Claudin18.2 specific molecular imaging probe as well as preparation method and application thereof

Abstract

The invention provides a Claudin18.2 specific molecular imaging probe as well as a preparation method and application thereof. The probe comprises a tumor targeting group and radionuclide, the tumor targeting group is selected from a CLDN18.2 specific nano antibody or a nano antibody fusion protein, the nano antibody has amino acid sequences as shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7, and the nano antibody fusion protein has an amino acid sequence as shown in SEQ ID No.9. The tumor targeting group is a CLDN18.2 specific nano antibody or a CLDN18.2 specific nano antibody. The probe is a positron nuclide emission type probe and can be used for immune PET imaging; in addition, non-invasive visualization of the malignant tumor cell surface CLDN18.2 can be achieved, expression level difference and dynamic evolution are monitored in vivo, the efficiency of the probe is further improved on the basis, and diagnosis and treatment integration is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular imaging probes, and particularly relates to a Claudin18.2-specific molecular imaging probe, a preparation method thereof, and an application thereof. Background Art

[0002] The first-line treatment for advanced gastric cancer is chemotherapy. With the increase in tumor drug resistance, patients cannot achieve good long-term survival. In tumor diagnosis and treatment strategies, molecular targeted therapies such as small molecule inhibitors and monoclonal antibodies, as well as immunotherapies (such as immune checkpoint inhibitors), are changing the treatment status of various solid tumors and hematological malignancies. Finding suitable gastric cancer diagnosis and treatment targets is of great significance for the benefit of gastric cancer patients.

[0003] Claudin 18 (CLDN18) is a transmembrane protein located in the tight junctions of epithelia and endothelia, and is an important component and functional structure that constitutes the tight junctions between cells. There are two alleles in the first exon of the human CLDN18 gene, and the differences form two different splicing mutants, namely CLDN18.1 protein and CLDN18.2 protein. CLDN18 is highly conserved in normal tissues. The CLDN18.2 protein is mainly distributed in the gastric mucosa with a short differentiation cycle and a high renewal rate (normal gastric glands, chief cells, parietal cells, endocrine cells), as well as in Paneth cells of the duodenum. It is generally believed that during malignant transformation of tumors, cell polarity changes, resulting in the widespread distribution of CLDN18.2 on the cell membrane surface. The positive rate of CLDN18.2 in gastric cancer, as well as the proportion of CLDN18.2-positive gastric cancer in gastric cancer, vary greatly in different studies. Currently, some studies have limited the expression rate of CLDN18.2 in gastric cancer to 42% - 86%, and CLDN18.2-positive gastric cancer accounts for about 16% - 73% of the gastric cancer population. The expression of CLDN18.2 has certain molecular pathological characteristics: diffuse gastric cancer is higher than intestinal-type gastric cancer, and Epstein-Barr virus (EBV)-positive gastric cancer is higher than EBV-negative gastric cancer (81.0% vs. 40.2%, P < 0.001), and the expression in the primary focus, surrounding lymph node metastases, and distant metastases is basically the same. Due to its abnormal activation and high expression in digestive system malignancies, CLDN18.2 is considered to be a very promising target in the treatment of digestive system malignancies.

[0004] Existing studies have shown that CLDN18.2 is a good target for solid tumor treatment. Zolbetuximab (IMAB362, claudixmab) is a chimeric IgG1 monoclonal antibody that specifically binds to CLDN18.2 on the surface of tumor cells, thereby triggering antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, and inhibiting cell proliferation. Currently, multiple phase I / II trials have evaluated its clinical efficacy and safety. On November 17, 2022, Astellas announced that the phase III clinical trial SPOTLIGHT of Claudin18.2 antibody Zolbetuximab + chemotherapy for the treatment of recurrent metastatic gastric cancer with Claudin18.2 positive and HER2 negative reached the primary endpoint. The study results showed that compared with placebo + mFOLFOX6, the progression-free survival (PFS) and overall survival (OS) of patients treated with zolbetuximab + mFOLFOX6 were statistically significant. Humanized anti-CLDN18.2 autologous CAR-T therapy has also shown anti-tumor activity and safety, and multiple ongoing clinical trials are also expected to provide more options for patients with CLDN18.2 positive tumors. CLDN18.2 is involved in targeted drugs, small molecule inhibitors, as well as CAR-T, immune cell therapy, antibody-drug conjugates, tumor vaccines, etc. The cancer types involve gastric cancer, gastroesophageal junction cancer, and pancreatic cancer. The personalized immunotherapy of CLDN18.2 may be the next research hotspot for digestive system malignancies.

[0005] Antibodies have received special attention in biomedical research due to their clear structure, relative stability, high specificity, and high affinity. The PET imaging carried out with probes based on antibodies is called immunological PET imaging, which belongs to a branch of molecular imaging. Probes targeting receptors of tumor cells and the tumor microenvironment can effectively reflect the occurrence and development of tumors in real time. Immunological PET probes based on monoclonal antibodies have the characteristics of high stability and strong specificity, and are relatively widely used in clinical practice. However, monoclonal antibodies (about 150 kD) have too large a molecular weight, low tissue penetration ability, poor imaging target-to-background ratio, and non-specific uptake is likely to occur in non-target organs. The spatial structure of monoclonal antibodies is complex, the expression and preparation costs are relatively high, and they have high immunogenicity. It is difficult for the modified antibodies to achieve the original affinity. Many factors limit their application and popularization in clinical practice.

