Bispecific antibody aiming at IL-6R and TL1A and application thereof

Abstract

The present disclosure relates to a bispecific antibody comprising a first domain that specifically binds to interleukin-6 receptor (IL-6R), and a second domain that specifically binds to TNF-like ligand 1a (TL1A). The present disclosure also relates to methods of using said bispecific antibodies in the treatment or prevention of diseases associated with IL-6R and / or TL1A.

Description

Technical Field

[0001] This disclosure relates to bispecific antibodies against IL-6R and TL1A and their applications. Background Technology

[0002] Interleukin-6 (IL-6) is a multifunctional cytokine discovered in the 1980s. It is produced by various cell types, such as T cells, B cells, monocytes, fibroblasts, osteoblasts, keratinocytes, endothelial cells, mesangial cells, and some tumor cells. IL-6 interacts with different IL-6 receptors on the cell surface and then transmits different biological signals to different tissues and cells via downstream signaling pathways.

[0003] Interleukin-6 (IL-6) binds to two receptors: the specific receptor IL-6R (an 80 kDa type I transmembrane protein) and gp130, a common receptor subunit of IL-6 family cytokine members. gp130 can be expressed in all cells, but the expression of the interleukin-6 receptor (IL-6R) is more restricted, primarily found in hepatocytes, neutrophils, monocytes, and CD4+ T cells. IL-6 plays important roles in immune regulation, hematopoiesis, inflammation, and tumorigenesis. IL-6 is involved in the treatment of diseases or conditions and has become a target for drug discovery.

[0004] TL1A, also known as TNFSF15 or VEGI, is a member of the tumor necrosis factor superfamily. First discovered in 2002, it is expressed in various cell types. TL1A regulates different immune cells, producing inflammatory cytokines and chemokines. TL1A activates the TRADD pathway by binding to its receptor DR3. TRADD can promote pro-inflammatory effects by regulating TRAF2 and RIP1, thereby inducing PI3K, MAPKs, and NF-κB. It also participates in apoptosis and necrotic cell death by activating the FADD, RIP3, and Caspase-8 / 3 / 7 pathways.

[0005] TL1A is associated with a variety of autoimmune diseases, including rheumatoid arthritis (RA), inflammatory bowel disease (IBD), psoriasis (PSA), primary biliary cirrhosis (PBC), systemic lupus erythematosus (SLE), and ankylosing spondylitis (AS), in which TL1A is abnormally expressed.

[0006] Ulcerative colitis and Crohn's disease are very common conditions with limited available treatments, and some patients do not respond to initial treatment or lose their response over time. Currently, the only treatment for patients who do not respond to first-line therapy is surgery, namely stricture repair (intestinal repair) or resection (intestinal removal). Surgical treatment of IBD is invasive, and some patients experience postoperative risks such as anastomotic leakage, infection, and bleeding after surgery. The pathogenesis of IBD is thought to involve an uncontrolled immune response that may be triggered by certain environmental factors in genetically susceptible hosts. The heterogeneity of disease pathogenesis and clinical course, combined with varying responses to treatment and its associated side effects, suggests that targeted therapies are an ideal treatment strategy for these diseases. Therefore, novel therapies that specifically target the pathogenesis of IBD, and drugs that inhibit TL1A activity, have become popular targets for IBD treatment development. IBD presents a significant clinical need and has broad market potential.

[0007] However, there are currently no similar dual-target drugs approved for marketing in China or abroad. Summary of the Invention

[0008] 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 simultaneously and specifically bind to two targets: interleukin-6 receptor (IL-6R) and TNF-like ligand 1a (TL1A). By binding to two different cell surface antigens, it potentially increases binding specificity and reduces off-target side effects. Compared to combination therapy with monoclonal antibodies, this can effectively reduce treatment costs.

[0009] One aspect of this disclosure provides a bispecific antibody comprising: a first domain that specifically binds to the interleukin-6 receptor (IL-6R), and a second domain that specifically binds to TNF-like ligand 1a (TL1A).

[0010] In some embodiments, the first domain is an antibody that specifically binds to IL-6R or a functional fragment thereof.

[0011] In some embodiments, the second domain is an antibody that specifically binds to TL1A or a functional fragment thereof.

[0012] 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 IL-6R.

[0013] 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 TL1A.

[0014] In some implementations, the first domain and the second domain are directly connected or connected through connectors.

[0015] 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.

[0016] In some embodiments, the bispecific antibody includes a first domain and a second domain connected in any manner.

[0017] In some embodiments, the bispecific antibody includes a first domain-second domain extending from the N-terminus to the C-terminus.

