CN116554322A - A kind of antibody targeting IL-18Rβ and its application - Google Patents

Abstract

The invention provides an antibody or an antigen binding fragment thereof aiming at IL-18 Rbeta, a preparation method and application thereof. In particular, the invention also provides nucleic acid molecules encoding the antibodies or antigen binding fragments thereof, corresponding expression vectors and host cells capable of expressing the antibodies or antigen binding fragments thereof, and methods for producing and uses of the antibodies or antigen binding fragments thereof. The antibody or antigen binding fragment thereof aiming at IL-18R beta can specifically bind to human IL-18R beta, has much higher affinity than a wild type antibody, and has good IL-18/IL-18R blocking activity. The antibody or the antigen binding fragment thereof provides a new technical means for preventing/diagnosing/treating diseases related to the abnormal expression of IL-18 receptor or related to the high activity of IL-18 signaling pathway.

Description

An antibody targeting IL-18Rβ and its application

Technical Field

The present invention belongs to the field of biomedicine, and in particular relates to an antibody specifically targeting IL-18Rβ and an antigen-binding fragment thereof, and a preparation method and application thereof.

Background Art

Interleukin-18 (IL-18), also known as interferon-γ inducing factor, regulates inflammatory responses mediated by various types of immune cells. IL-18 not only activates Th1 cells and NK cells to promote the release of IFN-γ, but also regulates the activation of Th2, Th17 and macrophages, and is an important inflammatory factor.

Many studies have found that 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.

The IL-18 receptor is a heterodimeric transmembrane protein composed of the IL-18R alpha (IL-18Rα) subunit, which binds the ligand, and the IL-18R beta (IL-18Rβ) subunit, which is essential for functional signaling.

After IL-18 is hydrolyzed into an active form by the protease Caspas-1, it acts on the IL-18 receptor on the cell surface and activates the downstream pro-inflammatory signaling pathway. IL-18 first binds to IL-18Rα with low affinity to form a dimer, but it cannot transmit signals. Only when the dimer binds to IL-18Rβ to form a high-affinity receptor complex can it transmit pro-inflammatory signals downstream. Blocking the formation of the ternary complex of IL-18 and its receptor and effectively inhibiting the downstream inflammatory signaling pathway of IL-18 is currently a very promising method for treating autoimmune and inflammatory diseases.

IL-18Rα is widely expressed and can also bind to the inflammatory inhibitor IL-37, and its role in regulating inflammation is relatively complex. IL-18Rβ is specifically expressed in immune cells and is a necessary receptor for IL-18 to transmit signals. Therefore, antibodies targeting IL-18Rβ can specifically and effectively inhibit IL-18 signals. Due to the natural presence of IL-18 high-affinity decoy receptor IL-18BP in the body, as well as the presence of membrane-bound IL-18, targeting IL-18 itself is affected by many factors and its mechanism of action is relatively complex. In general, antibodies targeting IL-18Rβ have advantages in specificity, effectiveness, and safety over other components of the IL-18 signaling pathway.

Therefore, there is an urgent need in the art to develop an antibody that specifically targets IL-18Rβ for the treatment of autoimmune diseases and inflammatory diseases.

Summary of the invention

The purpose of the present invention is to provide a new treatment method for autoimmune diseases and inflammatory diseases.

Another object of the present invention is to provide an antibody against IL-18Rβ and its application.

In a first aspect of the present invention, an antibody or an antigen-binding fragment thereof against IL-18Rβ is provided.

In another preferred embodiment, the antibody or antigen-binding fragment thereof can specifically bind to IL-18Rβ.

In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises:

(a) heavy chain complementary determining regions CDRH1, CDRH2, CDRH3, wherein the amino acid sequences of CDRH1, CDRH2, CDRH3 are shown in SEQ ID NOs: 2, 3, and 4, respectively, or in SEQ ID NOs: 10, 3, and 11, respectively, or in SEQ ID NOs: 2, 3, and 11, respectively; and

(b) light chain complementary determining regions CDRL1, CDRL2, CDRL3, the amino acid sequences of CDRL1, CDRL2, CDRL3 are shown in SEQ ID NOs: 6, 7 and 8, respectively, or as shown in SEQ ID NOs: 13, 7 and 8, respectively, or as shown in SEQ ID NOs: 16, 7 and 8, respectively, or as shown in SEQ ID NOs: 22, 7 and 8, respectively.

In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and/or substitution of at least one (such as 1-4) amino acids and can retain the ability to specifically bind to IL-18Rβ.

In another preferred example, the antibody or antigen-binding fragment thereof has 6 CDRs (CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3) of the A034, A035, A037, A038, or A039 antibodies in Table 1.

In another preferred embodiment, the derivative sequence that has been added, deleted, modified and/or substituted at least one amino acid and can retain the ability to specifically bind to IL-18Rβ is an amino acid sequence with a homology or 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%.

In another preferred example, the antibody or its antigen-binding fragment may include beneficial mutations selected from the following groups 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: F27L, F27I, F27V, S99A, S31F, A34D, A34E, or a combination thereof.

In another preferred example, the antibody or antigen-binding fragment thereof may include a beneficial mutation selected from the following group based on the wild-type heavy chain variable region shown in SEQ ID NO: 18: F27L, F27I, F27V, S99A, or a combination thereof.

In another preferred example, the antibody or antigen-binding fragment thereof may include a beneficial mutation selected from the following group based on the wild-type light chain variable region shown in SEQ ID NO: 20: S31F, A34D, A34E, or a combination thereof.

In another preferred embodiment, the antibody or antigen-binding fragment thereof has a beneficial mutation selected from the following group based on the wild-type heavy chain variable region shown in SEQ ID NO: 18 and the wild-type light chain variable region shown in SEQ ID NO: 20:

S31F, A34E, F27L;

S31F, A34E, F27I, S99A;

A34D, F27I, S99A;

S31F, A34D, F27L;

A34E, F27I;

A34D, F27I;

A34E, F27L;

S31F, A34D, F27V, S99A;

A34E, F27L, S99A; or

S31F, A34E, F27L.

In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4.

In another preferred embodiment, the antibody or antigen-binding fragment thereof further includes a framework region FR.

In another preferred example, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 1, 9 or 14, and a light chain variable region as shown in SEQ ID NO: 5, 12, 15 or 17.

In another preferred embodiment, the antibody or antigen-binding fragment thereof may include a monomer, a bivalent antibody, and/or a multivalent antibody.

In another preferred embodiment, the bivalent antibody may also be a bispecific antibody.

In another preferred embodiment, the multivalent antibody may also be a multispecific antibody.

In another preferred embodiment, the antigen-binding fragment is selected from scFv, Fab, Fab', F(ab') ₂ , Fv fragment, and disulfide-bonded Fv (dsFv).

In another preferred example, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof are shown as SEQ ID NO:9 and SEQ ID NO:5, respectively, or as SEQ ID NO:1 and SEQ ID NO:5, respectively, or as SEQ ID NO:9 and SEQ ID NO:12, respectively, or as SEQ ID NO:14 and SEQ ID NO:17, respectively, or as SEQ ID NO:14 and SEQ ID NO:15, respectively.