[0006] Nanobodies are derived from the smallest functional antigen-binding fragments of heavy-chain antibodies in adult camels and have a molecular weight of only 15 kDa. Nanobodies have high stability and high affinity for antigen binding and possess many unique properties compared with conventional antibodies: 1) The sequences encoding nanobodies have high homology with human VH families 3 and 4 and weak immunogenicity; 2) Nanobodies have a small molecular weight and simple structure, can be highly expressed in microbial systems, and are easy to purify; 3) Nanobodies can recognize a large number of antigen epitopes, including some epitopes hidden in molecular clefts; 4) Due to their small molecular weight, they are easy to penetrate tissues and reach sites where conventional antibodies are difficult to reach; 5) They can be matched with short half-life radionuclides to achieve same-day imaging. The various characteristics of nanobodies make them promising tools for disease diagnosis and treatment: As imaging tracers, nanobodies can obtain high-quality images at an early stage; as therapeutic agents, nanobodies can be conjugated with cytotoxic drugs and achieve precise targeted therapy through specific delivery to the target. In recent years, we have focused on the development and clinical translation of nanobody-derived tracers to exploit their superior molecular imaging properties. Although radiolabeled monovalent nanobodies are ideal companion diagnostic tools, their short in vivo half-life and high renal uptake still leave room for further improvement. To develop an integrated diagnostic and therapeutic platform, the albumin-binding domain (ABD) targeting human / mouse albumin was introduced into the nanobody to extend the in vivo half-life of nanobody derivatives. Studies have shown that bispecific nanobody derivatives targeting both tumor antigens and albumin improve the in vivo biodistribution and can be used as carriers for developing theranostic toolboxes.

[0007] Therefore, those skilled in the art are committed to developing a nanobody-based immuno-PET imaging probe with low preparation cost, small molecular weight, short in vivo circulation time, short imaging cycle, low radiation dose, and easy clinical translation and application.

[0008] In addition, the traditional screening of CLDN18.2-positive patient groups relies on the pathological results of biopsy or surgical specimens, which is highly invasive, has poor repeatability, and may lead to differences in staining results due to sampling errors and tumor heterogeneity. The commonly used CLAUDETECT TM 18.2 immunohistochemistry kit cannot distinguish between the two splice mutants of CLDN18 and may produce false-positive results. The commonly used non-specific imaging agent 18 The value of 18F-FDG PET / CT in the diagnosis of distant metastases (M stage) of gastric cancer has been widely recognized. However, it has no convincing advantages over other imaging methods in the diagnosis and evaluation of primary tumors and regional lymph nodes, especially in the diagnosis of early gastric cancer, signet ring cell carcinoma, or mucinous adenocarcinoma. There is an urgent need for tools that can non-invasively diagnose gastric cancer at an early stage and screen suitable patient groups for targeted therapy.

[0009] Molecular imaging plays an important role in disease screening, differential diagnosis, patient stratification and clinical staging, efficacy evaluation and prognosis. Developing novel probes for non-invasive visualization of specific targets in gastric cancer is crucial for early diagnosis and precise treatment to reduce the mortality rate and extend the survival period of gastric cancer patients. Summary of the Invention

[0010] To solve the above problems, the object of the present invention is to provide a Claudin18.2-specific molecular imaging probe, its preparation method and application. By constructing a novel Claudin 18.2 (CLDN18.2)-specific molecular imaging probe, the present invention realizes non-invasive visualization of CLDN18.2 on the surface of malignant tumor cells, monitors the difference in expression levels and dynamic evolution in vivo, and further improves the efficacy of the probe on this basis to promote the realization of integrated diagnosis and treatment.

[0011] Based on existing evidence and the applicant's previous findings, we hypothesized that an immune PET imaging probe targeting CLDN18.2 could non-invasively display the expression of CLDN18.2 in gastric cancer tumor cells, serve the diagnosis and treatment system of gastric cancer, and promote the development of individualized treatment in precision medicine.

[0012] Previous studies have shown that immune PET can better display the distribution and abundance of target sites of interest in vivo and better predict the response to targeted therapy compared to immunohistochemical staining or other traditional predictive markers. Traditional immune PET probes constructed based on monoclonal antibodies have the characteristics of high stability and strong specificity, and are relatively widely used in clinical applications. However, monoclonal antibodies (about 150 kDa) have too large a molecular weight, low tissue penetration ability, poor imaging target-to-background ratio, and non-specific uptake is likely to occur in non-target organs. The spatial structure of monoclonal antibodies is complex, the expression and preparation costs are relatively high, and they have high immunogenicity. It is difficult for the modified antibodies to achieve the original affinity. Many factors limit their application and popularization in clinical practice. Currently, the field of CLDN18.2 molecular imaging probes is still in the stage of being to be developed.

[0013] To fill the gap in this field, the applicant innovatively developed the construction of a CLDN18.2-targeted diagnosis and treatment pair derived from nanobodies and characterized its diagnostic and potential therapeutic value in cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models.

