CA2381327A1

CA2381327A1 - Human tumor necrosis factor receptors tr13 and tr14

Info

Publication number: CA2381327A1
Application number: CA002381327A
Authority: CA
Inventors: Steven M. Ruben; Jian Ni; Paul E. Young
Original assignee: Individual
Current assignee: Human Genome Sciences Inc
Priority date: 1999-07-16
Filing date: 2000-07-14
Publication date: 2001-01-25
Also published as: EP1203024A4; WO2001005834A1; AU6102500A; AU780060B2; JP2003505045A; EP1203024A1; WO2001005834A9

Abstract

The present invention relates to two novel members of the tumor necrosis factor family of receptors. More specifically, isolated nucleic acid molecules are provided encoding the novel human necrosis factor receptors, TR13 and TR14. TR13 and TR14 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of TR13 and/or TR14 activity.

Description

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Human Tumor Necrosis Factor Receptors TR13 and TR14 s Background of the Invention l0 Field of the Invention The present invention relates to two novel members of the tumor necrosis factor family of receptors. More specifically, isolated nucleic acid molecules are provided encoding the novel human tumor necrosis factor receptors, TR13 and TR14. TR13 and TR14 polypeptides are also provided, as are vectors, host cells, and recombinant methods for producing the same. The 15 invention further relates to screening methods for identifying agonists and antagonists of TR13 and/or TR14 activity.
Related Art Many biological actions, for instance, response to certain stimuli and natural biological 2o processes, are controlled by factors, such as cytokines. Many cytokines act through receptors by engaging the receptor and producing an infra-cellular response.
For example, tumor necrosis factors (TNF) alpha and beta are cytokines, which act through TNF receptors to regulate numerous biological processes, including protection against infection and induction of shock and inflammatory disease. The TNF molecules belong to the 2s "TNF-ligand" superfamily, and act together with their receptors or counter-ligands, the "TNF-receptor" superfamily. So far, nine members of the TNF ligand superfamily have been identified and ten members of the TNF-receptor superfamily have been characterized.
Among the ligands there are included TNF-«, lymphotoxin-« (LT-« , also known as TNF-(3), LT-[3 (found in complex heterotrimer LT-2-~), FasL, CD40L, CD27L, CD30L, 4-30 1BBL, OX40L and nerve growth factor (NGF). The superfamily of TNF receptors includes the p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS
antigen or APO-1, CD40, CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (A. Meager, Biologicals 22:291-295 ( 1994)).
Many members of the TNF-ligand superfamily are expressed by activated T-cells, 3s implying that they are necessary for T-cell interactions with other cell types which underlie cell ontogeny and functions. (A. Meager, 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 ligand cause lymphoproliferative disease (R. Watanabe-Fukunaga et al., Nature 356:314 (1992)), perhaps reflecting a failure of programmed cell death. Mutations of the CD40 ligand cause an X-linked immunodeficiency state characterized by high levels of immunoglobulin M
and low levels of immunoglobulin G in plasma, indicating faulty T-cell-dependent B-cell activation (R.C. Allen et al., Science 259:990 (1993)). Targeted mutations of the low affinity nerve growth factor receptor cause a disorder characterized by faulty sensory innovation of to peripheral structures (K.F. Lee et al., Cell 69:737 (1992)).
TNF- « and LT- « are capable of binding to two TNF receptors (the 55- and 75-kd TNF
receptors). A large number of biological effects elicited by TNF- « and LT- «, 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 deleterious effects of ionizing radiation. TNF-« and LT-« are involved in the pathogenesis of a wide range of diseases, including endotoxic shock, cerebral malaria, tumors, autoimmune disease, AIDS and graft-host rejection (B. Beutler and C. Von Huffel, Science 264:667-668 (1994)). Mutations in the p55 receptor cause increased susceptibility to microbial infection.
2o Moreover, an about 80 amino acid domain near the C-terminus of TNFR1 (p55) and Fas was reported as the "death domain," which is responsible for transducing signals for programmed cell death (Tartaglia et al., Cell 74:845 (1993)).
Apoptosis, or programmed cell death, is a physiologic process essential to the normal development and homeostasis of multicellular organisms (H. Steller, Science 267:1445-1449 (1995)). Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome (C.B. Thompson, Science 267:1456-1462 (1995)). Recently, much attention has focused on the signal transduction and biological function of two cell surface death receptors, Fas/APO-1 and TNFR-1 (J.L. Cleveland et al., Cell 81:479-482 (1995); A. Fraser et al., Cell 85:781-784 (1996); S. Nagata et al., Science 267:1449-56 (1995)). Both are members of the TNF receptor family, which also include TNFR-2, low affinity NGFR, CD40, and CD30, among others (C.A. Smith et al., Science 248: 1019-23 (1990); M. Tewari et al., in Modular Texts in Molecular and Cell Biology M. Purton, Heldin, Carl, Ed. (Chapman and Hall, London, 1995).
While family members are defined by the presence of cysteine-rich repeats in their extracellular domains, Fas/APO-1 and TNFR-1 also share a region of intracellular homology, appropriately designated the "death domain," which is distantly related to the Drosophila suicide gene, reaper (P. Golstein et al., Cell 81:185-6 (1995); K. White et al., Science 264:677-83 (1994)). This shared death domain suggests that both receptors interact with a related set of signal transducing molecules that, until recently, remained unidentified. Activation of Fas/APO-1 recruits the death domain-containing adapter molecule FADD/MORT1 (A.M.
Chinnaiyan et al., Cell 81:505-512 (1995); M. P. Boldin et al., J. Biol. Chem. 270:7795-8 (1995);
F.C.
Kischkel et al., EMBO 14:5579-5588 (1995)), which in turn binds and presumably activates FLICE/MACH1, a member of the ICE/CED-3 family of pro-apoptotic proteases (M.
Muzio et al., Cell 85: 817-827 (1996); M.P. Boldin et al., Cell 85:803-815 (1996)).
While the central role of Fas/APO-1 is to trigger cell death, TNFR-1 can signal an array of diverse biological activities-many of which stem from its ability to activate NF-kB (L.A.
Tartaglia et al., Immunol Today 13:151-153 (1992)). Accordingly, TNFR-1 recruits the multivalent adapter molecule TRADD, which like FADD, also contains a death domain (H. Hsu et al., Cell 81:495-504 (1995); H. Hsu et al., Cell 84:299-308 (1996)). Through its associations with a number of signaling molecules including FADD, TRAF2, and RIP, TRADD can signal both apoptosis and NF-kB activation(H. Hsu et al., Cell 84:299-308 (1996); H. Hsu et al., Immunity 4:387-396 (1996)).
Recently, a new apoptosis inducing TNF ligand has been discovered. S.R. Wiley et al., Immunity 3:673-682 (1995), named the new molecule, "TNF-related apoptosis-inducing ligand" or "TRAIL." R.M. Pitti et al., J. Biol. Chem. 271:12687-12690 (1996), named the molecule "Apo-2ligand" or "Apo-2L." This molecule was also disclosed in co-pending U.S.
2o provisional patent application no. 60/013405. For convenience, this molecule will be referred to herein as TRAIL.
Unlike FAS ligand, whose transcripts appear to be largely restricted to stimulated T-cells, significant levels of TRAIL are detected in many human tissues (e.g., spleen, lung, prostate, thymus, ovary, small intestine, colon, peripheral blood lymphocytes, placenta, kidney), and it is constitutively transcribed by some cell lines. It has been shown that TRAIL
acts independently from the FAS ligand (5.R. Wiley et al., supra). It has also been shown that TRAIL activates apoptosis rapidly, within a time frame that is similar to death signaling by Fas/Apo-1L, but much faster than TNF-induced apoptosis. S.A. Marsters et al., Current Biology 6:750-752 (1996). The inability of TRAIL to bind TNFR-1, Fas, or the recently 3o identified DR3, suggests that TRAIL may interact with a unique receptor(s).
Work to date suggests that there are several unique TNF receptors for TRAIL.
In co-pending U.S. provisional patent application no. 60/035,722, one novel death domain containing receptor for TRAIL, DR4, was disclosed. See, Pan et al., Science 276:111-113 (April 1997). In co-pending U.S. provisional patent application no.
60/040,846, a novel death domain containing receptor, DRS (TR7), was disclosed. This receptor has now been shown to bind TRAIL. In co-pending U.S. provisional patent application no. 60/035,496, another receptor, TRS, was disclosed. This receptor has also now been shown to bind TRAIL, however, TRS has been shown to be a non-signaling decoy receptor which antagonizes apoptos~s.
The effects of TNF family ligands and receptors are varied and influence numerous functions, both normal and abnormal, in the biological processes of the mammalian system.
There is a clear need, therefore, for identification and characterization of such receptors and ligands that influence biological activity, both normally and in disease states. In particular, there is a need to isolate and characterize additional novel receptors that bind TRAIL.
Summary of the Invention l0 The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the TR13 receptor having the amino acid sequence shown in SEQ ID
N0:2 (Figures lA-C), amino acid sequence shown in SEQ ID N0:40 (Figures 7A-D) or the amino acid sequence encoded by the cDNA clone deposited as American Type Culture Collection ("ATCC") Deposit No. PTA-349 (HWLHM70) on July 13, 1999, and/or the amino ~5 acid sequence encoded by the cDNA clone deposited as American Type Culture Collection ("ATCC") Deposit No. PTA-507 (HWLHN83) on August 12, 1999. The ATCC is located at 10801 University Boulevard, Manassas, Virginia 20110-2209. It would be apparent to the ' skilled artisan that the various methods of use, including but not limited to diagnostic and therapeutic uses described herein, for the TR13 receptor polynucleotides and polypeptides 2o would apply equally to all variants and fragments thereof (e.g., fragments of the TR13 receptor disclosed and described herein in Figures lA-C, Figure 7A-D, SEQ ID NO:1, SEQ
ID N0:2, SEQ ID N0:39, SEQ ID N0:40 and/or contained or encoded by one or both of the deposited cDNA clones HWLHM70 and HWLHN83).
The present invention also relates to recombinant vectors, which include the isolated 25 nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of TR13 polypeptides (e.g., the TR13 polypeptide sequence shown in Figures lA
C and/or Figures 7A-D, or a fragment thereof) by recombinant techniques.
The invention further provides an isolated TR13 polypeptide (e.g., the TR13 30 polypeptide sequence shown in Figures lA-C and/or Figures 7A-D or fragments thereof) having an amino acid sequence encoded by a polynucleotide described herein (e.g., the polynucleotide sequence shown in SEQ ID NO:1 and/or SEQ ID N0:39, or a fragment thereof).
The present invention also provides diagnostic assays such as quantitative and 35 diagnostic assays for detecting levels of TR13 polynucleotide and/or protein (e.g., the TR13 protein shown in Figures lA-C and/or Figures 7A-D, or fragments thereof).
Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of TR13, or soluble form thereof, compared to normal control tissue samples may be used to detect the presence of tumors.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the TR14 receptor having the amino acid sequence shown in SEQ ~

5 N0:61 (Figures l0A-H), and/or the amino acid sequence encoded by the cDNA
clone deposited as American Type Culture Collection ("ATCC") Deposit No. PTA-348 (HMSHK47) on July 13, 1999. While the sequence of SEQ ID N0:61 and Figures l0A-H are preferred embodiments of TR14 receptor protein, the present invention provides alternative isolated nucleic acid molecule embodiments comprising a polynucleotide encoding the TR14 receptor having the amino acid sequence shown in SEQ ID NO:S (Figures 4A-D). The sequence of amino acid residues T-78 to M-231 of SEQ ID N0:61 is identical to the sequence of amino acid residues T-73 to M-226 of SEQ ID N0:5. It would be apparent to the skilled artisan that the various methods of use, including, but not limited to, diagnostic and therapeutic uses described herein, for the TR13 receptor polynucleotides and polypeptides would apply equally to all variants and fragments thereof (e.g., fragments of the TR14 receptor disclosed and described in Figures l0A-H and SEQ ID NOS:60 and 61, or, alternatively, Figures 4A-D and SEQ ID
N0:4, SEQ ID N0:5 and/or contained or encoded by the deposited cDNA clone (HMSHK47)).
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant 2o vectors, as well as to methods of making such vectors and host cells and for using them for production of TR14 polypeptides by recombinant techniques.
The invention further provides an isolated TR14 polypeptide (e.g., the TR14 polypeptide sequence shown in Figures l0A-H or, alternatively, Figures 4A-D, or fragments thereof) having an amino acid sequence encoded by a polynucleotide described herein (e.g., the polynucleotide sequence shown in SEQ ID N0:60, or, alternatively SEQ ID N0:4, or fragments thereof).
The present invention also provides diagnostic assays such as quantitative and diagnostic assays for detecting levels of TR14 polynucleotide and/or protein (e.g., the TR14 polypeptide sequence disclosed in Figures l0A-H or 4A-D, or fragments thereof). Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of TR14, or soluble form thereof, compared to normal control tissue samples may be used to detect the presence of tumors.
Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cell proliferation, cytotoxicity, anti-viral activity, immunoregulatory activities, hematopoiesis, and the transcriptional regulation of several genes. Cellular response to TNF-family ligands include not only normal physiological responses, but also diseases associated with increased apoptosis or the inhibition of apoptosis. Apoptosis-programmed cell death is a physiological mechanism involved in the deletion of peripheral T lymphocytes of the immune system, and its dysregulation can lead to a number of different pathogenic processes. Diseases associated with increased cell survival, unregulated cell proliferation, or the inhibition of apoptosis, include cancers, autoimmune disorders, viral infections, inflammation, graft vs. host disease, acute graft rejection, and chronic graft rejection. Diseases associated with increased apoptosis include AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemic injury, toxin-induced liver disease, septic shock, cachexia, and anorexia.
Thus, the invention further provides a method for inhibiting TR13 mediated signaling and/or apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses the TR13 polypeptide (i.e., the TR13 polypeptide shown in Figures lA-C and/or Figures 7A-D, or a fragment thereof) an effective amount of a TR13 antagonist capable of decreasing TR13 mediated apoptosis and/or decreasing TR13 mediated signaling.
Preferably, TR13 mediated signaling is decreased to treat a disease wherein increased apoptosis is exhibited.
Thus, the invention further provides a method for promoting TR13 mediated signalling and/or apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses the TR13 polypeptide (e.g., the TR13 polypeptide shown in Figures lA-C and/or Figures 7A-D, or a fragment thereof) an effective amount of a TR13 agonist capable of 2o increasing TR13 mediated apoptosis and/or increasing TR13 mediated signaling. Preferably, TR13 mediated signaling is increased to treat a disease wherein decreased apoptosis is exhibited.
Thus, the invention further provides a method for inhibiting TR14 mediated signaling and/or apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses the TR14 polypeptide an effective amount of a TR14 antagonist capable of decreasing TR14 mediated apoptosis and/or capable of decreasing TR14 mediated signaling.
Preferably, TR14 mediated signaling is decreased to treat a disease wherein increased apoptosis is exhibited.
Thus, the invention further provides a method for promoting TR14 mediated signaling 3o and/or apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses the TR14 polypeptide an effective amount of a TR14 agonist capable of increasing TR14 mediated apoptosis and/or capable of increasing TR14 mediated signaling.
Preferably, TR14 mediated signaling is increased to treat a disease wherein decreased apoptosis is exhibited.
In a further aspect, the present invention is directed to a method for enhancing TR13 mediated signaling induced by a TNF-family ligand (e.g., APRIL and Neutrokine-alpha (International Application Publication No. WO 98/18921), which involves administering to a cell which expresses the TR13 polypeptide (e.g., the polypeptide shown in Figures lA-C
and/or Figures 7A-D, or a fragment thereof) an effective amount of an agonist capable of increasing TR13 mediated activity. Preferably, TR13 mediated activity is increased to treat a disease wherein decreased apoptosis is exhibited.
Whether any candidate "agonist" or "antagonist" of the present invention can enhance or inhibit TR13 mediated signaling can be determined using art-known TNF-family ligand/receptor cellular response assays, including those described in more detail below. Thus, in a further aspect, a screening method is provided for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response to a TR13 TNF-family to ligand. The method involves contacting cells which express the TR13 polypeptide (e.g., the polypeptide shown in Figures lA-C and/or Figures 7A-D, or a fragment thereof) with a candidate compound and a TNF-family ligand (e.g., Neutrokine-alpha or APRIL), assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the ligand/receptor signaling pathway and a decreased cellular response compared to the standard indicates that the candidate compound is an antagonist of the ligand/receptor signaling pathway. By the invention, a cell expressing a TR13 polypeptide (e.g., the polypeptide shown in Figures lA-C and/or Figures 7A-D, or a fragment thereof) can be contacted with either an endogenous or exogenously administered TNF-family ligand.
In a further aspect, the present invention is directed to a method for enhancing apoptosis TR14 mediated signaling induced by a TNF-family ligand, which involves administering to a cell which expresses the TR14 polypeptide (e.g., the polypeptide shown in Figures l0A-H, or, alternatively 4A-D, or a fragment thereof) an effective amount of an agonist capable of increasing TR14 mediated activity. Preferably, TR14 mediated activity is increased to treat a disease wherein decreased apoptosis is exhibited.
In specific, preferred embodiments, TR14 polynucleotides and polypeptides, as well as antibodies that agonize TR14 receptor (as described in the section on Antibodies, above), stimulate epithelial cell proliferation and/or development to ameliorate the diseases and disorders described in this section. Members of the TNF family of proteins are known to signal through the NF-KB singaling pathway. NF-KB is a transcription factor activated by a wide certain agents to stimulate cell activation and differentiation. It is believed that the TR14 receptor of the instant invention signals through the NF-xB pathway to activate proliferation and development of cells. Thus, TR14 polynucleotides and polypeptides of the invention as well as antibodies and peptides that agonize TR14 may be used in accordance with the invention to stimulate NF-xB-mediated epithelial cell proliferation, including but not limited to ectodermal dysplasia.
Whether any candidate "agonist" or "antagonist" of the present invention can enhance or inhibit TR14 mediated signaling can be determined using art-known TR14 TNF-family ligand/receptor cellular response assays, including those described in more detail below. Thus, in a further aspect, a screening method is provided for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand.
The method involves contacting cells which express the TR14 polypeptide with a candidate compound and a TNF-family ligand, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the ligand/receptor signaling pathway and a decreased cellular response compared to the standard indicates that the candidate compound is an antagonist of the ligand/receptor signaling pathway. By the invention, a cell expressing the TR14 polypeptide (e.g., the polypeptide shown in Figures l0A-H, or, alternatively 4A-D) can be contacted with either an endogenous or exogenously administered TNF-family ligand.
Brief Description of the Figures 2o Figures lA-C shows the nucleotide (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID N0:2) of the TR13 receptor. Predicted amino acids from about 105 to about 170, about 251 to about 265, about 331 to about 410, and about 580 to about 610 constitute the cysteine-rich domains (amino acid residues from about 105 to about 170, about 251 to about 265, about 331 to about 410, and about 580 to about 610 in SEQ ID N0:2) and are represented by the underlined amino acid regions; amino acids from about 139 to about 142, about 140 to about 143, about 153 to about 156, about 293 to about 296, about 325 to about 328, about 421 to about 424, about 466 to about 469, about 696 to about 699, and about 728 to about 731 constitute potential sites of N-glycosylation (amino acid residues from about 139 to about 142, about 140 to about 143, about 153 to about 156, about 293 to about 296, about 325 to about 328, about 421 to about 424, about 466 to about 469, about 696 to about 699, and about 728 to about 731 in SEQ ID N0:2) which are represented by the bolded amino acids;
amino acids from about 312 to about 315, and about 458 to about 461, constitute potential cAMP
phosphorylation sites (amino acid residues from about from about 312 to about 315, and about 458 to about 461 in SEQ ID N0:2) and are represented by asterisks (*) above the amino acid residues; amino acids from about 50 to about 53, about 66 to about 69, about 80 to about 83, about 276 to about 279, about 311 to about 314, about 438 to about 441, about 559 to about 562, about 564 to about 567, about 698 to about 701, and about 725 to about 728 constitute potential sites of protein kinase C (PKC) phosphorylation (amino acid residues from about 50 to about 53, about 66 to about 69, about 80 to about 83, about 276 to about 279, about 311 to about 314, about 438 to about 441, about 559 to about 562, about 564 to about 567, about 698 to about 701, and about 725 to about 728 in SEQ ID N0:2) and are represented by the italicized amino acid residues; amino acids from about 80 to about 83, about 89 to about 92, about 180 to about 183, about 198 to about 201, about 214 to about 217, about 272 to about 275, about 306 to about 309, about 510 to about 513, about 529 to about 532, about 584 to about 587, about 609 to about 312, about 642 to about 645, and about 698 to about 701 casein kinase II
1o phosphorylation sites (amino acid residues from about 80 to about 83, about 89 to about 92, about 180 to about 183, about 198 to about 201, about 214 to about 217, about 272 to about 275, about 306 to about 309, about 510 to about 513, about 529 to about 532, about 584 to about 587, about 609 to about 312, about 642 to about 645, and about 698 to about 701 in SEQ ID N0:2) and are represented by the double underlined amino acids; amino acids from about 69 to about 74, about 149 to about 154, about 154 to about 159, about 163 to about 168, about 212 to about 217, about 248 to about 253, about 365 to about 370, about 383 to about 388, about 393 to about 398, about 588 to about 593, about 623 to about 628, about 661 to about 666, and about 665 to about 670 N-myristoylation sites (amino acids from about 69 to about 74, about 149 to about 154, about 154 to about 159, about 163 to about 168, about 212 2o to about 217, about 248 to about 253, about 365 to about 370, about 383 to about 388, about 393 to about 398, about 588 to about 593, about 623 to about 628, about 661 to about 666, and about 665 to about 670 in SEQ ID N0:2) and are represented by the strikethrough amino acids (e.g. ~); and amino acids from about 456 to about 459 constitute a potential amidylation site (amino acid residues from about 456 to about 459 of SEQ ID N0:5) and is represented by the lowercase amino acids.
Figures 2A-C show the regions of similarity between the amino acid sequences of the TR13 receptor protein (SEQ ID N0:2), and the OX40 protein (SEQ ID N0:3).
Figure 3 shows an analysis of the TR13 amino acid sequence (SEQ ID N0:2).
Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. In the "Antigenic Index - Jameson-Wolf" graph, amino acid residues from about M1 to about A9, about K12 to about L20, about N47 to about T55, about H58 to about S66, about D63 to about S71, about P77 to about F85, about A90 to about Q98, about F136 to about Q144, about S152 to about C160, about 8159 to about A167, about A211 to about M219, about M235 to about V243, about V266 to about V274, about W277 to about S285, about I290 to about F298, about A310 to about V318, about E343 to about C351, about I360 to about H368, about 6391 to about I399, about F409 to about T417, about S436 to about Y444, about C453 to about S461, about I472 to about S480, about Y548 to about S556, about C557 to about 1565, about V567 to about V575, about T584 to about 6592, about 8632 to about 6640, about W680 to about Y688, about Q684 to about K692, about T698 to about A706, about 5726 to about S734, and about S734 to about L742 of SEQ ID N0:2 (Figures lA-C) correspond to the highly antigenic regions of the TR13 5 protein, predicted using the Jameson-Wolf antigenic index (See Figure 3 and Table I). These highly antigenic fragments correspond to the amino acid residues illustrated in Figures lA-C
and in SEQ ID N0:2.
Figures 4A-D shows the nucleotide (SEQ ID N0:4) and deduced amino acid sequence (SEQ ID N0:5) of the TR14 receptor. The predicted extracellular domain constitutes amino 10 acids from about 1 to about 133 (amino acid residues from 1 to 133 of SEQ
ID N0:5) and are represented by the underlined amino acids; amino acids from about 65 to about 85 constitute a conserved cysteine-rich domain (amino acid residues from about 65 to about 85 of SEQ ID
N0:5) and is represented by the italized amino acid residues; amino acids from about I34 to about 150 constitute the predicted transmembrane domain (amino acid residues from about 134 ~5 to about 150 in SEQ ID N0:5) which are represented by the double underlined amino acid residues; amino acid residues from about 151 to about 226 constitutes the predicted intracellular domain (amino acid residues from about 151 to about 226 of SEQ
ID N0:5) and are represented by the lower case amino acid residues; amino acids from about 178 to about 180 constitute potential protein kinase C (PKC) phosphorylation sites (amino acid residues 2o from about 178 to about 180 of SEQ ID N0:5) and are represented by asterisks (*) above the amino acid residues; amino acids from about 5 to about 8, about 118 to about 121, about 178 to about 181, and about 193 to about 196 constitute potential sites of casein kinase II
phosphorylation (amino acid residues from about 5 to about 8, about 118 to about 121, about 178 to about 181, about 193 to about 196 of SEQ ID N0:5) and are represented by the 25 strikethrough amino acid residues; and amino acids from about 9 to about 14 contitutes a potential N-myristoylation site (amino acid residues from about 9 to about 14 of SEQ ID N0:5) and is represented by the bold amino acids.
Figures 5A-B show the regions of similarity between the amino acid sequences of the TR14 receptor protein (SEQ ID N0:5), and the Tumor Necrosis Factor Receptor protein (SEQ
3o ID N0:6).
Figure 6 shows an analysis of the TR14 amino acid sequence (SEQ ID N0:5).
Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. In the "Antigenic Index - Jameson-Wolf" graph, amino acid residues from about T3 to about S 11, from about V 16 to about R24, 35 from about Q44 to about M52, from about F85 to about G93, from about T103 to about V 111, from about F161 to about 6169, from about V 187 to about A195, from about P218 to about M226 of SEQ ID N0:5 (Figures 4A-D) correspond to the highly antigenic regions of the TR14 protein, predicted using the Jameson-Wolf antigenic index (See Figure 6 and Table II). These highly antigenic fragments correspond to the amino acid residues illustrated in Figures 4A-D
and in SEQ ID N0:5.
Figures 7A-D shows the nucleotide (SEQ ID N0:39) and deduced amino acid sequence (SEQ ID N0:40) of the full-length TR13 receptor. The predicted signal sequence constitutes amino acids from about 1 to about 41 (amino acid residues from about 1 to about 41 of SEQ 117 N0:40) and are represented by the dotted underlined amino acids; amino acids from about 42 to about 906 constitutes the predicted extracellular domain (amino acid residues from 42 to 906 of SEQ ID N0:40) and are represented by the single underlined amino acids; amino acids from about 271 to about 421 and from about 585 to about 595 constitute conserved cysteine-rich domains (amino acid residues from about 271 to about 421 and from about 585 to about 595 of SEQ ID N0:40) and is represented by the italized amino acid residues; amino acids from about 907 to about 931 constitute the predicted transmembrane domain (amino acid residues from about 907 to about 931 in SEQ ID N0:40) which are represented by the double underlined amino acid residues; amino acid residues from about 932 to about 1001 constitutes the predicted intracellular domain (amino acid residues from about 932 to about 1001 of SEQ ID
N0:40) and are represented by the lower case amino acid residues; amino acids from about 11 to about 13, about 18 to about 20, 107 to about 109, about 156 to about 158, about 224 to about 226, about 301 to about 303, about 317 to about 319, about 331 to about 333, about 527 2o to about 529, about 562 to about 564, about 689 to about 691, about 810 to about 812, about 815 to about 817, about 949 to about 951, and about 976 to about 978 constitute potential protein kinase C (PKC) phosphorylation sites (amino acid residues from about 11 to about 13, about 18 to about 20, 107 to about 109, about 156 to about 158, about 224 to about 226, about 301 to about 303, about 317 to about 319, about 331 to about 333, about 527 to about 529, about 562 to about 564, about 689 to about 691, about 810 to about 812, about 815 to about 817, about 949 to about 951, and about 976 to about 978 of SEQ ID N0:40) and are represented by asterisks (*) above the amino acid residues; amino acids from about 42 to about 45, about 59 to about 62, about 81 to about 84, about 146 to about 149, about 282 to about 285, about 331 to about 334, about 340 to about 343, about 431 to about 434, about 449 to about 452, about 465 to about 468, about 523 to about 526, about 557 to about 560, about 761 to about 764, about 780 to about 783, about 780 to about 783, about 835 to about 838, about 860 to about 863, about 893 to about 896, and about 949 to about 952 constitute potential sites of casein kinase II phosphorylation (amino acid residues from about 42 to about 45, about 59 to about 62, about 81 to about 84, about 146 to about 149, about 282 to about 285, about 331 to about 334, about 340 to about 343, about 431 to about 434, about 449 to about 452, about 465 to about 468, about 523 to about 526, about 557 to about 560, about 761 to about 764, about 780 to about 783, about 780 to about 783, about 835 to about 838, about 860 to about 863, about 893 to about 896, and about 949 to about 952 of SEQ ID N0:40) and are represented by the strikethrough amino acid residues; amino acids from about 77 to about 82, about 88 to about 93, about 152 to about 157, about 268 to about 273, about 288 to about 293, about 320 to about 325, about 400 to about 405, about 414 to about 419, about 463 to about 468, about 599 to about 604, about 616 to about 621, about 634 to about 639, about 644 to about 649, about 839 to about 844, about 874 to about 879, about 912 to about 917, and about 916 to about 921 contitute potential N-myristoylation sites (amino acid residues from about 77 to about 82, about 88 to about 93, about 152 to about 157, about 268 to about 273, about 288 to about 293, about 320 to about 325, about 400 to about 405, about 414 to about 419, about 463 to about 468, about 599 to about 604, about 616 to about 621, about 634 to about 639, about 644 to about 649, about 839 to about 844, about 874 to about 879, about 912 to about 917, and about 916 to about 921 of SEQ ID N0:40) and are represented by a plus sign ("+") above the amino acids; amino acids from about 50 to about 56, and 109 to about 116 constitute potential tyrosine phosphorylation sites (amino acids from about 50 to about 56, and about 109 to about 116 of SEQ ID N0:40) are represented by the double strikethrough amino acids; and amino acids from about 153 to about 156, 390 to about 393, 391 to about 394, about 404 to about 407, about 544 to about 547, about 576 to about 579, about 672 to about 675, about 717 to about 720, about 947 to about 950, and about 979 to about 982 constitute potential N-glycosylation sites (amino acids from about 153 to about 156, 390 to about 393, 391 to about 394, about 404 to about 407, about 544 to about 547, about 576 to about 579, about 672 to about 675, about 717 to about 720, about 947 to about 950, and about 979 to about 982 of SEQ ID N0:40) which are represented by the shaded amino acids.
Figures 8A-B show the regions of similarity between the amino acid sequences of the full-length TR13 receptor protein (SEQ ID N0:40), and the Tumor Necrosis Factor Receptor II
homolog (gbIAAB94382.1) (SEQ ID NO: 41).
Figure 9 shows an analysis of the full-length TR13 amino acid sequence disclosed in Figures 7A-D (SEQ ID N0:40). Alpha, beta, turn and coil regions;
hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. In the "Antigenic Index - Jameson-Wolf" graph, amino acid residues from about 3o M1 to about H9, about V14 to about I22, about H47 to about H55, about C61 to about R69, about L82 to about E90, about D102 to about P110, about K109 to about 5117, about F124 to about H132, about M141 to about E149, about S146 to about C154, about S157 to about W 165, about F168 to about T 176, about N 182 to about N 190, about Q207 to about A215, about P213 to about M221, about M221 to about E229, about V233 to about V241, about T253 to about V261, about T282 to about S290, about N298 to about T306, about C308 to about Y316, about K315 to about S323, about P328 to about F336, about A341 to about Q349, about F387 to about Q395, about 5403 to about C411, about T409 to about P417, about F443 to about N451, about W451 to about Y459, about A462 to about M470, about 6478 to about M486, about A487 to about A495, about V517 to about V525, about T527 to about Q535, about I541 to about F549, about A561 to about V569, about E594 to about C602, about I611 to about H619, about 6643 to about I650, about P686 to about K694, about C704 to about S712, about 8722 to about I730, about E727 to about T735, about P746 to about 6754, about D776 to about L784, about Y799 to about S807, about C808 to about I816, about V818 to about V826, about T835 to about 6843, about 8883 to about 6891, about K932 to about K940, about Q935 to about K943, about T949 to about A957, about S977 to about S985, about S981 to about P989, and about N986 to about L994 of SEQ ID N0:40 (Figures 7A-D) correspond to the highly antigenic regions of the TR13 protein, predicted using the Jameson-Wolf antigenic index (See Figure 9 and Table III). These highly antigenic fragments correspond to the amino acid residues illustrated in Figures 7A-D and in SEQ
ID N0:40.
Figures l0A-H show a preferred nucleotide (SEQ ID N0:60) and deduced amino acid sequence (SEQ ID N0:61) of the TR14 receptor. The transmembrane domain from amino acids L-139 to L-155 is underlined.
Figure 11 shows an analysis of the full-length TR14 amino acid sequence disclosed in Figures l0A-H (SEQ ID N0:61). Alpha, beta, turn and coil regions;
hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. The data are presented in tabular form, amino acid by amino acid, in Table IV, 2o below.
Detailed Description of the Preferred Embodiments The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a TR13 polypeptide having the amino acid sequence shown in Figures lA-C (SEQ ID N0:2) and/or Figures 7A-D (SEQ ID N0:40) and/or fragments or variants thereof. The TR13 polypeptide of the present invention shares sequence homology with the human OX40 homologue (Figures 2A-C) and the tumor necrosis factor receptor II
homolog (Figures 8A-B). The nucleotide sequence shown in Figures lA-C (SEQ ID NO:1) was obtained by sequencing a cDNA clone (HWLHM70), which was deposited on July 13, 1999 at 3o the American Type Culture Collection, and given Accession Number PTA-349.
The nucleotide sequence shown in Figures 7A-D (SEQ ID N0:39) was obtained, in part, by sequencing a cDNA clone (HWLHN83), which was deposited on August 12, 1999 at the American Type Culture Collection, and given Accession Number PTA-507. The deposited clone is inserted in the pSportl clone (Life Technologies, Rockville, MD) using the SaII and NotI
restriction endonuclease cleavage sites.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a TR14 polypeptide having the amino acid sequence shown in Figures l0A-H (SEQ ID NO:51), or, alternatively 4A-D (SEQ ID NO:S) and/or fragments or variants thereof, which were determined by sequencing a cloned cDNA. The TR14 polypeptide of the present invention shares sequence homology with the Tumor Necrosis Factor Receptor (Figures SA-B). The nucleotide sequence shown in Figures l0A-H (SEQ ID N0:60) was obtained by sequencing a cDNA clone (HMSHK47), which was deposited on July 13, 1999 at the American Type Culture Collection, and given Accession Number PTA-348. The deposited clone is inserted in the pBluescript clone (Life Technologies, Rockville, MD) using the EcoRI
restriction endonuclease cleavage sites. While SEQ ID N0:60 is a preferred sequence for TR14, an alternative TR14 related sequence is shown in Figures 4A-D (SEQ ID
N0:4).
to Nucleic Acid Molecules Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA
molecule herein were determined using an automated DNA sequencer (such as the Model 3700 and Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA
sequence determined as above. Therefore, as is known in the art for any DNA
sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9%
identical to the 2o actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
Using the information provided herein, such as the nucleic acid sequence set out in SEQ
ID NO:1 and/or SEQ ID N0:39, a nucleic acid molecule of the present invention encoding a TR13 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in SEQ ID NO:1 was discovered in a cDNA
library derived from activated monocytes. Further, illustrative of the invention, the nucleic acid molecule described in SEQ ID N0:39 was discovered in a cDNA library derived from normal human colon. TR13 polynucleotides of the invention have also been identified in cDNA
libraries from the following tissues: pancreas tumor, endometrial tumor, adult small intestine, colon cancer, breast cancer cell line, resting T-cell, amygdala, rectum, T-cell helper, pineal gland, apoptotic T-cell, epididymus; greater omentum, prostate BPH, osteoclastoma, endometrial stromal cells, stromal cell, substantia nigra, activated T-cell, tonsil, and testes tissue.
The determined TR13 nucleotide sequence of SEQ ID NO:1 contains an open reading frame encoding a protein of about 750 amino acid residues, and a deduced molecular weight of 5 about 82 kDa. The amino acid sequence of the predicted TR13 receptor is shown in SEQ >D
N0:2 from amino acid residue about 1 to residue about 750. OF known members of the TNF
receptor family, this TR13 polypeptide shares the greatest degree of homology with human OX40 (See Figures 2A-C), including significant sequence homology over multiple cysteine rich domains.
to The determined TR13 nucleotide sequence of SEQ ID N0:39 contains an open reading frame encoding a protein of about 1001 amino acid residues, with a predicted signal encompassing amino acids about 1 to about 41, a predicted extracellular domain encompassing amino acids from about 42 to about 906, a transmembrane domain encompassing amino acids from about 907 to about 931, and an intracellular domain encompassing amino acids from 15 about 932 to 1001, of SEQ ID N0:40, and a deduced molecular weight of about 110 kDa. The amino acid sequence of the predicted TR13 receptor is shown in SEQ ID N0:40 from amino acid residue about 1 to residue about 1001. Of known members of the TNF
receptor family, this TR13 polypeptide shares the greatest degree of homology with the tumor necrosis factor receptor II homolog (See Figures 8A-B), including significant sequence homology over multiple cysteine rich domains.
Using the information provided herein, such as the nucleic acid sequence set out, preferably, in SEQ ID N0:60, or, alternatively SEQ ID N0:4, a nucleic acid molecule of the present invention encoding a TR14 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
Illustrative of the invention, the nucleic acid molecule contained in deposited clone HMSHK47 (described in SEQ ID N0:60) was discovered in a cDNA library derived from colon. The gene of the present invention has also been identified in cDNA libraries from the following tissues:
activated T-cell, endometrial tumor, thymus, and 12 week early stage human tissue.
The determined nucleotide sequence of the TR14 cDNA of SEQ ID N0:60 contains an open reading frame encoding a protein of about 231 amino acid residues, with a predicted extracellular domain encompassing amino acids from about 1 to about 138, a transmembrane domain encompassing amino acids from about 139 to about 155, and an intracellular domain encompassing amino acids from about 156 to about 231 of SEQ ID N0:61 and a deduced molecular weight of about 25 kDa.
The TR14 nucleotide sequence of SEQ ID N0:4 contains an open reading frame encoding a protein of about 226 amino acid residues, with a predicted extracellular domain encompassing amino acids from about 1 to about 133, a transmembrane domain encompassing amino acids from about 134 to about 150 (from about 139 to about 155 of SEQ ID
N0:61 ), and an intracellular domain encompassing amino acids from about 151 to about 226 of SEQ 117 N0:4 (acids from about 156 to about 231 of SEQ ID N0:61) and a deduced molecular weight of about 24.5 kDa. Of known members of the TNF receptor family, the TR14 polypeptide of the SEQ ID NO:S shares the greatest degree of homology with tumor necrosis factor receptor (See Figures SA-B).
As indicated, the present invention also encompasses mature forms) of the TR13 and/or TR14 polypeptides of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretory leader sequence which is to cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species on the protein.
Further, it has long been known that the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
Therefore, the present invention provides a nucleotide sequence encoding the mature form of the TR13 polypeptide having the amino acid sequence encoded by the cDNA clone identified as ATCC Deposit No. PTA-349 (HWLHM70), and/or of the amino acid sequence shown in Figures lA-C (SEQ ID N0:2). By the mature form of TR13 polypeptide having the amino acid sequence encoded by, for example, the cDNA clone identified as ATCC
Deposit No. PTA-349 (HWLHM70) is meant, the mature forms) of the TR13 receptor produced by expression in a mammalian cell (e.g., COS cells, as described below) of the complete open reading frame encoded by the human DNA sequence of the clone contained in the deposited vector. As indicated herein, the mature form of the TR13 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-349 (HWLHM70), may or may not differ from the predicted mature TR13 protein shown in SEQ
ID N0:2 (amino acids from about 1 to about 750) depending on the accuracy of the predicted cleavage site based on computer analysis. Polypeptides encoded by the nucleotide sequences are also encompassed by the invention.
Therefore, the present invention provides a nucleotide sequence encoding the mature form of the TR13 polypeptide having the amino acid sequence encoded by the cDNA clone identified as ATCC Deposit No. PTA-507 (HWLHN83), and/or of the amino acid sequence as shown in Figures 7A-D (SEQ ID N0:40). By the mature form of the TR13 polypeptide having the amino acid sequence encoded by, for example, the cDNA clone identified as ATCC Deposit No. PTA-507 (HWLHN83), is meant, the mature forms) of the TR13 receptor produced by expression in a mammalian cell (e.g., COS cells, as described below) of the complete open reading frame encoded by the human DNA sequence of the clone contained in the deposited vector. As indicated herein, the mature form of the TR13 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-507 (HWLHN83), may or may not differ from the predicted mature TR13 protein shown in SEQ
ID N0:40 (amino acids from about 42 to about 1001 ) depending on the accuracy of the predicted cleavage site based on computer analysis. Polypeptides encoded by these nucleotide sequences are also encompassed by the invention.
Methods for predicting whether a protein has a secretory leader as well as the cleavage point for that leader sequence are available. For instance, the method of McGeoch (Virus Res.
l0 3:271-286 (1985)) and von Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used.
The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. von Heinje, supra. However, the two methods do not always produce the same predicted cleavage points) for a given protein.
1n the present case, the predicted amino acid sequence of the TR13 polypeptide of the present invention was analyzed by a computer program ("PSORT"). See K. Nakai and M.
Kanehisa, Genomics 14:897-911 (1992). PSORT is an expert system for predicting the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated.
Thereafter, the complete amino acid sequences were further analyzed by visual inspection, applying a simple form of the (-1,-3) rule of von Heinje. von Heinje, supra.
Thus, the TR13 protein is predicted to consist of residues from about 1-750 in SEQ ID N0:2, and/or 1-1001 in SEQ ID N0:40. The mature form of the polypeptide sequence disclosed in SEQ ID
N0:40 is predicted to consist of residues from about 42 to 1001.
As one of ordinary skill would appreciate, due to the possibilities of sequencing errors, as well as the variability of cleavage sites for leaders in different known proteins, the predicted full-length TR13 polypeptide encoded by the deposited cDNA clones comprises about 1001 amino acids, but may be anywhere in the range of about 700 to about 1200 amino acids. It will further be appreciated that, the domains described herein have been predicted by computer analysis, and accordingly, depending on the analytical criteria used for identifying various 3o functional domains, the exact "address" of, for example, the extracelluar domain, intracelluar domain, cysteine-rich domains, and transmembrane domain of TR13 may differ slightly (e.g., the address may "shift" by about 1 to about 20 residues, more likely about 1 to about 5 residues). For example, the exact location of the TR13 cysteine-rich domains in Figures lA-C
(SEQ ID N0:2) and/or Figures 7A-D (SEQ ID N0:40) may vary slightly (e.g., the address may "shift" by about 1 to about 20 residues, more likely about 1 to about 5 residues) depending on the criteria used to define the motifs. In any event, as discussed further below, the invention further provides polypeptides having various residues deleted from the N-terminus and/or C-terminus of the full-length TR13, including polypeptides lacking one or more amino acids from the N-termini of the extracellular domain described herein, which constitute soluble forms of the extracellular domain of the TR13 polypeptides.
As one of ordinary skill would appreciate, due to the possibilities of sequencing errors, the preferred predicted full-length TR14 polypeptide encoded by the deposited cDNA clone comprises about 231 amino acids as shown in SEQ ID N0:61, but may be anywhere in the range of 175-275 amino acids. In an alternative embodiment, predicted full-length TR14 polypeptide comprises about 226 amino acids, but may be anywhere in the range of 175-275 amino acids, but may be anywhere in the range of about 45 to about 200 amino acids. It will to further be appreciated that, the domains described herein have been predicted by computer analysis, and accordingly, that depending on the analytical criteria used for identifying various functional domains, the exact "address" of, for example, the extracelluar domain, intracelluar domain, cysteine-rich domains, and transmembrane domain of TR14 may differ slightly (e.g., the address may "shift" by about 1 to about 20 residues, more likely about 1 to about 5 residues). For example, the exact location of the TR14 extracellular domain and/or cysteine-rich domains in Figures l0A-H (SEQ ID N0:61) or, alternatively Figures 4A-D
(SEQ ID
NO:S) may vary slightly (e.g., the address may "shift" by about 1 to about 20 residues, more likely about 1 to about 5 residues) depending on the criteria used to define the domain.
Additionally, in the event the polypeptide sequence of TR14 is longer than the sequence 2o depicted in Figures l0A-H or, alternatively Figures 4A-D, the skilled artisan would appreciate that the sequence could affect the ultimate location of the extracellular, transmembrane, or intracellular domain. In any event, as discussed further below, the invention further provides polypeptides having various residues deleted from the N-terminus and/or C-terminus of the full-length TR14, including polypeptides lacking one or more amino acids from the N-termini of the extracellular domain described herein, which constitute soluble forms of the extracellular domain of the TR14 polypeptides.
As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
By "isolated" nucleic acid molecules) is intended a nucleic acid molecule, DNA
or RNA, which has been removed from its native environment For example, recombinant DNA
molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA
molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or ire vitro RNA transcripts of the DNA

molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically. However, a nucleic acid molecule contained in a clone that is a member of a mixed clone library (e.g., a genomic or cDNA library) and that has not been isolated from other clones of the library (e.g., in the form of a homogeneous solution containing the clone without other members of the library) or a chromosome isolated or removed from a cell or a cell lysate (e.g., a "chromosome spread", as in a karyotype), is not "isolated" for the purposes of this invention.
Isolated nucleic acid molecules of the present invention include, for example, DNA
molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in Figures lA-C (SEQ ID NO:1), Figures 7A-D (SEQ ID N0:39) and/or contained in a deposited cDNA clone (e.g., HWLHM70 and HWLHN83); DNA molecules comprising, or alternatively consisting of, the coding sequence for the mature TR13 protein shown in Figures lA-C (SEQ
ID NO:1) and/or Figures 7A-D (SEQ ID N0:39) and/or contained in a deposited cDNA clone (e.g., HWLHM70 and HWLHN83); DNA molecules comprising, or alternatively consisting of, a fragment of the coding sequence for the full-length TR13 protein disclosed in Figures lA
C and/or Figures 7A-D and/or encoded by a deposited cDNA clone; and DNA
molecules which comprise, or alternatively consist of, a sequence substantially different from those described above, but which, due to the degeneracy of the genetic code, still encode TR13 polypeptides (including fragments of variants thereof). Of course, the genetic code is well known in the art.
Thus, it would be routine for one skilled in the art to generate such degenerate variants.
Isolated nucleic acid molecules of the present invention include, for example, DNA
molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown preferably in Figures l0A-H (SEQ ID N0:60) or, alternatively, in Figures 4A-D
(SEQ ID
N0:4) and/or contained in the deposited cDNA clone (HMSHK47); DNA molecules comprising, or alternatively consisting of, the coding sequence for the mature TR14 protein shown preferably in Figures l0A-H (amino acids 1-164 of SEQ ID N0:61), or alternatively, in Figures 7A-D (SEQ ID N0:4) and/or contained in the deposited cDNA clone (HMSHK47);
DNA molecules comprising, or alternatively consisting of, a fragment of the coding sequence for the full-length TR14 protein disclosed in preferably in Figures l0A-H or, alternatively, in 3o Figures 4A-D and/or encoded by the deposited cDNA clone (HMSHK47); and DNA
molecules which comprise a sequence substantially different from those described above, but which, due to the degeneracy of the genetic code, still encode TR14 polypeptides (including fragments or variants thereof). Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.
In another aspect, the invention provides isolated nucleic acid molecules encoding the TR13 polypeptide having an amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-349 (HWLHM70). In a further embodiment, nucleic acid molecules are provided that encode the mature form of the TR13 polypeptide disclosed in Figures lA-C
and/or encoded by the cDNA contained in ATCC Deposit No. PTA-349. In a further embodiment, nucleic acids are provided that the full-length TR13 polypeptide disclosed in Figures lA-C and/or encoded by the deposited cDNA clone, but lacking the N-terminal 5 methionine. In a further embodiment, nucleic acid molecules are provided that encode The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:1 or the nucleotide sequence of the TR I 3 cDNA contained in the above-described deposited cDNA clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA
molecules, are 1o useful, for example, as probes for gene mapping by in situ hybridization with chromosomes, and for detecting expression of the TR13 gene in human tissue, for instance, by Northern blot analysis.
In another aspect, the invention provides isolated nucleic acid molecules encoding the TR13 polypeptide having an amino acid sequence as encoded by the cDNA clone contained in 15 ATCC Deposit No. PTA-507 (HWLHN83). In a further embodiment, nucleic acid molecules are provided that encode the mature form of the TR13 polypeptide disclosed in Figures 7A-D, and/or encoded by the cDNA contained in ATCC Deposit No. PTA-507. In a further embodiment, nucleic acid molecules are provided that encode the full-length TR13 polypeptide disclosed in Figures 7A-D, and/or encoded by the deposited cDNA clone, but lacking the N-20 terminal methionine. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID N0:39 or the nucleotide sequence of the TR13 cDNA contained in the above-described deposited cDNA clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful, for example, as probes for gene mapping by in situ hybridization with chromosomes, and for detecting expression of the TR13 gene in human tissue, for instance, by Northern blot analysis.
In another aspect, the invention provides isolated nucleic acid molecules encoding the TR14 polypeptide having an amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-348 (HMSHK47). In a further embodiment, nucleic acid molecules are provided that encode the full-length TR14 polypeptide disclosed in Figures l0A-H or, alternatively, in Figures 4A-D, and/or encoded by the deposited cDNA clone, but lacking the N-terminal methionine. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown preferably in SEQ ID N0:60 or, alternatively, in SEQ ID N0:4 or the nucleotide sequence of the TR14 cDNA contained in the above-described deposited cDNA clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful, for example, as probes for gene mapping by in situ hybridization with chromosomes, and for detecting expression of the TR14 gene in human tissue, for instance, by Northern blot analysis.
In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:I which have been determined from the following related cDNA clones: HETAQ12R (SEQ ID N0:8), HETAK82R (SEQ ID N0:9), HETBH18R
(SEQ ID NO:10), HEPAB26R (SEQ ID NO:11), HETAN38R (SEQ ID N0:12), HPWDD30R
(SEQ ID N0:13), HETATOSR (SEQ ID N0:14), HETDQ39R (SEQ ID NO:15), HETEM84R
(SEQ ID N0:16), and HSIDV42R (SEQ ID N0:17).
In addition, the invention provides nucleic acid molecules having nucleotide sequences l0 related to extensive portions of SEQ ID N0:39 which have been determined from the following related cDNA clones: HETAQ12R (SEQ ID N0:48), HETAK82R (SEQ ID N0:49), HETBM71 R (SEQ ID NO:50), HETBH 18R (SEQ ID NO:51 ), HEPAB26R (SEQ ID N0:52), HETAN38R (SEQ ID N0:53), HPWDD30R (SEQ ID N0:54), HETATOSR (SEQ ID NO:55), HETDQ39R (SEQ ID N0:56), HPWBL93R (SEQ ID N0:57), HETEM84R (SEQ ID N0:58), and HSIDV42R (SEQ ID N0:59).
In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NOS:60 and 4 which have been determined from the following related cDNA clones: HSABDSOR (SEQ ID N0:18), HTXMX53R (SEQ
N0:19), HE20R74R (SEQ ID N0:20), HMSHK47R (SEQ ID N0:21 ), and HMSHK59R
(SEQ ID N0:22).
The present invention is further directed to fragments of the isolated TR13 nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of a deposited cDNA clone (e.g., HWLHN83 and/or HWLHM70), or the nucleotide sequence shown in Figures lA-C (SEQ ID NO:1) and/or Figures 7A-D (SEQ ID
N0:39), or the complementary strand thereto, is intended DNA fragments at least about l5nt, and more preferably at least about 20 nt, or at least 25 nt, still more preferably at least about 30 nt, or at least 35 nt, and even more preferably, at least about 40 nt, or at least about 50 nt in length which are useful, for example, as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, 3o as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:1 and/or SEQ ID N0:39. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA clone or the nucleotide sequence as shown in Figures lA-C (SEQ ID NO:1) and/or Figures 7A-D (SEQ ID N0:39). In this context "about"
includes the particularly recited size, or may be larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
The present invention is further directed to fragments of the isolated TR14 nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA clone (HMSHK47), or the nucleotide sequence shown preferably in Figures l0A-H (SEQ ID N0:60) or, alternatively in Figures 4A-D
(SEQ ID
N0:4), or the complementary strand thereto, is intended DNA fragments at least about l5nt, and more preferably at least about 20 nt, or at least 25 nt, still more preferably at least about 30 nt, or at least 35 nt, and even more preferably, at least about 40 nt, or at least 50 nt, in length which are useful, for example, as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown preferably in Figures l0A-H (SEQ ID N0:60) or, alternatively in Figures 4A-D (SEQ ID N0:4). By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in preferably in SEQ ID
N0:60, or, alternatively, in SEQ ID N0:4. In this context "about" includes the particularly recited size, or 1_5 may be larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Representative examples of TR13 polynucleotide fragments of the invention include, -for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 108, from about 109 to about 159, from about 160 to about 210, from about 211 to about 261, from about 262 to about 273, from about 274 to about 324, from about 325 to about 375, from about~376 to about 426, from about 427 to about 477, from about 478 to about 528, from about 529 to about 579, from about 580 to about 630, from about 631 to about 681, from about 682 to about 732, from about 733 to about 744, from about 745 to about 798, from about 799 to about 849, from about 850 to about 900, from about 901 to about 951, from about 952 to about 1002, from about 1003 to about 1053, from about 1054 to about 1104, from about 1105 to about 1155, from about 1156 to about 1164, from about 1165 to about 1197, from about 1198 to about 1248, from about 1249 to about 1266, from about 1267 to about 1317, from about 1318 to about 1368, from about 1369 to about 1419, from about 1420 to about 1470, from about 1471 to about 1521, 3o from about 1522 to about 1572, from about 1573 to about 1623, from about 1624 to about 1674, from about 1675 to about 1725, from about 1726 to about 1776, from about 1777 to about 1827, from about 1828 to about 1878, from about 1879 to about 1929, from about 1930 to about 1980, from about 1981 to about 2031, from about 2032 to about 2082, from about 2083 to about 2133, from about 2134 to about 2184, from about 2185 to about 2235, from about 2236 to about 2286, from about 2287 to about 2337, from about 2338 to about 2388, from about 2389 to about 2489, from about 2490 to about 2540, from about 2451 to about 2501, from about 2502 to about 2554 of the polynucleotide sequence shown in Figures lA-C

(SEQ ID NO: l ), or the complementary strand thereto, or the cDNA contained in the deposited clone (HWLHM70). Other representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively consist of, a sequence from about nucleotide 1 to about 362, from about 705 to about 830, from about 31 to about 2280, from about 343 to 414, from about 415 to about 459, from about 460 to about 540, 343 to about 540, from about 781 to about 804, from about 805 to about 830, from about 781 to about 822, from about 1021 to about 1260, from about 1768 to about 1812, from about 1813 to about 1866, from about 1768 to about 1866, from about 31 to about 540, from about 660 to about 984, from about 1057 to about 1470, from about 1672 to about 1806, and/or from about 1924 to about 2256 of the polynucleotide sequence shown in Figures lA-C (SEQ
ID NO:1 ), or the complementary strand thereto. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Polynucleotides which hybridize to any l, 2, 3, 4, 5 or more of these polynucleotide fragments are also encompassed by the invention. Moreover, polypeptides encoded by these polynucleotides and/or polynucleotide fragments are also encompassed by the invention.
Additional representative examples of TR13 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 108, from about 109 to about 159, from about 160 to about 210, from about 211 to about 261, from about 262 to about 273, from 2o about 274 to about 324, from about 325 to about 375, from about 376 to about 426, from about 427 to about 477, from about 478 to about 528, from about 529 to about 579, from about 580 to about 630, from about 631 to about 681, from about 682 to about 732, from about 733 to about 744, from about 745 to about 798, from about 799 to about 849, from about 850 to about 900, from about 901 to about 951, from about 952 to about 1002, from about 1003 to about 1053, from about 1054 to about 1104, from about 1105 to about 1155, from about 1156 to about 1164, from about 1165 to about 1197, from about 1198 to about 1248, from about 1249 to about 1266, from about 1267 to about 1317, from about 1318 to about 1368, from about 1369 to about 1419, from about 1420 to about 1470, from about 1471 to about 1521, from about 1522 to about 1572, from about 1573 to about 1623, from about 1624 to about 1674, from about 1675 to about 1725, from about 1726 to about 1776, from about 1777 to about 1827, from about 1828 to about 1878, from about 1879 to about 1929, from about 1930 to about 1980, from about 1981 to about 2031, from about 2032 to about 2082, from about 2083 to about 2133, from about 2134 to about 2184, from about 2185 to about 2235, from about 2236 to about 2286, from about 2287 to about 2337, from about 2338 to about 2388, from about 2389 to about 2489, from about 2490 to about 2540, from about 2451 to about 2501, from about 2502 to about 2554, about 2600 to about 2650, about 2651 to about 2700, about 2701 to about 2750, about 2751 to about 2800, about 2801 to about 2850, about 2851 to about 2900, about 2901 to about 2950, about 2951 to about 3000, about 3001 to about 3050, about 3051 to about 3100, about 3101 to about 3150, about 3151 to about 3200, about 3201 to about 3250, about 3251 to about 3300, and about 3301 to about 3334 of the polynucleotide sequence shown in Figures 7A-D (SEQ ID N0:39), or the complementary strand thereto, or the cDNA contained in the deposited clone (HWLHN83). Other representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively consist of, a sequence from about nucleotide 1 to about 42, from about 181 to about 2775, from about 984 to about 1142, from about 1485 to about 1610, from about 2361 to about 2718, from about 61 to about 3060, from about 58 to to about 3060, from about 58 to about 183, from about 58 to about 2775, from about 2776 to about 2850, from about 2851 to about 3060, from about 868 to about 1320, from about 868 to about 915, from about 925 to about 957, about 960 to about 1017, about 1042 to about 1140, about 1267 to about 1320, about 870 to about 1320, about 1810 to about 1842, about 2038 to about 2079, about 2185 to about 2289, about 2995 to about 3054, about 190 to about 237, about 418 to about 462, about 58 to about 843, about 847 to about 1326, about 1366 to about 2424, about 1812 to about 1842, about 2776 to about 2850, about 2428 to about 3060, about 2851 to about 3060, and/or from about 490 to about 537 of the polynucleotide sequence shown in Figures 7A-D (SEQ ID N0:39), or the complementary strand thereto. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) 2o nucleotides, at either terminus or at both termini. Polynucleotides which hybridize to any 1, 2, 3, 4, 5 or more of these polynucleotide fragments are also encompassed by the invention.
Moreover, polypeptides encoded by these polynucleotides and/or polynucleotide fragments are also encompassed by the invention. Moreover, polypeptides encoded by the polynucleotides and/or polynucleotide fragments are also encompassed by the invention.
Preferably, the polynucleotide fragments of the invention encode a polypeptide which demonstrates a TR13 functional activity. By a polypeptide demonstrating a TR13 "functional activity" is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) TR13 protein. Such functional activities include, but are not limited to, biological activity (e.g., cell proliferation activity), antigenicity (ability to 3o bind (or compete with a TR13 polypeptide for binding) to an anti-TR13 antibody), immunogenicity (ability to generate antibody which binds to a TR 13 polypeptide), ability to form multimers with TR13 polypeptides of the invention, and ability to bind to a receptor or ligand for a TR13 polypeptide.
In one embodiment where one is assaying for the ability to bind or compete with full-length TR13 polypeptide for binding to anti-TR13 antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, 5 immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are 1o within the scope of the present invention.
In another embodiment, where a TR13 ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting.
15 See generally, Phizicky, E., et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, physiological correlates of TR13 binding to its substrates (signal transduction) can be assayed.
In addition, assays described herein (see, for example, Examples 5, and 16-21) and those otherwise known in the art may routinely be applied to measure the ability of TR13 2o polypeptides and fragments, variants derivatives and analogs thereof to elicit a particular biological activity (e.g., to inhibit TRAIL induced apoptosis, to regulate (e.g., inhibit) B cell proliferation (see, e.g., Example 33), to regulate proliferation of other cells, and/or to inhibit hematopoiesis in vitro or in vivo). For example, techniques known in the art (such as for example assaying for thymidine incorporation), may be applied or routinely modified to assay 25 for the ability of the compositions of the invention to regulate (e.g., inhibit apoptosis) and/or to regulate (e.g., inhibit) proliferation of hematopoietic cells. Additionally, assays desribed herein (see e.g., Example 15 and Example 33) and otherwise known in the art may be applied or routinely modified to assay for the ability of the compositions of the invention to inhibit or stimulate B cell proliferation.
3o Representative examples of TR14 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 108, from about 109 to about 159, from about 160 to about 210, from about 211 to about 261, from about 262 to about 273, from about 274 to about 324, from about 325 to about 375, from about 376 to about 426, from about 427 to about 477, from about 478 to about 528, from about 529 to about 579, from about 580 to about 630, from about 631 to about 681, from about 682 to about 732, from about 733 to about 744, from about 745 to about 798, from about 799 to about 849, from about 850 to about 900, from about 901 to about 951, from about 952 to about 1002, from about 1003 to about 1053, from about 1054 to about 1104, from about 1105 to about 1155, from about 1156 to about 1164, from about 1165 to about 1197, from about 1198 to about 1248, from about 1249 to about 1266, from about 1267 to about 1317, from about 1318 to about 1368, from about 1369 to about 1419, from about 1420 to about 1470, from about 1471 to about 1521, from about 1522 to about 1572, from about 1573 to about 1623, from about 1624 to about 1674, from about 1675 to about 1725, from about 1726 to about 1776, from about 1777 to about 1827, from about 1828 to about 1878, from about 1879 to about 1929, from about 1930 to about 1980, from about 1981 to about 2031, from about 2032 to about 2082, from about 2083 to about 2133, from about 2134 to about 2184, from about 2185 to about 2235, from about 2236 to about 2286, from about 2287 to about 2337, from about 2338 to about 2388, from about 2389 to about 2489, from about 2490 to about 2540, from about 2451 to about 2501, from about 2502 to about 2552, from about 2553 to about 2603, from about 2604 to about 2654, from about 2655 to about 2705, from about 2706 to about 2756, from about 2806 to about 2856, from about 2857 to about 2907, from about 2908 to about 2958, from about 2959 to about 3009, from about 3010 to about 3060, from about 3061 to about 3111, and/or from about 3112 to about 3152 of the polynucleotide sequence shown in Figures l0A-H (SEQ
ID N0:60), or the complementary strand thereto, or the cDNA contained in the deposited clone (HMSHK47). Other representative examples of polynucleotide fragments of the invention 2o include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 1451, from about 1761 to about 2251, from about 89 to about 766, from about 89 to about 487, from about 488 to about 538, from about 539 to about 766, from about 92 to about 160, from about 212 to about 243, from about 281 to about 313, from about 314 to about 343, from about 281 to about 343, from about 325 to about 433, and/or 550 to about 766 of the polynucleotide sequence shown in Figures l0A-H (SEQ ID
N0:60), or the complementary strand thereto. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Polynucleotides which hybridize to any 1, 2, 3, 4, 5 or more of these polynucleotide fragments are also encompassed by the invention. Moreover, polypeptides encoded by these polynucleotides and/or polynucleotide fragments are also encompassed by the invention.
Alternative representative examples of TR14 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 108, from about 109 to about 159, from about 160 to about 210, from about 211 to about 261, from about 262 to about 273 , from about 274 to about 324, from about 325 to about 375, from about 376 to about 426, from about 427 to about 477, from about 478 to about 528, from about 529 to about 579, from about 580 to about 630, from about 631 to about 681, from about 682 to about 732, from about 733 to about 744, from about 745 to about 798, from about 799 to about 849, from about 850 to about 900, from about 901 to about 951, from about 952 to about 1002, from about 1003 to about 1053, from about 1054 to about 1104, from about 1105 to about 1155, from about 1156 to about 1164, from about 1165 to about 1197, from about 1198 to about 1248, from about 1249 to about 1266, from about 1267 to about 1317, from about 1318 to about 1368, from about 1369 to about 1419, from about 1420 to about 1470, from about 1471 to about 1521, from about 1522 to about 1572, from about 1573 to about 1623, from about 1624 to about 1674, from about 1675 to about 1725, from about 1726 to about 1776, from about 1777 to about 1827, from about 1828 to about 1878, from about 1879 to about 1929, to from about 1930 to about 1980, from about 1981 to about 2031, from about 2032 to about 2082, from about 2083 to about 2133, from about 2134 to about 2184, from about 2185 to about 2235, from about 2236 to about 2286, from about 2287 to about 2337, from about 2338 to about 2388, from about 2389 to about 2489, from about 2490 to about 2540, from about 2451 to about 2501, from about 2502 to about 2552, from about 2553 to about 2603, from about 2604 to about 2654, from about 2655 to about 2705, from about 2706 to about 2756, from about 2806 to about 2856, from about 2857 to about 2907, from about 2908 to about 2958, from about 2959 to about 3009, from about 3010 to about 3060, from about 3061 to about 3111, from about 3112 to about 3162, from about 3163 to about 3213, from about 3214 to about 3264, from about 3265 to about 3315, from about 3316 to about 3366, from about 3367 to about 3417, from about 3418 to about 3468, from about 3469 to about 3519, from about 3520 to about 3566, from about 3567 to about 3599, from about 3600 to about 3649, from about 3650 to about 3699, from about 3700 to about 3749, from about 3750 to about 3799, and/or from 3800 to about 3861 of the polynucleotide sequence shown in Figures 4A-D
(SEQ ID N0:4), or the complementary strand thereto, or the cDNA contained in the deposited clone (HMSHK47). Other representative examples . of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 1451, from about 1761 to about 2251, from about 3133 to about 3861, from about 89 to about 766, from about 89 to about 487, from about 488 to about 538, from about 539 to about 766, from about 92 to about 160, from about 212 to 3o about 243, from about 281 to about 313, from about 314 to about 343, from about 281 to about 343, from about 325 to about 433, and/or 550 to about 766 of the polynucleotide sequence shown in Figures 4A-D (SEQ ID N0:4), or the complementary strand thereto. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Polynucleotides which hybridize to any 1, 2, 3, 4, 5 or more of these polynucleotide fragments are also encompassed by the invention.
Moreover, polypeptides encoded by these polynucleotides and/or polynucleotide fragments are also encompassed by the invention.

