Preparation and application of Trop2-specific molecular imaging probe for integrated diagnosis and treatment

Abstract

The present invention provides a method for preparing and using a Trop2-specific integrated diagnostic and therapeutic molecular imaging probe. The Trop2-specific molecular imaging probe, constructed based on the Trop2-specific nanoantibodies WWD98 and WWD328, can be used for immuno-PET imaging. Immuno-PET imaging using the probe can non-invasively display Trop2 expression within tumors, achieving non-invasive visualization of human Trop2 molecular expression. This provides a better method for diagnosing and monitoring Trop2-positive solid tumors and can further enable non-invasive diagnosis of various tumors, such as pancreatic cancer and gastric cancer. The probe of the present invention has the advantages of a simple preparation process, low cost, high specificity, high stability, short imaging cycle, low radiation dose, and ease of clinical translation.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular imaging probes, and in particular to a preparation method and application of a Trop2-specific integrated diagnosis and treatment molecular imaging probe. Background Art

[0002] In 1993, Belgian scientists Hamers et al. reported for the first time in Nature magazine that there is a naturally missing light chain antibody in the peripheral blood of alpacas (Nature.1993;363(6428):446-8.). This antibody with a special structural domain is called a heavy chain antibody (HCAbs). By cloning the variable region of a heavy chain antibody through molecular biological methods, an antigen-binding fragment containing only the variable region of the heavy chain can be obtained, which is a nanobody (VHH, Variable Domain of Heavy Chain of Heavy Chain Antibody). The VHH crystal is 2.5nm wide and 4nm long, with a molecular weight of only 15kDa, so it is also called a nanobody ( A registered trade name of Ablynx. Nanobodies are the smallest known antibody units capable of binding to target antigens. They possess the advantages of high affinity, small molecular weight, low production cost (they can be expressed in Escherichia coli, yeast, Chinese hamster ovary cells, and other eukaryotic expression systems), and ease of clinical translation and widespread application.

[0003] Nanobodies are a popular target carrier for constructing molecular imaging probes in recent years (Theranostics. 2014; 4(4):386-98.; J Nucl Med. 2022 Oct; 63(10):1705-1709.). Currently, a variety of short-half-life radionuclides have been used to label nanobodies and prepare nanoantibody molecular imaging probes. Technetium-99m ( 99m Tc; T 1 / 2 =6.02h) labeled with a nanoantibody probe targeting programmed death ligand 1 (PD-L1) has been successfully translated into the clinic for non-invasive diagnosis of patients with non-small cell lung cancer (J Nucl Med. 2019; 60(9): 1213-1220.); Gallium-68 ( 68 Ga;T 1 / 2=1.1h) labeled with a nanoantibody probe targeting the human epidermal growth factor receptor (HER2) has also been successfully translated into clinical practice for the noninvasive diagnosis of breast cancer (J Nucl Med. 2016; 57(1):27-33.). The above examples show that radionuclide-labeled nanoantibody probes have great clinical application prospects and can be used for the early noninvasive diagnosis of human malignancies, visualization of key pathogenic targets, screening of patients for monoclonal antibody (mAb) treatment, and evaluation of the efficacy of monoclonal antibody treatment.

[0004] Trophoblast cell surface antigen 2 (Trop2) is a cell membrane glycoprotein composed of a 36 kDa nascent polypeptide modified by N-linked glycosylation. It regulates tumor proliferation, invasion, and migration through multiple signaling pathways and plays a role in stem cell biology. In a retrospective study of 197 paraffin-embedded pancreatic cancer primary tumor tissues, Trop2 expression was analyzed. Immunohistochemical results revealed overexpression of the antigen in 55% of cases, which was significantly associated with lymph node metastasis, poor tumor differentiation, and poor prognosis (P < 0.05). Trop2 expression has also been associated with the biological aggressiveness and poor prognosis of malignancies such as gastric cancer, female reproductive system tumors, prostate cancer, and colorectal cancer, suggesting that it promotes tumor development and progression. The differential expression of Trop2 in normal tissues and tumors makes it a promising tumor-specific biomarker, while avoiding potential side effects. Antibody-drug conjugates targeting Trop2 are currently in clinical trials. Therefore, there is an urgent need to develop a diagnostic tool targeting Trop2 to achieve non-invasive visualization and monitoring of Trop2 expression in solid tumors. Based on the research of companion diagnostic tools, new therapeutic methods targeting Trop2 can also be further developed.

