Optimized anti-TL1A antibody

Abstract

To provide novel therapeutics to treat IBD that specifically target enzymes involved in pathogenesis of inflammatory bowel disease (IBD), such as Crohns Disease (CD) and ulcerative colitis (UC).SOLUTION: The present invention provides humanized anti-TL1A antibodies and pharmaceutical compositions, wherein the antibodies possess superior therapeutic aspects compared to other tumor necrosis factor ligand 1A (TL1A) binding antibodies. The antibodies possess high sequence homology to human germline networks while still exhibiting high binding affinity, express at high levels in bacterial and mammalian culture, and possess fewer sequence liabilities, such as deamidation sites, that lead to increased degradation and reduced therapeutic effect.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits of U.S. Provisional Patent Application No. 62 / 662,605, filed on 25 April 2018, and U.S. Provisional Patent Application No. 62 / 756,494, filed on 6 November 2018, which are incorporated herein by reference. [Background technology]

[0002] Inflammatory bowel disease (IBD) refers to a group of intestinal diseases that cause inflammation in the gastrointestinal tract. The standard specimens of IBD are ulcerative colitis (UC) and Crohn's disease (CD). These diseases are prevalent, with approximately 1.86 million people diagnosed with UC and approximately 1.3 million people diagnosed with CD.

[0003] Each of these forms has a variety of asymptomatic phenotypic characteristics of severe IBD that are present in subpopulations of patients with CD and UC. One such disease is obstructive Crohn's disease, which can result from long-term inflammation that can lead to the formation of scar tissue (fibrostenosis) or bulging in the intestinal wall. Both outcomes can lead to narrowing (i.e., obstruction), known as either fibrotic stenosis or inflammatory stenosis. Severe stenosis can cause intestinal obstruction, leading to abdominal pain, bloating, nausea, and inability to defecate. Another example is the penetrating disease phenotype, characterized by intestinal obstruction or internal penetrating fistulas, or both, which often leads to complications associated with IBD, including, for example, intraperitoneal sepsis.

[0004] Unfortunately, the number of treatments available for IBD patients is limited, and the development of new treatments is hindered by suboptimal results in clinical trials. Existing anti-inflammatory treatments, such as steroids and tumor necrosis factor (TNF) inhibitors, are typically used as first-line treatments for IBD. Sadly, a significant number of patients experience a lack of or complete loss of response to existing anti-inflammatory treatments, particularly TNF-α inhibitors. Patients are treated with ineffective anti-inflammatory treatments, and their disease worsens. Surgical interventions in the form of strictureplasty (reshaping of the bowel) or resection (removal of the bowel) are the only treatment option for patients who do not respond to first-line treatment. Surgical treatment for IBD is invasive, and one-third of patients undergoing surgery experience postoperative risks, such as anastomotic leakage, infection, and bleeding.

[0005] The cause of IBD appears to involve an uncontrolled immune response that may be triggered by certain environmental factors in genetically susceptible hosts. Coupled with diverse responses to treatment and their associated side effects, the heterogeneity of etiology and clinical course suggests that targeted therapeutic approaches to the treatment of these diseases are the best course of action. However, there are few targeted therapies available for IBD patients, especially those who may be unresponsive to existing IBD treatments (e.g., anti-TNFα inhibitors). Therefore, there is a need for novel therapies to treat IBD that specifically target enzymes involved in the pathogenesis of IBD. [Overview of the project]

[0006] This disclosure provides antibodies useful for the treatment of IBD, including moderate to severe forms of IBD characterized by the asymptomatic phenotype disclosed herein (e.g., refractory disease, stenotic disease, penetrating disease). The antibodies described herein have superior therapeutic aspects compared to other tumor necrosis factor ligand 1A (TL1A) binding antibodies. First, the antibodies described herein exhibit high sequence homology to the human germline network while still showing high binding affinity, and have smaller sequence liabilities, such as deamidation sites, which are expressed at high levels in bacterial and mammalian cultures, leading to increased degradation and reduced therapeutic efficacy.

[0007] TL1A and the nucleic acid encoding TL1A (tumor necrosis factor ligand superfamily member 15 (TNFSF15)) are provided in Entrez Gene: 9966; UniProtKB: O95150. TL1A is an 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 be involved in the pathogenesis of IBD by bridging innate and adaptive immune responses and modulating adaptive immunity by enhancing Th1, Th2, and Th17 effector cell function, as well as in the immunopathology of T cell accumulation and inflammatory tissues.

[0008] Certain genotypes containing polymorphisms identified in the TNFSF15 gene are associated with an increased risk of developing IBD (e.g., UC or CD) or an asymptomatic phenotype of IBD, and therefore predict the risk of developing IBD (e.g., UC or CD) or an asymptomatic phenotype of IBD. TL1A mRNA expression is elevated in patients diagnosed with IBD who possess these risk genotypes. Therefore, inhibition of TL1A expression and / or activity is a promising therapeutic strategy for various T-cell-dependent autoimmune diseases, including IBD (e.g., UC and CD).

[0009] In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A is provided herein, the antibody or antigen-binding fragment comprising: a heavy chain variable region comprising: (a) HCDR1 comprising an amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising an amino acid sequence described by any one of SEQ ID NO: 554-564 or 574-577; (c) HCDR3 comprising an amino acid sequence described by any one of SEQ ID NO: 565-568 or 578-581; and a light chain variable region comprising: (d) LCDR1 comprising an amino acid sequence described by any one of SEQ ID NO: 569 or 570; (e) LCDR2 comprising an amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising an amino acid sequence described by any one of SEQ ID NO: 571-573 or 582-585. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 1 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 545. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 2 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 546. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 547 or 586-588. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 548. In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 1 which is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO:549.In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 2 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 550. In another embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 551. In yet another embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 552. In one embodiment, the antibody or antigen-binding fragment comprises: (a) a human heavy chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 545; (b) a human heavy chain framework region 2 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 546; (c) a human heavy chain framework region 3 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 547 or 586-588; (d) a human heavy chain framework region 4 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 548; (e) a human light chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 549; (f) SEQ ID (g) Human light chain framework region 2, which is at least 90%, 95%, 97%, or 98% identical to that described in NO:550; (h) Human light chain framework region 3, which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:551; and (g) Human light chain framework region 4, which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:552.In some embodiments, the antibody binds to human TL1A with stronger affinity, or twice as strong affinity, compared to the L8 clone, as determined by ELISA, where the L8 clone comprises a heavy chain variable region amino acid sequence described by SEQ ID NO: 491 and a light chain variable region amino acid sequence described by 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 comprising an amino acid sequence described by SEQ ID NO: 542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising an amino acid sequence described by SEQ ID NO: 542. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence described by SEQ ID NO: 544, comprising a light chain constant region. In one embodiment, the antibody or its antigen-binding fragment inhibits TL1A-induced secretion of interferon-γ from T lymphocytes. In one embodiment, 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 one embodiment, the pharmaceutical composition is formulated for intravenous administration. In one embodiment, the antibody or antigen-binding fragment, or the pharmaceutical composition, is intended for use in the treatment of inflammatory bowel disease, Crohn's disease, or colitis. In one embodiment, the antibody or antigen-binding fragment is encoded by a nucleic acid. In one embodiment, the cells contain nucleic acids. In one embodiment, the cells are eukaryotic cells. In one embodiment, the cells are Chinese hamster ovary (CHO) cells.In some embodiments, a method for treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising the step of administering an effective amount of an antibody or antigen-binding fragment or pharmaceutical composition to the individual, wherein the individual is diagnosed with or suspected of suffering from inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is for use in preventing or reducing interferon-gamma secretion by T lymphocytes. In some embodiments, a method for preventing or reducing interferon-gamma secretion by T lymphocytes in an individual is described herein, comprising the step of administering an effective amount of an antibody or antigen-binding fragment or pharmaceutical composition to the individual. In some embodiments, a method for preparing a treatment agent for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising the step of incubating cells containing nucleic acids encoding an antibody or antigen-binding fragment in a culture medium under conditions sufficient to secrete 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 one embodiment, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising the step of mixing an antibody or antigen-binding fragment with a pharmaceutically acceptable excipient, carrier, or diluent.

[0010] In other embodiments, an antibody or antigen-binding fragment that specifically binds to TL1A is provided herein, the antibody or antigen-binding fragment comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence described by SEQ ID NO: 559; (c) HCDR3 comprising the amino acid sequence described by SEQ ID NO: 567; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence described by any one of SEQ ID NO: 573. In one embodiment, the antibody or antigen-binding fragment comprises a human heavy chain framework region 1 which is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described by SEQ ID NO: 545. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 2 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 546. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 547 or 586-588. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 548. In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 1 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 549. In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 2 which is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO:550.In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 551. In another embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 552. In one embodiment, the antibody or antigen-binding fragment comprises: (a) a human heavy chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 545; (b) a human heavy chain framework region 2 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 546; (c) a human heavy chain framework region 3 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 547 or 586-588; (d) a human heavy chain framework region 4 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 548; (e) a human light chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 549; (f) SEQ ID (g) a human light chain framework region 2 which is at least 90%, 95%, 97%, or 98% identical to that described in NO:550; (h) a human light chain framework region 3 which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:551; and (g) a human light chain framework region 4 which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:552. In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A includes: (a) a heavy chain variable region containing an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:503; and (b) a light chain variable region containing an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:502.In some embodiments, the antibody binds to human TL1A with a stronger affinity or twice as strong an affinity compared to the L8 clone, as determined by ELISA, where the L8 clone comprises a heavy chain variable region amino acid sequence described by SEQ ID NO: 491 and a light chain variable region amino acid sequence described by 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 a 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 comprising an amino acid sequence described by SEQ ID NO: 542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising an amino acid sequence described by SEQ ID NO: 542. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence described by SEQ ID NO: 544, comprising a light chain constant region. In one embodiment, the antibody or its antigen-binding fragment inhibits TL1A-induced secretion of interferon-γ from T lymphocytes. In one embodiment, 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 one embodiment, the pharmaceutical composition is formulated for intravenous administration. In one embodiment, the antibody or antigen-binding fragment, or the pharmaceutical composition, is intended for use in the treatment of inflammatory bowel disease, Crohn's disease, or colitis. In one embodiment, the antibody or antigen-binding fragment is encoded by a nucleic acid. In one embodiment, the cells contain nucleic acids. In one embodiment, the cells are eukaryotic cells. In one embodiment, the cells are Chinese hamster ovary (CHO) cells.In some embodiments, a method for treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising the step of administering an effective amount of an antibody or antigen-binding fragment or pharmaceutical composition to the individual, wherein the individual is diagnosed with or suspected of suffering from inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is for use in preventing or reducing interferon-gamma secretion by T lymphocytes. In some embodiments, a method for preventing or reducing interferon-gamma secretion by T lymphocytes in an individual is described herein, comprising the step of administering an effective amount of an 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 the step of incubating cells containing nucleic acids encoding an antibody or antigen-binding fragment in a culture medium under conditions sufficient to secrete 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 one embodiment, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising the step of mixing an antibody or antigen-binding fragment with a pharmaceutically acceptable excipient, carrier, or diluent.

[0011] In other embodiments, antibodies or antigen-binding fragments that specifically bind to TL1A are provided herein, the antibodies or antigen-binding fragments comprising a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence described by SEQ ID NO: 563; and (c) HCDR3 comprising the amino acid sequence described by SEQ ID NO: 568; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence described by any one of SEQ ID NO: 572. In one embodiment, the antibodies or antigen-binding fragment comprises a human heavy chain framework region 1 which is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described by SEQ ID NO: 545. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 2 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 546. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 547 or 586-588. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 548. In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 1 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 549. In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 2 which is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO:550.In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 551. In another embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 552. In one embodiment, the antibody or antigen-binding fragment comprises: (a) a human heavy chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 545; (b) a human heavy chain framework region 2 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 546; (c) a human heavy chain framework region 3 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 547 or 586-588; (d) a human heavy chain framework region 4 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 548; (e) a human light chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 549; (f) SEQ ID (g) a human light chain framework region 2 which is at least 90%, 95%, 97%, or 98% identical to that described in NO:550; (h) a human light chain framework region 3 which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:551; and (g) a human light chain framework region 4 which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:552. In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A includes: (a) a heavy chain variable region containing an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:511; and (b) a light chain variable region containing an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:510.In some embodiments, the antibody binds to human TL1A with a stronger affinity or twice as strong an affinity compared to the L8 clone, as determined by ELISA, where the L8 clone comprises a heavy chain variable region amino acid sequence described by SEQ ID NO: 491 and a light chain variable region amino acid sequence described by 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 a 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 comprising an amino acid sequence described by SEQ ID NO: 542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising an amino acid sequence described by SEQ ID NO: 542. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence described by SEQ ID NO: 544, comprising a light chain constant region. In one embodiment, the antibody or its antigen-binding fragment inhibits TL1A-induced secretion of interferon-γ from T lymphocytes. In one embodiment, 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 one embodiment, the pharmaceutical composition is formulated for intravenous administration. In one embodiment, the antibody or antigen-binding fragment, or the pharmaceutical composition, is intended for use in the treatment of inflammatory bowel disease, Crohn's disease, or colitis. In one embodiment, the antibody or antigen-binding fragment is encoded by a nucleic acid. In one embodiment, the cells contain nucleic acids. In one embodiment, the cells are eukaryotic cells. In one embodiment, the cells are Chinese hamster ovary (CHO) cells.In certain embodiments, methods for treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis are described herein, including the step of administering to the individual an effective amount of an antibody or antigen-binding fragment or a pharmaceutical composition, where the individual has been diagnosed with or is suspected of having inflammatory bowel disease, Crohn's disease, or colitis. In certain embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is for use in preventing or reducing interferon-γ secretion by T lymphocytes. In certain embodiments, methods are described herein for preventing or reducing interferon-γ secretion by T lymphocytes in an individual, including the step of administering to the individual an effective amount of an antibody or antigen-binding fragment or a pharmaceutical composition. In certain embodiments, methods for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis are described herein, including the step of incubating a cell comprising a nucleic acid encoding an antibody or antigen-binding fragment in a medium under conditions sufficient for the antibody or antigen-binding fragment to be secreted into the medium. In certain embodiments, the method further comprises subjecting the medium to at least one purification step. In certain embodiments, methods for preparing a therapeutic agent for inflammatory bowel disease, Crohn's disease, or colitis are described herein, including the step of mixing an antibody or antigen-binding fragment and a pharmaceutically acceptable excipient, carrier, or diluent.

[0012] In other embodiments, antibodies or antigen-binding fragments that specifically bind to TL1A are provided herein, the antibodies or antigen-binding fragments comprising a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence described by SEQ ID NO: 555; and (c) HCDR3 comprising the amino acid sequence described by SEQ ID NO: 566; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence described by any one of SEQ ID NO: 572. In one embodiment, the antibodies or antigen-binding fragment comprises a human heavy chain framework region 1 which is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described by SEQ ID NO: 545. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 2 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 546. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 547 or 586-588. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 548. In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 1 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 549. In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 2 which is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO:550.In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 551. In another embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 552. In one embodiment, the antibody or antigen-binding fragment comprises: (a) a human heavy chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 545; (b) a human heavy chain framework region 2 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 546; (c) a human heavy chain framework region 3 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 547 or 586-588; (d) a human heavy chain framework region 4 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 548; (e) a human light chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 549; (f) SEQ ID (g) a human light chain framework region 2 which is at least 90%, 95%, 97%, or 98% identical to that described in NO:550; (h) a human light chain framework region 3 which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:551; and (g) a human light chain framework region 4 which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:552. In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A includes: (a) a heavy chain variable region containing an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:493; and (b) a light chain variable region containing an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:492.In some embodiments, the antibody binds to human TL1A with a stronger affinity or twice as strong an affinity compared to the L8 clone, as determined by ELISA, where the L8 clone comprises a heavy chain variable region amino acid sequence described by SEQ ID NO: 491 and a light chain variable region amino acid sequence described by 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 a 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 comprising an amino acid sequence described by SEQ ID NO: 542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising an amino acid sequence described by SEQ ID NO: 542. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence described by SEQ ID NO: 544, comprising a light chain constant region. In one embodiment, the antibody or its antigen-binding fragment inhibits TL1A-induced secretion of interferon-γ from T lymphocytes. In one embodiment, 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 one embodiment, the pharmaceutical composition is formulated for intravenous administration. In one embodiment, the antibody or antigen-binding fragment, or the pharmaceutical composition, is intended for use in the treatment of inflammatory bowel disease, Crohn's disease, or colitis. In one embodiment, the antibody or antigen-binding fragment is encoded by a nucleic acid. In one embodiment, the cells contain nucleic acids. In one embodiment, the cells are eukaryotic cells. In one embodiment, the cells are Chinese hamster ovary (CHO) cells.In some embodiments, a method for treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising the step of administering an effective amount of an antibody or antigen-binding fragment or pharmaceutical composition to the individual, wherein the individual is diagnosed with or suspected of suffering from inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is for use in preventing or reducing interferon-gamma secretion by T lymphocytes. In some embodiments, a method for preventing or reducing interferon-gamma secretion by T lymphocytes in an individual is described herein, comprising the step of administering an effective amount of an 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 the step of incubating cells containing nucleic acids encoding an antibody or antigen-binding fragment in a culture medium under conditions sufficient to secrete 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 one embodiment, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising the step of mixing an antibody or antigen-binding fragment with a pharmaceutically acceptable excipient, carrier, or diluent.

[0013] In other embodiments, antibodies or antigen-binding fragments that specifically bind to TL1A are provided herein, the antibodies or antigen-binding fragments comprising a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence described by SEQ ID NO: 558; and (c) HCDR3 comprising the amino acid sequence described by SEQ ID NO: 566; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence described by any one of SEQ ID NO: 572. In one embodiment, the antibodies or antigen-binding fragment comprises a human heavy chain framework region 1 which is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described by SEQ ID NO: 545. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 2 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 546. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 547 or 586-588. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 548. In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 1 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 549. In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 2 which is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO:550.In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 551. In another embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 552. In one embodiment, the antibody or antigen-binding fragment comprises: (a) a human heavy chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 545; (b) a human heavy chain framework region 2 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 546; (c) a human heavy chain framework region 3 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 547 or 586-588; (d) a human heavy chain framework region 4 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 548; (e) a human light chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 549; (f) SEQ ID (g) a human light chain framework region 2 which is at least 90%, 95%, 97%, or 98% identical to that described in NO:550; (h) a human light chain framework region 3 which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:551; and (g) a human light chain framework region 4 which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:552. In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A includes: (a) a heavy chain variable region containing an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:501; and (b) a light chain variable region containing an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:500.In some embodiments, the antibody binds to human TL1A with a stronger affinity or twice as strong an affinity compared to the L8 clone, as determined by ELISA, where the L8 clone comprises a heavy chain variable region amino acid sequence described by SEQ ID NO: 491 and a light chain variable region amino acid sequence described by 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 a 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 comprising an amino acid sequence described by SEQ ID NO: 542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising an amino acid sequence described by SEQ ID NO: 542. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence described by SEQ ID NO: 544, comprising a light chain constant region. In one embodiment, the antibody or its antigen-binding fragment inhibits TL1A-induced secretion of interferon-γ from T lymphocytes. In one embodiment, 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 one embodiment, the pharmaceutical composition is formulated for intravenous administration. In one embodiment, the antibody or antigen-binding fragment or the pharmaceutical composition is for use in the treatment of inflammatory bowel disease. In one embodiment, the antibody or antigen-binding fragment is encoded by a nucleic acid. In one embodiment, the cells contain nucleic acids. In one embodiment, the cells are eukaryotic cells. In one embodiment, the cells are Chinese hamster ovary (CHO) cells.In some embodiments, a method for treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising the step of administering an effective amount of an antibody or antigen-binding fragment or pharmaceutical composition to the individual, wherein the individual is diagnosed with or suspected of suffering from inflammatory bowel disease, Crohn's disease, or colitis. In some embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is for use in preventing or reducing interferon-gamma secretion by T lymphocytes. In some embodiments, a method for preventing or reducing interferon-gamma secretion by T lymphocytes in an individual is described herein, comprising the step of administering an effective amount of an 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 the step of incubating cells containing nucleic acids encoding an antibody or antigen-binding fragment in a culture medium under conditions sufficient to secrete 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 one embodiment, a method for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis is described herein, comprising the step of mixing an antibody or antigen-binding fragment with a pharmaceutically acceptable excipient, carrier, or diluent.

