Anti-TL1A antibody and its preparation method and application

Abstract

The present application discloses an anti-TL1A antibody in the field of biomedicine technology and its preparation method and application. It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 7 to 9; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 10 to 12. The anti-TL1A antibody has better biological activity characterization and its epitope is different from that of traditional anti-TL1A antibodies.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to an anti-TL1A antibody and a preparation method and application thereof. Background Art

[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 tumor necrosis factor superfamily (TNFSF) of ligands 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 a stable trimer through TNF homology domain (THD) interactions. TL1A is mainly expressed in a membrane-bound form (mTL1A) to form a stable trimer, which is subsequently cleaved by alternative splicing or metalloproteinases such as TNF-α converting enzyme (TACE) to produce soluble TL1A (sTL1A).

[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), providing stimulation signals for downstream signaling pathways, thereby regulating the proliferation, activation, apoptosis and production of cytokines and chemokines 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 are able to produce both transmembrane and soluble forms of DR3. In resting T cells, DR3 is expressed in a soluble form, which protects cells from apoptosis. Activated T cells express transmembrane DR3, which activates receptors, leading to apoptosis or activation of transcription factors such as NF-κB. sTL1A binds to DR3 (membrane-bound form) to activate downstream signaling cascades, thereby regulating immune responses and inflammation. The sTL1A / DR3 interaction can trigger two different signaling pathways, causing inflammation and apoptosis, respectively.

[0005] Inflammatory bowel diseases (IBD) are a group of chronic, relapsing, inflammatory intestinal disorders, which are typically characterized by enteritis and epithelial damage. Severe cases can lead to hospitalization and surgery, with a low mortality rate, but lifelong treatment is required. IBD mainly includes Crohn's disease (CD) and ulcerative colitis (UC), which have both overlapping and different clinical and pathological features. In patients with IBD, the expression of TL1A in serum and intestinal tissue is increased and correlated with disease activity. The expression of DR3 in the intestinal lamina propria lymphocytes, especially T cells, is also increased in these patients. In addition, studies have shown that TL1A in ileitis mice is mainly expressed on CD11c high dendritic cells in the ileum wall. The interaction between TL1A expressed on DCs and DR3 expressed on lymphocytes may play an important role in the pathogenesis of IBD by promoting excessive secretion of interferon-γ (IFN-γ). TL1A upregulates chemokine receptors leading to enhanced function of bone marrow-derived dendritic cells (BMDCs), including TL1A-dependent stimulation of T cells in BMDCs, which exacerbates dextran sulfate sodium (DSS)-induced colitis. The TL1A / DR3 pathway has emerged as an important module of mucosal immunity, which is involved in maintaining intestinal homeostasis but is also critically involved in the development and maintenance of chronic inflammatory responses that occur 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 are upregulated, and TL1A / DR3 signaling promotes disease progression.

[0006] Increased TL1A expression and / or TL1A gene polymorphisms have been implicated in the pathogenesis of various autoimmune and inflammatory diseases. Analysis of mouse models of autoimmune diseases and TL1A or DR3 transgenic mice has shown that TL1A-DR3 interactions play an important role in local inflammation in T cell-dependent autoimmune diseases. Therefore, TL1A links innate immune responses with adaptive immune responses and plays a key role in the induction of autoimmune and inflammatory diseases. These suggest that inhibition of TL1A-DR3 interactions is an effective therapeutic strategy to improve local inflammation in target organs of individuals with autoimmune diseases. In addition to inflammatory bowel diseases (including ulcerative colitis and Crohn's disease), inhibition of TL1A-DR3 may also be a therapeutic strategy for diseases such as psoriasis, psoriasis, primary biliary cirrhosis, as well as systemic lupus erythematosus and ankylosing spondylitis.

[0007] Therefore, it is necessary to develop anti-TL1A antibodies to meet clinical needs. Summary of the invention

[0008] Based on this, it is necessary to provide an anti-TL1A antibody and 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 amino acid sequences as shown in SEQ ID NOs: 7 to 9; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 10 to 12.

[0010] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2.

[0011] In some embodiments, the anti-TL1A antibody is a monoclonal antibody.

[0012] The second aspect of the present application provides a nucleic acid molecule, which encodes the anti-TL1A antibody according to the first aspect of the present application.

[0013] The third aspect of the present application provides an expression vector, which comprises the nucleic acid molecule described in the second aspect of the present application.