[0005] The applicant's previous series of basic and clinical studies have shown that by cleverly combining the extraordinary targeting specificity of antibodies with the superior sensitivity and resolution of positron emission tomography (PET), immuno-PET can better display the distribution and abundance of targets of interest in the body, especially heterogeneous expression, compared with immunohistochemistry (IHC) or other traditional predictive markers, and better predict the response to targeted therapy or immunotherapy (Chem Rev. 2020; 120 (8): 3787-3851.). For example, the value of immuno-PET imaging probes targeting human epidermal growth factor receptor 2 in breast cancer has been clinically verified. Based on the above evidence and our previous findings, we hypothesize that immuno-PET imaging probes targeting Trop2 can non-invasively display Trop2 expression in tumors and provide a better method for the diagnosis and monitoring of Trop2-positive solid tumors. In addition, there is evidence that radioimmunotherapy (RIT) and pre-targeted radioimmunotherapy (pRIT) may help cancer patients to alleviate their condition for a long time and even eradicate various types of cancer.

[0006] Currently, there is no Trop2-specific Nanobodies Reports on molecular imaging probes or radionuclide-labeled diagnostic and therapeutic probes. There are reports on molecular imaging probes based on Trop2 monoclonal antibodies (Eur J Nucl Med Mol Imaging. 2022 Feb; 49(3): 861-870.) and radionuclide-labeled diagnostic and therapeutic probes (Eur J Nucl Med Mol Imaging. 2022 Dec; 50(1): 168-183.). However, the application of radiolabeled monoclonal antibodies is seriously hindered by high cost, the necessity of using long half-life radionuclides, the cumbersome imaging process within a week, and the associated radiation exposure. In order to improve the application of antibody diagnosis in clinical practice, the field of molecular imaging is actively exploring pre-targeted imaging strategies or using antibody derivatives with smaller molecular weight to achieve same-day molecular imaging. Among small antibody forms, nanobodies or single-domain antibodies from Camelidae are the smallest antigen-binding moieties with a molecular weight of approximately 15 kDa. Small size, high affinity, and ease of engineering make nanobodies an excellent alternative for molecular imaging (J Nucl Med 2022 Oct; 63(10): 1705-1709). In recent years, we have focused on the development and clinical translation of nanobody-derived tracers to leverage their superior molecular imaging properties. Currently, there are no Trop2-specific nanobody molecular imaging probes available domestically or internationally. Summary of the Invention

[0007] In order to fill the gap in this field, the present invention provides a method for preparing and applying a Trop2-specific integrated diagnostic and therapeutic molecular imaging probe. The construction of a Trop2-targeted diagnostic and therapeutic pair derived from nanobodies is specifically described, and its diagnostic and therapeutic value in cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models is characterized. Therefore, the present invention has developed a nanobody 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 translation and application.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] In a first aspect, the present invention provides a Trop2 nanobody, including Trop2-specific nanobody WWD98, Trop2-specific nanobody WWD328 or Trop2-nonspecific nanobody WWD161;

[0010] The WWD98 has a sequence that is 70-100% homologous to the amino acid sequence shown in SEQ ID No. 1;

[0011] The WWD328 has a sequence with 70-100% homology to the amino acid sequence shown in SEQ ID No. 3;

[0012] The WWD161 has a sequence with 70-100% homology to the amino acid sequence shown in SEQ ID No. 5.

[0013] Preferably, the WWD98 has a sequence that is 70-100% homologous to the gene sequence shown in SEQ ID No. 2;

[0014] The WWD328 has a sequence with 70-100% homology to the gene sequence shown in SEQ ID No. 4;

[0015] The WWD161 has a sequence that is 70-100% homologous to the gene sequence shown in SEQ ID No. 6.

[0016] Preferably, the WWD98 has the same amino acid sequence as shown in SEQ ID No. 1, and the gene sequence as shown in SEQ ID No. 2;

[0017] The WWD328 has the same amino acid sequence as shown in SEQ ID No. 3 and the same gene sequence as shown in SEQ ID No. 4;

[0018] The WWD161 has the same amino acid sequence as shown in SEQ ID No.5 and the same gene sequence as shown in SEQ ID No.6.

