Preparation and application of CD70-specific nanoantibody molecular imaging probe

Abstract

The present invention provides a preparation method and application of a CD70-specific nano-antibody molecular imaging probe, wherein the probe includes a tumor-targeting group, a radionuclide, and a bifunctional chelator; the tumor-targeting group is a CD70-specific nano-antibody; the CD70-specific nano-antibody is R8B4, and the amino acid sequence of R8B4 is shown in SEQ ID No. 1. The probe of the present invention can realize non-invasive diagnosis of solid tumors such as renal clear cell carcinoma and hematological malignancies; and through charge adjustment, it effectively inhibits its reabsorption in the kidney, significantly reduces renal uptake, reduces nonspecific signals, improves the signal-to-noise ratio during targeted imaging or treatment, and improves imaging quality and treatment safety. 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.

Description

Technical Field

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

[0002] Cluster of differentiation antigen (CD70) is a type II transmembrane glycoprotein and a member of the tumor necrosis factor (TNF) superfamily. It is also a ligand for CD27. Binding of these two proteins can induce activation of multiple signaling pathways, promoting gene transcription, cell proliferation, and differentiation. Normally, CD70 is only transiently expressed on the surfaces of activated T cells, B cells, and mature dendritic cells. Recent studies have found elevated expression of CD70 in various hematological malignancies and solid tumors. Compared with normal renal tissue, CD70 expression is significantly elevated in renal cell carcinoma, particularly in clear cell renal cell carcinoma and sarcomatoid renal cell carcinoma, and high CD70 expression is associated with a poor prognosis. CD70 expressed on tumor cells can bind to CD27 on the surface of T cells, triggering the apoptosis protein Siva to induce cytotoxicity and apoptosis in immune cells, thereby achieving immune escape. The differential expression of CD70 in normal tissues and tumors makes it a highly promising tumor-specific marker, while also 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 has been used in a Phase I clinical trial in patients with metastatic renal cell carcinoma, achieving a clinical benefit rate of 78%. Therefore, there is an urgent need to develop a diagnostic tool targeting CD70 to visualize and monitor CD70 expression in solid tumors. Further development of a companion diagnostic tool could also lead to the development of novel CD70-targeted therapeutics.

[0003] The applicant team has submitted 68 National invention patent related to Ga-labeled CD70-specific nanoantibody probe (patent application name: Preparation method of CD70-specific integrated diagnostic and therapeutic molecular imaging probe; application publication number: CN115925951A; authorization announcement number: CN115925951B); and 18 National invention patent related to F-labeled CD70-specific immune PET imaging probe (patent application name: 18F-labeled nanoantibody probe, preparation method, and application thereof; Application Publication Number: CN117281928A; Application Number: 2023106075231; Status: Under Review). While the nanoantibody probe constructed based on the CD70-specific nanoantibody B6 covered by the above two patents has excellent tumor-targeting performance and can effectively and non-invasively visualize multiple metastatic lesions of renal cell carcinoma, its high renal accumulation makes it difficult to diagnose primary renal cancer lesions. Furthermore, the construction of a radionuclide immunotherapy probe based on B6 may be difficult due to its significant renal toxicity. Therefore, there is an urgent need to develop a new CD70 nanoantibody probe with reduced renal accumulation, which can facilitate the diagnosis of primary renal cancer lesions and the construction of a CD70-specific integrated diagnostic and therapeutic probe. Summary of the Invention

[0004] The present invention provides a preparation method and application of a CD70-specific nanoantibody molecular imaging probe. 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 cycle, low radiation dose, and easy clinical transformation. The probe effectively inhibits the reabsorption of nanoantibodies in the kidney through charge adjustment, reduces nonspecific signals, improves the signal-to-noise ratio during targeted imaging or treatment, and can accurately identify lesions. While reducing the potential risk of nephrotoxicity, it also reduces adverse reactions caused by immunogenicity, improves overall safety, and has high commercialization and clinical transformation value.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] In a first aspect, the present invention provides a CD70-specific nanoantibody, wherein the CD70-specific nanoantibody is R8B4, and the amino acid sequence of R8B4 is shown in SEQ ID No.1.

[0007] Preferably, the gene sequence of R8B4 is shown as SEQ ID No. 2.

[0008] In a second aspect, the present invention provides the use of the above-mentioned CD70-specific nanobody in the preparation of CD70-specific nanobody fusion protein.

[0009] In a third aspect, the present invention provides a CD70-specific nanoantibody fusion protein, which comprises the above-mentioned CD70-specific nanoantibody.

