Optimized anti-TL1A antibody

Abstract

Described herein are humanized anti-TL1A antibodies and pharmaceutical compositions for treating inflammatory bowel disease (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC).

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 662,605, filed April 25, 2018, and U.S. Provisional Application No. 62 / 756,494, filed November 6, 2018, which are incorporated herein by reference. Background Technology

[0003] Inflammatory bowel disease (IBD) is a general term for intestinal disorders that cause inflammation of the gastrointestinal tract. The main types of IBD are ulcerative colitis (UC) and Crohn's disease (CD). These diseases are very common, with approximately 1.86 million people diagnosed with UC and approximately 1.3 million people diagnosed with CD worldwide.

[0004] Each of these forms exhibits a variety of subclinical phenotypes characteristic of severe forms of IBD present in subgroups of CD and UC patients. One such condition is obstructive Crohn's disease, which may result from long-term inflammation that can lead to scarring (fibrous stenosis) or swelling of the intestinal wall. Both outcomes can result in narrowing or obstruction, and are referred to as fibrotic stenosis or inflammatory stenosis. Severe stenosis can lead to bowel obstruction, resulting in abdominal pain, bloating, nausea, and inability to pass stool. As another example, the penetrating disease phenotype, characterized by bowel obstruction or an internal penetrating fistula, or both, often leads to IBD-related complications, including, for example, intraperitoneal sepsis.

[0005] Unfortunately, the number of treatments available for IBD patients is limited, and suboptimal results in clinical trials have hindered the development of new therapies. Existing anti-inflammatory therapies, such as steroids and tumor necrosis factor (TNF) inhibitors, are typically used as first-line treatments for IBD. Regrettably, a significant number of patients do not respond to existing anti-inflammatory therapies, particularly TNF-α inhibitors, or lose their response. When patients are treated with ineffective anti-inflammatory therapies, their condition worsens. For patients who do not respond to first-line therapies, surgery in the form of structureplasty (intestinal reconstruction) or resection (intestinal removal) is the only treatment option. Surgical treatment of IBD is invasive, and it is estimated that approximately one-third of patients undergoing surgery will experience postoperative risks such as anastomotic leakage, infection, and bleeding.

[0006] The pathogenesis of IBD is thought to involve an uncontrolled immune response, which may be triggered by certain environmental factors in genetically susceptible hosts. The heterogeneity of disease pathogenesis and clinical course, coupled with variable responses to treatment and associated side effects, suggests that targeted therapies are the optimal treatment strategy for these diseases. However, there are few targeted therapies for IBD patients, especially those who may not respond to existing IBD therapies (e.g., anti-TNFα inhibitors). Therefore, there is a need for novel therapeutic agents that specifically target the enzymes involved in the pathogenesis of IBD. Summary of the Invention

[0007] This disclosure provides antibodies that can be used to treat IBD, including intermediate to severe forms of IBD characterized by the subclinical phenotypes disclosed herein (e.g., refractory disease, stenotic disease, penetrating disease). The antibodies described herein exhibit superior therapeutic aspects compared to other tumor necrosis factor ligand 1A (TL1A) binding antibodies. First, the antibodies described herein possess high sequence homology with the human germline framework while still exhibiting high binding affinity, high expression levels in bacterial and mammalian cultures, and fewer sequence liabilities, such as deamidation sites, which lead to increased degradation and reduced therapeutic efficacy.

[0008] TL1A and the nucleic acid encoding TL1A (tumor necrosis factor ligand superfamily member 15 (TNFSF15)) were provided from Entrez Gene:9966; UniProtKB:O95150. TL1A is a pro-inflammatory molecule that stimulates the proliferation and effector function of CD8(+) cytotoxic T cells, as well as Th1, Th2, and Th17 cells, in the presence of TCR stimulation. TL1A is thought to participate in the pathogenesis of IBD by modulating adaptive immunity through bridging innate and adaptive immune responses, enhancing the function of Th1, Th2, and Th17 effector cells, and T cell accumulation and immunopathology of inflamed tissues.

[0009] Certain genotypes containing polymorphisms identified at the TNFSF15 gene are associated with, and therefore predict, the risk of, developing IBD (e.g., UC or CD), or a subclinical phenotype of IBD. TL1A mRNA expression is enriched in patients diagnosed with IBD who possess these risk genotypes. Therefore, inhibiting TL1A expression and / or activity is a promising therapeutic strategy in various T-cell dependent autoimmune diseases, including IBD (e.g., UC and CD).

[0010] In one aspect, this document provides an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in any one of SEQ ID NO: 554 to 564 or 574 to 577; and (c) HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 565 to 568 or 578 to 581; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in any one of SEQ ID NO: 569 or 570; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 571 to 573 or 582 to 585. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 1 as shown in SEQ ID NO: 545. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 2 as shown in SEQ ID NO: 546. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 3 as shown in SEQ ID NO: 547 or 586 to 588. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 4 as shown in SEQ ID NO: 548. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 1 as shown in SEQ ID NO: 549. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 2 as shown in SEQ ID NO: 550. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 3 as shown in SEQ ID NO: 551. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 4 as shown in SEQ ID NO: 552.In some embodiments, the antibody or antigen-binding fragment comprises: (a) a human heavy chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545; (b) a human heavy chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 546; (c) a human heavy chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 547 or 586 to 588; (d) a human heavy chain frame region 4 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 548; (e) a human light chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 549; (f) a human light chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 550; and (g) a human light chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545. At least 90%, 95%, 97%, or 98% identical human light chain framework region 3 as shown in SEQ ID NO: 551; and (h) at least 90%, 95%, 97%, or 98% identical human light chain framework region 4 as shown in SEQ ID NO: 552. In some embodiments, as determined by ELISA, the antibody binds to human TL1A with a stronger affinity or twice the affinity of the L8 clone, wherein the L8 clone comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO: 491 and the light chain variable region amino acid sequence shown in SEQ ID NO: 490. In some embodiments, the antibody or antigen-binding fragment is chimeric or humanized. In some embodiments, the antibody or antigen-binding fragment is an IgG antibody. In some embodiments, the antibody or antigen-binding fragment comprises Fab, F(ab)2, single-domain antibody, single-chain variable fragment (scFv), or nanobody. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO: 542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:542. In some embodiments, the antibody or antigen-binding fragment comprises a light chain constant region containing the amino acid sequence shown in SEQ ID NO:544. In some embodiments, the antibody or antigen-binding fragment thereof inhibits TL1A-induced secretion of interferon-γ by T lymphocytes. In some embodiments, the antibody or antigen-binding fragment is a component of a pharmaceutical composition comprising the antibody or antigen-binding fragment and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the antibody or antigen-binding fragment or the pharmaceutical composition is used to treat inflammatory bowel disease, Crohn's disease, or colitis.In some embodiments, the antibody or antigen-binding fragment is encoded by a nucleic acid. In some embodiments, the cell contains nucleic acid. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell. In some embodiments, a method for treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising administering to the individual an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition, wherein the individual is diagnosed with or suspected of having inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is used to prevent or reduce interferon-γ secretion by T lymphocytes. In some embodiments, a method for preventing or reducing interferon-γ secretion by T lymphocytes in an individual is described herein, comprising administering to the individual an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition. In some embodiments, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising incubating cells containing nucleic acids encoding the antibody or antigen-binding fragment in a culture medium under conditions sufficient to allow the antibody or antigen-binding fragment to be secreted into the culture medium. In some embodiments, the method further includes subjecting the culture medium to at least one purification step. In some embodiments, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising mixing the antibody or antigen-binding fragment with a pharmaceutically acceptable excipient, carrier, or diluent.

[0011] On the other hand, this document provides an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 559; and (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 567; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 573. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 1 as shown in SEQ ID NO: 545. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 2 as shown in SEQ ID NO: 546. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 3 as shown in SEQ ID NO: 547 or 586 to 588. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 4 as shown in SEQ ID NO: 548. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 1 as shown in SEQ ID NO: 549. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 2 as shown in SEQ ID NO:550. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 3 as shown in SEQ ID NO:551. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 4 as shown in SEQ ID NO:552.In some embodiments, the antibody or antigen-binding fragment comprises: (a) a human heavy chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545; (b) a human heavy chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 546; (c) a human heavy chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 547 or 586 to 588; (d) a human heavy chain frame region 4 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 548; (e) a human light chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 549; (f) a human light chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 550; and (g) a human light chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545. At least 90%, 95%, 97%, or 98% identical to the human light chain framework region 3 shown in SEQ ID NO:551; and (h) at least 90%, 95%, 97%, or 98% identical to the human light chain framework region 4 shown in SEQ ID NO:552. In some embodiments, the antibody or antigen-binding fragment that specifically binds to TL1A comprises: (a) a heavy chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:503; and (b) a light chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:502. In some embodiments, as determined by ELISA, the antibody binds to human TL1A with a stronger affinity than or twice the affinity of the L8 clone, wherein the L8 clone comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:491 and the light chain variable region amino acid sequence shown in SEQ ID NO:490. In some embodiments, the antibody or antigen-binding fragment is chimeric or humanized. In some embodiments, the antibody or antigen-binding fragment is an IgG antibody. In some embodiments, the antibody or antigen-binding fragment comprises Fab, F(ab)2, a single-domain antibody, a single-chain variable fragment (scFv), or a nanobody. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:542. In some embodiments, the antibody or antigen-binding fragment comprises a light chain constant region containing the amino acid sequence shown in SEQ ID NO:544.In some embodiments, the antibody or its antigen-binding fragment inhibits TL1A-induced secretion of interferon-γ by T lymphocytes. In some embodiments, the antibody or antigen-binding fragment is a component of a pharmaceutical composition comprising the antibody or antigen-binding fragment and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is used to treat inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment is encoded by a nucleic acid. In some embodiments, the cell contains nucleic acid. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell. In some embodiments, a method of treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising administering to the individual an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition, wherein the individual is diagnosed with or suspected of having inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is used to prevent or reduce interferon-γ secretion by T lymphocytes. In some embodiments, a method for preventing or reducing interferon-γ secretion by T lymphocytes in an individual is described herein, comprising administering an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition to the individual. In some embodiments, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising incubating cells containing nucleic acids encoding the antibody or antigen-binding fragment in a culture medium under conditions sufficient to allow secretion of the antibody or antigen-binding fragment into the culture medium. In some embodiments, the method further comprises subjecting the culture medium to at least one purification step. In some embodiments, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising mixing the antibody or antigen-binding fragment with a pharmaceutically acceptable excipient, carrier, or diluent.

[0012] On the other hand, this document provides an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 563; and (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 568; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 572. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 1 as shown in SEQ ID NO: 545. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 2 as shown in SEQ ID NO: 546. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 3 as shown in SEQ ID NO: 547 or 586 to 588. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 4 as shown in SEQ ID NO: 548. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 1 as shown in SEQ ID NO: 549. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 2 as shown in SEQ ID NO:550. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 3 as shown in SEQ ID NO:551. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 4 as shown in SEQ ID NO:552.In some embodiments, the antibody or antigen-binding fragment comprises: (a) a human heavy chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545; (b) a human heavy chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 546; (c) a human heavy chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 547 or 586 to 588; (d) a human heavy chain frame region 4 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 548; (e) a human light chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 549; (f) a human light chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 550; and (g) a human light chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545. At least 90%, 95%, 97%, or 98% identical to the human light chain framework region 3 shown in SEQ ID NO:551; and (h) at least 90%, 95%, 97%, or 98% identical to the human light chain framework region 4 shown in SEQ ID NO:552. In some embodiments, the antibody or antigen-binding fragment that specifically binds to TL1A comprises: (a) a heavy chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:511; and (b) a light chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:510. In some embodiments, as determined by ELISA, the antibody binds to human TL1A with a stronger affinity than or twice the affinity of the L8 clone, wherein the L8 clone comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:491 and the light chain variable region amino acid sequence shown in SEQ ID NO:490. In some embodiments, the antibody or antigen-binding fragment is chimeric or humanized. In some embodiments, the antibody or antigen-binding fragment is an IgG antibody. In some embodiments, the antibody or antigen-binding fragment comprises Fab, F(ab)2, a single-domain antibody, a single-chain variable fragment (scFv), or a nanobody. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:542. In some embodiments, the antibody or antigen-binding fragment comprises a light chain constant region containing the amino acid sequence shown in SEQ ID NO:544.In some embodiments, the antibody or its antigen-binding fragment inhibits TL1A-induced secretion of interferon-γ by T lymphocytes. In some embodiments, the antibody or antigen-binding fragment is a component of a pharmaceutical composition comprising the antibody or antigen-binding fragment and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is used to treat inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment is encoded by a nucleic acid. In some embodiments, the cell contains nucleic acid. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell. In some embodiments, a method of treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising administering to the individual an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition, wherein the individual is diagnosed with or suspected of having inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is used to prevent or reduce interferon-γ secretion by T lymphocytes. In some embodiments, a method for preventing or reducing interferon-γ secretion by T lymphocytes in an individual is described herein, comprising administering an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition to the individual. In some embodiments, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising incubating cells containing nucleic acids encoding the antibody or antigen-binding fragment in a culture medium under conditions sufficient to allow secretion of the antibody or antigen-binding fragment into the culture medium. In some embodiments, the method further comprises subjecting the culture medium to at least one purification step. In some embodiments, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising mixing the antibody or antigen-binding fragment with a pharmaceutically acceptable excipient, carrier, or diluent.

[0013] On the other hand, this document provides an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 555; and (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 566; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 572. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 1 as shown in SEQ ID NO: 545. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 2 as shown in SEQ ID NO: 546. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 3 as shown in SEQ ID NO: 547 or 586 to 588. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 4 as shown in SEQ ID NO: 548. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 1 as shown in SEQ ID NO: 549. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 2 as shown in SEQ ID NO:550. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 3 as shown in SEQ ID NO:551. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 4 as shown in SEQ ID NO:552.In some embodiments, the antibody or antigen-binding fragment comprises: (a) a human heavy chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545; (b) a human heavy chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 546; (c) a human heavy chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 547 or 586 to 588; (d) a human heavy chain frame region 4 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 548; (e) a human light chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 549; (f) a human light chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 550; and (g) a human light chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545. At least 90%, 95%, 97%, or 98% identical to the human light chain framework region 3 shown in SEQ ID NO:551; and (h) at least 90%, 95%, 97%, or 98% identical to the human light chain framework region 4 shown in SEQ ID NO:552. In some embodiments, the antibody or antigen-binding fragment that specifically binds to TL1A comprises: (a) a heavy chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:493; and (b) a light chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:492. In some embodiments, as determined by ELISA, the antibody binds to human TL1A with a stronger affinity than or twice the affinity of the L8 clone, wherein the L8 clone comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:491 and the light chain variable region amino acid sequence shown in SEQ ID NO:490. In some embodiments, the antibody or antigen-binding fragment is chimeric or humanized. In some embodiments, the antibody or antigen-binding fragment is an IgG antibody. In some embodiments, the antibody or antigen-binding fragment comprises Fab, F(ab)2, a single-domain antibody, a single-chain variable fragment (scFv), or a nanobody. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:542. In some embodiments, the antibody or antigen-binding fragment comprises a light chain constant region containing the amino acid sequence shown in SEQ ID NO:544.In some embodiments, the antibody or its antigen-binding fragment inhibits TL1A-induced secretion of interferon-γ by T lymphocytes. In some embodiments, the antibody or antigen-binding fragment is a component of a pharmaceutical composition comprising the antibody or antigen-binding fragment and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is used to treat inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment is encoded by a nucleic acid. In some embodiments, the cell contains nucleic acid. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell. In some embodiments, a method of treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising administering to the individual an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition, wherein the individual is diagnosed with or suspected of having inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is used to prevent or reduce interferon-γ secretion by T lymphocytes. In some embodiments, a method for preventing or reducing interferon-γ secretion by T lymphocytes in an individual is described herein, comprising administering an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition to the individual. In some embodiments, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising incubating cells containing nucleic acids encoding the antibody or antigen-binding fragment in a culture medium under conditions sufficient to allow secretion of the antibody or antigen-binding fragment into the culture medium. In some embodiments, the method further comprises subjecting the culture medium to at least one purification step. In some embodiments, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising mixing the antibody or antigen-binding fragment with a pharmaceutically acceptable excipient, carrier, or diluent.

[0014] On the other hand, this document provides an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 558; and (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 566; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 572. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 1 as shown in SEQ ID NO: 545. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 2 as shown in SEQ ID NO: 546. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 3 as shown in SEQ ID NO: 547 or 586 to 588. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 4 as shown in SEQ ID NO: 548. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 1 as shown in SEQ ID NO: 549. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 2 as shown in SEQ ID NO:550. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 3 as shown in SEQ ID NO:551. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 4 as shown in SEQ ID NO:552.In some embodiments, the antibody or antigen-binding fragment comprises: (a) a human heavy chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545; (b) a human heavy chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 546; (c) a human heavy chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 547 or 586 to 588; (d) a human heavy chain frame region 4 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 548; (e) a human light chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 549; (f) a human light chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 550; and (g) a human light chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545. At least 90%, 95%, 97%, or 98% identical to the human light chain framework region 3 shown in SEQ ID NO:551; and (h) at least 90%, 95%, 97%, or 98% identical to the human light chain framework region 4 shown in SEQ ID NO:552. In some embodiments, the antibody or antigen-binding fragment that specifically binds to TL1A comprises: (a) a heavy chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:501; and (b) a light chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:500. In some embodiments, as determined by ELISA, the antibody binds to human TL1A with a stronger affinity than or twice the affinity of the L8 clone, wherein the L8 clone comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:491 and the light chain variable region amino acid sequence shown in SEQ ID NO:490. In some embodiments, the antibody or antigen-binding fragment is chimeric or humanized. In some embodiments, the antibody or antigen-binding fragment is an IgG antibody. In some embodiments, the antibody or antigen-binding fragment comprises Fab, F(ab)2, a single-domain antibody, a single-chain variable fragment (scFv), or a nanobody. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:542. In some embodiments, the antibody or antigen-binding fragment comprises a light chain constant region containing the amino acid sequence shown in SEQ ID NO:544.In some embodiments, the antibody or its antigen-binding fragment inhibits TL1A-induced interferon-γ secretion by T lymphocytes. In some embodiments, the antibody or antigen-binding fragment is a component of a pharmaceutical composition comprising the antibody or antigen-binding fragment and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is used to treat inflammatory bowel disease. In some embodiments, the antibody or antigen-binding fragment is encoded by a nucleic acid. In some embodiments, the cell contains nucleic acid. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell. In some embodiments, a method of treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising administering to the individual an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition, wherein the individual is diagnosed with or suspected of having inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is used to prevent or reduce interferon-γ secretion by T lymphocytes. In some embodiments, this document describes a method for preventing or reducing the secretion of interferon-γ by T lymphocytes in an individual, comprising administering an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition to the individual. In some embodiments, this document describes a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis, comprising incubating cells containing nucleic acids encoding the antibody or antigen-binding fragment in a culture medium under conditions sufficient to allow the antibody or antigen-binding fragment to be secreted into the culture medium. In some embodiments, the method further comprises subjecting the culture medium to at least one purification step. In some embodiments, this document describes a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis, comprising mixing the antibody or antigen-binding fragment with a pharmaceutically acceptable excipient, carrier, or diluent.

