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

Abstract

The present invention provides a Claudin18.2-specific molecular imaging probe and its preparation method and application, wherein the probe comprises a tumor targeting group and a radionuclide; the tumor targeting group is selected from a CLDN18.2-specific nanobody or a nanobody fusion protein, the nanobody has an amino acid sequence as shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, and SEQ ID No.7, and the nanobody fusion protein has an amino acid sequence as shown in SEQ ID NO.9. The probe is a positron nuclide emission probe and can be used for immune PET imaging; it can also realize non-invasive visualization of CLDN18.2 on the surface of malignant tumor cells, monitor the difference in expression levels and dynamic evolution in vivo, and on this basis further improve the effectiveness of the probe and promote the realization of integrated diagnosis and treatment.

Description

Technical Field

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

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

[0003] Claudin18 (CLDN18) is a transmembrane protein located in the tight junctions between epithelium and endothelium. It is an important component and functional structure of tight junctions between cells. There are two alleles in the first exon of the human CLDN18 gene, and their differences form two different splice mutants, namely CLDN18.1 protein and CLDN18.2 protein. CLDN18 is highly conserved in normal tissues. CLDN18.2 protein is mainly distributed in the gastric mucosa (normal gastric glands, chief cells, parietal cells, endocrine cells) with a short differentiation cycle and fast renewal rate, as well as Paneth cells in the duodenum. It is generally believed that the cell polarity of tumors changes during malignant transformation, resulting in the widespread distribution of CLDN18.2 on the cell membrane surface. The CLDN18.2 positive rate in gastric cancer and the proportion of CLDN18.2 positive gastric cancer in gastric cancer vary greatly in different studies. Some current studies limit the CLDN18.2 expression rate in gastric cancer to 42% to 86%, and CLDN18.2 positive gastric cancer accounts for about 16% to 73% of the gastric cancer population. CLDN18.2 expression has certain molecular pathological characteristics: diffuse gastric cancer is higher than intestinal gastric cancer, EBV virus (Epstein-Barr virus) positive gastric cancer is higher than negative gastric cancer (81.0% vs. 40.2%, P < 0.001), and the expression of primary lesions, peripheral lymph node metastasis and distant metastatic lesions is basically the same. Due to its abnormal activation and high expression in digestive system malignancies, CLDN18.2 is considered to be a very potential target in the treatment of digestive system malignancies.

[0004] Studies have shown that CLDN18.2 is a good target for the treatment of solid tumors. Zolbetuximab (IMAB362, claudixmab) is a chimeric IgG1 monoclonal antibody that specifically binds to CLDN18.2 on the surface of tumor cells, thereby inducing 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 SPOTLIGHT of Claudin18.2 antibody Zolbetuximab + chemotherapy combined with Claudin18.2-positive, HER2-negative recurrent metastatic gastric cancer reached the primary endpoint. The results showed that compared with placebo + mFOLFOX6, patients treated with zolbetuximab + mFOLFOX6 had statistically significant progression-free survival (PFS) and overall survival (OS). Humanized anti-CLDN18.2 autologous CAR-T therapy has also shown anti-tumor activity and safety, and the ongoing clinical trials are expected to provide more options for patients with CLDN18.2-positive tumors. CLDN18.2 involves targeted drugs, small molecule inhibitors, CAR-T, immune cell therapy, antibody-drug conjugates, tumor vaccines, etc. Cancer types include gastric cancer, gastroesophageal junction cancer and pancreatic cancer. Individualized immunotherapy of CLDN18.2 may be the next hot spot in the study of digestive system malignancies.

[0005] Antibodies have received special attention in biomedical research due to their clear structure, relative stability, high specificity and high affinity. PET imaging based on antibody-based probes is called immune PET imaging, which is a branch of molecular imaging. It uses probes targeting tumor cells and tumor microenvironment receptors to effectively reflect the occurrence and development of tumors in real time. ImmunoPET 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 150kD) have a large molecular weight, low tissue penetration, poor imaging targets, and are prone to non-specific uptake in non-target organs. Monoclonal antibodies have complex spatial structures, high expression and preparation costs, and high immunogenicity, but modified antibodies are difficult to achieve the original affinity. Many factors limit their application and popularization in clinical practice.

