Anti-Claudin18.2 humanized single-domain antibody

Abstract

The invention provides an anti-Claudin18.2 humanized single-domain antibody and a preparation method of the anti-Claudin18.2 humanized single-domain antibody. Specifically, the invention provides a Claudin18.2-targeted single-domain antibody, a humanized antibody thereof, a derivative protein, a gene sequence for encoding the Claudin18.2-targeted single-domain antibody, and an expression vector and an expression system for producing the Claudin18.2-targeted single-domain antibody. The single-domain antibody has high specificity, is combined with Claudin18.2 with high affinity and is basically not combined with Claudin18.1, and the single-domain antibody provides a research and development basis for development of antibody drugs, antibody coupling drugs and multi-specific antibody drugs.

Description

Anti-Claudin18.2 humanized single domain antibody Technical Field

[0001] The present invention belongs to the field of biomedicine technology. Specifically, the present invention relates to an anti-Claudin18.2 humanized single-domain antibody. Background Art

[0002] Gastric cancer ranks third in cancer-related mortality and is considered one of the most difficult cancers to cure worldwide. In patients with advanced or metastatic gastric cancer and gastroesophageal junction (GEJ) adenocarcinoma, the median overall survival (mOS) does not exceed 10 months. Although human epidermal growth factor receptor 2 (HER-2) targeted therapy and immune checkpoint inhibitors have certain efficacy, other targets are still needed in advanced gastric cancer. At the 2016 American Society of Clinical Oncology Annual Meeting (ASCO), Astellas's Phase II clinical study of IMAB362, an antibody drug targeting Claudin18.2, combined with chemotherapy for the treatment of patients with locally advanced or metastatic gastric cancer showed that both patients' progression-free survival and overall survival were prolonged. Phase III clinical data released at ASCO in 2023 also achieved positive results. However, no monoclonal antibodies, bispecific antibodies, or antibody-drug conjugates (ADCs) targeting Claudin18.2 have been approved for marketing.

[0003] Single-domain antibodies (SDAs) were first reported in Nature in 1993 by Belgian scientist Hamers-Casterman and his team. They found that some antibodies in the blood of camelids are heavy-chain antibodies lacking light chains. VHHs retain full antigen-binding capacity and are the smallest fragments that retain complete antigen binding. These fragments, known as single-domain antibodies (SDAs), have a molecular weight of only 15 kDa and offer advantages such as small size, high affinity, and excellent stability. They have been widely used in biopharmaceutical research and development.

[0004] Claudin18.1 is an alternative splice of CLDN18, expressed in the lungs. Claudin18.1 and claudin18.2 are structurally similar, differing only by eight amino acid residues in the ECL1 sequence in their extracellular domains. Therefore, developing antibodies that specifically recognize claudin18.2 but not claudin18.1 is a challenge in developing antibodies against claudin18.2.

[0005] Therefore, there is a need in this field to develop highly specific single-domain antibody drugs targeting Claudin18.2 to provide gastric cancer patients with wider and better medication options.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide an anti-Claudin18.2 single-domain antibody and a humanized antibody thereof.

[0008] In a first aspect of the present invention, a single-domain antibody targeting Claudin18.2 is provided, wherein the VHH chain of the single-domain antibody comprises complementary determining regions CDR1, CDR2 and CDR3 derived from the heavy chain variable region shown in SEQ ID NO: 6, 7 or 8, and the complementary determining regions CDR1, CDR2 and CDR3 are defined by Chothia, Abm, Kabat, or IMGT rules.

[0009] In another preferred embodiment, the CDR1, CDR2 and CDR3 are selected from the following groups:

[0010] (1) Based on the definition of Chothia rules:

[0011] CDR1 shown in SEQ ID NO:9,

[0012] CDR2 shown in SEQ ID NO: 10, and

[0013] CDR3 shown in SEQ ID NO: 11; or,

[0014] (2) Based on the definition of Abm rules:

[0015] CDR1 shown in SEQ ID NO: 12,

[0016] CDR2 shown in SEQ ID NO: 13, and

[0017] CDR3 shown in SEQ ID NO: 11; or,

[0018] (3) Based on the definition of Kabat rules:

[0019] CDR1 shown in SEQ ID NO: 14,

[0020] CDR2 shown in SEQ ID NO: 15, 19 or 20, and

[0021] CDR3 shown in SEQ ID NO: 11; or,

[0022] (4) Based on IMGT rules:

[0023] CDR1 shown in SEQ ID NO: 16,

[0024] CDR2 shown in SEQ ID NO: 17, and

[0025] CDR3 shown in SEQ ID NO:18.

[0026] In another preferred embodiment, the CDR region of the single-domain antibody VHH chain comprises an amino acid sequence having at least 80%, preferably at least 90%, and more preferably at least 95% sequence similarity to any of the above sequences.

[0027] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the binding affinity to Claudin18.2.

[0028] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-3, preferably 1-2, and more preferably 1.

[0029] In another preferred embodiment, the VHH chain of the single-domain antibody further includes a framework region (FR).

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

[0031] In another preferred embodiment, the framework region FR is of human, mouse, rabbit or camel origin.

[0032] In another preferred example, the VHH chain of the single-domain antibody targeting Claudin18.2 has an amino acid sequence with an amino acid sequence homology ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% to that of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0033] In another preferred example, the VHH chain of the single-domain antibody targeting Claudin18.2 has an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0034] In another preferred embodiment, the single domain antibody is humanized.

[0035] In another preferred embodiment, the antibody does not bind or weakly binds to human Claudin18.1.

[0036] In another preferred example, the single-domain antibody can specifically bind to Claudin18.2 of human, mouse, rat and monkey origin.

[0037] In another preferred embodiment, the single-domain antibody is capable of mediating the internalization of Claudin18.2.

[0038] In a second aspect, the present invention provides an antibody targeting Claudin18.2, wherein the antibody comprises one or more VHH chains of the single-domain antibody targeting Claudin18.2 as described in the first aspect of the present invention.

[0039] In another preferred example, the VHH chain of the single-domain antibody targeting Claudin18.2 has an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0040] In another preferred embodiment, the antibody is a monomer, a bivalent antibody, and / or a multivalent antibody.

[0041] In another preferred embodiment, the antibody is an animal-derived antibody, a humanized antibody, or a chimeric antibody.

[0042] In another preferred embodiment, the CDR region of the humanized antibody comprises 1, 2, or 3 amino acid changes.

[0043] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit, or camel.

[0044] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0045] In another preferred embodiment, the antibody is a monoclonal antibody.

[0046] In another preferred embodiment, the antibody is a partially or fully humanized antibody.

[0047] In another preferred embodiment, the antibody is a heavy chain antibody, and the heavy chain antibody comprises heavy chain constant regions CH2 and CH3 (Fc segment).

[0048] In another preferred embodiment, the heavy chain constant region is derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

[0049] In another preferred embodiment, the amino acid sequence of the Fc segment of human IgG is shown in SEQ ID NO: 21.

[0050] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the binding affinity to Claudin18.2.

[0051] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, and more preferably 10%.

[0052] In another preferred embodiment, the antibody does not bind or weakly binds to human Claudin18.1.

[0053] In another preferred embodiment, the antibody can specifically bind to Claudin18.2 of human, mouse, rat and monkey origin.

[0054] In another preferred embodiment, the antibody is capable of mediating the internalization of Claudin18.2.

[0055] In a third aspect, the present invention provides a multispecific antibody, which comprises: the single-domain antibody targeting Claudin18.2 according to the first aspect of the present invention or the antibody targeting Claudin18.2 according to the second aspect of the present invention.

[0056] In another preferred embodiment, the multispecific antibody comprises one or more second antigen binding regions that target additional antigen targets.

