WO2023133842A1

WO2023133842A1 - ANTIBODY TARGETING IL-18Rβ AND APPLICATION THEREOF

Info

Publication number: WO2023133842A1
Application number: PCT/CN2022/072143
Authority: WO
Inventors: 杜勇; 陈永锋; 刘淑素; 张玉华
Original assignee: 和径医药科技（上海）有限公司
Priority date: 2022-01-14
Filing date: 2022-01-14
Publication date: 2023-07-20

Abstract

Provided are an antibody targeting IL-18Rβ or an antigen-binding fragment thereof, a preparation method therefor, and an application thereof. Specifically, further provided are a nucleic acid molecule encoding the antibody or the antigen-binding fragments thereof, a corresponding expression vector, a host cell capable of expressing the antibody or the antigen-binding fragment thereof, and a production method for and an application of the antibody or the antigen-binding fragment thereof. The antibody targeting IL-18Rβ or the antigen-binding fragment thereof can specifically bind human IL-18Rβ, has affinity far higher than that of a wild-type antibody, and simultaneously has good IL-18/IL-18R blocking activity. The antibody or the antigen-binding fragments thereof provides a new technical means for the prevention/diagnosis/treatment of diseases related to abnormal expression of an IL-18 receptor or high activity of an IL-18 signaling pathway.

Description

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

technical field

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

Background technique

Interleukin-18 (IL-18), also known as interferon-γ-inducible factor, regulates inflammatory responses mediated by various types of immune cells. IL-18 not only activates Thl cells and NK cells to promote the release of IFN-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 ligand-binding IL-18R alpha (IL-18Rα) subunit and the IL-18R beta (IL-18Rβ ) subunit composition.

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

IL-18Rα is widely expressed and can also combine with the inflammatory inhibitor IL-37, which is more complicated in the regulation of inflammation. IL-18Rβ is specifically expressed in immune cells and is an essential receptor for IL-18 signaling, so antibodies targeting IL-18Rβ can specifically and effectively inhibit IL-18 signaling. Due to the natural presence of IL-18 high-affinity decoy receptor IL-18BP in the body, as well as the existence of the membrane-bound form of IL-18, targeting IL-18 itself is affected by many aspects, and the mechanism of action is relatively complicated. In summary, antibodies targeting IL-18Rβ have advantages in terms of specificity, efficacy and safety compared to other components of the IL-18 signaling pathway.

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

Contents of the invention

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

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

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

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

In another preferred example, the antibody or antigen-binding fragment thereof includes:

(a) Heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of the CDRH1, CDRH2, and CDRH3 are shown in SEQ ID NO: 2, 3, and 4, respectively, or shown in SEQ ID NO: 10, 3, and 11, respectively or as shown in SEQ ID NO:2, 3 and 11, respectively; and

(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 as SEQ ID NO: 13, 7 and 8 Shown, or respectively as shown in SEQ ID NO:16, 7 and 8, or respectively as shown in SEQ ID NO:22, 7 and 8.

In another preferred example, any amino acid sequence in the above amino acid sequence also includes at least one (such as 1-4) amino acids that are optionally added, deleted, modified and/or substituted and can retain the specificity for IL-18Rβ Derived sequences of binding capacity.

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 antibody in Table 1.

In another preferred example, the derivative sequence that has undergone addition, deletion, modification and/or substitution of at least one amino acid and can retain the specific binding ability to IL-18Rβ is homologous or has a sequence identity of at least 85% %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence.

In another preferred embodiment, the antibody or antigen-binding fragment thereof is based on the wild-type heavy chain variable region shown in SEQ ID NO: 18 and/or based on the wild-type light chain variable region shown in SEQ ID NO: 20 Beneficial mutations selected from the group consisting of F27L, F27I, F27V, S99A, S31F, A34D, A34E, or combinations thereof may be included.

In another preferred example, the antibody or antigen-binding fragment thereof may include beneficial mutations selected from the group consisting of F27L, F27I, F27V, S99A, or its combination.