In another preferred embodiment, 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.

In a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

(i) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

In another preferred embodiment, the recombinant protein specifically binds to IL-18Rβ.

In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

In the third aspect of the present invention, a nucleotide molecule is provided, wherein the polynucleotide encodes 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 recombinant protein as described in the second aspect of the present invention.

In another preferred embodiment, the nucleic acid of the present invention may be RNA, DNA or cDNA.

In the fourth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the nucleotide molecule described in the third aspect of the present invention.

In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or combinations thereof.

In another preferred embodiment, the expression vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

In another preferred embodiment, the expression vector further includes a member selected from the following group: a promoter, a transcription enhancing element WPRE, a long terminal repeat sequence LTR, etc.

In the fifth aspect of the present invention, a host cell is provided, wherein the 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 into its genome.

In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, yeast cells, and mammalian cells.

In a sixth aspect of the present invention, a chimeric antigen receptor CAR is provided, wherein the antigen binding region scFv segment of the CAR is a binding region that specifically binds to IL-18Rβ, and the heavy chain variable region of the scFv comprises:

Heavy chain complementary determining regions CDRH1, CDRH2, CDRH3, wherein the amino acid sequences of CDRH1, CDRH2, CDRH3 are shown in SEQ ID NOs: 2, 3 and 4, respectively, or are shown in SEQ ID NOs: 10, 3 and 11, respectively, or are shown in SEQ ID NOs: 2, 3 and 11, respectively; and/or

The light chain variable region of the scFv comprises:

The light chain complementary determining regions CDRL1, CDRL2, CDRL3, the amino acid sequences of CDRL1, CDRL2, CDRL3 are shown in SEQ ID NOs: 6, 7 and 8, respectively, or as shown in SEQ ID NOs: 13, 7 and 8, respectively, or as shown in SEQ ID NOs: 16, 7 and 8, respectively, or as shown in SEQ ID NOs: 22, 7 and 8, respectively.

In another preferred embodiment, the CAR further includes a signal peptide.

In another preferred embodiment, the CAR also includes other exogenous proteins.

In another preferred embodiment, the CAR has a structure shown in Formula Ia:

L-scFv-H-TM-C-CD3ζ (Ia)

In the formula,

L is none or a signal peptide sequence;

scFv is a domain that specifically binds to IL-18Rβ;

H is none or hinge region;

TM is the transmembrane domain;

C is the co-stimulatory signaling domain;

CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ (including wild type, or mutant/modified form thereof);

The "-" connects peptides or peptide bonds.

In another preferred embodiment, the L is selected from the signal peptides of the following proteins: CD8, GM-CSF, CD4, CD28, CD137, or mutants/modified forms thereof, or a combination thereof.

In another preferred embodiment, the scFv targets IL-18Rβ.

In another preferred embodiment, the scFv is an IL-18Rβ antibody or an antigen-binding fragment thereof.

In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

In another preferred embodiment, the TM is selected from the transmembrane region of the following group of proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or their mutants/modified forms, or their combinations.

In another preferred embodiment, the C is selected from the co-stimulatory domains of the following proteins: 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/modified forms thereof, or combinations thereof.

In the seventh aspect of the present invention, an engineered immune cell is provided, wherein the engineered immune cell expresses an exogenous CAR as described in the sixth aspect of the present invention.

In another preferred embodiment, the engineered immune cells are selected from the following group:

(i) Chimeric antigen receptor αβ T cells (CAR-T cells);

(ii) chimeric antigen receptor γδ T cells (CAR-T cells);

(iii) chimeric antigen receptor NKT cells (CAR-NKT cells);

(iv) Chimeric antigen receptor NK cells (CAR-NK cells).

In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or a combination thereof.

In another preferred embodiment, the engineered immune cells are CAR-T cells.

In an eighth aspect of the present invention, a method for producing an antibody or an antigen-binding fragment thereof against IL-18Rβ is provided, comprising the steps of:

(a) culturing the host cell according to the fifth aspect of the present invention under suitable conditions to obtain a culture containing the antibody or antigen-binding fragment thereof to IL-18Rβ;

(b) isolating and/or recovering the antibody or antigen-binding fragment thereof against IL-18Rβ from the culture; and

(c) Optionally, purifying and/or modifying the antibody against IL-18Rβ or the antigen-binding fragment thereof obtained in step (b).

In the ninth aspect of the present invention, an immunoconjugate is provided, wherein the immunoconjugate comprises:

(a) an antibody portion, the antibody portion comprising the antibody or antigen-binding fragment thereof according to the first aspect of the invention; and

(b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

In another preferred embodiment, the (a) part and the coupling part are coupled via a chemical bond or a linker.

In another preferred embodiment, the radioactive nuclides include:

(i) a diagnostic isotope 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 a combination thereof; and/or

(ii) therapeutic isotopes, wherein the therapeutic isotopes are 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 a combination thereof.

In another preferred embodiment, the coupling moiety is a drug or a toxin.

In another preferred embodiment, the drug is a drug for targeted treatment of IL-18 related diseases.

In another preferred embodiment, the IL-18-related diseases include diseases with high expression of IL-18, diseases associated with abnormal expression of IL-18 receptors or diseases associated with high activity of IL-18 signaling pathways.

In another preferred embodiment, the IL-18 related disease is an autoimmune disease or an inflammatory disease.

In another preferred embodiment, the IL-18 high expression disease is selected from: tumors, 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, Grave's disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, The invention relates to an inflammatory bowel disease, including but not limited to: arthritis, pernicious anemia, Sjögren's syndrome, type I diabetes mellitus, type II diabetes mellitus, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyoderma gangrenosum, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory bowel disease (IBD), adult-onset Still's disease (AOSD), chronic obstructive pulmonary disease (COPD) or pulmonary nodules, or a combination thereof.

In another preferred embodiment, 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, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.

In another preferred embodiment, the drug is a cytotoxic drug.

In another preferred embodiment, the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, Auristatins, Camptothecins, Duocarmycins, Etoposides, Maytansines and Maytansinoids (e.g., DM1 and DM4), Taxanes, Benzodiazepines or Benzodiazepine containing drugs (e.g., Pyrrolo[1,4]benzodiazepines (PBDs), Indolinobenzodiazepines and Oxazolidinobenzodiazepines), Vinca alkaloids, or combinations thereof.

In another preferred embodiment, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansyl, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyproline, succinimidyl, tadalafil, tadalafil, succinimidyl ... anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, Mitogellin, Retstrictocin, phenomycin, enomycin, curcin, crotonin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoids, or a combination thereof.

In another preferred embodiment, the coupling moiety is a detectable label.

In another preferred embodiment, the coupling portion is selected from the following group: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticles.

In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

In another preferred embodiment, the multivalency refers to the presence of multiple repeats of the same or different antibodies or antigen-binding fragments thereof as described in the first aspect of the present invention in the amino acid sequence of the immunoconjugate.

In the tenth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

(i) an active ingredient, wherein the active ingredient is selected from the group consisting 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

(ii) a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection and lyophilized agent.

In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or 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 a combination thereof and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.

In another preferred embodiment, 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.

