WO2023143535A1 - Antibody targeting il-18bp and use thereof - Google Patents

Abstract

Provided are an antibody targeting IL-18BP or an antigen-binding fragment thereof, a production method therefor, and use thereof. Specifically provided are a murine or chimeric monoclonal antibody and a humanized antibody targeting IL-18BP, a nucleic acid molecule encoding the antibody, a corresponding expression vector, and a host cell. The antibody targeting IL-18BP or the antigen-binding fragment thereof can specifically bind to IL-18BP, has high affinity, can release natural IL-18 in vivo, activate an IL-18 signal to enhance the ability of T cells and NK cells to kill tumors, and thus can be used for preventing/treating tumors, metabolic diseases, immune system diseases, neurodegenerative diseases, cardiovascular diseases, inflammatory diseases, or other diseases.

Description

A kind of antibody targeting IL-18BP and its application technical field

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

Background technique

Interleukin 18 (IL-18) can induce the proliferation of immune cells and enhance their activity, especially the activity of T cells and NK cells, and plays an important role in the regulation of tumor immunity. IL-18 activates downstream signaling pathways by acting on the heterodimeric receptor 18Rα/Rβ on the cell membrane surface to promote the activation and proliferation of immune cells. Most of IL-18 in the body binds to IL-18BP to form a complex. IL-18BP is a naturally occurring IL-18 pseudo-receptor that binds IL-18 with ultra-high affinity (pM level) and blocks its interaction with cell surface receptors. Somatosome interaction, thereby negatively regulating the IL-18 signaling pathway. Studies have found that IL-18BP protein level is up-regulated in the microenvironment of human tumor tissue, which is a new secreted immune checkpoint, which inhibits the killing ability of immune cells in the tumor microenvironment. IL-18BP is also significantly increased in PD-1 resistant non-small cell lung cancer, which is a major factor mediating PD-1 resistance.

Therefore, there is an urgent need in this field to develop high-affinity antibodies against IL-18BP to release natural IL-18 in vivo and activate IL-18 signaling to enhance the killing ability of T cells and NK cells on tumors.

Contents of the invention

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.

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

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

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

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

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

Or as shown in SEQ ID NO:10, 11 and 12 respectively,

Or as shown in SEQ ID NO:18, 19 and 20 respectively,

Or as shown in SEQ ID NO:26, 27 and 28 respectively,

Or as shown in SEQ ID NO:33, 34 and 20 respectively,

Or as shown in SEQ ID NO:38, 27 and 28 respectively,

Or as shown in SEQ ID NO:42, 43 and 44 respectively,

Or as shown in SEQ ID NO:50, 51 and 52 respectively,

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

(b) light chain complementarity determining regions CDRL1, CDRL2, CDRL3, the amino acid sequences of said CDRL1, CDRL2, CDRL3 are respectively shown in SEQ ID NO: 6, 7 and 8,

Or as shown in SEQ ID NO:14, 15 and 16 respectively,

Or as shown in SEQ ID NO:22, 23 and 24 respectively,

Or as shown in SEQ ID NO:30, 15 and 31 respectively,

Or as shown in SEQ ID NO:22, 36 and 24 respectively,

Or as shown in SEQ ID NO:40, 15 and 31 respectively,

Or as shown in SEQ ID NO:46, 47 and 48 respectively,

Or as shown in SEQ ID NO:54, 55 and 56 respectively,

Or respectively as shown in SEQ ID NO:62, 63 and 64;

Wherein, any amino acid sequence in the above amino acid sequence also includes a derivative sequence optionally undergoing addition, deletion, modification and/or substitution of at least one (such as 1-3) amino acids and retaining the ability to specifically bind IL-18BP .

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

In another preferred example, the derivative sequence that has undergone addition, deletion, modification and/or substitution of at least one amino acid and can retain the specific binding ability to IL-18BP has 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% of the amino acid sequence.

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

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

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

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

In another preferred example, the antibody or antigen-binding fragment thereof has heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and light chain complementarity determining regions CDRL1, CDRL2, and CDRL3, and the CDRH1, CDRH2, CDRH3, CDRL1, and CDRL2 The amino acid sequences of CDRL3 and CDRL3 are respectively shown in: SEQ ID NO: 2, 3, 4, 6, 7 and 8, or respectively shown in SEQ ID NO: 10, 11, 12, 14, 15 and 16, or respectively shown in SEQ ID NO: 18, 19, 20, 22, 23 and 24, or as shown in SEQ ID NO:26, 27, 28, 30, 15, 31 respectively, or as shown in SEQ ID NO: 33, 34, 20, 22, 36, 24 respectively, or respectively Shown in SEQ ID NO:38, 27, 28, 40, 15, 31, or shown in SEQ ID NO: 42, 43, 44, 46, 47, 48 respectively, or shown in SEQ ID NO: 50, 51 respectively , 52, 54, 55, 56, or shown in SEQ ID NO: 58, 59, 60, 62, 63, 64 respectively; wherein, any amino acid sequence in the above amino acid sequence also includes optionally added , Deleting, modifying and/or substituting at least one (eg 1-3) amino acid derivative sequence that can retain the specific binding ability to IL-18BP.

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

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

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

In another preferred example, the antibody or antigen-binding fragment thereof includes a murine antibody, a monkey antibody, a humanized antibody or a chimeric antibody.

