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

Antibodies targeting TL1A and their applications

[0001] This application claims priority to Chinese patent application No. 202410250612X, filed on March 5, 2024. This application incorporates the entire text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention belongs to the biological field, and particularly relates to an antibody targeting TL1A and an application thereof. Background Art

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

[0004] TL1A binds to its receptor, death receptor 3 (DR3), and activates downstream signaling, which then participates in innate and adaptive immune homeostasis.

[0005] Studies have shown that TL1A plays a crucial role in many autoimmune diseases, such as inflammatory bowel disease (IBD), asthma, multiple sclerosis, and arthritis. Overexpression of TL1A has been detected in the mucosa and serum of patients with IBDs such as ulcerative colitis (UC) and Crohn's disease (CD). TL1A binds to its functional receptor DR3, inducing stimulatory signals that activate the NF-κB and MAPK pathways, amplifying effector T cell responses, including Th1, Th17, Th2, and Th9.

[0006] Therefore, blocking the interaction between TL1A and DR3 can help achieve the goal of intervening in the early stages of inflammation, inhibit the release of pro-inflammatory cytokines by effector T cells and other immune cells, and provide more possibilities for the treatment of IBD patients or other TL1A-related diseases. Summary of the Invention

[0007] The present invention addresses the technical problem of the lack of existing drugs that utilize TL1A to treat related diseases. The present invention provides an antibody targeting TL1A and its use. The antibody of the present invention 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 invention solves the above technical problems through the following technical solutions.

[0009] The first aspect of the present invention provides an antibody or antigen-binding fragment thereof targeting TL1A, wherein the antibody or 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 set forth in SEQ ID NO:6, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:7, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:8, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:10, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12;

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

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

[0014] Alternatively, 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 as shown in any one of SEQ ID NOs: 13-19, and the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NOs: 20-23.

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

[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 heavy chain variable regions or light chain variable regions as shown in Table 1 below,

[0020] Table 1 Variable region combinations

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

[0022] In the present invention, 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 as shown in any one of SEQ ID NOs: 64-67.

[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 antigen-binding fragment thereof is a full-length antibody, Fab, Fab', F(ab')2, Fv, for example, a scFv.

[0026] In some specific embodiments, the heavy chain of the antibody or antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO: 24, and the light chain 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 antigen-binding fragment thereof has the amino acid sequence shown in SEQ ID NO:47, and the light chain has the amino acid sequence shown in SEQ ID NO:48.

[0028] The second aspect of the present invention provides an antibody combination, which comprises at least two antibodies or antigen-binding fragments thereof according to the first aspect.

[0029] The third aspect of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described in the first aspect or the antibody combination as described in the second aspect.

[0030] The fourth aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid as described in the third aspect.

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

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

[0033] The fifth aspect of the present invention provides a transformant, which comprises 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 species.

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

[0036] In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for producing antibodies or antigen-binding fragments thereof.

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

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

[0039] The seventh aspect of the present invention provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody combination as described in the second aspect, the isolated nucleic acid as described in the third aspect, the recombinant expression vector as described in the fourth aspect or the transformant as described in the fifth aspect, and a pharmaceutically acceptable excipient.

[0040] In an eighth aspect, the present invention provides a TL1A detection agent, comprising the antibody or 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] The ninth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody combination as described in the second aspect, the isolated nucleic acid as described in the third aspect, the recombinant expression vector as described in the fourth aspect, the transformant as described in the fifth aspect, or the pharmaceutical composition as described in the seventh aspect in the preparation of a diagnostic agent or a detection agent.

[0043] In some embodiments, the diagnostic agent or detection agent is used to diagnose or detect a disease mediated by TL1A.

[0044] The tenth aspect of the present invention provides use of the antibody or antigen-binding fragment thereof according to the first aspect or the antibody combination according to the second aspect in the preparation of a medicament for preventing or treating a disease mediated by TL1A.

