Monoclonal antibody against interleukin-6 protein and use thereof

Abstract

Disclosed in the present invention are a monoclonal antibody against an interleukin-6 protein and the use thereof. The present invention provides a nucleic acid molecule, a vector, a cell, a composition, a product, a bispecific antibody, a CAR, an antibody-drug conjugate and a pharmaceutical composition comprising an anti-IL-6 monoclonal antibody or an antigen-binding fragment thereof; and further provides the use of the monoclonal antibody or the antigen-binding fragment thereof, the nucleic acid molecule, the vector, the cell, the composition and the product in the detection of IL-6 or a nucleic acid fragment encoding same, in the preparation of a product for detecting IL-6, in a pharmaceutical composition for preventing or treating diseases related to abnormal IL-6 expression, and in the preparation of a pharmaceutical composition for regulating the expression level or activity of IL-6.

Description

Monoclonal antibodies targeting interleukin-6 protein and their applications Technical Field

[0001] The present disclosure belongs to the field of biotechnology and relates to monoclonal antibodies targeting interleukin-6 protein and applications thereof. Background Art

[0002] Interleukin-6 (IL-6) is a multi-effect cytokine that participates in many physiological and pathological processes in the body. Abnormal activation and dysfunction of the IL-6 signaling pathway are closely related to a variety of diseases, such as autoimmune diseases, chronic inflammation, and malignant tumors. In addition, abnormal expression of IL-6 also plays an important role in the cytokine storm syndrome (CSS) of the new coronavirus pneumonia (COVID-19). Unlike blocking common receptor molecules such as IL-6R or gp130, therapeutic monoclonal antibodies that block IL-6 protein are more specific. At present, only one monoclonal antibody drug targeting IL-6 protein has been approved for marketing by the US FDA and China NMPA ( Siltuximab for injection, Siltuximab), and more than 8 therapeutic monoclonal antibodies are in the clinical research stage. Siltuximab for injection is currently only approved for the treatment of adult patients with multicentric Castleman disease (MCD) who are human immunodeficiency virus (HIV) negative and human herpes virus type 8 (HHV-8) negative. In clinical studies, some therapeutic monoclonal antibodies targeting IL-6 protein have shown their unique therapeutic characteristics and beneficial efficacy (Clazakizumab for late antibody-mediated rejection, Doberer K, et al. JASN, 2021, 32(3): 708-722; Ziltivekimab for patients with chronic kidney disease (CKD) at high risk for atherosclerosis, Ridker PM, et al. The Lancet, 2021, 397(10289): 2060-2069.).

[0003] The development of high-affinity antibody drugs targeting IL-6 may be used to treat a variety of diseases related to abnormal activation and dysfunction of the IL-6 signaling pathway, such as autoimmune diseases, chronic inflammation, malignant tumors, cytokine storm syndrome (CSS), acute respiratory distress syndrome (ARDS), etc., and therefore may have very broad potential therapeutic effects and commercial prospects.

[0004] Summary of the Invention

[0005] In order to solve the technical problems existing in the related art, the present disclosure provides the following technical solutions:

[0006] In a first aspect, the present disclosure provides an anti-IL-6 monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof comprises CDR1-3 of a heavy chain variable region and CDR1-3 of a light chain variable region; the sequences of CDR1-3 of the heavy chain variable region are obtained by dividing the heavy chain variable region as shown in SEQ ID NO:8 using a CDR division scheme, and the sequences of CDR1-3 of the light chain variable region are obtained by dividing the light chain variable region as shown in SEQ ID NO:16 using a CDR division scheme, wherein the CDR division scheme includes a Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0007] Furthermore, the monoclonal antibody or its antigen-binding fragment is named 3C6, and the 3C6 includes the heavy chain variable region CDR1 shown in SEQ ID NO: 1, the heavy chain variable region CDR2 shown in SEQ ID NO: 2, the heavy chain variable region CDR3 shown in SEQ ID NO: 3, the light chain variable region CDR1 shown in SEQ ID NO: 9, the light chain variable region CDR2 shown in SEQ ID NO: 10, and the light chain variable region CDR3 shown in SEQ ID NO: 11.

[0008] The terms "CDR" and "complementarity determining region" as used herein are used interchangeably and refer to a portion of an antibody's variable chain that is involved in binding to an antigen. Thus, a CDR is a portion of an "antigen binding fragment" or an "antigen binding fragment." In some embodiments, a monoclonal antibody comprises three heavy chain variable region CDRs and three light chain variable region CDRs that together form an antigen binding fragment. "CDR" may refer to a CDR defined by any method known in the art (including a combination of methods).

[0009] In some embodiments, the CDRs are numbered using a system including, but not limited to, the Kabat, IMGT, Chothia, AbM, or Contact numbering systems.

[0010] Furthermore, the monoclonal antibody or antigen-binding fragment thereof further comprises heavy chain variable region framework regions FR1, FR2, FR3, and FR4, and light chain variable region framework regions FR1, FR2, FR3, and FR4.

[0011] Furthermore, the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity to the amino acid sequences described in SEQ ID NOs: 4-7; and the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity to the amino acid sequences described in SEQ ID NOs: 12-15.

[0012] Furthermore, the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 4-7, respectively; the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 12-15, respectively.

[0013] Furthermore, the sequence of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof has at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 8; the sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof has at least 90% sequence identity, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0014] Furthermore, the sequence of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 8; the sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 16.

[0015] As used herein, the term "IL-6" refers to the full-length IL-6 protein from human (UniProtKB P05231; GenBank Accession No. NP_000591.1) and cross-reactive full-length IL-6 proteins from other species, including macaque (UniProtKBAOA1D5QM02-1; GenBank Accession No. NP_001274245.1), mouse (UniProtKB P08505; GenBank Accession No. NP_112445.1), and rat (UniProtKB P20607; GenBank Accession No. NP_036721.1).

[0016] A second aspect of the present disclosure provides a nucleic acid molecule encoding the aforementioned monoclonal antibody or antigen-binding fragment thereof.

[0017] Furthermore, the nucleic acid molecule includes a nucleotide sequence encoding CDR1, CDR2, and CDR3 of the heavy chain variable region, and a nucleotide sequence encoding CDR1, CDR2, and CDR3 of the light chain variable region.

[0018] Furthermore, the nucleotide sequences encoding CDR1, CDR2, and CDR3 of the heavy chain variable region have at least 90% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 17-19, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity.

[0019] Furthermore, the nucleotide sequences encoding CDR1, CDR2, and CDR3 of the light chain variable region have at least 90% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 25-27, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity.

[0020] Furthermore, the nucleic acid molecule also includes nucleotide sequences encoding FR1, FR2, FR3, and FR4 of the heavy chain variable region framework region, and nucleotide sequences encoding FR1, FR2, FR3, and FR4 of the light chain variable region framework region.

[0021] Furthermore, the nucleotide sequences encoding FR1, FR2, FR3, and FR4 of the heavy chain variable region framework regions have at least 90% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 20-23, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity.

[0022] Furthermore, the nucleotide sequences encoding FR1, FR2, FR3, and FR4 of the light chain variable region framework region have at least 90% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 28-31, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity.

[0023] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the nucleic acid molecule has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO: 24, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity.

[0024] Furthermore, the nucleotide sequence encoding the light chain variable region of the nucleic acid molecule has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:32, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, preferably at least 98% sequence identity, preferably at least 99% sequence identity, and preferably at least 100% sequence identity.

[0025] Furthermore, the nucleic acid molecule includes DNA or mRNA.

[0026] The term "sequence identity" as used in this application refers to two polynucleotide sequences that are identical (i.e., on a nucleotide-by-nucleotide basis) over a comparison window. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, U, or I) occurs in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to produce the percentage of sequence identity.

[0027] The third aspect of the present disclosure provides a vector, which includes the nucleic acid molecule described above.

