CN115737845A - CD47 specific molecular imaging probe and preparation method and application thereof - Google Patents

Abstract

The invention discloses a CD47 specific molecular imaging probe and a preparation method and application thereof, relating to the technical field of molecular imaging, nuclear medicine and nano-antibody for tumor diagnosis and treatment, and comprising a human CD47 specific monovalent nano-antibody probe and a human CD47 specific nano-antibody fusion protein probe; the preparation method comprises the following steps: modifying the CD47 specific nano antibody C2 by a compound, synthesizing a radionuclide labeled precursor, 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 invention has the advantages of simple preparation process, low cost, high specificity, high stability, easy clinical transformation and the like. The non-invasive visualization of CD47 heterogeneous expression can be realized, patients with high CD47 expression can be screened, and CD47 specific radioimmunotherapy is further developed, namely, diagnosis and treatment integration is realized.

Description

CD47 specific molecular imaging probe and preparation method and application thereof

Technical Field

The invention relates to the technical field of molecular imaging, nuclear medicine and nano antibodies for tumor diagnosis and treatment, in particular to a CD47 specific molecular imaging probe and a preparation method and application thereof.

Background

The presence of a naturally light chain-deficient antibody in the peripheral blood of an alpaca was first reported in 1993 by the scientist Hamers et al in Nature journal (Nature.1993; 363 (6428): 446-8.) and this antibody with a specific domain is a Heavy chain antibody (HCAbs). Through molecular biology means, the Variable region of the Heavy Chain Antibody is cloned to obtain an antigen binding fragment only with the Heavy Chain Variable region, namely a nano Antibody (VHH). VHH crystals 2.5nm wide and 4nm long with a molecular weight of only 15KDa, and are therefore also called Nanobodies ((R))

Registered trade names of Ablynx corporation). The nano antibody is the minimum antibody unit which can be combined with a target antigen and is known at present, and has the advantages of high affinity, small molecular weight, low preparation cost (the expression can be carried out by using escherichia coli, and the expression can also be carried out by using eukaryotic expression systems such as yeast and Chinese hamster ovary cells), easy clinical transformation and popularization and application.

The nano antibody is a hot targeting carrier (Theranostics.2014; 4 (4): 386-98) for constructing a molecular imaging probe in recent years. At present, various nuclides with short half-life periods are used for marking nano antibodies and preparing nano antibody molecular imaging probes. Technetium-99 m ( ^99m Tc; t1/2 =6.02h) labeled programmed death ligand 1 (PD-L1) nanobody probes have been successfully transformed into clinics for noninvasive diagnosis of non-small cell lung cancer patients (J nuclear med.2019;60 (9) 1213 to 1220); gallium-68 ( ⁶⁸ Ga; t1/2= 1.1h) labeled nanobody probes targeting human epidermal growth factor receptor (HER 2) have also been successfully transformed into clinics for noninvasive diagnosis of breast cancer (J nuclear med.2016;57 (1):27-33.). The above examples show that the radionuclide-labeled nano antibody probe has great clinical transformation application prospect, and can be used for early noninvasive diagnosis of human malignant tumor,Visualization of key pathogenic targets, screening of monoclonal antibody (mAb) treated patients, and evaluation of efficacy of monoclonal antibody after treatment.

CD47 is the only known 5-transmembrane receptor in the immune system, which is widely expressed in different cell types in vivo. It exerts physiological effects by binding to signal-regulatory protein alpha (sirpa), affecting the homeostatic balance of red blood cells, platelets, and hematopoietic stem cells, and regulating synaptic pruning during neuronal development. However, tumor cells use this mechanism to send a "do not eat me" signal to macrophages to escape macrophage clearance. The research shows that the over-expression of CD47 on the surface of tumor cells is related to poor prognosis, such as acute myelogenous leukemia, colorectal cancer, lymphoma and the like. Clinical trials have demonstrated the therapeutic role of the targeting CD47 monoclonal antibody magrolimab in patients with non-hodgkin's lymphoma and other types of solid tumors. However, extensive expression of CD47 in normal tissues will lead to antigen-sinking effects of the targeting CD47 antibody in vivo, inevitably leading 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-CD 47 therapy. Based on the research of the accompanying diagnostic tools, novel treatment methods for CD47 can be further developed.

Currently, immunohistochemical staining (immunohistochemical staining) of surgically excised or needle biopsied tissue is the most common method for detecting CD47 expression. However, studies have shown that immuno-PET can better display the distribution and abundance of targets of interest in vivo and better predict response to targeted therapies compared to immunohistochemical staining or other conventional predictive markers.

By skillfully fusing the extraordinary targeting specificity of the antibody and the excellent sensitivity and resolution of Positron Emission Tomography (PET), the immune PET can noninvasively display the expression condition of the target point of interest in vivo (Chem Rev.2020;120 (8): 3787-3851.). For example, immunopet imaging probes targeting programmed cell death ligand-1 have been successfully applied in clinical practice and better predicted therapeutic efficacy for the PD-L1 specific monoclonal antibody atezolizumab than other traditional predictive biomarkers. In addition, it has been reported that the immuno-PET probe can be used to evaluate the dynamic changes of lymphocytes and myeloid cells before and after cancer immune checkpoint treatment, and further reveal the immune status inside tumors. Based on the above evidence and our previous findings, we hypothesized that CD 47-targeted immune PET imaging probes could non-invasively display intratumoral CD47 expression and provide a better approach for selecting patients who might benefit from CD47 phagocytosis checkpoint inhibition therapy. Furthermore, there is evidence that Radioimmunotherapy (RIT) and pre-targeted radioimmunotherapy (pRIT) may help patients with tumors to relieve disease for long periods of time, even eradicating a variety of cancer types.