[0014] In other embodiments, antibodies or antigen-binding fragments that specifically bind to TL1A are provided herein, the antibodies or antigen-binding fragments comprising a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence described by SEQ ID NO: 564; and (c) HCDR3 comprising the amino acid sequence described by SEQ ID NO: 568; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence described by any one of SEQ ID NO: 572. In one embodiment, the antibodies or antigen-binding fragment comprises a human heavy chain framework region 1 which is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described by SEQ ID NO: 545. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 2 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 546. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 547 or 586-588. In one embodiment, the antibody or antigen-binding fragment includes a human heavy chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 548. In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 1 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 549. In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 2 which is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO:550.In one embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 551. In another embodiment, the antibody or antigen-binding fragment includes a human light chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, and 99% identical to that described in SEQ ID NO: 552. In one embodiment, the antibody or antigen-binding fragment comprises: (a) a human heavy chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 545; (b) a human heavy chain framework region 2 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 546; (c) a human heavy chain framework region 3 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 547 or 586-588; (d) a human heavy chain framework region 4 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 548; (e) a human light chain framework region 1 that is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO: 549; (f) SEQ ID (g) a human light chain framework region 2 which is at least 90%, 95%, 97%, or 98% identical to that described in NO:550; (h) a human light chain framework region 3 which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:551; and (g) a human light chain framework region 4 which is at least 90%, 95%, 97%, or 98% identical to that described in SEQ ID NO:552. In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A includes: (a) a heavy chain variable region containing an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:515; and (b) a light chain variable region containing an amino acid sequence that is at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:514.In some embodiments, the antibody binds to human TL1A with a stronger affinity or twice as strong an affinity compared to the L8 clone, as determined by ELISA, where the L8 clone comprises a heavy chain variable region amino acid sequence described by SEQ ID NO: 491 and a light chain variable region amino acid sequence described by 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 a 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 comprising an amino acid sequence described by SEQ ID NO: 542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising an amino acid sequence described by SEQ ID NO: 542. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence described by SEQ ID NO: 544, comprising a light chain constant region. In one embodiment, the antibody or its antigen-binding fragment inhibits TL1A-induced secretion of interferon-γ from T lymphocytes. In one embodiment, 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 one embodiment, the pharmaceutical composition is formulated for intravenous administration. In one embodiment, the antibody or antigen-binding fragment, or the pharmaceutical composition, is intended for use in the treatment of inflammatory bowel disease, Crohn's disease, or colitis. In one embodiment, the antibody or antigen-binding fragment is encoded by a nucleic acid. In one embodiment, the cells contain nucleic acids. In one embodiment, the cells are eukaryotic cells. In one embodiment, the cells are Chinese hamster ovary (CHO) cells.In certain embodiments, methods for treating an individual suffering from inflammatory bowel disease, Crohn's disease, or colitis are described herein, comprising the step of administering to the individual an effective amount of an antibody or antigen-binding fragment or a pharmaceutical composition, where the individual is diagnosed with or suspected of having inflammatory bowel disease, Crohn's disease, or colitis. In certain embodiments, the antibody or antigen-binding fragment or pharmaceutical composition is for use in preventing or reducing interferon-γ secretion by T lymphocytes. In certain embodiments, methods are described herein for preventing or reducing interferon-γ secretion by T lymphocytes in an individual, comprising the step of administering to the individual an effective amount of an antibody or antigen-binding fragment or a pharmaceutical composition. In certain embodiments, methods for preparing a treatment for inflammatory bowel disease, Crohn's disease, or colitis are described herein, comprising the step of incubating a cell comprising a nucleic acid encoding an antibody or antigen-binding fragment in a medium under conditions sufficient for secretion of the antibody or antigen-binding fragment into the medium. In certain embodiments, the method further comprises subjecting the medium to at least one purification step. In certain embodiments, methods for preparing a therapeutic agent for inflammatory bowel disease, Crohn's disease or colitis are described herein, comprising the step of mixing an antibody or antigen-binding fragment and a pharmaceutically acceptable excipient, carrier, or diluent.

[0015] In other embodiments, antibodies or antigen-binding fragments that specifically bind to TL1A are provided herein, wherein the antibodies or antigen-binding fragments include: (a) 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 heavy chain variable region comprising HCDR1, HCDR2, and HCDR3; (b) SEQ ID NO: Includes a light chain variable region containing LCDR1, LCDR2, and LCDR3 from 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; where CDR is defined by the Kabat method, the Chothia method, or the IMGT method, or a combination thereof. In one embodiment, the antibody or antigen-binding fragment that specifically binds to TL1A is: (a) a heavy chain variable region containing an amino acid sequence that is at least approximately 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to one of the following SEQ ID NOs: 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) SEQ ID NO:Includes a light chain variable region containing an amino acid sequence that is at least approximately 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to 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.In some embodiments, the antibody binds to human TL1A with a stronger affinity or twice as strong an affinity compared to the L8 clone, as determined by ELISA, where the L8 clone comprises a heavy chain variable region amino acid sequence described by SEQ ID NO: 491 and a light chain variable region amino acid sequence described by 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 a 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 comprising an amino acid sequence described by SEQ ID NO: 542 or 543. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising an amino acid sequence described by SEQ ID NO: 542. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence described by SEQ ID NO: 544, comprising a light chain constant region. In one embodiment, 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 one embodiment, the pharmaceutical composition is formulated for intravenous administration.

[0016] In other embodiments, methods for treating a disease or illness in an individual harboring a risk variant associated with the disease or illness are described herein, the methods comprising the step of administering an effective amount of the antibody or antigen-binding fragment of this disclosure to the individual harboring the risk variant, wherein the disease or illness includes at least one of inflammatory bowel disease (IBD), Crohn's disease (CD), or colitis. In some embodiments, the individual harbors multiple risk variants. In some embodiments, the multiple risk variants are at least 3, 4, 5, or 10 risk variants. In some embodiments, one of the multiple risk variants is associated with an asymptomatic phenotype of the disease or illness. In some embodiments, the disease or illness is at least one severe form of IBD, CD, or colitis. [Brief explanation of the drawing]

[0017] Typical embodiments are illustrated in the reference drawings. The embodiments and drawings disclosed herein are intended to be considered illustrative, not limiting.

[0018] [Figure 1] The results of a filter-lift assay, performed to qualitatively evaluate chimeric 5C3D11 Fab expression and antigen binding, are shown. Filter portion A shows expression of heavy chain 5C3D11, filter portion B shows expression of light chain 5C3D11, and filter portion C shows binding of 5C3D11 Fab to the human TL1A antigen. [Figure 2] This shows the binding of chimeric 5C3D11 and humanized clone 12835 antibodies to human TL1A using enzyme-linked immunosorbent assay (ELISA). [Figure 3] The results of a capture filter lift assay demonstrate the high sensitivity and high binding strength of chimeric 5C3D11 to human TL1A. [Figure 4A]The results of ELISA showing the binding of CDR-transplant antibody clones 18-7, 21-3, and humanized clone 12835 to human TL1A are shown. [Figure 4B] The results of an ELISA showing the binding of CDR transplant antibody L8 to human TL1A, compared to the binding of humanized clone 12835 to human TL1A, are shown. [Figure 5] The results of ELISA demonstrate the strong binding of immobilized Fab (chimeric 5C3D11, humanized clone 12835, clone 18-7, clone 21-3, and CDR graft clone L8) to soluble human TL1A antigen. [Figure 6A] The ELISA results are shown, demonstrating increased affinity for human TL1A from anti-TL1A antibodies containing heavy chain CDR3 mutations 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, compared to the CDR graft (clone L8). [Figure 6B] The ELISA results are shown, demonstrating increased affinity for human TL1A from antibodies against heavy chain CDR3 mutation H3-7(V102W)-SEQ ID NO:46, 38, and humanized clone 12835 compared to CDR graft clone L8. [Figure 7A] The ELISA results are shown, demonstrating increased affinity for human TL1A from anti-TL1A antibodies containing light chain CDR3 mutations 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, compared to CDR graft clone L8. [Figure 7B]The ELISA results are shown, demonstrating increased affinity for human TL1A from antibodies against light chain CDR3 mutations L3-4(S92Q)-SEQ ID NO:51, 40, and humanized clone 12835, compared to CDR graft clone L8. [Figure 8] This paper presents an ELISA demonstrating the binding of Fab containing the 5C3D11 CDR mutant, transplanted onto human heavy chain germline IGH1-46*02 and human light chain germline IGKV3-20*01, to immobilized human TL1A. [Figure 9] This paper presents an ELISA demonstrating the binding of Fabs containing the 5C3D11 CDR mutant, transplanted onto human heavy chain germline IGH1-3*01 and human light chain germline IGKV3-20*01, to immobilized human TL1A. [Figure 10] This paper presents an ELISA demonstrating the binding of immobilized Fab containing the 5C3D11 CDR mutant, transplanted onto human heavy chain germline IGH1-46*02 and human light chain germline IGKV3-20*01, to soluble biotinylated human TL1A. [Figure 11] This paper presents an ELISA demonstrating the binding of immobilized Fab containing the 5C3D11 CDR mutant, transplanted onto human heavy chain germline IGH1-46*02 and human light chain germline IGKV3-20*01, to soluble biotinylated human TL1A. [Figure 12] This paper presents an ELISA demonstrating the binding of immobilized Fab containing the 5C3D11 CDR mutant, transplanted onto human heavy chain germline IGH1-46*02 and human light chain germline IGKV3-20*01, to soluble biotinylated human TL1A. [Figure 13] This paper presents an ELISA demonstrating the binding of immobilized Fab containing the 5C3D11 CDR mutant, transplanted onto human heavy chain germline IGH1-3*01 and human light chain germline IGKV3-20*01, to soluble biotinylated human TL1A. [Figure 14] This shows the binding of Fab containing the 5C3D11 CDR mutant to membrane-associated human TL1A. [Figure 15]This shows the lack of binding to TRAIL in Fabs containing the 5C3D11 CDR mutant transplanted onto human heavy chain germline IGH1-46*02 and human light chain germline IGKV3-20*01. [Figure 16] This shows the lack of binding to LIGHT in Fab containing the 5C3D11 CDR mutant transplanted onto human heavy chain germline IGH1-46*02 and human light chain germline IGKV3-20*01. [Figure 17] This shows the lack of binding to Fas in Fab containing the 5C3D11 CDR mutant transplanted onto human heavy chain germline IGH1-46*02 and human light chain germline IGKV3-20*01. [Figure 18] This shows the lack of binding to TRAIL in Fab containing the 5C3D11 CDR mutant transplanted onto human heavy chain germline IGH1-3*01 and human light chain germline IGKV3-20*01. [Figure 19] This shows the lack of binding to LIGHT in Fabs containing the 5C3D11 CDR mutant transplanted onto human heavy chain germline IGH1-3*01 and human light chain germline IGKV3-20*01. [Figure 20] This shows the lack of binding to Fas in Fab containing the 5C3D11 CDR mutant transplanted onto human heavy chain germline IGH1-3*01 and human light chain germline IGKV3-20*01. [Figure 21] This paper presents an ELISA demonstrating the binding of heavy chain variable regions and light chain variable regions, including 5C3D11 CDR mutants having either a modified IgG1 heavy chain and a κ light chain constant region (21A), or a IgG2 heavy chain and a κ light chain constant region (21B), to immobilized human TL1A. [Figure 22] This paper presents an ELISA demonstrating the binding of soluble biotinylated human TL1A to immobilized heavy chain variable regions and light chain variable regions, including 5C3D11 CDR mutants having either a modified IgG1 heavy chain and a κ light chain constant region (22A), or a IgG2 heavy chain and a κ light chain constant region (22B). [Figure 23]This study demonstrates the maintenance of binding of the IgG1 heavy chain (modified) and κ light chain constant region, or the heavy chain variable region and light chain variable region, including the 5C3D11 CDR mutant having the IgG2 heavy chain and κ light chain constant region, to the membrane-bound form of human TL1A. [Figure 24A] This paper presents an ELISA demonstrating the lack of binding of heavy chain variable regions and light chain variable regions to TNFSF family members Fas(24A), TRAIL(24B), or LIGHT(24C) from 5C3D11 CDR variants containing either a modified IgG1 heavy chain and a κ light chain constant region, or a IgG2 heavy chain and a κ light chain constant region. [Figure 24B] This paper presents an ELISA demonstrating the lack of binding of heavy chain variable regions and light chain variable regions to TNFSF family members Fas(24A), TRAIL(24B), or LIGHT(24C) from 5C3D11 CDR variants containing either a modified IgG1 heavy chain and a κ light chain constant region, or a IgG2 heavy chain and a κ light chain constant region. [Figure 24C] This paper presents an ELISA demonstrating the lack of binding of heavy chain variable regions and light chain variable regions to TNFSF family members Fas(24A), TRAIL(24B), or LIGHT(24C) from 5C3D11 CDR variants containing either a modified IgG1 heavy chain and a κ light chain constant region, or a IgG2 heavy chain and a κ light chain constant region. [Figure 25] This study demonstrates inhibition of TL1A-induced IFN-γ production in whole blood cynomolgus monkeys by humanized Ig constructs containing the 5C3D11 CDR mutant transplanted onto human heavy chain germline IGH1-46*02 and human light chain germline IGKV3-20*01, which have a modified IgG1 heavy chain and a constant κ light chain region. [Figure 26A] This specification illustrates the inhibition of TL1A-induced IFN-γ production in human whole blood by the antibodies described herein. Results obtained from three different donors (26A), (26B), and (26C) are shown, with antibody concentrations (nanomoles) indicated on the X axis. [Figure 26B]This specification illustrates the inhibition of TL1A-induced IFN-γ production in human whole blood by the antibodies described herein. Results obtained from three different donors (26A), (26B), and (26C) are shown, with antibody concentrations (nanomoles) indicated on the X axis. [Figure 26C] This specification illustrates the inhibition of TL1A-induced IFN-γ production in human whole blood by the antibodies described herein. Results obtained from three different donors (26A), (26B), and (26C) are shown, with antibody concentrations (nanomoles) indicated on the X axis. [Figure 27A] This specification illustrates the inhibition of TL1A-induced IFN-γ production in cynomolgus monkey whole blood by the antibodies described herein. Results obtained from three different donors (27A), (27B), and (27C) are shown, with antibody concentrations (nanomoles) indicated on the X axis. [Figure 27B] This specification illustrates the inhibition of TL1A-induced IFN-γ production in cynomolgus monkey whole blood by the antibodies described herein. Results obtained from three different donors (27A), (27B), and (27C) are shown, with antibody concentrations (nanomoles) indicated on the X axis. [Figure 27C] This specification illustrates the inhibition of TL1A-induced IFN-γ production in cynomolgus monkey whole blood by the antibodies described herein. Results obtained from three different donors (27A), (27B), and (27C) are shown, with antibody concentrations (nanomoles) indicated on the X axis. [Modes for carrying out the invention]

[0019] Tumor necrosis factor-like protein 1A (TL1A) is associated with the development and severity of severe inflammatory bowel disease (IBD), including colitis and severe forms of Crohn's disease (CD). In addition, preclinical and human genetic data suggest that TL1A is a potential therapeutic target for Crohn's disease. This disclosure describes an antibody optimized for TL1A, providing a novel therapeutic approach for the treatment of IBD.

[0020] In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence described by any one of SEQ ID NO: 554-564 or 574-577; (c) HCDR3 comprising the amino acid sequence described by any one of SEQ ID NO: 565-568 or 578-581; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by any one of SEQ ID NO: 569 or 570; (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence described by any one of SEQ ID NO: 571-573 or 582-585.

[0021] In another embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, wherein the antibody or antigen-binding fragment includes: (a) a heavy chain variable region having an amino acid sequence that is at least about 90% identical to 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) SEQ ID NO:Includes a light chain variable region containing an amino acid sequence that is at least approximately 90% identical to 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.

[0022] In some embodiments, an antibody refers to an immunoglobulin molecule that recognizes a target and specifically binds to that target via at least one antigen-recognizing site within the variable region of the immunoglobulin molecule, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof. In some embodiments, an antibody includes 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 antigen-determining portion of an antibody, and any other modified immunoglobulin molecule containing an antigen-recognizing site, insofar as 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), based on the identity of their heavy chain constant domains, which are called alpha, beta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different known subunit structures and three-dimensional arrangements. Antibodies may be naked or conjugated to other molecules such as toxins or radioisotopes.

[0023] In some embodiments, one or more amino acid modifications are introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. In this specification, the Fc region is the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The Fc region includes native sequence Fc regions and variant Fc regions. Fc region variants may include human Fc region sequences (e.g., the Fc regions of human IgG1, IgG2, IgG3, or IgG4) that contain amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0024] In some embodiments, the antibodies of this disclosure reduced antibody-dependent cell-mediated cytotoxicity (ADCC) or reduced complement fixation ability. While this is desirable in situations where inhibition of target function is desired, activation of downstream immune responses may cause undesirable side effects. Some Fc regions have a natural lack of effector function (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 reduced effector function. In some embodiments, the antibodies of this disclosure include the IgG2 constant region described in SEQ ID NO: 543. In some embodiments, the antibodies of this disclosure include the modified IgG1 constant region described in SEQ ID NO: 542.

[0025] In some embodiments, the antibodies of this disclosure are variants possessing some, but not all, of the effector functions, which makes the antibodies desirable candidates for applications where the half-life of the antibody in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays may be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. Non-limiting examples of in vitro assays for evaluating the ADCC activity of the molecule of interest are described in U.S. Patents 5,500,362 and 5,821,337. Alternatively, non-radioactive assay methods (e.g., ACTI® and CytoTox's 96® non-radioactive cytotoxicity assays) may be used. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs), macrophages, and natural killer (NK) cells.

[0026] Antibodies can increase half-life and / or improve binding to the neonatal Fc receptor (FcRn) (see, for example, U.S. Patent Application No. 2005 / 0014934). Such antibodies may contain an Fc region having one or more substitutions that improve binding of the Fc region to FcRn, and may contain an Fc region having substitutions in 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 scheme (see, for example, U.S. Patent Application No. 7,371,826). Other examples of Fc region variants are also considered (see, e.g., 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 exhibited increased serum half-life as a result of denaturation of the Fc region. In some embodiments, the denaturation includes M252Y / S254T / T256E mutations to IgG1, or M428L / N434S mutations to IgG1.

[0027] In some embodiments, the antibody includes an antigen-binding fragment that points to a portion of the antibody having an antigen that determines the variable region of the antibody. Examples of antibody fragments, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single-chain antibodies, and polyspecific antibodies formed from antibody fragments.

