Anti-TL1A antibody, method for preparing the same, and its use

Abstract

This invention discloses an anti-TL1A antibody, a method for preparing the same, and its use, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the anti-TL1A antibody has one or more of the following technical features: The heavy chain variable region comprises HCDR1 having the amino acid sequence shown in SEQ ID NO: 10 or 18; the heavy chain variable region comprises HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, 19, 39, 40, or 41; the heavy chain variable region comprises HCDR3 having the amino acid sequence shown in SEQ ID NO: 12, 20, or 42; the light chain variable region comprises LCDR1 having the amino acid sequence shown in SEQ ID NO: 13 or 21; the light chain variable region comprises LCDR2 having the amino acid sequence shown in SEQ ID NO: 14 or 22; and the light chain variable region comprises LCDR3 having the amino acid sequence shown in SEQ ID NO: 15 or 23. The antibody of the present invention can inhibit the secretion of IFN-γ and TNF-α by TL1A-induced PBMCs and inhibit TL1A-induced apoptosis. Therefore, it is suggested that the antibody or its composition can be used to treat diseases such as inflammatory bowel disease and gastrointestinal diseases associated with cystic fibrosis.

Description

[Technical Field]

[0001] This invention relates to the field of antibody pharmaceuticals, and more particularly to anti-TL1A antibodies, methods for preparing them, and their use. [Background technology]

[0002] Inflammatory bowel disease (IBD) is a specific type of chronic inflammatory bowel disease that includes Crohn's disease (CD) and ulcerative colitis (UC). While the etiology and pathogenesis of IBD are not yet fully understood, it is generally believed to be the result of the interaction of multiple factors, including dysregulation of intestinal mucosal immunity, persistent intestinal infection, defects in the intestinal mucosal barrier, and genetic and environmental factors. Both innate and adaptive immune responses in the intestinal mucosa contribute to the pathogenesis of IBD, and abnormal immune responses in the intestinal mucosa are the primary cause of mucosal tissue damage.

[0003] TL1A (also known as Tumor Necrosis Factor Superfamily Member 15 [TNFSF15]) is a member of the TNF ligand superfamily and, like other members, is a homotrimer. TL1A is a type II transmembrane protein that exists in both membrane-bound and soluble forms, and its receptors include two types: DR3 and soluble decoy receptor 3 (DcR3). DR3, also known as APO3, LARD, or WSL1, is a member of the tumor necrosis factor receptor superfamily (TNFSF). TL1A is a ligand for DR3, and when it binds to TL1A, it induces the activation of nuclear factor-kappa B (NF-κB), promotes the transcription of inflammatory cytokines, and activates apoptotic proteases, thereby exerting pro-inflammatory and pro-apoptotic effects. DcR3 competitively binds to TL1A, weakening the production of T cell costimulatory signals, exerting anti-apoptotic effects, and inhibiting the secretion of pro-inflammatory factors.

[0004] Recent studies have revealed that TL1A promotes Th1 cell polarization and efficacy by influencing the activation and proliferation of lamina propria T cells in the intestinal epithelium, thereby increasing IFN-γ expression and promoting inflammatory responses and fibrosis of the intestinal mucosa. Furthermore, TL1A acts on Th17 cells, increasing IL-17 secretion, causing an imbalance in intestinal mucosal immunity and inducing intestinal inflammatory responses. Chronic inflammatory responses are the primary cause of intestinal fibrosis, and these responses further activate intestinal fibroblasts, leading to the production of large amounts of extracellular matrix (ECM), which contributes to the formation of intestinal fibrosis. Therefore, TL1A may promote the development of chronic intestinal fibrosis associated with experimental colitis by regulating IL-17 and IFN-γ. Anti-TL1A antibodies effectively inhibit TL1A-induced IFN-γ and TNF-α secretion from human peripheral blood mononuclear cells (PBMCs) and inhibit TL1A-induced apoptosis, making them useful in the treatment of inflammatory bowel disease and related disorders. [Overview of the Initiative]

[0005] In view of the shortcomings of the prior art described above, the object of the present invention is to provide an anti-TL1A antibody, a method for preparing the same, and its use, in order to solve the problems of the prior art.

[0006] To achieve the aforementioned and other related objectives, the present invention provides an anti-TL1A antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the anti-TL1A antibody or antigen-binding fragment is <1> A heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 10 or 18, <2> A heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, 19, 39, 40, or 41. <3> A heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 12, 20, or 42, <4> A light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 13 or 21, <5> A light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 14 or 22, <6> Light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO:15 or 23, It has one or more of the following technical features.

[0007] The present invention also provides a recombinant protein comprising (i) the anti-TL1A antibody or its antigen-binding fragment, and (ii) a tag sequence that optionally contributes to expression and / or purification.

[0008] The present invention also provides isolated polynucleotides encoding the heavy chain variable region and / or light chain variable region or full-length amino acids of the anti-TL1A antibody or its antigen-binding fragment, or the recombinant protein.

[0009] The present invention also provides nucleic acid constructs comprising the aforementioned isolated polynucleotides.

[0010] The present invention also provides cells containing the construct or exogenous polynucleotides incorporated into its genome.

[0011] The present invention also provides a method for preparing an anti-TL1A antibody or its antigen-binding fragment, comprising the step of expressing the anti-TL1A antibody or its antigen-binding fragment by culturing the cells described in claim 11 under conditions suitable for the expression of the TL1A antibody or its antigen-binding fragment, further comprising the step of purifying and / or isolating the TL1A antibody or its antigen-binding fragment.

[0012] The present invention also provides the use of the anti-TL1A antibody or its antigen-binding fragment, or the recombinant protein, in the preparation of a disease therapeutic agent or a disease diagnostic agent.

[0013] In the present invention, the disease is selected from one or more of the following: inflammatory bowel disease, gastrointestinal disease associated with cystic fibrosis, colitis, irritable bowel syndrome, eosinophilic esophagitis, atopic dermatitis, eczema, scleroderma, arthritis, or rheumatoid arthritis. Here, inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0014] The present invention also provides a pharmaceutical composition comprising the anti-TL1A antibody or its antigen-binding fragment, or the recombinant protein.

[0015] The present invention also provides a method for treating a disease, comprising administering to a subject requiring such treatment an anti-TL1A antibody or its antigen-binding fragment, the recombinant protein, or the pharmaceutical composition described in the present invention.

[0016] As described above, the anti-TL1A antibody or antigen-binding fragment of the present invention has the following beneficial effects: it can effectively inhibit the secretion of IFN-γ and TNF-α by TL1A-induced human peripheral blood mononuclear cells (PBMCs), and it can inhibit TL1A-induced apoptosis. The antibody or its composition has been suggested to be usable in the treatment of inflammatory bowel disease, gastrointestinal disorders associated with cystic fibrosis, colitis, irritable bowel syndrome, eosinophilic esophagitis, atopic dermatitis, eczema, scleroderma, arthritis, or rheumatoid arthritis.

[0017] It should be understood that each of the above technical features of the present invention and each of the technical features specifically described below (for example, in the examples) can be combined with each other within the scope of the present invention to constitute novel or preferred technical solutions. Due to space limitations, each will not be explained in detail here. [Brief explanation of the drawing]

[0018] [Figure 1A] Figure 1A shows the binding ability of the test mouse antibody to the target antigen human TL1A-His in Example 1. [Figure 1B]Figure 1B shows the binding ability of the test mouse antibody to the target antigen human TL1A-His in Example 1. [Figure 1C] Figure 1C shows the binding ability of the test mouse antibody to the target antigen human TL1A-His in Example 1. [Figure 2A] Figure 2A shows the measurement results of the inhibitory activity of the mouse antibody against the binding of the stable expression cell line 293FT-DR3 to the TL1A protein in Example 1. [Figure 2B] Figure 2B shows the measurement results of the inhibitory activity of the mouse antibody against the binding of the stable expression cell line 293FT-DR3 to the TL1A protein in Example 1. [Figure 2C] Figure 2C shows the measurement results of the inhibitory activity of the mouse antibody against the binding of the stable expression cell line 293FT-DR3 to the TL1A protein in Example 1. [Figure 3A] Figure 3A shows the ELISA measurement results regarding the binding affinity of the candidate antibodies obtained after optimization of the physicochemical properties of the humanized antibodies 72851, 31122 and the humanized antibody 31122 to the antigen TL1A-his in Example 4. [Figure 3B] Figure 3B shows the measurement results of capillary isoelectric focusing electrophoresis of the candidate antibodies obtained after optimization of the physicochemical properties of the humanized antibodies 72851, 31122 and the humanized antibody 31122 in Example 4. [Figure 4] Figure 4 shows the experimental results that the anti-TL1A antibody inhibits the binding of TL1A and DR3 in Example 5. [Figure 5] Figure 5 shows the experimental results that the anti-TL1A antibody inhibits the NF-κB signaling pathway of the TL1A TF-1 cell line in Example 6. [Figure 6] Figure 6 shows the results that the anti-TL1A antibody inhibits TL1A-induced TF-1 apoptosis in Example 7. [Figure 7] Figure 7 shows the results that the anti-TL1A antibody inhibits the IFNγ secretion from TL1A-stimulated cells in Example 8. [Figure 8] Figure 8 shows the results that the anti-TL1A antibody inhibits the TNFα secretion from TL1A-stimulated cells in Example 9. [Figure 9A] Figure 9A shows the ELISA measurement results regarding the affinity of humanized antibody 72581 to human, cynomolgus monkey, rat, and mouse TL1A proteins in Example 10. [Figure 9B] Figure 9B shows the efficacy results of the anti-TL1A antibody in the DSS-induced rat chronic inflammatory bowel disease model in Example 10. [Figure 9C] Figure 9C shows the efficacy results of the anti-TL1A antibody in the DSS-induced rat chronic inflammatory bowel disease model in Example 10. [Figure 9D] Figure 9D shows the efficacy results of the anti-TL1A antibody in the DSS-induced rat chronic inflammatory bowel disease model in Example 10. [Figure 9E] Figure 9E shows the efficacy results of the anti-TL1A antibody in the DSS-induced rat chronic inflammatory bowel disease model in Example 10. [Figure 10A] Figure 10A shows the efficacy results of the anti-TL1A antibody in Example 11 in a rat model of TNBS-induced acute inflammatory bowel disease. [Figure 10B] Figure 10B shows the efficacy results of the anti-TL1A antibody in Example 11 in a rat model of TNBS-induced acute inflammatory bowel disease. [Modes for carrying out the invention]

