An antibody targeting IL-18BP and its application

Abstract

Provided are an antibody or antigen-binding fragment thereof targeting IL-18BP, as well as a method for producing and using the antibody or antigen-binding fragment thereof. Specifically provided are mouse or chimeric monoclonal antibodies, humanized antibodies, nucleic acid molecules encoding the antibodies, corresponding expression vectors, and host cells targeting IL-18BP. Antibodies or antigen-binding fragments thereof targeting IL-18BP can specifically bind to IL-18P with high affinity, release natural IL-18 in the body, activate IL-18 signals to enhance the tumor-killing ability of T cells and NK cells, and can be used to prevent / treat a variety of diseases such as tumors, metabolic diseases, immune system diseases, neurodegenerative diseases, cardiovascular diseases, or inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to an antibody specifically targeting IL-18BP and an antigen-binding fragment thereof, as well as a preparation method and application thereof. Background Art

[0002] Interleukin-18 (IL-18) can induce the proliferation of immune cells and enhance their activity, particularly T and NK cell activity, playing a crucial role in tumor immunoregulation. IL-18 activates downstream signaling pathways by acting on the heterodimeric receptor 18Rα / Rβ on the cell membrane, promoting the activation and proliferation of immune cells. The vast majority of IL-18 in the body binds to IL-18BP, a naturally occurring pseudo-receptor for IL-18. IL-18BP binds with ultra-high affinity (in the pM range) to IL-18 and blocks its interaction with cell surface receptors, thereby negatively regulating the IL-18 signaling pathway. Studies have found that IL-18BP protein levels are upregulated in the microenvironment of human tumor tissue, representing a novel secretory immune checkpoint that inhibits the cytotoxicity of immune cells within the tumor microenvironment. IL-18BP is also significantly elevated in PD-1-resistant non-small cell lung cancer, making it a major factor in mediating PD-1 resistance.

[0003] Therefore, there is an urgent need in this field to develop high-affinity antibodies against IL-18BP to release natural IL-18 in the body and activate IL-18 signals to enhance the tumor-killing ability of T cells and NK cells. Summary of the Invention

[0004] The purpose of the present invention is to provide a new treatment method for various diseases such as tumors, metabolic diseases, immune system diseases, neurodegenerative diseases, cardiovascular diseases or inflammatory diseases.

[0005] Another object of the present invention is to provide an antibody against IL-18BP and a preparation method and application thereof.

[0006] In a first aspect of the present invention, an antibody or an antigen-binding fragment thereof against IL-18BP is provided.

[0007] In another preferred embodiment, the antibody or antigen-binding fragment thereof can specifically bind to IL-18BP.

[0008] In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises:

[0009] (a) heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, wherein the amino acid sequences of CDRH1, CDRH2, and CDRH3 are shown in SEQ ID NOs: 2, 3, and 4, respectively;

[0010] or as shown in SEQ ID NO: 10, 11 and 12, respectively,

[0011] or as shown in SEQ ID NO: 18, 19 and 20, respectively,

[0012] or as shown in SEQ ID NO: 26, 27 and 28, respectively,

[0013] or as shown in SEQ ID NO: 33, 34 and 20, respectively,

[0014] or as shown in SEQ ID NO: 38, 27 and 28, respectively,

[0015] or as shown in SEQ ID NO: 42, 43 and 44, respectively,

[0016] or as shown in SEQ ID NO: 50, 51 and 52, respectively,

[0017] or as shown in SEQ ID NO: 58, 59 and 60, respectively; and / or

[0018] (b) light chain complementary determining regions CDRL1, CDRL2, and CDRL3, wherein the amino acid sequences of CDRL1, CDRL2, and CDRL3 are shown in SEQ ID NOs: 6, 7, and 8, respectively;

[0019] or as shown in SEQ ID NO: 14, 15 and 16, respectively,

[0020] or as shown in SEQ ID NO: 22, 23 and 24, respectively,

[0021] or as shown in SEQ ID NO:30, 15 and 31, respectively,

[0022] or as shown in SEQ ID NO: 22, 36 and 24, respectively,

[0023] or as shown in SEQ ID NO: 40, 15 and 31, respectively,

[0024] or as shown in SEQ ID NO: 46, 47 and 48, respectively,

[0025] or as shown in SEQ ID NO: 54, 55 and 56, respectively,

[0026] or as shown in SEQ ID NO: 62, 63 and 64, respectively;

[0027] Among them, any of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3) amino acids and can retain the ability to specifically bind to IL-18BP.

[0028] In another preferred embodiment, the antibody or antigen-binding fragment thereof has 6 CDRs (CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3) of Ab001, Ab002, Ab003, Ab009, humanized Ab009 (VH12VL9), humanized Ab009 (VH14VL9), Ab010, Ab012, Ab017, Ab018 or Ab020 antibody in Table 1.

[0029] In another preferred embodiment, the derivative sequence that has been subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the ability to specifically bind to IL-18BP is an amino acid sequence with a homology or sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0030] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4.

[0031] In another preferred embodiment, the antibody or antigen-binding fragment thereof further includes a framework region FR.

[0032] In another preferred example, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 1, 9, 17, 25, 32, 37, 41, 49, 57, 65 or 67, or a sequence identity thereof of at least 90%, 95% or 98%, and a light chain variable region as shown in SEQ ID NO: 5, 13, 21, 29, 35, 39, 45, 53, 61 or 66, or a sequence identity thereof of at least 90%, 95% or 98%.

[0033] In another preferred example, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 1, 9, 17, 25, 32, 37, 41, 49, 57, 65 or 67, and a light chain variable region as shown in SEQ ID NO: 5, 13, 21, 29, 35, 39, 45, 53, 61 or 66.

[0034] In another preferred embodiment, the antibody or antigen-binding fragment thereof has heavy chain complementary determining regions CDRH1, CDRH2, CDRH3, and light chain complementary determining regions CDRL1, CDRL2, CDRL3, and the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as shown in SEQ ID NOs: 2, 3, 4, 6, 7, and 8, respectively, or as shown in SEQ ID NOs: 10, 11, 12, 14, 15, and 16, respectively, or as shown in SEQ ID NOs: 18, 19, 20, 22, 23, and 24, respectively, or as shown in SEQ ID NOs: 26, 27, 28, 30, 15, and 31, respectively, or as shown in SEQ ID NOs: 33, 34, 20, 22, 36, and 24, respectively, or as shown in SEQ ID NOs: 38, 27, 28, 40, 15, and 31, respectively, or as shown in SEQ ID NOs: 39, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52, respectively. NO:42, 43, 44, 46, 47, 48, or as shown in SEQ ID NO:50, 51, 52, 54, 55, 56, or as shown in SEQ ID NO:58, 59, 60, 62, 63, 64, respectively; wherein, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (e.g., 1-3) amino acid and can retain the ability to specifically bind to IL-18BP.

[0035] In another preferred embodiment, the antibody or antigen-binding fragment thereof may include a monomer, a bivalent antibody, and / or a multivalent antibody.

[0036] In another preferred embodiment, the bivalent antibody may also be a bispecific antibody.

[0037] In another preferred embodiment, the multivalent antibody may also be a multispecific antibody.

[0038] In another preferred embodiment, the antibody or antigen-binding fragment thereof includes a mouse antibody, a monkey antibody, a humanized antibody or a chimeric antibody.

[0039] In another preferred embodiment, the murine antibody is derived from mice.

[0040] In another preferred embodiment, the monkey antibody is derived from cynomolgus monkey.

[0041] In another preferred example, the amino acid sequences of the heavy chain variable region and the light chain variable region of the humanized antibody are shown as SEQ ID NO: 65 and SEQ ID NO: 66, respectively, or as shown as SEQ ID NO: 67 and SEQ ID NO: 66, respectively.

[0042] In another preferred embodiment, the heavy chain variable region of the humanized antibody has a mutation selected from the following group based on SEQ ID NO: 25:

[0043] Q5V, D8G, L11V, V12K, A16S, I20V, V24S, Y25S, R40A, E42G, L70M, K74T, S76T, Q82E, N84R, N84S, T87R, E89D, S91T, and L112V;

[0044] The light chain variable region of the humanized antibody has a mutation selected from the following group based on SEQ ID NO: 29:

[0045] Q1E, A9D, I10F, M11Q, A13V, S14T, G16K, M21I, S39P, G40D, T41Q, W46L, T59S, S69D, S71T, S75N, M77L, A99Q, and L105I.

[0046] In another preferred embodiment, the constant region of the chimeric antibody is derived from the heavy chain constant region of human IgG1.

[0047] In another preferred embodiment, the antigen-binding fragment is selected from scFv, Fab, Fab', F(ab')2, Fv fragment, heavy chain antibody, and disulfide-linked Fv (dsFv).

