WO2000077191A1 - Tumor necrosis factor related receptor, tr6 - Google Patents

Abstract

TR6 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing TR6 polypeptides and polynucleotides in the design of protocols for the treatment of chronic and acute inflammation, arthritis, septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, restenosis, brain injury, AIDS, Bone diseases, cancer, atherosclerosis, and Alzheimer's disease, among others and diagnostic assays for such conditions.

Description

TUMORNECROSISFACTORRELATED RECEPTOR, TR6

This application is a contmuation-m-part of U.S. Serial No. 08/916,625, filed August 22, 1997 which in turn is a contmuation-in-part application of U.S. Serial No: 08/853,684, filed May 9, 1997, which claimed the benefit of U.S. Provisional Application No: 60/041,230, filed March 14, 1997. All three applications are herein incorporated by reference in their entirety.

FIELD OF INVENTION

This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to Tumor Necrosis Factor Related family, hereinafter referred to as TR6. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

Many biological actions, for instance, response to certain sUmuh and natural biological processes, are controlled by factors, such as cytokines. Many cytokines act through receptors by engaging the receptor and producing an mtracellular response.

For example, tumor necrosis factors (TNF) alpha and beta are cytokines which act through TNF receptors to regulate numerous biological processes, including m host defense processes such as protection against infections, and pathological conditions such as shock responses and inflammatory disease condistions. TNF-α belongs to the "TNF-ligand" superfamily of which 19 members have been identified so far. These hgands mediate their effects through interactions with cell surface or secreted, decoy, receptors, expressed by many different cell types, and which themselves now form a superfamily with 24 mdenUfied members to date.

Among the hgands there are included TNF-α, lymphotoxin-α (LT-α, also known as TNF-β), LT-β (found in heterotπmeπc complexes , LT-α2-β), FasL, CD40L, CD27L, CD30L, 4-lBBL, OX40L and nerve growth factor (NGF)). The receptor superfamily includes the p55 and p75 TNF receptor,

FAS APO-1, CD40, CD27, CD30, 4-1BB, OX40 and the low affinity p75 NGF-receptor (Meager, A., Biologicals, 22:291-295 (1994)).

Many members of the TNF- gand superfamily are expressed by cells of the immune and hematopoietic system which underscores their role in differentian of the cells of the immune cells and functional responses in host defense mechanisms (Meager, A , supra).

Considerable insight into the essential functions of several members of the TNF receptor family has been gained from the identification and creation of mutants that abolish the expression of these proteins. For example, naturally occurring mutations in the FAS antigen and its hgand cause lymphoproliferative disease (Watanabe-Fukunaga, R., et al, Nature 356.314 (1992)), perhaps reflecting a failure of programmed cell death. Mutations of the CD40 hgand cause an X-linked immunodeficiency state characteπzed by high levels of lmmunoglubuhn M and low levels of lmmunoglobulm G in plasma, indicating faulty T-cell-dependent B-cell activation (Allen, R.C. et al., Science 259:990 (1993)). Targeted mutations of the low affinity nerve growth factor receptor cause a disorder characteπzed by faulty sensory innovation of peπpheral structures (Lee, K.F. et al, Cell 69:737 (1992)). TNF-α and LT-α are capable of binding to two TNF receptors (the 55- and 75 -kd TNF receptors) A large number of biological effects are elicited by TNF-α and LT-α, acting through their receptors, include hemorrhagic necrosis of transplanted tumors, cytotoxicity, a role in endotoxic shock, inflammation, immunoregulation, proliferation and anti-viral responses, as well as protection against the deleteπous effects of ionizing radiation. TNF-α and LT-α are involved in the pathogenesis of a wide range of diseases, including endotoxic shock, cerebral malaπa, tumors, autoimmuine diseases, allergic disorders, AIDS and graft rejection (Beutler, B. and Von Huffel, C , Science 264:667-668 (1994)). Mutations in the p55 Receptor cause increased susceptibility to microbial infection.

Moreover, an about 80 amino acid domain near the C-termmus of TNFR1 (p55) and Fas was reported as the "death domain," which is responsible for transducing signals for programmed cell death (Tartagha et al , Cell 74:845 (1993)).

The effects of TNF hgand and TNF receptor families are vaπed and influence numerous functions, both normal and abnormal, in the biological processes of mammalian and non-mammalian species. There is a clear need, therefore, for identification and characteπzation of such receptors and hgands that influence biological activity, both normally and in disease states. In particular, there is a need to isolate and characteπze novel members of the TNF receptor family.

This indicates that these receptors have an established, proven history as therapeutic targets. Clearly there is a need for identification and characteπzation of further receptors which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, chronic and acute inflammation, arthπtis (including rheumatoid arthπtis), septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psoπasis), transplant rejection, graft vs host disease, infection, stroke, ischemia, acute respiratory disease syndrome, asthma, restenosis, bram injury, AIDS, Bone diseases, cancer , atheroschlerosis, and Alzheimers disease SUMMARY OF THE INVENTION

In one aspect, the invention relates to TR6 polypeptides and recombinant mateπals and methods for their production. Another aspect of the invention relates to methods for using such TR6 polypeptides and polynucleotides and recombinant mateπals. Such uses include the treatment of chronic and acute inflammation, arthπtis (including rheumatoid arthπtis), septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, asthma, restenosis, brain injury, AIDS, Bone diseases, cancer , atheroschlerosis, and Alzheimers disease, among others. Another aspect of the invention relates to methods of using such TR6 polynucleotides, polypeptides and recombinant mateπals for inhibiting angiogenesis and also inhibiting production of TNF-α and eicosanoids.

In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with TR6 imbalance with the identified compounds.

Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with mappropπate TR6 activity or levels.

DESCRIPTION OF THE INVENTION

Definitions

The following definitions are provided to facilitate understanding of certain terms used frequently herein. "TR6" refers, among others, to a polypeptide comprising the ammo acid sequence set forth in SEQ ID NO:2, or an allehc variant thereof.

"Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0 464 533 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokmetic properties [see, e.g., EP-A 0232 262]. On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified.

In the case of TR6 fusion protein, in one embodiment for example, the fusion protein can be a fusion of the extracellular portion of TR6 fused with the Fc portion of human IgG. In one exemplified construct of SEQ ID NO: 7 of Table 5, extra ammo acid residues were introduced within a hmge region between the TR6 and the IgG Fc portions of the molecule to facilitate cleavage of the protein by Factor Xa. This is sometimes desirable to facilitate the enzymatic cleavage of the IgG Fc portion of the recombinant protein from the TR6 part, either to facilitate binding studies or for generation of antibodies selectively to the TR6 portion of the recombmant protein. However, m clinical applications, although it may sometimes be desirable to introduce enzymatic cleavage sites withm the recombinant protein, it may not always be desirable to do so. To avoid cleavage of the recombinant protein Factor Xa, it may be desirable to construct a fusion protein of the extracellular portion of TR6 fused directly with the IgG Fc portion.

It may also be desirable to introduce intervening ammo acid sequences between the TR6 portion and the IgG Fc portion. Such intervening sequence may sometimes be desirable to modify the in vivo properties of the recombinant protein, such as by making the hmge region more rigid or more flexible. Such residues can also be added or removed to alter the effector function of the the IgG Fc portion of the recombinant protein. Examples of such effector functions include, but are not limited to, complement binding, Fc receptor binding, antibody dependent cellular cytotoxicity.

Other methods to alter the protein of a fusion protein is to use the Fc portions of different immunoglubulin isotypes. Examples of such constructs include, but are not limited to, fusion proteins with portions of IgGl, IgG2, IgG3, IgG4, IgA, IgM. Such constructs are expected to alter the in vivo properties of the recombinant fusion protein. For example, fusion proteins with the Fc portion of IgG4 would be expected to have reduced ability to bind to components of the complement cascade and to have reduced ability to bind to Fc receptor or to mediate ADCC. The Fc portion of IgA is known to facilitate transplacental transport.

Other single or multiple residue mutations can also alter the behaviour and function of such recombmant proteins. For example, single and multrpe residue mutations withm the Fc portion of lmmunoglobuhns can dramatically alter the Fc effector functions such as reduction in their ability to bind to one or more Fc receptor types. Some outcomes of such changes would be alterations of the pharmacokmetic and pharmacodynamic properties of the recombmant proteins and alterations in the lv vivo consequences such as effects on cells and tissues in vivo. Such alterations may sometimes be desirable in order to improve the clinical utility of the TR6 protein.

