Antibodies against tumor necrosis factor-like ligand 1a and uses thereof

Abstract

The present application discloses antibodies that bind to tumor necrosis factor-like ligand 1A (TL1A), nucleic acid molecules encoding the antibodies, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, methods of making and purifying the antibodies, and uses of the antibodies.

Description

Technical Field

[0001] This application generally relates to the fields of genetic engineering and antibody drugs; more specifically, it relates to antibodies that bind to tumor necrosis factor-like ligand 1A and their uses. Background Technology

[0002] Tumor necrosis factor-like ligand 1A (TL1A / VEGI-251 / TNFSF15) is a member of the tumor necrosis factor family, consisting of 251 amino acids, and is a type II transmembrane glycoprotein. As a type II transmembrane protein, TL1A, similar to other members of the TNF family, forms a stable non-covalent homotrimeric structure with a β-sandwich. TL1A is expressed in immune cells (such as monocytes, macrophages, dendritic cells, T lymphocytes, and plasma cells) and non-immune cells (such as endothelial cells and synovial fibroblasts). TL1A initially exists in a membrane-bound form and can be released as a soluble protein through alternative cleavage or tumor necrosis factor α-convertase at amino acid position 72 to perform its function. 1 The functional receptor for TL1A is death receptor 3 (DR3), and the non-functional receptor is decoy receptor 3 (DcR3). 2 TL1A induces DR3 receptor oligomerization by binding to DR3, and then recruits the adaptor protein TNFR-associated death domain protein (TRADD) through the intracellular death domain to regulate downstream pathways (such as TRAF2, RIP1, PI3K, MAPK, and NF-κB) to exert pro-inflammatory effects; or it promotes apoptosis through the FADD, RIP3, and Caspase-8 / -3 / -7 pathways; simultaneously, the NF-κB pathway can also activate c-IAP protein, thereby negatively regulating apoptosis. 1,3 .

[0003] Aberrant expression of TL1A is significantly associated with autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, and ankylosing spondylitis. 1 The key roles of the TL1A and DR3 signaling pathways in autoimmune and inflammatory diseases suggest that inhibiting the TL1A-DR3 interaction may be an effective therapeutic strategy for improving autoimmune diseases and local inflammation in target organs. Currently developed monoclonal antibodies targeting TL1A have clinical indications including inflammatory bowel disease and systemic sclerosis-related interstitial lung disease. Among them, RVT3101 and PRA023 have both shown significant clinical remission rates and endoscopic improvement rates in clinical trials for the treatment of inflammatory bowel disease.

[0004] Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a relapsing, inflammatory disease affecting the digestive tract, characterized by intestinal inflammation and epithelial damage. The etiology of IBD is not fully understood, but it may be related to a variety of factors, including genetics, environment, gut microbiota, and intestinal immunity. 4 Current IBD treatments include aminosalicylic acid derivatives, oral corticosteroids, oral small molecule Janus kinase inhibitors, tumor necrosis factor inhibitors, integrin receptor antagonists, and interleukin (IL) 12 / 23 antagonists. 5 However, the above treatment regimens still fail to elicit a sufficient response in IBD patients, indicating an unmet clinical need for IBD treatment.

[0005] Therefore, the development and application of new anti-human TL1A antibodies is needed in this field and has important biological and medical significance. Invention Overview

[0006] In a first aspect, this application provides an antibody that binds to tumor necrosis factor-like ligand 1A (TL1A), comprising a heavy chain variable region containing the amino acid sequences HCDR1, HCDR2, and HCDR3, and a light chain variable region containing the amino acid sequences LCDR1, LCDR2, and LCDR3, wherein...

[0007] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6; or

[0008] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 7, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 8, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 9, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 10, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 12; or

[0009] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 7, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 13, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 9, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 14, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 15, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; or

[0010] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 17, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 18, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 20, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 21, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 22.

[0011] The amino acid sequences of HCDR and LCDR are defined according to Kabat.

[0012] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 23, 24, 25 or 26.

[0013] In some embodiments of the first aspect, the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 27, 28, 29 or 30.

[0014] In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is as shown in SEQ ID NO: 23, and the amino acid sequence of the antibody light chain variable region is as shown in SEQ ID NO: 27; or

[0015] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 28; or

[0016] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 25, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 29; or

[0017] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 26, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 30.

[0018] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO: 23, 24, 25 or 26, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity with SEQ ID NO: 27, 28, 29 or 30.

[0019] In some embodiments of the first aspect, the antibody is a whole antibody, a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment (scFv).

[0020] In some embodiments of the first aspect, the antibody is a monoclonal antibody.

[0021] In some embodiments of the first aspect, the antibody further comprises a heavy chain constant region selected from IgG1, IgG2 or IgG4 subtypes.

[0022] In some embodiments of the first aspect, the antibody further comprises a light chain constant region selected from the κ or λ subtype.

[0023] In some embodiments of the first aspect, the antibody binds to TL1A in primates and / or rodents; and / or

[0024] The antibody inhibits the binding of TL1A to death receptor 3 (DR3); and / or

[0025] The antibody does not inhibit the binding of TL1A to decoy receptor 3 (DcR3); and / or

[0026] The antibody inhibits caspase activation in TL1A-stimulated cells; and / or

[0027] The antibody inhibits the NF-κB activation of TL1A-stimulated cells; and / or

[0028] The antibody inhibits the ability of TL1A to stimulate PBMCs to secrete interferon-γ.

[0029] Secondly, this application provides a nucleic acid molecule that encodes the antibody described in the first aspect.

[0030] Thirdly, this application provides a pharmaceutical composition comprising the antibody described in the first aspect and a pharmaceutically acceptable excipient, diluent, or carrier.

[0031] Fourthly, this application provides the use of the antibody described in the first aspect or the pharmaceutical composition described in the third aspect in the preparation of a medicament for the prevention or treatment of TL1A-related diseases.

[0032] Fifthly, this application provides a method for preventing or treating TL1A-related diseases, comprising administering to an individual in need the antibody described in the first aspect or the pharmaceutical composition described in the third aspect. Attached Figure Description

[0033] Figure 1 The results showed that the anti-TL1A mouse monoclonal antibody inhibited caspase activation in TL1A-stimulated TF-1 cells.

[0034] Figure 2 The results showed that the anti-TL1A mouse monoclonal antibody inhibited NF-κB activation in TL1A-stimulated 293T-hDR3-NF-κB-RE-luci cells.

[0035] Figure 3 The results showed that the anti-TL1A monoclonal antibody H3F1+L28E1 inhibited caspase activation in TL1A-stimulated TF-1 cells.

[0036] Figure 4 The study demonstrated that the anti-TL1A monoclonal antibody H3F1+L28E1 inhibited NF-κB activation in TL1A-stimulated 293T-hDR3-NF-κB-RE-luci cells.

[0037] Figure 5 The anti-TL1A monoclonal antibody H3F1+L28E1 was shown to inhibit the ability of TL1A-stimulated PBMCs to secrete interferon-γ.

[0038] Figure 6 The results of ELISA analysis show that the anti-human TL1A monoclonal antibody blocked the binding of human TL1A to human DR3.

[0039] Figure 7 The results of ELISA analysis show the blocking activity of anti-TL1A monoclonal antibody against the binding of human DcR3 and human TL1A.

[0040] Figure 8 The results of fecal scoring in rats with DNBS-induced acute ulcerative colitis are shown.

[0041] Figure 9 The results of macroscopic colonic damage scoring in rats with DNBS-induced acute ulcerative colitis are shown.

[0042] Sequence Description

[0043] SEQ ID NO:1-3 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the anti-TL1A single-chain antibody S4D3, respectively.

[0044] SEQ ID NO:4-6 show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the anti-TL1A single-chain antibody S4D3, respectively.

[0045] SEQ ID NO:7 shows the amino acid sequence of HCDR1 of anti-TL1A single-chain antibodies S9H9 and S7B11.

[0046] SEQ ID NO:8 shows the amino acid sequence of HCDR2 of the anti-TL1A single-chain antibody S9H9.

[0047] SEQ ID NO:9 shows the amino acid sequence of HCDR3 of anti-TL1A single-chain antibodies S9H9 and S7B11.

