Bispecific antibody aiming at TL1A and TNF-alpha and application thereof

Abstract

The present disclosure relates to a bispecific antibody comprising a first domain that specifically binds to TNF-like ligand 1a (TL1A), and a second domain that specifically binds to tumor necrosis factor alpha (TNF-alpha). The present disclosure also relates to methods of using said bispecific antibodies in the treatment or prevention of diseases associated with TL1A and / or TNF-alpha.

Description

Technical Field

[0001] This disclosure relates to bispecific antibodies against TL1A and TNF-α and their applications. Background Technology

[0002] TNF-like ligand 1a (TL1A, also known as TNF superfamily member 15 (TNFSF15)) is a member of the tumor necrosis factor superfamily. It is a type II membrane protein that is expressed by antigen-presenting cells (including dendritic cells, B cells, and macrophages), CD4+ and CD8+ T cells, and endothelial cells. It can be expressed on the cell surface or secreted in the form of soluble cytokines.

[0003] TL1A is associated with the development and severity of inflammatory bowel disease (IBD), the main types of which are ulcerative colitis (UC) and Crohn's disease (CD). These diseases are very common, and the number of available treatments is limited, with some patients not responding to initial treatment or losing their response over time. Currently, surgery is the only treatment option for patients who do not respond to first-line therapy. Surgical treatment of IBD is invasive, and approximately one-third of patients experience postoperative risks after surgery. The pathogenesis of IBD is believed to involve an uncontrolled immune response, and the heterogeneity of disease pathogenesis and clinical course suggests that targeted therapies are an ideal treatment strategy. Therefore, novel therapies specifically targeting the pathogenesis of IBD, and drugs that inhibit TL1A activity, have become popular targets for the development of IBD treatments.

[0004] Tumor necrosis factor-α (TNF-α) is a homotrimeric protein composed of 157 amino acids, mainly produced by activated macrophages, T lymphocytes, and natural killer cells. It plays an important role in multiple physiological processes such as immune response, inflammation, and apoptosis. Members of the TNF receptor family include TNFR1 and TNFR2. TNFR1 and TNFR2 are receptor proteins on the cell membrane surface that can bind to TNF-α and transmit cellular signals. Among them, TNFR1 mainly plays a pro-inflammatory role. When cells are subjected to inflammatory stimulation or stress, TNFR1 expression increases, and the TNF-α it binds to activates a series of signal transduction pathways, triggering biological processes such as apoptosis and inflammatory responses. Currently, blocking TNF-TNFR1 by binding to TNFR1 and its ligand TNF is one of the important therapeutic strategies for treating inflammation. Currently, there are various TNF-α inhibitors on the market, mainly divided into two categories: fusion proteins and monoclonal antibodies. Adalimumab, the world's largest-selling TNF-α inhibitor, has shown outstanding performance in the treatment of various diseases, such as rheumatoid arthritis, ankylosing spondylitis, and psoriasis.

[0005] However, there are currently no similar dual-target drugs approved for marketing in China or abroad. Summary of the Invention

[0006] To address one of the technical problems existing in the prior art, this disclosure provides a bispecific antibody that can better maintain the activity of its respective monoclonal antibody and can simultaneously and specifically bind to two targets, TNF-like ligand 1a (TL1A) and tumor necrosis factor α (TNF-α), exhibiting biological activity similar to or even superior to that of the monoclonal antibody.

[0007] One aspect of this disclosure provides a bispecific antibody comprising: a first domain that specifically binds to TNF-like ligand 1a (TL1A) and a second domain that specifically binds to tumor necrosis factor α (TNF-α).

[0008] In some embodiments, the first domain is an antibody that specifically binds to TL1A or a functional fragment thereof.

[0009] In some embodiments, the second domain is an antibody that specifically binds to TNF-α or a functional fragment thereof.

[0010] In some embodiments, the first domain includes an antibody Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, nanobody, heavy chain variable region (VH) fragment, and / or light chain variable region (VL) fragment that specifically binds to TL1A.

[0011] In some embodiments, the second domain includes an antibody Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, nanobody, heavy chain variable region (VH) fragment, and / or light chain variable region (VL) fragment that specifically binds to TNF-α.

[0012] In some implementations, the first domain and the second domain are directly connected or connected through connectors.

[0013] In some embodiments, the connector has a general formula (G) n S) m The amino acid sequence shown has n and m as integers from 1 to 10; more preferably, n is an integer from 1 to 4 and m is an integer from 1 to 3, or is consistent with the general formula (G n S) m The amino acid sequence shown is compared to an amino acid sequence having 1, 2, or 3 inserted, substituted, or deleted amino acids. In some embodiments of this disclosure, the link comprises at least 6 amino acids.

[0014] In some embodiments, the bispecific antibody includes a first domain and a second domain connected in any manner. In some embodiments, the bispecific antibody includes a first domain-second domain extending from the N-terminus to the C-terminus. In some embodiments, the bispecific antibody includes a second domain-first domain extending from the N-terminus to the C-terminus.

[0015] In some implementations, the first domain includes the following three heavy chain variable region complementarity-determining regions (HCDRs):

[0016] HCDR1, having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in SEQ ID NO:1 or 3, or having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR1 contained in the heavy chain variable region.

[0017] HCDR2, having the amino acid sequence of HCDR2 contained in the heavy chain variable region as shown in any of SEQ ID NO:1 or 3, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR2 contained in the heavy chain variable region.

[0018] HCDR3, having the amino acid sequence of HCDR3 contained in the heavy chain variable region as shown in any of SEQ ID NO:1 or 3, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR3 contained in the heavy chain variable region.

[0019] In some embodiments, the first domain includes the following three light chain variable region complementarity-determining regions (LCDRs):

[0020] LCDR1, having the amino acid sequence of LCDR1 contained in the light chain variable region as shown in SEQ ID NO:2 or 4, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR1 contained in the light chain variable region.

[0021] LCDR2, having the amino acid sequence of LCDR2 contained in the light chain variable region as shown in SEQ ID NO:2 or 4, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR2 contained in the light chain variable region.

[0022] LCDR3 having the amino acid sequence of LCDR3 contained in the light chain variable region as shown in SEQ ID NO:2 or 4, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR3 contained in the light chain variable region.

[0023] In some embodiments, the first structural domain includes HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:1, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:2.

[0024] In some embodiments, the first structural domain includes HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:3, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:4.

[0025] In some implementations, the first structural domain includes the HCDR1-3 and / or the LCDR1-3 is defined by the IMGT numbering system, the Kabat numbering system, the Chothia numbering system, the Contact numbering system, or a combination thereof.

[0026] In some embodiments, the first structural domain includes the following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the IMGT numbering system: (a) a heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:7, HCDR2 with sequence SEQ ID NO:8, and HCDR3 with sequence SEQ ID NO:9; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:10, LCDR2 with sequence YAT, and LCDR3 with sequence SEQ ID NO:11; or (b) a heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:25, HCDR2 with sequence SEQ ID NO:26, and HCDR3 with sequence SEQ ID NO:27; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:28, LCDR2 with sequence DAS, and LCDR3 with sequence SEQ ID NO:29. LCDR3, NO:29.

