Antibody targeting TL1a and use thereof

Abstract

Disclosed in the present invention are an antibody targeting TL1A and the use thereof. The antibody of the present invention can be used for blocking the interaction between TL1A and DR3, is helpful for achieving the purpose of intervention from the early stages of inflammation, inhibits immune cells such as effector T cells to release cytokines for promoting an inflammatory response, and provides more possibilities for treatment methods for IBD patients or other TL1A-related diseases.

Description

SPECIFICATION

[0001] The present application claims priority to Chinese patent application No. 202410250612X filed on March 5, 2024. The content of the above Chinese patent application is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present disclosure belongs to the biological field, and specifically relates to an antibody targeting TL1A and the use thereof. BACKGROUND

[0003] Tumor necrosis factor (TNF)-like ligand 1A (TL1A) is a member of the TNF superfamily of proteins, also known as TNFSF15, which is primarily expressed by antigen-presenting cells (monocytes, macrophages, and dendritic cells), T cells, and endothelial cells. TL1A can be expressed on the cell surface or secreted as a soluble cytokine, and is a ligand for death receptor 3 (DR3). DR3 is mainly expressed on T cells, NK cells, NKT cells, Treg cells, innate lymphoid cells (ILCs), fibroblasts, and epithelial cells.

[0004] TL1A binds to its receptor DR3, activates downstream signaling, and participates in innate and adaptive immune homeostasis.

[0005] Studies have shown that TL1A plays an important role in many autoimmune diseases, such as inflammatory bowel disease (IBD), asthma, multiple sclerosis, and arthritis. Overexpression of TL1A can be detected in the mucosa and serum of patients with IBD, such as ulcerative colitis (UC) and Crohn's disease (CD). TL1A-DR3 interaction provides stimulatory signal for downstream signaling pathways, such as NF-kB and MAPK pathways, and amplifies effector T cell responses, including those of Th1, Th17, Th2, and Th9 cells.

[0006] Therefore, blockade of the interaction between TL1A and DR3 is conducive to the facilitation of early intervention in the inflammatory process, thereby suppressing the release of pro-inflammatory cytokines by immune cells such as effector T cells. This provides more possibilities for the treatment of IBD patients or other TL1A-related diseases. SUMMARY

[0007] The technical problem to be solved by the present disclosure is the deficiency in the prior art due to the lack of drugs utilizing TL1A for the treatment of related diseases, thereby providing an antibody targeting TL1A and the use thereof. The antibody of the present disclosure can effectively bind to TL1A and modulate its activity, and has the ability to reduce or inhibit the binding of TL1A to its receptor DR3, thereby reducing or inhibiting downstream receptor signaling.

[0008] The present disclosure solves the above technical problem through the following technical solutions.

[0009] A first aspect of the present disclosure provides a TL1A-targeting antibody or an antigenbinding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region;

[0010] the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein

[0011] HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12;

[0012] or, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 28, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 30, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 31, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 32;

[0013] or, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 49, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 50, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 51, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 52, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 53, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 54;

[0014] or, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 49, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 57, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 58, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 59, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 60, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 61.

[0015] In some embodiments, the heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 13 to 19, and the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 20 to 23.

[0016] In some embodiments, the heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 33 to 39, and the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 40 to 46.

[0017] In some embodiments, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 55, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 56.

[0018] In some embodiments, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 62, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 63.

[0019] In some embodiments, the variable region has a combination of a heavy chain variable region and a light chain variable region shown in Table 1 below,

[0020] Table 1: Combination of variable regions Heavy chain variable region Light chain variable region SEQ ID NO: 13 SEQ ID NO: 20 SEQ ID NO: 13 SEQ ID NO: 21 SEQ ID NO: 13 SEQ ID NO: 22 SEQ ID NO: 13 SEQ ID NO: 23 SEQ ID NO: 14 SEQ ID NO: 20 SEQ ID NO: 14 SEQ ID NO: 21 SEQ ID NO: 14 SEQ ID NO: 22 SEQ ID NO: 14 SEQ ID NO: 23 SEQ ID NO: 15 SEQ ID NO: 20 SEQ ID NO: 15 SEQ ID NO: 21 SEQ ID NO: 15 SEQ ID NO: 22 SEQ ID NO: 15 SEQ ID NO: 23 SEQ ID NO: 16 SEQ ID NO: 20 SEQ ID NO: 16 SEQ ID NO: 21 SEQ ID NO: 16 SEQ ID NO: 22 SEQ ID NO: 16 SEQ ID NO: 23 SEQ ID NO: 17 SEQ ID NO: 20 SEQ ID NO: 17 SEQ ID NO: 21 SEQ ID NO: 17 SEQ ID NO: 22 SEQ ID NO: 17 SEQ ID NO: 23 SEQ ID NO: 18 SEQ ID NO: 20 SEQ ID NO: 18 SEQ ID NO: 21 SEQ ID NO: 18 SEQ ID NO: 22 SEQ ID NO: 18 SEQ ID NO: 23 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 19 SEQ ID NO: 21 SEQ ID NO: 19 SEQ ID NO: 22 SEQ ID NO: 19 SEQ ID NO: 23 SEQ ID NO: 33 SEQ ID NO: 40 SEQ ID NO: 33 SEQ ID NO: 41 SEQ ID NO: 33 SEQ ID NO: 42 SEQ ID NO: 33 SEQ ID NO: 43 SEQ ID NO: 33 SEQ ID NO: 44 SEQ ID NO: 33 SEQ ID NO: 45 SEQ ID NO: 33 SEQ ID NO: 46 SEQ ID NO: 34 SEQ ID NO: 40 SEQ ID NO: 34 SEQ ID NO: 41 SEQ ID NO: 34 SEQ ID NO: 42 SEQ ID NO: 34 SEQ ID NO: 43 SEQ ID NO: 34 SEQ ID NO: 44 SEQ ID NO: 34 SEQ ID NO: 45 SEQ ID NO: 34 SEQ ID NO: 46 SEQ ID NO: 35 SEQ ID NO: 40 SEQ ID NO: 35 SEQ ID NO: 41 SEQ ID NO: 35 SEQ ID NO: 42 SEQ ID NO: 35 SEQ ID NO: 43 SEQ ID NO: 35 SEQ ID NO: 44 SEQ ID NO: 35 SEQ ID NO: 45 SEQ ID NO: 35 SEQ ID NO: 46 SEQ ID NO: 36 SEQ ID NO: 40 SEQ ID NO: 36 SEQ ID NO: 41 SEQ ID NO: 36 SEQ ID NO: 42 SEQ ID NO: 36 SEQ ID NO: 43 SEQ ID NO: 36 SEQ ID NO: 44 SEQ ID NO: 36 SEQ ID NO: 45 SEQ ID NO: 36 SEQ ID NO: 46 SEQ ID NO: 37 SEQ ID NO: 40 SEQ ID NO: 37 SEQ ID NO: 41 SEQ ID NO: 37 SEQ ID NO: 42 SEQ ID NO: 37 SEQ ID NO: 43 SEQ ID NO: 37 SEQ ID NO: 44 SEQ ID NO: 37 SEQ ID NO: 45 SEQ ID NO: 37 SEQ ID NO: 46 SEQ ID NO: 38 SEQ ID NO: 40 SEQ ID NO: 38 SEQ ID NO: 41 SEQ ID NO: 38 SEQ ID NO: 42 SEQ ID NO: 38 SEQ ID NO: 43 SEQ ID NO: 38 SEQ ID NO: 44 SEQ ID NO: 38 SEQ ID NO: 45 SEQ ID NO: 38 SEQ ID NO: 46 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 39 SEQ ID NO: 41 SEQ ID NO: 39 SEQ ID NO: 42 SEQ ID NO: 39 SEQ ID NO: 43 SEQ ID NO: 39 SEQ ID NO: 44 SEQ ID NO: 39 SEQ ID NO: 45 SEQ ID NO: 39 SEQ ID NO: 46 SEQ ID NO: 55 SEQ ID NO: 56 SEQ ID NO: 62 SEQ ID NO: 63

[0021] In some embodiments, the antibody or the antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region.