[0028] Furthermore, the vector includes plasmid, lentivirus, adenovirus, adeno-associated virus and other vectors.

[0029] The term "vector" as used in this application refers to a nucleic acid molecule that is capable of transporting another nucleic acid molecule connected thereto. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other various polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which another DNA fragment can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which a virally derived DNA or RNA sequence is present in a vector for packaging into a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) are capable of autonomous replication in the host cells into which they are introduced. Other vectors (e.g., non-additional mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell and are thereby replicated together with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Common expression vectors used in recombinant DNA technology are typically in the form of plasmids.

[0030] A fourth aspect of the present disclosure provides a cell, which comprises the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, or the aforementioned vector.

[0031] Furthermore, the cells include any cells that can replicate, preserve, and express the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, or the aforementioned vector.

[0032] Furthermore, the cells include prokaryotic cells or eukaryotic cells, wherein prokaryotic cells include bacteria and eukaryotic cells include mammalian cells, insect cells, bird cells, and yeast cells.

[0033] As used herein, the terms "cell," "cell line," and "host cell" are used interchangeably and encompass plant and animal cells, and include invertebrate, non-mammalian vertebrate, and mammalian cells. All such designations include cell populations and progeny. Exemplary non-mammalian vertebrate cells include, for example, avian cells, reptilian cells, and amphibian cells. Exemplary invertebrate cells include, but are not limited to, insect cells, such as caterpillar (Spodoptera frugiperda) cells, mosquito (Aedes aegypti) cells, fruit fly (Drosophila melanogaster) cells, Schneider cells, and silkworm cells. The cells may be differentiated, partially differentiated, or undifferentiated, such as stem cells, including embryonic stem cells and pluripotent stem cells. Additional tissue samples derived from an organ or organ system may be used in accordance with the present disclosure. Exemplary mammalian cells include, for example, cells derived from humans, non-human primates, cats, dogs, sheep, goats, cows, horses, pigs, rabbits, rodents including mice, hamsters, rats, and guinea pigs, and any derivatives and progeny thereof.

[0034] A fifth aspect of the present disclosure provides a composition comprising the aforementioned monoclonal antibody or antigen-binding fragment thereof, and a detectable marker.

[0035] Furthermore, the monoclonal antibody or antigen-binding fragment thereof and the detectable marker form a complex by coupling.

[0036] Furthermore, the detectable label includes a direct label or an indirect label.

[0037] As used herein, the term "detectable label" refers to at least one label that is capable of producing a detectable signal, either directly or indirectly. A non-exhaustive list of labels includes enzymes that produce a detectable signal (e.g., by colorimetry, fluorescence, luminescence), such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase; chromophores, such as fluorescent, luminescent dyes; groups whose electron density is detected by electron microscopy or by their electrical properties, such as conductivity, amperometry, voltammetry, impedance; detectable groups, such as molecules having a sufficient size to induce a detectable modification of their physical and / or chemical properties, such detection being possible by optical methods such as diffraction, surface plasmon resonance, surface changes, contact angle changes, or physical methods such as atomic force spectroscopy, tunneling, or radioactive molecules such as 32 P. 35 S or 125 I come to finish.

[0038] A sixth aspect of the present disclosure provides a product, which includes the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, or the aforementioned vector, the aforementioned cell, or the aforementioned composition.

[0039] Furthermore, the product includes a test kit, a test paper, a nucleic acid membrane strip, a chip, a system, or a device.

[0040] A seventh aspect of the present disclosure provides a bispecific antibody, which comprises a first binding domain and a second binding domain that specifically target IL-6; the first binding domain and the second binding domain are both derived from the aforementioned monoclonal antibody or antigen-binding fragment thereof.

[0041] Furthermore, the first binding domain and the second binding domain are connected via a linker.

[0042] Furthermore, the linker includes a flexible linker, a rigid linker and a shearable linker.

[0043] The term "linker" used in the present disclosure refers to a polypeptide whose function is to prevent two adjacent peptide sequences from interfering with each other in terms of spatial structure.

[0044] An eighth aspect of the present disclosure provides an antibody-drug conjugate, wherein the antibody-drug conjugate comprises the aforementioned monoclonal antibody or antigen-binding portion thereof conjugated to at least one drug.

[0045] Furthermore, the at least one drug is selected from anti-apoptotic agents, mitotic inhibitors, anti-tumor antibiotics, immunomodulators, nucleic acids for gene therapy, alkylating agents, anti-angiogenic agents, anti-metabolites, boron-containing agents, chemotherapeutic protectants, hormone agents, anti-hormonal agents, corticosteroids, photosensitizing therapeutic agents, oligonucleotides, radionuclide agents, radiosensitizers, topoisomerase inhibitors, and tyrosine kinase inhibitors.

[0046] Furthermore, the radionuclide agent includes 131 I. 32 P. 89 Sr. 90 Y. 223 Ra, 125 I. 103 Pd.

[0047] Furthermore, the alkylating agents include chlorambucil, chlorambucil, melphalan, propranolol, phenylephrine, estramustine, cyclophosphamide, hexamethylmetazobactam, ifosfamide, fortepdin, triamine, carmustine, streptozotocin, improsulfan, dacarbazine, cisplatin, oxaliplatin, and carboplatin.

[0048] Furthermore, the antimetabolites include methotrexate, 5-fluorouracil, fluorouracil, 5-fluorodeoxyuracil, capecitabine, cytarabine, fludarabine, 6-mercaptopurine, 2-chlorodeoxyadenosine, 5-azacytidine, 2,2-difluorodeoxycytidine, cladribine, deoxycoformycin, and pentostatin.

[0049] Furthermore, the anti-tumor antibiotics include daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin C, valrubicin, mitoxantrone hydrochloride, bleomycin, dactinomycin, mithramycin, and procarbazine.

[0050] Furthermore, the mitotic inhibitors include docetaxel, vinblastine, paclitaxel, vincristine, vinblastine, and vinorelbine.

[0051] Furthermore, the anti-angiogenic agents include prinomastat, tanomastat, ilomastat, aproxilamide, marimastat, batimastat, CGS-27023A, bromochloropiperaqua, COL-3, neovastat, BMS-275291, and thalidomide.

[0052] Furthermore, the anti-hormonal agents include toremifene, raloxifene, tamoxifen, anastrozole, letrozole, droloxifene, odoxifene, exemestane, nilutamide, bicalutamide, spironolactone, flutamide, finasteride, cyproterone acetate, and cimetidine.

[0053] Furthermore, the immunomodulators include interleukin, tumor necrosis factor, interferon, mushroom polysaccharide, ciclosporin, roquine, pidomod, methoxypolyethylene glycol succinamide adenosine deaminase, and thymosin preparations.

[0054] A ninth aspect of the present disclosure provides a pharmaceutical composition, comprising the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, the aforementioned bispecific antibody, or the aforementioned antibody-drug conjugate.

[0055] Furthermore, the invention also includes a pharmaceutically acceptable adjuvant.

[0056] In the tenth aspect, the present disclosure provides a CAR, comprising: an extracellular binding domain scFv that specifically recognizes IL-6; the extracellular binding domain comprises the light chain variable region and heavy chain variable region of an antibody targeting IL-6 as defined in the aforementioned monoclonal antibody or antigen-binding fragment thereof.

[0057] Furthermore, the CAR further comprises one, multiple or all of a hinge domain, a transmembrane domain, a co-stimulatory intracellular domain, or a signaling domain.

[0058] Furthermore, the hinge domain is selected from the hinge region of one or more of the following molecules: CD8α, CD28, CD152, PD1 and / or IgG1 heavy chain.

[0059] Furthermore, the transmembrane domain is selected from one or more transmembrane regions of the following molecules: α, β, ζ chains of TCR, CD3ε, CD3ζ, CD8α, CD16, CD28, CD33, CD64, CD80, CD86, CD134, 4-1BB, CD152, and / or PD1.