[0006] Nanobodies are derived from the smallest functional antigen-binding fragment of heavy chain antibodies in adult camels, with a molecular weight of only 15kDa. Nanobodies are highly stable and have high affinity for binding to antigens. Compared with conventional antibodies, they have many unique properties: 1) The sequences encoded by nanobodies are highly homologous to human VH families 3 and 4, and have weak immunogenicity; 2) Nanobodies have a small molecular weight and a simple structure, can be expressed in large quantities in microbial systems, and are easy to purify; 3) Nanobodies can recognize a large number of antigenic epitopes, including some epitopes hidden in molecular cracks; 4) Due to their small molecular weight, they can easily penetrate tissues and reach sites that are difficult for conventional antibodies to reach; 5) They can be matched with short-half-life nuclides 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 early; as therapeutic agents, nanobodies can be coupled with cytotoxic drugs and delivered to targets specifically to achieve precise targeted therapy. In recent years, we have focused on the development and clinical transformation of nanobody-derived tracers to give full play to their superior molecular imaging properties. Although radiolabeled monovalent nanobodies are ideal companion diagnostic tools, their short in vivo half-life and high renal uptake leave room for further improvement. In order to develop an integrated diagnostic and therapeutic platform, the albumin binding domain (ABD) targeting human / mouse albumin was introduced into nanobodies to extend the half-life of nanobody derivatives in vivo. Studies have shown that bispecific nanobody derivatives that simultaneously target tumor antigens and albumin improve in vivo biodistribution and can be used as a carrier for the development of therapeutic diagnostic toolboxes.

[0007] Therefore, technicians in this field are committed to developing a nano-antibody immune PET imaging probe with low preparation cost, small molecular weight, short in vivo circulation time, short imaging cycle, low radiation dose, and easy clinical transformation and application.

[0008] In addition, traditional screening of CLDN18.2-positive patient groups relies on pathological results of biopsy or surgical specimens, which is more invasive and has poor reproducibility. In addition, sampling errors and tumor heterogeneity may lead to differences in staining results. TM 18.2 Immunohistochemistry kits cannot distinguish between the two splice mutants of CLDN18, and false positive results may occur. Commonly used nonspecific imaging agents 18 The value of F-FDG PET / CT in distant metastasis of gastric cancer, i.e., M staging, has been recognized, but it has no convincing advantages over other imaging methods in the diagnosis and evaluation of primary tumors and local lymph nodes, especially in the diagnosis of early gastric cancer, signet ring cell carcinoma, or mucinous adenocarcinoma. Currently, there is an urgent need for tools that can non-invasively diagnose gastric cancer at an early stage and screen suitable patients for targeted treatment.

[0009] Molecular imaging plays an important role in assisting disease screening, differential diagnosis, patient stratification and clinical staging, efficacy evaluation and prognosis. The development of new probes to achieve non-invasive visualization of gastric cancer-specific targets is crucial for early diagnosis and precise treatment to reduce mortality and prolong survival of gastric cancer patients. Summary of the invention

[0010] In order to solve the above problems, the purpose of the present invention is to provide a Claudin18.2-specific molecular imaging probe and its preparation method and application. The present invention constructs a new Claudin 18.2 (CLDN18.2)-specific molecular imaging probe to achieve non-invasive visualization of CLDN18.2 on the surface of malignant tumor cells, monitor the expression level differences and dynamic evolution in vivo, and further improve the effectiveness of the probe on this basis, promoting the realization of integrated diagnosis and treatment.

[0011] Based on the existing evidence and the applicant's previous findings, we hypothesize that the immunoPET imaging probe targeting CLDN18.2 can 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 personalized treatment in precision medicine.

