with A, I, L, S, T, M or V; replacement of H214 with K or R; replacing V215 with A, G, I, L, S, T or M; using A, G, L; s, T, M or V replaces I216; replacing Q217 with N; replacement of R218 with H or K; replacement of K219 with H or R; replacement of K220 with H or R; replacing V221 with A, G, I, L, S, T or M; replacement of H222 with K or R; replacing V223 with A, G, I, L, S, T or M; replacement of F224 with W or Y; replacing G225 with A, I, L, S, T, M or V; replacing D226 with E; replacing E227 with D; substitution of L228 with A, G, I, S, T, M or V; replacing S229 with a, G, I, L, T, M or V; replacing L230 with A, G, I, S, T, M or V; replacing V231 with A, G, I, L, S, T or M; replacing T232 with A, G, I, L, S, M or V; substitution of L233 with A, G, I, S, T, M or V; replacing F234 with W or Y; replacement of R235 with H or K; substitution of I237 with A, G, L, S, T, M or V; replacing Q238 with N; replacing N239 with Q; replacement of M240 with A, G, I, L, S, T or V; replacement of K242 with H or R; replacement of T243 with A, G, I, L, S, M or V; substitution of L244 with A, G, I, S, T, M or V; replacement of N246 with Q; replacing N247 with Q; replacing S248 with A, G, I, L, T, M or V; replacing Y250 with F or W; replacing S251 with A, G, I, L, T, M or V; replacing A252 with G, I, L, S, T, M or V; replacing G253 with A, I, L, S, T, M or V; replacing I254 with A, G, L, S, T, M or V; replacing A255 with G, I, L, S, T, M or V; replacement of R256 with H or K; replacement of L257 with A, G, I, S, T, M or V; replacing E258 with D; replacement of E259 with D; replacing G260 with A, I, L, S, T, M or V; replacing D261 with E; replacing E262 with D; substitution of I263 with A, G, L, S, T, M or V; substituting Q264 with N; replacing L265 with A, G, I, S, T, M or V; substitution of G, I, L, S, T, M or V for A266; replacement of I267 with A, G, L, S, T, M or V; replacement of R269 with H or K; replacing E270 with D; replacing N271 with Q; replacing A272 with G, I, L, S, T, M or V; replacing Q273 with N; replacement of I274 with A, G, L, S, T, M or V; replacing S275 with a, G, I, L, T, M or V; replacement of R276 with H or K; replacement of N277 with Q; replacing G278 with A, I, L, S, T, M or V; replacing D279 with E; replacing D280 with E; replacement of T281 with A, G, I, L, S, M or V; replacement of F282 with W or Y; replacing F283 with W or Y; substitution of G284 with A, I, L, S, T, M or V; replacing A285 with G, I, L, S, T, M or V; replacing L286 with A, G, I, S, T, M or V; replacement of K287 with H or R; replacement of L288 with A, G, I, S, T, M or V; and/or L289 is replaced with A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-alpha proteins of the invention may be routinely screened for Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties (e.g., enhanced or reduced stability and/or solubility). Preferably, the resulting proteins of the invention have increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha SV functional activity. More preferably, the resulting Neutrokine-alpha and/or Neutrokine-alpha SV protein of the invention has one or more increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties.

Amino acids in the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention that are essential for function may be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunnirgham and Wells, science 244: 1081-1085 (1989)). The latter approach is to make a single alanine mutation per residue in the molecule. The resulting mutants are then tested for functional activity, such as ligand binding and the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation and/or activation.

Of particular interest is the substitution of charged amino acids with other charged or neutral amino acids, which can result in proteins with desirable improved characteristics, such as low aggregation. Aggregation not only reduces activity but is also a problem in the preparation of pharmaceutical formulations, since aggregates can be immunogenic (Pinckard et al, clinical laboratory immunology 2: 331-340 (1967); Robbins et al, diabetes 36: 838-845 (1987); Cleland et al, Crit. Rev. therapeutic drug Systems 10: 307-377 (1993)).

In another embodiment, the present invention provides a polypeptide having an amino acid sequence comprising SEQ ID NO: 2, or a polypeptide of an amino acid sequence with non-conservative substitution of the amino acid sequence shown in the sequence table 2. For example, SEQ id no: 2, the non-conservative substitution of the Neutrokine-alpha protein sequence shown in the figure comprises: substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for M1; replacing D2 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing D3 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing S4 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of T5 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing E6 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing R7 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E8 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing Q9 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing S10 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R11 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L12 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of T13 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing S14 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of C15 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; substitution of L16 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K17 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K18 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing R19 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E20 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E21 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for M22; replacing K23 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L24 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K25 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E26 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacement of C27 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing V28 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S29 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of I30 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L31 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P32 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing R33 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K34 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E35 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing S36 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of P37 with D, E, H, K, R, A, G, I, L, ST, M, V, N, Q, F, W, Y, or C; replacing S38 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V39 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R40 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing S41 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S42 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K43 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing D44 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G45; replacing K46 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L47 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L48 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A49; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A50; replacement of T51 with D, E, H, K, R, N, Q, F, W, Y, P or C; substitution of L52 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L53 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L54 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A55; substitution of L56 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L57 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S58 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of C59 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacement of C60 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; substitution of L61 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of T62 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing V63 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V64 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S65 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F66 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacement of Y67 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing Q68 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing V69 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A70; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A71; substitution of L72 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q73 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G74; replacing D75 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of L76 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A77; replacing S78 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L79 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R80 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A81; replacing E82 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L83 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q84 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G85; replacing H86 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing H87 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A88; replacing E89 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K90 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L91 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P92 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A93; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G94; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A95; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G96; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A97; replacing P98 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing K99 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing A100 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing G101 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L102 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing E103 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E104 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing A105 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P106 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing A107 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V108 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of T109 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing A110 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of G111 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L112 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K113 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of I114 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F115 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing E116 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing P117 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing P118 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing A119 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P120 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G121; replacing E122 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing G123 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing N124 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing S125 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S126 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q127 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing N128 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing S129 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R130 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing N131 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing K132 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing R133 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing A134 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V135 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q136 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing G137 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P138 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing E139 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E140 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacement of T141 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing V142 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of T143 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing Q144 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing D145 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing C146 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing L147 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q148 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; substitution of L149 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing I150 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A151 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D152 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing S153 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing E154 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacement of T155 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing P156 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacement of T157 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing I158 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q159 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing K160 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G161; replacing S162 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C for Y163; replacement of T164 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing F165 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing V166 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P167 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of W168 with D, E, H, K, R, A, G, I, L, S, T, M, V, P or C; replacing L169 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L170 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S171 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F172 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing K173 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing R174 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G175; replacing S176 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A177 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L178 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing E179 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E180 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K181 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E182 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing N183 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing K184 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of I185 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L186 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V187 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K188 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E189 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; (ii) a Replacing T190 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G191; replacing Y192 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing F193 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing F194 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; substitution of I195 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y196 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G197; replacing Q198 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing V199 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L200 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y201 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing T202 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing D203 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing K204 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacement of T205 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing Y206 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing A207 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing M208 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing G209 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing H210 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing L211 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing I212 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q213 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing R214 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K215 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K216 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing V217 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing H218 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing V219 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F220 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing G221 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D222 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing E223 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L224 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S225 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L226 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V227 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of T228 with D, E, H, K, R, N, Q, F, W, Y, P or C; substitution of L229 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F230 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing R231 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing C232 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; substitution of I233 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q234 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing N235 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing M236 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of P237 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing E238 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for T239; replacing L240 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P241 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing N242 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing N243 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing S244 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of C245 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacement of Y246 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing S247 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A248 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G249; replacing I250 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A251 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K252 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing L253 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing E254 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E255 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing G256 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D257 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing E258 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L259 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q260 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing L261 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A262 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of I263 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P264 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing R265 with D, E, a, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E266 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing N267 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing A268 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C for Q269; replacing I270 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S271 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L272 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D273 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G274; replacing D275 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacement of T276 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacement of T277 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing F278 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing F279 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing G280 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A281; replacing L282 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K283 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing L284 with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L285 is replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-alpha proteins of the invention may be routinely screened for Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties (e.g., enhanced or reduced stability and/or solubility) as described herein and known in the art. Preferably, the resulting proteins of the invention have enhanced and/or reduced Neutrokine-alpha and/or Neutrokine-alpha SV functional activity. More preferably, the resulting Neutrokine-alpha and/or Neutrokine-alpha SV protein of the invention has one or more increased and/or decreased Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties.

In another embodiment, the Neutrokine-alpha polypeptide of the invention comprises more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, and 50) amino acid (conservative or non-conservative) substituted with the above-described amino acid.

In another embodiment of the invention, the amino acid sequence of SEQ ID NO: 19, non-conservative substitutions in the Neutrokine- α SV protein sequence set forth in seq id no: substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for M1; replacing D2 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing D3 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing S4 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of T5 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing E6 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing R7 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E8 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing Q9 with D, E, H, K, R, A, G, I, L, S, T, M, V, W, Y, P, or C; replacing S10 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R11 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L12 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of T13 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing S14 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of C15 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; substitution of L16 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K17 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K18 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing R19 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E20 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E21 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for M22; replacing K23 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L24 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K25 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E26 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacement of C27 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing V28 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S29 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of I30 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L31 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P32 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing R33 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K34 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E35 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing S36 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P37 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing S38 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V39 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R40 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing S41 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S42 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K43 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing D44 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G45; replacing K46 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L47 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L48 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A49; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A50; replacement of T51 with D, E, H, K, R, N, Q, F, W, Y, P or C; substitution of L52 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L53 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L54 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A55; substitution of L56 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L57 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S58 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of C59 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacement of C60 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; substitution of L61 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of T62 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing V63 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V64 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S65 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F66 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacement of Y67 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing Q68 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing V69 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A70; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A71; substitution of L72 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q73 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G74; replacing D75 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of L76 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A77; replacing S78 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L79 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R80 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A81; replacing E82 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L83 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q84 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G85; replacing H86 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing H87 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A88; replacing E89 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K90 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L91 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P92 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A93; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G94; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A95; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G96; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A97; replacing P98 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing K99 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing A100 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing G101 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L102 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing E103 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E104 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing A105 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P106 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing A107 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V108 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of T109 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing A110 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of G111 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L112 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K113 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of I114 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F115 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing E116 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing P117 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing P118 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing A119 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P120 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G121; replacing E122 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing G123 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing N124 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing S125 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S126 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q127 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing N128 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing S129 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R130 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing N131 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing K132 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing R133 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing A134 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V135 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q136 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing G137 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P138 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing E139 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E140 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacement of T141 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing G142 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S143 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y144 with D, E, H, K, R, A, G, I, L, S, T, M, V, P, or C; replacing T145 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F146 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing V147 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing P148 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of W149 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; replacing L150 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L151 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S152 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F153 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing K154 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing R155 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of G156 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S157 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A158 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L159 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing E160 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E161 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K162 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E163 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing N164 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing K165 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of I166 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for L167; replacing V168 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing K169 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E170 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacement of T171 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing G172 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of Y173 with D, E, H, K, R, A, G, I, L, S, T, M, V, P, or C; replacing F174 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing F175 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing I176 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y177 with D, E, H, K, R, A, G, I, L, S, T, M, V, P, or C; replacing G178 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q179 with D, E, H, K, R, A, G, I, L, S, T, M, V, M, V, P, or C; replacing V180 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing L181 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y182 with D, E, H, K, R, A, G, I, L, S, T, M, V, P, or C; replacement of T183 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D184 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing K185 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing T186 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y187 with D, E, H, K, R, A, G, I, L, S, T, M, V, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A188; substitution of M189 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing G190 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of H191 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing L192 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing I193 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q194 with D, E, H, K, R, A, G, I, L, S, T, M, V, M, V, P, or C; replacing R195 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K196 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K197 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for V198; substitution of D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C for H199; replacing V200 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing F201 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing G202 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D203 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing E204 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing L205 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S206 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L207 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V208 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing T209 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L210 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F211 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing R212 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacement of C213 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing I214 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q215 with D, E, H, K, R, A, G, I, L, S, T, M, V, M, V, P, or C; replacing N216 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing M217 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P218 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing E219 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing T220 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L221 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P222 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing N223 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing N224 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing S225 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing C226 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing Y227 with D, E, H, K, R, A, G, I, L, S, T, M, V, P, or C; replacing S228 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A229 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing G230 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing I231 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A232 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K233 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L234 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing E235 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E236 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G237; replacing D238 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing E239 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing L240 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q241 with D, E, H, K, R, A, G, I, L, S, T, M, V, M, V, P, or C; replacing L242 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A243 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of I244 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P245 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing R246 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E247 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing N248 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing A249 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q250 with D, E, H, K, R, A, G, I, L, S, T, M, V, M, V, P, or C; replacing I251 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S252 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L253 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D254 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing G255 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D256 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing V257 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing T258 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F259 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing F260 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing G261 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A262 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L263 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K264 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing L265 with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L266 is replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-alpha proteins of the invention may be routinely screened for Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties (e.g., enhanced or reduced stability and/or solubility) as described herein and known in the art. Preferably, the resulting proteins of the invention have enhanced and/or reduced Neutrokine-alpha and/or Neutrokine-alpha SV functional activity. More preferably, the resulting Neutrokine-alpha and/or Neutrokine-alpha SV protein of the invention has one or more enhanced and/or reduced Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties.

In another embodiment, the Neutrokine-alpha polypeptide of the invention comprises more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, and 50) amino acids substituted with the above-described substituted amino acids (conservative or non-conservative).

For example, SEQ ID NO: preferred non-conservative substitutions for the Neutrokine-alpha protein sequence shown in fig. 23 include: replacing R1 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for V2; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for V3; replacing D4 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of L5 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S6 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A7; replacing P8 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing P9 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A10; replacing P11 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacement of C12 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; substitution of L13 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P14 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G15; replacement of C16 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing R17 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacement of H18 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing S19 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q20 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacement of H21 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing D22 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing D23 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing N24 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G25; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for M26; replacing N27 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; substitution of L28 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R29 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing N30 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing R31 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacement of T32 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y33 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacement of T34 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F35 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for V36; replacing P37 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing W38 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; substitution of L39 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L40 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S41 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F42 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing K43 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing R44 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G45; replacing N46 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A47; substitution of L48 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing E49 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E50 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K51 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E52 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing N53 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing K54 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of I55 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for V56; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for V57; replacing R58 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing Q59 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacement of T60 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G61; replacing Y62 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing F63 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing F64 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; substitution of I65 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y66 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing S67 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q68 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for V69; substitution of L70 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y71 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacement of T72 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D73 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing P74 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of I75 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F76 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A77; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for M78; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G79; substitution of D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for H80; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for V81; substitution of I82 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q83 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing R84 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K85 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K86 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for V87; substitution of D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C for H88; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for V89; replacing F90 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G91; replacing D92 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing E93 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L94 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S95 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L96 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for V97; replacement of T98 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L99 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F100 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing R101 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing C102 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing I103 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q104 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing N105 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing M106 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing P107 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing K108 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing T109 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L110 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P111 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing N112 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing N113 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing S114 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing C115 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacement of Y116 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing S117 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A118 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing G119 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing I120 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A121 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R122 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing L123 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing E124 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E125 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of G126 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D127 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing E128 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of I129 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q130 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing L131 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A132 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing I133 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P134 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing R135 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E136 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing N137 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing A138 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q139 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing I140 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S141 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R142 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing N143 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing G144 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D145 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing D146 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing T147 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F148 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing F149 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing G150 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A151 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L152 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K153 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of L154 with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L155 is replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-alpha protein of the invention may be routinely screened for Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or solubility as described herein and known in the art. Preferably, the resulting proteins of the invention have enhanced or reduced Neutrokine-alpha and/or Neutrokine-alpha SV functional activity. More preferably, the resulting Neutrokine-alpha and/or Neutrokine-alpha SV protein of the invention has one or more enhanced or reduced Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties.

In another embodiment, the Neutrokine-alpha polypeptide of the invention comprises more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, and 50) amino acids substituted with the above-described substituted amino acids (conservative or non-conservative).

For example, SEQ ID NO: 38 include: substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for M1; replacing D2 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of E3 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing S4 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A5; replacing K6 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacement of T7 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L8 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P9 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing P10 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing P11 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing C12 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; substitution of L13 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing C14 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing F15 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing C16 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing S17 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of E18 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing K19 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G20; substitution of E21 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing D22 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for M23; replacing K24 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing V25 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G26; replacing Y27 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing D28 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing P29 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of I30 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of T31 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P32 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing Q33 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing K34 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of E35 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of E36 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G37; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A38; replacing W39 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing F40 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G41; substitution of I42 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing C43 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing R44 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing D45 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G46; replacing R47 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of L48 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L49 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A50; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A51; replacement of T52 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L53 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L54 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L55 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A56; substitution of L57 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L58 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S59 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S60 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S61 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F62 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacement of T63 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A64; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for M65; replacing S66 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L67 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y68 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing Q69 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; substitution of L70 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A71; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A72; substitution of L73 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q74 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A75; replacing D76 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of L77 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for M78; replacing N79 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; substitution of L80 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R81 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for M82; substitution of E83 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of L84 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q85 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing S86 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y87 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing R88 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G89; replacing S90 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A91; replacement of T92 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P93 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A94; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A95; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A96; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G97; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for A98; replacing P99 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing E100 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing L101 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing T102 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A103 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing G104 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V105 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K106 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing L107 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L108 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing T109 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P110 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing A111 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A112 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P113 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacement of R114 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing P115 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacement of H116 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing N117 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing S118 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S119 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R120 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G121; replacing H122 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing R123 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing N124 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing R125 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacement of R126 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing A127 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F128 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing Q129 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing G130 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P131 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing E132 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing E133 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing T134 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing E135 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing Q136 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing D137 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing V138 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D139 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing L140 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S141 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A142 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P143 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing P144 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing A145 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P146 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing C147 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing L148 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacement of P149 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing G150 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing C151 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing R152 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing H153 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing S154 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q155 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing H156 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing D157 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing D158 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing N159 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing G160 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for M161; replacing N162 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing L163 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R164 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing N165 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; substitution of I166 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing 1167 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q168 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing D169 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing C170 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; substitution of L171 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q172 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; substitution of L173 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of I174 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A175 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D176 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing S177 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D178 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing T179 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P180 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing A181 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L182 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of E183 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing E184 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing K185 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing E186 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing N187 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing K188 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; substitution of I189 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V190 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V191 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R192 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing Q193 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacement of T194 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G195; replacing Y196 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing F197 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing F198 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for I199; replacing Y200 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing S201 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q202 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing V203 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L204 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Y205 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing T206 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D207 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing P208 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing I209 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F210 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing A211 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P or C for M212; replacing G213 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing H214 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing V215 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of I216 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q217 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacement of R218 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing K219 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing K220 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing V221 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing H222 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing V223 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F224 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; substitution of G225 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D226 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing E227 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing L228 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S229 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L230 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing V231 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing T232 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L233 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F234 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing R235 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing C236 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; substitution of I237 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q238 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing N239 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacement of M240 with D, E, H, K, R, N, Q, F, W, Y, P or C; replacing P241 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing K242 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing T243 with D, E, H, K, R, N, Q, F, W, Y, P, or C; substitution of L244 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P245 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; replacing N246 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing N247 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing S248 with D, E, H, K, R, N, Q, F, W, Y, P, or C; c249 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; replacing Y250 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing S251 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A252 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing G253 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing I254 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A255 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R256 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of L257 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing E258 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing E259 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing G260 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D261 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing E262 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of I263 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q264 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; replacing L265 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing A266 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing I267 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing P268 with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; substitution of D, E, a, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C for R269; replacing E270 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing N271 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing A272 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing Q273 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; substitution of I274 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing S275 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing R276 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing N277 with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; replacing G278 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing D279 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; replacing D280 with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; substitution of T281 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing F282 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; replacing F283 with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; substitution of D, E, H, K, R, N, Q, F, W, Y, P, or C for G284; replacing A285 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing L286 with D, E, H, K, R, N, Q, F, W, Y, P, or C; replacing K287 with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; replacing L288 with D, E, H, K, R, N, Q, F, W, Y, P, or C; and/or L289 is replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The resulting Neutrokine-alpha proteins of the invention may be routinely screened for Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties (e.g., enhanced or reduced stability and/or solubility) as described herein and known in the art. Preferably, the resulting proteins of the invention have enhanced and/or reduced Neutrokine-alpha and/or Neutrokine-alpha SV functional activity. More preferably, the resulting Neutrokine-alpha and/or Neutrokine-alpha SV protein of the invention has one or more enhanced and/or reduced Neutrokine-alpha and/or Neutrokine-alpha SV functional activity and/or physical properties.

In another embodiment, the Neutrokine-alpha polypeptide of the invention comprises more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, and 50) amino acids substituted with the above-described substituted amino acids (conservative or non-conservative).

Amino acid substitutions may also alter selective binding of ligands to cell surface receptors. For example, Ostade et al, Nature 361: 266-268(1993) states that some mutations result in TNF- α binding selectively to only one of two known TNF receptors. Since Neutrokine-alpha and Neutrokine-alpha SV are members of the TNF polypeptide family, mutations similar to those in TNF-alpha have similar roles in Neutrokine-alpha and/or Neutrokine-alpha SV.

The key sites for ligand-receptor binding can also be determined by structural analysis, such as crystallization, nuclear magnetic resonance, or radio-affinity labeling (Smith et al, J. mol. biol. 224: 899-904(1992) and Uos et al, science 255: 306-312 (1992)).

Since Neutrokine-alpha is a member of the TNF-related family of proteins, the sequence encoding the amino acids in the TNF conserved domain, i.e., the amino acids within Gly191-Leu284 of FIGS. 1A and 1B (SEQ ID NO: 2), and preferably amino acid residues within this region that are not conserved in all, most or some of the TNF family members (e.g., TNF-alpha, TNF-beta, LT-3, and Fas ligand), may be mutated in order to modulate, but not completely eliminate, the functional activity (e.g., biological activity) of Neutrokine-alpha (see FIGS. 2A-B). By specifically mutating Neutrokine-alpha at the positions where such conserved amino acids are typically found in the relevant TNFs, Neutrokine-alpha muteins will act as antagonists and thus have the effect of inhibiting the proliferation, differentiation and/or activation of lymphocytes, such as B cells. Accordingly, the polypeptides of the invention include Neutrokine-alpha mutants. Such a Neutrokine-alpha mutant comprises or consists of a fragment, variant or derivative of the full-length or preferably the extracellular domain of the Neutrokine-alpha amino acid sequence as shown in FIGS. 1A and 1B (SEQ ID NO: 2). Polynucleotides encoding the aforementioned Neutrokine-alpha mutants are also encompassed by the present invention.

Since Neutrokine- α SV is a member of the TNF-related family of proteins, the sequence encoding the amino acids in the conserved domain of TNF, i.e., the amino acids within Gly172-Leu265 of FIGS. 5A and 5B (SEQ ID NO: 19), and preferably the amino acid residues within this region that are not conserved among all, most or some of the TNF family members (e.g., TNF- α, TNF- β, LT- β, and Fas ligand), may be mutated in order to modulate, but not completely eliminate, the functional activity of Neutrokine- α SV (see FIGS. 2A-B). By specifically mutating Neutrokine- α SV at the position typically found in the relevant TNFs for this amino acid, the Neutrokine- α SV mutein will act as an antagonist and thus have, for example, an inhibitory effect on the proliferation, differentiation and/or activation of lymphocytes such as B cells. Accordingly, the polypeptides of the invention include Neutrokine- α SV mutants. Such a mutant Neutrokine- α SV comprises or consists of a fragment, variant or derivative of the full-length or preferably the extracellular domain of the Neutrokine- α SV amino acid sequence as shown in FIGS. 5A and 5B (SEQ ID NO: 19). Polynucleotides encoding the aforementioned Neutrokine- α SV mutants are also encompassed by the present invention.

In addition, those skilled in the art will recognize that mutations directed to regions of the Neutrokine-alpha polypeptides of the invention that encompass 19 amino acid residue insertions not found in the Neutrokine-alpha SV polypeptide sequence (i.e., amino acid residues Val142-Lys160 of the sequences shown in FIGS. 1A and 1B and SEQ ID NO: 2) may affect the observed functional activity (e.g., biological activity) of the Neutrokine-alpha polypeptide. More particularly, such residues of the targetable mutated Neutrokine-alpha polypeptide sequence include, for example and without limitation, the following SEQ ID NOs: 2, amino acid residues of a Neutrokine-alpha polypeptide sequence shown in the specification: v142; t143; q144; d145; c146; l147; q148; l149; i150; a151; d152; s153; e154; t155; p156; t157; i158; q159; and K160.

Recombinant DNA methods known in the art (see, e.g., DNA shuffling, as described above) can be used to generate novel mutant proteins or muteins, including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides may exhibit, for example, enhanced activity or improved stability. In addition, they can be purified in high yields and exhibit better solubility than the corresponding native polypeptide, at least under certain purification and storage conditions.

Thus, the present invention also encompasses derivatives and analogs of Neutrokine-alpha and/or Neutrokine-alpha SV having one or more amino acid residue deletions, additions or substitutions to produce Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides that are more suitable for expression, amplification in a selected host cell. For example, cysteine residues may be deleted or substituted with other amino acid residues to eliminate disulfide bonds; the N-linked glycosylation sites can be altered or eliminated, for example, to achieve expression of homologous products that are more easily recovered and purified from yeast hosts that are known to have hyperglycosylation sites. Thus, various amino acid substitutions at or at the first or third amino acid position of one or more glycosylation recognition sequences in the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, and/or amino acid deletions at the second amino acid position of one or more such recognition sequences, will prevent glycosylation of Neutrokine-alpha and/or Neutrokine-alpha SV in modified tripeptide sequences (see Miyajimo et al, EMBO J.5 (6): 1193) and 1197).

In addition, one or more amino acid residues (e.g., arginine and lysine residues) of the polypeptides of the invention may be deleted or substituted with other residues to eliminate undesired processing by proteases such as furin or Kexins. One possible consequence of such mutations is that the Neutrokine-alpha polypeptide of the invention is not cleaved and released from the cell surface.

In a specific embodiment, SEQ ID NO: 2 to other amino acid residues or deleted together to prevent or reduce release of soluble forms of Neutrokine-alpha from cells expressing Neutrokine-alpha. In a more specific embodiment, SEQ ID NO: lys132 of the Neutrokine-alpha sequence shown in 2 was mutated to Ala 132. In another embodiment, SEQ ID NO: 2 to Ala 133. These mutated proteins and/or polynucleotides encoding these proteins have utility, for example, in vitro therapy or gene therapy to engineer cells that express Neutrokine-alpha polypeptides retained on the surface of the engineered cells.

In a specific embodiment, SEQ ID NO: 2 to other amino acid residues or deletions, e.g. to help prevent or reduce oligomerisation of the Neutrokine-alpha polypeptide mutant when expressed in an expression system (substantially as described in example 1). In a specific embodiment, Cys146 is replaced with a serine residue. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another specific embodiment, SEQ ID NO: 2 to other amino acid residues or deletions to help prevent or reduce oligomerisation of the Neutrokine-alpha polypeptide mutants when expressed in an expression system (substantially as described in example 1). In a specific embodiment, Cys232 is replaced with a serine residue. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another specific embodiment, SEQ ID NO: 2 to other amino acid residues or deletions, for example to help prevent or reduce oligomerisation of the Neutrokine-alpha polypeptide mutant when expressed in an expression system (substantially as described in example 1). In a specific embodiment, Cys245 is replaced with a serine residue. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

The polypeptides of the invention are preferably in isolated form, and are preferably substantially purified. Recombinantly produced Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides can be produced by Smith and Johnson at gene 67: 31-40(1988) in the one-step process.

The polypeptides of the present invention include the intact polypeptide encoded by the deposited cDNA (ATCC accession No. 97768), including the intracellular domain, transmembrane domain and extracellular domain of the polypeptide encoded by the deposited cDNA, the mature soluble polypeptide encoded by the deposited cDNA, the extracellular domain minus the intracellular domain and transmembrane domain of the protein, the intact polypeptide of FIGS. 1A and 1B (amino acid residues 1-285 of SEQ ID NO: 2), the mature soluble polypeptide of FIGS. 1A and 1B (amino acid residues 134-285 of SEQ ID NO: 2), the extracellular domain of FIGS. IA and 1B (amino acid residues 73-285 of SEQ ID NO: 2), minus the intracellular domain and transmembrane domain, and polypeptides having at least 80%, 85%, 90%, preferably at least 95%, more preferably at least 96%, 97%, 98% or 99% similarity to the above-mentioned polypeptides. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

The polypeptides of the present invention include the complete polypeptide encoded by the deposited cDNA (ATCC accession number 203518), including the intracellular, transmembrane and extracellular domains of the polypeptide encoded by the deposited cDNA, the mature soluble polypeptide encoded by the deposited cDNA, the extracellular domain minus the intracellular and transmembrane domains of the protein, the complete polypeptide of FIGS. 5A and 5B (amino acid residues 1-266 of SEQ ID NO: 19), the extracellular domain of FIGS. 5A and 5B (amino acid residues 73-266 of SEQ ID NO: 2) minus the intracellular and transmembrane domains, and polypeptides having at least 80%, 85%, 90%, preferably at least 95%, more preferably at least 96%, 97%, 98% or 99% similarity to the above polypeptides. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Other polypeptides of the invention include polypeptides which are at least 80% or 85%, preferably at least 90% or 95%, more preferably at least 96%, 97%, 98% or 99% identical to the polypeptides encoded by the deposited cDNA (ATCC No.97768) or represented in FIGS. 1A and 1B (SEQ ID NO: 2), and also include portions of such polypeptides having at least 30, preferably at least 50 amino acids. Polynucleotides encoding such polypeptides are also encompassed by the present invention.

Other polypeptides of the invention include polypeptides which are at least 80% or 85%, preferably at least 90% or 95%, more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA (ATCC No.203518) or represented in FIGS. 5A and 5B (SEQ ID NO: 19), and also include a portion of such polypeptides having at least 30, preferably at least 50 amino acids. Polynucleotides encoding such polypeptides are also encompassed by the present invention.

"percent similarity" between two polypeptides refers to the range of similarity that results from comparing the amino acid sequences of two polypeptides using the Bestfit program (Wisconsin sequence analysis software package, computer team genetics version 8 for Unix, university research park, 575 Science Drive, Madison, Wis.53711) and setting default parameters to determine similarity. Bestit used a local homology alignment of Smith and Waterman (Advances in applied Mathesics 2: 482-498, 1981) to find the best similar region between the two sequences.

A polypeptide having an amino acid sequence that is, for example, at least 95% "identical" to the amino acid sequence of a reference Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide means that the amino acid sequence of such polypeptide is identical to the reference sequence, except that such polypeptide sequence may include up to 5 changes per 100 amino acids of the amino acid sequence of the reference Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide. In other words, to obtain a polypeptide comprising an amino acid sequence that is at least 95% identical to a reference amino acid sequence, 5% of the amino acid residues in the reference sequence may be deleted or substituted with other amino acids, or 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the amino terminus or the carboxy terminus of the reference sequence, or at any position between these termini, interspersed between individual residues of the reference sequence or within one or more contiguous groups of the reference sequence.

Indeed, whether any particular polypeptide is at least 80%, 85%, 90%, 95% 96%, 97%, 98% or 99% identical to, for example, the amino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO: 2), the amino acid sequence encoded by the deposited cDNA clone HNEDU15(ATCC NO: 97768), or a fragment thereof, or, for example, the amino acid sequence shown in FIGS. 5A and 5B (SEQ ID NO: 19), the amino acid sequence encoded by the deposited cDNA clone HDPMC52(ATCCNO.203518), or a fragment thereof, can be determined using known computer programs such as the Bestfit program (supra). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, at least 95% identical to a reference sequence of the present invention, parameters are of course established such that the percent identity is calculated over the full length of the reference sequence and gaps in homology of 5% of the total amino acid residues in the reference sequence are allowed.

In a specific embodiment, the identity between the reference (reference) sequence (the sequence of the invention) and the target sequence, also referred to as global sequence alignment, is determined using the FASTDB computer program (Comp. App. Biosei.6: 237-, for N-and C-terminal truncations of the target sequence, the percent identity is adjusted by calculating the number of residues of the reference sequence that do not match the corresponding target sequence at the N-and C-terminus of the target sequence, as a percentage of the total base number of the reference sequence. Determination of whether a residue matches is determined by FASTDB sequence alignment. This percentage is then subtracted from the percentage of identity calculated by the FASTDB program using the specified parameters to obtain the final percentage identity range. This final percent identity is useful in the present invention. The percent identity need only be adjusted manually if the N-and C-terminal residues of the target sequence do not match the reference sequence. I.e., only the reference sequence residue positions are outside the most distal N-and C-terminal residues of the target sequence. For example, a 90 amino acid residue target sequence is compared to a 100 residue reference sequence to determine percent identity. Deletions occur at the N-terminus of the target sequence, and thus FASTDB comparisons do not show a match of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues unmatched at the N and C termini/total number of residues in the reference sequence), so this 10% is subtracted from the percent identity calculated by the FASTDB program. If the remaining 90 residues are sufficiently matched, the final percent identity is 90%. In another embodiment, a 90 residue target sequence is compared to a 100 residue reference sequence. This deletion is an intrinsic deletion, so there are no residues at the N-and C-termini of the target sequence that do not match the reference sequence. In this case, the percent identity calculated by FASTDB was adjusted manually. Furthermore, only residues outside the N-and C-termini of the target sequence do not match the reference sequence, as shown in the FASTDB alignment, which requires manual adjustment. The invention does not require other manual adjustments.

Uses of the polypeptides of the invention include, but are not limited to, use as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known in the art. In addition, as described in detail below, uses of the polypeptides of the invention include, but are not limited to, the production of polyclonal and monoclonal antibodies for detecting the expression of Neutrokine-alpha and/or Neutrokine-alpha SV, or as agonists and antagonists capable of enhancing or inhibiting the function of Neutrokine-alpha and/or Neutrokine-alpha SV. The polypeptides of the invention also have therapeutic effects as described below. In addition, such polypeptides can be used in yeast two-hybrid systems to "capture" Neutrokine-alpha and/or Neutrokine-alpha SV binding proteins that are also candidate agonists and antagonists of the present invention. This yeast two-hybrid system is found in Fields and Song, nature 340: 245-246 (1989). Transgenosis and "knock out"

The polypeptides of the invention may also be expressed in transgenic animals. Animals of any species, including but not limited to rats, mice, rabbits, hamsters, guinea pigs, piglets, goats, sheep, cows, and non-human primates such as baboons, monkeys, and chimpanzees, can be used to produce transgenic animals. In a specific embodiment, the methods described herein and other methods known in the art are used to express the polypeptides of the invention in humans as part of gene therapy.

Any method known in the art can be used to introduce a transgene (i.e., a polynucleotide of the invention) into an animal to produce an founder strain of transgenic animals. Such methods include, but are not limited to, pronuclear microinjection (Paterson et al, applied microbiology Biotechnology 40: 691-; retroviral-mediated gene transfer into germ cell lines (Vander potten et al, Proc. Natl. Acad. Sci. USA 82: 6148-6152(1985), blastocyst or embryo; gene targeting in embryonic stem cells (Thompson et al, cell 56: 313-321 (1989)); electroporation of cells or embryos (Lo, 1983, molecular cell biology 3: 1803-1814(1983)), introduction of polynucleotides of the invention using a gene gun (see Ulmer et al, science 259: 5 (1993)), introduction of nucleic acid constructs into embryonic pluripotent stem cells and re-migration of the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al, cell 57: 717-723 (1989); et al, for a review of these methods see Gordon, "transgenic animals", Int' l ReCytol.115: 171-229(1989), which is also incorporated by reference in its entirety, U.S.A., Capspe 5464764, and/36hipe. selection vectors (Captep 5631153), cells and non-human organisms containing a predetermined genomic modification and positive/negative selection methods and vectors for producing them); U.S. Pat. No.4736866(Leder et al, transgenic non-human animals); and U.S. Pat. No.4873191(Wagner et al, genetic transformation of fertilized eggs); all of which are incorporated by reference in their entirety.

Any method known in the art may be used to produce transgenic clones containing a polynucleotide of the invention, for example, by transferring the nucleus of a cell into an enucleated oocyte of a cultured embryo, fetal or adult cell induced to quiescence (Campell et al, Nature 380: 64-66 (1996); Wilmut et al, Nature 385: 810-813 (1997)).

The invention provides transgenic animals carrying a transgene in all of their cells, as well as animals carrying a transgene in some but not all of their cells, i.e., mosaic or chimeric animals. The transgene may be integrated as a single transgene or multiple copies such as concatemers, e.g., head-to-head or head-to-tail in tandem. The transgene may also be selectively introduced into and activated in particular cell types, for example, as taught by Lasko et al (Lasko et al, Proc. Natl. Acad. Sci. USA 89: 6232-. The regulatory sequences required for such cell type-specific activation depend on the particular cell type and are apparent to those skilled in the art. Gene targeting is preferred when it is desired to integrate a polynucleotide transgene into the chromosomal location of an endogenous gene. Briefly, when this method is used, a vector containing some nucleotide sequence homologous to an endogenous gene is designed, which is integrated into the nucleotide sequence of the endogenous gene and whose function is disrupted by homologous recombination with a chromosomal sequence. The transgene may also be selectively introduced into a particular cell type, thereby inactivating endogenous genes only in that cell type, as taught, for example, by Gu et al (Gu et al, science 265: 103-106 (1994)). The regulatory sequences required for such cell type-specific inactivation will depend on the particular cell type and will be apparent to those skilled in the art. In addition to expressing a polypeptide of the invention in a ubiquitous or tissue-specific manner in transgenic animals, it is also readily apparent to those skilled in the art to generate constructs that modulate the expression of the polypeptide by various other means (e.g., developmental or chemically modulated expression).

Once the transgenic animal has been produced, the expression of the recombinant gene can be analyzed using standard methods. Animal tissues can be initially examined by Southern blot analysis or PCR screening to verify that transgene integration has occurred. The level of mRNA expression of a transgene in the tissue of a transgenic animal can also be determined by methods including, but not limited to, Northern blot analysis, in vivo hybridization analysis, reverse transcriptase-PCR (rt-PCR) of tissue samples obtained from the animal; and TagMan PCR. Tissue samples expressing the transgene may also be evaluated immunocytochemically or immunohistochemically with antibodies specific for the transgene product.

Once founder animals are produced, they can be bred, inbred, distantly bred, or cross-bred to produce a particular animal community. Such breeding methods include, for example but are not limited to, distant breeding of founder animals with more than one integration site to create separate lines; inbreeding the segregating line to produce transgenic compounds that express the transgenes at high levels due to the additive effects of expression of each transgene; cross breeding heterozygote transgenic animals to produce animal homozygotes for a given integration site to increase expression and eliminate the need to screen animals by DNA analysis; cross-propagating the isolated homozygous lines to produce a compound of a heterozygous or homozygous line; propagating the transgene in different backgrounds appropriate for the test model; and breeding the transgenic animals into other animals carrying different transgenes or knockout mutations.

The use of the transgenic and "knock out" animals of the invention includes, but is not limited to, use as animal model systems in elucidating the biological functions of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, studying diseases associated with aberrant Neutrokine-alpha and/or Neutrokine-alpha SV expression, and screening compounds effective in ameliorating such diseases.

In a further embodiment of the invention, genetically engineered cells that express or do not express (e.g., knock out) a polypeptide of the invention are administered to a patient in vivo. Such cells may be obtained from a patient (i.e., an animal, including a human) or an MHC compatible donor, and may include, but are not limited to, fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, and the like. The cells are genetically engineered in vitro with recombinant DNA methods to introduce the coding sequence for the polypeptide of the invention into the cell, or to disrupt the coding sequence for the polypeptide of the invention and/or endogenous regulatory sequences associated therewith, such as by transduction (with viral vectors, preferably vectors that integrate transgenes into the genome of the cell), or transfection methods including, but not limited to, plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, and the like. The coding sequence for a polypeptide of the invention may be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to express, and preferably secrete, a polypeptide of the invention. Engineered cells expressing and preferably secreting a polypeptide of the invention can be introduced systemically, e.g., in the circulation or intraperitoneally, into a patient.

Alternatively, the cells may be incorporated into a matrix and implanted into the body, e.g., genetically engineered fibroblasts may be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft (see, e.g., Anderson et al, U.S. Pat. No. 5399349; and Mullingan and Wilson, U.S. Pat. No.5460959, all incorporated by reference).

Where the cells administered are non-self or non-MHC compatible cells, they may be administered by methods known to generate an immune response to the introduced cells using tissue hosts. For example, cells may be introduced in a capsule form, allowing the components to be immediately exchanged with the extracellular environment without allowing the introduced cells to be recognized by the host immune system.

Antibodies

Other polypeptides of the invention relate to antibodies and T cell antigen receptors (TCRs) that immunospecifically bind to the polypeptide of SEQ ID No: 2 and/or SEQ ID No: 19 (as determined by immunoassay analysis of specific antibody-antigen binding well known in the art). Antibodies of the invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab') fragments, fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies (including anti-idiotypic antibodies directed against antibodies of the invention) and epitope-binding fragments of any of the above. The term "antibody" refers to immunoglobulin molecules and immunologically active portions thereof, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen. The immunoglobulin molecules of the present invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass immunoglobulin molecules. The immunoglobulin may be heavy or light chain. A collection of IgG, IgE, IgM, IgD, IgA and IgY heavy chains can be paired with light chains in either the kappa or lambda form.

Most preferred antibodies are antibody fragments of the invention that bind human antigens, including but not limited to Fab, Fab ', and F (ab') 2, Fd, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), and fragments comprising VL or VH domains. Antigen-binding antibody fragments, including single chain antibodies, may comprise the variable regions alone or in combination with all or part of the following regions: the hinge region, CH1, CH2, and CH3 domains. The invention also includes antigen-binding fragments that also include any combination of the variable and hinge regions, CH1, CH2, and CH3 domains. The antibodies of the invention can be from any animal, including birds and mammals. Preferably, the antibody is human, murine (e.g., rat and mouse), donkey, rabbit, goat, guinea pig, camel, horse or chicken. "human" antibodies, as used herein, include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from a human immunoglobulin library or from an animal transgenic for one or more human immunoglobulins and which does not express endogenous immunoglobulins, as described previously and as described in Kucherlapati et al, U.S. Pat. No. 5939598.

The antibodies of the invention may be monospecific, bispecific, trispecific or more specific. Multispecific antibodies may be specific for different epitopes of a polypeptide of the invention, or may be specific for a polypeptide of the invention as well as heterologous epitopes such as a heterologous polypeptide or a solid support. See, for example, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; tutt et al, journal of immunology 147: 60-69 (1991); U.S. patent nos. 4474893; 4714681, respectively; 5573920, respectively; 5601819, respectively; kostelng et al, J Immunol 148: 1547-1553(1992).

Antibodies of the invention may be described in terms of epitopes or portions of polypeptides of the invention that they recognize or specifically bind. A portion of the epitope or polypeptide can be specified, for example, by the N-terminal and C-terminal positions, the size of consecutive amino acid residues, or listed in the table and figures. Antibodies that specifically bind to any epitope or polypeptide of the invention may also be excluded. Thus, the invention includes antibodies that specifically bind to the polypeptides of the invention and allows the exclusion of the same antibodies.

In specific embodiments, the antibodies of the invention bind to a polypeptide comprising the amino acid sequence: SEQ ID No: 2 Phe115-Leu147, Ile150-Tyr163, Ser171-Phe194, Glu223-Tyr246, and Ser271-Phe 278. In another specific embodiment, the antibody of the invention binds to a polypeptide comprising the amino acid sequence: SEQ ID No: 2 Phe115-Leu147, Ile150-Tyr163, Ser171-Phe194, Glu223-Tyr246, and Ser271-Phe 278. In a preferred embodiment, the antibody of the invention binds to a polypeptide comprising SEQ ID No: 2 Glu223-Tyr 246. In another preferred embodiment, the antibody of the invention binds to a polypeptide consisting of SEQ ID No: 2 Glu223-Tyr 246. In a more preferred embodiment, the antibody of the invention binds to a polypeptide consisting of SEQ ID No: 2 Phe230-Asn 242. In another preferred embodiment, an antibody of the invention inhibits one or more biological activities of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide of the invention by specific binding. In a more preferred embodiment, the antibodies of the invention inhibit Neutrokine-alpha and/or Neutrokine-alpha SV mediated B-cell proliferation.

The antibodies of the invention may also be illustrated or described for their cross-reactivity. The invention includes antibodies that do not bind to any other analog, ortholog, or homolog of the polypeptide of the invention. The invention also includes antibodies that bind polypeptides at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% and 50% identical (calculated by methods known in the art and described herein) to a polypeptide of the invention. In specific embodiments, the antibodies of the invention cross-react with rat, mouse and/or rabbit homologues of human proteins and their corresponding epitopes. The invention also includes antibodies that do not bind to a polypeptide that is less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% identical to a polypeptide of the invention (calculated using methods known in the art and described herein). In a specific embodiment, the cross-reactivity is with respect to any specific antigenic or immunogenic polypeptide, or a combination of 2, 3, 4, 5 or more specific antigenic and/or immunogenic polypeptides. The invention also includes antibodies that bind to a polypeptide encoded by a polynucleotide that hybridizes under hybridization conditions (as described herein) to a polynucleotide of the invention. The antibodies of the invention may also be illustrated or described for their binding affinity to the polypeptides of the invention. Preferred binding affinities include those with a dissociation constant or Kd of less than 5X 10 ^-5M，10^-5M，5×10^-6M，10^-6M，5×10^-7M，10^-7M，5×10^-8M，10^-8M，5×10^-9M，10^-9M，5×10^-10M，10^-10M，5×10^-11M，10^-11M，5×10^-12M，10^-12M，5×10^-13M，10^-13M，5×10^-14M，10^-14M，5×10^-15M, or 10^-15An antibody to M.

The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention, as determined by any method known in the art for determining competitive binding, e.g., the immunoassay described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, 90%, 85%, 80%, 75%, 70%, 60%, or 50%.

The antibodies of the invention may act as agonists or antagonists of the polypeptides of the invention. For example, the invention includes antibodies that partially or fully disrupt receptor/ligand interaction with the polypeptides of the invention. Preferably, the antibodies of the invention bind to an antigenic epitope, or a portion thereof, described herein. The invention includes receptor-specific antibodies and ligand-specific antibodies. The invention also includes receptor-specific antibodies that do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) can be determined by other methods described herein or known in the art. For example, receptor activation can be determined by detecting phosphorylation of the receptor or its substrate (e.g., tyrosine or serine/threonine) by immunoprecipitation followed by Western blot analysis (as described above). In specific embodiments, antibodies are provided that inhibit the activity of a ligand or receptor by at least 95%, 90%, 85%, 80%, 75%, 70%, 60%, or 50% as compared to the activity of the antibody in the absence of the antibody.