[0019] Preferably, the Trop2 nanobody comprises the Trop2-specific nanobody WWD98 or the Trop2-specific nanobody WWD328.

[0020] In a second aspect, the present invention provides a use of a nanobody in preparing a Trop2 molecular imaging probe; the Trop2 molecular imaging probe includes a Trop2-specific molecular imaging probe and a Trop2-nonspecific molecular imaging probe.

[0021] Preferably, the Trop2-specific molecular imaging probe is a Trop2-specific integrated diagnosis and treatment molecular imaging probe.

[0022] In a third aspect, the present invention provides a Trop2-specific molecular imaging probe, wherein the probe comprises a tumor targeting group and a radionuclide;

[0023] The tumor targeting group is selected from WWD98 or WWD328;

[0024] The radionuclide is selected from 68 Ga, 18 F. 64 Cu or 89 Zr; 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 DFO.

[0025] Preferably, the probe is specific for human Trop2 68 Ga-labeled monovalent nanoantibody probe[ 68 Ga]Ga-NOTA-WWD98 or [ 68 Ga]Ga-NOTA-WWD328.

[0026] In a fourth aspect, the present invention provides a Trop2 non-specific molecular imaging probe, characterized in that the probe comprises a tumor targeting group and a radionuclide;

[0027] The tumor targeting group is selected from WWD161;

[0028] The radionuclide is selected from 68 Ga, 18 F. 64 Cu or 89 Zr; when the radionuclide is selected from68 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 DFO.

[0029] Preferably, the probe is non-specific for human Trop2 68 Ga-labeled monovalent nanoantibody probe[ 68 Ga]Ga-NOTA-WWD161.

[0030] In a fifth aspect, the present invention provides a method for preparing the aforementioned Trop2-specific molecular imaging probe or Trop2-nonspecific molecular imaging probe, wherein the probe comprises a tumor-targeting gene, a chelating agent, and a radionuclide. 68 Ga, 64 Cu or 89 When Zr is used, the preparation method thereof comprises the following steps:

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

[0032] The probe comprises a tumor targeting gene and a radionuclide 18 F, wherein the preparation method comprises the following steps:

[0033] Using radionuclides 18 F labeled small molecule compound precursor, 18 F marks the precursor;

[0034] Preparation of DBCO randomly coupled nanobodies;

[0035] Will 18 The F-labeled precursor undergoes a click chemistry reaction with the nanoantibody randomly coupled to DBCO to obtain the probe.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1) The Trop2-specific molecular imaging probe constructed based on the Trop2-specific nanoantibodies WWD98 and WWD328 of the present invention can be used for immuno-PET imaging; immuno-PET imaging performed by the probe can non-invasively display Trop2 expression in tumors, realizing non-invasive visualization of human Trop2 molecular expression, providing a better method for the diagnosis and monitoring of Trop2-positive solid tumors, and further realizing non-invasive diagnosis of various tumors such as pancreatic cancer and gastric cancer.

[0038] 2) The probe described in this invention offers advantages such as simple preparation, low cost, high specificity, high stability, short imaging cycle, low radiation dose, and ease of clinical translation. Its clinical translational application is expected to enable non-invasive visualization of heterogeneous Trop2 expression, screening for patients with high Trop2 expression, and further development of Trop2-specific radioimmunotherapy, thereby achieving integrated target-specific diagnosis and treatment of Trop2-positive tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0040] Figure 1 The expression of nanoantibodies WWD98 and WWD161 was determined by SDS-PAGE; Figure 1 A is the WWD98 test result, Figure 1 B is the test results of bovine serum albumin (BSA), WWD98, and WWD161; R stands for reducing, NR stands for non-reducing, and BSA stands for bovine serum albumin (BSA);

[0041] Figure 2 The results of flow cytometric detection of Trop2 in pancreatic cancer cell lines BxPC-3 and AsPC-1 are shown;

[0042] Figure 3 Results of affinity determination of nanobodies WWD98 and WWD328 to human Trop2;

[0043] Figure 4 for[ 68 Ga]Ga-NOTA-WWD98, [ 68 Ga]Ga-NOTA-WWD328 and [ 68 Radiochemical purity (PCR) results of Ga]Ga-NOTA-WWD161; the left figure shows the RCP results of the unpurified probe; the right figure shows the RCP results of the purified probe;

