Application of Claudin18.2-targeting nano antibody in preparation of drugs for combined radiotherapy treatment of tumors or cancers

Abstract

The invention discloses an application of a Claudin18.2-targeted nano antibody in preparation of a medicine for combined radiotherapy treatment of tumors or cancers, and belongs to the technical field of biological medicines. The nano antibody comprises a heavy chain variable region, the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the amino acid sequence of the CDR1 is as shown in SEQ ID NO.1; the amino acid sequence of the CDR2 is as shown in SEQ ID NO. 2; and the amino acid sequence of the CDR3 is as shown in SEQ ID NO.3. The nano antibody as a monoclonal antibody drug is combined with carbon ion irradiation (with the energy of 80.55 MeV / u, the linear energy transfer value of 50KeV / mu m and the dosage of 4Gy) to be applied to gastric cancer treatment, so that Claudin18.2 positive tumor proliferation is remarkably inhibited, tumor cell death is promoted, and an excellent in-vivo tumor inhibition effect is shown in a subcutaneous transplantation tumor model of a nude mouse.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and in particular relates to an application of a nanobody targeting Claudin 18.2 in preparation of a medicament for combined radiotherapy for treating tumors or cancers. BACKGROUND

[0002] Tumors still seriously threaten human health, especially recurrent and metastatic tumors. Targeted therapy and immunotherapy have brought great changes to the clinical treatment of various types of cancers, but further improvements are still needed to improve the overall response rate and treatment effect.

[0003] Human Claudin 18.2 (CLDN18.2) protein is one of the subtypes of Claudin 18, a member of the tight junction protein family. The human CLDN18 gene locus on chromosome 3q22 covers about 35 kb, which is divided into 6 exons and 5 introns. Selective splicing of exons 1a and 1b forms two subtypes of CLDN18.1 and CLDN18.2. The CLDN18.2 gene encodes 261 amino acids with a relative molecular mass of 27700 Da, forming a transmembrane protein composed of four transmembrane domains, an N-terminal and a C-terminal in the cytoplasm, and two extracellular loop domains.

[0004] CLDN18.2 protein is a highly selective biomarker, which is only abnormally expressed in differentiated gastric mucosal epithelial cells and various primary malignant tumors such as gastric cancer / gastroesophageal junction cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, and is not expressed in undifferentiated gastric stem cells, and the expression level is limited in normal healthy tissues. Generally, CLDN18.2 exists in the tight junctions of gastric mucosal cells, maintaining the barrier function of the gastric mucosa to prevent H+ leakage in gastric acid through the paracellular pathway. In normal tissues, CLDN18.2 exists in the tight junction complex, and its antigen epitopes are difficult to be recognized by intravenously injected antibodies or immune cells; after malignant transformation of gastric epithelial tissue, disturbance of cell polarity leads to exposure of CLDN18.2 antigen epitopes to the cell surface, enabling them to be recognized by specific antibodies or immune cells targeting CLDN18.2, which makes CLDN18.2 a unique biomarker and target for targeted immunotherapy of various tumors, especially gastric cancer and gastroesophageal junction cancer.

[0005] Zolbetuximab, a monoclonal antibody targeting CLDN18.2, specifically recognizes and binds to the first extracellular domain of CLDN18.2 protein with high affinity, without non-specific binding to any other claudin family members. Zolbetuximab mediates antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against CLDN18.2-expressing gastric and pancreatic cancer cell lines, leading to the lysis of CLDN18.2-expressing gastric cancer cells and the apoptosis of pancreatic cancer cells. A large number of clinical trial results show that Zolbetuximab combined with chemotherapy slows down tumor growth and metastasis, improves the progression-free survival and overall survival of subjects, enhances T cell infiltration and induces the release of inflammatory cytokines, with good risk / benefit characteristics.

[0006] Chimeric antigen receptor T (CAR-T) cell therapy is based on the engineering of T lymphocytes to express chimeric antigen receptors, which enable the modified T lymphocytes to directly recognize tumor cells without the influence of major histocompatibility complex. A number of clinical trials have shown that CAR-T therapy targeting CLDN18.2 is safe and well-tolerated in subjects with advanced malignant solid tumors, with good anti-tumor effect. However, due to the heterogeneity of tumors, the suppression of the tumor microenvironment on immune cells, and the different response rates of different patients to CAR-T cell therapy, these defects significantly reduce the efficacy of CAR-T cells; in addition, as a personalized therapy, the production process of CAR-T cells is complex and costly, which is one of the reasons restricting its application.

[0007] Antibody conjugated drugs (ADC) are a class of potent therapeutic drugs that target and deliver drugs to tumor cells to directly kill cancer cells, which are composed of recombinant monoclonal antibodies combined with cytotoxic payloads through degradable or non-degradable linkers. SYSA-1801 is an ADC drug targeting CLDN18.2, which is a fully humanized monoclonal antibody conjugated with monomethyl auristatin E (MMAE), a microtubulin inhibitor. In vitro and in vivo animal experiments show that SYSA-1801 can effectively target tumor cells through CLDN18.2 antibodies and trigger endocytosis, allowing small molecule toxins MMAE to enter tumor cells to achieve anti-tumor effect, with good safety for gastric cancer, pancreatic cancer and lung cancer; it has good efficacy in clinical trials. RC118 is also an ADC drug of recombinant humanized CLDN18.2 monoclonal antibody conjugated with MMAE, which has been used to treat locally advanced unresectable or metastatic malignant solid tumors.