The invention also includes receptor-specific antibodies that prevent ligand binding and receptor activation, as well as antibodies that recognize receptor-ligand complexes, preferably antibodies that do not specifically recognize unbound receptors or unbound ligands. The invention also includes neutralized antibodies that bind to the ligand and prevent the ligand from binding to the receptor, as well as antibodies that bind to the ligand, thereby preventing activation of the receptor but not preventing the ligand from binding to the receptor. The invention additionally includes antibodies that activate the receptor. These antibodies may act as receptor agonists, i.e., enhance or activate all or part of the ligand-mediated biological activity of receptor activation, e.g., by inducing receptor dimerization. The antibodies may be described as agonists, antagonists or inverse agonists of biological activity, including the specific biological activity of the peptides of the invention. The above antibody agonists can be produced by methods known in the art. See, for example, PCT publication WO 92/40281; U.S. patent nos. 581109; deng et al, blood 92 (6): 1981-1988 (1998); cher et al, cancer research 58 (16): 3668-3678 (1998); harrop et al, J Immunol 161 (4): 1786-1794 (1998); zhu et al, cancer research 58 (15): 3209 and 3214 (1998); yoon et al, J Immunol 160 (7): 3170-3179(1998): prat et al, journal of cytoscience 111(Pt 2): 237- & lt247 & gt (1998); pitard et al, journal of immunological methods 205 (2): 177-190 (1997); liautard et al, cytokine 9 (4): 233-241 (1997); carlson et al, J. Biochem 272 (17): 11295-11301 (1997); taryman et al, neuron 14 (4): 755-; muller et al, Structure 6 (9): 1153-1167 (1998); bartunek et al, cytokine 8 (1): 14-20(1996) (all of which are incorporated by reference in their entirety).

Antibodies of the invention can be used, for example, but not limited to, purifying, detecting, and targeting polypeptides of the invention, including in vitro and in vivo diagnostic and therapeutic methods. For example, antibodies can be used in immunoassays to quantitatively and qualitatively determine the level of a polypeptide of the present invention in a biological sample. See, e.g., Harlow et al, handbook of antibody experiments (Cold spring harbor laboratory Press, Vol. II, 1988) (incorporated by reference herein in its entirety).

As described in more detail below, the antibodies of the invention can be used alone or in combination with other compositions. The antibody may be fused to a heterologous polypeptide at the N-or C-terminus, or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other composition. For example, the antibodies of the invention may be recombinantly fused or conjugated to molecules used as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides or toxins. See, for example, PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. patent nos. 5314995; and EP 396387.

The antibodies of the invention include modified derivatives, i.e., covalently attached to the antibody by any type of molecule such that the covalent attachment does not prevent the antibody from producing an anti-idiotypic response. Antibody derivatives include, for example and without limitation, antibodies modified by: for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any chemical modification can be carried out by known methods, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-canonical amino acids.

The antibodies of the invention can be produced by any method known in the art. Polyclonal antibodies to the corresponding antigen can be generated by various methods well known in the art. For example, the unique administration of the present invention can be to a variety of host animals including, but not limited to, rabbits, rats, mice, etc., to induce the production of serum containing polyclonal antibodies specific for the antigen. Depending on the host species, various adjuvants may be used to enhance the rabbit immune response, including but not limited to Freund's (complete or incomplete), mineral gels such as alumina, surfactants such as lysolecithin, polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin), and Corynebacterium parvum. Such adjuvants are well known in the art.

Monoclonal antibodies can be prepared by a variety of methods known in the art, including the use of hybridomas, recombinant, and phage display methods, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma methods, including those known in the art and such as Harlow et al, handbook of antibody experiments (Cold spring harbor laboratory Press, Vol. II, 1988); hammerling et al, monoclonal antibody and T cell hybridoma 563-681(Elsevier, N.Y.1981), which are incorporated herein by reference in their entirety. The term "monoclonal antibody" is not limited to antibodies produced by hybridoma methods. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not to the method of producing it.

A "monoclonal antibody" may comprise or consist of two proteins, a heavy chain and a light chain.

Methods for producing and screening specific antibodies using hybridoma methods are well known in the art and are described in detail in the examples (e.g., example 9). In a non-limiting example, cells unique to or expressing such unique of the present invention can be used for seeding. Once an immune response is detected, such as the detection of antibodies specific for the antigen in the mouse serum, the spleen of the mouse is removed and splenocytes isolated. The splenocytes are then fused by well known methods to any suitable myeloma cells, such as the cells of the SP20 cell line obtained from ATCC. Hybridomas were selected and cloned by limiting dilution. The hybridoma clones are then analyzed by methods known in the art for cells that secrete antibodies that bind to the unique antibodies of the present invention. Ascites fluid, which usually contains high levels of antibodies, can be produced by inoculating mice with positive hybridoma clones.

Thus, the present invention provides a method of producing monoclonal antibodies and antibodies produced by the method, comprising culturing hybridoma cells which secrete an antibody of the invention, wherein preferably the hybridoma is produced by fusing a spleen cell, isolated from a mouse immunized with an antigen of the invention, with a myeloma cell, and then screening the hybridomas from the fusion for selection of hybridoma clones which secrete antibodies which bind uniquely to the invention.

Antibody fragments that recognize specific epitopes can be generated by known methods. For example, Fab and F (ab ') 2 fragments of the invention may be produced by proteolytic cleavage of an immunoglobulin molecule using an enzyme such as papain (to produce Fab fragments), or trypsin (to produce F (ab') 2 fragments). The F (ab') 2 fragment contains the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the invention can also be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding them. In specific embodiments, such phage may be used to display antigen binding domains expressed from repertoires or combinatorial antibody libraries. Phage expressing antigen binding domains that bind to the corresponding antigen can be selected or identified with the antigen, e.g., with a labeled antigen or an antigen bound or captured to a solid surface or bead. The phage used in these methods are typically filamentous phage comprising fd and M13 binding domains expressed from phage cells with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to phage gene III or gene VIII proteins. Phage display methods that can be used to produce antibodies of the invention include, for example, those described in the following references: such as Brinkman et al, journal of immunization methods 182: 41-50 (1995); ames et al, journal of immunization methods 184: 177-186 (1995); kettleborough et al, European journal of immunology 24: 952 and 958 (1994); persic et al, Gene 187: 9-18 (1997); burton et al, immunological progression 57: 191-280 (1994); PCT application No. PCT/GB 91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/20401; and U.S. patent nos. 5698426, 5223409, 5403484, 5580717, 5427908, 5750753, 5821047, 5571698, 5427908, 5516637, 5780225, 5658727, 5733743, and 5969108, all of which are incorporated by reference in their entirety.

As described in the above references, following phage selection, the antibody coding regions in the phage can be isolated and used to produce whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, as described in detail below. For example, methods of recombinantly producing Fab, Fab ', and F (ab') 2 fragments can also be applied by methods known in the art, such as PCT publications WO 92/22324; mullinax et al, Biotechnology 12 (6): 864-869 (1992); and Sawai et al, AJRI 34: 26-34 (1995); and Better et al, science 240: 1041-1043(1988), which is incorporated by reference in its entirety.

Methods that can be used to produce single chain Fvs and antibodies include, for example, U.S. patents 4946778 and 5258498; huston et al, methods enzymology 203: 46-88 (1991); shu et al, PNAS 90: 7995-7999 (1993); and SKerra et al, science 240: 1038-1040 (1988). For some applications, including the use of antibodies in humans and in vitro detection assays, chimeric, humanized or human antibodies are preferred. A chimeric antibody is a molecule in which different portions of the antibody are derived from different species of animals, e.g., an antibody having variable regions derived from a murine monoclonal antibody and human immunoglobulin constant regions. Methods of producing chimeric antibodies are known in the art. See, e.g., Morrison, science 229: 1202 (1985); oi et al, Biotechnology 4: 214 (1986); gillies et al (1989), J Immunity methods 125: 191-202; as described in U.S. patent nos. 5807715, 4816567 and 4816397. Humanized antibodies are antibody molecules from non-human species antibodies that bind a desired antigen having one or more Complementarity Determining Regions (CDRs) of the non-human species and framework regions of a human immunoglobulin molecule. Typically, framework residues within the human framework region will be substituted with the corresponding residues of the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as by modeling the interaction of the CDRs with framework residues to identify framework residues important for antigen binding, and performing sequence alignments to identify unique framework residues at specific positions (see, e.g., Queen et al, U.S. Pat. No. 5585089; Riechmann et al, Nature 332: 323(1988), incorporated herein by reference in its entirety). Antibodies can be humanized by a variety of methods known in the art, including, for example, CDR translocation (EP 239400; PCT publication;/09967 U.S. Pat. Nos. 5225539, 5530101, and 5585089), Veenerg or resurfaced (EP 592106; EP 519596; Padlan, molecular immunology 28 (4/5): 489-498 (1991); Studnica et al, protein engineering 7 (6):; 814 (1994); Roguska et al, PNAS 91: 969-973(1994)), and chain shuffling (U.S. Pat. No. 5565332).

Fully human antibodies are particularly desirable for treatment of patients. Human antibodies can be produced by a variety of methods known in the art, including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See U.S. patent nos. 4444887, and 4716111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735 and WO 91/10741, each of which is incorporated by reference in its entirety.

Human antibodies can also be produced using transgenic mice that do not express functional endogenous immunoglobulins, but express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or homologously recombined into murine embryonic stem cells. Alternatively, in addition to the human heavy and light chain genes, the human variable, constant and diversity regions may be introduced into murine embryonic stem cells. Murine heavy and light chain immunoglobulin genes can be either non-functionally isolated or simultaneously introduced into the human immunoglobulin locus by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells were expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to produce homozygous progeny expressing human antibodies. Transgenic mice are immunized in the normal manner with all or a portion of a selected antigen, such as a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from transgenic mice immunized by conventional hybridoma methods. Human immunoglobulin transgenes are rearranged by transgenic mice during B cell differentiation, followed by class switching and somatic mutation. Accordingly, using this approach it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. General studies on this method of generating human antibodies are described in Lonberg and Huszar, immunological studies int.13: 65-93 (1995). Methods for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies are detailed, for example, in PCT publications WO 98/24893, WO 92/01047, WO96/34096, WO 96/33735; european patent No. 0598877; U.S. patent nos. 5413923, 5625126, 563425, 5569825, 5661016, 5545806, 5814318, 5885793, 5916771 and 5939598, all of which are incorporated herein by reference in their entirety. In addition, Abgenix corporation (Freemont, CA) and Genpharm corporation (San Jose, CA) can provide human antibodies directed against selected antigens, produced using methods similar to those described above.

Fully human antibodies that recognize selected epitopes can be generated using a method known as "guided selection". In this method, a non-human monoclonal antibody such as a murine antibody is selected for guidance in selecting a fully human antibody recognizing the same epitope (Jespers et al, Biotechnology 12: 899-903 (1988)).

In addition, antibodies to the polypeptides of the invention may be used to generate anti-idiotypic antibodies that "mimic" the polypeptides of the invention using methods well known in the art (see, e.g., Greenspan and Bona, FASEB journal 7 (5): 437-444, (1989) and Nissinoff, journal of immunology 147 (8): 2429-2438 (1991)). For example, antibodies that bind to and competitively inhibit multimerization of a polypeptide and/or binding of a polypeptide of the invention to a ligand can be used to generate an anti-idiotype that "mimics" the multimerization and/or binding domain of a polypeptide and thereby binds and neutralizes the polypeptide and/or its ligand. Such neutralizing anti-idiotype antibodies or Fab fragments of such anti-idiotype antibodies can be used in a therapeutic regimen to neutralize the polypeptide ligand. For example, such anti-idiotype antibodies can be used to bind to a polypeptide of the invention and/or to its ligand/receptor, thereby blocking its biological activity.

Polynucleotides encoding antibodies

The invention also provides polynucleotides comprising nucleotide sequences encoding the antibodies of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or low stringency hybridization conditions (as described above) to polynucleotides encoding antibodies that preferably specifically bind to a polypeptide of the invention, more preferably to a polypeptide having the amino acid sequence of SEQ ID NO: 2. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 19 amino acid sequence. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 23 amino acid sequence. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 28 amino acid sequence. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 30 amino acid sequence.

The polynucleotide may be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide may be determined by any method known in the art. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (as described by Kutmeier et al, Biotechnology 17: 242 (1994)), in brief, comprising synthesizing overlapping oligonucleotides comprising a portion of the sequence encoding the antibody, annealing and ligating the oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

Alternatively, the polynucleotide encoding the antibody may be generated from a nucleic acid of suitable origin. If clones containing nucleic acid encoding a particular antibody are not available, but the sequence of the antibody molecule is known, then the nucleic acid encoding the immunoglobulin can be chemically synthesized, or obtained from an appropriate source via PCR amplification using synthetic primers that hybridize to the particular gene sequence to be identified, e.g., the 3 'and 5' ends of cDNA clones from a cDNA library encoding the antibody, or cloned from an appropriate source using oligonucleotide probes specific for that sequence (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing the antibody, such as hybridoma cells selected for expression of the antibody of the invention, or a nucleic acid isolated from any tissue or cell expressing the antibody, preferably poly A + RNA). The amplified nucleic acid produced by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of an antibody have been determined, the nucleotide sequence of the antibody can be manipulated by methods well known in the art, such as recombinant DNA methods, site-directed mutagenesis, PCR, etc. (see, e.g., Sambrook et al, 1990, molecular cloning, A laboratory Manual, second edition, Cold spring harbor laboratory, Cold spring harbor, N.Y. and Ausubel et al, eds., 1998, general methods of molecular biology, John Wiley and Sons, NY, which are incorporated by reference in their entirety) to produce antibodies having different amino acid sequences, e.g., to produce amino acid substitutions, deletions and/or insertions.

In a specific embodiment, the amino acid sequences of the heavy and/or light chain variable domains are examined to identify the sequences of the Complementarity Determining Regions (CDRs), by methods well known in the art, such as comparing known amino acid sequences of other heavy and light chain variable regions, to determine regions of high sequence variability. Using conventional recombinant DNA methods, one or more CDRs can be inserted into a framework region, such as a human framework region, to humanize a non-human antibody, as previously described. The framework regions may be naturally occurring or consensus framework regions, and are preferably human framework regions (see, e.g., Chothia et al, J. mol. biol. 278: 457-. Preferably, the polynucleotides produced by combining the framework regions and the CDRs encode an antibody that specifically binds to a polypeptide of the invention. Preferably, as previously described, one or more amino acid substitutions may be made within the framework regions, and preferably, such amino acid substitutions improve binding of the antibody to its antigen. In addition, such methods can be used to generate amino acid substitutions or deletions of one or more variable region cysteine residues involved in an intrachain disulfide bond to generate an antibody molecule lacking one or more intrachain disulfide bonds. Other variations of polynucleotides are also encompassed by the present invention.

Alternatively, a method for producing a "chimeric antibody" (Morrison et al, Proc. Natl. Acad. Sci. USA 81: 851855 (1984); Neuberger et al, Nature 312: 604-454 (1984); Takeda et al, Nature 314: 452-454(1985)) can be used by splicing a gene derived from a murine antibody molecule of appropriate antigen specificity with a gene derived from a human antibody molecule of appropriate biological activity. As previously mentioned, a chimeric antibody is a molecule in which different portions are derived from different species of animals, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

Alternatively, a method of producing a single-chain antibody can be used (U.S. Pat. No. 4946778; Bird, science 242: 423-42 (1988); Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-.

Method for producing antibody

The antibodies of the invention may be produced by any synthetic antibody known in the art, in particular by chemical synthesis or preferably by recombinant expression methods. Recombinant expression of an antibody of the invention, or a fragment, derivative or analog thereof (e.g., a heavy or light chain of an antibody of the invention, or a single chain antibody of the invention) requires construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule of the invention or a heavy or light chain thereof, or a portion thereof (preferably a portion comprising a heavy or light chain variable domain) has been obtained, vectors for producing the antibody molecule can be produced by recombinant DNA methods using methods well known in the art. Thus, described herein are methods for producing proteins by expressing polynucleotides containing antibody coding sequences. Expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals can be constructed using methods well known in the art. These methods include, for example, in vitro recombinant DNA methods, synthetic methods, and in vivo genetic recombination. The invention thus provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention or its heavy or light chain, or its heavy or light chain variable region, operably linked to a promoter. Such vectors may include nucleotide sequences encoding the constant regions of the antibody molecule (see, e.g., PCT publication WO 86/05807; PCT publication WO 89/01036; and U.S. Pat. No.5122464), and the variable domains of the antibody may be cloned into such vectors to express the entire heavy or light chain.

The expression vector is transferred to a host cell by a conventional method, and the transfected cell is then cultured by a conventional method to produce the antibody of the present invention. Accordingly, the invention includes a host cell comprising a polynucleotide encoding an antibody of the invention, its heavy or light chain, or a single chain antibody of the invention, operably linked to a heterologous promoter. In a preferred embodiment of expressing a diabody, vectors encoding the heavy and light chains may be co-expressed in a host cell to express the entire immunoglobulin molecule, as described below.

Various host expression vector systems can be used to express the antibody molecules of the invention. Such a host expression system represents a vector by which the corresponding coding sequence can be produced and subsequently purified, but also represents a cell which, when transformed or transfected with the appropriate nucleotide coding sequence, can express the antibody molecule of the invention in situ. Such host expression systems include, but are not limited to, microorganisms such as bacteria (e.g., E.coli, Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., saccharomyces, pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant viral expression vectors containing antibody coding sequences (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus TMV), or transformed with recombinant plasmid expression vectors containing antibody coding sequences (e.g., Ti plasmid); or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) having recombinant expression constructs comprising promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or promoters derived from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5k promoter). Preferably, bacterial cells such as e.coli, more preferably eukaryotic cells expressing especially the whole recombinant antibody molecule are used for expression of the recombinant antibody molecule. For example, mammalian cells such as Chinese Hamster Ovary (CHO) cells are effective expression systems for antibodies in combination with vectors such as the major mid-early gene promoter factor of human cytomegalovirus (Foecking et al, Gene 45: 101 (1986); Cockett et al, Bio/technology 8: 2 (1990)).

In bacterial systems, a number of advantageous expression vectors may be selected depending on the antibody molecule to be expressed. For example, when large quantities of such proteins are produced, vectors expressing fusion protein products that are easily purified directly at high levels are required for the production of pharmaceutical compositions of antibody molecules. Such vectors include, but are not limited to, the E.coli expression vector pUR278(Ruther et al, EMBO J.2: 1791(1983)) in which the antibody coding sequence is ligated separately into a vector in frame with the LacZ coding region to produce a fusion protein; pIN vectors (Inouye and InouYe, nucleic acids Res. 13: 3101-. pGEX vectors are also useful for expressing foreign polypeptides as fusion proteins with glutathione-S-transferase (GST). Typically, such proteins are soluble and easily purified from lysed cells by adsorption and binding to the matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vector includes thrombin or factor Xo proteolytic sites so that the cloned target gene product may be released from the GST component.

In the insect system, autographa californica multinuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences can be cloned into nonessential regions of the virus (e.g., polyhedrin gene) alone and placed under the control of an AcNPV promoter (e.g., polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems can be used. In the case of an adenovirus used as an expression vector, the corresponding antibody-encoding curve can be linked to an adenovirus transcription/translation control complex, such as the late promoter and tripartite leader sequence. The chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertions in non-essential regions of the viral genome, such as the E1 region or the E3 region, result in recombinant viruses which are viable and which are capable of expressing antibody molecules in infected hosts (see, for example, Logan and Shenk, Proc. Natl. Acad. Sci. USA 81: 355-359 (1984)). Specific initiation signals may also be required to efficiently translate antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. In addition, the initiation codon must be in frame with the desired coding sequence to ensure translation of all inserts. These exogenous translational control signals and initiation codons can be of various origins, such as natural and synthetic. Expression efficiency can be enhanced by including appropriate transcription enhancer elements, transcription terminators, and the like (see Bitther et al, methods enzymology 153: 51-544 (1987)).

In addition, host cell strains may be selected which modulate the expression of the inserted sequences or modify and process the gene product in a specifically desired manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product are important to the function of the protein. Different host cells have specific and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected to ensure proper modification and processing of the foreign protein to be expressed. For this purpose, eukaryotic host cells can be used which have the cellular machinery for the correct processing of the primary transcript, glycosylation and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, in particular breast cancer cell lines and BT 483, Hs 578T, HTB2, HT20, and T47D, and normal breast cell lines such as CRL 7030 and Hs 578 Bst.

For long-term high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the antibody molecule can be genetically engineered. Unless an expression vector is used which contains the origin of viral replication, the host cell may be transformed with DNA under the control of appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After the introduction of the foreign DNA, the engineered cells are grown in a nutrient medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cell to stably integrate the plasmid into its chromatin and grow into colonies which can then be cloned and expanded into cell lines. This method can be used to genetically engineer cell lines that express antibody molecules. Such engineered cell lines are particularly useful for screening and evaluating compounds that interact directly or indirectly with antibody molecules.

A number of selection systems may be used, including but not limited to herpes simplex virus thymidine kinase (Wigler et al, cell 11: 223(1977)), hypoxanthine guanine phosphoribosyl transferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48: 202(1992)), and adenine phosphoribosyl transferase (Lowy et al, cell 22: 817 (1980)) genes can be used for tk-, hgprt-or aprt-cells, respectively, similarly, antimetabolite resistance can be used as the basis for the selection of dhfr, which confers methotrexate resistance (Wigler et al, Proc. Natl. Acad. Sci. USA 77: 357(1980), O' Hare et al, Proc. Natl. Acad. USA 78: 1527(1981)), gpt, which confers mycophenolic acid resistance (Mullingn & Berg, Proc. Acad. USA 78: 1981), neo-like 207505, Wolo glycoside resistance (W418; Wglu 418, biological treatment 3: 87-95 (1991); tolstshev, drug toxicity analysis study 32: 573-596 (1993); mulligan, science 260: 926-; and Morgan and Anderson, biochemical analysis study 62: 191-217 (1993); may, 1993, TIB, TECH 11 (5): 155-; and hygro, which awards hygromycin resistance (Santerre et al, Gene 30: 147 (1984.) the recombinant DNA methods known in the present invention are routinely used to select the desired recombinant clone, as described, for example, by Ausubel et al, (ed.), general methods in molecular biology, John Wiley & Sons, NY (1993); Kriegler, A Gene transfer and expression laboratory Manual, Stockton Press, NY (1990); and Dracopoli et al (ed.), common methods in human genetics, Chapter 12 and 13, John Wiley & Sons, NY (1994); Colbene-Garapin et al, J. mol. biol. 150: 1 (1981)), all of which are incorporated herein by reference in their entirety.

The expression level of antibody molecules can be increased by vector amplification (see Bebbington, and Hentschel, expression of cloned genes in mammalian cells using vectors based on gene amplification, DNA cloning, volume three (scientific press, new york, 1987)). When the marker in the vector system expressing the antibody is amplifiable, increased levels of inhibitor in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with an antibody gene, antibody production is also increased (Crouse et al, molecular cell biology 3: 257(1983)).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain-derived polypeptide and the second vector encoding a light chain-derived polypeptide. Both vectors may contain the same selectable marker that is capable of expressing both heavy and light chain polypeptides identically. Alternatively, one can be used, the vector encoding and capable of expressing both heavy and light chain polypeptides. In this case, the light chain should be placed before the heavy chain to avoid toxic free heavy chain overload (Proudfoot, Nature 322: 52 (1986); Kohler, Proc. Acad. Sci. USA 77: 2197 (1980.) the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once the antibody molecule of the invention has been produced by animal, chemical synthesis, or recombinant expression, it may be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly by specific antigen affinity chromatography following protein a, and size column chromatography), centrifugation, differential solubility, or by any other standard method for purifying proteins. In addition, the antibodies or fragments thereof of the invention can be fused to other heterologous sequences described herein or known in the art to facilitate purification.

The invention encompasses antibodies recombinantly fused or chemically conjugated (including covalent and non-covalent transformations) to a polypeptide of the invention (or a portion thereof, preferably a polypeptide of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids) to produce a fusion protein. The fusion need not be direct, but may also occur via a linker sequence. The antibody may be specific for an antigen different from the polypeptide of the invention (or a portion thereof, preferably a polypeptide of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids). For example, antibodies can be used to target a polypeptide of the invention to a particular cell type in vitro or in vivo by fusing or conjugating the polypeptide of the invention to an antibody specific for a particular cell surface receptor. Antibodies fused or conjugated to polypeptides of the invention may also be used in vitro immunoassay and purification methods. See, e.g., Harbor et al, supra, and PCT publication WO 93/21232; EP 439095; naramura et al, immunological communication, 39: 91-99 (1994); us patent 5474981; gillies et al, PNAS 89: 1428-1432 (1992); fecl et al, journal of immunology 146: 2446-2452 (1991); all of which are incorporated herein by reference in their entirety.

The invention also includes a composition comprising a polypeptide of the invention fused or conjugated to an antibody domain other than a variable region. For example, a polypeptide of the invention may be fused or conjugated to the Fc region of an antibody, or a portion thereof. The antibody portion fused to the polypeptide of the invention may include a constant region, a hinge region, a CH1 domain, a CH2 domain, and a CH3 domain, or any combination or portion of all of the domains. The polypeptide may also be fused or conjugated to a portion of an antibody as described above to form a multimer. For example, an Fc portion fused to a polypeptide of the invention may form a dimer through disulfide bonds between the Fc portions. Higher multimeric forms can be produced by fusion of polypeptides with portions of IgA and IgM. Methods of fusing or conjugating the polypeptides of the invention to antibodies are known in the art. See, e.g., U.S. patent nos. 5336603; 5622929, respectively; 5359046, respectively; 5349053, respectively; 5447851, respectively; 5112946, respectively; EP 307434; EP 367166; PCT publications WO 96/04388; WO 91/06570; ashkenazi et al, proceedings of the American academy of sciences 88: 10535-10539 (1991); zheng et al, journal of immunology 154: 5590-; and Vil et al, Proc. Natl Acad Sci USA 89: 11337, 11341(1992), which are incorporated herein by reference in their entirety.

As previously described, the sequences corresponding to SEQ ID NOs: 2 the polypeptide, polypeptide fragment or variant thereof may be fused or conjugated to an antibody moiety as described above to increase the in vivo half-life of the polypeptide, or used in an immunoassay. In addition, the sequences corresponding to SEQ ID NO: 19, fused or conjugated to an antibody moiety as described above to facilitate purification. For example, one report describes a chimeric protein consisting of the first two domains of the human CD 4-polypeptide and various domains of the constant region of a mammalian immunoglobulin light or heavy chain. (EP 394827; Traunecker et al, Nature 331: 84-86 (1988)). Polypeptides of the invention fused or conjugated to antibodies having disulfide-linked dimeric structures (due to IgG) may also bind and neutralize other molecules more efficiently than monomeric secreted proteins or fragments thereof (Foutoulakis et al, J. biol. chem. 270: 3958-3964 (1995). in many cases, the Fc portion of the fusion protein is advantageous for therapy and diagnosis and may thus, for example, improve pharmacokinetic properties (EP A232262.) or may be deleted after the fusion protein has been expressed, detected and purified. for example, if the fusion protein is used as an antigen for immunization, the Fc portion may hinder therapy and diagnosis. in drug development, for example, human proteins such as hIL-5 are fused to the Fc portion for high-throughput screening assays to identify antagonists of hIL-5 (see Bennett et al, J. mol. ID. 8: 52-58 (1995); Johanson et al, journal of biochemistry 270: 9459-9471(1995).

In addition, the antibodies or fragments thereof of the invention can be fused to a marker sequence, such as a peptide, to facilitate purification. In a preferred embodiment of the above protocol, the marker amino acid sequence is a 6-histidine peptide, such as the marker provided in the pQE vector (QIAGEN, inc., 9259 Eton Avenue, Chatsworth, CA, 91311), many of which are commercially available. Such as Gentz et al, Proc. Natl. Acad. Sci. USA 86: 821-824(1989), the 6-histidine can be routinely used for the purification of fusion proteins. Other peptide tags for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, cell 37: 767(1984)), and the "flag" tag.

The invention also encompasses antibodies or fragments thereof conjugated to diagnostic or therapeutic agents. Such antibodies can be used, for example, diagnostically to monitor tumor development or progression as part of a clinical test, e.g., to determine the effectiveness of a treatment regimen. Coupling the antibody to a detectable substance may facilitate detection. Detectable substances include, for example, various enzymes, prosthetic groups, fluorophores, luminescent substances, bioluminescent substances, radioactive substances, positron emitting metals using various positron emission computerized tomography, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated to the antibody (or fragment thereof) directly, or indirectly via an intermediate, such as a linker as is known in the art, using methods known in the art. See, e.g., U.S. patent No.4741900 for a description of metal ions that can be conjugated to antibodies, which can be used as diagnostic agents according to the present invention. Suitable enzymes include, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic group complexes include, for example, streptavidin/biotin and avidin/biotin; suitable fluorochromes include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, dansyl chloride or phycoerythrin; luminophores include, for example, luminol; bioluminescents include, for example, luciferase, luciferin and aequorin; suitable radioactive materials include ¹²⁵I，¹³¹I，¹¹¹In, or⁹⁹Tc。

In addition, the antibody or fragment thereof can be conjugated to a therapeutic component such as a cytotoxin, e.g., a cytostatic or cytocidal agent,therapeutic agents or radioactive metal ions, e.g. alpha-emitters, e.g.²¹³And (4) Bi. Cytotoxic or cytotoxic agents include any agent that is harmful to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin thiophenic glycosides, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dydroxyanthrax, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogues or homologues thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., dichloromethyldiethylamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cycloothostamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and dichlorodiamine cisplatin (II) (DDP), anthracyclines (e.g., daunorubicin, (pre-daunomycin) and doxorubicin), antibiotics (e.g., daunomycin (pre-actinomycin), bleomycin, mithramycin, Amitocin (AMC)), and antimitotics (e.g., vincristine and vinblastine).

The conjugates of the invention may be used to modify a given biological response, and the therapeutic agent or drug component need not be limited to conventional chemotherapeutic agents. For example, the pharmaceutical component may be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptosis agents such as alpha-TNF, beta-TNF, AIMI (see International publication No. WO 97/33899), AIMII (see International publication No. WO 97/34911), Fas ligand (Takahashi et al, Immunol 6: 1567-1574(1994)), VEGI (see International publication No. WO 99/23105), CD 40 ligand, thrombosis (thrombotic) agent or antiangiogenic agents such as angiostatin or endostatin; or biological response modifiers such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growth factors.

The antibodies may also be attached to a solid support, which is particularly useful for immunoassay or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

Methods for conjugating such therapeutic components to antibodies are well known, see, e.g., Arnon et al, "drug-immune directed monoclonal antibodies in cancer therapy," monoclonal antibodies to cancer therapy, Reisfeld et al (ed.). P243-56(Alan R. Liss, Inc. 1985); hellstrom et al, "antibody to drug delivery", controlled drug delivery (second edition), Robinson et al (ed.), 623-53(Marcel Dekker, Inc. 1987); thorpe, "antibody carrier for cytotoxic agents in cancer therapy", monoclonal antibody 84: biological and clinical applications, Pinchera et al (ed.), P475-506 (1985); "analysis, results and expected results of therapeutic use of radiolabeled antibodies in cancer treatment", monoclonal antibodies in cancer detection and treatment, Ballowin et al, P303-16 (scientific Press 1985), and Thorpe et al, "preparation and cytotoxicity of antibody-toxin conjugates", immunological studies 62: 119-58(1982).

Alternatively, the antibody may be conjugated to another antibody to form an antibody heteroconjugate, as described by Segal in U.S. patent No.4676980, which is incorporated herein by reference in its entirety.

The antibody, with or without a therapeutic component conjugated thereto, administered alone or in combination with cytotoxic factors and/or cytokines, may be used for therapy.

Immunotyping

The antibodies of the invention are useful for immunophenotyping of cell lines and biological samples. The translation products of the genes of the invention can be used as cell-specific markers, or more specifically as cell markers that are expressed differently at the various differentiation and/or maturation stages of a particular cell type. Monoclonal antibodies directed against specific epitopes or combinations of epitopes can be screened for marker-expressing cell populations. Various methods for screening populations of cells expressing markers with monoclonal antibodies are available, including magnetic separation with antibody-coated magnetic beads, panning with antibodies attached to a solid substrate (i.e., a plate), and flow cytometry (see, e.g., U.S. Pat. No. 5985660; and Morrison et al, cell 96: 737-49 (1999)).

These methods are used to screen specific populations of cells, such as blood cancers (i.e., MRDs in acute leukemic patients) and transplanted "non-self" cells, to prevent graft/host disease (GVHD). Alternatively, these methods can be used to screen hematopoietic stem and progenitor cells capable of proliferation and/or differentiation, which can be found in human cord blood.

Antibody binding assays

Antibodies of the invention can be subjected to immunospecific binding assays by any method known in the art. Immunoassays that may be used include, but are not limited to, competitive or non-competitive assay systems, such as those using Western blot analysis, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and the like. Such assays are common and well known in the art (see, e.g., Ausubel et al, eds., 1994, general methods of molecular biology, first volume, John Wiley & Sons, Inc., New York; incorporated herein by reference in its entirety). Some immunoassays are briefly exemplified below (but not limited to).

Immunoprecipitation methods generally include lysing cell populations in RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.01% SDS, 0.15M NaCl, 0.01M sodium phosphate, pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the corresponding antibodies to the cell lysate, incubating at 4 ℃ for a period of time (e.g., 1-4 hours), adding protein A and/or protein G sepharose beads to the cell lysate, incubating at 4 ℃ for about 1 hour or more, washing the beads in the lysate, and resuspending them in SDS/sample buffer. The ability of the corresponding antibody to immunoprecipitate a particular antigen can be determined by, for example, Western blot analysis. One skilled in the art will recognize that parameters can be modified to increase antibody binding to antigen and to decrease background (e.g., pre-washing cell lysates with agarose gel beads). Further details regarding immunoprecipitation methods are set forth, for example, in Ausubel et al, eds, 1994, general methods in molecular biology, first volume, John Wiley & Sons, Inc., New York, 10.16.1.

Westeyn blot analysis typically involves preparing a protein sample, running the protein sample on a polyacrylamide gel (e.g., 8% -20% SDS-PAGE based on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel onto a membrane, such as nitrocellulose, PVDF or nylon membrane, blocking the membrane in a blocking solution (e.g., PBS or skim milk with 3% BSA), washing the membrane in a wash buffer (e.g., PBS-Tween 20), blocking the membrane with a primary antibody (corresponding to the antibody) diluted in the blocking solution, washing the membrane in a wash buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., anti-human antibody), diluted in a blocking solution, conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (e.g., horseradish peroxidase or alkaline phosphatase), or the like³²P or¹²⁵I) The membrane is rinsed in a rinsing solution and tested for the presence of antigen. Those skilled in the art will recognize that parameters may be modified to improve the detected signal and reduce background interference. Further details regarding Western blotting procedures are provided, for example, in Ausubel et al, eds, 1994, general methods in molecular biology, first volume, John Wiley&Sons, inc, new york, set forth at 10.8.1.

ELISAs involve preparing antigens, coating 96-well microtiter plates with the antigens, adding corresponding antibodies conjugated to a detectable compound, such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase), to the wells, incubating for a period of time, and detecting the presence of the antigens. In ELISAs, the corresponding antibody need not necessarily be linked to a detectable compound; in contrast, a secondary antibody conjugated to a detectable compound (which recognizes the primary antibody) may be added to the well. Alternatively, instead of coating the wells with antigen, the wells may be coated with antibody. In this case, the secondary antibody conjugated to the detectable compound may be added after the addition of the corresponding antigen to the coated wells. Those skilled in the art will recognize that parameters may be modified to improve detection signals, as well as other variations of ELISAs known in the art. Further details regarding ELISA are set forth, for example, in Ausubel et al, editors, 1994, general methods in molecular biology, first volume, John Wiley & Sons, Inc., New York, 11.2.1.

The binding affinity of an antibody to an antigen and the rate of dissociation of antibody-antigen interactions can be determined by competitive binding assays. Competitive binding assays are, for example, radioimmunoassays which involve incubating a labeled antigen with a corresponding antibody in the presence of an increased amount of unlabeled antigen (e.g.³H or¹²⁵I) And detecting the antibody bound to the labeled antigen. The affinity and binding-to-detachment ratio of the corresponding antibody to a particular antigen can be determined from data obtained from Scatchard blot analysis. Competition with the second antibody can also be determined using radioimmunoassay. In this case, the antigen is conjugated to a labelled compound (e.g.³H or¹²⁵I) In the presence of an increased amount of another antibody that is not labeled.

Therapeutic uses

The invention also relates to an antibody-based therapeutic method comprising administering an antibody of the invention to an animal, preferably a mammal, more preferably a human, patient, for the treatment of one or more of said diseases, disorders or pathological conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof), and antibodies encoding the invention (including fragments, analogs and derivatives thereof, and said anti-idiotypic antibodies). The antibodies of the invention are useful for treating, inhibiting or preventing diseases associated with aberrant expression and/or activity of the polypeptides of the invention. A functional disorder or pathological condition, including but not limited to any one or more of the diseases, functional disorders or pathological conditions described herein (e.g., autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmune cytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, recurrent polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), multiple sclerosis, neuritis, uveitis, multiple endocrine diseases, purpura (e.g., Henloch-Scoenlein purpura), Reiter's disease, Stiff-Man syndrome, autoimmune pneumonia, Guillain-Pane syndrome, insulin dependent diabetes mellitus, and autoimmune ophthalmia, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis), systemic lupus erythematosus, Goodpasture's syndrome, pemphigus, receptor autoimmunity such as (a) Graves disease, (b) Myastenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigus, Sjogren's syndrome, diabetes, and epinephrine drug resistance (including adrenoceptor drug resistance to treat asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine adenopathies, vitiligo, vasculitis, post-myocardial syndrome, urticaria, hereditary allergic dermatitis, asthma, inflammatory myopathies, and other inflammations, grandifoliatous, degenerative, and atrophic dysfunctions).

In a specific embodiment, the antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent rheumatoid arthritis.

In another specific embodiment, the antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent systemic lupus erythematosus.

Treatment and/or prevention of diseases, disorders or pathological conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including but not limited to alleviation of symptoms associated with such diseases, disorders or pathological conditions. The antibodies of the invention may also be used to target and kill cells expressing Neutrokine-alpha on their surface, or cells having Neutrokine-alpha bound on their surface. The antibodies of the invention may be provided in pharmaceutically suitable compositions known in the art or described herein.

Methods for the therapeutic use of the antibodies of the invention include binding of a polynucleotide or polypeptide of the invention locally or systemically in vivo, or by direct cytotoxicity of the antibody, e.g., mediated by complement (CDC) or effector cells (ADCC). Some such methods are set forth in more detail below. From the teachings herein, one skilled in the art would know how to use the antibodies of the present invention for diagnosis, monitoring or treatment without undue experimentation.

The antibodies of the invention are advantageously used in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (e.g., IL-2, IL-3, and IL-7), e.g., to increase the number or activity of effector cells that interact with the antibody.

The antibodies of the invention can be administered alone or in combination with other types of therapeutic regimens (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy, antineoplastic agents, antibiotics, and immunoglobulins). Generally, it is preferred to administer a species source or species-reactive (in the case of antibodies) product of the same species as the patient. Thus, in a preferred embodiment, a human antibody, fragment derivative, analogue or nucleic acid is administered to a human patient for treatment or prophylaxis.

Antibodies against the polypeptides or polynucleotides of the invention are preferably used with high affinity and/or strong in vivo to inhibit and/or neutralize diseases associated with the polynucleotides or polypeptides of the invention, including fragments thereof. Such antibodies, fragments or regions preferably have affinity for the polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include dissociation constantsOr Kd is less than 5X 10^-5M，10^-5M，5×10^-6M，10^-6M，5×10^-7M，10^-7M，5×10^-8M，10^-8M，5×10^-9M，10^-9M，5×10^-10M，10^-10M，5×10^-11M，10^-11M，5×10^-12M，10^-12M，5×10^-13M，10^-13M，5×10^-14M，10^-14M，5×10^-15M, and 10^-15M。

Gene therapy

In a specific embodiment, a nucleic acid comprising a sequence encoding an antibody or functional derivative thereof is administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention by gene therapy. Gene therapy refers to treatment by administering an expressed or expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acid produces its encoded protein that mediates the therapeutic effect.

Any suitable method of gene therapy in the art may be used in accordance with the present invention. Such as the methods described below.

General studies on gene therapy are described by Golddspiel et al, clinical pharmacy 12: 488-505 (1993); wu and Wu, biotherapy 3: 87-95 (1991); tolstshev, pharmatoxicology analysis study 32: 573-596 (1993); mulligan, science 260: 926-; and Morgan and Anderson, biochemical analysis study 62: 191-217 (1993); may, TIB TECH 11 (5): 155-. Recombinant DNA techniques well known in the art can be used, see Ausubel et al (eds.), general methods of molecular biology, John Wiley & Sons, NY (1993); and Kriegler, A handbook of Gene transfer and expression experiments, Stockton Press, NY (1990).

In a preferred embodiment, the compound comprises a nucleic acid sequence encoding the antibody, which nucleic acid sequence is part of an expression vector for expressing the antibody or fragment thereof or chimeric protein or heavy or light chain in a suitable host. In particular, such nucleic acid sequences have a promoter operably linked to the antibody coding region, which promoter is inducible or constitutive, and optionally tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequence and any other desired sequences flank a region in the genome where homologous recombination is initiated (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); Zijlstra et al, Nature 342: 435-438 (1989)) so that the antibody-encoding nucleic acid is expressed in the chromosome (in a specific embodiment, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding the heavy and light chains of an antibody or fragments thereof.

Delivery of nucleic acids to a patient can be direct, in that the patient is directly exposed to the nucleic acid or to a vector carrying the nucleic acid, or indirect, in that the cells are first transformed with the nucleic acid in vitro and then transplanted into the patient. These two methods are known as in vivo or ex vivo gene therapy, respectively.

In a specific embodiment, the nucleic acid sequence is administered directly in vivo, and is expressed in vivo to produce the encoded product. This can be done by various methods known in the art, for example, constructing them as part of suitable nucleic acid expression vectors and administering them to render them intracellular material, for example by infection with defective or attenuated retroviruses or other viral vectors (see U.S. Pat. No.4980286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g.gene gun; Biolistic, Dupont), or coating with lipid or cell surface receptors or transfection agents, encapsulation with liposomes, microparticles or microcapsules, or by administration linked to peptides known to enter the nucleus, receptor-mediated endocytosis by administration linked to ligands (see, for example, Wu and Wu, J.Biochem.262: 4429-4432 (1987)) (which can be used to target cell types specifically expressing receptors). In another embodiment, a nucleic acid-ligand complex can be formed wherein the ligand comprises a fusogenic viral peptide to disrupt endosomes, protecting the nucleic acid from lysosomal degradation. In another embodiment, the nucleic acid can be targeted to cells specifically taken up and expressed in vivo by targeting a specific receptor (see, e.g., PCT publications WO 92/06180; WO 92/22635; WO 92/20316; WO 93/14188; WO 923/20221). Alternatively, the nucleic acid may be introduced into cells by homologous recombination and incorporated into host cell DNA for expression (Koller and Smities, Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); Zijlstra et al, Nature 342: 435-438 (1989)).

In a specific embodiment, a viral vector containing a nucleic acid sequence encoding an antibody of the invention is used. For example, retroviral vectors can be used (see Miller et al, methods in enzymology 217: 581-, general view in genetics and development 3: 110-114(1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are particularly useful vehicles for gene delivery to the airway epithelium. Adenoviruses naturally infect the respiratory epithelium, resulting in mild disease. Other targets for adenovirus-based delivery systems are liver, central nervous system, endothelial cells and muscle. Adenoviruses have the advantage of being able to infect undifferentiated cells. Kozansky and Wilson, general view of inheritance and development 3: 499-503(1993) proposes an adenovirus-based gene therapy. Bout et al, human gene therapy 5: 3-10(1994) describes an adenovirus vector for transferring genes to the airway epithelium of asthmatic monkeys using the adenovirus vector. Other references for use of adenovirus in gene therapy are found in Rosenfeld et al, science 252: 431-434 (1991); rosenfeld et al, cell 68: 143- & ltwbr/& gt 155 (1992); mastrangeli et al, J. Clin. Res. 91: 225-234 (1993); PCT publications WO 94/12649; and Wang et al, gene therapy 2: 775-783(1995). In a preferred embodiment, an adenoviral vector is used.

Adeno-associated virus (AAV) has been used for this purpose in gene therapy (Walsh et al, Proc. Natl. Acad. Sci. USA 204: 289-300 (1993); U.S. Pat. No. 5436146).

Another gene therapy approach involves gene transfer into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Typically, the transfer method involves transferring a detectable label to the cell. The treated cells expressing the transgene are then isolated. These cells are then administered to the patient.

In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction may be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or phage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, minicell-mediated gene transfer, spheroplast fusion, and the like. A number of methods for introducing foreign genes into cells are known in the art (see, for example, Loeffler and Beht, methods in enzymology 217: 599-618 (1993); Cohen et al, methods in enzymology 217: 618-644 (1993); clinical drug therapy 29: 69-92m (1985)), which can be used according to the invention without disrupting the essential developmental and physiological functions of the recipient cell.

The resulting recombinant cells can be administered to a patient by various methods known in the art. The recombinant blood cells (e.g., hematopoietic stem cells or progenitor cells) are preferably administered intravenously. The number of cells used depends on the desired effect, the patient's state, etc., and can be determined by one skilled in the art.

Cells into which the nucleic acid is introduced for gene therapy encompass any desired appropriate cell type, including but not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, myocytes, hepatocytes, blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, particularly hematopoietic stem or progenitor cells, such as from bone marrow. Cord blood, peripheral blood, fetal liver, and the like.

In a preferred embodiment, the cells used for gene therapy are of the patient's own.

In embodiments where recombinant cells are used for gene therapy, the nucleic acid sequences encoding the antibodies are introduced into the cells so that they can be expressed by the cells or cell progeny, followed by administration of the recombinant cells in vivo for therapy. In a specific embodiment, the cells are used in progenitor cells. Any stem and/or progenitor cell that can be isolated and maintained in vitro can be used according to this embodiment of the invention (see, e.g., PCT publication WO 94/08598; Stemple and Anderson, cell 71: 973-.

In a specific embodiment, the nucleic acid introduced for gene therapy comprises an inducible promoter operably linked to the coding region, whereby expression of the nucleic acid can be controlled by control with or without the presence of an appropriate transcriptional inducer.

Demonstration of therapeutic or prophylactic Activity

The compounds or pharmaceutical compositions of the invention are preferably tested in vitro and then in vivo for the desired therapeutic or prophylactic activity before being administered to humans. For example, in vitro assays demonstrating therapeutic or prophylactic activity of a compound or pharmaceutical composition include the effect of the compound on cell lines or tissue samples of a patient. The effect of a compound or composition on a cell line or tissue sample of a patient can be determined using methods known in the art, including, but not limited to, a rosette formation assay and a cell lysis assay. In accordance with the present invention, it is useful to determine whether a specific compound administered is an indicated in vitro assay, including an in vitro cell culture assay, wherein a patient tissue sample is grown in culture, exposed to or administered with the compound, and the effect of such compound on the tissue sample is observed.

Methods and compositions for therapeutic and/or prophylactic administration

The present invention provides methods for achieving therapeutic, inhibitory and prophylactic purposes by administering to a subject an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred embodiment, the compound is substantially purified (e.g., substantially free of materials that limit its action or produce undesirable side effects). The subject is preferably an animal, including but not limited to cows, pigs, horses, chickens, cats, dogs, etc., preferably a mammal, and most preferably a human.

When the compound comprises a nucleic acid or immunoglobulin, the administration formulations and methods that can be used are as described above; additional suitable formulations and routes of administration may be selected from those described below.

Various delivery systems are known and may be used to administer the compounds of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biochem. 262: 4429-4432(1987)), constructing nucleic acids as part of a retrovirus or other vector, and the like. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, sublingual, intranasal, epidermal or oral. The compounds or compositions may be administered by conventional routes, for example by fusion or bolus injection, absorbed through epithelial or mucosal to skin boundaries (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other biologically active agents. Can be administered systemically or locally. In addition, the pharmaceutical compounds or compositions of the present invention may be introduced into the central nervous system by any suitable route, including intravenous and intraarterial injection; intravenous injection is simplified by an intravenous catheter, for example, adsorbed in a fluid bath such as an Ommaya fluid bath. Pulmonary administration is also possible, e.g. using an inhaler or nebulizer, and formulations with an aerosol.

In a specific embodiment, it is desirable to administer the pharmaceutical compounds or compositions of the present invention topically at the site in need of treatment; this can be applied, for example, by local perfusion during surgery, topical application, such as with a wound dressing after surgery, by injection, by catheter, by suppository, or by implantation, the implant being porous, non-porous, or gelatinous, including membranes, such as sialastic membranes, or fibers. Preferably, when administering the proteins of the present invention, including antibodies, care must be taken to use substances that are not absorbed by the protein.

