Molecular probe of targeted tyrosine kinase receptor as well as preparation method and application of molecular probe

Abstract

The invention relates to the technical field of molecular probes, and particularly discloses a molecular probe of a targeted tyrosine kinase receptor as well as a preparation method and application of the molecular probe, the molecular probe comprises radioisotope, targeted polypeptide and a chelating group, the targeted polypeptide specifically targets the tyrosine kinase receptor, the chelating group is connected with the targeted polypeptide to form a targeted precursor, and the chelating group is connected with the targeted polypeptide to form the targeted precursor. The radioactive isotope labeled precursor forms a molecular probe, and the sequence of the targeted polypeptide is shown as any one of SEQ ID No.1-8. The targeting polypeptide has good targeting property on a tyrosine kinase receptor coded by an RET gene, can realize specific high uptake in a cell line of NEPC, has relatively good biological safety, can realize accurate imaging aiming at NEPC, and clearly and durably visualizes NEPC tumors in PET imaging; meanwhile, the molecular probe can effectively delay the growth capacity of NEPC, can provide a new treatment choice for NEPC patients, and has good clinical transformation potential.

Description

Technical Field

[0001] This application relates to the fields of molecular probes and biomedical technology, specifically to a molecular probe targeting a tyrosine kinase receptor, its preparation method, and its application. Background Technology

[0002] The RET proto-oncogene encodes the transmembrane glycoprotein Ret, a member of the receptor tyrosine kinase (RTK) family. Ret can activate various downstream signaling pathways, such as RAS, PI3K, and STAT, inducing cell proliferation. Under normal conditions, RET is involved in the maturation of the central and peripheral nervous systems, the development of the kidneys and urinary tract, and the self-renewal of spermatogonial stem cells. As a common signal transduction receptor for glial cell-derived neurotrophic factor (GDNF) family ligands (GFL), GF forms a complex with GDNF family receptor α (GFRα) protein and recruits receptor tyrosine kinase dimers, followed by phosphorylation and activation of pathways such as JAK-STAT, RAS-MAPK, and PI3K-AKT, which participate in cell migration, proliferation, and survival.

[0003] Prostate cancer (PC) is a health challenge faced by millions of men worldwide. In recent years, the treatment modality of androgen deprivation therapy (ADT) combined with analgesic signaling inhibitors (ARSI) has achieved remarkable success in PC treatment; however, a large proportion of patients still succumb to tumor recurrence and metastasis. ARSI has led to a higher incidence of neuroendocrine prostate cancer (t-NEPC), accounting for approximately 16-25% of castration-resistant prostate cancer (CRPC), compared to only 1-2% for de novo prostate cancer. As a highly aggressive subtype of prostate cancer, t-NEPC has a median overall survival of only about 2 years after diagnosis, with small cell carcinoma having an OS of less than 1 year.

[0004] The atypical clinical features and significant heterogeneity among patients severely hinder the management of NEPC. For example, there is a mismatch between PSA levels and tumor progression; the sensitivity and specificity of several serum neuroendocrine markers, such as synaptophysin (SYP), chromogranin A (CHGA), and neuron-specific enolase (NSE), vary considerably, making traditional imaging tests ineffective. The final diagnosis relies on tissue biopsy and immunohistochemistry (IHC) of neuroendocrine markers, but the ambiguous timing of biopsy, the positive rates of biopsy and staining, and the risks of invasive procedures remain challenging. Because prostate-specific membrane antigen (PSMA) is highly expressed on the surface of most PC cells, PSMA-targeted radionuclide molecular imaging is recommended for initial staging and metastasis detection in PC. 177Lu-PSMA-617 received FDA approval for the treatment of metastatic CRPC due to its significant improvement in radiographic progression-free survival and overall survival. However, PSMA expression exhibits significant inter- and intra-patient heterogeneity, particularly in NEPC where PSMA expression is absent. This leads to a lack of specific diagnostic methods and effective targeted therapy for NEPC. Another important reason is that both the lineage transition from luminal cancer cells to neuroendocrine cancer cells and the expansion of early neuroendocrine prostate cancer-like / castration-resistant prostate cancer-like cells (NEPC / CRPC-like cells) render the AR target ineffective. Response rates to chemotherapy combined with platinum-based regimens for small cell lung cancer (SCLC) are generally low, and adverse reactions are concerning. Furthermore, failures in clinical trials have undermined confidence in NEPC immunotherapy because NEPC cells lack immune infiltration. Therefore, there is an urgent need to find new targets for non-invasive diagnosis and targeted therapy of NEPC. Summary of the Invention

