NZ507293A - U4, a member of the hematopoietin receptor superfamily - Google Patents

Abstract

An isolated polynucleotide comprises a nucleotide selected from: a) The nucleotide sequence of SEQ ID NO:4 from nucleotide 242 to nucleotide 1396 b) The nucleotide sequence of SEQ ID NO:6 from nucleotide 71 to nucleotide 1225 c) The nucleotide sequence of SEQ ID NO:9 from nucleotide 136 to nucleotide 1401. An isolated protein comprises the amino acid sequence selected from: a) The amino acid sequence of SEQ ID NO:5 b) The amino acid sequence of SEQ ID NO:7 c) The amino acid sequence of SEQ ID NO:10 Also described are vectors and host cells comprising the nucleotide and pharmaceutical compositions comprising the protein or an antibody that specifically reacts with the protein.

Description

New Zealand Paient Spedficaiion for Paient Number 507293 507293 WO 99/53066 PCT/US99/07882 114, A MEMBER OF THE HEMATOPOIETIN RECEPTOR SUPERFAMILY This application is a continuation-in-part of application Ser. No. 08/784,863, filed January 16,1997.

Field of the Invention The present invention relates to new members of the mammalian hematopoietin superfamily of proteins (including without limitation human and murine receptor proteins), fragments thereof and recombinant polynucleotides and cells useful for expressing such proteins.

Background of the Invention 10 A variety of regulatory molecules, known as hematopoietins, have been identified which are involved in the development and proliferation of the various populations of hematopoietic or blood cells. Most hematopoietins exhibits certain biological activities by interacting with a receptor on the surface of target cells. Cytokine receptors are commonly composed of one, two or three chains. Many 15 cytokine receptors and some cytokines, such as IL-12 p40, are members of the hematopoietin receptor superfamily of proteins. Identification of new members of the hematopoietin receptor superfamily can be useful in regulation of hematopoiesis, in regulation of immune responses and in identification of other members of the hematopoietin superfamily, including cytokines and receptors. 20 It would be desirable to identify and determine the DNA and protein sequence for heretofore unknown members of the hematopoietin receptor superfamily.

Summary of the Invention In accordance with the present invention, polynucleotides encoding the U4 25 hematopoietin receptor superfamily chain are disclosed, including without limitation those from the murine and human sources. In certain embodiments, the invention provides an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO:4 from nucleotide 242 to 30 nucleotide 1396; 1 Printed from Mimosa 10/03/2000 10:18:39 page -3- WO 99/53066 PCT/US99/07882 (b) the nucleotide sequence of SEQ ID NO:6 from nucleotide 71 to nucleotide 1225; (c) the nucleotide sequence of SEQ ID NO:9 from nucleotide 136 to nucleotide 1401; (d) a nucleotide sequence varying from the sequence of the nucleotide sequence 10 specified in (a), (b) or (c) as a result of degeneracy of the genetic code; and (e) a nucleotide sequence encoding a species homologue of the sequence specified in (a), (b) or (c).

The nucleotide sequence may be operably linked to an expression control sequence. In preferred embodiments, 20 the polynucleotide comprises the nucleotide sequence of SEQ ID NO:4 from nucleotide 242 to nucleotide 1396; the nucleotide sequence of SEQ ID NO:4 from nucleotide 122 to nucleotide 1396; the nucleotide sequence of SEQ ID NO:6 from nucleotide 71 to nucleotide 1225; or the nucleotide sequence of SEQ ID NO:6 from nucleotide 11 to nucleotide 1225; or the nucleotide sequence of SEQ ID NO:9 from 25 nucleotide 136 to nucleotide 1401; or the nucleotide sequence of SEQ ID NO:9 from nucleotide 247 to nucleotide 1401.

The invention also provides isolated polynucleotides comprising a nucleotide sequence encoding a peptide or protein comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:5; (b) the amino acid sequence of SEQ ID NO:5 from amino acids 41 to 425; (c) the amino acid sequence of SEQ ID NO: 7; (d) the amino acid sequence of SEQ ID NO:7 from amino acids 24 to 408; INTELLECTUAL PROPERTY office of n.z. 2 5 JUL 2003 RECEIVED (e) the amino acid sequence of SEQ ID NO: 10; and (f) the amino acid sequence of SEQ ID NO: 10 from amino acids 38 to 421.

Other preferred embodiments encode the amino acid sequence of SEQ ID NO:5; the amino acid sequence of SEQ ID NO:5 from amino acids 41 to 425; the amino acid sequence of SEQ ID NO:7; the amino acid sequence of SEQ ID NO:7 from amino acids 24 to 408; the amino acid sequence of SEQ ID NO: 10; and the amino acid sequence of SEQ ID NO: 10 from amino acids 38 to 421.

Host cells, preferably mammalian cells, transformed with the polynucleotides are also provided.

In other embodiments, the invention provides a process for producing a protein. The process comprises: (a) growing a culture of the host cell of the present invention in a suitable culture medium; and (b) purifying the protein encoded by the transformed polynucleotide. Proteins produced according to these methods are also provided.

The present invention also provides for an isolated protein comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:5; (b) the amino acid sequence of SEQ ID NO:5 from amino acids 41 to 425; (c) the amino acid sequence of SEQ ID NO:7; (d) the amino acid sequence of SEQ ID NO:7 from amino acids 24 to 408; and (e) the amino acid sequence of SEQ ID NO: 10; and (f) the amino acid sequence of SEQ ID NO: 10 from amino acids 38 to 421. Preferably the protein comprises the amino acid sequence of SEQ ID NO:5; the amino acid sequence of SEQ ID NO:5 from amino acids 41 to 425; the amino acid sequence of SEQ ID NO:7; or the amino acid sequence of SEQ ID NO:7 from 3 intellectual PROPERTY office of n.z. 2 5 JUL 2003 RECEIVED amino acids 24 to 408. In other preferred embodiments, the specified amino acid sequence is part of a fusion protein. Preferred fusion proteins comprise an antibody fragment, such as an Fc fragment.

Pharmaceutical compositions comprising a protein of the present invention 10 and a pharmaceutical^ acceptable carrier are also provided.

The present invention further provides for compositions comprising an antibody which specifically reacts with a protein of the present invention.

Detailed Description of Preferred Embodiments The inventors of the present application have for the first time identified and provided murine and human polynucleotides encoding the U4 hematopoietin receptor superfamily chain (hereinafter "U4" or "U4 protein").