In another embodiment, the compound or composition may be delivered in vesicles, particularly liposomes (see Langer, science 249: 1527-.

In another embodiment, the compound or composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC crit. Ref. biomed. Eng.14: 201 (1987); Buchwald et al, science 88: 507 (1980); Saudek et al, N.Engl. J. Med.321: 574 (1989)). In another embodiment, polymers may be used (see controlled release medical applications, Langer and Wise (eds.), CRC Press, Boca Raton, Florida (1974); controlled drug bioavailability, drug design and manufacture, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol, Sci. Rev. Macromol. chem.23: 61 (1983); also seen in Levy et al, science 228: 190 (1985); During et al, neurology 25: 351 (1989); Howanol et al, Neurosurg 71: 105 (1989)). In another embodiment, the controlled release system may be placed in the vicinity of the target of treatment, i.e., the brain, such that only a fraction of the systemic dose is required (see, e.g., Goodson, medical applications of controlled release, supra, Vol. II, p115-138 (1984)).

Other controlled release systems are described in Langer (science 249: 1527-.

In a specific embodiment wherein the compound of the invention is a nucleic acid encoding a protein, the nucleic acid may be administered in vivo to initiate expression of the protein it encodes, by constructing it as part of a suitable nucleic acid expression vector and administering it to become part of the intracellular compartment, for example by using a retroviral vector (see U.S. Pat. No.4980286), or by direct injection, or by using microprojectile bombardment (e.g., gene gun; Biolistic, Dupont), or coating with lipids or cell surface receptors or transfection agents, or by ligation with a similar homeobox peptide known to enter the nucleus (see, for example, Joliot et al, Proc. Natl. Acad. Sci. USA 88: 1864-. Alternatively, the nucleic acid may be introduced into the cell by homologous recombination and incorporated into the host cell DNA for expression.

The invention also provides a pharmaceutical composition. Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically suitable carrier. In a specific embodiment, the term "pharmaceutically suitable" refers to those items permitted by federal or governmental agencies or listed by the U.S. drug administration agencies, or otherwise generally recognized as being useful in animals, particularly humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including petroleum, animal, vegetable or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline and dextrose and glycerol solutions may also be employed as liquid carriers. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, lime, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk, glycerol, propylene, glycol, water, ethanol and the like. The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The compositions may be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Suitable pharmaceutical carriers are described, for example, by e.w. martin in Remington's pharmaceutical science. Such compositions contain a therapeutically effective amount of the compound, preferably in purified form, and an appropriate amount of carrier to provide a dosage form for proper administration to a patient.

In a preferred embodiment, the composition is formulated according to conventional pharmaceutical procedures for preparing medicaments suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a sterile isotonic solution. If desired, the composition may also include stabilizers and local anesthetics such as lignocaine to reduce pain at the injection site. Generally, the ingredients will be provided separately or in unit dose mixtures, for example as anhydrous lyophilized powders or anhydrous concentrates in sealed containers such as ampoules or pouches indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be used to mix the ingredients prior to administration.

The compounds of the invention may be formulated in neutral or salt form. Pharmaceutically suitable salts include salts with cations such as those produced from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, or anions such as those produced from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histamine, procaine, and the like.

The amount of a compound of the invention that is effective to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The actual dosage employed in the formulation will also depend on the route of administration, the severity of the disease, and will depend on the diagnosis of the physician and the condition of the patient. Effective dosages can be determined by generating dose response curves from in vitro or animal model test systems.

In the case of antibodies, the dose administered to the patient is between 0.1 and 100mg/kg body weight, more preferably between 1 and 10mg/kg body weight. Generally, human antibodies have a longer half-life in humans than antibodies of other species, due to an immune response to the foreign polypeptide. Thus, smaller doses of human antibody can be administered, and the frequency of administration can also be longer. In addition, the dosage and frequency of administration of the antibodies of the invention are reduced by modification of the antibodies, e.g., by lipidation, to enhance antibody uptake and tissue permeability (e.g., into the brain).

The invention also provides a pharmaceutical pack or kit comprising one or more containers containing one or more ingredients of the pharmaceutical composition of the invention. Optionally associated with such container is a notice by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects the licensed manufacture, use or sale of biological products or pharmaceuticals for administration to humans.

Diagnosis and imaging

Labeled antibodies, and derivatives and analogs thereof, that specifically bind to the corresponding peptide, are useful for diagnostically detecting, diagnosing or monitoring a quantity of a disease and/or disorder associated with aberrant expression and/or activity of a polypeptide of the invention. The present invention provides methods for detecting abnormal expression of a corresponding polypeptide comprising (a) analyzing the expression of the corresponding polypeptide in cells or body fluids of an individual using one or more antibodies specific for the corresponding polypeptide, and (b) comparing the gene expression level to a standard gene expression level, whereby an increase or decrease in the analyzed polypeptide gene expression level compared to the standard gene expression level indicates abnormal expression.

The present invention provides diagnostic assays for diagnosing a dysfunction comprising (a) assaying the expression of a corresponding polypeptide in cells or body fluids of an individual using one or more antibodies specific for the corresponding polypeptide, and (b) comparing the gene expression level to a standard gene expression level, whereby an increase or decrease in the gene expression level of the assayed polypeptide compared to the standard expression level is indicative of a particular dysfunction, and in the case of cancer, the presence of a relatively high amount of transcript in the biopsy tissue of the individual may be indicative of a predisposition to the development of the disease, or may provide a means of detecting the disease prior to the onset of true clinical symptoms, and a more definitive diagnosis may allow a health practitioner to apply earlier preventive measures or aggressive treatment, thereby preventing the development or further progression of the cancer.

Antibodies of the invention can be used to analyze protein levels in biological samples using conventional immunohistological methods known to those skilled in the art (see, e.g., Jalkanen et al, J. cell biol. 101: 976-305 (1985); Jalkanen et al, J. cell biol. 105: 3087-3096 (1987)). Other antibody-based methods for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and Radioimmunoassays (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase; radioisotopes such as iodine (A), (B), (C), (D), (C), (D¹³¹I，¹²⁵I，¹²¹I) Carbon (C)¹⁴C) Sulfur (S) (S)³⁵S) tritium (³H) Indium (I)¹¹⁵mIn，¹¹³mIn，¹¹²In，¹¹¹In), and technetium (⁹⁹Tc，⁹⁹mTc), thallium (²⁰¹Ti), gallium (⁶⁸G^a，67Ga), palladium (A)¹⁰³Pd), molybdenum (C)⁹⁹Mo), xenon (¹³⁸Xe), fluorine (¹⁸F)，¹⁵³Sm，¹⁷⁷Lu，¹⁵⁹Gd，¹⁴⁹Pm，¹⁴⁰La，¹⁷⁵Yb，¹⁶⁶Ho，⁹⁰Y，⁴⁷Sc，¹⁸⁶Re，¹⁸⁸Re，¹⁴²Pr，¹⁰⁵Rh，⁹⁷Ru; luminescent labels such as lumineol; and fluorescent labels such as fluorescein, and rhodamine, and biotin.

The antibodies of the invention can be labeled using methods known in the art. Such methods include, but are not limited to, the use of bifunctional conjugating agents (see, e.g., U.S. Pat. Nos. 5756065; 5714631; 5996239; 5652361; 5505931; 5489425; 5435990; 5428139; 5342604; 5274119; 4994560; and 5808003, all of which are incorporated by reference in their entirety).

One embodiment of the present invention is the detection and diagnosis of diseases or disorders associated with aberrant expression of the corresponding polypeptide in an animal, preferably a mammal, more preferably a human. In one embodiment, the diagnostic method comprises (a) administering to a subject being diagnosed an effective amount of a labeled molecule that specifically binds to the corresponding polypeptide, such as by parenteral, subcutaneous, or intraperitoneal administration; (b) waiting a period of time after administration to allow the labeled molecules to preferentially concentrate at the site of polypeptide expression (and clear unlabeled molecules); (c) determining a background level; and (d) detecting the marker molecule in the subject, whereby detection of the marker molecule above background levels indicates that the subject has a particular disease or disorder associated with aberrant expression of the corresponding polypeptide. The background level can be determined by various methods, including comparing the amount of marker molecules detected to a standard value previously determined in a particular system. As described herein, a specific embodiment of the invention is the use of the antibodies of the invention to quantitatively or qualitatively concentrate cells of a B cell line or cells of a monocyte cell line.

As also described herein, the antibodies of the invention are useful for treating, diagnosing or preventing an individual having an immunodeficiency, and in a specific embodiment, the antibodies of the invention are useful for treating, diagnosing and/or preventing an individual having CVID or a subtype thereof. In another embodiment, the antibodies of the invention are used to diagnose, prevent, treat, or treat a disease characterized by a deficiency in serum immunoglobulin production, recurrent infection, and/or immune system dysfunction.

Also as described herein, the antibodies of the invention are useful for treating, diagnosing or prognosing an individual with an autoimmune disease or disorder. In a specific embodiment, the antibodies of the invention are used to treat, diagnose and/or prognose individuals having systemic lupus erythematosus or a subtype of this disease. In another specific embodiment, the antibodies of the invention are used to treat, diagnose and/or prognose individuals having rheumatoid arthritis or a subtype thereof.

In the field ofThe size of the subject and the imaging system used will determine the amount of imaging component needed to generate a diagnostic image. In the case of radioisotope compositions, the amount of radioactivity injected is normally in the range of about 5-20 milliCuries, for human purposes⁹⁹mTc. The labeled antibody or antibody fragment then preferentially accumulates in cells containing the specific protein. In vivo tumor imaging is described in S.W.Burchiel et al, "immunopharmacology of radiolabeled antibodies and fragments thereof" (Chapter 13 in tumor imaging: radiochemical detection of cancer, edited by S.W.Burchiel and B.A.Rhocles, Masson Publishing Inc. (1982)).

Depending on a number of variables, including the type of label used and the mode of administration, the time interval after administration is 6-48 hours, or 6-24 hours or 6-12 hours, with a time interval that allows the labeled molecules to preferentially concentrate at the target site and the unbound labeled molecules to be cleared to background levels. In another embodiment, the time interval after administration is 5-20 days or 5-10 days.

In one embodiment, the disease or disorder is monitored by repeating the method of diagnosing the disease, e.g., after one month of initial diagnosis, after 6 months, after one year, etc.

The presence or absence of marker molecules in a patient can be detected by in vivo scanning methods known in the art. These methods depend on the type of label used. The skilled artisan will be able to determine the appropriate method of detecting a particular marker. Methods and apparatus that may be used in the diagnostic methods of the present invention include, but are not limited to, Computerized Tomography (CT), whole body scans such as Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI), and ultrasound.

In a specific embodiment, the molecule is labeled with a radioisotope and detected in the patient using radiation responsive surgical instrumentation (Thurston et al, U.S. Pat. No. 5441050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in the patient with a fluorescence response scanner, and in another embodiment, the molecule is labeled with a positron emitting metal and detected in the patient with positron emission tomography. In another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient using Magnetic Resonance Imaging (MRI).

Reagent kit

The present invention provides a kit that can be used in the above method. In one embodiment, the kit comprises one or more containers comprising an antibody of the invention, preferably a purified antibody, and in a specific embodiment, the kit comprises a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with an antibody of the kit. Preferably, the kit of the invention further comprises a control antibody that is not reactive with the corresponding polypeptide. In another specific embodiment, the kit of the invention comprises two or more antibodies (monoclonal and/or polyclonal) recognizing the same and/or different sequences or regions of the polypeptide of the invention. In another specific embodiment, the kit of the invention comprises means for detecting the binding of the antibody to the corresponding polypeptide (e.g., the antibody may be conjugated to a detectable substrate, such as a fluorescent, enzymatic, radioactive, or luminescent compound, or a second antibody that recognizes the first antibody may be conjugated to a detectable substrate).

In another specific embodiment of the invention, the kit is a diagnostic kit for screening sera containing antibodies specific against proliferative and/or cancer polynucleotides and polypeptides. Such kits may include a control antibody that is not reactive with the corresponding polypeptide. Such kits can include a substantially isolated polypeptide antigen comprising an epitope that is specifically immunoreactive with at least one antibody directed against the polypeptide antigen. In addition, such kits include means for detecting binding of the antibody to the antigen (e.g., the antibody can be conjugated to a fluorescent compound such as fluorescein or rhodamine, which can be detected by flow cytometry). In specific embodiments, the kit can include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigens of the kit can also be attached to a solid support.

In a more specific embodiment, the detection means of the kit described above comprises a solid support to which the polypeptide antigen is attached, and such a kit may also comprise an unattached receptor-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen is detected by binding of the receptor-labeled antibody.

In another embodiment, the invention includes a diagnostic kit for screening sera containing the polypeptide antigens of the invention. The diagnostic kit comprises a substantially isolated antibody specifically immunoreactive with a polynucleotide or polypeptide antigen, and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support, and in a specific embodiment, the antibody can be a monoclonal antibody. The detection means in the kit may comprise a second labelled monoclonal antibody. Alternatively, the assay is a competitive antigen that may include a label.

In one diagnostic configuration, the test serum is reacted with a solid phase reagent having surface-bound antigens obtained by the method of the invention. After binding of antibodies to a specific antigen to the reagent and removal of bound serum components by washing, the reagent is reacted with a receptor-labeled anti-human antibody, the ratio of receptor to reagent binding being the amount of anti-antigen antibody on the solid support. The reagent is washed again to remove unbound labeled antibody and to determine the number of receptors associated with the reagent. In particular, the receptor is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorescent, luminescent or colorimetric substrate (Sigma, st.

The solid surface reagents for the above assays are prepared by methods known in the art for attaching proteins to solid supports, such as polymer beads, dipsticks, 96-well plates or filters. These attachment methods typically involve non-specific adsorption of the protein to a support, or covalent attachment of the protein to a chemically reactive gene on a solid support via a free amino group, such as an active carboxyl, hydroxyl or aldehyde group. Alternatively, streptavidin-coated plates can be used in conjugation with biotinylated antigens.

Accordingly, the present invention provides an assay system or kit for performing such a diagnostic method. Such kits generally comprise a support having a surface bound recombinant antigen, and a receptor-labeled anti-human antibody to detect the surface bound anti-antigen antibody.

The invention also relates to antibodies which are agonists or antagonists of the polypeptides of the invention. For example, the invention includes antibodies that partially or fully disrupt receptor/ligand interaction with the polypeptides of the invention, including both receptor-specific antibodies and ligand-specific antibodies. Wherein the receptor specific antibody does not prevent ligand binding but prevents receptor activation. Receptor activation (i.e., signaling) can be determined by methods described herein or known in the art. Receptor-specific antibodies that prevent both ligand binding and receptor activation are also included. Also included are neutral antibodies that bind to the ligand and prevent the ligand from binding to the receptor, and antibodies that bind to the ligand and thereby prevent activation of the receptor, but do not prevent the ligand from binding to the receptor, and antibodies that activate the receptor. These antibodies can act as agonists affecting all or part of the biological activity through ligand-mediated receptor activation. The antibodies may be specific as agonists or antagonists of biological activity comprising specific activity as described herein. Also included are antibodies that bind Neutrokine-alpha and/or Neutrokine-alpha SV, whether or not Neutrokine-alpha or Neutrokine-alpha SV binds to the Neutrokine-alpha receptor. These antibodies act as agonists of Neutrokine-alpha and/or Neutrokine-alpha SV and, in the presence of these antibodies, enhance cell proliferation in response to binding of Neutrokine-alpha and/or Neutrokine-alpha SV to the Neutrokine-alpha receptor. The above antibody agonists can be produced by methods known in the art. See, e.g., WO 96/40281; us patent 5811097; deng, b. isoblood 92 (6): 1981-1988 (1998); chen, z, et al, cancer research 58 (16): 3668-3678 (1998); harrop, j.a. et al, journal of immunology 161 (4): 1786-1794 (1998); zhu, z, et al, cancer study 58 (15): 3209 and 3214 (1998); yoon, d.y., et al, journal of immunology 160 (7): 3170-3179 (1998); prat, m, et al, journal of cytoscience 111(Pt 2): 237- & lt247 & gt (1998); ptard, V. et al, journal of immunological methods 205 (2): 177-190 (1997); laudard, j, et al, cytokine 9 (4): 233-241 (1997); carlson, n.g., et al, journal of biochemistry 272 (17): 11295-11301 (1997); taryman, r.e., et al, neuron 14 (4): 755-; muller, y.a., et al, structure 6 (9): 1153-1167 (1998); bartunek, p, et al, cytokine 8 (1): 14-20(1996) (which is incorporated by reference in its entirety).

At least 14 monoclonal antibodies against Neutrokine-alpha have been generated, which are designated 12D6, 2E5, 9B6, 1B8, 5F4, 9a5, 10G12, 11G12, 16B4, 3D4, 16C9, 13D5, 15C10, and 12C 5. Preliminary analysis of these antibodies showed that Neutrokine-alpha was bound both in Western blot analysis and when Neutrokine-alpha protein was bound to ELISA plates. However, further analysis of the 12D6, 2E5, 9B6, 1B8, 5F4, 9a5, 10G12, 11G12, and 16B4 antibodies revealed that only the 12D6, 9B6, 2E5, 10G12, 9a5, and 11G12 antibodies bound to the membrane bound form of Neutrokine- α. Thus, it has been determined that a subset of the monoclonal antibodies generated against Neutrokine-alpha bind only to the membrane bound form of Neutrokine-alpha (i.e., this subset does not bind to the soluble form of Neutrokine-alpha corresponding to amino acid 134-285 of SEQ ID NO: 2), which, as described herein, is restricted to expression on monocytes and dendritic cells.

It has been found that the 9B6 antibody specifically binds to the membrane-bound form of Neutrokine-alpha, but does not bind to the soluble form of Neutrokine-alpha.

The epitope map of antibody 9B6 indicates that this antibody specifically binds to the epitope contained in SEQ ID NO: 2 Ser171-Phe194 amino acid residues. More particularly, the epitope map indicates that antibody 9B6 specifically binds to a polypeptide comprising SEQ ID NO: 2 Lys 173-Lys188 amino acid residues.

In contrast, antibodies 16C9 and 15C10 have been found to bind to the soluble form of Neutrokine-alpha (i.e., amino acids 134-285 of SEQ ID NO: 2) and inhibit Neutrokine-alpha mediated B cell proliferation. See, e.g., example 10, where it has been found that the 15C10 antibody inhibits the binding of Neutrokine-alpha to its receptor. The epitope map of antibody 15C10 has shown that this antibody specifically binds to the epitope contained in SEQ ID NO: 2 Glu223-Tyr246 amino acid residue. More particularly, the epitope map indicates that antibody 15C10 specifically binds to a polypeptide comprising SEQ ID NO: 2 Val227-Asn 242. Antibody 15C10 also specifically binds to a polypeptide comprising SEQ ID NO: 2 Phe 230-Cys 245.

As described above, monoclonal antibodies against Neutrokine-alpha have now been prepared. Hybridomas producing the 9B6 and 15C10 antibodies were deposited with the ATCC as PTA-1158 and PTA-1159, respectively. In one embodiment, the antibody of the invention has one or more of the same biological properties as the antibody secreted by the hybridoma cell line having accession number PTA-1158 or PTA-1159. "biological property" refers to an in vitro or in vivo activity or property of an antibody, such as the ability to bind Neutrokine-alpha (e.g., the polypeptide of SEQ ID NO: 2, mature form of Neutrokine-alpha, membrane bound form of Neutrokine-alpha, soluble form of Neutrokine-alpha (amino acid 134-285 of SEQ ID NO: 2), and antigenic and/or epitope regions of Neutrokine-alpha), the ability to substantially block binding of Neutrokine-alpha to its receptor, or the ability to block Neutrokine-alpha mediated biological activity (e.g., stimulation of B cell proliferation and immune protein production). Optionally, the antibody of the invention binds to the same epitope as the at least one antibody specified. Such epitope binding can be routinely determined using methods known in the art.

Thus, in one embodiment, the invention provides antibodies that specifically bind to a membrane-bound form and do not bind to the soluble form of Neutrokine-alpha. The use of these antibodies includes, but is not limited to, as diagnostic probes, identifying and/or isolating monocyte cell lines expressing the membrane-bound form of Neutrokine-alpha. For example, on activated monocytes, the membrane-bound form of Neutrokine-alpha is expressed in elevated amounts, and thus, the antibodies encompassed by the present invention can be used to detect and/or determine the level of activated monocytes. In addition, antibodies that bind only the membrane-bound form of Neutrokine-alpha can be used to target the toxin to other cells (e.g., monocytes and dendritic cells) that are tumorigenic, pre-tumorigenic, and/or express the membrane-bound form of Neutrokine-alpha.

In another embodiment, the antibody of the invention specifically binds only soluble form of Neutrokine-alpha (amino acids 134-285 of SEQ ID NO: 2). Uses of these antibodies include, but are not limited to, use as diagnostic probes to analyze soluble forms of Neutrokine-alpha in biological samples, and as therapeutic agents to target toxins to cells expressing Neutrokine-alpha receptors (e.g., B cells), and/or to reduce or block Neutrokine-alpha mediated biological activities (e.g., stimulation of B cell proliferation and/or immunoglobulin production) in vitro or in vivo.

The invention also provides antibodies that specifically bind to membrane-bound and soluble forms of Neutrokine-alpha.

As noted above, the present invention encompasses antibodies that inhibit or reduce the ability of Neutrokine- α and/or Neutrokine- α SV to bind to its receptor in vivo and/or in vitro, in one specific embodiment, the antibodies of the present invention inhibit or reduce the ability of Neutrokine- α and/or Neutrokine- α SV to bind to its receptor in vitro, in another specific embodiment, the antibodies of the present invention inhibit or reduce the ability of Neutrokine- α and/or Neutrokine- α SV to bind to its receptor in vivo. Such inhibition can be assayed by methods described herein or known in the art.

The invention also encompasses antibodies that specifically bind Neutrokine-alpha and/or Neutrokine-alpha SV, but do not inhibit the ability of Neutrokine-alpha and/or Neutrokine-alpha SV to bind its receptor in vitro and/or in vivo. In a specific embodiment, the antibodies of the invention do not inhibit or reduce the ability of Neutrokine-alpha and/or Neutrokine-alpha SV to bind its receptor in vitro. In another specific embodiment, the antibodies of the invention do not inhibit or reduce the ability of Neutrokine-alpha and/or Neutrokine-alpha SV to bind its receptor in vivo.

As noted above, the present invention encompasses antibodies that inhibit or reduce Neutrokine-alpha and/or Neutrokine-alpha SV mediated biological activity in vitro and/or in vivo. In a specific embodiment, the antibodies of the invention inhibit or reduce Neutrokine-alpha and/or Neutrokine-alpha SV mediated B-cell proliferation in vitro. Such inhibition can be assayed by conventional methods of modifying B cell proliferation described herein or known in the art. In another specific embodiment, the antibodies of the invention inhibit or reduce B cell proliferation mediated by Neutrokine-alpha and/or Neutrokine-alpha SV in vivo. In a specific embodiment, the antibody of the invention is 15C10, or a humanized form thereof. In another preferred specific embodiment, the antibody is 16C9, or a humanized form thereof. Thus, in specific embodiments of the invention, the 16C9 and/or 15C10 antibodies, or humanized forms thereof, are used to bind Neutrokine-alpha and/or Neutrokine-alpha SV in soluble form, and/or agonists and/or antagonists thereof, thereby inhibiting (partially or fully) B cell proliferation.

Alternatively, antibodies that specifically bind Neutrokine-alpha and/or Neutrokine-alpha SV but do not inhibit or reduce Neutrokine-alpha and/or Neutrokine-alpha SV mediated biological activities in vitro and/or in vivo (e.g., stimulation of B-cell proliferation) are also encompassed by the invention. In a specific embodiment, the antibodies of the invention do not inhibit or reduce Neutrokine-alpha and/or Neutrokine-alpha SV mediated in vitro biological activity. In another specific embodiment, the antibodies of the invention do not inhibit or reduce Neutrokine-alpha and/or Neutrokine-alpha SV mediated in vivo biological activity. In a specific embodiment, the antibody of the invention is 9B6, or a humanized form thereof.

As mentioned above, the present invention encompasses antibodies that specifically bind to the same epitope as the at least one specified antibody in vitro and/or in vivo.

In a specific embodiment, the antibody of the invention specifically binds to a polypeptide comprised in SEQ ID NO: 2 Ser171-Phe 194. In another specific embodiment, the antibody of the invention specifically binds to a polypeptide comprised in seq id NO: 2 Ser171-Phe 194. In another specific embodiment, the antibody of the invention specifically binds to a polypeptide comprised within SEQ id no: 2 Lys173-Lys188 amino acid residues. In another specific embodiment, the antibody of the invention specifically binds to a polypeptide contained in SEQ id no: 2 Lys173-Lys188 amino acid residues.

In another specific embodiment, the antibody of the invention specifically binds to a polypeptide comprised in SEQ ID NO: 2 Glu223-Tyr 246 amino acid residue. In another specific embodiment, the antibody of the invention specifically binds to a polypeptide comprised in seq id NO: 2, Glu223-Tyr 246 amino acid residue. In another specific embodiment, the antibody of the invention specifically binds to a polypeptide comprised within SEQ id no: 2, Val227-Asn 242. In another specific embodiment, the antibody of the invention specifically binds to a polypeptide comprised in SEQ ID NO: 2 Val227-Asn 242. In another specific embodiment, the antibody of the invention specifically binds to a polypeptide comprised in SEQ ID NO: 2 Phe230-Cys245 amino acid group. In another specific embodiment, the antibody of the invention specifically binds to a polypeptide contained in SEQ id no: 2 Phe230-Cys245 amino acid residue.

The present invention also provides methods for competitively inhibiting the binding of the 9B6 monoclonal antibody produced by deposited PTA-1159 to a polypeptide of the present invention, preferably SEQ ID NO: 2, more preferably a polypeptide having seq id NO: 2, Ser171-Phe 194. Competitive inhibition can be determined by any method known in the art, for example, using the competitive binding assays described herein. In a preferred embodiment, the antibody competitively inhibits the interaction of the 9B6 monoclonal antibody with SEQ ID NO: 2, preferably having the amino acid sequence of SEQ ID NO: 2, at least 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50% of the polypeptide binding of the amino acid sequence Ser171-Phe 194.

The present invention also provides a 15C10 monoclonal antibody that competitively inhibits production by the deposited PTA-1158 hybridoma, which binds to a polypeptide of the present invention, preferably SEQ ID NO: 2, more preferably a polypeptide having the amino acid sequence of SEQ ID NO: 2, Glu223-Try246 amino acid sequence. In a preferred embodiment, this antibody competes with the inhibitory 15C10 monoclonal antibody for binding to SEQ ID NO: 2, preferably having the amino acid sequence of SEQ ID NO: 2, at least 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50% of the polypeptide binding of the amino acid sequence in position Glu223-Tyr 246.

Additional embodiments of the invention relate to the 9B6 antibody and hybridoma cell lines expressing the antibody. The hybridoma cell line expressing the 9B6 antibody was deposited with the ATCC as accession number PTA-1159 on 7/1/2000. In a preferred embodiment, antibody 9B6 is humanized.

Further embodiments of the invention relate to the 15C10 antibody and hybridoma cell lines expressing the antibody. The hybridoma cell line expressing antibody 15C10 was deposited with the ATCC at 2000 on 7/1 and accession number PTA-1158. In a preferred embodiment, antibody 15C10 is humanized.

In a specific embodiment, the specific antibodies are humanized using methods described herein or known in the art and then used as therapeutic agents.

In another specific embodiment, any of the above antibodies is used in soluble form.

In another specific embodiment, any of the above antibodies is conjugated to a toxin or label (as described above). Such conjugated antibodies are used to kill, or quantify, specific cell populations. In a preferred embodiment, such conjugated antibodies are used to kill B cells that express Neutrokine-alpha receptors on their surface. In another preferred embodiment, such conjugated antibodies are used to quantify B cells expressing Neutrokine-alpha receptors on their surface.

In another specific embodiment, any of the above antibodies is conjugated to a toxin or label (as described above). Such conjugated antibodies are used to kill or quantify specific cell populations. In a preferred embodiment, such conjugated antibodies are used to kill monocytes expressing a membrane-bound form of Neutrokine-alpha. In another preferred embodiment, such conjugated antibodies are used to quantify monocytes expressing a membrane-bound form of Neutrokine-alpha.

The antibodies of the invention also have therapeutic and/or prophylactic uses, including but not limited to, activating monocytes or blocking monocyte activation and/or killing of monocyte cell lines that express membrane-bound forms of Neutrokine-alpha on their surface (e.g., to treat, prevent and/or diagnose myeloid leukemia, monocyte-based leukemias and lymphomas, mononucleosis, rheumatoid arthritis, and other disorders or pathological conditions associated with activated monocytes). In a specific embodiment, the antibodies of the invention fix complement. In other specific embodiments, the antibodies (or fragments thereof) of the invention are conjugated to a heterologous polypeptide or nucleic acid (e.g., toxins such as compounds that bind to and activate endogenous cytotoxic effector systems, and radioisotopes; and cytotoxic drugs).

In another embodiment, one or more monoclonal antibodies are produced that recognize or bind Neutrokine-alpha and/or its muteins, but do not recognize or bind Neutrokine-alpha SV and/or its muteins. In a related embodiment, one or more monoclonal antibodies are generated, wherein they recognize or bind Neutrokine-alpha and/or a mutein thereof, but do not recognize or bind Neutrokine-alpha and/or a mutein thereof.

As described above, antibodies to Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention may be used to generate anti-idiotypic antibodies that "mimic" Neutrokine-alpha using methods well known to those of skill in the art (see, e.g., Greenspan & Bona, FASEB journal 7 (5): 437-444(1989), and Nissionff, J Immunol 147 (8): 2429-2438 (1991.) such as, for example, antibodies that bind Neutrokine-alpha and/or Neutrokine-alpha SV and competitively inhibit Neutrokine-alpha and/or Neutrokine-alpha SV and/or bind to ligands, antibodies that "mimic" Neutrokine-alpha TNF multimers and/or binding structures, thereby binding and neutralizing Neutrokine-alpha or Neutrokine-alpha and/or SV or binding fragments thereof, can be used in a therapeutic regimen to neutralize Neutrokine-alpha ligand. For example, such anti-idiotypic antibodies may be used to bind to Neutrokine-alpha and/or Neutrokine-alpha SV, or to Neutrokine-alpha and/or Neutrokine-alpha SV receptors on the surface of B cells, thereby blocking Neutrokine-alpha and/or Neutrokine-alpha SV mediated B cell activation, proliferation and/or differentiation.

Diagnosis of immune system related diseases

Neutrokine-alpha is expressed in kidney, lung, peripheral blood leukocytes, bone marrow, T-cell lymphomas, B-cell lymphomas, activated T-cells, gastric cancer, smooth muscle, macrophages, and cord blood tissues and specific cells of the monocyte lineage. In addition, Neutrokine- α SV was expressed in primary dendritic cells. In addition, Neutrokine- α is expressed on the cell surface of the following non-hematopoietic tumor cell lines: colon cancer HCT 116(ATCC accession No. NO. CCL-247) and HT-29(ATCC accession No. NO. HTB-38); colon adenocarcinoma Caco-2(ATCC accession No. HTB-37), COLO 201(ATCC accession No. CCL-224) and WiDr (ATCC accession No. CCL-218); breast cancer MDA-MB-231(ATCC accession No. HTB-26); bladder squamous carcinoma SCaBER (ATCC accession No. NO. HTB-3); bladder cancer HT-1197(ATCC accession No. CRL-1473); renal cancer A-498(ATCC accession No. HTB-44); caki-1(ATCC accession No. NO. HTB-46), and Caki-2(ATCC accession No. HTG-47); kidney Wilms tumor SK-NEP-1(ATCC accession No. htb-48); and pancreatic cancer Hs766T (ATCC accession No. HTB-134); MIA PaCa-2(ATCC accession No. CRL-1420) and SU.86.86(ATCC accession No. CRL-1837). For many immune system related diseases, changes (increases or decreases) in the expression level of Neutrokine-alpha and/or Neutrokine-alpha SV genes as compared to a standard level may be detected in immune system tissue or other cells or body fluids (e.g., serum, plasma, urine, synovial fluid or cerebrospinal fluid) taken from individuals with such a disease, and "standard" Neutrokine-alpha and/or Neutrokine-alpha SV gene expression levels are expression levels in immune system tissue or body fluids taken from individuals without an immune system disease. Accordingly, the present invention provides a diagnostic method for use during diagnosis of an immune system disorder, comprising determining the level of expression of a gene encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide in immune system tissue or other cells or body fluids taken from an individual, and comparing the level of expression of the gene with a standard Neutrokine-alpha and/or Neutrokine-alpha SV gene level, whereby an increase or decrease in the level of expression of the gene is indicative of an immune system disorder or normal activation, proliferation, differentiation and/or death.

In particular, it is believed that the expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides and the encoded Neutrokine-alpha and/or Neutrokine-alpha SV and mRNA are significantly increased or decreased in certain tissues of mammals with cancer as compared to "standard" levels. In addition, it is believed that increased or decreased expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides in some bodily fluids (e.g., serum, plasma, urine, and cerebrospinal fluid) or cells or tissues of such a mammal with cancer may be detected as compared to serum from the same species without cancer.

For example, Neutrokine-alpha is highly expressed in cells of the monocyte cell line as described herein. Thus, the polynucleotides of the invention (e.g., polynucleotide sequences complementary to all or a portion of Neutrokine- α mRNA and/or Neutrokine- α SV mRNA) and antibodies (and antibody fragments) directed against the polypeptides of the invention may be used to quantify or characterize the concentration of cells of a monocytic cell line expressing Neutrokine- α on their surface (e.g., monocytic leukemia cells). These antibodies are additionally of diagnostic use in detecting abnormal expression levels of the Neutrokine-alpha gene, or abnormalities in the structure and/or timing, tissue, cellular or subcellular localization of Neutrokine-alpha and/or Neutrokine-alpha SV. These diagnostic assays may be performed in vivo or in vitro, for example in blood samples, biopsies or autopsies.

In addition, as described herein, the Neutrokine-alpha receptor is initially expressed on cells of the B cell line. Accordingly, the Neutrokine-alpha polypeptides of the invention (including labeled Neutrokine-alpha polypeptides and Neutrokine-alpha fusion proteins), and anti-Neutrokine-alpha antibodies (including fragments of anti-Neutrokine-alpha antibodies) against the polypeptides of the invention, may be used to quantify or characterize the concentration of B cell line cells expressing Neutrokine-alpha receptors on their surface (e.g., B cell-associated leukemia or lymphoma cells). These Neutrokine-alpha polypeptides and antibodies are additionally of diagnostic use in detecting abnormal levels of Neutrokine-alpha receptor gene expression, or abnormalities in the structure and/or timing, tissue, cellular or subcellular localization of Neutrokine-alpha receptor, and/or in diagnosing activity/deficiencies in signaling pathways associated with Neutrokine-alpha. Such diagnostic assays may be performed in vivo or in vitro, for example, on blood samples or biopsies using methods described herein or known in the art.

In one embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists or antagonists of Neutrokine-alpha and/or Neutrokine-alpha SV (e.g., anti-Neutrokine-alpha and/or anti-Neutrokine-alpha SV antibodies), of the invention are used to treat, prevent, diagnose or predict individuals having an immune deficiency.

Immunodeficiency which may be treated, prevented, diagnosed and/or predicted by Neutrokine-alpha and/or Neutrokine-alpha polynucleotides or polypeptides, or Neutrokine-alpha and/or Neutrokine-alpha SV agonists or antagonists (e.g., antibodies against Neutrokine-alpha and/or Neutrokine-alpha SV) of the present invention include, but are not limited to, one or more immunodeficiency selected from: x-linked Severe Combined Immunodeficiency (SCID), autosomal SCID; adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia, (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult-onset agammaglobulinemia, late-onset agammaglobulinemia, abnormal gamma globulinemia, low gamma globulinemia, agammaglobulinemia, Common Variable Immunodeficiency (CVID) (acquired), Wiskott-Aldrich syndrome (WAS), X-linked high IgM immunodeficiency, non-X-linked high IgM immunodeficiency, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), normal or elevated IgS antibody deficiency, thymoma immunodeficiency, Ig heavy chain deficiency, k chain deficiency, B-cell lymphoproliferative disease (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticulocyte dysplasia, neonatal neutropenia, severe congenic leukopenia, immunodeficiency thymic lymphodysplasia-hypoplasia or dysplasia, ataxia-telangiectasia, short limb dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome combined with IgS immunodeficiency, purine nucleotide phosphatase deficiency (PNP), MHC class II deficiency (barrel lymphocyto syndrome), and severe combined immunodeficiency.

According to this embodiment, the level of Neutrokine-alpha and/or Neutrokine-alpha SV is abnormally low in individuals with immunodeficiency compared to individuals without immunodeficiency. Any of the methods described herein or known in the art may be used to detect the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention (e.g., FACS analysis or ELISA detection and hybridization or PCR detection), and to determine the expression pattern of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention in a biological sample.

Biological samples from persons with immunodeficiency are characterized by low expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV, as compared to the results observed in individuals without immunodeficiency. Accordingly, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention, and/or agonists or antagonists thereof, may be used for the diagnosis and/or prognosis of immunodeficiency according to the methods of the invention. For example, biological samples ("targets") obtained from a human suspected of having an immunodeficiency may be analyzed for the relative expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention. The expression level of one or more of these molecules of the invention is then compared to the expression level of the same molecule in a human known to be free of immunodeficiency. The expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention, and/or agonists and/or antagonists thereof, differ significantly between samples obtained from the target and the control, suggesting that the target is suffering from an immunodeficiency.

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention, or agonists or antagonists thereof (such as antibodies against Neutrokine-alpha and/or Neutrokine-alpha SV), are useful in treating, diagnosing and/or prognosing individuals with common variable immunodeficiency disorders, (CVID, also known as acquired agammaglobulinemia, and acquired hypogammaglobulinemia), or a subset thereof. According to this embodiment, individuals of a patient's CVID or a subset thereof exhibit abnormal levels of expression of Neutrokine-alpha and/or Neutrokine-alpha SV receptors on their B cells and/or monocytes when compared to individuals without CVID. Any of the methods described herein and known in the art may be used to detect a Neutrokine-alpha polynucleotide or polypeptide of the invention and/or its receptor polypeptide (e.g., FACS analysis or ELISA to detect the polypeptide, and hybridization or PCR methods to detect the polynucleotide), and to determine the expression pattern of a Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide of the invention and its receptor polypeptide, as distinct from that in a sample containing at least monocytes or some fraction thereof (e.g., RNA) and in a sample containing at least B cells or a fraction thereof (e.g., RNA). In determining that Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide expression is enhanced in a sample containing at least monocytes or some component thereof (e.g., RNA), and in determining that Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide expression is at a level below normal in a sample containing at least B cells or some component thereof (e.g., RNA), such a sample may be indicative of CVID (i.e., acquired agammaglobulinemia, or acquired hypogammaglobulinemia).

Persons with CVID are characterized by high levels of Neutrokine-alpha and its receptor (NAR) expression in peripheral or circulating blood B cells when compared to results observed in persons without CVID. In contrast, persons who do not suffer from CVID are characterized by low levels of Neutrokine-alpha expression and high levels of NAR expression in peripheral or circulating blood B cells. Thus, the Neutrokine-alpha, Neutrokine-alpha SV polypeptides and/or NAR polypeptides, polynucleotides, and/or agonists or antagonists thereof of the present invention may be used in accordance with the methods of the present invention in various diagnoses of this CVID subtype, e.g., in analyzing peripheral blood B cell samples obtained from a human (target) suspected of having CVID for the relevant expression levels of the Neutrokine-alpha, Neutrokine-alpha SV and/or NAR polynucleotides and/or polypeptides of the present invention. The expression level of one or more of these molecules of the invention is compared to the expression level of the same molecule in a human (control) known to be free of CVID. Significant differences in the expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention, and/or NAR polypeptides, between target and control samples, suggest that the target is afflicted with a CVID subtype of disease.

In a specific embodiment, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, or agonists or antagonists thereof (e.g., anti-Neutrokine-alpha and/or anti-Neutrokine-alpha SV antibodies), are used to diagnose, prognose, treat or prevent conditions characterized by serum immunoglobulin production deficiencies, recurrent infections and/or immune system dysfunction. In addition, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists or antagonists thereof (e.g., antibodies against Neutrokine-alpha and/or Neutrokine-alpha SV), are useful in diagnosing, prognosing, treating or preventing infection of joints, bones, skin and/or parotid, blood infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., as described herein), inflammation, and malignancies and/or any disease or disorder or pathological condition associated with such diseases, infections, disorders and/or malignancies, including but not limited to CVID, other primary immunodeficiency, HIV disease, CLL, recurrent bronchitis, fistulas, otitis media, connective tissue inflammation, pneumonia, hepatitis, meningitis, shingles (e.g. severe shingles) and/or pneumocystis scroni.

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention or agonists or antagonists thereof (e.g., anti-Neutrokine-alpha and/or Neutrokine-alpha SV antibodies) are used to treat, diagnose or prognose individuals having autoimmune diseases.

Autoimmune diseases that may be treated, diagnosed or predicted using Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention, or agonists or antagonists thereof (e.g., antibodies against Neutrokine-alpha and/or Neutrokine-alpha SV), include, but are not limited to, one or more of the following: autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmune cytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, recurrent polychondritis, rheumatic heart disease, glomerulonephritis (such as IgA nephropathy), multiple sclerosis, neuritis, uveitis, multiple endocrine diseases, purpura (such as Henloch-Scoenlein purpura), Reiter's disease, Stiff-Man syndrome, autoimmune pneumonia, Guillain-Pane syndrome, insulin-dependent diabetes mellitus, and autoimmune ophthalmia, autoimmune thyroiditis, hypothyroidism (i.e. Hashimoto's thyroiditis), systemic lupus erythematosus, Gopasture's syndrome, pemphigus, receptor autoimmunity such as (a) Graves disease, (b) myastenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigus, Sjogren's syndrome, diabetes, and adrenergic drug resistance (including adrenergic drug resistance for the treatment of asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland disorders, vitiligo, vasculitis, post MI, myocardial syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathy, and other inflammatory, grandilagomatous, degenerative, and atrophic dysfunction).

According to this embodiment, an individual having an autoimmune disease or disorder exhibiting an abnormally high level of expression of Neutrokine-alpha, Neutrokine-alpha SV and/or NAR as compared to an individual without an autoimmune disease or disorder, any of the methods described herein and known in the art can be used to detect Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention, and/or NAR polypeptides (e.g., FACS analysis or ELISA to detect polypeptides, and hybridization or PCR to detect polynucleotides), and can be used to determine the expression pattern of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention, and/or NAR polypeptides in a biological sample.

A human biological sample having an autoimmune disease is characterized by a high level of expression of Neutrokine-alpha, Neutrokine-alpha SV and/or NAR when compared to the results observed in individuals without the autoimmune disease or dysfunction. Accordingly, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention, and/or agonists or antagonists thereof, may be used in accordance with the methods of the invention for the diagnosis and/or prognosis of autoimmune diseases or dysfunctions. For example, a biological sample from a human (target) suspected of having an autoimmune disease or disorder may be analyzed for the associated expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides and/or NAR polypeptides. The expression level of one or more of these molecules of the invention is then compared to the expression level of the same molecule in a human known to be free of systemic disease. Significant differences in the expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention, and/or agonists and/or antagonists thereof, and/or NAR polypeptides, between samples obtained from the target and control indicate that the target has an autoimmune disease or disorder.

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention, or agonists or antagonists thereof (such as anti-Neutrokine-alpha and/or anti-NEUTROKINE-ASV antibodies), are used for the treatment, diagnosis or prognosis of individuals having systemic lupus erythematosus or a subtype thereof. According to this embodiment, an individual having systemic lupus erythematosus or a subtype thereof has an abnormally high level of Neutrokine- α and/or Neutrokine- α SV expression when compared to an individual without systemic lupus erythematosus. Any of the methods described herein and known in the art may be used to detect a Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide of the invention (e.g., FACS analysis or ELISA for detecting polypeptides, and hybridization or PCR methods for detecting polynucleotides), and to determine the expression pattern of a Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide and/or polypeptide of the invention in a biological sample.

A human biological sample having systemic lupus erythematosus is characterized by a high level of expression of Neutrokine-alpha and/or Neutrokine-alpha SV when compared to that observed in an individual having no systemic lupus erythematosus. Accordingly, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention, and/or agonists or antagonists thereof, may be used to diagnose and/or predict systemic lupus erythematosus or a subtype thereof, recurrence according to the invention. For example, biological samples obtained from patients (targets) suspected of having systemic lupus erythematosus are analyzed for the relative expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention. The expression level of one or more of these molecules of the invention is then compared to the expression level of the same molecule in a human known to have no systemic lupus erythematosus. A significant difference in the expression levels of the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention, and/or agonists and/or antagonists thereof, between samples obtained from the target and control indicates that the target has systemic lupus erythematosus or a subtype thereof.

In addition, there is a direct relationship between the degree of disease in systemic lupus erythematosus or a subtype thereof and the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides (RNAs) and/or polypeptides of the invention. Accordingly, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides (RNAs), polypeptides and/or agonists or antagonists of the invention, in accordance with the methods of the invention, may be used to predict the extent of systemic lupus erythematosus or a subtype thereof. For example, biological samples obtained from patients (subjects) suspected of having systemic lupus erythematosus may be analyzed for the relative expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention. The expression level of one or more of these molecules of the invention is then compared to the expression level of the same molecule in a group of diseases known to represent varying degrees of the disease. According to this method, which member of the group of patients the expression level corresponds to indicates the degree of having that member.

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention or agonists or antagonists thereof (such as antibodies against Neutrokine-alpha and/or Neutrokine-alpha SV) are used for the treatment, diagnosis or prognosis of individuals suffering from rheumatoid arthritis or a subtype thereof. According to this embodiment, the expression level of Neutrokine-alpha and/or Neutrokine-alpha SV is abnormally high in individuals suffering from rheumatoid arthritis or a subtype thereof as compared to individuals without rheumatoid arthritis or a subtype thereof. Any of the methods described herein and known in the art may be used to detect a Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide of the invention (e.g., FACS analysis or ELISA for detecting polypeptides, and hybridization or PCR methods for detecting polynucleotides), and may be used to determine the expression pattern of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention in a biological sample.

The biological sample of a patient suffering from rheumatoid arthritis is characterized by an increased level of expression of Neutrokine-alpha and/or Neutrokine-alpha SV when compared to the results observed in individuals without rheumatoid arthritis. Accordingly, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention, and/or agonists or antagonists thereof, may be used in accordance with the methods of the invention for the diagnosis and/or prognosis of rheumatoid arthritis or a subtype thereof. For example, biological samples from patients (subjects) suspected of having rheumatoid arthritis are analyzed for the relative expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention. The expression level of one or more of these molecules of the invention is then compared to the expression level of the same molecule in a human known to be free of rheumatoid arthritis. The expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides and/or polypeptides of the invention, and/or agonists and/or antagonists thereof, differ significantly between samples obtained from the target and control, suggesting that the target suffers from rheumatoid arthritis or a subtype thereof.

Accordingly, the present invention provides an effective diagnostic method in diagnosing immune system disorders, including cancers of the system, as well as immunodeficiency and/or autoimmune diseases, comprising determining the level of expression of a gene encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide, in the immune system tissue or other cells or body fluids of the individual, and comparing the level of expression with the level of expression of a standard Neutrokine-alpha and/or Neutrokine-alpha SV gene, whereby an increase or decrease compared to the standard level indicates the presence of an immune system disease.

Diagnosis of immune system disorders including, but not limited to, diagnosis of tumors, immunodeficiency, and/or autoimmune diseases has been performed according to conventional methods, and the present invention is used as a prognostic indicator, whereby patients exhibit enhanced or reduced expression levels of Neutrokine-alpha and/or Neutrokine-alpha SV genes, with more severe clinical outcomes than patients with gene expression levels approaching standard levels.

Analyzing or determining the expression level of a gene encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide refers to determining or estimating the expression level of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or mRNA encoding a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide, either directly or indirectly, in a first biological sample, determining or estimating the absolute protein level or mRNA level, indirectly, comparing the level of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or mRNA level in a second biological sample. Preferably, the level of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or mRNA in the first biological sample is determined or estimated and compared to the standard Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide level or mRNA level obtained from a second biological sample from a normal human, or by determining the average level in a normal human. One skilled in the art will recognize that once the standard Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide level or mRNA level is known, it can be reused as a control standard.

