A CD47-specific molecular imaging probe and its preparation method and application

Abstract

The present invention discloses a CD47-specific molecular imaging probe and its preparation method and application, which relates to the molecular imaging, nuclear medicine and nano-antibody technology fields of tumor diagnosis and treatment, including human CD47-specific monovalent nano-antibody probes and human CD47-specific nano-antibody fusion protein probes; its preparation method comprises: modifying CD47-specific nano-antibody C2 by compounds, synthesizing radionuclide labeling precursors, and preparing the CD47-specific molecular imaging probe by radionuclide labeling; the prepared probe is applied to immune PET imaging and radioimmunotherapy of malignant tumors. The present invention has the advantages of simple preparation process, low cost, high specificity, high stability, and easy clinical transformation. It can realize non-invasive visualization of heterogeneous expression of CD47, screen patients with high expression of CD47, and further carry out CD47-specific radioimmunotherapy, that is, realize integrated diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the field of molecular imaging, nuclear medicine and nano-antibody technology for tumor diagnosis and treatment, and in particular to a CD47-specific molecular imaging probe and a preparation method and application thereof. Background Art

[0002] In 1993, Belgian scientists Hamers et al. reported for the first time in Nature magazine that there is a naturally missing light chain antibody in the peripheral blood of alpacas (Nature. 1993; 363 (6428): 446-8.). This antibody with a special structural domain is called heavy chain antibody (HCAbs). Through molecular biological methods, the variable region of heavy chain antibodies can be cloned to obtain antigen-binding fragments with only the variable region of heavy chain, which is nano antibody (VHH, Variable Domain of Heavy Chain of Heavy Chain Antibody). VHH crystals are 2.5nm wide, 4nm long, and have a molecular weight of only 15KDa, so they are also called nano antibodies ( Ablynx registered trade name). Nanobodies are the smallest antibody units known to bind to target antigens, with the advantages of high affinity, small molecular weight, low preparation cost (can be expressed in E. coli, yeast, Chinese hamster ovary cells and other eukaryotic expression systems), and easy clinical transformation and promotion and application.

[0003] Nanobodies are popular target vectors for constructing molecular imaging probes in recent years (Theranostics. 2014; 4(4): 386-98.). Currently, a variety of short-half-life radionuclides have been used to label nanobodies and prepare nanoantibody molecular imaging probes. Technetium-99m ( 99m Tc; T1 / 2 = 6.02h) labeled nanoantibody probes targeting programmed death ligand 1 (PD-L1) have been successfully transformed into clinical practice for non-invasive diagnosis of patients with non-small cell lung cancer (J Nucl Med. 2019; 60(9): 1213-1220.); Gallium-68 ( 68 The nanoantibody probe labeled with radioactive isotope Ga; T1 / 2 = 1.1h) targeting human epidermal growth factor receptor (HER2) has also been successfully transformed into clinical practice for non-invasive diagnosis of breast cancer (J Nucl Med. 2016; 57(1): 27-33.). The above examples show that radionuclide-labeled nanoantibody probes have great clinical application prospects and can be used for early non-invasive diagnosis of human malignant tumors, visualization of key pathogenic targets, screening of patients for monoclonal antibody (mAb) treatment, and efficacy evaluation after monoclonal antibody treatment.

[0004] CD47 is the only known five-transmembrane receptor in the immune system, which is widely expressed in different cell types in the body. It plays a physiological role by binding to signal regulatory protein α (SIRPα), affecting the homeostasis of red blood cells, platelets and hematopoietic stem cells, and regulating synaptic pruning during neuronal development. However, tumor cells use this mechanism to transmit "don't eat me" signals to macrophages to escape macrophage clearance. Studies have shown that overexpression of CD47 on the surface of tumor cells is associated with poor prognosis, such as acute myeloid leukemia, colorectal cancer and lymphoma. Clinical trials have confirmed the therapeutic effect of magrolimab, a monoclonal antibody targeting CD47, in patients with non-Hodgkin's lymphoma and other types of solid tumors. However, the widespread expression of CD47 in normal tissues can lead to the antigen sinking effect of CD47-targeted antibodies in the body, which inevitably leads to damage to normal tissues. Therefore, there is an urgent need to develop a companion diagnostic tool targeting CD47 to help stratify patients who may benefit from anti-CD47 therapy. Based on the research of companion diagnostic tools, new treatments targeting CD47 can also be further developed.

[0005] Currently, immunohistochemical staining of surgically removed or biopsied tissue is the most commonly used method to detect CD47 expression. However, studies have shown that immuno-PET can better display the distribution and abundance of targets of interest in the body and better predict the response to targeted therapy compared with immunohistochemical staining or other traditional predictive markers.

[0006] By cleverly combining the extraordinary targeting specificity of antibodies with the superior sensitivity and resolution of positron emission tomography (PET), immuno-PET can noninvasively display the expression of targets of interest in vivo (Chem Rev. 2020; 120(8): 3787-3851.). For example, immuno-PET imaging probes targeting programmed cell death ligand-1 have been successfully applied in clinical practice and better predicted the therapeutic efficacy of atezolizumab, a monoclonal antibody specific for PD-L1, than other traditional predictive biomarkers. In addition, it has been reported that immuno-PET probes can be used to evaluate the dynamic changes of lymphocytes and myeloid cells before and after cancer immune checkpoint therapy and further reveal the immune status within the tumor. Based on the above evidence and our previous findings, we hypothesized that immuno-PET imaging probes targeting CD47 could noninvasively display CD47 expression within tumors and provide a better method for selecting patients who may benefit from CD47 checkpoint inhibition therapy. In addition, there is evidence that radioimmunotherapy (RIT) and pretargeted radioimmunotherapy (pRIT) may help cancer patients achieve long-term remission or even eradicate multiple cancer types.