In another preferred example, the murine antibody is derived from a mouse.

In another preferred example, the monkey-derived antibody is derived from cynomolgus monkeys.

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 in SEQ ID NO: 65 and SEQ ID NO: 66, respectively, or shown in SEQ ID NO :67 and shown in SEQ ID NO:66.

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

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

The light chain variable region of the humanized antibody has a mutation selected from the 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.

In another preferred example, the constant region of the chimeric antibody is derived from the heavy chain constant region of human IgG1.

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

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

In another preferred example, the heavy chain constant region of the antibody or its antigen-binding fragment is selected from human IgG1, IgG2, IgG3 Or the heavy chain constant region of IgG4, the light chain constant region of the antibody or an antigen-binding fragment thereof is selected from the constant region of human antibody kappa chain or lambda chain.

In another preferred example, the heavy chain constant region is the heavy chain constant region of human IgG1 or IgG4.

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 as shown in SEQ ID NO:71 and SEQ ID Shown in NO:72, or respectively as shown in SEQ ID NO:73 and SEQ ID NO:74, or respectively as shown in SEQ ID NO:75 and SEQ ID NO:76, or respectively as shown in SEQ ID NO:77 and SEQ ID NO:78, or respectively as shown in SEQ ID NO:79 and SEQ ID NO:80, or respectively as shown in SEQ ID NO:81 and SEQ ID NO:82, or respectively as shown in SEQ ID NO:83 and Shown in SEQ ID NO:84, or respectively as shown in SEQ ID NO:85 and SEQ ID NO:86, or respectively as shown in SEQ ID NO:87 and SEQ ID NO:88, or respectively as shown in SEQ ID NO:89 and shown in SEQ ID NO:90.

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

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

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

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

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

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

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

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

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

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

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

In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or combinations thereof. Preferably, the expression vectors include viral vectors, such as lentivirus, adeno Viruses, AAV viruses, retroviruses, or combinations thereof.

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

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

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

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

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

In another preferred example, the host cells are 293F cells or expi-293 cells.

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

Heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of the CDRH1, CDRH2, and CDRH3 are shown in SEQ ID NO: 2, 3, and 4, respectively, or shown in SEQ ID NO: 10, 11, and 12, respectively, Or as shown in SEQ ID NO:18, 19 and 20 respectively, or as shown in SEQ ID NO:26, 27 and 28 respectively, or as shown in SEQ ID NO:33, 34 and 20 respectively, or as shown in SEQ ID NO:33, 34 and 20 respectively Shown in NO:38, 27 and 28, or respectively as shown in SEQ ID NO:42, 43 and 44, or respectively as shown in SEQ ID NO:50, 51 and 52, or respectively as shown in SEQ ID NO:58,59 and 60; and/or

The light chain variable region of the scFv comprises:

Light chain complementarity determining regions CDRL1, CDRL2, CDRL3, the amino acid sequences of said CDRL1, CDRL2, CDRL3 are respectively shown in SEQ ID NO: 6, 7 and 8, or respectively shown in SEQ ID NO: 14, 15 and 16, Or as shown in SEQ ID NO:22, 23 and 24 respectively, or as shown in SEQ ID NO:30, 15 and 31 respectively, or as shown in SEQ ID NO:22, 36 and 24 respectively, or as shown in SEQ ID NO:22, 36 and 24 respectively Shown in NO:40,15 and 31, or respectively as shown in SEQ ID NO:46,47 and 48, or respectively as shown in SEQ ID NO:54,55 and 56, or respectively as shown in SEQ ID NO:62,63 and 64 are shown.

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

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

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

L-scFv-H-TM-C-CD3ζ (Ia)

In the formula,

L is nothing or a signal peptide sequence;

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

H is none or hinge region;

TM is the transmembrane domain;

C is costimulatory signal domain;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(b) a coupling moiety selected from the group consisting of detectable markers, therapeutic agents, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combination.

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

In another preferred example, the radionuclides include:

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

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

In another preferred example, the coupling moiety may contain at least one therapeutic agent.

In another preferred example, the coupling moiety is a therapeutic agent.

In another preferred example, 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.

In another preferred example, the therapeutic agent is PD-1 antibody and/or PD-L1 antibody.

In another preferred example, the immune checkpoint drug is an inhibitor or activator of the immune checkpoint.

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

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

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

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

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

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

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

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

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

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

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

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

In another preferred example, the dosage form of the pharmaceutical composition is selected from the group consisting of injections and freeze-dried preparations.

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

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

In another preferred example, the pharmaceutical composition further includes a second active ingredient: PD-1 antibody and/or PD-L1 antibody.

In another preference, the pharmaceutical composition includes:

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

(b) a second active ingredient, 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, preferably 3:1.

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

In another preferred example, the dose of the PD-1 antibody is 5-20 mg/kg, preferably 10 mg/kg.

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

(a) drugs for the prevention and/or treatment of diseases or conditions;

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

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

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

In another preferred example, 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.

In another preferred example, the disease or disorder is a disease or disorder with high expression of IL-18BP or abnormal IL-18 pathway.

In another preferred example, the diseases or conditions include: tumors, metabolic diseases, immune system diseases, neurodegenerative diseases, cardiovascular diseases or inflammatory diseases.