[0014] The fourth aspect of the present application provides a cell, which comprises the expression vector described in the third aspect of the present application.

[0015] The fifth aspect of the present application provides a method for preparing the anti-TL1A antibody described in the first aspect of the present application, comprising culturing the cell described in the fourth aspect of the present application, and obtaining the anti-TL1A antibody from the culture.

[0016] The sixth aspect of the present application provides a pharmaceutical composition, which comprises the anti-TL1A antibody described in the first aspect of the present application and a pharmaceutically acceptable excipient.

[0017] The seventh aspect of the present application provides a TL1A detection product, wherein the TL1A detection product comprises the anti-TL1A antibody described in the first aspect of the present application.

[0018] The eighth aspect of the present application provides a method for detecting TL1A, comprising reacting the anti-TL1A antibody described in the first aspect of the present application with a sample to be tested.

[0019] The ninth aspect of the present application provides the use of the anti-TL1A antibody described in the first aspect of the present application, the nucleic acid molecule described in the second aspect of the present application, the expression vector described in the third aspect of the present application, the cell described in the fourth aspect of the present application, or the pharmaceutical composition described in the sixth aspect of the present application in the preparation of a drug for treating a disease associated with TLA1.

[0020] The aforementioned anti-TL1A antibodies have better characterized biological activities and different epitopes from conventional anti-TL1A antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0022] Figure 1 In one embodiment of the present application, the anti-TL1A antibody blocks the binding of TL1A to DR3 cells;

[0023] Figure 2 In one embodiment of the present application, NFκB is inhibited by anti-TL1A antibody in Jurkat-hDR3-Luc cells;

[0024] Figure 3 In one embodiment of the present application, the anti-TL1A antibody inhibits the production of TL1A-induced CD4+T cell factor IFNγ;

[0025] Figure 4 Mapping the epitope of the anti-TL1A antibody in one embodiment of the present application;

[0026] Figure 5 In one embodiment of the present application, an anti-TL1A antibody blocks the binding of TL1A to DcR3;

[0027] Figure 6 The anti-TL1A antibody in one embodiment of the present application has binding specificity to TL1A and homologues. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0030] The terms "having", "containing", "including" and "comprising" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0031] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0032] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0033] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form a new technical solution.

[0034] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0035] The embodiments of the present application at least provide an anti-TL1A antibody and a preparation method and application thereof.

[0036] In some embodiments, the present application screens out a monoclonal antibody with better biological activity than competing antibodies through single B cell cloning technology. Balb / c mice and SJL mice were immunized with recombinantly expressed Human TL1A Protein, and serum titer was detected. After three immunizations, the spleen was taken, spleen cells were separated, and single specific B cells were separated by FACS sorting technology. Using PCR amplification technology, the antibody gene was cloned for sequencing, and the expression vector was constructed after initial screening of the linear expression transfection supernatant, and the chimeric antibody was expressed and purified. By screening the pure antibody, an antibody CR20M0681 was obtained. The epitope of CR20M0681 is different from that of the competing antibodies (RVT-3101, PRA023), and it shows significantly better activity than the competing antibodies in the characterization of biological activity (blocking, NF-κB signaling pathway, and IFNγ cytokine inhibition).

[0037] In this application, the amino acid sequences of the screened antibody CR20M0681 and competing antibodies (RVT-3101, PRA023) are shown in Table 1 below:

[0038] Table 1

[0039]

[0040] 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 with amino acid sequences as shown in SEQ ID NOs: 7 to 9; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 10 to 12.

[0041] In this application, unless otherwise specified, "TL1A" refers to a type II transmembrane protein that exists primarily in membrane-bound (mTL1A) or soluble (sTL1A) forms, including variants, homologs, orthologs and paralogs.

[0042] In the present application, unless otherwise specified, "anti-TL1A antibody" refers to an antibody or protein that specifically binds to TL1A. "Specific binding" refers to the binding of an antibody to an epitope on a predetermined antigen.

[0043] In this application, unless otherwise specified, "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 in inhibiting a specific 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 the 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.

[0044] In this application, unless otherwise specified, "antibody" or "Ab" refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, generally referring to a Y-shaped tetrameric protein comprising two heavy chains (H) and two light chains (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chain of an antibody can be divided into κ or λ light chains. The heavy chain can be divided into μ, δ, γ, α or ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA or IgE, respectively. In the light chain and heavy chain, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementary determining regions, referred to as CDRs) separated by relatively conserved regions (called framework regions, referred to as FRs). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites / parts, respectively. The distribution of amino acids in various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT or Chothia. In the specific embodiments of the present application, the CDR sequences are determined using the numbering definitions in the Kabat system.

