TL1a single-domain antibody and use thereof

Abstract

Provided are a TL1A single-domain antibody and the use thereof. Specifically provided are a single-domain antibody or antigen-binding fragment specifically binding to human TL1A, a multispecific antigen-binding molecule, a chimeric antigen receptor, an immune effector cell, a nucleic acid molecule, a vector, a cell, a preparation method, a pharmaceutical composition, a kit, a pharmaceutical use, and a disease treatment method.

Description

TL1A single-domain antibodies and uses thereof

[0001] Cross-reference to Related Applications

[0002] This disclosure claims priority to and the benefit of Chinese Patent Application No. 202411262437.2, filed September 10, 2024, and Chinese Patent Application No. 202510401859.1, filed March 31, 2025, the entire contents of both of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present disclosure relates to the field of medicine, in particular, to antibodies that specifically bind to human TNF-like ligand 1A (TL1A) and block its activity, and their use in the treatment of immune-mediated inflammatory diseases (IMIDs). BACKGROUND

[0004] Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), belongs to autoimmune diseases. Common symptoms are diarrhea, bloody stool, and long-term inflammation can involve systemic organs. As a chronic non-specific intestinal inflammatory disease, IBD is recurrent, and there is currently no complete cure, and the global disease burden is heavy, with increasing incidence and prevalence. Traditional treatment drugs for IBD include glucocorticoids, antibiotics, immunosuppressants, aminosalicylic acid, etc. After infliximab and adalimumab targeting TNF-a were approved for UC / CD indications, IBD has entered the “biological agent” era. However, there is still a considerable proportion of target patient groups who do not respond to existing biological agents or will lose their response over time, and there is still a great unmet medical need in the clinic.

[0005] Tumor necrosis factor-like cytokine 1A (TL1A) is a member of the tumor necrosis factor family. It is expressed in different immune cells, such as monocytes, macrophages, dendritic cells, T cells, and non-immune cells, such as synovial fibroblasts, endothelial cells, etc. The basal level of TL1A is low, but it will rapidly increase after immune activation. Studies have shown that the expression level of TL1A in the colon tissue of IBD patients is related to the severity of inflammation.

[0006] According to the foregoing, TL1A plays an important role in biological processes related to several important human diseases. As a representative of emerging antibody drugs, nanobodies are being widely used in new drug research and development. Nanobody drugs have many advantages, such as small molecular weight, good solubility, high stability, and strong tissue penetration, and will play a more important role in the immunotherapy of tumors. SUMMARY

[0007] The present disclosure contemplates obtaining TL1A nanobodies with high affinity to block the interaction of TL1A with its receptor DR3 using nanotechnology, thereby applying to therapeutic, prophylactic, diagnostic and prognostic uses.

[0008] In view of this, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to TL1A, multispecific antigen-binding molecules, chimeric antigen receptors, immune effector cells, nucleic acid fragments, vectors, host cells, pharmaceutical compositions, kits, methods of preparation, and their applications in treating immune-mediated inflammatory diseases (IMIDs), such as autoimmune diseases or inflammatory diseases.

[0009] In a first aspect, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to TL1A, comprising CDR1, CDR2 and CDR3, respectively having the amino acid sequence of CDR1, CDR2 and CDR3 as shown in any one of SEQ ID NOs: 12-19, 85-90, 93-98, 102-105 or 107-110.

[0010] In some specific embodiments, the CDR1, CDR2 and CDR3 are determined according to the Kabat numbering system, the IMGT numbering system or the Chothia numbering system; optionally, the CDR1, CDR2 and CDR3 are selected from Table 5.

[0011] In some specific embodiments, the CDR1 has an amino acid sequence as set forth in SEQ ID NO: 29, 32, 35, 43, 45, 46, 49, 52, 56, 59, 61, 64, 67, 69, 72, 75, 77, 80 or 83, or at least 70% identity thereto or at most 3 mutations;

[0012] In some specific embodiments, the CDR2 has an amino acid sequence as set forth in 30, 33, 36, 37, 39, 41, 47, 50, 53, 54, 57, 60, 62, 65, 68, 70, 73, 76, 78, 81, 84, 100 or 101, or at least 70% identity thereto or at most 3 mutations;

[0013] In some specific embodiments, the CDR3 has an amino acid sequence as set forth in SEQ ID NO: 31, 34, 38, 40, 42, 44, 48, 51, 55, 58, 63, 66, 71, 74, 79 or 82, or at least 70% identity thereto or at most 3 mutations.

[0014] Preferably, the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 29-31, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 32-34, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 35-36 and 31, respectively, according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system.

[0015] Preferably, the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 29 and 37-38, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 32 and 39-40, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 35, 41 and 38, respectively, according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system.

[0016] Preferably, the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 29, 37 and 42, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 43, 39 and 44, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 45 and 41-42, respectively, according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system.

[0017] Preferably, the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 46-48, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 49-51, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 52-53 and 48, respectively, according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system.

[0018] Preferably, the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 46 and 54-55, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 56-58, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 59-60 and 55, respectively, according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system.

[0019] Preferably, the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 61-63, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 64-66, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 67-68 and 63, respectively, according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system.

[0020] Preferably, the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 69-71, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 72-74, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 75-76 and 71, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 69, 100 and 71, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 69, 101 and 71, respectively, according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system.

[0021] Preferably, the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 77-79, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 80-82, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences as set forth in SEQ ID NOs: 83-84 and 79, respectively, according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system.

[0022] In some embodiments, the antibody or antigen binding fragment is a single domain antibody or antigen binding fragment, and the single domain antibody or antigen binding fragment comprises the aforementioned CDR1, CDR2 and CDR3.

[0023] In some embodiments, the antibody or antigen binding fragment comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 12-19, 21-28, 85-90, 93-98, 102-105 or 107-110, or a sequence having at least 70% identity or at most 15 mutations compared to the amino acid sequence as set forth in any one of SEQ ID NOs: 12-19, 21-28, 85-90, 93-98, 102-105 or 107-110.

[0024] In some embodiments, the antibody or antigen-binding fragment comprises FR regions in any one of SEQ ID NOs: 12-19, 21-28, 85-90, 93-98, 102-105, or 107-110, or FR regions having at least 70% identity or at most 15 amino acid mutations to the FR regions in any one of SEQ ID NOs: 12-19, 85-90, 93-98, 102-105, or 107-110.

[0025] In some embodiments, the antibody or antigen-binding fragment is: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or (3) a fully human antibody or fragment thereof. In some embodiments, the antibody or antigen-binding fragment comprises or does not comprise an antibody heavy chain constant region; optionally, the antibody heavy chain constant region can be selected from human, llama, mouse, rat, rabbit, or sheep; optionally, the antibody heavy chain constant region can be selected from IgG, IgM, IgA, IgE, or IgD, and the IgG can be selected from IgGl, IgG2, IgG3, or IgG4.

[0026] Optionally, the heavy chain constant region can be selected from an Fc region, a CH3 region, or a complete heavy chain constant region.

[0027] Preferably, the heavy chain constant region is a human Fc region.

[0028] Preferably, the antibody or antigen-binding fragment is a heavy chain antibody.

[0029] In some embodiments, the Fc region has an amino acid sequence as set forth in SEQ ID NO: 20, or an amino acid sequence having at least 70% identity or at most 15 amino acid mutations thereto.

[0030] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain HC, wherein,

[0031] the heavy chain HC has an amino acid sequence as set forth in any one of SEQ ID NOs: 21-28, or an amino acid sequence having at least 70% identity or at most 15 amino acid mutations to the amino acid sequence as set forth in any one of SEQ ID NOs: 21-28.

[0032] In some embodiments, the at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; the at most 3 amino acid mutations are preferably at most 3, 2, 1, or 0 amino acid mutations; the at most 15 amino acid mutations are at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid mutations.

[0033] Optionally, the mutation is selected from a substitution, deletion, or insertion mutation; optionally, the mutation is a back mutation or a hot spot mutation; preferably, the substitution is a conservative amino acid substitution.

[0034] In some embodiments, the antibody or antigen binding fragment of the present disclosure comprises:

[0035] (1) a chimeric antibody or fragment thereof; and / or

[0036] (2) a humanized antibody or fragment thereof; and / or,

[0037] (3) a fully human antibody or fragment thereof.

[0038] In some embodiments, the antibody or antigen binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, or a single domain antibody.

[0039] In some embodiments, the antibody or antigen binding fragment is further conjugated to a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent, or an immunomodulatory agent, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, or a photosensitizer.

[0040] In some embodiments, the antibody or antigen-binding fragment specifically binds to human TL1A, monkey TL1A, and / or murine TL1A, preferably, the antibody or antigen-binding fragment has a KD of less than 1E-5 M, 1E-6 M, 1E-7 M, 1E-8 M, 2E-8 M, 3E-8 M, 4E-8 M, 5E-8 M, 6E-8 M, 7E-8 M, 8E-8 M, 9E-8 M, 1E-9 M, 2E-9 M, 3E-9 M, 4E-9 M, 5E-9 M, 6E-9 M, 7E-9 M, 8E-9 M, 9E-9 M, 1E-10 M, 2E-10 M, 3E-10 M, 4E-10 M, 5E-10 M, 6E-10 M, 7E-10 M, 8E-10 M, 9E-10 M, or 1E-11 M to human TL1A, monkey TL1A, or murine TL1A.

[0041] In some embodiments, the antibody or antigen-binding fragment is further linked to another functional molecule, preferably, the another functional molecule can be selected from one or more of the following: a signal peptide, a protein tag, a cytokine, an angiogenesis inhibitor, or an immune checkpoint inhibitor.

[0042] In some embodiments, the antibody or antigen-binding fragment exhibits one or more effector functions selected from the group consisting of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

[0043] In some embodiments, the antibody or antigen-binding fragment is further conjugated to a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent, or an immunomodulatory agent, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, and a photosensitizer.

[0044] In a second aspect, the present disclosure provides a multispecific antigen-binding molecule comprising the aforementioned antibody or antigen-binding fragment; and another antigen-binding molecule that binds to another antigen other than TL1A, or another antigen-binding molecule that binds to a different epitope of TL1A than the aforementioned antibody or antigen-binding fragment; optionally, the other antigen is selected from the group consisting of a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), an immune checkpoint, and a target that recruits and / or activates an immune cell.

[0045] Optionally, the other antigen-binding molecule is an antibody or antigen-binding fragment.

[0046] Optionally, the multispecific antigen-binding molecule can be bispecific, trispecific, or tetraspecific.

[0047] Optionally, the multispecific antigen-binding molecule can be bivalent, tetravalent or hexavalent.

[0048] In a third aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen-binding domain comprising the aforementioned antibody or antigen-binding fragment.

[0049] In a fourth aspect, the present disclosure provides an immune effector cell expressing the aforementioned CAR or comprising a nucleic acid fragment encoding the aforementioned CAR; optionally, the immune effector cell is selected from a T cell, a NK cell, a natural killer cell, a NKT cell, a natural killer T cell, a monocyte, a macrophage, a dendritic cell or a mast cell;

[0050] Optionally, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

[0051] Preferably, the T cell is selected from a cytotoxic T cell, a regulatory T cell or a helper T cell.

[0052] In a fifth aspect, the present disclosure provides an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule or chimeric antigen receptor.

[0053] In a sixth aspect, the present disclosure provides a vector comprising the aforementioned nucleic acid fragment.