[0048] In another preferred example, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO: 1 and SEQ ID NO: 5, respectively, or SEQ ID NO: 9 and SEQ ID NO: 13, respectively, or SEQ ID NO: 17 and SEQ ID NO: 21, respectively, or SEQ ID NO: 25 and SEQ ID NO: 29, respectively, or SEQ ID NO: 65 and SEQ ID NO: 66, respectively, or SEQ ID NO: 67 and SEQ ID NO: 66, respectively, or SEQ ID NO: 32 and SEQ ID NO: 35, respectively, or SEQ ID NO: 37 and SEQ ID NO: 39, respectively, or SEQ ID NO: 41 and SEQ ID NO: 45, respectively, or SEQ ID NO: 49 and SEQ ID NO: 53, respectively, or SEQ ID NO: 57 and SEQ ID NO: 61, respectively.

[0049] In another preferred embodiment, the heavy chain constant region of the antibody or its antigen-binding fragment is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and the light chain constant region of the antibody or its antigen-binding fragment is selected from the constant region of human antibody κ chain or λ chain.

[0050] In another preferred embodiment, the heavy chain constant region is the heavy chain constant region of human IgG1 or IgG4.

[0051] In another preferred example, the heavy chain and light chain amino acid sequences of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively, or SEQ ID NO: 71 and SEQ ID NO: 72, respectively, or SEQ ID NO: 73 and SEQ ID NO: 74, respectively, or SEQ ID NO: 75 and SEQ ID NO: 76, respectively, or SEQ ID NO: 77 and SEQ ID NO: 78, respectively, or SEQ ID NO: 79 and SEQ ID NO: 80, respectively, or SEQ ID NO: 81 and SEQ ID NO: 82, respectively, or SEQ ID NO: 83 and SEQ ID NO: 84, respectively, or SEQ ID NO: 85 and SEQ ID NO: 86, respectively, or SEQ ID NO: 87 and SEQ ID NO: 88, respectively, or SEQ ID NO: 89 and SEQ ID NO: 90, respectively.

[0052] In a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0053] (i) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and

[0054] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0055] In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

[0056] In another preferred embodiment, the recombinant protein specifically binds to IL-18BP.

[0057] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0058] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0059] In the third aspect of the present invention, a nucleotide molecule is provided, which encodes a protein selected from the group consisting of the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention.

[0060] In another preferred embodiment, the nucleic acid of the present invention may be RNA, DNA or cDNA.

[0061] In another preferred example, the nucleotide molecule includes: a heavy chain nucleotide sequence as shown in SEQ ID NO:91 and a light chain nucleotide sequence as shown in SEQ ID NO:92; or a heavy chain nucleotide sequence as shown in SEQ ID NO:93 and a light chain nucleotide sequence as shown in SEQ ID NO:94; or a heavy chain nucleotide sequence as shown in SEQ ID NO:95 and a light chain nucleotide sequence as shown in SEQ ID NO:96; or a heavy chain nucleotide sequence as shown in SEQ ID NO:97 and a light chain nucleotide sequence as shown in SEQ ID NO:98; or a heavy chain nucleotide sequence as shown in SEQ ID NO:99 and a light chain nucleotide sequence as shown in SEQ ID NO:100; or a heavy chain nucleotide sequence as shown in SEQ ID NO:101 and a light chain nucleotide sequence as shown in SEQ ID NO:102.

[0062] In the fourth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the nucleotide molecule described in the third aspect of the present invention.

[0063] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or combinations thereof.

[0064] In another preferred embodiment, the expression vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

[0065] In another preferred embodiment, the expression vector further comprises a member selected from the group consisting of: a promoter, a transcription enhancer element WPRE, a long terminal repeat sequence LTR, and the like.

[0066] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell contains the expression vector described in the fourth aspect of the present invention, or the nucleotide molecule described in the third aspect of the present invention is integrated into its genome.

[0067] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0068] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0069] In another preferred embodiment, the host cell is a 293F cell or an expi-293 cell.

[0070] In a sixth aspect of the present invention, a chimeric antigen receptor CAR is provided, wherein the antigen binding region scFv segment of the CAR is a binding region that specifically binds to IL-18BP, and the heavy chain variable region of the scFv comprises:

[0071] Heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of which are shown in SEQ ID NOs: 2, 3, and 4, respectively, or SEQ ID NOs: 10, 11, and 12, respectively, or SEQ ID NOs: 18, 19, and 20, respectively, or SEQ ID NOs: 26, 27, and 28, respectively, or SEQ ID NOs: 33, 34, and 20, respectively, or SEQ ID NOs: 38, 27, and 28, respectively, or SEQ ID NOs: 42, 43, and 44, respectively, or SEQ ID NOs: 50, 51, and 52, respectively, or SEQ ID NOs: 58, 59, and 60, respectively; and / or

[0072] The light chain variable region of the scFv comprises:

[0073] The light chain complementary determining regions CDRL1, CDRL2, and CDRL3, the amino acid sequences of which are shown in SEQ ID NOs: 6, 7, and 8, respectively, or in SEQ ID NOs: 14, 15, and 16, respectively, or in SEQ ID NOs: 22, 23, and 24, respectively, or in SEQ ID NOs: 30, 15, and 31, respectively, or in SEQ ID NOs: 22, 36, and 24, respectively, or in SEQ ID NOs: 40, 15, and 31, respectively, or in SEQ ID NOs: 46, 47, and 48, respectively, or in SEQ ID NOs: 54, 55, and 56, respectively, or in SEQ ID NOs: 62, 63, and 64, respectively.

[0074] In another preferred embodiment, the CAR further comprises a signal peptide.

[0075] In another preferred embodiment, the CAR also includes other exogenous proteins.

[0076] In another preferred embodiment, the CAR has the structure shown in Formula Ia:

[0077] L-scFv-H-TM-C-CD3ζ (Ia)

[0078] Where,

[0079] L is none or a signal peptide sequence;

[0080] scFv is a domain that specifically binds to IL-18BP;

[0081] H is none or hinge region;

[0082] TM is the transmembrane domain;

[0083] C is the costimulatory signaling domain;

[0084] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type, or mutants / modified forms thereof);

[0085] The "-" connects peptides or peptide bonds.

[0086] In another preferred embodiment, the L is selected from the signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or a combination thereof.

[0087] In another preferred embodiment, the scFv targets IL-18BP.

[0088] In another preferred embodiment, the scFv is an IL-18BP antibody or an antigen-binding fragment thereof.

[0089] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0090] In another preferred embodiment, the TM is selected from the transmembrane region of the following histones: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutants / modified forms thereof, or combinations thereof.

[0091] In another preferred embodiment, the C is selected from the costimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants / modified forms thereof, or combinations thereof.

[0092] In the seventh aspect of the present invention, an engineered immune cell is provided, wherein the engineered immune cell expresses an exogenous CAR as described in the sixth aspect of the present invention.

[0093] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0094] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0095] (ii) chimeric antigen receptor γδ T cells (CAR-T cells);

[0096] (iii) chimeric antigen receptor NKT cells (CAR-NKT cells);

[0097] (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0098] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or a combination thereof.

[0099] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0100] In an eighth aspect of the present invention, a method for producing an antibody or an antigen-binding fragment thereof against IL-18BP is provided, comprising the steps of:

[0101] (a) culturing the host cell according to the fifth aspect of the present invention under suitable conditions to obtain a culture containing the antibody or antigen-binding fragment thereof to IL-18BP;

[0102] (b) isolating and / or recovering the antibody or antigen-binding fragment thereof against IL-18BP from the culture; and

[0103] (c) Optionally, purifying and / or modifying the antibody against IL-18BP or the antigen-binding fragment thereof obtained in step (b).

[0104] In a ninth aspect of the present invention, an immunoconjugate is provided, comprising:

[0105] (a) an antibody portion, said antibody portion comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and

[0106] (b) a conjugated moiety selected from the group consisting of a detectable label, a therapeutic agent, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0107] In another preferred embodiment, the (a) part and the coupling part are coupled via a chemical bond or a linker.

[0108] In another preferred embodiment, the radioactive nuclides include:

[0109] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0110] (ii) therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0111] In another preferred embodiment, the conjugated moiety may contain at least one therapeutic agent.

[0112] In another preferred embodiment, the conjugated moiety is a therapeutic agent.

[0113] In another preferred embodiment, the therapeutic agent is an immune checkpoint drug, and the immune checkpoint is selected from: PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR.

[0114] In another preferred embodiment, the therapeutic agent is a PD-1 antibody and / or a PD-L1 antibody.

[0115] In another preferred embodiment, the immune checkpoint drug is an inhibitor or activator of the immune checkpoint.

[0116] In another preferred embodiment, the therapeutic agent is a cytotoxic drug.

[0117] In another preferred embodiment, the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0118] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposide, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0119] In another preferred embodiment, the coupling moiety is a toxin.