In another preferred example, the disease or condition is a tumor.

In another preferred example, the tumor is a solid tumor or a hematological tumor.

In another preferred example, the tumor is selected from: lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colorectal cancer, nasopharyngeal cancer , brain tumor, cervical cancer, blood cancer, bone cancer, lymphatic cancer, and pancreatic cancer.

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

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

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

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

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

(1) 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

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

or,

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

In another preferred example, the secondary antibody in the second container is HRP-labeled goat anti-human IgGκ.

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

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

In another preferred example, the IL-18BP-related disease is a disease or disease with high expression of IL-18BP or abnormal IL-18 pathway.

In the fourteenth aspect of the present invention, there is provided a method of preventing and/or treating a disease or disorder, the method comprising: administering the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention to a subject in need , 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 tenth aspect of the present invention A pharmaceutical composition according to the aspect, or a combination thereof.

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

In another preferred example, the disease or disorder is a disease or disorder with high expression of IL-18BP or abnormal IL-18 pathway.

In another preferred example, the diseases or conditions include: tumors, metabolic diseases, immune system diseases, neurodegenerative diseases, cardiovascular diseases or inflammatory diseases.

In another preferred example, the disease or condition is a tumor.

In another preferred example, the tumor is a solid tumor or a hematological tumor.

In another preferred example, the tumor is selected from: lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colorectal cancer, nasopharyngeal cancer , brain tumor, cervical cancer, blood cancer, bone cancer, lymphatic cancer, and pancreatic cancer.

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

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

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

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

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

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

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

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

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

In another preferred example, the IL-18BP-related disease is a disease or disease with high expression of IL-18BP or abnormal IL-18 pathway.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the sequence information of humanized antibody Ab009 (VH12VL9) and humanized antibody Ab009 (VH14VL9).

Figure 2 shows that anti-IL-18BP antibody can restore IL-18-induced IFN-γ release activity of KG-1 cells.

Figure 3 shows that the humanized antibody VH12VL9 restores the IFN-γ release activity of KG-1 cells induced by IL-18.

Figure 4 shows that anti-IL-18BP antibody restores IL-18-induced IFN-γ release in human peripheral blood mononuclear cells.

Figure 5A shows the secretion of IL-18BP after SK-OV-3 cell stimulation, and Figure 5B shows the binding activity of IL-18BP antibody to human tumor cell-derived IL-18BP.

Figure 6 shows the changes in tumor volume of mice in different drug administration groups over time.

Fig. 7 shows the body weight change rate (%) of mice in different administration groups.

Detailed ways

Through extensive and in-depth research and a large number of screenings, the inventors unexpectedly developed a class of antibodies and antigen-binding fragments thereof with high specificity and high affinity for IL-18BP for the first time, and also unexpectedly obtained antibodies with excellent affinity and specificity Anti-IL-18BP humanized antibody. The antibody and its antigen-binding fragment of the present invention can block the combination of IL-18BP and IL-18, release natural IL-18 in vivo, and activate IL-18 signal to enhance the ability of T cells and NK cells to kill tumors. Therefore, the antibodies and antigen-binding fragments thereof of the present invention have the potential to be used in the prevention and/or treatment of various diseases such as tumors, inflammation-related diseases and metabolism-related diseases.

the term

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition within an acceptable error range for a 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.

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.

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

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

Table 1

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

Preferably, the numbers of the antibodies of the present invention and the corresponding sequences and numbers of the VH region and VL region, HC and LC are shown in Table 2 below.

Table 2

Preferably, the numbers of the antibodies of the present invention and the corresponding CDR sequences and numbers are shown in Table 3 below.

table 3

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

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

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

The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes, termed kappa and lambda, based on the amino acid sequence of their constant regions. Depending on the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be assigned to different classes. 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. Corresponding to different types of immune globules The heavy-chain constant regions of proteins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

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

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

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

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

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

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

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

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

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

"Sequence identity" in the present invention means the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with mutations such as appropriate substitutions, insertions or deletions. The sequence identity between the sequences described in the present invention and sequences to which they are 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% , 100%.

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

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

As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substitutions The replaced 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 mature polypeptide combined with another compound (such as an elongated polypeptide Compounds with a half-life, such as polyethylene glycol) fused to a polypeptide, or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or protein used to purify the polypeptide original sequence, or a fusion protein formed with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

In the present invention, antibodies include murine, monkey, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and nonhuman portions, which can be obtained by standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as a chimeric antibody having variable regions from a murine monoclonal antibody, and constant regions from a human immunoglobulin (see, e.g., U.S. Patent Nos. 4,816,567 and U.S. Patent 4,816,397, hereby incorporated by reference in its entirety). A humanized antibody is an antibody molecule derived from a non-human species that has one or more complementarity determining regions (CDRs) derived from a non-human species and framework regions derived from a human immunoglobulin molecule (see U.S. Patent No. 5,585,089, incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.

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

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

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

In the present invention, antibodies can be monospecific, bispecific, trispecific, or more multiple specific.

In the present invention, the antibody of the present invention also includes its conservative variant, which means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and optimally Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 4.

Table 4

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

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

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

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

The present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

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

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

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

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

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

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be done by electroporation if desired method is carried out. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

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

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

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

As used herein, "domain" refers to a region of a polypeptide that folds into a specific structure independently of other regions.