[0054] In a seventh aspect, the present disclosure provides a host cell comprising the aforementioned nucleic acid fragment or the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as a bacterium (E. coli), a fungus (yeast), an insect cell or a mammalian cell (CHO cell line or 293T cell line); optionally, the cell lacks a fucosyltransferase, such as FUT8.

[0055] In an eighth aspect, the present disclosure provides a method of producing the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, the method comprising culturing the aforementioned cell, and isolating the antibody or antigen-binding fragment expressed by the cell, or isolating the multispecific antigen-binding molecule expressed by the cell.

[0056] In a ninth aspect, the present disclosure provides a method of producing the aforementioned immune effector cell, the method comprising introducing into the immune effector cell a nucleic acid fragment encoding the aforementioned CAR, optionally, the method further comprises initiating the expression of the aforementioned CAR by the immune effector cell.

[0057] In a tenth aspect, the present disclosure provides a pharmaceutical composition comprising the foregoing antibody or antigen binding fragment, multispecific antigen binding molecule, chimeric antigen receptor, immune effector cell, nucleic acid fragment, vector, or cell; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent, or adjuvant; optionally, the pharmaceutical composition further comprises an additional therapeutic agent.

[0058] In an eleventh aspect, the present disclosure provides use of the foregoing antibody or antigen binding fragment, multispecific antigen binding molecule, chimeric antigen receptor, immune effector cell, nucleic acid fragment, vector, or cell in the manufacture of a medicament for treating an immune-mediated inflammatory disease (IMID); preferably, the immune-mediated inflammatory disease (IMID) can be selected from one or more of inflammatory bowel disease (such as Crohn’s disease and ulcerative colitis), multiple sclerosis, psoriasis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, diabetes (e.g., type 1 diabetes and type 2 diabetes), vasculitis, asthma, eczema, atopic dermatitis, fibrosis, transplant rejection, graft-versus-host disease, allergy, ankylosing spondylitis, celiac disease, chronic obstructive pulmonary disease (COPD), chronic peptic ulcer, cystic fibrosis, glomerulonephritis, gout, hepatitis (e.g., active hepatitis), myositis, osteoarthritis, pelvic inflammatory disease (PID), a neurodegenerative disease of aging, periodontal disease (e.g., periodontitis), pre-perfusion injury transplant rejection, psoriasis, pulmonary fibrosis, rheumatic disease, scleroderma, sinusitis, tuberculosis, arteriosclerosis, and uveitis.

[0059] In a twelfth aspect, the present disclosure provides a method of treating an immune-mediated inflammatory disease (IMID), the method comprising administering to a subject an effective amount of the foregoing antibody or antigen binding fragment, multispecific antigen binding molecule, chimeric antigen receptor, immune effector cell, nucleic acid fragment, vector, or cell; preferably, the immune-mediated inflammatory disease (IMID) can be selected from one or more of inflammatory bowel disease (such as Crohn’s disease and ulcerative colitis), multiple sclerosis, psoriasis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, diabetes (e.g., type 1 diabetes and type 2 diabetes), vasculitis, asthma, eczema, atopic dermatitis, fibrosis, transplant rejection, graft-versus-host disease, allergy, ankylosing spondylitis, celiac disease, chronic obstructive pulmonary disease (COPD), chronic peptic ulcer, cystic fibrosis, glomerulonephritis, gout, hepatitis (e.g., active hepatitis), myositis, osteoarthritis, pelvic inflammatory disease (PID), a neurodegenerative disease of aging, periodontal disease (e.g., periodontitis), pre-perfusion injury transplant rejection, psoriasis, pulmonary fibrosis, rheumatic disease, scleroderma, sinusitis, tuberculosis, arteriosclerosis, and uveitis.

[0060] In a thirteenth aspect, the present disclosure provides the aforementioned antibody or antigen binding fragment, multispecific antigen binding molecule, chimeric antigen receptor, immune effector cell, nucleic acid fragment, nucleic acid vector, or host cell for use in the treatment of an immune-mediated inflammatory disease (IMID); preferably, the immune-mediated inflammatory disease (IMID) can be selected from one or more of inflammatory bowel disease (such as Crohn’s disease and ulcerative colitis), multiple sclerosis, psoriasis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, diabetes (e.g., type 1 diabetes and type 2 diabetes), vasculitis, asthma, eczema, atopic dermatitis, fibrosis, transplant rejection, graft-versus-host disease, allergy, ankylosing spondylitis, celiac disease, chronic obstructive pulmonary disease (COPD), chronic peptic ulcer, cystic fibrosis, glomerulonephritis, gout, hepatitis (e.g., active hepatitis), myositis, osteoarthritis, pelvic inflammatory disease (PID), neurodegenerative disease of aging, periodontal disease (e.g., periodontitis), preperfusion injury transplant rejection, psoriasis, pulmonary fibrosis, rheumatism, scleroderma, sinusitis, tuberculosis, arteriosclerosis, and uveitis.

[0061] In a fourteenth aspect, the present disclosure provides a kit comprising the aforementioned antibody or antigen binding fragment, multispecific antigen binding molecule, immune effector cell, nucleic acid fragment, vector, product of manufacture or pharmaceutical composition prepared according to the aforementioned methods.

[0062] Definitions and explanations

[0063] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by

[0064] Further, unless otherwise noted, terms used herein shall include both singular and plural forms of the terms. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0065] The terms "comprise," "comprising," "include," "including," and "have," "having" are used interchangeably and mean that the scheme includes the elements that follow the word.

[0066] The term "and / or," when used herein, includes the meaning of "and", "or", and "any or all of the elements linked to the respective term", and any other combination of the elements.

[0067] The term "TLIA", also known as "TNF ligand-related molecule 1 and vascular endothelial growth inhibitor (VEGI)" or "tumor necrosis factor superfamily member 15 (TNFSF15)", belongs to the tumor necrosis factor family, and is a ligand of DR3 and decoy receptor TR6 / DcR3. "TL1A" herein includes TL1A full-length protein or its mutants (e.g. point mutation, insertion mutation or deletion mutation), splice variants, Orthologs and fragments of TL1A. "TL1A" herein can be derived from human, primates (e.g. cynomolgus monkey, rhesus monkey) and rodents (e.g. mouse, rat). Exemplarily, the human TL1A amino acid sequence can be found at Uniprot No. O95159, the cynomolgus monkey TL1A amino acid sequence can be found at Uniprot No. G7PRK8, and the mouse TL1A amino acid sequence can be found at Uniprot No. Q5UBV8.

[0068] The terms "specific binding", "immunobinding" and "immunobinding property" herein refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin has specificity, and is the property of an antigen-binding molecule (e.g. body) to specifically bind an antigen and essentially the same antigen usually with high affinity, but not to unrelated antigens with high affinity. The strength or affinity of an immunobinding interaction is usually reflected in the equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. The immunobinding property of a selected polypeptide can be quantitatively determined using methods well known in the art. Taking antibodies as an example, high affinity usually refers to a KD of about 10 -8 M or lower, about 1 x 10 -9 M or lower, about 1 x 10 -10 M or lower, 1 x 10 -11 M or lower or 1 x 10 -12 M or lower. The KD is calculated as follows: KD = Kd / Ka, where Kd represents the dissociation rate and Ka represents the association rate. The equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (e.g. Biacore) or equilibrium dialysis.

[0069] The term "antigen binding molecule" is used herein in the broadest sense, and refers to a molecule that specifically binds to an antigen. Exemplary, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. An "antibody mimetic" refers to an organic compound or binding domain that is capable of specifically binding to an antigen, but is not related to the structure of an antibody. Exemplary, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), aptamers, or Kunitz domain peptides.

[0070] The term "antibody" is used herein in the broadest sense, and refers to a polypeptide or combination of polypeptides that contains a sufficient sequence from a heavy chain variable region of an immunoglobulin and / or a sufficient sequence from a light chain variable region of an immunoglobulin to be capable of specifically binding to an antigen. "Antibody" herein encompasses various forms and various structures, as long as they exhibit the desired antigen binding activity. The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. "Antibody" herein includes, but is not limited to, a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a whole antibody, a fragment of a whole antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0071] The terms "antibody fragment" or "antigen binding fragment" are used herein interchangeably, and refer to a portion of an antibody that does not possess the full structure of an intact antibody, but only contains a portion or a local variant of an intact antibody that possesses the ability to bind to the same antigen recognized by the intact antibody. "Antibody fragment" or "antigen binding fragment" also includes any synthetic or genetically engineered protein that functions like an antibody by binding to a particular antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the light chain variable region, "Fv" fragments consisting of the heavy and light chain variable regions, recombinant single chain polypeptide molecules (scFv) in which the light and heavy chain variable regions are connected by a peptide linker, and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region. "Antigen binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single domain antibodies.

[0072] An "antibody" herein also includes surrogate protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which comprise mutations introduced to, for example, stabilize the three-dimensional structure of an antibody) as well as wholly synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds can also include non-antibody-derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to, tenascin, fibronectin, peptide aptamers, and the like.

[0073] An "antibody" herein includes a typical "four-chain antibody" which belongs to an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); a heavy chain refers to a polypeptide chain which consists of, in the direction from N-terminus to C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, when the full-length antibody is of an IgE isotype, a heavy chain constant region CH4 domain is optionally included; a light chain is a polypeptide chain which consists of, in the direction from N-terminus to C-terminus, a light chain variable region (VL) and a light chain constant region (CL); the heavy chains and the heavy chains, the heavy chains and the light chains are connected by disulfide bonds, forming a "Y" shape. Due to the difference in the amino acid composition and the order of arrangement of the immunoglobulin heavy chain constant region, the antigenicity is also different. Accordingly, the "immunoglobulin" herein can be divided into five categories, or called isotypes of immunoglobulin, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same category of Ig can be divided into different subcategories according to the difference in the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bond, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4, IgA can be divided into IgA1 and IgA2. The light chain is divided into κ chain or λ chain through the constant region. Each of the five categories of Ig can have κ chain or λ chain.

[0074] The term "antibody" herein also includes antibodies that do not comprise light chains, such as heavy-chain antibodies (HCAbs) produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and Vicugna pacos, and immunoglobulin new antigen receptor (IgNAR) found in cartilaginous fishes such as sharks.

[0075] The term "heavy-chain antibody" herein refers to antibodies that lack light chains of conventional antibodies. The term specifically includes, but is not limited to, homodimeric antibodies comprising a VHantigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

[0076] The terms "single domain antibody" (sdAb), "VHH", and "nanobody" have the same meaning and are used interchangeably herein to refer to the variable region of a heavy chain of an antibody, which is constructed into a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with full function. Usually, after obtaining an antibody naturally lacking light chains and heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody consisting of only one heavy chain variable region. The single domain antibody can be derived from a camelid heavy chain antibody or an IgNAR of cartilaginous fishes.

[0077] Further descriptions of "heavy chain antibodies" and "single domain antibodies", "VHH domains" and "nanobodies" can be found in: Hamers-Casterman et al., Nature. 1993; 363; 446-8; review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001); and the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; WO 03 / 050531; WO 01 / 90190; WO 03 / 025020; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 and other prior art mentioned in these applications.

[0078] The term "chimeric antibody" herein refers to an antibody in which a portion of the light or / and heavy chain is derived from one antibody (which can be derived from a particular species or belong to a particular antibody class or subclass), and the other portion of the light or / and heavy chain is derived from another antibody (which can be derived from the same or a different species or belong to the same or a different antibody class or subclass), but retains the binding activity to the target antigen. For example, the term "chimeric antibody" can include an antibody (e.g., a human mouse chimeric antibody) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody), while the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human antibody).

