Methods for preparing and using CD70-specific diagnostic and imaging probes

Abstract

The present invention provides a method for preparing and using a CD70-specific diagnostic-integrated molecular imaging probe, which comprises a tumor targeting group and a radionuclide, the tumor targeting group being selected from a CD70-specific nanobody or a CD70-specific nanobody fusion protein, the CD70-specific nanobody being B3 or B6 having the amino acid sequences set forth in SEQ ID NO. 1 and SEQ ID NO. 3, and the CD70-specific nanobody fusion protein being ABDB3 or ABDB6 having the amino acid sequences set forth in SEQ ID NO. 5 and SEQ ID NO. 7. The present invention achieves noninvasive visualization of human CD70 molecule expression and realizes noninvasive diagnosis of renal cell carcinoma. This probe has the advantages of simple preparation, low cost, high specificity, high stability, short imaging cycle, low radiation dose, and easy clinical translation.

Description

[Technical Field]

[0001] The present invention relates to the technical field of molecular imaging probes, and particularly to the preparation and use of CD70-specific diagnostic-integrated molecular imaging probes. [Background technology]

[0002] In 1993, Belgian scientists Hamers et al. reported for the first time in Nature the presence of antibodies naturally lacking light chains in the peripheral blood of alpacas (Nature. 1993;363(6428):446-8). Antibodies with such special domains are called heavy-chain antibodies (HCAbs). By cloning the variable domain of a heavy-chain antibody using molecular biological techniques, it is possible to obtain antigen-binding fragments containing only the heavy-chain variable domain, i.e., nanobodies (VHH, Variable Domain of Heavy Chain of Heavy Chain Antibody). VHH crystals are 2.5 nm wide, 4 nm long, and have a molecular weight of only 15 kDa, and are therefore also called nanobodies (Nanobodies®, a registered trademark of Ablynx). Nanobodies are currently known as the smallest antibody units capable of binding to target antigens, and have the advantages of high affinity, small molecular weight, low preparation costs (they can be expressed in E. coli or eukaryotic expression systems such as yeast or Chinese hamster ovary cells), and ease of clinical translation and widespread application.

[0003] Nanoantibodies have recently become a target vector for constructing molecular imaging probes (Theranostics. 2014;4(4):386~98.; J Nucl Med. 2022 Oct;63(10):1705~1709.). Currently, various short-half-life nuclides are used to label nanoantibodies and prepare nanoantibody molecular imaging probes. Technetium-99m ( 99m Tc;T 1 / 2A nano-antibody probe targeting programmed death-ligand 1 (PD-L1) labeled with gallium-68 ( ) has been successfully introduced into the clinic for the non-invasive diagnosis of patients with non-small cell lung cancer (J Nucl Med. 2019;60(9):1213~1220.), and gallium-68 ( 68 Ga;T 1 / 2 A radionuclide-labeled nanoantibody probe targeting human epidermal growth factor receptor (HER2) has also been successfully introduced into clinical practice for the noninvasive diagnosis of breast cancer (J Nucl Med. 2016;57(1):27-33). These examples demonstrate that radionuclide-labeled nanoantibody probes have promising applications for clinical translation, including early noninvasive diagnosis of human malignancies, visualization of important pathogenic targets, screening of monoclonal antibody (mAb)-treated patients, and evaluation of therapeutic efficacy after mAb treatment.

[0004] The cluster of differentiation antigen CD70 is a type II transmembrane glycoprotein and a member of the tumor necrosis factor superfamily. It is also a ligand for CD27. Binding of the two antigens induces activation of various signaling pathways, promoting gene transcription, cell proliferation, and differentiation. Normally, CD70 is transiently expressed only on the surface of activated T cells, B cells, and mature dendritic cells. Recent studies have demonstrated that CD70 expression is elevated in various hematological malignancies and solid tumors. Compared with normal renal tissue, CD70 expression is significantly elevated in renal cell carcinoma, particularly clear cell renal carcinoma and sarcomatoid renal cell carcinoma, and high CD70 expression is associated with poor prognosis. CD70 expressed on tumor cells binds to CD27 on the surface of T cells, activating the apoptotic protein Siva, which induces cytotoxicity and apoptosis in immune cells, thereby achieving immune evasion. The differential expression of CD70 between normal tissues and tumors makes it a promising tumor-specific marker, potentially avoiding potential side effects. Currently, drugs targeting CD70, such as monoclonal antibodies, antibody-drug conjugates, and chimeric antigen receptor T cells, have entered clinical trials. The anti-CD70 monoclonal antibody SGN-CD70A was used in a phase I clinical trial in patients with metastatic renal cell carcinoma, demonstrating a clinical benefit rate of 78%. Therefore, there is an urgent need to develop diagnostic tools targeting CD70 to visualize and monitor CD70 expression in solid tumors. Research into companion diagnostic tools may also lead to the further development of novel therapeutic approaches targeting CD70.

[0005] The applicant's previous series of basic and clinical studies have demonstrated that by skillfully combining the excellent target specificity of antibodies with the excellent sensitivity and resolution of positron emission tomography (PET), immuno-PET can better indicate the distribution and abundance of targets of interest in vivo, particularly heterogeneous expression, and better predict response to targeted therapy or immunotherapy compared with immunohistochemistry (IHC) or other conventional predictive markers (Chem Rev. 2020;120(8):3787-3851). However, the clinical application of monoclonal antibody immuno-PET imaging probes is significantly limited due to factors such as high preparation costs, large molecular weight, long circulation time in the body, long imaging cycle, high radiation dose, and significant toxic side effects. Currently, there are no reports of CD-specific molecular imaging probes or nuclide-labeled diagnostic probes in clinical practice or literature. To fill this gap, experts in the field are working to develop nanoantibody immunoPET imaging probes that are inexpensive to prepare, have small molecular weights, short circulation times in the body, have short imaging cycles, low radiation doses, and are easy to apply to clinical translation. Summary of the Invention [Problem to be solved by the invention]

[0006] To solve the above problems, the present invention aims to provide a method for preparing and using a CD70-specific diagnostic and imaging-integrated molecular probe. [Means for solving the problem]

[0007] The object of the present invention is achieved by the following technical solutions: In a first aspect, the present invention provides a CD70-specific Nanobody which is B3 having the amino acid sequence shown in SEQ ID NO. 1, or B6 having the amino acid sequence shown in SEQ ID NO. 3.