Fusion can also be made to the tail portions of proteins such as IgA and IgM which can facilitate expression of multivalent proteins Other embodiments may include fusion with ammo acid sequences which can facility dimer or tπmer formation (e.g. zmk finger proteins or the stock regions of collagen), heterbifunctional fusion proteins (e.g. with cytokines, immunoglobulin domains or other receptors and hgands) designed to facilitate recognition of more than one target, viral peptide sequences known to mediate protein transduction (e.g. HIV -tat, HSV-VP22), fusion to cytotoxms (e.g. staphyloccocal enterotoxms, πcm). Chemical conjugation of TR6 to cytotoxic, cytostatic or cytoprotective compounds is also possible.

It may also be desirable to alter the glycosylation sites on the recombinant protein to beneficial alter the pharmacokmetic and/or pharmcodynamic properties of the protein or improve manufacturing or stability of the recombmant protein.

"Receptor Activity" or "Biological Activity of the Receptor" refers to the metabolic or physiologic function of said TR6, including similar activities or improved activities or decreased undesirable side-effects of the whole or parts of TR6, including m a recombmatorial form fused with other molecules (e.g. in fusion with parts of lmmunoglobulms or other desirable polypeptides, such as as TR6-Ig fusion proteins). Also included are antigenic and lmmunogemc activities of said TR6.

"TR6 gene" refers to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: l or allehc variants thereof and/or their complements.

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeπc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present m a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein

"Polynucleotide" generally refers to any polyπbonucleotide or polydeoxπbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to tπple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tπtylated bases and unusual bases such as mosme A vaπety of modifications has been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteπstic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres "Polypeptide" refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins. Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids. "Polypeptides" include ammo acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and m more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side -chains and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites m a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-πbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as arginylation, and ubiquitmation. See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W.

H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al , "Analysis for protein modifications and nonprotem cofactors", Meth Enzymol (1990) 182:626- 646 and Rattan et al, "Protein Synthesis: Posttranslational Modifications and Aging", Ann NYAcad

Set (1992) 663:48-62.

"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the vaπant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those descπbed in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Gπffin,

H.G , eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gπbskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Caπllo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Prefeπed methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Prefeπed computer program methods to determine identity and similaπty between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894;

Altschul, S., et al., J. Mol. Biol. 215- 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following.

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl Acad. Sci. USA. 89: 10915-10919 (1992)

Gap Penalty- 12

Gap Length Penalty: 4 A program useful with these parameters is publicly available as the "gap" program from

Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = +10, mismatch = 0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The "gap" program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid comparisons. By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:l, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence. Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups with the reference sequence. The number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the numerical percent of the respective percent ιdentιty(dιvιded by 100) and subtracting that product from said total number of nucleotides in SEQ ID NO.1 , or: nn < xn - (xn • y), wherem nn is the number of nucleotide alterations, xn is the total number of nucleotides m SEQ ID NO: l, and y is, for instance, 0.70 for 70%, 0 80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc, and wherein any non-mteger product of xn and y is rounded down to the nearest integer prior to subtracting it from xn. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

Similarly, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is be 100% identical, or it may include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the % identity is less than 100%o. Such alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or m one or more contiguous groups withm the reference sequence. The number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids m SEQ ID NO.2 by the numerical percent of the respective percent ιdentιty(dιvιded by 100) and then subtracting that product from said total number of ammo acids in SEQ ID NO:2, or: na < xa - (xa • y), wherein na is the number of ammo acid alterations, xa is the total number of ammo acids m SEQ ID NO:2, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherem any non- mteger product of xa and y is rounded down to the nearest integer pπor to subtracting it from xa

Polypeptides of the Invention In one aspect, the present invention relates to TR6 polypeptides The TR6 polypeptides include the polypeptides of SEQ ID NOS:2 and 4; as well as polypeptides comprising the ammo acid sequence of SEQ ID NO:2; and polypeptides comprising the ammo acid sequence which have at least 80% identity to that of SEQ ID NO:2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2. Furthermore, those with at least 97-99% are highly prefeπed. Also included withm TR6 polypeptides are polypeptides having the ammo acid sequence which have at least 80% identity to the polypeptide having the ammo acid sequence of SEQ ID NO: 2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2. Furthermore, those with at least 97-99% are highly prefeπed. Preferably TR6 polypeptides exhibit at least one biological activity of the receptor.

The TR6 polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include additional ammo acid sequences which contain secretory or leader sequences, pro-sequences, sequences which aid in puπfication such as multiple histidme residues or the Fc portion of lmmunonoglobuhns, which may also improve in vivo half life, or an additional sequence for stability duπng recombinant production

The TR6 polypeptides also include fragments of the aforementioned polypeptides. More specifically, a fragment is a polypeptide having an ammo acid sequence that entirely is the same as part, but not all, of the ammo acid sequence of the aforementioned TR6 polypeptides An example of fragment is extracellular domain of the polypepitde having the ammo acid sequence of SEQ ID NO: 2. Amino acid sequence from 1 to 184 is predicted to be the extracellular domain sequence of SEQ ID NO: 2, which includes the leader sequence compnsrng the first 53 ammo acids; however, sequences longer or shorter than that of 1 to 184 is also possible for it to be extracellular. As with TR6 polypeptides, fragments may be "free-standmg," or compπsed withm a larger polypeptide of which they form a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about ammo acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of TR6 polypeptide. In this context "about" includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 ammo acid at either extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of TR6 polypeptides, except for deletion of a continuous seπes of residues that includes the ammo terminus, or a continuous seπes of residues that includes the carboxyl terminus or deletion of two continuous seπes of residues, one including the ammo terminus and one including the carboxyl terminus. Also prefeπed are fragments characteπzed by structural or functional attributes such as fragments that compπse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, high antigenic index regions or soluble forms of the receptor. Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate receptor activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or lmmunogenic in an animal, especially in a human.

Preferably, all of these polypeptide fragments retain the biological activity of the receptor, including antigenic activity. Among the most preferred fragment is that having the ammo acid sequence of SEQ ID NO. 4. Vaπants of the defined sequence and fragments also form part of the present invention Preferred vaπants are those that vary from the referents by conservative ammo acid substitutions - i.e., those that substitute a residue with another of like characteπstics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly prefeπed are vaπants in which several, 5-10, 1-5, or 1-2 am o acids are substituted, deleted, or added in any combination.

The TR6 polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurπng polypeptides, recombmantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for prepaπng such polypeptides are well understood in the art.

Polynucleotides of the Invention

Another aspect of the invention relates to TR6 polynucleotides. TR6 polynucleotides include isolated polynucleotides which encode the TR6 polypeptides and fragments, and polynucleotides closely related thereto. More specifically, TR6 polynucleotide of the invention include a polynucleotide compnsmg the nucleotide sequence set forth in SEQ ID NO: 1 encoding a TR6 polypeptide of SEQ ID NO: 2, and polynucleotides having the particular sequences of SEQ ID NOS: 1 and 3. TR6 polynucleotides further include a polynucleotide compnsmg a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the TR6 polypeptide of SEQ ID NO:2 over its entire length, and a polynucleotide that is at least 80% identical to that having SEQ ID NO:l over its entire length. In this regard, polynucleotides at least 90% identical are particularly preferred, and those with at least 95%> are especially preferred. Furthermore, those with at least 97% are highly prefeπed and those with at least 98-99% are most highly prefeπed, with at least 99% being the most prefeπed. Also included under TR6 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained m SEQ ID NO.1 to hybridize under conditions useable for amplification or for use as a probe or marker. The invention also provides polynucleotides which are complementary to such TR6 polynucleotides.

TR6 of the invention is structurally related to other proteins of the Tumor Necrosis Factor Receptor family, as shown by the results of sequencing the cDNA encodmg human TR6. The cDNA sequence of SEQ ID NO:l contains an open reading frame (nucleotide numbers 94 to 1329) encoding a polypeptide of 411 ammo acids of SEQ ID NO:2. The ammo acid sequence of Table 2 (SEQ ID NO:2) has about 58% identity (using GAP (From GCG )) in 411 ammo acid residues with DR4, the receptor for the hgand TRAIL. (Pan,G., 0'Rourke,K., Chmnaιyan,A.M., Gentz,R., Ebner,R., Nι,J. and DixitN.M., Science 276, 111-113 (1997)). The nucleotide sequence of Table 1 (SEQ ID ΝO:l) has about 70% identity (using GAP (from GCG)) in 1335 nucleotide residues with DR4, the receptor for the hgand TRAIL. TR6 contains a death domain (ammo acids 290 to 324 in SEQ ID NO:2) which is 64% identical to the death domain of the human Death receptor 4 (DR4) (Pan,G., 0'Rourke,K., Chιnnaιyan,A.M., Gentz,R., Ebner,R., Nι,J. and DixitN.M., Science 276, 111-113 (1997)), 35.7% identical to the death domain of the human Death receptor 3 (DR3) (A.M. Chinnaiyan, et al, Science 274 (5289), 990-992 (1996)), 32.7% identical to the death domain of human TNFRl, and 19.6% identical to the death domain of CD95 (Fas) (I. Cascino, J. Immunol. 154 (6), 2706-2713 (1995)).