Currently, there is no report in the literature on the diagnosis of CD 47-targeted therapies. Zoleznyak A et al demonstrate use ⁸⁹ The Zr-tagged targeting CD47 monoclonal antibody (mAb) showed feasibility of CD47 expression within tumors (Mol imaging. Nov-Dec 2013 (8). However, the use of radiolabeled monoclonal antibodies is hampered by the high cost, necessity of using long half-life radionuclides, cumbersome imaging procedures within a week, and associated radiation exposure. To improve the clinical applications of antibody diagnostics, the field of molecular imaging is actively exploring pre-targeted imaging strategies or using smaller antibody derivatives to achieve on-day molecular imaging (same-day imaging). In the minibody format, nanobodies or single domain antibodies from the family camelidae are the smallest antigen-binding part with a molecular weight of about 15 kDa. The small size, high affinity and easy engineering design make nanobodies an excellent alternative to 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 in vivo half-life is too short and renal uptake is high, leaving room for further improvement. To develop an integrated diagnostic therapeutic platform, an Albumin Binding Domain (ABD) targeting human/mouse albumin was introduced into nanobodies to extend the half-life of nanobody derivatives in vivo. Research shows that the bispecific nano antibody derivative simultaneously targeting tumor antigen and albumin improves the bodyThe internal biodistribution can be used as a carrier for developing a therapeutic and diagnostic tool kit. At present, no CD47 specific nano antibody molecular imaging probe exists at home and abroad.

To fill the gap in this field, we describe herein the construction of a nanobody-derived CD 47-targeted diagnostic pair and characterize its diagnostic and therapeutic value in cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models.

Therefore, those skilled in the art are devoted to develop a nano antibody immune PET imaging probe which has low preparation cost, small molecular weight, short in-vivo circulation time, short imaging period, low radiation dose and easy clinical transformation application.

Disclosure of Invention

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

In order to achieve the above purpose, the present invention provides a CD47 specific molecular imaging probe, comprising a human CD47 specific monovalent nanobody probe and a human CD47 specific nanobody fusion protein probe; the human CD47 specific monovalent nanometer antibody probe comprises a nanometer antibody C2 and a labeled radionuclide; the human CD47 specific nano antibody fusion protein probe comprises a nano antibody fusion protein ABDC2 and a labeled radionuclide.

Furthermore, the amino acid sequence of the nano antibody C2 is shown in a sequence table SEQ ID No.1, and the gene sequence of the nano antibody C2 is shown in a sequence table SEQ ID No. 2; the human CD47 specific monovalent nanometer antibody probe comprises human CD47 specificity ⁶⁸ Ga-labeled monovalent nanobody probe ⁶⁸ Ga]Ga-NOTA-C2 and human CD47 specificity ¹⁷⁷ Lu labeled monovalent nanobody probe ¹⁷⁷ Lu]Lu-DOTA-C2; the human CD47 specific nano antibody fusion protein probe comprises human CD47 specificity ⁶⁸ Ga-labeled nanobody fusion protein probe ⁶⁸ Ga]Ga-NOTA-ABDC2, human CD47 specificity ⁸⁹ Zr labeled nano antibody fusion protein probe ⁸⁹ Zr]Zr-DFO-ABDC2 andspecificity for human CD47 ¹⁷⁷ Lu labeled nano antibody fusion protein probe ¹⁷⁷ Lu]Lu-DOTA-ABDC2。

Further, the amino acid sequence of the nano antibody fusion protein ABDC2 is shown in a sequence table SEQ ID No.3, and the gene sequence of the nano antibody fusion protein ABDC2 is shown in a sequence table SEQ ID No. 4; the nano antibody fusion protein ABDC2 contains a linker, the amino acid sequence of the linker is shown as SEQ ID No.5 in a sequence table, and the gene sequence of the linker is shown as SEQ ID No.6 in the sequence table.

Furthermore, the radionuclide 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, 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-188, sm-186, ra-223, ru-106, na-24, sr-89, tb-149, xe-149, yb-227, yb-169 or Yb-177.

The invention also provides a preparation method of the CD47 specific molecular imaging probe, which comprises the following steps:

step 1, modifying a CD47 specific nano antibody C2 through a compound to synthesize a small molecule compound precursor, namely a radionuclide labeled precursor;

and 2, labeling the small molecular compound precursor obtained in the step 1 by using a radionuclide to prepare the CD47 specific molecular imaging probe.

Further, the compound in step 1 comprises 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-triazacyclonane-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-acetoxyamino) -6,11,17,22-tetraazaheptobiose ] thiourea (1- (4-isothiocyanatophenyl) -3- [6,17-dihydroxy-7,10,18,21-tetroxy-27- (N-acetylaminocarbonyl) -3525-tetraazaoxourea ] 3579); the p-SCN-Bn-DOTA is 1,4,7, 10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (S-2- (4-isothiocyanato-zyl) -1,4,7,10-tetraazacyclododecaneacetic acid).

The invention also provides application of the CD47 specific molecular imaging probe in an immune PET diagnostic reagent, wherein the diagnostic reagent is a noninvasive target spot specific immune PET diagnostic reagent for specific tumors based on CD47 differential expression noninvasive visualization in tumor tissues and normal tissues and organs.

Furthermore, the labeled radionuclide of 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.

The invention also provides application of the CD47 specific molecular imaging probe in a radioimmunotherapy reagent, wherein the radioimmunotherapy reagent is a CD47 expression positive tumor radioimmunotherapy reagent.

Furthermore, the marked radionuclide of the 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.