[0120] In another preferred embodiment, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansin, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxybenzoate, daptomycin, acetaminophen, chlortetracycline, chloramphenicol, chloramphenicol, daptomycin, chlortetracycline, chloramphenicol, daptomycin, chlortetracycline, chloramphenicol, chlortetracycline ... anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, Mitogellin, Retstrictocin, phenomycin, enomycin, curcin, crotin, calicheamicin, a Sapaonaria officinalis inhibitor, a glucocorticoid, or a combination thereof.

[0121] In another preferred embodiment, the coupling moiety is a detectable label.

[0122] In another preferred embodiment, the coupling portion is selected from the following group: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticles.

[0123] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

[0124] In another preferred embodiment, the multivalency refers to the presence of multiple repeats of the same or different antibodies or antigen-binding fragments thereof as described in the first aspect of the present invention in the amino acid sequence of the immunoconjugate.

[0125] In a tenth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0126] (i) an active ingredient selected from the group consisting of the antibody or antigen-binding fragment thereof according to the first aspect of the invention, the recombinant protein according to the second aspect of the invention, the engineered immune cell according to the seventh aspect of the invention, the immunoconjugate according to the ninth aspect of the invention, or a combination thereof; and

[0127] (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0128] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection and lyophilized preparation.

[0129] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention, or the engineered immune cell as described in the seventh aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention, or a combination thereof and 0.01 to 99.99% of a pharmaceutically acceptable carrier, where the percentages are the mass percentages of the pharmaceutical composition.

[0130] In another preferred embodiment, the concentration of the engineered immune cells in the active ingredient is 1×10 3 -1×10 8 cells / mL, preferably 1×10 4 -1×10 7 cells / mL.

[0131] In another preferred embodiment, the pharmaceutical composition further comprises a second active ingredient: PD-1 antibody and / or PD-L1 antibody.

[0132] In another preferred embodiment, the pharmaceutical composition comprises:

[0133] (a) a first active ingredient, such as the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0134] (b) a second active ingredient, a PD-1 antibody and / or a PD-L1 antibody; and

[0135] (c) a pharmaceutically acceptable carrier, diluent or excipient;

[0136] The ratio of the first active ingredient to the second active ingredient is 5:1-1:1, preferably 3:1.

[0137] In another preferred embodiment, the dosage of the active ingredient in the pharmaceutical composition is 5-60 mg / kg, preferably 10-30 mg / kg.

[0138] In another preferred embodiment, the dosage of the PD-1 antibody is 5-20 mg / kg, preferably 10 mg / kg.

[0139] In the eleventh aspect of the present invention, there is provided a use of an active ingredient, wherein the active ingredient is selected from the group consisting of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the engineered immune cell according to the seventh aspect of the present invention, the immunoconjugate according to the ninth aspect of the present invention, or a combination thereof, wherein the active ingredient is used to prepare:

[0140] (a) medicines for the prevention and / or treatment of diseases or conditions;

[0141] (b) Reagents for detecting IL-18BP-related diseases.

[0142] In another preferred embodiment, the reagent is a diagnostic reagent, preferably, the diagnostic reagent is a detection sheet or a detection plate.

[0143] In another preferred embodiment, the diagnostic reagent is used to detect IL-18BP protein or a fragment thereof in a sample.

[0144] In another preferred embodiment, the antibody or antigen-binding fragment thereof is used as the first active ingredient in combination with a second active ingredient; preferably, the second active ingredient is an anti-PD-1 / PD-L1 antibody.

[0145] In another preferred embodiment, the disease or disorder is a disease or disorder characterized by high expression of IL-18BP or abnormal IL-18 pathway.

[0146] In another preferred embodiment, the disease or condition includes: tumor, metabolic disease, immune system disease, neurodegenerative disease, cardiovascular disease or inflammatory disease.

[0147] In another preferred embodiment, the disease or disorder is a tumor.

[0148] In another preferred embodiment, the tumor is a solid tumor or a hematological tumor.

[0149] In another preferred embodiment, the tumor is selected from one or more of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal cancer, brain tumor, cervical cancer, blood cancer, bone cancer, lymphoma, and pancreatic cancer.

[0150] In a twelfth aspect of the present invention, a method for detecting IL-18BP protein or a fragment thereof in a sample in vitro is provided, the method comprising the steps of:

[0151] (1) contacting the sample in vitro with the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0152] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of the corresponding target of the IL-18BP protein or its fragment in the sample.

[0153] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0154] The thirteenth aspect of the present invention provides a kit, comprising:

[0155] (1) a first container containing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention; and / or

[0156] (2) a second container containing a secondary antibody against the contents of the first container;

[0157] or,

[0158] The kit contains a detection plate, which includes: a substrate (support plate) and a test strip, wherein the test strip contains the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention, or the engineered immune cell as described in the seventh aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention, or the pharmaceutical composition as described in the tenth aspect of the present invention, or a combination thereof.

[0159] In another preferred embodiment, the secondary antibody in the second container is HRP-labeled goat anti-human IgGκ.

[0160] In another preferred embodiment, the kit further comprises an instruction manual, and according to the instruction manual, the kit is used for non-invasively detecting the expression of IL-18BP in a subject.

[0161] In another preferred embodiment, the kit is used for detecting IL-18BP-related diseases.

[0162] In another preferred embodiment, the IL-18BP-related disease is a disease or condition in which IL-18BP is highly expressed or the IL-18 pathway is abnormal.

[0163] In the fourteenth aspect of the present invention, a method for preventing and / or treating a disease or condition is provided, the method comprising: administering to a subject in need thereof the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention, or the engineered immune cell as described in the seventh aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention, or the pharmaceutical composition as described in the tenth aspect of the present invention, or a combination thereof.

[0164] In another preferred embodiment, the subject includes mammals, such as humans.

[0165] In another preferred embodiment, the disease or disorder is a disease or disorder characterized by high expression of IL-18BP or abnormal IL-18 pathway.

[0166] In another preferred embodiment, the disease or condition includes: tumor, metabolic disease, immune system disease, neurodegenerative disease, cardiovascular disease or inflammatory disease.

[0167] In another preferred embodiment, the disease or disorder is a tumor.

[0168] In another preferred embodiment, the tumor is a solid tumor or a hematological tumor.

[0169] In another preferred embodiment, the tumor is selected from one or more of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal cancer, brain tumor, cervical cancer, blood cancer, bone cancer, lymphoma, and pancreatic cancer.

[0170] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from the subject (autologous cells).

[0171] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).

[0172] In another preferred embodiment, the method can be used in combination with other treatment methods.

[0173] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy and the like.

[0174] In another preferred embodiment, the method is used in combination with PD-1 antibody and / or PD-L1 antibody.

[0175] In a fifteenth aspect of the present invention, a method for diagnosing IL-18BP-related diseases is provided, comprising the steps of:

[0176] (i) obtaining a sample from a diagnostic subject, and contacting the sample with the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention; and

[0177] (ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates that the subject is a confirmed patient of an IL-18-related disease.

[0178] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues and organs.

[0179] In another preferred embodiment, the IL-18BP-related disease is a disease or condition in which IL-18BP is highly expressed or the IL-18 pathway is abnormal.

[0180] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0181] Figure 1 The sequence information of humanized antibody Ab009 (VH12VL9) and humanized antibody Ab009 (VH14VL9) are shown.

[0182] Figure 2 It was shown that anti-IL-18BP antibody could restore IL-18-induced IFN-γ release activity in KG-1 cells.

[0183] Figure 3 It was shown that the humanized antibody VH12VL9 restored the IL-18-induced IFN-γ release activity of KG-1 cells.

[0184] Figure 4 It was shown that anti-IL-18BP antibodies restored IL-18-induced IFN-γ release in human peripheral blood mononuclear cells.

[0185] Figure 5 A shows the secretion of IL-18BP after stimulation of SK-OV-3 cells. Figure 5 B shows the binding activity of IL-18BP antibody to IL-18BP derived from human tumor cells.

[0186] Figure 6 The changes in tumor volume over time in mice in different drug-treated groups are shown.

[0187] Figure 7 The body weight change rate (%) of mice in different drug administration groups is shown. DETAILED DESCRIPTION

[0188] Through extensive and intensive research and extensive screening, the inventors unexpectedly developed, for the first time, a class of antibodies and antigen-binding fragments thereof with high specificity and affinity for IL-18BP. They also unexpectedly obtained humanized antibodies against IL-18BP with excellent affinity and specificity. The antibodies and antigen-binding fragments of the present invention can block the binding of IL-18BP to IL-18, release natural IL-18 in the body, and activate IL-18 signaling to enhance the anti-tumor ability of T cells and NK cells. Therefore, the antibodies and antigen-binding fragments of the present invention have the potential to prevent and / or treat a variety of diseases, including tumors, inflammatory diseases, and metabolic diseases.

[0189] the term

[0190] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0191] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0192] The term "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0193] As used herein, the terms "antibody of the present invention", "antibodies of the present invention", "antibodies against IL-18BP of the present invention", "antibodies against IL-18BP", "antibodies against IL-18BP", and "IL-18BP antibodies" have the same meaning and can be used interchangeably, all referring to antibodies that specifically recognize and bind to IL-18BP protein (including human IL-18BP protein).