[0028] In some embodiments, a humanized antibody refers to a form of antibody from a non-human species (e.g., mouse) that has a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof containing minimal non-human (e.g., mouse) sequences. In non-limiting examples, a humanized antibody contains less than about 40% non-human sequences in its variable region. In some cases, a humanized antibody contains less than about 20% non-human sequences in its full-length antibody sequence. In some cases, a humanized antibody is a human immunoglobulin in which residues from a complementarity-determining region (CDR) having desired specificity, affinity, and capability are replaced by residues from a CDR from a non-human species (e.g., mouse, rat, rabbit, hamster).

[0029] In some embodiments, a chimeric antibody refers to an antibody whose immunoglobulin molecule sequence originates from two or more species. As a non-limiting example, both the light and heavy chain variable regions correspond to the variable regions of an antibody derived from a certain species of mammal (e.g., mouse, rat, rabbit) having desired specificity, affinity, and ability, while the constant region is homologous to the sequence in an antibody derived from another species (usually human) to avoid inducing an immune response in that species.

[0030] As used herein, the term "about" means within 10% of the stated quantity.

[0031] As used herein, “risk variant” means any gene sequence of an individual, typically a DNA sequence, that increases the risk of developing a phenotype (e.g., inflammatory bowel disease, Crohn's disease, colitis, or an asymptomatic phenotype thereof) in that individual. Risk variants include, but are not limited to, single nucleotide polymorphisms (SNPs), indels of any length, short tandem repeats (STRs), and chromosol translocations, duplications, or deletions. The risk variants include variants associated with severe forms of inflammatory bowel disease, Crohn's disease, or colitis. The risk variants include variants that may indicate that an individual is resistant to treatment with any current treatment for inflammatory bowel disease, Crohn's disease, or colitis. As intended herein, risk variants can be used to inform a decision on a treatment method using any of the antibodies described herein.

[0032] The terms "complementarity-determining region" and "CDR," which are synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids within the antibody variable region, which provide antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3) and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, each full-length heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) and each full-length light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4). The precise amino acid sequence boundaries of a given CDR or FR are as follows: 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.These can be readily determined using one of many well-known schemes, including those described by “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan; 27(1):55-77 ("IMGT" numbering); Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8; 309(3):657-70 ("Aho" numbering); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modeling antibodies on the WEB,” Protein Eng. 2000 Dec; 13(12):819-24 ("AbM" numbering). In one embodiment, the CDR of the antibodies described herein may be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0033] In some embodiments, antibodies that specifically bind to proteins indicate that the antibody reacts to or is associated with the protein more frequently, more rapidly, for a longer duration, with higher affinity, or in some combination of the above, than alternative substances containing unrelated proteins.

[0034] In some embodiments, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymers may be linear or branched, contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention, e.g., disulfide bond formation, glycosylation, lipid modification, acetylation, phosphorylation, or other operations or modifications, e.g., fusion with another polypeptide and / or bonding (e.g., using a labeling component). The definition also includes polypeptides containing, for example, one or more analogues of amino acids (e.g., non-natural amino acids), as with other modifications known in the art.

[0035] In some embodiments, “polynucleotide” or “nucleic acid” is used interchangeably herein and refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase. Polynucleotides may include modified nucleotides, e.g., methylated nucleotides and their analogs, or non-nucleotide components. Modification of the nucleotide structure may be given before or after the assembly of the polymer. Polynucleotides may be further modified after polymerization, such as by binding with labeling components.

[0036] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after the sequence has been aligned and gaps introduced, if necessary, to achieve the maximum percentage of sequence identity, and does not consider any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved using various known methods, such as publicly available computer software, including BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including algorithms required to achieve the maximum alignment over the entire length of the sequences being compared. However, for the purposes described herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the United States Copyright Office (Washington DC20559) and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from source code. The ALIGN-2 program must be compiled for use with UNIX® operating systems, including Digital UNIX® V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0037] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B, or to a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having a specific % amino acid sequence identity to certain amino acid sequences B, or to certain amino acid sequences B) is calculated as follows: 100 times fraction X / Y, where X is the number of amino acid residues scored as identical by the sequence alignment program ALIGN-2 in the alignment of A and B in that program, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0038] In some embodiments, the terms “individual” and “subject” are used interchangeably and are not limited to any animal that may be the recipient of a particular treatment, including humans, non-human primates, rodents, and domestic and game animals. Primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, e.g., rhesus macaques. Rodents include mice, rats, wild mice, ferrets, rabbits, and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cats), canid species (e.g., dogs, foxes, wolves), birds (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In various embodiments, a subject may be a person who has or has been previously diagnosed or identified as having a disease requiring treatment. In certain embodiments, the subject is a human. In various other embodiments, a subject who has a disease or has been previously diagnosed or identified as having a disease may or may not have received treatment for the disease. In yet another embodiment, a subject may not have been previously diagnosed with a disease (i.e., a subject exhibiting one or more risk factors for the disease). A “subject in need of treatment for a particular disease” may be a subject who has the disease, has been diagnosed with the disease, or is at risk of developing the disease. In some embodiments, a subject is a “patient” diagnosed with one of the diseases or diseases described herein.

[0039] In some embodiments, the term “therapeutically effective amount” refers to an amount of an antibody, polypeptide, polynucleotide, small organic molecule, or other drug that is effective in “treating” a disease or disorder 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 disorder. In some examples, the disease or disorder includes inflammatory bowel disease (IBD), Crohn's disease (CD), or ulcerative colitis (UC). In some examples, IBD, CD, and / or UC are severe or medically refractory forms 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, ulcers, nausea, vomiting, bleeding, bloody stools, loss of appetite, and weight loss.

[0040] In some embodiments, the terms “treat” and “treating” as used herein refer to both therapeutic and preventive measures, the purpose of which is to prevent or delay (mitigate) a targeted pathological disease, to prevent a pathological disease, to pursue or achieve good overall survival, or, even if the treatment is ultimately unsuccessful, to reduce the likelihood that the individual will progress to the disease. In some embodiments provided herein, subjects requiring treatment include those who already have a disease or illness, as well as those who are susceptible to developing a disease or illness, or those for whom a disease or illness should be prevented. Diseases or illnesses include inflammatory diseases or illnesses, fibrostenotic or fibrous diseases, thiopurine toxicity or diseases associated with thiopurine toxicity, and non-response to anti-TNF therapy, steroids, or immunomodulatory agents.

[0041] Anti-TL1A antibody 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 comprising a heavy chain containing four heavy chain framework regions (HCFRs) and three heavy chain complementarity-determining regions (HCDRs): HCFR1, HCDR1, HCFR2, HCDR2, HCFR3, HCDR3, and HCFR4; and a light chain containing four light chain framework regions (LCFRs) and three light chain complementarity-determining regions (LCDRs): LCFR1, LCDR1, LCFR2, LCDR2, LCFR3, LCDR3, and LCFR4. The anti-TL1A antibody may contain any region described herein, for example, provided in Tables 1, 2, 3, Examples, and SEQ ID NOs: 1-54, 490-588. In some embodiments, the anti-TL1A antibody comprises a variable domain provided herein having, for example, one or more CDR mutations shown in Table 2 or 19-22. In some embodiments, the anti-TL1A antibody comprises one or more CDRs containing sequences shown in Tables 19-22.

[0042] In one embodiment, the anti-TL1A antibody comprises a CDR corresponding to those described in Tables 19-22. In one embodiment, the anti-TL1A antibody or antigen-binding fragment comprises a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 484 (DTYMH); (b) HCDR2 comprising the amino acid sequence described by SEQ ID NO: 485 (PASGH); and (c) HCDR3 comprising the amino acid sequence described by SEQ ID NO: 486 (SGGLPD); and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by SEQ ID NO: 487 (ASSSVSYMY); (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488 (ATSNLAS); and (f) LCDR3 comprising the amino acid sequence described by SEQ ID NO: 489 (GNPRT).

[0043] In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising: (a) HCDR1 comprising an amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising an amino acid sequence described by any one of SEQ ID NO: 554-564 or 574-577; (c) HCDR3 comprising an amino acid sequence described by any one of SEQ ID NO: 565-568 or 578-581; and a light chain variable region comprising: (d) LCDR1 comprising an amino acid sequence described by any one of SEQ ID NO: 569 or 570; (e) LCDR2 comprising an amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising an amino acid sequence described by any one of SEQ ID NO: 571-573 or 582-585.

[0044] In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising: a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence described by SEQ ID NO: 559; (c) HCDR3 comprising the amino acid sequence described by SEQ ID NO: 567; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence described by any one of SEQ ID NO: 573. In some embodiments, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising: a heavy chain variable region having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 503; and a light chain variable region having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequence to 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.

[0045] In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence described by SEQ ID NO: 563; and (c) HCDR3 comprising the amino acid sequence described by SEQ ID NO: 568; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence described by any one of SEQ ID NO: 572. In some embodiments, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising: a heavy chain variable region having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 511; and a light chain variable region having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequence to 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.

[0046] In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence described by SEQ ID NO: 555; and (c) HCDR3 comprising the amino acid sequence described by SEQ ID NO: 566; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence described by any one of SEQ ID NO: 572. In some embodiments, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising: a heavy chain variable region having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO:493; and a light chain variable region having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequence to 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.

[0047] In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence described by SEQ ID NO: 558; and (c) HCDR3 comprising the amino acid sequence described by SEQ ID NO: 566; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence described by SEQ ID NO: 572. In some embodiments, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising: a heavy chain variable region having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 501; and a light chain variable region having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequence to 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.

[0048] In one embodiment, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising a heavy chain variable region comprising: (a) HCDR1 comprising the amino acid sequence described by SEQ ID NO: 553; (b) HCDR2 comprising the amino acid sequence described by SEQ ID NO: 564; and (c) HCDR3 comprising the amino acid sequence described by SEQ ID NO: 568; and a light chain variable region comprising: (d) LCDR1 comprising the amino acid sequence described by SEQ ID NO: 569; (e) LCDR2 comprising the amino acid sequence described by SEQ ID NO: 488; and (f) LCDR3 comprising the amino acid sequence described by any one of SEQ ID NO: 572. In some embodiments, an antibody or antigen-binding fragment that specifically binds to TL1A is described herein, the antibody or antigen-binding fragment comprising: a heavy chain variable region having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 515; and a light chain variable region having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequence to 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.

[0049] In one embodiment, the anti-TL1A antibody or antigen-binding fragment includes a heavy chain variable region comprising HCDR1, HCDR2, 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; SEQ ID NO: Includes a light chain variable region, including LCDR1, LCDR2, LCDR3 from 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, where CDR is defined by the Kabat method, IMGT method, Chothia method, or a combination thereof. In one embodiment, the anti-TL1A antibody or antigen-binding fragment contains a heavy chain variable region with an amino acid sequence that is at least approximately 85%, 90%, 95%, 97%, 98%, 99%, or 100% 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, and 541; SEQ ID NO:Includes a light chain variable region containing an amino acid sequence that is at least approximately 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to 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.

[0050] In one embodiment, the anti-TL1A antibody or antigen-binding fragment is a heavy chain variable region comprising: (a) Human heavy chain framework region 1 which is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the original described in SEQ ID NO: 545; (b) HCDR1 which includes the amino acid sequence described by SEQ ID NO: 484 (DTYMH); (c) Human heavy chain framework region 2 which is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the one described in SEQ ID NO: 546; (d) HCDR2 which includes the amino acid sequence described by SEQ ID NO: 485 (PASGH); (e) Human heavy chain framework region 3 which is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the one described in SEQ ID NO: 547 or 586-588; and (f) SEQ ID HCDR3 containing the amino acid sequence described by NO:486(SGGLPD);(g) Human heavy chain framework region 4, which is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that described by SEQ ID NO:548; and a light chain variable region, which is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that described by SEQ ID NO:549;(i) LCDR1 containing the amino acid sequence described by SEQ ID NO:487(ASSSVSYMY);(j) Human light chain framework region 2, which is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that described by SEQ ID NO:550;(k) LCDR2 containing the amino acid sequence described by SEQ ID NO:488(ATSNLAS);(l) SEQ ID It includes a light chain variable region, which includes a human light chain framework region 3 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that described in NO:551; an LCDR3 containing the amino acid sequence described by (m)SEQ ID NO:489 (GNPRT); and a human light chain framework region 4 that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to that described in (n)SEQ ID NO:552.

[0051] Table 1

[0052] Table 2

[0053] In various embodiments, the anti-TL1A antibody specifically binds to the same region of TL1A, or specifically binds to a region of TL1A that overlaps with the region of TL1A to which an antibody specifically binds to a In various embodiments, the anti-TL1A antibody specifically binds to the same region of TL1A, or specifically binds to a region of TL1A that overlaps with the region of TL1A to which an antibody specifically binds is bound. The antibody comprises a heavy chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO:36 and a light chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO:38. In various embodiments, the anti-TL1A antibody specifically binds to the same region of TL1A, or specifically binds to a region of TL1A that overlaps with the region of TL1A to which an antibody specifically binds is bound. The antibody comprises a heavy chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO:40 and a light chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO:42. In various embodiments, the anti-TL1A antibody specifically binds to the same region of TL1A, or specifically binds to a region of TL1A that overlaps with the region of TL1A to which an antibody specifically binds is located, comprising a heavy chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO:40 and a light chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO:38.In various embodiments, the anti-TL1A antibody specifically binds to the same region of TL1A, or specifically binds to a region of TL1A that overlaps with the region of TL1A to which an antibody specifically binds is located, comprising a heavy chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO: 503 and a light chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO: 502. In various embodiments, an anti-TL1A antibody specifically binds to the same region of TL1A, or specifically binds to a region of TL1A that overlaps with the region of TL1A to which an antibody specifically binds is provided, comprising a heavy chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO:511 and a light chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO:510. In various embodiments, the anti-TL1A antibody specifically binds to the same region of TL1A, or specifically binds to a region of TL1A that overlaps with the region of TL1A to which an antibody specifically binds is located, comprising a heavy chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO:493 and a light chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO:492. In various embodiments, an anti-TL1A antibody specifically binds to the same region of TL1A, or specifically binds to a region of TL1A that overlaps with the region of TL1A to which an antibody specifically binds is located, comprising a heavy chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO: 501 and a light chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO: 500. In various embodiments, an anti-TL1A antibody specifically binds to the same region of TL1A, or specifically binds to a region of TL1A that overlaps with the region of TL1A to which an antibody specifically binds is provided, comprising a heavy chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO: 515 and a light chain containing a sequence at least approximately 90%, 92%, 95%, 98%, or 100% identical to SEQ ID NO: 514.

[0054] In various embodiments, the anti-TL1A antibody or fragment is at least about 1E -7 , 1E -8 , 1E -9 , 1E -10 , or 1E -11 It has binding affinity to TL1A. In some cases, the binding affinity is approximately 1E. -9 From approximately 1E -11 That is the case.

[0055] Various embodiments provide reference antibodies, such as anti-TL1A antibodies that bind to the same region of the TL1A protein or a portion thereof as any anti-TL1A antibody described herein. In some embodiments, the reference antibody includes heavy chain CDRs with SEQ ID NO: 150, 12, and 152, and light chain CDRs with SEQ ID NO: 18, 21, and 155.

[0056] 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 to which the antibodies described herein bind. Exemplary embodiments include a competition assay. For example, the method includes the step of determining whether the test antibody can compete for binding between a reference antibody and the TL1A protein or a portion thereof, or whether the reference antibody can compete for binding between the test antibody and the TL1A protein or a portion thereof. An exemplary method includes the use of surface plasmon resonance to evaluate whether an anti-TL1A antibody can compete for binding between TL1A and other anti-TL1A antibodies. In some cases, surface plasmon resonance is utilized in a competition assay. Non-limiting methods are described in the examples.

[0057] The TL1A antibodies described herein bind to specific regions or epitopes of human TL1A. These regions are indicated herein as being useful for inhibiting interferon-γ secretion from T lymphocytes. In some embodiments, antibodies that compete with the antibodies described herein for binding to TL1A are disclosed herein. In some embodiments, antibodies that bind to the same epitope of TL1A bound by the antibodies described herein are disclosed herein. In some embodiments, antibodies that bind to an isolated epitope that overlaps with the epitope of TL1A bound by the antibodies described herein are disclosed herein. In some embodiments, antibodies that compete for binding to the epitope of TL1A are disclosed herein, including antibodies that bind to the same epitope of TL1A and overlap with the epitope of TL1A by one or more amino acid residues, or antibodies or fragments thereof that include a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 503 and a light chain variable region containing the amino acid of SEQ ID NO: 502, with the antibodies to which the TL1A epitope is bound. In some embodiments, antibodies are disclosed herein that compete for binding to the TL1A epitope with antibodies that bind to the same epitope of TL1A and overlap with the TL1A epitope by one or more amino acid residues, or antibodies or fragments thereof that include a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 511 and a light chain variable region containing the amino acid of SEQ ID NO: 510. In some embodiments, antibodies are disclosed herein that compete for binding to the TL1A epitope with antibodies that bind to the same epitope of TL1A and overlap with the TL1A epitope by one or more amino acid residues, or antibodies or fragments thereof that include a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 493 and a light chain variable region containing the amino acid of SEQ ID NO: 492. In one embodiment, antibodies are disclosed herein that compete for binding to the TL1A epitope with antibodies that bind to the same epitope of TL1A and overlap with the TL1A epitope by one or more amino acid residues, or antibodies or fragments thereof that include 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 of SEQ ID NO: 500.In one embodiment, antibodies are disclosed herein that compete for binding to the TL1A epitope with antibodies that bind to the same epitope of TL1A and overlap with the TL1A epitope by one or more amino acid residues, or antibodies or fragments thereof that include a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 515 and a light chain variable region containing the amino acid of SEQ ID NO: 514.

[0058] Method for generating antibodies Various embodiments provide antibodies produced using polypeptides 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.

[0059] The antibodies described herein may be analyzed for specific binding by any method known in the art. Immunological assays that can be used include, but are not limited to, competitive and non-competitive assay systems employing techniques such as BIAcore analysis, FACS analysis, immunofluorescence assays, immunocytochemistry, Western blotting, radioimmunoassays, ELISA, "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescence immunoassays, and protein A immunoassays. Such assays are provided, for example, in Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York.

[0060] In various embodiments, the anti-TL1A antibody is an antagonist of a TL1A receptor, including but not limited to DR3 and TR6 / DcR3. In certain 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 one or more activities of the bound TL1A receptor. In certain embodiments, the antibody inhibits TL1A activation as measured by interferon γ release in human blood. In certain embodiments, the antibody inhibits interferon γ release in human blood with an IC 50 between about 1 nanomolar and about 100 picomolar. In certain embodiments, the antibody inhibits interferon γ release in human blood with an IC 50 between about 500 picomolar and about 100 picomolar. In certain embodiments, the antibody inhibits interferon γ release in human blood with an IC 50 of about 500 picomolar. In certain embodiments, the antibody inhibits interferon γ release in human blood with an IC 50 of about 250 picomolar.

[0061] In various embodiments, monoclonal antibodies are prepared using methods known in the art, such as the hybridoma method, as described above, in which a host animal is immunized to induce the production of lymphocytes that produce antibodies that specifically bind to an immunizing antigen (Kohler and Milstein (1975) Nature 256:495). Hybridomas produce monoclonal antibodies that are specific for a selected antigen. Monoclonal antibodies are purified from the culture medium or ascites by techniques known in the art when grown either in vitro or in vivo.

[0062] In some embodiments, monoclonal antibodies are produced using the recombinant DNA method described in U.S. Patent No. 4,816,567. The polynucleotides encoding the monoclonal antibody 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 and transfected into host cells (e.g., E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or bone marrow cells) to produce the monoclonal antibody. The polynucleotides encoding the monoclonal antibody can be further modified in many different ways using recombinant DNA technology to produce alternative antibodies.