Table T

1 CTTTGCGCCC ACAAAATACA CCGACGATGC CCGATCTACT TTAAGGGCTG

51 AAACCCACGG GCCTGAGAGA CTATAAGAGC GTTCCCTACC GCCATGGAAC 101 AACGGGGACA GAACGCCCCG GCCGCTTCGG GGGCCCGGAA AAGGCACGGC

151 CCAGGACCCA GGGAGGCGCG GGGAGCCAGG CCTGGGCCCC GGGTCCCCAA

201 GACCCTTGTG CTCGTTGTCG CCGCGGTCCT GCTGTTGGTC TCAGCTGAGT

251 CTGCTCTGAT CACCCAACAA GACCTAGCTC CCCAGCAGAG AGCGGCCCCA

301 CAACAAAAGA GGTCCAGCCC CTCAGAGGGA TTGTGTCCAc cTGGACACCA 351 TATCTCAGAA GACGGTAGAG ATTGCATCTC CTGCAAATAT gGACAGGACT

401 ATAGCACTCA aTGGAATGAC CTCCTTTTCT GCTTGCGCTG CACCAGGTGT

451 GATTCAGGTG AAGTGGAGCT AAGTCCCTGC ACCACGACCA GAAACACAGT

501 GTGTCAGTGC GAAGAAgGCA CCTTCCGGGA AGAAGATTCT CCTGAGATGT

551 GCCGGAAGTG CCGCACAGGG TGTCCCAgAG GGATGGTCAA GGTCGGTGAT 601 TGTACACCCT GGAGTGACAT CGAATGTGTC CACAAAGAAT CAGGCATCAT

651 CATAgGAGTC ACAGTTGCAG CCGTAGTCTT GATTGTGGCT GTGTTTGTTT

701 GCaAgTCTTT ACTGTGGAAg AAAGTCCTTC CTTACCTGAA AGGCATCTGC

751 TCAGGTGGTG GTGGGGACCC TGAGCGTGTG GACAGAAGcT CACAACGACc

801 TGGGGCTGAG GACAATGTCC TCAATGAGAT CGTGAGTATC TTGCAGCCCA 851 CCCAGGTCCC TGAGCAGGAA ATGGAAGTCC AGGAGCCAGC AGAGCCAACA

901 GGTGTCAACA TGTTGTCCCC CGGGGAGTCA GAGCATCTGC TGGAACCGGC

951 AGAAGCTGAA AGGTCTCAGA GGAGGAGGCT GCTGGTTCCA GCAAATGAAG

1001 GTGATCCCAC TGAGACTCTG AGACAGTGCT TCGATGACTT TGCAGACTTG

1051 GTGCCCTTTG ACTCCTGGGA gCCgCTCATG AGGAAGTTGG GCCTCATGGA 1101 CAATgAGATa aaGGTGGCTA AAGCTGAGGC AGCGGGCCAC AGGGACACCT

1151 TGTACACGAT GCTGATAAAG TGGGTCAACA AAACCGGGCG AGATGCCTCT

1201 GTCCACACCC TGCTGGATGC CTTGGAGACG CTGGGAGAGA GACTTGCCAA

1251 GCAGAAGATT GAGGACCACT TGTTGAGCTC TGGAAAGTTC ATGTATCTAG

1301 AAGGTAATGC AGACTCTGCC ATGTCCTAAG TGTGATTCTC TTCAGGAAGT 1351 CAGACCTTCC CTGGTTTACC TTTTTTCTGG AAAAAGCCCA ACTGGACTCC

1401 AGTCAGTAGG AAAGTGCCAC AATTGTCACA TGACCGGTAC TGGAAGAAAC

1451 TCTCCCATCC AACATCACCC AGTGGATGGA ACATCCTGTA ACTTTTCACT

1501 GCACTTGGCA TTATTTTTAT AAGCTGAATG TGATAATAAG GACACTATGG 1551 AAATGTCTGG ATCATTCCGT TTGTGCGTAC TTTGAgATTT GGTTTGGGAT

1601 GTCATTGTTT TCACAGCACT TTTTTATCCT AATGTAAATG CTTTATTTAT

1651 TTATTTGGGC TACATTGTAA gATCCATCTA CACAGTCGTT GTCCGACTTC

1701 ACTTGATACT ATATGATATG AACCTTTTTT GGGTGGGGGG TGCGGGGCAg 1751 TTCACTCTGT CTCCCAGGCT GGAGTGCAAT GGTGCAATCT TGGCTCACTA

1801 TAGCCTTGAC CTCTCAGGCT CAAGCGATTC TCCCACCTCA GCCATCCAAA

1851 TAGCTGGGAC CACAGGTGTG CACCACCACG CCCGGCTAAT TTTTTGTATT

1901 TTGTCTAgAT ATAGGGGCTC TCTATGTTGC TCAGGGTGGT CTCgAATTCC

1951 TGGAcTCAAG CAGTCTGCCC ACcTCAGAcT CCCAAAGCGG TGGAATTAGA 2001 GGCGTGAGCC CCCATGcTTG gCCTTACcTT TcTACTTTTA TAATTCTGTA

2051 TGTTATTATT TTATGAACAT GAAGAAACTT TAGTAAATGT ACTTGTTTAC

2101 ATAGTTATGT GAATAGATTA GATAAACATA AAAGGAGGAG ACATACAATG

2151 GGGGAAGAAG AAGAAGTCCC CTGTAAGATG TCACTGTcTG GGTTCCAGCC

2201 CTCCCTCAGA TGTACTTTGG CTTCAATGAT TGGCAACTTC TACAGGGGCC 2251 AGTCTTTTGA ACTGGACAAC CTTACAAGTA TATGAGTATT ATTTATAGGT

2301 AGTTGTTTAC ATATGAGTCG GGACCAAAGA GAACTGGATC CACGTGAAGT

2351 CCTGTGTGTG GCTGGTCCCT ACCTGGGCAG TcTCATTTGC ACCCATAGCC

2401 CCCATCTATG GACAGGCTGG GACAGAGGCA GATGGGTTAG ATCACACATA 2451 ACAATAGGGT CTATGTCATA TCCCAAGTGA ACTTGAGCCC TGTTTGGGCT 2501 CAGGAGATAG AAGACAAAAT CTGTCTCCCC ACGTCTGCCA TGGCATCAAG

2551 GGGGAAGAGT AGATGGTGCT tGAGAATGGT GTGAAATGGT TGCCATCTCA

2601 GGAGTAGATG GCCCGGCTCA CTTCTGGTTA TCtGTCACCC TGAGCCCAtG

2651 AGCTGCcTTT TAGGGTACAG ATTGCCTACT TGAGGACCTT GGCCGCTCTG

2701 TAAGCATCTG ACTCATCTCA GAAATGTCAA TTCTTAAACA CTGTGGCAAC 2751 AGGACCTAGA ATGGCTGACG CATTAAGGTT TTCTTcTTGT GTCCTGTTCT

2801 ATTAtTGTTT TAAGACCTCA GTAACCATTT CAGCCTCTTT CCAGCAAACC

2851 CTTCTCCATA GTATTTCAGT CATGGAAGGA TCATTTATGC AGGTAGTCAT

2901 TCCAGGAGTT TTTGGTCTTT TCTGTCTCAA GGCATTGTGT GTTTTGTTCC

2951 GGGACTGGTT TGGGTGGGAC AAAGTTAGAA TTGCCTGAAG ATcAcACATT 3001 CAGACTGTtG TGTCTGTGGA GTTTTAGGAG TGGGGGGTGA CCTTTcTGGT

3051 CTTtGcAcTT CCATCcTcTC CCAcTTCCAT cTGGCATCCC CACGcGTTGT 3101 CCCcTGCAcT TcTGGAAGGC ACAGGGTGCT GCTGCTTCCT GGTCTTTGCC

3151 TTTGCTGGGC cTTCTGTGCA GGACGCTCAG CCTCAGGGCT CAGAAGGTGC

3201 CAGTCCGGTC CCAGGTCCCT TGTCCCTTCC ACAGAGGCCT TCcTAGAAGA 3251 TGCATCTAGA GTGTCAGCCT TATCAGTGTT TAAGATTTTT CTTTTATTTT

3301 TAATTTTTTT GAGACAGAAT CTCACTCTCT CGCCCAGGCT GGAGTGCAAC

3351 GGTACGATCT TGGCTCAGTG CAACCTCCGC CTCCTGGGTT CAAGCGATTC 3401 TCGTGCCTCA GCCTCCGGAG TAGCTGGGAT TGCAGGCACC CGCCACCACG 3451 CCTGGCTAAT TTTTGTATTT TTAGTAGAGA CGGGGTTTCA CCATGTTGGT