[0045] In some embodiments, the disease is selected from at least one of inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, allergies, 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, urinary intestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis, and scleroderma.

[0046] In the eleventh aspect, the present invention provides a method for preventing, ameliorating or treating a disease, disorder or condition mediated by TL1A, comprising administering to a subject in need thereof an effective amount of the antibody or 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.

[0047] In some embodiments, the disease is selected from at least one of inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, allergies, 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, urinary intestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis, and scleroderma.

[0048] The twelfth aspect of the present invention provides the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody combination as described in the second aspect, or the pharmaceutical composition as described in the seventh aspect, for preventing, ameliorating or treating a disease, disorder or condition mediated by TL1A.

[0049] In some embodiments, the disease is selected from at least one of inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, asthma, allergies, 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, urinary intestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis, and scleroderma.

[0050] In a thirteenth aspect, the present invention provides a method for detecting TL1A, comprising contacting the antibody or 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, for example, by detecting the antibody to quantify or quantify TL1A in the sample to be tested. In some embodiments, the sample to be tested is a tissue or a cell.

[0052] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

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

[0054] The positive progress effect of the present invention is:

[0055] The present invention relates to antibodies that bind to TL1A and modulate its activity. The antibodies or antigen-binding fragments thereof of the present invention 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] Figure 1 is a chemiluminescence image of TF-1-NFkB-Luc reporter cells.

[0057] Figure 2 shows the EC values ​​of hu7B5-1 and hu4G9 binding to hTL1A detected by ELISA. 50 .

[0058] Figure 3 shows the EC binding of hu7B5-1, hu7B5-2 and hu4G9 to hTL1A detected by flow cytometry. 50 .

[0059] Figure 4 shows the IC values ​​of hu7B5-1, hu7B5-2, and hu4G9 for blocking hTL1A-induced reporter cell activity. 50 .

[0060] Figure 5 shows the OD values ​​of hu7B5-1 and hu4G9 blocking the binding of hTL1A to DR3 identified by ELSIA method. 450 .

[0061] Figure 6 shows the IC values ​​of hu7B5-1, hu7B5-2 and hu4G9 for blocking hTL1A-induced IFN-γ release. 50 .

[0062] Figure 7A and Figure 7B are the OD values ​​of hu7B5-1 and hu4G9 binding to human TRAIL / TNFSF10, human LIGHT / TNFSF14, and Human Fas Ligand / TNFSF6, respectively. 450 . DETAILED DESCRIPTION

[0063] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0064] definition

[0065] "Antibody" is an immunoglobulin molecule that is capable of specifically binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, unless otherwise specified, the term includes not only complete polyclonal or monoclonal antibodies, but also any antigen binding portion thereof that competes with the complete antibody for specific binding, fusion proteins comprising the antigen binding portion, and any other modified configuration of the immunoglobulin molecule comprising the antigen recognition site. Antigen binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs, such as shark and camelid antibodies), fragments comprising complementary determining regions (CDRs), single-chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and double-scFvs, as well as polypeptides containing at least a portion of an immunoglobulin sufficient to confer antigen-specific binding to the polypeptide. Antibodies include antibodies of any kind or their antigen-binding fragments, such as IgG, IgA or IgM (or their subclasses), and antibodies do not need to be of any particular kind. Depending on the antibody amino acid sequence of the heavy chain constant region of the immunoglobulin, the immunoglobulin can be classified into different classes. There are five main types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these immunoglobulins can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known.

[0066] As used interchangeably herein, the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply, "antibody portion") refer 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 can 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] The "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 is known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FR) connected by three complementarity determining regions (CDRs) (also referred to as hypervariable regions), and contribute to the formation of the antigen binding site of the antibody. If a variant of the subject variable region is desired, particularly one with substitutions of amino acid residues outside the CDR region (i.e., in the framework region), appropriate amino acid substitutions, preferably conservative amino acid substitutions, can be identified by comparing the subject variable region with the variable regions of other antibodies containing CDR1 and CDR2 sequences in the same typical species as the subject variable region.