[0063] In some embodiments, chimeric antibodies, that is, molecules derived from various animal species, can be produced, in which different parts include a variable region derived from a mouse monoclonal antibody and a constant region of human immunoglobulin (e.g., a humanized antibody). Chimeric antibodies can be produced using various 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).

[0064] In some embodiments, anti-TL1A monoclonal antibodies are humanized antibodies that, when administered to human subjects, reduce antigenicity and HAMA (human anti-mouse antibody) response. Humanized antibodies can be produced using various techniques known in the art. For example, antibodies are humanized by (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, e.g., determining the antibody framework regions to be used during the humanization process; (3) actual humanization methodologies / technologies; and (4) transfection and expression of the humanized antibody (see, e.g., U.S. Patents 5,585,089; 6,835,823; 6,824,989). In various embodiments, humanized antibodies are optimized to reduce potential immunogenicity while maintaining functional activity for human therapeutics.

[0065] Humanized antibodies can also be produced in transgenic mice containing a human immunoglobulin locus that, during immunization, can produce a sufficient repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patents 5,545,806; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Humanized antibodies can also be obtained by novel genetic engineering approaches 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.)

[0066] Fully humanized antibodies can be produced by first designing a variable region amino acid sequence containing non-human, e.g., rodent-derived CDRs, which are embedded in a human-derived framework sequence. The non-human CDRs provide the desired specificity. Therefore, in some cases, these residues are included in the design of the essentially immutable remodeled variable region. In some cases, modifications should therefore be minimized, and changes in antibody specificity and affinity should be carefully monitored. On the other hand, the theoretical framework residues may be derived from any human variable region. To produce a remodeled antibody exhibiting one acceptable, and even improved, affinity, a human framework sequence should be selected that is equally suitable for creating the remodeled variable region and for maintaining antibody affinity. The human framework may be germline-derived or may be derived from a non-germline (e.g., mutated or affinity-mature) sequence. Genetic engineering techniques well known in the art, such as, but not limited to, phage display of human antibody libraries, transgenic mice, human-human hybridomas, hybrid hybridomas, B cell immortalization and cloning, single-cell RT-PCR, or HuRAb technology, may be used to generate humanized antibodies having DNA sequences containing human frameworks and non-human CDRs. Methods for obtaining “humanized antibodies” are well known to those skilled in the art (e.g., U.S. Patents 5,861,155, 6,479,284, 6,407,213, 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)).

[0067] In one embodiment, the anti-TL1A antibody is a human antibody. Human antibodies can be generated using various techniques known in the art. Immortalized human B lymphocytes, either immunized in vitro or isolated from immunized individuals, that produce antibodies directed towards a target antigen 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 US Pat. No. 5,750,373). Human antibodies may also be selected from a phage library. Techniques for generating and using antibody phage libraries are described in U.S. Patents No. 5,969,108, No. 6,172,197, No. 5,885,793, No. 6,521,404; No. 6,544,731; No. 6,555,313; No. 6,582,915; No. 6,593,081; No. 6,300,064; No. 6,653,068; No. 6,706,484; and No. 7,264,963; and Rothe et al., 2007, J. Mol. Bio., doi:10.1016 / j.jmb.2007.12.018.

[0068] Chimeric humanized antibodies and chimeric human antibodies can be produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression regulatory sequence operably ligated to the coding sequence of an antibody chain, which may contain naturally related or heterologous promoter regions. In some embodiments, it may be desirable to produce amino acid sequence variants of these humanized antibodies, particularly if they improve the antibody's binding affinity or other biological properties.

[0069] In some embodiments, antibody fragments are used to treat and / or induce remission of IBD. Various techniques for the production of antibody fragments are known. Typically, these fragments can be obtained by the protein 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). All Fab, Fv, and scFv antibody fragments can be expressed in E. coli or other host cells, and secreted from E. coli or other host cells, thereby enabling the production of large quantities of these fragments. Other techniques for the production of antibody fragments will become apparent to those skilled in the art.

[0070] In accordance with this disclosure, the technique may be adapted for the production of single-chain antibodies specific to TL1A (e.g., U.S. Patent No. 4,946,778). In addition, the method may be adapted for the construction of a Fab expression library (e.g., see Huse, et al., Science 246:1275-1281 (1989)) to enable the rapid and effective identification of monoclonal FAb fragments, or their derivatives, fragments, analogs, or homologs having desired specificity for TL1A. Antibody fragments may be generated by techniques of the art, and such antibody fragments include, but are not limited to, (a) F(ab')2 fragments generated by pepsin treatment of an antibody molecule; (b) FAb fragments generated by reducing the disulfide crosslinks of an F(ab')2 fragment; (c) FAb fragments generated by treatment of an antibody molecule with papain and a reducing agent; and (d) Fv fragments.

[0071] Furthermore, modified antibodies containing any type of variable region that provides association between TL1A and the antibody are provided herein. Those skilled in the art will understand that a modified antibody may include an antibody (e.g., a full-length antibody or its immunoreactive fragment) in which at least one portion of one or more constant region domains is deleted or otherwise modified to result in a desired biochemical property, such as a reduction in TL1A. In some embodiments, the variable region in both the heavy and light chains is modified by at least partial substitution of one or more CDRs, and, if necessary, by substitution and sequence modification of partial framework regions. In some embodiments, the substituted CDRs may be derived from antibodies of different classes, e.g., antibodies of the same class, same subclass, or from different species and / or combinations thereof. In some embodiments, the constant region of the modified antibody includes a human constant region. Modifications to the constant region that are compatible with this disclosure include the addition, deletion, or substitution of one or more amino acids in one or more domains.

[0072] In various embodiments, the expression of the antibodies or antigen-binding fragments described herein may occur in either prokaryotes or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungal, bird, and mammalian cells, either in vivo or in situ, or host cells of mammalian, insect, bird, or yeast origin. Mammalian cells or tissues may be derived from humans, primates, hamsters, rabbits, rodents, cows, pigs, sheep, horses, goats, dogs, or cats, but other mammalian cells may be used. In other embodiments, the antibodies or antigen-binding fragments described herein may be transfected into a host.

[0073] In some embodiments, the expression vector is transfected into a recipient cell line for the production of humanized or composite human antibodies of the chimeric cells described herein. In various embodiments, mammalian cells may be useful as hosts for antibody protein production and 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 may be used to express polypeptides include, but are not limited to, COS cells including COS 7 cells; 293 cells including 293-6E cells; CHO cells including CHO--S and DG44 cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, a specific eukaryotic host cell is selected based on its ability to perform desired post-translational modifications to the heavy and / or light chains.

[0074] Many suitable host cell lines capable of secreting intact heterologous proteins have been developed in this field, including, but are not limited to, CHO cell lines, various COS cell lines, HeLa cells, L cells, and multiple myeloma cell lines.

[0075] Chimeric humanized antibody constructs or conjugated human antibody constructs, expression vectors having the antibodies or antigen-binding fragments described herein, can be introduced into suitable host cells by any variety of suitable means known to those skilled in the art, including, but not limited to, transformation, transfection, lipofection, conjugation, electroporation, direct microinjection, and particle guns, based on the type of cell host. Expression vectors for these cells may include expression control sequences, e.g., replication origins, promoters, enhancers, and necessary information processing sites, e.g., ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences.

[0076] In various embodiments, yeast can also be used as a host for the production of antibody molecules or peptides described herein. In various other embodiments, bacterial species can also be used as a host for the production of antibody molecules or peptides described herein. Examples of bacterial species include, but are not limited to, Escherichia coli, Bacillus species, Enterobacteriaceae, and various Pseudomonas species.

[0077] In some embodiments, one or more antibodies or their antigen-binding fragments described herein can be produced in vivo in animals by being manipulated (transgenic) or transfected with one or more nucleic acid molecules encoding polypeptides according to any suitable method. When producing transgenic animals, the transgene can be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells, and the neural nuclei of such cells are transferred to enucleated oocytes. Once expressed, the antibodies can be purified according to standard procedures of the art, including HPLC purification, column chromatography, and gel electrophoresis (see, generally, Scope, Protein Purification (Springer-Verlag, NY, 1982)).

[0078] Once expressed in a host, the entire antibody, antibody fragments (e.g., individual light and heavy chains), or other immunoglobulin forms of the Disclosure may be collected and purified by known techniques (e.g., immunoadsorption, immunoaffinity chromatography, chromatographic methods such as HPLC (high-performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination thereof). See, for general information, Scope, Protein Purification (Springer-Verlag, NY, 1982). Substantially pure immunoglobulins with at least approximately 90%–95% homology are advantageous, and those with 98%–99% or more homology are also advantageous, especially for pharmaceutical use. Once partially or homogeneously purified as needed, humanized antibodies or compound human antibodies can be used therapeutically or in the development and implementation of assay procedures such as immunofluorescence staining. See, for general information, Vols. I & II Immunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, NY, 1979 and 1981).

[0079] Various embodiments provide gene constructs comprising nucleic acids encoding an anti-TL1A antibody or fragment provided herein. The antibody gene construct may be in the form of an expression cassette suitable for expression of the encoded anti-TL1A antibody or fragment. The gene construct can be introduced into host cells whether or not it is incorporated into a vector. For example, the gene construct may be incorporated into liposomes or viral particles. Alternatively, the purified nucleic acid molecule may be directly inserted into host cells by methods known in the art. The gene construct can be introduced directly into host subject cells by transfection, infection, electroporation, cell fusion, protoplast fusion, microinjection, or ballistic bombardment.

[0080] Various embodiments provide recombinant vectors comprising gene constructs of antibodies provided herein. Recombinant vectors may be plasmids, cosmids, or phages. Recombinant vectors may include other functional elements, such as a suitable promoter for initiating gene expression.

[0081] Various embodiments provide host cells comprising gene constructs and / or recombinant vectors described herein.

[0082] Various host systems can also be advantageously used to express recombinant proteins. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells and other cell lines capable of expressing suitable vectors, such as L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa cell lines, and BHK cell lines. Mammalian expression vectors include non-transcription elements, such as replication start sites, appropriate promoters and enhancers linked to the gene to be expressed, other 5' or 3' adjacent non-transcription sequences, and 5' or 3' untranslated sequences, such as the required ribosome binding sites, polyadenylation sites, splice donor and receptor sites, and transcription termination sequences.

[0083] Proteins produced by transformed hosts can be purified according to any suitable method. Such standard analytical methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for protein purification. Affinity tags such as hexahistidine, maltose-binding domains, influenza coat sequences, and glutathione-S-transferases bind to proteins, allowing for easy purification by subculturing on a suitable affinity column. 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 may be isolated. Known methods in the art for the purification of antibodies and other proteins include, for example, those described in U.S. Patent Application Nos. 2008 / 0177048 and 2009 / 0187005.

[0084] Those skilled in the art will recognize that individual substitutions, deletions, or additions to the sequence of a nucleic acid, peptide, polypeptide, or protein that alter one amino acid or a small proportion of amino acids in the encoded sequence are “conservatively modified variants” where the modification results in the substitution of an amino acid with a chemically similar amino acid, thereby retaining the ability to specifically bind to a target antigen. Such conservatively modified variants are, in addition to, pleomorphic variants, interspecies congeners, and alleles consistent with the present disclosure, and are not excluded.

[0085] A given amino acid can be replaced by a residue having similar physicochemical properties, for example, by substituting one aliphatic residue for another (e.g., He, Val, Leu, or Ala) or by substituting one polar residue for another (e.g., between Ly and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative substitutions (e.g., substitution of entire regions with similar hydrophobic properties) are well known. Polypeptides containing conservative amino acid substitutions can be tested with any of the assays described herein to confirm the desired activity (e.g., the antigen-binding activity and specificity of the native polypeptide or reference polypeptide are preserved).

[0086] Certain conservative substitutions occur, for example, from Ala to Gly or Ser; from Arg to Lys; from Asn to Gin or His; from Asp to Glu; from Cys to Ser; from Gin to Asn; from Glu to Asp; from Gly to Ala or Pro; from His to Asn or Gin; from lie to Leu or Val; from Leu to lie or Val; from Lys to Arg, Gin, or Glu; from Met to Leu, Tyr, or lie; from Phe to Met, Leu, or Tyr; from Ser to Thr; from Thr to Ser; from Trp to Tyr; from Tyr to Trp; and / or from Phe to Val, lie, or Leu.

[0087] In some embodiments, the antibodies and / or antigen-binding fragments described herein may be variants of the sequences described herein, for example, conserved substitutional variants of antibody polypeptides. In some embodiments, the variant is a conservedly modified variant. The variant may refer to a polypeptide that is substantially homologous to the natural polypeptide or reference polypeptide, but has an amino acid sequence that differs from the amino acid sequence of the natural or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant polypeptide contains one or more additions, deletions, or substitutions of nucleotides compared to the natural or reference DNA sequence, but contains a sequence that encodes a variant protein or fragment that retains activity (e.g., antigen-specific binding activity to the relevant target polypeptide).

[0088] Modification of natural amino acid sequences can also be carried out by any of the many techniques known to those skilled in the art. Mutations can be introduced at specific loci or into oligonucleotides by oligonucleotide-directed site-specific mutagenesis procedures. Techniques for making such modifications are very well established, including, for example, those described 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 Nos. 4,518,584 and 4,737,462.

[0089] Nucleic acid molecules encoding amino acid sequence variants of antibodies are prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and preparations by cassette mutagenesis of previously prepared variant or non-variant versions of antibodies. Nucleic acid sequences encoding at least one antibody, portion, or polypeptide described herein can be recombined with vector DNA according to conventional techniques including, but not limited to, blunt or overhanging 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 may be used to construct monoclonal antibody molecules or nucleic acid sequences encoding antigen-binding regions.

[0090] In some embodiments, the nucleic acid encoding the antibody or its antigen-binding fragment described herein constitutes a vector. In some embodiments described herein, the nucleic acid sequence encoding the antibody or its antigen-binding fragment described herein, or any module thereof, is operably ligated to a vector. The term “vector” as used herein refers to a nucleic acid construct designed for delivery to or transfer between different host cells. As used herein, a vector may be viral or nonviral. The term “vector” encompasses a genetic element that can replicate when associated with a regulatory element and can transfer a gene sequence into a cell. Examples of vectors include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, and virions.

[0091] As used herein, the term “expression vector” refers to a vector that directs the expression of RNA or polypeptides of a sequence ligated to a transcriptional regulatory sequence on the vector. The term “expression” refers to the intracellular processes involved in the production of RNA and proteins, and, if applicable, the secretion of proteins, which include, but are not limited to, transcription, transcriptional processing, translation and protein folding, modification, and processing, where applicable. “Expression product” includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term “gene” means a nucleic acid sequence (DNA) that, when operably ligated to an appropriate regulatory sequence, is transcribed to RNA in vitro or in vivo. A gene may or may not include regions before and after the coding region, e.g., the 5' untranslated (5'UTR) or “leader” sequence and the 3'UTR or “trailer” sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0092] As used herein, the term “viral vector” refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. Viral vectors may contain nucleic acids encoding antibodies or their antigen-binding portions as described herein, instead of non-essential viral genes. Vectors and / or particles may be used for the purpose of transferring any nucleic acid into cells in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0093] The term "recombinant vector" means that the vector contains a heterologous nucleic acid sequence or a "transgene" that can be expressed in vivo.

[0094] Pharmaceutical composition, dosage, and dosage The anti-TL1A antibody provided is useful for a variety of applications, including but not limited to therapeutic treatment methods such as the treatment of IBD. The method of use may be in vitro, ex vivo, or in vivo. In one embodiment, the anti-TL1A antibody is an antagonist for the TL1A receptor.

[0095] In one embodiment, the diseases treated with an anti-TL1A antibody or TL1A receptor antagonist are IBD, CD, UC, and / or MR-UC.

[0096] In various embodiments, pharmaceutical compositions are formulated for delivery via any route of administration. "Route of administration" can mean, but is not limited to, a route of administration known in the art, including, but is not limited to, spray, nasal, oral, transmucosal, transdermal, or parenteral.

[0097] "Transdermal" administration can be performed using topical medications or ointments, or by transdermal patches.

[0098] "Parerale" generally refers to routes of administration usually associated with injection, including intraorbital, intradrip, intraarterial, intrasacral, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intrasternal, intrasternal, intrasacral, intrauterine, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via parenteral routes, compositions may be in the form of solutions or suspensions for infusion or injection, or lyophilized powders.

[0099] When delivered via the intestinal pathway, the pharmaceutical composition may be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microparticles or nanoparticles, or lipid vesicles or polymer vesicles, allowing for controlled release.

[0100] When administered via topical routes, pharmaceutical compositions are formulated for the treatment of skin and mucous membranes and may take the form of ointments, creams, emulsions, ointments, powders, penetrating pads, liquids, gels, sprays, lotions, or suspensions. They may further include microspheres or nanospheres, or lipid vesicles or polymer vesicles, or polymer patches and hydrogels that allow for controlled release. These compositions for topical routes may be in anhydrous or aqueous form depending on the clinical indication.

[0101] When administered via the transocular route, they may be in the form of eye drops.

[0102] In various embodiments, the drug may be administered intravenously by injection or by a slow infusion over a long period of time. Considering a suitable formulation for a given route, for example, drugs useful in the methods and compositions described herein may be administered intravenously, intranasally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, and intrafoveal, and, if necessary, by peristaltic methods or other methods known to those skilled in the art. In certain embodiments, the compounds used herein are administered orally, intravenously, or intramuscularly to patients suffering from IBD, CD, UC, and / or MR-UC.

[0103] The pharmaceutical composition may also contain any pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” means a pharmaceutically acceptable substance, composition, or vehicle that is involved in transporting or carrying the compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. 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 “pharmaceutically acceptable” in that it must be compatible with the other components of the formulation. Each component of the carrier must also be suitable for use in contact with any tissue or organ it may come into contact with, and must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or other complications that disproportionately exceed the therapeutic effect.

[0104] In various embodiments, pharmaceutical compositions are provided comprising a therapeutically effective amount of anti-TL1A antibody along with pharmaceutically acceptable excipients. “Pharmaceutically acceptable excipients” generally means excipients that are safe, non-toxic, and helpful in preparing a desirable pharmaceutical composition, and include excipients acceptable for use in animals and for human pharmaceutical applications. The active ingredient may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, and may be in amounts suitable for use in the treatments described herein. Such excipients may be solid, liquid, semi-solid, or, in the case of aerosol compositions, gas. Suitable excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, water, saline, glucose, propylene glycol, glycerin, ethanol, mannitol, polysorbate, and combinations thereof. In addition, the composition may, if necessary, contain small amounts of auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc., to enhance or maintain the effect of the active ingredient. The therapeutic compositions described herein may contain pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts formed from inorganic acids (e.g., hydrogen chloride or phosphoric acid), organic acids (e.g., acetic acid, tartaric acid or mandelic acid), salts formed from inorganic salts such as sodium, potassium, ammonium, calcium or iron hydroxide, and salts formed from organic salts such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine. Liquid compositions may contain liquid phases with and without water, e.g., glycerin, vegetable oils such as cottonseed oil, and oil-water emulsions. Pharmaceutically acceptable carriers are well known in the art. The amount of active agent (i.e., antibody or fragment thereof) used that is effective in treating a particular disease or condition depends on the nature of the disease or condition and can be determined by those skilled in the art using standard clinical techniques.

[0105] The pharmaceutical composition may further be formulated for oral administration by encapsulating, forming tablets, or as an emulsion or syrup. A pharmaceutically acceptable solid or liquid carrier may be added to enhance and stabilize the composition, or to facilitate its preparation. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline solution, alcohol, and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, clay, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar, or gelatin. The carrier may further contain a sustained-release substance, such as glyceryl monostearate or glyceryl distearate, alone or in combination with wax.

