WO2023133842A1 - ANTICORPS CIBLANT IL-18Rβ ET SON UTILISATION - Google Patents

ANTICORPS CIBLANT IL-18Rβ ET SON UTILISATION Download PDF

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WO2023133842A1
WO2023133842A1 PCT/CN2022/072143 CN2022072143W WO2023133842A1 WO 2023133842 A1 WO2023133842 A1 WO 2023133842A1 CN 2022072143 W CN2022072143 W CN 2022072143W WO 2023133842 A1 WO2023133842 A1 WO 2023133842A1
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Prior art keywords
antibody
antigen
seq
binding fragment
variable region
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PCT/CN2022/072143
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English (en)
Chinese (zh)
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杜勇
陈永锋
刘淑素
张玉华
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和径医药科技（上海）有限公司
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Priority to PCT/CN2022/072143 priority Critical patent/WO2023133842A1/fr
Priority to PCT/CN2023/072444 priority patent/WO2023134767A1/fr
Priority to CN202380011140.6A priority patent/CN117177999A/zh
Publication of WO2023133842A1 publication Critical patent/WO2023133842A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to an antibody specifically targeting IL-18R ⁇ and its antigen-binding fragment, its preparation method and application.
  • Interleukin-18 also known as interferon- ⁇ -inducible factor, regulates inflammatory responses mediated by various types of immune cells. IL-18 not only activates Thl cells and NK cells to promote the release of IFN- ⁇ , but also regulates the activation of Th2, Th17 and macrophages, and is an important inflammatory factor.
  • IL-18 is closely related to a variety of autoimmune diseases, such as hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome, atopic dermatitis, idiopathic pulmonary fibrosis , scleroderma, systemic juvenile idiopathic arthritis (sJIA), systemic lupus erythematosus (SLE), and inflammatory bowel disease.
  • HHLH hemophagocytic lymphohistiocytosis
  • macrophage activation syndrome atopic dermatitis
  • idiopathic pulmonary fibrosis scleroderma
  • sJIA systemic juvenile idiopathic arthritis
  • SLE systemic lupus erythematosus
  • the IL-18 receptor is a heterodimeric transmembrane protein composed of the ligand-binding IL-18R alpha (IL-18R ⁇ ) subunit and the IL-18R beta (IL-18R ⁇ ) subunit composition.
  • IL-18 After IL-18 is hydrolyzed into an active form by protease Caspas-1, it acts on the IL-18 receptor on the cell surface and activates downstream pro-inflammatory signaling pathways. IL-18 first binds to IL-18R ⁇ with a low affinity to form a dimer, but it cannot transmit signals. Only the dimer combines with IL-18R ⁇ to form a high-affinity receptor complex to transmit pro-inflammatory signals downstream. Blocking the formation of IL-18 and its receptor ternary complex and effectively inhibiting the downstream inflammatory signaling pathway of IL-18 is a promising method for the treatment of autoimmune and inflammatory diseases.
  • IL-18R ⁇ is widely expressed and can also combine with the inflammatory inhibitor IL-37, which is more complicated in the regulation of inflammation.
  • IL-18R ⁇ is specifically expressed in immune cells and is an essential receptor for IL-18 signaling, so antibodies targeting IL-18R ⁇ can specifically and effectively inhibit IL-18 signaling. Due to the natural presence of IL-18 high-affinity decoy receptor IL-18BP in the body, as well as the existence of the membrane-bound form of IL-18, targeting IL-18 itself is affected by many aspects, and the mechanism of action is relatively complicated.
  • antibodies targeting IL-18R ⁇ have advantages in terms of specificity, efficacy and safety compared to other components of the IL-18 signaling pathway.
  • the purpose of the present invention is to provide a new treatment method for autoimmune diseases, inflammatory diseases and the like.
  • Another object of the present invention is to provide an antibody against IL-18R ⁇ and its application.
  • an antibody or antigen-binding fragment thereof against IL-18R ⁇ is provided.
  • the antibody or antigen-binding fragment thereof can specifically bind IL-18R ⁇ .
  • the antibody or antigen-binding fragment thereof includes:
  • any amino acid sequence in the above amino acid sequence also includes at least one (such as 1-4) amino acids that are optionally added, deleted, modified and/or substituted and can retain the specificity for IL-18R ⁇ Derived sequences of binding capacity.