In the preferred embodiment 1 of the present invention, a novel CD 47-specific nanobody C2 and nanobody fusion protein ABDC2 and a method for preparing the same are explained in detail;

in another preferred embodiment 2 of the present invention, the process of establishing a mouse model of tumor positive for CD47 expression is described in detail;

in another preferred embodiment 3 of the present invention, the preparation of the probe [ 2 ] ⁶⁸ Ga]Ga-NOTA-C2 and [68Ga ]]Ga-NOTA-ABDC2 and application thereof in diagnosing gastric cancer by immune PET imaging;

in another preferred embodiment 4 of the present invention, the preparation of the probe [ 2 ] ⁸⁹ Zr]Zr-DFO-ABDC2 and the application thereof in the diagnosis of gastric cancer by immune PET imaging;

in another preferred embodiment 5 of the present invention, the preparation [ preparation ] ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]Lu-DOTA-ABDC2 and its application in radioimmunotherapy.

The beneficial technical effects of the invention are as follows:

the invention realizes the noninvasive visualization of human CD47 molecular expression and further realizes the noninvasive diagnosis of colorectal cancer and gastric cancer, and the probe disclosed by the invention has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging period, low radiation dose, easy clinical transformation and the like.

The invention constructs a novel targeted CD47 treatment method, namely radioimmunotherapy. [ ¹⁷⁷ Lu]Lu-DOTA-ABDC2 has the advantages of high tumor site uptake, high signal to noise ratio and the like, and shows good anti-tumor capability in preliminary treatment experiments.

<xnotran> , [ </xnotran> ⁶⁸ Ga]Ga-NOTA-C2、[ ⁶⁸ Ga]Ga-NOTA-ABDC2、[ ⁸⁹ Zr]Zr-DFO-C2、[ ⁸⁹ Zr]The Zr-DFO-ABDC2 is a positron nuclide emission type probe and is used for immune PET imaging; by developing the immune PET imaging based on the probe, the noninvasive visualization of the expression of the CD47 in tumor tissues and normal tissues and organs can be realized, and the method is further used for noninvasive target specific diagnosis of specific types of tumors.

[ ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]Lu-DOTA-ABDC2 is a beta-ray and gamma-ray emission type probe, the emitted beta-ray can be used for radioimmunotherapy of CD47 expression positive tumor, and the gamma-ray can be used for Single Photon Emission Computed Tomography (SPECT). In other words, the term ¹⁷⁷ Lu]Lu-DOTA-C2 and [177Lu]Lu-DOTA-ABDC2 is a diagnosis and treatment integrated molecular imaging probe.

The conception, specific structure and technical effects of the present invention will be further described in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention.

Drawings

FIG. 1 is a diagram of the results of SDS-PAGE and Western blotting to determine the expression of Nanobody C2 according to a preferred embodiment of the present invention 1;

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

FIG. 3 shows the results of flow cytometry experiments using anti-human CD47 monoclonal antibody (MCA-911, clone BRIC126, BIO-RAD) as a positive anti-ovarian cell line SKOV-3 and colon adenocarcinoma cell line LS174T CD47 according to a preferred embodiment 2 of the present invention;

FIG. 4 shows the positive test results of SKOV-3 ovarian cancer, LS174T colon adenocarcinoma and human-derived xenograft model (PDX) No.490 CD47 expression by Immunohistochemical staining (IHC) using anti-human CD47 monoclonal antibody (B6H 12, sc-12730, santa Cruz) as a primary antibody in accordance with a preferred embodiment 2 of the present invention;

FIG. 5 shows the result of the determination of the affinity of the Nanobody C2 and the Nanobody fusion protein ABDC2 to human CD47 according to the preferred embodiment 2 of the present invention;

FIG. 6 shows the result of the affinity assay of the nanobody C2 and the nanobody fusion protein ABDC2 of the preferred embodiment 2 of the present invention with murine CD 47;

FIG. 7 is the term "2" according to a preferred embodiment of the present invention ⁶⁸ Ga]Ga-NOTA-C2 and [ 2 ] ⁶⁸ Ga]A Ga-NOTA-ABDC2 quality control chart;

FIG. 8 is [ 2 ] of preferred embodiment 3 of the present invention ⁶⁸ Ga]Ga-NOTA-C2 and [ 2 ] ⁶⁸ Ga]The result of the pharmacokinetic study of Ga-NOTA-ABDC2 in tumor-free Balb/c mice is shown;

FIG. 9 is the term "2" according to a preferred embodiment of the present invention ⁶⁸ Ga]Ga-NOTA-C2 and ⁶⁸ Ga]graph of the uptake value of the main tissue organ of Ga-NOTA-ABDC2 in tumor-free Balb/c mice along with the time change and a comparison graph of two probes (ROI);

FIG. 10 is the term "2" according to a preferred embodiment of the present invention ⁶⁸ Ga]The Ga-NOTA-C2 immune PET imaging diagnoses a colorectal cancer PET/CT imaging picture, an ROI and a biodistribution picture;

FIG. 11 is the term "2" according to a preferred embodiment of the present invention ⁶⁸ Ga]Ga-NOTA-C2 immune PET imaging diagnosisCutting off a stomach cancer PET/CT image display, an ROI and an in-vitro biological distribution diagram;

FIG. 12 is the probe of the preferred embodiment of the present invention 3 ⁶⁸ Ga]ROI data comparison graph and in vitro biodistribution data comparison graph of distribution condition of Ga-NOTA-C2 in main tissue organs in the colorectal cancer model;

FIG. 13 is a probe [ 2 ] according to a preferred embodiment of the present invention ⁶⁸ Ga]An ROI data comparison graph and an in vitro biodistribution data comparison graph of the distribution condition of the main tissues and organs of Ga-NOTA-C2 in a gastric cancer model body;