[0069] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising this population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that 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 as being obtained from a substantially homogeneous antibody population and should not be construed as requiring the antibody to be produced by any particular method.

[0070] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0071] The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as antibodies in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody, or vice versa. The term also encompasses antibodies comprising a V region from one individual of one 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, 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 fragment thereof and an antigen. The term "binding affinity" is used to describe a monovalent interaction (intrinsic activity).

[0074] As used herein, "vector" means a construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids 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 production cells.

[0075] "Host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector for incorporating a polynucleotide insert. Host cells include the 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 a polynucleotide of the invention.

[0076] The term "TL1A" refers to any naturally occurring form of TL1A (whether monomeric or polymeric), including dimers, trimers, etc., which can 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 generally be characterized as having the same type of activity as naturally occurring TL1A, such as the ability to bind to DR3 and the ability to induce receptor-regulated activity.

[0077] Antibodies useful in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising antibody portions (e.g., domain antibodies), humanized antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies 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 specific embodiments, the antibody is a human antibody.

[0078] In this application, 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 this application was RPMI 1640 culture medium (purchased from Gibco, 61870036).

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

[0081] 1.1 Immunity and Fusion

[0082] Balb / c mice (purchased from Beijing Weitong Lihua) were immunized with TL1A protein, and spleen cells from immunized mice with antibodies that showed strong specificity for the TL1A antigen were isolated. Splenocytes and myeloma cells were mixed at a ratio of 4:1 and prewarmed at 37°C. The mixture was centrifuged and the supernatant discarded. The pellet was gently tapped to loosen and evenly disperse the cells. The mixture was then incubated in a 37°C water bath. 1 mL of PEG was added to the mixed cells over 1 minute, gently mixing. Over 60 seconds, 2 mL of prewarmed SFM was added. 3 mL, 5 mL, and 10 mL to 30 mL were then added over 3 minutes. The mixture was allowed to stand at 37°C for 10 minutes. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and the cells were resuspended in MD2001Clone Easy medium containing HAT and hybridoma culture supplements. The cells were then added to a total volume of 240 mL of medium and mixed thoroughly. The cells were aliquoted into 96-well cell culture plates, with 0.2 mL per well. After 6 days, the medium was replaced with HAT medium. Observe the growth of hybridoma cells and when they grow to more than 1 / 10 of the bottom area of ​​the well, aspirate the supernatant 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] Take an enzyme-labeled plate (purchased from Suzhou Beaver Bio, 40304) and add TL1A protein diluted with PBS to each well at 1 μg / mL. Coat at 4°C overnight, wash the coated cell plate with PBST, and then add blocking solution (PBS + 2% BSA + 0.01% Tween 20) to block at room temperature for about 2 hours. Discard the blocking solution, and then add 90 μL of the fused hybridoma culture supernatant. Mark it when adding to correspond to the 96-well cell plate, then incubate at room temperature for 1 hour, wash three times with PBST, add 90 μL of secondary antibody diluted with antibody diluent (PBS + 0.5% BSA + 0.01% Tween20) (1:20000 dilution), stand at room temperature for 1 hour, wash three times with PBST, add 45 μL TMB solution for color development for about 5 minutes, then add stop solution to stop color development, and use OD on the enzyme reader. 450Reading value. According to the OD value, the hybridoma cell wells with higher reading value and stronger affinity with TL1A were selected for transwell culture. The specific results are shown in Table 2 and Table 3. According to Table 2, the OD value of the 2B9 cell line in the first hybridoma fusion cell was 450 The value was higher, reaching 1.1855, and the OD of 3A6 cell line in the second hybridoma fusion cell was 450 The value is higher, reaching 1.5874.