  • the antibody or antigen-binding fragment thereof has 6 CDRs (CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3) of the A034, A035, A037, A038, or A039 antibody in Table 1.
  • the derivative sequence that has undergone addition, deletion, modification and/or substitution of at least one amino acid and can retain the specific binding ability to IL-18R ⁇ is homologous or has a sequence identity of at least 85% %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence.
  • the antibody or antigen-binding fragment thereof is based on the wild-type heavy chain variable region shown in SEQ ID NO: 18 and/or based on the wild-type light chain variable region shown in SEQ ID NO: 20
  • Beneficial mutations selected from the group consisting of F27L, F27I, F27V, S99A, S31F, A34D, A34E, or combinations thereof may be included.
  • the antibody or antigen-binding fragment thereof may include beneficial mutations selected from the group consisting of F27L, F27I, F27V, S99A, or its combination.
  • the antibody or antigen-binding fragment thereof may include beneficial mutations selected from the group consisting of S31F, A34D, A34E, or combinations thereof based on the wild-type light chain variable region shown in SEQ ID NO: 20 .
  • the antibody or antigen-binding fragment thereof is based on the wild-type heavy chain variable region shown in SEQ ID NO: 18 and based on the wild-type light chain variable region shown in SEQ ID NO: 20.
  • the CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4.
  • the antibody or antigen-binding fragment thereof further includes a framework region FR.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 1, 9 or 14, and has a heavy chain variable region as shown in SEQ ID NO: 5, 12, 15 or 17 The light chain variable region shown.
  • the antibody or antigen-binding fragment thereof may include a monomer, a bivalent antibody, and/or a multivalent antibody.
  • the bivalent antibody can also be a bispecific antibody.
  • the multivalent antibody can also be a multispecific antibody.
  • the antigen-binding fragment is selected from scFv, Fab, Fab', F(ab') 2 , Fv fragments, and disulfide-linked Fv (dsFv).
  • amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 9 and SEQ ID NO: 5, respectively, or shown in SEQ ID NO: 5, respectively.
  • the heavy chain constant region of the antibody or antigen-binding fragment thereof is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.
  • a recombinant protein is provided, and the recombinant protein has:
  • the tag sequence includes Fc tag, HA tag, GGGS sequence, FLAG tag, Myc tag, 6His tag, or a combination thereof.
  • the recombinant protein specifically binds IL-18R ⁇ .
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • nucleotide molecule in the third aspect of the present invention, there is provided a nucleotide molecule, the polynucleotide encoding a protein selected from the group consisting of the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the second antibody of the present invention Aspects of the recombinant protein.
  • the nucleic acid of the present invention can be RNA, DNA or cDNA.
  • an expression vector containing the nucleotide molecule described in the third aspect of the present invention is provided.
  • the expression vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or combinations thereof.
  • the expression vector comprises a viral vector, such as lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • the expression vector is selected from the group consisting of pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV and the like.
  • the expression vector further includes a promoter, a transcriptional enhancer element WPRE, a long terminal repeat sequence LTR, etc. selected from the group.
  • a host cell contains the expression vector described in the fourth aspect of the present invention, or the nucleotide molecule described in the third aspect of the present invention is integrated in its genome .
  • the host cells include prokaryotic cells or eukaryotic cells.
  • the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.
  • a chimeric antigen receptor CAR is provided, the antigen-binding region scFv of the CAR is a binding region that specifically binds to IL-18R ⁇ , and the heavy chain variable region of the scFv include:
  • Heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of CDRH1, CDRH2, and CDRH3 are shown in SEQ ID NO: 2, 3, and 4, respectively, or shown in SEQ ID NO: 10, 3, and 11, respectively, or as shown in SEQ ID NO:2, 3 and 11 respectively; and/or
  • the light chain variable region of the scFv comprises:
  • CDRL1, CDRL2, CDRL3 Light chain complementarity determining regions CDRL1, CDRL2, CDRL3, the amino acid sequences of said CDRL1, CDRL2, CDRL3 are respectively shown in SEQ ID NO: 6, 7 and 8, or respectively shown in SEQ ID NO: 13, 7 and 8, Or as shown in SEQ ID NO: 16, 7 and 8 respectively, or as shown in SEQ ID NO: 22, 7 and 8 respectively.
  • the CAR further includes a signal peptide.
  • the CAR further includes other foreign proteins.
