CD70 specific nano antibody, molecular imaging probe as well as preparation method and application of molecular imaging probe

Abstract

The invention provides a CD70 specific nano antibody, a molecular imaging probe as well as a preparation method and application of the 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 RD06, and the amino acid sequence of the RD06 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 CD70-specific nanobody, a molecular imaging probe, and a preparation method and application thereof. Background Art

[0002] Cluster of differentiation CD70 is a type II transmembrane glycoprotein, a member of the tumor necrosis factor superfamily, and 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 induce 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 phase I clinical trials for patients with metastatic renal cell carcinoma, and the clinical benefit rate is 78%. Therefore, there is an urgent need to develop a diagnostic tool targeting CD70 to achieve the 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 team has previously submitted 68 a national invention patent related to a Ga-labeled CD70-specific nanobody probe (application patent 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 (application patent name: 18F-labeled nanobody probe, its preparation method and application; Application Publication No.: CN117281928A; Application No.: 2023106075231; Status: Under examination). 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 novel 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 CD70-specific nanobody, a molecular imaging probe, its preparation method and application. The present invention effectively reduces the uptake of the probe in the kidney, 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 probe effectively inhibits the reabsorption of the nanobody in the kidney, reduces non-specific signals, and improves the signal-to-noise ratio during targeted imaging or treatment, 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 RD06, and the amino acid sequence of the RD06 is shown in SEQ ID No.1.

[0006] Preferably, the gene sequence of the RD06 is shown in 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, an albumin binding domain and a linker.

[0009] Preferably, the CD70-specific nanobody fusion protein is ABDRD06, and the amino acid sequence of the ABDRD06 is shown in SEQ ID No.3.

[0010] Preferably, the gene sequence of ABDRD06 is as shown in SEQ ID No. 4.

[0011] Fourthly, the present invention provides an 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.

[0012] Fifthly, 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 CD70-specific nanobody RD06 or the above CD70-specific nanobody fusion protein ABDRD06.

[0013] Preferably, the tumor targeted by the tumor targeting gene is a tumor expressing CD70.

[0014] 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, renal cancer, urothelial cancer, skin cancer, melanoma, penile cancer, etc.

[0015] 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, Tb-161, Th-227, Xe-133, Yb-169 or Yb-177. More preferably, the radionuclide is Ga-68 or F-18.

[0016] 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, p -SCN-Bn-DOTA, iEDTA, p -SCN-Bn-DTPA, Iso -SGMIB or Boc2-SGMTB, etc.

[0017] Preferably, the bifunctional chelating agent used in the PET imaging probe is selected from (±) H3RESCA-TFP or p -SCN-Bn-NOTA.

[0018] In a sixth aspect, the present invention provides a method for preparing a CD70-specific nanobody molecular imaging probe, and the preparation method includes the following steps: (1) Modify the tumor targeting group with a bifunctional chelating agent to obtain a conjugated tumor targeting group; (2) Label the conjugated tumor targeting group with a radionuclide to obtain the probe.

[0019] The present invention will be described in more detail below.

[0020] The present invention provides a CD70-specific nanobody, and the CD70-specific nanobody is RD06, and the amino acid sequence of the RD06 is shown in SEQ ID No.1.

[0021] The present invention provides a CD70-specific nanobody fusion protein ABDRD06, and the CD70-specific nanobody fusion protein includes the above-mentioned CD70-specific nanobody RD06, an albumin binding domain ABD035, and a linker; the amino acid sequence of the ABDRD06 is shown in SEQ ID No.3; the gene sequence of the ABDRD06 is shown in SEQ ID No.4.

[0022] In some embodiments, the present invention also provides a variant of the CD70-specific nanobody as described herein, which has a sequence identity of 80%-99% or higher with the amino acid sequence of the CD70-specific nanobody, and substantially retains the biological function (such as the biological activity of specifically binding to a target) of the nanobody from which it is derived.

[0023] More specifically, the variant differs 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 residues) amino acid residues.

