Anti-tl1a antibodies, methods of making and using the same

Abstract

The application discloses an anti-TL1A antibody and a preparation method and application thereof in the technical field of biological medicine. The anti-TL1A antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. The antibody has high affinity, is better in biological activity characterization, and has a different epitope from traditional anti-TL1A antibodies.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an anti-TL1A antibody and a preparation method and application thereof. BACKGROUND

[0002] Tumor Necrosis Factor-like ligand 1A (TL1A), also known as Vascular Endothelial Growth Inhibitor (VEGI)-251 and TNFSF15 (Tumor Necrosis Factor Superfamily 15), is a member of the ligand tumor necrosis factor superfamily (TNFSF) and was discovered by Migone et al. in 2002. Human TL1A consists of 251 amino acids: 35 in the cytoplasmic domain, 24 in the transmembrane region, and 192 in the extracellular domain. TL1A is a type II transmembrane protein that self-assembles into stable trimers through TNF homology domain (THD) interactions. TL1A is mainly expressed in the form of membrane binding (mTL1A), forms stable trimers, and then produces soluble TL1A (sTL1A) through selective splicing or metalloproteinase (such as TNF-alpha converting enzyme (TACE)) cleavage.

[0003] TL1A is expressed in different immune cells (such as monocytes, macrophages, dendritic cells, T cells) and non-immune cells (such as synovial fibroblasts, endothelial cells). TL1A competitively binds to death receptor 3 (DR3) to provide a stimulating signal for the downstream signaling pathway, thereby regulating the proliferation, activation, apoptosis and cytokine, chemokine production of effector cells. Studies have found that TL1A is abnormally expressed in autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease (IBD), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, and ankylosing spondylitis.

[0004] DR3 is a type I transmembrane protein, and osteoblasts can produce transmembrane and soluble forms of DR3. In resting T cells, DR3 is expressed in a soluble form, which protects the cells from apoptosis. Activated T cells express transmembrane DR3, which activates the receptor, leading to cell apoptosis or activation of transcription factors such as NF-κB. sTL1A binds to DR3 (membrane-bound form) to activate the downstream signal cascade, thereby regulating immune responses and inflammation. sTL1A / DR3 interaction can trigger two different signaling pathways, which cause inflammation and cell apoptosis, respectively.

[0005] Inflammatory bowel diseases (IBD) are a group of chronic, relapsing, inflammatory intestinal disorders characterized by intestinal inflammation and epithelial damage. Severe cases can lead to hospitalization and surgery, with low mortality but lifelong treatment. IBD mainly includes Crohn's disease (CD) and ulcerative colitis (UC), two diseases that have both overlapping and distinct clinical and pathological features. In patients with IBD, the expression of TL1A is increased in both serum and intestinal tissue, and is associated with disease activity. The expression of DR3 is also increased in the intestinal lamina propria lymphocytes, particularly T cells, of these patients. In addition, studies have shown that TL1A is mainly expressed on CD11chigh dendritic cells in the ileal wall of ileitis mice. The interaction between TL1A expressed on DCs and DR3 expressed on lymphocytes can play an important role in the pathogenesis of IBD by promoting the excessive secretion of interferon-gamma (IFN-γ). TL1A up-regulates chemokine receptors, leading to enhanced function of bone marrow-derived dendritic cells (BMDCs), including TL1A-dependent stimulation of T cells in BMDCs, and exacerbation of dextran sulfate sodium (DSS)-induced colitis. The TL1A / DR3 pathway has become an important module of mucosal immunity, which is involved in maintaining intestinal homeostasis, but also plays a key role in the development and maintenance of chronic inflammatory reactions in patients with inflammatory bowel disease (IBD). Studies have reported that in inflammatory diseases such as IBD, rheumatoid arthritis and psoriasis, the expression of TL1A and its two receptors is up-regulated, and TL1A / DR3 signaling promotes disease progression.

[0006] TL1A expression increase and / or TL1A gene polymorphism are associated with the pathogenesis of various autoimmune diseases and inflammatory diseases. Analysis of mouse models of autoimmune diseases and TL1A or DR3 transgenic mice shows that TL1A-DR3 interaction plays an important role in local inflammation in T cell-dependent autoimmune diseases. Therefore, TL1A links innate and adaptive immune responses and plays a key role in the induction of autoimmune and inflammatory diseases. These suggest that inhibiting TL1A-DR3 interaction is an effective therapeutic strategy to improve local inflammation in target organs of individuals with autoimmune diseases. In addition to inflammatory bowel disease (including ulcerative colitis and Crohn's disease), inhibiting TL1A-DR3 can also be a treatment strategy for diseases such as psoriasis, psoriasis, primary biliary cirrhosis, and systemic lupus erythematosus, ankylosing spondylitis, etc.

[0007] Therefore, there is a need to develop anti-TL1A antibodies to meet the needs of the clinic. SUMMARY

[0008] Therefore, it is necessary to provide an anti-TL1A antibody, a preparation method and application thereof.

[0009] The first aspect of the present application provides an anti-TL1A antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 11-13, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 14-16; or,

[0010] the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 17-19, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 20-22; or,

[0011] the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 23-25, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 26-28; or,

[0012] the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 29-31, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 32-34; or,

[0013] the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 35-37, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 38-40.

[0014] In some embodiments, the heavy chain variable region comprises a heavy chain variable region framework region FR, and the light chain variable region comprises a light chain variable region framework region FR, wherein the heavy chain variable region framework region FR is a human or murine heavy chain variable region framework region FR, and / or the light chain variable region framework region FR is a human or murine light chain variable region framework region FR.

[0015] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; or,

[0016] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 4; or,

[0017] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 6; or,

[0018] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8; or,

[0019] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 10.

[0020] In some embodiments, the anti-TLlA antibody is a monoclonal antibody.

[0021] A second aspect of the present application provides a nucleic acid molecule encoding the anti-TLlA antibody of the first aspect of the present application.

[0022] A third aspect of the present application provides an expression vector comprising the nucleic acid molecule of the second aspect of the present application.

[0023] A fourth aspect of the present application provides a cell comprising the expression vector of the third aspect of the present application.

[0024] A fifth aspect of the present application provides a method for preparing an anti-TLlA antibody, comprising culturing the cell of the fourth aspect of the present application, and obtaining the anti-TLlA antibody from the culture.

[0025] A sixth aspect of the present application provides a pharmaceutical composition comprising the anti-TLlA antibody of the first aspect of the present application and a pharmaceutically acceptable excipient.