FIG. 14 is the term "2" according to a preferred embodiment of the present invention ⁶⁸ Ga]An optimized experiment result of the Ga-NOTA-C2 immune PET imaging diagnosis of ovarian cancer;

FIG. 15 is a graph showing the results of ROI data analysis of an experiment group injected with sodium maleate and a control group not injected with sodium maleate, which are contrast agents in accordance with a preferred embodiment 3 of the present invention;

FIG. 16 is [ 2 ] of preferred embodiment 3 of the present invention ⁶⁸ Ga]The Ga-NOTA-ABDC2 immune PET imaging diagnoses a gastric cancer PET/CT imaging picture;

FIG. 17 is [ 2 ] of preferred embodiment 3 of the present invention ⁶⁸ Ga]Graph of ROI data analysis of Ga-NOTA-ABDC2 in extended acquisition time in a gastric carcinoma subcutaneous tumor model;

FIG. 18 is [ 2 ] of preferred embodiment 3 of the present invention ⁶⁸ Ga]An enrichment result graph of Ga-NOTA-ABDC2 in a gastric cancer subcutaneous tumor model in-vitro distribution experiment at a tumor part;

FIG. 19 is the term "2" according to a preferred embodiment of the present invention ⁶⁸ Ga]An ROI data contrast graph and an in vitro biodistribution data contrast graph of the Ga-NOTA-C2 probe in the gastric cancer model PET/CT imaging are obtained;

FIG. 20 is the set forth term of preferred embodiment 3 of the present invention ⁶⁸ Ga]A contrast graph of ROI data and a contrast graph of in vitro biodistribution data of the Ga-NOTA-ABDC2 probe in the PET/CT imaging of a gastric cancer model;

FIG. 21 is the term "2" according to a preferred embodiment of the present invention ⁸⁹ Zr]Detecting a non-attenuation correction radiochemical yield map after the Zr-DFO-ABDC2 labeling reaction is finished;

FIG. 22 is a drawing of the inventionThe composition of preferred embodiment 4 ⁸⁹ Zr]A Zr-DFO-ABDC2 quality control chart;

FIG. 23 is the term "2" according to a preferred embodiment of the present invention ⁸⁹ Zr]Carrying out PET imaging diagnosis on gastric cancer by Zr-DFO-ABDC2 immune PET;

FIG. 24 is the term "2" according to a preferred embodiment of the present invention ⁸⁹ Zr]MIP (maximum Intensity Projection) map for diagnosing gastric cancer by Zr-DFO-ABDC2 immune PET imaging;

FIG. 25 is the value of [ 2 ], [ 2 ] of a preferred embodiment of the present invention ⁸⁹ Zr]A curve graph of the change of the uptake value of a main tissue and organ along with time is drawn by the CD47 specific nano antibody fusion protein ABDC2 through ROI analysis in the Zr-DFO-ABDC2 immune PET imaging;

FIG. 26 is the probe [ 2 ] in the in vitro biodistribution test of the preferred embodiment 4 of the present invention ⁸⁹ Zr]Distribution of Zr-DFO-ABDC2 in main tissues and organs in vivo;

FIG. 27 is a diagram of immunohistochemical staining of a gastric carcinoma subcutaneous tumor model using HPA044659 as a CD 47-specific antibody in accordance with a preferred embodiment of the present invention;

FIG. 28 is the set forth term of preferred embodiment 5 of the present invention ¹⁷⁷ Lu]Lu-DOTA-C2 quality control chart;

FIG. 29 is the term "2" according to a preferred embodiment of the present invention ¹⁷⁷ Lu]Lu-DOTA-ABDC2 quality control chart;

FIG. 30 is the term "2" according to a preferred embodiment of the present invention ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]A Lu-DOTA-ABDC2 in vitro biodistribution data comparison graph;

FIG. 31 is [ 2 ] of a preferred embodiment 5 of the present invention ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]Time-dependent curves of mouse body weight and tumor volume during Lu-DOTA-ABDC2 radioimmunotherapy;

FIG. 32 is the set forth term of preferred embodiment 5 of the present invention ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]A SPECT/CT image display of Lu-DOTA-ABDC2 radioimmunotherapy and a distribution image of main tissues and organs in a probe.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Example 1 novel CD 47-specific Nanobody C2 and Nanobody fusion protein ABDC2

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

The invention discloses a CD47 specific nano antibody fusion protein ABDC2, which has an amino acid sequence shown as SEQ ID No.3 in a sequence table and a gene sequence shown as SEQ ID No.4 in the sequence table. The novel CD47 specific nano antibody C2 is prepared according to the method (the invention and creation name: a novel molecular imaging probe for diagnosing multiple myeloma; application number: CN202011131233.7; publication number: CN112457401A; state: check) published by the inventor.

SDS-PAGE and Western blot analysis show that the expression of the nano antibody C2 is shown in figure 1, and the molecular weight of the nano antibody C2 is about 15KDa, and the purity can be as high as nearly 99%; the expression of the nano antibody fusion protein ABDC2 is measured by western blotting and high performance liquid chromatography (HPLC, agilent), as shown in figure 2, after C2 is fused with ABD, the molecular weight is about 20KDa, and the HPLC can display clear product peaks.