[0008] Preferably, the gene sequence of B3 is shown in SEQ ID NO.2, and the gene sequence of B6 is shown in SEQ ID NO.4.

[0009] In a second aspect, the present invention provides the use of a CD70-specific Nanobody as described above in the preparation of a CD70-specific Nanobody fusion protein.

[0010] The Nanobody fusion protein comprises GGGGS linkers of different amino acid lengths, bridging the serum protein binding domain (ABD) and the Nanobody (B3 or B6), and the linker "GGGGS" may have 1 to 10 pairs, specifically 1 pair, 2 pairs, 3 pairs, 4 pairs, 5 pairs, 6 pairs, 7 pairs, 8 pairs, 9 pairs or 10 pairs.

[0011] Preferably, the linker is triplicate and has the amino acid sequence shown in SEQ ID NO:9 (ie, GGGGSGGGGSGGGGS) and the gene sequence shown in SEQ ID NO:10.

[0012] In a third aspect, the present invention provides a CD70-specific Nanobody fusion protein which is ABDB3 having the amino acid sequence set forth in SEQ ID No.5, or ABDB6 having the amino acid sequence set forth in SEQ ID No.7.

[0013] Preferably, the gene sequence of ABDB3 is shown in SEQ ID NO.6, and the gene sequence of ABDB6 is shown in SEQ ID NO.8.

[0014] The method for preparing a CD70-specific Nanobody or a CD70-specific Nanobody fusion protein according to the present invention is as follows: the gene sequence of a CD70-specific Nanobody or a CD70-specific Nanobody fusion protein (as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8) is cloned into an expression vector, and then the gene sequence is transformed into an expression host strain, and the transformed strain is expanded, expression is induced and purified to obtain the CD70-specific Nanobody or CD70-specific Nanobody fusion protein.

[0015] In a fourth aspect, the present invention provides the use of a CD70-specific Nanobody as defined above or a CD70-specific Nanobody fusion protein as defined above in the preparation of a CD70-specific diagnostic-integrated molecular imaging probe. In a fifth aspect, the present invention provides a CD70-specific diagnostic-integrated molecular imaging probe comprising a tumor targeting group selected from the above CD70-specific Nanobody or the above CD70-specific Nanobody fusion protein, and a radionuclide. Preferably, the radionuclide is 68 Ga, 18 F, 64 Cu, or 89 Zr,

[0016] The radionuclide 68 Ga, 64 Cu, or 89 When selected from Zr, the probe further comprises a chelating agent selected from p-SCN-Bn-NOTA or p-SCN-Bn-Deferoxamine.

[0017] Preferably, the probe is 68 Ga-labeled monovalent nanoantibody probe [ 68 Ga]Ga-NOTA-B3 and [ 68 Ga]Ga-NOTA-B6, 68 Ga-labeled nanoantibody fusion protein probe [ 68 Ga]Ga-NOTA-ABDB3 and [ 68Ga]Ga-NOTA-ABDB6, 18 F-labeled monovalent nanoantibody probe [ 18 F]F-B3 and [ 18 F]F-B6, 89 Zr-labeled nanoantibody fusion protein probe [ 89 Zr]Zr-DFO-ABDB3 and [ 89 Zr]Zr-DFO-ABDB6.

[0018] Preferably, the probes include a tumor targeting gene, a chelating agent, and a radionuclide. 68 Ga, 64 Cu, or 89 When Zr is contained, the preparation method is as follows: The tumor-targeting gene is modified with a chelator to form a modified nanobody, and the modified nanobody is then coupled to a radionuclide. 68 Ga or 89 The method includes a step of labeling with Zr to obtain a probe. Preferably, the probe comprises a tumor targeting gene and a radionuclide. 18 When F is contained, the preparation method is Low molecular weight compound precursors are treated with radionuclides 18 Labeled with F, 18 obtaining an F-labeled precursor; preparing a DBCO randomly coupled nanobody; 18 and performing click chemistry on the nanoantibody randomly coupled with the F-labeled precursor and DBCO to obtain the probe. [Effects of the Invention]

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention realizes non-invasive visualization of human CD70 molecule expression and further realizes non-invasive diagnosis of renal cell carcinoma. The probe used in the present invention has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging cycle, low radiation dose, and easy clinical translation. [Brief explanation of the drawings]