[0106] Pharmaceuticals are prepared according to conventional pharmaceutical techniques, including, if necessary, crushing, mixing, granulation, and compression for tablet form, or crushing, mixing, and filling for hard capsule form. When liquid carriers are used, the preparations are in the form of syrups, elixirs, emulsions, or aqueous or non-aqueous suspensions. Such liquid formulations may be administered directly orally or filled into soft gelatin capsules.

[0107] Pharmaceutical compositions may be delivered in therapeutically effective doses. The precise therapeutically effective dose is the amount of composition that produces the most effective result in terms of the efficacy of the treatment in a given subject. This amount will vary, but is not limited, depending on various factors, including the properties of the therapeutic compound (including its action, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological state of the subject (including age, sex, type and stage of disease, general physical condition, responsiveness to a given dose, and type of drug therapy), the properties of the pharmaceutically acceptable carrier or carrier in the formulation, and the route of administration. Those skilled in the art in the clinical and pharmaceutical fields may determine the therapeutically effective dose through customary procedures, for example, by monitoring the subject's response to the 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).

[0108] Typical doses of effective anti-TL1A antibodies may be as indicated to those skilled in the art, based on in vitro responses or responses in animal models. Such doses can typically be reduced by up to approximately an order of magnitude in concentration or volume without loss of relevant biological activity. Therefore, the actual dose depends on the physician's judgment, the patient's condition, and the effectiveness of the treatment method based on the in vitro responsiveness of tissue-cultured tissue samples, such as relevant primary cultured cells or obtained biological samples, or the response observed in appropriate animal models.

[0109] For the treatment of a disease, the appropriate dosage of antibody depends on the type of disease being treated, the severity and course of the disease, the responsiveness to the disease, whether the antibody is administered for therapeutic or prophylactic purposes, previous treatments, and the patient's medical history. The dosage may be further adjusted by individual physicians and at the discretion of the administering physician if any complications occur. The administering physician can determine the optimal dose, administration method, and repetition rate. TL1A antibodies may be administered as a single dose, over a series of treatments lasting from several days to several months, or until a cure is achieved or a reduction in the disease state is achieved (e.g., treatment or improvement of IBD). The duration of treatment depends on the subject's clinical course and response to treatment. In some embodiments, the dosage is 0.01 μg to 100 mg per kg of body weight and may be administered daily, weekly, monthly, or annually, once or more times. For systemic administration, subjects may be administered therapeutic doses such as approximately 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 higher. In some embodiments, the therapeutic dose is selected from approximately 1, 3, 10, 30, 100, 300, 600, and 800 milligrams, administered as a flat dosage. In some embodiments, the therapeutic dose is approximately 1, 2, 3, 4, 5, 6, 7, 8, or 9 milligrams, administered as a flat dosage. In some embodiments, the therapeutic dose is approximately 10, 20, 30, 40, 50, 60, 70, 80, or 90 milligrams, administered as a flat dosage. In one embodiment, the therapeutic dose is approximately 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 milligrams, administered as a constant dose. In another embodiment, the therapeutic dose is approximately 5 to 30 milligrams per kilogram. In yet another embodiment, the therapeutic dose is approximately 5 to 30 milligrams per kilogram, administered weekly or bi-weekly. In yet another embodiment, the therapeutic dose is approximately 5, 10, 15, 20, 25, or 30 milligrams per kilogram.In one embodiment, the therapeutic dose is approximately 5, 10, 15, 20, 25, or 30 milligrams per kilogram, administered weekly or bi-weekly.

[0110] Treatment method Various embodiments provide methods for treating inflammatory bowel disease (IBD), the methods comprising the step of administering an antibody against TL1A described herein to a subject. In some embodiments, the subject comprises one or more risk genotypes. In some embodiments, IBD is a severe form of IBD. The severe form of IBD may be characterized by an asymptomatic phenotype described herein.

[0111] In various embodiments, methods for treating inflammatory bowel disease (IBD) in a subject are provided herein, the methods comprising: administering to the subject a therapeutically effective amount of antibody or antigen-binding fragment that specifically binds to TL1A. In some embodiments, the anti-TL1A antibody comprises HCFR1 including SEQ ID NO: 545, or a sequence differing from SEQ ID NO: 545 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises HCDR1 selected from SEQ ID NO: 9, 150, 484, and 553, or a sequence differing from a sequence selected from SEQ ID NO: 9, 150, 484, and 553 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody comprises HCFR2 including SEQ ID NO: 546, or a sequence differing from SEQ ID NO: 546 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody includes an HCDR2 selected from SEQ ID NO: 12, 574-564, and 554-577, or a sequence that differs from SEQ ID NO: 12, 574-564, and 554-577 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody includes an HCFR3 selected from SEQ ID NO: 547 and 586-588, or a sequence that differs from the sequence selected from SEQ ID NO: 547 and 586-588 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody contains HCDR3 selected from SEQ ID NO: 15, 152, 565-568, and 578-581, or a sequence that differs from a sequence selected from SEQ ID NO: 15, 152, 565-568, and 578-581 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody contains HCFR4 including SEQ ID NO: 548, or a sequence that differs from SEQ ID NO: 548 by up to approximately 5, 4, 3, or 2 amino acids.In some embodiments, the anti-TL1A antibody contains LCFR1 including SEQ ID NO: 549, or a sequence that differs from SEQ ID NO: 549 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody contains LCDR1 selected from SEQ ID NO: 487, 569, and 570, or a sequence that differs from SEQ ID NO: 487, 569, and 570 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody contains LCFR2 including SEQ ID NO: 550, or a sequence that differs from SEQ ID NO: 550 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody contains LCDR2 including SEQ ID NO: 488, or a sequence that differs from SEQ ID NO: 488 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody contains LCFR3 including SEQ ID NO: 551, or a sequence that differs from SEQ ID NO: 551 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody contains LCDR3 selected from SEQ ID NO: 571-573 and 582-585, or a sequence that differs from a sequence selected from SEQ ID NO: 571-573 and 582-585 by up to approximately 5, 4, 3, or 2 amino acids. In some embodiments, the anti-TL1A antibody contains LCFR4 including SEQ ID NO: 552, or a sequence that differs from SEQ ID NO: 552 by up to approximately 5, 4, 3, or 2 amino acids.

[0112] The subjects disclosed herein may be mammals, such as mice, rats, guinea pigs, rabbits, non-human primates, or livestock. In some cases, the subjects are human. In some cases, the subjects are patients diagnosed with IBD. In some cases, the subjects are not diagnosed with IBD. In some cases, the subjects suffer from symptoms associated with the diseases or illnesses disclosed herein (e.g., abdominal pain, muscle spasms, diarrhea, rectal bleeding, fever, weight loss, fatigue, loss of appetite, dehydration, and malnutrition, anemia, or ulcers).

[0113] In various embodiments, subjects are either unresponsive to induction of anti-TNF therapy (e.g., adalimumab, certolizumab, etanercept, golimumab, infliximab) or lose their response to said anti-TNF therapy after a certain period of time following treatment (anti-TNF response loss). In various embodiments, subjects are at risk of developing anti-TNF unresponsiveness or anti-TNF response loss. In some embodiments, subjects are treated by administering anti-TL1A antibodies disclosed herein, provided that the subjects are at risk of developing anti-TNF unresponsiveness or anti-TNF response loss, or suffer from anti-TNF unresponsiveness or anti-TNF response loss.

[0114] In various other embodiments, subjects are determined to have increased TL1A expression. In some embodiments, administration of a therapeutically effective amount of anti-TL1A antibody causes a decrease in TL1A in the treated subjects.

[0115] The methods disclosed herein provide a method for treating inflammatory bowel disease (IBD) in a subject by administering the subject an anti-TL1A antibody described herein. In various embodiments, IBD is Crohn's disease (CD) or ulcerative colitis (UC). In some embodiments, IBD is a severe form of IBD. In some embodiments, the severe form of IBD is characterized by an asymptomatic phenotype. In some embodiments, IBD is a moderate to severe form of IBD. In some embodiments, IBD is a moderate form of IBD.

[0116] Asymptomatic phenotypes of IBD include, but are not limited to, non-stenotic, stenotic, stenotic and osmotic, as well as isolated internal osmotic, disease, and perianal clonal disease (pCD). Stenotic is a progressive narrowing of the intestine. Disease with internal osmotic formation creates abnormal passages (fistulas) between the intestine and other structures. pCD is a form of Crohn's disease that causes inflammation around the anus.

[0117] IBD can be refractory. The term “medically refractory” or “refractory” refers to the failure of standard treatment to induce remission of the disease, as used herein. In some embodiments, the disease includes 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 treatment include glucocorticosteroids, anti-TNF therapy, anti-α4-B7 therapy (vedolizumab), anti-IL12p40 therapy (ustekinumab), thalidomide, and cytoxin. In some embodiments, UC is medically refractory UC (mrUC). In some embodiments, CD is refractory.

[0118] A method for administering an anti-TL1A antibody to a subject is disclosed herein. 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.

[0119] In various embodiments, an anti-TL1A antibody is administered to a subject for the treatment of IBD described herein. In various other embodiments, the anti-TL1A antibody is administered in a series of treatments. In some embodiments, the anti-TL1A antibody and the second IBD treatment agent may be administered in any order or simultaneously. In selected embodiments, the anti-TL1A antibody is administered to a patient who has previously received treatment with the second IBD treatment agent. In some other embodiments, the anti-TL1A antibody and the second IBD treatment agent are administered substantially simultaneously or concurrently. For example, a subject may be given the anti-TL1A antibody while receiving a series of treatments with the second IBD treatment agent. In some embodiments, the anti-TL1A antibody is administered within one year of treatment with the second IBD treatment agent. In some other embodiments, the anti-TL1A antibody is administered within 10, 8, 6, 4, or 2 months of any treatment with the second IBD treatment agent. In some other embodiments, the anti-TL1A antibody is administered within 4, 3, 2, or 1 week of any treatment with the second IBD treatment agent. In some embodiments, the anti-TL1A antibody is administered within 5, 4, 3, 2, or 1 day of any treatment with the second IBD treatment agent. It will be further understood that the two treatment agents may be administered to the subject within just a few hours or minutes (i.e., simultaneously).

[0120] Other IBD treatments include, but are not limited to: 1) anti-inflammatory drugs (e.g., sulfasalazine, azulfidine, 5-aminosalicylic acid, mesalamine, asacol, realda, Rowasa, Canasa, valsalazide, Colazal, and olsalazine, dipentum, etc., aminosalicylic acids; 2) corticosteroids (e.g., prednisone and hydrocortisone); 3) immunosuppressants (e.g., azathioprine, Azasan, Imuran, mercaptopurine, prinetol, Purixan, cyclosporine, Gengraf, Neoral and Sandimmune, infliximab, Remicade, adalimumab, Humira, golimumab, and Simponi), tumor necrosis factor (TNF)-α inhibitors (e.g., infliximab), methotrexate, rheumatoid arthritis Examples include: Torex, natalizumab, Tysabri, vedolizumab, Entyvio, ustekinumab, and Stelara; 4) Antibiotics (e.g., metronidazole, Flagyl, ciprofloxacin, Cipro); 5) Antidiarrheal agents (e.g., fiber supplements - Metamucil or Citrucel) or loperamide; 6) Analgesics (e.g., Tylenol, ibuprofen, naproxen sodium, and diclofenac sodium); and 7) Surgical procedures (e.g., colectomy, partial gastrointestinal resection, colectomy, corectomy, and / or strictureplasty). In some embodiments, these IBD treatments may be administered in combination with anti-TL1A antibodies. Antibody-based treatments may be administered before, concurrently with, or after the administration of IBD treatments. Concomitant administration may include simultaneous administration of a single drug formulation or separate formulations, or continuous administration in any order, but generally within a period in which all active agents can exert their biological activity simultaneously. Any medication schedule for such IBD treatments may also be used as determined by a skilled practitioner.

[0121] In some embodiments, the second IBD treatment includes an antibody. Therefore, the treatment may include the co-administration of the antibody provided herein with other antibodies against additional IBD-related antigens (but not limited to tumor necrosis factor (TNF)-α). The co-administration may include administration in a single pharmaceutical formulation, concurrent administration using separate formulations, or in any order, but generally, continuous administration over a period of time in which all active agents can exert their biological activity simultaneously.

[0122] kit Furthermore, a kit for treating IBD (e.g., CD, UC, and / or mrUC) is provided. The kit comprises the antibody described herein and can be used to carry out the method described herein. The kit is useful for carrying out the method of the present invention for treating IBD, CD, UC, and / or mrUC patients by administration of an antibody against TL1A. The kit is an assembly of substances or components comprising at least one of the compositions of the present invention. Thus, in some embodiments, the kit contains a composition comprising an antibody against TL1A for the treatment of IBD, CD, UC, and / or mrUC as described above. In other embodiments, the kit contains the necessary and / or sufficient components for carrying out a detection assay for TL1A, including all controls, instructions for carrying out the assay, and any necessary software for analysis and presentation of results.

[0123] The exact properties of the components comprised in the kit of the present invention depend on its 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 configured, in particular, for the purpose of treating mammalian subjects. In another embodiment, the kit is configured, in particular, for the purpose of treating human subjects. In further embodiments, the kit is configured for veterinary use, but is not limited to, treating subjects such as livestock, domestic animals, and laboratory animals.

[0124] Instructions for use may be included in the kit. These instructions typically include clear language describing the techniques used to achieve desired outcomes, such as treating or alleviating IBD, CD, UC, and / or MR-UC. Optionally, the kit may also include other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting tools, or measuring instruments, bandaging materials, or other useful tools readily recognizable to those skilled in the art.

[0125] The substances or components assembled in the kit may be stored in a convenient and appropriate manner that maintains their operability and usefulness and provided to the operator. For example, the components may be in a thawed, dehydrated, or lyophilized form; they may be provided at room temperature, refrigerated, or frozen temperature. The components are typically contained in suitable packaging materials. As used herein, the phrase “packaging materials” refers to one or more physical structures used to contain the contents of the kit, such as the compositions of the present invention. The packaging materials are preferably constructed in a well-known manner to provide a sterile, contaminant-free environment. The packaging materials used in the kit are those conventionally used for gene expression assays and the administration of treatment agents. As used herein, the term “packaging” refers to a suitable solid matrix or material, such as glass, plastic, paper, or foil, that can hold the components of the individual kits. Thus, for example, the packaging may be a glass vial or a pre-filled syringe used to contain a suitable amount of the compositions of the present invention containing anti-TL1A antibodies and / or primers and probes for TL1A. Packaging materials typically have external labels indicating the contents and / or the purpose of the kit and / or its components. [Examples]

[0126] The following examples are illustrative of embodiments described herein and should not be construed as limiting the scope of this disclosure. Where specific materials are mentioned, they are for illustrative purposes only and not intended to limit the scope. Those skilled in the art can develop equivalent means or reactants without exercising their inventive capacity and without departing from the scope of this disclosure.

[0127] Example 1: Production and characterization of humanized anti-TL1A antibodies The mouse anti-TL1A antibody was humanized to reduce its potential immunogenicity. A first mutant, 12835, was generated, consisting of mouse 5C3D11 CDRs (SEQ ID NO: 9, 12, 15, 18, 21, 24) transplanted into a human variable region framework to generate a heavy chain variable region containing EQ ID NO: 26 and a light chain variable region containing SEQ ID NO: 28. Unfortunately, clone 12835 contains nine framework reverse mutations (mouse framework residues), resulting in an incompletely humanized mutant. Complete humanization is crucial to reduce the likelihood of the subject eliciting an immune response to the administered antibody. Consequently, the goal was to generate a humanized antibody containing fewer mouse framework residues while retaining the functional activity of the parent 12835 antibody. Unfortunately, using visual sequencing, it is not easy to distinguish between mouse framework residues critical to antibody function and those that are not, and therefore, it is not easy to distinguish which amino acid residues can be replaced by their corresponding human framework residues. Therefore, as the first step, 12835 was re-humanized by CDR transplantation into the nearest complete human germline framework (IGV1-46 as determined by NCBI's igblast tool). * 02 and IGKV3-20 *01). This clone is L8 and contains 12835 CDRs defined by 5C3D11 and a combination of the Kabat, Chothia, and IMGT methods (HCDR1,GFDIQDTYMH;HCDR2,RIDPASGHTKYDPKFQV;HCDR3,SRSGGLPDV;LCDR1,RASSSVSYMY;LCDR2,ATSNLAS;LCDR3,QQWSGNPRT).

[0128] In this study, numerous mutants of 12835 were created and tested to identify more human-like antibodies that retained the functional activity of the parent 12835 antibody. In the first stage, mutants containing significantly fewer mouse framework residues were identified. Subsequently, the 12835 CDR library was combined with a single complete human germline framework to identify multiple mutants that lacked mouse framework residues but retained the functional activity and / or affinity of the parent 12835 antibody.

[0129] Cloning of mouse 5C3D11 and humanized construct 12835 into a phage expression system. DNA encoding the heavy chain and light chain variable regions of both mouse 5C3D11 and humanized 12835 was cloned into a phage expression vector containing the human κ light chain constant domain and the human G1 heavy chain constant domain 1. In addition, the vector contained a his- tag and a hemagglutinin A tag at the carboxy-terminal end of the heavy chain to facilitate purification and detection. Cloning the mouse variable regions into a phage expression vector containing the human constant domain resulted in the expression of chimeric 5C3D11.

[0130] The mouse 5C3D11 heavy chain variable region DNA (SEQ ID NO:1) and light chain variable region DNA (SEQ ID NO:4) were codons optimized for bacterial expression to produce SEQ ID NO:2 and 5, respectively. The humanized 12835 heavy chain variable region DNA was a codon optimized to produce SEQ ID NO:25, and the light chain variable region DNA was a codon optimized to produce SEQ ID NO:27.

[0131] Expression and quantification of Fab in the perimembrane space of E. coli Cloning was validated by expressing and quantifying Fab in the perimembranous space of E. coli cells. Briefly, XL-0 bacteria were grown in 2X YT medium at 37°C until the culture reached a density of 0.9–1.1 OD600, then isopropyl β-D-thiogalactoside was added to the cells to a final concentration of 1 mM, and 3.0 mL of the culture was transferred to a 14 mL snap-top tube. Each tube was transfected with 25 μL of high-titer phage stock, and the cultures were placed in a shaker (225 rpm) at 37°C. After 1 hour, the temperature was changed to 25°C and the cultures were grown for a further 14–16 hours. Cells were collected by centrifugation at 3900 rpm for 30 minutes in an Eppendorf 5810R centrifuge (~3,200xg), the supernatant was decanted, and the cells were re-mixed 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 placed on ice for 15 minutes. The cell suspension was transferred to a 1.5 mL tube, and the cell fragments were pelletized by centrifugation at 15,000 rpm for 15 minutes in an Eppendorf 5424 microcentrifuge tube (~21,000xg). The supernatant was carefully removed without disturbing the pellet, and the pellet was stored at 4°C until use.