[0086] Table 2 ELISA screening results of the first hybridoma fusion cell supernatant

[0087] Table 3 Second hybridoma fusion cell supernatant ELISA screening results

[0088] 1.3 Preliminary screening of positive hybridoma cells using the reporter gene method

[0089] To evaluate the antibody's ability to block intracellular signaling by inhibiting the TL1A-DR3 interaction, we constructed a TF-1-NFκB-Luc reporter cell line (NFκB-Luc reporter plasmid purchased from Beyotime Biosciences, D2206). TF-1 cells (purchased from Wuhan Pronocell, CL-0232) are a human erythroleukemia cell line that expresses the TL1A receptor DR3. TF-1 cells were transfected with the NFκB-Luc luciferase reporter gene to generate cells transduced with the NFκB-driven luciferase gene. Luciferase activity was measured to assess TL1A's activation of the NFκB pathway through DR3.

[0090] The TF-1-NFκB-Luc reporter gene cell line in the logarithmic growth phase was collected, washed twice with PBS, and starved overnight with GM-CSF-free RPMI 1640 medium (purchased from Gibco, 61870036). The hybridoma supernatant from the primary ELISA screening was added to a 96-well white-bottom plate, and diluted TL1A protein was added. The cells were incubated at 37°C for 30 minutes. The reporter cells that had been starved overnight were collected and counted, and 30,000 cells were added to each well. The cells were mixed and incubated at 37°C for 4.5 hours. 50 μL / well of One-lite luminescent substrate reagent (purchased from Norvegian, DD1203-03) was added. The cells were incubated at room temperature in the dark for 5 minutes, and the chemiluminescence was read on a microplate reader. As shown in Figure 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 identify hybridoma cell subclones

[0092] According to the ELISA primary screening and reporter gene method, cells from 16 positive cell wells were selected and subcloned for the first time using the limiting dilution method. The cells in the positive clone wells of the 96-well plate were blown evenly and transferred to a 24-well plate containing 1 mL of culture medium. 20 μL of suspended cell culture medium was aspirated and added to 80 μL of transfer medium, mixed, and then counted. According to the counting results, the diluted culture medium containing a total of 100 cells was added to 20 mL of transfer medium, mixed thoroughly, and then 200 μL per well was spread over the 96-well plate. After all the positive clones were plated in turn, they were cultured in a 37°C cell culture incubator. The 96-well plate was observed under a microscope for 4-5 days and the single clone wells were recorded. On the 7th day of culture, subclone ELISA screening was performed on a TL1A antigen-coated plate to select hybridoma cells that secrete anti-TL1A specific antibodies. According to OD 450 Based on the cell status and readings, select cells from wells 6H8F3, 7B5C11, 1B5C11, 3A6G8, 4G9E1, 12H2G4, 13C8G10, 15C4A11, 16F12A2, 18D10D11, 20D7E2, 17A3C8, and 19C9E4 for transwell culture. Once the cells reach the logarithmic growth phase in the 24-well culture dish, transfer them to a 10 cm diameter dish and freeze them when they cover more than 80% of the dish bottom.

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

[0094] After freezing, continue to culture the cells in serum-free medium in the dish. When the hybridoma cells grow to more than 80% of the cell dish bottom again, divide the cells into two dishes and culture them. Each clone is cultured for about 50 mL. After 5-7 days, harvest the supernatant for purification. Centrifuge the harvested hybridoma cell culture supernatant (300g, 10 min), filter and clarify, place on ice as the sample, equilibrate the protein A column with equilibration buffer for 5-8 column volumes, then pass the filtered supernatant through the column and collect the flow-through. After the sample is loaded, wash the chromatography column with equilibration buffer for 15-20 column volumes, or until the A280 absorbance value drops below 20 mAu. Elute the bound antibody with 20 mM sodium citrate elution buffer (pH = 3.0) to obtain about 2.5 mL of eluted protein. Immediately adjust the pH of the eluate to neutral. Detect the antibody concentration using nanodrop and record the results.

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

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

[0097] 3.2 Prometheus / Merck's anti-TL1A monoclonal antibody RPA-023 (MK-7240), the sequence of which is referenced from patent WO2022178158A1, the heavy chain amino acid sequence is shown in SEQ ID NO: 4, and the light chain amino acid sequence is shown in SEQ ID NO: 5.