[0019] The present invention provides an anti-TL1A antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the anti-TL1A antibody or its antigen-binding fragment is <1> A heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 10 or 18, <2> A heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, 19, 39, 40, or 41. <3> A heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 12, 20, or 42, <4> A light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 13 or 21, <5> A light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 14 or 22, <6> A light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO: 15 or 23, The present invention has one or more technical features. In some embodiments of the present invention, the anti-TL1A antibody or its antigen-binding fragment includes the following technical features. <1> The heavy chain variable region contains HCDR1 having the amino acid sequence shown in SEQ ID NO:10. <2> The heavy chain variable region contains HCDR2 having the amino acid sequence shown in SEQ ID NO:11. <3> The heavy chain variable region contains HCDR3 having the amino acid sequence shown in SEQ ID NO:12. <4> The light chain variable region contains LCDR1 having the amino acid sequence shown in SEQ ID NO:13. <5> The light chain variable region contains LCDR2 having the amino acid sequence shown in SEQ ID NO:14. <6> The light chain variable region is LCDR3, which has the amino acid sequence shown in SEQ ID NO:15.

[0020] In some embodiments of the present invention, the anti-TL1A antibody or its antigen-binding fragment comprises the following technical features. <1> The heavy chain variable region contains HCDR1 having the amino acid sequence shown in SEQ ID NO:18. <2> The heavy chain variable region contains HCDR2 having the amino acid sequence shown in SEQ ID NO:19. <3> The heavy chain variable region contains HCDR3 having the amino acid sequence shown in SEQ ID NO:20. <4> The light chain variable region contains LCDR1 having the amino acid sequence shown in SEQ ID NO:21. <5> The light chain variable region includes LCDR2 having the amino acid sequence shown in SEQ ID NO:22. <6> The light chain variable region contains LCDR3, which has the amino acid sequence shown in SEQ ID NO:23.

[0021] In some embodiments of the present invention, the anti-TL1A antibody or its antigen-binding fragment is <1> A heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO:18, <2> A heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO:39, <3> A heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO:20, <4> A light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO:21, <5> A light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO:22, <6> It includes a light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO:23.

[0022] In some embodiments of the present invention, the anti-TL1A antibody or its antigen-binding fragment is <1> A heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO:18, <2> A heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO:40, <3> A heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO:20, <4> A light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO:21, <5> A light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO:22, <6> It includes a light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO:23.

[0023] In some embodiments of the present invention, the anti-TL1A antibody or its antigen-binding fragment is <1> A heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO:18, <2> A heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO:41, <3> A heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO:20, <4> A light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO:21, <5> A light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO:22, <6> It includes a light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO:23.

[0024] In some embodiments of the present invention, the anti-TL1A antibody or its antigen-binding fragment is <1> A heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO:18, <2> A heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO:39, <3> A heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO:42, <4> A light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO:21, <5> A light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO:22, <6> It includes a light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO:23.

[0025] In some embodiments of the present invention, the anti-TL1A antibody or its antigen-binding fragment is <1> A heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO:18, <2> A heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO:40, <3> A heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO:42, <4> A light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO:21, <5> A light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO:22, <6> It includes a light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO:23.

[0026] In some embodiments of the present invention, the anti-TL1A antibody or its antigen-binding fragment is <1> A heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO:18, <2> A heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO:41, <3> A heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO:42, <4> A light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO:21, <5> A light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO:22, <6> It includes a light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO:23.

[0027] A CDR (complementarity determining region) typically refers to a region in an antibody that can form spatial complementarity with an antigenic determinant. Variableity in antibodies is usually not uniformly distributed throughout the entire variable region. Monoclonal antibodies typically have three hypervariable regions (HVRs) in both the heavy chain and light chain variable regions. These regions are spatially complementary to the antigenic determinant and are therefore also called complementarity determining regions (CDRs). Specifically, the heavy chain variable region typically contains three complementarity determining regions: HCDR1, HCDR2, and HCDR3, while the light chain variable region typically contains three complementarity determining regions: LCDR1, LCDR2, and LCDR3.

[0028] In some embodiments of the present invention, the heavy chain variable region and the light chain variable region may also include a framework region, which may be located between or at both ends of the complementarity-determining regions. In some specific embodiments of the present invention, the framework region sequence is a framework region sequence obtained by substituting, deleting, or adding one or more (specifically 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3) amino acids to the framework region sequence of a human monoclonal antibody or a mouse monoclonal antibody variable region, and the framework region sequence may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology to the framework region sequence of the variable region sequence of a human monoclonal antibody.

[0029] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown as one of SEQ ID NO:3, SEQ ID NO:16, SEQ ID NO:7, SEQ ID NO:24, or SEQ ID NO:26-31.

[0030] In some embodiments of the present invention, the amino acid sequences of the light chain variable region of the anti-TL1A antibody or its antigen-binding fragment are shown in SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:9, and SEQ ID NO:25.

[0031] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

[0032] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO:16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:17. In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:9.

[0033] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25.

[0034] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO:26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25.

[0035] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO:27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25.

[0036] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO:28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25.

[0037] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO:29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25.

[0038] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO:30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25.

[0039] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO:31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:25.

[0040] In some embodiments of the present invention, the anti-TL1A antibody or its antigen-binding fragment comprises a monovalent antibody, a bivalent antibody, and / or a polyvalent antibody.

[0041] In some embodiments of the present invention, the antigen-binding fragment is selected from scFv, Fab, Fab', F(ab')2, Fv fragment, heavy chain antibody, and disulfide-bound Fv (dsFv).

[0042] In some embodiments of the present invention, the heavy chain constant region of the anti-TL1A antibody or its antigen-binding fragment is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4, and is preferably the human IgG1 heavy chain constant region.

[0043] In some embodiments of the present invention, the light chain constant region of the anti-TL1A antibody or its antigen-binding fragment is selected from the constant regions of the human antibody κ chain or λ chain, and is preferably the κ chain constant region.

[0044] The amino acid sequence of the heavy chain variable region or light chain variable region of the anti-TL1A antibody or its antigen-binding fragment also includes a derivative sequence in which at least one amino acid is added, deleted, modified, and / or substituted in the sequence, and which maintains binding affinity to TL1A. In another preferred embodiment, the derivative sequence is one in which 1 to 5 amino acids, for example 1, 2, or 3 amino acids are added, deleted, modified, and / or substituted in the arbitrary amino acid sequence, and the derivative antibody consisting of VH and VL containing the derivative CDR sequence maintains binding affinity to TL1A.

[0045] The anti-TL1A antibody or its antigen-binding fragment is selected from a complete antibody, a single-chain antibody, or an antibody fragment.

[0046] A complete antibody comprises a variable region (V) and a constant region (C). The constant region comprises a light chain constant region (LC) and a heavy chain constant region (HC). The constant region may be a variant of the constant region of the natural sequence or its amino acid sequence. The constant region of the natural sequence is, for example, the constant region of a mammalian, e.g., human natural sequence. In some embodiments of the present invention, the heavy chain constant region is the IgG1 constant region and / or the light chain constant region is the kappa chain constant region (i.e., the κ chain constant region). In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:33 and / or the amino acid sequence of the light chain constant region is shown in SEQ ID NO:34.

[0047] An "antibody fragment" contains a portion of a complete antibody, preferably including its antigen-binding region or variable region. For example, antibody fragments include Fab, Fab', F(ab'), F(ab')2, and Fv fragments. An Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. This region is a structure in which one heavy chain variable region and one light chain variable region are closely bound, and this bond may be covalent (for example, in scFv). In such a conformation, the three CDRs of each variable region interact to determine the antigen-binding site on the surface of the VV dimer. A "Fab" fragment contains a light chain variable region and a constant region, a heavy chain variable region and a first constant region (CH1). A single F(ab') antibody fragment contains a pair of Fab fragments, which are usually covalently linked via hinge cysteine ​​near the carboxyl terminus.