[0026] A seventh aspect of the present application provides a TLlA detection product comprising the anti-TLlA antibody of the first aspect of the present application.

[0027] An eighth aspect of the present application provides a method for detecting TLlA, comprising reacting the anti-TLlA antibody of the first aspect of the present application with a sample to be tested.

[0028] The ninth aspect of the present application provides use of the anti-TL1A antibody of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the cell of the fourth aspect of the present application or the pharmaceutical composition of the sixth aspect of the present application in the preparation of a medicament for treating a disease related to TL1A.

[0029] The foregoing antibody has high affinity, better biological activity characterization, and a different epitope from traditional anti-TL1A antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments and examples of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the description of the embodiments or examples will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0031] Figure 1 A in the above table is that the CR20M0007 antibody and the CR20M0007hz13 antibody in an embodiment of the present application block the binding of TL1A to DcR3, B is that the CR20M0008 antibody and the CR20M0008hz1 antibody block the binding of TL1A to DcR3, C is that the CR20M0055 antibody and the CR20M0055hz2 antibody block the binding of TL1A to DcR3, D is that the CR20M0203 antibody and the CR20M0203hz4 antibody block the binding of TL1A to DcR3, and E is that the CR20M0666 antibody and the CR20M0666hz15 antibody block the binding of TL1A to DcR3;

[0032] Figure 2 is the binding specificity of the anti-TL1A antibody in an embodiment of the present application to TL1A and homologs;

[0033] Figure 3 is the epitope mapping of the anti-TL1A antibody in an embodiment of the present application;

[0034] Figure 4 A in the above table is that the CR20M0007 antibody and the CR20M0007hz13 antibody in an embodiment of the present application block the binding of TL1A to DcR3, B is that the CR20M0008 antibody and the CR20M0008hz1 antibody block the binding of TL1A to DcR3, C is that the CR20M0055 antibody and the CR20M0055hz2 antibody block the binding of TL1A to DcR3, D is that the CR20M0203 antibody and the CR20M0203hz4 antibody block the binding of TL1A to DcR3, and E is that the CR20M0666 antibody and the CR20M0666hz15 antibody block the binding of TL1A to DcR3;

[0035] Figure 5A is CR20M0007 antibody and CR20M0007 hz13 antibody inhibit NFkB, B is CR20M0008 antibody and CR20M0008 hz1 antibody inhibit NFkB, C is CR20M0055 antibody and CR20M0055 hz2 antibody inhibit NFkB, D is CR20M0203 antibody and CR20M0203 hz4 antibody inhibit NFkB, E is CR20M0666 antibody and CR20M0666 hz15 antibody inhibit NFkB;

[0036] Figure 6 A is CR20M0007 antibody and CR20M0007 hz13 antibody inhibit TL1A induced CD4+ T cell cytokine IFNy production, B is CR20M0008 antibody and CR20M0008 hz1 antibody inhibit TL1A induced CD4+ T cell cytokine IFNy production, C is CR20M0055 antibody and CR20M0055 hz2 antibody inhibit TL1A induced CD4+ T cell cytokine IFNy production, D is CR20M0203 antibody and CR20M0203 hz4 antibody inhibit TL1A induced CD4+ T cell cytokine IFNy production, E is CR20M0666 antibody and CR20M0666 hz15 antibody inhibit TL1A induced CD4+ T cell cytokine IFNy production. DETAILED DESCRIPTION

[0037] For the purposes of this application, the following description will be made with reference to the accompanying drawings in which preferred embodiments of the application are shown. The application may, however, be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. It is therefore contemplated that the application will include any and all combinations of the characteristics coming in the particular embodiments.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0039] As used herein, the terms "have," "having," "contain," "containing," "include," "including," and "comprise," "comprising" are open-ended, and do not exclude additional, unrecited members or features. Members or features, such as materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also include actions, conditions under which actions occur, timing, states, etc.

[0040] In the present application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of listed contents, and also include open technical features or technical solutions containing listed contents.

[0041] In the present application, unless there is an explicit different description herein, the execution of the steps involved in the method flow has no strict order limitation, and can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, which do not necessarily be executed at the same time, but can be executed at different times, and the execution order is not necessarily sequential, but can be executed alternately or simultaneously with other steps or sub-steps or stages of other steps.

[0042] In the present application, the exemplary description involving "in some embodiments (or examples)", "in an embodiment (or example)" and the like can cover but is not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0043] In the present application, in the terms "first aspect", "second aspect", "third aspect", "fourth aspect" and the like, the terms "first", "second", "third", "fourth" and the like are only for description purpose, and cannot be understood as indicating or implying relative importance or quantity, nor can be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0044] In the present application, at least one anti-TL1A antibody and a preparation method and application thereof are provided.

[0045] In some embodiments, the present application screened out 5 monoclonal antibodies with better bioactivity than the competitor antibodies by single B cell cloning technology. Balb / c mice and SJL mice were immunized with recombinant Human TL1A Protein, and serum titers were detected. After three immunizations, the spleen was taken out, and the spleen cells were separated. Single specific B cells were separated by FACS sorting technology. The antibody genes were cloned by PCR amplification technology, and sequenced. After preliminary screening by linear expression transfected supernatant, the expression vector was constructed, and the chimeric antibody was expressed and purified. Through the screening of the pure antibody, 5 antibodies (CR20M0007, CR20M0008, CR20M0055, CR20M0203, CR20M0666) were obtained, which were better than the competitor antibodies (RVT-3101, PRA023) in binding affinity, biological activity characterization (block, NF-κB signaling pathway and IFNγ cytokine inhibition). After humanization of the 5 antibodies, the activity of the parent was retained.

[0046] In the present application, the amino acid sequences of the competitor antibodies (RVT-3101, PRA023) are shown in Table 1 as follows:

[0047] Table 1

[0048]

[0049] In the first aspect of the present application, an anti-TL1A antibody is provided, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having an amino acid sequence as shown in SEQ ID NO: 11~13, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having an amino acid sequence as shown in SEQ ID NO: 14~16; or,

[0050] the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having an amino acid sequence as shown in SEQ ID NO: 17~19, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having an amino acid sequence as shown in SEQ ID NO: 20~22; or,

[0051] the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having an amino acid sequence as shown in SEQ ID NO: 23~25, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having an amino acid sequence as shown in SEQ ID NO: 26~28; or,

[0052] the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 35-37, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 38-40.

[0053] the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 35-37, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 38-40.