[0194] Preferably, the antibody numbers of the present invention and the corresponding sequence numbers are shown in Table 1 below.

[0195] Table 1

[0196]

[0197] Note: Each numerical value in the table represents a sequence number, i.e., "1" represents "SEQ ID NO: 1", and the sequence numbers of VH, CDRH1, CDRH2, CDRH3, VL, CDRL1, CDRL2, and CDRL3 shown in the table are the numbers of their amino acid sequences.

[0198] Preferably, the antibody numbers of the present invention and the corresponding sequences and numbers of the VH and VL regions, HC and LC are shown in Table 2 below.

[0199] Table 2

[0200]

[0201]

[0202]

[0203]

[0204]

[0205] Preferably, the antibody numbers of the present invention and the corresponding CDR sequences and numbers are shown in Table 3 below.

[0206] Table 3

[0207]

[0208] The term "antibody" as used herein is intended to include full-length antibodies and any antigen-binding fragments (i.e., antigen-binding portions) or single chains thereof. A full-length antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, 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. The VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs), which are separated by relatively conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

[0209] As used 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 to an antigen (e.g., IL-18BP protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Antibodies, antigen-binding fragments, or derivatives thereof disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, or chimeric antibodies, single-chain antibodies, and antigen-binding fragments. Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of VH and CH1; (iv) an Fv fragment consisting of the VL and VH arms of an antibody; (v) a dAb fragment consisting of VH; (vi) an isolated complementarity-determining region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be recombinantly linked via a synthetic linker that makes them a single protein chain, wherein the VL and VH regions pair to form a monovalent molecule) (referred to as single-chain Fc (scFv)). These single-chain antibodies are also intended to be included in the meaning of the term. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0210] As used herein, the term "variable" refers to certain portions of the variable region in an antibody that differ in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0211] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes, called kappa and lambda, based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0212] Generally, an antibody's antigen-binding properties are described by three specific regions within the variable region of the heavy chain, known as the variable regions (CDRs). This region is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0213] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0214] As used herein, the terms "light chain variable region" and "VL" are used interchangeably.

[0215] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0216] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDRH1, CDRH2, and CDRH3.

[0217] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0218] In a preferred embodiment of the present invention, the light chain variable region of the antibody includes three complementarity determining regions: CDRL1, CDRL2, and CDRL3.

[0219] In a preferred embodiment of the present invention, the light chain of the antibody comprises the above-mentioned light chain variable region and light chain constant region.

[0220] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in antigen binding. Thus, the present invention includes molecules having antibody heavy chain variable regions with CDRs that are 90% or more (preferably 95% or more, and most preferably 98% or more) homologous to the CDRs identified herein.

[0221] As used herein, "sequence identity" refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between a sequence described herein and a sequence to which it is identical may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0222] In the present invention, the terms "antibody of the present invention," "protein of the present invention," or "polypeptide of the present invention" are used interchangeably to refer to polypeptides that specifically bind to IL-18BP protein, such as proteins or polypeptides having a heavy chain variable region. These may or may not contain an initial methionine.

[0223] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0224] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0225] In the present invention, antibodies include murine, monkey, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, include human and non-human parts and can be obtained by standard DNA recombinant techniques, which are all useful antibodies. A chimeric antibody is a molecule in which different parts are derived from different animal species, such as a chimeric antibody having a variable region from a mouse monoclonal antibody and a constant region from a human immunoglobulin (see, for example, U.S. Patent No. 4,816,567 and U.S. Patent No. 4,816,397, which are hereby incorporated by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent No. 5,585,089, which is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant techniques well known in the art.

[0226] The antibodies of the present invention refer to polypeptides that have IL-18BP protein binding activity and include the aforementioned CDR regions. The term also encompasses variants of polypeptides that include the aforementioned CDR regions and have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as the addition of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter protein function. The term also encompasses active fragments and active derivatives of the antibodies of the present invention.

[0227] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0228] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention also encompasses fragments of the antibodies of the invention. Typically, the fragments comprise at least about 50 contiguous amino acids of the antibodies of the invention, 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.

[0229] In the present invention, the antibodies can be monospecific, bispecific, trispecific, or more multispecific.

[0230] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table 4.

[0231] Table 4

[0232]

[0233] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0234] Preferably, the nucleotide sequence encoding the antibody of the present invention is as follows:

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0242] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0243] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0244] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0245] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0246] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0247] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0248] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0249] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0250] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0251] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0252] As used herein, a "chimeric antigen receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is also referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)." The "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to an antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known to be a domain that transmits signals to activate or inhibit biological processes within a cell.

[0253] As used herein, "domain" refers to a region of a polypeptide that is independent of other regions and folds into a specific structure.

[0254] The antibodies of the present invention may be used alone or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0255] The present invention also provides other proteins or fusion expression products comprising the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) comprising a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0256] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by binding therapeutic agents, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the antibodies or fragments thereof targeting IL18BP.

[0257] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0258] Therapeutic agents that can be combined or coupled with the antibodies of the present invention include but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0259] In another preferred embodiment, the therapeutic agent is an immune checkpoint drug, which is an inhibitor or activator of an immune checkpoint selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR. Preferably, the immune checkpoint is PD-1 and / or PD-L1, and the therapeutic agent is a PD-1 antibody and / or a PD-L1 antibody.

[0260] As used herein, the terms "administer" and "treat" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer" and "treat" can refer to therapy, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, and contact of a fluid with a cell. "Administer" and "treat" also mean in vitro and ex vivo treatment by an agent, diagnostic, binding composition, or by another cell. "Treatment," when applied to a human, animal, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; including contact of an anti-IL-18BP antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.

[0261] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent, including any of the anti-human IL-18BP antibodies and compositions of the present invention, to a patient experiencing one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Generally, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (a therapeutically effective amount).

[0262] As used herein, the terms "optionally" or "optionally" mean that the subsequently described event or circumstance may occur but is not required to occur. For example, "optionally, the antibody against IL-18BP or its antigen-binding fragment obtained in step (b) is purified and / or modified" means that "the antibody against IL-18BP or its antigen-binding fragment obtained in step (b) is purified and / or modified." This step may occur but is not required.

[0263] IL-18 and IL-18BP

[0264] Interleukin-18 (IL18, also known as interferon-gamma-inducing factor) is part of the immune system's arsenal of cytokines. In humans, it is encoded by the IL-18 gene. The protein encoded by this gene is a pro-inflammatory cytokine. Many cell types, both hematopoietic and non-hematopoietic, have the potential to produce IL-18.

[0265] Recent studies have shown that IL-18 inhibits tumor growth primarily by enhancing the activity of effector T cells and NK cells and increasing the expression of anti-tumor effector molecules. Researchers analyzing the transcriptome-level expression characteristics of cytokines and related receptors in tumor-infiltrating CD8+ T cells found that IL-18 and its two receptor subunits (IL-18Rα / Rβ) were enriched in both activated and dysregulated tumor-infiltrating CD8+ T cells, suggesting that IL-18 stimulation can effectively activate anti-tumor immune responses.

[0266] In many types of cancer, there is a high level of a protein called interleukin-18 binding protein (IL-18 Binding Protein, IL-18BP), which acts as a "decoy receptor" to prevent IL-18 from binding to receptors on immune system cells and activating an immune response. Studies have found that after patients are treated with recombinant IL-18, IL-18 can be neutralized by IL-18BP. After IL-18 treatment, the concentration of IL-18BP in the patient's serum increases by 10-100 times. This is a secreted strong antagonist that binds to IL-18 with extremely strong affinity, and the affinity constant KD is less than 1nM. Therefore, IL-18BP in the tumor microenvironment may play the role of a secretory immune checkpoint, limiting the immunotherapy effect of IL-18.

[0267] Pharmaceutical composition

[0268] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-mentioned antibody, active fragment thereof, or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.

[0269] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0270] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight and, in most cases, does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0271] Preferably, the pharmaceutical composition further comprises a second active ingredient, PD-1 / PD-L1 antibody, and the ratio of the antibody or antigen-binding fragment thereof of the present invention as the first active ingredient to the second active ingredient is 5:1-1:1, preferably 3:1.

[0272] Typically, the dosage of the first active ingredient in the pharmaceutical composition is 5-60 mg / kg, preferably 10-30 mg / kg; the dosage of the PD-1 antibody is 5-20 mg / kg, preferably 10 mg / kg.

[0273] Antibodies against IL-18BP

[0274] In the present invention, the antibodies against IL-18BP include monomers, bivalents (bivalent antibodies), tetravalents (tetravalent antibodies), and / or multivalents (multivalent antibodies).