Preferably, the polynucleotide fragments of the invention encode a polypeptide which demonstrates a TR14 functional activity. By a polypeptide demonstrating a TR14 "functional activity" is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) TR14 protein. Such functional activities include, but are not limited to, biological activity, antigenicity (ability to bind (or compete with a TRI4 polypeptide for binding) to an anti-TR14 antibody), immunogenicity (ability to generate antibody which binds to a TR14 polypeptide), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a TR14 polypeptide.
In one embodiment where one is assaying for the ability to bind or compete with full-length TR14 polypeptide for binding to anti-TRI4 antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by. detecting a label on the primary antibody. In 2o another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
In another embodiment, where a TRI4 ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting.
See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, physiological correlates of TR14 binding to its substrates (signal transduction) can be assayed.
3o In addition, assays described herein (see, for example, Examples 5 and 15-21 and 33 and those otherwise known in the art may routinely be applied to measure the ability of TRI4 polypeptides and fragments, variants derivatives and analogs thereof to elicit a particular biological activity (e.g., to inhibit TRAIL induced apoptosis, to regulate (e.g., inhibit) B cell proliferation (see, e.g., Example 33), and/or to inhibit hematopoiesis in vitro or in vivo). For example, techniques known in the art (such as for example assaying for thymidine incorporation), may be applied or,routinely modified to assay for the ability of the compositions of the invention to regulate (e.g., inhibit apoptosis) and/or to regulate (e.g., inhibit) proliferation of hematopoietic cells. Additionally, assays desribed herein (see e.g., Example 15 and Example 33) and otherwise known in the art may be applied or routinely modified to assay for the ability of the compositions of the invention to inhibit or stimulate B
cell proliferation.
Other methods will be known to the skilled artisan and are within the scope of the invention.
Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of any combination of one, two, three or all four TR13 cysteine rich domains (amino acid residues from about 105 to about 170, from about 251 to about 264, from about 331 to about 410 and from about 580 to about 610 in Figures lA-C (amino acids from about 105 to about 170, from about 251 to about 265, from about 331 to about 410 and from about 580 to about 610 in SEQ ID NO:1). Since, as discussed above, the location of these domains have been predicted by computer analysis, one of ordinary skill would appreciate that the amino acid residues constituting these domains may be the particularly recited ranges for each domain or t5 may vary slightly (e.g., by about I , 2, 3, 4, 5, 10, or 15 residues at either extreme or at both extremes) depending on the criteria used to define each domain.
Additional preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of the TR13 receptor extracellular domain (amino acids 1 to 906 in 2o Figures 7A-D); a polypeptide comprising or alternatively, consisting of, the mature TR13 receptor extracellular domain (amino acids 42 to 906 in Figures 7A-D); a polypeptide comprising or alternatively, consisting of, one or more of the TR13 cysteine rich domains disclosed in Figures 7A-D (e.g., amino acid residues from about 271 to about 421, from about 271 to about 286, about 290 to about 300, about 301 to about 320, about 329 to about 361, 25 about 404 to about 421, and from about 585 to about 595 in Figures 7A-D
(amino acid residues from about 271 to about 421, from about 271 to about 286, about 290 to about 300, about 301 to about 320, about 329 to about 361, about 404 to about 421, and from about 585 to about 595 in SEQ ID N0:39 and SEQ ID N0:40); a polypeptide comprising, or alternatively, consisting of the TR13 transmembrane domain (amino acids 907 to 931 in Figures 7A-D); and 30 a polypeptide comprising, or alternatively consisting of the TR13 intracellular domain (amino acid 932 to 1001 in Figures 7A-D). As above, since the location of these domains have been predicted by computer analysis, one of ordinary skill would appreciate that the amino acid residues constituting these domains may be the particularly recited ranges for each domain or may vary slightly (e.g., by about 1, 2, 3, 4, 5, 10, or 15 residues at either extreme or at both 35 extremes) depending on the criteria used to define each domain.
It is believed that the cysteine rich motifs of TR13 disclosed in Figures lA-C
are important for interactions between TR13 and its ligands. Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of amino acid residues from about 105 to about 170, from about 251 to about 265, from about 331 to about 410, and from about 580 to about 610 of SEQ ID NO:S (corresponding to amino acid residues from about 105 to about 5 170, from about 251 to about 265, from about 331 to about 410, and from abour 580 to about 610 of Figures 4A-D). In a specific embodiment, the polynucleotides encoding polypeptides of the invention comprise or alternatively consist of, polynucleotide sequences encoding any combination of 2, 3, or all four of the cysteine-rich motifs of TR13. In this context, "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, 10 or 1) nucleotides, at either terminus or at both termini. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
Further, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of amino acid residues from about 271 to about 421, from about 271 to about 286, from about 290 to 15 about 300, from about 301 to about 320, about 329 to about 361, about 404 to about 421, and about 585 to about 595 of SEQ ID N0:40 (corresponding to amino acid residues from about 271 to about 421, from about 271 to about 286, from about 290 to about 300, from about 301 to about 320, about 329 to about 361, about 404 to about 421, and about 585 to about 595 of Figures 7A-D). In a specific embodiment, the polynucleotides encoding TR13 polypeptides of 2o the invention comprise or alternatively consist of, polynucleotide sequences encoding any combination of 2, 3, or all four of the cysteine-rich motifs of TR13 disclosed in Figures 7A-D.
In this context, "about" includes the particularly recited ranges, larger or smaller by several (S, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Polypeptides encoded by these polynucleotides are also encompassed by the invention.
25 Preferred nucleic acid fragments of the invention encode a full-length TR13 polypeptide lacking the nucleotides encoding the amino terminal methionine (e.g., nucleotides 34-750 in SEQ ID NO:1), as it is known that the methionine is cleaved naturally and such sequences may be useful in genetically engineering TR13 expression vectors. Polypeptides encoded by such nucleic acids are also contemplated by the invention.
3o Preferred nucleic acid fragments of the invention encode a full-length TR13 polypeptide lacking the nucleotides encoding the amino terminal methionine (e.g., nucleotides 61-1001 in Figures 7A-D and SEQ ID N0:39), as it is known that the methionine is cleaved naturally and such sequences may be useful in genetically engineering TR13 expression vectors.
Polypeptides encoded by such nucleic acids are also contemplated by the invention.
Preferred nucleic acid fragments of the present invention further include nucleic acid molecules encoding epitope-bearing portions of the TR13 receptor protein. In particular, such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising or alternatively consisting of, amino acid residues from about 1 to about 170 in Figures lA-C (corresponding to about amino acid 1 to about 170 in N0:2); a polypeptide comprising or alternatively consisting of, amino acid residues from about 210 to about 318 in Figures lA-C (corresponding to about amino acid 210 to about 318 in SEQ
ID N0:2); a polypeptide comprising or alternatively consisting of, amino acid residues from about 343 to about 480 in Figures lA-C (corresponding to about amino acid 343 to about 480 in SEQ ID N0:2); a polypeptide comprising or alternatively consisting of, amino acid residues from about 548 to about 592 in Figures lA-C (corresponding to about amino acid 548 to about 592 in SEQ ID N0:2); and a polypeptide comprising or alternatively consisting of, amino acid to residues from about 632 to about 742 in Figures lA-C (corresponding to about amino acid 632 to about 742 in SEQ ID N0:2). The inventors have determined that the above polypeptide fragments are antigenic regions of the TR13 protein. Methods for determining other such epitope-bearing portions of the TR13 protein are described in detail below.
Preferred nucleic acid fragments of the present invention further include nucleic acid molecules encoding antigenic fragments of the TR13 receptor protein. In particular, such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising or alternatively consisting of, amino acid residues from about M1 to about A9, about K12 to about L20, about N47 to about T55, about H58 to about S66, about D63 to about S71, about P77 to about F85, about A90 to about Q98, about F136 to about Q 144, about S 152 to about C 160, about 8159 to about A 167, about A211 to about M219, about M235 to about V243, about V266 to about V274, about W277 to about S285, about I290 to about F298, about A310 to about V318, about E343 to about C351, about I360 to about H368, about 6391 to about I399, about F409 to about T417, about S436 to about Y444, about C453 to about S461, about I472 to about S480, about Y548 to about S556, about C557 to about I565, about V567 to about V575, about T584 to about 6592, about 8632 to about 6640, about W680 to about Y688, about Q684 to about K692, about T698 to about A706, about S726 to about S734, and about S734 to about L742 of SEQ ID N0:2 (Figures lA-C) correspond to the highly antigenic regions of the TR13 protein, predicted using the Jameson-Wolf antigenic index (See Figure 3 and Table I). These highly antigenic fragments correspond to the amino acid residues illustrated in Figure lA-C and in SEQ ID N0:2. In this context, "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Methods for determining other such antigenic fragments of the TR13 protein are described in detail below.
Additional preferred nucleic acid fragments of the present invention further include nucleic acid fragments encoding: a polypeptide comprising or alternatively consisting of, amino acid residues from about 1 to about 262 in Figures 7A-D (corresponding to about amino acid 1 to about 262 in SEQ ID N0:40); a polypeptide comprising or alternatively consisting of, amino acid residues from about 264 to about 423 in Figures 7A-D (corresponding to about amino acid 264 to about 423 in SEQ ID N0:40); a polypeptide comprising or alternatively consisting of, amino acid residues from about 437 to about 789 in Figures 7A-D (corresponding to about amino acid 437 to about 789 in SEQ ID N0:40); and a polypeptide comprising or alternatively consisting of, amino acid residues from about 791 to about 1001 in Figures 7A-D
(corresponding to about amino acid 791 to about 1001 in SEQ ID N0:40). The inventors have determined that the above polypeptide fragments are antigenic regions of the TR13 protein.
Methods for determining other such epitope-bearing portions of the TR13 protein are described in detail below.
Additional preferred nucleic acid fragments of the present invention encoding antigenic fragments of the TR13 receptor protein include nucleic acid molecules encoding: a polypeptide comprising or alternatively consisting of, amino acid residues from about M1 to about H9, about V 14 to about I22, about H47 to about H55, about C61 to about R69, about L82 to about E90, about D102 to about P110, about K109 to about S117, about F124 to about H132, about M141 to about E149, about 5146 to about C154, about S157 to about W165, about F168 to about T 176, about N 182 to about N 190, about Q207 to about A215, about P213 to about M221, about M221 to about E229, about V233 to about V241, about T253 to about V261, about T282 to about S290, about N298 to about T306, about C308 to about Y316, about K315 to about S323, about P328 to about F336, about A341 to about Q349, about F387 to about Q395, about S403 to about C411, about T409 to about P417, about F443 to about N451, about W451 to about Y459, about A462 to about M470, about 6478 to about M486, about A487 to about A495, about V517 to about V525, about T527 to about Q535, about I541 to about F549, about A561 to about V569, about E594 to about C602, about I611 to about H619, about 6643 to about I650, about P686 to about K694, about C704 to about S712, about 8722 to about I730, about E727 to about T735, about P746 to about 6754, about D776 to about L784, about Y799 to about S807, about C808 to about I816, about V818 to about V826, about T835 to about 6843, about 8883 to about 6891, about K932 to about K940, about Q935 to about K943, about T949 to about A957, about S977 to about S985, about S981 to about P989, and about N986 to about L994 of SEQ ID N0:40 (Figures 7A-D) correspond to the highly antigenic regions of the TR13 protein, predicted using the Jameson-Wolf antigenic index (See Figure 9 and Table III). These highly antigenic fragments correspond to the amino acid residues illustrated in Figure 7A-D and in SEQ ID N0:40. In this context, "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Methods for determining other such antigenic fragments of the TR13 protein are described in detail below.
Additionally, it is believed that the extracellular cysteine rich motif of TR14 disclosed in Figures l0A-H or, alternatively, Figures 4A-D is important for interactions between TR14 and its ligands. Accordingly, specific embodiments of the invention are directed to nucleic acid molecules encoding polypeptides which comprise, or alternatively consist of, preferably amino acids Cys-31 to Cys-104 of Figures l0A-B and SEQ ID N0:61, or, alternatively, the amino acid sequence of amino acid residues from about 70 to about 90 of Figure 10A
and SEQ 1D
N0:61 (corresponding to amino acid residues from about 65 to about 85 of Figures 4A-D or SEQ ID NO:S). In this context, "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Polypeptides encoded by these polynucleotides are also encompassed by the invention.
Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of, the TR14 receptor extracellular domain (preferably amino acid residues from about 1 to 138 in Figures l0A-H or, alternatively, from about 1 to about 133 in Figures 4A-D);
a polypeptide comprising or alternatively, consisting of, the TR14 cysteine rich domain (preferably amino acid residues from about 31 to about 104 of Figures l0A-H, or amino acid residues from about 70 to 90 in Figures 10A, or, alternatively, from about 65 to about 85 in Figures 4A-D); a polypeptide comprising or alternatively, consisting of the transmembrane domain (preferably amino acid residues from about 139 to 155 in Figures l0A-H or, alternatively, 134 to about 150 in Figures 4A-D); and a polypeptide comprising or alternatively, consisting of, the TR14 intracellular domain (preferably amino acid residues from about 156 to about 231 in Figures l0A-H or, alternatively, amino acid residues from about 151 to about 226 in Figures 4A-D). Since the location of these domains have been predicted by computer analysis, one of ordinary skill would appreciate that the amino acid residues constituting these domains may be the particularly recited ranges for each domain or may vary slightly (e.g., by about 1, 2, 3, 4, 5, 10, or 15 residues at either extreme or at both extremes) depending on the criteria used to define each domain.
Preferred nucleic acid fragments of the invention encode a full-length TR14 polypeptide lacking the nucleotides encoding the amino terminal methionine (e.g., nucleotides 70-759 of Figures l0A-H or SEQ ID N0:60, or nucleotides 102-765 in SEQ ID N0:4), as it is known that the methionine is cleaved naturally and such sequences may be useful in genetically 3o engineering TR14 expression vectors. Polypeptides encoded by such nucleic acids are also contemplated by the invention.
Preferred nucleic acid fragments of the present invention further include nucleic acid molecules encoding epitope-bearing portions of the TR14 receptor protein. In particular, preferred epitope-bearing polypeptides of the present invention comprise, or alternatively consist of, one, two, three, four, five, six, or all six of the immunogenic epitopes of the TR14 protein shown in SEQ ID NO: 61 as residues: Asp-2 to Asp-10, Thr-17 to Asp-38, Pro-45 to Ser-52, Pro-88 to Arg-95, Thr-108 to Glu-115, Thr-131 to Glu-136, Phe-166 to Gly-174, Ala-180 to Ala-200, and Gln-224 to Met-231. Fragments and/or variants of these polypeptides, such as, for example, fragments and/or variants as described herein, are encompassed by the invention. Polynucleotides encoding these polypeptides (including fragments and/or variants) are also encompassed by the invention, as are antibodies that bind these polypeptides.
Alternatively, such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising or alternatively consisting of, amino acid residues from about 2 to about 24 in Figures 4A-D (corresponding to about amino acid 2 to about 24 in SEQ ID N0:5); a polypeptide comprising or alternatively consisting of, amino acid residues from about 42 to about 52 in Figures 4A-D (corresponding to about amino acid 42 to about 52 in SEQ ID NO:S); a polypeptide comprising or alternatively consisting of, amino acid residues from about 80 to about 115 in Figures 4A-D (corresponding to about amino acid 80 to about 115 in SEQ ID NO:S and about amino acid 85 to about 120 of SEQ ID
N0:61); and a polypeptide comprising or alternatively consisting of, amino acid residues from about 155 to about 226 in Figures 4A-D (corresponding to about amino acid 155 to about 226 in SEQ ID
N0:5 and about amino acid 160 to about amino acid 231 of SEQ ID N0:61). The inventors have determined that the above polypeptide fragments are antigenic regions of the TR14 protein. Methods for determining other such epitope-bearing portions of the TR14 protein are described in detail below.
Alternative nucleic acid fragments of the present invention further include nucleic acid molecules encoding antigenic fragments of the TR14 receptor protein. In particular, such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising or alternatively consisting of, amino acid residues of SEQ ID N0:5 (Figures 4A-D) from about T3 to about S 11, from about V 16 to about R24, from about Q44 to about M52, from about F85 to about G93 (about F90 to about G98 of SEQ ID
N0:61), from about T103 to about V 111 (about T108 to about V 116 of SEQ ID N0:61), from about F161 to about 6169 (about F165 to about 6174 of SEQ ID N0:61 ), from about V 187 to about A 195 (from about V 192 to about A200 of SEQ ID N0:61 ), from about P218 to about M226 (about P223 to about M231 of SEQ ID N0:61) correspond to the highly antigenic regions of the TR14 protein, predicted using the Jameson-Wolf antigenic index (See Figure 11 and Table IV).
3o These highly antigenic fragments correspond to the amino acid residues illustrated in Figure 4A-D and in SEQ ID NO:S (or Figures l0A-H and SEQ ID N0:61, as indicated above). In this context, "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Methods for determining other such antigenic fragments of the TR14 protein are described in detail below.
The data presented in Figure 3 are also represented in tabular form in Table I. The columns in Table I are labeled with the headings "Res", "Position", and Roman Numerals I-XIV. The column headings refer to the following features of the amino acid sequence presented in Figure 3 and Table I:

"Res": amino acid residue of SEQ ID N0:2 and Figures IA-1C; "Position":
position of the corresponding residue within SEQ ID N0:2 and Figures IA-C; I: Alpha, Regions - Gamier-Robson; II: Alpha, Regions Chou-Fasman; III: Beta, Regions - Gamier-Robson; IV: Beta, Regions - Chou-Fasman; V: Turn, Regiot - Gamier-Robson; VI: Turn, Regions - Chou-Fasman; VII: Coil, Regions - Gamier-Robson; VIII:
5 Hydrophilicity Plot - Kyte-Doolittle; IX: Hydrophobicity Plot - Hopp-Woods;
X: Alpha, Amphipathic Regions - Eisenberg; XI: Beta, Amphipathic Regions - Eisenberg; XII: Flexible Regions - Karplus-Schul XIII: Antigenic Index - Jameson-Wolf; and XIV: Surface Probability Plot -Emini.
The data presented in Figure 9 are also represented in tabular form in Table III. The columns in Table III are labeled with the headings "Res", "Position", and Roman Numerals I-XIV . The column t o headings refer to the following features of the amino acid sequence presented in Figure 9 and Table III:
"Res": amino acid residue of SEQ ID N0:40 and Figures 7A-D; "Position":
position of the corresponding residue within SEQ ID N0:40 and Figures 7A-D; I: Alpha, Regions - Garnier-Robson; II: Alpha, Regior - Chou-Fasman; III: Beta, Regions - Gamier-Robson; IV: Beta, Regions - Chou-Fasman; V: Turn, Regions - Garnier-Robson; VI: Turn, Regions - Chou-Fasman; VII: Coil, Regions -Gamier-Robson;
15 VIII: Hydrophilicity Plot - Kyte-Doolittle; IX: Hydrophobicity Plot - Hopp-Woods; X: Alpha, Amphipathic Regions - Eisenberg; XI: Beta, Amphipathic Regions - Eisenberg;
XII: Flexible Regions -Karplus-Schulz; XIII: Antigenic Index - Jameson-Wolf; and XIV: Surface Probability Plot - Emini..
In additional embodiments, the nucleic acid molecule of the invention encodes a polypeptide comprising, or alternatively consisting of, a functional attribute of TR13.
20 Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index 25 regions of TR13.
The data representing the structural or functional attributes of TR13 (SEQ ID
N0:40) set forth in Figure 9 and/or Table III, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table III
can be used to 30 determine regions of TR13 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, andlor XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

35 Certain preferred regions in these regards are set out in Figure 3, but may, as shown in Table I, be represented or identified by using tabular representations of the data presented in Figure 3. The DNA*STAR computer algorithm used to generate Figure 3 (set on the original default parameters) was used to present the data in Figure 3 in a tabular format (See Table I).
The tabular format of the data in Figure 3 may be used to easily determine specific boundaries of a preferred region.
Certain preferred regions in these regards are set out in Figure 9, but may, as shown in Table III, be represented or identified by using tabular representations of the data presented in Figure 9. The DNA*STAR computer algorithm used to generate Figure 9 (set on the original default parameters) was used to present the data in Figure 9, in a tabular format (See Table I11).
The tabular format of the data in Figure 9 may be used to easily determine specific boundaries of a preferred region.
l0 The above-mentioned preferred regions set out in Figure 3 and in Table I
include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figure 1. As set out in Figure 3 and in Table I, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and ~5 Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.
The above-mentioned preferred regions set out in Figure 9 and in Table III
include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid 20 sequence set out in Figure 9. As set out in Figure 9 and in Table III, such preferred regions include Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-25 forming regions.
The data presented in Figure 11 are also represented in tabular form in Table IV. The columns in Table IV are labeled with the headings "Res", "Pos", and Roman Numerals I-XIV.
The column heading refer to the following features of the amino acid sequence presented in Figures l0A-H, and 11: "Res":
amino acid residue of SEQ ID N0:61 and Figures l0A-H; "Pos": position of the corresponding residue 30 within SEQ ID N0:61 and Figures I OA-H; I: Alpha, Regions - Gamier-Robson;
II: Alpha, Regions -Chou-Fasman; III: Beta, Regions - Gamier-Robson; IV: Beta, Regions - Chou-Fasman; V: Turn, Region - Garnier-Robson; VI: Turn, Regions - Chou-Fasman; VII: Coil, Regions - Gamier-Robson; VIII:
Hydrophilicity Plot - Kyte-Doolittle; IX: Hydrophobicity Plot - Hopp-Woods; X:
Alpha, Amphipathic Regions - Eisenberg; XI: Beta, Amphipathic Regions - Eisenberg; XII: Flexible Regions - Karplus-Schul 35 XIII: Antigenic Index - Jameson-Wolf; and XIV: Surface Probability Plot -Emini.
The data presented in Figure 6 are also represented in tabular form in Table II. As above, the columns in Table II are labeled with the headings "Res", "Position", and Roman Numerals I-XIV. The column headings refer to the following features of the amino acid sequence presented in Figure 6 and Table II: "Res": amino acid residue of SEQ ID NO:S and Figures 4A-D;
"Position": position of the corresponding residue within SEQ ID NO:S and Figures 4A-D; I: Alpha, Regions -Gamier-Robson; II:
Alpha, Regions - Chou-Fasman; III: Beta, Regions - Gamier-Robson; IV: Beta, Regions - Chou-Fasmar V: Turn, Regions - Gamier-Robson; VI: Turn, Regions - Chou-Fasman; VII: Coil, Regions - Garnier-Robson; VIII: Hydrophilicity Plot - Kyte-Doolittle; IX: Hydrophobicity Plot -Hopp-Woods; X: Alpha, Amphipathic Regions - Eisenberg; XI: Beta, Amphipathic Regions - Eisenberg;
XII: Flexible Regions -Karplus-Schulz; XIII: Antigenic Index - Jameson-Wolf; and XIV: Surface Probability Plot - Emini.
In additional embodiments, the polynucleotides of the invention encode functional l0 attributes of TR14. Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of TR14.
The data representing the structural or functional attributes of TR13 (SEQ ID
N0:2) set forth in Figure 3 and/or Table I, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table I can be used to determine regions 2o of TR13 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV
by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
The data representing the structural or functional attributes of TR 14 (SEQ ID
N0:61, as set forth in Figure 11 and/or Table IV; or, alternatively, SEQ ID N0:5, as set forth in Figure 6 and/or Table II), as described above, were generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table II can be used to determine regions of TR14 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, 1X, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
Certain preferred regions in these regards are set out in Figure 6, but may, as shown in Table II, be represented or identified by using tabular representations of the data presented in Figure 6. The DNA*STAR computer algorithm used to generate Figure 6 (set on the original default parameters) was used to present the data in Figure 6 in a tabular format (See Table II).
The tabular format of the data in Figure 6 may be used to easily determine specific boundaries of a preferred region.
Certain even more preferred regions in these regards are set out in Figure 11, but may, as shown in Table IV, be represented or identified by using tabular representations of the data presented in Figure 11. The DNA*STAR computer algorithm used to generate Figure 11 (set on the original default parameters) was used to present the data in Figure 11 in a tabular format (See Table IV). The tabular format of the data in Figure 11 may be used to easily determine specific boundaries of a preferred region.
l0 The above-mentioned preferred regions set out in Figure 11 and in Table IV
include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figures l0A-H. As set out in Figure 11 and in Table IV, such preferred regions include Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.
The above-mentioned preferred regions set out in Figure 6 and in Table II
include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid 2o sequence set out in Figures 4A-D. As set out in Figure 6 and in Table II, such preferred regions include Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.

Table I

Res Pos II III IV V VI VII VIII IX X XI XII XIII XIV
I

Met 1 . . B . . . . 0.73 -0.31. . . 1.40 1.44 Asp 2 . . . . T T . 1.12 -0.26. . . 2.25 1.62 Gln 3 . . . . T T . 0.92 -0.29* . . 2.50 2.20 Ser 4 . . . . . T C 0.64 -0.21. . . 2.05 2.24 Thr 5 . . B . . T . 0.44 -0.26. . F 1.60 0.72 Gln 6 . A B . . . . 0.70 0.24. . F 0.35 0.42 Ala 7 . A B . . . . 0.70 0.27. . . -0.050.31 Cys 8 . A . . T . . 0.74 -0.11. . . 0.70 0.37 Ala 9 . A . . T . . 1.01 -0.60. . . 1.00 0.43 Gly 10 . A . . T . . 0.66 -0.50. . F 0.85 0.58 Glu 11 . A . . T . . 0.62 -0.43. . F 0.85 0.58 Lys 12 . A . . T . . 1.21 -0.50. . F 1.10 0.78 His 13 . A . . T . . 1.99 -0.60. * . 1.65 1.27 Cys 14 . A . . T . . 2.23 -1.03. . . 1.90 1.43 His 15 . . . . T . . 2.23 -0.60. * . 2.20 0.71 Asn 16 . . . . T T . 1.42 -0.17. * F 2.50 0.52 Arg 17 . . . . T T . 1.34 0.01* * F 1.65 0.79 Gly 18 . . . . T T . 0.68 -0.06* * F 2.00 0.79 Gly 19 . . . . T T . 1.46 0.23* * F 1.15 0.43 Leu 20 . A . . . . C 0.89 -0.17* * . 0.75 0.43 His 21 . A B . . . . 0.08 0.44* * . -0.600.43 Phe 22 . A B . . . . -0.240.70. * . -0.600.36 Arg 23 . A B . . . . -0.710.70. * . -0.600.67 Met 24 . A B . . . . -0.370.70. * . -0.600.40 Leu 25 . A B . . . . 0.13 0.60. * . -0.600.81 Pro 26 . A . . . . C -0.120.30. * . -0.100.60 Leu 27 . . . B T . . 0.54 1.21* . . -0.200.63 Gln 28 . . . B T . . -0.421.10. . . -0.051.05 Thr 29 . . . B T . . -0.491.06* . . -0.200.50 Trp 30 . . B B . . . 0.43 1.20* . . -0.600.33 His 31 . . B B . . . 0.64 0.51* . . -0.600.37 Val 32 . . B B . . . 0.87 0.51* . . -0.600.44 Cys 33 . . B B . . . 0.52 0.53* . . -0.600.43 Arg 34 . . B B . . . 0.02 0.04* . . -0.300.31 Gln 35 . . . B T . . -0.500.23* . . 0.10 0.34 Ala 36 . . . B T . . -1.170.27* . . 0.10 0.53 Gly 37 . . . B T . . -1.120.49* . . -0.200.23 Leu 38 . . B B . . . -0.461.17* . . -0.600.11 Leu 39 . . B B . . . -0.881.17* . . -0.600.19 Phe 40 . . B B . . . -1.691.16* . . -0.600.28 Leu 41 . . B B . . . -1.311.41* . . -0.600.28 Gln 42 . . B B . . . -1.271.16. . . -0.600.52 Thr 43 . . B B . . . -0.460.86. . F -0.450.81 Leu 44 . . B B . . . 0.06 0.47. . F -0.301.57 Pro 45 . . . . . T C 0.51 0.17* . F 0.60 1.22 Ser 46 . . . . T T . 1.02 0.53. . F 0.50 1.32 Asn 47 . . . . T T . 1.02 0.43. . F 0.84 2.15 Ser 48 . . . . . T C 1.38 0.14. . F 1.28 2.24 Tyr 49 . . . . T . . 1.84 -0.29. . F 2.22 3.34 Ser 50 . . . . . . C 2.06 -0.24. . F 2.36 2.05 Asn 51 . . . . T T . 2.04 -0.64. . F 3.40 2.65 Lys 52 . . . . T T . 1.74 -0.54. . F 3.06 2.44 Gly 53 . . . . T T . 1.38 -0.91. * F 2.72 2.44 Glu 54 . . . . T T . 1.59 -0.73* * F 2.23 0.81 Thr 55 . . . . T T . 1.89 -0.63* . F 1.89 0.55 Ser 56 . . B . . T . 1.22 -0.23* . F 0.85 0.97 Cys 57 . . B . . T . 1.18 -0.09. * . 1.04 0.30 His 58 . . B . . T . 1.31 -0.09. * . 1.38 0.35 Gln 59 . . . . T . . 1.31 -0.14 . . 1.920.40 .

Cys 60 . . . . T . . 1.67 -0.53 . . 2.711.25 .

Asp 61 . . . . T T . 1 -1.10 . F 3.401.84 .72 .

Pro 62 . . . . T T . 2.09 -0.84 . F 3.061.66 .

5 Asp 63 . . . . T T . 2.12 -0.86 . F 3.064.15 .

Lys 64 . . . . T T . 2.17 -1.43 * F 3.064.31 *

Tyr 65 . . B . . T . 2.49 -1.43 . F 2.665.57 *

Ser 66 . . B . . T . 2.19 -1.43 . F 2.663.30 *

Glu 67 . . . . T T . 2.10 -1.04 . F 3.402.21 *

10 Lys 68 . . . . T T . 1.80 -0.66 * F 3.061.89 *

Gly 69 . . . . T . . 1.09 -1.03 * F 2.52I
* .89 Ser 70 . . . . T T . 1.33 -0.84 * F 2.230.59 .

Ser 71 . . . . T T . 0.78 -0.44 * F 1.590.47 .

Ser 72 . . . . T T . 0.89 0.20 * F 0.650.35 .

15 Cys 73 . . . . T T . 0.63 -0.23 * F 1.250.52 .

Asn 74 . . . . T . . 0.39 -0.19 * . 0.900.60 .

Val 75 . . B . . . . 0.02 -0.07 * . 0.500.45 *

Arg 76 . . B . . T . 0.01 0.11 * . 0.100.45 .

Pro 77 . . B . . T . 0.31 0.03 * . 0.440.40 .

20 Ala 78 . . B . . T . 1.02 -0.37 * . 1.380.91 .

Cys 79 . . B . . T . 1.02 -1.01 * . 2.020.93 .

Thr 80 . . B . . . . 1.63 -1.01 * F 2.311.00 *

Asp 81 . . . . T T . 0.82 -0.69 . F 3.401.55 *

Lys 82 . . . . T T . 0.79 -0.40 . F 2.762.50 .

25 Asp 83 . . . . T T . 1.07 -0.21 . F 2.422.72 .

Tyr 84 . . B . . T . 1.70 -0.21 . . 1.532.35 .

Phe 85 . . B B . . . I.70 0.29 . . 0.191.60 .

Tyr 86 . . B B . . . 1.11 0.77 . . -0.451.38 .

Thr 87 . . B B . . . 0.40 1.27 . . -0.600.89 .

30 His 88 . . B B . . . 0.40 1.09 . . -0.600.55 .

Thr 89 . . B B . . . 0.06 0.30 . . -0.300..59 .

Ala 90 . . B B . . . 0.76 0.04 * . 0.000.41 .

Cys 91 . . . B T . . 0.66 -0.04 * . 1.300.49 .

Asp 92 . . . . T T . 0.97 -0.11 * . 2.000.33 .

35 Ala 93 . . . . . T C 0.69 -0.60 * F 2.550.57 .

Asn 94 . . . . . T C 1.00 -0.61 * F 3.001.54 .

Gly 95 . . . . . T C 0.78 -0.79 * F 2.701.60 .

Glu 96 . A . . . . C 0.84 -0.10 * F 1.701.30 .

Thr 97 . A B . . . . 0.60 0.01 * F 0.450.80 .

40 Gln 98 . A B . . . . 1.23 0.37 * F 0.301.27 *

Leu 99 . A B . . . . 0.94 -0.06 * . 0.451.47 *

Met 100. A B . . . . 0.70 0.86 * . -0.451.07 *

Tyr 101. A B . . . . 0.74 0.87 * . -0.600.62 *

Lys 102. A . . T . . 0.84 0.47 * . -0.051.51 *

Trp 103. A . . T . . 0.89 0.21 * . 0.252.36 *

Ala 104. A . . . . C 0.81 -0.40 * F 0.803.01 .

Lys 105. A . . . . C 0.74 -0.47 * F 0.801.06 *

Pro 106. A . . T . . 0.69 0.10 * F 0.250.54 .

Lys 107. . . . T . . 0.64 -0.43 * F 1.050.71 .

Ile 108. . B . . . . 0.93 -0.93 * . 0.800.62 .

Cys 109. . B . . T . 0.71 -0.93 * . 1.000.67 .

Ser I10. . B . . T . 0.67 -0.67 * F 1.150.28 .

Glu 111. . B . . T . 0.53 -0.67 . F 1.150.68 *

Asp 112A . . . . T . -0.10-0.93 * F 1.301.26 *

Leu 1 A A . . . . . -0.07-1.00 * F 0.750.95 13 *

Glu 114A A . . . . . 0.64 -0.74 * F 0.750.41 *

Gly 115A A . . . . . 0.13 -0.74 * F 0.750.49 *

Ala 116. A B . . . . -0.08-0.06 * . 0.300.49 *

Val 117. A B . . . . -0.67-0.31 * . 0.300.43 *

Lys 118. A B . . . . -0.160.19 * . -0.300.44 *

Leu 119 A B . . . -0.500.14* * . -0.300.59 . .

Pro 120 . B . T . -1.010.07* * . 0.10 0.78 . .

Ala 121 . . T T . -0.380.07* * F 0.65 0.29 . .

Ser 122 . . T T . 0.17 0.07* * F 0.65 0.70 . .

Gly 123 . . T T . 0.09 -0.13* * F 1.25 0.66 . .

Val 124 . B . . . 0.23 -0.06. . F 0.79 0.89 . .

Lys 125 . B . . . 0.23 O.OI. . F 0.33 0.35 . .

Thr 126 . B . . . 0.61 0.06. . F 0.47 0.55 . .

His 127 . B . . . 0.24 0.06* . . 0.61 1.15 . .

Cys 128 . B . T . 0.59 -0.01. . . 1.40 0.31 . .

Pro 129 . B . T . 1.23 0.39* * F 0.81 0.34 . .

Pro 130 . . T T . 0.84 0.33. . F 1.07 0.39 . .

Cys 131 . . T T . 0.46 0.26. . F 0.93 0.72 . .

Asn 132 . . . T C -0.210.47. . F 0.29 0.40 . .

Pro 133 . . T T . 0.50 0.83. . F 0.35 0.23 . .

Gly 134 . . T T . 0.40 0.40. . . 0.20 0.85 . .

Phe 135 . B . T . 0.61 0.31. . . 0.10 0.76 . .

Phe 136 . B . . . 1.28 0.31. . F 0.30 0.79 . .

Lys 137 . . T . . 0.98 0.29. . F 1.10 1.28 . .

Thr 138 . . T . . 0.88 0.24. . F 1.35 1.99 . .

Asn 139 . . T . . 0.56 -0.06* . F 2.20 3.31 . .

Asn 140 . . T T . 1.26 -0.27* . F 2.50 0.89 . .

Ser 141 . . T T . 1.74 0.13* * F 1.80 1.06 . .

Thr 142 . . T T . 1.03 0.07. . F 1.55 1.02 . .

Cys 143 . . T T . 1.13 0.24. . F 1.15 0.34 . .

Gln 144 . B . . . 0.89 0.27. . F 0.30 0.39 . .

Pro 145 . B . . . 0.54 0.64. . F -0.250.43 . .

Cys 146 . B . T . 0.54 0.59. . . -0.200.79 . .

Pro 147 . B . T . 0.61 0.40. . . -0.200.61 . .

Tyr 148 . . T T . 0.98 0.76. . . 0.20 0.62 . .

Gly 149 . . T T . 0.98 0.71. . . 0.35 1.55 . .

Ser 150 . . T . . 0.84 0.54. . F 0.30 1.61 . .

Tyr 151 . . T 'f . 1.21 0.54. . F 0.50 1.02 . .

Ser 152 . . T T . 1.42 0.17. . F 1.11 1.38 . .

Asn 153 . . T T . 1.00 -0.26. . F 2.02 1.71 . .

Gly 154 . . T T . 1.03 -0.07* . F 2.18 0.59 . .

Ser 155 . . T T . 1.44 -0.34* . F 2.49 0.63 . .

Asp 156 . . T T . 1.02 -0.73* . F 3.10 0.77 . .

Cys 157 . B . T . 1.11 -0.56* * F 2.39 0.42 . .

Thr 158 . B . T . 0.52 -0.56* * F 2.39 0.48 . .

Arg 159 . B . . . 0.52 -0.44* . F 1.89 0.29 . .

Cys 160 . B . T . 0.51 -0.01. . . 1.94 0.54 . .

Pro 161 . . T 'f . 0.51 -0.10. . . 2.34 0.54 . .

Ala 162 . . T T . 0.97 -0.59. * F 3.10 0.47 . .

Gly 163 . . . T C 0.69 -0.16* * F 2.44 1.37 . .

Thr 164 . . . . C -0.28-0.23. * F 1.78 0.89 . .

Glu 165 . B . . . 0.04 -0.01. . F 1.27 0.66 . .

Pro 166 . B . . . -0.44-0.09. * F 0.96 0.66 . .

Ala 167 . B . . . 0.14 0.27. * . -0.100.39 . .

Val 168 . B . . . 0.24 -0.21. * . 0.50 0.39 . .

Gly 169 . B . . . 0.60 0.54. * . -0.400.40 . .

Phe 170 A B . . . 0.31 0.11. * . -0.300.79 . .

Glu 171 A B . . . 0.23 0.53. * . -0.451.12 . .

Tyr 172 A . T . . 0.82 0.80* * . -0.051.19 . .

Lys 173 A . T . . 1.37 0.77* * . -0.052.21 . .

Trp 174 A . 'f . . 0.90 0.47* * . -0.051.84 . .

Trp 175 A . T . . 1.39 1.16* * . -0.200.97 . .

Asn 176 . . . . C 1.08 0.83* * . -0.200.75 . .

Thr 177 . . . . C 1.32 1.31* . F 0.10 1.03 . .

Leu 178 . . . . C 0.68 0.80* * F 0.10 1.57 . .

Pro 179 . . . . T C 0.97 0.50* . F 0.12 0.97 .

Thr 180 . . . . T C 0.94 0.10* . F 0.54 1.16 .

Asn 181 . . . . T C 0.63 0.10* * F 0.51 2.03 .

Met 182 . B . . T . 0.09 -0.10. * F 0.88 1.90 .

Glu 183 . B B . . . 0.09 0.11. * F -0.300.98 .

Thr 184 . B B . . . -0.000.31. . F -0.270.50 .

Thr 185 . B B . . . -0.030.30* . F -0.240.68 .

Val 186 . B B . . . -0.920.11* * F -0.210.39 .

Leu 187 . B B . . . -0.320.80* * F -0.480.19 .

Ser 188 . B B . . . -1.020.71. * F -0.450.21 .

Gly 189 . . B . . C -0.711.01. * . -0.400.24 .

Ile 190 A B B . . . -0.640.37* * . -0.300.51 .

Asn 191 A B B . . . 0.26 0.44* * . -0.600.60 .

Phe 192 A B B . . . 0.72 0.06* * . -0.151.21 .

Glu 193 A B B . . . 0.42 0.06* * . -0.151.71 . -Tyr 194 . B . . T . 0.46 -0.01* * . 0.85 1.05 .

Lys 195 . . . T T . 1.00 0.07* * F 0.80 1.76 .

Gly 196 . . . T T . 0.71 -0.29* * F 1.40 1.00 .

Met 197 . . . . T C 1.41 0.63. * F 0.15 0.67 .

Thr 198 . . . . . C 0.56 -0.13. * F 0.85 0.58 .

Gly 199 A . . . C 0.21 0.51. * . -0.400.44 .

Trp 200 A B . . . . -0.180.59* * . -0.420.45 .

Glu 201 A B . . . . 0.17 0.40* . . -0.240.31 .

Val 202 A B . . . . 0.73 -0.09* . . 0.84 0.52 .

Ala 203 A B . . . . 0.16 -0.01* . . 1.02 0.67 .

Gly 204 . . . T . . 0.26 -0.24* . . 1.80 0.27 .

Asp 205 . . B T . . 0.23 0.51* . . 0.52 0.57 .

His 206 . B B . . . -0.360.36* . . 0.24 0.82 .

Ile 207 . B B . . . -0.090.36. . . 0.06 0.83 .

Tyr 208 . B B . . . 0.16 0.43* . . -0.420.50 .

Thr 209 . B B . . . -0.090.86. . . -0.600.37 .

Ala 210 . B B . . . -0.390.86. . . -0.600.53 .

Ala 211 . B . . . . -0.360.56. . . -0.120.45 .

Gly 212 . . . . . C 0.53 -0.20. . . 1.26 0.52 .

Ala 213 . . . . . C 0.78 -0.29. . F 1.69 0.83 .

Ser 214 . . . . T C 0.39 -0.79. . F 2.62 1.38 .

Asp 215 . . . T T . 0.38 -0.50. . F 2.80 1.21 .

Asn 216 . . . . T C 0.08 -0.31. . F 2.32 1.18 .

Asp 217 . B . . T . -0.39-0.13. . F 1.69 0.62 .

Phe 218 . B B . . . -0.110.17. . . 0.26 0.30 .

Met 219 . B B . . . -0.620.66. . . -0.320.27 .

Ile 220 . B B . . . -1.480.94. . . -0.600.14 .

Leu 221 . B B . . . -2.331.59. . . -0.600.12 .

Thr 222 . B B . . . -2.541.44. . . -0.600.09 .

Leu 223 . B B . . . -2.191.26. . . -0.600.19 .

Val 224 . B B . . . -2.291.00* * . -0.600.23 .

Val 225 . B B . . . -1.291.10* * . -0.600.14 .

Pro 226 . B . . . . -0.690.61* * . -0.400.33 .

Gly 227 . . . T . . -0.590.36* . F 0.45 0.68 .

Phe 228 . B . . . . 0.22 0.14* . F 0.45 1.42 .

Arg 229 . . . . . C 0.78 -0.10* * F 1.50 1.59 .

Pro 230 . . . . T C 0.78 -0.14* . F 1.95 2.16 .

Pro 231 . . . T T . 0.39 0.07* * F 1.80 1.85 .

Gln 232 . . . T T . 0.14 -0.10* * F 2.50 0.93 .

Ser 233 . B . . 'I' . 0.84 0.40* * F 0.95 0.61 .

Val 234 . B . . . . 0.42 -0.03* * . 1.55 0.66 .

Met 235 . B . . . . 0.63 0.03. . . 1.00 0.55 .

Ala 236 . B . . . . 0.84 -0.37. * . 1.65 0.71 .

Asp 237 . B . . T . 0.89 -0.36. . F 2.20 1.54 .

Thr 238 . . . . T C 1.19 -1.00. . F 3.00 3.11 .

Glu 239 A . . . T . 1.19 -1.61. . F 2.50 5.33 .

Asn 240 A . . . T . 1.20 -1.47* * F 2.20 2.37 .

Lys 241 A . . . . . 1.90 -0.97* * F 1.70 1.66 .

Glu 242 A . . . . . I -1.46* * F 1 1.88 . .01 .40 Val 243 A . B . . . 1.01 -0.77* * . 0.60 0.82 .

Ala 244 . B B . . . 0.31 -0.69* * . 0.60 0.59 .

Arg 245 . B B . . . -0.540.10* * . -0.300.29 .

Ile 246 . B B . . . -1.290.74* * . -0.600.29 .

Thr 247 . B B . . . -1.290.89* * . -0.600.25 .

Phe 248 . B B . . . -0.740.39* * . -0.300.22 .

Val 249 . B B . . . -0.970.87* * . -0.600.46 .

Phe 250 . B B . . . -1.740.87* * . -0.600.26 .

Glu 251 . B B . . . -1.160.96* * . -0.600.16 .

Thr 252 . . B T . . -1.700.56* * . -0.200.29 .

IS Leu 253 . . B T . . -I.000.56* * . -0.200.25 .

Cys 254 . . B T . . -0.810.17* * . 0.10 0.23 .

Ser 255 . . . T T . -0.110.74* * . 0.20 0.09 .

Val 256 . . . T T . -0.920.26* * . 0.50 0.18 .

Asn 257 . B . 'f T . -0.860.26. * . 0.50 0.28 .

Cys 258 . B . . T . -0.740.44. * . -0.200.33 .

Glu 259 . B B . . . -0.680.84. * . -0.600.38 .

Leu 260 . B B . . . -1.230.81. * . -0.600.24 .

Tyr 261 . B B . . . -0.721.06. * . -0.600.33 .

Phe 262 . B B . . . -1.580.91. * . -0.600.19 .

Met 263 . B B . . . -0.911.56. * . -0.600.17 .

Val 264 . B B . . . -1.211.27. * . -0.600.17 .

Gly 265 . B B . . . -0.290.90. * . -0.320.27 .

Val 266 . B B . . . -0.360.11. * . 0.26 0.53 .

Asn 267 . . . . T C 0.34 -0.01. * F 2.04 1.03 .

Ser 268 . . . . T C 0.63 -0.26. * F 2.32 1.67 .

Arg 269 . . . T T . 1.28 -0.20. * F 2.80 3.24 .

Thr 270 . . . T T . 0.77 -0.41. * F 2.52 3.12 .

Asn 271 . . . . . C 1.62 -0.17* * F 1.84 1.73 .

Thr 272 . . . . . C 1.31 -0.56* * F 1.86 1.53 .

Pro 273 . B . . . . 1.32 -0.07* * F 1.08 1.53 .

Val 274 . B . . . . 1.26 0.36. * F 0.05 1.00 .

Glu 275 . B . . . . 1.22 -0.04* . F 0.80 1.38 .

Thr 276 . B . . . . 0.92 -0.10* . F 0.99 0.88 .

Trp 277 . B . . . . 1.28 -0.14. . F 1.48 1.60 .

Lys 278 . . . T . . 1.14 -0.79. . F 2.52 1.85 .

Gly 279 . . . T . . 2.04 -0.36. . F 2.56 1.27 .

Ser 280 . . . T T . 2.04 -0.84. . F 3.40 2.41 .

Lys 281 . . . . T C 2.06 -1.36. . F 2.86 2.08 .

Gly 282 . . . T T . 2.10 -0.97. . F 2.72 2.82 .

Lys 283 . . . T T . 1.74 -0.64. . F 2.38 3.30 .

Gln 284 . . B T . . 1.84 -0.54* . F 1.64 2.38 .

Ser 285 . B B . . . 1.26 0.21* * F 0.00 3.77 .

Tyr 286 . B B . . . 0.32 0.47* * . -0.451.32 .

Thr 287 . B B . . . 0.67 1.16* . . -0.600.53 .

Tyr 288 . B B . . . 0.62 0.76. . . -0.600.69 .

Ile 289 . B B . . . 0.62 0.37* . . -0.300.76 .

Ile 290 . B B . . . 0.61 0.01. . . -0.040.85 .

Glu 291 . B B . . . 0.54 0.01. . F 0.37 0.78 .

Glu 292 . B . . . . 0.54 -0.26. . F 1.58 1.61 .

Asn 293 . . B T . . 0.49 -0.46. . F 2.04 3.32 .

Thr 294 . . B T . ' 0.68 -0.76. . F 2.60 2.57 . .

Thr 295 . . B . . C 1.26 0.03. * F 1.24 1.29 .

Thr 296 . . B . . C 0.97 0.51. . F 0.68 1.15 .

Ser 297 . . B . . C 0.38 1.03* * F 0.27 0.84 .

Phe 298 . B B . . . -0.321.04* * . -0.340.59 .

Thr 299 . B B . . . -0.011.34* . . -0.600.35 .

Trp 300 . B B . . . 0.41 1.26* . . -0.600.46 .

Ala 301 . . B . . C 0.41 0.87* . . -0.251.03 .

Phe 302 . . B T . . 0.40 0.57* * . -0.051.03 .

Gln 303 . . B T . . 0.40 0.57* * . -0.051.42 .

Arg 304 . . B . . C 0.68 0.44. . F -0.101.21 .

Thr 305 . . B . . C 0.97 0.44. . F -0.101.91 .

Thr 306 . . B . . C 0.97 -0.34* . F 0.80 1.91 .

Phe 307 . . B . . C 1.37 -0.24* * . 0.50 0.98 .

His 308 . . B . . C 1.48 0.14* * . -0.100.91 .

Glu 309 . . B . . C 1.41 -0.34* * . 0.65 1.24 .

Ala 310 . . . T . C 1.48 -0.83* . F 1.84 2.86 .

Ser 311 . . . 1' . . 1.48 -0.86* . F 2.18 3.30 .

Arg 312 . . . T . . 2.18 -0.87* . F 2.52 2.75 .

Lys 313 . . . T . . 2.21 -0.47* . F 2.56 4.38 .

Tyr 314 . . . T T . 1.36 -0.97* . F 3.40 5.45 .

Thr 315 . . . T T . 1.36 -0.71* . F 3.06 2.07 .

Asn 316 . B . . T . 1.70 -0.21* . F 2.02 1.04 .

Asp 317 . B . . T . 0.70 -0.21* . F 1.68 1.33 .

Val 318 . B B . . . 0.41 -0.29* . F 0.79 0.65 .

Ala 319 . B B . . . 0.36 -0.01* . . 0.30 0.63 .

Lys 320 . B B . . . -0.22-0.03* . . 0.30 0.51 .

Ile 321 . B B . . . -0.220.66* . . -0.600.48 .

Tyr 322 . B B . . . -1.080.41. . . -0.600.76 .

Ser 323 . B B . . . -0.530.56. . . -0.600.28 .

Ile 324 . B B . . . 0.06 1.04. . . -0.600.58 .

Asn 325 . B B . . . -0.840.76. . . -0.600.60 .

Val 326 . B B . . . -0.560.64. . . -0.600.33 .

Thr 327 . B B . . . -0.310.87. * . -0.600.47 .

Asn 328 . B B . . . -0.360.59* * . -0.600.47 .

Val 329 . B . . T . -0.320.61* * . -0.200.62 .

Met 330 . B . . T . -0.910.61* . . -0.200.32 .

Asn 331 . B . . T . -0.360.63* . . -0.200.20 .

Gly 332 . B . . T . -0.290.61* . . -0.200.36 .

Val 333 . B . . . . -0.960.73* . . -0.400.57 .

Ala 334 . B . . T . 0.01 0.69* . . -0.200.19 .

Ser 335 . B . . T . 0.40 0.29* . . 0.10 0.38 .

Tyr 336 . B . . T . -0.270.29* * . 0.10 0.79 .

Cys 337 . B . . T . -0.510.21* . . 0.10 0.42 .

Arg 338 . B . . 1' . -0.470.21. . . 0.10 0.32 .

Pro 339 . B . . T . 0.12 0.51. . . -0.200.17 .

Cys 340 . B . . T . -0.17-0.24. . . 0.70 0.54 .

Ala 341 . B . . T . -0.22-0.31* . . 0.70 0.28 .

Leu 342 . B . . . . 0.44 0.07* . . -0.100.24 .

Glu 343 . B . . . . -0.52-0.36* . . 0.75 0.75 .

Ala 344 . B . . . . -0.66-0.29. . F 1.15 0.55 .

Ser 345 . B . . . . -0.29-0.36. . F 1.40 0.66 .

Asp 346 . . . T T . 0.00 -0.66. . F 2.55 0.51 .

Val 347 . . . T T . 0.14 -0.27. . F 2.50 0.68 .

Gly 348 . . . T T . -0.17-0.20* . F 2.25 0.27 .

Ser 349 . . . T T . 0.12 -0.10* . F 2.00 0.23 .

Ser 350 . . . T . . -0.240.29* . F 0.95 0.42 .

Cys 351 . B . . T . -0.460.21. . F 0.50 0.23 .

Thr 352 . B . . T . -0.190.21. . F 0.25 0.26 .

5$ Ser 353 . B . . T . -0.190.33. . F 0.25 0.20 .

Cys 354 . B . . T . -0.130.37. . . 0.10 0.37 .

Pro 355 . B . . T . -0.080.56. . . -0.200.40 .

Ala 356 . . . T T . -0.300.83. . . 0.20 0.47 .

Gly 357 . B . . T . 0.01 1.13* . . -0.200.61 .

Tyr 358 . B . . T . 0.42 0.56* . . -0.200.66 .

Tyr 359 . B B . . . 1.09 0.13* . . 0.19 1.28 .

Ile 360 . B B . . . I.00 -0.37* . . 1.13 2.16 .

Asp 361 . B B . . . 1.24 -0.41* . . 1.47 1.84 .

Arg 362 . . . T . . 1.28 -0.74* . F 2.86 1.16 .

5 Asp 363 . . . T T . 0.86 -1.01* . F 3.40 2.40 .

Ser 364 . . . 'C T . 1.07 -1.13* . F 2.91 0.77 .

Gly 365 . . . T T . 1.66 -0.63* . F 2.57 0.53 .

Thr 366 . . . T T . 0.99 -0.24. . F 1.93 0.43 .

Cys 367 . . . T T . 0.67 0.33. * . 0.84 0.17 .

10 His 368 . . . T T . 0.46 0.37. . . 0.50 0.27 .

Ser 369 . . . T T . 0.76 0.37. . . 0.50 0.29 .

Cys 370 . B . . T . 0.79 0.29. . . 0.10 0.86 .

Pro 371 . . . . T C 0.21 0.20. . F 0.45 0.91 .

Pro 372 . . . T T . 0.07 0.39* . F 0.65 0.48 .

15 Asn 373 . . . T T . 0.14 0.69* * F 0.35 0.74 .

Thr 374 . B . . T . -0.140.11* * F 0.25 0.95 .

Ile 375 A B . . . . 0.49 0.19* . . -0.300.62 .

Leu 376 A B . . . . 0.70 0.26* . . -0.300.53 .

Lys 377 A B . . . . 0.70 0.26* . . -0.300.63 .

20 Ala 378 A B . . . . 0.46Ø20* . . -O.151.39 .

His 379 A B . . . 0.42 0.27. . . -0.152.64 .

Gln 380 . B . . T . 0.46 0.01* * F 0.40 1.31 .

Pro 381 . . . T T . 1.27 0.66. * . 0.20 0.96 .

Tyr 382 . . . T T . 0.63 0.56. * . 0.35 1.22 .

25 Gly 383 . . . T T . 0.56 0.56. * . 0.20 0.71 .

Val 384 . B B . . . -0.270.73. * . -0.600.25 .

Gln 385 . B B . . . -0.480.94. * . -0.600.12 .

Ala 386 . B B . . . -0.930.61. * . -0.600.18 .

Cys 387 . B B . . . -1.030.76. * . -0.60O.13 .

30 Val 388 . B B . . . -0.900.54. * . -0.600.08 .

Pro 389 . B . . . . -0.390.57. * . -0.400.12 .

Cys 390 . B . . . . -0.700.50. . . -0.400.21 .

Gly 391 . B . . T . -0.070.41. . F 0.29 0.42 .

Pro 392 . . . T 'T . 0.60 -0.23. . F 1.93 0.54 .

35 Gly 393 . . . T T . 1.46 -0.26. . F 2.42 1.61 .

Thr 394 . . . T T . 1.71 -0.43. . F 2.76 2.62 .

Lys 395 . . . T T . 1.49 -0.86. . F 3.40 3.39 .

Asn 396 . . . T T . 1.80 -0.60. . F 3.06 2.40 .

Asn 397 . B . . T . 1.71 -0.53. . F 2.32 2.26 .

40 Lys 398 . B . . T . 1.24 -0.63. . F 1.98 1.52 .

Ile 399 . B . . . . 0.89 0.06. . . 0.24 0.78 .

His 400 . B . . T . 0.60 0.23. . . 0.10 0.26 .

Ser 401 . B . . T . 0.60 0.59. . . -0.200.20 .

Leu 402 . B . . T . 0.60 0.99* . . -0.200.47 .

45 Cys 403 . B . . T . -0.110.30* . . 0.10 0.57 .

Tyr 404 . . . T . . 0.47 0.37. . . 0.30 0.23 .

Asn 405 . . . T T . -0.200.47. . . 0.20 0.40 .

Asp 406 . . . T T . -0.200.57* . . 0.20 0.65 .

Cys 407 . B . . T . 0.72 0.39* . . 0.10 0.55 .

Thr 408 . B . . T . 1.39 -0.37. . . 0.70 0.67 .

Phe 409 . B . . . . 1.32 -0.37. . . 0.80 0.65 .

Ser 410 . . . T T . 1.11 0.11. . F 1.40 1.75 .

Arg 411 . . . T T . 0.80 -0.03. . F 2.30 1.87 .

Asn 412 . . . . T C 1.58 -0.03. . F 2.40 3.12 .

Thr 413 . . . . T C 1.58 -0.81. . F 3.00 4.55 .

Pro 414 . . . . T C 1.58 -0.71* . F 2.70 3.36 . ~

Thr 415 . . . T T . 1.88 0.07* . F 1.70 1.81 .

Arg 416 . B . . T . 1.52 0.07* . F 1.00 2.01 .

Thr 417 . B . . T . 1.52 0.34* * F 0.70 2.04 .

Phe 418 . B . . . . 1.13 0.31* * . 0.05 2.27 .

Asn 419 . B . . T . 1.04 0.61* * . -0.051.01 .

Tyr 420 . B . . T . 0.77 1.00* . . -0.200.93 .

Asn 421 . B . . T . -0.161.01* * . -0.051.09 .

Phe 422 . B . . T . -0.430.91* * . -0.200.56 .

Ser 423 A . . . . C 0.27 1.01* * . -0.400.36 .

Ala 424 A . . . . C -0.040.66* * . -0.400.36 .

Leu 425 A B . . . . -0.660.74* * . -0.600.60 .

Ala 426 A B . . . . -0.970.60. * . -0.600.33 .

Asn 427 A B . . . . -1.080.70* . . -0.600.47 .

Thr 428 . B B . . . -1.370.89* . . -0.600.47 .

Val 429 . B B . . . -1.120.70. . . -0.600.47 .

Thr 430 . B B . . . -0.660.63. . . -0.600.29 .

Leu 431 . B B . . . -0.280.66. . . -0.600.20 .

Ala 432 . B B . . . -0.580.60. . . -0.600.42 .

Gly 433 . . B . . C -0.970.34. * F 0.05 0.39 .

Gly 434 . . . . T C -0.420.64. . F 0.15 0.41 .

Pro 435 . . . . T C -0.410.44. * F 0.15 0.58 .

Ser 436 . . . . T C 0.44 0.33. * F 0.73 0.79 .

Phe 437 . B . . T . 0.69 -0.10. . F 1.56 1.59 .

Thr 438 . B . . . . 0.22 -0.10* . F 1.64 1.02 .

Scr 439 . B . . 'f . 0.61 0.16* . F 1.37 0.63 .

Lys 440 . . . T T . 0.58 -0.23* . F 2.80 1.45 .

Gly 441 . . . T T . 0.18 -0.26* . F 2.52 1.57 .

Leu 442 . . . . T C 0.84 0.04* . F 1.44 1.02 .

Lys 443 A B . . . . 1.12 0.16* . . 0.26 0.69 .

Tyr 444 A B . . . . 0.72 0.66* . . -0.320.95 .

Phe 445 A B . . . . 0.37 I.01* * . -0.601.00 .

His 446 A B . . . . -0.100.81* * . -0.600.72 .

His 447 A B . . . . 0.41 1.50* * . -0.600.38 .

Phe 448 A B . . . . -0.441.13* * . -0.600.59 .

Thr 449 A B . . . . -0.871.03. * . -0.600.36 .

Leu 450 A . . T . . -0.511.10. * . -0.200.14 .

Ser 451 . . B T . . -0.481.03. * . -0.200.16 .

Leu 452 . . B T . . -0.440.64. * . 0.14 0.18 .

Cys 453 . . B T . . -0.090.56. * . 0.48 0.37 .

Gly 454 . . B T . . 0.33 0.30* . F 1.27 0.28 .

Asn 455 . . . T T . 1.19 -0.09* . F 2.61 0.66 .

Gln 456 . . . T T . 0.89 -0.77* . F 3.40 2.45 .

Gly 457 . . . T T . 1.40 -0.73* . F 3.06 2.45 .

Arg 458 . . . T T . 1.21 -0.77* . F 2.72 2.04 .

Lys 459 . B B . . . 0.89 -0.53* . F 1.43 0.87 .

Met 460 . B B . . . 0.58 -0.36* . . 0.64 0.47 .

Ser 461 . B B . . . 0.58 -0.30* . . 0.30 0.35 .

Val 462 . B B . . . 0.92 -0.30* . . 0.30 0.29 .

Cys 463 . B . . T . -0.040.10* . . 0.10 0.47 .

Thr 464 . B . . T . -0.400.13* * . 0.10 0.26 .

Asp 465 . B . . T . 0.20 0.23* . F 0.25 0.51 .

Asn 466 . B . . T . -0.31-0.41* * F 1.00 1.59 .

Val 467 . B B . . . 0.66 -0.30. * F 0.45 0.91 .

Thr 468 . B B . . . 0.43 -0.79. * F 0.90 1.07 .

Asp 469 . B B . . . 0.53 -0.10. * F 0.45 0.46 .

Leu 470 . B B . . . 0.53 -0.07. * F 0.76 0.97 .

Arg 471 . B B . . . 0.19 -0.71. * F 1.52 1.16 .

Ile 472 . B . . T . 1.04 -0.77. * F 2.08 0.69 .

Pro 473 . B . . T . 1.06 -0.77. * F 2.54 1.44 .

Glu 474 . . . T T . 0.71 -1.07. * F 3 0.99 . .
I

Gly 475 . . . . T C 0.82 -0.64. * F 2.74 1.40 .

Glu 476 . . . T T . 0.41 -0.54* . F 2.48 0.78 .

Ser 477 . . . . T C 1.34 -0.59* . F 2.10 0.60 .

Gly 478 . . . T T . 1.26 -0.59* . F 2.27 1.22 .

Phe 479 . . . T T . 0.37 -0.63* . F 1.94 0.95 .

Ser 480 . . . . T C 0.40 0.06* . F 0.97 0.49 .

Lys 481 . . . T T . -0.190.16* . F 1.30 0.72 .

Ser 482 . . . T T . -0.130.23* . F 1.17 0.84 .

Ile 483 . B . . T . -0.640.20* . . 0.49 0.98 .

Thr 484 . B B . . . -0.610.46* . . -0.340.37 .

Ala 485 . B B . . . -0.311.03* . . -0.470.15 .

Tyr 486 . B B . . . -0.941.04* . . -0.600.36 .

Val 487 . B B . . . -1.500.86. . . -0.600.25 .

Cys 488 . B B . . . -1.501.01. . . -0.600.19 .

Gln 489 . B B . . . -2.081.20. . . -0.600.08 .

Ala 490 . B B . . . -1.701.13. . . -0.600.08 .

Val 491 . B B . . . -1.670.91. . . -0.600.23 .

Ile 492 . B B . . . -0.810.77. . . -0.600.20 .

Ile 493 . B B . . . -1.000.37. . . -0.300.35 .

Pro 494 . B . . T . -1.310.51. * . -0.200.35 .

Pro 495 . B . . T . -1.070.36. * F 0.42 0.71 .

Glu 496 . B . . T . -0.460.10. . F 0.74 1.01 .

Val 497 . B . . T . 0.48 0.17. . F 0.91 1.02 .

Thr 498 . B . . T . 0.78 -0.26. * F 1.68 1.32 .

Gly 499 . B . . T . 0.64 -0.19. . F 1.70 0.77 .

Tyr 500 . B . . T . 0.00 0.24. . F 1.08 1.03 .

Lys 501 . B . . T . -0.300.24. . F 0.76 0.53 .

Ala 502 . B . . . . 0.26 0.14. * F 0.39 0.71 .

Gly 503 . B . . . . 0.57 0.10. * F 0.22 0.61 .

Val 504 . B . . . . 0.70 -0.26. * F 0.65 0.53 .

Ser 505 . B . . . . 0.09 0.17. * F 0.05 0.81 .

Ser 506 . B . . . . -0.260.31. * F 0.05 0.61 .

Gln 507 . B . . . . -0.480.27. . F 0.20 1.10 .

Pro 508 . B . . . . -0.720.31. . F 0.05 0.67 .

Val 509 A B . . . . 0.13 0.43* * F -0.450.51 .

Ser 510 A B . . . . 0.54 0.04* * . -0.300.49 .

Leu 511 A B . . . . 0.03 -0.36* . . 0.30 0.62 .

Ala 512 A B . . . . -0.86-0.10* * . 0.30 0.69 .

Asp 513 A B B . . . -0.99-0.06* * . 0.30 0.36 .

Arg 514 A B B . . . -0.99-0.01* * . 0.30 0.43 .

Leu 515 . B B . . . -I.00-0.06* . . 0.30 0.32 .

Ile 516 . B B . . . -0.50-0.07* . . 0.30 0.28 .

Gly 517 . B B . . . 0.09 0.41* . . -0.600.20 .

Val 518 . B B . . . -0.510.41* . . -0.600.41 .

Thr 519 . B B . . . -0.930.34* * F -0.150.58 .

Thr 520 . B B . . . -0.930.14. * F -0.150.85 .

Asp 521 . B B . . . -0.040.40. * F -0.450.94 .

Met 522 . B B . . . -0.04-0.24. * F 0.60 1.09 .

Thr 523 . B B . . . -0.08-0.30. * . 0.30 0.75 .

Leu 524 . B B . . . -0.08-0.10* * F 0.45 0.31 .

Asp 525 . . B T . . -0.070.39* * F 0.25 0.46 .

Gly 526 . . B T . . -0.280.16* * F 0.34 0.42 .

Ile 527 . . B . . C -0.270.10* . F 0.23 0.79 .

Thr 528 . . B . . C 0.04 -0.09* * F 0.92 0.48 .

Ser 529 . . . . T C 0.04 -0.09* . F 1.41 0.84 .

Pro 530 . . . . T C -0.660.17* . F 0.90 0.99 .

Ala 531 . B . . T . -0.340.27* . F 0.61 0.59 .

Glu 532 . B . . T . -0.270.29. * . 0.37 0.60 .

Leu 533 A B . . . . 0.04 0.59. . . -0.420.32 .

Phe 534 A B . . . . 0.04 0.16. . . -0.210.55 .

His 535 A B . . . . -0.560.04. . . -0.300.43 .

Leu 536 A B . . . . -0.310.73. . . -0.600.43 .

Glu 537 A B . . . . -1.200.47. . . -0.600.49 .

Ser 538 . . . T . . -0.600.37. . . 0.30 0.25 .

Leu 539 . . . T . . 0.10 0.30 . . 0.30 0.47 . .

Gly 540 . . . . . C -0.72 -0.39 . . 0.70 0.45 . .

Ile 541 . . B . . C -0.80 0.26 . F 0.05 0.25 . .

Pro 542 . B B . . . -1.50 0.56 . F -0.450.21 . .

Asp 543 . B B . . . -1.90 0.66 . . -0.600.19 . .

Val 544 . B B . . . -1.33 1.01 * . -0.600.23 . *

Ile 545 . B B . . . -0.88 1.09 . . -0.600.23 . *

Phe 546 . B B . . . -0.29 0.66 * . -0.600.27 . *

Phe 547 . B B . . . -0.08 1.04 * . -0.600.50 . .

Tyr 548 . B . . . . -0.08 0.80 * . 0.09 1.14 . .

Arg 549 . . . T T . -0.08 0.11 . F 1.48 2.19 . .

Ser 550 . . . T T . 0.50 -0.03 . F 2.42'1.88 . .

Asn 551 . . . T T . 1.20 -0.33 . F 2.76 1.73 . *

Asp 552 . . . T T . 1.60 -0.69 . F 3.40 1.53 . *

Val 553 . . . T . . 1.18 -0.30 . F 2.56 1.53 . .

Thr 554 . B . . . . 0.77 -0.11 . F 1.67 0.51 . *

Gln 555 . B . . . . 0.77 -0.13 . F 1.33 0.41 . *

Ser 556 . B . . . . 0.42 0.26 * F 0.67 0.74 . *

Cys 557 . B . . T . 0.53 0.04 * F 0.81 0.51 . *

Ser 558 . . . T T . 1.09 -0.44 * F 2.09 0.57 . *

Ser 559 . . . T T . 1.09 -0.46 * F 2.37 0.57 . .

Gly 560 . . . T T . 0.78 -0.36 . F 2.80 1.54 . .

Arg 561 . . B T . . 0.19 -0.44 * F 2.12 1.66 . .

Ser 562 . . B T . . 0.97 -0.14 * F 1.69 0.87 . *

Thr 563 . B B . . . 0.41 -0.53 * F 1.46 1.72 . *

Thr 564 . B B . . . 0.82 -0.31 * F 0.73 0.65 . .

Ile 565 . B B . . . 0.50 -0.31 * F 0.45 0.95 . .

Arg 566 . B B . . . 0.09 -0.13 * . 0.30 0.35 . .

Val 567 . B B . . . 0.18 -0.23 * . 0.64 0.33 . .

Arg 568 . B B . . . 0.49 -0.29 * . 0.98 0.73 . .

Cys 569 . B B . . . 0.84 -0.57 * . 1.62 0.64 . .

Ser 570 . . . . T C 1.42 -0.57 * F 2.86 1.73 . *

Pro 571 . . . T T . 0.46 -0.73 * F 3.40 1.27 . *

Gln 572 . . . T T . 1.10 -0.09 * F 2.76 1.76 . *

Lys 573 . B . . T . 0.64 -0.23 * F 2.02 2.03 . .

Thr 574 . B . . . . 1.01 -0.19 . F 1.48 1.30 . .

Val 575 . B . . T . 0.50 -0.23 . F 1.34 1.01 . .

Pro 576 . B . . T . -0.10 0.06 . F 0.25 0.42 . .

Gly 577 . B . . T . -0.91 0.74 . F -0.050.24 . .

Ser 578 . B . . T . -1.17 0.94 . F -0.050.26 . .

Leu 579 . B . . . . -1.20 0.73 * F -0.250.26 . .

Leu 580 . B . . . . -0.66 0.73 * F -0.400.26 . .

Leu 581 . B . . T . -1.11 0.79 . F -0.050.28 . .

Pro 582 . B . . T . -1.07 0.97 . F -0.050.18 . .

Gly 583 . . . T T . -0.77 0.67 . F 0.35 0.30 . .

Thr 584 . B . . T . -0.30 -0.01 . F 1.16 0.61 . .

Cys 585 . . . T T . 0.20 -0.27 . F 1.87 0.39 . .

Ser 586 . . . T T . 0.34 -0.21 . F 2.18 0.57 . .

Asp 587 . . . T T . 0.56 -0.07 . F 2.49 0.21 . .

Gly 588 . . . T T . 0.56 -0.56 . F 3.10 0.66 . .

Thr 589 . . . T . . 0.20 -0.70 . F 2.59 0.48 . *

Cys 590 . . . T T . 0.87 -0.51 . F 2.48 0.16 . *

Asp 591 . . . T T . 0.47 -0.11 . F 1.87 0.25 . .

Gly 592 . . . T T . 0.43 0.24 * F 0.96 0.15 . .

Cys 593 . . . T T . 0.08 0.26 . . 0.50 0.38 . .

Asn 594 A B . . . . -0.42 0.47 . . -0.600.20 . .

Phe 595 A B . . . . -0.04 1.16 * . -0.600.17 . .

His 596 A B . . . . -0.04 1.64 * . -0.600.33 . .

Phe 597 A B . . . . 0.00 1.07 * . -0.600.35 . *

Leu 598 A . . T . . 0.08 1.06 * . -0.200.54 . .

Trp 599 A . . T . . -0.51 0.77. * . -0.200.40 .

Glu 600 A . . T . . -0.40 0.77. * . -0.200.47 .

Ser 601 A . . T . . -1.03 0.49. . . -0.200.58 .

Ala 602 A . . T . . -0.54 0.37. . . 0.100.29 .

Ala 603 A . . T . . -0.54 -O.II. . . 0.700.26 .

Ala 604 A . . T . . -0.92 0.57. . . -0.200.16 .

Cys 605 . . . . T C -1.22 0.76. . . 0.000.09 .

Pro 606 . B . . T . - I 0.64. . . -0.200.1 . .78 1 Leu 607 . B . . T . -1 0.79* . ' -0.200.08 . .78 .

10Cys 608 . B . . T . -1.19 0.79* . . -0.200.16 .

Ser 609 . B B . . . -0.84 0.21. . . -0.300.17 .

Val 610 . B B . . . -0.21 0.54. . . -0.600.32 .

Ala 611 . B B . . . -0.59 0.36. . . -0.300.82 .

Asp 612 . B . . . . -0.67 0.29. . . -0.100.62 .

15Tyr 613 . B B . . . -0.86 0.59. . . -0.600.58 .

His 614 . B B . . . -0.86 0.59. . . -0.600.43 .

Ala 615 . B B . . . -0.30 0.47. . . -0.600.34 .

Ile 616 . B B . . . -0.38 0.86. * . -0.600.29 .

Val 617 . B B . . . - I 0.67. . . -0.600.12 . .23 20Ser 618 . B B . . . -1.58 0.81. . . -0.600.09 .