[0045] In this application, unless otherwise specified, "epitope" refers to the position on the surface of an antigen molecule that binds to a single antibody molecule. 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. When an antibody recognizes the same or spatially overlapping epitopes as a reference antibody, the antibody binds to the "substantially the same epitope" as the reference antibody. The most commonly used and rapid method to determine whether two epitopes bind to the same or spatially overlapping epitopes is a competitive test, which can be configured in various forms with labeled antigens or labeled antibodies, for example, radioactive labels or enzyme labels can be used to determine the ability of unlabeled antibodies to block the binding of labeled antibodies.

[0046] 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, biclonal antibodies, multispecific antibodies, chimeric antibodies, and humanized antibodies.

[0047] In this application, unless otherwise specified, "monoclonal antibody" or "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody molecule preparation with a single molecular composition. The monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.

[0048] In this application, unless otherwise specified, "chimeric antibody" refers to an antibody having at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from one species; and at least a portion of a constant region derived from another species. For example, a chimeric antibody may comprise a murine variable region and a human constant region. "Humanized antibody" refers to an antibody containing a complementarity determining region (CDR) derived from a non-human antibody, and a framework region and a constant region derived from a human antibody.

[0049] In some embodiments, the anti-TL1A antibody of the present application comprises a human IgG1 heavy chain constant region and a human Kappa light chain constant region.

[0050] In some embodiments, the amino acid sequence of the anti-TL1A antibody heavy chain variable region is as shown in SEQ ID NO: 1 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: 1.

[0051] In some embodiments, the amino acid sequence of the anti-TL1A antibody light chain variable region is as shown in SEQ ID NO: 2 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: 2.

[0052] In the present application, if not otherwise specified, "identity" refers to the percentage of amino acid residues in the first sequence that are identical to the amino acid residues in the second sequence when comparing amino acid sequences (introducing gaps if necessary) to achieve maximum sequence identity percentage, and not considering any conservative substitutions as part of sequence identity. To measure the amino acid sequence identity percentage, the alignment can be achieved in a variety of ways within the scope of 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 the parameters suitable for measuring the alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequence.

[0053] In the second aspect of the present application, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the anti-TL1A antibody described in the first aspect of the present application.

[0054] In this application, unless otherwise specified, "nucleic acid molecules" refer primarily to isolated nucleic acid molecules. "Isolated" refers to molecules that are substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to refer to the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds described herein.

[0055] In the third aspect of the present application, an expression vector is provided, wherein the expression vector comprises the nucleic acid molecule described in the second aspect of the present application.

[0056] In some embodiments, the expression vector can be obtained by conventional methods in the art, for example, by connecting the nucleic acid molecules described in the present application to various expression vectors. The expression vector can accommodate the nucleic acid molecules described in the present application, for example, the expression vector can include a plasmid, a cosmid, a phage or a viral vector.

[0057] In the present application, if there is no other explanation, "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which another DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors with bacterial replication origins and free mammalian vectors). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, certain vectors can guide the expression of genes to which they are effectively connected. This vector is referred to herein as a "recombinant expression vector" (or simply "expression vector"). Generally, expression vectors useful in recombinant DNA technology are usually present in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (for example, replication-defective retroviruses, adenoviruses and adeno-associated viruses), which play equivalent functions.

[0058] In the fourth aspect of the present application, a cell is provided, wherein the cell comprises the expression vector described in the third aspect of the present application.

[0059] In this application, unless otherwise specified, "cell" or "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plants or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms 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.

[0060] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of passages. It should also be understood that all progeny may not be exactly identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, this is clear from the context.

[0061] In the fifth aspect of the present application, a method for preparing an anti-TL1A antibody is provided, which comprises culturing the cell described in the fourth aspect of the present application and obtaining the anti-TL1A antibody from the culture.

[0062] In some embodiments, the method of preparing anti-TL1A antibodies of the present application is obtained by biosynthesis, such as culturing the cells. Those skilled in the art will appreciate that, if the amino acid sequence of the anti-TL1A antibody is known, it can also be obtained by other methods such as solid phase synthesis.

[0063] In the sixth aspect of the present application, a pharmaceutical composition is provided, comprising the anti-TL1A antibody described in the first aspect of the present application and a pharmaceutically acceptable excipient.

