HUMAN ANTIBODIES TO HUMAN TNF-LIKE LIGAND 1A (TL1A)

Abstract

A fully human antibody or antigen-binding fragment of a human antibody that specifically binds and inhibits human TNF-like ligand 1A (hTL1A) is provided. The human anti-hTL1A antibodies are useful in treating diseases or disorders associated with TL1A, such as inflammatory diseases or disorders, e.g., inflammatory bowel diseases, including ulcerative colitis and Crohn's disease, rheumatoid arthritis, and the like; autoimmune diseases or disorders, such as multiple sclerosis, diabetes, and the like; and allergic reactions, such as asthma and allergic lung inflammation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of and claims the benefit of currently pending U.S. patent application Ser. No. 14/144,800, entitled "METHOD OF TREATING A TL1A-ASSOCIATED DISEASE OR DISORDER WITH HUMAN ANTIBODIES TO TL1A", filed Dec. 31, 2013, which is a continuation of U.S. patent application Ser. No. 13/291,145, filed Nov. 8, 2011, now U.S. Pat. No. 8,642,741, which claims the benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional Application Nos. 61/411,276 filed Nov. 8, 2010; and 61/478,309 filed Apr. 22, 2011, all of which are herein specifically incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention is related to human antibodies and antigen-binding fragments of human antibodies that specifically bind human TNF-like ligand 1A (hTL1A), and therapeutic methods of using those antibodies.

STATEMENT OF RELATED ART

[0003] TL1A is a type II cell membrane protein of the tumor necrosis factor superfamily (TNFSF) and also designated as TNFSF15. It is expressed on the surface of endothelial cells, and activated cells of the hematopoietic lineage, including monocytes, macrophages, lymphocytes, lamina propria mononuclear cells, dendritic cells and plasma cells (Tan, K. B. et aL, 1997, Gene 204:35-46; Prehn, J. L. et al., 2007, J Immunol 178:4033-4038). It is also expressed in kidney, lung, prostate and thymus (Tan et al., 1997, supra). In endothelial cells, expression of TL1A is upregulated by IL-1.alpha. and TNF.alpha. (Migone, T. S. et al., 2002, Immunity 16:479-492). In human fresh blood monocytes and monocyte-derived dendritic cells, TL1A expression is upregulated by Fc.gamma.R-mediated or Toll-like receptor (TLR) signaling (Prehn et al., 2007, supra; Meylan, F. et al., 2008, Immunity 29:79-89). TL1A can be cleaved from the cell membrane via a mechanism analogous to TNF.alpha. and a soluble ectodomain form of TL1A has been reported (Migone et al., 2002, supra; Kim, S. et al., 2005, J Immunol Methods 298:1-8; Yang, C. R. et al., 2004, Cancer Res 64:1122-1129). Protein sequencing has confirmed that this form of TL1A is liberated following cleavage of the membrane-anchored precursor between residues Ala-71 and Leu-72 (Migone et al., 2002, supra). Two variant cDNAs that potentially encode N-terminally truncated versions of TL1A have been identified: VEGI-174 (or TL1) (Zhai, Y. et al., 1999, FASEB J 13:181-189) and VEGI-192 (Chew, L. J. et al., 2002, FASEB J 16:742-744). The published data suggest the biologically active products of the TL1A gene are the full-length type II transmembrane protein (residues 1-251) and its proteolytically cleaved ectodomain (residues 72-251) (Migone et al., 2002, supra; Jin et al., 2007, Biochem Biophys Res Commun 364:1-6). A variant of hTL1A, designated as "Fhm", containing a single amino acid substitution of Gln-167 with Arg, is disclosed in U.S. Pat. No. 6,521,422.

[0004] TL1A mediates signals via its cognate receptor Death Receptor 3 (DR3; also known as TNFRSF25; the nucleic acid and amino acid sequences of SEQ ID NO:251 and 252, respectively), resulting in promoting cell survival and secretion of pro-inflammatory cytokines, or promoting apoptosis, in a context-dependent manner. TL1A is one of three known ligands (in addition to FasL and LIGHT) that are bound by the endogenous soluble decoy receptor, DcR3 (also known as TR6, NTR3 or TNFRSF21; the nucleic acid and amino acid sequences of SEQ ID NO:253 and 254, respectively) (Migone et al., 2002, supra; Yang C. R. et al., 2004, Cancer Res 64:1122-1129).

[0005] DR3 is a TNF receptor-related death-domain receptor expressed on the majority of activated T lymphocytes and NK cells (Migone et al., 2002, supra; Screaton G. R. et al., 1997, Proc Natl Acad Sci (USA) 94:4615-4619). TL1A engages DR3 on T cells, enhancing their responsiveness to IL-2 (Migone et al., 2002, supra), potentiating T cell proliferation and release of IFN.gamma. and GM-CSF under conditions of suboptimal costimulation (Migone et al., 2002, supra; Meylan et al., 2008, supra). TL1A has also been shown to synergize with suboptimal levels of IL-12/IL-18 to induce IFN.gamma. production by CD4.sup.+ T cells (Papadakis, K. A. et al., 2004, J Immunol. 172:7002-7007; Prehn, J. L. et al., 2004, Clin Immunol. 112:66-77; Papadakis, K. A. et al., 2005, J Immunol. 174:4985-4990; Cassatella, M. A. et al., 2007, J Immunol. 178:7325-7333).

[0006] TL1A has been implicated in various inflammatory diseases and/or auto immune diseases, including inflammatory bowel diseases [e.g., ulcerative colitis (UC) and Crohn's disease (CD)], rheumatoid arthritis, multiple sclerosis (MS), atherosclerosis, and the like (see Bayry, J., 2010, Nature Reviews/Rheumatology 6:67-68; Takedatsu, H. et al., 2008, Gastroenterology 135:552-567; Prehn et al., 2004, supra; Bamias, G. et al., 2008, Clin Immunol 129:249-255; Bull, M. J. et al., 2008, J Exp Med 205:2457-2464; Pappu, B. P. et al., 2008, J Exp Med 205:1049-1062; Bamias, G. et al., 2003, J Immunol 171:4868-4874; Kang, Y. et al., 2005, Cytokine 29:229-235). Although the majority of the published data are consistent with a pivotal role for TL1A in driving differentiation of T.sub.H1 and T.sub.H17 effector function, a recent study has proposed a role for the TL1A/DR3 interaction in development of T.sub.H2 T cell responses in asthma models (Fang, L. et al., 2008, J Exp Med 205:1037-1048). Thus, the use of TL1A inhibitors, such as fully human antibodies against TL1A with high affinities and neutralizing activity, alone or in combination with currently available anti-inflammatory agents, immunosuppresants (e.g., TNF-.alpha. antagonists, cortisone or steroids, and the like), and/or anti-allergy agents, provides effective treatment for these diseases and disorders.

[0007] The nucleic acid and the amino acid sequences of human TL1A are shown in SEQ ID NOS: 243 and 244, respectively, and those of Fhm are shown in SEQ ID NOS:245 and 246, respectively. Antibodies to TL1A are disclosed in, for example, U.S. Pat. No. 7,597,886, U.S. Pat. No. 7,820,798 and US 2009/0280116.

BRIEF SUMMARY OF THE INVENTION

[0008] In a first aspect, the invention provides fully human monoclonal antibodies (mAbs) and antigen-binding fragments thereof that specifically bind and neutralize human TL1A (hTL1A) activity.

[0009] The antibodies can be full-length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab').sub.2 or scFv fragment), and may be modified to affect functionality, e.g., to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933).

[0010] In one embodiment, the invention features an antibody or antigen-binding fragment of an antibody comprising a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NO:2, 18, 34, 50, 66, 82, 98, 114, 118, 134, 138, 154, 158, 174, 178, 194, 198, 214, 218 and 234, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In another embodiment, the antibody or an antigen-binding fragment thereof comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO:2, 18, 34, 50, 66, 134, 174 and 234. In yet another embodiment, the antibody or fragment thereof comprises a HCVR comprising SEQ ID NO:2, 18, 174 or 234.

[0011] In one embodiment, the antibody or fragment thereof further comprises a light chain variable region (LCVR) selected from the group consisting of SEQ ID NO:10, 26, 42, 58, 74, 90, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226 and 236, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In another embodiment, the antibody or antigen-binding portion of an antibody comprises a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO:10, 26, 42, 58, 74, 136, 176 and 236. In yet another embodiment, the antibody or fragment thereof comprises a LCVR comprising SEQ ID NO:10, 26, 176 or 236.

[0012] In further embodiments, the antibody or fragment thereof comprises a HCVR and LCVR (HCVR/LCVR) sequence pair selected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/116, 118/126, 134/136, 138/146, 154/156, 158/166, 174/176, 178/186, 194/196, 198/206, 214/216, 218/226 and 234/236. In one embodiment, the antibody or fragment thereof comprises a HCVR and LCVR selected from the amino acid sequence pairs of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 134/136, 174/176 and 234/236. In another embodiment, the antibody or fragment thereof comprises a HCVR/LCVR pair comprising SEQ ID NO:2/10, 18/26, 174/176 or 234/236.

[0013] In a second aspect, the invention features an antibody or antigen-binding fragment of an antibody comprising a heavy chain complementarity determining region 3 (HCDR3) amino acid sequence selected from the group consisting of SEQ ID NO:8, 24, 40, 56, 72, 88, 104, 124, 144, 164, 184, 204 and 224, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR3 (LCDR3) amino acid sequence selected from the group consisting of SEQ ID NO:16, 32, 48, 64, 80, 96, 112, 132, 152, 172, 192, 212 and 232, or substantially similar sequences thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the antibody or fragment thereof comprises a HCDR3/LCDR3 amino acid sequence pair comprising SEQ ID NO:8/16, 24/32, 40/48, 56/64, 72/80, 88/96, 104/112, 124/132, 144/152, 164/172, 184/192, 204/212 or 224/232. In another embodiment, the antibody or fragment thereof comprises a HCDR3/LCDR3 amino acid sequence pair comprising SEQ ID NO: 8/16, 24/32, 40/48, 56/64, 72/80, 124/132, 164/172 or 224/232. In yet another embodiment, the antibody or fragment thereof comprises a HCDR3/LCDR3 amino acid sequence pair comprising SEQ ID NO:8/16, 24/32, 164/172 or 224/232.

[0014] In a further embodiment, the invention features an antibody or fragment thereof further comprising a heavy chain CDR1 (HCDR1) amino acid sequence selected from the group consisting of SEQ ID NO:4, 20, 36, 52, 68, 84, 100, 120, 140, 160, 180, 200 and 220, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) amino acid sequence selected from the group consisting of SEQ ID NO:6, 22, 38, 54, 70, 86, 102, 122, 142, 162, 182, 202 and 222, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and/or a light chain CDR1 (LCDR1) amino acid sequence selected from the group consisting of SEQ ID NO:12, 28, 44, 60, 76, 92, 108, 128, 148, 168, 188, 208 and 228, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and/or a light chain CDR2 (LCDR2) amino acid sequence selected from the group consisting of SEQ ID NO:14, 30, 46, 62, 78, 94, 110, 130, 150, 170, 190, 210 and 230, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In one embodiment, the antibody or fragment thereof comprises a combination of HCDR1/HCDR2/HCDR3 selected from the group consisting of SEQ ID NO:4/6/8, 20/22/24, 36/38/40, 52/54/56, 68/70/72, 84/86/88, 100/102/104, 120/122/124, 140/142/144, 160/162/164, 180/182/184, 200/202/204 and 220/222/224; and/or a combination of LCDR1/LCDR2/LCDR3 selected from the group consisting of 6SEQ ID NO:12/14/16, 28/30/32, 44/46/48, 60/62/64, 76/78/80, 92/94/96, 108/110/112, 128/130/132, 148/150/152, 168/170/172, 188/190/192, 208/210/212 and 228/230/232. In another embodiment, the heavy and light chain CDR amino acid sequences comprise a CDR sequence combination selected from the group consisting of SEQ ID NO:4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48, 52/54/56/60/62/64, 68/70/72/76/78/80, 84/86/88/92/94/96, 100/102/104/108/110/112, 120/122/124/128/130/132, 140/142/144/148/150/152, 160/162/164/168/170/172, 180/182/184/188/190/192, 200/202/204/208/210/212 and 220/222/224/228/230/232. In another embodiment, the antibody or antigen-binding fragment thereof comprises heavy and light chain CDR sequences of SEQ ID NO:4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48, 52/54/56/60/62/64, 68/70/72/76/78/80, 120/122/124/128/130/132, 160/162/164/168/170/172 or 220/222/224/228/230/232. In yet another embodiment, the heavy and light chain CDR amino acid sequences comprise a CDR sequence combination of SEQ ID NO:4/6/8/12/14/16, 20/22/24/28/30/32, 160/162/164/168/170/172 or 220/222/224/228/230/232.

[0015] In a related embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody which specifically binds hTL1A, wherein the antibody or fragment thereof comprises heavy and light chain CDR domains contained within heavy and light chain sequence pairs selected from the group consisting of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/116, 118/126, 134/136, 138/146, 154/156, 158/166, 174/176, 178/186, 194/196, 198/206, 214/216, 218/226 and 234/236. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are known in the art and can be applied to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Conventional definitions that can be applied to identify the boundaries of CDRs include the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody. In one embodiment, the antibody or fragment thereof comprises CDR sequences contained within a HCVR and LCVR pair selected from the group consisting of the amino acid sequence pairs of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 134/136, 174/176 and 234/236. In another embodiment, the antibody or fragment thereof comprises CDR sequences contained within the HCVR and LCVR sequence pair of SEQ ID NO: 2/10, 18/26, 174/176 or 234/236.

[0016] In another related embodiment, the invention provides an antibody or antigen-binding fragment thereof that competes for specific binding to hTL1A with an antibody or antigen-binding fragment comprising heavy and light chain CDR sequences of SEQ ID NO:4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48, 52/54/56/60/62/64, 68/70/72/76/78/80, 120/122/124/128/130/132, 160/162/164/168/170/172 or 220/222/224/228/230/232. In one embodiment, the antibody or antigen-binding fragment thereof competes for specific binding to hTL1A with an antibody or antigen-binding fragment comprising heavy and light chain CDR sequences of SEQ ID NO:4/6/8/12/14/16, 20/22/24/28/30/32, 160/162/164/168/170/172 or 220/222/224/228/230/232. In another embodiment, the antibody or antigen-binding fragment of the invention competes for specific binding to hTL1A with an antibody or antigen-binding fragment comprising a HCVR/LCVR sequence pair of SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 134/136, 174/176 or 234/236. In yet another embodiment, the antibody or antigen-binding fragment thereof competes for specific binding to hTL1A with an antibody or antigen-binding fragment comprising a HCVR/LCVR sequence pair of SEQ ID NO:2/10, 18/26, 174/176 or 234/236.

[0017] In another related embodiment, the invention provides an antibody or antigen-binding fragment thereof that binds the same epitope on hTL1A that is recognized by an antibody or fragment thereof comprising heavy and light chain CDR sequences of SEQ ID NO:4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48, 52/54/56/60/62/64, 68/70/72/76/78/80, 120/122/124/128/130/132, 160/162/164/168/170/172 or 220/222/224/228/230/232. In one embodiment, the antibody or antigen-binding fragment thereof binds the same epitope on hTL1A that is recognized by an antibody or antigen-binding fragment thereof comprising heavy and light chain CDR sequences of SEQ ID NO:4/6/8/12/14/16, 20/22/24/28/30/32, 160/162/164/168/170/172 or 220/222/224/228/230/232. In another embodiment, the antibody or antigen-binding fragment of the invention recognizes the same epitope on hTL1A that is recognized by an antibody or antigen-binding fragment thereof comprising a HCVR/LCVR sequence pair of SEQ ID NO: SEQ ID NO:2/10, 18/26, 34/42, 50/58, 66/74, 134/136, 174/176 or 234/236. In yet another embodiment, the antibody or antigen-binding fragment thereof recognizes the same epitope on hTL1A that is recognized by an antibody or antigen-binding fragment thereof comprising a HCVR/LCVR sequence pair of SEQ ID NO:2/10, 18/26, 174/176 or 234/236.

[0018] In a third aspect, the invention provides nucleic acid molecules encoding anti-TL1A antibodies or fragments thereof described above. Recombinant expression vectors carrying the nucleic acids of the invention, and isolated host cells, e.g., bacterial cells, such as E. coli, or mammalian cells, such as CHO cells, into which such vectors have been introduced, are also encompassed by the invention, as are methods of producing the antibodies by culturing the host cells under conditions permitting production of the antibodies, and recovering the antibodies produced.

[0019] In one embodiment, the invention provides an antibody or fragment thereof comprising a HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, 17, 33, 49, 65, 81, 97, 113, 117, 133, 137, 153, 157, 173, 177, 193, 197, 213, 217 and 233, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof. In another embodiment, the antibody or fragment thereof comprises a HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, 17, 33, 49, 65, 133, 173 and 233. In yet another embodiment, the antibody or fragment thereof comprises a HCVR encoded by the nucleic acid sequence of SEQ ID NO:1, 17, 173 or 233.

[0020] In one embodiment, the antibody or fragment thereof further comprises a LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:9, 25, 41, 57, 73, 89, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225 and 235, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof. In another embodiment, the antibody or fragment thereof comprises a LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:9, 25, 41, 57, 73, 135, 175 and 235. In yet another embodiment, the antibody or fragment thereof comprises a LCVR encoded by the nucleic acid sequence of SEQ ID NO:9, 25, 175 or 235.

[0021] In further embodiments, the antibody or fragment thereof comprises a HCVR and LCVR (HCVR/LCVR) sequence pair encoded by a nucleic acid sequence pair selected from the group consisting of SEQ ID NO:1/9, 17/25, 33/41, 49/57, 65/73, 81/89, 97/105, 113/115, 117/125, 133/135, 137/145, 153/155, 157/165, 173/175, 177/185, 193/195, 197/205, 213/215, 217/225 and 233/235. In one embodiment, the antibody or fragment thereof comprises a HCVR/LCVR sequence pair encoded by a nucleic acid sequence pair selected from the group consisting of SEQ ID NO:1/9, 17/25, 33/41, 49/57, 65/73, 133/135, 173/175 and 233/235. In yet another embodiment, the antibody or fragment thereof comprises a HCVR/LCVR pair encoded by a nucleic acid sequence pair of SEQ ID NO:1/9, 17/25, 173/175 or 233/235.

[0022] In one embodiment, the invention features an antibody or antigen-binding fragment of an antibody comprising a HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:7, 23, 39, 55, 71, 87, 103, 123, 143, 163, 183, 203 and 223, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof; and a LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:15, 31, 47, 63, 79, 95, 111, 131, 151, 171, 191, 211 and 231, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof. In another embodiment, the antibody or fragment thereof comprises a HCDR3 and LCDR3 sequence pair encoded by the nucleic acid sequence pair of SEQ ID NO:7/15, 23/31, 39/47, 55/63, 71/79, 87/95, 103/111, 123/131, 143/151, 163/171, 183/191, 203/211 or 223/231. In another embodiment, the antibody or fragment thereof comprises a HCDR3 and LCDR3 sequence pair encoded by the nucleic acid sequence pair of SEQ ID NO:7/15, 23/31, 39/47, 55/63, 71/79, 123/131, 163/171 or 223/231. In yet another embodiment, the HCDR3/LCDR3 sequence pair is encoded by the nucleic acid sequence pair of SEQ ID NO:7/15, 23/31, 163/171 or 223/231.