  • the CAR has the structure shown in formula Ia:
  • L is nothing or a signal peptide sequence
  • scFv is a domain that specifically binds IL-18R ⁇ ;
  • H is none or hinge region
  • TM is the transmembrane domain
  • C is costimulatory signal domain
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ (including wild type, or mutants/modifiers thereof);
  • the "-" connects a peptide or a peptide bond.
  • the Ls are respectively selected from signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants/modifications thereof, or combinations thereof.
  • the scFv targets IL-18R ⁇ .
  • the scFv is an IL-18R ⁇ antibody or an antigen-binding fragment thereof.
  • the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.
  • the TM is selected from the transmembrane regions of the following histones: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutants/modifications thereof, or combinations thereof.
  • the C is selected from the co-stimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants/modifications thereof, or combinations thereof.
  • histones OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants/modifications thereof, or combinations thereof.
  • an engineered immune cell expressing the exogenous CAR as described in the sixth aspect of the present invention.
  • the engineered immune cells are selected from the following group:
  • CAR-T cells chimeric antigen receptor ⁇ T cells
  • CAR-T cells chimeric antigen receptor ⁇ T cells
  • CAR-NKT cells chimeric antigen receptor NKT cells
  • the engineered immune cells include autologous or allogeneic ⁇ T cells, ⁇ T cells, NKT cells, NK cells, or a combination thereof.
  • the engineered immune cells are CAR-T cells.
  • step (c) Optionally, purifying and/or modifying the IL-18R ⁇ antibody or antigen-binding fragment thereof obtained in step (b).
  • an immunoconjugate comprising:
  • a coupling moiety selected from the group consisting of detectable labels, drugs, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof.
  • the part (a) is coupled to the coupling part through a chemical bond or a linker.
  • the radionuclides include:
  • isotopes for diagnosis are selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or
  • the isotope for treatment is selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb- 177, or combinations thereof.
  • the coupling moiety is a drug or a toxin.
  • the drug is a drug for targeted treatment of IL-18-related diseases.
  • the IL-18-related diseases include diseases with high expression of IL-18, diseases related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway.
  • the disease with high expression of IL-18 is selected from: tumor, leukemia, diabetic nephropathy, Alzheimer's disease, Parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, silver Psoriasis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, Graves' disease , Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, pus Toxicity, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome,
  • the tumor is selected from: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethra cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or combinations thereof.
  • the drug is a cytotoxic drug.
  • the cytotoxic drugs are selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy A sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
  • cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, Auristatins, camptothecins, (Camptothecins), Duocarmycins/Duocarmycins, Etoposides, Maytansines and Maytansinoids (such as DM1 and DM4), Taxanes ( Taxanes), benzodiazepines, or benzodiazepine containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines Indolinobenzodiazepines and Oxazolidinobenzodiazepines), Vinca alkaloids, or combinations thereof.
  • Auristatins camptothecins, (Camptothecins), Duocarmycins/Duocarmycins, Etoposides, Maytansines and Maytansinoids (such as DM1 and DM4), Taxanes ( Taxa
  • the toxin is selected from the group consisting of auristatins (for example, auristatin E, auristatin F, MMAE, and MMAF), aureomycin, maytansinol, ricin, grate Anesthetic toxin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, Tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraxin diketone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, acacia Toxin, abrin A chain, lotus root toxin A chain, ⁇ -sarcinia, gelonin, Mitogellin, Retstrictocin,
  • the coupling moiety is a detectable label.
  • the coupling moiety is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer X-ray tomography) contrast agents, or capable of producing Detectable products of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles , prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), or nanoparticles in any form.
  • DTD DT-diaphorase
  • BPHL biphenylhydrolase-like protein
  • the immunoconjugate comprises: a multivalent (eg, bivalent) antibody or antigen-binding fragment thereof according to the first aspect of the present invention.
  • the multivalent means that the amino acid sequence of the immunoconjugate contains multiple repetitions of the same or different antibodies or antigen-binding fragments thereof according to the first aspect of the present invention.
  • a pharmaceutical composition which contains:
  • a pharmaceutically acceptable carrier, diluent or excipient (ii) A pharmaceutically acceptable carrier, diluent or excipient.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of injections and freeze-dried preparations.
  • the pharmaceutical composition includes 0.01-99.99% of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention, or The engineered immune cell according to the seventh aspect of the present invention, or the immunoconjugate according to the ninth aspect of the present invention, or a combination thereof and 0.01 to 99.99% of a pharmaceutical carrier, the percentage is the percentage of the drug The mass percent of the composition.