[0064] In this application, unless otherwise specified, "pharmaceutically acceptable" means suitable for administration to patients within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0065] In the present application, unless otherwise specified, "pharmaceutically acceptable excipient" refers to one or a combination of surfactants, solution stabilizers, isotonicity regulators or buffers, including but not limited to.

[0066] In the seventh aspect of the present application, a TL1A detection product is provided, comprising the anti-TL1A antibody described in the first aspect of the present application.

[0067] In the present application, the type of TL1A detection product is not particularly limited, for example, a diagnostic reagent, a test strip, a detection plate or a test kit. In the detection product, the anti-TL1A antibody binds to TLIA in the test product.

[0068] In an eighth aspect of the present application, a method for detecting TL1A is provided, comprising reacting the anti-TL1A antibody described in the first aspect of the present application with a sample to be tested.

[0069] In this application, unless otherwise specified, "reaction" refers to immunological binding, and "immunological binding" refers to 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 immunological binding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction, wherein the smaller the KD value, the higher the affinity. The immunological binding properties between two molecules can be quantified using methods known in the art. One method involves measuring the speed of formation and dissociation of antigen binding sites / antigen complexes.

[0070] The present application relates to a method for immunodetection or determination of a target antigen (e.g., TL1A), a reagent for immunodetection or determination of a target antigen (e.g., TL1A), a method for immunodetection or determination of cells expressing a target antigen (e.g., TL1A), and a diagnostic agent for diagnosing a disease associated with target antigen (e.g., TL1A)-positive cells, which comprises the antibody or antibody fragment of the present application that specifically recognizes the target antigen (e.g., TL1A) and binds to the amino acid sequence of the extracellular region or its three-dimensional structure as an active ingredient.

[0071] In the present application, the method for detecting or measuring the amount of the target antigen (e.g., TL1A) may be any known method. For example, it includes an immunodetection or determination method. An immunodetection or determination method is a method for detecting or determining the amount of an antibody or an antigen using a labeled antigen or antibody. Examples of immunodetection or determination methods include radioactive substance labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, protein immunoblotting, physicochemical methods, and the like.

[0072] To detect cells expressing the polypeptide, known immunodetection methods can be used, and preferably immunoprecipitation, fluorescent cell staining, immunohistostaining, etc. In addition, fluorescent antibody staining using FMAT8100 HTS system (Applied Biosystem) can be used.

[0073] In the present application, there is no particular limitation on the sample to be tested for detecting or measuring the target antigen (e.g., TL1A), as long as it has the 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.

[0074] Depending on the desired detection method, the test product may also contain reagents for performing antigen-antibody reactions or reagents for detecting reactions. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include reagents commonly used in immunoassays or assays, such as a labeled second antibody that recognizes the monoclonal antibody, its antibody fragment, or its conjugate, and a substrate corresponding to the label, etc.

[0075] In the ninth aspect of the present application, provided is a use of the anti-TL1A antibody described in the first aspect of the present application, the nucleic acid molecule described in the second aspect of the present application, the expression vector described in the third aspect of the present application, the cell described in the fourth aspect of the present application, or the pharmaceutical composition described in the sixth aspect of the present application in the preparation of a drug for treating a disease associated with TLA1.

[0076] In the tenth aspect of the present application, a treatment method is provided, comprising administering an effective dose of the anti-TL1A antibody or the pharmaceutical composition to a subject.

[0077] "Administering," "giving," and "treating" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0078] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments of the present application, to a patient who has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these 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 induce regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptom. Although the embodiments of the present application (e.g., treatment methods or products) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0079] "Effective amount" or "effective dose" refers to the amount of a drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For preventive use, beneficial or desired results include eliminating or reducing risk, reducing severity or delaying the onset of a condition, including biochemical, histological and / or behavioral symptoms of the condition, its complications and intermediate pathological phenotypes presented during the development of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related conditions of the present application or improving one or more symptoms of the condition, reducing the dose of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition of the present application in the patient.

[0080] Diseases treated are typically TL1A-mediated diseases, such as autoimmune diseases, inflammatory bowel disease (ulcerative colitis and Crohn's disease), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, and ankylosing spondylitis.

[0081] Some examples are provided below.