[0057] In another preferred embodiment, the second antigen binding region is an antibody or antibody fragment, and the antibody fragment includes: (i) Fab fragment; (ii) F(ab')2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single-chain Fv (scFv) molecule; (vi) dAb fragment.

[0058] In another preferred embodiment, the second antigen binding region is a single domain antibody.

[0059] In a fourth aspect, the present invention provides a recombinant protein, comprising:

[0060] (i) the single-domain antibody targeting Claudin18.2 as described in the first aspect of the present invention, or the antibody targeting Claudin18.2 as described in the second aspect of the present invention; and

[0061] (ii) optionally a polypeptide molecule or fragment having therapeutic function; and / or

[0062] (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

[0063] In another preferred embodiment, the improving the physicochemical properties or drugability of the protein includes prolonging the half-life of a single-domain antibody targeting Claudin18.2.

[0064] In another preferred embodiment, the recombinant protein further comprises: (iv) an optional tag sequence for facilitating expression and / or purification.

[0065] In another preferred embodiment, the tag sequence is selected from the following group: 6His tag, GGGS sequence, FLAG tag.

[0066] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0067] In another preferred embodiment, the polypeptide molecules or fragments with therapeutic functions include but are not limited to: insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, and cytokines.

[0068] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0069] In another preferred embodiment, the fusion protein includes a multispecific antibody or a chimeric antibody.

[0070] In another preferred embodiment, the functional domain for improving the physicochemical properties or drugability of a protein includes an Fc segment and human serum albumin (HSA).

[0071] In another preferred embodiment, the fusion protein has the following elements from the NC end:

[0072] AB;

[0073] Among them, the A component is a single-domain antibody targeting Claudin18.2; the B component is the Fc segment or human serum albumin (HSA);

[0074] “-” represents a peptide bond.

[0075] In another preferred example, the VHH chain of the single-domain antibody targeting Claudin18.2 has an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0076] In a fifth aspect, the present invention provides an immunoconjugate comprising:

[0077] (a) an antibody portion, wherein the antibody portion is the single domain antibody targeting Claudin18.2 according to the first aspect of the present invention or the antibody targeting Claudin18.2 according to the second aspect of the present invention; and

[0078] (b) a conjugated moiety conjugated to the single-domain antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

[0079] In another preferred embodiment, the immunoconjugate is a single domain antibody drug conjugate.

[0080] In another preferred embodiment, the single domain antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0081] In another preferred embodiment, the coupling moiety is a chemical label or a biological label.

[0082] In another preferred embodiment, the chemical label is an isotope, an immunotoxin and / or a chemical drug.

[0083] In another preferred embodiment, the biomarker is biotin, avidin or an enzyme label.

[0084] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0085] In another preferred embodiment, the drug is a cytotoxic drug.

[0086] In another preferred embodiment, the toxin is selected from the following group:

[0087] auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis officinalis) inhibitors, glucocorticoids, or a combination thereof.

[0088] In another preferred embodiment, the coupling moiety is MMAE or a topoisomerase inhibitor.

[0089] In another preferred embodiment, the coupling moiety is a detectable label.

[0090] In another preferred embodiment, the detectable marker comprises a radionuclide, and the radionuclide comprises:

[0091] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0092] (ii) therapeutic isotopes selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133 Yb-169, Yb-177, or a combination thereof.

[0093] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold single-domain particles / single-domain rods, viral particles, liposomes, single-domain magnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of single-domain particles, etc.

[0094] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) single-domain antibody targeting Claudin18.2 as described in the first aspect of the present invention or an antibody targeting Claudin18.2 as described in the second aspect of the present invention.

[0095] In another preferred embodiment, the multivalency means that the amino acid sequence of the immunoconjugate contains multiple repeats of the single domain antibody targeting Claudin18.2 as described in the first aspect of the present invention or the antibody targeting Claudin18.2 as described in the second aspect of the present invention.

[0096] In another preferred embodiment, the detection is in vivo detection or in vitro detection.

[0097] In another preferred embodiment, the immunoconjugate is used for diagnosing and / or treating tumors expressing CLAUDIN18.2 protein.

[0098] In another preferred embodiment, the immunoconjugate has the following molecular formula:

[0099] in:

[0100] nAb is a single-domain antibody targeting Claudin18.2, an antibody targeting Claudin18.2, or a multispecific antibody;

[0101] LU is the linker (also called connector);

[0102] D is for medicine;

[0103] Furthermore, the subscript p is a value selected from 1-10.

[0104] In another preferred embodiment, LU is selected from maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC) linkers connected to the antibody portion, and comprises one or more linkers of valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG).

[0105] In another preferred embodiment, the antibody portion is covalently bound to the linker by reacting with a portion selected from the group consisting of maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC), and the like.

[0106] In another preferred embodiment, D is selected from the following group of compounds having anti-tumor activity:

[0107] (i) Tubulin inhibitors, such as maytansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF);

[0108] (ii) DNA-acting toxins, such as duocarmycin and pyrrolobenzodiazepine (PBD);

[0109] (iii) Topoisomerase inhibitors, such as camptothecin, SN38, exitecan, and Dxd.

[0110] In a sixth aspect, the present invention provides a pharmaceutical composition comprising:

[0111] (i) the single-domain antibody targeting Claudin18.2 according to the first aspect of the present invention, the antibody targeting Claudin18.2 according to the second aspect of the present invention, the multispecific antibody according to the third aspect of the present invention, the recombinant protein according to the fourth aspect of the present invention, or the immunoconjugate according to the fifth aspect of the present invention;

[0112] (ii) a pharmaceutically acceptable carrier.

[0113] In another preferred embodiment, the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.

[0114] In another preferred embodiment, the pharmaceutical composition further comprises other biologically active substances, such as drugs for treating tumors.

[0115] In another preferred embodiment, the administration method of the pharmaceutical composition is selected from the following group: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spraying and aerosol inhalation.

[0116] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.

[0117] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0118] In another preferred embodiment, the pharmaceutical composition is an injection.

[0119] In a seventh aspect, the present invention provides a use of an active ingredient, wherein the active ingredient is selected from the following group: a single-domain antibody targeting Claudin18.2 as described in the first aspect of the present invention, an antibody targeting Claudin18.2 as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, a recombinant protein as described in the fourth aspect of the present invention, an immunoconjugate as described in the fifth aspect of the present invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a detection reagent, a detection plate or a kit; and / or (b) preparing a drug for preventing and / or treating CLAUDIN18.2-related diseases.

[0120] In another preferred embodiment, the detection reagent, detection plate or kit is used for:

[0121] (1) detecting CLAUDIN18.2 protein in a sample; and / or

[0122] (2) detecting endogenous CLAUDIN18.2 protein in tumor cells; and / or

[0123] (3) Detect tumor cells expressing CLAUDIN18.2 protein.

[0124] In another preferred embodiment, the detection types include but are not limited to flow cytometry, cell immunofluorescence detection, enzyme-linked immunosorbent assay, immunoblotting detection, etc.

[0125] In another preferred embodiment, the detection reagent, detection plate or kit is used to diagnose CLAUDIN18.2-related diseases.

[0126] In another preferred embodiment, the drug is used to treat or prevent tumors with high expression of CLAUDIN18.2, tumor migration, or tumor resistance.

[0127] In another preferred embodiment, the tumor drug resistance includes: resistance to tumor immunotherapy drugs, resistance to tumor targeted therapy drugs, resistance to conventional tumor chemotherapy, and insensitivity to radiotherapy.

[0128] In another preferred embodiment, the CLAUDIN18.2-related disease is selected from the following group: cancer.

[0129] In another preferred embodiment, the CLAUDIN18.2-related diseases include: tumor occurrence, growth and / or metastasis.

[0130] In another preferred embodiment, the cancer includes solid tumors and blood cancers.