3501 CAGGCTGGTC TCGAACTCCT GACCTCAGGT GATCCACNTT GGCCTCCGAA

3551 AGTGCTGGGa tatacaaggc GTGAGCCACC AGCCAGGCCA AGATATTNTT 3601 NTAAAGNNAG CTTCCGGANG ACATGAAATA ANGGGGGGTT TTGTTGTTTA

3651 GTAACATTNG GCTTTGATAT ATCCCCAGGC CAAATNGCAN GNGACACAGG

3701 ACAGCCATAG TATAGTGTGT CACTCGTGGT TGGTGTCCTT TCATGGTTcT

3751 GCCCTGTCAA AGGTCCCTAT TTGAAATGTG TTATAATACA AACAAGGAAG 3801 CACATTGTGT ACAAAATACT TATGTATTTA TGAATCCATG ACCAAATTAA 3851 ATATGAAACC TTATATAAAA AAAAAAAAAA A ^a A nucleotide sequence of a human TR6. (SEQ ID NO: 1).

Table 2^b

1 Met Glu Gin Arg Gly Gin Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys 16

17 Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Pro 32

33 Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 48 49 Val Ser Ala Glu Ser Ala Leu lie Thr Gin Gin Asp Leu Ala Pro Gin 64

65 Gin Arg Ala Ala Pro Gin Gin Lys Arg Ser Ser Pro Ser Glu Gly Leu 80

81 Cys Pro Pro Gly His His lie Ser Glu Asp Gly Arg Asp Cys lie Ser 96

97 Cys Lys Tyr Gly Gin Asp Tyr Ser Thr Gin Trp Asn Asp Leu Leu Phe 112

113 Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 128 129 Cys Thr Thr Thr Arg Asn Thr Val Cys Gin Cys Glu Glu Gly Thr Phe 144

145 Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys 160

161 Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp lie 176

177 Glu Cys Val His Lys Glu Ser Gly He He He Gly Val Thr Val Ala 192

193 Ala Val Val Leu He Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp 208 209 Lys Lys Val Leu Pro Tyr Leu Lys Gly He Cys Ser Gly Gly Gly Gly 224

225 Asp Pro Glu Arg Val Asp Arg Ser Ser Gin Arg Pro Gly Ala Glu Asp 240

241 Asn Val Leu Asn Glu He Val Ser He Leu Gin Pro Thr Gin Val Pro 256

257 Glu Gin Glu Met Glu Val Gin Glu Pro Ala Glu Pro Thr Gly Val Asn 272

273 Met Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala 288 289 Glu Arg Ser Gin Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp 304

305 Pro Thr Glu Thr Leu Arg Gin Cys Phe Asp Asp Phe Ala Asp Leu Val 320 321 Pro Phe Asp Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp 336

337 Asn Glu He Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr 352

353 Leu Tyr Thr Met Leu He Lys Trp Val Asn Lys Thr Gly Arg Asp Ala 368

369 Ser Val His Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu 384 385 Ala Lys Gin Lys He Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met 400

401 Tyr Leu Glu Gly Asn Ala Asp Ser Ala Met Ser End 411 ^b An ammo acid sequence of a human TR6. (SEQ ID NO: 2).

One polynucleotide of the present invention encoding TR6 may be obtained using standard cloning and screening, from a cDNA library deπved from m-RNA in cells of human thymus stromal cells, monocytes, peπpheral blood lymphocytes, pπmary dendπtic, and bone maπow cells using the expressed sequence tag (EST) analysis (Adams, M.D., et al Science (1991) 252: 1651-1656; Adams, M.D. et al , Nature, (1992) 355:632-634; Adams, M.D., et al, Nature (1995) 377 Supp:3-174).

Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbraπes or can be synthesized using well known and commercially available techniques.

The nucleotide sequence encoding TR6 polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 94 to 1329 of SEQ ID NO: 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.

When the polynucleotides of the invention are used for the recombinant production of TR6 polypeptide, the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which facilitates puπfication of the fused polypeptide can be encoded. In certain prefeπed embodiments of this aspect of the invention, the marker sequence is a hexa-histidme peptide, as provided in the pQE vector (Qiagen, Inc.) and descπbed in Gentz et al. , Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag The polynucleotide may also contain non-codmg 5 ' and 3 ' sequences, such as transcπbed, non-translated sequences, splicing and polyadenylation signals, πbosome binding sites and sequences that stabilize mRNA.

Further prefeπed embodiments are polynucleotides encoding TR6 vaπants compnsmg the ammo acid sequence of TR6 polypeptide of Table 1 (SEQ ID NO:2) m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acid residues are substituted, deleted or added, in any combmation. Among the prefeπed polynucleotides of the present invention is contamed in Table 3 (SEQ ID NO: 3) encoding the amino acid sequence of Table 4 (SEQ ID NO: 4).

Table 3^C

1 ATGACCTCCT TTTCTGCTTG CGCTGCACCA GGTGTGATTC AGGTGAAGTG

51 GAGCTAAGTC CCTGCACCAC GACCAGAAAC ACAGTGTGTC AGTGCGAAGA 101 AgGCACCTTC CGGGAAGAAG ATTCTCCTGA GATGTGCCGG AAGTGCCGCA

151 CAGGGTGTCC CAgAGGGATG GTCAAGGTCG GTGATTGTAC ACCCTGGAGT

201 GACATCGAAT GTGTCCACAA AGAATCAGGC ATCATCATAg GAGTCACAGT

251 TGCAGCCGTA GTCTTGATTG TGGCTGTGTT TGTTTGCaAg TCTTTACTGT

301 GGAAgAAAGT CCTTCCTTAC CTGAAAGGCA TCTGCTCAGG TGGTGGTGGG 351 GACCCTGAGC GTGTGGACAG AAGcTCACAA CGACcTGGGG CTGAGGACAA

401 TGTCCTCAAT GAGATCGTGA GTATCTTGCA GCCCACCCAG GTCCCTGAGC

451 AGGAAATGGA AGTCCAGGAG CCAGCAGAGC CAACAGGTGT CAACATGTTG

501 TCCCCCGGGG AGTCAGAGCA TCTGCTGGAA CCGGCAGAAG CTGAAAGGTC

551 TCAGAGGAGG AGGCTGCTGG TTCCAGCAAA TGAAGGTGAT CCCACTGAGA 601 CTCTGAGACA GTGCTTCGAT GACTTTGCAG ACTTGGTGCC CTTTGACTCC

651 TGGGAgCCgC TCATGAGGAA GTTGGGCCTC ATGGACAATg AGATaaaGGT

701 GGCTAAAGCT GAGGCAGCGG GCCACAGGGA CACCTTGTAC ACGATGCTGA

751 TAAAGTGGGT CAACAAAACC GGGCGAGATG CCTCTGTCCA CACCCTGCTG

801 GATGCCTTGG AGACGCTGGG AGAGAGACTT GCCAAGCAGA AGATTGAGGA 851 CCACTTGTTG AGCTCTGGAA AGTTCATGTA TCTAGAAGGT AATGCAGACT

901 CTGCCATGTC CTAAGTGTGA TTCTCTTCAG GAAGTCAGAC CTTCCCTGGT

951 TTACCTTTTT TCTGGAAAAA GCCCAACTGG ACTCCAGTCA GTAGGAAAGT

1001 GCCACAATTG TCACATGACC GGTACTGGAA GAAACTCTCC CATCCAACAT

1051 CACCCAGTGG AT

A partial nucleotide sequence of a human TR6. (SEQ ID NO: 3).

