Antibody binding to TL1a, and use thereof

Abstract

The present disclosure relates to an antibody binding to TL1A or an antigen-binding fragment thereof, and the use thereof in the treatment of inflammatory bowel disease.

Description

Antibodies combining TL1A and their applications

[0001] This application claims priority to Chinese Patent Application No. 202411288205.4, filed on September 14, 2024, entitled "Antibody Conjugated to TL1A and Its Application Thereof". The contents of that application are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the field of biomedicine. Specifically, it relates to a monoclonal antibody or an antigen-binding fragment thereof that binds to tumor necrosis factor-like cytokine 1A (TL1A), and its pharmaceutical uses. Background Technology

[0003] Inflammatory bowel disease (IBD) is a group of chronic diseases involving persistent inflammation of the intestine or its lining. Its main forms include ulcerative colitis (UC) and Crohn's disease (CD). Common symptoms of IBD include fatigue, decreased appetite, occasional fever, irregular bowel movements containing mucus and / or blood, severe diarrhea, abdominal pain, occult or hidden bleeding, and possible anemia. The pathophysiology of IBD involves complex genetic, environmental, epithelial, microbial, and immune factors. While ulcerative colitis lesions are confined to the colon and present as superficial mucosal inflammation, Crohn's disease can affect any part of the digestive tract, with lesions typically discontinuous and characterized by transmural inflammation. The recurrent flare-ups and symptoms of IBD severely impact patients' daily lives and quality of life.

[0004] Tumor necrosis factor-like cytokine 1A (TL1A, also known as TNFSF15) is a member of the tumor necrosis factor family. It is a type II transmembrane protein that self-assembles into a stable trimer through interaction with the TNF homologous domain (THD). It is primarily expressed in monocytes, macrophages, dendritic cells, T cells, and non-immune cells such as synovial fibroblasts and endothelial cells. It plays a role in various biological processes, including apoptosis, inflammatory responses, and the development of immune cells. TL1A can bind to its receptor DR3 (Death Receptor 3), thereby activating the apoptosis signaling pathway and participating in the regulation of cell death and survival.

[0005] In inflammatory bowel disease (IBD), TL1A binds to death receptor 3 (DR3), activating the TRADD pathway and subsequently regulating downstream pathways such as TRAF2, RIP1, PI3K, MAPKs, and NF-κB, exerting a pro-inflammatory effect and participating in the proliferation, activation, and apoptosis of immune cells, as well as the production of cytokines and chemokines. The TL1A / DR3 signaling pathway plays a crucial role in the pathogenesis of IBD. Studies have shown that TL1A is abnormally expressed in IBD patients, and its elevated levels are associated with the inflammatory activity of the disease. SUMMARY

[0006] The present disclosure obtains a TL1A antibody through mouse immunization and single B cell screening, and obtains a humanized TL1A antibody through a CDR grafting strategy. The TL1A antibody obtained by the present disclosure has excellent neutralization activity, affinity, TL1A and DR3 binding blocking activity, and in vivo IBD treatment effect.

[0007] The present disclosure provides, in a first aspect, an antibody or antigen-binding fragment thereof that binds to TL1A, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

[0008] The heavy chain variable region comprises:

[0009] HCDR1 comprising the HCDR1 of the heavy chain variable region sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to the HCDR1 of the heavy chain variable region sequence set forth in SEQ ID NO: 1,

[0010] HCDR2 comprising the HCDR2 of the heavy chain variable region sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to the HCDR2 of the heavy chain variable region sequence set forth in SEQ ID NO: 1,

[0011] HCDR3 comprising the HCDR3 of the heavy chain variable region sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to the HCDR3 of the heavy chain variable region sequence set forth in SEQ ID NO: 1;

[0012] The light chain variable region comprises:

[0013] LCDR1 comprising the LCDR1 of the light chain variable region sequence set forth in SEQ ID NO: 2 or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to the LCDR1 of the light chain variable region sequence set forth in SEQ ID NO: 2,

[0014] LCDR2 comprising the LCDR2 of the light chain variable region sequence set forth in SEQ ID NO: 2 or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to the LCDR2 of the light chain variable region sequence set forth in SEQ ID NO: 2,

[0015] LCDR3 comprising the LCDR3 of the light chain variable region sequence set forth in SEQ ID NO: 2, or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to the LCDR3 of the light chain variable region sequence set forth in SEQ ID NO: 2.

[0016] In some embodiments, the heavy chain variable region comprises:

[0017] HCDR1 comprising the amino acid sequence of HCDR1 of the heavy chain variable region sequence set forth in SEQ ID NO: 1,

[0018] HCDR2 comprising the amino acid sequence of HCDR2 of the heavy chain variable region sequence set forth in SEQ ID NO: 1,

[0019] HCDR3 comprising the amino acid sequence of HCDR3 of the heavy chain variable region sequence set forth in SEQ ID NO: 1;

[0020] the light chain variable region comprises:

[0021] LCDR1 comprising the amino acid sequence of LCDR1 of the light chain variable region sequence set forth in SEQ ID NO: 2,

[0022] LCDR2 comprising the amino acid sequence of LCDR2 of the light chain variable region sequence set forth in SEQ ID NO: 2,

[0023] LCDR3 comprising the amino acid sequence of LCDR3 of the light chain variable region sequence set forth in SEQ ID NO: 2.

[0024] In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the following definition systems: Kabat, AbM, Chothia, or IMGT.

[0025] In some embodiments, the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5;

[0026] the light chain variable region comprises: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15; LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16; LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; or,

[0027] the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6; a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7; and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8;

[0028] the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18; a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19; and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; or,

[0029] the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10; and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11;

[0030] the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21; a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22; and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or,

[0031] the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12; a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13; and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14;

[0032] the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24; a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25; and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a murine-derived antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0034] In some embodiments, the antigen-binding fragment comprises a Fab, Fab’, Fv fragment, F(ab’)2, scFv, or di-scFv.

[0035] In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 1;

[0036] The light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 2.

[0037] In some embodiments, the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 1 and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 2.

[0038] In some embodiments, the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 27 or 28.

[0039] In some embodiments, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 29 or 30.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of:

[0041] 1) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 27 and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 29;

[0042] 2) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 27 and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 30;

[0043] 3) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 28 and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 29; and

[0044] 4) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 28 and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 30.

[0045] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof further comprises a constant region of human IgGl, IgG2, IgG3 or IgG4 or a variant thereof; the constant region of human IgGl, IgG2, IgG3 or IgG4 or a variant thereof comprises an amino acid sequence set forth in any one of SEQ ID NOs: 39-51.

[0046] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof further comprises a constant region of human IgK or a variant thereof;

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain selected from the group consisting of:

[0048] 1) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 37 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 37, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 38 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 38;

[0049] 2) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 31 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 31, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 35 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 35;

[0050] 3) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 31 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 31, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 36 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 36;

[0051] 4) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 32 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 35 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 35;

[0052] 5) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 32 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 36 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 36;

[0053] 6) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 33 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 33, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 35 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 35;

[0054] 7) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 33 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 33, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 36 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 36;

[0055] 8) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 34 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 35 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 35;

[0056] 9) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 34 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 34, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 36 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 36; and

[0057] 10) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 63 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 63, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 35 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 35.

[0058] In some embodiments, the antibody or antigen binding fragment thereof has at least one of the following characteristics:

[0059] (1) has cell neutralization activity;

[0060] (2) binds to human TL1A with a KD value of less than 1╳10 -7 M, preferably less than 1╳10 -8 M, more preferably less than 1╳10 -9 M, even more preferably less than 1╳10 -10 M;

[0061] (3) is capable of blocking the binding of human TL1A to DR3;

[0062] (4) is capable of improving body weight loss, reducing DAI score and / or restoring colon length in a dextran sulfate sodium (DSS)-induced mammalian IBD model.

[0063] The second aspect of the present disclosure provides a fusion protein comprising the antibody or antigen binding fragment thereof of the first aspect of the present disclosure.

[0064] The third aspect of the present disclosure provides a bispecific or multispecific antibody comprising the antibody or antigen binding fragment of the first aspect of the present disclosure.

[0065] The fourth aspect of the present disclosure provides a nucleic acid molecule encoding the antibody or antigen binding fragment thereof of the first aspect of the present disclosure or the fusion protein of the second aspect of the present disclosure or the bispecific or multispecific antibody of the third aspect of the present disclosure; preferably, the nucleic acid is DNA or RNA.

[0066] The fifth aspect of the present disclosure provides a vector comprising the nucleic acid molecule of the fourth aspect of the present disclosure.

[0067] The sixth aspect of the present disclosure provides a recombinant cell comprising the nucleic acid molecule of the fourth aspect of the present disclosure and / or the vector of the fifth aspect of the present disclosure.

[0068] The seventh aspect of the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the first aspect of the present disclosure, and / or the fusion protein of the second aspect of the present disclosure, and / or the bispecific or multispecific antibody of the third aspect of the present disclosure, and / or the nucleic acid molecule of the fourth aspect of the present disclosure, and / or the vector of the fifth aspect of the present disclosure, and / or the recombinant cell of the sixth aspect of the present disclosure, and a pharmaceutically acceptable carrier.

[0069] The eighth aspect of the present disclosure provides a kit comprising the antibody or antigen-binding fragment thereof of the first aspect of the present disclosure, and / or the fusion protein of the second aspect of the present disclosure, and / or the bispecific or multispecific antibody of the third aspect of the present disclosure, and / or the nucleic acid molecule of the fourth aspect of the present disclosure, and / or the pharmaceutical composition of the seventh aspect of the present disclosure.

[0070] The ninth aspect of the present disclosure provides a method of treating and / or preventing a TL1A-associated disease or disorder, comprising administering to a patient in need thereof the antibody or antigen-binding fragment thereof of the first aspect of the present disclosure, and / or the fusion protein of the second aspect of the present disclosure, and / or the bispecific or multispecific antibody of the third aspect of the present disclosure, and / or the nucleic acid molecule of the fourth aspect of the present disclosure, and / or the vector of the fifth aspect of the present disclosure, and / or the pharmaceutical composition of the seventh aspect of the present disclosure.