[0018] In some embodiments, the bispecific antibody includes a second domain—a first domain—extending from the N-terminus to the C-terminus.

[0019] In some implementations, the first domain includes the following three heavy chain variable region complementarity determination regions (HCDRs):

[0020] 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.

[0021] 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.

[0022] 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.

[0023] In some implementations, the first domain includes the following three light chain variable region complementarity-determining regions (LCDRs):

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[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 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 YTS, and LCDR3 with sequence SEQ ID NO:47.

[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 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.

[0030] 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 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.

[0031] 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 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.

[0032] In some embodiments, the first 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.

[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: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.

[0035] 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.

[0036] 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.

[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: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.

[0039] 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.

[0040] 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.

[0041] In some embodiments, the second 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.

[0042] In some embodiments, the second 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.

[0043] 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.

[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 IMGT numbering system as follows: (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.

[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 Kabat numbering system: (a) a heavy chain variable region containing 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 containing 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 containing 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 containing 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:35, LCDR3 with sequence SEQ ID NO:36, and LCDR3 with sequence SEQ ID NO:37. LCDR2 with sequence number NO:34, and LCDR3 with sequence number NO:29.

[0046] 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: (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:34; and / or, a light chain variable region comprising the following three LCDRs: LCDR1 with sequence SEQ ID NO:35, LCDR2 with sequence SEQ ID NO:36, and LCDR3 with sequence SEQ ID NO:34. LCDR2 with sequence number NO:34, and LCDR3 with sequence number NO:29.

[0047] 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: (a) a heavy chain variable region containing 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 containing 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 containing 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 containing 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.

[0048] In some embodiments, the second 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.

[0049] In some embodiments, the second 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.

[0050] In some embodiments, the second 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.

[0051] 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. In some embodiments, the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:69 and 70, 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:71 and 72, 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.

[0052] Another aspect of this disclosure provides an isolated nucleic acid molecule that encodes the bispecific antibody of this disclosure.

[0053] 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. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO:83 and 84, 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:85 and 86, 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:87 and 88, 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:89 and 90, 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.

[0054] 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.

[0055] Another aspect of this disclosure provides an expression vector comprising the isolated nucleic acid molecules of this disclosure.

[0056] In some embodiments, the expression vector includes adenovirus, adeno-associated virus, lentivirus, or other acceptable expression vectors.

[0057] Another aspect of this disclosure provides a host cell comprising the isolated nucleic acid molecules or expression vectors described in this disclosure.

[0058] 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.

[0059] Another aspect of this disclosure provides a conjugate comprising the bispecific antibody described herein, and a conjugation portion.

[0060] 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.

[0061] Another aspect of this disclosure provides a pharmaceutical composition comprising the bispecific antibody described herein, and a pharmaceutically acceptable carrier.

[0062] In some embodiments, the pharmaceutical composition comprises the isolated nucleic acid molecule described in this disclosure, and a pharmaceutically acceptable carrier.

[0063] In some embodiments, the pharmaceutical composition includes the expression vector described in this disclosure, as well as pharmaceutically acceptable vectors.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] Another aspect of this disclosure provides a kit comprising the bispecific antibody described herein, and optionally, instructions for use.

[0070] 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 IL-6R-related diseases.

[0071] Another aspect of this disclosure provides a method for treating or preventing diseases associated with TL1A and / or IL-6R, 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.

[0072] In some implementations, the disease includes one or more of inflammatory diseases, immune system diseases, and cancer-related diseases.

[0073] 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.

[0074] This disclosure provides a bispecific antibody that can specifically bind to IL-6R and TL1A. This bispecific antibody simultaneously targets IL-6R and TL1A, and can block both the IL6 / IL6R signaling pathway and the TL1A / DR3 signaling pathway, exhibiting a synergistic effect. Detailed Implementation

[0075] 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.

[0076] Interleukin-6 (IL-6) is a multifunctional cytokine discovered in the 1980s, previously known as hepatocyte-stimulating factor, B-cell-stimulating factor 2, cytotoxic T-cell differentiation factor, B-cell differentiation factor, hybridoma / plasmacytoma growth factor, monocyte-granulocyte inducer type 2, and thrombopoietin. IL-6 is produced by various cell types, including T cells, B cells, monocytes, fibroblasts, osteoblasts, keratinocytes, endothelial cells, mesangial cells, and some tumor cells. IL-6 contains four α-helical domains with motifs of four cysteine ​​residues essential for its tertiary structure. IL-6 interacts with different IL-6 receptors on the cell surface, then transmits various biological signals to different tissues and cells via downstream signaling pathways.

