CN117327182A

CN117327182A - Preparation method and application of CLDN18.2 single domain antibody probe

Info

Publication number: CN117327182A
Application number: CN202311210180.1A
Authority: CN
Inventors: 魏伟军; 安淑娴; 刘建军
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2023-09-19
Filing date: 2023-09-19
Publication date: 2024-01-02

Abstract

The invention relates to the technical fields of molecular images, nuclear medicine and single-domain antibodies for tumor diagnosis and treatment, in particular to a preparation method and application of a CLDN18.2 single-domain antibody probe. The CLDN18.2 prepared by the invention has specificity ¹⁸ F marked monovalent nanometer antibody probe has simple preparation process, low cost, high specificity, high stability, short imaging period and radiation doseLow cost, easy clinical transformation, etc. And by developing an immune PET (positron emission tomography) image based on the probe prepared by the invention, the noninvasive visualization of the expression of the CLDN18.2 in tumor tissues and normal tissues and organs can be realized, and the method is further used for noninvasive target specific diagnosis of specific types of tumors.

Description

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

Technical Field

The invention relates to the technical fields of molecular images, nuclear medicine and single-domain antibodies for tumor diagnosis and treatment, in particular to a preparation method and application of a CLDN18.2 single-domain antibody probe.

Background

Gastric cancer is one of the most common malignant tumors in China, the incidence rate is the second most serious malignant tumor, and the death rate is only inferior to liver cancer and lung cancer. Surgery is the most common treatment, but most patients are already in advanced stages at the time of diagnosis and have poor overall prognosis. The first-line treatment mode of the progressive gastric cancer is chemotherapy, and patients cannot obtain better long-term survival along with the increase of tumor drug resistance. In tumor diagnosis and treatment strategies, molecular targeted therapies such as small molecule inhibitors and monoclonal antibodies and immunotherapy (such as immune checkpoint inhibitors) are changing the current state of treatment of various solid tumors and blood system tumors. Finding a proper stomach cancer diagnosis and treatment target has important significance for the benefit of stomach cancer patient groups.

The tight junction protein 18 (Claudin 18, CLDN 18) is a transmembrane protein located in the tight junction of the epithelium and endothelium, and is an important constituent and functional structure that constitutes the tight junction between cells. The first exon of the human CLDN18 gene has two alleles, the differences of which form two different splice mutants, CLDN18.1 protein and CLDN18.2 protein, respectively. CLDN18 is highly conserved in normal tissues, and CLDN18.2 proteins are mainly distributed in gastric mucosa (gastric normal glands, main cells, parietal cells, endocrine cells) with short differentiation cycle and rapid turnover, and in pannicol cells of the duodenum. It is currently widely believed that tumors undergo a change in cell polarity during malignant transformation, resulting in a broad distribution of CLDN18.2 across the cell membrane surface. The positive rate of CLDN18.2 in gastric cancer and the ratio of CLDN18.2 positive gastric cancer in gastric cancer are greatly different in different researches, the current partial researches limit the expression rate of the CLDN18.2 in gastric cancer to 42-86%, the CLDN18.2 positive gastric cancer accounts for about 16-73% of gastric cancer groups, and the CLDN18.2 expression has certain molecular pathological characteristics: diffuse gastric cancer is higher than intestinal gastric cancer, EBV virus (Epstein-Barr virus) positive gastric cancer is higher than negative gastric cancer (81.0% vs.40.2%, P < 0.001), primary foci, peripheral lymph node metastasis and distant metastasis are expressed substantially consistently. CLDN18.2 is considered to be a very potential target in the treatment of digestive system malignancies due to aberrant activation and high expression in digestive system malignancies.

CLDN18.2 has been shown to be a good target for solid tumor treatment. Zolbetuximab (IMAB 362, claudixmab) is a chimeric IgG1 monoclonal antibody that specifically binds to CLDN18.2 on the surface of tumor cells, thereby eliciting antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (complement dependent cytotoxicity, CDC), apoptosis, and inhibiting cell proliferation. A number of phase I/II trials are currently evaluating their clinical efficacy and safety. The company Claudin18.2 antibody Zolbetuximab+chemotherapy was announced by America, 11, 2022, 17, inc. (Astella) to reach the primary endpoint in the third-stage clinical SPOTLIGHT of Claudin18.2 positive, HER2 negative recurrent metastatic gastric cancer. The study results showed that the Progression Free Survival (PFS) and total survival (OS) of zolbetuximab+mfofox 6 treated patients were statistically significant compared to placebo+mfofox 6. Humanized anti-CLDN 18.2 autologous CAR-T therapy also shows anti-tumor activity and safety, and multiple clinical trials are underway to provide more options for CLDN18.2 positive tumor patients. CLDN18.2 is involved in targeting drugs, small molecule inhibitors, as well as CAR-T, immune cell therapies, antibody-coupled drugs, tumor vaccines, etc., cancer species are involved in gastric cancer, gastro-esophageal junction cancer as well as pancreatic cancer, personalized immunotherapy of CLDN18.2 or will be a hotspot for the next digestive system malignancy study.