[0098] Example 4: Detecting the blocking effect of candidate molecules in reporter gene assays

[0099] TF-1-NFkB-Luc reporter cells in the logarithmic growth phase were collected, washed twice with PBS, and starved overnight with GM-CSF-free medium. Purified hybridoma antibodies (starting at 7.5 μg / mL, 4-fold serial dilution, a total of 8 concentration gradients) were added to a 96-well white bottom plate, and diluted TL1A protein was added to a final concentration of 20 ng / mL. The cells were incubated at 37°C for 30 minutes, and the reporter cells that had been starved overnight were collected and counted. 30,000 cells were added to each well, mixed, and incubated at 37°C for 4.5 hours. 50 μL / well One-lite luminescent substrate reagent was added, and the cells were incubated at room temperature in the dark for 5 minutes before reading the chemiluminescence on a microplate reader. The results are shown in Table 4. According to whether the reporter gene activation activity and IC can be completely blocked, the expression of ... 50 Based on the expression of 4 clones, 4G9E1, 15C4A11, 18D10D11 and 7B5C11 were selected for the next step of blocking cytokine release activity detection.

[0100] Table 4 Blocking activity of hybridoma antibodies in reporter gene assays

[0101] Example 5: Detection of the blocking effect of candidate molecules on the release of IFN-γ from PBL (peripheral blood lymphocyte) cells induced by TL1A combined with IL12 / IL18

[0102] Resuscitated frozen PBMCs were placed in pre-warmed culture medium, centrifuged (300g, 10min), resuspended in culture flasks and cultured at 37°C. After 2 hours, non-adherent lymphocytes (PBL) were collected and counted, and 0.2M / well was plated into 96-well U-bottom plates. Add the dilution of the test antibody (10μg / mL, 3-fold serial dilution, 8 gradients) to the U-bottom plate, add 50ng / mL of TL1A protein to the 96-well U-bottom plate, add 0.5ng / mL IL-12 and 5ng / mL IL-18 protein to the U-bottom plate, and culture at 37°C. After incubation for 24 hours, centrifuge at 2000rpm for 15min, collect the culture supernatant, and detect the IFN-γ content in the supernatant by ELISA. The results are shown in Table 5. According to IC 50 Finally, 4G9E1 and 7B5C11 were selected as the final cell clones.

[0103] Table 5 Blocking activity of hybridoma antibodies in cytokine assays

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

[0105] Hybridoma cells 7B5C11 and 4G9E1, grown to the logarithmic growth phase, were counted, 500,000 to 1,000,000 cells were centrifuged, and the supernatant discarded. The cells were then washed once with sterile PBS. Total RNA was extracted using a kit (purchased from Takara, 9767). The concentration and purity of the extracted RNA were determined, and reverse transcription was performed using oligoDT primers and reverse transcriptase at 42°C for 45 minutes to synthesize cDNA. After PCR amplification, 10 μL of the PCR product was analyzed by gel electrophoresis. PCR products with the correct band size were linked to a T vector (purchased from Takara, 6013) at room temperature. After a 30-minute reaction, DH5α cells were transformed and incubated at 37°C with shaking for 30 minutes. The cells were then plated and incubated overnight for blue-white spot screening. Ten white spots were selected for sequencing. The sequencing results were analyzed and compared to 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] In addition, 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 sequence is humanized. First, the antibody variable region sequence is analyzed to determine the antibody CDR region and framework region. Then, the framework region is aligned with the human germline sequence. The variable region framework of the mouse candidate antibody is replaced with the most matching human germline antibody sequence variable region framework. Finally, the mouse candidate antibody constant region is also replaced with the human antibody constant region to obtain a humanized antibody with a mouse CDR region and human sequences in other regions. According to the 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 specificity-determining 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 the antibody spatial structure, the key amino acid sites in the humanized framework region need to be mutated back to the amino acids in the original mouse antibody sequence; if the back mutation exceeds two amino acids, permutation and combination are required before expression testing, and genes are synthesized based on the antibody heavy and light chain sequences after the back mutation.