[0275] In a preferred embodiment of the present invention, the antibody against IL-18BP comprises one or more heavy chains having a heavy chain variable region as shown in SEQ ID NO: 1, 9, 17, 25, 32, 37, 41, 49, 57, 65 or 67, or a heavy chain having at least 90%, 95% or 98% sequence identity thereto, and a light chain variable region as shown in SEQ ID NO: 5, 13, 21, 29, 35, 39, 45, 53, 61 or 66, or a light chain having at least 90%, 95% or 98% sequence identity thereto.

[0276] Labeled antibodies

[0277] In a preferred embodiment of the present invention, the antibody carries a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label, or a fluorescent label.

[0278] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, an antibody against IL-18BP protein is labeled with colloidal gold to obtain a colloidal gold-labeled antibody.

[0279] The antibody against IL-18BP of the present invention can effectively bind to IL-18BP protein.

[0280] Antibody preparation

[0281] Any method suitable for producing monoclonal antibodies can be used to produce the IL-18BP antibodies of the present invention. For example, animals can be immunized with a linked or naturally occurring IL-18BP protein or fragments thereof. Suitable immunization methods, including adjuvants, immunostimulants, and repeated booster immunizations, can be used, using one or more routes.

[0282] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al. Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. patent application 4,816,567), or phage antibody library technology (see, for example, Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).

[0283] For example, monoclonal antibodies can be prepared as follows. First, mice or other suitable host animals are immunized with an immunogen (with an adjuvant added if necessary). After immunization, the animal will produce lymphocytes that secrete antibodies that specifically bind to the immunogen. Alternatively, lymphocytes can also be obtained by in vitro immunization. The target lymphocytes are collected and fused with myeloma cells using a suitable fusing agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). The hybridoma cells prepared above can be inoculated into a suitable culture medium for growth, and the culture medium of the growing hybridoma cells is used to detect the production of monoclonal antibodies against the specific antigen. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). For example, the affinity of monoclonal antibodies can be determined using the Scatchard analysis described by Munson et al. in Anal. Biochem. 107:220 (1980). After determining the specificity, affinity, and reactivity of the antibodies produced by the hybridoma, the target cell line can be subcloned using the standard limiting dilution method described in (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). Suitable culture media include DMEM or RPMI-1640. Alternatively, hybridoma cells can be grown in animals as ascites tumors. The monoclonal antibodies secreted by the subcloned cells can be isolated from the cell culture medium, ascites, or serum using conventional immunoglobulin purification methods, such as protein A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0284] Monoclonal antibodies can also be obtained through genetic engineering and recombinant technology. Using nucleic acid primers that specifically bind to the heavy and light chain genes of the monoclonal antibody, PCR amplification can be performed to isolate DNA molecules encoding the heavy and light chain genes of the monoclonal antibody from hybridoma cells. The resulting DNA molecules are inserted into expression vectors, which are then transfected into host cells (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins) and cultured under appropriate conditions to obtain recombinantly expressed target antibodies.

[0285] Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G. Subsequently or alternatively, the specific antigen (the target molecule recognized by the antibody) or its antigenic epitope can be immobilized on a column and the immunospecific antibody can be purified by immunoaffinity chromatography. For purification of immunoglobulins, reference can be made, for example, to D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0286] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)).

[0287] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0288] Humanized antibodies are particularly advantageous because they can retain the expected properties of non-human donor antibodies (e.g., mouse antibodies) while effectively reducing the immunogenicity of non-human donor antibodies (e.g., mouse antibodies) in human subjects. However, due to the matching problem between the CDRs of the donor antibody and the FRs of the recipient antibody, the expected properties of humanized antibodies (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response) are generally lower than those of non-human donor antibodies (e.g., mouse antibodies).

[0289] Therefore, although researchers in this field have conducted in-depth research on antibody humanization and achieved some progress (see, for example, Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992); and Clark, Immunol. Today 21: 397-402 (2000)), the prior art does not provide detailed guidance on how to fully humanize a donor antibody so that the resulting humanized antibody has the highest possible degree of humanization while retaining the desired properties of the donor antibody as much as possible. Technicians need to explore, explore and modify specific donor antibodies and put in a lot of creative work to obtain humanized antibodies that have a high degree of humanization (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% humanization) while retaining the expected properties of the specific donor antibody.

[0290] In the present invention, in order to enable the humanized antibody to retain the properties of the donor antibody as much as possible (including, for example, antigen specificity, affinity, reactivity, the ability to increase immune cell activity and / or the ability to enhance immune response), the framework region (FR) in the humanized antibody of the present invention may contain both amino acid residues of the human recipient antibody and amino acid residues of the corresponding non-human donor antibody.

[0291] In this application, the properties of the antibodies of the present invention include: (1) specific recognition / binding to IL-18BP (particularly human IL-18BP); and (2) inhibition and / or blocking of the binding of IL-18BP to IL-18. The humanized antibodies of the present invention retain one or more of the above properties of the parent antibody (murine antibody or mouse-human chimeric antibody).

[0292] Chimeric or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal antibody prepared above. DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques.

[0293] To prepare chimeric antibodies, murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). For example, DNA encoding VH can be operably linked to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene, or DNA encoding VL can be operably linked to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (as well as a Fab light chain gene). The sequences of human heavy chain constant region genes and light chain constant region genes are known in the art (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be IgG1 (e.g., uniprot ID DP01857), IgG2 (e.g., uniprot ID P01859), IgG3 (e.g., uniprot ID P01860), IgG4 (e.g., uniprot ID P01861), IgA, IgE, IgM, or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region. The light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.

[0294] To create humanized antibodies, the murine CDR regions can be inserted into human framework sequences using methods known in the art (see U.S. Pat. No. 5,225,539 to Winter; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370, all by Queen et al.; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be used that are capable of producing no endogenous immunoglobulins upon immunization and that are capable of producing a full repertoire of human antibodies (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258; Lonberg et al. (1994) Nature 368(6474):856-859; WO 02 / 43478). Other methods for antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

[0295] As used herein, the term "humanization degree" is an indicator used to evaluate the number of non-human amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be, for example, predicted by using the IMGT website DomainGapAlign to determine the homology between the variable region sequence and the human V domain.

[0296] Any suitable form of IL-18BP can be used as an immunogen (antigen) to generate non-human antibodies specific for IL-18BP and screen the biological activity of the antibodies. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen can be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in origin. The DNA encoding the immunogen can be expressed using a suitable genetic vector, including but not limited to adenoviral vectors, baculoviral vectors, plasmids, and non-viral vectors.

[0297] Detection method

[0298] The present invention also relates to a method for detecting IL-18BP protein or a fragment thereof. The method generally comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of IL-18BP protein in the dissolved sample.

[0299] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage medium.

[0300] Reagent test kit

[0301] The present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc.

[0302] The present invention also provides a detection kit for detecting IL-18BP protein levels, which includes an antibody that recognizes IL-18BP protein, a lysis medium for dissolving the sample, and common reagents and buffers required for detection, such as various buffers, a detection marker, and a detection substrate. The detection kit can be an in vitro diagnostic device.

[0303] application

[0304] As described above, the antibodies of the present invention have broad biological and clinical applications, encompassing the treatment of IL-18-related diseases, diagnosis of IL-18-related diseases, basic medical research, biological research, and other fields. A preferred application is in the clinical prevention and treatment of IL-18BP protein.

[0305] The present invention also provides a method for stimulating an immune response mediated by T cells targeting a tumor cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cells of the present invention to the mammal.

[0306] In one embodiment, the present invention comprises a type of cell therapy in which a patient's own T cells (or those of an allogeneic donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then infused back into the same patient. This approach minimizes the likelihood of a graft-versus-host reaction, as antigens are recognized by T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0307] In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and can persist for months to years. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, wherein the CAR-T cells can induce a specific immune response against tumor cells that overexpress the antigen recognized by the CAR antigen binding domain. For example, the CAR-T cells of the present invention induce a specific immune response against tumor cells that overexpress IL-18BP.

[0308] Treatable cancers include tumors that are not vascularized or substantially not vascularized, as well as vascularized tumors. Cancer types treated with the CAR of the present invention include, but are not limited to, gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like.

[0309] Generally, cells activated and expanded as described herein can be used to treat and prevent diseases such as tumors. Therefore, the present invention provides a method for treating cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of the CAR-T cells of the present invention.

[0310] The CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may include a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0311] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or may be determined by clinical trials.

[0312] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. The pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 7The T cell composition can be administered at a dose of 10 cells / kg body weight (including all integer values within the range). The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a specific patient can be easily determined by a person skilled in the art of medicine by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0313] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous injection. The T cell composition can be injected directly into the tumor, lymph node or infection site.

[0314] In certain embodiments of the present 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 with (e.g., before, simultaneously, or after) any number of related treatment modalities, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or efavirenz treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0315] The dosage 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. Dosage ratios for human administration may be implemented according to practices accepted in the art. Typically, 1×10 5 to 1×10 10The modified T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0316] Those skilled in the art will appreciate that the above applications can be used in combination with other treatment methods, such as in combination with PD-1 / PD-L1 antibodies.

