Anti-human TL1a monoclonal antibody and use thereof

Abstract

Provided are an anti-human TL1A antibody and the use thereof. The anti-human TL1A antibody contains three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein (a) the amino acid sequence of CDR-H1 is as represented by SEQ ID NO: 1; (b) the amino acid sequence of CDR-H2 is as represented by SEQ ID NO: 2; (c) the amino acid sequence of CDR-H3 is as represented by SEQ ID NO: 3; (d) the amino acid sequence of CDR-L1 is as represented by SEQ ID NO: 4; (e) the amino acid sequence of CDR-L2 is as represented by SEQ ID NO: 5; and (f) the amino acid sequence of CDR-L3 is as represented by SEQ ID NO: 6. The anti-human TL1A antibody is expected to exhibit good clinical effects in the prevention and treatment of related diseases.

Description

Anti-human TL1A monoclonal antibodies and their applications Technical Field

[0001] This application relates to the field of antibody drugs. Specifically, this application relates to antibodies against human TL1A and their applications. Background Technology

[0002] Tumor necrosis factor-like ligand 1A (TL1A), also known as vascular endothelial growth inhibitory factor (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 domain, and 192 in the extracellular domain. TL1A is a type II transmembrane protein that self-assembles into a stable trimer through interactions with the TNF homologous domain (THD). TL1A is mainly expressed in its membrane-bound form (mTL1A), forming a stable trimer, which is then cleaved by selective splicing or metalloproteinases (such as TNF-α converting enzyme (TACE)) to produce soluble TL1A (sTL1A).

[0003] TL1A is expressed in various immune cells (such as monocytes, macrophages, dendritic cells (DCs), and T cells) and non-immune cells (such as synovial fibroblasts and endothelial cells). TL1A competitively binds to death domain receptor 3 (DR3), providing stimulatory signals to 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, a member of the TNF receptor family, also known as Wsl-1, Apo-3, TRAMP, and LARD, and now referred to as TNF receptor superfamily member 25 (TNFRSF25). DR3 is a death receptor that activates and induces both death and survival factors. Osteoblasts produce transmembrane and soluble forms of DR3. In resting T cells, DR3 is expressed in its soluble form, protecting cells from apoptosis. Activated T cells express transmembrane DR3, which activates the receptor, leading to apoptosis or activation of transcription factors such as NF-κB. sTL1A binds to DR3 (membrane-bound form), and their interaction triggers two distinct signaling pathways, activating downstream signaling cascades that induce inflammation and apoptosis, respectively.

[0005] Inflammatory bowel diseases (IBD) are a group of chronic, relapsing, inflammatory bowel disorders characterized by enteritis and epithelial damage. Severe cases can lead to hospitalization and surgery; the mortality rate is low, but lifelong treatment is required. The main types of IBD are ulcerative colitis (UC) and Crohn's disease (CD). These diseases are very common; approximately 1.86 million people worldwide are diagnosed with UC, and approximately 1.3 million are diagnosed with CD.

[0006] Ulcerative colitis and Crohn's disease present with different clinical manifestations. Ulcerative colitis generally only affects the large intestine, while Crohn's disease can cause inflammation and tissue destruction in any part of the gastrointestinal tract, from the mouth to the anus, most commonly the colon and ileum. The lesions of ulcerative colitis are mainly confined to the mucosa and submucosa, while the inflammation and damage in Crohn's disease can break through the submucosa and penetrate the entire intestinal wall, sometimes referred to as "transmural inflammation," extending from the mucosa to the serosa. Furthermore, unlike the continuous lesions of ulcerative colitis, the inflammatory areas in Crohn's disease are scattered, interspersed with healthy tissue, giving it a "pebbly" appearance.

[0007] Various subclinical phenotypes specific to severe forms of IBD may exist within subgroups of patients with CD and UC. One is obstructive Crohn's disease, which may result from long-term inflammation that can lead to scarring (fibrous stenosis) or swelling of the intestinal wall. Severe stenosis can cause intestinal obstruction, resulting in abdominal pain, bloating, nausea, and inability to pass stool. Additionally, the penetrating disease phenotype, characterized by intestinal obstruction or internal penetrating fistulas or both, often leads to associated complications such as intraperitoneal sepsis.

[0008] Currently, treatment options for IBD patients are limited. Existing anti-inflammatory therapies, such as steroids and TNF inhibitors, are typically used as first-line treatments for IBD. Unfortunately, a significant number of patients do not respond to existing anti-inflammatory therapies, especially TNF-α inhibitors, or lose their response. For patients who do not respond to first-line therapy, surgery in the form of stenosis repair or resection is the only treatment option. Surgical treatment of IBD is invasive, and it is estimated that approximately one-third of patients undergoing surgery will experience postoperative risks such as anastomotic leakage, infection, and bleeding.