[0060] Furthermore, the co-stimulatory intracellular domain is selected from the intracellular regions of one or more of the following molecules: CD27, CD28, CD30, CD40, OX40, CD134, 4-1BB, CD150, CD223, PD-L2, PD-L1, NKD2C and / or FcεRIγ.

[0061] Furthermore, the signaling domain is selected from the intracellular region of one or more of the following molecules: Igα, Igβ, TCRξ, FcR1γ, FcR1β, CD3γ, CD3δ, CD3ε, CD22, CD28, CD79a, CD79b and / or CD3ζ.

[0062] The eleventh aspect of the present disclosure provides the use of the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, and the aforementioned composition in detecting IL-6 or a nucleic acid fragment encoding the same.

[0063] The twelfth aspect of the present disclosure provides the use of the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned vector, and the aforementioned cell in the preparation of a product for detecting IL-6.

[0064] The thirteenth aspect of the present disclosure provides the use of the aforementioned monoclonal antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, the aforementioned bispecific antibody, the aforementioned antibody-drug conjugate, or the aforementioned CAR in the preparation of a pharmaceutical composition for preventing or treating diseases related to abnormal IL-6 expression.

[0065] Furthermore, the diseases associated with abnormal IL-6 expression include rheumatoid arthritis, glomerular proliferation of glomerulonephritis, Crohn's disease (CD), Castleman's disease, asthma, viral myocarditis, hypertension, cardiac hypertrophy, arterial wall damage, atherosclerosis, enteritis, systemic lupus erythematosus (SLE), psoriasis, type 2 diabetes, endometritis, hemophagocytic lymphocytosis, Epstein-Barr virus-associated hemophagocytic lymphocytosis, systemic or non-systemic juvenile idiopathic arthritis-associated macrophage activation syndrome, NLRC4 macrophage activation syndrome, cytokine release syndrome, sepsis, acute respiratory distress syndrome (ARDS), pneumonia, pulmonary embolism, pulmonary edema, and respiratory failure.

[0066] The term "IL-6 abnormal expression disease" used in this application refers to any disease regulated by IL-6, including but not limited to immune system diseases, respiratory system inflammatory diseases, circulatory system inflammatory diseases, digestive system inflammatory diseases and other types of inflammatory diseases. In the embodiments of the present disclosure, specific examples of the IL-6 abnormal expression disease include but are not limited to rheumatoid arthritis, glomerular proliferation of glomerulonephritis, Crohn's disease (CD), Castleman's disease, asthma, viral myocarditis, hypertension, cardiac hypertrophy, arterial wall damage, atherosclerosis, enteritis, systemic lupus erythematosus (SLE), psoriasis, type 2 diabetes, endometritis, hemophagocytic lymphocytosis, Epstein-Barr virus-associated hemophagocytic lymphocytosis, systemic or non-systemic juvenile idiopathic arthritis-associated macrophage activation syndrome, NLRC4 macrophage activation syndrome, cytokine release syndrome, sepsis, acute respiratory distress syndrome (ARDS), pneumonia, pulmonary embolism, pulmonary edema, respiratory failure.

[0067] The term "acute respiratory distress syndrome (ARDS)" as used herein refers to a severe acute inflammatory response in the lungs, accompanied by diffuse damage to the alveolar-capillary barrier and massive exudation of protein-rich edema fluid into the alveolar cavity, leading to severe gas exchange impairment and a high mortality rate exceeding 40%, particularly in the elderly. Currently, there is a lack of effective therapeutic drugs in clinical practice, and glucocorticoids are the most commonly used treatment.

[0068] The term "cytokine release syndrome (CRS)" used in this application is also called cytokine storm. It is a severe systemic inflammatory response syndrome caused by the activation of immune cells and the release of large amounts of cytokines. The clinical manifestations are characterized by fever and multiple organ dysfunction. Among them, multiple cytokines such as tumor necrosis factor α, interleukin (IL), and interferon (IFN) are rapidly produced in large quantities, forming a cytokine storm.

[0069] Furthermore, the diseases associated with abnormal IL-6 expression include cytokine release syndrome and acute respiratory distress syndrome.

[0070] In some specific embodiments, the pharmaceutical composition for treating diseases related to abnormal IL-6 expression can reduce the level of IFN-γ (Interferon-γ) in cytokine release syndrome. In some specific embodiments, the pharmaceutical composition for treating diseases related to abnormal IL-6 expression can reduce the mortality rate of ARDS.

[0071] Furthermore, the pharmaceutical composition includes the form of nasal spray, oral preparation, suppository or parenteral preparation.

[0072] The term "parenteral" as used herein may include subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0073] Furthermore, the pharmaceutical composition includes a pharmaceutically acceptable adjuvant.

[0074] The term "pharmaceutically acceptable" as used herein describes a material that is not biologically or otherwise undesirable, ie, does not cause unacceptable levels of undesired biological effects or interact in a deleterious manner.

[0075] In a fourteenth aspect, the present disclosure provides the use of the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, the aforementioned bispecific antibody, the aforementioned antibody-drug conjugate, or the aforementioned CAR in the preparation of a pharmaceutical composition for regulating IL-6 expression level or activity.

[0076] A fifteenth aspect of the present disclosure provides the use of the aforementioned monoclonal antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, the aforementioned composition, the aforementioned bispecific antibody, the aforementioned antibody-drug conjugate, the aforementioned pharmaceutical composition, or the aforementioned CAR in regulating immune system responses.

[0077] Furthermore, the immune system includes all genes, immune cells and immune organs / tissues of innate immunity, adaptive immunity, cellular immunity, humoral immunity, active immunity and passive immune responses.

[0078] The term "immune system" as used in this application is a term that includes all phenomena that affect the immune response in the body (i.e., participate in mechanisms that manifest new immune effects) through functional changes of manipulated immunoregulatory factors, and includes all substances, compositions, methods and uses that directly or indirectly participate in such immune system.

[0079] Furthermore, the immune cells include T cells, B cells, NK cells, NKT cells, DNT cells, hematopoietic cells, pluripotent stem cells, myeloid progenitor cells, lymphoid progenitor cells, and tumor-infiltrating lymphocytes.

[0080] Furthermore, the T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes.

[0081] Furthermore, the T cells include helper T cells, cytotoxic T cells, tumor-infiltrating cytotoxic T cells, CD4 + CD8 + T cells, CD4 - CD8 - T cells, αβ T cells expressing the α and β chains of the T cell receptor and / or γδ T cells expressing the γ and δ chains of the TCR.

[0082] The term "immune cell" as used in this application refers to cells that can trigger an immune response, including but not limited to T cells, B cells, NK cells, NKT cells, DNT cells, etc., their respective precursor cells and their progeny. Immune response cells can also refer to cells of the lymphoid or myeloid lineages.

[0083] As used herein, the term "immune cell" and its grammatical alternatives may refer to immune cells of any origin. For example, immune cells may be derived from blood, such as autologous T cells, allogeneic T cells, autologous NK cells, or allogeneic NK cells. They may also be derived from cell lines, such as NK cell lines prepared by infection with EBV, NK cells differentiated from embryonic stem cells and iPS cells, and the NK92 cell line. "Immune cells" may be human or non-human mammalian.

[0084] The terms "T cell" or "T lymphocyte" as used herein are art-recognized and are intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell can be a T helper (Th) cell, such as a T helper 1 (Th1) or T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 + T cells), cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells, αβT cells expressing the α and β chains of T cell receptors (TCR) and γδT cells expressing TCRγ and δ chains, or any other subset of T cells.Other illustrative T cell groups suitable for specific embodiments include memory T cells, suitably early memory T cells.The term "T cell" includes within its scope natural T cells (e.g., separated from an organism, such as a mammal, for example, people, for example, a subject), isolated T cells and genetically engineered T cells.The term T cell also includes T cells comprising T cell receptors (e.g., natural TCR, or recombinant TCR) and T cells (e.g., CAR-T cells) comprising artificial T cell receptors.