[0020] Other features, objects and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments, given with reference to the drawings, in which: [Figure 1] 1 shows the expression status of nanoantibodies B3 and B6 measured by SDS-PAGE. [Figure 2] 1 shows the expression status of nanobody fusion proteins ABDB3 and ABDB6 measured by SDS-PAGE. [Figure 3] This shows the results of CD70 staining of human renal cell carcinoma xenograft model No. 62 PDX. [Figure 4] The results show the affinity of Nanobodies B3 and B6, and Nanobody fusion proteins ABDB3 and ABDB6 to human CD70. The curves shown in each figure correspond to concentrations from lowest to highest, from bottom to top. [Figure 5] These are the results of measuring the affinity of nanobodies B3 and B6, and nanobody fusion proteins ABDB3 and ABDB6 with human serum albumin and mouse serum albumin. The curves shown in each figure correspond to concentrations from lowest to highest, from bottom to top. [Figure 6] The radiochemical purity of the probes [68Ga]Ga-NOTA-B3, [68Ga]Ga-NOTA-B6, [68Ga]Ga-NOTA-ABDB3, and [68Ga]Ga-NOTA-ABDB6 was measured using radioactive thin-layer chromatography. [Figure 7] 7A and 7B show experimental results of diagnosing renal cell carcinoma using [68Ga]Ga-NOTA-B3 immunoPET imaging. FIG. 7A shows a PET / CT image, FIG. 7B shows an ROI image, and FIG. 7C shows an in vitro biodistribution data map. [Figure 8] 8A and 8B show experimental results of diagnosing renal cell carcinoma using [68Ga]Ga-NOTA-B6 immunoPET imaging. FIG. 8A shows a PET / CT image, FIG. 8B shows an ROI image, and FIG. 8C shows an in vitro biodistribution data map. [Figure 9]These are the results of PET / CT images from a closed-loop experiment for diagnosing renal cell carcinoma using [68Ga]Ga-NOTA-B6 immunoPET imaging. [Figure 10] This shows the ROI results of a closure experiment for diagnosing renal cell carcinoma using [68Ga]Ga-NOTA-B6 immunoPET imaging. [Figure 11] These are in vitro biodistribution data results from a closure experiment for the diagnosis of renal cell carcinoma using [68Ga]Ga-NOTA-B6 immunoPET imaging. [Figure 12] PET / CT imaging results for the diagnosis of renal cell carcinoma using [68Ga]Ga-NOTA-ABDB3 and [68Ga]Ga-NOTA-ABDB6 immunoPET imaging. [Figure 13] ROI results for diagnosing renal cell carcinoma using [68Ga]Ga-NOTA-ABDB3 and [68Ga]Ga-NOTA-ABDB6 immunoPET imaging. [Figure 14] In vitro biodistribution data map results for the diagnosis of renal cell carcinoma using [68Ga]Ga-NOTA-ABDB3 and [68Ga]Ga-NOTA-ABDB6 immunoPET imaging. [Figure 15] Comparison of ROI and in vitro biodistribution data for [68Ga]Ga-NOTA-B3 and [68Ga]Ga-NOTA-ABDB3 in PET / CT images of a kidney cancer model. [Figure 16] Comparison of ROI and in vitro biodistribution data for [68Ga]Ga-NOTA-B6 and [68Ga]Ga-NOTA-ABDB6 in PET / CT images of a kidney cancer model. [Figure 17] The radiochemical purity of the probes [89Zr]Zr-DFO-ABDB3 and [89Zr]Zr-DFO-ABDB6 was measured by radioactive thin-layer chromatography. [Figure 18] PET / CT imaging results for the diagnosis of renal cell carcinoma using [89Zr]Zr-DFO-ABDB3 immunoPET imaging. [Figure 19] PET / CT imaging results for the diagnosis of renal cell carcinoma using [89Zr]Zr-DFO-ABDB6 immunoPET imaging. [Figure 20] ROI results for diagnosing renal cell carcinoma using [89Zr]Zr-DFO-ABDB3 immunoPET imaging. [Figure 21] ROI results for diagnosing renal cell carcinoma using [89Zr]Zr-DFO-ABDB6 immunoPET imaging. [Figure 22] In vitro biodistribution data of renal cell carcinoma using immunoPET imaging of [89Zr]Zr-DFO-ABDB3 and [89Zr]Zr-DFO-ABDB6. [Figure 23] This is the result of immunohistochemical staining of the tumor using the CD70-specific antibody E3Q1A. [Figure 24] These are PET / CT image results for diagnosing renal cell carcinoma using [18F]F-B6 immunoPET imaging. [Figure 25] ROI and in vitro biodistribution data for the diagnosis of renal cell carcinoma using [18F]F-B6 immunoPET imaging. DETAILED DESCRIPTION OF THE INVENTION

[0021] To facilitate understanding of the present invention, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments. It should be noted that the present invention is not limited to the specific methods, solutions, cell lines, constructs, and reagents described herein, and may similarly be modified. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of the present invention. The terms used in the present specification are intended to describe specific examples only and are not intended to limit the present invention.

[0022] The use of radiolabeled monoclonal antibodies is largely hindered by their high cost, the need for long-half-life radionuclides, the tedious imaging process spanning a week, and the associated radiation exposure. To improve the clinical application of antibody diagnostics, the field of molecular imaging is actively exploring pretargeted imaging strategies and the use of relatively small antibody derivatives for same-day imaging. Among small antibody forms, camelid-derived nanoantibodies or single-domain antibodies are the smallest antigen-binding moieties, with a molecular weight of approximately 15 kDa. Due to their small size, high affinity, and ease of engineering, nanoantibodies have become an excellent alternative for molecular imaging (J Nucl Med 2022 Oct;63(10):1705-1709). In recent years, the applicant has been devoting himself to the development and clinical translation of nanoantibody-derived tracers to demonstrate their excellent molecular imaging properties. Therefore, in this application, we developed CD70-specific nanoantibodies B3 and B6, and applied them to CD70-specific diagnostic and imaging integrated molecular imaging probes, and radiolabeled monovalent nanoantibody probes (e.g., 68 Ga]Ga-NOTA-B3 and [ 68 Ga]Ga-NOTA-B6, [ 18 F]F-B3 and [ 18 F]F-B6).