[0132] To quantify Fab expression, a 96-well Costar-3366 plate was coated overnight at 4°C with 50 μl / well of 2 μg / ml sheep anti-human Fd (Southern Biotech, Prod. # 2046-01, Lot # A7212-VJ06) in PBS. The plate was washed three times with PBS containing 0.05% Tween 20 (PBS-T), and 50 μl / well of sample diluent was added. Sample dilution was performed using PBS-T. A calibration curve was generated using human Fab (Rockland, Prod. # 009-01015, Lot # 38543), starting at 500 ng / ml and continuously diluted threefold. The above plates were incubated at 25°C for 1 hour, washed three times with PBS-T, and incubated with 50 μl / well of anti-κ HRP conjugate (Southern Biotech, Prod. # 2060-05, Lot # K3114-S506B) diluted 10,000-fold in PBS-T at 25°C for 1 hour. The above plates were washed three times with PBST and developed with 50 μl / well of 1-Step Ultra TMB-ELISA (Thermo Scientific, Prod. # 34028, Lot # SF2405221). The reaction was terminated by adding 2N H2SO4, and A650 and A450 were measured before and after H2SO4 addition using a Spectramax plate reader.

[0133] Characterization of chimera 5C3D11 and filter-lift assay of 12835 The filter-lift assay was developed to facilitate the characterization of heavy and light chain expression and to validate the functional activity of Fab constructs via binding to biotinylated antigens. Using the filter-lift assay, a bacterial community was infected with phages under conditions where each phage produced a different plaque (a zone of slower-growing bacteria). A nitrocellulose filter was placed over the community to capture the expressed Fab. The filter could then be examined with biotinylated antigens and / or reagents directed against immunoglobulins or peptide tags.

[0134] A diluent of high-titer phage stock (typically 10⁶-fold) was combined with 0.35 ml of confluent E. coli XL culture and 20 μg / ml of tetracycline. This mixture was conjugated with 3.5 ml of top agar (0.7% Bacto-agar in Luria broth) and placed on an LB agar plate (1.5% Bacto-agar in Luria broth). The plate was incubated at 37°C for 6–8 hours, at which point a nitrocellulose filter (Whatman 82 mm diameter, 0.45 μm pore size, GE Healthcare, Prod. #10401116) was placed on top, and the plate was incubated at 25°C for 12–15 hours. The filter was removed, the mixture was briefly rinsed in PBS, and transferred to 5% MP blocking solution at 25°C for 2 hours with constant stirring.

[0135] Subsequently, the filter was cut into three parts (one to evaluate light chain expression, one to evaluate heavy chain expression, and one to evaluate antigen binding). Each part was transferred to a primary detection reagent: goat anti-human κ, HRP conjugate diluted 1000-fold in 5% MP for light chain detection (Southern Biotech, Prod. # 2060-05, Lot # K3114-S506B), rat anti-HA, HRP conjugate diluted 1000-fold in 5% MP for heavy chain detection (Roche, Prod. # 12013819001), or biotinylated antigen at a desired concentration in 5% MP.

[0136] To label the antigen with biotin, 500 μg of human TL1A (Fitzgerald, Prod. # 30R-AT070, Lot # A13102302) was re-mixed in 1 mg / ml water. After suspension in water, the protein was added to 10 mM Tris (pH 8.5) with 75 mM arginine. Tris and arginine were removed by buffer exchange using a 7 K MW cutoff, 5 ml Zeba spin desalting column (Thermo Prod. # 89891) equilibrated with 10 mM phosphate buffer (pH 8.0) with 65 mM NaCl. Immediately after recovering the protein, it was biotinylated by combining it with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Prod. # 21327) at a 5:1 molar concentration at 25°C for 30 minutes. The reaction was terminated by adding 750 mM arginine to achieve a final concentration of 75 mM. The reaction mixture was transferred to ice and stored at 4°C.

[0137] The filters were incubated at 25°C for 2 hours with constant agitation and washed five times with PBS-0.05% Tween 20 (each wash for 2 minutes with constant agitation). The filters probed with biotinylated antigen were transferred to 10 ml of High Sensitivity Neutravidin and HRP conjugate (Thermo Scientific, Prod. #31030) diluted 5000-fold in 1% BSA in PBS, and incubated at 25°C for 1 hour. Subsequently, the filters were washed five times with PBS-0.05% Tween 20 (each wash for 2 minutes with constant agitation). All filters were developed with 1-Step Ultra TMB-Blotting (Thermo Scientific, Prod. #37574).

[0138] Using this approach, we demonstrated the expression of heavy chains (Figure 1A) and light chains (Figure 1B). Furthermore, when the filter was probed with 8 nM biotinylated human TL1A, staining indicating that the bacteria expressed functional Fab was observed (Figure 1C).

[0139] Characterization of Fab binding by ELISA The filter-lift assay provides a qualitative assessment of antigen binding activity. For a more quantitative method, an ELISA was developed to compare the binding activity of the chimeric 5C3D11 with that of the humanized construct 12835. 96-well Costar-3366 plates were coated overnight at 4°C with 2 μg / ml human TL1A (Fitzgerald, Prod. # 30R-AT070, Lot # A13102302) in 50 μl / well of PBS. The plates were rinsed once with PBS-T and blocked at 25°C for 1 hour with 1% BSA (1% BSA) in 100 μl / well. Fab samples were serially diluted 3-fold using 1% BSA and incubated at 25°C for 1 hour (50 μl / well). The above plates were washed three times with PBS-T, and 50 μl / well of anti-human κ and HRP conjugate diluted 10,000-fold in 1% BSA (Southern Biotech, Prod. # 2060-05, Lot # K3114-S506B) were added and incubated at 25°C for 1 hour. In a specific assay (expanded wash format), the above plates were placed in a large volume of PBS-T (up to 1 L), incubated with mixing for 2-5 hours, and then anti-human κ and HRP conjugate were added. The above plates were washed three times with PBS-T and developed with 50 μl / well of 1-Step Ultra TMB-ELISA (Thermo Scientific, Prod. # 34028, Lot # SF2405221). The reaction was terminated by the addition of 2N H2SO4, and A650 and A450 were measured before and after the addition of H2SO4 using a Spectramax plate reader. The same protocol was used to measure binding to mouse TL1A, but the plates were coated with 2 μg / ml mouse TL1A (BioLegend, Prod. #753004, Lot # B204691), and the Fab samples were serially diluted 2-fold.

[0140] The binding activity of chimeric 5C3D11 Fab was compared with that of humanized 12835 Fab (Figure 2). While the binding activity of 5C3D11 and humanized 12835 appeared similar in the IgG format (bivalent), the binding activity of 12835 in the Fab format (monovalent) was observed to be slightly reduced compared to the chimeric Fab (Figure 2). This discrepancy reflects the difference between true affinity (monovalent format) and similar avidity (bivalent format). Using the monovalent assay format, the chimeric Fab appears to have 2–3 times higher affinity than humanized 12835 Fab.

[0141] Lift assay capture A nitrocellulose filter (Whatman, 82 mm diameter, 0.45 μm pore size, GE Healthcare, Prod. #10401116) was floated on 10 ml of 10 mg / ml goat anti-human κ (Southern Biotech Prod. #2060-01) at 25°C for 2 hours. After briefly immersing the filter in water, it was removed and transferred to 10 ml of 5% MP at 25°C for 2 hours. The filter was removed from the 5% MP, briefly rinsed once with PBS, and air-dried. The filter was then treated in the same manner as the filter-lift assay described above, with minor modifications. In short, high-titer phage stocks (typically 10 6A diluent (1 / 2) was combined with 0.35 ml of confluent E. coli strain XL culture and 20 μg / ml tetracycline. This mixture was conjugated with 3.5 ml of top agar (0.7% Bacto-agar in Luria broth) and placed on an LB agar plate (1.5% Bacto-agar in Luria broth). The plate was incubated at 37°C for 6–8 hours, at which point a pre-treated nitrocellulose filter (described above) was placed on top, and the plate was incubated at 25°C for 12–15 hours. The filter was removed, the plate was briefly rinsed in PBS, and the biotinylated antigen was transferred to the desired concentration in 5% MP. The above filters were incubated at 25°C for 2 hours with constant agitation, washed five times with PBS-0.05% Tween 20 (each wash for 2 minutes with constant agitation), and transferred to 10 ml of highly sensitive neutraavidin, 5000-fold diluted HRP conjugate in 1% BSA in PBS (Thermo Scientific, Prod. #31030), and incubated at 25°C for 1 hour. Subsequently, the above filters were washed five times with PBS-0.05% Tween 20 (each wash for 2 minutes with constant agitation). All filters were unfolded with 1-Step Ultra TMB-Blotting (Thermo Scientific, Prod. #37574). The unfolded filters, as shown in Figure 3, demonstrate the high sensitivity and avidity of 5C3D11 against TL1A.

[0142] Removal of mouse framework residues from 12835 to identify multiple active humanized clones, including 18-7 and 21-3. Mouse framework residues were removed using Kunkel mutagenesis (Kunkel TA 1985. PNAS 82:488-492). Briefly, single-stranded M13 plasmids were isolated and stimulated for DNA replication with human-encoding mutagenic oligonucleotides instead of mouse framework residues. After elongation to complete the circle, bacterial transformation yielded a mixture of wild-type (unique mouse framework residue) plasmids and mutated (human framework residue) plasmids. Mutagenesis was performed simultaneously at multiple sites to generate a small combinatorial library containing a mixture of clones with various combinations of mouse and human framework mutations. The mixture was then plated and screened by capture lift to identify the most active framework combinations.

[0143] Library clones characterized by DNA sequencing Fab was expressed in E. coli, quantified by ELISA, and its binding activity was evaluated by titration against immobilized antigens using ELISA. Fab expression, isolation of the periplasmic fraction, quantification of Fab expression, and binding to the antigen by ELISA were all performed as described above.

[0144] Using this approach, we identified multiple active clones containing different numbers of mouse framework residues. Examples of active clones with varying numbers of mouse framework residues and their corresponding locations are summarized in Table 3.

[0145] [Table 3]

[0146] Synthesis of CDR transplant structures The two identified clones, 18-7 and 21-3, contained only two mouse framework reverse mutations. The light chain of clone 18-7 lacked mouse framework residues, and the heavy chain of clone 21-3 lacked mouse framework residues. Screening of the framework combinatorial library did not identify any CDR transplant mutants (lacking mouse framework residues in both the heavy and light chains). For comparison, CDR transplant mutants were synthesized using Kunkel mutagenesis, compared, and their binding activity was characterized by ELISA. The CDR transplant constructs bound to the antigen, but the humanized 12835 mutant consistently showed stronger binding to the antigen (Figure 4B).

[0147] After inducing reverse mutations in the mouse framework residues as shown in Table 3, the heavy chain variable region framework 1-3 became human germline IGHV1-46 * 01, IGHV1-46 * 02, and IGHV1-46 * It is identical to 03, and the light chain variable region framework is human germline IGKV3-20 * It was identical to 01.

[0148] In addition, a novel heavy chain variable region was found in human germline IGHV1-3 * Various reverse mutations were introduced into the third framework of the heavy chain variable region, homologous to 01 (see VH SEQ IDs 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, and 541). Collectively, clones containing these reverse mutations are called alternative framework variants.

[0149] Characterization of humanized mutants using alternative ELISA formats Multiple Fab variants were characterized by ELISA using an alternative format that allows for rapid and direct comparison of the relative affinity of different Fab clones isolated from different cultures, regardless of the relative expression levels of the clones (single-well determination without the need for dilution series) (Watkins et al. 1997, Analytical Biochemistry 253). This assay allows for a more quantitative comparison of the relative binding strength of the variants because the plates are saturated with different Fabs despite the different expression levels. Thus, slight differences in binding profiles caused by variance in Fab quantification assays are eliminated. Briefly, 96-well Costar-3366 plates were coated with 50 μl / well of 2 μg / ml goat anti-human κ (Southern Biotech Prod. #2060-01) at 25°C for 2 hours, washed once with PBS-0.05% Tween 20, and incubated with 50 μl / well of sample Fab at 25°C for 2 hours. The above plates were washed four times with PBS-0.05% Tween 20 and incubated at 25°C for 2 hours with a serial dilution of biotinylated antigen at 50 μl / well. The preparation of the biotinylated antigen is described above. The above plates were washed four times with PBS-0.05% Tween 20 and incubated at 25°C for 1 hour with 50 μl / well of highly sensitive neutraavidin and HRP conjugate (Thermo Scientific, Prod. #31030) diluted 5000-fold with 1% BSA in PBS. The above plates were washed three times with PBS-T and developed with 50 μl / well of 1-Step Ultra TMB-ELISA (Thermo Scientific, Prod. #34028, Lot #SF2405221). The reaction was terminated by adding 2N H2SO4, and A650 and A450 were measured before and after the addition of H2SO4 using a Spectramax plate reader.

[0150] Chimeric Fabs bind more strongly to the antigen than CDR-transplanted Fabs in the alternative ELISA format (Figure 5, compare white and black circles). Humanized clone 12835 shows slightly reduced binding compared to the chimeric clone (compare white triangles and white circles), followed by clones 18-7 and 21-3. The binding of clone 21-3 is most similar to that of the CDR-transplanted mutant, suggesting that one of the mouse heavy chain framework reverse mutations may be important for maintaining full binding activity with the parental (wild-type) CDR.

[0151] Example 2: Generation and characterization of an anti-TL1A antibody with optimized CDR. To identify CDR mutations that can restore and improve the binding activity of CDR transplant constructs (complete human, germline framework), each of the six CDR positions (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) was mutagenesized by Kunkel mutagenesis using degenerate oligonucleotides in which the codon encoding the target amino acid was replaced with an NNK. First, one library was synthesized at each of the HCDR3 and LCDR3 positions. These position libraries, with a theoretical diversity of 32 codons / 20 amino acids / 1 stop codon, were screened by capture lift. In some cases, each position was screened by itself (theoretical diversity of the library is equal to 32), while in other cases, specific CDR positions were pooled and screened as a CDR library (theoretical diversity of the library is equal to 32 times the number of pooled positions; for example, HCDR3 consists of 7 positions, and therefore the theoretical library size was 32 × 7 = 224 members). The library was screened with biotinylated human TL1A concentrations ranging from 15 to 1,000 pM. Positive plaques were selected and sequenced. As described above, Fab was expressed, isolated from the periplasmic fraction, and characterized by ELISA. Tables 4 and 5 summarize the capture-lift screening, DNA sequencing, and relative binding activity by ELISA for some of the initial screens of LCDR3 and HCDR3, respectively. The results of more thorough capture-lift screening for all six CDRs are summarized in Tables 6–12.

[0152] In addition, libraries of LCDR3 and HCDR3 constructed using an alternative heavy-chain variable-region framework were created and screened by capture lift (Tables 13 and 14). Screening of CDR libraries constructed on the alternative framework was performed using VH1-46. *In addition to identifying several mutations previously identified on the 01 framework, we also identified novel mutations that had not been identified before (e.g., heavy chain CDR3 L98S, V102H, V102F, and light chain CDR3 S92A, S92F, and S92Y).

[0153] [Table 4]

[0154] [Table 5]

[0155] [Table 6]

[0156] [Table 7]

[0157] [Table 8]

[0158] [Table 9]

[0159] [Table 10]

[0160] [Table 11]

[0161] [Table 12]

[0162] [Table 13]

[0163] [Table 14]

[0164] Example 3. Generation and characterization of an anti-TL1A antibody with optimized CDR using a mutated (S93) heavy chain. Clones containing the CDR-transplanted heavy chain (CDR-transplanted and 21-3) exhibited lower binding activity than clones containing the mouse reverse mutation (S) at heavy chain position 93. For this reason, a further HCDR3 library was constructed. The library was constructed as described above, except that the degenerate oligonucleotides for all positions were mixed prior to mutagenesis, and the library was synthesized and expressed as a pool, in contrast to examining each position separately. Similar to the HCDR3 position screening performed on the L8 heavy chain backbone (S93A), HCDR3 position 102 resulted in several mutations that enhanced antigen binding in the capture-lift format. Screening of the HCDR3 library based on the heavy chain S93 template identified several mutations identified on the heavy chain A93 template, but also identified novel mutations that had not been previously identified (e.g., heavy chain CDR3 V102I and V102Y). A summary of the capture-lift screening and DNA sequencing is shown in Table 15.

[0165] [Table 15]

[0166] Example 4. Identification and Engineering of Latent Sequence Liability A structural homology model of the variable region of the CDR transplant construct L8, based on known PDB antibody structures, was constructed using Molecular Engineering Environment (MOE) 2018.01 software (Chemical Computing Group, Montreal, Canada). Sequence liability evaluation was performed using the above model and the BioMOE prediction algorithm. In addition, the potential sequence liability of mutant 12835 and L8 was analyzed based on known potentially unstable sequence motifs (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 approaches, several residues were identified as potentially unstable, including: light chain M33, W35, W47, W91, and N94; heavy chain D31T32, M34, D52P52a, M69, and W103 (summarized in Table 17; FR indicates framework).

[0167] Thorough capture-lift screening was performed at all potentially unstable sites within the CDR to identify amino acid substitutions that can remove these residues while maintaining antigen binding. As an example, one screen was focused on light chain CDR3 N94 (a potential deamide site). Initial capture-lift screening at LCDR3 position 94 did not identify mutations that showed enhanced affinity to the wild-type sequence. Therefore, the conditions of the initial capture-lift screening were modified to identify acceptable mutations for removing these potentially unstable residues. In particular, instead of screening with antigen concentrations at which the wild-type sequence did not provide a signal, the antigen concentration was increased to make clones expressing the wild-type sequence visible on the lift. In this way, it is possible to identify mutants that bind with affinity similar to the wild-type sequence but remove problematic residues.

[0168] Approximately 3,000 clones were plated from the LCDR3 position 94 library and analyzed by capture lift. Capture lift was screened using 1000 pM human TL1A, and plaques showing eight of the darkest staining intensities and six of the brighter staining intensities were selected and sequenced. The results are summarized in Table 16. As expected, two of the darkest staining plaques expressed the wild-type residue N94. However, the other six dark staining plaques expressed T94, which indicates that the mutant N94T largely retains binding affinity to the wild-type sequence and removes the potential deamide site. In addition, five different sequences were identified from the brighter staining plaques. These included D, F, K, R, and S. These alternative sequences may also have slightly lower affinity than the wild-type sequence, but when bound to other, higher-affinity mutants identified elsewhere, all can function as substitutes for N94.

[0169] [Table 16]

[0170] [Table 17]

[0171] Example 5. Identification of mutations that result in enhanced expression in Escherichia coli. Certain mutations identified during capture-lift screening of the CDR library consistently expressed soluble Fab at higher levels in the perimembranous space of bacterial cells than other mutants, although they did not always show enhanced binding in the ELISA format. This phenomenon was particularly observed at heavy chain CDR2 position V65 (V65G, V65T, and V65K) and light chain CDR1 position R24 (R24G). These results were surprising because the capture-lift screening format is designed to minimize the effects of varying expression levels while maximizing the effect of affinity on signal intensity. Consequently, these mutations were focused on and integrated into a subsequent combinatorial library containing the affinity-enhanced mutations to determine whether they confer expression and / or thermal stability advantages to candidate immunoglobulins expressed as intact immunoglobulins in mammalian expression systems.

[0172] Example 6. Generation and characterization of anti-TL1A antibodies with combinatorial HCDR3 and LCDR3 mutations. Based on the initial identification of beneficial mutations in both HCDR3 and LCDR3, further libraries were synthesized, expressed, and screened to identify independent mutation combinations that could further improve binding affinity. The libraries were constructed by mutagenesis at two sites using oligonucleotides encoding subsets of mutations identified in site scanning. Oligonucleotides encoding wild-type residues were also included. This combinatorial library contained 30 different variants: wild-type (no mutation), 9 variants including single mutations (overlapping with variants identified by site screening as shown in Tables 4 and 5), and 20 unique combinations. Capture-lift screening with 200 pM antigen identified 21 active clones. DNA sequencing of the 21 clones identified certain combinations more frequently than others (Table 18).

