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 disclosed by the invention has high specificity, is combined with Claudin18.2 with high affinity and is basically not combined with Claudin18.1. The single-domain antibody provided by the invention provides a research and development basis for development of antibody drugs, antibody coupling drugs and multispecific antibody drugs.

Description

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 still need to be found in advanced gastric cancer. At the 2016 American Society of Clinical Oncology Annual Meeting (ASCO), Astellas's antibody drug IMAB362 targeting Claudin18.2 plus chemotherapy for the treatment of patients with locally advanced or metastatic gastric cancer showed that the patients' progression-free survival and overall survival were prolonged. The Phase III clinical data disclosed at ASCO in 2023 also achieved positive results. However, no monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates (ADCs) targeting Claudin18.2 have been approved for marketing.

[0003] Single-domain antibodies were first reported by Belgian scientist Hamers-Casterman and his team in Nature magazine in 1993. They found that some antibodies in the blood of camelids were "heavy chain antibodies" without light chains. VHH retains all antigen binding ability and is the smallest fragment that retains complete antigen binding. It is called single-domain antibodies, with a molecular weight of only 15kDa. It has the advantages of small molecular weight, high affinity, and good stability, and has been widely used in biopharmaceutical research and development.

[0004] Claudin18.1 is another splice of CLDN18, expressed in the lungs. Claudin18.1 is similar to Claudin18.2 in structure, and the extracellular domain only differs by 8 amino acid residues in the ECL1 sequence. Therefore, the difficulty in developing antibodies against Claudin18.2 lies in the development of antibodies against Claudin18.2 that specifically recognize Claudin18.2 but not Claudin18.1.

[0005] Therefore, there is a need in the art to develop highly specific single-domain antibody drugs targeting Claudin18.2 to provide gastric cancer patients with broader and better medication options. Summary of the invention

[0006] The object of the present invention is to provide an anti-Claudin18.2 single domain antibody and a humanized antibody thereof.

[0007] In the first aspect of the present invention, a single-domain antibody targeting Claudin18.2 is provided, the VHH chain of the single-domain antibody comprising 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.

[0008] In another preferred embodiment, the CDR1, CDR2 and CDR3 are selected from the following groups:

[0009] (1) Based on the definition of Chothia rules:

[0010] CDR1 shown in SEQ ID NO:9,

[0011] CDR2 shown in SEQ ID NO:10, and

[0012] CDR3 shown in SEQ ID NO:11; or,

[0013] (2) Based on the definition of Abm rules:

[0014] CDR1 shown in SEQ ID NO:12,

[0015] CDR2 shown in SEQ ID NO:13, and

[0016] CDR3 shown in SEQ ID NO:11; or,

[0017] (3) Based on the definition of Kabat rules:

[0018] CDR1 shown in SEQ ID NO:14,

[0019] CDR2 shown in SEQ ID NO: 15, 19 or 20, and

[0020] CDR3 shown in SEQ ID NO:11; or,

[0021] (4) Based on the definition of IMGT rules:

[0022] CDR1 shown in SEQ ID NO:16,

[0023] CDR2 shown in SEQ ID NO: 17, and

[0024] CDR3 shown in SEQ ID NO:18.

[0025] In another preferred example, 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.

[0026] In another preferred embodiment, any one of the above amino acid sequences also 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 for Claudin18.2.

[0027] 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.

[0028] In another preferred embodiment, the VHH chain of the single-domain antibody further includes a framework region (FR).

[0029] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0030] In another preferred embodiment, the framework region FR is of human, mouse, rabbit or camel origin.

[0031] 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% with that of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0032] 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.

[0033] In another preferred embodiment, the single domain antibody is humanized.

[0034] 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.

[0035] 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.

[0036] In another preferred embodiment, the antibody is a monomer, a bivalent antibody, and / or a multivalent antibody.

[0037] In another preferred embodiment, the antibody is an animal-derived antibody, a humanized antibody, or a chimeric antibody.

[0038] In another preferred embodiment, the CDR region of the humanized antibody comprises 1, 2, or 3 amino acid changes.

[0039] In another preferred embodiment, the animal is a non-human mammal, preferably a mouse, sheep, rabbit or camel.

[0040] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0041] In another preferred embodiment, the antibody is a monoclonal antibody.

[0042] In another preferred embodiment, the antibody is a partially or fully humanized antibody.

[0043] 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).

[0044] In another preferred embodiment, the heavy chain constant region is derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

[0045] In another preferred embodiment, the amino acid sequence of the Fc segment of human IgG is shown in SEQ ID NO:21.