[0010] In a fourth aspect, the present invention provides the use of the above-mentioned CD70-specific nanoantibody or the above-mentioned CD70-specific nanoantibody fusion protein in the preparation of a CD70-specific nanoantibody molecular imaging probe.

[0011] In the fifth aspect, the present invention provides a CD70-specific nanoantibody molecular imaging probe, which includes a tumor targeting group, a radionuclide and a bifunctional chelator; the tumor targeting group is the above-mentioned CD70-specific nanoantibody or the above-mentioned CD70-specific nanoantibody fusion protein.

[0012] Preferably, the tumor targeted by the tumor targeting gene is a tumor expressing CD70.

[0013] More preferably, the CD70-expressing tumor is a malignant tumor that highly expresses CD70; the malignant tumor that highly expresses CD70 includes at least one of renal clear cell carcinoma, nasopharyngeal carcinoma, lymphoma, multiple myeloma, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, oral cancer, pharyngeal cancer, esophageal cancer, bladder cancer, uterine cancer, ovarian cancer, brain glioma, glioblastoma, thyroid cancer, liver cancer, kidney cancer, urothelial carcinoma, skin cancer, melanoma, penile cancer, etc.

[0014] 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 More preferably, the radionuclide is Ga-68 or F-18.

[0015] 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.

[0016] Preferably, the bifunctional chelating agent is selected from (±) H3RESCA-TFP or p -SCN-Bn-NOTA.

[0017] 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:

[0018] (1) Using a bifunctional chelating agent to modify the tumor targeting group to obtain a coupled tumor targeting group;

[0019] (2) Use radioactive nuclides to label the coupled tumor targeting group to obtain a probe.

[0020] The present invention will be described in more detail below.

[0021] The present invention provides a CD70-specific nanoantibody, wherein the CD70-specific nanoantibody is R8B4, and the amino acid sequence of R8B4 is shown in SEQ ID No.1.

[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 70%-99% or higher with the amino acid sequence of the CD70-specific Nanobody and substantially retains the biological function of the Nanobody from which it is derived (e.g., biological activity of specifically binding to the target).

[0023] More specifically, the variants differ from the CD70-specific Nanobodies as described herein only by conservative substitutions of one or more (e.g., conservative substitutions of at most 20, at most 15, at most 10, at most 5 or at most 1) amino acid residues.

[0024] 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)azoctan-1-yl]phenyl]propanoate;

[0025] 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;

[0026] The NOTA is 1,4,7-triazacyclononane-1,4,7-triacetic acid;

[0027] 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;

[0028] described p -SCN-Bn-NOTA is 2-S-(4-isothiocyanatophenyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid;

[0029] described p -SCN-Bn-DFO is 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxyamino)-6,11,17,22-tetraazaheptanobiose]thiourea;

[0030] described p -SCN-NODA is 1,4,7-triazacyclooctane-1,4-diacetic acid-7-isothiocyanatobenzyl;

[0031] 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;

[0032] 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];

[0033] The DOTA-NHS is 2,2',2"-(10-(2-((2,5-dioxypyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-triazacyclododecane-1,4,7-triyl)triacetic acid;

[0034] The iEDTA is 1-(4-isothiocyanobenzyl)ethylenediamine-N,N,N',N'-tetraacetic acid;

[0035] described p -SCN-Bn-DTPA is 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid;

[0036] described p -SCN-Bn-DOTA is 1-(4-isothiocyanatophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0037] Compared with the prior art, the at least one technical solution adopted in the embodiments of this specification can achieve the following beneficial effects:

[0038] (1) Nanobody probes of the present invention [ 18 F]AlF-RESCA-R8B4 and [ 68 Ga]Ga-NOTA-R8B4 effectively inhibits its reabsorption in the kidney through charge adjustment, significantly reduces renal uptake, reduces nonspecific signals, improves the signal-to-noise ratio during targeted imaging or treatment, and improves imaging quality and treatment safety; at the same time, the present invention also fully protects the binding site of the nanoantibody with CD70, ensuring its high affinity and high specificity for CD70 in vivo, enabling the probe to accurately identify lesions and realize non-invasive diagnosis of renal cell carcinoma.

[0039] (2) The present invention effectively reduces the uptake of the probe in the kidney, thereby reducing the potential risk of nephrotoxicity and adverse reactions caused by immunogenicity, thereby improving overall safety. The method for preparing the probe of the present invention is easy to promote and apply, and has high commercial and clinical transformation value.