A 79 amino acid region of the human IL-5 receptor 20 (LMTNAFISIIDDLSKYDVQVRAAVSSMCREAGLWSEWSQPIYVGND F.HTCPT R F.WFVTVTM ATTPFTT T TT, SEQ ID NO:l) was used to search the GenBank EST database using the TBLASTN algorithm. EST W66776 was identified with homology to this region, suggesting that this might encode a novel hematopoietin receptor. Translation of the reverse-complement of this EST using 25 the GCG map program revealed a protein sequence in the second reading frame that contained the conserved WSXWS motif found in hematopoietin receptors. However, a stop codon was also present in this reading frame at nucleotide 227, indicating that this EST either was not a novel hematopoietin receptor, or that the DNA sequence in the EST was incorrect.

To determine whether this EST sequence might be related to a hematopoietin receptor, we screened a murine embryo library with an oligonucleotide probe of the sequence CTTGGCTTGG AAGAGGAAAT CCTTGAGAGC (SEQ ID NO:2). A full-length cDNA clone U6-3(1A) was identified and complete sequence was obtained. The DNA sequence and the predicted amino acid sequence for the murine protein are reported as SEQ ID NO:4 intellectual PROPERTY office of N.Z. 2 5 JUL 2003 RECEIVED WO 99/53066 PCT/US99/07882 and SEQ ID NO:5, respectively. The murine protein encodes a novel member of the hematopoietin receptor family. It has a leader sequence, and the conserved cysteine pairs, PP, and WSXWS motifs characteristic of this family. This clone has no transmembrane or cytoplasmic domains. Alignment of this clone with the EST in GenBank revealed that the EST did have a frame shift mutation. 10 SEQ ID NO:4 provides the nucleotide sequence of a cDNA encoding the murine U4. SEQ ID NO:5 provides predicted the amino acid sequence of the receptor chain, including a putative signal sequence from amino acids 1-40. The mature murine U4 is believed to have the sequence of amino acids 41-383 of SEQ ID NO:5.

To identify additional related sequences in GenBank, the W66776 sequence was used to search GenBank using the BLASTN algorithm. A closely related EST, H14009, derived from human genomic DNA was identified. An oligonucleotide derived from this EST CTGAGCGTGC GCTGGGTGTC GCCAC (SEQ ID NO:3) was then used to isolate a cDNA clone from a human cDNA library. A 20 cDNA clone (HU4-3B) encoding a full-length mature protein homolog was completely sequenced. This clone does not have a complete signal sequence, but does encode the entire predicted full-length mature protein. The human clone is 85% homologous at the DNA level with the mouse clone. The predicted amino acid sequences have 95% identity between human and mouse. The nucleotide and 25 amino acid sequence for human U4 are reported as SEQ ID NO:6 and SEQ ID NO:7, respectively.

SEQ ID NO:6 provides the nucleotide sequence of a cDNA encoding the human U4. SEQ ID NO:7 provides predicted the amino acid sequence of the receptor chain, including a putative signal sequence from amino acids 1-23. The 30 mature human U4 is believed to have the sequence of amino acids 24-380 of SEQ ID NO:7.

The murine and human clones were deposited with the American Type Culture Collection on January 15, 1997, as accession numbers ATCC 98305 and ATCC 98306, respectively.

Printed from Mimosa 10/03/2000 10:18:39 page -7- WO 99/53066 PCMJS99/07882 Human U4 protein can be expressed by replacing the human leader sequence with the sequence of the murine leader, or by extending the human leader sequence with amino acids 1-14 of the murine sequence (MPAGRPGPVA QSAR, SEQ ID NO:8). Additionally, a longer cDNA or genomic clone encoding the actual human leader can be isolated using the sequences disclosed herein as probes. 10 The sequence of the human U4 cDNA described above was used to isolate a longer cDNA encoding human U4 protein. The nucleotide sequence of this longer clone is reported in SEQ ID NO:9. The predicted amino acid sequence encoded thereby is reported in SEQ ID NO: 10. The longer cDNA clone was deposited with the American Type Culture Collection on March 10, 1998, as 15 accession number ATCC 98688.

Any forms of U4 proteins of less than full length are encompassed within the present invention and are referred to herein collectively with full length and mature forms as "U4" or "U4 proteins." U4 proteins of less than full length may be produced by expressing a corresponding fragment of the polynucleotide 20 encoding the full-length U4 protein (SEQ ID NO:4 or SEQ ID NO:6). These corresponding polynucleotide fragments are also part of the present invention. Modified polynucleotides as described above may be made by standard molecular biology techniques, including construction of appropriate desired deletion mutants, site-directed mutagenesis methods or by the polymerase chain reaction using 25 appropriate oligonucleotide primers.

For the purposes of the present invention, a protein has "a biological activity of the U4 hematopoietin receptor superfamily chain" if it possess one or more of the biological activities of the corresponding mature U4 protein.

U4 or active fragments thereof (U4 proteins) may be fused to carrier 30 molecules such as immunoglobulins. For example, soluble forms of the U4 may be fused through "linker" sequences to the Fc portion of an immunoglobulin. Other fusions proteins, such as those with GST, Lex-A or MBP, may also be used.

The invention also encompasses allelic variants of the nucleotide sequences as set forth in SEQ ID NO:4 or SEQ ID NO:6, that is, naturally-occurring 35 alternative forms of the isolated polynucleotide of SEQ ID NO:4 or SEQ ID NO:6 6 Printed from Mimosa 10/03/2000 10:18:39 page -8- WO 99/53066 PCT/US99/07882 which also encode U4 proteins, preferably those proteins having a biological activity of U4. Also included in the invention are isolated polynucleotides which hybridize to the nucleotide sequence set forth in SEQ ID NO:4 or SEQ ID NO:6 under highly stringent conditions (for example, O.lxSSC at 65°C). Isolated polynucleotides which encode U4 proteins but which differ from the nucleotide 10 sequence set forth in SEQ ID NO:4 or SEQ ID NO:6 by virtue of the degeneracy of the genetic code are also encompassed by the present invention. Variations in the nucleotide sequence as set forth in SEQ ID NO:4 or SEQ ID NO:6 which are caused by point mutations or by induced modifications are also included in the invention.

The present invention also provides polynucleotides encoding homologues of the murine and human U4 from other animal species, particularly other mammalian species. Species homologues can be identified and isolated by making probes or primers from the murine or human sequences disclosed herein and screening a library from an appropriate species, such as for example libraries 20 constructed from PBMCs, thymus or testis of the relevant species.

The isolated polynucleotides of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19,4485-4490 (1991), in order to produce the U4 protein recombinandy. Many suitable expression control sequences are 25 known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185.537-566 (1990). As defined herein "operably linked" means enzymatically or chemically ligated to form a covalent bond between the isolated polynucleotide of the invention and the expression control sequence, in such a way that the U4 protein 30 is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expression of the U4 protein. Any cell type capable of expressing functional U4 protein may be used. Suitable mammalian host cells include, for example, monkey COS cells, 35 Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal 7 Printed from Mimosa 10/03/2000 10:18:39 page -9- A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from m vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK, Rat2, BaF3, 32D, FDCP-1, PC12, Mix or C2C12 cells.

