EA042971B1 - ANTIBODIES THAT SPECIFICLY BIND TO TL1A - Google Patents

Description

Cross-reference to related applications

This application claims priority over U.S. Provisional Application No. 62/220442, filed September 2015, the contents of which are hereby incorporated by reference in their entirety and for all purposes.

Sequence listing

This application includes a sequence listing submitted electronically as a text file named TL1A_ST25 created on September 18, 2015 and 89,000 bytes in size. The sequence listing is incorporated herein by reference.

Technical field

This disclosure relates generally to the field of antibody engineering. More specifically, this disclosure relates to antibody variants that specifically bind to TL1A and that inhibit the interaction between TL1A and death receptor 3 (DR3). In some aspects, the antibodies also inhibit the interaction between TL1A and decoy receptor 3 (DcR3). The antibodies have improved activity compared to the parent antibody from which the variants were derived.

State of the art

Various publications, including patents, published applications, access numbers, technical articles and scientific articles are cited in the description. Each of these cited publications is incorporated by reference in its entirety and for all purposes in this document.

TNF-like ligand 1A (TL1A, syn. 15 member of the TNF superfamily (TNFSF15); TL1 and VEGI) is a member of the tumor necrosis factor superfamily that is expressed by antigen presenting cells (including dendritic cells, B cells, and macrophages), CD4+ and CD8+ T- cells and endothelial cells. TL1A can be expressed on the cell surface or secreted as a soluble cytokine. The receptor for TL1A, death receptor 3 (DR3), is expressed by a variety of cells, including CD4+ and CD8+ T cells, NK cells, NKT cells, and FOXP3+ regulatory T cells (Treg) and type 2 and type 3 innate lymphoid cells (ILC2 and ILC3).

TL1A can also bind a decoy receptor (DcR3), which is a competitive inhibitor of DR3. DcR3 also acts as a decoy receptor for Fas ligand (Fas-L) and a lymphotoxin-like inducible protein that competes with glycoprotein D for binding of the herpes virus entry mediator on T cells (LIGHT - lymphotoxin-like inducible protein). Accordingly, DcR3 is an important regulator of several signaling pathways.

The TL1A/DR3 signaling pathway has been implicated in several biological systems that are associated with human disease. For example, TL1A has been shown to play a role in immunity, angiogenesis, and barrier tissue homeostasis. Inhibition of the interaction of TL1A with DR3 has been shown to contribute to a therapeutic effect in several immune-mediated pathological conditions, such as experimental autoimmune encephalomyelitis (EAE, a multiple sclerosis model), colitis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, skin disease, asthma, and arthritis.

Accordingly, compounds which inhibit TL1A activity are suitable, for example, for their therapeutic, prophylactic, diagnostic and prognostic uses.

Brief description of the essence of the invention

Provided herein is a recombinant antibody comprising a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 28, a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: : 17, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 29, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 30, with provided that if the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 1, the light chain variable region does not contain the amino acid sequence of SEQ ID NO: 2.

In some aspects, the antibody comprises a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody comprises a heavy chain variable region CDR2 comprising the amino acid sequence SEQ ID NO: 21, and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 20. In some aspects, the antibody comprises a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 23, and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody comprises a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a light chain variable region CDR1 comprising amino acid sequence of SEQ ID NO: 24, and a light chain variable region CDR3 containing

- 1 042971 amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody comprises a heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 16, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 25, and a variable region CDR3 light chain comprising the amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody comprises a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 26, and a variable region CDR3 light chain region comprising the amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody comprises a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 22. B In some aspects, the antibody comprises a heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 21, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 24, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody comprises a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 21, a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 25, and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 22 In some aspects, the antibody comprises a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 21, a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 26, and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 22. In some aspects, the antibody comprises a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:27.

Also provided herein is a recombinant antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14, provided that if the variable the heavy chain region contains the amino acid sequence of SEQ ID NO: 1, the light chain variable region does not contain the amino acid sequence of SEQ ID NO: 2. In some aspects, the antibody contains a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1, and a light chain variable region , containing the amino acid sequence of SEQ ID NO: 4. In some aspects, the antibody contains a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. In some aspects, the antibody contains a variable region a heavy chain region comprising the amino acid sequence of SEQ ID NO: 1; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. In some aspects, an antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and the variable region light chain comprising the amino acid sequence of SEQ ID NO: 6. In some aspects, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7. In some aspects, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some aspects, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. In some aspects, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6. In some aspects, aspects, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7. In some aspects, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 , and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some aspects, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.

In some aspects, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 60. In some aspects, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 61. In some aspects, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 60 , and a light chain containing amino

- 2 042971 acid sequence SEQ ID NO: 61.

Such recombinant antibodies are preferably full length and are preferably monoclonal. Such recombinant antibodies bind to TL1A with increased affinity for an antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. Such recombinant antibodies have increased activity compared to with an antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. The increased activity can be at least about 10-fold higher than that of an antibody having a variable a heavy chain region containing the amino acid sequence of SEQ ID NO: 1; and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. The increased activity can be at least about 12-fold higher than that of an antibody having a heavy chain variable region containing amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. Increased activity can be at least about 13-fold higher than that of an antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: : 1, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. Increased activity can be at least about 15-fold higher than that of an antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1 and variable light chain region containing the amino acid sequence of SEQ ID NO: 2. The increased activity can be at least about 20-fold higher than that of an antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1 and a light chain variable region containing amino acid sequence of SEQ ID NO: 2. The increased activity can be at least about 25-fold higher than that of an antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence of SEQ ID NO: : 2. Increased activity can be at least about 27-fold higher than that of an antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. Increased activity may be at least about 40-fold higher than the activity of an antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. The fold increase in activity can be determined according to with analysis of TL1A-induced caspase activity in TF-1 cells.

Such recombinant antibodies may comprise a human IgG1 heavy chain constant region, a human IgG2 heavy chain constant region, or a human IgG4 heavy chain constant region, or any allotypes thereof. The human IgG1 heavy chain constant region may comprise SEQ ID NO: 42, or SEQ ID NO: 43 (human AK IgG1), or SEQ ID NO: 44 (human IgG1 with YTE), or SEQ ID NO: 64 (human IgG1 with YTE and AK), or SEQ ID NO: 63 (human IgG1 with L234A, L235A, G237A), or SEQ ID NO: 62 (human IgG1 with L234A, L235A, G237A and AK), or SEQ ID NO: 65 (human IgG1 with L235A and G237A), or SEQ ID NO: 66 (human IgG1 with L235A, G237A and AK). The human IgG2 heavy chain constant region may comprise SEQ ID NO: 67, or SEQ ID NO: 70 (human IgG2 AK), or SEQ ID NO: 71 (human IgG2 with A330S, P331S), or SEQ ID NO: 68 (human IgG2 with A330S, P331S and ΔΚ). The heavy chain IgG4 constant region of human IgG4 may comprise SEQ ID NO: 45 or SEQ ID NO: 46 (human IgG4 with S228P and ΔΚ) or SEQ ID NO: 47 (human IgG4 with S228P and YTE) or SEQ ID NO: 69 (Human IgG4 with S228P, YTE and ΔΚ). It is understood that an IgG4 heavy chain can be used without a stabilizing S228P substitution (eg, IgG4 with YTE only, or IgG4 with YTE and ΔΚ, or IgG4 with ΔΚ only).

Recombinant antibodies may contain a human lambda light chain constant region or an allotype thereof. The human lambda light chain constant region may comprise SEQ ID NO: 48.

Such recombinant antibodies bind to human TL1A and can bind to TL1A of a non-human primate or TL1A of a non-human mammal such as a mouse, rat, guinea pig, cat, dog, rabbit or pig.

Such recombinant antibodies can be used in a method of treating a respiratory disease, a method of treating a gastrointestinal disease, a method of treating a skin disease, or a method of treating arthritis, or can be used to treat a respiratory disease, a gastrointestinal disease, a skin disease, or arthritis. , or may be used to make a medicament for the treatment of a respiratory disease, a gastrointestinal disease, a skin disease, or arthritis. Respiratory disease may include one or more of asthma, chronic obstructive pulmonary disease (COPD),

- 3 042971 pulmonary fibrosis, pulmonary sarcoidosis, allergic rhinitis or cystic fibrosis. The gastrointestinal disease may include one or more of inflammatory bowel disease, Crohn's disease, colitis, ulcerative colitis, eosinophilic esophagitis, or irritable bowel syndrome, or a disease or condition of the gastrointestinal tract associated with cystic fibrosis. Arthritis may include rheumatoid arthritis. The skin disease may include one or more of atopic dermatitis, eczema, and scleroderma.

Humans, non-human primates, or non-human mammals in need of such therapies can be treated with antibodies or compositions containing antibodies, for example, by administering the antibodies or composition to a subject. Administration may be parenteral, eg subcutaneous and/or intravenous.

Such recombinant antibodies can be used in a method for detecting TL1A on the surface of peripheral blood mononuclear cells (PBMCs). The methods include contacting an antibody that binds to TL1A, as described or exemplified herein, with PBMCs obtained from a subject and detecting the TL1A-bound antibody on the surface of the PBMC. The methods may further include quantifying the level of TL1A in PBMCs. The methods may further include obtaining a PBMC from a subject.

Such recombinant antibodies can be used in a method for detecting TL1A in serum. The methods include contacting an antibody that binds to TL1A, as described or exemplified herein, with sera obtained from a subject and detecting an antibody bound to TL1A in the serum. The methods may further include quantifying the level of TL1A in serum. The methods may further include obtaining serum from blood obtained from the subject. The methods may further include obtaining blood from a subject.

Provided are polynucleotides encoding one or more heavy chain variable regions and light chain variable regions of such antibodies. The polynucleotides may further encode a heavy chain constant region and/or a light chain constant region.

In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3, such as a nucleic acid sequence comprising SEQ ID NO: 51 or SEQ ID NO: 58. In some aspects, the polynucleotide comprises a nucleic acid sequence an acid encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 2, for example, a nucleic acid sequence comprising SEQ ID NO: 50. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, such as a nucleic acid sequence comprising SEQ ID NO: 52 or SEQ ID NO: 59. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, such as the sequence a nucleic acid sequence comprising SEQ ID NO: 54. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 7, such as a nucleic acid sequence comprising SEQ ID NO: 55. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, e.g., a nucleic acid sequence comprising SEQ ID NO: 56. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an antibody light chain variable region, containing the amino acid sequence of SEQ ID NO: 10, for example, a nucleic acid sequence containing SEQ ID NO: 57.

In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, such as a nucleic acid sequence comprising SEQ ID NO: 49. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding a light variable region. an antibody chain comprising the amino acid sequence of SEQ ID NO: 4, e.g., a nucleic acid sequence comprising SEQ ID NO: 52. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, e.g. a nucleic acid sequence comprising SEQ ID NO: 53. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, such as a nucleic acid sequence comprising SEQ ID NO: 54. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an antibody light chain variable region comprising the amino acid sequence

- 4 042971

SEQ ID NO: 7, e.g., a nucleic acid sequence comprising SEQ ID NO: 55. In some aspects, the polynucleotide comprises a nucleic acid sequence encoding an antibody light chain variable region comprising the amino acid sequence

SEQ ID NO: 8, e.g. a nucleic acid sequence containing SEQ ID NO: 56.

Vectors containing one or more of these polynucleotides are provided. Cells transformed with one or more such polynucleotides or such vectors are provided. The transformed cells may be mammalian and are preferably mammalian expression host cells such as CHO cells, NSO cells, or HEK293 cells.

Brief description of the drawings

In FIG. 1 shows the positions and identities of each of the single amino acid substitutions made in the CDR1 or CDR2 regions, or to selected amino acid residues adjacent to the parent antibody heavy chain variable region CDR2 (320-179). The boxed regions are CDRs according to the AbM numbering system.

In FIG. 2 shows the positions and identities of each of the individual amino acid substitutions made in the CDR1 or CDR3 regions of the light chain variable region of the parent antibody (320-179). The boxed regions are CDRs according to the AbM numbering system.

In FIG. 3 shows the heavy chain alignment of anti-TL1A antibody variants.

In FIG. 4 shows the light chain alignment of anti-TL1A binding antibody variants.

In FIG. 5 shows a comparison of the dissociation phase of TL1A anti-TL1A antibody variants measured by SPR (surface plasmon resonance).

In FIG. 6 shows the results of the analysis of caspase activity of TF-1 cells with anti-TL1A antibody variants.

In FIG. 7 shows the results of assays of caspase activity of TF-1 cells using various anti-TL1A antibodies compared to the parent antibody 320-179.

In FIG. 8 shows that antibody 320-587 has superior TL1A activity in the TF-1 caspase activity assay compared to other known anti-TL1A antibodies in several different experiments.

In FIG. 9 shows various anti-TL1A antibodies that inhibit TL1A binding to DR3 compared to an isotype control as measured by ELISA format.

In FIG. 10 shows that the parent antibody 320-179 does not inhibit TL1A binding to DcR3, while anti-TL1A antibody variants inhibit TL1A-DcR3 interaction.

In FIG. 11 shows that antibody 320-587 cross-reacts and binds to different species of TL1A; said antibody binds to TL1A of all species tested.

In FIG. 12A-12D show the results of administration of antibody 320-587 in a TNBS-induced colitis (TNBS-trinitrobenzenesulfonic acid) rat model, demonstrating that the antibody significantly alleviates the symptoms of colitis.

In FIG. 13 shows that antibody 320-587 detects human TL1A isolated from immune complex stimulated human PBMCs in ELISA.

In FIG. 14 shows that antibody 320-587 detects a population of human PBMCs that express membrane TL1A on their surface in flow cytometric analysis.

In FIG. 15 shows that in rats with acute OVA-induced asthma (OVA-ovalbumin) treated with antibody 320-587, the level of eosinophils in bronchoalveolar lavage fluid (BALF) was significantly reduced.

In FIG. 16A-16D show that guinea pigs with acute OVA-induced asthma treated with the 320-587 antibody had improvements in eosinophils in the BAL (Fig. 16A), macrophages in the BAL (Fig. 16B), and upper airway hyperresponsiveness. after the early asthmatic reaction (FIG. 16C), and the severity of the early asthmatic response (FIG. 16D).

In FIG. 17A-17E show that rats with chronic OVA-induced asthma treated with antibody 320-587 had improvements in eosinophils in BAL (Fig. 17A), macrophages in BAL (Fig. 17B), IL-13 in BAL (FIG. 17C), goblet cell hyperplasia as assessed by PAS (FIG. 17D), and mucosal thickening as assessed on sections stained with H&E (hematoxylin and eosin) (FIG. 17E).

In FIG. 18 shows the dose of ovalbumin required to double the airway obstruction.

In FIG. 19 shows that rats with TNBS-induced colitis treated with antibody 320-587 had a positive effect on fibrosis in the area of ulceration.

In FIG. 20A-20E show a comparison of TNBS-induced colitis rats with DNBS-induced colitis (DNBS - dinitrobenzene sulfonic acid) treated with antibody 320-587 and the effect observed at days 7 and 14 on the colonic mass/length ratio (Fig. 20A), colonic fibrosis (FIG. 20B), colonic infiltrate (FIG. 20C) and colonic injury (FIG. 20D). In FIG. 20E shows representative sections of the area of ulceration on days 7 and 14.