[0015] On the other hand, this document provides an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 564; and (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 568; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 572. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 1 as shown in SEQ ID NO: 545. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 2 as shown in SEQ ID NO: 546. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 3 as shown in SEQ ID NO: 547 or 586 to 588. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human heavy chain framework region 4 as shown in SEQ ID NO: 548. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 1 as shown in SEQ ID NO: 549. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 2 as shown in SEQ ID NO:550. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 3 as shown in SEQ ID NO:551. In some embodiments, the antibody or antigen-binding fragment comprises at least 90%, 95%, 96%, 97%, 98%, and 99% of the same human light chain framework region 4 as shown in SEQ ID NO:552.In some embodiments, the antibody or antigen-binding fragment comprises: (a) a human heavy chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545; (b) a human heavy chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 546; (c) a human heavy chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 547 or 586 to 588; (d) a human heavy chain frame region 4 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 548; (e) a human light chain frame region 1 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 549; (f) a human light chain frame region 2 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 550; and (g) a human light chain frame region 3 that is at least 90%, 95%, 97%, or 98% identical to that shown in SEQ ID NO: 545. At least 90%, 95%, 97%, or 98% identical to the human light chain framework region 3 shown in SEQ ID NO:551; and (h) at least 90%, 95%, 97%, or 98% identical to the human light chain framework region 4 shown in SEQ ID NO:552. In some embodiments, the antibody or antigen-binding fragment that specifically binds to TL1A comprises: (a) a heavy chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:515; and (b) a light chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:514. In some embodiments, as determined by ELISA, the antibody binds to human TL1A with a stronger affinity than or twice the affinity of the L8 clone, wherein the L8 clone comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:491 and the light chain variable region amino acid sequence shown in SEQ ID NO:490. In some embodiments, the antibody or antigen-binding fragment is chimeric or humanized. In some embodiments, the antibody or antigen-binding fragment is an IgG antibody. In some embodiments, the antibody or antigen-binding fragment comprises Fab, F(ab)2, a single-domain antibody, a single-chain variable fragment (scFv), or a nanobody. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:542. In some embodiments, the antibody or antigen-binding fragment comprises a light chain constant region containing the amino acid sequence shown in SEQ ID NO:544.In some embodiments, the antibody or its antigen-binding fragment inhibits TL1A-induced secretion of interferon-γ by T lymphocytes. In some embodiments, the antibody or antigen-binding fragment is a component of a pharmaceutical composition comprising the antibody or antigen-binding fragment and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is used to treat inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment is encoded by a nucleic acid. In some embodiments, the cell contains nucleic acid. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell. In some embodiments, a method of treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising administering to the individual an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition, wherein the individual is diagnosed with or suspected of having inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is used to prevent or reduce interferon-γ secretion by T lymphocytes. In some embodiments, a method for preventing or reducing interferon-γ secretion by T lymphocytes in an individual is described herein, comprising administering an effective amount of the antibody or antigen-binding fragment or pharmaceutical composition to the individual. In some embodiments, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising incubating cells containing nucleic acids encoding the antibody or antigen-binding fragment in a culture medium under conditions sufficient to allow secretion of the antibody or antigen-binding fragment into the culture medium. In some embodiments, the method further comprises subjecting the culture medium to at least one purification step. In some embodiments, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising mixing the antibody or antigen-binding fragment with a pharmaceutically acceptable excipient, carrier, or diluent.

[0016] On the other hand, this article provides an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: (a) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 from any one of SEQ ID NO: 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, or 541; and (b) a light ... LCDR1, LCDR2, and LCDR3 of any one of NO: 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, or 540; wherein the CDR is defined by the Kabat, Chothia, or IMGT method or a combination thereof. In some embodiments, the antibody or antigen-binding fragment that specifically binds to TL1A comprises: (a) a heavy chain variable region comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, or 541; and (b) a light chain variable region comprising the same amino acid sequence as ... The amino acid sequences of at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, or 540 are specified. In some embodiments, as determined by ELISA, the antibody binds to human TL1A with a stronger affinity or twice the affinity of the L8 clone, wherein the L8 clone comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO: 491 and the light chain variable region amino acid sequence shown in SEQ ID NO: 490. In some embodiments, the antibody or antigen-binding fragment is chimeric or humanized. In some embodiments, the antibody or antigen-binding fragment is an IgG antibody. In some embodiments, the antibody or antigen-binding fragment comprises Fab, F(ab)2, single-domain antibodies, single-chain variable fragments (scFv), or nanobodies.In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO: 542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO: 542. In some embodiments, the antibody or antigen-binding fragment comprises a light chain constant region containing the amino acid sequence shown in SEQ ID NO: 544. In some embodiments, the antibody or antigen-binding fragment is a component of a pharmaceutical composition comprising the antibody or antigen-binding fragment and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0017] On the other hand, this document describes a method for treating a disease or condition in an individual having a risk variant associated with that disease or condition, the method comprising administering an effective amount of an antibody or antigen-binding fragment of this disclosure to the individual having the risk variant, wherein the disease or condition includes at least one of inflammatory bowel disease (IBD), Crohn's disease (CD), or colitis. In some embodiments, the individual has multiple risk variants. In some embodiments, the multiple risk variants are at least 3, 4, 5, or 10 risk variants. In some embodiments, the risk variants of the multiple risk variants are associated with a subclinical phenotype of the disease or condition. In some embodiments, the disease or condition is a severe form of at least one of IBD, CD, or colitis. Attached Figure Description

[0018] Exemplary embodiments are illustrated in the accompanying drawings. The embodiments and drawings disclosed herein are intended to be illustrative and not restrictive.

[0019] Figure 1 The results of a filter enhancement assay, performed as a qualitative assessment of chimeric 5C3D11 Fab expression and antigen binding, are depicted. Part A of the filter shows the expression of heavy chain 5C3D11, part B shows the expression of light chain 5C3D11, and part C shows the binding of 5C3D11 Fab to the human TL1A antigen.

[0020] Figure 2 The binding of chimeric 5C3D11 and humanized clone 12835 antibodies to human TL1A was described by enzyme-linked immunosorbent assay (ELISA).

[0021] Figure 3 The results of the capture filter enhancement test are described, which demonstrate the high sensitivity and high binding strength of the chimeric 5C3D11 to human TL1A.

[0022] Figure 4AThe results of the ELISA were depicted, showing the binding of CDR-transplanted antibody clones 18-7, 21-3 and humanized clone 12835 to human TL1A.

[0023] Figure 4B The results of the ELISA were described, showing that the binding of the CDR-transplanted antibody clone L8 to human TL1A was compared to the binding of the humanized clone 12835 to human TL1A.

[0024] Figure 5 The results of the ELISA were described, demonstrating the strong binding of immobilized Fab (chimeric 5C3D11, humanized clone 12835, clone 18-7, clone 21-3 and CDR transplanted clone L8) to soluble human TL1A antigen.

[0025] Figure 6A The results of the ELISA were described, demonstrating that the anti-TL1A antibodies H3-7(V102M)-SEQ ID NO:44, 38, H3-7(V102K)-SEQ ID NO:43, 38, and H3-7(V102Q)-SEQ ID NO:45, 38 and humanized clone 12835 with heavy chain CDR3 mutations had increased affinity for human TL1A compared to the CDR graft (clone L8).

[0026] Figure 6B The results of the ELISA were described, demonstrating that the anti-TL1A antibodies H3-7(V102W)-SEQ ID NO:46, 38 and humanized clone 12835 with heavy chain CDR3 mutations had increased affinity for human TL1A compared with CDR transplanted clone L8.

[0027] Figure 7A The results of the ELISA were described, demonstrating that the anti-TL1A antibodies L3-4(S92D)–SEQ ID NO:47, 40, L3-4(S92E)–SEQ ID NO:48, 40, L3-4(S92H)–SEQ ID NO:49, 40, L3-4(S92N)–SEQ ID NO:50, 40 and humanized clone 12835 with light chain CDR3 mutations had increased affinity for human TL1A compared with CDR transplanted clone L8.

[0028] Figure 7B The results of the ELISA were described, demonstrating that the anti-TL1A antibodies L3-4 (S92Q)-SEQ ID NO:51, 40 and humanized clone 12835 with light chain CDR3 mutations had increased affinity for human TL1A compared with CDR transplanted clone L8.

[0029] Figure 8A , 8B 8C describes an ELISA that demonstrates the binding of Fab, containing the 5C3D11 CDR variant transplanted onto human heavy chain line IGH1-46*02 and human light chain line IGKV3-20*01, to immobilized human TL1A.

[0030] Figure 9A and 9B An ELISA was described, demonstrating the binding of Fab, which includes the 5C3D11 CDR variant transplanted onto human heavy chain line IGH1-3*01 and human light chain line IGKV3-20*01, to immobilized human TL1A.

[0031] Figure 10A and 10B An ELISA was described, demonstrating the binding of immobilized Fab containing the 5C3D11 CDR variant transplanted onto the human heavy chain line IGH1-46*02 and the human light chain line IGKV3-20*01 to soluble biotinylated human TL1A.

[0032] Figure 11A and 11B An ELISA was described, demonstrating the binding of immobilized Fab containing the 5C3D11 CDR variant transplanted onto the human heavy chain line IGH1-46*02 and the human light chain line IGKV3-20*01 to soluble biotinylated human TL1A.

[0033] Figure 12A and 12B An ELISA was described, demonstrating the binding of immobilized Fab containing the 5C3D11 CDR variant transplanted onto the human heavy chain line IGH1-46*02 and the human light chain line IGKV3-20*01 to soluble biotinylated human TL1A.

[0034] Figure 13A and 13B An ELISA was described that demonstrated the binding of immobilized Fab containing the 5C3D11 CDR variant transplanted onto human heavy chain line IGH1-3*01 and human light chain line IGKV3-20*01 to soluble biotinylated human TL1A.

[0035] Figure 14A and 14B The binding of Fab containing the 5C3D11 CDR variant to membrane-associated human TL1A was demonstrated.

[0036] Figure 15A and 15BThe study depicted Fab with TRAIL lack of binding, including the 5C3D11 CDR variant transplanted onto the human heavy chain line IGH1-46*02 and the human light chain line IGKV3-20*01.

[0037] Figure 16A and 16B The Fab-LIGHT lack of binding was described, which includes the 5C3D11 CDR variant transplanted onto the human heavy chain line IGH1-46*02 and the human light chain line IGKV3-20*01.

[0038] Figure 17A and 17B The binding of Fab with Fas deficiency, including the 5C3D11 CDR variant transplanted onto the human heavy chain line IGH1-46*02 and the human light chain line IGKV3-20*01, was described.

[0039] Figure 18 The study depicted Fab with TRAIL lack of binding, including the 5C3D11 CDR variant transplanted onto the human heavy chain line IGH1-3*01 and the human light chain line IGKV3-20*01.

[0040] Figure 19 The Fab-LIGHT lack of binding was described, which includes the 5C3D11 CDR variant transplanted onto the human heavy chain line IGH1-3*01 and the human light chain line IGKV3-20*01.

[0041] Figure 20 The binding of Fab with Fas deficiency, including the 5C3D11 CDR variant transplanted onto human heavy chain line IGH1-3*01 and human light chain line IGKV3-20*01, was described.

[0042] Figure 21A and 21B An ELISA was described that demonstrated the binding of heavy chain and light chain variable regions containing the 5C3D11 CDR variant with either the IgG1 heavy chain (modified) and κ light chain constant region (21A) or the IgG2 heavy chain and κ light chain constant region (21B) to immobilized human TL1A.

[0043] Figure 22A and 22B An ELISA was described that demonstrated the binding of soluble biotinylated human TL1A to immobilized heavy and light chain variable regions containing the 5C3D11 CDR variant, which have either a constant region of IgG1 heavy chain (modified) and κ light chain (22A) or a constant region of IgG2 heavy chain and κ light chain (22B).

[0044] Figure 23It was demonstrated that the binding of heavy chain and light chain variable regions containing the 5C3D11 CDR variant with constant regions of IgG1 heavy chain (modified) and κ light chain or with constant regions of IgG2 heavy chain and κ light chain was maintained with membrane-associated human TL1A.

[0045] Figure 24A , 24B 24C describes an ELISA that demonstrates that heavy chain and light chain variable regions containing the 5C3D11 CDR variant, which have constant regions of IgG1 heavy chain (modified) and κ light chain or IgG2 heavy chain and κ light chain constant regions, lack binding to TNFSF family members Fas (24A), TRAIL (24B), or LIGHT (24C).

[0046] Figure 25 The study demonstrated that a humanized Ig construct inhibited IFN-γ production in whole blood induced by TL1A in cynomolgus monkeys. The humanized Ig construct comprised a 5C3D11CDR variant transplanted onto human heavy chain line IGH1-46*02 and human light chain line IGKV3-20*01, possessing constant regions of IgG1 heavy chain (modified) and κ light chain.

[0047] Figure 26A , 26B Figures 26A and 26C illustrate the antibody-induced inhibition of TL1A-induced IFN-γ production from human whole blood. Results from three different donors (26A), (26B), and (26C) are shown, with antibody concentrations (nanomoles) displayed on the x-axis.

[0048] Figure 27A , 27B Figures 27A and 27C illustrate the antibody-induced inhibition of TL1A-induced IFN-γ production from whole blood of cynomolgus monkeys. Results from three different donors (27A), (27B), and (27C) are shown, with antibody concentrations (nanomoles) displayed on the x-axis. Detailed Implementation

[0049] Tumor necrosis factor-like protein 1A (TL1A) is associated with the development and severity of severe inflammatory bowel disease (IBD)—including severe forms of colitis and Crohn's disease (CD). Furthermore, preclinical and human genetic correlation data suggest that TL1A is a potential therapeutic target for Crohn's disease. This disclosure describes an optimized antibody against TL1A and provides novel therapeutic agents for the treatment of IBD.

[0050] In one aspect, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in any one of SEQ ID NO: 554 to 564 or 574 to 577; and (c) HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 565 to 568 or 578 to 581; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in any one of SEQ ID NO: 569 or 570; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 571 to 573 or 582 to 585.

[0051] On the other hand, this article describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: (a) a heavy chain variable region comprising an amino acid sequence that is at least about 90% identical to any one of SEQ ID NO: 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, or 541; and (b) a light ... NO: Any one of 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, or 540, with at least about 90% identical amino acid sequences.

[0052] In some embodiments, an antibody is an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof through at least one antigen recognition site within the variable region of an immunoglobulin molecule. In some embodiments, antibodies include intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) mutants, CDR-transplanted antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigenic determinant of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, provided that the antibody exhibits the desired biological activity. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated α, δ, ε, γ, and μ, respectively, based on the identity of their heavy chain constant domains. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies can be naked or conjugated to other molecules such as toxins, radioactive isotopes, etc.

[0053] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby creating an Fc region variant. The Fc region as used herein is the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. Fc regions include native sequence Fc regions and variant Fc regions. Fc region variants may contain human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) with amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0054] In some embodiments, the antibodies of this disclosure have reduced antibody-dependent cell-mediated cytotoxicity (ADCC) or reduced complement fixation. This is ideal when inhibition of a target function is required, but activation of downstream immune responses can produce undesirable side effects. Some Fc regions naturally lack effector functions (e.g., IgG2, SEQ ID NO: 543), and some Fc regions may contain mutations that reduce effector function (e.g., modified IgG1, SEQ ID NO: 542). In some embodiments, the antibodies of this disclosure have reduced effector functions. In some embodiments, the antibodies of this disclosure contain an IgG2 constant region as shown in SEQ ID NO: 543. In some embodiments, the antibodies of this disclosure contain a modified IgG1 constant region as shown in SEQ ID NO: 542.

[0055] In some embodiments, the antibodies of this disclosure are variants possessing some, but not all, effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is critical while certain effector functions (such as complement and ADCC) are non-essential or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patents 5,500,362 and 5,821,337. Alternatively, non-radioactive assays (e.g., ACTI) can be employed. TM and CytoTox (Non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0056] Antibodies may have an increased half-life and / or improved binding to neonatal Fc receptors (FcRn) (see, for example, US 2005 / 0014934). Such antibodies may comprise an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn, and include antibodies having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, according to the EU numbering system (see, for example, US Patent 7,371,826). Other examples of Fc region variants are also considered (see, for example, Duncan & Winter, Nature 322:738-40 (1988); U.S. Patents 5,648,260 and 5,624,821; and WO94 / 29351). In some embodiments, the antibodies of this disclosure have an increased serum half-life due to alterations to the Fc region. In some embodiments, said alterations include M252Y / S254T / T256E mutations against IgG1, or M428L / N434S mutations against IgG1.

[0057] In some implementations, the antibody comprises an antigen-binding fragment, which is an antibody portion having an antigen-determining variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments.

[0058] In some embodiments, a humanized antibody refers to a form of non-human (e.g., mouse) antibody having a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof, containing a minimum amount of non-human (e.g., mouse) sequence. In non-limiting examples, the humanized antibody contains less than about 40% non-human sequence in the variable region. In some cases, the humanized antibody contains less than about 20% non-human sequence in the full-length antibody sequence. In some cases, the humanized antibody is a human immunoglobulin in which residues from the complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capability.

[0059] In some implementations, a chimeric antibody refers to an antibody in which the sequence of the immunoglobulin molecule is derived from two or more species. As a non-limiting example, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability, while the constant regions are homologous to sequences in an antibody derived from another species (typically human) to avoid evoking an immune response in that species.

[0060] As used herein, the term “about” means within 10% of the stated amount.

[0061] As used herein, a “risk variant” refers to any genetic sequence, typically a DNA sequence, that increases an individual’s risk of developing a phenotype (e.g., inflammatory bowel disease, Crohn’s disease, colitis, or a subclinical phenotype thereof). Risk variants include, but are not limited to, single nucleotide polymorphisms (SNPs), insertions or deletions of any length, short tandem repeats (STRs), and chromosomal translocations, duplications, or deletions. The risk variants include those associated with severe forms of inflammatory bowel disease, Crohn’s disease, or colitis. The risk variants include those that indicate an individual may be refractory to any current therapy for inflammatory bowel disease, Crohn’s disease, or colitis. As contemplated herein, risk variants can be used to inform treatment decisions using any of the antibodies described herein.

[0062] The terms “complementarity-determining region” and “CDR,” synonyms with “hypervariant region” or “HVR,” are known in the art and refer to the non-continuous amino acid sequence within the variable region of an antibody that confers antigen specificity and / or binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. The terms “frame region” and “FR,” known in the art, refer to the non-CDR portions of the variable regions of the heavy and light chains. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of many well-known schemes, including those described in the following literature: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745 (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp. Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, June 8, 2001; 309(3):657-70 (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modeling antibodies on the WEB,” Protein Eng. December 2000; 13(12):819-24 (“AbM” numbering scheme). In some embodiments, the CDR of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

[0063] In some implementations, an antibody that specifically binds to a protein means that, compared to alternatives, including unrelated proteins, the antibody reacts or associates with the protein more frequently, more rapidly, for a longer duration, has a higher affinity, or some combination thereof.

[0064] In some embodiments, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also include naturally modified or intervened amino acid polymers; for example, polymers modified by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as fusion and / or conjugation with another polypeptide, or conjugation with a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (e.g., non-natural amino acids, etc.), and other modifications known in the art.

[0065] In some embodiments, the terms "polynucleotide" or "nucleic acid," used interchangeably herein, refer to a nucleotide polymer of any length and include both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the polymer by a DNA or RNA polymerase. Polynucleotides may contain modified nucleotides, such as, but not limited to, methylated nucleotides and their analogs, or non-nucleotide components. Modifications to the nucleotide structure can be conferred before or after polymer assembly. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeled components.

[0066] The percentage of sequence identity (%) relative to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after sequence alignment and, where necessary, the introduction of vacancies to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignments aimed at determining the percentage of amino acid sequence identity can be performed in various known ways, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for sequence alignment can be determined, including the algorithm required to achieve maximum alignment across the full length of the sequences being compared. However, for the purposes of this paper, the amino acid sequence identity percentage is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and its source code has been submitted as user documentation to the U.S. Copyright Office (Washington D.C., 20559) and registered under U.S. Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., and can also be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0067] When using ALIGN-2 for amino acid sequence comparison, the percentage of amino acid sequence identity between a given amino acid sequence A and / or relative to a given amino acid sequence B (or can be expressed as: a given amino acid A has or contains a certain percentage of amino acid sequence identity with or relative to a given amino acid sequence B) is calculated as follows: score X / Y multiplied by 100, where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A. Unless otherwise specified, all amino acid sequence identity percentage values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0068] In some embodiments, the terms "individual" and "subject" are used interchangeably and refer to any animal, including but not limited to humans, non-human primates, rodents, and domesticated and hunting animals, that will become the recipient of a particular treatment. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domesticated and hunting animals include cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, and wolves, bird species such as chickens, emus, and ostriches, and fish such as trout, catfish, and salmon. In various embodiments, the subject may be a subject who has previously been diagnosed with or determined to have or has a condition requiring treatment. In some embodiments, the subject is a human. In various other embodiments, a subject who has previously been diagnosed with or determined to have or has a condition may or may not have received treatment for that condition. In other embodiments, the subject may also be a subject who has not previously been diagnosed with a condition (i.e., a subject exhibiting one or more risk factors for the condition). A “subject requiring treatment for a specific condition” may be a subject who has the condition, has been diagnosed with the condition, or is at risk of developing the condition. In some embodiments, the subject is a “patient” who has been diagnosed with the disease or condition described herein.

[0069] In some implementations, the term "therapeuticly effective amount" refers to the amount of antibody, peptide, polynucleotide, small organic molecule, or other drug that effectively "treats" a disease or condition in a subject or mammal. In some cases, a therapeutically effective amount of a drug reduces the severity of the symptoms of a disease or condition. In some cases, the disease or condition includes inflammatory bowel disease (IBD), Crohn's disease (CD), or ulcerative colitis (UC). In some cases, the IBD, CD, and / or UC is a severe or medically refractory form of IBD, CD, and / or UC. Non-limiting examples of symptoms of IBD, CD, and / or UC include, but are not limited to, diarrhea, fever, fatigue, abdominal pain, abdominal cramps, inflammation, ulceration, nausea, vomiting, bleeding, hematochezia, decreased appetite, and weight loss.