[0048] The present invention also provides a recombinant protein comprising (i) the anti-TL1A antibody or its antigen-binding fragment, and (ii) a tag sequence that optionally contributes to expression and / or purification.

[0049] In some embodiments of the present invention, the tag sequence includes Fc tags, HA tags, GGGS sequences, FLAG tags, Myc tags, 6His tags, or combinations thereof.

[0050] In some embodiments of the present invention, the recombinant protein includes a fusion protein.

[0051] In some embodiments of the present invention, the recombinant protein is a monomer, dimer, or polymer.

[0052] The present invention also provides isolated polynucleotides encoding the heavy chain variable region and / or light chain variable region or full-length amino acids of the anti-TL1A antibody or its antigen-binding fragment, or the recombinant protein.

[0053] In some embodiments of the present invention, the polynucleotide is selected from RNA (e.g., mRNA) or DNA (e.g., cDNA).

[0054] In some embodiments of the present invention, the polynucleotide sequences encoding the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment are shown in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:43-48.

[0055] In some embodiments of the present invention, the polynucleotide sequences encoding the light chain variable region of the anti-TL1A antibody or its antigen-binding fragment are shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:36, and SEQ ID NO:38.

[0056] The present invention also provides nucleic acid constructs comprising the aforementioned isolated polynucleotides.

[0057] In some embodiments of the present invention, the nucleic acid construct is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene delivery systems, or combinations thereof.

[0058] In some embodiments of the present invention, the nucleic acid construct includes a viral vector such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0059] In some embodiments of the present invention, the nucleic acid construct is a plasmid, a retrovirus, or a lentiviral vector.

[0060] In some embodiments of the present invention, the nucleic acid construct is selected from the group consisting of pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, and the like.

[0061] In some embodiments of the present invention, the nucleic acid construct also includes elements selected from the group consisting of promoters, transcription enhancer elements (WPREs), and long terminal repeat sequences (LTRs).

[0062] The term “nucleic acid construct” refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues, and such nucleic acid construct can be various expression vectors comprising a vector backbone (i.e., an empty vector) and an expression cassette. The term “expression cassette” refers to a sequence that may encode a protein.

[0063] The type of expression vector is not particularly limited. An expression vector is a nucleic acid molecule that enables the insertion of foreign nucleotides without impairing the vector's ability to replicate and / or integrate in host cells. An expression vector may contain nucleic acid sequences that enable replication within host cells, such as origins of replication. An expression vector may also contain one or more select marker genes and other genetic elements. An expression vector is a vector that contains regulatory sequences necessary for the transcription and translation of the inserted gene. Expression vectors are selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0064] The prokaryotic expression vector is selected from E. coli expression vectors, Bacillus subtilis expression vectors, or Streptomyces expression vectors. In certain preferred embodiments, the prokaryotic expression vector is selected from E. coli expression vectors. Compared to other expression systems, the E. coli expression system has a clear genetic background, short culture cycles, high target gene expression levels, and strong contamination resistance. The E. coli expression vector is, for example, a pET expression vector, specifically pET28a, pET32a, which can be stably expressed in E. coli. The expression vector may also be a pCW expression vector or a pUC expression vector.

[0065] The eukaryotic expression vector is selected from yeast expression vectors, insect expression vectors, or mammalian expression vectors. The mammalian expression vector is selected from retrovirus expression vectors, lentivirus expression vectors, adenovirus expression vectors, and adeno-associated virus expression vectors. In certain preferred embodiments, the eukaryotic expression vector is selected from retrovirus expression vectors that can be stably expressed in a cell line, and the retrovirus vector is, for example, pMSCV.

[0066] The host cells are selected from eukaryotic or prokaryotic host cells. Eukaryotic host cells are selected from fungi such as yeast, insects, birds, plants, Cynorhabdaitis elegans (C. elegans), nematodes, or mammalian host cells. A non-specific example of an insect cell is the fall armyworm (Spodopterafrugiperda, Sf) cell. Examples of yeast host cells include Saccharomyces cerevisiae, Kluyveromyces lactis (K. lactis), or Yarrowia lipolytica. Examples of mammalian cells include COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary cells (CHO), human fetal kidney cells (HEK), African green monkey cells, CV1 cells, Vero, or Hep-2 cells. Examples of prokaryotic host cells include bacterial cells such as Escherichia coli, Streptomyces, Bacillus subtilis, Salmonella typhi, or mycobacteria.

[0067] Those skilled in the art can obtain cells containing a gene encoding an anti-TL1A antibody or its antigen-binding fragment by transfecting host cells with an expression vector using methods well known in the art. For example, an expression vector can be introduced into eukaryotic cells by calcium phosphate coprecipitation, electroporation, microinjection, lipofection, or transfection using a polyamine transfection reagent.

[0068] The present invention also provides cells containing the construct or exogenous polynucleotides incorporated into its genome.

[0069] The cells described in the present invention are obtained by transforming host cells with nucleic acid constructs.

[0070] In some embodiments of the present invention, the cells include prokaryotic cells or eukaryotic cells.

[0071] In some embodiments of the present invention, the cells are selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0072] The present invention also provides a method for preparing an anti-TL1A antibody or its antigen-binding fragment, comprising the step of expressing the anti-TL1A antibody or its antigen-binding fragment by culturing the cells described in claim 11 under conditions suitable for the expression of the TL1A antibody or its antigen-binding fragment, further comprising the step of purifying and / or isolating the TL1A antibody or its antigen-binding fragment.

[0073] The present invention also provides the use of the anti-TL1A antibody or its antigen-binding fragment, or the recombinant protein, in the preparation of a disease therapeutic agent or a disease diagnostic agent.

[0074] In the present invention, the disease is selected from one or more of the following: inflammatory bowel disease, gastrointestinal disease associated with cystic fibrosis, colitis, irritable bowel syndrome, eosinophilic esophagitis, atopic dermatitis, eczema, scleroderma, arthritis, or rheumatoid arthritis. Here, inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0075] The present invention also provides a pharmaceutical composition comprising the anti-TL1A antibody or its antigen-binding fragment, or the recombinant protein.

[0076] In some embodiments of the present invention, the pharmaceutical composition comprises the anti-TL1A antibody of the present invention or its antigen-binding fragment, or the recombinant protein, and a pharmaceutically acceptable vector or additive.

[0077] The term "pharmaceutically acceptable" means that a drug, when administered appropriately to animals or humans, does not cause adverse reactions, allergic reactions, or other side effects.

[0078] A "pharmaceutically acceptable vector or additive" is one that is compatible with the active ingredient, meaning it can be mixed with the drug under normal circumstances without significantly reducing its efficacy. Specific examples of substances that may be pharmaceutically acceptable vectors or additives include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyhydric alcohols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; emulsifiers such as alginic acid and Tween; humectants such as sodium lauryl sulfate; colorants; flavorings; tableting agents; stabilizers; antioxidants; preservatives; pyrogenic substance-removed distilled water; isotonic saline; and phosphate buffers. These substances are used, as needed, to contribute to the stability of the formulation, enhance its activity or biological efficacy, or create an acceptable taste or aroma for oral administration.

[0079] The form of the pharmaceutical composition is not particularly limited and can be in various forms such as solid, liquid, gel, semi-fluid, or aerosol.

[0080] The pharmaceutical composition is primarily intended for mammals. Preferably, the mammals are rodents, artiodactyls, odd-toed ungulates, lagomorphs, primates, etc. Preferably, the primates are monkeys, apes, or humans.

[0081] The prescribed pharmaceutical composition may be administered by conventional routes of administration, including but not limited to intravenous injection, intravenous drip infusion, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (e.g., intraperitoneal injection), intracranial injection, or intracavitary injection.

[0082] The present invention also provides a method for treating a disease, comprising administering to a subject requiring such treatment an anti-TL1A antibody or its antigen-binding fragment, the recombinant protein, or the pharmaceutical composition described in the present invention.

[0083] In some embodiments of the present invention, the subject includes a mammal, such as a human.

[0084] In some embodiments of the present invention, the disease is selected from one or more of inflammatory bowel disease, gastrointestinal diseases associated with cystic fibrosis, colitis, irritable bowel syndrome, eosinophilic esophagitis, atopic dermatitis, eczema, scleroderma, arthritis, or rheumatoid arthritis. Here, inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0085] In some embodiments of the present invention, the method can be used in combination with other treatments.

[0086] In some embodiments of the present invention, the other treatment methods include treatments such as chemotherapy, radiotherapy, and molecular targeted therapy.

[0087] When treating a disease, a safe and effective amount of antibody or its antigen-binding fragment is administered to the subject. This safe and effective amount is typically at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight; preferably, the dose is about 10 μg / kg body weight to 10 mg / kg body weight. Of course, the specific dose should be determined considering factors such as the mode of administration and the patient's health condition, all within the scope of a skilled physician's expertise.