In FIG. 21A-21C show that rats with DSS-induced colitis treated with antibody 320-587 had improvements in weight change during DSS administration (FIG. 21A),

- 5 042971 clinical indicators during the introduction of DSS (Fig. 21B) and the mass/length ratio of the colon (Fig. 21C).

In FIG. 22 shows changes in TL1A-induced intraperitoneal cytokines in response to treatment 320-587.

Detailed Description of the Invention

Various terms relating to aspects of the disclosure are used throughout the specification and claims. Such terms shall have their ordinary meaning in the art, unless otherwise indicated. Other specifically defined terms should be interpreted in accordance with the definition provided in this document.

In some instances, the terms subject and patient are used interchangeably herein and include any animal. Preferred are mammals, including companion animals (eg, cat, dog) and agricultural mammals (eg, pigs, horses, cows), as well as rodents, including mice, rabbits and rats, guinea pigs, and other rodents. Non-human primates such as cynomolgus macaques are preferred and humans are most preferred.

A molecule, such as an antibody, has been isolated if it has been altered and/or removed from its natural environment by human hand.

As used herein, singular forms include plural forms unless expressly stated otherwise.

Specificity in the context of antibody-antigen interactions is not necessarily an absolute definition, but may be a relative term indicating the degree of selectivity of an antibody for an antigen-positive cell versus an antigen-negative cell. The specificity of an antibody for an antigen positive cell is mediated by the variable regions of the antibody and usually by the complementarity determining regions (CDRs) of the antibody. The construct may have 100 to 1000 fold specificity for antigen positive cells compared to antigen negative cells.

As used herein, the term recombinant includes the expression of genes obtained through genetic engineering or otherwise through laboratory manipulation.

The disclosure provides anti-TL1A antibody variants comprising a recombinantly altered heavy and/or light chain variable region of antibody 320-179 such that the antibody variants specifically bind to TL1A. These 320-179 antibody variants inhibit the ability of TL1A to interact with DR3 and in some aspects also with DcR3 and furthermore inhibit signaling induced by TL1A interaction with DR3. These antibodies are more effective than antibody 320-179. These antibodies have increased affinity for TL1A compared to antibody 320-179.

The increased activity may be at least about 10-fold higher than that of an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. The increased activity may be at least about 12 times the activity of an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. The increased activity may be at least about 13 times higher than the activity of an antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. The increased activity can be at least about 15 times higher than the activity of an antibody, having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. Increased activity can be at least about 20-fold higher than that of an antibody having a heavy chain variable region , containing the amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. Increased activity can be at least about 25 times higher than that of an antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. The increased activity can be at least about 27-fold higher than that of an antibody having a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. The increased activity can be at least about 40-fold higher than that of an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 2. The fold increase in activity can be determined, for example, by measuring the release of caspase in TL1A-induced apoptosis in the analysis of TF-1 cells.

- 6 042971

Antibody variants 320-179 are expressed recombinantly and specifically bind to TL1A. The parent antibody 320-179 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In some aspects, the 320-179 antibody variant comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 3, and the light chain variable region containing the amino acid sequence of SEQ ID NO: 14, provided that if the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 1, the variable region the light chain does not contain the amino acid sequence of SEQ ID NO: 2. Antibody variant 320-179 is able to inhibit the interaction of TL1A with DR3. The 320-179 antibody variant has increased activity compared to the 320-179 antibody and/or has an increased affinity for TL1A relative to the 320-179 antibody.

In some aspects, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 4. In some aspects, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 2. In some aspects, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 5. In some aspects, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: : 1 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 6. In some aspects, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 1 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 7. In some aspects, the variable region the heavy chain contains the amino acid sequence of SEQ ID NO: 1 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 8. In some most preferred aspects, the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 3 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 4. In some aspects, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 6. In some aspects, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 7. In some aspects, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. In some aspects, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 10.

In most preferred aspects, the 320-179 antibody variant comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and specifically binds to TL1A. In some aspects, the heavy chain variable region of SEQ ID NO: 3 is fused to a human IgG1 (AK) heavy chain constant region (e.g., SEQ ID NO: 43) such that the heavy chain comprises SEQ ID NO: 60. In some aspects, the light chain variable region chain of SEQ ID NO: 4 is fused to a human lambda light chain constant region (eg, SEQ ID NO: 48) such that the light chain contains SEQ ID NO: 61. Antibody variant 320-179 is able to inhibit the interaction of TL1A with DR3. The antibody variant has increased activity compared to antibody 320-179 and/or has increased affinity for TL1A relative to antibody 320-179.

In some aspects, antibody variants 320-179 are recombinantly expressed and specifically associated with TL1A and contain a heavy chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 28, and a variable heavy chain CDR3 a heavy chain region comprising the amino acid sequence of SEQ ID NO: 17. Antibodies may comprise a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a variable region CDR3 light chain containing the amino acid sequence of SEQ ID NO: 30. In aspects in which the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 1, the light chain variable region preferably does not contain the amino acid sequence of SEQ ID NO: 2. These antibody variants are 320- 179 are able to inhibit the interaction of TL1A with DR3. These variants of antibodies 320-179 have increased activity compared to antibody 320-179 and/or have increased affinity for TL1A compared to antibody 320-179.

In some aspects, the antibodies specifically bind to TL1A and comprise a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 16,

- 7 042971

CDR3 of the heavy chain variable region containing the amino acid sequence

SEQ ID NO: 17, a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 22.

In some aspects, the antibodies specifically bind to TL1A and comprise a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 23, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 22 .

In some aspects, the antibodies specifically bind to TL1A and comprise a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 24, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 22 .

In some aspects, the antibodies specifically bind to TL1A and comprise a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 25, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 22 .

In some aspects, the antibodies specifically bind to TL1A and comprise a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 18, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 20 .

In some aspects, the antibodies specifically bind to TL1A and comprise a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 26, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 22 .

In some aspects, the antibodies specifically bind to TL1A and comprise a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 18, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 22 .

In some aspects, the antibodies specifically bind to TL1A and comprise a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 24, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 22 .

In some aspects, the antibodies specifically bind to TL1A and contain the CDR1 variable

- 8 042971 heavy chain region containing the amino acid sequence of SEQ ID NO: 15, heavy chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 21, heavy chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 17, light chain variable region CDR1 a chain containing the amino acid sequence of SEQ ID NO: 25, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 22.

In some aspects, the antibodies specifically bind to TL1A and comprise a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 26, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 22 .

In some aspects, the antibodies specifically bind to TL1A and comprise a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region CDR1 containing the amino acid sequence of SEQ ID NO: 18, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 27 .

In some aspects, the antibodies specifically bind to TL1A and contain a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 1 and a light chain variable region, provided that the light chain variable region does not contain the amino acid sequence of SEQ ID NO: 2. In some aspects, antibodies specifically bind to TL1A and contain a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 3 and a light chain variable region or light chain. The light chain variable region may further comprise a lambda constant region.

In some aspects, the antibodies specifically bind to TL1A and contain a light chain variable region containing the amino acid sequence of SEQ ID NO: 2 and a heavy chain variable region, provided that the heavy chain variable region does not contain the amino acid sequence of SEQ ID NO: 1. In some aspects, antibodies specifically bind to TL1A and contain a light chain variable region containing the amino acid sequence of SEQ ID NO: 4 and a heavy chain variable region. In some aspects, the antibodies specifically bind to TL1A and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a heavy chain variable region. In some aspects, the antibodies specifically bind to TL1A and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a heavy chain variable region. In some aspects, the antibodies specifically bind to TL1A and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region. In some aspects, the antibodies specifically bind to TL1A and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region. In some aspects, the antibodies specifically bind to TL1A and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region. In some aspects, the antibodies specifically bind to TL1A and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a heavy chain variable region. In some aspects, the antibodies specifically bind to TL1A and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region. In some aspects, the antibodies specifically bind to TL1A and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12 and a heavy chain variable region. In some aspects, the antibodies specifically bind to TL1A and contain a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable region. In some aspects, antibodies specifically bind to TL1A and comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a heavy chain variable region, provided that if the light chain variable region contains the amino acid sequence of SEQ ID NO: 2, the variable region heavy chain does not contain the amino acid sequence of SEQ ID NO: 1. The heavy chain variable region may further comprise a heavy chain constant region, including any IgG1, IgG2, or IgG4 heavy chain constant region amino acid sequence described or illustrated herein.

- 9 042971

Antibody variants 320-179 bind specifically to TL1A. The antibodies bind to human TL1A and can bind to one or more of cynomolgus monkey TL1A, mouse TL1A, rat TL1A, guinea pig TL1A, cat TL1A, dog TL1A, pig TL1A, or rabbit TL1A. In some aspects, antibodies can bind to TL1A of several different species, for example, if the epitope is shared. In some aspects, human TL1A comprises the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO. 33. In some aspects, cynomolgus TL1A contains the amino acid sequence of SEQ ID NO: 34. In some aspects, murine TL1A contains the amino acid sequence of SEQ ID NO: 35. In some aspects, rat TL1A contains the amino acid sequence of SEQ ID NO: 36. In some aspects, marine TL1A mumps contains the amino acid sequence of SEQ ID NO: 37. In some aspects, feline TL1A contains the amino acid sequence of SEQ ID NO: 38. In some aspects, porcine TL1A contains the amino acid sequence of SEQ ID NO: 39. In some aspects, rabbit TL1A contains the amino acid sequence of SEQ ID NO: 40. In some aspects, dog TL1A contains the amino acid sequence of SEQ ID NO: 41.

Antibody variants 320-179 have a binding affinity for an epitope on TL1A that includes an equilibrium dissociation constant (KD) that can be measured using a kinetic exclusion assay such as the KINEXA® assay (Sapidyne Instruments Inc., Boise, Idaho).

The K _D for TL1A binding, as determined by kinetic exclusion assay, is preferably less than about 1000 pM. In some aspects, the K _D for TL1A binding, as determined by kinetic exclusion assay, is less than about 500 pM, or less than about 400 pM, or less than about 300 pM, or less than about 200 pM. In some preferred aspects, the KD for TL1A binding, as determined by kinetic exclusion assay, is less than about 100 pM.

The KD for TL1A binding, as determined by kinetic exclusion assay, can be from about 10 pM to about 100 pM. The K _D for TL1A binding, as determined by kinetic exclusion assay, may be from about 25 pM to about 75 pM. The K _D for TL1A binding, as determined by kinetic exclusion assay, may be from about 30 pM to about 60 pM. The K _D for TL1A binding, as determined by kinetic exclusion assay, may be from about 30 pM to about 50 pM. The KD for TL1A binding, as determined by kinetic exclusion assay, can be from about 35 pM to about 50 pM. The KD for TL1A binding, as determined by kinetic exclusion assay, can be from about 36 pM to about 46 pM. The K _D for TL1A binding, as determined by kinetic exclusion assay, may be from about 38 pM to about 44 pM. The KD for TL1A binding, as determined by kinetic exclusion assay, can be from about 39 pM to about 43 pM. The KD for TL1A binding, as determined by kinetic exclusion assay, can be from about 40 pM to about 45 pM. The KD for TL1A binding, as determined by kinetic exclusion assay, can be from about 35 pM to about 42 pM. The KD for TL1A binding, as determined by kinetic exclusion assay, may be about 40 pM. The KD for TL1A binding, as determined by kinetic exclusion assay, may be about 41 pM. The K _D for TL1A binding, as determined by kinetic exclusion analysis, may be about 42 pM. A kinetic exclusion assay can use an antibody molecule or a TL1A molecule as a constant binding partner and another molecule as a titrant.

Anti-TL1A antibody variants 320-179 are preferably capable of binding to TL1A positive cells. The antibody can bind to a TL1A positive cell with an EC ₅₀ value of less than about 100 nM. The antibody can bind to a TL1A positive cell with an EC ₅₀ value of less than about 75 nM. The antibody can bind to a TL1A positive cell with an EC ₅₀ value of less than about 50 nM. The antibody can bind to a TL1A positive cell with an EC ₅₀ value of less than about 30 nM. The antibody can bind to a TL1A positive cell with an EC ₅₀ value of less than about 25 nM. The antibody can bind to a TL1A positive cell with an EC ₅₀ value of less than about 20 nM. The antibody can bind to a TL1A positive cell with an EC ₅₀ value of less than about 18 nM. The antibody can bind to a TL1A positive cell with an EC ₅₀ value of less than about 15 nM. The antibody can bind to a TL1A positive cell with an EC ₅₀ value of less than about 13 nM. The antibody can bind to a TL1A positive cell with an EC ₅₀ value of less than about 10 nM.

Antibody variants 320-179 are preferably monoclonal and more preferably full length antibodies containing two heavy chains and two light chains. In some aspects, the antibodies contain derivatives or fragments or portions of antibodies that retain antigen-binding specificity and also preferably retain more or all of the affinity of the parent antibody molecule 320-179 (eg, for TL1A). For example, derivatives may contain at least one variable region (heavy chain or light chain variable region). Other examples of suitable antibody derivatives and fragments include, but are not limited to

- 10 042971 polyepitope specific antibodies, bispecific antibodies, multispecific antibodies, diabodies, single chain molecules, and FAb, F(Ab')2, Fd, Fabc and Fv molecules, single chain (Sc) antibodies, single chain Fv antibodies (scFv ), individual antibody light chains, individual antibody heavy chains, fusions between antibody chains and other molecules, heavy chain monomers or dimers, light chain monomers or dimers, single heavy and single light chain dimers, and other multimers. Single chain Fv antibodies may be multivalent. All isotypes of antibodies can be used to obtain derivatives, fragments and parts of antibodies. Derivatives, fragments and/or parts of an antibody can be recombinantly produced and expressed by any cell type, prokaryotic or eukaryotic.

In a full-length antibody, each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region consists of a single domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) that alternate with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In general, the antigen-binding properties of an antibody are less likely to be affected by changes in FR sequences than by changes in CDR sequences. Immunoglobulin molecules can be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

Antibody variants 320-179 are fully human. Fully human antibodies are those in which the entire molecule is human or otherwise of human origin, or contains an amino acid sequence identical to the human form of the antibody. Fully human antibodies include antibodies derived from a human V gene library, for example, where human genes encoding antibody variable regions are recombinantly expressed. Fully human antibodies can be expressed in other organisms (eg mice and XenoMouse technology) or cells of other organisms transformed with genes encoding human antibodies. Fully human antibodies may, however, contain amino acid residues not encoded by human sequences, such as mutations introduced by random or site-directed mutations.

In some aspects, antibody variants 320-179 may contain protein scaffolds that are not derived from immunoglobulin. For example, reference can be made to (Ku & Schutz, 1995, Proc. Natl. Acad. Sci., USA, 92:6552-6556), which describes the protein as a four helix bundle of cytochrome b562 having two loops randomly arranged for generating CDRs that have been selected for antigen binding.

Antibody variants 320-179 may contain post-translational modifications or fragments that may affect antibody activity or stability. These modifications or moieties include, without limitation, methylated, acetylated, glycosylated, sulfated, phosphorylated, carboxylated, and amidated moieties, and other moieties that are well known in the art. Fragments include any chemical group or combinations of groups normally found naturally in immunoglobulin molecules or otherwise added to antibodies using recombinant expression systems, including prokaryotic and eukaryotic expression systems.