In the eleventh aspect of the present invention, there is provided a use of an active ingredient, wherein the active ingredient is 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 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 a combination thereof, wherein the active ingredient is used to prepare:

(a) Drugs for preventing and/or treating IL-18 related diseases;

(b) Reagents for detecting IL-18-related diseases.

In another preferred embodiment, the reagent is a diagnostic reagent, preferably, the diagnostic reagent is a detection sheet or a detection plate.

In another preferred embodiment, the diagnostic reagent is used to detect IL-18Rβ protein or a fragment thereof in a sample.

In another preferred embodiment, the IL-18-related diseases include diseases with high expression of IL-18, diseases associated with abnormal expression of IL-18 receptors or diseases associated with high activity of IL-18 signaling pathways.

In another preferred embodiment, the IL-18 related disease is an autoimmune disease or an inflammatory disease.

In another preferred embodiment, the disease in which IL-18 is highly expressed is selected from the group consisting of: tumors, 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, Grave's disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myositis gravis, Asthenia, pernicious anemia, Sjögren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyoderma gangrenosum, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory bowel disease (IBD), adult-onset Still's disease (AOSD), chronic obstructive pulmonary disease (COPD) or pulmonary nodules, or a combination thereof.

In another preferred embodiment, 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, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.

In the twelfth aspect of the present invention, a method for detecting IL-18Rβ protein or a fragment thereof in a sample in vitro is provided, the method comprising the steps of:

(1) contacting the sample in vitro with the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of the corresponding target of the IL-18Rβ protein or its fragment in the sample.

In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

The thirteenth aspect of the present invention provides a kit, comprising:

(1) a first container, wherein the first container 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 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 the pharmaceutical composition according to the tenth aspect of the present invention, or a combination thereof; and/or

(2) a second container, wherein the second container contains a secondary antibody against the contents of the first container;

or,

The kit contains a detection plate, which includes: a substrate (support plate) and a test strip, wherein the test strip contains the antibody or its antigen-binding fragment as described in the first aspect of the present invention, or 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 the pharmaceutical composition as described in the tenth aspect of the present invention, or a combination thereof.

In another preferred embodiment, the kit further comprises an instruction manual, and according to the instruction manual, the kit is used for non-invasively detecting the expression of IL-18Rβ in a test subject.

In another preferred embodiment, the kit is used for detecting IL-18 related diseases.

In another preferred embodiment, the IL-18 related diseases include diseases with high expression of IL-18, including diseases associated with abnormal expression of IL-18 receptors or diseases associated with high activity of IL-18 signaling pathways.

In another preferred embodiment, the IL-18 high expression disease is selected from: tumors, 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, Grave's disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, The invention relates to an inflammatory bowel disease, including but not limited to: arthritis, pernicious anemia, Sjögren's syndrome, type I diabetes mellitus, type II diabetes mellitus, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyoderma gangrenosum, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory bowel disease (IBD), adult-onset Still's disease (AOSD), chronic obstructive pulmonary disease (COPD) or pulmonary nodules, or a combination thereof.

In another preferred embodiment, 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, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.

In the fourteenth aspect of the present invention, a method for preventing and/or treating IL-18 related diseases is provided, the method comprising: administering to a subject in need thereof the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or 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 the pharmaceutical composition as described in the tenth aspect of the present invention, or a combination thereof.

In another preferred embodiment, the subject includes mammals, such as humans.

In another preferred embodiment, the IL-18 related diseases include diseases with high expression of IL-18, diseases associated with abnormal expression of IL-18 receptors or diseases associated with high activity of IL-18 signaling pathways.

In another preferred embodiment, the IL-18 related disease is an autoimmune disease or an inflammatory disease.

In another preferred embodiment, the IL-18 high expression disease is selected from: tumors, 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, Grave's disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, The invention relates to an inflammatory bowel disease, including but not limited to: arthritis, pernicious anemia, Sjögren's syndrome, type I diabetes mellitus, type II diabetes mellitus, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyoderma gangrenosum, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory bowel disease (IBD), adult-onset Still's disease (AOSD), chronic obstructive pulmonary disease (COPD) or pulmonary nodules, or a combination thereof, or a combination thereof.

In another preferred embodiment, 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, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.

In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells (autologous cells) derived from the subject.

In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).

In another preferred embodiment, the method can be used in combination with other treatment methods.

In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy and the like.

In a fifteenth aspect of the present invention, a method for diagnosing IL-18 related diseases is provided, comprising the steps of:

(i) obtaining a sample from a diagnostic subject, and contacting the sample with 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 the pharmaceutical composition according to the tenth aspect of the present invention; and

(ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates that the subject is a confirmed patient of an IL-18-related disease.

In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues and organs.

In another preferred embodiment, the IL-18 related diseases include diseases with high expression of IL-18, diseases associated with abnormal expression of IL-18 receptors or diseases associated with high activity of IL-18 signaling pathways.

In another preferred embodiment, the IL-18 related disease is an autoimmune disease or an inflammatory disease.

In another preferred embodiment, the IL-18 high expression disease is selected from: tumors, 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, Grave's disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, The invention relates to an inflammatory bowel disease, a condition characterized by poor prognosis, poor prognosis, and poor prognosis. The invention relates to an inflammatory bowel disease, a condition characterized by poor prognosis, poor prognosis, and poor prognosis. The invention relates to an inflammatory bowel disease, a condition characterized by poor prognosis, poor prognosis, and poor prognosis. The invention relates to an inflammatory bowel disease, a condition characterized by poor prognosis, poor prognosis, and poor prognosis.

In another preferred embodiment, 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, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the sequence information of the IL-18RβWT antibody.

FIG2 shows the concentration-response curve of anti-IL-18Rβ antibody for inhibition of IFN-γ in KG-1 cells.

FIG3 shows the concentration-response curves of anti-IL-18Rβ antibodies for inhibition of IFN-γ in human PBMC cells.

DETAILED DESCRIPTION

After extensive and in-depth research and a large number of screenings, the inventors have developed for the first time a high-affinity antibody targeting IL-18Rβ and an antigen-binding fragment thereof. Specifically, the present invention uses the WT antibody of IL-18Rβ as a starting material for improving affinity to construct a precise mutation library, and introduces saturation mutations into all 73 residues in the six CDR regions of the WT antibody. After further screening, the selected mutants are sorted by the dissociation rate constant measured by surface plasmon resonance, and a combinatorial library is constructed with random combinations of beneficial mutations, thereby obtaining the IL-18Rβ antibody of the present invention and an antigen-binding fragment thereof with improved binding affinity. The antibody targeting IL-18Rβ and its antigen-binding fragment developed by the present invention can be used as a new therapeutic method for targeted treatment of diseases related to abnormal expression of IL-18 receptors or high activity of IL-18 signaling pathways.

the term

As used herein, the terms "antibody of the present invention", "antibodies of the present invention", "antibodies against IL-18Rβ of the present invention", "antibodies against IL-18Rβ", "antibodies against IL-18Rβ", and "IL-18Rβ antibodies" have the same meaning and can be used interchangeably, all referring to antibodies that specifically recognize and bind to IL-18Rβ protein (including human IL-18Rβ protein).