[0070] In some implementations, the terms “treatment” or “management” as used herein refer to therapeutic treatments and preventative or preventative measures aimed at preventing or slowing (alleviating) a target pathological condition, preventing the pathological condition, pursuing or achieving good overall survival, or reducing an individual’s chance of developing the condition, even if the treatment is ultimately unsuccessful. In some aspects provided herein, subjects requiring treatment include subjects who already have a disease or condition, subjects susceptible to the disease or condition, or subjects who require prevention of the disease or condition. The disease or condition may include inflammatory diseases or conditions, fibrotic stenosis or fibrotic diseases, mercaptopurine toxicity or diseases associated with mercaptopurine toxicity, and unresponsiveness to anti-TNF therapy, steroids, or immunomodulatory agents.

[0071] Anti-TL1A antibody

[0072] Various embodiments provide antibodies that bind to TL1A. In some embodiments, the antibody specifically binds to soluble TL1A. In some embodiments, the antibody specifically binds to membrane-bound TL1A. In some embodiments, an anti-TL1A antibody is provided having a heavy chain comprising four heavy chain framework regions (HCFR) and three heavy chain complementarity-determining regions (HCDR): HCFR1, HCDR1, HCFR2, HCDR2, HCFR3, HCDR3, and HCFR4; and a light chain comprising four light chain framework regions (LCFR) and three light chain complementarity-determining regions (LCDR): LCFR1, LCDR1, LCFR2, LCDR2, LCFR3, LCDR3, and LCFR4. The anti-TL1A antibody may contain any of the regions provided herein, such as those provided in Tables 1, 2, 3, Examples, and SEQ ID NOs: 1 to 54, 490 to 588. In some embodiments, the anti-TL1A antibody contains a variable domain, such as those provided herein, having one or more CDR mutations as shown in Tables 2 or 19 to 22. In some implementations, the anti-TL1A antibody comprises one or more CDRs containing the sequences shown in Tables 19 to 22.

[0073] In some embodiments, the anti-TL1A antibody comprises CDRs corresponding to those shown in Tables 19 to 22. In some embodiments, the anti-TL1A antibody or antigen-binding fragment comprises: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence (DTYMH) shown in SEQ ID NO:484; (b) HCDR2 comprising the amino acid sequence (PASGH) shown in SEQ ID NO:485; and (c) HCDR3 comprising the amino acid sequence (SGGLPD) shown in SEQ ID NO:486; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence (ASSSVSYMY) shown in SEQ ID NO:487; (e) LCDR2 comprising the amino acid sequence (ATSNLAS) shown in SEQ ID NO:488; and (f) LCDR3 comprising the amino acid sequence (GNPRT) shown in SEQ ID NO:489.

[0074] In some embodiments, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in any one of SEQ ID NO: 554 to 564 or 574 to 577; and (c) HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 565 to 568 or 578 to 581; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in any one of SEQ ID NO: 569 or 570; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 571 to 573 or 582 to 585.

[0075] In some embodiments, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 559; and (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 567; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 573. In some embodiments, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:503; and a light chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:502. In some embodiments, the antibody or antigen-binding fragment comprises a κ light chain constant region and an IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a κ light chain constant region and an IgG2 heavy chain constant region.

[0076] In some embodiments, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 563; and (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 568; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 572. In some embodiments, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:511; and a light chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:510. In some embodiments, the antibody or antigen-binding fragment comprises a κ light chain constant region and an IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a κ light chain constant region and an IgG2 heavy chain constant region.

[0077] In some embodiments, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 555; and (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 566; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 572. In some embodiments, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:493; and a light chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:492. In some embodiments, the antibody or antigen-binding fragment comprises a κ light chain constant region and an IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a κ light chain constant region and an IgG2 heavy chain constant region.

[0078] In some embodiments, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 558; and (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 566; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 572. In some embodiments, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:501; and a light chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:500. In some embodiments, the antibody or antigen-binding fragment comprises a κ light chain constant region and an IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a κ light chain constant region and an IgG2 heavy chain constant region.

[0079] In some embodiments, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 564; and (c) HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 568; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 572. In some embodiments, this document describes an antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:515; and a light chain variable region containing at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:514. In some embodiments, the antibody or antigen-binding fragment comprises a κ light chain constant region and an IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a κ light chain constant region and an IgG2 heavy chain constant region.

[0080] In some embodiments, the anti-TL1A antibody or antigen-binding fragment comprises: a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 from any one of SEQ ID NO: 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, or 541; and a light chain variable region comprising HCDR1, HCDR2, and HCDR3 from ... any one of SEQ ID NO: 541, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, or 541; LCDR1, LCDR2, and LCDR3 of any one of NO: 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, or 540, wherein the CDR is defined by the Kabat method, the IMGT method, the Chothia method, or a combination thereof. In some embodiments, the anti-TL1A antibody or antigen-binding fragment comprises: a heavy chain variable region comprising at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, or 541; and a light chain variable region comprising the same amino acid sequence as ... NO: Any one of 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538 or 540 has at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% identical amino acid sequences.

[0081] In some embodiments, the anti-TL1A antibody or antigen-binding fragment comprises: a heavy chain variable region comprising: (a) a human heavy chain frame region 1 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that shown in SEQ ID NO: 545; (b) an HCDR1 comprising the amino acid sequence (DTYMH) shown in SEQ ID NO: 484; (c) a human heavy chain frame region 2 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that shown in SEQ ID NO: 546; (d) an HCDR2 comprising the amino acid sequence (PASGH) shown in SEQ ID NO: 485; (e) a human heavy chain frame region 3 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that shown in SEQ ID NO: 547 or 586 to 588; (f) an HCDR3 comprising the amino acid sequence (SGGLPD) shown in SEQ ID NO: 486; and (g) a human heavy chain frame region 3 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that shown in SEQ ID NO: 545; (f) an HCDR3 comprising the amino acid sequence (SGGLPD) shown in SEQ ID NO: 486; and (g) a human heavy chain frame region 3 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that shown in SEQ ID NO: 545; The human heavy chain framework region 4 is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that shown in SEQ ID NO:548; and the light chain variable region comprises: (h) a human light chain framework region 1 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that shown in SEQ ID NO:549; (i) an LCDR1 comprising the amino acid sequence (ASSSVSYMY) shown in SEQ ID NO:487; (j) a human light chain framework region 2 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that shown in SEQ ID NO:550; (k) an LCDR2 comprising the amino acid sequence (ATSNLAS) shown in SEQ ID NO:488; (l) a human light chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that shown in SEQ ID NO:551; and (m) a light chain variable region comprising the amino acid sequence (ASSSVSYMY) shown in SEQ ID NO:487. LCDR3 of the amino acid sequence (GNPRT) shown in NO:489; and (n) the human light chain framework region 4 which is at least 90%, 95%, 96%, 97%, 98% or 99% identical to that shown in SEQ ID NO:552.

[0082] Table 1. Exemplary anti-TL1A antibodies

[0083]

[0084]

[0085] Table 2. Exemplary anti-TL1A CDR sequences

[0086]

[0087]

[0088] In various embodiments, the anti-TL1A antibody specifically binds to the same TL1A region, or specifically binds to a TL1A region overlapping with a TL1A region specifically bound by an antibody described below. The antibody comprises a heavy chain and a light chain, the heavy chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:3, and the light chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:6. In various embodiments, the anti-TL1A antibody specifically binds to the same TL1A region, or specifically binds to a TL1A region overlapping with a TL1A region specifically bound by an antibody described below. The antibody comprises a heavy chain and a light chain, the heavy chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:26, and the light chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:28. In various embodiments, the anti-TL1A antibody specifically binds to the same TL1A region, or specifically binds to a TL1A region overlapping with a TL1A region specifically bound by an antibody described below. The antibody comprises a heavy chain and a light chain, the heavy chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:36, and the light chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:38. In various embodiments, the anti-TL1A antibody specifically binds to the same TL1A region, or specifically binds to a TL1A region overlapping with a TL1A region specifically bound by an antibody described below. The antibody comprises a heavy chain and a light chain, the heavy chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:40, and the light chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:42. In various embodiments, the anti-TL1A antibody specifically binds to the same TL1A region, or specifically binds to a TL1A region that overlaps with the TL1A region specifically bound by the antibody below, the antibody comprising a heavy chain and a light chain, the heavy chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:40, and the light chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:38.In various embodiments, the anti-TL1A antibody specifically binds to the same TL1A region, or specifically binds to a TL1A region overlapping with a TL1A region specifically bound by an antibody described below. The antibody comprises a heavy chain and a light chain, the heavy chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:503, and the light chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:502. In various embodiments, the anti-TL1A antibody specifically binds to the same TL1A region, or specifically binds to a TL1A region overlapping with a TL1A region specifically bound by an antibody described below. The antibody comprises a heavy chain and a light chain, the heavy chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:511, and the light chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:510. In various embodiments, the anti-TL1A antibody specifically binds to the same TL1A region, or specifically binds to a TL1A region overlapping with a TL1A region specifically bound by an antibody described below. The antibody comprises a heavy chain and a light chain, the heavy chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:493, and the light chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:492. In various embodiments, the anti-TL1A antibody specifically binds to the same TL1A region, or specifically binds to a TL1A region overlapping with a TL1A region specifically bound by an antibody described below. The antibody comprises a heavy chain and a light chain, the heavy chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:501, and the light chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:500. In various embodiments, the anti-TL1A antibody specifically binds to the same TL1A region, or specifically binds to a TL1A region that overlaps with the TL1A region specifically bound by the antibody, which comprises a heavy chain and a light chain, the heavy chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:515, and the light chain comprising at least about 90%, 92%, 95%, 98%, or 100% of the sequence identical to that of SEQ ID NO:514.

[0089] In various embodiments, the anti-TL1A antibody or fragment has a binding affinity for TL1A of at least about 1E. -7 1E -8 1E -9 1E -10 Or 1E -11In some cases, the binding affinity is approximately 1E. -9 To approximately 1E -11 .

[0090] Various embodiments provide anti-TL1A antibodies that bind to the same region of the TL1A protein or a portion thereof as a reference antibody, such as any anti-TL1A antibody described herein. In some embodiments, the reference antibody comprises the heavy chain CDR of SEQ ID NO:150, 12, and 152 and the light chain CDR of SEQ ID NO:18, 21, and 155.

[0091] A non-limiting method is provided for determining whether an anti-TL1A antibody (i.e., a test antibody) binds to the same region of the TL1A protein or a portion thereof as described herein. Exemplary embodiments include a competitive assay. For example, the method includes determining whether the test antibody can compete with a reference antibody for binding to the TL1A protein or a portion thereof, or determining whether a reference antibody can compete with the test antibody for binding to the TL1A protein or a portion thereof. Exemplary methods include using surface plasmon resonance to evaluate whether an anti-TL1A antibody can compete with another anti-TL1A antibody for binding to TL1A. In some cases, surface plasmon resonance is utilized in this competitive assay. Non-limiting methods are described in the examples.

[0092] The TL1A antibodies described herein bind to specific regions or epitopes of human TL1A. These regions are demonstrated to be useful for inhibiting the secretion of interferon-γ from T lymphocytes. In some embodiments, antibodies are disclosed that compete with the antibodies described herein for binding to TL1A. In some embodiments, antibodies are disclosed that bind to the same epitope of TL1A as the antibodies described herein. In some embodiments, antibodies are disclosed that bind to discrete epitopes overlapping with the TL1A epitope bound by the antibodies described herein. In some embodiments, antibodies are disclosed that bind to the same epitope of TL1A, overlap one or more amino acid residues with the epitope of TL1A, or compete with antibodies or fragments thereof for binding to the epitope of TL1A that comprise a heavy chain variable region containing the amino acid sequence SEQ ID NO: 503; and a light chain variable region containing the amino acid sequence SEQ ID NO: 502. In some embodiments, this document discloses an antibody that binds to the same epitope of TL1A, overlaps with one or more amino acid residues of the epitope of TL1A, or competes with an antibody or fragment thereof for binding to the epitope of TL1A: the antibody or fragment thereof comprises a heavy chain variable region containing the amino acid sequence SEQ ID NO: 511; and a light chain variable region containing the amino acid sequence SEQ ID NO: 510. In some embodiments, this document discloses an antibody that binds to the same epitope of TL1A, overlaps with one or more amino acid residues of the epitope of TL1A, or competes with an antibody or fragment thereof for binding to the epitope of TL1A: the antibody or fragment thereof comprises a heavy chain variable region containing the amino acid sequence SEQ ID NO: 493; and a light chain variable region containing the amino acid sequence SEQ ID NO: 492. In some embodiments, this document discloses an antibody that binds to the same epitope of TL1A, overlaps with one or more amino acid residues of the epitope of TL1A, or competes with an antibody or fragment thereof for binding to the epitope of TL1A: the antibody or fragment thereof comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 501; and a light chain variable region containing the amino acid sequence of SEQ ID NO: 500. In some embodiments, this document discloses an antibody that binds to the same epitope of TL1A, overlaps with one or more amino acid residues of the epitope of TL1A, or competes with an antibody or fragment thereof for binding to the epitope of TL1A: the antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 515; and a light chain variable region containing the amino acid sequence of SEQ ID NO: 514.

[0093] Methods of generating antibodies

[0094] Various embodiments provide antibodies generated using peptide or nucleotide sequences. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human antibody or a humanized antibody. In some embodiments, the antibody is an antibody fragment. For example, the antibody is Fab, scFv, or (Fab)2. In some embodiments, the antibody is a chimeric antibody.

[0095] The specific binding of the antibodies described herein can be determined by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as BIAcore analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blotting, radioimmunoassay, ELISA, sandwich immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradioassay, fluorescence immunoassay, and protein A immunoassay. Such assays are provided, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, 1994.

[0096] In various embodiments, the anti-TL1A antibody is an antagonist of the TL1A receptor, such as, but not limited to, DR3 and TR6 / DcR3. In some embodiments, the antibody inhibits at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100% of the activity of one or more of the bound TL1A receptor. In some embodiments, the antibody inhibits TL1A activation, as measured by interferon-γ release in human blood. In some embodiments, the antibody is administered at an IC50 concentration of about 1 nanomolar to about 100 picomolars. 50 It inhibits the release of interferon-gamma in human blood. In some embodiments, the antibody is administered at an IC50 concentration of approximately 500 picoseconds to approximately 100 picoseconds. 50 It inhibits the release of interferon-gamma in human blood. In some embodiments, the antibody is administered at an IC50 concentration of approximately 500 picomol. 50 It inhibits the release of interferon-gamma in human blood. In some embodiments, the antibody has an IC50 concentration of approximately 250 picomol. 50 It inhibits the release of interferon-γ in human blood.

[0097] In various embodiments, monoclonal antibodies are prepared using methods known in the art, such as, but not limited to, hybridoma methods, wherein the host animal is immunized as described above to induce lymphocytes to produce antibodies that specifically bind to the immunoantigen (Kohler and Milstein (1975) Nature 256:495). The hybridoma produces monoclonal antibodies specific to the selected antigen. When proliferated in vitro or in vivo, the monoclonal antibodies are purified from the culture medium or ascites fluid using techniques known in the art.

[0098] In some embodiments, monoclonal antibodies are prepared using a recombinant DNA method, such as that described in U.S. Patent 4,816,567. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector, which generates monoclonal antibodies when transfected into host cells (e.g., *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells). The polynucleotides encoding monoclonal antibodies can be further modified in a variety of different ways using recombinant DNA technology to generate candidate antibodies.

[0099] In various implementations, chimeric antibodies can be generated, i.e., molecules in which different parts are derived from different animal species, such as those antibodies having a variable region derived from a mouse monoclonal antibody and a constant region of a human immunoglobulin (e.g., humanized antibodies). Chimeric antibodies can be generated using a variety of techniques, such as those described in Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); and Takeda et al., Nature 314:452-454 (1985).

[0100] In some embodiments, the anti-TL1A monoclonal antibody is a humanized antibody designed to reduce antigenicity and HAMA (human anti-mouse antibody) response when administered to human subjects. A variety of techniques known in the art can be used to generate humanized antibodies. For example, an antibody is humanized by the following steps: (1) determining the nucleotide and predicted amino acid sequences of the variable domains of the light and heavy chains of the starting antibody; (2) designing the humanized antibody, for example, determining which antibody framework region to use during the humanization process; (3) the actual humanization method / technique; and (4) transfection and expression of the humanized antibody (see, for example, U.S. Patents 5,585,089; 6,835,823; 6,824,989). In various embodiments, the humanized antibody can be further optimized to reduce potential immunogenicity while maintaining functional activity for human treatment.

[0101] Humanized antibodies can also be prepared in transgenic mice containing a human immunoglobulin locus that generates a complete repertoire of human antibodies without producing endogenous immunoglobulins after immunization. This method is described in U.S. Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Humanized antibodies can also be obtained through novel genetic engineering methods that enable the production of affinity-mature human-like polyclonal antibodies in large animals such as rabbits and mice (see, for example, U.S. Patent 6,632,976).

[0102] Fully humanized antibodies can be created by first designing a variable region amino acid sequence containing a non-human, such as rodent-derived, CDR embedded in a human framework sequence. The non-human CDR provides the desired specificity. Therefore, in some cases, these residues are included substantially unchanged in the design of the remodeled variable region. Thus, in some cases, modifications should be limited to a minimum, and changes in antibody specificity and affinity should be closely monitored. On the other hand, theoretically, framework residues can be derived from any human variable region. Human framework sequences should be chosen, as they are equally suitable for creating remodeled variable regions and maintaining antibody affinity in order to create remodeled antibodies exhibiting acceptable or even improved affinity. The human framework can be germline or derived from non-germline (e.g., mutated or affinity-mature) sequences. Genetic engineering techniques known to those skilled in the art, such as, but not limited to, phage display of human antibody libraries, transgenic mice, human-human hybridomas, heterozygous hybridomas, unlimited B cell proliferation and cloning, single-cell RT-PCR, or HuRAb technology, can be used to generate humanized antibodies with hybrid DNA sequences containing human frameworks and non-human CDRs. Methods for obtaining “humanized antibodies” are well known to those skilled in the art. (For example, U.S. Patent 5,861,155, U.S. Patent 6,479,284, U.S. Patent 6,407,213, U.S. Patent 5,624,821, US2003166871, US20020078757, Queen et al., Proc. Natl. Acad Sci USA, 86:10029-10032 (1989) and Hodgson et al., Bio / Technology, 9:421 (1991)).

[0103] In some embodiments, the anti-TL1A antibody is a human antibody. Human antibodies can be prepared directly using a variety of techniques known in the art. Unlimited proliferation of human B lymphocytes that produce antibodies against the target antigen, either immunized in vitro or isolated from an immunized individual, can be generated (see, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J. Immunol., 147(1): 86-95; and U.S. Patent 5,750,373). Human antibodies can be selected from a phage library. Techniques for generating and using antibody phage libraries are described in U.S. Patents 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., 2007, J.Mol.Bio., doi:10.1016 / j.jmb.2007.12.018.

[0104] Chimeric, humanized, and human antibodies can be generated through recombinant expression. Recombinant polynucleotide constructs typically contain expression control sequences operatively linked to the coding sequence of the antibody chain, including naturally associated or heterologous promoter regions. In some embodiments, it may be desirable to generate amino acid sequence variants of these humanized antibodies, particularly in cases where these improve antibody binding affinity or other biological properties.

[0105] In some implementations, antibody fragments are used to treat and / or improve IBD. Various techniques for generating antibody fragments are known. Typically, these fragments are derived by the proteolytic digestion of intact antibodies (e.g., Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods 24:107-117; Brennan et al., 1985, Science, 229:81). Fab, Fv, and scFv antibody fragments can be expressed and secreted from *E. coli* or other host cells, allowing for the production of large quantities of these fragments. Other techniques for generating antibody fragments will be readily apparent to those skilled in the art.

[0106] According to this disclosure, techniques can be modified to adapt to the generation of TL1A-specific single-chain antibodies (see, for example, U.S. Patent No. 4,946,778). Additionally, methods can be modified to adapt to the construction of Fab expression libraries (see, for example, Huse et al., Science 246:1275-1281 (1989)) to allow for rapid and efficient identification of monoclonal Fab fragments or their derivatives, fragments, analogs, or homologs having desired specificity for TL1A. Antibody fragments can be generated by techniques in the art, including but not limited to: (a) F(ab')2 fragments generated by digesting antibody molecules with pepsin; (b) Fab fragments generated by reducing the disulfide bonds of F(ab')2 fragments; (c) Fab fragments generated by treating antibody molecules with papain and a reducing agent; and (d) Fv fragments.