[0024] As used herein, (±) H3RESCA-TFP is 3-[4-[(2S,5R,8R,11S)-2,5,8-tris(carboxymethyl)-11-(4-(bis(2-hydroxyethyl)amino)benzyl)azacyclooctan-1-yl]phenyl]propanoic acid 2,3,5,6-tetrafluorophenyl ester; (±) 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; NOTA is 1,4,7-triazacyclononane-1,4,7-triacetic acid; 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-tetraazatricosaose]thiourea; The p -SCN-NODA is 1,4,7-triazacyclooctane-1,4-diacetic acid-7-p-isothiocyanatobenzyl; 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; 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-tetraazacyclododecane-1,4,7-triyl)triacetic acid; DOTA-NHS 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 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; The Iso -SGMIB is N-[4-(iodophenylcarbamoylmethyl)-succinimidyl] guanidinomethyl benzoate; The Boc2-SGMTB is N-succinimidyl-4-(N,N-di-tert-butoxycarbonylguanidinomethyl)-3-(tert-butoxycarbonylaminomethyl) benzoate.

[0025] 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 probes 18 F]AlF-RESCA-RD06 and 68 Ga]Ga-NOTA-RD06 of the present invention effectively inhibit their reabsorption in the kidneys through charge adjustment, significantly reduce kidney uptake, reduce non-specific signals, improve the signal-to-noise ratio during targeted imaging or treatment, improve 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 the lesion, and realizing non-invasive and accurate diagnosis of the target of primary and metastatic renal cell carcinoma.

[0026] (2) The present invention effectively reduces the uptake of the probe in the kidneys, reduces the potential risk of renal toxicity while also reducing the adverse reactions caused by immunogenicity, and improves the 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.

[0027] (3) The probe of the present invention is not only applicable to tumor molecular imaging diagnosis, but can also be combined with radioactive tracers or drugs for targeted radiotherapy and drug delivery, providing multiple possible combination schemes for precision medicine.

[0028] (4)The novel nanobody probe targeting CD70 with reduced renal uptake proposed by the present invention fully solves the key problems in the prior art, such as high renal uptake, complex preparation process, immunogenicity risk, and high cost. While ensuring high targeting, the present invention significantly reduces renal uptake, improves imaging quality and treatment safety through molecular site-directed modification and structural optimization. Looking ahead, this probe not only has broad application prospects in tumor diagnosis, but also provides new technical support for targeted therapy and personalized precision medicine, and has important scientific research and clinical application value. Brief Description of the Drawings

[0029] To more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required for the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to the drawings: Figure 1 It is the SDS-PAGE determination of the expression of nanobody RD06 and the experimental result diagram of HPLC; Figure 2 It is the SDS-PAGE determination of the expression of nanobody fusion protein ABDRD06 and the experimental result diagram of HPLC; Figure 3 It is the determination result of the affinity between nanobody RD06 and human CD70 protein; Figure 4 For the use of 18 The quality control diagram of the probe 18 F]AlF-RESCA-RD06 constructed by labeling nanobody RD06 with Figure 5 For the probe 18 The PET / CT imaging diagram, ROI and in vitro biodistribution diagram of the probe Figure 6 For the use of 68 The quality control diagram of the probe 68 Ga]Ga-NOTA-RD06 constructed by labeling nanobody RD06 with Figure 7 For the probe 68 The PET / CT imaging diagram, ROI and in vitro biodistribution diagram of the probe Detailed Embodiments

[0030] The following will describe the embodiments of the present application in detail with reference to the drawings.

[0031] The following specific examples illustrate the implementation manners of the present application. Those skilled in the art can easily understand 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 creative efforts belong to the scope protected by the present application.

[0032] Example 1 This example provides a preparation method of a CD70-specific nanobody RD06. The amino acid sequence of the nanobody RD06 is shown in SEQ ID No.1 (SEQ ID No.1: QLQLVESGGGLVQPGGSLRLSCKASIFTSDYYDIGWLRQAPGKEREGVSCTTSSTGKTDYLDSVKGRFTISRANSENTWYLQMDNLSPEDTGVYYCATKTSSGYECGYYSWQYDYWGQGTQVTVSS), and the gene sequence is shown in SEQ ID No.2. The nanobody fusion protein ABDRD06 includes the CD70-specific nanobody RD06, an albumin-binding domain ABD035, and a linker. Specifically, the amino acid sequence of the ABDRD06 is shown in SEQ ID No.3 (SEQ ID No.3: HHHHHHQLQLVESGGGLVQPGGSLRLSCKASIFTSDYYDIGWLRQAPGKEREGVSCTTSSTGKTDYLDSVKGRFTISRANSENTWYLQMDNLSPEDTGVYYCATKTSSGYECGYYSWQYDYWGQGTQVTVSSGGGGSGGGGSGGGGSLAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP), and the gene sequence is shown in SEQ ID No.4. The CD70-specific monovalent nanobody RD06 and the nanobody fusion protein ABDRD06 are both 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, next-generation sequencing, and recombinant expression.