Ser 619 . B B . . . -1.89 0.81. . . -0.600. I
. 2 Cys 620 . B B . . . -2.19 0.56* . . -0.600.16 .

Val 621 . B B . . . -1.30 0.60* . . -0.600.09 .

Ala 622 . B B . . . -0.40 0.61* . . -0.600.11 .

25Gly 623 . B B . . . -0.41 0.23* . . -0.300.41 .

Ile 624 . B B . . . -0.42 0.14* . . -0.300.80 .

Gln 625 . B B . . . 0.00 -0.01. . F 0.601.15 .

Lys 626 . B B . . . 0.00 0.24* . F 0.001.82 .

Thr 627 . B B . . . 0.30 0.46* * F -0.301.92 .

30Thr 628 . B B . . . 0.76 0.69* . F -0.301.17 .

Tyr 629 . B B . . . 1.64 0.29* . . -0.151.14 .

Val 630 A B B . . . 1.43 0.29* . . -0.151.37 .

Trp 631 A B B . . . 1.43 0.23* * . -0.151.47 .

Arg 632 A B B . . . 0.93 -0.26* . F 0.601.88 .

35Glu 633 A B B . . . 0.58 -0.33* . F 0.852.09 .

Pro 634 A . . T . . 0.52 -0.40* . F 1.501.06 .

Lys 635 A . . T . . 1.03 -0.93* . F 1.900.73 .

Leu 636 A . . T . . 0.98 -0.50* . F 1.850.42 . ' Cys 637 . . . T T . -0.02 -0.07* . F 2.500.27 .

40Ser 638 . . . T T . -0.32 0.19. * F 1.650.09 .

Gly 639 . . . T T . -0.92 0.57* * F 1.100.15 .

Gly 640 . . . T T . -1.18 0.57* . F 0.850.23 .

Ile 641 . . . . . C -0.37 0.43* . F 0.200.27 .

Ser 642 . . . . . C 0.30 0.04. * F 0.250.47 .

45Leu 643 . B . . . . 0.71 0.01. * F 0.050.82 .

Pro 644 . B . . . . 0.20 -0.41. * F 0.802.30 .

Glu 645 . B B . . . 0.23 -0.46. * F 0.601.27 .

Gln 646 . B B . . . 0.23 -0.36. * F 0.602.23 .

Arg 647 . B B . . . -0.13 -0.36. * F 0.601.01 .

50Val 648 . B B . . . 0.72 -0.21. * . 0.300.31 .

Thr 649 . B B . . . 0.62 -0.21. * . 0.300.36 .

Ile 650 . B B . . . -0.27 -0.13. * . 0.300.27 .

Cys 651 . B B . . . -0.27 0.56. * . -0.600.25 .

Lys 652 . B B . . . -1.08 -0.09* * . 0.300.29 .

55Thr 653 . B B . . . -0.51 0.21* * . -0.300.36 .

Ile 654 . B B . . . -1.01 0.44* * . -0.600.71 .

Asp 655 . B B . . . -0.08 0.56* * . -0.600.29 .

Phe 656 . B B . . . -0.27 0.56* * . -0.600.40 .

Trp 657 . B B . . . -0.66 0.71* * . -0.600.43 .

60Leu 658 . B B . . . -1.23 0.46* * . -0.600.25 .

Lys 659. . B B . . . -0.641.14* * . -0.600.20 Val 660. . . B T . . -1.230.74* * . -0.200.26 Gly 661. . . B T . . -0.880.33* * . 0.100.32 Ile 662. . . B T . . -0.900.07* * . 0.100.16 5 Ser 663. . . . . T C -0.760.56* * . 0.000.31 Ala 664. . . . T T . -1.110.49. * F 0.350.17 Gly 665. . . . T T . -0.840.54. . F 0.350.34 Thr 666. . B . . T . -1.390.36. . F 0.250.26 Cys 667. . B B . . . -1.310.66. . . -0.600.18 10 Thr 668. . B B . . . -1.820.84. . . -0.600.15 Ala 669. . B B . . . -1.541.10. . . -0.600.09 Ile 670. . B B . . . -2.061.10. . . -0.600.23 Leu 671. . B B . . . -2.561.17. . . -0.600.12 Leu 672. . B B . . . -2.201.37. . . -0.600.10 15 Thr 673. . B B . . . -2.561.36. . . -0.600.20 Val 674. . B B . . . -2.211.24. . . -0.600.13 Leu 675. . B B . . . -2.021.31. . . -0.600.25 Thr 676. . B B . . . -1.501.41* . . -0.600.15 Cys 677. . B B . . . -0.641.84* . . -0.600.21 20 Tyr 678. . B B . . . -0.291.20. . . -0.600.51 Phe 679. . . B T . . 0.57 0.51. . . -0.200.70 Trp 680. . . B T . . 1.38 0.43* . . 0.292.10 Lys 681. . . . . T C 1.73 0.26* . F 1.282.32 Lys 682. . . . T T . 1.59 -0.50* * F 2.425.37 25 Asn 683. . . . . T C 1.83 -0.60* * F 2.864.21 Gln 684. . . . T T . 2.29 -1.51* * F 3.403.65 Lys 685. . B . . . . 2.62 -0.76* * F 2.462.86 Leu 686. . B . . . . 2.33 -0.76* * F 2.323.55 Glu 687. . B . . . . 1.99 -0.40* * . 1.733.21 30 1'yr688. . B . . T . 2.03 -0.41* * . 1.792.15 Lys 689. . B . . T . 1.22 -0.41* * F 1.805.22 Tyr 690. . B . . T . 0.32 -0.41* * F 2.002.49 Ser 691. . B . . T . 0.53 0.23* * F 1.201.18 Lys 692. A B . . . . 0.53 0.09* * F 0.450.58 35 Leu 693. A B . . . . 0.19 0.49* * .. -0.200.60 Val 694. A B . . . . -0.170.23* * . -0.100.45 Met 695. A B . . . . -0.730.33* * . -0.300.33 Asn 696. A B . . . . -0.391.01. * . -0.600.33 Ala 697. A B . . . . -0.430.33* * . -0.300.88 40 Thr 698. A B . . . . -0.29-0.31. * . 0.651.48 Leu 699. A B . . . . 0.57 -0.36* . F 0.850.49 Lys 700. A B . . . . 0.36 -0.76. * F 1.350.82 Asp 701. . . . T T . 0.14 -0.57. * F 2.350.47 Cys 702. . B . . T . 0.14 -0.63. . . 2.000.87 45 Asp 703. . B . . T . -0.13-0.81. . . 1.800.44 Leu 704. . B . . 'C . 0.68 -0.31. . . 1.300.27 Pro 705. . B . . . . 0.33 -0.31. . . 0.900.83 Ala 706. . . . T . . -0.33-0.50. * . 1.100.67 Ala 707A . . . . . . -0.260.07. . . -0.100.43 50 Asp 708A . . . . T . -1.14-0.11. . . 0.700.28 Ser 709. . B . . T . -0.930.14. . . 0.100.20 Cys 710. . B . . T . -0.720.26. . . 0.100.19 Ala 711. . B . . 'f . -0.48-0.24. . . 0.700.20 Ile 712. A B . . . . 0.11 0.19. . . -0.300.15 Met 713. A B . . . . 0.11 -0.20. . . 0.300.48 Glu 714. A B . . . . -0.44-0.77. . F 0.750.79 Gly 715. A . . . . C 0.22 -0.63* . F 0.950.83 Glu 716A A . . . . . 0.81 - * . F 0.901.46 I
.31 Asp 717A A . . . . . 1.70 -1.93* . F 0.901.41 Val 718A A . . . . . 1.49 -1.93* . F 0.902.38 Glu 719 A A . . . . . 0.60 -1.67* . F 0.90 1.13 Asp 720 A A . . . . . 0.24 -0.99* . F 0.75 0.48 Asp 721 A A . . . . . -0.07-0.20. * F 0.45 0.55 Leu 722 A A . . . . . -0.37-0.36* * . 0.30 0.46 Ile 723 A A . . . . . 0.53 0.03. * . -0.300.37 Phe 724 . A B . . . . 0.53 0.03. . . -0.300.44 Thr 725 . A B . . . . 0.50 0.43. . F -0.450.87 Ser 726 . . . . . T C 0.20 0.24. . F 0.60 1.68 Lys 727 . . . . '1' T . 0.20 -0.06. . F 1.40 2.60 Asn 728 . . . . . T C 0.74 -0.16* * F 1.48 1.49 His 729 . . . . . T C 1.56 -0.21* * F 1.76 1.10 Ser 730 . . . . . . C 1.57 -0.60. * . 1.99 1.07 Leu 731 . . . . T . . 1.87 -0.21. * . 2.02 0.90 Gly 732 . . . . T T . 1.79 -0.21. . F 2.80 1.06 Arg 733 . . . . T T . 0.98 -0.21* . F 2.52 1.07 Ser 734 . . . . T T . 0.80 0.09* . F 1.88 1.07 Asn 735 . . . . T T . 0.89 -0.17* * F 2.44 1.68 His 736 . . . . . . C 1.81 -0.17* * F 2.00 1.33 Leu 737 . . . . . . C 1.81 -0.17* * F 1.96 1.94 Pro 738 . . . . . T C 0.89 -0.13* * F 2.40 1.19 Pro 739 . . . . T T . 0.38 0.16. * F 1.61 0.72 Arg 740 . . . . T T . -0.220.34. * F 1.37 0.72 Gly 741 . . B . . T . -0.190.27. * F 0.73 0.46 Leu 742 . A B . . . . -0.19-0.16. * . 0.54 0.50 Leu 743 . A B . . . . -0.290.10* . . -0.300.21 Met 744 . A B . . . . -0.080.59* . . -0.600.31 Asp 745 . A B . . . . -0.860.56* . . -0.600.64 Ixu 746 . A B . . . . -0.400.44. . . -0.600.42 Thr 747 . A B . . . . 0.02 -0.24. * . 0.30 0.83 Gln 748 . A B . . . . 0.44 -0.43. . F 0.45 0.63 Cys 749 . A B . . . . 0.66 0.00. . . -0.300.98 Arg 750 . A B . . . . 0.27 -0.26. . . 0.30 0.87 TABLE
II

Res Pos I II III IV V VI VII VIII IX X XI XII XIII XIV

Met 1 . . B . . . . 0.08 0.20. . . -0.100.71 Ser 2 . . B . . T . 0.47 0.26. . . 0.10 0.80 Thr 3 . . B . . T . 0.51 0.23. . . 0.50 1.01 Gly 4 . . . . . T C 0.90 0.23. * . 0.95 1.01 Thr 5 . . . . T T . 0.94 -0.39. . F 2.15 1.26 Asn 6 . . . . . T C 0.69 -0.34. . F 2.05 0.86 Gly 7 . . . . T T . 0.69 -0.19. . F 2.50 0.65 Asp 8 . . . . T T . 0.79 -0.23. . F 2.25 0.60 Gly 9 . . B . . T . 0.54 -0.29. . F 1.60 0.58 Val 10 . . B . . . . 0.86 -0.19. * F 1.15 0.59 Ser 11 . . B . . . . 0.51 -0.21. . F 0.90 0.57 Pro 12 . . B . . T . 0.00 0.21* . F 0.25 0.57 Ala 13 . . B . . T . -0.860.43* . F -0.050.57 Asn 14 . . B . . T . -1.320.43* . F -0.050.31 Gly I . . B . . T . -0.470.73. . . -0.200.17 S

Val 16 . . B B . . . -0.060.30. . . -0.300.28 Val 17 . . B B . . . -0.14-0.20. . . 0.60 0.34 Leu 18 . . B B . . . 0.20 -0.21. . . 0.90 0.46 Asp 19 . . B . . T . -0.010.11* * F 1.15 0.96 Arg 20 . . B . . '1' . 0.44 -0.10* * F 2.20 2.01 Ser 21 . . . . . T C 0.41 -0.74* * F 3.00 4.77 Tyr 22 . . . . . T C 0.41 -0.74* * F 2.70 2.00 Pro 23 . . B B . . . 0.37 -0.10* * F 1.35 0.76 Arg 24 . . B B . . . -0.23 0.54* * . 0.00 0.42 ~

Ile 25 . A B B . . . -0.34 0.77* * . -0.300.27 Val 26 . A B B . . . 0.07 0.01. * . -0.300.30 Val 27 . A B B . . . -0.54 -0.41. * . 0.30 0.30 Met 28 . A B B . . . -0.33 0.23. * . -0.300.31 Glu 29 . A B B . . . -1.04 -0.46. * . 0.30 0.73 Arg 30 A A . B . . . -0.37 -0.49. * . 0.30 0.98 Val 31 A A . B . . . 0.18 -0.70. * . 0.75 1.53 Glu 32 A A . B . . . 0.44 -0.83* * . 0.75 1.27 Met 33 A A . . . . . 1.04 -0.33* * . 0.30 0.66 Pro 34 A . . . . . . 0.83 0.07* * F 0.20 1.53 Thr 35 A . . . . . . 0.13 -0.14* * F 0.80 1.37 Ala 36 A A . . . . . 0.18 0.36. . F 0.00 1.40 Gln 37 A A . . . . . -0.63 0.43. . F -0.450.75 Pro 38 A A . . . . . -0.62 0.69. . F -0.450.43 Ala 39 A A . . . . . -1.27 0.70. . . -0.600.43 Leu 40 A A . . . . . -0.96 0.84* . . -0.600.18 Leu 41 A A . . . . . -0.32 0.84* . . -0.600.20 Ala 42 A A . . . . . -0.32 0.41* . . -0.600.40 Val 43 . A B . . . . -0.92 0.31* . . -0.300.85 Gln 44 . A B . . . . -0.68 0.31* . . -0.060.85 Lys 45 . A B . . . . -0.08 0.06* . F 0.33 0.83 Gln 46 . A . . T . . 0.52 -0.01. . F 1.72 1.74 lxu 47 . A . . T . . 1.11 -0.23. . F 1.96 1.55 Gly 48 . . . . . T C 1.37 -0.23. . F 2.40 1.34 Pro 49 . . . . . T C 0.70 0.39. . F 1.41 0.77 Pro 50 . . . . T T . 0.77 0.56* * F 1.07 0.50 Gln 51 . . . . T '1' . -0.09 -0.13* * . 1.58 0.99 Met 52 . . B B . . . 0.13 0.09* * . -0.060.47 Cys 53 . . B B . . . -0.19 0.16* * . -0.300.31 Arg 54 . . B B . . . -0.29 0.30* * . -0.300.10 Val 55 . . B B . . . -0.74 0.39* . . -0.300.14 Ala 56 . . B B . . . -1.33 0.34* * . -0.300.14 Cys 57 . . B B . . . -1.59 0.27* * . -0.300.07 Thr 5 . . B B . . . -1 .81 0.9. * . -0.600.07 8 l Cys 59 . . B B . . . -1.92 0.96* * . -0.600.05 Ala 60 . . B B . . . -0.96 0.86* . . -0.600.15 Val 61 . . B B . . . -1.22 0.29* * . -0.300.20 Ile 62 . . B B . . . -0.56 0.44* . . -0.600.28 Asn 63 . . B B . . . -0.20 0.27* . . -0.300.48 Arg 64 . . B B . . . -0.39 -0.23* . . 0.45 1.30 Val 65 . . B B . . . 0.20 -0.23* . F 0.60 1.38 Gln 66 . . B B . . . 0.39 -0.51* . F 0.90 1.38 Lys 67 . . B B . . . 0.97 -0.34* . F 0.45 0.38 Val 68 . . B B . . . 0.76 0.14* . . -0.300.73 Asn 69 . . B B . . . 0.33 -0.07* * . 0.30 0.65 Cys 70 . . B B . . . 0.89 0.01* * F -0.150.47 Thr 71 . . B . . T . 0.89 0.40. * F 0.25 0.85 Pro 72 . . . . T T . 0.26 0.16. * F 0.65 0.85 Thr 73 . . . . T T . 0.26 0.26. * F 0.80 1.61 Ser 74 . . . . T T . -0.41 0.33. . F 0.65 0.83 Asn 75 . . B . . . . -0.09 0.41. . F -0.250.29 Ala 76 . . B . . . . 0.22 0.41. . . -0.400.20 Val 77 . . B . . . . -0.23 -0.07. . . 0.50 0.24 Cys 78 . . B . . T . -0.73 0.11. . . 0.10 0.08 Gly 79 . . B . . T . -0.64 0.40* * . 0.10 0.07 Asp 80 . . B . . T . -0.53 0.33* * . 0.10 0.14 Cys 81 . . B . . T . -0.64 -0.31* * . 0.70 0.51 Leu 82 . . B . . . . -0.03 -0.10* * . 0.50 0.44 Pro 83 . . B . . T . 0.740.23 * . 0.100.42 *

Arg 84 . . B . . T . 1.130.23 * . 0.251.52 *

Phe 85 . . B . . T . 0.82-0.34 * . 0.853.69 *

Tyr 86 . . B . . 'f . 1.60-0.54 * . 1.383.44 *

Arg 87 . . B B . . . 1.52-0.97 * F 1.363.44 *

Lys 88 . . B B . . . 1.39-0.29 * F 1.292.79 *

Thr 89 . . B B . . . 0.93-0.64 * F 1.821.76 *

Arg 90 . . . B T . . 0.82-0.97 * F 2.300.89 *

Ile 91 . . . B T . . 1.07-0.29 . F 1.770.37 .

Gly 92 . . . . T . . 0.960.11 . F 1.140.44 .

Gly 93 . A . . T . . 0.91-0.37 * F 1.310.38 .

Leu 94 . A . . . . C 1.220.03 * F 0.280.93 *

Gln 95 . A . . T . . 0.44-0.66 * F 1.301.62 .

Asp 96 . A . . T . . 0.44-0.51 . F 1.150.88 .

Gln 97 . A B . . . . 0.58-0.26 . F 0.450.75 .

Glu 98 . A B . . . . 0.26-0.51 . F 0.750.67 .

Cys 99 . A B . . . . 0.76-0.34 . . 0.300.21 .

Ile 100. . B . . . . 0.800.14 . . -0.100.18 .

Pro 101. . . . T . . 0.80-0.26 . . 0.900.21 .

Cys 102. . . . T T . 0.490.14 . . 0.500.67 *

Thr 103. . . . T T . 0.280.06 . F 1.101.37 *

Lys 104. . . . T T . 0.63-0.20 . F 2.001.37 .

Gln 105. . . . . T C 1.22-0.14 . F 2.103.69 .

Thr 106. . . . . T C 1.43-0.33 . F 2.403.43 .

Pro 107. . . . . T C 1.24-0.81 * F 3.002.97 .

Thr 108. . . . T T . 1.56-0.17 * F 2.601.27 .

Ser 109. . B . . T . 0.84-0.17 * F 1.901.53 .

Glu 110. A B . . . . 0.26-0.09 * F 1.050.53 *

Val 111. A B . . . . -0.13-0.01 * . 0.600.37 *

Gln 112. A B . . . . 0.080.29 * . -0.300.24 *

Cys 113A A . . . . . -0.420.30 * . -0.300.24 *

Ala 114A A . . . . . -0.420.99 * . -0.600.27 *

Phe 1 A A . . . . . -1.230.73 * . -0.600.21 15 .

Gln 116A A . . . . . -1.231.01 * . -0.600.32 .

Leu 117A A . . . . . -1.231.09 * . -0.600.23 .

Ser 118. A B . . . . -1.160.59 * . -0.600.47 .

Leu 119. A B . . . . -0.570.30 * . -0.300.27 .

Val 120A A . . . . . -0.46-0.10 * . 0.300.55 .

Glu 121A A . . . . . -0.67-0.29 * . 0.300.41 .

Ala 122A A . . . . . -0.17-0.24 . . 0.300.78 .

Asp 123. A . . T . . -0.72-0.44 . . 0.851.51 .

Ala 124. A . . . . C -0.12-0.44 . F 0.650.65 .

Pro 125. A . . . . C 0.52-0.01 * F 0.650.99 .

Thr 126. . . . . . C 0.52-0.09 . F 0.850.92 .

Val 127. . . . . . C 1.110.31 . F 0.401.57 .

Pro 128. . . . . . C 0.52-0.19 . F 1.001.76 .

Pro 129A . . . . . . 0.80-0.11 . F 0.801.23 .

Gln 130A . . . . . . 0.20-0.11 . F 0.802.40 .

Glu 131A . . . . . . -0.34-0.07 . F 0.801.28 .

Ala 132A . . B . . . -0.080.14 . F -0.150.61 .

Thr 133A . . B . . . -0.680.21 . . -0.300.36 .

Leu 134A . . B . . . -1.320.50 . . -0.600.17 .

Val 135A . . B . . . -1.621.14 . . -0.600.13 .

Ala 136A . . B . . . -1.921.03 . . -0.600.12 .

Leu 137A . . B . . . -2.140.93 . . -0.600.19 *

Val 138A . . B . . . -2.640.93 . . -0.600.21 *

Ser 139A . . B . . . -2.690.97 . . -0.600.17 .

Scr 140. . B B . . . -2.691.11 . . -0.600.15 *

Leu 141. . B B . . . -2.801.07 . . -0.600.15 *

Leu 142. . B B . . . -2.301.21 . . -0.600.10 *

Val 143 . . B B . . . -2.261.31. . . -0.600.11 Val 144 . A B B . . . -2.541.61. * . -0.600.11 Phe 145 . A B B . . . -2.941.43. . . -0.600.13 Thr 146 . A B B . . . -2.941.53. . . -0.600.15 Leu 147 A A . B . . . -2.481.57. . . -0.600.17 Ala 148 A A . B . . . -2.431.36. . . -0.60O.19 Phe 149 A A . B . . . -2.281.26. . . -0.600.11 Leu 150 A A . B . . . -2.281.56. . . -0.600.12 Gly 151 A A . B . . . -2.781.66. . . -0.600.10 Leu 152 A A . B . . . -2.211.84. . . -0.600.10 Phe 153 A A . B . . . -2.291.81. . . -0.600.18 Phe 154 A A . B . . . -1.541.70* . . -0.600.10 Leu 155 A A . B . . . -0.731.27* . . -0.600.24 Tyr 156 A A . B .. . . -1.090.99* . . -0.600.48 Cys 157 . A . B T . . -0.980.99* . . -0.200.48 Lys 158 . A . B T . . -0.280.99* . . -0.200.50 Gln 159 . A . B T . . 0.530.70* . . -0.200.51 Phe 160 . A . B T . . 1.31-0.06* . . 0.85 1.88 Phe 161 . A . B T . . 0.89-0.13* . . 1.16 1.28 Asn 162 . . . . T T . 1.560.44* * . 0.82 0.39 Arg 163 . . . . 'I' T . 1.620.44* * . 1.13 0.79 His 164 . . . . 'f T . 1.28-0.34* * . 2.49 1.79 Cys 165 . . . . T 'f . 1.63-0.70* * . 3.10 1.10 Gln 166 . . . . T T . 1.52-0.67* . F 2.79 0.56 Arg 167 . . . . T T . 0.710.01* * F 1.58 0.34 Gly 168 . . . . T T . 0.600.20* * F 1.27 0.52 Gly l69 . . . . T T . -0.070.03* . F 0.96 0.52 Leu 170 . A . . . . C 0.600.41. * . -0.400.23 Leu 171 . A B . . . . 0.010.41. * . -0.600.40 Gln 172 . A B . . . . -0.100.49. * . -0.600.41 Phe 173 A A . . . . . 0.290.06. * . -0.300.83 Glu 174 A A . . . . . 0.32-0.63. * . 0.75 2.01 Ala 175 A A . . . . . 0.54-0.83* * F 0.90 1.67 Asp 176 A A . . . . . 1.40-0.73* * F 0.90 1.95 Lys 177 A A . . . . . 1.40-1.51. * F 0.90 2.26 Thr 178 A A . . . . . 2.10-1.51* * F 0.90 3 .87 Ala 179 A A . . . . . 1.80-2.01* . F 0.90 4.01 Lys 180 A A . . . . . 1.58-1.63* . F 0.90 2.69 Glu 181 A A . . . . . 0.88-0.94* . F 0.90 1.54 Glu 182 A A . . . . . 0.62-0.64. . F 0.90 1.32 Ser 183 A . . . . . . 0.08-0.71. * F 1.10 1.02 Leu 184 . . B . . . . 0.46-0.07. * . 0.50 0.44 Phe 185 . . B . . . . 0.200.36. . . 0.20 0.39 Pro 186 . . . . . . C -0.100.79. . . 0.40 0.45 Val 187 . . . . . . C -0.060.79. . F 0.85 0.73 Pro 188 . . . . . T C 0.240.10. . F 1.80 1.69 Pro 189 . . . . . T C 0.74-0.69. . F 3.00 1.89 Ser 190 . . . . . T C 1.14-0.63. . F 2.70 3.67 Lys 191 . . . . . T C 0.77-0.89. . F 2.40 3.18 Glu 192 A A . . . . . 1.62-0.81. . F 1.50 2.08 Thr 193 A A . . . . . 1.53-1.24. . F 1.20 2.69 Ser 194 A A . . . . . 1.74-1.24. * F 0.90 I
.80 Ala 195 A A . . . . . 1.19-0.84. * F 0.90 1.80 Glu 196 A A . . . . . 0.84-0.20. * F 0.45 0.93 Ser 197 A . . . . . . 0.56-0.30. * F 0.65 0.93 Gln 198 A . . . . . . 0.280.23. * F 0.05 0.96 Val 199 . . B . . . . 0.370.23. * . -0.100.56 Ser 200 . . B . . . . 0.610.66. * . -0.400.65 Trp 201 . . . . . . C 0.310.70. * . -0.200.37 Ala 202 . . . . . T C -0.200.69. . . 0.00 0.67 Pro 203 . . . . T C -0.790.73 . F 0.15 0.41 . *

Gly 204 . . . T T . 0.07 0.84 . F 0.35 0.40 . *

Ser 205 . . . . T C -0.440.33 . F 0.45 0.68 . *

Leu 206 . B . . . . -0.860.51 . . -0.400.36 . *

5 Ala 207 . B . . . . -0.570.87 . . -0.400.32 . *

Gln 208 . B . . . . -1.170.83 . . -0.400.32 . .

L.eu209 . B . . . . -0.821.13 . . -0.400.32 . .

Phe 210 . B . . . . -0.820.44 . . -0.400.52 . .

Ser 211 . B . . . . -0.870.33 . . -0.100.40 . .

10 L.cu212 . B . . . . -0.490.57 . . -0.400.36 . .

Asp 213 . . . T . . -1.380.31 . F 0.45 0.65 . .

Ser 214 . . . . . C -0.780.21 . F 0.25 0.34 . .

Val 215 . . . . . C -0.080.26 * F 0.25 0.64 . .

Pro 216 . . . . . C 0.22 -0.03 . F 0.85 0.66 . .

15 Ile 217 . B . . . . 1.03 0.37 . F 0.05 0.86 . .

Pro 218 . B . . . . 1.03 0.39 . F 0.46 2.00 . .

Gln 219 . B . . . . 0.99 0.14 * F 0.72 2.24 . .

Gln 220 . B . . . . 1.63 0.14 . F 0.98 3.16 . .

Gln 221 . . . . . C 1.84 -0.11 . F 2.04 3.16 . .

20 Gln 222 . . . . . C 2.13 -0.54 . F 2.60 3.16 . .

Gly 223 . . . . T C 1.96 -0.33 . F 2.24 1.80 . .

Pro 224 . . . . T C 1.57 -0.30 . F 1.98 1.33 . .

Glu 225 . B . . T . 1.18 -0.27 . . 1.22 0.98 . .

Met 226 . B . . T . 0.79 -0.24 . . 1.1l 1.27 . *

TableIII

Res Pos 11 III N V VI VII VIII IX XI XII XIII X1V
I X

30 Met 1 . . B . . . . 0.20 -0.17 . . 0.93 1.00 .

Ala 2 . . B . . . . 0.56 -0.17 . . 1.06 0.78 .

Glu 3 . . . . . T C 0.64 -0.10 . . 1.74 0.83 .

Pro 4 . . . . . T C 1.00 -0.14 . . 2.17 1.12 .

Gly 5 . . . . T T . 1.36 -0.26 . F 2.80 1.51 .

35 His 6 . . . . . T C 1.14 -0.26 . . 2.17 1.18 .

Ser 7 . A . . . . C 1.43 0.43 . . 0.44 0.63 .

His 8 . A . . . . C 0.84 0.39 * . 0.46 0.86 .

His 9 . A . . . . C 1.17 0.46 * . -0.120.64 .

Leu 10 . A B . . . . 0.66 -0.04 * . 0.30 0.93 .

40 Ser 11 . A B B . . . 0.80 0.21 * . -0.300.51 .

Ala 12 . A B B . . . 0.76 -0.29 * . 0.30 0.73 .

Arg 13 . A B B . . . 0.90 -0.36 * . 0.30 0.87 .

Val 14 . . B B . . . 0.62 -1.04 * F 1.16 1.28 .

Arg 15 . . B B . . . 1.43 -0.94 * F 1.42 1.83 *

45 Gly 16 . . . . T . . 1.84 -1.44 * F 2.28 1.61 *

Arg 17 . . B . . . . 2.54 -1.44 * F 2.14 4.26 *

Thr 18 . . . . . . C 1.54 -2.09 * F 2.60 4.26 *

Glu 19 . . B . . . . 2.19 -1.40 * F 2.14 3 * .02 Arg 20 . . B B . . . 2.19 -1.40 * F 1.68 2.38 *

50 Arg 21 . . B B . . . 1.72 -1.40 * F 1.42 3.23 *

Ile 22 . . B B . . . 1.32 -1.20 * F 1.16 1.54 *

Pro 23 . . B B . . . 1.74 -0.29 . F 0.45 0.83 *

Arg 24 . . . B T . . 0.93 -0.29 . F 0.85 0.83 *

L.eu25 . . B B . . . 0.01 0.40 . . -0.300.97 *

55 Trp 26 . . B B . . . -0.910.40 * . -0.300.52 *

Arg 27 . . B B . . . -0.310.66 . . -0.600.22 *

L.eu28 . . B B . . . -0.691.57 . . -0.600.28 *

L.eu29 . . B B . . . -1.141.39 . . -0.600.27 *

L.eu30 . . B B . . . -0.640.90 * . -0.600.14 *

Trp 31 . . . B . . C -0.941.39 * . -0.400.24 *

Ala 32 . . . B . . C -1.761.20 * . -0.400.29 *

Gly 33 . . . B . . C -0.941.30 . . -0.400.30 .

Thr 34 . . . B . . C -0.991.01 . . -0.400.50 .

Ala 35 . . B B . . . -0.490.74 . . -0.600.37 .

Phe 36 . . B B . . . -0.200.73 . . -0.600.54 .

Gln 37 . . B B . . . 0.04 0.70 . . -0.600.64 .

Val 38 . . B B . . . 0.08 0.64 . . -0.600.63 .

Thr 39 . . B B . . . 0.04 0.63 . F -0.301.05 .

Gln 40 . . . B T . . 0.42 0.27 . F 0.250.60 .

10Gly 41 . . . . T . . 1.12 0.30 . F 0.601.25 .

Thr 42 . . . . . . C 0.31 -0.34 . F 1.001.50 *

Gly 43 . . . . . T C 1.13 -0.14 . F 1.050.72 .

Pro 44 . . . . T C 0.86 -0.04 . F 1.050.98 .

Glu 45 . . B . . T . 0.19 0.03 . F 0.250.69 .

15Leu 46 . . B . . T . 0.58 0.11 . . 0.100.37 .

His 47 . . B . . . . 0.89 -0.31 . . 0.500.48 .

Ala 48 . . . . . . C 0.93 -0.74 . . 1.000.48 .

Cys 49 A . . . . T . 1.14 -0.36 . . 0.700.78 .

Lys 5 0 . . . . T . 0.90 -1.04 . F I 1.00 A . .15 20Glu 51 A . . . . T . 1.68 -0.79 . F 1.301.55 .

Ser 52 . . . . T T . 1.47 -0.79 . F 1.703.93 *

Glu 53 . A . . T . . 2.06 -0.60 . F 1.303.08 .

Tyr 54 . A . . 'f . . 2.48 -0.60 . . 1.153.08 .

His 55 . A . . T . . 2.12 0.16 * . 0.253.60 .

25'1'yr56 . A . . T . . 1.53 0.26 * . 0.253.00 .

Glu 57 . A B . . . . 1.17 0.76 . . -0.451.93 .

Tyr 58 . A B . . . . 1.17 0.57 . . -0.600.76 .

Thr 59 . A B . . . . 1.11 0.07 . . -0.300.81 .

Ala 60 . A B . . . . 0.83 -0.30 . . 0.640.63 .

30Cys 61 . A B . . . . 0.73 0.19 . . 0.380.58 .

Asp 62 . A . . T . . 0.43 -0.14 . F 1.870.40 .

Ser 63 . . . . T T . 0.79 -0.24 . F 2.610.53 *

Thr 64 . . . . T T . 0.81 -0.74 * F 3.401.92 *

Gly 65 . . . . T T . 1.51 -0.40 * F 2.761.21 .

35Ser 66 . . . . T T . 1.32 -0.40 * F 2.421.77 .

Arg 67 . . . B T . . 0.73 -0.14 * F 1.530.91 *

'1'rp68 . . B B . . . 0.18 -0.13 * . 0.640.93 .

Arg 69 . . B B . . . 0.28 0.09 * . -0.300.51 .

Val 70 . . B B . . . 0.59 0.13 * . -0.300.41 .

40Ala 71 . . B B . . . 0.58 0.63 * . -0.600.53 .

Val 72 . . B B . . . 0.26 0.20 * . -0.300.39 .

Pro 73 . . . . T . . 0.20 0.63 * . 0.000.81 *

His 74 . . . . T . . -0.720.41 * . 0.000.79 *

Thr 75 . . . . . T C -0.530.60 . F 0.150.88 .

45Pro 76 . . . . T T . -0.260.53 . F 0.350.30 .

Gly 77 . . . . T T . 0.30 0.59 . F 0.350.32 .

Leu 78 . . B . . T . -0.300.47 . -0.200.30 .

Cys 79 . . B . . . . -0.480.67 . . -0.400.16 .

Thr 80 . . B . . . . -0.170.67 . . -0.400.25 .

50Ser 81 . . B . . . . -0.170.24 . F 0.050.51 *

Leu 82 . . B . . T . -0.68-0.01 . F 1.301.46 *

Pro 83 . . B . . T . 0.18 0.06 * F 0.850.75 *

Asp 84 . . . . . T C 0.50 -0.43 * F 2.101.12 *

Pro 85 . . . . 'f T . 0.50 -0.39 . F 2.601.34 *

55Val 86 . . . . T . . 0.80 -0.59 . F 3.001.25 *

Lys 87 . . B . . . . 0.94 -1.01 . F 2.301.30 *

Gly 88 . . B . . . . 0.86 -0.44 . F 1.550.45 *

Thr 89 . . B . . . . 0.16 -0.49 . F 1.250.81 *

Glu 90 . . B . . . . 0.07 -0.34 . F 0.950.35 .

60Cys 91 . . B . . T . 0.26 0.04 * . 0.100.48 .

Ser 92 . . B . . T . 0.21 0.19 * . 0.10 0.18 .

Phe 93 . . B . . T . -0.030.10 * . 0.10 0.16 .

Ser 94 . . . . T T . -0.070.60 * . 0.20 0.31 .

Cys 95 . . . . 'f . . -0.070.46 * . 0.00 0.23 .

Asn 96 . . . . T T . -0.100.07 . . 0.50 0.46 *

Ala 97 . . . . . T C -0.610.07 . . 0.30 0.30 *

Gly 98 . . . . . T C 0.09 0.37 . F 0.45 0.46 *

Glu 99 A . . . . '1' . -0.21-0.20 * . 0.70 0.47 .

Phe 100 . A B . . . . 0.50 0.01 . . -0.300.46 .

Leu 101 . A B . . . . 0.50 -0.49 . . 0.30 0.94 .

Asp 102 A A . . . . . 1.09 -0.91 . . 0.94 0.90 .

Met 103 . A . . T . . 1.13 -0.51 . . 1.83 1.81 .

Lys 104 . A . . T . . 0.47 -0.91 . F 2.32 2.94 *

Asp 105 . . . . T T . 1.21 -1.03 . F 2.91 0.94 *

Gln 106 . . . . T T . 1.81 -1.03 * F 3.40 1.90 .

Ser 107 . . . . '1' T . 1 -1 . F 3 1 .14 .21 .06 .47 .

Cys 108 . . . . T T . 1.16 -0.64 * F 2.57 0.47 .

Lys 109 . . B . . T . I.11 -0.14 * F 1.53 0.28 .

Pro 110 . . B . . T . 0.77 -0.54 * F 1.69 0.36 .

Cys 111 . . B . . T . 0.88 -0.50 * . 1.10 0.66 .

Ala 112 . . B . . T . 0.93 -1.07 * F 1.75 0.64 .

Glu 113 . . B . . . . 1.30 -0.31 * F 1.45 0.65 .

Gly 114 . . B . . T . 0.44 -0.36 * F 2.00 1.63 .

Arg 115 . . B . . T . 0.31 -0.24 * F 1.80 1.33 .

Tyr 116 . . B . . T . 0.67 -0.31 * . 1.30 0.76 .

Ser 117 . . B . . T . 0.91 0.17 * . 0.65 1.11 .

Leu 118 . . B B . . . 0.02 0.17 * . -0.100.56 *

Gly 119 . . . B T . . 0.48 0.86 * F -0.050.25 *

Thr 120 . . B B . . . -0.330.10 * F -0.150.37 *

Gly 121 . . B B . . . -0.090.50 * F -0.450.39 *

Ile 122 . A B B . . . 0.21 -0.19 * . 0.30 0.65 *

Arg 123 . A B B . . . 0.73 -0.61 * . 0.60 0.78 .

Phe 124 . A B B . . . 1.08 -0.19 * . 0.30 0.83 .

Asp 125 . A . B . . C 1.39 -0.61 * . 0.95 1.97 .

Glu 126 . A . . T . . 0.92 -1.30 * F 1.30 1.75 .

Trp 127 . A . . T . . 1.60 -0.61 * F 1.30 1.66 .

Asp 128 . A . . . . C 1.46 -0.97 * F 1.10 1.54 .

Glu 129 . A . . . . C 1.81 -0.47 . F 0.80 1.21 *

Leu 130 . . . . . T C 1.11 -0.04 . . 1.05 1.14 *

Pro 131 . . . . . T C 0.52 -0.17 . . 0.90 0.59 *

His 132 . . . . T T . 0.51 0.33 . . 0.50 0.34 *

Gly 133 . . . . . T C -0.300.71 . . 0.00 0.56 *

Phe 134 . A . . . . C -0.600.71 . . -0.400.30 *

Ala 135 . A . . . . C -0.380.67 * . -0.400.29 *

Ser 136 . A . . . . C -0.170.67 * . -0.400.30 .

Leu 137 . A . . . . C -0.730.64 * . -0.400.56 .

Ser 138 . . . . . 'f C -0.390.47 * . 0.00 0.55 .

Ala 139 . . . . . T C -0.50-0.03 * . 0.90 0.71 .

Asn 140 . . B . . T . 0.09 0.27 * . 0.10 0.71 .

Met 141 . . B . . T . 0.39 -0.41 * . 0.70 0.88 .

Glu 142 . . B . . . . 0.90 -0.80 * . 0.95 1.45 .

Leu 143 A . . . . T . 0.61 -0.91 * . 1.15 1.21 .

Asp 144 A . . . . T . 0.61 -0.81 * F 1.30 1.24 .

Asp 145 A . . . . T . 0.61 -0.93 * F 1.15 0.72 .

Ser 146 A . . . . T . 0.91 -0.93 . F 1.58 1.51 .

Ala 147 A . . . . . . 0.60 -1.23 . F 1.66 1.22 .

Ala 148 A . . . . . . 1.07 -0.74 . F 1.94 1.05 .

Glu 149 . . . . T . . 1.07 -0.31 . F 2.17 0.78 .

Ser 150 . . . . T T . 0.40 -0.30 . F 2.80 1.23 *

Thr 151 . . . . T T . 0.39 -0.23 . F 2.37 0.66 .

Gly 152 . . . . T T . 0.68 -0.24. . F 2.09 0.55 Asn 153 . . . . T T . 0.97 0.14. . F 1.21 0.55 Cys 154 . . . . T T . 1.01 0.14. . F 0.93 0.51 Thr 155 . . . . T T . 1.02 -0.34. . F 1.40 1.02 Ser 156 . . . . T T . 0.48 0.14. . F 0.65 0.67 Ser 157 . . . . T T . 0.61 0.39* * F 0.88 0.93 Lys 158 . . . . T . . 0.72 0.24* * F 0.91 0.99 Trp 159 . . B . . . . 1.04 -0.24* * F 1.49 1.45 Val 160 . . B . . T . 1.36 -0.20* * F 1.92 1.07 Pro 161 . . B . . T . 1.41 -0.59* * F 2.30 0.90 Arg 162 . . . . T T . 0.82 0.17* * F 1.72 1.33 Gly 163 . . B . . T . 0.19 -0.06* * F 1.69 1.26 Asp 164 . . . . T . . -0.22-0.20* * F 1.51 0.82 Tyr 165 . . B . . . . 0.63 0.16* . . 0.13 0.36 lle 166 . . B . . . . 0.53 0.56. * . -0.400.59 Ala 167 . . B . . . . 0.42 0.61. * . -0.400.51 Phe 168 . . B . . . . 0.77 0.61. . . -0.090.54 Asn 169 . . B . . T . 0.10 -0.14. . . 1.47 1.35 Thr 170 . . . . . T C 0.03 -0.26. . F 1.98 0.71 Asp 171 . . . . T T . 0.33 -0.27. * F 2.64 1.19 Glu 172 . . . . T T . 0.61 -0.56. . F 3.10 0.75 Cys 173 . . . B T . . 0.50 -0.47. . . 1.94 0.75 Thr 174 . . B B . . . -0.10-0.27. . . 1.23 0.37 Ala 175 . . B B . . . -0.030.34. . . 0.32 0.21 Thr 176 . . B B . . . -0.621.10. . . -0.290.62 Leu 177 . . B B . . . -1.481 * * . -0.600.43 .03 Met 178 . . B B . . . -0.811.19. * . -0.600.32 Tyr 179 . . B B . . . -1.311.09. * . -0.600.35 Ala 180 . . B B . . . -0.681.29. * . -0.600.35 Val 181 . . B B . . . -0.370.60. * . -0.600.71 Asn 182 . . B B . . . 0.14 0.39. * . -0.300.79 Leu 183 . . B B . . . 0.40 0.01. * F 0.21 1.05 Lys 184 . . B B . . . 0.33 -0.06. * F 1.02 1.40 Gln 185 . . . . T T . 0.07 -0.21. * F 2.03 1.25 Ser 186 . . . . . T C 0.92 0.03. * F 1.44 1.13 Gly 187 . . . . . T C 0.22 -0.26. * F 2.10 0.91 Thr 188 . . B . . T . 1.03 0.53. * F 0.79 0.45 Val 189 . . B B . . . 0.74 0.13. * F 0.48 0.59 Asn 190 . . B B . . . 0.50 0.50. * . -0.180.93 Phe 191 . . B B . . . 0.56 0.83. * . -0.241.01 Glu 192 . . B B . . . 0.69 1.10. * . -0.452.13 Tyr 193 . . B B . . . 1.00 0.89. * . -0.452.04 Tyr 194 . . . . T . . 1.56 0.49. * . 0.15 3.94 Tyr 195 . . . . . T C 1.26 0.09. * . 0.45 3.05 Pro 196 . . . . T T . 1.07 0.47. . F 0.50 2.61 Asp 197 . . . . T T . 0.18 0.40. . F 0.50 1.17 Ser 198 . . . . . T C -0.280.33. . F 0.45 0.52 Ser 199 . . B B . . . -0.030.36. * F -0.150.29 Ile 200 . . B B . . . -0.49-0.07. * . 0.30 0.30 Ile 201 . . B B . . . -0.980.71. * . -0.600.20 Phe 202 . . B B . . . -1.831.11* * . -0.600.13 Glu 203 . . B B . . . -1.531.37. * . -0.600.13 Phe 204 . . B B . . . -1.231.09. * . -0.600.33 Phe 205 . . B B . . . -0.340.80* * . -0.600.62 Val 206 . . . B T . . 0.54 0.01* . . 0.10 0.59 Gln 207 . . . . T T . 0.58 0.41. * F 0.50 1.19 Asn 208 . . . . T T . 0.58 0.20. . F 0.65 0.73 Asp 209 . . . . T T . 1.07 -0.19. . F 1.40 1.71 Gln 210 . . . . 1' T . 1.77 -0.40. . F 1.74 1.53 Cys 211 . . . . T . . 2.03 -0.40. * F 1.88 1.53 Gln 212 . B . . T . 2.03-0.30 * F 1.870.93 . .

Pro 213 . B . . T . 2.03-0.30 * F 2.210.89 . .

Asn 214 . . . T T . 1.73-0.70 . F 3.402.78 . .

Ala 215 . . . T T . 1.84-0.89 * F 3.062.15 . .

Asp 216 . . . T . . 2.22-1.29 . F 2.752.73 . .

Asp 2l7 . . . T T . 1.62-0.80 . F 2.841.78 . *

Ser 218 . . . . T C 1.88-0.59 . F 2.531.75 . *

Arg 219 . . . T T . 1.57-1.09 . F 2.622.09 . *

Trp 220 . B . . T . 1.84-0.60 . . 2.301.81 . *

Met 221 A B . . . . 1.84-0.11 . . 1.371.95 . *

Lys 222 A B . . . . 1.89-0.50 * F 1.291.72 . *

Thr 223 A . . . . C 1.84-0.50 . F 1.263.27 . *

Thr 224 A . . . . C 1.44-0.99 . F 1.333.27 . *

Glu 225 A . . . . C 1.73-0.69 . F I.101.72 . *

Lys 226 A . . . . C 1.63-0.69 * F 1.102.06 . *

Gly 227 A . . T . . 1.56-0.39 . F 1.001.24 . *

Trp 228 A . . . . C 1.57-0.37 . . 0.500.97 . .

Glu 229 A . . . . C 1.020.01 . . -0.100.65 . .

Phe 230 A B . . . . 1.020.66 . . -0.600.49 . .

His 231 A B . . . . 0.170.23 . . -0.300.81 . .

Ser 232 A B . . . . 0.510.00 . . -0.300.38 . .

Val 233 A B . . . . 0.910.40 . . -0.260.71 . *

Glu 234 A . . . . C 0.57-0.39 . . 1.331.03 . .

Leu 235 A . . . . C 1.27-0.46 . . 1.520.76 . .

Asn 236 . . . T T . 1.30-0.44 . F 2.761.64 . .

Arg 237 . . . T T . 0.74-0.69 . F 3.401.52 . *

Gly 238 . . . 'f T . 0.79-0.04 . F 2.761.37 . *

Asn 239 . . . T T . 0.54-0.04 . F 2.270.70 . *

Asn 240 . . B . . C 1.070.31 * F 0.730.56 . *

Val 241 . B B . . . 1.181.23 * . -0.260.60 . .

Leu 242 . B B . . . 0.760.80 * . -0.600.73 . .

Tyr 243 . B B . . . 0.790.89 * . -0.600.65 . .

Trp 244 . B B . . . 0.200.97 * . -0.451.27 . .

Arg 245 . B B . . . -0.500.83 . . -0.451.56 . .

Thr 246 . B B . . . 0.060.93 . F -0.450.86 . .

Thr 247 . B B . . . 0.010.56 * . -0.451.10 . .

Ala 248 . B B . . . -0.030.29 . . -0.300.42 . .

Phe 249 . B B . . . -0.061.20 * . -0.600.30 . .

Ser 250 . B B . . . -0.121.20 * . -0.600.30 . .

Val 251 . . B T . . -0.670.71 * . -0.200.60 . *

Trp 252 . B B . . . -0.570.86 . . -0.360.51 . *

'fhr 253 . B B . . . 0.070.50 . F 0.030.59 . *

Lys 254 . . B . . C 0.560.11 * F 0.921.60 . ' *

Val 255 . . . . T C -0.00-0.10 . F 2.162.35 . *

Pro 256 . . . . T C 0.04-0.37 . F 2.401.21 . *

Lys 257 . B . . T . -0.52-0.17 * F 1.810.50 . *

Pro 258 . B . . T . -0.100.47 * F 0.670.50 . .

Val 259 . B B . . . -0.14-0.17 . . 0.780.63 . *

Leu 260 . B B . . . -0.18-0.20 . . 0.540.51 . .

Val 261 . B B . . . -0.560.49 . . -0.600.23 . .

Arg 262 . B B . . . -1.490.56 . . -0.600.31 . .

Asn 263 . B B . . . -1.590.60 . . -0.600.27 . *

Ile 264 . B B . . . -1.080.40 . . -0.600.52 . .

Ala 265 . B B . . . -1.120.19 . . -0.300.26 . *

Ile 266 . B B . . . -0.860.83 . . -0.600.12 . *

Thr 267 . B B . . . -1.210.93 . . -0.600.17 . .

Gly 268 . B B . . . - I .00 * . -0.600.27 . I .
.52 Val 269 . B B . . . -0.930.99 . . -0.600.56 . .

Ala 270 . B B . . . -0.340.69 . . -0.600.52 . .

'fyr 271 . B B . . . -0.120.20 . . -0.300.90 . *

Thr 272 . B . . T . -0.51 0.34 . F 0.25 0.65 . .

Ser 273 . B . T T . -0.38 0.49 . F 0.35 0.56 . .

Glu 274 . . . T T . -0.19 0.41 . F 0.35 0.55 . .

Cys 275 . B . . T . 0.44 0.23 . . 0.10 0.20 . .

5 Phe 276 . B . . . . 0.48 -0.26 . . 0.50 0.31 . .

Pro 277 . . . T . . 0.44 -0.21 . . 0.90 0.27 . .

Cys 278 . . . T . . 0.43 0.21 . . 0.30 0.50 . .

Lys 279 . . . . T C 0.19 0.13 . F 0.45 0.84 . .

Pro 280 . . . T T . 0.27 0.10 * F 0.65 0.85 . .

10 Gly 281 . . . T T . 0.97 0.17 * F 0.80 1.60 . *

1'hr282 . B . . T . 1.22 -0.40 . F 1.34 1.34 . *

Tyr 283 . B . . . . 1.89 -0.40 . F 1.48 1.73 . *

Ala 284 . B . . . .. 1.50 -0.43 . F 1.82 3.03 . *

Asp 285 . B . . T . 1.41 -0.43 . F 2.36 2.08 . *

15 I_ys286 . . . T 1' . 1.46 -0.53 . F 3.40 1.78 . *

Gln 287 . . . T T . 1.07 -0.90 . F 3.06 2.36 . *

Gly 288 . . . T T . 0.64 -0.61 . F 2.72 1.22 . *

Ser 289 . . . T T . 1.28 -0.04 . F 1.93 0.33 . *

Ser 290 . B . T T . 0.47 -0.04 . F 1.59 0.38 . *

20 Phe 291 . B . . T . -0.24 0.24 . . 0.10 0.32 . *

Cys 292 . B . . T . -0.46 0.39 . . 0.10 0.13 . *

Lys 293 . B . . . . -0.70 0.43 . . -0.400.15 . *

Leu 294 . B . . . . -0.40 0.54 . . -0.400.17 . .

Cys 295 . B . . . . -0.40 0.16 . . -0.100.51 . .

25 Pro 296 . B . . . . 0.06 -0.03 . . 0.50 0.34 . .

Ala 297 . . . T . . 0.42 0.73 . . 0.00 0.65 . .

Asn 298 . . . T T . 0.38 0.43 . F 0.84 1.62 . .

Ser 299 . . . T T . 1.23 0.26 . F 1.48 1.69 . *

Tyr 300 . . . T T . 1.56 -0.17 . F 2.42 3.34 . .

30 Ser 301 . . . . T C 1.77 -0.24 . F 2.56 2.05 . *

Asn 302 . . . T T . 2.04 -0.64 . F 3.40 2.65 . *

Lys 303 . . . T T . 1.74 -0.54 . F 3.06 2.44 . .

Gly 304 . . . T T . 1.38 -0.91 * F 2.72 2.44 . .

Glu 305 . . . T T . 1.59 -0.73 . F 2.23 0.81 . *

35 Thr 306 . . . T T . 1.89 -0.63 . F 1.89 0.55 . *

Ser 307 . B . . T . 1.22 -0.23 . F 0.85 0.97 . *

Cys 308 . B . . T . I.18 -0.09 * . 1.04 0.30 . .

His 309 . B . . T . 1.31 -0.09 . . 1.38 0.35 . .

Gln 310 . . . T . . 1.31 -0.14 . . 1.92 0.40 . .

40 Cys 311 . . . T . . 1.67 -0.53 . . 2.71 1.25 . .

Asp 312 . . . T T . 1.72 -I.10 . F 3.40 1.84 . .

Pro 3l3 . . . T T . 2.09 -0.84 . F 3.06 1.66 . .

Asp 314 . . . T T . 2.12 -0.86 . F 3.06 4.15 . .

Lys 315 . . . T T . 2.17 -1.43 . F 3.06 4.31 . *

45 Tyr 316 . B . . T . 2.49 -1.43 . F 2.66 5.57 . *

Ser 317 . B . . T . 2.19 -1.43 . F 2.66 3.30 . *

Glu 318 . . . T T . 2 .10 -1.04 . F 3 2.21 . * .40 Lys 319 . . . T T . 1.80 -0.66 . F 3.06 1.89 . *

Gly 320 . . . T . . 1.09 -1.03 * F 2.52 1.89 . *

50 Ser 321 . . . T T . 1.33 -0.84 * F 2.23 0.59 . .

Ser 322 . . . T T . 0.78 -0.44 * F 1.59 0.47 . .

Ser 323 . . . T T . 0.89 0.20 * F 0.65 0.35 . .

Cys 324 . . . T T . 0.63 -0.23 * F 1.25 0.52 . .

Asn 325 . . . T . . 0.39 -0.19 * . 0.90 0.60 . .

55 Val 326 . B . . . . 0.02 -0.07 * . 0.50 0.45 . .

Arg 327 . B . . T . 0.01 0.11 * . 0.10 0.45 . .

Pro 328 . B . . T . 0.31 0.03 * . 0.44 0.40 . .

Ala 329 . B . . T . 1.02 -0.37 * . 1.38 0.91 . .

Cys 330 . B . . T . 1.02 -1.01 * . 2.02 0.93 . .

60 Thr 331 . B . . . . 1.63 -1.01 * F 2.31 1.00 . *

Asp 332 . . . . T T . 0.82 -0.69* . F 3.40 1.55 Lys 333 . . . . T T . 0.79 -0.40. . F 2.76 2.50 Asp 334 . . . . T T . 1.07 -0.21. . F 2.42 2.72 Tyr 335 . . B . . T . 1.70 -0.21. . . 1.53 2.35 Phe 336 . . B B . . . 1.70 0.29. . . 0.19 1.60 Tyr 337 . . B B . . . I.11 0.77. . . -0.451.38 Thr 338 . . B B . . . 0.40 1.27. . . -0.600.89 His 339 . . B B . . . 0.40 1.09. . . -0.600.55 Thr 340 . . B B . . . 0.06 0.30. . . -0.300.59 Ala 341 . . B B . . . 0.76 0.04. * . 0.00 0.41 Cys 342 . . . B T . . 0.66 -0.04. * . 1.30 0.49 Asp 343 . . . . T T . 0.97 -0.11. * . 2.00 0.33 Ala 344 . . . . . T C 0.69 -0.60. * F 2.55 0.57 Asn 345 . . . . . T C 1.00 -0.61. * F 3.00 1.54 Gly 346 . . . . . T C 0.78 -0.79. * F 2.70 1.60 Glu 347 . A . . . . C 0.84 -0.10. * F 1.70 1.30 Thr 348 . A B . . . . 0.60 0.01. * F 0.45 0.80 Gln 349 . A B . . . . 1.23 0.37* *. F 0.30 1.27 Leu 350 . A B . . . . 0.94 -0.06* * . 0.45 1.47 Met 351 . A B . . . . 0.70 0.86* * . -0.451.07 Tyr 352 . A B . . . . 0.74 0.87* * . -0.600.62 Lys 353 . A . . T . . 0.84 0.47* * . -0.051.51 Trp 354 . A . . T . . 0.89 0.21* * . 0.25 2.36 Ala 355 . A . . . . C 0.81 -0.40. * F 0.80 3.01 Lys 356 . A . . . . C 0.74 -0.47* * F 0.80 1.06 Pro 357 . A . . T . . 0.69 0.10. * F 0.25 0.54 Lys 358 . . . . T . . 0.64 -0.43. * F 1.05 0.71 Ile 359 . . B . . . . 0.93 -0.93. * . 0.80 0.62 Cys 360 . . B . . T . 0.71 -0.93. * . 1.00 0.67 Ser 361 . . B . . T . 0.67 -0.67. * F 1.15 0.28 Glu 362 . . B . . T . 0.53 -0.67* . F 1.15 0.68 Asp 363 A . . . . T . -0.10-0.93* * F 1.30 1.26 Leu 364 A A . . . . . -0.07-1.00* * F 0.75 0.95 Glu 365 A A . . . . . 0.64 -0.74* * F 0.75 0.41 Gly 366 A A . . . . . 0.13 -0.74* * F 0.75 0.49 Ala 367 . A B . . . . -0.08-0.06* * . 0.30 0.49 Val 368 . A B . . . . -0.67-0.31* * . 0.30 0.43 Lys 369 . A B . . . . -0.160.19* * . -0.300.44 Leu 370 . A B . . . . -0.500.14* * . -0.300.59 Pro 371 . . B . . T . -1.010.07* * . 0.10 0.78 Ala 372 . . . . T T . -0.380.07* * F 0.65 0.29 Ser 373 . . . . T T . 0.17 0.07* * F 0.65 0.70 Gly 374 . . . . T T . 0.09 -0.13* * F 1.25 0.66 Val 375 . . B . . . . 0.23 -0.06. . F 0.79 0.89 Lys 376 . . B . . . . 0.23 0.01. . F 0.33 0.35 Thr 377 . . B . . . . 0.61 0.06. . F 0.47 0.55 His 378 . . B . . . . 0.24 0.06* . . 0.61 1.15 Cys 379 . . B . . T . 0.59 -0.01. . . 1.40 0.31 Pro 380 . . B . . T . 1.23 0.39* * F 0.81 0.34 Pro 381 . . . . T T . 0.84 0.33. . F 1.07 0.39 Cys 382 . . . . T T . 0.46 0.26. . F 0.93 0.72 Asn 383 . . . . . T C -0.210.47. . F 0.29 0.40 Pro 384 . . . . T T . 0.50 0.83. . F 0.35 0.23 Gly 385 . . . . T T . 0.40 0.40. . . 0.20 0.85 Phe 386 . . B . . T . 0.61 0.31. . . 0.10 0.76 Phe 387 . . B . . . . 1.28 0.31. . F 0.30 0.79 Lys 388 . . . . T . . 0.98 0.29. . F 1.10 1.28 Thr 389 . . . . T . . 0.88 0.24. . F 1.35 1.99 Asn 390 . . . . T . . 0.56 -0.06* . F 2.20 3.31 Asn 391 . . . . T T . 1.26 -0.27* . F 2.50 0.89 Ser 392 . . . T 'C . 1.74 0.13* . F 1.80 1.06 .

Thr 393 . . . T T . 1.03 0.07. . F 1.55 1.02 .

Cys 394 . . . 'f T . 1.13 0.24. . F 1.15 0.34 .

Gln 395 . B . . . . 0.89 0.27. . F 0.30 0.39 .

Pro 396 . B . . . . 0.54 0.64. . F -0.250.43 .

Cys 397 . B . . T . 0.54 0.59. . . -0.200.79 .

Pro 398 . B . . 'f . 0.61 0.40. . . -0.200.61 .

Tyr 399 . . . T T . 0.98 0.76. . . 0.20 0.62 .

Gly 400 . . . T T . 0.98 0.71. . . 0.35 1.55 .

Ser 401 . . . T . . 0.84 0.54. . F 0.30 1.61 .

Tyr 402 . . . T T . 1.21 0.54. . F 0.50 1.02 .

Ser 403 . . . T T . 1.42 0.17. . F 1.11 1.38 .

Asn 404 . . . T T . 1.00 -0.26. . F 2.02 1.71 .

Gly 405 . . . T T . 1.03 -0.07* . F 2.18 0.59 .

Ser 406 . . . T '1' . 1.44 -0.34* . F 2.49 0.63 .

Asp 407 . . . T 'f . 1.02 -0.73* . F 3.10 0.77 .

Cys 408 . B . . T . 1.11 -0.56* * F 2.39 0.42 .

Thr 409 . B . . T . 0.52 -0.56* * F 2.39 0.48 .

Arg 410 . B . . . . 0.52 -0.44* . F 1.89 0.29 .

Cys 411 . B . . 'f . 0.51 -0.01. . . 1.94 0.54 .

Pro 412 . . . T T . 0.51 -0.10. . . 2.34 0.54 .

Ala 413 . . . T T . 0.97 -0.59. * F 3.10 0.47 .

Gly 414 . . . . T C 0.69 -0.16* * F 2.44 1.37 .

Thr 415 . . . . . C -0.28-0.23. * F 1.78 0.89 .

Glu 416 . B . . . . 0.04 -0.01. . F 1.27 0.66 .

Pro 417 . B . . . . -0.44-0.09. . F 0.96 0.66 .

Ala 418 . B . . . . 0.14 0.27. * . -0.100.39 .

Val 419 . B . . . . 0.24 -0.21. * . 0.50 0.39 .

Gly 420 . B . . . . 0.60 0.54. * . -0.400.40 .

Phe 421 A B . . . . 0.31 0.11. * . -0.300.79 .

Glu 422 A B . . . . 0.23 0.53. * . -0.451.12 .

Tyr 423 A . . T . . 0.82 0.80* * . -0.051.19 .

Lys 424 A . . T . . 1.37 0.77* * . -0.052.21 .

Trp 425 A . . T . . 0.90 0.47* * . -0.051.84 .

Trp 426 A . . 'f . . 1.39 1.16* * . -0.200.97 .

Asn 427 . . . . . C 1.08 0.83* * . -0.200.75 .

'Chr428 . . . . . C 1.32 1.31* . F 0.10 1.03 .

Leu 429 . . . . . C 0.68 0.80* * F 0.10 1.57 .

Pro 430 . . . . T C 0.97 0.50* . F 0.12 0.97 .

Thr 431 . . . . T C 0.94 O.10* . F 0.54 1.16 .

Asn 432 . . . . T C 0.63 0.10* * F 0.51 2.03 .

Met 433 . B . . T . 0.09 -0.10. * F 0.88 1.90 .

Glu 434 . B B . . . 0.09 0.11. * F -0.300.98 .

Thr 43S . B B . . . -0.000.31. . F -0.270.50 .

Thr 436 . B B . . . -0.030.30* . F -0.240.68 .

Val 437 . B B . . . -0.920.11* * F -0.210.39 .

Leu 438 . B B . . . -0.320.80* * F -0.480.19 .

Ser 439 . B B . . . -1.020.71. * F -0.450.21 .

Gly 440 . . B . . C -0.711.01. * . -0.400.24 .

Ile 441 A B B . . . -0.640.37* * . -0.300.51 .

Asn 442 A B B . . . 0.26 0.44* * . -0.600.60 .

Phe 443 A B B . . . 0.72 0.06* * . -0.151.21 .

Glu 444 A B B . . . 0.42 0.06* * . -0.151.71 .

Tyr 445 . B . . T . 0.46 -0.01* * . 0.85 1.05 .

Lys 446 . . . T T . 1.00 0.07* * F 0.80 1.76 .

Gly 447 . . . T T . 0.71 -0.29* * F 1.40 1.00 .

Met 448 . . . . T C 1.41 0.63. * F 0.15 0.67 .

Thr 449 . . . . . C 0.56 -0.13. * F 0.85 0.58 .

Gly 450 A . . . . C 0.21 0.51. * . -0.400.44 .

'1'rp451 A B . . . . -0.180.59* * . -0.420.45 .

Glu 452 . A B . . . . 0.17 0.40* . . -0.240.31 Val 453 . A B . . . . 0.73 -0.09* . . 0.84 0.52 Ala 454 . A B . . . . 0.16 -O.OI* . . 1.02 0.67 Gly 455 . . . . T . . 0.26 -0.24* . . 1.80 0.27 Asp 456 . . . B T . . 0.23 0.51* . . 0.52 0.57 His 457 . . B B . . . -0.360.36* . . 0.24 0.82 Ile 458 . . B B . . . -0.090.36. . . 0.06 0.83 Tyr 459 . . B B . . 0.16 0.43* . . -0.420.50 Thr 460 . . B B . . . -0.090.86. . . -0.600.37 Ala 461 . . B B . . . -0.390.86. . . -0.600.53 Ala 462 . . B . . . . -0.360.56. . . -0.120.45 Gly 463 . . . . . . C 0.53 -0.20. . . 1.26 0.52 Ala 464 . . . . . . C 0.78 -0.29. . F 1.69 0.83 Ser 465 . . . . . T C 0.39 -0.79. . F 2.62 1.38 Asp 466 . . . . T 'C . 0.38 -0.50. . F 2.80 1.21 Asn 467 . . . . . T C 0.08 -0.31. . F 2.32 1.18 Asp 468 . . B . . T . -0.39-0.13. . F 1.69 0.62 Phe 469 . . B B . . . -0.110.17. . . 0.26 0.30 Met 470 . . B B . . . -0.620.66. . . -0.320.27 Ile 471 . . B B . . . -1.480.94. . . -0.600.14 Leu 472 . . B B . . . -2.331.59. . . -0.600.12 Thr 473 . . B B . . . -2.541.44. . . -0.600.09 Leu 474 . . B B . . . -2.191.26. . . -0.600.19 Val 475 . . B B . . . -2.291.00* * . -0.600.23 Val 476 . . B B . . . -1.291.10* . . -0.600.14 Pro 477 . . B . . . . -0.690.61* * . -0.400.3 Gly 478 . . . . T . . -0.590.36* . F 0.45 0.68 Phe 479 . . B . . . . 0.22 0.14* . F 0.45 1.42 Arg 480 . . . . . . C 0.78 -0.10* * F 1.50 1.59 Pro 481 . . . . . 'f C 0.78 -0.14* . F 1.95 2.16 Pro 482 . . . . T 'f . 0.39 0.07* * F 1.80 1.85 Gln 483 . . . . T T . 0.14 -0.10* * F 2.50 0.93 Ser 484 . . B . . T . 0.84 0.40* * F 0.95 0.61 Val 485 . . B . . . . 0.42 -0.03* * . 1.55 0.66 Met 486 . . B . . . . 0.63 0.03. . . 1.00 0.55 Ala 487 . . B . . . . 0.84 -0.37. * . 1.65 0.71 Asp 488 . . B . . 'f . 0.89 -0.36. . F 2.20 1.54 Thr 489 . . . . . T C 1.19 -1.00. . F 3.00 3.11 Glu 490 A . . . . T . 1.19 -1.61. . F 2.50 5.33 Asn 491 A . . . . T . 1.20 -1.47* * F 2.20 2.37 Lys 492 A . . . . . . 1.90 -0.97* * F 1.70 1.66 Glu 493 A . . . . . . 1.01 -1.46* * F 1.40 1.88 Val 494 A . . B . . . 1.01 -0.77* * . 0.60 0.82 Ala 495 . . B B . . . 0.31 -0.69* * . 0.60 0.59 Arg 496 . . B B . . . -0.540.10* * . -0.300.29 Ile 497 . . B B . . . -1.290.74* * . -0.600.29 Thr 498 . . B B . . . -1.290.89* * . -0.600.25 Phe 499 . . B B . . . -0.740.39* * . -0.300.22 Val 500 . . B B . . . -0.970.87* * . -0.600.46 Phe 501 . . B B . . . -1.740.87* * . -0.600.26 Glu 502 . . B B . . . -1.160.96* * . -0.600.16 Thr 503 . . . B T . . -1.700.56* . . -0.200.29 Leu 504 . . . B T . . -1.000.56* * . -0.200.25 Cys 505 . . . B T . . -0.810.17* * . 0.10 0.23 Ser 506 . . . . T T . -0.110.74* * . 0.20 0.09 Val 507 . . . . T T . -0.920.26* * . 0.50 0.18 Asn 508 . . B . T T . -0.860.26. * . 0.50 0.28 Cys 509 . . B . . T . -0.740.44. * . -0.200.33 Glu 510 . . B B . . . -0.680.84. * . -0.600.38 Leu 511 . . B B . . . -1.230.81. * . -0.600.24 Tyr 512 . B B . . . -0.721.06 * . -0.600.33 . .

Phe 513 . B B . . . -1.580.91 * . -0.600.19 . .

Met 514 . B B . . . -0.911.56 * . -0.600.17 . .

Val 515 . B B . . . -1.211.27 * . -0.600.17 . .

Gly 516 . B B . . . -0.290.90 * . -0.320.27 . .

Val 517 . B B . . . -0.360.11 * . 0.26 0.53 . .

Asn 518 . . . . T C 0.34 -0.01 * F 2.04 1.03 . .

Ser 519 . . . . T C 0.63 -0.26 * F 2.32 1.67 . .

Arg 520 . . . T T . 1.28 -0.20 * F 2.80 3.24 . .

Thr 521 . . . T T . 0.77 -0.41 * F 2.52 3.12 . .

Asn 522 . . . . . C 1.62 -0.17 * F 1.84 1.73 . *

Thr 523 . . . . . C 1.31 -0.56 * F 1.86 1.53 . *

Pro 524 . B . . . . 1.32 -0.07 * F 1.08 1.53 . *

Val 525 . B . . . . 1.26 0.36 * F 0.05 1.00 . .

Glu 526 . B . . . . 1.22 -0.04 . F 0.80 1.38 . *

Thr 527 . B . . . . 0.92 -0.10 . F 0.99 0.88 . *

Trp 528 . B . . . . 1.28 -0.14 . F 1.48 1.60 . .

Lys 529 . . . T . . 1.14 -0.79 . F 2.52 1.85 . .