[0079] The term "humanized antibody" herein refers to a non-human-derived antibody that has been genetically engineered to have an amino acid sequence that is modified to increase homology to the sequence of a human-derived antibody. Typically, a humanized antibody has all or a portion of the CDR regions from a non-human-derived antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., FRs in the variable region and / or constant regions) from a human-derived immunoglobulin (acceptor antibody). A humanized antibody typically retains or partially retains the desired properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, and the like.

[0080] The term "fully human antibody" herein refers to an antibody having a variable region in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. A "fully human antibody" herein can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo). However, a "fully human antibody" herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

[0081] The term "naked antibody" herein refers to an antibody that is not linked, fused, or conjugated to another agent or molecule (e.g., a label or a drug), a peptide, or a polypeptide. In particular embodiments, a naked antibody expressed by a mammalian host cell can be glycosylated by the glycosylation machinery (e.g., glycosylation enzymes) of the host cell. In certain embodiments, a naked antibody is not glycosylated when expressed by a host cell that does not have its own glycosylation machinery (e.g., glycosylation enzymes). In certain embodiments, a naked antibody is a whole antibody, while in other embodiments, a naked antibody is an antigen-binding fragment of a whole antibody.

[0082] An "antibody" herein can be derived from any animal, including but not limited to humans and non-human animals, which can be selected from primates, mammals, rodents, and vertebrates, such as a camelid, a llama, a vicuna, an alpaca, a sheep, a rabbit, a mouse, a rat, or a cartilaginous fish (e.g., a shark).

[0083] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that can arise during production of the antibody, such variants are typically present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and transgenic animals containing all or part of the human immunoglobulin loci, and other methods known in the art.

[0084] The term "natural antibody" as used herein refers to an antibody produced and paired by the immune system of a multicellular organism. The term "engineered antibody" as used herein refers to a non-natural antibody obtained by techniques of genetic engineering, antibody engineering, and the like. Exemplarily, "engineered antibodies" include chimeric antibodies, humanized antibodies, antibody fragments (e.g., scFv, sdAb, and the like), bispecific antibodies, and the like.

[0085] The term "monospecific" refers to having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.

[0086] The term "multispecific antibody" refers to having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," "tetraspecific," and the like refer to the number of different epitopes to which the antibody / antigen binding molecule can bind.

[0087] The term "valency" refers to the presence of a specified number of binding sites in an antibody / antigen binding molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen binding molecule.

[0088] The term "variable region" herein refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen, "variable region of the heavy chain" and "VH" or "HCVR" are used interchangeably, and "variable region of the light chain" and "VL" or "LCVR" are used interchangeably. The variable domains of the heavy chain and light chain (VH and VL, respectively) in a native antibody generally have similar structures and each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain can be sufficient to confer antigen-binding specificity. The term "complementarity determining region" and "CDR" are used interchangeably herein to refer to the hypervariable regions of a heavy chain variable region (VH) or light chain variable region (VL), which are also referred to as hypervariable loops (HVRs), because of their high degree of sequence variability in antibodies that specifically bind to antigens, and their ability to form structurally constrained, antigen binding sites that are specifically complementary to the surface of an antigen. The CDRs of a heavy chain variable region can be abbreviated as HCDRs, and the CDRs of a light chain variable region can be abbreviated as LCDRs. The term "framework region" or "FR region" is interchangeable and refers to those amino acid residues of a heavy chain variable region or light chain variable region that are not included in the CDRs. Typically, a canonical antibody variable region is composed of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0089] The CDRs herein can be annotated and defined in ways known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tools websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0090] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0091] The term "Chothia numbering system" herein generally refers to an immunoglobulin numbering system proposed by Chothia et al. that is based on the location of structural loops to identify CDR region boundaries (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0092] The term "IMGT numbering system" herein generally refers to a numbering system based on The international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0093] The term "heavy chain constant region" herein refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in binding of an antibody to an antigen, but exhibits effector functions such as interaction with Fc receptors, which has a more conserved amino acid sequence relative to the variable domain of the antibody. The "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The "heavy chain constant region" includes both "full length heavy chain constant region" and "heavy chain constant region fragment", the former has substantially similar structure as the native antibody constant region, while the latter only includes "a portion of the full length heavy chain constant region". Exemplarily, a typical "full length antibody heavy chain constant region" consists of CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it further includes CH4 domain; when the antibody is heavy chain antibody, it does not include CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" can be selected from CH1, Fc or CH3 domain.

[0094] The term "light chain constant region" herein refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding of an antibody to an antigen, which can be selected from constant kappa domain or constant lambda domain.

[0095] The term "Fc," as used herein, refers to the carboxy-terminal portion of an intact antibody that results from papain digestion of an antibody, typically containing the CH3 and CH2 domains of an antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the Fc region of human IgG heavy chains typically extends from an amino acid residue at about position Cys226, or from about Pro230 to the carboxy-terminus. The C-terminal lysine (residue 447 according to the Kabat numbering system) of the Fc region can be removed, for example, during production or purification of the antibody, or by recombinant engineering of nucleic acid encoding the antibody heavy chain, and therefore, the Fc region can include or not include the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447).

[0096] Typically, an IgG Fc region comprises IgG CH2 and IgG CH3 domains, optionally, on this basis, also the complete or partial hinge region, but not the CH1 domain. The "CH2 domain" of a human IgG Fc region extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the stretch of residues in the Fc region C-terminal to the CH2 domain (i.e., from an amino acid residue at about position 341 to an amino acid residue at about position 447 of IgG). The CH3 region herein can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with a "knob" introduced in one of its chains and a corresponding "hole" introduced in the other of its chains; see U.S. Patent No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains, as described herein.

[0097] Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0098] The term "epitope" herein includes any protein determinant capable of specific binding to an immunoglobulin, scFv or T-cell receptor. The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, an antibody can be raised against an N-terminal or C-terminal peptide of a polypeptide.

[0099] The term "mutation" herein includes genetic mutation and amino acid mutation, wherein the genetic mutation refers to deletion, insertion of heterologous nucleic acid, inversion or substitution, which can lead to change of amino acid sequence in the corresponding protein product; the amino acid mutation, also known as non-synonymous single nucleotide mutation, is due to the change of some single bases, resulting in the change of amino acid sequence in the protein product. The change of amino acid affects the stability, interaction and enzyme activity of the protein, thereby leading to the occurrence of diseases.

[0100] The term "amino acid" herein is the basic unit constituting a protein, which endows the protein with a specific molecular structure form, so that the molecule has biochemical activity. In chemistry, amino acid refers to an organic compound containing amino (-NH2) and carboxyl (-COOH) in its structure. According to the position of amino group connected to the carbon atom in carboxylic acid, amino acids can be divided into α, β, γ, δ, etc.: the amino group and carboxyl group of α-amino acid are connected to the same carbon atom, the amino group and carboxyl group of β-amino acid are connected to adjacent carbon atoms, and so on. In biology, amino acid usually specifically refers to α-amino acid, i.e. the amino acid whose amino group and carboxyl group are directly connected to the same -CH- structure, and its general formula is H2NCHRCOOH (R represents a certain organic substituent group). Exemplarily, the common 20 kinds of amino acids include glycine (Glycine), alanine (Alanine), valine (Valine), leucine (Leucine), isoleucine (Isoleucine), phenylalanine (Phenylalanine), tryptophan (Tryptophan), tyrosine (Tyrosine), aspartate (Aspartate), histidine (Histidine), asparagine (Asparagine), glutamate (Glutamate), lysine (Lysine), glutamine (Glutamine), methionine (Methionine), arginine (Arginine), serine (Serine), threonine (Threonine), cysteine (Cysteine), proline (Proline).

[0101] The term "amino acid substitution" herein refers to those where at least one amino acid residue in the native or starting sequence is removed and a different amino acid is inserted in its place. Substitutions can be single, where only one amino acid in a molecule has been substituted, or they can be multiple, where two or more amino acids in the same molecule have been substituted.

[0102] The term "conservative amino acid substitution" herein refers to the substitution of an amino acid that normally occurs in a sequence with a different amino acid that has similar size, charge, or polarity. Examples of conservative substitutions include substitution of non-polar (hydrophobic) residues such as isoleucine, valine, and leucine for one another. Likewise, examples of conservative substitutions include substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, substitution of a basic residue such as lysine, arginine, or histidine for another, or substitution of one acidic residue such as aspartic acid or glutamic acid for another is an additional example of a conservative substitution. Examples of non-conservative substitutions include substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or substitution of a polar residue for a non-polar residue.

[0103] The term "mutant" herein refers to a "variant" of the protein or peptide can have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid identity to the amino acid sequence of the protein or peptide.

[0104] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually denoted 5' to 3'. In the present context, the term nucleic acid molecule encompasses deoxyribonucleic acids (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acids (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. The nucleic acid molecule can be linear or circular. Furthermore, the term nucleic acid molecule includes both the sense and the antisense strand, as well as single- and double-stranded forms. Also, the nucleic acid molecules described herein can contain naturally-occurring or non-naturally-occurring nucleotides. Examples of non-naturally-occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. The nucleic acid molecule also encompasses DNA and RNA molecules which are suitable as vectors for the direct expression of an antibody of the present disclosure in vitro and / or in vivo, e.g. in a host or patient. Such DNA (e.g. cDNA) or RNA (e.g. mRNA) vectors can be unmodified or modified. For example, the mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that the mRNA can be injected into a subject to produce the antibody in vivo.

[0105] The term "identity" herein can be calculated by determining the percent "identity" of two amino acid sequences or two nucleic acid sequences, by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

[0106] The percent identity between two sequences is varied as a function of the number of identical positions shared by the sequences, taking into account the number of gaps required to achieve an optimal alignment of the two sequences and the length of each gap.

[0107] Sequence comparisons and percent identity calculations between two sequences can be accomplished using a mathematical algorithm. For instance, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap penalty of 16, 14, 12, 10, 8, 6, or 4, and a gap length penalty of 1, 2, 3, 4, 5, or 6. By way of further example, the percent identity between two nucleotide sequences can be determined using the GAP program (available at www.gcg.com), using a NWSgapdna.CMP matrix and gap weight of 40, 50, 60, 70, or 80, and a gap length penalty of 1, 2, 3, 4, 5, or 6. An especially preferred parameter set (and the one that should be used unless otherwise indicated) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0108] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0109] Additionally or alternatively, the nucleic acid sequences and protein sequences described in the present disclosure can be further used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. ((1990) J. Mol. Biol. 215:403-10). BLAST nucleotide searches can be performed with the NBLAST program. BLAST protein searches can be performed with the XBLAST program to achieve amino acid sequences homologous to the protein molecules of the present disclosure. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0110] The term "antigen chimeric receptor (CAR)" herein refers to an artificial cell surface receptor engineered to be expressed on an immune effector cell and specifically binds an antigen, which comprises at least (1) an extracellular antigen binding domain, such as a variable heavy chain or light chain of an antibody, (2) a transmembrane domain anchoring the CAR into the immune effector cell, and (3) an intracellular signaling domain. CARs are capable of redirecting T cells and other immune effector cells to a selected target, such as a cancer cell, in a non-MHC restricted manner using the extracellular antigen binding domain.