[0022] In the present disclosure, the heavy chain constant region and the light chain constant region are of human or murine origin.

[0023] In some specific embodiments, the heavy chain constant region has an amino acid sequence shown in any one of SEQ ID NOs: 64 to 67.

[0024] In some specific embodiments, the light chain constant region has the amino acid sequence shown in SEQ ID NO: 68 or 69.

[0025] In some embodiments, the antibody or the antigen-binding fragment thereof is a fulllength antibody, Fab, Fab', F(ab')2, or Fv, such as scFv.

[0026] In some specific embodiments, the heavy chain of the antibody or the antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO: 24, and the light chain of the antibody or the antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO: 25 or 26.

[0027] In some specific embodiments, the heavy chain of the antibody or the antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO: 47, and the light chain of the antibody or the antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO: 48.

[0028] A second aspect of the present disclosure provides an antibody combination comprising at least two antibodies or antigen-binding fragments thereof according to the first aspect.

[0029] A third aspect of the present disclosure provides an isolated nucleic acid encoding the antibody or the antigen-binding fragment thereof according to the first aspect or the antibody combination according to the second aspect.

[0030] A fourth aspect of the present disclosure provides a recombinant expression vector comprising the isolated nucleic acid according to the third aspect.

[0031] In some embodiments, the recombinant expression vector is a plasmid, a cosmid, a phage, or a viral vector.

[0032] In some specific embodiments, the plasmid is a pCDNA3.1 plasmid.

[0033] A fifth aspect of the present disclosure provides a transformant comprising the isolated nucleic acid according to the third aspect or the recombinant expression vector according to the fourth aspect in a host cell.

[0034] In some embodiments, the transformant is a non-animal or plant variety.

[0035] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0036] In some specific embodiments, the host cell is selected from a yeast cell, a mammalian cell, or other cells suitable for preparing an antibody or an antigen-binding fragment thereof.

[0037] In some specific embodiments, the mammalian cell is a HEK293 cell.

[0038] A sixth aspect of the present disclosure provides a method for preparing a TL1A- targeting antibody or an antigen-binding fragment thereof, comprising culturing the transformant according to the fifth aspect, and obtaining the TL1A-targeting antibody or the antigen-binding fragment thereof from the culture.

[0039] A seventh aspect of the present disclosure provides a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof according to the first aspect, the antibody combination according to the second aspect, the isolated nucleic acid according to the third aspect, the recombinant expression vector according to the fourth aspect, or the transformant according to the fifth aspect, and a pharmaceutically acceptable excipient.

[0040] An eighth aspect of the present disclosure provides a TL1A detection agent comprising the antibody or the antigen-binding fragment thereof according to the first aspect and / or the antibody combination according to the second aspect.

[0041] In some embodiments, the TL1A detection agent is used for detection for non-diagnostic purposes.

[0042] A ninth aspect of the present disclosure provides the use of the antibody or the antigenbinding fragment thereof according to the first aspect, the antibody combination according to the second aspect, the isolated nucleic acid according to the third aspect, the recombinant expression vector according to the fourth aspect, the transformant according to the fifth aspect, or the pharmaceutical composition according to the seventh aspect in the manufacture of a diagnostic agent or a detection agent.

[0043] In some embodiments, the diagnostic agent or the detection agent is used for diagnosing or detecting a TL1A-mediated disease.

[0044] A tenth aspect of the present disclosure provides the use of the antibody or the antigenbinding fragment thereof according to the first aspect or the antibody combination according to the second aspect in the manufacture of a medicament for preventing or treating a TL1A-mediated disease.

[0045] In some embodiments, the disease is selected from at least one of the following: inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, allergy, diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, transplant rejection, graft-versus-host disease (GVHD), primary biliary cirrhosis, atherosclerosis, bladder syndrome / interstitial cystitis, urointestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis, and scleroderma.

[0046] An eleventh aspect of the present disclosure provides a method for preventing, ameliorating, or treating a TL1A-mediated disease, disorder, or condition, comprising administering to a subject in need thereof an effective amount of the antibody or the antigen-binding fragment thereof according to the first aspect, the antibody combination according to the second aspect, or the pharmaceutical composition according to the seventh aspect of the present disclosure.

[0047] In some embodiments, the disease is selected from at least one of the following: inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, allergy, diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, transplant rejection, graft-versus-host disease (GVHD), primary biliary cirrhosis, atherosclerosis, bladder syndrome / interstitial cystitis, urointestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis, and scleroderma.

[0048] A twelfth aspect of the present disclosure provides the antibody or the antigen-binding fragment thereof according to the first aspect, the antibody combination according to the second aspect, or the pharmaceutical composition according to the seventh aspect, for use in preventing, ameliorating, or treating a TL1A-mediated disease, disorder, or condition.

[0049] In some embodiments, the disease is selected from at least one of the following: inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, allergy, diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, transplant rejection, graft-versus-host disease (GVHD), primary biliary cirrhosis, atherosclerosis, bladder syndrome / interstitial cystitis, urointestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis, and scleroderma.

[0050] A thirteenth aspect of the present disclosure provides a method for detecting TL1A, comprising contacting the antibody or the antigen-binding fragment thereof according to the first aspect or the antibody combination according to the second aspect with a sample to be tested.

[0051] In some embodiments, the detection is for non-diagnostic purposes, such as quantifying or qualifying TL1A in a sample to be tested by detecting the antibody. In some embodiments, the sample to be tested is a tissue or a cell.

[0052] On the basis of common knowledge in the art, the above preferred conditions can be arbitrarily combined to obtain preferred examples of the present disclosure.

[0053] The reagents and raw materials used in the present disclosure are commercially available.

[0054] The positive and progressive effects of the present disclosure are as follows:

[0055] The present disclosure relates to an antibody that binds to TL1A and modulates its activity. The antibody or the antigen-binding fragment thereof in the present disclosure may have the ability to reduce or inhibit the binding of TL1A to its receptor DR3 and thereby reduce or inhibit downstream receptor signaling. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG. 1 is the chemiluminescence value of TF-1-NFkB-Luc reporter cells.

[0057] FIG. 2 shows the EC50 values for hu7B5-1 and hu4G9 binding to hTL1A as determined by ELISA.

[0058] FIG. 3 shows the EC50 values for hu7B5-1, hu7B5-2, and hu4G9 binding to hTL1A as determined by flow cytometry.

[0059] FIG. 4 shows the IC50 values for hu7B5-1, hu7B5-2, and hu4G9 in blocking hTL1A-induced reporter cell activity.

[0060] FIG. 5 shows the OD450 values for hu7B5-1 and hu4G9 in blocking the binding of hTL1A to DR3 as determined by ELSIA.

[0061] FIG. 6 shows the IC50 values for hu7B5-1, hu7B5-2, and hu4G9 in blocking hTL1A-induced IFN-y release.

[0062] FIG. 7A and FIG. 7B show the OD450 values for hu7B5-1 and hu4G9 binding to human TRAIL / TNFSF10, human LIGHT / TNFSF14, and Human Fas Ligand / TNFSF6, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0063] The present disclosure is further described below by way of examples, but the present disclosure is not thereby limited to the scope of the described examples. Experimental methods for which specific conditions are not indicated in the following examples are selected according to conventional methods and conditions, or according to the commodity instructions.