[0173] [Table 18]

[0174] Subsequently, multiple combinatorial libraries were synthesized, expressed, and screened (details below). Generally, these libraries combined mutations identified as improving affinity (Examples 2 and 3) with mutations modifying potentially unstable residues (Example 4) and mutations potentially conferring enhanced thermal stability / expression (Example 5). The combinatorial libraries were screened using multiple ELISA formats to identify clones with the best characteristics (affinity, selectivity, binding to membrane-bound TL1A, and development potential) for further development. As summarized in Tables 19-22, multiple mutants with optimized and diverse CDR sequences were identified using various VH germline templates.

[0175] [Table 19]

[0176] [Table 20]

[0177] [Table 21]

[0178] [Table 22]

[0179] The Fab mutants with CDRs shown in Tables 19-22 were tested for binding to human TL1A in multiple formats. First, human TL1A was immobilized on the surface of an ELISA plate, and the soluble Fab mutants were titrated as shown in Figures 8 and 9. Next, a fixed (saturated) volume of soluble Fab mutants was captured on the surface of an ELISA plate, and soluble biotinylated human TL1A was titrated as shown in Figures 10-13. In both ELISA formats, all Fab mutants bound to human TL1A and showed significantly enhanced binding compared to the CDR-implanted mutant L8. In addition, all mutants bound well or better than variant 12835, while either having no mouse reverse mutations in the framework or having significantly fewer mouse reverse mutations. As a result, these experiments revealed a set of anti-TLIA variable regions that exhibited both high binding affinity and high homology to human germline Ig sequences.

[0180] All Fab variants were tested for binding to membrane-bound human TL1A. These tests used the HEK293 cell line transfected with human TL1A. 293 cells expressing membrane-bound human TL1A were maintained in a 37°C incubator with 5% CO2 in DMEM containing L-glutamine, glucose, sodium pyruvate, and phenol red (ThermoFisher cat#11995-065), 10% fetal bovine serum, 1X penicillin streptomycin (Fisher cat#15140122), and 2 μg / ml puromycin (Gibco cat#A11138-03). Three days prior to the assay, 3 × 10⁶ T-75 flasks were filled to ensure the flasks were 90–95% confluent on the day of the assay. 6 The cells were seeded. The culture medium was aspirated, and the cell monolayer was gently washed with 5 ml of PBS. Adherent cells were removed from the monolayer by repeatedly pipetting 10 ml of ice-cold 1% BSA / PBS. The cells were counted, and 5 × 10⁶ cells were analyzed for each sample. 5The cells were divided equally. The cells were collected by centrifugation at 300xg at 4°C for 5 minutes, and the washing solution was discarded. The cells were re-mixed in 100 μl of Fab or IgG diluted in 1% BSA / PBS and placed on ice for 30 minutes. Next, the cells were washed in 1 ml of 1% BSA / PBS and collected by centrifugation at 300xg at 4°C for 5 minutes. The washing solution was discarded, and 100 μl of secondary goat F(ab')2 anti-human κFITC (Southern Biotech cat#2062-02) or goat F(ab')2 anti-human IgG PE (Southern Biotech cat#2043-09) conjugate, diluted 1:200 in BSA / PBS, was added. The cells were placed on ice for 30 minutes. Finally, the cells were collected by centrifugation at 300xg at 4°C for 5 minutes and washed in 1 ml of BSA / PBS. The washing solution was removed, and the cells were re-mixed in 500 μl of 1% BSA / PBS. One drop of Sytox AADvanced ReadyFlow reagent (ThermoFisher cat#R37173) was added to each sample, and the samples were analyzed using an Attune NxT flow cytometer (ThermoFisher). As shown in Figure 14, all mutants bound to membrane-bound human TL1A.

[0181] Next, all Fab variants were characterized for their selectivity of human TL1A over other TNFSF members, TRAIL, LIGHT, and Fas. Briefly, ELISA plates were coated overnight at 4°C with 50 μl / well antigen at 1 μg / ml in PBS (Fas / TNFSF6, R&D Systems, cat. no. 126-FL / CF; TRAIL / TNFSF10, R&D Systems, cat. no. 375-TL / CF; LIGHT / TNFSF14, R&D Systems, cat. no. 664-LI / CF). The plates were washed three times with PBS-T and blocked with 100 μl of 1% BSA / PBS. The above block was discarded, and Fab variants or control antibodies (Fas / TNFSF6, R&D Systems, cat. no. AF126; TRAIL / TNFSF10, R&D Systems, cat. no. AF375; LIGHT / TNFSF14, R&D Systems, cat. no. AF664) were titrated in 50 μl of 1% BSA / PBS and incubated at 25°C for 1 hour. The plate was washed three times with PBS-T, and a secondary HRP-conjugated antibody (diluted 5,000-fold in 1% BSA / PBS) was added and incubated at 25°C for 1 hour. The plate was washed three times with PBST. As shown in Figures 15-20, none of the variants showed detectable binding to the relevant family members, indicating that selectivity for human TL1A versus other TNFSF family members was maintained while manipulating higher affinity using the human germline framework template.

[0182] Example 7: Characterization of selected humanized mutants expressed on various IgG constant regions. From Tables 19 and 20 above, the light chain variable regions and heavy chain variable regions of clones 14, 17L, 23, 34, 47, and 53 were cloned into the κ light chain constant region and the modified IgG1 or IgG2 heavy chain backbone, respectively. The modified IgG1 backbone and IgG2 were selected to reduce the potential effector function of the antibody. Transient expression and purification characteristics are shown in Table 23 below. In all of these mutants, modified IgG1 was better expressed than IgG2. Furthermore, the yields obtained were consistent with observations made regarding the effect of specific mutations on expression in bacteria (see Example 5). Specifically, the highest-expressing mutants 14, 34, and 47 all contained mutations in the heavy chain CDR2 V65G, V65T, or V65K, while the lowest-expressing mutants 17L, 23, and 53 did not contain mutations.

[0183] In general, binding of all mutants to human TL1A (modified IgG1 and IgG2) was preserved in both formats, as assessed by ELISA conjugation to antigen-coated plates (Figure 21) and ELISA capture of soluble biotinylated antigens (Figure 22). In addition, binding to membrane-bound human TL1A was preserved in all mutants, with the exception of mutant 53 when expressed as IgG2 (Figure 23). Finally, since none of the clones showed recognizable binding to the TNFSF family members Fas, TRAIL, or LIGHT, selectivity for human TL1A versus other TNFSF members was preserved (Figure 24).

[0184] [Table 23]

[0185] Example 8: Characterization of efficacy and species selectivity in whole blood assays The neutralizing activity and potency of the mutants described herein, expressed as IgG1 (modified) and IgG2, were tested in a human whole blood assay using healthy donors. 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 Jun 1; 178(11):7325-33, and measured IFN-γ production under conditions in which TL1A and its receptor DR3 are upregulated and activated. This assay produces soluble, membrane-bound TL1A. The results of this assay were 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.

[0186] In short, 96-well plates were coated with human gamma globulin in PBS overnight at 4°C, washed with PBS, and incubated with anti-human IgG (Fc fragment specific) at 25°C for at least 1 hour to generate immune complexes (ICs). Immediately before use, the plates were washed three times with PBS. Collected blood samples were treated with IL-12 and IL-18, the antibodies were titrated in the samples, and the samples were added to the plates and allowed to stand 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 then ~150 μl of diluted plasma was collected for IFN-γ measurement. PBS was added to facilitate the collection of cellular plasma from the plates due to the high proportion of blood (95%). All samples were diluted to ensure that the values ​​were within the linear range of the standard curve. All mutants, regardless of IgG format (modified IgG1 or IgG2), showed potent inhibition of IFN-γ production (Table 24). For comparison, typical ICs of mouse parental antibody 5C3D11 and humanized 12835 were observed. 50 The values ​​were 1.38±0.95 nM (donor n=16) and 9.28±10.71 nM (donor n=4), respectively.

[0187] [Table 24]

[0188] Next, to evaluate the cross-reactivity of optimized humanized mutants containing cynomolgus monkey TL1A, samples were evaluated using the same assay with blood obtained from cynomolgus monkeys. Instead of performing a complete titration, the assay was carried out similarly to the assay using human whole blood, except that the mutants were tested at a single concentration (10 nM). All mutants inhibited IFN-γ production, however, mutants 47 and 53 did not inhibit it to the same extent as mutants 14, 23, or 34, and mouse 5C3D11 (Figure 25). These data demonstrate that the optimized humanized mutants retained cross-reactivity to cynomolgus monkey TL1A.

[0189] The neutralization of the TL1A antibody was also formatted as an effector-less IgG1 molecule (indicated by SEQ ID NO: 542), expressed from CHO cells, purified, and tested for efficacy using whole blood from humans (Figure 26A-C) and cynomolgus monkeys (Figure 27A-C) as described above. The results are summarized in Table 25 below.

[0190] [Table 25]

[0191] These experiments demonstrated that all mutants were active and potent, with mutants 14 and 34 typically showing the greatest potency in human blood. All mutants became potent using cynomolgus monkey blood. Mutants 14, 23, and 34 showed similar potency to human and cynomolgus monkey TL1A, while mutant 53 showed approximately twice as potency as human TL1A.

[0192] Example 9: Competitive Assay A binding competition assay using surface plasmon resonance (SPR) was performed to evaluate whether the test anti-TL1A antibody bound to the same region on TL1A as any anti-TL1A antibody described herein.

[0193] Using a Biacore 2000 or 3000 instrument, the reference antibody was directly immobilized by amine coupling onto the carboxymethylated dextran sensor chip surface (CMS). To achieve a binding level of at least 100 response units (RUs) on the immobilized antibody, recombinant soluble human TL1A 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 RI / min for approximately 1 minute. Subsequently, the reference antibody was injected at 30 nM for 5 minutes to saturate all potential binding sites on the TL1A. This saturation was confirmed by repeated injections of the reference antibody. Next, the test antibody in PBS-NET or PBS-NET alone was injected at 30 nM for 5 minutes as a control. If the test antibody binds to TL1A saturated with the first antibody, this indicates that the test antibody binds to a non-competitive site on TL1A compared to the reference antibody. If the test antibody does not bind to saturated TL1A, this indicates that the two antibodies bind to the same region or compete for binding to TL1A. This strategy can be repeated by immobilizing the test antibody and injecting the reference antibody after the test antibody has bound to TL1A. Each cycle can be repeated. At the end of each cycle, the immobilized antibody surface is regenerated by a 30-second pulse of 3M MgCl2 or by two consecutive 15-second pulses of 0.1% TFA followed by PBS-NET. All injections are performed at 25°C with a collection rate of 10 Hz. All sensorgrams are double-referenced by using both a control surface and buffer injection.

[0194] Other binding competition assays using SPR are performed to evaluate whether the test anti-TL1A antibody binds to the same region on TL1A as the anti-TL1A antibody described herein. The reference antibody is immobilized on an SPR chip with amines bound at three or four different densities across the array. TL1A protein is injected in increasing concentration series to estimate kinetic parameters and appropriate concentrations for injection during the competition binning experiment. Once the optimal antigen concentration for the binning experiment is determined, regeneration conditions (typically short-duration, low-pH injections) are evaluated to establish optimal conditions for regeneration between cycles of the binning assay.

[0195] Binning is performed using a pre-mixed approach, in which TL1A is injected onto the array either alone at a moderate concentration or pre-conjugated with a test antibody at a saturated concentration (e.g., 30-50 μg / mL). The assay can be performed so that the test antibody is immobilized and the reference antibody is pre-conjugated with TL1A. Clones that bind to a specific region of the immobilized antibody result in an increased signal, while competitive clones decrease the antigen-binding signal. A competitive assay is performed so that all clones are tested as both ligand and analyte.

[0196] Example 10: Comparison of 5C3D11 binding to other anti-TL1A antibodies Two epitope binning tests were performed 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.

[0197] [Table 26]

[0198] To minimize avidity effects, a planar carboxymethyl dextran surface sensor tip was used in the first test (Xantec Prod. #SPMXCMDP), and an HC30M sensor tip was used in the second test (Xantec Prod. #SPMXHC30M). The running buffer for continuous flow microspotting was HBS-EP+ at a flow rate of 65 μl / min. The tips were activated for 7 minutes with 18 mM EDC and 4.5 nM sulfo-NHS in 100 mM MES, pH 5.5. The antibodies were then immobilized for 15 minutes on two replica prints. The antibodies were diluted to 10 μg / ml in 10 mM acetate (pH 4.5). The antibodies were titrated at three locations across the plate in a 3-fold serial dilution to establish concentration series with different spot densities. Each antibody was spotted eight times, i.e., twice for each of the four dilutions. This allowed for the preparation of a 10 × 8 array. The remaining active groups were neutralized with 1 M ethanolamine (pH 8.5) and quenched for 7 minutes. Epitope binning was performed using pre-mixed conditions because the antigen is a homotrimeric protein and the IgG is bivalent.

[0199] The first epitope binning test - TL1A was prepared at a final concentration of 50 nM (3.3 μg / ml) and mixed with 333 nM (50 μg / ml) analyte (solution-phase antibody) or electrophoresis buffer (control). In the IgG format sample, 50 μg / ml is 333 nM, and in the Fab format sample, 50 μg / ml is 1 μM. The mixed sample was injected onto the array for 5 minutes and regenerated for 30 seconds after each cycle using a 4:1 mixture of Pierce IgG elution buffer and 5 M NaCl (final concentration 1 M).

[0200] The second epitope binning test—TL1A—was prepared at a final concentration of 50 nM (3.3 μg / ml) and mixed with 1 μM (150 g / ml) IgG or 2 μM (200 μg / ml) Fab analyte (solution-phase antibody), or with electrophoresis buffer (control). The antibody sample was serially diluted 2-fold seven times (final 7.8 nM for IgG, final 15 nM for Fab). The mixed sample was injected onto the array for 5 minutes and regenerated for 30 seconds after each cycle using a 4:1 mixture of Pierce IgG elution buffer and 5 M NaCl (final concentration 1 M).

[0201] In epitope binning tests, a clear signal (sandwich) was observed using immobilized 5C3D11 and 12835, and all control antibodies were tested as analytes (Table 27, top two columns). These results indicate that 5C3D11 and 12835 bind to TL1A simultaneously with other antibodies and are therefore capable of recognizing distinct epitopes.

[0202] [Table 27]

[0203] Example 11: In vivo evaluation of anti-TL1A efficacy The efficacy of anti-TL1A antibodies was investigated in animal models of colitis. Anti-TL1A antibodies were used to treat acute colitis induced by rectal administration of di-nitrobenzenesulfonic acid or tri-nitrobenzenesulfonic acid (D / TNBS), or by administration of oxazolone and DSS in drinking water, or CD45RB. hiThe study was conducted in rodent models of chronic colitis induced by T cell transplantation. DNBS and oxazolone induce local ulceration and inflammation. DSS administration induces severe systemic inflammation of the gastrointestinal tract, characterized by erosive lesions and inflammatory infiltration. Symptoms in all these models typically include diarrhea, occult blood, weight loss, and occasionally rectal prolapse. In the prophylactic model, antibody treatment is initiated at the start of administration of the colitis-inducing compound. In the therapeutic model, antibody treatment is initiated a few days after induction begins. The effect of the treatment on weight, stool consistency, and occult blood, as well as microscopic effects on epithelial integrity and the degree of inflammatory infiltration, are determined. Daily clinical scoring is performed based on stool consistency and the presence of occult blood to obtain a Disease Activity Index (DAI) score.

[0204] Example 12: Phase 1 Clinical Trial A Phase 1 clinical trial will be conducted to evaluate the safety, tolerability, pharmacokinetics, and pharmacokinetics of the anti-TL1A antibody provided herein in subjects with Crohn's disease.

[0205] Single-dose escalation (SAD) group: Subjects in each group (subjects are grouped based on the presence or absence of risk variants) receive either a single dose of antibody or placebo. Exemplary doses are 1, 3, 10, 30, 100, 300, 600, and 800 mg of antibody, or between 5 and 30 milligrams per kilogram. Safety monitoring and PK evaluation are performed for a predetermined period. Based on the evaluation of PK data, and if the antibody is deemed well-tolerated, dose escalation is performed within the same group or in a further group of healthy subjects. Dose escalation is continued until the highest dose is reached, unless the predefined maximum exposure has not been reached or intolerances have not become apparent.

[0206] Multiple Dose Elevation (MAD) Group: Subjects in each group (subjects are grouped based on the presence or absence of risk variants) receive either multiple doses of antibody or placebo. Dosage levels and intervals are selected as those predicted to be safe based on SAD data. Dosage levels and frequencies are selected to achieve therapeutic drug levels in systemic circulation maintained at a steady state for several days, allowing appropriate safety parameters to be monitored. Samples are collected and analyzed to determine the PK profile.

[0207] Inclusion Criteria: Healthy subjects between 18 and 55 years of age with no potential for pregnancy. Health is defined as the absence of clinically relevant abnormalities identified by a detailed medical history, a thorough physical examination including blood pressure and pulse rate measurement, a 12-lead electrocardiogram, and clinical laboratory tests. Female subjects with no potential for childbirth must meet at least one of the following criteria: (1) have reached postmenopausal status as defined below: cessation of regular menstruation for at least 12 consecutive months without alternative pathological or physiological causes; and have serum follicle-stimulating hormone (FSH) levels within the laboratory's reference range for postmenopausal females; (2) have undergone a demonstrated hysterectomy and / or bilateral oophorectomy; or (3) have medically confirmed ovarian failure. All other female subjects (including females with tubal ligation, and females without a record of hysterectomy, bilateral oophorectomy, and / or ovarian failure) are considered to have potential for childbirth. Body Mass Index (BMI) between 17.5 and 30.5 kg / m2; and total weight > 50 kg (110 lbs). Proof of personally signed and dated informed consent document showing that the subject (or legal representative) was informed of all appropriate aspects of the study.

[0208] Two groups of healthy subjects were selected: one group of subjects with risk variants (whose presence is associated with increased susceptibility to Crohn's disease), and another group of subjects without risk variants.

[0209] Exclusion Criteria: Evidence or history of clinically significant hematological, renal, endocrine, pulmonary, gastrointestinal, cardiovascular, hepatic, psychiatric, neurological, or allergic disorders (including drug allergies, but excluding seasonal allergies that are untreated and asymptomatic at the time of administration). Subjects with a history of or current positive result on any of the following serological tests: 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 allergy or anaphylactic reaction to a therapeutic agent. Treatment with the investigational drug within 30 days (or whichever is longer as specified by site requirements), or treatment with a biologic with a 5 half-life or 180 days preceding the first dose of the investigational drug. Pregnant females; lactating females; and females capable of giving birth.

[0210] Primary endpoints: Incidence of adverse events (AEs) associated with dose-limiting or unacceptable treatments [timeframe: 12 weeks]. Incidence, severity, and causality of treatment-induced AEs (TEAEs), and withdrawals due to adverse events occurring during treatment [timeframe: 12 weeks]. Incidence and magnitude of abnormal laboratory findings [timeframe: 12 weeks]. Abnormal and clinically relevant changes in vital signs, blood pressure (BP), and electrocardiogram (ECG) parameters [timeframe: 12 weeks].