[0054] In the present application, "TL1A" refers to a type II transmembrane protein, which exists mainly in the form of membrane-bound (mTL1A) or soluble (sTL1A), including variants, homologs, orthologs and paralogs, unless otherwise specified.

[0055] In the present application, "anti-TL1A antibody" refers to an antibody or protein that specifically binds to TL1A. "Specifically binds" refers to the binding of an antibody to an epitope on a predetermined antigen. Specific binding refers to the binding of an antibody to an epitope on a predetermined antigen. Typically, an antibody binds with an affinity (KD) of about less than 10-8M, for example, about less than 10-9M, 10-10M, 10-11M, 10-12M or less.

[0056] The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the present application bind with an affinity of less than about 10-11M, and the affinity of an antibody of the present application for a cell surface antigen is determined by measuring the KD value using Biacore 8K.

[0057] In the present application, "IC50" refers to the half maximal inhibitory concentration of a drug or substance, or the concentration of the substance that induces 50% inhibition. IC50 is a measure of the potency of a substance to inhibit a particular biological or biochemical function. The lower the IC50, the greater the potency of the antagonist drug or substance as an inhibitor. For example, the IC50 of an anti-TL1A antibody of the present application is for blocking the binding of TL1A to DR3 cells, inhibiting the NFκB pathway, and inhibiting the production of the cytokine IFNγ.

[0058] In the present application, unless otherwise specified, "antibody" or "Ab" refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, and generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and noncovalent interactions. The light chain of an antibody can be classified as either kappa or lambda. The heavy chain can be classified as mu, delta, gamma, alpha, or epsilon, which defines the isotype of an antibody as IgM, IgD, IgG, IgA, or IgE, respectively. Within the light and heavy chains, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains (CH1, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are relatively conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, connected in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antigen binding site / portion, respectively. The distribution of amino acids in various regions or domains follows the numbering definitions of Kabat, IMGT, or Chothia, etc., and in the specific embodiments of the present application, the determination of CDR sequences uses the numbering definition in the Kabat system.

[0059] In the present application, unless otherwise specified, "epitope" refers to the location on the surface of an antigen molecule to which a single antibody molecule binds. Typically, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and epitopes with conformation. An antibody binds to "essentially the same epitope" as a reference antibody when the antibody recognizes the same or a spatially overlapping epitope as the reference antibody. The most common and rapid method for determining whether two epitopes bind the same or a spatially overlapping epitope is a competition test, which can be configured in various different forms using labeled antigen or labeled antibody, for example, a radioactively labeled or enzymatically labeled antibody can be used to determine the ability of an unlabeled antibody to block the binding of the labeled antibody.

[0060] In the present application, unless otherwise specified, anti-TL1A antibodies include, but are not limited to, monoclonal antibodies, F(ab')2 fragments, Fab' fragments, Fab fragments, Fv fragments, scFv fragments, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies.

[0061] In the present application, unless otherwise specified, "monoclonal antibody" or "mAb" or "monoclonal antibody composition" refers to an antibody molecule preparation of single molecular composition. The monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0062] In the present application, unless otherwise specified, "chimeric antibody" refers to an antibody having at least a portion of the heavy chain variable region and at least a portion of the light chain variable region derived from one species and at least a portion of the constant region derived from another species. For example, a chimeric antibody can include murine variable regions and human constant regions. "Humanized antibody" refers to an antibody containing complementarity determining regions (CDRs) derived from a non-human antibody, and framework regions and constant regions derived from a human antibody.

[0063] In some embodiments, the anti-TLl A antibodies of the present application comprise a human IgGl heavy chain constant region and a human Kappa light chain constant region.

[0064] In some embodiments, the heavy chain variable region further comprises a heavy chain variable region framework region FR, and the light chain variable region further comprises a light chain variable region framework region FR, wherein the heavy chain variable region framework region FR is a human-derived or murine-derived heavy chain variable region framework region FR, and the light chain variable region framework region FR is a human-derived or murine-derived light chain variable region framework region FR.

[0065] In some embodiments, the amino acid sequence of the anti-TLl A antibody heavy chain variable region is set forth in SEQ ID NO: 1 or an amino acid sequence that is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 1.

[0066] In some embodiments, the amino acid sequence of the anti-TLl A antibody light chain variable region is set forth in SEQ ID NO: 2 or an amino acid sequence that is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 2.

[0067] In some embodiments, the amino acid sequence of the anti-TLl A antibody heavy chain variable region is set forth in SEQ ID NO: 3 or an amino acid sequence that is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 3.

[0068] In some embodiments, the amino acid sequence of the anti-TLl A antibody heavy chain variable region is set forth in SEQ ID NO: 3 or an amino acid sequence that is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 3.

[0069] In some embodiments, the amino acid sequence of the anti-TLl A antibody heavy chain variable region is set forth in SEQ ID NO: 3 or an amino acid sequence that is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 3.

[0070] In some embodiments, the amino acid sequence of the anti-TLl A antibody heavy chain variable region is set forth in SEQ ID NO: 3 or an amino acid sequence that is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 3.

[0071] In some embodiments, the amino acid sequence of the anti-TLl A antibody heavy chain variable region is set forth in SEQ ID NO: 3 or an amino acid sequence that is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 3.

[0072] In some embodiments, the amino acid sequence of the anti-TLl A antibody heavy chain variable region is set forth in SEQ ID NO: 9 or the amino acid sequence is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 9.

[0073] In some embodiments, the amino acid sequence of the anti-TLl A antibody heavy chain variable region is set forth in SEQ ID NO: 9 or the amino acid sequence is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 9.

[0074] In some embodiments, the amino acid sequence of the anti-TLl A antibody heavy chain variable region is set forth in SEQ ID NO: 9 or the amino acid sequence is at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SEQ ID NO: 9.

[0075] In the present application, "identity" means the percentage of amino acid residues in a first sequence that are identical with the amino acid residues in a second sequence, after aligning the sequences, introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. To determine the percent sequence identity of two amino acid sequences, the sequences are aligned for maximum percent sequence identity, and the number of identical positions is counted. The percent sequence identity is then calculated by comparing the number of identical positions to the total number of positions in the reference sequence. Alignment can be achieved in various ways, which are within the scope of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve the maximum alignment over the entire length of the sequences being compared.

[0076] In a second aspect of the present application, there is provided a nucleic acid molecule encoding the anti-TLl A antibody of the first aspect of the present application.