[0040] (3) The probe of the present invention is not only suitable for tumor molecular imaging diagnosis, but can also be combined with radioactive tracers or drugs for targeted radiotherapy and drug delivery, providing a variety of possible combination solutions for precision medicine.

[0041] (4) The novel nanoantibody probe proposed in this invention that targets CD70 and has reduced renal uptake fully solves the key problems of the existing technology, such as high renal uptake, complex preparation process, immunogenicity risk, and high cost. While ensuring high targeting, the present invention significantly reduces renal uptake through molecular site-specific modification and structural optimization, thereby improving 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 personalized precision medicine, and has important scientific research and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on the drawings without inventive effort.

[0043] Figure 1 The figure shows the expression of nanobody R8B4 determined by SDS-PAGE and the experimental results of HPLC;

[0044] Figure 2 The results show the affinity test between nanobody R8B4 and human CD70 protein;

[0045] Figure 3 For use 18 F-labeled probe constructed by nanoantibody R8B4[ 18 F]Quality control chart of AlF-RESCA-R8B4;

[0046] Figure 4 For the probe 18 F] PET / CT images, region of interest (ROI), and in vitro biodistribution of AlF-RESCA-R8B4 in the Caki-1 clear cell renal cell carcinoma tumor model;

[0047] Figure 5 For the probe 18 F] PET / CT images, ROIs, and in vitro biodistribution of AlF-RESCA-R8B4 in the Caki-1 clear cell renal cell carcinoma tumor model after co-injection with 200 µg R8B4.

[0048] Figure 6 For the probe 18 F] Comparison of ROI and in vitro biodistribution of the blocking group co-injected with AlF-RESCA-R8B4 and 200 µg R8B4 and the unblocked group without co-injection of R8B4;

[0049] Figure 7 For use 68 Ga-labeled nanoantibody R8B4 constructed probe [ 68 Quality control chart of Ga]Ga-NOTA-R8B4;

[0050] Figure 8 For the probe 68 PET / CT images, ROI, and in vitro biodistribution of Ga]Ga-NOTA-R8B4 in the Caki-1 clear cell renal cell carcinoma tumor model;

[0051] Figure 9 The figure shows the results of HE staining and CD70 immunohistochemical staining of Caki-1 tumor in vitro;

[0052] Figure 10 For the probe 18 F]AlF-RESCA-R8B4 imaging and staining results in a patient with renal clear cell carcinoma after surgery; Figure 10 A in the figure is the imaging result; Figure 10 B in the figure is the HE staining result; Figure 10C in the figure shows the results of CD70 immunohistochemical staining;

[0053] Figure 11 For the probe 18 F]AlF-RESCA-R8B4 imaging results in a patient with newly diagnosed clear cell renal cell carcinoma;

[0054] Figure 12 For the probe 68 The imaging and staining results of Ga]Ga-NOTA-R8B4 in a patient with newly diagnosed renal clear cell carcinoma; Figure 12 A in the figure is the imaging result; Figure 12 B in the figure is the HE staining result; Figure 12 Panel C shows the results of CD70 immunohistochemical staining. DETAILED DESCRIPTION

[0055] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0056] The following describes the embodiments of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the features in the following embodiments and embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of this application.

[0057] Example 1

[0058] This example provides a method for preparing the CD70-specific nanobody R8B4, the amino acid sequence of which is shown in SEQ ID No. 1 (MHSSALLCCLVLLTGVRAEVQLV ESGGGFVQPGGSLRLSCAASGFTLDGYAVAWFRQAPGKEREGVSCISSSDGSTYYIDSVQGRFTITRNNAKNTVYLQMNSLKPEDTAVYYCTTDVLTSCRSDRWLEVWGQGTLVTVSS), and the gene sequence is shown in SEQ ID No. 2. The CD70-specific monovalent nanobody R8B4 was obtained by immunizing alpacas with human CD70 eukaryotic protein (Company: Biopsies Biotech Co., Ltd.; Catalog No.: CDL-H52Da), isolating peripheral blood lymphocytes, constructing a phage display library, screening the phages, performing next-generation sequencing, and recombinant expression.

[0059] The specific preparation steps of CD70-specific monovalent nanobody R8B4 are as follows:

[0060] 1) Using conventional molecular biological methods, the gene sequence shown in SEQ ID NO. 2 was cloned into the pET-30a(+) expression vector to obtain plasmid DNA containing the target antibody (R8B4).