Table 4^d

1 DLLFCLRCTR CDSGEVELSP CTTTRNTVCQ CEEGTFREED SPEMCRKCRT 51 GCPRGMVKVG DCTP SDIEC VHKESGIIIG VTVAAWLIV AVFVCKSLL 101 KKVLPYLKGI CSGGGGDPER VDRSSQRPGA EDNVLNEIVS ILQPTQVPEQ

151 EMEVQEPAEP TGVNMLSPGE SEHLLEPAEA ERSQRRRLLV PANEGDPTET

201 LRQCFDDFAD LVPFDSWEPL MRKLGLMDNE IKVAKAEAAG HRDTLYTMLI

251 KWVNKTGRDA SVHTLLDALE TLGERLAKQK IEDHLLSΞGK FMYLEGNADS

301 AMS* ^d A partial ammo acid sequence of a human TR6. (SEQ ID NO: 4).

Table 5 (SEQ ID NO: 7

1 MEQRGQNAPA ASGARKRHGP GPREARGARP GPRVPKTLVL WAAVLLLVS 51 AESALITQQD LAPQQRAAPQ QKRSSPSEGL CPPGHHISED GRDCISCKYG

101 QDYSTQWND LFC RCTRCD SGEVELSPCT TTRNTVCQCE EGTFREEDSP

151 EMCRKCRTGC PRG VKVGDC TPWSDIECVH KESGRSJ-EG-R GTEPKΞADKT

201 HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCW VDVSHEDPEV

251 KFN YVDGVE VHNAKTKPRE EQYNSTYRW SVLTVLHQD LNGKEYKCKV 301 SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV SLTCLVKGFY

351 PSDIAVE ES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF

401 SCSVMHEALH NHYTQKSLSL SPGK*

The present invention further relates to polynucleotides that hybπdize to the herein above- descπbed sequences. In this regard, the present invention especially relates to polynucleotides which hybπdize under stnngent conditions to the herein above-descπbed polynucleotides. As herein used, the term "stπngent conditions" means hybπdization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.

Polynucleotides of the invention, which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 or a fragment thereof, including that of SEQ ID NO:3, may be used as hybπdization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding TR6 and to isolate cDNA and genomic clones of other genes that have a high sequence similaπty to the TR6 gene. Such hybπdization techniques are known to those of skill m the art.

Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent. The probes generally will compnse at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly prefeπed probes will range between 30 and 50 nucleotides In one embodiment, to obtain a polynucleotide encoding TR6 polypeptide compnses the steps of screening an appropπate library under stπngent hybndization conditions with a labeled probe having the SEQ ID NO: 1 or a fragment thereof, including that of SEQ ID NO: 3, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybndization techniques are well known to those of skill in the art. Thus m another aspect, TR6 polynucleotides of the present invention further include a nucleotide sequence compnsmg a nucleotide sequence that hybπdize under stnngent condition to a nucleotide sequence having SEQ ID NO: 1 or a fragment thereof, including that of SEQ ID NO:3. Also included with TR6 polypeptides are polypeptide compnsmg ammo acid sequence encoded by nucleotide sequence obtained by the above hybndization condition. Stnngent hybndization conditions are as defined above or alternatively conditions under overnight incubation at

42°C m a solution compnsmg: 50% formamide, 5xSSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. lx SSC at about 65°C

The polynucleotides and polypeptides of the present invention may be employed as research reagents and mateπals for discovery of treatments and diagnostics to animal and human disease.

Vectors, Host Cells, Expression

The present invention also relates to vectors which compπse a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques. Cell- free translation systems can also be employed to produce such proteins using RNAs denved from the

DNA constructs of the present invention.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells can be effected by methods descπbed in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING. A LABORATORY MANUAL, 2nd Ed., Cold Spπng Harbor Laboratory Press, Cold Spnng Harbor, N . (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, catio c hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection. Representative examples of appropπate hosts include bactenal cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs cells; fungal cells, such as yeast cells and Aspergύlus cells; insect cells such as Drosophila S2 and Spodoptera Sf cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells. A great vanety of expression systems can be used. Such systems include, among others, chromosomal, episomal and virus-deπved systems, e.g., vectors denved from bacteπal plasrmds, from bacteπophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors denved from combinations thereof, such as those denved from plasmid and bacteπophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide m a host may be used. The appropnate nucleotide sequence may be inserted into an expression system by any of a vaπety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the penplasmic space or into the extracellular environment, appropnate secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.

If the TR6 polypeptide is to be expressed for use in screening assays, generally, it is prefeπed that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use m the screening assay. If TR6 polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced mtracellularly, the cells must first be lysed before the polypeptide is recovered

TR6 polypeptides can be recovered and puπfied from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for puπfication. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured dunng isolation and or puπfication.

Diagnostic Assays This invention also relates to the use of TR6 polynucleotides for use as diagnostic reagents.

Detection of a mutated form of TR6 gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under- expression, over-expression or altered expression of TR6. Individuals carrying mutations m the TR6 gene may be detected at the DNA level by a vanety of techniques Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, unne, saliva, tissue biopsy or autopsy matenal. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques pπor to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in companson to the normal genotype. Point mutations can be identified by hybπdizmg amplified DNA to labeled TR6 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denatuπng agents, or by direct DNA sequencing. See, e.g., Myers et al , Science (1985) 230:1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method. See Cotton et al , Proc NatlAcadSci USA (1985) 85 4397-4401. In another embodiment, an aπay of o gonucleotides probes comprising TR6 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Aπay technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining a susceptibility to chronic and acute inflammation, arthπtis (including rheumatoid arthπtis), septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, asthma, restenosis, brain injury, AIDS, Bone diseases, cancer (e.g lymphoproliferative disorders), atheroschlerosis, and Alzheimers disease, among others, through detection of mutation in the TR6 gene by the methods descπbed.

In addition, chronic and acute inflammation, arthπtis (including rheumatoid arthπtis), septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, asthma, restenosis, brain injury, AIDS, Bone diseases, cancer (e.g. lymphoproliferative disorders), atheroschlerosis, and Alzheimers disease, among others., can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of TR6 polypeptide or TR6 mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known m the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as an TR6, in a sample denved from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive- binding assays, Western Blot analysis and ELISA assays.

Chromosome Assays

The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybπdize with a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step in coπelating those sequences with gene associated disease Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be coπelated with genetic map data. Such data are found, for example, in V. McKusick, Mendehan Inheπtance in Man (available on line through Johns Hopkins

University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinhentance of physically adjacent genes) The differences m the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

The 3' untranslated region of TR6 matches the 295 bp nucleotide sequence of a mapped EST (Genbank ID: D20151). This EST has been mapped by the Whitehead Institute to chromosome 8, 97.68 cR from the top of the Chromosome 8 linkage group Antibodies

The polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies lmmunospecific for the TR6 polypeptides The term "lmmunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the pnor art. Antibodies generated against the TR6 polypeptides can be obtained by admmistenng the polypeptides or epitope-beaπng fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybndoma technique (Kohler, G. and Milstem, C , Nature (1975) 256:495-497), the tπoma technique, the human B-cell hybndoma technique (Kozbor et al , Immunology Today (1983) 4:72) and the EBV-hybπdoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Ine , 1985) Techmques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms including other mammals, may be used to express humanized antibodies.

The above-descnbed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to puπfy the polypeptides by affinity chromatography.

Antibodies against TR6 polypeptides may also be employed to treat chronic and acute inflammation, arthπtis (including rheumatoid arthntis), septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psonasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, asthma, restenosis, brain injury, AIDS, Bone diseases, cancer (e.g. lymphoproliferative disorders), atheroschlerosis, and Alzheimers disease, among others. , among others.

Vaccines

Another aspect of the invention relates to a method for inducing an m munological response in a mammal which comprises inoculating the mammal with TR6 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from chronic and acute inflammation, arthntis (including rheumatoid arthπtis), septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, asthma, restenosis, brain injury, AIDS, Bone diseases, cancer (e.g. lymphoproliferative disorders), atheroschlerosis, and Alzheimers disease, , among others Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering TR6 polypeptide via a vector directing expression of TR6 polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.

Further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a TR6 polypeptide wherem the composition comprises a TR6 polypeptide or TR6 gene. The vaccine formulation may further comprise a suitable earner. Since TR6 polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, mtradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous stenle injection solutions which may contain anti- oxidants, buffers, bacteπostats and solutes which render the formulation lnstomc with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dπed condition requiπng only the addition of the stenle liquid earner immediately pnor to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil- water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine expeπmentation.

Screening Assays

We have now discovered that TL2 of SEQ ID NO: 6 (otherwise known as TRAIL, Immunity (6):673-682 (1995)) is a hgand of TR6. Thus, the TR6 polypeptide of the present invention, and one of its hgands, TL2 may be employed in a screening process for compounds which bmd the receptor, or its hgand, and which activate (agonists) or inhibit activation of (antagonists) the receptor polypeptide of the present invention, or its hgand TL2. Thus, polypeptides of the invention may be used to assess the binding of small molecule substrates and hgands in, for example, cells, cell-free preparations, chemical braπes, and natural product mixtures. These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics. See Coligan et al , Current Protocols in Immunology l(2):Chapter 5 (1991).