[0211] Secondary endpoint: Single dose escalation: Maximum plasma concentration (C) max [Time frame: 12 weeks]. Single dose escalation: Maximum plasma concentration (T max Time to reach [Time frame: 12 weeks]. Single dose escalation: Area under the plasma concentration-time profile from 0 hours to 14 days (AUC 14 days) [Time frame: 12 weeks]. Single dose escalation: Estimated time from 0 hours to infinity (AUC inf Area under the plasma concentration-time profile [time frame: 12 weeks]. Single dose escalation: 0 hours to final quantifiable time (AUC). last)Area under the plasma concentration-time profile [time frame: 12 weeks]. Single-dose escalation: Dose normalized maximum plasma concentration (C max [dn]) [time frame: 12 weeks]. Single-dose escalation: Estimated area under the plasma concentration-time profile from 0 hour to infinity (AUC inf [dn]) [time frame: 12 weeks]. Single-dose escalation: Dose normalized area under the plasma concentration-time profile from 0 hour to the last quantifiable time (AUC last [dn]) [time frame: 12 weeks]. Single-dose escalation: Plasma decay half-life (t1 / 2) [time frame: 12 weeks]. The plasma decay half-life is the time measured for the plasma concentration to decrease by half. Single-dose escalation: Mean residence time (MRT) [time frame: 12 weeks]. Single-dose escalation: Volume of distribution at steady state (V ss ) [time frame: 6 weeks]. The volume of distribution is defined as the theoretical volume in which the total amount of the drug needs to be uniformly distributed to produce the desired blood concentration. Volume of distribution at steady state (V ss ) is the apparent volume of distribution at steady state. Single-dose escalation: Total body clearance (CL) [time frame: 6]. CL is a quantitative measure of the rate at which the parent drug is removed from the body.

[0212] First dose of multiple-dose escalation: Maximum plasma concentration (C max ) [time frame: 12 weeks]. First dose of multiple-dose escalation: Time to reach the maximum plasma concentration (T max ) [time frame: 12 weeks]. First dose of multiple-dose escalation: Area under the plasma concentration-time profile from 0 hour to τ hours, τ = dosing interval of 2 weeks (AUC τ ) [time frame: 12 weeks]. First dose of multiple-dose escalation: Dose normalized maximum plasma concentration (C max [dn]) [time frame: 12 weeks]. First dose of multiple-dose escalation: Dose normalized area under the plasma concentration-time profile from 0 hour to τ hours, τ = dosing interval of 2 weeks (AUC τ[dn]) [Time frame: 12 weeks]. Plasma decay half-life (t1 / 2) [Time frame: 12 weeks]. Plasma decay half-life is the time measured for the plasma concentration to decrease by half. First dose in multiple dose escalation: Mean residence time (MRT) [Time frame: 12 weeks]. Apparent volume of distribution (Vz / F) [Time frame: 12 weeks]. Volume of distribution is defined as the theoretical volume in which the total amount of drug must be uniformly distributed to produce the desired plasma concentration of the drug. The apparent volume of distribution (Vz / F) after oral administration is affected by the rate of absorption. First dose in multiple dose escalation: Steady state (V ss Volume of distribution (V) [Time frame: 12 weeks]. Volume of distribution is defined as the theoretical volume in which the total amount of a drug must be uniformly distributed to produce the desired blood concentration of the drug. Normal volume of distribution (V) ss ) is the apparent volume of the steady-state distribution. First dose in a multi-dose escalation: Apparent oral clearance (CL / F) [Time frame: 12 weeks]. Drug clearance is a measure of the rate at which a drug is metabolized or removed by normal biological processes. Clearance obtained after oral administration (apparent oral clearance) is influenced by the proportion of the absorbed dose. Clearance is estimated from population pharmacokinetic (PK) modeling. Drug clearance is a quantitative measure of the rate at which the active pharmaceutical ingredient is removed from the blood. First dose in a multi-dose escalation: Systemic clearance (CL) [Time frame: 12 weeks]. CL is a quantitative measure of the rate at which the active pharmaceutical ingredient is removed from the body.

[0213] Multiple doses in dose escalation: Maximum plasma concentration (C) max [Time frame: 12 weeks]. Multiple dose escalation: Maximum plasma concentration (T max Time to reach [Time frame: 12 weeks]. Multiple doses in multiple dose escalation: Area under the plasma concentration-time profile from 0 hours to τ hours, τ = 2-week dosing interval (AUC). τ [Time frame: 12 weeks]. Multiple dose escalation: Dose-normalized maximum plasma concentration (C max [dn])[Time frame: 12 weeks]. Multiple dose escalation: Dose-normalized area under plasma concentration-time profile from 0 hours to τ hours, τ = 2-week dosing interval (AUC) τ[dn]) [Time frame: 12 weeks]. Multiple doses of escalating doses: Plasma elimination half-life (t1 / 2) [Time frame: 12 weeks]. The plasma elimination half-life is the time measured for the plasma concentration to decrease by half. Multiple doses of escalating doses: Apparent volume of distribution (Vz / F) [Time frame: 12 weeks]. The volume of distribution is defined as the theoretical volume in which the total amount of drug needs to be uniformly distributed to produce the desired plasma concentration of the drug. The apparent volume of distribution (Vz / F) after oral administration is affected by the absorbed fraction. Multiple doses of escalating doses: Volume of distribution at steady state (V ss ) [Time frame: 12 weeks]. The volume of distribution is defined as the theoretical volume in which the total amount of drug needs to be uniformly distributed to produce the desired blood concentration of the drug. The steady-state volume of distribution (V ss ) is the apparent volume of distribution at steady state.

[0214] Multiple doses of escalating doses: Apparent oral clearance (CL / F) [Time frame: 12 weeks]. The clearance of a drug is a measure of the rate at which the drug is metabolized or removed by normal biological processes. The clearance obtained after oral administration (apparent oral clearance) is affected by the fraction of the absorbed dose. Clearance was estimated from population pharmacokinetic (PK) modeling. Drug clearance is a quantitative measure of the rate at which the parent drug is removed from the blood. Multiple doses of escalating doses: Total body clearance (CL) [Time frame: 12 weeks]. CL is a quantitative measure of the rate at which the parent drug is removed from the body. Multiple doses of escalating doses: Minimum plasma trough concentration (C min ) [Time frame: 12 weeks]. Multiple doses of escalating doses: Mean concentration at steady state (Cav) [Time frame: 12 weeks]. Multiple doses of escalating doses: Accumulation ratio (Rac) [Time frame: 12 weeks]. Multiple doses of escalating doses: Peak-to-trough fluctuation (PTF) [Time frame: 12 weeks]. Further parameter for multiple doses of escalating doses: Estimation of bioavailability (F) for subcutaneous administration with the corresponding intravenous dose [Time frame: 12 weeks]. Immunogenicity for both single-dose escalation and multiple-dose escalation: Development of anti-drug antibodies (ADA) [Time frame: 12 weeks].

[0215] Example 13: Phase 1b Clinical Trial A 1b open-label clinical trial will be conducted to evaluate the efficacy of the anti-TL1A antibody provided herein in patients with risk variants associated with Crohn's disease.

[0216] Group: Ten patients who tested positive for risk variants (whose presence is associated with increased susceptibility to Crohn's disease) received antibodies. Five to ten patients who tested negative for risk variants also received antibodies. Patients were monitored in real time. Central-ready procedures were used for endoscopy and biopsy, and readers were blinded to the procedure and endpoint times.

[0217] Inclusion criteria: Two groups of subjects will be selected: one group of subjects with risk variants (whose presence is associated with increased susceptibility to Crohn's disease), and one group of subjects without risk variants.

[0218] The primary endpoints reported were the simplified endoscopic score for Crohn's disease (SESCD), the Crohn's disease activity index (CDAI), and patient-reported outcomes (PROs). A Phase 2a clinical trial will be conducted if the risk variant-positive group shows a 50% reduction from baseline.

[0219] Inclusion Criteria: PRO Inclusion Criteria: Abdominal pain score of 2 or higher and / or bowel movement frequency score of 4 or higher. Primary outcome is a pain core of 0 or 1 and a bowel movement frequency score of 3 or lower, without deterioration from baseline. Endoscopic Inclusion Criteria: If the colon is involved, SESCD ileum should only be entered with scores of 4 and 6. Primary endoscopic outcome is a δ of 40-50% of the mean SESCD.

[0220] Example 14: Phase 2a Clinical Trial A Phase 2a clinical trial will be conducted to evaluate the efficacy of the anti-TL1A antibody provided herein in subjects with Crohn's disease.

[0221] Groups: Each group will consist of 40 patients (antibody and placebo groups) treated with either antibody or placebo for 12 weeks. 20 patients in each group will be treated with the highest dose, and an interim analysis will be conducted after examining a 40-50% δ (50% reduction from baseline in SESCD, CDAI, and PRO) between the placebo and treated groups in the primary endpoint.

[0222] The primary endpoints reported were the simplified endoscopic score for Crohn's disease (SESCD), the Crohn's disease activity index (CDAI), and patient-reported outcomes (PROs).

[0223] Inclusion Criteria: PRO Inclusion Criteria: Abdominal pain score of 2 or higher and / or bowel movement frequency score of 4 or higher. The primary endpoint is a pain core of 0 or 1 and a bowel movement frequency score of 3 or lower, without deterioration from baseline. Endoscopic Inclusion Criteria: If the colon is involved, only SESCD ileum is included with scores of 4 and 6. The primary endoscopic endpoint is δ of 40-50% of the mean SESCD.

[0224] Various embodiments are described in the detailed description above. While these descriptions directly illustrate the embodiments described above, it will be understood that those skilled in the art will be able to conceive of modifications and / or variations to the specific embodiments shown and described herein. Such modifications or variations within the scope of this description are also intended to be included therein. Unless specifically noted, the words and phrases in this specification and in the claims are intended by the inventors to have meanings that are common and familiar to those skilled in the applicable art.

[0225] The foregoing description of the various embodiments known to the applicant at the time of filing of this application is presented for purposes of illustration and description. This description is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed, and many modifications and variations are possible in light of the above teachings. The described embodiments are provided to explain the principles and practical applications and to enable one of ordinary skill in the art to utilize the embodiments with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the disclosure not be limited to the particular forms disclosed.

[0226] Although specific embodiments have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made without departing from the disclosure and its broader aspects, and therefore, the appended claims are to encompass within their scope all such changes and modifications as fall within the true spirit and scope of this disclosure. It is generally understood by those skilled in the art that the terms used herein are generally intended as "open" terms (e.g., the term "including" is to be interpreted as "including but not limited to," the term "having" is to be interpreted as "having at least," and the term "includes" is to be interpreted as "includes but not limited to").

[0227]

Table 28-1

[0228]

Table 28-2

[0229]

Table 28-3

[0230]

Table 28-4

[0231]

Table 28-5

[0232] SEQ ID NO: 490 (L8; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK SEQ ID NO: 491 (L8; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDVWGQGTTVTVSS SEQ ID NO: 492 (Clone 34; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK SEQ ID NO: 493 (Clone 34; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHIKYDPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDWWGQGTTVTVSS SEQ ID NO: 494 (Clone 2; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK SEQ ID NO: 495 (Clone 2; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHIKYSPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDWWGQGTTVTVSS SEQ ID NO: 496 (Clone 52; VL) EIVLTQSPGTLSLSPGERATLSCGASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK SEQ ID NO: 497 (Clone 52; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHIKYSPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDWWGQGTTVTVSS SEQ ID NO: 498 (Clone 46; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK SEQ ID NO: 499 (Clone 46; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHVKYSPKFQVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDWWGQGTTVTVSS SEQ ID NO: 500 (Clone 47; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK SEQ ID NO: 501 (Clone 47; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHVKYDPKFQTRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDWWGQGTTVTVSS SEQ ID NO: 502 (Clone 14; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK SEQ ID NO: 503 (Clone 14; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHiKYDPKFQkRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS SEQ ID NO: 504 (Clone 16L; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK SEQ ID NO: 505 (Clone 16L; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHvKiDPKFQVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS SEQ ID NO: 506 (Clone 17L; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK SEQ ID NO: 507 (Clone 17L; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQVRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS SEQ ID NO: 508 (Clone 17L-1; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK SEQ ID NO: 509 (Clone 17L-1; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQRRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS SEQ ID NO: 510 (Clone 23; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK SEQ ID NO: 511 (Clone 23; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDKWGQGTTVTVSS SEQ ID NO: 512 (Clone A1; VL) EIVLTQSPGTLSLSPGERATLSCGASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK SEQ ID NO: 513 (Clone A1; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDKWGQGTTVTVSS SEQ ID NO: 514 (Clone 53; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK SEQ ID NO: 515 (Clone 53; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHLKYDPKFQERVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDKWGQGTTVTVSS SEQ ID NO: 516 (Clone E1; VL) EIVLTQSPGTLSLSPGERATLSCGASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK SEQ ID NO: 517 (Clone E1; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIEPASGHLKYDPKFQERVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGGLPDKWGQGTTVTVSS SEQ ID NO: 518 (Clone 3-17L VA; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK SEQ ID NO: 519 (Clone 3-17L VA; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS SEQ ID NO: 520 (Clone 3-17L; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWQGNPRTFGGGTKLEIK SEQ ID NO: 521 (Clone 3-17L; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHLKYDPKFQGRVTITRDTSASTVYMELSSLRSEDTAVYYCARSGGLPDMWGQGTTVTVSS SEQ ID NO: 522 (Clone L8mod; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK SEQ ID NO: 523 (Clone L8mod; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDVWGQGTTVTVSS SEQ ID NO: 524 (Clone XV; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK SEQ ID NO: 525 (Clone XV; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQVRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS SEQ ID NO: 526 (Clone X; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK SEQ ID NO: 527 (Clone X; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS SEQ ID NO: 528 (Clone H3-1; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK SEQ ID NO: 529 (Clone H3-1; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDLWGQGTTVTVSS SEQ ID NO: 530 (Clone XL3-6; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCSQWSGNPRTFGGGTKLEIK SEQ ID NO: 531 (Clone XL3-6; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS SEQ ID NO: 532 (Clone XL3-10; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRSFGGGTKLEIK SEQ ID NO: 533 (Clone XL3-10; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS SEQ ID NO: 534 (Clone XL3-15; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSRNPRTFGGGTKLEIK SEQ ID NO: 535 (Clone XL3-15; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS SEQ ID NO: 536 (Clone L3-13; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWKGNPRTFGGGTKLEIK SEQ ID NO: 537 (Clone L3-13; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHTKYDPKFQGRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS SEQ ID NO: 538 (Clone H2-2; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK SEQ ID NO: 539 (Clone H2-2; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHSKYDPKFQVRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS SEQ ID NO: 540 (Clone H2-5; VL) EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRLLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSGNPRTFGGGTKLEIK SEQ ID NO: 541 (Clone H2-5; VH) QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVRQAPGQGLEWMGRIDPASGHYKYDPKFQVRATITTDTSASTAYLQLSSLRSEDTAVYYCARSGGLPDFWGQGTTVTVSS SEQ ID NO: 542 modified G1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 543 G2 constant domain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 544 κ constant domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 545 (L8 HFR1) QVQLVQSGAEVKKPGASVKVSCKAS SEQ ID NO: 546 (L8 HFR2) WVRQAPGQGLEWMG SEQ ID NO: 547 (L8 HFR3) RVTMTRDTSTSTVYMELSSLRSEDTAVYYC SEQ ID NO: 548 (L8 HFR4) WGQGTTVTVSS SEQ ID NO: 549 (L8 LFR1) EIVLTQSPGTLSLSPGERATLSC SEQ ID NO: 550 (L8 LFR2) WYQQKPGQAPRLLIY SEQ ID NO: 551 (L8 LFR3) GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC SEQ ID NO: 552 (L8 LFR4) FGGGTKLEIK SEQ ID NO: 586 (VH FR3) RATITTDTSASTAYLQLSSLRSEDTAVYYC SEQ ID NO: 587 (VH FR3) RVTITRDTSASTVYMELSSLRSEDTAVYYC SEQ ID NO: 588 (VH FR3) RVTITRDTSASTAYMELSSLRSEDTAVYYC

Claims

1. A method for preparing a treatment agent for inflammatory bowel disease (IBD), Crohn's disease, or colitis, the method comprising the step of incubating cells containing nucleic acids encoding an antibody or antigen-binding fragment that specifically binds to tumor necrosis factor ligand 1A (TL1A) in the culture medium under conditions sufficient to secrete the antibody or antigen-binding fragment into the culture medium, The antibody or antigen-binding fragment that specifically binds to TL1A is HCDR1 containing the amino acid sequence described by SEQ ID NO: 553, HCDR2 containing an amino acid sequence described by either SEQ ID NO: 554-564 or 574-577, HCDR3 containing an amino acid sequence described by either SEQ ID NO: 565-568 or 578-581, A heavy chain variable region including, LCDR1 containing the amino acid sequence described by either SEQ ID NO: 569 or 570, LCDR2 containing the amino acid sequence described by SEQ ID NO: 488, and LCDR3 containing an amino acid sequence described by one of the following SEQ ID NOs: 571-573 or 582-585, A light chain variable region including, Methods that include...

2. The method according to claim 1, wherein the cell is a eukaryotic cell.

3. The method according to claim 1, wherein the cells are Chinese hamster ovary (CHO) cells.

4. The method according to claim 1, wherein the cells are COS cells, HeLa cells, L cells, or multiple myeloma cells.

5. The method according to claim 1, wherein the cells are C127 cells, 3T3 cells, or BHK cells.

6. The antibody or antigen-binding fragment that specifically binds to TL1A is A heavy chain variable region containing an amino acid sequence that is at least 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, A light chain variable region containing an amino acid sequence that is at least 90% identical to any one of the following SEQ ID NOs: 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, including, The method according to claim 1.

7. An antibody or antigen-binding fragment that specifically binds to the aforementioned TL1A, A heavy-chain variable region including HCDR1, HCDR2, and HCDR3, selected from any one of the following 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, A light chain variable region including LCDR1, LCDR2, and LCDR3, selected from any one of the following 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, Here, CDR is defined by the Kabat method, Chothia method, or IMGT method. The method according to claim 1.

8. The method according to claim 1, further comprising the step of subjecting the culture medium to at least one purification step.

9. The method according to claim 8, wherein the purification step includes high-performance liquid chromatography (HPLC), column chromatography, gel electrophoresis, or any combination thereof.

10. The method according to claim 8, wherein the purification step comprises immunoadsorption, immunoaffinity chromatography, ammonium sulfate precipitation, or gel electrophoresis, or any combination thereof.

11. A method for preparing a treatment agent for inflammatory bowel disease (IBD), Crohn's disease, or colitis, wherein the method is: The process includes mixing an antibody or antigen-binding fragment that specifically binds to tumor necrosis factor ligand 1A (TL1A) with a pharmaceutically acceptable excipient, carrier, or diluent. An antibody or antigen-binding fragment that specifically binds to the aforementioned TL1A, HCDR1 containing the amino acid sequence described by SEQ ID NO: 553, HCDR2 containing an amino acid sequence described by either SEQ ID NO: 554-564 or 574-577, HCDR3 containing an amino acid sequence described by either SEQ ID NO: 565-568 or 578-581, A heavy chain variable region including, LCDR1 containing the amino acid sequence described by either SEQ ID NO: 569 or 570, LCDR2 containing the amino acid sequence described by SEQ ID NO: 488, and LCDR3 containing an amino acid sequence described by one of the following SEQ ID NOs: 571-573 or 582-585, A light chain variable region including, Methods that include...

12. The antibody or antigen-binding fragment that specifically binds to TL1A is A heavy chain variable region containing an amino acid sequence that is at least 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, A light chain variable region containing an amino acid sequence that is at least 90% identical to any one of the following SEQ ID NOs: 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, including, The method according to claim 11.

13. The antibody or antigen-binding fragment that specifically binds to TL1A is A heavy-chain variable region including HCDR1, HCDR2, and HCDR3, selected from any one of the following 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, A light chain variable region including LCDR1, LCDR2, and LCDR3, selected from any one of the following 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, Here, CDR is defined by the Kabat method, Chothia method, or IMGT method. The method according to claim 11.

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