[0111] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses, or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vectors of the present disclosure contain polynucleotide sequences as well as additional sequence elements, such as for example, for the expression of proteins and / or for the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of the antibodies and antibody fragments of the present disclosure include plasmids containing regulatory sequences that direct transcription of the gene, such as promoter and enhancer regions. Other useful vectors for the expression of antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA produced from the transcription of the genes. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites in order to direct efficient transcription of the genes carried on the expression vectors. The expression vectors of the present disclosure can also contain a polynucleotide that encodes a marker for the selection of cells containing such vectors. Examples of suitable markers include genes that encode antibiotic (e.g., ampicillin, chloramphenicol, kanamycin, or neomycin) resistance.

[0112] The term "host cell" herein refers to a cell into which foreign nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include both the primary transformed cell and progeny of the primary transformed cell that are not identical to the parent cell in having the same genotypic characteristics but can not necessarily include every single cell that arose after the mutation. Mutant progeny that have the same functional or biological activity as screened or selected for in the initially transformed cell are included herein.

[0113] The term "pharmaceutical composition" herein refers to a preparation which is in a form suitable for its administration to a subject in need thereof and which contains an active ingredient in an effective amount to achieve the desired biological activity without additional ingredients that are unacceptable with respect to toxicity to the subject to which the pharmaceutical composition is administered.

[0114] The term "pharmaceutically acceptable excipient," "pharmaceutically acceptable carrier," or "pharmaceutically acceptable carrier, diluent or adjuvant" as used herein refers to an excipient that does not stimulate an undesirable response in an organism, and does not substantially interfere with the biological activity and / or property of the active compound. Suitable excipients are well known to those skilled in the art, and include, for example, carbohydrates, waxes, water soluble and / or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0115] The term "treatment" as used herein refers to a surgical or therapeutic treatment whose purpose is to prevent, slow down (reduce), or halt the progression of an undesirable physiological change or condition, such as cancer, immune-mediated inflammatory diseases (IMIDs) (e.g., autoimmune and inflammatory diseases), and viral infections, in a subject. Beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. A subject in need of treatment includes a subject who has a condition or disease, as well as a subject who is predisposed to having a condition or disease or who is intended to prevent a condition or disease. When referring to the terms slow down, reduce, diminish, palliate, alleviate, and the like, the meaning also includes elimination, disappearance, nonoccurrence, and the like.

[0116] The term "subject," "object," "patient" as used herein refers to an organism that receives treatment for a particular disease or condition as described in the present disclosure. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals, that receive treatment for a disease or condition. "Patient" also refers to an organism that receives treatment for a particular disease or condition as described herein, such as an immune-mediated inflammatory disease (IMID) or cancer.

[0117] The term "effective amount" as used herein refers to that amount of a therapeutic agent, alone or in combination with another therapeutic agent, which is effective to prevent or alleviate a disease condition or the progression of the disease in a cell, tissue, or subject. An "effective amount" also refers to the amount of a compound which is sufficient to reduce symptoms, e.g., to treat, cure, prevent, or alleviate the relevant medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of these conditions. When the active ingredient is administered individually to an individual, the therapeutically effective dose refers to the amount of the ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined amount of the active ingredients that produces the therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0118] The term "immune-mediated inflammatory disease" or "IMID" herein refers to any group of conditions or diseases that lack a clear etiology but are characterized by common inflammatory pathways that lead to inflammation, and which can result from or be triggered by a dysregulation of the normal immune response. "Immune-mediated inflammatory disease" herein encompasses both autoimmune diseases and inflammatory diseases.

[0119] "Autoimmune disease" herein refers to a condition in which a subject mounts an immune response against its own cells, tissues and / or organs, resulting in damage to the cells, tissues and / or organs. "Inflammatory disease" herein refers to a condition in a subject characterized by inflammation, preferably chronic inflammation. An autoimmune condition can or can not be accompanied by inflammation. Furthermore, inflammation can or can not be caused by an autoimmune condition. The terms "autoimmune disease", "autoimmune disorder" and "autoimmune-related disease" can be used interchangeably herein.

[0120] In some embodiments, exemplary immune-mediated inflammatory diseases include inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), multiple sclerosis, psoriasis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, diabetes (e.g., type 1 diabetes and type 2 diabetes), vasculitis, asthma, eczema, atopic dermatitis, fibrosis, transplant rejection, graft-versus-host disease, allergy, ankylosing spondylitis, celiac disease, chronic obstructive pulmonary disease (COPD), chronic peptic ulcer, cystic fibrosis, glomerulonephritis, gout, hepatitis (e.g., active hepatitis), myositis, osteoarthritis, pelvic inflammatory disease (PID), neurodegenerative diseases of aging, periodontal disease (e.g., periodontitis), pre-perfusion injury transplant rejection, psoriasis, pulmonary fibrosis, rheumatic disease, scleroderma, sinusitis, tuberculosis, arteriosclerosis, uveitis, and the like.

[0121] The term "cancer" herein refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition is benign cancer and malignant cancer. The term "tumor" or "neoplasm" herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein. BRIEF DESCRIPTION OF DRAWINGS

[0122] Figure 1 shows the binding activity of anti-TLl A chimeric heavy chain antibodies to human TLl A fusion protein.

[0123] Figure 2 shows the binding activity of anti-TLl A chimeric heavy chain antibodies to cynomolgus TLl A fusion protein.

[0124] Figure 3 shows the binding activity of anti-TLl A chimeric heavy chain antibodies to mouse TLl A fusion protein.

[0125] Figure 4 shows the binding activity of anti-TLlA chimeric heavy chain antibodies to CHO-K1 -human TLlA high expression cell line.

[0126] Figure 5 shows the effect of anti-TLlA chimeric heavy chain antibodies on blocking human TLlA interaction with CHO-K1 -human DR3 cells.

[0127] Figure 6 shows the effect of anti-TLlA chimeric heavy chain antibodies on blocking human TLlA interaction with DcR3 protein.

[0128] Figure 7 shows the anti-TLlA chimeric heavy chain antibodies inhibit NFkB activity in TF-1-NFkB-luciferase cells.

[0129] Figure 8 shows the anti-TLlA chimeric heavy chain antibodies inhibit apoptosis activity in TF-1 cells.

[0130] Figure 9 shows the anti-TLlA chimeric heavy chain antibodies inhibit human TLlA and IL-12 and IL-18 stimulated PBMC production of IFN-g secretion.

[0131] Figure 10 shows the binding activity of anti-TLlA humanized heavy chain antibodies to human TLlA fusion protein.

[0132] Figure 11 shows the binding activity of anti-TLlA humanized heavy chain antibodies to cynomolgus TLlA fusion protein.

[0133] Figure 12 shows the binding activity of anti-TLlA humanized heavy chain antibodies to mouse TLlA fusion protein.

[0134] Figure 13 shows the binding activity of anti-TLlA humanized heavy chain antibodies to CHO-K1 -human TLlA high expression cell line.

[0135] Figure 14 shows the effect of anti-TLlA humanized heavy chain antibodies on blocking human TLlA interaction with CHO-K1 -human DR3 cells.

[0136] Figure 15 shows the effect of anti-TLlA humanized heavy chain antibodies on blocking human TLlA interaction with DcR3 protein.

[0137] Figure 16 shows the anti-TLlA humanized heavy chain antibodies inhibit NFkB activity in TF-1-NFkB-luciferase cells.

[0138] Figure 17 shows the anti-TLlA humanized heavy chain antibodies inhibit apoptosis activity in TF-1 cells.

[0139] Figure 18 shows the inhibition of IFN-g secretion from human PBMCs stimulated with IL-12 and IL-18 by anti-TL1A humanized heavy chain antibodies. DETAILED DESCRIPTION

[0140] The advantages and features of the present disclosure will become apparent from the description, which is given, by way of example, with reference to the accompanying drawings. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased on the market.

[0141] The embodiments of the present disclosure are exemplary only and do not constitute any limitation on the scope of the present disclosure. Those skilled in the art should understand that the details and forms of the technical solutions of the present disclosure can be modified or replaced without departing from the spirit and scope of the present disclosure, and such modifications and replacements all fall within the protection scope of the present disclosure.

[0142] Example 1 Preparation of TL1A antigen

[0143] The human TL1A protein extracellular region (UniProt sequence number: O95150, SEQ ID NO: 1), cynomolgus monkey TL1A protein extracellular region (UniProt sequence number: G7PRK8, SEQ ID NO: 2), and mouse TL1A protein extracellular region (UniProt sequence number: Q5UBV8, SEQ ID NO: 3) were used as templates for TL1A in the embodiments of the present disclosure (Table 1). The amino acid sequences of the antigens and detection proteins involved in the present disclosure were designed, and different tags were optionally fused to the TL1A protein. The sequences were cloned into the PTT5 vector (Invitrogen), expressed in 293 cells by transient transfection or expressed in CHO cells by stable transfection, and purified to obtain the antigens and detection proteins of the present disclosure.

[0144] Table 1 Amino acid sequences of TL1A proteins

[0145] Example 2 Construction of CHO-K1 engineering cell strain

[0146] The nucleotide sequences corresponding to the amino acid sequences of human TL1A full-length (UniProt sequence number: O95150, SEQ ID NO: 4) and human DR3 full-length (UniProt sequence number: Q93038, SEQ ID NO: 5) (Table 2) were cloned into the pcDNA3.1 vector (purchased from Clontech) to prepare plasmids. The CHO-K1 cell line (purchased from the Chinese Academy of Sciences) was transfected with the plasmids (purchased from Clontech) to prepare the CHO-K1 engineering cell strain. After transfection with the 4D-Nucleofector® X Kit (Lonza, Cat. No. V4XP-3022) and the 4D-Nucleofector® X One Touch® Electroporation System (Lonza, Cat. No. VST-4X00), the cells were cultured in DMEM / F12 medium containing 10% (w / w) fetal bovine serum for 2 weeks, and then single-cell clones were plated into 96-well plates and cultured at 37 °C, 5% (v / v) CO2. After about 2 weeks, some single-cell clones were selected for expansion. The expanded clones were screened by flow cytometry. The cell lines with good growth, high fluorescence intensity and single clones were selected for further expansion and cryopreservation in liquid nitrogen.

[0147] Table 2 Human TL1A and human DR3 full-length amino acid sequences

[0148] Example 3 Construction of TF1-NFκB luciferase reporter cell line

[0149] The NF-kBre-nanoLuc plasmid (purchased from Promega) was used to transfect the human erytholeukemia cell line TF-1 (Cell Biolabs, Cat. No. CBP60808) which constitutively expresses the TL1A receptor DR3 (transfection was performed using the 4D-Nucleofector® X Kit (Lonza, Cat. No. L3000-015) after transfection with the 4D-Nucleofector® X Kit (Lonza, Cat. No. L3000-015) and the 4D-Nucleofector® X One Touch® Electroporation System (Lonza, Cat. No. VST-4X00), the cells were cultured in DMEM / F12 medium containing 10% (w / w) fetal bovine serum for 2 weeks, and then single-cell clones were plated into 96-well plates and cultured at 37 °C, 5% (v / v) CO2. After about 2 weeks, some single-cell clones were selected for expansion. The expanded clones were screened by flow cytometry. The cell lines with good growth, high fluorescence intensity and single clones were selected for further expansion and cryopreservation in liquid nitrogen. 3000 Transfection Kit, purchased from Invitrogen, Cat. No. L3000-015) after transfection with the 4D-Nucleofector® X Kit (Lonza, Cat. No. L3000-015) and the 4D-Nucleofector® X One Touch® Electroporation System (Lonza, Cat. No. VST-4X00), the cells were cultured in DMEM / F12 medium containing 10% (w / w) fetal bovine serum for 2 weeks, and then single-cell clones were plated into 96-well plates and cultured at 37 °C, 5% (v / v) CO2. After about 2 weeks, some single-cell clones were selected for expansion. The expanded clones were screened by flow cytometry. The cell lines with good growth, high fluorescence intensity and single clones were selected for further expansion and cryopreservation in liquid nitrogen.