[0064] Definitions

[0065] An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, or polypeptide, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, unless otherwise specified, the term encompasses not only intact polyclonal or monoclonal antibodies, but also any antigen-binding portion thereof that competes with the intact antibody for specific binding, fusion proteins comprising the antigen-binding portion, and any other modified configuration of the immunoglobulin molecule comprising an antigen recognition site. Antigen-binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), fragments comprising complementarity determining regions (CDRs), single-chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv, as well as polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide. Antibodies include an antibody of any class or an antigen-binding fragment thereof, such as IgG, IgA, or IgM (or subclass thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the heavy chain constant region, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as a, 8, s, y, and u, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0066] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion"), as used interchangeably herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TL1A). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0067] Antibodies may be derived from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals such as birds (e.g., chickens), fish (e.g., sharks), and camelids (e.g., llamas).

[0068] A "variable region" of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. As known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs) (also known as hypervariable regions), and contribute to the formation of the antigen-binding site of antibodies. If variants of a variable region of interest are desired, particularly with substitution in amino acid residues outside of the CDR region (i.e., in the framework region), appropriate amino acid substitution, preferably conservative amino acid substitution, can be identified by comparing the variable region of interest to variable regions of other antibodies containing CDR1 and CDR2 sequences of the same canonical class as the variable region of interest.

[0069] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0070] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or prepared using any of the techniques for producing human antibodies as disclosed herein. The definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0071] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody, or vice versa. The term also encompasses antibodies comprising a V region from one individual of a species (e.g., a first mouse) and a constant region from another individual of the same species (e.g., a second mouse).

[0072] As known in the art, a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0073] The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules, such as an antibody or a fragment thereof and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity).

[0074] As used herein, "vector" means a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0075] A "host cell" includes an individual cell or cell culture that may be or has been a recipient of vectors for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present disclosure.

[0076] The term "TL1A" means any naturally occurring form of TL1A (whether monomeric or multimeric), including dimers, trimers, etc., which may be derived from any suitable organism. As used herein, "TL1A" refers to mammalian TL1A, such as human, rat, or mouse, as well as non-human primate, bovine, ovine, or porcine TL1A. The term "TL1A" also encompasses fragments, variants, isoforms, and other homologs of such TL1A molecules. Variant TL1A molecules will typically be characterized by having the same type of activity as naturally occurring TL1A, such as the ability to bind DR3 and the ability to induce receptor- modulated activity.

[0077] The antibodies useful in the present disclosure may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, and Fc), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single-chain antibodies (ScFv), mutants thereof, fusion proteins comprising an antibody portion (e.g., a domain antibody), humanized antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen-binding site of the desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody may be of murine, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the TL1A antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In a particular embodiment, the antibody is a human antibody.

[0078] In the present disclosure, HCDR1 represents VH complementarity determining region 1, HCDR2 represents VH complementarity determining region 2, HCDR3 represents VH complementarity determining region 3, LCDR1 represents VL complementarity determining region 1, LCDR2 represents VL complementarity determining region 2, and LCDR3 represents VL complementarity determining region 3.

[0079] The culture medium used in the present disclosure is RPMI 1640 medium (purchased from Gibco, 61870036).

[0080] Example 1: Discovery of monoclonal antibodies targeting TL1A

[0081] 1.1 Immunization and fusion

[0082] Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized with TL1A protein, and splenocytes from the immunized mice showing strong antibody-specific responses to TL1A antigen were isolated. Splenocytes and myeloma cells were mixed at a ratio of 4:1, pre-warmed at 37C, centrifuged, and the supernatant was discarded. The bottom of the tube was gently tapped to loosen and homogenize the cell pellets, followed by incubation in a 37C water bath. 1 mL of PEG was added to the mixed cells within 1 minute with gentle mixing. Subsequently, 2 mL of pre-warmed SFM medium was added within 60 seconds, followed by the addition of 3 mL, 5 mL, and 10 mL up to a total of 30 mL within 3 minutes. The mixture was allowed to stand at 37C for 10 minutes. The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in MD2001 Clone Easy medium containing HAT and hybridoma culture supplements, and the total volume was adjusted to 240 mL with thorough mixing. The cell suspension was dispensed into a 96-well cell culture plate at 0.2 mL per well. After 6 days, the original medium was replaced with HAT medium. The growth of hybridoma cells was monitored, and when the cells reached a confluence of over 1 / 10 on the bottom area of the well, the supernatant was aspirated for antibody detection.

[0083] The amino acid sequence of TL1A used is shown in SEQ ID NO: 1.

[0084] 1.2 Preliminary screening of positive hybridoma cells by ELISA binding assay

[0085] An ELISA plate (purchased from Suzhou Beaver Bio, 40304) was coated with TL1A protein diluted in PBS at 1 pg / mL per well and incubated overnight at 4C. The coated plate was washed once with PBST, followed by the addition of blocking solution (PBS + 2% BSA + 0.01% Tween 20), and blocked at room temperature for approximately 2 hours. The blocking solution was discarded, and 90 pL of hybridoma culture supernatant was added corresponding to the 96-well plate, followed by static incubation at room temperature for 1 hour. The plate was washed three times with PBST, followed by the addition of 90 pL of secondary antibody diluted in antibody dilution buffer (PBS + 0.5% BSA + 0.01% Tween 20) at a 1:20,000 dilution, and allowed to stand at room temperature 13 for 1 hour. The plate was washed three times with PBST, followed by the addition of 45 pL of TMB solution for color development for approximately 5 minutes. Stop solution was then added to terminate the reaction, and the OD450 was read using a microplate reader. Based on the OD values, hybridoma cell wells with higher readings and stronger affinity to TL1A were selected for subculture. Specific results are shown in Table 2 and Table 3. As shown in Table 2, in the first hybridoma fusion, the 2B9 cell line exhibited a higher OD450 value of 1.1855, while in the secondary hybridoma fusion, the 3A6 cell line exhibited a higher OD450 value of 1.5874.

[0086] Table 2: ELISA screening results of cell supernatants from the first hybridoma fusion Cell line OD value Cell line OD value Cell line OD value 1G8 0.4959 7A5 0.5582 12E6 0.6554 2B9 1.1855 7D1 0.7323 17A3 0.801 2B10 0.6345 7D10 0.5885 19C9 0.4836 3D1 0.7159 7B5 0.5169 21C11 0.2422 4C6 0.6759 7G10 0.5013 6H8 0.7573 10G5 0.5312

[0087] Table 3: ELISA screening results of cell supernatants from the secondary hybridoma fusion Cell line OD value Cell line OD value Cell line OD value Cell line OD value 1B5 0.7875 7E4 1.0192 12H2 1.4856 16F12 0.7728 2C11 1.1098 7D11 1.0592 13B9 0.6856 17C4 0.5888 2G10 1.0556 7H4 1.1045 13C8 0.8093 17C9 0.637 3A6 1.5874 8B9 0.8311 14D3 1.0512 18D10 1.023 3E5 1.1556 8C5 0.8104 14G1 0.6559 19H1 0.7691 3F9 1.1386 8F11 0.8357 14G4 0.6804 19F4 1.0813 4C1 1.1233 9B4 0.8168 14F9 0.5806 19F8 0.7326 4G9 1.2625 9E12 0.8155 15C4 0.6736 20D7 0.4339 5H4 1.381 10F8 0.6772 15C9 0.6526 6B4 0.5615 10F11 0.6955 15G11 1.1743 7A4 1.1252 11D9 0.4207 16F8 0.5074

[0088] 1.3 Preliminary screening of positive hybridoma cells by reporter gene assay

[0089] To evaluate the function of antibodies in blocking intracellular signaling by inhibiting the TL1A-DR3 interaction, a TF-1-NFkB-Luc reporter cell line was constructed (NFkB-Luc reporter plasmid was purchased from Beyotime Biotechnology, D2206). TF-1 cells (purchased from Procell, CL-0232) are a human erythroleukemia cell line expressing DR3, the receptor for TL1A. TF-1 cells were transfected with the NFkB-Luc luciferase reporter gene to obtain cells transduced with the NFKB-driven luciferase gene. By detecting luciferase activity, the activation of NFkB pathway by TL1A through DR3 can be evaluated.