[0044] Figure 5 for[ 68 Ga]Ga-NOTA-WWD98, [ 68 Ga]Ga-NOTA-WWD161 and conventional [ 18 PET / CT results of the experiment on diagnosis of pancreatic cancer using [F]-FDG immunoPET imaging;

[0045] Figure 6 for[ 68 Ga]Ga-NOTA-WWD98, [68 Ga]Ga-NOTA-WWD161 and conventional [ 18 ROI quantitative results of the experiment of [F]-FDG immunoPET imaging for the diagnosis of pancreatic cancer;

[0046] Figure 7 for[ 68 Ga]Ga-NOTA-WWD98 and [ 68 Ga]Ga-NOTA-WWD161 or regular [ 18 F]-FDG ROI comparison results in the BxPC-3 tumor model;

[0047] Figure 8 for[ 68 Ga]Ga-NOTA-WWD98, [ 68 In vitro biodistribution data of Ga]Ga-NOTA-WWD161 immunoPET imaging for pancreatic cancer diagnosis;

[0048] Figure 9 The results of immunohistochemical staining of BxPC-3 tumor tissue sections;

[0049] Figure 10 The results of immunohistochemical staining of As-PC-1 tumor tissue sections are shown;

[0050] Figure 11 for[ 68 PET / CT imaging results of the experiment using Ga]Ga-NOTA-WWD328 immunoPET imaging to diagnose pancreatic cancer;

[0051] Figure 12 for[ 68 Ga]Ga-NOTA-WWD98, [ 68 PET / CT imaging results of the experiment using Ga]Ga-NOTA-WWD161 immunoPET imaging to diagnose gastric cancer (No. 490PDX tumor);

[0052] Figure 13 for[ 68 Ga]Ga-NOTA-WWD98, [ 68 ROI quantitative results of the immunoPET imaging for gastric cancer diagnosis using Ga]Ga-NOTA-WWD161;

[0053] Figure 14 The immunohistochemical staining results of No.490PDX tumor sections. DETAILED DESCRIPTION

[0054] 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 examples. It should be noted that the present invention is not limited to the ad hoc method described herein, scheme, cell line, construct and reagent, and is equally changeable. Unless otherwise defined, all technical and scientific terms used in this specification sheet are identical with the meaning generally understood by those skilled in the art of the present invention. The terms used in this specification sheet in the specification sheet of the present invention are just for the purpose of describing specific embodiments and are not used to limit the present invention.

[0055] Before the present invention, antibody-drug conjugates targeting Trop2 had entered clinical trials. There are also reports that immunoPET (ImmunoPET), which organically combines the high sensitivity of PET imaging and the high affinity of antibodies, is a new type of molecular imaging modality that can be used for non-invasive visualization of tumor targets and target-specific diagnosis of tumors (Chem Rev. 2020; 120(8): 3787-3851.). However, there are no reports of Trop2-specific nanoantibody molecular imaging probes and radionuclide-labeled integrated diagnostic and therapeutic probes in clinical practice and literature reports. There are reports of molecular imaging probes based on Trop2 monoclonal antibodies (Eur J Nucl Med Mol Imaging. 2022Feb; 49(3): 861-870.) and radionuclide-labeled integrated diagnostic and therapeutic probes (Eur J Nucl Med Mol Imaging. 2022Dec; 50(1): 168-183.). However, the clinical translation and application of monoclonal antibody immuno-PET imaging probes are severely limited by factors such as high preparation cost, large molecular weight, long in vivo circulation time, long imaging cycle, high radiation dose, and significant toxic side effects. In comparison, nanoantibody immuno-PET imaging probes have the advantages of low preparation cost, small molecular weight, short in vivo circulation time, short imaging cycle, low radiation dose, and ease of clinical translation and application.

[0056] Based on this, the present invention aims to construct a Trop2-specific integrated diagnostic and therapeutic nanoantibody molecular imaging probe that can non-invasively visualize Trop2 expression within tumors, providing a better approach for diagnosing and monitoring Trop2-positive solid tumors. Further development of novel Trop2-targeted therapeutic approaches is planned.