"biological sample" refers to any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source containing Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or mRNAs. As noted above, biological samples include body fluids (e.g., serum, plasma, urine, synovial fluid and cerebrospinal fluid) that contain free extracellular domains of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, immune system tissues, and other tissues found to express intact or free Neutrokine-alpha and/or Neutrokine-alpha SV or extracellular domains of its receptors. Methods for obtaining biopsied tissue and body fluids from mammals are well known in the art. When the biological sample comprises mRNA, biopsy tissue is a preferred source.

The compounds of the invention are useful in the diagnosis, prognosis or treatment of various immune system related diseases in mammals, preferably humans. Such diseases include, but are not limited to, tumors (e.g., B cell and monocytic leukemias and lymphomas) and tumor metastases, bacterial, viral and other parasitic infections, immunodeficiency, inflammation, lymphadenopathy, autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, mixed connective tissue disease, and inflammatory myopathy), and graft/host disease.

Total cellular RNA can be isolated from a biological sample by any suitable method, such as Chomczynski and Sacchi in biochemical analysis 162: 156-159(1987)) in the one-step guanidinium thiocyanate-phenol-chloroform method. The mRNA levels encoding Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides are then analyzed by any suitable method. These methods include Northern blot analysis, S1 nuclease mapping, Polymerase Chain Reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), and reverse transcription-ligase chain reaction (RT-LCR).

Antibody-based methods can be used to analyze the levels of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide in a biological sample. For example, expression of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides in tissues can be studied using conventional immunohistological methods (Jalkane, M., et al, J. cell biol. 101: 976-30985 (1985); Jalkane, M., et al, J. cell biol. 105: 3087-3096 (1987)). Other antibody-based methods for detecting Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) Radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase, and radioisotopes, such as iodine: (a), (b), and (c) ¹³¹I，¹²⁵I，¹²³I，¹²¹I) Carbon (C)¹⁴C) Sulfur (S) (S)³⁵S), tritium (³H) Indium (I)^115mIn，^113mIn，¹¹²mIn，¹¹¹mln, and technetium: (m;,)⁹⁹Tc，⁹⁹mTc), titanium (²⁰¹Ti), gallium (⁶⁸Ga，⁶⁷Ga), palladium (A)¹⁰³Pd), molybdenum (C)⁹⁹Mo) xenon (¹³³Xe), fluorine(¹⁸F)，¹⁵³Sm，¹⁷⁷Lu，¹⁵⁹Gd，¹⁴⁹Pm，¹⁴⁰La，¹⁷⁵Yb，¹⁶⁶Ho，⁹⁰Y，⁴⁷Sc，¹⁸⁸Re，¹⁴²Pr，¹⁰⁵Rh，⁹⁷Ru; luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodamine, and biotin.

Methods known in the art can be used to label the antibodies of the invention. Such methods include, but are not limited to, the use of bifunctional conjugating agents (see, e.g., U.S. Pat. Nos. 5756065; 5714631; 5696239; 5652361; 55055931; 5489425; 5435990; 5428139; 5342604; 5274119; 4994560; and 5808003; the entire disclosures of which are incorporated herein by reference).

The tissue or cell types analyzed generally include cells or tissues known or suspected to express the Neutrokine-alpha gene (e.g., monocyte cell line cells), or cells or tissues known or suspected to express the Neutrokine-alpha receptor gene (e.g., B cell line and spleen cells). The protein isolation methods used in the present invention are, for example, the methods described by Harlow and Lane (Harlow, E and Lane, D., 1988, "antibody laboratory Manual", Cold spring harbor laboratory Press, Cold spring harbor, N.Y.), which are incorporated by reference in their entirety. The isolated cells may be derived from a cell culture or a patient. Analysis of cells taken from culture is an essential step in determining whether the cells can be used as part of a cell-based gene therapy method, or to test the effect of the expression of the compound Neutrokine-alpha gene or Neutrokine-alpha receptor gene.

For example, the antibodies or fragments thereof described herein can be used to quantitatively or qualitatively detect the presence of Neutrokine-alpha gene products or conserved variants or peptide fragments thereof. This can be done, for example, by immunofluorescence methods, with detection of fluorescently labeled antibodies using optical microscopy, flow cytometry or fluorimetry.

The antibodies (or fragments thereof) or Neutrokine-alpha polypeptides of the invention may additionally be used in histological assays such as immunofluorescent assays, immunoelectron microscopy or non-immunological assays to detect Neutrokine-alpha gene products or conserved variants or peptide fragments thereof in situ, or Neutrokine-alpha binding to Neutrokine-alpha receptors. In situ detection may be accomplished by taking a histological sample from a patient, or administering a labeled antibody of the invention or a Neutrokine-alpha polypeptide thereto. The antibody (or fragment) or Neutrokine-alpha polypeptide is preferably administered by overlaying the labeled antibody (or fragment) on the biological sample. By using this method, not only can the Neutrokine-alpha gene product, or a conserved variant or peptide fragment, or Neutrokine-alpha polypeptide binding be determined, but its distribution in the test tissue can also be determined. Using the present invention, one skilled in the art will readily appreciate that many histological methods (e.g., staining methods) can be modified for in situ detection.

Immunological and non-immunological assays for the Neutrokine-alpha gene product or a conserved variant or peptide fragment thereof involve incubating a sample such as biological fluid, a tissue extract, freshly harvested cells, or cell lysates incubated in cell culture in the presence of a detectably labeled antibody capable of identifying the Neutrokine-alpha gene product or a conserved variant or peptide fragment thereof and detecting the bound antibody by a number of methods well known in the art.

Immunological and non-immunological assays for a Neutrokine-alpha receptor gene product or a conserved variant or peptide fragment thereof comprise incubating a sample such as a biological fluid, a tissue extract, freshly harvested cells or cell lysates incubated in cell culture in the presence of a detectable or labeled Neutrokine-alpha polypeptide capable of identifying the Neutrokine-alpha receptor gene product or a conserved variant or peptide fragment thereof and detecting the bound Neutrokine-alpha polypeptide by a number of methods well known in the art.

The biological sample may be contacted with and immobilized on a solid support or carrier, such as nitrocellulose or other solid support capable of immobilizing cells, cell particles, or soluble proteins. This support is then washed with an appropriate buffer and subsequently treated with a detectably labeled anti-Neutrokine-alpha antibody or a detectable Neutrokine-alpha polypeptide. The solid support is then washed once more with buffer to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labelled. The amount of label bound to the solid support can then be detected by conventional means.

"solid phase support or carrier" refers to any support capable of binding an antigen or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gavite, and magnetite. The nature of the carrier may be soluble in some circumstances or insoluble for the purposes of the present invention. The solid support may have virtually any possible configuration, so long as the conjugated molecule is capable of binding an antigen or antibody. Thus, the support configuration may be spherical, such as a bead, or cylindrical, such as on the inner surface of a test tube, or on the outer surface of a rod. Alternatively, the surface may be flat such as a sheet, test paper, or the like. Preferred supports include polystyrene beads. Many other suitable carriers for binding antibodies or antigens are known to those skilled in the art or can be determined using routine experimentation.

In addition to analyzing the level of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or polynucleotides in a biological sample obtained from an individual, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or polynucleotides may also be detected in vivo by imaging. For example, in one embodiment of the invention, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides and/or anti-Neutrokine-alpha antibodies are used to image B-cell lymphomas. In another embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides and/or anti-Neutrokine-alpha antibodies and/or Neutrokine-alpha polynucleotides of the present invention (e.g., polynucleotides complementary to all or a portion of Neutrokine-alpha and/or Neutrokine-alpha SV mRNA) are used to image lymphomas (e.g., monocytes and B-cell lymphomas).

Antibody labels or markers for in vivo imaging of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides include labels detectable by X-ray, NMR, MRI, CAT scan, or ESR. In the case of X-rays, suitable labels include radioisotopes such as barium or cesium which emit detectable radiation but are not significantly harmful to the test subject. Suitable labels for NMR and ESR include labels with detectably characteristic spins such as tritium, which can be incorporated into the antibody by labeling the nutrients of the relevant hybridoma. Human antibodies or "humanized" chimeric monoclonal antibodies are preferably used when imaging in vivo for detecting enhanced levels of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides for diagnosis of humans. Such antibodies can be produced as described herein or as known in the art. For example, methods of producing chimeric antibodies are known in the art. See, e.g., Morrison, science 229: 1202 (1985); oi et al, Biotechnology 4: 214 (1986); cabilly et al, U.S. Pat. No. 4,16567; taniguchi et al, EP 171496; morrison et al, EP 173494; neuberger et al, WO 8601533; robinson et al, WO 8702671; boulianne et al, nature 312: 643 (1984); neuberger et al, Nature 314: 268(1985).

Alternatively, any Neutrokine-alpha polypeptide whose presence can be detected can be administered. For example, a Neutrokine-alpha polypeptide labeled with a radiopaque or other suitable compound may be administered and the labeled antibody evaluated in vivo as described above. In addition, the Neutrokine-alpha polypeptide can be used for in vitro diagnostic methods.

Antibodies or antibody fragments specific for Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, which have been labeled with a suitable detectable imaging component, such as a radioisotope (e.g., a radioisotope) (e.g., by parenteral, subcutaneous, or intraperitoneal administration), are introduced into a mammal for the detection of immune system disorders¹³¹I，¹¹²In，⁹⁹mTc，(¹³¹I，¹²⁵I，¹²³I，¹²¹I) Carbon (C)¹⁴C) Sulfur (S) (S)³⁵S), tritium (³H) Indium (I)¹¹⁵mIn，¹¹³mIn，¹¹²In，¹¹¹In, and technetium (C) ((C))⁹⁹Tc，⁹⁹mTc), titanium (²⁰¹Ti), gallium (⁶⁸Ga，⁶⁷Ga), palladium (A)¹⁰³Pd), molybdenum (C)⁹⁹Mo) xenon (¹³³Xe), fluorine (¹⁸F)，¹⁵³Sm，¹⁷⁷Lu，¹⁵⁹Gd，¹⁴⁹Pm，¹⁴⁰La，¹⁷⁵Yb，¹⁶⁶Ho，⁹⁰Y，⁴⁷Sc，¹⁸⁶Re，¹⁸⁸Re，¹⁴²Pr，¹⁰⁵Rh，⁹⁷Ru), radiopaque substances, or substances detectable by nuclear magnetic resonance. One skilled in the art will appreciate that the size of the test subject and the imaging system used will determine the imaging composition from which the diagnostic image is generated. In the case of radioisotopes, the radioactive quantity injected is generally about 5-20 milliCuries, for the human being⁹⁹mTc. The labeled antibody and antibody fragment then preferentially accumulate at cells containing Neutrokine-alpha protein. In vivo tumor imaging is found in S.W.Burchiel et al, "immunopharmacology of radiolabeled antibodies and fragments thereof" (Chapter 13, tumor imaging: radiochemical detection of cancer, edited by S.W.Burchiel and B.A.Rhodes, Masson Publishing Inc. (1982)).

In the case of antibodies, one of the ways in which an anti-Neutrokine-alpha antibody can be detectably labeled is by linking it to an enzyme and using the linked product in an Enzyme Immunoassay (EIA) (Voller, a. "enzyme linked immunosorbent assay (ELISA)", 1978, diagnostic levels 2: 1-7, microbiologically related quarterworks, walker ville, MD); voller et al, journal of clinical pathology 31: 507-520 (1978); butter, j.e. enzymology method 73: 482-523 (1981); maggio, e, et al (editors) 1980, enzyme immunoassay, CRC press, Boca Raton, FL; ishikawa, E.et al (eds.), 1981, enzyme immunoassay, Kgaku Shoin, Tokyo). The enzyme which is bound to the antibody structure will react with a suitable substrate, preferably a chromogenic substrate, in such a way as to produce a chemical component which can be detected, for example, spectrophotometrically, fluorimetrically or visually. Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. In addition, detection can be performed by a colorimetric method using a chromogenic substrate that reacts with an enzyme. The reaction of the substrate with the enzyme can also be detected visually by comparing it with a similarly prepared standard.

Any other immunoassay may also be used for detection. Neutrokine-alpha can be detected, for example, by Radioimmunoassay (RIA) by radiolabelling of The antibody or antibody fragment (see, e.g., Weintraub, B., radioimmunoassay principles, 7 th tracking Corse for radioligand assay, The Endocrine Society, March, 1986, incorporated herein by reference in its entirety). Radioisotopes can be detected by methods including, but not limited to, gamma counter, scintillation counter, or autoradiography.

Antibodies can also be labeled with fluorescent compounds. When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected due to fluorescein. Commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.

The antibody may also be a fluorescent emitting metal such as¹⁵²Eu, or other lanthanide metals. These metals can be attached to the antibody with a chelating group for such a metal, such as diethylenetriamine pentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

The antibody may also be detectably labeled by coupling it to a chemiluminescent compound. The chemiluminescent-labeled antibody is then identified by detecting the occurrence of luminescence during the chemical reaction. Particularly useful chemiluminescent labeling compounds are, for example, luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate salt.

In addition, bioluminescent compounds can be used to label the antibodies of the invention. Bioluminescence is a chemiluminescence found in biological systems in which a catalytic protein increases the efficacy of the chemiluminescent reaction. The presence of the bioluminescent protein can be detected by detecting the presence of the luminophore. Important bioluminescent compounds for labeling include, but are not limited to, luciferin, luciferase, and aequorin.

Treatment of immune system related diseases

As described above, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides and polynucleotides and anti-Neutrokine-alpha antibodies are useful for diagnosing diseases with abnormally high or low expression of Neutrokine-alpha and/or Neutrokine-alpha SV activity. Cells and tissues are provided in which Neutrokine-alpha and/or Neutrokine-alpha SV is expressed and its activity is modulated by Neutrokine-alpha and/or Neutrokine-alpha SV, the level of expression of Neutrokine-alpha and/or Neutrokine-alpha SV in an individual is altered (increased or decreased) compared to a standard or "normal" level, producing a pathological change associated with an organic system in which Neutrokine-alpha and/or Neutrokine-alpha SV is expressed and/or active.

It will also be appreciated by those skilled in the art that since the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the present invention are members of the TNF family, the extracellular domain of a representative protein may be released in soluble form from cells expressing Neutrokine-alpha and/or Neutrokine-alpha SV by proteolysis, and thus when Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide (particularly the representative extracellular domain in soluble form) is exogenously added to the tissue, cell or body of an individual, this polypeptide exhibits modulated activity on any of its target cells of the individual. Similarly, cells expressing this type II transmembrane protein may be added to cells, tissues or organisms of an individual such that the added cells bind to cells expressing the receptor for Neutrokine- α and/or Neutrokine- α SV, whereby the cells expressing Neutrokine- α and/or Neutrokine- α SV may have an effect (e.g., proliferation or cytotoxicity) on the target cells bearing the receptor.

In one embodiment, the invention provides methods of delivering a composition comprising a polypeptide of the invention (e.g., a composition comprising a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or an anti-Neutrokine-alpha and/or anti-Neutrokine-alpha SV antibody associated with a heterologous polypeptide, heterologous nucleic acid, toxin or prodrug) to a targeted cell, such as a B cell expressing a receptor for Neutrokine-alpha and/or Neutrokine-alpha SV or a monocyte expressing a cell surface-bound form of Neutrokine-alpha and/or Neutrokine-alpha SV. The Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or anti-Neutrokine-alpha and/or Neutrokine-alpha SV antibodies of the present invention bind to heterologous polypeptides, heterologous nucleic acids, toxins or prodrugs through hydrophilic, hydrophobic, ionic and/or covalent interactions.

In one embodiment, the invention provides a method of specifically delivering a composition of the invention to a cell by administering a polypeptide of the invention (e.g., a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or an anti-Neutrokine-alpha and/or anti-Neutrokine-alpha antibody) in association with a heterologous polypeptide or nucleic acid. In one embodiment, the invention provides a method of delivering a therapeutic protein to a targeted cell. In another embodiment, the invention provides a single-stranded nucleic acid (e.g., an antisense or ribozyme molecule), or a double-stranded nucleic acid (e.g., DNA that can be integrated into the genome of a cell or additionally replicated and transcribed).

In another embodiment, the invention provides a method of specifically destroying cells (e.g., destroying tumor cells) by administering a polypeptide of the invention (e.g., a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or an anti-Neutrokine-alpha and/or anti-Neutrokine-alpha SV antibody) in combination with a toxin or a cytotoxic prodrug.

In a specific embodiment, the invention provides a method of specifically destroying cells of a B cell line (e.g., a B cell-associated leukemia or lymphoma) by administering a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide in combination with a toxin or cytotoxic prodrug.

In another specific embodiment, the invention provides a method of specifically destroying cells of a monocytic cell line (e.g., a monocytic leukemia or lymphoma) by administering an anti-Neutrokine-alpha and/or anti-Neutrokine-alpha SV antibody in association with a toxin or a cytotoxic prodrug.

"toxin" refers to compounds, radioisotopes, holotoxins, modified toxins, catalysis of toxins that bind to and activate endogenous cytotoxic effector systemsA subunit, a cytotoxin (cytotoxic agent), or any molecule or enzyme that is not normally present or on the cell surface under defined conditions that result in cell death. Toxins that may be used in accordance with the methods of the present invention include, but are not limited to, radioisotopes, compounds such as antibodies that bind to (or contain a portion of) the complement fixation domain of) the intrinsic or induced endogenous cytotoxic effector system, thymidine kinase, endonucleases, RNase, alpha-toxin, ricin, ormosia toxin, pseudomonas exotoxin a, diphtheria toxin, saporin, momordicadine, gelonin, pokeweed antiviral protein, alpha-sarcin, and cholera toxin known in the art. "toxins" also include cytostatics or cytocides, therapeutic agents or radioactive metal ions, e.g. alpha-emitters such as ²¹³Bi, or other radioactive isotopes such as¹³³Xe，¹³¹I，⁶⁸Ge，⁵⁷Co，⁶⁵Zn，⁸⁵Sr，³²P，³⁵S，⁹⁰Y，¹⁵³Sm，¹⁵³Gd，¹⁶⁹Yb，⁵¹Cr，⁵⁴Mn，⁷⁵Se，¹¹³Sn，⁹⁰The yttrium is present in the form of yttrium,¹¹⁷the amount of tin is such that,¹⁸⁶the rhenium is used as a catalyst for the reaction of rhenium,¹⁶⁶holmium and¹⁸⁸rhenium, luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin.

Methods known in the art can be used to label the antibodies of the invention. Such methods include, but are not limited to, the use of bifunctional conjugating agents (see, e.g., U.S. Pat. Nos. 5756065; 5714631; 5696239; 5652361; 5505931; 5489425; 5435990; 5428139; 5342604; 5274119; 4994560; and 5808003; all of which are incorporated by reference in their entirety). Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucopyranoside, epipodophyllotoxin thiophenoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dydroxyanthrax dione mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogues or homologues thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., dichloromethyldiethylamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cycloothostamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and dichlorodiamine cisplatin (II) (DDP), anthracyclines (e.g., daunorubicin, (pre-daunomycin) and doxorubicin), antibiotics (e.g., daunorubicin (pre-actinomycin), bleomycin, mithramycin, anthranilic Acid (AMC)), and antimitotics (e.g., vincristine and vinblastine).

By "cytotoxic prodrug" is meant a non-toxic compound that is converted to a cytotoxic compound by enzymes normally present in the cell. Cytotoxic prodrugs that may be used in accordance with the method of the present invention include, but are not limited to, glutamyl derivatives of the mustard benzoate alkylating agent, phosphate derivatives of epipodophyllotoxin glucopyranoside or mitomycin C, cytarabine, daunorubicin and phenoxyacetamide derivatives of doxorubicin.

It will be appreciated that diseases, particularly immune system diseases, which result from a level of Neutrokine-alpha and/or Neutrokine-alpha SV activity in an individual that is below a standard or normal level may be treated by administration of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides (in the form of soluble extracellular domains or cells expressing intact proteins) or agonists. Accordingly, the present invention also provides a method of treating an individual in need of increasing the level of Neutrokine-alpha and/or Neutrokine-alpha SV activity comprising administering to such an individual a pharmaceutical composition comprising an amount of an isolated Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide of the present invention, or an agonist thereof, effective to increase the level of Neutrokine-alpha and/or Neutrokine-alpha SV activity in such an individual.

It will also be appreciated that diseases, particularly immune system diseases, resulting from an individual having a level of Neutrokine-alpha and/or Neutrokine-alpha SV activity above the standard or normal level may be treated by administering Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides (soluble extracellular domains or cellular forms expressing intact proteins) or antagonists thereof (e.g. antibodies against Neutrokine-alpha). The present invention thus also provides a method of treating an individual in need of reducing the activity of Neutrokine-alpha and/or Neutrokine-alpha SV comprising administering to such an individual a pharmaceutical composition comprising an amount of an isolated Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide of the invention, or an antagonist thereof, effective to reduce the level of Neutrokine-alpha and/or Neutrokine-alpha SV activity in such an individual.

The Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide or antagonist thereof can be used for treating infectious diseases. For example, infectious diseases can be treated by increasing the immune response, particularly B cell proliferation and differentiation. The immune response may be enhanced by enhancing an existing immune response, or by initiating a new immune response. Alternatively, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides or antagonists thereof may also directly inhibit the infectious agent without eliciting an immune response.

The virus is, for example, an infectious agent that produces a disease or clinical condition that can be treated by a Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide or an agonist thereof. Viruses include, for example, but are not limited to, the following DNA and RNA viruses and virus families: arbovirus, adenovirus, arenavirus, arterivirus, birnavirus, bunyavirus, calicivirus, Circoviridae, coronavirus, dengue virus, EBV, HIV, flavivirus, hepadnavirus (hepatitis virus), herpesvirus (e.g., cytomegalovirus, herpes simplex virus, herpes zoster virus), mononegavir (e.g., paramyxovirus, measles virus, rhabdovirus), orthomyxovirus (e.g., influenza a virus, influenza b virus, and parainfluenza virus), papilloma virus, papovavirus, parvovirus, picornavirus, poxvirus (e.g., smallpox or vaccinia virus), reovirus (e.g., rotavirus), retrovirus (HTLV-I, HTLV-II, lentivirus), and togavirus (e.g., rubella virus). Viruses in these families can cause a variety of diseases or clinical symptoms, including, but not limited to, arthritis, bronchitis, respiratory syncytial virus, encephalitis, ocular infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (a, B, c, e, chronic active, type δ), japanese encephalitis type B, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's lymphoma, varicella, hemorrhagic fever, measles, mumps, parainfluenza, rabies, the common cold poliomyelitis, leukemia, rubella, venereal diseases, skin diseases (e.g., Kaposi' warts), and viremia. Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides or agonists or antagonists thereof may be used for the treatment, prevention, diagnosis and/or detection of any of these conditions or diseases. In specific embodiments, the Neutrokine-alpha-polynucleotide, polypeptide or agonist is used for the treatment, prevention and/or diagnosis of: meningitis, dengue fever, EBV, and/or hepatitis (e.g. b). In another specific embodiment, the Neutrokine-alpha-polynucleotide, polypeptide or agonist is used to treat a patient who is not responsive to one or more other commercially available hepatitis vaccines. In another specific embodiment, the Neutrokine-alpha polynucleotide, polypeptide or agonist is used for the treatment, prevention and/or diagnosis of AIDS. In another specific embodiment, Neutrokine-alpha and/or Neutrokine-alpha SV and/or its receptor polynucleotides, polypeptides, agonists and/or antagonists are used for the treatment, prevention and/or diagnosis of cryptosporidiosis.

Similarly, bacterial or fungal etiologies that may cause disease or clinical symptoms and may be treated with Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists or antagonists thereof, include, but are not limited to, the following gram negative and gram positive bacteria and bacterial families and fungi: from the order of the actinomycetales (e.g., Corynebacterium, Mycobacterium, Nocardia), Cryptococcus neoformans, Aspergillus, Bacillaceae (e.g., Bacillus anthracis, Clostridium), Bacteroides, Bluetophyte, Borrelia (e.g., Borrelia Burgdorferi), Brucella, Candida, Campylobacter, Coccidioides, Cryptococcus, Dermatocystoses, Escherichia coli (e.g., enterotoxigenic and enterohemorrhagic Escherichia coli), Enterobacterium (e.g., Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella), Serratia, Yersinia), Erysipelothrix, helicobacter, Legionella, Leptospira, Listeria (e.g., Listerionychogenes), Mycoplasma, Martensis, Vibrio, Neisseria (e.g., Actinobacterium, Gomorrha), meningococcus, isseria meningitidis, Passeria, Pasteurella (e.g., Actinobacillus, Haemophilus B), pasteurella), Pseudomonas, Rickettsia, Chlamydia, syphilis, Shigella, Staphylococcus, meningococcus, pneumococcus, and Streptococcus (e.g., Streptococcus pneumoniae and group B streptococci). These bacterial or fungal families may produce diseases or conditions including, but not limited to bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (such as AIDS-related infections), paronychia, prosthesis-related infections, Reiter's disease, respiratory infections, such as pertussis or empyema, septicemia, Lyme disease, Cat-serratch disease, dysentery, paratyphoid fever, food poisoning, typhoid fever, pneumonia, gonorrhea, meningitis (such as type a, type b meningitis), chlamydia, syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, hot rheumatism, scarlet fever, venereal diseases, skin diseases (such as cellulitis, dermacytoses), toxemia, urinary tract infections, wound infections. Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists or antagonists thereof, may be used to treat, prevent, diagnose and/or detect any of these diseases or conditions. In specific embodiments, the Neutrokine-alpha polynucleotide, polypeptide or agonist thereof is used for the treatment, prevention and/or diagnosis of: tetanus, dipheria, botulism and/or meningitis type b.

In addition, parasitic causes that cause disease or symptoms and that can be treated with Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists thereof, include, but are not limited to, the following families or types: amebiasis, babesiosis, coccidiosis, cryptosporidiosis, dienarenoebiasis, dourine, ecoparopsitic, giardiasis, helminthiasis, leishmaniasis, theileriosis, toxoplasmosis, trypanosomiasis, and trichomonas and sporozoites (e.g., Plasmodium virax, Plasmodium falciparum, Plasmodium malariae, and Plasmodium ovale). These parasites can cause a variety of diseases or conditions, including but not limited to scabies, tsutsugamushi disease, eye infections, intestinal diseases (e.g., dysentery, giardiasis), liver diseases, lung diseases, opportunistic infections (e.g., AIDS-related infections), malaria, pregnancy complications, and toxoplasmosis. Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists or antagonists thereof, may be used for the treatment, prevention, diagnosis and/or detection of any of these conditions or diseases. In specific embodiments, the Neutrokine-alpha-polynucleotide, polypeptide or agonist thereof is used for the treatment, prevention and/or diagnosis of malaria.

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides and/or agonists and/or antagonists thereof of the present invention are useful for the treatment, prevention and/or diagnosis of inner ear infections (such as otitis media), as well as other infections characterized by Streptococcus pneumoniae and other pathogenic microorganisms.

In a specific embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists or antagonists thereof (e.g., anti-Neutrokine-alpha and/or Neutrokine-alpha SV antibodies), are used to treat or prevent conditions characterized by serum immunoglobulin production deficiency, recurrent infection and/or immune system dysfunction. Additionally, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists or antagonists thereof (e.g., antibodies against Neutrokine-alpha and/or Neutrokine-alpha SV), may be useful in the treatment or prevention of joint, bone, skin and/or parotid infections, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., as described herein), inflammation, and malignancies, and/or any disease or disorder or pathological change associated with such infections, diseases, functional disorders or malignancies, including but not limited to CVID, other primary immunodeficiency, HIV, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, shingles (e.g. severe shingles), and/or pneumocystis carinii.

Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists or antagonists thereof, are useful in diagnosing, prognosing, treating or preventing one or more of the following diseases or disorders, or pathological conditions: primary immunodeficiency, immune-mediated platelet anemia, Kawasaki syndrome, bone marrow transplantation (e.g., adult or child bone marrow transplantation), chronic B-cell lymphoid leukemia, HIV infection (e.g., adult or child HIV infection), chronic inflammatory demyelinating polyneuropathy, and post-transfusion purpura.

Additionally, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the present invention, or agonists or antagonists thereof, may be useful in diagnosing, prognosing, treating or preventing one or more of the following diseases, disorders or pathological conditions: Guillian-Barre syndrome, anemia (e.g., anemia associated with parvovirus B19, patients with stable multiple myeloma at high risk of infection (e.g., recurrent infection), autoimmune hemolytic anemia (e.g., febrile autoimmune hemolytic anemia), platelet anemia (e.g., neonatal platelet anemia), and immune-mediated neurological anemia, transplantation (e.g., CMV-negative receptors of CMV-positive organs), hypogammaglobulinemia (e.g., neonatal hypogammaglobulinemia with infectious or morbidity risk factors), epilepsy (e.g., refractory epilepsy), systemic vascular syndrome, muscle weakness (e.g., cardiac decompensation in muscle weakness), dermatomyositis, and polymyositis.

Additional preferred embodiments of the invention include, but are not limited to, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, polynucleotides and functional agonists thereof, for use in:

administration to an animal (e.g., mouse, rat, immune, hamster, guinea pig, piglet, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, preferably human) to aid the immune system in producing increased amounts of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), inducing higher affinity antibody production (e.g., IgG, IgA, IgM, and IgE), and/or enhancing immune response. In a specific embodiment, a Neutrokine-alpha polypeptide of the invention and/or an agonist thereof is administered to assist the immune system in producing increased amounts of IgG. In another specific embodiment, a Neutrokine-alpha polypeptide of the invention and/or an agonist thereof is administered to assist the immune system in producing increased amounts of IgA. In another specific embodiment, a Neutrokine-alpha polypeptide of the invention and/or an agonist thereof is administered to assist the immune system in producing increased amounts of IgM.

Administration to animals (including but not limited to those listed above and also including transgenic animals) that are incapable of producing functional endogenous antibody molecules, or have an endogenous immune system of additional composition, but are incapable of producing human immunoglobulin molecules by reconstituting or partially reconstituting the immune system from another animal (see, e.g., PCT publication nos. WO 98/24893, WO 96/34096, WO 96/33735 and WO 91/10741).

A vaccine adjuvant enhances the immune response to specific antigens. In a specific embodiment, the vaccine adjuvant is a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide as described herein. In another specific embodiment, the vaccine adjuvant is a Neutrokine-alpha and/or alpha SV polynucleotide as described herein (i.e., Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide is a genetic vaccine adjuvant). As described herein, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides of the present invention may be administered using methods known in the art, including but not limited to liposome delivery, recombinant vector delivery, injection of naked DNA, and biolistic delivery.

An adjuvant enhances tumor-specific immune responses.

An adjuvant enhances the antiviral immune response. Antiviral immune responses, including but not limited to viral-related diseases or conditions described herein or known in the art, can be enhanced using the compositions of the invention as adjuvants. In specific embodiments, the compositions of the invention are used as adjuvants to enhance the immune response to a viral disease or condition selected from the group consisting of: AIDS, meningitis, dengue fever, EBV, and hepatitis (e.g. hepatitis b). In another specific embodiment, the compositions of the invention are used as adjuvants to enhance the immune response to a virus, disease or condition selected from the group consisting of: HIV/AIDS, respiratory syncytial virus, dengue fever, rotavirus, Japanese encephalitis B, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley fever, herpes simplex, and yellow fever. In another specific embodiment, the compositions of the invention are used as adjuvants to enhance the immune response to the HIV gp120 antigen.

An adjuvant enhances the antibacterial or antifungal immune response. The antibacterial or antifungal immune response that may be enhanced using the compositions of the present invention as adjuvants includes bacteria or fungi and bacterial or fungal related diseases or conditions as described herein and known in the art. In specific embodiments, the compositions of the invention are used as adjuvants to enhance the immune response to a bacterial or fungal, disease or condition selected from: tetanus, diphtheria, botulism, and meningitis type b. In another specific embodiment, the composition of the invention is used as an adjuvant to enhance the immune response to a bacterial or fungal, disease or condition selected from the group consisting of: vibrio cholerae, Brevibacterium, Salmonella typhi, Salmonella paratyphi, Meisseria meningitis, Streptococcus pneumoniae, group B streptococci, Shigella, enterotoxigenic Escherichia coli, enterohemorrhagic Escherichia coli, Borreliaburgdorferi, Plasmodium (malaria).

An adjuvant enhances an anti-parasitic immune response. The compositions of the present invention can be used as adjuvants to enhance anti-parasitic immune responses, including parasites as described herein or known in the art and diseases or conditions associated with parasites. In specific embodiments, the compositions of the invention are used as adjuvants to enhance the immune response to parasites. In another specific embodiment, the compositions of the invention are used as adjuvants to enhance the immune response to plasmodium (malaria).

As a stimulator of the B cell response to pathogens.

As a factor in assessing the immune status of an individual prior to receiving immunosuppressive therapy.

As a factor inducing high affinity antibodies.

As a factor for increasing serum immunoglobulin concentration.

As a factor to accelerate the recovery of the immune containing individual.

As a factor assisting the immune response of the elderly.

As an enhancer of the immune system before, during or after bone marrow transplantation and/or other transplantations (e.g., allogeneic or xenogeneic organ transplantation). In view of transplantation, the compositions of the present invention may be administered prior to, concurrently with, and/or after transplantation. In a specific embodiment, the compositions of the invention are administered after transplantation, prior to the start of recovery of the T cells. In another specific embodiment, the compositions of the invention are administered first after the beginning of restoration of the T cell population following transplantation, but before the complete restoration of the B cells.

As a factor assisting the immune response of an individual with a B cell immunodeficiency, for example, a partially or fully splenectomy patient. B cell immunodeficiency disorders which may be ameliorated or treated by administration of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide, or agonist thereof, of the invention include, but are not limited to, X-linked Severe Combined Immunodeficiency (SCID), autosomal SCID, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglulinemia, acquired agammaglulinemia, adult onset agammaglulinemia, late onset agammaglulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia in infants, non-specific agammaglobulinemia, agammaglulinemia, Common Variant Immunodeficiency (CVID) (acquired), Wiskott-Aldrich syndrome (WAS), X-linked high IgM immunodeficiency, non-X linked high IgM immunodeficiency, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), normal or high IgS antibody deficiency, thymoma immunodeficiency, Ig heavy chain deletion, k chain deletion, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysplasia, neonatal neutropenia, severe congenital leukemia, immunodeficiency thymic lymphoid tissue dysplasia, dyskinetic telangiectasia, short limb dwarfism, X-linked lymphoproliferative syndrome (XLP), Nozelof syndrome combined with Igs immunodeficiency, Purine Nucleoside Phosphorylase (PNP) deficiency, mhc class ii deficiency (Bare lymphocyto syndrome), and severe combined immunodeficiency.

Diseases that result in acquired B cell loss that may be ameliorated or treated by administration of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide, or agonist thereof, of the invention as a booster immune response agent for individuals with acquired loss of B cell function include, but are not limited to, HIV infection, AIDS, bone marrow transplantation, and B cell Chronic Lymphocytic Leukemia (CLL).

As a factor in assisting the immune response in a transient immunocompromised individual. Diseases that result in temporary immune deficiencies that may be ameliorated or treated by administration of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide, or agonist thereof, of the invention include, but are not limited to, viral infections (e.g., influenza), malnutrition-related diseases, mononucleosis infections, or stress-related diseases, measles, blood transfusions, surgery.

As modulators of antigen presentation by monocytes, dendritic cells and/or B cells. In one embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide (soluble, membrane-bound or transmembrane form) or polynucleotide enhances antigen presentation or antagonizes antigen presentation in vitro or in vivo. In addition, in related embodiments, such enhancement or antagonism of antigen presentation may be useful in anti-tumor therapy or in modulating the immune system.

As a mediator of mucosal immune responses. Monocytes express Neutrokine-alpha and B cells respond to other cues, which may be involved in the exchange of signals between B cells and monocytes or between their differentiated progeny. This activity is in many ways identical to the CD40-CD154 signal between B cells and T cells. Therefore, Neutrokine-alpha may be an important regulator of the T cell-independent immune response to environmental pathogens. The non-conventional B cell population (CD5+), which is particularly associated with the mucosa and which responds to many innate immune responses in humans, responds to Neutrokine-alpha, thus enhancing the protective immune status of the individual.

As a factor directing the immune system of an individual to mount a humoral response (i.e., TH2) as opposed to a TH1 cellular response.

As a factor inducing tumor proliferation and thus making it more suitable as an antitumor agent. For example, multiple myeloma is a slowly differentiating disease and therefore does not work with virtually all anti-tumor approaches. If these cells were allowed to proliferate more rapidly, their ability to accept anti-tumor approaches would be improved.

As a B-cell specific binding protein, specific activators or inhibitors of cell growth may be attached thereto. The result is to focus the activity of such activators or inhibitors on normal, diseased or neoplastic B cell populations.

As a factor for detecting B cell line cells by its specificity. This application may require labeling of the protein with biotin or other agents (as described herein) for detection.

As a stimulator of B cell production in some diseases, such as AIDS, chronic lymphocyte dysfunction and/or general variable immunodeficiency.

As part of the method of selecting B cells, it functions to isolate B cells from a heterogeneous mixture of cell types. Neutrokine-alpha can be coupled to a solid support to which B cells then specifically bind. Unbound cells are washed away, followed by elution of bound cells. One non-limiting application of this option is to allow tumor cells to be cleared from, for example, bone marrow or peripheral blood prior to transplantation.

As a therapy for generating and/or regenerating lymphoid tissue following surgery, trauma or genetic defect.

As gene therapy for genetic diseases that result in anergy as observed in SCID patients.

As an antigen that generates a response that inhibits or enhances Neutrokine-alpha mediators.

As a substance for activating monocytes/macrophages against parasitic diseases acting on monocytes, such as leishmaniasis.

As a pretreatment for bone marrow samples prior to transplantation. This treatment enhances B cell re-presentation and thus accelerates recovery.

As a substance for regulating the secretion of cytokines, the secretion of the cytokines is stimulated by Neutrokine-alpha.

Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or polynucleotides of the invention, or agonists thereof, may be used to modulate IgE concentrations in vitro or in vivo.

In addition, the Neutrokine-alpha or Neutrokine-alpha SV polypeptides or nucleotides of the present invention, or agonists thereof, may be used for the treatment, prevention and/or diagnosis of IgE-mediated allergy. Such allergies include, but are not limited to, asthma, rhinitis, and eczema.

In a specific embodiment, a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or an agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate selective IgA deficiency.

In another specific embodiment, a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate dyskinetic telangiectasia.

In another specific embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or polynucleotides of the present invention, or agonists thereof, are administered to treat, prevent, diagnose and/or ameliorate the general variant of an immune deficiency.

In another specific embodiment, a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate X-linked agammaglobulinemia.

In another specific embodiment, a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or an agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate Severe Combined Immunodeficiency (SCID).

In another specific embodiment, a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate Wiskott-Aldrich syndrome.

In another specific embodiment, a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate the Ig deficit of X-linked high IgM.

In another specific embodiment, a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or an agonist or antagonist thereof (e.g., an anti-Neutrokine-alpha antibody), is administered to treat, prevent, diagnose and/or diagnose chronic myelogenous leukemia, acute myelogenous leukemia, hystoctic leukemia, monocytic leukemia (e.g., acute monocytic leukemia), reticulocytic leukemia, shaking-type monocytic leukemia, and/or other leukemias derived from monocytes and/or mononuclear cells and/or tissues.

In another specific embodiment, a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or agonist thereof, is administered to treat, prevent, diagnose and/or ameliorate a monocytic leukemic response, such as that seen in tuberculosis.

In another specific embodiment, a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or agonist thereof, is administered for the treatment, prevention, diagnosis and/or amelioration of mononucleosis, mononucleosis anemia and/or mononucleosis.

In a specific embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, and/or anti-Neutrokine-alpha antibodies and/or agonists or antagonists thereof, of the present invention are useful for the treatment, prevention, detection and/or diagnosis of primary B-lymphocyte dysfunction and/or diseases and/or pathological conditions associated therewith. In one embodiment, the primary B lymphocyte dysfunction, disease and/or pathological condition is characterized by a complete or partial loss of humoral immunity. Primary B lymphocyte dysfunction, disease and/or pathological conditions, including but not limited to X-linked hypogammaglobuline (XLA), Severe Combined Immunodeficiency (SCID), and selective IgA impairment, characterized by complete or partial loss of humoral immunity and which can be prevented, treated, detected and/or diagnosed with the compositions of the present invention.

In a preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides, polypeptides and/or agonists or antagonists thereof are used for the treatment, prevention and/or diagnosis of one or more various mucosal related diseases of the body. Such diseases include, but are not limited to, mucositis, mucodestructive surgery, mucositis colitis, mucocutaneous leishmaniasis (e.g., leishmaniasis, nasopharyngeal leishmaniasis, and new world leishmaniasis), mucocutaneous lymphotrophic syndrome (e.g., Kawasaki disease), mucositis enteritis, mucoid epidermoid carcinoma, mucoid epidermoid tumor, mucosal epithelial dysplasia, mucoid adenocarcinoma, mucoid degeneration; degeneration of myxoma; polymyxa, mucoid mesohigh [ e.g., thecal necrosis ], mucolipidosis (including mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, and mucolipidosis type IV), mucositis enteritis, mucopolysaccharidosis (e.g., mucopolysuria type I (i.e., Hurler's syndrome), mucopolysaccharidosis type IS (i.e., Scheie's syndrome or mucopolysaccharidosis type V), mucopolysaccharidosis type II (i.e., Hunter's syndrome), mucopolysaccharidosis type III (i.e., Sanfilippo's syndrome), mucopolysaccharidosis type IV (i.e., Morquio's syndrome), mucopolysaccharidosis type VI (i.e., Naroteux-Lamy syndrome), mucopolysaccharidosis type VII (i.e., mucopolysaccharidosis due to β -glucuronidase deficiency), and (mucopolysaccharidosis), mucomucomucosulfate mucositis, mucopurulent, mucosis, mucomycosis, mucormycosis, mucosis, mucositis (i.e.e.e.e.g., viral diarrhea), mucositis and mucositis), and pancreatic fibrocystic disease (e.g., pancreatic cystic fibrosis, Carke-Hadfield syndrome, pancreatic fibrocystic disease). In a particularly preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides, polypeptides and/or agonists and/or antagonists thereof are used for the treatment, prevention and/or diagnosis of mucositis, in particular mucositis associated with chemotherapy.

In a preferred embodiment, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides, polypeptides and/or agonists and/or antagonists thereof are used for the treatment, prevention and/or diagnosis of diseases associated with sinusitis.

Another disease or condition that may be treated, prevented and/or diagnosed by a Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide, or an agonist thereof, is osteomyelitis.

Another disease or condition that Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides, or agonists thereof, may treat, prevent and/or diagnose is endocarditis.

All of the above applications can be used in veterinary medicine.

Antagonists of Neutrokine-alpha include binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes, and Neutrokine-alpha polypeptides of the invention. These may be expected to reverse some of the activities of the above ligands and find some clinical or practical applications, such as:

for blocking various aspects of the immune response to foreign or self substances. Examples include autoimmune diseases such as lupus, and arthritis, as well as immune responses to skin allergies, inflammation, intestinal disease, injury, and pathogens. Although the current data directly illustrates the potential role of Neutrokine-alpha in B-cell and monocyte related pathologies, other cell types may also express or respond to Neutrokine-alpha. Thus, Neutrokine-alpha, like CD40 and its ligands, can be regulated by the status of the immune system and the microenvironment in which the cells are located.

For use in therapy to prevent B cell proliferation and Ig secretion associated with autoimmune diseases, such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and MS.

As an inhibitor of graft-versus-host disease or graft rejection.

Treatment of B cell malignancies such as ALL, Hodgkins disease, non-Hodgkins lymphoma, chronic lymphocytic leukemia, plasmacytoma, multiple myeloma, Burkitt's lymphoma, and EBV-transformed disease.

Treatment of chronic hypergammaglobulinemia, as in single cell gammopathy of undetermined importance (MGUS), Waldenstrom's disease, relatively idiopathic single cell gammopathy, and plasmacytoma.

Decreased cell proliferation for the treatment of large B-cell lymphoma.

B-cell and Ig-link associated with chronic myelogenous leukemia are reduced.

As an immunosuppressant.

The Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or polynucleotides of the present invention, or antagonists thereof, may be used to modulate IgE concentrations in vitro or in vivo.

In another embodiment, administration of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide or polynucleotide of the invention, or an antagonist thereof, may be used to treat, prevent and/or diagnose IgE-mediated allergic reactions, including but not limited to asthma, rhinitis and eczema.

As inhibitors of the signaling pathway involving ERK1, COX2 and Cyclin D2, which pathway is associated with Neutrokine-alpha induced B cell activation.

The above applications can be used in a wide variety of hosts. Such hosts include, but are not limited to, humans, mice, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, piglets, chickens, cows, sheep, dogs, cats, non-human primates, and humans. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog, or cat. In a preferred embodiment, the host is a mammal. In a more preferred embodiment, the host is a human.

The agonists and antagonists may be used in combination with a pharmaceutically suitable carrier.

Antagonists are useful, for example, in inhibiting Neutrokine-alpha mediated and/or Neutrokine-alpha SV mediated chemotaxis and activation of macrophages and their precursors, and neutrophils, basophils, B lymphocytes, and some T cell subsets such as activated and CD8 cytotoxic T cells, and natural killer cells, in some autoimmune and chronic inflammatory and infectious diseases. Autoimmune diseases, including, for example, multiple sclerosis, and insulin-dependent diabetes mellitus, antagonists may also be useful in the treatment, prevention and/or diagnosis of infectious diseases, including silicosis, sarcoidosis, primary pulmonary fibrosis, by preventing mononuclear phagocyte recruitment and activation. They may also be used for the treatment, prevention and/or diagnosis of primary eosinophilic syndrome by preventing the production and migration of eosinophils. Endotoxic shock can also be treated with such antagonists by preventing macrophage migration and its production of the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention. The antagonists are also useful in the treatment of arteriosclerosis by preventing infiltration of monocytes into the arterial wall. The antagonists are also useful in the treatment, prevention and/or diagnosis of histamine-mediated allergic reactions and immune disorders, including advanced allergies, chronic urticaria, and atopic dermatitis, by inhibiting chemokine-induced mast cell and basophil degranulation and histamine release. IgE-mediated allergic reactions such as allergic asthma, rhinitis and eczema may also be treated. Antagonists may also be useful in the treatment, prevention and/or diagnosis of chronic and acute inflammation by preventing the invasion of monocytes into the damaged area. They are also useful in regulating normal pulmonary macrophage populations because chronic and acute pulmonary inflammation are associated with the pooling of mononuclear macrophages in the lung. The antagonists may also be used in the treatment, prevention and/or diagnosis of rheumatoid arthritis by preventing the invasion of monocytes into the synovial fluid in the joints of a patient. Monocyte influx and activation play an important role in degenerative and inflammatory joint disease. Antagonists can be used to interfere with the deleterious threading response to the body caused by IL-1 and TNF by preventing the biosynthesis of other inflammatory cytokines. In this manner, antagonists may be used to prevent inflammation. Antagonists are also useful for inhibiting prostaglandin-independent fever induced by Neutrokine-alpha and/or Neutrokine-alpha SV. Antagonists may also be useful in the treatment, prevention and/or diagnosis of myelopathies, such as aplastic anemia and myelodysplastic syndrome. Antagonists may also be useful in the treatment, prevention and/or diagnosis of asthma and allergy by preventing the accumulation of eosinophils in the lung. Antagonists may also be useful in the treatment, prevention and/or diagnosis of sub-epithelial basement membrane fibrosis, a significant feature of asthmatic lungs. Antagonists may also be useful in the treatment, prevention and/or diagnosis of lymphoma (such as, but not limited to, one or more of the lymphomas listed herein).

All of the above applications can be used in veterinary medicine.

The Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides and/or agonists and/or antagonists thereof of the present invention may be used for the treatment, prevention, and/or diagnosis of various immune system related diseases in mammals, preferably humans. Many autoimmune diseases are caused by inappropriate recognition of self as a foreign by immune cells. This inappropriate recognition results in an immune response that destroys host tissue. Thus, administration of a Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide and/or agonist and/or antagonist thereof of the present invention may inhibit immune responses, particularly B cell proliferation and/or immunoglobulin production, and may be effective in the treatment and/or prevention of autoimmune diseases. Thus, in a preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV antagonists of the present invention (e.g., polypeptide fragments of Neutrokine-alpha and/or Neutrokine-alpha SV and anti-Neutrokine-alpha antibodies) are useful for the treatment, prevention and/or diagnosis of autoimmune diseases.