Gly 530 . . . T . . 2.04 -0.36 . F 2.56 1.27 . .

Ser 5 31 . . . T T . 2.04 -0.84 . F 3 2.41 . . .40 Lys 532 . . . . T C 2.06 -1.36 . F 2.86 2.08 . .

Gly 533 . . . T T . 2.10 -0.97 . F 2.72 2.82 . .

Lys 534 . . . T T . 1.74 -0.64 . F 2.38 3.30 . .

Gln 535 . . B T . . 1.84 -0.54 . F 1.64 2.38 . *

Ser 536 . B B . . . 1.26 0.21 * F 0.00 3.77 . *

Tyr 537 . B B . . . 0.32 0.47 * . -0.451.32 . *

Thr 538 . B B . . . 0.67 1.16 . . -0.600.53 . *

Tyr 539 . B B . . . 0.62 0.76 . . -0.600.69 . .

Ile 540 . B B . . . 0.62 0.37 . . -0.300.76 . *

Ile 541 . B B . . . 0.61 0.01 . . -0.040.85 . .

Glu 542 . B B . . . 0.54 0.01 . F 0.37 0.78 . .

Glu 543 . B . . . . 0.54 -0.26 . F 1.58 1.61 . .

Asn 544 . . B T . . 0.49 -0.46 . F 2.04 3.32 . .

Thr 545 . . B T . . 0.68 -0.76 . F 2.60 2.57 . .

Thr 546 . . B . . C 1.26 0.03 * F 1.24 1.29 . .

Thr 547 . . B . . C 0.97 0.51 . F 0.68 1.15 . .

Ser 548 . . B . . C 0.38 1.03 * F 0.27 0.84 . *

Phe 549 . B B . . . -0.321.04 * . -0.340.59 . *

Thr 550 . B B . . . -0.011.34 . . -0.600.35 . *

Trp 551 . B B . . . 0.41 1.26 . . -0.600.46 . *

Ala 552 . . B . . C 0.41 0.87 . . -0.251.03 . *

Phe 553 . . B T . . 0.40 0.57 * . -0.051.03 . *

Gln 554 . . B T . . 0.40 0.57 * . -0.051.42 . *

Arg 555 . . B . . C 0.68 0.44 . F -0.101.21 . .

Thr 556 . . B . . C 0.97 0.44 . F -0.101.91 . .

Thr 557 . . B . . C 0.97 -0.34 . F 0.80 1.91 . *

Phe 558 . . B . . C 1.37 -0.24 * . 0.50 0.98 . *

His 559 . . B . . C 1.48 0.14 * . -0.100.91 . *

Glu 560 . . B . . C 1.41 -0.34 * . 0.65 1.24 . *

Ala 561 . . . T . C 1.48 -0.83 . F 1.84 2.86 . *

Ser 562 . . . T . . 1.48 -0.86 . F 2.18 3.30 . *

Arg 563 . . . T . . 2.18 -0.87 . F 2.52 2.75 . *

Lys 564 . . . T . . 2.21 -0.47 . F 2.56 4.38 . *

Tyr 565 . . . T T . 1.36 -0.97 . F 3.40 5.45 . *

Thr 566 . . . T T . 1.36 -0.71 . F 3.06 2.07 . *

Asn 567 . B . . T . 1.70 -0.21 . F 2.02 1.04 . *

Asp 568 . B . . T . 0.70 -0.21 . F 1.68 1.33 . *

Val 569 . B B . . . 0.41 -0.29 . F 0.79 0.65 . *

Ala 570 . B B . . . 0.36 -0.01 . . 0.30 0.63 . *

Lys 571 . B B . . . -0.22-0.03 . . 0.30 0.51 . *

Ile 572 . B B . . . -0.22 0.66* . . -0.600.48 .

Tyr 573 . B B . . . -1.08 0.41. . . -0.600.76 .

Ser 574 . B B . . . -0.53 0.56. . . -0.600.28 .

Ile 575 . B B . . . 0.06 1.04. . . -0.600.58 .

5 Asn 576 . B B . . . -0.84 0.76. . . -0.600.60 .

Val 577 . B B . . . -0.56 0.64. . . -0.600.33 .

Thr 578 . B B . . . -0.31 0.87. * . -0.600.47 .

Asn 579 . B B . . . -0.36 0.59* * . -0.600.47 .

Val 580 . B . . T . -0.32 0.61* . . -0.200.62 .

10Met 581 . B . . T . -0.91 0.61* . . -0.200.32 .

Asn 582 . B . . T . -0.36 0.63* . . -0.200.20 .

Gly 583 . B . . T . -0.29 0.61* . . -0.200.36 .

Val 584 . B . . . . -0.96 0.73* . . -0.400.57 .

Ala 585 . B . . 'f . 0.01 0.69* . . -0.200.19 .

15Ser 586 . B . . T . 0.40 0.29* . . 0.10 0.38 .

Tyr 587 . B . . T . -0.27 0.29* * . 0.10 0.79 .

Cys 588 . B . . T . -0.51 0.21* . . 0.10 0.42 .

Arg 589 . B . . T . -0.47 0.21. . . 0.10 0.32 .

Pro 590 . B . . T . 0.12 0.51. . . -0.200.17 .

20Cys 591 . B . . T . -0.17 -0.24. . . 0.70 0.54 .

Ala 592 . B . . T . -0.22 -0.31* . . 0.70 0.28 .

Leu 593 . B . . . . 0.44 0.07* . . -0.100.24 .

Glu 594 . B . . . . -0.52 -0.36* . . 0.75 0.75 .

Ala 595 . B . . . . -0.66 -0.29. . F 1.15 0.55 .

25Ser 596 . B . . . . -0.29 -0.36. . F 1.40 0.66 .

Asp 597 . . . T T . 0.00 -0.66. . F 2.55 0.51 .

Val 598 . . . T T . 0.14 -0.27. . F 2.50 0.68 .

Gly 599 . . . T T . -0.17 -0.20* . F 2.25 0.27 .

Ser 600 . . . T T . 0.12 -0.10* . F 2.00 0.23 .

30Ser 601 . . . T . . -0.24 0.29* . F 0.95 0.42 .

Cys 602 . B . . T . -0.46 0.21. . F 0.50 0.23 .

Thr 603 . B . . T . -0.19 0.21. . F 0.25 0.26 .

Ser 604 . B . . T . -0.19 0.33. . F 0.25 0.20 .

Cys 605 . B . . T . -0.13 0.37. . . 0.10 0.37 .

35Pro 606 . B . . T . -0.08 0.56. . . -0.200.40 .

Ala 607 . . . T T . -0.30 0.83. . . 0.20 0.47 .

Gly 608 . B . . T . 0.01 1..13* . . -0.200.61 .

Tyr 609 . B . . T . 0.42 0.56* . . -0.200.66 .

Tyr 610 . B B . . . 1.09 0.13* . . 0.19 1.28 .

40Ilc 611 . B B . . . I.00 -0.37* . . 1.13 2.16 .

Asp 612 . B B . . . 1.24 -0.41* . . 1.47 1.84 .

Arg 613 . . . T . . 1.28 -0.74* . F 2.86 1.16 .

Asp 614 . . . T T . 0.86 -1.01* . F 3.40 2.40 .

Ser 615 . . . T T . 1.07 -1.13* . F 2.91 0.77 .

45Gly 616 . . . T T . 1.66 -0.63* . F 2.57 0.53 .

Thr 617 . . . T T . 0.99 -0.24. . F 1.93 0.43 .

Cys 618 . . . T T . 0.67 0.33. . . 0.84 0.17 .

His 619 . . . T T . 0.46 0.37. . . 0.50 0.27 .

Ser 620 . . . T T . 0.76 0.37. . . 0.50 0.29 .

50Cys 621 . B . . T . 0.79 0.29. . . 0.10 0.86 .

Pro 622 . . . . 'C C 0.21 0.20. . F 0.45 0.91 .

Pro 623 . . . T T . 0.07 0.39* . F 0.65 0.48 .

Asn 624 . . . T T . 0.14 0.69* . F 0.35 0.74 .

Thr 625 . B . . 'f . -0.14 0.11* * F 0.25 0.95 .

55Ile 626 A B . . . . 0.49 0.19* . . -0.300.62 .

Leu 627 A B . . . . 0.70 0.26* . . -0.300.53 .

Lys 628 A B . . . . 0.70 0.26* . . -0.300.63 .

Ala 629 A B . . . . 0.46 0.20* . . -0.151.39 .

His 630 A B . . . . 0.42 0.27. . . -0.152.64 .

60Gln 631 . B . . T . 0.46 0.01* . F 0.40 1.31 .

Pro 632 . . . T T . 1.27 0.66. * . 0.20 0.96 .

Tyr 633 . . . T T . 0.63 0.56. * . 0.35 1.22 .

Gly 634 . . . T T . 0.56 0.56. * . 0.20 0.71 .

Val 635 . B B . . . -0.270.73. . . -0.600.25 .

Gln 636 . B B . . . -0.480.94. * . -0.600.12 .

Ala 637 . B B . . . -0.930.61. * . -0.60O.18 .

Cys 638 . B B . . . -1.030.76. * . -0.600.13 .

Val 639 . B B . . . -0.900.54. . . -0.600.08 .

Pro 640 . B . . . . -0.390.57. * . -0.400.12 .

10Cys 641 . B . . . . -0.700.50. . . -0.400.21 .

Gly 642 . B . . T . -0.070.41. . F 0.29 0.42 .

Pro 643 . . . T T . 0.60 -0.23. . F 1.93 0.54 .

Gly 644 . . . T T . 1.46 -0.26. . F 2.42 1.61 .

Thr 645 . . . 'f T . 1.71 -0.43. . F 2.76 2.62 .

15Lys 646 . . . T ~1' . 1.49 -0.86. . F 3.40 3.39 .

Asn 647 . . . T T . 1.80 -0.60. . F 3.06 2.40 .

Asn 648 . B . . T . 1.71 -0.53. . F 2.32 2.26 .

Lys 649 . B . . T . 1.24 -0.63. . F 1.98 1.52 .

Ile 650 . B . . . . 0.89 0.06. . . 0.24 0.78 .

20His 651 . B . . T . 0.60 0.23. . . 0.10 0.26 .

Ser 652 . B . . T . 0.60 0.59. . . -0.200.20 .

Leu 653 . B . . T . 0.60 0.99* . . -0.200.47 .

Cys 654 . B . . T . -0.110.30* . . 0.10 0.57 .

Tyr 655 . . . T . . 0.47 0.37. . . 0.30 0.23 .

25Asn 656 . . . T T . -0.200.47. . . 0.20 0.40 .

Asp 657 . . . T T . -0.200.57* . . 0.20 0.65 .

Cys 658 . B . . T . 0.72 0.39* . . 0.10 0.55 .

Thr 659 . B . . T . 1.39 -0.37. . . 0.70 0.67 .

Phe 660 . B . . . . 1.32 -0.37. . . 0.80 0.65 .

30Ser 661 . . . T T . 1.11 0.11. . F 1.40 1.75 .

Arg 662 . . . T T . 0.80 -0.03. . F 2.30 1.87 .

Asn 663 . . . . T C 1.58 -0.03. . F 2.40 3.12 .

Thr 664 . . . . T C 1.58 -0.81. . F 3.00 4.55 .

Pro 665 . . . . T C 1.58 -0.71* . F 2.70 3.36 .

35Thr 666 . . . T T . 1.88 0.07* . F 1.70 1.81 .

Arg 667 . B . . T . 1.52 0.07* . F 1.00 2.01 .

'rhr 668 . B . . T . 1.52 0.34* * F 0.70 2.04 .

Phc 669 . B . . . . 1.13 0.31* * . 0.05 2.27 .

Asn 670 . B . . T . 1.04 0.61* * . -0.05I.01 .

40Tyr 671 . B . . T . 0.77 1.00* . . -0.200.93 .

Asn 672 . B . . T . -0.161.01* * . -0.051.09 .

Phc 673 . B . . T . -0.430.91* * . -0.200.56 .

Ser 674 A . . . . C 0.27 1.01* * . -0.400.36 .

Ala 675 A . . . . C -0.040.66* * . -0.400.36 .

45Leu 676 A B . . . . -0.660.74* * . -0.600.60 . ' Ala 677 A B . . . . -0.970.60. * . -0.600.33 .

Asn 678 A B . . . . -1.080.70* . . -0.600.47 .

Thr 679 . B B . . . -1.370.89* . . -0.600.47 .

Val 680 . B B . . . -1.120.70. . . -0.600.47 .

50Thr 681 . B B . . . -0.660.63. . . -0.600.29 .

Leu 682 . B B . . . -0.280.66. . . -0.600.20 .

Ala 683 . B B . . . -0.580.60. . . -0.600.42 .

Gly 684 . . B . . C -0.970.34. . F 0.05 0.39 .

Gly 685 . . . . T C -0.420.64. . F 0.15 0.41 .

55Pro 686 . . . . T C -0.410.44. * F 0.15 0.58 .

Ser 687 . . . . T C 0.44 0.33. * F 0.73 0.79 .

Phe 688 . B . . T . 0.69 -0.10. . F 1.56 1.59 .

Thr 689 . B . . . . 0.22 -0.10* . F 1.64 1.02 .

Ser 690 . B . . T . 0.61 0.16* . F 1.37 0.63 .

60Lys 691 . . . T T . 0.58 -0.23* . F 2.80 1.45 .

Gly 692 . . . T T . 0.18 -0.26* . F 2.52 1.57 .

Leu 693 . . . . T C 0.84 0.04* . F 1.44 1.02 .

Lys 694 A B . . . . 1.12 0.16* . . 0.26 0.69 .

Tyr 695 A B . . . . 0.72 0.66* . . -0.320.95 .

Phe 696 A B . . . . 0.37 1.01* * . -0.601.00 .

His 697 A B . . . . -0.100.81* * . -0.600.72 .

His 698 A B . . . . 0.41 1.50* * . -0.600.38 .

Phe 699 A B . . . . -0.441.13* * . -0.600.59 .

Thr 700 A B . . . . -0.871.03. * . -0.600.36 .

Leu 701 A . . T . . -0.511.10. * . -0.200.14 .

Ser 702 . . B T . . -0.481.03. * . -0.200.16 .

Leu 703 . . B T . . -0.440.64. * . 0.14 0.18 .

Cys 704 . . B T . . -0.090.56. * . 0.48 0.37 .

Gly 705 . . B T . . 0.33 0.30* . F 1.27 0.28 .

Asn 706 . . . T 'f . 1.19 -0.09* . F 2.61 0.66 .

Gln 707 . . . T T . 0.89 -0.77* . F 3.40 2.45 .

Gly 708 . . . T T . 1.40 -0.73* . F 3.06 2.45 .

Arg 709 . . . T T . 1.21 -0.77* . F 2.72 2.04 .

Lys 710 . B B . . . 0.89 -0.53* . F 1.43 0.87 .

Met 711 . B B . . . 0.58 -0.36* . . 0.64 0.47 .

Ser 712 . B B . . . 0.58 -0.30* . . 0.30 0.35 .

Val 713 . B B . . . 0.92 -0.30* . . 0.30 0.29 .

Cys 714 . B . . T . -0.040.10* . . 0.10 0.47 .

Thr 715 . B . . 1' . -0.400.13* * . 0.10 0.26 .

Asp 716 . B . . T . 0.20 0.23* . F 0.25 0.51 .

Asn 717 . B . . T . -0.31-0.41* * F 1.00 1.59 .

Val 718 . B B . . . 0.66 -0.30. * F 0.45 0.91 .

Thr 719 . B B . . . 0.43 -0.79. * F 0.90 1.07 .

Asp 720 . B B . . . 0.53 -0.10. * F 0.45 0.46 .

Leu 721 . B B . . . 0.53 -0.07. * F 0.76 0.97 .

Arg 722 . B B . . . 0.19 -0.71. * F 1.52 1.16 .

Ile 723 . B . . T . 1.04 -0.77. * F 2.08 0.69 .

Pro 724 . B . . T . 1.06 -0.77. * F 2.54 1.44 .

Glu 725 . . . T T . 0.71 -1 . * F 3.10 0.99 . .07 Gly 726 . . . . T C 0.82 -0.64. * F 2.74 1.40 .

Glu 727 . . . T 'f . 0.41 -0.54* . F 2.48 0.78 .

Ser 728 . . . . 'f C 1.34 -0.59* . F 2.10 0.60 .

Gly 729 . . . T T . 1.26 -0.59* . F 2.27 1.22 .

Phe 730 . . . T T . 0.37 -0.63* . F 1.94 0.95 .

Ser 731 . . . . T C 0.40 0.06* . F 0.97 0.49 .

Lys 732 . . . T T . -0.190.16* . F 1.30 0.72 .

Ser 733 . . . T T . -0.130.23* . F 1.17 0.84 .

Ile 734 . B . . T . -0.640.20* . . 0.49 0.98 .

Thr 735 . B B . . . -0.610.46* . . -0.340.37 .

Ala 736 . B B . . . -0.311.03* . . -0.470.15 .

Tyr 737 . B B . . . -0.941.04* . . -0.600.36 .

Val 738 . B B . . . -1.500.86. . . -0.600.25 .

Cys 739 . B B . . . -1.501.01. . . -0.600.19 .

Gln 740 . B B . . . -2.081.20. . . -0.600.08 .

Ala 741 . B B . . . -1.701.13. . . -0.600.08 .

Val 742 . B B . . . -1.670.91. . . -0.600.23 .

Ile 743 . B B . . . -0.810.77. . . -0.600.20 .

Ile 744 . B B . . . -1.000.37. . . -0.300.35 .

Pro 745 . B . . T . -1.310.51. * . -0.200.35 .

Pro 746 . B . . T . -1.070. . * F 0.42 0.71 . 3 Glu 747 . B . . T . -0.460.10. . F 0.74 1.01 .

Val 748 . B . . T . 0.48 0.17. . F 0.91 1.02 .

Thr 749 . B . . T . 0.78 -0.26. * F 1.68 1.32 .

Gly 750 . B . . T . 0.64 -0.19. . F 1.70 0.77 .

Tyr 751 . B . . T . 0.00 0.24. . F 1.08 1.03 .

Lys 752 . B . . T . -0.300.24. . F 0.76 0.53 .

Ala 753 . B . . . . 0.26 0.14. * F 0.39 0.71 .

Gly 754 . B . . . . 0.57 0.10. * F 0.22 0.61 .

Val 755 . B . . . . 0.70 -0.26. * F 0.65 0.53 .

Ser 756 . B . . . . 0.09 0.17. * F 0.05 0.81 .

Ser 757 . B . . . . -0.260.31. * F 0.05 0.61 .

Gln 758 . B . . . . -0.480.27. . F 0.20 I.10 .

Pro 759 . B . . . . -0.720.31. . F 0.05 0.67 .

Val 760 A B . . . . 0.13 0.43* * F -0.450.51 .

Ser 761 A B . . . . 0.54 0.04* * . -0.300.49 .

Leu 762 A B . . . . 0.03 -0.36* . . 0.30 0.62 .

Ala 763 A B . . . . -0.86-0.10* * . 0.30 0.69 .

Asp 764 A B B . . . -0.99-0.06* * . 0.30 0.36 .

Arg 765 A B B . . . -0.99-0.01* * . 0.30 0.43 .

Leu 766 . B B . . . -1.00-0.06* . . 0.30 0.32 .

Ile 767 . B B . . . -0.50-0.07* . . 0.30 0.28 .

Gly 768 . B B . . . 0.09 0.41* . . -0.600.20 .

Val 769 . B B . . . -0.510.41* . . -0.600.41 .

Thr 770 . B B . . . -0.930.34* * F -0.150.58 .

Thr 771 . B B . . . -0.930.14. * F -0.150.85 .

Asp 772 . B B . . . -0.040.40. * F -0.450.94 .

Met 773 . B B . . . -0.04-0.24. * F 0.60 1.09 .

Thr 774 . B B . . . -0.08-0.30. * . 0.30 0.75 .

Leu 775 . B B . . . -0.08-0.10* * F 0.45 0.31 .

Asp 776 . . B T . . -0.070.39* * F 0.25 0.46 .

Gly 777 . . B T . . -0.280.16* * F 0.34 0.42 .

Ile 778 . . B . . C -0.270.10* . F 0.23 0.79 .

Thr 779 . . B . . C 0.04 -0.09* * F 0.92 0.48 .

Ser 780 . . . . T C 0.04 -0.09* . F 1.41 0.84 .

Pro 781 . . . . T C -0.660.17* . F 0.90 0.99 , .

Ala 782 . B . . T . -0.340.27* . F 0.61 0.59 .

Glu 783 . B . . T . -0.270.29. * . 0.37 0.60 .

Leu 784 A B . . . . 0.04 0.59. . . -0.420.32 .

Phe 785 A B . . . . 0.04 0.16. . . -0.210.55 .

His 786 A B . . . . -0.560.04. . . -0.300.43 .

Leu 787 A B . . . . -0.310.73. . . -0.600.43 .

Glu 788 A B . . . . -1.200.47. . . -0.600.49 .

Ser 789 . . . T . . -0.600.37. . . 0.30 0.25 .

Leu 790 . . . T . . 0.10 0.30. . . 0.30 0.47 .

Gly 791 . . . . . C -0.72-0.39. . . 0.70 0.45 .

Ile 792 . . B . . C -0.800.26. . F 0.05 0.25 .

Pro 793 . B B . . . -1.500.56. . F -0.450.21 .

Asp 794 . B B . . . -1.900.66. . . -0.600.19 .

Val 795 . B B . . . -1.331.01* * . -0.600.23 .

Ile 796 . B B . . . -0.881.09* . . -0.600.23 .

Phe 797 . B B . . . -0.290.66* * . -0.600.27 .

Phe 798 . B B . . . -0.081.04. * . -0.600.50 .

Tyr 799 . B . . . . -0.080.80. . . 0.09 1.14 .

Arg 800 . . . T T . -0.080.11. . F 1.48 2.19 .

Ser 801 . . . T T . 0.50 -0.03. . F 2.42 1.88 .

Asn 802 . . . T T . 1.20 -0.33* . F 2.76 1.73 .

Asp 803 . . . T T . 1.60 -0.69* . F 3.40 1.53 .

Val 804 . . . T . . 1.18 -0.30. . F 2.56 1.53 .

Thr 805 . B . . . . 0.77 -0.11* . F 1.67 0.51 .

Gln 806 . B . . . . 0.77 -0.13* . F 1.33 0.41 .

Ser 807 . B . . . . 0.42 0.26* * F 0.67 0.74 .

Cys 808 . B . . T . 0.53 0.04* * F 0.81 0.51 .

Ser 809 . . . T T . 1.09 -0.44* * F 2.09 0.57 .

Ser 810 . . . T T . 1.09 -0.46. * F 2.37 0.57 .

Gly 811 . . . '1' T . 0.78 -0.36. . F 2.80 1.54 .

Arg 812 . . B T . . 0.19 -0.44 . F 2.12 1.66 . .

Ser 813 . . B T . . 0.97 -0.14 * F 1.69 0.87 . *

Thr 814 . B B . . . 0.41 -0.53 * F 1.46 1.72 . *

Thr 815 . B B . . . 0.82 -0.31 * F 0.73 0.65 . .

Ile 816 . B B . . . 0.50 -0.31 * F 0.45 0.95 . .

Arg 817 . B B . . . 0.09 -0.13 * . 0.30 0.35 . .

Val 818 . B B . . . 0.18 -0.23 * . 0.64 0.33 . .

Arg 819 . B B . . . 0.49 -0.29 * . 0.98 0.73 . .

Cys 820 . B B . . . 0.84 -0.57 * . 1.62 0.64 . .

Scr 821 . . . . T C 1.42 -0.57 * F 2.86 1.73 . *

Pro 822 . . . T T . 0.46 -0.73 * F 3.40 1.27 . *

Gln 823 . . . 'f T . 1.10 -0.09 * F 2.76 1.76 . *

Lys 824 . B . . T . 0.64 -0.23 * F 2.02 2.03 . .

Thr 825 . B . . . . 1.01 -0.19 . F 1.48 1.30 . .

Val 826 . B . . T . 0.50 -0.23 . F 1.34 1.01 . .

Pro 827 . B . . T . -0.10 0.06 . F 0.25 0.42 . .

Gly 828 . B . . T . -0.91 0.74 . F -0.050.24 . .

Ser 829 . B . . T . -1.17 0.94 . F -0.050.26 . .

Leu 830 . B . . . . -1.20 0.73 * F -0.250.26 . .

Leu 831 . B . . . . -0.66 0.73 * F -0.400.26 . .

Leu 832 . B . . T . -1.11 0.79 . F -0.050.28 . .

Pro 833 . B . . 'f . -1.07 0.97 . F -0.050.18 . .

Gly 834 . . . T T . -0.77 0.67 . F 0.35 0.30 . .

Thr 835 . B . . T . -0.30 -0.01 . F 1.16 0.61 . .

Cys 836 . . . T T . 0.20 -0.27 . F 1.87 0.39 . .

Ser 837 . . . T T . 0.34 -0.21 . F 2.18 0.57 . .

Asp 838 . . . T 'f . 0.56 -0.07 . F 2.49 0.21 . .

Gly 839 . . . T T . 0.56 -0.56 . F 3.10 0.66 . .

Thr 840 . . . T . . 0.20 -0.70 . F 2.59 0.48 . *

Cys 841 . . . T T . 0.87 -0.51 . F 2.48 0.16 . *

Asp 842 . . . T T . 0.47 -0.11 . F 1.87 0.25 . .

Gly 843 . . . T T . 0.43 0.24 * F 0.96 0.15 . .

Cys 844 . . . T T . 0.08 0.26 . . 0.50 0.38 . .

Asn 845 A B . . . . -0.42 0.47 . . -0.600.20 . .

Phe 846 A B . . . . -0.04 1.16 * . -0.600.17 . .

His 847 A B . . . . -0.04 1.64 * . -0.600.33 . .

Phe 848 A B . . . . 0.00 1.07 * . -0.600.35 . *

Leu 849 A . . T . . 0.08 1.06 * . -0.200.54 . .

Trp 850 A . . T . . -0.51 0.77 * . -0.200.40 . .

Glu 851 A . . T . . -0.40 0.77 * . -0.200.47 . .

Ser 852 A . . T . . -1.03 0.49 . . -0.200.58 . .

Ala 853 A . . T . . -0.54 0.37 . . 0.10 0.29 . .

Ala 854 A . . T . . -0.54 -0.11 . . 0.70 0.26 . .

Ala 855 A . . T . . -0.92 0.57 . . -0.200.16 . .

Cys 856 . . . . T C -1.22 0.76 . . 0.00 0.09 . .

Pro 857 . B . . T . -1.78 0.64 . . -0.200.1 . . 1 Leu 858 . B . . T . - I 0.79 . . -0.200.08 . .78 *

Cys 859 . B . . T . -1.19 0.79 . . -0.200.16 . *

Ser 860 . B B . . . -0.84 0.21 . . -0.300.17 . .

Val 861 . B B . . . -0.21 0.54 . . -0.600.32 . .

Ala 862 . B B . . . -0.59 0.36 . . -0.300.82 . .

Asp 863 . B . . . . -0.67 0.29 . . -0.100.62 . .

Tyr 864 . B B . . . -0.86 0.59 . . -0.600.58 . .

His 865 . B B . . . -0.86 0.59 . . -0.600.43 . .

Ala 866 . B B . . . -0.30 0.47 . . -0.600.34 . .

Ile 867 . B B . . . -0.38 0.86 . . -0.600.29 . .

Val 868 . B B . . . -1.23 0.67 . . -0.600.12 . .

Ser 869 . B B . . . -1.58 0.81 . . -0.600.09 . .

Ser 870 . B B . . . -1.89 0.81 . . -0.600.
. . I

Cys 871 . B B . . . -2.19 0.56 . . -0.600.16 . *

Val 872 . B B . . . -1.30 0.60* . . -0.600.09 .

Ala 873 . B B . . . -0.40 0.61* . . -0.600.11 .

Gly 874 . B B . . . -0.41 0.23* . . -0.300.41 .

Ile 875 . B B . . . -0.42 0.14* . . -0.300.80 .

5 Gln 876 . B B . . . 0.00 -0.01. . F 0.60 1.15 .

Lys 877 . B B . . . 0.00 0.24* . F 0.00 1.82 .

Thr 878 . B B . . . 0.30 0.46* * F -0.301.92 .

Thr 879 . B B . . . 0.76 0.69* . F -0.301.17 .

Tyr 880 . B B . . . 1.64 0.29* . . -0.151.14 .

10 Val 881 A B B . . . 1.43 0.29* . . -0.151.37 .

Trp 882 A B B . . . 1.43 0.23* * . -0.151.47 .

Arg 883 A B B . . . 0.93 -0.26* . F 0.60 1.88 .

Glu 884 A B B . . . 0.58 -0.33* . F 0.85 2.09 .

Pro 885 A . . 'I' . . 0.52 -0.40* . F 1.50 1.06 .

15 Lys 886 A . . T . . 1.03 -0.93* . F 1.90 0.73 .

Leu 887 A . . T . . 0.98 -0.50* . F 1.85 0.42 .

Cys 888 . . . T T . -0.02 -0.07* . F 2.50 0.27 .

Ser 889 . . . 'f T . -0.32 0.19. * F 1.65 0.09 .

Gly 890 . . . T T . -0.92 0.57* * F 1.10 0.15 .

20 Gly 891 . . . T T . -1.18 0.57* . F 0.85 0.23 .

Ile 892 . . . . . C -0.37 0.43* . F 0.20 0.27 .

Ser 893 . . . . . C 0.30 0.04. . F 0.25 0.47 .

Leu 894 . B . . . . 0.71 0.01. * F 0.05 0.82 .

Pro 895 . B . . . . 0.20 -0.41. * F 0.80 2.30 .

25 Glu 896 . B B . . . 0.23 -0.46. * F 0.60 1.27 .

Gln 897 . B B . . . 0.23 -0.36. * F 0.60 2.23 .

Arg 898 . B B . . . -0.13 -0.36. * F 0.60 1.01 .

Val 899 . B B . . . 0.72 -0.21. * . 0.30 0.31 .

Thr 900 . B B . . . 0.62 -0.21. * . 0.30 0.36 .

30 Ile 901 . B B . . . -0.27 -0.13. * . 0.30 0.27 .

Cys 902 . B B . . . -0.27 0.56. * . -0.600.25 .

Lys 903 . B B . . . -1.08 -0.09* * . 0.30 0.29 .

Thr 904 . B B . . . -0.51 0.21* * . -0.300.36 .

Ile 905 . B B . . . -1.01 0.44* * . -0.600.71 .

35 Asp 906 . B B . . . -0.08 0.56* * . -0.600.29 .

Phe 907 . B B . . . -0.27 0.56* * . -0.600.40 .

Trp 908 . B B . . . -0.66 0.71* * . -0.600.43 .

Leu 909 . B B . . . -1.23 0.46* * . -0.600.25 .

Lys 910 . B B . . . -0.64 1.14* * . -0.600.20 .

40 Val 911 . . B T . . -1.23 0.74* * . -0.200.26 .

Gly 912 . . B T . . -0.88 0.33* * . 0.10 0.32 .

Ile 913 . . B T . . -0.90 0.07* * . 0.10 0.16 .

Ser 914 . . . . T C -0.76 0.56* * . 0.00 0.31 .

Ala 915 . . . 'f T . -1.11 0.49. * F 0.35 0.17 .

45 Gly 916 . . . T T . -0.84 0.54. . F 0.35 0.34 .

Thr 917 . B . . T . -1.39 0.36. . F 0.25 0.26 .

Cys 918 . B B . . . -1.31 0.66. . . -0.600.18 .

Thr 919 . B B . . . -1.82 0.84. . . -0.600.15 .

Ala 920 . B B . . . - I 1.10. . . -0.600.09 . .54 50 Ile 921 . B B . . . -2.06 1.10. . . -0.600.23 .

Leu 922 . B B . . . -2.56 1.17. . . -0.600.12 .

Leu 923 . B B . . . -2.20 1.37. . . -0.600.10 .

Thr 924 . B B . . . -2.56 1.36. . . -0.600.20 .

Val 925 . B B . . . -2.21 1.24. . . -0.600.13 .

55 Ixu 926 . B B . . . -2.02 1.31. . . -0.600.25 .

Thr 927 . B B . . . -1.50 1.41* . . -0.600.
. I

Cys 928 . B B . . . -0.64 1.84* . . -0.600.21 .

Tyr 929 . B B . . . -0.29 1.20. . . -0.600.51 .

Phe 930 . . B T . . 0.57 0.51. . . -0.200.70 .

60 '1'rp931 . . B T . . 1.38 0.43* . . 0.29 2.10 .

Lys 932 . . . . T C 1.73 0.26* . F 1.28 2.32 .

Lys 933 . . . T 1' . 1.59 -0.50* * F 2.42 5.37 .

Asn 934 . . . . T C 1.83 -0.60* * F 2.86 4.21 .

Gln 935 . . . T T . 2.29 -1.51* * F 3.40 3.65 .

Lys 936 . . B . . . 2.62 -0.76* * F 2.46 2.86 .

Leu 937 . . B . . . 2.33 -0.76* * F 2.32 3.55 .

Glu 938 . . B . . . 1.99 -0.40* * . 1.73 3.21 .

Tyr 939 . . B . T . 2.03 -0.41* * . 1.79 2.15 .

Lys 940 . . B . T . 1.22 -0.41* * F 1.80 5.22 .

Tyr 941 . . B . T . 0.32 -0.41* * F 2.00 2.49 .

Ser 942 . . B . T . 0.53 0.23* * F 1.20 1.18 .

Lys 943 . A B . . . 0.53 0.09* * F 0.45 0.58 .

Leu 944 . A B . . . 0.19 0.49* . . -0.200.60 .

Val 945 . A B . . . -0.170.23* * . -0.100.45 .

Met 946 . A B . . . -0.730.33* * . -0.300.33 .

Asn 947 . A B . . . -0.391.01. * . -0.600.33 .

Ala 948 . A B . . . -0.430.33* * . -0.300.88 .

Thr 949 . A B . . . -0.29-0.31. * . 0.65 1.48 .

Leu 950 . A B . . . 0.57 -0.36* . F 0.85 0.49 .

Lys 951 . A B . . . 0.36 -0.76. * F 1.35 0.82 .

Asp 952 . . . T T . 0.14 -0.57. * F 2.35 0.47 .

Cys 953 . . B . T . 0.14 -0.63. . . 2.00 0.87 .

Asp 954 . . B . T . -0.13-0.81. . . 1.80 0.44 .

Leu 955 . . B . T . 0.68 -0.31. . . 1.30 0.27 .

Pro 956 . . B . . . 0.33 -0.31. . . 0.90 0.83 .

Ala 957 . . . T . . -0.33-0.50. * . 1.10 0.67 .

Ala 958 A . . . . . -0.260.07. . . -0.100.43 .

Asp 959 A . . . T . - -0.1. . . 0.70 0.28 . I 1 .14 Ser 960 . . B . T . -0.930.14. . . 0.10 0.20 . .

Cys 961 . . B . T . -0.720.26. . . 0.10 0.19 .

Ala 962 . . B . T . -0.48-0.24. . . 0.70 0.20 .

Ile 963 . A B . . . 0.11 0.19. . . -0.300.15 .

Met 964 . A B . . . 0.11 -0.20. . . 0.30 0.48 .

Glu 965 . A B . . . -0.44-0.77. . F 0.75 0.79 .

Gly 966 . A . . . C 0.22 -0.63* . F 0.95 0.83 .

Glu 967 A A . . . . 0.81 -1.31* . F 0.90 1 . .46 Asp 968 A A . . . . 1.70 -1.93* . F 0.90 1 . .41 Val 969 A A . . . . 1.49 -1.93* . F 0.90 2.38 .

Glu 970 A A . . . . 0.60 -1.67* . F 0.90 1.13 .

Asp 971 A A . . . . 0.24 -0.99* . F 0.75 0.48 .

Asp 972 A A . . . . -0.07-0.20. * F 0.45 0.55 .

Leu 973 A A . . . . -0.37-0.36* * . 0.30 0.46 .

Ile 974 A A . . . . 0.53 0.03. * . -0.300.37 .

Phe 975 . A B . . . 0.53 0.03. . . -0.300.44 .

Thr 976 . A B . . . 0.50 0.43. . F -0.450.87 .

Ser 977 . . . . T C 0.20 0.24. . F 0.60 1.68 .

Lys 978 . . . T T . 0.20 -0.06. . F 1.40 2.60 .

Asn 979 . . . . T C 0.74 -0.16* . F 1.48 1.49 .

His 980 . . . . T C 1.56 -0.21* * F 1.76 1.10 .

Ser 981 . . . . . C 1.57 -0.60. * . 1.99 1.07 .

Leu 982 . . . T . . 1.87 -0.21. . . 2.02 0.90 .

Gly 983 . . . T T . 1.79 -0.21. . F 2.80 1.06 .

Arg 984 . . . T T . 0.98 -0.21* . F 2.52 1.07 .

Ser 985 . . . T T . 0.80 0.09* . F 1.88 1.07 .

Asn 986 . . . T T . 0.89 -0.17* * F 2.44 1.68 .

His 987 . . . . . C 1.81 -0.17* * F 2.00 1.33 .

Leu 988 . . . . . C 1.81 -0.17* * F 1.96 1.94 .

Pro 989 . . . . T C 0.89 -0.13* * F 2.40 1.19 .

Pro 990 . . . T T . 0.38 0.16. * F 1.61 0.72 .

Arg 991 . . . T T . -0.220.34. * F 1.37 0.72 .

Gly 992 . B . . T . -0.19 0.27 . * F 0.73 0.46 .

Leu 993 A B . . . . -0.19 -0.16 . . 0.54 0.50 . *

Leu 994 A B . . . . -0.29 0.10 * . . -0.30 0.21 .

Met 995 A B . . . . -0.08 0.59 * . . -0.60 0.31 .

Asp 996 A B . . . . -0.86 0.56 * . . -0.60 0.64 .

Leu 997 A B . . . . -0.40 0.44 . . . -0.60 0.42 .

Thr 998 A B . . . . 0.02 -0.24 . . 0.30 0.83 . .

Gln 999 A B . . . . 0.44 -0.43 . F 0.45 0.63 . .

Cys 1000 A B : . . . 0.66 0.00 . . . -0.30 0.98 .

Arg 1001 A B . . . . 0.27 -0.26 . . 0.30 0.87 . .

Table IV

Res Pos II III N V VI VII VIII IX X XI XII XIII XIV
I

Met 1 . A . . . . C 0.68 -0.13. * . 0.50 0.57 Asp 2 . A . . T . . 1.07 -0.56. * . 1.00 0.78 Cys 3 . A . . T . . 1.46 -0.59. . . 1.34 0.98 Gln 4 . A . . T . . 1.60 -1.01. * . 1.83 1.71 Glu 5 . A . . 'f . . 1.70 -0.87. * F 2.32 1.61 Asn 6 . . . . T T . 2.30 0.04. * F 2.16 3.15 Glu 7 . . . . T T . 2.30 -0.53. . F 3.40 3.04 Tyr 8 . . . . T T . 2.68 -0.53* . F 3.06 3.04 Trp 9 . . . . T T . 2.33 0.39* * . 1.67 1.99 Asp 10 . . . . T 'f . 2.44 0.41* * . 1.03 1.14 Gln I1 . . . . T T . 1.78 0.41* . F 0.84 1.42 Trp 12 . . . . T T . 0.92 0.23* . . 0.50 0.72 Gly 13 . . . . T T . 0.86 -0.04* . . 1.10 0.32 Arg 14 . . . B T . . 0.48 0.44* . . -0.200.27 Cys 15 . . . B T . . 0.48 0.61* * . -0.200.14 Val 16 . . . B T . . 0.59 0.10* * . 0.35 0.24 Thr 17 . . . B T . . 0.21 -0.33* * . 1.20 0.24 Cys 18 . . . . T T . 0.21 0.24. * . 1.25 0.24 Gln 19 . . . . T T . -0.110.10. * . 1.50 0.32 Arg 20 . . . . T T . 0.21 -0.11. . F 2.50 0.34 Cys 21 . . . . T T . 1.07 -0.17. . F 2.25 0.63 Gly 22 . . . . . T C 1.38 -0.34. . F 1.80 0.63 Pro 23 . . . . T T . 1.23 -0.74. . F 2.05 0.56 Gly 24 . . . . T T . 0.93 -0.06. . F 1.81 0.86 Gln 25 . . . . T T . 0.87 -0.24. * F 2.02 1.16 Glu 26 . . . . T . . 1.53 -0.67* . F 2.43 1.50 Leu 27 . . . . T . . 1.21 -1.10* . F 2.74 2.54 Ser 28 . . . . T T . 1.08 -0.96* . F 3.10 0.79 Lys 29 . . . . T 'f . 1.18 -0.93* . F 2.79 0.45 Asp 30 . . . . T T . 0.83 -0.17* . F 2.18 0.85 Cys 31 . . . . T T . 0.83 -0.43. * F 2.21 0.63 Gly 32 . . . . T . . 1.30 -0.81. . . 2.19 0.55 .

Tyr 33 . . . . T . . 1.26 -0.39. . F 2.07 0.32 Gly 34 . . . . . T C 1.21 0.04. . F 1.81 0.60 Glu 35 . . . . T T . 0.62 -0.53. . F 3.40 1.01 Gly 36 . . . . T T . 1.04 -0.46. . F 2.61 0.65 Gly 37 . . . . T T . 1.10 -0.46* . F 2.42 1.03 Asp 38 . . . . T . . 1.31 0.03* . F 1.13 0.62 Ala 39 . . . . . . C 1.36 0.53* . . 0.14 0.86 Tyr 40 . . . . T . . 0.54 0.49* . . 0.15 1.16 Trp 41 . . B . . . . 0.68 0.74* . . -0.400.57 His 42 . . . . . . C 0.72 1.17* * . -0.200.88 Ser 43 . . . . . C 0.42 1.06. . . -0.200.75 .

Leu 44 . . . . T C I.01 0.69. . F 0.15 0.96 .

Pro 45 . . . T T . 1.0I 0.17* * F 1.04 1.22 .

Ser 46 . . . T T . 1.34 0.43* . F 0.98 1.42 .

Ser 47 . . . T T . 1.08 0.04. . F 1.52 3.45 .

Gln 48 . . . T . . 1.08 -0.26. * F 2.16 2.99 .

Tyr 49 . . . T . . 1.60 -0.30. * F 2.40 2.99 .

Lys 50 . . . T . . 1.47 0.23. * F 1.56 2.35 .

Ser 51 . . . T T . 1.73 0.27. * F 1.52 1.34 .

Ser 52 . . . T T . 2.00 0.37. * F 1.28 1.17 .

Trp 5 . . . T T . 2.04 0.1. * . 0.74 0.79 .

Gly 54 . . . T T . 1.62 0.11. * . 0.87 1.18 .

His 55 . . . T . . 1.58 0.30. * . 0.74 0.47 .

His 56 . . . T . . 1.58 0.31. . . 0.96 0.78 .

Lys 57 . . . T . . 1.21 -0.21. * . 1.93 1.06 .

Cys 58 . . . T T . 0.61 -0.07. * . 2.20 0.42 .

Gln 59 . . . T T . 0.64 0.11* * . 1.38 0.21 .

Ser 60 . . . T T . 0.01 0.10* * . 1.16 0.15 .

Cys 61 . . . T T . -0.540.67* * . 0.64 0.15 .

Ile 62 . B B . . . -1.440.60* * . -0.380.09 .

Thr 63 . B B . . . -1.670.84. * . -0.600.05 .

Cys 64 . B B . . . -1.671.14. . . -0.600.07 .

Ala 65 . B B . . . -1.260.97* * . -0.600.15 .

Val 66 . B B . . . -1.440.29* * . -0.300.20 .

Ile 67 . B B . . . -0.560.44* * . -0.340.28 .

Asn 68 . . B T . . -0.200.27* . . 0.62 0.48 .

Arg 69 . . B T . . -0.39-0.23* . . 1.63 1.30 .

Val 70 . . B 'r . . 0.20 -0.23* . F 2.04 i . .38 Gln 71 . . B T . . 0.39 -0.51* . F 2.60 1.38 .

Lys 72 . . B T . . 0.97 -0.34* . F 1.89 0.38 .

Val 73 . . B T . . 0.76 0.14* . . 0.88 0.73 .

Asn 74 . . B T . . 0.33 -0.07* * . 1.22 0.65 .

Cys 75 . . B T . . 0.89 0.01* * F 0.51 0.47 .

Thr 76 . . . . T C 0.89 0.40. * F 0.45 0.85 .

Pro 77 . . . T T . 0.26 0.16. * F 0.65 0.85 .

Thr 78 . . . T T . 0.26 0.26. * F 0.80 1.61 .

Ser 79 . . . T T . -0.410.33. . F 0.65 0.83 .

Asn 80 . . . T . . -0.090.41. . F 0.15 0.29 .

Ala 81 . . . T . . 0.22 0.41. . . 0.00 0.20 .

Val 82 . . . T . . -0.23-0.07. . . 0.90 0.24 .

Cys 83 . . . T T . -0.730.11. . . 0.50 0.08 .

Gly 84 . . . T T . -0.640.40* * . 0.50 0.07 .

Asp 85 . . . T T . -0.530.33* * . 0.50 0.14 .

Cys 86 . B . . T . -0.64-0.31* * . 0.70 0.51 .

Leu 87 . B . . . . -0.03-0.10* * . 0.81 0.44 .

Pro 88 . . . T T . 0.74 0.23* * . 1.12 0.42 .

Arg 89 . . . T T . 1.13 0.23* * . 1.58 1.52 .

Phe 90 . . . T T . 0.82 -0.34* * . 2.49 3.69 .

Tyr 91 . . . T T . 1.60 -0.54* * . 3.10 3.44 .

Arg 92 . . B T . . 1.52 -0.97* * F 2.54 3.44 .

Lys 93 . . B T . . 1.39 -0.29* * F 1.93 2.79 .

Thr 94 . . B T . . 0.93 -0.64* * F 1.92 1.76 .

Arg 95 . . B T . . 0.82 -0.97* * F 1.46 0.89 .

Ile 96 . . B T . . 1.07 -0.29. . F 0.85 0.37 .

Gly 97 . . . T . . 0.96 0.11. . F 0.45 0.44 .

Gly 98 A . . T . . 0.91 -0.37. * F 0.85 0.38 .

Leu 99 A . . . . C 1.22 0.03* * F 0.05 0.93 .

Gln 100 A . . T . . 0.44 -0.66. * F 1.30 1.62 .

Asp 101 A . . T . . 0.44 -0.51. . F 1.15 0.88 .

Gln 102 A . . T . . 0.58 -0.26. . F 0.85 0.75 .

Glu 103. A . . T . . 0.26-0.51. . F 1.15 0.67 Cys 104. A . . T . . 0.76-0.34. * . 0.70 0.21 Ile 105. . B . . . . 0.800.14. . . -0.100.18 Pro 106. . . . T . . 0.80-0.26. . . 0.90 0.21 Cys 107. . . . T T . 0.490.14* . . 0.50 0.67 Thr 108. . . . T T . 0.280.06* . F 1.10 1.37 Lys 109. . . . T T . 0.63-0.20. . F 2.00 1.37 Gln 110. . . . . 'f C 1.22-0.14. . F 2.10 3.69 Thr 111. . . . . T C 1.43-0.33. . F 2.40 3.43 Pro 112. . . . . T C 1.24-0.81. * F 3.00 2.97 Thr 113. . . . T T . 1.56-0.17. * F 2.60 1.27 Ser 114. . . . . T C 0.84-0.17. * F 2.10 1.53 Glu 115. A . . T . . 0.26-0.09* * F 1.45 0.53 Val 116. A B . . . . -0.13-0.01* * . 0.60 0.37 Gln 117. A B . . . . 0.080.29* * . -0.300.24 Cys 118. A B . . . . -0.420.30* * . -0.300.24 Ala 119A A . . . . . -0.420.99* * . -0.600.27 Phe 120A A . . . . . -1.230.73. * . -0.600.21 Gln 121A A . . . . . -1.231.01. * . -0.600.32 Leu 122. A . . . . C -1.231.09. * . -0.400.23 Ser 123. A . . . . C -1.160.59. * . -0.400.47 Leu 124. A . . . . C -0.570.30. * . -0.100.27 Val 125. A . . . . C -0.46-0.10. * . 0.50 0.55 Glu 126. A . . . . C -0.67-0.29. * . 0.50 0.41 Ala 127. A . . T . . -0.17-0.24. . . 0.70 0.78 Asp 128. A . . T . . -0.72-0.44. . . 0.85 1.51 Ala 129. A . . . . C -0.12-0.44. * F 0.65 0.65 Pro 130. A . . . . C 0.52-0.01. * F 0.85 0.99 Thr 131. . . . . . C 0.52-0.09. . F 1.25 0.92 Val 132. . . . . . C 1.110.31. . F 1.00 1.57 Pro 133. . . . . . C 0.52-0.19. . F 1.80 1.76 Pro 134. . . . . . C 0.80-0.11. . F 2.00 1.23 Gln 135. . . . . . C 0.20-0.11. . F 1.80 2.40 Glu 136A . B . . . . -0.34-0.07. . F 1.40 1.28 Ala 137. . B B . . . -0.080.14. . F 0.25 0.61 Thr 138. . B B . . . -0.680.21. . . -0.100.36 Leu 139. . B B . . . -1.320.50. . . -0.600.17 Val 140. . B B . . . -1 1.14. . . -0.600.13 .62 Ala 141. . B B . . . -1.921.03. . . -0.600.12 Leu 142. . B B . . . -2.140.93* . . -0.600.19 Val 143. . B B . . . -2.640.93* . . -0.600.21 Ser 144. . B B . . . -2.690.97. . ~ -0.600.17 .

Ser 145. . B B . . . -2.691.11* . . -0.600.15 Leu 146. . B B . . . -2.801.07* . . -0.600.15 Leu 147. . B B . . . -2.301.21* . . -0.600.10 Val 148. . B B . . . -2.261.31. . . -0.600.11 Val 149. A B B . . . -2.541.61. * . -0.600.11 Phe 150. A B B . . . -2.941.43. . . -0.600.13 Thr 151. A B B . . . -2.941.53. . . -0.600.15 Leu 152. A B B . . . -2.481.57. . . -0.600.17 Ala 153A A . B . . . -2.431.36. . . -0.600.19 Phe 154. A . B T . . -2.281.26. . . -0.200.11 Leu 155. A . B T . . -2.281.56. . . -0.200.12 Gly 156. A . B T . . -2.781.66. . . -0.200.10 Leu 157. A . B T . . -2.211.84. . . -0.200.10 Phe 158. A . B T . . -2.291.81. . . -0.200.18 Phe 159. A . B T . . -1.541.70* . . -0.200.10 Leu 160. A . B T . . -0.731.27* . . -0.200.24 Tyr 161. A . B T . . -1.090.99* . . -0.200.48 Cys 162. A . B T . . -0.980.99* . . -0.200.48 Lys 163 . A . B T . . -0.280.99* . . -0.200.50 Gln 164 . A . B T . . 0.53 0.70* . . -0.200.51 Phe 165 . A . B T . . 1.31 -0.06* . . 0.85 1.88 Phe 166 . A . B T . . 0.89 -0.13* . . 1.16 1.28 5 Asn 167 . . . . T T . 1.56 0.44* ~ . 0.82 0.39 *

Arg 168 . . . . 'C T . 1.62 0.44* * . 1.13 0.79 His 169 . . . . T T . 1.28 -0.34* * . 2.49 1.79 Cys 170 . . . . T T . 1.63 -0.70* * . 3.10 1.10 Gln 171 . . . . T T . 1.52 -0.67* . F 2.79 0.56 10 Arg 172 . . . . T T . 0.71 0.01* * F 1.58 0.34 Gly 173 . . . . T T . 0.60 0.20* * F 1.27 0.52 Gly 174 . . . . T T . -0.070.03* * F 0.96 0.52 Lcu 175 . A . . . . C 0.60 0.41* * . -0.400.23 Leu 176 . A . . . . C 0.01 0.41. * . -0.400.40 15 Gln 177 . A B . . . . -0.100.49. * . -0.600.41 Phe 178 . A B . . . . 0.29 0.06. * . -0.300.83 Glu 179 A A . . . . . 0.32 -0.63. * . 0.75 2.01 Ala 180 A A . . . . . 0.54 -0.83* * F 0.90 1.67 Asp 181 A A . . . . . 1.40 -0.73* * F 0.90 1.95 20 Lys 182 A A . . . . . 1.40 -1.5. * F 0.90 2.26 I

Thr I A A . . . . . 2.10 -1.51* * F 0.90 3 83 .87 Ala 184 A A . . . . . 1.80 -2.01* * F 1.20 4.01 Lys 185 A A . . . . . 1.58 -1.63* . F 1.50 2.69 Glu 186 A A . . . . . 0.88 -0.94* . F 1.80 1.54 25 Glu 187 . A . . T . . 0.62 -0.64. . F 2.50 1.32 Ser 188 . . . . T . . 0.08 -0.71. * F 3.00 1.02 Leu 189 . . . . T . . 0.46 -0.07. * . 2.10 0.44 Phe 190 . . . . . . C 0.20 0.36. . . 1.00 0.39 Pro 191 . . . . . . C -0.100.79. . . 0.70 0.45 30 Val 192 . . . . . . C -0.060.79. . F 0.85 0.73 Pro 193 . . . . . T C 0.24 0.10. . F 1.50 1.69 Pro 194 . . . . . T C 0.74 -0.69. . F 2.70 1.89 Scr 195 . . . . . T C 1.14 -0.63. . F 3.00 3.67 Lys 196 . . . . . T C 0.77 -0.89. . F 2.70 3.18 35 Glu 197 . A . . . . C 1.62 -0.81. . F 2.00 2.08 Thr 198 . A . . . . C 1.53 -1.24. . F 1.70 2.69 Ser 199 . A . . . . C 1.74 -1.24. * F 1.40 1.80 Ala 200 . A . . . . C 1.19 -0.84. * F 1.10 1.80 Glu 201 . A . . T . . 0.84 -0.20. * F 0.85 0.93 40 Ser 202 . . . . . . C 0.56 -0.30. * F 0.85 0.93 Gln 203 . . . . T . . 0.28 0.23. * F 0.45 0.96 Val 204 . . . . . . C 0.37 0.23. * . 0.10 0.56 Ser 205 . . . . T . . 0.61 0.66. * . 0.00 0.65 Trp 206 . . . . . . C 0.31 0.70. * . -0.200.37 45 Ala 207 . . . . . T C -0.200.69. . . 0.00 0.67 Pro 208 . . . . . T C -0.790.73* . F 0.15 0.41 Gly 209 . . . . T T . 0.07 0.84* . F 0.35 0.40 Ser 210 . . . . . T C -0.440.33* . F 0.45 0.68 Leu 211 . . . . . . C -0.860.51* . . -0.200.36 50 Ala 212 . . . . . . C -0.570.87* . . -0.200.32 Gln 213 . . B . . . . -1.170.83. . . -0.400.32 Leu 214 . . B . . . . -0.821.13. . . -0.400.32 Phe 215 . . B . . . . -0.820.44. . . -0.400.52 Ser 216 . . B . . . . -0.870.3 . . . -0.100.40 55 Leu 217 . . . T . . -0.490.57. . . 0.00 0.36 Asp 218 . . . . T . . -1.380.31. . F 0.45 0.65 Ser 219 . . . . . . C -0.780.21. . F 0.25 0.34 Val 220 . . . . . . C -0.080.26. * F 0.25 0.64 Pro 221 . . . . . . C 0.22 -0.03. . F 0.85 0.66 60 Ile 222 . . . . . . C 1.03 0.37. . F 0.25 0.86 Pro 223 . . . . . . C 1.03 0.39 . F 0.66 2.00 .

Gln 224 . . . . T . . 0.99 0.14 . F 1.12 2.24 *

Gln 225 . . . . . . C 1.63 0.14 . F 1.18 3.16 *

Gln 226 . . . . . . C 1.84 -0.11 . F 2.04 3.16 .

Gln 227 . . . . . . C 2.13 -0.54 . F 2.60 3.16 .

Gly 228 . . . . . T C 1.96 -0.33 . F 2.24 1.80 .

Pro 229 . . . . . T C 1.57 -0.30 . . 1.83 1.33 .

Glu 230 . . . . T T . 1.18 -0.27 . . 1.62 0.98 .

Met 231 . . . . . T C 0.79 -0.24 * . 1.31 1.27 .

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a TR13 nucleic acid molecule of the invention described above, for instance, the cDNA clone (HWLHM70) contained in ATCC Deposit No. PTA-349, the nucleic acid sequence disclosed in Figures 1 A-C or the complementary strand thereof, and fragments thereof (e.g., as described herein).
By "stringent hybridization conditions" is intended overnight incubation at 42°C in a solution comprising: 50% formamide, Sx SSC (750 mM NaCI, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), Sx Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to molecule of the invention described above, for instance, the TR13 cDNA clone (HWLHN83) contained in ATCC Deposit No. PTA-507, the nucleic acid sequence disclosed in figures 7A-D
or the complementary strand thereto, and fragments thereof (e.g., as described herein).
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more 2o preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below. In this context "about"
includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
By a portion of a polynucleotide of "at least 20 nt in length," for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., cDNA deposited as ATCC Deposit No. PTA-507, or the nucleotide sequence as shown in SEQ ID N0:39 or the complementary strand thereto, or a fragment thereof).
By a portion of a polynucleotide of "at least 20 nt in length," for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., cDNA desposited as ATCC Deposit No: PTA-349, or the nucleotide sequence as shown in SEQ ID NO:1 or the complementary strand thereto, or a fragment thereof).
Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of the TR13 cDNA shown in SEQ ID NO:1 or SEQ ID N0:39), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a TR14 nucleic acid molecule of the invention described above, for instance, a cDNA clone (HMSHK47) contained in ATCC Deposit No. PTA-348, the nucleic acid sequence disclosed in preferably in Figures l0A-H or, alternatively, in Figures 4A-D or the complementary strand thereto, and fragments thereof (e.g., as described herein).
By a portion of a polynucleotide of "at least 20 nt in length," for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., cDNA deposited as ATCC Deposit No: PTA-348, or the nucleotide sequence as shown preferably in SEQ ID N0:60 or, alternatively, in SEQ ID N0:4 or the complementary strand thereto, or a fragment thereof).
Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of the TR14 cDNA shown preferably in SEQ ID N0:60 or, alternatively, in SEQ ID N0:4), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
In specific embodiments, the polynucleotides of the invention are less than 100000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb, or 5 kb in length.
In further embodiments, nucleic acids of the invention comprise at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of TR13 coding sequence, but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA
that flanks the S' or 3' coding nucleotide set forth in Figures lA-C (SEQ ID
NO:1) or in Figures 7A-D (SEQ ID N0:39). In further embodiments, nucleic acids of the invention comprise at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of TR13 coding sequence, but do not comprise all or a portion of any TR13 intron. In another embodiment, the nucleic acid comprising TR13 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., S' or 3' to the TR13 gene in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
In further, nucleic acids of the invention comprise at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of TR14 coding sequence, but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA
that flanks the 5' or 3' coding nucleotide set forth preferably in Figures l0A-H (SEQ ID
N0:60) or, alternatively, in Figures 4A-D (SEQ ID N0:4). In further embodiments, nucleic acids of the invention comprise at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of TR14 coding sequence, but do not comprise all or a portion of any TR14 intron. In another embodiment, the nucleic acid comprising TR14 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the TR14 gene in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, to or 1 genomic flanking gene(s).
As indicated, nucleic acid molecules of the present invention which encode a polypeptide may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein ~5 sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA
processing -including splicing and polyadenylation signals, for example - ribosome binding and stability of 2o mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, for instance, the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many 25 of which are commercially available. As described in Gentz et al., Proc.
Natl. Acad. Sci.
USA 86: 821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37:767-778 (1984). As discussed below, other such fusion proteins 3o include, but are not limited to, the TR13 receptor fused to Fc at the N- or C-terminus.
As indicated, nucleic acid molecules of the present invention which encode a polypeptide may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein 35 sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA
processing -including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, for instance, the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide. In 5 certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl.
Acad. Sci.
USA 86: 821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The "HA" tag is another peptide useful for purification which corresponds to to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37:767-778 (1984). As discussed below, other such fusion proteins include, but are not limited to, the TR14 receptor fused to Fc at the N- or C-terminus.
The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs, or derivatives of the TR13 receptor.
15 Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions, deletions or additions 2o which may involve one or more nucleotides. The variants may be altered in coding or non coding regions or both. Alterations in the coding regions may produce conservative or non conservative amino acid substitutions, deletions,. or additions. Especially preferred among these are silent substitutions, additions, and deletions, which do not alter the properties and activities of the TR13 receptor or portions thereof. Also especially preferred in this regard are 25 conservative substitutions.
The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs, or derivatives of the TR14 receptor.
Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of 3o an organism. Genes 1l, Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions, deletions or additions which may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-35 conservative amino acid substitutions, deletions, or additions. Especially preferred among these are silent substitutions, additions, and deletions, which do not alter the properties and activities of the TR14 receptor or portions thereof. Also especially preferred in this regard are conservative substitutions.

Further embodiments of the invention include isolated nucleic acid molecules comprising or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98%, or 99%
identical to: (a) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID N0:2;
(b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID
N0:2, but lacking the amino terminal methionine (amino acid positions 2 - 750 of SEQ ID
N0:2); (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-349 (HWLHM70); (d) a nucleotide sequence encoding the mature TR13 polypeptide having the amino acid sequence to encoded by the cDNA clone contained in ATCC Deposit No. PTA-349 (HWLHM70);
(e) a nucleotide sequence encoding any combination of one, two, three or all four of the TR13 cysteine rich domains disclosed in Figures lA-C (amino acids 105 to 170, amino acids 251 to 265, amino acids 331 to 410, and/or amino acids 580 to 610 of SEQ ID N0:2);
(f) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 105 ~5 to about 170 of SEQ ID N0:2; (g) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 251 to about 265 of SEQ ID N0:2;
(h) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 331 to about 410 of SEQ ID N0:2; (i) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 580 to about 610 of SEQ
ID N0:2; and 20 (j) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Further embodiments of the invention include isolated nucleic acid molecules comprising or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 25 90% identical, and more preferably at least 95%, 96%, 97%, 98%, or 99%
identical to: (a) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID
N0:40; (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID N0:40, but lacking the amino terminal methionine (amino acid positions 2 - 1001 of SEQ ID N0:40); (c) a nucleotide sequence encoding the polypeptide having the amino acid 3o sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-507 (HWLHN83);
(d) a nucleotide sequence encoding the mature TR13 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-507 (HWLHN83);
(e) a nucleotide sequence encoding the TR13 receptor mature extracellular domain (amino acid positions from about 42 to about 906 of SEQ ID N0:40); (f) a nucleotide sequence encoding 35 the TR13 receptor transmembrane domain (amino acid positions from about 134 to about 150 of SEQ ID N0:40); (g) a nucleotide sequence encoding the TR13 receptor intracellular domain (amino acid positions 932 to about 1001 of SEQ ID N0:40); (h) a nucleotide seuqence encoding the TR13 receptor extracellular and intracellular domains with all or a part of the transmembrane domain deleted (amino acid positions from about 42 to about 96 and 932 to about 1001 of SEQ ID N0:40); (i) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 271 to about 421 of SEQ ID N0:40;
(j) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 271 to about 286 of SEQ ID N0:40; (k) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 290 to about 300 of SEQ
ID N0:40; (1) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 301 to about 320 of SEQ ID N0:40; (m) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 329 to about 361 of SEQ
ID N0:40; (n) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 404 to about 421 of SEQ ID N0:40; (o) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 585 to about 595 of SEQ ID N0:40; (p) a nucleotide sequence encoding any one of the TR13 conserved domains as shown in Figures 7A-D; (q) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 661 to about 674 of SEQ ID N0:40; (r) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 710 to about 744 of SEQ ID N0:40; (s) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 980 to about 991 of SEQ ID N0:40;
(t) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from 2o about 45 to about 60 of SEQ ID N0:40; (u) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 121 to about 135 of SEQ
ID N0:40;
(v) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 145 to about 160 of SEQ ID N0:40; and (w) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (1), (m), (n), (o), (p), (q), (r), (s), (t), (u) or (v) above. In this context "about"
includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
By a polynucleotide having a nucleotide sequence at least, for example, 95%
"identical"
to a reference nucleotide sequence encoding a TR13 polypeptide is intended that the nucleotide 3o sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five mismatches per each 100 nucleotides of the reference nucleotide sequence encoding the TR13 polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mismatches of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The reference (query) sequence may be the entire TR13 encoding nucleotide sequence shown in Figures lA-C (SEQ ID NO:1) or Figures 7A-D (SEQ ID N0:39) or any TR13 polynucleotide fragment (e.g., a polynucleotide encoding the amino acid sequence of any of the TR13 N- and/or C- terminal deletions described herein), variant, derivative or analog, as described herein.
As a practical matter, whether any particular polynucleotide sequence is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID N0:39 or to the nucleotide sequence of the deposited cDNA clone (HWLHM70 or HWLHN83) can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI
53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full-length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
2o Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90%
identical, and more preferably at least 95%, 96%, 97%, 98%, or 99% identical to: (a) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID N0:61; (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:
61, but lacking the amino terminal methionine (amino acid positions 2 - 231 of SEQ ID
N0:61 ); (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. HMSHK47; (d) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO:S; (e) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO: 5, but lacking the 3o amino terminal methionine (amino acid positions 2 - 226 of SEQ ID NO:S);
(f) a nucleotide sequence encoding the TR14 receptor extracellular domain (preferably preferably amino acid positions from about 1 to about 138 of SEQ ID N0:61 or, alternatively, amino acid positions from about 1 to about 133 of SEQ ID NO:S); (g) a nucleotide sequence encoding the TR14 cysteine rich domain (preferably amino acid positions from about 70 to about 90 of SEQ ID
N0:61 or, alternatively, amino acid positions from about 65 to about 85 of SEQ
ID NO:S); (h) a nucleotide sequence encoding the TR14 receptor transmembrane domain (preferably, amino acid positions from about 139 to about 155 of SEQ ID N0:61 or, alternatively, amino acid positions from about 134 to about 150 of SEQ ID NO:S) ; (i) a nucleotide sequence encoding the TR14 receptor intracellular domain (preferably, amino acid positions from about 156 to about 231 of SEQ ID N0:61 or, alternatively, from about amino acid positions 151 to about 226 of SEQ ID NO:S); (j) a nucleotide sequence encoding the TR14 receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted (preferably amino acid positions from about 1 to about 138 and 156 to about 231 of SEQ ID N0:61 or, alternatively, amino acid positions from about 1 to about 133 and 151 to about 226 of SEQ ~
NO:S); and (k) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) or (k) above. In this context "about" includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either to terminus or at both termini.
By a polynucleotide having a nucleotide sequence at least, for example, 95%
"identical"
to a reference nucleotide sequence encoding a TR14 polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five mismatches per each 100 nucleotides of the t5 reference nucleotide sequence encoding the TR14 polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mismatches of the 2o reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The reference (query) sequence may be the entire TR14 encoding nucleotide sequence shown preferably in Figures l0A-H (SEQ ID N0:60) or, alternatively, in Figures 25 4A-D (SEQ ID N0:4) or any TR14 polynucleotide fragment (e.g., a polynucleotide encoding the amino acid sequence of any of the TR14 N- and/or C- terminal deletions described herein), variant, derivative or analog, as described herein.
As a practical matter, whether any particular polynucleotide sequence is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown 30 preferably in SEQ ID N0:60 or, alternatively, in SEQ ID N0:4 or to the nucleotide sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI
53711 ). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in 35 Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full-length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
In a specific embodiment, the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence 5 alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are:
Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the to subject nucleotide sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction is made to the results to take into consideration the fact that the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ~5 ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A
determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB
sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above 2o FASTDB program using the specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of this embodiment. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB
alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score. For example, a 90 base subject sequence is aligned to a 25 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at S' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program.
If the remaining 30 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence 35 which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.
The present application is directed to nucleic acid molecules comprising a polynucleotide sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence for example, shown in SEQ ID NO:1 or SEQ ID N0:39, or to the nucleic acid sequence of the cDNA deposited as ATCC deposit No. PTA-349 or PTA-507, irrespective of whether they encode a polypeptide having TR13 receptor activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having TR13 functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having TR13 receptor activity include, inter alias (1) isolating the TR13 receptor gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal 1o spreads to provide precise chromosomal location of the TR13 receptor gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Technigues, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting TR13 receptor mRNA
expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to for example, the nucleic acid sequence shown in SEQ ~
NO:1 or SEQ ID N0:39, or to the nucleic acid sequence of the cDNA deposited as PTA-349 or PTA-507, which do, in fact, encode a polypeptide having TR13 receptor functional activity. By "a polypeptide having TR13 functional receptor activity" is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the TR13 receptor of the invention (either the full-length protein or, preferably, the mature protein), as measured, for example, in a particular biological assay.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in SEQ ID NO: I or SEQ ID N0:39 will encode a polypeptide "having TR13 receptor functional activity." In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.
It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate 3o variants, a reasonable number will also encode a polypeptide having TR13 receptor activity.
This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).
For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in J.U. Bowie et al., "Deciphering the Message in Protein Sequences:
Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein the authors indicate that proteins are surprisingly tolerant of amino acid substitutions.

The present application is directed to nucleic acid molecules comprising a polynucleotide sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence for example, shown preferably in SEQ ID N0:60 or, alternatively, in SEQ ID
N0:4, or to the nucleic acid sequence of the cDNA deposited as ATCC Deposit No. PTA-348, and even more preferably to the polypeptide coding regions of these sequences, irrespective of whether they encode a polypeptide having TRI4 receptor activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having TR14 functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the 1o nucleic acid molecules of the present invention that do not encode a polypeptide having TR14 receptor activity include, inter alias (1) isolating the TR14 receptor gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the TRI4 receptor gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting TR14 receptor mRNA
expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to for example, the nucleic acid sequence shown preferably in SEQ ID N0:60 or, alternatively, in SEQ ID N0:4, or to the nucleic acid sequence of the deposited cDNA, and even more preferably to the polypeptide coding regions of these sequences, which do, in fact, encode a polypeptide having TRI4 receptor functional activity.
By "a polypeptide having TR14 functional receptor activity" is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the TR14 receptor of the invention (either the full-length protein or, preferably, the mature protein), as measured, for example, in a particular biological assay.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown preferably in SEQ 1D
N0:60 or, 3o alternatively, in SEQ ID N0:4 will encode a polypeptide "having TR14 receptor functional activity." In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having TR14 receptor activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).

Polynucleotide assays This invention is also related to the use of TR13 polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent.
Detection of a mutated form of TR13 polynucleotide associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to a disease which results from under-expression over-expression or altered expression of TR13 or a soluble form thereof, such as, for example, tumors or autoimmune disease.
Individuals carrying mutations in the TR13 gene may be detected at the nucleic acid level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis. (Saiki et al., Nature 324:163-166 (1986)). RNA or cDNA may also be used in the same ways, or through routine modification of these polynucleotides. As an example, PCR
primers complementary to the nucleic acid encoding TR13 can be used to identify and analyze TR13 expression and mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
Point mutations can be identified using techniques known in the art, for example, by hybridizing amplified DNA to radiolabeled TR13 RNA or alternatively, radiolabeled TR13 antisense DNA
sequences.
Perfectly matched sequences can be distinguished from mismatched duplexes by, for example, RNase A digestion or by differences in melting temperatures.
This invention is also related to the use of TR14 polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent.
Detection of a mutated form of TR14 polynucleotide associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to a disease which results from under-expression over-expression or altered expression of TR14 or a soluble form thereof, such as, for example, tumors or autoimmune disease.
Individuals carrying mutations in the TR14 gene may be detected at the nucleic acid level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
The genomic 3o DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis. (Saiki et al., Nature 324:163-166 (1986)). RNA or cDNA may also be used in the same ways, or through routine modification of these polynucleotides. As an example, PCR
primers complementary to the nucleic acid encoding TR14 can be used to identify and analyze TR14 expression and mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
Point mutations can be identified by hybridizing amplified DNA to radiolabeled TR14 RNA or alternatively, radiolabeled TR14 antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by, for example, RNase A digestion or by differences in melting temperatures.
Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method. For example, a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures to with fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing ~5 formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science 230:1242 (1985)).
Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., 20 Proc. Natl. Acad. Sci. USA 85: 4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods such as, for example, hybridization, RNase protection, chemical cleavage, direct DNA
sequencing or the use of restriction enzymes, (e.g., restriction fragment length polymorphisms ("RFLP") and Southern blotting of genomic DNA.
25 In addition to more conventional gel-electrophoresis and DNA sequencing, mutations also can be detected by in situ analysis.
Vectors and Host Cells The present invention also relates to vectors which include the isolated DNA
molecules 30 of the present invention, host cells which are genetically engineered with the recombinant vectors and/or nucleic acids of the invention and the production of TR13 polypeptides or fragments thereof by recombinant techniques.
The present invention also relates to vectors which include the isolated DNA
molecules of the present invention, host cells which are genetically engineered with the recombinant 35 vectors and/or nucleic acids of the invention and the production of TR14 polypeptides or fragments thereof by recombinant techniques.
Host cells can be genetically engineered to incorporate nucleic acid molecules and express polypeptides of the present invention. The polynucleotides may be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention.
In accordance with the present invention the vector may be, for example, a clone vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA
viral vector.
5 Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells. Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.
Preferred among vectors, in certain respects, are those for expression of polynucleotides and polypeptides of the present invention. Generally, such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
15 The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a clone vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The DNA insert should be operatively linked to an appropriate promoter, such as the 2o phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will 25 preferably include a translation initiating at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other 3o bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells;
animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
35 Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNHBA, pNHl6a, pNHlBA, pNH46A, and pSport available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRlTS available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art.
The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a t0 bacterial cell. The host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired.
Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled.
Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins.
Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).
In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., TR13 coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous TR13 polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous TR13 polynucleotide sequences via homologous recombination (see, e.g., US Patent Number 5,641,670, issued June 24, 1997; International Publication Number WO 96/29411, published September 26, 1996; International Publication Number WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., TR14 coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous TR14 polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous TR14 polynucleotide sequences via homologous recombination (see, e.g., US Patent Number 5,641,670, issued June 24, 1997; International Publication Number WO 96/29411, published September 26, 1996; International Publication Number WO 94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
The TR13 polypeptides of the invention may be expressed in a modified form, such as a fusion protein (comprising the polypeptide joined via a peptide bond to a heterologous protein sequence (of a different protein)), and may include not only secretion signals but also additional heterologous functional regions. Alternatively, such a fusion protein can be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. Thus, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. For example, in one embodiment, polynucleotides encoding TR13 polypeptides of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency to expression and purification of such polypeptides in Gram-negative bacteria.
See, US Patent Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by reference in their entireties.
The TR14 polypeptides of the invention may also be expressed in a modified form, such as a fusion protein (comprising the polypeptide joined via a peptide bond to a heterologous protein sequence (of a different protein)), and may include not only secretion signals but also additional heterologous functional regions. Alternatively, such a fusion protein can be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. Thus, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. For example, in one embodiment, polynucleotides encoding TR14 polypeptides of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency to expression and purification of such polypeptides in Gram-negative bacteria. See, US Patent Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by reference in their entireties.
A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (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 advantageous manner described. This is the case when the Fc portion proves to be a hindrance to use in therapy and diagnosis, for example, when the fusion protein is to be used as an antigen for immunizations. In drug discovery, for example, human proteins, such as the hILS-receptor, have been fused W th Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
See, D. Bennett et al., Journal of Molecular Recognition 8:52-58 ( 1995) and K. Johanson et al., The Journal of Biological Chemistry 270:16:9459-9471 (1995).
2o Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
In addition, TR13 polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y. (1983), and Hunkapiller, et al., Nature 310:105-111 (1984)). For example, a TR13 polypeptide fragment of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the TR13 polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
In addition, TR14 polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y. (1983), and Hunkapiller, et al., Nature 310:105-111 (1984)). For example, a TR14 polypeptide fragment of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the TR14 polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino ~5 acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
The invention additionally, encompasses TR13 polypeptides (proteins) which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic 2o cleavage, linkage to an antibody molecule or other cellular ligand, etc.
Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin; etc.
25 The invention additionally, encompasses TR14 polypeptides (proteins) which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, 3o specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C
35 terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
Also provided by the invention are chemically modified derivatives of TR13 polypeptides (proteins) which may provide additional advantages such as increased solubility, 5 stability and circulating time of the polypeptide, or decreased immunogenicity (see U. S. Patent No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as, for example, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
Also provided by the invention are chemically modified derivatives of TR14 polypeptides which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.
S. Patent No.
15 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as, for example, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical 20 moieties.
The polymer may be of any molecular weight, and may be branched or unbranched.
For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and 25 manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between 3o about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the 35 polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
As noted above, the polyethylene glycol may have a branched structure.
Branched polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575;
Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
The polyethylene glycol molecules (or other chemical moieties) should be attached to to the polypeptide (proteins) with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a 2o reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
3o One may specifically desire polypeptides (proteins) chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.
9:249-304 (1992);
Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Patent No.
4,002,531; U.S. Patent 1o No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (C1SOZCHZCF3).
Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Patent No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO
98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
The number of polyethylene glycol moieties attached to each protein of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule.
Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev.
Thera. Drug Carrier Sys. 9:249-304 (1992).

As mentioned the TR13 and TR14 polypeptides (proteins) of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modiEcation techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given TR13 or TR14 polypeptide. TR13 or TR14 polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching.
Cyclic, branched, and branched cyclic TR13 or TR14 polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, 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 phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI
anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR
PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
2o Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 ( 1990); Rattan et al., Ann NY Acad Sci 663:48-62 ( 1992)).
As mentioned the TR14 polypeptides (proteins) of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given TR14 polypeptide. TR14 polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic TR14 polypeptides may result from posttranslation natural prpcesses or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, 3o 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 phosphotidylinosi~tol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA
mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.

Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL
COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990);
Rattan et al., Ann NY Acad Sci 663:48-62 ( 1992)).
The TR 13 polypeptides (proteins) of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or canon exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
The TR14 polypeptides (proteins) of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or canon exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
TR13 polynucleotides and polypeptides of the present invention, and agonsits or antogonists thereof, may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of TR13.
Among these are, for example, applications in treatment of tumors; resistance to parasites, bacteria and viruses; to regulate (i.e., induce) proliferation of T-cells, endothelial cells and hematopoietic cells; to treat restenosis, and graft vs. host disease; to regulate anti-viral responses; and to prevent certain autoimmune diseases after stimulation of TR13 by an 3o agonist. Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms. These aspects of the invention are discussed further below.
TR14 polynucleotides and polypeptides of the present invention, and agonsits or antogonists thereof, may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of TR 14.
Among these are, for example, applications in treatment of tumors; resistance to parasites, bacteria and viruses; to regulate (i.e., induce) proliferation of T-cells, endothelial cells and hematopoietic cells; to treat restenosis, and graft vs. host disease; to regulate anti-viral responses; and to prevent certain autoimmune diseases after stimulation of TR14 by an agonist. Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms. These aspects of the invention are discussed further below.
Transgenics and "knock-outs"
The TR13 polypeptides (proteins) of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express 1o polypeptides of the invention in humans, as part of a gene therapy protocol.
The TR14 polypeptides (proteins) of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific ~5 embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
Any technique known in the art may be used to introduce the transgene (i.e., nucleic acids of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl.
20 Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 ( 1993); Wright et al., Biotechnology (NY) 9:830-834 ( 1991 ); and Hoppe et al ., US Patent Number 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989));
electroporation of 25 cells or embryos (Lo, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993);
introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, "Transgenic Animals,"
30 Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.
Further, the contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety. See also, U.S. Patent No. 5,464,764 (Capecchi, et al., Positive-Negative Selection Methods and Vectors); U.S. Patent No. 5,631,153 (Capecchi, et al., Cells and Non-Human Organisms Containing Predetermined Genomic Modifications and Positive-35 Negative Selection Methods and Vectors for Making Same); U.S. Patent No.
4,736,866 (Leder, et al., Transgenic Non-Human Animals); and U.S. Patent No. 4,873,191 (Wagner, et al., Genetic Transformation of Zygotes); each of which is hereby incorporated by reference in its entirety.

Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each of which is herein incorporated by reference in its entirety).
The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric animals. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Proc. Natl. Acad. Sci.
USA 89:6232-6236 ( 1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
When it is desired that the polynucleotide transgene be integrated into the chromosomal site of ~5 the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene.
The transgene may also be selectively introduced into a particular cell type, thus inactivating the 2o endogenous gene in only that cell type, by following, for example, the teaching of Gu et al.
(Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.
25 Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited 3o to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
Once the founder animals are produced, they may be bred, inbred, outbred, or 35 crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of TR13 polypeptides, studying conditions and/or disorders associated with aberrant TR13 expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of TR14 polypeptides, studying conditions and/or disorders associated with aberrant TR14 expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
In further embodiments of the invention, cells that are genetically engineered to express the proteins of the invention, or alternatively, that are genetically engineered not to express the proteins of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC
compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, etc. The cells are genetically 2o engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of clones, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the 3o circulation, or intraperitoneally. Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. US Patent Number 5,399,349;
and Mulligan & Wilson, US Patent Number 5,460,959, each of which is incorporated by reference herein in its entirety).
When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
TRI3 Polypeptides The TR13 proteins (polypeptides) of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to monomers and multimers of the TRI3 proteins (polypeptides) of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them. In to specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term TR13 homomer, refers to a multimer containing only TR13 proteins of the invention (including TR13 fragments, variants, and fusion proteins, as described herein).
These homomers may contain TR13 proteins having identical or different polypeptide sequences. In a specific embodiment, a homomer of the invention is a multimer containing only TR13 proteins having an identical polypeptide sequence. In another specific embodiment, a homomer of the invention is a multimer containing TR13 proteins having different polypeptide sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing TR13 proteins having identical or different polypeptide sequences) or a homotrimer (e.g., containing TR13 proteins having identical or different polypeptide sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
As used herein, the term TR13 heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing only polypeptide sequences that do not correspond to a polypeptide sequences encoded by the TR13 gene) in addition to the TR13 proteins of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the 3o invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when proteins of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when proteins of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the TR13 proteins of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the protein (e.g., the polypeptide sequence recited in SEQ ID
N0:2 or SEQ ID N0:40 or the polypeptide encoded by the cDNA deposited in ATCC
Deposit No. PTA-349 or ATCC Deposit No. PTA-507. In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide.
In another instance, the covalent associations are the consequence of chemical or recombinant manipulation.
Alternatively, such covalent associations may involve one or more amino acid residues to contained in the heterologous polypeptide sequence in a TR13 fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a TR13-Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequences from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
In another embodiment, two or more TR13 polypeptides of the invention are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference).
Proteins comprising multiple TR13 polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.
Another method for preparing multimer TR13 polypeptides of the invention involves use of TR13 polypeptides fused to a leucine zipper or isoleucine polypeptide sequence.
Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are 3o naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric TR13 proteins are those described in PCT application WO 94/10308, hereby incorporated by reference.
Recombinant fusion proteins comprising a soluble TR13 polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric TR13 is recovered from the culture supernatant using techniques known in the art.
Certain members of the TNF family of proteins are believed to exist in trimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cel173:431, 1993).
Thus, trimeric TR13 may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS
Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference.
Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric TR13.
In further preferred embodiments, TR 13 polynucleotides of the invention are fused to a polynucleotide encoding a "FLAG" polypeptide. Thus, a TR13-FLAG fusion protein is encompassed by the present invention. The FLAG antigenic polypeptide may be fused to a TR13 polypeptide of the invention at either or both the amino or the carboxy terminus. In preferred embodiments, a TR13-to FLAG fusion protein is expressed from a pFLAG-CMV-Sa or a pFLAG-CMV-1 expression vector (available from Sigma, St. Louis, MO, USA). See, Andersson, S., et al., J.
Biol. Chem. 264:8222-29 (1989); Thomsen, D. R., et al., Proc. Natl. Acad. Sci. USA, 81:659-63 (1984); and Kozak, M., Nature 308:241 (1984) (each of which is hereby incorporated by reference). In further preferred embodiments, a TR13-FLAG fusion protein is detectable by anti-FLAG monoclonal antibodies (also available from Sigma).
In another example, proteins of the invention are associated by interactions between Flag~ polypeptide sequence contained in Flag~-TR13 fusion proteins of the invention. In a further embodiment, associated proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag~-TR13 fusion proteins of the invention and anti-Flag~ antibody.
The multimers of the invention may be generated using chemical techniques known in the art. For example, proteins desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Further, proteins of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., US
Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Additionally, techniques known in the art may be applied to generate liposomes containing the protein components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, proteins contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
The polypeptides (proteins) of the present invention are preferably provided in an isolated form. By "isolated polypeptide" is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an "isolated polypeptide" are polypeptides that have been purified, partially or substantially, from a recombinant host cell. For example, a recombinantly produced version of the 2o polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
Accordingly, in one embodiment, the invention provides an isolated TR13 polypeptide having the amino acid sequence encoded by the cDNA deposited in ATCC Deposit No. PTA
349 or ATCC Deposit No. PTA-507, or the amino acid sequence in SEQ ID N0:2 or SEQ ID
N0:40, or a peptide or polypeptide comprising a portion of the above polypeptides.
Polypeptide fragments of the present invention include polypeptides comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID N0:2, encoded by the cDNA contained in the clone deposited as ATCC Deposit No. PTA-349, or encoded by a nucleic acid which hybridizes (e.g., under stringent hybridization conditions) to the nucleotide 3o sequence contained in the deposited clone, or shown in Figures lA-C (SEQ ID
NO:1) or the complementary strand thereto, or polynucleotide fragments thereof (e.g., as disclosed herein).
Protein fragments may be "free-standing," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
Representative examples of polypeptide fragments of the invention, include, for example, fragments that comprise, or alternatively consist of, from about amino acid residues: 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 250, 251 to 300, 301 to 350, 351 to 400, 401 to 450, 451 to 500, 501 to 550, 551 to 600, 601 to 650, 651 to 700, and/or 701 to 750 of SEQ
ID N0:2. Moreover, polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length. In this context "about"
includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
Polypeptide fragments of the present invention include polypeptides comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID
N0:40, encoded by the cDNA contained in the clone deposited as ATCC Deposit No. PTA-507, or encoded by a nucleic acid which hybridizes (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone, or shown in Figures 7A-D (SEQ ID
N0:40) or the l0 complementary strand thereto or polynucleotide fragments thereof (e.g., as disclosed herein).
Protein fragments may be "free-standing," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
Representative examples of polypeptide fragments of the invention, include, for example, fragments that comprise, or alternatively consist of, from about amino acid residues: 1 to 50, ~5 51 to 100, 101 to 150, 151 to 200, 201 to 250, 251 to 300, 301 to 350, 351 to 400, 401 to 450, 451 to 500, 501 to 550, 551 to 600, 601 to 650, 651 to 700, 701 to 750, 751 to 800, 801 to 850, 851 to 900, 901 to 950, and/or 951 to 1001 of SEQ ID N0:2. Moreover, polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 20 1001 amino acids in length. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
Polypeptide fragments of the present invention include polypeptides comprising or alternatively, consisting of from about amino acid residues: 105 to about 170, from 251 to 25 about 265, from 331 to about 410, from 580 to about 610, from 139 to about 142, from 140 to about 143, from 153 to about 156, from 293 to about 296, from 325 to about 328, from 421 to about 424, from 466 to about 469, from 696 to about 699, from 728 to about 731, from 312 to about 315, from 454 to about 461, from 458 to about 461, from 50 to about 53, from 66 to about 69, from 80 to about 83, from 276 to about 279, from 311 to about 314, from 438 to 30 about 441, from 559 to about 562, from 564 to about 567, from 698 to about 701, from 725 to about 728, from 80 to about 83, from 89 to about 92, from 180 to about 183, from 198 to about 201, from 214 to about 217, from 272 to about 275, from 306 to about 309, from 510 to about 513, from 529 to about 532, from 584 to about 867, from 609 to about 612, from 642 to about 645, from 698 to about 701, from 69 to about 74, from 149 to about 154, from 154 to 35 about 159, from 163 to about 168, from 212 to about 217, from 248 to about 253, from 365 to about 370, from 383 to about 388, from 393 to about 398, from 588 to about 593, from 623 to about 628, from 661 to about 666, from 665 to about 670, and/or 456 to about 459 of SEQ ID
N0:2. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1 ) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
Polypeptide fragments of the present invention include polypeptides comprising or alternatively, consisting of from about amino acid residues: 42 to about 906, from 42 to about 1001, from 906 to 'about 931, from 932 to about 1001, from 271 to about 421, from 271 to about 286, from 290 to about 300, from 301 to about 320, from 329 to about 361, from 404 to about 421, from 585 to about 595, from 661 to about 674, from 710 to about 744, from 980 to about 991, from 45 to about 60, from 121 to about 135, from 145 to about 160, from I to about 262, from 264 to about 423, from 437 to about 789, from 791 to about 1001, from 310 to about 363, from 477 to about 519, from 769 to about 887, from 153 to about 156, from 11 to about 13, from about 18 to about 20, from 107 to about 109, from about 156 to about 158, from about 224 to about 226, from about 301 to about 303, from about 317 to about 319, from about 331 to about 333, from about 527 to about 529, from about 562 to about 564, from about 689 to about 691, from about 810 to about 812, from about 815 to about 817, from about 949 to about 951, from about 976 to about 978, from 42 to about 45, from about 59 to about 62, from about 81 to about 84, from about 146 to about 149, from about 282 to about 285, from about 331 to about 334, from about 340 to about 343, from about 431 to about 434, from about 449 to about 452, from about 465 to about 468, from about 523 to about 526, from about 557 to about 560, from about 761 to about 764, from about 780 to about 783, from about 780 to about 783, from about 835 to about 838, from about 860 to about 863, from about 893 to about 896, from about 949 to about 952, from from about 77 to about 82, from about 88 to about 93, from about 152 to about 157, from about 268 to about 273, from about 288 to about 293, from about 320 to about 325, from about 400 to about 405, from about 414 to about 419, from about 463 to about 468, from about 599 to about 604, from about 616 to about 621, from about 634 to about 639, from about 644 to about 649, from about 839 to about 844, from about 874 to about 879, from about 912 to about 917, from about 916 to about 921, from from about 50 to about 56, from from about 109 to about 116, from from about 153 to about 156, from 390 to about 393, from 391 to about 394, from about 404 to about 407, from about 544 to about 547, from about 576 to about 579, from about 672 to 3o about 675, from about 717 to about 720, from about 947 to about 950, from and about 979 to about 982 of SEQ ID N0:40. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
In additional embodiments, the polypeptide fragments of the invention comprise, or alternatively consist of, one or more domains of the TR13 polypeptide disclosed in Figures 1A
C. Preferred polypeptide fragments of the present invention include a member selected from the group: (a) a polypeptide comprising or alternatively, consisting of, any combination of one, two, three, or all four of the TR13 cysteine rich domains disclosed in Figures lA-C (predicted to constitute amino acid residues from about 105 to about 170, about 251 to about 265, about 331 to about 410, and about 580 to about 610 of SEQ ID N0:2); (b) a polypeptide comprising, or alternatively, consisting of, one, two, three, four or more, epitope bearing portions of the TR13 receptor protein disclosed in Figures lA-C (for example, those epitope bearing portions predicted to constitute amino acid residues from about 1 to about 170, or about 210 to about 318, or about 343 to about 480, or about 548 to about 592, or about 632 to about 742 of SEQ
ID N0:2); (c) any combination of polypeptides (a)-(c). Polynucleotides encoding these polypeptides are also encompassed by the invention. In this context "about"
includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1 ) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptide fragments are also to encompassed by the invention.
In additional embodiments, the polypeptide fragments of the invention comprise, or alternatively consist of, one or more domains of the TR13 polypeptide disclosed in Figure 7A-D. Preferred polypeptide fragments of the present invention include a member selected from the group: (a) a polypeptide comprising, or alternatively consisting of, amino acids 1 to about 41 of SEQ ID N0:40; (b) a polypeptide comprising, or alternatively consisting of, amino acids 42 to about 906 of SEQ ID N0:40; (c) a polypeptide comprising, or alternatively consisting of, amino acids 907 to about 931 of SEQ ID N0:40; (d) a polypeptide comprising, or alternatively consisting of, amino acids 932 to about 1001 of SEQ ID N0:40; (e) a polypeptide comprising or alternatively, consisting of, any combination of one, two, three, four or more of the TR13 2o cysteine rich domains disclosed in Figures 7A-D (predicted to constitute amino acid residues from about 271 to about 421, 271 to about 286, about 290 to about 300, about 301 to about 320, about 329 to about 361, about 404 to about 421, about 585 to about 595 of SEQ ID
N0:40); (f) a polypeptide comprising, or alternatively, consisting of, one, two, three, four or more, epitope bearing portions of the TR13 receptor protein disclosed in Figures 7A-D (for example, these epitope bearing portions predicted to constitute amino acid residues from about 1 to about 262, or about 264 to about 423, or about 437 to about 789, or about 791 to about 1001, of SEQ ID N0:40); and (g) any combination of polypeptides (a)-(f). In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these 3o polypeptides are also encompassed by the invention.
As discussed above, it is believed that the extracellular cysteine rich motifs of TR13 are important for interactions between TR13 and its ligands. Accordingly, in preferred embodiments, polypeptide fragments of the invention comprise, or alternatively consist of amino acid residues from about 105 to about 170, about 251 to about 265, about 331 to about 410 and/or about 580 to about 610 of the amino acid sequence disclosed in Figures lA-C (SEQ
ID N0:2). In a specific embodiment the polypeptides of the invention comprise, or alternatively consist of any combination of one, two, three or all four extracellular cysteine rich motifs disclosed in Figures lA-C. In this context "about" includes the particularly recited ranges, larger or smaller by several (S, 4, 3, 2, or 1 ) amino acids, at either extreme or at both extremes.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
As discussed above, it is believed that the extracellular cysteine rich motifs of TR13 are important for interactions between TR13 and its ligands. Accordingly, in preferred embodiments, polypeptide fragments of the invention comprise, or alternatively consist of amino acid residues from about 271 to about 421, or 271 to about 286, or about 290 to about 300, or about 301 to about 320, or about 329 to about 361, or about 404 to about 421, or about 585 to about 595 of the amino acid sequence disclosed in Figures 7A-D (SEQ ID
N0:40). In a specific embodiment the polypeptides of the invention comprise, or alternatively consist of any l0 combination of one, two, three, four or more of the extracellular cysteine rich motifs disclosed in Figures 7A-D. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
Among the especially preferred fragments of the invention are fragments characterized by structural or functional attributes of TR13 (SEQ ID N0:2 or SEQ ID N0:40).
Such fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic 2o regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) of complete (i.e., full-length) TR13 (SEQ ID N0:2 or SEQ ID N0:40). Certain preferred regions are those set out in Figure 3 (Table I) and Figure 9 (Table III) and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in Figures lA-C (SEQ ID N0:2) or Figures 7A-D (SEQ ID N0:40), respectively. Such preferred regions include; Gamier-Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions;
Chou-Fasman predicted alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle predicted hydrophilic and Hopp-Woods predicted hydrophobic regions; Eisenberg alpha and beta 3o amphipathic regions; Emini surface-forming regions; and Jameson-Wolf high antigenic index regions, as predicted using the default parameters of these computer programs.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
As mentioned above, even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind TR13 ligand) may still be retained. For example, the ability of shortened TR13 muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
It is not unlikely that an TR13 mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR13 amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TR13 amino acid sequence shown in Figures lA-C, up to the aspartic acid residue at position number 745 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n'-750 of Figures lA-C, where n' is an integer from 2 to 745 corresponding to the position of the amino acid residue in Figures lA-C (which is identical to the sequence shown as SEQ ID N0:2). In a specific embodiment, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n'-750 of Figures lA-C, where n' is an integer from 2 to 610 corresponding to the position of the amino acid residue in Figures lA-C.
Polynucleotides encoding these polypeptides are also encompassed.
In one embodiment, N-terminal deletions of the TR13 polypeptides of the invention can be described by the general formula n2-750, where n2 is a number from 2 to 745, corresponding to the position of amino acid identified in Figures lA-C (SEQ ID
N0:2). N-terminal deletions of the TR13 polypeptide of the invention shown as SEQ ID
N0:2 include polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: D-2 to R-750; Q-3 to R-750; S-4 to R-750; T-5 to R-750; Q-6 to R-750; A-7 to R-750; C-8 to 8-750; A-9 to R-750; G-10 to R-750; E-11 to R-750; K-12 to R-750; H-13 to R-750;
C-14 to R-750; H-15 to R-750; N-16 to R-750; R-17 to R-750; G-18 to R-750; G-19 to R-750; L-20 to R-750; H-21 to R-750; F-22 to R-750; R-23 to R-750; M-24 to R-750; L-25 to R-750; P-26 to R-750; L-27 to R-750; Q-28 to R-750; T-29 to R-750; W-30 to R-750; H-31 to R-750; V-32 to R-750; C-33 to R-750; R-34 to R-750; Q-35 to R-750; A-36 to R-750; G-37 to R-750; L-38 to R-750; L-39 to R-750; F-40 to R-750; L-41 to R-750; Q-42 to R-750; T-43 to R-750; L-44 to 3o R-750; P-45 to R-750; S-46 to R-750; N-47 to R-750; S-48 to R-750; Y-49 to R-750; S-50 to R-750; N-51 to R-750; K-52 to R-750; G-53 to R-750; E-54 to R-750; T-55 to R-750; S-56 to R-750; C-57 to R-750; H-58 to R-750; Q-59 to R-750; C-60 to R-750; D-61 to R-750; P-62 to R-750; D-63 to R-750; K-64 to R-750; Y-65 to R-750; S-66 to R-750; E-67 to R-750; K-68 to R-750; G-69 to R-750; S-70 to R-750; S-71 to R-750; S-72 to R-750; C-73 to R-750; N-74 to R-750; V-75 to R-750; R-76 to R-750; P-77 to R-750; A-78 to R-750; C-79 to R-750; T-80 to R-750; D-81 to R-750; K-82 to R-750; D-83 to R-750; Y-84 to R-750; F-85 to R-750; Y-86 to R-750; T-87 to R-750; H-88 to R-750; T-89 to R-750; A-90 to R-750; C-91 to R-750; D-92 to R-750; A-93 to R-750; N-94 to R-750; G-95 to R-750; E-96 to R-750; T-97 to R-750; Q-98 to R-750; L-99 to R-750; M-100 to R-750; Y-101 to R-750; K-102 to R-750; W-103 to R-750;
A-104 to R-750; K-105 to R-750; P-106 to R-750; K-107 to R-750; I-108 to R-750; C-109 to R-750; S-110 to R-750; E-111 to R-750; D-112 to R-750; L-113 to R-750; E-114 to R-750; 6-115 to R-750; A-116 to R-750; V-117 to R-750; K-118 to R-750; L-119 to R-750;
P-120 to R-750; A-121 to R-750; S-122 to R-750; G-123 to R-750; V-124 to R-750; K-125 to R-750; T-126 to R-750; H-127 to R-750; C-128 to R-750; P-129 to R-750; P-130 to R-750;
C-131 to R-750; N-132 to R-750; P-133 to R-750; G-134 to R-750; F-135 to R-750; F-136 to R-750; K-137 to R-750; T-138 to R-750; N-139 to R-750; N-140 to R-750; S-141 to R-750;
T-142 to 8-750; C-143 to R-750; Q-144 to R-750; P-145 to R-750; C-146 to R-750; P-147 to R-750; Y-148 to R-750; G-149 to R-750; S-150 to R-750; Y-151 to R-750; S-152 to R-750;
N-153 to R-750; G-154 to R-750; S-155 to R-750; D-156 to R-750; C-157 to R-750; T-158 to R-750; 8-159 to R-750; C-160 to R-750; P-161 to R-750; A-162 to R-750; G-163 to R-750;
T-164 to R-750; E-165 to R-750; P-166 to R-750; A-167 to R-750; V-168 to R-750; G-169 to R-750; F-170 to R-750; E-171 to R-750; Y-172 to R-750; K-173 to R-750; W-174 to R-750;
W-175 to R-750; N-176 to R-750; T-177 to R-750; L-178 to R-750; P-179 to R-750; T-180 to R-750; N-181 to R-750; M-182 to R-750; E-183 to R-750; T-184 to R-750; T-185 to R-750;
V-186 to R-750; L-187 to R-750; S-188 to R-750; G-189 to R-750; I-190 to R-750; N-191 to R-750; F-192 to R-750; E-193 to R-750; Y-194 to R-750; K-195 to R-750; G-196 to R-750;
M-197 to R-750; T-198 to R-750; G-199 to R-750; W-200 to R-750; E-201 to R-750; V-202 to R-750;
2o A-203 to R-750; G-204 to R-750; D-205 to R-750; H-206 to R-750; I-207 to R-750; Y-208 to R-750; T-209 to R-750; A-210 to R-750; A-211 to R-750; G-212 to R-750; A-213 to R-750;
S-214 to R-750; D-215 to R-750; N-216 to R-750; D-217 to R-750; F-218 to R-750; M-219 to R-750; I-220 to R-750; L-221 to R-750; T-222 to R-750; L-223 to R-750; V-224 to R-750; V-225 to R-750; P-226 to R-750; G-227 to R-750; F-228 to R-750; R-229 to R-750;
P-230 to R-750; P-231 to R-750; Q-232 to R-750; S-233 to R-750; V-234 to R-750; M-235 to R-750; A-236 to R-750; D-237 to R-750; T-238 to R-750; E-239 to R-750; N-240 to R-750;
K-241 to R-750; E-242 to R-750; V-243 to R-750; A-244 to R-750; R-245 to R-750; I-246 to R-750; T-247 to R-750; F-248 to R-750; V-249 to R-750; F-250 to R-750; E-251 to R-750;
T-252 to 8-750; L-253 to R-750; C-254 to R-750; S-255 to R-750; V-256 to R-750; N-257 to R-750; C-258 to R-750; E-259 to R-750; L-260 to R-750; Y-261 to R-750; F-262 to R-750;
M-263 to R-750; V-264 to R-750; G-265 to R-750; V-266 to R-750; N-267 to R-750; S-268 to R-750; R-269 to R-750; T-270 to R-750; N-271 to R-750; T-272 to R-750; P-273 to R-750;
V-274 to 8-750; E-275 to R-750; T-276 to R-750; W-277 to R-750; K-278 to R-750; G-279 to R-750; S-280 to R-750; K-281 to R-750; G-282 to R-750; K-283 to R-750; Q-284 to R-750;
S-285 to R-750; Y-286 to R-750; T-287 to R-750; Y-288 to R-750; I-289 to R-750; I-290 to R-750; E-291 to R-750; E-292 to R-750; N-293 to R-750; T-294 to R-750; T-295 to R-750;
T-296 to 8-750; S-297 to R-750; F-298 to R-750; T-299 to R-750; W-300 to R-750; A-301 to R-750; F-302 to R-750; Q-303 to R-750; R-304 to R-750; T-305 to R-750; T-306 to R-750;
F-307 to R-750; H-308 to R-750; E-309 to R-750; A-310 to R-750; S-311 to R-750; R-312 to R-750; K-313 to R-750; Y-314 to R-750; T-315 to R-750; N-316 to R-750; D-317 to R-750;
V-318 to 8-750; A-319 to R-750; K-320 to R-750; I-321 to R-750; Y-322 to R-750; S-323 to R-750; I-324 to R-750; N-325 to R-750; V-326 to R-750; T-327 to R-750; N-328 to R-750; V-329 to R-750;
M-330 to R-750; N-331 to R-750; G-332 to R-750; V-333 to R-750; A-334 to R-750; S-335 to R-750; Y-336 to R-750; C-337 to R-750; R-338 to R-750; P-339 to R-750; C-340 to R-750;
A-341 to R-750; L-342 to R-750; E-343 to R-750; A-344 to R-750; S-345 to R-750; D-346 to R-750; V-347 to R-750; G-348 to R-750; S-349 to R-750; S-350 to R-750; C-351 to R-750; T-352 to R-750; S-353 to R-750; C-354 to R-750; P-355 to R-750; A-356 to R-750;
G-357 to R-l0 750; Y-358 to R-750; Y-359 to R-750; I-360 to R-750; D-361 to R-750; R-362 to R-750; D-363 to R-750; S-364 to R-750; G-365 to R-750; T-366 to R-750; C-367 to R-750;
H-368 to R-750; S-369 to R-750; C-370 to R-750; P-371 to R-750; P-372 to R-750; N-373 to R-750; T-374 to R-750; I-375 to R-750; L-376 to R-750; K-377 to R-750; A-378 to R-750;
H-379 to 8-750; Q-380 to R-750; P-381 to R-750; Y-382 to R-750; G-383 to R-750; V-384 to R-750; Q-~5 385 to R-750; A-386 to R-750; C-387 to R-750; V-388 to R-750; P-389 to R-750; C-390 to R-750; G-391 to R-750; P-392 to R-750; G-393 to R-750; T-394 to R-750; K-395 to R-750; N-396 to R-750; N-397 to R-750; K-398 to R-750; I-399 to R-750; H-400 to R-750;
S-401 to R-750; L-402 to R-750; C-403 to R-750; Y-404 to R-750; N-405 to R-750; D-406 to R-750; C-407 to R-750; T-408 to R-750; F-409 to R-750; S-410 to R-750; R-411 to R-750;
N-412 to R-20 750; T-413 to R-750; P-414 to R-750; T-415 to R-750; R-416 to R-750; T-417 to R-750; F-418 to R-750; N-419 to R-750; Y-420 to R-750; N-421 to R-750; F-422 to R-750;
S-423 to 8-750; A-424 to R-750; L-425 to R-750; A-426 to R-750; N-427 to R-750; T-428 to R-750; V-429 to R-750; T-430 to R-750; L-431 to R-750; A-432 to R-750; G-433 to R-750;
G-434 to 8-750; P-435 to R-750; S-436 to R-750; F-437 to R-750; T-438 to R-750; S-439 to R-750; K-25 440 to R-750; G-441 to R-750; L-442 to R-750; K-443 to R-750; Y-444 to R-750; F-445 to 8-750; H-446 to R-750; H-447 to R-750; F-448 to R-750; T-449 to R-750; L-450 to R-750; S-451 to R-750; L-452 to R-750; C-453 to R-750; G-454 to R-750; N-455 to R-750;
Q-456 to 8-750; G-457 to R-750; R-458 to R-750; K-459 to R-750; M-460 to R-750; S-461 to R-750; V-462 to R-750; C-463 to R-750; T-464 to R-750; D-465 to R-750; N-466 to R-750;
V-467 to R-30 750; T-468 to R-750; D-469 to R-750; L-470 to R-750; R-471 to R-750; I-472 to R-750; P-473 to R-750; E-474 to R-750; G-475 to R-750; E-476 to R-750; S-477 to R-750;
G-478 to 8-750; F-479 to R-750; S-480 to R-750; K-481 to R-750; S-482 to R-750; I-483 to R-750; T-484 to R-750; A-485 to R-750; Y-486 to R-750; V-487 to R-750; C-488 to R-750;
Q-489 to 8-750; A-490 to R-750; V-491 to R-750; I-492 to R-750; I-493 to R-750; P-494 to R-750; P-495 35 to R-750; E-496 to R-750; V-497 to R-750; T-498 to R-750; G-499 to R-750; Y-500 to R-750;
K-501 to R-750; A-502 to R-750; G-503 to R-750; V-504 to R-750; S-505 to R-750; S-506 to R-750; Q-507 to R-750; P-508 to R-750; V-509 to R-750; S-510 to R-750; L-511 to R-750; A-512 to R-750; D-513 to R-750; R-514 to R-750; L-515 to R-750; I-516 to R-750;
G-517 to R-750; V-518 to R-750; T-519 to R-750; T-520 to R-750; D-521 to R-750; M-522 to R-750; T-523 to R-750; L-524 to R-750; D-525 to R-750; G-526 to R-750; I-527 to R-750;
T-528 to 8-750; S-529 to R-750; P-530 to R-750; A-531 to R-750; E-532 to R-750; L-533 to R-750; F-534 to R-750; H-535 to R-750; L-536 to R-750; E-537 to R-750; S-538 to R-750;
L-539 to R-750; G-540 to R-750; I-541 to R-750; P-542 to R-750; D-543 to R-750; V-544 to R-750; I-545 to R-750; F-546 to R-750; F-547 to R-750; Y-548 to R-750; R-549 to R-750; S-550 to R-750;
N-551 to R-750; D-552 to R-750; V-553 to R-750; T-554 to R-750; Q-555 to R-750; S-556 to R-750; C-557 to R-750; S-558 to R-750; S-559 to R-750; G-560 to R-750; R-561 to R-750; S-562 to R-750; T-563 to R-750; T-564 to R-750; I-565 to R-750; R-566 to R-750;
V-567 to R-l 0 750; R-568 to R-750; C-569 to R-750; S-570 to R-750; P-571 to R-750; Q-572 to R-750; K-573 to R-750; T-574 to R-750; V-575 to R-750; P-576 to R-750; G-577 to R-750;
S-578 to 8-750; L-579 to R-750; L-580 to R-750; L-581 to R-750; P-582 to R-750; G-583 to R-750; T-584 to R-750; C-585 to R-750; S-586 to R-750; D-587 to R-750; G-588 to R-750;
T-589 to 8-750; C-590 to R-750; D-591 to R-750; G-592 to R-750; C-593 to R-750; N-594 to R-750; F-595 to R-750; H-596 to R-750; F-597 to R-750; L-598 to R-750; W-599 to R-750;
E-600 to R-750; S-601 to R-750; A-602 to R-750; A-603 to R-750; A-604 to R-750; C-605 to R-750; P-606 to R-750; L-607 to R-750; C-608 to R-750; S-609 to R-750; V-610 to R-750;
A-611 to 8-750; D-612 to R-750; Y-613 to R-750; H-614 to R-750; A-615 to R-750; I-616 to R-750; V-617 to R-750; S-618 to R-750; S-619 to R-750; C-620 to R-750; V-621 to R-750;
A-622 to R-750; G-623 to R-750; I-624 to R-750; Q-625 to R-750; K-626 to R-750; T-627 to R-750; T-628 to R-750; Y-629 to R-750; V-630 to R-750; W-631 to R-750; R-632 to R-750;
E-633 to R-750; P-634 to R-750; K-635 to R-750; L-636 to R-750; C-637 to R-750; S-638 to R-750;
G-639 to R-750; G-640 to R-750; I-641 to R-750; S-642 to R-750; L-643 to R-750; P-644 to R-750; E-645 to R-750; Q-64b to R-750; R-647 to R-750; V-648 to R-750; T-649 to R-750; I-650 to R-750; C-651 to R-750; K-652 to R-750; T-653 to R-750; I-654 to R-750;
D-655 to 8-750; F-656 to R-750; W-657 to R-750; L-658 to R-750; K-659 to R-750; V-660 to R-750; G-661 to R-750; I-662 to R-750; S-663 to R-750; A-664 to R-750; G-665 to R-750;
T-666 to 8-750; C-667 to R-750; T-668 to R-750; A-669 to R-750; I-670 to R-750; L-671 to R-750; L-672 to R-750; T-673 to R-750; V-674 to R-750; L-675 to R-750; T-676 to R-750;
C-677 to R-750; Y-678 to R-750; F-679 to R-750; W-680 to R-750; K-681 to R-750; K-682 to R-750; N-683 to R-750; Q-684 to R-750; K-685 to R-750; L-686 to R-750; E-687 to R-750;
Y-688 to 8-750; K-689 to R-750; Y-690 to R-750; S-691 to R-750; K-692 to R-750; L-693 to R-750; V-694 to R-750; M-695 to R-750; N-696 to R-750; A-697 to R-750; T-698 to R-750;
L-699 to 8-750; K-700 to R-750; D-701 to R-750; C-702 to R-750; D-703 to R-750; L-704 to R-750; P-705 to R-750; A-706 to R-750; A-707 to R-750; D-708 to R-750; S-709 to R-750;
C-710 to R-750; A-711 to R-750; I-712 to R-750; M-713 to R-750; E-714 to R-750; G-715 to R-750; E-716 to R-750; D-717 to R-750; V-718 to R-750; E-719 to R-750; D-720 to R-750;
D-721 to 8-750; L-722 to R-750; I-723 to R-750; F-724 to R-750; T-725 to R-750; S-726 to R-750; K-727 to R-750; N-728 to R-750; H-729 to R-750; S-730 to R-750; L-731 to R-750;
G-732 to 8-750; R-733 to R-750; S-734 to R-750; N-735 to R-750; H-736 to R-750; L-737 to R-750; P-738 to R-750; P-739 to R-750; R-740 to R-750; G-741 to R-750; L-742 to R-750;
L-743 to 8-750; M-744 to R-750; D-745 to R-750; of SEQ ID N0:2. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TR13 amino acid sequence shown in Figures 7A-D, up to the aspartic acid residue at position number 996 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or 1o alternatively consisting of, the amino acid sequence of residues n'-1001 of Figures 7A-D, . where n' is an integer from 2 to 996 corresponding to the position of the amino acid residue in Figures 7A-C (which is identical to the sequence shown as SEQ ID N0:40). In a specific embodiment, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n'-906 of Figures 7A-D
where n' is an integer from 42 to 595 corresponding to the position of the amino acid residue in Figures 7A
D. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In another embodiment, N-terminal deletions of the TR13 polypeptide can be described by the general formula n2-1001, where n2 is a number from 2 to 996, corresponding to the position of amino acid identified in Figures 7A-D (SEQ ID N0:40). N-terminal deletions of the 2o TR13 polypeptide of the invention shown as SEQ ID N0:40 include polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: A-2 to R-1001; E-3 to 8-1001; P-4 to R-1001; G-5 to R-1001; H-6 toR-1001; S-7 to R-1001; H-8 to R-1001; H-9 to R-1001; L-10 to R-1001; S-11 to R-1001; A-12 to R-1001; R-13 to R-1001;V-14 to R-1001; 8-15 to R-1001; G-16 to R-1001; R-17 to R-1001; T-18 to R-1001; E-19 to R-1001;
R-20 to 8-1001;8-21 to R-1001; I-22 to R-1001; P-23 to R-1001; R-24 to R-1001; L-25 to R-1001; W-26 to R-1001; R-27 to R-1001; L-28to R-1001; L-29 to R-1001; L-3 0 to R-1001;
W-31 to 8-1001; A-32 to R-1001; G-33 to R-1001; T-34 to R-1001; A-35 toR-1001; F-36 to R-1001; Q-37 to R-1001; V -3 8 to R-1001; T-39 to R-1001; Q-40 to R-1001; G -41 to R-1001; T-42 toR-1001; G-43 to R-1001; P-44 to R-1001; E-45 to R-1001; L-46 to R-1001; H-47 to R-1001; A-48 to R-1001; C-49 toR-1001; K-50 to R-1001; E-51 to R-1001; S-52 to R-1001; E-53 to 8-1001; Y-54 to R-1001; H-55 to R-1001; Y-56 toR-1001; E-57 to R-1001; Y-58 to R-1001; T-59 to R-1001; A-60 to R-1001; C-61 to R-1001; D-62 to R-1001; S-63 toR-1001; T-64 to R-1001; G-65 to R-1001; S-66 to R-1001; R-67 to R-1001; W-68 to R-1001; R-69 to R-1001; V-70 to R-1001; A-71 to R-1001; V -72 to R-1001; P-73 to R-1001; H-74 to R-1001;
T-75 to 8-1001; P-76 to R-1001; G-77 toR-1001; L-78 to R-1001; C-79 to R-1001; T-80 to R-1001; S-81 to R-1001; L-82 to R-1001; P-83 to R-1001; D-84 toR-1001; P-85 to R-1001; V-86 to R-1001; K-87 to R-1001; G-8 8 to R-1001; T-89 to R-1001; E-90 to R-1001; C-91 toR-1001; S -92 to R-1001; F-93 to R-1001; S-94 to R-1001; C-95 to R-1001; N-96 to R-1001;
A-97 to R-I 001; G-98 toR-1001; E-99 to R-1001; F-100 to R-1001; L-1 O l to R-100 I ; D-102 to R-1001;
M-103 to R-1001; K-104 to R-1001; D-1 OS to R-1001; Q-106 to R-1001; S-107 to R- I 001; C-108 to R-1001; K-109 to R-1001; P-110 to R-1001; C-111 to R- l 001;A-112 to R-1001; E-113 to R-1001; G-114 to R-1001; R-115 to R-1001; Y-116 to R-1001; S-117 to R-1001;