[0005] To address the aforementioned technical problems, this application proposes a molecular probe targeting tyrosine kinase receptors, its preparation method, and its application. The aim is to provide a molecular probe that can specifically target the tyrosine kinase receptor encoded by the RET gene, enabling in vivo imaging of the expression level of the tyrosine kinase receptor. This allows for real-time in vivo imaging of tumors (NEPC) associated with the expression level of RET membrane proteins, and can be used for the imaging diagnosis of neuroendocrine prostate cancer or the preparation of targeted radiopharmaceuticals.

[0006] To achieve the above objectives, this application first provides a molecular probe targeting a tyrosine kinase receptor, comprising a radioactive isotope, a targeting polypeptide, and a chelating group. The targeting polypeptide can specifically target the tyrosine kinase receptor, the chelating group connects the targeting polypeptide to form a targeting precursor, and the radioactive isotope labels the targeting precursor to form a molecular probe. The sequence of the targeting polypeptide is shown in any one of SEQ ID No. 1 to SEQ ID No. 8, and the tyrosine kinase receptor is a membrane protein encoded by the RET gene.

[0007] Preferably, the chelating group is one of DOTA and NOA.

[0008] Preferably, the radioactive isotope is 68 Ga、 177 Lu、 18 F, 161 Tb, 223 Ra、 89 Sr、 99m Tc, 64 Cu、 89 Zr、 225 Any of Ac.

[0009] Preferably, the structure of the targeting precursor is shown in Formula I:

[0010]

[0011] Preferably, the structure of the molecular probe is shown in Formula II:

[0012]

[0013]

[0014] Where X is the radioactive isotope.

[0015] Based on a general inventive concept, this application also provides a method for preparing a molecular probe targeting a tyrosine kinase receptor, comprising the following steps: combining a targeting polypeptide with a chelating group to form a precursor compound, then mixing the precursor compound with a radioactive isotope, reacting under acidic conditions, and purifying to obtain the molecular probe.

[0016] Based on a general inventive concept, this application also provides the application of a molecular probe targeting a tyrosine kinase receptor in the preparation of a PET imaging agent for neuroendocrine prostate cancer.

[0017] Based on a general inventive concept, this application also provides the application of a molecular probe targeting a tyrosine kinase receptor in the preparation of a radiopharmaceutical for neuroendocrine prostate cancer.

[0018] Compared with the prior art, this application has the following beneficial effects:

[0019] 1. This application discovered high expression of RET-encoded tyrosine kinase receptor in neuroendocrine prostate cancer (NEPC) tissues through transcriptome sequencing and multiple databases. RET expression was observed in cell lines and extensively validated by immunohistochemistry in patient-derived samples. The expression level of tyrosine kinase receptor was significantly increased in cell lines, animal models, and NEPC patient tissues with NEPC characteristics. This study found and confirmed that RET-encoded tyrosine kinase receptor is an important target for imaging and treatment of NEPC.