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

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

As known to those skilled in the art, immunoconjugates and fusion expression products include: therapeutic agents, toxins, cytokines (Cytokine), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments thereof of the present invention formed conjugates. The present invention also includes cell surface markers or antigens that bind to the antibody against IL18BP or its fragments.

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

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

In another preferred example, the therapeutic agent is an immune checkpoint drug, and the immune checkpoint drug is an immune checkpoint drug. Inhibitors or activators of checkpoints, the immune checkpoints are selected from: 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 PD-1 antibody and/or PD-L1 antibody.

As used herein, the terms "administering" and "treating" 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. "Administering" and "treating" can refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contacting reagents with cells, reagents with fluids, and fluids with cells. "Administering" and "treating" also mean in vitro and ex vivo treatment of a cell by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to a human, animal or research subject means therapeutic treatment, prophylactic or preventive measures, research and diagnosis; , physiological compartments or contact with physiological fluids.

As used herein, the term "treatment" refers to the administration of internal or external therapeutic agents to a patient, who has one or more disease symptoms, and known The therapeutic agent has a therapeutic effect on these symptoms. Generally, an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount) is administered to a patient.

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance can but does not have to occur. For example, "optionally, purifying and/or modifying the antibody against IL-18BP or its antigen-binding fragment obtained in step (b)" means "purifying and/or modifying the antibody against IL-18BP or its antigen-binding fragment obtained in step (b) Antigen-binding fragments are purified and/or modified” This step is possible but not required.

IL-18 and IL-18BP

Interleukin-18 (IL18, also known as interferon-gamma inducible factor) is part of the immune system arsenal known as a cytokine, which in humans is encoded by the IL-18 gene. The protein encoded by this gene is a pro-inflammatory cytokine. Many cell types, both hematopoietic and non-hematopoietic, have the potential to produce IL-18.

Studies in recent years have shown that IL-18 inhibits tumor growth mainly by increasing the activity of effector T cells and NK cells and increasing the expression of anti-tumor effector molecules. The researchers analyzed the transcriptome-level expression characteristics of cytokines and related receptors in tumor-infiltrating CD8+ T cells and found that IL-18 and two subunits of its receptors (IL-18Rα/Rβ) are highly active in activated and dysregulated tumors. All infiltrating CD8+ T cells were enriched, suggesting that the stimulation of IL-18 can effectively activate the anti-tumor immune response.

In many types of cancer, there are high levels of a protein called IL-18 Binding Protein (IL-18BP), which acts as a "decoy receptor" that prevents IL-18 from interacting with cells on immune system cells. The ability of a receptor to bind and activate an immune response. Studies have found that after patients are treated with recombinant IL-18, IL-18 can be neutralized by IL-18BP, and after IL-18 treatment, the concentration of IL-18BP in the serum of patients increases by 10-100 times. This is a secreted strong antagonist, which has a very strong affinity with IL-18, and the affinity constant KD<1nM. Therefore, IL-18BP in the tumor microenvironment may act as a secreted immune checkpoint, limiting the immunotherapeutic effect of IL-18.

pharmaceutical composition

The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intraperitoneal, intravenous, or topical administration.

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

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

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

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

Antibodies against IL-18BP

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

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

labeled antibody

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

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

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

Antibody preparation

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

The monoclonal antibodies of the present invention can be prepared by various 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 No. 4,816,567), or bacteriophage Antibody library technology (see, eg, Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).

For example, monoclonal antibodies can be prepared as follows. Injected mice or other suitable host animals are first immunized with the immunogen (adding an adjuvant if necessary). After the animal is immunized, lymphocytes that secrete antibodies that specifically bind to the immunogen will be produced in the body. In addition, lymphocytes can also be obtained by in vitro immunization. The target lymphocytes are collected and fused with myeloma cells using a suitable fusion 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 to grow, and the culture medium of the growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). For example, the affinity of mAbs can be determined using the Scatchard assay described by Munson et al., Anal. Biochem. 107:220 (1980). After the specificity, affinity and reactivity of the antibody produced by the hybridoma are determined, the target cell line can be limited by the criteria described in (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996). Subcloning by dilution. A suitable culture medium can be DMEM or RPMI-1640, etc. Alternatively, hybridoma cells can also be grown in animals in the form of ascites tumors. Monoclonal antibodies secreted by subcloned cells can be purified from cell culture fluid, isolated from ascites or serum.

Monoclonal antibodies can also be obtained through genetic engineering and recombination techniques. The DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from hybridoma cells by using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody to carry out PCR amplification. Insert the obtained DNA molecule into the expression vector, then transfect host cells (such as E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin), and culture under appropriate conditions, Antibodies of interest can be obtained recombinantly expressed.

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 an epitope thereof can be immobilized on a column and the immunospecific antibody purified by immunoaffinity chromatography. The purification of immunoglobulin can refer to, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.14, No.8 (Apr.17, 2000), pp.25-28).

As used herein, the term "Chimeric antibody" refers to an antibody whose light chain and/or heavy chain are partly derived from an antibody (which may be derived from a particular species or belong to a certain a specific antibody class or subclass), and the other part of the light chain or/and 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 regardless However, it still retains the binding activity to the target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).