[0009] In IBD patients, TL1A expression is increased in both serum and intestinal tissues and is correlated with disease activity. DR3 expression is also increased in the intestinal lymphocytes of these patients, particularly T cells. The interaction between TL1A expressed on dendritic cells (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 upregulation of chemokine receptors leads to enhanced function of bone marrow-derived dendritic cells (BMDCs), including T cell-dependent stimulation of BMDCs by TL1A, exacerbating dextran sulfate sodium (DSS)-induced colitis. The TL1A / DR3 pathway has become an important module of mucosal immunity, involved in maintaining intestinal homeostasis, but also crucially involved in the development and maintenance of chronic inflammatory responses in patients with inflammatory bowel disease (IBD). Studies have reported that in inflammatory diseases such as IBD, rheumatoid arthritis, and psoriasis, TL1A expression and its receptor upregulation, and TL1A / DR3 signaling promote disease progression.

[0010] Increased TL1A expression and / or TL1A gene polymorphisms are associated with the pathogenesis of various autoimmune and inflammatory diseases. Inhibiting TL1A-DR3 interaction is an effective therapeutic strategy to improve local inflammation of target organs in individuals with autoimmune diseases. In addition to inflammatory bowel disease, inhibiting TL1A-DR3 may also be a therapeutic strategy for diseases such as psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, and ankylosing spondylitis.

[0011] Currently, the anti-TL1A antibody drugs under development internationally include Pfizer's RVT3101 (Phase 3 clinical trial), Teva Pharmaceutical's TEV48574 (Phase 2 clinical trial), and Merck's PRA023 (Phase 3 clinical trial). None of them are on the market yet. Therefore, there is a need to develop antibody drugs that bind to TL1A to address unmet clinical needs. Summary of the Invention

[0012] The purpose of this application is to provide a novel anti-human TL1A antibody, a pharmaceutical composition comprising the antibody, and the use of the antibody.

[0013] Specifically, this application relates to the following aspects:

[0014] 1. An isolated anti-human TL1A antibody comprising three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein:

[0015] (a) The amino acid sequence of CDR-H1 (in this specification, CDR-H1 represents heavy chain CDR1) is shown in SEQ ID NO:1 (GYVMS);

[0016] (b) The amino acid sequence of CDR-H2 (in this specification, CDR-H2 represents heavy chain CDR2) is shown in SEQ ID NO:2(VIATGGSAYYATWAKG);

[0017] (c) The amino acid sequence of CDR-H3 (in this specification, CDR-H3 represents heavy chain CDR3) is shown in SEQ ID NO:3 (GGPGYGTYDMDP);

[0018] (d) The amino acid sequence of CDR-L1 (in this specification, CDR-L1 represents light chain CDR1) is shown in SEQ ID NO:4 (QASEDIESYLA);

[0019] (e) The amino acid sequence of CDR-L2 (in this specification, CDR-L2 represents the light chain CDR2) is shown in SEQ ID NO:5 (TASDLAS); and

[0020] (f) The amino acid sequence of CDR-L3 (in this specification, CDR-L3 represents the light chain CDR3) is shown in SEQ ID NO:6 (QQGYTAANIPNA).

[0021] 2. The anti-human TL1A antibody according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein,

[0022] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and its amino acid sequence is EVQLVESGGGLVQPGGSLRLSCAASGFSLSGYVMSWVRQAPGKGLEWIGVIATGGSAYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARGGPGYGTYDMDPWGQGTLVTVSS; and / or

[0023] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:8, and its amino acid sequence is DIQMTQSPSSVSASVGDRVTITCQASEDIESYLAWYQQKPGKAPKLLIYTASDLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYTAANIPNAFGGGTKVEIK.

[0024] 3. The anti-human TL1A antibody according to item 1 or 2, comprising a heavy chain and a light chain, wherein,

[0025] The amino acid sequence of the heavy chain is shown in SEQ ID NO:10; and / or

[0026] The amino acid sequence of the light chain is shown in SEQ ID NO:11.

[0027] 4. An isolated nucleic acid encoding any of the aforementioned anti-human TL1A antibodies.

[0028] 5. A vector comprising the isolated nucleic acid described in item 4.

[0029] 6. A host cell comprising the nucleic acid described in item 4 or the vector described in item 5.

[0030] The nucleic acid can be present on a vector. The vector can be of any type, for example, a recombinant vector such as an expression vector. Any of a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, for example, *Escherichia coli* (E. coli). In another embodiment, the host cell is a eukaryotic cell, for example, a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

[0031] 7. A method for producing an antibody, the method comprising culturing the host cells described in item 6 to produce any of the aforementioned antibodies.