[0014] The object of the present invention is achieved by the following technical solutions:

[0015] In a first aspect, the present invention provides a series of CLDN18.2-specific nanobodies, namely hu19V3, 3E10, 3E11, and 3A12, which have amino acid sequences as shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, and SEQ ID No.7.

[0016] Preferably, hu19V3, 3E10, 3E11, and 3A12 have gene sequences as shown in SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, and SEQ ID No.8.

[0017] In a second aspect, the present invention provides an application of a nanobody in the preparation of a CLDN18.2-specific nanobody fusion protein.

[0018] The nanobody fusion protein is composed of bridging a serum protein binding domain (ABD) and a nanobody (hu19V3) through a GGGGS linker with different amino acid lengths. The linker "GGGGS" is in 1 - 10 groups, specifically 1 group, 2 groups, 3 groups, 4 groups, 5 groups, 6 groups, 7 groups, 8 groups, 9 groups, or 10 groups.

[0019] Preferably, the linker is in 3 groups and has an amino acid sequence as shown in GGGGSGGGGSGGGGS.

[0020] In a third aspect, the present invention provides a CLDN18.2-specific nanobody fusion protein, namely ABDCLDN18.2;

[0021] ABDCLDN18.2 has an amino acid sequence as shown in SEQ ID NO.9.

[0022] Preferably, ABDCLDN18.2 has a gene sequence as shown in SEQ ID NO.10.

[0023] The preparation method of the CLDN18.2-specific nanobody or CLDN18.2-specific nanobody fusion protein of the present invention is as follows: Clone the gene sequences of the CLDN18.2-specific nanobody or CLDN18.2-specific nanobody fusion protein (as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, and SEQ ID NO.10) onto an expression vector; then transform the gene sequences into an expression host strain, expand the culture of the transformed strain, induce expression, and obtain the CLDN18.2-specific nanobody or CLDN18.2-specific nanobody fusion protein after purification.

[0024] Fourthly, the present invention provides an application of a nanobody or a nanobody fusion protein in the preparation of a CLDN18.2-specific molecular imaging probe.

[0025] Fifthly, the present invention provides a CLDN18.2-specific molecular imaging probe, which comprises a tumor targeting group and a radionuclide;

[0026] The tumor targeting group is selected from a CLDN18.2-specific nanobody or a CLDN18.2-specific nanobody fusion protein.

[0027] Preferably, the radionuclide is selected from 68 Ga, 64 Cu or 89 Zr;

[0028] When the radionuclide is selected from 68 Ga, 64 Cu or 89 Zr, the probe further comprises a chelating agent, and the chelating agent is selected from p-SCN-Bn-NOTA, p-SCN-Bn-DOTA or p-SCN-Bn-Deferoxamine.

[0029] Preferably, the probe is 68 Ga-labeled monovalent nanobody probe 68 Ga]Ga-NOTA-hu19V3, 68 Ga]Ga-NOTA-3E10, 68 Ga]Ga-NOTA-3E11, 68 Ga]Ga-NOTA-3A12, 68 Ga-labeled nanobody fusion protein probe 68 Ga]Ga-NOTA-ABDCLDN18.2, 64 Cu-labeled nanobody probe 64 Cu]Cu-DOTA-hu19V3, 89 Zr-labeled nanobody fusion protein probe 89 Zr]Zr-DFO-ABDCLDN18.2.

[0030] Preferably, when the probe contains a tumor targeting gene, a chelating agent and a radionuclide 68 Ga, 64 Cu or 89 Zr, its preparation method comprises the following steps:

[0031] The tumor-targeting gene will be modified with a chelating agent to form a modified nanobody; then a radionuclide 68 Ga, 64 Cu or 89 Zr will be used to label the modified nanobody to obtain the probe.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) The molecular probes constructed in the present invention (such as 68 Ga]Ga-NOTA-hu19V3, 68 Ga]Ga-NOTA-3E10, 68 Ga]Ga-NOTA-3E11, 68 Ga]Ga-NOTA-3A12, 64 Cu]Cu-DOTA-hu19V3, 68 Ga]Ga-NOTA-ABDCLDN18.2 and 89 Zr]Zr-DFO-ABDCLDN18.2) are positron-emitting probes and can be used for immuno-PET imaging; by performing immuno-PET imaging based on the above probes, non-invasive visualization of the expression of CLDN18.2 in tumor tissues and normal tissues and organs can be achieved, and further used for non-invasive target-specific diagnosis of specific types of tumors.

[0034] 2) The nanobody fusion protein ABDCLDN18.2 constructed in the present invention not only optimizes the pharmacokinetic properties of the monovalent nanobody in the imaging results, but also shows unique value in the direction of tumor treatment as an important part of the antibody drug development strategy, with significant clinical significance. The nanobody fusion protein probe constructed in the present invention can provide more in-depth diagnostic information and quantitative analysis for CLDN18.2-positive tumors, providing an effective means and strong basis for antibody treatment.