Examples of side chain modifications discussed herein include amino modifications such as reductive alkylation with an aldehyde followed by reduction with NaBH ₄ ; amidation with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH ₄ .

The guanidine group of arginine residues can be modified by forming heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group can be modified by activation with a carbodiimide through the formation of an O-acylisourea followed by the formation of a derivative, for example the corresponding amide. Sulfidryl groups can be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; oxidation of performic acid to cysteic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; the formation of mercury compound derivatives using 4-chloromercury benzoate, 4-chloromercuryphenylsulfonic acid, phenylmercuric chloride, 2-chloromercury-4-nitrophenol, and other mercury compounds; carbamoylation with

- 11 042971 cyanate at alkaline pH. Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide or by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halides. On the other hand, tyrosine residues can be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative. The modification of the imidazole ring of the histidine residue can be carried out by alkylation with iodoacetic acid derivatives or by N-carbethoxylation with diethylpyrocarbonate.

Antibody variants 320-179 may include modifications that modulate serum half-life and biodistribution, including, without limitation, modifications that modulate the antibody's interaction with the neonatal Fc receptor (FcRn), a receptor that plays a key role in protecting IgG from catabolism and maintaining high concentration of antibodies in serum. Modifications that modulate serum half-life can occur in the Fc region of IgG1, IgG2, or IgG4, including the M252Y/S254T/T256E triple substitution (YTE substitutions numbered according to the EU numbering system (Edelman, G.M. et al. (1969) , Proc. Natl. Acad., USA, 63, 78-85)) as described in US Pat. No. 7,083,784. Other substitutions may occur at positions 250 and 428, see for example US Pat. 307, 380 and 434, see, for example, PCT Publication No. WO 00/042072. Examples of constant region amino acid substitutions that modulate binding to Fc receptors and subsequent function mediated by these receptors, including FcRn binding and serum half-life, are described in US Publication No. 2009/0142340, 2009/0068175 and 2009/0092599. Antibodies of any class may have the heavy chain C-terminal lysine omitted or removed to reduce heterogeneity (AK). The substitution S228P (EC numbering) in human IgG4 can stabilize the exchange of antibody Fab fragments in vivo (Labrin et al. (2009), Nature Biotechnology, 27:8, 767-773), and this substitution may be present concurrently with modifications to YTE and/ or AK.

Antibody variants 320-179 contain human constant domains. The heavy chain constant domains are preferably human IgG1, IgG2 or IgG4 constant domains. The light chain constant domains are preferably human lambda constant domains. A suitable human lambda domain comprises SEQ ID NO: 48.

Heavy chain constant regions of human IgG1 that can be used with antibody variants 320-179 can be selected from human IgG1 (SEQ ID NO: 42), human IgG1 (AK) (SEQ ID NO: 43), human IgG1 252Y/254T/ 256E (SEQ ID NO: 44), Human IgG1 252Y/254T/256E (AK) (SEQ ID NO: 64), Human IgG1 L234A/L235A/G237A (SEQ ID NO: 63), Human IgG1 L234A/L235A/G237A ( AK) (SEQ ID NO: 62), human IgG1 L235A/G237A (SEQ ID NO: 65) and human IgG1 L235A/G237A (AK) (SEQ ID NO: 66). Human IgG2 heavy chain constant regions that can be used with antibody variants 320-119 can be selected from human IgG2 with or without AK (SEQ ID NO: 67 and SEQ ID NO: 70) and human IgG2 A330S/P331S with or without (ΔΚ) (SEQ ID NO: 71 and SEQ ID NO: 68). Human IgG4 heavy chain constant regions that can be used with antibody variants 320-179 can be selected from human IgG4 S228P (SEQ ID NO: 45), human IgG4 S228P (AK) (SEQ ID NO: 46), human IgG4 228P /252Y/254T/256E (SEQ ID NO: 47) and human IgG4 228P/252Y/254T/256E (ΔΚ) (SEQ ID NO: 69).

Antibody variants 320-179 may be labeled, linked or conjugated to any chemical or biomolecular moieties. Labeled antibodies can be used in therapeutic, diagnostic or basic research. Such labels/conjugates can be detectable, such as fluorochromes, electrochemiluminescent probes, quantum dots, radioactive labels, enzymes, fluorescent proteins, luminescent proteins, and biotin. Labels/conjugates can be chemotherapeutic agents, toxins, isotopes, and other agents used to treat such pathological conditions, such as killing cancer cells. Chemotherapeutic agents can be any that are suitable for the purposes for which the antibody is used.

Antibodies can be derivatized with known protecting/blocking groups to prevent proteolytic cleavage or increase activity or stability.

Polynucleotide sequences that encode antibodies and their subdomains (eg, FR and CDR) are presented in the description. Polynucleotides include, but are not limited to, RNA, DNA, cDNA, RNA/DNA hybrids, and single, double or triple stranded RNA, DNA, or hybrids thereof. The polynucleotides may contain a nucleic acid sequence encoding a heavy chain variable region and/or a light chain variable region of the 320-179 antibody variant as described or exemplified herein. Complementary strands of polynucleotide sequences are also within the scope of the disclosure.

The polynucleotide may comprise a nucleic acid sequence encoding the antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3. The polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 may comprise the nucleic acid sequence of SEQ ID NO: 51.

- 12 042971

The polynucleotide may comprise a nucleic acid sequence encoding an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1.

The polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 may comprise the nucleic acid sequence of SEQ ID NO: 49.

The polynucleotide may comprise a nucleic acid sequence encoding the antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. The polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 may comprise the nucleic acid sequence of SEQ ID NO: 50.

The polynucleotide may comprise a nucleic acid sequence encoding the antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. The polynucleotide encoding the amino acid sequence of SEQ ID NO: 4 may comprise the nucleic acid sequence of SEQ ID NO: 52.

The polynucleotide may comprise a nucleic acid sequence encoding the antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. The polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 may comprise the nucleic acid sequence of SEQ ID NO: 53.

The polynucleotide may comprise a nucleic acid sequence encoding the antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 6. The polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 may comprise the nucleic acid sequence of SEQ ID NO: 54.

The polynucleotide may comprise a nucleic acid sequence encoding the antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 7. The polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 may comprise the nucleic acid sequence of SEQ ID NO: 55.

The polynucleotide may comprise a nucleic acid sequence encoding the antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. The polynucleotide encoding the amino acid sequence of SEQ ID NO: 8 may comprise the nucleic acid sequence of SEQ ID NO: 56.

The polynucleotide may comprise a nucleic acid sequence encoding the antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 10. The polynucleotide encoding the amino acid sequence of SEQ ID NO: 10 may comprise the nucleic acid sequence of SEQ ID NO: 57.

In some aspects, the polynucleotide comprises a first nucleic acid sequence encoding an antibody heavy chain variable region and a second nucleic acid sequence encoding an antibody light chain variable region. The first nucleic acid sequence may encode an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. A polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 may comprise the sequence nucleic acid sequence of SEQ ID NO: 51, and the polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 may contain the nucleic acid sequence of SEQ ID NO: 50.

The first nucleic acid sequence may encode an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. A polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 may comprise the sequence nucleic acid sequence of SEQ ID NO: 51, and the polynucleotide encoding the amino acid sequence of SEQ ID NO: 4 may contain the nucleic acid sequence of SEQ ID NO: 52.

The first nucleic acid sequence may encode an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6. A polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 may comprise the sequence nucleic acid sequence of SEQ ID NO: 51, and the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 may contain the nucleic acid sequence of SEQ ID NO: 54.

The first nucleic acid sequence may encode an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7. A polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 may comprise the sequence nucleic acid sequence of SEQ ID NO: 51, and the polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 may contain the nucleic acid sequence of SEQ ID NO: 55.

The first nucleic acid sequence may encode an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. A polynucleotide,

- 13 042971 encoding the amino acid sequence of SEQ ID NO: 3 may contain the nucleic acid sequence of SEQ ID NO: 51, and the polynucleotide encoding the amino acid sequence of SEQ ID NO: 8 may contain the nucleic acid sequence of SEQ ID NO: 56.

The first nucleic acid sequence may encode an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10. A polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 may comprise the sequence nucleic acid sequence of SEQ ID NO: 51, and the polynucleotide encoding the amino acid sequence of SEQ ID NO: 10 may contain the nucleic acid sequence of SEQ ID NO: 57.

The first nucleic acid sequence may encode an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. A polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 may comprise the sequence nucleic acid sequence of SEQ ID NO: 49, and the polynucleotide encoding the amino acid sequence of SEQ ID NO: 4 may contain the nucleic acid sequence of SEQ ID NO: 52.

The first nucleic acid sequence may encode a heavy chain variable region of an antibody comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. A polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 may comprise a nucleic acid sequence acids of SEQ ID NO: 49, and the polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 may contain the nucleic acid sequence of SEQ ID NO: 53.

The first nucleic acid sequence may encode an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6. A polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 may comprise the sequence nucleic acid sequence of SEQ ID NO: 49, and the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 may contain the nucleic acid sequence of SEQ ID NO: 54.

The first nucleic acid sequence may encode an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7. A polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 may comprise the sequence nucleic acid sequence of SEQ ID NO: 49, and the polynucleotide encoding the amino acid sequence of SEQ ID NO: 7 may contain the nucleic acid sequence of SEQ ID NO: 55.

The first nucleic acid sequence may encode an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. A polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 may comprise the sequence nucleic acid sequence of SEQ ID NO: 49, and the polynucleotide encoding the amino acid sequence of SEQ ID NO: 8 may contain the nucleic acid sequence of SEQ ID NO: 56.

In some aspects, the polynucleotide comprises a first nucleic acid sequence encoding an antibody heavy chain variable region and a second nucleic acid sequence encoding a heavy chain constant region. In preferred aspects, the polynucleotide comprises a first nucleic acid sequence encoding an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a second nucleic acid sequence encoding an IgG1 heavy chain constant region (AK) of SEQ ID NO: 43, e.g., a polynucleotide, containing the nucleic acid sequence of SEQ ID NO: 58.

In some aspects, the polynucleotide comprises a first nucleic acid sequence encoding an antibody light chain variable region and a second nucleic acid sequence encoding a light chain constant region. In preferred aspects, the polynucleotide comprises a first nucleic acid sequence encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a second nucleic acid sequence encoding a lambda light chain constant region of SEQ ID NO: 48, e.g. acids SEQ ID NO: 59.

Any of the polynucleotides described or exemplified herein may be placed into a vector. Thus, vectors containing polynucleotides are provided as part of the disclosure. The vectors may be expression vectors. Thus, recombinant expression vectors containing a sequence encoding a polypeptide of interest are provided. The expression vector may contain one or more additional sequences such as, but not limited to, regulatory sequences, a selectable marker, a purification tag, or a polyadenylation signal. Such regulatory elements may contain a transcriptional promoter, enhancers, mRNA ribosome binding sites, or sequences that control

- 14 042971 termination of transcription and translation.

Expression vectors, especially mammalian expression vectors, may contain one or more non-transcribed elements such as an origin of replication, an appropriate promoter and enhancer associated with the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3 '-untranslated sequences (such as essential ribosome binding sites), polyadenylation site, splicing donor and acceptor site, or transcription termination sequences. An origin of replication may also be included, which allows replication in a particular host.

Vectors can be used to transform any of a wide variety of host cells well known to those skilled in the art, and preferably host cells capable of expressing antibodies. Vectors include, but are not limited to, plasmids, phagemids, cosmids, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and baculovirus, as well as other bacterial, eukaryotic, yeast, and viral vectors. Suitable host cells include, but are not limited to, CHO cells, NS0 cells, HEK293 cells, or any eukaryotic cell line known or produced, and include bacteria, yeast, and insect cells.

Antibodies can also be produced by hybridoma cells; methods for producing hybridomas are well known and developed in the art.

The disclosure also provides compositions containing antibody variants 320-179. The compositions may contain any of the antibodies described and/or exemplified herein, and an acceptable carrier, such as a pharmaceutically acceptable carrier. Suitable carriers include any media that do not interfere with the biological activity of the antibody and preferably are not toxic to the host to which it is administered. The compositions may be formulated for administration to a subject in any suitable dosage form.

Antibody variants 320-179 can be used to treat respiratory disease, gastrointestinal disease, arthritis, or skin disease in a subject. Thus, the disclosure includes treatments. In general, the methods include administering the 320-179 antibody variant, or composition thereof, to a subject in need of treatment for a respiratory disease, gastrointestinal disease, arthritis, or skin disease, such that the respiratory disease, gastrointestinal disease, arthritis, or skin disease is treated. . Antibody variant 320-179 may contain any antibody described or exemplified herein. Administration may include subcutaneous administration of the antibody. Administration may include intravenous administration of the antibody. The subject is preferably a human. The subject may be a non-human primate, such as a cynomolgus monkey, or may be a mammal, such as a mouse, rat, guinea pig, cat, pig, rabbit, or dog.

In those aspects in which the respiratory disease is to be treated, the methods include administering the 320-179 antibody variant or composition thereof to a subject in need of treatment of the respiratory disease. The respiratory disease may include one or more of asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, pulmonary sarcoidosis, allergic rhinitis, or cystic fibrosis. Thus, for example, in some aspects, the methods include administering the 320-179 antibody variant or composition to a subject in need of asthma treatment such that the subject's asthma is treated. In some aspects, the methods include administering the 320-179 antibody variant, or composition thereof, to a subject in need of treatment for COPD such that the subject is treated for COPD. In some aspects, the methods include administering the 320-179 antibody variant or composition to a subject in need of treatment for pulmonary fibrosis such that the subject is treated for pulmonary fibrosis. In some aspects, the methods include administering the 320-179 antibody variant or composition thereof to a subject in need of treatment for pulmonary sarcoidosis such that the subject is treated for pulmonary sarcoidosis. In some aspects, the methods include administering the 320-179 antibody variant, or composition thereof, to a subject in need of treatment for allergic rhinitis such that the subject is treated for allergic rhinitis. In some aspects, the methods include administering the 320-179 antibody variant, or composition thereof, to a subject in need of treatment for cystic fibrosis such that the cystic fibrosis is treated in the subject.

In those aspects in which a disease of the gastrointestinal tract is to be treated, the methods include administering the 320-179 antibody variant or composition thereof to a subject in need of treatment of the disease of the gastrointestinal tract. The gastrointestinal disease may include one or more of the following: inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis, irritable bowel syndrome (IBS), eosinophilic esophagitis, or a disease or condition of the gastrointestinal tract, associated with cystic fibrosis. Thus, for example, in some aspects, the methods include administering the 320-179 antibody variant or composition to a subject in need of IBD treatment such that the subject is treated for IBD. In some aspects, the methods include administering the 320-179 antibody variant or composition to a subject in need of treatment for Crohn's disease such that the subject's Crohn's disease is treated. IN

- 15 042971 In some aspects, the methods include administering the 320-179 antibody variant, or composition thereof, to a subject in need of treatment for colitis such that the subject's colitis is treated. In some aspects, the methods include administering the 320-179 antibody variant or composition to a subject in need of treatment for ulcerative colitis such that the subject is treated for ulcerative colitis. In some aspects, the methods include administering the 320-179 antibody variant, or composition thereof, to a subject in need of IBS treatment such that the subject is treated for IBS. In some aspects, the methods include administering the 320-179 antibody variant or composition to a subject in need of treatment for eosinophilic esophagitis such that the subject is treated for eosinophilic esophagitis. In some aspects, the methods include administering the 320-179 antibody variant, or composition thereof, to a subject in need of treatment of a gastrointestinal disease or condition associated with cystic fibrosis such that the gastrointestinal disease or condition associated with cystic fibrosis , is treated in the subject.