[0012] Studies have shown that immuno-PET can better display the distribution and abundance of targets of interest in the body, and better predict the response to targeted therapy, compared with immunohistochemical staining or other traditional predictive markers. Traditional immuno-PET probes 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 a large molecular weight, low tissue penetration, poor imaging targets, and are prone to non-specific uptake in non-target organs. Monoclonal antibodies have complex spatial structures, high expression and preparation costs, and high immunogenicity, but modified antibodies are difficult to achieve the original affinity. Many factors limit its application and popularization in clinical practice. At present, the field of CLDN18.2 molecular imaging probes is still in the development stage.

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

[0014] The objective of the present invention is achieved through the following technical solutions:

[0015] In a first aspect, the present invention provides a series of CLDN18.2-specific nanobodies, wherein the nanobodies are hu19V3, 3E10, 3E11, 3A12, and 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, the 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 a use of a nanobody in preparing a CLDN18.2-specific nanobody fusion protein.

[0018] The nanobody fusion protein is composed of a GGGGS linker containing different amino acid lengths, bridging the serum protein binding domain (ABD) and the nanobody (hu19V3), and the linker "GGGGS" is 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 groups of 3 and has an amino acid sequence shown as GGGGSGGGGSGGGGS.

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

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

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

[0023] The preparation method of the CLDN18.2-specific nanoantibody or CLDN18.2-specific nanoantibody fusion protein of the present invention is: cloning the gene sequence of the CLDN18.2-specific nanoantibody or CLDN18.2-specific nanoantibody fusion protein (as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10) into an expression vector; then transforming the gene sequence into a strain of an expression host, expanding the transformed strain, inducing expression, and purifying to obtain the CLDN18.2-specific nanoantibody or CLDN18.2-specific nanoantibody fusion protein.

[0024] In a fourth aspect, the present invention provides a use of a nanobody or a nanobody fusion protein in the preparation of a CLDN18.2-specific molecular imaging probe.

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

[0026] The tumor targeting group is selected from CLDN18.2-specific nanoantibody or CLDN18.2-specific nanoantibody 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 When Zr is present, 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 nanoantibody probe [ 68 Ga]Ga-NOTA-hu19V3, [ 68 Ga]Ga-NOTA-3E10, [ 68 Ga]Ga-NOTA-3E11,[ 68 Ga]Ga-NOTA-3A12, 68 Ga-labeled nanoantibody fusion protein probe [ 68 Ga]Ga-NOTA-ABDC LDN18.2, 64 Cu-labeled nanoantibody probe 64 Cu]Cu-DOTA-hu19V3, 89 Zr-labeled nanoantibody fusion protein probe 89 Zr]Zr-DFO-ABDC LDN18.2.

[0030] Preferably, the probe comprises a tumor targeting gene, a chelator and a radionuclide 68 Ga, 64 Cu or 89 When Zr, the preparation method comprises the following steps:

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

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

[0033] 1) The molecular probe constructed by 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) is a positron emission probe that can be used for immuno-PET imaging. By conducting immuno-PET imaging based on the above probe, non-invasive visualization of CLDN18.2 expression 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 by the present invention not only optimizes the pharmacokinetic properties of monovalent nanobodies in imaging results, but also demonstrates its unique value in tumor treatment as an important part of the antibody drug strategy, with significant clinical significance. The nanobody fusion protein probe constructed by 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 therapy.