[0027] In some embodiments, the first structural domain includes the following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Kabat numbering system as follows: (a) a heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:12, HCDR2 with sequence SEQ ID NO:13, and HCDR3 with sequence SEQ ID NO:14; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:15, LCDR2 with sequence SEQ ID NO:16, and LCDR3 with sequence SEQ ID NO:11; or (b) a heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:30, HCDR2 with sequence SEQ ID NO:31, and HCDR3 with sequence SEQ ID NO:32; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:33, LCDR2 with sequence SEQ ID NO:30, LCDR2 with sequence SEQ ID NO:31, and LCDR3 with sequence SEQ ID NO:32; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:33, LCDR2 with sequence SEQ ID NO:30, LCDR3 with sequence SEQ ID NO:31, LCDR2 with sequence SEQ ID NO:32, and LCDR3 with sequence SEQ ID NO:33. LCDR2 with sequence number NO:34, and LCDR3 with sequence number NO:29.

[0028] In some embodiments, the first structural domain includes the following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Chothia numbering system: (a) a heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:17, HCDR2 with sequence SEQ ID NO:18, and HCDR3 with sequence SEQ ID NO:14; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:15, LCDR2 with sequence SEQ ID NO:16, and LCDR3 with sequence SEQ ID NO:11; or (b) a heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:35, HCDR2 with sequence SEQ ID NO:36, and HCDR3 with sequence SEQ ID NO:32; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:33, LCDR2 with sequence SEQ ID NO:34, LCDR3 with sequence SEQ ID NO:35, LCDR2 with sequence SEQ ID NO:36, and LCDR3 with sequence SEQ ID NO:35. LCDR2 with sequence number NO:34, and LCDR3 with sequence number NO:29.

[0029] In some embodiments, the first structural domain includes the following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Contact numbering system: (a) a heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:19, HCDR2 with sequence SEQ ID NO:20, and HCDR3 with sequence SEQ ID NO:21; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:22, LCDR2 with sequence SEQ ID NO:23, and LCDR3 with sequence SEQ ID NO:24; or (b) a heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:37, HCDR2 with sequence SEQ ID NO:38, and HCDR3 with sequence SEQ ID NO:39; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:40, LCDR2 with sequence SEQ ID NO:39, LCDR3 with sequence SEQ ID NO:39, and LCDR3 with sequence SEQ ID NO:39. LCDR2 with sequence number NO:41, and LCDR3 with sequence number NO:42.

[0030] In some embodiments, the first structural domain comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1 or 3, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the heavy chain, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the heavy chain variable region; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:2 or 4, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the light chain variable region, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the light chain variable region.

[0031] In some embodiments, the first domain includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:2.

[0032] In some embodiments, the first domain includes a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:4.

[0033] In some embodiments, the second structural domain includes the following three heavy chain variable region complementarity-determining regions (HCDRs):

[0034] HCDR1, having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in SEQ ID NO:5, or having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR1 contained in the heavy chain variable region.

[0035] HCDR2, having the amino acid sequence of HCDR2 contained in the heavy chain variable region as shown in SEQ ID NO:5, or having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR2 contained in the heavy chain variable region.

[0036] HCDR3, having the amino acid sequence of HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:5, or having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR3 contained in the heavy chain variable region.

[0037] In some embodiments, the second structural domain includes the following three light chain variable region complementarity-determining regions (LCDRs):

[0038] LCDR1, having the amino acid sequence of LCDR1 contained in the light chain variable region as shown in SEQ ID NO:6, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR1 contained in the light chain variable region.

[0039] LCDR2, having the amino acid sequence of LCDR2 contained in the light chain variable region as shown in SEQ ID NO:6, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR2 contained in the light chain variable region.

[0040] LCDR3 having the amino acid sequence of LCDR3 contained in the light chain variable region as shown in SEQ ID NO:6, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR3 contained in the light chain variable region.

[0041] In some embodiments, the second structural domain includes the HCDR1-3 and / or the LCDR1-3 is defined by the IMGT numbering system, the Kabat numbering system, the Chothia numbering system, the Contact numbering system, or a combination thereof.

[0042] In some embodiments, the second structural domain includes the following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the IMGT numbering system as follows: heavy chain variable regions containing the following three HCDRs: HCDR1 with sequence SEQ ID NO:43, HCDR2 with sequence SEQ ID NO:44, and HCDR3 with sequence SEQ ID NO:45; and / or light chain variable regions containing the following three LCDRs: LCDR1 with sequence SEQ ID NO:46, LCDR2 with sequence DAS, and LCDR3 with sequence SEQ ID NO:47.

[0043] In some embodiments, the second structural domain includes the following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Kabat numbering system as follows: heavy chain variable regions containing the following three HCDRs: HCDR1 with sequence SEQ ID NO:48, HCDR2 with sequence SEQ ID NO:49, and HCDR3 with sequence SEQ ID NO:50; and / or light chain variable regions containing the following three LCDRs: LCDR1 with sequence SEQ ID NO:51, LCDR2 with sequence SEQ ID NO:52, and LCDR3 with sequence SEQ ID NO:47.

[0044] In some embodiments, the second structural domain includes the following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Chothia numbering system as follows: heavy chain variable regions comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:53, HCDR2 with sequence SEQ ID NO:54, and HCDR3 with sequence SEQ ID NO:50; and / or light chain variable regions comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:51, LCDR2 with sequence SEQ ID NO:52, and LCDR3 with sequence SEQ ID NO:47.

[0045] In some embodiments, the second structural domain includes the following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Contact numbering system as follows: heavy chain variable regions containing the following three HCDRs: HCDR1 with sequence SEQ ID NO:55, HCDR2 with sequence SEQ ID NO:56, and HCDR3 with sequence SEQ ID NO:57; and / or light chain variable regions containing the following three LCDRs: LCDR1 with sequence SEQ ID NO:58, LCDR2 with sequence SEQ ID NO:59, and LCDR3 with sequence SEQ ID NO:60.

[0046] In some embodiments, the second structural domain comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:5, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the heavy chain, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the heavy chain variable region; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:6, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the light chain variable region, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the light chain variable region.

[0047] In some embodiments, the bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 61 and 62, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the bispecific antibody, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the bispecific antibody. In some embodiments, the bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 63 and 64, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the bispecific antibody, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the bispecific antibody. In some embodiments, the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:65 and 66, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the bispecific antibody, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the bispecific antibody. In some embodiments, the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:67 and 68, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the bispecific antibody, or an amino acid sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with the bispecific antibody.

[0048] Another aspect of this disclosure provides an isolated nucleic acid molecule that encodes the bispecific antibody of this disclosure.

[0049] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO:75 and 76, or a nucleotide sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with it. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO:77 and 78, or a nucleotide sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with it. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO:79 and 80, or a nucleotide sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with it. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO:81 and 82, or a nucleotide sequence having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0050] In some embodiments, the bispecific antibody of this disclosure forms a dimer. In some embodiments, the bispecific antibody of this disclosure forms a homodimer. In some embodiments, the bispecific antibody of this disclosure forms a heterodimer.

[0051] Another aspect of this disclosure provides an expression vector comprising the isolated nucleic acid molecules of this disclosure.

[0052] In some embodiments, the expression vector includes adenovirus, adeno-associated virus, lentivirus, or other acceptable expression vectors.

[0053] Another aspect of this disclosure provides a host cell comprising the isolated nucleic acid molecules or expression vectors described in this disclosure.

[0054] Another aspect of this disclosure provides a chimeric antigen receptor comprising an extracellular antigen-binding fragment, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding fragment comprises the bispecific antibody described in this disclosure.

[0055] Another aspect of this disclosure provides a conjugate comprising the bispecific antibody described herein, and a conjugation portion.