[0085] In a sixteenth aspect, the present disclosure provides the use of the aforementioned monoclonal antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, the aforementioned composition, the aforementioned bispecific antibody, the aforementioned antibody-drug conjugate, the aforementioned pharmaceutical composition, or the aforementioned CAR in regulating cell phosphorylation, inhibiting inflammatory response, regulating cell apoptosis, proliferation, or migration.

[0086] Furthermore, the phosphorylation of cells includes phosphorylation of immune cells.

[0087] Furthermore, the immune cells include T cells, B cells, NK cells, NKT cells, DNT cells, hematopoietic cells, pluripotent stem cells, myeloid progenitor cells, lymphoid progenitor cells, and tumor-infiltrating lymphocytes.

[0088] Furthermore, the T cells include helper T cells, cytotoxic T cells, tumor-infiltrating cytotoxic T cells, CD4 + CD8 + T cells, CD4 - CD8 - T cells, αβ T cells expressing the α and β chains of the T cell receptor and / or γδ T cells expressing the γ and δ chains of the TCR.

[0089] In one embodiment, the T cells are helper T cells. In one embodiment, the T cells are cytotoxic T cells. In one embodiment, the T cells are tumor-infiltrating cytotoxic T cells. In one embodiment, the T cells are CD4 + CD8 + In one embodiment, the T cells are CD4 - CD8 - T cells. In one embodiment, the T cells are αβ T cells expressing the α and β chains of the T cell receptor. In one embodiment, the T cells are γδ T cells expressing the γ and δ chains of the TCR.

[0090] Furthermore, the phosphorylation includes phosphorylation of STAT3 protein.

[0091] In some embodiments, the inflammatory response is an inflammatory response in a subject.

[0092] The term "inflammation" used in this application generally refers to the biological response of tissue to harmful stimuli such as pathogens, damaged cells (for example, wounds) and irritants.Term "inflammatory response" refers to the specific mechanism of achieving and regulating inflammation, including (only by way of example) immune cell activation or migration, migration, autoimmunity and autoimmune diseases, cytokine production, vasodilation, including kinin release, fibrinolysis and coagulation, and other methods as described herein and known in the art.Ideally, inflammation is the protective attempt of the body to remove harmful stimuli and start the healing process of infected one or more tissues.In the absence of inflammation, wounds and infections will not heal, and the continued destruction of the resulting tissue will endanger the situation of survival.

[0093] In the seventeenth aspect of the present disclosure, the monoclonal antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the cell, the bispecific antibody, the antibody-drug conjugate, or the CAR described above are used in the preparation of a pharmaceutical composition for treating tumors.

[0094] Furthermore, the tumor includes a tumor associated with STAT3 phosphorylation.

[0095] Furthermore, the tumor includes solid tumors and non-solid tumors.

[0096] The terms "tumor", "cancer" or "cancerous disease" as used in this application refer to malignant diseases, which are preferably selected from the group consisting of: adenocystic carcinoma, adenoid cystic carcinoma, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma, cerebellar astrocytoma / malignant glioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor, optic pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma lymphoma, childhood carcinoid tumors, gastrointestinal carcinoid tumors, carcinoma of unknown primary, childhood cerebellar astrocytoma, childhood cerebellar astrocytoma / glioblastoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, colon cancer, cutaneous T-cell lymphoma (including mycosis fungoides and Sezary syndrome), Syndrome), desmoplastic small round cell tumor, endometrial cancer, esophageal cancer, Ewing sarcoma (a member of the Ewing family of tumors), extracranial germ cell tumors in children, extragonadal germ cell tumors, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor (GIST), extracranial, extragonadal, or ovarian germ cell tumors, gestational trophoblastic tumor, brainstem glioma, juvenile astrocytoma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma (liver cancer), Hodgkin lymphoma, human papillomavirus (HPV)-associated cancers, hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, lip and oral cavity cancer, Liposarcoma, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, Burkitt lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, malignant fibrous histiocytoma of bone / osteosarcoma, childhood medulloblastoma, Merkel cell carcinoma Carcinoma), adult malignant mesothelioma, childhood mesothelioma, childhood multiple endocrine neoplasia syndrome (MSN), multiple myeloma / plasma cell neoplasms, multiple myeloma (bone marrow cancer), nasal and sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, sinus and nasal cancer, parathyroid cancer, penile cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, childhood pinealocyte tumor, pituitary adenoma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter cancer, childhood rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma),Small bowel cancer, squamous cell carcinoma, metastatic occult primary squamous cell carcinoma of the neck, primitive neuroectodermal tumors in children, testicular cancer (seminoma and nonseminoma), pharyngeal cancer, thymoma in children, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, gestational trophoblastic tumor, urethral cancer, endometrial cancer, vaginal cancer, gliomas of the visual pathway and hypothalamus in children, vulvar cancer, and Wilms tumor (kidney cancer) in children.

[0097] In an eighteenth aspect, the present disclosure provides a method for producing the aforementioned monoclonal antibody or its antigen-binding fragment, the method comprising the following steps: transforming the aforementioned nucleic acid molecule or the aforementioned vector into cells, culturing the cells, and isolating and purifying the aforementioned monoclonal antibody or its antigen-binding fragment from the cell culture fluid.

[0098] In a nineteenth aspect, the present disclosure provides a method for inhibiting IL-6, comprising introducing the aforementioned monoclonal antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, or the aforementioned vector into a cell of an organism, and inhibiting the activity of IL-6 by expressing the aforementioned monoclonal antibody or its antigen-binding fragment.

[0099] The twentieth aspect of the present disclosure provides a method for detecting IL-6 in a test sample, the method comprising contacting the test sample with the aforementioned monoclonal antibody or its antigen-binding fragment, or contacting the test sample with the aforementioned composition; and detecting the binding of IL-6 to the aforementioned monoclonal antibody or its antigen-binding fragment, or detecting the binding of IL-6 to the aforementioned composition.

[0100] The twenty-first aspect of the present disclosure provides a method for treating a disease with abnormal IL-6 expression, the method comprising the step of using the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, or the aforementioned composition for treatment.

[0101] Furthermore, the diseases associated with abnormal IL-6 expression include rheumatoid arthritis, glomerular proliferation of glomerulonephritis, Crohn's disease, Castleman's disease, asthma, viral myocarditis, hypertension, cardiac hypertrophy, arterial wall damage, atherosclerosis, enteritis, systemic lupus erythematosus, psoriasis, type 2 diabetes, endometritis, hemophagocytic lymphocytosis, Epstein-Barr virus-associated hemophagocytic lymphocytosis, systemic or non-systemic juvenile idiopathic arthritis-associated macrophage activation syndrome, NLRC4 macrophage activation syndrome, cytokine release syndrome, sepsis, acute respiratory distress syndrome, pneumonia, pulmonary embolism, pulmonary edema, and respiratory failure.

[0102] The twenty-second aspect of the present disclosure provides a method for treating a disease with abnormal IL-6 expression, the method comprising: 1) diagnosing the disease with abnormal IL-6 expression using the aforementioned monoclonal antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, or the aforementioned composition; and 2) administering a drug to the patient based on the diagnosis result.

[0103] Furthermore, the diseases associated with abnormal IL-6 expression include rheumatoid arthritis, glomerular proliferation of glomerulonephritis, Crohn's disease, Castleman's disease, asthma, viral myocarditis, hypertension, cardiac hypertrophy, arterial wall damage, atherosclerosis, enteritis, systemic lupus erythematosus, psoriasis, type 2 diabetes, endometritis, hemophagocytic lymphocytosis, Epstein-Barr virus-associated hemophagocytic lymphocytosis, systemic or non-systemic juvenile idiopathic arthritis-associated macrophage activation syndrome, NLRC4 macrophage activation syndrome, cytokine release syndrome, sepsis, acute respiratory distress syndrome, pneumonia, pulmonary embolism, pulmonary edema, and respiratory failure.