TR6 polypeptides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate TR6 on the one hand and which can inhibit the function of TR6 or remove TR6 expressing cells on the other hand. Antagonists, or agents which remove TR6 expressing cells, may be employed for a vanety of therapeutic and prophylactic purposes for such conditions as chronic and acute inflammation, arthntis (including rheumatoid arthntis), septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psonasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, asthma, restenosis, bram injury, AIDS, Bone diseases, cancer (e.g. lymphoproliferative disorders), atheroschlerosis, and Alzheimers disease, among others. Agonists can be employed for therapeutic and prophylactic purposes for such conditions responsive to activation of T cells and other components of the immune system, such as for treatment of cancer and AIDS. However, agonists can also be employed for mappropnate stimulation of T cells and other components of the immune system which leads to down modulation of immune activity with therapeutic or prophylactic application for conditions such , as chronic and acute inflammation, arthntis (including rheumatoid arthπtis), septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psoπasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, asthma, restenosis, bram injury, AIDS, Bone diseases, cancer (e.g. lymphoproliferative disorders), atheroschlerosis, and Alzheimers disease, among others. Candidate compounds may be identified using assays to detect compounds which inhibit binding of TL2 to TR6 in either cell-free or cell based assays. Suitable cell- free assays may be readily determined by one of skill in the art. For example, an ELISA format may be used in which puπfied TR6, or a punfied deπvative of TR6, containing the extracellular domain of TR6, is immobilized on a suitable surface, either directly or indirectly (e.g., via an antibody to TR6) and candidate compounds are identified by their ability to block binding of puπfied TL2 to TR6. The binding of TL2 to TR6 could be detected by using a label directly or indirectly associated with TL2.

Suitable detection systems include the streptavidin horseradish peroxidase conjugate, or direct conjugation by a tag, e.g., fluorescem. Conversely, punfied TL2 may be immobilized on a suitable surface, and candidate compounds identified by their ability to block binding of punfied TR6 to TL2. The binding of TR6 to TL2 could be detected by using a label directly or indirectly associated with TR6. Many other assay formats are possible that use the TR6 protein and its hgands.

Suitable cell based assays may be readily determined by one of skill the art. In general, such screening procedures involve producing appropπate cells which express the receptor polypeptide of the present invention on the surface thereof. Such cells include cells from mammals, yeast, Drosophila or E coh. Cells expressing the receptor (or cell membrane containing the expressed receptor) are then contacted with a known hgand, such as TL2, or test compound to observe binding, or stimulation or inhibition of a functional response. The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the receptor is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor, such as the hgand TL2 Further, these assays may test whether the candidate compound results in a signal generated by activation of the receptor or its hgand (e.g. TL2)usιng detection systems appropriate to the cells bearing the receptor or its hgand and fusion proteins thereof at their surfaces. Typical fusion partners include fusing the extracellular domain of the receptor or hgand with the lntracellular tyrosine kmase domain of a second receptor. Inhibitors of activation are generally assayed in the presence of a known agonist, such as the hgand TL2, and the effect on activation by the agonist by the presence of the candidate compound is observed. Standard methods for conducting such screening assays are well understood in the art.

Examples of potential TR6 antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the hgand of the TR6, e.g., a fragment of the hgand TL2, or small molecules which bind to the receptor, or its hgand, but do not elicit a response, so that the activity of the receptor is prevented. Examples of potential TR6 agonists include antibodies that bind to TR6, its hgand, such as TL2, or denvatives thereof, and small molecules that bmd to TR6. These agonists will elicit a response mimicking all or part of the response induced by contacting the native hgand. The nucleotide sequence of TL2 (SEQ ID NO: 5) (published by Immunex Research and Development Corporation, Seattle, Washington as TNF-related apoptosis-mducmg hgand (TRAIL) TWiley SR, et al. Immunity (6):673-682 (1995)) is as follows.

1 CCTCACTGAC TATAAAAGAA TAGAGAAGGA AGGGCTTCAG TGACCGGCTG

51 CCTGGCTGAC TTACAGCAGT CAGACTCTGA CAGGATCATG GCTATGATGG

101 AGGTCCAGGG GGGACCCAGC CTGGGACAGA CCTGCGTGCT GATCGTGATC

151 TTCACAGTGC TCCTGCAGTC TCTCTGTGTG GCTGTAACTT ACGTGTACTT

201 TACCAACGAG CTGAAGCAGA TGCAGGACAA GTACTCCAAA AGTGGCATTG 251 CTTGTTTCTT AAAAGAAGAT GACAGTTATT GGGACCCCAA TGACGAAGAG

301 AGTATGAACA GCCCCTGCTG GCAAGTCAAG TGGCAACTCC GTCAGCTCGT

351 TAGAAAGATG ATTTTGAGAA CCTCTGAGGA AACCATTTCT ACAGTTCAAG

401 AAAAGCAACA AAATATTTCT CCCCTAGTGA GAGAAAGAGG TCCTCAGAGA

451 GTAGCAGCTC ACATAACTGG GACCAGAGGA AGAAGCAACA CATTGTCTTC 501 TCCAAACTCC AAGAATGAAA AGGCTCTGGG CCGCAAAATA AACTCCTGGG

551 AATCATCAAG GAGTGGGCAT TCATTCCTGA GCAACTTGCA CTTGAGGAAT

601 GGTGAACTGG TCATCCATGA AAAAGGGTTT TACTACATCT ATTCCCAAAC

651 ATACTTTCGA TTTCAGGAGG AAATAAAAGA AAACACAAAG AACGACAAAC

701 AAATGGTCCA ATATATTTAC AAATACACAA GTTATCCTGA CCCTATATTG 751 TTGATGAAAA GTGCTAGAAA TAGTTGTTGG TCTAAAGATG CAGAATATGG

801 ACTCTATTCC ATCTATCAAG GGGGAATATT TGAGCTTAAG GAAAATGACA

851 GAATTTTTGT TTCTGTAACA AATGAGCACT TGATAGACAT GGACCATGAA

901 GCCAGTTTTT TCGGGGCCTT TTTAGTTGGC TAACTGACCT GGAAAGAAAA

951 AGCAATAACC TCAAAGTGAC TATTCAGTTT TCAGGATGAT ACACTATGAA 1001 GATGTTTCAA AAAATCTGAC CAAAACAAAC AAACAGAAAA CAGAAAACAA

1051 AAAAACCTCT ATGCAATCTG AGTAGAGCAG CCACAACCAA AAAATTCTAC

1101 AACACACACT GTTCTGAAAG TGACTCACTT ATCCCAAGAA AATGAAATTG

1151 CTGAAAGATC TTTCAGGACT CTACCTCATA TCAGTTTGCT AGCAGAAATC

1201 TAGAAGACTG TCAGCTTCCA AACATTAATG CAATGGTTAA CATCTTCTGT 1251 CTTTATAATC TACTCCTTGT AAAGACTGTA GAAGAAAGCG CAACAATCCA

1301 TCTCTCAAGT AGTGTATCAC AGTAGTAGCC TCCAGGTTTC CTTAAGGGAC

1351 AACATCCTTA AGTCAAAAGA GAGAAGAGGC ACCACTAAAA GATCGCAGTT

1401 TGCCTGGTGC AGTGGCTCAC ACCTGTAATC CCAACATTTT GGGAACCCAA 1451 GGTGGGTAGA TCACGAGATC AAGAGATCAA GACCATAGTG ACCAACATAG

1501 TGAAACCCCA TCTCTACTGA AAGTGCAAAA ATTAGCTGGG TGTGTTGGCA

1551 CATGCCTGTA GTCCCAGCTA CTTGAGAGGC TGAGGCAGGA GAATCGTTTG

1601 AACCCGGGAG GCAGAGGTTG CAGTGTGGTG AGATCATGCC ACTACACTCC

1651 AGCCTGGCGA CAGAGCGAGA CTTGGTTTCA AAAAAAAAAA AAAAAAAAAA

1701 CTTCAGTAAG TACGTGTTAT TTTTTTCAAT AAAATTCTAT TACAGTATGT

1751 CAAAAAAAAA AAAAAAAAA

The ammo acid sequence of TL2 (SEQ ID NO: 6) (published by Immunex Research and Development Corporation, Seattle, Washington as TNF-related apoptosis-inducmg hgand (TRAIL)

TWiley SR, et al. Immunity (6):673-682 (1995)) is as follows:

1 Met Ala Met Met Glu Val Gin Gly Gly Pro Ser Leu Gly Gin Thr Cys 16 17 Val Leu He Val He Phe Thr Val Leu Leu Gin Ser Leu Cys Val Ala 32