[0032] The method includes producing the antibody by expressing a recombinant vector encoding the anti-human TL1A antibody in a suitable host cell. In some embodiments, the method includes culturing host cells containing nucleic acids encoding the anti-human TL1A antibody to express the nucleic acids. The method may further include recovering the anti-human TL1A antibody from a host cell culture or host cell culture medium.

[0033] 8. A pharmaceutical composition comprising any of the aforementioned antibodies and a pharmaceutically acceptable carrier.

[0034] The pharmaceutical composition may further comprise additional therapeutic agents (e.g., different anti-human TL1A antibodies).

[0035] 9. The pharmaceutical composition according to item 8, for treating diseases related to TL1A-mediated signal transduction.

[0036] 10. The pharmaceutical composition according to claim 9, wherein the diseases associated with the TL1A-mediated signal transduction are allergies, asthma, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus (SLE), irritable bowel syndrome, systemic sclerosis, systemic sclerosis-associated interstitial lung disease, psoriasis, type 1 diabetes, and transplant rejection.

[0037] 11. Use of any of the aforementioned antibodies in the preparation of medicaments for the treatment of diseases related to TL1A-mediated signal transduction.

[0038] 12. The use according to item 11, wherein the diseases associated with the TL1A-mediated signal transduction are selected from allergies, asthma, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus (SLE), irritable bowel syndrome, systemic sclerosis, systemic sclerosis-associated interstitial lung disease, psoriasis, type 1 diabetes, and transplant rejection.

[0039] 13. A method for treating diseases related to TL1A-mediated signal transduction, comprising:

[0040] Administer a therapeutically effective amount of any of the preceding anti-human TL1A antibodies or any of the preceding pharmaceutical compositions to the subject.

[0041] 14. The method according to claim 13, wherein the diseases associated with the TL1A-mediated signal transduction are selected from allergies, asthma, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus (SLE), irritable bowel syndrome, systemic sclerosis, systemic sclerosis-associated interstitial lung disease, psoriasis, type 1 diabetes, and transplant rejection.

[0042] 15. Any of the aforementioned antibodies may be used to treat diseases related to TL1A-mediated signal transduction.

[0043] 16. The use according to item 15, wherein the diseases associated with the TL1A-mediated signal transduction are selected from allergies, asthma, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus (SLE), irritable bowel syndrome, systemic sclerosis, systemic sclerosis-associated interstitial lung disease, psoriasis, type 1 diabetes, and transplant rejection.

[0044] The beneficial effects of this application are:

[0045] The anti-human TL1A antibody of this application can specifically bind to human TL1A.

[0046] The anti-human TL1A antibody of this application exhibits superiority over similar antibody products under development in numerous biological activities. These activities include, for example, neutralizing TL1A-induced TF-1 apoptosis and neutralizing TL1A-induced NF-κB signaling activity in DR3-Jurkat cells. Therefore, the anti-human TL1A antibody of this application is expected to demonstrate good clinical efficacy in the prevention and treatment of related diseases. Attached Figure Description

[0047] Figure 1 shows the electrophoresis results of the constructed HZD306-12 transient transfection expression plasmid. In the figure, M: Marker; Band 1: PCR product 306VH-Hu3; Band 2: pQXHC, HindIII / EcoRI; Band 3: PCR product 306VK-Hu1; Band 4: pQXLC, HindIII / EcoRI.

[0048] Figure 2 is a flowchart of the instantaneous transition expression.

[0049] Figure 3 shows the electrophoretic detection results of HZD306-12.

[0050] Figure 4 shows the binding spectrum of HZD306-12 to membrane TL1A.

[0051] Figure 5 is a map of TF-1 cell apoptosis induced by HZD306-12 and TL1A.

[0052] Figure 6 is a map of NF-κB signaling activity in DR3-Jurkat cells induced by HZD306-12 and TL1A.

[0053] Figure 7 shows the binding activity of HZD306-12 with TL1A from different species. Detailed Implementation

[0054] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0055] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0056] Generally speaking, the terms used in this specification have the following meanings.

[0057] In this specification, the term "antibody" is used in the broadest sense to encompass various immunoglobulin structures, including but not limited to monoclonal antibodies, multispecific (e.g., bispecific) antibodies, and antibody fragments, provided that such substances exhibit the desired antigen-binding activity. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a monoclonal monospecific antibody. In some embodiments, the antibody is a monoclonal multispecific antibody. In some embodiments, the antibody is a monoclonal bispecific antibody.

[0058] In this specification, an “isolated” antibody is an antibody that has been separated from components of its native environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, which is determined by, for example, electrophoresis (e.g., SDS-PAGE isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0059] In this specification, "monoclonal antibody" means an antibody derived from a group of substantially homologous antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, and such variants are typically present in trace amounts, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles). Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous group of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibody used according to this application can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein. For clarity, it should be understood that the term "monoclonal antibody" as used herein encompasses multispecific antibodies. For example, the term “monoclonal antibody” as used in this specification also covers a class of multispecific antibodies, including the anti-human TL1A monoclonal antibody described herein, and another monoclonal antibody with different binding specificity, namely another monoclonal antibody that can specifically bind to proteins other than TL1A.