toR-1001; G-119 to R-1001; T-120 to R-1001; G-121 to R-1001; I-122 to R-1001;
R-123 to R-1001; F-124 to R- l 001;D- I 25 to R-1001; E-126 to R-1001; W-127 to R-1001;
D-128 to R-1001; E-129 to R-1001; L-130 to R-1001; P-131 toR-1001; H-132 to R-1001; G-133 to R-1001; F-134 to R-1001; A-135 to R-1001; S-136 to R-1001; L-137 to R-1001;5-138 to R-1001; A-139 to R-1001; N-140 to R-1001; M-141 to R-1001; E-142 to R-1001; L-143 to 8-1001; D-144 toR-1001; D-145 to R-1001; S-146 to R-1001; A-147 to R-1001; A-148 to R-1001; E-149 to R-1001; S-150 to R-1001sT-151 to R-1001; G-152 to R-1001; N-153 to R-1001; C-154 to R-1001; T-155 to R-1001; S-156 to R-1001; S-157 toR-1001; K-158 to 8-1001; W-159 to R-1001; V-160 to R-1001; P-161 to R-1001; R-162 to R-1001; G-163 to R-1001;D-164 to R-1001; Y-165 to R-1001; I-166 to R-1001; A-167 to R-1001; F-168 to R-1001; N-169 to R-1001; T-170 toR-1001; D-171 to R-1001; E-172 to R-1001; C-173 to 8-1001; T-174 to R-1001; A-175 to R-1001; T-176 to R-1001;L-177 to R-1001; M-178 to R-1001; Y-179 to R-1001; A-180 to R-1001; V-181 to R-1001; N-182 to R-1001; L-183 toR-1001; K-184 to R- I 001; Q-185 to R-1001; S-186 to R-1001; G-187 to R-1001; T-18 8 to 8-1001; V -189 to R-1001; N-190 to R-1001; F-191 to R-1001; E-192 to R-1001; Y-193 to R-1001; Y-194 to R-1001; Y-195 to R- I 001; P- I 96 toR-1001; D-197 to R-1001; S-198 to R-I 001; S-199 to R-1001; I-200 to R-1001; I-201 to R-1001; F-202 to R-100 I ; E-203 to R-1001;
F-204 to R-1001; F-205 to R-1001; V -206 to R-1001; Q-207 to R-1001; N-208 to R-1001; D-209 to R- l 001;Q-210 to R-1001; C-211 to R-1001; Q-212 to R-1001; P-213 to R-1001; N-214 to R-1001; A-215 to R-1001; D-216 toR-1001; D-217 to R-1001; S-218 to R-1001;
R-219 to R-1001; W-220 to R-1001; M-221 to R-1001; K-222 to R- l 001sT-223 to R-1001; T-224 to R-1001; E-225 to R-1001; K-226 to R-1001; G-227 to R-1001; W-228 to R-1001; E-229 toR-1001; F-230 to R-1001; H-231 to R-1001; S-232 to R-1001; V-233 to R-1001; E-234 to R-1001; L-235 to R-1001;N-236 to R-1001; R-237 to R-1001; G-238 to R-1001; N-239 to R-1001; N-240 to R-1001; V -241 to R-1001; L-242 toR-1001; Y-243 to R-1001; W-244 to 8-1001; R-245 to R-1001; T-246 to R-1001; T-247 to R-1001; A-248 to R- l 001;F-249 to 8-1001; S-250 to R-1001; V-251 to R-1001; W-252 to R-1001; T-253 to R-1001; K-254 to R-1001; V-255 toR-1001; P-256 to R-1001; K-257 to R-1001; P-258 to R-1001; V-259 to R-1001; L-260 to R-1001; V -261 to R-1001;8-262 to R-1001; N-263 to R-1001; I-264 to R-1001; A-265 to R-1001; I-266 to R-1001; T-267 to R-1001; G-268 toR-1001; V-269 to 8-1001; A-270 to R-1001; Y-271 to R-1001; T-272 to R-1001; S-273 to R-1001; E-274 to 8-1001; C-275 to R-1001; F-276 to R- I 001; P-277 to R-1001; C-278 to R-1001; K-279 to R-1001; P-280 to R-1001; G-281 toR-1001; T-282 to R-1001; Y-283 to R-1001; A-284 to 8-1001; D-285 to R-1001; K-286 to R-1001; Q-287 to R-1001;6-288 to R-1001; S-289 to R-1001; S-290 to R-1001; F-291 to R-1001; C-292 to R-1001; K-293 to R-1001; L-294 toR-1001; C-295 to R-1001; P-296 to R-1001; A-297 to R-1001; N-298 to R-1001; S-299 to R-1001; Y-300 to R-1001;S-301 to R-1001; N-302 to R-1001; K-303 to R-1001; G-304 to R-1001; E-305 to R-1001; T-306 to R-1001; S-307 toR-1001; C-308 to R-1001; H-309 to R-1001; Q-310 to R-1001; C-311 to R-1001; D-312 to R-100 I ; P-313 to R-1001; D-314 to 8-1001; K-315 to R-1001; Y-316 to R-1001; S-317 to R-1001; E-318 to R-1001; K-319 to 8-1001; G-320 toR-1001; S-321 to R-1001; S-322 to R-1001; S-323 to R-1001; C-324 to R-1001; N-325 to R-1001; V-326 to R-1001;8-327 to R-1001; P-328 to R-1001; A-329 to R-1001; C-330 to R-1001; T-331 to R-1001; D-332 to R-1001; K-333 toR-1001; D-334 to R-1001; Y-335 to R-1001; F-336 to R-1001; Y-337 to R-1001; T-338 to R-1001; H-339 to R-l 001sT-340 to R-1001; A-341 to R-1001; C-342 to R-1001; D-343 to R-1001; A-344 to R-1001; N-345 to R-1001; G-346 toR-1001; E-347 to R-1001; T-348 to R-1001; Q-349 to 8-1001; L-350 to R-1001; M-351 to R-1001; Y-352 to R-1001;x-353 to R-1001; W-354 to 8-1001; A-355 to R-1001; K-356 to R-1001; P-357 to R-1001; K-358 to R-1001; I-359 toR-1001; C-360 to R-1001; S-361 to R-1001; E-362 to R-1001; D-363 to R-1001; L-364 to 8-1001; E-365 to R-1001;6-366 to R-1001; A-367 to R-1001; V-368 to R-1001; K-369 to R-1001; L-370 to R-1001; P-371 to R-1001; A-372 toR-1001; S-373 to R-1001; G-374 to R-1001; V-375 to R-1001; K-376 to R-1001; T-377 to R-1001; H-378 to R-1001;C-379 to 8-1001; P-380 to R-1001; P-381 to R-1001; C-382 to R-1001; N-383 to R-1001; P-384 to R-1001; G-385 toR-1001; F-386 to R-1001; F-387 to R-1001; K-388 to R-1001; T-389 to 8-1001; N-390 to R-1001; N-391 to R-1001;S-392 to R-1001; T-393 to R-1001; C-394 to 8-1001; Q-395 to R-1001; P-396 to R-1001; C-397 to R-1001; P-398 toR-1001; Y-399 to 8-1001; G-400 to R-1001; S-401 to R-1001; Y-402 to R-1001; S-403 to R-1001; N-404 to 8-1001; G-405 to R-1001; S-406 to R-1001; D-407 to R-1001; C-408 to R-1001; T-409 to 8-1001; R-410 to R-1001; C-411 toR-1001; P-412 to R-1001; A-413 to R-1001; G-414 to R-1001; T-415 to R-1001; E-416 to R-1001; P-417 to R- l 001;A-418 to R-1001; V -419 to 8-1001; G-420 to R- I 001; F-421 to R-1001; E-422 to R-1001; Y -423 to R-1001; K-424 toR-1001; W-425 to R-1001; W-426 to R-1001; N-427 to R-1001; T-428 to R-1001; L-429 to R-1001; P-430 to R- l 001sT-431 to R-1001; N-432 to R-1001; M-433 to R-1001; E-434 to R-1001; T-435 to R-1001; T-436 to R-1001; V-437 toR-1001; L-438 to R-1001; S-439 to 8-1001; G-440 to R-1001; I-441 to R-1001; N-442 to R-1001; F-443 to R-1001; E-444to 8-1001; Y-445 to R-1001; K-446 to R-1001; G-447 to R-1001; M-448 to R-1001; T-449 to R-1001; G-450 to R-1001; W-451 to R-1001; E-452 to R- I 001; V -45 3 to R-1001;
A-454 to R-1001; G-455 to R-I 001; D-456 to R-1001; H-457 toR-1001; I-458 to R-1001; Y-459 to 8-1001; T-460 to R-1001; A-461 to R-1001; A-462 to R-1001; G-4.63 to R-1001;A-464 to 8-1001; S-4.65 to R-1001; D-466 to R-1001; N-467 to R-1001; D-4.68 to R-1001; F-469 to 8-1001; M-470 toR-1001; I-471 to R-1001; L-472 to R-1001; T-473 to R-1001; L-474 to R-1001; V -475 to R-1001; V -476 to R-1001; P-477 to R-1001; G-47 8 to R-1001; F-479 to R-1001; R-480 to R-1001; P-481 to R-1001; P-482 to R-1001; Q-483 to R-1001; S-484 to 8-1001; V -485 to R-1001; M-486 to R-1001; A-487 to R-1001; D-48 8 to R-1001; T-489 to 8-1001; E-490 toR-1001; N-491 to R-1001; K-492 to R-1001; E-493 to R-1001; V -494 to 8-1001; A-495 to R-1001; R-496 to R-1001; I-497 to R-1001; T-498 to R-1001; F-499 to R-1001; V-500 to R-1001; F-501 to R-1001; E-502 to R-1001; T-503 toR-1001; L-504 to R-1001; C-505 to R-1001; S-506 to R-1001; V-507 to R-1001; N-508 to R-1001; C-509 to R-l 001;E-510 to R-1001; L-511 to R-1001; Y -512 to R-1001; F-513 to R-1001; M-514 to 8-1001; V-515 to R-1001; G-516 toR-1001; V-517 to R-1001; N-518 to R-1001; S-519 to 8-1001; R-520 to R-1001; T-521 to R-1001; N-522 to R- l 001sT-523 to R-1001; P-524 to R-1001; V-525 to R-1001; E-526 to R-1001; T-527 to R-1001; W-528 to R-1001; K-529 toR-1001; G-530 to R-1001; S-531 to R-1001; K-532 to R-1001; G-533 to R-1001; K-534 to 8-1001; Q-535 to R-1001;5-536 to R-1001; Y-537 to R-1001; T-538 to R-1001; Y-539 to 8-1001; I-540 to R-1001; I-541 to R-1001; E-542 toR-1001; E-543 to R-1001; N-544 to R-1001;
T-545 to R-1001; T-546 to R-1001; T-547 to R-1001; S-548 to R-1001; F-549 to R-1001; T-550 to R-1001; W-551 to R-1001; A-552 to R-1001; F-553 to R-1001; Q-554 to R-1001; 8-555 toR-1001; T-556 to R-1001; T-557 to R-1001; F-558 to R-1001; H-559 to R-1001; E-560 to R-1001; A-561 to R-1001;S-562 to R-1001; R-563 to R-1001; K-564 to R-1001;
Y-565 to R-1001; T-566 to R-1001; N-567 to R-1001; D-568 toR-1001; V-569 to R-1001; A-570 to R-1001; K-571 to R-1001; I-572 to R-1001; Y-573 to R-1001; S-574 to R-1001; I-575to R-1001;
N-576 to R-1001; V-577 to R-1001; T-578 to R-1001; N-579 to R-1001; V-580 to R-1001; M-581 to R-1001;N-582 to R-1001; G-583 to R-1001; V-584 to R-1001; A-585 to R-1001; S-586 to R-1001; Y-587 to R-1001; C-588 toR-1001; R-589 to R-1001; P-590 to R-1001;
C-591 to R-1001; A-592 to R-1001; L-593 to R-1001; E-594 to R-1001;A-595 to R-1001; S-596 to R-1001; D-597 to R-1001; V-598 to R-1001; G-599 to R-1001; S-600 to R-1001; S-601 toR-1001; C-602 to R-1001; T-603 to R-1001; S-604 to R-1001; C-605 to R-1001; P-606 to 8-1001; A-607 to R-1001;6-608 to R-1001; Y-609 to R-1001; Y-610 to R-1001; I-611 to R-1001; D-612 to R-1001; R-613 to R-1001; D-614 toR-1001; S-615 to R-1001; G-616 to R-1001; T-617 to R-1001; C-618 to R-1001; H-619 to R-1001; S -620 to R- l 001;C-621 to 8-1001; P-622 to R-1001; P-623 to R-1001; N-624 to R-1001; T-625 to R-1001; I-626 to R-1001; L-627 toR-1001; K-628 to R-1001; A-629 to R-1001; H-63 0 to R-1001; Q-631 to 8-1001; P-632 to R-1001; Y-633 to R-1001;6-634 to R-1001; V-635 to R-1001; Q-636 to R-1001; A-637 to R-1001; C-63 8 to R-1001; V -639 to R-1001; P-640 toR-1001; C-641 to 8-1001; G-642 to R-1001; P-643 to R-1001; G-644 to R-1001; T-645 to R-1001; K-646 to R-l 001;N-647 to R-1001; N-648 to R-1001; K-649 to R-1001; I-650 to R-1001; H-651 to R-1001; S-652 to R-1001; L-653 toR-1001; C-654 to R-1001; Y-655 to R-1001; N-656 to R-1001; D-657 to R-1001; C-65 8 to R-1001; T-659 to R-1001; F-660 to R-1001; S-661 to 8-1001; R-662 to R-1001; N-663 to R-1001; T-664 to R-1001; P-665 to R-1001; T-666 toR-1001; R-667 to R-1001; T-668 to R-1001; F-669 to R-1001; N-670 to R-1001; Y-671 to R-1001; N-672 to R- l 001;F-673 to R-1001; S-674 to R-1001; A-675 to R-1001; L-676 to 8-1001; A-677 to R-1001; N-67 8 to R-1001; T-679 toR-1001; V -680 to R-1001; T-681 to 8-1001; L-682 to R-1001; A-683 to R-1001; G-684 to R-1001; G-685 to R-1001; P-686 to 8-1001; S-687 to R-1001; F-688 to R-1001; T-689 to R-1001; S-690 to R-1001; K-691 to R-1001; G-692 toR-1001; L-693 to R-1001; K-694 to R-1001; Y-695 to R-1001; F-696 to 8-1001; H-697 to R-1001; H-698 to R- l 001;F-699 to R-1001; T-700 to R-1001; L-701 to 8-1001; S-702 to R-1001; L-703 to R-1001; C-704 to R-1001; G-705 toR-1001; N-706 to 8-1001; Q-707 to R-1001; G-708 to R-1001; R-709 to R-1001; K-710 to R-1001; M-711 to 8-1001; S-712 to R-1001; V -713 to R-1001; C-7 I 4 to R-1001; T-715 to R-1001; D-716 to 8-1001; N-717 to R-1001; V -718 toR-1001; T-719 to R-1001; D-720 to R-1001; L-721 to 8-1001; R-722 to R-1001; I-723 to R-1001; P-724 to R-1001; E-725to R-1001; G-726 to 8-100 I ; E-727 to R-1001; S-728 to R-1001; G-729 to R-1001; F-730 to R-1001; S-731 to R-1001;K-732 to R-1001; S-733 to R-1001; I-734 to R-1001; T-735 to R-1001; A-736 to R-1001; Y-737 to R-1001; V-738 toR-1001; C-739 to R-1001; Q-740 to R-1001; A-741 to 8-1001; V -742 to R-1001; I-743 to R-1001; I-744 to R-1001; P-745to R-1001; P-746 to R-1001;
E-747 to R-1001; V-748 to R-1001; T-749 to R-1001; G-750 to R-1001; Y-751 to R-1001;K-752 to R-1001; A-753 to R-1001; G-754 to R-1001; V-755 to R-1001; S-756 to R-1001; 5-757 to R-1001; Q-758 toR-1001; P-759 to R-1001; V-760 to R-1001; S-761 to R-1001; L-762 to R-1001; A-763 to R-1001; D-764 to R-1001;8-765 to R-1001; L-766 to R-1001;
I-767 to 8-1001; G-768 to R-1001; V-769 to R-1001; T-770 to R-1001; T-771 toR-1001; D-772 to 8-1001; M-773 to R-1001; T-774 to R-1001; L-775 to R-1001; D-776 to R-1001; G-777 to R-1001;I-778 to R-1001; T-779 to R-1001; S-780 to R-1001; P-781 to R-1001; A-782 to 8-1001; E-783 to R-1001; L-784 toR-1001; F-785 to R-1001; H-786 to R-1001; L-787 to 8-1001; E-788 to R-1001; S-789 to R-1001; L-790 to R-1001;6-791 to R-1001; I-792 to R-1001; P-793 to R-1001; D-794 to R-1001; V -795 to R-1001; I-796 to R-1001; F-797 toR-1001; F-798 to R-1001; Y-799 to R-1001; R-800 to R-1001; S-801 to R-1001; N-802 to 8-1001; D-803 to R-1001;V-804 to R-1001; T-805 to R-1001; Q-806 to R-1001; S-807 to R-1001; C-808 to R-1001; S-809 to R-1001; S-810 toR-1001; G-811 to R-1001; R-812 to 8-1001; S-813 to R-1001; T-814 to R-1001; T-815 to R-1001; I-816 to R-1001; R-8 l7to 8-1001; V-818 to R-1001; R-819 to R-1001; C-820 to R-1001; S-821 to R-1001; P-822 to 8-1001; Q-823 to R-1001;x-824 to R-1001; T-825 to R-1001; V-826 to R-1001; P-827 to 8-1001; G-828 to R-1001; S-829 to R-1001; L-830 toR-1001; L-831 to R-1001; L-832 to 8-1001; P-833 to R-1001; G-834 to R-1001; T-835 to R-1001; C-836 to R-1001;S-837 to 8-1001; D-838 to R-1001; G-8 3 9 to R-1001; T-840 to R-1001; C-841 to R-1001; D-842 to 8-1001; G-843 toR-1001; C-844 to R-1001; N-845 to R-1001; F-846 to R- I 001; H-847 to R-1001; F-848 to R-1001; L-849 to R-1001; W-850 to R-1001; E-851 to R-1001; S-852 to 8-1001; A-853 to R-1001; A-854 to R-1001; A-855 to R-1001; C-856 toR-1001; P-857 to 8-1001; L-858 to R-1001; C-859 to R-1001; S-860 to R-1001; V-861 to R-1001; A-862 to R-l 001;D-863 to R-1001; Y-864 to R-1001; H-865 to R-1001; A-866 to R-1001; I-867 to 8-1001; V -868 to R-1001; S-869 toR-1001; S-870 to R-1001; C-871 to R-1001; V-872 to 8-1001; A-873 to R-1001; G-874 to R-1001; I-875 to R- l 001;Q-876 to R- I 001; K-877 to 8-1001; T-878 to R-1001; T-879 to R-1001; Y-880 to R-1001; V-881 to R-1001; W-882 toR-1001; R-883 to R-1001; E-884 to R-1001; P-885 to R-1001; K-886 to R-1001; L-887 to 8-1001; C-888 to R-1001;S-889 to R-1001; G-890 to R-1001; G-89-1 to R-1001; I-892 to 8-1001; S-893 to R-1001; L-894 to R-1001; P-895 toR-1001; E-896 to R-1001; Q-897 to 8-1001; R-898 to R-1001; V-899 to R-1001; T-900 to R-1001; I-901 to R-1001; C-902to 8-1001; K-903 to R-1001; T-904 to R-1001; I-905 to R-1001; D-906 to R-1001; F-907 to R-100 I ; W-908 to R-1001; L-909 to R-1001; K-910 to R-1001; V -911 to R-1001; G-912 to 8-1001; I-913 to R-1001; S-914 to R-1001; A-915 toR-1001; G-916 to R-1001; T-917 to 8-1001; C-918 to R-1001; T-919 to R-1001; A-920 to R-1001; I-921 to R-1001; L-922to 8-1001; L-923 to R-1001; T-924 to R-1001; V -925 to R-1001; L-926 to R-1001; T-927 to 8-1001; C-928 to R- l 001;Y-929 to R-1001; F-93 0 to R-1001; W-931 to R-1001; K-932 to R-~5 1001; K-933 to R-1001; N-934 to R-1001; Q-935 toR-1001; K-936 to R-1001; L-937 to 8-1001; E-93 8 to R-1001; Y-939 to R-1001; K-940 to R-1001; Y-941 to R-1001; S-942 to 8-1001; K-943 to R-1001; L-944 to R-1001; V -945 to R-1001; M-946 to R-1001; N-947 to 8-1001; A-948 toR-1001; T-949 to R-1001; L-950 to R-1001; K-951 to R-1001; D-952 to 8-1001; C-95 3 to R-1001; D-954 to R-1001; L-955 to R-1001; P-956 to R-1001; A-957 to 8-1001; A-958 to R-1001; D-959 to R-1001; S-960 to R-1001; C-961 toR-1001; A-962 to R-1001; I-963 to R-1001; M-964 to R-1001; E-965 to R-1001; G -966 to R-1001; E-967 to R-l 001;D-968 to R-1001; V -969 to R-1001; E-970 to R-1001; D-971 to R-1001; D-972 to 8-1001; L-973 to R-1001; I-974 toR-1001; F-975 to R-1001; T-976 to R-1001; S-977 to R-1001;
K-978 to R-1001; N-979 to R-1001; H-980 to R-1001; S-981 to R-1001; L-982 to R-1001; G-983 to R-1001; R-984 to R-1001; S-985 to R-1001; N-986 to R-1001; H-987 toR-1001; L-988 to R-1001; P-989 to R-1001; P-990 to R-1001; R-991 to R-1001; G-992 to R-1001;
L-993 to R- l 001;L-994 to R-1001; M-995 to R-1001; D-996 to R-1001; of S EQ ID N0:40.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C-3o terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind TR13 ligand) may still be retained. For example the ability of the shortened TR13 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an TR13 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR13 amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR13 polypeptide shown in Figures lA-C (SEQ ID N0:2), up to the glutamine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m' of Figures 1 A-C, where m' is an integer from 6 to 749 corresponding to the position of the amino acid residue in Figures lA-C.
1o Moreover, the invention provides TR13 polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: Q-6 to C-749; Q-6 to Q-748; Q-6 to T-747;
Q-6 to L-746; Q-6 to D-745; Q-6 to M-744; Q-6to L-743; Q-6 to L-742; Q-6 to G-741; Q-6 to R-740; Q-6 to P-739; Q-6 to P-738; Q-6 to L-737; Q-6 to H-736; Q-6 to N-735; Q-6to S-734;
Q-6 to R-733; Q-6 to G-732; Q-6 to L-731; Q-6 to S-730; Q-6 to H-729; Q-6 to N-728; Q-6 to K-727; Q-6 to S-726;Q-6 to T-725; Q-6 to F-724; Q-6 to I-723; Q-6 to L-722; Q-6 to D-721;
Q-6 to D-720; Q-6 to E-719; Q-6 to V-718; Q-6 to D-717;Q-6 to E-716; Q-6 to G-715; Q-6 to E-714; Q-6 to M-713; Q-6 to I-712; Q-6 to A-711; Q-6 to C-710; Q-6 to S-709; Q-6 to D-708;Q-6 to A-707; Q-6 to A-706; Q-6 to P-705; Q-6 to L-704; Q-6 to D-703; Q-6 to C-702; Q-6 to D-701; Q-6 to K-700; Q-6 to L-699;Q-6 to T-698; Q-6 to A-697; Q-6 to N-696;
Q-6 to M-695; Q-6 to V-694; Q-6 to L-693; Q-6 to K-692; Q-6 to S-691; Q-6 toY-690; Q-6 to K-689; Q-6 to Y-688; Q-6 to E-687; Q-6 to L-686; Q-6 to K-685; Q-6 to Q-684; Q-6 to N-683; Q-6 to K-682; Q-6to K-681; Q-6 to W-680; Q-6 to F-679; Q-6 to Y-678; Q-6 to C-677; Q-6 to T-676; Q-6 to L-675; Q-6 to V-674; Q-6 to T-673;Q-6 to L-672; Q-6 to L-671; Q-6 to I-670; Q-6 to A-669; Q-6 to T-668; Q-6 to C-667; Q-6 to T-666; Q-6 to G-665; Q-6 to A-664;Q-6 to S-663; Q-6 to I-662; Q-6 to G-661; Q-6 to V-660; Q-6 to K-659; Q-6 to L-658; Q-6 to W-657; Q-6 to F-656; Q-6 to D-655;Q-6 to I-654; Q-6 to T-653; Q-6 to K-652; Q-6 to C-651; Q-6 to I-650; Q-6 to T-649; Q-6 to V-648; Q-6 to R-647; Q-6 to Q-646;Q-6 to E-645; Q-6 to P-644;
Q-6 to L-643; Q-6 to S-642; Q-6 to I-641; Q-6 to G-640; Q-6 to G-639; Q-6 to S-638; Q-6 to C-637;Q-6 to L-636; Q-6 to K-635; Q-6 to P-634; Q-6 to E-633; Q-6 to R-632; Q-6 to W-631; Q-6 to V-630; Q-6 to Y-629; Q-6 toT-628; Q-6 to T-627; Q-6 to K-626; Q-6 to Q-625; Q-6 to I-624; Q-6 to G-623; Q-6 to A-622; Q-6 to V-621; Q-6 to C-620; Q-6 toS-619; Q-6 to S-618;
Q-6 to V-617; Q-6 to I-616; Q-6 to A-615; Q-6 to H-614; Q-6 to Y-613; Q-6 to D-612; Q-6 to A-611; Q-6 toV-610; Q-6 to S-609; Q-6 to C-608; Q-6 to L-607; Q-6 to P-606; Q-6 to C-605; Q-6 to A-604; Q-6 to A-603; Q-6 to A-602; Q-6 toS-601; Q-6 to E-600; Q-6 to W-599; Q-6 to L-598; Q-6 to F-597; Q-6 to H-596; Q-6 to F-595; Q-6 to N-594; Q-6 to C-593; Q-6to G-592; Q-6 to D-591; Q-6 to C-590; Q-6 to T-589; Q-6 to G-588; Q-6 to D-587; Q-6 to S-586; Q-6 to C-585; Q-6 to T-584;Q-6 to G-583; Q-6 to P-582; Q-6 to L-581; Q-6 to L-580; Q-6 to L-579; Q-6 to S-578; Q-6 to G-577; Q-6 to P-576; Q-6 to V-575;Q-6 to T-574; Q-6 to K-573; Q-6 to Q-572; Q-6 to P-571; Q-6 to S-570; Q-6 to C-569; Q-6 to R-568; Q-6 to V-567; Q-6 to R-566;Q-6 to I-565; Q-6 to T-564; Q-6 to T-563; Q-6 to S-562; Q-6 to R-561; Q-6 to G-560; Q-6 to S-559; Q-6 to S-558; Q-6 to C-557;Q-6 to S-556; Q-6 to Q-555; Q-6 to T-554; Q-6 to V-553; Q-6 to D-552; Q-6 to N-551; Q-6 to S-550; Q-6 to R-549; Q-6 to Y-548;Q-6 to F-547; Q-6 to F-546; Q-6 to I-545; Q-6 to V-544; Q-6 to D-543; Q-6 to P-542; Q-6 to I-541; Q-6 to G-540; Q-6 to L-539;Q-6 to S-538; Q-6 to E-537; Q-6 to L-536; Q-6 to H-535; Q-6 to F-534; Q-6 to L-533; Q-6 to E-532; Q-6 to A-531; Q-6 to P-530;Q-6 to S-529; Q-6 to T-528; Q-6 to I-527;
Q-6 to G-526;
Q-6 to D-525; Q-6 to L-524; Q-6 to T-523; Q-6 to M-522; Q-6 to D-521;Q-6 to T-520; Q-6 to T-519; Q-6 to V-518; Q-6 to G-517; Q-6 to I-516; Q-6 to L-515; Q-6 to R-514; Q-6 to D-513;
1o Q-6 to A-512;Q-6 to L-511; Q-6 to S-510; Q-6 to V-509; Q-6 to P-508; Q-6 to Q-507; Q-6 to S-506; Q-6 to S-505; Q-6 to V-504; Q-6 to G-503;Q-6 to A-502; Q-6 to K-501; Q-6 to Y-500;
Q-6 to G-499; Q-6 to T-498; Q-6 to V-497; Q-6 to E-496; Q-6 to P-495; Q-6 to P-494;Q-6 to I-493; Q-6 to I-492; Q-6 to V-491; Q-6 to A-490; Q-6 to Q-489; Q-6 to C-488; Q-6 to V-487; Q-6 to Y-486; Q-6 to A-485;Q-6 to T-484; Q-6 to I-483; Q-6 to S-482; Q-6 to K-481; Q-6 to S-480; Q-6 to F-479; Q-6 to G-478; Q-6 to S-477; Q-6 to E-476;Q-6 to G-475; Q-6 to E-474; Q-6 to P-473; Q-6 to I-472; Q-6 to R-471; Q-6 to L-470; Q-6 to D-469; Q-6 to T-468; Q-6 to V-467;Q-6 to N-466; Q-6 to D-465; Q-6 to T-464; Q-6 to C-463; Q-6 to V-462; Q-6 to S-461; Q-6 to M-460; Q-6 to K-459; Q-6 toR-458; Q-6 to G-457; Q-6 to Q-456; Q-6 to N-455; Q-6 to 6-454; Q-6 to C-453; Q-6 to L-452; Q-6 to S-451; Q-6 to L-450; Q-6to T-449; Q-6 to F-448; Q-6 to H-447; Q-6 to H-446; Q-6 to F-445; Q-6 to Y-444; Q-6 to K-443; Q-6 to L-442; Q-6 to G-441;Q-6 to K-440; Q-6 to S-439; Q-6 to T-438; Q-6 to F-437; Q-6 to S-436; Q-6 to P-435; Q-6 to G-434; Q-6 to G-433; Q-6 to A-432;Q-6 to L-431; Q-6 to T-430; Q-6 to V-429;
Q-6 to T-428; Q-6 to N-427; Q-6 to A-426; Q-6 to L-425; Q-6 to A-424; Q-6 to S-423;Q-6 to F-422; Q-6 to N-421; Q-6 to Y-420; Q-6 to N-419; Q-6 to F-418; Q-6 to T-417; Q-6 to R-416; Q-6 to T-415; Q-6 to P-414;Q-6 to T-413; Q-6 to N-412; Q-6 to R-411; Q-6 to S-410; Q-6 to F-409; Q-6 to T-408; Q-6 to C-407; Q-6 to D-406; Q-6 to N-405;Q-6 to Y-404; Q-6 to C-403;
Q-6 to L-402; Q-6 to S-401; Q-6 to H-400; Q-6 to I-399; Q-6 to K-398; Q-6 to N-397; Q-6 to N-396;Q-6 to K-395; Q-6 to T-394; Q-6 to G-393; Q-6 to P-392; Q-6 to G-391; Q-6 to C-390; Q-6 to P-389; Q-6 to V-388; Q-6 toC-387; Q-6 to A-386; Q-6 to Q-385; Q-6 to V-384; Q-6 to G-383; Q-6 to Y-382; Q-6 to P-381; Q-6 to Q-380; Q-6 to H-379; Q-6to A-378; Q-6 to K-377; Q-6 to L-376; Q-6 to I-375; Q-6 to T-374; Q-6 to N-373; Q-6 to P-372; Q-6 to P-371; Q-6 to C-370; Q-6to S-369; Q-6 to H-368; Q-6 to C-367; Q-6 to T-366; Q-6 to G-365; Q-6 to S-364; Q-6 to D-363; Q-6 to R-362; Q-6 to D-361;Q-6 to I-360; Q-6 to Y-359; Q-6 to Y-358; Q-6 to G-357; Q-6 to A-356; Q-6 to P-355; Q-6 to C-354; Q-6 to S-353; Q-6 to T-352;Q-6 to C-351;
Q-6 to S-350; Q-6 to S-349; Q-6 to G-348; Q-6 to V-347; Q-6 to D-346; Q-6 to S-345; Q-6 to A-344; Q-6 to E-343;Q-6 to L-342; Q-6 to A-341; Q-6 to C-340; Q-6 to P-339; Q-6 to R-338; Q-6 to C-337; Q-6 to Y-336; Q-6 to S-335; Q-6 to A-334;Q-6 to V-333; Q-6 to G-332; Q-6 to N-331; Q-6 to M-330; Q-6 to V-329; Q-6 to N-328; Q-6 to T-327; Q-6 to V-326; Q-6 toN-325; Q-6 to I-324; Q-6 to S-323; Q-6 to Y-322; Q-6 to I-321; Q-6 to K-320; Q-6 to A-319; Q-6 to V-318; Q-6 to D-317; Q-6 toN-316; Q-6 to T-315; Q-6 to Y-314; Q-6 to K-313; Q-6 to R-312; Q-6 to S-311; Q-6 to A-310; Q-6 to E-309; Q-6 to H-308; Q-6to F-307; Q-6 to T-306; Q-6 to T-305; Q-6 to R-304; Q-6 to Q-303; Q-6 to F-302; Q-6 to A-301; Q-6 to W-300; Q-6 to T-299; Q-6to F-298; Q-6 to S-297; Q-6 to T-296; Q-6 to T-295; Q-6 to T-294; Q-6 to N-293; Q-6 to E-292; Q-6 to E-291; Q-6 to I-290; Q-6to I-289; Q-6 to Y-288; Q-6 to T-287; Q-6 to Y-286; Q-6 to S-285; Q-6 to Q-284; Q-6 to K-283; Q-6 to G-282; Q-6 to K-281; Q-6to S-280; Q-6 to G-279; Q-6 to K-278; Q-6 to W-277; Q-6 to T-276; Q-6 to E-275; Q-6 to V-274; Q-6 to P-273; Q-6 to T-272;Q-6 to N-271; Q-6 to T-270; Q-6 to R-269; Q-6 to S-268; Q-6 to N-267; Q-6 to V-266; Q-l0 6 to G-265; Q-6 to V-264; Q-6 toM-263; Q-6 to F-262; Q-6 to Y-261; Q-6 to L-260; Q-6 to E-259; Q-6 to C-258; Q-6 to N-257; Q-6 to V-256; Q-6 to S-255; Q-6to C-254; Q-6 to L-253; Q-6 to T-252; Q-6 to E-251; Q-6 to F-250; Q-6 to V-249; Q-6 to F-248; Q-6 to T-247; Q-6 to I-246; Q-6to R-245; Q-6 to A-244; Q-6 to V-243; Q-6 to E-242; Q-6 to K-241; Q-6 to N-240; Q-6 to E-239; Q-6 to T-238; Q-6 to D-237;Q-6 to A-236; Q-6 to M-235; Q-6 to V-234; Q-6 to S-233; Q-6 to Q-232; Q-6 to P-231; Q-6 to P-230; Q-6 to R-229; Q-6 to F-228;Q-6 to G-227; Q-6 to P-226; Q-6 to V -225 ; Q-6 to . V -224; Q-6 to L-223 ; Q-6 to T-222; Q-6 to L-221; Q-6 to I-220;
Q-6 to M-219;Q-6 to F-218; Q-6 to D-217; Q-6 to N-216; Q-6 to D-215; Q-6 to S-214; Q-6 to A-213; Q-6 to G-212; Q-6 to A-211; Q-6 toA-210; Q-6 to T-209; Q-6 to Y-208; Q-6 to I-207;
Q-6 to H-206; Q-6 to D-205; Q-6 to G-204; Q-6 to A-203; Q-6 to V-202; Q-6 toE-201; Q-6 to 2o W-200; Q-6 to G-199; Q-6 to T-198; Q-6 to M-197; Q-6 to G-196; Q-6 to K-195; Q-6 to Y-194; Q-6 to E-193; Q-6to F-192; Q-6 to N-191; Q-6 to I-190; Q-6 to G-189; Q-6 to S-188; Q-6 to L-187; Q-6 to V-186; Q-6 to T-185; Q-6 to T-184; Q-6to E-183; Q-6 to M-182;
Q-6 to N-181; Q-6 to T-180; Q-6 to P-179; Q-6 to L-178; Q-6 to T-177; Q-6 to N-176; Q-6 to W-175;Q-6 to W-174; Q-6 to K-173; Q-6 to Y-172; Q-6 to E-171; Q-6 to F-170; Q-6 to G-169; Q-6 to V-168; Q-6 to A-167 ; Q-6 toP-166; Q-6 to E-165 ; Q-6 to T-164; Q-6 to G-163 ; Q-6 to A-162; Q-6 to P-161; Q-6 to C-160; Q-6 to R-159; Q-6 to T-158; Q-6 toC-157; Q-6 to D-156;
Q-6 to S-155; Q-6 to G-154; Q-6 to N-153; Q-6 to S-152; Q-6 to Y-151; Q-6 to S-150; Q-6 to G-149; Q-6to Y -148 ; Q-6 to P-147 ; Q-6 to C-146; Q-6 to P-145 ; Q-6 to Q-144; Q-6 to C-143 ; Q-6 to T-142; Q-6 to S-141; Q-6 to N-140; Q-6to N-139; Q-6 to T-138; Q-6 to K-137; Q-6 to F-136; Q-6 to F-135; Q-6 to G-134; Q-6 to P-133; Q-6 to N-132; Q-6 to C-131;Q-6 to P-130; Q-6 to P-129; Q-6 to C-128; Q-6 to H-127; Q-6 to T-126; Q-6 to K-125; Q-6 to V-124; Q-6 to G-123;
Q-6 toS-122; Q-6 to A-121; Q-6 to P-120; Q-6 to L-119; Q-6 to K-118; Q-6 to V-117; Q-6 to A-116; Q-6 to G-115; Q-6 to E- I 14; Q-6 toL-113; Q-6 to D-112; Q-6 to E-111;
Q-6 to S-110;
Q-6 to C-109; Q-6 to I-108; Q-6 to K-107; Q-6 to P-106; Q-6 to K-105; Q-6 toA-104; Q-6 to W-103; Q-6 to K-102; Q-6 to Y-101; Q-6 to M-100; Q-6 to L-99; Q-6 to Q-98; Q-6 to T-97; Q-6 to E-96; Q-6 toG-95; Q-6 to N-94; Q-6 to A-93; Q-6 to D-92; Q-6 to C-91; Q-6 to A-90; Q-6 to T-89; Q-6 to H-88; Q-6 to T-87; Q-6 to Y-86; Q-6to F-85; Q-6 to Y-84; Q-6 to D-83; Q-6 to K-82; Q-6 to D-81; Q-6 to T-80; Q-6 to C-79; Q-6 to A-78; Q-6 to P-77; Q-6 to R-76;Q-6 to V-75; Q-6 to N-74; Q-6 to C-73; Q-6 to S-72; Q-6 to S-71; Q-6 to S-70; Q-6 to G-69; Q-6 to K-68; Q-6 to E-67; Q-6 toS-66; Q-6 to Y-65; Q-6 to K-64; Q-6 to D-63; Q-6 to P-62; Q-6 to D-61;
Q-6 to C-60; Q-6 to Q-59; Q-6 to H-58; Q-6 to C-57; Q-6to S-56; Q-6 to T-55; Q-6 to E-54; Q-6 to G-53; Q-6 to K-52; Q-6 to N-51; Q-6 to S-50; Q-6 to Y-49; Q-6 to S-48; Q-6 to N-47;Q-6 to S-46; Q-6 to P-45; Q-6 to L-44; Q-6 to T-43; Q-6 to Q-42; Q-6 to L-41; Q-6 to F-40; Q-6 to L-39; Q-6 to L-38; Q-6 toG-37; Q-6 to A-36; Q-6 to Q-35; Q-6 to R-34; Q-6 to C-33; Q-6 to V-32; Q-6 to H-31; Q-6 to W-30; Q-6 to T-29; Q-6 to Q-28;Q-6 to L-27; Q-6 to P-26; Q-6 to L-25; Q-6 to M-24; Q-6 to R-23; Q-6 to F-22; Q-6 to H-21; Q-6 to L-20; Q-6 to G-19; Q-6 toG-18 ; Q-6 to R-17 ; Q-6 to N-16; Q-6 to H- I 5 ; Q-6 to C-14; Q-6 to H-13 ; Q-6 to K-12; of S EQ ID
N0:2. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR13 polypeptide shown in Figures 7A-D (SEQ ID N0:40), up to the histidine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m' of Figures 7A-D, where m' is an integer from 6 to 1001 corresponding to the position of the amino acid residue in Figures 7A-D.
Moreover, the invention provides TR13 polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: H-6 to C-1000; H-6 to Q-999; H-6 to T-998;H-6 to L-997; H-6 to D-996; H-6 to M-995; H-6 to L-994; H-6 to L-993; H-6 to G-992;
H-6 to R-991; H-6 toP-990; H-6 to P-989; H-6 to L-988; H-6 to H-987; H-6 to N-986; H-6 to S-985; H-6 to R-984; H-6 to G-983;H-6 to L-982; H-6 to S-981; H-6 to H-980; H-6 to N-979;
H-6 to K-978; H-6 to S-977; H-6 to T-976; H-6 toF-975; H-6 to I-974; H-6 to L-973; H-6 to D-972; H-6 to D-971; H-6 to E-970; H-6 to V-969; H-6 to D-968;H-6 to E-967; H-6 to G-966;
H-6 to E-965; H-6 to M-964; H-6 to I-963; H-6 to A-962; H-6 to C-961; H-6 toS-960; H-6 to D-959; H-6 to A-958; H-6 to A-957; H-6 to P-956; H-6 to L-955; H-6 to D-954; H-6 to C-953;H-6 to D-952; H-6 to K-951; H-6 to L-950; H-6 to T-949; H-6 to A-948; H-6 to N-947;
H-6 to M-946; H-6 toV-945; H-6 to L-944; H-6 to K-943; H-6 to S-942; H-6 to Y-941; H-6 to K-940; H-6 to Y-939; H-6 to E-938;H-6 to L-937; H-6 to K-936; H-6 to Q-935; H-6 to N-934;
3o H-6 to K-933; H-6 to K-932; H-6 to W-931; H-6to F-930; H-6 to Y-929; H-6 to C-928; H-6 to T-927; H-6 to L-926; H-6 to V-925; H-6 to T-924; H-6 toL-923; H-6 to L-922;
H-6 to I-921; H-6 to A-920; H-6 to T-919; H-6 to C-918; H-6 to T-917; H-6 to G-916;H-6 to A-915;
H-6 to S-914; H-6 to I-913; H-6 to G-912; H-6 to V-911; H-6 to K-910; H-6 to L-909; H-6 toW-908; H-6 to F-907; H-6 to D-906; H-6 to I-905; H-6 to T-904; H-6 to K-903;
H-6 to C-902; H-6 to I-901;H-6 to T-900; H-6 to V-899; H-6 to R-898; H-6 to Q-897; H-6 to E-896; H-6 to P-895; H-6 to L-894; H-6 toS-893; H-6 to I-892; H-6 to G-891; H-6 to G-890; H-6 to S-889; H-6 to C-888; H-6 to L-887; H-6 to K-886;H-6 to P-885; H-6 to E-884; H-6 to R-883;
H-6 to W-882; H-6 to V-881; H-6 to Y-880; H-6 to T-879; H-6 toT-878; H-6 to K-877; H-6 to Q-876; H-6 to I-875; H-6 to G-874; H-6 to A-873; H-6 to V-872; H-6 to C-871;H-6 to S-870;
H-6 to S-869; H-6 to V-868; H-6 to I-867; H-6 to A-866; H-6 to H-865; H-6 to Y-864; H-6 toD-863; H-6 to A-862; H-6 to V-861; H-6 to S-860; H-6 to C-859; H-6 to L-858;
H-6 to P-857; H-6 to C-856;H-6 to A-855; H-6 to A-854; H-6 to A-853; H-6 to S-852; H-6 to E-851;
H-6 to W-850; H-6 to L-849; H-6 toF-848; H-6 to H-847; H-6 to F-846; H-6 to N-845; H-6 to C-844; H-6 to G-843; H-6 to D-842; H-6 to C-841;H-6 to T-840; H-6 to G-839; H-6 to D-838;
H-6 to S-837; H-6 to C-836; H-6 to T-835; H-6 to G-834; H-6 toP-833; H-6 to L-832; H-6 to L-831; H-6 to L-830; H-6 to S-829; H-6 to G-828; H-6 to P-827; H-6 to V-826;H-6 to T-825;
H-6 to K-824; H-6 to Q-823; H-6 to P-822; H-6 to S-821; H-6 to C-820; H-6 to R-819; H-6 toV-818; H-6 to R-817; H-6 to I-816; H-6 to T-815; H-6 to T-814; H-6 to S-813;
H-6 to 8-812; H-6 to G-811;H-6 to S-810; H-6 to S-809; H-6 to C-808; H-6 to S-807; H-6 to Q-806;
H-6 to T-805; H-6 to V-804; H-6 toD-803; H-6 to N-802; H-6 to S-801; H-6 to R-800; H-6 to Y-799; H-6 to F-798; H-6 to F-797; H-6 to I-796;H-6 to V-795; H-6 to D-794; H-6 to P-793;
H-6 to I-792; H-6 to G-791; H-6 to L-790; H-6 to S-789; H-6 toE-788; H-6 to L-787; H-6 to t5 H-786; H-6 to F-785; H-6 to L-784; H-6 to E-783; H-6 to A-782; H-6 to P-781;H-6 to S-780;
H-6 to T-779; H-6 to I-778; H-6 to G-777; H-6 to D-776; H-6 to L-775; H-6 to T-774; H-6 toM-773; H-6 to D-772; H-6 to T-771; H-6 to T-770; H-6 to V-769; H-6 to G-768;
H-6 to I-767; H-6 to L-766;H-6 to R-765; H-6 to D-764; H-6 to A-763; H-6 to L-762; H-6 to S-761; H-6 to V-760; H-6 to P-759; H-6 toQ-758; H-6 to S-757; H-6 to S-756; H-6 to V-755; H-6 to G-754; H-6 to A-753; H-6 to K-752; H-6 to Y-751;H-6 to G-750; H-6 to T-749; H-6 to V-748;
H-6 to E-747; H-6 to P-746; H-6 to P-745; H-6 to I-744; H-6 toI-743; H-6 to V-742; H-6 to A-741; H-6 to Q-740; H-6 to C-739; H-6 to V-738; H-6 to Y-737; H-6 to A-736;H-6 to T-735;
H-6 to I-734; H-6 to S-733; H-6 to K-732; H-6 to S-731; H-6 to F-730; H-6 to G-729; H-6 toS-728; H-6 to E-727; H-6 to G-726; H-6 to E-725; H-6 to P-724; H-6 to I-723;
H-6 to R-722; H-6 to L-721;H-6 to D-720; H-6 to T-719; H-6 to V-718; H-6 to N-717; H-6 to D-716;
H-6 to T-715; H-6 to C-714; H-6 toV-713; H-6 to S-712; H-6 to M-711; H-6 to K-710; H-6 to R-709; H-6 to G-708; H-6 to Q-707; H-6 toN-706; H-6 to G-705; H-6 to C-704; H-6 to L-703; H-6 to S-702; H-6 to L-701; H-6 to T-700; H-6 to F-699;H-6 to H-698; H-6 to H-697;
H-6 to F-696; H-6 to Y-695; H-6 to K-694; H-6 to L-693; H-6 to G-692; H-6 toK-691; H-6 to 3o S-690; H-6 to T-689; H-6 to F-688; H-6 to S-687; H-6 to P-686; H-6 to G-685; H-6 to G-684;H-6 to A-683; H-6 to L-682; H-6 to T-681; H-6 to V-680; H-6 to T-679; H-6 to N-678;
H-6 to A-677; H-6 toL-676; H-6 to A-675; H-6 to S-674; H-6 to F-673; H-6 to N-672; H-6 to Y-671; H-6 to N-670; H-6 to F-669;H-6 to T-668; H-6 to R-667; H-6 to T-666; H-6 to P-665;
H-6 to T-664; H-6 to N-663; H-6 to R-662; H-6 toS-661; H-6 to F-660; H-6 to T-659; H-6 to C-658; H-6 to D-657; H-6 to N-656; H-6 to Y-655; H-6 to C-654;H-6 to L-653; H-6 to S-652;
H-6 to H-651; H-6 to I-650; H-6 to K-649; H-6 to N-648; H-6 to N-647; H-6 toK-646; H-6 to T-645; H-6 to G-644; H-6 to P-643; H-6 to G-642; H-6 to C-641; H-6 to P-640; H-6 to V-639;H-6 to C-638; H-6 to A-637; H-6 to Q-636; H-6 to V-635; H-6 to G-634; H-6 to Y-633;

H-6 to P-632; H-6 toQ-631; H-6 to H-630; H-6 to A-629; H-6 to K-628; H-6 to L-627; H-6 to I-626; H-6 to T-625; H-6 to N-624;H-6 to P-623; H-6 to P-622; H-6 to C-621; H-6 to S-620;
H-6 to H-619; H-6 to C-618; H-6 to T-617; H-6 toG-616; H-6 to S-615; H-6 to D-614; H-6 to R-613; H-6 to D-612; H-6 to I-61 l; H-6 to Y-610; H-6 to Y-609;H-6 to G-608; H-6 to A-607;
s H-6 to P-606; H-6 to C-605; H-6 to S-604; H-6 to T-603; H-6 to C-602; H-6 toS-601; H-6 to S-600; H-6 to G-599; H-6 to V-598; H-6 to D-597; H-6 to S-596; H-6 to A-595; H-6 to E-594;H-6 to L-593; H-6 to A-592; H-6 to C-591; H-6 to P-590; H-6 to R-589; H-6 to C-588;
H-6 to Y-587; H-6 toS-586; H-6 to A-585; H-6 to V-584; H-6 to G-583; H-6 to N-582; H-6 to M-581; H-6 to V-580; H-6 toN-579; H-6 to T-578; H-6 to V-577; H-6 to N-576; H-6 to I-575;
1o H-6 to S-574; H-6 to Y-573; H-6 to I-572;H-6 to K-571; H-6 to A-570; H-6 to V-569; H-6 to D-568; H-6 to N-567; H-6 to T-566; H-6 to Y-565; H-6 toK-564; H-6 to R-563; H-6 to S-562;
H-6 to A-561; H-6 to E-560; H-6 to H-559; H-6 to F-558; H-6 to T-557;H-6 to T-556; H-6 to R-555; H-6 to Q-554; H-6 to F-553; H-6 to A-552; H-6 to W-551; H-6 to T-550; H-6 toF-549;
H-6 to S-548; H-6 to T-547; H-6 to T-546; H-6 to T-545; H-6 to N-544; H-6 to E-543; H-6 to 15 E-542;H-6 to I-541; H-6 to I-540; H-6 to Y-539; H-6 to T-538; H-6 to Y-537;
H-6 to S-536;
H-6 to Q-535; H-6 toK-534; H-6 to G-533; H-6 to K-532; H-6 to S-531; H-6 to G-530; H-6 to K-529; H-6 to W-528; H-6 toT-527; H-6 to E-526; H-6 to V-525; H-6 to P-524; H-6 to T-523;
H-6 to N-522; H-6 to T-521; H-6 to R-520;H-6 to S-519; H-6 to N-518; H-6 to V-517; H-6 to G-516; H-6 to V-515; H-6 to M-514; H-6 to F-513; H-6 toY-512; H-6 to L-511; H-6 to E-510;
20 H-6 to C-509; H-6 to N-508; H-6 to V-507; H-6 to S-506; H-6 to C-SOS;H-6 to L-504; H-6 to T-503; H-6 to E-502; H-6 to F-501; H-6 to V-500; H-6 to F-499; H-6 to T-498; H-6 toI-497;
H-6 to R-496; H-6 to A-495; H-6 to V-494; H-6 to E-493; H-6 to K-492; H-6 to N-491; H-6 to E-490;H-6 to T-489; H-6 to D-488; H-6 to A-487; H-6 to M-486; H-6 to V-485;
H-6 to 5-484; H-6 to Q-483; H-6 toP-482; H-6 to P-481; H-6 to R-480; H-6 to F-479; H-6 to G-478;
2s H-6 to P-477; H-6 to V-476; H-6 to V-475;H-6 to L-474; H-6 to T-473; H-6 to L-472; H-6 to I-471; H-6 to M-470; H-6 to F-469; H-6 to D-468; H-6 toN-467; H-6 to D-466; H-6 to S-465;
H-6 to A-464; H-6 to G-463; H-6 to A-462; H-6 to A-461; H-6 to T-460;H-6 to Y-459; H-6 to I-458; H-6 to H-457; H-6 to D-456; H-6 to G-455; H-6 to A-454; H-6 to V-453; H-6 toE-452;
H-6 to W-451; H-6 to G-450; H-6 to T-449; H-6 to M-448; H-6 to G-447; H-6 to K-446; H-6 30 toY-445; H-6 to E-444; H-6 to F-443; H-6 to N-442; H-6 to 1-441; H-6 to G-440; H-6 to S-439; H-6 to L-438;H-6 to V-437; H-6 to T-436; H-6 to T-435; H-6 to E-434; H-6 to M-433;
H-6 to N-432; H-6 to T-431; H-6 toP-430; H-6 to L-429; H-6 to T-428; H-6 to N-427; H-6 to W-426; H-6 to W-425; H-6 to K-424; H-6 toY-423; H-6 to E-422; H-6 to F-421; H-6 to 6-420; H-6 to V-419; H-6 to A-418; H-6 to P-417; H-6 to E-416;H-6 to T-415; H-6 to G-414;
3s H-6 to A-413; H-6 to P-412; H-6 to C-411; H-6 to R-410; H-6 to T-409; H-6 toC-408; H-6 to D-407; H-6 to S-406; H-6 to G-405; H-6 to N-404; H-6 to S-403; H-6 to Y-402; H-6 to S-401;H-6 to G-400; H-6 to Y-399; H-6 to P-398; H-6 to C-397; H-6 to P-396; H-6 to Q-395;
H-6 to C-394; H-6 toT-393; H-6 to S-392; H-6 to N-391; H-6 to N-390; H-6 to T-389; H-6 to K-388; H-6 to F-387; H-6 to F-386;H-6 to G-385; H-6 to P-384; H-6 to N-383; H-6 to C-382;
H-6 to P-381; H-6 to P-380; H-6 to C-379; H-6 toH-378; H-6 to T-377; H-6 to K-376; H-6 to V-375; H-6 to G-374; H-6 to S-373; H-6 to A-372; H-6 to P-371;H-6 to L-370; H-6 to K-369;
H-6 to V-368; H-6 to A-367; H-6 to G-366; H-6 to E-365; H-6 to L-364; H-6 toD-363; H-6 to s E-362; H-6 to S-361; H-6 to C-360; H-6 to I-359; H-6 to K-358; H-6 to P-357;
H-6 to K-356;H-6 to A-355; H-6 to W-354; H-6 to K-353; H-6 to Y-352; H-6 to M-351; H-6 to L-350;
H-6 to Q-349; H-6 toT-348; H-6 to E-347; H-6 to G-346; H-6 to N-345; H-6 to A-344; H-6 to D-343; H-6 to C-342; H-6 to A-341;H-6 to T-340; H-6 to H-339; H-6 to T-338; H-6 to Y-337;
H-6 to F-336; H-6 to Y-335; H-6 to D-334; H-6 toK-333; H-6 to D-332; H-6 to T-331; H-6 to C-330; H-6 to A-329; H-6 to P-328; H-6 to R-327; H-6 to V-326;H-6 to N-325; H-6 to C-324;
H-6 to S-323; H-6 to S-322; H-6 to S-321; H-6 to G-320; H-6 to K-319; H-6 toE-318; H-6 to S-317; H-6 to Y-316; H-6 to K-315; H-6 to D-314; H-6 to P-313; H-6 to D-312; H-6 to C-311;H-6 to Q-310; H-6 to H-309; H-6 to C-308; H-6 to S-307; H-6 to T-306; H-6 to E-305;
H-6 to G-304; H-6 toK-303; H-6 to N-302; H-6 to S-301; H-6 to Y-300; H-6 to S-299; H-6 to N-298; H-6 to A-297; H-6 to P-296;H-6 to C-295; H-6 to L-294; H-6 to K-293; H-6 to C-292;
H-6 to F-291; H-6 to S-290; H-6 to S-289; H-6 toG-288; H-6 to Q-287; H-6 to K-286; H-6 to D-285; H-6 to A-284; H-6 to Y-283; H-6 to T-282; H-6 to G-281;H-6 to P-280; H-6 to K-279;
H-6 to C-278; H-6 to P-277; H-6 to F-276; H-6 to C-275; H-6 to E-274; H-6 toS-273; H-6 to T-272; H-6 to Y-271; H-6 to A-270; H-6 to V-269; H-6 to G-268; H-6 to T-267; H-6 to I-266;H-6 to A-265; H-6 to I-264; H-6 to N-263; H-6 to R-262; H-6 to V-261; H-6 to L-260; H-6 to V-259; H-6 toP-258; H-6 to K-257; H-6 to P-256; H-6 to V-255; H-6 to K-254; H-6 to T-253; H-6 to W-252; H-6 to V-251;H-6 to S-250; H-6 to F-249; H-6 to A-248; H-6 to T-247;
H-6 to T-246; H-6 to R-245; H-6 to W-244; H-6 toY-243; H-6 to L-242; H-6 to V-241; H-6 to N-240; H-6 to N-239; H-6 to G-238; H-6 to R-237; H-6 to N-236;H-6 to L-235; H-6 to E-234; H-6 to V-233; H-6 to S-232; H-6 to H-231; H-6 to F-230; H-6 to E-229; H-6 toW-228;
H-6 to G-227; H-6 to K-226; H-6 to E-225; H-6 to T-224; H-6 to T-223; H-6 to K-222; H-6 toM-221; H-6 to W-220; H-6 to R-219; H-6 to S-218; H-6 to D-217; H-6 to D-216;
H-6 to A-215; H-6 toN-214; H-6 to P-213; H-6 to Q-212; H-6 to C-211; H-6 to Q-210; H-6 to D-209;
H-6 to N-208; H-6 to Q-207;H-6 to V-206; H-6 to F-205; H-6 to F-204; H-6 to E-203; H-6 to 3o F-202; H-6 to I-201; H-6 to I-200; H-6 toS-199; H-6 to S-198; H-6 to D-197;
H-6 to P-196;
H-6 to Y-195; H-6 to Y-194; H-6 to Y-193; H-6 to E-192;H-6 to F-191; H-6 to N-190; H-6 to V-189; H-6 to T-188; H-6 to G-187; H-6 to S-186; H-6 to Q-185; H-6 toK-184; H-6 to L-183;
H-6 to N-182; H-6 to V-181; H-6 to A-180; H-6 to Y-179; H-6 to M-178; H-6 to L-177;H-6 to T-176; H-6 to A-175; H-6 to T-174; H-6 to C-173; H-6 to E-172; H-6 to D-171; H-6 to T-170;
H-6 toN-169; H-6 to F-168; H-6 to A-167; H-6 to I-166; H-6 to Y-165; H-6 to D-164; H-6 to G-163; H-6 to R-162;H-6 to P-161; H-6 to V-160; H-6 to W-159; H-6 to K-158; H-6 to S-157; H-6 to S-156; H-6 to T-155; H-6 toC-154; H-6 to N-153; H-6 to G-152; H-6 to T-151;
H-6 to S-150; H-6 to E-149; H-6 to A-148; H-6 to A-147;H-6 to S-146; H-6 to D-145; H-6 to D-144; H-6 to L-143 ; H-6 to E-142; H-6 to M-141; H-6 to N-140; H-6 toA-139; H-6 to S-13 8;
H-6 to L-137; H-6 to S-136; H-6 to A-135; H-6 to F-134; H-6 to G-133; H-6 to H-132;H-6 to P-131; H-6 to L-130; H-6 to E-129; H-6 to D-128; H-6 to W-127; H-6 to E-126; H-6 to D-125; H-6 toF-124; H-6 to R-123; H-6 to I-122; H-6 to G-121; H-6 to T-120; H-6 to G-119; H-6 to L-118; H-6 to S-117;H-6 to Y-116; H-6 to R-115; H-6 to G-114; H-6 to E-113; H-6 to A-112; H-6 to C-111; H-6 to P-110; H-6 toK-109; H-6 to C-108; H-6 to S-107; H-6 to Q-106;
H-6 to D-105; H-6 to K-104; H-6 to M-103; H-6 to D-102;H-6 to L-101; H-6 to F-100; H-6 to E-99; H-6 to G-98; H-6 to A-97; H-6 to N-96; H-6 to C-95; H-6 to S-94;H-6 to F-93; H-6 to S-92; H-6 to C-91; H-6 to E-90; H-6 to T-89; H-6 to G-88; H-6 to K-87; H-6 to V-86;H-6 to to P-85; H-6 to D-84; H-6 to P-83; H-6 to L-82; H-6 to S-81; H-6 to T-80; H-6 to C-79; H-6 to L-78; H-6to G-77; H-6 to P-76; H-6 to T-75; H-6 to H-74; H-6 to P-73; H-6 to V-72; H-6 to A-71; H-6 to V-70; H-6 toR-69; H-6 to W-68; H-6 to R-67; H-6 to S-66; H-6 to G-65; H-6 to T-64; H-6 to S-63; H-6 to D-62; H-6 toC-61; H-6 to A-60; H-6 to T-59; H-6 to Y-58; H-6 to E-57; H-6 to Y-56; H-6 to H-55; H-6 to Y-54; H-6 toE-53; H-6 to S-52; H-6 to E-51; H-6 to t5 K-50; H-6 to C-49; H-6 to A-48; H-6 to H-47; H-6 to L-46; H-6 toE-45; H-6 to P-44; H-6 to G-43; H-6 to T-42; H-6 to G-41; H-6 to Q-40; H-6 to T-39; H-6 to V-38; H-6 toQ-37; H-6 to F-36; H-6 to A-35; H-6 to T-34; H-6 to G-33; H-6 to A-32; H-6 to W-31; H-6 to L-30; H-6 toL-29; H-6 to L-28; H-6 to R-27; H-6 to W-26; H-6 to L-25; H-6 to R-24; H-6 to P-23; H-6 to I-22; H-6 toR-21; H-6 to R-20; H-6 to E-19; H-6 to T-18; H-6 to R-17; H-6 to G-16; H-6 to 2o R-15; H-6 to V-14; H-6 toR-13; H-6 to A-12; of SEQ ID N0:4~0.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
In another embodiment, N-terminal deletions of the predicted extracellular domain of the predicted mature TR13 protein, with the amino acid sequence shown in Figures 7A-D, can be described by the general formula n2-906, where n2 is a number from 2 to 900, 25 corresponding to the position of amino acid identified in Figures 7A-D (SEQ
ID N0:40). N-terminal deletions of the TR13 polypeptide of the invention shown as SEQ ID
N0:4~0 include polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: T-42 to D-906; G-43 to D-906; P-44 to D-906; E-45 to D-906; L-46 to D-906; H-47 to D-906; A-48 to D-906; C-49 to D-906; K-50 to D-906; E-51 to D-906; S-52 to D-906; E-53 to D-906; Y-3o 54 to D-906; H-55 to D-906; Y-56 toD-906; E-57 to D-906; Y-58 to D-906; T-59 to D-906; A-60 to D-906; C-61 to D-906; D-62 to D-906; S-63 toD-906; T-64 to D-906; G-65 to D-906; S-66 to D-906; R-67 to D-906; W-68 to D-906; R-69 to D-906; V -70 toD-906; A-71 to D-906; V-72 to D-906; P-73 to D-906; H-74 to D-906; T-75 to D-906; P-76 to D-906; G-77 toD-906; L-78 to D-906; C-79 to D-906; T-80 to D-906; S-81 to D-906; L-82 to D-906; P-83 to D-906; D-35 84 to D-906; P-85 to D-906; V-86 to D-906; K-87 to D-906; G-88 to D-906; T-89 to D-906; E-90 to D-906; C-91 toD-906; S-92 to D-906; F-93 to D-906; S-94 to D-906; C-95 to D-906; N-96 to D-906; A-97 to D-906; G-98 toD-906; E-99 to D-906; F-100 to D-906; L-101 to D-906;
D-102 to D-906; M-103 to D-906; K-104 to D-906; D-105to D-906; Q-106 to D-906;
S-107 to D-906; C-108 to D-906; K-109 to D-906; P-110 to D-906; C-111 to D-906;A-112 to D-906; E-113 to D-906; G-114 to D-906; R-115 to D-906; Y-116 to D-906; S-117 to D-906;
L-118 toD-906; G-119 to D-906; T-120 to D-906; G-121 to D-906; I-122 to D-906; R-123 to D-906; F-124 to D-906;D-125 to D-906; E-126 to D-906; W-127 to D-906; D-128 to D-906; E-129 to D-906; L-130 to D-906; P-131 toD-906; H-132 to D-906; G-133 to D-906; F-134 to D-906; A-135 to D-906; S-136 to D-906; L-137 to D-906;S-138 to D-906; A-139 to D-906; N-140 to D-906; M-141 to D-906; E-142 to D-906; L-143 to D-906; D-144 toD-906; D-145 to D-906; S -146 to D-906; A-147 to D-906; A-148 to D-906; E-149 to D-906; S-150 to D-906;T-151 to D-906; G-152 to D-906; N-153 to D-906; C-154 to D-906; T-155 to D-906; S-156 to D-906; S-157 toD-906; K-158 to D-906; W-159 to D-906; V-160 to D-906; P-161 to D-906; R-162 to D-906; G-163 to D-906;D-164 to D-906; Y-165 to D-906; I-166 to D-906; A-167 to D-906; F-168 to D-906; N-169 to D-906; T-170 toD-906; D-171 to D-906; E-172 to D-906; C-173 to D-906; T-174 to D-906; A-175 to D-906; T-176 to D-906;L-177 to D-906; M-178 to D-906; Y-179 to D-906; A-180 to D-906; V-181 to D-906; N-182 to D-906; L-183 toD-906; K-184 to D-906; Q-185 to D-906; S-186 to D-906; G-187 to D-906; T-188 to D-906; V-189 to D-906;N-190 to D-906; F-191 to D-906; E-192 to D-906; Y-193 to D-906; Y-194 to D-906;
Y-195 to D-906; P-196 toD-906; D-197 to D-906; S-198 to D-906; S-199 to D-906; I-200 to D-906; I-201 to D-906; F-202 to D-906; E-203to D-906; F-204 to D-906; F-205 to D-906; V-206 to D-906;
Q-207 to D-906; N-208 to D-906; D-209 to D-906;Q-210 to D-906; C-211 to D-906;
Q-212 to 2o D-906; P-213 to D-906; N-214 to D-906; A-215 to D-906; D-216 toD-906; D-217 to D-906; 5-218 to D-906; R-219 to D-906; W-220 to D-906; M-221 to D-906; K-222 to D-906;T-223 to D-906; T-224 to D-906; E-225 to D-906; K-226 to D-906; G-227 to D-906; W-228 to D-906;
E-229 toD-906; F-230 to D-906; H-231 to D-906; S-232 to D-906; V-233 to D-906;
E-234 to D-906; L-235 to D-906;N-236 to D-906; R-237 to D-906; G-238 to D-906; N-239 to D-906;
N-240 to D-906; V-241 to D-906; L-242 toD-906; Y-243 to D-906; W-244 to D-906;
R-245 to D-906; T-246 to D-906; T-247 to D-906; A-248 to D-906;F-249 to D-906; S-250 to D-906; V-251 to D-906; W-252 to D-906; T-253 to D-906; K-254 to D-906; V-255 toD-906; P-256 to D-906; K-257 to D-906; P-258 to D-906; V-259 to D-906; L-260 to D-906; V-261 to D-906;R-262 to D-906; N-263 to D-906; I-264 to D-906; A-265 to D-906; I-266 to D-906;
T-267 to D-906; G-268 toD-906; V-269 to D-906; A-270 to D-906; Y-271 to D-906; T-272 to D-906; S-273 to D-906; E-274 to D-906;C-275 to D-906; F-276 to D-906; P-277 to D-906; C-278 to D-906; K-279 to D-906; P-280 to D-906; G-281 toD-906; T-282 to D-906; Y-283 to D-906; A-284 to D-906; D-285 to D-906; K-286 to D-906; Q-287 to D-906;6-288 to D-906; S-289 to D-906; S-290 to D-906; F-291 to D-906; C-292 to D-906; K-293 to D-906; L-294 toD-906; C-295 to D-906; P-296 to D-906; A-297 to D-906; N-298 to D-906; S-299 to D-906;
Y-300 to D-906;S-301 to D-906; N-302 to D-906; K-303 to D-906; G-304 to D-906; E-305 to D-906; T-306 to D-906; S-307 toD-906; C-308 to D-906; H-309 to D-906; Q-310 to D-906; C-311 to D-906; D-312 to D-906; P-313 to D-906;D-314 to D-906; K-315 to D-906; Y-316 to D-906; S-317 to D-906; E-318 to D-906; K-319 to D-906; G-320 toD-906; S-321 to D-906; S-322 to D-906; S-323 to D-906; C-324 to D-906; N-325 to D-906; V-326 to D-906;8-327 to D-906; P-328 to D-906; A-329 to D-906; C-330 to D-906; T-331 to D-906; D-332 to D-906;
K-333 toD-906; D-334 to D-906; Y-335 to D-906; F-336 to D-906; Y-337 to D-906; T-338 to D-906; H-339 to D-906;T-340 to D-906; A-341 to D-906; C-342 to D-906; D-343 to D-906; A-344 to D-906; N-345 to D-906; G-346 toD-906; E-347 to D-906; T-348 to D-906; Q-349 to D-906; L-350 to D-906; M-351 to D-906; Y-352 to D-906;x-353 to D-906; W-354 to D-906; A-355 to D-906; K-356 to D-906; P-357 to D-906; K-358 to D-906; I-359 toD-906; C-360 to D-906; S-361 to D-906; E-362 to D-906; D-363 to D-906; L-364 to D-906; E-365 to D-906;6-366 to D-906; A-367 to D-906; V-368 to D-906; K-369 to D-906; L-370 to D-906; P-371 to D-906; A-372 toD-906; S-373 to D-906; G-374 to D-906; V-375 to D-906; K-376 to D-906; T-377 to D-906; H-378 to D-906;C-379 to D-906; P-380 to D-906; P-381 to D-906; C-382 to D-906; N-383 to D-906; P-384 to D-906; G-385 toD-906; F-386 to D-906; F-387 to D-906; K-388 to D-906; T-389 to D-906; N-390 to D-906; N-391 to D-906;S-392 to D-906; T-393 to D-906; C-394 to D-906; Q-395 to D-906; P-396 to D-906; C-397 to D-906; P-398 toD-906; Y-399 to D-906; G-400 to D-906; S-401 to D-906; Y-402 to D-906; S-403 to D-906; N-404 to D-906;G-405 to D-906; S-406 to D-906; D-407 to D-906; C-408 to D-906; T-409 to D-906;
R-410 to D-906; C-411 toD-906; P-412 to D-906; A-413 to D-906; G-414 to D-906; T-415 to D-906; E-416 to D-906; P-417 to D-906;A-418 to D-906; V-419 to D-906; G-420 to D-906; F-421 to D-906; E-422 to D-906; Y-423 to D-906; K-424 toD-906; W-425 to D-906; W-426 to D-906; N-427 to D-906; T-428 to D-906; L-429 to D-906; P-430 to D-906;T-431 to D-906; N-432 to D-906; M-433 to D-906; E-434 to D-906; T-435 to D-906; T-436 to D-906; V-437 toD-906; L-438 to D-906; S-439 to D-906; G-440 to D-906; I-441 to D-906; N-442 to D-906;
F-443 to D-906; E-444to D-906; Y-445 to D-906; K-446 to D-906; G-447 to D-906; M-448 to D-906; T-449 to D-906; G-450 to D-906;W-451 to D-906; E-452 to D-906; V-453 to D-906; A-454 to D-906; G-455 to D-906; D-456 to D-906; H-457 toD-906; I-458 to D-906; Y-459 to D-906; T-460 to D-906; A-461 to D-906; A-4b2 to D-906; G-463 to D-906;A-464 to D-906; S-465 to D-906; D-466 to D-906; N-467 to D-906; D-468 to D-906; F-469 to D-906; M-470 toD-906; I-471 to D-906; L-472 to D-906; T-473 to D-906; L-474 to D-906; V-475 to D-906;
V-476 to D-906; P-477to D-906; G-478 to D-906; F-479 to D-906; R-480 to D-906; P-481 to D-906; P-482 to D-906; Q-483 to D-906;5-484 to D-906; V-485 to D-906; M-486 to D-906; A-487 to D-906; D-488 to D-906; T-489 to D-906; E-490 toD-906; N-491 to D-906; K-492 to D-906; E-493 to D-906; V-494 to D-906; A-495 to D-906; R-496 to D-906;I-497 to D-906; T-498 to D-906; F-499 to D-906; V-500 to D-906; F-501 to D-906; E-502 to D-906; T-503 toD-906; L-504 to D-906; C-505 to D-906; S-506 to D-906; V-507 to D-906; N-508 to D-906; C-509 to D-906;E-510 to D-906; L-511 to D-906; Y-512 to D-906; F-513 to D-906; M-514 to D-906; V-S 15 to D-906; G-516 toD-906; V-517 to D-906; N-518 to D-906; S-519 to D-906;
R-520 to D-906; T-521 to D-906; N-522 to D-906;T-523 to D-906; P-524 to D-906; V-525 to D-906; E-526 to D-906; T-527 to D-906; W-528 to D-906; K-529 toD-906; G-530 to D-906; S-531 to D-906; K-532 to D-906; G-533 to D-906; K-534 to D-906; Q-535 to D-906;S-536 to D-906; Y-537 to D-906; T-538 to D-906; Y-539 to D-906; I-540 to D-906; I-541 to D-906;
E-542 toD-906; E-543 to D-906; N-544 to D-906; T-545 to D-906; T-546 to D-906; T-547 to D-906; S-548 to D-906;F-549 to D-906; T-550 to D-906; W-551 to D-906; A-552 to D-906; F-553 to D-906; Q-554 to D-906; R-555 toD-906; T-556 to D-906; T-557 to D-906; F-558 to D-906; H-559 to D-906; E-560 to D-906; A-561 to D-906;S-562 to D-906; R-563 to D-906; K-564 to D-906; Y-565 to D-906; T-566 to D-906; N-567 to D-906; D-568 toD-906; V-569 to D-906; A-570 to D-906; K-571 to D-906; I-572 to D-906; Y-573 to D-906; S-574 to D-906;
I-575to D-906; N-576 to D-906; V-577 to D-906; T-578 to D-906; N-579 to D-906; V-580 to D-906; M-581 to D-906;N-582 to D-906; G-583 to D-906; V-584 to D-906; A-585 to D-906; S-586 to D-906; Y-587 to D-906; C-588 toD-906; R-589 to D-906; P-590 to D-906; C-591 to D-906; A-592 to D-906; L-593 to D-906; E-594 to D-906;A-595 to D-906; S-596 to D-906; D-597 to D-906; V-598 to D-906; G-599 to D-906; S-600 to D-906; S-601 toD-906; C-602 to D-906; T-603 to D-906; S-604 to D-906; C-605 to D-906; P-606 to D-906; A-607 to D-906;6-608 to D-906; Y-609 to D-906; Y-610 to D-906; I-611 to D-906; D-612 to D-906; R-613 to D-906; D-614 toD-906; S-615 to D-906; G-616 to D-906; T-617 to D-906; C-618 to D-906; H-619 to D-906; S-620 to D-906;C-621 to D-906; P-622 to D-906; P-623 to D-906; N-624 to D-906; T-625 to D-906; I-626 to D-906; L-627 toD-906; K-628 to D-906; A-629 to D-906; H-630 to D-906; Q-631 to D-906; P-632 to D-906; Y-633 to D-906;6-634 to D-906; V-635 to D-906; Q-636 to D-906; A-637 to D-906; C-638 to D-906; V-639 to D-906; P-640 toD-906; C-641 to D-906; G-642 to D-906; P-643 to D-906; G-644 to D-906; T-645 to D-906; K-646 to D-906;N-647 to D-906; N-648 to D-906; K-649 to D-906; I-650 to D-906; H-651 to D-906;
S-652 to D-906; L-653 toD-906; C-654 to D-906; Y-655 to D-906; N-656 to D-906; D-657 to D-906; C-658 to D-906; T-659 to D-906;F-660 to D-906; S-661 to D-906; R-662 to D-906; N-663 to D-906; T-664 to D-906; P-665 to D-906; T-666 toD-906; R-667 to D-906; T-668 to D-906; F-669 to D-906; N-670 to D-906; Y-671 to D-906; N-672 to D-906;F-673 to D-906; S-674 to D-906;
A-675 to D-906; L-676 to D-906; A-677 to D-906; N-678 to D-906; T-679 toD-906;
V-680 to D-906; T-681 to D-906; L-682 to D-906; A-683 to D-906; G-684 to D-906; G-685 to D-906;P-686 to D-906; S-687 to D-906; F-688 to D-906; T-689 to D-906; S-690 to D-906;
K-691 to D-906; G-692 toD-906; L-693 to D-906; K-694 to D-906; Y-695 to D-906; F-696 to D-906; H-697 to D-906; H-698 to D-906;F-699 to D-906; T-700 to D-906; L-701 to D-906; S-702 to D-906; L-703 to D-906; C-704 to D-906; G-705 toD-906; N-706 to D-906; Q-707 to D-906; 6-708 to D-906; R-709 to D-906; K-710 to D-906; M-711 to D-906;S-712 to D-906; V-713 to D-906; C-714 to D-906; T-715 to D-906; D-716 to D-906; N-717 to D-906; V-718 toD-906; T-719 to D-906; D-720 to D-906; L-721 to D-906; R-722 to D-906; I-723 to D-906;
P-724 to D-906; E-725to D-906; G-726 to D-906; E-727 to D-906; S-728 to D-906; G-729 to D-906; F-730 to D-906; S-731 to D-906;x-732 to D-906; S-733 to D-906; I-734 to D-906; T-735 to D-906; A-736 to D-906; Y-737 to D-906; V-738 toD-906; C-739 to D-906; Q-740 to D-906; A-741 to D-906; V-742 to D-906; I-743 to D-906; I-744 to D-906; P-745to D-906; P-746 to D-906; E-747 to D-906; V-748 to D-906; T-749 to D-906; G-750 to D-906; Y-751 to D-906;K-752 to D-906; A-753 to D-906; G-754 to D-906; V-755 to D-906; S-756 to D-906;
S-757 to D-906; Q-758 toD-906; P-759 to D-906; V-760 to D-906; S-761 to D-906; L-762 to D-906; A-763 to D-906; D-764 to D-906;8-765 to D-906; L-766 to D-906; I-767 to D-906; G-768 to D-906; V-769 to D-906; T=770 to D-906; T-771 toD-906; D-772 to D-906; M-773 to D-906; T-774 to D-906; L-775 to D-906; D-776 to D-906; G-777 to D-906;I-778 to D-906; T-779 to D-906; S-780 to D-906; P-781 to D-906; A-782 to D-906; E-783 to D-906; L-784 toD-906; F-785 to to D-906; H-786 to D-906; L-787 to D-906; E-788 to D-906; S-789 to D-906; L-790 to D-906;6-791 to D-906; I-792 to D-906; P-793 to D-906; D-794 to D-906; V-795 to D-906; I-796 to D-906; F-797 toD-906; F-798 to D-906; Y-799 to D-906; R-800 to D-906; S-801 to D-906;
N-802 to D-906; D-803 to D-906;V-804 to D-906; T-805 to D-906; Q-806 to D-906;
S-807 to D-906; C-808 to D-906; S-809 to D-906; S-810 toD-906; G-811 to D-906; R-812 to D-906; S-t5 813 to D-906; T-814 to D-906; T-815 to D-906; I-816 to D-906; R-817to D-906; V-818 to D-906; R-819 to D-906; C-820 to D-906; S-821 to D-906; P-822 to D-906; Q-823 to D-906;K-824 to D-906; T-825 to D-906; V-826 to D-906; P-827 to D-906; G-828 to D-906;
S-829 to D-906; L-830 toD-906; L-831 to D-906; L-832 to D-906; P-833 to D-906; G-834 to D-906; T-835 to D-906; C-836 to D-906;S-837 to D-906; D-838 to D-906; G-839 to D-906; T-840 to D-20 906; C-841 to D-906; D-842 to D-906; G-843 toD-906; C-844 to D-906; N-845 to D-906; F-846 to D-906; H-847 to D-906; F-848 to D-906; L-849 to D-906;W-850 to D-906; E-851 to D-906; S-852 to D-906; A-853 to D-906; A-854 to D-906; A-855 to D-906; C-856 toD-906; P-857 to D-906; L-858 to D-906; C-859 to D-906; S-860 to D-906; V-861 to D-906;
A-862 to D-906;D-863 to D-906; Y-864 to D-906; H-865 to D-906; A-866 to D-906; I-867 to D-906; V-25 868 to D-906; S-869 toD-906; S-870 to D-906; C-871 to D-906; V-872 to D-906; A-873 to D-906; G-874 to D-906; I-875 to D-906;Q-876 to D-906; K-877 to D-906; T-878 to D-906; T-879 to D-906; Y-880 to D-906; V-881 to D-906; W-882 toD-906; R-883 to D-906; E-884 to D-906; P-885 to D-906; K-886 to D-906; L-887 to D-906; C-888 to D-906;5-889 to D-906; G-890 to D-906; G-891 to D-906; I-892 to D-906; S-893 to D-906; L-894 to D-906;
P-895 toD-30 906; E-896 to D-906; Q-897 to D-906; R-898 to D-906; V-899 to D-906; and T-900 to D-906 of SEQ ID N0:40. Polynucleotides encoding these polypeptides are also encompassed by the mvent~on.
The present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the predicted extracellular domain of the predicted mature 35 TR13 protein, with the amino acid sequence shown in Figures 7A-D (SEQ ID
N0:40), up to the alanine residue at position number 48, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 42-m' of Figures 7A-D, where m' is an integer from 48 to 906 corresponding to the position of the amino acid residue in Figures 7A-D.
Thus, the invention provides TR13 polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: T-42 to D-906; T-42 to I-905; T-42 to T-904; T-42 to K-903; T-42 to C-902; T-42 to I-901sT-42 to T-900; T-42 to V-899; T-42 to R-898; T-42 to Q
897; T-42 to E-896; T-42 to P-895; T-42 to L-894; T-42 toS-893; T-42 to I-892;
T-42 to 6-891; T-42 to G-890; T-42 to S-889; T-42 to C-888; T-42 to L-887; T-42 to K-886;T-42 to P-885; T-42 to E-884; T-42 to R-883; T-42 to W-882; T-42 to V-881; T-42 to Y-880; T-42 to T-879; T-42 toT-878; T-42 to K-877; T-42 to Q-876; T-42 to I-875; T-42 to G-874;
T-42 to A-t0 873; T-42 to V-872; T-42 to C-871sT-42 to S-870; T-42 to S-869; T-42 to V-868; T-42 to I-867; T-42 to A-866; T-42 to H-865; T-42 to Y-864; T-42 toD-863; T-42 to A-862;
T-42 to V-861; T-42 to S-860; T-42 to C-859; T-42 to L-858; T-42 to P-857; T-42 to C-856;T-42 to A-855; T-42 to A-854; T-42 to A-853; T-42 to S-852; T-42 to E-851; T-42 to W-850; T-42 to L-849; T-42 toF-848; T-42 to H-847; T-42 to F-846; T-42 to N-845; T-42 to C-844;
T-42 to G-843; T-4.2 to D-842; T-42 to C-841sT-42 to T-840; T-42 to G-839; T-42 to D-838; T-42 to S-837; T-42 to C-836; T-42 to T-835; T-42 to G-834; T-42 toP-833; T-42 to L-832;
T-42 to L-831; T-42 to L-830; T-42 to S-829; T-42 to G-828; T-42 to P-827; T-42 to V-826;T-42 to T-825; T-42 to K-824; T-42 to Q-823; T-42 to P-822; T-42 to S-821; T-42 to C-820; T-42 to 8-819; T-42 toV-818; T-42 to R-817; T-42 to I-816; T-42 to T-815; T-42 to T-814;
T-42 to S-813; T-42 to R-812; T-42 to G-811;T-42 to S-810; T-42 to S-809; T-42 to C-808;
T-42 to S-807; T-42 to Q-806; T-42 to T-805; T-42 to V-804; T-42 toD-803; T-42 to N-802;
T-42 to S-801; T-42 to R-800; T-42 to Y-799; T-42 to F-798; T-42 to F-797; T-42 to I-796;T-42 to V-795; T-42 to D-794; T-42 to P-793; T-42 to I-792; T-42 to G-791; T-42 to L-790; T-42 to S-789; T-42 toE-788; T-42 to L-787; T-42 to H-786; T-42 to F-785; T-42 to L-784;
T-42 to E-783; T-42 to A-782; T-42 to P-781sT-42 to S-780; T-42 to T-779; T-42 to I-778;
T-42 to 6-777; T-42 to D-776; T-42 to L-775; T-42 to T-774; T-42 toM-773; T-42 to D-772;
T-42 to T-771; T-42 to T-770; T-42 to V-769; T-42 to G-768; T-42 to I-767; T-42 to L-766;T-42 to R-765; T-42 to D-764; T-42 to A-763; T-42 to L-762; T-42 to S-761; T-42 to V-760; T-42 to P-759; T-42 toQ-758; T-42 to S-757; T-42 to S-756; T-42 to V-755; T-42 to G-754;
T-42 to A-753; T-42 to K-752; T-42 to Y-751sT-42 to G-750; T-42 to T-749; T-42 to V-748;
T-42 to ~
747; T-4.2 to P-746; T-42 to P-745; T-42 to I-744; T-42 toI-743; T-42 to V-742; T-42 to A-741; T-42 to Q-740; T-42 to C-739; T-42 to V-738; T-42 to Y-737; T-42 to A-736;T-42 to T-735; T-42 to I-734; T-42 to S-733; T-42 to K-732; T-42 to S-731; T-42 to F-730; T-42 to 6-729; T-42 toS-728; T-42 to E-727; T-42 to G-726; T-42 to E-725; T-42 to P-724;
T-42 to I-723; T-42 to R-722; T-42 to L-721sT-42 to D-720; T-42 to T-719; T-42 to V-718;
T-42 to N-717; T-42 to D-716; T-42 to T-715; T-42 to C-714; T-42 toV-713; T-42 to S-712;
T-42 to M-711; T-42 to K-710; T-42 to R-709; T-42 to G-708; T-42 to Q-707; T-42 toN-706;
T-42 to 6-705; T-42 to C-704; T-42 to L-703; T-42 to S-702; T-42 to L-701; T-42 to T-700; T-42 to F-699;T-42 to T-4298; T-42 to T-4297; T-42 to F-696; T-42 to Y-695; T-42 to K-694; T-42 to L-693; T-42 to G-692; T-42 toK-691; T-42 to S-690; T-42 to T-689; T-42 to F-688;
T-42 to S-687; T-42 to P-686; T-42 to G-685; T-42 to G-684;T-42 to A-683; T-42 to L-682;
T-42 to T-681; T-42 to V-680; T-42 to T-679; T-42 to N-678; T-42 to A-677; T-42 toL-676;
T-42 to A-675; T-42 to S-674; T-42 to F-673; T-42 to N-672; T-42 to Y-671; T-42 to N-670; T-42 to F-669;T-42 to T-668; T-42 to R-667; T-42 to T-666; T-42 to P-665; T-42 to T-664;
T-42 to N-663; T-42 to R-662; T-42 toS-661; T-42 to F-660; T-42 to T-659; T-42 to C-658;
T-42 to D-657; T-42 to N-656; T-42 to Y-655; T-42 to C-654;T-42 to L-653; T-42 to S-652;
T-42 to T-4251; T-42 to I-650; T-42 to K-649; T-42 to N-648; T-42 to N-647; T-42 toK-646; T-42 to T-645; T-42 to G-644; T-42 to P-643; T-42 to G-642; T-42 to C-641; T-42 to P-640; T-42 to V-639;T-42 to C-638; T-42 to A-637; T-42 to Q-636; T-42 to V-635; T-42 to G-634;
T-42 to Y-633; T-42 to P-632; T-42 toQ-631; T-42 to T-4230; T-42 to A-629; T-42 to K-628; T-42 to L-627; T-42 to I-626; T-42 to T-625; T-42 to N-624;T-42 to P-623; T-42 to P-622;
T-42 to C-621; T-42 to S-620; T-42 to T-4219; T-42 to C-618; T-42 to T-617; T-42 toG-616; T-42 to S-615; T-42 to D-614; T-42 to R-613; T-42 to D-612; T-42 to I-611; T-42 to Y-610; T-42 to Y-609;T-42 to G-608; T-42 to A-607; T-42 to P-606; T-42 to C-605; T-42 to S-604;
T-42 to T-603; T-42 to C-602; T-42 toS-601; T-42 to S-600; T-42 to G-599; T-42 to V-598;
T-42 to D-597; T-42 to S-596; T-42 to A-595; T-42 to E-594;T-42 to L-593; T-42 to A-592;
T-42 to C-591; T-42 to P-590; T-42 to R-589; T-42 to C-588; T-42 to Y-587; T-42 toS-586;
T-42 to A-585; T-42 to V-584; T-42 to G-583; T-42 to N-582; T-42 to M-581; T-42 to V-580; T-42 toN-579; T-42 to T-578; T-42 to V-577; T-42 to N-576; T-42 to I-575; T-42 to S-574; T-42 to Y-573; T-42 to I-572;T~2 to K-571; T-42 to A-570; T-42 to V-569; T-42 to D-568;
T-42 to N-567; T-42 to T-566; T-42 to Y-565; T-42 toK-564; T-42 to R-563; T-42 to S-562;
T-42 to A-561; T-42 to E-560; T-42 to H-559; T-42 to F-558; T-42 to T-557;T-4.2 to T-556; T-42 to R-555; T-42 to Q-554; T-42 to F-553; T-42 to A-552; T-42 to W-551; T-42 to T-550; T-42 toF-549; T-42 to S-548; T-42 to T-547; T-42 to T-546; T-42 to T-545; T-42 to N-544; T-42 to E-543; T-42 to E-542;T-42 to I-541; T-42 to I-540; T-42 to Y-539; T-42 to T-538;
T-42 to Y-537; T-42 to S-536; T-42 to Q-535; T-42 toK-534; T-42 to G-533; T-42 to K-532;
T-42 to S-531; T-42 to G-530; T-42 to K-529; T-42 to W-528; T-42 toT-527; T-42 to E-526;
T-42 to V-525; T-42 to P-524; T-42 to T-523; T-42 to N-522; T-42 to T-521; T-42 to R-520;T-42 to S-S 19; T-42 to N-518; T-42 to V-517; T-42 to G-516; T-42 to V-S 15; T-42 to M-514; T-42 to F-513; T-42 toY-512; T-42 to L-511; T-42 to E-510; T-42 to C-509; T-42 to N-508;
T-42 to V-507; T-42 to S-506; T-42 to C-SOS;T-42 to L-504; T-42 to T-503; T-42 to E-502;
T-42 to F-501; T-42 to V-500; T-42 to F-499; T-42 to T-498; T-42 toI-497; T-42 to R-496;
T-42 to A-495; T-42 to V-494; T-42 to E-493; T-42 to K-492; T-42 to N-491; T-42 to E-490;T-42 to T-489; T-42 to D-488; T-42 to A-487; T-42 to M-486; T-42 to V-485; T-42 to S-484; T-42 to Q-483; T-42 toP-482; T-42 to P-481; T-42 to R-480; T-42 to F-479; T-42 to G-478;
T-42 to P-477; T-42 to V-476; T-42 to V-475;T-42 to L-474; T-42 to T-473; T-42 to L-472;
T-42 to I-471; T-42 to M-470; T-42 to F-469; T-42 to D-468; T-42 toN-467; T-42 to D-466;
T-42 to S-465; T-42 to A-464; T-42 to G-463; T-42 to A-462; T-42 to A-461; T~2 to T-460;T-42 to Y-459; T-42 to I-458; T-42 to H-457; T-42 to D-456; T-42 to G-455; T-42 to A-454; T-42 to V-453; T-42 toE-452; T-42 to W-451; T-42 to G-450; T-42 to T-449; T-42 to M~48;
T-42 to G-447; T-42 to K-446; T-42 toY~45; T-42 to E-444; T-42 to F-443; T-42 to N-442;
T-42 to I-441; T-42 to G-440; T-42 to S-439; T-42 to L-438;T-42 to V-437; T-42 to T-436;
T-42 to T-435; T-42 to E-434; T-42 to M-433; T-42 to N-432; T-42 to T-431; T-42 toP-430;
T-42 to L-429; T-42 to T-428; T-42 to N-427; T-42 to W-426; T-42 to W-425; T-42 to K-424; T-42 toY-423; T-42 to E-422; T-42 to F-421; T-42 to G-420; T-42 to V-419; T-42 to A-4.18; T-42 to P-417; T-42 to E-416;T-42 to T-415; T-42 to G-414; T-42 to A-413; T-42 to P-412;
T-42 to C-411; T-42 to R-410; T-42 to T-409; T-42 toC-408; T-42 to D-407; T-42 to S-406;
T-42 to G-405; T-42 to N-404; T-42 to S-403; T-42 to Y-402; T-42 to S-4.O1;T-42 to G-400; T-42 to Y-399; T-42 to P-398; T-42 to C-397; T-42 to P-396; T-42 to Q-395; T-42 to C-394; T-42 toT-393; T-42 to S-392; T-42 to N-391; T-42 to N-390; T-42 to T-389; T-42 to K-388; T-42 to F-387; T-42 to F-386;T-42 to G-385; T-42 to P-384; T-42 to N-383; T-42 to C-382;
T-42 to P-381; T-42 to P-380; T-42 to C-379; T-42 toH-378; T-42 to T-377; T-42 to K-376;
T-42 to V-375; T-42 to G-374; T-42 to S-373; T-42 to A-372; T-42 to P-371sT-42 to L-370;
T-42 to K-369; T-42 to V-368; T-42 to A-367; T-42 to G-366; T-42 to E-365; T-42 to L-364; T-42 toD-363; T-42 to E-362; T-42 to S-361; T-42 to C-360; T-42 to I-359; T-42 to K-358; T-42 to P-357; T-42 to K-356;T-42 to A-355; T-42 to W-354; T-42 to K-353; T-42 to Y-352;
T-42 to M-351; T-42 to L-350; T-42 to Q-349; T-42 toT-348; T-42 to E-347; T-42 to G-34b;
T-42 to N-345; T-42 to A-344; T-42 to D-343; T-42 to C-342; T-42 to A-341sT-42 to T-340;
T-42 to H-339; T-42 to T-338; T-42 to Y-337; T-42 to F-336; T-42 to Y-335; T-42 to D-334; T-42 toK-333; T-42 to D-332; T-42 to T-331; T-42 to C-330; T-42 to A-329; T-42 to P-328; T-42 to R-327; T-42 to V-326;T-42 to N-325; T-42 to C-324; T-42 to S-323; T-42 to S-322;
T-42 to S-321; T-42 to G-320; T-42 to K-319; T-42 toE-318; T-42 to S-317; T-42 to Y-316;
T-42 to K-315; T-42 to D-314; T-42 to P-313; T-42 to D-312; T-42 to C-311;T-42 to Q-310;
T-42 to H-309; T-42 to C-308; T-42 to S-307; T-42 to T-306; T-42 to E-305; T-42 to G-304; T-42 toK-303; T-42 to N-302; T-42 to S-301; T-42 to Y-300; T-42 to S-299; T-42 to N-298; T-42 to A-297; T-42 to P-296;T-42 to C-295; T-42 to L-294; T-42 to K-293; T-42 to C-292;
T-42 to F-291; T-42 to S-290; T-42 to S-289; T-42 toG-288; T-42 to Q-287; T-42 to K-286;
T-42 to D-285; T-42 to A-284; T-42 to Y-283; T-42 to T-282; T-42 to G-281sT-42 to P-280;
T-42 to K-279; T-42 to C-278; T-42 to P-277; T-42 to F-276; T-42 to C-275; T-42 to E-274; T-42 toS-273; T-42 to T-272; T-42 to Y-271; T-42 to A-270; T-42 to V-269; T-42 to G-268; T-42 to T-267; T-42 to I-266;T-42 to A-265; T-42 to I-264; T-42 to N-263; T-42 to R-262;
T-42 to V-261; T-42 to L-260; T-42 to V-259; T-42 toP-258; T-42 to K-257; T-42 to P-256;
T-42 to V-255; T-42 to K-254; T-42 to T-253; T-42 to W-252; T-42 to V-251sT-42 to S-250;
T-42 to F-249; T-42 to A-248; T-42 to T-247; T-42 to T-24b; T-4.2 to R-245; T-42 to W-244; T-42 toY-243; T-42 to L-242; T-42 to V-241; T-42 to N-240; T-42 to N-239; T-42 to G-238; T-42 to 8-237; T-42 to N-236;T-42 to L-235; T-42 to E-234; T-42 to V-233; T-42 to S-232;
T-42 to H-231; T-42 to F-230; T-42 to E-229; T-42 toW-228; T-42 to G-227; T-42 to K-226;
T-42 to E-225; T-42 to T-224; T-42 to T-223 ; T-42 to K-222; T-42 toM-221; T-42 to W-220; T-42 to R-219; T-42 to S-218; T-42 to D-217; T-42 to D-216; T-4.2 to A-215; T-42 toN-214; T-42 to P-213; T-42 to Q-212; T-42 to C-211; T-42 to Q-210; T-42 to D-209; T-42 to N-208; T-42 to Q-207;T-42 to V-206; T-42 to F-205; T-42 to F-204; T-42 to E-203; T-42 to F-202;
T-42 to I-201; T-42 to I-200; T-42 toS-199; T-42 to S-198; T-42 to D-197; T-42 to P-196;
T-42 to Y-195; T-42 to Y-194; T-42 to Y-193; T-42 to E-192;T-42 to F-191; T-42 to N-190;
T-42 to V-189; T-42 to T-188; T-42 to G-187; T-42 to S-186; T-42 to Q-185; T-42 toK-184;
T-42 to L-183; T-42 to N-182; T-42 to V-181; T-42 to A-180; T-42 to Y-179; T-42 to M-178; T-42 to L-177;T-42 to T-176; T-42 to A-175; T-42 to T-174; T-42 to C-173; T-42 to E-172;
T-42 to D-171; T-42 to T-170; T-42 toN-169; T-42 to F-168; T-42 to A-167; T-42 to I-166;
T-42 to Y-165 ; T-42 to D-164; T-42 to G-163 ; T-42 to R-162;T-42 to P-161; T-42 to V -160; T-42 to W-159; T-42 to K-158; T-42 to S-157; T-42 to S-156; T-42 to T-155; T-42 toC-154;
T-42 to N-153; T-42 to G-152; T-42 to T-151; T-42 to S-150; T-42 to E-149; T-42 to A-148; T-42 to A-147;T-42 to S-146; T-42 to D-145; T-42 to D-144; T-42 to L-143; T-42 to E-142;
T-42 to M-141; T-42 to N-140; T-42 toA-139; T-42 to S-138; T-42 to L-137; T-42 to S-136;
T-42 to A-135 ; T-42 to F-134; T-42 to G-13 3 ; T-42 to H-132;T-42 to P-131; T-42 to L-130; T-42 to E-129; T-42 to D-128; T-42 to W-127; T-42 to E-126; T-42 to D-125; T-42 toF-124;
T-42 to 8-123; T-42 to I-122; T-42 to G-121; T-42 to T-120; T-42 to G-119; T-42 to L-118; T-42 to S-117;T-42 to Y-116; T-42 to R-115; T-42 to G-114; T-42 to E-113; T-42 to A-112;
T-42 to C-111; T-42 to P-110; T-42 toK-109; T-42 to C-108; T-42 to S-107; T-42 to Q-106;
T-42 to D-105; T-42 to K-104; T-42 to M-103 ; T-42 to D-102;T-42 to L-1 O 1; T-42 to F-100; T-42 to E-99; T-42 to G-98; T-42 to A-97; T-42 to N-96; T-42 to C-95; T-42 to S-94;T-42 to F-93; T-42 to S-92; T-42 to C-91; T-42 to E-90; T-42 to T-89; T-42 to G-88; T-42 to K-87;
T-42 to V-86;T-42 to P-85; T-42 to D-84; T-42 to P-83; T-42 to L-82; T-42 to S-81; T-42 to T-80; T-42 to C-79; T-42 to L-78; T-42to G-77; T-42 to P-76; T-42 to T-75; T-42 to H-74;
T-42 to P-73;
T-42 to V-72; T-42 to A-71; T-42 to V-70; T-42 toR-69; T-42 to W-68; T-42 to R-67; T-42 to S-66; T-42 to G-65; T-42 to T-64; T-42 to S-63; T-42 to D-62; T-42 toC-61; T-42 to A-60; T-42 to T-59; T-42 to Y-58; T-42 to E-57; T-42 to Y-56; T-4.2 to H-55; T-42 to Y-54; T-42 toE-53; T-42 to S-52; T-42 to E-51; T-42 to K-50; T-42 to C-49; and T-42 to A-48 of SEQ ID
N0:40. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues n'- m' and/or n2- m' of Figures lA-C (i.e., SEQ ID N0:2), where n', n2, and m' are integers as described above. Thus, any of the above listed N- or C-terminal deletions can be combined to produce an N- and C-terminal deleted TR13 polypeptide.