[0077] In the present application, unless otherwise specified, "nucleic acid molecule" refers primarily to an isolated nucleic acid molecule. "Isolated" means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cellular debris and growth media. Generally, the term "isolated" is not intended to mean completely free from these materials or free from water, buffers, or salts, unless they are present in an amount that significantly interferes with experimental or therapeutic uses of the compounds as described herein.

[0078] In a third aspect of the present application, an expression vector is provided, which comprises the nucleic acid molecule of the second aspect of the present application.

[0079] In some embodiments, the expression vector can be obtained by conventional methods in the art, for example, by constructing the nucleic acid molecule of the present application linked to various expression vectors. The expression vector can accommodate the nucleic acid molecule of the present application, for example, the expression vector can include a plasmid, a cosmid, a phage or a viral vector.

[0080] In the present application, unless otherwise specified, "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA technologies are often in the form of plasmids. However, other forms of expression vectors are available (e.g., viral vectors, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0081] In a fourth aspect of the present application, a cell is provided, which comprises the expression vector of the third aspect of the present application.

[0082] As used herein, and unless otherwise indicated, the term "cell" also means "host cell" and refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Bacteria that are readily transformed include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as strains of Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microbial hosts include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0083] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny can not necessarily be identical to the parental cell since there can be, as a result of mutation, known or unknown, deliberate or inadvertent changes. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended to be clearly apparent from the context.

[0084] In a fifth aspect of the application, a method of producing an anti-TLlA antibody is provided, comprising culturing the cell of the fourth aspect of the application, and obtaining the anti-TLlA antibody from the culture.

[0085] In some embodiments, the method of producing an anti-TLlA antibody of the application is obtained by biosynthesis, such as culturing the cell. It is understood by one skilled in the art that, given the amino acid sequence of the anti-TLlA antibody, it can also be obtained by other means, such as solid phase synthesis.

[0086] In a sixth aspect of the application, a pharmaceutical composition is provided, comprising the anti-TLlA antibody of the first aspect of the application and a pharmaceutically acceptable excipient.

[0087] As used herein, and unless otherwise indicated, "pharmaceutically acceptable" means suitable for administration to a patient and commensurate with a reasonable benefit / risk ratio.

[0088] In the present application, "pharmaceutically acceptable excipient" means, unless otherwise indicated, one or a combination of more than one of a surface active agent, a solution stabilizer, an isotonicity adjusting agent or a buffer, but is not limited thereto.

[0089] In a seventh aspect of the present application, there is provided a TL1A detection product comprising the anti-TL1A antibody according to the first aspect of the present application.

[0090] In the present application, the kind of the TL1A detection product is not particularly limited, and for example, it is a diagnostic reagent, a test strip, a detection plate or a kit. In the detection product, the anti-TL1A antibody binds to TL1A in a sample to be measured.

[0091] In an eighth aspect of the present application, there is provided a method for detecting TL1A, comprising reacting the anti-TL1A antibody according to the first aspect of the present application with a sample to be measured.

[0092] In the present application, "reacting" means, unless otherwise indicated, immunobinding, and "immunobinding" means a specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific. The strength or affinity of the immunobinding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction, wherein a smaller value of KD indicates a higher affinity. The immunobinding properties between two molecules can be quantified using methods known in the art. One method involves measuring the rate of formation and dissociation of antigen binding site / antigen complexes.

[0093] The present application relates to a method for immunologically detecting or assaying a target antigen (e.g., TL1A), a reagent for immunologically detecting or assaying a target antigen (e.g., TL1A), a method for immunologically detecting or assaying a cell expressing a target antigen (e.g., TL1A), and a diagnostic agent for diagnosing a disease associated with a cell positive for a target antigen (e.g., TL1A), which comprises an antibody or antibody fragment of the present application that specifically recognizes a target antigen (e.g., TL1A) and binds to an amino acid sequence or a three-dimensional structure of an extracellular region as an active ingredient.

[0094] In the present application, the method for detecting or assaying the amount of a target antigen (e.g., TL1A) can be any known method. For example, it includes an immunological detection or assay method. The immunological detection or assay method is a method for detecting or assaying the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunological detection or assay method include a radioisotope-labeled immunological antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a fluorescent immunoassay (FIA), a luminescent immunoassay, a Western blotting method, a physical-chemical method, etc.

[0095] For detecting the cells expressing the polypeptide, known immunodetection methods can be used, and preferably, immunoprecipitation, fluorescent cell staining, immunohistological staining, etc. can be used. In addition, fluorescent antibody staining using FMAT8100 HTS system (Applied Biosystem) or the like can be used.

[0096] In the present application, the sample to be tested for detecting or assaying the target antigen (e.g., TL1A) is not particularly limited as long as it has a possibility of containing cells expressing the target antigen (e.g., TL1A), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

[0097] The detection product can further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction, according to the desired detection method. The reagent for performing an antigen-antibody reaction includes a buffer, a salt, etc. The reagent for detection includes a reagent generally used for an immunodetection or assay method, such as a labeled second antibody recognizing the monoclonal antibody, an antibody fragment thereof, or a conjugate thereof, and a substrate corresponding to the label, etc.

[0098] In a ninth aspect of the present application, there is provided use of the anti-TL1A antibody of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the cell of the fourth aspect of the present application, or the pharmaceutical composition of the sixth aspect of the present application in the manufacture of a medicament for treating a disease associated with TL1A.

[0099] In a tenth aspect of the present application, there is provided a therapeutic method, which comprises administering to a subject an effective amount of the anti-TL1A antibody or the pharmaceutical composition.

[0100] "Administering", "administered", and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, means the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering", "administered", and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contact of a reagent with the cell, as well as contact of a reagent with a fluid that is in contact with the cell. "Administering", "administered", and "treatment" also mean treatment by a reagent, diagnostic, binding composition, or by another cell in vitro and ex vivo, for example. "Treatment" when applied to a human, veterinary, or research subject, means therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0101] "Treatment" means the administration of an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments of the application, to a patient who has one or more symptoms of a disease, where the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, to the extent that such symptoms are induced to regress or inhibited from progressing to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any particular symptom of a disease (also referred to as a "therapeutically effective amount") can vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired therapeutic effect in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical test method used by a physician or other health care professional to assess the severity or progression of the symptom. While embodiments of the application (e.g., a method of treatment or article of manufacture) can not be effective in alleviating every symptom of a disease of interest, it is determined that a statistically significant number of patients should have alleviation of the disease symptom of interest according to any statistical test method known in the art, such as the Student t-test, the Chi-square test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[0102] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition that is necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include eliminating or reducing risk, lessening severity, or delaying onset of a disease or disorder, including biochemical, histological, and / or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes presenting during development of the disorder. For therapeutic use, beneficial or desired results include clinical results such as decreasing the incidence of various disorders associated with the target antigens of the application, or ameliorating one or more symptoms of the disorder, reducing the dose of other medications required to treat the disorder, enhancing effect of another medication, and / or delaying the progression of the disorder in patients with the target antigens of the application.

