Preparation method and application of CD70 specific nano antibody molecular imaging probe

Abstract

The invention provides a preparation method and application of a CD70 specific nano antibody molecular imaging probe. The probe comprises a tumor targeting group, radionuclide and a bifunctional chelating agent, the tumor targeting group is a CD70 specific nano antibody; the CD70 specific nano antibody is R8B4, and the amino acid sequence of the R8B4 is as shown in SEQ ID No. 1. The probe provided by the invention can realize noninvasive diagnosis of solid tumors such as renal clear cell carcinoma and malignant tumors of a blood system; reabsorption of the compound in the kidney is effectively inhibited through charge adjustment, kidney uptake is remarkably reduced, non-specific signals are reduced, the signal-to-noise ratio during targeted imaging or treatment is increased, and the imaging quality and treatment safety are improved. The probe not only has a wide application prospect in tumor diagnosis, but also provides a new technical support for targeted therapy and individualized precision medical treatment, and has important scientific research and clinical application values.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular imaging probes, and particularly relates to a preparation method and application of a CD70-specific nanobody molecular imaging probe. Background Art

[0002] Cluster of differentiation antigen CD70 is a type II transmembrane glycoprotein, a member of the tumor necrosis factor superfamily, and also the ligand of CD27. The binding of the two can induce the activation of multiple signaling pathways, promoting gene transcription, cell proliferation and differentiation. Under normal circumstances, CD70 is only transiently expressed on the surface of activated T cells, B cells and mature dendritic cells. Recent studies have found that CD70 is highly expressed in a variety of malignant hematological tumors and solid tumors. Compared with normal renal tissue, in renal cell carcinoma, especially in clear cell renal cell carcinoma and sarcomatoid renal cell carcinoma, the expression of CD70 is significantly increased, and the high expression of CD70 is associated with poor prognosis. CD70 expressed on tumor cells can bind to CD27 on the surface of T cells, initiate the apoptosis protein Siva to cause cytotoxic effects in immune cells and apoptosis, achieving the effect of immune escape. The expression difference of CD70 in normal tissues and tumors makes it a highly potential tumor-specific marker and can avoid potential side effects. Currently, drugs such as monoclonal antibodies, antibody-drug conjugates and chimeric antigen receptor T cells targeting CD70 have entered clinical trials. The anti-CD70 monoclonal antibody SGN-CD70A has been used in patients with metastatic renal cell carcinoma in a phase I clinical trial, with a clinical benefit rate of 78%. Therefore, there is an urgent need to develop a diagnostic tool targeting CD70 to achieve visualization and monitoring of CD70 expression in solid tumors. On the basis of studying companion diagnostic tools, new treatment methods targeting CD70 can be further developed.

[0003] The applicant's team has previously submitted 68 a national invention patent related to a Ga-labeled CD70-specific nanobody probe (patent application name: Preparation method of a CD70-specific integrated diagnosis and treatment molecular imaging probe; application publication number: CN115925951A; authorization announcement number: CN115925951B); and 18 a national invention patent related to an F-labeled CD70-specific immunological PET imaging probe (patent application name: 18F-labeled nanobody probe, its preparation method and application; Publication No.: CN117281928A; Application No.: 2023106075231; Status: Under review). Although the nanobody probe constructed based on the CD70-specific nanobody B6 involved in the above two patents has excellent tumor targeting performance and can better non-invasively visualize multiple metastatic foci of renal cell carcinoma, its renal accumulation is relatively high, making it difficult to diagnose the primary focus of renal cancer. Moreover, it may be difficult to construct a radionuclide immunotherapy probe based on B6 due to relatively high renal toxicity. Therefore, there is an urgent need to develop a new type of CD70 nanobody probe with reduced renal accumulation, so as to achieve the diagnosis of the primary focus of renal cancer and the construction of a CD70-specific diagnosis and treatment integrated probe. Summary of the Invention

[0004] The present invention provides a preparation method and application of a CD70-specific nanobody molecular imaging probe. The present invention effectively reduces the uptake of the probe in the kidneys, and has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging period, low radiation dose, easy clinical transformation, etc.; through charge adjustment, the nanobody's reabsorption in the kidneys is effectively inhibited, non-specific signals are reduced, and the signal-to-noise ratio during targeted imaging or treatment is improved, enabling accurate identification of lesions; while reducing the potential risk of renal toxicity, it also reduces the adverse reactions caused by immunogenicity, improves the overall safety, and has high commercialization and clinical transformation value.