[0077] Interleukin-6 (IL-6) binds to two receptors: the specific receptor IL-6R (an 80 kDa type I transmembrane protein) and gp130, a common receptor subunit of IL-6 family cytokine members. gp130 can be expressed in all cells, but the expression of the interleukin-6 receptor (IL-6R) is more restricted, primarily found in hepatocytes, neutrophils, monocytes, and CD4+ T cells. Dimerization of the interleukin-6 receptor gp130 initiates cellular events including activation of the JAK-STAT3 pathway and ras-mediated MAP kinase signaling.

[0078] TL1A, also known as TNFSF15 or VEGI, is a member of the tumor necrosis factor superfamily. First discovered in 2002, it is expressed in various cell types. These cells, such as monocytes (Mo) and macrophages, endothelial cells, dendritic cells, chondrocytes, and synovial fibroblasts, produce TL1A. TL1A regulates different immune cells, producing inflammatory cytokines and chemokines. TL1A activates the TRADD pathway by binding to its receptor DR3. TRADD can promote pro-inflammatory effects by regulating TRAF2 and RIP1, thereby inducing PI3K, MAPKs, and NF-κB. It also participates in apoptosis and necrotic cell death by activating the FADD, RIP3, and Caspase-8 / 3 / 7 pathways.

[0079] TL1A, a member of the tumor necrosis factor superfamily, mediates inflammation and fibrosis and is a key mediator of intestinal diseases. In the development of inflammatory bowel disease (IBD), TL1A affects epithelial-mesenchymal transition (EMT) in IBD patients through the TGF-β / Smad3 pathway, leading to colonic fibrosis and inflammatory responses. TL1A / DR3 can maintain the homeostasis of intestinal Treg cells, thereby exerting an immunosuppressive effect. A recent study suggests that DR3 / TL1A can also increase DR3 expression through Th1, Th2, and Th17 pathways. αDR3 activates the p38 signaling pathway of ILC3, thereby promoting GM-CSF secretion. Increased GM-CSF secretion further recruits pro-inflammatory myeloid cells to the intestine, and the intestinal infiltration of inflammatory myeloid cells ultimately leads to increased IL-23 expression and exacerbation of intestinal inflammation. As a central regulator of mucosal immune responses, allergies, and autoimmunity, TL1A / DR3 plays a crucial role in autoimmune and autoinflammatory diseases, and inhibiting TL1A is also an effective strategy in the treatment of autoimmune and inflammatory diseases.

[0080] TL1A is associated with a variety of autoimmune diseases, including rheumatoid arthritis (RA), inflammatory bowel disease (IBD), psoriasis (PSA), primary biliary cirrhosis (PBC), systemic lupus erythematosus (SLE), and ankylosing spondylitis (AS). TL1A is abnormally expressed in all of these diseases. Currently, the main indication for drug research is inflammatory bowel disease, including Crohn's disease and ulcerative colitis.

[0081] This disclosure, based on screened monoclonal antibodies targeting IL-6R and TL1A, further describes the expression of bispecific antibodies through co-transfection of host cells with dual plasmids. After routine purification to obtain the 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 IL-6R and TL1A. The bispecific antibodies disclosed in this disclosure target both TL1A and IL-6R-p19. CN2024104525229 and CN2024104373245 are incorporated herein by reference in their entirety.

[0082] 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.

[0083] 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.

[0084] Table 1

[0085]

[0086]

[0087] Table 2

[0088]

[0089]

[0090]

[0091] definition

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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).

[0096] 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 IL-6R and TL1A. In some embodiments, the bispecific antibody provided by this disclosure binds to IL-6R and TL1A at a Kk D All are 10 -7 M or lower, for example, 10 -10 M to 10 -11 M.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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).

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] Example 1: Construction of expression plasmid

[0111] The bispecific antibodies in this embodiment target IL-6R and TL1A. The anti-TL1A monoclonal antibody is the complete molecule of VDJ013 or VDJ013-PLT344, and the anti-IL-6R monoclonal antibody is the complete molecule of VDJ020-14. The anti-IL-6R×TL1A bispecific antibody is constructed by linking the complete molecules of VDJ013 or VDJ013-PLT344 or their scFv with the complete molecule of VDJ020-14 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.

[0112] Table 3

[0113]

[0114] Example 2: Transient expression of bispecific antibodies

[0115] 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. 6 Cells 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.

[0116] Example 3: Purification and Identification of Bispecific Antibodies

[0117] Six bispecific antibodies and a control monoclonal antibody 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.