[0317] The main advantages of the present invention include:

[0318] 1. The antibody of the present invention is a monoclonal antibody with high affinity, high specificity and high titer, and the affinity can reach the pM level.

[0319] 2. The antibodies and antigen-binding fragments thereof of the present invention not only specifically recognize and bind to IL-18BP but also block the binding of IL-18BP to IL-18. Therefore, the antibodies and antigen-binding fragments thereof of the present invention have the potential to prevent and / or treat a variety of diseases, including tumors, inflammatory diseases, and metabolic diseases.

[0320] 3. The antibody or antigen-binding fragment thereof against IL-18BP of the present invention can restore the IFN-γ release activity induced by IL-18.

[0321] 4. The IL-18BP antibody or antigen-binding fragment thereof of the present invention has good binding activity to IL-18BP secreted by human tumor cells.

[0322] 5. The humanized antibodies of the present invention can be safely administered to human subjects without triggering an immunogenic response. Therefore, the antibodies of the present invention have significant clinical value.

[0323] 6. The IL-18BP antibody or antigen-binding fragment thereof of the present invention can release natural IL-18 in the body and activate IL-18 signaling to enhance the tumor-killing ability of T cells and NK cells.

[0324] 7. The antibodies and antigen-binding fragments thereof of the present invention can be used in combination with PD-1 antibodies / PD-L1 antibodies to enhance the inhibitory effect on tumors.

[0325] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0326] Example 1. Preparation of high-affinity anti-IL-18BP antibodies

[0327] The purified human IL-18BP recombinant protein (antigen sequence Human IL-18BP (NP_001034748): TPVSQTTTAATASVRSTKDPCPSQPPVFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSILYWLGNGSFIEHLPGRLWEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVLVDPEQVVQRHVVLAQLWAGLRATLPPTQEALPSSHSSPQQQG, SEQ ID NO: 68) was mixed with an equal volume of complete Freund's adjuvant and used to immunize 8-10 week old SJL female mice.

[0328] Afterwards, the mice were given a booster immunization at intervals of 2 weeks, 3 weeks, and 3 weeks. After 4 immunizations, the serum titer of the mice detected by ELISA reached 1:10. 5 At 4 hr, the immunized mice were sacrificed by orbital blood sampling and cervical dislocation. The abdomen was disinfected with 75% alcohol, and the spleen and lymph nodes were surgically removed to remove excess connective tissue.

[0329] Collect spleens and lymph nodes to prepare a cell suspension, and collect single lymphocyte suspensions for later use. One day before cell fusion, prepare mouse peritoneal macrophages as feeder cells and culture them in a cell culture incubator at 37°C, 5% CO2. Resuscitate Sp2 / 0-Ag14 myeloma cells, ensuring they are in the logarithmic growth phase at the time of fusion. Wash SP2 / 0 cells twice with incomplete RPMI-1640 medium and resuspend in incomplete RPMI-1640 medium for later use.

[0330] The lymphocyte suspension and Sp2 / 0-Ag14 cells were fused using 50% PEG4000 (pH 8.0-8.2) and electroporated. The fused cells were cultured in HAT medium for 1-2 weeks, and the supernatant was collected and assayed for antibody binding activity using ELISA.

[0331] Subcloning was performed using the limiting dilution method, and positive hybridoma cell lines were obtained after multiple screenings. Total RNA from the positive cloned hybridomas was extracted, and cDNA containing the variable region sequence was obtained by RT-PCR using 3' RACE and 5' RACE techniques. The VH and VL genes were cloned into a T vector and sequenced. The sequences were then compared and analyzed to determine the variable region gene sequence.

[0332] The variable region sequences were combined with the human IgG1 / IgG4 constant region to construct chimeric antibody expression vectors. These expression vectors were transfected into 293F cells and purified to obtain large quantities of chimeric antibodies. The resulting chimeric antibodies were designated Ab001, Ab002, Ab003, Ab009, Ab010, Ab012, Ab017, Ab018, and Ab020.

[0333] Example 2. Humanized modification of anti-IL-18BP antibody

[0334] The humanization of antibody Ab009 was performed using CDR grafting and backmutation. The FR and CDR regions were defined using the CDR numbering convention. An IGBLAST search was performed on the VL and VH sequences of Ab009, respectively, against the antibody sequence database.

[0335] Based on the search results, IGHV1-18*01 was selected as the VH template, and IGKV6-21*01 as the VL template. The antibody CDRs were transplanted into the VH and VL templates, respectively, for homology modeling. Based on factors such as three-dimensional structure, immunogenicity, and physicochemical properties, a few residues were mutated back to their mouse counterparts, and the sequences were further optimized.

[0336] The sequence information of humanized antibody Ab009 (VH12VL9) and humanized antibody Ab009 (VH14VL9) is as follows: Figure 1 Among them, underlined regions represent CDR regions, double-underlined and bolded regions represent mutation sites based on IGHV1-18*01 (VH template) or IGKV6-21*01 (VL template), and wavy and bolded regions represent mutation sites based on Ab009.

[0337] The VH region of the humanized antibody Ab009 (VH12VL9) sequence was mutated to M at position 37, to K at position 38, to I at position 48, to K at position 67, to A at position 68, to A at position 72, and to F at position 95 based on the IGHV1-18*01 template.

[0338] The VL region of the humanized antibody Ab009 (VH12VL9) sequence was mutated to R at position 45, to Y at position 48, and to Y at position 70 based on the IGKV6-21*01 template.

[0339] The VH region of the humanized antibody Ab009 (VH14VL9) sequence was mutated to V at position 24, to M at position 37, to K at position 38, to I at position 48, to K at position 67, to A at position 68, to L at position 70, and to F at position 95 based on the IGHV1-18*01 template.

[0340] The VL region of the humanized antibody Ab009 (VH14VL9) sequence was mutated to R at position 45, to Y at position 48, and to Y at position 70 based on the IGKV6-21*01 template.

[0341] The VH region of the humanized antibody Ab009 (VH12VL9) sequence is based on the VH region of Ab009 (SEQ ID NO: 25) with the following mutations:

[0342] Q5V, D8G, L11V, V12K, I20V, V24S, Y25S, R40A, E42G, L70M, K74T, S76T, Q82E, N84R, T87R, E89D, S91T, and L112V.

[0343] The VL region of the humanized antibody Ab009 (VH12VL9) sequence has the following mutations based on the VL region of Ab009 (SEQ ID NO: 29):

[0344] Q1E, A9D, I10F, M11Q, A13V, S14T, G16K, M21I, S39P, G40D, T41Q, W46L, T59S, S69D, S71T, S75N, M77L, A99Q, and L105I.

[0345] The VH region of the humanized antibody Ab009 (VH14VL9) sequence is based on the VH region of Ab009 (SEQ ID NO: 25) with the following mutations:

[0346] Q5V, D8G, L11V, V12K, A16S, I20V, Y25S, R40A, E42G, S76T, Q82E, N84S, T87R, S91T, and L112V.

[0347] The VL region of the humanized antibody Ab009 (VH14VL9) sequence has the following mutations based on the VL region of Ab009 (SEQ ID NO: 29):

[0348] Q1E, A9D, I10F, M11Q, A13V, S14T, G16K, M21I, S39P, G40D, T41Q, W46L, T59S, S69D, S71T, S75N, M77L, A99Q, and L105I.

[0349] The humanized modified molecules designed above were constructed into the IgG1 / IgG4 constant region and transfected into expi-293 cells to express and purify the humanized antibody molecules.

[0350] The affinity of the humanized antibody was determined using Biacore 8K with an association time of 120 s, a dissociation time of 360 s, and a regeneration buffer of 10 mM glycine-HCl pH 1.5.

[0351] As shown in Table 5, the affinity of the humanized Ab009 antibody is consistent with that of the mouse molecule.

[0352] Table 5 Binding kinetics of Ab009 humanized antibody (SPR)

[0353]

[0354] Example 3. Affinity evaluation of anti-IL-18BP antibodies

[0355] The diluent was used to prepare 1 μg / mL of human IL-18BP protein, mouse IL-18BP protein, and cynomolgus monkey IL-18BP protein, which were then coated on 96-well ELISA plates, respectively, at 4°C overnight.

[0356] IL-18BP antibody was serially diluted three-fold starting at 100 nM into 11 concentrations and reacted with coated human, mouse, and cynomolgus monkey IL-18BP proteins. HRP-conjugated goat anti-human IgGκ was used as a secondary antibody for color development. OD450 nm was plotted against antibody concentration to calculate EC50.

[0357] The binding activities of these 10 antibodies to IL-18BP proteins from different species are summarized in Table 6. IL-18BP antibodies showed excellent affinity for human IL-18BP, with Ab002, Ab003, Ab009, Ab010, Ab012, Ab017, Ab018, and Ab020 all having affinities at the pM level.