Example 2 establishment of CD47 expression-positive tumor-bearing mouse model

The establishment of the CD47 expression positive tumor-bearing mouse model comprises the following steps: with anti-human CD47 monoclonal antibody (MCA-911, clone BRIC126, BIO-RAD) as a primary antibody, positive expression of ovarian cancer cell line SKOV-3 and colon cancer cell line LS174T CD47 was found by flow cytometry experiments, as shown in FIG. 3; SKOV-3 ovarian cancer, LS174T colon adenocarcinoma and human-derived xenograft model (PDX) No.490 CD47 expression were found to be positive by Immunohistochemical staining (IHC) using anti-human CD47 monoclonal antibody (B6H 12, sc-12730, santa Cruz) as a primary antibody, as shown in FIG. 4. Suspending 2X 106SKOV-3 and LS174T cells in PBS and matrigel (Corning), wherein the ratio of the two is 1:1, and injecting the cells into the right shoulder of a Balb/c nude mouse with the age of 4-5 weeks to establish a subcutaneous ovarian cancer and colon adenocarcinoma model; no.490PDX tissue blocks of 2mm x 2mm size were inoculated in the right shoulder of NCG (NOD-Prkdcem 26Cd52Il2rgem26Cd 22/Nju) mice to establish subcutaneous gastric carcinoma PDX model.

The affinity of the nano antibody C2 and the nano antibody fusion protein ABDC2 and human CD47 is determined as shown in FIG. 5, K _D Values of 23.5pM and 84.57pM, respectively; the affinity assay of the nano antibody C2 and the nano antibody fusion protein ABDC2 and the murine CD47 is shown in FIG. 6, which shows that the affinity assay of the C2 and the ABDC2 and the murine CD47 is not good.

Example 3 preparation of the Probe [ 2 ] ⁶⁸ Ga]Ga-NOTA-C2 and [ 2 ] ⁶⁸ Ga]Ga-NOTA-ABDC2 and application thereof in immune PET imaging diagnosis of colorectal cancer, gastric cancer and ovarian cancer

And preparing intermediates NOTA-C2 and NOTA-ABDC2 by modifying C2 and ABDC2 with NOTA. The method comprises the following specific steps: 1mgC2 or ABDC2 was dissolved in 1mL Phosphate Buffer (PBS), 0.1mL 0.1M sodium carbonate (Na 2CO3, pH = 11.4) buffer to adjust the pH of the nanobody solution to 9.0-10, and the volume of the reaction system was 1.1mL. p-SCN-Bn-NOTA (CAS Number: 147597-66-8) freshly dissolved in dimethyl sulfoxide (DMSO) was added to the above nanobody solution at a molar ratio of p-SCN-Bn-NOTA/C2 or ABDC2= 10. Placing the reaction system at room temperature for reacting for 2 hours, then using PBS as a mobile phase, purifying the NOTA-modified nano antibody by using a pre-balanced PD-10 desalting column (GE Healthcare), and collecting NOTA-C2 or NOTA-ABDC2; then concentrated using an ultrafiltration tube (Merck Millipore) with a cut-off of 10kDa, and the NOTA-C2 or NOTA-ABDC2 concentration was measured using a NanoDrop and stored at-20 ℃ until use.

⁶⁸ Preparation of Ga-labeled NOTA-C2 and NOTA-ABDC2 ⁶⁸ Ga]Ga-NOTA-C2 and [ 2 ] ⁶⁸ Ga]Ga-NOTA-ABDC2. The method comprises the following specific steps: germanium gallium generator (Eckert) rinsed with 4mL of 0.05M hydrochloric acid solution (HCl)&Ziegler Radiopharma Inc), collecting 68Ga leachates of equivalent volume activity of about 370-555 MBq; taking 2mL of the middle section 68Ga leacheate with the highest activity, and adding 0.1mL of 1M sodium acetate solution (NaoAc) to adjust the pH value of the 68Ga leacheate to 4.0-4.5; taking 100-200 μ g of coupled NOTA-C2 and NOTA-ABDC2Adding 68Ga eluent and the volume of the reaction system<2.5mL; placing the reaction system in a constant temperature oscillator to react for 5-10 minutes at room temperature; after the labeling reaction, the free DNA was separated again by using a pre-equilibrated PD-10 desalting column using PBS as a mobile phase ⁶⁸ Ga. Purifying the final product; the unattenuated corrected Radiochemical yield (RCY) obtained according to the above procedure>50％。

[ ⁶⁸ Ga]Ga-NOTA-C2 and [ 2 ] ⁶⁸ Ga]And controlling the quality of Ga-NOTA-ABDC2. Suction of 10 [ mu ] L [ alpha ], [ solution ] ⁶⁸ Ga]Ga-NOTA-C2 or [ 2 ] ⁶⁸ Ga]Ga-NOTA-ABDC2 was spotted on a silica gel plate using 0.1M sodium citrate solution (pH = 5) as the mobile phase and radioactive thin layer chromatography (Radio-TLC, eckert)&Ziegler Radiopharma Inc) measures the Radiochemical purity (RCP) of the probes. <xnotran> 7 , [ </xnotran> ⁶⁸ Ga]Ga-NOTA-C2 and [68Ga ]]Ga-NOTA-ABDC 2RCP is more than 99%.