[0131] In another preferred embodiment, the cancer is selected from the following group: gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer (such as lung adenocarcinoma and non-small cell lung cancer), breast cancer (such as triple-negative breast cancer), malignant glioma, liver cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer, endometrial cancer, cervical cancer, leukemia, bone marrow cancer, angiosarcoma, or a combination thereof.

[0132] In an eighth aspect, the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:

[0133] (1) the single-domain antibody targeting Claudin18.2 as described in the first aspect of the present invention, the antibody targeting Claudin18.2 as described in the second aspect of the present invention, or the multispecific antibody as described in the third aspect of the present invention; or

[0134] (2) The recombinant protein as described in the fourth aspect of the present invention.

[0135] In another preferred embodiment, the polynucleotide includes RNA, DNA or cDNA.

[0136] The ninth aspect of the present invention provides a vector comprising the polynucleotide as described in the eighth aspect of the present invention.

[0137] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0138] The tenth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the ninth aspect of the present invention or the polynucleotide described in the eighth aspect of the present invention is integrated into its genome.

[0139] In an eleventh aspect, the present invention provides a method for in vitro detection (including diagnostic or non-diagnostic) of CLAUDIN18.2 in a sample, the method comprising the steps of:

[0140] (1) in vitro, contacting the sample with the single domain antibody targeting Claudin18.2 described in the first aspect of the present invention, the antibody targeting Claudin18.2 described in the second aspect of the present invention, or the immunoconjugate described in the fifth aspect of the present invention;

[0141] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CLAUDIN18.2 in the sample.

[0142] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0143] In a twelfth aspect, the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

[0144] (a) culturing the host cell according to the tenth aspect of the present invention under conditions suitable for expression;

[0145] (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is a single-domain antibody targeting Claudin18.2 as described in the first aspect of the present invention, an antibody targeting Claudin18.2 as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, or a recombinant protein as described in the fourth aspect of the present invention.

[0146] In a thirteenth aspect, the present invention provides a method for treating a CLAUDIN18.2-related disease, the method comprising: administering to a subject in need thereof a single-domain antibody targeting Claudin18.2 as described in the first aspect of the present invention, an antibody targeting Claudin18.2 as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, a recombinant protein as described in the fourth aspect of the present invention, an immunoconjugate as described in the fifth aspect of the present invention, or a pharmaceutical composition as described in the sixth aspect of the present invention, or a combination thereof.

[0147] In another preferred embodiment, the method further comprises: administering other drugs or treatment methods to a subject in need for combined treatment.

[0148] In another preferred embodiment, the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.

[0149] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] FIG1 shows the positive rate detection of HEK293T-Claudin18.2 (human) and HEK293T-Claudin18.1 (human) stable cell lines.

[0151] FIG2 shows the binding of camel-derived VHH-Fc to human Claudin 18.1 detected by flow cytometry (FACS).

[0152] FIG3 shows the binding of camel-derived VHH-Fc to human Claudin 18.2 detected by flow cytometry (FACS).

[0153] FIG4 shows the cell affinity detection of anti-human Claudin18.2 humanized single domain antibody and HEK293T-Claudin18.2 (human) (flow cytometry).

[0154] FIG5 shows the binding detection of anti-human Claudin18.2 humanized single domain antibody to NUGC4-Claudin18.2 (human) cells (flow cytometry).

[0155] FIG6 shows the binding detection of anti-human Claudin18.2 humanized single domain antibody to HEK293T-Claudin18.1 (human) cells (flow cytometry).

[0156] FIG7 shows the binding detection of anti-human Claudin18.2 humanized single domain antibody to HEK293T-Claudin18.2 (monkey) cells (flow cytometry).

[0157] FIG8 shows the binding detection of anti-human Claudin18.2 humanized single domain antibody to HEK293T-Claudin18.2 (rat) cells (flow cytometry).

[0158] FIG9 shows the binding detection of anti-human Claudin18.2 humanized single domain antibody to HEK293T-Claudin18.2 (mouse) cells (flow cytometry).

[0159] FIG10 shows the results of antibody-dependent cell-mediated cytotoxicity (ADCC) assay of anti-human Claudin18.2 humanized single-domain antibody.

[0160] FIG11 shows the endocytic activity determination (flow cytometry) of anti-human Claudin18.2 humanized single-domain antibody.

[0161] FIG12 shows the endocytic activity (killing method) assay of anti-human Claudin18.2 humanized single-domain antibody. DETAILED DESCRIPTION

[0162] After extensive and in-depth research, the present inventors have developed for the first time a single-domain antibody and a humanized version thereof that specifically target claudin 18.2. The single-domain antibody and humanized version thereof of the present invention have high specific affinity for claudin 18.2, but substantially do not bind to its isoform, claudin 18.1. This work has led to the present invention.

[0163] the term

[0164] As used herein, the terms "single domain antibody of the present invention", "anti-Claudin18.2 nanobody of the present invention", and "CLAUDIN18.2 single domain antibody of the present invention" are used interchangeably and refer to single domain antibodies that specifically recognize and bind to CLAUDIN18.2 (including human CLAUDIN18.2). Particularly preferred are single domain antibodies whose VHH chain amino acid sequences are shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0165] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0166] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning and refer to the variable region of the heavy chain of a monoclonal antibody. A single-domain antibody (VHH) consisting solely of a single heavy chain variable region is constructed, which is the smallest antigen-binding fragment with complete function. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting solely of a single heavy chain variable region.

[0167] As used herein, the term "heavy chain antibody" refers to an antibody containing only a heavy chain. A portion of antibodies found in the blood of camelids are "heavy chain antibodies" that lack light chains. The heavy chain antibodies of the present invention comprise a heavy chain variable region (VHH) and heavy chain constant regions CH2 and CH3. The heavy chain antibodies of the present invention may be antibodies isolated from animals (such as camel-derived) that naturally lack light chains and heavy chain constant region 1 (CH1); or they may be recombinant antibodies obtained by recombining a single domain antibody (VHH) of the present invention with a heavy chain constant region. The heavy chain antibodies of the present invention may comprise a constant region derived from, for example, IgG1, IgG2, IgG3 or IgG4, preferably a constant region derived from IgG1.

[0168] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0169] As known to those skilled in the art, immunoconjugates and fusion products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the anti-Claudin18.2 protein antibodies or fragments thereof.

[0170] As used herein, the terms "heavy chain variable region" and "V H ” can be used interchangeably.

[0171] As used herein, the terms "hypervariable region" and "complementarity determining region (CDR)" are used interchangeably.

[0172] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.

[0173] 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.

[0174] In the present invention, the terms "recombinant protein of the present invention," "protein of the present invention," or "polypeptide of the present invention" are used interchangeably to refer to polypeptides that specifically bind to Claudin18.2, such as proteins or polypeptides comprising the VHH chains of the single-domain antibodies of the present invention. These may or may not contain an initial methionine.

[0175] The present invention also provides other proteins or fusion expression products comprising the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) comprising a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0176] Generally, an antibody's antigen-binding properties are described by three specific regions within the heavy chain variable region, known as hypervariable regions (CDRs). This region is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a looped structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0177] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in antigen binding. Thus, the present invention includes molecules having antibody heavy chain variable regions with CDRs that are 90% or more (preferably 95% or more, and most preferably 98% or more) homologous to the CDRs identified herein.

[0178] Claudin18.2 and anti-Claudin18.2 single domain antibodies

[0179] As used herein, the term "Claudin18.2" refers to the Claudin18.2 protein in the Claudins protein family. The role of the Claudins protein family is to maintain tight junctions that control the exchange of molecules between cells. The Claudin18.2 (CLDN 18.2) isoform is a gastric-specific isoform that is usually buried in the gastric mucosa. The occurrence of malignant tumors can lead to the destruction of tight junctions, exposing the Claudin18.2 epitope on the surface of tumor cells, making it a specific target. Claudin18.2 is abnormally activated and overexpressed in a variety of primary malignant tumors, especially in digestive system malignancies, including gastric cancer (70%), pancreatic cancer (50%), esophageal cancer (30%), etc. CLDN 18.2 activation can also be seen in esophageal cancer, ovarian cancer and lung adenocarcinoma. In tumors, Claudin18.2 is involved in the proliferation, differentiation and migration of tumor cells.

[0180] As used herein, "Chothia", "Kabat", "IMGT", and "AbM" refer to the determination of complementary binding determining regions (CDRs) under the above-mentioned different assignment systems. The assignment systems include, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International Immuno GeneTics database (IMGT), and Chothia definitions based on loop structural positions.

[0181] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0182] Preferably, any one of the above amino acid sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the binding affinity to CLAUDIN18.2.

[0183] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0184] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a human-animal chimeric antibody, and preferably a humanized antibody.

[0185] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other antibody derivatives known in the art, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0186] Wherein, the animal is preferably a mammal, such as a mouse or a camel.

[0187] In a preferred embodiment, the present invention discloses a variety of camel-derived and humanized nanobodies with high specificity and high affinity targeting Claudin18.2, which only include heavy chains, and the heavy chains contain heavy chain variable region (VHH) amino acid sequences and optional constant regions CH2 and CH3.

[0188] Recombinant protein (or fusion protein)

[0189] In the present invention, a recombinant protein (or fusion protein) containing the Claudin18.2 single domain antibody of the present invention is also included. A preferred fusion protein is a multispecific antibody (e.g., a bispecific antibody). The multispecific antibody further includes a second antigen binding region targeting a target selected from the group consisting of BCMA, CD73, GPC3, HER2, PMSA, 4-1BB, OX40, GLP-1, Trop2, FGL1, LFA-3, 2B4, 5T4, α-4 integrin, α-V integrin, α4β7 integrin, α4β7 integrin, α-SMA, AGR2, Apelin J receptor, APRIL, B7-H3, B7-H4, BAFF, BTLA, C5 complement, C-242, CA9, CA19-9, carbonic anhydrase 9, CD2, CD3, CD6, CD9, CD11a, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD33, CD38, CD40, CD40L, CD41, CD44, CD44v6, CD47, CD 51. CD52, CD56, CD64, CD69, CD70, CD71, CD74, CD80, CD81, CD86, CD95, CD107a, CD117, CD123, CD12 5. CD132(IL-2Rg), CD133, CD137, CD138, CD160, CD166, CD172A, CD248, CEACAM5(CEA), CEACAM6(NC A-90), CLAUDIN-3, CLAUDIN-4, cMet, collagen, Cripto, CSFR, CSFR-1, CTLA-4, CTGF, CXCL10, CXCL13, CXCR1, CXCR2, CXCR4, CYR61, DL44, DLK1, DLL4, DPP-4, DSG1, EDA, EDB, EGFR, EGFRviii, endothelin B receptor (ETBR), ENPP3, EpCAM, EPHA2, EPHB2, ERBB3, RSV F protein, FAP, FGF-2, FGF8, FGFR1, FGFR2, FGFR3, FGFR4, FLT-3, folate receptor alpha (FRα), FSP-1, GAL3ST1, G-CSF, G-CSFR, GD2, GITR, GLUT1, GLUT4, GM-CSF, GM-CSFR, GPIlb / IIIa receptor, Gpl30, GPIIB / IIIA, GPNMB, GRP78, HER2 / neu, HER3, HER4, HGF, hGH, HLA-DR, HVEM, hyaluronidase, ICOS, IFNα, IFNβ, IFNγ, IgE, IgE receptor (FceRI), IGF, IGF1R, IL1B, IL1R, IL2, IL11, IL12, IL12p40, IL -12R, IL-12Rβl, IL13, IL13R, IL13Ra2, IL15, IL17, IL18, IL21, IL23, IL23R, IL27 / IL27R(wsxl), IL29, IL-31R, IL31 / IL31R, IL2R, IL4, IL4R, IL6, IL6R, IL1 receptor accessory protein (IL1RAP), insulin receptor, Jagged ligand, Jagged 1, Jagged 2, KISS1-R, KLRG1, LAG-3, LIF-R, Lewis X, LIGHT, LRP4, LRRC26, Ly6G6D, LyPD1, MCSP, mesothelin, MRP4, MUC1, mucin-16 (MUC16, CA-125), Na / K ATPase, NGF, Nicastrin, Notch receptor, Notch 1, Notch 2, Notch 3, Notch 4. NOV, OSM-R, OX-40, PAR2, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, PD-1, PD-L1, PD-L2, phosphatidylserine, P1GF, PSCA, PSMA, PS GR, RAAG12, RAGE, SLC44A4, Siglecl5, STEAP1, STEAP2, TAG-72, TAPA1, TEM-8, TGFβ, TIGIT, TIM-3, TLR2, TLR4, TL R6, TLR7, TLR8, TLR9, TMEM31, TNFα, TNFR, TNFRS12A, TRAIL-R1, TRAIL-R2, transferrin, transferrin receptor, TRK-A, TRK-B, uPAR, VAP1, VCAM-1, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, or a combination thereof.

[0190] Preferably, the multispecific antibody comprises one or more second antigen-binding regions, or further comprises a third antigen-binding region.

[0191] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0192] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0193] The antibodies of the present invention refer to polypeptides that have binding activity to the CLAUDIN18.2 protein and include the aforementioned CDR regions. The term also encompasses variants of polypeptides comprising the aforementioned CDR regions that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as the addition of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter protein function. The term also encompasses active fragments and active derivatives of the antibodies of the present invention.

[0194] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0195] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0196] Table A

[0197] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0198] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0199] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0200] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0201] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0202] Antibody preparation

[0203] The DNA sequence of the antibody or fragment thereof of the present invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences for the light and heavy chains can be fused together to form a single-chain antibody, or the coding sequences for a single-domain antibody and the constant region can be fused together to form a heavy-chain antibody.

[0204] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0205] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0206] Currently, DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0207] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0208] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0209] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0210] The resulting monoclonal antibodies can be characterized using conventional methods. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0211] The antibodies of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0212] Immunoconjugates

[0213] The present invention also provides an immunoconjugate (ADC) based on the antibody of the present invention, preferably a single-domain antibody-drug conjugate (NDC).

[0214] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, preferably chemically coupled. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, an agonist small molecule (STING, TLR7, TLR8, etc.), or a radionuclide.

[0215] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0216] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0217] Single-domain antibodies can be conjugated to drugs to form antibody-drug conjugates (NDCs). Typically, NDCs contain a linker positioned between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers typically readily degrade in the intracellular environment, for example, at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptide linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0218] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0219] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0220] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0221] The immunoconjugate drug of the present invention may also be a radionuclide drug conjugate (RDC), which is composed of the antibody of the present invention conjugated to a radionuclide.

[0222] In the present invention, drug-linkers can be used to form NDCs in a single step. In other embodiments, bifunctional linker compounds can be used to form NDCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug to form an NDC.

[0223] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0224] The present invention also provides a method for preparing NDC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (NDC).

[0225] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0226] In some embodiments, the structure of the immunoconjugate, preferably a single domain antibody drug conjugate (NDC), is shown in the following molecular formula:

[0227] in:

[0228] nAb is the above-mentioned single-domain antibody targeting Claudin18.2, heavy chain antibody targeting Claudin18.2, or multispecific antibody, and LU is a linker / connector;

[0229] D is for medicine;

[0230] And the subscript p is a value selected from 1 to 10.