[0082] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0083] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0084] Example 1

[0085] 1. Immunization

[0086] Balb / c mice and SJL mice were immunized using various strategies using recombinant human and mouse TL1A trimer protein and CHO-K1 cells stably transfected with an expression vector stably encoding human TL1A (CHO-hTL1A cells). Some mice were immunized with human TL1A protein alone, 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 protein was used at a dose of 50 μg / mouse for the first immunization and 25 μg / mouse for the remaining immunizations. The amount of CHO-hTL1A cells used for immunization was 1×10 7 During the immunization process, serum samples were collected by tail vein or retro-orbital bleeding to monitor the immune response.

[0087] 2. Specific single B cell sorting

[0088] 2.1 Immune cell collection

[0089] Dissect the mouse, take fresh spleen and place it in sterile PBS at 4℃, grind the spleen in serum-free DMEM, filter the ground cell suspension with a 100μm filter and collect the cell precipitate by centrifugation. Add 1-2mL ACK lysis buffer to the cells and gently pipette to mix, let stand for 2min, wash with 20mL PBS and collect the cells by centrifugation, resuspend the cells with PBS containing 3% FBS and count them.

[0090] 2.2 B cell sorting

[0091] Biotinylated Human TL1A trimer protein was labeled with SA-BV421 and SA-PE-Cy7, respectively. Mouse BD Fc Block was added to the mouse spleen cell suspension and blocked at 4°C for 5 minutes; 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, and Benzonase were then added and incubated at 4°C for 30 minutes. The cells were washed twice with PBS containing 4% FBS. After fluorescent labeling, single B cells that specifically bound to Human TL1A protein were sorted into a 96-well PCR plate containing cell lysate using a 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+.

[0092] 3. Single B cell antibody gene amplification and analysis

[0093] First, single B cells are reverse transcribed to synthesize cDNA, and then the antibody heavy and light chain genes are amplified.

[0094] Reverse transcription cDNA synthesis method: cDNA was synthesized in a 96-well PCR plate with a 10 μl reaction system, 1 μM OligodT30, 1 μM TSO Primer, 1 mM dNTPs, and 100 U HiScript III Reverse Transcriptase, at 42°C, in a PCR instrument for 1 h;

[0095] Nested PCR amplification of antibody IgH, Igκ and Igλ variable region genes, method: first round PCR: 20μl system contains 2μl RT reaction product, 10μl 2×Phanta Max Master Mix enzyme, upstream primer is 0.25μM ISPCR, downstream primer is 0.25μM antibody constant region primer mIgHGCR or mIgκCR or mIgλCR. Reaction conditions: pre-denaturation at 95℃ for 3min, then 30 PCR cycles, each cycle is: 95℃×15sec, 57℃×15sec, 72℃×35sec, and finally extended at 72℃ for 5min. Second round of PCR: 20μl system contains 2μl of the first round of PCR reaction product, 10μl of 2×Phanta MaxMaster Mix enzyme, upstream primer is 0.25μM FR1 region primer mVH-mix or mVκ-mix or mVλ-mix, downstream primer is 0.25μM variable region J region primer mJH-mix or mJκ-mix or mJλ-mix. Reaction conditions: pre-denaturation at 95℃ for 3min, then 35 PCR cycles, each cycle is: 95℃×15sec, 57℃×15sec, 72℃×30sec, and finally extension at 72℃ for 5min.

[0096] The antibody genes identified as positive and with matching heavy and light chains were sequenced and analyzed using the IMGT online service (https: / / www.imgt.org / IMGT_vquest / ).

[0097] 4. Production of recombinant antibodies

[0098] The obtained antibody heavy and light chain variable region genes were cloned into expression vectors containing human IgG1 heavy chain constant region and human kappa light chain constant region, respectively, to prepare antibody expression plasmids. According to the instructions of ExpiCHO™ ExpressionSystem transfection kit (ThermoFisher, A29133), the obtained recombinant plasmid was co-transfected with the transfection reagent ExpiFectamine into ExpiCHO-S cells, and cultured in an incubator at 37°C and 8% CO2. After 5 days of culture, the supernatant was collected, 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.