The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues n'- m' and/or n2- m' of Figures 7A-D (SEQ ID N0:40), where n', n2, and m' are integers as described above. Thus, any of the above listed N- or C-terminal deletions can be combined to produce an N- and C-terminal deleted TR13 polypeptide.
It will be recognized in the art that some amino acid sequences of TR13 polypeptides can be varied without significant effect on the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. Thus, the invention further includes variations of to the TR13 polypeptide, which show substantial TR13 activity or which include regions of TR13 polypeptides, such as the polypeptide portions discussed herein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions. As indicated above, guidance concerning which amino acid changes are likely to be phenotypically silent can be found in J.U. Bowie et al., Science 247:1306-1310 (1990).
Thus, the fragment, derivative, or analog of the polypeptide of SEQ ID N0:2 or SEQ
ID N0:40, or that encoded by the cDNA deposited as ATCC Deposit No. PTA-349 or ATCC
Deposit No. PTA-507, may be (i) one in which at least one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue(s), and more preferably at least one but less than ten conserved amino acid 2o residues) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
Of particular interest are substitutions of charged amino acids with another charged 3o amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the TR13 polypeptide. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).
The replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 361:266-268 (1993), describes certain mutations resulting in selective binding of TNF-a to only one of the two known types of TNF
receptors. Thus, the TR13 polypeptides of the present invention may include one or more amino acid substitutions, deletions, or additions, either from natural mutations or human manipulation.
As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table V).
TABLE V. Conservative Amino Acid Substitutions Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine Polar ~ Glutamine Asparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Seri ne Threonine Methionine G1 cine In specific embodiments, the number of substitutions, additions or deletions in the amino acid sequence of Figures lA-C or Figures 7A-D and/or any of the polypeptide fragments described herein (e.g., one or more of the cysteine rich domains, the mature extracellular domain, etc.) is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, S, 4, 3, 2, 1 or 30-20, 20-15, 20-10, 15-10, 10-1, 5-10, 1-5, 1-3 or 1-2.
Amino acids in the TR13 polypeptides of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in 2o vitro proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).
To improve or alter the characteristics of TR13 polypeptides, protein engineering may be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res. 13:4331 ( 1986); and Zoller et al., Nucl. Acids Res. 10:6487 ( 1982)), cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (see e.g., Wells et al., Philos. Traps. R. Soc. London SerA 317:415 (1986)).
Thus, the invention also encompasses TR13 derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate TR13 polypeptides that are better suited for expression, scale up, etc., in the host cells chosen. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate 2o disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites. To this end, a variety of amino acid substitutions at one or both of the first or third amino acid positions on any one or more of the glycosylation recognitions sequences in the TR13 polypeptides of the invention, and/or an amino acid deletion at the second position of any one or more such recognition sequences will prevent glycosylation of the TR13 at the modified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J 5(6):1193-1197). Additionally, one or more of the amino acid residues of the polypeptides of the invention (e.g., arginine and lysine residues) may be deleted or substituted with another residue to eliminate undesired processing by proteases such as, for 3o example, furins or kexins.
The polypeptides of the present invention include a polypeptide comprising, or alternatively, consisting of the polypeptide encoded by the cDNA deposited as ATCC Deposit No. PTA-349, including the leader; a polypeptide comprising, or alternatively, consisting of the mature polypeptide encoded by the deposited cDNA minus the leader (i.e., the mature protein); a polypeptide comprising, or alternatively, consisting of amino acids from about 1 to about 750 of SEQ ID N0:2; a polypeptide comprising, or alternatively, consisting of amino acids from about 2 to about 750 of SEQ ID N0:2; a polypeptide comprising, or alternatively, consisting of amino acids from about 1 to about 331 in SEQ ID N0:2; a polypeptide comprising, or alternatively, consisting of any one or more of the four cysteine rich domains disclosed in Figures lA-C (predicted to constitute amino acids from about 105 to about 170, 251 to about 265, about 331 to about 410, and about 580 to about 610 of SEQ ID
N0:2); as well as polypeptides which are at least 80% identical, more preferably at least 90% or 95%
identical, still more preferably at least 96%, 97%, 98%, or 99% identical to the polypeptides described above (e.g., the polypeptide encoded by the deposited cDNA clone, the polypeptide of Figures lA-C (SEQ ID N0:2) and polypeptide fragments thereof such as disclosed herein), and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2,or 1) amino acids, at either extreme or at both extremes.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
The polypeptides of the present invention include a polypeptide comprising, or alternatively, consisting of the polypeptide encoded by the cDNA deposited as ATCC Deposit No. PTA-507, including the leader; a polypeptide comprising, or alternatively, consisting of the mature polypeptide encoded by the deposited cDNA minus the leader (i.e., the mature protein); a polypeptide comprising, or alternatively, consisting of amino acids from about 1 to about 1001 of SEQ ID N0:40; a polypeptide comprising, or alternatively, consisting of amino acids from about 2 to about 1001 of SEQ ID N0:40; a polypeptide comprising, or alternatively, consisting of amino acids from about 1 to about 906 in SEQ ID N0:40; a polypeptide 2o comprising, or alternatively, consisting of amino acids from about 42 to about 1001 in SEQ ID
N0:40; a polypeptide comprising, or alternatively, consisting of amino acids from about 42 to about 906 in SEQ ID N0:40; a polypeptide comprising, or alternatively, consisting of amino acids from about 907 to about 931 in SEQ ID N0:40; a polypeptide comprising, or alternatively, consisting of amino acids from about 932 to about 1001 in SEQ
ID N0:40; a polypeptide comprising, or alternatively, consisting of any of the seven cysteine rich domains disclosed in Figures 7A-D (predicted to constitute amino acids from about 271 to about 421, 271 to about 286, about 290 to about 300, about 301 to about 320, about 329 to about 361, about 404 to about 421, and about 585 to about 595 of SEQ ID N0:40); as well as polypeptides which are at least 80% identical, more preferably at least 90% or 95% identical, 3o still more preferably at least 96%, 97%, 98%, or 99% identical to the polypeptides described above (e.g., the polypeptide encoded by the deposited cDNA clone, the polypeptide of Figures 7A-D (SEQ ID N0:40) and polypeptide fragments thereof, such as those disclosed herein), and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2,or 1) amino acids, at either extreme or at both extremes.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
By a polypeptide (protein) comprising, or alternatively, consisting of, an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a TR13 polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the TR13 polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal to positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, for instance, the amino acid sequence shown in SEQ ID N0:2 or SEQ ID N0:40, or to the amino acid sequence encoded by the cDNA clone deposited as ~5 ATCC Deposit No. PTA-349 or ATCC Deposit No. PTA-507, can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95%
20 identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full-length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
In a specific embodiment, the identity between a reference (query) sequence (a sequence 25 of the present invention) and a subject sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence 30 length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction is made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-terminal truncations of the subject 35 sequence when calculating global percent identity. For subject sequences truncated at the N-and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine to percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program.
If the remaining ~5 90 residues were perfectly matched the final percent identity would be 90%.
In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N-or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside 2o the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.
The present application is also directed to proteins comprising, or alternatively consisting of, a polypeptide sequence at least 90%, 95%, 96%, 97%, 98% or 99%
identical to 25 the TR13 polypeptide sequence set forth as n'-m', and/or n2- ml for polypeptide sequence shown in Figure lA-C or Figure 7A-D herein. In preferred embodiments, the application is directed to proteins comprising, or alternatively consisting of, a polypeptide sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific TR13 N- and C-terminal deletions recited herein. Additional preferred 3o embodiments are directed to fusion proteins comprising these polypeptide sequences.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide described herein. An 35 "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope." The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes.
See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 87:3998- 4002 (1983).
As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, J.G. Sutcliffe et al., "Antibodies That React With Predetermined Sites on Proteins,"
Science 219:660-666 (1983). Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple 1o chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful, for example, to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777. Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least 9, at least 20, at least 25, at least 30, at least 40, at least 50 and most preferably between at least about 55 to about 100 amino acids contained within the amino acid sequence of a polypeptide of the invention. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2,or 1) amino acids, at either extreme or at both extremes.
Non-limiting examples of predicted antigenic polypeptides that can be used to generate TR13 receptor-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about 1 to about 170 in Figures lA-C
(corresponding to about amino acid 1 to about 170 in SEQ ID N0:2); a polypeptide comprising amino acid residues from about 210 to about 318 in Figures lA-C (corresponding to about amino acid 210 to about 318 in SEQ ID N0:2); a polypeptide comprising amino acid residues from about 343 to about 480 in Figures lA-C (corresponding to about amino acid 343 to about 480 in SEQ
ID N0:2); a polypeptide comprising amino acid residues from about 548 to about 592 in Figures lA-C
(corresponding to about amino acid 548 to about 592 in SEQ ID N0:2); and a polypeptide comprising amino acid residues from about 632 to about 742 in Figures lA-C
(corresponding to about amino acid 632 to about 742 in SEQ ID N0:2). As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the TR13 receptor protein. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
Additional non-limiting examples of predicted antigenic polypeptides that can be used to generate TR13 receptor-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about about M1 to about A9, about K12 to about L20, about N47 to about T55, about H58 to about S66, about D63 to about S71, about P77 to about F85, about A90 to about Q98, about F136 to about Q144, about S152 to about C160, about 8159 to about A167, about A211 to about M219, about M235 to about V243, about V266 to about V274, about W277 to about S285, about I290 to about F298, about A310 to about V318, about E343 to about C351, about I360 to about H368, about 6391 to about I399, about F409 to about T417, about S436 to about Y444, about C453 to about S461, about I472 to about 5480, about Y548 to about S556, about C557 to about I565, about V567 to about V575, about T584 to about 6592, about 8632 to about 6640, about W680 to about Y688, about Q684 to about K692, about T698 to about A706, about S726 to about S734, and about S734 to to about L742 of SEQ ID N0:2 (Figures lA-C) correspond to the highly antigenic regions of the TR13 protein, predicted using the Jameson-Wolf antigenic index (See Figure 3 and Table I).
These highly antigenic fragments correspond to the amino acid residues illustrated in Figure lA-C and in SEQ ID N0:2. As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the TR13 receptor protein. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Non-limiting examples of predicted antigenic polypeptides that can be used to generate TR13-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about 1 to about 262 in Figures 7A-D (corresponding to about amino acid 1 to about 262 in SEQ ID N0:40); a polypeptide comprising amino acid residues from about 264 to about 423 in Figures 7A-D (corresponding to about amino acid 264 to about 423 in SEQ ID N0:40); a polypeptide comprising amino acid residues from about 437 to about 789 in Figures 7A-D (corresponding to about amino acid 437 to about 789 in SEQ ID
N0:40); and a polypeptide comprising amino acid residues from about 791 to about 1001 in Figures 7A-D
(corresponding to about amino acid 791 to about 1001 in SEQ ID N0:40). As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the TR13 receptor protein. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
3o Polynucleotides encoding these polypeptides are also encompassed by the invention.
Additional non-limiting examples of predicted antigenic polypeptides that can be used to generate TR13 receptor-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about about Ml to about H9, about V 14 to about I22, about H47 to about H55, about C61 to about R69, about L82 to about E90, about D102 to about P110, about K109 to about S117, about F124 to about H132, about M141 to about E149, about S146 to about C154, about S157 to about W165, about F168 to about T176, about N 182 to about N 190, about Q207 to about A215 , about P213 to about M221, about M221 to about E229, about V233 to about V241, about T253 to about V261, about T282 to about S290, about N298 to about T306, about C308 to about Y316, about K315 to about S323, about P328 to about F336, about A341 to about Q349, about F387 to about Q395, about S403 to about C411, about T409 to about P417, about F443 to about N451, about W451 to about Y459, about A462 to about M470, about 6478 to about M486, about A487 to about A495, about V517 to about V525, about T527 to about Q535, about I541 to about F549, about A561 to about V569, about E594 to about C602, about 1611 to about H619, about 6643 to about I650, about P686 to about K694, about C704 to about 5712, about 8722 to about 1730, about E727 to about T735, about P746 to about 6754, about D776 to about L784, about Y799 to about 5807, about C808 to about I816, about V818 to about V826, about T835 to about 6843, about 8883 to about 6891, about K932 to about K940, about Q935 to about K943, about T949 to about A957, about S977 to about S985, about 5981 to about P989, and about N986 to about L994 of SEQ ID N0:40 (Figures 7A-D) correspond to the highly antigenic regions of the TR13 protein, predicted using the Jameson-Wolf antigenic index (See Figure 9 and Table III). These highly antigenic fragments correspond to the amino acid residues illustrated in Figure 7A-D and in SEQ ID N0:40. As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the TR13 receptor protein. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. R.A. Houghten, "General Method for the Rapid Solid-phase Synthesis of Large Numbers of Peptides: Specificity of Antigen-Antibody Interaction at the Level of Individual Amino Acids," Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986).
As one of skill in the art will appreciate, TR13 receptor polypeptides of the present invention and the epitope-bearing fragments thereof, described herein (e.g., corresponding to a portion of the extracellular domain, such as, for example, amino acid residues 105 to about 170, about 251 to about 265, about 331 to about 410, and/or about 580 to about 610 of SEQ
ID N0:2), can be combined with heterologous polypeptide sequences, for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHI, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric TR13 protein or protein fragment alone (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
As one of skill in the art will appreciate, TR13 receptor polypeptides of the present invention and the epitope-bearing fragments thereof, described herein (e.g., corresponding to a portion of the extracellular domain, such as, for example, amino acid residues 1 to about 262, about 264 to about 423, about 437 to about 789, about 271 to about 421, and/or about 585 to 599 of SEQ ID N0:40), can be combined with heterologous polypeptide sequences, for to example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric TR13 protein or protein fragment alone (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In 2o this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
Preferred TR13 Fc fusions of the present invention include, but are not limited to constructs comprising, or alternatively consisting of, amino acid residues 1 to 750, 10 to 750, to 750, 30 to 750, 40 to 750, 1 to 740, 1 to 730, 1 to 720, 1 to 710, 10 to 740, 10 to 730, and/or 10 to 720 of SEQ ID N0:2. Polynucleotides encoding these TR13 fusions are also encompassed by the invention.
Additional preferred TR13 Fc fusions of the present invention include, but are not limited to constructs comprising, or alternatively consisting of, amino acid residues 1 to 906, 42 to 906, 271 to 421, 585 to 595, 1 to 1001, 10 to 1001, 20 to 1001, 30 to 1001, 42 to 1001, 42 to 906, 1 to 990, 1 to 980, 1 to 970, 1 to 960, 10 to 990, 10 to 980, and/or 10 to 970 of SEQ ID N0:2. Polynucleotides encoding these TR13 fusions are also encompassed by the invention.
The polypeptides of the present invention have uses which include, but are not limited to, as sources for generating antibodies that bind the polypeptides of the invention, and as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
TRl4 Polypeptides The TR14 proteins (polypeptides) of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to monomers and multimers of the TR14 proteins (polypeptides) of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term TR14 homomer, refers to a multimer containing only TR14 proteins of the 1o invention (including TR14 fragments, variants, and fusion proteins, as described herein).
These homomers may contain TR14 proteins having identical or different polypeptide sequences. In a specific embodiment, a homomer of the invention is a multimer containing only TR14 proteins having an identical polypeptide sequence. In another specific embodiment, a homomer of the invention is a multimer containing TR14 proteins having different polypeptide sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing TR14 proteins having identical or different polypeptide sequences) or a homotrimer (e.g., containing TR14 proteins having identical or different polypeptide sequences). In additional embodiments, the homomeric multimer of the invention is at least a, homodimer, at least a homotrimer, or at least a homotetramer.
2o As used herein, the term TR14 heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing only polypeptide sequences that do not correspond to a polypeptide sequences encoded by the TR14 gene) in addition to the TR14 proteins of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when proteins of the invention contact one another in solution. In 3o another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when proteins of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the TR14 proteins of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the protein (e.g., the polypeptide sequence recited preferably in SEQ ID N0:61 or, alternatively, in SEQ ID NO:S or the polypeptide encoded by the deposited cDNA clone). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a TR14 fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US
Patent Number 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a TR14-Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention l0 are between heterologous polypeptide sequences from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more TR14 polypeptides of the invention are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described in U.S. Pat.
No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.
Another method for preparing multimer TR14 polypeptides of the invention involves 2o use of TR14 polypeptides fused to a leucine zipper or isoleucine polypeptide sequence.
Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric TR14 proteins are those described in PCT application WO 94/10308, hereby incorporated by reference.
Recombinant fusion proteins comprising a soluble TR14 polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric TR14 is recovered from the culture supernatant using techniques known in the art.
Certain members of the TNF family of proteins are believed to exist in trimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cel173:431, 1993).
Thus, trimeric TR14 may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS
Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference.
Other peptides derived from naturally occurnng trimeric proteins may be employed in preparing trimeric TR14.

In further preferred embodiments, TR14 polynucleotides of the invention are fused to a polynucleotide encoding a "FLAG" polypeptide. Thus, a TR14-FLAG fusion protein is encompassed by the present invention. The FLAG antigenic polypeptide may be fused to a TR14 polypeptide of the invention at either or both the amino or the carboxy terminus. In preferred embodiments, a TR14-FLAG fusion protein is expressed from a pFLAG-CMV-Sa or a pFLAG-CMV-1 expression vector (available from Sigma, St. Louis, MO, USA).
See, Andersson, S., et al., J. Biol. Chem. 264:8222-29 (1989); Thomsen, D. R., et al., Proc. Natl.
Acad. Sci. USA, 81:659-63 (1984); and Kozak, M., Nature 308:241 (1984) (each of which is hereby incorporated by reference). In further preferred embodiments, a TR14-FLAG fusion protein is detectable by anti-FLAG monoclonal antibodies (also available from Sigma).
In another example, proteins of the invention are associated by interactions between Flag~ polypeptide sequence contained in Flag~-TR14 fusion proteins of the invention. In a further embodiment, associated proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag~-TR14 fusion proteins of the invention and anti-Flag~ antibody.
The multimers of the invention may be generated using chemical techniques known in the art. For example, proteins desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein 2o incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). Further, proteins of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., US
Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Additionally, techniques known in the art may be applied to generate liposomes containing the protein components 3o desired to be contained in the multimer of the invention (see, e.g., US
Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, proteins contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
The polypeptides (proteins) of the present invention are preferably provided in an 1o isolated form. By "isolated polypeptide" is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an "isolated polypeptide" are polypeptides that have been purified, partially or substantially, from a recombinant host cell. For example, a recombinantly produced version of the polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
Accordingly, in one embodiment, the invention provides an isolated TR14 polypeptide having the amino acid sequence encoded by the cDNA deposited as ATCC Deposit No. PTA
348, or the amino acid sequence shown preferably in SEQ ID N0:61 or, alternatively, in SEQ
2o ID NO:S, or a polypeptide comprising a portion of the above polypeptides.
The polypeptides of the present invention are preferably provided in an isolated form.
By "isolated polypeptide" is intended a polypeptide removed from its native environment.
Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an "isolated polypeptide" are polypeptides that have been purified, partially or substantially, from a recombinant host cell.
For example, a recombinantly produced version of the TR14 polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
Polypeptide fragments of the present invention include polypeptides comprising or alternatively, consisting of, an amino acid sequence contained preferably in SEQ ID N0:61 or, alternatively, in SEQ ID NO:S, encoded by the cDNA contained in the clone deposited as ATCC Deposit No. PTA-348, or encoded by a nucleic acid which hybridizes (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone, or shown preferably in Figures l0A-H (SEQ ID N0:61) or, alternatively, in Figures 4A-D
(SEQ ID N0:4) or the complementary strand thereto, or polynucleotide fragments thereof (e.g., as disclosed herein). Protein fragments may be "free-standing," or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Preferred representative examples of polypeptide fragments of the invention, include, for example, fragments that comprise, or alternatively consist of, from about amino acid residues: 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 231 of SEQ >D
N0:61. Alternative, less preferred representative examples of polypeptide fragments of the invention, include, for example, fragments that comprise, or alternatively consist of, from about amino acid residues: 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 226 of SEQ ID
NO:S, and the corresponding amino acid residues of SEQ ID N0:61 (as the sequence of amino acid residues T-78 to M-231 of SEQ ID N0:61 is identical to the sequence of amino acid residues T-73 to M-226 of SEQ ID NO:S). Moreover, polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length. In this context "about" includes the particularly recited ranges, larger or smaller by to several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Polypeptide fragments of the present invention include polypeptides comprising or alternatively, consisting of amino acid residues from about: 178 to about 180, 118 to about 121, 178 to about 181, 193 to about 196, 9 to about 14, and/or 65 to about 85 of SEQ >D
N0:2. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
Polynucleotides encoding the polypeptide fragments are also encompassed by the invention.
In specific embodiments, polypeptide fragments of the invention comprise, or alternatively consist of, amino acid residues from about: 1 to about 138, 139 to about 155, 2o and/or 156 to about 231 as depicted in SEQ ID N0:61; or, alternativley, about 1 to about 133, 134 to about 150, and/or 151 to about 226 as depicted in SEQ ID NO:S. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In additional embodiments, the polypeptide fragments of the invention comprise, or alternatively consist of, one or more TR14 domains. Preferred polypeptide fragments of the present invention include a member selected from the group: (a) a polypeptide comprising or alternatively, consisting of, the TR14 extracellular domain (predicted to constitute preferably amino acid residues from about 1 to about 138 in Figures l0A-H and SEQ ID
N0:61, or, 3o alternatively, from about 1 to about 133 of SEQ ID NO:S and Figures 4A-D, or from about 1 to about 133 of SEQ ID NO:S); (b) a polypeptide comprising or alternatively, consisting of, the TR14 cysteine rich domain (predicted to constitute preferably amino acids Cys-31 to Cys-104 of SEQ ID N0:61, or, alternatively, amino acid residues from about 65 to about 88 of Figures 4A-D, or from about 65 to about 85 in SEQ ID NO:S); (c) a polypeptide comprising or alternatively, consisting of, the TR14 transmembrane domain (predicted to constitute amino acid residues from about 139 to about 155 of Figures l0A-H and SEQ ID N0:61 or from about 134 to about 150 of Figures 4A-D and SEQ ID NO:S); (d) a polypeptide comprising or alternatively, consisting of, the TR14 intracellular domain (predicted to constitute amino acid residues from about 155 to about 231 of Figures l0A-H and SEQ ID N0:61 or amino acid residues from about 151 to about 226 of Figures 4A-D and SEQ ID NO:S); (e) a polypeptide comprising, or alternatively, consisting of, one, two, three, four or more, epitope bearing portions of the TR14 polypeptide (predicted to constitute preferably Asp-2 to Asp-10, Thr-17 to Asp-38, Pro-45 to Ser-52, Pro-88 to Arg-95, Thr-108 to Glu-I15, Thr-131 to Glu-136, Phe-166 to Gly-174, Ala-180 to Ala-200, and Gln-224 to Met-231 of SEQ ID
N0:61, or the corresponding amino acid sequences in SEQ ID NO:S, as the sequence of amino acid residues T-78 to M-231 of SEQ ID N0:61 is identical to the sequence of amino acid residues T-73 to M-226 of SEQ ID NO:S. Additional epitope bearing TR14 polypeptides comprise or, 1o alternatively, consist of amino acid residues from about 2 to about 24, 42 to about 52, 80 to about 115, and I55 to about 226 of SEQ ID NO:S (or the corresponding amino acid sequences in SEQ ID N0:61, as the sequence of amino acid residues T-78 to M-231 of SEQ
ID N0:61 is identical to the sequence of amino acid residues T-73 to M-226 of SEQ ID
NO:S); and (f) any combination of polypeptides (a)-(e). In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.
As discussed above, it is believed that the extracellular cysteine rich motifs of TR14 is important for interactions between TR14 and its ligands. Accordingly, in a specific embodiment, polypeptide fragments of the invention comprise, or alternatively consist of amino 2o acid residues 31 to 104 of SEQ ID N0:61 or 65 to 85 of SEQ ID NO:S.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
Among the especially preferred fragments of the invention are fragments characterized by structural or functional attributes of TR14 (preferably SEQ ID N0:61 or, alternatively, SEQ
ID NO:S). Such fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or 3o equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) of complete (i.e., full-length) TR14 (preferably SEQ ID N0:61 or, alternatively, SEQ ID NO:S).
Certain preferred regions are those set out in Figure 6 and Table II and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted preferably Figures l0A-H (SEQ ID N0:61) or, alternatively, in Figures 4A-D (SEQ ID NO:S), such preferred regions include; Gamier-Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle predicted hydrophilic and Hopp-Woods predicted hydrophobic regions; Eisenberg alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson-Wolf high antigenic index regions, as predicted using the default parameters of these computer programs. Polynucleotides encoding these polypeptides are also encompassed by the invention.
As mentioned above, even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind TR14 ligand) may still be retained. For example, the ability of shortened TR14 muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature to polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N
terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
It is not unlikely that an TR14 mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR14 amino acid residues may often evoke an immune response.
Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TR14 amino acid sequence shown depicted preferably Figures l0A-H (SEQ ID N0:61) or, alternatively, in Figures 4A-D
(SEQ ID NO:S), up to the methionine residue at position number 231 of SEQ ID N0:61 (or, number 226 of 2o SEQ ID NO:S) and polynucleotides encoding such polypeptides. In particular preferred embodiments for TR14, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n'-231 of Figures l0A-H, where n' is an integer from 1 to 231 corresponding to the position of the amino acid residue in Figures l0A-H. In alternative embodiments, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n'-226 of Figures 4A-D, where n' is an integer from 1 to 226 corresponding to the position of the amino acid residue in Figures 4A-D.
1n specific embodiments, N-terminal deletions of the TR14 polypeptides of the invention can be described by the general formula n2-231, where n2 is a number from 2 to 226, corresponding to the position of amino acid identified in Figures l0A-H (SEQ
ID N0:61 ). N-terminal deletions of the TR14 polypeptide of the invention shown as SEQ ID
N0:61 include polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: D-2 to M-231; C-3 to M-231; Q-4 to M-231; E-5 to M-231; N-6 to M-231; E-7 to M-231; Y-8 to M-231; W-9 to M-231; D-10 to M-231; Q- I 1 to M-231; W-12 to M-231; G-13 to M-231; R-14 to M-231; C-15 to M-231; V -16 to M-231; T-17 to M-231; C-18 to M-231; Q-19 to M-231; R-20 to M-231; C-21 to M-231; G-22 to M-231; P-23 to M-231; G-24 to M-231; Q-25 to M-231;
E-26 to M-23 I ; L-27 to M-231; S-28 to M-231; K-29 to M-231; D-30 to M-231; C-31 to 1VI-231; G-32 to M-231; Y-33 to M-231; G-34 to M-231; E-35 to M-231; G-36 to M-23 I ; G-37 to M-231; D-38 to M-231; A-39 to M-231; Y-40 to M-231; W-41 to M-231; H-42 to M-231; S-43 to M-231; L-44 to M-231; P-45 to M-231; S-46 to M-231; S-47 to M-231; Q-48 to M-231; Y-49 to M-23 I ; K-50 to M-231; S-51 to M-231; S-52 to M-231; W-53 to M-231; G-54 to M-23 I ;
H-55 to M-231; H-56 to M-231; K-57 to M-231; C-5 8 to M-231; Q-59 to M-231; S-60 to M-231; C-61 to M-231; I-62 to M-231; T-63 to M-231; C-64 to M-231; A-65 to M-231; V -66 to M-231; I-67 to M-231; N-68 to M-231; R-69 to M-231; V -70 to M-231; Q-71 to M-231; K-72 to M-231; V-73 to M-231; N-74 to M-231; C-75 to M-231; T-76 to M-231; P-77 to M-231; T-78 to M-231; S-79 to M-231; N-80 to M-231; A-81 to M-231; V -82 to M-231; C-83 to M-231;
G-84 to M-231; D-85 to M-231; C-86 to M-231; L-87 to M-231; P-8 8 to M-231; R-89 to M-231; F-90 to M-231; Y-91 to M-231; R-92 to M-231; K-93 to M-231; T-94 to M-231; R-95 to M-231; I-96 to M-231; G-97 to M-231; G-98 to M-231; L-99 to M-231; Q-100 to M-231; D-1 O l to M-231; Q-102 to M-231; E-103 to M-231; C-104 to M-231; I-105 to M-231; P-106 to M-231; C-107 to M-231; T-108 to M-231; K-109 to M-231; Q-110 to M-231; T-111 to M-231;
P-112 to M-231; T-113 to M-231; S-114 to M-231; E-115 to M-231; V-116 to M-231; Q-117 to M-231; C-118 to M-231; A-119 to M-231; F-120 to M-231; Q-121 to M-231; L-122 to M-231; S-123 to M-23 I ; L-124 to M-231; V -125 to M-231; E-126 to M-231; A-127 to M-231; D-128 to M-231; A-129 to M-231; P-130 to M-231; T-131 to M-231; V-132 to M-231;
P-133 to M-231; P-134 to M-231; Q-135 to M-231; E-136 to M-231; A-137 to M-231; T-138 to M-231;
L-139 to M-231; V-140 to M-231; A-141 to M-231; L-142 to M-231; V-143 to M-231; S-144 2o to M-231; S-145 to M-23 I ; L-146 to M-231; L-147 to M-231; V -148 to M-231; V -149 to M-231; F-150 to M-231; T-151 to M-231; L-152 to M-231; A-153 to M-231; F-154 to M-231; L-155 to M-231; G-156 to M-231; L-157 to M-231; F-158 to M-231; F-159 to M-231;
L-160 to M-231; Y - I 61 to M-231; C-162 to M-231; K-163 to M-231; Q-164 to M-231; F-165 to M-231;
F- I 66 to M-231; N-167 to M-23 I ; R-168 to M-231; H-169 to M-231; C-170 to M-231; Q-171 to M-231; R-172 to M-231; G-173 to M-231; G-174 to M-231; L-175 to M-231; L-176 to M-231; Q-177 to M-231; F-178 to M-231; E-179 to M-231; A-180 to M-231; D-181 to M-231; K-182 to M-231; T-183 to M-231; A-184 to M-231; K-185 to M-231; E-186 to M-231;
E-187 to M-231; S-188 to M-231; L-189 to M-231; F-190 to M-231; P-191 to M-231; V-192 to M-231;
P-193 to M-231; P-194 to M-231; S-195 to M-231; K-196 to M-231; E-197 to M-231; T-198 3o to M-231; S-199 to M-231; A-200 to M-231; E-201 to M-231; S-202 to M-231; Q-203 to M-231; V-204 to M-231; S-205 to M-231; W-206 to M-231; A-207 to M-231; P-208 to M-231;
G-209 to M-231; S-210 to M-231; L-211 to M-231; A-212 to M-231; Q-213 to M-231; L-214 to M-231; F-215 to M-231; S-216 to M-231; L-217 to M-231; D-218 to M-231; S-219 to M-231; V-220 to M-231; P-221 to M-23 I ; I-222 to M-23 I ; P-223 to M-231; Q-224 to M-231; Q-225 to M-231 and Q-226 to M-231 of SEQ ID N0:61.
In additional embodiments, N-terminal deletions of the TR14 polypeptides of the invention can be described by the general formula n2-226, where n2 is a number from 2 to 221, corresponding to the position of amino acid identified in Figures 4A-D (SEQ ID
NO:S). N-terminal deletions of the TR14 polypeptide of the invention shown as SEQ ID
NO:S include polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: S-2 to M-226; T-3 to M-226; G-4 to M-226; T-5 to M-226; N-6 to M-226; G-7 to M-226; D-8 to M-226; G-9 to M-226; V-10 to M-226; S-11 to M-226; P-12 to M-226; A-13 to M-226; N-14 to M-226; G-15 to M-226; V -16 to M-226; V -17 to M-226; L-18 to M-226; D-19 to M-226; R-20 to M-226; S-21 to M-226; Y-22 to M-226; P-23 to M-226; R-24 to M-226; I-25 to M-226;
V-26 to M-226; V-27 to M-226; M-28 to M-226; E-29 to M-226; R-30 to M-226; V-31 to M-226; E-32 to M-226; M-33 to M-226; P-34 to M-226; T-35 to M-226; A-36 to M-226; Q-37 to M-226; P-38 to M-226; A-39 to M-226; L-40 to M-226; L-41 to M-226; A-42 to M-226; V-43 to M-226; Q-44 to M-226; K-45 to M-226; Q-46 to M-226; L-47 to M-226; G-48 to M-226; P-49 to M-226; P-50 to M-226; Q-51 to M-226; M-52 to M-226; C-53 to M-226; R-54 to M-226;
V-55 to M-226; A-56 to M-226; C-57 to M-226; T-58 to M-226; C-59 to M-226; A-60 to M-226; V-61 to M-226; I-62 to M-226; N-63 to M-226; R-64 to M-226; V-65 to M-226; Q-66 to M-226; K-67 to M-226; V-68 to M-226; N-69 to M-226; C-70 to M-226; T-71 to M-226; P-72 to M-226; T-73 to M-226; S-74 to M-226; N-75 to M-226; A-76 to M-226; V-77 to M-226; C-78 to M-226; G-79 to M-226; D-80 to M-226; C-81 to M-226; L-82 to M-226; P-83 to M-226;
R-84 to M-226; F-85 to M-226; Y-86 to M-226; R-87 to M-226; K-88 to M-226; T-89 to M-226; R-90 to M-226; I-91 to M-226; G-92 to M-226; G-93 to M-226; L-94 to M-226; Q-95 to M-226; D-96 to M-226; Q-97 to M-226; E-98 to M-226; C-99 to M-226; I-100 to M-226; P-2o 101 to M-226; C-102 to M-226; T-103 to M-226; K-104 to M-226; Q-105 to M-226; T-106 to M-226; P-107 to M-226; T-108 to M-226; S-109 to M-226; E-110 to M-226; V-11 I
to M-226;
Q-112 to M-226; C-113 to M-226; A-114 to M-226; F-115 to M-226; Q-116 to M-226; L-117 to M-226; S-118 to M-226; L-119 to M-226; V -120 to M-226; E-121 to M-226; A-122 to M-226; D-123 to M-226; A-124 to M-226; P-125 to M-226; T-126 to M-226; V-127 to M-226; P-128 to M-226; P-129 to M-226; Q-130 to M-226; E-131 to M-226; A-132 to M-226;
T-133 to M-226; L-134 to M-226; V-135 to M-226; A-136 to M-226; L-137 to M-226; V-138 to M-226;
S-139 to M-226; S-140 to M-226; L-141 to M-226; L-142 to M-226; V-143 to M-226; V-144 to M-226; F-145 to M-226; T-146 to M-226; L-147 to M-226; A-148 to M-226; F-149 to M-226; L-150 to M-226; G-151 to M-226; L-152 to M-226; F-153 to M-226; F-154 to M-226; L-155 to M-226; Y-156 to M-226; C-157 to M-226; K-158 to M-226; Q-159 to M-226;
F-160 to M-226; F-161 to M-226; N-162 to M-226; R-163 to M-226; H-164 to M-226; C-165 to M-226;
Q-166 to M-226; R-167 to M-226; G-168 to M-226; G-169 to M-226; L-170 to M-226; L-171 to M-226; Q-172 to M-226; F-173 to M-226; E-174 to M-226; A-175 to M-226; D-176 to M-226; K-177 to M-226; T-178 to M-226; A-179 to M-226; K-180 to M-226; E-181 to M-226; E-182 to M-226; S-183 to M-226; L-184 to M-226; F-185 to M-226; P-186 to M-226;
V-187 to M-226; P-188 to M-226; P-189 to M-226; S-190 to M-226; K-191 to M-226; E-192 to M-226;
T-193 to M-226; S-194 to M-226; A-195 to M-226; E-196 to M-226; S-197 to M-226; Q-198 to M-226; V-199 to M-226; S-200 to M-226; W-201 to M-226; A-202 to M-226; P-203 to M-226; G-204 to M-226; S-205 to M-226; L-206 to M-226; A-207 to M-226; Q-208 to M-226; L-209 to M-226; F-210 to M-226; S-211 to M-226; L-212 to M-226; D-213 to M-226;
S-214 to M-226; V-215 to M-226; P-216 to M-226; I-217 to M-226; P-218 to M-226; Q-219 to M-226;
Q-220 to M-226; Q-221 to M-226; of SEQ ID NO:S. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In another embodiment, N-terminal deletions of the extracellular domain of the polypeptide can be described by the general formula n2-133, where n2 is a number from 1 to 128, corresponding to the position of amino acids identified in Figures 4A-D.
N-terminal deletions of the extracellular domain of the TR14 polypeptide of the invention shown as SEQ
t0 ID N0:7 include polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: S-2 to T-133; T-3 to T-133; G-4 to T-133; T-5 to T-133;
N-6 to T-133;
G-7 to T-133; D-8 to T-133; G-9 to T-133; V-10 to T-133; S-11 to T-133; P-12 to T-133; A-13 to T-133; N-14 to T-133; G-15 to T-133; V-16 to T-133; V-17 to T-133; L-18 to T-133; D-19 to T-133; R-20 to T-133; S-21 to T-133; Y-22 to T-133; P-23 to T-133; R-24 to T-133; I-25 to T-133; V-26 to T-133; V-27 to T-133; M-28 to T-133; E-29 to T-133; R-30 to T-133; V-31 to T-133; E-32 to T-133; M-33 to T-133; P-34 to T-133; T-35 to T-133; A-36 to T-133; Q-37 to T-133; P-38 to T-133; A-39 to T-133; L-40 to T-133; L-41 to T-133; A-42 to T-133; V-43 to T-133; Q-44 to T-133; K-45 to T-133; Q-46 to T-133; L-47 to T-133; G-48 to T-133; P-49 to T-133; P-SO to T-133; Q-51 to T-133; M-52 to T-133; C-53 to T-133; R-54 to T-133; V-55 to 2o T-133; A-56 to T-133; C-57 to T-133; T-58 to T-133; C-59 to T-133; A-60 to T-133; V-61 to T-133; I-62 to T-133; N-63 to T-133; R-64 to T-133; V-65 to T-133; Q-66 to T-133; K-67 to T-133; V-68 to T-133; N-69 to T-133; C-70 to T-133; T-71 to T-133; P-72 to T-133; T-73 to T-133; S-74 to T-133; N-75 to T-133; A-76 to T-133; V-77 to T-133; C-78 to T-133; G-79 to T-133; D-80 to T-133; C-81 to T-133; L-82 to T-133; P-83 to T-133; R-84 to T-133; F-85 to T-133; Y-86 to T-133; R-87 to T-133; K-88 to T-133; T-89 to T-133; R-90 to T-133; I-91 to T-133; G-92 to T-133; G-93 to T-133; L-94 to T-133; Q-95 to T-133; D-96 to T-133; Q-97 to T-133; E-98 to T-133; C-99 to T-133; I-100 to T-133; P-101 to T-133; C-102 to T-133; T-103 to T-133; K-104 to T-133; Q-105 to T-133; T-106 to T-133; P-107 to T-133; T-108 to T-133; S-109 to T-133; E-110 to T-133; V-111 to T-133; Q-112 to T-133; C-113 to T-133;
A-114 to T-133; F-115 to T-133; Q-116 to T-133; L-117 to T-133; S-118 to T-133; L-119 to T-133; V-120 to T-133; E-121 to T-133; A-122 to T-133; D-123 to T-133; A-124 to T-133; P-125 to T-133;
T-126 to T-133; V-127 to T-133; P-128 to T-133; of SEQ ID N0:7 (or the corresponding amino acid sequences in SEQ ID N0:61, as the sequence of amino acid residues T-78 to T-138 of SEQ ID N0:61 is identical to the sequence of amino acid residues T-73 to T-133 of SEQ ID
N0:7). Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities), ability to multimerize, ability to bind TR14 ligand) may still be retained. For example the ability of the shortened TR14 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an TR14 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR14 amino acid residues may often evoke an immune response.
1o Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR14 polypeptide shown in Figures IOA-H (SEQ ID N0:61), up to the glutamic acid residue at position number 7, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m' of Figures l0A-H, where m' is an integer from 7 to 231 corresponding to the position of the amino acid residue in Figures l0A-H (which is identical to the sequence shown as SEQ ID N0:61).
Moreover, the invention provides polypeptides comprising, or alternatively consisting of, the amino acid residues: M-1 to E-230; M-1 to P-229; M-1 to G-228; M-1 to Q-227; M-1 to Q-226; M-1 to Q-225; M-1 to Q-224; M-I to P-223; M-1 to I-222; M-1 to P-221; M-1 to V-220; M-1 to S-219; M-1 to D-218; M-1 to L-217; M-1 to S-216; M-1 to F-215; M-1 to L-214;
M-1 to Q-213; M-1 to A-212; M-1 to L-211; M-1 to S-210; M-1 to G-209; M-1 to P-208; M-1 to A-207; M-1 to W-206; M-1 to S-205; M-1 to V-204; M-1 to Q-203; M-1 to S-202; M-1 to E-201; M-1 to A-200; M-1 to S-199; M-1 to T-198; M-1 to E-197; M-1 to K-196; M-1 to S-195;
M-1 to P-194; M-1 to P-193; M-1 to V-192; M-1 to P-191; M-1 to F-190; M-1 to L-189; M-1 to S-188; M-1 to E-187; M-1 to E-186; M-1 to K-185; M-1 to A-184; M-1 to T-183; M-1 to K-182; M-1 to D-181; M-1 to A-180; M-1 to E-179; M-1 to F-178; M-1 to Q-177; M-1 to L-176;
M-1 to L-175; M-I to G-174; M-I to G-173; M-1 to R-172; M-1 to Q-171; M-1 to C-170; M-1 to H-169; M-1 to R-168; M-t to N-167; M-1 to F-166; M-1 to F-165; M-1 to Q-164; M-1 to K-163; M-1 to C-162; M-1 to Y-161; M-1 to L-160; M-1 to F-159; M-1 to F-158; M-1 to L-157;
M-1 to G-156; M-1 to L-155; M-1 to F-154; M-1 to A-153; M-1 to L-152; M-1 to T-151; M-1 to F-150; M-1 to V-149; M-1 to V-148; M-1 to L-147; M-1 to L-146; M-1 to S-145; M-1 to S-144; M-1 to V-143; M-1 to L-142; M-1 to A-141; M-1 to V-140; M-1 to L-139; M-1 to T-138;
M-1 to A-137; M-1 to E-136; M-1 to Q-135; M-1 to P-134; M-1 to P-133; M-1 to V-132; M-1 to T-131; M-1 to P-130; M-1 to A-129; M-1 to D-128; M-1 to A-127; M-1 to E-126; M-1 to V-125; M-1 to L-124; M-1 to S-123; M-1 to L-122; M-1 to Q-121; M-1 to F-120; M-1 to A-119;
M-1 to C-118; M-1 to Q-117; M-1 to V-116; M-1 to E-115; M-1 to S-114; M-1 to T-113; M-1 to P-112; M-1 to T-111; M-1 to Q-110; M-1 to K-109; M-1 to T-108; M-1 to C-107; M-1 to P-106; M-1 to I-105; M-1 to C-104; M-1 to E-103; M-1 to Q-102; M-1 to D-101; M-1 to Q-100;
M-1 to L-99; M-1 to G-98; M-1 to G-97; M-1 to I-96; M-1 to R-95 ; M-1 to T-94;
M-1 to K-93 ;
M-1 to R-92; M-1 to Y-91; M-1 to F-90; M-1 to R-89; M-1 to P-88; M-1 to L-87;
M-1 to C-86;
M-1 to D-85; M-1 to G-84; M-1 to C-83; M-1 to V-82; M-1 to A-81; M-1 to N-80;
M-1 to S-79; M-1 to T-78; M-1 to P-77; M-1 to T-76; M-1 to C-75; M-1 tv N-74; M-1 to V-73; M-1 to K-72; M-1 to Q-71; M-1 to V-70; M-1 to R-69; M-1 to N-68; M-1 to I-67; M-1 to V-66; M-1 to A-65 ; M-1 to C-64; M-1 to T-63 ; M-1 to I-62; M-1 to C-61; M-1 to S-60; M-1 to Q-59; M-1 to C-58; M-1 to K-57; M-1 to H-56; M-1 to H-55; M-1 to G-54; M-1 to W-53; M-1 to S-52; M-1 to S-51; M-1 to K-50; M-1 to Y-49; M-1 to Q-48; M-1 to S-47; M-1 to S-4b; M-1 to P-45; M-1 1o to L-44; M-1 to S-43; M-1 to H-42; M-1 to W-41; M-1 to Y-40; M-1 to A-39; M-1 to D-38; M-1 to G-37; M-1 to G-36; M-1 to E-35; M-1 to G-34; M-1 to Y-33; M-1 to G-32; M-1 to C-31;
M-1 to D-30; M-1 to K-29; M-1 to S-28; M-1 to L-27; M-1 to E-26; M-1 to Q-25;
M-1 to G-24;
M-1 to P-23 ; M-1 to G-22; M-1 to C-21; M-1 to R-20; M-1 to Q-19; M-1 to C-18;
M-1 to T-17;
M-1 to V-16; M-1 to C-15; M-1 to R-14; M-1 to G-13; M-1 to W-12; M-1 to Q-11;
M-1 to D-10; M-1 to W-9; M-1 to Y-8 and M-1 to E-7 of SEQ ID N0:61. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Alternatively, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR14 polypeptide shown in Figures 4A-D (SEQ ID NO:S), up to the asparagine residue at position number 6, and 2o polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m' of Figures 4A-D, where m' is an integer from 6 to 226 corresponding to the position of the amino acid residue in Figures 4A-D (which is identical to the sequence shown as SEQ ID
NO:S).
Moreover, the invention provides polypeptides comprising, or alternatively consisting of, the amino acid residues: M-I to E-225; M-1 to P-224; M-1 to G-223; M-1 to Q-222; M-1 to Q-221; M-1 to Q-220; M-1 to Q-219; M-1 to P-218; M-1 to I-217; M-1 to P-216; M-1 to V-215 ; M-1 to S-214; M-1 to D-213 ; M-1 to L-212; M-1 to S-211; M-1 to F-210; M-1 to L-209;
M-1 to Q-208 ; M-1 to A-207 ; M-1 to L-206; M-1 to S-205 ; M-1 to G-204; M-1 to P-203 ; M-1 to A-202; M-1 to W-201; M-1 to S-200; M-1 to V -199; M-1 to Q-198; M-1 to S-197; M-1 to E-196; M-1 to A-195; M-1 to S-194; M-1 to T-193; M-1 to E-192; M-1 to K-191; M-1 to S-190;
M-1 to P-189; M-1 to P-188; M-1 to V-187; M-1 to P-186; M-1 to F-185; M-1 to L-184; M-1 to S-183; M-1 to E-182; M-1 to E-181; M-1 to K-180; M-1 to A-179; M-1 to T-178; M-1 to K-177; M-1 to D-176; M-1 to A-175; M-1 to E-174; M-1 to F-173; M-1 to Q-172; M-1 to L-171;
M-1 to L-170; M-1 to G-169; M-1 to G-168; M-1 to R-167; M-I to Q-166; M-1 to C-165; M-1 to H-164; M-1 to R-163; M-1 to N-162; M-1 to F-161; M-1 to F-160; M-1 to Q-159; M-1 to K-158; M-1 to C-157; M-1 to Y-156; M-1 to L-155; M-1 to F-154; M-1 to F-153; M-1 to L-152;
M-1 to G-151; M-1 to L-150; M-1 to F-149; M-1 to A-148; M-1 to L-147; M-1 to T-146; M-1 to F-145; M-I to V-144; M-1 to V-143; M-1 to L-142; M-I to L-141; M-1 to S-140; M-1 to S-139; M-1 to V-138; M-1 to L-137; M-1 to A-136; M-I to V-135; M-1 to L-134; M-1 to T-133;
M-1 to A-132; M-1 to E-131; M-1 to Q-130; M-1 to P-129; M-1 to P-128; M-1 to V-127; M-1 to T-126; M-1 to P-125; M-1 to A-124; M-1 to D-123; M-1 to A-122; M-1 to E-121; M-1 to V-120; M-1 to L-119; M-1 to S-I 18; M-I to L-117; M-1 to Q-116; M-1 to F-115; M-1 to A-114;
M-1 to C-113; M-1 to Q-112; M-1 to V-111; M-1 to E-110; M-1 to S-109; M-1 to T-108; M-1 to P-107; M-1 to T-106; M-1 to Q-105; M-1 to K-104; M-1 to T-103; M-I to C-102; M-1 to P-101; M-1 to I-100; M-1 to C-99; M-1 to E-98; M-1 to Q-97; M-1 to D-96; M-1 to Q-95; M-1 to L-94; M-1 to G-93; M-1 to G-92; M-1 to I-91; M-1 to R-90; M-1 to T-89; M-I to K-88; M-1 to to R-87; M-1 to Y-86; M-1 to F-85; M-1 to R-84; M-1 to P-83; M-1 to L-82; M-1 to C-81; M-I to D-80; M-1 to G-79; M-1 to C-78; M-1 to V-77; M-1 to A-76; M-1 to N-75; M-1 to S-74; M-I
to T-73; M-1 to P-72; M-1 to T-71; M-1 to C-70; M-1 to N-69; M-1 to V-68; M-1 to K-67; M-1 to Q-66; M-1 to V-65; M-1 to R-64; M-1 to N-63; M-1 to I-62; M-1 to V-61; M-1 to A-60; M-1 to C-59; M-1 to T-58; M-1 to C-57; M-1 to A-56; M-1 to V-55; M-1 to R-54; M-1 to C-53; M-1 to M-52; M-1 to Q-51; M-1 to P-50; M-1 to P-49; M-1 to G-48; M-1 to L-47; M-1 to Q-46; M-1 to K-45; M-1 to Q-44; M-1 to V-43; M-1 to A-42; M-1 to L-41; M-1 to L-40; M-1 to A-39;
M-1 to P-38; M-1 to Q-37; M-1 to A-36; M-1 to T-35; M-1 to P-34; M-1 to M-33;
M-1 to E-32;
M-1 to V-31; M-1 to R-30; M-I to E-29; M-1 to M-28; M-1 to V-27; M-1 to V-26;
M-1 to I-25;
M-1 to R-24; M-1 to P-23 ; M- I to Y-22; M-1 to S-21; M-1 to R-20; M-1 to D-19; M-1 to L-18 ;
M-1 to V-17; M-1 to V-16; M-1 to G-15; M-1 to N-14; M-1 to A-13; M-1 to P-12;
M-1 to S-11; M-1 to V-10; M-1 to G-9; M-1 to D-8; M-1 to G-7; M-1 to N-6; of SEQ ID
NO:S.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities), ability to multimerize, ability to bind TR14 ligand) may still be retained. For example the ability of the shortened TR14 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular 3o polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an TR14 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. 1n fact, peptides composed of as few as six TR14 amino acid residues may often evoke an immune response.
The present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR14 polypeptide shown in Figures 4A-D (SEQ ID NO:S) and polynucleotides encoding such polypeptides.
In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 133-m' of Figures 4A-D, where m' is an integer from 6 to 132 corresponding to the position of the amino acid residue in Figures 4A-D (which is identical to the sequence shown as SEQ ID NO:S).
Moreover, the invention provides TR14 polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: M-1 to A-132; M-1 to E-131; M-1 to Q-130; M-1 to P-129; M-1 to P-128; M-1 to V-127; M-1 to T-126; M-1 to P-125; M-1 to A-124;
M-1 to D-123; M-1 to A-122; M-1 to E-121; M-1 to V-120; M-1 to L-119; M-1 to S-118; M-1 to L-117; M-1 to Q-116; M-1 to F-115; M-1 to A-114; M-1 to C-113; M-1 to Q-I
12; M-1 to V-111; M-1 to E-110; M-1 to S-109; M-1 to T-108; M-1 to P-107; M-1 to T-106; M-1 to Q-105;
t0 M-1 to K-104; M-1 to T-103; M-1 to C-102; M-1 to P-101; M-1 to I-100; M-1 to C-99; M-1 to E-98; M-1 to Q-97; M- I to D-96; M-1 to Q-95; M-1 to L-94; M-1 to G-93 ; M-1 to G-92; M-1 to I-91; M-1 to R-90; M-1 to T-89; M-1 to K-88; M-1 to R-87; M-1 to Y-86; M-1 to F-85; M-1 to R-84; M-1 to P-83; M-1 to L-82; M-1 to C-81; M-1 to D-80; M-1 to G-79; M-1 to C-78; M-1 to V-77; M-1 to A-76; M-1 to N-75; M-1 to S-74; M-1 to T-73; M-1 to P-72; M-1 to T-71; M-1 to C-70; M-1 to N-69; M-1 to V-68; M-1 to K-67; M-1 to Q-66; M-1 to V-65; M-1 to R-64; M-1 to N-63; M-1 to I-62; M-1 to V-61; M-1 to A-60; M-1 to C-59; M-1 to T-58; M-1 to C-57; M-1 to A-56; M-1 to V-55; M-1 to R-54; M-1 to C-53; M-1 to M-52; M-1 to Q-51; M-1 to P-50;
M-1 to P-49; M-1 to G-48; M-1 to L-47; M-1 to Q-46; M-1 to K-45; M-1 to Q~4; M-1 to V-43 ; M-1 to A-42; M-1 to L-41; M-1 to L-40; M-1 to A-39; M-1 to P-3 8 ; M-1 to Q-37; M-1 to 2o A-36; M-1 to T-35; M-1 to P-34; M-1 to M-33; M-1 to E-32; M-1 to V-31; M-1 to R-30; M-1 to E-29; M-1 to M-28; M-1 to V-27; M-1 to V-26; M-1 to I-25; M-1 to R-24; M-1 to P-23; M-1 to Y-22; M-1 to S-21; M-1 to R-20; M-1 to D-19; M-1 to L-18; M-1 to V-17; M-1 to V-16; M-1 to G-15; M-1 to N-14; M-1 to A-13; M-1 to P-12; M-1 to S-11; M-1 to V-10; M-1 to G-9; M-1 to D-8; M-1 to G-7; M-1 to N-6; of SEQ ID N0:7. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues n'- m', n2- m', n'-mz and/or n2-mZ, where n', n2, m', and m2 are integers as described above.
Thus, any of the above listed N- or C-terminal deletions can be combined to produce an N- and C-terminal deleted TR14 polypeptide.
It will be recognized in the art that some amino acid sequences of TR14 polypeptides can be varied without significant effect on the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. Thus, the invention further includes variations of the TR14 polypeptide, which show substantial TR14 receptor activity or which include regions of TR14 polypeptides, such as the polypeptide portions discussed herein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions. As indicated above, guidance concerning which amino acid changes are likely to be phenotypically silent can be found in J.U. Bowie et al., Science 247:1306-1310 (1990).
Thus, the fragment, derivative, or analog of the polypeptide encoded by the cDNA
deposited as ATCC Deposit No. PTA-348 or shown, preferably, in SEQ ID N0:61 or, alternatively, in SEQ ID N0:5, may be (i) one in which at least one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue(s), and more preferably at least one but less than ten conserved amino acid residues) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a to substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the TR14 polypeptide. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 70:307-377 (1993)).
The replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 361:266-268 (1993), describes certain mutations resulting in selective binding of TNF-a to only one of the two known types of TNF
receptors. Thus, the TR14 receptor of the present invention may include one or more amino acid substitutions, deletions, or additions, either from natural mutations or human manipulation.
3o As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table IV).
In specific embodiments, the number of substitutions, additions or deletions in the amino acid sequence of Figures l0A-H (SEQ ID N0:61) and/or any of the polypeptide fragments described herein (e.g., the cysteine-rich domain, the extracellular domain, or intracellular domain) is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20-15, 20-10, 15-10, 10-1, 5-10, 1-5, 1-3 or 1-2.