[0090] TF-1-NFkB-Luc reporter cells in the logarithmic growth phase were collected, washed twice with PBS, and starved overnight in GM-CSF-free RPMI 1640 medium (purchased from Gibco, 61870036). Hybridoma supernatants from the preliminary ELISA screening were added to a 96-well plate with a white bottom, followed by the addition of diluted TL1A protein, and incubated at 37OC for 30 minutes. Starved reporter cells were collected and counted, added at 30,000 cells per well, mixed well, and incubated at 37OC for 4.5 hours. 50 pL / well of One-lite luminescence substrate reagent (purchased from Vazyme, DD1203-03) was then added, followed by incubation at room temperature in the dark for 5 minutes, and chemiluminescence was read using a microplate reader. As shown in FIG. 1, 1G8, 6H8, 7B5, 7D10, 17A3, 19C9, 1B5, 3A6, 4G9, 5H4, 12H2, 13C8, 15C4, 15G11, 16F12, 18D10, and 20D7 were cells with inhibitory activity.

[0091] 1.4 ELISA assay to confirm subcloning of hybridoma cells

[0092] Based on the preliminary ELISA screening and the reporter gene assay, cells from 16 positive cell wells were selected for the first subcloning using the limited dilution method. The cells from the positive clone wells in the 96-well plate were resuspended and transferred to a 24-well plate containing 1 mL of medium. 20 pL of the suspended cell culture medium was aspirated into 80 pL of transfer medium, mixed well, followed by counting. According to the counting results, a diluted medium containing a total of 100 cells was added to 20 mL of transfer medium, mixed well, and seeded into the 96-well plate at 200 pL per well. After all positive clones were sequentially seeded, the 96-well plate was incubated in a cell incubator at 37OC, followed by observation under a microscope on days 4 to 5, and monoclonal wells were recorded. On day 7, subcloning ELISA screening was performed on the TL1A antigen-coated plate to select hybridoma cells secreting anti-TL1A specific antibodies. Based on the OD450 reading results and cell status, cells from wells 6H8F3, 7B5C11, 1B5C11, 3A6G8, 4G9E1, 12H2G4, 13C8G10, 15C4A11, 16F12A2, 18D10D11, 20D7E2, 17A3C8, and 19C9E4 were selected for subculture. After the cells transferred to the 24-well plate reached the logarithmic growth phase, they were transferred to a 10 cm diameter dish for culture. The cells were cryopreserved when they reached a confluence of over 80% on the bottom area of the dish.

[0093] Example 2: Expression and purification of hybridoma antibodies

[0094] After cryopreservation, the cells were continued to be cultured in serum-free medium in the dish. When the hybridoma cells again reached a confluence of over 80% on the bottom area of the dish, the cells were subcultured into multiple dishes. Each clone was cultured in approximately 50 mL total. After 5 to 7 days, the supernatant was harvested for purification. The harvested hybridoma cell culture supernatant was centrifuged (300 g, 10 minutes), filtered for clarification, and placed on ice as the loading sample. The Protein A column was equilibrated with equilibration buffer for 5 to 8 column volumes. The filtered supernatant was then passed through the column, and the flow-through was collected. After sample loading, the chromatography column was washed with equilibration buffer for 15 to 20 column volumes, or until the A280 absorbance dropped below 20 mAu. The bound antibodies were eluted with 20 mM sodium citrate elution buffer (pH = 3.0) to obtain approximately 2.5 mL of eluted protein. The eluate pH was immediately adjusted to neutral, and the antibody concentration was measured using Nanodrop and recorded.

[0095] Example 3: Preparation of TL1A-targeting monoclonal antibodies from Pfizer and Prometheus as reference antibodies

[0096] 3.1 The anti-TL1A monoclonal antibody PF-06480605 (RVT-3101) from Pfizer, with sequences referenced from patent US9683998B2, has the heavy chain amino acid sequence shown in SEQ ID NO: 2 and the light chain amino acid sequence shown in SEQ ID NO: 3.

[0097] 3.2 The anti-TL1A monoclonal antibody RPA-023 (MK-7240) from Prometheus / Merck & Co., with sequences referenced from patent WO2022178158A1, has the heavy chain amino acid sequence shown in SEQ ID NO: 4 and the light chain amino acid sequence shown in SEQ ID NO: 5.

[0098] Example 4: Detection of blocking effect of candidate molecules in reporter gene assay

[0099] TF-1-NFkB-Luc reporter cells in the logarithmic growth phase were collected, washed twice with PBS, and starved overnight in GM-CSF-free medium. Purified hybridoma antibodies (starting at 7.5 pg / mL, 4-fold serial dilution, a total of 8 concentration gradients) were added to a 96-well plate with a white bottom, followed by the addition of diluted TL1A protein to a final concentration of 20 ng / mL, and incubated at 37OC for 30 minutes. Starved reporter cells were collected and counted, added at 30,000 cells per well, mixed well, and incubated at 37OC for 4.5 hours. 50 pL well of One-lite luminescence substrate reagent was then added, followed by incubation at room temperature in the dark for 5 minutes, and chemiluminescence was read using a microplate reader. The results are shown in Table 4. Based on whether the reporter gene activation activity was completely blocked and the IC50 values, four clones, 4G9E1, 15C4A11, 18D10D11, and 7B5C11, were selected for further detection of the blocking effect on cytokine release activity.

[0100] Table 4: Blocking activity of hybridoma antibodies in reporter gene assay Hybridoma antibody IC50 (gg / mL) 1B5C11 1.314 3A6G8 0.1036 4G9E1 0.01192 12H2G4 0.07535 13C8G10 0.0186 15C4A11 0.01702 16F12A2 0.01585 18D10D11 0.01594 20D7E2 0.0267 6H8F3 0.0465 7B5C11 0.01019 17A3C8 3.619 19C9E4 5.356

[0101] Example 5: Detection of blocking effect of candidate molecules on IFN-y release from PBLs (peripheral blood lymphocytes) induced by TL1A combined with IL12 / IL18

[0102] Cryopreserved PBMCs were thawed in pre-warmed medium, centrifuged (300 g, 10 minutes), resuspended, and added to a culture flask for incubation at 37OC. After 2 hours, non-adherent lymphocytes (PBLs) were collected and counted, and seeded into a 96-well U-bottom plate at 0.2 M / well. Serially diluted antibodies to be tested (10 ugmL. 3-fold serial dilution, 8 gradients) were added to the 96-well U-bottom plate, followed by the addition of 50 ng / mL TL1A protein. 0.5 ng / mL IL-12 and 5 ng / mL IL-18 proteins were then added to the U-bottom plate, followed by incubation at 37OC. After 24 hours of incubation, the plate was centrifuged at 2000 rpm for 15 minutes, culture supernatants were collected, and IFN-y levels in the supernatants were measured by ELISA. The results are shown in Table 5. Based on the IC50 values, 4G9E1 and 7B5C11 were ultimately selected as the final cell clones.

[0103] Table 5: Blocking activity of hybridoma antibodies in cytokine assay Hybridoma antibody IC50 (pg / mL) 4G9E1 0.07510 15C4A11 0.1648 18D10D11 0.1409 7B5C11 0.07167

[0104] Example 6: Cloning and sequencing of murine antibodies 7B5C11 and 4G9E1

[0105] Hybridoma cells 7B5C11 and 4G9E1 in the logarithmic growth phase were counted. 500,000 to 1,000,000 cells were centrifuged, and the supernatant was discarded. The cells were then washed once with sterile PBS, and total RNA was extracted using a kit (purchased from Takara, 9767). The concentration and purity of the extracted RNA were measured and then reverse transcription was performed using oligo DT primers and reverse transcriptase at 42'"C for 45 minutes to synthesize cDNA. After PCR amplification, 10 pL of the PCR product was subjected to gel electrophoresis for identification. The PCR product with the correct band size was ligated to a T-vector (purchased from Takara, 6013) at room temperature for 30 minutes, then transformed into DH5a cells, and incubated with shaking at 37OC for 30 minutes. The plate was coated and then incubated overnight for bluewhite screening. Ten white colonies were selected for sequencing. After obtaining the sequencing results, analysis and alignment were performed to finally obtain the heavy and light chain sequences of the candidate antibodies. The amino acid sequence of 7B5C11 VH is shown in SEQ ID NO: 13, the amino acid sequence of 7B5C11 VL is shown in SEQ ID NO: 20, the amino acid sequence of 4G9E1 VH is shown in SEQ ID NO: 33, and the amino acid sequence of 4G9E1 VL is shown in SEQ ID NO: 40.