[0088] Embodiments of the present invention will be described below through specific examples. Those skilled in the art will readily understand other advantages and benefits of the present invention from what is disclosed herein. The present invention can be carried out or applied through various other specific embodiments, and the details herein can be modified or altered on different viewpoints and uses without departing from the spirit of the invention.

[0089] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below. It should also be understood that the terms used in the examples are intended to describe specific embodiments and are not intended to limit the scope of protection of the present invention. In this specification and in the claims, unless otherwise specified, the singular forms "one," "one," and "this" include the plural forms.

[0090] Where numerical ranges are indicated in the examples, it should be understood that, unless otherwise specified herein, values ​​at both ends of each numerical range, and any values ​​between those ends, may be used. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, apparatus, and materials used in the examples, similar or equivalent prior art methods, apparatus, and materials used in the examples may also be used to implement the present invention, based on the knowledge of those skilled in the art and the disclosure of the present invention. Unless otherwise specified, percentages and parts refer to weight percentages and parts by weight.

[0091] Example 1: Preparation and screening of antigen-immunized animals and hybridomas 1.1 Preparation of antigen protein and positive control antibody The human TL1A extracellular domain (TL1A-ECD) sequence used as the antigen was obtained from the UniProt database (Entry: O95150), and its amino acid sequence is shown in SEQ ID NO: 1. A 6×His tag was added to the N-terminus, and the sequence was incorporated into a pcDNA 3.4 expression vector. HEK-293F cells were transfected with this vector and expressed for 5 days. After expression, the cell culture supernatant was collected and purified to obtain the TL1A-His protein. Similarly, the 6×His tag was replaced with the Fc sequence of human IgG1, and HEK-293F cells were transfected with this protein. Expression and purification were then performed to obtain the TL1A-Fc protein. The sequence of the positive control antibody pra023 was obtained from patent WO-2021081365-A1. SEQ ID NO: 1 LKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL

[0092] 1.2 Mouse Immunity In this experiment, female Balb / c mice aged 6-8 weeks were used. After purchasing the mice, they were reared for one week in a laboratory environment with a 12 / 12-hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. The human TL1A-His protein expressed in Example 1.1 was used as the immunoantigen, and Balb / c mice were immunized three times by intraperitoneal injection (IP) at a dose of 50 μg / mouse / 0.2 ml, as usual. Immunization was performed on days 0, 14, and 28, and blood was collected on days 21 and 35. Antibody titers in the mouse serum were analyzed by ELISA. After the three immunizations, mice with high antibody titers in their serum were stimulated by intraperitoneal injection (IP) of TL1A-His protein at a dose of 50 μg / mouse / 0.2 ml. Three to four days later, the spleens of the mice were collected and fusion experiments were performed.

[0093] 1.3 Preparation and screening of hybridoma cells Three to four days after final immunization of mice, PEG fusion of mouse spleen cells and mouse myeloma cells SP2 / 0 was performed using standard hybridoma technology. The fused cells were homogeneously suspended in complete medium consisting of 1% penicillin-streptomycin, 20% fetal bovine serum (FBS), and 1*HAT, supplemented with RPMI1640-GLUMAX. The fused cells were then divided into 4*10 4 Individual cells were seeded at 200 μl / well into 60 96-well plates. The supernatant was collected after 7-12 days, and hybridoma wells positive for human TL1A binding activity were screened using ELISA.

[0094] The screening method for human TL1A-binding activity-positive hybridoma wells using ELISA is as follows: TL1A-His was diluted to 1 μg / ml using PBS buffer, added to a plate at 100 μl / well, and incubated overnight at 4°C. The supernatant was discarded the next day, 5% skim milk powder was added, the plate was blocked at 37°C for 1 hour, and the plate was washed three times with PBST. The hybridoma supernatant collected at 100 μl / well was sequentially added to the blocked plate and left at 37°C for 1 hour. The plate was washed three times with PBST, HRP-labeled goat anti-mouse IgG secondary antibody was added, and the plate was left at 37°C for 30 minutes. After washing the plate three times with PBST, any remaining droplets were wiped off as much as possible on absorbent paper, TMB was added at 100 μl / well, and the plate was left at room temperature (20±5°C) in the dark for 5 minutes. The substrate reaction was stopped by adding 2M H2SO4 stop solution at 50 μl / well, and the OD value at 450 nm was read using a microplate reader to analyze the binding ability of the test antibody to the target antigen TL1A. Hybridoma cell lines obtained by screening were grown in serum-containing complete medium, centrifuged, and then replaced with serum-free culture medium, Hybridoma-SFM medium, and the cell density was increased to 1-2 × 10⁶. 6 The cells were cultured at 8% CO2 and 37°C for 1 week. The culture supernatant was collected by centrifugation and purified by Protein G affinity chromatography to obtain anti-human TL1A monoclonal antibody protein. A total of 60 hybridoma cell lines were obtained by screening. All antibodies obtained from the hybridoma cell lines of the present invention can be obtained by genetic engineering techniques.

[0095] 1.4 Binding ability of mouse antibodies to human TL1A-His protein The binding ability of mouse antibodies to human TL1A-His protein was analyzed using enzyme-linked immunosorbent assay (ELISA). The specific method is as follows. TL1A-His protein was diluted to 1 μg / ml using PBS buffer, added to a plate at 100 μl / well, incubated overnight at 4°C, blocked with 5% skim milk powder, and incubated at 37°C for 1 hour. After washing the plate three times with PBST, laboratory-prepared anti-human TL1A mouse antibody was serially diluted 3-fold from 10 μg / ml to an 11-fold gradient using 1% BSA-PBS buffer. 1% BSA-PBS was used as a blank control and added to a plate of TL1A-His pre-coated at 100 μl / well, incubated at 37°C for 1 hour. After washing the plate three times with PBST, HRP-labeled goat anti-mouse IgG secondary antibody was added and left at 37°C for 30 minutes. After washing the plate three times with PBST, any remaining droplets were wiped off as much as possible on absorbent paper, 100 μl / well of TMB was added, and the plate was left at room temperature (20±5°C) in the dark for 5 minutes. The substrate reaction was stopped by adding 2M H2SO4 stop solution at 50 μl / well, and the OD value at 450 nm was read using a microplate reader to analyze the binding ability of the test antibody to the target antigen, human TL1A-His. The obtained data were fitted and analyzed using GraphPad Prism 9 software. The results are shown in Figures 1A, 1B, and 1C. As can be seen from Figures 1A, 1B, and 1C, most mouse antibodies showed good binding activity to the target antigen TL1A-His, and EC 50 These are shown in Tables 1-1 to 1-3. [Table 1-1] [Table 1-2] [Table 1-3]

[0096] 1.5 Measurement of inhibitory activity of mouse antibodies against 293FT-DR3 binding to TL1A protein in cell lines that stably express TL1A protein. In this example, the inhibitory activity of a mouse antibody against the binding of 293FT-DR3 to the human TL1A protein was measured using the fluorescence-activated cell sorting (FACS) method.

[0097] In this experiment, 293FT-DR3 (a modified cell line that highly expresses human DR3, constructed in the laboratory using a lentiviral vector) was used as the target cell. TL1A was bound to the 647 fluorescent tag using Alexa Fluor® 647 NHSEster reagent (thermofisher), 293FT-DR3 cells were counted, and the cells were washed once with 1% BSA-PBS. 2 × 10 5 The cells were plated at 100 μl / well, and the supernatant was removed by centrifugation. The purified mouse antibody was serially diluted 3-fold from 12 μg / ml to 11 gradients using 1% BSA-PBS. Using 1% BSA-PBS as a blank control, 100 μl of each antibody dilution was mixed with TL1A-his-647 fluorescent protein (in-house prepared, final concentration 100 ng / ml, 20 μl / well) and incubated at 37°C for 30 minutes. Then, 100 μl of the mixture was resuspended in wells of 293FT-DR3 cells and incubated at 4°C for 1 hour. The cells were washed twice with 1% BSA-PBS, and the cells were resuspended in 200 μl of PBS. The inhibitory activity of the mouse antibody against the binding of the TL1A-his protein to the cells was measured using a flow cytometer, and the obtained data were fitted and analyzed using GraphPad Prism 9 software. The results are shown in Figures 2A, 2B, and 2C. As a result, most mouse antibodies can inhibit the binding of 293FT cell surface DR3 to TL1A-his protein, and IC 50 This is shown in Tables 2-1 to 2-3. [Table 2-1] [Table 2-2] [Table 2-3]

[0098] Example 2 Humanization of mouse anti-human TL1A monoclonal antibody 2.1 Identification of Variable Region Sequences of Mouse Anti-Human TL1A Monoclonal Antibodies Hybridoma clones 728H4D9 and 311C9E10 were selected as candidate antibodies, and total RNA was extracted from the corresponding hybridoma monoclonal cell lines using Trizol (purchased from Life Technologies). mRNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from Takara), and PCR was performed using combination primers reported in the literature (Antibody Engineering, Volume 1, Edited by Roland Kontermann and Stefan Dubel, the sequences of the combination primers are on page 323). The resulting PCR products were sequenced and analyzed in the Kabat database, confirming that the obtained sequences were the variable region sequences of the mouse antibodies. The heavy chain variable region gene sequence of 728H4D9 is 357 bp in length, encoding 119 amino acid residues. The nucleotide sequence is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:3. The light chain variable region gene sequence is 318 bp in length and codes for 106 amino acid residues. The nucleotide sequence is shown in SEQ ID NO:4, and the amino acid sequence is shown in SEQ ID NO:5. The heavy chain variable region gene sequence of 311C9E10 is 357 bp in length and codes for 119 amino acid residues. The nucleotide sequence is shown in SEQ ID NO:6, and the amino acid sequence is shown in SEQ ID NO:7. The light chain variable region gene sequence is 318 bp in length and codes for 106 amino acid residues. The nucleotide sequence is shown in SEQ ID NO:8, and the amino acid sequence is shown in SEQ ID NO:9.