[0107] This document also provides modified antibodies comprising any type of variable region that enables the antibody to associate with TL1A. Those skilled in the art will understand that modified antibodies may comprise antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) in which at least a portion of one or more constant region domains has been deleted or otherwise altered to provide desired biochemical properties, such as reduced TL1A. In some embodiments, the variable regions in both the heavy and light chains are altered by at least partially replacing one or more CDRs, and, if necessary, by partial frame region substitution and sequence alteration. In some embodiments, the substituted CDRs may be derived from antibodies of the same class or subclass, derived from antibodies of different classes, such as those derived from different species, and / or combinations thereof. In some embodiments, the constant region of the modified antibody will comprise a human constant region. Modifications of the constant region compatible with this disclosure include the addition, deletion, or substitution of one or more amino acids in one or more domains.

[0108] In various embodiments, the expression of the antibody or its antigen-binding fragment as described herein can occur in prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including in vivo or in situ yeast, insect, fungal, avian, and mammalian cells, or host cells derived from mammals, insects, birds, or yeast. Mammalian cells or tissues may be derived from humans, primates, hamsters, rabbits, rodents, cattle, pigs, sheep, horses, goats, dogs, or cats, but any other mammalian cells may be used. In other embodiments, the antibody or its antigen fragment as described herein may be transfected into a host.

[0109] In some embodiments, the expression vector is transfected into a recipient cell line to generate the chimeric, humanized, or composite human antibodies described herein. In various embodiments, mammalian cells may be used as hosts for antibody protein production, which may include, but are not limited to, fibroblast-derived cells such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells, HeLa cells, and L cells. Exemplary eukaryotic cells that can be used to express the peptide include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PER.C6 cells; and others. TM Crucell; and NSO cells. In some implementations, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modifications on the heavy chain and / or light chain.

[0110] Many suitable host cell lines capable of secreting complete heterologous proteins have been developed in this field, including but not limited to CHO cell lines, various COS cell lines, HeLa cells, L cells, and multiple myeloma cell lines.

[0111] Depending on the type of cell host, an expression vector carrying the chimeric, humanized, or composite human antibody constructs, antibodies, or their antigen-binding fragments described herein can be introduced into suitable host cells using any of a variety of suitable methods, including but not limited to transformation, transfection, lipid transfection, conjugation, electroporation, direct microinjection, and microparticle bombardment, which are known to those skilled in the art. Expression vectors used in these cells may contain expression control sequences, such as origin of replication, promoters, enhancers, and essential processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences.

[0112] In various embodiments, yeast may also be used as a host to produce the antibody molecules or peptides described herein. In various other embodiments, bacterial strains may also be used as a host to produce the antibody molecules or peptides described herein. Examples of bacterial strains include, but are not limited to, species of *Escherichia coli*, *Bacillus*, *Enterobacteria*, and various species of *Pseudomonas*.

[0113] In some embodiments, one or more antibodies or antigen-binding fragments thereof described herein can be produced in animals that have been engineered (transgenic) or transfected with one or more nucleic acid molecules encoding the polypeptide, according to any suitable method. To produce transgenic animals, the transgene can be microinjected into fertilized oocytes, or it can be introduced into the genome of embryonic stem cells, and the nucleus of such cells can be transferred into enucleated oocytes. Once expressed, the antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. (see Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0114] Once expressed in a host, the complete antibody, antibody fragments (e.g., single light and heavy chains), or other immunoglobulin forms disclosed herein can be recovered and purified using known techniques, such as immunoadsorption or immunoaffinity chromatography, chromatographic methods like HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination of these methods. See generally Scopes, PROTEIN PURIF. (Springer-Verlag, NY, 1982). Substantially pure immunoglobulins with at least about 90% to 95% homogeneity are advantageous, as are those with 98% to 99% or higher homogeneity, particularly for pharmaceutical applications. Once partially purified or purified to the desired homogeneity, humanized antibodies or composite human antibodies can then be used therapeutically or for developing and performing assays, immunofluorescence staining, etc. See generally Immunol. Meth. (editors Lefkovits & Pernis, Acad. Press, NY, 1979 and 1981), Volumes I and II.

[0115] Various embodiments provide genetic constructs containing nucleic acids encoding the anti-TL1A antibody or fragment provided herein. The antibody genetic construct may be in the form of an expression cassette adapted to express the encoded anti-TL1A antibody or fragment. The genetic construct may be introduced into host cells with or without being incorporated into a vector. For example, the genetic construct may be incorporated into liposomes or viral particles. Alternatively, purified nucleic acid molecules may be directly inserted into host cells using methods known in the art. The genetic construct may be directly introduced into the host subject's cells via transfection, infection, electroporation, cell fusion, protoplast fusion, microinjection, or ballistic bombardment.

[0116] Various embodiments provide recombinant vectors containing genetic constructs of the antibodies provided herein. The recombinant vector may be a plasmid, a granule, or a phage. The recombinant vector may include other functional elements; for example, a suitable promoter to initiate gene expression.

[0117] Each implementation provides a host cell containing the genetic constructs and / or recombinant vectors described herein.

[0118] Various host systems are also advantageous for expressing recombinant proteins. Examples of suitable mammalian host cell lines include the COS-7 monkey kidney cell line, and other cell lines capable of expressing appropriate vectors, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors may contain non-transcriptional elements such as the origin of replication, suitable promoters and enhancers linked to the gene to be expressed, as well as other 5' or 3' flanking non-transcriptional sequences and 5' or 3' non-translational sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences.

[0119] Proteins produced by the transformed host can be purified using any suitable method. Standard methods of this kind include chromatography (e.g., ion-exchange chromatography, affinity chromatography, and large and small column chromatography), centrifugation, differential solubility, or any other standard technique used for protein purification. Affinity tags such as hexahistine, maltose-binding domains, influenza shell sequences, and glutathione S-transferases can be attached to proteins to facilitate purification by suitable affinity columns. The isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography. Recombinant proteins produced in bacterial cultures can be isolated. Methods known in the art for purifying antibodies and other proteins also include, for example, those described in U.S. Patent Publications 2008 / 0177048 and 2009 / 0187005.

[0120] Those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences to alter one or a small subset of amino acids in the coding sequence constitute a "conserved variant of modification," in which the alteration results in the replacement of the amino acid with a chemically similar amino acid, while retaining the ability to specifically bind to a target antigen. Such conserved variants of modification are complementary to, and not excluded from, polymorphic variants, interspecies homologs, and alleles consistent with this disclosure.

[0121] A given amino acid can be replaced with a residue having similar physicochemical properties, for example, replacing one aliphatic residue with another (e.g., He, Val, Leu, or Ala replacing each other), or replacing one polar residue with another (e.g., between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conserved substitutions, such as substitutions of entire regions with similar hydrophobic properties, are well known. Peptides containing conserved amino acid substitutions can be tested in any of the assays described herein to confirm that the desired activities, such as antigen-binding activity and specificity, of the native or reference peptide are preserved.

[0122] Specific conservative substitutions include, for example: Ala is substituted to Gly or Ser; Arg is substituted to Lys; Asn is substituted to Gin or His; Asp is substituted to Glu; Cys is substituted to Ser; Gin is substituted to Asn; Glu is substituted to Asp; Gly is substituted to Ala or Pro; His is substituted to Asn or Gin; lie is substituted to Leu or Val; Leu is substituted to lie or Val; Lys is substituted to Arg, Gin, or Glu; Met is substituted to Leu, Tyr, or lie; Phe is substituted to Met, Leu, or Tyr; Ser is substituted to Thr; Thr is substituted to Ser; Trp is substituted to Tyr; Tyr is substituted to Trp; and / or Phe is substituted to Val, lie, or Leu.

[0123] In some embodiments, the antibodies and / or antigen-binding fragments described herein may be variants of the sequences described herein, such as conserved substitution variants of antibody peptides. In some embodiments, the variant is a conserved modified variant. A variant may refer to a peptide substantially homologous to a native or reference peptide, but having an amino acid sequence different from that of the native or reference peptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant peptide includes sequences containing one or more additions, deletions, or substitutions of nucleotides compared to the native or reference DNA sequence, but encoding a variant protein or fragment thereof that retains activity against the relevant target peptide, such as antigen-specific binding activity.

[0124] Alterations to the natural amino acid sequence can be accomplished using any of a variety of techniques known to those skilled in the art. Mutations can be introduced at specific loci or via site-specific mutagenesis procedures guided by oligonucleotides. Techniques for performing such alterations are well-established and include, for example, those disclosed in Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patents 4,518,584 and 4,737,462.

[0125] Nucleic acid molecules encoding amino acid sequence variants of antibodies can be prepared using a variety of methods known in the art. These methods include, but are not limited to, preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared antibody variants or non-variant forms. Nucleic acid sequences encoding at least one antibody, fraction, or polypeptide described herein can be recombined with vector DNA using conventional techniques, including but not limited to blunt or staggered ends for ligation and restriction enzyme digestion. Techniques for such operations are disclosed, for example, in Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and can be used to construct nucleic acid sequences encoding monoclonal antibody molecules or antigen-binding regions.

[0126] In some embodiments, the nucleic acid encoding the antibody or antigen-binding fragment thereof described herein is contained in a vector. In some aspects described herein, a nucleic acid sequence or any module thereof encoding the antibody or antigen-binding fragment thereof described herein is operatively ligated to a vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector may be a viral vector or a non-viral vector. The term "vector" encompasses any genetic element that, when associated with appropriate control elements, is capable of replicating and can transfer a gene sequence into a cell. Vectors may include, but are not limited to, cloning vectors, expression vectors, plasmids, bacteriophages, transposons, granules, chromosomes, viruses, virions, etc.

[0127] As used herein, the term “expression vector” refers to a vector that directs the expression of RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence. The term “expression” refers to the cellular process involving the production of RNA and proteins, and, where appropriate, the secretion of proteins, including, where applicable, transcription, transcript processing, translation, and protein folding, modification, and processing. “Expression product” includes RNA transcribed from a gene and polypeptides obtained through the translation of mRNA transcribed from a gene. The term “gene” refers to a nucleic acid sequence that, when operatively linked to a suitable regulatory sequence, is transcribed (DNA) into RNA in vitro or in vivo. The gene may or may not include regions preceding and following the coding region, such as a 5' untranslated (5'UTR) or “leader” sequence and a 3'UTR or “tail” sequence, as well as intercalation sequences (introns) between the individual coding regions (exons).

[0128] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one virally derived element and has the ability to be packaged into viral vector particles. Viral vectors may contain nucleic acids encoding antibodies or their antigen-binding moieties as described herein, instead of non-essential viral genes. Vectors and / or particles can be used for the purpose of transferring any nucleic acid into cells in vitro or in vivo. Various forms of viral vectors are known in the art.

[0129] The term "recombinant vector" refers to a vector that contains a heterologous nucleic acid sequence or "transgenic gene" that can be expressed in vivo.

[0130] Pharmaceutical composition, administration and dosage

[0131] The provided anti-TL1A antibody can be used in a variety of applications, including but not limited to therapeutic treatments such as IBD. The method of use can be in vitro, ex vivo, or in vivo. In some embodiments, the anti-TL1A antibody is an antagonist of the TL1A receptor.

[0132] In some implementations, the diseases treated with anti-TL1A antibodies or TL1A receptor antagonists are IBD, CD, UC, and / or MR-UC.

[0133] In various embodiments, the pharmaceutical composition is formulated for delivery via any route of administration. "Route of administration" may refer to any route of administration known in the art, including but not limited to aerosol, nasal, oral, mucosal, dermal, or parenteral administration.

[0134] Transdermal drug delivery can be accomplished using topical creams or ointments or transdermal patches.

[0135] "Parenteral" refers to routes of administration typically associated with injection, including intraorbital, infusion, intra-arterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrasheath, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal routes. Through the parenteral route, the composition can be in the form of a solution or suspension for infusion or injection, or a lyophilized powder.

[0136] via the intestinal route, the pharmaceutical composition may be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles, thereby allowing controlled release.

[0137] Through a local route, pharmaceutical compositions are formulated for the treatment of skin and mucous membranes and are in the form of ointments, creams, emulsions, powders, dipping pads, solutions, gels, sprays, lotions, or suspensions. They may also be in the form of microspheres or nanospheres or liposomes or polymeric vesicles or polymeric patches and hydrogels, thereby allowing controlled release. Depending on the clinical indication, these local route compositions may be in anhydrous or aqueous forms.

[0138] They can be taken as eye drops via the ocular route.

[0139] In various embodiments, the agent may be administered intravenously by injection or by gradual infusion over time. Given a suitable formulation for a given route, for example, an agent that can be used in the methods and compositions described herein may be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavitarily, and, if desired, by peristalsis or by other means known to those skilled in the art. In particular embodiments, the compounds used herein are administered orally, intravenously, or intramuscularly to patients with IBD, CD, UC, and / or MR-UC.

[0140] Pharmaceutical compositions may also contain any pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” is a pharmaceutically acceptable material, composition, or medium involved in carrying or transporting the target compound from one tissue, organ, or body part to another. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutical acceptable” because it must be compatible with the other components of the formulation. It must also be suitable for contact with any tissue or organ it may come into contact with, meaning it must not carry the risk of toxicity, irritation, allergic reactions, immunogenicity, or any other complications that far outweigh its therapeutic benefit.

[0141] In various embodiments, pharmaceutical compositions comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of anti-TL1A antibody are provided. "Pharmaceutically acceptable excipient" means a generally safe, non-toxic, and desirable excipient that can be used to prepare the pharmaceutical composition, and includes excipients acceptable for both veterinary and human pharmaceutical applications. The active ingredient can be mixed with a pharmaceutically acceptable and compatible excipient in an amount suitable for the treatment methods described herein. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. Suitable excipients are, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, water, saline, dextran, propylene glycol, glycerin, ethanol, mannitol, polysorbate, and combinations thereof. Additionally, if desired, the composition may contain small amounts of excipients, such as wetting agents or emulsifiers, pH buffers, etc., which enhance or maintain the effectiveness of the active ingredient. The therapeutic compositions described herein may comprise pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, acid addition salts formed with organic acids such as acetic acid, tartaric acid, or mandelic acid, salts formed from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and salts formed from organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Liquid compositions may also comprise a liquid phase other than water, such as glycerol, vegetable oils such as cottonseed oil, and water-in-oil emulsions, including compositions containing water and those not containing water. Physiologically tolerable carriers are well known in the art. The amount of an active agent (i.e., an antibody or fragment thereof) effective in the treatment of a particular condition or disease will depend on the nature of the condition or disease and can be determined by those skilled in the art using standard clinical techniques.

[0142] The pharmaceutical composition may also be encapsulated, tableted, or prepared as an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition or to facilitate its preparation. Liquid carriers include syrups, peanut oil, olive oil, glycerin, saline, alcohol, and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, kaolin, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar, or gelatin. The carrier may also include a sustained-release material, either alone or with a wax, such as glyceryl monostearate or glyceryl distearate.

[0143] The pharmaceutical product is prepared using conventional pharmaceutical techniques. For tablets, these techniques include grinding, mixing, granulation, and, if necessary, compression; or for hard gelatin capsules, grinding, mixing, and filling. When a liquid carrier is used, the product will be in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspension. Such liquid formulations can be administered orally directly or filled into soft gelatin capsules.

[0144] The pharmaceutical composition may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is the amount of composition that will produce the most effective result in terms of therapeutic efficacy in a given subject. This amount will vary depending on a number of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the subject's physiological condition (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the nature of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. Those skilled in the art in the clinical and pharmacological fields will be able to determine the therapeutically effective amount through routine experiments, such as by monitoring the subject's response to administration of the compound and adjusting the dose accordingly. For further guidance, see Remington: The Science and Practice of Pharmacy (Gennaro, ed., 20th edition, Williams & Wilkins PA, USA) (2000).

[0145] A typical dose of an effective anti-TL1A antibody can be provided to technicians based on in vitro reactions or reactions in animal models. Such doses can typically be reduced in concentration or amount by up to about an order of magnitude without loss of the associated biological activity. Therefore, the actual dose will depend on the physician's judgment, the patient's condition, and the effectiveness of the treatment, such as based on the in vitro reactivity of the obtained biological sample from relevant primary cultured cells or tissue cultures, or based on the response observed in an appropriate animal model.

[0146] For the treatment of disease, the appropriate dosage of the antibody depends on the type of disease being treated, the severity and duration of the disease, the responsiveness to the disease, whether the antibody is administered for therapeutic or preventative purposes, previous treatments, and the patient's clinical history. In the event of any complications, the dosage may also be adjusted by a separate physician at the attending physician's discretion. The prescribing physician can determine the optimal dosage, method of administration, and frequency of repetition. TL1A antibodies can be administered as a single dose or over a series of treatments lasting from several days to several months, or until a cure or remission of the disease state is achieved (e.g., treatment or improvement in IBD). The duration of treatment depends on the subject's clinical progress and responsiveness to treatment. In some implementations, the dosage is from 0.01 μg to 100 mg per kg of body weight and can be given once or multiple times daily, weekly, monthly, or annually. For systemic administration, therapeutic doses may be administered to the subject, for example, about 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more. In some embodiments, the therapeutic dose is selected from about 1, 3, 10, 30, 100, 300, 600, and 800 mg as a steady-state dose. In some embodiments, the therapeutic dose is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 mg as a steady-state dose. In some embodiments, the therapeutic dose is about 10, 20, 30, 40, 50, 60, 70, 80, or 90 mg as a steady-state dose. In some embodiments, the therapeutic dose is about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 mg as a steady-state dose. In some embodiments, the therapeutic dose is about 5 to about 30 mg / kg. In some embodiments, the therapeutic dose is about 5 to about 30 mg / kg, administered weekly or every other week. In some embodiments, the therapeutic dose is about 5, 10, 15, 20, 25, or 30 mg / kg. In some embodiments, the therapeutic dose is about 5, 10, 15, 20, 25, or 30 mg / kg, administered weekly or every other week.

[0147] Treatment

[0148] Various implementations provide methods for treating inflammatory bowel disease (IBD) that include administering the anti-TL1A antibody described herein to a subject in need. In some implementations, the subject includes one or more risk genotypes. In some implementations, the IBD is a severe form of IBD. Severe forms of IBD may be characterized by the subclinical phenotype described herein.

[0149] In various embodiments, this document provides a method for treating inflammatory bowel disease (IBD) in a subject of need, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment that specifically binds to TL1A. In some embodiments, the anti-TL1A antibody comprises HCFR1, which comprises SEQ ID NO:545, or a sequence differing from SEQ ID NO:545 by a maximum of about 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises HCDR1, which is selected from SEQ ID NO:9, 150, 484, and 553, or a sequence differing from sequences selected from SEQ ID NO:9, 150, 484, and 553 by a maximum of about 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises HCFR2, which comprises SEQ ID NO:546, or a sequence differing from SEQ ID NO:546 by a maximum of about 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises HCDR2, which is selected from SEQ ID NO:12, 554 to 564 and 574 to 577, or a sequence differing from the sequence selected from SEQ ID NO:12, 554 to 564 and 574 to 577 by a maximum of about 5, 4, 3 or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises HCFR3, which comprises a sequence selected from SEQ ID NO:547 and 586 to 588, or a sequence differing from the sequence selected from SEQ ID NO:547 and 586 to 588 by a maximum of about 5, 4, 3 or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises HCDR3, which is selected from SEQ ID NO:15, 152, 565 to 568 and 578 to 581, or a sequence differing from the sequence selected from SEQ ID NO:15, 152, 565 to 568 and 578 to 581 by a maximum of about 5, 4, 3 or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises HCFR4, which is selected from SEQ ID NO:548, or a sequence differing from SEQ ID NO:548 by a maximum of about 5, 4, 3 or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises LCFR1, which is selected from SEQ ID NO:549, or a sequence differing from SEQ ID NO:549 by a maximum of about 5, 4, 3 or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises LCDR1 selected from SEQ ID NO:487, 569 and 570, or sequences differing from SEQ ID NO:487, 569 and 570 by a maximum of about 5, 4, 3 or 2 amino acids.In some embodiments, the anti-TL1A antibody comprises LCFR2, which comprises SEQ ID NO:550, or a sequence differing from SEQ ID NO:550 by a maximum of about 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises LCDR2, which comprises SEQ ID NO:488, or a sequence differing from SEQ ID NO:488 by a maximum of about 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises LCFR3, which comprises SEQ ID NO:551, or a sequence differing from SEQ ID NO:551 by a maximum of about 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises LCDR3, which is selected from SEQ ID NO:571 to 573 and 582 to 585, or a sequence differing from sequences selected from SEQ ID NO:571 to 573 and 582 to 585 by a maximum of about 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises LCFR4, which contains SEQ ID NO:552, or a sequence that differs from SEQ ID NO:552 by a maximum of about 5, 4, 3 or 2 amino acids.