As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, and the like. Donor antibodies can be mouse, rat, rabbit or non-human primate with desired properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and/or ability to enhance immune response) Animal-like (eg, cynomolgus monkey) antibodies.

Humanized antibodies can not only retain the expected properties of non-human donor antibodies (such as murine antibodies), but also effectively reduce the immunogenicity of non-human donor antibodies (such as murine antibodies) in human subjects, Therefore, it is particularly advantageous. However, due to matching issues between the CDRs of the donor antibody and the FRs of the recipient antibody, the desired properties of the humanized antibody (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and/or The ability to enhance the immune response) is generally lower than that of non-human donor antibodies (such as murine antibodies).

Therefore, although researchers in this field have carried out in-depth research on the humanization of antibodies and made 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)), however, how to The donor antibody is fully humanized so that the resulting humanized antibody has as high a degree of humanization as possible and can retain the expected properties of the donor antibody as much as possible. The prior art does not provide detailed guidance. Technologists need to explore, explore and modify specific donor antibodies, and only after a lot of creative work can it be obtained, which has a high degree of humanization (such as 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% degree of humanization), while retaining the expectation of a specific donor antibody properties of humanized antibodies.

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

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

The chimeric antibody or humanized antibody of the present invention can be prepared according to the sequence of the mouse monoclonal antibody prepared above. DNA encoding the heavy and light chains can be obtained from the murine hybridoma of interest and engineered to contain non-murine (eg, human) immunoglobulin sequences using standard molecular biology techniques.

To make chimeric antibodies, the murine immunoglobulin variable regions can be linked to human immunoglobulin constant regions using methods known in the art (see, eg, US Patent No. 4,816,567 to Cabilly et al.). For example, DNA encoding VH is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full-length heavy chain gene, or DNA encoding VL is operably linked to another DNA molecule encoding a light chain constant region CL. DNA molecules to obtain the full-length light chain gene (as well as the Fab light chain gene). The sequences of the human heavy and light chain constant region genes are known in the art (see, e.g., Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242), DNA fragments comprising these regions can be amplified by standard PCR. The heavy chain constant region can be an IgG1 (e.g. uniprot ID P01857), 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 typically Preferably an IgGl or IgG4 constant region. The light chain constant region can be a kappa or lambda constant region, but is generally preferred to be a kappa constant region.

To prepare humanized antibodies, the murine CDR regions can be inserted into human framework sequences using methods known in the art (see U.S. Patent No. 5,225,539 to Winter; U.S. Patent Nos. 5,530,101 to Queen et al.; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals that do not produce endogenous immunoglobulins following immunization and are capable of producing a fully human antibody repertoire (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; WO02/43478). 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).

As used herein, the term "degree of humanization" is an index 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 predicted, for example, by using DomainGapAlign on the IMGT website to predict the homology between the variable region sequence and the human V domain.

Any suitable form of IL-18BP can be used as an immunogen (antigen) for producing non-human antibodies specific to IL-18BP, and screening the biological activity of the antibodies. Immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art. Immunogens can be purified from natural sources, or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic in origin (eg cDNA). 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.

Detection method

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

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

Reagent test kit

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

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

application

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

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

In one embodiment, the present invention includes a type of cell therapy, in which a patient's own T cells (or a heterologous donor) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient. In this way, the probability of graft-versus-host reaction is extremely low, and the antigen is recognized by T cells without MHC restriction. Furthermore, a single CAR-T can treat all cancers that express 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.

In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and last for several months to several years. Alternatively, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-T cells can induce a specific immune response to tumor cells that overexpress the antigen recognized by the CAR antigen-binding domain. For example, the CAR-T cells of the present invention elicit a specific immune response against tumor cells with high expression of IL-18BP.

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

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

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

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

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

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

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

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

Those skilled in the art know that the above application can be used in combination with other treatment methods, such as PD-1/PD-L1 antibody.

The main advantages of the present invention include:

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

2. The antibodies and antigen-binding fragments thereof of the present invention can not only specifically recognize/bind IL-18BP, but also block the binding between IL-18BP and IL-18. Therefore, the antibodies and antigen-binding fragments thereof of the present invention have the potential to be used in the prevention and/or treatment of various diseases such as tumors, inflammation-related diseases and metabolism-related diseases.

3. The antibody to IL-18BP of the present invention or its antigen-binding fragment can restore the IFN-γ release activity induced by IL-18.

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

5. The humanized antibody of the present invention can be safely administered to a human subject without causing an immunogenic reaction. Therefore, the antibodies of the present invention have great clinical value.

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

7. The antibody of the present invention and its antigen-binding fragment can be used in combination with PD-1 antibody/PD-L1 antibody to enhance the inhibitory effect on tumors.

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

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

The purified human IL-18BP recombinant protein (antigen sequence Human IL-18BP (NP_001034748): TPVSQTTTAATASVRSTKDPPCSQPPVFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSILYWLGNGSFIEHLPGRLWEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVLVDPEQVVQRHV VLAQLWAGLRATLPPTQEALPSSHSSPQQQG, SEQ ID NO:68) was mixed with an equal volume of complete Freund's adjuvant, and immunized SJL female mice aged 8-10 weeks.