In those aspects in which arthritis is to be treated, the methods include administering the 320-179 antibody variant, or composition thereof, to a subject in need of arthritis treatment. Arthritis may include rheumatoid arthritis. Thus, for example, in some aspects, the methods include administering the 320-179 antibody variant or composition thereof to a subject in need of treatment for rheumatoid arthritis such that the subject is treated for rheumatoid arthritis.

In those aspects in which a skin disease is to be treated, the methods include administering the 320-179 antibody variant, or composition thereof, to a subject in need of treatment for the skin disease. The skin disease may include one or more of atopic dermatitis, eczema, or scleroderma. Thus, for example, in some aspects, the methods include administering the 320-179 antibody variant, or composition thereof, to a subject in need of atopic dermatitis treatment such that the subject is treated for atopic dermatitis. In some aspects, the methods include administering the 320-179 antibody variant or composition to a subject in need of treatment for eczema such that the subject's eczema is treated. In some aspects, the methods include administering the 320-179 antibody variant or composition to a subject in need of treatment for scleroderma such that the subject is treated for scleroderma.

Antibody variants 320-179 described or exemplified herein may be used in the preparation of a medicament for use in the treatment of respiratory disease. Antibody variants 320-179 described or exemplified herein may be used in the preparation of a medicament for use in the treatment of gastrointestinal disease. Antibody variants 320-179 described or exemplified herein can be used in the preparation of a medicament for use in the treatment of arthritis. Antibody variants 320-179 described or exemplified herein may be used in the preparation of a medicament for use in the treatment of skin disease. Antibody variants 320-179 described or exemplified herein can be used in the preparation of a medicament for use in the treatment of any disease from asthma, COPD, pulmonary fibrosis, pulmonary sarcoidosis, allergic rhinitis, cystic fibrosis, inflammatory bowel disease, Crohn's disease, colitis, ulcerative colitis, irritable bowel syndrome, eosinophilic esophagitis, gastrointestinal disease or condition associated with cystic fibrosis, arthritis, rheumatoid arthritis, atopic dermatitis, eczema, or scleroderma.

Antibody variants 320-179 described or exemplified herein can be used to treat respiratory disease. Antibody variants 320-179 described or exemplified herein can be used to treat disease of the gastrointestinal tract. Antibody variants 320-179 described or exemplified herein can be used to treat asthma. Antibody variants 320-179 described or exemplified herein can be used to treat skin disease. Antibody variants 320-179 described or exemplified herein can be used to treat COPD. Antibody variants 320-179 described or exemplified herein can be used to treat pulmonary fibrosis. Antibody variants 320-179 described or exemplified herein can be used to treat lung sarcoidosis. Antibody variants 320-179 described or exemplified herein can be used to treat allergic rhinitis. Antibody variants 320-179 described or exemplified herein can be used to treat cystic fibrosis. Antibody variants 320-179 described or exemplified herein can be used to treat inflammatory bowel disease. Antibody variants 320-179 described or exemplified herein can be used to treat Crohn's disease. Antibody variants 320-179 described or exemplified herein can be used to treat colitis. Antibody Options 320-179 as described or

- 16 042971 given as an example in this document, can be used to treat ulcerative colitis. Antibody variants 320-179 described or exemplified herein can be used for eosinophilic esophagitis. Antibody variants 320-179 described or exemplified herein can be used to treat a disease or condition of the gastrointestinal tract associated with cystic fibrosis. Antibody variants 320-179 described or exemplified herein can be used to treat irritable bowel syndrome. Antibody variants 320-179 described or exemplified herein can be used to treat rheumatoid arthritis. Antibody variants 320-179 described or exemplified herein can be used to treat atopic dermatitis. Antibody variants 320-179 described or exemplified herein can be used to treat eczema. Antibody variants 320-179 described or exemplified herein can be used to treat scleroderma.

Also provided is an in vitro method for detecting TL1A in a tissue sample isolated from a subject, comprising contacting an antibody according to any one of claims 1 to 19 with a tissue sample isolated from a subject to form an antibody-TL1A complex, and detecting the complex in the tissue sample. .

The 320-179 antibody variant can be used to detect TL1A positive cells, for example, in a tissue sample obtained from a subject. The antibodies can be used to detect TL1A positive peripheral blood mononuclear cells (PBMCs), such as PBMCs, obtained from a subject. Antibodies can be used to detect TL1A in serum. Such methods can be carried out in vivo, ex vivo, in vitro or in situ. In general, the methods include contacting any of the antibody variants 320-179 described or illustrated herein with tissue or cells, such as PBMCs, isolated from a subject to form an antibody-TL1A complex and detecting the complex in the tissue or on the cells. The antibody may be labeled with a detectable label. The antibody can be detected by a secondary antibody that is labeled with a detectable label. The tissue may contain or be a biological fluid such as blood or blood serum. The tissue may or may be respiratory tract tissues such as lung tissue, sputum, bronchoalveolar lavage fluid, gastrointestinal tissue, or gastrointestinal lavage fluid. The fabric may or may be leather or skin tissue. The tissue may comprise or be tissue from any joint in the body. The method may further include isolating tissue from the subject. Such methods can be quantitative, such as quantifying the level of TL1A in tissue, quantifying the level of TL1A positive cells, or quantifying the level of TL1A on cells, or quantifying the level of TL1A in serum.

The disclosure also provides kits containing any of the antibody variants 320-179 described and exemplified herein. The kits can be used to provide antibodies and other agents for use in diagnostics, basic research, or therapeutic methods, among others. In some aspects, kits contain any one or more of the antibody variants 320-179 described or exemplified herein and instructions for using one or more antibodies in a method of treating a respiratory disease, in a method of treating a gastrointestinal disease, or in a method of treating arthritis.

The following examples are provided to describe the disclosure in more detail. They are intended to be illustrative and not to limit disclosure.

Example 1. Materials and methods.

The amino acid positions in these examples are numbered according to the Kabat numbering system. CDRs are determined according to the AbM method of the CDR determination system in this document.

1.1. Generation of variants of beams.

Amino acid sequences of the variable region of the heavy and light chains of antibodies 320-179 (SEQ ID NO: 1 and 2, respectively) were used as templates for the development of these variants. 320-179 was previously described in US Publication No. 2014/0255302 (VH is SEQ ID NO: 186 and VL is SEQ ID NO: 199 in this publication) as 320-179 (also described as C320-179). This antibody had favorable biophysical properties, was a potent inhibitor of TL1A, and had a low predictive immunogenicity profile.

Antibody variants 320-179 were made by substituting one of a group of nine typical amino acids - A, S, Q, D, H, K, L, W, Y - one at each amino acid position of the CDR (as defined using AbM nomenclature) in light chain CDR1 (CDR-L1), light chain CDR3 (CDR-L3), heavy chain CDR1 (CDR-H1) and heavy chain CDR2 (CDR-H2). Antibody variants including A, S, Q, D, H, K, L, W, Y were also made at positions 59 and 60 in the heavy chain variable region and at position 79 in the light chain variable region. A complete listing of all single substituted antibody variants generated is shown in FIG. 1 (heavy chain variable region) and FIG. 2 (light chain variable region), respectively.

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1.2. Construction of vectors expressing antibodies.

Variable region variants were generated by reverse transcription of amino acid sequences into DNA sequences, which were subsequently synthesized de novo by assembling synthetic oligonucleotides. The VH variants were subcloned into a mammalian expression vector containing a human constant region to generate full-length antibody heavy chains (human IgG1 heavy chain CH1, hinge, CH2 and CH3 domains) (eg, UniProt # P01857). Similarly, V _L variants were subcloned into a mammalian expression vector containing a human lambda light chain constant region to generate full length antibody lambda chains (SwissProt # P0CG05.1). In some cases, the full-length heavy chain and the light chain alone were retranslated into DNA sequences and subsequently synthesized de novo by assembling synthetic oligonucleotides.

1.3. Expression of antibody variants.

Antibodies were generated by co-transfection of antibody heavy and light chains into EXPI293® cells (Life Technologies, Carlsbad, CA). One day before transfection, the number of cells required for the experiment was determined. For each 20 ml transfection, 3.6x10 ⁷ cells were required in 20 ml of EXPI293® Expression Medium. The day before transfection, cells were seeded at a density of 0.9x106 viable cells/ml and incubated overnight at 37° C. in a humidified atmosphere with 8% CO ₂ in air on an orbital shaker rotating at 200 rpm. On the day of transfection, the number of cells and their viability was determined using an automated cell counter. Cultures with more than 98% viable cells were used. For each 20 ml transfection, lipid-DNA complexes were prepared by diluting 10 μg heavy chain DNA and 10 μg light chain DNA in OPTI-MEM® I Reduced Serum Medium (Life Technologies, Carlsbad, CA) (#31985-062) to total volume 1.0 ml. 54 µl of EXPIFECTAMINE® 293 Reagent (Life Technologies, Carlsbad, CA) was diluted in OPTI-MEM® I medium to a total volume of 1.0 ml. Both vials were gently mixed and incubated for 5 min at room temperature. After incubation, diluted DNA was mixed with diluted EXPIFECTAMINE® 293 Reagent and DNA-EXPIFECTAMINE® 293 Reagent reaction mix and incubated for an additional 20 min at room temperature to form DNA-EXPIFECTAMINE® 293 Reagent complexes. After incubation, 2 ml of DNA-EXPIFECTAMINE® 293 Reagent complex was added to each 50 ml bioreactor tube (TPP Techno Plastic Products AG). In the negative control tube, 2 ml of OPTI-MEM® I medium (Life Technologies, Carlsbad, CA) was added instead of the DNA-EXPIFECTAMINE® 293 Reagent complex.

Cells were incubated in an incubator at 37°C with a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 200 rpm. Approximately 16-18 hours after transfection, 100 μl EXPIFECTAMINE® 293 Transfection Enhancer 1 and 1.0 ml EXPIFECTAMINE® 293 Transfection Enhancer 2 were added to each bioreactor. Antibodies were collected approximately 72 hours after transfection.

1.4. Purification of antibody variants.

Each antibody variant was expressed in EXPI293® cells in 20 ml of cell culture. The cultures were centrifuged in 50 ml Falcon tubes at 3000xg for 20 min and the supernatants were filtered using a 0.22 µm filter (Corning). Supernatants were purified using a Gilson ASPEC GX274 robot. Briefly, SPE (solid phase extraction) cartridges (Agilent, 12131014) filled with 1.2 ml of MABSELECT SURE® protein A resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were pre-equilibrated with 3 column volumes of 1X PBS. 18 ml of the supernatant was passed through the columns and then washed with 4 ml of 1X PBS. Each column was pre-eluted with 0.9 ml of 0.1 M citric acid at pH 2.9. Purified antibodies were eluted with 2 ml of 0.1 M citric acid, pH 2.9. Antibodies were desalted in PBS S0rensens (59.5 mM KH2PO4, 7.3 mM Na ₂ HPO ₄ -2H ₂ O, 145.4 mM NaCl (pH ~5.8)) using PD-10 columns (GE Healthcare).

1.5. Antibody expression and antigen binding as determined by SPR (Surface Plasmon Resonance).

Using CM5 sensor chips (GE Healthcare), protein A (Pierce) was coupled to the surface of the chip using an amine coupling kit (GE Healthcare). Protein A was coupled to flow cell 1 and 2 (or alternatively 3 and 4) using BIACORE® T200. Supernatants from EXPI-293® cells containing antibody or alternatively purified antibodies (as described in 1.4) were passed over the surface of flow cell 2, while buffer (HBS-EP) was passed through flow cell 1. Amount of supernatant or purified protein (as well as concentration) administered during the capture step varied between runs and is indicated in the header of the table. 3-11. At the end of administration of the supernatant or purified antibody, the change in response units was measured. This value was indicated as the level of capture in the table. 3-11. To determine if TL1A is antibody bound, TL1A is then passed through flow cells 1 and 2 and response units are measured until the end of TL1A administration (association phase). This value is referred to as the level of binding TL1A (early) in table. 3-11.

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Response units were measured until the end of the dissociation phase. This value is labeled as the level of binding TL1A (late) in table. 3-11 and is a measure of the amount of antibody that has been lost from the surface of the chip as a result of dissociation of the TL1A-antibody complex. Sensorgrams were double standardized (flow cell 2 subtracted from flow cell 1 and control buffer). Since a large number of antibody variants were available for screening, they were tested in different runs (Tables 3-11). In each case (except run 3), the parent antibody 320-179 was included for comparison. Below is a brief description of the conditions used in each run.

Table 1

Run # Supernatant Protein Approximate Capture Level (EO) conc, sample TL1A (µg/ml) TL1A binding level (early) at time point TL1A (late) binding level at time point Notes 1 X 400 10 59 590 Antibody diluted to 2 µg/mL, variable uptake time 2 X 1000 10 44 220 Supernatant diluted in running buffer, variable capture time 2 X 400 10 44 590 Antibody diluted to 2 µg/mL, variable uptake time 3 X variable 10 59 590 Supernatant diluted in running buffer, capture 60 s 4 X 500 5 44 220 Antibody diluted to 2 µg/ml in running buffer 5 X 100 5 44 220 Supernatant diluted in running buffer 6 X variable 5 44 170 Antibody diluted to 2 µg/mL in running buffer and captured within 45 s y* X 400 5 44 220 Antibody diluted to approx. 2 µg/ml 8 X 400 5 44 590 Antibody diluted to 2 mcg/mL, variable capture time 9 X 400 5 44 590 Antibody diluted to 2 µg/mL, variable uptake time

* This run was performed on Biacore® A100.

Binding of anti-TL1A antibody to various TL1A species was also determined using SPR. Anti-TL1A antibody was captured on the surface of protein A. TL1A from human, rat, mouse, rabbit, guinea pig, monkey, pig, dog, cat, or cynomolgus monkey was passed over the surface and response units were measured.

1.6. Getting TL1A.

Human TL1A was generated in the EXPI293® mammalian expression system using a DNA expression construct encoding the extracellular domain (ECD) of human TL1A with an N-terminal HIS and

- 19 042971 labeled FLAG. Other types of TL1A forms have been created based on sequence listings from public databases. They are listed below.

table 2

Types TL1A Link to public database SEQID NO: Human UniProt: 095150 31 Javanese macaque SEQ ID NO: 125 of US Publication No. 2014/0255302 34 Mouse UniProt: Q5UBV8 35 Rat UniProt: Q8K3Y7 36 Guinea pig UniProt: H0VFN8 37 Cat NCBI: XP_003995828.1 38 Pig UniProt: I3EE00 39 Rabbit UniProt: G1T1T1 40 Dog UniProt: J9P221 41

The culture supernatant containing secreted TL1A protein was collected by centrifugation at 2000xg for 10 min to remove cells. The TL1A protein was purified from the supernatant using an HP HISTRAP® column (GE Healthcare). The eluted protein was buffer exchanged in PBS using a HILOAD® 16/60 Superdex 200 column for prep grade (GE Healthcare) and a ~70 kDa fraction, separated by gel filtration on a HILOAD® 26/60 SUPERDEX® 200 column for prep grade ( GE Healthcare).