[0023] Although radiolabeled monovalent nanobody is an ideal companion diagnostic tool, its in vivo half-life is too short and its renal uptake is high, so there is still room for further improvement. In order to develop an integrated diagnostic and therapeutic platform, the applicant further introduced an albumin binding domain (ABD) targeting human / mouse albumin into the monovalent nanobody to obtain a CD70-specific nanobody fusion protein (ABDB3 or ABDB6), which was then applied to a CD70-specific diagnostic and therapeutic integrated molecular imaging probe, and radiolabeled nanobody fusion protein probe (e.g., [ 68 Ga]Ga-NOTA-ABDB3 and [ 68 Ga]Ga-NOTA-ABDB6, [ 89 Zr]Zr-DFO-ABDB3, [ 89Zr]Zr-DFO-ABDB6, [ 18 F]F-ABDB3, and [ 18 [F]F-ABDB6), thereby significantly prolonging the half-life of the monovalent nanobody derivative in vivo and further optimizing the pharmacokinetics and pharmacodynamics of the molecular imaging probe. Research has shown that bispecific nanobody derivatives that simultaneously target tumor antigens and albumin can improve biodistribution and serve as carriers for the development of therapeutic diagnostic kits. The CD70-specific diagnostic-integrated molecular imaging probe constructed by the present invention can noninvasively display CD70 expression in tumors and provide an improved method for the diagnosis and monitoring of CD70-positive solid tumors. [Example]

[0024] This example provides a method for preparing CD70-specific nanobody B3 and B6, the amino acid sequence of which is shown in SEQ ID NO. 1 and the nucleotide sequence of which is shown in SEQ ID NO. 2, and the amino acid sequence of which is shown in SEQ ID NO. 3 and the nucleotide sequence of which is shown in SEQ ID NO. 4, of the CD70-specific nanobody B6. The specific steps are as follows: 1) Using conventional molecular biology techniques, the base sequences shown in SEQ ID NO. 2 and SEQ ID NO. 4 were each cloned into the pET-30a(+) expression vector to obtain plasmid DNA containing the target antibody (B3 or B6).

[0025] 2) The above target antibody was expressed in E. coli. 2.1 Transformation of E. coli First, BL21(DE3) competent cells were removed from -80°C and placed on ice to thaw. 100 ng of plasmid DNA containing each antibody of interest was added to the BL21(DE3) competent cells and mixed gently. The competent cells were then placed on ice and incubated for 30 minutes. The competent cells were then heat-shocked at 42°C in a stationary state for 90 seconds. The competent cells were then placed on ice for 3 minutes. 100 μl of room temperature LB medium was added to the competent cells and incubated at 200 rpm and 37°C for 60 minutes. The cells were then plated on an LB agar plate containing 50 μg / ml kanamycin. The agar plate was then inverted and incubated overnight at 37°C.

[0026] 2.2 Small-scale test expression Monoclonals that were relatively well dispersed were randomly selected from the agar plate and inoculated into LB medium containing 50 μg / ml kanamycin. Each was then cultured and incubated at 200 rpm and 37°C. When the OD600 measurement value reached 0.6 to 0.8 hours, isopropylthiogalactoside (IPTG) was added to the culture tube to a concentration of 0.5 mM, and the tube was then incubated at 15°C for 16 hours or at 37°C for 4 hours (both culture conditions were possible).

[0027] 2.3 Measuring protein expression levels by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) After incubation, 450 μl of the culture medium was collected and the cell pellet was centrifuged. 300 μl of lysis solution (50 mM Tris, 150 mM NaCl, 5% glycerol, pH 8.0) was added, and the cells were sonicated for 1 minute to lyse. The lysed cell sample was then heated at 100°C for 10 minutes and centrifuged at 15,000 rpm for 5 minutes before poplar gene detection. The expression profiles of the target antibodies B3 and B6 obtained are shown in Figure 1. In Figure 1, lane 1 is Western blot labeling, lane 2 is reduced B3 or B6, and lane 3 is unreduced B3 or B6.

[0028] 3) Measurement of the affinity of target antibodies B3 and B6 to human CD70 Human recombinant CD40 extracellular domain was immobilized on a Biacore chip, and different concentrations of monovalent nanoantibodies (B3 or B6) were loaded as the flow phase and eluted. The results are shown in Figure 4. The affinity K values ​​for B3 and B6 were 5.688 nM and 3.732 nM, respectively. [Example]

[0029] This example provides a method for preparing CD70-specific Nanobody fusion proteins ABDB3 and ABDB6, which contain GGGGS linkers of different amino acid lengths linking the serum protein binding domain (ABD) and the Nanobody (B3 or B6), and the linker "GGGGS" can have one, two, three, four, five, six, seven, eight, nine or ten pairs. The Nanobody fusion proteins ABDB3 and ABDB6 prepared in this example employ three pairs of linkers, and have the amino acid sequences shown in SEQ ID NO.9 and the gene sequences shown in SEQ ID NO.10.

[0030] The CD70-specific Nanobody fusion protein ABDB3 has an amino acid sequence shown in SEQ ID NO. 5 and a nucleotide sequence shown in SEQ ID NO. 6, and the CD70-specific Nanobody fusion protein ABDB6 has an amino acid sequence shown in SEQ ID NO. 7 and a nucleotide sequence shown in SEQ ID NO. 8.

[0031] The specific preparation steps are as follows. 1) Using conventional molecular biology techniques, the base sequences shown in SEQ ID NO. 6 and SEQ ID NO. 8 were each cloned into the pET-30a(+) expression vector to obtain plasmid DNA containing the target fusion protein (ABDB3 or ABDB6).

[0032] 2) The above target fusion protein was expressed in E. coli. The specific procedure for step 2) was the same as that for step 2) in Example 1. The expression status of the obtained target fusion proteins ABDB3 and ABDB6 is shown in Figure 2, where lane 1 is Western blot labeling, lane 2 is ABDB3 or ABDB6 under reduced conditions, and lane 3 is ABDB3 or ABDB6 under non-reduced conditions.