[0005] The object of the present invention is achieved through the following technical solutions: In the first aspect, the present invention provides a CD70-specific nanobody, and the CD70-specific nanobody is R8B4, and the amino acid sequence of R8B4 is shown as SEQ ID No.1.

[0006] Preferably, the gene sequence of R8B4 is shown as SEQ ID No.2.

[0007] In the second aspect, the present invention provides the application of the above CD70-specific nanobody in the preparation of a CD70-specific nanobody fusion protein.

[0008] In the third aspect, the present invention provides a CD70-specific nanobody fusion protein, and the CD70-specific nanobody fusion protein includes the above CD70-specific nanobody.

[0009] In the fourth aspect, the present invention provides the application of the above CD70-specific nanobody or the above CD70-specific nanobody fusion protein in the preparation of a CD70-specific nanobody molecular imaging probe.

[0010] Fifth aspect, the present invention provides a CD70-specific nanobody molecular imaging probe, which comprises a tumor targeting group, a radionuclide and a bifunctional chelating agent; the tumor targeting group is the above-mentioned CD70-specific nanobody or the above-mentioned CD70-specific nanobody fusion protein.

[0011] Preferably, the tumor targeted by the tumor targeting gene is a tumor expressing CD70.

[0012] More preferably, the tumor expressing CD70 is a malignant tumor with high expression of CD70; the malignant tumor with high expression of CD70 includes at least one of renal clear cell carcinoma, nasopharyngeal carcinoma, lymphoma, multiple myeloma, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, prostate cancer, cervical cancer, head and neck cancer, lung cancer, oral cancer, laryngeal cancer, esophageal cancer, bladder cancer, uterine cancer, ovarian cancer, glioma, glioblastoma, thyroid cancer, liver cancer, kidney cancer, urothelial cancer, skin cancer, melanoma, penile cancer, etc.

[0013] Preferably, the radionuclide is selected from Tc-99m, Ga-68, F-18, I-123, I-125, I-131, I-124, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, Y-86, Mn-52, Sc-44, Y-90, Ac-225, At-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra-223, Ru-106, Na-24, Sr-89, Tb-149, Th-227, Xe-133, Yb-169 or Yb-177. More preferably, the radionuclide is Ga-68 or F-18.

[0014] Preferably, the bifunctional chelating agent is selected from (±) H3RESCA-TFP, (±) H3RESCA-Mal, NOTA, MAA-NOTA, p -SCN-Bn-NOTA, p -SCN-Bn-DFO, p -SCN-NODA, MAA-GA-NODA, MAA-DOTA, DOTA-NHS, iEDTA or p -SCN-Bn-DTPA.

[0015] Preferably, the bifunctional chelator is selected from (±) H3RESCA-TFP or p -SCN-Bn-NOTA.

[0016] In a sixth aspect, the present invention provides a method for preparing a CD70-specific nanobody molecular imaging probe, the preparation method comprising the following steps: (1) Modifying a tumor targeting group with a bifunctional chelator to obtain a conjugated tumor targeting group; (2) Labeling the conjugated tumor targeting group with a radionuclide to obtain the probe.

[0017] The present invention will be described in more detail below.

[0018] The present invention provides a CD70-specific nanobody, the CD70-specific nanobody being R8B4, and the amino acid sequence of the R8B4 being as shown in SEQ ID No.1.