[0109] The heavy and light chain combinations were then cloned into vectors containing the human kappa constant region and the human IgG1-LALA heavy chain constant region, respectively. The plasmids containing the heavy and light chains were then transfected into CHO cells for expression. The affinity of the humanized antibodies was measured using the supernatant of the expressed cells. The chimeric antibody was used as a control. The affinity of the humanized antibodies must not be lower than that of the control antibody. Finally, the humanized heavy and light chain combinations with the best affinity were selected: hu7B5-1, hu7B5-2, and hu4G9, 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] Take the enzyme-labeled plate and add the antibody diluted with PBS at 1 μg / mL per well, coat it at 4°C overnight, wash the coated cell plate three times with PBST, then add blocking solution (PBS + 2% BSA + 0.01% Tween 20) and block at room temperature for about 1 hour. Discard the blocking solution, then add 100 μL of TL1A protein diluted in multiple ratios (200 nM starting, 5-fold serial dilution, 8 concentration gradients), incubate at room temperature for 1 hour, wash three times with PBST, add 100 μL of secondary antibody diluted with antibody diluent (PBS + 0.5% BSA + 0.01% Tween 20) (1:20000 dilution), incubate at room temperature for 1 hour, wash three times with PBST, add 100 μL of TMB solution for color development for about 5 minutes, then add stop solution to stop color development, and read OD on the enzyme reader. 450 The results are shown in Figure 2. The EC values ​​of hu7B5-1 and hu4G9 for hTL1A binding are 50 The EC values ​​of PF-06480605 and PRA-023 for hTL1A binding were 0.5710 nM and 0.7765 nM, respectively. 50 0.5180nM and 0.7454nM respectively.

[0113] 8.2 Biofilm Interferometry

[0114] The affinity of hu7B5-1, hu7B5-2 and hu4G9 to human TL1A was tested based on biofilm interferometry (BLI) technology. Using a Gator instrument, the antibodies were first immobilized on the Protein A probe, and then the TL1A protein was diluted to 200nM, 100nM, 50nM, 25nM, 12.5nM, and 6.25nM. The binding and dissociation curve test procedures for the detection antibodies are shown in Table 6. The dissociation constant results after the TL1A protein binds to hu7B5-1, hu7B5-2, and hu4G9 antibodies are shown in Table 7.

[0115] Table 6 uses Binding and dissociation curve assay procedures for detection antibodies

[0116] Table 7 Dissociation constants of TL1A protein binding to hu7B5-1, hu7B5-2 and hu4G9 antibodies

[0117] 8.3 Flow cytometry

[0118] HEK293-hTL1A cells in the logarithmic growth phase were collected, resuspended in staining buffer (PBS + 2% FBS) and counted, and 50,000 / well were added to a 96-well V-bottom plate. The antibody to be tested was diluted in multiples (starting at 30 μg / mL, 5-fold serial dilution, a total of 8 concentration gradients) and added to a 96-well V-bottom plate and incubated at 4°C for 1 hour. Washed twice with staining buffer, and then FITC-labeled goat anti-human IgG secondary antibody (purchased from Abcam, ab7149) was added and incubated at 4°C for 1 hour. Washed twice with staining buffer and analyzed using a CytoFLEX flow cytometer. The results are shown in Figure 3. The EC values ​​of hu7B5-1, hu7B5-2 and hu4G9 for binding to hTL1A were 50 The EC values ​​of PF-06480605 and PRA-023 for hTL1A binding were 0.2995 μg / mL, 0.3252 μg / mL, and 0.2332 μg / mL, respectively. 50 They were 0.3182 μg / mL and 0.6265 μg / mL respectively.