The U4 protein may also be produced by operably linking the isolated 10 polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and 15 Smith, Texas Agricultural Experiment Station Bulletin No. 1555 n987~). incorporated herein by reference. Soluble forms of the U4 protein may also be produced in insect cells using appropriate isolated polynucleotides as described above.

Alternatively, the U4 protein may be produced in lower eukaryotes such as 20 yeast or in prokaiyotes such as bacteria. Suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. 25 Expression in bacteria may result in formation of inclusion bodies incorporating the recombinant protein. Thus, refolding of the recombinant protein may be required in order to produce active or more active material. Several methods for obtaining correctly folded heterologous proteins from bacterial inclusion bodies are known in the art. These methods generally involve 30 solubilizing the protein from the inclusion bodies, then denaturing the protein completely using a chaotropic agent. When cysteine residues are present in the primary amino acid sequence of the protein, it is often necessary to accomplish the refolding in an environment which allows correct formation of disulfide bonds (a redox system). General methods of refolding are disclosed in Kohno, Meth.

Printed from Mimosa 10/03/2000 10:18:39 page -10- WO 99/53066 PCT/US99/07882 Enzvm.. 185:187-195 (1990). EP 0433225 and copending application USSN 08/163,877 describe other appropriate methods.

The U4 protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a 10 polynucleotide sequence encoding the U4 protein.

The U4 protein of the invention may be prepared by growing a culture transformed host cells under culture conditions necessary to express the desired protein. The resulting expressed protein may then be purified from the culture medium or cell extracts. Soluble forms of the U4 protein of the invention can be 15 purified from conditioned media. Membrane-bound forms of U4 protein of the invention can be purified by preparing a total membrane fraction from the expressing cell and extracting the membranes with a non-ionic detergent such as Triton X-100.

The U4 protein can be purified using methods known to those skilled in the 20 art. For example, the U4 protein of the invention can be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or 25 substrate having pendant diethylaminoethyl (DEAE) or polyetheyleneimine (PEI) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred 30 (e.g., S-Sepharose® columns). The purification of the U4 protein from culture supernatant may also include one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; or by hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or by immunoaffinity chromatography. 35 Finally, one or more reverse-phase high performance liquid chromatography (RP- 9 Printed from Mimosa 10/03/2000 10:18:39 page -11- WO 99/53066 PCT/US99/07882 HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the U4 protein. Affinity columns including antibodies to the U4 protein can also be used in purification in accordance with known methods. Some or all of the foregoing purification steps, in various combinations or with other known methods, can also 10 be employed to provide a substantially purified isolated recombinant protein. Preferably, the isolated U4 protein is purified so that it is substantially free of other mammalian proteins.

U4 proteins of the invention may also be used to screen for agents which are capable of binding to U4. Binding assays using a desired binding protein, 15 immobilized or not, are well known in the art and may be used for this purpose using the U4 protein of the invention. Purified cell based or protein based (cell free) screening assays may be used to identify such agents. For example, U4 protein may be immobilized in purified form on a carrier and binding or potential ligands to purified U4 protein may be measured.

U4 proteins, purified from cells or recombinantly produced, may be used as a pharmaceutical composition when combined with a pharmaceutical^ acceptable carrier. Such a composition may contain, in addition to U4 or inhibitor and carrier, various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" 25 means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.

The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, 30 IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14, IL-15, G-CSF, stem cell factor, and erythropoietin. The pharmaceutical composition may also include anti-cytokine antibodies. The pharmaceutical composition may contain thrombolytic or anti-thrombotic factors such as plasminogen activator and Factor VIII. The pharmaceutical composition may further contain other anti-35 inflammatory agents. Such additional factors and/or agents may be included in the Printed from Mimosa 10/03/2000 10:18:39 page -12- WO 99/53066 PCT/US99/07882 pharmaceutical composition to produce a synergistic effect with isolated U4 protein, or to minimize side effects caused by the isolated U4 protein. Conversely, isolated U4 protein may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, 10 other hematopoietic factor, thrombolytic or anti-thrombotic factor, or antiinflammatory agent.

The pharmaceutical composition of the invention may be in the form of a liposome in which isolated U4 protein is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which 15 exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for 20 example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein by reference.

As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method 25 that is sufficient to show a meaningful patient benefit, e.g., amelioration of symptoms of, healing of, or increase in rate of healing of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether 30 administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, a therapeutically effective amount of isolated U4 protein is administered to a mammal. Isolated U4 protein may be administered in accordance with the method of the invention either alone or in combination with other therapies such as 35 treatments employing cytokines, lymphokines or other hematopoietic factors. 11 Printed from Mimosa 10/03/2000 10:18:39 page -13- WO 99/53066 PCT/US99/07882 When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, U4 protein may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or antithrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering U4 protein in 10 combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.

Administration of U4 protein used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, or cutaneous, subcutaneous, 15 or intravenous injection. Intravenous administration to the patient is preferred.

When a therapeutically effective amount of U4 protein is administered orally, U4 protein will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, 20 capsule, and powder contain from about 5 to 95% U4 protein, and preferably from about 25 to 90% U4 protein. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, 25 dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of U4 protein, and preferably from about 1 to 50% U4 protein.

When a therapeutically effective amount of U4 protein is administered by 30 intravenous, cutaneous or subcutaneous injection, U4 protein will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, 35 in addition to U4 protein an isotonic vehicle such as Sodium Chloride Injection, 12 Printed from Mimosa 10/03/2000 10:18:39 page -14- Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additive known to those of skill in the art.

The amount of U4 protein in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of U4 protein with which to treat each individual patient. Initially, the attending physician will administer low doses 15 of U4 protein and observe the patient's response. Larger doses of U4 protein may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not generally increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.1 jig to about 100 mg of U4 protein per kg body 20 weight.

The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient It is contemplated that the duration of each application of the U4 protein 25 will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.

The polynucleotide and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited 30 herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).

Cytokine and Cell Proliferation/Differentiation Activity 13 Printed from Mimosa 10/03/2000 10:18:39 page -15- A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient 10 confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.

The activity of a protein of the invention may, among other means, be measured 15 by the following methods: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kraisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 20 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992; Bowman etal., J.Immunol. 152:1756-1761,1994.

Assays for cytokine production and/or proliferation of spleen cells, lymph node 25 cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon y. Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 30 1994.

Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and 35 Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al.. Nature 336:690-692,1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of 14 Printed from Mimosa 10/03/2000 10:18:39 page -16- human Interleukin 11 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience 15 (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411,1981; Takai etal., J.Immunol. 137:3494-3500,1986; Takai et al., J. Immunol. 140:508-512, 1988.

Immune Stimulating or Suppressing Activity A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies 25 and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, 30 bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer. 35 Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the Printed from Mimosa 10/03/2000 10:18:39 page -17- present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.

Using the proteins of the invention it may also be possible to immune responses, 10 in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires 15 continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent. 20 Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue 25 transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a 30 monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an 35 immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a 16 Printed from Mimosa 10/03/2000 10:18:39 page -18- subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.

The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats 10 and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to 15 determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.

Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production 20 of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block costimulation of T cells by disrupting receptonligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may 25 be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, 30 systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Upregulation of an antigen function (preferably a B lymphocyte antigen function), 35 as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis 17 Printed from Mimosa 10/03/2000 10:18:39 page -19- might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.

Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a 10 stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells 15 into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.

In another application, up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, 20 carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or 25 in conjunction with a peptide having B7-1-like activity and/or B7-3-like activity. The transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo.

The presence of the peptide of the present invention having the activity of a B 30 lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class n molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain 35 truncated portion) of an MHC class I a chain protein and P2 microglobulin protein or an MHC class n a chain protein and an MHC class n p chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected 18 Printed from Mimosa 10/03/2000 10:18:39 page -20- tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to 10 overcome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. 15 Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492,1981; Herrmann et al., J. Immunol. 128:1968-1974,1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; 20 Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Nad. Acad. Sci. USA 78:2488-2492,1981; Herrmann et al., J. Immunol. 128:1968-1974,1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 25 153:3079-3092, 1994.

Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033,1990; and Assays for B cell function: In vitro 30 antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Thl and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, 35 D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1 -3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992. 19 Printed from Mimosa 10/03/2000 10:18:39 page -21- Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgadoret al., Journal of Experimental Medicine 182:255-260, 10 1995; Nair et al., Journal of Virology 67:4062-4069,1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640,1990.

Assays for lymphocyte survival/apoptosis (which will identify, among others, 15 proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670,1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 20 14:891-897,1993; Gorczyca et al., International Journal of Oncology 1:639-648,1992.

Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122,1994; Galy et al., Blood 85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,1991.

Hematopoiesis Regulating Activity A protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell 30 lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as 35 granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to Printed from Mimosa 10/03/2000 10:18:39 page -22- platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell 0 compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, be measured by the following methods: S Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embiyonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., 0 Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915,1993.

Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic 5 Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New Yoric, NY. 1994; Hirayamaet al., Proc. Natl. Acad. Sci. USA 89:5907-5911,1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R. A. In Culture of Hematopoietic Cells. R.L Freshney, etal. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; 0 Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay, Sutherland, 5 H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.

Research Uses and Utilities 21 Printed from Mimosa 10/03/2000 10:18:39 page -23- polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight 0 markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of 5 discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a 0 receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occur or to identify inhibitors of the binding interaction.

The proteins provided by the present invention can similarly be used in assay to 5 deteimine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue D differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or 5 small molecule inhibitors or agonists of the binding interaction.

Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular Cloning: 22 Printed from Mimosa 10/03/2000 10:18:39 page -24- A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.

Nutritional Uses Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid 15 preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.

U4 proteins of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the U4 20 protein and which may inhibit binding of ligands to the receptor. Such antibodies may be obtained using the entire U4 as an immunogen, or by using fragments of U4. Smaller fragments of the U4 may also be used to immunize animals. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin 25 (KLH). Additional peptide immunogens may be generated by replacing tyrosine residues with sulfated tyrosine residues. Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J.Amer.Chem.Soc. 85. 2149-2154 (1963); J.L. Krstenansky, et al., FEBS Lett. 2U, 10 (1987).

Neutralizing or non-neutralizing antibodies (preferably monoclonal 30 antibodies) binding to U4 protein may also be useful therapeutics for certain tumors and also in the treatment of conditions described above. These neutralizing monoclonal antibodies may be capable of blocking ligand binding to the U4 receptor chain.

Example Expression of U4 Protein 23 Printed from Mimosa 10/03/2000 10:18:39 page -25-

Claims (18)

WO 99/53066 PCT/US99/07882 5 DNA encoding the full-length murine U4 protein was fused to a spacer sequence encoding Gly-Ser-Gly by PCR and ligated in frame with sequences encoding the hinge CH2 CH3 regions of human IgGl in the COS-1 expression vector pED.Fc . The DNA was transfected into Cos cells and expression of the fusion protein was detected by ELISA using antibodies that detected the IgGl 10 portion of the protein. This demonstrated that the protein could be expressed and secreted in Cos cells. All patent and literature references cited herein are incorporated by reference as if fully set forth. 24 Printed from Mimosa 10/03/2000 10:18:39 page -26- What is claimed is:

1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO:4 from nucleotide 242 to nucleotide 1396; (b) the nucleotide sequence of SEQ ID NO:6 from nucleotide 71 to nucleotide 1225; (c) the nucleotide sequence of SEQ ID NO:9 from nucleotide 136 to nucleotide 1401; and (d) a nucleotide sequence varying from the sequence of the nucleotide sequence specified in (a), (b) or (c) as a result of degeneracy of the genetic code; and (e) a nucleotide sequence encoding a species homologue of the sequence specified in (a), (b) or (c)

2. The polynucleotide of claim 1 wherein said nucleotide sequence is operably linked to an expression control sequence.

3. The polynucleotide of claim 1 comprising the nucleotide sequence of SEQ ID NO:4 from nucleotide 122 to nucleotide 1396.

4. The polynucleotide of claim 1 comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide 11 to nucleotide 1225.

5. A host cell transformed with the polynucleotide of claim 2, with the proviso that if the cell is a human cell, it is transformed ex vivo.