[0008] Nanobody (Nb), also known as variable domain of heavy chain of heavy-chain antibody (VHH), has biological and pharmacological characteristics such as small volume, high solubility, high stability, strong tissue penetration ability, etc. Radionuclide conjugated drug (RDC), 68Ga-labeled nanobody 68Ga-NC-BCH, is a kind of PET / CT imaging agent based on single domain antibody. Clinical phase in vivo experiments and human experiments show that the preparation of 68Ga-NC-BCH is stable, and it shows good radiochemical performance, rapid blood clearance, high affinity for CLDN18.2 and high specific uptake in CLDN18.2 positive cells and xenotransplantation mouse models, and can be used as a supporting diagnostic tool for targeted CLDN18.2 treatment in tumors.

[0009] ADC drugs targeting CLDN18.2 have some advantages in avoiding drug resistance of monoclonal antibodies, but they also face common ADC-related obstacles. Future research will focus on screening monoclonal antibodies with high binding affinity to target antigens, minimal immunogenicity and appropriate anti-tumor activity half-life to avoid unnecessary side effects, including off-target toxicity and premature elimination from circulation due to immunogenicity. Therefore, it is of great significance to provide nanobodies targeting CLDN18.2 with high penetration ability, high stability, low immunogenicity and high affinity for targeted therapy of malignant tumors with high expression of CLDN18.2.

[0010] Heavy ion (such as carbon ion) radiotherapy has the advantages of superior dose distribution and strong killing ability to tumor cells (especially to radioresistant cells) due to its high linear energy transfer (LET) characteristics, but its single application may still be affected by the radioresistance of tumor cells. There is no report on the combination of nanobodies targeting CLDN18.2 and carbon ion irradiation for the treatment of gastric cancer. Based on this, the present application aims to provide a treatment scheme of nanobodies targeting CLDN18.2 combined with carbon ion irradiation, which can enhance the treatment effect of gastric cancer through synergistic effect, and provide a new strategy for clinical treatment of gastric cancer.

[0011] The information disclosed in this BACKGROUND section is only intended to increase an understanding of the general context in which the present application can be practiced. It is not admitted that any of the information provided herein constitutes prior art against the present application. SUMMARY

[0012] The application aims to overcome the poor effect of radiotherapy or targeted therapy alone in the existing gastric cancer treatment, and provides an application of a nano-antibody targeting CLDN18.2 in the preparation of a drug for combined radiotherapy for treating tumors or cancers. The nano-antibody is used in combination with carbon ion irradiation for gastric cancer treatment, which significantly improves the radiosensitivity of gastric cancer cells, inhibits tumor cell proliferation, promotes tumor cell death, and enhances the tumor inhibition effect in mice, and has a good application prospect.

[0013] The application provides an application of a nano-antibody targeting Claudin18.2 in the preparation of a drug for combined radiotherapy for treating tumors or cancers, wherein the nano-antibody comprises a heavy chain variable region, and the heavy chain variable region comprises CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 3.

[0014] In an embodiment of the application, the amino acid sequence of the nano-antibody is shown as SEQ ID NO. 4.

[0015] In an embodiment of the application, the radiotherapy is carbon ion irradiation.

[0016] In an embodiment of the application, the irradiation dose of the carbon ion irradiation is 4 Gy.

[0017] In an embodiment of the application, the parameters of the carbon ion irradiation are: energy 80.55 MeV / u, linear energy transfer value 50 KeV / μm, and irradiation dose 4 Gy.

[0018] In an embodiment of the application, the radiotherapy is performed before, simultaneously and / or after the administration of the nano-antibody.

[0019] In an embodiment of the application, the radiotherapy is performed once.

[0020] In an embodiment of the application, the administration dose of the nano-antibody is 1-3 mg / kg of body weight.

[0021] In an embodiment of the application, the administration object of the drug is a mammal.

[0022] In an embodiment of the application, the administration object of the drug is a rodent or a primate.

[0023] In an embodiment of the application, the administration object of the drug is a human.

[0024] In an embodiment of the application, the tumor or cancer is gastric cancer.

[0025] The second aspect of the present application provides a method for treating gastric cancer by using the above-mentioned nanobody in combination with carbon ion irradiation.

[0026] Compared with the prior art, the present application has the following technical effects: The present application adopts Claudin18.2 nanobody drug combined with carbon ion irradiation therapy, through the synergistic effect of the two, significantly inhibits the proliferation of Claudin18.2 positive gastric cancer cells, promotes cell death, and enhances the tumor inhibition effect in gastric cancer model animals, and improves the effectiveness of gastric cancer treatment. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the affinity curve graph of the anti-Claudin18.2 nanobody detected by SPRm 200; Figure 2 is a survival rate graph of Claudin18.2 nanobody combined with carbon ion irradiation of Claudin18.2 positive tumor cells; Figure 3 is a death rate graph of Claudin18.2 nanobody combined with carbon ion irradiation of Claudin18.2 positive tumor cells; Figure 4 is a graph of Claudin18.2 nanobody combined with carbon ion irradiation to inhibit tumor growth in mice. DETAILED DESCRIPTION

[0028] Unless otherwise specifically defined herein, all terms are to be given their broadest possible interpretation including synonyms and equivalents in connection with the applicable statutes. It must also be understood that the description and specific examples, while indicating preferred embodiments, are given by way of illustration only and not by way of limitation. Various substitutions and modifications can be made thereto by those skilled in the art, without departing from the broadest scope of the present application as set forth in the claims.