[0099] SEQ ID NO: 2 GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTAATCCTTACAGCGGTGGTACTAACTACAACCAGAAGTTCAAGGGCAAGGCCACATTAACTGTAGACAAGACATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCGTCCATCTACCAAAGGCACGACGGGATTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO: 3 EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYSGGTNYNQKFKGKATLTVDKTSSTAYMELLSLTSEDSAVYYCASIYQRHDGIAYWGQGTLVTVSA SEQ ID NO: 4 CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAATTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCAACTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAGCCCATACACGTTCGGAGGGGGGACCAAGCTGGAAATCAAA SEQ ID NO: 5 QIVLTQSPAIMSASPGEKVTMTCSASSSVNYMHWYQQKSGTSPKRWIYDTSKLASGVPTRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSSPYTFGGGTKLEIK SEQ ID NO: 6 GAAGTGAAACTTGAGGAGTCTGGAGGAGGCTTAGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGGAATTAGATTGAAATCTAATAATTATACAACACAGTATGCGGAGTCTGTGAAAGGGAGGTTCACCATTTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTATTGTACCCCATTACTCCTCAATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO: 7 EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAGIRLKSNNYTTQYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTPLLLNGMDYWGQGTSVTVSS SEQ ID NO: 8 GAAAATGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAAAGGTCATCATGACCTGTAGTGCCAGCTCAAGTGTAAGCTACATGCACTGGTACCAGCGGAAGTCAAACACCTCCCCAAACTCTGGATTTATGACACATCCAATCTG GCTTCTGGAGTCCCAGGTCGCTTCAGTGGCACTGGGTCTGGAAACTCTTACTCTCTCACGATCAGCAGCATGGAGGCTGAAGATGTTGCCACTTATTACTGTTTTCAGGAGAGTGGGTACCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA SEQ ID NO: 9 ENVLTQSPAIMSASPGEKVIMTCSASSSVSYMHWYQRKSNTSPKLWIYDTSNLASGVPGRFSGTGSGNSYSLTISSMEAEDVATYYCFQESGYPFTFGSGTKLEIK

[0100] 2.2 Humanization of mouse anti-human TL1A monoclonal antibody The amino acid sequences of the 728H4D9 heavy and light chain variable regions were analyzed, and three antigen complementarity-determining regions (CDRs) and four framework regions (FRs) were determined according to Kabat rules. Of these, the amino acid sequences of the heavy chain complementarity-determining regions were HCDR1:GYTMN (SEQ ID NO:10), HCDR2:LINPYSGGTNYNQKFKG (SEQ ID NO:11), and HCDR3:IYQRHDGIAY (SEQ ID NO:12), while the amino acid sequences of the light chain complementarity-determining regions were LCDR1:SASSSVNYMH (SEQ ID NO:13), LCDR2:DTSKLAS (SEQ ID NO:14), and LCDR3:QQWSSSPYT (SEQ ID NO:15).

[0101] By comparing homology with human IgG germline sequences (Germlines) using NCBI IgBlast, IGHV1-2*02 was selected as the heavy chain CDR transplantation template and IGKV6-21*01 as the light chain CDR transplantation template. The CDR region of the 728H4D9 antibody was transplanted into the selected humanized template, replacing the CDR region of the humanized template. The heavy chain variable region was recombined with the human IgG1 constant region (amino acid sequence shown in SEQ ID NO:33), and the light chain variable region was recombined with the human kappa chain constant region (amino acid sequence shown in SEQ ID NO:34). Furthermore, based on the three-dimensional structure of the antibody, reverse mutations were performed on embedding residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of the antibody's VL and VH regions, yielding multiple humanized antibodies. Affinity screening was used to determine the amino acid sequences of the heavy chain variable region (SEQ ID NO: 16) and light chain variable region (SEQ ID NO: 17) of humanized antibody 72851. The nucleotide sequences are shown in SEQ ID NO: 35 and SEQ ID NO: 36, respectively.

[0102] SEQ ID NO: 16 QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQNLEWIGLINPYSGGTNYNQKFKGRATLTVDTSISTAYMELSRLRSDDTAVYYCASIYQRHDGIAYWGQGTLVTVSS SEQ ID NO: 17 EIVLTQSPDFQSVTPKEKVTITCSASSSVNYMHWYQQKPDQSPKLLIKDTSKLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQWSSSPYTFGGGTKVEIK SEQ ID NO: 33 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 34 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 35 CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCTGGCGCTAGCGTGAAGGTGAGCTGCAAGGCTAGCGGCTACAGCTTCACCGGCTACACCATGAACTGGGTGAGACAAGCCCCTGGACAGAACCTGGAGTGGATCGGCCTGATCAACCCTTACAGCGGCGGAACCAACTACAATCAGAAGTTCAAGGGCAGAGCCACACTGACCGTGGACACAAGCATCAGCACCGCCTACATGGAGCTGAGCAGACTGAGAAGCGACGACACCGCCGTGTATTATTGCGCCTCCATCTATCAGAGACACGACGGCATCGCCTACTGGGGCCAAGGCACCCTGGTGACCGTGAGCAGC SEQ ID NO: 36 GAGATCGTGCTGACACAGAGCCCTGACTTTCAGAGCGTGACCCCTAAGGAGAAGGTGACCATTACCTGCAGCGCTAGCAGCAGCGTGAACTACATGCACTGGTATCAGCAGAAGCCTGATCAGAGCCCTAAGCTGCTGATCAAGGACACAAGCAAGCTG GCTAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAGCCTGGAGGCCGAGGACGCCGCCACCTACTACTGTCAGCAGTGGAGCAGCAGCCCTTACACCTTCGGCGGAGGCACCAAGGTGGAGATCAAA

[0103] The amino acid sequences of the heavy and light chain variable regions of 311C9E10 were analyzed, and three antigen complementarity-determining regions (CDRs) and four framework regions (FRs) were determined according to Kabat rules. Of these, the amino acid sequences of the heavy chain complementarity-determining regions were HCDR1:NYWMN (SEQ IDNO:18), HCDR2:GIRLKSNNYTTQYAESVKG (SEQ IDNO:19), and HCDR3:LLLNGMDY (SEQ IDNO:20), and the amino acid sequences of the light chain complementarity-determining regions were LCDR1:SASSSVSYMH (SEQ IDNO:21), LCDR2:DTSNLAS (SEQ IDNO:22), and LCDR3:FQESGYPFT (SEQ IDNO:23).

[0104] By comparing homology with human IgG germline sequences (Germlines) using NCBI IgBlast, IGHV1-2*01 was selected as the heavy chain CDR transplantation template and IGKV6-21*01 as the light chain CDR transplantation template. The CDR region of the 311C9E10 antibody was transplanted into the selected humanized template, replacing the CDR region of the humanized template. The heavy chain variable region was recombined with the human IgG1 constant region (amino acid sequence shown in SEQ ID NO:33), and the light chain variable region was recombined with the human kappa chain constant region (amino acid sequence shown in SEQ ID NO:34). Furthermore, based on the three-dimensional structure of the antibody, reverse mutations were performed on embedding residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of the antibody's VL and VH regions, yielding multiple humanized antibodies. Affinity screening was used to determine the heavy chain variable region sequence (SEQ ID NO:24) and light chain variable region sequence (SEQ ID NO:25) of humanized antibody 31122. The nucleotide sequences are shown in SEQ ID NO:37 and SEQ ID NO:38, respectively.