[0035] 3) The present invention realizes non-invasive visualization and dynamic evaluation of CLDN18.2 expression levels, and further realizes in vivo diagnosis of gastric cancer. 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 transformation. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0037] Figure 1 The expression of nanobody hu19V3 and nanobody fusion protein ABDCLDN18.2 was determined by SDS-PAGE;

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

[0039] Figure 3 To determine the probe using radioactive thin layer chromatography [ 68 The radiochemical purity results of Ga]Ga-NOTA-hu19V3;

[0040] Figure 4 for[ 68 PET / CT imaging results of Ga]Ga-NOTA-hu19V3 for subcutaneous tumors of different sizes (CHO-CLDN18.2 tumor model);

[0041] Figure 5 ROI diagram and in vitro biodistribution data diagram results of the blocking experiment using hu19V3;

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

[0043] Figure 7 For the closed group using ABDCLDN18.2 [ 68 Ga]Ga-NOTA-hu19V3 immunoPET / CT imaging results;

[0044] Figure 8 The ROI diagram and in vitro biodistribution data of the ABDCLDN18.2 blocked and unblocked groups;

[0045] Fig. 9 for[ 68 PET / CT imaging results of CT26-CLDN18.2 tumor model by Ga]Ga-NOTA-hu19V3;

[0046] Fig.10 for[ 68 ROI map results of Ga]Ga-NOTA-hu19V3 on CT26-CLDN18.2 tumor model;

[0047] Fig.11 Results of immunohistochemical staining for two gastric cancer (NO.144 and NO.490) PDX tissues;

[0048] Fig.12 for[ 68 PET / CT imaging results of NO.144PDX tumor model by Ga]Ga-NOTA-hu19V3;

[0049] Fig.13 for[68 ROI map results of Ga]Ga-NOTA-hu19V3 on NO.144PDX tumor model;

[0050] Fig.14 To determine the probe using radioactive thin layer chromatography [ 64 Radiochemical purity results of Cu]Cu-DOTA-hu19V3;

[0051] Fig.15 for[ 64 Cu]Cu-DOTA-hu19V3 PET / CT imaging results at multiple time points in the CHO-CLDN18.2 lung metastasis model;

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

[0053] Fig.17 To determine the probe using radioactive thin layer chromatography [ 68 Ga]Ga-NOTA-3E10, [ 68 Ga]Ga-NOTA-3E11,[ 68 The radiochemical purity results of Ga]Ga-NOTA-3A12;

[0054] Fig.18 for[ 68 Ga]Ga-NOTA-3E10 imaging results in normal Balb / c mice;

[0055] Fig.19 for[ 68 In vitro biodistribution results of Ga]Ga-NOTA-3E10 in normal Balb / c mice;

[0056] Fig. 20 for[ 68 Ga]Ga-NOTA-3E11 imaging results in normal Balb / c mice;

[0057] Fig.21 for[ 68 In vitro biodistribution results of Ga]Ga-NOTA-3E11 in normal Balb / c mice;

[0058] Fig. 22 for[ 68 Ga]Ga-NOTA-3A12 imaging results in normal Balb / c mice;

[0059] Fig.23 for[ 68In vitro biodistribution results of Ga]Ga-NOTA-3A12 in normal Balb / c mice. DETAILED DESCRIPTION

[0060] For ease of understanding of the present invention, the present invention will be described in more detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the present invention is not limited to the ad hoc methods, schemes, cell lines, constructs and reagents described herein, and can be changed equally. Unless otherwise defined, all technical and scientific terms used in this specification are identical with the meanings generally understood by those skilled in the art belonging to the technical field of the present invention. The terms used in this specification in the specification of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention.

[0061] Example 1

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

[0063] The specific steps are as follows:

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

[0065] 2) Expressing the above-mentioned target nanobody in Escherichia coli (E.coli):

[0066] 2.1 E. coli transformation: 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 mix gently; incubate the competent cells on ice for 30 minutes; in a static state, 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 LB agar plates containing 50 μg / ml kanamycin; invert the agar plates and incubate at 37 ° C overnight.