[0029] The technical solutions of the present application are described below through specific examples. It should be understood that the one or more steps mentioned in the present application do not exclude other methods and steps before and after the combination steps, or other methods and steps can be inserted between these explicitly mentioned steps. It should also be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. Unless otherwise specified, the numbering of each method step is only for the purpose of identifying each method step, and is not limited to the arrangement order of each method or the scope of the implementation of the present application. Changes or adjustments of the relative relationship, without substantial technical content changes, can also be considered as the implementation scope of the present application.

[0030] The raw materials and instruments used in the examples have no specific restrictions on their sources, and can be purchased on the market or prepared according to the conventional methods well known to those skilled in the art.

[0031] As used herein, the terms "single domain antibody", "heavy chain variable region domain of heavy chain antibody", "VHH", "nanobody", "single variable domain" are used interchangeably and all refer to a single domain polypeptide or protein that specifically recognizes and binds to an antigen. Single domain antibody is the variable region of a heavy chain antibody. Typically, a single domain antibody contains three CDRs and four FRs. Single domain antibody is the smallest functional antigen binding fragment. Usually, after obtaining an antibody naturally lacking light chain and heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0032] A "heavy chain antibody" as described herein is an antibody derived from a Camelid or a Chondrichthyan. Compared to the above 4-chain antibody, a heavy chain antibody lacks light chain and heavy chain constant region 1 (CH1) and contains only 2 heavy chains consisting of a variable region (VHH) and other constant regions, the variable region is connected to the constant region by a hinge-like region structure. Each heavy chain of a Camelid heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of a Chondrichthyan heavy chain antibody contains 1 variable region and 5 constant regions (CH1-CH5). The antigen binding fragment of a heavy chain antibody includes VHH and single chain heavy chain antibody. By fusing with the constant region of human IgG Fc, a heavy chain antibody can have CH2 and CH3 of human IgG Fc.

[0033] A binding molecule comprising two or more single domain antibodies is a multivalent single domain antibody; a binding molecule comprising two or more single domain antibodies of different specificity is a multispecific single domain antibody. A multivalent single domain antibody or a multispecific single domain antibody connects multiple single domain antibodies through a linker. The linker usually consists of 1-15 amino acids selected from G and S.

[0034] Herein, heavy chain antibody and antibody are intended to distinguish different combination ways of antibodies. Due to the similarity of the structures of both, the following structural description of antibodies applies to heavy chain antibodies as well, except for the light chain.

[0035] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of an antibody. The variable domains of the heavy and light chains can be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding sites.

[0036] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies, and are used to confer antigen binding specificity and particular antigenic specificity of a particular antibody. However, the variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in three segments called hypervariable regions (HVRs) both in the light chain and the heavy chain variable domains, i.e., HCDR1, HCDR2, HCDR3 in the variable region of the heavy chain (which can be referred to as CDR1, CDR2, CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 in the variable region of the light chain. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of the naturally occurring heavy and light chains each comprise four FR regions, FR1, FR2, FR3, and FR4, mostly from beta-sheet formation, connected by three HVRs. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies. Generally, the structure of a light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of a heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains do not participate directly in binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0037] The "Fc region" (fragment, crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of each of the two heavy chains; the Fc region of IgM and IgE comprises three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. While the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino-terminus at heavy chain position C226 or P230 to the carboxy-terminus of the heavy chain, with numbering according to the EU index as in Kabat. Each heavy chain of a camelid heavy chain antibody comprises one variable region (VHH) and two constant regions (CH2 and CH3). By fusion with the constant region of human IgG Fc, the heavy chain antibody can have CH2 and CH3 of human IgG Fc.

[0038] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding and / or variable region of the intact antibody. Antibody fragments preferably are antigen binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragments capable of increasing the half-life of the antibody by chemical modification or by incorporation into a liposome. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, which contains the antibody's epitope recognition site for its Fc receptors. Fab fragments consist of an entire light chain and the variable region of a heavy chain, and one heavy chain constant region domain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen combining site. Pepsin treatment yields a F(ab')2 fragment that roughly corresponds to two disulfide linked Fab fragments having different antigen binding activities and that is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having an additional few residues at the carboxy terminus of the CH1 domain including the one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined in the Fc region, which is also the region recognized by Fc receptors (FcR) found on certain types of cells. Antigen binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies.

[0039] "Fv" is the minimum antibody fragment that contains a complete antigen- recognition and binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute most of the antigen- binding specificity and diversity of an antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. "Single-chain Fv" also is abbreviated "sFv" or "scFv" are antibody fragments that comprise the VHand VLdomains of antibody, linked by a synthetic linker from one to the other. Preferably, the sFv polypeptides are expressed from a recombinant DNA molecule. The sFv is often what is referred to as a "dual chain" antibody. Fv of heavy chain antibodies is VHH.