[0104] The term "antibody" as used in this application is used in the broadest sense and includes various antibody structures, including but not limited to structures known from monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0105] The term "monoclonal antibody" as used in this application is intended to be used in the art and refers to an antibody obtained from a population of substantially homologous antibodies that bind to the same antigenic determinant (epitope). "Substantially homologous" means that the individual antibodies are identical except for possible naturally occurring mutations that may be present in small amounts. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against a variety of different antigenic determinants (epitopes). The term "monoclonal antibody" encompasses complete and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. "Monoclonal antibodies" and antigen-binding fragments thereof are made in many ways, including but not limited to, by hybridomas, phage selection, recombinant expression, and transgenic animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] FIG1 is an SDS-PAGE electrophoresis diagram of antibody 3C6;

[0107] FIG2 is a graph showing the results of an ELISA affinity assay of antibody 3C6 binding to IL-6;

[0108] FIG3 is a graph showing the results of the inhibition of IL-6 binding to IL-6R by antibodies 3C6 and Siltuximab;

[0109] FIG4 is a graph showing the specific binding of antibody 3C6 to IL-6;

[0110] Figure 5 shows that antibody 3C6 inhibits IL-6-induced human CD4 + T cell STAT3 molecule phosphorylation results diagram;

[0111] FIG6 is a graph showing the pharmacodynamics of antibody 3C6 in treating the ARDS model of human IL-6 / IL-6R double transgenic mice;

[0112] FIG7 is a graph showing the results of the inhibition of cytokines in cytokine release syndrome by antibody 3C6. DETAILED DESCRIPTION

[0113] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features that can be readily separated or combined with features from any of the other several aspects without departing from the scope or spirit of this disclosure.

[0114] It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the specification and the relevant art, and should not be interpreted as idealized or overly formal unless expressly defined herein.

[0115] Example 1 Screening of fully human monoclonal antibody 3C6 against human interleukin 6 (hIL-6) IL-6

[0116] 1. Construction of phage antibody library

[0117] A fully human antibody library targeting hIL-6 protein was constructed using computer-aided molecular design, and candidate antibodies were screened using phage display technology. The specific steps are as follows: (1) The displayed phage library adheres to human interleukin-6; (2) Non-specific binding is removed by repeated washing, and phage displaying human interleukin-6 is eluted and collected; (3) Escherichia coli is re-infected, and single clones are selected for display on the phage surface. ELISA is used to screen positive clones that recognize human interleukin-6.

[0118] 2. Determination and analysis of antibody gene sequences

[0119] The positive clones were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for antibody gene sequencing, and the sequencing results were retrieved and analyzed using DNAMAN and database.

[0120] 3. Results

[0121] Through the above steps, a fully human monoclonal antibody 3C6 capable of recognizing human interleukin-6 was screened out, and the specific sequence is shown in Table 1.

[0122] Table 1 Antibody sequences

[0123] Example 2 Preparation of Antibody 3C6

[0124] 1. Experimental methods

[0125] The antibody 3C6 sequence was amplified using PCR and cloned into an expression vector using molecular cloning. The expression vector was transfected into mammalian cells for expression. The supernatant was collected and purified using a Cytiva (formerly GE) ProteinAFF column.

[0126] The solution was eluted with pH 3.0 citric acid buffer, and the effluent was collected and immediately neutralized with 1 mol / L pH 8.5 Tris-HCl buffer, dialyzed with pH 7.2, 0.01 mol / L PBS for 72 h, and sterilized by filtration with a 0.22 μm filter membrane.

[0127] The expression and purification of the antibody were verified by SDS-PAGE, and the concentration of the purified antibody was detected by BCA method. The antibody was stored at 4°C.

[0128] 2. Experimental results

[0129] The experimental results are shown in FIG1 , which shows an SDS-PAGE electrophoresis diagram of antibody 3C6 (reduced). From left to right, lane 1 is a protein molecular weight marker, and lane 2 is purified 3C6.

[0130] Example 3 ELISA detection of the binding of 3C6 to human IL-6

[0131] 1. Experimental methods

[0132] Use carbonate buffer (Sigma Cat#C3041) to dilute IL-6 to 0.5μg / mL, 100μL / well, coat at room temperature for 1h, 500rpm. After washing the plate 3 times, use SuperBlock (Thermo Fisher Scientific Inc, Cat.#37515) 150μL / well, 750rpm, incubate at room temperature for 1h. After patting the ELISA plate dry, add 3C6 at concentrations ranging from 0.1ng / mL to 10000ng / mL, 500rpm, and incubate at room temperature for 1h. After washing the plate 6 times, add 1:60000 diluted GoatAnti-Human IgG HRP (SouthernBiotech Cat.#2049-5), 500rpm, and incubate at room temperature for 1h. After washing the plate 6 times, add TMB colorimetric solution and terminate the reaction with dilute sulfuric acid. OD value was detected by dual wavelength OD 450 / 630nm. Use Pro software fits and calculates EC 50 value.

[0133] 2. Experimental results

[0134] The results of ELISA detection are shown in Figure 2 and Table 2, which show that 3C6 has an affinity close to that of Siltuximab.

[0135] Table 2

[0136] Example 4 3C6 inhibits the binding of human IL-6 to human IL-6R

[0137] 1. Experimental methods

[0138] IL-6 was diluted to 0.5 μg / mL using carbonate buffer (Sigma Cat.#C3041) and coated at 100 μL / well for 1 hour at room temperature at 500 rpm. After washing the plate three times, SuperBlock (Thermo Fisher Scientific Inc, Cat.#37515) was used at 150 μL / well at 500 rpm for 1 hour at room temperature. After patting the plate dry, serial dilutions of 3C6 were added, ranging from 0.1 nM to 33.3 nM, and IL-6R-Biotin (250 ng / mL) was incubated at 500 rpm for 1 hour at room temperature. After washing the plate six times, Streptavidin HRP (Sigma Cat.#85878) was added at a 1:20,000 dilution at 500 rpm for 1 hour at room temperature. After washing the plate six times, TMB colorimetric solution was added and the reaction was terminated with dilute sulfuric acid. OD values ​​were measured at a dual wavelength of 450 / 630 nm.

[0139] 2. Experimental results

[0140] 3C6 can significantly inhibit the interaction between IL-6 and its receptor IL-6R. As shown in Figure 3, the inhibitory activity is comparable to that of siltuximab. Therefore, the antibody 3C6 may be used to treat a variety of diseases caused by IL-6 imbalance, such as autoimmune diseases, chronic inflammation, malignant tumors, and cytokine storm syndrome (CSS).

[0141] Example 5 Specificity of Antibody 3C6

[0142] 1. Experimental methods

[0143] IL-6, VEGF, CD19, CD3, C5a, and Sars-Cov-2 S1 proteins (purchased from Sino Biological Technologies, Beijing, China) were diluted to 0.5 μg / mL using carbonate buffer (Sigma, Cat#C3041). Coating was performed at 100 μL / well for 1 hour at room temperature at 500 rpm. The plate was washed three times and then incubated with 150 μL / well of SuperBlock (Thermo Fisher Scientific, Inc, Cat.#37515) at 500 rpm for 1 hour at room temperature. After patting the plate dry, 2000 ng / mL of 3C6 was added at 500 rpm for 1 hour at room temperature. The plate was washed six times, and then Goat Anti-Human IgG HRP (Southern Biotech, Cat.#2049-5) was added at a dilution of 1:60,000 at 500 rpm for 1 hour at room temperature. The plate was washed six times, and then TMB color development solution was added and the reaction was terminated with dilute sulfuric acid. OD value was detected by dual wavelength of 450 / 630nm.