[0019] In some embodiments, the present invention also provides variants of the CD70-specific nanobody as described herein, which have 70%-99% or higher sequence identity with the amino acid sequence of the CD70-specific nanobody and substantially retain the biological functions (such as the bioactivity of specifically binding to a target) of the nanobody from which they are derived.

[0020] More specifically, the variants differ from the CD70-specific nanobody as described herein only by conservative substitutions of one or more (e.g., up to 20, up to 15, up to 10, up to 5, or up to 1 amino acid) amino acid residues.

[0021] As used herein, the (±) H3RESCA-TFP is 2,3,5,6-tetrafluorophenyl 3-[4-[(2S,5R,8R,11S)-2,5,8-tris(carboxymethyl)-11-(4-(bis(2-hydroxyethyl)amino)benzyl)azacyclooctan-1-yl]phenyl]propionate; The (±) H3RESCA-Mal is (±)-2,2',2''-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; The NOTA is 1,4,7-triazacyclononane-1,4,7-triacetic acid; The MAA-NOTA is (2,2'-(7-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7-triazacyclononane-1,4-diyl)diacetic acid; The p -SCN-Bn-NOTA is 2-S-(4-isothiocyanatophenyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; The p -SCN-Bn-DFO is 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxyamino)-6,11,17,22-tetraazatricosanoyl]thiourea; The p -SCN-NODA is 1,4,7-triazacyclooctane-1,4-diacetic acid-7-p-isothiocyanatobenzyl; The MAA-GA-NODA is 2,2'-(7-(1-carboxy-4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutyl)-1,4,7-triazacyclononane-1,4-diyl)diacetic acid; The MAA-DOTA is 2,2',2″-(10-(1-carboxy-4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutyl)-1,4,7,10-triazacyclododecane-1,4,7-triyl)triacetic acid]; The DOTA-NHS is 2,2',2”-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-triazacyclododecane-1,4,7-triyl)triacetic acid; The iEDTA is 1-(4-isothiocyanatobenzyl)ethylenediamine-N,N,N',N'-tetraacetic acid; The p -SCN-Bn-DTPA is 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid; The p -SCN-Bn-DOTA is 1-(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0022] Compared with the prior art, the beneficial effects that can be achieved by at least one of the above technical solutions adopted in the embodiments of this specification at least include: (1) The nanobody probe of the present invention 18 F]AlF-RESCA-R8B4 and 68Ga]Ga-NOTA-R8B4 effectively inhibits its renal reabsorption through charge adjustment, significantly reduces renal uptake, decreases non-specific signals, improves the signal-to-noise ratio during targeted imaging or treatment, enhances imaging quality and treatment safety; at the same time, the present invention also fully protects the binding site of the nanobody to CD70, ensuring its high affinity and high specificity for CD70 in vivo, enabling the probe to accurately identify lesions and achieving non-invasive diagnosis of renal cell carcinoma.

[0023] (2) The present invention effectively reduces the uptake of the probe in the kidney, while reducing the potential risk of renal toxicity, also reducing the adverse reactions caused by immunogenicity, and improving overall safety; the method for preparing the probe of the present invention is easy to promote and apply, and has high commercialization and clinical transformation value.

[0024] (3) The probe of the present invention is not only applicable to tumor molecular imaging diagnosis, but also can be combined with radioactive tracers or drugs for targeted radiotherapy and drug delivery, providing various possible combination schemes for precision medicine.