In additional embodiments, the number of substitutions, additions or deletions in the amino acid sequence of Figures 4A-D (SEQ ID NO:S) and/or any of the polypeptide fragments described herein (e.g., the cysteine-rich domain, the extracellular domain, or intracellular domain) is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20 15, 20-10, 15-10, 10-1, 5-10, 1-S, 1-3 or 1-2.
Amino acids in the TR14 polypeptides of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The to resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).
To improve or alter the characteristics of TR14 polypeptides, protein engineering may be employed. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show 2o better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)), cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (see e.g., Wells et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)).
Thus, the invention also encompasses TR14 derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate TR14 polypeptides that are 3o better suited for expression, scale up, etc., in the host cells chosen. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites. To this end, a variety of amino acid substitutions at one or both of the first or third amino acid positions on any one or more of the glycosylation recognitions sequences in the TR14 polypeptides of the invention, and/or an amino acid deletion at the second position of any one or more such recognition sequences will prevent glycosylation of the TR14 at the modified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J 5(6):1193-1197). Additionally, one or more of the amino acid residues of the polypeptides of the invention (e.g., arginine and lysine residues) may be deleted or substituted with another residue to eliminate undesired processing by proteases such as, for example, furins or kexins.
The polypeptides of the present invention include a polypeptide comprising, or alternatively, consisting of the polypeptide encoded by the cDNA deposited as ATCC Deposit No. PTA-348; a polypeptide comprising, or alternatively, consisting of amino acids from 1 to about 231 of SEQ ID N0:61 or from 1 to about 226 of SEQ ID NO:S; a polypeptide comprising, or alternatively, consisting of amino acids from about from 2 to about 231 of SEQ
1o ID N0:61 or 2 to about 226 of SEQ ID NO:S; a polypeptide comprising, or alternatively, consisting of amino acids from from 1 to about 138 of SEQ ID N0:61 or from 1 to about 133 of SEQ ID NO:S; a polypeptide comprising, or alternatively, consisting of the extracellular domain of the polypeptide encoded by the cDNA deposited as ATCC Deposit No.
PTA-348; a polypeptide comprising, or alternatively, consisting of the cysteine rich domain of the polypeptide encoded by the cDNA deposited as ATCC Deposit No. PTA-348, or as shown in amino acids about 31 to about 104 of SEQ ID N0:61, or shown in amino acids from about 65 to about 85 of SEQ ID NO:S; a polypeptide comprising, or alternatively, consisting of the transmembrane domain of the polypeptide encoded by the cDNA deposited as ATCC
Deposit No. PTA-348 (predicted to constitute amino acids from about 139 to about 155 of SEQ ID
2o N0:61 or from 134 to about 150 of SEQ ID NO:S); a polypeptide comprising, or alternatively, consisting of the intracellular domain (predicted to constitute amino acids from about 155 to about 231 of SEQ ID N0:61 or from about 151 to about 226 of SEQ ID NO:S); a polypeptide comprising, or alternatively, consisting of the extracellular and intracellular domains with all or part of the transmembrane domain deleted; as well as polypeptides which are at least 80%
identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98%, or 99% identical to the polypeptides described above (e.g., the polypeptide encoded by the cDNA in ATCC Deposit No. PTA-348, the polypeptide of Figures l0A-H
(SEQ ID N0:61); or the polypeptide of Figures 4A-D (SEQ ID NO:S)) or polypeptide fragments thereof, such as those disclosed herein), and also include portions of such 3o polypeptides with at least 30 amino acids and more preferably at least 50 amino acids. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1 ) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.
By a polypeptide (protein) comprising, or alternatively consisting of, an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a TR14 polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the TR14 polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, for instance, the amino acid sequence shown in SEQ ID NO:S, or to the amino acid sequence encoded by the cDNA deposited as ATCC Deposit No. PTA
348, can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full-length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
2o In a specific embodiment, the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction is made to the results to take into consideration the fact 3o that the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N-and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program.
If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N-or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity t5 calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB
alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.
The present application is also directed to proteins cotaining polypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to the TR14 polypeptide sequence set forth as n'-m', n2- m', n'- m2, and/or n2-m2 described herein. In preferred embodiments, the application is directed to proteins comprising, or alternatively consisting of, polypeptide sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific TR14 N- and C-terminal deletions recited herein.
Polynucleotides encoding these polypeptides are also encompassed by the invention.
In certain preferred embodiments, TR14 proteins of the invention comprise fusion proteins as described above wherein the TR14 polypeptides are those described as n'-m', and/or n2- m' herein. In preferred embodiments, the application is directed to nucleic acid molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences 3o encoding polypeptides having the amino acid sequence of the specific N- and C-terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.
In another aspect, the invention provides a TR14 polypeptide comprising an epitope-bearing portion of a polypeptide of the invention.. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide described herein. An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope." The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 87:3998- 4002 (1983).
As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, J.G. Sutcliffe et al., "Antibodies That React With Predetermined Sites on Proteins,"
Science 219:660-666 (1983). Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al., Cell 37:767-778 ( 1984) at 777.
Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine, at least 20, at least 25, at least 30, at least 40, at least 50 and most preferably between at least about SS to about 100 amino acids contained within the amino acid sequence of a polypeptide of the invention. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) 2o amino acids, at either extreme or at both extremes.
Non-limiting examples of predicted antigenic polypeptides that can be used to generate TR14-specific antibodies include polypeptides comprising about: Asp-2 to Asp-10, Thr-17 to Asp-38, Pro-45 to Ser-52, Pro-88 to Arg-95, Thr-108 to Glu-115, Thr-131 to Glu-136, Phe-166 to Gly-174, Ala-180 to Ala-200, and Gln-224 to Met-231 of SEQ ID N0:61.
Fragments and/or variants of these polypeptides, such as, for example, fragments and/or variants as described herein, are encompassed by the invention. Polynucleotides encoding these polypeptides (including fragments and/or variants) are also encompassed by the invention, as are antibodies that bind these polypeptides.
Additional non-limiting examples of predicted antigenic polypeptides that can be used to generate TR14-specific antibodies include: a polypeptide comprising, or alternatively consisting of amino acid residues from about 2 to about 24 in Figures 4A-D (corresponding to about amino acid 2 to about 24 in SEQ ID NO:S); a polypeptide comprising amino acid residues from about 42 to about 52 in Figures 4A-D (corresponding to about amino acid 42 to about 52 in SEQ ID NO:S); a polypeptide comprising amino acid residues from about 80 to about 115 in Figures 4A-D (corresponding to about amino acid 80 to about 115 in SEQ ID
NO:S); and a polypeptide comprising amino acid residues from about 155 to about 226 in Figures 4A-D
(corresponding to about amino acid 155 to about 226 in SEQ ID NO:S), and the corresponding amino acid sequences of SEQ ID N0:61, as the sequence of amino acid residues T-78 to M-231 of SEQ ID N0:61 is identical to the sequence of amino acid residues T-73 to M-226 of SEQ ID NO:S. As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the TR14 receptor protein. In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Additional non-limiting examples of predicted antigenic polypeptides that can be used to generate TR14-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about T3 to about S11, from about V 16 to about R24, from about to Q44 to about M52, from about F85 to about G93, from about T103 to about V 1 I 1, from about F161 to about 6169, from about V 187 to about A 195, from about P218 to about M226 of SEQ
ID NO:S (Figures 4A-D, and the corresponding amino acid sequences of SEQ ID
N0:61, as the sequence of amino acid residues T-78 to M-231 of SEQ ID N0:61 is identical to the sequence of amino acid residues T-73 to M-226 of SEQ ID NO:S) correspond to the highly antigenic regions of the TR14 protein, predicted using the Jameson-Wolf antigenic index (See Figure 6 and Table II). These highly antigenic fragments correspond to the amino acid residues illustrated in Figure 4A-D and in SEQ ID NO:S. In this context "about"
includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.
The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. R.A. Houghten, "General Method for the Rapid Solid-phase Synthesis of Large Numbers of Peptides: Specificity of Antigen-Antibody Interaction at the Level of Individual Amino Acids," Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986).
As one of skill in the art will appreciate, TR14 receptor polypeptides of the present invention and the epitope-bearing fragments thereof, described herein (e.g., corresponding to a portion of the extracellular domain, such as, for example, amino acid residues 1 to about 149, from about 2 to about 24, from about 42 to about 52, from about 80 to about 115, and/or from about 155 to about 226 of SEQ ID N0:5), can be combined with heterologous polypeptide sequences, for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827;
Traunecker et al., Nature 337:84-86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric TR14 protein or protein fragment alone (Fountoulakis et al., J.
Biochem.
270:3958-3964 (1995)). In this context "about" includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
Preferred TR14 Fc fusions of the present invention include, but are not limited to constructs comprising, or alternatively consisting of, amino acid residues: 1 to 138, 50 to 138, 70 to 90, 1 to 231, 10 to 231, 20 to 231, 30 to 231, 40 to 231, 1 to 221, 1 to 211, 1 to 201, 1 to 191, 10 to 221, 10 to 201, and/or 10 to 191 of SEQ ID N0:61.
Polynucleotides encoding these TR14 fusions are also encompassed by the invention.
Additonal TR14 Fc fusions of the present invention include, but are not limited to constructs comprising, or alternatively consisting of, amino acid residues: 1 to 133, 50 to 133, 65 to 85, 1 to 226, 10 to 226, 20 to 226, 30 to 226, 40 to 226, 1 to 216, 1 to 206, 1 to 196, 1 to 186, 10 to 216, 10 to 206, and/or 10 to 196 of SEQ ID NO:S. Polynucleotides encoding t5 these TR14 fusions are also encompassed by the invention.
The polypeptides of the present invention have uses which include, but are not limited to, as sources for generating antibodies that bind the polypeptides of the invention, and as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
Diagnostic Assays The compounds of the present invention are useful for diagnosis or treatment of various immune system-related disorders in mammals, preferably humans. Such disorders include but are not limited to tumors. (e.g., T cell, B cell and monocytic cell leukemias and lymphomas) and tumor metastasis, infections by bacteria, viruses and other parasites, immunodeficiencies, inflammatory diseases, lymphadenopathy, autoimmune diseases, and graft versus host disease.
TR13 and TR14 are expressed in immune cells and tissue. For a number of immune system-related disorders, substantially altered (increased or decreased) levels TR13 and/or TR14 gene expression can be detected in immune system tissue or other cells or bodily fluids (e.g., sera, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a "standard" TR13 and/or TR14 gene expression level, that is, the TR13 and/or TR14 expression level in immune system tissues or bodily fluids from an individual not having the immune system disorder. Thus, the invention provides a diagnostic method useful during diagnosis of an immune system disorder, which involves measuring the expression level of the gene encoding the TR13 and/or TR14 polypeptide in immune system tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard TR13 and/or TR14 gene expression level, respectively, whereby an increase or decrease in the gene expression level compared to the standard is indicative of an immune system disorder or normal activation, proliferation, differentiation, and/or death.
In particular, it is believed that certain tissues in mammals with cancer (such as, for example, cancer of cells or tissue of the immune system) express significantly enhanced or reduced levels of normal or altered TR13 and/or TR14 polypeptide and mRNA
encoding the TR13 and/or TR14 polypeptide when compared to a corresponding "standard"
level. Further, it is believed that enhanced or depressed levels of the TR13 and/or TR14 polypeptide can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) or cells or tissue from mammals with such a cancer when compared to sera from mammals of the same species not having the cancer.
to For example, polynucleotides of the invention (e.g., polynucleotide sequences complementary to all or a portion of TR13 and/or TR14 mRNA) and antibodies (and antibody fragments) directed against the polypeptides of the invention may be used to quantitate or qualitate concentrations of cells of T cell lineage and/or B cell lineage (e.g., B cell leukemia cells) expressing TR13 and/or TR14 on their cell surfaces. These antibodies additionally have diagnostic applications in detecting abnormalities in the level of TR13 and/or TR14 gene expression, or abnormalities in the structure and/or temporal, tissue, cellular, or subcellular location of TR13 and/or TR14. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
For example, as disclosed herein, TR13 or TR14 is expressed in T cells.
Accordingly, 2o polynucleotides of the invention (e.g., polynucleotide sequences complementary to all or a portion of TR13 or TR14 mRNA) and antibodies (and antibody fragments) directed against the polypeptides of the invention may be used to quantitate or qualitate concentrations of cells of T
cell lineage (e.g., T cell leukemia cells) expressing TR13 or TR14 on their cell surfaces. These polypeptides and antibodies additionally have diagnostic applications in detecting abnormalities in the level of TR13 or TR14 gene expression, or abnormalities in the structure and/or temporal, tissue, cellular, or subcellular location of TR13 or TR14. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
Thus, the invention provides a diagnostic method useful during diagnosis of a immune system disorder (including cancers of this system) and/or cell proliferation disorder (e.g., cancer, such as a cancer disclosed herein) which involves measuring the expression level of the gene encoding the TR13 and/or TR14 polypeptide in immune system tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard TR13 and/or TR14 gene expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of an immune system disorder and/or cell proliferation disorder.
Where a diagnosis of a disorder in the immune system (including diagnosis of a tumor) and/or diagnosis of a cell proliferation disorder has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed TR13 and/or TR14 gene expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
By "assaying the expression level of the gene encoding the TR13 and/or TR14 polypeptide" is intended qualitatively or quantitatively measuring or estimating the level of the TR13 and/or TR14 polypeptide or the level of the mRNA encoding the TR13 and/or polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the TR13 and/or to TR14 polypeptide level or mRNA level in a second biological sample).
Preferably, the TR13 and/or TR14 polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard TR13 and/or TR14 polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder of the immune system. As will be appreciated in the art, once a standard TR13 and/or TR14 polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing TR13 and/or TR14 receptor protein (including portions thereof) or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain free extracellular domains of the TR13 and/or TR14 polypeptide, immune system tissue, and other tissue sources found to express complete or free extracellular domain of the TR13 and/or TR14 receptor. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
Total cellular RNA can be isolated from a biological sample using any suitable technique such as, for example, the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987).
Levels of mRNA encoding the TR13 and/or TR14 polypeptide are then assayed using any appropriate method. These include Northern blot analysis, S1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).
The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of TR13 polypeptide, or the soluble form thereof, in cells and tissues, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of TR13, or soluble form thereof, compared to normal control tissue samples may be used to detect the presence of tumors, for example. Assay techniques that can be used to determine levels of a protein, such as a TR13 polypeptide of the present invention, or a soluble form thereof, in a biological sample derived 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. Preferred for assaying TR13 polypeptide levels in a biological sample are antibody-based techniques. For example, TR13 polypeptide expression in tissues can be studied with classical immunohistological methods. (M. Jalkanen et al., J.
Cell. Biol.
101:976-985 (1985); M. Jalkanen et al., J. Cell. Biol. 705:3087-3096 (1987)).
Other antibody-based methods useful for detecting TR13 gene expression include immunoassays, such as the enzyme linked immunosorbent assay (EL1SA) and the radioimmunoassay (RIA).
The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of TR14 polypeptide, or the soluble form thereof, in cells and tissues, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of TR14, or ~5 soluble form thereof, compared to normal control tissue samples may be used to detect the presence of tumors, for example. Assay techniques that can be used to determine levels of a protein, such as a TR14 polypeptide of the present invention, or a soluble form thereof, in a biological sample derived 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 2o ELISA assays. Preferred for assaying TR14 polypeptide levels in a biological sample are antibody-based techniques. For example, TR14 polypeptide expression in tissues can be studied with classical immunohistological methods. (M. Jalkanen et al., J.
Cell. Biol.
101:976-985 (1985); M. Jalkanen et al., J. Cell. Biol. 705:3087-3096 (1987)).
Other antibody-based methods useful for detecting TR14 gene expression include immunoassays, 25 such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase, and radioisotopes, such as iodine ('3'I~ 125I~ 123I~ lz~I)~
carbon ('4C), sulfur (3s5), tritium (3H), indium ("smln, "3'"In, "zln, "'In), and technetium (99Tc, 99mTC), thallium (zo~Ti), gallium (6gGa, 6'Ga), palladium ('°3Pd), molybdenum (99Mo), xenon ('33Xe), fluorine 30 (~sF)> >s3sm~ »~Lu~ 159Gd' 149Pm' ~aoLa~ ms~,b~ 166Ho~ 9oY~ a~Sc~ is6Re~
~ggRe~ iazPr~ iosRh~
9'Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the TR13 and/or TR14 gene (such as, for example, cells of T cell 35 lineage) or cells or tissue which are known, or suspected, to express the TR13 ligand and/or TR14 ligand gene (such as, for example, cells of monocytic lineage and the spleen). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the TR13 and/or TR14 gene or TR13 ligand and/or TR14 ligand gene.
For example, antibodies, or fragments of antibodies, such as those described herein, may be used to quantitatively or qualitatively detect the presence of TR13 and/or TR14 gene products or conserved variants or peptide fragments thereof. This can be accomplished, for to example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
The antibodies (or fragments thereof) or TR13 and/or TR14 polypeptides or TR13 ligand and/or TR14 ligand polypeptides of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of TR13 and/or TR14 gene products or conserved variants or polypeptide fragments thereof, or for TR13 and/or TR14 binding to TR13 and/or TR14 ligand, respectively. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody, TR13 polypeptide, or TR14 polypeptide of the present invention. The antibody (or fragment) or TR13 and/or TR14 polypeptide is 2o preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
Through the use of such a procedure, it is possible to determine not only the presence of the TR13 and/or TR14 gene product, or conserved variants or peptide fragments, or TR13 and/or TR14 polypeptide binding, but also its distribution in the examined tissue.
Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
Immunoassays and non-immunoassays for TR13 and/or TR14 gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of TR13 and/or TR14 gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
Immunoassays and non-immunoassays for TR13 ligand and/or TR14 ligand gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectable or labeled TR13 and/or TR14 polypeptide capable of identifying TR13 ligand and/or TR14 ligand gene products or conserved variants or polypeptide fragments thereof, and detecting the bound TR13 and/or TR14 polypeptide by any of a number of techniques well-known in the art.
The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled anti-TR13 and/or TR14 antibody or detectable TR13 and/or TR14 polypeptide. The solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled. The amount of bound label on solid support may then be detected by conventional means.
1o By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surFace of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
Assaying TR13 or TR14 protein levels in a biological sample can occur using any art-known method.
The binding activity of a given lot of anti-TR13 and/or anti-TR14 antibody or and/or TR14 polypeptide may be determined according to well known methods.
Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
In addition to assaying TR13 and/or TR14 polypeptide levels or polynucleotide levels in a biological sample obtained from an individual, TR13 and/or TR14 polypeptide or polynucleotide can also be detected in vivo by imaging. For example, in one embodiment of 3o the invention, TR13 and/or TR14 polypeptide is used to image monocytic leukemias or lymphomas. In another embodiment, TR13 and/or TR14 polynucleotides of the invention (e.g., polynucleotides complementary to all or a portion of TR13 and/or TR14 mRNA) is used to image T cell leukemias or lymphomas.
Antibody labels or markers for in vivo imaging of TR13 and/or TR14 polypeptide include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma. Where in vivo imaging is used to detect enhanced levels of TR13 and/or TR14 polypeptide for diagnosis in humans, it may be preferable to use human antibodies or "humanized"
chimeric monoclonal antibodies. Such antibodies can be produced using techniques described herein or otherwise known in the art. For example methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechnigues 4:214 (1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP
171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).
Additionally, any TR13 and/or TR14 polypeptide whose presence can be detected, can be administered. For example, TR13 and/or TR14 polypeptides labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further such TR13 and/or TR14 polypeptides can be utilized for in vitro diagnostic procedures.
A TR13 and/or TR14 polypeptide-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example,'3'I, "zln, ~'mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or 2o intraperitoneally) into the mammal to be examined for an immune system disorder and/or cell proliferation disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of ~'"'Tc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain TR13 and/or TR14 protein. In vivo tumor imaging is described in S.W.
Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments"
(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B.
A. Rhodes, eds., Masson Publishing Inc. ( 1982)).
3o With respect to antibodies, one of the ways in which the anti-TR13 and/or anti-TR14 antibody can be detectably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Volley, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Volley et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J.E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC
Press, Boca Raton, FL,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase,asparaginase, glucose oxidase,beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme.
Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in l0 comparison with similarly prepared standards.
Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect TR13 and/or TR14 through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can 2o then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
The antibody can also be detectably labeled using fluorescence emitting metals such as ~szEu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
3o Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

TR13 and TR14 Binding Peptides and Other Molecules The invention also encompasses screening methods for identifying polypeptides and nonpolypeptides that bind TR13 or TR14, and the TR13 or TR14 binding molecules identified thereby. These binding molecules are useful, for example, as agonists and antagonists of the TR13 or TR14 receptor proteins. Such agonists and antagonists can be used, in accordance with the invention, in the therapeutic embodiments described in detail, below.
This method comprises the steps of:
a. contacting a TR13 or TR14 protein or TR13 or TR14-like protein with a plurality of molecules; and l0 b. identifying a molecule that binds the TR13 or TR14 protein or TR13 or TR14-like protein.
The step of contacting the TR13 or TR14 protein or TR13 or TR14-like protein with the plurality of molecules may be effected in a number of ways. For example, one may contemplate immobilizing the TR13 or TR14 protein or TR13 or TR14-like protein on a solid 15 support and bringing a solution of the plurality of molecules in contact with the immobilized TR13 or TR14 protein or TR13 or TR14-like protein. Such a procedure would be akin to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized TR13 or TR14 protein or TR13 or TR14-like protein. The molecules having a selective affinity for the TR13 or TR14 protein or TR13 or TR14-like protein can then be purified by 20 affinity selection. The nature of the solid support, process for attachment of the TR13 or TR14 protein or TR13 or TR14-like protein to the solid support, solvent, and conditions of the affinity isolation or selection are largely conventional and well known to those of ordinary skill in the art.
Alternatively, one may also separate a plurality of polypeptides into substantially 25 separate fractions comprising a subset of or individual polypeptides. For instance, one can separate the plurality of polypeptides by gel electrophoresis, column chromatography, or like method known to those of ordinary skill for the separation of polypeptides.
The individual polypeptides can also be produced by a transformed host cell in such a way as to be expressed on or about its outer surface (e.g., a recombinant phage). Individual isolates can then be 30 "probed" by the TR13 or TR14 protein or TR13 or TR14-like protein, optionally in the presence of an inducer should one be required for expression, to determine if any selective affinity interaction takes place between the TR13 or TR14 protein or TR13 or TR14-like protein and the individual clone. Prior to contacting the TR13 or TR14 protein or TR13 or TR14-like protein with each fraction comprising individual polypeptides, the polypeptides could first be transferred to a solid support for additional convenience. Such a solid support may simply be a piece of filter membrane, such as one made of nitrocellulose or nylon. In this manner, positive clones could be identified from a collection of transformed host cells of an expression library, which harbor a DNA construct encoding a polypeptide having a selective affinity for TR13 or TR14 protein or TR13 or TR14-like protein. Furthermore, the amino acid sequence of the polypeptide having a selective affinity for the TR13 or TR14 protein or TR13 or TR14-like protein can be determined directly by conventional means or the coding sequence of the DNA
encoding the polypeptide can frequently be determined more conveniently. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid to sequence is to be determined from the polypeptide itself, one may use microsequencing techniques. The sequencing technique may include mass spectroscopy.
In certain situations, it may be desirable to wash away any unbound TR13 or TR14 protein or TR13 or TR14-like protein, or alterntatively, unbound polypeptides, from a mixture of the TR13 or TR14 protein or TR13 or TR14-like protein and the plurality of polypeptides prior to attempting to determine or to detect the presence of a selective affinity interaction. Such a wash step may be particularly desirable when the TR13 or TR14 protein or TR13 or TR14-like protein or the plurality of polypeptides is bound to a solid support.
The plurality of molecules provided according to this method may be provided by way of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind to TR13 or TR14. Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries. Examples of chemically synthesized libraries are described in Fodor et al., 1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710;Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251;
Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc.
Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412;
Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242;
and 3o Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.
Examples of phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R. B., et al., 1992, J.
Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.
In vitro translation-based libraries include but are not limited to those described in PCT
Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl.

Acad. Sci. USA 91:9022-9026.
By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be adapted for use.
Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used.
Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).
The variety of non-peptide libraries that are useful in the present invention is great. For example, Ecker and Crooke, 1995, Bio/Technology 13:351-360 list benzodiazepines, 1o hydantoins, piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, and oxazolones as among the chemical species that form the basis of various libraries.
Non-peptide libraries can be classified broadly into two types: decorated monomers and oligomers. Decorated monomer libraries employ a relatively simple scaffold structure upon which a variety functional groups is added. Often the scaffold will be a molecule with a known useful pharmacological activity. For example, the scaffold might be the benzodiazepine structure.
Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in ways that create new shapes that depend on the order of the monomers. Among the 2o monomer units that have been used are carbamates, pyrrolinones, and morpholinos. Peptoids, peptide-like oligomers in which the side chain is attached to the alpha amino group rather than the alpha carbon, form the basis of another version of non-peptide oligomer libraries. The first non-peptide oligomer libraries utilized a single type of monomer and thus contained a repeating backbone. Recent libraries have utilized more than one monomer, giving the libraries added flexibility.
Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries:
Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S.
Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673; and CT Publication No. WO
94/18318.
In a specific embodiment, screening to identify a molecule that binds TR13 or TR14 can be carried out by contacting the library members with a TR13 or TR14 protein or TR13 or TR14-like protein immobilized on a solid phase and harvesting those library members that bind to the TR13 or TR14 protein or TR13 or TR14-like protein. Examples of such screening methods, termed "panning" techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT
Publication No. WO 94/18318; and in references cited herein.
In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al., 1991, Proc. Natl.
Acad. Sci. USA
88:9578-9582) can be used to identify molecules that specifically bind to TR13 or TR14 or TR13 or TR14-like proteins.
Where the TR13 or TR14 binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, to combinatorial peptide libraries, or biased peptide libraries. The term "biased" is used herein to mean that the method of generating the library is manipulated so as to restrict one or more parameters that govern the diversity of the resulting collection of molecules, in this case peptides.
Thus, a truly random peptide library would generate a collection of peptides in which 15 the probability of finding a particular amino acid at a given position of the peptide is the same for all 20 amino acids. A bias can be introduced into the library, however, by specifying, for example, that a lysine occur every fifth amino acid or that positions 4, 8, and 9 of a decapeptide library be fixed to include only arginine. Clearly, many types of biases can be contemplated, and the present invention is not restricted to any particular bias.
Furthermore, the present 2o invention contemplates specific types of peptide libraries, such as phage displayed peptide libraries and those that utilize a DNA construct comprising a lambda phage vector with a DNA
insert.
As mentioned above, in the case of a TR13 or TR14 binding molecule that is a polypeptide, the polypeptide may have about 6 to less than about 60 amino acid residues, 25 preferably about 6 to about 10 amino acid residues, and most preferably, about 6 to about 22 amino acids. In another embodiment, a TR13 or TR14 binding polypeptide has in the range of 15-100 amino acids, or 20-SO amino acids.
The selected TR13 or TR14 binding polypeptide can be obtained by chemical synthesis or recombinant expression.
Enitopes The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the TR13 and TR14 polypeptides described in detail above or encoded by a polynucleotide that hybridizes to the complement of the sequence of TR13 and TR14 coding sequences described in detail above, under stringent hybridization conditions or lower stringency hybridization conditions as defined supra.

The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the, polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
The term "epitopes," as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses 1o a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An "immunogenic epitope," as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-(1983)). The term "antigenic epitope," as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not 2o necessarily be immunogenic.
Fragments that function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).
In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 ( 1983)).
Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA
82:910-914;
and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as, for example, rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., 1o Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
For instance, peptides containing cysteine residues may be coupled to a carrier using a 15 linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as, for example, rabbits, rats, and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or 20 carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody that can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
The titer of anti-peptide antibodies in serum from an immunized animal may be 25 increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope 3o can be fused to other polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for 35 chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone.
See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA
88:8972- 897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix-binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Niz+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the 2o polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238;
5,830,721;
5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J.
Mol.
Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308- 13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ
ID NO:l or 60 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA
segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide coding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. (each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of TR13 and/or TR14 polynucleotides and corresponding polypeptides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired TR13 and/or TR14 molecule by homologous, or site-specific, recombination. In another embodiment, TR13 and/or TR14 polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of TR13 and/or TR14 may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are receptors for TNF-alpha, TNF-beta, lymphotoxin-alpha, lymphotoxin-beta, FAS
ligand, and APRIL. In further preferred embodiments, the heterologous molecules are any member of the TNF family.
Antibodies The present invention further relates to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, preferably an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments 2o produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, 3o single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable regions) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable regions) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT' publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
Antibodies of the present invention may be described or specified in terms of ~5 the epitope(s) or portions) of a polypeptide of the present invention that they recognize or specifically bind. The epitope(s) or polypeptide portions) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies that specifically bind any epitope or polypeptide of the present invention may also be excluded.
2o Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind 25 polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%
identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, 30 less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
Further included in the present invention are antibodies that bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under 35 stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than SX10-ZM, 10-2M, 5X10-3M, 10-3M, SX10-4M, 4M, 5X10~5M, 10-5M, SX10-6M, 10-6M, SX10-'M, 10-'M, SX10-$M, 10-8M, 5X10-9M, 10-9M, SX10-'°M, 10-'°M, SX10-"M, 10-"M, 5X10-'ZM, 10-'2M, 5X10-'3M, 10-'3M, SX10-'4M, 10''°M, 5X10-'SM, and 10-'5M.
The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
Antibodies of the present invention may act as agonists or antagonists of the 1o polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation.
15 Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand 20 or receptor activity by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or 25 the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, 30 i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281;
35 U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen, et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-(1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon, et al., J.
Immunol.
160(7):3170-3179 ( 1998); Prat et al ., J. Cell . Sci . 11 I (Pt2):237-247 ( 1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998);
Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).
Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring to levels of the polypeptides of the present invention in biological samples.
See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins.
See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
The antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be 1o produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not 15 limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
Methods for producing and screening for specific antibodies using hybridoma technology are routine and well-known in the art and are discussed in detail in Example 20 5, below. Briefly, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the 25 ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
3o Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas 35 resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular, such phage can be utilized to display antigen-binding domains expressed from a repertoire or to combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv t5 antibody domains recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J.
Immunol.
Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 20 18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737;
WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908;
5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;
25 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and 3o expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to ~recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO
92/22324;
Mullinax et al., BioTechniques 12(6):864-869 ( 1992); and Sawai et al., AJRI
34:26-35 34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol.
Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entireties. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539;
5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the ~5 transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int.
Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735;
U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;
5,545,806; 5,814,318; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.
(Jespers et al., Biotechnology 12:899-903 (1988)).
Further, antibodies~to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand.
Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
Polynucleotides Encoding Antibodies The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to TR13 or TR14 polypeptide of the invention, as described above.
~ 5 The polynucleotides may be obtained, and the nucleotide sequence of the po(ynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonuc(eotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis 2o of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding 25 a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by 3o PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
35 Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.

(see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or is consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid 2o substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen.
Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
Other 25 alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;
Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing 3o genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized 35 antibodies.
Alternatively, techniques described for the production of single chain antibodies (U.S. Patent No. 4,694,778; Bird, 1988, Science 242:423- 42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038- 1041).
Methods of Producing Antibodies The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody of the invention, requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a ~5 heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA
technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide 2o sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors 25 comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO
86/05807;
PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464.) and the variable 3o domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells 35 containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences;
yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences;
plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K
promoter).
Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101;
Cockett et al., 1990, Bio/Technology 8:2).
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E.
coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791 ), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye &
Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.
Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned 1o individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the ~5 antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of 2o expressing the antibody molecule in infected hosts. (e.g., see Logan &
Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences.
These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to 25 ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).
3o In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
Different host cells have characteristic and specific mechanisms for the post-35 translational processing and modification of proteins and gene products.
Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
2o A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl.
Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA
78:1527);
gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc.
Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside 3o G-418 Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.
62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley &
Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated by reference herein in their entireties.
The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA
cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene.
Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Grouse et al., 1983, Mol. Cell. Biol. 3:257).
The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
Antibody Conjugates The present invention encompasses antibodies recombinantly fused or 3o chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention.
For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO
93/21232;
EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.
146:2446-2452(1991), which are incorporated by reference in their entireties.
The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH 1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046;
5,349,053;
5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388;
WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991);
Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl.
Acad.
Sci. USA 89:11337- 11341(1992) (said references incorporated by reference in their entireties).
As discussed, supra, the polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art.
Further, the polypeptides of the present invention may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A

232,262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)0.
Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitates their purification. In 1o preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein.
Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.
The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S.
Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or 3o acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine ('3'I,'zsl,'z3I,'z'1), carbon ('4C), sulfur (35S), tritium (3H), indium ("smln, "3mln, "zln, "'In), and technetium (~''Tc, ~''"Tc), thallium (z°'Ti), gallium (6~Ga, 6'Ga), palladium ('°3Pd), molybdenum (~''Mo), xenon ('33Xe), fluorine ('8F), 153Sm' 177Lu' 159Gd' 149Pm' l4oLa~ l7sYb' 166HO' 90Y' 47SC' 186Re' 188Re' 142Pr' lOSRh, 97Ru .
Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
The conjugates of the invention can be used for modifying a given 2o biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon,13-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-I
("IL-1 "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.
Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.
623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62:119-( 1982).
to Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No.
4,676,980, which is incorporated herein by reference in its entirety.
An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factors) and/or cytokine(s) can be used as a therapeutic.
Assays For Antibody Binding The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but 2o are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
3o Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1%
sodium deoxycholate, 0.1% SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1%
Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C, adding protein A
and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF
or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can 2o be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes 3o the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art.
For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest is 1o conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
Therapeutic Uses The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the described disorders. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases and disorders associated with aberrant expression and/or activity of a polypeptides of the invention, including, but not limited to, immune disorders. The treatment and/or prevention of diseases and disorders associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases and disorders. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
Antibodies that agonize the TR14 receptor can be used to ameliorate or treat biological activities associated with epithelial cell proliferation, tooth development, growth of 3o mucosal layers, and the growth of epithelial surfaces, including hair folicles, sweat glands, basal cells, and dermis. Accordingly, TR14 agonistic antibodies may be used in the treatment of diseases and/or disorders relating to the epithelium (e.g., anhidrotic ectodermal dysplasia, hidrotic ectodermal dysplasia, sweat gland disorders, venous ulcers, psoriasis, prickly heat disorder, wounds healing, cancers of epithelial origins, male pattern baldness, and/or as described under "Epithelial Cell Proliferation and Wound Healing" below).
A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or 2o neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10-6 M, 10-6 M, 5 X 10-7 M, 10-7 M, 5 X 10-8 M, 10-8 M, 5 X 10-9 M, 10-9 M, 5 X 10-10 M, 10-10 M, 5 X 10-11 M, 10-11 M, 5 X 10-12 M, 10-12 M, 5 X 10-13 M, 10- 13 M, 5 X 10-14 M, 10-14 M, 5 X 10-15 M, and 10-15 M.
3o Gene Therapy In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.
62:191-217; May, 1993, TIBTECH 11(5):155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY;
and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.
In a preferred aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue- specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody nucleic acids (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA
86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as 3o in vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product.
This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun;
Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT
to Publications WO 92/06180 dated April 16, 1992 (Wu et al.); WO 92/22635 dated December 23, 1992 (Wilson et al.); W092/20316 dated November 26, 1992 (Findeis et al.); W093/14188 dated July 22, 1993 (Clarke et al.), WO 93/20221 dated October 14, 1993 (Young)). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination ~5 (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935;
Zijlstra et al., 1989, Nature 342:435-438).
In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviral 20 vectors have been to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use 25 of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin.
Invest.
93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in 3o Genetics and Devel. 3:110-114.
Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central 35 nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys.
Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143- 155;
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT Publication W094/12649;
and Wang, et al., 1995, Gene Therapy 2:775-783. In a preferred embodiment, adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S.
Patent No. 5,436,146).
to Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those is cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector 2o containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be 25 used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
3o The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
35 Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes;
blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous to the patient.
In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or to progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598, dated April 28, 1994;
Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio.
21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).
1n a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
2o Demonstration of Therapeutic or Prophylactic Activity The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample.
The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific 3o compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
Therapeutic/Prophylactic Administration and Composition The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above;
additional appropriate formulations and routes of administration can be selected from among those described herein below.
Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, to microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or ~5 compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the 2o invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
25 In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a 3o suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a 35 vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533;
Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.) In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974);
Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev.
Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J.Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533).
In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic 2o acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox- like peptide which is known to enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.
Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA
for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a 3o pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier"
refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, ,can also contain minor amounts of wetting or emulsifying agents, or pH
buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W.
Martin.
Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a 1o patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
Further, the ~5 dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical 20 compositions of the invention. Optionally associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
25 Diagnosis and Imaging Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the 30 detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard 35 expression level is indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosising a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to~ those of skill in the art (e.g., see Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985);
Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ('3'I,'ZSI, "~I,'z'I), carbon ('4C), sulfur (35S), tritium (3H), indium ("5'"In, "3"'In, "ZIn, "'In), and technetium (9~'Cc, 99"'Tc), thallium (Z°'Ti), gallium (~Ga, 6'Ga), palladium ('°3Pd), molybdenum (~'Mo), 2o xenon (~ssXe)~ Buorine (~sF)~ ls~sm~ »~Lu~ ~s9Gd~ ~a9Pm~ ~a°La~
mshb~ ~66Ho~ 9oY~
4'Sc,'g6Re,'~Re,'42Pr,'osRh, 9'Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of the interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at 3o sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level;
and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest.
Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson to Publishing Inc. (1982).
Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or t5 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, 20 etc.
Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic 25 methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et 3o al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic 35 label and is detected in a patient using magnetic resonance imaging (MRI).
Kits The present invention provides kits that can be used in the above methods.
In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a 1o fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not~react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-po(ypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.
In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached.
Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention.
The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or 1o colorimetric substrate (Sigma, St. Louis, MO).
The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
Therapeutics The Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes (D.V. Goeddel et al., "Tumor Necrosis Factors: Gene Structure and Biological Activities," Symp. Quant. Biol. 51:597- 609 (1986), Cold Spring Harbor; B.
Beutler and A.
Cerami, Annu. Rev. Biochem. 57:505-518 (1988); L.J. Old, Sci. Am. 258:59-75 (1988); W.
Fiers, FEBS Lett. 285:199-224 (1991)). The TNF-family ligands induce such various cellular responses by binding to TNF-family receptors.
E~ithilial Disorder-Related Therapeutic Embodiments for TR14 TR14 polynucleotides or polypeptides, or agonists or antagonists of the present invention, can be used in assays to test for one or more biological activities. If these polynucleotides or polypeptides, or agonists or antagonists of the present invention, do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides, and agonists or antagonists could be used to treat the associated disease.
TR14 polynucleotides and translation products are believed to be involved in biological activities associated with epithelial cell proliferation, tooth development, growth of mucosal layers, and the growth of epithelial surfaces, including hair folicles, sweat glands, basal cells, and dermis. Accordingly, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, detection and/or treatment of diseases and/or disorders associated with aberrant TR14 activity. In preferred embodiments, compositions of the invention (including TR14 polynucleotides, polypeptides and TR14 agonists, including peptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, detection and/or treatment of diseases and/or disorders relating to the epithelium (e.g., anhidrotic ectodermal dysplasia, hidrotic ectodermal dysplasia, sweat gland disorders, venous ulcers, psoriasis, prickly heat disorder, wounds healing, cancers of epithelial origins, male pattern baldness, and/or as described under "Epithelial Cell Proliferation and Wound Healing" below).
Thus, polynucleotides, translation products and antibodies of the invention are useful in the diagnosis, detection and/or treatment of diseases and/or disorders associated with activities that include, but are not limited to, diseases and/or disorders of the epithelium and epithelial cell proliferation diseases and/or disorders.
2o More generally, polynucleotides, translation products and antibodies corresponding to this gene may be useful for the diagnosis, detection and/or treatment of diseases and/or disorders associated with the following systems.
Epithelial Cell Proliferation and Wound Healing In accordance with yet a further aspect of the present invention, there is provided a process for utilizing polynucleotides or translation products, as well as agonists or antagonists of the present invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as 3o agonists or antagonists of the present invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associted with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote dermal reestablishment subsequent to dermal loss.

In specific, preferred embodiments, TR14 polynucleotides and polypeptides, as well as antibodies that agonize TR14 receptor (as described in the section on Antibodies, above), stimulate epithelial cell proliferation and/or development to ameliorate the diseases and disorders described in this section. Members of the TNF family of proteins are known to signal through the NF-xB singaling pathway. NF-xB is a transcription factor activated by a wide certain agents to stimulate cell activation and differentiation. It is believed that the TR14 receptor of the instant invention signals through the NF-xB pathway to activate proliferation and development of cells. Thus, TR14 polynucleotides and polypeptides of the invention as well as antibodies and peptides that agonize TR14 may be used in accordance with the invention to stimulate NF-xB-mediated epithelial cell proliferation, and thereby treat the epithelial disorders described above.
It is believed that polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intesting, and large intestine.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. Polynucleotides or polypeptides, agonists or antagonists of the present invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are types of grafts that polynucleotides or polypeptides, agonists or antagonists of the present invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Oilier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, 3o split skin graft, thick split graft. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, can be used to promote skin strength and to improve the appearance of aged skin.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may have a cytoprotective effect on the small intestine mucosa. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in to frequent, open and painful blisters by accelerating reepithelialization of these lesions.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflamamatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease.
Treatment with polynucleotides or polypeptides, agonists or antagonists of the present invention, is expected to 2o have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat diseases associate with the under expression.
Moreover, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to prevent and heal damage to the lungs due to various pathological states. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using polynucleotides or polypeptides, agonists or antagonists of the present invention. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.
Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).
In addition, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.
Additional Therapeutic Embodiments TR13 nucleic acids, polypeptides (proteins), agonists and/or antagonists of the invention may be administered to a patient (e.g., mammal, preferably human) afflicted with any disease or disorder mediated (directly or indirectly) by defective, or deficient levels of, TR13.
Alternatively, a gene therapy approach may be applied to treat such diseases or disorders. In one embodiment of the invention, TR13 polynucleotide sequences are used to detect mutein 2o TR13 genes, including defective genes. Mutein genes may be identified in in vitro diagnostic assays using techniques known in the art, and by comparison of the TR13 nucleotide sequence disclosed herein with that of a TR13 gene obtained from a patient suspected of harboring a defect in this gene. Defective genes may be replaced with normal TRI3-encoding genes using techniques known to one skilled in the art.
In another embodiment, the TR13 polypeptides, nucleic acids, agonists and/or antagonists of the present invention are used as research tools for studying the phenotypic effects that result from inhibiting TR13/TR13 ligand interactions on various cell types. TR13 polypeptides and antagonists (e.g. monoclonal antibodies to TR13) also may be used in in vitro assays for detecting TR13 or TR13 ligand(s) or the interactions thereof.
3o TR14 nucleic acids, polypeptides (proteins), agonists and/or antagonists of the invention may be administered to a patient (e.g., mammal, preferably human) afflicted with any disease or disorder mediated (directly or indirectly) by defective, or deficient levels of, TR14.
Alternatively, a gene therapy approach may be applied to treat such diseases or disorders. In one embodiment of the invention, TR14 polynucleotide sequences are used to detect mutein TR14 genes, including defective genes. Mutein genes may be identified in in vitro diagnostic assays using techniques known in the art, and by comparison of the TR14 nucleotide sequence disclosed herein with that of a TR14 gene obtained from a patient suspected of harboring a defect in this gene. Defective genes may be replaced with normal TR14-encoding genes using techniques known to one skilled in the art.
In another embodiment, the TR14 polypeptides, nucleic acids, agonists and/or antagonists of the present invention are used as research tools for studying the phenotypic effects that result from inhibiting TR14/TR14 ligand interactions on various cell types. TR14 polypeptides and antagonists (e.g. monoclonal antibodies to TR14) also may be used in in vitro assays for detecting TR14 or TR14 ligand(s) or the interactions thereof.
Cells which express the TR13 polypeptide and are believed to have a potent cellular response to TR13 ligands include pancrease tumor, endometrial tumor, adult small intestine, colon cancer, breast cancer cell line, resting T-cell, amygdala, rectum, T-cell helper, pineal gland, apoptotic T-cell, epididymus, greater omentum, prostate BPH, osteoclastoma, endometrial stromal cells, stromal cell, substantia nigra, activated T-cell, tonsil, and testes tissue. By "a cellular response to a TNF-family ligand" is intended any genotypic, phenotypic, and/or morphologic change to a cell, cell line, tissue, tissue culture or patient that is induced by IS a TNF-family ligand (such as, for example, a TNF-ligand disclosed herein).
As indicated, such cellular responses include not only normal physiological responses to TNF-family ligands, but also diseases associated with increased apoptosis or the inhibition of apoptosis.
Apoptosis-programmed cell death is a physiological mechanism involved in the deletion of peripheral T lymphocytes of the immune system, and its dysregulation can lead to a number of 2o different pathogenic processes (J.C. Ameisen, AIDS 8:1197-1213 (1994); P.H.
Krammer et al., Curr. Opin. Immunol. 6:279-289 ( 1994)).
Cells which express TR14 polypeptide and that are believed to have a potent cellular response to TR14 ligands include activated T-cell, endometrial, thymus, and 12 week early stage human tissue. By "a cellular response to a TNF-family ligand" is intended any genotypic, 25 phenotypic, and/or morphologic change to a cell, cell line, tissue, tissue culture or patient that is induced by a TNF-family ligand (such as, for example, a TNF-ligand described herein). As indicated, such cellular responses include not only normal physiological responses to TNF-family ligands, but also diseases associated with dysregulation of these physiological responses, such as, for example, diseases associated with increased apoptosis or the inhibition 30 of apoptosis. Apoptosis-programmed cell death is a physiological mechanism involved in the deletion of peripheral T lymphocytes of the immune system, and its dysregulation can lead to a number of different pathogenic processes (J.C. Ameisen, AIDS 8:1197-1213 (1994); P.H.
Krammer et al., Curr. Opin. Immunol. 6:279-289 (1994)).
Diseases associated with increased cell survival, or the inhibition of apoptosis, and that 35 may be treated or prevented by the TR13 polynucleotides, polypeptides and/or agonists or antagonists of the invention include, but are not limited to, cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostrate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis rheumatoid arthritis);
viral infections (such as herpes viruses, pox viruses and adenoviruses); inflammation; graft vs. host disease;
acute graft rejection and chronic graft rejection. In preferred embodiments, TR13 nucleic acids, polypeptides, agonists and/or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above, or in the paragraphs that follow.
Additional diseases or conditions associated with increased cell survival and that may be treated or prevented by the TR13 polynucleotides, polypeptides, agonists and/or antagonists of the invention include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's 2o macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. In preferred embodiments, TR13 nucleic acids, polypeptides, agonists and/or antagonists are used to treat the diseases and disorders listed above.
In additional embodiments, TR13 nucleic acids, polypeptides, agonists and/or antagonists are used to treat pancreas tumor, endometrial tumor, colon cancer, breast cancer, prostate BPH and/or osteosarcoma.

Thus, in preferred embodiments TR13 polynucleotides or polypeptides of the invention and agonists or antagonists thereof, are used to treat or prevent autoimmune diseases and/or inhibit the growth, progression, and/or metastasis of cancers, including, but not limited to, those cancers disclosed herein, such as, for example, pancreatic cancer, endometrial cancer, colon cancer, breast cancer, osteocarcoma, and lymphocytic leukemias (including, for example, MLL and chronic lymphocytic leukemia (CLL)) and follicular lymphomas. In another embodiment TR13 polynucleotides or polypeptides of the invention and/or agonists or antagonists thereof, are used to activate, differentiate or proliferate cancerous cells or tissue (e.g., T cell lineage related cancers and B cell lineage related cancers (e.g., CLL and MLL), to lymphocytic leukemia, or lymphoma) and thereby render the cells more vulnerable to cancer therapy (e.g., chemotherapy or radiation therapy).
Diseases associated with increased apoptosis and that may be treated or prevented by the polynucleotides, polypeptides and/or agonists or antagonists of the invention include, but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis); myelodysplastic syndromes (such as aplastic 2o anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (such as hepatitis related liver injury, ischemialreperfusion injury, cholestosis (bile duct injury) and liver cancer);
toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
In preferred embodiments, TR13 nucleic acids, polypeptides, agonists and/or antagonists are used to treat the diseases and disorders listed above.
Diseases associated with increased cell survival, or the inhibition of apoptosis, and that may be treated or prevented by the TR14 polynucleotides, polypeptides and/or agonists or antagonists of the invention include, but are not limited to, cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but 3o not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostrate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis rheumatoid arthritis);
viral infections (such as herpes viruses, pox viruses and adenoviruses); inflammation; graft vs. host disease;
acute graft rejection and chronic graft rejection. In preferred embodiments, TR14 nucleic acids, polypeptides, agonists and/or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above, or in the paragraphs that follow.
Additional diseases or conditions associated with increased cell survival and that may be treated or prevented by the TR14 polynucleotides, polypeptides and/or agonists or antagonists of the invention include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. In preferred embodiments, TR14 nucleic acids, polypeptides, agonists and/or antagonists are used to treat the diseases and disorders listed above.
Thus, in preferred embodiments TR14 polynucleotides or polypeptides of the invention and agonists or antagonists thereof, are used to treat or prevent autoimmune diseases and/or inhibit the growth, progression, and/or metastasis of cancers, including, but not limited to, those cancers disclosed herein, such as, for example, lymphocytic leukemias (including, for example, MLL and chronic lymphocytic leukemia (CLL)) and follicular lymphomas.
In another embodiment TRI4 polynucleotides or polypeptides of the invention and/or agonists or antagonists thereof, are used to activate, differentiate or proliferate cancerous cells or tissue (e.g., T cell lineage cancers and B cell lineage related cancers (e.g., CLL
and MLL), lymphocytic leukemia, or lymphoma) and thereby render the cells more vulnerable to cancer therapy (e.g., chemotherapy or radiation therapy).
Diseases associated with increased apoptosis and that may be treated or prevented by the TR14 polynucleotides, polypeptides and/or agonists or antagonists of the invention include, but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amvotrophic lateral sclerosis, Retinitis pi~mentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis); myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (such as hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer);
toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
In preferred embodiments, TR14 nucleic acids, polypeptides, agonists and/or antagonists are used to treat the diseases and disorders listed above.
Many of the pathologies associated with HIV are mediated by apoptosis, including HIV-induced nephropathy and HIV encephalitis. Thus, in additional preferred embodiments, ~5 TR13 nucleic acids, polypeptides, and/or TR13 agonists or antagonists of the invention are used to treat AIDS and pathologies associated with AIDS.
Many of the pathologies associated with HIV are mediated by apoptosis, including HIV-induced nephropathy and HIV encephalitis. Thus, in additional preferred embodiments, TR14 nucleic acids, polypeptides, and/or TR14 agonists or antagonists of the invention are 2o used to treat AIDS and pathologies associated with AIDS.
Another embodiment of the present invention is directed to the use of TR13 to reduce cell death dependent upon a TNF family member, of T cells in HN-infected patients. The state of immunodeficiency that defines AIDS is secondary to a decrease in the number and function of CD4+ T-lymphocytes. Recent reports estimate the daily loss of CD4+ T cells to be between 25 3.5 x 10' and 2 x 109 cells (Wei et al., Nature 373:117-122 (1995)). One cause of CD4+ T cell depletion in the setting of HIV infection is believed to be HIV-induced apoptosis (see, for example, Meyaard et al., Science 257:217-219, 1992; Groux et al., J Exp. Med., 175:331, 1992; and Oyaizu et al., in Cell Activation and Apoptosis in HIV Infection, Andrieu and Lu, Eds., Plenum Press, New York, 1995, pp. 101-114). Indeed, HIV-induced apoptotic cell 3o death has been demonstrated not only in vitro but also, more importantly, in infected individuals (J.C. Ameisen, AIDS 8:1197-1213 (1994); T.H. Finkel and N.K.
Banda, Curr.
Opin. Immunol. 6:605-615(1995); C.A. Muro-Cacho et al., J. Immunol. 154:5555-(1995)). Furthermore, apoptosis and CD4+ T-lymphocyte depletion is tightly correlated in different animal models of AIDS (T. Brunner et al., Nature 373:441-444 (1995);
M.L.
35 Gougeon et al., AIDS Res. Hum. Retroviruses 9:553-563 (1993)) and, apoptosis is not observed in those animal models in which viral replication does not result in AIDS. Id.
Further data indicates that uninfected but primed or activated T lymphocytes from HIV-infected individuals undergo apoptosis after encountering the TNF-family ligand Fast.
Using monocytic cell lines that result in death following HIV infection, it has been demonstrated that infection of U937 cells with HIV results in the de novo expression of Fast and that Fast mediates HIV-induced apoptosis (A.D. Badley et al., J. Virol. 70:199-206 (1996)). Further, the TNF-family ligand was detectable in uninfected macrophages and its expression was upregulated following HIV infection resulting in selective killing of uninfected CD4 T-lymphocytes. Id. Thus, by the invention, a method for treating HIV+
individuals is provided which involves administering TR13 polynucleotides, polypeptides and/or TR13 agonists or antagonists of the present invention to reduce selective killing of CD4+ T-lymphocytes. Modes of administration and dosages are discussed in detail below.
Activated human T cells are induced to undergo programmed cell death (apoptosis) upon triggering through the CD3/T cell receptor complex, a process termed activated-induced cell death (AICD). AICD of CD4+ T cells isolated from HIV-Infected asymptomatic individuals has been reported (Groux et al., supra). Thus, A1CD may play a role in the depletion of CD4+
T cells and the progression to AIDS in HIV-infected individuals. Thus, the present invention provides a method of inhibiting a tumor-necrosis factor family member-mediated T cell death in HIV patients, comprising administering a TR13 polypeptide of the invention (preferably, a soluble TR13 polypeptide) to the patients. In one embodiment, the patient is asymptomatic when treatment with TR13 commences. If desired, prior to treatment, peripheral blood T cells may be extracted from an HIV patient, and tested for susceptibility to cell death mediated by a tumor necrosis factor family member, by procedures known in the art. In one embodiment, a patient's blood or plasma is contacted with TR13 ex vivo. The TR13 may be bound to a suitable chromatography matrix by procedures known in the art. The patient's blood or plasma flows through a chromatography column containing TR13 bound to the matrix, before being returned to the patient. In the event the immobilized TR13 bound to TRAIL, or another TNF
family member(s), TRAIL and/or other TNF family member protein would be removed from the patient's blood.
In additional embodiments a TR13 polypeptide, polynucleotide, and/or agonist or antagonist of the invention is administered in combination with inhibitors of T cell apoptosis.
For example, TRAIL-mediated apoptosis and Fas-mediated apoptosis have been implicated in loss of T cells in HIV individuals (See, e.g., Katsikis et al., J. Exp. Med.
181:2029-2036 (1995)). Thus, a patient susceptible to both Fas ligand mediated and/or TRAIL
mediated T cell death may be treated as an agent that blocks TRAILJTRAIL receptor interactions and/or an agent that blocks Fas-ligand/Fas interactions. Suitable agents for blocking binding ,of Fas-ligand to Fas include, but are not limited to, soluble Fas polypeptides;
mulitmeric forms of soluble Fas polypeptides (e.g., dimers of sFas/Fc); anti-Fas antibodies that bind Fas without transducing the biological signal that results in apoptosis; anti-Fas-ligand antibodies that block binding of Fas-ligand to Fas; and muteins of Fas-ligand that bind Fas but do not transduce the biological signal that results in apoptosis. Preferably, the antibodies employed according to this method are monoclonal antibodies. Examples of suitable agents for blocking Fas-ligand/Fas interactions, including blocking anti-Fas monoclonal antibodies, are described in International application publication number WO 95/10540, hereby incorporated by reference.
Suitable agents, which block binding of TRAIL to a TRAIL receptor or FAS
ligand to FAS that may be administered with the nucleic acids, polypeptides, and/or agonists or antagonists of the present invention include, but are not limited to, soluble TRAIL receptor polypeptides (e.g., a soluble form of OPG, DR4 (International application publication number WO 98/32856); TR5 (International application publication number WO 98/30693);
and DRS
(International application publication number WO 98/41629)); multimeric forms of soluble to TRAIL receptor polypeptides; and TRAIL receptor antibodies that bind the TRAIL receptor without transducing the biological signal that results in apoptosis, anti-TRAIL antibodies that block binding of TRAIL to one or more TRAIL receptors, and muteins of TRAIL
that bind TRAIL receptors but do not transduce the biological signal that results in apoptosis. Preferably, the antibodies employed according to this method are monoclonal antibodies.
Another embodiment of the present invention is directed to the use of TR14 to reduce cell death dependent upon a TNF family member, of T cells in HIV-infected patients. The state of immunodeficiency that defines AIDS is secondary to a decrease in the number and function of CD4+ T-lymphocytes. Recent reports estimate the daily loss of CD4+ T cells to be between 3.5 x 10' and 2 x 109 cells (Wei et al., Nature 373:117-122 (1995)). One cause of CD4+ T cell 2o depletion in the setting of HIV infection is believed to be HIV-induced apoptosis (see, for example, Meyaard et al., Science 257:217-219, 1992; Groux et al., J Exp. Med., 175:331, 1992; and Oyaizu et al., in Cell Activation and Apoptosis in HIV Infection, Andrieu and Lu, Eds., Plenum Press, New York, 1995, pp. 101-114). Indeed, HIV-induced apoptotic cell death has been demonstrated not only in vitro but also, more importantly, in infected individuals (J.C. Ameisen, AIDS 8:1197-1213 (1994); T.H. Finkel and N.K.
Banda, Curr.
Opin. Immunol. 6:605-615(1995); C.A. Muro-Cacho et al., J. Immunol. 154:5555-(1995)). Furthermore, apoptosis and CD4+ T-lymphocyte depletion is tightly correlated in different animal models of AIDS (T. Brunner et al., Nature 373:441-444 (1995);
M.L.
Gougeon et al., AIDS Res. Hum. Retroviruses 9:553-563 (1993)) and, apoptosis is not observed in those animal models in which viral replication does not result in AIDS. Id.
Further data indicates that uninfected but primed or activated T lymphocytes from HIV-infected individuals undergo apoptosis after encountering the TNF-family ligand Fast.
Using monocytic cell lines that result in death following HIV infection, it has been demonstrated that infection of U937 cells with HIV results in the de novo expression of Fast and that Fast mediates HIV-induced apoptosis (A.D. Badley et al., J. Virol. 70:199-206 (1996)). Further, the TNF-family ligand was detectable in uninfected macrophages and its expression was upregulated following HIV infection resulting in selective killing of uninfected CD4 T-lymphocytes. Id. Thus, by the invention, a method for treating HIV+
individuals is provided which involves administering TR14 polynucleotides, polypeptides, and/or TR14 agonists or antagonists of the present invention to reduce selective killing of CD4+ T-lymphocytes. Modes of administration and dosages are discussed in detail below.
Activated human T cells are induced to undergo programmed cell death (apoptosis) upon triggering through the CD3/T cell receptor complex, a process termed activated-induced cell death (AICD). AICD of CD4+ T cells isolated from HIV-Infected asymptomatic individuals has been reported (Groux et al., supra). Thus, AICD may play a role in the depletion of CD4+
T cells and the progression to AIDS in HIV-infected individuals. Thus, the present invention provides a method of inhibiting a tumor-necrosis factor family member-mediated T cell death in 1o HIV patients, comprising administering a TRI4 polypeptide of the invention (preferably, a soluble TR14 polypeptide) to the patients. In one embodiment, the patient is asymptomatic when treatment with TR14 commences. If desired, prior to treatment, peripheral blood T cells may be extracted from an HIV patient, and tested for susceptibility to cell death mediated by a member of the TNF-family, by procedures known in the art. In one embodiment, a patient's blood or plasma is contacted with TR14 ex vivo. The TR14 may be bound to a suitable chromatography matrix by procedures known in the art. The patient's blood or plasma flows through a chromatography column containing TR14 bound to the matrix, before being returned to the patient. In the event the immobilized TR14 bound to TRAIL, or another TNF family member(s), TRAIL and/or other TNF family member protein would be removed from the patient's blood.
In additional embodiments a TR14 polypeptide, polynucleotide, and/or agonist or antagonist of the invention is administered in combination with inhibitors of T cell apoptosis.
For example, TRAIL-mediated apoptosis and Fas-mediated apoptosis have been implicated in loss of T cells in HIV individuals (See e.g., Katsikis et al., J. Exp. Med.
181:2029-2036 (1995)). Thus, a patient susceptible to both Fas ligand mediated and TRAIL
mediated T cell death may be treated as an agent that blocks TRAILITRAIL receptor interactions and/or an agent that blocks Fas-ligand/Fas interactions. Suitable agents for blocking binding of Fas-ligand to Fas include, but are not limited to, soluble Fas polypeptides;
mulitmeric forms of soluble Fas polypeptides (e.g., dimers of sFas/Fc); anti-Fas antibodies that bind Fas without transducing the biological signal that results in apoptosis; anti-Fas-ligand antibodies that block binding of Fas-ligand to Fas; and muteins of Fas-(igand that bind Fas but do not transduce the biological signal that results in apoptosis. Preferably, the antibodies employed according to this method are monoclonal antibodies. Examples of suitable agents for blocking Fas-ligand/Fas interactions, including blocking anti-Fas monoclonal antibodies, are described in International application publication number WO 95/10540, hereby incorporated by reference.
Suitable agents, which block binding of TRAIL to a TRAIL receptor or FAS
ligand to FAS that may be administered with the nucleic acids, polypeptides, and/or agonists or antagonists of the present invention include, but are not limited to, soluble TRAIL receptor polypeptides (e.g., a soluble form of OPG, DR4 (International application publication number WO 98/32856); TR5 (International application publication number WO 98/30693);
and DRS
(International application publication number WO 98/41629)); multimeric forms of soluble TRAIL receptor polypeptides; and TRAIL receptor antibodies that bind the TRAIL
receptor without transducing the biological signal that results in apoptosis, anti-TRAIL antibodies that block binding of TRAIL to one or more TRAIL receptors, and muteins of TRAIL
that bind TRAIL receptors but do not transduce the biological signal that results in apoptosis. Preferably, the antibodies employed according to this method are monoclonal antibodies.
TR13 polypeptides, nucleic acids, and/or agonists or antagonists of the invention may 1o be used to treat cardiovascular disorders, including peripheral artery disease, such as limb ischemia.
TR14 polypeptides, nucleic acids, and/or agonists or antagonists of the invention may be used to treat cardiovascular disorders, including peripheral artery disease, such as limb ischemia.
Cardiovascular disorders include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects, and conditions characterized by clotting of small blood vessels.
Cardiovascular disorders also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT
syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic functional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mural valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, to hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, thrombotic microangiopathies (e.g., thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS)), and venous insufficiency.
Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart 3o aneurysms, and iliac aneurysms.
Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
Cerebrovascular disorders include carotid artery diseases, cerebral amyloid angiopathy, ~ cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral DEMANDES OU BREVETS VOLUMINEUX
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JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.

NOTE: For additional valumes please contact the Canadian Patent Office.

Claims

What Is Claimed Is:

1. An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 750 in SEQ ID NO:1;

(b) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 750 in SEQ ID NO:1;

(c) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 750 in SEQ ID NO:1;

(d) a nucleotide sequence encoding a polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-349;

(e) a nucleotide sequence encoding the mature TR13 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.
PTA-349;

(f) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 231 in SEQ ID NO:61;

(g) a nucleotide sequence encoding a polypeptide comprising amino acids from about 2 to about 231 in SEQ ID NO:61;

(h) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 138 in SEQ ID NO:61;

(i) a nucleotide sequence encoding a polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-348;

(j) a nucleotide sequence encoding a polypeptide comprising amino acids 1 to 226 of SEQ ID NO:5;

(k) a nucleotide sequence encoding the TR14 extracellular domain;

(l) a nucleotide sequence encoding the TR14 transmembrane domain;

(m) a nucleotide sequence encoding the TR14 intracellular domain;

(n) a nucleotide sequence encoding the TR14 receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted;

(o) a nucleotide sequence encoding a polypeptide comprising amino acids from about 1 to about 1001 in SEQ ID NO:40;

(p) a nucleotide sequence encoding a polypeptide comprising amino acids from about 2 to about 1001 in SEQ ID NO:40;

(q) a nucleotide sequence encoding a polypeptide comprising amino acids from about 42 to about 1001 in SEQ ID NO:40;

(r) a nucleotide sequence encoding a polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-507;

(s) a nucleotide sequence encoding the mature TR13 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.
PTA-507;

(t) a nucleotide sequence encoding the TR13 extracellular domain;

(u) a nucleotide sequence encoding the TR13 transmembrane domain;

(v) a nucleotide sequence encoding the TR13 intracellular domain;

(w) a nucleotide sequence encoding the TR13 receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted;
and (x) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (P), (q), (r), (s), (t), (u), (v), or (w).

2. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence in SEQ ID NO:1.

3. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence in SEQ ID NO:60.

4. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence in SEQ ID NO:39.

5. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence in SEQ ID NO 1: encoding the TR13 receptor having the amino acid sequence in SEQ ID NO:2.

6. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence in SEQ ID NO:60 encoding the TR14 receptor having the amino acid sequence in SEQ ID NO:61.

7. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence in SEQ ID NO:39 encoding the TR14 receptor having the amino acid sequence in SEQ ID NO:40.

8. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence in SEQ ID NO:1 encoding the mature TR13 receptor having the amino acid sequence in SEQ ID NO:2.

9. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence in SEQ ID NO:60 encoding the extracellular domain of the TR14 receptor having the amino acid sequence in SEQ ID NO:61.

10. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence in SEQ ID NO:39 encoding the mature TR13 receptor having the amino acid sequence in SEQ ID NO:40.

11. The nucleic acid molecule of claim 1, wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in ATCC Deposit No.
PTA-349.

12. The nucleic acid molecule of claim 1, wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in ATCC Deposit No.
PTA-348.

13. The nucleic acid molecule of claim 1, wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in ATCC Deposit No.
PTA-507.

14. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence encoding the TR13 receptor having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-349.

15. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence encoding the TR14 receptor having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-348.

16. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence encoding the TR13 receptor having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-507.

17. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence encoding the mature TR13 receptor having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-349.

18. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence encoding the extracellular domain of the TR14 receptor having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-348.

19. The nucleic acid molecule of claim 1, wherein said polynucleotide has the nucleotide sequence encoding the mature TR13 receptor having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-507.

20. An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x) of claim 1, wherein said polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T
residues.

21. An isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a TR13 receptor having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (r), (s);
(t), (u), (v), (w), or (x) of claim 1.

22. An isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a TR14 receptor having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), or (x) of claim 1.

23. The isolated nucleic acid molecule of claim 21, which encodes an epitope-bearing portion of a TR13 receptor selected from the group consisting of: a polypeptide comprising amino acid residues from about 2 to about 170 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 210 to about 318 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 343 to about 480 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 548 to about 592 in SEQ ID NO:2; or a polypeptide comprising amino acid residues from about 632 to about 742 in SEQ ID NO:2, or a polypeptide comprising amino acid residues from about 1 to about 262 in SEQ ID NO:40, or a polypeptide comprising amino acid residues from about 264 to about 423 in SEQ ID NO:40, or a polypeptide comprising amino acid residues from about 437 to about 789 in SEQ
ID NO:40, or a polypeptide comprising amino acid residues from about 791 to about 1001 in SEQ ID NO:40.

24. The isolated nucleic acid molecule of claim 22, which encodes an epitope-bearing portion of a TR14 receptor selected from the group consisting of: a polypeptide comprising amino acid residues from about 2 to about 24 in SEQ ID NO:5; a polypeptide comprising amino acid residues from about 42 to about 52 in SEQ ID NO:5; a polypeptide comprising amino acid residues from about 80 to about 115 in SEQ ID NO:5; or a polypeptide comprising amino acid residues from about 155 to about 226 in SEQ ID NO:5.

25. The isolated nucleic acid molecule of claim 1, which encodes the TR14 receptor extracellular domain.

26. The isolated nucleic acid molecule of claim 1, which encodes the TR14 receptor transmembrane domain.

27. The isolated nucleic acid molecule of claim 1, which encodes the TR14 receptor intracellular domain.

28. The isolated nucleic acid molecule of claim 1, which encodes the TR13 receptor extracellular domain.

29. The isolated nucleic acid molecule of claim 1, which encodes the TR13 receptor transmembrane domain.

30. The isolated nucleic acid molecule of claim 1, which encodes the TR13 receptor intracellular domain.

31. An isolated nucleic acid molecule comprising a polynucleotide having a sequence at least 95% identical to a sequence selected from the group consisting of:

(a) the nucleotide sequence of clone HETAQ12R (SEQ ID NO:8);

(b) the nucleotide sequence of clone HETAK82R (SEQ ID NO:9);

(c) the nucleotide sequence of clone HETBH18R (SEQ ID NO:10);

(d) the nucleotide sequence of clone HEPAB26R (SEQ ID NO:11);

(e) the nucleotide sequence of clone HETAN38R (SEQ ID NO:12);

(f) the nucleotide sequence of clone HPWDD30R (SEQ ID NO:13);

(g) the nucleotide sequence of clone HETAT05R (SEQ ID NO:14);

(h) the nucleotide sequence of clone HETDQ39R (SEQ ID NO:15);

(i) the nucleotide sequence of clone HETEM84R (SEQ ID NO:16);

(j) the nucleotide sequence of clone HSIDV42R (SEQ ID NO:17); and (k) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), c), (d), (e), (f), (g), or (h), (i), or (j) above.

32. An isolated nucleic acid molecule comprising a polynucleotide having a sequence at least 95% identical to a sequence selected from the group consisting of:

(a) the nucleotide sequence of clone HSABDSOR (SEQ ID NO:18);

(b) the nucleotide sequence of clone HTXMX53R (SEQ ID NO:19);

(c) the nucleotide sequence of clone HE20R74R (SEQ ID NO:20);

(d) the nucleotide sequence of clone HMSHK47R (SEQ ID NO:21);

(e) the nucleotide sequence of clone HMSHK59R (SEQ ID NO:22); and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e) above.

33. An isolated nucleic acid molecule comprising a polynucleotide having a sequence at least 95% identical to a sequence selected from the group consisting of:

(a) the nucleotide sequence of clone HETAQ12R (SEQ ID NO:48);
(b) the nucleotide sequence of clone HETAK82R (SEQ ID NO:49);
(c) the nucleotide sequence of clone HETBM71R (SEQ ID NO:50);
(d) the nucleotide sequence of clone HETBH18R (SEQ ID NO:51);
(e) the nucleotide sequence of clone HEPAB26R (SEQ ID NO:52);
(f) the nucleotide sequence of clone HETAN38R (SEQ ID NO:53);
(g) the nucleotide sequence of clone HPWDD30R (SEQ ID NO:54);
(h) the nucleotide sequence of clone HETATO5R (SEQ ID NO:55);
(i) the nucleotide sequence of clone HETDQ39R (SEQ ID NO:56);
(j) the nucleotide sequence of clone HPWBL93R (SEQ ID NO:57);
(k) the nucleotide sequence of clone HETEM84R (SEQ ID NO:58);
(l) the nucleotide sequence of clone HSIDV42R (SEQ ID NO:59); and (m) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l) or (m) above.

34. A method for making a recombinant vector comprising inserting an isolated nucleic acid molecule of claim 1 into a vector.

35. A recombinant vector produced by the method of claim 34.

36. A method of making a recombinant host cell comprising introducing the recombinant vector of claim 35 into a host cell.

37. A recombinant host cell produced by the method of claim 36.

38. A recombinant method for producing a TR13 polypeptide, comprising culturing the recombinant host cell of claim 37 under conditions such that said polypeptide is expressed, and recovering said polypeptide.

39. A recombinant method for producing a TR14 polypeptide, comprising culturing the recombinant host cell of claim 37 under conditions such that said polypeptide is expressed, and recovering said polypeptide.

40. An isolated TR13 polypeptide having an amino acid sequence at least 95%
identical to a sequence selected from the group consisting of:

(a) amino acids from about 1 to about 750 in SEQ ID NO:2;
(b) amino acids from about 2 to about 750 in SEQ ID NO:2;
(c) amino acids from about 1 to about 1001 in SEQ ID NO:40;
(d) amino acids from about 2 to about 1001 in SEQ ID NO:40;
(e) the amino acid sequence of the TR13 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.PTA-349, (f) the amino acid sequence of the TR13 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No.PTA-507, (g) the amino acid sequence of the mature TR13 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit No. PTA-349;
(h) the amino acid sequence of the mature TR13 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit No. PTA-507; and (i) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), (c), (d), (e), (f), (g), or (h).

41. An isolated TR14 polypeptide having an amino acid sequence at least 95%
identical to a sequence selected from the group consisting of:
(a) amino acids from about 1 to about 231 in SEQ ID NO:61;
(b) amino acids from about 2 to about 231 in SEQ ID NO:61;
(c) amino acids from about 1 to about 138 in SEQ ID NO:61;
(d) amino acids from about 156 to about 231 in SEQ ID NO:61;
(e) the amino acid sequence of the TR14 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-348;

(f) amino acids 73 to 226 of SEQ ID NO:5;

(g) the amino acid sequence of the TR14 receptor extracellular domain;

(h) the amino acid sequence of the TR14 receptor transmembrane domain;

(i) the amino acid sequence of the TR14 receptor intracellular domain;

(j) the amino acid sequence of the TR14 receptor intracellular and extracellular domains with all or part of the transmembrane domain deleted;
and (k) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j).

42. An isolated polypeptide comprising an epitope-bearing portion of the TR13 receptor protein, wherein said portion is selected from the group consisting of: a polypeptide comprising amino acid residues from about 2 to about 170 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 210 to about 318 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 343 to about 480 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 545 to about 592 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 632 to about 742 in SEQ ID NO:2; a polypeptide comprising amino acid residues from about 1 to about 262 in SEQ ID NO:40; a polypeptide comprising amino acid residues from about 264 to about 423 in SEQ ID NO:40; a polypeptide comprising amino acid residues from about 437 to about 789 in SEQ ID NO:40; a polypeptide comprising amino acid residues from about 791 to about 1001 in SEQ ID NO:40.

43. An isolated polypeptide comprising an epitope-bearing portion of the TR14 receptor protein, wherein said portion is selected from the group consisting of: a polypeptide comprising amino acid residues from about 2 to about 24 in SEQ ID NO:5; a polypeptide comprising amino acid residues from about 42 to 52 in SEQ ID NO:5; a polypeptide comprising amino acid residues from about 80 to about 115 in SEQ ID NO:5; and a polypeptide comprising amino acid residues from about 155 to about 226 in SEQ ID NO:5.

44. An isolated antibody that binds specifically to a TR13 receptor polypeptide of claim 40.

45. An isolated antibody that binds specifically to a TR14 receptor polypeptide of claim 41.

46. A method of treating diseases and disorders associated with the inhibition of apoptosis comprising administering an effective amount of the polypeptide as claimed in claim 40, or an agonist thereof to a patient in need thereof.

47. A method of treating diseases and disorders associated with the inhibition of apoptosis comprising administering an effective amount of the polypeptide as claimed in claim 41, or an agonist thereof to a patient in need thereof.

48. A method of treating diseases and disorders associated with increased apoptosis comprising administering to a patient in need thereof an effective amount of an antagonist of the polypeptide as claimed in claim 40 to a patient in need thereof.

49. A method of treating diseases and disorders associated with increased apoptosis comprising administering to a patient in need thereof an effective amount of an antagonist of the polypeptide as claimed in claim 41 to a patient in need thereof.

50. A method of treating inflammatory diseases and disorders comprising administering to a patient in need thereof an effective amount of an antagonist of the polypeptide as claimed in claim 40.

51. A method of treating inflammatory diseases and disorders comprising administering to a patient in need thereof an effective amount of an antagonist of the polypeptide as claimed in claim 41.

52. A method of treating, preventing, or ameliorating an epithelial disorder comprising administering a TR14 polynucleotide, polypeptide, or agonist to a patient in need of such treatment, prevention, or amelioration.

53. The method of claim 52, wherein said disorder is hypohidrotic ectodermal dysplasia.