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

In another preferred example, the antibody or antigen-binding fragment thereof is based on the wild-type heavy chain variable region shown in SEQ ID NO: 18 and based on the wild-type light chain variable region shown in SEQ ID NO: 20. Beneficial mutations from the following groups:

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 example, the CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4.

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

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

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

In another preferred example, the bivalent antibody can also be a bispecific antibody.

In another preferred example, the multivalent antibody can also be a multispecific antibody.

In another preferred example, the antigen-binding fragment is selected from scFv, Fab, Fab', F(ab') ₂ , Fv fragments, and disulfide-linked 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 its antigen-binding fragment are shown in SEQ ID NO: 9 and SEQ ID NO: 5, respectively, or shown in SEQ ID NO: 5, respectively. 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 shown in SEQ ID NO: 14 and SEQ ID NO: 17, or respectively as shown in Shown in SEQ ID NO:14 and SEQ ID NO:15.

In another preferred example, 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, and the recombinant protein has:

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

(ii) Optional tag sequences to aid in expression and/or purification.

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

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

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

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

In the third aspect of the present invention, there is provided a nucleotide molecule, the polynucleotide encoding a protein selected from the group consisting of the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the second antibody of the present invention Aspects of the recombinant protein.

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

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

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

In another preferred example, the expression vector is selected from the group consisting of pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV and the like.

In another preferred example, the expression vector further includes a promoter, a transcriptional enhancer element WPRE, a long terminal repeat sequence LTR, etc. selected from the group.

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

In another preferred example, the host cells include prokaryotic cells or eukaryotic cells.

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

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

Heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of CDRH1, CDRH2, and CDRH3 are shown in SEQ ID NO: 2, 3, and 4, respectively, or shown in SEQ ID NO: 10, 3, and 11, respectively, or as shown in SEQ ID NO:2, 3 and 11 respectively; and/or

The light chain variable region of the scFv comprises:

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.

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

In another preferred example, the CAR further includes other foreign proteins.

In another preferred example, the CAR has the structure shown in formula Ia:

L-scFv-H-TM-C-CD3ζ (Ia)

In the formula,

L is nothing or a signal peptide sequence;

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

H is none or hinge region;

TM is the transmembrane domain;

C is costimulatory signal domain;

CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type, or mutants/modifiers thereof);

The "-" connects a peptide or a peptide bond.

In another preferred example, the Ls are respectively selected from signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants/modifications thereof, or combinations thereof.

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

In another preferred example, 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 example, the TM is selected from the transmembrane regions of the following histones: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutants/modifications thereof, or combinations thereof.

In another preferred example, the C is selected from the co-stimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants/modifications thereof, or combinations thereof.

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

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

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

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

(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).

In the ninth aspect of the present invention, there is provided an immunoconjugate comprising:

(a) an antibody portion comprising an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and

(b) A coupling moiety selected from the group consisting of detectable labels, drugs, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof.

In another preferred example, the part (a) is coupled to the coupling part through a chemical bond or a linker.

In another preferred example, the radionuclides include:

(i) isotopes for diagnosis, the isotopes for diagnosis are selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or

(ii) isotope for treatment, the isotope for treatment is selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb- 177, or combinations thereof.

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

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

In another preferred example, the IL-18-related diseases include diseases with high expression of IL-18, diseases related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway.

In another preferred example, the disease with high expression of IL-18 is selected from: tumor, leukemia, diabetic nephropathy, Alzheimer's disease, Parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, silver Psoriasis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, Graves' disease , Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, pus Toxicity, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome, systemic lupus erythematosus, septic arthritis, pyoderma gangrenosum, or combinations thereof.

In another preferred example, the tumor is selected from: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethra cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, or combinations thereof.

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

In another preferred example, the cytotoxic drugs are selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy A sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.

