WO2025051070A1 - Anti-claudin 18.2 humanized single-domain antibody - Google Patents

Abstract

Provided is an anti-Claudin 18.2 humanized single-domain antibody. Specifically, provided are a single-domain antibody targeting Claudin 18.2, a humanized antibody and derived protein thereof, a gene sequence encoding same, an expression vector for producing same, and an expression system. The single-domain antibody has high specificity, binds Claudin 18.2 with high affinity and basically does not bind Claudin 18.1, and the single-domain antibody provides a research and development basis for development of antibody drugs, antibody-drug conjugates and multispecific antibody drugs.

Description

Anti-Claudin18.2 Humanized Single Domain Antibody Technical Field

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

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.

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.

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.

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

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

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 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.

In another preferred embodiment, 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.

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.

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.

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.

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

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

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

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

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.

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

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

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

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

In a second aspect, the present invention provides an antibody targeting Claudin18.2, wherein the antibody comprises one or more The VHH chain of the single-domain antibody targeting Claudin18.2 according to the first aspect of the invention.

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.

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

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

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

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

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

In another preferred embodiment, the antibody is a monoclonal antibody.

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

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).

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

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

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.

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%.

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

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

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

The third aspect of the present invention provides a multispecific antibody, which 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.

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

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.

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

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

(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

(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.

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.

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

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

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

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.

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

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

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

In another preferred embodiment, the fusion protein has the following elements from the N-C terminus:

A-B;

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);

“-” represents a peptide bond.

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.

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

(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

(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.

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

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

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

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

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

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

In another preferred embodiment, the drug is a cytotoxic drug.

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

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, related Clonogenic toxin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotonin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoid, or a combination thereof.

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

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

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

(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

(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.

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.

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.

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.

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

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

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

in:

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

LU is a linker (also called a connector);

D is for medicine;

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

In another preferred embodiment, LU is selected from maleimidocaproyl (MC), maleimide (MAL), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) (SMCC) linker connected to the antibody portion, and comprises valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine The invention can be used as a linker of one or more of amino-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), and polyethylene glycol (PEG).

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.

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

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

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

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

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

(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;

(ii) a pharmaceutically acceptable carrier.

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

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

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.

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

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

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

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.

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

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

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

(3) Detect tumor cells expressing CLAUDIN18.2 protein.

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.

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

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

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.

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

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

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

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.

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

(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

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

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

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.

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.

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.

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:

(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;

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

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

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

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

(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.

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.

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

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

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

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

Figure 2 shows the binding of camel-derived VHH-Fc to human Claudin 18.1 detected by flow cytometry (FACS).

Figure 3 shows the binding of camel-derived VHH-Fc to human Claudin 18.2 detected by flow cytometry (FACS).

FIG. 4 shows the cell affinity detection of anti-human Claudin18.2 humanized single domain antibody and HEK293T-Claudin18.2 (human) (flow cytometry).

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

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

FIG. 7 shows the binding detection of anti-human Claudin18.2 humanized single domain antibody to HEK293T-Claudin18.2 (monkey) cells (flow cytometry).

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

FIG. 9 shows the binding detection of anti-human Claudin18.2 humanized single domain antibody to HEK293T-Claudin18.2 (mouse) cells (flow cytometry).

FIG. 10 shows the results of antibody-dependent cell-mediated cytotoxicity (ADCC) assay of anti-human Claudin18.2 humanized single domain antibody.

FIG. 11 shows the determination of the endocytic activity (flow cytometry) of the anti-human Claudin18.2 humanized single domain antibody.

FIG. 12 shows the endocytic activity (killing method) assay of anti-human Claudin18.2 humanized single domain antibody.

DETAILED DESCRIPTION

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.

the term

As used herein, the terms "single domain antibody of the present invention", "anti-Claudin18.2 nanoantibody 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.

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.

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.

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.

As used herein, the term "variable" refers to certain portions of the variable region of an antibody that differ in sequence, which accounts for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments of the variable region of both the light and heavy chains called the complementarity determining regions (CDRs) or hypervariable regions. 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 generally 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, pp. 647-669 (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-dependent cytotoxicity of the antibody.

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.

As used herein, the term "heavy chain variable region" is used interchangeably with " _VH ."

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

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

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

In 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.

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.

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 hypervariable regions (CDRs), which are divided into four framework regions (FRs). 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.

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.

Claudin18.2 and single domain antibodies against Claudin18.2

As used herein, the term "Claudin18.2" refers to the Claudin18.2 protein in the Claudins protein family. The function 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 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.

As used herein, "Chothia", "Kabat", "IMGT", "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 antibodies and the topology of 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 the variability of antibody sequences (Kabat et al., Sequence s of Proteins of Immunological Interest, 4th ed., U.S. 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 definitions based on loop structure position.

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.

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.

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%.

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.

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.

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

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.

Recombinant protein (or fusion protein)

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, GP Ilb/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.

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

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.

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 in which one or more amino acid residues have a non-conservative amino acid residue. (iii) a polypeptide having a substitution group, or (iv) a polypeptide formed by fusing a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (v) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secretory sequence or a sequence or a 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 belong to the scope known to those skilled in the art.

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.

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.

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.

Table A

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.

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.

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

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 lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the 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.

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.

Antibody preparation

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.

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.

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.

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.

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.

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

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 procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, and other conventional separation and purification methods well known to those skilled in the art to obtain the antibodies of the present invention.

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).

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.

Immunoconjugates

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

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.

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).

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.

Single domain antibodies can be conjugated to drugs to form antibody drug conjugates (NDCs). Typically, NDCs contain The 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 linkers containing peptidyl that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as linkers containing glucuronides that can be degraded by glucuronidase. 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 bond linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

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.

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.

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.

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.

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.

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.

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

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.

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

in:

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;

D is for medicine;

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

application

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.

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, and more preferably gastric cancer.

Pharmaceutical composition

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.

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.

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.

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.

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.

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.

The main advantages of the present invention include:

(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.

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

(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.

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

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 used to limit the scope of the present invention. The experimental methods in the following examples where detailed conditions are not specified are generally carried out according to 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.

Abbreviations

CDR: complementarity determining region

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

VH: antibody heavy chain variable region

VL: antibody light chain variable region

IgG: Immunoglobulin G

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

Chothia: Immunoglobulin numbering system proposed by Chothia et al.

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

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

ELISA: Enzyme-linked immunosorbent assay

FACS: Flow Cytometry

PCR: polymerase chain reaction

HRP: Horseradish peroxidase

ADCC: Antibody-dependent cell-mediated cytotoxicity

CDC: Complement-dependent cytotoxicity

DMEM: Dulbecco's Modified Eagle Medium

RPMI1640: Roswell Park Memorial Institute 1640 medium

FBS: Fetal bovine serum

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

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.

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.

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

Example 2 Alpaca immunization and serum titer detection

CHO-Claudin 18.2 stable cells were used to immunize a healthy adult alpaca (Alpaca) at a volume of 2.0×10 ⁷ 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 Claudin 18.2 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.

Table 1 FACS detection of alpaca serum titer

Example 3 Phage library construction and screening

3.1 Phage library construction

50 mL of alpaca peripheral blood was collected after four immunizations, and PBMCs were separated according to the instructions of lymphocyte separation solution (Tianjin Haoyang Huake Biotechnology Co., Ltd.). Total RNA was extracted using the Trizol method, and the extracted total RNA was reverse transcribed using PrimeScript ^TM II 1st Strand cDNA Synthesis Kit (Takara, Cat. No.: 6210A) to synthesize cDNA. Single-domain antibody gene fragments were amplified using nested PCR: First round PCR:

Upstream primer: 5’-CTTGGTGGTCCTGGCTGC-3’ (SEQ ID NO: 22)

Downstream primer: 5’-GGTACGTGCTGTTGAACTGTTCC-3’ (SEQ ID NO: 23)

Second round of PCR:

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

Upstream primer:

Downstream Primer-1:

Downstream Primer-2:

The VHH nucleic acid fragment product of the second round of PCR amplification was recovered, digested with SfiI (NEB, catalog number: R0123L), digested 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. The next day, there were 217 clones on the plate with a ^10-4 dilution gradient to determine the number of library capacity, so the library capacity was 2.17× ¹⁰⁹ . At the same time, 48 clones were randomly selected from the library capacity measurement plate for bacterial liquid PCR identification, and the results showed that the library insertion rate was 100%.

3.2 Screening of anti-human Claudin 18.2 single domain antibodies

Take 5×10 ⁶ HEK293T-Claudin 18.2 cells, centrifuge at 500g for 5min, resuspend and wash the cell pellet twice with 5% serum-PBS; add 500μL 3% OVA-PBS to the centrifuge tube containing the cells, and block with slight shaking at 4℃ for 1h; centrifuge after cell blocking, remove the supernatant, add phage library diluent, and shake gently at 4℃ for 1h; centrifuge to remove unbound phages, and wash with 5% serum-PBS 6 times; add 100μL Gly-HCl eluent, incubate at 37℃ for 8min, and elute the specifically bound phages; transfer the eluate to a 1.5mL sterile centrifuge tube and quickly neutralize with 10μL Tris-HCl neutralization buffer; take 10μL for gradient dilution, determine the titer, calculate the panning recovery rate, and mix the remaining eluates for amplification and purification for the next round of affinity panning.

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 covered; washed twice with PBS; 100 μL 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 5% skim milk was added to each well, and blocked at 37°C for 1 h; washed once with PBST, 50 μL phage culture supernatant and 50 μL 5% skim milk were added to each well, and 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 1:10,000) was added, 100 μL/well, and 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.

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

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 extracted by shaking, transfected into HEK293E cells 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.

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

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 are as follows: digest HEK293T-Claudin18.2 and HEK293T-Claudin18.1 cells, collect cells by centrifugation, and wash three times with pre-cooled PBS; resuspend cells with 1% BSA (in PBS), add cells to 96-well pointed bottom plates, add 3×10 ⁵ cells to each well, and the volume is 50 μl; dilute the antibody to be tested to 10ug/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 to the cells, mix well, and incubate at 4°C for 1 hour; collect 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 cells by centrifugation, wash three times with pre-cooled PBS, and use 200 μl The cells were resuspended in PBS and detected by flow cytometry (Sony, model: SA3800), and the data were analyzed by FlowJo software. The results are shown in Figures 2-3 and Table 2. BS002-41, BS002-52 and BS002-376 can effectively bind to human Claudin 18.2 cells, and have no cross-binding with human Claudin 18.1 cells. BS002-5 and BS002-82 can bind to human Claudin 18.2, but have cross-binding with human Claudin 18.1 cells. Based on the binding affinity, BS002-376 is used as a candidate molecule for subsequent humanization.

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

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

Using the CDR transplantation method, we first used the conventional BLAST method to find the original camel sequence BS002-376 The human germline sequence with the highest homology was used as a template; the CDR of the camel-derived single-domain antibody was transplanted onto the human template to construct a chimera; based on 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 were backmutated to camel-derived amino acids to maintain the original affinity; the constructed humanized antibodies were calculated and immunogenicity analyzed to find the 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.

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

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 extracted by shaking, transfected into HEK293E cells 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.

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

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

Instrument: Waters Alliance e2695 HPLC;

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

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

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.

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

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

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

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 membrane, centrifuge instantly; The results were detected by Q-PCR instrument (Hangzhou Jingge, CG-05), and the Q-PCR parameter settings were as follows: Target (ROX), program (25°C, 3 min; 1% rate, 95°C; 95°C, 2 min).

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.

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

The affinity of anti-human Claudin18.2 humanized single domain antibody was detected by flow cytometry (FACS). The experimental steps are as follows: digest HEK293T-Claudin18.2 (human) and NUGC4-Claudin18.2 (human) cells, collect the cells by centrifugation, and 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, add 3×10 ⁵ cells to each well, and the volume is 50 μl; dilute the antibody to be tested with 1% BSA (in PBS), starting at 40ug/mL, 3-fold dilution, 10 concentration points, take 50 μl/well and add it to the cells, so that the final antibody concentration is 20ug/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 50μl 1% BSA (in PBS), add 1μl 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, and use 200μl 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.

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, and their EC50 values are 208.3 ng/mL, 355.7 ng/mL, and 308.2 ng/mL, respectively, and the maximum fluorescence signal values are 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.

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, and their EC50 values are 620.5 ng/mL, 576.1 ng/mL, and 528.9 ng/mL, respectively, and the maximum fluorescence signal values are 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.

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.

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

The specificity of the humanized single-domain antibody against human Claudin18.2 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 precooled PBS, resuspended with 1% BSA (in PBS), 3×10 ⁵ cells per well were plated in 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, incubated at 4°C for 1 hour, washed three times with precooled PBS, 1μl of fluorescent secondary antibody was added to each well, incubated at 4°C for 0.5 hours, washed three times with precooled PBS, resuspended, and detected by flow cytometer (Beckman, model: CytoFLEX), and the detection values were imported into Graph-Prism for graphing and calculation.

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

Example 12 Species Cross-Evaluation of Anti-Human Claudin18.2 Humanized Single Domain Antibody

The amino acid sequence of cynomolgus monkey Claudin18.2 refers to XP_015300615.1 in the protein database of NCBI. The amino acid sequence of mouse Claudin18.2 refers to NP_001181850.1 in the protein database of NCBI. The amino acid sequence of rat Claudin18.2 refers to NP_001014118.1 in the protein database of NCBI. The above amino acid sequences were codon-optimized by GenScript, synthesized on the lentiviral vector pLVX, and the virus was prepared by the lentiviral packaging system. After the virus was obtained, HEK293T was infected respectively, and the stable cell lines of HEK293T-Claudin18.2 (monkey), HEK293T-Claudin18.2 (rat), and HEK293T-Claudin18.2 (mouse) were obtained by puromycin selection.

The affinity of 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: digest HEK293T-Claudin18.2 (monkey), HEK293T-Claudin18.2 (rat), and HEK293T-Claudin18.2 (mouse) cells, collect the cells by centrifugation, and 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, add 3×10^5 cells to each well, and the volume is 50μL; dilute the antibody to be tested with 1% BSA (in PBS), starting at 40μg/mL, 3-fold dilution, 10 concentrations. At 4 o'clock, take 50 μL/well and add it to the cells, so that the final antibody concentration is 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 value into Graph-Prism for graphing and calculation.

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 it, and their EC50 values are 62.33 ng/mL, 103.9 ng/mL, and 85.11 ng/mL, respectively, and the maximum fluorescence signal values are 1325843, 1651947, and 1552783, respectively.

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 it, and their EC50 values are 167.1 ng/mL, 180.7 ng/mL, and 175.9 ng/mL, respectively, and the maximum fluorescence signal values are 2726433, 2768686, and 2661951, respectively.

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 it, and their EC50 values are 113.8 ng/mL, 131.7 ng/mL, and 115.9 ng/mL, respectively, and the maximum fluorescence signal values are 2415543, 2466115, and 2177835, respectively.

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

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

The ADCC activity of humanized single domain antibody against human Claudin18.2 was detected by fluorescein reporter system. The experimental steps are as follows: HEK293T-Claudin18.2 cells were collected by digestion and centrifugation, and the cells were resuspended with DEME + 2% FBS and 100,000 cells were plated in 50 μL per well; Jurkat-NFAT-CD16a cells were collected by centrifugation, and the cells were resuspended with 1640 + 2% FBS and 50000 cells were plated in each well. 100,000 cells were plated with 100 μL of the antibody to be tested; the antibody to be tested was diluted with DEME + 2% FBS, starting at 100 μg/mL, 5-fold dilution, 11 concentration points, 100 μL/well was added to the cells, so that the final antibody concentration was 50 μg/mL, and the antibody to be tested was added with gradient dilutions to mix, 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 reading was carried out using an enzyme reader (MD, i3x).

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.

Example 14 Determination of the Endocytic Activity (Killing Method) of Anti-Human Claudin18.2 Humanized Single Domain Antibody

The endocytic activity of humanized single-domain antibody against human Claudin18.2 was detected by cell killing method. The experimental steps are as follows: HEK293T-Claudin18.2 cells were collected by trypsin digestion and centrifugation, and the cells were resuspended in DMEM + 2% FBS and counted. The volume of each well was 50 μL, and a total of 5000 cells were spread in each well of 96-well plates for standby use; the antibody to be tested was diluted with DMEM + 2% FBS, with a starting concentration of 40 μg/mL, 4-fold gradient dilution, a total of 11 concentration points, and 100 μL was added to the cell well to make the final concentration of 20 μg/mL; Anti-human IgG-MMAE (Apark Bio) was diluted with DMEM + 2% FBS to a concentration of 8 μg/mL, and 50 μL was added to the cell well to make a final concentration of 2 μg/mL, and control wells were set: naked anti-control (antibody + cells), MMAE control (Anti-human IgG-MMAE + cells), blank control (only cells). 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. OD450nm was detected by an enzyme reader (MD, model: Spectramax ABS Plus) and the detection values were imported into Graph-Prism for graphical calculation.

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

Example 15 Determination of Endocytosis Activity of Anti-Human Claudin18.2 Humanized Single Domain Antibody (Flow Cytometry)

The endocytic activity of 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 detected at a concentration of 10ug/mL at 4°C for 1 hour, 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 with 1μl fluorescent secondary antibody, incubated at 4°C for 0.5h, washed three times with PBS, resuspended and put on the machine. 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.

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

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

Table 4 Antibody sequences

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 (13)

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. The single domain antibody according to claim 1, wherein the CDR1, CDR2 and CDR3 are selected from the following group: (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. The single domain antibody as described in claim 1 is 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% with the amino acid sequence. The single domain antibody according to claim 1, wherein the single domain antibody is humanized. The single domain antibody as described in claim 4 is 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 with a homology of ≥85% to the amino acid sequence. 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. The antibody according to claim 6, characterized in that the antibody is a heavy chain antibody, and the heavy chain antibody comprises heavy chain constant regions CH2 and CH3 (Fc segment). 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. 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. 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. 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. The immunoconjugate according to claim 11, characterized in that the conjugated moiety is a radionuclide. 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.