[0023] In a further embodiment, the antibody or fragment thereof further comprises, a HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:3, 19, 35, 51, 67, 83, 99, 119, 139, 159, 179, 199 and 219, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof; a HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:5, 21, 37, 53, 69, 85, 101, 121, 141, 161, 181, 201 and 221, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof; a LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:11, 27, 43, 59, 75, 91, 107, 127, 147, 167, 187, 207 and 227, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof; and a LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:13, 29, 45, 61, 77, 93, 109, 129, 149, 169, 189, 209 and 229, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereof. In one embodiment, the antibody or fragment thereof comprises a combination of HCDR1/HCDR2/HCDR3 encoded by SEQ ID NO:3/5/7, 19/21/23, 35/37/39, 51/53/55, 67/69/71, 83/85/87, 99/101/103, 119/121/123, 139/141/143, 159/161/163, 179/181/183, 199/201/203 or 219/221/223; and a combination of LCDR1/LCDR2/LCDR3 encoded by SEQ ID NO:11/13/15, 27/29/31, 43/45/47, 59/61/63, 75/77/79, 91/93/95, 107/109/111, 127/129/131, 147/149/151, 167/169/171, 187/189/191, 207/209/211 or 227/229/231. In one embodiment, the antibody or fragment thereof comprises heavy and light chain CDR sequences encoded by a nucleic acid sequence combination selected from the group consisting of SEQ ID NO:3/5/7/11/13/15, 19/21/23/27/29/31, 35/37/39/43/45/47, 51/53/55/59/61/63, 67/69/71/75/77/79, 83/85/87/91/93/95, 99/101/103/107/109/111, 119/121/123/127/129/131, 139/141/143/147/149/151, 159/161/163/167/169/171, 179/181/183/187/189/191, 199/201/203/207/209/211 and 219/221/223/227/229/231. In another embodiment, the antibody or antigen-binding portion thereof comprises heavy and light chain CDR sequences encoded by a nucleic acid sequence combination of SEQ ID NO:3/5/7/11/13/15, 19/21/23/27/29/31, 35/37/39/43/45/47, 51/53/55/59/61/63, 67/69/71/75/77/79, 119/121/123/127/129/131, 159/161/163/167/169/171 or 219/221/223/227/229/231. In yet another embodiment, the antibody or antigen-binding portion thereof comprises heavy and light chain CDR sequences encoded by a nucleic acid sequence combination of SEQ ID NO: 3/5/7/11/13/15, 19/21/23/27/29/31, 159/161/163/167/169/171 or 219/221/223/227/229/231.

[0024] In a fourth aspect, the invention features an isolated antibody or antigen-binding fragment of an antibody that specifically binds hTL1A, comprising a HCDR3 and a LCDR3, wherein the HCDR3 comprises an amino acid sequence of the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-X- .sup.10X.sup.11X.sup.12X.sup.13X.sup.14X.sup.15X.sup.16(SEQ ID NO:239), wherein X.sup.1 is Thr or Ala, X.sup.2 is Lys, Arg or absent, X.sup.3 is Glu, Gly or absent, X.sup.4 is Asp, Pro or absent, X.sup.5 is Leu or absent, X.sup.6 is Arg, Tyr, Glu or absent, X.sup.7 is Gly, Asp, Ala or absent, X.sup.8 is Asp, Ser or Tyr, X.sup.9 is Tyr or Trp, X.sup.10 is Tyr or Asp, X.sup.11 is Tyr, Lys or Ile, X.sup.12 is Gly, Tyr, Asn, or Ser, X.sup.13 is Val, Gly or Ser, X.sup.14 is Phe or Met, X.sup.15 is Asp, and X.sup.16 is Tyr or Val; and the LCDR3 comprises an amino acid sequence of the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9 (SEQ ID NO:242), wherein X.sup.1 is Gln, X.sup.2 is Gln, X.sup.3 is Tyr, Leu or Phe, X.sup.4 is His, Tyr or Asn, X.sup.5 is Arg or Ser, X.sup.6 is Ser, Thr or Tyr, X.sup.7 is Trp or Pro, X.sup.8 is Phe, Leu or absent, and X.sup.9 is Thr.

[0025] In a further embodiment, the antibody or fragment thereof further comprises a HCDR1 sequence comprising an amino acid sequence of the formula X.sup.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8 (SEQ ID NO:237), wherein X.sup.1 is Gly, X.sup.2 is Phe, X.sup.3 is Thr, X.sup.4 is Phe, X.sup.5 is Ser, X.sup.6 is Thr, Ser or Asn, X.sup.7 is Tyr, and X.sup.8 is Gly, Trp, Val or Ala; a HCDR2 sequence comprising an amino acid sequence of the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8 (SEQ ID NO:238), wherein X.sup.1 is Ile or Val, X.sup.2 is Ser or Lys, X.sup.3 is Gly or Glu, X.sup.4 is Thr, Asp, Ser or Arg, X.sup.5 is Gly, X.sup.6 is Arg, Ser or Gly, X.sup.7 is Thr, Glu or Ser, and X.sup.8 is Thr or Lys; a LCDR1 sequence comprising an amino acid sequence of the formula X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-X- .sup.10-X.sup.11-X.sup.12 (SEQ ID NO:240), wherein X.sup.1 is Gln, X.sup.2 is Thr, Ser, Ala or Gly, X.sup.3 is Ile, X.sup.4 is Ser or Leu, X.sup.5 is Tyr or absent, X.sup.6 is Ser or absent, X.sup.7 is Ser or absent, X.sup.8 is Asn or absent, X.sup.9 is Asn or absent, X.sup.10 is Lys or absent, X.sup.11 is Ser, Asn or Thr, and X.sup.12 is Trp or Tyr; and a LCDR2 sequence comprising an amino acid sequence of the formula X.sup.1-X.sup.2-X.sup.3 (SEQ ID NO:241) wherein X.sup.1 is Ala, Trp or Ser, X.sup.2 is Ala or Thr, and X.sup.3 is Ser.

[0026] In a fifth aspect, the invention features a human anti-TL1A antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) encoded by nucleotide sequence segments derived from V.sub.H, D.sub.H and J.sub.H germline sequences, and a light chain variable region (LCVR) encoded by nucleotide sequence segments derived from V.sub.K and J.sub.K germline sequences. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the HCVR and the LCVR encoded by nucleotide sequence segments derived from a germline gene combination selected from the group consisting of: (i) V.sub.H3-23, D.sub.H2-21, J.sub.H4, V.sub.K1-5 and J.sub.K1; (ii) V.sub.H3-7, D.sub.H1-7, J.sub.H6, V.sub.K4-1 and J.sub.K3; (iii) V.sub.H3-23, D.sub.H2-2, J.sub.H6, V.sub.K1-9 and J.sub.K2; (iv) V.sub.H3-23, D.sub.H6-6, J.sub.H4, V.sub.K1-9 and J.sub.K4; (v) V.sub.H1-2, D.sub.H2-15, J.sub.H3, V.sub.K1-12 and J.sub.K4; (vi) V.sub.H4-34, D.sub.H3-9, J.sub.H4, V.sub.K3-20 and J.sub.K4; (vii) V.sub.H4-34, D.sub.H1-1, J.sub.H4, V.sub.K3-20 and J.sub.K4; and (viii) V.sub.H4-34, D.sub.H3-3, J.sub.H4, V.sub.K2-24 and J.sub.K4.

[0027] In a sixth aspect, the invention features an antibody or antigen-binding fragment thereof that specifically binds to hTL1A or Fhm with an equilibrium dissociation constant (K.sub.D) of about 1 nM or less, as measured by surface plasmon resonance assay (for example, BIACORETM). In certain embodiments, the antibody of the invention exhibits a K.sub.D of about 800 pM or less; about 700 pM or less; about 600 pM or less; about 500 pM or less; about 400 pM or less; about 300 pM or less; about 200 pM or less; about 150 pM or less; about 100 pM or less; about 90 pM or less; about 80 pM or less; about 50 pM or less; or 30 pM or less.

[0028] In a seventh aspect, the present invention provides an anti-hTL1A antibody or antigen-binding fragment thereof that binds hTL1A protein of SEQ ID NO:244, but does not cross-react with a variant thereof, such as Fhm of SEQ ID NO:246, as determined by, for example, ELISA, surface plasmon resonance assay, or Luminex.RTM. xMAP.RTM. Technology, as described herein. Fhm contains a single amino acid substitution at position 167, corresponding to Gln in hTL1A, with Arg (see U.S. Pat. No. 6,521,422). In related embodiments, the invention also provides an anti-hTL1 A antibody or antigen-binding fragment thereof that binds a hTL1A protein and cross-reacts with an Fhm. In another related embodiment, the invention provides an anti-hTL1A antibody or antigen binding fragment thereof that does not cross-react with mouse TL1A (mTL1A: SEQ ID NO:250, encoded by the nucleotide sequence of SEQ ID NO:249) but does cross-react with TL1A of cynomolgus monkey (Macaca fascicularis, or MfTL1A: SEQ ID NO:248, encoded by the nucleotide sequence of SEQ ID NO:247) or rhesus monkey (Macaca mulatta: the same amino acid sequence as MfTL1A). In further related embodiments, the invention provides an anti-hTL1A antibody or antigen-binding fragment thereof that cross-reacts with both mTL1A and MfTL1A.

[0029] The invention encompasses anti-hTL1A antibodies having a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or e.g., removal of a fucose moiety to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, removal of N-glycosylation site may reduce undesirable immune reactions against the therapeutic antibodies, or increase affinities of the antibodies. In yet other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

[0030] In an eighth aspect, the invention features a pharmaceutical composition comprising a recombinant human antibody or fragment thereof which specifically binds hTL1A and a pharmaceutically acceptable carrier. In one embodiment, the invention features a composition which is a combination of an antibody or antigen-binding fragment thereof of the invention, and a second therapeutic agent. The second therapeutic agent may be one or more of any agent such as immunosuppressants, anti-inflammatory agents, analgesic agents, anti-allergy agents, and the like, many of which may have overlapping therapeutic effects of one another. Suitable immunosuppressants to be used in combination with the anti-hTL1A antibodies of the invention include, but are not limited to, glucocorticoids, cyclosporin, methotrexate, interferon .beta. (IFN-.beta.), tacrolimus, sirolimus, azathioprine, mercaptopurine, opioids, mycophenolate, TNF-binding proteins, such as infliximab, eternacept, adalimumab, and the like, cytotoxic antibiotics, such as dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, and the like, antibodies targeting immune cells, such as anti-CD20 antibodies, anti-CD3 antibodies, and the like. Suitable anti-inflammatory agents and/or analgesics for combination therapies with anti-hTL1A antibodies include, corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, naproxen, Cox-2 inhibitors, and the like, TNF-.alpha. antagonists, IL-1 antagonists, IL-6 antagonists, acetaminophen, morphinomimetics, and the like. Suitable anti-allergy agents include antihistamines, glucocorticoids, epinephrine (adrenaline), theophylline, cromolyn sodium and anti-leukotrienes, as well as anti-cholinergics, decongestants, mast cell stabilizers, and the like.

[0031] In a ninth aspect, the invention features methods for inhibiting hTL1A activity using the anti-hTL1A antibody or antigen-binding portion of the antibody of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody of the invention and, optionally, one or more additional therapeutic agents described above. The disease or disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented, or its occurrence rate reduced compared to that without anti-hTL1A antibody treatment, by removal, inhibition or reduction of TL1A activity. Examples of diseases or disorders treatable by the methods of the invention include, but are not limited to, inflammatory diseases and/or autoimmune diseases, such as inflammatory bowel diseases (IBD) including UC and CD, RA, MS, type 1 and type 2 diabetes, psoriasis, psoriatic arthritis, ankylosing spondylitis, atopic dermatitis, and the like; allergic reactions or conditions, including asthma, allergic lung inflammation, and the like; cancers atherosclerosis, infections, neurodegenerative diseases, graft rejection, graft vs. host diseases (GVHD), cardiovascular disorders/diseases, and the like.

[0032] Other embodiments will become apparent from a review of the ensuing detailed description.

DETAILED DESCRIPTION

[0033] Before the present invention is described in detail, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

Definitions

[0035] The term "human TNF-like ligand 1A" or "hTL1A", as used herein, refers to hTL1A having the nucleic acid sequence shown in SEQ ID NO:243 and the amino acid sequence of SEQ ID NO:244, or a biologically active fragment thereof, as well as hTL1A variants, including Fhm having the nucleic acid sequence shown in SEQ ID NO:245 and the amino acid sequence of SEQ ID NO:246, or a biologically active fragment thereof, unless specifically indicated otherwise.

[0036] The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (HCVR) and a heavy chain constant region (C.sub.H; comprised of domains C.sub.H1, C.sub.H2 and C.sub.H3). Each light chain is comprised of a light chain variable region (LCVR) and a light chain constant region (C.sub.1). The HCVR and LCVR can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each HCVR and LCVR is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0037] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed with for binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens, based on published crystal structures, and concluded that only about one fifth to one third of CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs had no amino acids in contact with an antigen (see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

[0038] CDR residues not contacting antigen can be identified based on previous studies (for example, residues H60-H65 in CDRH2 are often not required), from regions of Kabat CDRs lying outside Chothia CDRs, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, it is usually substituted with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[0039] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse), have been grafted onto human FR sequences.

[0040] The fully-human anti-TL1A antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residues(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline back-mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the V.sub.H and/or V.sub.L domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residues of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

[0041] The present invention also includes anti-TL1A antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-TL1A antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 2 or 1, conservative amino acid substitution(s) relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.

[0042] Unless specifically indicated otherwise, the term "antibody" (Ab), as used herein, shall be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "full antibody molecules") as well as antigen-binding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-display antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0043] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0044] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a V.sub.H domain associated with a V.sub.L domain, the V.sub.H and V.sub.L domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V.sub.H-V.sub.H, V.sub.H-V.sub.L or V.sub.L-V.sub.L dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V.sub.H or V.sub.L domain.

[0045] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) V.sub.H-C.sub.H1; (ii) V.sub.H-C.sub.H2; (iii) V.sub.H-C.sub.H3; (iv) V.sub.H- C.sub.H1-C.sub.H2; (V) V.sub.H-C.sub.H1-C.sub.H2-C.sub.H3; (Vi) V.sub.H-C.sub.H2-C.sub.H3; V.sub.H-C.sub.L; V.sub.L-C.sub.H1; (ix) V.sub.L-C.sub.H2; (X) V.sub.L-C.sub.H3; (xi) V.sub.L-C.sub.H1-C.sub.H2; (xii) V.sub.L-C.sub.H1-C.sub.H2-C.sub.H3; (xiii) V.sub.L-C.sub.H2-C.sub.H3; and (xiv) V.sub.L-C.sub.L. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric V.sub.H or V.sub.L domain (e.g., by disulfide bond(s)).

[0046] As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

[0047] In certain embodiments, antibody or antibody fragments of the invention may be conjugated to a therapeutic moiety ("immunoconjugate"), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.

[0048] The term "specifically binds," or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant (K.sub.D) of about 3000 nM or less (i.e., a smaller K.sub.D denotes a tighter binding), about 2000 nM or less, about 1000 nM or less; about 500 nM or less; about 300 nM or less; about 200 nM or less; about 100 nM or less; about 50 nM or less; about 1 nM or less; or about 0.5 nM or less. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. An isolated antibody that specifically binds hTL1A may, however, exhibit cross-reactivity to other antigens, such as TL1A molecules from other species, for example, cynomolgus monkey TL1A (SEQ ID NO:248), and/or mouse TL1A (SEQ ID NO:250), and/or a TL1A variant, such as Fhm (SEQ ID NO:246). Moreover, multi-specific antibodies (e.g., bispecifics) that bind to hTL1A and one or more additional antigens are nonetheless considered antibodies that "specifically bind` hTL1A, as used herein.

[0049] The term "high affinity" antibody refers to those antibodies having a binding affinity to hTL1A, expressed as K.sub.D, of about 1.times.10.sup.-9 M or less, about 0.5.times.10.sup.-9 M or less, about 0.25.times.10.sup.-9 M or less, about 1.times.10.sup.-10 M or less, or about 0.5.times.10.sup.-10 M or less, as measured by surface plasmon resonance, e.g., BIACORE.TM. or solution-affinity ELISA.

[0050] The term "K.sub.D", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0051] By the term "slow off rate", "Koff" or "k.sub.d" is meant an antibody that dissociates from hTL1A with a rate constant of 1.times.10.sup.-3 s.sup.-1 or less, preferably 1.times.10.sup.-4 s.sup.-1 or less, as determined by surface plasmon resonance, e.g., BIACORE.TM..

[0052] By the term "intrinsic affinity constant" or "k.sub.a" is meant an antibody that associates with hTL1A at a rate constant of about 1.times.10.sup.3 M.sup.-1 s.sup.-1 or higher, as determined by surface plasmon resonance, e.g., BIACORE.TM..

[0053] An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other mAbs having different antigenic specificities (e.g., an isolated antibody that specifically binds hTL1A is substantially free of Abs that specifically bind antigens other than hTL1A). An isolated antibody that specifically binds hTL1A may, however, have cross-reactivity to other antigens, such as TL1A molecules from other species, such as cynomolgus monkey and mouse, and/or hTL1A variants, such as Fhm.

[0054] A "neutralizing antibody", as used herein (or an "antibody that neutralizes TL1A activity"), is intended to refer to an antibody whose binding to TL1A results in inhibition of at least one biological activity of TL1A. This inhibition of the biological activity of TL1A can be assessed by measuring one or more indicators of TL1A biological activity by one or more of several standard in vitro or in vivo assays known in the art (also see examples below).

[0055] The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE.TM. system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

[0056] The term "epitope" is a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.

[0057] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below.

[0058] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0059] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 2000, supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403 410 and, 1997, Nucleic Acids Res. 25:3389 402, each of which is herein incorporated by reference.

[0060] By the phrase "therapeutically effective amount" is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, the age and the size of a subject treated, the route of administration, and the like, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

Preparation of Human Antibodies

[0061] Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to TL1A.

[0062] Using VELOCIMMUNE.TM. technology or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies to TL1A are initially isolated having a human variable region and a mouse constant region. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, and the like.

[0063] In general, the antibodies of the instant invention possess very high affinities, typically possessing K.sub.D of from about 10.sup.-12 M through about 10.sup.-9 M, when measured by binding to antigen either immobilized on solid phase or in solution phase. The mouse constant regions are replaced with desired human constant regions, for example, wild-type IgG1 (SEQ ID NO:255) or IgG4 (SEQ ID NO:256), or modified IgG1 or IgG4 (for example, IgG4 with Ser-108 substituted with Pro as shown in SEQ ID NO:257), to generate the fully human antibodies of the invention. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics of the antibodies reside in the variable region.