[0103] The disease to be treated is typically a TL1A-mediated disease, such as autoimmune diseases, inflammatory bowel disease (ulcerative colitis and Crohn's disease), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, and ankylosing spondylitis, among others.

[0104] Some embodiments are provided below.

[0105] The embodiments of the present application will be described in detail below with reference to the embodiments. It should be understood that these embodiments are only used to illustrate but not to limit the scope of the present application. The experimental methods in the following examples without specified conditions, the priority is given to the guidance given in the present application, but also can be in accordance with the experimental manual or conventional conditions in the art, but also in accordance with the manufacturer's recommended conditions, or reference to the known experimental methods in the art.

[0106] In the following examples, the amount of raw material components involved in the parameter, such as no particular mention, there may be a slight deviation in the range of weighing accuracy. Involving temperature and time parameters, allow the instrument test accuracy or operation accuracy caused by acceptable deviation.

[0107] Example 1

[0108] 1. Immunization

[0109] Using recombinant human and mouse TL1A trimeric protein and stable transfection of CHO-K1 cells (CHO-hTL1A cells) stably transfected with expression vector encoding human TL1A, Balb / c mice and SJL mice were immunized using a variety of strategies. Some mice were immunized with human TL1A protein only, some mice were cross immunized with human TL1A protein and mouse TL1A protein, and some mice were cross immunized with human TL1A protein and CHO-hTL1A cells. The first time of protein immunization used a dose of 50 μg per mouse, and the rest of the time 25 μg per mouse. The amount of CHO-hTL1A cells used for immunization was 1 x 10 7 During the process of immunization, serum samples were obtained by tail vein or retro-orbital blood sampling to monitor the immune response.

[0110] 2. Specific single B cell sorting

[0111] 2.1 Immunocyte collection

[0112] The mice were dissected, and the fresh spleen was taken in 4°C sterile PBS, and the spleen was grinded in serum-free DMEM, and the cell suspension after grinding was filtered with 100 μm filter and centrifuged to collect the cell precipitate. 1-2 ml ACK lysis solution was added to the cells, and the mixture was gently blown and mixed, and then it was placed for 2 min, 20 ml PBS was washed and centrifuged to collect the cells, and the cells were resuspended in PBS containing 3% FBS and counted.

[0113] 2.2 Single B cell sorting

[0114] SA-BV421 and SA-PE-Cy7 labeled Biotinylated Human TL1A trimeric protein, respectively. Mouse splenocyte suspension was added with Mouse BD Fc Block, blocked at 4°C for 5 min; then added with CD4-FITC, CD8a-FITC, GR1-FITC, F4 / 80-FITC, B220-APC-H7, IgD-PE, IgM-PerCP-Cy5.5, IgG1-APC, IgG2a / b-APC, LIVE / DEAD Fixable Aqua, Human TL1A-BV421, Human TL1A-PE-Cy7, Benzonase, incubated at 4°C for 30 min. Cells were washed twice with PBS containing 4% FBS. After fluorescence labeling, single B cells specifically binding to human TL1A protein were sorted into 96-well PCR plates containing cell lysis solution using BD FACSAria™ III flow cytometer. The sorting strategy was: AqVD-Amycan- / CD4- / CD8a- / GR1- / F4 / 80-FITC- / B220-APC-H7+ / IgD-PE- / IgM-PerCP-Cy5.5- / IgG1&IgG2a / b-APC+ / TL1A-BV421+ / TL1A-PE-Cy7+.

[0115] 3. Single B cell antibody gene amplification and analysis

[0116] First, single B cells were reverse transcribed to synthesize cDNA, and then the antibody heavy and light chain genes were amplified.

[0117] The method for reverse transcription to synthesize cDNA: cDNA was synthesized in a 10-μl reaction system in a 96-well PCR plate, 1 μM of OligodT30, 1 μM of TSO Primer, 1 mM of dNTPs, and 100 U of HiScript III Reverse Transcriptase, and the reaction was carried out at 42°C for 1 h on a PCR instrument;

[0118] Nested PCR to amplify antibody IgH, IgK and IgL variable region genes, method: first round of PCR: 20 μl system contains 2 μl of RT reaction product, 10 μl of 2x Phanta Max Master Mix enzyme, 0.25 μM of ISPCR as upstream primer, 0.25 μM of antibody constant region primer mlgHGCR or mlgKCR or mlgLCR as downstream primer. Reaction conditions: pre-denaturation 95℃, 3 min, then 30 cycles of PCR, each cycle: 95℃ x 15 sec, 57℃ x 15 sec, 72℃ x 35 sec, finally extend at 72℃ for 5 min. Second round of PCR: 20 μl system contains 2 μl of first round of PCR reaction product, 10 μl of 2x Phanta Max Master Mix enzyme, 0.25 μM of FR1 region primer mVH-mix or mVκ-mix or mVλ-mix as upstream primer, 0.25 μM of variable region J region primer mJH-mix or mJκ-mix or mJλ-mix as downstream primer. Reaction conditions: pre-denaturation 95℃, 3 min, then 35 cycles of PCR, each cycle: 95℃ x 15 sec, 57℃ x 15 sec, 72℃ x 30 sec, finally extend at 72℃ for 5 min.

[0119] Sequence the antibody genes identified as positive and heavy chain and light chain can be matched pairs, analyzed by IMGT online service (https: / / www.imgt.org / IMGT_vquest / ).

[0120] 4. Production of recombinant antibodies

[0121] The obtained antibody heavy and light chain variable region genes were cloned into expression vectors containing human IgG1 heavy chain constant region and expression vectors containing human kappa light chain constant region, respectively, to prepare antibody expression plasmids. The obtained recombinant plasmids were co-transfected with transfection reagent ExpiFectamine according to the instructions of ExpiCHO™ Expression System Transfection Kit (ThermoFisher, A29133), and ExpiCHO-S cells were cultured in a 37℃, 8% CO2 incubator for 5 days. The supernatant was collected after culture, the cell debris was discarded by centrifugation, and the supernatant was purified by Protein A affinity chromatography. The purified antibody was tested by SDS-PAGE and SEC-HPLC purity detection.