[0061] 2) Express the above target antibody in Escherichia coli (E. coli)

[0062] 2.1 E. coli transformation: First, remove BL21 (DE3) competent cells from -80°C and thaw on ice; add 100 ng of plasmid DNA containing the target antibody to the BL21 (DE3) competent cells and gently mix; incubate the competent cells on ice for 30 minutes; heat shock the competent cells at 42°C for 90 seconds in a static state; place the competent cells on ice for 3 minutes; add 100 μl of room temperature LB medium to the competent cells; incubate at 200 rpm and 37°C for 60 minutes; plate the competent cells on LB agar plates containing 50 μg / ml kanamycin; invert the agar plates and incubate at 37°C overnight.

[0063] 2.2 Small-scale expression test: Randomly select well-dispersed single clones from the agar plate and inoculate them into LB medium containing 50 μg / ml kanamycin for culture. Incubate at 200 rpm and 37°C. When the OD600 value reaches 0.6–0.8, add isopropylthiogalactoside (IPTG) to the culture tube to a concentration of 0.5 mM. Incubate at 15°C for 16 hours or 37°C for 4 hours (both incubation conditions are acceptable).

[0064] The expression of nanoantibody R8B4 was determined by SDS-PAGE. The specific steps are as follows: First, prepare a 1.5mm 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 after incubation in the above step 2.2) for 5 minutes; after the SDS-PAGE gel is assembled, add 500ml of 1x SDS-PAGE buffer, slowly spot the protein sample into the loading well, and place it in an 80V constant voltage electric bath for about 30 minutes. After the bromophenol blue indicator passes through the concentrated gel, adjust the voltage to 120V, electrophoresed to the bottom of the gel, remove the gel, heat and stain it in Coomassie blue dye for 50 minutes, then take it out, and decolorize it with decolorizing solution until the background is clean and the bands are clear, and then take pictures. Figure 1 As shown in the left picture.

[0065] The expression of nanobody R8B4 was determined by HPLC, and the results were as follows: Figure 1 As shown in the right figure. Figure 1 It can be seen that the molecular weight of the nanobody R8B4 is about 15 kDa and the purity can be as high as nearly 99%.

[0066] Affinity determination of nanobody R8B4 and human CD70: Surface plasmon resonance determination of the affinity of nanobody R8B4 and human CD70 Figure 2 As shown in the figure, it can be seen that the KD value of nanobody R8B4 is 9.583 nM.

[0067] Example 2

[0068] This embodiment provides a 18 F-labeled CD70-specific nanoantibody probe [ 18 Preparation method of F]AlF-RESCA-R8B4. The specific steps are as follows:

[0069] (1) Modification of R8B4 with (±)-H3RESCA-TFP to prepare the intermediate RESCA-R8B4

[0070] A nanobody solution was prepared by dissolving 1 mg of R8B4 in 0.05 M NaHCO₃ solution (pH = 8.6). Freshly dissolved (±)-H3RESCA-TFP in dimethyl sulfoxide (DMSO) was added to the nanobody solution at a molar ratio of (±)-H3RESCA-TFP: nanobody of 12:1. The reaction was allowed to react at room temperature for 2 h. The (±)-H3RESCA-TFP-modified nanobody was then purified using a pre-equilibrated PD-10 desalting column (GE Healthcare) with 0.1 M CH₃COONH₄ solution (pH = 4.6) as the mobile phase. RESCA-R8B4 was collected and concentrated using ultrafiltration tubes with a cutoff of 10 kDa (Merck Millipore). The RESCA-R8B4 concentration was determined using a NanoDrop filter and stored at -80°C until use.

[0071] (2) 18 Preparation of F-labeled RESCA-R8B4 [ 18 F]AlF-RESCA-R8B4

[0072] 500 μL 18 F solution (about 100 mCi) was added to a QMA column (Waters GmbH, Germany), and the QMA column was rinsed with 500 μL of saline and the 18 F solution, add 16μL 2mM aluminum chloride solution (pH 4.4-4.6) to it, and let it stand at room temperature for 5 minutes. Add 200μg of RESCA-R8B4 ready for coupling to the reaction system, add 800μL 0.1M CH3COONH4 solution (pH=4.6), place the reaction system on a constant temperature oscillator and react at room temperature for 12 minutes. After the labeling reaction is completed, physiological saline is used as the mobile phase, and the free 18 F. Purification of the final product 18 F]AlF-RESCA-R8B4; the unattenuated radiochemical yield (RCY) obtained according to the above steps was >50%.