  • the concentration of the engineered immune cells in the active ingredient is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/mL, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells /mL.
  • an active ingredient selected from the group consisting of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the antibody according to the second aspect of the present invention
  • the recombinant protein, or the engineered immune cell as described in the seventh aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention, or a combination thereof, the active ingredient is used to prepare:
  • the reagent shown is a diagnostic reagent, preferably, the diagnostic reagent is a detection chip or a detection plate.
  • the diagnostic reagent is used for: detecting IL-18R ⁇ protein or fragments thereof in a sample.
  • the IL-18-related diseases include diseases with high expression of IL-18, diseases related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway.
  • the disease with high expression of IL-18 is selected from: tumor, leukemia, diabetic nephropathy, Alzheimer's disease, Parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, Psoriasis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, Sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome, systemic
  • the tumor is selected from: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethra cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or combinations thereof.
  • a method for in vitro detection of IL-18R ⁇ protein or fragments thereof in a sample comprising the steps of:
  • the detection includes diagnostic or non-diagnostic.
  • a kit comprising:
  • the first container which contains the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention, or the seventh aspect of the present invention
  • the test kit contains a detection plate, the detection plate includes: a substrate (support plate) and a test strip, and the test strip contains the antibody or its antigen-binding fragment as described in the first aspect of the present invention, or such as The recombinant protein as described in the second aspect of the present invention, or the engineered immune cell as described in the seventh aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention, or as described in the tenth aspect of the present invention A pharmaceutical composition, or a combination thereof.
  • the kit also contains an instruction, and according to the instruction, the kit is used to non-invasively detect the expression of IL-18R ⁇ in the subject.
  • the kit is used for the detection of IL-18-related diseases.
  • the IL-18-related diseases include diseases with high expression of IL-18, including diseases related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway.
  • the disease with high expression of IL-18 is selected from: tumor, leukemia, diabetic nephropathy, Alzheimer's disease, Parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, silver Psoriasis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, Graves' disease , Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, pus Toxicity, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome,
  • the tumor is selected from: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethra cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or combinations thereof.
  • a method for preventing and/or treating IL-18-related diseases comprising: administering the antibody or its antigen as described in the first aspect of the present invention to a subject in need Binding fragments, or recombinant proteins as described in the second aspect of the present invention, or engineered immune cells as described in the seventh aspect of the present invention, or immunoconjugates as described in the ninth aspect of the present invention, or as described in the present invention
  • the pharmaceutical composition of the tenth aspect, or a combination thereof comprising: administering the antibody or its antigen as described in the first aspect of the present invention to a subject in need Binding fragments, or recombinant proteins as described in the second aspect of the present invention, or engineered immune cells as described in the seventh aspect of the present invention, or immunoconjugates as described in the ninth aspect of the present invention, or as described in the present invention.
  • the subject includes mammals, such as humans.
  • the IL-18-related diseases include diseases with high expression of IL-18, diseases related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway.
  • the disease with high expression of IL-18 is selected from: tumor, leukemia, diabetic nephropathy, Alzheimer's disease, Parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, silver Psoriasis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, Graves' disease , Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, pus Toxicity, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome,
  • the tumor is selected from: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethra cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or combinations thereof.
  • the engineered immune cells or the CAR immune cells included in the pharmaceutical composition are cells derived from the subject (autologous cells).
  • the engineered immune cells or the CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).
  • the above method can be used in combination with other treatment methods.
  • the other treatment methods include chemotherapy, radiotherapy, targeted therapy and other methods.
  • a diagnostic method for IL-18-related diseases comprising the steps of:
  • the sample is a blood sample or a throat swab sample, or a sample from other tissues and organs.
  • the IL-18-related diseases include diseases with high expression of IL-18, diseases related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway.
  • the disease with high expression of IL-18 is selected from: tumor, leukemia, diabetic nephropathy, Alzheimer's disease, Parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, silver Psoriasis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, Graves' disease , Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, pus Toxicity, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome,
  • the tumor is selected from: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethra cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or combinations thereof.
  • Figure 1 shows the sequence information of IL-18R ⁇ WT antibody.
  • Figure 2 shows the concentration-response curves of anti-IL-18R ⁇ antibody for the inhibition of IFN- ⁇ in KG-1 cells.