[0060] In this specification, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K0). D Affinity can be measured using methods known in the art.

[0061] In this specification, "identity" in the context of amino acid sequences refers to the number of identical residues in two sequences when aligned for maximum correspondence. Many different algorithms known in the art exist for measuring the percentage of amino acid identity (i.e., the Basic Local Alignment Tool or...). Unless otherwise specified, use the default parameters for the specific program or algorithm.

[0062] In this specification, "isolated" nucleic acid means a nucleic acid molecule that has been isolated from components of its native environment. Isolated nucleic acid includes nucleic acid molecules that are normally found in cells containing nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations other than their native chromosomal locations.

[0063] In this specification, "isolated nucleic acid encoding anti-human TL1A monoclonal antibody" means one or more nucleic acid molecules encoding the antibody heavy and light chains, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in the host cell.

[0064] In this specification, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0065] In this specification, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny (regardless of passage number). Progeny may not be identical to parental cells in terms of nucleic acid contents, but may contain mutations. Mutant progeny with the same function or biological activity selected from the initially transformed cells are included in this specification.

[0066] In this specification, "pharmaceutical composition" means an article which is in a form that enables the bioactivity of the active ingredient contained therein to exert its effect, and which does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation is to be administered.

[0067] In this specification, "pharmaceuticalally acceptable carrier" means a component of the pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0068] In this specification, "therapeuticly effective amount" means an amount of drug or pharmaceutical agent that will elicit a biological or medical response in a tissue, system, or animal, such response as is desired by a researcher or clinician. Furthermore, the term "therapeuticly effective amount" means any amount that, compared to a corresponding control who did not receive such an amount, results in the treatment, cure, prevention, or relief of a disease, disorder, or side effect, or a decrease in the rate of progression of the disease or disorder. The term also includes, within its scope, amounts that effectively enhance normal physiological function.

[0069] In this specification, human TL1A (Human Tumor Necrosis Factor-like ligand 1A, hTL1A) refers to a protein derived from humans, with the amino acid sequence shown in SEQ ID NO:9 (full length).

[0070] SEQ ID NO:9:

[0071] In this specification, "anti-human TL1A monoclonal antibody" means a monoclonal antibody that binds to human TL1A with sufficient affinity, such that the monoclonal antibody can be used as a diagnostic and / or therapeutic agent targeting human TL1A. For clarity, the term "anti-human TL1A monoclonal antibody" as used herein should be understood to encompass multispecific antibodies comprising the anti-human TL1A monoclonal antibody described herein and another monoclonal antibody with a different binding specificity, i.e., another monoclonal antibody capable of specifically binding to proteins other than TL1A.

[0072] This application provides an isolated anti-human TL1A antibody comprising three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein: (a) the amino acid sequence of CDR-H1 is shown in SEQ ID NO:1 (GYVMS); (b) the amino acid sequence of CDR-H2 is shown in SEQ ID NO:2 (VIATGGSAYYATWAKG); (c) the amino acid sequence of CDR-H3 is shown in SEQ ID NO:3 (GGPGYGTYDMDP); (d) the amino acid sequence of CDR-L1 is shown in SEQ ID NO:4 (QASEDIESYLA); (e) the amino acid sequence of CDR-L2 is shown in SEQ ID NO:5 (TASDLAS); and (f) the amino acid sequence of CDR-L3 is shown in SEQ ID NO:6 (QQGYTAANIPNA).

[0073] The antibody comprises a variable region of the antibody heavy chain and a variable region of the antibody light chain.

[0074] In one specific embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7, or has at least 90% identity with SEQ ID NO:7.

[0075] SEQ ID NO:7

[0076] In one specific embodiment, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8, or has at least 90% identity with SEQ ID NO:8.

[0077] SEQ ID NO:8

[0078] In one specific embodiment, the amino acid sequence of the antibody 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.

[0079] In one specific embodiment, the amino acid sequence of the antibody heavy chain is as shown in SEQ ID NO:10, or has at least 90% identity with SEQ ID NO:10.

[0080] SEQ ID NO:10

[0081] In one specific embodiment, the amino acid sequence of the antibody light chain is as shown in SEQ ID NO:11, or has at least 90% identity with SEQ ID NO:11.

[0082] SEQ ID NO:11

[0083] Among them, SEQ ID NO:10 and SEQ ID NO:11 are both humanized sequences.

[0084] In one specific embodiment, the amino acid sequence of the antibody heavy chain is shown in SEQ ID NO:10, and the amino acid sequence of the light chain is shown in SEQ ID NO:11.

