Preparation method for and use of CLDN18.2 single-domain antibody probe

Abstract

The present invention relates to the technical field of molecular imaging, nuclear medicine and single-domain antibodies for tumor diagnosis and treatment, in particular to a method for preparing a CLDN18.2 single-domain antibody probe and the use of the probe. The prepared CLDN18.2-specific 18F labeled monovalent nanobody probe has the advantages of a simple preparation process, a low cost, high specificity, high stability, a short imaging period, a low radiation dose, easy clinical implementation, etc. Moreover, by means of performing immune PET imaging on the basis of the prepared probe, non-invasive visualization of the expression of CLDN18.2 in tumor tissue and normal tissue and organs can be realized, which can further be used for non-invasive target-specific diagnosis of specific types of tumors.

Description

Preparation method and application of CLDN18.2 single-domain antibody probe Technical Field

[0001] The present invention relates to the fields of molecular imaging, nuclear medicine and single-domain antibody technology for tumor diagnosis and treatment, and in particular to a preparation method and application of a CLDN18.2 single-domain antibody probe. Background Art

[0002] Gastric cancer is one of the most common malignant tumors in my country, with the second highest incidence among malignant tumors and the highest mortality rate after liver cancer and lung cancer. Surgery is the most common treatment method, but most patients are already in the advanced stage when diagnosed, and the overall prognosis is poor. The first-line treatment for advanced gastric cancer is chemotherapy, but as tumor resistance increases, patients cannot achieve good long-term survival. In tumor diagnosis and treatment strategies, molecular targeted therapies such as small molecule inhibitors and monoclonal antibodies, as well as immunotherapy (such as immune checkpoint inhibitors), are changing the treatment status of a variety of solid tumors and hematological tumors. Finding suitable targets for the diagnosis and treatment of gastric cancer is of great significance for the benefit of gastric cancer patients.

[0003] Claudin 18 (CLDN18) is a transmembrane protein located in the tight junctions between epithelial and endothelial cells. It is a crucial component and functional structure of intercellular tight junctions. The first exon of the human CLDN18 gene has two alleles, resulting in two distinct splice variants: CLDN18.1 and CLDN18.2. CLDN18 is highly conserved in normal tissues. CLDN18.2 is primarily distributed in the gastric mucosa (normal gastric glands, chief cells, parietal cells, and endocrine cells), which have a short differentiation cycle and rapid turnover rate, as well as in Paneth cells of the duodenum. It is currently believed that changes in cell polarity during malignant transformation in tumors lead to the widespread distribution of CLDN18.2 on the cell membrane surface. The prevalence and proportion of CLDN18.2-positive gastric cancers in gastric cancer vary significantly across studies. Some current studies limit the CLDN18.2 expression rate in gastric cancer to 42% to 86%, with CLDN18.2-positive gastric cancers accounting for approximately 16% to 73% of gastric cancer patients. CLDN18.2 expression exhibits distinct molecular pathological characteristics: higher in diffuse-type gastric cancers than intestinal-type cancers and higher in Epstein-Barr virus (EBV)-positive cancers than in Epstein-Barr virus (EBV)-negative cancers (81.0% vs. 40.2%, P < 0.001). CLDN18.2 expression is generally consistent across primary lesions, surrounding lymph node metastases, and distant metastatic lesions. Due to its aberrant activation and high expression in digestive system malignancies, CLDN18.2 is considered a promising therapeutic target.

[0004] Studies have shown that CLDN18.2 is a promising target for solid tumor treatment. Zolbetuximab (IMAB362, claudixmab) is a chimeric IgG1 monoclonal antibody that specifically binds to CLDN18.2 on the tumor cell surface, triggering antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, and inhibition of cell proliferation. Its clinical efficacy and safety are currently being evaluated in multiple Phase I / II trials. On November 17, 2022, Astellas announced that the Phase III SPOTLIGHT trial, a combination of the claudin18.2 antibody zolbetuximab and chemotherapy for the treatment of claudin18.2-positive, HER2-negative recurrent metastatic gastric cancer, achieved its primary endpoint. The results showed that patients treated with zolbetuximab plus mFOLFOX6 had statistically significant improvements in progression-free survival (PFS) and overall survival (OS) compared to placebo plus mFOLFOX6. Humanized anti-CLDN18.2 autologous CAR-T therapy has also demonstrated anti-tumor activity and safety, and multiple ongoing clinical trials are expected to provide more options for patients with CLDN18.2-positive tumors. CLDN18.2 is being investigated in a variety of cancer types, including targeted drugs, small molecule inhibitors, CAR-T, immune cell therapy, antibody-drug conjugates, and tumor vaccines. Therapeutics are targeting gastric cancer, gastroesophageal junction cancer, and pancreatic cancer. Personalized immunotherapy targeting CLDN18.2 may become the next research hotspot for digestive system malignancies.