[0033] 3) Measurement of the affinity of the target fusion proteins ABDB3 and ABDB6 to human CD70 Human recombinant CD40 extracellular domain was immobilized on a Biacore chip, and different concentrations of monovalent nanoantibodies (ABDB3 or ABDB6) were loaded as the flow phase and eluted. The K values ​​were 110.3 pM and 134.9 pM, respectively, as shown in Figure 4.

[0034] 4) Measurement of the affinity of the target fusion proteins ABDB3 and ABDB6 to human serum albumin and mouse serum albumin Recombinant human serum albumin or mouse serum albumin was immobilized on a Biacore chip, and different gradients of ABDB3 and ABDB6 were loaded as the mobile phase and then eluted. The measurement results are shown in Figure 5. The KD values ​​for ABDB3 with human serum albumin and ABDB6 with mouse serum albumin were 74.19 pM and 380.1 pM, respectively, and the KD values ​​for ABDB6 with human serum albumin and ABDB6 with mouse serum albumin were 88.46 pM and 454.1 pM, respectively. [Example]

[0035] In this example, human CD70-specific 68 Ga-labeled monovalent nanoantibody probe [ 68 Ga]Ga-NOTA-B3 and [ 68 Ga]Ga-NOTA-B6, human CD70 specific 68 Ga-labeled nanoantibody fusion protein probe [ 68 Ga]Ga-NOTA-ABDB3 and [ 68 A method for the preparation of [Ga]Ga-NOTA-ABDB6 is provided, and the detailed procedures are as follows.

[0036] 1) Modifying B3, B6, ABDB3, and ABDB6 with p-SCN-Bn-NOTA to prepare intermediates NOTA-B3, NOTA-B6, NOTA-ABDB3, and NOTA-ABDB6. 1 mg of B3, B6, ABDB3, or ABDB6 was dissolved in 1 mL of phosphate buffer solution (PBS) and 0.1 mL of 0.1 M sodium carbonate (NaCO, pH = 11.4) buffer to adjust the pH of the nanoantibody solution to 9.0-10 and the reaction volume to 1.1 mL. Freshly dissolved p-SCN-Bn-NOTA (CAS Number: 170597-66-8; Macrocyclics) in dimethyl sulfoxide (DMSO) was added to the nanoantibody solution at a molar ratio of p-SCN-Bn-NOTA:nanoantibody or fusion protein = 10:1. The resulting reaction system was reacted at room temperature for 2 hours, and then the NOTA-modified nanoantibodies were purified using a pre-equilibrated PD-10 desalting column (GE Healthcare) with PBS as the mobile phase to collect NOTA-B3, NOTA-B6, NOTA-ABDB3, or NOTA-ABDB6, which were then concentrated using an ultrafiltration tube with a cutoff value of 10 kDa (Merck Millipore). The concentrations of NOTA-B3, NOTA-B6, NOTA-ABDB3, or NOTA-ABDB6 were then measured using NanoDrop, and each was stored at -20°C for use.

[0037] 2) 68 NOTA-B3, NOTA-B6, NOTA-ABDB3, and NOTA-ABDB6 were labeled with Ga and [ 68 Ga]Ga-NOTA-B3,[ 68 Ga]Ga-NOTA-B6, [ 68 Ga]Ga-NOTA-ABDB3 and [ 68 [Ga]Ga-NOTA-ABDB6 was prepared

[0038] A germanium gallium generator (Eckert & Ziegler Radiopharma Inc.) was washed with 4 mL of 0.05 M hydrochloric acid solution (HCl) and charged with an equivalent volume of HCl with an activity of approximately 370–555 MBq. 68 The Ga eluent is collected and the most active intermediate stage is 68Take 2 mL of the Ga eluent and add 0.1 mL of 1 M sodium acetate solution (NaoAc). 68 Adjust the pH of the Ga eluent to 4.0-4.5, and take 200 μg of NOTA-B3, NOTA-B6, NOTA-DBAB3, and NOTA-ABDB6 for coupling. 68 The reaction mixture was added to the Ga eluent, and the resulting volume of the reaction mixture was less than 2.5 mL. The reaction mixture was placed in a thermostatic shaker and reacted at room temperature for 5-10 minutes. After the labeling reaction was completed, the mixture was again separated using a pre-equilibrated PD-10 desalting column with PBS as the mobile phase. 68 The final product was purified by separating Ga and obtained according to the procedure described above with an undecayed corrected radiochemical yield (RCY) of >50%.

[0039] 3) 68 Ga]Ga-NOTA-B3,[ 68 Ga]Ga-NOTA-B6, [ 68 Ga]Ga-NOTA-ABDB3 and [ 68 Quality control of [Ga]Ga-NOTA-ABDB6 The [ 68 Ga]Ga-NOTA-B3,[ 68 Ga]Ga-NOTA-B6, [ 68 Ga]Ga-NOTA-ABDB3 and [ 68 Ten μl of [Ga]Ga-NOTA-ABDB6 was spotted onto a silica gel plate, and the radiochemical purity (RCP) of the probe was measured by radio-thin layer chromatography (Radio-TLC, Eckert & Ziegler Radiopharma Inc.) using 0.1 M sodium citrate solution (pH = 5) as the mobile phase. As shown in Figure 6, the freshly prepared [Ga]Ga-NOTA-ABDB6 was 68 Ga]Ga-NOTA-B3,[ 68 Ga]Ga-NOTA-B6, [ 68 Ga]Ga-NOTA-ABDB3 and [ 68 The RCPs of Ga]Ga-NOTA-ABDB6 are all greater than 99%. [Example]