[0122] 5. Characterization of binding and blocking of chimeric antibodies

[0123] 5.1 Chimeric antibody affinity detection

[0124] The affinities of anti-TL1A chimeric antibodies binding to human TL1A protein and monkey TL1A protein were detected by Biacore 8K. Each antibody was diluted to 1 μg / ml with EP+Buffer buffer; each antigen was diluted to 2.5, 1.25, 0.625, 0.3125, 0.156, 0 with EP+Buffer buffer; wherein the main method parameters are as follows: Capture: 30 s, Association: 120 s, Dissociation: 600 s, Regeneration: Glycine 1.5, 30 s; the experimental data were processed using Biacore 8K Evaluation Software, and the kinetics fitting was performed using a 1:1 model to calculate the association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD). The results are shown in Table 2, and all antibodies bind to human and monkey TL1A with high affinity.

[0125] Table 2: Binding affinities of anti-TL1A chimeric antibodies

[0126]

[0127] 5.2 Anti-TL1A antibodies block TL1A-DcR3 binding

[0128] 96-well plates were coated with Human DcR3 Protein (1 μg / ml) diluted in coating buffer (0.05 M carbonate, pH 9.6) at 50 μl per well at 4°C overnight; wash the plates 3 times with 250 μl / well PBST and tap off the residual liquid; add 100 μl of 5% skim milk to each well, and block at room temperature for 1 h, then tap off the residual liquid; add 25 μl of antibody (initial concentration 180 μg / ml, 3-fold dilution, a total of 7 concentration gradient points) diluted with PBS to the corresponding wells; add 25 μl of ligand (concentration 5 μg / ml, final concentration 2.5 μg / ml) diluted with 3% BSA to the corresponding wells, and incubate at 37°C for 30 min; wash the plates 3 times with 250 μl / well PBST and tap off the residual liquid; add 50 μl of HRP secondary antibody (diluted with PBS, antibody dilution ratio 1:3000) to each well, and incubate at room temperature for 1 h; wash the plates 4 times with 250 μl / well PBST and tap off the residual liquid; add 50 μl of TMB color developing solution to each well, and react at room temperature for 10 min; add 50 μl of stop solution to each well to stop the color development, and read the OD450 value. The results are shown in Figures 5A and 5B, and similar to the reference antibody, all antibodies do not block TL1A-DcR3 at low antibody concentration (3 μg / mL). Figure 1

[0129] 5.3 Binding specificity of anti-TL1A antibodies against TL1A and homologs​

[0130] Human TL1A Protein and homologous proteins (TNF-β, TNF-α, Fas Ligand, TRAIL, LIGHT) were coated in 96-well enzyme-labeled plates, 1 μg / mL, 50 μl / well, 4°C overnight incubation, the first four rows were coated with homologous proteins, and the last four rows were coated with Human TL1A Protein; after washing with PBST for 3 times, 100 μl of 3% BSA blocking solution was added, and incubated at 37°C for 1 h; after washing with PBST for 3 times, the diluted antibody to be tested (100 ng / μl) was added, and incubated at 37°C for 1 h; after washing with PBST for 4 times, 50 μl of Anti-human IgG HRP (1:10000 dilution) was added, and incubated at 37°C for 1 h; after washing with PBST for 4 times, 50 μl of TMB color developing liquid was added, and developed at 37°C for 5 min, 50 μl of ELISA stop solution was added per well to stop the reaction, and the absorbance value was determined at 450 nm wavelength by an enzyme-labeled instrument. The results are shown in Figure 2 All antibodies do not cross-bind with homologous proteins TNF-β, TNF-α, Fas Ligand, TRAIL, and LIGHT.

[0131] 6. Epitope mapping of anti-TL1A antibodies

[0132] The antigen human TL1A protein was diluted to 2 μg / ml with PBST buffer; each antibody was diluted to 100 nM with PBST buffer; the secondary antibody was diluted to 100 nM with PBST buffer; the diluted antigen, antibody, Regeneration Buffer, and Kinetics buffer were added to a 96-well sample plate; the HIS1K probe and sample plate were placed in the corresponding positions in the instrument; after setting the sample, probe, method, and file information using Octet BLI discovery software, the method was run for affinity detection. The main method parameters are as follows: Baseline1: 30 s, Baseline2: 30 s, Antigen Association: 0.2 nm, Antibody1 Association: 200 s, Antibody2 Association: 200 s; the experimental data were processed using OctetAnalysis Studio, and the antibody epitope grouping was analyzed. The results are shown in Figure 3 CR20M0007 cross-reacts with RVT-3101 and does not cross-react with PRA023; CR20M0008, CR20M0055, and CR20M0666 cross-react with RVT-3101 and partially cross-react with PRA023; CR20M0203 does not cross-react with RVT-3101 and PRA023.

[0133] 7. Antibody humanization

[0134] In order to reduce the immunogenicity of the murine antibodies, the antibodies CR20M0007, CR20M0008, CR20M0055, CR20M0203, CR20M0666 were humanized. The strategy for antibody humanization was to select human germline antibody sequences according to homology, to perform CDR grafting and back mutations, to replace the murine antibody constant region with a human antibody constant region, and to obtain a humanized antibody.

[0135] IGHV3-23*04 / IGHJ6*01 was selected as the CR20M0007 humanized VH template, IGKV1-39*01 / IGKJ4*01 was selected as the CR20M00007 humanized VL template, CDR grafting and back mutation were performed to obtain the CR20M0007hz13 humanized antibody variable region. The sequence of the humanized heavy chain variable region of CR20M0007hz13 is shown as SEQ ID NO: 1; the sequence of the humanized light chain variable region is shown as SEQ ID NO: 2. IGHV4-38-2*01 / IGHJ6*01 was selected as the CR20M0008 humanized VH template, IGKV1-33*01 / IGKJ2*01 was selected as the CR20M0008 humanized VL template, CDR grafting and back mutation were performed to obtain the CR20M0008hz1 humanized antibody variable region. The sequence of the humanized heavy chain variable region of CR20M0008hz1 is shown as SEQ ID NO: 3; the sequence of the humanized light chain variable region is shown as SEQ ID NO: 4. IGHV1-69*02 / IGHJ6*01 was selected as the CR20M0055 humanized VH template, IGKV1-33*01 / IGKJ4*01 was selected as the CR20M0055 humanized VL template, CDR grafting and back mutation were performed to obtain the CR20M0055hz2 humanized antibody variable region. The sequence of the humanized heavy chain variable region of CR20M0055hz2 is shown as SEQ ID NO: 5; the sequence of the humanized light chain variable region is shown as SEQ ID NO: 6. IGHV3-7*01 / IGHJ6*01 was selected as the CR20M0203 humanized VH template, IGKV4-1*01 / IGKJ4*01 was selected as the CR20M0203 humanized VL template, CDR grafting and back mutation were performed to obtain the CR20M0203hz4 humanized antibody variable region. The sequence of the humanized heavy chain variable region of CR20M0203hz4 is shown as SEQ ID NO: 7; the sequence of the humanized light chain variable region is shown as SEQ ID NO: 8. IGHV1-69*02 / IGHJ1*01 was selected as the CR20M0666 humanized VH template, IGKV1-33*01 / IGKJ2*01 was selected as the CR20M0666 humanized VL template, CDR grafting and back mutation were performed to obtain the CR20M0666hz15 humanized antibody variable region. The sequence of the humanized heavy chain variable region of CR20M0666hz15 is shown as SEQ ID NO: 9; the sequence of the humanized light chain variable region is shown as SEQ ID NO: 10.