[ ⁶⁸ Ga]Ga-NOTA-C2 and ⁶⁸ Ga]the pharmacokinetic studies of Ga-NOTA-ABDC2 in tumor-free Balb/c mice are shown in FIG. 8. The method comprises the following steps: the PET/CT Imaging acquisitions of the small animals involved in this study were all done using an IRIS small animal PET/CT scanner (Inviscan Imaging Systems). Each non-tumor Balb/c mouse is injected with 3.7-7.4MBq [ 2 ] by tail vein ⁶⁸ Ga]Ga-NOTA-C2 and ⁶⁸ Ga]Ga-NOTA-ABDC2 (4 per group), anesthetizing the mice with isoflurane (concentration of 2%) mixed with oxygen at 0.5 hour, 2 hours and 4 hours after injection, respectively, placing the mice under deep anesthesia on a PET/CT scanning bed in a supine position, continuously acquiring PET and CT images, and completing image reconstruction with an IRIS system with software, as shown in FIG. 8, wherein the upper part of FIG. 8 ⁶⁸ Ga]The Ga-NOTA-C2 probe mainly accumulates in the kidney and the bladder at 0.5 hour, while the lower part of FIG. 8 [ partially ], [ 2 ] ⁶⁸ Ga]The Ga-NOTA-ABDC2 probe is mainly focused on the heart and the kidney is relatively ingested ⁶⁸ Ga]Ga-NOTA-C2 is obviously reduced. Delineating regions of interest (ROI) such as heart and major tissue organs (liver, lung, kidney, muscle) on the reconstructed PET image by using a OsiriX Lite image processing workstation (Pixmeo SARL), and calculating the percentage of the important tissue organs by taking the% ID/g (percentage of the excluded tissue per gram) as a unitThe radioactivity uptake value is taken, a curve of the uptake value of the main tissue and organs along with the time is drawn, the pharmacokinetic difference of the two probes is further compared, and the curve diagrams at the left side and the middle part of the graph of FIG. 9 respectively show ⁶⁸ Ga]Ga-NOTA-C2 and [ 2 ] ⁶⁸ Ga]The uptake of Ga-NOTA-ABDC2 in vivo in the major tissues and organs was shown as a trend with time, and the right graph in FIG. 9 shows the difference in the uptake of the two probes in the different tissues and organs at 1H, and it can be seen that ⁶⁸ Ga]In vivo circulation time of Ga-NOTA-ABDC2 probe is set to be longer ⁶⁸ Ga]Ga-NOTA-C2 is prolonged remarkably.

[ ⁶⁸ Ga]The imaging of Ga-NOTA-C2 immune PET is used for diagnosing colorectal cancer and gastric cancer, and the experimental result is shown in figure 10 and figure 11. The left side of the two panels shows PET/CT images, the middle shows ROI images, and the right side shows in vitro biodistribution data images. The two sets of figures can show the CD47 specific nano antibody probe ⁶⁸ Ga]Ga-NOTA-C2 has higher uptake in tumor tissues and higher non-specific uptake in main excretion (kidney) and metabolism (liver) tissues. By delineating the ROI analysis ⁶⁸ Ga]The distribution of Ga-NOTA-C2 in vivo, and in addition, the in vitro biodistribution experiment result further reveals the distribution of the probe in main tissues and organs in vivo. By analyzing the ROI data and biodistribution data developed by two tumor models, it can be shown that ⁶⁸ Ga]The diagnostic efficacy of Ga-NOTA-C2 was not significantly different for these two tumor models, and the results are shown in fig. 12 and 13. The above result indicates that ⁶⁸ Ga]The Ga-NOTA-C2 probe can non-invasively visualize CD47 expression.

[ ⁶⁸ Ga]In an optimized experiment for diagnosing ovarian cancer by Ga-NOTA-C2 immune PET imaging, sodium maleate (465 mg/kg) is injected in an experimental group within 5 minutes before the imaging agent is injected, a control group is not injected, and PET images are acquired after the imaging agent is injected for 0.5h and 2h respectively. The PET/CT results are shown in fig. 14, and the upper and lower parts respectively show the imaging results of the control group and the sodium maleate group, indicating that sodium maleate can significantly reduce the uptake of the monovalent nanobody probe in the kidney tissue. By statistical analysis of the ROI data, it was found that the renal uptake of the sodium maleate group was significantly lower than that of the control group, and the results are shown in fig. 15.

[ ⁶⁸ Ga]Ga-NOTA-ABDC2 immune PET imaging diagnosis stomachThe cancer, the test result is shown in FIG. 16 ⁶⁸ Ga]Ga-NOTA-ABDC2 multi-time-point PET/CT imaging in No.490 gastric cancer model. CD47 specific nano antibody fusion protein probe ⁶⁸ Ga]Ga-NOTA-ABDC2 still has higher uptake in tumor tissues. Pass [ 2 ] ⁶⁸ Ga]The imaging study of Ga-NOTA-ABDC2 in tumor-free Balb/c mice shows that the circulation time is remarkably prolonged, so that the Ga-NOTA-ABDC2 is injected ⁶⁸ Ga]After the Ga-NOTA-ABDC2 probe, the collection time is prolonged to 8h. As shown in the ROI data of fig. 17, the uptake at the tumor site gradually increased with time within 8h, while the uptake at the heart and the like gradually decreased. The in vitro distribution data shown in figure 18 further confirms the enrichment of the probe at the tumor site. By further comparison of [68Ga ]]Ga-NOTA-C2 and [68Ga]ROI and in-vitro biodistribution data of the Ga-NOTA-ABDC2 probes in the gastric cancer model PET/CT imaging show that the fusion protein probes do not influence the capability of non-invasive visualization of CD47 in tumors, and the experimental results are shown in FIG. 19 and FIG. 20.

Example 4 preparation of probe [ 2 ] ⁸⁹ Zr]Zr-DFO-ABDC2 and application thereof in immune PET imaging diagnosis of gastric cancer

The preparation of the intermediate DFO-ABDC2 by modifying ABDC2 with DFO comprises the following steps: 3mg of ABDC2 was dissolved in 1mL Phosphate Buffer Solution (PBS), 0.1mL 0.1M sodium carbonate (Na) ₂ CO ₃ PH = 11.4) buffer PH of the nanobody solution was adjusted to 8.9-9.1, and the volume of the reaction system was 1.1mL. DFO (CAS Number: 147597-66-8) freshly dissolved in dimethyl sulfoxide (DMSO) was added to the above nanobody solution at a molar ratio DFO/ABDC2= 5:1. Placing the reaction system at room temperature for reaction for 30 minutes, then using PBS as a mobile phase, purifying the nano antibody modified by DFO by using a pre-balanced PD-10 desalting column (GE Healthcare), and collecting DFO-ABDC2; then concentrated using an ultrafiltration tube (Merck Millipore) with a cut-off of 10kDa, and the concentration of DFO-ABDC2 was measured by NanoDrop, and the resulting solution was aliquoted at-20 ℃ for further use.