[0150] Example 4 Preparation of control antibodies and detection antibodies

[0151] Using the control antibody RVT3101 sequence (sequence source: CN113150144A), PRA023 sequence (sequence source: CN114901311A / WO2022178159A1) and Teva-C320-168 sequence (sequence source: US20140255302A1) as templates, they were constructed into the PPT5-hIgG1 LALAGA vector (provided by General Biotech (Anhui) Co., Ltd.) for expression in Expi293F cells or stable expression in CHO cells for purification to obtain positive control antibodies (Table 3).

[0152] Table 3 Control antibody sequences

[0153] Generation of anti-human TL1A single-domain heavy chain antibodies

[0154] Anti-human TL1A single-domain heavy chain antibodies were obtained by immunizing lama or camel, and screened by yeast library screening or phage library screening, respectively. The detailed description is as follows.

[0155] 5.1. Llama immunization and yeast library construction and screening

[0156] Two llamas were immunized with human TL1A-his protein (Acro, item # TLA-H5243), and after 4 or 5 immunizations, the lymphocytes of 50 mL peripheral blood of the llama were extracted and total RNA was extracted using an RNA extraction kit (Takara, item # 9109). The extracted RNA was reverse transcribed into cDNA using PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, item # 6210A) according to the instructions. The nucleic acid fragments encoding the variable region of the heavy chain antibody were amplified by nested PCR, and the target single-domain heavy chain antibody nucleic acid fragments were recovered. The product and the yeast display vector pYDC3 (ApexBio) were co-electrotransformed into yeast cells to construct a single-domain heavy chain antibody yeast display library against the TL1A target and the library was tested. By gradient dilution plating, the size of the library capacity was calculated to be 10 8 The above. To detect the insertion rate of the library, 48 clones were randomly selected for colony PCR, and the results showed that the insertion rate had reached 100%.

[0157] Single-domain heavy chain antibody screening against TL1A: The antigen human TL1A-Biotin (biotin) (prepared in Example 1) was bound to magnetic beads, and the antigen-specific yeast was first enriched with magnetic beads. The yeast cells bound to the magnetic beads were further amplified for 15-16 hours, and the magnetic beads in the culture fluid were removed for preservation. The enriched yeast was stained with biotinylated antigen (concentration of 33.3 nM) and Streptavidin Protein (streptavidin), DyLight TM 650 (Thermo, item # 84547) and Anti-HA-AF488 Tag Antibody (Critical Point Bio, item # 003-101-007). Double positive cells (AF488 + , DyLight 650 +After sorting, the products were amplified and cultured in 2 mL of medium at 30°C for 16-18 hours, then plated and incubated at 30°C for 48 hours. Single clones were selected for colony identification and colony PCR, and then sent for sequencing. After single-clone colony expression, the colonies were reacted with the target protein human TL1A-biotin (using three concentrations: 33.3 nM, 11.1 nM, and 3.7 nM) and with Streptavidin Protein, DyLight, respectively. TM 650 was incubated at 4°C for 1 hour, followed by incubation at 4°C for 1 hour with Anti-HA-AF488. After washing, the cells were identified using flow cytometry. Based on sequencing results and monoclonal identification results, a subset of sequences was selected and constructed into a eukaryotic expression vector for further identification.

[0158] 5.2. Camel Immunology and Phage Library Construction and Screening

[0159] Two Xinjiang camels (Qingdao Antisai Biotechnology Co., Ltd.) were immunized with the human TL1A-his protein prepared in Example 1. After four or five immunizations, peripheral blood samples from the fifth and sixth immunizations were collected, and peripheral blood mononuclear cells (PBMCs) were separated according to the instructions for use of the lymphocyte separation medium. Total RNA was extracted from the fifth and sixth immunization PBMCs using Trizol reagent. PrimeScript was used. TM II. Reverse transcription was performed using the 1st Strand cDNA Synthesis Kit (Takara, catalog number #6210A) to prepare cDNA according to the manufacturer's instructions. Nested amplification was performed using 5-immune and 6-immune cDNA as templates, and the VHH fragment was recovered by gel excision. The vector and target fragment were digested with SfiI (NEB, catalog number #R0123L) overnight at 50°C and then recovered. The vector and target fragment were ligated at a molar ratio of 1:3 overnight at 6°C. The ligation product was desalted and concentrated, then gently mixed with TG1 electroporation competent cells (lucigen, catalog number #60502-2), incubated on ice for 5 min, and subjected to 5 electroporations. Immediately after each electroporation, 1 mL of SOC medium (preheated to 37°C) was added to the electroporation vessel for recovery. The electroporation product was aspirated, and the product was incubated at 37°C at 180 rpm for 45 min. 100 μL of the product was serially diluted to 10⁻⁶. -3 and 10 -4 The number of transformants in the reservoir was determined and coated onto a 90 mm plate, while the remainder was coated onto five 150 mm plates. The reservoir capacity was counted and calculated the following day; the reservoir capacities for the two camels were 8.75 × 10⁻⁶. 8 and 1.9×10 9 Ninety-six clones were randomly selected from each library, cultured, and sequenced. The correct insertion rates were 96.5% and 93%, respectively.

[0160] The E. coli library was inoculated into 400 mL of 2YT-AG (containing ampicillin: 100 μg / mL, glucose: 1%) medium to an initial OD600=0.1-0.2, and cultured at 37°C, 220 rpm until OD600=0.8 or more. The amount of helper phage M13K07 added was calculated according to OD600 (M13K07: E. coli = 20: 1), and after standing at 37°C for 30 min, the culture was incubated at 37°C, 180 rpm for 30 min. The supernatant was removed by centrifugation at 5000 rpm, and the 2YT-AK (containing 100 μg / mL ampicillin + 50 μg / mL kanamycin) culture was incubated at 30°C, 220 rpm overnight. The phage library was precipitated according to the NEB website procedure, and its titer was determined.

[0161] The human TL1A-his protein was selected by solid phase and liquid phase methods, respectively, and the input amount of the first round of phage library was 4.5 x 10 11 The antigen coating amount was 1 μg / well and 0.8 μg / well, respectively, the phage was eluted, its titer was determined, and 96 clones were randomly picked for preparation of phage supernatant, ELISA identification of phage binding to human TL1A protein, and the positive rate of one round of binding activity was higher than 78%; Subsequently, the antigen coating amount was reduced and the washing times were increased to enrich the dominant clones. In addition, the phage supernatant was identified by blocking experiment of TL1A and DR3 binding, and finally the clones with binding activity and blocking activity were selected for sequencing, and some sequences were constructed into eukaryotic expression vectors for further identification.

[0162] Example 6 Identification of Anti-human TL1A Chimeric Heavy Chain Antibody

[0163] After selection and preliminary identification, 49 single domain antibody sequences were selected to construct human-mouse chimeric heavy chain antibodies, and 8 chimeric heavy chain antibodies were identified: HcAb1, HcAb2, HcAb3, HcAb4, HcAb5, HcAb6, HcAb7 and HcAb8. Table 4 shows the sequences of the chimeric heavy chain antibodies, and Table 5 shows the CDR analysis results of the chimeric heavy chain antibodies.

[0164] Table 4 Chimeric Heavy Chain Antibody Sequences

[0165] Table 5 CDR Sequences of Chimeric Heavy Chain Antibodies

[0166] 6.1 Anti-TL1A Chimeric Heavy Chain Antibody Binding Activity Detection with Human TL1A-his Protein, Cynomolgus Monkey TL1A-his Protein and Mouse TL1A-his Protein

[0167] Human TL1A-his protein / cynomolgus monkey TL1A-his protein or mouse TL1A-his protein, prepared based on the extracellular region of TL1A protein in Example 1, was diluted to a final concentration of 2 μg / mL with PBS, and then added at 50 μL / well to a 96-well ELISA plate. The plate was sealed with plastic film and incubated overnight at 4°C. The next day, the plate was washed twice with PBST, and blocking buffer [PBS + 5% (w / w) skim milk] was added, blocking at room temperature for 1 hour. The plate was washed three times with PBST, and 100 nM of an 8-fold serially diluted anti-TL1A mouse monoclonal antibody or control antibody was added at 50 μL / well. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. HRP (horseradish peroxidase)-labeled secondary antibody (Jackson Immuno, catalog number #115-035-003) was added, and after incubation at 37°C for 1 hour, the plate was washed five times with PBST. TMB substrate was added at 50 μL / well. After incubation at room temperature for 4 minutes, stop solution (1.0N HCl) was added at 50 μL / well. OD450 nm values ​​were read using an ELISA plate reader (Multimode Plate Reader, EnSight, Perkin Elmer). Data were then analyzed using software (GraphPad Prism9) to perform data fitting and calculate EC50 values. The experimental results showed that all anti-TL1A chimeric heavy chain antibodies could effectively bind to human TL1A-his protein and cynomolgus monkey TL1A-his protein (Figure 1, Figure 2, and Table 6), and the HcAb1 antibody could also bind to mouse TL1A-his protein (Figure 3 and Table 6).

[0168] 6.2 FACS method for detecting the binding activity of anti-TL1A chimeric heavy chain antibody to CHO-K1 human TL1A-overexpressing cell line.

[0169] The CHO-K1-human TL1A cells (Clone 1F2, a human TL1A high-expressing cell line) prepared in Example 2 were cultured in T-75 cell culture flasks until 90% confluence. The culture medium was aspirated, and the cells were washed twice with PBS buffer. Then, the cells were digested with trypsin, and after stopping the digestion, they were washed twice with PBS buffer. After counting the cells from the previous step, the cell pellet was resuspended in [PBS + 2% (w / w) FBS] blocking solution to a concentration of 2 × 10⁻⁶ cells / mL. 6Cells were added to 96-well reaction plates at 5,000 cells / mL, 50 μL / well. 50 μL / well of 200 nM starting 4-fold dilution of the test sample was added to the plates and incubated on ice for 1 hour. Centrifugal washing with PBS buffer was performed 3 times, and 50 μL / well of Alexa Fluor 647-labeled secondary antibody (Jackson Immuno, Cat. #115-605-003) was added and incubated on ice for 1 hour. Centrifugal washing with PBS buffer was performed 5 times, and the results were detected and analyzed by flow cytometry (FACS CantoTM, purchased from BD company). Data analysis was performed by software (FlowJo), and the mean fluorescence intensity (MFI) of the cells was obtained. Analysis by software (GraphPad Prism9) was performed for data fitting, and the EC50 value was calculated. The experimental results showed that all anti-TL1A chimeric heavy chain antibodies could effectively bind to CHO-K1 human TL1A high expression cell strains (Figure 4, Table 6).