[0007] Currently, there are no reports of CD47-targeted therapeutic diagnosis in the literature. Zheleznyak A et al. demonstrated the use of 89 Zr-labeled monoclonal antibodies (mAbs) targeting CD47 have demonstrated the feasibility of intratumoral CD47 expression (Mol Imaging. Nov-Dec 2013; 12(8)). However, the application of radiolabeled mAbs is hampered by high cost, the necessity of using long half-life radionuclides, cumbersome imaging procedures within a week, and associated radiation exposure. In order to improve the clinical application of antibody diagnostics, the field of molecular imaging is actively exploring pre-targeted imaging strategies or the use of smaller antibody derivatives to achieve same-day molecular imaging. Among the small antibody formats, nanobodies or single-domain antibodies from the camelid family are the smallest antigen-binding moieties with a molecular weight of approximately 15 kDa. The small size, high affinity, and ease of engineering make nanobodies an excellent alternative for molecular imaging. In recent years, we have focused on the development and clinical transformation of nanobody-derived tracers to exert their superior molecular imaging properties. Although radiolabeled monovalent nanobodies are ideal companion diagnostic tools, their short in vivo half-life and high renal uptake leave room for further improvement. In order to develop an integrated diagnostic and therapeutic platform, the albumin binding domain (ABD) targeting human / mouse albumin was introduced into nanobodies to extend the half-life of nanobody derivatives in vivo. Studies have shown that bispecific nanobody derivatives that simultaneously target tumor antigens and albumin improve the biodistribution in vivo and can be used as a carrier for the development of therapeutic diagnostic toolboxes. At present, there are no CD47-specific nanobody molecular imaging probes at home and abroad.

[0008] To fill this gap in the field, we describe here the construction of a CD47-targeting theranostic pair derived from a nanobody and characterize its diagnostic and therapeutic value in cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models.

[0009] Therefore, technicians in this field are committed to developing a nano-antibody immune PET imaging probe with low preparation cost, small molecular weight, short in vivo circulation time, short imaging cycle, low radiation dose, and easy clinical transformation and application. Summary of the invention

[0010] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to prepare a nano-antibody immune PET imaging probe with low cost, small molecular weight, short in vivo circulation time, short imaging cycle, low radiation dose, and easy clinical transformation and application.

[0011] To achieve the above-mentioned object, the present invention provides a CD47-specific molecular imaging probe, including a human CD47-specific monovalent nanoantibody probe and a human CD47-specific nanoantibody fusion protein probe; the human CD47-specific monovalent nanoantibody probe includes nanoantibody C2 and a labeled radionuclide; the human CD47-specific nanoantibody fusion protein probe includes nanoantibody fusion protein ABDC2 and a labeled radionuclide.

[0012] Further, the amino acid sequence of Nanobody C2 is shown in the sequence list SEQ ID No. 1, and the gene sequence of Nanobody C2 is shown in the sequence list SEQ ID No. 2; the human CD47-specific monovalent Nanobody probe includes human CD47-specific 68 Ga-labeled monovalent nanoantibody probe [ 68 Ga]Ga-NOTA-C2 and human CD47 specificity 177 Lu-labeled monovalent nanoantibody probe [ 177 Lu]Lu-DOTA-C2; human CD47-specific nanoantibody fusion protein probe includes human CD47-specific 68 Ga-labeled nanoantibody fusion protein probe [ 68 Ga]Ga-NOTA-ABDC2, human CD47 specific 89 Zr-labeled nanoantibody fusion protein probe 89 Zr]Zr-DFO-ABDC2 and human CD47 specificity 177 Lu-labeled nanoantibody fusion protein probe 177 Lu]Lu-DOTA-ABDC2.

[0013] Furthermore, the amino acid sequence of the nanobody fusion protein ABDC2 is shown in SEQ ID No.3 in the sequence listing, and the gene sequence of the nanobody fusion protein ABDC2 is shown in SEQ ID No.4 in the sequence listing; the nanobody fusion protein ABDC2 contains a linker, the linker amino acid sequence is shown in SEQ ID No.5 in the sequence listing, and the linker gene sequence is shown in SEQ ID No.6 in the sequence listing.

[0014] Furthermore, the radionuclides are 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, Lu-177, 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, I-125, I-131, 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.

[0015] The present invention also provides a method for preparing a CD47-specific molecular imaging probe, comprising the following steps:

[0016] Step 1, modifying the CD47-specific nanobody C2 with a compound to synthesize a small molecule compound precursor, i.e., a radionuclide labeled precursor;

[0017] Step 2: Use radioactive nuclides to label the small molecule compound precursor obtained in step 1 to prepare a CD47-specific molecular imaging probe.