[0025] (4) The novel nanobody probe targeting CD70 with reduced renal uptake proposed by the present invention fully solves the key problems such as high renal uptake, complex preparation process, immunogenic risk and high cost in the prior art. While ensuring high targeting, through molecular site-directed modification and structural optimization, the present invention significantly reduces renal uptake, improves imaging quality and treatment safety. Looking ahead, this probe not only has broad application prospects in tumor diagnosis, but also provides new technical support for targeted therapy and individualized precision medicine, and has important scientific research and clinical application value. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained according to the drawings without creative efforts: Figure 1 It is the SDS-PAGE determination of the expression of nanobody R8B4 and the experimental result diagram of HPLC; Figure 2 It is the determination result of the affinity of nanobody R8B4 with human CD70 protein; Figure 3 It is for using 18 The quality control diagram of the probe 18 F]AlF-RESCA-R8B4 constructed with F-labeled nanobody R8B4; Figure 4 It is for the probe 18PET / CT imaging, region of interest (ROI), and in vitro biodistribution maps of [¹⁸F]AlF-RESCA-R8B4 in the Caki-1 renal clear cell carcinoma tumor model; Figure 5 For the probe 18 PET / CT imaging, ROI, and in vitro biodistribution maps of [¹⁸F]AlF-RESCA-R8B4 after co-injection with 200 μg of R8B4 in the Caki-1 renal clear cell carcinoma tumor model; Figure 6 For the probe 18 Comparison maps of ROI and in vitro biodistribution between the blocked group co-injected with [¹⁸F]AlF-RESCA-R8B4 and 200 μg of R8B4 and the unblocked group not co-injected with R8B4; Figure 7 For use 68 Probe constructed using the [⁶⁸Ga]Ga-labeled nanobody R8B4 68 Quality control map of [⁶⁸Ga]Ga-NOTA-R8B4; Figure 8 For the probe 68 PET / CT imaging, ROI, and in vitro biodistribution maps of [⁶⁸Ga]Ga-NOTA-R8B4 in the Caki-1 renal clear cell carcinoma tumor model; Figure 9 Results of HE staining and CD70 immunohistochemical staining of Caki-1 tumors in vitro; Figure 10 For the probe 18 Imaging and staining results of [¹⁸F]AlF-RESCA-R8B4 in a patient after surgery for renal clear cell carcinoma; among them, Figure 10 A in [] is the imaging result; Figure 10 B in [] is the HE staining result map; Figure 10 C in [] is the CD70 immunohistochemical staining result map; Figure 11 For the probe 18 Imaging results of [¹⁸F]AlF-RESCA-R8B4 in a newly diagnosed patient with renal clear cell carcinoma; Figure 12 For the probe 68 Imaging and staining results of [⁶⁸Ga]Ga-NOTA-R8B4 in a newly diagnosed patient with renal clear cell carcinoma; among them, Figure 12 A in [] is the imaging result; Figure 12 B in [] is the HE staining result map; Figure 12 C in [] is the CD70 immunohistochemical staining result map. Detailed implementation methods

[0027] The embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0028] The following specific examples illustrate the implementation manners of the present application. Those skilled in the art can easily understand the other advantages and effects of the present application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. The present application can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, without conflict, the following embodiments and the features in the embodiments can be combined with each other. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without making creative efforts belong to the scope of protection of the present application.

[0029] Example 1 This example provides a preparation method of a CD70-specific nanobody R8B4. The amino acid sequence of the nanobody R8B4 is shown in SEQ ID No.1 (SEQ ID No.1: MHSSALLCCLVLLTGVRAEVQLV ESGGGFVQPGGSLRLSCAASGFTLDGYAVAWFRQAPGKEREGVSCISSSDGSTYYIDSVQGRFTITRNNAKNTVYLQMNSLKPEDTAVYYCTTDVLTSCRSDRWLEVWGQGTLVTVSS), and the gene sequence is shown in SEQ ID No.2. The CD70-specific monovalent nanobody R8B4 is obtained by immunizing alpacas with human CD70 eukaryotic protein (company: Absin Bioscience Inc.; product number: CDL-H52Da), isolating peripheral blood lymphocytes, constructing a phage display library, phage screening, second-generation sequencing, and recombinant expression.

[0030] The specific preparation steps of the CD70-specific monovalent nanobody R8B4 are as follows: 1) Using conventional molecular biology methods, the gene sequence shown in SEQ ID NO.2 is cloned into the pET-30a(+) expression vector respectively to obtain plasmid DNA containing the target antibody (R8B4).