Epitope Mapping and Related Technologies

[0064] To screen for antibodies that bind to a particular epitope, a routine cross-blocking assay such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.) can be performed. Other methods include alanine scanning mutants, peptide blots (Reineke, 2004, Methods Mol Biol 248:443-63) (herein specifically incorporated by reference in its entirety), or peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9: 487-496) (herein specifically incorporated by reference in its entirety).

[0065] The term "epitope" refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

[0066] Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of mAbs directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (US 2004/0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical mAbs, such that characterization can be focused on genetically distinct mAbs. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the anti-TL1A mAbs of the invention into groups of mAbs binding different epitopes.

[0067] The present invention includes hTL1A antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein. Likewise, the present invention also includes anti-hTL1A antibodies that compete for binding to hTL1A or a hTL1A fragment with any of the specific exemplary antibodies described herein.

[0068] One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti-hTL1A antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference anti-hTL1A antibody of the invention, the reference antibody is allowed to bind to a hTL1A protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the hTL1A molecule is assessed. If the test antibody is able to bind to hTL1A following saturation binding with the reference anti-hTL1A antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-hTL1A antibody. On the other hand, if the test antibody is not able to bind to the hTL1A molecule following saturation binding with the reference anti-hTL1A antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-hTL1A antibody of the invention.

[0069] To determine if an antibody competes for binding with a reference anti-hTL1A antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to a hTL1A molecule under saturating conditions followed by assessment of binding of the test antibody to the hTL1A molecule. In a second orientation, the test antibody is allowed to bind to a hTL1A molecule under saturating conditions followed by assessment of binding of the reference antibody to the TL1A molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the TL1A molecule, then it is concluded that the test antibody and the reference antibody compete for binding to hTL1A. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

[0070] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0071] Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.

Immunoconjugates

[0072] The invention encompasses a human anti-TL1A monoclonal antibody conjugated to a therapeutic moiety ("immunoconjugate"), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxin agents include any agent that is detrimental to cells. Examples of suitable cytotoxin agents and chemotherapeutic agents for forming immunoconjugates are known in the art, (see for example, WO 05/103081, herein specifically incorporated by reference).

Bispecifics

[0073] The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific mAbs may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69. The human anti-hTL1A mAbs can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or a multispecific antibody with a second binding specificity.

[0074] An exemplary bi-specific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) C.sub.H3 domain and a second Ig C.sub.H3 domain, wherein the first and second Ig C.sub.H3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig C.sub.H3 domain binds Protein A and the second Ig C.sub.H3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C.sub.H3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second C.sub.H3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and V821 (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the bi-specific antibody format described above are contemplated within the scope of the present invention.

Bioequivalents

[0075] The anti-hTL1A antibodies and antibody fragments of the present invention encompass proteins having amino acid sequences that vary from those of the described mAbs, but that retain the ability to bind human TL1A. Such variant mAbs and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described mAbs. Likewise, the hTL1A mAb-encoding DNA sequences of the present invention encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an anti-hTL1A antibody or antibody fragment that is essentially bioequivalent to an anti-hTL1A antibody or antibody fragment of the invention. Examples of such variant amino acid and DNA sequences are discussed above.

[0076] Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single does or multiple dose. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied. In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0077] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

[0078] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[0079] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0080] Bioequivalent variants of anti-hTL1A antibodies of the invention may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.

Therapeutic Administration and Formulations

[0081] The invention provides therapeutic compositions comprising the anti-hTL1A antibodies or antigen-binding fragments thereof of the present invention and the therapeutic methods using the same. The administration of therapeutic compositions in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN.TM.), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA, 1998, J Pharm Sci Technol 52:238-311.

[0082] The dose may vary depending upon the age and the size of a subject to be administered, target disease, the purpose of the treatment, conditions, route of administration, and the like. When the antibody of the present invention is used for treating various conditions and diseases directly or indirectly associated with TL1A, including inflammatory diseases/disorders, autoimmune diseases/disorders, allergic reactions, and the like, in an adult patient, it is advantageous to intravenously or subcutaneously administer the antibody of the present invention at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, to about 100 mg, or to about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0083] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition 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.

[0084] The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., 1989, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0085] In certain situations, the pharmaceutical 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). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, 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).

[0086] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule. A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0087] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN.TM. (Owen Mumford, Inc., Woodstock, UK), DISETRONIC.TM. pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25.TM. pen, HUMALOG.TM. pen, HUMALIN 70/30.TM. pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN.TM. I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR.TM. (Novo Nordisk, Copenhagen, Denmark), BD.TM. pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN.TM., OPTIPEN PRO.TM., OPTIPEN STARLET.TM., and OPTICLIK.TM. (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR.TM. pen (sanofi-aventis), the FLEXPEN.TM. (Novo Nordisk), and the KWIKPEN.TM. (Eli Lilly).

[0088] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 0.1 to about 800 mg per dosage form in a unit dose; especially in the form of injection, the aforesaid antibody is contained in about 1 to about 500 mg, in about 5 to 300 mg, in about 8 to 200 mg, and in about 10 to about 100 mg for the other dosage forms.

Combination Therapies

[0089] The invention further provides therapeutic methods for treating diseases or disorders, which is directly or indirectly associated with hTL1A, by administering the hTL1A mAb or fragment thereof of the invention in combination with one or more additional therapeutic agents. The additional therapeutic agent may be one or more of any agent that is advantageously combined with the antibody or fragment thereof of the invention, including immunosuppressants, anti-inflammatory agents, analgesic agents, anti-allergy agents, and the like. Suitable immunosuppressants include, but are not limited to, glucocorticoids, cyclosporin, methotrexate, interferon .beta. (IFN-.beta.), tacrolimus, sirolimus, azathioprine, mercaptopurine, opioids, mycophenolate, TNF-binding proteins, such as infliximab, eternacept, adalimumab, and the like, cytotoxic antibiotics, such as dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, and the like, antibodies targeting immune cells, such as anti-CD20 antibodies, anti-CD3 antibodies, and the like. Suitable anti-inflammatory agents and/or analgesics for combination therapies with the anti-hTL1A antibodies include, corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, naproxen, Cox-2 inhibitors, and the like, TNF-.alpha. antagonists (e.g., Infliximab or REMICADE.RTM. by Centocor Inc.; golimumab by Centocor Inc.; etanercept or ENBREL.RTM. by Amgen/Wyeth; adalimumab or HUMIRA.RTM. by Abbott Laboratories, and the like), IL-1 antagonists (e.g., IL-1-binding fusion proteins, for example, ARCALYST.RTM. by Regeneron Pharmaceuticals, Inc., see U.S. Pat. No. 6,927,044; KINERET.RTM. by Amgen, and the like), IL-6 antagonists (e.g., anti-IL-6 receptor antibodies as disclosed in U.S. Pat. No. 7,582,298, and ACTEMRA.RTM. by Roche), acetaminophen, morphinomimetics, and the like. Suitable anti-allergy agents, which can block the action of allergic mediators, or to prevent activation of cells and degranulation processes, include antihistamines, glucocorticoids, epinephrine (adrenaline), theophylline, cromolyn sodium and anti-leukotrienes, such as montelukast (SINGULAIR.RTM. by Merck) or zafirlukast (ACCOLATE.RTM. by AstraZeneca), as well as anti-cholinergics, decongestants, mast cell stabilizers, and other compounds that can impair eosinophil chemotaxis.

[0090] The hTL1A mAb or fragment thereof of the invention and the additional therapeutic agent(s) can be co-administered together or separately. Where separate dosage formulations are used, the antibody or fragment thereof of the invention and the additional agents can be administered concurrently, or separately at staggered times, i.e., sequentially, in appropriate orders.

EXAMPLES

[0091] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used but some experimental errors and deviations should be accounted for. Unless indicated otherwise, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1

Generation of Human Antibodies to Human TL1A

[0092] VELOCIMMUNE.TM. mice were immunized with human TL1A, and the antibody immune response monitored by antigen-specific immunoassay using serum obtained from these mice. Anti-hTL1A antibody-expressing B cells were harvested from the spleens of immunized mice shown to have elevated anti-hTL1A antibody titers and were fused with mouse myeloma cells to form hybridomas. The hybridomas were screened and selected to identify cell lines expressing hTL1A-specific antibodies using assays as described below. The assays identified several cell lines that produced chimeric anti-hTL1A antibodies designated as H2M1681N, H2M1704N, H2M1804N, H2M1805N, H2M1817N and H2M1818N. These antibodies were later converted to hIgG4 isotype by replacing the respective mouse constant regions with the hIgG4 amino acid sequence of SEQ ID NO:257, which contains a S108P mutation in the hinge region, and designated as H4H1681N, H4H1704N, H4H1804N, H4H1805N, H4H1817N and H4H1818N, respectively.

[0093] Human TL1A-specific antibodies were also isolated directly from antigen-immunized B cells without fusion to myeloma cells, as described in U.S. Pat. No. 7,582,298, which is hereby incorporated by reference in its entirety. Heavy and light chain variable regions were cloned to generate fully human anti-hTL1A antibodies designated as H4H1719P, H4H1725P, H4H1738P, H4H1742P, H4H1745P, H4H1750P and H4H1752P. Stable recombinant antibody-expressing CHO cell lines were established.

Example 2

Variable Gene Utilization Analysis

[0094] To analyze the structure of antibodies produced, the nucleic acids encoding antibody variable regions were cloned and sequenced. From the nucleic acid sequence and predicted amino acid sequence of the antibodies, gene usage was identified for each Heavy Chain Variable Region (HCVR) and Light Chain Variable Region (LCVR). Table 1 shows the gene usage for selected antibodies in accordance with the invention.

TABLE-US-00001 TABLE 1 HCVR LCVR Antibody V.sub.H D.sub.H J.sub.H V.sub.K J.sub.K H2M1704 3-7 1-7 6 4-1 3 H2M1681 3-23 2-21 4 1-5 1 H2M1817 4-34 3-9 4 3-20 4 H2M1804 4-34 1-1 4 3-20 4 H2M1818 3-11 4-17 6 4-1 1 H2M1805 4-34 3-3 4 2-24 4 H4H1719 3-9 3-3 6 2-28 2 H4H1725 1-2 2-15 3 1-12 4 H4H1738 3-15 4-4 6 2-28 2 H4H1742 3-23 2-2 6 1-9 2 H4H1745 3-23 6-6 4 1-9 4 H4H1750 3-30 4-17 6 1-17 1 H4H1752 3-23 1-7 4 1-5 1

[0095] Table 2 shows the heavy and light chain variable region amino acid sequence pairs of selected anti-hTL1A antibodies and their corresponding antibody identifiers. The N and P designations refer to antibodies having heavy and light chains with identical CDR sequences but with sequence variations in regions that fall outside of the CDR sequences (i.e., in the framework regions). Thus, N and P variants of a particular antibody have identical CDR sequences within their heavy and light chain variable regions but contain modifications within the framework regions.

TABLE-US-00002 TABLE 2 mAb Name HCVR/LCVR (H2M- or H4H-) SEQ ID NOS 1704N 2/10 1681N 18/26 1804N 34/42 1805N 50/58 1817N 66/74 1818N 82/90 1719N 98/106 1719P 114/116 1725N 118/126 1725P 134/136 1738N 138/146 1738P 154/156 1745N 158/166 1745P 174/176 1750N 178/186 1750P 194/196 1752N 198/206 1752P 214/216 1742N 218/226 1742P 234/236

Example 3

TL1A Binding Affinity Determination

[0096] Binding affinities and kinetic constants were determined by surface plasmon resonance at 25.degree. C. and 37.degree. C. as indicated in Tables 3-5 for human monoclonal anti-TL1A antibodies binding to the following TL1A species variants: human (h) (CHO-expressed, residues 72-251 of SEQ ID NO:244, with N-terminal His.sub.6-tag), cynomolgus monkey (Mf) (E. coli-expressed, residues 72-251 of SEQ ID NO:248, with or without N-terminal Met), cynomolgus monkey (CHO-expressed, residues 72-251 of SEQ ID NO:248, with N-terminal His.sub.6-tag), mouse (m) (E. coli-expressed, residues 76-252 of SEQ ID NO:250, with or without N-terminal Met), mouse (CHO-expressed, residues 76-252 of SEQ ID NO:250, with N-terminal His.sub.6-tag), and rat (CHO cell-expressed; residues 76-252 of SEQ ID NO:258, with N-terminal His.sub.6-tag). Binding constants were also determined for the hTL1A variant, Fhm (E. coli-expressed, residues 72-251 of SEQ ID NO:246 containing Q167R substitution, with or without N-terminal Met). Measurements were conducted on a T100 BIACORE.TM. instrument. Antibodies, expressed with either mouse Fc (designated with prefix "H2M") or human IgG4(S108P) Fc (designated with prefix "H4H"), were captured onto an anti-Fc sensor surface, and at least three different concentrations of the soluble TL1A proteins ranging from 1.25 nM to 100 nM were injected over the sensor surface. Kinetic association (k.sub.a) and dissociation (k.sub.d) rate constants were determined by fitting the data to a 1:1 binding model using BIAevaluation 4.1 curve fitting software (BIAcore Life Sciences). Molar concentrations of TL1A/Fhm used in the data fitting assumed a monomeric state for TL1A in solution. Binding dissociation equilibrium constants (K.sub.D) and dissociative half-lives (t.sub.12) were calculated from the kinetic rate constants as: K.sub.D(M)=k.sub.d/k.sub.a; and t.sub.1/2 (min)=[In2/(60*k.sub.d)]. NB: No binding under the conditions tested; NT: Not tested in this experiment; *: Fitted k.sub.d values below 1.times.10.sup.-6 (1/s) are slower than the detection limit under these experimental conditions; therefore, k.sub.d values were set at 1.times.10.sup.-6 (1/s) for the purpose of approximating K.sub.D and t.sub.1/2; **: Equilibrium dissociation constants for antibodies were determined under steady state conditions.

[0097] As shown in Tables 3 and 4, antibodies bound with high affinity to CHO-expressed forms of both human and monkey TL1A proteins at 25.degree. C. (13 and 12 antibodies with K.sub.D<1 nM, respectively) and at 37.degree. C. (13 and 12 antibodies with K.sub.D<1 nM, respectively). H4H1750P bound significantly weaker to the monkey compared to the human TL1A protein. H4H1704N bound to CHO-expressed mTL1A with K.sub.D<2 nM at both 25.degree. C. and 37.degree. C. H4H1818N bound CHO-expressed mTL1A at 25.degree. C. (K.sub.D.about.7 nM) but not at 37.degree. C. Five antibodies, H4H1681N, H4H1738P, H4H1750P, H4H1752P and H4H1805N, did not bind to CHO-expressed rat TL1A at either 25.degree. C. or 37.degree. C.; the other eight antibodies bound to rat TL1A at both temperatures with K.sub.D ranging from .about.0.6 .mu.M to .about.16 nM.

[0098] As shown in Table 5, three antibodies (H2M1681N, H4H1752P and H2M1805N) did not demonstrate binding to the E. coli-expressed Fhm variant [hTL1A(Q167R)] under the conditions tested. Three antibodies (H2M1704N, H4H1725P, and H2M1818N) demonstrated weak binding (K.sub.D ranging from .about.60 nM to .about.170 nM) to mouse TL1A expressed in E. coli as assessed under steady-state conditions, while all other tested antibodies did not bind to the mouse TL1A protein under the tested conditions.

TABLE-US-00003 TABLE 3 CHO-expressed TL1A at 25.degree. C. hTL1A hTL1A MfTL1A MfTL1A mTL1A mTL1A rTL1A rTL1A K.sub.D t.sub.1/2 K.sub.D t.sub.1/2 K.sub.D t.sub.1/2 K.sub.D t.sub.1/2 mAb (pM) (min) (pM) (min) (pM) (min) (pM) (min) H4H1681N 263 36 404 25 NB NB NB NB H4H1704N 39.2 453 59.9 276 194 46 404 25 H4H1719P 481 44 417 45 NB NB 364 38 H4H1725P 63.6 185 346 64 NB NB 317 26 H4H1738P 608 64 361 93 NB NB NB NB H4H1742P 60.4 755 115 577 NB NB 78 144 H4H1745P 164 172 115 231 NB NB 2.7 (nM) 4 H4H1750P 15.8* 11550* 8.6 (nM) 21 NB NB NB NB H4H1752P 156 197 213 139 NB NB NB NB H4H1804N 291 51 264 49 NB NB 321 43 H4H1805N 365 73 342 74 NB NB NB NB H4H1817N 321 103 356 92 NB NB 2.5 (nM) 25 H4H1818N 124 120 119 122 7.1 (nM) 12 88 92

TABLE-US-00004 TABLE 4 CHO-expressed TL1A at 37.degree. C. hTL1A hTL1A MfTL1A MfTL1A mTL1A mTL1A rTL1A rTL1A K.sub.D t.sub.1/2 K.sub.D t.sub.1/2 K.sub.D t.sub.1/2 K.sub.D t.sub.1/2 mAb (pM) (min) (pM) (min) (pM) (min) (pM) (min) H4H1681N 254 31 226 32 NB NB NB NB H4H1704N 1.00* 11550* 0.93* 11550* 1.3 (nM) 27 46 133 H4H1719P 7.61 1912 2.01 5784 NB NB 78 86 H4H1725P 23.9 758 17.7 888 NB NB 411 15 H4H1738P 571 51 465 60 NB NB NB NB H4H1742P 4.37* 11550* 4.45* 11550* NB NB 653 27 H4H1745P 177 129 173 124 NB NB 16.5 (nM) 11 H4H1750P 13.8* 11550* 17.1 (nM) 11 NB NB NB NB H4H1752P 225 96 223 91 NB NB NB NB H4H1804N 45.8 286 78 167 NB NB 127 88 H4H1805N 299 80 352 68 NB NB NB NB H4H1817N 27.8 1098 25.6 1150 NB NB 1.6 (nM) 26 H4H1818N 0.925* 11550* 0.804* 11550* NB NB 0.61* 11550*

TABLE-US-00005 TABLE 5 E. coli-expressed Fhm/TL1A 25.degree. C. 37.degree. C. 25.degree. C. 37.degree. C. 25.degree. C. Fhm Fhm Fhm Fhm MfTL1A MfTL1A MfTL1A MfTL1A mTL1A K.sub.D t.sub.1/2 K.sub.D t.sub.1/2 K.sub.D t.sub.1/2 K.sub.D t.sub.1/2 K.sub.D mAb (pM) (min) (pM) (min) (pM) (min) (pM) (min) (pM)** H2M1681N NB NB NT NT 546 54 1.5 (nM) 14 NB H2M1704N 282 52 NT NT 285 57 751 16 127 (nM) H4H1719P 109 96 174 42 242 79 289 40 NB H4H1725P 28.9 447 43.6 194 63.7 292 62.2 174 62 (nM) H4H1738P 1020 19 3100 4 1360 18 4.2 (nM) 4 NB H4H1742P 437 129 696 45 593 97 945 35 NB H4H1745P 115 226 207 71 141 221 281 74 NB H4H1750P 204 623 244 335 52.7 (nM) 4 456 (nM) 1 NB H4H1752P NB NB NB NB 274 122 1320 29 NB H2M1804N 192 108 NT NT 176 172 218 118 NB H2M1805N NB NB NT NT 345 67 439 36 NB H2M1817N 451 57 NT NT 1250 37 1.0 (nM) 30 NB H2M1818N 1080 17 NT NT 1840 10 4.1 (nM) 3 171 (nM)

Experiment 4: Inhibition of TL1A by Anti-hTL1A Antibodies

[0099] HEK293 cell lines (CRK01573, ATCC) were generated to stably express human DR3 (full-length; SEQ ID NO:252) or mouse DR3 (full-length; SEQ ID NO:259) along with a luciferase reporter [NH.kappa.B response element (5.times.)-luciferase-IRES-GFP]. NF.kappa.B activation by TL1A has been shown previously (Migone et al., 2002, Immunity 16:479-492). In order to test the membrane-bound form of TL1A and TL1A variants, HEK293 cell lines were generated that stably express full length human TL1A (SEQ ID NO:244), full-length human TL1A with Gln-167 substituted by Arg [Fhm; TL1A(Q167R); SEQ ID NO:246], full-length TL1A from cynomolgus monkey, Macaca fascicularis (MfTL1A; SEQ ID NO:248), full length mouse TL1A (SEQ ID NO:250), and full length rat TL1A (SEQ ID NO:258). The stable cell lines were isolated and maintained in 10% fetal bovine serum (FBS; Irvine Scientific), Dulbecco's Modified Eagle Medium (DMEM; Irvine Scientific), non-essential amino acids (NEAA; Irvine Scientific), Penicillin/Streptomycin (Invitrogen), and G418 (Invitrogen).