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

[0068] The expression of nano-antibodies was determined by SDS-PAGE. The specific steps are as follows: first, prepare a 1.5 mm thick, 15 well gel according to the method of the SDS-PAGE gel kit, preheat the metal bath to 100°C, and heat the above expressed nano-antibody (containing 5X loading buffer) protein sample for 5 minutes; after the SDS-PAGE gel is assembled, add 500 ml of 1xSDS-PAGE buffer, slowly spot the protein sample into the loading well, and place it in an 80V constant voltage bath for about 30 minutes. After the bromophenol blue indicator passes over the concentrated gel, adjust the voltage to 120V, electrophoresed to the bottom of the gel, remove the gel, heat and stain it in Coomassie blue dye for 50 minutes, then take it out, and take pictures when the decolorization solution is decolorized until the background is clean and the bands are clear. Figure 1 As shown, it can be seen that the molecular weight of the nanoantibody hu19V3 is about 10KDa and has a high purity; after hu19V3 is fused with ABD, the molecular weight is about 20KDa.

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

[0070] Example 2

[0071] This embodiment provides a method for preparing a CLDN18.2-specific nanobody fusion protein ABDCLDN18.2, which is composed of a serum protein binding domain (ABD) and a nanobody (B3 or B6) bridged by a GGGGS linker containing different amino acid lengths, and 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 CLDN18.2-specific nanobody fusion protein ABDCLDN18.2 prepared in this embodiment uses 3 groups of linkers, and its amino acid sequence is shown in SEQ ID NO.9 and the base sequence is shown in SEQ ID NO.10.

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

[0073] 2) Express the above target fusion protein in 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 nano antibody fusion protein ABDCLDN18.2 was determined by SDS-PAGE, and the specific steps were the same as those in Example 1. Figure 1 As shown, it can be seen that the molecular weight of ABDCLDN18.2 after fusion of hu19V3 and ABD is about 20KDa.

[0076] Example 3

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

[0078] 1) NOTA modification of hu19V3, 3E10, 3E11, 3A12 and ABDCLDN18.2 to prepare intermediates NOTA-hu19V3, NOTA-3E10, NOTA-3E11, NOTA-3A12 and NOTA-ABDCLDN18.2: 1 mg of hu19V3, 3E10, 3E11, 3A12 or ABDCLDN18.2 nanobodies in PBS solution was taken, 0.1 M Na2CO3 (pH ≈ 11) was added to adjust the solution pH to 9.0-10.0, and the chelating agent p-SCN-Bn-NOTA (CAS Number: 147597-66-8; Macrocyclics) was dissolved in anhydrous DMSO and added to the antibody solution at a molar ratio of 10:1 of chelating agent: antibody. The mixed solution was placed on a shaker at room temperature and incubated at 600 rpm for 2 hours, then purified using a PD-10 desalting column equilibrated with PBS in advance, eluted with PBS solution, and the collected purified antibody solution was centrifuged and concentrated at 4°C using an Amicon centrifugal filter column (Merck Millipore) with a cutoff value of 10KDa. The obtained antibody concentrate was measured for the concentration of proteins NOTA-hu19V3, NOTA-3E10, NOTA-3E11, NOTA-3A12 and NOTA-ABDCLDN18.2 using Nanodrop 2000 and stored at -20°C.

[0079] 2) 68 Preparation of Ga-labeled NOTA-hu19V3, NOTA-3E10, NOTA-3E11, NOTA-3A12 and NOTA-ABDC LDN18.2 [ 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: Elute the germanium gallium generator (Eckert & Ziegler Radiopharma Inc) with 4 mL of 0.05 M hydrochloric acid solution (HCl) and collect an equal volume of about 370-555 MBq of activity 68 Ga eluent; take the middle section with the highest activity 68 2 mL of Ga eluent, add 0.1 mL of 1 M sodium acetate solution (NaoAc) to adjust 68Ga eluent pH = 4.0-4.5; add about 2 ml of eluent with pH = 4.0-4.5 to the antibody concentrate containing 100-200 μg NOTA-hu19V3 or NOTA-ABDCLDN18.2 prepared in step 1) (specifically, add 200 μg), place the reaction system in a constant temperature oscillator and react at room temperature for 5-10 minutes; after the labeling reaction is completed, use PBS as the mobile phase and use the pre-equilibrated PD-10 desalting column to separate the free 68 Ga, purification of 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 was >50%.