[0144] 2. Experimental results

[0145] The experimental results are shown in FIG4 , which show that antibody 3C6 specifically binds to IL-6 without obvious cross-reaction with other tested proteins, indicating that antibody 3C6 has good specificity.

[0146] Example 6 Antibody 3C6 against CD4 + Inhibition of T cell STAT3 phosphorylation

[0147] 1. Experimental methods

[0148] Human leukocyte samples (Milestone Biotechnologies) were stimulated with IL-6 (5 ng / mL) at 37°C for 15 minutes as a positive control. In the experimental groups, IL-6 was pre-incubated with various concentrations of 3C6 or siltuximab (ST) at 37°C for 30 minutes and then stimulated for 15 minutes. An unstimulated group served as a negative control. Subsequently, 2 mL of cold erythrocyte lysis / fixation buffer (Biolegend, Cat. #422401) was added to lyse erythrocytes and fix the phosphorylation status of STAT3, followed by incubation on ice for 0.5 hours. After washing, anti-CD3 and anti-CD4 fluorescent antibodies (Biolegend, Cat. 300306 and 357420) were added and incubated on ice for 0.5 hours. The cells were then washed and permeabilized with 1 mL of pre-cold Perm buffer III (BD, Cat. 558050) on ice for 30 minutes. Subsequently, cells were stained with anti-pSTAT3 (pY705)-PE (BD, Cat, 559321) and anti-CD45 fluorescent antibody (Biolegend, Cat, 368506) at room temperature for 30 minutes. The stained cells were washed twice with 2 mL of staining buffer (BD, Cat, 554656) and analyzed by flow cytometry (DxFLEX, Beckman Coulter).

[0149] 2. Experimental results

[0150] The results are shown in Figure 5. The results show that antibody 3C6 can effectively inhibit CD4 + Phosphorylation of STAT3 in T cells was comparable to that of siltuximab.

[0151] Example 7 Pharmacodynamics of Antibody 3C6 in the ARDS Model of IL-6 / IL-6R Double Transgenic Mice

[0152] 1. Experimental methods

[0153] Experimental animals: 6-8 weeks old, human IL-6 / IL-6R double transgenic mice (purchased from Shanghai Model Organisms Technology Co., Ltd.);

[0154] Model establishment: All experimental groups of mice were intraperitoneally injected with lipopolysaccharide. The normal control group received an intraperitoneal injection of sodium chloride injection. Approximately 6 hours (±30 minutes) after intraperitoneal injection of LPS, animals in each group were anesthetized with isoflurane inhalation, and lipopolysaccharide was administered via intratracheal aerosolization. The normal control group received intratracheal aerosolization of sodium chloride injection (1 mL / kg). All groups received intravenous administration. The normal control group received normal saline, the model control group received normal saline, the 3C6 low-dose group received 3C6 (1.1 mg / kg), the 3C6 high-dose group received 3C6 (11 mg / kg), and the tocilizumab group received the antibody at a dose of 12 mg / kg. Drug mortality and other indicators were observed on the second day after administration.

[0155] 2. Experimental results

[0156] The results are shown in Figure 6. High-dose 3C6 effectively inhibited death in ARDS model animals, achieving a survival rate of 75%, superior to the 50% survival rate of tocilizumab. Low-dose 3C6 was comparable to tocilizumab in inhibiting death in ARDS models.

[0157] Example 8: Antibody 3C6 alleviates cytokine release syndrome

[0158] 1. Experimental methods

[0159] Human PBMCs were resuspended in RPMI 1640 complete medium (supplemented with 20% FBS, 2% penicillin and streptomycin antibiotics, and 1% glutamate supplement) and the concentration was adjusted to 1×10 6 / mL. Antibodies 3C6 and tocilizumab were added to a 96-well cell culture plate at a final concentration of 150 μg / mL, LPS was added to a final concentration of 10 ng / mL, and 50 μL of the cell suspension was incubated at 37°C, 5% CO₂ for 24 hours. The supernatant was collected by centrifugation at 1000 g for 10 minutes. IFN-γ concentration in the supernatant was measured using a kit from Meso Scale Discovery.

[0160] 2. Experimental results

[0161] The results are shown in Figure 7, which show that both antibody 3C6 and tocilizumab can effectively inhibit the IFN-γ level in the LPS-induced cytokine release model, and there is no significant difference between antibody 3C6 and tocilizumab.

[0162] Having benefited from the teachings given in the foregoing description and the associated drawings, those skilled in the art to which the disclosed technical solutions pertain will recognize many modifications and other aspects disclosed herein. Therefore, it should be understood that the present disclosure is not limited to the specific aspects disclosed, and modifications and other aspects are intended to be included within the scope of the appended claims. Those skilled in the art will recognize many variations and adaptations of the various aspects described herein. These variations and adaptations are intended to be included in the teachings of the present disclosure and are covered by the claims herein. Unless otherwise indicated, various aspects of the present disclosure will employ techniques of molecular biology, microbiology, organic chemistry, biochemistry, physiology, cell biology, vascular biology, etc., which are within the technical scope of the art. Such techniques are fully explained in the literature.

[0163] The description of the above embodiments is only for understanding the method and core concept of the present disclosure. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present disclosure without departing from the principles of the present disclosure, and these improvements and modifications will also fall within the scope of protection of the claims of the present disclosure.

Claims

1. An anti-IL-6 monoclonal antibody or antigen-binding fragment thereof, comprising CDRs 1-3 of a heavy chain variable region and CDRs 1-3 of a light chain variable region; the sequences of the CDRs 1-3 of the heavy chain variable region are obtained by dividing the heavy chain variable region as shown in SEQ ID NO: 8 using a CDR demarcation system, and the sequences of the CDRs 1-3 of the light chain variable region are obtained by dividing the light chain variable region as shown in SEQ ID NO: 16 using a CDR demarcation system, wherein the CDR demarcation system includes a Kabat, IMGT, Chothia, AbM, or Contact numbering system.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region CDR1 set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 set forth in SEQ ID NO: 2, a heavy chain variable region CDR3 set forth in SEQ ID NO: 3, a light chain variable region CDR1 set forth in SEQ ID NO: 9, a light chain variable region CDR2 set forth in SEQ ID NO: 10, and a light chain variable region CDR3 set forth in SEQ ID NO:

11.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity with the amino acid sequences of SEQ ID NOs: 4-7, respectively.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 95% sequence identity with the amino acid sequences of SEQ ID NOs: 4-7, respectively.

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 4, wherein the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 96% sequence identity with the amino acid sequences of SEQ ID NOs: 4-7, respectively.

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 5, wherein the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 97% sequence identity with the amino acid sequences of SEQ ID NOs: 4-7, respectively.

7. The monoclonal antibody or antigen-binding fragment thereof according to claim 6, wherein the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 98% sequence identity with the amino acid sequences of SEQ ID NOs: 4-7, respectively.

8. The monoclonal antibody or antigen-binding fragment thereof according to claim 7, wherein the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 99% sequence identity with the amino acid sequences of SEQ ID NOs: 4-7, respectively.

9. The monoclonal antibody or antigen-binding fragment thereof according to claim 8, wherein the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have 100% sequence identity with the amino acid sequences of SEQ ID NOs: 4-7, respectively.

10. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 90% sequence identity with the amino acid sequences of SEQ ID NOs: 12-15, respectively.

11. The monoclonal antibody or antigen-binding fragment thereof according to claim 10, wherein the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 95% sequence identity with the amino acid sequences of SEQ ID NOs: 12-15, respectively.

12. The monoclonal antibody or antigen-binding fragment thereof according to claim 11, wherein the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 96% sequence identity with the amino acid sequences of SEQ ID NOs: 12-15, respectively.

13. The monoclonal antibody or antigen-binding fragment thereof according to claim 12, wherein the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 97% sequence identity with the amino acid sequences of SEQ ID NOs: 12-15, respectively.