[0046] In another preferred embodiment, any one of the above amino acid sequences also 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 for Claudin18.2.

[0047] 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%, more preferably 10%.

[0048] In another preferred embodiment, the antibody does not bind or weakly binds to human Claudin18.1.

[0049] The third aspect of the present invention provides a multispecific antibody, wherein the multispecific antibody comprises: the single domain antibody targeting Claudin18.2 described in the first aspect of the present invention or the antibody targeting Claudin18.2 described in the second aspect of the present invention.

[0050] In another preferred embodiment, the multispecific antibody comprises one or more second antigen binding regions targeting additional antigen targets.

[0051] In another preferred embodiment, the second antigen binding region is an antibody or an 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.

[0052] In another preferred embodiment, the second antigen binding region is a single domain antibody.

[0053] In a fourth aspect, the present invention provides a recombinant protein, wherein the recombinant protein comprises:

[0054] (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

[0055] (ii) optional polypeptide molecules or fragments having therapeutic functions; and / or

[0056] (iii) Optional functional domains that enhance the physicochemical properties or druggability of the protein.

[0057] In another preferred embodiment, the improving the physicochemical properties or drugability of the protein includes prolonging the half-life of the single domain antibody targeting Claudin18.2.

[0058] In another preferred embodiment, the recombinant protein further comprises: (iv) an optional tag sequence for facilitating expression and / or purification.

[0059] In another preferred embodiment, the tag sequence is selected from the following group: 6His tag, GGGS sequence, FLAG tag.

[0060] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0061] In another preferred embodiment, the polypeptide molecules or fragments having therapeutic functions include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, and cytokines.

[0062] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0063] In another preferred embodiment, the fusion protein includes a multispecific antibody and a chimeric antibody.

[0064] In another preferred embodiment, the functional domains for improving the physicochemical properties or drugability of proteins include Fc segments and human serum albumin (HSA).

[0065] In another preferred embodiment, the fusion protein has the following elements from the NC end:

[0066] AB;

[0067] 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);

[0068] “-” represents a peptide bond.

[0069] 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.

[0070] In a fifth aspect, the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0071] (a) an antibody portion, wherein the antibody portion is 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

[0072] (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.

[0073] In another preferred embodiment, the immunoconjugate is a single domain antibody drug conjugate.

[0074] In another preferred embodiment, the single domain antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0075] In another preferred embodiment, the coupling moiety is a chemical marker and a biological marker.

[0076] In another preferred embodiment, the chemical label is an isotope, an immunotoxin and / or a chemical drug.

[0077] In another preferred embodiment, the biological marker is biotin, avidin or an enzyme marker.

[0078] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0079] In another preferred embodiment, the drug is a cytotoxic drug.

[0080] In another preferred embodiment, the toxin is selected from the following group:

[0081] auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, 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, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotonin, calicheamicin, saponin, officinalis) inhibitors, glucocorticoids, or a combination thereof.

[0082] In another preferred embodiment, the coupling moiety is a detectable label.

[0083] In another preferred embodiment, the detectable marker comprises a radionuclide, and the radionuclide comprises:

[0084] (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

[0085] (ii) therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133 Yb-169, Yb-177, or a combination thereof.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] In another preferred embodiment, the detection is in vivo detection or in vitro detection.

[0090] In another preferred embodiment, the immunoconjugate is used for diagnosing and / or treating tumors expressing CLAUDIN18.2 protein.

[0091] In another preferred embodiment, the immunoconjugate has the following molecular formula:

[0092]

[0093] in:

[0094] nAb is a single-domain antibody targeting Claudin18.2, an antibody targeting Claudin18.2, or a multispecific antibody;

[0095] LU is a linker (also called a connector);

[0096] D is for medicine;

[0097] Furthermore, the subscript p is a value selected from 1-10.

[0098] 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 contains 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).

[0099] In another preferred embodiment, the antibody portion is covalently bound to the linker by reacting with a portion selected from the following group: maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC), etc.

[0100] In another preferred embodiment, D is selected from the following group of compounds having anti-tumor activity:

[0101] (i) Tubulin inhibitors, such as maytansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF);

[0102] (ii) Acting on DNA toxins, such as duocarmycin and pyrrolobenzodiazepine (PBD);

[0103] (iii) Topoisomerase inhibitors, such as camptothecin, SN38, exitecan, and Dxd.

[0104] In a sixth aspect, the present invention provides a pharmaceutical composition, comprising:

[0105] (i) 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, the multispecific antibody as described in the third aspect of the present invention, the recombinant protein as described in the fourth aspect of the present invention, or the immunoconjugate as described in the fifth aspect of the present invention;

[0106] (ii) a pharmaceutically acceptable carrier.

[0107] In another preferred embodiment, the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.

[0108] In another preferred embodiment, the pharmaceutical composition further comprises other biologically active substances, such as drugs for treating tumors.

[0109] 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.

[0110] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: liquid, solid, or gel.

[0111] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0112] In another preferred embodiment, the pharmaceutical composition is an injection.

[0113] In the seventh aspect of the present invention, a use of an active ingredient is provided, 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.

[0114] In another preferred embodiment, the detection reagent, detection plate or kit is used for:

[0115] (1) detecting CLAUDIN18.2 protein in a sample; and / or

[0116] (2) detecting endogenous CLAUDIN18.2 protein in tumor cells; and / or

[0117] (3) Detect tumor cells expressing CLAUDIN18.2 protein.

[0118] In another preferred embodiment, the detection type includes but is not limited to flow cytometry, cell immunofluorescence detection, enzyme-linked immunosorbent assay, immunoblotting detection, etc.

[0119] In another preferred embodiment, the detection reagent, detection plate or kit is used to diagnose CLAUDIN18.2-related diseases.

[0120] In another preferred embodiment, the drug is used to treat or prevent tumors with high expression of CLAUDIN18.2, tumor migration, or tumor resistance.

[0121] In another preferred embodiment, the tumor resistance includes: resistance to tumor immunotherapy drugs, resistance to tumor targeted therapy drugs, resistance to conventional tumor chemotherapy, and insensitivity to radiotherapy.

[0122] In another preferred embodiment, the CLAUDIN18.2-related disease is selected from the following group: cancer.

[0123] In another preferred embodiment, the CLAUDIN18.2-related diseases include: tumor occurrence, growth and / or metastasis.

[0124] In another preferred embodiment, the cancer includes solid tumors and blood cancers.

[0125] 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.

[0126] In an eighth aspect, the present invention provides a polynucleotide encoding a polypeptide selected from the following group:

[0127] (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

[0128] (2) The recombinant protein as described in the fourth aspect of the present invention.

[0129] In another preferred embodiment, the polynucleotide includes RNA, DNA or cDNA.

[0130] The ninth aspect of the present invention provides a vector, wherein the vector contains the polynucleotide as described in the eighth aspect of the present invention.

[0131] 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.

[0132] 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.

[0133] In an eleventh aspect of the present invention, a method for in vitro detection (including diagnostic or non-diagnostic) of CLAUDIN18.2 in a sample is provided, the method comprising the steps of:

[0134] (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;

[0135] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CLAUDIN18.2 in the sample.

[0136] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0137] In a twelfth aspect, the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

[0138] (a) culturing the host cell according to the tenth aspect of the present invention under conditions suitable for expression;

[0139] (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.

[0140] In the thirteenth aspect of the present invention, a method for treating CLAUDIN18.2-related diseases is provided, 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.

[0141] In another preferred embodiment, the method further comprises: administering other drugs or treatment methods to a subject in need for combined treatment.

[0142] In another preferred embodiment, the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.

[0143] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] Figure 1The positive rate detection of HEK293T-Claudin18.2 (human) and HEK293T-Claudin18.1 (human) stable cell lines is shown.

[0145] Figure 2 The binding of camel-derived VHH-Fc to human Claudin 18.2 was detected by flow cytometry (FACS).

[0146] Figure 3 Flow cytometry (FACS) detection of camelid VHH-Fc binding to human Claudin 18.1 is shown.

[0147] Figure 4 The cell affinity detection (flow cytometry) of anti-human Claudin18.2 humanized single domain antibody and HEK293T-Claudin18.2 (human) is shown.

[0148] Figure 5 The binding detection of anti-human Claudin18.2 humanized single domain antibody to HEK293T-Claudin18.1 (human) cells (flow cytometry) is shown.

[0149] Figure 6 The endocytic activity assay of anti-human Claudin18.2 humanized single domain antibody is shown. DETAILED DESCRIPTION

[0150] After extensive and in-depth research, the inventors have developed for the first time a single domain antibody and a humanized antibody thereof that specifically targets Claudin 18.2. The single domain antibody and the humanized antibody thereof of the present invention have a specific high affinity for Claudin 18.2, but basically do not bind to its isomer Claudin 18.1. On this basis, the present invention was completed.

[0151] the term

[0152] 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), and 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.

[0153] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 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 between the heavy chains of different immunoglobulin isotypes varies. 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 to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0154] As used herein, the terms "single domain antibody (VHH)" and "nanobody" have the same meaning, referring to the variable region of the heavy chain of a monoclonal antibody, and constructing a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, 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 of only one heavy chain variable region.

[0155] 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 antibody of the present invention comprises a heavy chain variable region (VHH) and heavy chain constant regions CH2 and CH3. The heavy chain antibody of the present invention may be an antibody isolated from an animal (such as camel-derived) that naturally lacks light chains and heavy chain constant region 1 (CH1); or it may be a recombinant antibody obtained by recombining a single domain antibody (VHH) of the present invention with a heavy chain constant region. The heavy chain antibody 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.

[0156] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms 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 fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0157] As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments of the present invention to form conjugates. The present invention also includes cell surface markers or antigens combined with the anti-Claudin18.2 protein antibodies or fragments thereof.

[0158] As used herein, the term "heavy chain variable region" is synonymous with "V H ” can be used interchangeably.

[0159] As used herein, the terms "hypervariable region" and "complementarity determining region (CDR)" are used interchangeably.

[0160] 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.

[0161] 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.

[0162] 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, and all refer to polypeptides that specifically bind to Claudin18.2 protein, such as proteins or polypeptides having a single domain antibody VHH chain of the present invention. They may or may not contain an initial methionine.

[0163] The present invention also provides other proteins or fusion expression products having 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) having 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.

[0164] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the hypervariable region (CDR). This segment is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0165] 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 binding to antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0166] Claudin18.2 and single domain antibodies against Claudin18.2

[0167] 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) subtype is a gastric-specific subtype 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 and becoming 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.

[0168] As used herein, "Chothia", "Kabat", "IMGT", "AbM" refer to the determination of complementary binding determining regions CDR 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 loop (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), International Immuno GeneTics database (IMGT), and Chothia definition based on loop structure position.

[0169] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above-mentioned ways.

[0170] Preferably, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and is able to retain the binding affinity to CLAUDIN18.2.

[0171] 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%.

[0172] 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.

[0173] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0174] Wherein, the animal is preferably a mammal, such as a mouse or a camel.

[0175] 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.

[0176] Recombinant protein (or fusion protein)

[0177] 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 also 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, CDlla, 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.

[0178] Preferably, the multispecific antibody comprises one or more second antigen binding regions, or further comprises a third antigen binding region.

[0179] 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.

[0180] 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. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as 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). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0181] The antibody of the present invention refers to a polypeptide having CLAUDIN18.2 protein binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0182] 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.

[0183] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids 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 amino acid substitution according to Table A.

[0184] Table A

[0185]

[0186] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0187] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0188] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0189] The present invention also relates to polynucleotides that hybridize with 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 can hybridize with 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) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0190] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0191] Antibody preparation

[0192] The sequence of the DNA molecule of the antibody or its fragment of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody; or the coding sequences of the single-domain antibody and the constant region can be fused together to form a heavy-chain antibody.

[0193] 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.

[0194] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0195] At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The 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 sequence of the present invention by chemical synthesis.

[0196] 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.

[0197] 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, HEK-293 cells.

[0198] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.

[0199] The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by 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, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0200] The antibodies of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, the recombinant protein can be separated and purified by various separation methods using 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 protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0201] Immunoconjugates

[0202] The present invention also provides an immunoconjugate (ADC) based on the antibody of the present invention, preferably a single domain antibody-drug conjugate (NDC).

[0203] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, and preferably chemically coupled. Wherein, the effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule can 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.

[0204] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent can be 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 can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0205] 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 agents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0206] Single domain antibodies can be coupled to drugs to form antibody drug conjugates (NDCs). Typically, NDCs contain a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. 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 (such as linkers that are hydrolyzed when the pH is less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by proteases.

[0207] 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 through the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimide compounds, halogenated amides (e.g., iodinated, brominated or chlorinated); halogenated esters (e.g., iodinated, brominated or chlorinated); halogenated methyl ketones (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, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.

[0208] The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.

[0209] 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.

[0210] 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 with a radionuclide.

[0211] In the present invention, drug-linkers can be used to form NDC in one simple step. In other embodiments, bifunctional linker compounds can be used to form NDC 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.

[0212] Typically, the functional groups on the linker are selected to react specifically with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety can be used to react specifically with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group. 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 attachment 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 of the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0213] The present invention also provides a method for preparing NDC, which may further include: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (NDC).

[0214] In certain embodiments, the methods of the invention include: combining the antibody with the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further include: combining the antibody-linker conjugate with the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0215] In some embodiments, the structure of the immunoconjugate, preferably a single domain antibody drug conjugate NDC, is shown in the following molecular formula:

[0216]

[0217] in:

[0218] nAb is the above-mentioned single domain antibody targeting Claudin18.2, heavy chain antibody targeting Claudin18.2 or multispecific antibody,

[0219] LU is the linker / connector;

[0220] D is for medicine;

[0221] And the subscript p is a value selected from 1 to 10.

[0222] application

[0223] The present invention also provides uses of the antibodies of the present invention, such as for preparing diagnostic preparations, or for preparing drugs for preventing and / or treating CLAUDIN18.2-related diseases. The CLAUDIN18.2-related diseases include tumor occurrence, growth and / or metastasis, tumor resistance-related diseases, inflammation, metabolism-related diseases, etc.

[0224] 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 occurrence, growth and / or metastasis, especially tumors with high expression of CLAUDIN18.2. The tumors include (but 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, etc.; especially gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung adenocarcinoma, more preferably gastric cancer.

[0225] Pharmaceutical composition

[0226] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its NDC, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0227] The pharmaceutical composition of the present invention can be directly used to 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.

[0228] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention 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, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. 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 microgram / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0229] 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 usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0230] 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 protein (antigen) on the cell surface, the drug is released 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, and the antibody-drug conjugate mentioned above can be administered to a subject (e.g., a human) in a therapeutically effective amount through a suitable route. The subject in need of treatment may 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 by routine physical examination.

[0231] When treated with the single domain antibody-drug conjugate of the present invention, it can be delivered by conventional methods in the art. For example, it can be introduced into cells by 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.

[0232] The main advantages of the present invention include:

[0233] (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.

[0234] (2) The present invention provides a humanized single-domain antibody, which reduces the immunogenicity of the antibody and improves its in vivo safety.

[0235] (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.

[0236] (4) The single domain antibody of the present invention has high purity and thermal stability.

[0237] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0238] Abbreviations

[0239] CDR: complementarity determining region

[0240] FR: amino acid residues in the variable region of an antibody other than CDR residues

[0241] VH: antibody heavy chain variable region

[0242] VL: antibody light chain variable region

[0243] IgG: Immunoglobulin G

[0244] Kabat: Immunoglobulin alignment and numbering system proposed by Elvin A. Kabat

[0245] Chothia: immunoglobulin numbering system proposed by Chothia et al.

[0246] IMGT: numbering system based on the International Immunogenetics Information System initiated by Lefranc et al.

[0247] EC50: Half maximal effect concentration, which is the concentration that can cause 50% of the maximum effect

[0248] ELISA: Enzyme-linked immunosorbent assay

[0249] FACS: Flow Cytometry

[0250] PCR: polymerase chain reaction

[0251] HRP: Horseradish peroxidase

[0252] ADCC: Antibody-dependent cell-mediated cytotoxicity

[0253] CDC: Complement-dependent cytotoxicity

[0254] DMEM: Dulbecco's Modified Eagle Medium

[0255] RPMI1640: Roswell Park Memorial Institute 1640 Medium

[0256] FBS: Fetal bovine serum

[0257] Example 1 Construction and identification of Claudin18.2 overexpressing cell lines

[0258] The amino acid sequence of human Claudin18.2 refers to P56856-2 in the Uniprot protein database. The amino acid sequence of human Claudin18.1 refers to P56856-1 in the Uniprot protein database. The above amino acid sequences were codon-optimized by Anhui General Biotechnology Co., Ltd. and synthesized on the lentiviral vector pLVX.

[0259] The virus was prepared by the lentiviral packaging system, and the obtained virus was used to infect HEK293T cells (Nanjing Kebai, CBP60439) and CHO cells (ECACC). After puromycin selection, HEK293T-Claudin18.1 (human) and HEK293T-Claudin18.2 (human) stable cell lines were obtained. After successful construction, the positive rate of Claudin18.2 or 18.1 expression was determined by flow cytometry (FACS) for subsequent experiments.

[0260] The positive rate of flow cytometry test is as follows Figure 1 As shown, the positive rates of the above stable cell lines were all high (>95%).

[0261] Example 2 Alpaca immunization and serum titer detection

[0262] CHO-Claudin 18.2 stably transfected cells were cultured at 2.0×10 7 A healthy adult alpaca (Alpaca) was immunized with 100 cells. A total of four immunizations were performed, with an interval of 21 days between each immunization. Seven days after the fourth immunization, peripheral blood was collected and serum was separated. HEK293T-Claudin 18.2 and HEK293T-Claudin 18.1 stable cells and HEK293T cells (Nanjing Kebai, CBP60439) were used to detect the titer and specificity of antibodies against human Claudin18.2 in the serum using a flow cytometer (Sony, model: SA3800). The MFI value of the immunized serum against Claudin 18.2 cells was 10 times higher than that of the negative serum, and there was also a difference in binding to Claudin 18.1 cells.

[0263] Table 1 FACS detection of alpaca serum titer

[0264]

[0265] Example 3 Phage library construction and screening

[0266] 3.1 Phage library construction

[0267] After four immunizations, 50 mL of alpaca peripheral blood was collected and PBMCs were separated according to the instructions of lymphocyte separation medium (Tianjin Haoyang Huake Biotechnology Co., Ltd.). Total RNA was extracted using the Trizol method and PrimeScript TM II 1st StrandcDNA Synthesis Kit (Takara, Cat. No.: 6210A) was used to reverse transcribe the extracted total RNA and synthesize cDNA. Nested PCR was used to amplify the single domain antibody gene fragment: First round PCR:

[0268] Upstream primer: 5'-CTTGGTGGTCCTGGCTGC-3' (SEQ ID NO: 22)

[0269] Downstream primer: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO: 23)

[0270] Second round of PCR:

[0271] The second round of PCR uses the recovered product of the first round of PCR as a template.

[0272] Upstream primer:

[0273] 5'-CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC-3'(SEQ ID NO:24)

[0274] Downstream Primer-1:

[0275] 5'-CATGCCATGACTCGCGGCCGGCCTGGCCGCTGGGGTCTTCGCTGTGGTGCG-3'(SEQ ID NO:25)

[0276] Downstream Primer-2:

[0277] 5'-CATGCCATGACTCGCGGCCGGCCTGGCCTGGTTGTGGTTTTGGTGTCTTGGG-3'(SEQ ID NO:26)

[0278] 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. 100 μL of the product after electroporation was plated by gradient dilution and plated at 10 -4 There are 217 clones on the plate for the dilution gradient determination of the library capacity, so the library capacity is 2.17×10 9 At the same time, 48 clones were randomly selected from the library capacity test plate for bacterial liquid PCR identification, and the results showed that the library insertion rate was 100%.

[0279] 3.2 Screening of anti-human Claudin 18.2 single domain antibodies

[0280] 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 mixture was blocked with slight shaking for 1 hour at 4°C; after the cells were blocked, the supernatant was removed, and the phage library diluent was added, and the mixture was slightly shaken for 1 hour at 4°C; the mixture was centrifuged to remove unbound phages, and the cells were washed 6 times with 5% serum-PBS; 100μL Gly-HCl eluent was added, and the mixture was incubated at 37°C for 8 minutes to elute the 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 gradient dilution, the titer was determined, and the panning recovery rate was calculated. The remaining eluates were mixed and amplified and purified for the next round of affinity panning.

[0281] After three rounds of screening, 192 clones were randomly selected from the third round of titer determination plates for monoclonal phage supernatant ELISA identification. HEK293T-Claudin 18.2 and HEK293T-Claudin 18.1 cells were cultured until the entire plate was confluent; washed twice with PBS; 100 μL of 4% paraformaldehyde was added to fix the cells, and the cells were treated at 25°C for 20-30 min; washed twice with PBS, 300 μL of 5% skim milk was added to each well, and the cells were blocked at 37°C for 1 h; washed once with PBST, 50 μL of phage culture supernatant and 50 μL of 5% skim milk were added to each well, and the cells were incubated at 37°C for 1 h; washed five times with PBST, and horseradish peroxidase-labeled anti-M13 antibody (Chengdu Critical Point Biotechnology Co., Ltd., used at a dilution of 1:10000) was added, 100 μL / well, and the cells were treated at 37°C for 1 h; washed six times with PBST. TMB colorimetric solution was added for color development, 100 μL / well, 37°C, 7 min, and 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, and the sequencing was performed by the Chengdu Branch of Beijing Qingke Biotechnology Co., Ltd. The sequencing results were compared and sorted, and after removing similar sequences, 5 specific clones with different sequences were screened and obtained, and their sequence CDRs were analyzed using KABAT, Chothia and IMGT software respectively.

[0282] Example 4 Expression and purification of anti-human Clau18.2 camel-derived VHH-Fc

[0283] The anti-Clau18.2 VHH fused human constant region IgG1 CH2-CH3 (SEQ NO: 21) obtained by screening was constructed on the PTT5-IgG1 vector. After obtaining the recombinant plasmid, it was directly shaken and extracted, and HEK293E cells were transfected with PEImax. After expression for about 7 days, the supernatant was collected by centrifugation. The supernatant was purified using MabSelectSure affinity filler. All purified antibodies were ultrafiltered into PBS buffer, the concentration was determined, and stored at -20 degrees.

[0284] Example 5 Detection of anti-human Clau18.2 camel-derived VHH-Fc antibody

[0285] The binding of camel-derived VHH-Fc to cells expressing Claudin 18.2 and Claudin 18.1 was detected by flow cytometry (FACS). 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-cooled PBS; the cells were resuspended with 1% BSA (in PBS), and the cells were added to a 96-well pointed bottom plate, with 3×10 5cells, with a volume of 50 μl; dilute the antibody to be tested to 10 ug / mL with 1% BSA (in PBS) (blank cells: no antibody added; isotype control: anti-chicken lysozyme antibody from Critical Point Biotechnology Co., Ltd.), take 50 μl / well and add it to the cells, mix well, and incubate at 4°C for 1 hour; collect the cells by centrifugation, wash three times with pre-cooled PBS, add 50 μl Alexa Fluor 647-labeled secondary antibody (Jackson, 109-605-003), 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, and detect them by flow cytometer (Sony, model: SA3800), and the data are analyzed by FlowJo software. The results are shown in Figure 2. Figure 2-Figure 3 As shown in Table 2, BS002-41, BS002-52 and BS002-376 can effectively bind to human Claudin 18.2 cells, and have no cross-binding to human Claudin 18.1 cells. BS002-5 and BS002-82 can bind to human Claudin18.2, but have cross-binding to human Claudin 18.1 cells. Based on the binding affinity, BS002-376 is used as a candidate molecule for subsequent humanization.

[0286] Table 2 Anti-Clau18.2 camel-derived VHH-Fc binding to human Claudin 18.2 and human Claudin 18.1 cells

[0287]

[0288]

[0289] Example 6 Humanization of anti-human Claudin18.2 single domain antibody

[0290] Using the CDR transplantation method, first use the conventional BLAST method to find the human germline sequence with the highest homology to the original camel sequence BS002-376 as a template; transplant the CDR of the camel-derived single-domain antibody to the human template to construct a chimera; according to the structural analysis of the FR amino acids in the camel-derived single-domain antibody that can retain its original conformation, the corresponding amino acids in the chimera are backmutated to camel-derived amino acids to maintain the original affinity; the constructed humanized antibodies are calculated and immunogenicity analyzed to find the highly immunogenic fragments, and replace the low immunogenic fragments to form three humanized single-domain antibodies named LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3.

[0291] Example 7 Preparation of anti-human Claudin18.2 humanized single domain antibody

[0292] 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 Biology for gene synthesis, and after codon optimization, they were constructed on the PTT5-IgG1 vector, including human constant region IgG1 CH2-CH3 (SEQ NO:21). After the plasmid was synthesized, it was directly shaken and extracted, and HEK293E cells were transfected by PEImax, expressed for about 7 days, and the supernatant was collected by centrifugation. The supernatant was purified using MabSelectSure affinity filler. All purified antibodies were ultrafiltered into PBS buffer, the concentration was determined, and stored at -20 degrees. The reference antibody IMAB362 was prepared in the same way, and the sequence came from patent: CN 101312989 B.

[0293] Example 8 Purity Detection of Anti-Human Claudin18.2 Humanized Single Domain Antibody

[0294] The purity of the anti-human Claudin18.2 humanized single domain antibody was tested by SEC-HPLC as follows:

[0295] Instrument: Waters Alliance e2695 HPLC;

[0296] Chromatographic column: Thermo MabPac SEC-1, 5um, 7.8*300mm;

[0297] Mobile phase: 61mmol / L Na2HPO4, 39mmol / L NaH2PO4, 200mmol / L NaCl, 5% IPA;

[0298] 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.

[0299] The experimental results are shown in Table 3. LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 all have high purity (>97%).