[0080] 3) CLDN18.2 nanoantibody probe quality control: Use a capillary glass tube to draw a small amount of 68 Ga]Ga-NOTA-hu19V3, [ 68 Ga]Ga-NOTA-3E10, [ 68 Ga]Ga-NOTA-3E11,[ 68 Ga]Ga-NOTA-3A12 was spotted on a silica gel plate, and 0.1 M sodium citrate solution (pH = 4) was used as the mobile phase. Radiochemical purity (RCP) of the probe was determined by radio-thin layer chromatography (Radio-TLC, Eckert & Ziegler Radiopharma Inc). Figure 3 , Fig.17 As shown, freshly prepared [ 68 Ga]Ga-NOTA-hu19V3, [ 68 Ga]Ga-NOTA-3E10, [ 68 Ga]Ga-NOTA-3E11,[ 68 Ga]Ga-NOTA-3A12 RCP is greater than 99%.

[0081] Example 4

[0082] This embodiment provides a CLDN18.2 specific 64 Cu labeled monovalent nanoantibody probe [ 64The preparation method of Cu]Cu-DOTA-hu19V3, the specific steps are as follows:

[0083] 1) DOTA modification of hu19V3: 3 mg hu19V3 was dissolved in 1 mL phosphate buffer (PBS), 0.08 ml 0.1 M sodium carbonate (Na2CO3, pH = 11) buffer to adjust the pH of the nanoantibody solution to 9.0-10.0, and the reaction system volume was 1.1 mL. Freshly dissolved p-SCN-Bn-DOTA in dimethyl sulfoxide (DMSO) was added to the above nanoantibody solution at a molar ratio of DOTA / hu19V3 = 10:1. The resulting reaction system was placed at room temperature for 30 minutes, and then PBS was used as the mobile phase to purify the DOTA-modified nanoantibody with a pre-equilibrated PD-10 desalting column (GE Healthcare), collect DOTA-hu19V3 and concentrate it with an ultrafiltration tube (Merck Millipore) with a cutoff value of 10 KDa, and use NanoDrop to determine the concentration of DOTA-hu19V3, and the devices were stored at -20 ° C for use.

[0084] 2) 64 Preparation of Cu-labeled DOTA-hu19V3 64 Cu]Cu-DOTA-hu19V3: Take 160 μL of 44.92 MBq 64 Cu solution, to which 800 μL 1M sodium acetate (pH = 5) solution was added, and the final pH of the solution was 5 ± 0.5. 64 The Cu solution was added to the concentrate containing 200 μg DOTA-hu19V3 prepared in step 1) and incubated at 37°C with shaking at 400 rpm for 30 minutes. Finally, the final product was purified by elution using a PD-10 desalting column pre-equilibrated with PBS. 64 Cu]Cu-DOTA-hu19V3. Use a capillary glass tube to draw a small amount of [ 64 Cu]Cu-DOTA-hu19V3 was spotted on a silica gel plate, and 0.1 M sodium citrate solution (pH = 5) was used as the mobile phase. The radiochemical purity (RCP) of the probe was determined by radio-thin layer chromatography (Radio-TLC, Eckert & Ziegler Radiopharma Inc). The unattenuated radiochemical yield (RCY) obtained according to the above steps was 61.54%, and the RCP was 100%. Fig.14 shown.

[0085] Verification Example

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

[0087] The stomach, liver, kidney, lung, spleen and pancreas of normal nude mice were obtained and fixed with tissue fixative, and immunohistochemical staining was performed using 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. Figure 2 As shown, immunohistochemical experiments revealed that the gastric glandular epithelium of mice expressed positively, while other organs such as the liver, lung, kidney, pancreas, and spleen were negative.