[0231] application

[0232] The present invention also provides uses of the antibodies of the present invention, such as for preparing diagnostic preparations or medicaments for preventing and / or treating CLAUDIN18.2-related diseases, including tumor development, growth, and / or metastasis, tumor resistance-related diseases, inflammation, and metabolic diseases.

[0233] The antibodies, NDCs, RDCs, and bispecific antibodies of the present invention can be used for a variety of purposes, including (but not limited to): diagnosis, prevention, and / or treatment of tumor development, growth, and / or metastasis, particularly tumors with high CLAUDIN18.2 expression. Such tumors include (but are not limited to): gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer (such as lung adenocarcinoma and non-small cell lung cancer), breast cancer (such as triple-negative breast cancer), malignant glioma, liver cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer, endometrial cancer, cervical cancer, leukemia, bone marrow cancer, angiosarcoma, and the like; particularly gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung adenocarcinoma, and more preferably gastric cancer.

[0234] Pharmaceutical composition

[0235] The present invention also provides a composition. In preferred embodiments, the composition is a pharmaceutical composition comprising the aforementioned antibody, active fragment thereof, fusion protein thereof, or NDC, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0236] The pharmaceutical composition of the present invention can be used to directly bind to Claudin18.2 protein molecules, and thus can be used to prevent and treat diseases such as tumors. In addition, other therapeutic agents can also be used simultaneously.

[0237] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0238] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0239] For NDC, since the single domain antibody-drug conjugate provided by the present invention can target a specific cell population and bind to a specific cell surface protein (antigen), thereby releasing the drug into the cell in an active form through endocytosis of the conjugate or drug penetration, the single domain antibody-drug conjugate of the present invention can be used to treat target diseases. The above-mentioned antibody-drug conjugate can be administered to a subject (e.g., a human) in a therapeutically effective amount through an appropriate route. The subject in need of treatment can be a patient who is at risk or suspected of having a disease related to the activity or expression of a specific antigen. Such patients can be identified through routine physical examinations.

[0240] When treating with the single domain antibody-drug conjugates of the present invention, delivery can be performed by conventional methods in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or vector can be delivered locally by direct injection or by using an infusion pump.

[0241] The main advantages of the present invention include:

[0242] (1) The single-domain antibody of the present invention has a high affinity for CLAUDIN18.2, but does not substantially bind to CLAUDIN18.1, which is highly similar to CLAUDIN18.2, and has high specificity.

[0243] (2) The present invention provides a humanized single-domain antibody, which reduces the immunogenicity of the antibody and improves its in vivo safety.

[0244] (3) The single-domain antibody of the present invention has a high internalization rate and can mediate the internalization of the target, which is beneficial to the construction and use of ADC.

[0245] (4) The single domain antibody of the present invention has high purity and thermal stability.

[0246] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0247] Abbreviations

[0248] CDR: complementarity determining region

[0249] FR: amino acid residues in the antibody variable region excluding CDR residues

[0250] VH: antibody heavy chain variable region

[0251] VL: antibody light chain variable region

[0252] IgG: Immunoglobulin G

[0253] Kabat: Immunoglobulin alignment and numbering system proposed by Elvin A. Kabat

[0254] Chothia: Immunoglobulin numbering system proposed by Chothia et al.

[0255] IMGT: numbering system based on the International Immunogenetics Information System initiated by Lefranc et al.

[0256] EC50: Half-maximal effect concentration, the concentration that can cause 50% of the maximum effect

[0257] ELISA: enzyme-linked immunosorbent assay

[0258] FACS: flow cytometry

[0259] PCR: polymerase chain reaction

[0260] HRP: horseradish peroxidase

[0261] ADCC: Antibody-dependent cell-mediated cytotoxicity

[0262] CDC: Complement-dependent cytotoxicity

[0263] DMEM: Dulbecco's Modified Eagle Medium

[0264] RPMI1640: Roswell Park Memorial Institute 1640 medium

[0265] FBS: Fetal bovine serum

[0266] Example 1 Construction and identification of Claudin18.2 overexpressing cell lines

[0267] The amino acid sequence of human claudin18.2 is referenced to P56856-2 in the Uniprot protein database. The amino acid sequence of human claudin18.1 is referenced to P56856-1 in the Uniprot protein database. These amino acid sequences were codon-optimized by Anhui General Biotechnology Co., Ltd. and synthesized in the lentiviral vector pLVX.

[0268] Viruses were prepared using a lentiviral packaging system and then infected with HEK293T cells (Nanjing Kebai, CBP60439) and CHO cells (ECACC). Stable HEK293T-Claudin18.1 (human) and HEK293T-Claudin18.2 (human) cell lines were generated by puromycin selection. Following successful construction, the expression rate of Claudin18.2 or 18.1 was determined by flow cytometry (FACS) for subsequent experiments.

[0269] The results of flow cytometry positive rate are shown in FIG1 . The positive rates of the above stable cell lines were all high (>95%).

[0270] Example 2 Alpaca Immunization and Serum Titer Detection

[0271] CHO-Claudin 18.2 stably transfected cells were cultured at 2.0×10 7 A healthy adult alpaca (Alpaca) was immunized with 100 cells of the immunization kit. Four immunizations were performed, with 21 days between each. Seven days after the fourth immunization, peripheral blood was collected and serum was isolated. Serum titers and specificities for antibodies against human claudin 18.2 were determined using flow cytometry (Sony, model: SA3800) using HEK293T cells (Nanjing Kebai, CBP60439) and HEK293T cells (Claudin 18.2 and Claudin 18.1 stably transfected cells). The MFI of the immunized serum against claudin 18.2 cells was 10-fold higher than that of the negative serum, and there was also a difference in binding to claudin 18.1 cells.

[0272] Table 1 FACS detection of alpaca serum titer

[0273] Example 3 Phage library construction and screening

[0274] 3.1 Phage library construction

[0275] After four immunizations, 50 mL of alpaca peripheral blood was collected and PBMCs were isolated according to the instructions of lymphocyte separation medium (Tianjin Haoyang Huake Biotechnology Co., Ltd.). Total RNA was extracted using Trizol method and PrimeScript TM The extracted total RNA was reverse transcribed using II 1st Strand cDNA Synthesis Kit (Takara, Cat. No. 6210A) to synthesize cDNA. The single-domain antibody gene fragment was amplified by nested PCR: First round PCR:

[0276] Upstream primer: 5'-CTTGGTGGTCCTGGCTGC-3' (SEQ ID NO: 22)

[0277] Downstream primer: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO: 23)

[0278] Second round of PCR:

[0279] The second round of PCR uses the recovered product from the first round of PCR as a template.

[0280] Upstream primer:

[0281] Downstream primer-1:

[0282] Downstream primer-2:

[0283] The VHH nucleic acid fragment product of the second round of PCR amplification was recovered and digested with SfiI (NEB, catalog number: R0123L) at 50°C overnight, and then the target fragment was recovered. The VHH nucleic acid fragment after digestion was inserted into the phage display vector pComb3xss (Chengdu Critical Point Biotechnology Co., Ltd.) and connected using T4 DNA Ligase (NEB, catalog number: M0202L). The ligation product was electroporated into Escherichia coli TG1 competent cells to construct a single domain antibody phage library against Claudin 18.2. After electroporation, 100 μL of the product was plated by gradient dilution and plated the next day at 10 -4 There were 217 clones on the plate for the dilution gradient determination of the library capacity, so the library capacity was 2.17×10 9 At the same time, 48 clones were randomly picked from the library capacity test plate for bacterial liquid PCR identification, and the results showed that the library insertion rate was 100%.