1.7. Analysis of the activity of the TF-1 cell line.

To determine which anti-TL1A antibodies functionally neutralize the biological activity of TL1A, the antibodies were tested for their ability to neutralize TL1A-induced apoptosis in the TF-1 cell line. The human erythroleukemia cell line TF-1 (ATCC: CRL-2003) was maintained in culture under standard conditions. TF-1 cells (7.5x10 ⁴ /well) were incubated in opaque 96-well plates (Greiner) with 100 ng/ml human TL1A and 10 μg/ml cycloheximide to induce apoptosis. Test antibodies at a concentration of 10 μg/ml (66.7 nM) or less were added to the plates and incubated for 4-5 hours. Apoptosis induction was then assessed using the Homogeneous Caspases Kit (Roche) according to the manufacturer's instructions.

The data were normalized for expression as a percentage of maximum apoptosis (levels of apoptosis achieved by human TL1A plus cycloheximide in the absence of anti-TL1A antibody).

1.8. Receptor selectivity for the most active antibodies.

TL1A binds to both its cognate signaling receptor DR3 and the decoy receptor DcR3, which also serves as a decoy receptor for TNF family members Fas-L and LIGHT. Antibodies were evaluated for their ability to inhibit the binding of TL1A to its receptors in a competitive ELISA. DR3/Fc chimera (R&D Systems) or DcR3/Fc chimera (R&D Systems) were applied to a 96-well plate (Maxisorp, Nunc) at a concentration of 2 μg/ml. Serially diluted test antibodies were pre-incubated with 1 μg/ml biotinylated at one site human TL1A for 30 min, then added to wells coated with DR3/Fc or DcR3/Fc. Bound TL1A was detected using streptavidin-horseradish peroxidase 1:2000 (BD Pharmingen). Data were normalized for expression as a percentage of maximum TL1A receptor binding in the absence of anti-TL1A antibody.

1.9. Kinetic exclusion analysis.

This assay measures the free concentration of one of the binding partners without disturbing the equilibrium. Solutions can be prepared offline using unmodified proteins in solution, and affinity measurements can be read several days after mixing to ensure equilibrium is reached. In a kinetic exclusion assay, one interactant (called a constant binding partner, or CBP) is held at a constant concentration and the other (called a titrant) is diluted sequentially. Kinetic exclusion assays can be used to determine the dissociation constant (K _D ) and affinity of the antibody-antigen interaction. In a typical kinetic exclusion assay, a titrant is immobilized onto beads (eg Sepharose or PMMA (polymethyl methacrylate) beads) and used to capture free CBP in solution. A secondary labeled probe is then used to quantify the amount of captured CBP. Kinetic exclusion analysis is described in Darling,

RK et al. (2004), ASSAY and Drug Development Technol., 2(6):647-57.

The components were combined and allowed to reach equilibrium. Exclusion kinetic analysis was then used to quantify the free fraction of CBP. Equilibrium curves with several concentrations of CBP were analyzed using the n-curve analysis method in the KinExA® Pro software (version 4.1.11, Sapidyne) to obtain reliable K _D values. Interaction 320-587 for human TL1A was investigated using two directions:

(1) CBP is 320-587, titrant is TL1A;

(2) CBP is TL1A, titrant is 320-587.

Example 2. Results of the experiment.

2.1. Selection of TL1A-binding variants with equivalent or improved dissociation rate compared to C320-179.

Antibody variants 320-179 were constructed and expressed as described above. The EXPI293® (Life Technologies Corp.) supernatants of each variant were evaluated by BIACORE® (GE Healthcare) and compared with the parent antibody 320-179. In a separate experiment, antibodies were purified using protein A chromatography (see 1.4) and purified antibodies were used in the BIACORE® experiments (GE Healthcare). In table. 3-11 shows the level of expression of each variant, as well as their binding to TL1A at an early and late time point. In later series (Tables 10 and 11), antibody variants containing more than one amino acid substitution were tested.

Table 3

SPR experiment - run 1 anti-TL1A antibody binding to TL1A

Antibody Replacement in VH regarding 320-179 Replacement in VL regarding 320-179 Capture level TL1A binding level (early) TL1A binding level (late) Purified antibody 320-179 Absent Absent 410 140 117 320- 184* Absent L79A 414 141 118 320- 185* Absent L79S 401 137 114 320- 186* Absent L79Q 398 136 113 320- 187* Absent L79D 388 134 112 320- 188* Absent L79H 397 136 113 320- 189* Absent L79K 393 130 107 320- 190* Absent L79W 395 136 113 320- 191* Absent L79Y 408 140 117

* indicates the antibodies that were selected for the activity assay.

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Table 4

SPR experiment - run 2 - anti-TL1A antibodies binding to TL1A

Antibody Replacement in VH regarding 320-179 Replacement in VL regarding 320-179 Uro- wreath of capture TL1A binding level (early) TL1A binding level (late) Capture level TL1A binding level (early) TL1A binding level (late) Supernatant Purified antibody 320-179 Absent Absences 1133 287 261 410 140 117 320-192 Absent T24A 960 238 218 - - 320-193 Absent T24S 990 249 227 409 103 91 320-194 Absent T24Q 974 247 226 398 76 64 320-195 Absent T24D 992 249 227 - - 320-196 Absent T24N 995 250 228 - - 320-197 Absent T24K 1017 255 232 401 105 94 320-198* Absent T24L 1023 262 241 - - 320-199 Absent T24W 1050 251 233 404 97 85 320-200 Absent T24Y 1027 251 231 320-201* Absent S25A 990 274 252 393 73 57 320-202* Absent S25Q 1034 234 214 - - 320-203 Absent S25D 977 61 24 407 65 35 320-204 Absent S25H 1003 193 155 - - 320-205* Absent S25K 1025 290 271 401 70 51 320-206 Absent S25L 1000 257 235 - - 320-207* Absent S25W 1022 268 243 - - 320-208 Absent S25Y 1034 222 199 - - 320-209* Absent S26A 1007 222 200 - - 320-210 Absent S26Q 1033 225 202 - - 320-211* Absent S26D 1025 206 164 392 80 64 320-212 Absent S26H 1013 226 205 - - 320-213* Absent S26K 1052 218 194 422 79 65 320-214 Absent S26L 1038 221 196 - - 320-215 Absent S26W 1021 220 199 - - 320-216 Absent S26Y 1010 171 147 - - 320-217 Absent S27A 1076 258 235 - - 320-218 Absent S27Q 1081 239 218 412 92 80 320-219* Absent S27D 1061 243 220 - - 320-220 Absent S27H 1049 253 230 - - 320-221 Absent S27K 1063 225 207 413 99 89 320-222 Absent S27L 1037 156 96 - - - 320-223 Absent S27W 1061 143 66 - - - 320-224 Absent S27Y 1055 198 153 - - - 320-225 Absent S27aA 1066 267 243 - - - 320-226* Absent S27aQ 1035 250 228 - - - 320-227 Absent S27aD 1022 242 218 - - - 320-228 Absent S27aH 1072 259 235 - - - 320-229* Absent S27aK 1057 268 251 - - - 320-230 Absent S27aL 1087 245 223 - - - 320-231 Absent S27aW 1078 271 243 - - - 320-232 Absent S27AY 1051 261 235 - - - 320-233 Absent D27bA 1118 255 235 409 48 16 320-234 Absent D27bS 1089 262 239 - - - 320-235 Absent D27bQ 1110 256 236 - - - 320-236 Absent D27bH 1085 254 232 415 5 6 320-237 Absent D27bK 1073 240 221 398 10 8 320-238 Absent D27bL 1106 221 191 - - - 320-239 Absent D27bW 1079 212 163 - - - 320-240 Absent D27bY 1089 230 196 - - - 320-241* Absent I27cA 1092 179 122 - - - 320-242 Absent I27cS 1076 124 49 - - - 320-243 Absent I27cQ 1065 71 26 - - -

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320-244* Absent I27cD 1083 21 20 - - - 320-245* Absent 127cN 1084 59 26 - - - 320-246 Absent 127sK 1089 32 23 - - - 320-247 Absent I27cL 1078 205 162 - - - 320-248 Absent I27cW 1110 96 thirty - - - 320-249 Absent I27cY 1096 77 28 - - - 320-250 Absent G28A 1104 180 115 - - - 320-251 Absent G28S 1085 134 50 - - -

* indicates the antibodies that were selected for the activity assay.

Table 5

SPR experiment - run 3 anti-TL1A antibodies binding to TL1A

Antibody Replacement in UN regarding 320-179 Replacement in VL regarding 320-179 Capture level Binding level TL1A (early) Supernathan Binding level TL1A (late) T 320-252 Absent G28Q 996 20 9 320-253 Absent G28D 1073 6 6 320-254 Absent G28H 1021 17 8 320-255 Absent G28K 842 24 8 320-256 Absent G28L 1055 thirty 10 320-257 Absent G28W 981 83 18 320-258 Absent G28Y 823 9 6 320-259 Absent A29S 824 194 179 320-260 Absent A29Q 836 182 165 320-261 Absent A29D 956 86 29 320-262 Absent A29N 891 209 190 320- 263* Absent A29K 848 121 123 320-264 Absent A29L 831 199 184 320-265 Absent A29W 1057 168 69 320-266 Absent A29Y 934 218 192 320- 267* Absent G30A 1148 299 297 320-268 Absent G30S 392 28 7 320-269 Absent G30Q 764 126 93 320-270 Absent G30D 511 9 5 320-271 Absent G30H 1067 106 thirty 320-272 Absent G30K 935 5 5 320-273 Absent G30L 710 4 4 320-274 Absent G30W 796 12 7 320-275 Absent G30Y 751 44 10 320-276 Absent L31A 1273 244 215 320- 277* Absent L31S 1741 387 361 320- 278* Absent L31Q 1696 389 374 320-279 Absent L31D 561 144 128 320- 280* Absent L31H 881 228 211 320-281 Absent L31K 321 22 6 320-282 Absent L31W 811 75 16 320-283 Absent L31Y 317 2 3 320-284 Absent G32A 374 1 2 320-285 Absent G32S 400 1 2 320-286 Absent G32Q 576 1 4 320-287 Absent G32D 339 0 2

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320-288 Absent G32H 422 1 3 320-289 Absent G32K 463 1 3 320-290 Absent G32L 361 1 2 320-291 Absent G32W 414 2 3 320-292 Absent G32Y 423 111 94 320-293 Absent V33A 421 104 86 320-294 Absent V33S 419 79 61 320-295 Absent V33Q 414 109 94 320-296 Absent V33D 428 118 104 320- 297* Absent V33H 420 95 77 320-298 Absent V33K 416 114 100 320-299 Absent V33L 420 51 13 320-300 Absent H34W 417 111 95 320-301 Absent H34Y 456 108 96 320- 302* Absent H34A 423 99 74 320-303 Absent H34S 408 71 32 320-304 Absent H34Q 401 103 82 320-305 Absent H34D 424 34 10 320-306 Absent H34K 452 147 133 320- 307* Absent H34L 873 216 200 320-308 Absent H34W 458 106 73 320-309 Absent H34Y 255 47 40 320-310 Absent Q89A 348 70 62 320-311 Absent Q89S 0 0 0

* indicates the antibodies that were selected for the activity assay.

Table 6

SPR experiment - run 4 anti-TL1A antibodies binding to TL1A

Antibody Replacement in VH regarding 320-179 Replacement in VL regarding 320-179 Capture level Binding level TL1A (early) TL1A binding level (late) Purified antibody 320-179 Absent Absent 616 177 163 320-312 Absent Q89D 529 10 10 320- 313* Absent Q89H 502 141 132 320- 314* Absent Q89K 598 165 158 320-315 Absent Q89L 515 85 82

- 24 042971

320-316 Absent Q89W 535 65 62 320-317 Absent Q89Y 570 86 83 320- 318* Absent S90A 578 160 149 320-319 Absent S90Q 523 18 14 320-320 Absent S90D 534 31 17 320-321 Absent S90H 491 16 13 320-322 Absent S90K 450 17 14 320-323 Absent S90W 490 14 13 320-324 Absent S90Y 528 13 12 320-325 Absent Y91A 491 95 83 320- 326* Absent Y91S 550 126 120 320-327 Absent Y91Q 493 130 119 320- 328* Absent Y91H 542 155 146 320-329 Absent Y91K 600 9 AND 320-330 Absent Y91L 600 139 119 320- 331* Absent Y91W 615 173 176 320-332 Absent D92A 531 66 44 320-333 Absent D92S 559 95 67 320-334 Absent D92Q 543 15 14 320-335 Absent D92H 581 15 14 320-336 Absent D92K 484 AND AND 320-337 Absent D92L 475 21 15 320-338 Absent D92W 548 9 AND 320-339 Absent D92Y 509 10 AND 320-340 Absent G93A 548 145 132 320-341 Absent G93S 560 149 135 320-342 Absent G93Q 560 152 139 320-343 Absent G93D 545 121 90 320-344 Absent G93H 529 117 87 320- 345* Absent G93K 637 163 154 320-346 Absent G93L 526 126 107 320-347 Absent G93W 560 149 130 320-348 Absent G93Y 541 144 127 320-349 Absent T94A 556 112 88 320-350 Absent T94S 502 130 118 320-351 Absent T94Q 553 112 83

- 25 042971

320-352 Absent Γ94Ό 568 101 63 320-353 Absent G94N 558 97 73 320-354 Absent G94K 536 53 thirty 320-355 Absent T94L 526 102 76 320-356 Absent T94W 486 109 86 320-357 Absent Γ94Υ 563 119 91 320-358 Absent L95A 501 82 50 320-359 Absent L95S 503 57 27 320-360 Absent L95Q 554 86 53 320- 361* Absent L95D 477 12 eleven 320-362 Absent L95H 522 8 9 320-363 Absent L95Y 566 17 16 320-364 Absent S95aA 516 124 109 320-365 Absent S95aQ 536 141 128 320-366 Absent S95aD 487 62 31 320-367 Absent S95aH 595 119 116 320-368 Absent S95aK 602 50 26 320-369 Absent S95aL 503 89 60 320-370 Absent S95aW 570 66 39 320-371 Absent S95AY 576 92 70

* indicates the antibodies that were selected for the activity assay.