[0099] 5. Anti-TL1A antibody blocks the binding of TL1A to DR3 cells

[0100] The anti-human TL1A antibody in this application blocked the binding of TL1A to DR3 cells by FACS. HEK293 cells overexpressing DR3 (HEK293-DR3 cells) were diluted with DPBS containing 3% FBS and the density was adjusted to 0.5×10 6 / mL, added to a 96-well plate at 100 μl / well. The cells were centrifuged at 300g for 5 minutes and the supernatant was discarded. 50μl of the gradient dilution of the anti-TL1A antibody or control in this application (starting at 15μg / ml, 3-fold gradient dilution) was added to each well, and then 50 μl of 0.5μg / ml Biotinylated Human TL1A was added, and incubated at 2-8°C for 30 minutes. The cells were centrifuged at 300g for 5 minutes and the supernatant was discarded. After washing three times with DPBS containing 3% FBS, SA-PE (Biolenged, 405204) was added at 100 μl / well and incubated at 2-8°C for 30 minutes. The cells were centrifuged at 300g for 5 minutes and the supernatant was discarded. After adding 100 μl of DPBS containing 3% FBS to each well to resuspend the cells, the average fluorescence intensity of the cells was detected using a Cytoflex flow cytometer, and the IC50 value of the antibody blocking TL1A binding to DR3 overexpressing cells was calculated. The results are shown in Tables 2 and Figure 1 As shown, the antibodies in the present application can strongly inhibit the binding of Human TL1A to DR3 overexpressing cells.

[0101] Table 2 TL1A antibody blocks TL1A binding to DR3 cell activity

[0102]

[0103] 6. Inhibition of NFκB by anti-TL1A antibody in Jurkat-hDR3-Luc cells

[0104] Dilute Jurkat-hDR3-Luc cells with RPMI 1640 medium containing 1% FBS to adjust the density to 4 × 10 6 / ml, add 50μl / well to a 96-well plate. Add 25μl of gradient dilution of the anti-TL1A antibody or control in this application (starting at 30μg / ml, 3-fold gradient dilution) to each well, then add 25μl 0.5μg / ml Biotinylated HumanTL1A, and incubate at 37°C for 5h. Add 50μl of colorimetric solution (Biyuntian, RG052M) to each well, shake and mix, and incubate at room temperature in the dark for 5-10min. Use the SpectraMax i3X microplate reader to detect LUM luminescence, and calculate the IC50 value of the antibody inhibiting Human TL1A-induced Jurkat-hDR3-Luc cell DR3 downstream signal activation. The results are shown in Tables 3 and Figure 2 As shown, the antibodies in the present application can significantly inhibit the activation of DR3 downstream signals in Jurkat-hDR3-Luc cells induced by Human TL1A.

[0105] Table 3 Anti-TL1A antibodies inhibit NFκB

[0106]

[0107] 7. Anti-TL1A antibody inhibits TL1A-induced CD4+ T cell IFNγ production

[0108] CD4+ T cells were isolated from PBMCs (peripheral blood mononuclear cells) cultured overnight after thawing using EasySep™ Human CD4+ T Cell Isolation Kit (Stemcell, 17952). The isolated CD4+ T cells were washed once with assay buffer (RPMI 1640 medium containing 10% FBS) and the cell density was adjusted to 1×10 6 / ml, add 50µl / well to a 96-well U-bottom plate. Add 25 μl of diluted IL12 solution (1 ng / ml), 25 μl of diluted IL18 solution (25 ng / ml), and 50 μl of diluted IL18 solution (40 ng / ml) to each well. Add 50 μl of the anti-TL1A antibody or control in this application (starting at 15 μg / ml, 3-fold gradient dilution) to each well and incubate at 37°C for 24 h. Use HumanInterferon-γ ELISA Kit (Acro, CRS-A017) to detect the IFNγ content in the cell culture supernatant, and calculate the IC50 value of the antibody inhibiting TL1A-induced CD4+T cell secretion of IFNγ. The results are shown in Tables 4 and Figure 3 As shown, the antibodies in the present application all significantly inhibited the production of IFNγ, a CD4+ T cell factor induced by TL1A.

[0109] Table 4 Anti-TL1A antibody inhibits IFNγ production

[0110]

[0111] 8. Anti-TL1A Antibody Epitope Mapping

[0112] Dilute the antigen Human TL1A protein to 2μg / ml with PBST buffer; dilute each antibody to 100nM with PBST buffer; dilute the second antibody to 100nM with PBST buffer; add the diluted antigen, antibody, RegenerationBuffer, and Kinetics buffer to the 96-well sample plate respectively; place the HIS1K probe and sample plate in the corresponding positions in the instrument; use Octet BLI discovery software to set the sample, probe, method, and file information, and run the method for affinity detection. The main method parameters are as follows: Baseline1: 30s, Baseline2: 30s, AntigenAssociation: 0.2nm, Antibody1 Association: 200s, Antibody2 Association: 200s; use Octet Analysis Studio to analyze the experimental data, calculate the percentage of the second antibody signal / the second antibody signal when the first antibody is not present, and analyze the antibody epitope grouping. The results are shown in Tables 5 and Figure 4 As shown, CR20M0681 has a different epitope from PRA023 and RVT-3101.