Autoimmune diseases that can be treated, prevented and/or diagnosed with a Neutrokine-alpha polynucleotide, polypeptide and/or antagonist (e.g., an anti-Neutrokine-alpha antibody) of the invention include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmune cytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, recurrent polychondritis, rheumatic heart disease, glomerulonephritis (i.e., IgA nephropathy), multiple sclerosis, neuritis, uveitis, multiple endocrinopathies, purpura (e.g., henoch-sconelein purpura), Reiter's disease, Stiff-Man syndrome, autoimmune pneumonia, Guillain-Barre syndrome, insulin-dependent diabetes mellitus and autoimmune ocular inflammation.

Additional autoimmune diseases that can be treated, prevented and/or diagnosed with the compositions of the present invention include, but are not limited to, autoimmune thyroiditis, methyleneate (i.e., Hashimoto's thyroiditis), (generally characterized by cell-mediated and humoral thyrotoxicities), systemic lupus erythematosus (generally characterized by blood circulation and local production of immune complexes), Goodpasture's syndrome (generally characterized by anti-basement membrane antibodies), receptor autoimmunity such as (a) Grave's disease (generally characterized by TSH receptor antibodies), (b) yasthenia Gravis (generally characterized by acetylcholine receptor antibodies), and (c) insulin resistance (generally characterized by insulin receptor antibodies), autoimmune hemolytic anemia (generally characterized by phagocytosis by antibody-sensitized RBCs), autoimmune thrombocytopenic purpura (generally characterized by phagocytosis by antibody-sensitized platelets).

Additional autoimmune diseases that can be treated, prevented and/or diagnosed with the compositions of the present invention include, but are not limited to, rheumatoid arthritis (often characterized by immune complexes in the joints), schleroderma (often characterized by nucleolar antibodies and other nuclear antibodies) with anti-collagen antibodies, mixed connective tissue diseases (often characterized by antibodies that can extract nuclear antigens (such as ribonucleoproteins)), polymyositis/epidermyositis (often characterized by non-group ANA), pernicious anemia (often characterized by anti-parietal cells, microsomes, and intrinsic factor antibodies) primary Addison's disease (often characterized by humoral and cell-mediated adrenal toxicity), infertility (often characterized by anti-sperm antibodies), glomerulonephritis (often characterized by glomerular antibodies or immune complexes) such as primary glomerulonephritis and IgA nephropathy, bullous pemphigus (often characterized by IgG and compensation in the basement membrane), Sjogren's syndrome (often characterized by multi-tissue antibodies and/or non-group ANA (SS-B)), diabetes (often characterized by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including asthma or cystic fibrosis adrenergic drug resistance) (often characterized by β adrenergic receptor antibodies).

Additional autoimmune diseases that can be treated, prevented and/or diagnosed with the compositions of the present invention include, but are not limited to, chronic active hepatitis (often characterized by smooth muscle antibodies), primary biliary cirrhosis (often characterized by mitochondrial antibodies), other endocrine gland disorders (often characterized by specific tissue antibodies in some cases), vitiligo (often characterized by melanocytic antibodies), vasculopathy (often characterized by Ig and complement in the vessel wall and/or low serum complement), MI sequelae (characterized by myocardial antibodies), post-cardiac surgery syndrome (often characterized by myocardial antibodies), urticaria (often characterized by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized by IgG and IgM antibodies to IgE), asthma (often characterized by IgG and IgM antibodies to IgE), inflammatory myopathy and many other inflammatory diseases, grandifoliatous, degenerative and other atrophic diseases.

In a preferred embodiment, the above autoimmune diseases and disorders and/or pathological conditions are treated, prevented and/or diagnosed with anti-Neutrokine-alpha antibodies and/or anti-Neutrokine-alpha SV antibodies.

In a particularly preferred embodiment, rheumatoid arthritis is treated, prevented and/or diagnosed with an anti-Neutrokine-alpha antibody and/or an anti-Neutrokine-alpha SV antibody and/or other antagonist of the present invention.

In a specific preferred embodiment, lupus is treated, prevented and/or diagnosed with an anti-Neutrokine-alpha antibody and/or a Neutrokine-alpha SV antibody and/or other antagonist of the invention.

In a specific preferred embodiment, lupus-associated nephritis is treated, prevented and/or diagnosed with an anti-Neutrokine-alpha antibody and/or a Neutrokine-alpha SV antibody and/or other antagonist of the invention.

In a specific embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide, or an antagonist thereof (e.g., an anti-Neutrokine-alpha antibody and/or a Neutrokine-alpha SV antibody), is used to treat or prevent systemic lupus erythematosus and/or a disease associated therewith. Lupus-associated diseases that may be treated with a Neutrokine-alpha antibody and/or a Neutrokine-alpha SV polynucleotide or polypeptide, or antagonist thereof, of the present invention include, but are not limited to, hematological diseases (e.g., hemolytic anemia, leukemia, lymphocytic anemia, and platelet anemia), immunological diseases (e.g., anti-DNA antibodies, and anti-Sm antibodies), skin rash, photosensitivity, oral ulcers, arthritis, fever, fatigue, weight loss, serous diseases (e.g., pleuritis), renal diseases (e.g., nephritis), neuropathy (e.g., episodic peripheral neuropathy, CNS-associated diseases), gastrointestinal diseases, Raynaud's phenomenon, such as pericarditis. In a preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide, or an antagonist thereof (e.g., an anti-Neutrokine-alpha and/or Neutrokine-alpha SV antibody), is used to treat or prevent renal disease associated with systemic lupus erythematosus. In a more preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide, or an antagonist thereof (e.g., an anti-Neutrokine-alpha and/or Neutrokine-alpha SV antibody), is used to treat or prevent nephritis associated with systemic lupus erythematosus.

Similarly, allergic diseases such as asthma (particularly allergic asthma) or other respiratory diseases may also be treated with Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof. In addition, these molecules can be used to treat, prevent and/or diagnose allergy, hypersensitivity, or incompatibility of blood groups to the antigen molecule.

The Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention, and/or agonists and/or antagonists thereof, may also be useful in the treatment, prevention and/or diagnosis of organ rejection or graft-host rejection disease (GVHD) and/or diseases associated therewith. The transplanted tissue undergoes organ rejection by destruction of host immune cells by the immune response. Similarly, an immune response is also included in GVHD, but in this case, the foreign transplanted immune cells destroy host tissues. Administration of Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides and/or agonists/antagonists thereof of the invention that inhibit immune responses, particularly T cell proliferation, differentiation or chemotaxis, may be effective in preventing organ rejection or GVHD.

Similarly, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof may also be useful in modulating inflammation. For example, Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides and/or agonists and/or antagonists thereof of the present invention may inhibit proliferation and differentiation of cells in an inflammatory response. These molecules are useful for treating, preventing and/or diagnosing acute and chronic inflammation, including chronic prostatitis, granulomatous prostatitis, and malacia, inflammation associated with infection (e.g., septic shock, or Systemic Inflammatory Response Syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethal disease, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine-or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease, or disease due to overproduction of cytokines (e.g., TNF or IL-1).

In a specific embodiment, the anti-Neutrokine-alpha and/or Neutrokine-alpha SV antibodies of the present invention are used for the treatment, prevention, modulation, detection and/or diagnosis of inflammation.

In a specific embodiment, the anti-Neutrokine-alpha and/or Neutrokine-alpha SV antibodies of the present invention are used for the treatment, prevention, modulation, detection and/or diagnosis of inflammatory diseases.

In another specific embodiment, the anti-Neutrokine-alpha and/or Neutrokine-alpha SV antibodies of the present invention are used for the treatment, prevention, modulation, detection and/or diagnosis of allergy and/or hypersensitivity disorders.

anti-Neutrokine-alpha and/or Neutrokine-alpha SV antibodies may be used to bind to and inhibit Neutrokine-alpha and/or Neutrokine-alpha SV activity for the treatment, prevention and/or diagnosis of ARDS by preventing neutrophil infiltration into the lung after injury. The agonists and antagonists of the present invention may be used in combination with pharmaceutically suitable carriers, as described hereinafter.

The Neutrokine-alpha and/or Neutrokine-alpha SV and/or Neutrokine-alpha receptor polynucleotides or polypeptides of the invention, and/or agonists and/or antagonists thereof, are useful in the treatment, prevention and/or diagnosis of pulmonary diseases (such as bronchial diseases, e.g., sinopulmonary and bronchial infections, and diseases associated therewith and other respiratory diseases). In specific embodiments, such diseases include, but are not limited to, bronchial adenoma, bronchial asthma, pneumonia (e.g., bronchopneumonia, and tuberculous bronchopneumonia), Chronic Obstructive Pulmonary Disease (COPD), bronchial polyps, bronchiectasis (e.g., dry bronchiectasis, barrel bronchiectasis, and saccular bronchiectasis), bronchial adenocarcinoma, bronchial carcinoma, bronchiolitis (e.g., exudative bronchiolitis, fibromatosis-occlusive bronchiolitis, and proliferative bronchitis), broncho-alveolar carcinoma, bronchial asthma, bronchitis (e.g., asthmatic bronchitis, Castellani's bronchitis, chronic bronchitis, croup-bronchitis, fibrinous bronchitis, hemorrhagic bronchitis, infectious avian bronchitis, occlusive bronchitis, plastic bronchitis, pseudomembranous bronchitis, putrefactive bronchitis, and helminthic bronchitis), bronchioal granulomatosis, bronchial edema, bronchoesophageal disease, bronchogenic carcinoma, bronchogenic cysts, broncholithiasis, bronchomalacia, bronchial mycosis (such as bronchopulmonary aspergillosis), bronchopulmonary spiropathy, hemorrhagic bronchitis, bronchial myxoma, bronchospasm, bronchial hemorrhage, bronchial stenosis, Biot's respiratory sounds, bronchial respiratory sounds, Kussmaul-kien respiratory sounds, respiratory acidosis, respiratory alkalosis, neonatal respiratory distress syndrome, respiratory insufficiency, respiratory scleroses, respiratory syncytial virus, and the like.

In a specific embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention, and/or agonists and/or antagonists thereof, are useful for the treatment, prevention, and/or diagnosis of Chronic Obstructive Pulmonary Disease (COPD).

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides and/or agonists and/or antagonists thereof of the present invention are used for the treatment, prevention and/or diagnosis of pathologies associated with fibrosis, such as, but not limited to, cystic fibrosis (including pancreatic cystic fibrosis, Clarke-Hadfield syndrome, pancreatic fibrocysts, and mucinous lesions), cardiac fibrosis, primary retroperitoneal fibrosis, flexo-meningeal fibrosis, mediastinal fibrosis, subcortical nodule fibrosis, central peripheral fibrosis, perimyofibrosis, vocal cord fibrosis, subclinical fibrosis and Symmer's clay fibrosis.

TNF family ligands are known to be the most potent cytokines, inducing a number of cellular responses including cytotoxicity, antiviral activity, immunomodulatory activity, and transcriptional regulation of several genes (D.V.Goeddet al, "tumor necrosis factor: Gene Structure and biological Activity", Symp. Quant. biol.51: 597-609(1986), Cold spring harbor; B.Beutler and A.Cerami, Biochemical analysis Res 57: 505-5181 (1988); L.J.Old.Sci.Am.258: 59-75 (1988); W.Fiers, FEBs communication 284: 199-224 (1991)). TNF family ligands, including Neutrokine-alpha and/or Neutrokine-alpha SV of the present invention, induce such diverse cellular responses by binding to TNF family receptors. Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides are believed to elicit potent cellular responses, including cells, cell lines, tissues. A change in genotype, phenotype and/or morphology of the tissue culture or patient. As noted above, such cellular responses include not only normal physiological responses to TNF family ligands, but also diseases associated with increased or inhibited apoptosis. Apoptosis is a physiological mechanism involved in the loss of peripheral B and/or T lymphocytes of the immune system and its dysfunction can lead to a number of different pathological changes (J.C. Ameisen, AIDS 8: 1197-1213, (1994); P.H. Kramer et al, Immunol. general 6: 279-289 (1994)).

Diseases associated with increased cell viability or inhibition of apoptosis that may be diagnosed, treated or prevented using the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention, as well as agonists and antagonists thereof, include cancers (e.g., cystic lymphoma, cancers due to p53 mutations, and hormone-dependent tumors including, but not limited to, colon cancer, myocardial tumor, pancreatic duct cancer, melanoma, retinal glioma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, bone cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteogenic C tumor, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma, and ovarian cancer); autoimmune diseases (e.g., systemic lupus erythematosus, and immune-related glomerulonephritis, rheumatoid arthritis); viral infections (e.g., herpes viruses, pox viruses, and adenovirus); inflammation; graft versus host; acute transplant rejection and chronic transplant rejection. Thus, in preferred embodiments, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention and/or agonists or antagonists thereof, for the treatment, prevention and/or diagnosis of autoimmune diseases and/or suppression, growth, progression and/or metastasis of cancers, including but not limited to those described herein, such as lymphocytic leukemia (including, for example, MLL, and Chronic Lymphocytic Leukemia (CLL) and lymphofollicular tumors, hi another embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention are useful for activating cancer cells or tissues such as B-cell line associated cancers (e.g., CLL and MLL), lymphocytic leukemia, or lymphoma) to render the cells more receptive to cancer treatment (e.g., chemotherapy or radiation therapy).

In addition, in other embodiments, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides or agonists or antagonists thereof of the present invention are useful for inhibiting the growth, progression and/or metastasis of malignancies and related diseases such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myelogenous C-leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia)) and chronic leukemias (e.g., chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (Hodgkin's disease and non-Hdgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid-state tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, myeloma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilm's tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytic cancer, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, melanoma, neuroblastoma and retinoblastoma.

Diseases associated with increased apoptosis, including AIDS, that may be diagnosed, treated or prevented using the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention and agonists and antagonists thereof; neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis, cerebellar degeneration); myelodysplastic syndromes (e.g., aplastic anemia), ischemic injury (e.g., myocardial infarction, stroke, and reperfusion injury), toxin-induced liver disease (e.g., due to ethanol), septic shock, cachexia, and anorexia. Thus, in a preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention and/or agonists or antagonists thereof are used for the treatment, prevention and/or diagnosis of the above mentioned diseases.

In a preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides and/or agonists or antagonists thereof (e.g., anti-Neutrokine-alpha antibodies) of the present invention inhibit the growth of human histiocytic lymphoma U-937 cells in a dose-dependent manner. In another preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides and/or agonists or antagonists thereof (e.g., anti-Neutrokine-alpha antibodies) of the present invention inhibit the growth of PC-3 cells, HT-29 cells, Hela cells, MCF-7 cells and A293 cells. In another more preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides or polypeptides of the invention and/or agonists thereof (e.g., anti-Neutrokine-alpha antibodies) are used to inhibit the growth, progression and/or metastasis of prostate, colon, cervical and breast cancer.

Thus, in another preferred embodiment, the invention relates to a method of increasing apoptosis induced by a TNF family ligand comprising administering to a cell expressing a receptor for Neutrokine-alpha and/or Neutrokine-alpha SV an effective amount of Neutrokine-alpha and/or Neutrokine-alpha SV, or an agonist or antagonist thereof, that increases or decreases Neutrokine-alpha and/or Neutrokine-alpha SV mediated signaling. Preferably, increased or decreased Neutrokine-alpha and/or Neutrokine-alpha SV mediated signaling may treat, prevent and/or diagnose diseases in which apoptosis is decreased or expression of cytokines and adsorbed molecules is decreased. Agonists or antagonists may include soluble forms of Neutrokine-alpha and/or Neutrokine-alpha SV and monoclonal antibodies directed against Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides.

In another aspect, the invention relates to a method of inhibiting apoptosis induced by a TNF family ligand comprising administering to a cell expressing a receptor for Neutrokine-alpha and/or Neutrokine-alpha SV an effective amount of an agonist or antagonist that increases or decreases Neutrokine-alpha and/or Neutrokine-alpha SV mediated signaling. Preferably, increased or decreased Neutrokine-alpha and/or Neutrokine-alpha SV mediated signaling may treat, prevent and/or diagnose diseases in which apoptosis or NF-KB expression is increased. Agonists or antagonists may include soluble forms of Neutrokine-alpha and/or Neutrokine-alpha SV and monoclonal antibodies directed against Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides.

Since Neutrokine-alpha and/or Neutrokine-alpha SV belongs to the TNF superfamily, this polypeptide should also modulate angiogenesis. In addition, the polypeptide has a wide range of anti-inflammatory activity due to Neutrokine-alpha and/or Neutrokine-alpha SV suppressing immune cell function. Neutrokine-alpha and/or Neutrokine-alpha SV may be used as anti-neoangiogenesis agents for the treatment, prevention and/or diagnosis of solid tumors, by stimulating host defense cell invasion and activation, such as cytotoxic T cells and macrophages, and by inhibiting angiogenesis of tumors. One skilled in the art can identify other non-cancerous indications in which vascular proliferation is undesirable. They may also be used to enhance host resistance to chronic and acute infections, for example against myobacterial infections by attracting and activating microbicidal leukocytes. Neutrokine-alpha and/or Neutrokine-alpha SV may also inhibit T cell proliferation by inhibiting IL-2 biosynthesis for the treatment of T cell-mediated autoimmune diseases and lymphocytic leukemias, including, for example, Chronic Lymphocytic Leukemia (CLL). Neutrokine-alpha and/or Neutrokine-alpha SV may also be used to stimulate wound healing by recruiting connective tissue for debris clearance and initiating inflammatory cells. In the same manner, Neutrokine-alpha and/or Neutrokine-alpha SV may also be used in the treatment, prevention and/or diagnosis of other fibrotic diseases, including cirrhosis of the liver, osteoarthritis and pulmonary fibrosis. Neutrokine-alpha and/or Neutrokine-alpha SV also increases the content of eosinophils, which have the unique function of killing a large number of parasites, such as schistosomes, trichinella and roundworms, that infest tissues. It can also be used to regulate hematopoiesis by regulating the activation and differentiation of various hematopoietic progenitor cells, such as the release of mature leukocytes from bone marrow after chemotherapy, i.e., in stem cell metastasis. Neutrokine-alpha and/or Neutrokine-alpha SV may also be used for the treatment, prevention and/or diagnosis of sepsis.

The polynucleotides or/and polypeptides of the invention and/or agonists and/or antagonists thereof are useful in the diagnosis and treatment or prevention of a number of diseases and/or pathological changes. Such diseases and pathological changes include, but are not limited to, cancer (e.g., immune cell-related cancer, breast cancer, prostate cancer, ovarian cancer, lymphoid follicular tumor, cancer associated with p53 mutations or alterations, brain tumor, bladder cancer, cervical cancer, colon cancer, colorectal cancer, non-small cell lung cancer, gastric cancer, etc.), lymphoproliferative disease (e.g., lymphadenopathy), microbial (e.g., viral, bacterial, etc.) infection, (e.g., HIV-1 infection, HIV-2 infection, herpes viral infection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenoviral infection, poxvirus infection, human papilloma viral infection, hepatitis viral infection (e.g., HAV, HBV, HCV, etc.), helicobacter pylori infection, staphylococcus, etc.), parasitic infection, nephritis, bone disease (e.g., osteoporosis), atherosclerosis, pain, cardiovascular diseases (e.g., neovascularization, angiogenesis reduction, or reduced circulating blood volume (e.g., ischemic diseases) such as myocardial infarction, stroke, etc.)), AIDS, allergy, inflammation, neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration, etc.), transplant rejection (acute and chronic), graft-versus-host disease, diseases caused by myelodysplasia (e.g., aplastic anemia, etc.), joint tissue destruction in rheumatism, liver diseases (e.g., acute and chronic hepatitis, liver injury and cirrhosis), autoimmune diseases (e.g., multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis, etc.), cardiomyopathy (such as dilated cardiomyopathy), diabetes, diabetic complications (such as diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephritis, septic shock and ulcerative colitis.

The polynucleotide and/or polypeptide of the present invention and/or agonists and/or antagonists thereof are useful for promoting angiogenesis, wound healing (e.g., wounds, burns and bone fractures). The polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof, also act as adjuvants to enhance the immune response to specific antigens, anti-viral immune response.

Typically, the polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are used to modulate (i.e. provoke or reduce) an immune response. For example, the polynucleotides and/or polypeptides of the invention may be used in preparation for or recovery from surgery, trauma, radiation therapy, chemotherapy and transplantation, or may be used to assist immune response and/or recovery in the elderly and immunocompromised. Alternatively, the polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful as immunosuppressive agents, e.g. in the treatment or prevention of autoimmune diseases. In specific embodiments, the polynucleotides and/or polypeptides of the invention are used to treat or prevent chronic inflammatory, allergic, or autoimmune diseases, such as those described herein and others known in the art.

Preferably, an effective amount of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide, or agonist or antagonist thereof, of the present invention, is administered to a patient, using a Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotide or polypeptide, and/or agonist or antagonist thereof (e.g., an anti-Neutrokine-alpha antibody); or removing the cells from the patient, feeding the cells with Neutrokine-alpha and/or Neutrokine-alpha SV polynucleotides, and then re-administering the engineered cells to the patient [ ex vivo therapy ]. In addition, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides or polynucleotides as described further herein may be used as adjuvants in vaccines to generate immune responses against infectious diseases.

Formulations and methods of administration

The Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide compositions (preferably containing polypeptides that are soluble forms of Neutrokine-alpha and/or Neutrokine-alpha SV ectodomain) are formulated and dosed for good medical use, taking into account the clinical condition of the individual patient (especially the side effects of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide alone), the site of delivery of the Neutrokine-alpha and/or polypeptide composition, the method of administration, the administration procedure, and other known factors. An "effective amount" of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide is thus determined by such conditions.

Generally, the total pharmaceutically effective amount of each dose of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide administered parenterally will range from about 1 μ g/kg day to 10mg/kg body weight/day, although therapeutic decisions will be made as described above. Preferably the dose is at least 0.01mg/kg body weight/day, more preferably between 0.01 and 1mg/kg body weight/day.

In another embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide of the invention is administered to a human in a dose of between 0.0001-0.045 mg/kg body weight/day, preferably between 0.0045-0.045 mg/kg body weight/day, more preferably 45 μ g/kg body weight/day; the dose administered to mice was approximately 3mg/kg body weight/day.

If used continuously, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides are administered in a dose of about 1. mu.g/kg body weight/hour to 50. mu.g/kg body weight/hour, injected 1-4 times per day or continuously subcutaneously infused, e.g., with a micropump. Intravenous infusion may also be used.

The treatment time is determined according to the effect.

In a specific embodiment, the total pharmaceutically effective amount of each dose of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide administered parenterally ranges from about 0.1 μ g/kg body weight/day to 45 μ g/kg body weight/day, although therapeutic decisions are made as described above. Preferably, the dosage is at least 0.1 μ g/kg body weight/day, more preferably the dosage is between 0.01 to 50 μ g/kg body weight/day for administration to a human. Neutrokine-alpha and/or Neutrokine-alpha SV may be infused continuously, injected multiple times per day (e.g., three or more times per day, or twice per day, once per day, or injected intermittently (e.g., twice per day, once per day, every other day, twice per week, once a month, once in two months, once in three months.) if administered continuously, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides are administered at a dose of about 0.001 to 10 μ g/kg body weight/hour to about 50 μ g/kg body weight/hour, injected 1 to 4 times per day, or continuously subcutaneously, e.g., using a micro-pump.

Effective dosages of the compositions of the present invention to be administered can be determined by methods known in the art, noting parameters such as biological half-life, biological efficacy, and toxicity. Such assays are well known to those skilled in the art.

Biological exposure of the body to Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides during treatment also plays an important role in a definitive and/or pharmaceutically effective amount. Variations in dosage, such as repeated administration of a relatively low dose of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide for a relatively long period of time, and repeated administration of a relatively high dose of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide for a relatively short period of time, have different therapeutic and/or pharmacological effects. See, e.g., the serum immunoglobulin level assay shown in example 6.

Using equivalent surface area dose conversion factors provided by Freirich, E.J., et al (cancer chemotherapy reports 50 (4): 219-44(1966)), one skilled in the art can routinely convert the data obtained from using Neutrokine-alpha and/or Neutrokine-alpha SV in a given assay into an accurate estimate of the pharmaceutically effective amount of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide per dose administered in another assay line. From experimental data obtained by administering Neutrokine-alpha in mice (see, e.g., example 6), the pharmaceutically effective amount of Neutrokine-alpha in rats, monkeys, dogs and humans can be accurately assessed by a conversion factor provided by Freiich et al. The following translation table (Table 3) summarizes the data provided by Freirich et al. Table 3 provides an approximation factor that converts the dose in mg/kg body weight in one species to an equivalent surface area dose in mg/kg body weight in another species.

TABLE 3 equivalent surface area dose conversion factor

From now on	Mouse (20g)	Rat (150g)	Monkey (3.5kg)	Dog (8kg)	Human (60kg)
						Mouse, rat, monkey, dog and human	124612	1/21247	1/41/215/33	1/61/43/512	1/121/71/31/21

Thus, for example, using the conversion factors provided in table 3, a dose of 50mg/kg in mice was converted to an appropriate dose of 12.5mg/kg in monkeys, since (50mg/kg) × 1/4 is 12.5 mg/kg. In addition, doses of 0.02, 0.08, 0.8, 2 and 8mg/kg in mice are equivalent to the effect of doses of 1.667. mu.g/kg, 6.67. mu.g/kg, 66.7. mu.g/kg, 166.7. mu.g/kg and 0.667mg/kg in humans.

Pharmaceutical compositions containing Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention may be administered orally, rectally, parenterally, subcutaneously, intracistemally, intravaginally, intraperitoneally, topically (in the form of powders, ointments, drops or patches), buccally, or by oral or nasal spray (e.g. vapor or powder inhalation), and the like. In one embodiment, "pharmaceutically suitable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulate, or formulation auxiliary of any type. In a specific embodiment, "pharmaceutically suitable" refers to an article that is approved by a governmental regulatory agency, the U.S. department of medicine, for use in animals, particularly humans. Suitable pharmaceutical carriers according to this embodiment are provided by e.w. martin in "Remington's pharmaceutical sciences" for example, without limitation, and include sterile liquids such as water and oils, including those derived from petroleum, animal, vegetable or synthetic sources such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is injected intravenously. Saline and dextrose and glycerol solutions are useful as liquid carriers, particularly for injectable solutions, and the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.

The term "parenteral" refers to modes of administration that include intravenous, intraperitoneal, subcutaneous and intraarterial, and gluteal injections and infusions.

In a preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV compositions (including polypeptides, polynucleotides and antibodies, agonists and/or antagonists thereof) of the present invention are administered subcutaneously.

In another preferred embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV compositions (including polypeptides, polynucleotides and antibodies, agonists and/or antagonists thereof) of the present invention are administered intravenously.

Neutrokine-alpha and/or Neutrokine-alpha SV compositions of the present invention may also be suitably administered via a sustained release system. Sustained release compositions include, for example, a suitable polymer (e.g., a semipermeable polymeric matrix such as a film or microcapsule), a suitable hydrophobe (e.g., a suitable oil emulsion), or an ion exchange resin, and a small amount of a soluble derivative (e.g., a small amount of a soluble salt).

Sustained release matrices include polylactic acid (U.S. Pat. No.3773919, EP58481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, V. et al, biopolymer 22: 547-: 98-105(1982), ethyl vinyl acetate (R.Langer et al, Id.) or poly-D-3-hydroxybutyric acid (EP 133988).

Sustained release compositions also include liposome-entrapped compositions of the invention (see Langer, science 249: 1527) -1533 (1990)); treat et al, liposomes in the treatment of infectious diseases and cancer, Lopez-Berestein and Fidler (ed.), Liss, New York, p317-327 and 353-. Liposomes containing Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides may be prepared by known methods, see DE 3218121; epstein et al, proceedings of the national academy of sciences USA 82: 3688-3692 (1985); hwang et al, Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980); EP 52322; EP 36676; EP 88046; EP 143949; EP 142641; japanese patent application 83-118008; U.S. patent nos. 4485045 and 4544545; and EP 102324. Typically, the liposomes are small unilamellar (approximately 200-800Angstroms) with lipid content greater than 30mol cholesterol, and the selection ratios are adjusted for optimal Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide therapy.

In another embodiment, the sustained release composition of the present invention comprises a crystalline formulation known in the art.

In another embodiment, the compositions of the invention are delivered by a pump (see Langer, supra; Sefton, CRC Crit Ref. biomed. Eng.14: 201 (1987); Buchwald et al, science 88: 507 (1980); Saudek et al, N.Engl. J.Med 321: 574 (1989)).

Other controlled release systems are described in Langer (science 249: 1527-.

For parenteral administration, in one embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides are generally in the desired purity, in unit dose injectable form (solution, suspension or emulsion), with a pharmaceutically suitable carrier, i.e., a carrier that is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients in the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds known to be detrimental to polypeptides.

In general, formulations are formulated by uniformly and intimately bringing into contact the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides with liquid carriers or well-separated solid carriers or both. The product is then formed into the desired shape, if desired. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Such carriers include, for example, water, saline, ringer's solution, and dextrose solution. Non-aqueous carriers such as fixed lipids and ethyl oleic acid and liposomes may also be used herein.

The carrier suitably contains minor amounts of additives such as to enhance isotonicity and chemical stability. Such substances are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphoric acid, citric acid, succinic acid, acetic acid and other organic acids or salts thereof; antioxidants such as ascorbic acid; low molecular weight (less than 10 residues) polypeptides such as polyarginine or tripeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides, or other carbohydrates including cellulose or its derivatives, glucose, mannose, sucrose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counter ions such as sodium ions; preservatives such as cresols, phenol, chlorobutanol, benzylethanol, and parabens and/or nonionic surfactants such as polysorbates, poloxamers or PEG.

The concentration of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide formulated in such a carrier is about 0.001mg/ml to 100mg/ml, or 0.1mg/ml to 100mg/ml, preferably 1-10mg/ml, or 1-10mg/ml, and the pH is about 3-10, or 2-8, preferably 5-8, more preferably 6-7. It will be appreciated that the use of some of the aforementioned receptors, carriers or stabilizers will result in the formation of salts of Neutrokine-alpha and/or Neutrokine-alpha SV.

Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides for therapeutic administration must be sterile. Sterility can be achieved by filtration through sterile filtration membranes (e.g., 0.2micron membranes). Therapeutic Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide compositions are typically placed in a container having a sterile passageway, such as an intravenous injection solution bag or a vial having a stopper pierceable by a hypodermic injection needle.

Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides are typically stored in unit-dose or multi-dose containers, such as sealed ampoules or vials, in aqueous solution or in reconstitutable lyophilized form. The lyophilized formulation is, for example, a 10ml vial containing 5ml of sterile filtered 1% (w/v) aqueous Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide solution and the resulting mixture is lyophilized. The perfusate is prepared by reconstituting the lyophilized Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide with sterile water for injection.

Alternatively, Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide is stored in lyophilized form in single dose containers. The perfusate is reconstituted with sterile injectable vehicle.

The invention also provides a pharmaceutical package or kit. Comprising one or more containers filled with one or more ingredients of the pharmaceutical composition of the invention. Such containers are also accompanied by a notice of approval for human use by a governmental agency responsible for the approval of the manufacture, use or sale of pharmaceuticals or biological products. In addition, the polypeptides of the invention may be used in combination with other therapeutic compounds.

The compositions of the invention may be administered alone or in combination with other adjuvants. Adjuvants that may be used in combination with the compositions of the present invention include, but are not limited to, alum plus deoxycholate (Immuno Ag), MTP-PE (Biocine Corp.) QS21(Genentech, Inc.), BCG and MPI. In a specific embodiment, the compositions of the invention are administered in combination with QS-21. Other adjuvants that may be used in combination with the compositions of the present invention include, but are not limited to, lipid monophosphoryl immunomodulators, AdjuVax 100a,QS-21, QS-18, CRL1005, aluminium salts, MF-59 and viral adjuvants. Vaccines that may be used with the compositions of the present invention include, but are not limited to, MMR (measles, mumps, rubella) vaccines, polio vaccines, varicella vaccines, tetanus vaccines, diphtheria vaccines, hepatitis A vaccines, hepatitis B vaccines, Haemophilus influenza B vaccines, pertussis vaccines, pneumonia vaccines, influenza vaccines, Lyme's disease vaccines, cholera vaccines, yellow fever vaccines, epidemic encephalitis vaccines, rabies vaccines, tetanus vaccines, pertussis vaccines and/or EUPNMOVAX-23 ^TM. Administration may be concomitant, e.g., as a mixture, separately but simultaneously; or may be administered in a sequential combination, which includes presentation, wherein the combined agents are administered together as a therapeutic mixture, and also includes separate but simultaneous injections of the combined agents into the same body, e.g., via separate intravenous routes. "combination" administration also includes administration of one compound followed by another, respectively.

In another specific embodiment, the compositions of the invention are administered with PNEVMOVAX-23^TMIn combination, treat, prevent and/or diagnose infectious diseases and/or any diseases associated therewith. In one embodiment, the composition of the invention is combined with PNEVMOVAX-23^TMCombined administration, treatment, prevention and/or diagnosis of any gram-positive bacterial infection and/or any disease associated therewith. In another embodiment, the compositions of the invention are combined with PNEVMOVAX-23^TMCombined administration, treatment prevention and/or diagnosis of infections associated with one or more members of the enterobacter and/or streptomyces genera. In another embodiment, the compositions of the invention are administered with PNEOMOVAX-23^TMIn combination, treat, prevent and/or diagnose a disease associated with one or more members of the genus Streptococcus B. In another embodiment, the compositions of the invention are administered with PNEOMOVAX-23 ^TMIn combination, for the treatment, prevention and/or diagnosis of a disease associated with streptococcus pneumoniae.

The compositions of the present invention may be administered alone, or in combination with other therapeutic agents, including but not limited to chemotherapeutic agents, antibiotics, antiviral agents, steroidal and nonsteroidal anti-inflammatory agents, conventional immunotherapeutic agents, and cytokines. The combined administration may be concomitant administration, e.g., as a mixture, separately but simultaneously; or administered sequentially. Including the administration of the combination agents together as a therapeutic mixture, and also including the separate but simultaneous administration of the combination agents, e.g., by simultaneous infusion into the same individual via separate intravenous routes, "combined" administration also includes the separate administration of one compound followed by the other.

In one embodiment, the compositions of the invention are administered in combination with other members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered in combination with the compositions of the invention include, but are not limited to, soluble forms of TNF- α and/or α -lymphotoxin (α -LT, also known as TNF- β), LT- β (found in the heterotrimeric LT- α 2- β complex), OPGL, FasL, CD27L, CD30L, CD40L, 40-1BBL, DcR3, OX40L, γ -TNF (International publication WO 96/14328), AIM-I (International publication WO 97/33899), AIM-II (International publication WO 97/34911), APRIL (J.Exp.Med.188 (6): 1185 OX 1190), α -intrinsic factor (International publication WO98/07880), TR6 (International publication WO98/30694), OPG and Neutrokine- α (International publication WO 98/18921), 40 and Nerve Growth Factor (NGF) and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International publication WO98/34095), DR3 (International publication WO 97/33904), DR4 (International publication WO98/32856), TR5 (International publication WO 98/30693), TR6 (International publication WO98/30694) TR7 (International publication WO 98/41629), TRANK, TR9 (International publication WO 98/56892), TR10 (International publication WO 98/54202), 312C2 (International publication WO 98/06842) and TR 12.

In a preferred embodiment, the compositions of the invention are combined with a CD40 ligand (CD40L), a soluble form of CD40L (e.g., AVREND)^TM) A biologically active fragment, variant or derivative of CD40L, an anti-CD 40L antibody (e.g., an agonistic or antagonistic antibody), and/or an anti-CD 40 antibody (e.g., an agonistic or antagonistic antibody).

In some embodiments, the compositions of the present invention are administered in combination with an antiretroviral agent, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, and/or protease inhibitor. Nucleoside reverse transcriptases that may be administered in combination with the compositions of the invention include, but are not limited toNot restricted to RETROVIR^TM(zidovudine/AZT)，VIDEX^TM(didanosine/ddI)，HIVID^TM(zalcitabine/ddC)，ZERIT^TM(stavudine/d4T)，EPIVIR^TM(lamivudine/3 TC), and COMBIVIR^TM(zidovudine/lamivudine). Non-nucleoside reverse transcriptases that may be administered in combination with the compositions of the invention include, but are not limited to, VIRAMUNE^TM(nevirapine)，RESCRIPTOR^TM(delavirdine), and SUSTIVA^TM(efavirenz). Protease inhibitors that can be administered in combination with the compositions of the invention include, but are not limited to, CRXIVAN^TM(indinavir)，NORVIR^TM(ritonavir)，INVIRASE^TM(saquinavir), and ViRACEPT^TM(nelfinavir). In a specific embodiment, antiviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be combined with any of the compositions of the present invention to treat, prevent and/or diagnose AIDS and/or treat, prevent and/or diagnose infection by HIV.

In other embodiments, the compositions of the present invention may be used in combination with an anti-opportunistic infection agent. Anti-opportunistic infection agents that can be administered in combination with the compositions of the present invention include, but are not limited to, TRIMETHOPRIM-sulfomethoxazole^TM，DAPSONE^TM，PENTAMIDINE^TM，ATOVAQUONE^TM，ISONIAZID^TM，RIFAMPIN^TM，PYRAZINAMIDE^TM，ETHAMBUTOL^TM，RIFABUTIN^TM，CLARITHROMYCIN^TM，AZITHROMYCIN^TM，GANCICLOVIR^TM，FOSCARNET^TM，CIDOFOVIR^TM，FLUCONAZOLE^TM，ITRACONAZOLE^TM，KETOCONAZOLE^TM，ACYCLOVIR^TM，FAMCICOLVIR^TM，PYRIMETHAMINE^TM，LEUCOVORIN^TM，NEVPOGEN^TM(filigram/G-CSF), and Leukine^TM(sargramostim/GM-CSF). In a specific embodiment, the composition of the invention may be combined with TRIMETHOPRIM-sulfethyoxazole^TM，DAPSONE^TM，PENTAMIDINE^TMAnd/or ATOVAQUONE^TMAny combination to prophylactically treat, prevent and/or diagnose opportunistic pneumococcistis carinii infection. In another specific embodiment, the compositions of the invention are administered with ISONIAZID^TM，RIFAMPIN^TM，PYRAZINAMIDE^TMAnd/or ETHAMBUTOL^TMAny combination thereof, for the prophylactic treatment, prevention and/or diagnosis of opportunistic infection by a Mycobacterium avium complex. In another specific embodiment, the compositions of the invention are administered with RIFABUTIN^TM，CLARITHROMYCIN^TMAnd AZITHROMYCIN^TMAny combination thereof, for prophylactic treatment, prevention and/or diagnosis of opportunistic tubercle bacillus infection. In another specific embodiment, the compositions of the invention are administered with GANCICLOVIR^TM，FOSCARNET^TMAnd/or CIDOFOVIR^TMAny combination to prophylactically treat, prevent and/or diagnose an opportunistic cytomegalovirus infection. In another specific embodiment, the compositions of the invention are administered with fluconizole ^TM，ITRACONAZOLE^TMAnd/or KETOCONAZOLE^TMAny combination thereof, for prophylactic treatment, prevention and/or diagnosis of opportunistic fungal infections. In another specific embodiment, the compositions of the invention are administered with acyclovir^TMAnd/or FAMCICOLVIR^TMAny combination thereof, for prophylactic treatment, prevention and/or diagnosis of opportunistic type I or type II herpes simplex virus infections. In another specific embodiment, the compositions of the invention are admixed with PYRIMETHAMINE^TMAnd/or Leucomorbin^TMAny combination, to prophylactically treat, prevent and/or diagnose opportunistic Toxoplasma gondii infections. In another specific embodiment, the compositions of the invention are administered with Leucomorborin^TMAnd/or NEUPOGEN^TMAny combination thereof, for prophylactic treatment, prevention and/or diagnosis of opportunistic bacterial infections.

In another embodiment, the compositions of the present invention are administered in combination with an antiviral agent. Antiviral agents that may be administered in combination with the compositions of the present invention include, but are not limited to, acyclovir, ribavirin, amantadine, and rematidine.

In another embodiment, the compositions of the invention are administered in combination with an antibiotic. Antibiotics that may be administered in combination with the compositions of the present invention include, but are not limited to, amoxicillin, aminoglycoside antibiotics, beta-lactams (glycopeptides), beta-lactamases, clindamycin, chloramphenicol, cephalosporins, tendomycin, erythromycin, fluorescein quinomycin, macrolides, 2-methyl-5-nitro-1-imidazolylethanol, penicillins, quinomycin, rifampin, streptomycin, sulfonamides, tetracyclines, trimethoprim-thiamine imidazole, and vancomycin.

Conventional non-specific immunosuppressive agents that may be administered in combination with the compositions of the present invention include, but are not limited to, steroids, cyclosporine analogs cyclophosphamide, cyclophosphamide IV, methylprednisolone, prednisolone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that inhibit responsive T cell function.

In specific embodiments, the compositions of the invention are administered in combination with an immunosuppressive agent. Immunosuppressant preparations which can be administered in combination with the compositions of the present invention include, but are not limited to, orthioclone^TM(OKT3)，SANDIMMUNE^TM/NEORAL^TM/SANGDYA^TM(Cyclosporin), PROGRAF^TM(tacrolimus)，CELLCEPT^TM(mycophenolate salts), azathioprine, glucortheroides, and RAPAMUNE^TM(sirolimus). In a specific embodiment, the immunosuppressive agent is useful for preventing rejection in organ or bone marrow transplants.

In a preferred embodiment, the composition of the invention is administered in combination with steroid therapy. Steroids that may be used in combination with the present invention include, but are not limited to, oral cortisone, prednisone, and methylprednisolone (e.g., IV methylprednisolone). In a specific embodiment, the composition of the invention is administered in combination with prednisone. In another specific embodiment, the composition of the invention is administered in combination with prednisone and an immunosuppressive agent. Immunosuppressive agents that can be administered in combination with the compositions of the invention and prednisone are as described herein, including but not limited to azathioprine, cyclophosphamide and cyclophosphamide IV. In another specific embodiment, the composition of the invention is administered in combination with methylprednisolone. In another specific embodiment, the composition of the invention is used in combination with methylprednisolone and an immunosuppressive agent. Immunosuppressive agents that can be administered in combination with the compositions of the invention and methylprednisolone are as described herein, including but not limited to azathioprine, cyclophosphamide and cyclophosphamide IV.

In a preferred embodiment, the composition of the invention is administered in combination with an antimalarial agent. Antimalarial agents that may be administered in combination with the compositions of the present invention include, but are not limited to, hydroxychloroquine, chloroquine, and/or quinacrine.

In a preferred embodiment, the compositions of the present invention are administered in combination with an NSAID.

In one embodiment, the compositions of the present invention are administered in combination with one, two, three, four, five, ten or more of the following: NRD-101(Hoechst Marion Roussel), cyfluazin (Dimetaloid), oxaprozin potassium (Monsanto), mecastemin (Chiron), T-614(Toyama), pemetrexed disodium (Eli Lilly), atrileuton (Abbott), valdecoxib (Monsanto), eletenac (Byk Gulden), campath, AGM-1470(Takeda), CDP-571(Celltech Chiroscience), CM101(CarboMed), ML-3000(Merckle), CB-2431(KS Biomedix), CBF-BS2(KS Biomedix), IL-1 gene therapy (Valentis), JTE-522 (JapanTotoco), paclitaxel (Angelica Biohraphe DW-166), Whithromy Dohrfa, IL-1Ra gene therapy (Boshimage), Boshimehrne (I-1), Bogeemve-284 (I), Bogeemx-Michel), Pixel-1 (I), Pigeemx-Michel-11 (Pixel), Pigeffex-166), Pigeherx-Hadame-Michel-11 (I), Pigeherx), Pigeffe-Micex, Pigeherx-Micex (I-Micex), Pigeherx Micex Hadame-Micex, Pigeherx (I-Micex), Pigeherz-Micex (I-Micex) and Pigeherz) or Pigeherz et Micex (I), Pigeherz et Micex receptor (I), Pigeherz) or Micex receptor (I), Pi, ST-1482(Sigma-Tau), and butixocort propionate (Warner Lam bent).

In a preferred embodiment, the composition of the invention is administered in combination with one, two, three, four, five or more of the following drugs: methotrexate, sulfasalazine, gold sulfomalate, acethioglucamide, cyclosporine, penicillamine, azathioprine, antimalarial (as described herein), cyclophosphamide, meconine, gold, ENBREL^TM(Etanercept), anti-TNF antibodies, and prednisolone.

In a more preferred embodiment, the compositions of the invention are administered with an anti-malarial drug, methotrexate, an anti-TNF antibody, ENBREL^TMAnd/or sulfasalazine. In one embodiment, the composition of the invention is administered in combination with methotrexate. In another embodiment, the compositions of the invention are administered in combination with an anti-TNF antibody. In another embodiment, the compositions of the invention are administered in combination with methotrexate and an anti-TNF antibody. In another embodiment, the composition of the invention is administered in combination with sulfasalazine. In another specific embodiment, the compositions of the invention are used with ENBREL^TMThe administration is combined. In another embodiment, the composition of the invention is combined with ENBREL^TMMethotrexate, and sulfasalazine. In other embodiments, one or more anti-malarial drugs are administered in combination with one of the combinations described above. In a specific embodiment, the compositions of the invention are administered in combination with an anti-malarial drug (e.g., hydroxychloroquine), sulfasalazine, an anti-TNF antibody, and methotrexate.

In another embodiment, the compositions of the invention are administered alone or in combination with one or more intravenous immunoglobulin. Intravenous immunoglobulins that can be administered in combination with the compositions of the present invention include, but are not limited to, GAMMA^TM，IVEEGAM^TM，SANDOGLOBULIN^TM，GAMMAGARD S/D ^TMAnd GAMMA-methyl^TM. In a specific embodiment, the compositions of the invention are administered in combination with intravenous immunoglobulin in a transplantation therapy (e.g., bone marrow transplantation).

CD40 ligand (CD40L), a soluble form of CD40L (e.g., AUREND @)^TM) A biologically active fragment, variant or derivative of CD40L, an anti-CD 40L antibody (e.g., an agonistic or antagonistic antibody), and/or an anti-CD 40 antibody (e.g., an agonistic or antagonistic antibody).

In another embodiment, the compositions of the present invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered in combination with the compositions of the present invention include, but are not limited to, glucocorticoids and non-hormonal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acid derivatives, arylpropionic acid derivatives, pyrazoles, pyrazolines, salicylic acid derivatives, carbamothiazine, e-acetamidohexanoic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, benzathine, 6-benzyladenine, bucolone, bisphenylacetamine, dibenzazole, emorfazone, guaiazole, nabumetone, nimesulide, orgotein, oxaprostol, paranyline, perinox, pizoxine, proquazone, proxazole, and tenidapa.

In another embodiment, the compounds of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered in combination with the compositions of the present invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin and daunorubicin); antiestrogens (such as tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, 5-fluorouracil deoxynucleoside, alpha-interferon 2b, glutamic acid, mithramycin, mercaptopurine, and b-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, 1-lomustine, CCNU, cytarabine, cyclophosphamide, estramustine phosphate, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); hormones (e.g., megestrol, estramustine sodium phosphate, ethylestradiol, estradiol, pregnenone acetate, methyltestosterone, diethylstilbestrol diphosphate, tris-methoxychloroethylene, and testosterone); nitrogen mustard derivatives (e.g., mephalen, chlorambucil, mechlorethamine, and thiotepa); steroids and compositions (e.g., sodium betamethasone phosphate); and other substances (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and epipodophyllotoxin glucopyranoside).

In a specific embodiment, the composition of the invention is administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) or any combination of CHOP components. In another embodiment, the composition of the invention is administered in combination with Rituximab. In another embodiment, the compositions of the present invention are administered in combination with Rituximab and CHOP, or any combination of Rituxamab and CHOP components.