[0150] The subjects disclosed herein may be mammals, such as mice, rats, guinea pigs, rabbits, non-human primates, or farm animals. In some cases, the subject is a human. In some cases, the subject is a patient diagnosed with IBD. In some cases, the subject has not been diagnosed with IBD. In some cases, the subject suffers from symptoms related to the disease or condition disclosed herein (e.g., abdominal pain, cramps, diarrhea, rectal bleeding, fever, weight loss, fatigue, loss of appetite, dehydration and malnutrition, anemia, or ulcers).

[0151] In various embodiments, the subject exhibits either an induction of non-response to anti-TNF therapy (e.g., adalimumab, sertozumab, etanercept, golimumab, infliximab) or a loss of response to said anti-TNF therapy after a period of time during treatment (loss of anti-TNF response). In various embodiments, the subject is at risk of developing anti-TNF non-response or loss of anti-TNF response. In some embodiments, the subject is treated by administering the anti-TL1A antibody disclosed herein to the subject if the subject is at risk of developing anti-TNF non-response or loss of anti-TNF response or already has anti-TNF non-response or loss of anti-TNF response.

[0152] In various other implementations, the subject is identified as having increased TL1A expression. In some implementations, administration of a therapeutically effective amount of anti-TL1A antibody results in a reduction of TL1A in the treated subject.

[0153] The methods disclosed herein provide a way to treat a subject with inflammatory bowel disease (IBD) by administering the anti-TL1A antibody described herein. In various embodiments, the IBD is Crohn's disease (CD) or ulcerative colitis (UC). In some embodiments, the IBD is a severe form of IBD. In some embodiments, the severe form of IBD is characterized by a subclinical phenotype. In some embodiments, the IBD is a moderate to severe form of IBD. In some embodiments, the IBD is a moderate form of IBD.

[0154] Subclinical phenotypes of IBD can include, but are not limited to, non-stenotic, stenotic, narrowed, and penetrating lesions, as well as isolated internal penetrating lesions and perianal CD (pCD). Stenosis is a progressive narrowing of the intestine. Internal penetrating lesions create abnormal channels (fistulas) between the intestine and other structures. pCD is a form of Crohn's disease that causes inflammation around the anus.

[0155] The IBD may be refractory. As used herein, the term "medically refractory" or "refractory" means that standard treatments fail to induce disease remission. In some embodiments, the disease includes the inflammatory diseases disclosed herein. Non-limiting examples of refractory inflammatory diseases include refractory Crohn's disease and refractory ulcerative colitis (e.g., mrUC). Non-limiting examples of standard treatments include glucocorticoids, anti-TNF therapy, anti-α4-β7 therapy (vedolizumab), anti-IL12p40 therapy (ustekinumab), thalidomide, and cytotoxic agents. In some embodiments, the UC is medically refractory UC (mrUC). In some embodiments, the CD is refractory.

[0156] This document discloses a method for administering an anti-TL1A antibody to a subject in need. In various embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human antibody. In various embodiments, the antibody is a humanized antibody. In various embodiments, the antibody is a neutralizing antibody.

[0157] In various embodiments, the anti-TL1A antibody is administered to the subject for the treatment of the IBD described herein. In various other embodiments, the anti-TL1A antibody is administered as part of a series of treatments. In some embodiments, the anti-TL1A antibody and the second IBD treatment may be administered in any order or simultaneously. In selected embodiments, the anti-TL1A antibody is administered to a patient who has previously received second IBD treatment. In some other embodiments, the anti-TL1A antibody and the second IBD treatment will be administered substantially simultaneously or concurrently. For example, the subject may be given the anti-TL1A antibody during the course of treatment for the second IBD. In some embodiments, the anti-TL1A antibody will be administered within 1 year of the second IBD treatment. In some alternative embodiments, the anti-TL1A antibody will be administered within 10, 8, 6, 4, or 2 months of any treatment with the second IBD treatment. In some other embodiments, the anti-TL1A antibody will be administered within 4, 3, 2, or 1 week of any treatment with the second IBD treatment. In some embodiments, the anti-TL1A antibody will be administered within 5, 4, 3, 2, or 1 day of any treatment with the second IBD treatment. It should be further understood that the two treatments can be administered to the subject within approximately several hours or minutes (i.e., simultaneously).

[0158] Other IBD treatments include, but are not limited to, 1) anti-inflammatory drugs (e.g., aminosalicylates, such as but not limited to sulfasalazine (Azulfidine), 5-aminosalicylate, mesalazine, Asacol, Lialda, Rowasa, Canasa, balsalazine (Colazal), and oxalazine (Dipentum); 2) corticosteroids (e.g., prednisone and hydrocortisone); and 3) immunosuppressants (e.g., azathioprine, Azasan, Imuran, purinetol, Purixan, cyclosporine, Gengraf, Neural, and Sandimmune, infliximab, Remicade, adalimumab, Humi). The following medications are indicated: 1) antidiarrheal drugs, including ra, golimumab and Simponi, tumor necrosis factor (TNF)-α inhibitors (e.g., infliximab), methotrexate, rheumatrex, natezumab, Tysabri, vedolizumab, Entyvio, ustekinumab and Stelara; 2) antibiotics (e.g., metronidazole, Flagyl, ciprofloxacin, Cipro); 3) antidiarrheal drugs (e.g., fiber supplements—Metamucil or Citrucel) or loperamide; 4) analgesics (e.g., Tylenol, ibuprofen, naproxen sodium and diclofenac sodium); and 5) surgery (e.g., colectomy, partial gastrointestinal resection, colectomy, rectocolic resection and / or stricture repair). In some implementations, these IBD treatments may be administered in combination with anti-TL1A antibodies. Antibody treatment may be administered before, concurrently with, or after IBD treatment. Combination administration may include co-administration with a single drug formulation or using separate formulations, or sequential administration in either order, but typically over a period of time, so that all active agents can exert their biological activity simultaneously. Any dosing schedule for this type of IBD treatment may also be used, as determined by a skilled technician.

[0159] In some implementations, the second IBD treatment comprises an antibody. Therefore, treatment may involve co-administering the antibody described herein with other antibodies targeting additional IBD-associated antigens, such as, but not limited to, tumor necrosis factor (TNF)-α. Co-administration may include co-administration with a single drug formulation or using separate formulations, or sequential administration in either order, but typically over a period of time, so that all active agents can exert their biological activity simultaneously.

[0160] Reagent test kit

[0161] A kit for treating IBD (e.g., CD, UC, and / or mrUC) is further provided. This kit contains antibodies described herein that can be used to perform the methods described herein. This kit can be used to implement the methods of the present invention for treating patients with IBD, CD, UC, and / or mrUC by administering anti-TL1A antibodies. The kit is a collection of materials or components, including at least one composition of the present invention. Therefore, in some embodiments, the kit contains a composition containing anti-TL1A antibodies as described above for treating IBD, CD, UC, and / or MR-UC. In other embodiments, the kit contains all components necessary and / or sufficient to perform the TL1A assay, including all controls, instructions for performing the assay, and any necessary software for analyzing and presenting the results.

[0162] The exact properties of the components configured in the kit of the present invention depend on their intended purpose. For example, some embodiments are configured for the purpose of treating IBD, CD, UC, and / or MR-UC. In one embodiment, the kit is specifically configured for the purpose of treating mammalian subjects. In another embodiment, the kit is specifically configured for the purpose of treating human subjects. In a further embodiment, the kit is configured for veterinary applications to treat subjects, such as, but not limited to, farm animals, domestic animals, and laboratory animals.

[0163] The kit may include instructions for use. Instructions for use typically include a tangible representation of the techniques employed to achieve the desired outcome (e.g., treatment or relief of IBD, CD, UC, and / or MR-UC) using the components of the kit. Optionally, the kit may also contain other useful components such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipettes or measuring instruments, dressing materials, or other useful instruments readily recognized by those skilled in the art.

[0164] Materials or components assembled in the kit can be provided to practitioners in any convenient and suitable manner that maintains its operability and usability. For example, the components may be in dissolved, dehydrated, or lyophilized form; they may be provided at room temperature, refrigerated, or frozen temperatures. These components are typically contained in suitable packaging materials. As used herein, the phrase "packaging material" refers to one or more physical structures for containing the contents of the kit, such as the compositions of the present invention. Packaging materials are constructed using known methods to preferably provide a sterile, uncontaminated environment. The packaging materials used in the kit are those conventionally used in gene expression assays and therapeutic administration. As used herein, the term "packaging" refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, etc., capable of containing the individual kit components. Thus, for example, packaging may be a glass vial or a pre-filled syringe used to contain an appropriate amount of the composition of the present invention containing anti-TL1A antibody and / or primers and probes against TL1A. Packaging materials typically have external labels indicating the contents and / or purpose and / or components of the kit.

[0165] Example

[0166] The following examples illustrate the embodiments described herein and should not be construed as limiting the scope of this disclosure. References to specific materials are for illustrative purposes only and are not intended to be limiting. Those skilled in the art can develop equivalent means or reactants without inventive step and without departing from the scope of this disclosure.

[0167] Example 1: Generation and characterization of humanized anti-TL1A antibody

[0168] The mouse anti-TL1A antibody was humanized to reduce potential immunogenicity. A first variant, 12835, was generated, consisting of mouse 5C3D11 CDRs (SEQ ID NO: 9, 12, 15, 18, 21, 24) transplanted into the human variable region framework to generate a heavy chain variable region containing SEQ ID NO: 26 and a light chain variable region containing SEQ ID NO: 28. Unfortunately, clone 12835 contained nine (9) framework reversion mutations (mouse framework residues), resulting in an incompletely humanized variant. Full humanization is crucial to reducing the chance of an immune response to the administered antibody in subjects. Therefore, the goal was to generate a humanized antibody containing fewer mouse framework residues while retaining the functional activity of the parental 12835 antibody. Unfortunately, by visually inspecting the sequence, it was not possible to directly distinguish between mouse framework residues crucial for antibody function and non-critical mouse framework residues, making it difficult to determine which amino acid residues could be replaced by the corresponding human framework residues. Therefore, as a first step, 12835 was rehumanized by transplanting the CDRs into the closest fully human phylogenetic framework (IGV1-46*02 and IGKV3-20*01 identified by NCBI's igblast tool). This clone was L8 and contained 5C3D11 and 12835 CDRs (HCDR1, GFDIQDTYMH; HCDR2, RIDPASGHTKYDPKFQV; HCDR3, SRSGGLPDV; LCDR1, RASSSVSYMY; LCDR2, ATSNLAS; LCDR3, QQWSGNPRT) defined by a combination of Kabat, Chothia, and IMGT methods.

[0169] In this study, numerous variants of 12835 were prepared and tested to identify more human-like antibodies that retained the functional activity of the parental 12835 antibody. In the first phase, variants containing significantly fewer mouse framework residues were identified. Subsequently, a CDR library of 12835 was combined with a fully human germline framework to identify multiple variants that did not contain any mouse framework residues but still retained the functional activity and / or affinity of the parental 12835 antibody.

[0170] Mouse 5C3D11 and humanized construct 12835 were cloned into a phage expression system.

[0171] DNA encoding the heavy and light chain variable regions of mouse 5C3D11 and humanized 12835 was cloned into a phage expression vector containing the human κ light chain constant region and the human G1 heavy chain constant region 1. Additionally, the vector contained a his tag and a hemagglutinin A tag at the C-terminus of the heavy chain to facilitate purification and detection. Cloning the mouse variable region into the phage expression vector containing the human constant domain resulted in the expression of chimeric 5C3D11.

[0172] Mouse 5C3D11 heavy chain variable region DNA (SEQ ID NO:1) and light chain variable region DNA (SEQ ID NO:4) were codon-optimized for bacterial expression to generate SEQ ID NO:2 and 5, respectively. Humanized 12835 heavy chain variable region DNA was codon-optimized to generate SEQ ID NO:25, and light chain variable region DNA was codon-optimized to generate SEQ ID NO:27. Expression and quantification of Fab in the periplasmic space of *E. coli* were also performed.

[0173] Cloning was validated by the expression and quantification of Fab in the periplasmic interstitial space of *E. coli*. Briefly, XL-0 bacteria were grown at 37°C in 2X YT medium until the culture density reached 0.9–1.1 at OD600. Isopropyl β-D-thiogalactoside was then added to the cells to a final concentration of 1 mM, and 3.0 mL of the culture was transferred to 14 mL snap-top tubes. Each tube was transfected with 25 μL of high-titer phage stock solution, and the culture was placed in a shaker (225 rpm) at 37°C. After one hour, the temperature was changed to 25°C, and the culture was allowed to regrow for 14–16 h. Cells were collected by centrifugation at 3900 rpm for 30 min in an Eppendorf 5810R centrifuge (approximately 3,200 x g). The supernatant was decanted, and the cells were resuspended in 0.3 mL of lysis buffer (30 mM Tris, pH 8.0, 2 mM EDTA, 20% sucrose, 2 mg / mL lysozyme, 5 U / mL DNase I) and incubated on ice for 15 min. The cell suspension was transferred to 1.5 mL tubes, and cell debris was precipitated by centrifugation at 15,000 rpm for 15 min in an Eppendorf 5424 microcentrifuge (approximately 21,000 x g). The supernatant was carefully removed without disturbing the precipitate and stored at 4°C until use.

[0174] To quantify Fab expression, 96-well Costar-3366 plates were coated overnight at 4°C with 50 μl / well of 2 μg / ml sheep anti-human Fd (Southern Biotech, Item No. 2046-01, Lot No. A7212-VJ06) in PBS. The plates were washed three times with PBS-T containing 0.05% Tween 20, and 50 μl / well of sample diluent was added. Sample dilutions were performed using PBS-T. Standard curves were generated using serially 3-fold dilutions of human Fab (Rockland, Item No. 009-01015, Lot No. 38543) starting at 500 ng / ml. The plates were incubated at 25°C for 1 h, washed three times with PBS-T, and then incubated at 25°C for 1 h with 50 μl / well of an anti-κHRP conjugate (Southern Biotech, product number 2060-05, batch number K3114-S506B) diluted 10,000 times in PBS-T. The plates were washed three times with PBS-T and developed with 50 μl / well of a 1-Step Ultra TMB-ELISA (ThermoScientific, product number 34028, batch number SF2405221). The reaction was terminated by adding 2N H2SO4, and A650 and A450 were determined using a Spectramax microplate reader before and after the addition of H2SO4.

[0175] Characterization of the hybrid 5C3D11 and 12835 – Filter Enhancement Test

[0176] A filter enhancement assay was developed to facilitate the characterization of heavy and light chain expression and to validate the functional activity of the Fab construct by binding to biotinylated antigens. For the filter enhancement assay, bacterial motility was improved by infecting the motility with phages under conditions where each phage produced different plaques (regions of slower bacterial growth). A nitrocellulose filter was placed on the motility to capture the expressed Fab. The filter was then probed with biotinylated antigens and / or reagents targeting immunoglobulins or peptide tags.

[0177] High-titer phage stock solution (usually 10) 6A 1:1 dilution was combined with 0.35 ml of a confluent *E. coli* strain XL culture and 20 μg / ml tetracycline. This mixture was then combined with 3.5 ml of top agar (0.7% Bacto-agar in Luria broth) and spread onto LB agar plates (1.5% Bacto-agar in Luria broth). The plates were incubated at 37°C for 6–8 h, during which time a nitrocellulose filter (Whatman 82-mm diameter, 0.45 μm pore size, GE Healthcare, product number 10401116) was placed on top, and the plates were incubated at 25°C for 12–15 h. The filter was removed, the plates were briefly rinsed in PBS, and transferred to 5% MP blocking solution and incubated at 25°C with constant stirring for 2 h.

[0178] The filter was then cut into three parts: one for assessing light chain expression, one for assessing heavy chain expression, and one for assessing antigen binding. Each part was transferred to the primary assay reagents: goat anti-human κ,HRP conjugate (Southern Biotech, Item No. 2060-05, Lot No. K3114-S506B) diluted 1000-fold in 5% MP for light chain detection, rat anti-HA,HRP conjugate (Roche, Item No. 12013819001) diluted 1000-fold in 5% MP for heavy chain detection, or biotinylated antigen at the desired concentration in 5% MP.

[0179] To biotinylate the antigen, 500 μg of human TL1A (Fitzgerald, product number 30R-AT070, batch number A13102302) was resuspended in water to 1 mg / ml. After resuscitation, the protein was in 10 mM Tris containing 75 mM arginine at pH 8.5. Tris and arginine were removed by buffer exchange using a 7K MW cutoff 5 ml Zeba rotary desalting column (Thermo product number 89891) equilibrated with 10 mM phosphate buffer containing 65 mM NaCl at pH 8.0. After protein recovery, it was immediately biotinylated by mixing it with EZ-Link Sulfo-NHS-LC-Biotin (Thermo product number 21327) at a 5:1 molar ratio at 25°C for 30 min. The reaction was terminated by adding 750 mM arginine to achieve a final concentration of 75 mM. The reaction was transferred to ice and stored at 4°C.

[0180] The filters were incubated at 25°C for 2 hours with constant stirring, and washed five times (2 minutes each time with constant stirring) in PBS-0.05% Tween 20. The filters probed with the biotinylated antigen were then transferred to 10 ml of High Sensitivity Neutravidin, HRP conjugate (Thermo Scientific, product number 31030) diluted 5000-fold in PBS containing 1% BSA, and incubated at 25°C for 1 hour. The filters were then washed five times (2 minutes each time with constant stirring) in PBS-0.05% Tween 20. All filters were developed using 1-Step Ultra TMB-Blotting (Thermo Scientific, product number 37574).

[0181] Using this method, the heavy chain ( Figure 1 A) and light chains ( Figure 1 Expression of B). Furthermore, when the filter was probed with 8 nM biotinylated human TL1A, staining was observed (B). Figure 1 C) indicates that the bacterium expresses functional Fab.

[0182] Characterization of Fab binding by ELISA

[0183] Filter enhancement assays provide a qualitative assessment of antigen binding. To compare the binding activity of chimeric 5C3D11 with the humanized construct 12835 in a more quantitative manner, an ELISA was developed. 96-well Costa-3366 plates were coated overnight at 4°C with 50 μl / well of 2 μg / ml human TL1A (Fitzgerald, product number 30R-AT070, lot number A13102302) in PBS. The plates were washed once with PBS-T and blocked at 25°C for 1 h with 100 μl / well of 1% BSA in PBS. Fab samples were serially diluted 3-fold with 1% BSA and incubated at 25°C (50 μl / well) for 1 h. Wash the plate three times with PBS-T and add 50 μl / well of anti-human κ,HRP conjugate diluted 10,000 times in 1% BSA (Southern Biotech, Product No. 2060-05, Lot No. K3114-S506B), incubating at 25°C for 1 h. In some assays (extended wash forms), place the plate in a large volume (up to 1 L) of PBS-T and incubate with mixing for 2–5 h, followed by the addition of the anti-human κ,HRP conjugate. Wash the plate three times with PBS-T and develop with 50 μl / well of 1-Step Ultra TMB-ELISA (Thermo Scientific, Product No. 34028, Lot No. SF2405221). Terminate the reaction by adding 2N H2SO4, and determine A650 and A450 before and after H2SO4 addition using a Spectramax microplate reader. To measure the binding to mouse TL1A, the same protocol was used, but the plate was coated with 2 μg / ml mouse TL1A (BioLegend, product number 753004, lot number B204691), and the Fab sample was serially diluted 2-fold.

[0184] The binding activity of chimeric 5C3D11 Fab was compared with that of humanized 12835 Fab. Figure 2 Although the binding activities of 5C3D11 and the humanized 12835 in IgG form (bivalent) appeared similar, a slightly lower binding activity was observed in the Fab form of 12835 (monovalent) compared to the chimeric Fab. Figure 2 This difference likely reflects the discrepancy between true affinity (monovalent form) and similar affinity (divalent form). Using the monovalent determination form, the affinity of the chimeric Fab appears to be 2 to 3 times that of the humanized 12835 Fab.