[0048] SEQ ID NO:10-12 show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the anti-TL1A single-chain antibody S9H9, respectively.

[0049] SEQ ID NO:13 shows the amino acid sequence of HCDR2 of the anti-TL1A single-chain antibody S7B11.

[0050] SEQ ID NO:14-16 show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the anti-TL1A single-chain antibody S7B11, respectively.

[0051] SEQ ID NO:17-19 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the fully human anti-TL1A monoclonal antibody H3F1+L28E1, respectively.

[0052] SEQ ID NO:20-22 show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the fully human anti-TL1A monoclonal antibody H3F1+L28E1, respectively.

[0053] SEQ ID NO:23 shows the amino acid sequence of the heavy chain variable region of the anti-TL1A single-chain antibody S4D3.

[0054] SEQ ID NO:24 shows the amino acid sequence of the heavy chain variable region of the anti-TL1A single-chain antibody S9H9.

[0055] SEQ ID NO:25 shows the amino acid sequence of the heavy chain variable region of the anti-TL1A single-chain antibody S7B11.

[0056] SEQ ID NO:26 shows the amino acid sequence of the heavy chain variable region of the fully human anti-TL1A monoclonal antibody H3F1+L28E1.

[0057] SEQ ID NO:27 shows the amino acid sequence of the light chain variable region of the anti-TL1A single-chain antibody S4D3.

[0058] SEQ ID NO:28 shows the amino acid sequence of the light chain variable region of the anti-TL1A single-chain antibody S9H9.

[0059] SEQ ID NO:29 shows the amino acid sequence of the light chain variable region of the anti-TL1A single-chain antibody S7B11.

[0060] SEQ ID NO:30 shows the amino acid sequence of the light chain variable region of the fully human anti-TL1A monoclonal antibody H3F1+L28E1.

[0061] SEQ ID NO:31 shows the amino acid sequence of recombinant human TL1A (hTL1A).

[0062] SEQ ID NO:32 shows the amino acid sequence of recombinant cynomolgus monkey TL1A (mfTL1A).

[0063] SEQ ID NO:33 shows the amino acid sequence of recombinant mouse TL1A (mTL1A).

[0064] SEQ ID NO:34 shows the amino acid sequence of recombinant rat TL1A (rTL1A).

[0065] SEQ ID NO:35 shows the amino acid sequence of the Foldon domain (Fib Foldon) of T4 phage fibrin.

[0066] SEQ ID NO:36 shows the amino acid sequence of the His tag.

[0067] SEQ ID NO:37 shows the amino acid sequence of the heavy chain constant region (CH-IgG1) of the human (homo sapiens) IgG1 subtype.

[0068] SEQ ID NO:38 shows the amino acid sequence of the human IgG1 subtype heavy chain constant region mutant IgG1m3 (CH-IgG1m3).

[0069] SEQ ID NO:39 shows the amino acid sequence of the human IgG1 subtype heavy chain constant region mutant IgG1m3-YTE (CH-IgG1m3-YTE).

[0070] SEQ ID NO:40 shows the amino acid sequence of the constant region of the light chain of the human (homo sapiens) κ subtype.

[0071] SEQ ID NO:41 shows the amino acid sequence of the constant region of the light chain of the human (homo sapiens) λ subtype.

[0072] SEQ ID NO:42 shows the amino acid sequence of the recombinant protein His-fib-hTL1A.

[0073] SEQ ID NO:43 shows the amino acid sequence of the recombinant protein His-fib-mfTL1A.

[0074] SEQ ID NO:44 shows the amino acid sequence of the recombinant protein His-fib-mTL1A.

[0075] SEQ ID NO:45 shows the amino acid sequence of the recombinant protein His-fib-rTL1A.

[0076] SEQ ID NO:46 shows the nucleotide sequence of primer PmCGR.

[0077] SEQ ID NO:47 shows the nucleotide sequence of primer PmCKR.

[0078] SEQ ID NO:48 shows the amino acid sequence of the single-chain antibody S4D3.

[0079] SEQ ID NO:49 shows the amino acid sequence of the single-chain antibody S9H9.

[0080] SEQ ID NO:50 shows the amino acid sequence of the single-chain antibody S7B11.

[0081] SEQ ID NO:51 shows the amino acid sequence of the heavy chain variable region of the anti-TL1A monoclonal antibody RVT-3101.

[0082] SEQ ID NO:52 shows the amino acid sequence of the light chain variable region of the anti-TL1A monoclonal antibody RVT-3101.

[0083] SEQ ID NO:53 shows the amino acid sequence of the heavy chain variable region of the anti-TL1A monoclonal antibody PRA023.

[0084] SEQ ID NO:54 shows the amino acid sequence of the light chain variable region of the anti-TL1A monoclonal antibody PRA023.

[0085] SEQ ID NO:55 shows the amino acid sequence of the heavy chain variable region of the negative control antibody DP47.

[0086] SEQ ID NO:56 shows the amino acid sequence of the light chain variable region of the negative control antibody DP47. Invention Details

[0087] The inventors of this application have obtained a novel antibody that binds to tumor necrosis factor-like ligand 1A (TL1A) through antibody engineering technology. In various aspects of this application, novel antibodies against tumor necrosis factor-like ligand 1A (TL1A) are provided, along with nucleic acid molecules encoding said antibodies, vectors containing said nucleic acid molecules, host cells containing said nucleic acid molecules or vectors, methods for preparing and purifying said antibodies, and medical and biological applications of said antibodies. Based on the amino acid sequence of the variable region of the antibody provided in this application, full-length antibody molecules can be constructed as drugs for the prevention or treatment of TL1A-related diseases.

[0088] Unless otherwise specified, this application is implemented using conventional molecular biology, microbiology, cell biology, biochemistry and immunology techniques in the art.

[0089] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0090] definition

[0091] As used herein, an "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, peptides, etc. The term "antibody" as used herein includes not only complete (i.e., full-length) antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), their variants, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, biantibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules containing antigen recognition sites of desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0092] Typically, a full-length or complete antibody consists of two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). Full-length antibodies can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses mentioned above), but the antibody does not need to belong to any specific class. Immunoglobulins can be assigned to different classes based on the antibody's amino acid sequence of the heavy chain constant domain. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0093] As used herein, the term "antigen-binding fragment or antigen-binding region" refers to a portion or region of the complete antibody molecule responsible for binding the antigen. The antigen-binding domain may contain a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically contains three complementarity-determining regions, CDR1, CDR2, and CDR3.

[0094] It is well known to those skilled in the art that complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the variable regions that have the greatest impact on antibody affinity and specificity. There are two common definitions for the CDR amino acid sequence of VH or VL: the Chothia definition and the Kabat definition. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, MD. (1991). 7 ;A1-Lazikani et al., J.Mol.Biol.273:927-948 (1997) 8 ; and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989) 9 For a given antibody's variable region amino acid sequence, the CDR amino acid sequence in the VH and VL amino acid sequences can be determined according to the Chothia or Kabat definition. In the embodiments of this application, the Kabat definition of the CDR amino acid sequence is used.

[0095] For a given antibody's variable region amino acid sequence, the CDR amino acid sequence can be analyzed in various ways, such as using the online software Abysis (http: / / www.abysis.org / ).

[0096] Examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having VL-CL chains and VH-CH1 chains; (2) F(ab')2 fragments, which may be divalent fragments having two Fab' fragments connected by disulfide bridges (i.e., Fab' dimers) in the hinge region; (3) Fv fragments having a single arm of antibody with VL and VH domains; (4) single-chain Fv(scFv), which may be a single multipeptide chain consisting of VH and VL domains connected by peptide linkers; and (5) (scFv)2, which may contain two VH domains and two VL domains connected by peptide linkers, the two VL domains being combined with the two VH domains via disulfide bridges.

[0097] As used in this article, "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.

[0098] As used in this article, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies that make up the population are identical, except that naturally occurring mutations may exist in a small number of individuals.

[0099] In a first aspect, this application provides an antibody that binds to tumor necrosis factor-like ligand 1A (TL1A), comprising a heavy chain variable region containing the amino acid sequences HCDR1, HCDR2, and HCDR3, and a light chain variable region containing the amino acid sequences LCDR1, LCDR2, and LCDR3, wherein...

[0100] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6; or

[0101] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 7, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 8, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 9, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 10, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 12; or

[0102] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 7, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 13, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 9, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 14, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 15, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 16; or

[0103] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 17, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 18, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 20, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 21, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 22.

[0104] The amino acid sequences of HCDR and LCDR are defined according to Kabat.

[0105] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 23, 24, 25 or 26.

[0106] In some embodiments of the first aspect, the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID NO: 27, 28, 29 or 30.

[0107] In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 23, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 27.

[0108] In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 24, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 28.

[0109] In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 25, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 29.

[0110] In some embodiments of the first aspect, the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 26, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO: 30.

[0111] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO: 23, 24, 25 or 26, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity with SEQ ID NO: 27, 28, 29 or 30.

[0112] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO: 23, 24, 25 or 26.

[0113] In some embodiments of the first aspect, the amino acid sequence of the variable region of the light chain of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with SEQ ID NO: 27, 28, 29 or 30.

[0114] In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody differs from the amino acid sequence shown in SEQ ID NO: 23, 24, 25 or 26 by substitution, deletion and / or addition of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0115] In some embodiments of the first aspect, the amino acid sequence of the variable region of the light chain of the antibody differs from the amino acid sequence shown in SEQ ID NO: 27, 28, 29 or 30 by substitution, deletion and / or addition of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0116] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 23, 24, 25 or 26 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of a similar heavy chain variable region of the antibody.

[0117] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 23, 24, 25 or 26, and the resulting amino acid sequence still retains the function of the heavy chain variable region similar to that of the antibody.

[0118] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 23, 24, 25 or 26, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the antibody.

[0119] In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 27, 28, 29 or 30 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining the function of a similar light chain variable region of the antibody.

[0120] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 27, 28, 29 or 30, and the resulting amino acid sequence still retains the function of the light chain variable region similar to that of the antibody.

[0121] In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted in regions other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 27, 28, 29 or 30, as long as the altered amino acid sequence substantially maintains the function of the light chain variable region of the antibody.

[0122] In some embodiments of the first aspect, the antibody is a whole antibody, a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment (scFv).

[0123] In some embodiments of the first aspect, the antibody is a fully human antibody.

[0124] In some embodiments of the first aspect, the antibody is a monoclonal antibody.

[0125] In some embodiments of the first aspect, the antibody further comprises a heavy chain constant region selected from IgG1, IgG2 or IgG4 subtypes.

[0126] In some embodiments of the first aspect, the heavy chain constant region is the IgG1 subtype.

[0127] In some embodiments of the first aspect, the heavy chain constant region comprises the Fc segment sequence of the IgG1 subtype heavy chain constant region, and the amino acid sequences at positions 234, 235, and 331 of the Fc segment sequence are F, E, and S, respectively; wherein the amino acid sequence of the antibody constant region is determined according to EU numbering.

[0128] In some embodiments of the first aspect, the heavy chain constant region comprises the Fc segment sequence of the IgG1 subtype heavy chain constant region, and the amino acid sequences at positions 252, 254, and 256 of the Fc segment sequence are Y, T, and E, respectively; wherein the amino acid sequence of the antibody constant region is determined according to EU numbering.

[0129] In some embodiments of the first aspect, the heavy chain constant region comprises the Fc segment sequence of the IgG1 subtype heavy chain constant region, and the amino acid sequences at positions 234, 235, and 331 of the Fc segment sequence are F, E, and S, respectively, and the amino acid sequences at positions 252, 254, and 256 of the Fc segment sequence are Y, T, and E, respectively; wherein the amino acid sequence of the antibody constant region is determined according to EU numbering.

[0130] In some embodiments of the first aspect, the antibody further comprises a light chain constant region selected from the κ or λ subtype.

[0131] In some embodiments of the first aspect, the antibody binds to TL1A of a primate. In some embodiments, the primate is a human, or a monkey (e.g., a cynomolgus monkey).

[0132] In some embodiments of the first aspect, the antibody binds to TL1A in rodents. In some embodiments, the rodent is a murine animal, such as a rat or mouse.

[0133] In some embodiments of the first aspect, the antibody binds to recombinant human TL1A (SEQ ID NO: 31).

[0134] In some embodiments of the first aspect, the antibody binds to recombinant monkey (e.g., cynomolgus monkey) TL1A (SEQ ID NO: 32).

[0135] In some embodiments of the first aspect, the antibody binds to recombinant mouse TL1A (SEQ ID NO:33).

[0136] In some embodiments of the first aspect, the antibody binds to recombinant rat TL1A (SEQ ID NO: 34).

[0137] In some embodiments of the first aspect, the antibody inhibits the binding of TL1A to death receptor 3 (DR3).

[0138] In some embodiments of the first aspect, the antibody does not inhibit the binding of TL1A to decoy receptor 3 (DcR3).

[0139] In some embodiments of the first aspect, the antibody inhibits caspase activation in TL1A-stimulated cells (such as tumor cells, e.g., TF-1 cells).

[0140] In some embodiments of the first aspect, the antibody inhibits NF-κB activation in TL1A-stimulated cells (e.g., 293T-hDR3-NF-κB-RE-luci cells).

[0141] In some embodiments of the first aspect, the antibody inhibits the ability of TL1A to stimulate PBMCs to secrete interferon-γ.

[0142] Secondly, this application provides a nucleic acid molecule that encodes the antibody described in the first aspect.

[0143] In some implementations, the nucleic acid molecule is operatively linked to a regulatory sequence that can be recognized by host cells transformed with the vector.

[0144] Thirdly, this application provides a pharmaceutical composition comprising the antibody described in the first aspect and a pharmaceutically acceptable excipient, diluent, or carrier.

[0145] In some embodiments of the third aspect, the pharmaceutical composition is used for the prevention or treatment of TL1A-related diseases.

[0146] In some implementations of the third aspect, the TL1A-related diseases are rheumatoid arthritis, inflammatory bowel disease (e.g., Crohn's disease (CD) and ulcerative colitis (UC)), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, ankylosing spondylitis, and interstitial lung disease associated with systemic sclerosis.

[0147] In some embodiments of the third aspect, the pharmaceutical composition may further comprise one or more of the following: lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives, such as benzoic acid, sorbic acid, and calcium propionate; sweeteners and / or flavoring agents, etc.

[0148] In some embodiments of the third aspect, the pharmaceutical composition of this application may be formulated as tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.

[0149] In some embodiments of the third aspect, the pharmaceutical composition of this application may be delivered using any physiologically acceptable method of administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.

[0150] In some embodiments of the third aspect, a pharmaceutical composition for therapeutic use can be formulated for storage by mixing a reagent of the desired purity with, as appropriate, a pharmaceutically acceptable carrier, excipient, etc., in the form of a lyophilized formulation or an aqueous solution.

[0151] Fourthly, this application provides the use of the antibody described in the first aspect and the pharmaceutical composition described in the third aspect in the preparation of a medicament for the prevention or treatment of TL1A-related diseases.

[0152] In some implementations of the fourth aspect, the TL1A-related diseases are rheumatoid arthritis, inflammatory bowel disease (e.g., Crohn's disease (CD) and ulcerative colitis (UC)), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, ankylosing spondylitis, and interstitial lung disease associated with systemic sclerosis.

[0153] Fifthly, this application provides a method for preventing or treating TL1A-related diseases, comprising administering to an individual in need the antibody described in the first aspect, or the pharmaceutical composition described in the third aspect.

[0154] In some implementations of the fifth aspect, the TL1A-related diseases are rheumatoid arthritis, inflammatory bowel disease (e.g., Crohn's disease (CD) and ulcerative colitis (UC)), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, ankylosing spondylitis, and interstitial lung disease associated with systemic sclerosis.

[0155] In other aspects, this application also provides a vector comprising a nucleic acid molecule encoding an antibody of the present invention or its light or heavy chain, a host cell comprising the nucleic acid molecule or the vector, and a method for producing the antibody. In some embodiments, the nucleic acid molecule is operatively linked to a regulatory nucleotide sequence that can be recognized by a host cell transformed with the vector. In some embodiments, the method of producing the antibody includes culturing the host cell to express the nucleic acid. In some embodiments, the method of producing the antibody further includes recovering the antibody from the host cell culture medium.

[0156] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments. Example

[0157] The following examples are for illustrative purposes only and are not intended to limit the scope of this application.

[0158] Example 1: Preparation of recombinant protein

[0159] The preparation and identification of anti-TL1A antibodies require various recombinant proteins, including human TL1A (hTL1A, SEQ ID NO: 31), cynomolgus monkey TL1A (mfTL1A, SEQ ID NO: 32), mouse TL1A (mTL1A, SEQ ID NO: 33), and rat TL1A (rTL1A, SEQ ID NO: 34). Adding the Foldon domain (Fib Foldon, SEQ ID NO: 35) of T4 phage fibrin to the N-terminus of these recombinant proteins helps maintain the native trimer conformation of TL1A and enhances its stability. Simultaneously, adding a His tag (His, SEQ ID NO: 36) to the N-terminus of these recombinant proteins facilitates their purification and functional identification. When preparing recombinant antibodies, the constant region of the antibody heavy chain can be human IgG1 subtype (CH-IgG1, SEQ ID NO: 37) or various mutants of the defined human IgG1 subtype, such as IgG1m3 (CH-IgG1m3, SEQ ID NO: 38) and IgG1m3-YTE (CH-IgG1m3-YTE, SEQ ID NO: 39); the constant region of the light chain can be human κ subtype (CK, SEQ ID NO: 40) or human λ subtype (CL, SEQ ID NO: 41).

[0160] Based on the amino acid sequences of recombinant proteins in the Uniprot database, genes for the various recombinant proteins (including His tags and Fib Foldon domains) were designed and synthesized. Using conventional molecular biology techniques, the synthesized recombinant protein genes were cloned into suitable eukaryotic expression vectors (such as Invitrogen's pcDNA3.1). Then, using liposomes (such as Invitrogen's 293fectin) or other cationic transfection reagents (such as PEI), the prepared recombinant protein expression plasmids were transfected into HEK293 cells (such as Invitrogen's HEK293F). The cells were cultured in serum-free suspension for 3-4 days, and the culture supernatant was harvested by centrifugation.

[0161] Recombinant proteins His-fib-hTL1A (SEQ ID NO: 42), His-fib-mfTL1A (SEQ ID NO: 43), His-fib-mTL1A (SEQ ID NO: 44), and His-fib-rTL1A (SEQ ID NO: 45) expressed by His-tag fusion were purified in one step from the culture supernatant using a metal chelate affinity chromatography column (such as GE's HisTrap FF). The recombinant antibody was purified in one step using a Protein A / G affinity chromatography column (such as GE's Mabselect SURE). Then, the recombinant protein storage buffer was replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (such as GE's Hitrap desaulting). After filtration and sterilization, the proteins were aliquoted and stored at -20°C for later use.

[0162] Example 2: Preparation and Screening of Immune Libraries

[0163] 2.1 Mouse immunization and preparation of antibody library

[0164] BALB / c mice aged 6-8 weeks were used. Blood was collected from the tail vein of each mouse before immunization to obtain baseline serum. For the first immunization, the His-fib-hTL1A fusion protein was emulsified with Freund's complete adjuvant, and 50 μg of the fusion protein was injected into each mouse. Two weeks later, booster immunizations were performed. For the first and third booster immunizations, the His-fib-hTL1A fusion protein was emulsified with Freund's incomplete adjuvant, and 50 μg of the fusion protein was injected into each mouse. Blood was collected from the tail of each mouse before injection. For the second and fourth booster immunizations, the His-fib-mfTL1A fusion protein was emulsified with Freund's incomplete adjuvant, and 100 μg of the fusion protein was injected into each mouse. For the sixth immunization, the His-fib-hTL1A recombinant antigen without adjuvant was used as the immunogen, and 50 μg of the fusion protein was injected into each mouse. Three days after the initial immunization, the mice were sacrificed, and spleen cells were collected.

[0165] Mouse spleen lymphocytes were isolated using mouse lymphocyte separation medium (Beijing Dakowei Biotechnology Co., Ltd., CAT#DKW33-R0100), and total RNA was extracted from the lymphocytes using a total RNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., CAT#DP430). Using the extracted total RNA as a template, cDNA from the variable regions of the heavy and light chains was synthesized using a first-strand cDNA synthesis kit (Thermoscientific, CAT#K1621). Gene-specific primers were used for reverse transcription, with primer pairing regions located in the constant regions of the antibody heavy and light chains, respectively, with specific sequences PmCGR:TGCATTTGAACTCCTTGCC (SEQ ID NO: 46) and PmCKR:CCATCAATCTTCCACTTGAC (SEQ ID NO: 47). The synthesized cDNA was immediately stored at -70℃ for later use. Then, using the cDNA obtained from reverse transcription as a template, cDNA was synthesized according to the references. 6 Primers were synthesized, and mouse antibody VH and VK genes were amplified separately using PCR. Then, single-chain antibody (scFv) genes were constructed using overlap extension PCR. Finally, the prepared mouse single-chain antibody genes were cloned into the vector pADSCFV-S (see Chinese Patent Application No. 201510097117.0). 7 A scFv library was constructed. This antibody library has a size of 6.0E+8 and an accuracy of 70%.

[0166] 2.2 Screening of mouse single-chain antibody libraries

[0167] Using His-fib-hTL1A and His-fib-mfTL1A prepared in Example 1 as antigens, a solid-phase screening strategy was employed (experimental protocol referenced Phage Display: A Universal Guide to Experimentation, edited by Clackson, T., and Lowman, HB; translated by Ma Lan et al., Chemical Industry Press, May 2008). 8 The phage library displaying mouse single-chain antibodies constructed above was screened through three rounds of screening: binding, elution, neutralization, infection, and amplification. Finally, three single-chain antibodies with different sequences, S4D3 (SEQ ID NO: 48), S9H9 (SEQ ID NO: 49) and S7B11 (SEQ ID NO: 50), were obtained.

[0168] Using conventional molecular biology techniques, single-chain antibodies S4D3, S9H9, and S7B11 were prepared into IgG1m3 subtype full antibodies, respectively. This was done in accordance with US Patent No. US20150132311A1. 9The heavy chain variable region (SEQ ID NO: 51) and light chain variable region (SEQ ID NO: 52) of RVT-3101 were synthesized; refer to US Patent No. US20210122828A1. 10 The heavy chain variable region (SEQ ID NO: 53) and light chain variable region (SEQ ID NO: 54) of PRA023 were synthesized. RVT-3101 and PRA023 were used to prepare IgG1m3 subtype full antibodies as positive controls. DP47 (germline gene antibody, according to US Patent No. US20160200833A1) was used. 11 DP47VH, SEQ ID NO: 55; DP47VK, SEQ ID NO: 56) were prepared as IgG1m3 subtype full antibodies for use as negative controls.

[0169] Example 3: Identification of anti-TL1A mouse monoclonal antibody

[0170] 3.1 Affinity analysis of anti-TL1A mouse monoclonal antibodies S4D3, S9H9, and S7B11

[0171] The affinity of mouse anti-human TL1A monoclonal antibody was determined using surface plasmon resonance (SPR) technology with a Biacore T200. All reagents and consumables, including the amino-coupled antibody kit (BR-1000-50), human antibody capture kit (BR-1008-39), S-series CM5 chip (14100530), and 10×HBS-EP (BR100669) at pH 7.4, were purchased from GE Healthcare. Following the kit instructions, the carboxylated CM5 chip surface was activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The anti-human IgG (Fc) antibody (capture antibody) was diluted to 25 μg / mL with 10 mM pH 5.0 sodium acetate and then injected at a flow rate of 10 μL / min to achieve a conjugation volume of approximately 10,000 response units (RU). After injection of the capture antibody, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, the anti-TL1A monoclonal antibody was diluted to 1 μg / mL and injected at a flow rate of 10 μL / min, ensuring that approximately 200 RU of antibody was captured by the anti-human Fc antibody. A series of concentration gradients (e.g., 1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM) of His-fib-hTL1A, His-fib-mTL1A, and His-fib-mfTL1A were injected at a flow rate of 30 μL / min from low to high concentrations, with a binding time of 90 s and a dissociation time of 1200 s. The chip surface was then regenerated by injecting 3M MgCl2 at a flow rate of 10 μL / min for 30 s. The binding rate (K0) was calculated using Biacore T200 evaluation software version 3.2.1 by fitting binding and dissociation sensor maps using a 1:1 binding model. a ) and dissociation rate (K d ). Using ratio K d / K a Calculate the dissociation equilibrium constant (K) D The fitting results are shown in Tables 1, 2 and 3.

[0172] Table 1. Affinity constants of TL1A monoclonal antibody binding to human TL1A (His-fib-hTL1A)

[0173] Antibody <![CDATA[K a (M -1 s -1 ))]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> RVT-3101 6.521E+5 1.001E-4 1.536E-10 PRA023 4.755E+5 1.686E-4 3.546E-10 S4D3 5.323E+5 1.313E-4 2.467E-10 S9H9 1.340E+7 5.012E-4 3.742E-11 S7B11 4.704E+5 1.101E-4 2.341E-10

[0174] Table 2. Affinity constants of TL1A monoclonal antibody binding to mouse TL1A (His-fib-mTL1A)

[0175] Antibody <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> RVT-3101 9.039E+5 4.358E-5 4.821E-11 PRA023 NB / / S4D3 NB / / S9H9 6.857E+4 2.551E-3 3.719E-8 S7B11 6.832E+4 3.657E-3 5.353E-8

[0176] NB: No binding detected

[0177] Table 3. Affinity constants of TL1A monoclonal antibody binding to monkey TL1A (His-fib-mfTL1A)

[0178] Antibody <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> RVT-3101 1.643E+6 4.515E-5 2.748E-11 PRA023 1.124E+6 9.014E-5 8.023E-11 S4D3 1.290E+6 6.309E-5 4.893E-11 S9H9 2.900E+6 1.749E-4 6.031E-11 S7B11 1.322E+6 6.930E-5 5.202E-11

[0179] 3.2 The effect of anti-TL1A mouse monoclonal antibody on inhibiting TL1A-stimulated caspase activation in TF-1 cells

[0180] TF-1 cells (ATCC, CRL-2003, human erythroid leukemia cells) were purchased from ATCC. Cell growth was entirely dependent on IL-3 or GM-CSF and unresponsive to IL-5. These cells expressed the TL1A receptor DR3, and TL1A stimulated caspase 3 / 7 activation. Cycloheximide (CHX) is an inhibitor of eukaryotic protein synthesis, inhibiting cellular protein synthesis. TF-1 cells were resuspended in test medium containing 2 μM Cycloheximide at a concentration of 2 × 10⁻⁶ cells using RPMI 1640 + 5% inactivated FBS as the test medium. 5 The antibody was seeded in 96-well plates at 50 μL / well using test medium containing 800 ng / mL His-fib-hTL1A. The antibody was initially diluted at 40 nM and serially diluted 1.7-fold for a total of 10 concentrations. 50 μL of the diluted antibody was added to each well of TF-1 cells, for a total volume of 100 μL / well. Cells were incubated at 37°C and 5% CO2 for 5 hours. Caspase 3 / 7 activity was detected using a Caspase-glo 3 / 7 kit (Promega, G8093) and a multi-mode microplate reader (Molecular Devices). I3X) was used for full-wavelength scanning, and the chemiluminescence readings were fitted and analyzed. The results ( Figure 1 As shown in Table 4, the anti-TL1A mouse monoclonal antibodies S4D3, S9H9 and S7B11 can effectively inhibit His-fib-hTL1A-induced caspase activation, and their activity is comparable to that of RVT-3101.

[0181] Table 4. Results of the inhibition of caspase activation in TL1A-stimulated TF-1 cells by anti-TL1A mouse monoclonal antibody.

[0182] Antibody <![CDATA[IC 50 (nM)]]> RVT-3101 3.129 S4D3 3.047 S9H9 2.686 S7B11 2.686

[0183] 3.3 Anti-TL1A mouse monoclonal antibody inhibits NF-κB activation in TL1A-stimulated 293T-hDR3-NF-κB-RE-luci cells

[0184] 293T-NF-κB-RE-luci (catalog number CS025) was purchased from Yingmaoshengye Biotechnology Co., Ltd., and is an NF-κB reporter gene cell line. Based on this cell line, hDR3 expression was expressed via liposome transfection, and a stable hDR3-expressing 293T-hDR3-NF-κB-RE-luci cell line was selected for evaluating the activity of anti-TL1A antibodies. DMEM + 2% FBS was used as the test medium. The test antibody was diluted with test medium containing 240 ng / mL His-fib-hTL1A, starting at 20 nM, and serially diluted 1.7-fold for a total of 10 concentration points. 293T-hDR3-NF-κB-RE-luci cells were diluted to 4 × 10⁻⁶ cells / mL with test medium. 6 Cells were cultured at 50 μL / mL, with 50 μL of antibody mixed and seeded into 96-well plates. The cells were then incubated at 37°C and 5% CO2 for 20 hours. The activities of firefly luciferase and renal luciferase were detected sequentially using a Dual-glo luciferase assay kit (Promega, E2920). A multi-functional microplate reader (Molecular Devices) was used. I3X) was used for full-wavelength scanning, and the ratio of the luminescence signal of firefly luciferase to that of sea cucumber luciferase was used for fitting analysis. Results ( Figure 2 As shown in Table 5, the anti-TL1A mouse monoclonal antibodies S4D3, S9H9 and S7B11 can effectively inhibit NF-κB signaling induced by His-fib-hTL1A, and their activity is comparable to that of RVT-3101.

[0185] Table 5. Results of the inhibition of NF-κB activation in TL1A-stimulated 293T-hDR3-NF-κB-RE-luci cells by anti-TL1A mouse monoclonal antibody.

[0186] Antibody <![CDATA[IC 50 (nM)]]> S9H9 1.284 S7B11 1.346 RVT-3101 1.278 S4D3 1.128

[0187] Example 4: Screening of a fully human Fab recombinant library

[0188] Using the recombinant His-fib-hTL1A and His-fib-mfTL1A prepared in Example 1 as antigens, a fully human Fab phage library was screened using a solid-phase screening strategy (experimental protocol referenced Phage Display: A Universal Guide to Experimentation, edited by Clackson, T., and Lowman, HB; translated by Ma Lan et al., Chemical Industry Press, 2008.5) (see Chinese Patent Application No. 202210871809.6). Three rounds of screening were performed through binding, elution, neutralization, infection, and amplification, ultimately yielding a monoclonal antibody H3F1+L28E1 that specifically binds to TL1A in humans, cynomolgus monkeys, mice, and rats (the amino acid sequence of H3F1VH is shown in SEQ ID NO: 26; the amino acid sequence of L28E1VK is shown in SEQ ID NO: 30).

[0189] Using conventional molecular biology techniques, H3F1+L28E1 was prepared into a complete antibody against the IgG1m3 subtype.

[0190] Example 5: Identification of fully human anti-TL1A monoclonal antibody

[0191] 5.1 Affinity analysis of anti-TL1A monoclonal antibody H3F1+L28E1

[0192] Referring to Example 3.1, the affinity of the TL1A monoclonal antibody H3F1+L28E1 for binding to TL1A of different species was analyzed using Biacore T200. The results are shown in Tables 6, 7, 8 and 9.

[0193] Table 6. Affinity constants of anti-TL1A monoclonal antibody H3F1+L28E1 binding to human TL1A (His-fib-hTL1A)

[0194] Antibody <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> RVT-3101 7.063E+5 1.016E-4 1.438E-10 PRA023 4.755E+5 1.686E-4 3.546E-10 H3F1+L28E1 5.381E+6 1.562E-4 2.093E-11

[0195] Table 7. Affinity constants of anti-TL1A monoclonal antibody H3F1+L28E1 binding to mouse TL1A (His-fib-mTL1A)

[0196] Antibody <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M) <!-- 13 -->]]> RVT-3101 7.635E+5 5.92E-5 7.753E-11 PRA023 NB / / H3F1+L28E1 9.643E+5 1.831E-3 1.898E-9

[0197] NB: No binding detected

[0198] Table 8. Affinity constants of anti-TL1A monoclonal antibody H3F1+L28E1 binding to rat TL1A (His-fib-rTL1A)

[0199] Antibody <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> RVT-3101 9.778E+5 4.243E-4 4.34E-10 PRA023 NB / / H3F1+L28E1 8.037E+6 1.170E-3 1.456E-10

[0200] NB: No binding detected

[0201] Table 9. Affinity constants of anti-TL1A monoclonal antibody H3F1+L28E1 binding to monkey TL1A (His-fib-mfTL1A)

[0202] Antibody <![CDATA[K a (M -1 s -1 )]]> <![CDATA[K d (s -1 )]]> <![CDATA[K D (M)]]> RVT-3101 1.340E+6 6.700E-5 4.998E-11 PRA023 1.124E+6 9.014E-5 8.023E-11 H3F1+L28E1 4.191E+6 1.330E-4 3.173E-11

[0203] 5.2H3F1+L2831 inhibits caspase activation in TL1A-stimulated TF-1 cells.

[0204] Referring to Example 3.2, the inhibitory effect of H3F1+L2831 on caspase activation in TL1A-stimulated TF-1 cells was evaluated. The antibody was diluted with test medium containing 800 ng / mL His-fib-hTL1A. All test antibodies were initially diluted 1.7-fold at 40 nM, for a total of 10 concentration points. Results ( Figure 3 As shown in Table 10, H3F1+L28E1 can effectively inhibit caspase signaling induced by His-fib-TL1A, and its activity is superior to that of the control antibodies RVT-3101 and PRA023.

[0205] Table 10. Results of H3F1+L2831 inhibiting caspase activation in TL1A-stimulated TF-1 cells.

[0206] Antibody <![CDATA[IC 50 (nM)]]> RVT-3101 3.464 PRA023 6.523 H3F1+L28E1 1.734

[0207] 5.3H3F1+L28E1 inhibits NF-κB activation in TL1A-stimulated 293T-hDR3-NF-κB-RE-luci cells.

[0208] Referring to Example 3.3, the inhibitory effect of H3F1+L28E1 on NF-κB activation in TL1A-stimulated 293T-hDR3-NF-κB-RE-luci cells was evaluated. The antibody was diluted with test medium containing 240 ng / mL His-fib-hTL1A, starting at 20 nM, and serially diluted 1.7-fold for a total of 10 concentration points. Results ( Figure 4 As shown in Table 11, H3F1+L28E1 can effectively inhibit His-fib-hTL1A-stimulated NF-κB signaling, and its activity is superior to that of RVT-3101 and PRA023.

[0209] Table 11. Inhibition of NF-κB activation in TL1A-stimulated 293T-hDR3-NF-κB-RE-luci cells by H3F1+L28E1

[0210] Antibody <![CDATA[IC 50 (nM)]]> RVT-3101 1.082 PRA023 2.813 H3F1+L28E1 0.574

[0211] 5.4H3F1+L28E1 inhibits the ability of TL1A to stimulate PBMCs to secrete interferon-γ.

[0212] NK cells or activated T cells express the TL1A receptor DR3. With the help of co-stimulatory factors such as IL-12 / IL-18, TL1A binds to NK cells or activated T cells, activating downstream signaling pathways and ultimately leading to the secretion of interferon-γ. The inhibitory effect of anti-TL1A antibodies on TL1A can be evaluated by detecting the level of interferon-γ in cell supernatant.

[0213] Blood (50 mL) was collected from healthy volunteers, with the blood provided by the inventors and their colleagues as volunteers. All volunteers signed informed consent forms. The inclusion criteria for volunteers were:

[0214] 1. Must be over 18 years old;

[0215] 2. No HIV or HBV infection;

[0216] 3. Blood routine test results were normal;

[0217] 4. Women who are not pregnant or breastfeeding.

[0218] Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy individuals using Ficoll density gradient centrifugation. PBMCs were resuspended in test medium containing 0.5 ng / mL IL-12 and 2 ng / mL IL-18 to a final volume of 6 × 10⁻⁶. RPMI 1640 + 5% inactivated FBS was used as the test medium. 5 Interferon-γ (IFN-γ) content in cell supernatants was determined by seeding 100 μL / well into 96-well plates and incubating at 37°C and 5% CO2 for 3 hours. After 3 hours, the antibody was diluted with test medium containing 2.4 μg / mL His-fib-hTL1A. The antibody was serially diluted 2.3-fold from 200 nM to 8 concentration points. 100 μL / well of the diluted antibody was added to each PBMC, for a total volume of 200 μL / well, and incubated at 37°C and 5% CO2 for 20 hours. The interferon-γ content in the cell supernatant was detected using a human IFN-γ pre-coated ELISA kit (Dayou, 1110002). Values ​​were read at OD 450 nm using an ELISA reader (800TS, Biotek). A standard curve of concentration and absorbance was plotted using the interferon-γ standard provided in the kit. The interferon-γ content in the supernatant was calculated and analyzed. Results ( Figure 5 As shown in Table 12, H3F1+L28E1 can effectively inhibit the secretion of interferon-γ by His-fib-hTL1A-stimulated PBMCs, and its activity is superior to that of RVT-3101 and PRA023.

[0219] Table 12. Ability of H3F1+L28E1 to inhibit TL1A-stimulated interferon-γ secretion from PBMCs

[0220] Antibody <![CDATA[IC 50 (nM)]]> RVT-3101 13.61 PRA023 6.545 H3F1+L28E1 5.232

[0221] Example 6: Anti-TL1A monoclonal antibody selectively neutralizes functional receptor DR3

[0222] Use the biotin-labeled kit (EZ-Link) TM Sulfo-NHS-Biotin, No-Weigh TM Biotinylated human DR3 (Recombinant Human DR3 / TNFRSF25 Fc Chimera Protein, CF, R&D SYSTEMS, 943-D3-050) was prepared using Format, ThermoFisher, A39256. His-fib-hTL1A was coated onto 96-well ELISA plates at 4 μg / mL, 100 μL / well, and incubated overnight at 4°C. Blocking was performed at 37°C for 1 hour using blocking buffer (3% skim milk-PBST). Serial dilutions of 10 μg / mL biotinylated human DR3 with anti-human TL1A monoclonal antibodies (RVT-3101 and H3F1+L28E1) were performed, starting at 200 μg / mL and followed by 3-fold dilutions for a total of 8 concentrations. 100 μL / well was added to each well of the blocked 96-well ELISA plate and incubated at 37°C for 1 hour. Wash the ELISA plate with PBST, then add horseradish peroxidase-labeled streptavidin (Streptavidin / HRP, Biosen, bs-0437P-HRP) and incubate at 37°C for 1 hour. Wash the ELISA plate with PBST again, add OPD substrate chromogenic solution, and stop the color development with 1M H2SO4 after 5-10 minutes. Measure the single-wavelength optical density at 490 nm using a microplate reader. ELISA analysis results are as follows. Figure 6 As shown: RVT-3101 and H3F1+L28E1 can block the binding of human DR3 to human TL1A.

[0223] Human DcR3 (Recombinant Human DcR3 / TNFRSF6B Fc Chimera Protein, CF, R&DSYSTEMS, 142-DC-100) was coated onto 96-well ELISA plates at 3 μg / mL, 100 μL / well, and incubated overnight at 4°C. Blocking was performed at 37°C for 1 hour using blocking buffer (3% skim milk-PBST). Anti-human TL1A monoclonal antibodies (RVT-3101, PRA023, S4D3, S9H9, S7B11, and H3F1+L28E1) were serially diluted with 3 μg / mL His-fib-hTL1A, starting at 100 μg / mL, with 11 3-fold serial dilutions, and 100 μL / well was added to each well of the blocked 96-well ELISA plate. The plates were incubated at 37°C for 1 hour. Wash the ELISA plate with PBST, then add HRP-labeled anti-His-tagged mouse monoclonal antibody (Kangwei Century, cw0285M) and incubate at 37°C for 1 hour. Wash the ELISA plate with PBST again, add OPD substrate chromogenic solution, and stop the color development with 1M H2SO4 after 5-10 minutes. Measure the single-wavelength optical density at 490 nm using a microplate reader. ELISA analysis results are as follows. Figure 7 As shown: RVT-3101 and PRA023 can block human DcR3 from binding to human TL1A, while S4D3, S9H9, S7B11 and H3F1+L28E1 do not block human DcR3 from binding to human TL1A.

[0224] DcR3, as a common decoy receptor for TNF family cytokines (Fas-L, LIGHT, and TL1A), possesses a natural antagonistic function in vivo. S4D3, S9H9, S7B11, and H3F1+L28E1 can neutralize DR3 but not DcR3, indicating that these antibodies do not disrupt the natural antagonistic activity of DcR3, helping to maintain DcR3 homeostasis in vivo and exhibiting better safety.

[0225] Example 7: Effect of anti-TL1A monoclonal antibody on DNBS-induced acute ulcerative colitis in rats

[0226] Male Wistar rats with no prior drug administration history were purchased from Shanghai Slack Laboratory Animal Breeding Co., Ltd., and acclimatized to the animal facility environment 3 days in advance. Before DNBS modeling, the rats were fasted for 40 hours, during which they were subcutaneously injected with 5% glucose injection (10 mL / kg) as an energy supplement. DNBS powder was dissolved in 30% ethanol to a final concentration of 60 mg / mL. On day 0, the fasted rats were anesthetized with chlorpheniramine (intraperitoneal injection, 25-50 mg / kg) and toluidine (intraperitoneal injection, 5-10 mg / kg). Specific animal grouping and administration regimens are shown in Table 13. In groups G2-G4, a soft tube was inserted into the colon through the anus to induce colitis in rats via DNBS enema. Group G1 underwent enema with 30% ethanol using the same method. The isotype control was DP47 antibody.

[0227] Table 13. Grouping and Dosing Regimens

[0228]

[0229] During the experiment, the characteristics of the experimental animals' feces were scored daily (0 = normal, 1 = moist / sticky, 2 = loose, 3 = liquid). The weight of the experimental animals was measured and recorded daily. On day 6, all experimental animals were euthanized by asphyxiation with excessive carbon dioxide. Afterwards, the abdominal cavity was opened, the colon was removed, longitudinally dissected, and thoroughly cleaned. The colonic ulceration surface was observed, and the colonic length, weight, and ulcer area were recorded. A macroscopic colonic damage score was calculated. Results ( Figure 8 and Figure 9 The results showed that the fecal characteristics and intestinal damage of the experimental animals in the G4 group (H3F1+L28E1) were significantly improved compared with those in the G2 group (model-isotype control group).

[0230] All patents, patent application publications, and non-patent documents mentioned and / or listed in this application are incorporated herein by reference in their entirety. Exemplary embodiments of the inventions described above have been described; however, those skilled in the art can modify or improve the exemplary embodiments described herein without departing from the spirit and scope of this application, and such variations or equivalents also fall within the scope of this application.

[0231] Sequence information

[0232] SEQ ID NO:1

[0233] KYDIN

[0234] SEQ ID NO:2

[0235] WIFPGDGRTDYNEKFKG

[0236] SEQ ID NO:3

[0237] YGYALDY

[0238] SEQ ID NO:4

[0239] RSSQNIVHSNGDTYLE

[0240] SEQ ID NO:5

[0241] KVSNRFS

[0242] SEQ ID NO:6

[0243] FQGSHFPYT

[0244] SEQ ID NO:7

[0245] NYWLG

[0246] SEQ ID NO:8

[0247] DIHPGRGNIFYNEKFKG

[0248] SEQ ID NO:9

[0249] GYDTFDY

[0250] SEQ ID NO:10

[0251] KASQDVSTAVA

[0252] SEQ ID NO:11

[0253] SASYRYT

[0254] SEQ ID NO:12

[0255] QQHYSTPWT

[0256] SEQ ID NO:13

[0257] DIHPGRGNIYYNEKFKG

[0258] SEQ ID NO:14

[0259] RASKSISKYLA

[0260] SEQ ID NO:15

[0261] SGSTLQS

[0262] SEQ ID NO:16

[0263] QQHNEYPYT

[0264] SEQ ID NO:17

[0265] SYDNEY

[0266] SEQ ID NO:18

[0267] WLNPNSGNTGYAQKFQG

[0268] SEQ ID NO:19

[0269] EIPESAAIEY

[0270] SEQ ID NO:20

[0271] TSSSSDIGAGLGVH

[0272] SEQ ID NO:21

[0273] GYYNRPS

[0274] SEQ ID NO:22

[0275] QSYDASLTGI

[0276] SEQ ID NO:23

[0277] QVQLKESGSDLATPGASVKLSCKVSGYTFTKYDINWVRQRPEQGLEWIGWIFPGDGRTDYNEKFKGKATLTIDTSSSTAYMQLSRLTSEDASAVYFCARYGYALDYWGQGTSVTVSS

[0278] SEQ ID NO:24

[0279] QVQLKQSGAELVRPGTSVKISCKASGYAFTNYWLGWIKQRPGHGLEWIGDIHPGRGNIFYNEKFKGKATLTADKSSSTAYMQLSLSTSEDASAVYFCAEGYDTFDYWGQGTTLTVSA

[0280] SEQ ID NO:25

[0281] QVKLQQSGGELVRPGTSVKISCKASGYAFTNYWLGWIKQRPGHGLEWIGDIHPGRGNIYYNEKFKGKATLTADKSSSTAYMQLSLSTSEDASAVYFCAEGYDTFDYWGQGTTLTVSS

[0282] SEQ ID NO:26

[0283] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWLNPNSGNTGYAQKFQGRVTMTADRSTSTAYMELSSLRSEDTAVYYCAREIPESAAIEYWGQGTLVTVSS

[0284] SEQ ID NO:27

[0285] DVVMTQTPLSLPVSLGDQASISCRSSQNIVHSNGDTYLEWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHFPYTFGGGTKLEMK

[0286] SEQ ID NO:28

[0287] DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEMK

[0288] SEQ ID NO:29

[0289] DIVMTQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPYTFGGGTKLEIK

[0290] SEQ ID NO:30

[0291] QSVLTQPPSVSGAPGQRVTISCTSSSSDIGAGLGVHWYQQLPGTAPKLLIEGYYNRPSGVPDRFSGSKSGTSASLTITGLLPEDEGDYYCQSYDASLTGIFGGGTKLTVL

[0292] SEQ ID NO:31

[0293] LKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL

[0294] SEQ ID NO:32

[0295] LKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHLKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFVYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL

[0296] SEQ ID NO:33

[0297] ITEERSEPSPQQVYSPPRGKPRAHLTIKKQTPAPHLKNQLSALHWEHDLGMAFTKNGMKYINKSLVIPESGDYFIYSQITFRGTTSVCGDISRGRRPNKPDSITMVITKVADSYPEPARLLTGSKSVCEISNNWFQSLYLGATFSLEEGDRLMVNVSDISLVDYTKEDKTFFGAFLL

[0298] SEQ ID NO:34

[0299] VTEERSAPSAQPVYTPSRDKPKAHLTIMRQTPVPHLKNELAALHWENNLGMAFTKNRMNYTNKFLVIPESGDYFIYSQITFRGTTSECGDISRVRRPKKPDSITVVITKVADSYPEPAHLLTGTKSVCEISSNWFQPIYLGAMFSLEEGDRLMVNVSDISLVDYTKEDKTFFGAFLI

[0300] SEQ ID NO:35

[0301] GYIPEAPRDGQAYVRKDGEWVLLSTFL

[0302] SEQ ID NO:36

[0303] HHHHHH

[0304] SEQ ID NO:37

[0305] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0306] SEQ ID NO:38

[0307] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0308] SEQ ID NO:39

[0309] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0310] SEQ ID NO:40

[0311] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0312] SEQ ID NO:41

[0313] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0314] SEQ ID NO:42

[0315] HHHHHHGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGGSLKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCEVGSN

[0316] WFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL

[0317] SEQ ID NO:43

[0318] HHHHHHGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGGSLKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHLKNQFPALHWEHELGLAFTKNRMNYTNKFL LIPESGDYFVYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL

[0319] SEQ ID NO:44

[0320] HHHHHHGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGGSITEERSEPSPQQVYSPPRGKPRAHLTIKKQTPAPHLKNQLSALHWEHDLGMAFTKNGMKYINKSLVI PESGDYFIYSQITFRGTTSVCGDISRGRRPNKPDSITMVITKVADSYPEPARLLTGSKSVCEISNNWFQSLYLGATFSLEEGDRLMVNVSDISLVDYTKEDKTFFGAFLL

[0321] SEQ ID NO:45

[0322] HHHHHHGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGGSVTEERSAPSAQPVYTPSRDKPKAHLTIMRQTPVPHLKNELAALHWENNLGMAFTKNRMNYTNKFLVI PESGDYFIYSQITFRGTTSECGDISRVRRPKKPDSITVVITKVADSYPEPAHLLTGTKSVCEISSNWFQPIYLGAMFSLEEGDRLMVNVSDISLVDYTKEDKTFFGAFLI

[0323] SEQ ID NO:46

[0324] TGCATTTGAACTCCTTGCC

[0325] SEQ ID NO:47

[0326] CCATCAATCTTCCACTTGAC

[0327] SEQ ID NO:48

[0328] QVQLKESGSDLATPGASVKLSCKVSGYTFTKYDINWVRQRPEQGLEWIGWIFPGDGRTDYNEKFKGKATLTIDTSSSTAYMQLSRLTSEDSAVYFCARYGYALDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQNIVHSNGDTYLEWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHFPYTFGGGTKLEMK

[0329] SEQ ID NO:49

[0330] QVQLKQSGAELVRPGTSVKISCKASGYAFTNYWLGWIKQRPGHGLEWIGDIHPGRGNIFYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAEGYDTFDYWGQGTTLTVSAGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEMK

[0331] SEQ ID NO:50

[0332] QVKLQQSGGELVRPGTSVKISCKASGYAFTNYWLGWIKQRPGHGLEWIGDIHPGRGNIYYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAEGYDTFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPYTFGGGTKLEIK

[0333] SEQ ID NO:51

[0334] QVQLVQSGAEVKKPGASVKVSCKASGYDFTYYGISWVRQAPGQGLEWMGWISTYNGNTHYARMLQGRVTMTTDTSTRTAYMELRSLRSDDTAVYYCARENYYGSGAYRGGMDVWGQGTTVTVSS

[0335] SEQ ID NO:52

[0336] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPWTFGQGTKVEIK

[0337] SEQ ID NO:53QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVKQRPGQGLEWMGRIDPASGHTKYDPKFQVRVTITRDTSTSTVYLELSSLRSEDTAVYYCARSGGLPDVWGQGTTVTVSS

[0338] SEQ ID NO:54

[0339] EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRPLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK

[0340] SEQ ID NO:55

[0341] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS

[0342] SEQ ID NO:56

[0343] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK

[0344] References

[0345] 1.Xu WD,Li R,Huang AF.Role of TL1A in Inflammatory Autoimmune Diseases:A Comprehensive Review.Front Immunol.2022Jul 14:13:8

[0346] 2.Migone TS,Zhang J,Luo X,Zhuang L,Chen C,Hu B,Hong JS,Perry JW,ChenSF,Zhou JX,Cho YH,Ullrich S,Kanakaraj P,Carrell J,Boyd E,Olsen HS,Hu G,PukacL,Liu D,Ni J,Kim S,Gentz ​​R,Feng P,Moore PA,Ruben SM,Wei P.TL1A is a TNF-like ligand for DR3 and TR6 / DcR3 and functions as a T cell costimulator.Immunity.2002Mar16(3):479-9

[0347] 3.Valatas V,Kolios G,Bamias G.TL1A(TNFSF15)and DR3(TNFRSF25):A Co-stimulatory System of Cytokines With Diverse Functions in Gut Mucosal Immunity.Front Immunol.2019Mar27:10:583.

[0348] 4.Ramos GP,Papadakis KA.Mechanisms of Disease:Inflammatory Bowel Diseases.Mayo Clin Proc.2019Jan94(1):155-1

[0349] 5.Li N,Ye M.Advances in biological agents in the treatment ofinflammatory bowel disease[J].Yixue Xinzhi Zazhi,2022,32(4):310-3

[0350] 6. Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Plückthun A. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods. 1997 Feb 14; 201 (1): 35-55.

[0351] 7.CN201510097117.0.

[0352] 8. Phage Display: A Universal Laboratory Guide / Edited by (US) Clackson, T., and (US) Lowman, HB; Translated by Ma Lan et al. Chemical Industry Press, May 2008.

[0353] 9.US20150132311A1

[0354] 10.US20210122828A1

[0355] 11.US20160200833A1.

Claims

1. An antibody that binds to tumor necrosis factor-like ligand 1A (TL1A), comprising a heavy chain variable region containing the amino acid sequences HCDR1, HCDR2, and HCDR3, and a light chain variable region containing the amino acid sequences LCDR1, LCDR2, and LCDR3, wherein... The amino acid sequence of HCDR1 is shown in SEQ ID NO: 17, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 18, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 19, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 20, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 21, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

22. in, The amino acid sequences of HCDR and LCDR are defined according to Kabat.

2. The antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:

26.

3. The antibody according to claim 1, wherein the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:

30.

4. The antibody according to any one of claims 1-3, wherein The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO: 26, and the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO:

30.

5. The antibody according to any one of claims 1-3, wherein the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity with SEQ ID NO: 26, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity with SEQ ID NO:

30.

6. The antibody according to any one of claims 1-3, wherein The antibody is a whole antibody, a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment (scFv); and / or The antibody is a monoclonal antibody; and / or The antibody further comprises a heavy chain constant region selected from IgG1, IgG2, or IgG4 subtypes; and / or The antibody also contains a light chain constant region selected from the κ or λ subtype.

7. The antibody according to claim 6, wherein the antibody is a fully human antibody.

8. The antibody according to claim 6, wherein the constant region of the antibody heavy chain is the IgG1 subtype.

9. The antibody according to claim 6, wherein the antibody heavy chain constant region comprises the Fc segment sequence of the IgG1 subtype heavy chain constant region, and the amino acid sequences at positions 234, 235, and 331 of the Fc segment sequence are F, E, and S, respectively, and / or the amino acid sequences at positions 252, 254, and 256 of the Fc segment sequence are Y, T, and E, respectively; wherein the amino acid sequence of the antibody constant region is determined according to EU numbering.

10. The antibody according to any one of claims 1-3, wherein The antibody binds to TL1A in primates and / or rodents; and / or The antibody inhibits the binding of TL1A to death receptor 3 (DR3); and / or The antibody does not inhibit the binding of TL1A to decoy receptor 3 (DcR3); and / or The antibody inhibits caspase activation in TL1A-stimulated cells; and / or The antibody inhibits the NF-κB activation of TL1A-stimulated cells; and / or The antibody inhibits the ability of TL1A to stimulate PBMCs to secrete interferon-γ.

11. A nucleic acid molecule encoding an antibody as described in any one of claims 1-10.

12. A pharmaceutical composition comprising the antibody according to any one of claims 1-10 and a pharmaceutically acceptable excipient.

13. The pharmaceutical composition according to claim 12, wherein the excipient is a diluent or a carrier.

14. Use of the antibody of any one of claims 1-10, or the pharmaceutical composition of claim 12, in the preparation of a medicament for the prevention or treatment of TL1A-related diseases; wherein the TL1A-related diseases are rheumatoid arthritis, inflammatory bowel disease, psoriasis, and systemic sclerosis-associated interstitial lung disease.

15. The use according to claim 14, wherein the inflammatory bowel disease is Crohn's disease and ulcerative colitis.

Images