[0035] 3) The present invention realizes non-invasive visualization and dynamic evaluation of the expression level of CLDN18.2, further realizes in vivo diagnosis of gastric cancer, and the designed probe has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging cycle, low radiation dose, and easy clinical translation. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] By reading the following detailed description of the non-limiting embodiments with reference to the accompanying drawings, other features, objects, and advantages of the present invention will become more apparent:

[0037] Figure 1 For SDS-PAGE to determine the expression of nanobody hu19V3 and nanobody fusion protein ABDCLDN18.2;

[0038] Figure 2 It is the immunohistochemical CLDN18.2 staining result of normal nude mice;

[0039] Figure 3 It is the result of measuring the radiochemical purity of the probe 68 Ga]Ga-NOTA-hu19V3 by a radio thin layer chromatograph;

[0040] Figure 4 It is 68 the PET / CT imaging result of

[0041] Figure 5 Ga]Ga-NOTA-hu19V3 for subcutaneous tumors of different sizes (CHO-CLDN18.2 tumor model);

[0042] Figure 6 It is the HE (A) and immunohistochemical staining (B) results of CHO tumor sections;

[0043] Figure 7 It is for the 68 Ga]Ga-NOTA-hu19V3 immunological PET / CT imaging result of the blocking group using ABDCLDN18.2;

[0044] Figure 8 It is the ROI map and in vitro biodistribution data map results of the ABDCLDN18.2 blocking group and the non-blocking group;

[0045] Figure 9 It is 68 the PET / CT imaging result of

[0046] Figure 10 Ga]Ga-NOTA-hu19V3 for the CT26-CLDN18.2 tumor model; 68 It is the ROI map result of

[0047] Figure 11 Ga]Ga-NOTA-hu19V3 for the CT26-CLDN18.2 tumor model;

[0048] Figure 12 It is 68 the PET / CT imaging result of

[0049] Figure 13 Ga]Ga-NOTA-hu19V3 for the NO.144 PDX tumor model;

[0049] Figure 13 It is68 ROI map results of Ga]Ga-NOTA-hu19V3 for the NO.144 PDX tumor model;

[0050] Figure 14 For the determination of the probe by radio thin layer chromatography 64 Radiochemical purity results of Cu]Cu-DOTA-hu19V3;

[0051] Figure 15 For 64 PET / CT multi-time point imaging results of Cu]Cu-DOTA-hu19V3 for the CHO-CLDN18.2 lung metastasis model;

[0052] Figure 16 For 64 ROI map results of Cu]Cu-DOTA-hu19V3 for the CHO-CLDN18.2 lung metastasis model;

[0053] Figure 17 For the determination of the probe by radio thin layer chromatography 68 Ga]Ga-NOTA-3E10, 68 Ga]Ga-NOTA-3E11, 68 Radiochemical purity results of Ga]Ga-NOTA-3A12;

[0054] Figure 18 For 68 Imaging results of Ga]Ga-NOTA-3E10 in normal Balb / c mice;

[0055] Figure 19 For 68 Ex vivo biodistribution results of Ga]Ga-NOTA-3E10 in normal Balb / c mice;

[0056] Figure 20 For 68 Imaging results of Ga]Ga-NOTA-3E11 in normal Balb / c mice;

[0057] Figure 21 For 68 Ex vivo biodistribution results of Ga]Ga-NOTA-3E11 in normal Balb / c mice;

[0058] Figure 22 For 68 Imaging results of Ga]Ga-NOTA-3A12 in normal Balb / c mice;

[0059] Figure 23 For 68Biodistribution results of [Ga]Ga-NOTA-3A12 in normal Balb / c mice. Detailed implementation manners

[0060] To facilitate the understanding of the present invention, the present invention will be described in more detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the present invention is not limited to the specific methods, schemes, cell lines, constructs and reagents described herein, and can be changed accordingly. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in this specification in the description of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention.

[0061] Example 1

[0062] This example provides a method for preparing CLDN18.2-specific nanobodies hu19V3, 3E10, 3E11, 3A12. The amino acid sequences of the CLDN18.2-specific nanobodies hu19V3, 3E10, 3E11, 3A12 are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, and the base sequences are shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8.

[0063] The specific steps are as follows:

[0064] 1) Using conventional molecular biology methods, the gene sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 are respectively cloned into the pET-30a(+) expression vector to obtain plasmid DNA containing the target antibodies (hu19V3, 3E10, 3E11, 3A12).

[0065] 2) Express the above target nanobodies in Escherichia coli (E. coli):

[0066] 2.1 Transformation of Escherichia coli: First, take out the BL21(DE3) competent cells from -80°C and thaw them on ice; add 100 ng of plasmid DNA containing the target antibody to the BL21(DE3) competent cells and gently mix; incubate the competent cells on ice for 30 minutes; under static conditions, heat shock the competent cells at 42°C for 90 seconds; place the competent cells on ice for 3 minutes; add 100 μl of room temperature LB medium to the competent cells; incubate at 200 rpm and 37°C for 60 minutes; plate on an LB agar plate containing 50 μg / ml kanamycin; invert the agar plate and incubate overnight at 37°C.

[0067] 2.2 Small-scale expression: Randomly select well-dispersed monoclonal antibodies from the agar plate and inoculate them into LB medium containing 50 μg / ml kanamycin for culture respectively; incubate at 200 rpm and 37 °C; when the OD600 measurement reaches 0.6 - 0.8, add isopropylthiogalactoside (IPTG) to the culture tube to make its concentration reach 0.5 mM, and then incubate under the incubation conditions of 15 °C for 16 hours or 37 °C for 4 hours (both of these two incubation conditions are acceptable).