[0118] The results in Table 4 show that the expression levels and purity of the bispecific antibodies VDJ013-9, VDJ013-10, VDJ013-11, VDJ013-12, VDJ013-23, and VDJ013-24 are all high.

[0119] Table 4. Antibody expression levels and SEC-HPLC purity identification

[0120] serial number Expression level (mg / L) purity(%) VDJ013-9 90.3 87.8 VDJ013-10 86.7 92.4 VDJ013-11 85.5 95.3 VDJ013-12 82.7 92.7 VDJ013-23 90.2 94.6 VDJ013-24 87.7 98.2 VDJ020-14 57.8 99.8 VDJ013 37.1 99.1 VDJ013-PLT344 105 98.0

[0121] Example 4: Detection of biological activity of TL1A target in bispecific antibodies

[0122] 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.

[0123] The Human TL1A gene (containing 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).

[0124] 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.

[0125] The results in Table 5 show that, compared with monoclonal antibodies VDJ013 and VDJ013-PLT344, bispecific antibodies VDJ013-9, VDJ013-10, VDJ013-11, VDJ013-12, VDJ013-23, and VDJ013-24 have comparable biological activity against the TL1A target.

[0126] Table 5. Detection of TL1A target biological activity of bispecific antibodies

[0127] serial number <![CDATA[IC 50 (nM)]]> Biological activity (reference standard / analyte * 100%) VDJ013 0.241-0.248 NA VDJ013-9 0.301 80 VDJ013-10 0.228 106 VDJ013-23 0.264 107 VDJ013-24 0.255 116 VDJ013-PLT344 0.427 NA VDJ013-11 0.572 75 VDJ013-12 0.451 95

[0128] Example 5: Detection of biological activity of IL-6R target in bispecific antibodies

[0129] The pGL4.47[luc2P SIE Hygro] plasmid from Promega was transformed into HEK293 cells (ATCC) to construct the HEK293-IL6 luciferase reporter gene viability test cell line. HEK293-IL6-res reporter gene cells were digested with trypsin, gently mixed, centrifuged, and the supernatant was discarded. The cells were resuspended and counted, and the cell density was adjusted to 5 × 10⁶ cells / year. 5Cells / mL were added at a rate of 80 μL / well to the corresponding wells. hIL-6 (R&D, 206-IL-010 / CF) was diluted to 50 ng / mL and added at 10 μL / well to the sample and positive control wells. 10 μL / well of basal medium was added to the negative wells. Antibodies were diluted to different concentration gradients with culture medium and added at 10 μL / well to the corresponding wells of a 96-well plate, with two replicates for each concentration. 10 μL / well of basal medium was added to both the positive and negative control wells. The cell culture plates were incubated at 37°C in a 5% CO2 incubator for 22 h. The next day, 100 μL / well of the melted and mixed One Lite assay reagent (brewed to room temperature) was added to the 96-well plates. The plates were shaken for 3 min to mix, and the chemiluminescence values ​​were read using a multi-mode microplate reader. Prism 5 (GraphPad) software was used to plot the data, calculate the IC50 value and relative biological activity. The results are shown in Table 6.

[0130] The results in Table 6 show that, compared with the biological activity of monoclonal antibodies, bispecific antibodies VDJ013-9, VDJ013-10, VDJ013-11, VDJ013-12, VDJ013-23, and VDJ013-24 have comparable biological activity against the IL-6R target.

[0131] Table 6. Detection of IL-6R target biological activity of bispecific antibodies

[0132] serial number <![CDATA[IC 50 (nM)]]> Biological activity (reference standard / analyte * 100%) VDJ020-14 0.1096～0.1278 NA VDJ013-9 0.1401 91 VDJ013-10 0.133 96 VDJ013-11 0.134 98 VDJ013-12 0.155 85 VDJ013-23 0.117 94 VDJ013-24 0.119 92

[0133] Example 6: Detection of relative binding activity of TL1A target in bispecific antibodies

[0134] 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.

[0135] The results in Table 7 show that the bispecific antibodies VDJ013-9, VDJ013-10, VDJ013-11, VDJ013-12, VDJ013-23 and VDJ013-24 all exhibited high binding activity to the TL1A target.

[0136] Table 7. Detection of relative binding activity of the bispecific antibody to the TL1A target

[0137] serial number EC50(nM) VDJ013-9 0.1871 VDJ013-10 0.1760 VDJ013-23 0.1602 VDJ013-24 0.210 VDJ013-11 0.294 VDJ013-12 0.371

[0138] Example 7: Detection of relative binding activity of IL-6R target in bispecific antibodies

[0139] The human IL-6R gene (NP_000556.1) was cloned into the pCDNA3.4 vector to construct a human IL-6R plasmid with the his tag. The plasmid was expressed by HEK293 transient transduction and purified by nickel column to obtain human IL-6R (hIL-6R).