[0119] Example 9: Identification of the 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 with GM-CSF-free medium. hu7B5-1, hu7B5-2, and hu4G9 were added to a 96-well white-bottom plate, diluted TL1A protein was added, and the cells were incubated at 37°C for 30 minutes. The reporter cells that were starved overnight were collected and counted, and 30,000 cells were added to each well. The cells were mixed and incubated at 37°C for 4.5 hours. 50 μL / well One-lite luminescent substrate reagent was added, and the cells were incubated at room temperature in the dark for 5 minutes before reading the chemiluminescence on a microplate reader. The results are shown in Figure 4. The IC values ​​of hu7B5-1, hu7B5-2, and hu4G9 for blocking TL1A-induced reporter cell activity were 0. 50 The IC values ​​of PF-06480605 and PRA-023 were 0.008743 μg / mL, 0.01073 μg / mL, and 0.01146 μg / mL, respectively. 50 They are 0.007735μg / mL and 0.04554μg / mL respectively.

[0121] Example 10: ELSIA method to identify the blocking effect of humanized antibodies on the binding of TL1A to DR3

[0122] Take the ELISA plate and add DR3 protein diluted with PBS (purchased from Biopsies, TN5-H5258) at 2 μg / mL per well. Coat at 4°C overnight. Wash the coated cell plate three times with PBST, then add blocking solution (PBS + 2% BSA + 0.01% Tween 20) and block at room temperature for about 1 hour. Discard the blocking solution, then mix the diluted TL1A protein (final concentration 0.5 μg / mL) and the serially diluted antibody to be tested, add 100 μL to each well, and incubate at room temperature for 2 hours. Wash three times with PBST, add 100 μL of secondary antibody diluted with antibody diluent (PBS + 0.5% BSA + 0.01% Tween 20) (1:30000 dilution), and let it stand at room temperature for 1 hour. Wash three times with PBST, add 100 μL of TMB solution to develop color for about 5 minutes, then add stop solution to stop color development, and read the OD on the ELISA reader. 450 The specific results are shown in Figure 5. The IC values ​​of hu7B5-1 and hu4G9 for blocking the binding of TL1A to DR3 are 50 The IC values ​​of PF-06480605 and PRA-023 were 0.551 μg / mL and 1.05 μg / mL, respectively. 50 They were 1.14 μg / mL and 4.492 μg / mL respectively.

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

[0124] Resuscitated frozen PBMCs were placed in pre-warmed culture medium, centrifuged (300 g, 10 min), resuspended in culture flasks and cultured at 37°C. After 2 hours, non-adherent PBL cells were collected and counted, and 0.2 M / well was plated into 96-well U-bottom plates. Added the dilution of the test antibody (10 μg / mL, 3-fold serial dilution, 8 gradients) to the U-bottom plate, added 50 ng / mL of TL1A protein to the 96-well U-bottom plate, added 0.5 ng / mL of IL-12 and 5 ng / mL of IL-18 protein to the U-bottom plate, and cultured at 37°C. After incubation for 24 hours, centrifuged at 2000 rpm for 15 min, collected the culture supernatant, and the IFN-γ content in the supernatant was detected by ELISA. The results are shown in Figure 6. The IC values ​​of hu7B5-1, hu7B5-2 and hu4G9 for blocking TL1A-induced IFN-γ release were 0. 50 The values ​​were 0.1393 μg / mL, 0.1006 μg / mL, and 0.1391 μg / mL, respectively. The IC 50 It is 0.1298μg / mL.

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

[0126] The affinity of hu7B5-1 and hu4G9 for cynomolgus macaque TL1A was tested using biomembrane interferometry (BLI). Using a Gator instrument, the antibodies were first immobilized onto a Protein A probe, and then the cynomolgus macaque TL1A protein was diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM. The binding and dissociation curves for the antibodies using the Gator instrument are shown in Table 8. The dissociation constants for the binding of cynomolgus macaque TL1A protein to hu7B5-1 and hu4G9 are shown in Table 9. Both hu7B5-1 and hu4G9 cross-reacted with cynomolgus macaque TL1A, with K values ​​of 2.52E-09 M and 1.87E-09 M, respectively.