[0113] Table 5 Percentage of cross-binding signals of anti-TL1A antibodies

[0114]

[0115] 9. Anti-TL1A antibody blocks the binding of TL1A to DcR3

[0116] Human DcR3 Protein (1 μg / ml) diluted with coating solution (0.05M carbonate, pH 9.6) was added to the 96-well plate, 50 μl per well, and coated overnight at 4°C; the plate was washed 3 times with 250 μl / well PBST, and the residual liquid was patted dry; 100 μl of 5% skim milk was added to each well, blocked at room temperature for 1 hour, and the residual liquid was patted dry; 25 μl of the antibody diluted with PBS gradient (initial concentration 180 μg / ml, 3-fold dilution, a total of 7 concentration gradient points) was added to the corresponding wells; the ligand diluted with 3% BSA gradient (concentration 5 μg / ml) was added to the corresponding wells, 25 μl per well, and incubated at 37°C for 30 minutes; 250 μl / well PBST was added to wash the plate 3 times, and the residual liquid was patted dry; 50 μl of HRP secondary antibody (diluted with PBS, antibody dilution factor is 1:3000) was added to each well, and incubated at room temperature for 1 hour; 250 μl / well Wash the plate 4 times with PBST, pat dry the residual liquid; add 50μl TMB colorimetric solution to each well, react at room temperature for 10min; add 50μl stop solution to each well, stop colorimetric development, and read the OD450 value. Figure 5As shown, similar to the reference antibody, at low antibody concentrations, the CR20M0681 antibody did not block the binding of TL1A to DcR3.

[0117] 10. Binding specificity of anti-TL1A antibodies against TL1A and homologs

[0118] Human TL1A Protein and homologous proteins (TNF-β, TNF-α, Fas Ligand, TRAIL, LIGHT) were coated on a 96-well ELISA plate (1μg / mL, 50μl / well) and incubated overnight at 4°C, with the first four rows coated with homologous proteins and the last four rows coated with Human TL1A Protein; after washing 3 times with PBST, 100μl 3% BSA blocking solution was added and incubated at 37°C for 1h; after washing 3 times with PBST, the diluted test antibody (100ng / μl) was added and incubated at 37°C for 1h; after washing 3 times with PBST, 50μl Anti-human IgG HRP (1:10000 dilution) was added and incubated at 37°C for 1h; after washing 4 times with PBST, 50μl TMB colorimetric solution was added and color developed at 37°C for 5min, 50μl / well stop solution was added to terminate the reaction, and the absorbance was measured at a wavelength of 450nm using an ELISA reader. The results are as follows Figure 6 As shown, the CR20M0681 antibody does not cross-bind with homologous proteins TNF-β, TNF-α, Fas Ligand, TRAIL, and LIGHT.

[0119] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0120] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the scope of the claims.

Claims

1. An anti-TL1A 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 whose amino acid sequences are respectively shown in SEQ ID NOs: 7 to 9, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are respectively shown in SEQ ID NOs: 10 to 12.

2. The anti-TL1A antibody according to claim 1, 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.

3. The anti-TL1A antibody according to claim 1 or 2, characterized in that The anti-TL1A antibody is a monoclonal antibody.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-TL1A antibody according to any one of claims 1 to 3.

5. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 4.

6. A cell, characterized in that The cell comprises the expression vector of claim 5.

7. A method for preparing the anti-TL1A antibody according to any one of claims 1 to 3, characterized in that: The method comprises culturing the cell according to claim 6, and obtaining the anti-TL1A antibody from the culture.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-TL1A antibody according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient.

9. A TL1A detection product, characterized in that: The TL1A detection product comprises the anti-TL1A antibody according to any one of claims 1 to 3.

10. Use of the anti-TL1A antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the cell according to claim 6 or the pharmaceutical composition according to claim 8 in the preparation of a drug for treating a disease associated with TL1A; the disease associated with TL1A is selected from one or more of inflammatory bowel disease, psoriasis, primary biliary cirrhosis, systemic lupus erythematosus and ankylosing spondylitis.

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