[0031] 2) Express the above target antibody in Escherichia coli (E. coli) 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 gently mix. Incubate the competent cells on ice for 30 minutes. Under static conditions, subject the competent cells to heat shock treatment at 42°C for 90 seconds. Place the competent cells on ice for 3 minutes. Add 100 μl of room temperature LB medium to the competent cells. Incubate at 200 rpm and 37°C for 60 minutes. Plate on an LB agar plate containing 50 μg / ml kanamycin. Invert the agar plate and incubate overnight at 37°C.

[0032] 2.2 Small-scale expression: Randomly select well-dispersed monoclonal colonies from the agar plate and inoculate them into LB medium containing 50 μg / ml kanamycin for separate culture. Incubate at 200 rpm and 37°C. When the OD600 measurement reaches 0.6 - 0.8, add isopropyl β-D-thiogalactopyranoside (IPTG) to the culture tube to a concentration of 0.5 mM, and then incubate under the incubation conditions of 15°C for 16 hours or 37°C for 4 hours (either of these two incubation conditions is acceptable).

[0033] The expression of nanobody R8B4 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 protein sample containing loading buffer (5X) (i.e., the incubation solution obtained from the previous step 2.2) for 5 min. After assembling the SDS-PAGE gel, add 500 ml of 1x SDS-PAGE buffer. Slowly load the protein sample into the wells. Run at a constant voltage of 80 V in an electrophoresis bath for about 30 min. After the bromophenol blue indicator passes through the stacking gel, adjust the voltage to 120 V and electrophorese until the bottom of the gel. Take down the gel, heat and stain it in Coomassie blue staining solution for 50 min, then take it out and decolorize it with decolorizing solution until the background is clean and the bands are clear, and then take a picture. Respectively as Figure 1 shown in the left figure below.

[0034] The expression of nanobody R8B4 was determined by HPLC. The results are respectively as Figure 1 shown in the right figure below. It can be seen from Figure 1 that the molecular weight of nanobody R8B4 is about 15 kDa and the purity can be as high as nearly 99%.

[0035] Determination of the affinity of nanobody R8B4 for human CD70: The results of surface plasmon resonance for determining the affinity of nanobody R8B4 for human CD70 are as Figure 2 shown. It can be seen from the figure that the KD value of nanobody R8B4 is 9.583 nM.

[0036] Example 2 This example provides a 18 method for preparing an 18 F-labeled CD70-specific nanobody probe F]AlF-RESCA-R8B4. The specific steps are as follows: (1) Preparation of intermediate RESCA-R8B4 by modifying R8B4 with (±)-H3RESCA-TFP

[0037] (2) 18 F-labeling of RESCA-R8B4 to prepare 18 F]AlF-RESCA-R8B4 Add 500 μL 18 F solution (about 100 mCi) to a QMA column (Waters GmbH, Germany), rinse the QMA column with 500 μL of normal saline and collect the 18 F solution. Add 16 μL of 2 mM aluminum chloride solution (pH 4.4 - 4.6) thereto and let stand at room temperature for 5 min. Add 200 μg of RESCA-R8B4 reserved for coupling to the reaction system, add 800 μL of 0.1 M CH3COONH4 solution (pH = 4.6), place the reaction system in a thermostatic shaker and react at room temperature for 12 min. After the labeling reaction is completed, use normal saline as the mobile phase and separate the free 18 F and purify the final product 18 F]AlF-RESCA-R8B4; the unattenuated corrected radiochemical yield (RCY) obtained according to the above steps is > 50%.

[0038] 18 Quality control of ​Pipette 10 µL [ 18 F]AlF-RESCA-R8B4 was spotted on a silica gel plate, and physiological saline 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 As shown, freshly prepared [ 18 F]AlF-RESCA-R8B4 RCP is greater than 99%.

[0039] Example 3 This embodiment is [ 18 F]AlF-RESCA-R8B4 immunoPET imaging for diagnosis of renal cell carcinoma. The specific steps are as follows: 1) Construction of Caki-1 tumor model: Through database consultation and immunohistochemical staining, it was found that the human renal clear cell carcinoma cell line Caki-1 expressed positive CD70. 6 Caki-1 cells were inoculated into the right shoulder of Balb / c nu mice to establish a subcutaneous renal clear cell carcinoma cell line transplant tumor model.

[0040] 2) The small animal PET / CT imaging acquisition involved in this example was completed using the IRIS small animal PET / CT scanner (Inviscan Imaging Systems). 18 F]AlF-RESCA-R8B4 was injected with 200 µg R8B4, while the unblocked group was not injected. Each model mouse was injected with 3.7-7.4 MBq [ 18 F]AlF-RESCA-R8B4 (3 mice in each group), anesthetized mice with isoflurane mixed with oxygen (concentration of 2%) 30 minutes after injection, and placed the mice in a deep anesthesia state in a supine position on the PET / CT scanning bed, and continued to acquire PET and CT images, and image reconstruction was completed using the IRIS system's own software. The OsiriX Lite image processing workstation (Pixmeo SARL) was used to outline the regions of interest (ROI) such as the heart and major tissue organs (liver, lung, kidney, muscle) on the reconstructed PET images, and the radioactive uptake values of important tissues and organs were calculated in %ID / g (percent of injected dose per gram). The PET / CT results, ROI, and in vitro biodistribution of the unsealed group are shown in Figure 2. Figure 4 As shown in the figure, it can be seen that the CD70-specific nanoantibody probe [ 18F]AlF-RESCA-R8B4 has a high uptake in tumor tissues and a slightly higher non-specific uptake in the main excretory (kidney) tissues. The PET / CT results, ROI, and in vitro biodistribution of the blocking group are as Figure 5 shown. It can be seen that co-injection of R8B4 to block CD70 on the surface of tumor cells can significantly reduce the uptake of the monovalent nanobody probe in tumor tissues. Through statistical analysis of the ROI data and in vitro biological data, it can be seen that the tumor uptake of the blocking group is significantly lower than that of the unblocked group, and the results are as Figure 6 shown. The left side shows the ROI map, and the right side shows the in vitro biodistribution data map. The above results indicate that 18 the F]AlF-RESCA-R8B4 probe can non-invasively visualize CD70 expression.

[0041] Example 4 This example provides a 68 method for preparing a 68 Ga-labeled CD70-specific nanobody probe (1) p Modify R8B4 with -SCN-Bn-NOTA to prepare the intermediate NOTA-R8B4 Dissolve 1 mg of R8B4 in 1 mL of phosphate buffer (PBS), adjust the pH of the nanobody solution to 9.0–10 with 0.1 mL of 0.1 M sodium carbonate (Na2CO3, PH = 11.4) buffer, and the volume of the reaction system is 1.1 mL. With p the molar ratio of -SCN-Bn-NOTA to the nanobody being 10:1, add freshly dissolved p -SCN-Bn-NOTA (CAS Number: 147597-66-8; Macrocyclics) in dimethyl sulfoxide (DMSO) to the above nanobody solution. Place the reaction system at room temperature for 2 h, then use PBS as the mobile phase and purify the nanobody modified with p -SCN-Bn-NOTA with a pre-equilibrated PD-10 desalting column (GE Healthcare) to collect NOTA-R8B4; then concentrate it with an ultrafiltration tube with a cut-off value of 10 KDa (Merck Millipore), measure the concentration of NOTA-R8B4 with a NanoDrop, and aliquot and store at -80 °C for later use.