[0068] The expression of nanobodies was determined by SDS-PAGE. The specific steps are as follows: First, prepare a 1.5 mm thick and 15-well gel according to the method of the SDS-PAGE gel kit, preheat the metal bath to 100 °C, and heat the protein sample of the above-expressed nanobody (containing 5X loading buffer) for 5 min; after assembling the SDS-PAGE gel, add 500 ml of 1x SDS-PAGE buffer, slowly load the protein sample into the loading wells, perform constant voltage electrophoresis at 80 V for about 30 min, adjust the voltage to 120 V after the bromophenol blue indicator crosses the stacking gel, electrophorese until the bottom of the gel, take down the gel, heat and stain it in Coomassie blue staining solution for 50 min and then take it out, and decolorize it with decolorizing solution until the background is clean and the bands are clear for photography. As Figure 1 shown, it can be seen that the molecular weight of the nanobody hu19V3 is about 10 KDa and the purity is relatively high; after hu19V3 is fused with ABD, the molecular weight is about 20 KDa.

[0069] The expression and purification methods of 3E10, 3E11, and 3A12 nanobody proteins refer to hu19V3.

[0070] Example 2

[0071] This example provides a preparation method of a CLDN18.2-specific nanobody fusion protein ABDCLDN18.2. The nanobody fusion protein is composed of bridging a serum protein binding domain (ABD) and a nanobody (B3 or B6) through a GGGGS linker with different amino acid lengths. The linker "GGGGS" can be 1 group, 2 groups, 3 groups, 4 groups, 5 groups, 6 groups, 7 groups, 8 groups, 9 groups or 10 groups. The linker used for the CLDN18.2-specific nanobody fusion protein ABDCLDN18.2 prepared in this example is 3 groups, and its amino acid sequence is as shown in SEQ ID NO.9 and the base sequence is as shown in SEQ ID NO.10.

[0072] 1) Using conventional molecular biology methods, the nucleotide sequence shown in SEQ ID NO.10 was cloned into the pET-30a(+) expression vector to obtain plasmid DNA containing the target fusion protein (ABDCLDN18.2).

[0073] 2) Express the above-mentioned target fusion protein in Escherichia coli (E. coli).

[0074] The specific operation method of step 2) is the same as that of step 2) in Example 1.

[0075] The expression of the nanobody fusion protein ABDCLDN18.2 was determined by SDS-PAGE, and the specific steps were the same as those in Example 1. As Figure 1 shown, it can be seen that the molecular weight of ABDCLDN18.2 after the fusion of hu19V3 and ABD is about 20KDa.

[0076] Example 3

[0077] This example provides a preparation method for a CLDN18.2-specific 68 68Ga-labeled monovalent nanobody probe 68 68Ga]Ga-NOTA-hu19V3, 68 68Ga]Ga-NOTA-3E10, 68 68Ga]Ga-NOTA-3E11, 68 68Ga]Ga-NOTA-3A12, and a preparation method for a CLDN18.2-specific 68 68Ga-labeled nanobody fusion protein probe 68 68Ga]Ga-NOTA-ABDCLDN18.2, the specific steps are as follows:

[0078] 1) Preparation of intermediates NOTA-hu19V3, NOTA-3E10, NOTA-3E11, NOTA-3A12 and NOTA-ABDCLDN18.2 by modifying hu19V3, 3E10, 3E11, 3A12 and ABDCLDN18.2 with NOTA: Take 1 mg of the nanobodies hu19V3, 3E10, 3E11, 3A12 or ABDCLDN18.2 with PBS as the solution, add 0.1 M Na2CO3 (pH≈11) to adjust the solution pH to 9.0 - 10.0. Dissolve the chelator p-SCN-Bn-NOTA (CAS Number: 147597-66-8; Macrocyclics) in anhydrous DMSO, and add it to the antibody solution at a chelator:antibody molar ratio of 10:1. Place the mixed solution on a shaker and incubate it at room temperature with a shaking speed of 600 rpm for 2 h. Then purify it using a PD-10 desalting column pre-equilibrated with PBS, elute it with PBS solution, and concentrate the collected purified antibody solution using an Amicon centrifugal filter column (Merck Millipore) with a cut-off value of 10 KDa at 4°C. Determine the concentrations of the antibody concentrates NOTA-hu19V3, NOTA-3E10, NOTA-3E11, NOTA-3A12 and NOTA-ABDCLDN18.2 using Nanodrop 2000, and store them at -20°C.

[0079] 2) 68 Preparation of 68 Ga-labeled NOTA-hu19V3, 68 Ga-labeled NOTA-3E10, 68 Ga-labeled NOTA-3E11, 68 Ga-labeled NOTA-3A12 and 68 Ga-labeled NOTA-ABDCLDN18.2: Rinse the germanium-gallium generator (Eckert&Ziegler Radiopharma Inc) with 4 mL of 0.05 M hydrochloric acid solution (HCl), and collect the eluate of 68 Ga with an activity of approximately 370 - 555 MBq in the same volume; Take the middle section with the highest activity of 68 Ga eluate, add 0.1 mL of 1 M sodium acetate solution (NaoAc) to adjust 68Adjust the pH of the Ga eluent to 4.0–4.5; Add about 2 ml of the eluent with pH = 4.0–4.5 to the antibody concentrate containing 100-200 μg of NOTA-hu19V3 or NOTA-ABDCLDN18.2 prepared in step 1) (taking the addition of 200 μg as an example), and place the reaction system in a thermostatic oscillator to react at room temperature for 5–10 minutes; After the labeling reaction, use PBS as the mobile phase, and separate the free 68 Ga and purify the final product 68 Ga]Ga-NOTA-hu19V3, 68 Ga]Ga-NOTA-3E10, 68 Ga]Ga-NOTA-3E11, 68 Ga]Ga-NOTA-3A12 and 68 Ga]Ga-NOTA-ABDCLDN18.2; The unattenuated corrected radiochemical yield (RCY) obtained according to the above steps is > 50%.

[0080] 3) Quality control of CLDN18.2 nanobody probe: Use a capillary glass tube to aspirate a small amount of 68 Ga]Ga-NOTA-hu19V3, 68 Ga]Ga-NOTA-3E10, 68 Ga]Ga-NOTA-3E11, 68 Spot Ga]Ga-NOTA-3A12 on a silica gel plate, use 0.1 M sodium citrate solution (pH = 4) as the mobile phase, and use a radio-thin layer chromatograph (Radio-TLC, Eckert&Ziegler Radiopharma Inc) to determine the radiochemical purity (RCP) of the probe. As Figure 3 、 Figure 17 shown, freshly prepared 68 Ga]Ga-NOTA-hu19V3, 68 Ga]Ga-NOTA-3E10, 68 Ga]Ga-NOTA-3E11, 68 Ga]Ga-NOTA-3A12 has an RCP greater than 99%.

[0081] Example 4

[0082] This example provides a CLDN18.2-specific 64 Cu-labeled monovalent nanobody probe 64Preparation method of Cu]Cu-DOTA-hu19V3, the specific steps are as follows:

[0083] 1) Modification of hu19V3 with DOTA: Dissolve 3 mg of hu19V3 in 1 mL of phosphate buffer (PBS). Adjust the pH of the nanobody solution to 9.0 - 10.0 with 0.08 ml of 0.1 M sodium carbonate (Na2CO3, pH = 11) buffer, and the volume of the reaction system is 1.1 mL. Add freshly dissolved p-SCN-Bn-DOTA in dimethyl sulfoxide (DMSO) to the above nanobody solution at a molar ratio of DOTA / hu19V3 = 10:1. Place the obtained reaction system at room temperature for 30 minutes, then use PBS as the mobile phase and purify the DOTA-modified nanobody with a pre-equilibrated PD-10 desalting column (GE Healthcare). Collect DOTA-hu19V3 and concentrate it with an ultrafiltration tube (Merck Millipore) with a cut-off value of 10 KDa. Measure the concentration of DOTA-hu19V3 with NanoDrop, and aliquot and store it at -20 °C for later use.

[0084] 2) 64 Preparation of 64 Cu-labeled DOTA-hu19V3 64 Cu]Cu-DOTA-hu19V3: Take 160 μL of 64 Cu solution with an activity of 44.92 MBq, add 800 μL of 1 M sodium acetate (pH = 5) solution to it, and the final pH of the solution is 5 ± 0.5. Add the pH-adjusted 64 Cu solution to the concentrated solution containing 200 μg of DOTA-hu19V3 prepared in step 1), and shake and incubate at 37 °C at 400 rpm for 30 minutes. Finally, elute and purify with a PD-10 desalting column pre-equilibrated with PBS to obtain the final product 64 Cu]Cu-DOTA-hu19V3. Use a capillary glass tube to absorb a small amount of Figure 14 Cu]Cu-DOTA-hu19V3 and spot it on a silica gel plate. Use 0.1 M sodium citrate solution (pH = 5) as the mobile phase and measure the radiochemical purity (RCP) of the probe with a radio-thin layer chromatograph (Radio-TLC, Eckert&Ziegler RadiopharmaInc). The unattenuated corrected radiochemical yield (RCY) obtained according to the above steps is 61.54%, and the RCP is 100%, as

[0085] Verification examples

[0086] 1) Verify the expression of CLDN18.2 in the main tissues and organs of normal nude mice

[0087] Obtain and fix the stomach, liver, kidney, lung, spleen and pancreas of normal nude mice using tissue fixative. Use recombinant CLDN18.2 monoclonal antibody (EPR19202, ab222512, Abcam) as the primary antibody and horseradish peroxidase-conjugated rabbit anti-human IgG H&L (HRP-labeled rabbit anti-human IgG H&L; ab6759; Abcam) as the secondary antibody to perform immunohistochemical staining. As Figure 2 shown, through immunohistochemical experiments, positive expression was found in the gastric gland epithelium of mice, while negative expression was found in other organs such as the liver, lung, kidney, pancreas and spleen.