[0018] Furthermore, the compound in step 1 includes a macrocyclic ligand p-SCN-Bn-NOTA, p-SCN-Bn-Deferoxamine or p-SCN-Bn-DOTA; p-SCN-Bn-NOTA is 1,4,7-triazacyclononane-1,4,7-triacetic acid (2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid), p-SCN-Bn-Deferoxamine is deferoxamine, i.e., 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxyamino)-6,11,17, 22-tetraazaheptaeicosine]thiourea (1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosine]thiourea); p-SCN-Bn-DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid).

[0019] The present invention also provides an application of a CD47-specific molecular imaging probe in an immune PET diagnostic reagent, wherein the diagnostic reagent is a non-invasive target-specific immune PET diagnostic reagent for a specific type of tumor based on non-invasive visualization of the differential expression of CD47 in tumor tissues and normal tissues and organs.

[0020] Furthermore, the radionuclide labeled with the CD47-specific molecular imaging probe is 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 or Sc-44.

[0021] The present invention also provides an application of a CD47-specific molecular imaging probe in a radioimmunotherapy reagent, wherein the therapeutic reagent is a radioimmunotherapy reagent for CD47-positive tumors.

[0022] Furthermore, a radionuclide labeled with a CD47-specific molecular imaging probe is Lu-177, 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, I-125, I-131, 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.

[0023] In preferred embodiment 1 of the present invention, novel CD47-specific nanobody C2 and nanobody fusion protein ABDC2 and methods for preparing the same are described in detail;

[0024] In another preferred embodiment 2 of the present invention, the process of establishing a CD47 expression-positive tumor-bearing mouse model is described in detail;

[0025] In another preferred embodiment 3 of the present invention, the preparation of the probe [ 68 Ga]Ga-NOTA-C2 and [68Ga]Ga-NOTA-ABDC2 and their application in immunoPET imaging for the diagnosis of gastric cancer;

[0026] In another preferred embodiment 4 of the present invention, the preparation of the probe [ 89 Zr]Zr-DFO-ABDC2 and its application in the diagnosis of gastric cancer by immunoPET imaging;

[0027] In another preferred embodiment 5 of the present invention, the preparation of [ 177 Lu]Lu-DOTA-C2 and [ 177 Lu]Lu-DOTA-ABDC2 and its application in radioimmunotherapy.

[0028] The beneficial technical effects of the present invention are as follows:

[0029] The present invention realizes non-invasive visualization of human CD47 molecule expression, and further realizes non-invasive diagnosis of colorectal cancer and gastric cancer. The probe disclosed in the present invention has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging cycle, low radiation dose, and easy clinical transformation.

[0030] The present invention constructs a novel CD47-targeted treatment method, namely radioimmunotherapy. 177Lu]Lu-DOTA-ABDC2 has the advantages of high uptake in tumor sites and high signal-to-noise ratio, and preliminary therapeutic experiments have shown good anti-tumor ability.

[0031] in,[ 68 Ga]Ga-NOTA-C2, [ 68 Ga]Ga-NOTA-ABDC2, [ 89 Zr]Zr-DFO-C2, [ 89 Zr]Zr-DFO-ABDC2 is a positron emission probe used for immuno-PET imaging. By conducting immuno-PET imaging based on the above probe, non-invasive visualization of CD47 expression in tumor tissues and normal tissues and organs can be achieved, and further used for non-invasive target-specific diagnosis of specific types of tumors.

[0032] [ 177 Lu]Lu-DOTA-C2 and [ 177 Lu]Lu-DOTA-ABDC2 is a β-ray and γ-ray emitting probe. The β-rays it emits can be used for radioimmunotherapy of CD47-positive tumors, and the γ-rays it emits can be used for single photon emission computed tomography (SPECT). 177 [177Lu]Lu-DOTA-C2 and [177Lu]Lu-DOTA-ABDC2 are themselves integrated molecular imaging probes for diagnosis and treatment.

[0033] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a graph showing the experimental results of SDS-PAGE and protein blotting to determine the expression of Nanobody C2 in a preferred embodiment 1 of the present invention;

[0035] Figure 2 This is a diagram of the expression of nano antibody fusion protein ABDC2 measured by Western blotting and high performance liquid chromatography according to a preferred embodiment 1 of the present invention;

[0036] Figure 3 The results of a flow cytometric experiment of a preferred embodiment 2 of the present invention using anti-human CD47 monoclonal antibody (MCA-911, CloneBRIC126; BIO-RAD) as an antibody against ovarian cancer cell line SKOV-3 and colon adenocarcinoma cell line LS174T CD47 positive expression;

[0037] Figure 4The preferred embodiment 2 of the present invention is to use anti-human CD47 monoclonal antibody (B6H12, sc-12730, Santa Cruz) as the primary antibody, and to find positive experimental results of CD47 expression in SKOV-3 ovarian cancer, LS174T colon adenocarcinoma and gastric cancer human xenograft model (Patient-derived tumor xenograft, PDX) No. 490 by immunohistochemical staining (IHC);

[0038] Figure 5 It is the result of the affinity determination between the Nanobody C2 and the Nanobody fusion protein ABDC2 of a preferred embodiment 2 of the present invention and human CD47;

[0039] Figure 6 It is the result of the affinity determination of Nanobody C2 and Nanobody fusion protein ABDC2 with mouse CD47 in a preferred embodiment 2 of the present invention;