[0170] 6.3 FACS method for detecting anti-TL1A chimeric heavy chain antibody blocking human TL1A and CHO-K1 human DR3 cell interaction

[0171] CHO-K1 human DR3 cells prepared in Example 2 were expanded in a T-75 cell culture flask to 90% confluence, the culture medium was aspirated, washed with PBS buffer for 2 times, and then the cells were trypsinized, washed with PBS buffer for 2 times after stopping the digestion. After cell counting, the cells were diluted to 2 x 10 6 Cells were added to 96-well reaction plates at 5,000 cells / mL, 50 μL / well. The test sample was started at 400 nM, 3-fold dilution, mixed with hTL1A-his (prepared in Example 1) diluted to 0.4 μg / mL with PBS at 1:1, and incubated at room temperature for 30 minutes. After the incubation was completed, 50 μL of the mixture was added to the plate and incubated on ice for 1 hour. Centrifugal washing with PBS buffer was performed 3 times, and 50 μL / well of Alexa Fluor 647-labeled secondary antibody (GenScript, Cat. #A01802) was added and incubated on ice for 1 hour. Centrifugal washing with PBS buffer was performed 5 times, and the results were detected and analyzed by flow cytometry (FACS CantoTM, purchased from BD company). Data analysis was performed by software (FlowJo), and the mean fluorescence intensity (MFI) of the cells was obtained. Analysis by software (GraphPad Prism9) was performed for data fitting, and the IC50 value was calculated. The experimental results showed that the anti-TL1A chimeric heavy chain antibodies could effectively block the binding of TL1A protein to CHO-K1-human DR3 cells (Figure 5 and Table 6).

[0172] 6.4 ELISA method for detecting anti-TL1A chimeric heavy chain antibody inhibiting human TL1A and DcR3 interaction

[0173] Human DcR3 / TNFRSF6B, Fc Tag protein (Acro, Cat# TNB-H5255) was diluted with PBS to a final concentration of 2 pg / mL, then added to a 96-well ELISA plate at 50 pL / well. Incubate overnight at 4°C with plastic film, the next day wash the plate twice with PBST, add blocking solution [PBS + 5% (w / w) skim milk] and incubate for 1 hour at room temperature, wash the plate 3 times with PBST. Mix 400 nM of the antibody to be tested diluted in a 4-fold gradient with 1 pg / mL of biotinylated hTL1A protein (prepared in Example 1) at 1:1, and incubate for 30 min. After incubation, add 50 pL of the mixture to the ELISA plate, incubate for 1 hour at 37°C, then wash the plate 3 times with PBST. Add HRP (horseradish peroxidase) labeled secondary antibody (Sigma, Cat# S2438), incubate for 1 hour at 37°C, then wash the plate 5 times with PBST. Add TMB substrate at 50 pL / well. Incubate for 4 minutes at room temperature, then add stop solution (1.0 N HC1) at 50 pL / well. Read the OD450nm values with an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). Then analyze the data by software (GraphPad Prism 9) for data fitting, and calculate the IC50value. The experimental results show that the anti-TL1A chimeric heavy chain antibody can block the binding of TL1A to DcR3 (Figure 6 and Table 6). In addition, if the effect of the TL1A antibody on the binding of TL1A to the DcR3 decoy receptor is weak, this selectivity can bring better efficacy and safety.

[0174] 6.5 Detection of anti-TL1A chimeric heavy chain antibody inhibiting NFkB activity in TF-1-NFkB-luciferase cells

[0175] The TF-1-NFkB-luciferase cells prepared in Example 3 were expanded in a T-75 cell culture flask to the logarithmic growth phase, and the culture medium supernatant was directly centrifuged and discarded, and the cell pellet was washed twice with PBS. The cells were adjusted to a density of 8 x 105cells / mL with [1640 + 2% (w / w) FBS] medium, and 100 pL of the cell suspension was added to each well of a 96-well plate. Incubate at 37°C for 4-6 hours, then discard the medium supernatant and wash the cells twice with PBS. Add 100 pL of the test antibody solution to each well, and incubate at 37°C for 24 hours. After incubation, add 100 pL of lysis buffer to each well, and incubate at room temperature for 10 minutes. Then add 100 pL of luciferase substrate to each well, and incubate at room temperature for 10 minutes. Finally, read the luminescence value of each well with a microplate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The experimental results show that the anti-TL1A chimeric heavy chain antibody can inhibit the NFkB activity in TF-1-NFkB-luciferase cells (Figure 7 and Table 7). 5Each milliliter, 50 μL per well into white transparent reaction plate (Cavie Biological, item #62096), 37 ℃ cell incubator starvation for 24 hours. Human TL1A-his protein (preparation of Example 1) was diluted with [1640+2% (w / w) FBS] diluent to a final concentration of 0.6 μg / mL, 1:1 added to 4-fold gradient dilution of the test sample, pre-incubated at room temperature for 30 minutes. After pre-incubation, 50 μL of the mixture was added to the starved cell plate, and incubated in a 37 ℃ incubator for five hours. Detection was performed by Nano-Light luciferase reporter gene detection kit (Meridian Biological, item #MA0521-2), 50 μL / well of reagent was added, and the instrument PE EnSight microplate reader was used for reading. The results showed that the anti-TL1A chimeric heavy chain antibody could inhibit the downstream NFKB signaling pathway caused by the combination of human TL1A and its receptor (Fig. 7 and Table 6).

[0176] 6.6 Detection of anti-TL1A chimeric heavy chain antibody inhibition of TF-1 cell apoptosis activity

[0177] The total volume of the test system used in this example was 100 μL. Human TL1A protein (preparation of Example 1) was prepared with detection medium (RPMI1640+10% FBS+1x Penicillin / Streptomycin) to a final concentration of 100 ng / mL, and 20 μL / well was added to a 96-well flat-bottom white plate (Corning, item #3917). The TL1A antibody to be tested was diluted with the detection medium to a maximum final concentration of 32 nM, 2-fold dilution, 8 concentration gradients, and 20 μL / well was added to the 96-well flat-bottom white plate to mix with the TL1A protein, and incubated at room temperature for 30 minutes. Logarithmic growth phase TF-1 cells were collected and resuspended with detection medium, and CHX (MCE, item #HY-12320) was added to a final concentration of 10 μg / mL, 20000 cells / well of TF-1 cells were inoculated into the above-mentioned 96-well flat-bottom white plate, and mixed evenly. The cell plate was placed in a 37 ℃, 5% CO2 incubator for 24 hours. After 24 hours, the cell plate was taken out of the incubator and equilibrated to room temperature, 100 μL / well of equilibrated cell viability detection reagent Cell Counting-Lite 2.0 (Nanjing Nvzhan Biotech, item #DD1101-03) was added, placed on a shaker for 5 minutes, and incubated at room temperature for 10 minutes. The fluorescence signal was detected by a microplate reader (PerkinElmer, Envision). The results showed that the anti-TL1A chimeric heavy chain antibody could inhibit the apoptosis activity of TF-1 cells (Fig. 8 and Table 6).

[0178] Table 6 Identification of TL1A chimeric heavy chain antibody Note: PB: Partial binding; NB: No binding; Table 6 corresponds to isotype in Figure 1-7 as negative control, represented as N / A (Not Applicable).

[0179] 6.7 Detection of inhibition of IFN-γ secretion from PBMC stimulated by TL1A and IL-12 and IL-18 by anti-TL1A chimeric heavy chain antibodies

[0180] Isolation of CD4+T cells from PBMC of healthy human donors + T cells (Stemcell, Cat# 17952) were adjusted to a cell density of 1 million / mL, and CD4+T cells were transferred to 96-well U-bottom cell culture plates, corresponding to 100 μL per well. Appropriate amount of recombinant human interleukin 18 (rhIL-18, R&D, Cat# 9124-IL-050, working concentration 50 ng / mL) and recombinant human interleukin 12 (rhIL-4, Peprotech, Cat# 200-12, working concentration 2 ng / mL) and recombinant human TL1A (rhTL1A, prepared in Example 1, working concentration 50 ng / mL) were prepared, and 50 μL of rhIL-18 / rhIL-4 / rhTL1A cytokine mixture was added to each well to induce CD4+T cell activation. The tested TL1A antibodies or isotype control antibodies were prepared, corresponding to working concentrations of 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 and 0.078125 nM, and 50 μL of TL1A antibodies were added to each well. After mixing, the cells were incubated at 37 °C in a 5% CO2incubator for 2 days, and the cell culture supernatant was collected. The content of IFN-γ in the supernatant was detected using Human IFN-γ Precoated ELISA Kit (Dakewe, Cat# 1110003) according to the instructions. As shown in Figure 9, the anti-TL1A chimeric heavy chain antibodies of the present disclosure can effectively inhibit the secretion of IFN-γ from PBMC stimulated by TL1A and IL-12 and IL-18.

[0181] 6.8 BIAcore detection of the affinity of anti-TL1A chimeric heavy chain antibodies to human, cynomolgus monkey and mouse TL1A proteins

[0182] The specific binding between the tested anti-TL1A chimeric heavy chain antibodies and human, cynomolgus monkey TL1A protein and mouse TL1A was detected by BIAcore 8K. The experiment used a Protein A chip (purchased from cytiva, 29-1275-56) to capture the antibody, and gradient dilution of human TL1A-His, cynomolgus monkey TL1A-His or mouse TL1A-His protein based on the extracellular region of Example 1 was prepared, and the affinity of the antibody and the antigen was measured by multi-cycle kinetics. In each cycle, after capturing the antibody, gradient concentration of TL1A protein was injected, and the binding and dissociation process of the antigen and the antibody was recorded. After each cycle, the Protein A chip was regenerated (remove the protein on the chip) with Glycine pH 1.5 (purchased from cytiva, BR-1003-54). Finally, the data was analyzed by applying a 1:1 binding model to fit the antibody-antigen binding kinetics parameters, including the association rate constant ka, the dissociation rate constant kd, the equilibrium dissociation constant KD, and the maximum binding signal Rmax. The results in Table 7 show that all the tested anti-TL1A antibodies have specific binding with human or cynomolgus protein, and the affinity level is high.

[0183] Table 7 Affinity of anti-TL1A chimeric heavy chain antibodies to human, cynomolgus monkey, mouse TL1A protein

[0184] Example 7 Humanization of anti-human TL1A chimeric heavy chain antibodies

[0185] By comparing the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region gene database, the heavy chain variable region gene with high homology to the VHH single domain antibody was selected as a template, and the CDR of the VHH single domain antibody was transplanted into the corresponding human template to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. According to the needs, the key amino acids in the framework sequence are back-mutated to the corresponding amino acids of the VHH single domain antibody to ensure the original affinity, that is, to obtain a humanized anti-TL1A single domain antibody. The CDR amino acid residues of the antibody are usually determined and annotated by the Kabat numbering system.

[0186] 7.1 Humanization of HcAb1

[0187] The humanization template of the VHH single-domain antibody HcAb1 is IGHV3-7*01 and IGHJ6*01, and the CDRs of HcAb1 are transplanted into the humanized template, respectively, to obtain the corresponding humanized version. According to the needs, the key amino acids in the FR region sequence of the HcAb1 humanized antibody are back-mutated to the corresponding amino acids of the HcAb1 single-domain antibody to ensure the original affinity. The specific back-mutation design is shown in Table 8.

[0188] Table 8 Back-mutation design of humanized single-domain antibody of HcAb1 Note: Graft represents the transplantation of VHH single-domain antibody CDR into human germline template FR region sequence; V37F represents the mutation of V at position 37 of Graft to F, and the others are similar. The numbering of the back-mutation amino acids is the natural order numbering.