[0085] In this specification, having at least 90% identity means having at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity.

[0086] The anti-human TL1A antibody of this application does not bind to proteins unrelated to the target. Here, "unrelated protein" refers to proteins other than human TL1A, which is the target; and "does not bind" means that, if the binding ability of the anti-human TL1A antibody of this application to human TL1A, which is its target, is taken as 100%, the binding ability of the anti-human TL1A antibody of this application to the unrelated protein is less than 10%, for example, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001%, 0.0000001%, 0.0000001%, or 0.

[0087] The anti-human TL1A antibody of this application may or may not bind to TL1A of other animal species.

[0088] The anti-human TL1A antibody of this application has equilibrium dissociation constants (K0) of ≤1 μM, ≤100 nM, ≤50 nM, and ≤40 nM. D ).

[0089] Experimental results show that the anti-human TL1A antibody of this application can specifically bind to human TL1A.

[0090] The anti-human TL1A antibody of this application is superior to similar antibody products under investigation in many biological activities. Such biological activities include, for example, neutralizing TL1A-induced TF-1 apoptosis activity, neutralizing TL1A-induced NF-κB signaling transduction activity in DR3-Jurkat cells, and neutralizing TL1A-induced HEL (human erythroleukemia cell) apoptosis activity.

[0091] This application also provides a multispecific protein (e.g., a bispecific protein (antibody)) comprising a first antigen-binding region and a second antigen-binding region; wherein the first antigen-binding region comprises the anti-human TL1A antibody described herein, and the second antigen-binding region comprises an antibody capable of specifically binding to antigens other than TL1A.

[0092] This application also provides an isolated nucleic acid molecule encoding the aforementioned anti-human TL1A antibody. In some embodiments, the nucleic acid molecule is an RNA or DNA molecule.

[0093] This application also provides a vector comprising the aforementioned isolated nucleic acids. The vector can be of any type, for example, a recombinant vector such as an expression vector.

[0094] This application also provides a host cell comprising the aforementioned isolated nucleic acid or vector. In one embodiment, the host cell is a prokaryotic cell, such as *Escherichia coli* (E. coli). In another embodiment, the host cell is a eukaryotic cell, such as a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

[0095] This application also provides a method for producing antibodies, the method comprising culturing the aforementioned host cells to produce any of the aforementioned anti-human TL1A antibodies.

[0096] The method includes producing the antibody by expressing a recombinant vector encoding the anti-human TL1A antibody in a suitable host cell. In some embodiments, the method includes culturing host cells containing nucleic acids encoding the anti-human TL1A antibody to express the nucleic acids. The method may further include recovering the anti-human TL1A antibody from a host cell culture or host cell culture medium.

[0097] This application also provides a pharmaceutical composition comprising any of the aforementioned anti-human TL1A antibodies and a pharmaceutically acceptable carrier.

[0098] Those skilled in the art will understand that the pharmaceutical composition of this application may further comprise other therapeutic agents, such as different anti-human TL1A antibodies.

[0099] This application also provides the use of any of the aforementioned antibodies in the treatment of diseases related to TL1A-mediated signal transduction.

[0100] This application also provides the use of any of the aforementioned anti-human TL1A antibodies in the preparation of medicaments for treating diseases related to TL1A-mediated signal transduction.

[0101] This application also provides a method for treating TL1A-mediated signal transduction-related diseases, comprising: administering to a subject a therapeutically effective amount of any of the aforementioned anti-human TL1A antibodies or any of the aforementioned pharmaceutical compositions.

[0102] The effective therapeutic dose can be adjusted according to the subject's age and body size, target disease, symptoms, route of administration, etc.

[0103] The subjects were mammals, such as humans.

[0104] In the above uses or methods, the diseases associated with the TL1A-mediated signal transduction are known in the art, such as those selected from allergies, asthma, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus (SLE), irritable bowel syndrome, systemic sclerosis, systemic sclerosis-associated interstitial lung disease, psoriasis, type 1 diabetes, and transplant rejection.

[0105] Example

[0106] The present application will be described in more detail below through embodiments. It should be understood that the present application is not limited to these embodiments.

[0107] Example 1: Preparation of anti-human TL1A monoclonal antibody HZD306-12

[0108] Human TL1A rabbit Fc fusion protein was obtained by transient transfection of HEK293F cells and used as an immunogen to immunize New Zealand rabbits. B cell cloning technology was used to obtain antigen-binding specific antibody clones, and then monoclonal antibodies binding to TL1A and exhibiting TL1A inhibitory activity were screened. First, B cell culture supernatant was detected by Binding ELISA to select clones binding to TL1A; then, reporter gene assay was used to select clones with TL1A inhibitory activity.