[0040] Figure 7 It is a preferred embodiment 3 of the present invention [ 68 Ga]Ga-NOTA-C2 and [ 68 Ga]Ga-NOTA-ABDC2 quality control chart;

[0041] Figure 8 It is a preferred embodiment 3 of the present invention [ 68 Ga]Ga-NOTA-C2 and [ 68 Pharmacokinetic study results of Ga]Ga-NOTA-ABDC2 in tumor-free Balb / c mice;

[0042] Fig. 9 It is a preferred embodiment 3 of the present invention [ 68 Ga]Ga-NOTA-C2 and [ 68 The curve of the uptake value of Ga]Ga-NOTA-ABDC2 in the main tissues and organs of tumor-free Balb / c mice over time and the comparison of the two probes (Region of interest, ROI);

[0043] Fig.10 It is a preferred embodiment 3 of the present invention [ 68 Ga]Ga-NOTA-C2 immunoPET imaging for diagnosis of colorectal cancer PET / CT imaging, ROI and biodistribution map;

[0044] Fig.11 It is a preferred embodiment 3 of the present invention [ 68Ga]Ga-NOTA-C2 immunoPET imaging for diagnosis of gastric cancer PET / CT imaging, ROI and in vitro biodistribution;

[0045] Fig.12 It is a probe of a preferred embodiment 3 of the present invention [ 68 Comparison of ROI data and in vitro biodistribution data of the distribution of Ga]Ga-NOTA-C2 in the main tissues and organs of the colorectal cancer model;

[0046] Fig.13 A probe of a preferred embodiment 3 of the present invention [ 68 Comparison of ROI data and in vitro biodistribution data of the distribution of Ga]Ga-NOTA-C2 in the main tissues and organs of the gastric cancer model;

[0047] Fig.14 It is a preferred embodiment 3 of the present invention [ 68 Optimization experimental results of Ga]Ga-NOTA-C2 immunoPET imaging for the diagnosis of ovarian cancer;

[0048] Fig.15 This is a graph showing the ROI data analysis results of an experimental group of an injection imaging agent injected with sodium maleate and a control group of a group not injected with sodium maleate according to a preferred embodiment 3 of the present invention;

[0049] Fig.16 It is a preferred embodiment 3 of the present invention [ 68 Ga]Ga-NOTA-ABDC2 immunoPET imaging for diagnosis of gastric cancer PET / CT imaging;

[0050] Fig.17 It is a preferred embodiment 3 of the present invention [ 68 ROI data analysis of the extended acquisition time of Ga]Ga-NOTA-ABDC2 in the subcutaneous gastric cancer tumor model;

[0051] Fig.18 It is a preferred embodiment 3 of the present invention [ 68 The enrichment result of Ga]Ga-NOTA-ABDC2 in the tumor site in the in vitro distribution experiment of gastric cancer subcutaneous tumor model;

[0052] Fig.19 It is a preferred embodiment 3 of the present invention [ 68 Comparison of ROI data and in vitro biodistribution data of Ga]Ga-NOTA-C2 probe in PET / CT imaging of gastric cancer model;

[0053] Fig. 20 It is a preferred embodiment 3 of the present invention [ 68Comparison of ROI data and in vitro biodistribution data of Ga]Ga-NOTA-ABDC2 probe in PET / CT imaging of gastric cancer model;

[0054] Fig.21 It is a preferred embodiment 4 of the present invention [ 89 After the Zr]Zr-DFO-ABDC2 labeling reaction was completed, the undecayed corrected radiochemical yield graph was detected;

[0055] Fig. 22 It is a preferred embodiment 4 of the present invention [ 89 Zr]Zr-DFO-ABDC2 quality control chart;

[0056] Fig.23 It is a preferred embodiment 4 of the present invention [ 89 Zr]Zr-DFO-ABDC2 immunoPET imaging for diagnosis of gastric cancer PET / CT coronal images;

[0057] Fig.24 It is a preferred embodiment 4 of the present invention [ 89 Zr]Zr-DFO-ABDC2 immunoPET imaging for diagnosis of gastric cancer MIP (Maximal Intensity Projection) image;

[0058] Fig.25 It is a preferred embodiment 4 of the present invention [ 89 In Zr]Zr-DFO-ABDC2 immunoPET imaging, CD47-specific nanoantibody fusion protein ABDC2 was used to draw a curve of the uptake value of major tissues and organs over time through ROI analysis;

[0059] Fig.26 It is a probe in the in vitro biodistribution experiment of a preferred embodiment 4 of the present invention [ 89 Distribution of Zr]Zr-DFO-ABDC2 in major tissues and organs in the body;

[0060] Fig. 27 This is an immunohistochemical staining diagram of a subcutaneous gastric cancer tumor model using CD47-specific antibody HPA044659 in a preferred embodiment 4 of the present invention;

[0061] Fig.28 It is a preferred embodiment 5 of the present invention [ 177 Lu]Lu-DOTA-C2 quality control chart;

[0062] Fig.29 It is a preferred embodiment 5 of the present invention [ 177Lu]Lu-DOTA-ABDC2 quality control chart;