[0140] The binding ability of bispecific antibodies to hIL-6R was detected using an ELISA method. hIL-6R 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 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 developed at 37°C for 10 minutes. Finally, 50 μL of H2SO4 was added to stop the reaction. OD450 values ​​were read using a TECAN multi-mode microplate reader, and the data were plotted using Prism 5 (GraphPad) software. EC50 values ​​and relative binding activities were calculated. The results are shown in Table 8.

[0141] The results in Table 8 show that the bispecific antibodies VDJ013-9, VDJ013-10, VDJ013-11, VDJ013-12, VDJ013-23 and VDJ013-24 all exhibited high binding activity to the IL-6R target.

[0142] Table 8. Detection of relative binding activity of the bispecific antibody to the IL-6R target

[0143] serial number EC50(nM) VDJ013-9 0.1599 VDJ013-10 0.1607 VDJ013-11 0.181 VDJ013-12 0.179 VDJ013-23 0.119 VDJ013-24 0.126

[0144] Example 8: Affinity determination of TL1A target in bispecific antibodies

[0145] Bio-Layer Interferometry (BLI) was used to determine the affinity of six 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.

[0146] The results in Table 9 show that the bispecific antibodies VDJ013-9, VDJ013-10, VDJ013-11, VDJ013-12, VDJ013-23 and VDJ013-24 all have high affinity for hTL1A.

[0147] Table 9. Affinity determination of antibody with hTL1A

[0148] serial number koff(1 / s) kon(1 / Ms) KD(M) VDJ013-9 4.44E-004 2.79E+006 1.59E-10 VDJ013-10 2.78E-04 4.12E+06 6.75E-11 VDJ013-23 1.26E-005 1.12E+006 1.12E-11 VDJ013-24 1.77E-005 1.29E+006 1.36E-011 VDJ013-11 <1.00E-006 6.00E+005 ＜1.0E-12 VDJ013-12 <1.00E-006 4.84E+005 ＜1.0E-12

[0149] Example 9: Affinity determination of IL-6R target in bispecific antibodies

[0150] The affinity of six bispecific antibodies for hIL-6R (as in Example 7) was determined using bio-layer interferometry (BLI). All antibodies were diluted to 15 μg / mL, and hIL-6R protein was serially diluted from 50 nM to 1.56 nM. Antibodies were added to hFc probes at 400 rpm / min, and then different gradients of diluted hIL-6R 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 bound analytes, allowing for regeneration and storage of the hFc probes. Using the built-in analysis software of Gator, the fitted lines of each concentration were integrated to obtain affinity data, and the results are shown in Table 10.

[0151] The results in Table 10 show that the bispecific antibodies VDJ013-9, VDJ013-10, VDJ013-11, VDJ013-12, VDJ013-23 and VDJ013-24 all have high affinity for hIL-6R.

[0152] Table 10. Affinity determination of antibody with IL-6R

[0153] serial number koff(1 / s) kon(1 / Ms) KD(M) VDJ013-9 2.07E-005 7.59E+005 2.73E-011 VDJ013-10 5.12E-005 8.23E+005 6.22E-011 VDJ013-11 2.73E-005 7.75E+005 3.53E-011 VDJ013-12 8.73E-005 1.05E+006 8.34E-011 VDJ013-23 2.20E-005 6.12E+005 3.60E-011 VDJ013-24 9.09E-005 8.75E+005 1.04E-010

[0154] 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 the interleukin-6 receptor (IL-6R) and a second domain that specifically binds to TNF-like ligand 1a (TL1A).

2. The bispecific antibody according to claim 1, characterized in that, The first domain is an antibody that specifically binds to IL-6R or a functional fragment thereof; and / or The second domain is an antibody that specifically binds to TL1A 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 IL-6R; and / or The second domain includes an antibody Fab fragment that specifically binds to TL1A, a Fab' fragment, and FF(ab'). 22 Fragments, Fv fragments, scFv fragments, nanobodies, heavy chain variable region (VH) fragments and / or light chain variable region (VL) fragments.

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 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.

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: 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 YTS, 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.

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: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.

8. The bispecific antibody according to any one of claims 1 to 7, characterized in that, The second 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 second 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.

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: (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.

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: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 second 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 second 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.

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 IL-6R-related diseases.

20. A method of treating or preventing diseases associated with TL1A and / or IL-6R, 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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