[0185] Capture Enhancement Trial

[0186] A nitrocellulose filter (Whatman 82-mm diameter, 0.45μm pore size, GE Healthcare, product number 10401116) was floated on top of 10 ml of 10 mg / ml goat anti-human κ (Southern Biotech product number 2060-01) at 25°C for 2 hours. The filter was briefly submerged, then removed and transferred to 10 ml of 5% MP, and incubated at 25°C for 2 hours. The filter was removed from the 5% MP, briefly rinsed once with PBS, and air-dried. Subsequently, the filter was treated in the same manner as the filter lift test described above, with minor modifications. In short, a high-titer phage stock solution (typically 10...) was used... 6 A 1:1 (fold) dilution was combined with 0.35 ml of confluent *E. coli* strain XL culture and 20 μg / ml tetracycline. This mixture was then combined with 3.5 ml of top agar (0.7% Bacto-agar in Luria broth) and spread onto LB agar plates (1.5% Bacto-agar in Luria broth). The plates were incubated at 37°C for 6–8 h, during which time a pretreated nitrocellulose filter (as described above) was placed on top, and the plates were incubated at 25°C for 12–15 h. The filter was removed, briefly rinsed in PBS, and transferred to 5% MP at the desired concentration of biotinylated antigen. The filters were incubated at 25°C for 2 hours with constant stirring, washed five times with PBS-0.05% Tween 20 (2 minutes each time with constant stirring), and transferred to 10 ml of High Sensitivity Neutravidin, HRP conjugate (Thermo Scientific, product number 31030) diluted 5000-fold in PBS containing 1% BSA, and incubated at 25°C for 1 hour. The filters were then washed five times with PBS-0.05% Tween 20 (2 minutes each time with constant stirring). All filters were developed using 1-Step Ultra TMB-Blotting (Thermo Scientific, product number 37574). Figure 3 The color-developing filter shown demonstrates the high sensitivity and affinity of 5C3D11 for TL1A.

[0187] Mouse framework residues were removed from 12835 to identify several active humanized clones, including 18-7 and 21-3.

[0188] Mouse framework residues were removed using Kunkel mutagenesis (Kunkel TA 1985. PNAS 82:488-492). In short, single-stranded M13 plasmids were isolated, and DNA replication was initiated using mutagenic oligonucleotides encoding human framework residues instead of those encoding mouse framework residues. After extension to circularization, bacterial transformation yielded a mixture of wild-type (unpaired mouse framework residues) and mutant (human framework residues) plasmids. Mutagenesis was simultaneously performed at multiple sites to generate small combinatorial libraries containing a mixture of clones with various combinations of mouse and human framework mutations. The mixtures were then inoculated onto plates and screened by capture lift to identify the most active framework combinations. The library clones were characterized by DNA sequencing.

[0189] Fab was expressed in E. coli, quantified by ELISA, and its binding activity was assessed by ELISA using titration against immobilized antigens. Fab expression, periplasmic fragment isolation, quantification of Fab expression, and binding to antigens via ELISA were all performed as described above.

[0190] Using this method, multiple active clones containing different numbers of mouse framework residues were identified. Table 3 summarizes examples of active clones with different numbers and positions of mouse framework residues.

[0191] Table 3. Active humanized 5C3D11 constructs with different amounts of framework reversion mutations

[0192]

[0193]

[0194] Synthesis of CDR Porting Constructs

[0195] The two identified clones—18-7 and 21-3—contained only two mouse framework reversion mutations. The light chain of clone 18-7 contained no mouse framework residues, while the heavy chain of clone 21-3 contained no mouse framework residues. Screening of the framework composite library did not identify any CDR-transplanted variants (lacking mouse framework residues on either the heavy or light chain). For comparison, CDR-transplanted variants were synthesized using Kunkel mutagenesis and their binding activity was characterized by ELISA. Although the CDR-transplanted constructs bound the antigen, the humanized 12835 variant consistently showed stronger binding to the antigen. Figure 4B ).

[0196] As shown in Table 3, after reversion mutation of mouse framework residues, the heavy chain variable region frameworks 1-3 are the same as those of human lines IGHV1-46*01, IGHV1-46*02 and IGHV1-46*03, while the light chain variable region framework is the same as that of human line IGKV3-20*01.

[0197] Furthermore, different reversion mutations were introduced into the third frame of the heavy chain variable region, making the new heavy chain variable region homologous to the human germline IGHV1-3*01 (see VH SEQ ID 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, and 541). In general, clones containing these reversion mutations are referred to as alternative frame variants.

[0198] Characterization of humanized variants in alternative ELISA formats

[0199] Multiple Fab variants were characterized by ELISA using a method that allows for rapid and direct comparison (single-well assays without dilution series) of the relative affinity of different Fab clones isolated from different cultures, without regard to alternative forms of relative expression levels of clones (Watkins et al. 1997, Analytical Biochemistry 253). This assay enables a more quantitative comparison of the relative binding strength of variants because the plates are saturated with different Fabs despite varying expression levels. Therefore, subtle differences in binding profiles caused by variations in Fab quantification are eliminated. In short, 96-well Costa-3366 plates were coated with 50 μl / well of 2 μg / ml goat anti-human κ (Southern Biotech product number 2060-01) at 25°C for 2 h, washed once with PBS-0.05% Tween 20, and incubated at 50°C with 50 μl / well of sample Fab for 2 h. The plate was washed four times with PBS-0.05% Tween 20 and incubated at 25°C for 2 h with serially diluted biotinylated antigen (50 μl / well). The preparation of the biotinylated antigen was described above. The plate was washed four times with PBS-0.05% Tween 20 and incubated at 25°C for 1 h with 50 μl / well of a highly sensitive Neutravidin, HRP conjugate (Thermo Scientific, product number 31030) diluted 5000-fold in PBS containing 1% BSA. The plate was washed three times with PBS-T and developed using a 1-Step Ultra TMB-ELISA (Thermo Scientific, product number 34028, batch number SF2405221) with 50 μl / well. The reaction was terminated by adding 2N H2SO4, and A650 and A450 were determined using a Spectramax microplate reader before and after the addition of H2SO4.

[0200] Among the alternative ELISA formats, chimeric Fab binds to the antigen more strongly than CDR-transplanted Fab. Figure 5(Comparison of hollow and solid circles). Compared to the chimera, the binding of humanized clone 12835 was slightly weakened (comparison of hollow triangles and hollow circles), followed by clones 18-7 and 21-3. The binding of clone 21-3 was most similar to the CDR transplanted variant, suggesting that one of the mouse heavy chain framework reversion mutations may be crucial for maintaining full binding activity with the parental (wild-type) CDR.

[0201] Example 2: Generation and characterization of anti-TL1A antibody with optimized CDR

[0202] To identify CDR mutations that could restore and improve binding activity to the CDR transplant construct (complete human germline framework), mutagenesis was performed at each position of all six CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) via Kunkel mutagenesis, using degenerate oligonucleotides in which the codons encoding the target amino acid were replaced by NNK. Initially, a library was synthesized at each position of HCDR3 and LCDR3. These positional libraries were screened by capture boosting, with a theoretical diversity of 32 codons / 20 amino acids / 1 stop codon. In some cases, each position was screened by itself (theoretical library diversity equals 32), while in others, positions of specific CDRs were merged and screened as a CDR library (theoretical library diversity equals 32 times the number of merged positions; for example, HCDR3 consists of 7 positions, so the theoretical library size is 32 x 7 equals 224 members). Libraries were screened at concentrations of biotinylated human TL1A from 15 to 1,000 pM. Positive plaques were picked and sequenced. As previously described, Fab expression was isolated from the periplasmic region and characterized by ELISA. Tables 4 and 5 show a summary of capture-enhancing screening, DNA sequencing, and relative binding activity by ELISA for some initial screenings of LCDR3 and HCDR3. Tables 6 through 12 summarize the more detailed capture-enhancing screening results for all six CDRs.

[0203] In addition, LCDR3 and HCDR3 libraries constructed using alternative heavy chain variable region frameworks were prepared and screened using capture enhancement (Tables 13 and 14). Screening of CDR libraries constructed on alternative frameworks identified some mutations identified on the VH1-46*01 framework, as well as novel mutations previously unidentified (e.g., heavy chain CDR3 L98S, V102H, V102F and light chain CDR3 S92A, S92F, and S92Y).

[0204] Table 4. Light chain CDR3 position scanning (screened with 200pM antigen)

[0205]

[0206] Table 5. Heavy chain CDR3 position scanning (screened with 500pM antigen)

[0207]

[0208] Table 6. Summary of Heavy Chain CDR1 Screening (L8 Template; 1-46*02 Framework)

[0209]

[0210]

[0211] Table 7. Summary of Heavy Chain CDR2a Screening (L8 Template; 1-46*02 Framework)

[0212]

[0213] Table 8. Summary of Heavy Chain CDR2b Filtering (Various Templates; 1-46*02 Framework)

[0214]

[0215]

[0216] Table 9. Summary of Heavy Chain CDR3 Screening (L8 Template; 1-46*02 Framework)

[0217]

[0218] Table 10. Summary of CDR1 screening for light chains (L8 template; paired with heavy chain 1-46*02 frame)

[0219]

[0220] Table 11. Summary of CDR2 screening for light chains (L8 template; paired with heavy chain 1-46*02 frame)

[0221]

[0222] Table 12. Summary of CDR3 screening for light chains (various templates; paired with heavy chain 1-46*02 framework)

[0223]

[0224]

[0225] Table 13. Summary of Heavy Chain CDR3 Filtering (L8mod template; related framework for heavy chains 1-3*01)

[0226]

[0227] Table 14. Summary of CDR3 screening for light chains (L8mod template; paired with heavy chain 1-3*01 related framework)

[0228]

[0229] Example 3. Generation and characterization of anti-TL1A antibodies with optimized CDR using a mutant (S93) heavy chain.

[0230] Clones containing CDR transplanted heavy chains (CDR transplanted and 21-3) exhibited lower binding activity than clones containing a mouse reversion mutation (S) at position 93 of the heavy chain. Therefore, another HCDR3 library was constructed. The library was constructed as described above, except that degenerate oligonucleotides from all positions were mixed prior to mutagenesis, and the library was synthesized and expressed as a pool, unlike examining each position individually. Similar to the HCDR3 position scan on the L8 heavy chain backbone (S93A), position 102 of HCDR3 yielded several mutations that enhanced antigen binding in the capture-enhanced form. Screening the HCDR3 library based on the heavy chain S93 template identified several mutations identified on the heavy chain A93 template, as well as novel mutations previously unidentified (e.g., heavy chain CDR3 V102I and V102Y). Table 15 shows a summary of the capture-enhanced screening and DNA sequencing.

[0231] Table 15. Capture boosting screening of HCDR3 library pools on heavy chain templates with S93

[0232] mutation frequency V102K 10 V102M 7 V102Y 4 V102L 2 V102I 1 V102E 1 V102T 1

[0233] Example 4. Identification and Engineering of Potential Sequence Libraries

[0234] Using Molecular Operating Environment (MOE) 2018.01 software (Chemical Computing Group, Montreal, Canada), a structural homology model of the L8 variable region of the CDR transplantation construct was established based on known PDB antibody structures. This model and the BioMOE prediction algorithm were used to assess sequence liabilities. In addition, based on known potentially unstable sequence motifs, the potential sequence liabilities of variant 12835 and L8 were analyzed (Jarasch et al., J. Pharm. Sci. 104:1885-1898 (2015); Sydow et al., PLOS ONE 9:e100736 (2014); Vlasak and Ionescu mAbs 3:253-263 (2011)). Using these methods, several residues were identified as potentially unstable, including: light chains M33, W35, W47, W91, and N94; heavy chains D31T32, M34, D52P52a, M69, and W103 (summarized in Table 17; FR denotes frames).

[0235] Extensive capture-boost screening was performed at all potentially unstable sites within the CDR to identify amino acid substitutions that could eliminate these residues while preserving antigen binding. For example, one screening focused on light chain CDR3 N94 (a potential deamidation site). Initial capture-boost screening at LCDR3 position 94 failed to identify mutations showing enhanced affinity relative to the wild-type sequence. Therefore, to identify acceptable mutations to eliminate these potentially unstable residues, the conditions of the initial capture-boost screening were modified. Specifically, instead of screening with the antigen at a concentration where the wild-type sequence did not provide a signal, the antigen concentration was increased so that clones expressing the wild-type sequence became visible on boost. In this way, variants that bind with similar affinity to the wild-type sequence but eliminate the problematic residues can be identified.

[0236] For the LCDR3 position 94 library, approximately 3,000 clones were plated and analyzed by capture boost. Capture boost was screened using 1000 pM human TL1A, and plaques exhibiting eight darkest and six lighter staining intensities were picked and sequenced. The results are summarized in Table 16. As expected, the two darkest stained plaques expressed the wild-type residue N94. However, the other six dark stained plaques expressed T94, indicating that the variant N94T largely retains the binding affinity of the wild-type sequence while eliminating a potential deamidation site. Additionally, five distinct sequences were identified from the lighter stained plaques. These included D, F, K, R, and S. Although the affinity of these candidate sequences may be slightly lower than that of the wild-type sequence, all of these candidate sequences could also serve as substitutes for N94 when combined with other higher affinity mutations identified elsewhere.

[0237] Table 16. Identification of candidate residues at the potential unstable site LCDR3 N94 (screened with 1000pM antigen)

[0238]

[0239] Table 17. Summary of potentially unstable residues and active variants that eliminate instability

[0240]

[0241]

[0242] Example 5. Identification of mutations leading to enhanced expression in Escherichia coli

[0243] Some mutations identified during CDR library screening via capture-enhanced expression do not always show enhanced binding in the ELISA format, but instead exhibit persistent expression of soluble Fab at higher levels in the bacterial periplasmic space than other variants. This phenomenon was specifically observed at position V65 (V65G, V65T, and V65K) of the heavy chain CDR2 and position R24 (R24G) of the light chain CDR1. These results are surprising because the capture-enhanced screening format was configured to minimize the effects of different expression levels while maximizing the effect of affinity on signal intensity. Therefore, these mutations were noted and incorporated into subsequent combinatorial libraries that included affinity-enhancing mutations to determine whether these mutations would confer expression and / or thermostability benefits to candidates expressed as intact immunoglobulins in mammalian expression systems.

[0244] Example 6. Generation and characterization of anti-TL1A antibody with combined HCDR3 and LCDR3 mutations.

[0245] Based on the preliminary identification of beneficial mutations in HCDR3 and LCDR3, another library was synthesized, expressed, and screened to identify combinations of independent mutations that could further improve binding affinity. The library was constructed using oligonucleotides encoding the subset of mutations identified in the positional screening via two-site mutagenesis. Oligonucleotides encoding wild-type residues were also included. This combinatorial library contained 30 different variants: wild-type (no mutations), 9 variants containing single mutations (with redundancy from the variants identified in the positional screening, as shown in Tables 4 and 5), and 20 unique combinations. Capture-enhanced screening with 200 pM antigen identified 21 active clones. DNA sequencing of these 21 clones identified certain combinations more frequently than other clones (Table 18).

[0246] Table 18. Summary of Combinatorial Library Screening and DNA Sequences

[0247]

[0248] Subsequently, multiple combinatorial libraries were synthesized, expressed, and screened (detailed below). Typically, these library combinations were identified as mutations that improved affinity (Examples 2 and 3), mutations that altered potentially unstable residues (Example 4), and mutations that might confer enhanced thermostability / expression (Example 5). Combinatorial libraries were screened using various ELISA formats to identify clones with optimal properties (affinity, selectivity, binding to membrane-associated TL1A, and developability) for further development. As summarized in Tables 19 through 22, multiple variants with optimized and diverse CDR sequences utilizing different VH germline templates were identified.

[0249] Heavy chain CDRs on the heavy chain template in Table 19.1-46*02

[0250]

[0251]

[0252] Light Chain CDR on Table 20.3-20*01 Light Chain Template

[0253]

[0254] Heavy chain CDRs on Table 21.1-3*01 Heavy Chain Template

[0255]

[0256]

[0257] Light Chain CDR on Table 22.3-20*01 Light Chain Template

[0258]

[0259] The binding of Fab variants with CDRs shown in Tables 19–22 to human TL1A was tested in multiple formats. First, human TL1A was immobilized on the surface of an ELISA plate, and soluble Fab variants were titrated, as shown in Figures 8 and 9. Next, a uniform (saturated) amount of soluble Fab variants was captured on the surface of the ELISA plate, and soluble biotinylated human TL1A was titrated, as shown in Figures 10–13. In both ELISA formats, all Fab variants bound human TL1A, and showed significantly enhanced binding relative to the CDR-transplanted variant L8. Furthermore, these variants bound variant 12835 as well or better, while having no or significantly fewer mouse reversion mutations in the frame. As a result, these experiments elucidate a set of anti-TL1A variable regions that exhibit high binding affinity and high homology with human germline Ig sequences.

[0260] The binding of all Fab variants to membrane-associated TL1A was tested. For these studies, the HEK293 cell line, already transfected with human TL1A, was used. 293 cells expressing membrane-bound human TL1A were maintained in a 37°C incubator containing 5% CO2 in DMEM containing L-glutamine, glucose, sodium pyruvate, and phenol red (Thermo Fisher catalog 11995-065) plus 10% fetal bovine serum, 1X penicillin-streptomycin (Fisher catalog 15140122), and 2 μg / ml puromycin (Gibco catalog A11138-03). Three days prior to assay, 3 x 10 6 Seed cells into T-75 culture flasks to ensure 90-95% confluence on the day of assay. Aspirate the culture medium and gently wash the cell monolayer with 5 ml of PBS. Remove adherent cells by repeatedly aspirating 10 ml of ice-cold 1% BSA / PBS onto the monolayer. Count the cells and aliquot each sample into 5 x 10⁻⁶ aliquots. 5Cells were collected by centrifugation at 300 x g for 5 min at 4 °C, and the washing buffer was discarded. Cells were resuspended in 100 μl of Fab or IgG diluted in 1% BSA / PBS and placed on ice for 30 min. Next, cells were washed with 1 ml of 1% BSA / PBS and collected by centrifugation at 300 x g for 5 min at 4 °C. The washing buffer was discarded, and 100 μl of a second goat F(ab')2 anti-human κFITC (Southern Biotech catalog number 2062-02) or goat F(ab')2 anti-human IgG PE (Southern Biotech catalog number 2043-09) conjugate diluted 1:200 in BSA / PBS was added. Cells were placed on ice for 30 min. Finally, cells were washed with 1 ml of BSA / PBS and collected by centrifugation at 300 x g for 5 min at 4 °C. The washing buffer was discarded, and cells were resuspended in 500 μl of 1% BSA / PBS. One drop of Sytox AAD Advanced ReadyFlow Reagent (ThermoFisher catalog number R37173) was added to each sample, and the samples were analyzed on an Attune NxT flow cytometer (ThermoFisher). All variants bound to membrane-associated human TL1A, as shown in Figure 14.

[0261] Next, the selectivity of all Fab variants for human TL1A relative to other TNFSF members TRAIL, LIGHT, and Fas was characterized. Briefly, ELISA plates were coated overnight at 4°C with 50 μl / well of antigen at a concentration of 1 μg / ml in PBS (Fas / TNFSF6, R&D Systems, catalog number 126-FL / CF; TRAIL / TNFSF10, R&D Systems, catalog number 375-TL / CF; LIGHT / TNFSF14, R&D Systems, catalog number 664-LI / CF). The plates were washed three times with PBS-T and blocked with 100 μl of 1% BSA / PBS. Discard the blocking solution and titrate 50 μl of 1% BSA / PBS with either the Fab variant or control antibody (Fas / TNFSF6, R&D Systems, catalog number AF126; TRAIL / TNFSF10, R&D Systems, catalog number AF375; LIGHT / TNFSF14, R&D Systems, catalog number AF664), and incubate at 25°C for 1 h. Wash the plate three times with PBS-T and add the HRP-conjugated secondary antibody (diluted 5,000 times in 1% BSA / PBS), incubate at 25°C for 1 h. Wash the plate three times with PBS-T and develop the color. See Figure 15 to... Figure 20As shown, none of the variants exhibited detectable binding to related family members, suggesting that when using ethno-based framework templates to design higher affinity, selectivity for human TL1A relative to other TNFSF family members is preserved.

[0262] Example 7: Characterization of selected humanized variants expressed on different IgG constant regions

[0263] The light and heavy chain variable regions of clones 14, 17L, 23, 34, 47, and 53 from Tables 19 and 20 above were cloned into the κ light chain constant region and the modified IgG1 or IgG2 heavy chain backbone, respectively. Modified IgG1 and IgG2 backbones were selected to reduce the potential effector function of the antibodies. Transient expression and purification characteristics are shown in Table 23 below. For these variants, expression as modified IgG1 was superior to expression as IgG2. Furthermore, the yields obtained were consistent with observations regarding the effect of certain mutations on bacterial expression (see Example 5). Specifically, the highest-expressing variants 14, 34, and 47 all contained mutations at CDR2 V65G, V65T, or V65K in the heavy chain, while the lowest-expressing variants 17L, 23, and 53 did not.