[0063] Fig.30 It is a preferred embodiment 5 of the present invention [ 177 Lu]Lu-DOTA-C2 and [ 177 Comparison of in vitro biodistribution data of Lu]Lu-DOTA-ABDC2;

[0064] Fig.31 It is a preferred embodiment 5 of the present invention [ 177 Lu]Lu-DOTA-C2 and [ 177 Curve diagram of the changes in mouse body weight and tumor volume over time during Lu]Lu-DOTA-ABDC2 radioimmunotherapy;

[0065] Fig.32 It is a preferred embodiment 5 of the present invention [ 177 Lu]Lu-DOTA-C2 and [ 177 Lu]Lu-DOTA-ABDC2 radioimmunotherapy SPECT / CT images and the distribution of the probe in major tissues and organs in the body. DETAILED DESCRIPTION

[0066] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0067] Example 1 Novel CD47-specific nanobody C2 and nanobody fusion protein ABDC2

[0068] The present invention discloses a CD47-specific nano antibody C2, which has an amino acid sequence as shown in SEQ ID No. 1 in the sequence table and a gene sequence as shown in SEQ ID No. 2 in the sequence table.

[0069] The present invention discloses a CD47-specific nano-antibody fusion protein ABDC2, which has an amino acid sequence as shown in SEQ ID No. 3 in the sequence table and a gene sequence as shown in SEQ ID No. 4 in the sequence table. According to the method previously published by the inventor (name of the invention: a new molecular imaging probe for diagnosing multiple myeloma; application number: CN202011131233.7; publication number: CN112457401A; status: under review), a new CD47-specific nano-antibody C2 is prepared.

[0070] The expression of nanobody C2 was determined by SDS-PAGE and Western blotting. Figure 1As shown, the molecular weight of nanobody C2 is about 15KDa, and the purity can be as high as nearly 99%; the expression of nanobody fusion protein ABDC2 was determined by Western blotting and high performance liquid chromatography (HPLC, Agilent). Figure 2 As shown, after C2 was fused with ABD, the molecular weight was about 20 KDa, and HPLC showed a clear product peak.

[0071] Example 2 Establishment of CD47-positive tumor-bearing mouse model

[0072] The establishment of a CD47-positive tumor-bearing mouse model included the following steps: using anti-human CD47 monoclonal antibody (MCA-911, Clone BRIC126; BIO-RAD) as the primary antibody, flow cytometry experiments revealed that the ovarian cancer cell line SKOV-3 and the colon adenocarcinoma cell line LS174T expressed positive CD47, such as Figure 3 As shown; using anti-human CD47 monoclonal antibody (B6H12, sc-12730, Santa Cruz) as the primary antibody, immunohistochemical staining (IHC) revealed that SKOV-3 ovarian cancer, LS174T colon adenocarcinoma and gastric cancer human xenograft model (Patient-derived tumor xenograft, PDX) No.490 expressed positive CD47, as shown Figure 4 2×106 SKOV-3 and LS174T cells were suspended in PBS and Matrigel (Corning) at a ratio of 1:1 and injected into the right shoulder of 4-5 week-old Balb / c nude mice to establish subcutaneous ovarian cancer and colon adenocarcinoma models; 2mm*2mm*2mm No.490PDX tissue blocks were inoculated into the right shoulder of NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22 / Nju) mice to establish a subcutaneous gastric cancer PDX model.

[0073] Determination of affinity of nanobody C2 and nanobody fusion protein ABDC2 to human CD47 Figure 5 As shown, K D The values ​​were 23.5pM and 84.57pM respectively; the affinity of nanobody C2 and nanobody fusion protein ABDC2 to mouse CD47 was determined as follows Figure 6 As shown, it was shown that C2 and ABDC2 had no affinity for mouse CD47.

[0074] Example 3 Preparation of probe 68 Ga]Ga-NOTA-C2 and [ 68 Ga]Ga-NOTA-ABDC2 and its application in immuno-PET imaging for the diagnosis of colorectal cancer, gastric cancer and ovarian cancer

[0075] NOTA modified C2 and ABDC2 to prepare intermediates NOTA-C2 and NOTA-ABDC2. The specific steps are as follows: 1 mg C2 or ABDC2 was dissolved in 1 mL phosphate buffer (PBS), 0.1 mL 0.1 M sodium carbonate (Na2CO3, PH = 11.4) buffer was used to adjust the pH of the nanobody solution to 9.0-10, and the reaction system volume was 1.1 mL. At a molar ratio of p-SCN-Bn-NOTA / C2 or ABDC2 = 10:1, p-SCN-Bn-NOTA (CAS Number: 147597-66-8; Macrocyclics) freshly dissolved in dimethyl sulfoxide (DMSO) was added to the above nanobody solution. The reaction system was placed at room temperature for 2 hours, and then PBS was used as the mobile phase. The NOTA-modified nanoantibodies were purified using a pre-equilibrated PD-10 desalting column (GE Healthcare) to collect NOTA-C2 or NOTA-ABDC2; they were then concentrated using an ultrafiltration tube with a cutoff value of 10KDa (Merck Millipore), and the concentration of NOTA-C2 or NOTA-ABDC2 was determined using NanoDrop, and the separate devices were stored at -20°C for later use.

[0076] 68 Preparation of Ga-labeled NOTA-C2 and NOTA-ABDC2 68 Ga]Ga-NOTA-C2 and [ 68 Ga]Ga-NOTA-ABDC2. The specific steps are as follows: elute the gallium germanium generator (Eckert & Ziegler Radiopharma Inc) with 4 mL of 0.05 M hydrochloric acid solution (HCl), and collect an equal volume of 68Ga eluent with an activity of about 370–555 MBq; take 2 mL of the middle section 68Ga eluent with the highest activity, add 0.1 mL of 1 M sodium acetate solution (NaoAc) to adjust the pH of the 68Ga eluent to 4.0–4.5; take 100–200 μg of NOTA-C2 and NOTA-ABDC2 that have been coupled and add them to the 68Ga eluent, and the volume of the reaction system is <2.5 mL; place the reaction system in a constant temperature oscillator and react at room temperature for 5–10 minutes; after the labeling reaction, use PBS as the mobile phase, and use the pre-equilibrated PD-10 desalting column again to separate the free 68 Ga. Purify the final product; the unattenuated corrected radiochemical yield (RCY) obtained according to the above steps is >50%.

[0077] [ 68 Ga]Ga-NOTA-C2 and [ 68 Ga]Ga-NOTA-ABDC2 quality control. Pipette 10 μL [ 68Ga]Ga-NOTA-C2 or [ 68 Ga]Ga-NOTA-ABDC2 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). Figure 7 As shown, freshly prepared [ 68 The RCP of [Ga]Ga-NOTA-C2 and [68Ga]Ga-NOTA-ABDC2 are both greater than 99%.

[0078] [ 68 Ga]Ga-NOTA-C2 and [ 68 Pharmacokinetics of Ga]Ga-NOTA-ABDC2 in tumor-free Balb / c mice Figure 8 The following steps are included: The small animal PET / CT imaging acquisition involved in this study was completed using the IRIS small animal PET / CT scanner (Inviscan Imaging Systems). Each tumor-free Balb / c mouse was injected with 3.7-7.4MBq[ 68 Ga]Ga-NOTA-C2 and [ 68 Ga]Ga-NOTA-ABDC2 (4 mice in each group), the mice were anesthetized with isoflurane mixed with oxygen (concentration of 2%) at 0.5 hours, 2 hours and 4 hours after injection, and the mice in deep anesthesia were placed in supine position on the PET / CT scanning bed, and 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, Figure 8 Upper part 68 The Ga]Ga-NOTA-C2 probe mainly accumulated in the kidney and bladder at 0.5 h, while Figure 8 Lower part 68 Ga]Ga-NOTA-ABDC2 probe mainly accumulates in the heart, and the kidney takes up less 68The OsiriX Lite image processing workstation (Pixmeo SARL) was used to outline the regions of interest (ROI) such as the heart and major tissues and organs (liver, lungs, kidneys, muscles) 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), and the uptake values ​​of major tissues and organs were plotted over time to further compare the pharmacokinetic differences of the two probes. Fig. 9 The left and middle graphs represent [ 68 Ga]Ga-NOTA-C2 and [ 68 The uptake trend of Ga]Ga-NOTA-ABDC2 in main tissues and organs over time, Fig. 9 The figure on the right shows the difference in uptake of the two probes in different tissues and organs at 1H. 68 The in vivo circulation time of Ga]Ga-NOTA-ABDC2 probe is longer than that of 68 Ga]Ga-NOTA-C2 is significantly extended.

[0079] [ 68 Ga]Ga-NOTA-C2 immunoPET imaging for the diagnosis of colorectal cancer and gastric cancer. Fig.10 and Fig.11 As shown. The left side of the two groups of figures shows the PET / CT image, the middle shows the ROI image, and the right side shows the in vitro biodistribution data image. Both groups of figures show that the CD47-specific nanoantibody probe [ 68 Ga]Ga-NOTA-C2 has a higher uptake in tumor tissues and a higher nonspecific uptake in major excretion (kidney) and metabolic (liver) tissues. 68 In addition, the results of in vitro biodistribution experiments further revealed the distribution of the probe in major tissues and organs in vivo. By analyzing the ROI data and biodistribution data of the two tumor models, it can be shown that [ 68 There was no significant difference in the diagnostic efficacy of Ga]Ga-NOTA-C2 for these two tumor models. Fig.12 and Fig.13 The above results show that [ 68 The Ga]Ga-NOTA-C2 probe can noninvasively visualize CD47 expression.

[0080] [ 68In the optimization experiment of Ga]Ga-NOTA-C2 immunoPET imaging for ovarian cancer diagnosis, the experimental group was injected with sodium maleate (465 mg / kg) within 5 minutes before the injection of the imaging agent, while the control group was not injected. PET images were collected 0.5h and 2h after the injection of the imaging agent. Fig.14 As shown in the figure, the upper and lower parts show the imaging results of the control group and the sodium maleate group, respectively, indicating that sodium maleate can significantly reduce the uptake of the monovalent nanoantibody probe in kidney tissue. By statistical analysis of the ROI data, it can be seen that the kidney uptake of the sodium maleate group is significantly lower than that of the control group. Fig.15 shown.