[0005] Antibodies have received special attention in biomedical research due to their well-defined structure, relative stability, high specificity, and high affinity. PET imaging performed using probes constructed based on antibodies is called immuno-PET imaging, a branch of molecular imaging. Using probes targeting tumor cells and tumor microenvironment receptors, it can effectively reflect the occurrence and development of tumors in real time. Immuno-PET probes constructed based on monoclonal antibodies have the characteristics of high stability and strong specificity, and are relatively widely used in clinical practice. However, monoclonal antibodies (approximately 150kD) have a large molecular weight, low tissue penetration, poor imaging targets, and are prone to non-specific uptake by non-target organs. Monoclonal antibodies have a complex spatial structure, high expression and preparation costs, and are highly immunogenic, but modified antibodies are difficult to achieve the original affinity. Many factors limit their application and popularization in clinical practice.

[0006] Nanobodies are derived from the smallest functional antigen-binding fragment of heavy-chain antibodies in adult camels, with a molecular weight of only 15 kDa. Nanobodies are highly stable and bind antigens with high affinity, possessing many unique properties compared to conventional antibodies: 1) The sequences encoded by nanobodies share high homology with human VH families 3 and 4, resulting in weak immunogenicity; 2) their small molecular weight and simple structure allow for large-scale expression in microbial systems and ease of purification; 3) nanobodies can recognize a wide range of antigenic epitopes, including those hidden within molecular crevices; 4) their small molecular weight allows them to easily penetrate tissues, reaching sites inaccessible to conventional antibodies; and 5) they can be paired with short-half-life radionuclides, enabling same-day imaging. These properties make nanobodies promising diagnostic and therapeutic tools: as imaging tracers, nanobodies enable early acquisition of high-quality images; and as therapeutic agents, nanobodies can be conjugated to cytotoxic drugs for precise, targeted delivery. In recent years, we have focused on the development and clinical translation of nanobody-derived tracers to leverage their superior molecular imaging properties. Based on the above evidence and our previous findings, we hypothesize that an immunoPET imaging probe targeting CLDN18.2 can noninvasively display CLDN18.2 expression in gastric cancer tumor cells, serve the diagnosis and treatment system of gastric cancer, and promote the development of personalized treatment in precision medicine.

[0007] Currently, the field of CLDN18.2 molecular imaging probes is still in the development stage. Technicians in this field are committed to developing a nano-antibody immune PET imaging probe with low preparation cost, small molecular weight, short in vivo circulation time, short imaging cycle, low radiation dose, and easy clinical translation and application.

[0008] Summary of the Invention

[0009] To fill this gap in the field, we describe the construction of a nanobody-derived CLDN18.2-targeted diagnostic probe and characterize its biodistribution in normal Balb / c mice, with the goal of exploring its diagnostic and potential therapeutic value for gastric cancer. Specifically, the present invention provides a method for preparing and using a CLDN18.2 nanobody probe.

[0010] CLDN18.2-specific nanoantibodies

[0011] In one aspect, the present invention provides a CLDN18.2-specific Nanobody comprising:

[0012] (1) a CDR1 having the amino acid sequence shown in SEQ ID No. 1, a CDR2 having the amino acid sequence shown in SEQ ID No. 2, and a CDR3 having the amino acid sequence shown in SEQ ID No. 3,

[0013] (2) a CDR1 having the amino acid sequence shown in SEQ ID No. 6, a CDR2 having the amino acid sequence shown in SEQ ID No. 7, and a CDR3 having the amino acid sequence shown in SEQ ID No. 8,

[0014] (3) CDR1 having the amino acid sequence shown in SEQ ID No. 11, CDR2 having the amino acid sequence shown in SEQ ID No. 12, and CDR3 having the amino acid sequence shown in SEQ ID No. 13.

[0015] More specifically, the CLDN18.2-specific Nanobody of the present invention has the amino acid sequence shown in SEQ ID No. 4, 9 or 14.

[0016] In the present invention, for the sake of simplicity, the CLDN18.2-specific Nanobodies having the amino acid sequence shown in SEQ ID No. 4, 9 or 14 are referred to as 3E10, 3E11 or 3A12, respectively.

[0017] As used herein, the term "nanobody" is also referred to as "single-domain antibody (sdAb)" or VHH (Variable Domain of Heavy Chain of Heavy Chain Antibody), which are used interchangeably. Nanobodies have the meaning generally understood by those skilled in the art and refer to antibody fragments consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, nanobodies consist of four framework regions and three complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus so that they contain only part of FR1 and / or FR4, or lack one or two of those framework regions, as long as they substantially maintain antigen binding and specificity.

[0018] In some embodiments, the invention also encompasses antigen-binding fragments of the CLDN18.2-specific Nanobodies as described herein.

[0019] As used herein, the term "antigen-binding fragment" refers to a polypeptide comprising a fragment of a Nanobody that retains the ability to specifically bind to the same antigen to which the Nanobody binds, and / or competes with the Nanobody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the antibodies of the invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the Nanobodies of the invention. In some embodiments, the "antigen-binding fragment" of such a Nanobody may be truncated at the N-terminus or C-terminus compared to the full-length Nanobody so that it only comprises part of FR1 and / or FR4, or lacks one or both of those framework regions, as long as it substantially retains antigen binding and specificity.