[0088] 2) Construct a stable CLDN18.2-expressing cell line and a CLDN18.2-expressing tumor-bearing mouse model

[0089] Extract the plasmid of pCDNA3.1 vector (Life Technologies) containing the full-length gene of CLDN18.2 using a plasmid large-scale extraction kit (Biomiga). After sterile filtration of the expression plasmid, electrotransfect CHO-S cells and add G418 (sigma) for 96-well plating to construct a stable CHO-CLDN18.2 cell line. Pick the stable cell line on the 96-well plate and gradually scale up under the culture conditions with G418. Construct CT26-CLDN18.2 cells in the same way. The CT26 mouse colon cancer cell line was purchased from Shanghai Jiman Biotechnology Co., Ltd., and this cell line did not show positive expression of CLD18A2 before transfection. Suspend 2×10 6 CHO-CLDN18.2 and CT26-CLDN18.2 cells in PBS and Matrigel (Corning) at a ratio of 1:1, and inject each into the right shoulder of 4- to 5-week-old Balb / c nude mice to establish subcutaneous tumor models (i.e., CHO-CLDN18.2 tumor model and CT26-CLDN18.2 tumor model). For the CHO-CLDN18.2 lung metastasis model, resuspend 50x10 4 CHO-CLDN18.2 cells with 100 - 150 μL PBS and inject them into nude mice via the tail vein.

[0090] 3) 68Imaging of Ga]Ga-NOTA-hu19V3 in CHO-CLDN18.2 tumor-bearing nude mice. The small animal PET / CT imaging acquisition involved in this experiment was completed using an IRIS small animal PET / CT scanner (Inviscan Imaging Systems). Each tumor-bearing nude mouse (i.e., CHO-CLDN18.2 tumor model) was injected via the tail vein with 3.7-7.4 MBq of [Ga]Ga-NOTA-hu19V3 prepared in Example 1. 68 Ga]Ga-NOTA-hu19V3 (5 mice in each group), the mice were anesthetized with isoflurane (concentration of 2%) 0.5 hours after injection, and the mice in a deep anesthesia state were placed in a supine position on the PET / CT scanning bed, and PET and CT images were continuously acquired, and image reconstruction was completed using the IRIS system's own software, such as Figure 4 As shown, 68 The Ga]Ga-NOTA-hu19V3 probe was mainly accumulated in the kidney and bladder at 0.5 hours. The OsiriX Lite image processing workstation (Pixmeo SARL) was used to outline the regions of interest (ROI) such as the heart and major tissues and organs (liver, lungs, kidneys, muscles) on the reconstructed PET images, and the radioactive uptake values ​​of important tissues and organs were calculated in units of %ID / g (percent of injected dose per gram). The uptake value of the main tissues and organs was plotted over time to further compare the pharmacokinetic differences of the two probes. Figure 4 They show [ 68 Ga]Ga-NOTA-hu19V3 for subcutaneous tumors of different sizes, it can be seen that [ 68 The high uptake of Ga]Ga-NOTA-hu19V3 in tumors indicates that it has good sensitivity for detecting tumors of different sizes.

[0091] In addition, each tumor-bearing nude mouse was injected with 1 mg hu19V3 in advance and then injected with 68 Ga]Ga-NOTA-hu19V3, as the blocking group (n=5), was injected only with [ 68 The nude mice bearing Ga]Ga-NOTA-hu19V3 tumors were used as the unblocking group (unblocking group, n = 5). The in vitro experimental results were as follows Figure 5 shown. Figure 5 The left side shows the ROI diagram, and the right side shows the in vitro biodistribution data diagram. Both sets of diagrams show that the CLDN18.2-specific nanoantibody probe [ 68Ga]Ga-NOTA-hu19V3 has a high uptake in tumor tissues and also has a high non-specific uptake in the main excretory (kidney) and metabolic (liver) tissues. In addition, a high uptake was also observed in the stomach of mice, which is due to the high homology of the expression of CLDN18.2 among different species including humans, mice, rabbits, etc. Therefore, hu19V3 can also bind to CLDN18.2 in the mouse gastric epithelium. The results of ROI delineation and in vitro biodistribution experiments showed that although not statistically significant, the tumor and gastric uptake in the blocking group were lower than those in the non-blocking group, which may be due to the faster hemodynamics of the nanobody. The fixed staining results of subcutaneous tumors of CHO-CLDN18.2 are as Figure 6 shown, and immunohistochemical staining showed positive expression of CLDN18.2 on the surface of tumor cells.

[0092] At the same time, this experiment also carried out a blocking experiment using ABDCLDN18.2, that is, each tumor-bearing nude mouse was injected with 500 μg of ABDCLDN18.2 in advance, and then 68 Ga]Ga-NOTA-hu19V3 was injected. The experimental results of the blocking group (n = 5) are as Figure 7 shown. After injecting 500 μg of ABDCLDN18.2 into each mouse in advance, 68 Ga]Ga-NOTA-hu19V3 PET / CT imaging was performed, showing that the subcutaneous tumor of CHO-CLDN118.2 did not show a negative uptake result. The ROI and in vitro biodistribution results showed that there was no significant difference in the uptake of major tissue organs including tumors and stomachs between the blocking group and the non-blocking group. The results are as Figure 8 shown. The above results show that 68 Ga]Ga-NOTA-hu19V3 has a good diagnostic effect on CHO subcutaneous tumors with positive expression of CLDN18.2.