[0071] The tenth aspect of the present disclosure provides the use of the antibody or antigen-binding fragment thereof of the first aspect of the present disclosure, and / or the fusion protein of the second aspect of the present disclosure, and / or the bispecific or multispecific antibody of the third aspect of the present disclosure, and / or the nucleic acid molecule of the fourth aspect of the present disclosure, and / or the vector of the fifth aspect of the present disclosure, in the manufacture of a medicament for treating a TL1A-associated disease or disorder.

[0072] In some embodiments, the TL1A-associated disease or disorder is an inflammatory bowel disease, such as ulcerative colitis and / or Crohn’s disease. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 shows the cellular neutralizing activity of anti-TL1A antibodies.

[0074] Figure 2 shows the blocking activity of antibody KNR4-M0038 on TL1A and DR3 binding: KNR4-M0038, PRA-023, RVT-3101.

[0075] Figures 3a-3c show the effect of antibody KNR4-M0038 on body weight (a), DAI score (b) and colon length (c) in a mouse model of IBD.

[0076] Figure 4 shows the sequence alignment of antibody VH and VL before and after humanization. Figure 4a, sequence alignment of antibody VH before and after humanization; Figure 4b, sequence alignment of antibody VL before and after humanization.

[0077] Figure 5 shows the cell neutralization activity of humanized antibodies. Figure 5a, cell neutralization activity of humanized antibodies M0038-hu22, M0038-hu11, M0038-hu12 and parental antibody KNR4-M0038; Figure 5b, cell neutralization activity of humanized antibody M0038-hu21 and parental antibody KNR4-M0038; Figure 5c, cell neutralization activity of humanized antibody M0038-hu11 and control antibodies PRA-023, RVT-3101.

[0078] Figure 6 shows the blocking activity of humanized antibodies on human TL1A and DR3 binding: o M0038-hu11, D M0038-hu12, · M0038-hu21, M0038-hu22, A KNR4-M0038.

[0079] Figure 7 shows the blocking activity of M0038-hu11-AALS on human TL1A and DR3 binding: A M0038-hu11-AALS, o RVT-3101, D PRA-023.

[0080] Figure 8 shows the inhibitory activity of humanized antibodies on TL1A stimulated CD4+T cells releasing IFN-γ.

[0081] Figure 9 shows the pharmacokinetic properties of humanized antibodies in FcRn humanized mice. DETAILED DESCRIPTION

[0082] Definitions of terms

[0083] Unless otherwise indicated or defined, all terms used have the ordinary meaning that would be understood by one of skill in the art, which meaning will be apparent to those skilled in the art. Reference is made to, for example, standard handbooks, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nded.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nded.), Gower Medical Publishing, London, New York (1989), as well as the general prior art cited herein; and further, all methods, procedures, techniques and manipulations not specifically detailed are performed and known in the art per se, unless otherwise stated, which will be apparent to those skilled in the art. Reference is also made to, for example, standard handbooks, the above general prior art, and other references cited therein.

[0084] As used herein, the term "tumor necrosis factor-like cytokine 1A" or "TL1A" or "TNFSF15" refers to any variant, functional fragment, isoform and species homolog of human TL1A. Exemplary human TL1A can refer to the protein disclosed at Uniprot ID NO: 095150, and can also be a protein having the amino acid sequence set forth in any one of SEQ ID NOs: 54-57.

[0085] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether naturally occurring or produced in whole or in part synthetically (e.g., recombinantly), including any fragment thereof that retains the ability of a full-length immunoglobulin to bind with specificity to an immunoglobulin antigen. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen binding domain (antibody binding site). Antibodies include antibody fragments, such as antibody fragments against TL1A. As used herein, the term antibody thus includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. Antibodies provided herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0086] As used herein, an "antibody fragment" or "antigen binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length, but which at least contains a portion of the variable region (e.g., one or more CDRs and / or one or more antigen binding sites) of the antibody that binds antigen, and thus retains the binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen binding fragment refers to an antibody fragment that contains an antigen binding portion that binds the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, e.g., recombinantly produced derivatives. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments can include multiple chains linked together, e.g., by disulfide bonds and / or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. An antigen binding fragment includes any antibody fragment that, when inserted into an antibody framework (e.g., by substitution of the corresponding region), acquires the ability to immunospecifically bind (i.e., exhibit at least or at least about 10 7 -10 8 M -1 fold increase in Ka) an antigen.

[0087] As used herein, a "monoclonal antibody" refers to a population of identical antibodies, indicating that each individual antibody molecule in the population of monoclonal antibodies is identical to the other antibody molecules. This property is in contrast to polyclonal populations of antibodies, which contain antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalizing B cells, e.g., by fusion with myeloma cells to produce hybridoma cell lines or by infection of B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from clonal populations of host cells by transforming host cells with plasmids carrying artificial sequences encoding the nucleotides of the antibodies.

[0088] As used herein, a "conventional antibody" refers to an antibody comprising two heavy chains (which can be designated H and H') and two light chains (which can be designated L and L') and two antigen binding sites, wherein each heavy chain can be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen binding ability (e.g., a heavy chain includes but is not limited to V H heavy chain, V H -C H 1 heavy chain, and V H -C H 1-C H 2-C H 3 heavy chain), and each light chain can be a full-length light chain or any functional region thereof (e.g., a light chain includes but is not limited to V L heavy chain, and V L -C L heavy chain). Each heavy chain (H and H') pairs with one light chain (L and L', respectively).

[0089] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., V H -C H 1-C H 2-C H 3 or V H -C H 1-C H 2-C H 3-C H 4) and two full-length light chains (V L -C L ) and a hinge region, such as an antibody naturally produced by an antibody secreting B cell and an antibody synthetically produced with the same domains.

[0090] As used herein, a dsFv refers to an Fv with an engineered intermolecular disulfide bond stabilizing the V H -V L pair.

[0091] As used herein, a Fab fragment is an antibody fragment obtained by digestion of a full-length immunoglobulin with papain, or a fragment with the same structure synthetically produced, e.g., by recombinant methods. A Fab fragment comprises a light chain (comprising V L and C L ) and another chain comprising a variable domain of a heavy chain (V H ) and one constant region domain of a heavy chain (C H 1).

[0092] As used herein, the F(ab')2 fragment is an antibody fragment resulting from the digestion of immunoglobulins with pepsin at pH 4.0–4.5, or a fragment with the same structure synthesized, for example, by a recombinant method. The F(ab')2 fragment essentially comprises two Fab fragments, each heavy chain containing several additional amino acids, including cysteine ​​residues that form the disulfide bond connecting the two fragments.

[0093] As used in this article, the Fab' fragment is a fragment that contains half of the F(ab')2 fragment (one heavy chain and one light chain).

[0094] As used herein, scFv fragments refer to variable light chains (V) covalently linked in any order via peptide linkers. L ) and variable heavy chain (V H The antibody fragment has a linker length that allows the two variable domains to bridge with minimal interference. An exemplary linker is (Gly-Ser) with some Glu or Lys residues dispersed to increase solubility. n Residues.

[0095] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0096] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing a minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit.

[0097] Furthermore, in humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated to improve one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, through PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conserved mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs typically do not exceed one or two. Therefore, the humanized antibodies described in this disclosure also cover antibodies containing one or two amino acid mutations within the CDRs.

[0098] As used herein, a variable domain or variable region is a particular Ig domain of an antibody heavy or light chain that comprises an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has one variable region domain, V L and V H The variable domain provides antigen specificity and is thus responsible for antigen recognition. Each variable region comprises CDRs and framework regions (FRs), the CDRs being part of the antigen binding site domain.

[0099] As used herein, "antigen binding domain" and "antigen binding site" are used synonymously to refer to the domain within an antibody that recognizes and physically interacts with a corresponding antigen. A native, conventional full-length antibody molecule has two conventional antigen binding sites, each comprising a heavy chain variable region portion and a light chain variable region portion. A conventional antigen binding site comprises loops connecting antiparallel beta strands within the variable region domain. An antigen binding site can comprise other portions of the variable region domain. Each conventional antigen binding site comprises 3 hypervariable regions from the heavy chain and 3 hypervariable regions from the light chain. Hypervariable regions are also known as complementarity determining regions (CDRs).

[0100] As used herein, "hypervariable region," "HV," "complementarity determining region," and "CDR," and "antibody CDR," are used interchangeably to refer to one of a number of portions within each variable region that together form an antigen binding site of an antibody. Each variable region domain comprises 3 CDRs, designated CDR1, CDR2, and CDR3. For example, a light chain variable region domain comprises 3 CDRs, designated VL CDR1, VL CDR2, and VL CDR3; a heavy chain variable region domain comprises 3 CDRs, designated VH CDR1, VH CDR2, and VH CDR3. The 3 CDRs in a variable region are non-contiguous along the linear amino acid sequence, but are proximate in the folded polypeptide. The CDRs are located within loops connecting the parallel beta strands of the variable domain's beta sheet.

[0101] As described herein, those skilled in the art know and can identify CDRs based on Kabat, Chothia, or IMGT numbering (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J.Mol.Biol. 196:901-917). Alternative methods for numbering the amino acid residues of CDRs are also known in the art. For example, AbM CDRs represent a compromise between the Kabat hypervariable region and the Chothia structural loop and are used in Oxford Molecular's AbM antibody modeling software; for example, IMGT CDRs are defined according to the unique numbering system of IMGT (International ImMunoGeneTics Information System). The residue descriptions of CDRs from each method are as follows:

[0102] However, it should be noted that, as is known in the art, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence.

[0103] As used in this article, the frame region (FR) is a domain located within the antibody variable region domain within the β-sheet; in terms of amino acid sequence, the FR region is relatively more conserved than the hypervariable region.

[0104] As used in this article, the "constant region" domain is a domain in the antibody heavy or light chain that contains a more conserved amino acid sequence than the variable region domain. In a typical full-length antibody molecule, each light chain has a single light chain constant region (C0). L ) structural domains, and each heavy chain contains one or more heavy chain constant regions (C H ) structural domain, including C H 1. C H 2. C H 3 and C H 4. Full-length IgA, IgD, and IgG isotypes include C.H 1, C H 2, C H 3 and hinge region, while IgE and IgM contain C H 1, C H 2, C H 3 and C H 4. C H 1 and C L The constant regions of the antibodies can serve effector functions, such as, but not limited to, clearing antigens, pathogens, and toxins to which the antibodies specifically bind, for example, by interacting with various cells, biomolecules, and tissues.