[0057] Example 1

[0058] 1) The Trop2-specific nanobody described in this patent is produced by the extracellular domain of human Trop2 protein (TR2-H5223S;

[0059] The specific immunization protocol is as follows:

[0060]

[0061]

[0062] 2) The phage screening library was further prepared by conventional laboratory methods such as separation of collected peripheral blood lymphocytes, RNA extraction, nested PCR, ligation of vector and target fragment, electroporation, colony PCR verification of insertion rate and library diversity analysis, and phage library packaging.

[0063] 3) Using the extracellular domain of the Trop2 protein (TR2-H5223S; Acrobiosystems) as the target protein, affinity panning, post-panning library amplification, identification and analysis of specific phage clones, monoclonal ELISA, and phylogenetic tree analysis of the monoclonal sequences were performed. After four rounds of screening, 96 clones were identified on the first titer plate, 96 clones on the second titer plate, and 192 clones on the third titer plate (for a total of 384 clones), resulting in 106 positive clones. WWD98, WWD328, and WWD161 were further recombinantly expressed for the construction of immuno-PET imaging probes.

[0064] Example 2

[0065] This embodiment provides a method for preparing a Trop2-specific nanobody WWD98, wherein the amino acid sequence of the Trop2-specific nanobody WWD98 is shown in SEQ ID NO.1 and the gene sequence is shown in SEQ ID NO.2.

[0066] The specific steps are as follows:

[0067] 1) Using conventional molecular biological methods, the gene sequence shown in SEQ ID NO. 2 was cloned into the pET-30a(+) expression vector to obtain plasmid DNA containing the target antibody (WWD98).

[0068] 2) Expressing the above-mentioned target antibody in Escherichia coli (E. coli).

[0069] 2.1 E. coli transformation: First, remove BL21 (DE3) competent cells from -80°C and thaw 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 the competent cells on LB agar plates containing 50 μg / ml kanamycin; invert the agar plates and incubate at 37°C overnight.

[0070] 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 value reaches 0.6–0.8, add isopropylthiogalactoside (IPTG) to the culture tube to a concentration of 0.5 mM. Incubate at 15°C for 16 hours or 37°C for 4 hours (both incubation conditions are acceptable).

[0071] The expression of nanoantibody WWD98 was determined by SDS-PAGE. The specific steps are as follows: first, a 1.5 mm thick, 15 well gel was prepared according to the method of the SDS-PAGE gel kit, and the metal bath was preheated to 100 ° C. The protein sample containing loading buffer (5X) (i.e., the incubation solution obtained after incubation in the above step 2.2) was heated for 5 minutes; after the SDS-PAGE gel was assembled, 500 ml of 1x SDS-PAGE buffer was added, and the protein sample was slowly spotted into the loading well. The protein sample was placed in an 80V constant voltage electric bath for about 30 minutes. After the bromophenol blue indicator passed through the concentrated gel, the voltage was adjusted to 120V. Electrophoresis was performed to the bottom of the gel, and the gel was removed. After heating and staining in Coomassie blue dye for 50 minutes, the gel was removed and decolorized with decolorizing solution until the background was clean and the bands were clear, and then the gel was photographed. Figure 1 As shown, Figure 1 A is the WWD98 test result, Figure 1 B is the test results of bovine serum albumin (BSA), WWD98 and WWD161.

[0072] The results of the affinity determination between the nanobody WWD98 and human Trop2 are as follows Figure 3 As shown, its K D The value is 768pM.

[0073] Example 3

[0074] This example provides a method for preparing a Trop2-specific nanoantibody WWD328, wherein the amino acid sequence of the Trop2-specific nanoantibody WWD328 is shown in SEQ ID NO.3 and the gene sequence is shown in SEQ ID NO.4.

[0075] The specific steps are as follows:

[0076] 1) Using conventional molecular biological methods, the gene sequence shown in SEQ ID NO. 4 was cloned into the pET-30a(+) expression vector to obtain plasmid DNA containing the target antibody (WWD328).

[0077] 2) Express the above target antibody in E. coli

[0078] Same as step 2) in Example 1.

[0079] The affinity determination results of the nanobody WWD328 and human Trop2 are as follows Figure 3 As shown, its K D The value is 9nM.

[0080] Example 4

[0081] This example provides a method for preparing a Trop2 non-specific nanoantibody WWD161, wherein the amino acid sequence of the Trop2 non-specific nanoantibody WWD161 is shown in SEQ ID NO.5 and the gene sequence is shown in SEQ ID NO.6.