[0056] In some embodiments, the coupling portion is selected from detectable markers, radioisotopes, fluorescent substances, luminescent substances, colored substances, enzymes, polyethylene glycol (PEG), radionuclides, nucleic acids, small molecule toxins, polypeptides with binding activity, proteins, receptors, ligands, and other active substances that inhibit tumor cell growth or promote tumor cell apoptosis or necrosis.

[0057] Another aspect of this disclosure provides a pharmaceutical composition comprising the bispecific antibody described herein, and a pharmaceutically acceptable carrier.

[0058] In some embodiments, the pharmaceutical composition comprises the isolated nucleic acid molecule described in this disclosure, and a pharmaceutically acceptable carrier.

[0059] In some embodiments, the pharmaceutical composition includes the expression vector described in this disclosure, as well as pharmaceutically acceptable vectors.

[0060] In some embodiments, the pharmaceutical composition is in the form of tablets, powders, granules, pills, injections, suspensions, powders, emulsions, aerosols, gels, eye drops, sustained-release formulations, or sustained-release implants. In some embodiments, the pharmaceutical composition may be formulated as an injectable preparation. In some embodiments, the preparation is suitable for intravitreal injection, subcutaneous, intradermal, intramuscular, intravenous, intrathecal, or epidural administration.

[0061] Another aspect of this disclosure provides a medicine box comprising the pharmaceutical composition described in this disclosure, the pharmaceutical composition being encapsulated in a container.

[0062] In some embodiments, the container is a glass ampoule, a glass bottle, a plastic ampoule, a plastic bottle, a plastic bag, or a pre-filled syringe.

[0063] In some embodiments, this disclosure relates to pharmaceutical unit dosage forms suitable for parenteral administration to humans, the pharmaceutical unit dosage form comprising a pharmaceutical composition as described herein in a suitable container. In some embodiments, the suitable container is a pre-filled syringe. In some embodiments, the pre-filled syringe includes an injection needle.

[0064] Another aspect of this disclosure provides a kit comprising the bispecific antibody described herein, and optionally, instructions for use.

[0065] Another aspect of this disclosure provides the use of the bispecific antibody, the isolated nucleic acid molecule, the expression vector, the chimeric antigen receptor, the conjugate, or the pharmaceutical composition described herein in the preparation of a medicament for the prevention and / or treatment of TL1A and / or TNF-α related diseases.

[0066] Another aspect of this disclosure provides a method for treating or preventing diseases associated with TL1A and / or TNF-α, the method comprising administering to a subject a therapeutically effective amount of the bispecific antibody of this disclosure, the isolated nucleic acid molecule, the expression vector, the chimeric antigen receptor, the conjugate, or the pharmaceutical composition thereof.

[0067] In some implementations, the disease includes one or more of inflammatory diseases, immune system diseases, and cancer-related diseases.

[0068] In some embodiments, the disease is selected from the following: inflammatory bowel disease, allergies, asthma, arthritis, rheumatoid arthritis (including, for example, moderate to severe adult active rheumatoid arthritis), psoriatic arthritis, systemic juvenile idiopathic arthritis (JIA), osteoarthritis, psoriatic arthritis, ankylosing spondylitis (including, for example, active ankylosing spondylitis), spondyloarthritis, psoriasis, inflammatory bowel disease (IBD), ulcerative colitis (UC).This includes conditions such as moderate to severe ulcerative colitis, Crohn's disease (CD), Lyme arthritis, graft-versus-host disease (GVHD), meningoencephalitis, immune-mediated inflammatory diseases of the central and peripheral nervous systems, pancreatitis, graft-versus-host disease, arteritis, cardiovascular inflammation, neuromyelitis optica, Castleman's disease, systemic juvenile giant cell arteritis, giant cell arteritis, arteriosclerosis, eosinophilic esophagitis, atopic dermatitis, primary sclerosing cholangitis, vasculitis, autoimmune thyroiditis, eczema, scleroderma, infection-associated endotoxic shock, aortitis, transplant rejection, heart disease, bone resorption, and Paget's disease. Osteoporosis, primary biliary cirrhosis, atherosclerosis, sepsis, periodontitis, hypochlorhydria, cytokine release syndrome (CRS), systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, immune-related adverse reactions (irAE), thyroid ophthalmopathy (TED), chronic kidney disease (CKD), Sjögren's syndrome, multiple sclerosis, systemic sclerosis-associated interstitial lung disease, polymyalgia rheumatica, myocardial infarction, uveitis, encephalomyelitis, myasthenia gravis, familial Mediterranean fever, Schnitzler syndrome, transplant rejection, graft-versus-host disease, hematopoietic stem cell transplantation, kidney transplantation, heart transplantation Implantation, bladder syndrome / interstitial cystitis, urinary and bowel dysfunction, systemic sclerosis-associated interstitial lung disease (SSc-ILD), novel coronavirus infection-related cytokine storm (CRS), severe or life-threatening cytokine storm (CRS) caused by CAR-T cells, acute myeloid leukemia, Behçet's syndrome, esophageal adenocarcinoma, liver cancer, pancreatic cancer, non-small cell lung cancer, lung cancer, kidney cancer, colorectal cancer, cervical cancer, glioma, metastatic esophageal squamous cell carcinoma, esophageal cancer, oral squamous cell carcinoma, urothelial carcinoma, head and neck tumors, nervous system cancers, B-cell malignancies and aging, breast cancer, ovarian cancer, urothelial carcinoma, prostate cancer, rectal cancer The following are considered as potential causes of cancer: colon cancer, bladder cancer, solid tumors (renal cell carcinoma), hematopoietic and lymphocytic tumors, malignant solid tumors, ameloblastic craniopharyngioma, melanoma, recurrent glioblastoma, leukemia and lymphoma, amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis (ALS), schizophrenia, major depressive disorder, diabetes, fibrous dysplasia of bone, obesity, hemophagocytic lymphohistiocytosis, idiopathic retroperitoneal fibrosis, alopecia areata, pulmonary fibrosis, cystic fibrosis, gastrointestinal diseases associated with cystic fibrosis, irritable bowel syndrome, pelvic inflammatory disease, Alzheimer's disease, Castleman's disease, dermatomyositis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal syndrome, peritonitis, surgical adhesions, surgical trauma, stroke, eczematous dermatitis, psoriasis, and Sjögren's syndrome.

[0069] This disclosure provides a bispecific antibody that specifically binds to TL1A and TNF-α. This bispecific antibody simultaneously targets TL1A and TNF-α, blocking both the TL1A / DR3 and TNF-α / TNFR signaling pathways, exhibiting a synergistic effect. TL1A and TNF-α are from the same family, and dual-targeting therapy has a potential additive effect. Detailed Implementation

[0070] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure.

[0071] TNF-like ligand 1a (TL1A) is a member of the tumor necrosis factor superfamily and is a type II membrane protein expressed by antigen-presenting cells (including dendritic cells, B cells, and macrophages), CD4+ and CD8+ T cells, and endothelial cells. It can be expressed on the cell surface or secreted as a soluble cytokine. TL1A is upregulated by the pro-inflammatory cytokines TNF and IL-1, and is also upregulated by immune complexes (ICs). TL1A can be cleaved from the cell membrane via a mechanism similar to TNF-α, thereby enabling signal transduction. TL1A binds with high affinity to death receptor 3 (DR3), a member of the TNF receptor family, also known as Wsl-1, Apo-3, TRAMP, and LARD, and now referred to as TNF receptor superfamily member 25 (TNFRSF25). DR3 is expressed by a variety of cells, including CD4+ and CD8+ T cells, NK cells, and FOXP3+ regulatory T (Treg) cells. TL1A, by binding to DR3, mediates signaling pathways that promote the secretion of pro-inflammatory cytokines or induce apoptosis. Depending on the cellular environment, TL1A binding to DR3 can trigger one of two signaling pathways: activation of the transcription factor NF-κB or activation of caspase and apoptosis.