[0105] The term "Fc region" herein defines a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, i.e., CH2, CH3, and optionally CH4 domains. For example, in native antibodies, an immunoglobulin Fc domain comprises the second and third constant domains of two heavy chains (CH2 and CH3 domains) from an antibody of the IgG, IgA, and IgD classes; or the second, third, and fourth constant domains of two heavy chains (CH2, CH3, and CH4 domains) from an antibody of the IgM and IgE classes. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or the heavy chain constant region is according to the EU numbering scheme (also called the EU index) as set forth in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The term "Fc region" herein does not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, and the heavy chain constant region CH1 and light chain constant region CL, but can in some cases include the hinge region N-terminal to the heavy chain constant region. In some embodiments, the Fc region of the present disclosure is from IgGl, IgG2, IgG3, or IgG4.

[0106] As used herein, "specific binding" or "immunospecific binding" in reference to an antibody or antigen-binding fragment thereof are used interchangeably herein, and refer to the ability of the antibody or antigen-binding fragment to form one or more noncovalent bonds with the same antigen through noncovalent interactions between the antibody combining site of the antibody and the antigen. Typically, an antibody that immunospecifically binds to (or specifically binds) an antigen is one that binds to the antigen with an affinity of about or 1 x 10 7 M-1 or 1 x 10 8 M -1 or a larger affinity constant Ka (or 1 x 10 -7 M or 1 x 10 -8 M or lower dissociation constant (K d )) for the antigen. Affinity constants can be determined by standard kinetic methods of antibody reactions, e.g., immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11 :54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989)). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0107] As used herein, "polypeptide" refers to two or more amino acids covalently linked. The terms "polypeptide" and "protein" are used interchangeably herein.

[0108] "Isolated protein," "isolated polypeptide," or "isolated antibody" refers to the protein, polypeptide, or antibody (1) not associated with naturally occurring components that accompany it in its natural state, (2) not containing other proteins from the same species, (3) expressed by a cell from a different species, or (4) not occurring in nature. Thus, a polypeptide chemically synthesized or synthesized in a cell system other than the cell system from which the polypeptide naturally originates will be "isolated" from its naturally associated components. A protein can also be isolated by being rendered substantially free of naturally associated components using protein purification techniques well known in the art.

[0109] In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. In general, the skilled artisan recognizes that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. co., p. 224).

[0110] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer of at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that are typically linked together by phosphodiester bonds.

[0111] As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules present in a natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, can be produced by recombinant technology substantially free of other cellular material, or culture medium when produced by chemical synthesis, or substantially free of chemical precursors or other chemicals when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules that encode the provided antibodies or antigen binding fragments.

[0112] Sequence "identity" has the conventional meaning in the art and can be calculated using published techniques. The percent sequence identity between two nucleic acid or polypeptide molecules or regions can be determined using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide, or along a region of that molecule. (See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are a number of methods for measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled artisan (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0113] As used herein, "operably linked" with respect to nucleic acid sequences, regions, elements, or domains means that the nucleic acid regions are functionally related to one another. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide, such that the promoter regulates or mediates transcription of the nucleic acid.

[0114] As used herein, "expression" refers to the process by which polypeptides are produced by transcription and translation of a polynucleotide. The level of expression of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from a host cell. Such methods can include, but are not limited to, quantifying a polypeptide in a cell lysate by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0115] As used herein, a "host cell" is a cell that is used to accept, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by a vector. As a host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. A host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells, such as HEK 293 cells.

[0116] As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Vectors include those into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction enzyme digestion and ligation. Vectors also include those comprising a nucleic acid encoding a polypeptide. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell, for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically episomal, but can be designed to integrate a gene or portion thereof into a chromosome of the genome. Vectors of artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles is well known to those skilled in the art.

[0117] As used herein, a vector also includes a "viral vector." A viral vector is an engineered virus that is operably linked to a foreign gene to transfer (as a vehicle or shuttle) the foreign gene into a cell.

[0118] As used herein, an "expression vector" includes a vector capable of expressing DNA operably linked to control sequences, such as a promoter region, capable of affecting expression of such DNA fragments. Such additional fragments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic and / or prokaryotic cells, as well as expression vectors that are maintained episomally or integrated into the host cell genome.

[0119] As used herein, "treatment" of an individual having a disease or condition means partial or complete alleviation of symptoms of the individual, or remaining unchanged after treatment. Thus, treatment includes prevention, therapy, and / or cure. Prevention refers to preventing an underlying disease and / or preventing symptoms from worsening or disease progression. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided herein, and any composition provided herein.

[0120] As used herein, "efficacy" means an effect resulting from the treatment of a subject that alters, usually improves or ameliorates symptoms of, or cures a disease or condition.

[0121] As used herein, "therapeutically effective amount" or "therapeutically effective dose" means the amount of a substance, compound, material, or composition containing a compound that, after administration to a subject, is sufficient to at least elicit a therapeutic effect. It is, therefore, the amount necessary to prevent, cure, improve, retard or partially retard symptoms of a disease or disorder.

[0122] As used herein, the term "and / or" means either or both of the items so conjoined.

[0123] As used herein, the term "comprising" or "including," or "having" means including but not limited to, and thus specifies the presence of what follows the term, but does not preclude the presence of additional, unrecited items. When used in this document, the terms "comprising" or "including" or "having" also encompass the case where the recited elements, integers or steps are combined.

[0124] The term "administering" generally refers to the method of giving a dose of a compound or pharmaceutical composition to a subject (e.g., a patient). Administration can be by any suitable means, including parenterally, intrapulmonary, and intranasally, as well as intralesionally if local treatment is desired. Parenteral infusions include, e.g., intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In the present application, the term "about" generally means a variance within 0.5-10% above or below the indicated numerical values, e.g., a variance within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the indicated numerical value. Unless otherwise specified, all numerical values mentioned in the present application are to be considered as modified by "about." If in doubt, or for error ranges not generally recognized in the art for a particular value or parameter, "about" means ±5% of the value or parameter.

[0125] The term "pharmaceutical adjuvant" refers to diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), excipients, carriers, or stabilizers, etc., administered with the active substance.

[0126] The term "pharmaceutical composition" refers to a composition that is in a form that is effective for the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that would render the administration of the composition to a subject unacceptable.

[0127] As used herein, "treat" refers to slowing, interrupting, arresting, ameliorating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0128] The term "therapeutic agent" as described herein encompasses any active substance or active agent that is effective in the prevention or treatment of the disease to be treated.

[0129] The term "pharmaceutical combination" or "combination product" refers to either a non-fixed combination product or a fixed combination product, including but not limited to a kit, pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody or antigen-binding fragment thereof of the present disclosure, and (ii) another therapeutic agent) are presented in a separate entity and are administered to a patient simultaneously, without specific time limitations relative to each other, or sequentially, with or without intention of having a mutual action. The term "fixed combination" means that the two or more active agents are present in a single entity, which is administered to a patient simultaneously. Preferably, the dosage amounts and / or time intervals of the two or more active agents are selected so that a combined use of the parts results in an effect which is greater than the effect achievable with either part alone in the treatment of a disease or condition. The parts can each be in a separate formulation, which formulations can be the same or different.

[0130] DETAILED DESCRIPTION

[0131] Antibodies binding to TL1A

[0132] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof binding to TL1A, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

[0133] the heavy chain variable region comprises:

[0134] HCDR1 comprising the HCDR1 of the heavy chain variable region sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to the HCDR1 of the heavy chain variable region sequence set forth in SEQ ID NO: 1,

[0135] HCDR2 comprising the HCDR2 of the heavy chain variable region sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to the HCDR2 of the heavy chain variable region sequence set forth in SEQ ID NO: 1,

[0136] HCDR3 comprising the HCDR3 of the heavy chain variable region sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to the HCDR3 of the heavy chain variable region sequence set forth in SEQ ID NO: 1.

[0137] the heavy chain variable region comprises:

[0138] HCDR1 comprising the amino acid sequence of HCDR1 of the heavy chain variable region sequence set forth in SEQ ID NO: 1,

[0139] HCDR2 comprising the amino acid sequence of HCDR2 of the heavy chain variable region sequence set forth in SEQ ID NO: 1,

[0140] HCDR3 comprising the amino acid sequence of HCDR3 of the heavy chain variable region sequence set forth in SEQ ID NO: 1;

[0141] the light chain variable region comprises:

[0142] the light chain variable region comprises:

[0143] LCDR1 comprising the amino acid sequence of LCDR1 of the light chain variable region sequence set forth in SEQ ID NO: 2,

[0144] LCDR2 comprising the amino acid sequence of LCDR2 of the light chain variable region sequence set forth in SEQ ID NO: 2,

[0145] LCDR3 comprising the amino acid sequence of LCDR3 of the light chain variable region sequence set forth in SEQ ID NO: 2.

[0146] the light chain variable region comprises:

[0147] the light chain variable region comprises:

[0148] LCDR2 comprising the amino acid sequence of LCDR2 of the light chain variable region sequence set forth in SEQ ID NO: 2,

[0149] LCDR3 comprising the amino acid sequence of LCDR3 of the light chain variable region sequence set forth in SEQ ID NO: 2.

[0150] the light chain variable region comprises:

[0151] the heavy chain variable region comprises:

[0152] HCDR1 consisting of the amino acid sequence of HCDR1 of the heavy chain variable region sequence set forth in SEQ ID NO: 1,

[0153] HCDR2 consisting of the amino acid sequence of HCDR2 of the heavy chain variable region sequence set forth in SEQ ID NO: 1,

[0154] HCDR3 consisting of the amino acid sequence of HCDR3 of the heavy chain variable region sequence set forth in SEQ ID NO: 1;

[0155] the light chain variable region comprises:

[0156] LCDR1 consisting of the amino acid sequence of LCDR1 of the light chain variable region sequence set forth in SEQ ID NO: 2,

[0157] LCDR2 consisting of the amino acid sequence of LCDR2 of the light chain variable region sequence set forth in SEQ ID NO: 2,

[0158] LCDR3 consisting of the amino acid sequence of LCDR3 of the light chain variable region sequence set forth in SEQ ID NO: 2.