Table 7

SPR experiment - run 5 anti-TL1A antibodies binding to TL1A

Antibody Replacement in VH regarding 320-179 Replacement in VL regarding 320-179 Capture level Binding level TL1A (early) Supernathan Binding level TL1A (late) T 320-179 Absent Absent 83 AND 10 320-372 Absent A96S 106 1 0 320-373 Absent A96Q 99 -1 -1 320-374 Absent A96D 98 1 0 320-375 Absent A96N 108 -2 -1 320-376 Absent A96K 98 3 1 320-377 Absent A96L 101 -1 -1 320-378 Absent A96W 105 -1 -1 320-379 Absent A96Y 105 3 1 320-380 Absent L97A 104 1 1 320-381 Absent L97S 97 3 1 320-382 Absent L97Q 106 0 0

- 26 042971

320-383 Absent L97D 102 3 1 320-384 Absent L97H 98 3 2 320-385 Absent L97K 108 1 1 320-386 Absent L97W 104 1 0 320-387 Absent L97Y 97 AND 10 320-388 G26A Absent 110 14 13 320-389 G26S Absent 105 12 And 320-390 G26Q Absent 100 12 And 320-391 G26D Absent 106 14 13 320-392 G26H Absent 109 12 And 320-393 G26K Absent 100 12 And 320-394 G26L Absent 106 13 12 320-395 G26W Absent 104 13 12 320-396 G26Y Absent 104 12 AND 320-397 Y27A Absent 109 12 12 320-398 Y27S Absent 110 12 AND 320-399 Y27Q Absent 105 10 10 320-400 Y27D Absent 106 AND AND 320-401 Y27H Absent 100 AND 10 320-402 Y27L Absent 98 AND 10 320-403 Y27W Absent 84 AND 10 320-404 T28A Absent 84 AND 10 320-405 T28S Absent 79 10 9 320-406 T28Q Absent 82 8 7 320-407 T28D Absent 88 12 AND 320-408 T28N Absent 81 13 12 320-409 T28K Absent 83 10 10 320-410 T28L Absent 86 AND 10 320-411 T28W Absent 82 10 9 320-412 T28Y Absent 89 10 9 320-413 F29A Absent 89 9 8 320-414 F29S Absent 78 7 7 320-415 F29Q Absent 85 5 4 320-416 F29D Absent 81 9 8 320-417 F29H Absent 85 9 8 320-418 F29K Absent 85 10 9 320-419 F29L Absent 83 AND 9 320-420 F29W Absent 85 AND 10 320-421 F28Y Absent 81 9 8 320-422 T30A Absent 80 10 9 320-423 T30S Absent 86 10 10 320-424 T30Q Absent 86 AND 10 320-425 T30D Absent 89 14 12 320-426 TZON Absent 84 10 9 320-427 TZOK Absent 88 AND 10 320-428 T30L Absent 88 AND 10 320-429 T30W Absent 90 12 AND 320-430 T30Y Absent 85 6 6 320-431 S31A Absent 85 6 6

- 27 042971

Table 8

SPR experiment - run 6 anti-TL1A antibodies binding to TL1A

Antibody Replacement in VH regarding 320-179 Replacement in VL regarding 320-179 Capture level ( TL1A binding level (early) purified anti Binding level TL1A (late) [body 320-179 Absent Absent 449 111 102 320-432 S31Q Absent 466 116 106 320-433 S31D Absent 412 90 83 320-434 S31K Absent 473 125 113 320-435 S31L Absent 384 92 83 320-436 S31W Absent 525 129 118 320-437 S31Y Absent 501 119 109 320-438 Y32A Absent 468 126 116 320-439 Y32S Absent 464 119 108 320-440 Y32Q Absent 408 108 97 320-441 Y32D Absent 388 80 67 320-442 Y32H Absent 490 124 113 320- 443* Y32K Absent 438 114 103 320-444 Y32L Absent 458 107 95 320- 445* Y32W Absent 442 116 106 320-446 D33A Absent 469 18 8 320-447 D33S Absent 503 37 13 320-448 D33Q Absent 482 7 6 320-449 D33H Absent 483 76 59 320-450 D33K Absent 536 1 3 320-451 D33L Absent 497 19 8 320-452 D33W Absent 445 84 78 320-453 D33Y Absent 449 111 104 320-454 I34A Absent 189 43 37 320-455 I34S Absent 144 27 23 320-456 I34Q Absent 214 52 46 320-457 I34D Absent 55 6 4 320-458 I34H Absent 239 59 53 320-459 I34K Absent 93 20 17 320-460 I34L Absent 441 114 105 320-461 I34W Absent 465 87 82 320-462 I34Y Absent 373 58 52 320- 463* N35A Absent 462 102 101 320- 464* N35S Absent 600 130 124 320-465 N35Q Absent 476 92 78 320-466 N35D Absent 360 93 86 320-467 N35H Absent 350 44 24 320-468 N35K Absent 200 2 2 320-469 N35L Absent 315 69 61 320-470 N35W Absent 329 2 3 320-471 N35Y Absent 312 3 4 320-472 Absent S90L 467 23 10 320-473 Absent Y91D 453 69 55 320-474 Absent L95K 596 2 4 320-475 Absent L95W 727 8 8 320-476 Y27K Absent 560 128 118

- 28 042971

zgo477* S31H Absent 576 148 136 320-478 A60L Absent 379 85 77 320-479 A60W Absent 277 61 54 320-480 A60Y Absent 319 79 73 320-483 W50A Absent 492 4 6 320-484 W50S Absent 555 3 6 320-485 W50Q Absent 460 3 5 320-486 W50D Absent 178 1 2 320-487 W50H Absent 237 2 3 320-488 W50K Absent 293 1 2 320-489 W50L Absent 337 4 5 320-490 W50Y Absent 393 14 8 320-491 L51S Absent 356 85 76

* indicates the antibodies that were selected for the activity assay.

Table 9

SPR experiment - run 7 anti-TL1A antibodies binding to TL1A

Antibody Replacement in VH regarding 320-179 Replacement in VL regarding 320-179 Capture level TL1A binding level (early) TL1A binding level (late) Purified antibody 320-179 Absent Absent 301 61 56 320-492 L51Q Absent 439 89 82 320-493 L51D Absent 242 48 46 320-494 L51H Absent 811 150 138 320-495 L51K Absent 466 84 77 320-496 L51W Absent 581 93 87 320-497 N52A Absent 727 135 125 320-498 N52S Absent 485 90 82 320-499 N52Q Absent 551 122 FROM 320-500 N52D Absent 472 69 53 320-501 N52H Absent 533 118 108 320-502 N52K Absent 659 88 59 320-503 N52W Absent 484 41 6 320-504 N52Y Absent 500 88 65 320-505 R52aA Absent 570 118 110 320-506 P52aS Absent 444 97 89 320-507 P52aQ Absent 181 27 24 320-508 P52aD Absent 203 19 12 320-509 R52aH Absent 290 52 47 320-510 R52aK Absent 289 44 33 320-511 P52aL Absent 581 106 98 320-512 P52aW Absent 746 126 118 320-513 P52ay Absent 585 99 90 320-514 N53A Absent 516 96 88 320-515 N53S Absent 375 73 67 320-516 N53Q Absent 461 92 86 320-517 N53D Absent 493 55 50 320-518 N53H Absent 882 169 153 320-519 N53K Absent 993 217 196 320-520 N53L Absent 1016 174 162 320-521 N53W Absent 830 166 152 320-522 N53Y Absent 693 141 129

- 29 042971

320-523 S54A Absent 476 88 82 320-524 S54Q Absent 292 55 49 320-525 S54D Absent 437 33 17 320-526 S54H Absent 672 134 124 320-527 S54K Absent 578 146 136 320-528 S54L Absent 829 121 108 320-529 S54W Absent 605 94 83 320-530 S54Y Absent 425 77 67 320-531 G55A Absent 331 66 61 320-532 G55S Absent 648 10 7 320-533 G55Q Absent 441 93 86 320-534 G55D Absent 647 109 102 320-535 G55H Absent 637 126 115 320-536 G55K Absent 553 115 105 320-537 G55L Absent 717 133 123 320-538 G55W Absent 318 54 49 320-539 N56A Absent 859 172 159 320-540 N56S Absent 500 100 92 320-541 N56Q Absent 504 94 84 320-542 N56D Absent 839 97 86 320-543 N56H Absent 597 138 126 320-544 N56K Absent 690 120 112 320-545 N56L Absent 518 109 100 320-546 N56W Absent 341 67 61 320- 547* N56Y Absent 318 77 75 320-548 T57A Absent 335 74 68 320-549 T57S Absent 269 50 45 320-550 T57Q Absent 635 112 103 320-551 T57D Absent 280 44 36 320-552 T57H Absent 595 104 97 320-553 T57K Absent 494 84 78 320-554 T57L Absent 506 66 51 320-555 T57W Absent 674 62 thirty 320-556 T57Y Absent 634 100 90 320-557 G58A Absent 378 26 6 320-558 G58S Absent 463 3 2 320-559 G58Q Absent 535 9 5 320-560 G58D Absent 907 36 9 320-561 G58H Absent 539 14 6

- 30 042971

320-562 G58K Absent 326 2 1 320-563 G58L Absent 258 2 2 320-564 G58W Absent 345 72 67 320-565 G58Y Absent 545 43 12 320-566 Y59A Absent 720 120 110 320-567 Y59S Absent 590 96 86 320-568 Y59Q Absent 688 115 107 320-569 Y59D Absent 408 79 73 320-570 Y59H Absent 435 79 72 320-571 Y59K Absent 394 78 72 320-572 A60S Absent 459 98 90 320-573 A60Q Absent 338 67 62 320-574 A60D Absent 693 140 130 320-575 A60N Absent 581 121 110 320-576 A60K Absent 479 90 83 320-577 L51A Absent 479 98 90 320-578 L51Y Absent 415 83 77 320-579 N52L Absent 214 46 41 320-580 G55Y Absent 261 51 47 320-581 Y59L Absent 337 70 65 320-582 Y59W Absent 453 67 52

* indicates the antibodies that were selected for the activity assay.

Table 10

SPR experiment - run 8 - anti-TL1A antibodies binding to TL1A

Antibody Replacement in VH regarding 320-179 Replacement in VL regarding 320-179 Capture level TL1A binding level (early) TL1A binding level (late) Purified antibody 320179 Absent Absent 402 98 86 320- 583* Absent G30A, Y91W 404 108 103 320- 584* Absent L31S, Y91W 399 103 98 320- 585* Absent L31Q, Y91W 406 116 109 320- 586* Absent H34L, Y91W 405 108 103 320- 587* N56Y Y91W 402 123 122 320- 588* N56Y G30A 409 128 123 320- 589* N56Y L31S 399 120 109 320- 590* N56Y L31Q 405 130 121 320- 591* N56Y H34L 399 132 124 320- 592* N56Y G30A, Y91W 408 128 129 320- 593* N56Y L31S, Y91W 405 126 124 320- 594* N56Y L31Q, Y91W 407 134 132 320- 595* N56Y H34L, Y91W 403 129 128

* indicates the antibodies that were selected for the activity assay.

- 31 042971

Table 11

SPR experiment - run 9 anti-TL1A antibodies binding to TL1A

Antibody Replacement in VH regarding 320-179 Replacement in VL regarding 320-179 Capture level TL1A binding level (early) TL1A binding level (late) Purified antibody 320-179 Absent Absent 402 108 98 320-596 Absent Y91F 405 95 85 320-597 Absent L31Q, Y91F 401 115 105 320-598 Absent H34L, Y91F 403 112 101 320-599 Absent L31S, Y91F 411 FROM 103 320-600 Absent G30A, Y91F 399 111 104 320- 601* N56Y Y91F 409 121 120 320-602 N56Y L31Q, Y91F 399 131 125 320-603 N56Y H34L, Y91F 394 126 118 320-605 N56Y G30A, Y91F 399 125 120 320- 611* N56Y Y91W, G93K 397 125 127 320-612 Absent Y91W, G93Y 400 126 120 320- 613* N56Y Y91W, G93Y 408 135 131 320-614 N56Y G93K 401 122 111 320-615 N56Y G93Y 393 FROM 108 320-616 N56Y G93A 396 120 112

* indicates the antibodies that were selected for the activity assay.

Antibodies that had capture levels similar to or better than 320-179, as well as TL1A binding level (early) and TL1A binding level (late) values that were in a similar range, were taken for activity analysis. These options are indicated by shading in Table. 3-11. A comparison of dissociation rates measured by SPR for several antibodies is shown in FIG. 5. Some of the antibodies dissociated at a slower rate than the parent antibody 320-179.

2.2. Anti-TL1A antibodies with improved activity in cell assay.

To determine if the improved dissociation rate correlates with increased activity of the purified antibody, the variants were examined in a TL1A-induced caspase activity assay in TF-1 cells. Effective antibodies act by binding to TL1A and inhibit TL1A activation of the DR3 receptor. This receptor triggers an apoptotic pathway in which caspases are activated and can be detected using commercial reagents. In each experiment, the antibody variant was compared with 320-179 fold improvement in activity. The results are shown in table. 12.

Table 12

Analysis of TL1A-induced caspase activity in TF-1 cells. Inhibition by anti-TL1A antibodies

Antibody Replacement in VH regarding 320-179 Replacement in VL regarding 320-179 IR ₅₀ antibodies (µg/ml) IR ₅₀ 320-179 (µg/ml) Multiplicity of improvement 320-184 Absent L79A 0.010 0.030 3 320-185 Absent L79S 0.020 0.030 2 320-186 Absent L79Q 0.030 0.030 1 320-187 Absent L79D 0.020 0.040 2 320-188 Absent L79H 0.010 0.040 4 320-189 Absent L79K 0.110 0.040 0 320-190 Absent L79W 0.020 0.040 2 320-191 Absent L79Y 0.020 0.040 2 320-198 Absent T24L 0.010 0.010 1

- 32 042971

320-201 Absent S25A 0.005 0.010 2 320-202 Absent S25Q 0.010 0.010 1 320-205 Absent S25K 0.020 0.030 2 320-207 Absent S25W 0.007 0.020 3 320-209 Absent S26A 0.040 0.020 1 320-211 Absent S26D 0.010 0.030 3 320-213 Absent S26K 0.070 0.030 0 320-219 Absent S27D 0.020 0.030 2 320-226 Absent S27aQ 0.030 0.020 1 320-229 Absent S27aK 0.040 0.060 2 320-241 Absent I27cA 0.020 0.010 1 320-244 Absent I27cD 5.030 0.010 0 320-245 Absent I27CH 0.540 0.010 0 320-263 Absent A29K 0.003 0.010 3 320-267 Absent G30A 0.010 0.040 4 320-277 Absent L31S 0.003 0.010 3 320-278 Absent L31Q 0.010 0.020 2 320-280 Absent L31H 0.006 0.020 3 320-297 Absent V33H 0.005 0.020 4 320-302 Absent H34A 0.010 0.020 2 320-307 Absent H34L 0.010 0.040 4 320-313 Absent Q89H 0.020 0.030 2 320-314 Absent Q89K 0.020 0.030 2 320-318 Absent S90A 0.020 0.050 3 320-326 Absent Y91S 0.030 0.050 2 320-328 Absent Y91H 0.020 0.020 1 320-331 Absent Y91W 0.002 0.020 10 320-345 Absent G93K 0.020 0.010 1 320-361 Absent L95D 0.010 0.010 1 320-443 Y32K Absent 0.060 0.060 1 320-445 Y32W Absent 0.030 0.060 2 320-463 N35A Absent 0.020 0.050 3 320-464 N35S Absent 0.030 0.050 2 320-477 S31H Absent 0.030 0.040 1 320-547 N56Y Absent 0.004 0.040 10 320-583 Absent G30A, Y91W 0.003 0.080 27 320-584 Absent L31S, Y91W 0.008 0.080 10 320-585 Absent L31Q, Y91W 0.003 0.040 13 320-586 Absent H34L, Y91W 0.002 0.050 25 320-587 N56Y Y91W 0.001 0.040 40 320-588 N56Y G30A 0.004 0.020 5 320-589 N56Y L31S 0.020 0.020 1 320-590 N56Y L31Q 0.006 0.020 3 320-591 N56Y H34L 0.002 0.020 10 320-592 N56Y G30A, Y91W 0.003 0.020 7 320-593 N56Y L31S, Y91W 0.004 0.060 15 320-594 N56Y L31Q, Y91W 0.005 0.060 12 320-595 N56Y H34L, Y91W 0.003 0.060 20 320-601 N56Y Y91F 0.002 0.020 10 320-611 N56Y Y91W, G93K 0.005 0.040 8 320-613 N56Y Y91W, G93Y 0.010 0.040 4

** For several antibodies with a fold improvement greater than a 10-fold increase in activity, up to n=7 analyzes were performed. The results were consistent with the data given in this table. In FIG. 7 shows n=4 repeats of several antibodies with improved activity compared to 320-179.

As shown in Table. 12 and in FIG. 6, some of the single substitution antibodies tested have superior activity compared to 320-179. Of all single substitution antibody variants tested, two had more than a 10-fold increase in potency compared to 320-179. These variants were 320-331 (which contained the Y91W substitution in the light chain variable region) and 320-547 (which contained the N56Y substitution in the heavy chain variable region) (FIG. 6). This result is unexpected because, as a rule, the VH CDR3 of the antibody is predominantly involved in antibody binding, while, conversely, the N56Y substitution has a significant effect on binding to the VH CDR2 of the antibody. When variants were made with Y91W from VL or N56Y alone in VH with other substitutions that improved activity, highly active antibodies were obtained. When the Y91W change in VL was combined with the N56Y change in VH in one antibody, 320-587, the fold increase in activity compared to 320-179 was 40. FIG. 7 shows four different replicate experiments demonstrating an increase in the activity of four antibodies 320-587, 320-591, 320-592 and 320-601 compared to

320-179. The sequences of these antibodies with increased activity compared to 320-179 are shown in FIG. 3 (heavy chain variable region) and FIG. 4 (light chain variable region).

The activity of 320-587 was compared with other previously described antibodies against TL1A. These previously described antibodies include the 1681N antibody described in US Pat. ID NO: 24, VL is SEQ ID NO: 17), humanized 1B4 as described in US Pat. No. 8,263,743 (VH is SEQ ID NO: 74, VL is SEQ ID NO: 75) and referred to as C320-168) as described in US Publication No. 2014/0255302A1 (VH is SEQ ID NO: 181, VL is SEQ ID NO: 194). In FIG. 8 shows that 320.587 has superior cell-based assay performance over these previously described antibodies, making it the most effective anti-TL1A antibody.

2.3. Analysis of competitive binding to DR3 and DcR3 receptors.

Antibodies that showed increased activity compared to 320-179 were screened for their ability to inhibit TL1A binding to its DR3 signaling receptor or DcR3 decoy receptor. All anti-TL1A antibodies tested showed inhibition of TL1A binding to DR3 compared to the isotype control (FIG. 9). This confirms that the antibodies inhibit TL1A activity by blocking the TL1A-DR3 interaction.

In previous experiments described in US Publication No. 2014/0255302A1 (Example 4), antibody 320-179 (320-179) was tested in a competitive receptor binding assay and shown to selectively inhibit TL1A binding to DR3 but not DcR3 ( Fig. 10). In the experiments presented herein in FIG. 10 again shows that 320-179 does not inhibit TL1A-DcR3 interaction. This is in contrast to the improved anti-TL1A antibodies tested. Antibodies with improved activity in the TF-1 cell assay (as described in section 2.2), including antibody 320-587, consistently inhibited TL1A-DcR3 interaction.

Thus, the parent antibody 320-179 was able to inhibit the TL1A-DR3 interaction but not the TL1A-DcR3 interaction. Several antibodies with improved potency such as 320-267 (VH is SEQ ID NO: 1, VL is SEQ ID NO: 11), 320-277 (VH is SEQ ID NO: 1, VL is SEQ ID NO: 1 : 12), 320-278 (VH is SEQ ID NO: 1, VL is SEQ ID NO: 13) and 320-591 (VH is SEQ ID NO: 3, VL is SEQ ID NO: 9) inhibits interaction TL1a-DR3 but not TL1A-DcR3 interaction. Several antibodies with enhanced activity such as 320-331, 320-547, 320-583, 320-584, 320-585, 320-586, 320-587 and variants of these antibodies are able to inhibit both TL1A-DR3 and TL1A-DcR3.

2.4. The species cross-reactivity of 320-587 was tested for their ability to bind to recombinantly produced TL1A from different species. The antibody bound TL1A of all tested species (Fig. 11). Binding of 320-587 to human, rat, guinea pig, dog, cat, cynomolgus monkey TL1A had a slow dissociation rate, indicating a high affinity interaction. The antibody bound mouse and rabbit TL1A and had a fast dissociation rate.

2.5. Preclinical performance models for testing anti-TL1A antibodies in the following animal models of the disease.

Asthma - allergen-induced asthma. A rodent (mouse, rat, or guinea pig) was sensitized with an intradermal injection of ovalbumin (OVA), especially egg-derived OVA supplemented with alum, and then stimulated at least 2 weeks later by spraying with nebulized OVA, causing asthma symptoms, including respiratory hyperreactivity. pathways, eosinophil influx and increased cytokine production (eg, Hylkema et al., 2002, Clin. Exp. Immunol., 129:390-96). Such a pattern can be modified by restimulation to provide a more chronic disease profile with increased airway remodeling and fibrosis induction (eg, Bos et al., 2007, Eur. Respir. J., 30:653-661). Alternative allergens such as dust mites can also be used (eg, Lambert et al., 1998, Am. J. Respir. Crit. Care Med., 157:1991-9). Alternatively, a non-human primate (e.g., cynomolgus monkey) can be sensitized and challenged with an exogenous antigen such as Ascaris suum, resulting in airway hyperreactivity, eosinophil influx, and increased cytokine production (e.g., Wegner et al., 1991, J. Allergy Clinic Immunol., 87:835-41).

COPD is an inflammation of the airways caused by smoke. A rodent (mouse, rat, or guinea pig) will be exposed to cigarette smoke 3-7 times a week for at least 4 weeks, resulting in a pulmonary disease similar to COPD, characterized by accumulation of neutrophils in the lungs, increased production of inflammatory cytokines, fibrosis lungs and pulmonary hypertension (eg, Davis et al. (2012), PLoS One, 7:e33304). A more severe form of the disease may be caused by re-introduction of a bacterial or viral infection into the lungs during smoke exposure (eg, Li et al. (2012), Biol. Pharm. Bull., 35:1752-60). Rodents with smoke-induced COPD will be treated with anti-TL1A antibodies and tested for treatment efficacy.

Pulmonary fibrosis - bleomycin-induced pulmonary fibrosis. The rodent (mouse, rat or guinea pig) will be treated with bleomycin either by intratracheal/nasal instillation or by intravenous injection once or twice a week for at least 3 weeks. This technique induces significant and stable pulmonary fibrosis (eg, Pinart et al. (2009), Resp. Physiol., Neurobiol., 166:41-46). Rodents with bleomycin-induced pulmonary fibrosis will be treated with anti-TL1A antibodies and will be tested for treatment efficacy.

Cystic fibrosis is a CFTR (transmembrane regulator of cystic fibrosis) knockout ferret model. Ferrets homozygous for a knockout gene or with known mutations associated with the disease, CFTR (the gene that causes cystic fibrosis), spontaneously develop a cystic fibrosis-like disease characterized by airway mucosal obstruction, atelectasis, interstitial pneumonia, and recurrent lung infections with progressive bacterial infection. lung colonization (e.g., Sun et al. (2014), Am. J. Respir. Cell Mol. Biol., 50:502-12). CFTR’/’ ferrets will be treated with anti-TL1a antibodies and will be tested for effectiveness of the treatment.

Irritable bowel syndrome - stress-induced visceral hypersensitivity. Stress will be induced in rats by either separation of the neonate from the mother (eg, Coutinho et al. (2002), Am. J. Physiol. Gastrointest. Liver Physiol., 282:G307-16), or limitation of adult motor functions (eg, Shen et al. al (2010) J Neurogastroenterol Motil 16:281-90). This is expected to result in altered colonic motility and visceral hypersensitivity similar to that seen in IBS patients. Stressed rats will receive anti-TL1A antibody treatment and will be tested for effectiveness of the treatment.

Rheumatoid arthritis - collagen-induced arthritis. The rodent (mouse, rat or guinea pig) will be immunized and stimulated with collagen in the adjuvant. Animals develop bilateral leg edema and erythema, joint inflammatory infiltrate, and joint damage (eg, Bendele et al. (1999) Toxicol. Pathol., 27:134-42). Rodents with collagen-induced arthritis will be treated with anti-TL1A antibodies and will be tested for the effectiveness of the treatment.

Eosinophilic esophagitis is intranasal eosinophilic esophagitis induced by Aspergillus fumigatus. Mice subjected to repeated intranasal instillation of A. fumigatis develop marked esophageal eosinophilia, epithelial dysplasia and hyperplasia, and free eosinophilic granules (eg, Mishra et al. (2001), J. Clin. Invest., 107:83-90). Similarly, repeated exposure to ovalbumin aerosol for two weeks in sensitized guinea pigs causes esophageal eosinophilia with infiltration of both eosinophils and mast cells into the epithelial layer (e.g., Liu et al. (2015), Am. J. Physiol. Gastrointest. Liver Physiol., 308:G482-488). Mice or guinea pigs with eosinophilic esophagitis will be treated with anti-TL1A antibodies and tested for efficacy.

2.6. Use of anti-TL1A antibodies in detecting samples containing TL1A, the antibodies of the disclosure can be used to detect TL1A in human samples. 320-587 were used to detect human TL1A secreted from immune complex stimulated human PBMCs (FIG. 13) by ELISA. 320-587 were also used to detect populations of human PBMCs that express membrane TL1A on their surface in flow cytometry experiments (FIG. 14).

2.7. Determination of the binding affinity of an anti-TL1A antibody to human TL1A using a kinetic exclusion assay.

Time to reach equilibrium from 320-587 as SVR. First, the K _on rate for the 320-587/TL1A interaction was measured. Briefly, a solution was prepared by mixing 320-587 and TL1A and aliquots removed at various times over 3 hours. Free 320-587 was captured by passing the solution through a column packed with Sepharose beads coated with 20 μg/ml TL1A. Captured 320-587 was detected using Alexa Fluor® 647-conjugated anti-human immunoglobulin antibody (0.5 μg/ml). This analysis was repeated twice, yielding K _on rates of 8.35x105 and 7.45x105 Ms' ¹ with an average Kon rate of 7.90x105 Ms' ¹ . The Kon rate was then used to estimate the time required to reach equilibrium at various concentrations of 320-587 using the Theoretical Binding Curve tool provided on the Sapidyne website (www. Sapidyne.com).

Determination of K _D using 320-587 as CBP. CBP, 320-587, were diluted in assay buffer (DPBS (Dulbecco's phosphate buffered saline) supplemented with 1 mg/ml BSA (bovine serum albumin)) to final concentrations of 15, 50 and 150 pM. Human TL1A titrant was diluted in assay buffer to create a series of concentrations of 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 and 3000 pM. Using the equilibration time defined above, curves that contained either 50 or 150 pM 320-587 were allowed to equilibrate in an incubator at 25° C. for 2 days. Curves that

- 35 042971 contained 15 pM 320-587, allowed to equilibrate in an incubator at 25°C for 3 days.

After an equilibration period, the free fraction 320-587 in each reaction was quantified as described above. KD values were determined using n-curve analysis of equilibrium curves generated with 15, 50 and 150 pM 320-587.

Time to reach equilibrium with TL1A as CVR. The time to equilibrate in this orientation was estimated using K _on for the 320-587/TL1A interaction determined as described in 2.8 above. In this format, the free fraction of TL1A was captured by passing the solution through a column packed with PMMA beads coated with 30 μg/ml 320-587. Captured TL1A was detected using anti-6x-his DyLight 650 antibody (0.75 μg/ml). This analysis was repeated two times, obtaining speeds K _o n 6.11x105 and 5.74x105 Ms ^-1 with an average speed K _on 5.93x105 Ms ^-1 .

Determination of K _D using TL1A as CVR. CBP, human TL1A, was diluted in assay buffer to final concentrations of 30, 100 and 300 pM. Titrant 320-587 was diluted in assay buffer to create a series of concentrations of 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 pM.

Using the equilibration time defined above, all curves were allowed to equilibrate in an incubator at 25° C. for 3 days. After an equilibration period, the free fraction of TL1A in each reaction was quantified as described above. K _D values were determined using n-curve analysis of equilibrium curves generated with 30, 100 and 300 pM TL1A.

Good agreement was observed for the two KinExA methods, as well as a relatively low % error. The K _D value for the interaction of TL1A with 320-587, determined using 320-587 as the CBP, was 40.97 ± 8.33 pM (Table 13), while the K _D obtained using TL1A as the CBP was 41.52±13.5 pM (Table 14).

Table 13

Affinity. Sepharose beads coated with TL1A; 320-587 as SVR

Analysis No. K _D (pM) % error 1 40.43 4.57 2 52.03 5.61 3 46.31 4.76 4 32.42 3.74 5 33.66 3.9 Average 40.97 SD (standard deviation) 8.33 % CV (Coefficient of Variation) 20

Table 14

Affinity. PMMA balls coated 320-587; TL1A as SVR

Analysis No. K _D (pM) % error 1 33.39 5.33 2 57.1 5.51 3 34.07 3.9 Average 41.52 SD (standard deviation) 13.5 % CV (Coefficient of Variation) 33

.0. Preclinical performance models for testing antibodies against TL1a.

.0.1. Asthma.

Acute ovalbumin-induced asthma in rats. Gray rats were sensitized with OVA by i/p injection on day 0 and then stimulated with OVA aerosol daily on days 35-42. Rats were treated with antibody 320-587 or vehicle by iv injection on days 14, 21, 28 and 35. Bronchoalveolar lavage fluid (BALF) was assessed for total and differentiated cells on the 43rd day. Treatment was found to significantly reduce the number of eosinophils in the BAL (FIG. 15).

Chronic ovalbumin-induced asthma in rats. Rats were sensitized with alum-supplemented OVA by i/p injection on days 0 and 7, and then stimulated with OVA aerosol twice a week for 3 weeks from days 14 to 31, and 5 consecutive days from 37 to 31. 42 days. Animals were treated with antibody 320-587 or vehicle by intravenous injection on days 24, 29, 34 and 39. JBAL was assessed for total and differentiated cells and a panel of cytokines on the 43rd day. Lung sections were stained with hematoxylin and eosin (H&E) and schiff-iodic acid (PAS) and evaluated for a number of pathologies. Treatment with 320-587 significantly reduced eosinophils and macrophages in BALF (Figs. 17A and 17B), IL-4 and IL-13 in BALF (Fig. 17C), goblet cell hyperplasia (Fig. 17D), and bronchial epithelial layer thickness (Fig. 17E). compared to the carrier.