[0033] In the specific preparation process of the CD70-specific monovalent nanobody RD06 and the CD70-specific nanobody fusion protein ABDRD06, to increase the production yield of RD06 and ABDRD06, a signal peptide (the amino acid sequence of the signal peptide used in this example is shown in SEQ ID NO.5: MHSSALLCCLVLLTGVRA; the gene sequence is shown in SEQ ID NO.6) is first linked to the ends of RD06 and ABDRD06. During the preparation process, the signal peptide will be cleaved in the periplasmic space of the cell, and finally RD06 and ABDRD06 without the signal peptide are obtained. The specific preparation steps are as follows: 1) Using conventional molecular biology methods, after ligating the 5' ends of the gene sequences shown in SEQ ID NO.2 and SEQ ID NO.4 to the gene sequence shown in SEQ ID NO.6 respectively, they are cloned into the pET-30a(+) expression vector respectively to obtain plasmid DNA containing the target antibody (RD06) and plasmid DNA containing the target antibody fusion protein (ABDRD06).

[0034] 2) Express the above target antibody in Escherichia coli (E. coli). 2.1 Escherichia 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 and plasmid DNA containing the target antibody fusion protein to the BL21(DE3) competent cells respectively, and gently mix; incubate the competent cells on ice for 30 minutes; under static conditions, heat shock the competent cells at 42°C for 90 seconds; place the competent cells on ice for 3 minutes; add 100 μl of room temperature LB medium to the competent cells; incubate at 200 rpm and 37°C for 60 minutes; plate on an LB agar plate containing 50 μg / ml kanamycin; invert the agar plate and incubate overnight at 37°C.

[0035] 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 culture respectively; incubate at 200 rpm and 37°C; when the OD600 measurement reaches 0.6 - 0.8, add isopropyl thiogalactoside (IPTG) to the culture tube to make its concentration reach 0.5 mM, and then incubate under the incubation conditions of 15°C for 16 hours or 37°C for 4 hours (both of these two incubation conditions are acceptable).

[0036] The expression of nanobody RD06 and nanobody fusion protein ABDRD06 was determined by SDS-PAGE. The specific steps are as follows: First, prepare a 1.5 mm thick and 15-well gel according to the method of the SDS-PAGE gel kit. Preheat the metal bath to 100 °C and heat the protein sample containing loading buffer (5X) (i.e., the incubation solution obtained from the incubation in the aforementioned 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 sample wells, perform constant voltage electrophoresis at 80 V for about 30 min, adjust the voltage to 120 V after the bromophenol blue indicator crosses the stacking gel, electrophorese until the bottom of the gel, remove the gel, heat and stain it in Coomassie blue staining solution for 50 min, and then take it out and decolorize it with decolorizing solution until the background is clean and the bands are clear, and then take a picture. Respectively as shown in the left figures of Figure 1 and Figure 2 .

[0037] The expression of nanobody RD06 and nanobody fusion protein ABDRD06 was determined by HPLC. The results are respectively as shown in the right figures of Figure 1 and Figure 2 . It can be seen from Figure 1 that the molecular weight of nanobody RD06 is about 15 kDa and the purity can be as high as 96%. It can be seen from Figure 2 that the molecular weight of nanobody fusion protein ABDRD06 is about 20 kDa and the purity can be as high as 96%.

[0038] Determination of the affinity of nanobody RD06 for human CD70: The results of determining the affinity of nanobody RD06 for human CD70 by surface plasmon resonance are as shown in Figure 3 . It can be seen from the figure that the K D value of nanobody RD06 is 2.876 nM.

[0039] Example 2 This example provides a method for preparing a 18 18F-labeled CD70-specific nanobody probe 18 18F]AlF-RESCA-RD06. The specific steps are as follows: (1) Preparation of intermediate RESCA-RD06 by modifying RD06 with (±)-H3RESCA-TFP Dissolve 1 mg of RD06 in 0.05 M NaHCO3 solution (pH = 8.6) to obtain a nanobody solution. Add freshly dissolved (±)-H3RESCA-TFP in dimethyl sulfoxide (DMSO) to the above nanobody solution at a molar ratio of (±)-H3RESCA-TFP:nanobody = 12:1. Place the reaction system at room temperature for 2 h, and then use 0.1 M CH3COONH4 solution (pH = 4.6) as the mobile phase to purify the (±)-H3RESCA-TFP-modified nanobody with a pre-equilibrated PD-10 desalting column (GE Healthcare), and collect RESCA-RD06; then concentrate it with an ultrafiltration tube with a cut-off value of 10 KDa (MerckMillipore), measure the concentration of RESCA-RD06 with NanoDrop, and store it in aliquots at -80 °C for later use.

[0040] (2) 18 Preparation of 18F-labeled RESCA-RD06 18 [18F]AlF-RESCA-RD06 Add 500 μL 18 18F 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 18F solution, add 16 μL of 2 mM aluminum chloride solution (pH 4.4 - 4.6) to it, and let it stand at room temperature for 5 min. Add 200 μg of RESCA-RD06 prepared 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 oscillator and react at room temperature for 12 min. After the labeling reaction, use normal saline as the mobile phase and separate the free 18 18F and purify the final product 18 [18F]AlF-RESCA-RD06; The unattenuated corrected radiochemical yield (RCY) obtained according to the above steps is > 50%.

[0041] (3) 18 Quality control of [18F]AlF-RESCA-RD06 Pipette 10 μL of 18 [18F]AlF-RESCA-RD06 and spot it on a silica gel plate. Use normal saline as the mobile phase and measure the radiochemical purity (RCP) of the probe with a radio-thin layer chromatograph (Radio-TLC, Eckert&Ziegler Radiopharma Inc). AsFigure 4 As shown, freshly prepared [ 18 F]AlF-RESCA-RD06 RCP is greater than 99%.

[0042] Example 3 This embodiment is [ 18 F]AlF-RESCA-RD06 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.

[0043] 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). Each model mouse was injected with 3.7-7.4 MBq [ 18 F]AlF-RESCA-RD06 (3 mice in each group), anesthetized the mice with isoflurane mixed with oxygen (concentration of 2%) 1 hour 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 completed image reconstruction 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 results are shown in the figure. Figure 5 As shown, 18 F]AlF-RESCA-RD06 has a higher uptake in tumor tissues.

[0044] Example 4 This embodiment provides a 68 Ga-labeled CD70-specific nanoantibody probe 68 Preparation method of Ga]Ga-NOTA-RD06. The specific steps are as follows: (1) p -SCN-Bn-NOTA modified RD06 to prepare intermediate NOTA-RD06 Dissolve 1 mg of RD06 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. p Add -SCN-Bn-NOTA to the nanobody solution with a molar ratio of -SCN-Bn-NOTA to nanobody of 10:1. p Add freshly dissolved -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, and then use PBS as the mobile phase to purify the nanobody modified with p -SCN-Bn-NOTA using a pre-equilibrated PD-10 desalting column (GE Healthcare), and collect NOTA-RD06; then concentrate it using an ultrafiltration tube with a cut-off value of 10 KDa (Merck Millipore), measure the concentration of NOTA-RD06 with NanoDrop, and aliquot and store at -80 °C for later use.

[0045] (2) 68 Preparation of 68 Ga]Ga-NOTA-RD06 by labeling NOTA-RD06 with Wash the germanium-gallium generator (Eckert&Ziegler RadiopharmaInc) with 4 mL of 0.05 M hydrochloric acid solution (HCl), and collect about 370 - 555 MBq of 68 Ga eluate with the same volume; take the middle section with the highest activity of 68 2 mL of 68 Ga eluate, add 0.1 mL of 1 M sodium acetate solution (NaoAc) to adjust the pH of the 68 Ga eluate to 4.0 - 4.5; add 100 - 200 μg of NOTA-RD06 prepared for coupling to the 68 Ga eluate, and the volume of the reaction system is <2.5 mL; place the reaction system in a thermostatic oscillator and react at room temperature for 5 - 10 min; after the labeling reaction, use PBS as the mobile phase and use a pre-equilibrated PD-10 desalting column to separate free 68 Ga and purify the final product

[0046] (3) 68 Quality control of Pipette 10 μL of 68Ga]Ga-NOTA-RD06 was spotted on a silica gel plate, and 0.1 M sodium citrate solution (pH = 5) was used as the mobile phase. The radiochemical purity (RCP) of the probe was determined by radio-thin layer chromatography (Radio-TLC, Eckert & Ziegler Radiopharma Inc). 68 The radiochemical purity of Ga]Ga-NOTA-RD06 is greater than 99% ( Figure 6 ).

[0047] Example 5 This embodiment is for 68 The value of non-invasive visualization of CD70 expression by Ga]Ga-NOTA-RD06 immunoPET imaging in further diagnosing renal cancer was verified. The specific steps are as follows: This embodiment involves 68 Ga-labeled probes were collected by small animal PET / CT using an IRIS small animal PET / CT scanner (Inviscan Imaging Systems). Each Balb / c nude mouse with subcutaneous Caki-1 renal cancer tumor (Caki-1 tumor model, constructed in the same way as in Example 3) was injected with 3.7-7.4 MBq of the successfully prepared [ 68 Ga]Ga-NOTA-RD06, anesthetize the mice with isoflurane mixed with oxygen (concentration of 2%) 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 7 As shown, 68 The Ga]Ga-NOTA-RD06 probe is mainly excreted through the kidneys, and the probe is significantly enriched in the tumor site. 68 The Ga]Ga-NOTA-RD06 probe can also non-invasively visualize CD70 expression inside tumors.

[0048] In summary, the new renal low-accumulation CD70-specific nanoantibody molecular imaging probe constructed by the present invention overcomes the defects of monoclonal antibody molecular imaging probes such as long imaging cycle, large radiation dose and high renal accumulation of nanoantibodies, and realizes more convenient stratification of patients receiving targeted CD70 treatment, monitoring of the efficacy of targeted CD70 treatment, and CD70-targeted radionuclide therapy and CAR-T therapy.

[0049] The above embodiments are only for illustrating the technical concept and features 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. It should not 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, characterized in that, The CD70-specific nanobody is RD06, and the amino acid sequence of RD06 is as shown in SEQ ID No.1 or has a homologous sequence with a similarity of more than 80% to the sequence shown in SEQ ID No.

1.

2. The CD70-specific nanobody according to claim 1, wherein The gene sequence of RD06 is as shown in SEQ ID No.2 or has a homologous sequence with a similarity of more than 80% to the sequence shown in SEQ ID No.

2.

3. Use of a CD70-specific nanobody according to any one of claims 1-2 in the preparation of a CD70-specific nanobody fusion protein.

4. A CD70-specific nanobody fusion protein, characterized in that, The CD70-specific nanobody fusion protein comprises a CD70-specific nanobody according to any one of claims 1-2, an albumin-binding domain and a linker.

5. The CD70-specific nanobody fusion protein according to claim 4, characterized in that, The CD70-specific nanobody fusion protein is ABDRD06, and the amino acid sequence of ABDRD06 is as shown in SEQ ID No.3 or has a homologous sequence with a similarity of more than 80% to the sequence shown in SEQ ID No.

3.

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 nanobody molecular imaging probe.

7. A CD70-specific nanobody molecular imaging probe, characterized in that, The probe comprises a tumor targeting group, a radionuclide and a bifunctional chelator; the tumor targeting group is 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.

8. The CD70-specific nanobody molecular imaging probe according to claim 7, characterized in that, The tumor targeted by the tumor targeting gene is a tumor expressing CD70.

9. The CD70-specific nanobody molecular imaging probe according to claim 7, wherein 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 multiforme, thyroid cancer, liver cancer, renal cancer, urothelial cancer, skin cancer, melanoma, penile cancer, etc.

10. The CD70-specific nanobody molecular imaging probe according to claim 7, wherein 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, Tb-161, Th-227, Xe-133, Yb-169 or Yb-177; 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, p -SCN-Bn-DOTA, iEDTA, p -SCN-Bn-DTPA, Iso -SGMIB or Boc2-SGMTB.

11. A preparation method of the CD70-specific nanobody molecular imaging probe according to any one of claims 7 to 10, characterized in that, The preparation method includes the following steps: (1) Modifying the tumor targeting group with a bifunctional chelating agent to obtain a conjugated tumor targeting group; (2) Labeling the conjugated tumor targeting group with a radionuclide to obtain the probe.

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