[0040] In this example, human CD70-specific 89 Zr-labeled nanoantibody fusion protein probe [ 89 Zr]Zr-DFO-ABDB3 and [ 89 A method for preparing Zr-DFO-ABDB6 is provided, the specific steps of which are as follows:

[0041] 1) ABDB3 and ABDB6 were modified with p-SCN-Bn-Deferoxamine to prepare the intermediates DFO-ABDB3 and DFO-ABDB6. 3 mg of ABDB3 or ABDB6 was dissolved in 1 mL of phosphate buffered saline (PBS), and the nanoantibody solution was adjusted to pH 8.9-9.1 with 0.1 mL of 0.1 M sodium carbonate (NaCO, pH 11.4) buffer to a reaction volume of 1.1 mL. Freshly dissolved DFO (CAS Number: 170597-66-8; Macrocyclics) in dimethyl sulfoxide (DMSO) was added to the nanoantibody solution at a molar ratio of DFO / ABDB3 or ABDB6 = 5:1. The reaction mixture was incubated at room temperature for 30 minutes, and then the DFO-modified nanoantibodies were purified using a pre-equilibrated PD-10 desalting column (GE Healthcare) with PBS as the mobile phase to collect DFO-ABDB3 and DFO-ABDB6. These were then concentrated using an ultrafiltration tube with a cutoff value of 10 kDa (Merck Millipore), and the concentrations of DFO-ABDB3 and DFO-ABDB6 were measured using a NanoDrop. The mixture was then aliquoted and stored at -20°C for use.

[0042] 2) 89 DFO-ABDB3 and DFO-ABDB6 were labeled with Zr and [ 89 Zr]Zr-DFO-ABDB3 and [ 89 Zr]Zr-DFO-ABDB6 was prepared.

[0043] 100MBq in 450μl 89A Zr oxalate solution was prepared and adjusted to pH 7 using 1M Na2CO3 buffer solution. Next, 500 μl of 0.5M HEPES solution (pH 7.1-7.3) and 200 μg of DFO-ABDB3 or DFO-ABDB6 were gradually added to the reaction solution. The reaction system was placed in a thermostatic shaker and reacted at room temperature for 1 hour. After the labeling reaction was completed, the eluates were separated using a pre-equilibrated PD-10 desalting column again using PBS as the mobile phase. 89 After the labeling reaction was completed, the final product was purified by separating Zr. After the labeling reaction was completed, the product was separated by a pre-equilibrated PD-10 desalting column using PBS as the mobile phase. 89 Zr was separated to obtain the final product [ 89 Zr]Zr-DFO-ABDB3 and [ 89 Zr]Zr-DFO-ABDB6 was purified.

[0044] 3) 89 Zr]Zr-DFO-ABDB3 and [ 89 Quality control of Zr]Zr-DFO-ABDB6 The [ 89 Zr]Zr-DFO-ABDB3 and [ 89 Ten μl of [Zr]Zr-DFO-ABDB6 was spotted onto a silica gel plate, and the radiochemical purity (RCP) of the probe was measured by radio-thin layer chromatography (Radio-TLC, Eckert & Ziegler Radiopharma Inc.) using 0.1 M sodium citrate solution (pH = 5) as the mobile phase. As shown in Figure 17, the freshly prepared [ 89 Zr]Zr-DFO-ABDB3 and [ 89 The RCP of Zr]Zr-DFO-ABDB6 exceeded 90%. [Example]

[0045] In this example, human CD70-specific 18 F-labeled monovalent nanoantibody probe [ 18 F]F-B3 and [ 18 A method for preparing [F]F-B6 is provided, the specific steps of which are as follows: 1) 18Preparation of F[F]-RJDJ01 It was prepared according to the method described in Example 2 of the previously published patent document CN113476619A.

[0046] 2) Preparation of DBCO-B3 and DBCO-B6 1 mg of B3 or B6 was dissolved in phosphate buffered saline (PBS) to a volume of approximately 1 mL, and 80–100 μL of 0.1 M sodium carbonate (Na2CO3) buffer was added to adjust the pH of the nanoantibody solution to 9.0–10. Freshly dissolved DBCO-NHS ester in dimethyl sulfoxide (DMSO) was added to the nanoantibody solution so that the molar ratio of DBCO-NHS ester (CAS#: 1353016-71-3, MeloPEG) to B3 / B6 was 10:1. The resulting reaction system was reacted at room temperature for 2 hours, and then the nanoantibodies randomly coupled with DBCO were purified using a pre-equilibrated PD-10 desalting column (GE Healthcare) with PBS as the mobile phase, and DBCO-B3 or DBCO-B6 were collected. The nanoantibody samples were further concentrated using an ultrafiltration tube with a cutoff value of 10 KDa (Merck Millipore). Finally, the concentration of DBCO-B3 or DBCO-B6 was measured using NanoDrop, and DBCO-B3 and DBCO-B6 were placed in a refrigerator at 4°C for use.

[0047] 3) Click chemistry reaction [ 18 F]F-B3 and [ 18 F]F-B6 was prepared Take 332 μL (320 μg) of DBCO-B3 or DBCO-B6 and add it to 20 mL of 18 F[F]-RJDJ01 was added to the DBCO-B3 or DBCO-B6 nanoantibody solution, and the reaction system was placed in a thermostatic shaker at 45°C for 45 minutes. After the reaction was completed, the unreacted 18F-RJDJ01 was separated again using a pre-equilibrated PD-10 desalting column with PBS as the mobile phase, and the final product [ 18 F]F-B3 and [ 18 F]F-B6 was purified and collected.

[0048] The final product [ 18 F]F-ABDB3 and [ 18 F]F-ABDB6 can also be prepared.