[0109] Thirty-six clones were selected for recombinant expression and sequencing. The B cell recombinant supernatant was detected using Binding ELISA and reporter gene assay. Ten clones were selected and their antibody proteins purified to further verify the neutralizing activities of the antibodies against DR3 and DcR3. Finally, clone D306 was selected for humanization. Homology comparison of human IgG germline sequences was performed using NCBI IgBlast. IGHV3-66*01 was selected as the heavy chain CDR transplantation template. The CDR regions of the heavy chain of clone D306 (i.e., CDR-H1 (SEQ ID NO:1), CDR-H2 (SEQ ID NO:2), and CDR-H3 (SEQ ID NO:3)) were transplanted into the backbone region of IGHV3-66*01. IGKV1-12*01 was selected as the light chain CDR transplantation template. The CDR regions (i.e., CDR-L1 (SEQ ID NO:4), CDR-L2 (SEQ ID NO:5), and CDR-L3 (SEQ ID NO:6)) of the D306 clone light chain were transplanted into the backbone region of IGKV1-12*01. Reverse mutations were then performed at specific sites in the backbone region to obtain the variable region of the monoclonal antibody of this application. Finally, the amino acid sequence of the humanized heavy chain variable region is shown in SEQ ID NO:7; the amino acid sequence of the humanized light chain variable region is shown in SEQ ID NO:8.

[0110] The heavy chain gene (SEQ ID NO:10) was synthesized by General Biotechnology (Anhui) Co., Ltd., and then amplified by PCR; the light chain gene (SEQ ID NO:11) was synthesized by Jiangsu Sofei Biotechnology Co., Ltd., and then amplified by PCR. The heavy chain expression plasmid pQXHC was digested with HindIII and EcoRI; the light chain expression plasmid pQXLC was digested with HindIII and EcoRI; the PCR-amplified genes were inserted into the corresponding expression plasmids using Infusion recombinase to construct the heavy chain expression plasmid pQXHC-306VH-Hu3 and the light chain expression plasmid pQXLC-306VK-Hu1.

[0111] Figure 1 shows the results of double enzyme digestion of the plasmids detected by nucleic acid electrophoresis. As can be seen from the results in Figure 1, the PCR amplification results of the antibody heavy chain gene and light chain gene, as well as the results of double enzyme digestion of the heavy chain and light chain expression plasmids, show that the heavy chain plasmid is approximately 5000 bp in size, the light chain plasmid is approximately 7500 bp in size, the light chain gene is approximately 750 bp, and the heavy chain gene is approximately 1450 bp.

[0112] The correct heavy chain expression plasmid and light chain expression plasmid were co-transfected into ExpiCHO-S cells. One day before transfection, ExpiCHO-S cells were diluted to 3 × 10⁻⁶ cells / mL. 6 Cells were passaged at a density of 10 cells / ml before transfection. On the day of transfection, the cell density was diluted to 6 × 10⁻⁶ cells / ml. 6 Cells / ml, 25ml of cells were placed in a 125ml shake flask and awaited transfection. The transfection and expression process is shown in Figure 2.

[0113] On days 4-6 post-transfection, the culture supernatant was harvested and purified in one step using Protein A. The purified antibody was detected by SDS-PAGE electrophoresis and named HZD306-12. The results of protein electrophoresis of this antibody are shown in Figure 3. Protein electrophoresis was performed using a denaturing reducing gel. The results in Figure 3 show two bands, with sizes of approximately 50 kDa and 25 kDa, which are consistent with the theoretical molecular weights of the heavy chain (48.8 kDa) and light chain (23.4 kDa).

[0114] Example 2 Equilibrium dissociation constant (K) D Determination of )

[0115] The affinity of HZD306-12, TEV48574, PRA023, and RVT3101 for TL1A was detected using a Biacore T200 instrument. All procedures were performed at 25°C: Antibodies (1 μg / ml) were first captured using a Protein A chip for 60 s at a flow rate of 10 μl / min; then, serially diluted recombinant human TL1A (amino acid sequence as shown in SEQ ID NO:9) (starting at 50 nM, serially diluted at a 1:2 ratio of antigen volume to total diluted volume, for a total of 8 concentrations) was flowed sequentially through the Protein A chip from low to high concentration, with a binding time of 60 s, a dissociation time of 400 s, and a flow rate of 30 μl / min; regeneration was then performed using 10 nM glycine for 30 s at a flow rate of 30 μl / min. The binding rate constant k was calculated using the Kinetics option in the instrument's built-in analysis software. a dissociation rate constant k d and the dissociation equilibrium constant K D value.

[0116] Table 1 shows the K values ​​of HZD306-12 and TL1A. D The value was 2.88 ± 2.33E-11, while the K value of the control antibody was... D The values ​​are as follows: PRA023 is 7.28±0.630E-11, TEV48574 is 3.51±0.223E-11, and RVT3101 is 9.26±0.106E-11.