[0264] Typically, as assessed by ELISA binding to antigen-coated plates (Figure 21) and by ELISA capturing soluble biotinylated antigen (Figure 21), binding to human TL1A was preserved in both forms (modified IgG1 and IgG2). Additionally, binding to membrane-associated human TL1A was preserved for all variants, except when variant 53 expressed as IgG2. Figure 23 Finally, since no clones showed significant binding to TNFSF family members Fas, TRAIL, or LIGHT, the selectivity for human TL1A relative to other TNFSF members was maintained (Figure 24).

[0265] Table 23. Selected CDR variants expressing human IgG1 (modified) and IgG2

[0266]

[0267] Example 8: Characterization of potency and species selectivity in whole blood assays.

[0268] The neutralizing activity and potency of the described variants of IgG1 (modified) and IgG2 expressed as healthy donors were tested in human whole blood using a assay. This assay was a modification of Cassatella et al., “Soluble TNF-like cytokine (TL1A) production by immune complexes stimulated monocytes in rheumatoid arthritis” J Immunol. 2007 1J; 178(11):7325-33; and IFN-γ production was measured under conditions of upregulation and activation of TL1A and its receptor DR3. Soluble and membrane-associated TL1A were produced in this assay. The results of this assay have been shown to correlate with in vivo results in a mouse model of colitis. See Takedatsu, “TL1A (TNFSF15) regulates the development of chronic colitis by modulating both T-helper 1 and T-helper 17 activation. Gastroenterology. 2008 Aug;135(2):552–567.

[0269] In short, 96-well plates were coated overnight with human gamma globulin in PBS at 4°C, washed with PBS, and then incubated with anti-human IgG (Fc fragment specific) at 25°C for at least 1 hour to generate immune complexes (IC). The plates were washed three times with PBS immediately before use. Collected blood samples were treated with IL-12 and IL-18, antibodies were titrated in the samples, and the samples were added to the plates and incubated at 37°C for 24 hours. Next, 100 μl of PBS / 5% BSA was added to each well and mixed. The plates were centrifuged at 500 g for 5 minutes, and approximately 150 μl of diluted plasma was collected for IFN-γ measurement. PBS was added to facilitate collection of cellular plasma from the plates due to the high blood percentage (95%). All samples were diluted to ensure their values ​​were within the linear range of the standard curve. All variants, regardless of IgG form (modified IgG1 or IgG2), showed effective inhibition of IFN-γ production (Table 24). For comparison, the typical IC50 values ​​for the mouse parent antibody 5C3D11 and the humanized 12835 were 1.38 ± 0.95 nM (donor n = 16) and 9.28 ± 10.71 nM (donor n = 4), respectively.

[0270] Table 24. Efficacy in human whole blood assays

[0271]

[0272]

[0273] Next, samples were evaluated in the same assay using blood obtained from cynomolgus monkeys to assess the cross-reactivity of the optimized humanized variants with cynomolgus monkey TL1A. This assay was performed similarly to the assay using whole human blood, except that the variants were tested at a single concentration (10 nM) instead of a complete titration. All variants inhibited IFN-γ production, but variants 47 and 53 did not inhibit it to the extent that variants 14, 23, or 34 and mouse 5C3D11 did. Figure 25 These data demonstrate that the optimized humanized variant retains cross-reactivity with cynomolgus monkey TL1A.

[0274] Neutralizing TL1A antibodies were also formalized as effectorless IgG1 molecules (as shown in SEQ ID NO:542), expressed and purified from CHO cells, and used in human (as described above) Figure 26A -C) and cynomolgus monkeys ( Figure 27A The efficacy of whole blood was tested. The results are summarized in Table 25 below.

[0275] Table 25. Potential of CHO-expressing variants in whole blood assays in humans and cynomolgus monkeys (IC50, nM)

[0276]

[0277] These experiments confirmed that all variants were active and effective, with variants 14 and 34 typically showing the greatest potency when using human blood. All variants were effective when using cynomolgus monkey blood. Variants 14, 23, and 34 showed similar potency against human and cynomolgus monkey TL1A, while variant 53 showed approximately twice the potency against human TL1A.

[0278] Example 9: Competition Measurement

[0279] Binding competition assays were performed using surface plasmon resonance (SPR) to evaluate whether the tested anti-TL1A antibody bound to the same region on TL1A as any of the anti-TL1A antibodies described herein.

[0280] Using a Biacore 2000 or 3000 instrument, a reference antibody was directly immobilized onto the surface of a carboxymethylated dextran sensor chip (CMS) via amine coupling. Recombinant soluble human or mouse TL1A diluted to 10 nM in 8.1 mM Na₂HPO₄, 1.47 mM KF₂PO₄, pH 7.2, 237 mM NaCl, 2.7 mM KCl, 3.4 mM EDTA, and 0.01% Tween 20 (PBS-NET) was injected at a flow rate of 10 R⁻¹ / min for approximately 1 minute to achieve a binding level of at least 100 response units (RU) on the immobilized antibody. The reference antibody was then injected at 30 nM for 5 minutes to saturate all potential binding sites on TL1A. The injection of the reference antibody was repeated to confirm this saturation. Next, the test antibody in PBS-NET or PBS-NET-only as a control was injected at 30 nM for 5 minutes. If the test antibody binds to TL1A saturated with the first antibody, it indicates that the test antibody binds to a non-competitive region on TL1A compared to the reference antibody. If the test antibody does not bind to saturated TL1A, it indicates that both antibodies bind to the same region or compete for binding to TL1A. This strategy can be repeated using a fixed test antibody and a reference antibody injected after the test antibody binds to TL1A. Each cycle can be repeated. At the end of each cycle, the fixed antibody surface is regenerated by a 30-second pulse of 3M MgCl2 or by 0.1% TFA followed by two consecutive 15-second pulses of PBS-NET. All injections are performed at a collection rate of 10 Hz at 25°C. All sensor maps are double-referenced by using control surfaces and buffer injections.

[0281] Another binding competition assay was performed using SPR to evaluate whether the test anti-TL1A antibody bound to the same region on TL1A as any of the anti-TL1A antibodies described herein. Reference antibodies were immobilized on the SPR chip by amines coupled to an array at three or four different densities. TL1A protein was injected serially at incremental concentrations to estimate kinetic parameters and the appropriate concentration to inject during competitive binning experiments. Once the optimal antigen concentration for binning experiments was determined, regeneration conditions (typically brief low-pH injections) were evaluated to establish optimal conditions for regeneration between binning assay cycles.

[0282] Binning is performed using a premixing method, in which a moderate concentration of TL1A, either alone or pre-complexed with the test antibody at a saturated antibody concentration (e.g., 30–50 μg / mL), is injected onto the entire array. This assay can be performed to immobilize the test antibody, with a reference antibody pre-complexed with TL1A. Clones that bind to a unique region of the immobilized antibody provide signal enhancement, while competitive clones reduce the antigen-binding signal. A competitive assay is run so that all clones are tested as both ligands and analytes.

[0283] Example 10: Comparison of binding of 5C3D11 with other anti-TL1A antibodies

[0284] Two epitope binning studies were conducted to compare the epitopes recognized by 5C3D11 and 12835 with those recognized by other TL1A antibodies, including 1D1, 1681, 1B4 and 1A9, as shown in Table 26.

[0285] Table 26. Antibody variable region sequences used in epitope binning studies

[0286]

[0287] To minimize affinity effects, a planar carboxymethyl dextran surface sensor chip (Xantec product number SPMXCMDP) was used in the first study, while the HC30M sensor chip (Xantec product number SPMXHC30M) was used in the second study. The run buffer for continuous flow micro-spotting was HBS-EP+, with a flow rate of 65 μl / min. The chip was activated for 7 min in 100 mM MES, pH 5.5 with 18 mM EDC and 4.5 nM sulfo-NHS. The antibody was then fixed with two replicate prints for 15 min. The antibody was diluted to 10 μg / ml in 10 mM acetate at pH 4.5. Antibodies were titrated at three points on the plate at three-fold serial dilutions to establish a concentration series with different spot densities. Each antibody was spotted eight times—twice at each of the four dilutions. This created a 10 x 8 array. Remaining active groups were neutralized using 1 M ethanolamine (pH 8.5) with quenching for 7 min. Since the antigen is a homotrimeric protein and IgG is divalent, premixed conditions are used for epitope binning.

[0288] The first epitope binning study – TL1A was prepared at a final concentration of 50 nM (3.3 μg / ml) and mixed with either 333 nM (50 μg / ml) of analyte (solution phase antibody) or run buffer (control). For the IgG form sample, 50 μg / ml was 333 nM, while for the Fab form sample, 50 μg / ml was 1 μM. The mixed sample was injected onto the array for 5 min and regenerated for 30 sec after each cycle using a 4:1 mixture of Pierce IgG elution buffer and 5 M NaCl (1 M final concentration).

[0289] The second epitope binning study – TL1A was prepared at a final concentration of 50 nM (3.3 μg / ml) and mixed with 1 μM (150 μg / ml) IgG or 2 μM (200 μg / ml) Fab analyte (solution phase antibody) or run buffer (control). The antibody samples were serially diluted 2-fold seven times (7.8 nM for IgG and 15 nM for Fab). The mixed samples were injected onto the array for 5 min and regenerated for 30 sec after each cycle using a 4:1 mixture of Pierce IgG elution buffer and 5 M NaCl (1 M final concentration).

[0290] In the epitope binning study, clear signals (sandwich) were observed using fixed 5C3D11 and 12835, with all control antibodies tested as analytes (Table 27, first two rows). These results demonstrate that 5C3D11 and 12835 can simultaneously bind to TL1A with other antibodies, thus recognizing different epitopes.

[0291] Table 27. Summary of the ability of antibodies to form sandwich structures (yes)

[0292]

[0293] Example 11: In vivo evaluation of anti-TL1A efficacy

[0294] The efficacy of anti-TL1A antibodies in animal models of colitis was evaluated. This was assessed in rodent models of acute colitis induced by intrarectal administration of dinitrobenzenesulfonic acid or trinitrobenzenesulfonic acid (D / TNBS) or oxazolone, as well as in drinking water models induced by DSS or CD45RB transfer. hi Anti-TL1A antibodies were tested in a rodent model of T-induced chronic colitis. DNBS and oxazolone induced localized ulceration and inflammation. DSS administration induced severe generalized intestinal inflammation characterized by erosive lesions and inflammatory infiltration. Symptoms in all these models typically included diarrhea, hematuria, weight loss, and occasional rectal prolapse. In prophylactic models, antibody treatment began with the initial administration of the colitis-inducing compound. In therapeutic models, antibody treatment began a few days after induction. The effects of this treatment on body weight, stool consistency, and hematuria, as well as its microscopic effects on epithelial integrity and the extent of inflammatory infiltration, were determined. Daily clinical scores were given based on stool consistency and the presence of hematuria, resulting in a Disease Activity Index (DAI) score.

[0295] Example 12: Phase 1 Clinical Trial

[0296] A phase 1 clinical trial was conducted to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of the anti-TL1A antibody presented in this study in Crohn's disease subjects.

[0297] Single-Average-Dose (SAD) Arm: Subjects in each group (grouped according to the presence or absence of the risk variant) receive a single dose of the antibody or placebo. Exemplary doses are 1, 3, 10, 30, 100, 300, 600, and 800 mg of antibody, or 5 to 30 mg / kg. Safety monitoring and pharmacokinetic (PK) assessments are performed over a predetermined period. Based on the evaluation of PK data, if the antibody is deemed well-tolerated, dose escalation is performed in the same group of subjects or in another group of healthy subjects. Dose escalation continues until the maximum dose is reached, unless the predetermined maximum exposure is reached or significant intolerance occurs.

[0298] Multiple escalation dose (MAD) arm: Subjects in each group (grouped according to the presence or absence of risk variants) receive multiple doses of either antibody or placebo. Dosage levels and dosing intervals are selected based on dose levels and dosing intervals predicted to be safe according to SAD data. Dosage levels and dosing frequencies are selected to achieve therapeutic drug levels that maintain a steady state in systemic circulation for several days, allowing for monitoring of appropriate safety parameters. Samples are collected and analyzed to determine the pharmacokinetic profile.

[0299] Inclusion criteria: Healthy, infertile subjects aged 18 to 55 years. "Healthy" was defined as the absence of clinically relevant abnormalities detected by detailed medical history, comprehensive physical examination (including blood pressure and pulse rate measurements), 12-lead ECG, and clinical laboratory tests. Infertile female subjects must meet at least one of the following criteria: (1) postmenopausal status, defined as: cessation of regular menstruation for at least 12 consecutive months without other pathological or physiological causes; serum follicle-stimulating hormone (FSH) levels within the laboratory reference range for postmenopausal women; (2) a documented hysterectomy and / or bilateral oophorectomy; (3) medically confirmed ovarian failure. All other female subjects (including those with tubal ligation and those without a documented hysterectomy, bilateral oophorectomy, and / or ovarian failure) will be considered fertile. Body mass index (BMI) of 17.5 to 30.5 kg / m²; weight >50 kg (110 lbs). Evidence of a signed and dated informed consent form demonstrates that the subject (or legal representative) was informed of all relevant aspects of the study.

[0300] Two groups of healthy subjects were selected: subjects with a risk variant, the presence of which was associated with increased susceptibility to Crohn's disease, and subjects without a risk variant.

[0301] Exclusion criteria: Clinically significant evidence or history of hematological, renal, endocrine, pulmonary, gastrointestinal, cardiovascular, hepatic, psychiatric, neurological, or allergic disorders (including drug allergies, but excluding untreated, asymptomatic, or seasonal allergies at the time of administration). Subjects with a history or current positive serological test result for any of the following: hepatitis B surface antigen (HBsAg), hepatitis B core antibody (HBcAb), anti-hepatitis C antibody (HCV Ab), or human immunodeficiency virus (HIV). Subjects with a history of allergic or hypersensitivity reactions to the treatment drug. Subjects treated with the investigational drug within 30 days prior to the first dose of the investigational drug (or as determined by local requirements, whichever is longer) or, for biologics, within 5 half-lives or 180 days prior. Pregnant women; lactating women; and women of childbearing age.

[0302] Primary outcome measures: Incidence of dose-limiting or intolerance-related treatment-related adverse events (AEs) [time range: 12 weeks]. Incidence, severity, and causation of treatment-emergent AEs (TEAEs), and discontinuation of treatment due to treatment-emergent adverse events [time range: 12 weeks]. Incidence and severity of abnormal laboratory test results [time range: 12 weeks]. Abnormal and clinically relevant changes in vital signs, blood pressure (BP), and electrocardiogram (ECG) parameters [time range: 12 weeks].

[0303] Secondary outcome measure: Single escalation dose: Maximum observed plasma concentration (C0) max [Time range: 12 weeks]. Single escalation dose: Time to reach maximum observed plasma concentration (T0). max [Time range: 12 weeks]. Single escalation dose: Plasma concentration-time area under the curve (AUC14 days) from time zero to day 14 [Time range: 12 weeks]. Single escalation dose: Plasma concentration-time area under the curve (AUC14 days) extrapolated from time zero to infinity. inf [Time range: 12 weeks]. Single escalation dose: Area under the plasma concentration-time curve (AUC) from time zero to the final quantifiable concentration. las (t) [Time range: 12 weeks]. Single escalation dose: maximum plasma concentration normalized to dose (C max [dn]) [Time range: 12 weeks]. Single escalation dose: Area under the plasma concentration-time curve (AUC) normalized from time zero extrapolated to infinity. inf [dn]) [Time range: 12 weeks]. Single escalation dose: Area under the plasma concentration-time curve (AUC) normalized from time zero to the final quantifiable concentration. last[dn]) [Time range: 12 weeks]. Single increment dose: Plasma decay half-life (t1 / 2) [Time range: 12 weeks]. Plasma decay half-life is the time it takes for plasma concentration to decrease by half. Single increment dose: Mean residence time (MRT) [Time range: 12 weeks]. Single increment dose: Steady-state distribution volume (V ss [Time range: 6 weeks]. Distribution volume is defined as the theoretical volume at which the total amount of drug needs to be uniformly distributed to produce the desired blood drug concentration. Steady-state distribution volume (V0) ss The apparent volume of distribution at steady state is 0. Single incremental dose: Systemic clearance (CL) [time range: 6]. CL is a quantitative measure of the rate at which the active pharmaceutical ingredient is removed from the body.

[0304] Multiple escalation initial dose: maximum observed plasma concentration (C max [Time range: 12 weeks]. Multiple escalation initial dose: time to reach maximum observed plasma concentration (T0). max [Time range: 12 weeks]. Multiple escalation initial dose: Area under the plasma concentration-time curve (AUC) from time zero to time τ (dosing interval, where τ = 2 weeks). τ [Time range: 12 weeks]. Multiple escalation initial dose: dose-normalized maximum plasma concentration (C0). max [dn]) [Time range: 12 weeks]. Multiple escalation initial dose: Dose-normalized area under the plasma concentration-time curve (AUC) from time zero to time τ (dosing interval, where τ = 2 weeks). τ [dn]) [Time range: 12 weeks]. Plasma decay half-life (t1 / 2) [Time range: 12 weeks]. Plasma decay half-life is the time it takes for the plasma concentration to decrease by half. Multiple escalation first dose: Mean residence time (MRT) [Time range: 12 weeks]. Apparent volume of distribution (Vz / F) [Time range: 12 weeks]. Volume of distribution is defined as the theoretical volume at which the total amount of drug needs to be uniformly distributed to produce the desired blood drug concentration. The apparent volume of distribution (Vz / F) after an oral dose is affected by the fraction of absorption. Multiple escalation first dose: Steady-state volume of distribution (Vz / F) ss [Time range: 12 weeks]. Distribution volume is defined as the theoretical volume at which the total amount of drug needs to be uniformly distributed to produce the desired blood drug concentration. Steady-state distribution volume (V0) ssThe apparent volume of distribution at steady state is 1 / 3. Multiple escalation dose first dose: Apparent oral clearance (CL / F) [Time range: 12 weeks]. Drug clearance is a measure of the rate at which a drug is metabolized or eliminated through normal biological processes. The clearance obtained after an oral dose (apparent oral clearance) is affected by the fraction of dose absorbed. Clearance is estimated using population pharmacokinetic (PK) modeling. Drug clearance is a quantitative measure of the rate at which the active pharmaceutical ingredient (API) is removed from the bloodstream. Multiple escalation dose first dose: Systemic clearance (CL) [Time range: 12 weeks]. CL is a quantitative measure of the rate at which the API is removed from the body.

[0305] Multiple escalation doses: Maximum observed plasma concentration (C max [Time range: 12 weeks]. Multiple escalation doses: Time to reach maximum observed plasma concentration (T0). max [Time range: 12 weeks]. Multiple escalation doses: Area under the plasma concentration-time curve (AUC) from time zero to time τ (dosing interval, where τ = 2 weeks). τ [Time range: 12 weeks]. Multiple escalation doses: maximum plasma concentration normalized to dose (C0). max [dn]) [Time range: 12 weeks]. Multiple escalation doses: Dose-normalized area under the plasma concentration-time curve (AUC) from time zero to time τ (dosing interval, where τ = 2 weeks). τ [dn]) [Time range: 12 weeks]. Multiple escalation doses: Plasma decay half-life (t1 / 2) [Time range: 12 weeks]. Plasma decay half-life is the time it takes for plasma concentration to decrease by half. Multiple escalation doses: Apparent volume of distribution (Vz / F) [Time range: 12 weeks]. Volume of distribution is defined as the theoretical volume at which the total amount of drug needs to be uniformly distributed to produce the desired blood drug concentration. The apparent volume of distribution (Vz / F) after an oral dose is affected by the fraction of absorption. Multiple escalation doses: Steady-state volume of distribution (V ss [Time range: 12 weeks]. Distribution volume is defined as the theoretical volume at which the total amount of drug needs to be uniformly distributed to produce the desired blood drug concentration. Steady-state distribution volume (V0) ss ) is the apparent distributed volume under steady state.

[0306] Multiple Elevation-Dose Multiple Dose: Apparent Oral Clearance (CL / F) [Time Range: 12 Weeks]. Drug clearance is a measure of the rate at which a drug is metabolized or eliminated through normal biological processes. The clearance obtained after an oral dose (apparent oral clearance) is affected by the fraction of dose absorbed. Clearance is estimated using population pharmacokinetic (PK) modeling. Drug clearance is a quantitative measure of the rate at which the active pharmaceutical ingredient (API) is removed from the bloodstream. Multiple Elevation-Dose Multiple Dose: Systemic Clearance (CL) [Time Range: 12 Weeks]. CL is a quantitative measure of the rate at which the API is removed from the body. Multiple Elevation-Dose Multiple Dose: Minimum Observed Plasma Trough Concentration (C min [Time range: 12 weeks]. Multiple escalation doses: Steady-state mean concentration (Cav) [Time range: 12 weeks]. Multiple escalation doses: Observed cumulative ratio (Rac) [Time range: 12 weeks]. Multiple escalation doses: Peak-to-trough fluctuation (PTF) [Time range: 12 weeks]. Additional parameters for multiple escalation doses: Estimate of bioavailability (F) at the corresponding intravenous dose [Time range: 12 weeks]. Immunogenicity of single and multiple escalation doses: Generation of anti-drug antibodies (ADA) [Time range: 12 weeks].