[0082] The specific steps are as follows:

[0083] 1) Using conventional molecular biological methods, the gene sequence shown in SEQ ID NO. 6 was cloned into the pET-30a(+) expression vector to obtain plasmid DNA containing the target antibody (WWD161).

[0084] 2) Express the above target antibody in E. coli

[0085] Same as step 2) in Example 1.

[0086] The expression of nanobody WWD161 was determined by SDS-PAGE, and the specific steps were the same as those described in Example 1. Figure 1 As shown in B, the results show that the purity of WWD161 is >95%, which meets the basic requirements of molecular imaging experiments.

[0087] The results of affinity determination between the nanobody WWD161 and human Trop2 showed that WWD161 had no affinity for human Trop2.

[0088] Example 5

[0089] This example provides a Trop2-specific 68 Ga-labeled monovalent nanoantibody probe[ 68 Ga]Ga-NOTA-WWD98, Trop2 specificity 68 Ga-labeled nanoantibody probes[ 68 Ga]Ga-NOTA-WWD328 and Trop2 nonspecific 68 Ga-labeled nanoantibody probes[ 68 The preparation method of Ga]Ga-NOTA-WWD161 is as follows:

[0090] 1) NOTA modification of WWD98, WWD328, and WWD161 to prepare intermediates NOTA-WWD98, NOTA-WWD328, and NOTA-WWD161: 1 mg of each WWD98, WWD328, and WWD161 was dissolved in 1 mL of phosphate-buffered saline (PBS) to prepare a nanobody solution. The pH of each nanobody solution was then adjusted to 9.0–10 by adding 0.1 mL of 0.1 M sodium carbonate (Na2CO3, pH = 11.4) buffer. The resulting reaction volume was 1.1 mL. Freshly dissolved p-SCN-Bn-NOTA (CAS Number: 170597-66-8; Macrocyclics) in dimethyl sulfoxide (DMSO) was added to the reaction system at a molar ratio of p-SCN-Bn-NOTA:nanobody = 10:1. The reaction system was left to react at room temperature for 2 hours, and then PBS was used as the mobile phase. The NOTA-modified nanoantibodies were purified using a pre-equilibrated PD-10 desalting column (GE Healthcare) to collect NOTA-WWD98, NOTA-WWD328 and NOTA-WWD161 respectively; then concentrated using an ultrafiltration tube with a cutoff value of 10 kDa (Merck Millipore), and the concentrations of NOTA-WWD98, NOTA-WWD328 and NOTA-WWD161 were determined using NanoDrop, and the separate devices were stored at -20°C for use.

[0091] 2) 68 Preparation of Ga-labeled NOTA-WWD98, NOTA-WWD328 and NOTA-WWD161 68 Ga]Ga-NOTA-WWD98, [ 68 Ga]Ga-NOTA-WWD328 and [ 68 Ga]Ga-NOTA-WWD161: Elute the GaGe generator (Eckert & Ziegler Radiopharma Inc) with 4 mL of 0.05 M hydrochloric acid solution (HCl) and collect an equal volume of Ga]Ga-NOTA-WWD161 with an activity of approximately 370–555 MBq. 68 Ga eluent; take the middle section with the highest activity 68 2 mL of Ga eluent, adjusted with 0.1 mL of 1 M sodium acetate solution (NaOAc) 68 Ga eluent pH was adjusted to 4.0–4.5; 200 μg of NOTA-WWD98, NOTA-WWD328 and NOTA-WWD161, which had been coupled, were added to 68The reaction system volume was <2.5 mL. The reaction system was placed in a constant temperature oscillator at room temperature for 5–10 minutes. After the labeling reaction, PBS was used as the mobile phase and the free 68 Ga, purified to obtain the final product [ 68 Ga]Ga-NOTA-WWD98, [ 68 Ga]Ga-NOTA-WWD328 and [ 68 Ga]Ga-NOTA-WWD161; the undecayed corrected radiochemical yield (RCY) obtained according to the above steps was >50%.