Acute ovalbumin-induced asthma in guinea pigs. Male Dunkin Hart-36 042971 ley guinea pigs were sensitized to ovalbumin and underwent surgery to insert a balloon catheter to measure lung function and early and late asthmatic reactions. On days 16, 20, 24, and 28, animals received i/p injections with 320-587 antibody or vehicle. Aerosol stimulation with ovalbumin (0.05-0.1%) was performed 30 minutes after the last treatment. Airway reactivity (AHR) to histamine was measured 24 hours before stimulation, 6 hours after stimulation (immediately after an early asthmatic response) and 24 hours after stimulation (immediately after a late asthmatic response). The nature and duration of early and late asthmatic reactions were also recorded by online lung function recording over the entire 24-hour period. Animals were sacrificed 25 h after stimulation and bronchoalveolar lavage was performed. JBAL was evaluated for common and differentiated cells. Treatment with 320-587 significantly reduced both eosinophils and macrophages in the BAL (FIGS. 16A and 16B), as well as improvement in AHR after early asthmatic response (FIG. 16C) and overall magnitude of early asthmatic response (FIG. 16D) by compared to the carrier.

Chronic ovalbumin-induced asthma in guinea pigs. Male Dunkin Hartley guinea pigs were sensitized with ovalbumin and ovalbumin stimulated weekly for 12 weeks 4 weeks thereafter. Stimulation with ovalbumin (0.05-0.5%) was performed by inhalation of an aerosol solution until airway obstruction was determined. Animals were treated with antibody 320-587 or vehicle by i/p injections every 5 days starting from the 8th week of ovalbumin stimulation. Airway function by airway reactivity to histamine was measured before the initial stimulation, 24 hours before the final stimulation, and 6 hours after the final stimulation. Although no effect on AHR induced by histamine stimulation was observed, antibody 320-587 significantly reduced the allergic response to OVA, as gradually increasing doses of OVA were needed to induce airway obstruction (FIG. 18).

The differences in antibody therapeutic effect observed in guinea pig models of acute and chronic asthma are thought to be a consequence of the model itself. It is believed that in the chronic model, the degree of AHR decreases with time and, accordingly, becomes less responsive to treatment. In this field, the acute model is usually used to observe the complex effects of airway reactivity, and the chronic model is usually used to observe the complex effects of airway remodeling. Remodeling evaluations are ongoing. However, it was surprising to observe antibodies having an effect on the allergen response, although at this stage the antibody had little to no effect on the absolute AHR (histamine response), this greatly reduced the direct allergic response to the antigen.

3.0.2. Inflammatory bowel disease.

TNBS-induced colitis in rats. Rats received a single dose of trinitrobenzenesulfonic acid in ethanol by intrarectal instillation. Control animals received an equivalent volume of ethanol. Within 7 days, the animals developed focal colitis characterized by colonic ulceration with an inflammatory infiltrate and varying degrees of fibrosis (eg, in Wirtz et al. (2007), Nat. Protoc., 2:541-546). Administration of 320-587 significantly reduced multiple symptoms of the disease, including colonic thickness (Fig. 12A), number and severity of adhesions (Fig. 12B), and number and severity of strictures (Fig. 12C), resulting in a significantly milder course of the disease. than in animals treated with either vehicle or coupled to an isotype-like irrelevant antibody (FIG. 12D). Reduced colonic fibrosis (FIG. 19) was also observed in animals treated with 320-587.

Comparison of disease after 7 and 14 days in DNBS-induced colitis in rats. Colitis was induced as described above using dinitrobenzenesulfonic acid (DNBS) instead of TNBS, and the rats used in the DNBS experiments were Wistar rats. Animals were treated with antibody 320-587 or vehicle by intravenous injection on days 1 and 8. Groups were assessed for colitis on days 7 and 14 after DNBS administration and disease severity between two time points. Treatment with antibody 320-587 had limited effect on day 7, but on day 14, animals treated with antibody 320-587 showed significant improvement in colonic mass and length (Fig. 20A), fibrosis (Fig. 20B) , inflammatory infiltrate (Fig. 20C), and colonic injury (Fig. 20D). Representative sections of a colonic ulcer (FIG. 20E) demonstrate the extent of damage repair and fibrosis reduction after 14 days. On the 7th and 14th days, the animals treated with the vehicle had an extensive inflammatory infiltrate and fibrosis with a significant loss of intestinal architecture. In contrast, animals treated with 320-587 showed significant inflammatory infiltrate, fibrosis, and loss of intestinal architecture by day 7, but these effects were largely reversed over 14 days.

The differences observed in the therapeutic effects of the antibodies observed in the TNBS and DNBS models are believed to have arisen due to the use of different strains of rats (Sprague-Dawley for TNBS vs. Wistar for DNBS). Each line of rats has differences in their responses to immunization, so it is believed that the kinetics of their response in these models is different. Also, TNBS and DNBS are different

- 37 042971 structurally and are believed to cause changes in the course of the disease.

Chronic (21-day) DSS-induced colitis in rats. Rats were given dextran sodium sulfate (DSS) at a concentration of 5% w/v. in drinking water for 7 days, then 2% wt./about. in drinking water for another 14 days. The animals developed diarrhea, diffuse inflammation of the colon, goblet cell hyperplasia, and damage to the crypt and ulcer epithelium (eg, Randhawa et al. (2014), Korean J. Physiol. Pharmacol., 18:279-88). Rats were treated with antibody 320-587 or vehicle by intravenous injection on days 5, 12 and 19. Animals were weighed and assessed for clinical manifestations of the disease (diarrhea and occult blood) daily, and the weight and length of the colon was assessed on day 21. Treatment with antibody 320-587 significantly altered DSS-induced weight gain retardation (FIG. 21A), improved clinical symptoms of the disease (FIG. 21B), and improved colonic mass and length (FIG. 21C).

Induction of intraperitoneal cytokines with recombinant human TL1A. Intraperitoneal injection of recombinant mouse TL1A can induce the production of inflammatory cytokines such as IL-5. In this study, mice received a single dose of either 320-587 antibody or vehicle, and an hour later received 40 μg/mouse of recombinant human TL1A (rhTL1A). Six hours after administration of rhTL1A, peritoneal lavage was obtained and peritoneal fluid was assessed for cytokines and chemokines by multiplex analysis. Treatment with antibody 320-587 significantly reduced peritoneal concentrations of the cytokines G-CSF (granulocyte colony stimulating factor), IL-1b, IL-5, IL-6, IL-17 and chemokines IP-10, KC (keratinocyte chemokine), MHB-1 ( monocytic chemoattractant protein-1), MMB-1a, MMB-1b, MMB-2 (MBV - macrophage inflammatory protein) (Fig. 22).

The disclosure of the invention is not limited to the embodiments described and illustrated by the examples above, but is open to changes and modifications within the scope of the appended claims.

Claims (47)

1. Recombinant antibody containing a heavy chain variable region containing a CDR1 containing the amino acid sequence of SEQ ID NO: 15, a CDR2 containing the amino acid sequence of SEQ ID NO: 28, a CDR3 containing the amino acid sequence of SEQ ID NO: 17, and a light chain variable region , containing a CDR1 containing the amino acid sequence of SEQ ID NO: 29, a CDR2 containing the amino acid sequence of SEQ ID NO: 19, and a CDR3 containing the amino acid sequence of SEQ ID NO: 30, provided that the amino acid sequences of the heavy chain variable region and the light chain variable region the chains are not simultaneously the amino acid sequence of the heavy chain variable region of SEQ ID NO: 1, containing the indicated sequences of the indicated heavy chain variable region CDR1, CDR2, CDR3, and the light chain variable region sequence of SEQ ID NO: 2, containing the indicated sequences of the indicated CDR1, CDR2, CDR3 of the variable region of the light chain, and the specified antibody specifically binds to TNF-like ligand 1A (TL1A). 2. The recombinant antibody of claim 1, wherein the heavy chain variable region CDR2 contains the amino acid sequence of SEQ ID NO: 21, the light chain variable region CDR1 contains the amino acid sequence of SEQ ID NO: 18, and the light chain variable region CDR3 contains the amino acid sequence SEQ ID NO: 22. 3. A recombinant antibody according to claim 1 or 2, containing a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3. 4. A recombinant antibody according to claim 1 or 2, containing a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. 5. The recombinant antibody of claim 1, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. 6. A recombinant antibody according to any one of claims 1 to 3, containing a heavy chain variable region comprising tryptophan (W) at residue 91. 7. The recombinant antibody of claim 1, wherein (a) the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 22; (b) the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 21, and the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 20; (c) The heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 16, the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 23, and the light chain variable region CDR3 comprises the amino acid sequence - - 38 042971 SEQ ID NO: 22; (d) The heavy chain variable region CDR2 includes the amino acid sequence SEQ ID NO: 16, Light chain variable region CDR1 includes the amino acid sequence SEQ ID NO: 24, a light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 22; (e) The heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 16, the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 22; (f) The heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 16, the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 22; (g) The heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 21, the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 24, and the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 22; (h) the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 21, the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 22; (i) the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 21, the light chain variable region CDR1 comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 22; or (j) the heavy chain variable region CDR2 comprises the amino acid sequence of SEQ ID NO: 21 and the light chain variable region CDR3 comprises the amino acid sequence of SEQ ID NO: 27. 8. A recombinant antibody that specifically binds to TNF-like ligand 1A (TL1A), wherein said antibody comprises a heavy chain variable region and a light chain variable region, wherein (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1, and the light chain variable sequence comprises the amino acid sequence of SEQ ID NO: 4; (b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable sequence comprises the amino acid sequence of SEQ ID NO: 2; (c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable sequence comprises the amino acid sequence of SEQ ID NO: 5; (d) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable sequence comprises the amino acid sequence of SEQ ID NO: 6; (e) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable sequence comprises the amino acid sequence of SEQ ID NO: 7; (f) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 and the light chain variable sequence comprises the amino acid sequence of SEQ ID NO: 8; (g) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable sequence comprises the amino acid sequence of SEQ ID NO: 6; (h) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable sequence comprises the amino acid sequence of SEQ ID NO: 7; (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable sequence comprises the amino acid sequence of SEQ ID NO: 8; or (j) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable sequence comprises the amino acid sequence of SEQ ID NO: 10. 9. A recombinant antibody according to any one of claims 1 to 8, characterized in that it is capable of inhibiting - 39 042971 interaction of TL1A with death receptor 3 (DR3). 10. A recombinant antibody according to any one of claims 1 to 9, characterized in that the antibody is a monoclonal antibody. 11. A recombinant antibody according to any one of claims 1 to 10, characterized in that the antibody contains a human IgG1 heavy chain constant region. 12. The recombinant antibody of claim 11, wherein the human IgG1 heavy chain constant region comprises SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 66. 13. A recombinant antibody according to any one of claims 1 to 10, characterized in that the antibody contains a human IgG4 heavy chain constant region. 14. The recombinant antibody of claim 13, wherein the human IgG4 heavy chain constant region comprises SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 69. 15. A recombinant antibody according to any one of claims 1 to 8, characterized in that the antibody contains a human IgG2 heavy chain constant region. 16. The recombinant antibody of claim 15, wherein the human IgG2 heavy chain constant region comprises SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 71. 17. A recombinant antibody according to any one of claims 1 to 16, characterized in that the antibody contains a light chain constant region of a human lambda antibody. 18. The recombinant antibody of claim 17, wherein the light chain constant region of the human lambda antibody contains SEQ ID NO: 48. 19. Recombinant antibody according to claim 17, characterized in that the antibody contains a light chain containing the amino acid sequence of SEQ ID NO: 61. 20. A recombinant antibody according to claim 11 or 19, characterized in that the recombinant antibody contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 60. 21. A recombinant antibody according to any one of claims 1 to 20, wherein the K _D for TL1A is from about 30 pM to about 60 pM, as measured by a kinetic exclusion assay. 22. A recombinant antibody according to any one of claims 1 to 21, wherein the K _D for TL1A is from about 35 pM to about 50 pM, as measured by a kinetic exclusion assay. 23. A recombinant antibody according to any one of claims 1 to 22, wherein the K _D for TL1A is from about 35 pM to about 42 pM, as measured by a kinetic exclusion assay. 24. A recombinant antibody according to any one of claims 1 to 23, wherein TL1A is human TL1A. 25. A recombinant antibody according to any one of claims 1 to 23, wherein TL1A is a non-human primate TL1A. 26. A recombinant antibody according to any one of claims 1 to 23, wherein TL1A is non-human mammalian TL1A. 27. The recombinant antibody of claim 26, wherein the non-human mammal is a mouse, rat, rabbit, guinea pig, cat, dog, or pig. 28. A TL1A binding composition comprising a recombinant antibody according to any one of claims 1-27 and a pharmaceutically acceptable carrier. 29. The use of a recombinant antibody according to any one of claims 1 to 27 for the treatment of a respiratory disease. 30. The use of claim 29 wherein the respiratory disease is asthma, COPD, lung sarcoidosis, allergic rhinitis, or pulmonary fibrosis. 31. The use of a recombinant antibody according to any one of claims 1 to 27 for the treatment of cystic fibrosis. 32. The use of a recombinant antibody according to any one of claims 1 to 27 for the treatment of a disease of the gastrointestinal tract. 33. Use according to claim 32, wherein the gastrointestinal disease is inflammatory bowel disease, colitis or ulcerative colitis, irritable bowel syndrome, or Crohn's disease. 34. Use according to claim 32, wherein the gastrointestinal disease is eosinophilic esophagitis. 35. Use according to claim 31 or 32 for the treatment of gastrointestinal disease associated with cystic fibrosis. 36. The use of a recombinant antibody according to any one of claims 1 to 27 for the treatment of arthritis or rheumatoid arthritis. 37. The use of a recombinant antibody according to any one of claims 1 to 27 for the treatment of a skin disease. 38. Use according to claim 37, wherein the skin disease is atopic dermatitis, eczema, or scleroderma. 39. A transformed cell that expresses an antibody according to any one of claims 1 to 27, wherein the cell contains an expression vector containing a polynucleotide encoding said antibody. 40. The transformed cell according to claim 39, characterized in that the cell is a cell - 40 042971 mammal. 41. A transformed cell according to claim 39, wherein the mammalian cell is a Chinese hamster ovary cell. 42. A transformed cell according to claim 39, wherein the mammalian cell is a HEK 293 cell. 43. The use according to any one of claims 29-38, characterized in that the antibody is intended for intravenous or subcutaneous administration. 44. A polynucleotide encoding a heavy chain variable region of an antibody according to claim 1, comprising a nucleic acid sequence encoding a heavy chain variable region of an antibody according to claim 1, and this heavy chain variable region contains the amino acid sequence of SEQ ID NO: 3. 45. The polynucleotide of claim 44, wherein the polynucleotide further comprises a second nucleic acid sequence encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 4. 46. An expression vector containing a polynucleotide according to claim 44 or 45. 47. A transformed cell for producing an antibody heavy chain variable region containing the amino acid sequence of SEQ ID NO: 3 and an antibody light chain variable region containing the amino acid sequence of SEQ ID NO: 4, wherein said cell contains the vector of claim 46 containing the polynucleotide of item 45.