[0358] Table 6 IL-18BP antibody affinity test

[0359]

[0360] Example 4. Anti-IL-18BP antibody can restore IL-18-induced IFN-γ release activity in KG-1 cells

[0361] 3×10 5 KG-1 cells (ATCC, #CCL-246) were seeded into each well of a 96-well plate. Serial dilutions of the antibody prepared in Example 1 were added to the wells. After 30 minutes, 0.01 μg / mL of human IL-18BP was added. Another 30 minutes later, KG-1 cells were stimulated with 10 ng / mL IL-18 and 20 ng / mL TNFα.

[0362] After 24 hours of incubation, cells were pelleted by centrifugation, and IFN-γ production was measured from the supernatant using an ELISA kit (R&D Systems, #VAL104C) according to the manufacturer's instructions.

[0363] Representative experimental results are shown in Figure 2 As shown in Table 7, Ab002, Ab003, Ab009, Ab010, Ab012 and Ab017 can restore the IL-18BP-inhibited IFN-γ release in KG-1 cells. The average EC50 of the two experiments is shown in Table 7.

[0364] Table 7 EC50 of anti-IL-18BP antibodies in restoring IL-18-induced IFN-γ release

[0365]

[0366] In addition, if Figure 3 As shown, the KG-1 functional assay showed that the humanized antibody also had strong activity (EC50 = 12.15 nM).

[0367] Example 5. Anti-IL-18BP antibody restores IL-18-induced IFN-γ release activity in human peripheral blood mononuclear cells sex

[0368] Human peripheral blood mononuclear cells (PBMCs) (TPCS, #PB025C-W) were cultured at 1×10 5 Cells were seeded at a concentration of 10 cells / well in a 96-well plate, and serial dilutions of mAb003 and mAb009 antibodies of mouse origin, IgG1 subtype and IgG4 subtype, respectively, were added to the wells.

[0369] After 30 minutes, 0.01 μg / mL of human IL-18BP was added, and after another 30 minutes, PBMC cells were stimulated with 50 ng / mL IL-18 and 5 ng / mL IL-12.

[0370] After incubation for 48 hours, the amount of IFN-γ produced was measured from the supernatant using an ELISA kit (R&D Systems, #VAL104C) according to the manufacturer's instructions.

[0371] The results are as follows Figure 4 As shown, IL-18BP antibodies can restore IL-18-induced IFN-γ release in PBMC cells.

[0372] Example 6. Binding activity of anti-IL-18BP antibodies to IL-18BP derived from human tumor cells

[0373] Human ovarian cancer cell lines can secrete a large amount of IL-18BP under IFN-g stimulation. 6SK-OV-3 cells were seeded into 10 cm culture dishes and stimulated with 50 ng / mL human IFN-g. After 24 hours, the supernatant was collected to obtain conditioned medium containing IL-18BP, and the concentration of IL-BPa in the medium was quantified using a human IL-18BPa ELISA detection kit.

[0374] IL-18BP antibody was prepared at 1 μg / mL using the diluent and coated onto 96-well ELISA plates overnight at 4°C. Conditioned medium containing IL-18BP was diluted to 1200 pg / mL, 400 pg / mL, and 133.3 pg / mL and reacted with the coated anti-IL-18BP antibody. Color was developed using HRP-labeled anti-human IL-18BP antibody, and the OD 450 nm was plotted against the antibody concentration.

[0375] like Figure 5 The results showed that Ab002, Ab012, Ab018, Ab009, etc. had good binding activity to IL-18BP secreted by human tumor cells.

[0376] Example 7. In vivo efficacy test of PD-1 antibody and IL-18BP antibody and the effect of combined administration on tumor Tumor suppressive effect

[0377] Cell source: ATCC

[0378] PD-1 Antibody: BioXcell BE0146 Cat#:BP0146, Clone:RMP1-14

[0379] Animals: BALB / c mice, 6-8 weeks old, female, purchased from Weitonglihua Laboratory Animal Technology Co., Ltd.

[0380] Tumor-bearing mice were measured for tumor size, grouped, and dosed. The specific steps are as follows:

[0381] a) Based on tumor growth, the tumor was measured 7-9 days after inoculation and the tumor size was calculated. The tumor volume was calculated according to the following formula: tumor volume (mm 3 ) = length (mm) × width (mm) × width (mm) / 2.

[0382] b) Tumor-bearing mice were randomly divided into groups according to their weight and tumor size.

[0383] c) Based on the grouping results, the test drug was started. Specific dosing information is shown in Table 8.

[0384] d) Tumors were measured and weighed three times a week after the start of administration of the test drug.

[0385] e) Euthanize the animals after the experiment.

[0386] f) Data were processed using Excel or other software. The tumor inhibition rate (TGI) of the test compound was calculated according to the following formula: TGI% = [1 - (tumor volume of the treated group on the day of evaluation - tumor volume of the treated group) / (tumor volume of the control group on the day of evaluation - tumor volume of the control group)] × 100.

[0387] g) Experimental data are shown in Table 8, and the rate of change of tumor volume and body weight during the experiment are shown in Figure 6 and Figure 7 .

[0388] Table 8 Tumor inhibition rate (%) and body weight change rate (%) of different doses of Ab002 combined with PD-1 antibody

[0389]

[0390] h) Experimental results

[0391] The above results demonstrate that Ab002, administered at various doses (including doses equivalent to PD-1 alone), exhibited superior tumor inhibition compared to PD-1 alone. Furthermore, Ab002 combined with PD-1 also significantly enhanced tumor inhibition compared to PD-1 alone, demonstrating that combined therapy enhances tumor inhibition. Furthermore, regardless of whether the drug was administered alone or in combination, the animals maintained weight gain, demonstrating that Ab002 exhibits a favorable safety profile.

[0392] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An antibody or antigen-binding fragment thereof against IL-18BP, characterized in that: include: (a) heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of which are shown in SEQ ID NOs: 10, 11, and 12, respectively; and light chain complementary determining regions CDRL1, CDRL2, and CDRL3, the amino acid sequences of which are shown in SEQ ID NOs: 14, 15, and 16, respectively; or (b) heavy chain complementary determining regions CDRH1, CDRH2, CDRH3, the amino acid sequences of which are shown in SEQ ID NOs: 26, 27, and 28, respectively; and light chain complementary determining regions CDRL1, CDRL2, CDRL3, the amino acid sequences of which are shown in SEQ ID NOs: 30, 15, and 31, respectively; or (c) heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of which are shown in SEQ ID NOs: 38, 27, and 28, respectively; and light chain complementary determining regions CDRL1, CDRL2, and CDRL3, the amino acid sequences of which are shown in SEQ ID NOs: 40, 15, and 31, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO: 9 and SEQ ID NO: 13, respectively, or in SEQ ID NO: 37 and SEQ ID NO: 39, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO: 25 and SEQ ID NO: 29, respectively, or shown in SEQ ID NO: 65 and SEQ ID NO: 66, respectively, or shown in SEQ ID NO: 67 and SEQ ID NO: 66, respectively.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibodies or antigen-binding fragments thereof include monomers, bivalent antibodies, and / or multivalent antibodies.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein The bivalent antibody is a bispecific antibody.

6. The antibody or antigen-binding fragment thereof according to claim 4, wherein The multivalent antibody is a multispecific antibody.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region of the antibody or antigen-binding fragment thereof has a mutation selected from the following group based on SEQ ID NO: 25: Q5V, D8G, L11V, V12K, I20V, V24A, Y25S, R40A, E42G, L70M, K74T, S76T, Q82E, N84R, T87R, E89D, S91T, and L112V; and The light chain variable region of the antibody or antigen-binding fragment thereof has a mutation selected from the following group based on SEQ ID NO: 29: Q1E, A9D, I10F, M11Q, A13V, S14T, G16K, M21I, S39P, G40D, T41Q, W46L, T59S, S69D, S71T, S75N, M77L, A99Q, and L105I; or The heavy chain variable region of the antibody or antigen-binding fragment thereof has a mutation selected from the following group based on SEQ ID NO: 25: Q5V, D8G, L11V, V12K, A16S, I20V, Y25S, R40A, E42G, S76T, Q82E, N84S, T87R, S91T, and L112V; and The light chain variable region of the antibody or antigen-binding fragment thereof has a mutation selected from the following group based on SEQ ID NO: 29: Q1E, A9D, I10F, M11Q, A13V, S14T, G16K, M21I, S39P, G40D, T41Q, W46L, T59S, S69D, S71T, S75N, M77L, A99Q, and L105I.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein The amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO: 65 and SEQ ID NO: 66, respectively, or shown in SEQ ID NO: 67 and SEQ ID NO: 66, respectively; and the antibody or antigen-binding fragment thereof is a humanized antibody.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain and light chain amino acid sequences of the antibody or antigen-binding fragment thereof are shown in SEQ ID NO: 71 and SEQ ID NO: 72, respectively, or in SEQ ID NO: 75 and SEQ ID NO: 76, respectively, or in SEQ ID NO: 77 and SEQ ID NO: 78, respectively, or in SEQ ID NO: 79 and SEQ ID NO: 80, respectively, or in SEQ ID NO: 83 and SEQ ID NO: 84, respectively.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a murine antibody, a humanized antibody or a chimeric antibody.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain constant region of the antibody or antigen-binding fragment thereof is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain constant region of the antibody or antigen-binding fragment thereof is selected from the heavy chain constant region of human IgG1 or IgG4.