14. The monoclonal antibody or antigen-binding fragment thereof according to claim 13, wherein the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 98% sequence identity with the amino acid sequences of SEQ ID NOs: 12-15, respectively.

15. The monoclonal antibody or antigen-binding fragment thereof according to claim 14, wherein the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have at least 99% sequence identity with the amino acid sequences of SEQ ID NOs: 12-15, respectively.

16. The monoclonal antibody or antigen-binding fragment thereof according to claim 15, wherein the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof have 100% sequence identity with the amino acid sequences of SEQ ID NOs: 12-15, respectively.

17. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the sequences of the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof are respectively shown in SEQ ID NOs: 4-7; and the sequences of the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the monoclonal antibody or antigen-binding fragment thereof are respectively shown in SEQ ID NOs: 12-15.

18. A nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

19. The nucleic acid molecule according to claim 18, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the heavy chain variable region of the nucleic acid molecule has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 17-19.

20. The nucleic acid molecule according to claim 19, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the heavy chain variable region of the nucleic acid molecule has at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 17-19.

21. The nucleic acid molecule according to claim 20, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the heavy chain variable region of the nucleic acid molecule has at least 96% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 17-19.

22. The nucleic acid molecule according to claim 21, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the heavy chain variable region of the nucleic acid molecule has at least 97% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 17-19.

23. The nucleic acid molecule according to claim 22, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the heavy chain variable region of the nucleic acid molecule has at least 98% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 17-19.

24. The nucleic acid molecule according to claim 23, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the heavy chain variable region of the nucleic acid molecule has at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 17-19.

25. The nucleic acid molecule according to claim 24, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the heavy chain variable region of the nucleic acid molecule has 100% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 17-19.

26. The nucleic acid molecule according to claim 18, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the light chain variable region of the nucleic acid molecule has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 25-27.

27. The nucleic acid molecule according to claim 26, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the light chain variable region of the nucleic acid molecule has at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 25-27.

28. The nucleic acid molecule according to claim 27, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the light chain variable region of the nucleic acid molecule has at least 96% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 25-27.

29. The nucleic acid molecule according to claim 28, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the light chain variable region of the nucleic acid molecule has at least 97% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 25-27.

30. The nucleic acid molecule according to claim 29, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the light chain variable region of the nucleic acid molecule has at least 98% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 25-27.

31. The nucleic acid molecule according to claim 30, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the light chain variable region of the nucleic acid molecule has at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 25-27.

32. The nucleic acid molecule according to claim 31, wherein the nucleotide sequence encoding CDR1, CDR2, and CDR3 of the light chain variable region of the nucleic acid molecule has 100% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 25-27.

33. The nucleic acid molecule according to claim 18, wherein the nucleotide sequence encoding the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 20-23.

34. The nucleic acid molecule according to claim 33, wherein the nucleotide sequence encoding the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 20-23.

35. The nucleic acid molecule according to claim 34, wherein the nucleotide sequence encoding the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has at least 96% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 20-23.

36. The nucleic acid molecule according to claim 35, wherein the nucleotide sequence encoding FR1, FR2, FR3, and FR4 of the heavy chain variable region framework region of the nucleic acid molecule has at least 97% sequence identity with the nucleotide sequence shown in SEQ ID NO: 20-23.

37. The nucleic acid molecule according to claim 36, wherein the nucleotide sequence encoding FR1, FR2, FR3, and FR4 of the heavy chain variable region framework region of the nucleic acid molecule has at least 98% sequence identity with the nucleotide sequence shown in SEQ ID NO: 20-23.

38. The nucleic acid molecule according to claim 37, wherein the nucleotide sequence encoding the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 20-23.

39. The nucleic acid molecule according to claim 38, wherein the nucleotide sequence encoding the heavy chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has 100% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 20-23.

40. The nucleic acid molecule according to claim 18, wherein the nucleotide sequence encoding the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 28-31.

41. The nucleic acid molecule according to claim 40, wherein the nucleotide sequence encoding the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has at least 95% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 28-31.

42. The nucleic acid molecule according to claim 41, wherein the nucleotide sequence encoding the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has at least 96% sequence identity with the nucleotide sequence shown in SEQ ID NO: 28-31.

43. The nucleic acid molecule according to claim 42, wherein the nucleotide sequence encoding the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has at least 97% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 28-31.

44. The nucleic acid molecule according to claim 43, wherein the nucleotide sequence encoding the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has at least 98% sequence identity with the nucleotide sequence shown in SEQ ID NO: 28-31.

45. The nucleic acid molecule according to claim 44, wherein the nucleotide sequence encoding the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 28-31.

46. ​​The nucleic acid molecule according to claim 45, wherein the nucleotide sequence encoding the light chain variable region framework regions FR1, FR2, FR3, and FR4 of the nucleic acid molecule has 100% sequence identity with the nucleotide sequence shown in SEQ ID NOs: 28-31.

47. A vector comprising the nucleic acid molecule of any one of claims 18-46.

48. The vector according to claim 47, wherein the vector comprises a plasmid, a lentivirus, an adenovirus, or an adeno-associated virus.

49. A cell comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-17, the nucleic acid molecule of any one of claims 18-46, or the vector of claim 47 or 48.

50. The cell of claim 49, comprising a prokaryotic cell or a eukaryotic cell.

51. A composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, and a detectable label. The composition according to claim 51 , wherein the monoclonal antibody or antigen-binding fragment thereof and the detectable label form a complex by coupling.

53. The composition of claim 51, wherein the detectable label comprises a direct label or an indirect label.

54. A product comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-17, the nucleic acid molecule of any one of claims 18-46, or the vector of claim 47 or 48, the cell of claim 49 or 50, or the composition of any one of claims 51-53.

55. The product according to claim 54, comprising a kit, a test paper, a nucleic acid membrane strip, a chip, a system, or a device.

56. A bispecific antibody, comprising a first binding domain and a second binding domain that specifically target IL-6; the first binding domain and the second binding domain are both derived from the monoclonal antibody or antigen-binding fragment thereof according to claims 1-17. The bispecific antibody according to claim 56 , wherein the first binding domain and the second binding domain are connected by a linker. The bispecific antibody according to claim 57 , wherein the linker comprises a flexible linker, a rigid linker and a cleavable linker.

59. An antibody drug conjugate comprising at least one drug-conjugated monoclonal antibody or antigen-binding portion thereof according to any one of claims 1 to 17.

60. The antibody drug conjugate of claim 59, wherein the at least one drug is selected from the group consisting of anti-apoptotic agents, mitotic inhibitors, anti-tumor antibiotics, immunomodulators, nucleic acids for gene therapy, alkylating agents, anti-angiogenic agents, anti-metabolites, boron-containing agents, chemoprotectants, hormone agents, anti-hormonal agents, corticosteroids, photosensitizing therapeutic agents, oligonucleotides, radionuclide agents, radiosensitizers, topoisomerase inhibitors, and tyrosine kinase inhibitors.

61. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-17, the nucleic acid molecule of any one of claims 18-46, the vector of claim 47 or 48, the cell of claim 49 or 50, the bispecific antibody of any one of claims 56-58, or the antibody-drug conjugate of claim 59 or 60.

62. The pharmaceutical composition according to claim 61, further comprising a pharmaceutically acceptable adjuvant.

63. A CAR comprising: An extracellular binding domain scFv that specifically recognizes IL-6; the extracellular binding domain comprises the light chain variable region and heavy chain variable region of an antibody targeting IL-6 as defined in the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 17.

64. The CAR of claim 63, further comprising one, more or all of a hinge domain, a transmembrane domain, a co-stimulatory intracellular domain, or a signaling domain.

65. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, the nucleic acid molecule according to any one of claims 18 to 46, the vector according to claim 47 or 48, the cell according to claim 49 or 50, or the composition according to any one of claims 51 to 53 in detecting IL-6 or a nucleic acid fragment encoding the same.

66. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, the nucleic acid molecule according to any one of claims 18 to 46, the vector according to claim 47 or 48, or the cell according to claim 49 or 50 in the preparation of a product for detecting IL-6.

67. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-17, the nucleic acid molecule according to any one of claims 18-46, the vector according to claim 47 or 48, the cell according to claim 49 or 50, the bispecific antibody according to any one of claims 56-58, the antibody-drug conjugate according to claim 59 or 60, or the CAR according to claim 63 or 64 in the preparation of a pharmaceutical composition for preventing or treating diseases related to abnormal IL-6 expression.

68. The use according to claim 67, wherein the diseases associated with abnormal IL-6 expression include rheumatoid arthritis, glomerular proliferation in glomerulonephritis, Crohn's disease, Castleman's disease, asthma, viral myocarditis, hypertension, cardiac hypertrophy, arterial wall damage, atherosclerosis, enteritis, systemic lupus erythematosus, psoriasis, type 2 diabetes, endometritis, hemophagocytic lymphocytosis, Epstein-Barr virus-associated hemophagocytic lymphocytosis, systemic or non-systemic juvenile idiopathic arthritis-associated macrophage activation syndrome, NLRC4 macrophage activation syndrome, cytokine release syndrome, sepsis, acute respiratory distress syndrome, pneumonia, pulmonary embolism, pulmonary edema, and respiratory failure.

69. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-17, the nucleic acid molecule according to any one of claims 18-46, the vector according to claim 47 or 48, the cell according to claim 49 or 50, the bispecific antibody according to any one of claims 56-58, the antibody-drug conjugate according to claim 59 or 60, or the CAR according to claim 63 or 64 in the preparation of a pharmaceutical composition for regulating IL-6 expression level or activity.

70. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-17, the nucleic acid molecule according to any one of claims 18-46, or the vector according to claim 47 or 48, the cell according to claim 49 or 50, the composition according to any one of claims 51-53, the bispecific antibody according to any one of claims 56-58, the antibody-drug conjugate according to claim 59 or 60, the pharmaceutical composition according to claim 61 or 62, or the CAR according to claim 63 or 64 in regulating immune system responses.

71. The use according to claim 70, wherein the immune system comprises all genes, immune cells and immune organs / tissues of innate immunity, adaptive immunity, cellular immunity, humoral immunity, active immunity and passive immune responses.

72. The use according to claim 71, wherein the immune cells include T cells, B cells, NK cells, NKT cells, DNA damage receptor (DNRT) cells, hematopoietic cells, pluripotent stem cells, myeloid progenitor cells, lymphoid progenitor cells, and tumor-infiltrating lymphocytes.

73. The use according to claim 72, wherein the T cells include helper T cells, cytotoxic T cells, tumor-infiltrating cytotoxic T cells, CD4 + CD8 + T cells, CD4 - CD8 - T cells, αβ T cells expressing the α and β chains of the T cell receptor and / or γδ T cells expressing the γ and δ chains of the TCR.

74. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-17, the nucleic acid molecule according to any one of claims 18-46, or the vector according to claim 47 or 48, the cell according to claim 49 or 50, the composition according to any one of claims 51-53, the bispecific antibody according to any one of claims 56-58, the antibody-drug conjugate according to claim 59 or 60, the pharmaceutical composition according to claim 61 or 62, or the CAR according to claim 63 or 64 in regulating cell phosphorylation, inhibiting inflammatory response, regulating cell apoptosis, proliferation, or migration.

75. The use according to claim 74, wherein the phosphorylation of cells comprises phosphorylation of immune cells.

76. The use according to claim 75, wherein the immune cells include T cells, B cells, NK cells, NKT cells, DNA damage receptor (DNRT) cells, hematopoietic cells, pluripotent stem cells, myeloid progenitor cells, lymphoid progenitor cells, and tumor-infiltrating lymphocytes.

77. The use according to claim 76, wherein the T cells include helper T cells, cytotoxic T cells, tumor-infiltrating cytotoxic T cells, CD4 + CD8 + T cells, CD4 - CD8 - T cells, αβ T cells expressing the α and β chains of the T cell receptor and / or γδ T cells expressing the γ and δ chains of the TCR.

78. The use according to claim 74, wherein the phosphorylation comprises phosphorylation of STAT3 protein.

79. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-17, the nucleic acid molecule according to any one of claims 18-46, or the vector according to claim 47 or 48, the cell according to claim 49 or 50, the bispecific antibody according to any one of claims 56-58, the antibody-drug conjugate according to claim 59 or 60, or the CAR according to claim 63 or 64 in the preparation of a pharmaceutical composition for treating tumors.

80. The use according to claim 79, wherein the tumor comprises a tumor associated with STAT3 phosphorylation.

81. The use according to claim 79, wherein the tumor comprises a solid tumor and a non-solid tumor.

82. A method for producing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-17, the method comprising the steps of: transforming the nucleic acid molecule according to any one of claims 18-46 or the vector according to claim 47 or 48 into cells, culturing the cells, and isolating and purifying the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-17 from the cell culture fluid.

83. A method for inhibiting the activity of IL-6, the method comprising introducing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-17, the nucleic acid molecule according to any one of claims 18-46, or the vector according to claim 47 or 48 into a cell of an organism, and inhibiting the activity of IL-6 by expressing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-17.

84. A method for detecting IL-6 in a test sample, the method comprising contacting the test sample with the monoclonal antibody or antigen-binding fragment thereof described in any one of claims 1-17, or contacting the test sample with the composition described in any one of claims 51-53; and detecting the binding of IL-6 to the monoclonal antibody or antigen-binding fragment thereof described in any one of claims 1-17, or detecting the binding of IL-6 to the composition described in any one of claims 51-53.

85. A method for treating a disease caused by abnormal expression of IL-6, the method comprising administering to a patient the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, the nucleic acid molecule according to any one of claims 18 to 46, the vector according to claim 47 or 48, the cell according to claim 49 or 50, or the composition according to any one of claims 51 to 53.

86. The method according to claim 85, wherein the diseases associated with abnormal IL-6 expression include rheumatoid arthritis, glomerular proliferation of glomerulonephritis, Crohn's disease, Castleman's disease, asthma, viral myocarditis, hypertension, cardiac hypertrophy, arterial wall damage, atherosclerosis, enteritis, systemic lupus erythematosus, psoriasis, type 2 diabetes, endometritis, hemophagocytic lymphocytosis, Epstein-Barr virus-associated hemophagocytic lymphocytosis, systemic or non-systemic juvenile idiopathic arthritis-associated macrophage activation syndrome, NLRC4 macrophage activation syndrome, cytokine release syndrome, sepsis, acute respiratory distress syndrome, pneumonia, pulmonary embolism, pulmonary edema, and respiratory failure.

87. A method for treating a disease characterized by abnormal IL-6 expression, the method comprising: 1) diagnosing the disease characterized by abnormal IL-6 expression using the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, the nucleic acid molecule according to any one of claims 18 to 46, the vector according to claim 47 or 48, the cell according to claim 49 or 50, or the composition according to any one of claims 51 to 53; and 2) administering a drug to the patient according to the diagnosis result.

88. The method according to claim 87, wherein the diseases associated with abnormal IL-6 expression include rheumatoid arthritis, glomerular proliferation of glomerulonephritis, Crohn's disease, Castleman's disease, asthma, viral myocarditis, hypertension, cardiac hypertrophy, arterial wall damage, atherosclerosis, enteritis, systemic lupus erythematosus, psoriasis, type 2 diabetes, endometritis, hemophagocytic lymphocytosis, Epstein-Barr virus-associated hemophagocytic lymphocytosis, systemic or non-systemic juvenile idiopathic arthritis-associated macrophage activation syndrome, NLRC4 macrophage activation syndrome, cytokine release syndrome, sepsis, acute respiratory distress syndrome, pneumonia, pulmonary embolism, pulmonary edema, and respiratory failure.

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