[0189] Table 9 CDR sequences (Kabat numbering) of the humanized single-domain antibody of HcAb1

[0190] The specific sequences of the variable regions of the HcAb1 humanized single-domain antibodies are as follows:

[0191] The amino acid sequence of HcAb1-H1 is shown as SEQ ID NO: 85:

[0192] The amino acid sequence of HcAb1-H2 is shown as SEQ ID NO: 86:

[0193] The amino acid sequence of HcAb1-H3 is shown as SEQ ID NO: 87:

[0194] The amino acid sequence of HcAb1-H4 is shown as SEQ ID NO: 88:

[0195] The amino acid sequence of HcAb1-H5 is shown as SEQ ID NO: 89:

[0196] The amino acid sequence of HcAb1-H6 is shown as SEQ ID NO: 90:

[0197] The amino acid sequence of the humanized heavy chain template IGHV3-7*01 is shown as SEQ ID NO: 91:

[0198] The amino acid sequence of the humanized heavy chain template IGHJ3*01 is shown as SEQ ID NO: 92:

[0199] 7.2 Humanization of HcAb5

[0200] The humanization template of VHH single domain antibody HcAb5 is IGHV3-7*01 and IGHJ6*01, and the CDRs of HcAb5 are transplanted into the human template respectively, that is, the corresponding humanized version is obtained. According to the needs, the key amino acids in the FR region sequence of the HcAb5 humanized antibody are back-mutated to the corresponding amino acids of the HcAb5 single domain antibody to ensure the original affinity. The specific back-mutation design is shown in Table 10.

[0201] Table 10 Back-mutation design of humanized single domain antibody of HcAb5 Note: Graft represents the transplantation of VHH single domain antibody CDR into human germline template FR region sequence; V37F indicates that the V at position 37 of Graft is mutated to F, and the others are similar. The numbering of the back-mutation amino acids is the natural order numbering.

[0202] Table 11 CDR sequences of humanized single domain antibody of HcAb5 (Kabat numbering)

[0203] The specific sequences of the variable regions of the humanized single domain antibodies of HcAb5 are as follows:

[0204] The amino acid sequence of HcAb5-H1 is shown as SEQ ID NO: 93:

[0205] The amino acid sequence of HcAb5-H2 is shown as SEQ ID NO: 94:

[0206] The amino acid sequence of HcAb5-H3 is shown as SEQ ID NO: 95:

[0207] The amino acid sequence of HcAb5-H4 is shown as SEQ ID NO: 96:

[0208] The amino acid sequence of HcAb5-H5 is shown as SEQ ID NO: 97:

[0209] The amino acid sequence of HcAb5-H6 is shown as SEQ ID NO: 98:

[0210] The amino acid sequence of the humanized heavy chain template IGHV3-7*01 is shown as SEQ ID NO: 91:

[0211] The amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown as SEQ ID NO: 99:

[0212] 7.3 Humanization of HcAb7

[0213] The humanized heavy chain template of VHH single domain antibody HcAb7 is IGHV3-30*01 and IGHJ3*01, and the CDRs of HcAb7 are respectively grafted into the human templates, i.e. the corresponding humanized versions are obtained. According to the needs, the key amino acids in the FR region sequence of the humanized antibody of HcAb7 are back-mutated to the corresponding amino acids of the HcAb7 single domain antibody to ensure the original affinity. The specific back-mutation design is shown in Table 12.

[0214] Table 12: Back-mutation design of humanized antibody of HcAb7 Note: Graft represents the grafting of CDR of VHH single domain antibody into FR region sequence of human germline template; V34F means mutating V at position 37 of Graft to F, and the others are similar. The numbering of back-mutation amino acids is the natural order numbering.

[0215] Table 13: CDR sequences (Kabat numbering) of humanized single domain antibody of HcAb7

[0216] The specific sequences of the variable region of HcAb7 humanized antibody are as follows:

[0217] The amino acid sequence of HcAb7.H5 is shown as SEQ ID NO: 102:

[0218] The amino acid sequence of HcAb7.H5a is shown as SEQ ID NO: 103:

[0219] The amino acid sequence of HcAb7.H5b is shown as SEQ ID NO: 104:

[0220] The amino acid sequence of HcAb7.H5c is shown as SEQ ID NO: 105:

[0221] The amino acid sequence of the humanized heavy chain template IGHV3-30*01 is shown as SEQ ID NO: 106:

[0222] The amino acid sequence of the humanized heavy chain template IGHJ3*01 is shown as SEQ ID NO: 92:

[0223] 7.4 Humanization of HcAb8

[0224] The humanized heavy chain template of VHH single domain antibody HcAb8 is IGHV3-7*01 and IGHJ3*01, and the CDRs of HcAb8 are transplanted into the humanized template respectively, i.e. the corresponding humanized version is obtained. According to the needs, the key amino acids in the FR region sequence of the humanized antibody of HcAb8 are back-mutated to the corresponding amino acids of the HcAb8 single domain antibody to ensure the original affinity. The specific back-mutation design is shown in Table 14.

[0225] Table 14 Back-mutation design of humanized single domain antibody of HcAb8 Note: Graft represents the transplantation of VHH single domain antibody CDR into human germline template FR region sequence; V37F indicates that the V at position 37 of Graft is mutated to F, and the others are similar. The numbering of the back-mutation amino acids is the natural order numbering.

[0226] Table 15 CDR sequences of humanized single domain antibody of HcAb8 (Kabat numbering)

[0227] The specific sequences of the variable regions of the humanized antibodies of HcAb8 are as follows:

[0228] The amino acid sequence of HcAb8.H2 is shown as SEQ ID NO: 107:

[0229] The amino acid sequence of HcAb8.H3 is shown as SEQ ID NO: 108:

[0230] The amino acid sequence of HcAb8.H8 is shown as SEQ ID NO: 109:

[0231] The amino acid sequence of HcAb8.H4a is shown as SEQ ID NO: 110:

[0232] The amino acid sequence of the humanized heavy chain template IGHV3-7*01 is shown as SEQ ID NO: 91:

[0233] The amino acid sequence of the humanized heavy chain template IGHJ1*01 is shown as SEQ ID NO: 111:

[0234] Example 8 Identification of anti-TL1A humanized heavy chain antibodies

[0235] After expression, protein binding and blocking identification of all humanized molecules of antibodies, part of the humanized molecules of each molecule are selected for further identification.

[0236] 8.1 Detection of binding activity of anti-TLlA humanized heavy chain antibodies to human, cynomolgus and mouse TLlA-His proteins

[0237] The specific method is referred to Example 6.1. As shown in Table 16 and Figures 10-12, all the anti-TLlA humanized heavy chain antibodies can effectively bind to human or cynomolgus TLlA-His proteins; the HcAb1 humanized antibody can also bind to mouse TLlA-his protein.

[0238] 8.2 Detection of binding activity of anti-TLlA humanized heavy chain antibodies to CHO-K1-human TLlA high expression cell strain by FACS method

[0239] The specific method is referred to Example 6.2. As shown in Table 16 and Figure 13, the results show that all the humanized heavy chain antibodies can effectively bind to CHO-K1-human TLlA high expression cell strain.

[0240] 8.3 Detection of anti-TLlA humanized heavy chain antibodies inhibiting the interaction of human TLlA with CHO-K1-human DR3 cells by FACS method

[0241] The specific method is referred to Example 6.3. As shown in Table 16 and Figure 14, the experimental results show that the humanized heavy chain antibodies can effectively block the binding of TLlA protein to CHO-K1-human DR3 cells.

[0242] 8.4 Detection of anti-TLlA humanized heavy chain antibodies inhibiting the interaction of human TLlA with DcR3 by ELISA method

[0243] The specific method is referred to Example 6.4. As shown in Table 16 and Figure 15, the experimental results show that the humanized heavy chain antibodies can partially block the binding of TLlA protein to DcR3 protein. As mentioned before, the weaker effect of TLlA antibodies on the binding of TLlA to DcR3 pseudo-receptor can bring better efficacy and safety.

[0244] 8.5 Detection of anti-TLlA humanized heavy chain antibodies inhibiting the NFκB activity in TF-1-NFκB-luciferase cells.

[0245] The specific method is referred to Example 6.5. As shown in Table 16 and Figure 16, the experimental results show that the humanized heavy chain antibodies can effectively inhibit the NFκB activity in TF-1-NFκB-luciferase cells.

[0246] 8.6 Detection of anti-TLlA humanized heavy chain antibodies inhibiting the apoptosis activity of TF-1 cells.

[0247] The specific method is referred to Example 6.6. As shown in Table 16 and Figure 17, the experimental results show that HcAb1, HcAb5, HcAb7 and HcAb8 humanized heavy chain antibodies can all inhibit the apoptosis activity of TF-1 cells.

[0248] Table 16 Identification of TL1A humanized heavy chain antibodies Note: Table 16 corresponds to isotype in Figures 10-17 of the accompanying drawings, which is shown as N / A (Not Applicable)

[0249] 8.7 Detection of the inhibition of IFN-γ secretion by anti-TL1A humanized heavy chain antibodies on human TL1A and IL-12 and IL-18 stimulated PBMC.

[0250] The specific method is referred to Example 6.7. As shown in Figure 18, the results show that the humanized heavy chain antibodies can all effectively inhibit the IFN-γ secretion by human TL1A and IL-12 and IL-18 stimulated PBMC.

[0251] 8.8 BIAcore detection of the affinity of anti-TL1A humanized heavy chain antibodies to human, cynomolgus monkey and mouse TL1A proteins

[0252] The specific method is referred to Example 6.8. The results in Table 17 show that all the tested anti-TL1A antibodies have specific binding between human or cynomolgus monkey proteins, and the affinity level is high. Among them, the HcAb1 chimeric heavy chain antibody and the humanized antibody can bind to mouse TL1A, while the HcAb5, HcAb7, HcAb8 chimeric heavy chain antibodies and humanized antibodies do not bind to mouse TL1A.

[0253] Table 17 Affinity of anti-TL1A humanized heavy chain antibodies to human, cynomolgus monkey and mouse TL1A proteins

[0254] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0255] While example embodiments have been particularly shown and described, a person skilled in the art will understand that various changes in form and detail can be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

1. An antibody or antigen binding fragment that specifically binds TL1A, wherein, the antibody or antigen-binding fragment comprises a CDR1, a CDR2, and a CDR3, the CDR1, CDR2, and CDR3 each has an amino acid sequence as set forth in a CDR1, CDR2, and CDR3, respectively, of any one of SEQ ID NOs: 12-19, 85-90, 93-98, 102-105, or 107-110.

2. The antibody or antigen-binding fragment of claim 1, wherein, the CDR1 has an amino acid sequence as set forth in SEQ ID NO: 29, 32, 35, 43, 45, 46, 49, 52, 56, 59, 61, 64, 67, 69, 72, 75, 77, 80, or 83; and / or the CDR2 has an amino acid sequence as set forth in 30, 33, 36, 37, 39, 41, 47, 50, 53, 54, 57, 60, 62, 65, 68, 70, 73, 76, 78, 81, 84, 100, or 101; and / or the CDR3 has an amino acid sequence as set forth in SEQ ID NO: 31, 34, 38, 40, 42, 44, 48, 51, 55, 58, 63, 66, 71, 74, 79, or 82; Preferably, the CDR1-CDR3 are determined according to the Kabat, IMGT, or Chothia method.