[0093] 4) 68 Experiment of

[0094] Ga]Ga-NOTA-hu19V3 immunological PET imaging in diagnosing mouse colon cancer 6 Each tumor-bearing nude mouse (i.e., CT26-CLDN18.2 tumor model) was injected with Figure 9 8Ga]Ga-NOTA-hu19V3, and the specific method was the same as that in step 3) above. The experimental results are as 6 shown. The subcutaneous tumor model constructed from the mouse colon cancer cell line CT26 overexpressing CLDN18.2 had a high uptake of Figure 10 8Ga]Ga-NOTA-hu19V3 (4 mice in each group). The uptake of major organs and tumors obtained by ROI delineation matched the imaging results. The results are as

[0095] 5) 68 Experimental study on the diagnosis of gastric cancer by

[0096] Immunohistochemical staining was performed on PDX tissues of two gastric cancer tissues (NO.144 and NO.490). The experimental results are Figure 11 shown as follows. The tumor mass of NO.144 showed positive staining for CLDN18.2, while the tumor mass of NO.490 showed negative staining. A subcutaneous tumor model (i.e., NO.144 PDX tumor model) was constructed using the positive NO.144 PDX tissue, and 68 Ga]Ga-NOTA-hu19V3 was injected using the same method as in step 3) described above. Then, immunological PET imaging was performed (3 mice per group). The experimental results are Figure 12 shown as follows. The CLDN18.2-specific nanobody probe 68 Ga]Ga-NOTA-hu19V3 rapidly and clearly showed the NO.144 PDX tumor, and there was a high uptake within the tumor. The ROI results showed in detail the uptake of the main tissues, organs, and tumors. The results are Figure 13 shown as follows. After comparison, 68 the uptake value of

[0097] 6) 64 Immunological PET imaging of

[0098] The experimental results are Figure 15 shown as follows. For the CHO-CLDN18.2 lung metastasis model obtained 18 days after intravenous injection of CHO-CLDN18.2 cells, the probe 64 Cu]Cu-DOTA-hu19V3 was injected using the same method as in step 3) described above. Then, PET / CT imaging was performed. PET / CT images were collected 1, 24, and 48 hours after injection of the probe. It was observed that the kidneys showed a high uptake of the imaging agent at the first hour and the uptake gradually decreased with time delay. The data obtained from the ROI delineation analysis Figure 16 shown as follows (from left to right: heart, liver, lung, kidney, muscle) showed that the uptake of the main organs gradually decreased with time prolongation. Although no early lung metastasis foci were detected (perhaps because no detectable metastatic tumors were formed), 64 Cu]Cu-DOTA-hu19V3 demonstrated excellent metabolic characteristics in vivo.

[0099] 7) 68 Ga]Ga-NOTA-3E10, 68 Ga]Ga-NOTA-3E11, 68 Biodistribution study of Ga]Ga-NOTA-3A12 in Balb / c mice

[0100] As Figure 18 、 Figure 20 、 Figure 22 shown, after intravenous injection of 68 Ga]Ga-NOTA-3E10, 68 Ga]Ga-NOTA-3E11, 68 Ga]Ga-NOTA-3A12 (3 mice per group) at 45 minutes, PET / CT imaging was performed. After the imaging was completed, blood and normal tissue organs were taken for biodistribution experiments (such as Figure 19 、 Figure 21 、 Figure 23 ).

[0101] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A CLDN18.2-specific molecular imaging probe, characterized in that, The probe comprises a tumor targeting group and a radionuclide; the tumor targeting group is selected from any one of the following: a CLDN18.2-specific nanobody or a CLDN18.2-specific nanobody fusion protein, The CLDN18.2-specific nanobody is 3E10, which has an amino acid sequence as shown in SEQ ID No.3; The CLDN18.2-specific nanobody is 3E11, which has an amino acid sequence as shown in SEQ ID No.5; The CLDN18.2-specific nanobody is 3A12, which has an amino acid sequence as shown in SEQ ID No.7; The radionuclide is selected from 68 Ga or 64 Cu, and the probe further comprises a chelating agent selected from p-SCN-Bn-NOTA or p-SCN-Bn-DOTA.

2. The CLDN18.2-specific diagnostic and therapeutic integrated molecular imaging probe according to claim 1, wherein The nanobody is 3E10, and the 3E10 has a gene sequence as shown in SEQ ID No.

4.

3. The CLDN18.2-specific diagnostic and therapeutic integrated molecular imaging probe according to claim 1, characterized in that, The nanobody is 3E11, and the 3E11 has a gene sequence as shown in SEQ ID No.

6.

4. The CLDN18.2-specific diagnostic and therapeutic integrated molecular imaging probe according to claim 1, wherein The probe is 68 a Ga-labeled monovalent nanobody probe 68 Ga]Ga-NOTA-3E10, 68 Ga]Ga-NOTA-3E11, or 68 Ga]Ga-NOTA-3A12.

5. The CLDN18.2-specific diagnostic and therapeutic integrated molecular imaging probe according to any one of claims 1-4, characterized in that, The probe contains a tumor-targeting gene, a chelating agent, and a radionuclide 68 When it is 64 Ga or Cu, its preparation method comprises the following steps: The tumor-targeting gene will be modified with a chelating agent to form a modified nanobody or nanobody fusion protein; then, the modified nanobody or nanobody fusion protein will be labeled with a radionuclide 68 Ga or 64 Cu to obtain the probe.

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