[0105] SEQ ID NO: 24 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGGIRLKSNNYTTQYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTPLLLNGMDYWGQGTTVTVSS SEQ ID NO: 25 EIVLTQSPDFQSVTPKEKVTITCSASSSVSYMHWYQQKPDQSPKLWIYDTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCFQESGYPFTFGQGTKLEIK SEQ ID NO: 37 GAGGTGCAGCTGGTCGAGTCCGGAGGCGGACTGGTCCAACCTGGCGGAAGCCTGAAGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCAACTACTGGATGAACTGGGTGAGACAAGCTAGCGGCAAGGGCCTGGAGTGGGTGGGCGGCATCAGACTGAAGAGCAACAACTACACCACACAGTACGCCGAGAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAAGACCGAGGACACCGCCGTGTACTACTGCACACCTCTGCTCCTGAACGGCATGGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC SEQ ID NO: 38 GAGATCGTGCTGACACAGAGCCCTGACTTTCAGAGCGTGACCCCTAAGGAGAAGGTGACCATCACCTGCAGCGCTAGCAGCAGCGTGAGCTACATGCACTGGTATCAGCAGAAGCCTGATCAGAGCCCTAAGCTGTGGATCTACGACACAAGCAACCTGGCTAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAGCCTGGAGGCCGAGGACGCCGCCACCTACTACTGCTTCCAAGAGAGCGGCTACCCTTTCACCTTCGGCCAAGGCACCAAGCTGGAGATCAAA

[0106] Example 3 Measurement of the affinity dissociation constant KD of the humanized antibody The kinetic parameters for the binding and dissociation of candidate humanized antibodies 72851 and 31122, and the positive control antibody PRA023, with the antigen TL1A-his were measured using a capture method with a Biacore 8K intermolecular interaction analyzer. Antibodies were diluted to 2 μg / ml using HBS-EP+, pH 7.4 buffer and captured with a Protein A tip. The antigen was diluted with HBS-EP+, pH 7.4 buffer, bound to the antibody at a maximum concentration of 50 nM in a 6-step concentration gradient, and dissociated in HBS-EP+, pH 7.4 buffer. The experimental results are shown in Table 3, showing that the affinity of humanized antibodies 72851 and 31122 for TLIA-his is superior to that of the positive antibody PRA023. [Table 3] Note: KD represents the affinity constant, ka represents the association rate constant, and kd represents the dissociation rate constant.

[0107] Example 4: Optimization of the physicochemical properties of humanized antibody 31122 Due to the high glycosylation modification and charge heterogeneity of humanized antibody 31122, a mutant library (without altering the light chain or heavy chain constant region) was constructed by introducing single-site saturation mutations and multiple stacking dominant mutations into potential glycosylation and deamination sites within the CDR sequence of the heavy chain variable region. Candidate antibodies with superior affinity and charge heterogeneity compared to the parent antibody 31122 were screened using ELISA affinity analysis and capillary isoelectric focusing electrophoresis.

[0108] The heavy chain variable region (CDR) of each modified candidate antibody is shown in Table 4. [Table 4]

[0109] The amino acid sequences of the heavy chain variable region of each modified candidate antibody are shown in SEQ ID NO:26-31, and the nucleotide sequences are shown in SEQ ID NO:43-48, respectively. 311V25-HC (SEQ IDNO: 26) EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGGIRLKSNNYATQYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTPLLLNGMDYWGQGTTVTVSS 311V31-HC (SEQ ID NO: 27) EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGGIRLKSNNYKTQYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTPLLLNGMDYWGQGTTVTVSS 311V34-HC (SEQ ID NO: 28) EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGGIRLKSNNYRTQYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTPLLLNGMDYWGQGTTVTVSS 311V25-7-HC (SEQ ID NO: 29) EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGGIRLKSNNYATQYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTPLLLSGMDYWGQGTTVTVSS 311V31-7-HC (SEQ ID NO: 30) EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGGIRLKSNNYKTQYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTPLLLSGMDYWGQGTTVTVSS 311V34-7-HC (SEQ ID NO: 31) EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGGIRLKSNNYRTQYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTPLLLSGMDYWGQGTTVTVSS SEQ ID NO: 43 GAGGTGCAGCTGGTCGAGTCCGGAGGCGGACTGGTCCAACCTGGCGGAAGCCTGAAGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCAACTACTGGATGAACTGGGTGAGACAAGCTAGCGGCAAGGGCCTGGAGTGGGTGGGCGGCATCAGACTGAAGAGCAACAACTACGCCACACAGTACGCCGAGAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAAGACCGAGGACACCGCCGTGTACTACTGCACACCTCTGCTCCTGAACGGCATGGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC SEQ ID NO: 44 GAGGTGCAGCTGGTCGAGTCCGGAGGCGGACTGGTCCAACCTGGCGGAAGCCTGAAGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCAACTACTGGATGAACTGGGTGAGACAAGCTAGCGGCAAGGGCCTGGAGTGGGTGGGCGGCATCAGACTGAAGAGCAACAACTACAAGACACAGTACGCCGAGAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAAGACCGAGGACACCGCCGTGTACTACTGCACACCTCTGCTCCTGAACGGCATGGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC SEQ ID NO: 45 GAGGTGCAGCTGGTCGAGTCCGGAGGCGGACTGGTCCAACCTGGCGGAAGCCTGAAGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCAACTACTGGATGAACTGGGTGAGACAAGCTAGCGGCAAGGGCCTGGAGTGGGTGGGCGGCATCAGACTGAAGAGCAACAACTACAGAACACAGTACGCCGAGAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAAGACCGAGGACACCGCCGTGTACTACTGCACACCTCTGCTCCTGAACGGCATGGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC SEQ ID NO: 46 GAGGTGCAGCTGGTCGAGTCCGGAGGCGGACTGGTCCAACCTGGCGGAAGCCTGAAGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCAACTACTGGATGAACTGGGTGAGACAAGCTAGCGGCAAGGGCCTGGAGTGGGTGGGCGGCATCAGACTGAAGAGCAACAACTACGCCACACAGTACGCCGAGAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAAGACCGAGGACACCGCCGTGTACTACTGCACACCTCTGCTCCTGAGCGGCATGGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC SEQ ID NO: 47 GAGGTGCAGCTGGTCGAGTCCGGAGGCGGACTGGTCCAACCTGGCGGAAGCCTGAAGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCAACTACTGGATGAACTGGGTGAGACAAGCTAGCGGCAAGGGCCTGGAGTGGGTGGGCGGCATCAGACTGAAGAGCAACAACTACAAGA CACAGTACGCCGAGAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAAGACCGAGGACACCGCCGTGTACTACTGCACACCTCTGCTCCTGAGCGGCATGGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC SEQ ID NO: 48 GAGGTGCAGCTGGTCGAGTCCGGAGGCGGACTGGTCCAACCTGGCGGAAGCCTGAAGCTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCAACTACTGGATGAACTGGGTGAGACAAGCTAGCGGCAAGGGCCTGGAGTGGGTGGGCGGCATCAGACTGAAGAGCAACAACTACAGAA CACAGTACGCCGAGAGCGTGAAGGGCAGATTCACCATCAGCAGAGACGACAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGAAGACCGAGGACACCGCCGTGTACTACTGCACACCTCTGCTCCTGAGCGGCATGGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC

[0110] The ELISA experimental results are shown in Figure 3A. These results indicate that the binding affinity of candidate antibodies 72851, 31122, 311V25, 311V31, 311V34, 311V25-7, 311V31-7, and 311V34-7 to the antigen TL1A-his was superior to that of the positive control PRA023.

[0111] The results of capillary isoelectric focusing analysis of the anti-TL1A candidate antibody are shown in Figure 3B. The experimental results showed that the charge heterogeneity of the modified 311V25-7 and 311V31-7 antibodies was superior to that of the parent antibody 31122 and equivalent to that of candidate antibody 72851.

[0112] Example 5: Experiment on inhibition of TL1A and DR3 binding by anti-TL1A antibody This example measured the inhibitory effect of an anti-TL1A antibody on the binding of TL1A to its cell surface receptor using the fluorescence-activated cell sorting (FACS) method. The target cells used for the experiment were a laboratory-constructed 293FT modified cell line (293FT-DR3 cell line) that highly expresses human DR3. The sequence of DR3 was obtained from the UniProt database (Entry: Q93038), and the amino acid sequence is shown in SEQ ID NO: 32.

[0113] SEQ ID NO: 32 MEQRPRGCAAVAAALLLVLLGARAQGGTRSPRCDCAGDFHKKIGLFCCRGCPAGHYLKAPCTEPCGNSTCLVCPQDTFLAWENHHNSECARCQACDEQASQVALENCSAVADTRCGCKPGWFVECQVSQCVSSSPFYCQPCLDCGALHRHTRLLCSRRDTDCGTCLPGFYEHGDGCVSCPTSTLGSCPERCAAVCGWRQMFWVQVLLA GLVVPLLLGATLTYTYRHCWPHKPLVTADEAGMEALTPPPATHLSPLDSAHTLLAPPDSSEKICTVQLVGNSWTPGYPETQEALCPQVTWSWDQLPSRALGPAAAPTLSPESPAGSPAMMLQPGPQLYDVMDAVPARRWKEFVRTLGLREAEIEAVEVEIGRFRDQQYEMLKRWRQQQPAGLGAVYAALERMGLDGCVEDLRSRLQRGP

[0114] TL1A-his antigen was labeled with Alexa Fluor® 647 fluorescent dye (purchased from invitrogen, catalog number A20006), and the fluorescently labeled antigen TL1A-his-647 was diluted to 200 ng / ml using 1% BSA-PBS. Anti-TL1A antibody was diluted to 200 nM using 1% BSA-PBS and then diluted fourfold until eight concentration gradients were obtained. 200 ng / ml of TL1A-his-647 was added to the antibodies of the different concentration gradients and incubated at 4°C for 1 hour. Cell supernatant was removed by centrifugation and the cells were placed on a 293FT-DR3 cell plate (1 × 10⁶). 5 In addition to the solution ( / well), the cells were incubated at 4°C for 1 hour. To remove unbound antigen, the cells were washed twice with PBS, and finally resuspended in 200 μL of PBS. The mean fluorescence intensity was measured by flow cytometry. The obtained data were fitted and analyzed using GraphPad Prism 9 software. The results are shown in Figure 4. According to the experimental results, candidate antibodies 72851, 31122, 311V25, 311V31, 311V34, 311V25-7, 311V31-7, 311V34-7, and the positive control antibody PRA023 were all able to inhibit the binding of TL1A to DR3, and the inhibitory ability of the candidate antibodies was superior to that of PRA023.