[0100] For the bioassay, human or mouse DR3 reporter cells were seeded into 96-well assay plates at 1.times.10.sup.4 cells/well in low serum media, i.e., 0.1% FBS and OPTIMEM.RTM. (Invitrogen), and incubated at 37.degree. C. and 5% CO.sub.2 overnight. The next day, soluble TL1A or FHM (sTL1A or sFHM) was serially diluted at 1:3 and added to cells at concentrations ranging from 0.002 nM to 100 nM (plus a buffer control containing no TL1A). For inhibition, antibodies were serially diluted at 1:3 and added to cells at concentrations ranging from 0.002 nM to 100 nM (plus a buffer control containing no antibody) in the presence of constant concentrations of TL1A or Fhm: 800 pM hTL1A (CHO cell-expressed; residues 72-251 of SEQ ID NO:244, with N-terminal His.sub.6-tag), 100 pM hTL1A (E. coli-expressed; residues 72-251 of SEQ ID NO:244, with or without N-terminal Met), 500 pM Fhm (CHO cell-expressed; residues 72-251 of SEQ ID NO:246), 400 pM MfTL1A (CHO cell-expressed; residues 72-251 of SEQ ID NO:248, with N-terminal His.sub.6-tag), 400 pM MfTL1A (E. coli-expressed; residues 72-251 of SEQ ID NO:248, with or without N-terminal Met), 50 pM mouse TL1A (CHO cell-expressed; residues 76-252 of SEQ ID NO:250, with N-terminal His.sub.6-tag), 20 pM mouse TL1A (E. coli-expressed; residues 76-252 of SEQ ID NO:250, with or without N-terminal Met), and 50 pM rat TL1A (CHO cell-expressed; residues 76-252 of SEQ ID NO:258, with N-terminal His.sub.6-tag). Luciferase activity was detected after 5.5 hours of incubation at 37.degree. C. and 5% CO.sub.2. The results are shown in Table 6. Control mAb1: Positive control (an anti-hTL1A antibody with heavy and light chain variable domains having the amino acid sequences corresponding to SEQ ID NOS:21 and 27 of US 2009/0280116); control mAb2: Negative control (irrelevant antibody); NB: No binding under the conditions tested; NT: Not tested in this assay; *: Inhibition is not to baseline at the highest antibody concentration of 100 nM.

TABLE-US-00006 TABLE 6 sTL1A or sFhm hTL1A hTL1A Fhm MfTL1A MfTL1A mTL1A mTL1A rTL1A (CHO) (E. Coli) (CHO) (CHO) (E. Coli) (CHO) (E. Coli) (CHO) EC50 (nM) 0.63 0.12 0.32 0.86 2.02 0.08 0.01 0.06 Constant TL1A 800 100 500 400 400 50 20 50 or Fhm (pM) IC50 H4H1681N 0.17 0.02 NB 0.03 0.02 NB NB NB [nM] H4H1704N 0.13 0.06 0.17 0.01 0.03 NB NB 0.40 H4H1804N 0.07 0.03 0.10 0.03 0.02 NB NB 3.64 H4H1805N 0.10 0.04 185.50* 0.03 0.02 NB NB NB H4H1817N 0.11 0.04 0.12 0.04 0.02 NB NB 23.87 H4H1818N 0.37 0.29 0.62 0.13 0.06 NB NB 1.10 H4H1719P 0.06 0.02 0.08 0.01 0.02 NT NB NT H4H1725P 0.05 0.02 0.07 0.01 0.02 NB NB 63.43 H4H1738P 0.39 0.16 0.33 0.39 0.07 NT NB NT H4H1742P 0.31 0.19 0.53 0.26 0.07 NB NB 6.12 H4H1745P 0.09 0.06 0.15 0.05 0.03 NB NB NB H4H1750P 0.90 2.17 3.10 154.70 32.47* NT NB NT H4H1752P 0.36 0.21 NB 0.12 0.05 NT NB NT Control mAb1 NB 0.74 NB NB 3.25 NT NB NT Control mAb2 NB NB NB NB NB NB NT NB

[0101] As shown in Table 6, thirteen anti-TL1A antibodies were shown to inhibit soluble human TL1A (CHO and E. coli-expressed) stimulation of the human DR3 receptor expressed on HEK293 cells as determined using a luciferase reporter for NH.kappa.B activation. A positive control antibody (control mAb1) inhibited E. coli-expressed, but not CHO-expressed, hTL1A. Ten antibodies also inhibited stimulation of hDR3-expressing cells by Fhm (hTL1A with Q167R). H4H1681N, H4H1805N and H4H1752P did not fully inhibit Fhm at the highest antibody concentration of 100 nM. All thirteen antibodies also blocked MfTL1A (Table 6). Mouse TL1A (produced from both CHO and E. coli) stimulated NH.kappa.B activation in the hDR3 reporter cells; however, none of the 13 anti-human TL1A antibodies inhibited E. coli-expressed mouse TL1A in this assay (Table 6). Nine selected antibodies were further tested and did not demonstrate blocking of CHO-expressed mouse TL1A in this assay (Table 6).

[0102] To test the ability of TL1A expressed on cells to stimulate signaling in the hDR3 reporter system, bioassays were performed as described above for soluble TL1A with the following changes: Adherent HEK293/TL1A cells were dissociated using Enzyme-Free Dissociation Solution (Chemicon) and added to adherent hDR3 reporter cells after serially diluting the TL1A cells at 1:2 starting from 2.times.10.sup.5 cells to 195 cells (plus a no-cell control). For inhibition by antibodies, 1.times.10.sup.4 cells were added together with serially diluted antibodies from 100 nM to 0.002 nM (plus a control containing no antibody). The results are shown in Table 7. Control mAb1 and mAb2: Same as the assays above. NB: No binding under the conditions tested; NT: Not tested in this assay; *: Inhibition is not to baseline at the highest antibody concentration of 100 nM.

TABLE-US-00007 TABLE 7 Cell-Bound TL1A or Fhm HEK293/ HEK293/ HEK293/ HEK293/ HEK293/ hTL1A Fhm MfTL1A mTL1A rTL1A EC50 (cells) 23474 47921 8465 12366 9773 Constant TL1A or 10,000 Fhm (# cells) IC50 H4H1681N 0.66 NB 3.07 NT NT [nM] H4H1704N 1.11 1.58 3.76 NB NT H4H1804N 0.56 1.23 2.23 NB 3.99 H4H1805N 0.82 54.10* 1.93 NB NB H4H1817N 1.11 0.62 5.04 NB 94.96* H4H1818N 1.54 1.42 4.29 NT NT H4H1719P 0.82 0.84 3.00 NT NT H4H1725P 0.66 0.94 2.82 NB 190.30* H4H1738P 7.47 7.75 20.25 NT NT H4H1742P 6.55 8.39 18.45 NB NT H4H1745P 0.76 2.12 4.28 NT NT H4H1750P 12.82 29.52* 107.40* NT NT H4H1752P 1.99 138.10* 11.67 NT NT Control mAb1 NB NB NB NB NT Control mAb2 NB NB NB NB NB

[0103] As shown in Table 7, all thirteen antibodies blocked the stimulation of hDR3-expressing cells by hTL1A expressed on cells. With cell-bound Fhm, all antibodies inhibited significantly except H4H1681N, H4H1805N, H4H1750P and H4H1752P, which did not inhibit fully at the highest tested antibody concentration of 100 nM. With cell-bound MfTL1A, all antibodies inhibited, except H4H1750 that did not inhibit completely at the highest tested antibody concentration of 100 nM. Six of the antibodies H4H1704N, H4H1804N, H4H1805N, H4H1817N, H4H1725P, and H4H1742P were tested for blocking cell-surface mTL1A stimulation of mDR3 cells; and none showed inhibition. Reporter cells expressing mouse DR3 could also be stimulated by rTL1A-expressing 293 cells, with an observed EC.sub.50 of 9773 cells (Table 7). Four antibodies were tested in the rTL1A/mDR3 assay: three antibodies H4H1804N, H4H1817N, H4H1725P blocked while H4H1805N did not block stimulation of mDR3 cells by cell-surface rTL1A (Table 7). Control mAb1 blocked E. coli-expressed soluble hTL1A and MfTL1A stimulation of hDR3 cells, but failed to block the CHO-expressed forms of hTL1A and MfTL1A under all tested conditions (Table 6). Control mAb1 also did not inhibit stimulation of hDR3-expressing cells by any of the cell-surface expressed TL1A and Fhm under all tested conditions (Table 7).

Experiment 5: Blocking of TL1A to hDR3 and DcR3 by Anti-TL1A Antibodies

[0104] The ability of antibodies to block human TL1A binding to its cognate receptors, the DR3 and DcR3 receptors, was measured using a competition sandwich ELISA. In addition, blocking of a human TL1A variant FHM (human TL1A Q167R) and the cynomolgus monkey (Macaca fascicularis) TL1A (MfTL1A) protein binding to the human DR3 or DcR3 receptors was measured in the same manner. Constant amounts of biotinylated human TL1A or FHM (both expressed with a 6-His tag in CHO cells) or biotinylated MfTL1A (expressed in CHO cells) were separately titrated with varying amounts of antibodies. The antibody-protein complexes were incubated in solution (1 hr, 25.degree. C.) and then transferred to microtiter plates coated with human DR3 (hDR3) or human DcR3 (hDcR3) expressed as human IgG1 Fc fusion proteins. After one hour at 25.degree. C. the wells were washed, and bound human or monkey TL1A was detected with streptavidin conjugated with horseradish peroxidase (HRP). Wells were developed with a TMB solution to produce a colorimetric reaction and quenched with aqueous sulfuric acid before reading absorbance at 450 nm on a Perkin-Elmer Victor X5 plate reader. A sigmoidal dose-response curve was fit to the data using the Prism.TM. data analysis package. The calculated IC50 value, defined as the concentration of antibody required to block 50% of TL1A binding to hDR3 or hDcR3, was used as an indicator of blocking potency. Both fully human anti-hTL1A mAbs and comparator antibodies, i.e., control mAb1 (an anti-hTL1A antibody with heavy and light chain variable domains having the amino acid sequences corresponding to SEQ ID NOS:21 and 27, respectively, of US 2009/0280116) and control mAb3 (an anti-hTL1A antibody with heavy and light chain variable domains having the amino acid sequences corresponding to SEQ ID NOS:57 and 48, respectively, of US 2009/0280116), were included in the study. The results are shown in Table 8. NB: No binding under the conditions tested; NT: Not tested. Concentrations of biotinylated soluble ligands: (1) 150 pM; (2) 500 pM; (3) 10 pM; and (4) 50 pM.

[0105] As shown in Table 8, most of the fully human mAbs show effective blocking of the TL1A/hDR3 and TL1A/hDcR3 binding interaction, with several showing IC50 values below 50 pM. Two of the antibodies, H4H1752P and H4H1805N, strongly blocked binding of both human and monkey TL1A binding to both hDR3 and hDcR3 but failed to block binding of FHM to either hDR3 or hDcR3, suggesting that the binding epitope for these two antibodies may involve the region near the FHM mutation site (hTL1A with Q167R). The crystal structure of hTL1A shows that residue Q167 occurs within a surface-exposed loop (Zhan et al., 2009, Biochemistry 48: 7636-7645).

TABLE-US-00008 TABLE 8 hDR3 hDR3 hDR3 DcR3 DcR3 DcR3 hTL1A FHM MfTL1A hTL1A FHM MfTL1A (CHO) .sup.1 (CHO) .sup.1 (CHO) .sup.2 (CHO) .sup.3 (CHO) .sup.3 (CHO) .sup.4 mAb ID IC.sub.50 (pM) IC.sub.50 (pM) IC.sub.50 (pM) IC.sub.50 (pM) IC.sub.50 (pM) IC.sub.50 (pM) H4H1681N 60 >10000 141 17 90 93 H2M1681N 37 >10000 61 13 234 149 H4H1704N 30 44 42 77 170 110 H2M1704N NT NT NT NT NT NT H4H1719P 22 23 46 44 45 61 H4H1725P 15 18 16 68 85 145 H4H1738P 69 152 117 122 150 68 H4H1742P 64 214 240 181 231 127 H4H1745P 18 44 50 58 85 118 H4H1750P 341 589 5209 656 626 NB H4H1752P 104 NB 110 31 NB 56 H4H1804N 40 69 71 175 >10000 34 H2M1804N 46 102 81 120 >10000 9 H4H1805N 14 NB 26 33 436 313 H2M1805N 6 NB 13 12 2241 1138 H4H1817N 114 235 101 270 NT 322 H2M1817N 154 249 137 890 666 776 H4H1818N 119 202 123 232 NT 1102 H2M1818N 154 317 239 396 NB 55 Control mAb1 >10000 NB 5300 >1000 NT 21000 Control mAb3 >10000 NB 17000 8600 NT NT

Example 6

Cell Surface Binding Competition of Anti-TL1A Antibodies with Soluble hTL1A

[0106] Human embryonic kidney 293 cells stably transfected to over-express cell-surface hTL1A were first stained in a flow cytometric experiment with eight anti-hTL1A antibodies at four concentrations (1, 0.1, 0.01, and 0.003 .mu.g/ml). Bound human antibodies were detected using an allophycocyanin-labeled goat F(ab').sub.2 specific for human Fc.gamma. [or anti-hFc.gamma.-APC F(ab').sub.2, Jackson ImmunoResearch, #109-136-170]. The lowest antibody concentration providing significant staining levels was then used in a competition binding experiment. A negative isotype control antibody (human IgG4) was used at 1 .mu.g/ml to define the background signal. For the competition experiment, eight antibody samples, at the minimal concentrations identified above, were first treated with soluble hTL1A expressed from CHO cells at concentrations ranging from 0.03 .mu.g/ml to 10 .mu.g/ml. After pre-incubation for 30 min on ice, the antibody/hTL1A mixture was added to 293/HEK-hTL1A cells that had been isolated by centrifugation in a 96-well conical plate. After incubation for an additional 10 minutes on ice, the cells were washed. The secondary reagent, anti-hFc.gamma.-APC F(ab').sub.2, was added to all wells at a 200-fold dilution to detect bound antibodies. Samples were incubated for 15 minutes on ice, away from light, and then washed. Cells were processed on an BD.TM. LSR II Flow Cytometer (BD Biosciences) to detect anti-hTL1A antibodies bound to the cell surface, and data were analyzed using FlowJo software (version 8.8.6; Tree Star Inc.). The results are shown in Table 9. Maximum signal: Anti-hTL1A antibody binding in the absence of soluble hTL1A; Minimum signal: Signal recorded when 1 .mu.g /ml of isotype control antibody was added in place of the anti-hTL1A antibody. NT: Not tested.

TABLE-US-00009 TABLE 9 Mean Fluorescence Intensity for Soluble anti-hTL1A antibodies (H4H) binding to cell-surface hTL1A hTL1A 1704N 1725P 1742P 1804N 1805N 1817N 1681N 1745P (.mu.g/ml) 0.1 .mu.g/ml 0.1 .mu.g/ml 1 .mu.g/ml 0.1 .mu.g/ml 0.1 .mu.g/ml 1 .mu.g/ml 1 .mu.g/ml 1 .mu.g/ml 10 NT NT NT NT NT NT 16.9 15 3 22.6 34.9 19.9 28.9 23.6 25.2 28.5 19.5 1 26 29.2 32.7 33.7 25.1 29.1 80.9 26.9 0.3 31.5 40.7 44.7 33.5 36.8 23.6 236 115 0.1 132 84.5 79.4 51.1 60.2 97.2 327 93.1 0.03 163 207 126 126 156 85.3 318 80 Maximum 116 211 126 127 158 110 320 87.2 signal Minimum 27.5 27.5 27.5 27.5 27.5 27.5 17.9 17.9 signal

[0107] As shown in Table 9, the signals from the eight tested antibodies could be competed down to baseline levels by the addition of excess soluble hTL1A, demonstrating the specificity of binding of the antibodies to cell-surface hTL1A.

Example 7

Blocking of hTL1A-Dependent CD4.sup.+ T-cell Stimulation by Anti-TL1A Antibodies

[0108] To determine the ability of anti-hTL1A antibodies to block hTL1A-dependent stimulation of human CD4.sup.+ T-cells, an in vitro assay was developed in which hTL1A/anti-CD3/anti-CD28-stimulated release of IFN-gamma (IFN-y) was measured in the presence or absence of antibodies. Human CD4.sup.+ T-cells were isolated from fresh buffy coats prepared from human blood samples obtained from the New York Blood Center. Cells from a single donor were kept separate from other donor cells for each assay. The CD4.sup.+ T-cells were added to the wells of a 96-well plate at 3.5.times.10.sup.5 cells per well. To each well was then added soluble hTL1A (residues 72-251 of NP_005109.2 with an N-terminal hexa-histidine tag, expressed from CHO cells) to a final concentration of 1 .mu.g/ml (16 nM, assuming hTL1A forming trimers in solution) in RPMI+10% FBS, L-glutamine and penicillin/streptomycin. To each well was also added the anti-hTL1A antibodies or an isotype control antibody to final concentrations of 1.0 .mu.g/ml or 3.0 .mu.g/ml (6.7 nM or 20 nM, respectively). The samples were incubated for 15 minutes at 4.degree. C. in the dark, followed by the additions of anti-hCD3 (BD Pharmingen, cat #555336) and anti-hCD28 (BD Pharmingen, cat #555725) to each well to final concentrations of 1.0 .mu.g/ml. Samples were incubated for 24 hours at 37.degree. C., the supernatants harvested, and IFN-y levels determined by ELISA. The blocking effect (average from two separate wells for each condition) of each antibody on each human CD4.sup.+ T-cell donor sample is represented as the reduction from maximal signal divided by maximal response window; i.e., % Blocking=[(Max-Inhib)/(Max-Min)].times.100, where "Max", "Inhib", and "Min" are concentrations of IFN-.gamma. measured for CD4.sup.+ human T-cells treated as follows: "Max"--treated with [hTL1A+anti-hCD3+anti-hCD28+ isotype control mAb]; "Min: "--treated with [anti-hCD3 + anti-hCD28+ isotype control mAb]; and "Inhib" --treated with [hTL1A+anti-hCD3+anti-hCD28+anti-hTL1A test mAb]. Antibodies for which IFN-.gamma. blockade surpassed the "Min" baseline level are represented as 100% blockade. Ratio (Max/Min) is the ratio of the IFN-.gamma. concentration produced from human CD4.sup.+ T-cells treated under Max and Min conditions as defined above.