[0106] Additionally, the amino acid sequence of 15C4A11 VH is shown in SEQ ID NO: 55, the amino acid sequence of 15C4A11 VL is shown in SEQ ID NO: 56, the amino acid sequence of 18D10D11 VH is shown in SEQ ID NO: 62, and the amino acid sequence of 18D10D11 VL is shown in SEQ ID NO: 63.

[0107] Example 7: Construction of humanized antibodies

[0108] The obtained candidate antibody sequences were humanized by first analyzing the antibody variable region sequences to determine the antibody CDR regions and framework regions. The framework regions were then aligned with human germline sequences, and the murine candidate antibody variable region frameworks were replaced with the most closely matched human germline antibody variable region frameworks. Finally, the murine candidate antibody constant regions were also replaced with human antibody constant regions, resulting in humanized antibodies with murine CDR regions and human sequences in the other regions. Based on literature (Hwang WY, et al. (2005) Use of human germline genes in a cdr homology-based approach to antibody humanization; Tamura M, et al. (2000) Structural correlates of an anticarcinoma antibody: Identification of specificitydetermining residues (sdrs) and development of a minimally immunogenic antibody variant by retention of sdrs only; Morrow JK, Zhang S. (2012) Computational prediction of protein hot spot residues) and antibody spatial structure, key amino acid sites in the humanized framework region needed to be back-mutated to the amino acids in the original murine antibody sequence. If more than two amino acids were back-mutated, expression testing was performed after permutation and combination, and genes were synthesized based on the heavy and light chain sequences after back mutation.

[0109] The heavy and light chain combinations were then cloned into vectors containing the human k constant region and the human IgGl-LALA, heavy chain constant region, respectively. A plasmid combination containing the heavy and light chains was transfected into CHO cells for expression. After expression, the cell supernatant was collected to measure the affinity of the humanized antibodies. The chimeric antibody was used as a control, and the affinity of the humanized antibodies could not be lower than that of the control antibody. Finally, the humanized heavy and light chain combinations with the best affinity: hu7B5-1, hu7B5-2, and hu4G9 were selected as the final humanized antibodies. The heavy chain amino acid sequence of hu7B5 is shown in SEQ ID NO: 24, the light chain amino acid sequence of hu7B5-1 is shown in SEQ ID NO: 25, the light chain amino acid sequence of hu7B5-2 is shown in SEQ ID NO: 26, the heavy chain amino acid sequence of hu4G9 is shown in SEQ ID NO: 47, and the light chain amino acid sequence of hu4G9 is shown in SEQ ID NO: 48.

[0110] Example 8: Identification of affinity of humanized antibodies for TL1A protein

[0111] 8.1 ELISA method

[0112] An ELISA, plate was coated with antibody diluted in PBS at 1 pg / mL per well and incubated overnight at 4C. The coated plate was washed three times with PBST, followed by the addition of blocking solution (PBS + 2% BSA + 0.01% Tween 20), and blocked at room temperature for approximately 1 hour. The blocking solution was discarded, and 100 pL of serially diluted TL1A protein (starting at 200 nM, 5-fold serial dilution, a total of 8 concentration gradients) was added, followed by static incubation at room temperature for 1 hour. The plate was washed three times with PBST, followed by the addition of 100 uL of secondary antibody diluted in antibody dilution buffer (PBS + 0.5% BSA + 0.01% Tween 20) at a 1:20,000 dilution, and allowed to stand at room temperature for 1 hour. The plate was washed three times with PBST, followed by the addition of 100 uL of TMB solution for color development for approximately 5 minutes. Stop solution was then added to terminate the reaction, and the OD450 was read using a microplate reader. As shown in FIG. 2, the EC50 values for hu7B5-1 and hu4G9 binding to hTL1A were 0.5710 nM and 0.7765 nM, respectively, while the EC50 values for PF-06480605 and PRA-023 binding to hTL1A were 0.5180 nM and 0.7454 nM, respectively.

[0113] 8.2 Bio-layer interferometry

[0114] The affinity of hu7B5-1, hu7B5-2, and hu4G9 for human TL1A was measured based on bio-layer interferometry (BLI) technology. Using a Gator instrument, the antibodies were first immobilized onto Protein A probes, and the TL1A protein was diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM. The procedure for detecting antibody association and dissociation curves using Gator® is shown in Table 6. The dissociation constants for TL1A protein binding to hu7B5-1, hu7B5-2, and hu4G9 are shown in Table 7.

[0115] Table 6: Procedure for detecting antibody association and dissociation curves using Gator® Step name Detection time (s) Sample Activation 120 K buffer Fixation 120 Antibody Equilibration 60 K buffer Association 180 TL1A protein Dissociation 180 K buffer Probe regeneration Repeat 3 times Regeneration buffer / neutralization buffer

[0116] Table 7: Dissociation constants for TL1A protein binding to hu7B5-1, hu7B5-2, and hu4G9 koff (1 / s) kon (1 / Ms) Kd (M) hu7B5-1 0.000509 2.72E+05 1.87E-09 hu7B5-2 0.00039 2.39E+05 1.63E-09 hu4G9 0.000476 3.70E+05 1.28E-09 PF-06480605 0.000609 2.47E+05 2.46E-09 koff (1 / s) kon (1 / Ms) Kd (M) PRA-023 0.000494 1.48E+05 3.33E-09

[0117] 8.3 Flow cytometry

[0118] HEK293-hTL1A cells in the logarithmic growth phase were collected, resuspended in staining buffer (PBS + 2% FBS), counted, and added to a 96-well V-bottom plate at 50,000 cells / well. Serially diluted antibodies to be tested (starting at 30 pg / mL, 5-fold serial dilution, a total of 8 concentration gradients) were added to the 96-well V-bottom plate and incubated at 4C for 1 hour. The cells were washed twice with staining buffer, followed by the addition of FITC-labeled goat antihuman IgG secondary antibody (purchased from Abcam, ab7149), and incubated at 4C for 1 hour. After two washes with staining buffer, analysis was performed using a CytoFLEX flow cytometer. As shown in FIG. 3, the EC50 values for hu7B5-1, hu7B5-2, and hu4G9 binding to hTL1A were 0.2995 pg / mL, 0.3252 pg / mL, and 0.2332 pg / mL, respectively, while the EC50 values for PF-06480605 and PRA-023 binding to hTL1A were 0.3182 pgmL and 0.6265 pgmL, respectively.

[0119] Example 9: Identification of blocking effect of humanized antibodies on TL1A-induced TF-1-NFkB-Luc reporter cell activity

[0120] TF-1-NFkB-Luc reporter cells in the logarithmic growth phase were collected, washed twice with PBS, and starved overnight in GM-CSF-free medium. hu7B5-1, hu7B5-2, and hu4G9 were added to a 96-well plate with a white bottom, followed by the addition of diluted TL1A protein, and incubated at 37C for 30 minutes. Starved reporter cells were collected and counted, added at 30,000 cells per well, mixed well, and incubated at 37C for 4.5 hours. 50 pL / well of One-lite luminescence substrate reagent was then added, followed by incubation at room temperature in the dark for 5 minutes, and chemiluminescence was read using a microplate reader. As shown in FIG. 4, the IC50 values for hu7B5-1, hu7B5-2, and hu4G9 in blocking TL1A-induced reporter cell activity were 0.008743 pg / mL, 0.01073 pg / mL, and 0.01146 pg / mL, respectively, while the IC50 values for PF-06480605 and PRA-023 were 0.007735 pg / mL and 0.04554 pg / mL, respectively.