13. The antibody or antigen-binding fragment thereof according to claim 10, wherein The constant region of the chimeric antibody is derived from the heavy chain constant region of human IgG1.

14. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antigen binding fragment is selected from scFv, Fab, Fab', F(ab')2, Fv fragment, and disulfide-linked Fv (dsFv).

15. The antibody or antigen-binding fragment thereof according to claim 1, wherein The light chain constant region of the antibody or antigen-binding fragment thereof is selected from the constant region of a human antibody κ chain or λ chain.

16. A recombinant protein, characterized in that The recombinant protein has: (i) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15; and (ii) Tag sequences to facilitate expression and / or purification.

17. The recombinant protein according to claim 16, wherein The tag sequence includes an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

18. The recombinant protein according to claim 16, wherein The recombinant protein includes a fusion protein.

19. The recombinant protein according to claim 16, wherein The recombinant protein is a monomer, a dimer, or a polymer.

20. A nucleotide molecule, characterized in that The nucleotide molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the recombinant protein according to any one of claims 16 to 19.

21. The nucleotide molecule according to claim 20, wherein The nucleotide molecules are RNA and DNA.

22. The nucleotide molecule according to claim 20, wherein The nucleotide molecule is cDNA.

23. The nucleotide molecule according to claim 20, wherein The nucleotide molecule includes: a heavy chain nucleotide sequence as shown in SEQ ID NO: 93 and a light chain nucleotide sequence as shown in SEQ ID NO: 94; or a heavy chain nucleotide sequence as shown in SEQ ID NO: 97 and a light chain nucleotide sequence as shown in SEQ ID NO: 98; or a heavy chain nucleotide sequence as shown in SEQ ID NO: 101 and a light chain nucleotide sequence as shown in SEQ ID NO:

102.

24. An expression vector, characterized in that The expression vector contains the nucleotide molecule according to any one of claims 20 to 23.

25. The expression vector according to claim 24, wherein The expression vector is selected from the group consisting of viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

26. The expression vector according to claim 24, wherein The expression vector includes a viral vector.

27. The expression vector according to claim 26, wherein The viral vector is selected from the group consisting of lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

28. The expression vector according to claim 24, wherein The expression vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, and pLV.

29. The expression vector according to claim 24, wherein The expression vector further comprises elements selected from the following group: a promoter, a transcription enhancing element WPRE, and a long terminal repeat sequence LTR.

30. A host cell, characterized in that The host cell contains the expression vector according to any one of claims 24 to 29, or the nucleotide molecule according to any one of claims 20 to 23 is integrated into its genome.

31. The host cell according to claim 30, wherein The host cells include prokaryotic cells or eukaryotic cells.

32. The host cell according to claim 30, wherein The host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

33. The host cell according to claim 30, wherein The host cell is a 293F cell or an expi-293 cell.

34. A chimeric antigen receptor CAR, characterized in that The antigen binding region scFv segment of the CAR is a binding region that specifically binds to IL-18BP, and the heavy chain variable region and light chain variable region of the scFv include: (a) heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of which are shown in SEQ ID NOs: 10, 11, and 12, respectively; and light chain complementary determining regions CDRL1, CDRL2, and CDRL3, the amino acid sequences of which are shown in SEQ ID NOs: 14, 15, and 16, respectively; or (b) heavy chain complementary determining regions CDRH1, CDRH2, CDRH3, the amino acid sequences of which are shown in SEQ ID NOs: 26, 27, and 28, respectively; and light chain complementary determining regions CDRL1, CDRL2, CDRL3, the amino acid sequences of which are shown in SEQ ID NOs: 30, 15, and 31, respectively; or (c) heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of which are shown in SEQ ID NOs: 38, 27, and 28, respectively; and light chain complementary determining regions CDRL1, CDRL2, and CDRL3, the amino acid sequences of which are shown in SEQ ID NOs: 40, 15, and 31, respectively.

35. A method for producing an antibody or an antigen-binding fragment thereof against IL-18BP, characterized in that: Including steps: (a) culturing the host cell according to any one of claims 30 to 33 under suitable conditions to obtain a culture containing an antibody or antigen-binding fragment thereof to IL-18BP; (b) isolating and / or recovering the antibody or antigen-binding fragment thereof against IL-18BP from the culture.

36. The method of claim 35, wherein: The method further comprises the steps of: (c) purifying and / or modifying the antibody or antigen-binding fragment thereof against IL-18BP obtained in step (b).

37. An immunoconjugate, characterized in that The immunoconjugate contains: (a) an antibody portion, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15; and (b) a conjugated moiety selected from the group consisting of a detectable label, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle, a gold nanorod, a nanomagnetic particle, a viral coat protein, or a combination thereof.

38. An immunoconjugate, characterized in that The immunoconjugate contains: (a) an antibody portion, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15; and (b) Therapeutic agents.

39. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (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 15, or a recombinant protein according to any one of claims 16 to 19, or an immunoconjugate according to claim 37 or 38, or a combination thereof; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

40. The pharmaceutical composition according to claim 39, wherein The pharmaceutical composition further comprises a second active ingredient: PD-1 antibody and / or PD-L1 antibody.

41. The pharmaceutical composition of claim 39, wherein The dosage of the active ingredient in the pharmaceutical composition is 5-60 mg / kg.

42. The pharmaceutical composition of claim 39, wherein The dosage of the active ingredient in the pharmaceutical composition is 10-30 mg / kg.

43. The pharmaceutical composition according to claim 40, wherein The dosage of the PD-1 antibody is 5-20 mg / kg.

44. The pharmaceutical composition of claim 40, wherein The dose of the PD-1 antibody is 10 mg / kg.

45. A use of an active ingredient, characterized in that The active ingredient is selected from the group consisting of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, a recombinant protein according to any one of claims 16 to 19, an immunoconjugate according to claim 37 or 38, or a combination thereof, and is used to prepare: (a) medicines for the prevention and / or treatment of diseases; (b) Reagents for detecting IL-18BP-related diseases; The disease is selected from the group consisting of colorectal cancer, renal carcinoma, melanoma, ovarian cancer, lymphoma, or a combination thereof.

46. The use according to claim 45, characterized in that The reagent is a diagnostic reagent.

47. The use according to claim 46, wherein The diagnostic reagent is a detection sheet or a detection plate.

48. The use according to claim 45, wherein The antibody or antigen-binding fragment thereof is used as the first active ingredient in combination with a second active ingredient, and the second active ingredient is an anti-PD-1 antibody and / or an anti-PD-L1 antibody.

49. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (a) a first active ingredient, wherein the first active ingredient is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15; (b) a second active ingredient, which is a PD-1 antibody and / or a PD-L1 antibody; and (c) a pharmaceutically acceptable carrier, diluent or excipient; Wherein, the ratio of the first active ingredient to the second active ingredient is 5:1-1:

1.

50. The pharmaceutical composition of claim 49, wherein the ratio of the first active ingredient to the second active ingredient is 3:

1.

51. The pharmaceutical composition of claim 49, wherein The dosage of the first active ingredient in the pharmaceutical composition is 5-60 mg / kg.

52. The pharmaceutical composition of claim 49, wherein The dosage of the first active ingredient in the pharmaceutical composition is 10-30 mg / kg.

53. The pharmaceutical composition of claim 49, wherein The dosage of the PD-1 antibody is 5-20 mg / kg.

54. The pharmaceutical composition of claim 49, wherein The dose of the PD-1 antibody is 10 mg / kg.

55. A non-diagnostic in vitro method for detecting IL-18BP protein or a fragment thereof in a sample, characterized in that: The method comprises the steps of: (1) contacting the sample in vitro with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of the corresponding target of the IL-18BP protein or its fragment in the sample.

56. A kit, characterized in that The kit includes: a first container containing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or the recombinant protein according to any one of claims 16 to 19; or, The kit contains a detection plate, which includes: a substrate and a test strip, wherein the test strip contains the antibody or antigen-binding fragment thereof as described in any one of claims 1-15, or the recombinant protein as described in any one of claims 16-19, or the immunoconjugate as described in any one of claims 37 or 38, or the pharmaceutical composition as described in any one of claims 39-44, 49-54, or a combination thereof.

57. The kit according to claim 56, wherein The kit further comprises a second container, wherein the second container contains a secondary antibody against the content of the first container, and the secondary antibody in the second container is HRP-labeled goat anti-human IgGκ.

Images