In another embodiment, the composition of the invention is administered in combination with a cytokine. Cytokines that can be administered in combination with the compositions of the invention include, but are not limited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD 40, CD40L, IFN- α, IFN- β, IFN- γ, TNF- α, TNF- β. In another embodiment, the composition of the invention can be administered in combination with any interleukin, including but not limited to IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, and IL-22. In a preferred embodiment, the composition of the invention is administered in combination with IL4 and IL 10. The inventors observed that both IL4 and IL10 enhanced Neutrokine-alpha mediated B cell proliferation.

In another embodiment, the compositions of the invention are administered in combination with a chemokine. In another embodiment, the compositions of the invention are administered in combination with chemokine beta-8, chemokine beta-1, and/or macrophage inflammatory protein 4. In a preferred embodiment, the compositions of the invention are administered in combination with chemokine beta-8.

In another embodiment, the compositions of the invention are administered in combination with an IL-4 antagonist. IL-4 antagonists that may be administered in combination with the compositions of the present invention include, but are not limited to, soluble IL-4 receptor polypeptides, multimeric forms of soluble IL-4 receptor polypeptides; an anti-IL-4 receptor antibody that binds to the IL-4 receptor but does not transduce a biological signal stimulated by IL-4; anti-IL-4 antibodies that block IL-4 binding to one or more IL4 receptors, and IL4 muteins that bind IL4 receptors but do not transduce a biological signal stimulated by IL 4. Preferably, the antibodies used according to this method are monoclonal antibodies (including antibody fragments, as described herein).

In another embodiment, the compositions of the invention are administered in combination with hematopoietic growth factors. Can be combined with the compositions of the present inventionHematopoietic growth factors administered include, but are not limited to, leukin^TM(SARGRAMOSTIM^TM) And NEUPOGEN^TM(FILGRASTIM^TM)。

In another embodiment, the compositions of the invention are administered in combination with a fibroblast growth factor. Fibroblast growth factors that may be administered in combination with the compositions of the present invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

In addition, the compositions of the present invention may be administered alone or in combination with other therapeutic methods, including but not limited to radiation therapy. Such combination therapy may be administered sequentially and/or concomitantly.

Agonist and antagonist-assays and molecules

The invention also provides a method of screening compounds to identify compounds that enhance or block the effect of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide on a cell, such as its interaction with a Neutrokine-alpha and/or Neutrokine-alpha SV binding molecule, such as a receptor molecule. An agonist is a compound that enhances the natural biological function of Neutrokine-alpha and/or Neutrokine-alpha SV, or functions in a similar manner to Neutrokine-alpha and/or Neutrokine-alpha SV, while an antagonist is a compound that reduces or eliminates this function.

In another embodiment, the invention provides a method of specifically binding a receptor protein or other ligand binding protein of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide. For example, a cellular compartment, such as a cell membrane or a preparation thereof, may be prepared from a cell that expresses a molecule that binds Neutrokine-alpha and/or Neutrokine-alpha SV. This preparation is incubated with labeled Neutrokine-alpha and/or Neutrokine-alpha SV, and complexes of receptor-binding Neutrokine-alpha and/or Neutrokine-alpha SV or other binding proteins are isolated and characterized according to conventional methods known in the art. Alternatively, Neutrokine-alpha and/or Neutrokine-alpha SV may be bound to a solid support so that the binding molecules solubilized from the cells are bound to a chromatographic column and then eluted and characterized according to conventional methods.

In the agonist or antagonist assays of the invention, a cellular compartment, such as a cell membrane or a preparation thereof, may be prepared from cells expressing a molecule that binds Neutrokine-alpha and/or Neutrokine-alpha SV, such as a signaling or regulatory pathway molecule modulated by Neutrokine-alpha and/or Neutrokine-alpha SV. This preparation is incubated with labeled Neutrokine-alpha and/or Neutrokine-alpha SV with or without a candidate molecule which may be an agonist or antagonist of Neutrokine-alpha and/or Neutrokine-alpha SV. The ability of the candidate molecule to bind to the binding molecule is reflected in a decrease in labeled ligand binding. Unilaterally bound molecules, i.e., molecules that do not induce the binding effect of Neutrokine-alpha on Neutrokine-alpha and/or Neutrokine-alpha SV binding molecules, are good antagonists. Molecules that bind well and elicit effects similar to or closely related to Neutrokine-alpha and/or Neutrokine-alpha SV are agonists.

Neutrokine-alpha and/or Neutrokine-alpha SV-like effects of potential agonists and antagonists can be determined, for example, by determining the activity of the second messenger system following interaction of the candidate molecule with a cell or an appropriate cell preparation and comparing the effects of Neutrokine-alpha and/or Neutrokine-alpha SV or a molecule that elicits the same effect as Neutrokine-alpha and/or Neutrokine-alpha SV. Second messenger systems that can be used herein include, but are not limited to, AMP guanylate cyclase, ion channels or phospholytic second messenger systems.

Another assay for antagonists of Neutrokine-alpha and/or alpha SV is, for example, a competitive inhibition assay, which is performed under appropriate conditions by combining Neutrokine-alpha and/or Neutrokine-alpha SV with a potential antagonist, with a membrane-bound receptor molecule or a recombinant Neutrokine-alpha and/or Neutrokine-alpha SV receptor molecule. Neutrokine-alpha and/or Neutrokine-alpha SV may be labeled, for example, by radioactivity, so that the number of Neutrokine-alpha and/or Neutrokine-alpha SV molecules that bind to the receptor molecule may be accurately determined to confirm the efficacy of the potential antagonist.

Potential antagonists include small organic molecules, peptides, polypeptides (e.g., IL-13) and antibodies that bind to the polypeptides of the invention, thereby inhibiting or inactivating their activity. A potential antagonist may also be a small organic molecule, peptide, polypeptide such as a closely related protein or antibody that binds at the same site on a binding molecule such as a receptor molecule, without inducing Neutrokine-alpha and/or Neutrokine-alpha SV induced activity, thereby preventing the effects of Neutrokine-alpha and/or Neutrokine-alpha SV by excluding Neutrokine-alpha and/or Neutrokine-alpha SV binding.

Other potential antagonists include antisense molecules. Antisense methods can be used to control gene expression by antisense DNA or RNA or by triple helix formation. Antisense approaches are exemplified by Okara, journal of neurochemistry 56: 560 (1991); "oligodeoxynucleotides as antisense inhibitors of gene expression", as described by CRC Press, Boca Raton, F1 (1988). Triple helix formation is described, for example, by Lee et al, nucleic acids research 6: 3073 (1979); cooney et al, science 241: 456 (1988); and Dervan et al, science 251: 1360(1991). The method is based on the binding of a polynucleotide to complementary DNA or RNA. For example, the 5' coding portion of a polynucleotide encoding an extracellular domain of a polypeptide of the invention can be used to design antisense RNA oligonucleotides of about 10-40 base pairs in length. A DNA oligonucleotide complementary to a region of a gene involved in transcription is designed so as to prevent transcription and production of Neutrokine-alpha and/or Neutrokine-alpha SV. The antisense RNA oligonucleotide hybridizes to mRNA in vivo and blocks translation of mRNA into Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide. The oligonucleotides described above may also be delivered to cells such that antisense RNA or DNA may be expressed in vivo to inhibit production of Neutrokine-alpha and/or Neutrokine-alpha SV.

In one embodiment, the Neutrokine-alpha and/or Neutrokine-alpha SV antisense nucleic acids of the invention are produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof that transcribes to produce an antisense nucleic acid (RNA) of the invention. Such vectors contain sequences encoding Neutrokine-alpha and/or Neutrokine-alpha SV antisense nucleic acids. Such vectors may remain episomal or become chromosomally integrated until they can be transcribed to produce the desired antisense DNA. Such vectors can be constructed by recombinant DNA methods standard in the art. The vector may be a plasmid, virus, or other material known in the art for replication and expression in a vertebrate cell. The sequences encoding Neutrokine-alpha and/or Neutrokine-alpha SV, or fragments thereof, may be expressed in vertebrate, especially human, cells by any promoter known in the art. Such promoters may be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29: 304-310(1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al, cell 22: 787-797(1980)), the herpes thymidine promoter (Wagner et al, Proc. Natl. Acad. Sci. USA 78: 1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinster et al, Nature 296: 39-42(1982)), and the like.

The antisense nucleic acids of the invention comprise sequences that are complementary to at least a portion of an RNA transcript of a Neutrokine-alpha and/or Neutrokine-alpha SV gene. However, absolute complementarity, while preferred, is not required. A sequence "complementary to at least a portion of an RNA" refers to a sequence that has good complementarity and is capable of hybridizing to an RNA to form a stable duplex; in the case of double-stranded Neutrokine-alpha and/or Neutrokine-alpha SV antisense nucleic acids, single-stranded duplex DNA may be tested, or triplex formation, ability to hybridize may be analyzed depending on the degree of complementarity of the antisense nucleic acid and the length of the antisense nucleic acid. Generally, larger hybridizing nucleic acids, the more base mismatches with Neutrokine-alpha and/or Neutrokine-alpha SV RNA, may contain and still form a stable duplex (or triplex). One skilled in the art can ascertain the permissible degree of mismatch using standard methods to determine the melting point of the hybridization complex.

Oligonucleotides complementary to 5 'untranslated sequences at the 5' end of the messenger, e.g., near and including the AUG start codon, should inhibit translation more effectively. However, sequences complementary to the 3' untranslated sequence of mRNA have been shown to effectively inhibit translation of mRNA. See Wagner, r.1994, nature 372: 333-335. Thus, oligonucleotides complementary to the 5 'or 3' untranslated, non-coding regions of Neutrokine-alpha and/or Neutrokine-alpha SV, respectively, as shown in FIGS. 1A-B and 5A-B, may be used in antisense approaches to inhibit translation of endogenous Neutrokine-alpha and/or Neutrokine-alpha SV mRNA. The oligonucleotide complementary to the 5' untranslated region of the mRNA should include the complementary sequence of the AUG initiation codon. Antisense oligonucleotides complementary to mRNA coding regions are potent translational inhibitors, but may be used in accordance with the invention. The antisense nucleic acid should be 6 nucleotides in length, preferably 6-50 nucleotides in length, regardless of whether it hybridizes to the 5 ', 3' or coding region of Neutrokine-alpha and/or Neutrokine-alpha SVmRNA. In particular, the oligonucleotide is at least 10, 17, 25 or at least 50 nucleotides in length.

The polynucleotides of the invention may be DNA or RNA or chimeric mixtures or derived or modified forms thereof, and may be single-stranded or double-stranded. Oligonucleotides may be modified in the base moiety, sugar moiety or phosphate backbone, for example to improve the stability of the molecule, hybridization, etc. Such oligonucleotides may include other attached groups such as peptides (e.g., targeting host cell receptors in vivo), or agents that promote transport across membranes (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. USA 86: 6553-6556; Lemaitre et al, Proc. Natl. Acad. Sci. USA 84: 648-652 (1987); PCT publication No. WO 88/09810, 15.12.1988) or agents that cross the blood-brain barrier (see, e.g., PCT publication WO 89/10134, 25.4.1988), hybridization-initiated lytic agents (see, e.g., Krol et al, Biotechnology 6: 958-976(1988)) or intercalating agents (see, e.g., Zon, pharmaceutical research 5: 539-549 (1988)). Thus, the oligonucleotide may be conjugated to another molecule, such as a peptide, a hybridization-initiated cross-linking agent, a transport agent, a hybridization-initiated cleavage agent, and the like.

The antisense oligonucleotide may comprise a modified base composition selected from the group consisting of, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxyhydroxymethyluracil, 5-carboxymethylaminomethyl-2-thiopurine, 5-carboxymethylaminomethyluracil, dihydrouracil, β -D-galactosoqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-Methoxamethyl-2-thiouracil,. beta. -D-mannosylqueosine, 5-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), Wybutoxinosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-carboxypropyl) uracil, (acp3) w, and 2, 6-diaminopurine.

The antisense oligonucleotide may further comprise at least one modified sugar component selected from the group consisting of, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexoses.

In another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of, but not limited to, phosphorodithioates, phosphorothioates, phosphoramidites, phosphorodiamidates, methylphosphonates, alkylphosphotriesters, formacetal, or analogs thereof.

In another embodiment, the antisense oligonucleotide is an α -anomeric oligonucleotide. The alpha-terminal oligonucleotide forms a specific double-stranded hybrid with complementary RNA in which the strands are parallel to each other, as opposed to the normal beta units (Gautier et al, nucleic acids Res. 15: 6625-6641 (1987)). The oligonucleotide is a 2-O-methyl ribonucleotide (Inoue et al, nucleic acids Res 15: 6131-.

Polynucleotides of the invention can be synthesized by standard methods known in the art, e.g., using an automated DNA synthesizer) such as are commercially available from Biosearch, Applied biosystems, etc.). For example, phosphorothioates can be synthesized by the method of Stein et al (nucleic acids Res. 16: 3209(1988)), methylphosphonate oligonucleotides can be prepared by controlled pore glass polymer supports (Sarin et al, Proc. Natl. Acad. Sci. USA 85: 7448-7451(1988)), and the like.

Antisense nucleotides complementary to the Neutrokine-alpha and/or Neutrokine-alpha SV coding region sequences, preferably complementary to transcribed untranslated regions, may be used.

Potential antagonists of the invention also include catalytic RNA or ribozymes (see, e.g., PCT International publication WO 90/11364, published 1990, 10.4; Sarver et al, science 247: 1222-1225 (1990). ribozymes that cleave mRNA at site-specific recognition sequences can be used to disrupt Neutrokine-alpha and/or Neutrokine-alpha SV mRNA, preferably hammerhead ribozymes are used. the only requirement is that the target mRNA has a 5 '-UG-3' sequence for the cleavage of mRNAS located on both sides of the region that forms a complementary base pair with the target mRNA. construction and production of hammerhead ribozymes are well known in the art and are described in Haseloff and Gerlach, Nature 334: 585-591 (1988.) Neutrokine-alpha and/or Neutrokine-alpha SV nucleotide sequences have a number of potential hammerhead ribozyme cleavage sites (see FIGS. 1A-B and 5A-B, respectively.) preferably, the ribozymes are engineered such that the cleavage recognition site is located near the 5' end of Neutrokine-alpha and/or Neutrokine-alpha SVmRNA, i.e., to increase potency and minimize intracellular accumulation of non-functional mRNA transcripts.

In antisense approaches, the ribozymes of the invention may consist of modified oligonucleotides (e.g., improved stability, targeting, etc.) and should be delivered to cells that express Neutrokine-alpha and/or Neutrokine-alpha SV in vivo. A DNA construct encoding a ribozyme can be introduced into a cell in the same manner as described above to introduce antisense encoding DNA. Preferred methods of delivery include the use of DNA constructs that "encode" ribozymes under the control of strong constitutive promoters, such as polIII, or polII promoters, such that transfected cells will produce sufficient ribozymes to destroy the endogenous Neutrokine-alpha and/or Neutrokine-alpha SV message and inhibit translation. Since ribozymes are not catalytic like antisense molecules, lower intracellular concentrations are required to increase potency.

Endogenous gene expression may also be reduced by inactivation or "knocking out" of the Neutrokine-alpha and/or Neutrokine-alpha SV genes and/or their promoters using targeted homologous recombination (e.g., Smities et al, Nature 317: 230-24 (1985); Thomas and Capeahi, cell 51: 503-. For example, a mutated non-functional polynucleotide of the invention (or a complementary unrelated DNA sequence) flanked by DNA homologous to endogenous polynucleotide sequences (coding or regulatory regions of a gene) with or without a selectable marker and/or a negative selectable marker may be used to transfect cells that express a polypeptide of the invention in vivo. In another embodiment, the knockdown is generated in a cell that contains, but does not express, the corresponding gene using methods known in the art. Insertion of the DNA construct by targeted homologous recombination results in targeted gene inactivation. This method is particularly suitable for research and agricultural fields where modifications to embryonic stem cells can be used to produce animal progeny having inactivated targeted genes (see Thomas and Capechi1987 and Thompson1989 supra). However, this method can be routinely applied to humans, and it will be apparent to those skilled in the art that the recombinant DNA construct provided can be directly administered or targeted to the desired site in vivo using an appropriate viral vector. All cited documents are incorporated by reference in their entirety.

In other embodiments, antagonists of the invention include soluble forms of Neutrokine-alpha and/or Neutrokine-alpha SV (fragments of Neutrokine-alpha as shown in FIGS. 1A-B, including the ligand binding domain, the TNF-conserved domain, and/or the extracellular domain of Neutrokine-alpha and/or Neutrokine-alpha SV, and fragments of Neutrokine-alpha SV as shown in FIGS. 5A-B, including the ligand binding domain, the TNF-conserved domain, and/or the extracellular domain of Neutrokine-alpha and/or Neutrokine-alpha SV). Such soluble forms of Neutrokine-alpha and/or Neutrokine-alpha SV, which may be naturally occurring or synthetic, antagonize Neutrokine-alpha and/or Neutrokine-alpha SV mediated signals by competing with the natural Neutrokine-alpha and/or Neutrokine-alpha SV for binding to Neutrokine-alpha and/or Neutrokine-alpha SV receptors (e.g., DR5 (see International publication WO 98/41629), TR10 (see International publication WO 98/54202), 312C2 (see International publication WO 98/06842) and TR11, TR11SV1 and TR11SV2 (see U.S. Pat. No.09/176200)), or by forming multimers that may or may not bind to the receptors but are incapable of inducing signal transduction. Preferably, these antagonists inhibit the proliferation, differentiation and/or activation of stimulated lymphocytes (e.g., B cells) mediated by Neutrokine-alpha and/or Neutrokine-alpha SV. Antagonists of the invention also include antibodies specific for TNF family ligands (e.g., CD30) and Neutrokine- α -Fc and/or Neutrokine- α SV-Fc fusion proteins.

"TNF family ligand" refers to naturally occurring, recombinant, and synthetic ligands that bind to TNF receptor family members and induce and/or block ligand/receptor signaling pathways. TNF ligand family members include, but are not limited to TNF- α, lymphotoxin- α (LT- α, also known as TNF- β), LT- β (found in the heterotrimeric LT- α -2- β complex), FasL, CD40L, (TNF- γ (International publication WO 96/14328), ATIM-I (International publication WO 97/33899), AIM-II (International publication WO 97/34911), APRIL (J.Exp.Med.188 (6): 1185-1190), α -intrinsic factor (International publication WO 98/07880), α -neurogenic factor (International publication WO 97/18921), CD27L, CD30L, 4-1BBL, OX40L, CD27, CD30, 4-1BB, OX40 and Nerve Growth Factor (NGF). in a preferred embodiment, Nekinote- α and/or Nekinine- α 5 of the invention are Nekinine- α and/or Nekinine- α 5 family ligands (see International publication WO 98/41629), TR10 (see International publication WO 98/54202), 312C2 (see International publication WO 98/06842) and TR11, TR11SV1 and TR11SV2 (see U.S. patent application No. 09/176200).

Antagonists of the invention also include antibodies specific for TNF family receptors or the Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of the invention. Antibodies of the invention can be prepared by various standard methods using Neutrokine-alpha and/or Neutrokine-alpha SV immunogens of the invention. Such Neutrokine-alpha and/or Neutrokine-alpha SV immunogens comprise intact Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides (which may or may not include a leader sequence), and fragments of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides comprising, for example, ligand binding domains, TNF conserved domains, extracellular domains, transmembrane domains, and/or intracellular domains, or any combination thereof, as shown in FIGS. 1A-B (SEQ ID NO: 2) and 5A-B (SEQ ID NO: 19), respectively.

Polyclonal and monoclonal antibody agonists or antagonists of the invention may be administered according to Tartaglia and Goeddel, journal of biochemistry 267 (7): 4304-; tartaglia et al, cell 73: 213-216(1993) and PCT application WO 94/09137, and preferably is specific for (i.e. binds only to) a polypeptide having the sequence shown in SEQ ID NO: 2 amino acid sequence of the polypeptide of the invention. The term "antibody" (Ab) or "monoclonal antibody" (mAb) is meant to include intact molecules as well as fragments thereof) such as Fb and F (Ab') fragments, which are capable of binding antigen. Fab, Fab 'and F (ab') fragments lack the Fc fragment intact antibody, clear more rapidly from circulation, and can have less nonspecific tissue binding of intact antibody (Wahl et al, J. nucleic acid methods 24: 316-325 (1983)).

In a preferred method, the antibodies of the invention are mAbs. Such mAbs can be prepared by the hybridoma method (Kohler and Millstein, Nature 256: 495-497(1975) and U.S. Pat. No. 4376110; Harlow et al, antibodies laboratory Manual, Cold spring harbor laboratory Press, Cold spring harbor, NY, 1988; monoclonal antibodies and hybridomas: New developments in biological assays, Plenium Press, New York, NY, 1980; Campbell, "monoclonal antibody technology", Experimental procedures in biochemistry and molecular biology, Vol.13 (edited by Burdor et al), Elsevier, Amsterdam (1984)).

Proteins and other compounds that bind Neutrokine-alpha and/or Neutrokine-alpha SV domain are also candidate agonists and antagonists of the invention. Such binding compounds can be "captured" using the yeast two-hybrid system (Fields and Song, Nature 340: 245-246 (1989)). The yeast two-hybrid system has been modified by Roger Brent and co-workers (Gyuris, cell 75: 791-803 (1993); Zervos et al, cell 72: 223-232 (1993)). Preferably, compounds that bind the ligand binding domain, extracellular domain, intracellular domain, transmembrane domain, and death domain of Neutrokine-alpha and/or Neutrokine-alpha SV are captured using a yeast two-hybrid system according to the present invention. Such compounds are good candidates for agonists and antagonists of the present invention.

For example, using the two-hybrid assay described above, the extracellular domain intracellular domain of Neutrokine-alpha and/or Neutrokine-alpha SV receptor, or a portion thereof, may be used to identify cellular proteins that interact with Neutrokine-alpha and/or Neutrokine-alpha SV receptor in vivo. This assay can also be used to identify cellular proteins that have Neutrokine-alpha and/or Neutrokine-alpha SV receptor interactions in vivo. Such assays may also be used to identify ligands with potential agonistic or antagonistic activity for Neutrokine-alpha and/or Neutrokine-alpha SV receptor function. This screening assay was previously used to identify proteins that interact with the cytoplasmic region of mouse TNF-RII, and to identify two receptor-associated proteins. Rothe et al, cell 78: 681(1994). Such proteins and amino acid sequences that bind to the cytoplasmic region of Neutrokine-alpha and/or Neutrokine-alpha SV receptors are good candidates for agonists and antagonists of the present invention.

Other screening methods include using cells expressing a polypeptide of the invention (e.g., transfected CHO cells) to measure extracellular pH changes resulting from receptor activation, such as scientific 246: 181-296 (1989). In another example, a potential agonist or antagonist can be contacted with a cell expressing a polypeptide of the invention and a second messenger response, such as signal transduction, can be assayed to determine if the potential antagonist or agonist is effective.

Agonists of the invention include naturally occurring and synthetic compounds such as peptide fragments of TNF family ligands, transforming growth factors, neurotransmitters (e.g., glutamate, dopamine, N-methyl-D-aspartate), tumor suppressor factors (p53), cytolytic T cells and antimetabolites. Preferred agonists include chemotherapeutic agents and cisplatin, doxorubicin, bleomycin, cytarabine, mechlorethamine, methotrexate and vincristine. Other agonists include ethanol and amylin (science 267: 1457-.

Preferred agonists are fragments of Neutrokine-alpha and/or Neutrokine-alpha SV of the invention that stimulate proliferation, differentiation and/or activation of lymphocytes, such as B cells. Further preferred agonists include polyclonal and monoclonal antibodies raised against Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, or fragments thereof, of the present invention. Such agonist antibodies raised against TNF family receptors are found in Tartaglia et al, proceedings of the american academy of sciences, 88: 9292-9296 (1991); and Tartaglia et al, J. Biochem 267: 4304-. Also described in PCT application WO 94/09137.

In another embodiment, immunomodulatory molecules such as IL2, IL3, II4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD 40, CD40L, IFN- γ, and TNF- α, can be used as agonists of the Neutrokine- α and/or Neutrokine- α SV polypeptides of the invention that stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and/or activation. In a specific embodiment, IL4 and/or IL10 are used to proliferate Neutrokine-alpha and/or Neutrokine-alpha SV mediated B cell proliferation.

In another embodiment of the invention, cells engineered to express a polypeptide of the invention, or cells engineered not to express a polypeptide of the invention (e.g., knockdown), are administered to a patient in vivo. Such cells may be obtained from a patient (i.e., an animal, including a human), or from an MHC-compatible donor, and may include, but are not limited to, fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, and the like. The cells are genetically engineered in vitro with recombinant DNA methods to introduce the coding sequence for a polypeptide of the invention into the cell, or to disrupt the coding sequence and/or endogenous regulatory sequences associated with a polypeptide of the invention, such as by transduction (with viral vectors, and preferably vectors that integrate transgenes into the genome of the cell), or transfection methods, including but not limited to, with plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, and the like. The coding sequence for a polypeptide of the invention may be placed under the control of a strong constitutive promoter or an inducible promoter or promoter/multiplier to express, and preferably secrete, the polypeptide of the invention. Genetically engineered cells expressing and preferably secreting a polypeptide of the invention can be introduced systemically, e.g., by circulation or intraperitoneal injection, into a patient.

Alternatively, the cells may be incorporated into a matrix and implanted into the body, e.g., genetically engineered fibroblasts may be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft (see, e.g., Anderson et al, U.S. Pat. No. 5399349; and Mullgan and Wilson, U.S. Pat. No.5460959, all incorporated by reference in their entirety).

Where the cells to be administered are non-self or non-MHC compatible cells, they may be administered by well known methods of preventing the host from generating an immune response to the introduced cells. For example, cells may be introduced in a capsule form that allows for immediate exchange of components into the extracellular environment, but does not allow for recognition of the introduced cells by the host immune system.

In another embodiment of the invention, the activity of a Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide may be reduced by a "dominant negative". To this end, constructs encoding defective Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, such as mutants lacking all or part of the TNF conserved domain, may be used in gene therapy to attenuate the activity of Neutrokine-alpha and/or Neutrokine-alpha SV on appropriate target cells. For example, a nucleotide sequence directing expression of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide by a host cell in which all or part of the TNF-conserved domain has been altered or deleted may be introduced into monocytes or other cells or tissues (by in vivo or ex vivo gene therapy or other methods known in the art). Alternatively, targeted homologous recombination may be used to introduce such deletions or mutants into the endogenous Neutrokine-alpha and/or Neutrokine-alpha SV genes in the monocytes of an individual subject. The engineered cells will express non-functional Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides (i.e., ligands (e.g., multimers) that bind but cannot induce signal transduction).

Chromosome analysis

The nucleic acid molecules of the invention may also be used for chromosome identification. This sequence is specifically targeted and can hybridize to a specific location of an individual's human chromosome. In addition, it is often desirable to identify specific sites on the chromosome. Few chromosome labeling reagents based on true sequence data (repeat polymorphisms) are suitable for labeling chromosomal locations. DNA mapping of the chromosomes of the present invention is an important first step in determining the relevance of these sequences to disease-associated genes.

In some preferred embodiments, the cDNAs and/or polynucleotides set forth herein are used to clone the genomic DNA of the Neutrokine-alpha and/or Neutrokine-alpha SV gene. This can be done using various well known methods and commercially available libraries. This genomic DNA is then used for in situ chromosome mapping using well known methods.

In addition, in some cases, the sequence can be mapped on the chromosome by preparing PCR primers (preferably 15-25bp) from the cDNA. In silico analysis of the 3' untranslated region of a gene is used to rapidly select primers that span no more than one exon within the genome, otherwise complicating amplification. These primers are then used for PCR screening of somatic hybrids containing individual human chromosomes. Fluorescence In Situ Hybridization (FISH) of cDNA clones to mid-stage chromosomal smears can be used to provide accurate chromosomal localization in one step. This method may use probes of short length, e.g.50 or 60bp, taken from cDNA. This method is described in Verma et al, basic technical Manual of human chromosomes, Pergamon Press, New York (1988).

Once the sequence is precisely located on the chromosome, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data are available, for example, from Johns Hopkins university, Welch medical library, v.mckusick, mendelian inheritance In Man. The relationship between genes and diseases that have been mapped to the same chromosomal region is then identified by lineage analysis (a joint genetic profile naturally in the neighbourhood of genes).

It is next necessary to determine the differences in cDNA or genomic sequence between diseased and non-diseased individuals. If a mutation is observed in some or all of the diseased individuals and no mutation is observed in normal individuals, then the mutation is responsible for the disease.

The precise location of cDNA on chromosome is related to diseases caused by 50-500 kinds of potential pathogenic genes through the resolution of physical diagram and genetic diagram method. (assuming 1 megabase map resolution, and one gene per 20 kb).

Using the above method, it is possible to determine with great certainty on chromosome 13g34 using a combination of somatic hybrids and radiation hybrids for the chromosomal location of Neutrokine-alpha and/or Neutrokine-alpha SV.

Examples

The invention will be readily understood by reference to the following examples, which are intended to illustrate and not to limit the invention. Many of the following examples are specifically illustrative of Neutrokine-alpha polynucleotides and polypeptides of the present invention. Each of the examples is also applicable to the production and/or detection of Neutrokine- α SV polynucleotides and polypeptides of the invention. The examples for Neutrokine- α SV can be easily performed by those skilled in the art through the following examples. Example 1 a: expression and purification of "His-tagged" Neutrokine-alpha in E.coli bacterial expression was performed in this example using the bacterial expression vector pQE9(pD 10).

(QIAGEN, supra). pQE9 encodes ampicillin resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site ("RBS"), 6 codons encoding histidine residues which can be affinity purified using the nickel-nitrilotriacetic acid (Ni-NTA) affinity resin available from QIAGEN, and appropriate single restriction enzyme cleavage sites. The factors are arranged such that the inserted DNA segment encoding the polypeptide encodes a polypeptide having 6 histidine residues (i.e., 6X His tag) covalently attached to the amino terminus of the polypeptide.

The DNA sequence encoding the desired portion of the Neutrokine-alpha protein comprising the ectodomain sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers that anneal to the amino-terminal sequence of the desired portion of the protein, and 3' of the cDNA coding sequence in the deposited construct sequence, respectively. Additional nucleotides containing restriction sites in the pQE9 vector that facilitate cloning were added to the 5 'and 3' primer sequences, respectively.

To clone the ectodomain of the protein, the 5 ' primer was of sequence 5'-GTG GGA TCCAGC CTC CGG GCA GAG CTG-3' (SEQ ID NO: 10) containing the underlined Bam HI restriction site followed by 18 nucleotides of the amino-terminal coding sequence of the ectodomain of the sequences shown in FIGS. 1A and 1B. Of course, one skilled in the art will recognize that the position of the 5' primer start in the protein coding sequence can be varied to amplify a segment of DNA encoding any desired portion of the intact Neutrokine-alpha protein, which is longer or shorter than the extracellular domain. The 3 'primer has the sequence 5'-GTG AAG CTT TTA TTACAG CAG TTT CAA TGC ACC-3'(SEQ ID NO: 11) containing an underlined Hind III restriction site followed by 2 stop codons and 18 nucleotides complementary to the 3' coding sequence of the DNA sequence shown in FIGS. 1A and 1B.

The amplified DNA fragment and the vector pQE9 were digested with BamHI and Hind III, and the digested DNAs were ligated together. The DNA inserted in the restricted pQE9 vector places the protein coding region downstream of the IPTG inducible promoter and in-frame with the initial AUG and 6 histidine codons.

The ligation mixture is transformed into suitable E.coli cells using standard methods, such as Sambrook et al, molecular cloning, A laboratory Manual, second edition; cold spring harbor laboratory Press, Cold spring harbor, NY (1989). Coli strain M15/rep4 containing multiple copies of plasmid pREP4 expressing the lac repressor and conferring kanamycin resistance (Kan 4) was used to carry out the examples illustrated herein^r). This strain is only one of many strains suitable for expression of proteins, and is commercially available from QIAGEN corporation. Transformants were identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA was isolated from resistant clones and the cloned DNA was determined by restriction analysis, PCR and DNA sequencing. Clones containing the desired construct were grown overnight (O/N) in liquid LB medium supplemented with ampicillin (100. mu.g/ml) and kanamycin (25. mu.g/ml). The O/N culture was used to inoculate a large culture at a dilution of about 1: 25 to 1: 250. Cells were grown to an optical density at 600nm (OD600) between 0.4-0.6.

IPTG was then added to a final concentration of 1mM to induce transcription from the lac repressor sensitive promoter by inactivating the lac repressor. The cells were then incubated for a further 3-4 hours. Cells were then collected by centrifugation.

The cells were then agitated at 4 ℃ for 3-4 hours in 6M guanidine hydrochloride, pH 8. Cell debris was removed by centrifugation and the supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA) affinity resin chromatography column (available from QIAGEN). The protein with the 6 × His tag binds with high affinity to Ni-NTA resin and can be purified in a simple one-step procedure (see QIAexpressinst, 1995, QIAGEN). Briefly, the supernatant was loaded onto a chromatography column in 6M guanidine hydrochloride, pH8, which was first washed with 10 volumes of 6M guanidine hydrochloride, pH6, and finally eluted with 6M guanidine hydrochloride, pH5 for neutrokine-alpha and neutrokine-alpha SV polypeptides.

The purified protein was then renatured by dialysis against Phosphate Buffered Saline (PBS) or 50mM sodium acetate, pH6 buffer plus 200mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on a Ni-NTA chromatography column. The conditions applicable are as follows: renaturation with a linear 6M-1M urea gradient in 500mM NaCl, 20% glycerol, 20mM Tris/HCl pH7.4 containing protease inhibitors. Renaturation should be carried out for 1.5 hours or more. After renaturation, the protein can be eluted by adding 250mM imidazole. Imidazole was removed by final dialysis against PBS or 50mM sodium acetate pH6 buffer plus 200mM NaCl. The purified protein was stored at 4 ℃ or frozen at-80 ℃.

Example 1 b: expression and purification of neutrokinine-alpha SV in E.coli

Bacterial expression was performed in this example using the bacterial expression vector pQE 60. (QIAGEN, 9259 Eton Avenue, Chatsworth, CA, 91311). pQE60 encodes ampicillin resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site ("RBS"), 6 codons encoding histidine residues which can be affinity purified using the nickel-nitrilotriacetic acid (Ni-NTA) affinity resin available from QIAGEN, and appropriate single restriction enzyme cleavage sites. The elements are arranged such that the polypeptide-encoding DNA fragment, upon insertion, encodes a polypeptide having 6 histidine residues (i.e., a 6 His tag), the 6 histidine residues being covalently attached to the amino terminus of the polypeptide. However, in this example, the polypeptide coding sequence was inserted, thereby preventing translation of the 6 histidine codons and thus producing a polypeptide without the 6 histidine tags.

The DNA sequence encoding the desired portion of the protein comprising the ectodomain sequence is amplified from the deposited cDNA clone using PCR oligonucleotide primers that anneal to the amino-terminal sequence of the desired portion of the protein and 3' of the cDNA coding sequence in the sequence of the deposited construct, respectively. Additional nucleotides containing restriction sites in the pQE9 vector that facilitate cloning were added to the 5 'and 3' primer sequences, respectively.

To clone the extracellular domain of a protein, the 5 'primer was sequenced as 5' -GTGTCA TGAGCC TCC GGG CAG AGC TG-3' (SEQ ID NO: 12) which contains an underlined Bsp HI restriction site followed by 17 nucleotides of the amino-terminal coding sequence of the extracellular domain of the sequences shown in FIGS. 1A and 1B. Of course, one skilled in the art will recognize that the position in the protein coding sequence where the 5' primer starts can be varied to amplify any desired portion of the encoded intact protein, which is longer or shorter than the extracellular domain. The sequence of the 3 'primer is 5' -GTGAAG CTTTTA TTA CAG CAG TTT CAA TGC ACC-3 '(SEQ ID NO: 13), which contains an underlined Hind III restriction site followed by 2 stop codons, and 18 nucleotides complementary to the coding sequence at the 3' end of the DNA sequence shown in FIGS. 1A and 1B.

The amplified DNA fragment and the vector pQE60 were digested with Bsp HI and Hind III, and the digested DNAs were ligated together. The DNA inserted in the restricted pQE60 vector places the protein coding region downstream of the IPTG inducible promoter and in-frame with the initial AUG and 6 histidine codons. The relevant stop codon prevented translation of the 6 histidine codons downstream of the insertion point.

The ligation mixture is transformed into suitable E.coli cells using standard methods, such as Sambrook et al, molecular cloning, A laboratory Manual, second edition; cold spring harbor laboratory press, cold spring harbor,NY (1989). Coli strain M15/rep4 containing multiple copies of plasmid pREP4 expressing the lac repressor and conferring kanamycin resistance (Kan 4) was used to carry out the examples illustrated herein^r). This strain is one of many strains suitable for expression of proteins, and is available from QIAGEN corporation. Transformants were identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA was isolated from resistant clones and the cloned DNA was determined by restriction analysis, PCR and DNA sequencing.

Those skilled in the art will recognize that any of a number of bacterial expression vectors may be used in place of pQE9 and pQE60 for the expression methods used in this example. For example, a novel pHE4 bacterial expression vector family, in particular pHE4-5 vector, can be used for bacterial expression in this example (ATCC No. 209311; and variants thereof). The plasmid DNA designated pHE4-5/MPIFD23, deposited under ATCC accession number 209311, is a vector plasmid DNA containing an insert encoding another ORF. This construct was deposited at the American type culture Collection at 30/9/1997, located at 20110-. Using Nde I and Asp 718 restriction sites flanking an unrelated MPIF ORF insert, one skilled in the art can readily use current molecular biology methods to replace an unrelated ORF in the pHE4-5 vector with the Neutrokine-alpha ORF of the present invention.

The pHE4-5 bacterial expression vector included the neomycin phosphotransferase gene for selection, an E.coli origin of replication, a T5 phage promoter sequence, two lac operator sequences, a Shine-Delgarno sequence, and a lactose operator repressor gene (1 acIq). The elements are arranged so that upon insertion of a DNA fragment encoding the polypeptide, a polypeptide is expressed having 6 histidine residues (i.e., a 6 xhis tag), the 6 histidine residues being covalently attached to the amino terminus of the polypeptide. The promoter and operator sequences of the pHE4-5 vector were generated synthetically. Methods for the synthetic generation of nucleic acid sequences are well known in the art (CLONEECH 95/96 Catalog, p215-216, CLONEECH, 1020 East Meadow Circle, Palo Alto, CA 94303).

Clones containing the desired Neutokine- α SV construct were grown overnight (O/N) in liquid LB medium supplemented with ampicillin (100 μ g/ml) and kanamycin (25 μ g/ml). The O/N culture was used to inoculate a large culture at a dilution of about 1: 25 to 1: 250. Cells were grown to an optical density at 600nm (OD600) between 0.4-0.6. IPTG was then added to a final concentration of 1mM to induce transcription from the lac repressor sensitive promoter by inactivating the lac repressor. The cells were then incubated for a further 3-4 hours. Cells were then collected by centrifugation.

The cells were then agitated at 4 ℃ for 3-4 hours in 6M guanidine hydrochloride, pH 8. Cell debris was removed by centrifugation and the supernatant containing Neutrokine-alpha was dialyzed against 50mM sodium acetate pH6 buffer supplemented with 200mM NaCl. Alternatively, the protein can be successfully refolded by dialysis against 500mM NaCl, 20% glycerol, 25mM Tris/HCl pH7.4 containing a protease inhibitor. After renaturation, proteins can be purified by ion exchange, hydrophobic interaction and size exclusion chromatography. Alternatively, affinity chromatography such as an antibody column can be used to obtain the purified protein. The purified protein was stored at 4 ℃ or frozen at-80 ℃.

In some embodiments, it is preferred to generate an expression construct as described in this example to mutate one or more of the 3 cysteine residues in the Neutrokine-alpha polypeptide sequence. The cysteine residue in the Neutrokine-alpha polypeptide sequence is located in SEQ ID NO: 2 at positions 147, 232, and 245 as set forth in SEQ ID NO: positions 213 and 226 as indicated in FIG. 19 (the Neutrokine- α SV polypeptide sequence does not correspond to the cysteine of Cys-147 in the Neutrokine- α polypeptide sequence, since amino acid residues 143 and 160 of the Neutrokine- α polypeptide sequence are not present in the Neutrokine- α SV polypeptide sequence).

Example 2: cloning, expression, and purification of Neutrokine-alpha protein in baculovirus expression systems

In this example, plasmid shuttle vector pA2GP was used to insert cloned DNA encoding the extracellular domain of a protein, lacking its naturally associated intracellular and transmembrane sequences, into baculoviruses to express the extracellular domain of the Neutrokine-alpha protein, using the baculovirus leader sequence and methods as described by Summers et al, handbook of baculovirus and insect cell culture methods, Texas efficient Experimental Station Bulletin No.1555 (1987). This expression vector contains the strong polyhedrin promoter of AcMNPV, the secretion signal peptide (leader sequence) of the rear baculovirus gp67 protein, and conventional restriction sites such as BamHI, Xba I and Asp 718 restriction sites. The polyadenylation site of monkey virus 40(SV40) is used for efficient polyadenylation. For ease of selection of recombinant viruses, the plasmid contains the β -galactosidase gene from E.coli in the same orientation under the control of a weak Drosophila promoter, followed by a polyadenylation signal for the polyhedrin gene. The inserted genes flank the viral sequence for cell-mediated homologous recombination with wild-type viral DNA to produce live viruses expressing the cloned polynucleotides.

Many other baculovirus vectors may be used in place of the above vectors, such as pAc373, pVL941, and pAcIM1, provided those skilled in the art recognize that this construct provides transcription, translation, secretion, etc. signals in the appropriate location, including the desired signal peptide and in-frame AUG. Such vectors are, for example, Luckow et al, virology 170: 31-39 (1989).

The cDNA sequence encoding the extracellular domain of the N-terminally deleted form of Neutrokine-alpha protein in the deposited clones, lacking the AUG start codon, the naturally associated intracellular and transmembrane domain sequences, and the Gln-73 to Leu-79 amino acids shown in FIGS. 1A and 1B (SEQ ID NO: 2), was amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene. The 5 'primer sequence is 5' -GTGGGA TCCCCG GGC AGA GCTGCA GGG C-3' (SEQ ID NO: 14), which contains an underlined Bam HI restriction enzyme site followed by 18 nucleotides of the ectodomain sequence of the Neutrokine-alpha protein shown in FIGS. IA and 1B, starting at the N-terminus of the ectodomain of the protein shown. The 3 'primer sequence is 5' -GTGGGA TCCTTA TTA CAG CAG TTT CAA TGC ACC-3 '(SEQ ID NO: 15) which contains an underlined Bam HI restriction enzyme site followed by two stop codons, and 18 nucleotides which are complementary to the 3' coding sequence shown in FIGS. 1A and 1B.

In some other embodiments, constructs expressing the entire putative extracellular domain of Neutrokine- α (i.e., amino acid residues Gln-73 to Leu-285) are preferred. Those skilled in the art can use SEQ ID NO: 1 and SEQ ID NO: 2 to generate such clones, polynucleotide primers were designed to generate the polynucleotide and polypeptide sequences provided, respectively.

In a preferred embodiment, the pA2GP expression construct encodes the amino acid sequence of SEQ ID NO: 2 from Leu-112 to Leu-285 of the Neutrokine-alpha polypeptide sequence.

In another preferred embodiment, the pA2GP expression construct encodes the amino acid sequence of SEQ ID NO: 2 from Ser-78 to Leu-285 of the Neutrokine-alpha polypeptide sequence.

The amplified fragments were separated from 1% agarose gel using a commercially available kit (Geneclean, BIO 101, La Jolla, Ca.). This fragment was then digested with BamHI and purified again in a 1% agarose gel. This fragment is referred to herein as F1.

The plasmid was digested with the restriction enzyme BamHI and optionally dephosphorylated with calf intestinal phosphatase using conventional methods known in the art. The DNA was then separated from the 1% agarose gel using a commercially available kit (Geneclean, BIO 101, La Jolla, Ca.). This vector DNA is referred to herein as V1.

Fragment F1 and dephosphorylated plasmid V1 were ligated with T4 DNA ligase. Coli HB101 or other suitable E.coli hosts such as XL-1 Blue cells (Stagene Cloning Systems, La Jolla, Ca.) were transformed with this ligation mixture and plated onto culture plates. Bacteria containing plasmids having human genes were identified by digesting the DNA of each colony with BamHI, and then analyzing the digestion products by gel electrophoresis. The sequence of the cloned fragment was determined by DNA sequencing. This plasmid is referred to herein as pA2 GP-Neutrokine-alpha.

Mu.g of plasmid pA2 GP-Neutrokine-. alpha.was mixed with 1.0. mu.g of commercially available linear baculovirus DNA (Baculogold)^TMBaculovirus DNA, Pharmingen, San Diego, CA), using, for example, Felgner et al, proceedings of the american academy of sciences 84: 7413-7417 (1987). 1.0 mu g of Baculogold^TMViral DNA was mixed with 5. mu.g of plasmid pA2 GP-Neutrokine-. alpha.in sterile wells of microtiter plates containing 50. mu.l of serum-free Grace's medium (Life Technologies, Gaithersburg, Md.). Thereafter, 10. mu.l Lipofectin and 90. mu.l Grace's medium were added, mixed and incubated for 15 minutes at room temperature. The transfection mixture was then added dropwise to Sf9 insect cells (ATCC CRL 1711) and Sf9 insect cells were plated in 35mm tissue culture plates containing 1ml of serum-free Grace's medium. The plate was then incubated at 27 ℃ for 5 hours. The transfection solution was then removed from the plates and 1ml Grace's insect medium supplemented with 10% calf serum was added. Then, the culture was continued at 27 ℃ for 4 days.

After 4 days, supernatants were collected and plaque analyzed as described by Summers and Smith. Agarose gels with "Blue Gal" (Life Technologies, Rockville, Maryland) were used to facilitate identification and isolation of Gal expression clones producing Blue-stained plaques. (details of this type of plaque assay, also found in the instructions for use of insect cell culture and baculovirus distribution by Life Technologies, Rockville, Maryland, p 9-10). After appropriate incubation, the blue-stained plaques are picked up with the tip of a micropipette (e.g., Eppendorf). Agar containing the recombinant virus was then resuspended in a microfuge tube containing 200. mu.l Grace's medium and the suspension containing the recombinant baculovirus used to infect Sf9 cells seeded on 35mm plates. After 4 days, the supernatants from these plates were collected and then stored at 4 ℃. This recombinant virus is designated V-Neutrokine-alpha.

To examine the expression of the Neutrokine-alpha gene, Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells were infected with the recombinant baculovirus V-Neutrokine-alpha at a multiplicity of infection (MOI) of approximately 2. If radiolabeled protein is desired, the medium is removed after 6 hours and SF900 II medium (purchased from Life T) minus methionine and cysteine is used Biotechnology, Rockville, Maryland). After 42 hours, 5 microcurie of³⁵S-methionine and 5 microcurie³⁵S-cysteine (purchased from Amersham). After incubating the cells for 16 hours, they were collected by centrifugation. The proteins in the supernatant, as well as intracellular proteins, were analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Minisequencing of the amino acid sequence of the amino terminus of a purified protein can be used to determine the amino-terminal sequence of the extracellular domain of the protein, and thus the cleavage site and the length of the secretory signal peptide.

In a specific example, recombinant Neutrokine-alpha was purified from baculovirus-infected Sf9 cell supernatant as described below. Insect cells were grown in EXCEL401 medium (JRH Scientific) with 1% (v/v) calf serum. After 92 hours of infection, the collected supernatant was clarified by centrifugation at 18000 Xg, followed by filtration at 0.45m depth. A defatting filtration step may also be used to remove lipid contaminants and subsequently improve the initial capture of Neutrokine-alpha protein.

Supernatants were randomly loaded into a series of Poros HS-50/H-50. Alternatively, Toyopearl QAE, Toyopearl Super Q (Tosohass), Q-Sepharose (pharmacia), and equivalent resins may be used. This step serves as a negative purification step to remove strongly anion bound contaminants. The HS/HQ flow through the feed was adjusted to pH7.5 with 1M Tris-HCl pH8, diluted with an equal volume of 50mM Tris-HCl pH8, and loaded onto either a multi-well PI-20 or PI-50 chromatography column. The PI columns were first washed with 4 volumes of 75mM NaCl in 50mM Tris-HCl pH7.5 and then eluted with 3-5 column volumes of 300mM, 750mM, 1500mM NaCl in 50mM Tris-HCl pH 7.5. The Neutrokine-alpha protein presents a 17KD band on simplified SDS-PAGE and is present in the 0.75M-1.5M NaCl fraction.