[0117] Table 1. Affinity of antibody to human TL1A

[0118] Example 3: Detection of the binding activity of HZD306-12 to membrane TL1A

[0119] The binding activity of HZD306-12 and TEV48574 cells to membrane TL1A (HEK293-TL1A) was detected using FACS: HEK293-TL1A cells were incubated at 2 × 10⁻⁶ cells / year. 4 Cells / well were added to a 96-well plate. Serially diluted antibodies HZD306-12 and TEV48574 (diluted to 20 μg / ml, using a 1:6 antibody-to-total-volume ratio) were added to each well at a ratio of 1:6. The mixture was then incubated at 2-8°C for 30 min. After washing twice with 1*PBS, 2 μg / ml of Goat anti-Human IgG (H+L) Antibody Alexa Fluor was added. TM488 μL / well was incubated at 2-8℃ for 30 min, followed by two washes with 1*PBS. Then, 200 μL / well of 1*PBS was added before analysis. Four-parameter curves were fitted using GraphPad Prism software to calculate the sample IC50. 50 The value was analyzed to determine the binding activity with membrane TL1A.

[0120] The results in Figure 4 and Table 2 show that HZD306-12 exhibits superior binding activity to membrane TL1A compared to the control drug TEV48574.

[0121] Table 2. Binding activity of HZD306-12 with membrane TL1A

[0122] Example 4: HZD306-12 neutralizes TL1A-induced TF-1 apoptosis activity

[0123] Detection of the inhibitory activity of HZD306-12 and PRA023 on TL1A-induced apoptosis using TF-1 cells: TF-1 cells (treated with 50 μg / ml cycloacetyl) were injected at 1×10⁻⁶ cells per ... 5 Cells were seeded at a density of 100 cells / well in 96-well plates. HZD306-12 was diluted to 1 μg / ml, and the control antibody PRA023 was diluted to 5 μg / ml. These were then serially diluted at a 1:2 ratio of antibody volume to total diluted volume. The serially diluted antibodies were mixed with an equal volume of 50 ng / ml TL1A and incubated in a CO2 incubator (37℃, 5% CO2) for 1 h. An equal volume of the antigen-antibody complex was then added to TF-1 cells, and the cells were incubated in a CO2 incubator (37℃, 5% CO2) for 48 h. Cell suspensions were analyzed using the Cell Counting-Lite 2.0 Luminescent Cell Viability Assay, and fluorescence values ​​were read using a multi-mode microplate reader. Four-parameter curves were fitted using GraphPad Prism software to calculate the IC50 values ​​of the samples. 50 The values ​​were analyzed to neutralize the TL1A-induced apoptosis activity of TF-1.

[0124] Figure 5 and Table 3 show that HZD306-12 inhibits TL1A-induced apoptosis in TF-1 cells, with an IC50 value of [missing information]. 50 The concentration was 36.99 ± 0.020 ng / ml; the IC50 of PRA023 inhibited the TL1A-induced apoptosis activity of TF-1 cells. 50 The concentration was 194.30 ± 0.066 ng / ml. HZD306-12 showed superior inhibitory activity against TL1A-induced apoptosis in TF-1 cells compared to PRA023.

[0125] Table 3. HZD306-12 neutralizing TL1A-induced apoptosis activity in TF-1 cells

[0126] Example 5: Neutralization of TL1A-induced NF-κB signaling activity in HZD306-12-mediated DR3-Jurkat cells

[0127] Using DR3-Jurkat cells to detect the inhibition of TL1A-induced NF-κB signaling transduction activity in HZD306-12 and RVT3101: DR3-Jurkat cells were inoculated at a concentration of 5 × 10⁻⁶ cells / cells. 4 Cells were seeded at a density of 100 cells / well in 96-well plates. HZD306-12 and RVT3101 were diluted to 1 μg / ml, followed by serial dilutions at a 1:3 ratio of antibody volume to total diluted antibody volume. The serially diluted antibody was mixed with an equal volume of 100 ng / ml TL1A, and the antigen-antibody complex was added in equal volumes to DR3-Jurkat cells. Cells were incubated in a CO2 incubator (37℃, 5% CO2) for 6 h. Cell suspensions were analyzed using the Cell Counting-Lite 2.0 Luminescent Cell Viability Assay, and fluorescence values ​​were read using a multi-mode microplate reader. Four-parameter curves were fitted using GraphPad Prism software to calculate the IC50 of the samples. 50 The values ​​were analyzed to neutralize TL1A-induced intracellular NF-κB signaling activity in DR3-Jurkat cells.