[0159] In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the following definition systems: Kabat, AbM, Chothia, or IMGT.

[0160] (Kabat)

[0161] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

[0162] the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to SEQ ID NO: 3; HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to SEQ ID NO: 4; HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having 1 or 2 amino acid substitutions, deletions, or additions relative to SEQ ID NO: 5.

[0163] the heavy chain variable region comprises: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3; HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4; and HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5.

[0164] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

[0165] the heavy chain variable region comprises: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3; HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4; and HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5.

[0166] the light chain variable region comprises: LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15; LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16; and LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17.

[0167] (AbM)

[0168] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

[0169] the heavy chain variable region comprises: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6; HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7; and HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[0170] the heavy chain variable region comprises: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6; HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7; and HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[0171] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

[0172] the heavy chain variable region comprises: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6; HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7; and HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[0173] the light chain variable region comprises: LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18; LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19; and LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 20.

[0174] (Chothia)

[0175] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

[0176] the heavy chain variable region comprises: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 6; HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7; and HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[0177] the heavy chain variable region comprises: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9; HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10; and HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11.

[0178] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

[0179] the heavy chain variable region comprises: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9; HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10; and HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11.

[0180] the light chain variable region comprises: LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21; LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 22; and LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 23.

[0181] (IMGT)

[0182] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

[0183] the heavy chain variable region comprises: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9; HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10; and HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11.

[0184] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO: 1. In some embodiments, the heavy chain variable region comprises an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 1.

[0185] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,

[0186] the heavy chain variable region comprises: HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 12; HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13; and HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14.

[0187] the light chain variable region comprises: LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 24; LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 25; and LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 26.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO: 1. In some embodiments, the heavy chain variable region comprises an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 1.

[0189] In some embodiments, the heavy chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO: 1. In some embodiments, the heavy chain variable region consists of an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 1.

[0190] In some embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO: 2. In some embodiments, the light chain variable region comprises an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 2.

[0191] In some embodiments, the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO: 2. In some embodiments, the light chain variable region consists of an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 2.

[0192] In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2; in some embodiments, the heavy chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 1 and the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0193] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is humanized.

[0194] In some embodiments, the antibody or antigen-binding fragment thereof is humanized, and the heavy chain variable region comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO: 1; for example, the heavy chain variable region comprises or consists of an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 1; for another example, the heavy chain variable region comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to SEQ ID NO: 1.

[0195] In some embodiments, the antibody or antigen-binding fragment thereof is humanized, and the heavy chain variable region comprises or consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO: 1; for example, the heavy chain variable region comprises or consists of an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 1; for another example, the heavy chain variable region comprises or consists of an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to SEQ ID NO: 1.

[0196] In some embodiments, the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27 or 28.

[0197] In some embodiments, the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29 or 30.

[0198] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 29; or the heavy chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 27 and the light chain variable region consists of the amino acid sequence set forth in SEQ ID NO: 29.

[0199] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 30; or a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 27 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 30.

[0200] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 28 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 29; or a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 29.

[0201] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 28 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 30; or a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 30.

[0202] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of the present disclosure further comprises a constant region or a variant thereof, e.g., derived from murine or human constant regions. In some embodiments, the heavy chain comprises a constant region of human IgGl, IgG2, IgG3, or IgG4 or a variant thereof, e.g., the heavy chain comprises a constant region which can comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 39-42. In some embodiments, the heavy chain comprises a constant region which can comprise or consist of the amino acid sequence set forth in SEQ ID NO: 43.

[0203] In some embodiments, the Fc region of the heavy chain of the antibody or antigen-binding fragment thereof can further comprise a mutation that reduces or abrogates effector function. The effector function can be antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC), etc. Specifically, the mutation that reduces or abrogates effector function attenuates or abrogates the binding between the immunoglobulin and FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa, FcyRIIIb, and / or Clq.

[0204] The mutation that attenuates or abolishes effector function can be any mutation or combination of mutations known in the art, such as described in Esohe E.I., J Immunol 2000; 164: 4178-4184; Hutchins, J.T., Proc. Natl. Acad. Sci. USA 1995, 92, 11980-11984; Xu D., Cell Immunol. 2000, 200, 16-26; Hezareh, M., J. Virol. 2001, 75, 12161-12168; Schlothauer, T., Protein Eng. Des. Sel. 2016, 29, 457-466; Chu, S.Y., Mol. Immunol. 2008, 45, 3926-3933; Sazinsky, S.L., Proc. Natl. Acad. Sci. USA 2008, 105, 20167-20172; Oganesyan, V., Acta Crystallogr. Sect. D Biol. Crystallogr. 2008, 64 Pt 6, 700-704; An, Z., MAbs 2009, 1, 572-579; Moore, G.L., Methods 2019, 154, 38-50; Schlothauer, T., Protein Eng. Des. Sel. 2016, 29, 457-466; Strohl, W., US20150337053; Engelberts, P.J., EBioMedicine 2020, 52, 102625, and the like.

[0205] In some embodiments, the Fc region can further comprise a mutation that reduces or abrogates effector function selected from the group consisting of D265A, D270A, N297A, N297Q, N297G, N297D, K322A, P329A, P331G, D265A / P331G, L235A / G237A / E318A, L234A / L235A, S228P / L235E, G236R / L328R, S298G / T299A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, E233P / L234V / L235A / G236del / S267K, L234A / L235A / P329G, L234F / L235E / D265A, or V234A / G237A / P238S / H268A / V309L / A330S / P331S. Preferably, the Fc region can further comprise a mutation that reduces or abrogates effector function selected from the group consisting of L234A / L235A (hereinafter can be abbreviated as AA) or L234F / L235E / P331S (hereinafter can be abbreviated as FES).

[0206] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a constant region comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 44 or 45.

[0207] In some embodiments, the Fc region of the heavy chain of the antibody or antigen-binding fragment thereof can further comprise a mutation that modulates its ability to bind to FcRn. For example, the Fc region variant improves the binding to FcRn at acidic pH conditions without affecting the binding to FcRn at neutral pH conditions, thereby obtaining a longer half-life compared to the parent.

[0208] The mutation that modulates the ability to bind to FcRn and / or prolongs the half-life can be any known mutation or combination of mutations in the art, for example, described in Dall’Acqua W, J. Immunol. 2002; 169: 5171-5180; Dall’Acqua W, J. Biol. Chem. 2006b; 281: 23514-23524; Hinton PR, J. Immunol. 2006; 176: 346-356; Petkova, J. Exp. Med. 2006; 203: 275-280; Yeung, J. Immunol. 2009; 182: 7663-7671; Zalevsky, Nat. Biotechnol. 2010; 28: 157-159; Monnet, MAbs. 2014; 6: 422-436, etc.

[0209] In some embodiments, the Fc region can comprise a mutation selected from the group consisting of M252Y / S254T / T256E, T250Q / M428L, N434A, M428L / N434S, N315D / A330V / N361D / A378V / N434Y, E294D / T307P / N434Y, V259I / N315D / N434Y, T307A / N315D / A330V / E382V / N389T / N434Y, or L234F / L235E / D265A that modulates the ability to bind to FcRn and / or prolongs half-life. Preferably, the immunoglobulin Fc region variant can comprise a mutation selected from the group consisting of M252Y / S254T / T256E (hereinafter can be abbreviated as YTE) or M428L / N434S (hereinafter can be abbreviated as LS) that modulates the ability to bind to FcRn and / or prolongs half-life.

[0210] In some embodiments, the heavy chain of the antibody or antigen binding fragment thereof comprises or consists of the amino acid sequence set forth in SEQ ID NO: 46 or 47.

[0211] In some embodiments, the heavy chain of the antibody or antigen binding fragment thereof comprises a constant region, and the Fc region in the constant region can further comprise both the aforementioned mutations that reduce or eliminate effector function and the mutations that modulate the ability to bind to FcRn. In some embodiments, the Fc region in the constant region comprises both AA / LS, AA / YTE, FES / LS, or FES / LS mutations. In some embodiments, the heavy chain of the antibody or antigen binding fragment thereof comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 48-51.

[0212] In some embodiments, the heavy chain of the antibody or antigen binding fragment thereof comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31-34 or 37 or 63.

[0213] In some embodiments, the light chain of the antibody or antigen binding fragment thereof of the present disclosure further comprises a constant region or a variant thereof, e.g., a constant region derived from murine or human. In some embodiments, the light chain comprises a constant region of human Ig kappa or a variant thereof, e.g., the light chain comprises a constant region that can comprise or consist of the amino acid sequence set forth in SEQ ID NO: 52. In other embodiments, the light chain comprises a constant region that can comprise or consist of the amino acid sequence set forth in SEQ ID NO: 53.

[0214] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof of the present disclosure comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35-36 or 38.

[0215] In some embodiments, the antibody of the present disclosure that binds to TL1A comprises a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 37 and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 38. In other embodiments, the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35. In other embodiments, the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36. In other embodiments, the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35. In other embodiments, the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36. In other embodiments, the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35. In other embodiments, the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36. In other embodiments, the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35. In other embodiments, the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36. In other embodiments, the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35.

[0216] In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure has at least one of the following characteristics:

[0217] (1) has a cell neutralization activity, for example, the cell is a TF-1 cell, and for example, the cell is a TF1-NFkB-RE polyclonal cell; preferably, the antibody or antigen-binding fragment thereof has a half neutralization concentration of no higher than 5 pg / ml, more preferably no higher than 1 pg / ml, even more preferably no higher than 0.5 pg / ml; the antibody or antigen-binding fragment has a neutralization rate of higher than 20%, higher than 30%, higher than 40%, higher than 50% for a cell expressing TF1 (such as TF1-NFkB-RE) at a use concentration of 0.3 ug / L; has a neutralization rate of higher than 5%, 10%, 15%, 20% for a cell expressing TF1 (such as TF1-NFkB-RE) at a use concentration of 0.1 ug / L.