Antibodies are of particular interest in biomedical research due to their defined structure, relative stability, high specificity and high affinity. PET imaging developed based on probes constructed by antibodies is called immune PET imaging, belongs to one branch of molecular imaging, and can effectively reflect tumorigenesis development in real time by adopting probes targeting tumor cells and tumor microenvironment receptors. The immune PET probe constructed based on the monoclonal antibody has the characteristics of high stability and strong specificity, and has relatively wide clinical application, but the monoclonal antibody (about 150 kD) has overlarge molecular weight, low tissue penetration capability, poor imaging target cost and easy non-specific uptake of non-target organs. The monoclonal antibody has complex space structure, higher expression and preparation cost and high immunogenicity, and the modified antibody is difficult to achieve the original affinity. Many factors limit their clinical use and popularity.

Nanobodies are derived from the smallest functional antigen-binding fragment of heavy chain antibodies in adult camelids, with a molecular weight of only 15kDa. Nanobodies have a high stability and high affinity for binding to antigen, and have many unique properties compared to conventional antibodies: 1) The sequence coded by the nano antibody has high homology with human VH families 3 and 4 and weak immunogenicity; 2) The nano antibody has small molecular weight and simple structure, can be expressed in a large amount in a microorganism system, and is easy to purify; 3) The nanobody can recognize a large number of antigen epitopes, including some epitopes hidden in molecular cracks; 4) Due to their small molecular weight, they readily penetrate tissues to sites that are difficult for conventional antibodies to reach; 5) Can be matched with nuclides with short half-life periods to realize the current day imaging. The diverse properties of nanobodies make them a promising tool for disease diagnosis and treatment: as an imaging tracer, the nanobody can acquire high-quality images as soon as possible; as a therapeutic agent, nanobodies can be conjugated to cytotoxic drugs and delivered specifically to a target site to achieve precise targeted therapy. In recent years, we have focused on the development and clinical transformation of nanobody-derived tracers to exert their superior molecular imaging properties. Based on the evidence and our previous findings, we hypothesize that the immune PET imaging probe targeting CLDN18.2 can noninvasively display the expression of gastric cancer tumor cells CLDN18.2, serve the diagnosis and treatment system of gastric cancer system, and promote the development of accurate medical individuation treatment.

At present, the field of CLDN18.2 molecular image probes is still under development, and the person skilled in the art is dedicated to develop a nano antibody immune PET imaging probe which has the advantages of low preparation cost, small molecular weight, short in vivo circulation time, short imaging period, low radiation dose and easy clinical transformation application.

Disclosure of Invention

To fill the gap in this field, we describe herein the construction of nanobody-derived CLDN18.2 targeted diagnostic probes and characterize their biodistribution in normal Balb/c mice in an effort to explore their diagnostic and potential therapeutic value for gastric cancer. In particular to a preparation method and application of a CLDN18.2 nanometer antibody probe.

CLDN18.2 specific nanobodies

In one aspect, the invention provides a CLDN 18.2-specific nanobody comprising:

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

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

(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.

More specifically, the CLDN 18.2-specific nanobody of the invention has the amino acid sequence shown in SEQ ID No.4, 9 or 14.

In the present invention, CLDN 18.2-specific nanobodies having the amino acid sequence shown in SEQ ID nos. 4, 9 or 14 are referred to as 3E10, 3E11 or 3a12, respectively, for the sake of simplicity.

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 commonly understood by those skilled in the art, which refers 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 4 framework regions and 3 complementarity determining regions, having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Nanobodies may be truncated at the N-or C-terminus such that they comprise only a portion of FR1 and/or FR4, or lack one or both of those framework regions, so long as they substantially retain antigen binding and specificity.

In some embodiments, the invention also encompasses antigen-binding fragments of CLDN 18.2-specific nanobodies as described herein.

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 an antigen, also referred to as an "antigen-binding portion. Generally, see Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes, antigen binding fragments of the present antibodies may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the present nanobodies.

Antigen-binding fragments of nanobodies can be obtained from a given nanobody (e.g., a nanobody provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and specifically screened in the same manner as for whole nanobodies.

In this context, 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.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs are contained in the nanobody, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, e.g., as in 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, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, for example, lefranc et al, dev. Comparat. Immunol.27:55-77,2003).

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

As used herein, the term "CLDN18.2 specific" refers to specifically binding CLDN18.2.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be determined by the equilibrium dissociation constant (K _D ) And (3) representing. In the present invention, the term "K _D "refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen.