  • Figure 3 shows the concentration-response curves of anti-IL-18R ⁇ antibody for the inhibition of IFN- ⁇ in human PBMC cells.
  • the inventors After extensive and in-depth research and extensive screening, the inventors first developed a high-affinity IL-18R ⁇ -targeting antibody and its antigen-binding fragment. Specifically, the present invention uses the WT antibody of IL-18R ⁇ as the starting material for improving affinity to construct a precise mutation library, and introduces saturation mutations into all 73 residues of the six CDR regions of the WT antibody. After further screening, the selected mutants were sorted by dissociation rate constants determined by surface plasmon resonance, and a combinatorial library was constructed with a random combination of beneficial mutations, thereby obtaining the IL-18R ⁇ antibody of the present invention with improved binding affinity and its Antigen-binding fragments.
  • the IL-18R ⁇ -targeting antibody and its antigen-binding fragment developed in the present invention can be used as a new therapeutic means for targeted treatment of diseases related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway.
  • the terms “antibody of the invention”, “antibody of the invention”, “antibody against IL-18R ⁇ of the invention”, “antibody against IL-18R ⁇ ”, “antibody against IL-18R ⁇ ”, “IL-18R ⁇ ” “18R ⁇ antibody” has the same meaning and can be used interchangeably, both refer to antibodies that specifically recognize and bind to IL-18R ⁇ protein (including human IL-18R ⁇ protein).
  • the numbers of the antibodies of the present invention and the corresponding sequence numbers are shown in Table 1 below.
  • Each value in the table represents the sequence number, that is, "1" represents “SEQ ID NO: 1", and the sequence numbers of VH, CDRH1, CDRH2, CDRH3, VL, CDRL1, CDRL2, and CDRL3 shown in the table are the sequence numbers of their amino acid sequences. serial number.
  • antibody herein is intended to include full-length antibodies and any antigen-binding fragment (ie, antigen-binding portion) or single chains thereof.
  • Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs), which are separated by more conserved framework region (FR) regions.
  • CDRs complementarity determining regions
  • FR conserved framework region
  • Each VH and VL consists of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxyl terminal.
  • the variable regions of the heavy and light chains contain the binding domains that interact with the antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, IL-18R ⁇ protein). It has been demonstrated that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
  • binding fragments contained in the "antigen-binding portion" of an antibody include (i) Fab fragments, a monovalent fragment composed of VL, VH, CL, and CH1; (ii) F(ab') 2 fragments, comprising the hinge region II; Bivalent fragment of two Fab fragments connected by sulfur bridge; (iii) Fd fragment composed of VH and CH1; (iv) Fv fragment composed of antibody single arm VL and VH; (v) dAb fragment composed of VH; (vi) isolated complementarity determining regions (CDRs); and (vii) a Nanobody, a heavy chain variable region comprising a single variable domain and two constant domains.
  • the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked by recombinant methods via a synthetic linker that makes the two a single protein chain, where the VL and VH regions pair to form a monovalent molecule) (called is a single-chain Fc (scFv)).
  • scFv single-chain Fc
  • These single chain antibodies are also intended to be included within the meaning of the term.
  • These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as whole antibodies.
  • variable means that certain portions of the variable regions among antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable domains are called the framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the variable domains of native heavy and light chains each contain four FR regions in a roughly ⁇ -sheet configuration connected by three CDRs forming connecting loops and, in some cases, partial b-sheet structures.
  • the CDRs in each chain are in close proximity through the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines (Cytokine), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments thereof of the present invention to form of conjugates.
  • the present invention also includes cell surface markers or antigens that bind to the antibodies or fragments thereof against the novel coronavirus.
  • VL light chain variable region
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDRH1, CDRH2, and CDRH3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
  • the light chain variable region of the antibody includes three complementarity determining regions CDRL1, CDRL2, and CDRL3.
  • the light chain of the antibody includes the above-mentioned light chain variable region and light chain constant region.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to a polypeptide that specifically binds IL-18R ⁇ protein, such as a protein with a heavy chain variable region or peptides. They may or may not contain starting methionine.
  • the invention also provides other proteins or fusion expression products having the antibodies of the invention.