[0073] [ 18 F]AlF-RESCA-R8B4 quality control

[0074] Pipette 10 µL [ 18F]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%.

[0075] Example 3

[0076] This embodiment is [ 18 F]AlF-RESCA-R8B4 immunoPET imaging for the diagnosis of renal cell carcinoma. The specific steps are as follows:

[0077] 1) Construction of Caki-1 tumor model: Through database search and immunohistochemical staining, it was found that 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 xenograft tumor model.

[0078] 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 200µg 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 per group), 30 minutes after injection, the mice were anesthetized with isoflurane mixed with oxygen (concentration of 2%), and the mice in a deep anesthesia state were placed in a supine position on the PET / CT scanning bed, and PET and CT images were continuously acquired. Image reconstruction was completed using the software provided by the IRIS system. 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 units of %ID / g (percent of injected dose per gram). The PET / CT results, ROI and in vitro biodistribution of the unblocked 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 higher uptake in tumor tissue and a slightly higher nonspecific uptake in the main excretion tissue (kidney). Figure 5 As shown in the figure, it can be seen that the blocking of CD70 on the surface of tumor cells by co-injection of R8B4 can significantly reduce the uptake of monovalent nanoantibody probes in tumor tissues. By statistical analysis of 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 non-blocking group. Figure 6 As shown, the left side shows the ROI map and the right side shows the in vitro biodistribution data map. The above results show that [ 18 F]AlF-RESCA-R8B4 probe can noninvasively visualize CD70 expression.

[0079] Example 4

[0080] This embodiment provides a 68 Ga-labeled CD70-specific nanoantibody probe[ 68 Preparation method of Ga]Ga-NOTA-R8B4. The specific steps are as follows:

[0081] (1) p -SCN-Bn-NOTA modification of R8B4 to prepare intermediate NOTA-R8B4

[0082] 1 mg of R8B4 was dissolved in 1 mL of phosphate buffered saline (PBS), and the pH of the nanobody solution was adjusted to 9.0–10 with 0.1 mL of 0.1 M sodium carbonate (Na2CO3, pH = 11.4) buffer. The reaction volume was 1.1 mL. p The molar ratio of -SCN-Bn-NOTA to nanobody was 10:1, and freshly dissolved in dimethyl sulfoxide (DMSO) p -SCN-Bn-NOTA (CAS Number: 147597-66-8; Macrocyclics) was added to the above nanobody solution. The reaction system was left to react at room temperature for 2 h, and then purified using a pre-equilibrated PD-10 desalting column (GE Healthcare) with PBS as the mobile phase. p -SCN-Bn-NOTA modified nanoantibodies were collected to collect NOTA-R8B4; then concentrated using an ultrafiltration tube with a cutoff value of 10 KDa (Merck Millipore), and the concentration of NOTA-R8B4 was determined using NanoDrop, and the devices were stored at -80°C for later use.

[0083] (2) 68 Preparation of Ga-labeled NOTA-R8B4 68 Ga]Ga-NOTA-R8B4

[0084] The gallium germanium generator (Eckert & Ziegler Radiopharma Inc) was eluted with 4 mL of 0.05 M hydrochloric acid solution (HCl) to collect an equivalent volume of approximately 370–555 MBq of activity. 68 Ga eluent; take the middle section with the highest activity 68 2 mL of Ga eluent, adjusted with 0.1 mL of 1 M sodium acetate solution (NaoAc) 68 Ga eluent pH 4.0-4.5; take 100-200 μg of NOTA-R8B4 prepared for coupling and add to 68 The Ga eluent was used, and the reaction volume was <2.5 mL. The reaction system was placed in a constant temperature oscillator and reacted at room temperature for 5–10 min. After the labeling reaction, PBS was used as the mobile phase, and a pre-equilibrated PD-10 desalting column was used to separate the free 68 Ga, purified final product [ 68 Ga]Ga-NOTA-R8B4; the unattenuated corrected radiochemical yield (RCY) obtained according to the above steps was >50%.

[0085] (3) [ 68 Ga]Ga-NOTA-R8B4 Quality Control

[0086] Pipette 10 µL [ 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. 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-R8B4 is greater than 99% ( Figure 7 ).

[0087] Example 5

[0088] This embodiment is for 68 The value of noninvasive visualization of CD70 expression using Ga]Ga-NOTA-R8B4 immunoPET imaging for further diagnosis of renal cancer was verified. The specific steps are as follows:

[0089] This embodiment involves 68Ga-labeled probes were acquired using an IRIS small animal PET / CT scanner (Inviscan Imaging Systems). Each Balb / c nude mouse with a subcutaneous Caki-1 renal cancer tumor (Caki-1 tumor model, constructed in the same manner as in Example 3) was injected via the tail vein with 3.7-7.4 MBq of the successfully prepared [ 68 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. PET and CT images were acquired continuously, and image reconstruction was completed using the software provided by the IRIS system, 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 sacrificed, the subcutaneous Caki-1 tumors were removed and HE and CD70 immunohistochemical staining were performed. It can be seen that the tumors highly expressed CD70 ( Figure 9 ).

[0090] Example 6

[0091] 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:

[0092] The PET / CT images of the patients involved in this embodiment were acquired using a Total-body PET / CT scanner (uExplorer, United Imaging Healthcare). 18F]AlF-RESCA-R8B4. Images were acquired 1 hour after injection. Patients were placed in the supine position with their arms elevated overhead. Patients maintained steady breathing during the scan to minimize image fusion errors. Acquisition time was 10 minutes. Two nuclear medicine physicians evaluated PET / CT images. Regions of interest (ROIs) were defined as areas with increased uptake of the specific nanoantibody probe or tumors indicated by CT images. ROIs were delineated based on CT scans, and the maximum standardized uptake value (SUVmax) of the ROI was calculated using the following formula: SUV = [average radioactivity in the local ROI (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 metastases in the left 11th posterior rib and right adrenal gland of this patient, whereas the CD70-specific nanobody probe [ 18 In F]AlF-RESCA-R8B4, this patient had high uptake in the lung metastases, right adrenal metastases, left 11th posterior rib, and pancreatic metastases, as well as high nonspecific uptake in excretory (right kidney) tissues. Figure 10 B and C in the figure are HE staining and CD70 staining of the primary lesion of renal cancer of the patient, respectively, which confirmed that the lesion was CD70 positive. Figure 11 This is a patient with newly diagnosed renal cancer. The upper part of the figure shows the results of different imaging examinations of the patient's left kidney primary lesion, and the lower part of the figure shows the results of different imaging examinations of the patient's left perirenal metastasis. It can be seen that the new CD70-specific probe [ 18 F]AlF-RESCA-R8B4 can clearly show the primary and metastatic lesions of renal cancer patients. The above results show that [ 18 F]AlF-RESCA-R8B4 probe can noninvasively visualize CD70 expression.

[0093] Figure 12 A in [ 68 ImmunoPET imaging of Ga]Ga-NOTA-R8B4 in patients with renal cell carcinoma. Figure 12 This is a patient with newly diagnosed renal cancer, as shown in the figure. 68 The Ga]Ga-NOTA-R8B4 probe can clearly show the primary lesion in the patient's right kidney, while [ 18 In the F]FDG imaging, no obvious uptake was observed in the primary lesion of the right kidney of the patient. The patient's subsequent surgical pathology also confirmed the positive expression of CD70 in the lesion ( Figure 12Figures B and C are HE staining and CD70 staining of the primary renal cancer lesion of the patient, respectively).

[0094] In summary, the novel 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 for targeted CD70 treatment, monitoring of the efficacy of targeted CD70 treatment, and CD70-targeted radionuclide therapy and CAR-T treatment.

[0095] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the essence of the present invention are intended to be covered by the scope of protection 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 nanoantibody; The CD70-specific nanobody is R8B4, and the amino acid sequence of R8B4 is shown in SEQ ID No. 1; The radionuclide is Ga-68 and the bifunctional chelating agent is p -SCN-Bn-NOTA; or The radionuclide is F-18 and The bifunctional chelating agent is (±) H3RESCA-TFP.

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

2.

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

4. The CD70-specific nanobody molecular imaging probe according to claim 3, wherein: The CD70-expressing tumor is a malignant tumor that highly expresses CD70.

5. The CD70-specific nanobody molecular imaging probe according to claim 4, wherein: The malignant tumor that highly expresses CD70 includes at least one of renal clear cell carcinoma, nasopharyngeal carcinoma, pancreatic cancer, glioma, and melanoma.

6. The CD70-specific nanobody molecular imaging probe according to claim 5, wherein: The brain glioma includes glioblastoma.

7. A method for preparing a CD70-specific nanobody molecular imaging probe according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: (1) Using a bifunctional chelating agent to modify the tumor targeting group to obtain a coupled tumor targeting group; (2) Use radioactive nuclides to label the coupled tumor targeting group to obtain a probe.

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