[0109] As shown in Table 10, the antibodies H4H1725P, H4H1805N, H4H1817N, and H4H1804N significantly blocked hTL1A-stimulated IFN-.gamma. release at both 1 .mu.g/ml and 3 .mu.g/ml concentrations, with nearly complete blockade (>80%) observed for most donors at the higher antibody concentration. The results for blockade of IFN-.gamma. secretion by thirteen different anti-hTL1A antibodies against CD4.sup.+ T-cells from 10 different human donors are further summarized in Table 11. SD: Standard deviation.

TABLE-US-00010 TABLE 10 % Blocking of IFN-.gamma. production in human T-cells from 10 donors Donor # D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 Ratio (Max/Min) mAb ID 5 4 10 10 4 3 4 3 8 2 H4H1725P mAb 90 100 70 85 45 80 60 85 50 95 H4H1805N 1 .mu.g/ml 100 100 90 100 100 100 100 100 90 100 H4H1817N (6.7 nM) 100 100 90 90 100 45 55 100 55 80 H4H1804N 95 100 80 90 100 50 10 100 50 0 H4H1725P mAb 95 100 95 100 90 80 90 100 90 100 H4H1805N 3 .mu.g/ml 100 100 95 100 80 100 100 100 70 100 H4H1817N (20 nM) 100 100 100 100 100 100 100 100 95 85 H4H1804N 100 100 95 100 100 100 100 100 100 80

TABLE-US-00011 TABLE 11 Average % Average % Average % Blocking (SD) Blocking (SD) Blocking (SD) mAb ID 0.1 .mu.g/ml mAb 1 .mu.g/ml mAb 3 .mu.g/ml mAb H4H1681N 20% (24) 45% (30) 95% (7) H4H1704N 22% (29) 53% (27) 98% (5) H4H1719P 10% (22) 37% (23) 78% (30) H4H1725P 13% (14) 80% (20) 94% (7) H4H1738P 25% (30) 41% (35) 81% (18) H4H1742P 10% (22) 58% (33) 83% (16) H4H1745P 22% (27) 36% (36) 91% (7) H4H1750P 11% (14) 42% (35) 89% (15) H4H1752P 18% (25) 52% (35) 71% (30) H4H1804N 26% (30) 68% (38) 98% (6) H4H1805N 21% (28) 98% (4) 94% (10) H4H1817N 25% (33) 81% (22) 98% (5) H4H1818N 25% (33) 42% (33) 71% (35) Isotype Control 16% (21) 26% (33) 28% (27)

[0110] IFN-.gamma. levels were also measured at six different antibody concentrations (ranging from 0.03 .mu.g/ml to 10 .mu.g/ml) for each of six different antibodies added to CD4.sup.+ T-cells from twelve human donors. Curve fitting to the data allowed estimation of the antibody concentration at which half-maximal inhibition was achieved for each antibody for each donor cell sample. The average (.+-.SD) concentrations for achieving half-maximal inhibition are provided in Table 12.

TABLE-US-00012 TABLE 12 mAb IC.sub.50 (nM) Donor # H4H1725P H4H1742P H4H1805N H4H1817N H4H1804N H4H1704N D1 3.4 24 2.4 6.6 6.8 -- D2 3.2 4.6 5.5 3.1 8.6 -- D3 5.4 10 3.4 4.7 8.6 -- D4 4.7 13 2.5 2.4 7.7 -- D5 7.0 12 2.9 8.5 6.7 17 D6 5.4 10 3.4 4.7 8.6 11 D7 13 31 8.0 7.0 12 12 D8 12 27 5.1 7.0 11 19 D9 6.4 56 5.9 9.5 8.1 7.5 D10 4.1 12 2.8 7.3 12 10 D11 6.1 27 3.0 7.3 7.6 11 D12 6.4 14 3.1 9.4 7.5 8.0 Average 6.4 (3.1) 20 (14) 4.0 (1.7) 6.5 (2.3) 8.8 (1.9) 12 (4.1) (.+-.SD)

[0111] Four of the antibodies, H4H1725P, H4H1804N, H4H1805N, H4H1817N exhibited average half-maximal inhibition concentrations below 10 nM (ranging approximately 4-9 nM).

Sequence CWU 1

1

2591366DNAArtificial SequenceSynthetic 1gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag tctctggatt cacctttagt agttattgga tgagctgggt ccgccaggct 120ccagggaagg ggctggaatg ggtggccaac ataaaggaag atggaagtga gaaaaactat 180gtggactctg tgaagggccg attcaccctc tccagcgaca acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac acggctgttt attactgtgc gagagaggac 300tatgactcct actacaagta cggtatggac gtttggggcc aagggaccgc ggtcatcgtc 360tcctca 3662122PRTArtificial SequenceSynthetic 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Glu Asp Gly Ser Glu Lys Asn Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Leu Ser Ser Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Tyr Asp Ser Tyr Tyr Lys Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Ala Val Ile Val Ser Ser 115 120 324DNAArtificial SequenceSynthetic 3ggattcacct ttagtagtta ttgg 2448PRTArtificial SequenceSynthetic 4Gly Phe Thr Phe Ser Ser Tyr Trp1 5 524DNAArtificial SequenceSynthetic 5ataaaggaag atggaagtga gaaa 2468PRTArtificial SequenceSynthetic 6Ile Lys Glu Asp Gly Ser Glu Lys1 5 745DNAArtificial SequenceSynthetic 7gcgagagagg actatgactc ctactacaag tacggtatgg acgtt 45815PRTArtificial SequenceSynthetic 8Ala Arg Glu Asp Tyr Asp Ser Tyr Tyr Lys Tyr Gly Met Asp Val 1 5 10 15 9339DNAArtificial SequenceSynthetic 9gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60atcaactgca agtccagcca gagtatttta tacagctcca acaataagaa ctacttagct 120tggtatcagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 180gaatccgggg tccctgaccg attcagtggc agcgggtctg gaacagattt cactctcacc 240atcagcagcc tgcaggctga agatgtgtca gtttattact gtcaacaata ttatagtact 300ccattcactt tcggccctgg gaccaaagtg gatatcaaa 33910113PRTArtificial SequenceSynthetic 10Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Ile Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ser Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile 100 105 110 Lys1136DNAArtificial SequenceSynthetic 11cagagtattt tatacagctc caacaataag aactac 361212PRTArtificial SequenceSynthetic 12Gln Ser Ile Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 139DNAArtificial SequenceSynthetic 13tgggcatct 9143PRTArtificial SequenceSynthetic 14Trp Ala Ser1 1527DNAArtificial SequenceSynthetic 15caacaatatt atagtactcc attcact 27169PRTArtificial SequenceSynthetic 16Gln Gln Tyr Tyr Ser Thr Pro Phe Thr1 5 17363DNAArtificial SequenceSynthetic 17gaggtgcaac tattggagtc tgggggaggc ttggtgcagc ctgggaagtc ccttagactc 60tcctgtgcag tctctggatt cacctttagt acctatggca tgaattgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcaagt attagtggta ctggtcgtac cacataccat 180gcagactccg tgcagggccg gttcaccgtc tccagagaca attccaagaa cattctatat 240ttacagatga acagtctgcg agccgacgac acggccgtat atttctgtac gaaagagcgg 300ggagattact actacggggt ttttgactac tggggccagg gaaccctggt caccgtctcc 360tca 36318121PRTArtificial SequenceSynthetic 18Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Thr Gly Arg Thr Thr Tyr His Ala Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Ile Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Lys Glu Arg Gly Asp Tyr Tyr Tyr Gly Val Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 1924DNAArtificial SequenceSynthetic 19ggattcacct ttagtaccta tggc 24208PRTArtificial SequenceSynthetic 20Gly Phe Thr Phe Ser Thr Tyr Gly1 5 2124DNAArtificial SequenceSynthetic 21attagtggta ctggtcgtac caca 24228PRTArtificial SequenceSynthetic 22Ile Ser Gly Thr Gly Arg Thr Thr1 5 2342DNAArtificial SequenceSynthetic 23acgaaagagc ggggagatta ctactacggg gtttttgact ac 422414PRTArtificial SequenceSynthetic 24Thr Lys Glu Arg Gly Asp Tyr Tyr Tyr Gly Val Phe Asp Tyr 1 5 10 25318DNAArtificial SequenceSynthetic 25gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gactattagt tcctggttgg cctggtatca gcagacacca 120gagaaagccc ctaagctcct gatctatgcg gcgtctaatt tacaaagtgg agtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctacagcct 240gatgattttg caacttatta ctgccagcag tatcatcgtt cttggacgtt cggccaaggg 300accaaggtgg aaatcaca 31826106PRTArtificial SequenceSynthetic 26Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Thr Pro Glu Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Arg Ser Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Thr 100 105 2718DNAArtificial SequenceSynthetic 27cagactatta gttcctgg 18286PRTArtificial SequenceSynthetic 28Gln Thr Ile Ser Ser Trp1 5 299DNAArtificial SequenceSynthetic 29gcggcgtct 9303PRTArtificial SequenceSynthetic 30Ala Ala Ser1 3124DNAArtificial SequenceSynthetic 31cagcagtatc atcgttcttg gacg 24328PRTArtificial SequenceSynthetic 32Gln Gln Tyr His Arg Ser Trp Thr1 5 33351DNAArtificial SequenceSynthetic 33caggtgcagc tacaccagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60acctgcgctg tctctggtgt gtccttcagt gattatcact gggcctggat ccgccagtcc 120ccagggaagg ggctggagtg gattggggat atcaatcatc gtggaaggac caactacaac 180ccgtccctca agagtcgagt caccatatca cttgacacgt ccgggaaccc gttctccctg 240aagctgacct ctgtgaccgc cgcggacacg gctgtttatt actgtgcgag agattttccc 300aactgggttt ttgactcctg gggccaggga atcctagtca ccgtctcctc a 35134117PRTArtificial SequenceSynthetic 34Gln Val Gln Leu His Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Val Ser Phe Ser Asp Tyr 20 25 30 His Trp Ala Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn His Arg Gly Arg Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Leu Asp Thr Ser Gly Asn Pro Phe Ser Leu65 70 75 80 Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Phe Pro Asn Trp Val Phe Asp Ser Trp Gly Gln Gly Ile Leu 100 105 110 Val Thr Val Ser Ser 115 3524DNAArtificial SequenceSynthetic 35ggtgtgtcct tcagtgatta tcac 24368PRTArtificial SequenceSynthetic 36Gly Val Ser Phe Ser Asp Tyr His1 5 3721DNAArtificial SequenceSynthetic 37atcaatcatc gtggaaggac c 21387PRTArtificial SequenceSynthetic 38Ile Asn His Arg Gly Arg Thr1 5 3933DNAArtificial SequenceSynthetic 39gcgagagatt ttcccaactg ggtttttgac tcc 334011PRTArtificial SequenceSynthetic 40Ala Arg Asp Phe Pro Asn Trp Val Phe Asp Ser 1 5 10 41324DNAArtificial SequenceSynthetic 41gaaattgtat tgtcgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagcttct tagcctggta ccaacaatat 120cctggccagg ctcccaggct cctcatctat ggtgcatcca gaagggccac tggcatccca 180gacaggttcc gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcaa caatatggta gttcaccgct caccttcggc 300ggagggacca aggtggagaa caaa 32442108PRTArtificial SequenceSynthetic 42Glu Ile Val Leu Ser Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Phe Leu Ala Trp Tyr Gln Gln Tyr Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Arg 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Asn Lys 100 105 4321DNAArtificial SequenceSynthetic 43cagagtgtta gcagcagctt c 21447PRTArtificial SequenceSynthetic 44Gln Ser Val Ser Ser Ser Phe1 5 459DNAArtificial SequenceSynthetic 45ggtgcatcc 9463PRTArtificial SequenceSynthetic 46Gly Ala Ser1 4727DNAArtificial SequenceSynthetic 47caacaatatg gtagttcacc gctcacc 27489PRTArtificial SequenceSynthetic 48Gln Gln Tyr Gly Ser Ser Pro Leu Thr1 5 49357DNAArtificial SequenceSynthetic 49caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60acctgcgctg tctatggtgg gtccttcact ggtttctact ggagctggat ccgccagccc 120cccgggaagg ggctggagtg gattggggaa atcaatcatc gtggaaacac caactacaat 180ccgtccctca agagtcgagt caccatgtca gtagacacgt ccaagaacca gttctccctg 240aacatgatct ctgtgaccgc cgcggacacg gctatgtatt tctgtgcgag tcctttttac 300gatttttgga gtggttccga ctactggggc cagggaaccc tggtcaccgt ctcctca 35750119PRTArtificial SequenceSynthetic 50Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr Gly Phe 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Arg Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80 Asn Met Ile Ser Val Thr Ala Ala Asp Thr Ala Met Tyr Phe Cys Ala 85 90 95 Ser Pro Phe Tyr Asp Phe Trp Ser Gly Ser Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 5124DNAArtificial SequenceSynthetic 51ggtgggtcct tcactggttt ctac 24528PRTArtificial SequenceSynthetic 52Gly Gly Ser Phe Thr Gly Phe Tyr1 5 5321DNAArtificial SequenceSynthetic 53atcaatcatc gtggaaacac c 21547PRTArtificial SequenceSynthetic 54Ile Asn His Arg Gly Asn Thr1 5 5539DNAArtificial SequenceSynthetic 55gcgagtcctt tttacgattt ttggagtggt tccgactac 395613PRTArtificial SequenceSynthetic 56Ala Ser Pro Phe Tyr Asp Phe Trp Ser Gly Ser Asp Tyr 1 5 10 57336DNAArtificial SequenceSynthetic 57gacattatgt tgacccagac tccactcacc tcacctgtca cccttgggca gccggcctcc 60atctcctgca agtctagtca aagcctcgta cacagtgatg gaaacaccta cttgagttgg 120cttcagcaga ggccaggcca gcctccaaga ctcctatttt ataagatttc taaccggttc 180tctggagtcc cagacagatt cagtggcagt ggggcaggga cagatttcac actgaaaatc 240agcagggtgg aagctgagga tgtcggggtt tattactgca tgcaagctac acaatttcct 300ctcactttcg gcggagggac caaggtagag atcaaa 33658112PRTArtificial SequenceSynthetic 58Asp Ile Met Leu Thr Gln Thr Pro Leu Thr Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro 35 40 45 Pro Arg Leu Leu Phe Tyr Lys Ile Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Thr Gln Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 5933DNAArtificial SequenceSynthetic 59caaagcctcg tacacagtga tggaaacacc tac 336011PRTArtificial SequenceSynthetic 60Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr 1 5 10 619DNAArtificial SequenceSynthetic 61aagatttct 9623PRTArtificial SequenceSynthetic 62Lys Ile Ser1 6327DNAArtificial SequenceSynthetic 63atgcaagcta cacaatttcc tctcact 27649PRTArtificial SequenceSynthetic 64Met Gln Ala Thr Gln Phe Pro Leu Thr1 5 65351DNAArtificial SequenceSynthetic 65caggtgcaac tacaacagtg gggcgcagga ctgttgaagc cctcggagac cctgtccctc 60acctgcgctg tctctggtgg gtccttcagt gattacttct ggacctggat ccgccagccc 120cccgggaagg ggctggagtg gattggggaa atcagtcata gtggaagaac caactacaac 180ccgtccctca agagtcgagt caccatatca gttgacacgt ccatgagcca gttctccctg 240aagatgacct ctgtgaccgc cgcggactcg gctgtatatt actgtgcgag agattatccc 300aactgggttt ttgactactg gggccaggga accctggtca ccgtctcctc a 35166117PRTArtificial SequenceSynthetic 66Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Phe Ser Asp Tyr 20 25 30 Phe Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Ser His Ser Gly Arg Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Met Ser Gln Phe Ser Leu65 70 75 80 Lys Met Thr Ser Val Thr Ala Ala Asp Ser Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Tyr Pro Asn Trp Val Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100