[0072] Tumor necrosis factor-α (TNF-α) is a homotrimeric protein composed of 157 amino acids, mainly produced by activated macrophages, T lymphocytes, and natural killer cells. It plays an important role in multiple physiological processes, including immune responses, inflammatory responses, and apoptosis. Members of the TNF receptor family include TNFR1 and TNFR2. TNFR1 and TNFR2 are receptor proteins on the cell membrane surface that can bind to TNF-α and transmit cellular signals. Among them, TNFR1 mainly plays a pro-inflammatory role. When cells are subjected to inflammatory stimuli or stress, TNFR1 expression increases, and the TNF-α it binds to activates a series of signal transduction pathways, triggering biological processes such as apoptosis and inflammatory responses.

[0073] This disclosure, based on screened monoclonal antibodies targeting TL1A, further describes the expression of bispecific antibodies through co-transfection of host cells with dual plasmids. After routine purification to obtain a bifunctional fusion protein, the activity and affinity of the target protein obtained by transient transfection are analyzed to screen for bispecific antibodies that can simultaneously target TL1A and TNF-α. The bispecific antibodies disclosed in this disclosure target both TL1A and TNF-α. CN2024104373245 is incorporated herein by reference in its entirety.

[0074] Compared to monoclonal antibodies, bispecific antibodies have an additional specific antigen-binding site, thus exhibiting stronger specificity, more accurate targeting of tumor cells, and reduced adverse reactions caused by off-target toxicity. They can perform special functions that are difficult for monoclonal antibody drugs to achieve. Moreover, compared to combination therapy with monoclonal antibodies, they can also effectively reduce treatment costs and break through the current upper limit of efficacy in treating autoimmune diseases such as IBD and rheumatoid arthritis.

[0075] The sequences of the heavy chain variable region, light chain variable region, and their CDRs involved in the bispecific antibody disclosed herein are shown in Tables 1 and 2.

[0076] Table 1

[0077]

[0078] Table 2

[0079]

[0080]

[0081]

[0082] definition

[0083] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0084] Unless the context clearly indicates otherwise, the terms “a,” “an,” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0085] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0086] As used in this article, the term "affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a bispecific antibody or bispecific binding molecule) and its binding ligand (e.g., an antigen). Binding affinity is typically expressed as the dissociation constant (K0). D ) represents the dissociation constant (K) D ) is the dissociation rate (k d ) and binding rate (k a The ratio of ), i.e., K D =k d / k a Affinity can be measured using conventional methods known in the art, such as surface plasmon resonance (SPR).

[0087] As used herein, the terms "specific recognition" or "specific binding" refer to the selective recognition and binding of an antibody to a ligand or receptor, distinguishing it from unwanted or nonspecific binding. In one embodiment, for example, as measured by SPR, the bispecific antibody of this disclosure binds to unrelated proteins to less than about 10% of its binding to TL1A and TNF-α. In some embodiments, the bispecific antibody provided by this disclosure binds to TL1A and TNF-α at a Kc D All are 10 -7 M or lower, for example, 10 -10 M to 10 -11 M.

[0088] The term "substitution" as used herein for amino acids refers to the replacement of at least one amino acid residue in an amino acid sequence with another different "substituted" amino acid residue. The term "insertion" as used herein for amino acids refers to the incorporation of at least one additional amino acid into an amino acid sequence. While inserts typically consist of one or two inserted amino acid residues, larger "peptide inserts" can also be prepared, for example, inserts of about three to five or even up to about ten, fifteen, or twenty amino acid residues. As disclosed above, the inserted residues can be naturally occurring or non-naturally occurring. The term "deletion" as used herein for amino acids refers to the removal of at least one amino acid residue from an amino acid sequence.

[0089] The bispecific antibodies or fragments thereof disclosed herein may contain conserved amino acid substitutions at one or more amino acid residues, such as essential or non-essential amino acid residues. A “conserved amino acid substitution” is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, in this document, essential or non-essential amino acid residues in bispecific antibodies or linkers are preferably replaced with another amino acid residue from the same side chain family. In some embodiments, amino acid segments can be replaced with segments that are structurally similar but have different order and / or composition of side chain family members. Alternatively, in some embodiments, mutations can be randomly introduced along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be incorporated into the bispecific antibodies of this disclosure and screened for the ability of these peptides to bind to desired targets.

[0090] As used herein, the term "antibody" includes complete antibodies and any antigen-binding fragments (i.e., "antigen-binding moieties," "antigen-binding polypeptides," or "immunobinding agents"), or their single chains. An antibody is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) linked together by disulfide bonds, or its antigen-binding moieties. Each heavy chain includes a variable heavy chain (VH) region and a constant heavy chain (CH) region. Each light chain includes a variable light chain (VL) region and a constant light chain (CL) region. Each VH and VL region contains three regions with highly variable amino acid composition and sequence, called hypervariable regions or complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3. The amino acid composition and sequence of regions in the VH and VL regions other than the CDR regions are relatively conserved and are called framework regions (FRs). Each VH or VL region has four framework regions, denoted as FR1, FR2, FR3, and FR4, respectively.

[0091] Immunoglobulins exist in five main classes: IgA, IgD, IgE, IgG, and IgM. These main classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant heavy chain domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The CL length of different types (κ or λ) of immunoglobulins is generally consistent, but the CH lengths of different classes of immunoglobulins differ. For example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4. The hinge region, located between CH1 and CH2, is rich in proline and easily extends and bends, thereby altering the distance between antigen-binding sites and facilitating antibody binding to antigenic epitopes located at different positions. The hinge region is easily hydrolyzed by papain, pepsin, etc., producing different hydrolyzed fragments. Papain hydrolyzes Ig near the N-terminus of the two heavy chains linked by disulfide bonds in the hinge region, cleaving Ig into two identical Fab fragments and one Fc fragment. The Fab fragment is an antigen-binding fragment, consisting of a complete light chain and VH and CHI domains of the heavy chain.

[0092] As used herein, the term "homodimer" refers to a bispecific antibody formed by the present disclosure, including two identical bispecific antibodies of the present disclosure. The term "heterodimer" as used herein refers to a bispecific antibody formed by the present disclosure, including two different bispecific antibodies of the present disclosure.

[0093] As used herein, the term “individual” or “subject” refers to mammals, including but not limited to humans and non-human mammals. For example, mammals include, but are not limited to, domesticated animals (such as cattle, horses, dogs, sheep, goats, cats, and dogs), primates (such as humans and monkeys), and rodents (such as rabbits, mice, and rats).