  • the present invention includes any protein or protein conjugates and fusion expression products (i.e., immunoconjugates and fusion expression products) having a heavy chain containing a variable region, as long as the variable region is compatible with the heavy chain of the antibody of the present invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR), which is separated into four framework regions (FR), four FR amino acids
  • CDR variable region
  • FR framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a ring structure, and the ⁇ sheets formed by the FRs in between are close to each other in the spatial structure.
  • the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody.
  • Which amino acids constitute FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen. Therefore, the present invention includes those molecules having antibody heavy chain variable regions with CDRs, as long as the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein sex.
  • the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention.
  • the polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • the antibody of the present invention refers to a polypeptide that has IL-18R ⁇ protein binding activity and includes the above CDR region.
  • the term also includes variant forms of polypeptides comprising the above CDR regions that have the same function as the antibodies of the present invention. These variations include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • substitutions with amino acids with similar or similar properties generally do not change the function of the protein.
  • adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA hybrids that can hybridize with the DNA encoding the antibody of the present invention under high or low stringency conditions
  • the encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof.
  • the invention also includes fragments of the antibodies of the invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • “conservative variants of the antibody of the present invention” refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acid sequences compared with the amino acid sequence of the antibody of the present invention.
  • An amino acid is replaced by an amino acid with similar or similar properties to form a polypeptide.
  • These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 2.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • a polynucleotide of the invention may be in the form of DNA or RNA.
  • Forms of DNA include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either the coding strand or the non-coding strand.
  • a polynucleotide encoding a mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein.
  • biomolecules nucleic acid, protein, etc.
  • the biomolecules involved in the present invention include biomolecules in an isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another way is to use MgCl2. Transformation can also be performed by electroporation, if desired.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional media according to the host cells used.
  • the culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
  • the recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or substances capable of producing a detectable product. enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2. Biological toxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), etc.
  • DTD DT-diaphorase
  • BPHL biphenylhydrolase-like protein
  • Interleukin-18 (IL18, also known as interferon-gamma inducible factor) is a protein that in humans is encoded by the IL-18 gene.
  • the protein encoded by this gene is a pro-inflammatory cytokine.
  • the IL-18 receptor consists of an inducible component, IL-18R ⁇ , which binds mature IL-18 with low affinity, and a constitutively expressed co-receptor, IL-18R ⁇ . Binding of IL-18 to the ligand receptor IL-18R ⁇ induces the recruitment of IL-18R ⁇ to form a high-affinity complex that signals through the toll/interleukin-1 receptor (TIR) domain. This signaling domain activates pro-inflammatory programs and the MyD88 adapter protein of the NF- ⁇ B pathway.
  • TIR toll/interleukin-1 receptor
  • This signaling domain activates pro-inflammatory programs and the MyD88 adapter protein of the NF- ⁇ B pathway.
  • IL-18R ⁇ is specifically expressed in immune cells and is an essential receptor for IL-18 signaling, so antibodies targeting IL-18R ⁇ can specifically and effectively inhibit IL-18 signaling. Effectively inhibiting the pro-inflammatory signaling pathway downstream of IL-18 is currently a promising approach for the treatment of autoimmune and inflammatory diseases.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody (or its conjugate) of the present invention and a pharmaceutically acceptable acceptable carrier or excipient.
  • a pharmaceutically acceptable acceptable carrier or excipient include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 10 micrograms/kg to about 50 mg/kg body weight per day.
  • the polypeptides of the invention can also be used
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the antibody against IL-18R ⁇ includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).
  • the antibody against IL-18R ⁇ comprises one or more heavy chains having heavy chain variable regions as shown in SEQ ID NO: 1, 9 or 14, and having a heavy chain as shown in SEQ ID
  • the light chain of the light chain variable region represented by NO: 5, 12, 15 or 17.
  • the antibody has a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels or fluorescent labels.
  • Colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the antibody against IL-18R ⁇ protein is labeled with colloidal gold to obtain a colloidal gold-labeled antibody.
  • the antibody against IL-18R ⁇ of the present invention can effectively bind IL-18R ⁇ protein.
  • the present invention also relates to methods for detecting IL-18R ⁇ protein or fragments thereof.
  • the steps of the method are roughly as follows: obtain a cell and/or tissue sample; dissolve the sample in a medium; detect the level of IL-18R ⁇ protein in the dissolved sample.
  • the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or its fragment) or detection plate of the present invention.
  • the kit further includes a container, instructions for use, buffer and the like.