[0092] 3)[ 68 Ga]Ga-NOTA-WWD98, [ 68 Ga]Ga-NOTA-WWD328 and [ 68 Ga]Ga-NOTA-WWD161 quality control: aspirate 10 μL [ 68 Ga]Ga-NOTA-WWD98, [ 68 Ga]Ga-NOTA-WWD328 and [ 68 Ga]Ga-NOTA-WWD161 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 each probe was determined by radio-thin layer chromatography (Radio-TLC, Eckert & Ziegler Radiopharma Inc). Figure 4 As shown, the left figure is the RCP result of the unpurified probe; the right figure is the RCP result of the purified probe, and it can be seen that the freshly prepared [ 68 Ga]Ga-NOTA-WWD98, [ 68 Ga]Ga-NOTA-WWD328 and [ 68 The RCP of Ga]Ga-NOTA-WWD161 is greater than 90%.

[0093] Verification Example

[0094] 1) Construction of Trop2-positive tumor-bearing mouse model

[0095] Using anti-human Trop2 monoclonal antibody (10428-MM01-F, Sino Biological Inc.) as the primary antibody, flow cytometry experiments revealed that pancreatic cancer cell lines BxPC-3 (high Trop2 expression) and AsPC-1 (low Trop2 expression), such as Figure 2 As shown. 2×10 6BxPC-3 and AsPC-1 cells were suspended in a mixture of PBS and Matrigel (Corning) (PBS:Matrigel ratio:1) and then injected into the right shoulder of 4–5-week-old Balb / c nude mice to establish subcutaneous pancreatic cancer models (i.e., BxPC-3 and AsPC-1 tumor models). A 2 mm x 2 mm x 2 mm No.490 PDX tissue explant was inoculated into the right shoulder of NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22 / Nju) mice to establish a subcutaneous gastric cancer PDX model (i.e., No.490 PDX tumor model).

[0096] 2)[ 68 Ga]Ga-NOTA-WWD98, [ 68 Ga]Ga-NOTA-WWD161 and conventional [ 18 Experimental Study on Diagnosis of Pancreatic Cancer by [F]-FDG ImmunoPET Imaging

[0097] The small animal PET / CT imaging acquisition involved in this study was completed using the IRIS small animal PET / CT scanner (Inviscan Imaging Systems). Each BxPC-3 tumor model mouse and AsPC-1 tumor model mouse were injected with 3.7-7.4 MBq [ 68 Ga]Ga-NOTA-WWD98, [ 68 Ga]Ga-NOTA-WWD161 and [ 18 F]-FDG (3 mice per group), anesthetized mice with isoflurane mixed with oxygen (concentration of 2%) 1 hour after injection, and placed the deeply anesthetized mice in a supine position on the PET / CT scanning bed, and PET and CT images were acquired continuously, and image reconstruction was completed using the IRIS system's built-in software. It is worth noting that [ 18 Mice in the [F]-FDG group were fasted and deprived of water the day before imaging. After injection of the imaging agent, they were kept warm and placed under low-flow anesthesia. Regions of interest (ROIs), including the heart and major organs (liver, lungs, kidneys, and muscles), were delineated on reconstructed PET images using the OsiriX Lite image processing workstation (Pixmeo SARL). Radioactivity uptake in these organs was calculated as %ID / g (percent of injected dose per gram). Figure 5 Display PET / CT imaging results. Figure 6 and Figure 7 Display ROI quantitative results, Figure 8 Graphs showing in vitro biodistribution data. Figure 5It can be seen that the Trop2-specific nanobody probe [ 68 Ga]Ga-NOTA-WWD98 has a higher uptake in BxPC-3 tumor tissues that highly express Trop2, and also has a higher nonspecific uptake in major excretion (kidney) and metabolic (liver) tissues. 68 The distribution of Ga]Ga-NOTA-WWD98 in vivo. In addition, the results of in vitro biodistribution experiments further revealed the distribution of the probe in major tissues and organs in vivo. By analyzing the ROI data and biodistribution data of tumor models with two different Trop2 expression levels, and comparing [ 68 Ga]Ga-NOTA-WWD98 and nonspecific probes [ 68 ROI data and biodistribution data of Ga]Ga-NOTA-WWD161 imaging and comparison [ 68 Ga]Ga-NOTA-WWD98 and regular[ 18 F]-FDG PET / CT imaging ROI data showed that [ 68 The [Ga]Ga-NOTA-WWD98 probe can noninvasively visualize Trop2 expression.