[0049] Verification example 1) Construction of a CD70-positive tumor mouse model As shown in Figure 3, immunohistochemistry using anti-human CD70 monoclonal antibody (E3Q1A, 69209, Cell Signaling Technology) as the primary antibody revealed that the patient-derived tumor xenograft (PDX) model No. 62 of renal cell carcinoma (RCC) was positive for CD70 expression. A 2mm x 2mm x 2mm fragment of No. 62 PDX tissue was inoculated into the right shoulder of an NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22 / Nju) mouse to establish a subcutaneous RCC PDX model.

[0050] 2) 68 Ga]Ga-NOTA-B3 and [ 68 Diagnostic experiment of renal cell carcinoma using Ga]Ga-NOTA-B6 immunoPET imaging

[0051] All small animal PET / CT images for this experiment were collected using an IRIS small animal PET / CT scanner (Inviscan Imaging Systems). 68 Ga]Ga-NOTA-B3 and [ 68[Ga]Ga-NOTA-B6 was injected via the tail vein (three mice per group). One hour after injection, the mice were anesthetized with isoflurane (2%) mixed with oxygen. Deeply anesthetized mice were placed in a supine position on the PET / CT scanning platform. PET and CT images were collected continuously. Image reconstruction was completed using the IRIS system's proprietary software, as shown in Figures 7 and 8. Regions of interest (ROIs), including the heart and major tissues and organs (liver, lung, kidney, and muscle), were drawn on the PET images reconstructed using an OsiriX Lite image processing workstation (Pixmeo SARL). Radioactive uptake values ​​for the major tissues and organs were calculated in units of %ID / g (percent of injected dose per gram). Figures 7A and 8A show PET / CT images, Figures 7B and 8B show ROI images, and Figures 7C and 8C show in vitro biodistribution data maps. By drawing ROIs, [Ga]Ga-NOTA-B6 was calculated. 68 Ga]Ga-NOTA-B3 and [ 68 The distribution of Ga]Ga-NOTA-B6 in vivo was analyzed, and both were confirmed to be CD70-specific nanoantibody probe [ 68 Ga]Ga-NOTA-B3 and [ 68 We found that [Ga]Ga-NOTA-B6 showed relatively high uptake in tumor tissues and relatively high nonspecific uptake in the main excretory (kidney) and metabolic (liver) tissues. Furthermore, in vitro biodistribution experiments further clarified the distribution of the probe in major tissues and organs in the body. By analyzing the ROI and biodistribution data visualized for the two tumor models, we confirmed the diagnostic efficacy of [Ga]Ga-NOTA-B6 in the renal cell carcinoma model. 68 Ga]Ga-NOTA-B3 and [ 68 The above results showed that there was no significant difference between [Ga]Ga-NOTA-B6 and [ 68 Ga]Ga-NOTA-B3 and [ 68 This shows that the [Ga]Ga-NOTA-B6 probe can visualize CD70 expression noninvasively.

[0052] 3) 68 A closed-loop experiment for the diagnosis of renal cell carcinoma using Ga]Ga-NOTA-B6 immunoPET imaging The experimental group was treated with developer ([ 68 ABDB6 (20 mg / kg) was injected 48 hours before the injection of [Ga]Ga-NOTA-B6. The non-occlusion group (i.e., the control group) was not injected, and PET images were collected 1 hour after the injection of the respective developer. The PET / CT results are shown in Figure 9, where the upper and lower panels show the imaging results for the non-occlusion group and the occlusion group, respectively, demonstrating that occlusion of ABDB6 to tumor cell surface CDs can significantly reduce the uptake of monovalent nanobody probes in tumor tissue. Statistical analysis of the ROI data and in vitro biodata revealed that tumor uptake in the occlusion group was significantly lower than that in the non-occlusion group, as shown in Figures 10 and 11.

[0053] 4) 68 Ga]Ga-NOTA-ABDB3 and [ 68 Diagnostic experiment of renal cell carcinoma using Ga]Ga-NOTA-ABDB6 immunoPET imaging The specific operation is the same as in step 2) above. As a result of the experiment, as shown in Figure 12, 68 Ga]Ga-NOTA-ABDB3 and [ 68 PET / CT imaging results at multiple time points (2 and 4 hours after injection) of the CD70-specific nanoantibody fusion protein probe [Ga]Ga-NOTA-ABDB6 in the No. 62 PDX renal cell carcinoma model showed that 68 Ga]Ga-NOTA-ABDB3 and [ 68 [Ga]Ga-NOTA-ABDB6 still had relatively high uptake in tumor tissue. As shown in the ROI data in Figure 13, uptake at the tumor site gradually increased over time within 4 hours. The in vitro distribution data shown in Figure 14 further demonstrated enrichment of the probe at the tumor site. 68 Ga]Ga-NOTA-B3,[ 68 Ga]Ga-NOTA-B6 and [ 68 Ga]Ga-NOTA-ABDB3, [ 68Further comparison of the ROI and in vitro biodistribution data of the two probes, Ga]Ga-NOTA-ABDB6, in PET / CT images of a kidney cancer model showed that the nanoantibody fusion protein probe did not affect the ability to noninvasively visualize intratumoral CD70 and significantly reduced renal uptake. The experimental results are shown in Figures 15 and 16.