Preferably, the antibody numbers of the present invention and the corresponding sequence numbers are shown in Table 1 below.

Table 1

Note: Each numerical value in the table represents a 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 numbers of their amino acid sequences.

The term "antibody" herein is intended to include full-length antibodies and any antigen-binding fragments (i.e., antigen-binding portions) or single chains thereof. Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy chains and light chains being linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely CH1, CH2, and CH3. Each light chain consists 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 conservative framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may 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 (CIq) of the classical complement system.

The term "antigen-binding fragment" (or antigen-binding portion) of an antibody herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IL-18Rβ protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1; (ii) a F(ab') ₂ fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) a Fd fragment consisting of VH and CH1; (iv) a Fv fragment consisting of a single-arm VL and VH of an antibody; (v) a dAb fragment consisting of VH; (vi) an isolated complementarity determining region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by a recombinant method via a synthetic linker that makes the two into a single protein chain, wherein the VL and VH regions are paired to form a monovalent molecule) (referred to as single-chain Fc (scFv)). These single-chain antibodies are also intended to be included in 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 intact antibodies.

As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region 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. I, pp. 647-669 (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

One skilled in the art can define the amino acid sequence boundaries of a CDR using any of a variety of known numbering schemes, including those described in Kabat et al., supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme).

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (Cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments thereof of the present invention to form conjugates. The present invention also includes cell surface markers or antigens combined with the antibodies or fragments thereof against the new coronavirus.

As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

As used herein, the term "light chain variable region" is used interchangeably with "VL."

As used herein, the term "variable region" and "complementarity determining region (CDR)" are used interchangeably.

In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDRH1, CDRH2, and CDRH3.

In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

In a preferred embodiment of the present invention, the light chain variable region of the antibody includes three complementarity determining regions CDRL1, CDRL2, and CDRL3.

In a preferred embodiment of the present invention, the light chain of the antibody comprises the above-mentioned light chain variable region and light chain constant region.

In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, and all refer to polypeptides that specifically bind to IL-18Rβ protein, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.

The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

Generally, 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). This segment is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

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. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

The antibody of the present invention refers to a polypeptide having IL-18Rβ protein binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides containing the above-mentioned CDR region and having the same function as the antibody of the present invention. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids to 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 present invention.

Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

In the present invention, "conservative variants of the antibody of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 2.

Table 2

The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include a polynucleotide further including additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present 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) the addition of denaturing agents during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, 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 fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation 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. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

The antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination 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 enzymes capable of producing a detectable product.

Therapeutic agents that can be combined or coupled to the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

IL-18, IL-18R, and IL-18Rβ

Interleukin-18 (IL18, also known as interferon-gamma inducing factor) is a protein that in humans is encoded by the IL-18 gene. The protein encoded by this gene is a proinflammatory cytokine. Many cell types, both hematopoietic and non-hematopoietic, have the potential to produce IL-18.

The IL-18 receptor consists of an inducible component, IL-18Rα, which binds to mature IL-18 with low affinity, and a constitutively expressed co-receptor, IL-18Rβ. IL-18 binds to the ligand receptor IL-18Rα, inducing the recruitment of IL-18Rβ to form a high-affinity complex that signals through the toll/interleukin-1 receptor (TIR) domain. This signaling domain activates the pro-inflammatory program and the MyD88 adaptor protein of the NF-κB pathway. IL-18Rβ is specifically expressed in immune cells and is a necessary receptor for IL-18 signaling, so antibodies targeting IL-18Rβ can specifically and effectively inhibit IL-18 signals. Effective inhibition of IL-18 downstream inflammatory signaling pathways is currently a promising approach for the treatment of autoimmune and inflammatory diseases.

Pharmaceutical composition

The present invention also provides a composition. Preferably, 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. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance 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 carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 milligrams/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 10 milligrams/kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

Antibodies against IL-18Rβ

In the present invention, the antibody against IL-18Rβ includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).

In a preferred example of the present invention, the antibody against IL-18Rβ comprises one or more heavy chains having a heavy chain variable region as shown in SEQ ID NO: 1, 9 or 14, and a light chain having a light chain variable region as shown in SEQ ID NO: 5, 12, 15 or 17.

Labeled Antibodies

In a preferred embodiment of the present invention, the antibody carries a detectable marker. More preferably, the marker is selected from the group consisting of an isotope, a colloidal gold marker, a colored marker or a fluorescent marker.

Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, an antibody against IL-18Rβ protein is labeled with colloidal gold to obtain a colloidal gold-labeled antibody.

The antibody against IL-18Rβ of the present invention can effectively bind to IL-18Rβ protein.

Detection Methods

The present invention also relates to a method for detecting IL-18Rβ protein or a fragment thereof. The method generally comprises the following steps: obtaining a cell and/or tissue sample; dissolving the sample in a medium; and detecting the level of IL-18Rβ protein in the dissolved sample.

In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage solution.

Reagent test kit

The present invention also provides a kit containing the antibody (or fragment thereof) or the detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, and the like.

The present invention also provides a detection kit for detecting the level of IL-18Rβ protein, which includes an antibody that recognizes IL-18Rβ protein, a lysis medium for dissolving the sample, and universal reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

application

As described above, the antibodies of the present invention have a wide range of biological and clinical application values, and their applications involve the treatment of diseases related to high expression of IL-18, as well as the diagnosis of high expression of IL-18, basic medical research, biological research, and other fields. A preferred application is for clinical prevention and treatment of IL-18Rβ protein.

The present invention also provides a method for stimulating an immune response mediated by T cells targeting a tumor cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cells of the present invention to the mammal.

In one embodiment, the present invention includes a type of cell therapy that separates the patient's autologous T cells (or allogeneic donors), activates and genetically modifies them to produce CAR-T cells, which are then injected into the same patient. This approach makes the probability of graft-versus-host reaction extremely low, and the antigen is recognized by the T cell in an MHC-free manner. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and can last for months to years. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-T cells can induce a specific immune response to tumor cells that highly express the antigen recognized by the CAR antigen binding domain. For example, the CAR-T cells of the present invention cause a specific immune response to tumor cells that highly express IL-18Rβ.

Treatable cancers include tumors that are not vascularized or substantially not vascularized, as well as vascularized tumors. The types of cancers 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, etc.

Generally, cells activated and expanded as described herein can be used to treat and prevent diseases such as tumors. Therefore, the present invention provides a method for treating cancer, which comprises administering a therapeutically effective amount of the CAR-T cells of the present invention to a subject in need thereof.

The CAR-T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. In short, the pharmaceutical composition of the present invention may include 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.

When an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibitory effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, which takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. The pharmaceutical composition comprising the T cells described herein can be administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably 10 ⁵ to 10 ⁷ cells/kg body weight (including all integer values within the range). The T cell composition can also be administered multiple times at these doses. The cells can be administered using well-known infusion techniques in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a particular patient can be easily determined by a technician in the medical field by monitoring the patient's disease signs and adjusting the treatment accordingly.

Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous injection. The composition of the T cell can be directly injected into the tumor, lymph node or infection site.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to patients in combination with any number of related treatment forms (e.g., before, simultaneously or after), including but not limited to treatment with the following agents: agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to patients in combination with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, simultaneously or after). For example, in one embodiment, the subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered prior to or after surgery.

The dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 ⁵ to 1×10 ¹⁰ modified T cells of the present invention can be administered to the patient, for example, by intravenous reinfusion, per treatment or per course of treatment.

The main advantages of the present invention include:

1. The IL-18Rβ antibody or antigen-binding fragment thereof of the present invention can specifically bind to IL-18Rβ with high affinity, which is more than ten times higher than that of WT, and up to 22 times higher.

2. The IL-18Rβ antibody or antigen-binding fragment thereof of the present invention has good IL-18/IL-18R blocking activity and can effectively inhibit the downstream inflammatory signaling pathway of IL-18.

3. The IL-18Rβ antibody or antigen-binding fragment thereof of the present invention has advantages in specificity, effectiveness and safety compared with other components of the IL-18 signaling pathway.

4. The IL-18Rβ antibody or antigen-binding fragment thereof of the present invention can effectively block the release of IFN-γ in KG-1 cells stimulated by IL-18, and the activity efficacy is 20 times that of WT.

5. The IL-18Rβ antibody or antigen-binding fragment thereof of the present invention can effectively block the release of IFN-γ from human peripheral blood mononuclear cells stimulated by IL-18, and its activity is significantly higher than that of WT.

6. The IL-18Rβ antibody or antigen-binding fragment thereof of the present invention contributes to the prevention/diagnosis/treatment of diseases associated with abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway, and provides new methods and technical means.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

Amino acid sequence

SEQ ID NO: 1 A035 heavy chain variable region

EVQLVESGGGLVQPGGSLRLSCAASGINLYYSSMHWVRQAPGKGLEWVASIYSSNGRTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARASFSHGYGWYGLDYWGQGTLVTVSS

SEQ ID NO:2 A035, A039, A038 heavy chain complementary determining region CDRH1

GINLYYSSMH

SEQ ID NO:3 A035, A037, A034, A039, A038, C056-WT heavy chain complementary determining region CDRH2

SIYSSNGRTYYADSVKG

SEQ ID NO:4 A035 heavy chain complementary determining region CDRH3

ASFSHGYGWYGLDY

SEQ ID NO:5 A035, A034 light chain variable region

DIQMTQSPSSSLSASVGDRVTITCRASQSVSFAVEWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITTFGQGTKVEIK

SEQ ID NO:6 A035, A034 light chain complementary determining region CDRL1

RASQSVSFAVE

SEQ ID NO:7 A035, A037, A034, A039, A038, C056-WT light chain complementary determining region CDRL2

SASSLYS

SEQ ID NO:8 A035, A037, A034, A039, A038, C056-WT light chain complementary determining region CDRL3

QQYGYHDAGLIT

SEQ ID NO:9 A037, A034 heavy chain variable region

EVQLVESGGGLVQPGGSLRLSCAASGLNLYYSSMHWVRQAPGKGLEWVASIYSSNGRTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYWGQGTLVTVSS

SEQ ID NO:10 A037, A034 heavy chain complementary determining region CDRH1

GLNLYYSSMH

SEQ ID NO: 11A037, A034, A039, A038, C056-WT heavy chain complementary determining region CDRH3

SSFSHGYGWYGLDY

SEQ ID NO:12 A037 light chain variable region

DIQMTQSPSSSLSASVGDRVTITCRASQSVSFAVDWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITTFGQGTKVEIK

SEQ ID NO:13 A037 light chain complementary determining region CDRL1

RASQSVSFAVD

SEQ ID NO: 14 A039, A038 heavy chain variable region

EVQLVESGGGLVQPGGSLRLSCAASGINLYYSSMHWVRQAPGKGLEWVASIYSSNGRTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYWGQGTLVTVSS

SEQ ID NO:15 A039 light chain variable region

DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVDWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITTFGQGTKVEIK

SEQ ID NO:16 A039 light chain complementary determining region CDRL1

RASQSVSSAVD

SEQ ID NO: 17 A038 light chain variable region

DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVEWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITTFGQGTKVEIK

SEQ ID NO:18 C056-WT heavy chain variable region

EVQLVESGGGLVQPGGSLRLSCAASGFNLYYSSMHWVRQAPGKGLEWVASIYSSNGRTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYWGQGTLVTVSS

SEQ ID NO:19 C056-WT heavy chain complementary determining region CDRH1

GFNLYYSSMH

SEQ ID NO:20 C056-WT light chain variable region

DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITTFGQGTKVEIK

SEQ ID NO:21 C056-WT light chain complementary determining region CDRL1

RASQSVSSAVA

SEQ ID NO:22 A038 light chain complementary determining region CDRL1

RASQSVSSAVE

SEQ ID NO:23 IL-18Rβ antigen sequence

1 MLCLGWIFLW LVAGERIKGF NISGCSTKKL LWTYSTRSEE EFVLFCDLPE PQKSHFCHRN

61 RLSPKQVPEH LPFMGSNDLS DVQWYQQPSN GDPLEDIRKS YPHIIQDKCT LHFLTPGVNN

121 SGSYICRPKM IKSPYDVACC VKMILEVKPQ TNASCEYSAS HKQDLLLGST GSISCPSLSC

181 QSDAQSPAVT WYKNGKLLSV ERSNRIVVDE VYDYHQGTYV CDYTQSDTVS SWTVRAVVQV

241 RTIVGDTKLK PDILDPVEDT LEVELGKPLT ISCKARFGFE RVFNPVIKWY IKDSDLEWEV

301 SVPEAKSIKS TLKDEIIERN IILEKVTQRD LRRKFVCFVQ NSIGNTTQSV QLKEKRGVVL

361 LYILLGTIGT LVAVLAASAL LYRHWIEIVL LYRTYQSKDQ TLGDKKDFDA FVSYAKWSSF

421 PSEATSSLSE EHLALSLFPD VLENKYGYSL CLLERDVAPG GVYAEDIVSI IKRSRRGIFI

481 LSPNYVNGPS IFELQAAVNL ALDDQTLKLI LIKFCYFQEP ESLPHLVKKA LRVLPTVTWR

541 GLKSVPPNSR FWAKMRYHMP VKNSQGFTWN QLRITSRIFQ WKGLSRTETT GRSSQPKEW

Example 1. Preparation of high affinity anti-IL-18Rβ antibody

As shown in FIG1 , the inventors used the WT antibody of IL-18Rβ (i.e., wild-type antibody, C056-WT in Table 1) as the starting material for improving affinity. An accurate 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 sorted by the dissociation rate constant determined by surface plasmon resonance (SPR). A combinatorial library was subsequently constructed with a random combination of beneficial mutations. Lead Fab clones were also determined by SPR sorting. All SPR screenings were performed on 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 onto the SASA biosensor. After equilibrium, the antigen was injected for 120 seconds (binding period), followed by the injection of the running buffer for 360 seconds (dissociation period). The sensor surface was regenerated before injecting other selected clones. The experimental data were locally fitted to a 1:1 interaction model using Biacore 8K evaluation software to obtain the off-rate of the Fab clones. Finally, full-length IgG from the lead clones were expressed in HEK293 cells and evaluated in binding and functional assays.