[0121] Example 10: Identification of blocking effect of humanized antibodies on binding of TL1A to DR3 by ELSIA

[0122] An ELISA plate was coated with DR3 protein (purchased from ACROBiosystems, TN5-H5258) diluted in PBS at 2 pg / mL per well and incubated overnight at 4C. The coated plate was washed three times with PBST, followed by the addition of blocking solution (PBS + 2% BSA + 0.01% Tween 20), and blocked at room temperature for approximately 1 hour. The blocking solution was discarded, and diluted TL1A protein (final concentration of 0.5 pg / mL) was mixed with serially diluted antibodies to be tested. The mixture was added at 100 pL per well, followed by static incubation at room temperature for 2 hours. The plate was washed three times with PBST, followed by the addition of 100 pL of secondary antibody diluted in antibody dilution buffer (PBS + 0.5% BSA + 0.01% Tween 20) at a 1:30,000 dilution, and allowed to stand at room temperature for 1 hour. The plate was washed three times with PBST, followed by the addition of 100 pL of TMB solution for color development for approximately 5 minutes. Stop solution was then added to terminate the reaction, and the OD450 was read using a microplate reader. As shown in FIG. 5, the IC50 values for hu7B5-1 and hu4G9 in blocking the binding of TL1A to DR3 were 0.551 pg / mL and 1.05 pg / mL, respectively, while the IC50 values for PF-06480605 and PRA-023 were 1.14 pg / mL and 4.492 pg / mL, respectively.

[0123] Example 11: Identification of blocking effect of humanized antibodies on IFN-y release from PBLs induced by TL1A combined with IL12 / IL18

[0124] Cryopreserved PBMCs were thawed in pre-warmed medium, centrifuged (300 g, 10 minutes), resuspended, and added to a culture flask for incubation at 37OC. After 2 hours, non-adherent PBLs were collected and counted, and seeded into a 96-well U-bottom plate at 0.2 M / well. Serially diluted antibodies to be tested (10 pg / mL, 3-fold serial dilution, 8 gradients) were added to the 96-well U-bottom plate, followed by the addition of 50 ng / mL TL1A protein. 0.5 ng / mL IL-12 and 5 ng / mL IL-18 proteins were then added to the U-bottom plate, followed by incubation at 37OC. After 24 hours of incubation, the plate was centrifuged at 2000 rpm for 15 minutes, culture supernatants were collected, and IFN-y levels in the supernatants were measured by ELISA. As shown in FIG. 6, the IC50 values for hu7B5-1, hu7B5-2, and hu4G9 in blocking TLlA-induced IFN-y release were 0.1393 pg / mL, 0.1006 pg / mL, and 0.1391 pg / mL, respectively, while the IC50 value for PF-06480605 was 0.1298 pg / mL.

[0125] Example 12: Identification of affinity of humanized antibodies for cynomolgus monkey TL1A

[0126] The affinity of hu7B5-1 and hu4G9 for cynomolgus monkey TL1A was measured based on bio-layer interferometry (BLI) technology. Using a Gator instrument, the antibodies were first immobilized onto Protein A probes, and the cynomolgus monkey TL1A protein was diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM. The procedure for detecting antibody association and dissociation curves using gator is shown in Table 8. The dissociation constants for cynomolgus monkey TL1A protein binding to hu7B5-1 and hu4G9 are shown in Table 9. Both hu7B5-1 and hu4G9 showed cross-reactivity with cynomolgus monkey TL1A, with KD values of 2.52E-09 M and 1.87E-09 M, respectively.

[0127] Table 8: Procedure for detecting antibody association and dissociation curves using Gator® Step name Detection time (s) Sample Activation 120 K buffer Fixation 120 Antibody Equilibration 60 K buffer Association 180 TL1A protein Dissociation 180 K buffer Probe regeneration Repeat 3 times Regeneration buffer / neutralization buffer

[0128] Table 9: Dissociation constants for cynomolgus monkey TL1A protein binding to hu7B5- 1 and hu4G9 koff (1 / s) kon (1 / Ms) KD (M) hu7B5-1 0.000598 2.38E+05 2.52E-09 hu4G9 0.00064 3.43E+05 1.87E-09 PF-06480605 0.000518 2.19E+05 2.37E-09 PRA-023 0.000452 1.72E+05 2.62E-09

[0129] Example 13: Identification of binding specificity of humanized antibodies to hTL1A

[0130] An ELISA plate was coated with hu7B5-1 or hu4G9 diluted in PBS at 1 pg / mL per well and incubated overnight at 4C. The coated plate was washed three times with PBST, followed by the addition of blocking solution (PBS + 2% BSA + 0.01% Tween 20), and blocked at room temperature for approximately 1 hour. The blocking solution was discarded, and diluted TNFSF6, TNFSF10, and TNFSF14 proteins were added at 100 pL per well, followed by static incubation at room temperature for 1 hour. The plate was washed three times with PBST, followed by the addition of 100 pL of secondary antibody diluted in antibody dilution buffer (PBS + 0.5% BSA + 0.01% Tween 20) at a 1:20,000 dilution, and allowed to stand at room temperature for 1 hour. The plate was washed three times with PBST, followed by the addition of 100 uL of TMB solution for color development for approximately 5 minutes. Stop solution was then added to terminate the reaction, and the OD450 was read using a microplate reader. As shown in FIG. 7A and FIG. 7B, hu7B5-1 and hu4G9 showed no binding reaction with the three proteins of the same family.

[0131] The sequences used in the present disclosure are shown in Table 10.