[0088] 2) Construction of CLDN18.2 stable cell lines and CLDN18.2 positive tumor-bearing mouse models

[0089] The pCDNA3.1 vector (Life Technologies) containing the full-length CLDN18.2 gene was extracted with a plasmid extraction kit (Biomiga), and the expression plasmid was sterile filtered and electrotransfected into CHO-S cells, and G418 (Sigma) was added for 96-well plating to construct a CHO-CLDN18.2 stable cell line. The stable cell line on the 96-well plate was picked up and gradually amplified under the culture condition of adding G418. The CT26-CLDN18.2 cell was constructed in the same way. The CT26 mouse colon cancer cell line was purchased from Shanghai Jiman Biotechnology Co., Ltd., which did not show positive expression of CLD18A2 before transfection. 2×10 6 CHO-CLDN18.2 and CT26-CLDN18.2 cells were suspended in PBS and Matrigel (Corning) at a ratio of 1:1 and injected into the right shoulder of 4-5 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, 50x10 4 CHO-CLDN18.2 cells were injected 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 higher uptake in tumor tissues, and also has a higher nonspecific uptake in major excretory (kidney) and metabolic (liver) tissues. In addition, a higher uptake was observed in the mouse stomach. This is due to the high homology of CLDN18.2 expression in different species including humans, mice, rabbits, etc., so 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 there was no statistical significance, the tumor and gastric uptake in the blocked group was lower than that in the unblocked group, which may be due to the faster hemodynamics of the nanoantibody. The subcutaneous tumor fixation staining results of CHO-CLDN18.2 are shown in the figure. Figure 6 As shown, immunohistochemical staining showed that CLDN18.2 was positively expressed on the surface of tumor cells.

[0092] At the same time, this study also conducted a blocking experiment using ABDCLDN18.2, that is, each tumor-bearing nude mouse was injected with 500ug ABDCLDN18.2 in advance, and then injected with [ 68 Ga]Ga-NOTA-hu19V3, as a blocking group (Blocking group, n = 5) experimental results are as follows Figure 7 As shown, each mouse was injected with 500ug ABDCLDN18.2 in advance. 68 Ga]Ga-NOTA-hu19V3PET / CT imaging showed that CHO-CLDN118.2 subcutaneous tumors did not show negative uptake results. ROI and in vitro biodistribution results showed that there was no significant difference in uptake of major tissues and organs including tumors and stomach between the blocked and unblocked groups. Figure 8 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 Experimental study on the diagnosis of colon cancer in mice using immunoPET imaging of Ga]Ga-NOTA-hu19V3

[0094] Each tumor-bearing nude mouse (i.e., CT26-CLDN18.2 tumor model) was injected with [ 6 8Ga]Ga-NOTA-hu19V3, the specific method is the same as the method in the above step 3). The experimental results are as follows Fig. 9 As shown, the subcutaneous tumor model constructed by the mouse colon cancer cell line CT26 overexpressing CLDN18.2 was effective for [ 6 8Ga]Ga-NOTA-hu19V3 had a higher uptake (4 in each group). By outlining ROI, the uptake of major organs and tumors matched the imaging results. The results are shown in Fig.10 shown.

[0095] 5)[ 68 Experimental study on the diagnosis of gastric cancer using Ga]Ga-NOTA-hu19V3 immunoPET imaging

[0096] Immunohistochemical staining was performed on two gastric cancer (NO.144 and NO.490) PDX tissues. The experimental results are as follows Fig.11 As shown, the tumor mass numbered gastric NO.144 was positive for CLDN182 staining, and the tumor mass numbered NO.490 was negative for gastric staining. A subcutaneous tumor model (i.e., NO.144 PDX tumor model) was constructed using the positive NO.144 PDX tissue, and the same method as in step 3) was used to inject [ 68 Ga]Ga-NOTA-hu19V3, and then immunoPET imaging was performed (3 mice in each group). The experimental results are shown in Fig.12 As shown, the CLDN18.2-specific nanoantibody probe [ 68 Ga]Ga-NOTA-hu19V3 quickly and clearly displayed the NO.144PDX tumor and had a high uptake in the tumor. The ROI results showed the uptake of major tissues and organs and tumors in detail. Fig.13 As shown. By comparison, [ 68 The uptake value of Ga]Ga-NOTA-hu19V3 in NO.144 tumor was higher than that in CT26 (P=0.0168), and because PDX is a tumor tissue with natural CLDN18.2 expression, it is more suitable and representative for imaging and treatment.