Afterwards, the immunization was boosted once at intervals of 2 weeks, 3 weeks, and 3 weeks. After a total of 4 times of immunization, when the serum titer of the mice detected by ELISA reached 1: ¹⁰⁵ , the above-mentioned immunized mice were sacrificed by dislocation of the neck after blood collection from the orbit. The abdomen was disinfected with 75% alcohol, the spleen and lymph nodes were surgically removed, and excess connective tissue was removed.

The spleen and lymph nodes were collected to prepare cell suspension, and the single lymphocyte suspension was collected for future use. One day before cell fusion, mouse peritoneal macrophages were prepared as feeder cells and placed in a cell incubator at 37°C and 5% CO ₂ for culture. Resuscitate the Sp2/0-Ag14 myeloma cells to ensure that the cells are in the logarithmic growth phase at the time of fusion. The SP2/0 cells were washed twice with incomplete RPMI-1640 medium, then resuspended and resuspended in incomplete RPMI-1640 medium for use.

The above lymphocyte suspension and Sp2/0-Ag14 cells were electroporated using 50% PEG4000 (pH=8.0-8.2) Induce fusion. The fused cells were cultured in HAT medium for 1-2 weeks, the supernatant was aspirated, and the antibody binding activity in the supernatant was detected by ELISA.

The limited dilution method was used for subcloning, and positive hybridoma cell lines were obtained after multiple screenings. Extract the total RNA of the positive cloned hybridoma, use 3'RACE technology and 5'RACE technology, RT-PCR to obtain cDNA containing the sequence of the variable region, clone the VH and VL genes into the T vector for sequencing, and compare the sequences Analysis to determine the gene sequence of the variable region.

The variable region sequence and human IgG1/IgG4 constant region are combined to construct a chimeric antibody expression vector. The above expression vectors were transfected into 293F cells, and a large amount of chimeric antibodies were purified. The chimeric antibodies obtained above were named as Ab001, Ab002, Ab003, Ab009, Ab010, Ab012, Ab017, Ab018 and Ab020.

Example 2. Humanized transformation of anti-IL-18BP antibody

Humanization of antibody Ab009 used CDR grafting and back mutation. Apply the CDRs numbering convention to define FRs and CDRs regions. According to the antibody sequence database, IGBLAST searches were performed on the VL and VH of the Ab009 sequence, respectively.

According to the retrieval results, IGHV1-18*01 was selected as the template of VH, and IGKV6-21*01 was selected as the template of VL. Homology modeling was performed by grafting antibody CDRs into VH and VL templates, respectively. For factors such as three-dimensional structure, immunogenicity, and physical and chemical properties, a few residues were mutated back to mouse-derived residues, and the sequence was further optimized.

The sequence information of humanized antibody Ab009 (VH12VL9) and humanized antibody Ab009 (VH14VL9) is shown in FIG. 1 . Among them, the underline represents the CDR region, the double underline and bold represents the mutation site based on IGHV1-18*01 (VH template) or IGKV6-21*01 (VL template), and the wavy line and bold represents the mutation site based on Ab009 .

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

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

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

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

The VH region of the humanized antibody Ab009 (VH12VL9) sequence has the following mutations based on the VH region (SEQ ID NO: 25) of Ab009:

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

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

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

The VH region of the humanized antibody Ab009 (VH14VL9) sequence has the following mutations based on the VH region (SEQ ID NO: 25) of Ab009:

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

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

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

The humanized engineered molecules designed above were constructed into IgG1/IgG4 constant regions, and transfected into expi-293 cells to express purified humanized antibody molecules.

Use Biacore 8K to detect the affinity of the humanized antibody, the binding time is 120 seconds, the dissociation time is 360 seconds, and the regeneration buffer is 10mM glycine-HCl pH 1.5.

As shown in Table 5, the affinity of the Ab009 humanized antibody is consistent with that of the murine molecule.

Table 5 Ab009 Humanized Modified Antibody Binding Kinetics (SPR)

Example 3. Affinity Evaluation of Anti-IL-18BP Antibody

Use the diluent to prepare 1 μg/mL human IL-18BP protein, mouse IL-18BP protein and cynomolgus monkey IL-18BP protein, and coat 96-well ELISA plates respectively, overnight at 4°C.

Starting from 100nM, the IL-18BP antibody was serially diluted to 11 concentrations by 3 times, and reacted with the coated human IL-18BP protein, mouse IL-18BP protein and cynomolgus monkey IL-18BP protein. The color was developed after using HRP-labeled goat anti-human IgGκ as the secondary antibody. The EC50 was calculated by plotting the OD 450nm against the concentration of the antibody.

As shown in Table 6, the binding activities of these 10 antibodies to IL-18BP proteins of different species are summarized. The IL-18BP antibody shows excellent affinity to human IL-18BP, among which Ab002, Ab003, Ab009, Ab010, Ab012, Ab017, Ab018 and Ab020 all have affinities at the pM level.

Table 6 IL-18BP antibody affinity test

Example 4. Anti-IL-18BP antibody can restore the IFN-γ release activity of KG-1 cells induced by IL-18

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

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.

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

Table 7 Anti-IL-18BP antibody restores the EC50 of IL-18-induced IFN-γ release

In addition, as shown in Figure 3, the KG-1 functional test showed that the humanized antibody also had strong activity (EC50=12.15nM).