[0040] Antibodies herein also include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0041] Herein, a "binding molecule" of an antigen is a protein that specifically binds to an antigen, including but not limited to, antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affibodies, target-binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines. Herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single-chain molecules, as well as antibody fragments, particularly antigen-binding fragments, such as Fab, F(ab')2, and Fv. Herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0042] A "Claudin 18.2 binding molecule" described herein comprises an anti-Claudin 18.2 single-domain antibody, the complementarity determining regions (CDRs) of which comprise CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence set forth in SEQ ID NO: 1, CDR2 comprises the sequence set forth in SEQ ID NO: 2, and CDR3 comprises the sequence set forth in SEQ ID NO: 3.

[0043] "Kd" as used herein, i.e., Koff, represents the dissociation rate constant, representing the speed of dissociation between molecules, and has a unit of S -1 In the SPRm 200 assay affinity experiment, the greater the Koff represents the slower the rate of RU drop, and the flatter the curve slope. Therefore, high affinity is manifested as fast association and slow dissociation.

[0044] "Ka" as used herein, i.e., Kon, is the association rate constant, representing the speed of association between molecules, and has a unit of M -1 ∙S -1 In the SPRm 200 assay affinity experiment, the greater the Kon represents the shorter the time to reach maximum RU, and the steeper the curve slope.

[0045] "KD" as used herein represents the dissociation constant (KD), a specific type of equilibrium constant that measures the tendency of a larger object to separate (dissociate) from a smaller component, is the reciprocal of the association constant, and is expressed in units of mol / L (M) or nmol / L (nM). The smaller the KD value, the stronger the binding ability of the two substances. KD = Kd / Ka.

[0046] Example 1 This embodiment constructs a phage nanobody library and uses ELISA and molecular docking to perform preliminary screening, and the specific steps are as follows: (1) Construction of phage nanobody library After immunizing the alpaca 5 times with the Claudin18.2 virus (Human Claudin 18.2 Protein-VLP, Kailv Biological) expressed by HEK293 cells, and detecting the serum titer by ELISA, peripheral blood was extracted to isolate lymphocytes, total RNA was extracted, and then cDNA was reverse transcribed, and then the VHH gene was amplified by nested PCR. The steps of obtaining IgG2 and IgG3 heavy chain variable region sequences (heavy chain variable region VHH) by nested PCR are as follows: 1) design a pair of specific nested outer primers, use cDNA as a template for first-round PCR amplification, and amplify the llama heavy chain antibody gene, with product sizes of 750 bp and 900 bp, respectively; cut the gel and recover the 750 bp PCR product by DNA gel electrophoresis; 2) design nested inner primers, use the 750 bp first-round PCR product as a template for second-round PCR amplification, and the amplification product is the heavy chain antibody variable region VHH fragment, with a product size of 500 bp; use a PCR product purification kit to purify and recover the second-round PCR product. The VHH target gene and the vector pComb3xss are digested with SfiI, and the digested VHH and pComb3xss are ligated with T4 DNA ligase, and then the VHH target gene is cloned into the pHEN1 phagemid, electroporated into TG1 competent cells, and a VHH gene library is constructed.

[0047] (2) Screening of phage nanobody library The library screening was performed by phage display technology, and the specificity and binding strength of each clone were ensured by precise monoclonal phage ELISA, to obtain phages that can specifically bind to Claudin18.2 virus and Claudin18.2 overexpression stable cell strains but not to Claudin18.1 recombinant protein, and obtain several Claudin18.2 polypeptide nanobody sequences by sequencing; then, through Alpha Fold 3 software, Claudin18.2 polypeptide nanobody sequences that specifically bind to the extracellular domain of Claudin18.2 protein but not to the extracellular domain of Claudin18.1 protein are obtained by simulating molecular docking. The intersection of the nanobody sequences obtained by the two-step experiment is identified as the nanobody amino acid sequence that specifically binds to Claudin18.2, as shown in SEQ ID NO. 4 (QLQLVESGGGLVQPGGSLRLSCAASGSALDYYHITWIRQAPGKEREGVSCILNSGGSLYADSVKGRFSISRDKNTVYLQMNSLTPEDTAVYYCAADYVPGKFNFGCSLYIQRAYDLWGQGTQVTVSSEPKTPKPQDGQAGQ), and the screening number is ICV2-21. The nanobody comprises a heavy chain variable region, the heavy chain variable region comprises CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO. 1 (YYHIT); the amino acid sequence of CDR2 is shown in SEQ ID NO. 2 (CILNSGGSLYADSVKG); and the amino acid sequence of CDR3 is shown in SEQ ID NO. 3 (DYVPGKFNFGCSLYIQRAYDL).

[0048] Example 2 In this embodiment, the anti-Claudin18.2 nanobody (ICV2-21) screened in Example 1 is prokaryotic expressed and purified, and the antibody affinity is determined. The specific steps are as follows: (1) Construct the ICV2-21 VHH sequence prokaryotic expression vector with His tag.

[0049] Nde I and Xho I restriction sites were added at both ends of the nanobody (ICV2-21) protein coding gene sequence (SEQ ID NO. 5: CAGCTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTTCAGCCGGGTGGTAGTCTGCGCCTGAGCTGTGCAGCCAGTGGCAGTGCACTGGATTATTATCATATTACCTGGATTCGTCAGGCACCGGGTAAAGAACGTGAAGGTGTTAGCTGCATTCTGAATAGCGGTGGCAGTCTGTATGCCGATAGCGTGAAAGGCCGCTTTAGCATTAGTCGCGATAAAAATACCGTGTATCTGCAGATGAATAGTCTGACCCCGGAAGATACCGCAGTGTATTATTGCGCCGCCGATTATGTGCCGGGTAAATTCAATTTTGGCTGTAGCCTGTATATTCAGCGCGCATACGATCTGTGGGGCCAGGGTACACAGGTTACCGTTAGCAGTGAACCGAAAACCCCGAAACCGCAGGATGGTCAGGCCGGTCAG), and the nucleotide sequence (CAGCTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTTCAGCCGGGTGGTAGTCTGCGCCTGAGCTGTGCAGCCAGTGGCAGTGCACTGGATTATTATCATATTACCTGGATTCGTCAGGCACCGGGTAAAGAACGTGAAGGTGTTAGCTGCATTCTGAATAGCGGTGGCAGTCTGTATGCCGATAGCGTGAAAGGCCGCTTTAGCATTAGTCGCGATAAAAATACCGTGTATCTGCAGATGAATAGTCTGACCCCGGAAGATACCGCAGTGTATTATTGCGCCGCCGATTATGTGCCGGGTAAATTCAATTTTGGCTGTAGCCTGTATATTCAGCGCGCATACGATCTGTGGGGCCAGGGTACACAGGTTACCGTTAGCAGTGAACCGAAAACCCCGAAACCGCAGGATGGTCAGGCCGGTCAG) was recombined into Nde I and Xho I endonuclease linearized pET-22b vector, and DH5α competent cells were transformed. CAT ATG CAGCTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTTCAGCCGGGTGGTAGTCTGCGCCTGAGCTGTGCAGCCAGTGGCAGTGCACTGGATTATTATCATATTACCTGGATTCGTCAGGCACCGGGTAAAGAACGTGAAGGTGTTAGCTGCATTCTGAATAGCGGTGGCAGTCTGTATGCCGATAGCGTGAAAGGCCGCTTTAGCATTAGTCGCGATAAAAATACCGTGTATCTGCAGATGAATAGTCTGACCCCGGAAGATACCGCAGTGTATTATTGCGCCGCCGATTATGTGCCGGGTAAATTCAATTTTGGCTGTAGCCTGTATATTCAGCGCGCATACGATCTGTGGGGCCAGGGTACACAGGTTACCGTTAGCAGTGAACCGAAAACCCCGAAACCGCAGGATGGTCAGGCCGGTCAG CTC GAG ) was recombined into Nde I and Xho I endonuclease linearized pET-22b vector, and DH5α competent cells were transformed.

[0050] The single colony was picked from the transformed plate, and cultured at 37°C overnight. The PCR sample loading system was shown in Table 1. The PCR conditions were as follows: 95°C for 3 min; 95°C for 30 s, 50°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 5 min, and 4°C for storage. The single colony with expected band size was sent to a sequencing company for sequencing identification, and the single colony with correct sequencing result was stored.

[0051] Table 1: PCR sample loading system

[0052] (2) ICV2-21 VHH prokaryotic expression and purification Transformation: The plasmid with correct sequencing result was extracted, and was added to E. coli competent BL21 (DE3) for ice bath for 30 min, heat shock at 42°C for 60 s, and then ice bath for 3 min. 500 μl of LB medium was added and incubated at 37°C for 30 min. The plate containing 100 μg / ml ampicillin was coated, and incubated at 37°C overnight.

[0053] PCR identification: The single colony was picked from the transformed plate, and cultured at 37°C overnight. The PCR sample loading system and reaction conditions were as described above. The single colony with expected band size was detected by agarose gel electrophoresis, and was stored.

[0054] Induced expression: The BL21 (DE3) containing ICV2-21 VHH plasmid was cultured at 37°C. When the OD value reached 0.6, IPTG was added to a final concentration of 1 mM, and the culture was continued to be incubated at 37°C overnight to induce the expression of the fusion protein. After the expression was completed, the culture was centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. The bacterial cells were collected.

[0055] Protein purification: Crushing of bacterial cells: The bacterial cells were dissolved with binding buffer (0.5 mol / L NaCl, 5 mmol / L imidazole, 20 mmol / L Tris-Cl, pH 8.0), and were ultrasonically broken. The supernatant crude protein was collected by centrifugation at 10000 rpm at 4°C for 10 min. Equilibrium: The Ni-NTA filler was packed into a column, and the column was washed with binding buffer to equilibrate the column; Binding: The crude protein was naturally flowed through the equilibrated column filler, and the effluent was collected; Equilibrium: The Ni-NTA column was washed with 10 times the column volume of binding buffer; Washing: The Ni-NTA column was washed with 6 column volumes of washing buffer (0.5 mol / L NaCl, 60 mmol / L imidazole, 20 mmol / L Tris-Cl, pH 8.0), and the effluent was collected; Elution: The column was eluted with elution buffer (0.5 mol / L NaCl, 250 mmol / L imidazole, 20 mmol / L Tris-Cl, pH 8.0), and the effluent was collected.