6. The host cell of claim 5, wherein said cell is a mammalian cell.

7. A process for producing a protein, said process comprising: (a) growing a culture of the host cell of claim 6 in a suitable culture medium; and (b) purifying the protein encoded by the transformed polynucleotide from the culture. 25 intellectual PROPERTY OFFICE OF N.Z. 2 0 OCT 28 RECEIVE

8. An isolated protein comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:5; (b) the amino acid sequence of SEQ ID NO:5 from amino acids 41 to 425; (c) the amino acid sequence of SEQ ID NO:7; (d) the amino acid sequence of SEQ ID NO:7 from amino acids 24 to 408; (e) the amino acid sequence of SEQ ID NO: 10; and (f) the amino acid sequence of SEQ ID NO: 10 from amino acids 38 to 421;

9. The protein of claim 8 comprising the amino acid sequence of SEQ ID NO:5.

10. The protein of claim 8 comprising the sequence from amino acid 41 to 425 of SEQ ID NO:5.

11. The protein of claim 8 comprising the amino acid sequence of SEQ ID NO:7.

12. The protein of claim 8 comprising the sequence from amino acid 24 to 408 of SEQ ID NO:7.

13. A pharmaceutical composition comprising a protein of claim 8 and a pharmaceutically acceptable carrier.

14. A protein produced according to the process of claim 7.

15. A composition comprising an antibody which specifically reacts with a protein of claim 8.

16. An isolated polynucleotide comprising a nucleotide sequence encoding a peptide or protein comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:5; 26 intellectual PROPERTY. OFFICE OF N.Z. 2 5 JUL 2003 RECEIVED (b) the amino acid sequence of SEQ ID NO:5 from amino acids 41 to 425; (c) the amino acid sequence of SEQ ID NO:7; and (d) the amino acid sequence of SEQ ID NO:7 from amino acids 24 to 408.