105 110 Val Thr Val Ser Ser 115 6724DNAArtificial SequenceSynthetic 67ggtgggtcct tcagtgatta cttc 24688PRTArtificial SequenceSynthetic 68Gly Gly Ser Phe Ser Asp Tyr Phe1 5 6921DNAArtificial SequenceSynthetic 69atcagtcata gtggaagaac c 21707PRTArtificial SequenceSynthetic 70Ile Ser His Ser Gly Arg Thr1 5 7133DNAArtificial SequenceSynthetic 71gcgagagatt atcccaactg ggtttttgac tac 337211PRTArtificial SequenceSynthetic 72Ala Arg Asp Tyr Pro Asn Trp Val Phe Asp Tyr 1 5 10 73324DNAArtificial SequenceSynthetic 73gaaattgagt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aggagccacc 60ctctcctgca gggccagtca gagtgttttc aacagctact tagcgtggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca ggagggccac tggcatccct 180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240cctggagatt ttgcagtata tttctgtcag cagtatggta actcaccgct cactttcggc 300ggagggacca aggtggagat caaa 32474108PRTArtificial SequenceSynthetic 74Glu Ile Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Gly Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Phe Asn Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80 Pro Gly Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Gly Asn Ser Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 7521DNAArtificial SequenceSynthetic 75cagagtgttt tcaacagcta c 21767PRTArtificial SequenceSynthetic 76Gln Ser Val Phe Asn Ser Tyr1 5 779DNAArtificial SequenceSynthetic 77ggtgcatcc 9783PRTArtificial SequenceSynthetic 78Gly Ala Ser1 7927DNAArtificial SequenceSynthetic 79cagcagtatg gtaactcacc gctcact 27809PRTArtificial SequenceSynthetic 80Gln Gln Tyr Gly Asn Ser Pro Leu Thr1 5 81366DNAArtificial SequenceSynthetic 81cggttgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgaaactc 60tcctgtgcag cctctggatt caccatcagt gactactaca tgagctggat ccgccaggct 120ccagggaagg ggctggagtg ggtttcgtac attggtggta gtggtagtac catatattac 180gcagactctg tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gagagaggac 300gctgactcct actactacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360tcctca 36682122PRTArtificial SequenceSynthetic 82Arg Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Gly Gly Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Ala Asp Ser Tyr Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 8324DNAArtificial SequenceSynthetic 83ggattcacca tcagtgacta ctac 24848PRTArtificial SequenceSynthetic 84Gly Phe Thr Ile Ser Asp Tyr Tyr1 5 8524DNAArtificial SequenceSynthetic 85attggtggta gtggtagtac cata 24868PRTArtificial SequenceSynthetic 86Ile Gly Gly Ser Gly Ser Thr Ile1 5 8745DNAArtificial SequenceSynthetic 87gcgagagagg acgctgactc ctactactac tacggtatgg acgtc 458815PRTArtificial SequenceSynthetic 88Ala Arg Glu Asp Ala Asp Ser Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 89339DNAArtificial SequenceSynthetic 89gatattgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ccacttatct 120tggtatcagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 180gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240atcagcagcc tgcaggctga agatgtggca gtttattact gtcaacaata ttatagtact 300cctcggacgt tcggccaagg gaccaaggtg gaaatcaaa 33990113PRTArtificial SequenceSynthetic 90Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn His Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys9136DNAArtificial SequenceSynthetic 91cagagtgttt tatacagctc caacaataag aaccac 369212PRTArtificial SequenceSynthetic 92Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn His 1 5 10 939DNAArtificial SequenceSynthetic 93tgggcatct 9943PRTArtificial SequenceSynthetic 94Trp Ala Ser1 9527DNAArtificial SequenceSynthetic 95caacaatatt atagtactcc tcggacg 27969PRTArtificial SequenceSynthetic 96Gln Gln Tyr Tyr Ser Thr Pro Arg Thr1 5 97366DNAArtificial SequenceSynthetic 97gaagtgcaac tggtggagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60tcctgtgtag cctctggatt cacctttcaa gattatgcca tgcactgggt ccggcaagct 120ccaggaaagg gcctggagtg ggtctcaggt attaattgga ttagtgatga catgggctat 180gcggactctg tgatgggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatgg acagtctgag agctgaggac acggccttgt attactgtgc aaaagataag 300ggattacgat ttttggacta tgttatggac gtctggggcc aagggaccac ggtcaccgtc 360tcctca 36698122PRTArtificial SequenceSynthetic 98Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Gln Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Asn Trp Ile Ser Asp Asp Met Gly Tyr Ala Asp Ser Val 50 55 60 Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Lys Gly Leu Arg Phe Leu Asp Tyr Val Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 9924DNAArtificial SequenceSynthetic 99ggattcacct ttcaagatta tgcc 241008PRTArtificial SequenceSynthetic 100Gly Phe Thr Phe Gln Asp Tyr Ala1 5 10124DNAArtificial SequenceSynthetic 101attaattgga ttagtgatga catg 241028PRTArtificial SequenceSynthetic 102Ile Asn Trp Ile Ser Asp Asp Met1 5 10345DNAArtificial SequenceSynthetic 103gcaaaagata agggattacg atttttggac tatgttatgg acgtc 4510415PRTArtificial SequenceSynthetic 104Ala Lys Asp Lys Gly Leu Arg Phe Leu Asp Tyr Val Met Asp Val 1 5 10 15 105339DNAArtificial SequenceSynthetic 105gacatccagt tgacccagtc tcctctctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctactca gagcctcctg cataggaatg gccacaacta tttgcattgg 120tacctacata agccagggca gtctccacaa ctcctgattc atctgggttc taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggggtc tattactgca tgcaagctct acagactccg 300tacacttttg gccaggggac caaggtggag atcaaacga 339106113PRTArtificial SequenceSynthetic 106Asp Ile Gln Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Thr Gln Ser Leu Leu His Arg 20 25 30 Asn Gly His Asn Tyr Leu His Trp Tyr Leu His Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile His Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg10733DNAArtificial SequenceSynthetic 107cagagcctcc tgcataggaa tggccacaac tat 3310811PRTArtificial SequenceSynthetic 108Gln Ser Leu Leu His Arg Asn Gly His Asn Tyr 1 5 10 1099DNAArtificial SequenceSynthetic 109ctgggttct 91103PRTArtificial SequenceSynthetic 110Leu Gly Ser1 11127DNAArtificial SequenceSynthetic 111atgcaagctc tacagactcc gtacact 271129PRTArtificial SequenceSynthetic 112Met Gln Ala Leu Gln Thr Pro Tyr Thr1 5 113363DNAArtificial SequenceSynthetic 113gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60tcctgtgtag cctctggatt cacctttcaa gattatgcca tgcactgggt ccggcaagct 120ccaggaaagg gcctggagtg ggtctcaggt attaattgga ttagtgatga catgggctat 180gcggactctg tgatgggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240ctgcaaatgg acagtctgag agctgaggac acggccttgt attactgtgc aaaagataag 300ggattacgat ttttggacta tgttatggac gtctggggcc aagggaccac ggtcaccgtc 360tcc 363114121PRTArtificial SequenceSynthetic 114Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Gln Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Asn Trp Ile Ser Asp Asp Met Gly Tyr Ala Asp Ser Val 50 55 60 Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asp Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Lys Gly Leu Arg Phe Leu Asp Tyr Val Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 115336DNAArtificial SequenceSynthetic 115gacatcgtga tgacccagtc tcctctctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctactca gagcctcctg cataggaatg gccacaacta tttgcattgg 120tacctacata agccagggca gtctccacaa ctcctgattc atctgggttc taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggggtc tattactgca tgcaagctct acagactccg 300tacacttttg gccaggggac caagctggag atcaaa 336116112PRTArtificial SequenceSynthetic 116Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Thr Gln Ser Leu Leu His Arg 20 25 30 Asn Gly His Asn Tyr Leu His Trp Tyr Leu His Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile His Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 117354DNAArtificial SequenceSynthetic 117caggtgcagc tggtggagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcaaaccta aaagtggtgg cacaaactat 180gtacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcaa cacagcctac 240atggaactaa acaatctgaa atctgacgac acggccgttt attactgtgc gactggaggg 300agtcaagatg cttctgattt ctggggccaa gggacaatgg tcaccgtctc ttca 354118118PRTArtificial SequenceSynthetic 118Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Lys Pro Lys Ser Gly Gly Thr Asn Tyr Val Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr65 70 75 80 Met Glu Leu Asn Asn Leu Lys Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Gly Ser Gln Asp Ala Ser Asp Phe Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 11924DNAArtificial SequenceSynthetic 119ggatacacct tcaccggcta ctat 241208PRTArtificial SequenceSynthetic 120Gly Tyr Thr Phe Thr Gly Tyr Tyr1 5 12124DNAArtificial SequenceSynthetic 121atcaaaccta aaagtggtgg caca 241228PRTArtificial SequenceSynthetic 122Ile Lys Pro Lys Ser Gly Gly Thr1 5 12333DNAArtificial SequenceSynthetic 123gcgactggag ggagtcaaga tgcttctgat ttc 3312411PRTArtificial SequenceSynthetic 124Ala Thr Gly Gly Ser Gln Asp Ala Ser Asp Phe 1 5 10 125324DNAArtificial SequenceSynthetic 125gacatccagt tgacccagtc tccatcttcc gtgtctgcat ctgtaggagg cagagtcacc 60atcacttgtc gggcgagtca ggatattagc atctggttag cctggtatca acagaaacca 120gggaaagccc ctaacctcct gatctatgct gcatccagtt tacaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttacta ttgtcaacag gctgacagtt tcccgctcac tttcggcggt 300gggaccaagc tggagatcaa acga 324126108PRTArtificial SequenceSynthetic 126Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Gly Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ile Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asp Ser Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 12718DNAArtificial SequenceSynthetic 127caggatatta gcatctgg 181286PRTArtificial SequenceSynthetic 128Gln Asp Ile Ser Ile Trp1 5 1299DNAArtificial SequenceSynthetic 129gctgcatcc 91303PRTArtificial SequenceSynthetic 130Ala Ala Ser1 13127DNAArtificial

SequenceSynthetic 131caacaggctg acagtttccc gctcact 271329PRTArtificial SequenceSynthetic 132Gln Gln Ala Asp Ser Phe Pro Leu Thr1 5 133354DNAArtificial SequenceSynthetic 133caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcaaaccta aaagtggtgg cacaaactat 180gtacagaagt ttcagggcag ggtcaccatg accagggaca cgtccatcaa cacagcctac 240atggaactaa acaatctgaa atctgacgac acggccgttt attactgtgc gactggaggg 300agtcaagatg cttctgattt ctggggccaa gggacaatgg tcaccgtctc ttca 354134118PRTArtificial SequenceSynthetic 134Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Lys Pro Lys Ser Gly Gly Thr Asn Tyr Val Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr65 70 75 80 Met Glu Leu Asn Asn Leu Lys Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Gly Ser Gln Asp Ala Ser Asp Phe Trp Gly Gln Gly Thr 100 105 110 Met Val Thr Val Ser Ser 115 135321DNAArtificial SequenceSynthetic 135gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggagg cagagtcacc 60atcacttgtc gggcgagtca ggatattagc atctggttag cctggtatca acagaaacca 120gggaaagccc ctaacctcct gatctatgct gcatccagtt tacaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttacta ttgtcaacag gctgacagtt tcccgctcac tttcggcggt 300gggaccaagg tggagatcaa a 321136107PRTArtificial SequenceSynthetic 136Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Gly Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ile Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asp Ser Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 137378DNAArtificial SequenceSynthetic 137caggtgcagc tggtggagtc tgggggagcc ttggtaaagc ctggggggtc ccttagactc 60tcctgtgcag tctctggatt cactttcatt aatgactgga tgaactgggt ccgccaggct 120ccagggaagg ggctggaatg ggttggccgt attaaaagca caactgatgg tgggacaaca 180gacgacgctg cacccgtgaa aggcagattc accatctcaa gagatgactc aaaaaacacg 240ctatatctgc aaatgaatag cctgaaaacc gaggacacag ccgtgtatta ctgtaccaga 300gataggaacc gacagagaaa ttacttctat gacggtatgg acgtctgggg ccaagggacc 360acggtcaccg tctcctca 378138126PRTArtificial SequenceSynthetic 138Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ile Asn Asp 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Ser Thr Thr Asp Gly Gly Thr Thr Asp Asp Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Asp Arg Asn Arg Gln Arg Asn Tyr Phe Tyr Asp Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 13924DNAArtificial SequenceSynthetic 139ggattcactt tcattaatga ctgg 241408PRTArtificial SequenceSynthetic 140Gly Phe Thr Phe Ile Asn Asp Trp1 5 14130DNAArtificial SequenceSynthetic 141attaaaagca caactgatgg tgggacaaca 3014210PRTArtificial SequenceSynthetic 142Ile Lys Ser Thr Thr Asp Gly Gly Thr Thr 1 5 10 14351DNAArtificial SequenceSynthetic 143accagagata ggaaccgaca gagaaattac ttctatgacg gtatggacgt c 5114417PRTArtificial SequenceSynthetic 144Thr Arg Asp Arg Asn Arg Gln Arg Asn Tyr Phe Tyr Asp Gly Met Asp 1 5 10 15 Val145339DNAArtificial SequenceSynthetic 145gacatccagt tgacccagtc tccagtctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gaggctcctg cataataatg gaaacaacta tttggattgg 120tacctgcaga agccagggca gtctccacaa ctcctgatct atttgggctc taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagatttcac actgaaagtc 240agtagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300tacacttttg gccaggggac caagctggag atcaaacga 339146113PRTArtificial SequenceSynthetic 146Asp Ile Gln Leu Thr Gln Ser Pro Val Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Arg Leu Leu His Asn 20 25 30 Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Val65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg14733DNAArtificial SequenceSynthetic 147cagaggctcc tgcataataa tggaaacaac tat 3314811PRTArtificial SequenceSynthetic 148Gln Arg Leu Leu His Asn Asn Gly Asn Asn Tyr 1 5 10 1499DNAArtificial SequenceSynthetic 149ttgggctct 91503PRTArtificial SequenceSynthetic 150Leu Gly Ser1 15127DNAArtificial SequenceSynthetic 151atgcaagctc tacaaactcc gtacact 271529PRTArtificial SequenceSynthetic 152Met Gln Ala Leu Gln Thr Pro Tyr Thr1 5 153375DNAArtificial SequenceSynthetic 153gaggtgcagc tggtggagtc tgggggagcc ttggtaaagc ctggggggtc ccttagactc 60tcctgtgcag tctctggatt cactttcatt aatgactgga tgaactgggt ccgccaggct 120ccagggaagg ggctggaatg ggttggccgt attaaaagca caactgatgg tgggacaaca 180gacgacgctg cacccgtgaa aggcagattc accatctcaa gagatgactc aaaaaacacg 240ctatatctgc aaatgaatag cctgaaaacc gaggacacag ccgtgtatta ctgtaccaga 300gataggaacc gacagagaaa ttacttctat gacggtatgg acgtctgggg ccaagggacc 360acggtcaccg tctcc 375154125PRTArtificial SequenceSynthetic 154Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ile Asn Asp 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Ser Thr Thr Asp Gly Gly Thr Thr Asp Asp Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Asp Arg Asn Arg Gln Arg Asn Tyr Phe Tyr Asp Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 155336DNAArtificial SequenceSynthetic 155gacatcgtga tgacccagtc tccagtctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gaggctcctg cataataatg gaaacaacta tttggattgg 120tacctgcaga agccagggca gtctccacaa ctcctgatct atttgggctc taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagatttcac actgaaagtc 240agtagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300tacacttttg gccaggggac caagctggag atcaaa 336156112PRTArtificial SequenceSynthetic 156Asp Ile Val Met Thr Gln Ser Pro Val Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Arg Leu Leu His Asn 20 25 30 Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Val65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 157351DNAArtificial SequenceSynthetic 157gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagc agttatgcca tgacctgggt ccgccaggct 120ccagggaagg ggcttgagtg ggtctcaggt gtaagtggaa gaggtggtag tacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240cttcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gtattgggat 300atcagctcgt ttgactactg gggccaggga accctggtca ctgtctcctc a 351158117PRTArtificial SequenceSynthetic 158Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Val Ser Gly Arg Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Tyr Trp Asp Ile Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 15924DNAArtificial SequenceSynthetic 159ggattcacct ttagcagtta tgcc 241608PRTArtificial SequenceSynthetic 160Gly Phe Thr Phe Ser Ser Tyr Ala1 5 16124DNAArtificial SequenceSynthetic 161gtaagtggaa gaggtggtag taca 241628PRTArtificial SequenceSynthetic 162Val Ser Gly Arg Gly Gly Ser Thr1 5 16330DNAArtificial SequenceSynthetic 163gcgtattggg atatcagctc gtttgactac 3016410PRTArtificial SequenceSynthetic 164Ala Tyr Trp Asp Ile Ser Ser Phe Asp Tyr 1 5 10 165324DNAArtificial SequenceSynthetic 165gacatccagt tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgct gggccagtca gggcattagc acttatttag cctggtatca gcaaaaacca 120gggaaaaccc ctaaactcct gatctatgct gcatccactt tgcaaagtgg agtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtcaacag tttaatagtt acccgctcac tttcggcgga 300gggaccaagc tggagatcaa acga 324166108PRTArtificial SequenceSynthetic 166Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Trp Ala Ser Gln Gly Ile Ser Thr Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 16718DNAArtificial SequenceSynthetic 167cagggcatta gcacttat 181686PRTArtificial SequenceSynthetic 168Gln Gly Ile Ser Thr Tyr1 5 1699DNAArtificial SequenceSynthetic 169gctgcatcc 91703PRTArtificial SequenceSynthetic 170Ala Ala Ser1 17127DNAArtificial SequenceSynthetic 171caacagttta atagttaccc gctcact 271729PRTArtificial SequenceSynthetic 172Gln Gln Phe Asn Ser Tyr Pro Leu Thr1 5 173351DNAArtificial SequenceSynthetic 173gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagc agttatgcca tgacctgggt ccgccaggct 120ccagggaagg ggcttgagtg ggtctcaggt gtaagtggaa gaggtggtag tacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240cttcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gtattgggat 300atcagctcgt ttgactactg gggccaggga accctggtca ctgtctcctc a 351174117PRTArtificial SequenceSynthetic 174Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Val Ser Gly Arg Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Tyr Trp Asp Ile Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 175321DNAArtificial SequenceSynthetic 175gacatccagt tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgct gggccagtca gggcattagc acttatttag cctggtatca gcaaaaacca 120gggaaaaccc ctaaactcct gatctatgct gcatccactt tgcaaagtgg agtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtcaacag tttaatagtt acccgctcac tttcggcgga 300gggaccaagg tggagatcaa a 321176107PRTArtificial SequenceSynthetic 176Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Trp Ala Ser Gln Gly Ile Ser Thr Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Thr Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 177369DNAArtificial SequenceSynthetic 177gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggagatc cctgagactc 60tcctgtgtgg cctctggatt caccttcagt aactatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggaatg ggtggcagtt atatcatatg atggaaataa tgatttgtat 180tcagactccg tgaagggccg attcaccatc tccagagaca atgccaagaa cgcgctgtct 240ctgcaaatga ccagcctgag agctgaggac acggctgtct attactgtgc gagagatagt 300acgatgactc cctactacta ccacggtata gacgtctggg gccaagggac cacggtcacc 360gtctcctca 369178123PRTArtificial SequenceSynthetic 178Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met His Trp Val