[0136] >SEQ ID NO: 1

[0137] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWARQAPGKRLEWVAAIRSGGSYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIYDSTGTMDYWGQGTTVTVSS

[0138] SEQ ID NO: 11

[0139] HCDR1: SYGMS

[0140] SEQ ID NO: 12

[0141] HCDR2: AIRSGGSYTYYPDSVKG

[0142] SEQ ID NO: 13

[0143] HCDR3: YDSTGTMDY

[0144] >SEQ ID NO: 2

[0145] DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGDTLPWTFGGGTKVEIK

[0146] SEQ ID NO: 14

[0147] LCDR1: RASQDISNYLN

[0148] SEQ ID NO: 15

[0149] LCDR2: YTSRLHS

[0150] SEQ ID NO: 16

[0151] LCDR3: QQGDTLPWT

[0152] >SEQ ID NO: 3

[0153] QVQLQESGPGLVKPSETLSLTCAVSGYSISSNFYANWIRQPPGKKLEWMGYISYEGTNDYNPFLRSRITISRDTSKNQFSLKLSSVTAADTAVYYCARQRLRDVMDYWGQGTTVTVSS

[0154] SEQ ID NO: 17

[0155] HCDR1 : SNFYAN

[0156] SEQ ID NO: 18

[0157] HCDR2: YISYEGTNDYNPFLRS

[0158] SEQ ID NO: 19

[0159] HCDR3: QRLRDVMDY

[0160] >SEQ ID NO: 4

[0161] DIQMTQSPSSLSASVGDRVTITCKAGQDVSTAVAWYQQKPGKAPKLLIYSTSFRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQGTKLEIK

[0162] SEQ ID NO: 20

[0163] LCDR1 : KAGQDVSTAVA

[0164] SEQ ID NO: 21

[0165] LCDR2: STSFRYT

[0166] SEQ ID NO: 22

[0167] LCDR3: QQHYTTPLT

[0168] >SEQ ID NO: 5

[0169] QVQLVQSGAEVKKPGSSVKVSCKASGYTFSNYWMHWVRQAPGQGLEWMGEITPSTGRSSYDEKFKSRATLTVDKSTSTAYMELSSLRSEDTAVYYCARLDYFGYTLDYWGQGTTVTVSS

[0170] SEQ ID NO: 23

[0171] HCDR1 : NYWMH

[0172] SEQ ID NO: 24

[0173] HCDR2: EITPSTGRSSYDEKFKS

[0174] SEQ ID NO: 25

[0175] HCDR3: LDYFGYTLDY

[0176] >SEQ ID NO: 6

[0177] DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSASYRFTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPYTFGGGTKVEIK

[0178] SEQ ID NO: 26

[0179] LCDR1: KASQDVSTAVA

[0180] SEQ ID NO: 27

[0181] LCDR2: SASYRFT

[0182] SEQ ID NO: 28

[0183] LCDR3: QQHYTTPYT

[0184] >SEQ ID NO: 7

[0185] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHDMSWVRQAPGRRLEWVATISGGGRSTDYVDSVKGRFTISRDNVENSLYLQMNSLRAEDTAVYYCSRHAHYGGAYDYAMDYWGQGTTVTVSS

[0186] SEQ ID NO: 29

[0187] HCDR1: SHDMS

[0188] SEQ ID NO: 30

[0189] HCDR2: TISGGGRSTDYVDSVKG

[0190] SEQ ID NO: 31

[0191] HCDR3: HAHYGGAYDYAMDY

[0192] >SEQ ID NO: 8

[0193] DIVMTQSPDSLAVSLGERATINCKSSQSLFHSGTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLWTFGGGTKVEIK

[0194] SEQ ID NO: 32

[0195] LCDR1: KSSQSLFHSGTRKNYLA

[0196] SEQ ID NO: 33

[0197] LCDR2: WASTRES

[0198] SEQ ID NO: 34

[0199] LCDR3: KQSYNLWT

[0200] >SEQ ID NO: 9

[0201] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDFYMNWVRQAPGQGLEWMGEINPSTGGTTYNQKFKARATLTVDKSTSTAYMELSSLRSEDTAVYYCARERAQAWFAYWGQGTLVTVSS

[0202] SEQ ID NO: 35

[0203] HCDR1: DFYMN

[0204] SEQ ID NO: 36

[0205] HCDR2: EINPSTGGTTYNQKFKA

[0206] SEQ ID NO: 37

[0207] HCDR3: ERAQAWFAY

[0208] >SEQ ID NO: 10

[0209] DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVVWYQQKPGKAPKLLIYSASYRYIGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPLTFGQGTKLEIK

[0210] SEQ ID NO: 38

[0211] LCDR1: KASQDVSTAVV

[0212] SEQ ID NO: 39

[0213] LCDR2: SASYRYI

[0214] SEQ ID NO: 40

[0215] LCDR3: QQHYSTPLT

[0216] 8. Affinity and biological activity characterization of humanized antibodies

[0217] 8.1 Affinity detection of anti-TL1A humanized antibodies

[0218] The affinity of anti-human TL1A antibodies in this application to bind human TL1A protein was detected by Biacore 8K. The antibodies to be tested were captured by Protein A chip (cytiva, 29127556), flowed through TL1A antigen (TLA-H5243) and diluted HBS-EP+ Buffer (cytiva, BR100669), and the binding and dissociation curves were obtained by detecting the reaction signal. The data were fitted by BIAevaluation, and the affinity values were calculated, as shown in Table 3. The results showed that the humanized antibodies in this application all bound to human TL1A with high affinity.