[0081] [ 68 Ga]Ga-NOTA-ABDC2 immunoPET imaging for diagnosis of gastric cancer, the experimental results are as follows Fig.16 As shown, 68 Multi-time point PET / CT imaging of Ga]Ga-NOTA-ABDC2 in No.490 gastric cancer model. CD47-specific nanoantibody fusion protein probe [ 68 Ga]Ga-NOTA-ABDC2 still has a high uptake in tumor tissue. 68 The imaging study of Ga]Ga-NOTA-ABDC2 in tumor-free Balb / c mice showed that its circulation time was significantly prolonged. 68 Ga]Ga-NOTA-ABDC2 probe, the acquisition time was extended to 8h. Fig.17 As shown in the ROI data, the uptake in the tumor site gradually increased with time within 8 hours, while the uptake in the heart and other parts gradually decreased. Fig.18 The in vitro distribution data shown further confirmed the enrichment of the probe in the tumor site. By further comparing the ROI and in vitro biodistribution data of the two probes [68Ga]Ga-NOTA-C2 and [68Ga]Ga-NOTA-ABDC2 in gastric cancer model PET / CT imaging, it was shown that the fusion protein probe did not affect its ability to non-invasively visualize CD47 inside the tumor. The experimental results are shown in Fig.19 and Fig. 20 shown.

[0082] Example 4 Preparation of probe 89 Zr]Zr-DFO-ABDC2 and its application in the diagnosis of gastric cancer using immunoPET imaging

[0083] DFO modified ABDC2 to prepare the intermediate DFO-ABDC2, the specific steps are as follows: 3 mg ABDC2 was dissolved in 1 mL phosphate buffer (PBS), 0.1 mL 0.1 M sodium carbonate (Na2CO3, PH = 11.4) buffer to adjust the pH of the nanoantibody solution to 8.9-9.1, and the reaction system volume was 1.1 mL. DFO (CAS Number: 147597-66-8; Macrocyclics) freshly dissolved in dimethyl sulfoxide (DMSO) was added to the above nanoantibody solution at a molar ratio of DFO / ABDC2 = 5:1. The reaction system was placed at room temperature for 30 minutes, and then PBS was used as the mobile phase, and the DFO-modified nanoantibody was purified with a pre-equilibrated PD-10 desalting column (GE Healthcare), and DFO-ABDC2 was collected; it was then concentrated with an ultrafiltration tube (Merck Millipore) with a cutoff value of 10 KDa, and the concentration of DFO-ABDC2 was determined with NanoDrop, and the device was stored at -20°C for use.

[0084] 89 Preparation of Zr-labeled DFO-ABDC2 89 Zr]Zr-DFO-ABDC2. The specific steps are as follows: prepare 450μl, 100MBq89Zr oxalic acid solution, and adjust the pH to 7 using 1M Na2CO3 buffer solution. Then gradually add 500μl 0.5M HEPES solution (pH 7.1–7.3) and 200μl DFO-ABDC2 (360μg) to the reaction solution. Place the reaction system in a constant temperature oscillator and react at room temperature for 1 hour. After the labeling reaction is completed, PBS is used as the mobile phase, and the free 89 Zr, purified final product; the unattenuated corrected radiochemical yield (RCY) obtained according to the above steps is > 99%, such as Fig.21 shown.

[0085] [ 89 Zr]Zr-DFO-ABDC2 quality control. Pipette 10 μl [ 89 Zr]Zr-DFO-ABDC2 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). Fig. 22 As shown, freshly prepared [ 89 Zr]Zr-DFO-ABDC2 RCP is greater than 99%.

[0086] [ 89 Zr]Zr-DFO-ABDC2 immunoPET imaging for diagnosis of gastric cancer, 89 Zr]Zr-DFO-ABDC2 immunoPET imaging further confirmed the enrichment ability of CD47-specific nanoantibody fusion protein ABDC2 in tumor sites. PET / CT images were collected 1, 6, 12, 24, 48, 72, 96, 120, and 144 hours after probe injection. Fig.23 Coronal PET / CT images show tumor uptake at 6h, 72h, and 144h. Fig.24 The MIP images after PET / CT fusion showed the probe uptake of the tumor and major tissues and organs at all time points, indicating that the uptake of the probe in the tumor site gradually increased over time, reached a peak at 72 hours, and then gradually decreased. The uptake value curve of the tumor and major tissues and organs (heart, liver, lung, kidney, muscle, spleen and bone) was drawn over time through ROI analysis, indicating that [ 89 Zr]Zr-DFO-ABDC2 has a good signal-to-noise ratio in vivo and good stability in vivo, such as Fig.25 In vitro biodistribution experiments further revealed that the probe [ 89 The distribution of Zr]Zr-DFO-ABDC2 in tumors and major tissues and organs in vivo, such as Fig.26 Furthermore, immunohistochemical staining of the tumor using CD47-specific antibody HPA044659 confirmed the expression of CD47 inside the tumor, as shown in Fig. 27 shown.