[0056] Polyacrylamide gel electrophoresis detection: A polyacrylamide gel was prepared, and samples of the crude protein and the effluent components were prepared for polyacrylamide gel electrophoresis detection. The results showed that the fusion protein was successfully purified after purification, and SDS-PAGE electrophoresis analysis showed that there were obvious bands near the theoretical molecular weight, which indicated that the fusion protein was successfully purified.

[0057] Dialysis: The purified components were dialyzed into a protein storage buffer (PBS, 300 mM NaCl, 10% glycerol, 0.3% SKL, pH 7.4), concentrated, filtered, and sterilized; the protein concentration was quantified using a protein quantification kit, the antibody purity was determined by SEC-HPLC, and the protein was stored at -80°C.

[0058] In addition, the affinity of the purified VHH antibody was determined by SPRm 200 in this embodiment. SPRm is a biological analysis sensing technology developed based on surface plasmon resonance (SPR), which can detect and track the entire change process of the binding and dissociation of molecules in the solution with the molecules fixed on the chip surface, record it in the form of a sensing graph, and provide kinetic and affinity data.

[0059] During the determination process, first, the cell polymer well was installed on the J1 chip, and the chip surface was coated with protease; then, under sterile conditions, the cultivated Claudin18.2 positive cells were transferred to the cell polymer well chip for culture for 1-2 days, and the cell coverage was observed to be 30%-70%, and then the cells were fixed with 4% PFA; PBST was used as the buffer, and different concentrations of recombinant nanobody protein solution were used as the mobile phase, and the determination results are shown in Figure 1 The affinity of the antibody reached the sub-micromolar level, KD = 1.979E-07 M, Ka = 4.263E+04 M -1 ∙S -1 , Kd = 9.528E-03 S -1 .

[0060] Example 3 The present embodiment carries out cell proliferation experiment on ICV2-21 nanobody combined with carbon ion irradiation constructed in embodiment 2, including the following steps: (1) Claudin18.2 positive tumor cell inoculation NCI-N87 cells stably overexpressing Claudin18.2 (NCI-N87 CLDN18.2 ) as Claudin18.2 positive tumor cells, were divided into 4 groups (IgG control group, ICV2-21 nanobody group, IgG+carbon ion irradiation group, ICV2-21 nanobody+carbon ion irradiation group), each group with 3 parallels, each parallel inoculated 6×10 5 NCI-N87 CLDN18.2 cells in a 35mm cell culture dish, and cultured in RPMI-1640+10%FBS+1% penicillin-streptomycin double-antibiotic culture medium at 37℃, 5%CO2 incubator for 24 hours until the cells adhered.

[0061] (2) Nanobody combined with carbon ion irradiation treatment IgG control group, ICV2-21 nanobody group, IgG+carbon ion irradiation group, and ICV2-21 nanobody+carbon ion irradiation group were added with 4μg / mL of IgG or ICV2-21 nanobody, respectively, and cultured at 37℃, 5%CO2 incubator for 24 hours, then the IgG+carbon ion irradiation group and the ICV2-21 nanobody+carbon ion irradiation group were subjected to carbon ion irradiation (energy 80.55MeV / u, linear energy transfer value 50KeV / μm, irradiation dose 4Gy) at the Institute of Modern Physics, Chinese Academy of Sciences. After irradiation, the cells of different treatment groups were immediately trypsinized and collected, and the cell concentration was adjusted to 1×10 6 / ml, and inoculated in a 96-well plate at a density of 8000 cells per well.

[0062] (3) Detection method The medium was removed at 24h, 48h and 72h, 110μL of medium containing 10%CCK-8 was added to each well, and incubated at 37℃, 5%CO2 incubator for 2 hours, and the absorbance was measured at wavelength 450nm by enzyme label instrument.

[0063] (4) Cell survival rate calculation formula:

[0064] The results are as follows Figure 2As shown, after 24 hours of treatment, the cell survival rate of the IgG control group was 100.00±0.13%, the cell survival rate of the ICV2-21 nanobody group was 79.47±1.97%, the cell survival rate of the IgG+carbon ion irradiation group was 71.26±1.85%, and the cell survival rate of the ICV2-21 nanobody+carbon ion irradiation group was 58.90±1.32%; after 48 hours of treatment, the cell survival rate of the IgG control group was 100.00±0.61%, the cell survival rate of the ICV2-21 nanobody group was 67.70±3.98%, the cell survival rate of the IgG+carbon ion irradiation group was 62.54±4.44%, and the cell survival rate of the ICV2-21 nanobody+carbon ion irradiation group was 52.14±0.80%; after 72 hours of treatment, the cell survival rate of the IgG control group was 100.00±1.56%, the cell survival rate of the ICV2-21 nanobody group was 82.58±1.15%, the cell survival rate of the IgG+carbon ion irradiation group was 55.78±1.31%, and the cell survival rate of the ICV2-21 nanobody+carbon ion irradiation group was 46.35±0.89%. The results showed that the use of ICV2-21 nanobody alone or carbon ion irradiation alone can inhibit tumor cell proliferation, and the combination of ICV2-21 nanobody and carbon ion irradiation can further enhance the inhibitory effect. Notably, the cell survival rate of the ICV2-21 nanobody group showed a trend of first decreasing and then increasing over time, and after 72 hours, the cell survival rate of the ICV2-21 nanobody group was 82.58±1.15%, which was not only higher than the value after 48 hours, but also higher than the value after 24 hours, indicating that the tumor cells may have developed a certain degree of drug resistance to ICV2-21 nanobody. However, after combined carbon ion irradiation, the cell survival rate of the ICV2-21 nanobody+carbon ion irradiation group after 72 hours was 46.35±0.89%, which was lower than 52.14±0.80% after 48 hours and 55.78±1.31% of the IgG+carbon ion irradiation group, indicating that the combination of ICV2-21 nanobody and carbon ion irradiation not only overcame the problem of drug resistance, but also enhanced the therapeutic effect of carbon ion irradiation.