[0284] 3.2 Screening of anti-human Claudin 18.2 single domain antibodies

[0285] Take 5×10 6 HEK293T-Claudin 18.2 cells were centrifuged at 500g for 5 minutes, and the cell pellet was resuspended and washed twice with 5% serum-PBS; 500μL 3% OVA-PBS was added to the centrifuge tube containing the cells, and the cells were blocked with gentle shaking at 4°C for 1 hour; after blocking, the cells were centrifuged, the supernatant was removed, and the phage library diluent was added, and the cells were gently shaken at 4°C for 1 hour; centrifuged to remove unbound phages, and the cells were washed 6 times with 5% serum-PBS; 100μL Gly-HCl eluent was added, and the cells were incubated at 37°C for 8 minutes to elute specifically bound phages; the eluate was transferred to a 1.5mL sterile centrifuge tube and quickly neutralized with 10μL Tris-HCl neutralization buffer; 10μL was taken for serial dilution, the titer was determined, and the panning recovery rate was calculated. The remaining eluates were mixed, amplified and purified, and used for the next round of affinity panning.

[0286] After three rounds of screening, 192 clones were randomly selected from the third round of titer determination plates for monoclonal phage supernatant ELISA. HEK293T-Claudin 18.2 and HEK293T-Claudin 18.1 cells were cultured until confluent, washed twice with PBS, fixed with 100 μL of 4% paraformaldehyde, and incubated at 25°C for 20-30 min. The plates were washed twice with PBS, and 300 μL of 5% skim milk was added to each well and blocked at 37°C for 1 h. The plates were washed once with PBST, and 50 μL of phage culture supernatant and 50 μL of 5% skim milk were added to each well and incubated at 37°C for 1 h. The plates were washed five times with PBST, and horseradish peroxidase-conjugated anti-M13 antibody (Chengdu Critical Point Biotechnology Co., Ltd., used at a 1:10,000 dilution) was added at 100 μL / well and incubated at 37°C for 1 h. The plates were then washed six times with PBST. TMB colorimetric solution was added for color development (100 μL / well) at 37°C for 7 min. The reaction was terminated by adding stop solution (50 μL / well), and the optical density was measured at 450 nm. Human Claduin 18.2-positive and human Claduin 18.1-negative clones were selected for sequencing by the Chengdu Branch of Beijing Qingke Biotechnology Co., Ltd. The sequencing results were aligned and sorted, and after removing similar sequences, five specific clones with different sequences were screened. Their CDRs were analyzed using KABAT, Chothia, and IMGT software, respectively.

[0287] Example 4 Expression and Purification of Anti-Human Clau18.2 Cameloid VHH-Fc

[0288] The screened anti-Clau18.2 VHH fused to the human constant region IgG1 CH2-CH3 (SEQ NO: 21) was constructed and incorporated into the PTT5-IgG1 vector. The recombinant plasmid was directly shaken and extracted, then transfected into HEK293E cells using PEImax. After expression for approximately 7 days, the supernatant was collected by centrifugation. The supernatant was then purified using MabSelectSure affinity media. The purified antibody was ultrafiltered into PBS buffer, the concentration was determined, and the antibody was stored at -20°C.

[0289] Example 5 Detection of anti-human Clau18.2 camel-derived VHH-Fc antibodies

[0290] Flow cytometry (FACS) was used to detect the binding of camelid VHH-Fc to cells expressing Claudin 18.2 and Claudin 18.1. The experimental steps were as follows: HEK293T-Claudin18.2 and HEK293T-Claudin18.1 cells were digested, collected by centrifugation, and washed three times with pre-chilled PBS. The cells were resuspended in 1% BSA (in PBS) and added to a 96-well conical bottom plate at 3×10 cells per well. 5 cells in a volume of 50 μl; the test antibody was diluted to 10 ug / mL with 1% BSA (in PBS) (blank cells: no antibody was added; isotype control: anti-chicken lysozyme antibody was from Critical Point Biotechnology Co., Ltd.), 50 μl / well was added to the cells, mixed, and incubated at 4°C for 1 hour; the cells were collected by centrifugation, washed three times with pre-cooled PBS, and 50 μl of Alexa Fluor 647-labeled secondary antibody (Jackson, 109-605-003) was added, mixed, and incubated at 4°C for 0.5 hour; the cells were collected by centrifugation, washed three times with pre-cooled PBS, and resuspended in 200 μl PBS. The cells were detected by flow cytometry (Sony, model: SA3800), and the data were analyzed by FlowJo software. As shown in Figures 2-3 and Table 2, BS002-41, BS002-52, and BS002-376 all effectively bind to human Claudin 18.2 cells, while exhibiting no cross-binding to human Claudin 18.1 cells. BS002-5 and BS002-82 bind to human Claudin 18.2, but exhibit cross-binding to human Claudin 18.1 cells. Based on binding affinity, BS002-376 is considered a candidate molecule for subsequent humanization.

[0291] Table 2 Binding of anti-Clau18.2 camel-derived VHH-Fc to human Claudin 18.2 and human Claudin 18.1 cells

[0292] Example 6 Humanization of Anti-Human Claudin18.2 Single Domain Antibody

[0293] Using the CDR transplantation method, the conventional BLAST method was first used to find the human germline sequence with the highest homology to the original camel-derived sequence BS002-376, which was used as a template; the CDRs of the camel-derived single-domain antibody were transplanted onto the human template to construct a chimera; based on structural analysis, the FR amino acids in the camel-derived single-domain antibody that can retain its original conformation were identified, and the corresponding amino acids in the chimera were backmutated to camel-derived amino acids to maintain the original affinity; the constructed humanized antibodies were subjected to calculations and immunogenicity analysis to find highly immunogenic fragments, and the low-immunogenic fragments were replaced to form three humanized single-domain antibodies named LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3.

[0294] Example 7 Preparation of Humanized Single Domain Antibody Against Human Claudin18.2

[0295] The variable region amino acid sequences of LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 (SEQ NO:6, SEQ NO:7, and SEQ NO:8, respectively) were submitted to Anhui General Biotechnology for gene synthesis. After codon optimization, they were constructed onto the PTT5-IgG1 vector, containing the human constant region IgG1 CH2-CH3 (SEQ NO:21). After the plasmid was synthesized, it was directly shaken and extracted. HEK293E cells were transfected with PEImax and expressed for about 7 days, and the supernatant was collected by centrifugation. The supernatant was purified using MabSelectSure affinity filler. The purified antibodies were all ultrafiltered into PBS buffer, the concentration was determined, and the cells were stored at -20 degrees. The reference antibody IMAB362 was prepared in the same manner, and its sequence is derived from patent: CN 101312989 B.

[0296] Example 8 Purity Detection of Anti-Human Claudin18.2 Humanized Single Domain Antibody

[0297] The purity of the anti-human Claudin18.2 humanized single domain antibody was tested by SEC-HPLC as follows:

[0298] Instrument: Waters Alliance e2695 HPLC;

[0299] Chromatographic column: Thermo MabPac SEC-1, 5 μm, 7.8*300 mm;

[0300] Mobile phase: 61 mmol / L Na2HPO4, 39 mmol / L NaH2PO4, 200 mmol / L NaCl, 5% IPA;

[0301] Instrument parameters: sample chamber temperature: 8°C; column temperature: 30°C; flow rate: 0.5 ml / min; injection volume: 20 μg; detection wavelength: 280 nm; isocratic operation: 30 min.

[0302] The experimental results are shown in Table 3. LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 all have high purity (>97%).

[0303] Table 3 Purity of anti-human Claudin18.2 humanized single domain antibody

[0304] Example 9 Tm value detection of anti-human Claudin18.2 humanized single domain antibody

[0305] The Tm value of humanized single-domain antibodies was determined using DSF to reflect the thermal stability of the antibody. The experimental steps are as follows: the antibody sample to be tested was diluted to 1 mg / mL with PBS; the dye SYPRO Orange dye (Thermo #56651) was diluted to 40X with ddH2O; the reaction system was: 12.5uL sample + 2.5uL 40X dye + 5uL ddH2O; the membrane was sealed and the reaction was centrifuged briefly; the reaction was detected by Q-PCR instrument (Hangzhou Jingge, CG-05) with the following Q-PCR parameters: Target (ROX) and program (25°C, 3 min; 1% rate, 95°C; 95°C, 2 min).