[0098] In this experiment, we also used Trop2-specific antibody (sc-376746, Santa Cruz Biotechnology) to perform immunohistochemical staining to confirm the expression of Trop2 in the tumor ( Figure 9 and Figure 10 ).

[0099] 3)[ 68 Experimental Study on the Diagnosis of Pancreatic Cancer by ImmunoPET Imaging with Ga]Ga-NOTA-WWD328

[0100] The imaging steps and data processing of this experiment are basically the same as those in step 2). The only difference is that half an hour after the injection, the deeply anesthetized mice were placed in a supine position on the PET / CT scanner bed, and PET and CT images were continuously acquired. Image reconstruction was completed using the software provided by the IRIS system. 68 The experimental results of Ga]Ga-NOTA-WWD328 immunoPET imaging for the diagnosis of pancreatic cancer (BxPC-3 tumor model) are as follows Figure 11 The results show that: 68 Ga]Ga-NOTA-WWD328 immunoPET imaging has the same 68 Ga]Ga-NOTA-WWD98 has similar diagnostic efficacy.

[0101] 4)[ 68Experimental Study on the Diagnosis of Gastric Cancer by ImmunoPET Imaging with Ga]Ga-NOTA-WWD98

[0102] The imaging steps and data processing of this experiment are basically the same as the experimental method in step 2), the only difference is that this experiment uses No. 490PDX tumor model mice. Figure 12 Display PET / CT imaging results. Figure 13 Display ROI quantitative results. Figure 12 As shown, compared with nonspecific probes [ 68 Ga]Ga-NOTA-WWD161 compared to [ 68 Ga]Ga-NOTA-WWD98 can successfully delineate subcutaneous PDX gastric cancer tumors. ROI quantitative analysis results also further confirmed [ 68 The tumor uptake value of Ga]Ga-NOTA-WWD98 was significantly higher than that of [ 68 Ga]Ga-NOTA-WWD161, and [ 68 Ga]Ga-NOTA-WWD98 has a higher target-to-substance ratio ( Figure 13 ).

[0103] This study also used Trop2-specific antibody sc-376746 for immunohistochemical staining to confirm the expression of Trop2 in the tumor ( Figure 14 ).

[0104] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should be understood that ordinary technicians in this field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in this field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the existing technology should be within the scope of protection determined by the claims. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the description of the present invention; further, for ordinary technicians in this field, they can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A nanobody WWD98, characterized in that The WWD98 has the amino acid sequence shown in SEQ ID No.

1.

2. A gene encoding the Nanobody WWD98 according to claim 1, characterized in that Its gene sequence is shown as SEQ ID No.

2.

3. Use of the nanobody WWD98 according to claim 1 or the gene encoding the nanobody WWD98 according to claim 2 in the preparation of a Trop2 molecular imaging probe.

4. A Trop2-specific molecular imaging probe, characterized in that: The probe includes a tumor targeting group and a radionuclide; The tumor targeting group is WWD98 as described in claim 1; The radionuclide is selected from 68 Ga, 18 F. 64 Cu or 89 Zr; when the radionuclide is selected from 68 Ga, 64 Cu or 89 When Zr, the probe further comprises a chelating agent, which is selected from p -SCN-Bn-NOTA, p -SCN-Bn-DOTA or DFO.

5. The Trop2-specific molecular imaging probe according to claim 4, characterized in that The probe is specific for human Trop2 68 Ga-labeled monovalent nanoantibody probe[ 68 Ga]Ga-NOTA-WWD98.

6. A method for preparing a Trop2-specific molecular imaging probe according to claim 4, characterized in that: The probe comprises the tumor targeting group, the chelating agent and the radionuclide as claimed in claim 4 68 Ga, 64 Cu or 89 When Zr is used, the preparation method thereof comprises the following steps: The tumor targeting group is modified with a chelating agent to form a modified nanobody; then a radionuclide is used 68 Ga, 64 Cu or 89 The modified nanobody is labeled with Zr to obtain a probe; The probe comprises a tumor targeting group and a radionuclide 18 F, wherein the preparation method comprises the following steps: Using radionuclides 18 F labeled small molecule compound precursor, 18 F marks the precursor; Preparation of DBCO randomly coupled tumor targeting groups; Will 18 The F-labeled precursor undergoes a click chemistry reaction with a tumor-targeting group randomly coupled to DBCO to obtain the probe.

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