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

Human peripheral blood mononuclear cells (PBMC) (TPCS, #PB025C-W) were seeded in 96-well plates at a concentration of 1×10 ⁵ cells/well. Serial dilutions of mAb003 and mAb009 antibodies of mouse origin, IgG1 subtype and IgG4 subtype, respectively, were added to the wells.

After 30 minutes, 0.01 μg/mL 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.

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

The results are shown in Figure 4, IL-18BP antibody can restore IL-18-induced IFN-γ release in PBMC cells.

Example 6. Anti-IL-18BP antibody binding activity to human tumor cell-derived IL-18BP

The human ovarian cancer cell line can secrete a large amount of IL-18BP under the stimulation of IFN-g. 5×10 ⁶ SK-OV-3 cells were inoculated into a 10 cm culture dish, and the cells were stimulated with 50 ng/mL human IFN-g, 24 Hours later, the supernatant was collected to obtain a conditioned medium containing IL-18BP, and the concentration of IL-BPa therein was quantified using a human IL-18BPa ELISA detection kit.

Use the diluent to prepare 1 μg/mL IL-18BP antibody, coat 96-well ELISA plates respectively, and leave overnight at 4°C. The conditioned medium containing IL-18BP was diluted to 1200pg/mL, 400pg/mL and 133.3pg/mL to react with the coated anti-IL-18BP antibody. The color was developed with HRP-labeled anti-human IL-18BP antibody, and the OD 450nm was plotted against the concentration of the antibody.

The results shown in Figure 5 show that Ab002, Ab012, Ab018, Ab009, etc. have better human tumor cell-secreted IL-18BP binding activity.

Example 7. Test the anti-tumor effect of PD-1 antibody and IL-18BP antibody through in vivo drug efficacy and the effect of combined administration on tumor inhibition effect

Cell source: ATCC

PD-1 antibody: BioXcell BE0146 Cat#: BP0146, Clone: RMP1-14

Animals: BALB/c mice, 6-8 weeks old, female, purchased from Weitong Lihua Experimental Animal Technology Co., Ltd.

Tumor-bearing mice were subjected to tumor measurement, grouping, and administration, and the specific steps were as follows:

a) According to the tumor growth, measure the tumor 7-9 days after inoculation, and calculate the tumor size. The tumor volume is calculated according to the following formula: tumor volume (mm ³ ) = length (mm) × width (mm) × width (mm) /2.

b) According to the body weight and tumor size of the tumor-bearing mice, the mice were randomly divided into groups.

c) According to the grouping results, start to administer the test drug, see Table 8 for the specific administration information.

d) Tumors were measured and weighed three times a week after the administration of the test drug was started.

e) Euthanize the animals after the experiment.

f) Process data with software such as excel. The calculation of the test compound tumor inhibition rate TGI (%) is according to the following formula: TGI%=[1-(the tumor volume on the day of evaluation of the treatment group-the tumor volume of the treatment group)/(the tumor volume on the day of evaluation of the control group-contrast Group tumor volume)]×100.

g) The experimental data are shown in Table 8, and the tumor volume and weight change rate during the experiment are shown in Figure 6 and Figure 7 .

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

h) Experimental results

It can be seen from the above results that the administration of Ab002 at different doses (including the same dose as PD-1 single drug) has better tumor inhibitory effects than PD-1 single drug; the Ab002 antibody of the present invention combined with PD-1 After antibody treatment, the tumor inhibitory effect was also significantly better than that of PD-1 single drug, which indicated that combined administration enhanced the tumor inhibitory effect. In addition, whether it is single drug or combined drug administration, the body weight of the animals keeps increasing, indicating that Ab002 also shows good safety.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (17)