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Asn Asn Asp Leu Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ala Leu Ser65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Thr Met Thr Pro Tyr Tyr Tyr His Gly Ile Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 17924DNAArtificial SequenceSynthetic 179ggattcacct tcagtaacta tggc 241808PRTArtificial SequenceSynthetic 180Gly Phe Thr Phe Ser Asn Tyr Gly1 5 18124DNAArtificial SequenceSynthetic 181atatcatatg atggaaataa tgat 241828PRTArtificial SequenceSynthetic 182Ile Ser Tyr Asp Gly Asn Asn Asp1 5 18348DNAArtificial SequenceSynthetic 183gcgagagata gtacgatgac tccctactac taccacggta tagacgtc 4818416PRTArtificial SequenceSynthetic 184Ala Arg Asp Ser Thr Met Thr Pro Tyr Tyr Tyr His Gly Ile Asp Val 1 5 10 15 185324DNAArtificial SequenceSynthetic 185gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacattaga aatgatttag gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagac ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacag cataatagtt accctccgac gttcggccaa 300gggaccaagg tggagatcaa acga 324186108PRTArtificial SequenceSynthetic 186Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 18718DNAArtificial SequenceSynthetic 187caggacatta gaaatgat 181886PRTArtificial SequenceSynthetic 188Gln Asp Ile Arg Asn Asp1 5 1899DNAArtificial SequenceSynthetic 189gctgcatcc 91903PRTArtificial SequenceSynthetic 190Ala Ala Ser1 19127DNAArtificial SequenceSynthetic 191ctacagcata atagttaccc tccgacg 271929PRTArtificial SequenceSynthetic 192Leu Gln His Asn Ser Tyr Pro Pro Thr1 5 193366DNAArtificial SequenceSynthetic 193caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggagatc cctgagactc 60tcctgtgtgg cctctggatt caccttcagt aactatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggaatg ggtggcagtt atatcatatg atggaaataa tgatttgtat 180tcagactccg tgaagggccg attcaccatc tccagagaca atgccaagaa cgcgctgtct 240ctgcaaatga ccagcctgag agctgaggac acggctgtct attactgtgc gagagatagt 300acgatgactc cctactacta ccacggtata gacgtctggg gccaagggac cacggtcacc 360gtctcc 366194122PRTArtificial SequenceSynthetic 194Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Asn Asn Asp Leu Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ala Leu Ser65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Thr Met Thr Pro Tyr Tyr Tyr His Gly Ile Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 195321DNAArtificial SequenceSynthetic 195gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca ggacattaga aatgatttag gctggtatca gcagaaacca 120gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagac ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacag cataatagtt accctccgac gttcggccaa 300gggaccaagg tggagatcaa a 321196107PRTArtificial SequenceSynthetic 196Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 197363DNAArtificial SequenceSynthetic 197gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc cctgagactc 60tcctgttcag cctctggatt cacctttagt aaatatgtca tgacctgggt ccgccaggct 120ccagggaagg gactggagtg ggtctcagct attggtccta ctggtcgtac cacggaatac 180gcagactccg tgcagggccg cttcaccatc tccagagaca attccatgaa cacggttttt 240cttcacttga acagtctgac agccgaggac acggccgaat attattgtgc gaagatgttt 300gactggaatt acgacgtgta ctttgactcc tggggccagg gaaccctggt cactgtctcc 360tca 363198121PRTArtificial SequenceSynthetic 198Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Val Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Pro Thr Gly Arg Thr Thr Glu Tyr Ala Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Met Asn Thr Val Phe65 70 75 80 Leu His Leu Asn Ser Leu Thr Ala Glu Asp Thr Ala Glu Tyr Tyr Cys 85 90 95 Ala Lys Met Phe Asp Trp Asn Tyr Asp Val Tyr Phe Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 19924DNAArtificial SequenceSynthetic 199ggattcacct ttagtaaata tgtc 242008PRTArtificial SequenceSynthetic 200Gly Phe Thr Phe Ser Lys Tyr Val1 5 20124DNAArtificial SequenceSynthetic 201attggtccta ctggtcgtac cacg 242028PRTArtificial SequenceSynthetic 202Ile Gly Pro Thr Gly Arg Thr Thr1 5 20342DNAArtificial SequenceSynthetic 203gcgaagatgt ttgactggaa ttacgacgtg tactttgact cc 4220414PRTArtificial SequenceSynthetic 204Ala Lys Met Phe Asp Trp Asn Tyr Asp Val Tyr Phe Asp Ser 1 5 10 205321DNAArtificial SequenceSynthetic 205gacatccaga tgacccagtc tccctccatc ttgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc ggaccagtca gagtattagt aattggttgg cctggtatca gcagaaacca 120gggaaagccc ctaacctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgtcaacag tattatagat attggacgtt cggccaaggg 300accaaggtgg agatcaaacg a 321206107PRTArtificial SequenceSynthetic 206Asp Ile Gln Met Thr Gln Ser Pro Ser Ile Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Arg Tyr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 20718DNAArtificial SequenceSynthetic 207cagagtatta gtaattgg 182086PRTArtificial SequenceSynthetic 208Gln Ser Ile Ser Asn Trp1 5 2099DNAArtificial SequenceSynthetic 209aaggcgtct 92103PRTArtificial SequenceSynthetic 210Lys Ala Ser1 21124DNAArtificial SequenceSynthetic 211caacagtatt atagatattg gacg 242128PRTArtificial SequenceSynthetic 212Gln Gln Tyr Tyr Arg Tyr Trp Thr1 5 213363DNAArtificial SequenceSynthetic 213gaggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc cctgagactc 60tcctgttcag cctctggatt cacctttagt aaatatgtca tgacctgggt ccgccaggct 120ccagggaagg gactggagtg ggtctcagct attggtccta ctggtcgtac cacggaatac 180gcagactccg tgcagggccg cttcaccatc tccagagaca attccatgaa cacggttttt 240cttcacttga acagtctgac agccgaggac acggccgaat attattgtgc gaagatgttt 300gactggaatt acgacgtgta ctttgactcc tggggccagg gaaccctggt caccgtctcc 360tca 363214121PRTArtificial SequenceSynthetic 214Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Val Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Gly Pro Thr Gly Arg Thr Thr Glu Tyr Ala Asp Ser Val 50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Met Asn Thr Val Phe65 70 75 80 Leu His Leu Asn Ser Leu Thr Ala Glu Asp Thr Ala Glu Tyr Tyr Cys 85 90 95 Ala Lys Met Phe Asp Trp Asn Tyr Asp Val Tyr Phe Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 215318DNAArtificial SequenceSynthetic 215gacatccaga tgacccagtc tccctccatc ttgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc ggaccagtca gagtattagt aattggttgg cctggtatca gcagaaacca 120gggaaagccc ctaacctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgtcaacag tattatagat attggacgtt cggccaaggg 300accaaggtgg agatcaaa 318216106PRTArtificial SequenceSynthetic 216Asp Ile Gln Met Thr Gln Ser Pro Ser Ile Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Arg Tyr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 217369DNAArtificial SequenceSynthetic 217caggttcagc tggtgcagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagt aattatgtca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagtt ataagtggta gtggcggtag cacacactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaaaga cgcgctgtat 240ctacaaatga acagcctgag agccgaggac acggccgttt attactgtgc gaaaggcccg 300ctggaggctt actactacta caacggtatg gacgtctggg gccaagggac cacggtcacc 360gtctcctca 369218123PRTArtificial SequenceSynthetic 218Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Val Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Ala Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Pro Leu Glu Ala Tyr Tyr Tyr Tyr Asn Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 21924DNAArtificial SequenceSynthetic 219ggattcacct ttagtaatta tgtc 242208PRTArtificial SequenceSynthetic 220Gly Phe Thr Phe Ser Asn Tyr Val1 5 22124DNAArtificial SequenceSynthetic 221ataagtggta gtggcggtag caca 242228PRTArtificial SequenceSynthetic 222Ile Ser Gly Ser Gly Gly Ser Thr1 5 22348DNAArtificial SequenceSynthetic 223gcgaaaggcc cgctggaggc ttactactac tacaacggta tggacgtc 4822416PRTArtificial SequenceSynthetic 224Ala Lys Gly Pro Leu Glu Ala Tyr Tyr Tyr Tyr Asn Gly Met Asp Val 1 5 10 15 225321DNAArtificial SequenceSynthetic 225gccatccaga tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgct gggccagtca ggccattagc agttatttag cctggtatca gcacaaacca 120gggagagccc ctaagctcct gatctattct acatccactt tgcaaagtgg agtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtcaacag cttaatagtt accccacctt cggccaaggg 300accaagctgg agatcaaacg a 321226107PRTArtificial SequenceSynthetic 226 Ala Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Trp Ala Ser Gln Ala Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln His Lys Pro Gly Arg Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 22718DNAArtificial SequenceSynthetic 227caggccatta gcagttat 182286PRTArtificial SequenceSynthetic 228Gln Ala Ile Ser Ser Tyr1 5 2299DNAArtificial SequenceSynthetic 229tctacatcc 92303PRTArtificial SequenceSynthetic 230Ser Thr Ser1 23124DNAArtificial SequenceSynthetic 231caacagctta atagttaccc cacc 242328PRTArtificial SequenceSynthetic 232Gln Gln Leu Asn Ser Tyr Pro Thr1 5 233369DNAArtificial SequenceSynthetic 233gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagt aattatgtca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagtt ataagtggta gtggcggtag cacacactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaaaga cgcgctgtat 240ctacaaatga acagcctgag agccgaggac acggccgttt attactgtgc gaaaggcccg 300ctggaggctt actactacta caacggtatg gacgtctggg gccaagggac cacggtcacc 360gtctcctca

369234123PRTArtificial SequenceSynthetic 234Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Val Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Ala Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Pro Leu Glu Ala Tyr Tyr Tyr Tyr Asn Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 235318DNAArtificial SequenceSynthetic 235gacatccagt tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgct gggccagtca ggccattagc agttatttag cctggtatca gcacaaacca 120gggagagccc ctaagctcct gatctattct acatccactt tgcaaagtgg agtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtcaacag cttaatagtt accccacctt cggccaaggg 300accaagctgg agatcaaa 318236106PRTArtificial SequenceSynthetic 236Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Trp Ala Ser Gln Ala Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln His Lys Pro Gly Arg Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 2378PRTArtificial SequenceSynthetic 237Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 2388PRTArtificial SequenceSynthetic 238Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 23916PRTArtificial SequenceSynthetic 239Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 24012PRTArtificial SequenceSynthetic 240Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 2413PRTArtificial SequenceSynthetic 241Xaa Xaa Xaa1 2429PRTArtificial SequenceSynthetic 242Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 243756DNAHomo sapiens 243atggccgagg atctgggact gagctttggg gaaacagcca gtgtggaaat gctgccagag 60cacggcagct gcaggcccaa ggccaggagc agcagcgcac gctgggctct cacctgctgc 120ctggtgttgc tccccttcct tgcaggactc accacatacc tgcttgtcag ccagctccgg 180gcccagggag aggcctgtgt gcagttccag gctctaaaag gacaggagtt tgcaccttca 240catcagcaag tttatgcacc tcttagagca gacggagata agccaagggc acacctgaca 300gttgtgagac aaactcccac acagcacttt aaaaatcagt tcccagctct gcactgggaa 360catgaactag gcctggcctt caccaagaac cgaatgaact ataccaacaa attcctgctg 420atcccagagt cgggagacta cttcatttac tcccaggtca cattccgtgg gatgacctct 480gagtgcagtg aaatcagaca agcaggccga ccaaacaagc cagactccat cactgtggtc 540atcaccaagg taacagacag ctaccctgag ccaacccagc tcctcatggg gaccaagtct 600gtatgcgaag taggtagcaa ctggttccag cccatctacc tcggagccat gttctccttg 660caagaagggg acaagctaat ggtgaacgtc agtgacatct ctttggtgga ttacacaaaa 720gaagataaaa ccttctttgg agccttctta ctatag 756244251PRTHomo sapiens 244Met Ala Glu Asp Leu Gly Leu Ser Phe Gly Glu Thr Ala Ser Val Glu 1 5 10 15 Met Leu Pro Glu His Gly Ser Cys Arg Pro Lys Ala Arg Ser Ser Ser 20 25 30 Ala Arg Trp Ala Leu Thr Cys Cys Leu Val Leu Leu Pro Phe Leu Ala 35 40 45 Gly Leu Thr Thr Tyr Leu Leu Val Ser Gln Leu Arg Ala Gln Gly Glu 50 55 60 Ala Cys Val Gln Phe Gln Ala Leu Lys Gly Gln Glu Phe Ala Pro Ser65 70 75 80 His Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg 85 90 95 Ala His Leu Thr Val Val Arg Gln Thr Pro Thr Gln His Phe Lys Asn 100 105 110 Gln Phe Pro Ala Leu His Trp Glu His Glu Leu Gly Leu Ala Phe Thr 115 120 125 Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe Leu Leu Ile Pro Glu Ser 130 135 140 Gly Asp Tyr Phe Ile Tyr Ser Gln Val Thr Phe Arg Gly Met Thr Ser145 150 155 160 Glu Cys Ser Glu Ile Arg Gln Ala Gly Arg Pro Asn Lys Pro Asp Ser 165 170 175 Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser Tyr Pro Glu Pro Thr 180 185 190 Gln Leu Leu Met Gly Thr Lys Ser Val Cys Glu Val Gly Ser Asn Trp 195 200 205 Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu Gln Glu Gly Asp 210 215 220 Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp Tyr Thr Lys225 230 235 240 Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 245 250 245756DNAHomo sapiens 245atggccgagg atctgggact gagctttggg gaaacagcca gtgtggaaat gctgccagag 60cacggcagct gcaggcccaa ggccaggagc agcagcgcac gctgggctct cacctgctgc 120ctggtgttgc tccccttcct tgcaggactc accacatacc tgcttgtcag ccagctccgg 180gcccagggag aggcctgtgt gcagttccag gctctaaaag gacaggagtt tgcaccttca 240catcagcaag tttatgcacc tcttagagca gacggagata agccaagggc acacctgaca 300gttgtgagac aaactcccac acagcacttt aaaaatcagt tcccagctct gcactgggaa 360catgaactag gcctggcctt caccaagaac cgaatgaact ataccaacaa attcctgctg 420atcccagagt cgggagacta cttcatttac tcccaggtca cattccgtgg gatgacctct 480gagtgcagtg aaatcagacg agcaggccga ccaaacaagc cagactccat cactgtggtc 540atcaccaagg taacagacag ctaccctgag ccaacccagc tcctcatggg gaccaagtct 600gtatgcgaag taggtagcaa ctggttccag cccatctacc tcggagccat gttctccttg 660caagaagggg acaagctaat ggtgaacgtc agtgacatct ctttggtgga ttacacaaaa 720gaagataaaa ccttctttgg agccttctta ctatag 756246251PRTHomo sapiens 246Met Ala Glu Asp Leu Gly Leu Ser Phe Gly Glu Thr Ala Ser Val Glu 1 5 10 15 Met Leu Pro Glu His Gly Ser Cys Arg Pro Lys Ala Arg Ser Ser Ser 20 25 30 Ala Arg Trp Ala Leu Thr Cys Cys Leu Val Leu Leu Pro Phe Leu Ala 35 40 45 Gly Leu Thr Thr Tyr Leu Leu Val Ser Gln Leu Arg Ala Gln Gly Glu 50 55 60 Ala Cys Val Gln Phe Gln Ala Leu Lys Gly Gln Glu Phe Ala Pro Ser65 70 75 80 His Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg 85 90 95 Ala His Leu Thr Val Val Arg Gln Thr Pro Thr Gln His Phe Lys Asn 100 105 110 Gln Phe Pro Ala Leu His Trp Glu His Glu Leu Gly Leu Ala Phe Thr 115 120 125 Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe Leu Leu Ile Pro Glu Ser 130 135 140 Gly Asp Tyr Phe Ile Tyr Ser Gln Val Thr Phe Arg Gly Met Thr Ser145 150 155 160 Glu Cys Ser Glu Ile Arg Arg Ala Gly Arg Pro Asn Lys Pro Asp Ser 165 170 175 Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser Tyr Pro Glu Pro Thr 180 185 190 Gln Leu Leu Met Gly Thr Lys Ser Val Cys Glu Val Gly Ser Asn Trp 195 200 205 Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu Gln Glu Gly Asp 210 215 220 Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp Tyr Thr Lys225 230 235 240 Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 245 250 247756DNAMacaca fascicularis 247atggccgagg atctgggact gagctttggg gagacagcca gtgtggaaat gctgccagag 60cacggcagct gcaggcccaa ggccaggagc agcagcgcat gctgggctct cacctgctgc 120ctggtgttgc tccccttcct tgcagggctc accacctacc tgcttgtcag ccagctccgg 180gcccaaggag aggcctgtgt gcagctccag gatctaaaag gacaggagtt tgcaccttca 240catcagcaag tttatgcacc tcttagagca gatggagata agccaagggc acacctgaca 300gttgtgagac aaactcccac acagcactta aaaaatcagt tcccagctct gcactgggaa 360catgaactag gcctggcctt caccaagaac cgaatgaact acaccaacaa attcctgctg 420atcccagagt cgggagacta cttcgtttac tcccaggtca cattccgtgg gatgacctct 480gagtgcagtg aaatcagaca agcaggccga ccaaacaagc cagactccat cactgtggtc 540atcaccaagg taacagacag ctaccctgag ccaacccagc tcctcatggg gaccaagtct 600gtgtgtgaag taggcagtaa ctggttccag cccatctacc tcggagccat gttctccttg 660caagaagggg acaagctcat ggtgaacgtc agtgacatct ctttggtgga ttacacaaaa 720gaagataaaa ccttctttgg agccttctta ctatag 756248251PRTMacaca fascicularis 248Met Ala Glu Asp Leu Gly Leu Ser Phe Gly Glu Thr Ala Ser Val Glu 1 5 10 15 Met Leu Pro Glu His Gly Ser Cys Arg Pro Lys Ala Arg Ser Ser Ser 20 25 30 Ala Cys Trp Ala Leu Thr Cys Cys Leu Val Leu Leu Pro Phe Leu Ala 35 40 45 Gly Leu Thr Thr Tyr Leu Leu Val Ser Gln Leu Arg Ala Gln Gly Glu 50 55 60 Ala Cys Val Gln Leu Gln Asp Leu Lys Gly Gln Glu Phe Ala Pro Ser65 70 75 80 His Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg 85 90 95 Ala His Leu Thr Val Val Arg Gln Thr Pro Thr Gln His Leu Lys Asn 100 105 110 Gln Phe Pro Ala Leu His Trp Glu His Glu Leu Gly Leu Ala Phe Thr 115 120 125 Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe Leu Leu Ile Pro Glu Ser 130 135 140 Gly Asp Tyr Phe Val Tyr Ser Gln Val Thr Phe Arg Gly Met Thr Ser145 150 155 160 Glu Cys Ser Glu Ile Arg Gln Ala Gly Arg Pro Asn Lys Pro Asp Ser 165 170 175 Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser Tyr Pro Glu Pro Thr 180 185 190 Gln Leu Leu Met Gly Thr Lys Ser Val Cys Glu Val Gly Ser Asn Trp 195 200 205 Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu Gln Glu Gly Asp 210 215 220 Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp Tyr Thr Lys225 230 235 240 Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 245 250 249759DNAMus musculus 249atggcagagg agctggggtt gggcttcgga gaaggagtcc cagtggaagt gctgccggaa 60ggctgtagac acaggccaga ggccagggcc gggctagctg ccaggagcaa agcctgcctg 120gctctcacct gctgcctgtt gtcatttccc atcctcgcag gacttagcac cctcctaatg 180gctggccagc tccgggtccc cggaaaagac tgtatgcttc gggccataac agaagagaga 240tctgagcctt caccacagca agtttactca cctcccagag gcaagccgag agcacacctg 300acaattaaga aacaaacccc agcaccacat ctgaaaaatc agctctctgc tctacactgg 360gaacatgacc tagggatggc cttcaccaag aacgggatga agtacatcaa caaatccctg 420gtgatcccag agtcaggaga ctatttcatc tactcccaga tcacattccg agggaccaca 480tctgtgtgtg gtgacatcag tcgggggaga cgaccaaaca agccagactc catcaccatg 540gttatcacca aggtagcaga cagctaccct gagcctgccc gcctactaac agggtccaag 600tctgtgtgtg aaataagcaa caactggttc cagtccctct accttggggc cacgttctcc 660ttggaagaag gagacagact aatggtaaac gtcagtgaca tctccttggt ggattacaca 720aaagaagata aaactttctt tggagctttc ttgctataa 759250252PRTMus musculus 250Met Ala Glu Glu Leu Gly Leu Gly Phe Gly Glu Gly Val Pro Val Glu 1 5 10 15 Val Leu Pro Glu Gly Cys Arg His Arg Pro Glu Ala Arg Ala Gly Leu 20 25 30 Ala Ala Arg Ser Lys Ala Cys Leu Ala Leu Thr Cys Cys Leu Leu Ser 35 40 45 Phe Pro Ile Leu Ala Gly Leu Ser Thr Leu Leu Met Ala Gly Gln Leu 50 55 60 Arg Val Pro Gly Lys Asp Cys Met Leu Arg Ala Ile Thr Glu Glu Arg65 70 75 80 Ser Glu Pro Ser Pro Gln Gln Val Tyr Ser Pro Pro Arg Gly Lys Pro 85 90 95 Arg Ala His Leu Thr Ile Lys Lys Gln Thr Pro Ala Pro His Leu Lys 100 105 110 Asn Gln Leu Ser Ala Leu His Trp Glu His Asp Leu Gly Met Ala Phe 115 120 125 Thr Lys Asn Gly Met Lys Tyr Ile Asn Lys Ser Leu Val Ile Pro Glu 130 135 140 Ser Gly Asp Tyr Phe Ile Tyr Ser Gln Ile Thr Phe Arg Gly Thr Thr145 150 155 160 Ser Val Cys Gly Asp Ile Ser Arg Gly Arg Arg Pro Asn Lys Pro Asp 165 170 175 Ser Ile Thr Met Val Ile Thr Lys Val Ala Asp Ser Tyr Pro Glu Pro 180 185 190 Ala Arg Leu Leu Thr Gly Ser Lys Ser Val Cys Glu Ile Ser Asn Asn 195 200 205 Trp Phe Gln Ser Leu Tyr Leu Gly Ala Thr Phe Ser Leu Glu Glu Gly 210 215 220 Asp Arg Leu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp Tyr Thr225 230 235 240 Lys Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 245 250 2511254DNAHomo sapiens 251atggagcagc ggccgcgggg ctgcgcggcg gtggcggcgg cgctcctcct ggtgctgctg 60ggggcccggg cccagggcgg cactcgtagc cccaggtgtg actgtgccgg tgacttccac 120aagaagattg gtctgttttg ttgcagaggc tgcccagcgg ggcactacct gaaggcccct 180tgcacggagc cctgcggcaa ctccacctgc cttgtgtgtc cccaagacac cttcttggcc 240tgggagaacc accataattc tgaatgtgcc cgctgccagg cctgtgatga gcaggcctcc 300caggtggcgc tggagaactg ttcagcagtg gccgacaccc gctgtggctg taagccaggc 360tggtttgtgg agtgccaggt cagccaatgt gtcagcagtt cacccttcta ctgccaacca 420tgcctagact gcggggccct gcaccgccac acacggctac tctgttcccg cagagatact 480gactgtggga cctgcctgcc tggcttctat gaacatggcg atggctgcgt gtcctgcccc 540acgagcaccc tggggagctg tccagagcgc tgtgccgctg tctgtggctg gaggcagatg 600ttctgggtcc aggtgctcct ggctggcctt gtggtccccc tcctgcttgg ggccaccctg 660acctacacat accgccactg ctggcctcac aagcccctgg ttactgcaga tgaagctggg 720atggaggctc tgaccccacc accggccacc catctgtcac ccttggacag cgcccacacc 780cttctagcac ctcctgacag cagtgagaag atctgcaccg tccagttggt gggtaacagc 840tggacccctg gctaccccga gacccaggag gcgctctgcc cgcaggtgac atggtcctgg 900gaccagttgc ccagcagagc tcttggcccc gctgctgcgc ccacactctc gccagagtcc 960ccagccggct cgccagccat gatgctgcag ccgggcccgc agctctacga cgtgatggac 1020gcggtcccag cgcggcgctg gaaggagttc gtgcgcacgc tggggctgcg cgaggcagag 1080atcgaagccg tggaggtgga gatcggccgc ttccgagacc agcagtacga gatgctcaag 1140cgctggcgcc agcagcagcc cgcgggcctc ggagccgttt acgcggccct ggagcgcatg 1200gggctggacg gctgcgtgga agacttgcgc agccgcctgc agcgcggccc gtga 1254252417PRTHomo sapiens 252Met Glu Gln Arg Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu Leu 1 5 10 15 Leu Val Leu Leu Gly Ala Arg Ala Gln Gly Gly Thr Arg Ser Pro Arg 20 25 30 Cys Asp Cys Ala Gly Asp Phe His Lys Lys Ile Gly Leu Phe Cys Cys 35 40 45 Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro Cys Thr Glu Pro 50 55 60 Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gln Asp Thr Phe Leu Ala65 70 75 80 Trp Glu Asn His His Asn Ser Glu Cys Ala Arg Cys Gln Ala Cys Asp 85 90 95 Glu Gln Ala Ser Gln Val Ala Leu Glu Asn Cys Ser Ala Val Ala Asp 100 105 110 Thr Arg Cys Gly Cys Lys Pro Gly Trp Phe Val Glu Cys Gln Val Ser 115 120 125 Gln Cys Val Ser Ser Ser Pro Phe Tyr Cys Gln Pro Cys Leu Asp Cys 130 135 140 Gly Ala Leu His Arg His Thr Arg Leu Leu Cys Ser Arg Arg Asp Thr145 150 155 160 Asp Cys Gly Thr Cys Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys 165 170 175 Val Ser Cys Pro Thr Ser Thr Leu Gly Ser Cys Pro Glu Arg Cys Ala 180 185 190 Ala Val Cys Gly Trp Arg Gln Met Phe Trp Val Gln Val Leu Leu