33 Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gin Met Gin Asp Lys 48

49 Tyr Ser Lys Ser Gly He Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 64

65 Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gin Val 80

81 Lys Trp Gin Leu Arg Gin Leu Val Arg Lys Met He Leu Arg Thr Ser 96 97 Glu Glu Thr He Ser Thr Val Gin Glu Lys Gin Gin Asn He Ser Pro 112

113 Leu Val Arg Glu Arg Gly Pro Gin Arg Val Ala Ala His He Thr Gly 128

129 Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu 144

145 Lys Ala Leu Gly Arg Lys He Asn Ser Trp Glu Ser Ser Arg Ser Gly 160

161 His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val He 176 177 His Glu Lys Gly Phe Tyr Tyr He Tyr Ser Gin Thr Tyr Phe Arg Phe 192

193 Gin Glu Glu He Lys Glu Asn Thr Lys Asn Asp Lys Gin Met Val Gin 208

209 Tyr He Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro He Leu Leu Met Lys 224

225 Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr 240

241 Ser He Tyr Gin Gly Gly He Phe Glu Leu Lys Glu Asn Asp Arg He 256 257 Phe Val Ser Val Thr Asn Glu His Leu He Asp Met Asp His Glu Ala 272

273 Ser Phe Phe Gly Ala Phe Leu Val Gly End 281

Prophylactic and Therapeutic Methods This invention provides methods of treating abnormal conditions such as, chronic and acute inflammation, arthntis, septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psonasis, rheumatoid arthntis), transplant rejection, graft vs. host disease, mfection, stroke, ischemia, congestive heart failure, restenosis, acute respiratory disease syndrome, asthma, restenosis, bram injury, AIDS, Bone diseases, cancer (e.g. lymphoproliferative disorders), atheroschlerosis, and Alzheimers disease, among others, related to both an excess of or insufficient amounts of TR6 activity. Another aspect of the invention relates to methods of using such TR6 polypeptides and polynucleotides for inhibiting angiogenesis and also inhibiting production of TNF-α and eicosanoids. thereof expressing a TR6 polypeptide. In one prefeπed embodiment, the present invention contemplates a method of inhibiting angiogenesis in an individual in need thereof comprising administering extracellular domain of TR6 or soluble TR6 fusion protein to the individual. In yet another embodiment, the present invention also relates to a method of lowenng the production of eicosanoid or TNF-α m an individual in need thereof comprising admmistenng extracellular domain of TR6 or soluble TR6 fusion protein to the individual.

If the activity of TR6 is in excess, several approaches are available. One approach compnses admmistenng to a subject an inhibitor compound (antagonist) as heremabove descnbed along with a pharmaceutically acceptable earner m an amount effective to inhibit activation by blocking binding of hgands to the TR6, or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of TR6 polypeptides still capable of binding the hgand in competition with endogenous TR6 may be administered. Typical embodiments of such competitors comprise fragments of the TR6 polypeptide. In still another approach, expression of the gene encoding endogenous TR6 can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Alternatively, ohgonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al , Nucleic Acids Res (1979) 6:3073; Cooney et al , Science (1988) 241:456; Dervan et al , Science (1991) 251:1360. These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression of TR6 and its activity, several approaches are also available. One approach comprises admmistenng to a subject a therapeutically effective amount of TR6 polypeptides or a compound which activates TR6, i.e., an agonist as descnbed above, in combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of TR6 by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct may then be isolated and mtroduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engmeenng cells in vivo and expression of the polypeptide in vivo. For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based

Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). Another approach is to administer a therapeutic amount of TR6 polypeptides m combination with a suitable pharmaceutical earner

Formulation and Administration Peptides, such as the soluble form of TR6 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical earner. Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient. Such earners include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof Formulation should suit the mode of administration, and is well withm the skill of the art. The invention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds Prefeπed forms of systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection Other injection routes, such as subcutaneous, intramuscular, or mtrapentoneal, can be used Alternative means for systemic administration include transmucosal and transdermal administration using penefrants such as bile salts or fusidic acids or other detergents In addition, if properly formulated in entenc or encapsulated formulations, oral administration may also be possible. Admmisfration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are m the range of 0 1-100 μg kg of subject Wide vanations m the needed dosage, however, are to be expected m view of the vanety of compounds available and the diffeπng efficiencies of vanous routes of administration For example, oral administration would be expected to require higher dosages than administration by intravenous injection Vanations in these dosage levels can be adjusted using standard empmcal routines for optimization, as is well understood in the art Polypeptides used in treatment can also be generated endogenously m the subject, in treatment modalities often refeπed to as "gene therapy" as descnbed above. Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.

Examples

The examples below are earned out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise descnbed in detail. The examples illustrate, but do not limit the invention. Example 1

Two ESTs (EST#1760054 and EST#1635744) with sequence similaπty to the human TNF receptor were discovered in a commercial EST database. Analysis of the two nucleotide sequences (3,466 bp and 2,641 bp respectively), revealed each was a partial sequence of the complete cDNA sequence, overlapping, with 100%> identity, 2,226 bp at the nucleotide level. Together, the two sequences encompassed the complete predicted cDNA sequence of 3,881 bp, and encoded an open reading frame for a novel member of the TNF receptor superfamily and named TR6. The predicted protein is 411 am o acids long with a hydrophobic membrane spanning region indicating that at least one form of TR6 is expressed as a membrane bound protein Compaπson of TR6 protein sequence, with other TNF receptor family proteins indicates that it has two of the cysteme-nch repeats characteπstic of the extracellular domains of this family, and an mtracellular death domain.

Northern blot of TR6.

Vanous tissues and cell lines were screened for mRNA expression by Northern blot.

RNA was prepared from cells and cell lines using Tπ-Reagent (Molecular Research Center Inc., Cincinnati, OH), run m denatuπng agarose gels (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spπng Harbor Lab Press, NY (1989)) and transfered to Zeta-probe nylon membrane (Biorad, Hercules, CA.) via vacuum blotting m 25mM NaOh for 90 mm. After neutralization for 5-10 minutes with IM tns-HCl, pH 7.5 contammg 3M NaCl, the blots were prehybndized with 50% formamide, 8% dextran sulfate, 6XSSPE, 0.1%SDS and lOOmg/ml of sheared and dentured salmon sperm DNA for at least 30 mm. At 42°C. cDNA probes were labeled with 32P-CTP by random pπmmg (Statagene, La Jolla, CA), bnefly denatured with 0.25M NaOH and added to the prehybπdization solution. After a further incubation for at least 24h at 42°C, the blots were washed in high stnngency conditions and exposed to X-ray film. Very high expression of TR6 RNA was detected m aortic endothehal cells. High expression was also detected in monocytes. Low expression was detected in bone maπow and CD4+ activated PBLs. Very low, but detectable levels of TR6 RNA was expressed in CD19+ PBLs, CD8+ PBLs (both activated and unstimulated), and unstimulated CD4+ PBLs. In hematopoietic cell lines, low levels of TR6 RNA was expressed in HL60 (promyelocyte),

KGla (promyeloblast) and KG1 (myeloblast) cell lines. Very low but detectable levels of TR6 RNA was expressed in U937 (monoblast) and THP-1 (monocyte) cell lines.

The major RNA form is 3.8 kb in size

Expression and TR6-Ig Fusion Protein Purification: TR6 was expressed as Fc chimera by fusing the N terminal ammo acids 1-184 (which includes leader sequence) to a Factor Xa protease cleavage site and the hmge Fc region of a human IgG-γl heavy cvhain in COSFcLink (S. Kumar et al, J. Biol. Chem. 270:27905-27913 (1995); K. Johanson et al. J. Biol. Chem. 270:9459-9471 (1995)). This protein was expressed by transfection into CHO cells. 29.5L of conditioned media from CHO cells expressing TR6-Ig (also described herein as

TR6-Fc) was applied to a 2.6 X 11cm (216ml) Protein A Sepahrose, fast flow column (Pharmacia) equilibrated in 20mM sodium phosphate, 150mM sodium chloride, pH 7 (PBS). The column was washed with PBS and eluted with lOOmM glycme, pH 2.5. The eluate (pH 3.3) was immediately adjusted to pH 7 with 2M Tns, pH 8 and dialyzed vs. PBS. 1.3g of TR6-Ig was recovered at -95% purity by SDS-PAGE gel.

N-terminal sequence of the final mature processed protein, after the cleavage of the leader sequence was expenmentally determined to be ALITQQDLAP ( SEQ ID NO: 8). The MW was determined to be 110,528 Da by MALDI Mass Spec. Consistent with this, TR6-Ig ran as the expected dimer in non-denaturmg conditions on SDS-PAGE and size exclusion chromatography. The endotoxm level was 5.6eU/mg (gel clot assay). Thus the biology of TR6-Ig refers to the biology of SEQ ID NO:7 polypeptide without the leader sequence.

