Targeting Claudin18.2 nano antibody as well as related product and application thereof

Abstract

The invention discloses a Claudin18.2-targeting nano antibody as well as a related product and application thereof, 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 can be specifically combined with a Claudin18.2 antigen, so that the affinity is relatively high; when being used as a monoclonal antibody drug, the compound can inhibit clone formation, migration and invasion of Claudin18.2 positive tumor cells, and has very good targeted therapeutic activity on Claudin18.2 positive tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a nanobody targeting Claudin18.2 and related products and applications. Background Technology

[0002] Cancer remains a serious threat to human health, especially recurrent and metastatic cancer. Targeted therapy and immunotherapy have brought benefits to the clinical treatment of many types of cancer, but further improvements are still needed to enhance overall response rates and treatment outcomes.

[0003] The human Claudin18.2 (CLDN18.2) protein is a subtype of Claudin18, a member of the tight junction protein family. The human CLDN18 gene locus on chromosome 3q22 covers approximately 35 kb and consists of 6 exons and 5 introns. Alternative splicing of exons 1a and 1b forms two subtypes: CLDN18.1 and CLDN18.2. The CLDN18.2 gene encodes 261 amino acids with a relative molecular mass of 27,700 Da, forming a transmembrane protein composed of four transmembrane domains, an N-terminal and a C-terminal domain in the cytoplasm, and two extracellular loop domains.

[0004] CLDN18.2 protein is a highly selective biomarker, aberrantly expressed only in differentiated gastric mucosal epithelial cells and various primary malignant tumors, such as gastric / gastroesophageal junction cancer, breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer. It is not expressed in undifferentiated gastric stem cells and has limited expression levels in normal healthy tissues. Normally, CLDN18.2 is present at tight junctions of gastric mucosal cells, maintaining the barrier function of the gastric mucosa and preventing H+ leakage into gastric acid via the paracellular pathway. In normal tissues, CLDN18.2 is located in tight junction complexes, and its antigenic epitopes are difficult for intravenously injected antibodies or immune cells to recognize. However, after malignant transformation of gastric epithelial tissue, perturbation of cell polarity leads to the exposure of the CLDN18.2 antigenic epitope on the cell surface, making it recognizable by specific antibodies or immune cells targeting CLDN18.2. These characteristics make 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 the CLDN18.2 protein with high affinity, without non-specific binding to any other claudin family members. Against gastric and pancreatic cancer cell lines expressing CLDN18.2, Zolbetuximab mediates antibody-dependent cytotoxicity and complement-dependent cytotoxicity, leading to lysis of CLDN18.2-expressing gastric cancer cells and apoptosis in pancreatic cancer cells. Numerous clinical trial results demonstrate that Zolbetuximab in combination with chemotherapy slows tumor growth and metastasis, improves progression-free survival and overall survival, enhances T cell infiltration, and induces the release of inflammatory cytokines, exhibiting a favorable risk / benefit profile.

[0006] Chimeric antigen receptor T (CAR-T) cell therapy is based on engineering T lymphocytes to express chimeric antigen receptors, enabling the modified T lymphocytes to directly recognize tumor cells without being affected by the major histocompatibility complex. Multiple clinical trials have demonstrated that CAR-T therapy targeting CLDN18.2 is safe and well-tolerated in patients with advanced malignant solid tumors, exhibiting good anti-tumor effects. However, tumor heterogeneity, the inhibitory effect of the tumor microenvironment on immune cells, and varying response rates to CAR-T cell therapy among different patients significantly reduce the efficacy of CAR-T cells. Furthermore, as a personalized therapy, the complex and costly production process of CAR-T cells is also a limiting factor in its application.

[0007] Antibody-drug conjugates (ADCs) consist of a recombinant monoclonal antibody and a cytotoxic payload linked by a degradable or non-degradable linker. They are potent therapeutic agents that target and deliver drugs directly to tumor cells, killing cancer cells. SYSA-1801 is a fully humanized monoclonal antibody targeting CLDN18.2, conjugated with the microtubule inhibitor monomethyl auristatin E (MMAE). In vitro and in vivo animal studies have shown that SYSA-1801 effectively targets tumor cells via the CLDN18.2 antibody and triggers endocytosis, allowing the small molecule toxin MMAE to enter tumor cells and achieve an anti-tumor effect. It exhibits good safety in gastric, pancreatic, and lung cancers and demonstrates good efficacy in clinical trials. RC118 is also a recombinant humanized CLDN18.2 monoclonal antibody conjugated with MMAE and has been used to treat locally advanced unresectable or metastatic malignant solid tumors.

[0008] Antibody-addictive drugs (ADCs) targeting CLDN18.2 offer some advantages in avoiding monoclonal antibody resistance, but they also face common ADC-related obstacles. Future research will focus on screening monoclonal antibodies with high binding affinity to the target antigen, minimal immunogenicity, and an appropriate antitumor activity half-life to avoid unnecessary side effects, including off-target toxicity and premature elimination from circulation due to immunogenicity. Therefore, providing nanobodies that target CLDN18.2 with high penetration, high stability, low immunogenicity, and high affinity is of great significance for targeted therapy of malignant tumors that highly express CLDN18.2.

[0009] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a nanobody targeting Claudin18.2, as well as related products and applications. The nanobody possesses high affinity and effectively inhibits the proliferation and invasion of Claudin18.2-positive tumor cells, showing promising application prospects in tumor therapy.

[0011] The first aspect of the present invention provides a nanobody targeting Claudin18.2, which includes a heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO.1; the amino acid sequence of CDR2 is shown in SEQ ID NO.2; and the amino acid sequence of CDR3 is shown in SEQ ID NO.3.

[0012] In one embodiment of the present invention, the amino acid sequence of the nanobody is shown in SEQ ID NO.4.

[0013] A second aspect of the present invention provides a nucleic acid molecule that encodes the above-mentioned nanobody.

[0014] In one embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.5.

[0015] A third aspect of the present invention provides an expression vector comprising the aforementioned nucleic acid molecule. The expression vector may be a prokaryotic expression vector or a eukaryotic expression vector.

[0016] A fourth aspect of the present invention provides a host cell comprising the above-described expression vector, or having the above-described nucleic acid molecules integrated into its genome, or expressing the above-described nanobody.

[0017] A fifth aspect of the present invention provides a pharmaceutical composition comprising the above-described nanobody, the above-described nucleic acid molecule, the above-described expression vector, or the above-described host cell, and at least one pharmaceutically acceptable excipient.

[0018] A sixth aspect of the present invention provides a detection kit comprising the above-mentioned nanobody.

[0019] The seventh aspect of the present invention provides the use of the above-mentioned nanobody, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned host cell, or the above-mentioned pharmaceutical composition in the preparation of a tumor therapeutic drug, wherein the tumor is a tumor that highly expresses Claudin18.2.

[0020] The eighth aspect of the present invention provides the application of the above-described nanobody in the preparation of Claudin18.2 detection reagent.

[0021] In one embodiment of the present invention, the detection reagent is used for ELISA detection, chemiluminescent immunoassay (CLIA), immunofluorescence (IF) detection, immunohistochemistry (IHC) detection, flow cytometry detection, or circulating tumor cell detection.

[0022] Compared with the prior art, the technical effects achieved by the present invention are as follows: This invention uses Claudin18.2 virus-like virus as an antigen to immunize alpacas in order to construct a VHH phage library. After forward and reverse screening using Claudin18.2 virus-like virus and Claudin18.2 positive cells, and reverse screening using Claudin18.1 recombinant expression protein, as well as Alpha Fold 3-simulated molecular docking, anti-Claudin18.2 nanobodies were obtained. These nanobodies can specifically bind to the Claudin18.2 antigen with high affinity. When used as an antibody drug, they have good targeting effects and inhibitory activity against Claudin18.2 positive tumors. Attached Figure Description

[0023] Figure 1 This is an affinity curve of the anti-Claudin18.2 nanobody detected using SPRm 200; Figure 2 This is a diagram showing the inhibition of Claudin18.2 positive tumor cell clone formation by Claudin18.2 nanobody. Figure 3 This is a diagram showing the inhibition of Claudin18.2-positive tumor cell migration and invasion by Claudin18.2 nanobody. Figure 4 This is a diagram showing the inhibition of tumor growth in mice by Claudin18.2 nanobody. Detailed Implementation

[0024] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0025] The technical solution of the present invention is illustrated below through specific embodiments. It should be understood that the one or more steps mentioned in the present invention do not preclude the existence of other methods and steps before or after the combined steps, or that other methods and steps may be inserted between these explicitly mentioned steps. It should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise stated, the numbering of each method step is only for the purpose of identifying each method step, and not for limiting the order of each method or limiting the scope of the present invention. Changes or adjustments to their relative relationships, without substantial changes to the technical content, can also be considered as within the scope of the present invention.

[0026] The raw materials and instruments used in the examples are not subject to any specific restrictions on their source; they can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0027] As used herein, the terms "single-domain antibody," "heavy chain variable region domain of a heavy chain antibody," "VHH," "nanobody," and "single variable domain" are used interchangeably and all refer to a single-domain polypeptide or protein that specifically recognizes and binds to an antigen. A single-domain antibody is the variable region of a heavy chain antibody. Typically, a single-domain antibody contains three CDRs and four FRs. A single-domain antibody is the smallest functional antigen-binding fragment. Typically, an antibody lacking both the light chain and the heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0028] The "heavy chain antibody" described in this article refers to antibodies derived from camelid or cartilaginous fish. Compared to the aforementioned four-chain antibodies, heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), containing only two heavy chains composed of a variable region (VHH) and other constant regions. The variable region is linked to the constant region via a hinge-like structure. Each heavy chain of camelid heavy chain antibodies contains one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of cartilaginous fish heavy chain antibodies contains one variable region and five constant regions (CH1-CH5). The antigen-binding fragment of heavy chain antibodies includes VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, heavy chain antibodies can possess the CH2 and CH3 regions of human IgG Fc.

[0029] A binding molecule containing two or more single-domain antibodies is a multivalent single-domain antibody; a binding molecule containing two or more single-domain antibodies with different specificities is a multispecific single-domain antibody. Multivalent or multispecific single-domain antibodies are linked together by a linker. The linker typically consists of 1-15 amino acids selected from G and S.

[0030] In this article, heavy chain antibodies and antibodies are used to distinguish different combinations of antibodies. Due to their structural similarities, the structural descriptions of antibodies below, except for those involving light chains, also apply to heavy chain antibodies.

[0031] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0032] The term "variable" refers to the wide variation in certain segments within a variable domain within an antibody sequence. Variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across all amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (present in both light and heavy chain variable domains): HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain (simply referred to as CDR1, CDR2, and CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 in the light chain variable domain. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of both the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation and are linked by three HVRs that form a ring connection and, in some cases, part of a β-sheet structure. The HVRs in each chain are held together very closely by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site. Typically, the structure of the variable region in the light chain is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the variable region in the heavy chain is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cell-mediated cytotoxicity.

[0033] The “Fc region” (crystallizable fragment region), “Fc domain”, or simply “Fc” refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 and CH3 domains of the two antibody heavy chains; the Fc regions of IgM and IgE contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where the numbering is based on the EU index, as in Kabat. Each heavy chain of a camelid heavy chain antibody contains one variable region (VHH) and two constant regions (CH2 and CH3). By fusing with the constant regions of human IgG Fc, the heavy chain antibody can possess the CH2 and CH3 regions of human IgG Fc.

[0034] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region and / or variable region of the complete antibody. Antibody fragments are preferably antigen-binding fragments of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragment whose half-life should be increased through chemical modification or incorporation into liposomes. Digestion of the antibody with papain produces two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, the name reflecting its ease of crystallization. Fab fragments consist of a complete light chain and a variable domain (VH) of the heavy chain, and a first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen-binding site. Treatment of the antibody with pepsin produces a larger F(ab')2 fragment, which roughly corresponds to two Fab fragments linked by disulfide bonds, possessing different antigen-binding activities and still capable of cross-linking antigens. The Fab' fragment differs from the Fab fragment due to the addition of several additional residues (including one or more cysteine ​​residues from the antibody hinge region) at the carboxyl terminus of the CH1 domain. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments, with a hinge cysteine ​​residue between them. Other chemical couplings of antibody fragments are also known. The Fc fragment contains the carboxyl-terminal portions of two heavy chains held together by disulfide bonds. The effector function of the antibody is determined by the sequence in the Fc region, which is also recognized by the Fc receptor (FcR) found on certain cell types. Antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies.

[0035] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly bound, non-covalently linked heavy chain variable domain and a light chain variable domain. Six hypervariable rings (three from the heavy chain and three from the light chain) protrude from the folds of these two domains, contributing the amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only the three antigen-specific HVRs) can recognize and bind antigens, although with lower affinity than a complete binding site. A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing antibody VH and VL domains linked together into a single polypeptide chain. Preferably, the sFv polypeptide also contains a polypeptide linker between the VH and VL domains, allowing the sFv to form the desired antigen-binding structure. The Fv of a heavy chain antibody is VHH.

[0036] Antibodies in this article also include “chimeric” antibodies, wherein a portion of the heavy chain and / or light chain is identical or homologous to the 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 is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity.

[0037] In this document, an antigen-binding molecule is a protein that specifically binds to an antigen, including but not limited to antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, microbodies, affinity molecules, target-binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines. In this document, the term "antibody" includes monoclonal antibodies (including full-length antibodies having the immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies and single-chain molecules, and antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, and Fv). In this document, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0038] The “Claudin18.2 binding molecule” described herein comprises an anti-Claudin18.2 single-domain antibody, wherein the complementarity-determining region (CDR) of the single-domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO:1, CDR2 comprises the sequence shown in SEQ ID NO:2 and CDR3 comprises the sequence shown in SEQ ID NO:3.

[0039] In this article, "Kd" stands for Koff, which represents the dissociation rate constant, indicating the speed of intermolecular dissociation, and is measured in seconds (s). -1 In the SPRm 200 affinity assay, a larger Koff indicates a slower rate of RU decrease and a flatter curve slope. Therefore, high affinity is characterized by rapid binding and slow separation.

[0040] In this article, "Ka" stands for Kon, which is the association rate constant, representing the speed at which molecules bind together, and its unit is M. -1 ∙S -1 In the SPRm 200 affinity assay, a larger Kon value indicates a shorter time to reach maximum RU and a steeper curve slope.

[0041] The "KD" mentioned in this article represents the dissociation constant, a specific type of equilibrium constant used to measure the tendency of a larger substance to separate (dissociate) from another smaller component. It is the reciprocal of the association constant, and its unit is mol / L (M) or nmol / L (nM). The smaller the KD value, the stronger the binding affinity between the two substances. KD = Kd / Ka.

[0042] Example 1 This embodiment constructs a phage nanobody library and uses a combination of ELISA and molecular docking for preliminary screening. The specific steps are as follows: (1) Construction of phage nanobody library Alpacas were immunized five times with human Claudin 18.2 protein-VLP (expressed in eukaryotic cells by Kaika Biotechnology) in HEK293 cells. After serum titers were detected by ELISA, peripheral blood was collected to isolate lymphocytes, total RNA was extracted, reverse transcribed into cDNA, and then the VHH gene was amplified by nested PCR. The steps for obtaining the variable region sequences (VHH) of the IgG2 and IgG3 heavy chains using nested PCR are as follows: 1) Design a pair of specific nested outer primers and perform the first round of PCR amplification using cDNA as a template. The amplified region is the alpaca heavy chain antibody gene, and the product sizes are 750 bp and 900 bp, respectively. The 750 bp PCR product is recovered by gel electrophoresis. 2) Design nested inner primers and perform the second round of PCR amplification using the 750 bp first-round PCR product as a template. The amplified product is the VHH fragment of the heavy chain antibody variable region, with a product size of 500 bp. The second-round PCR product is purified and recovered using a PCR product purification kit. The target gene VHH and the vector pComb3xss are digested with SfiI enzyme. The digested VHH and pComb3xss are ligated using T4 DNA ligase, and the VHH target gene is cloned into pHEN1 phage particles. The phage particles are then transformed into TG1 competent cells by electroporation to construct a VHH gene library.

[0043] (2) Screening of phage nanobody libraries Library screening was performed using phage display technology, and precise monoclonal phage ELISA was used to ensure the specificity and binding strength of each clone. Phages that specifically bound to Claudin18.2 viroids and Claudin18.2 overexpressing stable cell lines but not to Claudin18.1 recombinant protein were obtained. Sequencing yielded several Claudin18.2 peptide nanobody sequences. Subsequently, Alpha Fold 3 software was used to simulate molecular docking to obtain Claudin18.2 peptide nanobody sequences that specifically bound to the extracellular domain of Claudin18.2 protein but not to the extracellular domain of Claudin18.1 protein. The intersection of the nanobody sequences obtained from the two-step experiment was used to identify the amino acid sequence of the nanobody that specifically binds to Claudin18.2, as shown in SEQ ID NO.4 (QLQLVESGGGLVQPGGSLRLSCAASGSALDYYHITWIRQAPGKEREGVSCILNSGGSLYADSVKGRFSISRDKNTVYLQMNSLTPEDTAVYYCAADYVPGKFNFGCSLYIQRAYDLWGQGTQVTVSSEPKTPKPQDGQAGQ), with the screening number ICV2-21. The nanobody contains a heavy chain variable region, which includes 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).

[0044] Example 2 This embodiment describes the prokaryotic expression and purification of the anti-Claudin18.2 nanobody (ICV2-21) screened in Example 1, followed by determination of antibody affinity. The specific steps are as follows: (1) Construct a prokaryotic expression vector of ICV2-21 VHH sequence with His tag.

[0045] In the Nanobody (ICV2-21) protein coding gene sequence (SEQ ID NO.5: CAGCTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTTCAGCCGGGTGGTAGTCTGCGCCTGAGCTGTGCAGCCAGTGGCAGTGCACTGGATTATCATATTACCTGGATTCGTCAGGCACCGGGTAAAGAACGTGAAGGTGTTAGCTGCATTCTGAATAGCGGTGGCAGTCTGTATGCCGATAGCGTGAAAGGCCGCTTTAGCATTAGTCGC Add Nde to both ends of GATAAAAATACCGTGTATCTGCAGATGAATAGTCTGACCCCGGAAGATACCGCATGGTATTATTGCGCCGCCGATTATGTGCCGGGTAAATTCAATTTTGGCTGTAGCCTGTATATTCAGCGCGCATACGATCTGTGGGGCCAGGGTACACAGGTTACCGTTAGCAGTGAACCGAAAACCCCGAAACCGCAGGATGGTCAGGCCGGTCAG) I and Xho I restriction sites were used to obtain the nucleotide sequence. CATATG CAGCTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTTCAGCCGGGTGGTAGTCTGCGCCTGAGCTGTGCAGCCAGTGGCAGTGCACTGGATTATTCATATTACCTGGATTCGTCAGGCACCGGGTAAAGAACGTGAAGGTGTTAGCTGCATTCTGAATAGCGGTGGCAGTCTGTATGCCGATAGCGTGAAAGGCCGCTTTAGCATTAGTC GCGATAAAAATACCGTGTATCTGCAGATGAATAGTCTGACCCCGGAAGATACCGCATGGTATTATTGCGCCGCCGATTATGTGCCGGGTAAATTCAATTTTGGCTGTAGCCTGTATATTCAGCGCGCATACGATCTGTGGGGCCAGGGTACACAGGTTACCGTTAGCAGTGAACCGAAAACCCCGAAACCGCAGGATGGTCAGGCCGGTCAG CTCGAG The cells were synthesized and recombined into the pET-22b vector, which was linearized with Nde I and Xho I endonucleases, and then transformed into DH5α competent cells.

[0046] Single clones were picked from transformation plates and incubated overnight at 37°C with shaking. The PCR loading system is shown in Table 1. The PCR conditions were: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min; and storage at 4°C. Single clones whose PCR product band size met the expectations were sent to a sequencing company for sequencing identification, and the single clones with correct sequencing results were stored.

[0047] Table 1: PCR sample loading system

[0048] (2) Prokaryotic expression and purification of ICV2-21 VHH Transformation: Extract the correctly sequenced plasmid, add it to E. coli competent cells BL21(DE3), incubate on ice for 30 min, heat shock at 42℃ for 60 s, continue incubation on ice for 3 min, add 500 μl LB medium and incubate at 37℃ for 30 min, spread on a plate containing 100 µg / ml ampicillin, and incubate at 37℃ overnight.

[0049] PCR identification: Single clones were picked from the transformation plate and cultured overnight at 37°C with shaking. The PCR loading system and reaction conditions were as described above. Single clones whose PCR product band size met the expectations were stored and detected by agarose gel electrophoresis.

[0050] Induction of expression: BL21(DE3) containing ICV2-21 VHH plasmid was cultured at 37℃ with shaking. When the OD value reached 0.6, IPTG was added to a final concentration of 1 mM. The culture was continued at 37℃ with shaking overnight to induce the expression of the fusion protein. After the expression was completed, the cells were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the cells were collected.

[0051] Protein purification: Cell lysis: The cells were dissolved in binding buffer (0.5 mol / L NaCl, 5 mmol / L imidazole, 20 mmol / L Tris-Cl, pH 8.0), sonicated, and centrifuged at 4℃ and 10000 rpm for 10 min to collect the supernatant crude protein. Equilibration: Pack the Ni-NTA packing material into the column and wash the column with binding buffer to equilibrate it; Combined: Allow crude protein to flow naturally through the equilibrated column packing and collect the effluent; Equilibration: Wash the Ni-NTA column with 10 column volumes of binding buffer; Washing: Wash the Ni-NTA column 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 collect the effluent; Elution: Elute with elution buffer (0.5 mol / L NaCl, 250 mmol / L imidazole, 20 mmol / L Tris-Cl, pH 8.0) and collect the eluent.

[0052] Polyacrylamide gel electrophoresis (PAGE) detection: Polyacrylamide gels were prepared, and crude protein and effluent components were processed separately for PAGE detection. The results showed that the fusion protein was purified; SDS-PAGE analysis revealed a clear band near the theoretical molecular weight, indicating that the fusion protein was successfully purified.

[0053] Dialysis: The purified fraction was dialyzed into protein preservation buffer (PBS, 300 mM NaCl, 10% Glycerol, 0.3% SKL, pH 7.4), concentrated, filtered and sterilized; protein concentration was quantified using a protein quantification kit, antibody purity was determined by SEC-HPLC, and stored at -80°C.

[0054] In addition, in this embodiment, the purified VHH antibody was also subjected to affinity determination using SPRm 200. SPRm is a bioanalytical sensing technology based on surface plasmon resonance (SPR). It can detect and track the entire process of binding and dissociation between molecules in solution and molecules immobilized on the chip surface, record it in the form of a sensing map, and provide kinetic and affinity data.

[0055] During the assay, the cell aggregate wells were first installed on the J1 chip, and protease was coated onto the chip surface. Then, on a sterile operating table, cultured Claudin18.2 positive cells were transferred to the cell aggregate well chip and cultured for 1-2 days, observing the cell coverage rate to be 30%-70%. Cells were then fixed with 4% PFA. PBST was used as the buffer, and solutions containing different concentrations of recombinant nanobody protein were used as the mobile phase. The assay results are as follows: Figure 1 As shown, the antibody's affinity reaches the sub-micromolar level, with KD = 1.979E-07 M and Ka = 4.263E+04 M. -1 ∙S -1 Kd = 9.528E-03 S -1 .

[0056] Example 3 This embodiment performs a cell clone formation experiment on the ICV2-21 nanobody constructed in Example 2, including the following steps: (1) Claudin18.2 positive tumor cell inoculation HGC-27 gastric cancer cells that normally express Claundin 18.2 (HGC-27-NC) and HGC-27 cells that overexpress Claundin 18.2 (HGC-27... CLDN18.2 As Claudin18.2 positive tumor cells, 3 × 10⁶ cells were seeded in 35 mm cell culture dishes. 5 HGC-27 CLDN18.2 Cells and HGC-27-NC cells were cultured for 24 hours until the cells adhered.

[0057] (2) Antibody treatment Add 0, 2, 4, and 6 μg / mL of ICV2-21 nanobody to each well, and continue culturing in a 37℃ 5% CO2 incubator for 48 hours; trypsin digest the cells, resuspend them in complete culture medium, and adjust the cell concentration to 1×10⁻⁶. 4 1500 cells were seeded into a 60 mm cell culture dish, 3 ml of complete culture medium was added, and the cells were cultured in a 37 °C 5% CO2 incubator for 10-14 days.

[0058] (3) Detection method When clones are visible to the naked eye, stop the culture, wash once with PBS, discard the liquid, add an appropriate amount of 4% paraformaldehyde for fixation, wash the residual fixative with PBS after 30 minutes, stain with crystal violet for 20 minutes, gently wash under running water, and then let it air dry in a well-ventilated, cool place. Calculate the number of clones formed.

[0059] (4) The formula for calculating the clone formation rate is: Clonal formation rate (%) = (Number of clones / Number of inoculated cells) × 100% The results are as follows Figure 2 As shown, the ICV2-21 nanobody constructed in this invention can inhibit the colony formation of Claudin18.2 positive tumor cells: when the concentration of ICV2-21 nanobody is 0 μg / mL, the colony formation rate of HGC-27-NC cells is 12.47 ± 0.10%, and HGC-27 CLDN18.2 The cell colony formation rate was 8.78 ± 0.12%; when the concentration of ICV2-21 nanobody was 2 μg / mL, the colony formation rate of HGC-27-NC cells was 10.80 ± 0.57%. CLDN18.2The cell colony formation rate was 6.98 ± 0.37%; when the concentration of ICV2-21 nanobody was 4 μg / mL, the colony formation rate of HGC-27-NC cells was 7.71 ± 0.14%. CLDN18.2 The cell colony formation rate was 4.84 ± 0.43%; when the concentration of ICV2-21 nanobody was 8 μg / mL, the colony formation rate of HGC-27-NC cells was 4.29 ± 0.36%. CLDN18.2 The cell colony formation rate was 2.42 ± 0.12%. Using the colony formation rate at an ICV2-21 nanobody concentration of 0 μg / mL as a baseline, the colony formation rate of HGC-27-NC cells decreased by 13.39%, 38.17%, and 65.60% at nanobody concentrations of 2 μg / mL, 4 μg / mL, and 8 μg / mL, respectively. CLDN18.2 The percentages of cells with decreased colony formation rate were 20.50%, 44.87%, and 72.44%, respectively. The results indicate that the colony formation rate of Claudin18.2 positive tumor cells decreased in a concentration-dependent manner with increasing ICV2-21 nanobody concentration in the culture environment. Furthermore, after treatment with different concentrations of ICV2-21 nanobody, the colony formation rate of HGC-27 cells decreased. CLDN18.2 The degree of reduction in cell clone formation was greater in both cells than in HGC-27-NC cells.

[0060] Example 4 This embodiment performs cell migration and invasion experiments on the ICV2-21 nanobody constructed in Example 2, including the following steps: (1) Claudin18.2 positive tumor cell inoculation HGC-27 gastric cancer cells that normally express Claundin 18.2 (HGC-27-NC) and HGC-27 cells that overexpress Claundin 18.2 (HGC-27... CLDN18.2 As Claudin18.2 positive tumor cells, 3 × 10⁶ cells were seeded in 35 mm cell culture dishes. 5 HGC-27 CLDN18.2 Cells and HGC-27-NC cells were cultured for 24 hours until the cells adhered.

[0061] (2) Antibody treatment Add 0, 2, 4, and 6 μg / mL of ICV2-21 nanobody to each well, and continue culturing in a 37°C, 5% CO2 incubator for 48 hours. Trypsin digest the cells, resuspend them in complete culture medium, and adjust the cell concentration to 1.5 × 10⁻⁶. 5 per mL.

[0062] (3) Detection method Cell migration: Place the Transwell chambers into 24-well plates, add 800 μL of culture medium containing 10% fetal bovine serum to the lower chamber, and add 200 μL of cell suspension to the upper chamber (3 × 10⁶ cells per well). 4 (1 cell), incubate at 37°C, 5% CO2 for 48 hours; remove the Transwell chamber, discard the supernatant, and gently wash the chamber twice with PBS; place the chamber in a culture dish containing 4% paraformaldehyde and fix at room temperature for 15-30 minutes; discard the fixative, wash twice with PBS, and then place the chamber in 0.1% crystal violet staining solution and stain at room temperature for 15-30 minutes; gently rinse the chamber with PBS, and gently wipe the upper layer of the chamber membrane with a cotton swab to completely remove any cells that have not penetrated the membrane; place the fixed and stained chamber under an inverted microscope and take pictures of 5 random fields of view to count the number of migrating cells.

[0063] Cell invasion: Dilute Matrigel (thawed at 4°C) with pre-cooled serum-free medium (concentration 1:8-1:10). Add 70 μL of diluted Matrigel to the upper layer of the Transwell chamber, ensuring the matrix gel evenly covers the membrane surface and avoiding air bubbles at the edges. Incubate the prepared chambers at 37°C for 30-60 minutes to allow the Matrigel to solidify into a gel. The remaining steps are the same as for cell migration.

[0064] The results are as follows Figure 3 As shown, the ICV2-21 nanobody constructed in this invention can inhibit the migration and invasion of Claudin18.2 positive tumor cells: when the concentration of ICV2-21 nanobody is 0 μg / mL, the number of migrating HGC-27-NC cells is 1458.00±53.38, and the number of HGC-27 cells is significantly lower than that of NC cells. CLDN18.2 The number of migrating cells was 1568.00±59.50; when the concentration of ICV2-21 nanobody was 2 μg / mL, the number of migrating HGC-27-NC cells was 1007.67±33.91. CLDN18.2 The number of migrating cells was 990.33±12.77; when the concentration of ICV2-21 nanobody was 4 μg / mL, the number of migrating HGC-27-NC cells was 704.00±5.51. CLDN18.2 The number of migrating cells was 718.00±31.79; when the concentration of ICV2-21 nanobody was 6 μg / mL, the number of migrating HGC-27-NC cells was 512.67±46.62. CLDN18.2The cell migration count was 364.33 ± 14.86. Using the cell migration count at an ICV2-21 nanobody concentration of 0 μg / mL as a baseline, the migration count of HGC-27-NC cells decreased by 30.89%, 51.71%, and 64.84% at nanobody concentrations of 2 μg / mL, 4 μg / mL, and 8 μg / mL, respectively. CLDN18.2 The percentages of decreased cell migration were 36.84%, 54.21%, and 76.76%, respectively. When the concentration of ICV2-21 nanobody was 0 μg / mL, the invasive number of HGC-27-NC cells was 2271.33 ± 24.18. CLDN18.2 The cell invasion number was 2347.33±42.44; when the ICV2-21 nanobody concentration was 2 μg / mL, the invasion number of HGC-27-NC cells was 1155.67±65.01. CLDN18.2 The cell invasion number was 1159.33±45.56; when the concentration of ICV2-21 nanobody was 4 μg / mL, the invasion number of HGC-27-NC cells was 849.33±31.47. CLDN18.2 The cell invasion number was 701.33±28.04; when the concentration of ICV2-21 nanobody was 6 μg / mL, the invasion number of HGC-27-NC cells was 635.33±66.09. CLDN18.2 The cell invasion number was 374.00 ± 28.36. Using the cell invasion number at an ICV2-21 nanobody concentration of 0 μg / mL as a baseline, the reduction rates of HGC-27-NC cell invasion number at nanobody concentrations of 2 μg / mL, 4 μg / mL, and 8 μg / mL were 49.12%, 62.61%, and 72.03%, respectively. CLDN18.2 The percentages of invasive cells decreased by 50.61%, 70.12%, and 84.07%, respectively. Treatment with different concentrations of ICV2-21 nanobody resulted in HGC-27 cells... CLDN18.2 The reduction in the number of cells migrating and invading was lower than that in HGC-27-NC cells, indicating that the ICV2-21 nanobody had a more significant inhibitory effect on cells with high Claudin18.2 expression.

[0065] Example 5 This embodiment presents an in vivo tumor inhibition experiment on the ICV2-21 nanobody constructed in Example 2. The specific steps are as follows: (1) Laboratory animals Male BALB / c nude mice aged 4-6 weeks were selected and temporarily housed for one week in an SPF-grade laboratory animal center at a temperature of 20-24℃ and humidity of 40-70%, with a light-dark cycle of 12 hours of light / 12 hours of darkness. The experiment was approved by the institution's ethics committee, approval number: 2023(013).

[0066] (2) Claudin18.2 positive tumor cell inoculation HGC-27 cells overexpressing Claundin18.2 (HGC-27) CLDN18.2 As Claudin18.2 positive tumor cells, they were cultured in a 37°C, 5% CO2 incubator, passaged every 2-3 days, and cells in the logarithmic growth phase were harvested. Cells were trypsinized, resuspended in complete culture medium, and the cell concentration was adjusted to 5 × 10⁻⁶. 7 cells / mL. HGC-27 on ice. CLDN18.2 Cells were mixed with Matrigel at a volume ratio of 1:1.

[0067] (3) Construction of subcutaneous tumor-bearing model Disinfect the right back of BALB / c nude mice with an alcohol swab, lift the skin pad with a 1mL syringe needle, and slowly inject 100µL of cell suspension; leave the needle under the skin for 3 seconds and then withdraw it to prevent leakage.

[0068] (4) Tail vein administration Preparation of ICV2-21 nanobody: Dilute with sterile PBS to three concentrations (50 μg / mL, 100 μg / mL, and 300 μg / mL), filter through a 0.22 µm filter membrane, store at 4°C protected from light, and prepare fresh before use. Tail vein injection: Subcutaneous injection of HGC-27 CLDN18.2 One week after cell culture, tumor-bearing BALB / c nude mice were placed in a 37°C heat box for 5 minutes to dilate their tail veins. The tails were 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, 100 µL of ICV2-21 nanoantibody at different concentrations was slowly injected, with 6-8 mice for each concentration. Immediately after needle removal, hemostasis was achieved by applying pressure with a dry cotton ball for 30 seconds.

[0069] Frequency and cycle: Repeat injection every 3 days, for a total of 4 injections (days 0, 3, 6, and 9).

[0070] (5) Detection method 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. Experimental endpoint: 72 hours after the last administration (day 12), death due to overdose anesthesia after measurement, with complete removal of tumor tissue. 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 should be performed.

[0071] The results are as follows Figure 4 As shown, on day 12, in animal models treated with ICV2-21 nanobody, the tumor volume was 1271.51±132.28 μm when the ICV2-21 nanobody concentration was 0 mg / kg body weight; 939.08±106.88 μm when the concentration was 0.25 mg / kg body weight; 752.93±135.50 μm when the concentration was 0.5 mg / kg body weight; and 190.95±69.70 μm when the concentration was 1.5 mg / kg body weight. Compared to mice treated with 0 mg / kg body weight, mice treated with 0.25 mg / kg body weight had approximately 26.14% less tumor volume, mice treated with 0.5 mg / kg body weight had approximately 40.78% less tumor volume, and mice treated with 1.5 mg / kg body weight had approximately 84.79% less tumor volume. The results showed that with the increase of the total injection concentration of ICV2-21 nanobody in the animal model, Claudin18.2 positive tumors exhibited inhibitory growth, and the tumor inhibition effect varied greatly among different concentrations. When the concentration of ICV2-21 nanobody was 1.5 mg / kg body weight, the tumor volume of mice decreased by as much as 84.79%.

[0072] In addition, we also paid attention to the changes in mouse weight. During the treatment period, the mouse weight did not fluctuate significantly, and the liver and kidney function tests were within the normal range, proving that nanobodies have high efficacy and safety.

[0073] In summary, this invention screens and prepares anti-Claudin18.2 nanobodies that can specifically bind to Claudin18.2. When used as antibody drugs, these nanobodies can inhibit the clonal formation, migration, and invasion of Claudin18.2-positive tumor cells and inhibit tumor growth in mice. This demonstrates that the nanobodies of this invention can be effectively applied to immunotherapy and are of great significance for the development of tumor therapeutic drugs.

[0074] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A nanobody targeting Claudin18.2, comprising a heavy chain variable region, said heavy chain variable region comprising CDR1, CDR2 and CDR3, characterized in that, The amino acid sequence of CDR1 is shown in SEQ ID NO.1; the amino acid sequence of CDR2 is shown in SEQ ID NO.2; and the amino acid sequence of CDR3 is shown in SEQ ID NO.

3.

2. The nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nanobody as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

5.

5. An expression carrier, characterized in that, It contains the nucleic acid molecule as described in claim 3 or 4.

6. A host cell, characterized in that, It comprises the expression vector as described in claim 5, or the nucleic acid molecule as described in claim 3 or 4 integrated into the genome, or expressing the nanobody as described in claim 1 or 2.

7. A pharmaceutical composition, characterized in that, It comprises a nanobody as described in claim 1 or 2, a nucleic acid molecule as described in claim 3 or 4, a carrier as described in claim 5, or a host cell as described in claim 6, and at least one pharmaceutically acceptable excipient.

8. A test kit, characterized in that, It contains the nanobody as described in claim 1 or 2.

9. The use of the nanobody of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the carrier of claim 5, the host cell of claim 6, or the pharmaceutical composition of claim 7 in the preparation of a tumor therapeutic drug, wherein, The tumor was a tumor that highly expressed Claudin18.

2.

10. The use of the nanobody according to claim 1 or 2 in the preparation of Claudin 18.2 detection reagent.

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