[0065] Example 4 In this embodiment, cell death rate detection was performed on the ICV2-21 nanobody combined with carbon ion irradiation constructed in Example 2, including the following steps: (1) Claudin18.2 positive tumor cell inoculation NCI-N87 cells stably overexpressing Claudin18.2 (NCI-N87 CLDN18.2) As Claudin18.2 positive tumor cells, they were divided into 4 groups (IgG control group, ICV2-21 nanobody group, IgG + carbon ion irradiation group, ICV2-21 nanobody + carbon ion irradiation group), 3 parallels in each group, 6x10 5 NCI-N87 CLDN18.2 cells in each parallel were inoculated in a 35mm cell culture dish and cultured in RPMI-1640 + 10% FBS + 1% penicillin-streptomycin double-antibiotic medium at 37°C in a 5% CO2 incubator for 24 hours until the cells adhered.

[0066] (2) Nanobody combined with carbon ion irradiation treatment IgG control group, ICV2-21 nanobody group, IgG + carbon ion irradiation group, ICV2-21 nanobody + carbon ion irradiation group were added with 4 μg / mL of IgG or ICV2-21 nanobody, and then cultured at 37°C in a 5% CO2 incubator for 24 hours. Then, the IgG + carbon ion irradiation group and the ICV2-21 nanobody + carbon ion irradiation group were subjected to carbon ion irradiation (energy 80.55 MeV / u, linear energy transfer value 50 KeV / μm, irradiation dose 4 Gy) at the Institute of Modern Physics, Chinese Academy of Sciences. After irradiation, the cells in different treatment groups were immediately trypsinized and collected, and the cell concentration was adjusted to 1x10 6 / ml, and then inoculated in a 24-well plate at a cell density of 1x10 5 / ml per well, and cultured for another 48 hours.

[0067] (3) Detection method At the end of the culture, the culture medium and trypsinized cells in each treatment group were collected, combined, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were washed twice with pre-cooled PBS, and then resuspended in staining buffer. 5 μL of PI (propidium iodide) and 10 μL of Annexin V-FITC (fluorescein isothiocyanate labeled annexin V) were added to each sample, and the samples were stained in an ice bath for 15 minutes. The samples were detected by flow cytometry, and the data obtained by flow cytometry were analyzed and processed using Flowjo software.

[0068] (4) The formula for calculating cell death rate is: Cell death rate = cell early apoptosis rate (Q3) + cell late apoptosis / death rate (Q2) The results are as follows: Figure 3As shown, the cell death rate of the IgG control group was 20.91 ± 1.14%, the cell death rate of the ICV2-21 nanobody group was 32.83 ± 3.91%, the cell death rate of the IgG + carbon ion irradiation group was 38.27 ± 0.42%, and the cell death rate of the ICV2-21 nanobody + carbon ion irradiation group was 50.58 ± 2.86%. The results showed that ICV2-21 nanobody or carbon ion irradiation can directly promote tumor cell death, and ICV2-21 nanobody combined with carbon ion irradiation can further increase the tumor cell death rate.

[0069] Example 5 This example carries out in vivo tumor inhibition experiment of ICV2-21 nanobody combined with carbon ion irradiation constructed in Example 2, and the specific steps are as follows: (1) Experimental animals 4-6 week old male BALB / c nude mice were selected and temporarily raised in the SPF level experimental animal center for 1 week, with temperature of 20-24°C, humidity of 40-70%, and light and dark cycle of 12 hours light / 12 hours dark. The experiment was approved by the ethics committee of the institution, approval number: 2023(013).

[0070] (2) Claudin18.2 positive tumor cell inoculation NCI-N87 cells overexpressing Claudin18.2 (NCI-N87 CLDN18.2 ) were used as Claudin18.2 positive tumor cells, cultured in a 37°C, 5% CO2 incubator, passaged every 2-3 days, and log phase cells were taken. Cells were trypsinized, resuspended in complete culture medium and adjusted to a cell concentration of 5 × 10 7 NCI-N87 CLDN18.2 cells were mixed with Matrigel matrix glue on ice at a volume ratio of 1:1.

[0071] (3) Construction of subcutaneous tumor-bearing model The right side of the BALB / c nude mouse was disinfected with alcohol and cotton ball, the skin pad was picked up with a 1 mL syringe needle, and 100 µL of cell suspension was slowly injected; the needle was left in the subcutaneous for 3 s before being pulled out to prevent leakage.

[0072] (4) ICV2-21 nanobody combined with carbon ion irradiation Carbon ion irradiation: NCI-N87 CLDN18.2Two weeks after cell culture, tumor-bearing BALB / c nude mice were anesthetized with tribromoethanol to reduce inter-group differences. The mice were then secured with adhesive tape to perforated lead plates, exposing the tumors. The lead plates ensured that all areas of the mice, except for the tumors, were completely shielded from radiation. The mice were then irradiated using a carbon ion therapy system with a carbon ion beam energy of 80.55 MeV / u, a linear energy transfer value of 50 keV / μm, and an irradiation dose of 4 Gy. After irradiation, the mice were placed in a 37°C constant temperature room to prevent hypothermia. Once the mice regained consciousness, they were administered drugs according to their respective groups.

[0073] Preparation of IgG and ICV2-21 nanobodies: Dilute with sterile PBS to 300 μg / mL, filter through a 0.22 µm filter membrane, store at 4°C protected from light, and prepare fresh before use.

[0074] Tail vein injection: Nude mice were placed in a 37°C heated box for 5 minutes to dilate the tail vein; the tail was wiped with alcohol, and a 1 mL syringe was inserted into the vein parallel to the tip third of the tail. After confirming blood return, about 100 µL of antibody (1.5 mg / kg body weight) was slowly injected; immediately after needle removal, hemostasis was achieved by applying pressure with a dry cotton ball for 30 seconds.

[0075] Frequency and cycle: Repeat injection every 3 days, for a total of 5 injections (on days 0, 3, 6, 9, and 12).

[0076] (5) Detection methods Observe the patient's mental state, diet, activity level, and weight daily. Once the tumor is visible, measure the tumor's long diameter (a) and short diameter (b) every 3 days, using the formula V = 0.5 × a × b. 2 Estimate volume. Ethical endpoint: Single diameter ≥ 20 mm, or volume ≥ 1500 mm². 3 If the patient experiences a weight loss of ≥20%, or develops ulcers or necrosis, immediate euthanasia will be administered. Experimental endpoint: Day 33 after carbon ion irradiation, death will occur due to overdose anesthesia, and the tumor tissue will be completely removed.

[0077] The results are as follows Figure 4 As shown, on day 33, the tumor volume in the IgG control group was 1060.84 ± 233.03 mm. 3 The tumor volume in the ICV2-21 nanobody group was 656.06 ± 56.54 mm. 3 The tumor volume in the IgG+ carbon ion irradiation group was 432.99±68.38 mm. 3 The tumor volume in the ICV2-21 nanobody + carbon ion irradiation group was 261.92±35.53 mm. 3 The results showed that tumor volume was significantly reduced by either nanobody or carbon ion irradiation, and the combination of nanobody and carbon ion irradiation had a more significant effect in inhibiting tumor growth.

[0078] In addition, we also pay attention to the change of mouse weight, and the fluctuation of mouse weight in each group has no obvious difference in the whole experiment.

[0079] In conclusion, the anti-Claudin18.2 nanobody prepared by the application can specifically bind to Claudin18.2, and the combination of carbon ion irradiation can significantly inhibit the proliferation of gastric cancer cells and promote cell death compared with single treatment; in the subcutaneous tumor-bearing nude mouse animal model, the tumor inhibition rate of the combination therapy is higher, which can break through the "efficacy ceiling" of single treatment method, reduce the dosage of administration, and the safety is not obviously affected, indicating that the application provides a new scheme for the precision treatment of gastric cancer, and can be popularized to the combined treatment research of other solid tumors, and has important clinical transformation value.

[0080] The foregoing description of specific exemplary embodiments of the application is intended to be illustrative only and is not intended to limit the application to the precise forms described. Many modifications and variations are possible in light of the above teachings without departing from the spirit or essential characteristics of the application. The exemplary embodiments were chosen and described in order to explain the principles of the application and its practical application and to allow others skilled in the art to understand the application for various exemplary embodiments with various modifications being applicable. The scope of the application is intended to be defined by the claims and their equivalents.

Claims

1. Use of a Claudin 18.2-targeting nanobody in the preparation of a medicament for treating a tumor or a cancer in combination with radiotherapy, wherein the nanobody comprises a heavy chain variable region comprising a CDR1, a CDR2 and a CDR3, characterized in that, the amino acid sequence of the CDR1 is shown as SEQ ID NO. 1; the amino acid sequence of the CDR2 is shown as SEQ ID NO. 2; and the amino acid sequence of the CDR3 is shown as SEQ ID NO.

3. the amino acid sequence of the nanobody is shown as SEQ ID NO.

4.

2. Use according to claim 1, characterized in that, the radiotherapy is carbon ion irradiation.

3. Use according to claim 1, characterized in that, the irradiation dose of the carbon ion irradiation is 4 Gy.

4. Use according to claim 3, characterized in that, the parameters of the carbon ion irradiation are: energy 80.55 MeV / u, linear energy transfer value 50 KeV / μm, and irradiation dose 4 Gy.

5. Use according to claim 4, characterized in that, the radiotherapy is performed before, simultaneously with and / or after the administration of the nanobody.

6. Use according to claim 1, characterized in that, the number of treatments of the radiotherapy is 1.

7. The use according to claim 1, characterized in that, the administration dose of the nanobody is 1-3 mg / kg body weight.

8. The use according to claim 1, characterized in that, the administration subject of the medicament is a human.

9. The use according to claim 1, characterized in that, the tumor or the cancer is gastric cancer.

10. The use according to claim 1, characterized in that, ​

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