[0020] Antigen-binding fragments of Nanobodies can be obtained from a given Nanobody (such as the Nanobodies provided herein) using conventional techniques known to those skilled in the art (for example, recombinant DNA techniques or enzymatic or chemical cleavage methods), and screened for specificity in the same manner as for intact Nanobodies.

[0021] In this document, unless the context clearly indicates otherwise, when referring to the term "Nanobody" it includes not only whole Nanobodies but also antigen-binding fragments of Nanobodies.

[0022] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. In nanobodies, there are three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0023] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0024] As used herein, the term "CLDN18.2-specific" refers to specific binding to CLDN18.2.

[0025] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0026] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate at which the antigen binding site / antigen complex forms and dissociates. The "association rate constant" (ka or k on ) and the “dissociation rate constant” (k dis or k off ) Both can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). dis / k on The ratio is equal to the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D 、k on and k dis In certain embodiments, the dissociation constant can be measured using surface plasmon resonance (SPR) in Biacore. Alternatively, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0027] In some embodiments, the present invention also provides variants of the CLDN18.2-specific Nanobodies as described herein, which have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID No. 4, 9 or 14, and substantially retain the biological function of the Nanobody from which it is derived (e.g., biological activity that specifically binds to CLDN18.2).

[0028] More specifically, the variants differ from the CLDN18.2-specific Nanobodies as described herein only by conservative substitutions of one or more (for example, conservative substitutions of up to 20, up to 15, up to 10, up to 5 or up to 1 amino acid residues).

[0029] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0030] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0031] polynucleotides

[0032] In another aspect, the present invention also provides a polynucleotide encoding the above-mentioned Nanobody or antigen-binding fragment thereof.

[0033] More specifically, the polynucleotide has the nucleotide sequence shown in SEQ ID No. 5, 10 or 15.

[0034] More specifically, the polynucleotide encoding the CLDN18.2-specific Nanobody as described herein has the nucleotide sequence shown in SEQ ID No. 5, 10 or 15.

[0035] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0036] The term "polynucleotide encoding a polypeptide / protein / antibody" may include a polynucleotide encoding the polypeptide / protein / antibody, or may also include additional coding and / or non-coding sequences.

[0037] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% identity between the two sequences, and the polypeptides / proteins / antibodies encoded by them have substantially the same functions and activities. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%.

[0038] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0039] carrier

[0040] In another aspect, the present invention also provides a vector comprising a polynucleotide encoding the above-mentioned CLDN18.2 antibody or an antigen-binding fragment thereof.

[0041] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain an origin of replication.

[0042] host cells

[0043] In another aspect, the present invention also provides a host cell comprising the vector as described herein.

[0044] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or other human cells. A host cell may include a single cell or a cell population.

[0045] Vectors can be introduced into host cells using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0046] The Nanobodies of the invention can be used alone or in combination or coupled to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety or a combination of any of these.

[0047] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product. Preferred detectable labels are radionuclides.

[0048] Therapeutic agents that can be combined or coupled with the antibodies of the present invention include but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0049] The binding or coupling of a detectable label or therapeutic agent to an antibody can be performed by conventional methods well known to those skilled in the art. For example, a detectable label can be directly or indirectly bound to a Nanobody, for example via a cleavable or non-cleavable linker peptide, or incorporated into a Nanobody. The detectable label can be bound to a Nanobody, in particular by replacement (for example by replacing H with I at the level of a tyrosine residue), by complexation or by chelation. For example, a therapeutic agent can be conjugated to a Nanobody via a cleavable linker (for example a peptidyl, disulfide or hydrazone linker).

[0050] In a preferred embodiment, as described in more detail below, the Nanobodies of the invention are conjugated to a radionuclide for use as CLDN18.2-specific molecular imaging probes.

[0051] CLDN18.2-specific molecular imaging probe

[0052] In another aspect, the present invention provides a CLDN18.2-specific 18 F-labeled monovalent Nanobody probe comprising a radionuclide-labeled CLDN18.2-specific Nanobody as described herein.

[0053] More specifically, the CLDN18.2-specific Nanobodies as described herein are radionuclide-labeled via a bifunctional chelator.

[0054] As used herein, a bifunctional chelator is a chelator having both a metal chelating end and a protein anchoring end. The bifunctional chelator can be selected from at least one of (±)-H3RESCA-TFP and (±)H3RESCA-Mal.

[0055] As used herein, the (±)-H3RESCA-TFP is a tetrafluorophenyl ester derivative of a constrained complexing agent (RESCA) that can be used to couple the chelator to biomolecules via amine coupling (e.g., the N-terminus and / or the ε-amino group of lysine);

[0056] (±)H3RESCA-Mal is (±)H3RESCA-maleimide, a tetrafluorophenyl ester derivative of a constrained complexing agent (RESCA), which can be used to couple the chelator to biomolecules via amine coupling (e.g., N-terminus and / or ε-amino group of lysine).