[0307] Example 13: Phase 1b Clinical Trial

[0308] A phase 1b open-label clinical trial will be conducted to evaluate the efficacy of the anti-TL1A antibody presented in this paper in patients with risk variants associated with Crohn's disease.

[0309] Arm: Administer antibodies to 10 patients positive for the risk variant, the presence of which is associated with increased susceptibility to Crohn's disease. Administer antibodies to 5–10 patients negative for the risk variant. Monitor patients in real time. Utilize centrally prepared endoscopy and biopsy, with the reader unaware of the time points and endpoints of treatment.

[0310] Inclusion criteria: Two groups of subjects were selected: subjects with a risk variant, the presence of which was associated with increased susceptibility to Crohn's disease, and subjects without a risk variant.

[0311] Primary outcome measures: Simple Endoscopic Examination of Crohn's Disease (SESCD) score, Crohn's Disease Activity Index (CDAI), and patient-reported outcomes (PRO). A phase 2a clinical trial was initiated if the risk variant-positive group showed a 50% reduction from baseline.

[0312] Inclusion criteria: PRO inclusion criteria: Abdominal pain score of 2 or higher, and / or bowel frequency score of 4 or higher. Primary outcome: Pain score of 0 or 1, bowel frequency score of 3 or lower, and no worsening compared to baseline. Endoscopic inclusion criteria: If the colon is involved, the ileum is included only at scores of 4 and 6. Primary endoscopic outcome: 40-50% difference in mean SESCD.

[0313] Example 14: Phase 2a Clinical Trial

[0314] A phase 2a clinical trial will be conducted to evaluate the efficacy of the anti-TL1A antibody presented in this paper in subjects with Crohn's disease.

[0315] Arms: Forty patients in each arm (antibody and placebo arm) were treated with either antibody or placebo for 12 weeks. An interim analysis was performed after 20 patients in each group were treated with the highest dose to look for 40-50% differences between the placebo and treatment groups in the primary outcomes (50% reduction in SESCD, CDAI, and PRO relative to baseline).

[0316] Primary outcome measures: Simple Endoscopic Examination of Crohn's Disease (SESCD) score, Crohn's Disease Activity Index (CDAI), and patient-reported outcomes (PRO).

[0317] Inclusion criteria: PRO inclusion criteria: Abdominal pain score of 2 or higher, and / or bowel frequency score of 4 or higher. Primary outcome: Pain score of 0 or 1, bowel frequency score of 3 or lower, and no worsening compared to baseline. Endoscopic inclusion criteria: If the colon is involved, the ileum is included only at scores of 4 and 6. Primary endoscopic outcome: 40-50% difference in mean SESCD.

[0318] Various embodiments have been described above in the detailed description. While these descriptions directly depict the above embodiments, it should be understood that modifications and / or alterations to the specific embodiments shown and described herein will occur to those skilled in the art. Any such modifications or alterations falling within the scope of this description are also intended to be included herein. Unless specifically indicated, it is the inventor's intention that the words and phrases in this specification and claims have their common and customary meanings to those skilled in the art.

[0319] The foregoing description of various embodiments known to the applicant at the time of filing this application is presented and is intended for illustrative and descriptive purposes. The description of the invention is not exhaustive, nor is it limited to the precise forms disclosed, and many modifications and alterations can be made in accordance with the foregoing teachings. The described embodiments are intended to explain the principles and practical applications, and to enable others skilled in the art to utilize the various embodiments, optionally with various modifications, and to suit a particular intended use. Therefore, this disclosure is intended to be limited to the specific embodiments disclosed.

[0320] While specific embodiments have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made based on the teachings herein without departing from the scope and broader aspects of this disclosure, and therefore the appended claims are intended to cover all such changes and modifications within their scope and to be covered by the true spirit and scope of this disclosure. Those skilled in the art will understand that, generally, the terms used herein refer to the term "openness" (e.g., the term "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "at least having," the term "comprising" should be interpreted as "including but not limited to," etc.).

[0321] sequence

[0322]

[0323]

[0324]

[0325]

[0326]

[0327] SEQ ID NO:490(L8; VL)

[0328] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK

[0329] SEQ ID NO:491(L8; VH)

[0330] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDVWGQGTTVTVSS

[0331] SEQ ID NO:492 (Clone 34; VL)

[0332] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK

[0333] SEQ ID NO:493 (Clone 34; VH)

[0334] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHIKYDPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDWWGQGTTVTVSS

[0335] SEQ ID NO:494 (Clone 2; VL)

[0336] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK

[0337] SEQ ID NO:495 (Clone 2; VH)

[0338] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHIKYSPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDWWGQGTTVTVSS[[ID=2�]]

[0339] SEQ ID NO:496 (Clone 52; VL)

[0340] EIVLTQSPGTLSLSPGERATLSCGASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK

[0341] SEQ ID NO:497 (Clone 52; VH)

[0342] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHIKYSPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDWWGQGTTVTVSS

[0343] SEQ ID NO:498 (Clone 46; VL)

[0344] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK

[0345] SEQ ID NO:499 (Clone 46; VH)

[0346] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHVKYSPKFQVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDWWGQGTTVTVSS

[0347] SEQ ID NO:500 (Clone 47; VL)

[0348] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK

[0349] SEQ ID NO:501 (Clone 47; VH)

[0350] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHVKYDPKFQTRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDWWGQGTTVTVSS

[0351] SEQ ID NO:502 (Clone 14; VL)

[0352] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK<0000l867>

[0353] SEQ ID NO:503 (Clone 14; VH)

[0354] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHiKYDPKFQkRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS

[0355] SEQ ID NO:504 (Clone 16L; VL)

[0356] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK

[0357] SEQ ID NO:505 (Clone 16L; VH)

[0358] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHvKiDPKFQVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS

[0359] SEQ ID NO:506 (Clone 17L; VL)

[0360] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK

[0361] SEQ ID NO:507 (Clone 17L; VH)

[0362] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS

[0363] SEQ ID NO:508 (Clone 17L-1; VL)

[0364] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK

[0365] SEQ ID NO:509 (Clone 17L-1; VH)

[0366] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQRRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS

[0367] SEQ ID NO:510 (Clone 23; VL)

[0368] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK

[0369] SEQ ID NO:511 (Clone 23; VH)

[0370] It should be noted that there is a small error in the original text where "0000890" and "0000898" should probably be "0000890" and "0000898" respectively. I have translated them as they are in the original but this might be a typo in the source.QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDKWGQGTTVTVSS

[0371] SEQ ID NO:512 (Clone A1; VL)

[0372] EIVLTQSPGTLSLSPGERATLSCGASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK

[0373] SEQ ID NO:513 (Clone A1; VH)

[0374] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDKWGQGTTVTVSS

[0375] SEQ ID NO:514 (Clone 53; VL)

[0376] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK

[0377] SEQ ID NO:515 (Clone 53; VH)

[0378] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHLKYDPKFQERVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDKWGQGTTVTVSS

[0379] SEQ ID NO:516 (Clone E1; VL)

[0380] EIVLTQSPGTLSLSPGERATLSCGASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK

[0381] SEQ ID NO:517 (Clone E1; VH)

[0382] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHLKYDPKFQERVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDKWGQGTTVTVSS

[0383] SEQ ID NO:518 (Clone 3 - 17L V - A; VL)

[0384] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK

[0385] SEQ ID NO:519 (Clone 3 - 17L V - A; VH)

[0386] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS

[0387] SEQ ID NO:520 (Clone 3 - 17L; VL)

[0388] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK

[0389] SEQ ID NO:521 (Clone 3 - 17L; VH)

[0390] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQGRVTITRDTSASTVYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS

[0391] SEQ ID NO:522 (Clone L8mod; VL)

[0392] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK

[0393] SEQ ID NO:523 (Clone L8mod; VH)

[0394] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDVWGQGTTVTVSS

[0395] SEQ ID NO:524 (Clone X-V; VL)

[0396] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK

[0397] SEQ ID NO:525 (Clone X-V; VH)[[ID=二十二]]

[0398] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQVRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS

[0399] SEQ ID NO:526 (Clone X; VL)

[0400] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK

[0401] SEQ ID NO:527 (Clone X; VH)

[0402] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS

[0403] SEQ ID NO:528 (Clone H3-1; VL)

[0404] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK

[0405] SEQ ID NO:529 (Clone H3-1; VH)

[0406] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDLWGQGTTVTVSS

[0407] SEQ ID NO:530 (Clone XL3-6; VL)

[0408] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCSQWSGNPRTFGGGTKLEIK

[0409] SEQ ID NO:531 (Clone XL3-6; VH)

[0410] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS

[0411] SEQ ID NO:532 (Clone XL3-10; VL)

[0412] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRSFGGGTKLEIK

[0413] SEQ ID NO:533 (Clone XL3-10; VH)

[0414] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS

[0415] SEQ ID NO:534 (Clone XL3-15; VL)

[0416] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSRNPRTFGGGTKLEIK

[0417] SEQ ID NO:535 (Clone XL3-15; VH)

[0418] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS

[0419] SEQ ID NO:536 (Clone L3-13; VL)

[0420] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWKGNPRTFGGGTKLEIK

[0421] SEQ ID NO:537 (Clone L3-13; VH)

[0422] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS

[0423] SEQ ID NO:538 (Clone H2-2; VL)

[0424] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK

[0425] SEQ ID NO:539 (Clone H2-2; VH)

[0426] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHSKYDPKFQVRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS

[0427] SEQ ID NO:540 (Clone H2-5; VL)

[0428] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK

[0429] SEQ ID NO:541 (Clone H2-5; VH)

[0430] QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHYKYDPKFQVRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS

[0431] G1 modified by SEQ ID NO:542

[0432] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0433] Constant region of G2 of SEQ ID NO:543

[0434] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0435] Constant region of Kappa of SEQ ID NO:544

[0436] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0437] SEQ ID NO:545(L8 HFR1)

[0438] QVQLVQSGAEVKKPGASVKVSCKAS

[0439] SEQ ID NO:546(L8 HFR2)

[0440] WVRQAPGQGLEWMG

[0441] SEQ ID NO:547(L8 HFR3)

[0442] RVTMTRDTSTSTVYMELSSLRSEDTAVYYC

[0443] SEQ ID NO:548(L8 HFR4)

[0444] WGQGTTVTVSS

[0445] SEQ ID NO:549(L8 LFR1)

[0446] EIVLTQSPGTLSLSPGERATLSC

[0447] SEQ ID NO:550(L8 LFR2)

[0448] WYQQKPGQAPRLLIY

[0449] SEQ ID NO:551(L8 LFR3)

[0450] GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC

[0451] SEQ ID NO:552(L8 LFR4)

[0452] FGGGTKLEIK

[0453] SEQ ID NO:586(VH FR3)

[0454] RATITTDTSASTAYLQLSSLRSEDTAVYYC

[0455] SEQ ID NO:587(VH FR3)

[0456] RVTITRDTSASTVYMELSSLRSEDTAVYYC

[0457] SEQ ID NO:588(VH FR3)

[0458] RVTITRDTSASTAYMELSSLRSEDTAVYYC

Claims

1. An antibody or antigen-binding fragment that specifically binds to TL1A, comprising: Heavy chain variable region, which contains: (a) HCDR1 containing the amino acid sequence shown in SEQ ID NO:553; (b) HCDR2 comprising the amino acid sequence shown in any one of SEQ ID NO: 554 to 564 or 574 to 577; and (c) HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 565 to 568 or 578 to 581; and The light chain variable region, which includes: (d) LCDR1 containing the amino acid sequence shown in either SEQ ID NO:569 or 570; (e) LCDR2 comprising the amino acid sequence shown in SEQ ID NO:488; and (f) LCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO:571 to 573 or 582 to 585.

2. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 1, wherein: (a) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:553; (b) The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:559; (c) The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:567; (d) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO:569; (e) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:488; and (f) The LCDR3 contains the amino acid sequence shown in SEQ ID NO:

573.

3. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 1, wherein: (a) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:553; (b) The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:563; (c) The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:568; (d) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO:569; (e) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:488; and (f) The LCDR3 contains the amino acid sequence shown in SEQ ID NO:

572.

4. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 1, wherein: (a) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:553; (b) The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:555; (c) The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:566; (d) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO:569; (e) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:488; and (f) The LCDR3 contains the amino acid sequence shown in SEQ ID NO:

572.

5. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 1, wherein: (a) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:553; (b) The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:558; (c) The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:566; (d) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO:569; (e) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:488; and (f) The LCDR3 contains the amino acid sequence shown in SEQ ID NO:

572.

6. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 1, wherein: (a) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:553; (b) The HCDR2 comprises the amino acid sequence shown in SEQ ID NO:564; (c) The HCDR3 comprises the amino acid sequence shown in SEQ ID NO:568; (d) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO:569; (e) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO:488; and (f) The LCDR3 contains the amino acid sequence shown in SEQ ID NO:

572.

7. An antibody or antigen-binding fragment that specifically binds to TL1A, comprising: a heavy chain variable region, comprising: (a) HCDR1, HCDR2, and HCDR3 selected from any one of SEQ ID NO: 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, or 541; and Light chain variable region, which contains (b) LCDR1, LCDR2 and LCDR3 selected from any one of SEQ ID NO: 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538 or 540; The CDR is defined by the Kabat, Chothia, or IMGT method or a combination thereof.

8. The antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 7, comprising: (a) Human heavy chain framework region 1, which is at least 95% identical to that shown in SEQ ID NO:545; (b) Human heavy chain framework region 2, which is at least 95% identical to that shown in SEQ ID NO:546; (c) Human heavy chain framework region 3, which is at least 95% identical to that shown in SEQ ID NO: 547 or 586 to 588; (d) Human heavy chain framework region 4, which is at least 95% identical to that shown in SEQ ID NO:548; (e) Human light chain framework region 1, which is at least 95% identical to that shown in SEQ ID NO:549; (f) Human light chain framework region 2, which is at least 95% identical to that shown in SEQ ID NO:550; (g) a human light chain framework region 3 that is at least 95% identical to that shown in SEQ ID NO:551; and (h) is at least 95% identical to the human light chain framework region 4 shown in SEQ ID NO:

552.

9. The antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 8, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 90% identical to any one of SEQ ID NO: 503, 511, 493, 501 or 515; and the light chain variable region comprises an amino acid sequence that is at least about 90% identical to any one of SEQ ID NO: 502, 510, 492, 500 or 514.

10. The antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 8, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO: 503, 511, 493, 501 or 515; and the light chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO: 502, 510, 492, 500 or 514.

11. An antibody or antigen-binding fragment that specifically binds to TL1A, comprising: (a) A heavy chain variable region comprising an amino acid sequence that is at least about 90% identical to any one of SEQ ID NO: 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, or 541; and (b) A light chain variable region comprising an amino acid sequence that is at least about 90% identical to any one of SEQ ID NO: 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538 or 540.

12. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 11, wherein the heavy chain variable region comprises at least about 90% of the same amino acid sequence as SEQ ID NO:503; and wherein the light chain variable region comprises at least about 90% of the same amino acid sequence as SEQ ID NO:

502.

13. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 12, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:503; and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

502.

14. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 11, wherein the heavy chain variable region comprises at least about 90% of the same amino acid sequence as SEQ ID NO:511; and wherein the light chain variable region comprises at least about 90% of the same amino acid sequence as SEQ ID NO:

510.

15. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 14, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:511; and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

510.

16. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 11, wherein the heavy chain variable region comprises at least about 90% of the same amino acid sequence as SEQ ID NO:493; and wherein the light chain variable region comprises at least about 90% of the same amino acid sequence as SEQ ID NO:

492.

17. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 16, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:493; and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

492.

18. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 11, wherein the heavy chain variable region comprises at least about 90% of the same amino acid sequence as SEQ ID NO:501; and wherein the light chain variable region comprises at least about 90% of the same amino acid sequence as SEQ ID NO:

500.

19. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 18, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:501; and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

500.

20. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 11, wherein the heavy chain variable region comprises at least about 90% of the same amino acid sequence as SEQ ID NO:515; and wherein the light chain variable region comprises at least about 90% of the same amino acid sequence as SEQ ID NO:

514.

21. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 20, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:515; and wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

514.

22. The antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 21, wherein, as determined by ELISA, the antibody binds to human TL1A with a stronger affinity than the L8 clone, wherein the L8 clone comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:491 and the light chain variable region amino acid sequence shown in SEQ ID NO:

490.

23. The antibody or antigen-binding fragment that specifically binds to TL1A according to claim 22, wherein, as determined by ELISA, the antibody binds to human TL1A with an affinity at least 2 times stronger than that of the L8 clone, wherein the L8 clone comprises the heavy chain variable region amino acid sequence shown in SEQ ID NO:491 and the light chain variable region amino acid sequence shown in SEQ ID NO:

490.

24. The antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment is chimeric or humanized.

25. The antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment is an IgG antibody.

26. The antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 25, wherein the antibody or antigen-binding fragment comprises Fab, F(ab)2, a single-domain antibody, a single-chain variable fragment (scFv), or a nanobody.

27. The antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 26, comprising a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO: 542 or 543.

28. The antibody or antigen-binding fragment according to any one of claims 1 to 26, comprising a heavy chain constant region containing the amino acid sequence shown in SEQ ID NO:

542.

29. The antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 26, comprising a light chain constant region containing the amino acid sequence shown in SEQ ID NO:

544.

30. An antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 29, wherein the antibody or antigen-binding fragment thereof inhibits TL1A-induced secretion of interferon γ by T lymphocytes.

31. A pharmaceutical composition comprising an antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 30, and a pharmaceutically acceptable excipient, carrier, or diluent.

32. The pharmaceutical composition according to claim 31, which is formulated for intravenous administration.

33. An antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 30, or a pharmaceutical composition according to claim 31 or 32, for the treatment of inflammatory bowel disease, Crohn's disease, or colitis.

34. A method of treating an individual with inflammatory bowel disease, Crohn's disease, or colitis, comprising administering to the individual an effective amount of an antibody or antigen-binding fragment specifically binding to TL1A according to any one of claims 1 to 30, or a pharmaceutical composition according to claim 31 or 32, wherein the individual is diagnosed with or suspected of having inflammatory bowel disease, Crohn's disease, or colitis.

35. An antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 30, or a pharmaceutical composition according to claim 31 or 32, for preventing or reducing interferon-γ secretion by T lymphocytes.

36. A method for preventing or reducing the secretion of interferon-γ by T lymphocytes in an individual, comprising administering to the individual an effective amount of an antibody or antigen-binding fragment specifically binding to TL1A according to any one of claims 1 to 30, or a pharmaceutical composition according to claim 31 or 32.

37. A nucleic acid encoding an antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 30.

38. A cell comprising the nucleic acid of claim 37.

39. The cell of claim 38, wherein the cell is a eukaryotic cell.

40. The cell according to claim 38, wherein the cell is a Chinese hamster ovary (CHO) cell.

41. A method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis, comprising incubating cells of any one of claims 38 to 40 in a culture medium under conditions sufficient to secrete an antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 30 into the culture medium.

42. The method of claim 41, further comprising subjecting the culture medium to at least one purification step.

43. A method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis, comprising mixing an antibody or antigen-binding fragment that specifically binds to TL1A according to any one of claims 1 to 30 with a pharmaceutically acceptable excipient, carrier, or diluent.

44. A method of treating a disease or condition in an individual having a risk variant associated with the disease or condition, the method comprising administering to the individual having a risk variant of the disease or condition an effective amount of an antibody or antigen-binding fragment specifically binding to TL1A of any one of claims 1 to 30 or a pharmaceutical composition of claim 31 or 32, wherein the disease or condition includes at least one of inflammatory bowel disease (IBD), Crohn's disease (CD), or colitis.

45. The method of claim 44, wherein the individual has multiple risk variants.

46. ​​The method of claim 45, wherein the plurality of risk variants is at least 3, 4, 5 or 10 risk variants.

47. The method of any one of claims 44 to 46, wherein the risk variants of the plurality of risk variants are associated with a subclinical phenotype of the disease or condition.

48. The method of claim 44, wherein the disease or condition is a severe form of at least one of IBD, CD, or colitis.

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