[0054] 5) 89 Zr]Zr-DFO-ABDB3 and [ 89 Zr]Zr-DFO-ABDB6 immunoPET imaging for diagnosing renal cell carcinoma

[0055] The specific procedure was the same as in step 2) above. PET / CT images were collected 1, 12, 24, 48, 72, 96, 120, and 144 hours after probe injection. As shown in Figures 18 and 19, the MIP images after PET / CT fusion showed the probe uptake in the tumor and major tissues and organs at all time points. The uptake in the tumor gradually increased over time, peaked at 72 hours, and then gradually decreased. The time-dependent uptake curves of the tumor and major tissues and organs (heart, liver, lungs, kidneys, muscle, spleen, and bone) were plotted by ROI analysis, revealing [ 89 Zr]Zr-DFO-ABDB3 and [ 89 [Zr]Zr-DFO-ABDB6 showed good signal-to-noise ratio and good stability in vivo, as shown in Figures 20 and 21. In vitro biodistribution experiments demonstrated the high activity of the probe [Zr]Zr-DFO in tumors and major tissues and organs in vivo, as shown in Figure 22. 89 Zr]Zr-DFO-ABDB3 and [ 89 The distribution of Zr-DFO-ABDB6 was further clarified. 89 Zr]Zr-DFO-ABDB3 and [ 89The results of [Zr]Zr-DFO-ABDB6 immunoPET imaging further demonstrated the enrichment ability of the CD70-specific nanobody fusion proteins ABDB3 and ABDB6 at tumor sites. Furthermore, immunohistochemical staining of tumors with the CD70-specific antibody E3Q1A demonstrated the expression of CD70 within the tumors, as shown in Figure 23.

[0056] 6) 18 F]F-B3 and [ 18 Diagnostic experiment of renal cell carcinoma using [F]F-B6 immunoPET imaging The specific operation is the same as in step 2) above. As a result of the experiment, as shown in Figure 24, 18 PET / CT images of the No. 62 PDX renal cell carcinoma model 30 minutes after injection of [F]F-B6 showed that the CD70-specific nanoantibody probe [ 18 The uptake of [F]F-B6 in tumor tissues was significant. As shown in the ROI data in Figure 25, the tumor site showed relatively high uptake 30 minutes after injection, with significant uptake also observed in the excretory organs, kidney and gallbladder. The in vitro distribution data shown in Figure 25 further demonstrated the enrichment of the probe in tumor sites.

[0057] The above description of the embodiments is intended to facilitate understanding and use of the present invention by those skilled in the art. It is apparent that those skilled in the art can easily make various modifications to these embodiments without creative effort, and can apply the general principles described herein to other embodiments. Therefore, the present invention is not limited to the above embodiments, and any improvements or 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 included within the scope of protection of the present invention.

Claims

1. A CD70-specific nanobody, characterized in that it is B3 having the amino acid sequence shown in SEQ ID NO. 1 or B6 having the amino acid sequence shown in SEQ ID NO.

3.

2. 2. The CD70-specific nanobody of claim 1, wherein the gene sequence of B3 is shown in SEQ ID NO. 2 and the gene sequence of B6 is shown in SEQ ID NO.

4.

3. 10. Use of a CD70-specific Nanobody according to claim 1 or 2 in the preparation of a CD70-specific Nanobody fusion protein.

4. A CD70-specific Nanobody fusion protein, characterized in that it is ABDB3 having the amino acid sequence shown in SEQ ID No. 5 or ABDB6 having the amino acid sequence shown in SEQ ID No.

7.

5. The CD70-specific nanobody fusion protein of claim 4, wherein the amino acid sequence of ABDB3 is set forth in SEQ ID NO. 6 and the amino acid sequence of ABDB6 is set forth in SEQ ID NO.

8.

6. Use of a CD70-specific Nanobody according to any one of claims 1-2 or a CD70-specific Nanobody fusion protein according to any one of claims 4-5 in the preparation of a CD70-specific diagnostic-integrated molecular imaging probe.

7. A CD70-specific diagnostic integrated molecular imaging probe comprising a tumor targeting group selected from the CD70-specific Nanobody of any one of claims 1-2 or the CD70-specific Nanobody fusion protein of any one of claims 4-5, and a radionuclide.

8. The radionuclide is ６８ Ga, １８ F. ６４ Cu or ８９ Zr, The radionuclide ６８ Ga, １８ F. ６４ Cu or ８９ The CD70-specific diagnostic and imaging integrated molecular imaging probe of claim 7, wherein when the chelating agent is selected from Zr, the probe further comprises a chelating agent selected from p-SCN-Bn-NOTA or p-SCN-Bn-Deferoxamine.

9. The probe is ６８ Ga-labeled monovalent nanoantibody probe [ ６８ Ga]Ga-NOTA-B3 and [ ６８ Ga]Ga-NOTA-B6, ６８ Ga-labeled nanoantibody fusion protein probe [ ６８ Ga]Ga-NOTA-ABDB3 and [ ６８ Ga]Ga-NOTA-ABDB6, １８ F-labeled monovalent nanoantibody probe [ １８ F]F-B3 and [ １８ F]F-B6, ８９ Zr-labeled nanoantibody fusion protein probe [ ８９ Zr]Zr-DFO-ABDB3 and [ ８９ 9. The CD70-specific molecular imaging probe for medical treatment according to claim 7 or 8, characterized in that the probe is any one of the following: Zr]Zr-DFO-ABDB6.

10. The probes may contain tumor-targeting genes, chelators, and radionuclides. ６８ Ga, ６４ Cu, or ８９ When Zr is contained, the preparation method thereof is as follows: The tumor-targeting gene is modified with a chelator to form a modified nanobody, and the modified nanobody is then coupled to a radionuclide. ６８ Ga, ６４ Cu, or ８９ labeling with Zr to obtain a probe; The probe comprises a tumor-targeting gene and a radionuclide. １８ When F is contained, the preparation method is Low molecular compound precursors are treated with radionuclides １８ Labeled with F, １８ obtaining an F-labeled precursor; preparing randomly coupled nanobodies with DBCO; １８ The CD70-specific diagnostic integrated molecular imaging probe of claim 8, further comprising a step of performing a click chemistry reaction on the nanobody randomly coupled with an F-labeled precursor and DBCO to obtain the probe.

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