An antibody against IL-18BP or an antigen-binding fragment thereof, characterized in that it comprises: (a) heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, the amino acid sequences of the CDRH1, CDRH2, and CDRH3 are shown in SEQ ID NO: 2, 3, and 4, respectively, Or as shown in SEQ ID NO:10, 11 and 12 respectively, Or as shown in SEQ ID NO:18, 19 and 20 respectively, Or as shown in SEQ ID NO:26, 27 and 28 respectively, Or as shown in SEQ ID NO:33, 34 and 20 respectively, Or as shown in SEQ ID NO:38, 27 and 28 respectively, Or as shown in SEQ ID NO:42, 43 and 44 respectively, Or as shown in SEQ ID NO:50, 51 and 52 respectively, or as shown in SEQ ID NO:58, 59 and 60 respectively; and/or (b) light chain complementarity determining regions CDRL1, CDRL2, CDRL3, the amino acid sequences of said CDRL1, CDRL2, CDRL3 are respectively shown in SEQ ID NO: 6, 7 and 8, Or as shown in SEQ ID NO:14, 15 and 16 respectively, Or as shown in SEQ ID NO:22, 23 and 24 respectively, Or as shown in SEQ ID NO:30, 15 and 31 respectively, Or as shown in SEQ ID NO:22, 36 and 24 respectively, Or as shown in SEQ ID NO:40, 15 and 31 respectively, Or as shown in SEQ ID NO:46, 47 and 48 respectively, Or as shown in SEQ ID NO:54, 55 and 56 respectively, Or respectively as shown in SEQ ID NO:62, 63 and 64; The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof has the following SEQ ID NO: 1, 9, 17, 25, 32, 37, 41, 49, 57, The heavy chain variable region shown in 65 or 67 or has at least 90% or 95% or 98% sequence identity therewith, and has such as SEQ ID NO:5,13,21,29,35,39,45, 53, 61 or 66 or have at least 90% or 95% or 98% sequence identity thereto. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof has heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, and light chain complementarity determining regions CDRL1, CDRL2, and CDRL3, The amino acid sequences of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are respectively shown in: SEQ ID NO: 2, 3, 4, 6, 7 and 8, or respectively shown in SEQ ID NO: 10, 11, 12, 14, 15 and 16, or respectively as shown in SEQ ID NO: 18, 19, 20, 22, 23 and 24, or respectively as shown in SEQ ID NO: 26, 27, 28, 30, 15, 31, Or shown in SEQ ID NO:33, 34, 20, 22, 36, 24 respectively, or shown in SEQ ID NO: 38, 27, 28, 40, 15, 31 respectively, or shown in SEQ ID NO: 42 respectively , 43, 44, 46, 47, 48 or as shown in SEQ ID NO:50, 51, 52, 54, 55, 56 respectively, or as shown in SEQ ID NO: 58, 59, 60, 62, 63, 64 respectively. The antibody or its antigen-binding fragment according to claim 1, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment are as SEQ ID NO: 1 and SEQ ID NO: 1 and SEQ ID NO: 1 respectively. ID NO:5, or respectively as shown in SEQ ID NO:9 and SEQ ID NO:13, or respectively as shown in SEQ ID NO:17 and SEQ ID NO:21, or respectively as shown in SEQ ID NO:25 and as shown in SEQ ID NO:29, or as shown in SEQ ID NO:65 and SEQ ID NO:66 respectively, or as shown in SEQ ID NO:67 and SEQ ID NO:66 respectively, or as shown in SEQ ID NO:32 respectively and SEQ ID NO:35, or respectively as shown in SEQ ID NO:37 and SEQ ID NO:39, or respectively as shown in SEQ ID NO:41 and SEQ ID NO:45, or respectively as SEQ ID NO: 49 and SEQ ID NO:53, or respectively as shown in SEQ ID NO:57 and SEQ ID NO:61. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region of the antibody or antigen-binding fragment thereof has a mutation selected from the group based on SEQ ID NO: 25: Q5V, D8G, L11V, V12K, I20V, V24S, 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 group based on SEQ ID NO: 29: or The heavy chain variable region of the antibody or antigen-binding fragment thereof has a mutation selected from the 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 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. The antibody or its antigen-binding fragment according to claim 5, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment are as SEQ ID NO: 65 and SEQ ID NO: 65 and SEQ ID NO: 65 respectively. Shown in ID NO:66, or as shown in SEQ ID NO:67 and SEQ ID NO:66 respectively; And the antibody or antigen-binding fragment thereof is a humanized antibody. 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 respectively shown in SEQ ID NO:69 and SEQ ID NO:70, Or as shown in SEQ ID NO:71 and SEQ ID NO:72 respectively, or as shown in SEQ ID NO:73 and SEQ ID NO:74 respectively, or as shown in SEQ ID NO:75 and SEQ ID NO:76 respectively , or as shown in SEQ ID NO:77 and SEQ ID NO:78 respectively, or as shown in SEQ ID NO:79 and SEQ ID NO:80 respectively, or as SEQ ID NO:81 and SEQ ID NO:82 respectively or as shown in SEQ ID NO:83 and SEQ ID NO:84 respectively, or as shown in SEQ ID NO:85 and SEQ ID NO:86 respectively, or as shown in SEQ ID NO:87 and SEQ ID NO: 88, or as shown in SEQ ID NO:89 and SEQ ID NO:90, respectively. 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. 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, preferably IgG1 or IgG4. A nucleotide molecule, characterized in that the nucleotide molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1-9. An expression vector, characterized in that the expression vector contains the nucleotide molecule according to claim 10. A host cell, characterized in that the host cell contains the expression vector according to claim 11, or the nucleotide molecule according to claim 10 is integrated in its genome. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-9 in the preparation of a medicament for treating and/or preventing diseases or disorders caused by high expression of IL-18BP or abnormal IL-18 pathway. The use according to claim 13, wherein 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 anti-PD-1/ PD-L1 antibody. The use according to claim 13, characterized in that the diseases or diseases caused by high expression of IL-18BP or abnormal IL-18 pathway include: tumors, metabolic diseases, immune system diseases, neurodegenerative diseases, cardiovascular diseases or inflammatory diseases; Preferably, the disease or condition is a tumor; preferably, the tumor is a solid tumor or a hematological tumor. The use according to claim 15, wherein the tumor is selected from the group consisting of: lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, One or more of colorectal cancer, nasopharyngeal cancer, brain tumor, cervical cancer, blood cancer, bone cancer, lymphatic cancer, and pancreatic cancer. A kind of pharmaceutical composition, is characterized in that, described pharmaceutical composition contains: (a) a first active ingredient, which is the antibody or antigen-binding fragment thereof according to any one of claims 1-9; (b) optionally a second active ingredient that is a PD-1 antibody and/or a PD-L1 antibody; and (c) a pharmaceutically acceptable carrier, diluent or excipient; Wherein, when the pharmaceutical composition includes a second active ingredient, the ratio of the first active ingredient to the second active ingredient is 5:1-1:1, preferably 3:1.