[0218] (2) has a KD value of less than 1 ╳ 10 -7 M, preferably less than 1 ╳ 10 -8 M, more preferably less than 1 ╳ 10 -9 M, even more preferably less than 1 ╳ 10 -10 M;

[0219] (3) is capable of blocking the binding of human TL1A to DR3, for example, the antibody or antigen-binding fragment thereof has a half blocking concentration of no higher than 200 nM, preferably no higher than 100 nM, more preferably no higher than 80 nM, even more preferably no higher than 50 nM;

[0220] (4) is capable of improving the weight loss, reducing the DAI score and / or restoring the colon length in a DSS-induced mammalian IBD model.

[0221] Nucleic acids, vectors and host cells comprising the same

[0222] In one aspect, the present disclosure provides nucleic acids encoding any of the above antibodies or antigen-binding fragments thereof. The present disclosure also encompasses nucleic acids that hybridize to the above-mentioned nucleic acids under stringent conditions, nucleic acids that have one or more substitutions (e.g., conservative substitutions), deletions or insertions compared to the above-mentioned nucleic acids, or nucleic acid sequences that have at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity compared to the above-mentioned nucleic acids.

[0223] For example, the nucleic acids of the present disclosure comprise a nucleic acid encoding an amino acid sequence selected from the amino acid sequence set forth in any one of SEQ ID NOs: 1-53, 63, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence selected from the amino acid sequence set forth in any one of SEQ ID NOs: 1-53, 63.

[0224] As will be appreciated by those skilled in the art, due to codon degeneracy, each antibody or antigen-binding fragment thereof amino acid sequence can be encoded by a variety of nucleic acid sequences. Nucleic acid sequences encoding molecules of the disclosure can be produced using methods well known in the art, for example, by de novo solid phase DNA synthesis, or by PCR amplification.

[0225] In one embodiment, nucleic acids encoding antibodies or antigen-binding fragments thereof of the disclosure can be in the same vector or in different vectors. In yet another embodiment, nucleic acids encoding each chain of an antibody or antigen-binding fragment thereof of the disclosure can be introduced into the same or different host cells for expression. Thus, in some embodiments, a method of producing an antibody or antigen-binding fragment thereof of the disclosure comprises the step of culturing a host cell comprising nucleic acids encoding each chain of said molecule under conditions suitable for expression of said each chain to produce an antibody or antigen-binding fragment thereof of the disclosure.

[0226] In another aspect, the disclosure provides vectors comprising the above-mentioned nucleic acids. The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host and which transfers nucleic acid molecules to and / or between host cells. The vector can include a vector primarily for insertion of DNA or RNA into a cell, a vector primarily for replication of DNA or RNA, and a vector primarily for transcription and / or translation of expression of DNA or RNA. The vector also includes a vector having a plurality of the above-mentioned functions. The vector can be a polynucleotide that is capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell comprising the vector.

[0227] In a preferred embodiment, the vector is an expression vector. The expression vector generally comprises at least one nucleic acid of the disclosure operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). Selection of the elements and their sequences for expression in a particular host is a matter of choice to one skilled in the art. Specific examples of control elements and other elements useful or necessary for expression of the fusion proteins of the disclosure include, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes. It is well within the understanding of one skilled in the art that vectors commonly employed in the art to which the disclosure pertains can be applied to the disclosure.

[0228] In one embodiment, the disclosure provides host cells comprising the nucleic acid or the vector.

[0229] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of the subject cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. The progeny can not be identical to the parent cell in nucleic acid content due to, e.g., mutation, but are still included, unless otherwise excluded. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein. Host cells are any type of cell system that can be used to produce the antibody molecules of the present disclosure, including eukaryotic cells, e.g., mammalian cells (e.g., CHO cells or HEK293 cells), insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cells in culture as well as cells inside a transgenic animal, transgenic plant, or cultured plant or animal tissue.

[0230] Compositions

[0231] In another aspect, the present disclosure provides a composition, e.g., preferably, a pharmaceutical composition, containing one or a combination of the antibodies or antigen-binding fragments thereof of the present disclosure formulated together with a pharmaceutically acceptable carrier.

[0232] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers and isotonic and absorption delaying agents, etc. which are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical (e.g., by injection or infusion) administration. Depending on the route of administration, the active compound, i.e., the antibody molecule, can be coated in a material to protect the compound from the action of acids and other natural conditions which can inactivate the compound.

[0233] The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material for a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent (100%), this amount will range from about 0.01% to about 99% of active ingredient, such as from about 0.1% to about 70%, or about 1% to about 30% of active ingredient, in combination with a pharmaceutically acceptable carrier.

[0234] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure can be varied to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular compositions of the present disclosure applied or its ester, salt or amide thereof; the route of administration; the time of administration; the rate of excretion of the particular compound being applied; the duration of the treatment; other drugs, compounds, and / or materials used in combination with the particular compositions employed; the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0235] The compositions of the present disclosure can be administered using one or more methods known in the art by one or more routes of administration. The skilled artisan will appreciate that the route and / or mode of administration will vary depending upon the desired results. Preferred routes of administration of the antibodies of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes, such as by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0236] Pharmaceutical combinations and kits

[0237] The present disclosure also provides a pharmaceutical combination or pharmaceutical combination product comprising a molecule of the present disclosure. Optionally, the pharmaceutical combination or pharmaceutical combination product further comprises one or more other therapeutic agents.

[0238] The present disclosure also provides a kit-of-parts comprising said pharmaceutical combination, e.g. said kit-of-parts comprises in the same package:

[0239] - a first container containing a pharmaceutical composition comprising a molecule of the present disclosure;

[0240] - optionally, a second container containing a pharmaceutical composition comprising one or more other therapeutic agents (in some embodiments, two or more other therapeutic agents are in the same container, or in separate containers, respectively).

[0241] In some embodiments, the other therapeutic agents encompass any therapeutic agent used in the treatment of the diseases described in the present disclosure.

[0242] Methods of treatment and uses

[0243] In yet another aspect, the present disclosure also provides a method of treating and / or preventing a TL1A-associated disease or disorder, comprising administering to a patient in need thereof one or more of the antibody or antigen-binding fragment thereof, nucleic acid, vector, pharmaceutical composition or drug combination of the present disclosure.

[0244] In some embodiments, the TL1A-associated disease or disorder refers to an indication associated with abnormal expression or activity of TL1A, or a disease or disorder associated with abnormal activation of TL1A / DR3. In some embodiments, the TL1A-associated disease or disorder is inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and / or Crohn’s disease (CD).

[0245] Accordingly, the present disclosure also provides a method of treating and / or preventing inflammatory bowel disease, such as ulcerative colitis and / or Crohn’s disease, comprising administering to a patient in need thereof one or more of the antibody or antigen-binding fragment thereof, nucleic acid, vector and pharmaceutical composition of the present disclosure.

[0246] In another aspect, the present disclosure also provides use of the antibody or antigen-binding fragment thereof, nucleic acid, vector and pharmaceutical composition in the manufacture of a medicament for treating and / or preventing a TL1A-associated disease or disorder, such as inflammatory bowel disease, e.g., ulcerative colitis and / or Crohn’s disease.

[0247] EMBODIMENTS

[0248] The present disclosure is further illustrated by the following examples, but any example or combination thereof should not be interpreted as limiting the scope or implementation of the present disclosure. The scope of the present disclosure is defined by the appended claims, and those skilled in the art can clearly understand the scope defined by the claims in combination with the present specification and general common knowledge in the art. Any modification or change to the technical solutions of the present disclosure made by those skilled in the art without departing from the spirit and scope of the present disclosure is also included in the scope of the present disclosure.

[0249] Example 1 Immunization and antibody screening

[0250] 1.1 Immunization

[0251] Immunize 10 Balb / c mice, each mouse with a single immunization dose of 50 μg, the first immunization emulsified with an equal volume of complete Freund's adjuvant, subcutaneous injection in multiple points on the abdomen; 2-3 weeks later, the same dose of immunogen and an equal volume of incomplete Freund's adjuvant were emulsified to subcutaneously inject in multiple points on the abdomen. One week after three immunizations, blood was taken, and the serum binding titer was determined by indirect ELISA method: 5 μg / mL coated immunogen protein, 1:8000 diluted serum, OD450-Blank>1.0 as the titer qualified standard.

[0252] 1.2 Screening

[0253] Screening was performed using Beacon (Berkeley Lights): immune tissues such as spleen, bone marrow, and lymph nodes of mice with qualified titer after immunization were ground into single cell suspension, red blood cells were lysed, mouse CD138 magnetic beads were added for incubation, and CD138+ plasma cells were obtained by positive selection. The separated plasma cells were added to the chip (cat: 750-00021, Berkeley Lights), and the cells were introduced into the NanoPen chamber by optical electroposition (OEP). The microspheres coated with antigens and fluorescent secondary antibodies (Goat anti-Mouse IgG Fc Cross-Adsorbed Secondary Antibody, FITC Conjugate (invitrogen, item number 31630) were introduced into the chip channel, and the positive cells secreting specific antibodies were screened by multiple rounds of photography. The data were analyzed to determine the location of the positive cells in the NanoPen, and the cells were introduced from the NanoPen to the 96-well plate containing cell lysis solution by OEP.

[0254] 1.3 Single cell PCR and vector construction

[0255] After the Beacon-derived single cells were lysed, cDNA was obtained by reverse transcription using a reverse transcription kit, and the antibody heavy and light chain variable region sequences were obtained by nest PCR amplification. The heavy and light chain variable region fragments were constructed into expression vectors to obtain sequencing-corrected heavy and light chain expression plasmids, which were transformed into HEK293 for transient expression, and a total of 224 strains of clones were cultured. The shake flask culture conditions were 5% CO2, temperature 37°C, and shaking speed 175 rpm. The culture was incubated for 3-7 days, and the culture supernatant was taken for ELISA detection.