As shown in Table 3 , 44 hits were analyzed by SPR, and 7 beneficial mutants were identified that exhibited better binding affinity (i.e., >2-fold the affinity of the WT antibody, as shown in Table 4 ).

The top 10 combined mutations with improved binding affinity (>10-fold of WT antibody affinity) are listed in Table 5. Five lead clones (A034, A035, A037, A038, and A039) were selected for full-length antibody production. The sequences are provided below.

Table 3 Affinity and kinetics of IL-18Rβ of 44 hit mutants

Table 4 Beneficial mutants identified by saturation mutagenesis screening

Table 5 Top 10 beneficial mutant combinations with enhanced affinity

Serial number Clone number ka(1/Ms) kd(1/s) KD(M) Ratio (kd) Sequence analysis 1 A034 1.31E+05 4.24E-05 3.22E-10 28.07 VL-S31F/VL-A34E/VH-F27L 2 A035 1.43E+05 3.19E-05 2.24E-10 37.30 VL-S31F/VL-A34E/VH-F27I/VH-S99A 3 A036 1.07E+05 6.91E-05 6.48E-10 17.22 VL-A34D/VH-F27I/VH-S99A 4 A037 1.18E+05 1.00E-06 8.47E-12 1190.00 VL-S31F/VL-A34D/VH-F27L 5 A038 7.82E+04 5.67E-05 7.25E-10 20.99 VL-A34E/VH-F27I 6 A039 1.15E+05 3.00E-05 2.60E-10 39.67 VL-A34D/VH-F27I 7 A040 7.70E+04 8.00E-05 1.04E-09 14.88 VL-A34E/VH-F27L 8 A041 1.53E+05 6.35E-05 4.15E-10 18.74 VL-S31F/VL-A34D/VH-F27V/VH-S99A 9 A042 7.90E+04 1.06E-04 1.34E-09 11.23 VL-A34E/VH-F27L/VH-S99A 10 A043 1.28E+05 6.29E-05 4.91E-10 18.92 VL-S31F/VL-A34E/VH-F27L C056-WT 6.66E+04 1.19E-03 1.78E-08 1.00

Example 2. Affinity evaluation of anti-IL-18Rβ antibodies

The affinity of anti-IL-18Rβ antibodies to human IL-18Rβ protein was determined using the surface plasmon resonance biosensor Biacore 8K (GE Healthcare). The measurements were performed at 25°C with HBS-EP+ running buffer. The antibody was injected as a capture onto the Series S sensor chip protein A. Diluted IL-18Rβ (400, 200, 100, 50, 25, 12.5, 6.25 nM) was then injected into the surface of flow cells 1 and 2 as the binding phase. The binding time was 120 seconds. The buffer flow was maintained for 420 seconds for separation. The surface was regenerated by injecting 10 mM glycine-HCl pH 1.5 for 30 seconds. The data for the dissociation (kd) rate constant and the association (ka) rate constant were obtained using the Biacore evaluation software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka.

The binding kinetic data of anti-IL-18Rβ antibodies are summarized in Table 6. The binding affinities of A035, A037, and A039 were in the range of 0.72 to 0.98 nm, which were 16-22 times higher than the parent antibody WT (15.8 nm).

Table 6 Binding kinetics of anti-IL-18Rβ antibodies

Example 3. Activity of anti-IL-18Rβ antibody in blocking IFN-γ release in KG-1 cells stimulated by IL-18

3×10 ⁵ KG-1 cells (ATCC, #CCL-246) were seeded into each well of a 96-well plate. Serial dilutions of the antibodies prepared in Example 1 were added to the wells before stimulation with 10 ng/mL IL-18 for 1 h. After 24 h incubation, cells were pelleted by centrifugation, and IFN-γ was measured from the supernatant using an ELISA kit (R&D Systems, #VAL104C) according to the manufacturer's instructions. The above anti-IL-18Rβ antibodies were subjected to at least 3 replicates, and the IC50 of each antibody is summarized in Table 7.

Table 7 IC50 of anti-IL-18Rβ antibody in KG-1 assay

Antibody IC50(nM) WT 8.14 A035 0.26 A037 0.27 A039 0.59

Representative experimental results are shown in Figure 2 , and the potency of A035, A037, and A039 antibodies is approximately 20 times that of the WT antibody.

Example 4. Anti-IL-18Rβ antibody blocks the activity of IL-18 stimulated human peripheral blood mononuclear cell IFN-γ release sex

Human peripheral blood mononuclear cells (PBMC) (TPCS, #PB025C-W) were seeded in 96-well plates at a concentration of 1×10 ⁵ cells/well. The cells were then incubated with different concentrations of anti-IL-18Rβ antibodies at 50 ng/mL IL-18 and 5 ng/mL IL-12 for 24 hours at 37°C and 5% CO _2. The production of IFN-γ was measured using a human ELISA kit (R&D Systems, #VAL104C) according to the manufacturer's instructions.

As shown in Figure 3, the A035, A037 and A039 antibodies showed higher potency than the WT antibody.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Sequence Listing

<110> Hejing Pharmaceutical Technology (Shanghai) Co., Ltd.

<120> An antibody targeting IL-18Rβ and its application

<130> P2021-3129

<160> 23

<170> PatentIn version 3.5

<210> 1

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> A035 heavy chain variable region

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Asn Leu Tyr Tyr Ser

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Tyr Ser Ser Asn Gly Arg Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Ser Phe Ser His Gly Tyr Gly Trp Tyr Gly Leu Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 2

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRH1 of A035, A039, A038

<400> 2

Gly Ile Asn Leu Tyr Tyr Ser Ser Met His

1 5 10

<210> 3

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRH2 of A035, A037, A034, A039, A038, C056-WT

<400> 3

Ser Ile Tyr Ser Ser Asn Gly Arg Thr Tyr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 4

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRH3 of A035

<400> 4

Ala Ser Phe Ser His Gly Tyr Gly Trp Tyr Gly Leu Asp Tyr

1 5 10

<210> 5

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> A035, A034 light chain variable region

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Phe Ala

20 25 30

Val Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Tyr His Asp Ala

85 90 95

Gly Leu Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 6

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRL1 of A035 and A034

<400> 6

Arg Ala Ser Gln Ser Val Ser Phe Ala Val Glu

1 5 10

<210> 7

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRL2 of A035, A037, A034, A039, A038, C056-WT

<400> 7

Ser Ala Ser Ser Leu Tyr Ser

1 5

<210> 8

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRL3 of A035, A037, A034, A039, A038, C056-WT

<400> 8

Gln Gln Tyr Gly Tyr His Asp Ala Gly Leu Ile Thr

1 5 10

<210> 9

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> A037, A034 heavy chain variable region

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Asn Leu Tyr Tyr Ser

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Tyr Ser Ser Asn Gly Arg Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ser Phe Ser His Gly Tyr Gly Trp Tyr Gly Leu Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRH1 of A037 and A034