[0042] (2) 68 Label NOTA-R8B4 with 68 Ga to prepare The germanium-gallium generator (Eckert&Ziegler Radiopharma Inc) was rinsed with 4 mL of 0.05 M hydrochloric acid solution (HCl), and the 68 Ga eluate with an equal volume and an activity of about 370–555 MBq was collected; the middle section with the highest activity of the 68 Ga eluate (2 mL) was taken, and 0.1 mL of 1 M sodium acetate solution (NaoAc) was added to adjust the 68 pH of the Ga eluate to 4.0 - 4.5; 100–200 μg of NOTA-R8B4 that had been coupled and reserved was added to the 68 Ga eluate, and the volume of the reaction system was <2.5 mL; the reaction system was placed in a thermostatic oscillator and reacted at room temperature for 5–10 min; after the labeling reaction was completed, PBS was used as the mobile phase, and a pre-equilibrated PD-10 desalting column was used to separate the free 68 Ga and the purified final product 68 Ga]Ga-NOTA-R8B4; the radiochemical yield (RCY) obtained according to the above steps was >50%.

[0043] (3) 68 Quality control of Ga]Ga-NOTA-R8B4 68 10 μL of 68 Ga]Ga-NOTA-R8B4 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 measured using a radio-thin layer chromatograph (Radio-TLC, Eckert&Ziegler Radiopharma Inc). The radiochemical purity of the probe Figure 7 Ga]Ga-NOTA-R8B4 prepared in the present invention was greater than 99% (

[0044] Example 5 This example verified the value of 68 Ga]Ga-NOTA-R8B4 in non-invasive visualization of CD70 expression by immune PET imaging for further diagnosis of renal cancer. The specific steps were as follows: The small animal PET / CT imaging acquisitions of the 68 Ga-labeled probe involved in this example were all completed using an IRIS small animal PET / CT scanner (Inviscan Imaging Systems). Each subcutaneous Caki-1 renal cancer tumor Balb / c nude mouse (Caki-1 tumor model, constructed in the same way as in Example 3) was injected via the tail vein with 3.7 - 7.4 MBq of the successfully prepared68 Ga]Ga-NOTA-R8B4, anesthetize the mice with isoflurane (concentration of 2%) mixed with oxygen 1 hour after injection, and place the deeply anesthetized mice in a supine position on the PET / CT scanning bed, and continue to acquire PET and CT images, and complete image reconstruction using the IRIS system's own software, such as Figure 8 As shown, 68 The Ga]Ga-NOTA-R8B4 probe is mainly excreted through the kidneys, and the probe is significantly enriched in the tumor site. 68 The Ga]Ga-NOTA-R8B4 probe can also non-invasively visualize the expression of CD70 in tumors. At the same time, after the experimental mice were killed, the subcutaneous Caki-1 tumors were removed and HE and CD70 immunohistochemical staining was performed, which showed that CD70 was highly expressed in the tumor ( Figure 9 ).

[0045] Example 6 This example is a specific nanoantibody probe [ 18 F]AlF-RESCA-R8B4 and [ 68 ImmunoPET imaging analysis of Ga]Ga-NOTA-R8B4 in patients with renal cell carcinoma. The specific steps are as follows: The PET / CT images of the patients involved in this embodiment were acquired using a Total-body PET / CT (uExplorer, United Imaging Healthcare) scanner. The patients were injected with 3.7-5.55 MBq / kg of [ 18 F]AlF-RESCA-R8B4, image acquisition was performed 1 hour after injection, the patient was in a supine position with the arm raised above the head. During the scan, the patient kept breathing steady to avoid image fusion errors as much as possible, and the acquisition time was 10 minutes. The PET / CT images were evaluated by two nuclear medicine doctors. The tumor area with increased uptake of specific nanoantibody probes or CT images was the region of interest (ROI). The region of interest was outlined according to CT and the maximum standardized uptake value (SUVmax) of the ROI was calculated by the following formula: SUV = [average radioactivity in the local region of interest (MBq / mL)] / [injected radioactivity (MBq) / body weight (g)]. Figure 10 This is a case of a patient with multiple metastases after surgery for left renal clear cell carcinoma. Figure 10 As shown in A, [ 18 F]FDG only showed metastatic lesions in the left 11th posterior rib and right adrenal gland of this patient, while the CD70-specific nanoantibody probe [18 In [¹⁸F]AlF-RESCA-R8B4, there was high uptake in the lung metastases, right adrenal metastases, left 11th posterior rib and pancreatic metastases of this patient. In addition, there was high non-specific uptake in the excretory (right kidney) tissue. Figure 10 B and C in it were the HE staining and CD70 staining of the primary focus of renal cancer in this patient, respectively, confirming that the focus was positive for CD70. Figure 11 This was a patient newly diagnosed with renal cancer. Above in the figure were the results of different imaging examinations of the primary focus in the left kidney of the patient, and below in the figure were the results of different imaging examinations of the metastatic foci around the left kidney of this patient. It could be seen that the novel CD70-specific probe 18 [¹⁸F]AlF-RESCA-R8B4 could clearly show the primary focus and metastatic foci of patients with renal cancer. The above results indicated that 18 [¹⁸F]AlF-RESCA-R8B4 probe could non-invasively visualize the expression of CD70.