The PI fraction was further purified by Sephacryl S100 HR (pharmacia) size exclusion chromatography column equilibrated with 0.15M NaCl, 50mM sodium acetate, pH 6. The S200 fraction was mixed with NaCl to a final concentration of 3M and applied to a toyopearHexyl 650C (Tosohass) column. Hexyl chromatography column was eluted with a linear gradient of 3M to 0.05M NaCl in 50mM sodium acetate pH6 in 5-10 volumes. This NaCl gradient in the Hexy1 chromatography was also replaced with a 1M-0M gradient of ammonium sulfate in 50mM sodium acetate pH 6. Fractions containing Neutrokine-alpha purified by SDS-PAGE analysis were combined and dialyzed against pH6 containing 150mM NaCl, 50mM sodium acetate.

The final purified Neutrokine-alpha protein expressed in the baculovirus system described herein has the amino acid sequence set forth in SEQ ID NO: 2, Ala-134 amino acid residue. RP-HPLC analysis showed a single peak above 95% purity. Endotoxin levels were below the detection limit of the LAL assay.

In another example, recombinant Neutrokine-alpha was purified from baculovirus-infected Sf9 cells containing 0.25% bovine serum, as described below.

Sf9 supernatant was collected by centrifugation at 18000 Xg. The supernatant was then washed with 10mM CaCl₂Treatment under weak base conditions for 10-15 minutes, followed by centrifugation and 0.22 μm depth filtration. The resulting Sf9 cell supernatant was then diluted 2-fold and loaded onto a Poros PI-50 chromatography column (purchased from PE biosystems). The column was equilibrated with 50mM Tris (pH 7.4). The PI-50 column was washed with 1CV of 50mM Tris (pH 7.4) and then eluted with 3CV or more of 1.5M NaCl in 50mM NaOAc (pH 6). The PI fraction was loaded onto a SephacrylS200 chromatography column equilibrated with 50mM NaOAc (pH 6), 125mM NaCl. The S200 fraction was mixed with salt to a final concentration of 0.7M ammonium sulfate and 0.6M NaCl and loaded onto a Toyopearl Hexyl 650C chromatography column (purchased from Toso Haas) equilibrated with a buffer containing 0.6M NaCl, 0.7M ammonium sulfate in 50mM NaOAc (pH 6). The column was then washed with 2CV of the same buffer. Recombinant Neutrokine- α was then eluted with 3CV of 50mM NaOAc (pH 6) followed by 2CV of 20% ethanol. The recombinant Neutrokine-alpha protein was then eluted at the end of an ammonium sulfate gradient (0.3-0M). The appropriate fractions were pooled and dialyzed against a buffer containing 50mM NaOAc (pH 6) and then passed through a porous 50HQ chromatography column. HQ flow-through was diluted 4-fold and loaded onto Toyopearl DEAD 6 50M column, then eluted with 25mM sodium citrate, 125M NaCl.

In another embodiment, recombinant Neutrokine-alpha is expressed and purified using a baculovirus vector system in Sf + insect cells.

First, a polynucleotide encoding Ser-78 to Leu-285 amino acid residues of the Neutrokine-alpha polypeptide sequence shown in FIGS. 1A and 1B (which are equal to Ser-78 to Leu-285 amino acid residues of the Neutrokine-alpha polypeptide sequence shown in SEQ ID NO: 2) was subcloned into the baculovirus transfer construct PSC to generate a baculovirus expression plasmid. The pA2GP transfer vector, derived from pVL941, contained a gp67 signal peptide, a modified multiple cloning site, and a lac gene cloned downstream of the Drosophila heat shock promoter to select for blue-stained plaques. Using the Neutrokine-alpha (SEQ ID NO: 2) sequence, and the pA2GP vector sequence, a cloning strategy was designed to insert the PSC signal peptide coding sequence closely fused to Ala-134 of the Neutrokine-alpha coding sequence (SEQ ID NO: 2 and FIGS. 1A and 1B) into the PSC baculovirus transfer plasmid. This method involves the use of two Polymerase Chain Reactions (PCR). First, primers for amplifying a Neutrokine-alpha sequence are designed. The 5 ' primer consists of the sequence encoding Ala-134 and residues (5'-GGT CGC CGT TTC TAA CGC GGCCGT TCA GGG TCC AGA AG-3'; SEQ ID NO: 31) preceding the sequence encoding the C-terminus of the PSC signal peptide. The 3 ' primer (5'-CTG GTTCGG CCC AAG GTA CCA AGC TTG TAC CTT AGA TCT TTT CTAGAT C-3'), consisting of the reverse complement of the pA2GP vector sequence located immediately downstream of the Neutrokine-alpha coding sequence, precedes the Kpn I restriction endonuclease site and the spacer sequence (to increase the Kpn I cleavage efficiency). PCR was performed using pA2GP containing the Neutrokine-. alpha.plasmid template and primers O-1887 and O-1888, and the resulting PCR product was purified by standard methods.

Another PCR reaction was performed using the PSC baculovirus transfer plasmid pMGS12 as a template. This pMGS12 plasmid consists of an AcNPV EcoRI "I" fragment inserted into pUC8, the polyhedrin coding sequence after the ATG start codon being replaced with a PSC signal peptide and a polylinker site. The PCR reaction was templated using pMGS12, with a 5 ' primer (5'-CTGGTA GTT CTT CGG AGT GTG-3'; SEQ ID NO: 33) annealed in the AcNPV ORF603 upstream of the unique NgoMIV and EcoRV sites, and a 3 ' primer (5'-CGCGTT AGA AAC GGC GAC C-3'; SEQ ID NO: 34) annealed to the 3 ' end of the sequence encoding the PSC signal peptide.

To generate a PCR product in which the PSC signal peptide is intimately fused to the Neutrokine-alpha coding sequence Ala-134, this PCR product was combined with the PSC signal peptide-polyhedrin upstream region PCR product and subjected to another PCR cycle. Since the 3 'end of the PSC signal peptide PCR product (pMGS12/O-959/O-1044) overlapped the 5' end of the Neutrokine-. alpha.PCR product prepared with primers O-1887/O-1888, the two PCR products were combined and overlap-extended by PCR with primers O-959 and O-1888.

The resulting overlap-extended PCR product containing the PSC signal peptide fused to the Neutrokine-alpha sequence was inserted into the baculovirus transfer plasmid pMGS 12. This PCR product was digested with NgoM IV and Kpn I, the fragment was purified and ligated into NgoM IV-Kpn I cut pMGS 12. After transformation of competent E.coli DH 5. alpha. cells with this ligation mixture, colonies were picked and plasmid DNA was prepared in minute quantities. Some positive clones for each ligation reaction were identified by restriction digest analysis of plasmid DNA and 3 clones (pAcC9669, pAcC9671, pAcC9672) were selected for large scale plasmid purification. The resulting plasmid DNA was subjected to DNA sequence analysis to determine and sequence the Neutrokine-alpha insert.

The following procedure illustrates the recovery and purification of recombinant Neutrokine-alpha from Sf + insect cells. Unless otherwise specified, the process is carried out at 2-8 ℃.

Recovering

The first step is as follows: CaCl₂Treatment of

Sf + cell supernatants were collected by centrifugation at 8000 Xg. Buffer-1 (1 MCaCl) will be recovered₂) Adding to the supernatant, thereby obtaining CaCl₂Was 10 mM. (in a preferred embodiment, 1M ZnCl is used₂In place of 1M CaCl₂). The pH of this solution was measured using recovery buffer-2 (1M Tris pH)8 (+ -0.2)) was adjusted to 7.7 + -. The solution was incubated for 15 minutes and then centrifuged at 8000 Xg.

Purification of

The first step is as follows: chromatography on a porous PI-50 column

The Sf + cell supernatant was applied to a multi-well PI-50 column (PE Biosystem) equilibrated with PI-1 buffer (50mM Tris, 50mM NaCl, pH7.4 (+ -0.2)). The PI-50 column was washed with 1-2CV of PI-1 buffer and then eluted with a 3CV linear gradient of PI-2 buffer (50mM sodium citrate, pH6 (+ -0.2)). The eluate was monitored for Ultraviolet (UV) absorbance at 280 nm. Fractions that crossed the elution peak were collected and analyzed by SDS-PAGE. The appropriate components are pooled.

The second step is that: chromatography on a Toyopearl Hexyl 650C column

Mixing the aggregated PI with salt to (NH) ₄)₂SO₄Was added to a Toyopearl Hexyl 650C column using HIC-1 buffer (50mM NaOAc, 0.6M NaCl, 0.7M (NH)₄)₂SO₄pH6(± 0.2)). The column was then washed with 2CV of HIC-1 buffer. Next, recombinant Neutrokine-alpha was eluted with 3-5CV HIC-2 buffer (50mM NaOAc pH6 (+ -0.2)) followed by 2CV of 20% ethanol. The eluate was monitored for Ultraviolet (UV) absorbance and conductivity at 280 nm. Fractions of the eluted peaks were collected and analyzed by SDS-PAGE. The appropriate components are pooled.

The third step: chromatography on SP Sepharose FF

Hexy1 fractions were dialyzed and the pH was adjusted to 4.5 with SP-1 buffer (50mM sodium acetate, pH4.5 (+ -0.2)), diluted 4-fold and loaded onto a SP Sepharose (cation exchanger, Pharmacia) column equilibrated with SP-1 buffer (50mM sodium acetate, pH4.5 (+ -0.2)). The recombinant Neutrokine-alpha protein was then eluted from the SP column at pH5.5 with SP-2 buffer (50mM sodium acetate, pH5.5 (+ -0.2)). The eluate was then monitored for Ultraviolet (UV) absorbance at 280 nm. Fractions that crossed the elution peak were collected and analyzed by SDS-PAGE. The appropriate components are pooled.

The fourth step: dialysis recombinant Neutrokine-alpha

The SP fractions were placed in a 6-8kd cut-off membrane set-up and then dialyzed or diafiltered overnight in dialysis buffer (10mM sodium citrate, 140mM NaCl, pH6 (+ -0.2)).

The fifth step: filtration and filling

The protein concentration of the recombinant Neutrokine-alpha solution of the sixth step was determined by bicinchoninic acid (BCA) protein assay. Recombinant Neutrokine-alpha is adjusted to final protein concentration with appropriate buffer and filtered under controlled conditions. The filtrate was stored in a suitable sterile container at below-20 ℃.

In a specific embodiment, the Neutrokine-alpha protein of the invention produced as described above is adjusted to a final protein concentration of 1-5mg/ml and buffered with 10mM sodium citrate, 140mM NaCl, pH 6.0(± 0.4) and stored in type 1 glass vials below-20 ℃.

During chromatography, the Ultraviolet (UV) absorbance at 280nm was monitored. When applicable, the chromatographic intermediates were tested for conductivity, pH and monitored by SDS and/or RP-HPLC.

The column and purification equipment were cleaned and washed with 0.2 or 0.5M NaOH and deionized water, followed by 0.1 or 0.5M acetic acid. The column and purification equipment are rinsed with deionized water and, if necessary, stored in a suitable storage solution. Prior to use, the device is equilibrated with an appropriate buffer (such as those described herein or well known in the art).

In a preferred embodiment, in the first step of the recovery described above, 1M ZnCl is used₂In place of 1M CaCl₂. Also, in this embodiment, a combination of ZnCl may be used₂And CaCl₂。0.1M ZnCl₂And 0.9M CaCl₂Some combinations of (a) may be used in the recovery process of recombinant Neutrokine-alpha protein, such as, but not limited to, 0.1M ZnCl₂And 0.9M CaCl₂，0.2M ZnCl₂And 0.8M CaCl₂，0.3M ZnCl₂And 0.7M CaCl₂，0.4M ZnCl₂And 0.6M CaCl₂，0.5M ZnCl₂And 0.5M CaCl₂，0.6M ZnCl₂And 0.4M CaCl₂，0.7M ZnCl₂And 0.3M CaCl₂，0.8M ZnCl₂And 0.2M CaCl₂，0.9M ZnCl₂And 0.1M CaCl₂And the like. However, the presence of EDTA will inhibit the recovery process. In addition, ZnCl was present in recovery buffer 1₂And/or CaCl₂Will lead to the formation of a large number of high molecular weight Neutrokine-alpha multimers.

Example 3: cloning and expression of Neutrokine-alpha in mammalian cells

Typical mammalian expression vectors contain a promoter element that mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for transcription termination and polyadenylation of the transcript. Other elements include enhancers, Kozak sequences and intervening sequences flanked by RNA splice donor and acceptor sites. High transcription efficiency is achieved with the early and late promoters of SV40, the Long Terminal Repeats (LTRs) of retroviruses such as RSV, HTLVI, HIVI and the early promoter of Cytomegalovirus (CMV). However, cytokines (e.g., the human actin promoter) may also be used. Suitable expression vectors for use in the present invention include, for example, pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that can be used include human HeLa, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, frightening QC1-3 cells, mouse L cells, Chinese Hamster Ovary (CHO) cells, and HEK 293 cells.

Alternatively, the gene may be expressed in a suitable cell line containing the gene integrated into the chromosome. Transfected cells can be identified and isolated by co-transfection with selectable markers such as dhfr, gpt, neomycin, hygromycin.

The transfected gene may also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is used to show cell lines carrying hundreds or even thousands of copies of the corresponding gene. Another useful selectable marker is Glutamine Synthase (GS) (Murphy et al, J. biochem. 227: 277-279 (1991); Bebbington et al, Bio/technology 10: 169-175 (1992)). Using these markers, cells were grown in selective media and the highest resistant cells were selected. These cell lines contain amplified genes integrated into the chromosome. Chinese Hamster Ovary (CHO) cells and NSO cells are commonly used for the production of proteins.

Expression vectors pC1 and pC4 contain a strong promoter (LTR) of Rous sarcoma virus (Cullen et al, molecular and cellular biology, 438-447 (3 months 1985)) plus a CMV-enhancer fragment (Boshart et al, cell 41: 521-530 (1985)). Multiple cloning sites, for example with restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the corresponding genes. The vector additionally contains the 3' intron of the murine preproinsulin gene, polyadenylation and termination signals.

Example 3 (a): cloning and expression in COS cells

The expression plasmid pNeutrokine-alpha-HA was generated by cloning a portion of the cDNA deposited encoding the extracellular domain of the protein into the expression vector pcDNAI/Amp or pcDNAIII (which is available from Invitrogen). To produce a soluble, secreted form of the polypeptide, the extracellular domain is fused to the secretory leader sequence of the human IL-6 gene.

The expression vector pcDNAI/Amp contains: (1) an E.coli origin of replication for efficient propagation in E.coli and other prokaryotic cells; (2) selecting an ampicillin resistance gene of a prokaryotic cell containing a plasmid; (3) SV40 origin of replication propagated in eukaryotic cells; (4) CMV promoter, polylinker, SV40 intron; (5) codons encoding the hemagglutinin fragment (i.e., the HA tag facilitating purification) are followed by a stop codon and a polyadenylation signal, whereby the cDNA is conveniently placed under the expression control of the CMV promoter and is operably linked to the SV40 intron and polyadenylation signal by restriction sites within the polylinker. This HA tag corresponds to an epitope derived from the influenza hemagglutinin protein, such as Wilson et al, cell 37: 767 (1984). Fusion of the HA tag to the target protein allows for easy detection and recovery of recombinant proteins having antibodies recognizing the HA epitope. In addition, pcDNAIII contains a selectable neomycin marker.

A DNA fragment encoding the extracellular domain of the Neutrokine-alpha polypeptide was cloned into the polylinker region of the vector so that the recombinant protein was expressed by the CMV promoter. The plasmid construction strategy is as follows. The Neutrokine-alpha cDNA of the deposited clone is amplified using primers containing conventional restriction sites, most as described above for the construction of vectors expressing Neutrokine-alpha in E.coli. Suitable primers include those used in this example below. A 18 nucleotide 5 'primer containing an underlined BamHI site, a Kozak sequence, an AUG start codon, a sequence encoding a secretory leader peptide of the human IL-6 gene, and a 5' coding sequence for the ectodomain of Neutrokine-alpha protein, having the following sequence: 5' -GCGGGA TCCGCC ACC ATG AAC TCC TTC TCC ACA AGC GCC TTCGGT CCA GTT GCC TTC TCC CTG GGG CTG CTC CTG GTG TTGCCT GCT GCC TTC CCT GCC CCA GTT GTG AGA CAA GGG GACCTG GCC AGC-3' (SEQ ID NO: 16). A BamHI restriction site, underlined, and a 3 'primer complementary to 18 nucleotides of the 3' coding sequence preceding the stop codon, having the following sequence: 5' -GTGGGA TCC TTA CAG CAG TTTCAA TGC ACC-3’(SEQ ID NO：17)。

The PCR-amplified DNA fragment and the vector pcDNAI/Amp were digested with BamHI and ligated. This ligation mixture was transformed into the E.coli strain SURE (purchased from Stratagene Cloning Systems, 11099 North torry pipes Road, La Jolla, CA 92037), and the transformed culture was plated on ampicillin medium, and the plate was incubated to grow ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and tested for the presence of a fragment encoding the extracellular domain of Neutrokine-alpha by restriction analysis or other means.

For expression of recombinant Neutrokine-alpha, COS cells are transfected with an expression vector as described above using DEAE-DEXTRAN, e.g., as described in Sambrook et al, A molecular cloning laboratory Manual, Cold spring harbor laboratory Press, Cold spring harbor, N.Y. (1989). The cells are incubated under conditions in which Neutrokine-alpha is expressed by the vector.

The expression of Neutrokine-alpha-HA fusion protein was detected by radiolabelling and immunoprecipitation using, for example, Harlow et al, antibody laboratory Manual, second edition; cold spring harbor laboratory Press, Cold spring harbor, New York (1988). Two days after transfection, cells were passed in a medium containing³⁵The S-cysteine was labeled by incubation in medium for 8 hours. Cells and medium were collected, washed with RIPA buffer containing detergent and lysed: 150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM TRIS, pH7.5, as described by Wilson et al, supra. Proteins were precipitated from cell lysates and culture media with HA-specific monoclonal antibodies. The precipitated proteins were then analyzed by SDS-PAGE and autoradiography. Expression products of the expected size were found in cell lysates and not in the negative control.

Example 3 (b): cloning and expression in CHO cells

The vector pC4 is used for expressing Neutrokine-alpha protein. Plasmid pC4 is a derivative of plasmid pSV2-dhfr (ATCC accession No: 37146). To produce a soluble, secreted form of the polypeptide, the extracellular domain is fused to the secretory leader sequence of the human IL-6 gene. The vector plasmid contained the murine DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary or other cells transfected with these plasmids that lack dihydrofolate activity can be selected by growing the cells in selective media (α minus MEM, life technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of the DHFR gene in cells resistant to Methotrexate (MTX) has been well documented (see, e.g., Alt, F.W., Kellems, R.M., Bertino, J.R., and Schimke, R.T., 1978, J.Biochem.253: 1357-. Cells grown in increasing concentrations of MTX are resistant to the drug by overproducing the targeting enzyme DHFR, resulting in the amplification of the DHFR gene. If another gene is linked to the DHFR gene, it is usually co-amplified and overexpressed. This method is known in the art to be useful for generating cell lines carrying more than 1000 copies of the amplified gene. Next, when methotrexate is removed, a cell line is obtained that contains the amplified gene integrated into one or more chromosomes of the host cell.

Plasmid pC4 contains the strong promoter of the Long Terminal Repeat (LTR) of Rouse sarcoma virus expressing the corresponding gene (Cullen et al, molecular and cell biology, 3.438-447, 1985), plus a fragment of an enhancer isolated from the immediate early gene of human Cytomegalovirus (CMV) (Boshart et al, cell 41: 521-530 (1985)). Downstream of the promoter is one of the following restriction enzyme cleavage sites that allow gene integration: BamHI, XbaI, and Asp 718. Following these cloning sites, the plasmid contains the 3' intron of the murine preproinsulin gene and a polyadenylation site. Other high efficiency promoters may also be used for expression, such as the human β -actin promoter, the SV40 early or late promoter, or the long terminal repeats of other retroviruses such as HIV and HTLVI. The Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express Neutrokine-alpha in a regulated manner in mammalian cells (Gossen, M., & Bujard, H.1992, Proc. Natl. Acad. Sci. USA 89: 5547-. For example, human growth hormone or globulin genes can be used as other signals for polyadenylation of mRNA. Stable cell lines carrying the corresponding gene integrated into the chromosome can also be selected on the basis of cotransfection with selectable markers such as gpt, G418 or hygromycin. It may be advantageous to use more than one marker in the beginning, such as G418 plus methotrexate.

Plasmid pC4 was digested with the restriction enzyme BamHI and then dephosphorylated with bovine small intestine phosphatase by methods known in the art. The vector was then separated from the 1% agarose gel.

The DNA sequence encoding the ectodomain of the Neutrokine-alpha protein was amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene. Underlined BamHI site, Kozak sequence, AUG originA start codon, a sequence encoding a secretory leader peptide of the human IL-6 gene, and a 5 'primer of 18 nucleotides of the 5' coding sequence of the ectodomain of Neutrokine-alpha protein, having the following sequence: 5' -GCGGGA TCCGCC ACC ATGAAC TCC TTC TCC ACA AGC GCC TTC GGT CCA GTT GCC TTCTCC CTG GGG CTG CTC CTG GTG TTG CCT GCT GCC TTC CCTGCC CCA GTT GTG AGA CAA GGG GAC CTG GCC AGC-3' (SEQ ID NO: 16). A BamHI restriction site, underlined, and a 3 'primer complementary to 18 nucleotides of the 3' coding sequence preceding the stop codon, having the following sequence: 5' -GTGGGA TCC TTA CAG CAG TTT CAA TGC ACC-3’SEQ IDNO：17)。

The amplified fragment was digested with endonuclease BamHI and then repurified on 1% agarose gel. The isolated fragment and dephosphorylated vector were then ligated using T4 DNA ligase. Coli HB101 or XL-1 Blue cells are then transformed and bacteria containing the fragment of the insert plasmid pC4 are identified, for example by restriction enzyme analysis.

Chinese hamster ovary cells lacking the active DHFR gene were used for transfection. Mu.g of expression plasmid pC4 was co-transfected with 0.5. mu.g of plasmid pSVneo by lipofection (Felgner et al, supra). Plasmid pSV2-neo contains a dominant selectable marker encoding the neo gene encoding the enzyme conferring antibiotic resistance, including Tn5 of G418. Cells were seeded in α minus MEM medium supplemented with 1mg/ml G418. After 2 days, cells were trypsinized and plated on hybridoma clone plates (Greiner, Germany) in α minus MEM medium supplemented with 10, 25, or 50ng/ml methotrexate and 1mg/ml G418. After about 10-14 days, each clone was trypsinized and plated with various concentrations of methotrexate (50, 100, 200, 400, 800Nm) on 6-well plates or 10ml flasks. Clones growing at the highest concentration of MTX were then transferred to new 6-well plates containing higher concentrations of MTX (1, 2, 5, 10, 20. mu.M). The same procedure was repeated until clones grown at a concentration of 100-200. mu.M were obtained. Expression of the desired gene product is analyzed by, for example, SDS-PAGE and Western blotting or by reverse phase HPLC.

The inventors have generated at least 6 Neutrokine-alpha expression constructs to facilitate the production of Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides of different specifications and in some systems. This expression construct is as follows: (1) pNa.A71-L285 (expressing the amino acid residue at Ala71-Leu 285), (2) pNa.A81-L285 (expressing the amino acid residue at Ala81-Leu 285), (3) pNa.L112-L285 (expressing the amino acid residue at Leu112-Leu 285), (4) pNa.A134-L285 (expressing the amino acid residue at Ala134-Leu 285), (5) pNa.L147-L285 (expressing the amino acid residue at Leu147-Leu 285), (6) pNa.G161-L285 (expressing the amino acid residue at Gly161-Leu 285).

In a preferred embodiment, the expression construct is used to express various Neutrokine-alpha muteins of bacterial, baculovirus and mammalian systems.

In other preferred embodiments, the construct expresses a fragment of Neutrokine-alpha polypeptide fused at the N-or C-terminus to a heterologous polypeptide, such as the signal peptide of human IL-6, the signal peptide of CK-beta 8 (amino acids-21 to-1 of the CK-beta 8 sequence disclosed in PCT publication PCT/US 95/09058), or the Fc region of human IgG. Other sequences that may be used are known to those skilled in the art.

Example 4: tissue distribution of Neutrokine-alpha mRNA expression

Northern blot analysis was performed to detect expression of the Neutrokine-alpha gene in human tissues using methods such as those described in Sambrook et al. cDNA Probe containing the entire nucleotide sequence of Neutrokine-alpha protein (SEQ ID NO: 1) Using rediprime^TMDNA labeling System (Amersham Life Science), according to the manufacturer's instructions³²And P is marked. After labeling, the probes were labeled with CHROMA SPIN-100^TMChromatography column (Clontech laboratories, Inc.), according to the manufacturer's protocol number PT1200-1 and purification. The purified labeled probe is then used to detect Neutrokine-alpha and/or Neutrokine-alpha SV mRNA from various human tissues.

Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM)From Clontech using ExpressHyb^TMThe hybridization solution (Clontech) was detected with a labeled probe according to the manufacturer's protocol No. PT 1190-1. After hybridization and washing, the blot was exposed overnight at-70 ℃ to a film, which was developed according to standard methods.

To determine the expression pattern of Neutrokine-alpha and/or Neutrokine-alpha SV, a panel of multi-tissue Northern blots was probed. It was shown that a 2.6kb mRNA was significantly expressed in peripheral blood leukocytes, spleen, lymph nodes and bone marrow, and was detectable in placenta, heart, lung, fetal liver, thymus and pancreas. Analysis of a panel of cell lines showed that Neutrokine-alpha and/or Neutrokine-alpha SV was highly expressed in HL60 cells, with detectable expression in K562, but not in Raji, HeLa, or MOLT-4 cells. All analyses showed that Neutrokine-alpha and/or Neutrokine-alpha SV mRNA expression was enriched in the immune system.

Example 5: gene therapy with endogenous Neutrokine-alpha gene

Another gene therapy according to the present invention involves the operative association of the endogenous Neutrokine-alpha sequence with a promoter by homologous recombination, as in U.S. Pat. No. 6/24 of 1997: 5641670, respectively; international publication WO 96/29411, published 26.9.1996; international publication WO 94/12650, published on 8/4 of 1994; koller et al, proceedings of the national academy of sciences USA 86: 8932-8935 (1989); and Zijlstra et al, nature 342: 435 and 438 (1989). The method involves activating a gene that is present in the target cell, but is not expressed or is expressed at a low level in the cell. A polynucleotide construct is generated containing a promoter and targeting sequences that are homologous to the 5' non-coding sequence of endogenous Neutrokine-alpha on both sides of the promoter. The targeting sequence is near the 5' end of Neutrokine-alpha, whereby the promoter is operably linked to the endogenous sequence by homologous recombination. The promoter and targeting sequences can be amplified by PCR. Preferably, the amplified promoter contains unique restriction enzyme sites at the 5 'and 3' ends. Preferably, the 3 'end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5 'end of the second targeting sequence contains the same restriction enzyme site as the 3' end of the amplified promoter.

The amplified promoter and amplified targeting sequence are digested with appropriate restriction enzymes and then treated with calf intestinal phosphatase. The digested promoter was added with the digested targeting sequence in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of the two fragments. The constructs were fractionated by size on an agarose gel and then purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct is administered as a naked polynucleotide by electroporation. However, the polynucleotide construct may also be administered with a transfection facilitating agent, such as liposomes, viral sequences, viral particles, precipitating agents, and the like. Such delivery is known in the art.

Once the cells are transfected, homologous recombination is performed, allowing the promoter to be operably linked to the endogenous Neutrokine-alpha sequence. This allows Neutrokine-alpha to be expressed in the cell. Expression may be detected by immunostaining or any other method known in the art.

Fibroblasts were obtained from skin biopsies of subjects. The resulting tissue was placed in DMEM + 10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts were trypsinized and washed from the plastic surface with nutrient medium. A portion of the cell suspension was removed for counting and the remaining cells were centrifuged. The supernatant was aspirated off and the pellet resuspended in 5ml of electroporation buffer (20mM HEPES pH7.3, 137mM NaCl, 5mM KCl, 0.7mM Na) ₂HPO₄6mM glucose). The cells were recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation containing 1mg/ml acetylated bovine serum albumin. The final cell suspension contained approximately 3X 10⁶Individual cells/ml. Electroporation should be performed immediately after resuspension.

Plasmid DNA was prepared according to standard methods. For example, to construct a plasmid directed to the Neutrokine-alpha locus, plasmid pUC18(MBI enzymes, Amherst, N.Y.) was digested with HindIII. The CMV promoter was amplified by PCR to contain an XbaI site at the 5 'end and a BamHI site at the 3' end. Two Neutrokine-alpha non-coding sequences were amplified by PCR: a Neutrokine-alpha non-coding sequence (Neutrokine-alpha fragment 1) containing a HindIII site at the 5 'end and an XbaI site at the 3' end after amplification; another Neutrokine-alpha noncoding sequence (Neutrokine-alpha fragment 2) contains a BamHI site at the 5 'end and a HindIII site at the 3' end after amplification. The CMV promoter and the Neutrokine-alpha fragment were digested with the appropriate enzymes (XbaI and BamHI for the CMV promoter; XbaI for Neutrokine-alpha fragment 1; BamHI for Neutrokine-alpha fragment 2), and ligated together. The resulting ligation product was digested with HindIII and ligated with HindIII-digested pUC18 plasmid.

Plasmid DNA was added to a sterile tube (Bio-Rad) with a 0.4cm electrode gap. The final DNA concentration is at least 120. mu.g/ml. Then 0.5ml of the cell suspension (containing about 1.5X 10)⁶Individual cells) were added to the tube and the cell suspension and DNA solution were gently mixed. Electroporation was carried out using a Gene-Pulser apparatus (Bio-Rad). The capacitance and voltage were set to 960 μ F and 250-300V, respectively. With increasing voltage, cell survival decreased, but the percentage of surviving cells stably incorporating the introduced DNA into their genome increased significantly. Given these parameters, a pulse time of about 14-20 milliseconds should be observed.

The electroporated cells were kept at room temperature for approximately 5 minutes, and then the contents of the tube were gently removed with a sterile pipette. Cells were added directly to 10ml of pre-warmed nutrient medium (DMEM with 15% calf serum) in 10cm dishes and incubated at 37 ℃. The following day, the medium was aspirated and replaced with 10ml of fresh medium, and incubated for a further 16-24 hours.

The genetically engineered fibroblasts are then injected into the host, either alone or after growing to confluence with cytodex 3 microcarrier beads. The fibroblasts now produce a protein product. The fibroblasts can then be introduced into the patient, as described above.

Example 6: neutrokine-alpha, a novel member of the tumor necrosis factor ligand family as B lymphocyte stimulator

In the human neutrophil/monocyte derived cDNA library, a 285 amino acid protein was identified which displayed significant homology in its putative extracellular receptor-ligand binding domain to APRIL (28.7%) (Hahne, M. et al, J. Exp. methods 188, 1185-90(1998)), TNF-. alpha. (16.2%) (Pennica, D. et al, Nature 312, 724-jar 729(1984)) and LT-. alpha. (14.1%) (Gray, Nature 312, 721-jar 724(1984)) (FIG. 7A). We refer to this cytokine as Neutrokine-alpha (also called B lymphocyte stimulator (BlyS) based on its biological activity). Hydrophobicity analysis of the Neutrokine-alpha protein sequence showed that a potential transmembrane domain between amino acid residues 47-73 precedes the non-hydrophobic amino acids, suggesting that Neutrokine-alpha is a type II membrane-bound protein, as is the case with other TNF ligand families (Cosman, D., Stem cell 12: 440-55 (1994)). Expression of this cDNA in mammalian cells (HEK293 and chinese hamster ovary cells) and Sf9 insect cells identified a soluble form of 152 amino acids with the N-terminal sequence from alanine at position 134 (fig. 7A). For the reconstruction of mass and charge ratios, the molecular weight of Neutrokine-alpha was determined to be 17038 Daltons, which is consistent with the predicted molecular weight of the 152 amino acid protein with a disulfide bond (17037.5 Daltons).

Using human/hamster somatic and radiation hybrid mapping, it was found that the gene encoding Neutrokine-alpha was linked to marker SHGC-36171, which was located in human chromosome 13q34, a region unrelated to any other member of the TNF superfamily of genes (Cosman, D. Stem cell 12: 440-55 (1994)).

The expression pattern of Neutrokine-alpha was determined by Northern blot (FIG. 7B) and flow cytometric analysis (Table 5 and FIG. 8). Neutrokine-alpha is encoded by a 2.6kb mRNA, which is found to be expressed at high levels in peripheral blood leukocytes, spleen, lymph nodes and bone marrow. Low levels of expression were detected in placenta, heart, lung, fetal liver, thymus and pancreas. In this set of cell lines, Neutrokine- α mRNA was detected in HL-60 and K562, but not in Raji, HeLa, or MOLT-4 cells. These results were confirmed by flow cytometric analysis using Neutrokine-alpha specific mAb 2E 5. As shown in FIG. 5, Neutrokine-alpha expression was not detected on T or B cell lines, but was restricted to cells of myeloid origin. Further analysis of normal blood cells showed significant expression on quiescent monocytes, up-regulated approximately 4-fold after exposing the cells to IFN- γ (100U/ml) for 3 days (FIG. 8A). A concomitant increase in Neutrokine-alpha specific mRNA was also detected (FIG. 8B). In contrast, Neutrokine-alpha is not expressed in newly isolated peripheral blood granulocytes, T cells, B cells or NK cells.

The ability of purified recombinant Neutrokine-alpha (rNeutrokine-alpha) to induce activation, proliferation, differentiation or death of cells, including B cells, T cells, monocytes, NK cells, hematopoietic progenitor cells, and cells of various endothelial and epithelial origin, in various cell-based assays is determined. Among these assays, Neutrokine-alpha was found to enhance B cell proliferation in a standard combination stimulation assay in which purified tonsil B cells were cultured in the presence of formalin-fixed Staphylococcus aureus Cowan I (SAC) or fixed anti-human IgM as an initiator (Sieckmann, D.G., et al, J.Exp. 147: 814-29 (1978); Ringden, O. et al, Scand.J.Immunol.6: 1159-69 (1977)). As shown in FIG. 9A, recombinant Neutrokine- α induced tonsil B-cell dose-dependent proliferation. This response was similar to that of rIL2 at a dose range of 0.1-10000 ng/ml. Neutrokine-alpha also induced B cell proliferation when cell culture was co-stimulated with immobilized anti-IgM antibody (fig. 9B). In the presence of a fixed concentration of IL2 or rNeutrokine-. alpha.a dose-dependent response is readily observed with increasing amounts of cross-linker.

In an attempt to correlate specific biological activity on B cells with receptor expression, purified Neutrokine-alpha was biotinylated. The resulting biotinylated Neutrokine-alpha retains biological function in standard B cell proliferation assays. Lineage specific analysis of whole blood cells surrounding humans showed that biotinylated Neutrokine-alpha binds to T cells, monocytes No detection on cells, NK cells and granulocytes was determined by CD3, CD14, CD56 and CD66b, respectively (fig. 10A). In contrast, biotinylated Neutrokine-alpha binds to peripheral blood CD20⁺B cells. Receptor expression was also detected in B cell tumor lines REH, ARH-77, Raji, Namalwa, RPMI8226 and IM-9, but not in any of the bone marrow derived cell lines tested including THP-1, HL-60, K-562 and U-937. Representative flow cytometry patterns for myeloma cell line IM-9 and tissue cell line U-937 are shown in FIG. 10B. Similar results were obtained with biologically active FLAG-labeled Neutrokine-alpha protein instead of chemically modified biotinylated Neutrokine-alpha. These results demonstrate that Neutrokine-alpha exhibits significant B-cell tropism in both its receptor profile and biological activity. These results also show whether cellular activation can induce the expression of Neutrokine-alpha receptor in peripheral blood cells, other normal cells or established cell lines.

To test the species specificity of Neutrokine-alpha, mouse splenic B cells were cultured in the presence of human Neutrokine-alpha and SAC. The results indicate that rNeutrokine-alpha induces murine splenic B cells to proliferate in vitro and bind to cell surface receptors on these cells. Interestingly, immature surface Ig negative B cell precursors isolated from mouse bone marrow neither proliferate in response to Neutrokine-alpha nor bind ligand.

To determine the in vivo activity of rNeutrokine- α, BALB/c mice (3/group) were injected (i.p.) twice daily with buffer, or 0.08mg/kg, 0.8mg/kg, 2mg/kg or 8mg/kg of rNeutrokine- α. Mice were subjected to this treatment for 4 consecutive days, after which they were sacrificed and various tissues and sera were collected for analysis. In another embodiment, BALB/c mice are injected twice daily (i.p.) with any dose of rNeutrokine-alpha in the range of 0.01-10 mg/kg. In a preferred embodiment, BALB/c mice are injected twice daily (i.p.) with any dose of rNeutrokine-alpha in the range of 0.01-3mg/kg (particularly preferred doses in this embodiment include, but are not limited to, 0.01mg/kg, 0.02mg/kg, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.8mg/kg, 1.9mg/kg, 1.0mg/kg, 1.1.1.1 mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.8mg/kg, 1.9mg/kg, 2.0mg/kg, 2.1mg/kg, 2.2mg/kg, 2.3mg/kg, 2.4mg/kg, 2.5mg/kg, 2.6mg/kg, 2.7mg/kg, 2.8mg/kg, 2.9mg/kg, and 3.0 mg/kg). In another preferred embodiment, BALB/c mice are injected twice daily (i.p.) with any dose of rNeutrokine-alpha in the range of 0.02-2mg/kg (particularly preferred doses in this embodiment include, but are not limited to, 0.02mg/kg, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/g, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/g, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.6mg/kg, 1.8mg/kg, 1.9mg/kg, 2.0 mg/kg).

The effect of administration of Neutrokine-alpha was evident in spleen tissue sections stained microscopically with HE and immunohistochemically with mAb specific for CD45R (B220) (fig. 11A). Normal spleen architecture was altered, white matter marginal zones were clearly extended, and cellular architecture of red marrow was significantly increased (fig. 11A). The marginal zone extension was due to an increased number of lymphocytes expressing the B cell marker CD45R (B220). In addition, the periarterial lymphatic sheath (PALS) region with dense T cells was also infiltrated by appropriate amounts of CD45R (B220) positive cells. This suggests that white matter changes are due to an increase in B cell numbers. Densely packed cell populations that normally populate the red marrow are not stained by CD45R (B220). Additional experiments were performed to characterize all cell types involved and to further determine the mechanism by which 0.08mg/kg, 0.8mg/kg, 2mg/kg altered spleen structure.

Flow cytometric analysis of spleen tissue from mice treated with 2mg/kg Neutrokine-alpha showed that Neutrokine-alpha increased maturation compared to control mice (CD45R (B220)^dull，ThB^bright) The proportion of B cells was approximately 10-fold (fig. 11B). Additional analyses performed in which mice were treated with buffer, 0.08mg/kg, 0.8mg/kg, 2mg/kg, or 8mg/kg Neutrokine-alpha showed 0.08mg/kg, 0.8mg/kg, 2mg/kg based on control mice kg of Neutrokine-alpha all increased maturation (CD45R (B220)^dull，ThB^bright) The proportion of B cells is approximately 10-fold, while buffer and 8mg/kg Neutrokine-alpha produce approximately the same proportion of mature B cells. See table 4.

Table 4: FACS analysis of mouse splenic B cell populations

Neutrokine-α(mg/kg)	Percentage of mature B cells (R2)	Percentage positive CD45R (R1)
			Control (buffer) 0.08mg/kg0.8mg/kg2mg/kg8mg/kg	1.2616.1518.5416.541.24	52.1756.5357.5657.5561.42

A potential consequence of increased expression of mature B cells in vivo is a relative increase in serum Ig titers. Thus, serum IgA, IgG and IgM levels were compared between buffer and Neutrokine-alpha treated mice (FIG. 11C). Administration of Neutrokine-alpha increased IgA and IgM levels in serum 2 and 5 fold, respectively. It is also noted that circulating levels of IgG do not increase.

In addition, a dose-dependent response was observed in serum IgA titration of mice treated with various amounts of Neutrokine-alpha for 4 days, whereas no significant dose-dependence was observed with the same amount of Neutrokine-alpha for 2 days. Serum IgA titration by administration of Neutrokine-alpha at 8, 2, 0.8, 0.08 and 0mg/kg were approximately 800. mu.g/ml, 700. mu.g/ml, 400. mu.g/ml, 200. mu.g/ml, and 200. mu.g/ml in the case of 4 days of administration. I.e., 4 days after administration of 8, 2, 0.8, and 0.08mg/kg of Neutrokine-alpha, IgA serum levels increased by approximately 4, 3.75, 2, and a minimal fold, respectively, compared to the background or basal IgA serum levels observed with buffer administration alone. In a further embodiment, these tests can be performed with any amount of rNeutrokine-alpha in the range of 0.01 to 10 mg/kg. In a preferred embodiment, Neutrokine- α is administered in the range of 0.01-3mg/kg (particularly preferred doses in this embodiment include, but are not limited to, for example, 0.01mg/kg, 0.02mg/kg, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.6mg/kg, 1.7mg/kg, 1.9mg/kg, 1.8mg/kg, 1.2mg/kg, 2.1mg/kg, 2.2mg/kg, 2.3mg/kg, 2.4mg/kg, 2.5mg/kg, 2.6mg/kg, 2.7mg/kg, 2.8mg/kg, 2.9mg/kg, 3.0 mg/kg). In another preferred embodiment, Neutrokine- α is administered in the range of 0.02-2mg/kg (in this embodiment, particularly preferred doses include, for example, but are not limited to, 0.02mg/kg, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.06mg/kg, 0.07mg/kg, 0.08mg/kg, 0.09mg/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.6mg/kg, 1.7mg/kg, 1.8mg/kg, 1.9mg/kg, 1.2mg/kg, 2 mg/kg).

The argument presented herein demonstrates that Neutrokine-alpha is a novel member of the TNF-ligand superfamily, which induces B cell proliferation and differentiation both in vivo and in vitro. Through its monocyte-specific gene/protein expression pattern, and its specific receptor distribution and biological activity on B lymphocytes, Neutrokine- α differs from other B cell growth and differentiation factors such as IL2(Metzger, D.W. et al, Immunol Studies 146: 499-505(1995)), IL4(Armitage, R.J. et al, Prov. biol. Ex. 292: 121-30 (1991); Yokota, T. et al, Proc. Natl. Acad. Sci. USA 83: 5894-98 (1986)); IL5(Takatsu, K., et al, Proc. Natl. Acad. Sci. USA 84: 4234-38 (1987); Bertolini, J.N.Etc., european journal of immunology 23: 398, 402(1993)), IL6 (poulart, p. et al, EMBO journal 6: 1219-24 (1987); hirano, T, nature 324: 73-76(1986)), IL7(Goodwin, R.G., et al, Proc. Natl. Acad. Sci. USA 86: 302-06 (1989); namen, a.e., et al, nature 333: 571-73(1988)), IL13(Punnonen, J. et al, allergy 49: 576-86(1994)), IL15(Armitage, r.j. et al, journal of immunology 154: 483-90(1995)), CD40L (Armitage, r.j. et al, nature 357: 80-82 (1992); van kootecn, c. and Banchereau, j.int, arch.allergy.immunol 113: 393-99(1997)) or CD27L (CD70) (Oshima, h. et al, int. immunol.10: 517-26 (1998); lens, s.m. et al, seminin.immunol.10: 517-26(1998)). These arguments suggest that Neutrokine-alpha is involved in the exchange of signals between B cells and monocytes or their differentiated progeny. Although this signaling pattern is available to all B cells, this restricted expression pattern and Ig secretion suggest that Neutrokine-alpha is activating CD5 ⁺Or "non-conventional" B cell responses. These B cells provide an important component of the innate immune system and provide protection against environmental pathogens by secreting multireactive IgM and IgA antibodies (Pennell, C.A. et al, European J Immunol 19: 1289-95 (1989); Hayakawa, K. et al, Proc. Sci. USA 81: 2494-98 (1984)). Alternatively, Neutrokine-alpha can act as a modulator of T cell independent responses in the same manner as CD40 and CD40L in T cell dependent antigen activation (Eertwegh, A.J. et al, J. Exp. 178: 1555-65 (1993); Grabstein, K.H. et al, J. Immunol 150: 3141-47 (1993)). Thus, Neutrokine-alpha, its receptor or a related antagonist is useful for the treatment of B-cell disorders associated with autoimmune, neoplasia and/or immunodeficiency syndromes.

Method of producing a composite material

Mice: BALB/cAnNCR (6-8 weeks) was purchased from Charles River laboratories and raised IN small isolation cages (Harlan Sprague Dawley, Indianapolis, IN) with recycled paper pads according to recommended standards (national research Committee, and Instructions on use of laboratory animals (1999)), and provided pelleted rodent chow (Harlan Sprague Dawley, Inc.), and bottled drinking water was placed ad libitum on the bottom. The animal protocol used in this study was referenced and approved by the HGS protocol of the animal feeding and use committee.

Isolating the cDNA of full-length Neutrokine-alpha: the human genome science, ltd, Expressed Sequence Tag (EST) database was probed using the BLAST program for sequences homologous to the receptor binding domain of the TNF family. Full-length Neutrokine-alpha clones were identified, sequenced and submitted to the gene bank (accession No. AF 132600). The Neutrokine-alpha open reading frame was PCR amplified using a 5 'primer (5'-CAGACT GGA TCC GCC ACC ATG GAT GAC TCC ACA GAA AG-3') that anneals at a predetermined start codon and a 3' primer (5'-CAG ACT GGT ACC GTCCTG CGT GCA CTA CAT GGC-3') that anneals at a predetermined downstream stop codon. The resulting amplicon was tailed with BamHI and Asp718 restriction sites and subcloned into a mammalian expression vector. Neutrokine-alpha is also expressed in p-CMV-1(Sigma Chemicals).

Purifying the recombinant human Neutrokine-alpha: the full-length cDNA encoding Neutrokine-alpha was subcloned into the baculovirus expression vector pA2 and transfected into Sf9 insect cells (Pater. V.P.et al, J. expression methods 185: 1163-72 (1997)). Recombinant Neutrokine-alpha was purified from the cell supernatant 92 hours after infection using a combination of anion exchange, size exclusion and hydrophobic interaction chromatography. The purified protein was formulated in a buffer containing 0.15M NaCl, 50mM NaOAc, pH6, sterile filtered and stored at 4 ℃ until required. Both SDS-PAGE and RP-HPLC analysis indicated that rNeutrokine-alpha was more than 95% purified. Endotoxin levels were below the detection limit in the LAL assay (associates cap Cod, Falmouth, MA). The sequence of the N-terminal of the finally purified Neutrokine-alpha protein is Ala-Val-Gln-Gly-Pro. This corresponds to the sequence of soluble Neutrokine-alpha derived from a CHO cell line stably transfected with the full-length Neutrokine-alpha gene.

Production of monoclonal antibodies: BALB/cAnNCR mice were immunized with 50 μ g of His-tagged Neutrokine- α suspended in complete Freund's adjuvant, followed by 2 challenges in incomplete Freund's adjuvant. Hybridomas and single gram antibodies were prepared as described (Gefter, M.L., et al, social. cbll Genet.3: 231-36 (1977); Akerstrom, B. et al, J. Immunol 135: 2589-92 (1985)).

Cell line: all human cell lines were purchased from ATCC (american type culture collection, manassas, VA).

FACS analysis: neutrokine-alpha expression was assessed on human cell lines, newly isolated normal peripheral blood nucleated cells, and monocytes cultured in vitro, mouse anti-human Neutrokine-alpha mAb 2E5(IgG1), and PE-conjugated F (ab') 2 goat antibody to mouse IgG (CALTAG laboratory, Burlingame, Calif.). Cells were analyzed by FACScan (Becton Dickinson immune cell Meter System, San Jose, Calif.) and dead cells were excluded by propidium iodide. Neutrokine- α binding was determined using rNeutrokine- α biotinylated with N-hydroxysuccinimide biotin reagent (Pierce, Rockford, IL), and PE conjugated streptavidin (Dako Corp, Glostrup, Denmark).