<400> 10

Gly Leu Asn Leu Tyr Tyr Ser Ser Met His

1 5 10

<210> 11

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRH3 of A037, A034, A039, A038, C056-WT

<400> 11

Ser Ser Phe Ser His Gly Tyr Gly Trp Tyr Gly Leu Asp Tyr

1 5 10

<210> 12

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> A037 light chain variable region

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Phe Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Tyr His Asp Ala

85 90 95

Gly Leu Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 13

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRL1 of A037

<400> 13

Arg Ala Ser Gln Ser Val Ser Phe Ala Val Asp

1 5 10

<210> 14

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> A039, A038 heavy chain variable region

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Asn Leu Tyr Tyr Ser

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Tyr Ser Ser Asn Gly Arg Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ser Phe Ser His Gly Tyr Gly Trp Tyr Gly Leu Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> A039 light chain variable region

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Tyr His Asp Ala

85 90 95

Gly Leu Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 16

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRL1 of A039

<400> 16

Arg Ala Ser Gln Ser Val Ser Ser Ala Val Asp

1 5 10

<210> 17

<211> 110

<212> PRT

<213> Artificial Sequence

<220>

<223> A038 light chain variable region

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala

20 25 30

Val Glu Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Tyr His Asp Ala

85 90 95

Gly Leu Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 18

<211> 123

<212> PRT

<213> Homo sapiens

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Leu Tyr Tyr Ser

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Tyr Ser Ser Asn Gly Arg Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ser Phe Ser His Gly Tyr Gly Trp Tyr Gly Leu Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 10

<212> PRT

<213> Homo sapiens

<400> 19

Gly Phe Asn Leu Tyr Tyr Ser Ser Met His

1 5 10

<210> 20

<211> 110

<212> PRT

<213> Homo sapiens

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Tyr His Asp Ala

85 90 95

Gly Leu Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 21

<211> 11

<212> PRT

<213> Homo sapiens

<400> 21

Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala

1 5 10

<210> 22

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> CDRL1 of A038

<400> 22

Arg Ala Ser Gln Ser Val Ser Ser Ala Val Glu

1 5 10

<210> 23

<211> 599

<212> PRT

<213> Homo sapiens

<400> 23

Met Leu Cys Leu Gly Trp Ile Phe Leu Trp Leu Val Ala Gly Glu Arg

1 5 10 15

Ile Lys Gly Phe Asn Ile Ser Gly Cys Ser Thr Lys Lys Leu Leu Trp

20 25 30

Thr Tyr Ser Thr Arg Ser Glu Glu Glu Phe Val Leu Phe Cys Asp Leu

35 40 45

Pro Glu Pro Gln Lys Ser His Phe Cys His Arg Asn Arg Leu Ser Pro

50 55 60

Lys Gln Val Pro Glu His Leu Pro Phe Met Gly Ser Asn Asp Leu Ser

65 70 75 80

Asp Val Gln Trp Tyr Gln Gln Pro Ser Asn Gly Asp Pro Leu Glu Asp

85 90 95

Ile Arg Lys Ser Tyr Pro His Ile Ile Gln Asp Lys Cys Thr Leu His

100 105 110

Phe Leu Thr Pro Gly Val Asn Asn Ser Gly Ser Tyr Ile Cys Arg Pro

115 120 125

Lys Met Ile Lys Ser Pro Tyr Asp Val Ala Cys Cys Val Lys Met Ile

130 135 140

Leu Glu Val Lys Pro Gln Thr Asn Ala Ser Cys Glu Tyr Ser Ala Ser

145 150 155 160

His Lys Gln Asp Leu Leu Leu Gly Ser Thr Gly Ser Ile Ser Cys Pro

165 170 175

Ser Leu Ser Cys Gln Ser Asp Ala Gln Ser Pro Ala Val Thr Trp Tyr

180 185 190

Lys Asn Gly Lys Leu Leu Ser Val Glu Arg Ser Asn Arg Ile Val Val

195 200 205

Asp Glu Val Tyr Asp Tyr His Gln Gly Thr Tyr Val Cys Asp Tyr Thr

210 215 220

Gln Ser Asp Thr Val Ser Ser Trp Thr Val Arg Ala Val Val Gln Val

225 230 235 240

Arg Thr Ile Val Gly Asp Thr Lys Leu Lys Pro Asp Ile Leu Asp Pro

245 250 255

Val Glu Asp Thr Leu Glu Val Glu Leu Gly Lys Pro Leu Thr Ile Ser

260 265 270

Cys Lys Ala Arg Phe Gly Phe Glu Arg Val Phe Asn Pro Val Ile Lys

275 280 285

Trp Tyr Ile Lys Asp Ser Asp Leu Glu Trp Glu Val Ser Val Pro Glu

290 295 300

Ala Lys Ser Ile Lys Ser Thr Leu Lys Asp Glu Ile Ile Glu Arg Asn

305 310 315 320

Ile Ile Leu Glu Lys Val Thr Gln Arg Asp Leu Arg Arg Lys Phe Val

325 330 335

Cys Phe Val Gln Asn Ser Ile Gly Asn Thr Thr Gln Ser Val Gln Leu

340 345 350

Lys Glu Lys Arg Gly Val Val Leu Leu Tyr Ile Leu Leu Gly Thr Ile

355 360 365

Gly Thr Leu Val Ala Val Leu Ala Ala Ser Ala Leu Leu Tyr Arg His

370 375 380

Trp Ile Glu Ile Val Leu Leu Tyr Arg Thr Tyr Gln Ser Lys Asp Gln

385 390 395 400

Thr Leu Gly Asp Lys Lys Asp Phe Asp Ala Phe Val Ser Tyr Ala Lys

405 410 415

Trp Ser Ser Phe Pro Ser Glu Ala Thr Ser Ser Leu Ser Glu Glu His

420 425 430

Leu Ala Leu Ser Leu Phe Pro Asp Val Leu Glu Asn Lys Tyr Gly Tyr

435 440 445

Ser Leu Cys Leu Leu Glu Arg Asp Val Ala Pro Gly Gly Val Tyr Ala

450 455 460

Glu Asp Ile Val Ser Ile Ile Lys Arg Ser Arg Arg Gly Ile Phe Ile

465 470 475 480

Leu Ser Pro Asn Tyr Val Asn Gly Pro Ser Ile Phe Glu Leu Gln Ala

485 490 495

Ala Val Asn Leu Ala Leu Asp Asp Gln Thr Leu Lys Leu Ile Leu Ile

500 505 510

Lys Phe Cys Tyr Phe Gln Glu Pro Glu Ser Leu Pro His Leu Val Lys

515 520 525

Lys Ala Leu Arg Val Leu Pro Thr Val Thr Trp Arg Gly Leu Lys Ser

530 535 540

Val Pro Pro Asn Ser Arg Phe Trp Ala Lys Met Arg Tyr His Met Pro

545 550 555 560

Val Lys Asn Ser Gln Gly Phe Thr Trp Asn Gln Leu Arg Ile Thr Ser

565 570 575

Arg Ile Phe Gln Trp Lys Gly Leu Ser Arg Thr Glu Thr Thr Gly Arg

580 585 590

Ser Ser Gln Pro Lys Glu Trp

595

Claims (25)