[0046] Figure 12 A in it was 68 Immune PET imaging of [⁶⁸Ga]Ga-NOTA-R8B4 in patients with renal cell carcinoma. Figure 12 This was a patient newly diagnosed with renal cancer. As shown in the figure, 68 [⁶⁸Ga]Ga-NOTA-R8B4 probe could clearly show the primary focus of the right kidney of the patient, while in 18 [¹⁸F]FDG imaging, there was no obvious uptake in the primary focus of the right kidney of the patient. The subsequent surgical pathology of the patient also confirmed the positive expression of CD70 in the focus ( Figure 12 B and C in it were the HE staining and CD70 staining of the primary focus of renal cancer in this patient, respectively).

[0047] In summary, the novel kidney-low-accumulation CD70-specific nanobody molecular imaging probe constructed by the present invention overcomes the defects of long imaging cycle, large radiation dose and high kidney accumulation of monoclonal antibody molecular imaging probes, and realizes more convenient stratification of patients for targeted CD70 treatment, monitoring of the efficacy of targeted CD70 treatment, and radionuclide treatment and CAR-T treatment targeting CD70.

[0048] The above embodiments are only for illustrating the technical concept and characteristics of the present invention, and the purpose is to enable those of ordinary skill in the art to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the essence of the content of the present invention should be covered within the protection scope of the present invention.

Claims

1. A CD70-specific nanobody molecular imaging probe, characterized in that, The probe includes a tumor targeting group, a radionuclide, and a bifunctional chelator; the tumor targeting group is a CD70-specific nanobody; The CD70-specific nanobody is R8B4, and the amino acid sequence of R8B4 is shown as SEQ ID No.1; The radionuclide is selected from Ga-68 or F-18; The bifunctional chelating agent is selected from (±) H3RESCA-TFP or p -SCN-Bn-NOTA.

2. The CD70-specific nanobody molecular imaging probe according to claim 1, wherein The gene sequence of R8B4 is shown as SEQ ID No.

2.

3. The CD70-specific nanobody molecular imaging probe according to claim 1, wherein The tumor targeted by the tumor targeting gene is a tumor expressing CD70.

4. The CD70-specific nanobody molecular imaging probe according to claim 3, characterized in that The tumor expressing CD70 is a malignant tumor with high expression of CD70; the malignant tumor with high expression of CD70 includes at least one of renal clear cell carcinoma, nasopharyngeal carcinoma, lymphoma, multiple myeloma, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, prostate cancer, cervical cancer, head and neck cancer, lung cancer, oral cancer, pharyngeal cancer, esophageal cancer, bladder cancer, uterine cancer, ovarian cancer, glioma, glioblastoma, thyroid cancer, liver cancer, renal cancer, urothelial cancer, skin cancer, melanoma, penile cancer, etc.

5. A method for preparing a CD70-specific nanobody molecular imaging probe according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: (1) Modify the tumor targeting group with a bifunctional chelator to obtain a conjugated tumor targeting group; (2) Label the conjugated tumor targeting group with a radionuclide to obtain the probe.

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