[0094] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0095] As used herein, the term "linker" refers to a (peptide) linker of natural and / or synthetic origin, composed of linear amino acids. Domains in the bispecific antibodies of this disclosure can be linked by linkers, wherein each linker is fused to and / or otherwise linked (e.g., via peptide bonds) with at least two polypeptides or domains. In some embodiments, the amino acid sequences of all linkers present in the bispecific antibodies of this disclosure are identical. In other embodiments, the amino acid sequences of at least two linkers present in the bispecific antibodies of this disclosure are different. Linkers should have a length suitable for linking two or more monomeric domains in this manner, ensuring that the different domains to which they are linked fold correctly and are properly presented to perform their biological activities. In various embodiments, linkers have a flexible conformation. Suitable flexible linkers include, for example, those having glycine, glutamine, and / or serine residues. In some embodiments, the amino acid residues in the linker can be arranged in small repeating units of up to five amino acids.

[0096] The "percentage (%) sequence identity" relative to a reference amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the reference amino acid sequence after sequence alignment and (if necessary) introducing gaps to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, alignment can be performed in various ways within the art, such as using BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0097] As used herein, the term "pharmaceuticalally acceptable carrier" refers to a component of a pharmaceutical preparation that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0098] As used herein, the term "treatment" refers to the reduction and / or improvement of the disorder and / or related illness or symptoms, as well as the prevention of the worsening of disorder symptoms. Desired therapeutic effects include, but are not limited to, prevention of the onset or recurrence of the disease, symptom relief, reduction of any direct or indirect pathological outcome of the disease, prevention of metastasis, slowing of disease progression, improvement or relief of symptoms, and relief or improvement of prognosis. However, it should be understood that treating a disease or symptom does not require the complete elimination of the disease or related symptoms.

[0099] The term "effective dose" as used in this article refers to the effective dose and time period required to achieve the desired therapeutic or preventative effect.

[0100] The following embodiments are provided to aid in understanding this disclosure. However, it should be understood that these embodiments are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of this disclosure is set forth in the claims. It should be understood that any modifications and changes may be made without departing from the spirit of this disclosure.

[0101] Example 1: Construction of expression plasmid

[0102] The bispecific antibodies in this embodiment target TL1A and TNF-α. The anti-TL1A monoclonal antibody is the complete molecule of VDJ013 or VDJ013-PLT344, and the anti-TNF-α monoclonal antibody is the complete molecule of VDJ013-18. The anti-TL1A×TNF-α bispecific antibody is constructed by linking the complete molecules of VDJ013 or VDJ013-PLT344 or their scFv with the complete molecule of VDJ013-18 or its scFv via a linker. The heavy chain and light chain genes of the bispecific antibody are cloned into the pCDNA3.4 vector, respectively, to construct the heavy chain recombinant plasmid and light chain recombinant plasmid of the bispecific antibody. Expression of the bispecific antibody is achieved by co-transfection of host cells with the two plasmids, as shown in Table 3.

[0103] Table 3

[0104]

[0105] Example 2: Transient expression of bispecific antibodies

[0106] With a density of 2×10 6 Expi-CHO-S cells per mL were placed in Erlenmeyer flasks and cultured at 100 rpm, 37°C, and 5% CO2 for 20-25 h until the cell density reached 5 × 10⁶ cells / mL. 6Cells with a density of approximately [number] cells / mL, in logarithmic growth phase, and a viability of over 95% were used for transfection experiments. The recombinant plasmid obtained in Example 1 was added to Trans Buffer A (Maibang Biotechnology) and gently mixed. Polyetherimide (PEI) was then added to Trans Buffer A and gently mixed. The diluted PEI solution was added to the corresponding diluted plasmid, gently mixed, and incubated at room temperature for 5 min. The mixture was then added to an Erlenmeyer flask containing Expi-CHO-S cells and cultured at 100 rpm, 37°C, and 5% CO2 in a shaker. 24 h after transfection, the transfected cells were cultured at 100 rpm, 32°C, and 5% CO2 in a shaker. 10–14 days after transfection, the cell culture medium was collected by centrifugation at 3000 rpm for 15 min.

[0107] Example 3: Purification and Identification of Bispecific Antibodies

[0108] Four bispecific antibodies and three control monoclonal antibodies were purified and their purity was identified. The cell culture supernatant obtained in Example 1 was first subjected to affinity chromatography using a HiTrap MabSelect Sure pre-packed column. After washing the column with pure water and equilibrating it with PB buffer, the supernatant was loaded onto the column for binding. After loading, the column was washed with PB buffer to baseline, and then the protein was eluted with 0.1M citrate elution buffer. The eluted protein was neutralized with 1M Tris-HCl, appropriately concentrated, and then loaded onto a PBS-equilibrated Superdex 200 for further purification. The collected bispecific antibodies were concentrated to a certain concentration and identified by SDS-PAGE electrophoresis. The expression levels and SEC-HPLC identification results are shown in Table 4.

[0109] Table 4. Antibody expression levels and SEC-HPLC purity identification

[0110] serial number Expression level (mg / L) purity(%) VDJ013-13 68.93 84.5 VDJ013-14 53.4 89.3 VDJ013-15 167 77.1 VDJ013-16 96.9 92.0 VDJ013-18 140 95.7 VDJ013 37.1 99.1 VDJ013-PLT344 105 98.0

[0111] Example 4: Detection of biological activity of TL1A target in bispecific antibodies

[0112] Using pNFκB-TA-luc-CP (Beyond the Clouds) and pGL4.48 (Promega) as templates, we modified the plasmid containing the NF-KB Luc2p Hygromycin gene by replacing the SBE gene of pGL4.48 with the NF-KB gene of pNFκB-TA-luc-CP.

[0113] The Human TL1A gene (encoding amino acids Leu72-Leu251, Uniprot: O95150-1) was cloned into the pCDNA3.4 vector to construct a human TL1A plasmid with the his tag. The plasmid was expressed by transient transduction using HEK293 and purified by nickel column to obtain the human TL1A stimulatory factor (hTL1A).

[0114] A plasmid containing the NF-κB Luc2p Hygromycin gene was transfected into TF-1 cells to construct the TF-1-NF-κB luciferase reporter gene viability test cell line. TF-1-NF-κB luciferase reporter gene cells were plated at 60,000 cells / well and then starved overnight. The next day, 1.0 μg / mL human TL1A stimulating factor working solution was prepared and used to dilute the antibody sample. Different concentration gradients of antibody were added to the positive wells containing the plated cells, and the cells were incubated at 37°C for 4 hours. Finally, 100 μL of One-Lite reagent (Novazia) was added to each well, and after incubation at room temperature for 5 minutes, the chemiluminescence values ​​were read using a TECAN multi-mode microplate reader. The data were plotted using Prism 5 (GraphPad) software, and the IC50 was calculated. 50 The values ​​are shown in Table 5.

[0115] The results in Table 5 show that, compared with monoclonal antibodies VDJ013 and VDJ013-PLT344, bispecific antibodies VDJ013-13 to VDJ013-16 have comparable biological activity against the TL1A target.

[0116] Table 5. Detection of TL1A target biological activity of bispecific antibodies

[0117] serial number <![CDATA[IC 50 (nM)]]> Biological activity (reference standard / analyte * 100%) VDJ013 0.2461 NA VDJ013-13 0.2510 98 VDJ013-14 0.273 90 VDJ013-PLT344 0.427 NA VDJ013-15 0.572 75 VDJ013-16 0.451 95

[0118] Example 5: Detection of biological activity of TNF-α target in bispecific antibodies

[0119] The L929 cell density was adjusted to 1×10⁻⁶. 6 Add 50 μL of the antibody solution to each well at a concentration of 1.25 ng / mL. Prepare a 1.25 ng / mL hTNFα stimulating factor (Acrobiosystems, C25CP1-22A5W1-172) working solution and add 40 μL / well to both positive and sample wells. Dilute the antibody samples and add 10 μL / well of different concentration gradients of antibody to the cell-coated positive wells. Incubate at 37°C for 20 hours. Then add 20 μL of MTS reagent (Promega) to each well, vortex to mix, and incubate for 0.5 hours. After incubation, vortex to mix, read the values ​​at 490 nm using a microplate reader, and plot the data using Prism 5 (GraphPad) software to calculate the IC50. 50The values ​​are shown in Table 6.