Examples of particularly useful classes of cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, Auristatins, camptothecins, (Camptothecins), Duocarmycins/Duocarmycins, Etoposides, Maytansines and Maytansinoids (such as DM1 and DM4), Taxanes ( Taxanes), benzodiazepines, or benzodiazepine containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines Indolinobenzodiazepines and Oxazolidinobenzodiazepines), Vinca alkaloids, or combinations thereof.

In another preferred embodiment, the toxin is selected from the group consisting of auristatins (for example, auristatin E, auristatin F, MMAE, and MMAF), aureomycin, maytansinol, ricin, grate Anesthetic toxin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, Tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraxin diketone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, acacia Toxin, abrin A chain, lotus root toxin A chain, α-sarcinia, gelonin, Mitogellin, Retstrictocin, phenomycin, enomycin, hemp Curicin, crotonin, calicheamicin, a Sapaonaria officinalis inhibitor, a glucocorticoid, or a combination thereof.

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

In another preferred embodiment, the coupling moiety is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer X-ray tomography) contrast agents, or capable of producing Detectable products of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles , prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), or nanoparticles in any form.

In another preferred embodiment, the immunoconjugate comprises: a multivalent (eg, bivalent) antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

In another preferred example, the multivalent means that the amino acid sequence of the immunoconjugate contains multiple repetitions of the same or different antibodies or antigen-binding fragments thereof according to the first aspect of the present invention.

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

(i) an active ingredient 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 recombinant protein as described in the second aspect of the present invention The engineered immune cell according to the seventh aspect, 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 example, the dosage form of the pharmaceutical composition is selected from the group consisting of injections and freeze-dried preparations.

In another preferred example, the pharmaceutical composition includes 0.01-99.99% of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention, or The engineered immune cell according to the seventh aspect of the present invention, or the immunoconjugate according to the ninth aspect of the present invention, or a combination thereof and 0.01 to 99.99% of a pharmaceutical carrier, the percentage is the percentage of the drug The mass percent of the composition.

In another preferred example, 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 selected from the group consisting of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the antibody according to the second aspect of the present invention The recombinant protein, or the engineered immune cell as described in the seventh aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention, or a combination thereof, the active ingredient is used to prepare:

(a) Drugs for the prevention and/or treatment of IL-18-related diseases;

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

In another preferred example, the reagent shown is a diagnostic reagent, preferably, the diagnostic reagent is a detection chip or a detection plate.

In another preferred example, the diagnostic reagent is used for: detecting IL-18Rβ protein or fragments thereof in a sample.

In another preferred example, the disease with high expression of IL-18 is selected from: tumor, leukemia, diabetic nephropathy, Alzheimer's disease, Parkinson's disease, arthritis, rheumatoid arthritis, multiple sclerosis, Psoriasis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, lupus, systemic lupus erythematosus, juvenile rheumatoid arthritis, juvenile idiopathic arthritis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis, pernicious anemia, Sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, Sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, macrophage activation syndrome, systemic lupus erythematosus, septic arthritis, pyoderma gangrenosum, or combinations thereof.

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

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

(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.

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

In a thirteenth aspect of the present invention, a kit is provided, comprising:

(1) The first container, which contains the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention, or the seventh aspect of the present invention The engineered immune cell according to the aspect, 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 containing a secondary antibody against the contents of the first container;

or,

The test kit contains a detection plate, the detection plate includes: a substrate (support plate) and a test strip, and the test strip contains the antibody or its antigen-binding fragment as described in the first aspect of the present invention, or such as The recombinant protein as described in the second aspect of the present invention, or the engineered immune cell as described in the seventh aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention, or as described in the tenth aspect of the present invention A pharmaceutical composition, or a combination thereof.

In another preferred example, the kit also contains an instruction, and according to the instruction, the kit is used to non-invasively detect the expression of IL-18Rβ in the subject.

In another preferred example, the kit is used for the detection of IL-18-related diseases.

In another preferred example, the IL-18-related diseases include diseases with high expression of IL-18, including diseases related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway.

In the fourteenth aspect of the present invention, there is provided a method for preventing and/or treating IL-18-related diseases, the method comprising: administering the antibody or its antigen as described in the first aspect of the present invention to a subject in need Binding fragments, or recombinant proteins as described in the second aspect of the present invention, or engineered immune cells as described in the seventh aspect of the present invention, or immunoconjugates as described in the ninth aspect of the present invention, or as described in the present invention The pharmaceutical composition of the tenth aspect, or a combination thereof.

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

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

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

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

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

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

(i) Obtain a sample from a diagnostic subject, combine said sample with 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, 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; and

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

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

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 in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

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

Figure 2 shows the concentration-response curves of anti-IL-18Rβ antibody for the inhibition of IFN-γ in KG-1 cells.

Figure 3 shows the concentration-response curves of anti-IL-18Rβ antibody for the inhibition of IFN-γ in human PBMC cells.

Detailed ways

After extensive and in-depth research and extensive screening, the inventors first developed a high-affinity IL-18Rβ-targeting antibody and its antigen-binding fragment. Specifically, the present invention uses the WT antibody of IL-18Rβ as the starting material for improving affinity to construct a precise mutation library, and introduces saturation mutations into all 73 residues of the six CDR regions of the WT antibody. After further screening, the selected mutants were sorted by dissociation rate constants determined by surface plasmon resonance, and a combinatorial library was constructed with a random combination of beneficial mutations, thereby obtaining the IL-18Rβ antibody of the present invention with improved binding affinity and its Antigen-binding fragments. The IL-18Rβ-targeting antibody and its antigen-binding fragment developed in the present invention can be used as a new therapeutic means for targeted treatment of diseases related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway.

the term

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

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

Table 1

抗体编号Antibody number	VHVH	CDRH1CDRH1	CDRH2CDRH2	CDRH3CDRH3	VLVL	CDRL1CDRL1	CDRL2CDRL2	CDRL3CDRL3
A034A034	99	1010	33	1111	55	66	77	88
A035 A035	11	22	33	44	55	66	77	88
A037A037	99	1010	33	1111	1212	1313	77	88
A038A038	1414	22	33	1111	1717	22twenty two	77	88
A039A039	1414	22	33	1111	1515	1616	77	88
C056-WTC056-WT	1818	1919	33	1111	2020	21twenty one	77	88

Note: Each value in the table represents the sequence number, that is, "1" represents "SEQ ID NO: 1", and the sequence numbers of VH, CDRH1, CDRH2, CDRH3, VL, CDRL1, CDRL2, and CDRL3 shown in the table are the sequence numbers of their amino acid sequences. serial number.

The term "antibody" herein is intended to include full-length antibodies and any antigen-binding fragment (ie, antigen-binding portion) or single chains thereof. Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs), which are separated by more conserved framework region (FR) regions. Each VH and VL consists of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxyl terminal. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

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 an antigen (eg, IL-18Rβ protein). It has been demonstrated that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments contained in the "antigen-binding portion" of an antibody include (i) Fab fragments, a monovalent fragment composed of VL, VH, CL, and CH1; (ii) F(ab') ₂ fragments, comprising the hinge region II; Bivalent fragment of two Fab fragments connected by sulfur bridge; (iii) Fd fragment composed of VH and CH1; (iv) Fv fragment composed of antibody single arm VL and VH; (v) dAb fragment composed of VH; (vi) isolated complementarity determining regions (CDRs); and (vii) a Nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked by recombinant methods via a synthetic linker that makes the two a single protein chain, where the VL and VH regions pair to form a monovalent molecule) (called is a single-chain Fc (scFv)). These single chain antibodies are also intended to be included within the meaning of the term. These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as whole antibodies.

As used herein, the term "variable" means that certain portions of the variable regions among antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each contain four FR regions in a roughly β-sheet configuration connected by three CDRs forming connecting loops and, in some cases, partial b-sheet structures. The CDRs in each chain are in close proximity through the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

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

As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

As used herein, the terms "light chain variable region" and "VL" are used interchangeably.

As used herein, the terms "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 includes the above-mentioned light chain variable region and light chain constant region.

In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably, and all refer to a polypeptide that specifically binds IL-18Rβ protein, such as a protein with a heavy chain variable region or peptides. They may or may not contain starting methionine.

The invention also provides other proteins or fusion expression products having the antibodies of the invention. Specifically, the present invention includes any protein or protein conjugates and fusion expression products (i.e., immunoconjugates and fusion expression products) having a heavy chain containing a variable region, as long as the variable region is compatible with the heavy chain of the antibody of the present invention The variable regions are identical or at least 90% homologous, preferably at least 95% homologous.

In general, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR), which is separated into four framework regions (FR), four FR amino acids The sequence is relatively conservative and does not directly participate in the binding reaction. These CDRs form a ring structure, and the β sheets formed by the FRs in between are close to each other in the spatial structure. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. Which amino acids constitute FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.

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

The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of said antibodies.

As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

The antibody of the present invention refers to a polypeptide that has IL-18Rβ protein binding activity and includes the above CDR region. The term also includes variant forms of polypeptides comprising the above CDR regions that have the same function as the antibodies of the present invention. These variations include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

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

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

In the present invention, "conservative variants of the antibody of the present invention" refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acid sequences compared with the amino acid sequence of the antibody of the present invention. An amino acid is replaced by an amino acid with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 2.

Table 2

最初的残基initial residue	代表性的取代representative replacement	优选的取代preferred substitution
Ala(A)Ala(A)	Val；Leu；IleVal; Leu; Ile	ValVal
Arg(R)Arg(R)	Lys；Gln；AsnLys; Gln; Asn	LysLys
Asn(N)Asn(N)	Gln；His；Lys；ArgGln; His; Lys; Arg	GlnGln
Asp(D)Asp(D)	GluGlu	GluGlu
Cys(C)Cys(C)	SerSer	SerSer
Gln(Q)Gln(Q)	AsnAsn	AsnAsn
Glu(E)Glu(E)	AspAsp	AspAsp
Gly(G)Gly(G)	Pro；AlaPro;	AlaAla
His(H)His(H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile (I)	Leu；Val；Met；Ala；PheLeu; Val; Met; Ala; Phe	LeuLeu
Leu(L)Leu(L)	Ile；Val；Met；Ala；PheIle; Val; Met; Ala; Phe	IleIle
Lys(K)Lys(K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met(M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu
Phe(F)Phe(F)	Leu；Val；Ile；Ala；TyrLeu; Val; Ile; Ala; Tyr	LeuLeu
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	SerSer	SerSer
Trp(W)Trp(W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val(V)	Ile；Leu；Met；Phe；AlaIle; Leu; Met; Phe;	LeuLeu

The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof. A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand.

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

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

The present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. 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) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods. The biomolecules (nucleic acid, protein, etc.) involved in the present invention include biomolecules in an isolated form.

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

The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another way is to use MgCl2. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The antibodies of the invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.

Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or substances capable of producing a detectable product. enzyme.

Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2. Biological toxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), etc.

IL-18, IL-18R and IL-18Rβ

Interleukin-18 (IL18, also known as interferon-gamma inducible factor) is a protein that in humans is encoded by the IL-18 gene. The protein encoded by this gene is a pro-inflammatory cytokine. 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 mature IL-18 with low affinity, and a constitutively expressed co-receptor, IL-18Rβ. Binding of IL-18 to the ligand receptor IL-18Rα induces 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 pro-inflammatory programs and the MyD88 adapter protein of the NF-κB pathway. IL-18Rβ is specifically expressed in immune cells and is an essential receptor for IL-18 signaling, so antibodies targeting IL-18Rβ can specifically and effectively inhibit IL-18 signaling. Effectively inhibiting the pro-inflammatory signaling pathway downstream of IL-18 is currently a promising approach for the treatment of autoimmune and inflammatory diseases.

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 materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody (or its conjugate) of the present invention and a pharmaceutically acceptable acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions. The active ingredient is administered in a therapeutically effective amount, eg, about 10 micrograms/kg to about 50 mg/kg body weight per day. In addition, the polypeptides of the invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

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

labeled antibody

In a preferred embodiment of the present invention, the antibody has a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels or fluorescent labels.

Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, the 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 IL-18Rβ protein.

Detection method

The present invention also relates to methods for detecting IL-18Rβ protein or fragments thereof. The steps of the method are roughly as follows: obtain a cell and/or tissue sample; dissolve the sample in a medium; detect the level of IL-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 cell-containing sample present in a cell preservation solution.

Reagent test kit

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

The present invention also provides a detection kit for detecting the level of IL-18Rβ protein, which includes an antibody for recognizing IL-18Rβ protein, a lysis medium for dissolving samples, general reagents and buffers required for detection, such as various buffer, detection label, detection substrate, etc. The test kit may be an in vitro diagnostic device.

application

As mentioned above, the antibody of the present invention has a wide range of biological and clinical application values, and its application involves the treatment of diseases related to the high expression of IL-18, taking into account the diagnosis of high expression of IL-18, basic medical research, biological research, etc. multiple fields. A preferred application is for clinical prevention and treatment against IL-18Rβ protein.

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

In one embodiment, the present invention includes a type of cell therapy, in which a patient's own T cells (or a heterologous donor) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient. In this way, the probability of graft-versus-host reaction is extremely low, and the antigen is recognized by T cells without MHC restriction. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in 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 last for several months to several years. Alternatively, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-T cells can induce a specific immune response to tumor cells that overexpress the antigen recognized by the CAR antigen-binding domain. For example, the CAR-T cells of the present invention elicit a specific immune response against tumor cells with high expression of IL-18Rβ.

Treatable cancers include tumors that are not or substantially not vascularized, as well as vascularized tumors. Cancer types treated with the CAR of the present invention include, but are not limited to: gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like.

Generally, cells activated and expanded as described herein can be used for the treatment and prevention of diseases such as tumors. Accordingly, the present invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell of the present invention.

The CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition with a diluent and/or in combination with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or may be determined by clinical trials.

When an "immunologically effective amount", "antitumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the precise amount of a composition of the invention to be administered can be determined by a physician, taking into account the patient (subject ) with individual differences in age, weight, tumor size, degree of infection or metastasis, and disease. Pharmaceutical compositions comprising T cells described herein may be administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably at a dose of 10 ⁵ to 10 ⁷ cells/kg body weight (including all integer values within the range). T cell compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical art by monitoring the patient for signs of disease, and adjusting treatment accordingly.

Administration of the compositions to a subject may be by any convenient means, including by nebulization, injection, swallowing, infusion, implantation or implantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by intravenous injection. Compositions of T cells can be injected directly into tumors, lymph nodes or sites of infection.

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 combined with any number of relevant treatment modalities (e.g., previously , simultaneously or subsequently) to the patient in a form of treatment including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for PML patients. In a further embodiment, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, the use of chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient. For example, in one embodiment, a subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, following transplantation, the subject receives an infusion of expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.

Dosages administered to a patient for the above treatments will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented according to practice accepted in the art. Usually, 1×10 ⁵ to 1×10 ¹⁰ modified T cells of the present invention can be administered to the patient for each treatment or each course of treatment, for example, through intravenous infusion.

The main advantages of the present invention include:

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

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

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

5. The IL-18Rβ antibody or antigen-binding fragment thereof of the present invention can efficiently block IL-18-stimulated release of IFN-γ from human peripheral blood mononuclear cells, and the activity efficiency 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 related to abnormal expression of IL-18 receptor or high activity of IL-18 signaling pathway, providing New methods and technical means.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

amino acid sequence

SEQ ID NO: 1A035 heavy chain variable region

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

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

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

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

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

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

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

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

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

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

SEQ ID NO: 12A037 light chain variable region

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

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

SEQ ID NO:15A039 light chain variable region

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

SEQ ID NO:17A038 light chain variable region

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

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

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

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

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

SEQ ID NO:23 IL-18Rβ antigen sequence

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

As shown in FIG. 1 , the inventors used the WT antibody of IL-18Rβ (ie, the wild-type antibody, C056-WT in Table 1) as the starting material for improving the affinity. A precision mutation library was constructed to introduce saturation mutations into all 73 residues of the six CDR regions of the WT antibody. After further screening, the selected mutants were ranked by their dissociation rate constants measured by surface plasmon resonance (SPR). A combinatorial library is then constructed with random combinations of beneficial mutations. Lead Fab clones were also identified by SPR sequencing. All SPR screens were performed on a Biacore 8K. The running buffer was HBS-EP (10mM HEPES, 500mM NaCl, 3mM EDTA, 0.05% Tween 20, pH 6.0). A secreted Fab was adsorbed to the SASA biosensor. After equilibration, antigen was injected for 120 seconds (association period), followed by running buffer for 360 seconds (dissociation period). Regenerate the sensor surface before injecting other selected clones. The off-rate of Fab clones was obtained by locally fitting the experimental data to a 1:1 interaction model using Biacore 8K evaluation software. Finally, full-length IgG from the lead clone was expressed in HEK293 cells and evaluated in binding and functional assays.

As shown in Table 3, 44 hits were analyzed by SPR, and 7 beneficial mutants were identified to exhibit better binding affinity (ie >2 times the affinity of the WT antibody, as shown in Table 4).

The top 10 combinatorial mutations with improved binding affinity (>10-fold the affinity of the WT antibody) are listed in Table 5. Five lead clones (A034, A035, A037, A038 and A039) were selected for full-length antibody production. 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 combinations of beneficial mutants with enhanced affinity

序号serial number	克隆编号clone number	ka(1/Ms)ka(1/Ms)	kd(1/s)kd(1/s)	KD(M)KD(M)	比值(kd)Ratio (kd)	序列分析Sequence analysis
11	A034A034	1.31E+051.31E+05	4.24E-054.24E-05	3.22E-103.22E-10	28.0728.07	VL-S31F/VL-A34E/VH-F27LVL-S31F/VL-A34E/VH-F27L
22	A035A035	1.43E+051.43E+05	3.19E-053.19E-05	2.24E-102.24E-10	37.3037.30	VL-S31F/VL-A34E/VH-F27I/VH-S99AVL-S31F/VL-A34E/VH-F27I/VH-S99A
33	A036A036	1.07E+051.07E+05	6.91E-056.91E-05	6.48E-106.48E-10	17.2217.22	VL-A34D/VH-F27I/VH-S99AVL-A34D/VH-F27I/VH-S99A
44	A037A037	1.18E+051.18E+05	1.00E-061.00E-06	8.47E-128.47E-12	1190.001190.00	VL-S31F/VL-A34D/VH-F27LVL-S31F/VL-A34D/VH-F27L
55	A038A038	7.82E+047.82E+04	5.67E-055.67E-05	7.25E-107.25E-10	20.9920.99	VL-A34E/VH-F27IVL-A34E/VH-F27I
66	A039A039	1.15E+051.15E+05	3.00E-053.00E-05	2.60E-102.60E-10	39.6739.67	VL-A34D/VH-F27IVL-A34D/VH-F27I
77	A040A040	7.70E+047.70E+04	8.00E-058.00E-05	1.04E-091.04E-09	14.8814.88	VL-A34E/VH-F27LVL-A34E/VH-F27L
88	A041A041	1.53E+051.53E+05	6.35E-056.35E-05	4.15E-104.15E-10	18.7418.74	VL-S31F/VL-A34D/VH-F27V/VH-S99AVL-S31F/VL-A34D/VH-F27V/VH-S99A
99	A042A042	7.90E+047.90E+04	1.06E-041.06E-04	1.34E-091.34E-09	11.2311.23	VL-A34E/VH-F27L/VH-S99AVL-A34E/VH-F27L/VH-S99A
1010	A043A043	1.28E+051.28E+05	6.29E-056.29E-05	4.91E-104.91E-10	18.9218.92	VL-S31F/VL-A34E/VH-F27LVL-S31F/VL-A34E/VH-F27L
the	C056-WTC056-WT	6.66E+046.66E+04	1.19E-031.19E-03	1.78E-081.78E-08	1.001.00	the

Example 2. Affinity Evaluation of Anti-IL-18Rβ Antibody

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

Table 6 summarizes the binding kinetics data for anti-IL-18Rβ antibodies. The binding affinities of A035, A037 and A039 were in the range of 0.72-0.98nm, which was 16-22 times higher than that of the parental antibody WT (15.8nm).

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

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

3×10 ⁵ KG-1 cells (ATCC, #CCL-246) were seeded into each well of a 96-well plate. Serial dilutions of the antibody prepared in Example 1 were added to the wells prior to stimulation with 10 ng/mL IL-18 for 1 h. After 24 hours of incubation, cells were pelleted by centrifugation and IFN-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 tested in at least 3 replicates and the IC50 for each antibody is summarized in Table 7.

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

抗体Antibody	IC50(nM)IC50(nM)
WTWT	8.148.14
A035A035	0.260.26
A037A037	0.270.27
A039A039	0.590.59

Representative experimental results are shown in Figure 2, A035, A037 and A039 antibodies were approximately 20 times more potent than WT antibody.

Example 4. Anti-IL-18Rβ Antibody Blocks IL-18-Stimulated Activity of IFN-γ Release from Human Peripheral Blood Mononuclear Cells

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

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

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content 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 appended claims of the present application.

Claims

An antibody against IL-18Rβ or an antigen-binding fragment thereof, characterized in that it comprises:

(a) Heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of the CDRH1, CDRH2, and CDRH3 are shown in SEQ ID NO: 2, 3, and 4, respectively, or shown in SEQ ID NO: 10, 3, and 11, respectively or as shown in SEQ ID NO:2, 3 and 11 respectively; and/or

(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 as SEQ ID NO: 13, 7 and 8 Shown, or respectively as shown in SEQ ID NO:16, 7 and 8, or respectively as shown in SEQ ID NO:22, 7 and 8.

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.
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 heavy chain variable region as shown in SEQ ID NO: 1, 9 or 14. The light chain variable region shown in ID NO: 5, 12, 15 or 17.
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: The wild-type light chain variable region shown at 20 may include beneficial mutations selected from the group consisting of F27L, F27I, F27V, S99A, S31F, A34D, A34E, or combinations thereof.
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 based on SEQ ID NO: 20 The wild-type light chain variable region shown 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.
The antibody or antigen-binding fragment thereof according to 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.
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 ).
The antibody or its antigen-binding fragment 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 its antigen-binding fragment are respectively as SEQ ID NO:9 and SEQ ID NO:9. 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 shown in SEQ ID NO:14 and as shown in SEQ ID NO: 17, or as shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively.
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.
A kind of recombinant protein, it is characterized in that, described 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.
A nucleotide molecule, characterized in that the nucleotide molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1-8, and/or the recombinant protein according to claim 9 .
An expression vector, characterized in that the expression vector contains the nucleotide molecule according to claim 10.
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.
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 includes:

Heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of CDRH1, CDRH2, and CDRH3 are shown in SEQ ID NO: 2, 3, and 4, respectively, or shown in SEQ ID NO: 10, 3, and 11, respectively, or as shown in SEQ ID NO:2, 3 and 11 respectively; and/or

The light chain variable region of the scFv comprises:

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.
An engineered immune cell, characterized in that the engineered immune cell expresses an exogenous CAR according to claim 13.
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).
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 labels, drugs, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof.
A kind of pharmaceutical composition, is characterized in that, described 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.
A use of 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 9 The engineered immune cell according to claim 14, or the immunoconjugate according to 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.
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.
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.