[0219] Table 3: Affinity results of antibodies binding to human TL1A

[0220]

[0221] 8.1 Anti-human TL1A antibodies block the binding of human TL1A to DR3 cells

[0222] The effect of anti-human TL1A antibodies in this application on blocking the binding of TL1A to DR3 cells was detected by FACS. DR3 overexpressing HEK293 cells were diluted with DPBS containing 3% FBS, and the density was adjusted to 0.5x10 6Jurkat-hDR3-Luc cells were diluted with RPMI 1640 containing 1% FBS to a density of 4 x 105cells / ml, and added to a 96-well plate at 50 μl / well. 25 μl of gradient-diluted anti-TL1A antibodies or controls (starting concentration of 30 μg / ml, 3-fold gradient dilution) were added to each well, followed by 25 μl of 0.5 μg / ml Biotinylated Human TL1A, and incubated at 37°C for 5 h. 50 μl of color developing solution (Biyuntian, RG052M) was added to each well, shaken to mix, and incubated at room temperature in the dark for 5-10 min. The LUM luminescence was detected using a SpectraMax i3X microplate reader, and the IC50value of the antibody for inhibiting human TL1A-induced activation of the DR3 downstream signal in Jurkat-hDR3-Luc cells was calculated. The results are shown in Table 5 and Figure 4 Table 5: Activity of antibodies in inhibiting TL1A binding to DR3 cells Figure 4 Table 5: Activity of antibodies in inhibiting TL1A binding to DR3 cells Figure 4 Table 5: Activity of antibodies in inhibiting TL1A binding to DR3 cells Figure 4 Table 5: Activity of antibodies in inhibiting TL1A binding to DR3 cells Figure 4 Table 5: Activity of antibodies in inhibiting TL1A binding to DR3 cells

[0223] Table 5: Activity of antibodies in inhibiting TL1A binding to DR3 cells

[0224]

[0225] 8.2 Inhibition of NFκB by anti-TL1A antibodies in Jurkat-hDR3-Luc cells

[0226] Jurkat-hDR3-Luc cells were diluted with RPMI 1640 containing 1% FBS to a density of 4 x 105 6 Jurkat-hDR3-Luc cells were diluted with RPMI 1640 containing 1% FBS to a density of 4 x 105 Figure 5 Table 5: Activity of antibodies in inhibiting TL1A binding to DR3 cells Figure 5 Table 5: Activity of antibodies in inhibiting TL1A binding to DR3 cells Figure 5 Table 5: Activity of antibodies in inhibiting TL1A binding to DR3 cells Figure 5 Table 5: Activity of antibodies in inhibiting TL1A binding to DR3 cellsFigure 5 As shown in Example E, the antibodies in the present application can significantly inhibit human TL1A-induced Jurkat-hDR3-Luc cell DR3 downstream signal activation.

[0227] Table 5: TL1A antibody inhibits NFKB

[0228]

[0229] 8.3 Anti-human TL1A antibody inhibits human TL1A-induced CD4+ T cell factor IFNy production

[0230] CD4+ T cells were extracted from the resuscitated overnight PBMC cells using EasySep™ Human CD4+ T Cell Isolation Kit (Stemcell, 17952). The extracted CD4+ T cells were washed once with assay buffer (1640 + 10% FBS) and adjusted the cell density to 1 x 10 6 6 cells / ml, added to 96-well U-bottom plate at 50 μl / well. 25 μl 1 ng / ml IL12 solution, 25 μl 25 ng / ml IL18 solution, 50 μl 40 ng / ml IL18 solution were added to each well. 50 μl gradient-diluted anti-TL1A antibodies in the present application or controls (starting concentration at 15 μg / ml, 3-fold gradient dilution) were added to each well, incubated at 37°C for 24 h. The content of IFNy in the cell culture supernatant was detected using Human Interferon-γ ELISA Kit (Acro, CRS-A017), and the IC50 value of the antibody inhibiting human TL1A-induced CD4+ T cell secretion of IFNy was calculated. The results are shown in Table 5. Figure 6 Example A, Figure 6 Example B, Figure 6 Example C, Figure 6 Example D, and Figure 6 As shown in Example E, the antibodies in the present application can significantly inhibit human TL1A-induced CD4+ T cell factor IFNy production.

[0231] Each technical feature of the above-described embodiments can be combined arbitrarily, and for the sake of brevity, all possible combinations of each technical feature in the above-described embodiments are not described, however, as long as the combination of the technical features does not exist contradictory, it should be considered as the scope of the present disclosure.

[0232] The above embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the patent scope of the application. It should be pointed out that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description and drawings can be used to explain the scope of the claims.

Claims

1. An anti-TLl A antibody, characterized in that, It comprises a heavy chain variable region and a light chain variable region, wherein, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 11-13, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 14-16, respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 17-19, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 20-22, respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 23-25, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 26-28, respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 29-31, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 32-34, respectively; or, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 35-37, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 38-40, respectively.

2. The anti-TLl A antibody of claim 1, wherein, the heavy chain variable region comprises a heavy chain variable region framework region FR, and the light chain variable region comprises a light chain variable region framework region FR, wherein the heavy chain variable region framework region FR is a human-derived or murine-derived heavy chain variable region framework region, and / or the light chain variable region framework region FR is a human-derived or murine-derived light chain variable region framework region.

3. The anti-TLl A antibody of claim 2, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; or, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; or, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6; or, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8; or, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

10.

4. The anti-TLl A antibody according to any one of claims 1 to 3, characterized in that, The anti-TL1A antibody is a monoclonal antibody.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-TL1A antibody according to any one of claims 1-4.

6. An expression vector, characterized by, The expression vector comprises the nucleic acid molecule according to claim 5.

7. A cell, characterized in that, The cell comprises the expression vector according to claim 6.

8. A method of preparing the anti-TLl A antibody according to any one of claims 1 to 4, characterized in that, It comprises culturing the cell according to claim 7, and obtaining the anti-TLl A antibody from the culture.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the anti-TLl A antibody according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient.

10. A TLlA detection product characterized by, The TLl A detection product comprises the anti-TLl A antibody according to any one of claims 1 to 4.

11. Use of the anti-TLl A antibody according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the cell according to claim 7 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating a disease associated with TLl A; the disease associated with TLl A is one or more of inflammatory bowel disease, psoriasis, psoriatic arthritis, primary biliary cirrhosis, systemic lupus erythematosus and ankylosing spondylitis.

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