[0120] The results in Table 6 show that the TNFα target biological activity of bispecific antibodies VDJ013-13 to VDJ013-16 is comparable to that of monoclonal antibody VDJ013-18.

[0121] Table 6. Detection of TNF-α target biological activity of bispecific antibodies

[0122] serial number <![CDATA[IC 50 (nM)]]> Biological activity (reference standard / analyte * 100%) VDJ013-18 0.0246-0.0263 NA VDJ013-13 0.024 103 VDJ013-14 0.023 106 VDJ013-15 0.027 93 VDJ013-16 0.020 132

[0123] Example 6: Detection of relative binding activity of TL1A target in bispecific antibodies

[0124] The binding ability of bispecific antibodies to hTL1A was detected using an ELISA method. hTL1A (same as in Example 4) was diluted to 3 μg / mL, 100 μL / well, and coated overnight at 4°C. The next day, the plates were washed and blocked with 5% skim milk for 2 hours, then washed again and antibody samples of different concentration gradients were added, and incubated at 37°C for 1 hour. After washing, 100 μL of HRP-labeled goat anti-human IgG secondary antibody (Solarbio, K0031G-HRP) was added to each plate, and incubated at 37°C for 30 minutes. TMB chromogenic buffer was then added, and the reaction was incubated at 37°C for 5 minutes. Finally, 50 μL of H2SO4 was added to stop the reaction, and the OD values ​​were read using a TECAN multi-mode microplate reader. 450 The values ​​were calculated, and Prism 5 (GraphPad) software was used to plot the data. The results are shown in Table 7.

[0125] The results in Table 7 show that the bispecific antibodies VDJ013-13 to VDJ013-16 all exhibit high binding activity to the TL1A target.

[0126] Table 7. Detection of relative binding activity of the bispecific antibody to the TL1A target

[0127] serial number <![CDATA[EC 50 (nM)]]> VDJ013-13 0.308 VDJ013-14 0.314 VDJ013-15 0.283 VDJ013-16 0.225

[0128] Example 7: Detection of relative binding activity of TNFα target in bispecific antibodies

[0129] The binding ability of bispecific antibodies to TNFα was detected using an ELISA method. hTNFα (same as in Example 5) was diluted to 1 μg / mL, 100 μL / well, and coated overnight at 4°C. The next day, the plates were washed and blocked with 5% skim milk for 2 hours. Then, the plates were washed again and antibody samples of different concentrations were added, and incubated at 37°C for 1 hour. After washing, 100 μL of HRP-labeled goat anti-human IgG secondary antibody (Solarbio, K0031G-HRP) was added to each plate, and incubated at 37°C for 30 minutes. TMB chromogenic buffer was then added, and the reaction was incubated at 37°C for 5 minutes. Finally, 50 μL of H2SO4 was added to stop the reaction, and the OD values ​​were read using a TECAN multi-mode microplate reader. 450 The values ​​were calculated, and Prism 5 (GraphPad) software was used to plot the data. The results are shown in Table 8.

[0130] The results in Table 8 show that the bispecific antibodies VDJ013-13 to VDJ013-16 all exhibit good binding activity to the TNFα target.

[0131] Table 8. Detection of relative binding activity of bispecific antibodies against TNFα target

[0132] serial number <![CDATA[EC 50 (nM)]]> VDJ013-13 0.03986 VDJ013-14 0.04706 VDJ013-15 0.03422 VDJ013-16 0.0399

[0133] Example 8: Affinity determination of TL1A target in bispecific antibodies

[0134] Bio-Layer Interferometry (BLI) was used to determine the affinity of four bispecific antibodies for hTL1A protein. All antibodies were diluted to 5 μg / mL, and hTL1A (same as in Example 4) was serially diluted from 50 nM to 3.125 nM. The antibodies were added to hFc probes (Gator) at 400 rpm / min, and then different gradients of hTL1A were bound to the antibody-coated hFc probes. The hFc probes bound with the antigen-antibody complex were placed in analyte-free Q buffer (10 nM PBS (pH 7.4) + 0.02% Tween + 0.2% BSA) to dissociate the bound antigen-antibody complex. The hFc probes were then immersed in regeneration buffer (10 nM glycine at pH 1.7) to remove residual bound analytes, regenerating and storing the probes. The affinity data were obtained by integrating the fitted lines for each concentration using the Gator's built-in analysis software. The results are shown in Table 9.

[0135] The results in Table 9 show that the bispecific antibodies VDJ013-13 to VDJ013-16 all have high affinity for hTL1A.

[0136] Table 9. Affinity determination of antibody with hTL1A

[0137] serial number koff(1 / s) kon(1 / Ms) KD(M) VDJ013-13 1.05E-04 1.09E+06 9.63E-11 VDJ013-14 1.06E-004 1.13E+006 9.42E-11 VDJ013-15 1.79E-005 1.24E+006 1.44E-11 VDJ013-16 1.79E-005 1.24E+006 ＜1.0E-12

[0138] Example 9: Affinity determination of TNF-α target in bispecific antibodies

[0139] The affinity of four bispecific antibodies for hTNF-α (as in Example 5) was determined using bio-layer interferometry (BLI). All antibodies were diluted to 15 μg / mL, and TNF-α protein was serially diluted from 6.25 nM to 0.391 nM. Antibodies were added to hFc probes at 400 rpm / min, and then different gradients of diluted TNF-α were bound to the antibody-coated hFc probes (Gator). The hFc probes containing the antigen-antibody complex were placed in analyte-free Q buffer (10 nM PBS (pH 7.4) + 0.02% Tween + 0.2% BSA) to dissociate the bound antigen-antibody complex. The probes were then immersed in regeneration buffer (10 nM glycine at pH 1.7) to remove residual analyte, allowing for regeneration and storage. Affinity data were obtained by integrating the fitted lines for each concentration using the Gator's built-in analysis software; the results are shown in Table 10.

[0140] The results in Table 10 show that the bispecific antibodies VDJ013-13 to VDJ013-16 have high affinity for TNFα.

[0141] Table 10. Determination of the affinity between the antibody and TNFα

[0142] serial number koff(1 / s) kon(1 / Ms) KD(M) VDJ013-13 6.01E-05 1.51E+06 3.98E-11 VDJ013-14 6.16E-005 1.49E+006 4.13E-11 VDJ013-15 1.69E-004 1.35E+006 1.26E-10 VDJ013-16 2.32E-005 1.49E+006 1.56E-11

[0143] The technical solutions disclosed herein are not limited to the specific embodiments described above. Any technical modifications made based on the technical solutions disclosed herein shall fall within the protection scope of this disclosure.

Claims

1. A bispecific antibody, comprising: It has a first domain that specifically binds to TNF-like ligand 1a (TL1A) and a second domain that specifically binds to tumor necrosis factor α (TNF-α).

2. The bispecific antibody according to claim 1, characterized in that, The first domain is an antibody that specifically binds to TL1A or a functional fragment thereof; and / or The second domain is an antibody that specifically binds to TNF-α or a functional fragment thereof.

3. The bispecific antibody according to claim 1 or 2, characterized in that, The first domain includes an antibody Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, nanobody, heavy chain variable region (VH) fragment, and / or light chain variable region (VL) fragment that specifically binds to TL1A; and / or The second domain includes antibody Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, nanobodies, heavy chain variable region (VH) fragments, and / or light chain variable region (VL) fragments that specifically bind to TNF-α.

4. The bispecific antibody according to any one of claims 1 to 3, characterized in that, The first structural domain and the second structural domain are directly connected or connected through connectors. Preferably, the connector has a general formula (G) n S) m The amino acid sequence shown has n and m as integers from 1 to 10; more preferably, n is an integer from 1 to 4 and m is an integer from 1 to 3, or is consistent with the general formula (G n S) m The amino acid sequence shown is compared to an amino acid sequence with 1, 2, or 3 inserted, substituted, or deleted amino acids.

5. The bispecific antibody according to any one of claims 1 to 4, characterized in that, The first structural domain includes: (1) the following three heavy chain variable region complementarity-determining regions (HCDRs): HCDR1, having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in SEQ ID NO:1 or 3, or having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR1 contained in the heavy chain variable region. HCDR2, having the amino acid sequence of HCDR2 contained in the heavy chain variable region as shown in any of SEQ ID NO:1 or 3, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR2 contained in the heavy chain variable region. HCDR3, having the amino acid sequence of HCDR3 contained in the heavy chain variable region as shown in any of SEQ ID NO:1 or 3, or having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of HCDR3 contained in the heavy chain variable region; and / or (2) The following three light chain variable region complementarity-determining regions (LCDRs): LCDR1, having the amino acid sequence of LCDR1 contained in the light chain variable region as shown in SEQ ID NO:2 or 4, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR1 contained in the light chain variable region. LCDR2, having the amino acid sequence of LCDR2 contained in the light chain variable region as shown in SEQ ID NO:2 or 4, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR2 contained in the light chain variable region. LCDR3, having the amino acid sequence of LCDR3 contained in the light chain variable region as shown in SEQ ID NO:2 or 4, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR3 contained in the light chain variable region. Preferably, the first structural domain includes: HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:1, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:2; or HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:3, and LCDR1, LCDR2 and LCDR3 contained in the light chain variable region as shown in SEQ ID NO:4; Preferably, the HCDR1-3 and / or the LCDR1-3 are defined by the IMGT numbering system, the Kabat numbering system, the Chothia numbering system, the Contact numbering system, or a combination thereof.

6. The bispecific antibody according to any one of claims 1-5, characterized in that, The first structural domain includes: (1) The following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the IMGT numbering system: (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:7, HCDR2 with sequence SEQ ID NO:8, and HCDR3 with sequence SEQ ID NO:9; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:10, LCDR2 with sequence YAT, and LCDR3 with sequence SEQ ID NO:11; or (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:25, HCDR2 with sequence SEQ ID NO:26, and HCDR3 with sequence SEQ ID NO:27; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:28, LCDR2 with sequence DAS, and LCDR3 with sequence SEQ ID NO:29; or (2) The following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Kabat numbering system: (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 of sequence SEQ ID NO:12, HCDR2 of sequence SEQ ID NO:13, and HCDR3 of sequence SEQ ID NO:14; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of sequence SEQ ID NO:15, LCDR2 of sequence SEQ ID NO:16, and LCDR3 of sequence SEQ ID NO:11; or (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:30, HCDR2 with sequence SEQ ID NO:31, and HCDR3 with sequence SEQ ID NO:32; and / or a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:33, LCDR2 with sequence SEQ ID NO:34, and LCDR3 with sequence SEQ ID NO:29; or (3) The following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Chothia numbering system: (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:17, HCDR2 of SEQ ID NO:18, and HCDR3 of SEQ ID NO:14; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:15, LCDR2 of SEQ ID NO:16, and LCDR3 of SEQ ID NO:11; or (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:35, HCDR2 with sequence SEQ ID NO:36, and HCDR3 with sequence SEQ ID NO:32; and / or a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:33, LCDR2 with sequence SEQ ID NO:34, and LCDR3 with sequence SEQ ID NO:29; or (4) The following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Contact numbering system: (a) A heavy chain variable region comprising the following three HCDRs: HCDR1 of SEQ ID NO:19, HCDR2 of SEQ ID NO:20, and HCDR3 of SEQ ID NO:21; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 of SEQ ID NO:22, LCDR2 of SEQ ID NO:23, and LCDR3 of SEQ ID NO:24; or (b) A heavy chain variable region comprising the following three HCDRs: HCDR1 with sequence SEQ ID NO:37, HCDR2 with sequence SEQ ID NO:38, and HCDR3 with sequence SEQ ID NO:39; and / or a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:40, LCDR2 with sequence SEQ ID NO:41, and LCDR3 with sequence SEQ ID NO:

42.

7. The bispecific antibody according to any one of claims 1-6, characterized in that, The first structural domain includes: The heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:1 or 3, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; and The light chain variable region has an amino acid sequence as shown in SEQ ID NO:2 or 4, an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to it, or an amino acid sequence with at least 80% sequence identity with it. Preferably, the first structural domain comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:1 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:2; Preferably, the first structural domain comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:3 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

4.

8. The bispecific antibody according to any one of claims 1-7, characterized in that, The second structural domain includes the following three heavy chain variable region complementarity-determining regions (HCDRs): HCDR1, having the amino acid sequence of HCDR1 contained in the heavy chain variable region as shown in SEQ ID NO:5, or having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR1 contained in the heavy chain variable region. HCDR2, having the amino acid sequence of HCDR2 contained in the heavy chain variable region as shown in SEQ ID NO:5, or having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of HCDR2 contained in the heavy chain variable region. HCDR3, having the amino acid sequence of HCDR3 contained in the heavy chain variable region as shown in SEQ ID NO:5, or having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence of HCDR3 contained in the heavy chain variable region; and / or The following three light chain variable region complementarity-determining regions (LCDRs): LCDR1, having the amino acid sequence of LCDR1 contained in the light chain variable region as shown in SEQ ID NO:6, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR1 contained in the light chain variable region. LCDR2, having the amino acid sequence of LCDR2 contained in the light chain variable region as shown in SEQ ID NO:6, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR2 contained in the light chain variable region. LCDR3, having the amino acid sequence of LCDR3 contained in the light chain variable region as shown in SEQ ID NO:6, or having an amino acid sequence with one or more amino acid substitutions, deletions or additions compared to the amino acid sequence of LCDR3 contained in the light chain variable region. Preferably, the HCDR1-3 and / or the LCDR1-3 are defined by the IMGT numbering system, the Kabat numbering system, the Chothia numbering system, the Contact numbering system, or a combination thereof.