1. An antibody against IL-18Rβ or an antigen-binding fragment thereof, characterized in that it comprises: (a) heavy chain complementarity determining regions CDRH1, CDRH2, CDRH3, the amino acid sequences of said CDRH1, CDRH2, CDRH3 are respectively shown in SEQ ID NO: 2, 3 and 4, or respectively shown in SEQ ID NO: 10, 3 and 11 Shown, or as shown in SEQ ID NO:2,3 and 11 respectively; And/or (b) 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. Preferably, any amino acid sequence in the above amino acid sequence also includes a derivative that optionally undergoes addition, deletion, modification and/or substitution of at least one (such as 1-4) amino acids and can retain the ability to specifically bind IL-18Rβ sequence. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein 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 light chain variable region as set forth in SEQ ID NO:5, 12, 15 or 17. 3. The antibody or antigen-binding fragment thereof according to claim 1, wherein 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 SEQ ID NO: 18 The wild-type light chain variable region represented by NO:20 may include beneficial mutations selected from the group consisting of F27L, F27I, F27V, S99A, S31F, A34D, A34E, or combinations thereof. 4. The antibody of claim 1 or an antigen-binding fragment thereof, wherein the antibody or an antigen-binding fragment thereof is based on the wild-type heavy chain variable region shown in SEQ ID NO: 18 and based on SEQ ID NO: The wild-type light chain variable region shown at 20 has beneficial mutations selected from the group consisting of: S31F, A34E, F27L; S31F, A34E, F27I, S99A; A34D, F27I, S99A; S31F, A34D, F27L; A34E, F27I; A34D, F27I; A34E, F27L; S31F, A34D, F27V, S99A; A34E, F27L, S99A; or S31F, A34E, F27L. 5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof may comprise a monomer, a bivalent antibody, and/or a multivalent antibody. Preferably, the bivalent antibody can also be a bispecific antibody; preferably, the multivalent antibody can also be a multispecific antibody. 6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of scFv, Fab, Fab', F(ab') ₂ , Fv fragments, disulfide-bonded Fv (dsFv). 7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof are respectively as shown in SEQ ID NO: 9 and SEQ ID NO:5, or respectively as shown in SEQ ID NO:1 and SEQ ID NO:5, or respectively as shown in SEQ ID NO:9 and SEQ ID NO:12, or respectively as SEQ ID NO: 14 and SEQ ID NO: 17, or as shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively. 8. The antibody or antigen-binding fragment thereof according to claim 1, wherein 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. 9. A recombinant protein, characterized in that, said recombinant protein comprises: (i) the antibody or antigen-binding fragment thereof of any one of claims 1-8; and (ii) Optional tag sequences to aid in expression and/or purification. 10. A nucleotide molecule, characterized in that, the nucleotide molecule encodes the antibody or antigen-binding fragment thereof as claimed in any one of claims 1-8, and/or the antibody or antigen-binding fragment thereof as claimed in claim 9. Recombinant protein. 11. An expression vector, characterized in that the expression vector contains the nucleotide molecule according to claim 10. 12. A host cell, characterized in that the host cell contains the expression vector according to claim 11, or the nucleotide molecule according to claim 10 is integrated in its genome. 13. A chimeric antigen receptor CAR, characterized in that the antigen-binding region scFv segment of the CAR is a binding region specifically binding to IL-18Rβ, and the heavy chain variable region of the scFv comprises: Heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of the CDRH1, CDRH2, and CDRH3 are respectively shown in SEQ ID NO: 2, 3, and 4, or respectively shown in SEQ ID NO: 10, 3, and 11, or respectively as shown in SEQ ID NO:2, 3 and 11; and/or The light chain variable region of the scFv comprises: 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. 14. An engineered immune cell, characterized in that the engineered immune cell expresses an exogenous CAR according to claim 13. 15. A method for producing an antibody against IL-18Rβ or an antigen-binding fragment thereof, comprising the steps of: (a) under suitable conditions, culturing the host cell as claimed in claim 12, thereby obtaining a culture containing IL-18Rβ antibodies or antigen-binding fragments thereof; (b) isolating and/or recovering said antibody against IL-18Rβ or an antigen-binding fragment thereof from said culture; and (c) Optionally, purifying and/or modifying the IL-18Rβ antibody or antigen-binding fragment thereof obtained in step (b). 16. An immunoconjugate, characterized in that, the immunoconjugate contains: (a) an antibody portion comprising the antibody or antigen-binding fragment thereof of any one of claims 1-8; and (b) a coupling moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof . 17. A pharmaceutical composition, characterized in that, said pharmaceutical composition contains: (i) an active ingredient selected from the group consisting of the antibody or antigen-binding fragment thereof as claimed in any one of claims 1-8, or the recombinant protein as claimed in claim 9, or as claimed in claim The engineered immune cell of claim 14, or the immunoconjugate of claim 16, or a combination thereof; and (ii) A pharmaceutically acceptable carrier, diluent or excipient. 18. A use of an active ingredient selected from the group consisting of the antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the recombinant protein of claim 9, or The engineered immune cell as claimed in claim 14, or the immunoconjugate as claimed in claim 16, or a combination thereof, wherein the active ingredient is used to prepare: (a) drugs for the prevention and/or treatment of IL-18-related diseases; (b) Reagents for detecting IL-18-associated diseases. 19. A method for in vitro detection of IL-18Rβ protein or fragments thereof in a sample, said method comprising the steps of: (1) In vitro, contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1-8; (2) Detecting whether the antigen-antibody complex is formed, wherein the formation of the complex indicates that the corresponding target of IL-18Rβ protein or its fragment exists in the sample. 20. A test kit comprising: (1) a first container containing the antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the recombinant protein according to claim 9, or the recombinant protein according to claim 14 The engineered immune cell, or the immunoconjugate as claimed in claim 16, or the pharmaceutical composition as claimed in claim 17; and/or (2) a second container containing a secondary antibody against the contents of the first container; and optional instruction manual. 21. A method for preventing and/or treating IL-18-related diseases, said method comprising: administering the antibody or antigen-binding fragment thereof as claimed in claims 1-8 to a subject in need, or as claimed in claim 9 or the engineered immune cell as claimed in claim 14, or the immunoconjugate as claimed in claim 16, or the pharmaceutical composition as claimed in claim 17, or a combination thereof. 22. The use according to claim 18, characterized in that the IL-18-related diseases include diseases with high expression of IL-18, which are related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway disease. 23. The use according to claim 18, wherein the IL-18-related disease is an autoimmune disease or an inflammatory disease. 24. The use according to claim 18, wherein the IL-18-related diseases are selected from the group consisting of tumors, leukemia, diabetic nephropathy, Alzheimer's disease, Parkinson's disease, arthritis, and 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, Sjögren's syndrome, type 1 diabetes, type 2 diabetes, vascular inflammation, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome, systemic lupus erythematosus, septic arthritis, pyoderma gangrenosum atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory bowel disease (IBD), adult-onset Still's disease (AOSD) , chronic obstructive pulmonary disease (COPD), or pulmonary nodules, or a combination thereof. 25. The use according to claim 24, wherein the tumor is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, Small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof.