CN117177999A - Antibody targeting IL-18 Rbeta and application thereof - Google Patents

Antibody targeting IL-18 Rbeta and application thereof Download PDF

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CN117177999A
CN117177999A CN202380011140.6A CN202380011140A CN117177999A CN 117177999 A CN117177999 A CN 117177999A CN 202380011140 A CN202380011140 A CN 202380011140A CN 117177999 A CN117177999 A CN 117177999A
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antibody
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binding fragment
antigen
cancer
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CN117177999B (en
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杜勇
陈永锋
刘淑素
张玉华
孔德升
卢小容
吴瑶
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Hejing Pharmaceutical Technology Shanghai Co ltd
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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

Antibody targeting IL-18 Rbeta and application thereof
The invention belongs to the field of biological medicine, and particularly relates to an antibody specifically targeting IL-18RP, an antigen binding fragment thereof, a preparation method and application thereof. Background art Interleukin 18 (IL-18), also known as an interferon-Y inducer, regulates many types of immune cell-mediated inflammatory responses oIL-18 not only activates Thl cells and NK cells to promote IFN-Y release, but also regulates Th2, thl7 and macrophage activation, which is an important inflammatory promoter. IL-18 has been found by many studies to be closely associated with various autoimmune diseases such as hemophagocytic lymphoproliferative disorder (Hemophagocytic lymphohistiocytosis, HLH), macrophage activation syndrome, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (Systemic juvenile idiopathic arthritis, sJIA), systemic red Bai Langchuang (Systemic lupus erythematosus, SLE), and inflammatory enteritis.
IL-18 receptors are heterodimeric transmembrane proteins that consist of a ligand-binding IL-18R alpha (IL-18 Ra) subunit and an IL-18R beta (IL-18R &) subunit that is critical for functional signaling.
After IL-18 is hydrolyzed to an active form by the protease Caspas-1, the IL-18 receptor acting on the cell surface activates the downstream pro-inflammatory signaling pathway oIL-18 to bind with lower affinity to IL-18Ra first to form a dimer, but does not transmit a signal, and only this dimer binds to IL-18RP to form a high affinity receptor complex that transmits a pro-inflammatory signal downstream. Blocking the formation of ternary complexes of IL-18 and its receptor, and effectively inhibiting the inflammatory signaling pathway downstream of IL-18, is a promising approach to the current treatment of autoimmune and inflammatory diseases.
IL-18Ra is widely expressed and can also bind to the inflammation inhibitor IL-37, which is complex in the regulation of inflammation. IL-18RP is specifically expressed in immune cells and is a receptor necessary for IL-18 signaling, and thus, IL-18 RP-targeted antibodies are capable of specifically and effectively inhibiting IL-18 signaling. Targeting IL-18 itself is affected in many ways and the mechanism of action is complex due to the high affinity decoy receptor IL-18 BP naturally occurring in vivo, as well as the presence of membrane-bound forms of IL-18. In combination, targeting IL-18RP antibodies has advantages in specificity, efficacy and safety over other components of the IL-18 signaling pathway. Thus, there is a strong need in the art to develop antibodies that specifically target IL-18RP for the treatment of autoimmune and inflammatory diseases, among others. The present invention aims to provide a novel therapeutic means for autoimmune diseases, inflammatory diseases, and the like. It is a further object of the present invention to provide an antibody against IL-18RP and uses thereof. In a first aspect of the invention, an antibody or antigen binding fragment thereof is provided against IL-18 RP. In another preferred embodiment In (3), the antibody or antigen binding fragment thereof is capable of specifically binding IL-18Rp o In another preferred embodiment, the antibody or antigen binding fragment thereof may comprise a beneficial mutation selected from the group consisting of the wild-type heavy chain variable region shown in SEQ ID NO. 18 and/or the wild-type light chain variable region shown in SEQ ID NO. 20: F27L, F27L F V, S99A, S31F, A34D, A34E, or a combination thereof. In another preferred embodiment, the antibody or antigen binding fragment thereof further comprises optionally added, deleted, modified andin another preferred embodiment, the antibody or antigen binding fragment thereof may comprise a beneficial mutation based on the wild-type heavy chain variable region shown in SEQ ID NO. 18 selected from the group consisting of: F27L, F27L F V, S99A, or a combination thereof. In another preferred embodiment, the antibody or antigen binding fragment thereof may comprise a beneficial mutation based on the wild-type light chain variable region shown in SEQ ID NO. 20 selected from the group consisting of: S31F, A34D, A E, or a combination thereof. In another preferred embodiment, the antibody or antigen binding fragment thereof has a beneficial mutation selected from the group consisting of the wild-type heavy chain variable region shown in SEQ ID NO. 18 and/or the wild-type light chain variable region shown in SEQ ID NO. 20, or a combination thereof:
S31F、 A34E、 F27L；
S31F、 A34E、 F27L S99A；
A34D 、 F27I、 S99A；
S31F、 A34D、 F27L；
A34E 、 F27I；
A34D 、 F27I；
A34E 、 F27L；
S31F、 A34D、 F27V、 S99A；
a34E, F27L, S a; or (b)
In another preferred embodiment, S31F, A34E, F Lo, said antibody or antigen binding fragment thereof has a beneficial mutation selected from the group consisting of: F27I, S99A, or a combination thereof; and/or the wild-type light chain variable region shown based on SEQ ID NO. 20 has a beneficial mutation selected from the group consisting of: S31F, A E, or a combination thereof. In another preferred embodiment, the antibody or antigen binding fragment thereof has a beneficial mutation based on the wild-type heavy chain variable region shown in SEQ ID NO. 18 selected from the group consisting of: F27L; and/or the wild-type light chain variable region shown based on SEQ ID NO. 20 has a beneficial mutation selected from the group consisting of: S31F, A D, or a combination thereof. In another preferred embodiment, the antibody or antigen binding fragment thereof has a beneficial mutation based on the wild-type heavy chain variable region shown in SEQ ID NO. 18 selected from the group consisting of: F27I; and/or the wild-type light chain variable region shown based on SEQ ID NO. 20 has a beneficial mutation selected from the group consisting of: a34D. In another preferred embodiment, the antibody or antigen binding fragment thereof comprises: (a) The amino acid sequences of the heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3 are respectively shown as SEQ ID NOs 0:2, 3 and 4, or are respectively shown as SEQ ID NOs 10.3 and 11, or are respectively shown as SEQ ID NOs 0:2, 3 and 11; and
(b) The amino acid sequences of the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 are shown as SEQ ID NOs 0:6, 7 and 8 respectively or as SEQ ID NOs 13 respectively>7 and 8, or SEQ ID NOS 16, 7 and 8, respectively, or SEQ ID NOS 22, 7 and 8, respectively. In another preferred embodiment, any of the amino acid sequences described above further comprises a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one (e.g., 1-4) amino acid residue and that retains the ability to specifically bind to IL-18 RP. In another preferred embodiment, the antibody or antigen binding fragment thereof has 6 CDRs (CDRH 1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL 3) of an a034, a035, a037, a038, or a039 antibody in table 1. In another preferred embodiment, the derivative sequence having at least one amino acid added, deleted, modified and/or substituted and capable of retaining the ability to specifically bind to IL-18RP is an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or sequence identity . In another preferred embodiment, the CDRK CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR 4. In another preferred embodiment, the antibody or antigen binding fragment thereof further comprises a framework region FRo 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 light chain variable region as shown in SEQ ID NO. 5, 12, 15 or 17. In another preferred embodiment, the amino acids of the heavy chain variable region and the light chain variable region of the antibody or antigen binding fragment thereofIn another preferred embodiment, the antibody or antigen binding fragment thereof is a murine antibody, chimeric antibody, humanized antibody or fully human antibody. In another preferred example, the antibody or antigen binding fragment thereof may comprise 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, disulfide-linked Fv (dsFv), or sdAbo in a second aspect of the invention, a recombinant protein is provided having:
The antibody or antigen-binding fragment thereof according to the first aspect of the invention; and
(ii) Optionally a tag sequence to assist expression and/or purification. In another preferred embodiment, the tag sequence comprises an Fc tag, an HA tag, a GGGS sequence ruffian U, FLAG tag, a Myc tag, a 6His tag, or a combination thereof. In another preferred embodiment, the recombinant protein specifically binds IL-18Rp 0 In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein. In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer. In a third aspect of the present invention, there is provided a nucleic acid molecule encoding a protein selected from the group consisting of: an antibody according to the first aspect of the inventionA body or antigen binding fragment thereof, or a recombinant protein according to the second aspect of the invention. In another preferred embodiment, the nucleic acid of the invention may be RNA, DNA or cDNA. In another preferred example, the nucleic acid molecule comprises a heavy chain nucleic acid sequence and a light chain nucleic acid sequence. In another preferred embodiment, the heavy chain nucleic acid sequence is selected from the group consisting of SEQ ID NOs 36, 38, 40, 42, 44, 46, or a combination thereof. In another preferred embodiment, the light chain nucleic acid sequence is selected from the group consisting of SEQ ID NOs 37, 39, 41, 43, 45, 47, or a combination thereof. In another preferred embodiment, the nucleic acid molecule comprises: a heavy chain nucleic acid sequence shown as S EQ ID NO. 36 and a light chain nucleic acid sequence shown as SEQ ID NO. 37; or a heavy chain nucleic acid sequence shown as SEQ ID NO. 38 and a light chain nucleic acid sequence shown as SEQ ID NO. 39; or a heavy chain nucleotide sequence shown as SEQ ID NO. 40 and a light chain nucleotide sequence shown as SEQ ID NO. 41; or a heavy chain nucleotide sequence shown as SEQ ID NO. 42 and a light chain nucleotide sequence shown as SEQ ID NO. 43; or a heavy chain nucleic acid sequence shown as SEQ ID NO. 44 and a light chain nucleic acid sequence shown as SEQ ID NO. 45; or a heavy chain nucleotide sequence shown as SEQ ID NO. 46 and a light chain nucleotide sequence shown as SEQ ID NO. 47. In a fourth aspect of the present invention, there is provided an expression vector comprising the alfalfa molecule of 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 a combination thereof. In another preferred embodiment, the expression vector is selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJMK pHCMV, pLBS. CAG, pHR, pLV, etc. In another preferred embodiment, the expression vector further comprises a gene selected from the group consisting of: promoters, transcription enhancing elements WPRE, long terminal repeat LTR, and the like. In a fifth aspect of the invention there is provided a host cell comprising an expression vector according to the fourth aspect of the invention, or a whole genome thereof The nucleic acid molecules of the third aspect of the invention are incorporated. In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell. In another preferred embodiment, the host cell is selected from the group consisting of: coli, yeast cells, mammalian cells. In a sixth aspect of the invention there is provided a chimeric antigen receptor CAR, the antigen binding region scFv fragment of the CAR being a binding region that specifically binds to IL-18RP, and the heavy chain variable region of the scFv comprising: the amino acid sequence of the CDRHk CDRH2 and CDRH3 is shown as SEQ ID NO. 2>3 and 4, or respectively as 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: the amino acid sequences of the light chain complementarity determining regions CDRL1, CDRL2 and CDRL3 are shown as SEQ ID NO. 6, 7 and 8, respectively, or as SEQ ID NO. 13, 7 and 8, respectively, or as SEQ ID NO. 16, 7 and 8, respectively, or as SEQ ID NO. 22, 7 and 8, respectively. In another preferred embodiment, the CAR further comprises a signal peptide. In another preferred embodiment, the CAR further comprises an additional exogenous protein. In another preferred embodiment, the CAR has a structure represented by formula la:
In the formula L-scFv-H-TM-C-CD3] (la),
l is a none or signal peptide sequence; scFv is a domain that specifically binds IL-18R 3;
h is a none or dumpling chain region;
TM is a transmembrane domain;
c is a costimulatory signaling domain;
CD3] is a cytoplasmic signaling sequence derived from CD3 (including wild type, or mutant/modified versions thereof); the linker peptide or peptide bond. In another preferred embodiment, the L is selected from the group consisting of signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or a mutant/modification thereof, or a combination thereof. In another preferred embodiment, the scFv targets IL-18R6. In another preferred embodiment, the scFv is an IL-18R0 antibody or antigen-binding fragment thereof.In another preferred embodiment, the H is selected from the string 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: CD28, CD3 epsilon>CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or a mutant/modification thereof, or a combination thereof. In another preferred embodiment, the C is selected from the group consisting of the costimulatory domains of: 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD-1, daplo, LIGHT, NKG2C, In a seventh aspect of the invention, there is provided an engineered immune cell expressing an exogenous CAR according to the sixth aspect of the invention. In another preferred embodiment, the engineered immune cell is selected from the group consisting of:
(i) Chimeric antigen receptor Qiu T cells (CAR-T cells);
(ii) Chimeric antigen receptor-modifying 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 comprise autologous or allogeneic T cells, secondary T cells, NKT cells, NK cells, or a combination thereof. In another preferred embodiment, the engineered immune cell is a CAR-T cell. In an eighth aspect of the invention, there is provided a method of producing an antibody or antigen binding fragment thereof directed against IL-18RP, comprising the steps of:
(a) Culturing a host cell according to the fifth aspect of the invention under suitable conditions, thereby obtaining a culture comprising an antibody or antigen binding fragment thereof to IL-18 RP;
(b) Isolating and/or recovering the antibody or antigen binding fragment thereof directed against IL-18RP from the culture; and
(c) Optionally, purifying and/or modifying the antibody or antigen binding fragment thereof against IL-18RP obtained in step (b). In a ninth aspect of the invention there is provided an immunoconjugate comprising:
(a) An antibody moiety comprising an antibody or antigen binding fragment thereof according to the first aspect of the invention, and
(b) A coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, radionuclide, enzyme, gold nanoparticle/nanorod, nanomagnetic particle, viral coat protein, or VLP, or a combination thereof. In another preferred embodiment, said moiety (a) is coupled to said coupling moiety by a chemical bond or a linker. In another preferred embodiment, the radionuclide comprises:
(i) A diagnostic isotope selected from the group consisting of: tc-99m, ga-68, F-18, 1-123, I-125, 1-131, in-111, ga-67, cu-64, zr-89, C-l l, lu-177, re-188, or a combination thereof; and/or
(ii) A therapeutic isotope selected from the group consisting of: lu-177, Y-90, ac-225, as-21K Bi-212, bi-213, cs-137, cr-5K Co-60, dy-165, er-169, fm-255, au-198, ho-166, I-125, 1-131, 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 embodiment, the coupling moiety is a drug or a toxin. In another preferred embodiment, the agent is a drug targeted to treat an IL-18 related disorder. In another preferred embodiment, the IL-18 related disorder comprises a disorder of high IL-18 expression, a disorder associated with aberrant IL-18 receptor expression or a disorder associated with high activity of IL-18 signaling pathway, or a disorder mediated by IL-18 RP. In another preferred embodiment, the IL-18 related disorder is an autoimmune disorder or an inflammatory disorder. In another preferred embodiment, the IL-18 related disorder 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, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemopoietic lymphocytosis, giant cell activation syndrome, systemic lupus erythematosus, suppurative arthritis, gangrene, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sa), inflammatory disease (IBD), stevens disease (aoil), sarcoidosis (aoil), chronic pulmonary disease, or a combination thereof. In another preferred embodiment, 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, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal 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 agent is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, 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 including, for example, auristatin (Auristatins), camptothecins (Camptothecins), duocarmycin/duocarmycin (Duocarmycins), etoposides, maytansinoids (Maytansines) and Maytansinoids (Maytansinoids) (e.g., DM1 and DM 4), taxanes (Taxanes), benzodiazepines (Benzodiazepines) or Benzodiazepines-containing drugs (Benzodiazepine containing drugs) (e.g., benzoo [1,4] Benzodiazepines (PBDs), oronoise H Benzodiazepines (indoxazediazepines) and benzobenzodiazepines (oxazodiazepines), vinca alkaloids (vilos), 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), aureomycin, mestaneol, ricin a-chain, combretastatin, docamicin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cis-box, cc1065, deserialized ethidium, mitomycin, etoposide or tenopof (Tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthrax-dione, actinomycin, diphtheria toxin, pseudomonas Exotoxin (PE) A, PE, abrin a chain, cucurbitacin a chain, a-sarcina, gelonin, mitoxin (Mitogellin), restrictocin (retctocin), phenomycin, enomycin, curcin (Curicin), crotonin, calicheamicin, saporin (Sapaonaria officinalis), or a combination thereof. In another preferred embodiment, the coupling moiety is a detectable label. In another preferred embodiment, the coupling moiety is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)) or any form of nanoparticle. In another preferred embodiment, the immunoconjugate comprises: multivalent (e.g. bivalent) antibodies or antigen binding fragments thereof according to the first aspect of the invention. In another preferred embodiment, the multivalent means that the same or different antibodies or antigen binding fragments thereof according to the first aspect of the invention comprise a plurality of repeats in the amino acid sequence of the immunoconjugate. In a tenth aspect of the present invention, there is provided a pharmaceutical composition comprising:
The method comprises the following steps of (1) preparing active ingredients: an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an engineered immune cell according to the seventh aspect of the invention, or an immunoconjugate according to the ninth aspect of the 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 group consisting of: injection and freeze-dried preparation. In another preferred embodiment, the pharmaceutical composition comprises 0.01-99.99% of the antibody or antigen binding fragment thereof according to the first aspect of the invention, or the recombinant protein according to the second aspect of the invention, or the engineered immune cell according to the seventh aspect of the invention, or the immunoconjugate according to the ninth aspect of the invention, or a combination thereof, and 0.01-99.99% of a pharmaceutically acceptable carrier, said percentages being mass percentages of the pharmaceutical composition. In another preferred embodiment, the concentration of the engineered immune cells in the active ingredient is 1x 103-1 x 108 cells/mL, preferably 1x104-1 x1 () 7 cells/mL. In an eleventh aspect of the invention there is provided the 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 invention, or the recombinant protein according to the second aspect of the invention, or the engineered immune cell according to the seventh aspect of the invention, or the immunoconjugate according to the ninth aspect of the invention, or a combination thereof, the active ingredients are used for the preparation of:
(a) Medicaments for preventing and/or treating IL-18 related diseases;
(b) Reagents for detecting IL-18 related disorders. In another preferred embodiment, the reagent is a diagnostic reagent, preferably a test strip or a test plate. In another preferred embodiment, the diagnostic reagent is for: detecting the IL-18RP protein or fragment thereof in the sample. In another preferred embodiment, the IL-18 related disorder comprises a disorder of high IL-18 expression, a disorder associated with aberrant IL-18 receptor expression or a disorder associated with high activity of IL-18 signaling pathway, or a disorder mediated by IL-18 RP. In another preferred embodiment, the IL-18 related disorder is an autoimmune disorder or an inflammatory disorder. In another preferred embodiment, the IL-18-expressing disorder 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, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytic lymphohistiocytosis, giant cell activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyodermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (jia), inflammatory Bowel Disease (IBD), adult steven's disease (aoil-disease), chronic Obstructive Pulmonary Disease (COPD), or a combination thereof in the market of chronic stage. In another preferred embodiment, 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, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof. In a twelfth aspect of the invention, there is provided a method for detecting IL-18RP protein or fragments thereof in a sample in vitro, said method comprising the steps of:
(1) Contacting the sample in vitro with an antibody or antigen binding fragment thereof according to the first aspect of the invention;
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of a corresponding target of the IL-18RP protein or fragment thereof in the sample. In another preferred embodiment, the detection comprises diagnostic or non-diagnostic. In a thirteenth aspect of the invention, there is provided a kit comprising (1) a first container comprising an antibody or antigen binding fragment thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an engineered immune cell according to the seventh aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention, or a pharmaceutical composition according to the tenth aspect of the invention, or a combination thereof; and/or
(2) A second container containing a second antibody against the contents of the first container; alternatively, the kit contains a test plate comprising: a substrate (support) and a test strip comprising an antibody or antigen binding fragment thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an engineered immune cell according to the seventh aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention, or a pharmaceutical composition according to the tenth aspect of the invention, or a combination thereof. In another preferred embodiment, the kit further comprises a kit for non-invasively detecting IL-18 expression in a subject according to the instructions. In another preferred embodiment, the kit is used for detection of IL-18 related diseases. In another preferred embodiment, the IL-18 related disorder comprises a disorder of high IL-18 expression, a disorder associated with aberrant IL-18 receptor expression or a disorder associated with high activity of IL-18 signaling pathway, or a disorder mediated by IL-18 RP. In another preferred embodiment, the IL-18 related disorder 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, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemopoietic lymphocytosis, giant cell activation syndrome, systemic lupus erythematosus, suppurative arthritis, gangrene, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sa), inflammatory disease (IBD), steven's disease (aoil-sd's), chronic pulmonary disease, or a combination thereof. In another preferred embodiment, 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, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof. In a fourteenth aspect of the invention, there is provided a method of preventing and/or treating an IL-18-associated disease, the method comprising: administering to a subject in need thereof an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, or a recombinant protein as described in the second aspect of the invention, or an engineered immune cell as described in the seventh aspect of the invention, or an immunoconjugate as described in the ninth aspect of the invention, or a pharmaceutical composition as described in the tenth aspect of the invention, or a combination thereof. In another preferred embodiment, the subject comprises a mammal, such as a human. In another preferred embodiment, the IL-18 related disorder comprises a disorder of high IL-18 expression, a disorder associated with aberrant IL-18 receptor expression or a disorder associated with high activity of IL-18 signaling pathway, or a disorder mediated by IL-18R 0. In another preferred embodiment, the IL-18 related disorder is an autoimmune disorder or an inflammatory disorder. In another preferred embodiment, the IL-18 related disorder 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, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytosis, giant cell activating syndrome, systemic lupus erythematosus, suppurative arthritis, gangrene, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sa), inflammatory disease (IBD), stevens disease (aoil), sarcoidosis (stevens), chronic Obstructive Pulmonary Disease (COPD), or a combination thereof. In another preferred embodiment, 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, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof. In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from the subject (autologous cells). In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from a healthy individual (allogeneic cells). In another preferred embodiment, the methods described can be used in combination with other therapeutic 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, there is provided a diagnostic method for an IL-18-associated disease comprising the steps of:
Obtaining a sample from a subject, contacting said sample with an antibody or antigen-binding fragment thereof according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or an engineered immune cell according to the seventh aspect of the invention, or an immunoconjugate according to the ninth aspect of the invention, or a pharmaceutical composition according to the tenth aspect of the invention; and
(ii) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates that the subject is a definitive patient for an IL-18 related disorder. In another preferred embodiment, the sample is a blood sample or a pharyngeal swab sample, or a sample in another tissue organ. In another preferred embodiment, the IL-18 related disorder comprises a disorder of high IL-18 expression, a disorder associated with aberrant IL-18 receptor expression or a disorder associated with high activity of IL-18 signaling pathway, or a disorder mediated by IL-18 RP. In another preferred embodiment, the IL-18 related disorder is an autoimmune disorder or an inflammatory disorder. In another preferred embodiment, the IL-18 related disorder 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, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II diabetes, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemopoietic lymphocytosis, giant cell activation syndrome, systemic lupus erythematosus, suppurative arthritis, gangrene, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sa), inflammatory disease (IBD), stevens disease (aoil), sarcoidosis (aoil), chronic pulmonary disease, or a combination thereof. In another preferred embodiment, the tumor is selected from the group consisting of: bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, and lymph Tumors, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, thyroid cancer, or a combination thereof. It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein. FIG. 1 shows sequence information for an IL-18R0 WT antibody. FIG. 2 shows a 24h concentration-response curve of anti-IL-18 RP antibody (IgGl) inhibiting IFN-y in KG-1 cells. FIG. 3 shows a 24h concentration-response curve of anti-IL-18 RP antibody (IgG 4) inhibiting IFN-y in KG-1 cells. FIG. 4 shows a 24h concentration-response curve (parallel assay) of anti-IL-18 RP antibody (IgGl) inhibiting IFN-y in KG-1 cells. FIG. 5 shows a 48h concentration-response curve of anti-IL-18 RP antibody (IgGl) inhibiting IFN-y in human PBMC cells. FIG. 6 shows a 72h concentration-response curve of anti-IL-18 RP antibody (IgG 4) inhibiting IFN-y in human PBMC cells. Detailed description of the inventionthe present inventors have conducted extensive and intensive studies and, through a large number of screening, developed for the first time a high affinity IL-18 RP-targeting antibody and antigen binding fragments thereof. Specifically, the present invention constructs a precise mutation library using a WT antibody of IL-18RP as a starting material for improving affinity, and introduces saturation mutations into all 73 residues of the six CDR regions of the WT antibody. After further screening, selected mutants were ranked by dissociation rate constants determined by surface plasmon resonance, and combinatorial libraries were constructed with random combinations of beneficial mutations, resulting in antibodies of the invention and antigen binding fragments thereof with increased binding affinity. The antibody and antigen binding fragment thereof for targeting IL-18R0, which are developed by the invention, can be used as novel therapeutic means for targeted therapy of diseases related to abnormal expression of IL-18 receptor or related to high activity of IL-18 signaling pathway. The term as used herein, the terms "antibody of the invention", "antibody of the invention against IL-18 RP", "antibody against IL-18 RP", "IL-18 Antibodies to RJ3 and "IL-18RJ3 antibodies" have the same meaning and are used interchangeably to refer to antibodies that specifically recognize and bind to IL-18RP proteins, including human IL-18RP proteins. Preferably, the antibody numbering and corresponding sequence numbering of the invention is shown in table 1 below.Preferably, the a035 antibody, a037 antibody, a039 antibody of the present invention have two types of IgGl and IgG4, respectively, wherein:
a035 The antibody (IgGl) has a heavy chain sequence shown as SEQ ID NO. 30 and a light chain sequence shown as SEQ ID NO. 31; the A037 antibody (IgGl) has a heavy chain sequence shown as SEQ ID NO. 32 and a light chain sequence shown as SEQ ID NO. 33; the A039 antibody (IgGl) has the heavy chain sequence shown in SEQ ID NO: 34 and the light chain sequence shown in SEQ ID NO: 35. The term "antibody" herein is intended to include full length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. Full length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being 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 is composed of three domains, namely CHI, CH2 and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The light chain constant region is composed of one domain CL. VH and VL regions can also be divided into hypervariable regions called Complementarity Determining Regions (CDRs) which are separated by more conserved Framework Regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to blanket-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody can mediate binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the classical complement system (Clq) o, the techniques herein The term "antigen-binding fragment" (or antigen-binding portion) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., an IL-18RP protein). It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of full length antibodies. Examples of binding fragments included in an "antigen-binding portion" of an antibody include a first Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH 1; (ii) F (ab) , ) 2 A fragment comprising a bivalent fragment of two Fab fragments disulfide-bridged in the region of the Bright chain; (iii) an Fd fragment consisting of VH and CH 1; (iv) Fv fragments consisting of antibody single arm VL and VH; (v) a dAb fragment consisting of VH; (vi) an isolated Complementarity Determining Region (CDR); and (vii) nanobodies, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, by recombinant methods, via a synthetic linker that makes them single protein chains, in which the VL and VH regions pair to form monovalent molecules) (known as single chain Fc (scFv)). These single chain antibodies are also intended to be included in the term meaning. 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 the whole antibody. As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are in a substantially 3-folded configuration, joined by three CDRs forming a linker loop, which in some cases may form part b of the folded structure. The CDRs in each chain are held closely together by 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 public No. 91-3242, vol. I, pp. 647-669 (1991)). Constant regions are not directly Involved in binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cytotoxicity, of the antibodies. Those skilled in the art can define the amino acid sequence boundaries of CDRs using any of a variety of known numbering schemes, including those set forth in the following documents: kabat et al, supra ("Kabat" numbering scheme); al-Lazikani et Al, 1997, J.mol.biol.,. 273:927-948 ("Chothia" numbering scheme); lefhmc et al, dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme). Immunoconjugates and fusion expression products include, as known to those of skill in the art: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules with antibodies or fragments thereof of the present invention. The invention also includes cell surface markers or antigens that bind to the antibodies or fragments thereof directed against IL-18 RP. 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" is used interchangeably with "complementarity determining region (Complementarity determining region, CDR). In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDRH2, and CDRH3. In a preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above. In a preferred embodiment of the invention, the light chain variable region of the antibody comprises three complementarity determining regions CDRL1, CDRL2, and CDRL3. In a preferred embodiment of the invention, the light chain of the antibody comprises the light chain variable region and the light chain constant region described above. In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably to refer to a polypeptide that specifically binds to an IL-18RP protein, such as a protein or polypeptide having a heavy chain variable region. They may or may not contain an initiating methionine. The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the invention includes a tool There are any proteins or protein conjugates and fusion expression products (i.e., immunoconjugates and fusion expression products) comprising a heavy chain of a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention. In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the variable region of the heavy chain, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the 3 folds formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody. The variable regions of the heavy chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to antigens. Thus, the invention includes those molecules having antibody heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein. The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the invention. The polypeptide fragments, derivatives or analogues of the invention may be polypeptides which comprise a substitution of one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, which may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound, such as a compound which increases the half-life of the polypeptide, for example polyethylene glycol, or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence for purifying the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein. The antibodies of the invention refer to polypeptides having IL-18RP protein binding activity that include the CDR regions described above. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids that are closely related or similar in performance typically does not alter the function of the protein. As another example, adding one or more amino acids at the C-terminal and/or N-terminal end will not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention. The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutant variants, proteins encoded by DNA capable of hybridizing to the DNA encoding the antibodies of the invention under conditions of high or low stringency, and polypeptides or proteins obtained using antisera raised against the antibodies of the invention. The invention also provides other polypeptides, such as fusion proteins comprising an antibody or fragment thereof. In addition to nearly 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 the antibody of the invention. In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table 2. TABLE 2The invention also provides polynucleic acid molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The alfalfa of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. Polynucleic acids encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences. The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include polynucleotides to which coding and/or non-coding sequences are added. The invention also relates to polynucleic acids which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to a polynucleic alfalfa which hybridizes under stringent conditions with the polynucleic acids described in the present invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, such as 0.2XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ℃ and the like during hybridization; or the hybridization occurs only when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleic acid has the same biological function and activity as the mature polypeptide. The full-length nucleic acid sequence of the antibody or a fragment thereof of the present invention can be usually obtained by PCR amplification, recombinant methods or artificial synthesis. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the coding sequences and tables of the heavy chains can also be used The tags (e.g., 6 His) are 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. This is usually accomplished by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form. At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis. The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; a bacterial cell of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, C0S7, animal cells of 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 prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaC12 method using procedures well known in the art. Another method is to use MgC12o and if desired transformation can also be carried out by electroporation. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc. The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. When the host is fine After cells are grown to the appropriate cell density, the selected promoters are induced by a suitable method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time. The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, 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 coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above. Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product. Therapeutic agents that may be conjugated or coupled to an antibody of the invention include, but are not limited to: firstly, radionuclide; 2, biotoxin; third, cytokines such as IL-2, etc.; 4, gold nanoparticles/nanorods; fifth, virus particles; sixth, liposome; seventhly, nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and the like.
IL-18, IL-18R and I L-18RP interleukin-18 (IL 18, also known as interferon-y inducer) are proteins that are encoded by the IL-18 gene in humans. The protein encoded by the gene is a pro-inflammatory cytokine. Many cell types, including hematopoietic and non-hematopoietic cells, have the potential to produce IL-18.
The IL-18 receptor consists of an inducible component IL-18Ra (which binds mature IL-18 with low affinity) and a constitutively expressed co-receptor IL-18 RP. IL-18 and ligand receptorsThe binding of bulk IL-18Ra induces the recruitment of IL-18Rp to form a high affinity complex that signals through the toll/interlukin-1 receptor (TIR) domain. The signal domain activates pro-inflammatory processes and MyD88 adaptor proteins of the NF-KB pathway. IL-18 is specifically expressed in immune cells and is a receptor necessary for IL-18 signaling, thus targeting IL-18RP antibodies specifically and effectively inhibits IL-18 signaling. Effective inhibition of the downstream inflammatory signaling pathway of IL-18 is a promising approach to the current treatment of autoimmune and inflammatory diseases. Pharmaceutical compositions the present invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a sterile, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending upon the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intraperitoneal, intravenous, or topical administration. The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibodies (or conjugates thereof) of the invention as described above, together with a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical composition, such as injection and solution, is preferably manufactured under aseptic conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents. When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight And in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner. In the present invention, the antibody or antigen-binding fragment thereof against IL-18RP includes monomers, bivalent bodies (bivalent antibodies), tetravalent bodies (tetravalent antibodies), and/or multivalent bodies (multivalent antibodies), and may be murine, chimeric, humanized or fully human. Typically, the antibodies or antigen binding fragments thereof of the invention comprise a beneficial mutation selected from the group consisting of the heavy chain variable region shown in SEQ ID NO: 18 and/or the light chain variable region shown in SEQ ID NO: 20: f27L, F27I, F V, S99A, S31F, A34D, A34E; the antibody or antigen binding fragment thereof also includes an antibody or antigen binding fragment thereof that is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-4) amino acid and that retains the ability to specifically bind to IL-18 RP. In a preferred embodiment of the invention, 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 shown in SEQ ID NO. 5, 12, 15 or 17. In a preferred embodiment of the invention, the antibody or antigen binding fragment thereof directed against IL-18RP comprises one or more heavy chains having the sequence shown as SEQ ID NO. 24, 26, 28, 30, 32 or 34 and light chains having the sequence shown as SEQ ID NO. 25, 27, 29, 31, 33 or 35. Labeled antibodies in a preferred embodiment of the invention, the antibodies bear a detectable label. More preferably, the marker is selected from the group consisting of: isotopes, colloidal gold labels, colored labels, or fluorescent labels. Colloidal gold labelling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the invention, the antibodies directed against IL-18RP protein are labeled with colloidal gold to provide colloidal gold-labeled antibodies. The antibodies of the invention directed against IL-18RP are capable of binding IL-18RP proteins efficiently. Detection methods the present invention also relates to Methods for detecting IL-18RP proteins or fragments thereof. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving a sample in a medium; detecting the level of IL-18RP protein in the solubilized 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. Kits the present invention also provides a kit comprising an antibody (or fragment thereof) or assay plate of the present invention, which in a preferred embodiment of the present invention further comprises a container, instructions for use, buffers, and the like. The invention also provides a detection kit for detecting the level of IL-18RP protein, which comprises a detection kit for recognizing IL-18R&Antibodies to proteins are used to solubilize the lysis medium of the sample, and universal reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, and the like. The detection kit may be an in vitro diagnostic device. As described above, the antibody of the present invention has a wide range of biological and clinical applications, and its application relates to the treatment of diseases associated with IL-18, and has a plurality of fields such as diagnosis of IL-18 high expression, basic medical research, biological research, etc. One preferred application is for clinical prophylaxis and treatment against IL-18RP proteins. The present invention also provides a method of stimulating an immune response mediated by T cells targeted to a mammalian tumor cell population or tissue, comprising the steps of: administering the CAR-T cells of the invention to a mammal. In one embodiment, the invention includes a class of cell therapies in which autologous T cells (or heterologous donors) from a patient are isolated, activated and genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. This approach results in a very low probability of occurrence of the graft anti-host response, and antigen is recognized by T cells in a non-MHC restricted manner. Furthermore, a CAR-T can treat all cancers that express this antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to sustained control of tumors. In one embodiment, the CAR-T cells of the invention can undergo stable in vivo expansion and can last from months to years. In addition, CAR-mediated immune response The response may be part of an adoptive immunotherapy step in which the CAR-T cells may induce a specific immune response to the highly expressing tumor cells of the antigen recognized by the CAR antigen binding domain. For example, the CAR-T cells of the invention elicit a specific immune response against tumor cells that are highly expressed by IL-18 RP. Treatable cancers include tumors that are not vascularized or have not been substantially vascularized, as well as vascularized tumors. Types of cancers treated with the CARs of the invention include, but are not limited to: gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, etc. In general, cells activated and expanded as described herein are useful in the treatment and prevention of diseases such as tumors. Accordingly, the 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 invention. The CAR-T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or 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, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients 41=1. The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease, or may be determined by clinical trials. When referring to an "immunologically effective amount", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). The pharmaceutical composition comprising the T cells described herein may be 1.4 to IO. A dose of individual cells/kg body weight, preferably 1. A dose of 5 to IO. T cell compositions may also be administered multiple times at these doses. Cells can be prepared by using injection techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng. J. OfMed. 319:1676, 1988) And (3) application. The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly. Administration of the subject compositions may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell compositions of the invention are 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. The composition of T cells can be injected directly into the tumor, lymph node or site of infection. In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously with, or after) any number of relevant therapeutic modalities, including, but not limited to, treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or ertapelizumab therapy for psoriasis patients or other therapy for PML patients. In a further embodiment, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressants such as, for example, huperzine, thiowa , methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously or after) with bone marrow transplantation, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives injection of expanded immune cells of the invention after transplantation. In an additional embodiment, the expanded filaments The cells are administered either pre-operatively or post-operatively. The dose of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio administered to humans may be carried out according to accepted practices in the art. Typically, 1 x 105 to iyio can be administered per treatment or per course of treatment 10 The modified T cells of the invention are administered to a patient, for example, by intravenous infusion. The main advantages of the invention include:
1. the antibody or antigen binding fragment thereof of the IL-18RP can specifically bind to the IL-18RP, has high affinity, and improves the affinity by more than ten times compared with the affinity of WT by 22 times at most.
2. The antibody or antigen binding fragment thereof of the IL-18RP has good IL-18/IL-18R blocking activity and can effectively inhibit the downstream inflammatory signal path of IL-18.
3. The antibodies or antigen binding fragments thereof to IL-18RP of the invention have advantages in specificity, efficacy and safety over other components of the IL-18 signaling pathway.
4. The antibody or antigen binding fragment thereof of the IL-18RP can effectively block the release of IFN-Y in KG-1 cells stimulated by IL-18, and the activity efficacy is 20 times that of WT.
5. The antibody or antigen binding fragment thereof of the IL-18RP can effectively block IFN-y release of human peripheral blood mononuclear cells stimulated by IL-18, and the activity is obviously higher than that of WT.
6. The antibodies or antigen binding fragments thereof to IL-18RP of the invention provide novel methods and techniques for the prevention/diagnosis/treatment of IL-18 related diseases, including but not limited to diseases in which IL-18 is highly expressed, diseases associated with aberrant expression of IL-18 receptors or with high activity of IL-18 signaling pathways, IL-18RP mediated diseases. The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. Amino acid sequence
SEQ ID NO. 1A 035 antibody heavy chain variable region
EVQLVESGGGLVQPGGSLRLSCAASGINLYYSSMHWVRQAPGKGLEWVASIYSSNGR TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARASFSHGYGWYGLDYW GQGTLVTVSS
SEQ ID NO. 2A 035, A039, A038 antibody heavy chain complementarity determining region CDRH1 GINLYYSSMH
SEQ ID NO. 3A 035, A037, A034, A039, A038, C056-WT antibody heavy chain complementarity determining region CDRH2 SIYSSNGRTYYADSVKG
SEQ ID NO. 4A 035 antibody heavy chain complementarity determining region CDRH3
ASFSHGYGWYGLDY
SEQ ID NO. 5A 035, A034 antibody light chain variable region
DIQMTQSPSSLSASVGDRVTITCRASQSVSFAVEWYQQKPGKAPKLLIYSASSLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIK
SEQ ID NO. 6A 035, A034 antibody light chain complementarity determining region CDRL1 RASQSVSFAVE
SEQ ID NO. 7A 035, A037, A034, A039, A038, C056-WT antibody light chain complementarity determining region CDRL2 SASSLYS
SEQ ID NO. 8A 035, A037, A034, A039, A038, C056-WT antibody light chain complementarity determining region CDRL3 QQYGYHDAGLIT
SEQ ID NO. 9A 037, A034 antibody heavy chain variable region
EVQLVESGGGLVQPGGSLRLSCAASGLNLYYSSMHWVRQAPGKGLEWVASIYSSNGR TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYW GQGTLVTVSS SEQ ID NO. 10A 037, A034 antibody heavy chain complementarity determining region CDRH1 GLNLYYSSMH
11A 037, A034, A039, A038, C056-WT antibody heavy chain complementarity determining region CDRH3 SSFSHGYGWYGLDY
SEQ ID NO. 12A 037 antibody light chain variable region
DIQMTQSPSSLSASVGDRVTITCRASQSVSFAVDWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIK
CDRL1 of the light chain complementarity determining region of the antibody of SEQ ID NO. 13A 037
RASQSVSFAVD
SEQ ID NO. 14A 039, A038 antibody heavy chain variable region
EVQLVESGGGLVQPGGSLRLSCAASGINLYYSSMHWVRQAPGKGLEWVASIYSSNGR
TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYW GQGTLVTVSS
SEQ ID NO. 15A 039 antibody light chain variable region
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVDWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIK
CDRL1 of the light chain complementarity determining region of the 16A 039 antibody
RASQSVSSAVD
SEQ ID NO. 17A 038 antibody light chain variable region
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVEWYQQKPGKAPKLLIYSASSLYSGVPS
RFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIK
SEQ ID NO. 18C 056-WT antibody heavy chain variable region
EVQLVESGGGLVQPGGSLRLSCAASGFNLYYSSMHWVRQAPGKGLEWVASIYSSNGR
TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYW GQGTLVTVSS
SEQ ID NO. 19C 056-WT antibody heavy chain complementarity determining region CDRH1
GFNLYYSSMH
SEQ ID NO. 20C 056-WT antibody light chain variable region
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIK
SEQ ID NO. 21C 056-WT antibody light chain complementarity determining region CDRL1
RASQSVSSAVA
CDRL1 of the light chain complementarity determining region of the 22A 038 antibody of SEQ ID NO. 22
RASQSVSSAVE
SEQ ID NO. 23 IL-18Rp 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
SEQ ID NO. 24A 035 antibody (IgG 4) heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGINLYYSSMHWVRQAPGKGLEWVASIYSSNGR
TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARASFSHGYGWYGLDYW
GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP
CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN
AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO. 25A 035 antibody (IgG 4) light chain
DIQMTQSPSSLSASVGDRVTITCRASQSVSFAVEWYQQKPGKAPKLLIYSASSLYSGVPS
RFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO. 26A 037 antibody (IgG 4) heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGLNLYYSSMHWVRQAPGKGLEWVASIYSSNGR
TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYW
GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP
CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN
AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO. 27A 037 antibody (IgG 4) light chain
DIQMTQSPSSLSASVGDRVTITCRASQSVSFAVDWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 28A 039 antibody (IgG 4) heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGINLYYSSMHWVRQAPGKGLEWVASIYSSNGR
TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYW
GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP
CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN
AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO. 29A 039 antibody (IgG 4) light chain
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVDWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO. 30A 035 antibody (IgGl) heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGINLYYSSMHWVRQAPGKGLEWVASIYSSNGR
TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARASFSHGYGWYGLDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO. 31A 035 antibody (IgGl) light chain
DIQMTQSPSSLSASVGDRVTITCRASQSVSFAVEWYQQKPGKAPKLLIYSASSLYSGVPS
RFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO. 32A 037 antibody (IgGl) heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGLNLYYSSMHWVRQAPGKGLEWVASIYSSNGR
TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
33A 037 antibody (IgGl) light chain of SEQ ID NO
DIQMTQSPSSLSASVGDRVTITCRASQSVSFAVDWYQQKPGKAPKLLIYSASSLYSGVP
SRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
34A 039 antibody (IgGl) heavy chain of SEQ ID NO
EVQLVESGGGLVQPGGSLRLSCAASGINLYYSSMHWVRQAPGKGLEWVASIYSSNGR
TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSFSHGYGWYGLDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO. 35A 039 antibody (IgGl) light chain
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVDWYQQKPGKAPKLLIYSASSLYSGVP SRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGYHDAGLITFGQGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO. 36A 035 antibody (IgG 4) heavy chain nucleic acid sequence
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGAGGTGCAGCTGGTGGAGA GCGGGGGGGGACTGGTGCAGCCAGGAGGAAGCCTGAGACTGAGCTGTGCCGCAAG CGGGATTAACCTGTATTACAGCAGCATGCACTGGGTGAGACAGGCACCTGGCAAA GGACTGGAGTGGGTGGCAAGCATATACAGCAGCAACGGAAGAACCTACTACGCCG ACAGTGTGAAGGGCAGATTCACTATCAGCGCCGACACCAGCAAAAACACCGCCTA CCTGCAGATGAACAGCCTGAGAGCCGAAGACACCGCCGTGTACTACTGCGCCAGG GCCTCCTTCAGCCATGGCTACGGCTGGTACGGCCTGGACTACTGGGGACAAGGAAC ACTGGTGACAGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGC CCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACA AGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATG CCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCC AAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGG
TGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGT GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTA CAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCA AAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGA GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGT GGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG GCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGATT CTAGA
SEQ ID NO. 37A 035 antibody (IgG 4) light chain nucleic acid sequence
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGACATTCAGATGACCCAGA GCCCCAGCAGCCTGAGCGCCAGCGTGGGAGACAGAGTGACCATAACCTGCAGAGC CAGCCAAAGCGTTAGCTTTGCCGTTGAGTGGTATCAGCAGAAACCTGGCAAAGCCC CCAAGCTGCTCATCTACAGCGCCAGTAGCCTGTATAGCGGCGTGCCCAGCAGATTT AGCGGAAGCAGATCAGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAACCCGA AGACTTCGCCACCTACTACTGCCAGCAGTACGGATACCACGACGCAGGCCTCATCA CCTTCGGACAAGGAACCAAAGTGGAGATTAAGCGTACGGTGGCTGCACCATCTGTC TTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGC CTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAG
CTTCAACAGGGGAGAGTGTTGATTCTAGA
SEQ ID NO. 38A 037 antibody (IgG 4) heavy chain nucleic acid sequence
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGAGGTGCAGCTGGTGGAGA
GCGGGGGGGGACTGGTGCAGCCAGGAGGAAGCCTGAGACTGAGCTGTGCCGCAAG CGGGCTGAACCTGTACTATAGTAGCATGCACTGGGTGAGACAGGCACCCGGCAAA GGACTGGAGTGGGTGGCTAGCATTTACAGCAGCAATGGAAGAACATACTACGCCG
ATAGCGTGAAGGGCAGATTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTA CCTGCAGATGAACAGCCTGAGAGCCGAAGACACAGCAGTGTACTACTGCGCCAGG
AGCAGCTTTTCCCATGGCTATGGCTGGTACGGCCTGGACTACTGGGGCCAGGGCAC CCTCGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGC
CCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACA AGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATG CCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCC
AAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGG TGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGT GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTA CAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCA AAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGT GGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG GCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGATT CTAGA
SEQ ID NO. 39A 037 antibody (IgG 4) light chain nucleic acid sequence
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGACATTCAGATGACCCAGA
GCCCCAGCAGCCTGAGCGCCAGCGTGGGAGACAGAGTGACCATAACCTGCAGAGC CAGCCAAAGCGTTAGCTTTGCCGTTGACTGGTATCAGCAGAAACCCGGCAAGGCCC CAAAACTGCTGATCTACAGCGCAAGTAGCCTGTACTCCGGCGTGCCCTCCCGCTTC AGCGGATCCAGATCCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAACCCGA AGACTTCGCCACCTACTACTGCCAGCAATACGGATATCACGACGCTGGCCTCATCA
CCTTTGGCCAAGGCACCAAGGTGGAAATTAAGCGTACGGTGGCTGCACCATCTGTC TTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGC CTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAG CTTCAACAGGGGAGAGTGTTGATTCTAGA
SEQ ID NO. 40A 039 antibody (IgG 4) heavy chain nucleic acid sequence
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT
GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGAGGTGCAGCTGGTGGAGA
GCGGGGGGGGACTGGTGCAGCCAGGAGGAAGCCTGAGACTGAGCTGTGCCGCAAG
CGGGATTAACCTGTATTACAGCAGCATGCACTGGGTGAGACAGGCACCTGGCAAA
GGACTGGAGTGGGTGGCAAGCATATACAGCAGCAACGGAAGAACCTACTACGCCG
ACAGTGTGAAGGGCAGATTCACTATCAGCGCCGACACCAGCAAAAACACCGCCTA
CCTGCAGATGAACAGCCTGAGAGCCGAAGACACCGCCGTGTACTACTGCGCCAGG
AGCTCCTTCAGCCATGGCTACGGCTGGTACGGCCTGGACTACTGGGGACAAGGAAC
ACTGGTGACAGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGC
CCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT
GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACA
AGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATG
CCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCC
AAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGG
TGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGT
GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTA
CAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCA
GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC
CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGT
GGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG
GCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGATT CTAGA
SEQ ID NO. 41A 039 antibody (IgG 4) light chain nucleic acid sequence
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT
GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGACATTCAGATGACCCAGA
GCCCCAGCAGCCTGAGCGCCAGCGTGGGAGACAGAGTGACCATAACCTGCAGAGC
CAGCCAAAGCGTTAGCAGCGCCGTGGACTGGTATCAGCAGAAACCCGGAAAGGCC
CCAAAACTGCTGATCTACAGCGCAAGTAGCCTGTACTCAGGAGTGCCCAGCAGATT
CAGCGGCAGCAGATCCGGAACCGACTTCACCCTGACCATCAGCAGCCTGCAACCTG
AGGATTTCGCCACCTACTACTGCCAGCAATACGGATACCACGACGCTGGTCTCATC
ACCTTCGGCCAAGGAACCAAGGTGGAAATTAAGCGTACGGTGGCTGCACCATCTGT
CTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTG
CCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC
GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACA
GCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGA GCTTCAACAGGGGAGAGTGTTGATTCTAGA
SEQ ID NO. 42A 035 antibody (IgGl) heavy chain nucleotidic acid sequence
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT
GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGAGGTGCAGCTGGTGGAGA GCGGGGGGGGACTGGTGCAGCCAGGAGGAAGCCTGAGACTGAGCTGTGCCGCAAG CGGGATTAACCTGTATTACAGCAGCATGCACTGGGTGAGACAGGCACCTGGCAAA
GGACTGGAGTGGGTGGCAAGCATATACAGCAGCAACGGAAGAACCTACTACGCCG
ACAGTGTGAAGGGCAGATTCACTATCAGCGCCGACACCAGCAAAAACACCGCCTA
CCTGCAGATGAACAGCCTGAGAGCCGAAGACACCGCCGTGTACTACTGCGCCAGG
GCCTCCTTCAGCCATGGCTACGGCTGGTACGGCCTGGACTACTGGGGACAAGGAAC
ACTGGTGACAGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC
CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT
GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACA
AGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAAC
TCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC
TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACA
TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC
CATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG
CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC
AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC
AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGATTCTAGA
SEQ ID NO. 43A 035 antibody (IgGl) light chain nucleotidic acid sequence
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT
GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGACATTCAGATGACCCAGA
GCCCCAGCAGCCTGAGCGCCAGCGTGGGAGACAGAGTGACCATAACCTGCAGAGC
CAGCCAAAGCGTTAGCTTTGCCGTTGAGTGGTATCAGCAGAAACCTGGCAAAGCCC
CCAAGCTGCTCATCTACAGCGCCAGTAGCCTGTATAGCGGCGTGCCCAGCAGATTT
AGCGGAAGCAGATCAGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAACCCGA
AGACTTCGCCACCTACTACTGCCAGCAGTACGGATACCACGACGCAGGCCTCATCA
CCTTCGGACAAGGAACCAAAGTGGAGATTAAGCGTACGGTGGCTGCACCATCTGTC
TTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGC
CTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG
CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAG CTTCAACAGGGGAGAGTGTTGATTCTAGA
SEQ ID NO. 44A 037 antibody (IgGl) heavy chain nucleotidic acid sequence
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT
GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGAGGTGCAGCTGGTGGAGA
GCGGGGGGGGACTGGTGCAGCCAGGAGGAAGCCTGAGACTGAGCTGTGCCGCAAG
CGGGCTGAACCTGTACTATAGTAGCATGCACTGGGTGAGACAGGCACCCGGCAAA
GGACTGGAGTGGGTGGCTAGCATTTACAGCAGCAATGGAAGAACATACTACGCCG
ATAGCGTGAAGGGCAGATTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTA
CCTGCAGATGAACAGCCTGAGAGCCGAAGACACAGCAGTGTACTACTGCGCCAGG AGCAGCTTTTCCCATGGCTATGGCTGGTACGGCCTGGACTACTGGGGCCAGGGCAC
CCTCGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACA AGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACA
TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC CATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC
AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGATTCTAGA
SEQ ID NO. 45A 037 antibody (IgGl) light chain nucleotidic acid sequence
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGACATTCAGATGACCCAGA GCCCCAGCAGCCTGAGCGCCAGCGTGGGAGACAGAGTGACCATAACCTGCAGAGC CAGCCAAAGCGTTAGCTTTGCCGTTGACTGGTATCAGCAGAAACCCGGCAAGGCCC CAAAACTGCTGATCTACAGCGCAAGTAGCCTGTACTCCGGCGTGCCCTCCCGCTTC AGCGGATCCAGATCCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAACCCGA AGACTTCGCCACCTACTACTGCCAGCAATACGGATATCACGACGCTGGCCTCATCA
CCTTTGGCCAAGGCACCAAGGTGGAAATTAAGCGTACGGTGGCTGCACCATCTGTC TTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGC CTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAG CTTCAACAGGGGAGAGTGTTGATTCTAGA
SEQ ID NO. 46A 039 antibody (IgGl) heavy chain nucleic acid sequence
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGAGGTGCAGCTGGTGGAGA GCGGGGGGGGACTGGTGCAGCCAGGAGGAAGCCTGAGACTGAGCTGTGCCGCAAG CGGGATTAACCTGTATTACAGCAGCATGCACTGGGTGAGACAGGCACCTGGCAAA GGACTGGAGTGGGTGGCAAGCATATACAGCAGCAACGGAAGAACCTACTACGCCG ACAGTGTGAAGGGCAGATTCACTATCAGCGCCGACACCAGCAAAAACACCGCCTA CCTGCAGATGAACAGCCTGAGAGCCGAAGACACCGCCGTGTACTACTGCGCCAGG
AGCTCCTTCAGCCATGGCTACGGCTGGTACGGCCTGGACTACTGGGGACAAGGAAC ACTGGTGACAGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCAC CCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACA AGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAAC
TCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGTCACA TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC CATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGATTCTAGA
Preparation of light chain nucleotide sequence GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACT GCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGACATTCAGATGACCCAGA GCCCCAGCAGCCTGAGCGCCAGCGTGGGAGACAGAGTGACCATAACCTGCAGAGC CAGCCAAAGCGTTAGCAGCGCCGTGGACTGGTATCAGCAGAAACCCGGAAAGGCC CCAAAACTGCTGATCTACAGCGCAAGTAGCCTGTACTCAGGAGTGCCCAGCAGATT CAGCGGCAGCAGATCCGGAACCGACTTCACCCTGACCATCAGCAGCCTGCAACCTG AGGATTTCGCCACCTACTACTGCCAGCAATACGGATACCACGACGCTGGTCTCATC ACCTTCGGCCAAGGAACCAAGGTGGAAATTAAGCGTACGGTGGCTGCACCATCTGT CTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTG CCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACA GCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGA GCTTCAACAGGGGAGAGTGTTGATTCTAGA of antibody (IgGl) of SEQ ID NO. 47A 039 example 1 anti-IL-18R 8 high affinity antibody As shown in FIG. 1, the inventors used the WT antibody of IL-18RP (i.e., wild type antibody, C056-WT in Table 1) as starting material for improving affinity. A library of exact mutations was constructed to introduce saturation mutations into all 73 residues of the six CDR regions of the WT antibody. Further screen After selection, the selected mutants were ranked by dissociation rate constant (Dissociation rate constant) determined by surface plasmon resonance (Surface plasmon resonance, SPR). A combinatorial library is then constructed using random combinations of beneficial mutations. The lead Fab clones were also determined by SPR sequencing. All SPR screens were performed on Biacore 8K. The running buffer was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 6.0). A secreted Fab was adsorbed onto the SASA biosensor. After equilibration, antigen was injected for 120 seconds (binding period) and then running buffer was injected for 360 seconds (dissociation period). The sensor surface was regenerated before injection of other selected clones. Experimental data were fitted locally to the 1:1 interaction model using Biacore 8K assessment software to obtain the off-rate o end of Fab clones, expressing full length IgG from the lead clones in HEK293 cells and assessed in binding and functional assays. As shown in Table 3, 44 hits were analyzed by SPR and 7 beneficial mutants were determined to exhibit better binding affinity (i.e., 2-fold greater than the affinity of the WT antibody, as shown in Table 4). The first 10 combinatorial mutations (> 10-fold of WT antibody affinity) with improved binding affinity are listed in table 5. 5 pilot clones (a 034, a035, a037, a038 and a 039) were selected for full length antibody production. The sequence is provided below. TABLE 3 affinity and kinetics of IL-18 for 44 hit mutants TABLE 4 beneficial mutant region mutations identified by saturation mutagenesis screening
VL-31 S F
VL-34A D,E
VH-27F L,I,V
VH-99S A table 5 top 10 human affinity enhanced beneficial mutant combinationsExample 2 evaluation of affinity of anti-IL-18R 8 antibodies the affinity of anti-IL-18R 0 antibodies to human IL-18RP protein was determined using the surface plasmon resonance biosensor Biacore 8K (GE Healthcare). Measurements were performed at 25℃with running buffer HBS-EP+. Antibodies were injected as a capture onto Series S sensor chip protein a. The diluted IL-18Rp (400, 200, 100, 50, 25, 12.5, 6.25, nM) was then injected as a binding phase into the surface of the flow cells 1 and 2. The bonding time was 120 seconds. The buffer stream was maintained for 420 seconds for separation. The surface was regenerated by injection of 10 mM glycine-HCl pH 1.5 seconds. Data for dissociation (kd) rate constants and association (ka) rate constants were obtained using Biacore evaluation software. The equilibrium dissociation constant (KD) is calculated from the ratio of KD to ka. Table 6 summarizes the binding kinetics data for anti-IL-18 RP antibodies. The binding affinities of A035, A037 and A039 were in the range of 0.72-0.98 nm, 16-22 fold improvement compared to the parent antibody WT (15.8 nm). TABLE 6 anti-IL-18R &Binding kinetics of antibodies antibody Chi 2 (RU 2 ) ka (1/Ms) KD (1/s) KD (M) Rmax (RU) ratio
WT 2.00E+00 7.10E+04 1.12E-03 1.58E-08 98.3 1.0
A035 2.27E+00 1.13E+05 1.12E-04 9.87E-10 135.4 16.0
A037 1.55E+00 1.03E+05 7.44E-05 7.19E-10 132.4 22.0
A039 2.32E+00 1.08E+05 9.31E-05.8.63E-10.148.2.18.3 example 3. Activity of anti-IL-18 RB antibodies against KG-1 intracellular IFN-y Release blocking IL-18 stimulation will be 3xl0 5 KG-1 cells (ATCC, # CCL-246) were seeded into each well of the 96-well plate. In use 10 ng/mL IL-
Serial dilutions of the antibodies prepared in example 1 were added to the wells before 18 stimulation of lh. After 24 hours incubation, cells were pelleted by centrifugation and, according to manufacturer's instructions, using an ELISA kit (R&Measurement of IFN-yo from supernatant from D Systems, # VAL 104C) at least 3 replicates of the anti-IL-18 RP antibodies described above (including both IgGl and IgG4 types), each of which is summarized in tables 7 and 8IC50 of the body. TABLE 7 IC50 of anti-IL-18 RR antibodies in KG-1 assay (24 hours)TABLE 8 IC50 of anti-IL-18R [3 ] antibodies in KG-1 assay (24 hours)The results of the corresponding data are plotted as shown in figures 2 and 3, with the a035, a037 and a039 antibodies being about 20-fold more potent than the WT antibody. In a further set of repeated experiments at a later time, antibodies a035, a037 and a039 were also demonstrated to have excellent cell inhibitory activity comparable to the above experiments, the results are shown in table 9 and fig. 4. TABLE 9 IC50 of anti-IL-18 RB antibodies in KG-1 repeat assay (24 hours) EXAMPLE 4 anti-IL-18R 8 antibody blocking IL-18 stimulated human peripheral blood mononuclear cells IFN-y Release active human peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC) (TPCS, #PB025C-W) were treated with Ix lO 5 The individual cell/well concentrations were seeded in 96-well plates. The cells were then incubated with different concentrations of anti-IL-18 RP antibody at 37℃and 5% CO under conditions of 50 ng/mL IL-18 and 5 ng/mL IL-12 2 For 48 hours (IgGl antibody) and 72 hours (IgG 4 antibody), respectively. According to the manufacturer's instructions, a human ELISA kit (R&D Systems, TABLE 10 IC50 of anti-IL-18 RP antibodies in human peripheral blood mononuclear cell assays (48 hours)TABLE 11 anti-IL-18 RP antibodies inIC50 in human peripheral blood mononuclear cell assay (72 hours)All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each was individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (1)

  1. Claim and claim
    1. An IL-18RP antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises one or more beneficial mutations selected from the group consisting of the heavy chain variable region set forth in SEQ ID No. 18 and/or the light chain variable region set forth in SEQ ID No. 20: f27L, F27I, F V, S99A, S31F, A34D, A34E; more specifically, the antibody or antigen binding fragment thereof further comprises an antibody or antigen binding fragment thereof that is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-4) amino acid residue and that retains the ability to specifically bind to IL-18 RP.
    The antibody or antigen-binding fragment thereof of claim 1, wherein the one or more beneficial mutations are each selected from one or more of the following:
    S31F、 A34E、 F27L；
    S31F、 A34E、 F27L S99A；
    A34D 、 F27L S99A；
    S31F、 A34D、 F27L；
    A34E、 F27I；
    A34D 、 F27I；
    A34E、 F27L；
    S31F、 A34D、 F27V、 S99A；
    a34E, F27L, S a; or (b)
    S31F、 A34E、 F27Lo
    The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the heavy chain complementarity determining region comprising:the synthetic fragment has a heavy chain variable region as shown in SEQ ID NO: K9 or 14, and has a light chain variable region as shown in SEQ ID NO: 5, 12, 15 or 17; preferably, the antibody or antigen binding fragment thereof has:
    36. A heavy chain variable region as shown in SEQ ID NO. 9 and a light chain variable region as shown in SEQ ID NO. 5; or a heavy chain variable region as shown in SEQ ID NO. 1 and a light chain variable region as shown in SEQ ID NO. 5; or a heavy chain variable region as shown in SEQ ID NO. 9 and a light chain variable region as shown in SEQ ID NO. 12; or a heavy chain variable region as shown in SEQ ID NO. 14 and a light chain variable region as shown in S EQ ID NO. 17; or a heavy chain variable region as shown in SEQ ID NO. 14 and a light chain variable region as shown in SEQ ID NO. 15.
    5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof has a heavy chain sequence as set forth in SEQ ID NOs 24, 26, 28, 30, 32 or 34, and has a light chain sequence as set forth in SEQ ID NOs 25, 27, 29, 31, 33 or 35; preferably, the antibody or antigen binding fragment thereof has: a heavy chain sequence as shown in SEQ ID NO. 24 and a light chain sequence as shown in SEQ ID NO. 25; or a heavy chain sequence as shown in SEQ ID NO. 26 and a light chain sequence as shown in SEQ ID NO. 27; or a heavy chain sequence as shown in SEQ ID NO. 28 and a light chain sequence as shown in SEQ ID NO. 29; or a heavy chain sequence as shown in SEQ ID NO. 30 and a light chain sequence as shown in SEQ ID NO. 31; or a heavy chain sequence as shown in SEQ ID NO. 32 and a light chain sequence as shown in SEQ ID NO. 33; or a heavy chain sequence as shown in SEQ ID NO. 34 and a light chain sequence as shown in SEQ ID NO. 35.
    6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
    7. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antigen-binding fragment is selected from scFv, fab, fab \f (ab') 2, fv > disulfide-linked Fv (dsFv), or sdAb.
    8. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the heavy chain constant region of the antibody or antigen-binding fragment thereof is selected from human IgGl, igG2, igG3 or IgG4, preferably IgGl or IgG4.
    9. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-8.
    10. An expression vector comprising the nucleic acid molecule of claim 9.
    11. A host cell comprising the expression vector of claim 10, or having incorporated into its genome the nucleic acid molecule of claim 9.
    12. A method of producing an antibody or antigen binding fragment thereof directed against IL-18RP, comprising the steps of:
    (a) Culturing the host cell of claim 11 under suitable conditions, thereby obtaining a culture comprising an antibody or antigen binding fragment thereof to IL-18 RP;
    (b) Isolating and/or recovering the antibody or antigen binding fragment thereof directed against IL-18RP from the culture; and
    (c) Optionally, purifying and/or modifying the antibody or antigen binding fragment thereof against IL-18RP obtained in step (b).
    13. A pharmaceutical composition comprising:
    (i) An active ingredient selected from the group consisting of: an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a nucleic acid molecule according to claim 9, or an expression vector according to claim 10, or
    37. The host cell of claim 11, or a combination thereof; and
    (ii) A pharmaceutically acceptable carrier, diluent or excipient.
    14. 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 nuclear alfalfa molecule according to claim 9, or the expression vector according to claim 10, or the host cell according to claim 11, or the pharmaceutical composition according to claim 13, or a combination thereof, characterized in that the active ingredient is used for the preparation of:
    (a) Medicaments for preventing and/or treating IL-18 related diseases;
    (b) Reagents for detecting IL-18 related disorders.
    15. A method of preventing and/or treating an IL-18-associated disease, the method comprising: administering to a subject in need thereof an antibody or antigen-binding fragment thereof of claims 1-8, or a nucleic acid molecule of claim 9, or an expression vector of claim 10, or a host cell of claim 11, or a pharmaceutical composition of claim 13, or a combination thereof.
    16. The use according to claim 14, wherein the IL-18 related disorder comprises a disorder of high IL-18 expression, a disorder associated with aberrant expression of IL-18 receptors or a disorder associated with high activity of IL-18 signaling pathways, an IL-18R & mediated disorder.
    17. The use according to claim 14, wherein the IL-18 related disorder is an autoimmune disorder or an inflammatory disorder.
    18. The use according to claim 14, wherein the IL-18 related disorder 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, myasthenia gravis, pernicious anemia, sjogren's syndrome, type I diabetes, type II glycouria, vasculitis, uveitis, sepsis, atherosclerosis, ankylosing spondylitis, hemophagocytosis, macrophage activation syndrome, systemic lupus erythematosus, suppurative arthritis, pyoderma, atopic dermatitis, idiopathic pulmonary fibrosis, scleroderma, systemic juvenile idiopathic arthritis (sJIA), inflammatory Bowel Disease (IBD), adult steven-ston's disease (aoil-stel), chronic pulmonary disease (COPD), or a combination thereof.
    19. The use of claim 18, 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, non-small cell lung cancer, breast cancer, urinary tract cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, parathyroid cancer, or a combination thereof.
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