[0097] 6)[ 64 Cu]Cu-DOTA-hu19V3 immunoPET imaging

[0098] The experimental results are as follows Fig.15 As shown. The CHO-CLDN18.2 lung metastasis model was obtained 18 days after the tail vein injection of CHO-CLDN18.2 cells, and then the probe was injected in the same way as in step 3) above. 64 After the injection of Cu]Cu-DOTA-hu19V3, PET / CT imaging was performed. PET / CT images were collected 1, 24, and 48 hours after the probe injection, respectively. It was observed that the kidneys showed high uptake of the imaging agent in the first hour, and the uptake gradually decreased with time. Fig.16 The data obtained from the delineated ROI analysis shown (from left to right: heart, liver, lung, kidney, muscle) show that the uptake of major organs gradually decreased over time. Although no early lung metastases were detected (perhaps because no detectable metastases were formed), [ 64 Cu]Cu-DOTA-hu19V3 exhibited excellent metabolic characteristics in vivo.

[0099] 7)[ 68 Ga]Ga-NOTA-3E10, [ 68 Ga]Ga-NOTA-3E11,[ 68 Study on the biodistribution of Ga]Ga-NOTA-3A12 in Balb / c mice

[0100] like Fig.18 , Fig. 20 , Fig. 22 As shown, injection via tail vein [ 68 Ga]Ga-NOTA-3E10, [ 68 Ga]Ga-NOTA-3E11,[ 68 PET / CT imaging was performed 45 minutes after the administration of Ga]Ga-NOTA-3A12 (3 mice in each group). After the imaging, blood and normal tissues and organs were collected for biodistribution tests (such as Fig.19 , Fig.21 , Fig.23 ).

[0101] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. 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 work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection 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 CLDN18.2-specific nanoantibodies or CLDN18.2-specific nanoantibody fusion proteins: The CLDN18.2-specific nanobody is hu19V3, having the amino acid sequence shown in SEQ ID No. 1; The CLDN18.2-specific nanobody fusion protein is ABDCLDN18.2; the ABDCLDN18.2 has an amino acid sequence as shown in SEQ ID NO.9; The radionuclide is selected from 68 Ga or 64 Cu, the probe further comprises a chelating agent, the chelating agent is selected from p -SCN-Bn-NOTA or p -SCN-Bn-DOTA.

2. The CLDN18.2-specific integrated diagnostic and therapeutic molecular imaging probe according to claim 1, characterized in that The nano antibody is hu19V3, and the hu19V3 has a gene sequence as shown in SEQ ID No.

2.

3. The CLDN18.2-specific integrated diagnosis and treatment molecular imaging probe according to claim 1, characterized in that: The CLDN18.2-specific nanoantibody fusion protein is ABDCLDN18.2; the ABDCLDN18.2 has a gene sequence as shown in SEQ ID NO.

10.

4. The CLDN18.2-specific integrated diagnosis and treatment molecular imaging probe according to claim 1, characterized in that: The probe is 68 Ga-labeled monovalent nanoantibody probe [ 68 Ga]Ga-NOTA-hu19V3, 68 Ga-labeled nanoantibody fusion protein probe [ 68 Ga]Ga-NOTA-ABDC LDN18.2, 64 Cu-labeled nanoantibody probe 64 Cu]Cu-DOTA-hu19V3.

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

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