The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. "binding Rate constant" (k) _a Or k _on ) And "dissociation rate constant" (k) _dis Or k _off ) Both can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, nature,1993, 361:186-187). k (k) _dis /k _on Is equal to the dissociation constantK _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 Values. In certain embodiments, the dissociation constant may be measured in Biacore using Surface Plasmon Resonance (SPR). In addition to this, bioluminescence interferometry or Kinexa can be used to measure the dissociation constant.

In some embodiments, the invention also provides variants of CLDN 18.2-specific nanobodies as described herein that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID nos. 4, 9 or 14 and substantially retain the biological function of the nanobody from which they are derived (e.g., the biological activity of specifically binding CLDN 18.2).

More specifically, the variants differ only in conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, up to 5, or up to 1 amino acids) amino acid residues as compared to a CLDN 18.2-specific nanobody as described herein.

As used herein, the term "identity" is used to refer to the match of sequences 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 two DNA molecules is occupied by adenine, or a position in each of 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 matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of 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 in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. ApplBiosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences may be determined using the Needleman and Wunsch (J mobiol. 48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum 62 matrix or PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. 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 substitutions of amino acids 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).

Polynucleotide

In another aspect, the invention also provides polynucleotides encoding the nanobodies described above or antigen-binding fragments thereof.

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

More specifically, the polynucleotide encoding a CLDN 18.2-specific nanobody as described herein has the nucleotide sequence set forth in SEQ ID No.5, 10 or 15.

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

The term "polynucleotide encoding a polypeptide/protein/antibody" may include polynucleotides encoding such polypeptide/protein/antibody, as well as polynucleotides further comprising additional coding and/or non-coding sequences.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80%, most preferably at least 90% identity between the two sequences, and which encode polypeptides/proteins/antibodies having substantially the same function and activity. The invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

Carrier body

In another aspect, the invention also provides a vector comprising a polynucleotide encoding the CLDN18.2 antibody or antigen-binding fragment thereof described above.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may 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, the vector may also contain a replication origin.

Host cells

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

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or other human cells. Host cells may include single cells or cell populations.

The vector may be introduced into the host cell by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The nanobodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.

Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product. The preferred detectable label is a radionuclide.

Therapeutic agents that may be conjugated or coupled to an antibody of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic 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 nanoparticle, and the like.

Binding or coupling of the detectable label or therapeutic agent to the antibody can be performed by conventional methods well known to those skilled in the art. For example, the detectable label may be directly or indirectly bound to the nanobody, e.g., via a cleavable or non-cleavable linker peptide, or incorporated into the nanobody. The detectable label may be bound to the nanobody, in particular by substitution (e.g. by substituting H with I at the tyrosine residue level), by complexation or by chelation. For example, the therapeutic agent may be conjugated to the nanobody via a cleavable linker (e.g., a peptidyl, disulfide, or hydrazone linker).

In a preferred embodiment, the nanobody of the invention is conjugated with a radionuclide for use as CLDN 18.2-specific molecular imaging probe, as described in more detail below.

CLDN18.2 specific molecular imaging probe

In another aspect, the invention provides a CLDN18.2 specificity ¹⁸ F-labeled monovalent nanobody probes comprising radionuclide-labeled CLDN 18.2-specific nanobodies as described herein.

More specifically, CLDN 18.2-specific nanobodies as described herein are labeled with radionuclides via bifunctional chelators.

As used herein, a bifunctional chelating agent is a class of chelating agents having both a metal chelating end and a protein anchoring end. The bifunctional chelating agent may be at least one selected from (+ -) -H3RESCA-TFP, (+ -) H3 RESCA-Mal.

As used herein, the (+ -) -H3RESCA-TFP is a tetrafluorophenyl ester derivative of a constrained complexing agent (RESCA) that can be used to couple a chelator to a biomolecule by amine coupling (e.g., N-terminal and/or epsilon-amino group of lysine);

the (+ -) H3RESCA-Mal is (+ -) H3 RESCA-maleimide, a tetrafluorophenyl ester derivative of a constrained complexing agent (RESCA), useful for coupling chelators to biomolecules via amine coupling (e.g., N-terminal and/or epsilon-amino groups of lysine).

More specifically, CLDN 18.2-specific nanobodies as described herein are labeled with radionuclides via (±) -H3RESCA-TFP or (±) H3 RESCA-Mal.

More specifically, the radionuclide is selected from F-18.

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

More specifically, the CLDN18.2 specificity ¹⁸ F labeling monovalent nanobody probe [ ¹⁸ F]F-H3RESCA-3A12, the specific nanobody of CLDN18.2 with the amino acid sequence shown in SEQ ID No.14 is labeled with F-18 via (+ -) -H3RESCA-TFP or (+ -) -H3 RESCA-Mal according to CDR1 with the amino acid sequence shown in SEQ ID No.11, CDR2 with the amino acid sequence shown in SEQ ID No.12 and CDR3 with the amino acid sequence shown in SEQ ID No. 13. It has better imaging effect in normal stomach tissue.

In another aspect, the invention also provides a method for preparing CLDN18.2 specificity ¹⁸ F, a method for labeling a monovalent nanobody probe comprises the steps of modifying a CLDN18.2 specific nanobody through a bifunctional chelating agent to obtain a radionuclide labeling precursor; and labeling the radionuclide-labeled precursor with a radionuclide to obtain CLDN18.2 specificity ¹⁸ F labeling monovalent nanobody probes.

More specifically, when the bifunctional chelating agent is (±) H3RESCA-Mal, a peptide fragment containing cysteine (GGGGS) nC, n=1-10, is expressed at the end of CLDN 18.2-specific nanobody before the preparation of radionuclide-labeled precursor; in particular n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Composition and method for producing the same

In another aspect, the invention provides a composition comprising a CLDN 18.2-specific nanobody, polynucleotide, vector, host cell or molecular imaging probe as described herein. The composition can be used to detect expression levels of CLDN18.2, diagnose CLDN 18.2-associated tumors, predict the therapeutic effect of CLDN 18.2-associated tumors, or treat CLDN 18.2-associated tumors.

In some embodiments, the composition may be a pharmaceutical composition.

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

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

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

In some embodiments, the CLDN 18.2-specific nanobody, polynucleotide, vector, host cell, or molecular imaging probe as described herein and the additional pharmaceutically active agent in the pharmaceutical composition can be provided as separate components or as a mixed component. Thus, a CLDN 18.2-specific nanobody, polynucleotide, vector, host cell, or molecular imaging probe as described herein and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

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 liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of a CLDN 18.2-specific nanobody, polynucleotide, vector, host cell, or molecular imaging probe as described herein. "prophylactically effective amount" means an amount sufficient to prevent, arrest or delay the onset of a disease. By "therapeutically effective amount" is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on the factors: the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

Kit for detecting a substance in a sample

The invention also provides a kit comprising a CLDN 18.2-specific nanobody, polynucleotide, vector, host cell or molecular imaging probe as described herein.

The kit can be used for detecting the expression level of the CLDN18.2, diagnosing the tumor associated with the CLDN18.2, predicting the treatment effect of the tumor associated with the CLDN18.2 or treating the tumor associated with the CLDN 18.2.

The kit may further comprise further containers, instructions for use, and other reagents and buffers required for the actual application, such as lysis media for lysing the sample, various buffers, detection labels, detection substrates, etc.

Diagnostic and therapeutic applications

The CLDN18.2 specific nano antibody has extremely high affinity to the CLDN18.2, so that the nano antibody can be used for detecting the expression level of the CLDN18.2, diagnosing the tumor related to the CLDN18.2, predicting the treatment effect of the tumor related to the CLDN18.2 or treating the tumor related to the CLDN 18.2.

Particularly, the CLDN18.2 specific molecular image probe prepared by the CLDN18.2 specific nano antibody has the characteristics of obviously improved affinity, obviously reduced non-specific uptake of normal tissue uptake and obviously improved image quality, and can be used for noninvasively, accurately and efficiently detecting the expression of human CLDN18.2, so that the CLDN18.2 specific molecular image probe is particularly suitable for diagnosing the tumor related to the CLDN18.2 and predicting the treatment effect of the tumor related to the CLDN 18.2. After the appropriate radionuclide is selected for coupling, the method can also be used for accurately treating the CLDN18.2 related tumor.

Thus, in another aspect, the invention also relates to the use of a CLDN 18.2-specific nanobody, polynucleotide, vector, host cell or molecular imaging probe as described herein in the manufacture of a kit or medicament for detecting expression levels of CLDN18.2, diagnosing a CLDN 18.2-associated tumor, predicting the therapeutic effect of a CLDN 18.2-associated tumor or treating a CLDN 18.2-associated tumor.

As used herein, CLDN 18.2-associated tumors can include various tumors or cancers well known in the art. For example, CLDN 18.2-related tumors can include digestive tract tumors such as gastric cancer, pancreatic cancer, esophageal cancer, cholangiocarcinoma, gall bladder cancer, etc.; and breast cancer, colon cancer, liver cancer, head and neck cancer, bronchus cancer, non-small cell lung cancer, etc.

The beneficial effects of the invention are that

1) CLDN18.2 specific molecular probe prepared by the invention ¹⁸ F]F-H3RESCA-3E10、[ ¹⁸ F]F-H3RESCA-3E11 [ [ ¹⁸ F]F-H3RESCA-3A12 is a positron nuclide emission type probe and is used for immune PET imaging; by developing the immune PET imaging based on the probe, the noninvasive visualization of the expression of the CLDN18.2 in tumor tissues and normal tissues and organs can be realized, and the method is further used for noninvasive target specific diagnosis of specific types of tumors.

2) The probe prepared by the invention also has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging period, low radiation dose, easy clinical transformation and the like.

Drawings

FIG. 1 shows the results of SDS-PAGE determination of nanobody 3E10, 3E11 and 3A12 expression;

FIG. 2 shows the results of histochemical staining of CLDN 18.2;

FIG. 3 shows [ [ ¹⁸ F]Radiochemical purity determination of F-H3RESCA-3E 10;

FIG. 4 shows [ [ ¹⁸ F]Radiochemical purity measurement of F-H3RESCA-3E 11;

FIG. 5 shows [ [ ¹⁸ F]Radiochemical purity measurement of F-H3RESCA-3A 12;

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

FIG. 7 shows [ [ ¹⁸ F]ROI analysis results of F-H3RESCA-3E10 after 45 min of normal Balb/c mice injection;

FIG. 8 shows [ sic ] ¹⁸ F]In vitro biodistribution results of F-H3RESCA-3E10 in normal Balb/c mice;

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

FIG. 10 shows [ sic ] ¹⁸ F]ROI map knot of F-H3RESCA-3E11 in normal Balb/c miceFruit;

FIG. 11 shows [ sic ] ¹⁸ F]In vitro biodistribution results of F-H3RESCA-3E11 in normal Balb/c mice;

FIG. 12 shows [ sic ] ¹⁸ F]PET/CT imaging results of F-H3RESCA-3A12 in normal Balb/c mice;

FIG. 13 shows [ sic ] ¹⁸ F]ROI map results of F-H3RESCA-3A12 in normal Balb/c mice;

FIG. 14 shows [ sic ] ¹⁸ F]In vitro biodistribution results of F-H3RESCA-3A12 in normal Balb/c mice;

FIG. 15 shows the chemical structure of chelator (+ -) H3 RESCA-TFP;

FIG. 16 shows the chemical structure of chelator (+ -) H3 RESCA-Mal.

Detailed Description

In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. It is noted that the invention is not limited to the particular methods, protocols, cell lines, constructs, and reagents described herein, and may vary as well. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Traditional screening of CLDN18.2 positive patient populations relies on pathology results of biopsy or surgical specimens, is highly traumatic, poorly reproducible, and may lead to differences in staining results due to sampling errors and tumor heterogeneity. Common CLAUDETECT ^TM 18.2 immunohistochemical kit failed to distinguish between the two splice mutants of CLDN18, false positive results were possible. Commonly used nonspecific imaging agents ¹⁸ The value of F-FDG PET/CT in the distant metastasis of gastric cancer, i.e., in the M stage, has been acknowledged, while there is no convincing advantage over other imaging methods in the diagnosis, evaluation of primary tumors and regional lymph nodes, especially in the diagnosis of early gastric cancer, print-stop cell carcinoma or mucous adenocarcinoma. There is a need for noninvasive early diagnosis of gastric cancer and screening methodsMeans for targeted treatment of a patient population are suitable.

Molecular imaging plays a non-negligible role in assisting in disease screening, differential diagnosis, patient stratification and clinical staging, efficacy assessment and prognosis. The development of a novel probe for realizing noninvasive visualization of a stomach cancer specific target is important for early diagnosis and accurate treatment for reducing the death rate of stomach cancer patients and prolonging the survival time.

Studies have shown that immune PET can better display the distribution and abundance of targets of interest in vivo and better predict response to targeted therapies than immunohistochemical staining or other traditional predictive markers. The traditional immune PET probe constructed based on the monoclonal antibody has the characteristics of high stability and strong specificity, and has relatively wide clinical application, but the monoclonal antibody (about 150 kDa) has overlarge molecular weight, low tissue penetration capability, poor imaging target cost and easy non-specific uptake of non-target organs. The monoclonal antibody has complex space structure, higher expression and preparation cost and high immunogenicity, and the modified antibody is difficult to achieve the original affinity. Many factors limit their clinical use and popularity. Currently, the field of CLDN18.2 molecular image probes is still under development.

Based on the method, a novel Claudin 18.2 (CLDN 18.2) specific molecular image probe is researched and constructed so as to realize noninvasive visualization of the CLDN18.2 on the surface of malignant tumor cells, monitor the expression level difference and dynamic evolution in vivo, further improve the efficiency of the probe on the basis, and promote the implementation of diagnosis and treatment integration.

Example 1: preparation of CLDN 18.2-specific nanobodies

The novel CLDN 18.2-specific nanobodies 3E10, 3E11, 3a12 were prepared according to the method previously published by the inventors (refer to the patent No. ZL202011131233.7 entitled "molecular imaging probe for diagnosing multiple myeloma" which is incorporated herein by reference in its entirety). The amino acid sequence of the nano antibody 3E10 is shown as SEQ ID NO.4 (wherein the CDR1 sequence is GNIVSINy (SEQ ID NO. 1), the CDR2 sequence is ITNGGSA (SEQ ID NO. 2), the CDR3 sequence is HASSVITTASLWGTDY (SEQ ID NO. 3)), and the gene sequence is shown as SEQ ID NO. 5; the amino acid sequence of the nanometer antibody 3E11 is shown as SEQ ID NO.9 (wherein CDR1 sequence is GRIFMINN (SEQ ID NO. 6), CDR2 sequence is ITRGGST (SEQ ID NO. 7), CDR3 sequence is NVNDTMPWRLQNDY (SEQ ID NO. 8)), and the gene sequence is shown as SEQ ID NO. 10; the amino acid sequence of the nanometer antibody 3A12 is shown as SEQ ID NO.14 (wherein CDR1 sequence is GRTFSDYN (SEQ ID NO. 11), CDR2 sequence is ITWSGSIR (SEQ ID NO. 12), CDR3 sequence is AANRLAMHRGLNYDY (SEQ ID NO. 13)), and the gene sequence is shown as SEQ ID NO. 15.

SDS-PAGE (SDS-PAGE) is used for determining the expression of the nanobodies 3E10, 3E11 and 3A12, and the specific steps are as follows: firstly, preparing a 1.5mm thick gel, namely 15well gel according to a SDS-PAGE gel kit method, preheating a metal bath to 100 ℃, and heating a protein sample containing loading buffer (5X) for 5min; after SDS-PAGE gel is assembled, adding 500ml of 1x SDS-PAGE buffer, slowly spotting a protein sample into the upper sample hole, carrying out constant-voltage electric bath for about 30min at 80V, adjusting the voltage to 120V after a bromophenol blue indicator passes through a concentrated gel, carrying out electrophoresis to the bottom of the gel, taking the gel out, heating and dyeing in a coomassie blue dye solution for 50min, taking out, decoloring the decolored solution until the background is clean, and carrying out photographing when the strip is clear. As shown in fig. 1, it can be seen that nanobodies 3E10, 3E11, 3a12 have a molecular weight of about 14KDa.

The expression condition of CLDN18.2 in the main tissues and organs of normal nude mice is verified, which comprises the following steps: the stomach, liver, kidney, lung, spleen and pancreas of normal nude mice were obtained and fixed with tissue fixative, recombinant CLDN18.2 monoclonal antibodies (EPR 19202, ab222512, abcam) as primary antibodies, horseradish peroxidase conjugated rabbit anti-human IgG H & L (HRP-labeled rabbit anti-human IgG H & L; ab6759; abcam) as secondary antibodies, and immunohistochemical staining was performed. As a result, as shown in FIG. 2, the mice were found to be positive for gastric gland epithelial expression and negative for other organs such as liver, lung, kidney, pancreas, spleen expression by immunohistochemical experiments.

Example 2: preparation of CLDN18.2 specific nanobody probe

1) The preparation of the intermediate H3RESCA-3E10, H3RESCA-3E11 or H3RESCA-3A12 comprises the following specific steps: with 4X 5mL of 0.05M NaHCO ₃ The solution pre-equilibrates the PD-10 column. 1mg of nanobody (3E 10, 3E11 or 3A 12) solution was applied to PD-10 column, 0.05M NaHCO was added ₃ The solution was replenished to a volume of 2.5mL. Using 0.05M NaHCO ₃ The solution was eluted and 2.5mL of eluate was collected. 12 times of the molar amount of the nanobody (. + -.) -H3RESCA-TFP (CAS number:1919794-40-3; having the chemical formula shown in FIG. 15) or (. + -.) H3RESCA-Mal (having the chemical formula shown in FIG. 16) was weighed out, and when the bifunctional chelating agent was used, a peptide fragment (GGGGS) containing cysteine was expressed at the end of the nanobody _n C, n=1 to 10, in this example n=3 is used, i.e. the peptide is GGGGSGGGGSGGGGSC, the end of which contains cysteine (GGGGS) _n The nanobody of C was obtained by the method for preparing nanobody described in reference example 1), and dissolved in 40. Mu.L of DMSO. And (3) respectively adding (+/-) -H3RESCA-TFP or (+/-) -H3 RESCA-Mal solution into the nano antibody solution, and fully homogenizing. The reaction was allowed to proceed for 2 hours at room temperature on a shaker. With 4X 5mL of 0.1M CH ₃ COONH ₄ The solution pre-equilibrates the PD-10 column. The reaction solution was applied to a PD-10 column using 0.1M CH ₃ COONH ₄ The solution was eluted and 3mL of eluent was collected. The centrifugal filter was rinsed with DD water. And concentrated using an Amicon ultracentrifuge filter, and the concentration of the product (H3 RESCA-3E10, H3RESCA-3E11, or H3RESCA-3A 12) was measured using NanoDrop.

2) ¹⁸ F, preparing marks H3RESCA-3E10, H3RESCA-3E11 and H3RESCA-3A12, wherein the specific steps are as follows: the QMA column was activated with 5mL of sterile injectable water-5 mL of air-0.9% 5mL of physiological saline-5 mL of air in sequence. mu.L of 18F ion-containing rich fraction was taken from the accelerator ¹⁸ O]The water was passed through a QMA column, and the filtrate was discarded, 500. Mu.L of 0.9% physiological saline was passed through the QMA column, and the filtrate was collected to measure the activity as 24mCi. mu.L of 2mM AlCl was added ₃ (in 0.1M CH ₃ COONH ₄ ) The solution was allowed to stand for 5 minutes. 160. Mu.L (7.6 mCi) ¹⁸⁺¹⁹ F-/Al3+ solution, 150. Mu.L (200) g, 690. Mu.L 0.1M CH ₃ COONH ₄ The solutions were mixed and reacted at room temperature for 12 minutes. During this time, the PD-10 column was pre-equilibrated with 4X 5mL of 0.9% NaCl eluent (pH 5.9-6.1) containing 5mg/mL of ascorbic acid. The reaction solution was added to PD-10, and the eluate was used to make up to 2.5mL, followed by 0.5mL each timeElution was performed, 5 tubes total, and the products were measured separately ([ solution ]) ¹⁸ F]F-H3RESCA-3E10、[ ¹⁸ F]F-H3RESCA-3E11、[ ¹⁸ F]F-H3RESCA-3A 12).

3)[ ¹⁸ F]F-H3RESCA-3E10、[ ¹⁸ F]F-H3RESCA-3E11、[ ¹⁸ F]Quality control of F-H3RESCA-3A 12. The method comprises the following specific steps: suction of small amounts using capillary glass tubes ¹⁸ F]F-H3RESCA-3E10、[ ¹⁸ F]F-H3RESCA-3E11 or [ ¹⁸ F]F-H3RESCA-3A12 was spotted on a silica gel plate using physiological saline as the mobile phase, and was purified by Radio-thin layer chromatography (Radio-TLC, eckert)&Ziegler Radiopharma Inc) the radiochemical purity of the probe was determined (Radiochemical purity, RCP). Freshly prepared [ as shown in FIGS. 3, 4, 5 ] ¹⁸ F]F-H3RESCA-3E10、[ ¹⁸ F]F-H3RESCA-3E11、[ ¹⁸ F]F-H3RESCA-3A12 RCP is more than 99%.

4)[ ¹⁸ F]Visualization of F-H3RESCA-3E10 in normal Balb/c mice. The method comprises the following specific steps: the PET/CT imaging acquisitions of the animals involved in this study were all done using an IRIS small animal PET/CT scanner (Inviscan Imaging Systems). Each mouse was injected via the tail vein with 3.7-7.4MBq [ ¹⁸ F]F-H3RESCA-3E10 (3 in each group), mice were anesthetized with isoflurane (concentration 2%) at 0.5 hours after injection, and the mice entering a deep anesthesia state were placed on a PET/CT scanning bed in a supine position, PET and CT images were continuously acquired, image reconstruction was completed with the IRIS system self-contained software, the image processing workstation (Pixmeo SARL) was used to delineate regions of interest (Region of interest, ROI) such as heart and major tissue organs (liver, lung, kidney, muscle) on the reconstructed PET images, the radioactive uptake values of the major tissue organs were calculated in units of% ID/g (percent of injected dose per gram), and the change of the major tissue organ uptake values with time was plotted. As shown in FIGS. 6 and 7, the stomach had significant uptake at 45 minutes, probe ([ [ solution ]) ¹⁸ F]F-H3RESCA-3E 10) is mainly excreted via the urinary system. The in vitro results are shown in FIG. 8 [ ¹⁸ F]F-H3RESCA-3E10 uptake in the kidneys was highest at 213.5+ -33.3% ID/g due to the need for tracer clearance in the kidneys. At the same time, it can be seen that in the normal stomach groupIn weaving [ ¹⁸ F]F-H3RESCA-3E10 was taken up significantly at 26.8.+ -. 4.4% ID/g.

5)[ ¹⁸ F]Visualization of F-H3RESCA-3E11 in normal Balb/c mice. The development step is as described in step 4) above. The PET/CT imaging results are shown in FIG. 9; the uptake of the major organ by delineating the ROI was matched to the imaging results, which are shown in fig. 10. The in vitro results are shown in FIG. 11 [ ¹⁸ F]F-H3RESCA-3E11 was taken up highest in the kidneys at 251.3.+ -. 65.2% ID/g. In normal stomach tissue [ ¹⁸ F]F-H3RESCA-3E11 uptake was 84.8.+ -. 50.3% ID/g.

6)[ ¹⁸ F]Visualization of F-H3RESCA-3A12 in normal Balb/c mice. The development step is as described in step 4) above. The PET/CT imaging results are shown in FIG. 12; the uptake of the major organ by delineating the ROI was matched to the imaging results, which are shown in fig. 13. The in vitro test results are shown in FIG. 14, and [ and ] ¹⁸ F]F-H3RESCA-3E10、[ ¹⁸ F]F-H3RESCA-3E11 is different [ ¹⁸ F]F-H3RESCA-3A12 was taken up highest in stomach tissue at 269.8.+ -. 162.9% ID/g. Uptake in the kidneys was 204.7.+ -. 107.4% ID/g, which is lower than in stomach tissue.

The above results show that, ¹⁸ f-labeled anti-CLDN 18.2 nanobody probe [ ¹⁸ F]F-H3RESCA-3E10、[ ¹⁸ F]F-H3RESCA-3E11、[ ¹⁸ F]F-H3RESCA-3A12 can be specifically combined with CLDN18.2 distributed in normal gastric epithelial cells, wherein ¹⁸ F]The F-H3RESCA-3A12 probe has better imaging effect in normal stomach tissue ¹⁸ F]F-H3RESCA-3E10、[ ¹⁸ F]F-H3RESCA-3E11 is more preferred. The above data indicate that ¹⁸ The F-labeled anti-CLDN 18.2 nanobody probe is an in-vivo noninvasive diagnosis and treatment tool with certain potential for targeting the CLDN 18.2.

It should be noted that the description of the present invention and the accompanying drawings illustrate preferred embodiments of the present invention, but the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, which are not to be construed as additional limitations of the invention, but are provided for a more thorough understanding of the present invention. The above-described features are further combined with each other to form various embodiments not listed above, and are considered to be the scope of the present invention described in the specification; further, modifications and variations of the present invention may be apparent to those skilled in the art in light of the foregoing teachings, and all such modifications and variations are intended to be included within the scope of this invention as defined in the appended claims.

Claims

1. A CLDN 18.2-specific nanobody comprising:

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

2. The CLDN 18.2-specific nanobody of claim 1, wherein the CLDN 18.2-specific nanobody has the amino acid sequence set forth in SEQ ID No.4, 9 or 14.

3. A polynucleotide encoding a CLDN 18.2-specific nanobody according to claim 1 or 2;

preferably, the polynucleotide has the nucleotide sequence shown in SEQ ID No.5, 10 or 15.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell comprising the vector of claim 4.

6. CLDN18.2 specificity ¹⁸ F-labeled monovalent nanobody probe, characterized in that it comprises a radionuclide-labeled CLDN 18.2-specific nanobody according to claim 1 or 2.

7. A CLDN18.2 specificity according to claim 6 ¹⁸ F-labeled monovalent nanobody probe, characterized in that CLDN 18.2-specific nanobody according to claim 1 or 2 is labeled with a radionuclide via a bifunctional chelator.

Preferably, the bifunctional chelating agent is selected from at least one of (+ -) -H3RESCA-TFP, (+ -) H3RESCA-Mal,

preferably, the radionuclide is selected from F-18.

8. CLDN18.2 specificity according to claim 7 ¹⁸ F-labeled monovalent nanobody probe, characterized in that CDR1 with the amino acid sequence shown in SEQ ID No.11, CDR2 with the amino acid sequence shown in SEQ ID No.12 and CDR3CLDN18.2-specific nanobody with the amino acid sequence shown in SEQ ID No.13 according to claim 1 or CLDN 18.2-specific nanobody with the amino acid sequence shown in SEQ ID No.14 according to claim 2 is labeled with F-18 via (±) -H3RESCA-TFP or (±) H3 RESCA-Mal.

9. A kit or composition for visualizing expression of CLDN18.2, diagnosing CLDN 18.2-related tumors, predicting progression and prognosis of CLDN 18.2-related tumors, predicting therapeutic effect of CLDN 18.2-related tumors or treating CLDN 18.2-related tumors, characterized in comprising BCMA-specific nanobodies according to claim 1 or 2, polynucleotides according to claim 3, vectors according to claim 4, host cells according to claim 5, probes according to any of claims 6-8.

10. Use of a CLDN 18.2-specific nanobody 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 of claims 6-8 in the preparation of a kit or composition for visualizing expression of CLDN18.2, diagnosing a CLDN 18.2-associated tumor, predicting progression and prognosis of a CLDN 18.2-associated tumor, predicting the therapeutic effect of a CLDN 18.2-associated tumor or treating a CLDN 18.2-associated tumor.