17. The protein of claim 8 wherein said amino acid sequence is part of a fusion protein.

18. The protein of claim 17 comprising an Fc fragment. INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 0 OCT 2003 RECEIVED 27 WO 99/53066 PC7YUS99/07882 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Donaldson, Debra Collins, Mary Neben, Tamlyn Whitters, Matthew (ii) TITLE OF INVENTION: CYTOKINE RECEPTOR CHAIN (iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Genetics Institute, Inc. (B) STREET: 87 CambridgePark Drive (C) CITY: Cambridge (D) STATE: MA (E) COUNTRY: USA (F) ZIP: 02140 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Brown, Scott A. (B) REGISTRATION NUMBER: 32,724 (C) REFERENCE/DOCKET NUMBER: GI5287 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (617) 498-B224 (B) TELEFAX: (617) 876-5851 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 70 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Leu Met Thr Asn Ala Phe lie Ser lie lie Asp Asp Leu Ser Lys Tyr 15 10 15 Asp Val Gin Val Arg Ala Ala Val Ser Ser Met Cys Arg Glu Ala Gly 20 25 30 Leu Trp Ser Glu Trp Ser Gin Pro lie Tyr Val Gly Asn Asp Glu His 35 40 45 1 Printed from Mimosa 10/03/2000 10:18:39 page -31- WO 99/53066 PCT/US99/07882 Lys Pro Leu Arg Glu Trp Phe Val He Val lie Met Ala Thr lie Cys 50 55 60 Phe lie Leu Leu lie Leu 65 70 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3 0 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "probe" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CTTGGCTTGG AAGAGGAAAT CCTTGAGAGC 30 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "probe" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CTGAGCGTGC GCTGGGTGTC GCCAC 25 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1656 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GTCGACCTTC GCTGTCCGCG CCCAGTGACG CGCGTGAGGA CCCGAGCCCC AATCTGCACC 60 CCGCAGACTC GCCCCCGCCC CATACCGGCG TTGCAGTCAC CGCCCGTTGC GCGCCACCCC 120 CATGCCCGCG GGTCGCCCGG GCCCCGTCGC CCAATCCGCG CGGCGGCCGC CGCGGCCGCT 180 GTCCTCGCTG TGGTCGCCTC TGTTGCTCTG TGTCCTCGGG GTGCCTCGGG GCGGATCGGG 240 AGCCCACACA GCTGTAATCA GCCCCCAGGA CCCCACCTTT CTCATCGGCT CCTCCCTGCA 300 AGCTACCTGC TCTATACATG GAGACACACC TGGGGCCACC GCTGAGGGGC TCTACTGGAC 360 2 Printed from Mimosa 10/03/2000 10:18:39 page -32- WO 99/53066 PCT/US99/07882 CCTCAATGGT CGCCGCCTGC CCTCTGAGCT GTCCCGCCTC CTTAACACCT CCACCCTGGC 420 CCTGGCCCTG GCTAACCTTA ATGGGTCCAG GCAGCAGTCA GGAGACAATC TGGTGTGTCA 480 CGCCCGAGAT GGCAGCATTC TGGCTGGCTC CTGCCTCTAT GTTGGCTTGC CCCCTGAGAA 540 GCCTTTTAAC ATCAGCTGCT GGTCCCGGAA CATGAAGGAT CTCACGTGCC GCTGGACACC 600 GGGTGCACAC GGGGAGACAT TCTTACATAC CAACTACTCC CTCAAGTACA AGCTGAGGTG 660 GTACGGTCAG GATAACACAT GTGAGGAGTA CCACACTGTG GGCCCTCACT CATGCCATAT 720 CCCCAAGGAC CTGGCCCTCT TCACTCCCTA TGAGATCTGG GTGGAAGCCA CCAATCGCCT 780 AGGCTCAGCA AGATCTGATG TCCTCACACT GGATGTCCTG GACGTGGTGA CCACGGACCC 840 CCCACCCGAC GTGCACGTGA GCCGCGTTGG GGGCCTGGAG GACCAGCTGA GTGTGCGCTG 900 GGTCTCACCA CCAGCTCTCA AGGATTTCCT CTTCCAAGCC AAGTACCAGA TCCGCTACCG 960 CGTGGAGGAC AGCGTGGACT GGAAGGTGGT GGATGACGTC AGCAACCAGA CCTCCTGCCG 1020 TCTCGCGGGC CTGAAGCCCG GCACCGTTTA CTTCGTCCAA GTGCGTTGTA ACCCATTCGG 1080 GATCTATGGG TCGAAAAAGG CGGGAATCTG GAGCGAGTGG AGCCACCCCA CCGCTGCCTC 1140 CACCCCTCGA AGTGAGCGCC CGGGCCCGGG CGGCGGGGTG TGCGAGCCGC GGGGCGGCGA 1200 GCCCAGCTCG GGCCCGGTGC GGCGCGAGCT CAAGCAGTTC CTCGGCTGGC TCAAGAAGCA 1260 CGCATACTGC TCGAACCTTA GTTTCCGCCT GTACGACCAG TGGCGTGCTT GGATGCAGAA 1320 GTCACACAAG ACCCGAAACC AGGACGAGGG GATCCTGCCC TCGGGCAGAC GGGGTGCGGC 1380 GAGAGGTCCT GCCGGCTAAA CTCTAAGGAT AGGCCATCCT CCTGCTGGGT CAGACCTGGA 1440 GGCTCACCTG AATTGGAGCC CCTCTGTACC ATCTGGGCAA CAAAGAAACC TACCAGAGGC 1500 TGGGGCACAA TGAGCTCCCA CAACCACAGC TTTGGTCCAC ATGATGGTCA CACTTGGATA 1560 TACCCCAGTG TGGGTAGGGT TGGGGTATTG CAGGGCCTCC CAAGAGTCTC TTTAAATAAA 1620 TAAAGGAGTT GTTCAGGTCC CGAAAAAAAA GTCGAC 1656 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 425 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Met Pro Ala Gly Arg Pro Gly Pro Val Ala Gin Ser Ala Arg Arg Pro 15 10 15 Pro Arg Pro Leu Ser Ser Leu Trp Ser Pro Leu Leu Leu Cys Val Leu 20 25 30 Gly Val Pro Arg Gly Gly Ser Gly Ala His Thr Ala Val lie Ser Pro 35 40 45 Gin Asp Pro Thr Phe Leu lie Gly Ser Ser Leu Gin Ala Thr Cys Ser 3 Printed from Mimosa 10/03/2000 10:18:39 page -33- WO 99/53066 PCT/US99/07882 50 55 60 lie His Gly Asp Thr Pro Gly Ala Thr Ala Glu Gly Leu Tyr Trp Thr 65 70 75 80 Leu Asn Gly Arg Arg Leu Pro Ser Glu Leu Ser Arg Leu Leu Asn Thr 85 90 95 Ser Thr Leu Ala Leu Ala Leu Ala Asn Leu Asn Gly Ser Arg Gin Gin 100 105 110 Ser Gly Asp Asn Leu Val Cys His Ala Arg Asp Gly Ser lie Leu Ala 115 120 125 Gly Ser Cys Leu Tyr Val Gly Leu Pro Pro Glu Lys Pro Phe Asn lie 130 135 140 Ser Cys Trp Ser Arg Asn Met Lys Asp Leu Thr Cys Arg Trp Thr Pro 145 150 155 160 Gly Ala His Gly Glu Thr Phe Leu His Thr Asn Tyr Ser Leu Lys Tyr 165 170 175 Lys Leu Arg Trp Tyr Gly Gin Asp Asn Thr Cys Glu Glu Tyr His Thr 180 185 190 Val Gly Pro His Ser Cys His lie Pro Lys Asp Leu Ala Leu Phe Thr 195 200 205 Pro Tyr Glu lie Trp Val Glu