In vitro biological activity of TR6-Ig.

The effect of TR6-Ig on induction cytokme and eicosanoid production by monocytes was evaluated Freshly isolated and punfied monocytes were stimulated with LPS and evaluated for the production TNF-α, IL-1 β, IL-6 and IL-8 and PGE2. TR6-Ig inhibits TNF-α and PGE production from LPS stimulated monocytes, slightly enhances IL-6 production in some donors, and has little effect on IL-1 β and IL-8 production. In vivo biological activity of TR6-Ig.

Single Dose Pharmacokinetics ofTR6-Fc in Rats (iv andsc)

With a view to evaluating the in vivo biological activities of TR6, the pharmacokinetics of TR6-Ig were studied in male Sprague-Dawley Rats following iv (1 mg/kg) or sc (3 mg/kg) administration. Rat plasma was assayed for TR6-Ig using a time-resolved fluorescence lmmunoassay method. In the assay, plasma TR6-Ig was captured on a microtiter plate with goat anti-human polyclonal antibody (IgG Fc specific) and the complex was detected with rabbit antι-TR6 polyclonal antibody. The lower limit of quantification of the assay was 1.0 ng/mL.

Following iv administration, TR6-Ig plasma concentrations declined in a bi-phasic manner. The majonty of the area under the plasma concentration versus time curve (88%) was associated with the secondary phase. This kmetically dominant phase was characterized by a half-hfe of 84 hrs.

Following sc administration (3 mg/kg), maximal plasma concentrations of approximately 19 ug/mL were observed 48 hours after dosing. Thereafter, TR6-Ig declined monoexponentially to one week when concentrations exceeded 9.6 ug/mL. Comparison of the exposure following iv and sc administration suggested TR6-Fc was well absorbed from the subcutaneous injection site into the systemic circulation.

TR6-Ig was evaluated in the mouse air-pouch granuloma model of inflammation and angiogenesis as described using the method based on Colville-Nash et al (J. Pharmacol. Exp. Therap., Vol: 274: 1463-1472) and also recently described in J. Jackson et al (J. Pharmacol. Exp.

Therap , Vol. 284: 687-692). Bnefly, granulomatous tissue was induced through the introduction of 3 ml of air into the dorsal subcutaneous tissue m anesthesized animals (day 0) followed one day later (day 1) by the injection of 0.5 ml of 0.1% croton oil m complete freundt's adjuvant. Animal were dosed with TR6-Ig on days 1 and 3 at the indicated doses. Cytokme and eicosanoid level in the granuloma were measured on day 6 as previously described. The granuloma tissue dry and wet weights were also evaluated. Angiogenesis was measured by two methods and represented as vascular index: a) by FITC-dextran or carmine dye uptake or b) by immunohistochemical method using CD31 (PECAM-1) as a marker of vessel wall endothehal cells.

In this model, TR6-Ig strongly inhibited LTB4 and LTC4 and PGE and TNF-α but not IL- lβ production. It also strongly inhibited angiogenesis, pnmaπly the formation of microvasculature, but had no effect on the tissue dry and wet weights

The ability of TR6-Ig to inhibit the production of inflammatory mediators and cytokines both in vitro and in vivo, combined with its anti-angiogemc properties in vivo, suggest that it may find utility as a protein therapeutic for chronic inflammatory, autoimmune or allergic conditions such as rheumatoid arthntis, psonasis and asthma. In addition, its anti-angiogenic properties indicate that it may have utility in the treatment of various ailments, such as cancer or arthritis, by starving the tumor or diseased tissue of the oxygen and nutrients it would otherwise receive through newly formed blood vessels.

Claims

What is claimed is:

1. An isolated polynucleotide comprising a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the TR6 polypeptide of SEQ ID NO:2 over its entire length; or a nucleotide sequence complementary to said nucleotide sequence.

2. The polynucleotide of claim 1 which is DNA or RNA.

3 The polynucleotide of claim 1 wherem said nucleotide sequence is at least 80% identical to that contained in SEQ ID NO: 1.

4. The polynucleotide of claim 3 wherem said nucleotide sequence comprises the TR6 polypeptide encoding sequence contained in SEQ ID NO:l

5. The polynucleotide of claim 3 which is polynucleotide of SEQ ID NO: 1.

6. A DNA or RNA molecule comprising an expression system, wherem said expression system is capable of producing a TR6 polypeptide compnsmg an ammo acid sequence, which has at least 80%. identity with the polypeptide of SEQ ID NO:2 when said expression system is present m a compatible host cell.

7. A host cell comprising the expression system of claim 6.

8 A process for producing a TR6 polypeptide comprising cultuπng a host of claim 7 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.

9. A process for producing a cell which produces a TR6 polypeptide thereof comprising transforming or transfecting a host cell with the expression system of claim 6 such that the host cell, under appropriate culture conditions, produces a TR6 polypeptide.

10. A TR6 polypeptide comprising an ammo acid sequence which is at least 80% identical to the ammo acid sequence of SEQ ID NO:2 over its entire length.

11. The polypeptide of claim 10 which comprises the ammo acid sequence of SEQ ID NO:2.

12. An antibody lmmunospecific for the TR6 polypeptide of claim 10.

13 A method for the treatment of a subject m need of enhanced activity or expression of TR6 polypeptide of claim 10 comprising: (a) admmistenng to the subject a therapeutically effective amount of an agonist to said receptor; and/or

(b) providing to the subject an isolated polynucleotide compnsmg a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the TR6 polypeptide of SEQ ID NO:2 over its entire length; or a nucleotide sequence complementary to said nucleotide sequence m a form so as to effect production of said polypeptide activity in vivo.

14 A method for the treatment of a subject having need to inhibit activity or expression of TR6 polypeptide of claim 10 comprising:

(a) admmistenng to the subject a therapeutically effective amount of an antagonist to said receptor; and/or

(b) admmistenng to the subject a nucleic acid molecule that inhibits the expression of the nucleotide sequence encoding said receptor; and/or

(c) admmistenng to the subject a therapeutically effective amount of a polypeptide that competes with said receptor for its hgand

15. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of TR6 polypeptide of claim 10 in a subject comprising:

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said TR6 polypeptide in the genome of said subject; and/or

(b) analyzing for the presence or amount of the TR6 polypeptide expression in a sample derived from said subject.

16 A method for identifying agonists to TR6 polypeptide of claim 10 compnsmg: (a) contacting a cell which produces a TR6 polypeptide with a candidate compound, and

(b) determining whether the candidate compound affects a signal generated by activation of the TR6 polypeptide.

17. An agonist identified by the method of claim 16, 21, 22, 23 or 24.

18. The method for identifying antagonists to TR6 polypeptide of claim 10 comprising:

(a) contacting said a cell which produces a TR6 polypeptide with an agonist; and

(b) determining whether the signal generated by said agonist is affected in the presence of a candidate compound.

19. The method of claim 18 m which agonist is TL2.

20. An antagonist identified by the method of claims 18, 19 21, 22, 23 or 24.

21 A method for identifying agonists or antagonists to TR6 polypeptides comprising:

(a) contacting a candidate compound with a TR6 polypeptide in the presence of labeled or unlabeled TL2 hgand; and (b)assessmg the ability of said candidate compound to compete with TL2 binding to said TR6 polypeptide

22. The method of claim 21 which the TR6 polypeptide is on the surface of a host cell, on a cell membrane or on a solid support.

23. The present invention further relates to a method for identifying agonists or antagonists to a TL2 comprising: (a) contacting a candidate compound with TL2 m the presence of a labeled or unlabeled TR6 polypeptide; and (b) assessing the ability of said candidate compound to compete with TR6 polypeptide binding to TL2.

24. The method of claim 23 m which TL2 is on the surface of a host cell, on a cell membrane or on a solid support

25. A recombinant host cell produced by the process of claim 9 or a membrane thereof expressing a TR6 polypeptide.

26. A method of inhibiting angiogenesis m an individual in need thereof comprising admmistenng extracellular domain of TR6 or soluble TR6 fusion protein to the individual.

27. A method of lowenng the production of eicosanoids or TNF-α in an individual m need thereof comprising admmistenng extracellular domain of TR6 or soluble TR6 fusion protein to the individual

28. TR6 fusion protein of claim 26 or 27 that is polypeptide of SEQ ID NO. 7.

29. TR6 fusion protein of claim 28 that is without the leader seqeunce.

30. The polypeptide of SEQ ID NO: 7.

31 The polypeptide of SEQ ID NO: 7 without the leader sequence