[0115] Example 6: Inhibition experiment of NF-κB activation by TL1A using an anti-TL1A antibody. When TL1A binds to its receptor DR3, it activates the intracellular NF-κB signaling pathway and induces apoptosis. In this study, a TF-1 cell line (TF-1-NF-κB-Luc cells) that stably expresses NF-κB-regulated luciferase was constructed, and the ability of an anti-TL1A antibody to inhibit the activation of the TL1A-induced NF-κB pathway was measured.

[0116] The TL1A-his protein was diluted to 800 ng / mL (final concentration: 200 ng / mL) using RPMI 1640 + 10% FBS medium and added to a flat-bottom 96-well plate at 25 μL / well. The anti-TL1A antibody was diluted to 200 nM using RPMI 1640 + 10% FBS medium and serially diluted 2-fold until an 8-point concentration gradient was obtained. Then 25 μL of it was added to the TL1A-his wells, with the starting and final concentrations of the anti-TL1A antibody being 50 nM. TF-1-NF-κB-Luc cells were centrifuged and counted, and the cells with adjusted density were added to the 96-well plate at 1×10 5 / well and 50 μL / well, and incubated in an incubator at 37 °C for 24 hours. The next day, the cell plate was taken out and equilibrated at room temperature for about 15 minutes. 100 μL of Bio-Lite Luciferase (purchased from Nanjing Novoprotein Scientific Co., Ltd., product number: DD1201-02) was added to each well, incubated at room temperature for 10 minutes, and then the Luminescence (RLU) value was read using a microplate reader. Data analysis and plotting were performed using GraphPad Prism9 to calculate the IC 50 . The results are shown in Figure 5. According to the experimental results, candidate antibodies 72851, 311V25-7, 311V31-7, and the positive control antibody PRA023 can all effectively inhibit NF-κB activation by TL1A, and the inhibitory ability of the candidate antibodies is superior to that of the positive control antibody PRA023.

[0117] Example 7 Experiment on inhibition of TL1A-induced TF-1 apoptosis by anti-TL1A antibody TL1A is an effective and specific inhibitor of endothelial cell proliferation and induces apoptosis through binding to DR3. In this example, the method of Fluorescence activated Cell Sorting (FACS) was used to measure TL1A-induced TF-1 apoptosis by anti-TL1A antibody. TF-1 cells were centrifuged and counted, and the cell density was adjusted to 1×10 5Cells were plated into 3599 cell plates at 100 μL / well. TL1A-his was diluted to 800 ng / mL using 1640 medium (containing 10% FBS and 1% PS), cycloheximide (CHX, purchased from CST, catalog number: 2112) was added to a centrifuge tube to a final concentration of 40 μg / mL, and after homogeneous mixing, 50 μL / well was added to the 3599 cell plates. Positive control antibody PRA023 and other humanized TL1A antibodies were diluted to 800 nM using the medium, 3-fold dilutions were made until nine concentration gradients were achieved, and 50 μL / well was added to the 3599 cell plates. That is, the final concentration of TL1A-his was 200 ng / mL, the final concentration of CHX was 10 μg / mL, and the initial final concentration of TL1A antibody was 200 nM. The cell plates were gently tapped to homogeneous mix and left in an incubator at 37°C. After 6 hours, the cell plates were removed, centrifuged at 500g for 5 minutes, the supernatant was discarded, and the cells were washed twice with 1% BSA-PBS. They were then stained with the Annexin V-FITC / PI Apoptosis Kit (purchased from Annexin Biotechnology (Shanghai) Co., Ltd., catalog number: 40302ES60). The cells were resuspended in 1x Binding buffer, 5 μL of Annexin V-FITC and 10 μL of PI Staining Solution were added to each well at 100 μL / well, and allowed to stand at room temperature in the dark for 10-15 minutes. After mixing uniformly, the cells were measured using a flow cytometer. Data analysis and plotting were performed using GraphPad Prism9. 50 The following was calculated. The results are shown in Figure 6. According to the experimental results, candidate antibodies 311V25-7, 311V31-7, 311V34-7, and the positive control antibody PRA023 were all able to inhibit TL1A-induced TF-1 apoptosis, and the inhibitory ability of the candidate antibodies was superior to that of the positive control antibody PRA023.

[0118] Example 8: Inhibition of TL1A-induced IFNγ secretion by cells by anti-TL1A antibody TL1A stimulates IFN-γ secretion by binding to DR3. In this example, we measured that the anti-TL1A antibody inhibits the IFN-γ secretion-inducing activity of PBMCs induced by TL1A.

[0119] Dilute PBMCs with RPMI 1640 + 10% FBS medium and set the cell density to 3 × 10⁻⁶ 6 The medium was adjusted to 100 μl / well to prepare a cell suspension. Using the culture medium, TL1A-his was diluted to 400 ng / ml and the antibody to 200 nM using a 3-fold gradient. The TL1A-his and antibody were mixed in a 1:1 volume ratio and incubated at room temperature for 30 minutes. The mixture was then added to the PBMC cell plate at a rate of 50 μl / well. Recombinant IL12 protein was diluted to 4 ng / ml and recombinant IL18 protein to 40 ng / ml using the culture medium. 25 μl / well of each was added to the cell plate, and the mixture was incubated at 37°C in a 5% CO2 incubator for 48 hours. The cell supernatant was then collected, and the human IFNγ expression level in the supernatant was measured. The results are shown in Figure 7. Experimental results show that candidate antibodies 72851, 311V25-7, 311V34-7, and the positive control antibody PRA023 can all inhibit IFN-γ secretion from PBMCs, and the inhibitory activity of the candidate antibodies is superior to that of the positive control PRA023.

[0120] Example 9: Inhibition of TL1A-induced TNFα secretion by cells by anti-TL1A antibody TL1A, a Th1 polarization factor, stimulates Th1 cells to activate the secretion of IFN-γ and TNF-α cytokines, and therefore may play a crucial role in the onset and progression of inflammatory bowel disease (IBD). This study measured the inhibition of TL1A-induced TNF-α secretion activity in PBMCs by an anti-TL1A antibody.

[0121] Dilute PBMCs with RPMI 1640 + 10% FBS medium and set the cell density to 3 × 10⁻⁶ 6The medium was adjusted to 100 μl / well to prepare a cell suspension. Using the culture medium, TL1A-his was diluted to 400 ng / ml and the antibodies to 200 nM and 80 nM using a 3-fold gradient. The TL1A-his and antibodies were mixed in a 1:1 volume ratio and incubated at room temperature for 30 minutes. The mixture was then added to the PBMC cell plate at 50 μl / well. Recombinant IL23 protein was diluted to 400 ng / ml and recombinant IL2 protein to 80 U / ml using the culture medium. 25 μl / well of each was added to the cell plate, and the mixture was incubated at 37°C in a 5% CO2 incubator for 72 hours. The cell supernatant was then collected, and the human TNF-α expression level in the supernatant was measured. The results are shown in Figure 8. Experimental results show that candidate antibodies 72851, 311V25-7, 311V34-7, and the positive control antibody PRA023 can all inhibit TNF-α secretion from PBMCs, and the inhibitory activity of the candidate antibodies is superior to that of the positive control PRA023.

[0122] Example 10: Efficacy study of anti-TL1A antibody in a DSS-induced rat model of chronic inflammatory bowel disease. When DSS is dissolved in rat drinking water, its highly negatively charged sulfate groups disrupt the integrity of the intestinal mucosal barrier, triggering an inflammatory response. The animals exhibit marked weight loss, loose stools, bloody stools, and granulocyte infiltration, with clinical symptoms and pathological features very similar to those of human ulcerative colitis. The candidate anti-TL1A antibody molecule 72851 shows equivalent binding activity to human (human-TL1A), cynomolgus monkey (which has the same sequence as rhesus monkeys; Cynomolgus(Rhesus)-TL1A), and rat (Rat-TL1A) TL1A homologous proteins (ELISA experimental results are shown in Figure 9A). Therefore, an in vivo efficacy study was conducted using a DSS-induced rat model of chronic inflammatory bowel disease.

[0123] In the experiment, the animals were divided into three groups: a blank control group, a model group, and a 72851 treatment group. The blank control group received regular drinking water, while the model group and the 72851 treatment group received drinking water containing 5% DSS for 5 days and purified water for 2 days, with each cycle lasting 7 days, for a total of 4 cycles. The dose of 7285 was 5 mg / kg, administered every two days. At the end of the experiment, blood was collected from the anesthetized animals and serum was obtained. After euthanasia, the entire intestinal tract from the cecum to the distal rectum was dissected and its length was measured. The spleen was removed and its weight was measured. Serum IL-6 and IL-1β levels were measured using the ELISA method.