9. The bispecific antibody according to any one of claims 1-8, characterized in that, The second structural domain includes: (1) The following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the IMGT numbering system: The heavy chain variable region contains the following three HCDRs: HCDR1 with sequence SEQ ID NO:43, HCDR2 with sequence SEQ ID NO:44, and HCDR3 with sequence SEQ ID NO:45; and / or, the light chain variable region contains the following three LCDRs: LCDR1 with sequence SEQ ID NO:46, LCDR2 with sequence DAS, and LCDR3 with sequence SEQ ID NO:47; or (2) The following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Kabat numbering system: The heavy chain variable region contains the following three HCDRs: HCDR1 with sequence SEQ ID NO:48, HCDR2 with sequence SEQ ID NO:49, and HCDR3 with sequence SEQ ID NO:50; and / or, the light chain variable region contains the following three LCDRs: LCDR1 with sequence SEQ ID NO:51, LCDR2 with sequence SEQ ID NO:52, and LCDR3 with sequence SEQ ID NO:47; or (3) The following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Chothia numbering system: The heavy chain variable region contains the following three HCDRs: HCDR1 with sequence SEQ ID NO:53, HCDR2 with sequence SEQ ID NO:54, and HCDR3 with sequence SEQ ID NO:50; and / or, the light chain variable region contains the following three LCDRs: LCDR1 with sequence SEQ ID NO:51, LCDR2 with sequence SEQ ID NO:52, and LCDR3 with sequence SEQ ID NO:47; or (4) The following heavy chain variable regions and / or light chain variable regions, wherein HCDR1-3 and / or LCDR1-3 are defined according to the Contact numbering system: The heavy chain variable region contains the following three HCDRs: HCDR1 with sequence SEQ ID NO:55, HCDR2 with sequence SEQ ID NO:56, and HCDR3 with sequence SEQ ID NO:57; and / or, the light chain variable region contains the following three LCDRs: LCDR1 with sequence SEQ ID NO:58, LCDR2 with sequence SEQ ID NO:59, and LCDR3 with sequence SEQ ID NO:

60.

10. The bispecific antibody according to any one of claims 1-9, characterized in that, The second structural domain includes: The heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:5, an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it; and The light chain variable region has an amino acid sequence as shown in SEQ ID NO:6, an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to it, or an amino acid sequence having at least 80% sequence identity with it.

11. An isolated nucleic acid molecule encoding a bispecific antibody as described in any one of claims 1 to 10.

12. An expression vector comprising the isolated nucleic acid of claim 10.

13. A host cell comprising the isolated nucleic acid molecule as claimed in claim 11 or the expression vector as claimed in claim 12.

14. A chimeric antigen receptor comprising an extracellular antigen-binding fragment, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding fragment comprises a bispecific antibody according to any one of claims 1-10.

15. A conjugate comprising: the bispecific antibody according to any one of claims 1-10; and a conjugation portion; Preferably, the coupling portion is selected from detectable markers, radioisotopes, fluorescent substances, luminescent substances, colored substances, enzymes, polyethylene glycol (PEG), radionuclides, nucleic acids, small molecule toxins, polypeptides with binding activity, proteins, receptors, ligands, and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

16. A pharmaceutical composition comprising a bispecific antibody as described in any one of claims 1 to 10, an isolated nucleic acid molecule as described in claim 11, or an expression vector as described in claim 12, and a pharmaceutically acceptable vector; Preferably, the pharmaceutical composition is selected from the form of tablets, powders, granules, pills, injections, suspensions, powders, emulsions, aerosols, gels, eye drops, sustained-release preparations, or sustained-release implants.

17. A pillbox comprising the pharmaceutical composition of claim 16, wherein the pharmaceutical composition is encapsulated in a container. Preferably, the container is selected from glass ampoules, glass bottles, plastic ampoules, plastic bottles, plastic bags, or pre-filled syringes.

18. A kit comprising the bispecific antibody according to any one of claims 1 to 10, and optionally, instructions for use.

19. Use of the bispecific antibody of any one of claims 1 to 10, the isolated nucleic acid molecule of claim 11, the expression vector of claim 12, the chimeric antigen receptor of claim 14, the conjugate of claim 15, or the pharmaceutical composition of claim 16 in the preparation of a medicament for the prevention and / or treatment of TL1A and / or TNF-α related diseases.

20. A method of treating or preventing a disease associated with TL1A and / or TNF-α, comprising administering to a subject a therapeutically effective amount of any one of claims 1 to 10, an isolated nucleic acid molecule as claimed in claim 11, an expression vector as claimed in claim 12, a chimeric antigen receptor as claimed in claim 14, a conjugate as claimed in claim 15, or a pharmaceutical composition as claimed in claim 16.

21. The method according to claim 20, characterized in that, The diseases include one or more of the following: inflammatory diseases, immune system diseases, and cancer-related diseases. Preferably, the diseases include inflammatory bowel disease, allergies, asthma, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic juvenile idiopathic arthritis (JIA), osteoarthritis, psoriatic arthritis, ankylosing spondylitis, spondyloarthritis, psoriasis, inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), Lyme arthritis, graft-versus-host disease (GVHD), meningoencephalitis, immune-mediated inflammatory diseases of the central and peripheral nervous systems, pancreatitis, graft-versus-host disease, arteritis, cardiovascular inflammation, neuromyelitis optica, Castleman's disease, systemic juvenile giant cell arteritis, giant cell arteritis, arteriosclerosis, eosinophilic esophagitis, atopic dermatitis, primary sclerosing cholangitis, vasculitis, autoimmune thyroiditis, eczema, scleroderma, infection-associated endotoxic shock, aortitis, transplant rejection, heart disease, bone resorption, Paget's disease.Diseases, osteoporosis, primary biliary cirrhosis, atherosclerosis, sepsis, periodontitis, hypochlorhydria, cytokine release syndrome (CRS), systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, immune-related adverse reactions (irAE), thyroid eye disease (TED), chronic kidney disease (CKD), Sjögren's syndrome, multiple sclerosis, systemic sclerosis-associated interstitial lung disease, polymyalgia rheumatica, myocardial infarction, uveitis, encephalomyelitis, myasthenia gravis, familial Mediterranean fever, Schnitzler syndrome, transplant rejection, graft-versus-host disease, hematopoietic stem cell transplantation, kidney transplantation, heart transplantation, bladder syndrome / interstitial cystitis, urinary and bowel dysfunction, systemic sclerosis-associated interstitial lung disease (SSc-ILD), novel coronavirus infection-related cytokine storm (CRS), severe or life-threatening cytokine storm (CRS) caused by CAR-T cells, acute myeloid leukemia, Behcet's disease. Syndrome, esophageal adenocarcinoma, liver cancer, pancreatic cancer, non-small cell lung cancer, lung cancer, kidney cancer, colorectal cancer, cervical cancer, glioma, metastatic esophageal squamous cell carcinoma, esophageal cancer, oral squamous cell carcinoma, urothelial cell carcinoma, head and neck tumors, nervous system cancers, B-cell malignancies and aging, breast cancer, ovarian cancer, urothelial carcinoma, prostate cancer, rectal cancer, colon cancer, bladder cancer, solid tumors (renal cell carcinoma), hematopoietic and lymphocytic tumors, malignant solid tumors, ameloblastic craniopharyngioma, melanoma, recurrent glioma Plasmoblastoma, leukemia and lymphoma, amyotrophic lateral sclerosis, ALS, schizophrenia, major depressive disorder, diabetes, fibrous dysplasia of bone, obesity, hemophagocytic lymphohistiocytosis, idiopathic retroperitoneal fibrosis, alopecia areata, pulmonary fibrosis, cystic fibrosis, gastrointestinal diseases associated with cystic fibrosis, irritable bowel syndrome, pelvic inflammatory disease, Alzheimer's disease, Castleman's disease, dermatomyositis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, surgical adhesions, surgical trauma, stroke, eczematous dermatitis, psoriasis, Sjögren's syndrome, or one or more of these conditions.

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