[0256] 1.4 Culture supernatant ELISA detection

[0257] ELISA binding assay: 1) coating: coating antigen 0.1 μg / mL, 1 μg / mL, 100 μL / well, 4°C coating overnight; 2) blocking: shake off the liquid in the plate and dry, 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h; 3) washing plate: 300 μL / well washing solution, wash the plate twice, and dry at last; 4) supernatant dilution: dilute the supernatant 2 times, 100 μL each to the corresponding well plate, mix well, and react at room temperature for 2 h; 5) washing plate: 300 μL / well washing solution, wash the plate 3 times, and dry at last; 6) add secondary antibody: dilute Rabbit Anti-Mouse IgG F(ab)2 / HRP secondary antibody to the use concentration, 100 μL / well, mix well, and incubate at room temperature for 1 h; 7) washing plate: 300 μL / well washing solution, wash the plate 3 times, and dry at last; 8) color development: mix color development A and color development B at 1:1, add 200 μL per well, and incubate at room temperature for 20 min in the dark; 9) termination: add 50 μL per well, and immediately measure the OD value at 450 nm wavelength.

[0258] ELISA competition assay: 1) coating: coating target protein hDR3 (purchased from Acro, Lot#P4432-2254F1-1AP) 0.6 μg / mL, 100 μL / well, 4°C coating overnight; 2) blocking: shake off the liquid in the plate and dry, 2% BSA, 300 μL / well, seal and incubate at room temperature for 1 h; 3) washing plate: 300 μL / well washing solution, wash the plate twice, and dry at last; 4) supernatant and immunogen protein dilution: dilute the supernatant 80 times, dilute the immunogen protein to 0.5 μg / mL, mix them at 1:1, incubate at room temperature for 0.5 h, 100 μL each to the corresponding well plate, mix well, and react at room temperature for 1 h; 5) washing plate: 300 μL / well washing solution, wash the plate 3 times, and dry at last; 6) add secondary antibody: dilute SA / HRP secondary antibody to the use concentration, 100 μL / well, mix well, and incubate at room temperature for 1 h; 7) washing plate: 300 μL / well washing solution, wash the plate 3 times, and dry at last; 8) color development: mix color development A and color development B at 1:1, add 200 μL per well, and incubate at room temperature for 20 min in the dark; 9) termination: add 50 μL per well, and immediately measure the OD value at 450 nm wavelength.

[0259] 1.5 Purification and detection of antibodies

[0260] The ELISA binding assay positive cell supernatant was centrifuged by a desktop centrifuge, the centrifugation condition was 4000g, the centrifugation time was 30 min, and the centrifuged cell supernatant was collected; the centrifuged cell supernatant was filtered by using a 0.45 μM filter membrane. The centrifuged cell culture supernatant was purified by using a protein A affinity purification method, and the monoclonal antibody was harvested.

[0261] Using micro-spectrophotometer, the sample was detected by detecting the sample corresponding buffer, and the purified antibody sample was sequentially spotted, and the absorbance value was recorded. The detection data was divided by the extinction coefficient of IgG (1.414), and the obtained value was the concentration (unit mg / mL) of the detection sample. The purity of the purified antibody was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). According to the different molecular weights of the proteins in the test sample, the proteins were separated in the electrophoresis gel, so as to verify the purity of the antibody.

[0262] It was detected that the concentration of positive clone KNR4-M0038 (VH: SEQ ID NO: 1, VL: SEQ ID NO: 2; HC: SEQ ID NO: 37, LC: SEQ ID NO: 38) was about 4.5 mg / mL, and the purity was 96.7%.

[0263] Detection of biological activity of anti-TL1A antibody of Example 2

[0264] 2.1 Cell neutralization activity

[0265] The experimental culture medium was RPMI 1640 containing 10% FBS and 10 ng / ml GM-CSF. After collecting the TF1-NFkB-RE polyclonal cells (TF-1 cells were infected with a virus, then passaged twice by adding 5 μg / mL puromycin, and sorted after flow detection), 50000 cells / 25 μL were added to each well of a 96-well cell culture plate (Corning, item number 3599). Nhis-TL1A-ECD (SEQ ID NO: 57) was diluted with the experimental culture medium to 0.9 μg / mL, 25 μL was added to each well, and the working concentration was 0.3 μg / mL. The test sample was diluted with the experimental culture medium to 0.9 and 0.3 μg / mL, and 25 μL was added to each well. After being placed in an incubator for 5 h, 50 μL of luciferase substrate (One-Lite Luciferase Assay System, Promega, VAZDD1203-03) was added, and the luciferase activity was detected. The RLUS signal was detected by an enzyme-labeled instrument, and the neutralization rate was calculated according to the formula: [RLUS(TL1A)-RLUS(Sample)] / RLUS(TL1A)*100. The results are shown in Figure 1. The neutralization activity of antibody KNR4-M0038 at low and high concentrations was higher than that of positive controls RVT-3101 (HC: SEQ ID NO: 59; LC: SEQ ID NO: 60) and PRA-023 (HC: SEQ ID NO: 61; LC: SEQ ID NO: 62).

[0266] 2.2 Affinity

[0267] The binding kinetics of KNR4-M0038 to Nhis-TL1A-ECD-biotin (self-produced Nhis-TL1A-ECD protein, labeled by biotin labeling kit EZ-Link™ Sulfo-NHS-Biotin (thermo, Cat. No. 21217)) were detected by Biolayer interferometry (BLI) technology, and compared with PRA-023 and RVT-3101. Nhis-TL1A-ECD-biotin was directly immobilized on the SA sensor, and then the antibody protein was diluted to 5 concentrations (25, 12.5, 6.25, 3.13, 1.56 nM), and combined with the immobilized protein. The equilibrium dissociation constant (KD), association rate (ka) and dissociation rate (kdis) were calculated using Data Analysis HT 12.0.2.59 software. The results are shown in Table 1, and the affinity of antibody KNR4-M0038 to TLA1 is similar to that of positive controls PRA-023 and RVT-3101.

[0268] Table 1 Affinity of anti-TL1A antibodies

[0269] 2.3 Blocking activity to TL1A and DR3 binding

[0270] 96-well plates were coated with 3 μg / mL hDR3-ECD-Fc-Chis (SEQ ID NO: 58), 3% BSA was blocked for 2 h, KNR4-M0038, PRA-023, RVT-3101 were diluted (5-fold gradient dilution of 8 concentrations) in a solution containing 5 μg / mL Nhis-TL1A-ECD-Biotin, after adding gradient-diluted samples (final concentration 0.002-700 nM) and incubating thoroughly, 1:4000 diluted Streptavidin (HRP) (abcam, CAT. AB7403, Lot: 1047334-3) was added, and KPL TMB Microwell Peroxidase Substrate (1-Component) (Cat: 5120-0074, Lot: 10662425) was used for color development, and then detected on a microplate reader. The results are shown in Figure 2, and the blocking activity of KNR4-M0038 to TL1A and DR3 binding is better than that of PRA-023 and RVT-3101.

[0271] 2.4 Mouse IBD model

[0272] 20 eight to ten weeks old male C57BL / 6 mice (purchased from Zhejiang Vantoll Life Experimental Animal Technology Co., Ltd.) were randomly divided into four groups according to the non-fasting body weight of the animals, 5 in each group. They were G1: normal group + PBS, G2: model group + PBS, G3: model group + KNR4-M0038 10 mg / kg, G4: model group + RVT-3101 10 mg / kg, single intravenous (iv) administration. The day of grouping and administration was recorded as D0, and from D0, the G2-G4 groups were given drinking water containing 3% DSS (changed every 2 days) every day to induce an acute inflammatory bowel disease (IBD) model, while the G1 group was given normal drinking water. The endpoint was D7. The body weight, loose stool and bloody stool of the mice were measured every day, and the DAI score was calculated. The colon tissue was taken out at the end of the experiment, and the colon length was measured. The results are shown in Figures 3a-3c. KNR4-M0038 can significantly improve the body weight loss, DAI score and colon length in this model, and the effect is better than that of the control molecule RVT-3101.

[0273] Example 3 Humanization of the antibody

[0274] The humanization of the variable region of the murine antibody KNR4-M0038 was performed using the CDR grafting strategy. The CDR sequences of the heavy chain and the light chain of the mouse M0038 were grafted into the human framework sequence VH1-2*02 and VL6D-41*01, respectively. The heavy chain constant region used IgG1 CH1, hinge region and CH2-CH3 region, and the light chain contained IgKappa CL domain. Then, back mutations introduced to the murine antibody sequence in the framework region of both VH and VL were introduced, and the complete recovery of binding activity was evaluated. Finally, the humanized heavy chain variable region M0038-hVHv1 (SEQ ID NO: 27) and M0038-hVHv2 (SEQ ID NO: 28), and the humanized light chain variable region M0038-hVLv1 (SEQ ID NO: 29) and M0038-hVLv2 (SEQ ID NO: 30) were obtained. Figures 4a and 4b show the sequence alignment results of the antibody VH and VL before and after humanization.

[0275] The above humanized sequences were gene synthesized, the VH was fused with a DNA fragment encoding human IgGl constant region, and cloned into a conventional mammalian expression vector to obtain the M0038 antibody heavy chain M0038-hHCvl (SEQ ID NO: 31), M0038-hHCv2 (SEQ ID NO: 32), M0038-hHCvl-AALS (SEQ ID NO: 33), M0038-hHCv2-AALS (SEQ ID NO: 34) and M0038-hHCvl-AA (SEQ ID NO: 63). The VL was fused with a DNA fragment encoding human Ig kappa constant region, and cloned into a conventional mammalian expression vector to obtain the M0038 antibody light chain M0038-hLCvl (SEQ ID NO: 35) and M0038-hLCv2 (SEQ ID NO: 36). The sequences of the final constructs were verified by DNA sequencing.

[0276] The mixture of antibody heavy chain and antibody light chain plasmids (combination mode see Table 2) was transfected into HEK293 cells for transient expression of the antibody. The recombinant expression plasmid was diluted with Freestyle293 medium and added to the required PEI (Polyethylenimine) solution, and each group of plasmid / PEI mixture was added to the HEK293 cell suspension, which was then placed in a 37°C, 5% CO2, 130 rpm incubator. Four hours later, EX293 medium and 2 mM glutamine were added and incubated at 130 rpm. After 24 hours, 3.8 mM valproic acid was added, and 4 g / L glucose was added after 72 hours. After 5-6 days of culture, the transient expression culture supernatant was collected, and the target protein was purified by Protein A affinity chromatography. The purity of the protein was evaluated by SDS-PAGE.