[0128] The results in Figure 6 and Table 4 show that HZD306-12 inhibits TL1A-induced NF-κB signaling activity in DR3-Jurkat cells, with an IC50 value of [missing information]. 50 The concentration was 37.95 ± 0.034 ng / ml; RVT3101 inhibited TL1A-induced intracellular NF-κB signaling activity in DR3-Jurkat cells, with an IC50 concentration of 37.95 ± 0.034 ng / ml. 50 The concentration was 73.41 ± 0.059 ng / ml. HZD306-12 showed superior inhibitory activity against TL1A-induced intracellular NF-κB signaling in DR3-Jurkat cells compared to RVT3101.

[0129] Table 4. NF-κB signaling activity in DR3-Jurkat cells induced by HZD306-12 and TL1A neutralization

[0130] Example 6: Binding characteristics of HZD306-12 with TL1A in different species

[0131] The binding characteristics of HZD306-12 to TL1A in different species were detected using Binding ELISA: 1 μg / ml TL1A protein from rats, rabbits, marmosets, rhesus monkeys, pigs, mice, guinea pigs, cats, dogs, and cynomolgus monkeys was coated into ELISA plates at 50 μl / well and incubated overnight. After washing, the plates were blocked with 0.5% BSA-PBS at room temperature for 2 h. After washing, 50 μl / well of serially diluted HZD306-12 (initial concentration 50 μg / ml, antibody volume to total volume ratio 1:5) was added and incubated at room temperature for 2 h. After washing, 50 μl / well of Goat anti-human IgG-HRP was added and incubated at room temperature in the dark for 1 h. Finally, the substrate was added for color development, and the color development reaction was terminated by adding stop solution. After termination, the microplate was placed in a microplate reader to read the absorbance at 450 nm (the absorbance at 650 nm was used as a reference). The binding characteristics of HZD306-12 with TL1A of different species were analyzed by fitting a four-parameter curve using SoftMax.

[0132] The results in Figure 7 show that HZD306-12 can bind to TL1A in cynomolgus monkeys, rhesus monkeys, marmosets, and rats.

Claims

1. An isolated anti-human TL1A antibody comprising three heavy chain complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3) and three light chain complementarity-determining regions (CDR-L1, CDR-L2, and CDR-L3), wherein: (a) The amino acid sequence of CDR-H1 is shown in SEQ ID NO:1; (b) The amino acid sequence of CDR-H2 is shown in SEQ ID NO:2; (c) The amino acid sequence of CDR-H3 is shown in SEQ ID NO:3; (d) The amino acid sequence of CDR-L1 is shown in SEQ ID NO:4; (e) The amino acid sequence of CDR-L2 is shown in SEQ ID NO:5; and (f) The amino acid sequence of CDR-L3 is shown in SEQ ID NO:

6.

2. The anti-human TL1A antibody according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and / or The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

8.

3. The anti-human TL1A antibody according to claim 1 or 2, comprising a heavy chain and a light chain, wherein, The amino acid sequence of the heavy chain is shown in SEQ ID NO:10; and / or The amino acid sequence of the light chain is shown in SEQ ID NO:

11.

4. An isolated nucleic acid encoding an anti-human TL1A antibody as described in any one of claims 1-3.

5. A vector comprising the isolated nucleic acid as described in claim 4.

6. A host cell comprising the isolated nucleic acid of claim 4 or the vector of claim 5.

7. A method for producing an antibody, the method comprising culturing the host cell of claim 6 to produce the anti-human TL1A antibody of any one of claims 1-3.

8. A pharmaceutical composition comprising the anti-human TL1A antibody according to any one of claims 1-3 and a pharmaceutically acceptable carrier.

9. Use of the anti-human TL1A antibody according to any one of claims 1-3 in the preparation of a medicament for treating TLIA-mediated signal transduction-related diseases.

10. The use according to claim 9, wherein, The diseases associated with TLIA-mediated signal transduction are selected from allergies, asthma, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus (SLE), irritable bowel syndrome, systemic sclerosis, systemic sclerosis-associated interstitial lung disease, psoriasis, type 1 diabetes, and transplant rejection.

11. The anti-human TL1A antibody according to any one of claims 1-3 is used to treat TL1A-mediated signal transduction-related diseases.

12. The use according to claim 11, wherein, The diseases associated with TLIA-mediated signal transduction are selected from allergies, asthma, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus (SLE), irritable bowel syndrome, systemic sclerosis, systemic sclerosis-associated interstitial lung disease, psoriasis, type 1 diabetes, and transplant rejection.

13. A method for treating TLIA-mediated signal transduction-related diseases, comprising: The subject is given a therapeutically effective amount of the anti-human TL1A antibody according to any one of claims 1-3 or the pharmaceutical composition according to claim 8.

14. The method according to claim 13, wherein, The diseases associated with TLIA-mediated signal transduction are selected from allergies, asthma, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, systemic lupus erythematosus (SLE), irritable bowel syndrome, systemic sclerosis, systemic sclerosis-associated interstitial lung disease, psoriasis, type 1 diabetes, and transplant rejection.

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