Ala Thr Asn Arg Leu Gly Ser Ala Arg 210 215 220 Ser Asp Val Leu Thr Leu Asp Val Leu Asp Val Val Thr Thr Asp Pro 225 230 235 240 Pro Pro Asp Val His Val Ser Arg Val Gly Gly Leu Glu Asp Gin Leu 245 250 255 Ser Val Arg Trp Val Ser Pro Pro Ala Leu Lys Asp Phe Leu Phe Gin 260 265 270 Ala Lys Tyr Gin lie Arg Tyr Arg Val Glu Asp Ser Val Asp Trp Lys 275 280 285 Val Val Asp Asp Val Ser Asn Gin Thr Ser Cys Arg Leu Ala Gly Leu 290 295 300 Lys Pro Gly Thr Val Tyr Phe Val Gin Val Arg Cys Asn Pro Phe Gly 305 310 315 320 lie Tyr Gly Ser Lys Lys Ala Gly lie Trp Ser Glu Trp Ser His Pro 325 330 335 Thr Ala Ala Ser Thr Pro Arg Ser Glu Arg Pro Gly Pro Gly Gly Gly 340 345 350 Val Cys Glu Pro Arg Gly Gly Glu Pro Ser Ser Gly Pro Val Arg Arg 355 360 365 Glu Leu Lys Gin Phe Leu Gly Trp Leu Lys Lys His Ala Tyr Cys Ser 370 375 380 Asn Leu Ser Phe Arg Leu Tyr Asp Gin Trp Arg Ala Trp Met Gin Lys 385 390 395 400 Ser His Lys Thr Arg Asn Gin Asp Glu Gly lie Leu Pro Ser Gly Arg 405 410 415 Arg Gly Ala Ala Arg Gly Pro Ala Gly 4 Printed from Mimosa 10/03/2000 10:18:39 page -34- WO 99/53066 PCT/US99/07882 420 425 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1579 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GCGGCCGCCG CCGTTGCTGC CCCTGCTGCT GCTGCTCTGC GTCCTCGGGG CGCCGCGAGC 60 CGGATCAGGA GCCCACACAG CTGTGATCAG TCCCCAGGAT CCCACGCTTC TCATCGGCTC 120 CTCCCTGCTG GCCACCTGCT CAGTGCACGG AGACCCACCA GGAGCCACCG CCGAGGGCCT 180 CTACTGGACC CTCAACGGGC GCCGCCTGCC CCCTGAGCTC TCCCGTGTAC TCAACGCCTC 240 CACCTTGGCT CTGGCCCTGG CCAACCTCAA TGGGTCCAGG CAGCGGTCGG GGGACAACCT 300 CGTGTGCCAC GCCCGTGACG GCAGCATCCT GGCTGGCTCC TGCCTCTATG TTGGCCTGCC 360 CCCAGAGAAA CCCGTCAACA TCAGCTGCTG GTCCAAGAAC ATGAAGGACT TGACCTGCCG 420 CTGGACGCCA GGGGCCCACG GGGAGACCTT CCTCCACACC AACTACTCCC TCAAGTACAA 480 GCTTAGGTGG TATGGCCAGG ACAACACATG TGAGGAGTAC CACACAGTGG GGCCCCACTC 540 CTGCCACATC CCCAAGGACC TGGCTCTCTT TACGCCCTAT GAGATCTGGG TGGAGGCCAC 600 CAACCGCCTG GGCTCTGCCC GCTCCGATGT ACTCACGCTG GATATCCTGG ATGTGGTGAC 660 CACGGACCCC CCGCCCGACG TGCACGTGAG CCGCGTCGGG GGCCTGGAGG ACCAGCTGAG 720 CGTGCGCTGG GTGTCGCCAC CCGCCCTCAA GGATTTCCTC TTTCAAGCCA AATACCAGAT 780 CCGCTACCGA GTGGAGGACA GTGTGGACTG GAAGGTGGTG GACGATGTGA GCAACCAGAC 840 CTCCTGCCGC CTGGCCGGCC TGAAACCCGG CACCGTGTAC TTCGTGCAAG TGCGCTGCAA 900 CCCCTTTGGC ATCTATGGCT CCAAGAAAGC CGGGATCTGG AGTGAGTGGA GCCACCCCAC 960 AGCCGCCTCC ACTCCCCGCA GTGAGCGCCC GGGCCCGGGC GGCGGGGCGT GCGAACCGCG 1020 GGGCGGAGAG CCGAGCTCGG GGCCGGTGCG GCGCGAGCTC AAGCAGTTCC TGGGCTGGCT 1080 CAAGAAGCAC GCGTACTGCT CCAACCTCAG CTTCCGCCTC TACGACCAGT GGCGAGCCTG 1140 GATGCAGAAG TCGCACAAGA CCCGCAACCA GGACGAGGGG ATCCTGCCCT CGGGCAGACG 1200 GGGCACGGCG AGAGGTCCTG CCAGATAAGC TGTAGGGGCT CAGGCCACCC TCCCTGCCAC 1260 GTGGAGACGC AGAGGCCGAA CCCAAACTGG GGCCACCTCT GTACCCTCAC TTCAGGGCAC 1320 CTGAGCCACC CTCAGCAGGA GCTGGGGTGG CCCCTGAGCT CCAACGGCCA TAACAGCTCT 1380 GACTCCCACG TGAGGCCACC TTTGGGTGCA CCCCAGTGGG TGTGTGTGTG TGTGTGAGGG 1440 TTGGTTGAGT TGCCTAGAAC CCCTGCCAGG GCTGGGGGTG AGAAGGGGAG TCATTACTCC 1500 CCATTACCTA GGGCCCCTCC AAAAGAGTCC TTTTAAATAA ATGAGCTATT TAGGTGCTGT 1560 5 Printed from Mimosa 10/03/2000 10:18:39 page -35- WO 99/53066 PCT/US99/07882 GAAAAAAAAA AAAAAAAAA 1579 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 408 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Arg Pro Pro Pro Leu Leu Pro Leu Leu Leu Leu Leu Cys Val Leu Gly 15 10 15 Ala Pro Arg Ala Gly Ser Gly Ala His Thr Ala Val lie Ser Pro Gin 20 25 30 Asp Pro Thr Leu Leu lie Gly Ser Ser Leu Leu Ala Thr Cys Ser Val 35 40 45 His Gly Asp Pro Pro Gly Ala Thr Ala Glu Gly Leu Tyr Trp Thr Leu 50 55 60 Asn Gly Arg Arg Leu Pro Pro Glu Leu Ser Arg Val Leu Asn Ala Ser 65 70 75 80 Thr Leu Ala Leu Ala Leu Ala Asn Leu Asn Gly Ser Arg Gin Arg Ser 85 90 95 Gly Asp Asn Leu Val Cys His Ala Arg Asp Gly Ser lie Leu Ala Gly 100 105 110 Ser Cys Leu Tyr Val Gly Leu Pro Pro Glu Lys Pro Val Asn lie Ser 115 120 125 Cys Trp Ser Lys Asn Met Lys Asp Leu Thr Cys Arg Trp Thr Pro Gly 130 135 140 Ala His Gly Glu Thr Phe Leu His Thr Asn Tyr Ser Leu Lys Tyr Lys 145 150 155 160 Leu Arg Trp Tyr Gly Gin Asp Asn Thr Cys Glu Glu Tyr His Thr Val 165 170 175 Gly Pro His Ser Cys His lie Pro Lys Asp Leu Ala Leu Phe Thr Pro 180 185 190 Tyr Glu lie Trp Val Glu Ala Thr Asn Arg Leu Gly Ser Ala Arg Ser 195 200 205 Asp Val Leu Thr Leu Asp lie Leu Asp Val Val Thr Thr Asp Pro Pro 210 215 220 Pro Asp Val His Val Ser Arg Val Gly Gly Leu Glu Asp Gin Leu Ser 225 230 235 240 Val Arg Trp Val Ser Pro Pro Ala Leu Lys Asp Phe Leu Phe Gin Ala 245 250 255 Lys Tyr Gin lie Arg Tyr Arg Val Glu Asp Ser Val Asp Trp Lys Val 260 265 270 Val Asp Asp Val Ser Asn Gin Thr Ser Cys Arg Leu Ala Gly Leu Lys Printed from Mimosa 10/03/2000 10:18:39 page -36- WO 99/53066 PCT/US99/07882 275 280 285 Pro Gly Thr Val Tyr Phe Val Gin Val Arg Cys Asn Pro Phe Gly lie 290 295 300 Tyr Gly Ser Lys Lys Ala Gly lie Trp Ser Glu Trp Ser His Pro Thr 305 310 315 320 Ala Ala Ser Thr Pro Arg Ser Glu Arg Pro Gly Pro Gly Gly Gly Ala 325 330 335 Cys Glu Pro Arg Gly Gly Glu Pro Ser Ser Gly Pro Val Arg Arg Glu 340 345 350 Leu Lys Gin Phe Leu