  • the present invention also provides a detection kit for detecting the level of IL-18R ⁇ protein, which includes an antibody for recognizing IL-18R ⁇ protein, a lysis medium for dissolving samples, general reagents and buffers required for detection, such as various buffer, detection label, detection substrate, etc.
  • the test kit may be an in vitro diagnostic device.
  • the antibody of the present invention has a wide range of biological and clinical application values, and its application involves the treatment of diseases related to the high expression of IL-18, taking into account the diagnosis of high expression of IL-18, basic medical research, biological research, etc. multiple fields.
  • a preferred application is for clinical prevention and treatment against IL-18R ⁇ protein.
  • the present invention also provides a method for stimulating an immune response mediated by T cells targeting mammalian tumor cell populations or tissues, comprising the following steps: administering the CAR-T cells of the present invention to mammals.
  • the present invention includes a type of cell therapy, in which a patient's own T cells (or a heterologous donor) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient.
  • a patient's own T cells or a heterologous donor
  • CAR-T can treat all cancers that express the antigen.
  • CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells of the present invention can undergo stable in vivo expansion and last for several months to several years.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-T cells can induce a specific immune response to tumor cells that overexpress the antigen recognized by the CAR antigen-binding domain.
  • the CAR-T cells of the present invention elicit a specific immune response against tumor cells with high expression of IL-18R ⁇ .
  • Treatable cancers include tumors that are not or substantially not vascularized, as well as vascularized tumors.
  • Cancer types treated with the CAR of the present invention include, but are not limited to: gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like.
  • the present invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell of the present invention.
  • the CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition with a diluent and/or in combination with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • the pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or may be determined by clinical trials.
  • compositions of the invention to be administered can be determined by a physician, taking into account the patient (subject ) with individual differences in age, weight, tumor size, degree of infection or metastasis, and disease.
  • Pharmaceutical compositions comprising T cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably at a dose of 10 5 to 10 7 cells/kg body weight (including all integer values within the range). T cell compositions can also be administered multiple times at these doses.
  • Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical art by monitoring the patient for signs of disease, and adjusting treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection or intraperitoneally.
  • the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by intravenous injection.
  • Compositions of T cells can be injected directly into tumors, lymph nodes or sites of infection.
  • cells activated and expanded using the methods described herein, or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant treatment modalities (e.g., previously , simultaneously or subsequently) to the patient in a form of treatment including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for PML patients.
  • the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents.
  • the cell composition of the invention is administered in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, the use of chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • a subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • Dosages administered to a patient for the above treatments will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented according to practice accepted in the art. Usually, 1 ⁇ 10 5 to 1 ⁇ 10 10 modified T cells of the present invention can be administered to the patient for each treatment or each course of treatment, for example, through intravenous infusion.
  • the IL-18R ⁇ antibody or antigen-binding fragment thereof of the present invention can specifically bind IL-18R ⁇ with high affinity, which is more than ten times higher than that of WT, and the highest is 22 times higher.
  • the IL-18R ⁇ antibody or antigen-binding fragment thereof of the present invention has good IL-18/IL-18R blocking activity and can effectively inhibit IL-18 downstream inflammatory signaling pathways.
  • the IL-18R ⁇ antibody or antigen-binding fragment thereof of the present invention has advantages in terms of specificity, effectiveness and safety.
  • the IL-18R ⁇ antibody or antigen-binding fragment thereof of the present invention can efficiently block the release of IFN- ⁇ in KG-1 cells stimulated by IL-18, and the activity efficiency reaches 20 times that of WT.
  • the IL-18R ⁇ antibody or antigen-binding fragment thereof of the present invention can efficiently block IL-18-stimulated release of IFN- ⁇ from human peripheral blood mononuclear cells, and the activity efficiency is significantly higher than that of WT.
  • the IL-18R ⁇ antibody or antigen-binding fragment thereof of the present invention contributes to the prevention/diagnosis/treatment of diseases related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway, providing New methods and technical means.
  • the inventors used the WT antibody of IL-18R ⁇ (ie, the wild-type antibody, C056-WT in Table 1) as the starting material for improving the affinity.
  • a precision mutation library was constructed to introduce saturation mutations into all 73 residues of the six CDR regions of the WT antibody. After further screening, the selected mutants were ranked by their dissociation rate constants measured by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • a combinatorial library is then constructed with random combinations of beneficial mutations. Lead Fab clones were also identified by SPR sequencing. All SPR screens were performed on a Biacore 8K.
  • the running buffer was HBS-EP (10mM HEPES, 500mM NaCl, 3mM EDTA, 0.05% Tween 20, pH 6.0).
  • a secreted Fab was adsorbed to the SASA biosensor. After equilibration, antigen was injected for 120 seconds (association period), followed by running buffer for 360 seconds (dissociation period). Regenerate the sensor surface before injecting other selected clones.
  • the off-rate of Fab clones was obtained by locally fitting the experimental data to a 1:1 interaction model using Biacore 8K evaluation software. Finally, full-length IgG from the lead clone was expressed in HEK293 cells and evaluated in binding and functional assays.
  • the affinity of anti-IL-18R ⁇ antibody to human IL-18R ⁇ protein was determined using surface plasmon resonance biosensor Biacore 8K (GE Healthcare). Measurements were performed at 25°C and the running buffer was HBS-EP+. Antibody was injected as capture onto the Series S Sensor Chip Protein A. Diluted IL-18R ⁇ (400, 200, 100, 50, 25, 12.5, 6.25 nM) was then injected on the surface of flow cells 1 and 2 as the binding phase. The binding time was 120 seconds. Buffer flow was maintained for 420 seconds for separation. The surface was regenerated by injecting 10 mM glycine-HCl pH 1.5 for 30 s. Data for dissociation (kd) rate constants and association (ka) rate constants were obtained using Biacore evaluation software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka.
  • KD equilibrium dissociation constant
  • Table 6 summarizes the binding kinetics data for anti-IL-18R ⁇ antibodies.
  • the binding affinities of A035, A037 and A039 were in the range of 0.72-0.98nm, which was 16-22 times higher than that of the parental antibody WT (15.8nm).
  • 3 ⁇ 10 5 KG-1 cells (ATCC, #CCL-246) were seeded into each well of a 96-well plate. Serial dilutions of the antibody prepared in Example 1 were added to the wells prior to stimulation with 10 ng/mL IL-18 for 1 h. After 24 hours of incubation, cells were pelleted by centrifugation and IFN- ⁇ was measured from the supernatant using an ELISA kit (R&D Systems, #VAL104C) according to the manufacturer's instructions. The above anti-IL-18R ⁇ antibodies were tested in at least 3 replicates and the IC50 for each antibody is summarized in Table 7.
  • PBMC peripheral blood mononuclear cells
  • TPCS Human peripheral blood mononuclear cells

Abstract

L'invention concerne un anticorps ciblant IL-18Rβ ou un fragment de liaison à l'antigène de celui-ci, son procédé de préparation et son utilisation. En particulier, l'invention concerne en outre une molécule d'acide nucléique codant pour l'anticorps ou des fragments de liaison à l'antigène de celui-ci, un vecteur d'expression correspondant, une cellule hôte capable d'exprimer l'anticorps ou un fragment de liaison à l'antigène de celui-ci, un procédé de production et une utilisation de l'anticorps ou du fragment de liaison à l'antigène de celui-ci. L'anticorps ciblant IL-18Rβ ou un fragment de liaison à l'antigène de celui-ci peut se lier de manière spécifique à l'IL-18Rβ humaine, a une affinité bien supérieure à celle d'un anticorps de type sauvage, et a simultanément une bonne activité de blocage d'IL-18/IL-18R. L'anticorps ou des fragments de liaison à l'antigène de celui-ci fournit un nouveau moyen technique pour la prévention/le diagnostic/le traitement de maladies associées à une expression anormale d'un récepteur d'IL-18 ou d'une activité élevée d'une voie de signalisation d'IL-18.
PCT/CN2022/072143 2022-01-14 2022-01-14 ANTICORPS CIBLANT IL-18Rβ ET SON UTILISATION WO2023133842A1 (fr)

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PCT/CN2022/072143 WO2023133842A1 (fr) 2022-01-14 2022-01-14 ANTICORPS CIBLANT IL-18Rβ ET SON UTILISATION
PCT/CN2023/072444 WO2023134767A1 (fr) 2022-01-14 2023-01-16 ANTICORPS CIBLANT IL-18Rβ ET SES UTILISATIONS
CN202380011140.6A CN117177999A (zh) 2022-01-14 2023-01-16 一种靶向IL-18Rβ的抗体及其应用

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