[0277] Table 2 Heavy chain and light chain combination of target antibody

[0278] Example 4 Detection of the biological activity of humanized antibody

[0279] 4.1 Cell neutralization activity

[0280] RPMI 1640 + 2 ng / ml GM-CSF containing 10% FBS was the experimental medium, and 25000 cells / 25 μL were added to each well after the collection of TF1-NFkB-RE polyclonal cells. Nhis-TL1A-ECD 0.9 μg / mL was prepared, 25 μL was added to each well, and the working concentration was 0.3 μg / mL; 30 μg / mL samples were prepared, 3-fold dilution, 7 concentrations, and 25 μL was added to each well; after 5 h in the incubator, 50 μL of substrate was added, and the Luciferase activity was detected. The results are shown in Figures 5a and 5b, and the humanized antibody showed equal or slightly better cell neutralization activity than KNR4-M0038.

[0281] The cell activity of M0038-hu11 and the positive controls PRA-023 and RVT-3101 were compared using a similar method, and the results are shown in Figure 5c. The cell neutralization activity of M0038-hu11 was equivalent to that of RVT-3101 and better than that of PRA-023.

[0282] 4.2 Affinity

[0283] The binding kinetics of M0038 humanized antibodies to human TL1A were detected according to the method of Example 2.2. The results are shown in Table 3, and the humanized antibodies maintained high affinity to human TL1A, with M0038-hu11 having better affinity than the parent KNR4-M0038.

[0284] Table 3 Affinity of humanized antibodies

[0285] Based on the BLI technology, the affinity of M0038-hu11-AALS to human, cynomolgus monkey, rat and mouse TL1A was also detected. M0038-hu11-AALS was first diluted to 10 μg / ml and immobilized on the Protein A biosensor, and then human TL1A, cynomolgus monkey TL1A (purchased from ACRO, item number: TLA-C5241) and rat TL1A (purchased from ACRO, item number: TLA-R5249) were diluted to 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM 5 concentration gradients; mouse TL1A (Nhis-mTL1A-ECD, mouse TL1A extracellular region protein, Uniprot ID: Q5UBV8) was diluted to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM 5 concentration gradients, and 0 nM was set as a control, which was combined with M0038-hu11-AALS respectively, and different intensity of binding and dissociation signals could be detected. The detection results of M0038-hu11-AALS to human, cynomolgus monkey, rat and mouse TL1A were fitted by using 1:1 model, and the dissociation time of mouse TL1A was fitted by 60 s, and the dissociation time of the rest species TL1A was fitted by 1200 s, and the equilibrium dissociation constant (KD) value of the sample was calculated. The results are shown in Table 4, M0038-hu11-AALS has obvious binding to various species of TL1A, and the affinity to human, cynomolgus monkey and rat TL1A is stronger.

[0286] Table 4 Affinity of M0038-hu11-AALS to various species of TL1A

[0287] 4.3 Blocking activity to human TL1A and DR3 binding

[0288] Referring to the method of Example 2.3, the blocking activity of M0038 humanized antibody to human TL1A and DR3 binding was detected. The results are shown in Figure 6, and the humanized antibody maintains the blocking activity to human TL1A and DR3 binding.

[0289] The blocking activity of M0038-hu11-AALS on human TL1A and DR3 binding was also tested: 96-well plates were coated with 3 pg / mL Nhis-TL1A-ECD, blocked with 3% BSA for 2 h, M0038-hu11-AALS, PRA-023, RVT-3101 were diluted (5-fold gradient dilution for 10 concentrations) in a solution containing 5 pg / mL hDR3-ECD-Fc-Chis-biotin, after adding the gradient-diluted samples, incubated thoroughly, then 1:4000 diluted Streptavidin (HRP) was added, KPL SureBlue™ TMB Microwell Peroxidase Substrate (1-Component, purchased from Thermo, item number: 34029) was used for color development, and then detected on a microplate reader. The results are shown in Figure 7, the blocking activity of M0038-hu11-AALS (IC 50 = 3.48 nM) was slightly better than the positive controls PRA-023 (IC 50 = 11 nM) and RVT-3101 (IC 50 = 7.8 nM).

[0290] 4.4 Blocking activity of TL1A stimulating CD4+T cells to release IFN-γ

[0291] CD4+T cells were sorted from PBMC cells using CD4+T Cell Isolation Kit (purchased from Stemcell, item number: 17952), and the cell density was adjusted to 3 x 10 6 / ml using 10% FBS + 1640 + Human CD3 / CD28 T Cell Activator medium (purchased from Stemcell, item number: 10991), and cultured in a 6-well plate for two days. After two days, CD4+T cells were collected, and the density was adjusted to 2 x 10 6 / ml using 10% FBS + 1640 medium. Nhis-TL1A-ECD was prepared to a concentration of 300 ng / ml using 10% FBS + 1640 medium. RVT-3101, PRA-023 and M0038-hu11 were prepared to concentrations of 7500, 1500, 300, 60 and 12 ng / ml, respectively, using 10% FBS + 1640 medium. In a 96-well plate, 50 mΐ of CD4+T cell suspension, 50 mΐ of Nhis-TL1A-ECD protein solution, and 50 mΐ of each concentration of drug were added in turn, and incubated in an incubator for 24 h, with 3 replicates for each group. After incubation, the supernatant was collected by centrifuging the 96-well plate at 1500 rpm for 5 min, and the content of IFN-γ in the supernatant was detected using a commercial ELISA kit according to the instructions (purchased from Dyou, item number: 1110003).

[0292] The ELISA detection procedure is as follows:

[0293] 1. Add 100 μΐ of sample (undiluted) to the enzyme-labeled plate of the kit, and then add 50 μΐ of biotin antibody (50x dilution) to each well. Incubate at 25°C for 2 h on a shaker (about 300 rpm).

[0294] 2. Wash the enzyme-labeled plate with 300 μΐ of Wash Buffer for 3 times, and then add 100 μΐ of HRP-linked antibody (100x dilution) to each well. Incubate at 25°C for 30 min.

[0295] 3. Wash the enzyme-labeled plate with 300 μΐ of Wash Buffer for 3 times, and then add 100 μΐ of TMB Substrate to each well. Incubate at 25°C for 15 min.

[0296] 4. Add 100 μΐ of Stop Solution to each well, mix, and then detect the absorbance at 450 nm using an enzyme-labeled instrument (SpectraMax iD5 Reader, MD).

[0297] The results are shown in Figure 8. M0038-hu11 significantly inhibited the release of IFN-γ from CD4+ T cells stimulated by TL1A, and was superior to the positive control PRA-023.

[0298] 4.5 Pharmacokinetic properties in FcRn humanized mice

[0299] Eighteen qualified 6-8 week old male FcRn humanized mice (B-hFcRn mice, strain name: C57BL / 6-Fcgrttm1(FCGRT)Bcgen / Bcgen, purchased from Bao Saigu Jiangsu Gene Biotechnology Co., Ltd., item number: 110001) were randomly divided into 3 groups according to body weight, 6 in each group. The animals in G1-G3 groups were intravenously administered with 5 mg / kg of M0038-hu11-AA, M0038-hu11-AALS and RVT-3101, respectively. About 100 μΐ of blood was collected from the orbital vein before administration, and at 2 min, 0.5 h, 4 h, 24 h, 48 h, 72 h, 96 h (4 days), 168 h (7 days), 240 h (10 days), 336 h (14 days) and 504 h (21 days) after administration. The blood collection of each group of animals was alternated at different time points, i.e. n = 3 at each time point. The collected blood was added to a coagulation blood collection tube containing separation gel, and then placed on an ice bag. Within about 2 hours, the serum was separated by centrifugation at 4°C and 4000 rpm for 10 minutes, and used for detection of blood drug concentration and ADA.

[0300] Blood drug concentration and PK detection method:

[0301] Coating: Dilute Nhis-TL1A-ECD to 3 μg / ml in 50 mM CB, 100 μl / well, place in 2-8°C refrigerator for 12-24 h. Discard liquid from plate, PBST20 (0.05%, v / v) 300 μl / well, wash 3 times. Add 3% BSA blocking solution to block enzyme-linked plate, 250 μl / well, 25°C ± 2°C for 2 h. Discard liquid from plate, PBST20 (0.05%, v / v) 300 μl / well, wash 3 times.

[0302] Sample addition: M0038-hu11-AA, M0038-hu11-AALS and RVT-3101 were used as standard respectively to prepare standard curve, standard, quality control, serum samples were diluted with the same sample diluent, the diluent was PBST20 (0.05%, v / v) containing 5% mouse blank serum and 1% BSA. M0038-hu11-AA, M0038-hu11-AALS standard was diluted by 2.5 times from 20 ng / ml, a total of 8 dilutions. Quality control was set at 3 concentrations of 16 ng / ml, 2 ng / ml, 0.2 ng / ml. RVT-3101 standard was diluted by 2.5 times from 16 ng / ml, a total of 8 dilutions. Quality control was set at 3 concentrations of 12.8 ng / ml, 1.6 ng / ml, 0.16 ng / ml. Standard, quality control, sample were set with two repeats. The sample was diluted at the estimated concentration to make the concentration after dilution within the detection range of the standard curve. 100 μl / well, 25°C±2°C incubation for 2h, blank control was set at the same time, sample and control were set with two repeats, 100 μl / well, 25°C±2°C incubation for 2h. Discard the liquid in the plate, wash 3 times with PBST20 (0.05%, v / v) 300 μl / well. Dilute Goat Anti-Human IgG, Monkey ads-HRP (purchased from SouthernBiotech, item number: 2049-05) with 1% BSA PBST20 (0.05%, v / v) diluent at 1:8000, add to the enzyme-labeled plate, 100 μl / well, 25°C±2°C incubation for 2h. Discard the liquid in the plate, wash 3 times with PBST20 (0.05%, v / v) 300 μl / well. Add 1-StepTM Ultra TMB-ELISA (purchased from Thermo, item number: 34029), 100 μl / well, color development at room temperature in the dark. Add 1M H2SO4 100 μl / well to stop the color development reaction. After termination, place the enzyme-labeled plate on the enzyme-labeled instrument to read the absorbance value at 450nm and 650nm wavelengths. The final absorbance value is the absorbance value at 450nm wavelength minus the absorbance value at 650nm wavelength. Use software Softmax Pro for editing and data processing analysis. Calculate and analyze the blood drug concentration in the sample according to the standard curve and sample absorbance value. Pharmacokinetic parameter analysis uses Pheonix WinNonlin (Certara company, version 8.1.0.3530) software non-compartment model calculation. Calculate the area under the concentration-time curve (AUC0-last) from 0 to t hours after drug administration by linear rising logarithmic descending trapezoidal rule. When calculating pharmacokinetic parameters, BLQ is set to 0 before Tmax and ND after Tmax. The calculated parameters include but are not limited to: T 1 / 2 , Cmax, Tmax, AUClast, etc.