[0087] Example 5 Preparation 177 Lu]Lu-DOTA-C2 and [ 177 Lu]Lu-DOTA-ABDC2 and its application in radioimmunotherapy

[0088] DOTA modified C2 and ABDC2 to prepare intermediates DOTA-C2 and DOTA-ABDC2. The specific steps are as follows:

[0089] 177 Preparation of Lu-labeled DOTA-C2 and DOTA-ABDC2 177 Lu]Lu-DOTA-C2 and [ 177 Lu]Lu-DOTA-ABDC2. The specific steps are as follows:

[0090] [ 177 Lu]Lu-DOTA-C2 and [ 177 Lu]Lu-DOTA-ABDC2 quality control. Pipette 10μl [ 177Lu]Lu-DOTA-C2 and [ 177 Lu]Lu-DOTA-ABDC2 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). Fig.28 and Fig.29 Freshly prepared [ 177 Lu]Lu-DOTA-C2 and [ 177 The RCP of Lu]Lu-DOTA-ABDC2 was greater than 99%, and the two probes remained stable in PBS solution for 72 hours, that is, the RCP was still greater than 90% after 72 hours.

[0091] [ 177 Lu]Lu-DOTA-C2 and [ 177 To investigate the efficacy of CD47-specific nanobody fusion protein ABDC2 as a therapeutic carrier, we used 177 Lu conducted the treatment experiment. Before conducting the treatment experiment, we injected [ 177 Lu]Lu-DOTA-C2 and [ 177 Lu]Lu-DOTA-ABDC2, and the mice were killed 7 days after injection for in vitro biodistribution experiments to clarify the distribution of the two probes in the main tissues and organs in vivo. Fig.30 As shown, 1 week after injection of the imaging agent, [ 177 The uptake of Lu]Lu-DOTA-ABDC2 in tumor sites was significantly higher than that of [ 177 Lu]Lu-DOTA-C2, while the uptake in the kidney was significantly lower than that in 177 Lu]Lu-DOTA-C2. Afterwards, we divided the treatment experiment into 5 groups, namely control group, [ 177 Lu]Lu-DOTA-C2 group, low dose [177Lu]Lu-DOTA-ABDC2 group, high dose [ 177 The body weight and tumor volume of the five groups of mice were monitored every 2 days after administration for a total of 1 month, and the curves of changes over time were drawn as shown in Fig.31 As shown. Among them, one week after administration, the injection of 177 Lu]Lu-DOTA-C2 and [ 177The three groups of Lu]Lu-DOTA-ABDC2 were subjected to SPECT / CT imaging, and the main tissue and organ ROIs were delineated to analyze the distribution of the probe in the body. Fig.32 The left coronal SPECT / CT image shows the probe uptake in the tumor and kidney. Fig.32 The right side shows the distribution differences of the three groups of probes in tumors and major organs in the body. Fig.30 The above data indicate that ABDC2 is a potential CD47-targeted therapeutic vector.

[0092] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A CD47-specific molecular imaging probe, characterized in that: The probe includes a human CD47-specific monovalent nanobody probe and a human CD47-specific nanobody fusion protein probe; the human CD47-specific monovalent nanobody probe includes nanobody C2 and a labeled radionuclide; the human CD47-specific nanobody fusion protein probe includes nanobody fusion protein ABDC2 and the labeled radionuclide; the amino acid sequence of the nanobody C2 is shown in the sequence table SEQ ID No.1, and the gene sequence of the nanobody C2 is shown in the sequence table SEQ ID No.2; the human CD47-specific monovalent nanobody probe includes human CD47-specific 68 Ga-labeled monovalent nanoantibody probe [ 68 Ga]Ga-NOTA-C2 and human CD47 specificity 177 Lu-labeled monovalent nanoantibody probe[ 177 Lu]Lu-DOTA-C2; the human CD47-specific nanoantibody fusion protein probe includes human CD47-specific 68 Ga-labeled nanoantibody fusion protein probe [ 68 Ga]Ga-NOTA-ABDC2, human CD47 specific 89 Zr-labeled nanoantibody fusion protein probe 89 Zr]Zr-DFO-ABDC2 and human CD47 specificity 177 Lu-labeled nanoantibody fusion protein probe 177 Lu]Lu-DOTA-ABDC2; the amino acid sequence of the nano antibody fusion protein ABDC2 is shown in SEQ ID No.3 in the sequence listing, and the gene sequence of the nano antibody fusion protein ABDC2 is shown in SEQ ID No.4 in the sequence listing; the nano antibody fusion protein ABDC2 contains a linker, the linker amino acid sequence is shown in SEQ ID No.5 in the sequence listing, and the linker gene sequence is shown in SEQ ID No.6 in the sequence listing.

2. The method for preparing the CD47-specific monovalent nanobody probe according to claim 1, characterized in that: The method comprises the following steps: Step 1, modifying the CD47-specific nanobody C2 with a compound to synthesize a small molecule compound precursor, i.e., a radionuclide labeled precursor; Step 2: labeling the small molecule compound precursor obtained in step 1 with a radionuclide to prepare the CD47-specific monovalent nanobody probe.

3. The preparation method according to claim 2, characterized in that: The compound in step 1 includes a macrocyclic ligand p-SCN-Bn-NOTA, p-SCN-Bn-Deferoxamine or p-SCN-Bn-DOTA; the p-SCN-Bn-NOTA is 1,4,7-triazacyclononane-1,4,7-triacetic acid (2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid), the p-SCN-Bn-Deferoxamine is deferoxamine, i.e. 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosine]thiourea The p-SCN-Bn-DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid).

4. A reagent comprising the molecular imaging probe according to claim 1.

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