Chromosome mapping: to determine the chromosomal location of the Neutrokine-alpha gene, a set of single-chromosome somatic hybrids (Quantum Biotechnology, Canada) retaining individual chromosomes was screened by PCR with Neutrokine-alpha specific primers (5 'primer: 5'-TGG TGT CTT TCT ACC AGG TGG-3'), 3' primer: 5'-TTTCTT CTG GAC CCT GAA CGG-3'). The putative 233bp PCR product was detected only in the human chromosome 13 hybrid. A set of 83 radiation hybrids (St. Louis, Mo.) and Stanford human genome Central database were used ( http://www.shgc.stanford.edu.RH/rhserver). Neutrokine-alpha was found to be linked to the SHGC-36171 marker on chromosome 13. The human Neutrokine-alpha is determined on chromosome band 13q34 by hypermapping this map with the cytogenesis map of human chromosome 13.

B lymphocyte proliferation assay: human tonsil B cells were purified by depleting CD3 positive cells with magnetic beads (MACS). The resulting cell population was more than 95% B cells as determined by expression of CD19 and CD 20. Various dilutions of human rNeutrokine-alpha or control protein recombinant human IL2 were placed in each well of a 96-well plateTo this was added 10 suspended in culture broth⁵B cells in a total volume of 150. mu.l, in a culture medium containing 10% FBS at 5X 10^-5M of 2ME, 100U/ml penicillin, 100. mu.g/ml streptomycin, and 10^-5Pansorbin (SAC) or RPMI1640 of an anti-IgM antibody. 72 hours after the addition of the above-mentioned factors, by³Proliferation was quantified by 20-hour pulsing (1. mu. Ci/well) with H-thymidine (6.7 Ci/Mm).

Histological analysis: spleen tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, cut into 5 μm sections, mounted on slides, and stained with HE or by indirect immunohistochemistry methods with enzymatic labeling of CD45R (B220) (Hilbert, d.m., european journal of immunology 23: 2412-18 (1993)).

Table 5: cell surface expression of Neutrokine-alpha

Cell lines	Morphology of cells	Neutrokine-alpha cell surface expression
			Monocyte series U-937BL-60K-562THP-1	Lymphoma, histiocytic/macrophage leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute monocytic leukemia	++++
T cell line JurkatSUP-T13MOLT-4	Leukemia, T-lymphocytic leukemia, T-lymphoblastic leukemia	---
			B cell line Daudi Namalwal RajiRehARH-77IM9RPMI 8226	Burkitt's, lymphoblast Burkitt's, lymphocyte Burkitt's, lymphocytic leukemia, plasma cell myeloma	-------

Example 7: assays for detecting stimulation or inhibition of B cell proliferation and differentiation

The generation of a functional humoral immune response requires soluble and associative signals between the B cell line and its microenvironment. The signal may be given a positive stimulus to allow the B cell line cells to continue their programmed development, or a negative stimulus to direct the cells to inhibit their current developmental pathway. A number of stimulatory and inhibitory signals have been found to affect B cell responses, including IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13, IL14 and IL 15. Interestingly, these signals are via weak effectors of their own but can be combined with various costimulatory proteins to induce activation, proliferation, differentiation, homing, tolerance and death of B cell populations. One of the best B cell costimulatory proteins is the TNF superfamily. CD40, CD27 and CD30 in this family and their respective ligands CD154, CD70 and CD153 have been found to modulate various immune responses. Assays that detect and/or observe the proliferation and differentiation of these B cell populations and their precursors are useful in determining the effect various proteins may have on these B cell populations during proliferation and differentiation. Listed below are two assays for detecting differentiation, proliferation or inhibition of B cell populations and their precursors.

In vitro analysis: determining the ability of the purified Neutrokine-alpha and/or Neutrokine-alpha SV protein, or truncated forms thereof, to induce activation, proliferation, differentiation or inhibition and/or death of B cell populations and precursors thereof. Neutrokine-alpha and/or NeutThe effect of rokine- α SV protein on purified human tonsil B cells, quantitatively determined in a dose range of 0.1-10000ng/ml, was determined in standard B lymphocyte co-stimulation, wherein purified tonsil B cells were cultured in the presence of formalin-fixed staphylococcus aureus Cowan I (SAC), or fixed anti-human IgM antibody as an initiator. Secondary signals such as IL-2 and IL-15 in conjunction with SAC and IgM cross-linking stimulate B cell proliferation as measured by incorporation of tritiated thymidine. The new synergists can be easily identified using this assay. This assay involves the isolation of human tonsil B cells by magnetic bead (MACS) depletion of CD3 positive cells. The obtained cell population was confirmed to be B cells in more than 95% by expression of CD45R (B220). Various dilutions of each sample were placed in individual wells of a 96-well plate, to which 10 suspended in culture broth was added⁵B cells in a total volume of 150. mu.l, in a culture medium containing 10% FBS at 5X 10^-5M of 2ME, 100U/ml penicillin, 100. mu.g/ml streptomycin, and 10 ^-5RPMI 1640 from Pansorbin (SAC). 72 hours after the addition of the above-mentioned factors, by³Proliferation was quantified by 20-hour pulsing (1. mu. Ci/well) with H-thymidine (6.7 Ci/Mm). The positive and negative control groups were IL2 and medium, respectively.

Agonists (including Neutrokine-alpha and/or Neutrokine-alpha SV polypeptide fragments) demonstrate increased B cell proliferation when compared to that observed when the same number of B cells are contacted with the same concentration of an initiating agent. Antagonists according to the invention exhibit reduced B cell proliferation when compared to a control group containing the same number of B cells, the same concentration of initiator, and the same concentration of soluble form of Neutrokine-alpha (e.g.amino acids 71-285, 81-285, 112-285 or 134-285 of the Neutrokine-alpha polypeptide of SEQ ID NO: 2) with enhanced B cell proliferation activity, without antagonist.

In vivo analysis: BALB/c mice were injected twice daily (i.p.) with buffer, or 2mg/kg of Neutrokine-alpha and/or Neutrokine-alpha SV protein, or truncated forms thereof. Mice received this treatment for 4 consecutive days, then sacrificed, and various tissues and sera were collected for analysis. The results of identifying the effect of Neutrokine-alpha and/or Neutrokine-alpha SV protein on splenocytes, such as the spread of periarterial lymphatic sheaths, and/or a significant increase in nucleated cell structure in the erythroid region, in comparison to normal HE tissue sections and spleen tissue treated with Neutrokine-alpha and/or Neutrokine-alpha SV protein, may indicate activation of differentiation and proliferation of B cell populations. Immunohistochemical studies using B cell markers, anti-CD 45R (B220), were used to determine if any physiological changes in spleen cells, such as changes in spleen tissue architecture, were due to increased B cell expression within loosely defined B cell bands of infiltrating established T cell zones.

Flow cytometric analysis of spleen tissue from mice treated with Neutrokine-alpha and/or Neutrokine-alpha SV protein was used to indicate whether Neutrokine-alpha and/or Neutrokine-alpha SV specifically increased the proportion of ThB +, CD45R (B220) empty B cells compared to control mice.

In addition, a putative result of increased expression of mature B cells in vivo is a relative increase in serum Ig titers. Thus, serum IgM and IgA levels were compared between buffer and mice treated with Neutrokine-alpha and/or Neutrokine-alpha SV proteins.

Example 8: neutrokine-alpha and its agonist in treating graft versus host disease related lymphoatrophy and hypoplasia in mice

Analysis of treatment, prevention and/or diagnosis of Graft Versus Host Disease (GVHD) -associated lymphoid atrophy and hypoplasia in mice using Neutrokine-alpha was performed by transplantation into a (BALB/C X57C 57BL/6) F1(CBF1) mouse model using the C57BL/6 parent. This parental entry into the F1 mouse model is well characterized and is a reproducible animal model of GVHD in bone marrow transplant patients, well known to those skilled in the art (see Gleichemann et al, modern immunology 5: 324, 1984). Soluble Neutrokine-alpha is expected to induce B-lymphocyte proliferation and differentiation and correct lymphoid atrophy and hypoplasia observed in animal models of GVHD (Piguet et al, J.Exp.Med.166: 1280 (1987); Hattori et al, blood 90: 542 (1997)).

GVHD begins by mixing approximately 1-5X 10⁸C57BL/6 splenocytes were injected intravenously (BALB/C X57C 57BL/6) into F1 mice (all purchased from Jackson Lab, Bar Harbor, Maine). After parental cell injection, 6-8 mice were initially injected intraperitoneally, intramuscularly or intradermally daily with 0.1-5.0mg/kg Neutrokine-alpha or control buffer when lymphoid atrophy and dysplasia was mild (about 5 days), moderate (about 12 days) or severe (about 20 days). The effects of Neutrokine-alpha on lymphoatrophy and hypoplasia of the spleen were analyzed by FACS and histopathology at various time points (3-4) between 10-30 days. Briefly, splenocytes were prepared from normal CBF1, GVHD, or Neutrokine-alpha treated mice, anti-H-2 Kb antibody conjugated with fluorescein phycoerythrin, anti-H-2 Kd antibody conjugated with biotin, and FITC-conjugated anti-CD 4 antibody, anti-CD 8 antibody, or anti-B220 antibody, followed by staining with CyChrome conjugated avidin. All these antibodies were purchased from PharMingen (San Diego, CA). Cells were then analyzed on a FACScan (Becton Dickinson, San Jose, Calif.). Recipient and donor lymphocytes were identified as H-2Kb + Kd + and H-2Kb + Kd-cells, respectively. The numbers of recipient or donor CD4+ T, CD8+ T and B220+ B cells were calculated from the total number of recovered splenocytes and the percentage of each subpopulation was determined by trichrome analysis. Histological evaluation of the relative extent of tissue damage in other GVHD related organs (liver, skin and small intestine) was performed after sacrifice of the animals.

Finally, animals treated with Neutrokine-alpha and buffer were evaluated clinically every other day to determine cachexia, body weight and lethality.

Agonists and antagonists of Neutrokine-alpha may also be tested in this mouse model of acute GVHD.

Example 9: isolation of antibody fragments against Neutrokine-alpha polypeptide from scFvs library

Naturally occurring V genes isolated from human PBLs are constructed as large libraries of antibody fragments that contain reactivity against Neutrokine-alpha and/or Neutrokine-alpha SV, with or without contact with donors (see U.S. patent 5885793, incorporated herein by reference in its entirety).

Rescue of libraries

The scFvs library was constructed from RNA from human PBLs as described in WO 92/01047 (incorporated herein by reference in its entirety). Approximately 10 for rescue of phage display antibody fragments⁹Coli carrying phagemids were inoculated with 50ml of 2 XTY (2 XTY-AMP-GLU) containing 1% glucose and 100. mu.g/ml ampicillin and grown with shaking to an O.D. of 0.8. 5ml of this culture was used to inoculate 50ml of 2 × TY-AMP-GLU, 2 × 10 was added⁸Delta gene 3 helper gene for TU (M13 delta gene III, see WO 92/01047) and cultures were incubated at 37 ℃ for 45 minutes without shaking and then at 37 ℃ for 45 minutes with shaking. This culture was centrifuged at 4000r.p.m. for 10 min and the pellet was resuspended in 2L of 2 XTY containing 100. mu.g/ml ampicillin and 50. mu.g/ml kanamycin and grown overnight. Phage were prepared as described in WO 92/01047.

The M13 δ gene III was prepared as follows: m13 delta gene III helper phage does not encode gene III protein, however phage (particle) display antibody fragments have a strong ability to bind antigen. Infected M13 delta gene III particles were generated by growing helper phage in cells carrying pUC19 derivatives that provide wild type gene III proteins during phage morphogenesis. The culture was incubated at 37 ℃ for 1 hour without shaking, and then at 37 ℃ for 1 hour with shaking. The cells were inverted (IEC-Centra 84000 revs/min, 10 min), resuspended in 300ml of 2 XTY broth (2 XTY-AMP-KAN) containing 100. mu.g/ml ampicillin and 25. mu.g/ml kanamycin, and grown overnight at 37 ℃ with shaking. Phage particles were purified and concentrated from the culture medium by two PEG precipitations (Sambrook et al, 1990), resuspended in 2ml PBS and filtered through a 0.45 μm filter (MinisartNML; Sartorius) to a final concentration of about 10¹³One transduction unit/ml (ampicillin resistant clone).

Panning libraries

An immune test tube (Nunc) was coated with 4ml of 100. mu.g/ml or 10. mu.g/ml of the polypeptide of the invention in PBS overnight. The tubes were blocked with 2% Marvel-PBS for 2 hours at 37 ℃ and then with PBS was washed 3 times. Add about 10 to the tube¹³TU phage and incubated at room temperature for 30 minutes with tumbling, then allowed to stand for 1.5 hours. The tubes were washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. The phage was eluted by adding 1ml of 100mM triethylamine, vortexed up and down for 15 minutes, immediately before being neutralized with 0.5ml of 1.0M Tris-HCl, pH 7.4. The eluted phage was then used to infect 10ml of mid-log E.coli TG1 by incubating the phage with the bacteria for 30 minutes at 37 ℃. Coli were plated on TYE plates containing 1% glucose and 100. mu.g/ml ampicillin. The resulting bacterial library was then panned with the delta gene 3 helper phage described above to prepare phage for subsequent selection. This procedure was repeated 4 times, increasing the affinity purification of 20 washes with PBS, 0.1% Tween-20 to 20 washes with PBS.

Qualitative conjugates

The eluted phages from the third and fourth selection were used to infect E.coli HB2151 and to generate soluble scFv from single colonies (Marks et al, 1991). ELISAs were performed using 10pg/ml microtiter plates coated with the polypeptide of the invention in 50mM bicarbonate, pH 9.6. Positive clones in ELISA were further characterized by PCR fingerprinting (see WO 92/01047) and then sequenced.

Example 10: neutralizing the interaction between Neutrokine-alpha/Neutrokine-alpha receptors with anti-Neutrokine-alpha monoclonal antibodies

Monoclonal antibodies against Neutrokine-alpha protein were produced according to the following method. Briefly, mice were injected subcutaneously with 50 μ g of His-tagged Neutrokine- α produced by the method of example 2 in 100 μ l PBS emulsified in 100 μ l complete Freunds adjuvant. 25 μ g of Neutrokine- α in incomplete Freunds' adjuvant was injected subcutaneously once two weeks apart, three times in total. The animals were rested for one month and finally 25 μ g of Neutrokine- α in PBS was pushed intraperitoneally. Four days later, the animals were sacrificed and splenocytes were taken for fusion.

The "fusion" procedure was performed by fusing cells taken from the spleen with 2X 10E7 P3X63Ag8.653 plasmacytoma cells using PEG1500(Boehringer Mannheim) according to the manufacturer's instructions (see Gefter, M.L. et al, matic Cell Genet 3: 231-36 (1977); Boehringer Mannheim PEG1500(Cat. No.783641, product description).

After fusion, cells were resuspended in 400ml HAT medium supplemented with 20% FBS and 4% hybridoma supplement (Boehringer Mannheim) and distributed in 96-well plates at a density of 200 μ Ι/well. After 7 days of fusion, 100. mu.l of the medium was aspirated and replaced with 100. mu.l of fresh medium. After 14 days of fusion, hybridomas were screened for antibody production.

Hybridoma supernatants were screened by ELISA for binding to Neutrokine-alpha protein immobilized on plates. Plates were coated with Neutrokine-alpha by incubating Neutrokine-alpha overnight at a concentration of 2. mu.g/ml in PBS at 100. mu.l/well. Hybridoma supernatants were diluted 1: 10 with PBS, placed in each well of a Neutrokine-alpha coated plate, and incubated overnight at 4 ℃. The next day, the plates were washed 3 times with PBS containing 0.1% Tween-20 and developed with an anti-mouse IgG ABC system (Vector Laboratories). 2M H was added at 25 ml/well₂SO₄The color reaction was terminated. Plates were read at 450 nm.

Ig isotypes of hybridoma supernatants were detected with Isotrichps. Cloning was performed by limiting dilution on HT medium. Approximately 3 × 10E6 cells in 0.9ml HBSS were injected into pristane primed mice. After 7-9 days, ascites were collected with a 19g syringe. All antibodies were purified by protein G affinity chromatography using the Acta FPLC system (Pharmacia).

All three mice exhibited strong immune responses after the initial and two consecutive subcutaneous injections; serum titration was determined to be 10E-7 by ELISA on Neutrokine-alpha coated plates.

In one experiment, more than 1000 primary hybridomas were generated using splenocytes from positive mice. Out of these 917 were screened for production of anti-Neutrokine-alpha antibody. Screening was performed with 1: 1 diluted supernatants to detect all positive clones. Of the 917 hybridomas screened, 76 were found to be positive, 17 of which were producers of IgG. After affinity testing and cloning, 9 of these were selected for further expansion and purification.

All purified monoclonal antibodies were able to bind to different forms of Neutrokine-alpha (including His-tagged and baculovirus system-produced proteins (see example 2)) in Western blot analysis and ELISA. 6 of these 9 clones were also able to bind Neutrokine-alpha on the THP-1 cell surface. However, none of the antibodies tested captured Neutrokine-alpha from solution.

A high affinity anti-Neutrokine-alpha monoclonal antibody is generated that recognizes Neutrokine-alpha expressed on the cell surface, but cannot recognize Neutrokine-alpha in solution, which can be used for neutralization studies in vivo, and monocyte and B cell assays in vitro. These antibodies are also used for sensitive detection of Neutrokine-alpha in Western blot analysis.

In one experiment, more than 1000 primary hybridomas were generated using splenocytes from positive mice. 729 of these were screened for production of anti-Neutrokine-alpha antibody. The supernatants were screened at 1: 10 dilution under stringent conditions to pick the most suitable clones of higher affinity. Of the 729 hybridomas screened, 23 were positive, including 16 IgM and 7 IgG producers (4 of the latter providing a strong IgM background). In this assay, isotype profiles of IgG hybridomas are biased towards the IgG2 subclass. The 7 IgG hybridomas produced 3 antibodies of the IgG2a subclass, 2 antibodies of the IgG2b subclass, and the remaining 2 were IgG1 producers.

Supernatants from all positive hybridomas produced in the second assay were tested for their ability to inhibit Neutrokine-alpha mediated B cell proliferation. In the first screening assay, two hybridomas producing IgG neutralizing antibodies (these are antibodies 16C9 and 12C5) were detected. In another experiment, IgG neutralizing activity was determined for hybridomas (i.e., 16C9 and 12C5), and strongly neutralized supernatants were not identified for the other two hybridomas 15C10 and 4a 6.

These 3 clones were then expanded in vivo (one clone, 15C10, was also expanded in a hollow fiber system) to provide affinity chromatography purification of the antibody. All three clones were able to bind Neutrokine-alpha on the THP-1 cell surface and also to bind (i.e., capture) Neutrokine-alpha from solution.

In particular, an assay was performed using the anti-Neutrokine-alpha monoclonal antibody described in the second assay above to determine whether the antibody neutralizes the binding of Neutrokine-alpha/Neutrokine-alpha receptors. Briefly, Neutrokine-alpha protein was biotinylated with EZ-linked TNHS-biotin reagent (Pierce, Rockford, IL). Biotinylated Neutrokine-alpha is then used to identify cell surface proteins that bind Neutrokine-alpha. Preliminary experiments indicate that Neutrokine-alpha binds to receptors on B lymphocytes.

The anti-Neutrokine-alpha antibody produced in the second assay described above neutralizes the binding of Neutrokine-alpha to the Neutrokine-alpha receptor. In a specific embodiment, anti-Neutrokine-alpha antibody 15C10 neutralizes the binding of Neutrokine-alpha to the Neutrokine-alpha receptor.

Thus, the anti-Neutrokine-alpha antibody (antibody 15C10 in particular) generated in the second assay described above recognizes and binds membrane-bound and soluble Neutrokine-alpha protein and neutralizes binding of Neutrokine-alpha to the Neutrokine-alpha receptor in vitro.

It should be clear that the invention can be practiced otherwise than as specifically described and illustrated. Various modifications may be made to the present invention in light of the above teachings and within the purview of the appended claims.

All publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited are hereby incorporated by reference in their entirety.

In addition, the sequence listing filed herein, as well as the application 09/005874 filed on 12.1.1998, US 60/036100 filed on 14.1.1997, and the sequence listing filed on 25.10.1996 in PCT/US96/17957, are all incorporated by reference in their entirety.

Sequence listing

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a, t, g, or c

<400>6

Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp

1 5 10 15

Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys

20 25 30

Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro

35 40 45

Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro

50 55 60

Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly

65 70 75 80

Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly

85 90 95

Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala

100 105 110

Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu

115 120 125

Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg

130 135 140

Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu

145 150 155 160

Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr

165 170 175

Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr

180 185 190

Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Ash Leu Pro Leu Ser

195 200 205

His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met

210 215 220

Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala

225 230 235 240

Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His

245 250 255

Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser

260 265 270

Gln Thr Phe Phe Gly Leu Tyr Lys Leu

275 280

<210>7

<211>337

<212>DNA

<213>Homo sapiens

<220>

<223> description of combined DNA/RNA molecules: n is equal to

a, t, g, or c

<220>

<221> misc _ feature

<222>(3)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(58)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(67)..(71)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(212)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(255)

<223> n equal to a, t, g or c

<220>

<221> misc _ feature

<222>(297)

<223> n equal to a, t, g or c

<220>

<221> misc _ feature

<222>(300)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(320)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(335)

<223> n is equal to a, t, g, or c

<400>7

ggntaactct cctgaggggt gagccaagcc ctgccatgta gtgcacgcag gacatcanca 60

aacacannnn ncaggaaata atccattccc tgtggtcact tattctaaag gccccaacct 120

tcaaagttca agtagtgata tggatgactc cacagaaagg gagcagtcac gccttacttc 180

ttgccttaag aaaagagaag aaatgaaact gnaaggagtg tgtttccatc ctcccacgga 240

aggaaagccc ctctntccga tcctccaaag acggaaagct gctggctgca accttgntgn 300

tggcattgtg ttcttgctgn ctcaaggtgg tgttntt 337

<210>8

<211>509

<212>DNA

<213>Homo sapiens

<220>

<223> description of combined DNA/RNA molecules: n is equal to

a, t, g, or c

<220>

<221> misc _ feature

<222>(10)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(13)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(209)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(315)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(322)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(325)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(334)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(343)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(347)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(351)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(356)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(409)..(410)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(416)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(422)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(424)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(426)..(427)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(429)

<222> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(431)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(433)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(438)..(439)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(443)..(444)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(446)..(447)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(449)..(450)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(452)..(453)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(458)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(461)..(462)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(466)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(469)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(471)..(472)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(474)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(478)..(481)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(496)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(498)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(504)

<223> n is equal to a, t, g, or c

<400>8

aattcggcan agnaaactgg ttactttttt atatatggtc aggttttata tactgataag 60

acctacgcca tgggacatct agttcagagg aagaaggtcc atgtctttgg ggatgaattg 120

agtctggtga ctttgtttcg atgtattcaa aatatgcctg aaacactacc caataattcc 180

tgctattcag ctggcattgc aaaactggna ggaaggagat gaactccaac ttgcaatacc 240

aggggaaaat gcacaattat cactgggatg gagatgttca cattttttgg gtgccattga 300

aactgctgtg acctncttac ancangtgct gttngctatt ttncctncct nttctntggt 360

aacctcttag gaaggaagga ttcttaactg ggaaataacc caaaaaaann ttaaangggt 420

angngnnana ngnggggnng ttnncnngnn gnnttttngg nntatnttnt nntngggnnn 480

ngtaaaaatg gggccnangg gggnttttt 509

<210>9

<211>497

<212>DNA

<213>Homo sapiens

<220>

<221> misc _ feature

<222>(168)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(213)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(288)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(325)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(346)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(406)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(415)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(419)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(437)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(442)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(467)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(473)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(476)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(481)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(483)..(484)

<223> n is equal to a, t, g, or c

<220>

<221> misc _ feature

<222>(494)

<223> n is equal to a, t, g, or c

<400>9

aattcggcac gagcaaggcc ggcctggagg aagctccagc tgtcaccgcg ggactgaaaa 60

tctttgaacc accagctcca ggagaaggca actccagtca gaacagcaga aataagcgtg 120

ccgttcaggg tccagaagaa acagtcactc aagactgctt gcaactgntt gcagacagtg 180

aaacaccaac tatacaaaaa ggctcccttc tgntgccaca tttgggccaa ggaatggaga 240

gatttcttcg tctggaaaca ttttgccaaa ctcttcagat actctttnct ctctgggaat 300

caaaggaaaa tctctactta gattnacaca tttgttccca tgggtntctt aagttttaaa 360

aggggagtgc ccttaggagg aaaaggggat aaatattggc caaggnactg gttantttnt 420

aaatatggtc aggtttntat anctggtagg cctcgccatg ggcattnatt canggngagg 480

ncnntctttt gggntga 497

<210>10

<211>27

<212>DNA

<213>Homo sapiens

<220>

<223> description of combined DNA/RNA molecules: x ═ x

Deoxyinosine

<400>10

gtgggatcca gcctccgggc agagctg 27

<210>11

<211>33

<212>DNA

<213>Homo sapiens

<400>11

gtgaagcttt tattacagca gtttcaatgc acc 33

<210>12

<211>26

<212>DNA

<213>Homo sapiens

<400>12

gtgtcatgag cctccgggca gagctg 26

<210>13

<211>33

<212>DNA

<213>Homo sapiens

<400>13

gtgaagcttt tattacagca gtttcaatgc acc 33

<210>14

<211>28

<212>DNA

<213>Homo sapiens

<400>14

gtgggatccc cgggcagagc tgcagggc 28

<210>15

<211>33

<212>DNA

<213>Homo sapiens

<400>15

gtgggatcct tattacagca gtttcaatgc acc 33

<210>16

<211>129

<212>DNA

<213>Homo sapiens

<400>16

gcgggatccg ccaccatgaa ctccttctcc acaagcgcct tcggtccagt tgccttctcc 60

ctggggctgc tcctggtgtt gcctgctgcc ttccctgccc cagttgtgag acaaggggac 120

ctggccagc 129

<210>17

<211>30

<212>DNA

<213>Homo sapiens

<400>17

gtgggatcct tacagcagtt tcaatgcacc 30

<210>18

<211>903

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(798)

<400>18

atg gat gac tcc aca gaa agg gag cag tca cgc ctt act tct tgc ctt 48

Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu

1 5 10 15

aag aaa aga gaa gaa atg aaa ctg aag gag tgt gtt tcc atc ctc cca 96

Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro

20 25 30

cgg aag gaa agc ccc tct gtc cga tcc tcc aaa gac gga aag ctg ctg 144

Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu

35 40 45

gct gca acc ttg ctg ctg gca ctg ctg tct tgc tgc ctc acg gtg gtg 192

Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Lcu Thr Val Val

50 55 60

tct ttc tac cag gtg gcc gcc ctg caa ggg gac ctg gcc agc ctc cgg 240

Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg

65 70 75 80

gca gag ctg cag ggc cac cac gcg gag aag ctg cca gca gga gca gga 288

Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

85 90 95

gcc ccc aag gcc ggc ctg gag gaa gct cca gct gtc acc gcg gga ctg 336

Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

100 105 110

aaa atc ttt gaa cca cca gct cca gga gaa ggc aac tcc agt cag aac 384

Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

115 120 125

agc aga aat aag cgt gcc gtt cag ggt cca gaa gaa aca gga tct tac 432

Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Gly Ser Tyr

130 135 140

aca ttt gtt cca tgg ctt ctc agc ttt aaa agg gga agt gcc cta gaa 480

Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu

145 150 155 160

gaa aaa gag aat aaa ata ttg gtc aaa gaa act ggt tac ttt ttt ata 528

Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile

165 170 175

tat ggt cag gtt tta tat act gat aag acc tac gcc atg gga cat cta 576

Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu

180 185 190

att cag agg aag aag gtc cat gtc ttt ggg gat gaa ttg agt ctg gtg 624

Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val

195 200 205

act ttg ttt cga tgt att caa aat atg cct gaa aca cta ccc aat aat 672

Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn

210 215 220

tcc tgc tat tca gct ggc att gca aaa ctg gaa gaa gga gat gaa ctc 720

Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu

225 230 235 240

caa ctt gca ata cca aga gaa aat gca caa ata tca ctg gat gga gat 768

Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp

245 250 255

gtc aca ttt ttt ggt gca ttg aaa ctg ctg tgacctactt acaccatgtc 818

Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

260 265

tgtagctatt ttcctccctt tctctgtacc tctaagaaga aagaatctaa ctgaaaatac 878

caaaaaaaaa aaaaaaaaaa aaaaa 903

<210>19

<211>266

<212>PRT

<213>Homo sapiens

<400>19

Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu

1 5 10 15

Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro

20 25 30

Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu

35 40 45

Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val

50 55 60

Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg

65 70 75 80

Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

85 90 95

Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

100 105 110

Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

115 120 125

Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Gly Ser Tyr

130 135 140

Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu

145 150 155 160

Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile

165 170 175

Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu

180 185 190

Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val

195 200 205

Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn

210 215 220

Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu

225 230 235 240

Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp

245 250 255

Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

260 265

<210>20

<211>136

<212>PRT

<213>Homo sapiens

<400>20

His Ser Val Leu His Leu Val Pro Ile Asn Ala Thr Ser Lys Asp Asp

1 5 10 15

Ser Asp Val Thr Glu Val Met Trp Gln Pro Ala Leu Arg Arg Gly Arg

20 25 30

Gly Leu Gln Ala Gln Gly Tyr Gly Val Arg Ile Gln Asp Ala Gly Val

35 40 45

Tyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp Val Thr Phe Thr Met

50 55 60

Gly Gln Val Val Ser Arg Glu Gly Gln Gly Arg Gln Glu Thr Leu Phe

65 70 75 80

Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg Ala Tyr Asn Ser

85 90 95

Cys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp Ile Leu Ser

100 105 110

Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn Leu Ser Pro His Gly

115 120 125

Thr Phe Leu Gly Phe Val Lys Leu

130 135

<210>21

<211>462

<212>DNA

<213>Homo sapiens

<400>21

atggctgttc agggtccgga agaaaccgtt actcaggact gccttcagct gatcgcagac 60

tctgaaactc cgaccatcca gaaaggttct tacacctttg ttccttggct gctttctttc 120

aaacgtggtt ctgccctgga agagaaagaa aacaaaatcc tggttaaaga aactggttac 180

ttctttatct acggtcaggt tctttacact gataagacct acgccatggg tcacctgatt 240

cagcgtaaga aagttcacgt tttcggtgac gagctgtctc tggttactct gtttcgctgc 300

attcagaaca tgccggaaac tcttcctaac aactcctgct actctgctgg catcgcaaaa 360

ctggaagagg gtgatgaact gcagctggca attcctcgtg aaaacgcaca aatttctctg 420

gacggtgatg taaccttctt tggtgcactg aaacttctgt aa 462

<210>22

<211>1040

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(1)..(468)

<400>22

cgc gtg gta gac ctc tca gct cct cct gca cca tgc ctg cct gga tgc 48

Arg Val Val Asp Leu Ser Ala Pro Pro Ala Pro Cys Leu Pro Gly Cys

1 5 10 15

cgc cat tct caa cat gat gat aat gga atg aac ctc aga aac aga act 96

Arg His Ser Gln His Asp Asp Asn Gly Met Asn Leu Arg Asn Arg Thr

20 25 30

tac aca ttt gtt cca tgg ctt ctc agc ttt aaa aga gga aat gcc ttg 144

Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu

35 40 45

gag gag aaa gag aac aaa ata gtg gtg agg caa aca ggc tat ttc ttc 192

Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe Phe

50 55 60

atc tac agc cag gtt cta tac acg gac ccc atc ttt gct atg ggt cat 240

Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly His

65 70 75 80

gtc atc cag agg aag aaa gta cac gtc ttt ggg gac gag ctg agc ctg 288

Val Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu

85 90 95

gtg acc ctg ttc cga tgt att cag aat atg ccc aaa aca ctg ccc aac 336

Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro Asn

100 105 110

aat tcc tgc tac tcg gct ggc atc gcg agg ctg gaa gaa gga gat gag 384

Asn Ser Cys Tyr Ser Ala Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu

115 120 125

att cag ctt gca att cct cgg gag aat gca cag att tca cgc aac gga 432

Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly

130 135 140

gac gac acc ttc ttt ggt gcc cta aaa ctg ctg taa ctcacttgct 478

Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu

145 150 155

ggagtgcgtg atccccttcc ctcgtcttct ctgtacctcc gagggagaaa cagacgactg 538

gaaaaactaa aagatgggga aagccgtcag cgaaagtttt ctcgtgaccc gttgaatctg 598

atccaaacca ggaaatataa cagacagcca caaccgaagt gtgccatgtg agttatgaga 658

aacggagccc gcgctcagaa agaccggatg aggaagaccg ttttctccag tcctttgcca 718

acacgcaccg caaccttgct ttttgccttg ggtgacacat gttcagaatg cagggagatt 778

tccttgtttt gcgatttgcc atgagaagag ggcccacaac tgcaggtcac tgaagcattc 838

acgctaagtc tcaggattta ctctcccttc tcatgctaag tacacacacg ctcttttcca 898

ggtaatacta tgggatacta tggaaaggtt gtttgttttt aaatctagaa gtcttgaact 958

ggcaatagac aaaaatcctt ataaattcaa gtgtaaaata aacttaatta aaaaggttta

1018

agtgtgaaaa aaaaaaaaaa aa

1040

<210>23

<211>155

<212>PRT

<213>Homo sapiens

<400>23

Arg Val Val Asp Leu Ser Ala Pro Pro Ala Pro Cys Leu Pro Gly Cys

1 5 10 15

Arg His Ser Gln His Asp Asp Asn Gly Met Asn Leu Arg Asn Arg Thr

20 25 30

Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu

35 40 45

Glu Glu Lys Glu Asn Lys Ile Val Val Arg Gln Thr Gly Tyr Phe Phe

50 55 60

Ile Tyr Ser Gln Val Leu Tyr Thr Asp Pro Ile Phe Ala Met Gly His

65 70 75 80

Val Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu

85 90 95

Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Lys Thr Leu Pro Asn

100 105 110

Asn Ser Cys Tyr Ser Ala Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu

115 120 125

Ile Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly

130 135 140

Asp Asp Thr Phe Phe Gly Ala Leu Lys Leu Leu

145 150 155

<210>24

<211>26

<212>DNA

<213>Homo sapiens

<400>24

ccaccagctc caggagaagg caactc 26

<210>25

<211>19

<212>DNA

<213>Homo sapiens

<400>25

accgcgggac tgaaaatct 19

<210>26

<211>23

<212>DNA

<213>Homo sapiens

<400>26

cacgcttatt tctgctgttc tga 23

<210>27

<211>657

<212>DNA

<213>Homo sapiens

<400>27

taccaggtgg cggccgtgca aggggacctg gccagcctcc gggcagagct gcagggccac 60

cacgcggaga agctgccagc aagagcaaga gcccccaagg ccggtctggg ggaagctcca 120

gctgtcaccg caggactgaa aatctttgaa ccaccagctc caggagaagg caactccagt 180

cagagcagca gaaataagcg tgctattcag ggtgcagaag aaacagtcat tcaagactgc 240

ttgcaactga ttgcagacag tgaaacacca actatacaaa aaggatctta cacatttgtt 300

ccatggcttc tcagctttaa aaggggaagt gccctagaag aaaaagagaa taaaatattg 360

gtcaaagaaa ctggttactt ttttatatat ggtcaggttt tatacactga taagacctat 420

gccatgggac atctaattca gaggaaaaaa gtccatgtct ttggggatga attgagtctg 480

gtgactttgt ttcgatgtat tcaaaatatg cctgaaacac tacccaataa ttcctgctat 540

tcagctggca ttgcaaaact ggaagaagga gatgaacttc aacttgcaat accacgagaa 600

aatgcacaaa tatcactgga tggagatgtc acattttttg gtgccctcaa actgctg 657

<210>28

<211>219

<212>PRT

<213>Homo sapiens

<400>28

Tyr Gln Val Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg Ala Glu

1 5 10 15

Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Arg Ala Arg Ala Pro

20 25 30

Lys Ala Gly Leu Gly Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile

35 40 45

Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Ser Ser Arg

50 55 60

Asn Lys Arg Ala Ile Gln Gly Ala Glu Glu Thr Val Ile Gln Asp Cys

65 70 75 80

Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser

85 90 95

Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu

100 105 110

Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe

115 120 125

Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His

130 135 140

Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu

145 150 155 160

Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn

165 170 175

Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu

180 185 190

Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly

195 200 205

Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

210 215

<210>29

<211>657

<212>DNA

<213>Homo sapiens

<400>29

taccaggtgg cggccgtgca aggggacctg gccagcctcc gggcagagct gcagagccac 60

cacgcggaga agctgccagc aagagcaaga gcccccaagg ccggtctggg ggaagctcca 120

gctgtcaccg cgggactgaa aatctttgaa ccaccagctc caggagaagg caactccagt 180

cagagcagca gaaataagcg tgctattcag ggtgcagaag aaacagtcat tcaagactgc 240

ttgcaactga ttgcagacag tgaaacacca actatacaaa aaggatctta cacatttgtt 300

ccatggcttc tcagctttaa aaggggaagt gccctagaag aaaaagagaa taaaatattg 360

gtcaaagaaa ctggttactt ttttatatat ggtcaggttt tatacactga taagacctat 420

gccatgggac atctaattca gaggaaaaaa gtccatgtct ttggggatga attgagtctg 480

gtgactttgt ttcgatgtat tcaaaatatg cctgaaacac tacccaataa ttcctgctat 540

tcagctggca ttgcaaaact ggaagaaggg gatgaacttc aacttgcaat accacgagaa 600

aatgcacaaa tatcactgga tggagatgtc acattttttg gtgccctcaa actgctg 657

<210>30

<211>219

<212>PRT

<213>Homo sapiens

<400>30

Tyr Gln Val Ala Ala Val Gln Gly Asp Leu Ala Ser Leu Arg Ala Glu

1 5 10 15

Leu Gln Ser His His Ala Glu Lys Leu Pro Ala Arg Ala Arg Ala Pro

20 25 30

Lys Ala Gly Leu Gly Glu Ala Pro Ala Val Thr Ala Gly Leu Lys Ile

35 40 45

Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Ser Ser Arg

50 55 60

Asn Lys Arg Ala Ile Gln Gly Ala Glu Glu Thr Val Ile Gln Asp Cys

65 70 75 80

Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser

85 90 95

Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu

100 105 110

Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe

115 120 125

Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His

130 135 140

Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu

145 150 155 160

Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn

165 170 175

Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu

180 185 190

Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly

195 200 205

Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

210 215

<210>31

<211>38

<212>DNA

<213>Homo sapiens

<400>31

ggtcgccgtt tctaacgcgg ccgttcaggg tccagaag 38

<210>32

<211>49

<212>DNA

<213>Homo sapiens

<400>32

ctggttcggc ccaaggtacc aagcttgtac cttagatctt ttctagatc 49

<210>33

<211>21

<212>DNA

<213>Homo sapiens

<400>33

ctggtagttc ttcggagtgt g 21

<210>34

<211>19

<212>DNA

<213>Homo sapicns

<400>34

cgcgttagaa acggcgacc 19

<210>35

<211>22

<212>DNA

<213>Homo sapiens

<220>

<221> misc _ feature

<222>(7)

<223> n is equal to deoxyinosine

<220>

<221> misc _ feature

<222>(12)

<223> n is equal to deoxyinosine

<220>

<221> misc _ feature

<222>(16)

<223> n is equal to deoxyinosine

<400>35

taccagntgg cngccntgca ag 22

<210>36

<211>22

<212>DNA

<213>Homo sapiens

<220>

<221> misc _ feature

<222>(3)

<223> n is equal to deoxyinosine

<220>

<221> misc _ feature

<222>(14)

<223> n is equal to deoxyinosine

<220>

<221> misc _ feature

<222>(16)..(17)

<223> n is equal to deoxyinosine

<400>36

gtnacagcag tttnanngca cc 22

<210>37

<211>866

<212>DNA

<213>Mus musculus

<400>37

atggatgagt ctgcaaagac cctgccacca ccgtgcctct gtttttgctc cgagaaagga 60

gaagatatga aagtgggata tgatcccatc actccgcaga aggaggaggg tgcctggttt 120

gggatctgca gggatggaag gctgctggct gctaccctcc tgctggccct gttgtccagc 180

agtttcacag cgatgtcctt gtaccagttg gctgccttgc aagcagacct gatgaacctg 240

cgcatggagc tgcagagcta ccgaggttca gcaacaccag ccgccgcggg tgctccagag 300

ttgaccgctg gagtcaaact cctgacaccg gcagctcctc gaccccacaa ctccagccgc 360

ggccacagga acagacgcgc cttccaggga ccagaggaaa cagaacaaga tgtagacctc 420

tcagctcctc ctgcaccatg cctgcctgga tgccgccatt ctcaacatga tgataatgga 480

atgaacctca gaaacatcat tcaagactgt ctgcagctga ttgcagacag cgacacgccg 540

gccttggagg agaaagagaa caaaatagtg gtgaggcaaa caggctattt cttcatctac 600

agccaggttc tatacacgga ccccatcttt gctatgggtc atgtcatcca gaggaagaaa 660

gtacacgtct ttggggacga gctgagcctg gtgaccctgt tccgatgtat tcagaatatg 720

cccaaaacac tgcccaacaa ttcctgctac tcggctggca tcgcgaggct ggaagaagga 780

gatgagattc agcttgcaat tcctcgggag aatgcacaga tttcacgcaa cggagacgac 840

accttctttg gtgccctaaa actgct 866

<210>38

<211>177

<212>DNA

<213>Mus musculus

<400>38

mdsaktcccs kgdmkvgydt kgawgcrdgr aatassstam syaaadmnrm syrgsataaa 60

gatagvktaa rhnssrghrn rragtdvdsa acgcrhshdd ngmnrndcad sdtaknkvvr 120

tgyysvytda mghvrkkvhv gdsvtrcnmk tnnscysaga rgdarnasrn gddtgak 177

Claims (28)

1. An isolated protein consisting of a Neutrokine-alpha protein fused to a toxin protein, wherein said Neutrokine-alpha protein consists of the amino acid sequence of SEQ ID NO: 2, amino acid sequence of amino acid residue 134-285.

2. An in vitro method of killing B lymphocytes comprising contacting said B lymphocytes with a protein of claim 1.

3. Use of a protein according to claim 1 in the manufacture of a medicament for killing B lymphocytes.

4. Use of a protein of claim 1 in the manufacture of a medicament for treating a B cell malignancy or an autoimmune disease in a patient in need thereof.

5. The use of claim 4, wherein the B cell malignancy is selected from the group consisting of:

(a) non-Hodgkin's lymphoma;

(b) multiple myeloma;

(c) chronic Lymphocytic Leukemia (CLL);

(d) acute Lymphocytic Leukemia (ALL);

(e) a plasmacytoma; and

(f) waldenstrom's macroglobulinemia.

6. The use of claim 4, wherein the autoimmune disease is selected from the group consisting of:

(a) rheumatoid arthritis;

(b) systemic lupus erythematosus;

(c) multiple sclerosis;

(d) myasthenia gravis;

(e) sjogren's syndrome;

(f) type 1 diabetes mellitus;

(g) idiopathic thrombocytopenic purpura;

(h) Guillian-Barre syndrome;

(i) hashimoto's thyroiditis;

(j) graves' disease; and

(k) vasculitis.

7. Use of a protein according to claim 1 for the preparation of a medicament for the treatment, prevention or alleviation of graft or transplant rejection.

8. The protein of claim 1, wherein the toxin protein is selected from the group consisting of:

(a) a ribosome inactivating protein;

(b) (ii) a cytotoxin; and

(c) a cytotoxic prodrug.

9. An in vitro method of killing a B lymphocyte comprising contacting the B lymphocyte with a protein of claim 8.

10. Use of a protein according to claim 8 in the manufacture of a medicament for killing B lymphocytes.

11. Use of a protein according to claim 8 in the manufacture of a medicament for the treatment of a B cell malignancy or an autoimmune disease in a patient in need thereof.

12. The use of claim 11, wherein the B cell malignancy is selected from:

(a) non-Hodgkin's lymphoma;

(b) multiple myeloma;

(c) chronic Lymphocytic Leukemia (CLL);

(d) acute Lymphocytic Leukemia (ALL);

(e) a plasmacytoma; and

(f) waldenstrom's macroglobulinemia.

13. The use of claim 11, wherein the autoimmune disease is selected from the group consisting of:

(a) Rheumatoid arthritis;

(b) systemic lupus erythematosus;

(c) multiple sclerosis;

(d) myasthenia gravis;

(e) sjogren's syndrome;

(f) type 1 diabetes mellitus;

(g) idiopathic thrombocytopenic purpura;

(h) Guillian-Barre syndrome;

(i) hashimoto's thyroiditis; and

(j) graves' disease; and

(k) vasculitis.

14. Use of a protein according to claim 8 for the preparation of a medicament for the treatment, prevention or alleviation of graft or transplant rejection.

15. The protein of claim 1, wherein the toxin protein is thymidine kinase, an endonuclease, an rnase, an alpha toxin, ricin, abrin, pseudomonas exotoxin a, diphtheria toxin, saporin, solithromycin, gelonin, pokeweed antiviral protein, alpha sarcina, or cholera toxin.

16. An in vitro method of killing a B lymphocyte, comprising contacting said B lymphocyte with a protein of claim 15.

17. Use of a protein according to claim 15 in the manufacture of a medicament for killing B lymphocytes.

18. Use of a protein of claim 15 in the manufacture of a medicament for treating a B cell malignancy or an autoimmune disease in a patient in need thereof.

19. The use of claim 18, wherein the B cell malignancy is selected from:

(a) non-Hodgkin's lymphoma;

(b) multiple myeloma;

(c) chronic Lymphocytic Leukemia (CLL);

(d) acute Lymphocytic Leukemia (ALL);

(e) a plasmacytoma; and

(f) waldenstrom's macroglobulinemia.

20. The use of claim 18, wherein the autoimmune disease is selected from the group consisting of:

(a) rheumatoid arthritis;

(b) systemic lupus erythematosus;

(c) multiple sclerosis;

(d) myasthenia gravis;

(e) sjogren's syndrome;

(f) type 1 diabetes mellitus;

(g) idiopathic thrombocytopenic purpura;

(h) Guillian-Barre syndrome;

(i) hashimoto's thyroiditis;

(j) graves' disease; and

(k) vasculitis.

21. Use of a protein according to claim 15 for the preparation of a medicament for the treatment, prevention or alleviation of graft or transplant rejection.

22. An isolated protein consisting of a Neutrokine-alpha protein fused to a toxin protein, wherein said Neutrokine-alpha protein consists of the amino acid sequence of SEQ ID NO: 2, amino acid sequence of amino acid residue 134-285; and wherein the toxin protein consists of an amino acid sequence selected from the group consisting of:

(i) a polypeptide consisting of a full-length gelonin polypeptide;

(ii) A polypeptide consisting of amino acids 47-297 of a full-length gelonin polypeptide;

(iii) a polypeptide consisting of amino acids 47-297 of a full length gelonin polypeptide, wherein the aspartic acid at position 297 is changed to a cysteine.

23. An in vitro method of killing a B lymphocyte, comprising contacting said B lymphocyte with a protein of claim 22.

24. Use of a protein according to claim 22 in the manufacture of a medicament for killing B lymphocytes.

25. Use of a protein of claim 22 in the manufacture of a medicament for treating a B cell malignancy or an autoimmune disease in a patient in need thereof.

26. The use of claim 25, wherein the B cell malignancy is selected from:

(a) non-Hodgkin's lymphoma;

(b) multiple myeloma;

(c) chronic Lymphocytic Leukemia (CLL);

(d) acute Lymphocytic Leukemia (ALL);

(e) a plasmacytoma; and

(f) waldenstrom's macroglobulinemia.

27. The method of claim 25, wherein the autoimmune disease is selected from the group consisting of:

(a) rheumatoid arthritis;

(b) systemic lupus erythematosus;

(c) multiple sclerosis;

(d) myasthenia gravis;

(e) sjogren's syndrome;

(f) type 1 diabetes mellitus;

(g) idiopathic thrombocytopenic purpura;

(h) Guillian-Barre syndrome;

(i) hashimoto's thyroiditis;

(j) graves' disease; and

(k) vasculitis.

28. Use of a protein according to claim 22 in the manufacture of a medicament for the treatment, prevention or alleviation of graft or transplant rejection.