[0300] Table 3 Purity of anti-human Claudin18.2 humanized single domain antibody

[0301]

[0302] Example 9 Tm value detection of anti-human Claudin18.2 humanized single domain antibody

[0303] DSF was used to determine the Tm value of humanized single-domain antibodies to reflect the thermal stability of antibodies. The experimental steps are as follows: dilute the antibody sample to be tested to 1 mg / mL with PBS; dilute the dye SYPRO Orange dye (Thermo #56651) to 40X with ddH2O; reaction system: sample 12.5uL + 40X dye 2.5uL + ddH2O 5uL; seal the film, centrifuge instantly; detect by Q-PCR instrument (Hangzhou Jingge, CG-05), Q-PCR parameter settings: Target (ROX), program (25°C, 3min; 1% rate, 95°C; 95°C, 2min).

[0304] The experimental results show that the Tm values ​​of LJD003-HZ1, LJD003-HZ2 and LJD003-HZ3 are 68.6℃, 67.1℃ and 69.1℃ respectively, indicating that all three have good thermal stability.

[0305] Example 10 Affinity Evaluation of Anti-Human Claudin18.2 Humanized Single Domain Antibody

[0306] The affinity of the humanized single domain antibody against human Claudin18.2 was detected by flow cytometry (FACS). The experimental steps are as follows: digest HEK293T-Claudin18.2 (human) cells, collect the cells by centrifugation, wash three times with pre-cooled PBS; resuspend the cells with 1% BSA (in PBS), add the cells to a 96-well pointed bottom plate, and add 3×10 5 cells, with a volume of 50 μl; dilute the antibody to be tested with 1% BSA (in PBS), starting at 100 ug / mL, 3-fold dilution, 10 concentration points, take 50 μl / well and add it to the cells, so that the final antibody concentration is 50 ug / 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 values ​​into Graph-Prism for graphing and calculation.

[0307] The experimental results are as follows Figure 4As shown, for HEK293T-Claudin18.2 (human) cells, LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 all have high affinity with them, and their EC50 values ​​are 96.30 ng / mL, 101.8 ng / mL, and 104.1 ng / mL, respectively, and the maximum fluorescence signal values ​​are 498997, 503325, and 491900, respectively; while the EC50 value of the reference antibody IMAB362 is 185.1 ng / mL, and the maximum fluorescence signal value is 393941. The above results show that the affinity of LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 to HEK293T-Claudin18.2 (human) cells is better than that of IMAB362.

[0308] Example 11 Specificity Evaluation of Anti-Human Claudin18.2 Humanized Single Domain Antibody

[0309] The specificity of the anti-human Claudin18.2 humanized single domain antibody was determined by flow cytometry (FACS). The experimental steps are as follows: HEK293T-Claudin18.1 (human) cells were collected by digestion and centrifugation, washed three times with pre-cooled PBS, resuspended with 1% BSA (in PBS), and 3×10 cells were added to each well. 5 The cells were plated into a 96-well pointed bottom plate, and the antibodies to be detected were added at a final concentration of 2ug / mL and 10ug / mL, respectively. The cells were incubated at 4°C for 1 hour, washed three times with pre-cooled PBS, and 1μl 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 then the cells were resuspended and detected by flow cytometer (Beckman, model: CytoFLEX). The detection values ​​were imported into Graph-Prism for graphical calculation.

[0310] The experimental results are as follows Figure 5 As shown, LJD003-HZ1, LJD003-HZ2, and LJD003-HZ3 do not non-specifically recognize human Claudin18.1.

[0311] Example 12 Evaluation of the endocytic activity of anti-human Claudin18.2 humanized single domain antibody

[0312] 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 at 4°C for 1 hour with the antibody to be detected at a concentration of 10ug / mL, washed 3 times with PBS, resuspended with DMEM+10% FBS, divided into 4 parts and incubated at 37°C for 0, 1, 2, and 4 hours, washed three times with PBS, added 1μl fluorescent secondary antibody, incubated at 4°C for 0.5h, and resuspended with PBS after washing three times. The endocytic rate was calculated according to the following formula: Endocytic rate (%) = [1-(average fluorescence value of the sample detected at this time point-average fluorescence value of the negative control sample at this time point) / (average fluorescence value of the sample detected at 0 hours-average fluorescence value of the negative control sample at 0 hours)]*100.

[0313] The experimental results are as follows Figure 6 As shown, LJD003-HZ3 was able to mediate the internalization of Claudin18.2, and the internalization rate was 20.47% at 4 hours.

[0314] The antibody sequences obtained by the present invention are shown in Table 4 below:

[0315] Table 4 Antibody sequences

[0316]

[0317]

[0318] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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) a single domain antibody targeting Claudin18.2 according to any one of claims 1 to 5, or an 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.

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