⁸⁹ Preparation of Zr-labeled DFO-ABDC2 ⁸⁹ Zr]Zr-DFO-ABDC2. The method comprises the following specific steps: mu.l of 100MBq 89Zr oxalic acid solution was prepared using 1M Na ₂ CO ₃ Buffer adjusted PH =7. Then 500. Mu.l of 0.5M HEPES solution was added stepwise (pH 7.1-7.3) and 200. Mu.l of DFO-ABDC2 (360. Mu.g) were added to the reaction solution. The reaction system was placed in a constant temperature oscillator and reacted at room temperature for 1 hour. After the labeling reaction, the free DNA was separated again by using a pre-equilibrated PD-10 desalting column using PBS as a mobile phase ⁸⁹ Zr, purifying the final product; the unattenuated corrected Radiochemical yield (RCY) obtained according to the above procedure>99% as shown in fig. 21.

[ ⁸⁹ Zr]And controlling the quality of the Zr-DFO-ABDC2. Suction of 10. Mu.l of ⁸⁹ Zr]Zr-DFO-ABDC2 was spotted on silica gel plates using 0.1M sodium citrate solution (pH = 5) as the mobile phase and using a radioactive thin layer chromatograph (Radio-TLC, eckert)&Ziegler Radiopharma Inc) measures the Radiochemical purity (RCP) of the probes. <xnotran> 22 , [ </xnotran> ⁸⁹ Zr]The RCP of Zr-DFO-ABDC2 is more than 99 percent.

[ ⁸⁹ Zr]The 2 [ deg. ] of Zr-DFO-ABDC2 immune PET imaging diagnosis gastric cancer ⁸⁹ Zr]The Zr-DFO-ABDC2 immune PET imaging further proves the enrichment capacity of the CD47 specific nano antibody fusion protein ABDC2 at the tumor site. PET/CT images are acquired respectively after 1,6,12,24, 48 and 72,96,120,144 hours of injection probes, the coronal PET/CT image in FIG. 23 shows the uptake of tumor parts of 6h, 72h and 144h, and the MIP image after PET/CT fusion in FIG. 24 shows the uptake of probes of tumors and major tissues and organs at all time points, which shows that the uptake of the probes at the tumor parts gradually increases with time, reaches a peak value in 72 hours and then gradually decreases. The time-dependent curve of the uptake values of the tumor and the major tissues and organs (heart, liver, lung, kidney, muscle, spleen and bone) was prepared by ROI analysis, which indicated that ⁸⁹ Zr]Good signal-to-noise ratio in vivo and good stability in vivo for Zr-DFO-ABDC2 as shown in figure 25. The in vitro biodistribution experiment further reveals the probe ⁸⁹ Zr]Distribution of Zr-DFO-ABDC2 in vivo tumor and major tissues and organs is shown in FIG. 26. Furthermore, immunohistochemical staining of the tumors was performed using the CD 47-specific antibody HPA044659, confirming the expression of CD47 inside the tumors, as shown in fig. 27.

Example 5 preparation of ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]Lu-DOTA-ABDC2 and its use in radioimmunoassayApplication of epidemic treatment

And (3) preparing intermediates DOTA-C2 and DOTA-ABDC2 by modifying C2 and ABDC2 with DOTA. The method comprises the following specific steps:

¹⁷⁷ preparation of Lu labeled DOTA-C2 and DOTA-ABDC2 ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]Lu-DOTA-ABDC2. The method comprises the following specific steps:

[ ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]And controlling the quality of Lu-DOTA-ABDC2. Respectively absorb 10 mul of the blood pressure ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]Lu-DOTA-ABDC2 was spotted on a silica gel plate using 0.1M sodium citrate solution (pH = 5) as the mobile phase and using a radioactive thin layer chromatograph (Radio-TLC, eckert)&Ziegler Radiopharma Inc) measures the Radiochemical purity (RCP) of the probes. <xnotran> 28 29 [ </xnotran> ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]Both Lu-DOTA-ABDC2 RCPs were greater than 99%, and both probes remained stable in PBS solution for 72 hours, i.e., RCPs remained greater than 90% at 72 hours.

[ ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]Lu-DOTA-ABDC2 radioimmunotherapy. To explore the efficacy of CD 47-specific nanobody fusion protein ABDC2 as a therapeutic vector, we used ¹⁷⁷ Lu developed therapeutic trials. Before the development of the treatment experiment, we injected the [ 2 ] separately into 3 mice as a model of gastric cancer PDX ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]Lu-DOTA-ABDC2, and mice were sacrificed 7 days after injection for ex vivo biodistribution test to clarify the distribution of the two probes in the major tissues and organs in vivo, as shown in FIG. 30, after 1 week of injection of imaging agent, [ 2 ] ¹⁷⁷ Lu]The uptake of Lu-DOTA-ABDC2 at the tumor part is obviously higher than that of [ 2 ] ¹⁷⁷ Lu]Lu-DOTA-C2, and the uptake in the kidney region is significantly lower than that of [ 2 ] ¹⁷⁷ Lu]Lu-DOTA-C2. Next, we divided the treatment experiment into 5 groups, which were control group, respectively ¹⁷⁷ Lu]Lu-DOTA-C2 group, low dose [177Lu]Lu-DOTA-ABDC2 group, high dose [ solution ] ¹⁷⁷ Lu]Lu-DOTA-ABDC2 group and ABDC2 group. The weight and tumor volume changes of five groups of mice were monitored every 2 days after the administration for 1 month, and a time-dependent change curve was plotted as shown in fig. 31. Wherein the injection is administered 1 week after the administration ¹⁷⁷ Lu]Lu-DOTA-C2 and [ 2 ] ¹⁷⁷ Lu]SPECT/CT imaging is carried out on three groups of Lu-DOTA-ABDC2, ROI of main tissues and organs are sketched, distribution conditions of probes in vivo are analyzed, the SPECT/CT image of the coronal position on the left side of the graph 32 shows probe uptake conditions of a tumor part and a kidney part, distribution differences of the three groups of probes in the tumor and the main organs in vivo are shown on the right side of the graph 32, and the result is consistent with biodistribution data of the graph 30. The above data indicate that ABDC2 is a targeted CD47 therapeutic vector with some potential.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concept. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims (10)

1. The CD47 specific molecular imaging probe is characterized by comprising a human CD47 specific monovalent nano antibody probe and a human CD47 specific nano antibody fusion protein probe; the human CD47 specific monovalent nanobody probe comprises a nanobody C2 and a labeled radionuclide; the human CD47 specific nano antibody fusion protein probe comprises a nano antibody fusion protein ABDC2 and the labeled radionuclide.

2. The molecular imaging probe of claim 1, wherein the amino acid sequence of the nanobody C2 is shown in SEQ ID No.1 of the sequence table, and the gene sequence of the nanobody C2 is shown in SEQ ID No.2 of the sequence table; the human CD47 specific monovalent nanometer antibody probe comprises specificity of human CD47 ⁶⁸ Ga-labeled monovalent nanobody probe ⁶⁸ Ga]Ga-NOTA-C2 and human CD47 specificity ¹⁷⁷ Lu labeled monovalent nanobody probe ¹⁷⁷ Lu]Lu-DOTA-C2; the human CD47 specific nano antibody fusion protein probe comprises human CD47 specificity ⁶⁸ Ga-labeled nanobody fusion protein probe ⁶⁸ Ga]Ga-NOTA-ABDC2, human CD47 specificity ⁸⁹ Zr marked nano antibody fusion protein probe ⁸⁹ Zr]Zr-DFO-ABDC2 and human CD47 specificity ¹⁷⁷ Lu labeled nano antibody fusion protein probe ¹⁷⁷ Lu]Lu-DOTA-ABDC2。

3. The molecular imaging probe of claim 1, wherein the amino acid sequence of the nano antibody fusion protein ABDC2 is shown in SEQ ID No.3 of the sequence table, and the gene sequence of the nano antibody fusion protein ABDC2 is shown in SEQ ID No.4 of the sequence table; the nano antibody fusion protein ABDC2 contains a linker, the amino acid sequence of the linker is shown as SEQ ID No.5 in a sequence table, and the gene sequence of the linker is shown as SEQ ID No.6 in the sequence table.

4. The molecular imaging probe of claim 1, wherein the radionuclide 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, 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, th-149, yb-227, yb-169, or Xe-177.

5. The method for preparing a CD 47-specific molecular imaging probe according to any one of claims 1 to 4, wherein the method comprises the steps of:

step 1, modifying a CD47 specific nano antibody C2 through a compound to synthesize a small molecule compound precursor, namely a radionuclide labeled precursor;

and 2, labeling the small molecular compound precursor obtained in the step 1 by using a radioactive nuclide, and preparing the CD47 specific molecular imaging probe.

6. The method of claim 5, wherein said compound of step 1 comprises the macrocyclic ligand p-SCN-Bn-NOTA, p-SCN-Bn-deferroxamine 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-triazacyclonane-1,4,7-triacetic acid), the p-SCN-Bn-Deferoxamine is Deferoxamine, i.e., 1- (4-isothiocyanatophenyl) -3- [ 4234 zxft 34-dihydroxy-7,10,18,21-tetraoxo-27- (N-acetoxyamino) -6,11,17,22-tetraazaheptobiose ] thiourea (1- (4-isothiocyanatophenyl) -3- [6,17-dihydroxyphenyl-7,10,18,21-tetroxy-27- (N-acetoxy) -3525-tetraazaoxine ] thionine); the p-SCN-Bn-DOTA is 1,4,7, 1-tetraazacyclododecane-1, 4,7, 1-tetraacetic acid (S-2- (4-Isothiocyanatobenzyl) -1,4,7,10-tetraazacyclodecane tetraacetic acid).

7. The use of the molecular imaging probe of any one of claims 1 to 4 in an immunoPET diagnostic reagent, wherein the diagnostic reagent is a noninvasive target-specific immunoPET diagnostic reagent for specific types of tumors based on the non-invasive visualization of CD47 differential expression in tumor tissues and normal tissues and organs.

8. The use of the reagent for immunoPET diagnosis according to claim 7, wherein the labeled radionuclide of the 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.

9. Use of the molecular imaging probe according to any one of claims 1 to 4 as a radioimmunotherapy agent, wherein the therapeutic agent is a radioimmunotherapy agent for CD 47-expressing positive tumors.

10. The use of the agent for radioimmunotherapy according to claim 9, wherein the labeled radionuclide of said 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, sm-106, na-24, sr-89, tb-149, th-227, xe-133, yb-169, or Yb-177.

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