3. The antibody or antigen-binding fragment of claim 1 or 2, wherein, the antibody or antigen-binding fragment comprises a CDR1, a CDR2, and a CDR3, (1) the CDR1, CDR2, and CDR3 each has an amino acid sequence as set forth in SEQ ID NO: 29-31; the CDR1, CDR2, and CDR3 each has an amino acid sequence as set forth in SEQ ID NO: 32-34; or the CDR1, CDR2, and CDR3 each has an amino acid sequence as set forth in SEQ ID NO: 35-36 and 31; and / or (2) the CDR1, CDR2, and CDR3 each has an amino acid sequence as set forth in SEQ ID NO: 29 and 37-38; the CDR1, CDR2, and CDR3 each has an amino acid sequence as set forth in SEQ ID NO: 32 and 39-40; or the CDR1, CDR2, and CDR3 each has an amino acid sequence as set forth in SEQ ID NO: 35, 41, and 38; and / or (3) the CDR1, CDR2, and CDR3 each has an amino acid sequence as set forth in SEQ ID NO: 29, 37, and 42; the CDR1, CDR2, and CDR3 each has an amino acid sequence as set forth in SEQ ID NO: 43, 39, and 44; or the CDR1, CDR2, and CDR3 each has an amino acid sequence as set forth in SEQ ID NO: 45 and 41-42; (4) the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 46-48, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 49-51, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 52-53 and 48, respectively; (5) the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 46 and 54-55, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 56-58, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 59-60 and 55, respectively; (6) the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 61-63, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 64-66, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 67-68 and 63, respectively; (7) the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 69-71, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 72-74, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 75-76 and 71, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 69, 100 and 71, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 69, 101 and 71, respectively; (8) the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 77-79, respectively; the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 80-82, respectively; or the CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs: 83-84 and 79, respectively.

4. The antibody or antigen binding fragment of any one of claims 1-3, wherein, the antibody or antigen binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 12-19, 85-90, 93-98, 102-105 or 107-110; or an amino acid sequence that has at least 70% identity or at most 15 amino acid mutations compared to the amino acid sequence of any one of SEQ ID NOs: 12-19, 85-90, 93-98, 102-105 or 107-110.

5. The antibody or antigen-binding fragment of any one of claims 1-4, wherein, The antibody or antigen-binding fragment comprises a FR region in any one of SEQ ID NOs: 12-19, 85-90, 93-98, 102-105, or 107-110, or a FR region having at least 70% identity or at most 15 amino acid mutations compared to a FR region in any one of SEQ ID NOs: 12-19, 85-90, 93-98, 102-105, or 107-110.

6. The antibody or antigen-binding fragment of any one of claims 1-5, comprising or not comprising an antibody heavy chain constant region; optionally, the antibody heavy chain constant region is selected from human, llama, mouse, rat, rabbit, or sheep; optionally, the antibody heavy chain constant region is selected from IgG, IgM, IgA, IgE, or IgD, the IgG is selected from IgGl, IgG2, IgG3, or IgG4; optionally, the heavy chain constant region is selected from an Fc region, a CH3 region, or a complete heavy chain constant region, preferably, the heavy chain constant region is a human Fc region; Preferably, the antibody or antigen-binding fragment is a heavy chain antibody. Preferably, the Fc region has an amino acid sequence as set forth in SEQ ID NO: 20, or an amino acid sequence having at least 70% identity or at most 15 amino acid mutations compared thereto.

7. The antibody or antigen-binding fragment of any one of claims 1-6, wherein, The antibody or antigen-binding fragment comprises a heavy chain HC having an amino acid sequence as set forth in any one of SEQ ID NOs: 21-28, or an amino acid sequence having at least 70% identity or at most 15 amino acid mutations compared to an amino acid sequence as set forth in any one of SEQ ID NOs: 21-28.

8. The antibody or antigen-binding fragment of any one of claims 1-7, wherein, The at least 70% identity is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; the at most 15 amino acid mutations is at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid mutations; optionally, the mutations are selected from substitution, deletion, or insertion mutations; optionally, the mutations are back mutations or hot spot mutations; preferably, the substitutions are conservative amino acid substitutions.

9. The antibody or antigen-binding fragment of any one of claims 1-8, wherein, The antibody or antigen-binding fragment comprises: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; and / or (3) a fully human antibody or fragment thereof.

10. The antibody or antigen-binding fragment of any one of claims 1-9, wherein, The antibody or antigen-binding fragment is capable of binding to human TL1A, monkey TL1A, and / or murine TL1A.

11. The antibody or antigen-binding fragment of any one of claims 1-10, wherein, The antibody or antigen-binding fragment further comprises a conjugate; optionally, the conjugate is selected from a therapeutic agent or a tracer, optionally, the therapeutic agent is selected from a radioisotope, a chemotherapeutic agent, or an immunomodulatory agent, optionally, the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent, or a photosensitizer.

12. The antibody or antigen-binding fragment of any one of claims 1-11, wherein, The antibody or antigen-binding fragment binds to human TL1A, monkey TL1A, or murine TL1A with a KD value of less than 1E-6 M, 1E-7 M, 1E-8 M, 1E-9 M, 1E-10 M, or 1E-11 M.

13. The antibody or antigen-binding fragment of any one of claims 1-12, wherein, The antibody or antigen-binding fragment is further linked to another functional molecule, preferably the another functional molecule is selected from one or more of the following: a signal peptide, a protein tag, a cytokine, an angiogenesis inhibitor, or an immune checkpoint inhibitor.

14. A multispecific antigen-binding molecule, wherein, The multispecific antigen-binding molecule comprises the antibody or antigen-binding fragment of any one of claims 1-13; and another antigen-binding molecule that binds to another antigen other than TL1A, or another antigen-binding molecule that binds to a different epitope of TL1A than the aforementioned antibody or antigen-binding fragment; preferably the another antigen-binding molecule is an antibody or antigen-binding fragment; Optionally, the another antigen is selected from the group consisting of: (1) a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA); (2) an immune checkpoint; and (3) a target that recruits and / or activates an immune cell; Optionally, the multispecific antigen-binding molecule is bispecific, trispecific, or tetraspecific; Optionally, the multispecific antigen-binding molecule is bivalent, tetravalent, or hexavalent.

15. A chimeric antigen receptor (CAR), wherein, The chimeric antigen receptor comprises at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, the extracellular antigen-binding domain comprises the antibody or antigen-binding fragment of any one of claims 1-13 or the multispecific antigen-binding molecule of claim 14.

16. An immune effector cell, wherein, The immune effector cell expresses and / or comprises a nucleic acid molecule encoding the chimeric antigen receptor of claim 15. Optionally, the immune effector cell is selected from a T cell, an NK cell, an NKT cell, a monocyte, a macrophage, a dendritic cell, or a mast cell; Optionally, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell. Preferably, the T cell is selected from a cytotoxic T cell, a regulatory T cell, or a helper T cell.

17. An isolated nucleic acid molecule, wherein, The nucleic acid molecule encodes the antibody or antigen-binding fragment of any one of claims 1-13, the multispecific antigen-binding molecule of claim 14, or the chimeric antigen receptor of claim 15.

18. A vector, wherein, The vector comprises the nucleic acid molecule of claim 17.

19. A cell, wherein, The cell comprises the nucleic acid molecule of claim 17 or the vector of claim 18.

20. A method of making the antibody or antigen binding fragment of any one of claims 1-13, or the multispecific antigen binding molecule of claim 14, wherein, The method comprises: (1) culturing the cell of claim 19 and / or (2) isolating the antibody or antigen-binding fragment, or the multispecific antigen-binding molecule, expressed by the cell.

21. A method of making the immune effector cell of claim 16, wherein, The method comprises introducing into the immune effector cell a nucleic acid molecule encoding the chimeric antigen receptor of claim 15, and / or initiating expression of the chimeric antigen receptor by the immune effector cell.

22. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the antibody or antigen binding molecule of any one of claims 1 to 13, or the multispecific antigen binding molecule of claim 14, or the immune effector cell of claim 16, or the nucleic acid molecule of claim 17, or the vector of claim 18, or the cell of claim 19, or the product produced according to the method of any one of claims 20 to 21 ; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition further comprises an additional anti-neoplastic agent.

23. Use of the antibody or antigen binding molecule of any one of claims 1 to 13, or the multispecific antigen binding molecule of claim 14, or the immune effector cell of claim 16, or the nucleic acid molecule of claim 17, or the vector of claim 18, or the cell of claim 19, the pharmaceutical composition of claim 22, or the product produced according to the method of any one of claims 20 to 21 for the manufacture of a medicament for the treatment of an immune-mediated inflammatory disease (IMID); preferably, the immune-mediated inflammatory disease (IMID) is an autoimmune and / or inflammatory disease; more preferably, the immune-mediated inflammatory disease (IMID) is selected from one or more of inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, diabetes, vasculitis, asthma, eczema, atopic dermatitis, fibrosis, transplant rejection, graft-versus-host disease, allergy, ankylosing spondylitis, celiac disease, chronic obstructive pulmonary disease (COPD), chronic peptic ulcer, cystic fibrosis, glomerulonephritis, gout, hepatitis, myositis, osteoarthritis, pelvic inflammatory disease (PID), neurodegenerative diseases of aging, periodontal disease, preperfusion injury transplant rejection, psoriasis, pulmonary fibrosis, rheumatic disease, scleroderma, sinusitis, tuberculosis, arteriosclerosis, and uveitis.

24. A method of treating an immune-mediated inflammatory disease (IMID), wherein, The method comprises administering to the subject an effective amount of the antibody or antigen binding molecule of any one of claims 1-13, or the multispecific antigen binding molecule of claim 14, or the immune effector cell of claim 16, or the nucleic acid molecule of claim 17, or the vector of claim 18, or the cell of claim 19, the pharmaceutical composition of claim 22, or the product prepared according to the method of any one of claims 20-21, preferably, the immune-mediated inflammatory disease (IMID) is an autoimmune disease and / or an inflammatory disease; more preferably, the immune-mediated inflammatory disease (IMID) is selected from one or more of inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, diabetes, vasculitis, asthma, eczema, atopic dermatitis, fibrosis, transplant rejection, graft-versus-host disease, allergy, ankylosing spondylitis, celiac disease, chronic obstructive pulmonary disease (COPD), chronic peptic ulcer, cystic fibrosis, glomerulonephritis, gout, hepatitis, myositis, osteoarthritis, pelvic inflammatory disease (PID), a neurodegenerative disease of aging, periodontal disease, pre-perfusion injury transplant rejection, psoriasis, pulmonary fibrosis, rheumatic disease, scleroderma, sinusitis, tuberculosis, arteriosclerosis, and uveitis.

25. The antibody or antigen binding molecule of any one of claims 1-13, or the multispecific antigen binding molecule of claim 14, or the immune effector cell of claim 16, or the nucleic acid molecule of claim 17, or the vector of claim 18, or the cell of claim 19, the pharmaceutical composition of claim 22, or the product prepared according to the method of any one of claims 20-21, for use in the treatment of an immune-mediated inflammatory disease (IMID) disease, preferably, the immune-mediated inflammatory disease (IMID) is an autoimmune disease and / or an inflammatory disease; more preferably, the immune-mediated inflammatory disease (IMID) is selected from one or more of inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, diabetes, vasculitis, asthma, eczema, atopic dermatitis, fibrosis, transplant rejection, graft-versus-host disease, allergy, ankylosing spondylitis, celiac disease, chronic obstructive pulmonary disease (COPD), chronic peptic ulcer, cystic fibrosis, glomerulonephritis, gout, hepatitis, myositis, osteoarthritis, pelvic inflammatory disease (PID), a neurodegenerative disease of aging, periodontal disease, pre-perfusion injury transplant rejection, psoriasis, pulmonary fibrosis, rheumatic disease, scleroderma, sinusitis, tuberculosis, arteriosclerosis, and uveitis.

Images