[0306] The experimental results showed that the Tm values ​​of LJD003-HZ1, LJD003-HZ2 and LJD003-HZ3 were 68.6℃, 67.1℃ and 69.1℃, respectively, indicating that all three have good thermal stability.

[0307] Example 10 Affinity Evaluation of Humanized Single Domain Antibodies Against Human Claudin18.2

[0308] The affinity of the humanized single-domain antibody against human Claudin18.2 was determined by flow cytometry (FACS). The experimental steps were as follows: HEK293T-Claudin18.2 (human) and NUGC4-Claudin18.2 (human) cells were digested, collected by centrifugation, and washed three times with pre-chilled PBS; the cells were resuspended in 1% BSA (in PBS) and added to a 96-well conical bottom plate at 3×10 cells per well. 5cells in a volume of 50 μl; dilute the test antibody with 1% BSA (in PBS) starting at 40 μg / mL, dilute 3-fold, and 10 concentration points, taking 50 μl / well and adding it to the cells to make the final antibody concentration of 20 μg / mL, mix well, and incubate at 4°C for 1 hour; collect the cells by centrifugation, wash three times with pre-cooled PBS, resuspend the cells with 50 μl 1% BSA (in PBS), add 1 μl fluorescent secondary antibody to each well, mix well, and incubate at 4°C for 0.5 hour; collect the cells by centrifugation, wash three times with pre-cooled PBS, resuspend the cells with 200 μl PBS, detect by flow cytometer (Beckman, model: CytoFLEX), and import the detection data into Graph-Prism for graphical calculation.

[0309] For HEK293T-Claudin18.2 (human) cells, the experimental results are shown in Figure 4. LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 all have high affinity with them, with EC50 values ​​of 208.3 ng / mL, 355.7 ng / mL, and 308.2 ng / mL, respectively, and maximum fluorescence signal values ​​of 1362190, 1378434, and 1178882, respectively; while the EC50 value of the reference antibody IMAB362 is 438.9 ng / mL, and the maximum fluorescence signal value is 1044124.

[0310] For NUGC4-Claudin18.2 (human) cells, the experimental results are shown in Figure 5. LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 all have high affinity with it, with EC50 values ​​of 620.5 ng / mL, 576.1 ng / mL, and 528.9 ng / mL, respectively, and maximum fluorescence signal values ​​of 1575179, 1325627, and 1105701, respectively; while the EC50 value of the reference antibody IMAB362 is 687.5 ng / mL, and the maximum fluorescence signal value is 879801.

[0311] The above results show that the affinity of LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 to HEK293T-Claudin18.2 (human) and NUGC4-Claudin18.2 (human) cells is better than that of IMAB362.

[0312] Example 11 Specificity Evaluation of Anti-Human Claudin18.2 Humanized Single Domain Antibody

[0313] The specificity of the humanized single-domain antibody against human Claudin18.2 was determined by flow cytometry (FACS). The experimental steps were as follows: HEK293T-Claudin18.1 (human) cells were collected by digestion and centrifugation, washed three times with pre-cooled PBS, and resuspended in 1% BSA (in PBS). 3×10 cells were added to each well. 5 Cells were plated into 96-well pointed-bottom plates, and the antibodies to be detected were added at final concentrations of 2 μg / mL and 10 μg / mL, respectively. The cells were incubated at 4°C for 1 hour, washed three times with pre-cooled PBS, and 1 μl of fluorescent secondary antibody was added to each well. The cells were incubated at 4°C for 0.5 hour, washed three times with pre-cooled PBS, and resuspended. The cells were detected by flow cytometry (Beckman, model: CytoFLEX), and the detection values ​​were imported into Graph-Prism for graphical calculation.

[0314] The experimental results are shown in Figure 6. LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 do not non-specifically recognize human Claudin18.1.

[0315] Example 12 Species Cross-Evaluation of Humanized Single Domain Antibodies Against Human Claudin18.2

[0316] The amino acid sequence of cynomolgus macaque Claudin18.2 is referenced to XP_015300615.1 in the NCBI protein database. The amino acid sequence of mouse Claudin18.2 is referenced to NP_001181850.1 in the NCBI protein database. The amino acid sequence of rat Claudin18.2 is referenced to NP_001014118.1 in the NCBI protein database. The above amino acid sequences were codon-optimized by GenScript and synthesized in the lentiviral vector pLVX. Viruses were prepared using the lentiviral packaging system and the obtained viruses were used to infect HEK293T cells. After selection with puromycin, HEK293T-Claudin18.2 (monkey), HEK293T-Claudin18.2 (rat), and HEK293T-Claudin18.2 (mouse) stable cell lines were obtained.

[0317] The affinity of the anti-human Claudin18.2 humanized single-domain antibody to monkey Claudin18.2, rat Claudin18.2, and mouse Claudin18.2 was detected by flow cytometry (FACS). The experimental steps are as follows: HEK293T-Claudin18.2 (monkey), HEK293T-Claudin18.2 (rat), and HEK293T-Claudin18.2 (mouse) cells were digested, collected by centrifugation, and washed three times with pre-cold PBS; the cells were resuspended with 1% BSA (in PBS) and added to a 96-well conical bottom plate, with 3×10^5 cells added to each well in a volume of 50 μL; the test antibody was diluted with 1% BSA (in PBS) starting at 40 μg / mL, diluted 3-fold, 10 concentration points, and 50 μL / well was added to the cells to make the final antibody concentration of 20 μg / mL, mixed, and incubated at 4°C for 1 hour; the cells were collected by centrifugation, washed three times with pre-cold PBS, resuspended with 50 μL 1% BSA (in PBS), 1 μL fluorescent secondary antibody was added to each well, mixed, and incubated at 4°C for 0.5 hour; the cells were collected by centrifugation, washed three times with pre-cold PBS, and resuspended with 50 μL 1% BSA (in PBS). 1 μL fluorescent secondary antibody was added to each well, mixed, and incubated at 4°C for 0.5 hour; the cells were collected by centrifugation, washed three times with pre-cold PBS, and The cells were resuspended in PBS and detected by flow cytometry (Beckman, model: CytoFLEX). The detected values ​​were imported into Graph-Prism for graphing and calculation.

[0318] For HEK293T-Claudin18.2 (monkey) cells, the experimental results are shown in Figure 7. LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 all have high affinity with them, with EC50 values ​​of 62.33 ng / mL, 103.9 ng / mL, and 85.11 ng / mL, respectively, and maximum fluorescence signal values ​​of 1325843, 1651947, and 1552783, respectively.

[0319] For HEK293T-Claudin18.2 (rat) cells, the experimental results are shown in Figure 8. LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 all have high affinity with them, with EC50 values ​​of 167.1 ng / mL, 180.7 ng / mL, and 175.9 ng / mL, respectively, and maximum fluorescence signal values ​​of 2726433, 2768686, and 2661951, respectively.

[0320] For HEK293T-Claudin18.2 (mouse) cells, the experimental results are shown in Figure 9. LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 all have high affinity with them, with EC50 values ​​of 113.8 ng / mL, 131.7 ng / mL, and 115.9 ng / mL, respectively, and maximum fluorescence signal values ​​of 2415543, 2466115, and 2177835, respectively.

[0321] The above results show that LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 can all recognize Claudin18.2 in crab-eating macaques, rats, and mice.