[0127] Table 8 uses Binding and dissociation curve assay procedures for detection antibodies

[0128] Table 9 Dissociation constants of cynomolgus monkey TL1A protein binding to hu7B5-1 and hu4G9 antibodies

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

[0130] Take the ELISA plate and add hu7B5-1 or hu4G9 antibody diluted in PBS to each well at 1 μg / mL. Coat overnight at 4°C. Wash the coated cell plate three times with PBST, then add blocking solution (PBS + 2% BSA + 0.01% Tween 20) and block at room temperature for about 1 hour. Discard the blocking solution, then add 100 μL of diluted TNFSF6, TNFSF10 and TNFSF14 protein to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add 100 μL of secondary antibody diluted in antibody diluent (PBS + 0.5% BSA + 0.01% Tween 20) (1:20000 dilution) and incubate at room temperature for 1 hour. Wash three times with PBST, add 100 μL of TMB solution to develop color for about 5 minutes, then add stop solution to stop color development and read the OD on the ELISA reader. 450 The results are shown in Figures 7A and 7B , showing that hu7B5-1 and hu4G9 had no binding reaction with the three proteins in the same family.

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

[0132] Table 10 Sequence Listing

[0133] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof targeting TL1A, characterized in that: 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 HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:6, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:7, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:8, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:10, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:11, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:12; Alternatively, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:27, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:28, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:29, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:30, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:31, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:32; Alternatively, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:49, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:50, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:51, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:52, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:53, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:54; Alternatively, 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 antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NOs: 13-19, and the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NOs: 20-23; Alternatively, the heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NOs: 33-39, and the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NOs: 40-46; Alternatively, 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; Alternatively, 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 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; Alternatively, 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; Alternatively, 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; Alternatively, 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 antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody or 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 the amino acid sequence shown in any one of SEQ ID NOs: 64-67, and the light chain constant region has the amino acid sequence shown in SEQ ID NO: 68 or 69.

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

6. An antibody combination, characterized in that 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, characterized in that The isolated nucleic acid encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or the antibody combination according to claim 6.

8. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid of claim 7; Preferably, the recombinant expression vector is a plasmid, cosmid, phage or viral vector; More preferably, the plasmid is pCDNA3.

1.

9. A transformant, characterized in that The transformant comprises the isolated nucleic acid of claim 7 or the recombinant expression vector of 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 yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof; the mammalian cells are, for example, HEK293 cells.

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

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody or 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, characterized in that The TL1A detection agent comprises the antibody or antigen-binding fragment thereof according to claims 1-5, and / or the antibody combination according to claim 6.

13. Use of the antibody or 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 preparation of a diagnostic agent or a detection agent; Preferably, the diagnostic agent or detection agent is used for diagnosing or detecting a disease mediated by TL1A.

14. A method for detecting TL1A, characterized in that: The method comprises contacting the antibody or 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 antigen-binding fragment thereof according to any one of claims 1 to 5 or the antibody combination according to claim 6 in the preparation of a medicament for preventing or treating a disease mediated by TL1A; Preferably, the disease is selected from at least one of the following: inflammatory bowel disease IBD, Crohn's disease, ulcerative colitis, asthma, allergies, 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, urinary intestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis and scleroderma.

16. A method for preventing, ameliorating or treating a disease, disorder or condition mediated by TL1A, comprising administering to a subject in need thereof an effective amount of the antibody or 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, allergies, 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, urinary intestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis and scleroderma.

17. The antibody or 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 disease, disorder or condition mediated by TL1A; Preferably, the disease is selected from at least one of the following: inflammatory bowel disease IBD, Crohn's disease, ulcerative colitis, asthma, allergies, 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, urinary intestinal dysfunction, uveitis, systemic lupus erythematosus, atopic dermatitis, eczematous dermatitis, psoriasis and scleroderma.