[0303] ADA analysis method (take M0038-hu11-AALS as an example):

[0304] Dilute M0038-hu11-AALS to 1 pg / mL with Coating Buffer pH 9.6, 100 pL / well, and place in a 2-8 °C refrigerator for 12-24 h. Discard the liquid in the plate, add PBST20 (0.05%, v / v) 300 pL / well, and wash 3 times. Add 3% BSA blocking solution to block the enzyme-labeled plate, 250 pL / well, and incubate at 25 °C ± 2 °C for 2 h. Discard the liquid in the plate, add PBST20 (0.05%, v / v) 300 pL / well, and wash 3 times. Dilute the B-hFcRn mouse serum samples of the administration group 10-fold with 1% BSA-containing PBST20 (0.05%, v / v) diluent, and load the samples. The B-hFcRn mouse serum before administration is the negative control and is also diluted, and muFc-GAP-TL1A-ECD and Nhis-TL1A-ECD are the positive controls and are diluted to 10 pg / mL. Load the diluted serum samples and control samples. Load 100 pL / well of each sample and control, and incubate at 25 °C ± 2 °C for 2 h, while setting up a blank control. Discard the liquid in the plate, add PBST20 (0.05%, v / v) 300 pL / well, and wash 3 times. Dilute M0038-hu11-AALS-Biotin to 0.2 pg / mL with 1% BSA-containing PBST20 (0.05%, v / v) diluent, mix well, load, 100 pL / well, and incubate at 25 °C ± 2 °C for 1.5 h. Discard the liquid in the plate, add PBST20 (0.05%, v / v) 300 pL / well, and wash 3 times. Dilute Streptavidin HRP 1:2000 with 1% BSA-containing PBST20 (0.05%, v / v) diluent, 100 pL / well, and incubate at 25 °C ± 2 °C for 1.5 h.

[0305] Discard the liquid in the plate, add PBST20 (0.05%, v / v) 300 pL / well, and wash 3 times. Add 1-Step™ Ultra TMB-ELISA, 100 pL / well, and develop at room temperature. Add 1 M H2SO4 100 pL / well to stop the color development reaction. Place the enzyme-labeled plate on a microplate reader to read the absorbance values at 450 nm and 650 nm wavelengths. The final absorbance value is the absorbance value at 450 nm wavelength minus the absorbance value at 650 nm wavelength. According to the test data analysis and judgment, determine whether the sample has immunogenicity.

[0306] The results are shown in Table 5 and Figure 9. The half-life of the humanized antibodies of the present disclosure is longer than that of the control molecule RVT-3101, and the half-life of the antibody molecule can be further prolonged after introducing the LS mutation in the Fc. The half-life of the G2 group is about 1.76 times that of the G3 group. The results of the ADA detection on day 21 show that 2 mice in the G3 group were positive for ADA (2 / 3), and no mice in the G2 group were positive for ADA (0 / 3), indicating that the TL1A molecules of the present disclosure have low immunogenicity.

[0307] Table 5 PK parameters in FcRn humanized mice

[0308] (*PK parameters of the G3 group after deleting the D21 concentration point)

[0309] SEQUENCE LISTING:

Claims

1. An antibody or antigen-binding fragment thereof that binds TL1A, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises: a HCDR1 comprising the HCDR1 of the heavy chain variable region sequence set forth in SEQ ID NO: 1 or an amino acid sequence having one or two amino acid substitutions, deletions, or additions relative to the HCDR1 of the heavy chain variable region sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the HCDR2 of the heavy chain variable region sequence set forth in SEQ ID NO: 1 or an amino acid sequence having one or two amino acid substitutions, deletions, or additions relative to the HCDR2 of the heavy chain variable region sequence set forth in SEQ ID NO: 1, a HCDR3 comprising the HCDR3 of the heavy chain variable region sequence set forth in SEQ ID NO: 1 or an amino acid sequence having one or two amino acid substitutions, deletions, or additions relative to the HCDR3 of the heavy chain variable region sequence set forth in SEQ ID NO: 1; the light chain variable region comprises: a LCDR1 comprising the LCDR1 of the light chain variable region sequence set forth in SEQ ID NO: 2 or an amino acid sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR1 of the light chain variable region sequence set forth in SEQ ID NO: 2, a LCDR2 comprising the LCDR2 of the light chain variable region sequence set forth in SEQ ID NO: 2 or an amino acid sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR2 of the light chain variable region sequence set forth in SEQ ID NO: 2, a LCDR3 comprising the LCDR3 of the light chain variable region sequence set forth in SEQ ID NO: 2 or an amino acid sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR3 of the light chain variable region sequence set forth in SEQ ID NO:

2.

2. The antibody or antigen-binding fragment thereof of claim 1, the heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence of the HCDR1 of the heavy chain variable region sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of the HCDR2 of the heavy chain variable region sequence set forth in SEQ ID NO: 1, a HCDR3 comprising the amino acid sequence of the HCDR3 of the heavy chain variable region sequence set forth in SEQ ID NO: 1; the light chain variable region comprises: a LCDR1 comprising the amino acid sequence of the LCDR1 of the light chain variable region sequence set forth in SEQ ID NO: 2, a LCDR2 comprising the amino acid sequence of the LCDR2 of the light chain variable region sequence set forth in SEQ ID NO: 2, a LCDR3 comprising the amino acid sequence of the LCDR3 of the light chain variable region sequence set forth in SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the following definition system: Kabat, AbM, Chothia, or IMGT.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein, the heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3; a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4; a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5; the light chain variable region comprises: a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15; a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16; a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17; or, the heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6; a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7; a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; the light chain variable region comprises: a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18; a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19; a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; or, the heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10; a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; the light chain variable region comprises: a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21; a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22; a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or, the heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12; a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13; a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14; the light chain variable region comprises: a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 24; a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25; a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

26.

5. The antibody or antigen-binding fragment of any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

6. The antibody or antigen-binding fragment of any one of claims 1-5, wherein the antigen-binding fragment comprises a Fab, Fab’, Fv fragment, F(ab’)2, scFv, or di-scFv.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 1 ; the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more sequence identity to SEQ ID NO:

2.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:

2.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 27 or 28.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 29 or 30.

11. The antibody or antigen-binding fragment thereof according to claim 9 or 10, which is selected from the group consisting of: 1) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 27 and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 29; 2) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 27 and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 30; 3) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 28 and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 29; and 4) the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 28 and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:

30.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1-11, wherein the heavy chain further comprises a constant region of human IgGl, IgG2, IgG3 or IgG4 or a variant thereof; preferably, the constant region or variant thereof comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 39-51.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1-12, wherein the light chain further comprises a constant region of human IgK or a variant thereof; preferably, the constant region or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO:

52.

14. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain and a light chain, which are selected from the group consisting of: 1) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 37 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 37, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 38 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 38; 2) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 31 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 31, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 35 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 35; 3) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 31 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 31, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 36 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 36; 4) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 32 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 35 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 35; 5) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 32 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 32, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 36 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO: 36; 6) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:33 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO:33, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:35 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO:35; 7) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:33 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO:33, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:36 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO:36; 8) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:34 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO:34, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:35 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO:35; and 9) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:34 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO:34, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:36 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO:36; 10) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO:63 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO:63, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:35 or an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or greater sequence identity to SEQ ID NO:

35.

15. The antibody or antigen-binding fragment thereof of any one of claims 1-14, which has at least one of the following characteristics: (1) has cellular neutralization activity; (2) a KD value of less than 1 for binding to human TL1A ╳ 10 -7 M, preferably less than 1 ╳ 10 -8 M, more preferably less than 1 ╳ 10 -9 M, even more preferably less than 1 ╳ 10 -10 M; (3) is capable of blocking the binding of human TL1A to DR3; (4) is capable of ameliorating body weight loss, reducing DAI score and / or restoring colon length in a dextran sulfate sodium (DSS)-induced mammalian IBD model.

16. A fusion protein comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15.

17. A bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15.

18. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-15 or the fusion protein of claim 16 or the bispecific or multispecific antibody of claim 17; preferably, the nucleic acid is DNA or RNA.

19. A vector comprising the nucleic acid molecule of claim 18.

20. A recombinant cell comprising the nucleic acid molecule of claim 18 and / or the vector of claim 19.

21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15 and / or the fusion protein of claim 16, and / or the bispecific or multispecific antibody of claim 17, and / or the nucleic acid molecule of claim 18, and / or the vector of claim 19, and / or the recombinant cell of claim 20, and a pharmaceutically acceptable carrier.

22. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15 and / or the fusion protein of claim 16, the bispecific or multispecific antibody of claim 17, and / or the nucleic acid molecule of claim 18, and / or the pharmaceutical composition of claim 21.

23. A method of treating and / or preventing a TL1A-associated disease or disorder, comprising administering to a patient in need thereof the antibody or antigen-binding fragment thereof of any one of claims 1-15 and / or the fusion protein of claim 16, the bispecific or multispecific antibody of claim 17, and / or the nucleic acid molecule of claim 18, and / or the vector of claim 19, and / or the pharmaceutical composition of claim 21.

24. The method of claim 23, wherein the TL1A-associated disease or disorder is an inflammatory bowel disease, such as ulcerative colitis and / or Crohn’s disease.

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