Gly Trp Leu Lys Lys His Ala Tyr Cys Ser Asn 355 360 365 Leu Ser Phe Arg Leu Tyr Asp Gin Trp Arg Ala Trp Met Gin Lys Ser 370 375 380 His Lys Thr Arg Asn Gin Asp Glu Gly lie Leu Pro Ser Gly Arg Arg 385 390 395 400 Gly Thr Ala Arg Gly Pro Ala Arg 405 (2) INFORMATION FOR SEQ ID NO: 8: £i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Pro Ala Gly Arg Pro Gly Pro Val Ala Asn Ser Ala Arg 15 10 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1787 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GAATTCCGGC GCGTCCGCGC CCAGCGACGT GCGGGCGGCC TGGCCCGCGC CCTCCCGCGC 60 CCGGCCTGCG TCCCGCGCCC TGCGCCACCG CCGCCGAGCC GCAGCCCGCC GCGCGCCCCC 120 GGCAGCGCCG GCCCCATGCC CGCCGGCCGC CGGGGCCCCG CCGCCCAATC CGCGCGGCGG 180 CCGCCGCCGT TGCTGCCCCT GCTGCTGCTG CTCTGCGTCC TCGGGGCGCC GCGAGCCGGA 240 TCAGGAGCCC ACACAGCTGT GATCAGTCCC CAGGATCCCA CGCTTCTCAT CGGCTCCTCC 300 7 Printed from Mimosa 10/03/2000 10:18:39 page -37- WO 99/53066 PCT/US99/07882 CTGCTGGCCA CCTGCTCAGT GCACGGAGAC CCACCAGGAG CCACCGCCGA GGGCCTCTAC 360 TGGACCCTCA ACGGGCGCCG CCTGCCCCCT GAGCTCTCCC GTGTACTCAA CGCCTCCACC 420 TTGGCTCTGG CCCTGGCCAA CCTCAATGGG TCCAGGCAGC GGTCGGGGGA CAACCTCGTG 480 TGCCACGCCC GTGACGGCAG CATCCTGGCT GGCTCCTGCC TCTATGTTGG CCTGCCCCCA 540 GAGAAACCCG TCAACATCAG CTGCTGGTCC AAGAACATGA AGGACTTGAC CTGCCGCTGG 600 ACGCCAGGGG CCCACGGGGA GACCTTCCTC CACACCAACT ACTCCCTCAA GTACAAGCTT 660 AGGTGGTATG GCCAGGACAA CACATGTGAG GAGTACCACA CAGTGGGGCC CCACTCCTGC 720 CACATCCCCA AGGACCTGGC TCTCTTTACG CCCTATGAGA TCTGGGTGGA GGCCACCAAC 780 CGCCTGGGCT CTGCCCGCTC CGATGTACTC ACGCTGGATA TCCTGGATGT GGTGACCACG 840 GACCCCCCGC CCGACGTGCA CGTGAGCCGC GTCGGGGGCC TGGAGGACCA GCTGAGCGTG 900 CGCTGGGTGT CGCCACCCGC CCTCAAGGAT TTCCTCTTTC AAGCCAAATA CCAGATCCGC 960 TACCGAGTGG AGGACAGTGT GGACTGGAAG GTGGTGGACG ATGTGAGCAA CCAGACCTCC 1020 TGCCGCCTGG CCGGCCTGAA ACCCGGCACC GTGTACTTCG TGCAAGTGCG CTGCAACCCC 1080 TTTGGCATCT ATGGCTCCAA GAAAGCCGGG ATCTGGAGTG AGTGGAGCCA CCCCACAGCC 1140 GCCTCCACTC CCCGCAGTGA GCGCCCGGGC CCGGGCGGCG GGGCGTGCGA ACCGCGGGGC 1200 GGAGAGCCGA GCTCGGGGCC GGTGCGGCGC GAGCTCAAGC AGTTCCTGGG CTGGCTCAAG 1260 AAGCACGCGT ACTGCTCCAA CCTCAGCTTC CGCCTCTACG ACCAGTGGCG AGCCTGGATG 1320 CAGAAGTCGC ACAAGACCCG CAACCAGGAC GAGGGGATCC TGCCCTCGGG CAGACGGGGC 1380 ACGGCGAGAG GTCCTGCCAG ATAAGCTGTA GGGGCTCAGG CCACCCTCCC TGCCACGTGG 1440 AGACGCAGAG GCCGAACCCA AACTGGGGCC ACCTCTGTAC CCTCACTTCA GGGCACCTGA 1500 GCCACCCTCA GCAGGAGCTG GGGTGGCCCC TGAGCTCCAA CGGCCATAAC AGCTCTGACT 1560 CCCACGTGAG GCCACCTTTG GGTGCACCCC AGTGGGTGTG TGTGTGTGTG TGAGGGTTGG 1620 TTGAGTTGCC TAGAACCCCT GCCAGGGCTG GGGGTGAGAA GGGGAGTCAT TACTCCCCAT 1680 TACCTAGGGC CCCTCCAAAA GAGTCCTTTT AAATAAATGA GCTATTTAGG TGCAAAAAAA 1740 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAACC 1787 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 422 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:10: Met Pro Ala Gly Arg Arg Gly Pro Ala Ala Gin Ser Ala Arg Arg Pro 15 10 15 Pro Pro Leu Leu Pro Leu Leu Leu Leu Leu Cys Val Leu Gly Ala Pro 8 Printed from Mimosa 10/03/2000 10:18:39 page -38- WO 99/53066 PCT/US99/07882 20 25 30 Arg Ala Gly Ser Gly Ala His Thr Ala Val lie Ser Pro Gin Asp Pro 35 40 45 Thr Leu Leu lie Gly Ser Ser Leu Leu Ala Thr Cys Ser Val His Gly 50 55 60 Asp Pro Pro Gly Ala Thr Ala Glu Gly Leu Tyr Trp Thr Leu Asn Gly 65 70 75 80 Arg Arg Leu Pro Pro Glu Leu Ser Arg Val Leu Asn Ala Ser Thr Leu 85 90 95 Ala Leu Ala Leu Ala Asn Leu Asn Gly Ser Arg Gin Arg Ser Gly Asp 100 105 110 Asn Leu Val Cys His Ala Arg Asp Gly Ser lie Leu Ala Gly Ser Cys 115 120 125 Leu Tyr Val Gly Leu Pro Pro Glu Lys Pro Val Asn lie Ser Cys Trp 130 135 140 Ser Lys Asn Met Lys Asp Leu Thr Cys Arg Trp Thr Pro Gly Ala His 145 150 155 160 Gly Glu Thr Phe Leu His Thr Asn Tyr Ser Leu Lys Tyr Lys Leu Arg 165 170 175 Trp Tyr Gly Gin Asp Asn Thr Cys Glu Glu Tyr His Thr Val Gly Pro 180 185 190 His Ser Cys His lie Pro Lys Asp Leu Ala Leu Phe Thr Pro Tyr Glu 195 200 205 lie Trp Val Glu Ala Thr Asn Arg Leu Gly Ser Ala Arg Ser Asp Val 210 215 220 Leu Thr Leu Asp lie Leu Asp Val Val Thr Thr Asp Pro Pro Pro Asp 225 230 235 240 Val His Val Ser Arg Val Gly Gly Leu Glu Asp Gin Leu Ser Val Arg 245 250 255 Trp Val Ser Pro Pro Ala Leu Lys Asp Phe Leu Phe Gin Ala Lys Tyr 260 265 270 Gin lie Arg Tyr Arg Val Glu Asp Ser Val Asp Trp Lys Val Val Asp 275 280 285 Asp Val Ser Asn Gin Thr Ser Cys Arg Leu Ala Gly Leu Lys Pro Gly 290 295 300 Thr Val Tyr Phe Val Gin Val Arg Cys Asn Pro Phe Gly lie Tyr Gly 305 310 315 320 Ser Lys Lys Ala Gly lie Trp Ser Glu Trp Ser His Pro Thr Ala Ala 325 330 335 Ser Thr Pro Arg Ser Glu Arg Pro Gly Pro Gly Gly Gly Ala Cys Glu 340 345 350 Pro Arg Gly Gly Glu Pro Ser Ser Gly Pro Val Arg Arg Glu Leu Lys 355 360 365 Gin Phe Leu Gly Trp Leu Lys Lys His Ala Tyr Cys Ser Asn Leu Ser 370 375 380 Phe Arg Leu Tyr Asp Gin Trp Arg Ala Trp Met Gin Lys Ser His Lys 9 Printed from Mimosa 10/03/2000 10:18:39 page -39- WO 99/53066 PCT/US99/07882 385 390 395 400 Thr Arg Asn Gin Asp Glu Gly lie Leu Pro Ser Gly Arg Arg Gly Thr 405 410 415 Ala Arg Gly Pro Ala Arg 420 10 Printed from Mimosa 10/03/2000 10:18:39 page -40-