[0132] Table 10: Sequence listing SEQ ID NO Name Sequence SEQ ID NO: 1 TL1A LKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQF PALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFRGM TSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCE VGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFF GAFLL SEQ ID NO: 2 PF-06480605 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYDFTYYGISWVRQAPGQ GLEWMGWISTYNGNTHYARMLQGRVTMTTDTSTRTAYMELRS LRSDDTAVYYCARENYYGSGAYRGGMDVWGQGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG SEQ ID NO: 3 PF-06480605 light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR LLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS NWPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 4 RPA-023 heavy chain QVQLVQSGAEVKKPGASVKLSCKASGFDIQDTYMHWVRQAPG QGLEWMGRIDPASGHTKYDPKFQVRVTMTTDTSTSTVYMELSS LRSEDTAVYYCSRSGGLPDVWGQGTTVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK SEQ ID NO: 5 RPA-023 light chain EIVLTQSPGTLSLSPGERVTMSCRASSSVSYMYWYQQKPGQAPR PWIYATSNLASGVPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQ WSGNPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID hu7B5 HCDR1 AYEIH NO: 6 SEQ ID NO: 7 hu7B5 HCDR2 GIDPDTGGTAYNQKFKD SEQ ID NO: 8 hu7B5 HCDR3 FRLLRYYTLDY SEQ ID NO: 9 hu7B5-2 LCDR1 KSSQSLLDADAKTYLN SEQ ID NO: 10 hu7B5-1 LCDR1 KSSQSLLDGDGKTYLN SEQ ID NO: 11 hu7B5 LCDR2 LVSKLDS SEQ ID NO: 12 hu7B5 LCDR3 LQATHLPWT SEQ ID NO: 13 7B5C11 VH EVKLQESGAELVRPGASVKLSCKASGYAFTAYEIHWVKQTPVHG LDWIGGIDPDTGGTAYNQKFKDKATLTADKSSNTAYMELHSLTS EDSAVYYCTRFRLLRYYTLDYWGQGTSVTVSS SEQ ID NO: 14 7B5 VH-0 QVQLVQSGAEVKKPGASVKVSCKASGYTFTAYEIHWVRQAPGQ GLEWMGGIDPDTGGTAYNQKFKDRVTMTRDTSTSTVYMELSSL RSEDTAVYYCARFRLLRYYTLDYWGQGTLVTVSS SEQ ID NO: 15 7B5 VH-4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTAYEIHWVRQAPGQ GLEWIGGIDPDTGGTAYNQKFKDRVTLTADKSTSTVYMELSSLR SEDTAVYYCARFRLLRYYTLDYWGQGTLVTVSS SEQ ID NO: 16 7B5 VH-5 QVQLVQSGAEVKKPGASVKVSCKASGYAFTAYEIHWVRQAPGQ GLEWIGGIDPDTGGTAYNQKFKDRVTLTADKSTSTVYMELSSLR SEDTAVYYCARFRLLRYYTLDYWGQGTLVTVSS SEQ ID NO: 17 7B5 VH-6 QVQLVQSGAEVKKPGASVKVSCKASGYAFTAYEIHWVRQAPGQ GLEWIGGIDPDTGGTAYNQKFKDRVTLTADKSTSTVYMELSSLR SEDTAVYYCTRFRLLRYYTLDYWGQGTLVTVSS SEQ ID NO: 18 7B5 VH-8 QVQLVQSGAEVKKPGASVKVSCKASGYAFTAYEIHWVRQAPGQ GLEWIGGIDPDTGGTAYNQKFKDRATLTADKSTSTAYMELSSLRS EDTAVYYCTRFRLLRYYTLDYWGQGTLVTVSS SEQ ID NO: 19 hu7B5 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTAYEIHWVRQAPGQ GLEWIGGIDPDTGGTAYNQKFKDRVTMTRDTSTSTVYMELSSLR SEDTAVYYCARFRLLRYYTLDYWGQGTLVTVSS SEQ ID NO: 20 7B5C11 VL DIVMTQSPLTLSVTLGQPASISCKSSQSLLDGDGKTYLNWLLQRP GESPKLLMYLVSKLDSGVPDRFTGSGSGTDFTLQIRRVEAEDLGI YYCLQATHLPWTFGGGTKLEIK SEQ ID NO: 21 7B5 VL-1 DIVMTQTPLSSPVTLGQPASISCKSSQSLLDGDGKTYLNWLQQRP GQPPRLLMYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVG VYYCLQATHLPWTFGQGTKVEIK SEQ ID hu7B5-1 VL DIVMTQTPLSSPVTLGQPASISCKSSQSLLDGDGKTYLNWLQQRP NO: 22 GQPPRLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGV YYCLQATHLPWTFGQGTKVEIK SEQ ID NO: 23 hu7B5-2 VL DIVMTQTPLSSPVTLGQPASISCKSSQSLLDADAKTYLNWLQQRP GQPPRLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGV YYCLQATHLPWTFGQGTKVEIK SEQ ID NO: 24 hu7B5 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTAYEIHWVRQAPGQ GLEWIGGIDPDTGGTAYNQKFKDRVTMTRDTSTSTVYMELSSLR SEDTAVYYCARFRLLRYYTLDYWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO: 25 hu7B5-1 light chain DIVMTQTPLSSPVTLGQPASISCKSSQSLLDGDGKTYLNWLQQRP GQPPRLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGV YYCLQATHLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 26 hu7B5-2 light chain DIVMTQTPLSSPVTLGQPASISCKSSQSLLDADAKTYLNWLQQRP GQPPRLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGV YYCLQATHLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 27 hu4G9 HCDR1 DFYMN SEQ ID NO: 28 hu4G9 HCDR2 EIDPNNGGTKYNQKFKD SEQ ID NO: 29 hu4G9 HCDR3 ERIQGWFAY SEQ ID NO: 30 hu4G9 LCDR1 KASQNVGTAVA SEQ ID NO: 31 hu4G9 LCDR2 SVSNRYT SEQ ID NO: 32 hu4G9 LCDR3 QQYSDSPFT SEQ ID NO: 33 4G9E1 VH EVKLEESGPELVKPGASVKISCKASGYTFTDFYMNWVKQSHGK SLEWIGEIDPNNGGTKYNQKFKDKATLTVDKSSSTAYMELRSLTS EDSGVYYCARERIQGWFAYWGQGTLVTVSA SEQ ID NO: 34 4G9 VH-0 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFYMNWVRQAPG QGLEWMGEIDPNNGGTKYNQKFKDRVTMTTDTSTSTAYMELRS LRSDDTAVYYCARERIQGWFAYWGQGTLVTVSS SEQ ID NO: 35 4G9 VH-1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFYMNWVRQAPG QGLEWIGEIDPNNGGTKYNQKFKDRVTMTTDTSTSTAYMELRSL RSDDTAVYYCARERIQGWFAYWGQGTLVTVSS SEQ ID NO: 36 4G9 VH-2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFYMNWVRQAPG QSLEWIGEIDPNNGGTKYNQKFKDRVTMTTDTSTSTAYMELRSL RSDDTAVYYCARERIQGWFAYWGQGTLVTVSS SEQ ID NO: 37 4G9 VH-4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFYMNWVRQAPG QGLEWMGEIDPNNGGTKYNQKFKDRATLTVDKSTSTAYMELRS LRSDDTAVYYCARERIQGWFAYWGQGTLVTVSS SEQ ID NO: 38 4G9 VH-5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFYMNWVRQAPG QSLEWIGEIDPNNGGTKYNQKFKDRVTLTVDKSTSTAYMELRSL RSDDTAVYYCARERIQGWFAYWGQGTLVTVSS SEQ ID NO: 39 hu4G9 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFYMNWVRQAPG QGLEWMGEIDPNNGGTKYNQKFKDRVTLTVDKSTSTAYMELRS LRSDDTAVYYCARERIQGWFAYWGQGTLVTVSS SEQ ID NO: 40 4G9E1 VL DIVLTQSTKFMSTSVGDRVSITCKASQNVGTAVAWYQQKPGQSP KIMIYSVSNRYTGVPDRFTGSRSGTDFTLTISNIQSEDLADYFCQQ YSDSPFTFATGTKLEIK SEQ ID NO: 41 4G9 VL-0 DIQMTQSPSSLSASVGDRVTITCKASQNVGTAVAWYQQKPGKVP KLLIYSVSNRYTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQ YSDSPFTFGQGTKLEIK SEQ ID NO: 42 4G9 VL-1 DIQMTQSPSSLSASVGDRVTITCKASQNVGTAVAWYQQKPGKVP KLMIYSVSNRYTGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQ QYSDSPFTFGQGTKLEIK SEQ ID NO: 43 4G9 VL-2 DIQMTQSPSSLSASVGDRVTITCKASQNVGTAVAWYQQKPGKVP KLMIYSVSNRYTGVPSRFSGSRSGTDFTLTISSLQPEDVATYYCQQ YSDSPFTFGQGTKLEIK SEQ ID NO: 44 4G9 VL-3 DIQMTQSPSSLSASVGDRVTITCKASQNVGTAVAWYQQKPGKVP KIMIYSVSNRYTGVPSRFSGSRSGTDFTLTISSLQPEDVATYYCQQ YSDSPFTFGQGTKLEIK SEQ ID NO: 45 4G9 VL-4 DIVLTQSPSSLSASVGDRVTITCKASQNVGTAVAWYQQKPGKVP KLMIYSVSNRYTGVPSRFSGSRSGTDFTLTISSLQPEDVATYYCQQ YSDSPFTFGQGTKLEIK SEQ ID NO: 46 hu4G9 VL DIQMTQSPSSLSASVGDRVTITCKASQNVGTAVAWYQQKPGKVP KLMIYSVSNRYTGVPSRFSGSRSGTDFTLTISSLQPEDVATYFCQQ YSDSPFTFGQGTKLEIK SEQ ID NO: 47 hu4G9 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDFYMNWVRQAPG QGLEWMGEIDPNNGGTKYNQKFKDRVTLTVDKSTSTAYMELRS LRSDDTAVYYCARERIQGWFAYWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO: 48 hu4G9 light chain DIQMTQSPSSLSASVGDRVTITCKASQNVGTAVAWYQQKPGKVP KLMIYSVSNRYTGVPSRFSGSRSGTDFTLTISSLQPEDVATYFCQQ YSDSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 49 15C4A11 / 18D1 0D11 HCDR1 DYAMS SEQ ID NO: 50 15C4A11 HCDR2 TISSGGTYTYYPDSVKG SEQ ID NO: 51 15C4A11 HCDR3 QDRYDVRVWFPY SEQ ID NO: 52 15C4A11 LCDR1 RASQDISNYLN SEQ ID NO: 53 15C4A11 LCDR2 YASRLHS SEQ ID NO: 54 15C4A11 LCDR3 QQGYSLP SEQ ID NO: 55 15C4A11 VH EVKLEESGGVLVKPGGSLKLSCAASGFTFSDYAMSWVRQTPEKR LEWVATISSGGTYTYYPDSVKGRFTMSRDNAKNTLYLQMSSLRS EDTAMYYCARQDRYDVRVWFPYWGQGTLVTVSA SEQ ID NO: 56 15C4A11 VL DIVITQTPSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVK LLIYYASRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDIATYFCQQG YSLPWTFGGGTKLEFK SEQ ID NO: 57 18D10D11 HCDR2 TISSGGTYPYYPDSVKG SEQ ID NO: 58 18D10D11 HCDR3 QDRSDVRVWFPH SEQ ID NO: 59 18D10D11 LCDR1 RASQDIAKYLN SEQ ID NO: 60 18D10D11 LCDR2 YTSRLHL SEQ ID NO: 61 18D10D11 LCDR3 QQGYTLP SEQ ID NO: 62 18D10D11 VH EVKLEQSGGGLVKPGGSLKLSCAASGFTFSDYAMSWVRQIPEQR LEWVATISSGGTYPYYPDSVKGRFAISRDNAKNTLYLQMSSLRSE DTAIYFCARQDRSDVRVWFPHWGQGTLVTVSA SEQ ID NO: 63 18D10D11 VL DIVLTQSTSSLSASLGDRVTISCRASQDIAKYLNWYQQKPDGTVK LLIFYTSRLHLEVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQG YTLPWTFGGGTNLEIK SEQ ID NO: 64 CH-1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL YITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQ ID NO: 65 CH-2 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQ ID NO: 66 CH-3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK SEQ ID NO: 67 CH-4 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLY ITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK SEQ ID NO: 68 CL-1 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC SEQ ID NO: 69 CL-2 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECS

[0133] Although specific embodiments of the present disclosure have been described above, those skilled in the art should understand that these are merely examples and that various changes or modifications can be made to these embodiments without departing from the principle and essence of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.

Claims

1. A TL1A-targeting antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region,the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein:HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12;or, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 28, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 30, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 31, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 32;or, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 49, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 50, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 51, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 52, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 53, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 54;or, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 49, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 57, HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 58, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 59, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 60, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 61.

2. The antibody or the antigen-binding fragment thereof according to claim 1, wherein,the heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 13 to 19, and the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 20 to 23;or, the heavy chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 33 to 39, and the light chain variable region has an amino acid sequence shown in any one of SEQ ID NOs: 40 to 46;or, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 55, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 56;or, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 62, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 63.

3. The antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein,the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 20;or, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 22 or 23;or, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 33, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 40;or, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 39, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 46.

4. The antibody or the antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or the antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region and the light chain constant region are of human or murine origin;more preferably, the heavy chain constant region has an amino acid sequence shown in any one of SEQ ID NOs: 64 to 67, and the light chain constant region has the amino acid sequence shown in SEQ ID NO: 68 or 69.

5. The antibody or the antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or the antigen-binding fragment thereof is a full-length antibody, Fab, Fab', F(ab')2, or Fv, such as scFv.

6. An antibody combination, wherein the antibody combination comprises at least two antibodies or antigen-binding fragments thereof according to any one of claims 1 to 5.

7. An isolated nucleic acid, wherein the isolated nucleic acid encodes the antibody or the antigenbinding fragment thereof according to any one of claims 1 to 5 or the antibody combination according to claim 6.

8. A recombinant expression vector, wherein the recombinant expression vector comprises the isolated nucleic acid according to claim 7;preferably, the recombinant expression vector is a plasmid, a cosmid, a phage, or a viral vector;more preferably, the plasmid is pCDNA3.1.

9. A transformant, wherein the transformant comprises the isolated nucleic acid according to claim 7 or the recombinant expression vector according to claim 8 in a host cell;preferably, the host cell is a prokaryotic cell or a eukaryotic cell;more preferably, the host cell is selected from a yeast cell, a mammalian cell, or other cells suitable for preparing an antibody or an antigen-binding fragment thereof; the mammalian cell is, for example, a HEK293 cell.

10. A method for preparing a TL1A-targeting antibody or an antigen-binding fragment thereof, wherein the method comprises culturing the transformant according to claim 9, and obtaining the TL1A-targeting antibody or the antigen-binding fragment thereof from the culture.

11. A pharmaceutical composition, wherein the pharmaceutical composition comprises the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody combination according to claim 6, the isolated nucleic acid according to claim 7, the recombinant expression vector according to claim 8, or the transformant according to claim 9; and a pharmaceutically acceptable excipient.

12. A TL1A detection agent, wherein the TL1A detection agent comprises the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 5 and / or the antibody combination according to claim 6.

13. Use of the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody combination according to claim 6, the isolated nucleic acid according to claim 7, the recombinant expression vector according to claim 8, the transformant according to claim 9, or the pharmaceutical composition according to claim 11 in the manufacture of a diagnostic agent or a detection agent;preferably, the diagnostic agent or the detection agent is used for diagnosing or detecting a TL1A-mediated disease.

14. A method for detecting TL1A, wherein the method comprises contacting the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 5 or the antibody combination according to claim 6 with a sample to be tested;preferably, the detection is for non-diagnostic purposes.

15. Use of the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 5 or the antibody combination according to claim 6 in the manufacture of a medicament for preventing or treating a TL1A-mediated disease;preferably, the disease is selected from at least one of the following: inflammatory bowel disease IBD, Crohn's disease, ulcerative colitis, asthma, allergy, diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, transplant rejection, graft-versus-host disease GVHD, primary biliary cirrhosis, atherosclerosis, bladder syndrome / interstitial cystitis, urointestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis, and scleroderma.

16. A method for preventing, ameliorating, or treating a TL1A-mediated disease, disorder, or condition, comprising administering to a subject in need thereof an effective amount of the antibody or the antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody combination according to claim 6, or the pharmaceutical composition according to claim 11;preferably, the disease is selected from at least one of the following: inflammatory bowel disease IBD, Crohn's disease, ulcerative colitis, asthma, allergy, diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, transplant rejection, graft-versus-host disease GVHD, primary biliary cirrhosis, atherosclerosis, bladder syndrome / interstitial cystitis, urointestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis, and scleroderma.

17. The antibody or the antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody combination according to claim 6, or the pharmaceutical composition according to claim 11, for use in preventing, ameliorating, or treating a TL1A-mediated disease, disorder, or condition;preferably, the disease is selected from at least one of the following: inflammatory bowel disease IBD, Crohn's disease, ulcerative colitis, asthma, allergy, diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, transplant rejection, graft-versus-hostdisease GVHD, primary biliary cirrhosis, atherosclerosis, bladder syndrome / interstitial cystitis, urointestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis, and scleroderma.

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