[0124] Experimental results showed that, compared to the model group (5% DSS), the candidate antibody 72851 effectively suppressed weight loss (shown in Figure 9B) and colon shortening (shown in Figure 9C) in rats, and also effectively suppressed increases in inflammatory cytokines IL-6 and IL-1β in rats (shown in Figures 9D and 9E).

[0125] Example 11: Efficacy of anti-TL1A antibody in a TNBS-induced rat acute inflammatory bowel disease model. TNBS is a low-molecular-weight hapten that, when administered rectally, binds to host proteins, inducing an immune response that leads to whole-wall necrosis and widespread inflammatory cell infiltration in the colon of animals, accompanied by weight loss and intestinal obstruction. It is therefore a commonly used drug to simulate a clinical model of acute enteritis in animals with Crohn's disease. Using this model, the efficacy of the anti-TL1A antibody 72851 against acute enteritis in rats was validated and compared with Roche's RVT-3101.

[0126] In the experiment, animals were divided into a blank control group, a model group, an RVT-3101-treated group, and a 72851-treated group. Before the start of the experiment, the animals were fasted for 18 hours. RVT-3101 and 72851 were administered at a dose of 5 mg / kg before the model was constructed. Next, under anesthesia, 400 μl of 1.5% TNBS ethanol solution (50% ethanol) was perfused into the colon of the model group, the RVT-3101-treated group, and the 72851-treated group via a plastic catheter. To prevent leakage of the perfusion fluid, the rats were held inverted for 3 minutes. The animals were observed during anesthesia. After awakening, they were returned to their cages and given free access to food and water. RVT-3101 and 72851 were administered intraperitoneally every two days. The model group was injected with PBS at a dose volume of 5 ml / kg every two days. On the seventh day of the experiment, blood was drawn from the anesthetized animals, and after euthanasia, the entire intestinal tract from the cecum to the distal rectum was dissected and its length was measured. The spleen was removed and its weight was measured.

[0127] Experimental results showed that, compared to the model group (1.5% TNBS), candidate antibody 72851 effectively suppressed colon shortening (shown in Figure 10A) and spleen weight increase (shown in Figure 10B) in rats treated with 72851. Its efficacy was superior to that of the positive control group treated with RVT-3101 (1.5% TNBS + RVT3101).

[0128] The above embodiments are intended to illustrate embodiments of the present invention and should not be construed as limiting the invention. Furthermore, various modifications and changes in the methods described herein will be obvious to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in detail with reference to various preferred embodiments, it should be understood that the present invention is not limited to these specific embodiments. In fact, various modifications of the present invention that are obvious to those skilled in the art are considered to be within the scope of the invention.

Claims

1. The anti-TL1A antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the anti-TL1A antibody or its antigen-binding fragment is <1> Heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 10 or 18, <2> Heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, 19, 39, 40 or 41, <3> Heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 12, 20, or 42, <4> Light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 13 or 21, <5> Light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 14 or 22, <6> Light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO: 15 or 23, An anti-TL1A antibody or its antigen-binding fragment, characterized by having one or more of the following technical features.

2. The anti-TL1A antibody or its antigen-binding fragment is <1> Heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 10, <2> Heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, <3> Heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 12, <4> Light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, <5> Light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, <6> Light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO: 15, and / or the anti-TL1A antibody is <1> Heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 18, <2> Heavy chain variable region containing HCDR2 having an amino acid sequence shown in any one of SEQ ID NO: 19, 39, 40, or 41, <3> Heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 20 or 42, <4> Light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, <5> Light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, <6> Light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO: 23, The anti-TL1A antibody or antigen-binding fragment according to claim 1, characterized by containing the above.

3. The anti-TL1A antibody or its antigen-binding fragment also, <1> Heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 18, <2> Heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO: 19, <3> Heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 20, <4> Light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, <5> Light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, <6> Light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO: 23, and / or the anti-TL1A antibody or its antigen-binding fragment, <1> Heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 18, <2> Heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO: 39, <3> Heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 20, <4> Light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, <5> Light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, <6> Light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO: 23, and / or the anti-TL1A antibody or its antigen-binding fragment, <1> Heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 18, <2> Heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO: 40, <3> Heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 20, <4> Light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, <5> Light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, <6> Light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO: 23, and / or the anti-TL1A antibody or its antigen-binding fragment, <1> Heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 18, <2> Heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO: 41, <3> Heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 20, <4> Light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, <5> Light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, <6> Light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO: 23, and / or the anti-TL1A antibody or its antigen-binding fragment, <1> Heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 18, <2> Heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO: 39, <3> Heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 42, <4> Light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, <5> Light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, <6> Light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO: 23, and / or the anti-TL1A antibody or its antigen-binding fragment, <1> Heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 18, <2> Heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO: 40, <3> Heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 42, <4> Light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, <5> Light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, <6> Light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO: 23, and / or the anti-TL1A antibody or its antigen-binding fragment, <1> Heavy chain variable region containing HCDR1 having the amino acid sequence shown in SEQ ID NO: 18, <2> Heavy chain variable region containing HCDR2 having the amino acid sequence shown in SEQ ID NO: 41, <3> Heavy chain variable region containing HCDR3 having the amino acid sequence shown in SEQ ID NO: 42, <4> Light chain variable region containing LCDR1 having the amino acid sequence shown in SEQ ID NO: 21, <5> Light chain variable region containing LCDR2 having the amino acid sequence shown in SEQ ID NO: 22, <6> Light chain variable region containing LCDR3 having the amino acid sequence shown in SEQ ID NO: 23, The anti-TL1A antibody or antigen-binding fragment according to claim 1 or 2, characterized by containing the above.

4. The anti-TL1A antibody or antigen-binding fragment according to claim 1, characterized in that the amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or antigen-binding fragment is shown in any one of SEQ ID NO: 3, SEQ ID NO: 16, SEQ ID NO: 7, SEQ ID NO: 24, or SEQ ID NO: 26-31, and / or the amino acid sequence of the light chain variable region of the anti-TL1A antibody or antigen-binding fragment is shown in SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 9, or SEQ ID NO:

25.

5. 1) The amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

5. 2) The amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

17. 3) The amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

9. 4) The amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO: 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

25. 5) The amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

25. 6) The amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

25. 7) The amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

25. 8) The amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

25. 9) The amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

25. 10) The amino acid sequence of the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

25. The anti-TL1A antibody or antigen-binding fragment according to claim 1, characterized by comprising any of the following features.

6. The anti-TL1A antibody or its antigen-binding fragment according to claim 1, wherein the anti-TL1A antibody or its antigen-binding fragment is selected from a complete antibody, a single-chain antibody, or an antibody fragment, and preferably the heavy chain constant region of the complete antibody is an IgG1 constant region, and / or the light chain constant region is a kappa chain constant region.

7. The anti-TL1A antibody or antigen-binding fragment according to claim 1, characterized in that the amino acid sequence of the heavy chain constant region of the anti-TL1A antibody or antigen-binding fragment is shown in SEQ ID NO: 33, and / or the amino acid sequence of the light chain constant region of the anti-TL1A antibody or antigen-binding fragment is shown in SEQ ID NO:

34.

8. An isolated polynucleotide characterized by encoding a heavy chain variable region and / or a light chain variable region or a full-length amino acid of an anti-TL1A antibody or its antigen-binding fragment according to any one of claims 1 to 7.

9. The polynucleotide according to claim 8, characterized in that the polynucleotide sequences encoding the heavy chain variable region of the anti-TL1A antibody or its antigen-binding fragment are shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 43-48, and / or the polynucleotide sequences encoding the light chain variable region of the anti-TL1A antibody or its antigen-binding fragment are shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 36, and SEQ ID NO:

38.

10. A nucleic acid construct comprising the isolated polynucleotide described in claim 8 or 9.

11. A cell comprising the construct described in claim 10, or characterized in that the genome incorporates the exogenous polynucleotide described in claim 8 or 9.

12. A method for preparing an anti-TL1A antibody or its antigen-binding fragment according to any one of claims 1 to 7, characterized by comprising the step of expressing the anti-TL1A antibody or its antigen-binding fragment by culturing the cells according to claim 11 under conditions suitable for the expression of the TL1A antibody or its antigen-binding fragment.

13. The use of an anti-TL1A antibody or its antigen-binding fragment according to any one of claims 1 to 7 in the preparation of a drug for treating a disease or a drug for diagnosing a disease, wherein the disease is selected from one or more of inflammatory bowel disease, gastrointestinal diseases associated with cystic fibrosis, colitis, irritable bowel syndrome, eosinophilic esophagitis, atopic dermatitis, eczema, scleroderma, arthritis, or rheumatoid arthritis.

14. The use according to claim 13, characterized in that the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

15. A pharmaceutical composition characterized by comprising an anti-TL1A antibody or its antigen-binding fragment according to any one of claims 1 to 7.

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