Ala 195 200 205 Gly Leu Val Val Pro Leu Leu Leu Gly Ala Thr Leu Thr Tyr Thr Tyr 210 215 220 Arg His Cys Trp Pro His Lys Pro Leu Val Thr Ala Asp Glu Ala Gly225 230 235 240 Met Glu Ala Leu Thr Pro Pro Pro Ala Thr His Leu Ser Pro Leu Asp 245 250 255 Ser Ala His Thr Leu Leu Ala Pro Pro Asp Ser Ser Glu Lys Ile Cys 260 265 270 Thr Val Gln Leu Val Gly Asn Ser Trp Thr Pro Gly Tyr Pro Glu Thr 275 280 285 Gln Glu Ala Leu Cys Pro Gln Val Thr Trp Ser Trp Asp Gln Leu Pro 290 295 300 Ser Arg Ala Leu Gly Pro Ala Ala Ala Pro Thr Leu Ser Pro Glu Ser305 310 315 320 Pro Ala Gly Ser Pro Ala Met Met Leu Gln Pro Gly Pro Gln Leu Tyr 325 330 335 Asp Val Met Asp Ala Val Pro Ala Arg Arg Trp Lys Glu Phe Val Arg 340 345 350 Thr Leu Gly Leu Arg Glu Ala Glu Ile Glu Ala Val Glu Val Glu Ile 355 360 365 Gly Arg Phe Arg Asp Gln Gln Tyr Glu Met Leu Lys Arg Trp Arg Gln 370 375 380 Gln Gln Pro Ala Gly Leu Gly Ala Val Tyr Ala Ala Leu Glu Arg Met385 390 395 400 Gly Leu Asp Gly Cys Val Glu Asp Leu Arg Ser Arg Leu Gln Arg Gly 405 410 415 Pro253900DNAHomo sapiens 253atgagggcgc tggaggggcc aggcctgtcg ctgctgtgcc tggtgttggc gctgcctgcc 60ctgctgccgg tgccggctgt acgcggagtg gcagaaacac ccacctaccc ctggcgggac 120gcagagacag gggagcggct ggtgtgcgcc cagtgccccc caggcacctt tgtgcagcgg 180ccgtgccgcc gagacagccc cacgacgtgt ggcccgtgtc caccgcgcca ctacacgcag 240ttctggaact acctggagcg ctgccgctac tgcaacgtcc tctgcgggga gcgtgaggag 300gaggcacggg cttgccacgc cacccacaac cgtgcctgcc gctgccgcac cggcttcttc 360gcgcacgctg gtttctgctt ggagcacgca tcgtgtccac ctggtgccgg cgtgattgcc 420ccgggcaccc ccagccagaa cacgcagtgc cagccgtgcc ccccaggcac cttctcagcc 480agcagctcca gctcagagca gtgccagccc caccgcaact gcacggccct gggcctggcc 540ctcaatgtgc caggctcttc ctcccatgac accctgtgca ccagctgcac tggcttcccc 600ctcagcacca gggtaccagg agctgaggag tgtgagcgtg ccgtcatcga ctttgtggct 660ttccaggaca tctccatcaa gaggctgcag cggctgctgc aggccctcga ggccccggag 720ggctggggtc cgacaccaag ggcgggccgc gcggccttgc agctgaagct gcgtcggcgg 780ctcacggagc tcctgggggc gcaggacggg gcgctgctgg tgcggctgct gcaggcgctg 840cgcgtggcca ggatgcccgg gctggagcgg agcgtccgtg agcgcttcct ccctgtgcac 900254300PRTHomo sapiens 254Met Arg Ala Leu Glu Gly Pro Gly Leu Ser Leu Leu Cys Leu Val Leu 1 5 10 15 Ala Leu Pro Ala Leu Leu Pro Val Pro Ala Val Arg Gly Val Ala Glu 20 25 30 Thr Pro Thr Tyr Pro Trp Arg Asp Ala Glu Thr Gly Glu Arg Leu Val 35 40 45 Cys Ala Gln Cys Pro Pro Gly Thr Phe Val Gln Arg Pro Cys Arg Arg 50 55 60 Asp Ser Pro Thr Thr Cys Gly Pro Cys Pro Pro Arg His Tyr Thr Gln65 70 75 80 Phe Trp Asn Tyr Leu Glu Arg Cys Arg Tyr Cys Asn Val Leu Cys Gly 85 90 95 Glu Arg Glu Glu Glu Ala Arg Ala Cys His Ala Thr His Asn Arg Ala 100 105 110 Cys Arg Cys Arg Thr Gly Phe Phe Ala His Ala Gly Phe Cys Leu Glu 115 120 125 His Ala Ser Cys Pro Pro Gly Ala Gly Val Ile Ala Pro Gly Thr Pro 130 135 140 Ser Gln Asn Thr Gln Cys Gln Pro Cys Pro Pro Gly Thr Phe Ser Ala145 150 155 160 Ser Ser Ser Ser Ser Glu Gln Cys Gln Pro His Arg Asn Cys Thr Ala 165 170 175 Leu Gly Leu Ala Leu Asn Val Pro Gly Ser Ser Ser His Asp Thr Leu 180 185 190 Cys Thr Ser Cys Thr Gly Phe Pro Leu Ser Thr Arg Val Pro Gly Ala 195 200 205 Glu Glu Cys Glu Arg Ala Val Ile Asp Phe Val Ala Phe Gln Asp Ile 210 215 220 Ser Ile Lys Arg Leu Gln Arg Leu Leu Gln Ala Leu Glu Ala Pro Glu225 230 235 240 Gly Trp Gly Pro Thr Pro Arg Ala Gly Arg Ala Ala Leu Gln Leu Lys 245 250 255 Leu Arg Arg Arg Leu Thr Glu Leu Leu Gly Ala Gln Asp Gly Ala Leu 260 265 270 Leu Val Arg Leu Leu Gln Ala Leu Arg Val Ala Arg Met Pro Gly Leu 275 280 285 Glu Arg Ser Val Arg Glu Arg Phe Leu Pro Val His 290 295 300 255330PRTArtificial SequenceSynthetic 255Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 256327PRTArtificial SequenceSynthetic 256Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 257327PRTArtificial SequenceSynthetic 257Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 258252PRTRattus norvegicus 258Met Ala Glu Glu Leu Gly Leu Gly Phe Gly Glu Ala Val Pro Val Glu 1 5 10 15 Met Leu Pro Glu Gly Cys Arg His Arg Arg Glu Ala Arg Thr Gly Leu 20 25 30 Ala Ala Arg Ser Lys Ala Cys Leu Ala Leu Thr Cys Cys Leu Leu Ser 35 40 45 Phe Pro Ile Leu Ala Gly Leu Ser Thr Leu Leu Met Thr Gly Gln Leu 50 55 60 Arg Ile Pro Gly Lys Asp Cys Met Phe Pro Thr Val Thr Glu Glu Arg65 70 75 80 Ser Ala Pro Ser Ala Gln Pro Val Tyr Thr Pro Ser Arg Asp Lys Pro 85 90 95 Lys Ala His Leu Thr Ile Met Arg Gln Thr Pro Val Pro His Leu Lys 100 105 110 Asn Glu Leu Ala Ala Leu His Trp Glu Asn Asn Leu Gly Met Ala Phe 115 120 125 Thr Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe Leu Val Ile Pro Glu 130 135 140 Ser Gly Asp Tyr Phe Ile Tyr Ser Gln Ile Thr Phe Arg Gly Thr Thr145 150 155 160 Ser Glu Cys Gly Asp Ile Ser Arg Val Arg Arg Pro Lys Lys Pro Asp 165 170 175 Ser Ile Thr Val Val Ile Thr Lys Val Ala Asp Ser Tyr Pro Glu Pro 180 185 190 Ala His Leu Leu Thr Gly Thr Lys Ser Val Cys Glu Ile Ser Ser Asn 195 200 205 Trp Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu Glu Glu Gly 210 215 220 Asp Arg Leu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp Tyr Thr225 230 235 240 Lys Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Ile 245 250 259411PRTMus musculus 259Met Glu Ala Arg Leu Leu Arg Gly Cys Val Val Glu Pro Leu Phe Leu 1 5 10 15 Pro Leu Leu Leu Leu Leu Leu Leu Leu Leu Gly Gly Gln Gly Gln Gly 20 25 30 Gly Met Ser Gly Arg Cys Asp Cys Ala Ser Glu Ser Gln Lys Arg Tyr 35 40 45 Gly Pro Phe Cys Cys Arg Gly Cys Pro Lys Gly His Tyr Met Lys Ala 50 55 60 Pro Cys Ala Glu Pro Cys Gly Asn Ser Thr Cys Leu Pro Cys Pro Ser65 70 75 80 Asp Thr Phe Leu Thr Arg Asp Asn His Phe Lys Thr Asp Cys Thr Arg 85 90 95 Cys Gln Val Cys Asp Glu Glu Ala Leu Gln Val Thr Leu Glu Asn Cys 100 105 110 Ser Ala Lys Ser Asp Thr His Cys Gly Cys Gln Ser Gly Trp Cys Val 115 120 125 Asp Cys Ser Thr Glu Pro Cys Gly Lys Ser Ser Pro Phe Ser Cys Val 130 135 140 Pro Cys Gly Ala Thr Thr Pro Val His Glu Ala Pro Thr Pro Arg Pro145 150 155 160 Cys Leu Pro Gly Phe Tyr Ile Arg Gly Asn Asp Cys Thr Ser Cys Pro 165 170 175 Thr Gly Phe Ser Ser Val Cys Pro Lys Ala Cys Thr Ala Val Cys Gly 180 185 190 Trp Lys Gln Met Phe Trp Val Gln Val Leu Leu Gly Val Ala Phe Leu 195 200 205 Phe Gly Ala Ile Leu Ile Cys Ala Tyr Cys Arg Trp Gln Pro Cys Lys 210 215 220 Ala Val Val Thr Ala Asp Thr Ala Gly Thr Glu Thr Leu Ala Ser Pro225 230 235 240 Gln Thr Ala His Leu Ser Ala Ser Asp Ser Ala His Thr Leu Leu Ala 245 250 255 Pro Pro Ser Ser Thr Gly Lys Ile Cys Thr Thr Val Gln Leu Val Gly 260 265 270 Asn Asn Trp Thr Pro Gly Leu Ser Gln Thr Gln Glu Val Val Cys Gly 275 280 285 Gln Ala Ser Gln Pro Trp Asp Gln Leu Pro Asn Arg Thr Leu Gly Thr 290 295 300 Pro Leu Ala Ser Pro Leu Ser Pro Ala Pro Pro Ala Gly Ser Pro Ala305 310 315 320 Ala Val Leu Gln Pro Gly Pro Gln Leu Tyr Asp Val Met Asp Ala Val 325 330 335 Pro Ala Arg Arg Trp Lys Glu Phe Val Arg Thr Leu Gly Leu Arg Glu 340 345 350 Ala Glu Ile Glu Ala Val Glu Val Glu Ile Cys Arg Phe Arg Asp Gln 355 360 365 Gln Tyr Glu Met Leu Lys Arg Trp Arg Gln Gln Gln Pro Ala Gly Leu 370 375 380 Gly Ala Ile Tyr Ala Ala Leu Glu Arg Met Gly Leu Glu

Gly Cys Ala385 390 395 400 Glu Asp Leu Arg Ser Arg Leu Gln Arg Gly Pro 405 410

Claims

1. A pharmaceutical composition comprising (a) an isolated human antibody or antigen-binding fragment thereof that specifically binds human TNF-like ligand 1A (hTL1A) and neutralizes hTL1A and (b) a pharmaceutically acceptable carrier.

2. The composition of claim 1, wherein the antibody or antigen-binding fragment thereof binds to hTL1A having the amino acid sequence of SEQ ID NO:244 with an equilibrium dissociation constant (KD) of about 700 pM or less, about 300 pM or less, about 80 pM or less, or 50 pM or less.

3. The composition of claim 1, wherein the antibody or antigen-binding fragment cross-reacts with cynomolgus monkey TL1A (MfTL1A).

4. The composition of claim 1, wherein the antibody or antigen-binding fragment comprises a heavy and light chain CDR amino acid sequence combination selected from the group consisting of SEQ ID NOs: 4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48, 52/54/56/60/62/64, 68/70/72/76/78/80, 84/86/88/92/94/96, 100/102/104/108/110/112, 120/122/124/128/130/132, 140/142/144/148/150/152, 160/162/164/168/170/172, 200/202/204/208/210/212, and 220/222/224/228/230/232.

5. The composition of claim 1, wherein the antibody or fragment cross-reacts with Fhm having the amino acid sequence of SEQ ID NO:246.

6. The composition of claim 5, wherein the antibody or antigen-binding fragment comprises a heavy and light chain CDR amino acid sequence combination selected from the group consisting of SEQ ID NOs: 4/6/8/12/14/16, 36/38/40/44/46/48, 68/70/72/76/78/80, 84/86/88/92/94/96, 100/102/104/108/110/112, 120/122/124/128/130/132, 140/142/144/148/150/152, 160/162/164/168/170/172, 180/182/184/188/190/192, and 220/222/224/228/230/232.

7. The composition of claim 1, wherein the antibody or fragment does not cross-react with Fhm having the amino acid sequence of SEQ ID NO: 246.

8. The composition of claim 7, wherein the antibody or antigen-binding fragment comprises a heavy and light chain CDR amino acid sequence combination selected from the group consisting of SEQ ID NOs: 20/22/24/28/30/32, 52/54/56/60/62/64, and 200/202/204/208/210/212.

9. The composition of claim 1, wherein the antibody or antigen-binding fragment comprises a heavy and light chain CDR amino acid sequence combination selected from the group consisting of SEQ ID NOs:4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48, 52/54/56/60/62/64, 68/70/72/76/78/80, 84/86/88/92/94/96, 100/102/104/108/110/112, 120/122/124/128/130/132, 140/142/144/148/150/152, 160/162/164/168/170/172, 180/182/184/188/190/192, 200/202/204/208/210/212, and 220/222/224/228/230/232.

10. The composition of claim 1, further comprising one or more additional therapeutic agents selected from the group consisting of glucocorticoids, cyclosporin, methotrexate, interferon .beta. (IFN-.beta.), tacrolimus, sirolimus, azathioprine, mercaptopurine, opioids, mycophenolate, TNF-binding proteins, infliximab, eternacept, adalimumab, cytotoxic antibiotics, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, antibodies targeting immune cells, anti-CD20 antibodies, anti-CD3 antibodies, corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), aspirin, ibuprofen, naproxen, Cox-2 inhibitors, TNF-.alpha. antagonists, IL-1 antagonists, IL-6 antagonists, acetaminophen, morphinomimetics, antihistamines, glucocorticoids, epinephrine (adrenaline), theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergics, decongestants, and mast cell stabilizers.

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