[0322] Example 13 Evaluation of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Activity of Humanized Anti-Human Claudin18.2 Single Domain Antibodies

[0323] ADCC activity of a humanized single-domain antibody against human claudin18.2 was measured using a luciferase reporter assay. The following experimental steps were used: HEK293T-Claudin18.2 cells were harvested by digestion and centrifugation, resuspended in DEME+2% FBS, and 100,000 cells were plated in 50 μL per well. Jurkat-NFAT-CD16a cells were harvested by centrifugation, resuspended in 1640+2% FBS, and 100,000 cells were plated in 50 μL per well. The test antibody was diluted in DEME+2% FBS, starting at 100 μg / mL, and diluted 5-fold over 11 concentrations. 100 μL / well was added to the cells to a final concentration of 50 μg / mL. Serially diluted test antibodies were added, mixed, and incubated at 37°C for 5 hours. 30 μL of Bio-Glory One-Step Luciferase Substrate (Suzhou Ruian, RA-GO04) was added to each well, and the results were read using a microplate reader (MD, i3x).

[0324] The experimental results are shown in Figure 10. LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 all have strong ADCC activity, with EC50 of 5.432 ng / mL, 4.572 ng / mL, and 3.158 ng / mL, respectively, which are better than the reference antibody IMAB362, whose EC50 is 37.50 ng / mL.

[0325] Example 14 Determination of Endocytic Activity (Killing Method) of Humanized Single Domain Antibodies Against Human Claudin18.2

[0326] The endocytic activity of a humanized single-domain antibody against human claudin18.2 was assessed by cell killing. The following experimental steps were performed: HEK293T-Claudin18.2 cells were harvested by trypsinization and centrifugation. The cells were resuspended in DMEM + 2% FBS and counted. A total of 5,000 cells were plated per well of a 96-well plate in a 50 μL volume. The test antibody was diluted in DMEM + 2% FBS to a starting concentration of 40 μg / mL. A four-fold serial dilution was performed over 11 concentrations, and 100 μL was added to the cell wells for a final concentration of 20 μg / mL. Anti-human IgG-MMAE (Apac Biotech) was diluted in DMEM + 2% FBS to a concentration of 8 μg / mL. 50 μL was added to the cell wells for a final concentration of 2 μg / mL. Control wells included a naked antibody control (antibody + cells), an MMAE control (anti-human IgG-MMAE + cells), and a blank control (cells alone). After 4 days of culture, 20 μL of CCK8 detection reagent was added to each well. The 96-well plate was placed in an incubator and incubated for 0.5-4 hours. OD450 nm was measured using a microplate reader (MD, model: Spectramax ABS Plus), and the detection values ​​were imported into Graph-Prism for graphical calculation.

[0327] The experimental results are shown in Figure 11. The signal value range of the naked antibody control wells is 1.5523-1.7328, the signal value range of the MMAE control wells is 1.2881-1.3091, the signal value range of the blank control wells is 1.6843-1.7405, the TOP value range of the detection wells is 1.285-1.359, and the Bottom value range is 0.9931-1.088. It can be seen that LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 can mediate the endocytosis of Claudin18.2, with EC50 of 26.09 ng / mL, 24.63 ng / mL, and 41.29 ng / mL, respectively, which are all better than the control antibody IMAB362, whose EC50 is 54.93 ng / mL.

[0328] Example 15 Determination of Endocytic Activity of Humanized Anti-Human Claudin18.2 Single Domain Antibody (Flow Cytometry)

[0329] The endocytic activity of the humanized single-domain antibody against human Claudin18.2 was detected by flow cytometry (FACS). The experimental steps are as follows: HEK293T-Claudin18.2 cells were collected by digestion and centrifugation, incubated with the antibody to be tested at a concentration of 10ug / mL at 4°C for 1 hour, washed three times with PBS, resuspended in DMEM + 10% FBS, divided into four parts and incubated at 37°C for 0, 1, 2, and 4 hours, respectively. After washing three times with PBS, 1μl of fluorescent secondary antibody was added, incubated at 4°C for 0.5h, washed three times with PBS, and resuspended in the microscope. The endocytic rate was calculated according to the following formula: Endocytic rate (%) = [1-(average fluorescence value of the test sample at that time point - average fluorescence value of the negative control sample at that time point) / (average fluorescence value of the test sample at 0 hour - average fluorescence value of the negative control sample at 0 hour)] * 100.

[0330] The experimental results are shown in FIG12 . LJD003-HZ3 can mediate the internalization of Claudin18.2, and the internalization rate is 20.47% after 4 hours.

[0331] The antibody sequences obtained by the present invention are shown in Table 4 below:

[0332] Table 4 Antibody sequences

[0333] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A single-domain antibody targeting Claudin18.2, wherein the VHH chain of the single-domain antibody comprises complementary determining regions CDR1, CDR2 and CDR3 derived from the heavy chain variable region shown in SEQ ID NO: 6, 7 or 8, and the complementary determining regions CDR1, CDR2 and CDR3 are defined by Chothia, Abm, Kabat, or IMGT rules.

2. The single domain antibody according to claim 1, characterized in that The CDR1, CDR2 and CDR3 are selected from the following groups: (1) Based on the definition of Chothia rules: CDR1 shown in SEQ ID NO:9, CDR2 shown in SEQ ID NO:10, and CDR3 shown in SEQ ID NO:11; or, (2) Based on the definition of Abm rules: CDR1 shown in SEQ ID NO:12, CDR2 shown in SEQ ID NO:13, and CDR3 shown in SEQ ID NO:11; or, (3) Based on the definition of Kabat rules: CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO: 15, 19 or 20, and CDR3 shown in SEQ ID NO:11; or, (4) Based on the definition of IMGT rules: CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO:

18.

3. The single domain antibody according to claim 1, characterized in that The VHH chain of the single-domain antibody has SEQ ID NO: 5 or an amino acid sequence with a homology of ≥ 85% to the amino acid sequence.

4. The single domain antibody according to claim 1, characterized in that The single domain antibody is humanized.

5. The single domain antibody according to claim 4, characterized in that The VHH chain of the single-domain antibody has an amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or an amino acid sequence having a homology of ≥ 85% with the amino acid sequence.

6. An antibody targeting Claudin18.2, characterized in that: The antibody comprises one or more VHH chains of the single-domain antibody targeting Claudin18.2 according to any one of claims 1 to 5.

7. The antibody according to claim 6, characterized in that The antibody is a heavy chain antibody, which comprises heavy chain constant regions CH2 and CH3 (Fc segment).

8. The antibody according to claim 7, characterized in that The heavy chain constant region is derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

9. A multispecific antibody, characterized in that The multispecific antibody comprises: a single domain antibody targeting Claudin18.2 as described in any one of claims 1 to 5 or an antibody targeting Claudin18.2 as described in any one of claims 6 to 8.

10. A recombinant protein, characterized in that The recombinant protein comprises: (i) the single domain antibody targeting Claudin18.2 according to any one of claims 1 to 5, or the antibody targeting Claudin18.2 according to any one of claims 6 to 8; and (ii) optional polypeptide molecules or fragments having therapeutic functions; and / or (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

11. An immunoconjugate, characterized in that: The immunoconjugate contains: (a) an antibody portion, which is a single domain antibody targeting Claudin18.2 as described in any one of claims 1 to 5 or an antibody targeting Claudin18.2 as described in any one of claims 6 to 8; and (b) a conjugated moiety conjugated to the single domain antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

12. The immunoconjugate according to claim 11, characterized in that The coupling part is a radionuclide.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (i) a single domain antibody targeting Claudin18.2 according to any one of claims 1 to 5, an antibody targeting Claudin18.2 according to any one of claims 6 to 8, a multispecific antibody according to claim 9, a recombinant protein according to claim 10, or an immunoconjugate according to any one of claims 11 to 12; (ii) a pharmaceutically acceptable carrier.