[0057] More specifically, the CLDN18.2-specific Nanobodies as described herein were radionuclide-labeled via (±)-H3RESCA-TFP or (±)H3RESCA-Mal.

[0058] More particularly, the radionuclide is selected from F-18.

[0059] More specifically, as described herein [ 18 F]F-H3RESCA-3E10, [ 18 F]F-H3RESCA-3E11 or [ 18 F]F-H3RESCA-3A12.

[0060] More specifically, the CLDN18.2 specific 18 F-labeled monovalent nanoantibody probe is [ 18 F]F-H3RESCA-3A12, a CLDN18.2-specific Nanobody having a CDR1 with the amino acid sequence shown in SEQ ID No. 11, a CDR2 with the amino acid sequence shown in SEQ ID No. 12, and a CDR3 with the amino acid sequence shown in SEQ ID No. 13, or having an amino acid sequence shown in SEQ ID No. 14, was labeled with F-18 via (±)-H3RESCA-TFP or (±)H3RESCA-Mal. Its imaging effect in normal gastric tissue is better.

[0061] In another aspect, the present invention also provides a method for preparing a CLDN18.2-specific 18 The method for labeling a monovalent nanobody probe with F comprises modifying a CLDN18.2-specific nanobody with a bifunctional chelator to obtain a radionuclide-labeled precursor; and labeling the radionuclide-labeled precursor with a radionuclide to obtain a CLDN18.2-specific nanobody. 18 F-labeled monovalent nanobody probe.

[0062] More specifically, when the bifunctional chelator is (±)H3RESCA-Mal, before preparing the radionuclide-labeled precursor, a cysteine-containing peptide segment (GGGGS)nC, n=1-10, needs to be expressed at the end of the CLDN18.2-specific nanobody; specifically, n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0063] Composition

[0064] In another aspect, the present invention provides a composition comprising a CLDN18.2-specific Nanobody, polynucleotide, vector, host cell, or molecular imaging probe as described herein. The composition can be used to detect CLDN18.2 expression levels, diagnose CLDN18.2-associated tumors, predict the therapeutic efficacy of CLDN18.2-associated tumors, or treat CLDN18.2-associated tumors.

[0065] In some embodiments, the composition may be a pharmaceutical composition.

[0066] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient.

[0067] In some embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0068] In some embodiments, the additional pharmaceutically active agent is an anti-inflammatory drug or an immunosuppressant.

[0069] In some embodiments, in the pharmaceutical composition, the CLDN18.2-specific Nanobodies, polynucleotides, vectors, host cells or molecular imaging probes as described herein and the additional pharmaceutically active agent can be provided as separate components or as mixed components. Thus, the CLDN18.2-specific Nanobodies, polynucleotides, vectors, host cells or molecular imaging probes as described herein and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0070] In some embodiments, the pharmaceutically acceptable carrier and / or excipient may comprise a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0071] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of a CLDN18.2-specific Nanobody, polynucleotide, vector, host cell, or molecular imaging probe as described herein. A "prophylactically effective amount" refers to an amount sufficient to prevent, inhibit, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered therapies.

[0072] Reagent test kit

[0073] The present invention also provides a kit comprising the CLDN18.2-specific nanobody, polynucleotide, vector, host cell or molecular imaging probe as described herein.

[0074] The kit can be used to detect the expression level of CLDN18.2, diagnose CLDN18.2-related tumors, predict the treatment effect of CLDN18.2-related tumors, or treat CLDN18.2-related tumors.

[0075] The kit may also include a container, instructions for use, and other reagents and buffers required for practical applications, such as a lysis medium for dissolving the sample, various buffers, detection labels, detection substrates, etc.

[0076] Diagnostic and therapeutic applications

[0077] The CLDN18.2-specific nanoantibodies of the present invention have extremely high affinity for CLDN18.2 and can therefore be used to detect the expression level of CLDN18.2, diagnose CLDN18.2-related tumors, predict the therapeutic effect of CLDN18.2-related tumors, or treat CLDN18.2-related tumors.

[0078] In particular, the CLDN18.2-specific molecular imaging probes prepared from the CLDN18.2-specific Nanobodies of the present invention exhibit significantly enhanced affinity, significantly reduced nonspecific uptake in normal tissues, and significantly improved image quality. They can be used for noninvasive, precise, and efficient detection of human CLDN18.2 expression, making them particularly suitable for diagnosing CLDN18.2-associated tumors and predicting treatment efficacy for CLDN18.2-associated tumors. After conjugation with an appropriate radionuclide, they can also be used for precision treatment of CLDN18.2-associated tumors.

[0079] Therefore, in another aspect, the present invention also relates to the use of CLDN18.2-specific nanobodies, polynucleotides, vectors, host cells or molecular imaging probes as described herein in the preparation of kits or drugs for detecting the expression level of CLDN18.2, diagnosing CLDN18.2-related tumors, predicting the treatment effect of CLDN18.2-related tumors or treating CLDN18.2-related tumors.

[0080] As used herein, CLDN18.2-associated tumors may include various tumors or cancers well known in the art. For example, CLDN18.2-associated tumors may include digestive tract tumors, such as gastric cancer, pancreatic cancer, esophageal cancer, bile duct cancer, gallbladder cancer, etc.; and may also include breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer.

[0081] Beneficial effects of the present invention

[0082] 1) CLDN18.2-specific molecular probe prepared by the present invention [ 18 F]F-H3RESCA-3E10, [ 18 F]F-H3RESCA-3E11 and [ 18 F]F-H3RESCA-3A12 is a positron-emitting radionuclide probe used for immuno-PET imaging. By conducting immuno-PET imaging based on the above-mentioned probe, the expression of CLDN18.2 in tumor tissues and normal tissues and organs can be non-invasively visualized, and further used for non-invasive target-specific diagnosis of specific types of tumors.

[0083] 2) The probe prepared by the present invention also has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging cycle, low radiation dose, and easy clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 shows the results of SDS-PAGE determination of the expression of Nanobodies 3E10, 3E11, and 3A12;

[0085] Figure 2 shows the histochemical staining results of CLDN18.2;

[0086] Figure 3 shows [ 18 F]F-H3RESCA-3E10 radiochemical purity determination results;

[0087] Figure 4 shows [ 18 F]F-H3RESCA-3E11 radiochemical purity determination results;

[0088] Figure 5 shows [ 18 F]F-H3RESCA-3A12 radiochemical purity determination results;

[0089] Figure 6 shows [ 18 F] PET / CT imaging results of normal Balb / c mice 45 minutes after injection of F-H3RESCA-3E10;

[0090] Figure 7 shows [ 18 F] ROI analysis results of F-H3RESCA-3E10 in normal Balb / c mice 45 minutes after injection;

[0091] Figure 8 shows [ 18 F]F-H3RESCA-3E10 in vitro biodistribution results in normal Balb / c mice;

[0092] Figure 9 shows [ 18 F] PET / CT imaging results of F-H3RESCA-3E11 in normal Balb / c mice;

[0093] Figure 10 shows the [ 18 F]F-ROI image of H3RESCA-3E11 in normal Balb / c mice;

[0094] Figure 11 shows the [ 18 F]F-H3RESCA-3E11 in vitro biodistribution results in normal Balb / c mice;

[0095] Figure 12 shows the [ 18 F] PET / CT imaging results of F-H3RESCA-3A12 in normal Balb / c mice;

[0096] Figure 13 shows the [ 18 F]F-ROI image of H3RESCA-3A12 in normal Balb / c mice;

[0097] Figure 14 shows the [ 18 F]F-H3RESCA-3A12 in vitro biodistribution results in normal Balb / c mice;

[0098] Figure 15 shows the chemical structure of the chelating agent (±)H3RESCA-TFP;

[0099] FIG16 shows the chemical structure of the chelating agent (±)H3RESCA-Mal. DETAILED DESCRIPTION

[0100] For ease of understanding of the present invention, the present invention will be described in more detail below in conjunction with the accompanying drawings and specific examples. It should be noted that the present invention is not limited to the ad hoc method described herein, scheme, cell line, construct and reagent, and is equally changeable. Unless otherwise defined, all technical and scientific terms used in this specification sheet are identical with the meaning generally understood by those skilled in the art of the present invention. The terms used in this specification sheet in the specification sheet of the present invention are just for the purpose of describing specific embodiments and are not used to limit the present invention.

[0101] Traditional screening of CLDN18.2-positive patient populations relies on pathological results of biopsy or surgical specimens, which is highly invasive and has poor reproducibility. In addition, sampling errors and tumor heterogeneity may lead to differences in staining results. TM 18.2 Immunohistochemistry kits cannot distinguish between the two splice mutants of CLDN18, and false positive results may occur. Commonly used nonspecific imaging agents 18 While F-FDG PET / CT is widely recognized for its value in determining distant metastasis (M) in gastric cancer, it lacks a compelling advantage over other imaging methods in diagnosing and evaluating primary tumors and regional lymph nodes. Its diagnostic value for early-stage gastric cancer, signet ring cell carcinoma, or mucinous adenocarcinoma is particularly unsatisfactory. Currently, there is an urgent need for tools that can noninvasively diagnose gastric cancer early and screen patients for appropriate targeted therapy.

[0102] Molecular imaging plays an essential role in assisting disease screening, differential diagnosis, patient stratification, clinical staging, therapeutic efficacy assessment, and prognosis. The development of novel probes that enable non-invasive visualization of gastric cancer-specific targets is crucial for early diagnosis and precise treatment, reducing mortality and prolonging survival in gastric cancer patients.

[0103] Studies have shown that immuno-PET can better display the distribution and abundance of targets of interest in the body than immunohistochemical staining or other traditional predictive markers, and can better predict the response to targeted therapy. Traditional immuno-PET probes based on monoclonal antibodies are characterized by high stability and strong specificity, and are relatively widely used in clinical practice. However, monoclonal antibodies (approximately 150 kDa) have a large molecular weight, low tissue penetration, poor imaging targets, and are prone to non-specific uptake in non-target organs. Monoclonal antibodies have a complex spatial structure, high expression and preparation costs, and are highly immunogenic, while modified antibodies are difficult to achieve the original affinity. Many factors limit their application and popularization in clinical practice. Currently, the field of CLDN18.2 molecular imaging probes is still in the development stage.

[0104] Based on this, the present invention studies and constructs a novel Claudin 18.2 (CLDN18.2)-specific molecular imaging probe to achieve non-invasive visualization of CLDN18.2 on the surface of malignant tumor cells, monitor expression level differences and dynamic evolution in vivo, and further improve the effectiveness of the probe on this basis to promote the realization of integrated diagnosis and treatment.

[0105] Example 1: Preparation of CLDN18.2-specific Nanobodies

[0106] According to the method previously published by the inventors (refer to the invention patent No. ZL202011131233.7 entitled "A molecular imaging probe for diagnosing multiple myeloma", which is incorporated herein by reference in its entirety), new CLDN18.2-specific nanoantibodies 3E10, 3E11, and 3A12 were prepared. The amino acid sequence of the nanobody 3E10 is shown in SEQ ID NO.4 (wherein the CDR1 sequence is GNIVSINY (SEQ ID NO.1), the CDR2 sequence is ITNGGSA (SEQ ID NO.2), and the CDR3 sequence is HASSVITTASLWGTDY (SEQ ID NO.3)), and the gene sequence is shown in SEQ ID NO.5; the amino acid sequence of the nanobody 3E11 is shown in SEQ ID NO.9 (wherein the CDR1 sequence is GRIFMINN (SEQ ID NO.6), the CDR2 sequence is ITRGGST (SEQ ID NO.7), and the CDR3 sequence is NVNDTMPWRLQNDY (SEQ ID NO.8)), and the gene sequence is shown in SEQ ID NO.10; the amino acid sequence of the nanobody 3A12 is shown in SEQ ID NO.14 (wherein the CDR1 sequence is GRTFSDYN (SEQ ID NO.11), the CDR2 sequence is ITWSGSIR (SEQ ID NO.12), and the CDR3 sequence is AANRLAMHRGLNYDY (SEQ ID NO.13)). NO.13)), the gene sequence is shown in SEQ ID NO.15.

[0107] The expression of nanobodies 3E10, 3E11, and 3A12 was determined by SDS-PAGE. The following steps were used: First, a 1.5 mm thick, 15-well gel was prepared according to the SDS-PAGE gel kit protocol. A preheated metal bath to 100°C was used to heat the protein sample in 5X loading buffer for 5 minutes. After the SDS-PAGE gel was assembled, 500 ml of 1x SDS-PAGE buffer was added and the protein sample was slowly spotted into the loading wells. The gel was then electrophoresed at 80 V for approximately 30 minutes. Once the bromophenol blue indicator crossed the stacking gel, the voltage was adjusted to 120 V. Electrophoresis was performed to the bottom of the gel. The gel was removed and heat-stained in Coomassie blue for 50 minutes. The gel was then removed and destained with a destaining solution until the background was clear and the bands were distinct, and then photographed. As shown in Figure 1, the molecular weight of nanobodies 3E10, 3E11, and 3A12 was approximately 14 kDa.

[0108] Verification of CLDN18.2 expression in major tissues and organs of normal nude mice involved the following steps: The stomach, liver, kidney, lung, spleen, and pancreas of normal nude mice were obtained and fixed using tissue fixative. Immunohistochemical staining was performed using a recombinant CLDN18.2 monoclonal antibody (EPR19202, ab222512, Abcam) as the primary antibody and a horseradish peroxidase-conjugated rabbit anti-human IgG H&L (HRP-labeled rabbit anti-human IgG H&L; ab6759; Abcam) as the secondary antibody. As shown in Figure 2, immunohistochemical staining revealed positive expression in the gastric glandular epithelium of mice, while negative expression was observed in other organs, including the liver, lung, kidney, pancreas, and spleen.

[0109] Example 2: Preparation of CLDN18.2-specific nanobody probe

[0110] 1) Preparation of intermediate H3RESCA-3E10, H3RESCA-3E11, or H3RESCA-3A12, specifically as follows: Pre-equilibrate a PD-10 column with 4×5 mL of 0.05 M NaHCO3 solution. Add 1 mg of the nanobody (3E10, 3E11, or 3A12) solution to the PD-10 column and add 0.05 M NaHCO3 solution to bring the volume to 2.5 mL. Elute with 0.05 M NaHCO3 solution, collecting 2.5 mL of the eluate. Weigh 12 times the molar amount of the nanobody (±)-H3RESCA-TFP (CAS number: 1919794-40-3; having the chemical formula shown in Figure 15) or (±)H3RESCA-Mal (having the chemical formula shown in Figure 16; when using this bifunctional chelator, a peptide containing cysteine ​​(GGGGS) must be expressed at the end of the nanobody). nC, n = 1-10, in this embodiment, n = 3, that is, the peptide segment is GGGGSGGGGSGGGGSC, the peptide segment containing cysteine ​​at the end (GGGGS) n C nanoantibody was obtained according to the preparation method of the nanoantibody described in Reference Example 1) and dissolved in 40 μL DMSO. After adding (±)-H3RESCA-TFP or (±)H3RESCA-Mal solution to the above nanoantibody solution, mix thoroughly. Place on a shaker at room temperature for 2 hours. Pre-equilibrate the PD-10 column with 4×5mL 0.1M CH3COONH4 solution. Add the reaction solution to the PD-10 column, elute with 0.1M CH3COONH4 solution, and collect 3mL of eluate. Rinse the centrifugal filter with DD water. Concentrate using an Amicon ultracentrifugal filter and determine the concentration of the product (H3RESCA-3E10, H3RESCA-3E11 or H3RESCA-3A12) using NanoDrop.

[0111] 2) 18 The preparation of F-labeled H3RESCA-3E10, H3RESCA-3E11, and H3RESCA-3A12 was carried out as follows: 5 mL of sterile water for injection, 5 mL of air, 5 mL of 0.9% saline, and 5 mL of air were used to activate the QMA column. 500 μL of 18F-rich solution was taken from the accelerator. 18 O] water was passed through the QMA column, the filtrate was discarded, 500 μL of 0.9% saline was passed through the QMA column, the filtrate was collected, and the activity was 24 mCi. 8 μL of 2mM AlCl3 (in 0.1M CH3COONH4) solution was added and allowed to stand for 5 minutes. 160 μL (7.6 mCi) 18+19 F– / Al3+ solution, 150 μL (200) μg, 690 μL 0.1M CH3COONH4 solution were mixed and reacted at room temperature for 12 minutes. During this period, 4 × 5 mL of 0.9% NaCl eluent (pH 5.9-6.1) containing 5 mg / mL ascorbic acid was used to pre-equilibrate the PD-10 column. The reaction solution was added to the PD-10 column and the eluent was added to 2.5 mL. Then, 0.5 mL was added each time for elution. A total of 5 tubes were used to measure the product ([ 18 F]F-H3RESCA-3E10, [ 18 F]F-H3RESCA-3E11, [ 18 F]F-H3RESCA-3A12) activity.

[0112] 3)[ 18 F]F-H3RESCA-3E10, [ 18 F]F-H3RESCA-3E11, [18 F]F-H3RESCA-3A12 quality control. The specific steps are as follows: use a capillary glass tube to draw a small amount of [ 18 F]F-H3RESCA-3E10, [ 18 F]F-H3RESCA-3E11 or [ 18 F]F-H3RESCA-3A12 was spotted on a silica gel plate, and physiological saline was used as the mobile phase. Radiochemical purity (RCP) of the probe was determined by radio-thin layer chromatography (Radio-TLC, Eckert & Ziegler Radiopharma Inc). As shown in Figures 3, 4, and 5, freshly prepared [ 18 F]F-H3RESCA-3E10, [ 18 F]F-H3RESCA-3E11, [ 18 F] F-H3RESCA-3A12 RCP is greater than 99%.

[0113] 4)[ 18 Imaging of F]F-H3RESCA-3E10 in normal Balb / c mice. The specific steps are as follows: All small animal PET / CT images in this study were acquired using an IRIS small animal PET / CT scanner (Inviscan Imaging Systems). Each mouse was injected via the tail vein with 3.7-7.4 MBq [ 18 F]F-H3RESCA-3E10 (3 mice per group), 0.5 hours after injection, mice were anesthetized with isoflurane (concentration of 2%) and placed in a supine position on the PET / CT scanner bed in a deeply anesthetized state. PET and CT images were acquired continuously. Image reconstruction was completed using the IRIS system's built-in software. Regions of interest (ROIs) such as the heart and major tissue organs (liver, lung, kidney, muscle) were delineated on the reconstructed PET images using the OsiriX Lite image processing workstation (Pixmeo SARL). The radioactive uptake values ​​of important tissues and organs were calculated in %ID / g (percent of injected dose per gram), and the uptake values ​​of major tissues and organs over time were plotted. As shown in Figures 6 and 7, there was significant uptake in the stomach at 45 minutes, and the probe ([ 18 F]F-H3RESCA-3E10) is mainly excreted through the urinary system. The in vitro experimental results are shown in Figure 8. 18The highest uptake of F]F-H3RESCA-3E10 was in the kidney, which was 213.5±33.3% ID / g. This is due to the clearance of the tracer in the kidney. 18 F]F-H3RESCA-3E10 showed significant uptake, at 26.8±4.4% ID / g.

[0114] 5)[ 18 Imaging of F-H3RESCA-3E11 in normal Balb / c mice. The imaging procedure was as described in step 4 above. The PET / CT imaging results are shown in Figure 9; by delineating the ROI, the uptake of major organs was matched to the imaging results, as shown in Figure 10. The in vitro experimental results are shown in Figure 11. 18 The highest uptake of F]F-H3RESCA-3E11 was in the kidney, which was 251.3±65.2% ID / g. 18 F] F-H3RESCA-3E11 uptake was 84.8±50.3% ID / g.

[0115] 6)[ 18 Imaging of F-H3RESCA-3A12 in normal Balb / c mice. The imaging procedure was as described in step 4). The PET / CT imaging results are shown in Figure 12; by delineating the ROI, the uptake of major organs was matched to the imaging results, as shown in Figure 13. The in vitro experimental results are shown in Figure 14, which are consistent with [ 18 F]F-H3RESCA-3E10, [ 18 F]F-H3RESCA-3E11 is different, [ 18 The highest uptake of F]F-H3RESCA-3A12 was in the stomach, at 269.8±162.9% ID / g, while the uptake in the kidney was 204.7±107.4% ID / g, which was lower than that in the stomach.

[0116] The above results show that 18 F-labeled anti-CLDN18.2 nanobody probe [ 18 F]F-H3RESCA-3E10, [ 18 F]F-H3RESCA-3E11, [ 18 F]F-H3RESCA-3A12 can specifically bind to CLDN18.2 distributed in normal gastric epithelial cells, among which [ 18 The imaging effect of F-H3RESCA-3A12 probe in normal gastric tissue is better than that of [ 18 F]F-H3RESCA-3E10, [ 18F]F-H3RESCA-3E11 is better. The above data show that 18 F-labeled anti-CLDN18.2 nanoantibody probe is a potential in vivo non-invasive diagnostic and therapeutic tool targeting CLDN18.2.

[0117] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features can be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the description of the present invention. Furthermore, it is obvious to those skilled in the art that improvements or changes can be made based on the above description, and all such improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A specific nanobody that binds to the CLDN18.2 antigen, characterized in that: Include: (1) CDR1 of the amino acid sequence shown in SEQ ID No. 1, CDR2 of the amino acid sequence shown in SEQ ID No. 2, and CDR3 of the amino acid sequence shown in SEQ ID No. 3, (2) CDR1 of the amino acid sequence shown in SEQ ID No. 6, CDR2 of the amino acid sequence shown in SEQ ID No. 7, and CDR3 of the amino acid sequence shown in SEQ ID No. 8, or (3) CDR1 of the amino acid sequence shown in SEQ ID No.11, CDR2 of the amino acid sequence shown in SEQ ID No.12, and CDR3 of the amino acid sequence shown in SEQ ID No.

13.

2. The specific nanobody binding to the CLDN18.2 antigen according to claim 1, characterized in that The specific nanobody that binds to the CLDN18.2 antigen has an amino acid sequence as shown in SEQ ID No. 4, 9 or 14.

3. A polynucleotide, characterized in that Encoding a specific Nanobody that binds to the CLDN18.2 antigen according to claim 1 or 2; The polynucleotide has a nucleotide sequence as shown in SEQ ID No. 5, 10 or 15.

4. A carrier, characterized in that Comprising the polynucleotide according to claim 3.

5. A host cell, characterized in that Comprising the vector according to claim 4.

6. A CLDN18.2 specificity 18 F-labeled monovalent nanoantibody probe, characterized in that Comprising a specific Nanobody that binds to the CLDN18.2 antigen according to claim 1 or 2 that is labeled with a radionuclide.

7. The CLDN18.2-specific 18 F-labeled monovalent nanoantibody probe, characterized in that The specific Nanobody binding to the CLDN18.2 antigen according to claim 1 or 2 is labeled with a radionuclide via a bifunctional chelator; The bifunctional chelating agent is selected from at least one of (±)-H3RESCA-TFP and (±)H3RESCA-Mal, The radionuclide is selected from F-18.

8. A kit or composition for visualizing the expression of CLDN18.2, characterized in that Comprising a specific Nanobody that binds to the CLDN18.2 antigen according to claim 1 or 2, a polynucleotide according to claim 3, a vector according to claim 4, a host cell according to claim 5, and a probe according to any one of claims 6-7.

9. Use of a specific Nanobody that binds to the CLDN18.2 antigen according to claim 1 or 2, a polynucleotide according to claim 3, a vector according to claim 4, a host cell according to claim 5, or a probe according to any one of claims 6-7 in the preparation of a kit or composition for visualizing the expression of CLDN18.2.

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