[0020] 2. This application utilizes phage display technology to screen for targeting peptides with high affinity for RET-encoded tyrosine kinase receptors. These small peptides exhibit good pharmacokinetics, low toxicity, and immunogenicity, and are easily chemically modified and radiolabeled. A novel therapeutic radionuclide probe targeting the tyrosine kinase receptor was successfully synthesized. This targeting peptide demonstrates good targeting specificity for the RET-encoded tyrosine kinase receptor, achieving high uptake in NEPC cell lines and exhibiting good biocompatibility. In summary, this novel therapeutic radionuclide probe possesses the ability to target tumors with high tyrosine kinase receptor expression, enabling precise imaging of neuroendocrine prostate cancer and clear, persistent visualization of NEPC tumors on PET imaging.

[0021] 3. The lack of expression or low expression of PSMA in NEPC leads to a lack of response to existing radionuclide therapy targeting PSMA. This application provides a new therapeutic target and a tumor-specific killing radionuclide probe that chelates a radioisotope of radiating β particles with a precursor, enabling it to specifically deliver therapeutic radiation to RET-positive tumors and effectively delay the growth of neuroendocrine prostate cancer. It can become a new treatment option for NEPC and PSMA-negative prostate cancer and has good potential for clinical translation.

[0022] 4. The molecular probe preparation method provided in this application is simple and stable, with mild conditions. The prepared molecular probe has a radiochemical purity of over 99% and good in vitro stability (greater than 95%), which is conducive to clinical application. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This study validates the high expression of RET in the neuroendocrine prostate gland as described in Example 1 of this application. Figure 1 ,in Figure 1 A is a graph showing the screening results of NEPC upregulated genes in the RNA-seq data (n=5) based on patient-derived samples and the datasets of SU2C-PCF Dream Team (ADPC, n=326; NEPC, n=41) and Beltran (ADPC, n=70; NEPC, n=44). Figure 1 B is a heatmap based on the Beltran dataset, showing the relationship between RET and neuroendocrine characteristic genes and AR characteristic genes. Figure 1Figure C shows the results of quantifying the mRNA levels of ADPC-related genes, NEPC-related genes, and RET expression using the SU2C-PCF Dream Team dataset; Figure 1 D represents the quantitative analysis of RET mRNA levels using the Prostate Cell Atlas dataset. Each point represents a single patient sample: NORMAL (prostate tissue; n = 173); PRIMARY (primary prostate cancer n = 708); ARPC (androgen receptor-positive castration-resistant prostate cancer; n = 428); DNPC (double-negative prostate cancer; n = 22); NEPC (n = 34). Figure 1 Figure E shows the results of qPCR analysis of high RET expression in the neuroendocrine cell line NCI-H660 (n=3 independent experiments); Figure 1 F is the result of Western blot showing that RET-encoded protein is specifically expressed in the NCI-H660 cell line (Non-PC, non-prostate cancer; CSPC, castration-sensitive prostate cancer); Figure 1 Figure G shows the RNA-seq data analysis results from genetically engineered mice, indicating high RET expression in DKO and TKO mice, (WT)PBCre4 wild-type; (SKO)PBCre4:Pten f / f :Rb1 f / + (DKO)PBCre4:Pten f / f :Rb1 f / f (TKO)PBCre4:Pten f / f :Rb1 f / f Trp53 f / f ; Figure 1 H represents typical H&E and RET IHC diagrams for different types of patients; Figure 1 I is a comparative statistical graph of the h-score of RET IHC in patients; Figure 1 J is a representative diagram of RET IHC in normal human tissue. Figure 1 K represents the expression of RET on the cell membranes of PC3 and NCI-H660 cells captured by confocal microscopy. *P<0.05,**P<0.01,***P<0.001,****p<0.0001. Data are presented as mean ± SD.

[0025] Figure 2 This study validates the high expression of RET in the neuroendocrine prostate gland as described in Example 1 of this application. Figure 2 ,in Figure 2 Figure A shows the t-SNE dimensionality reduction analysis results of RNA-seq data based on patient-derived samples; Figure 2 B is a differential gene volcano plot of NEPC versus adenocarcinoma tissue in RNA-seq data based on patient-derived samples; Figure 2 C is the differential gene volcano plot of NEPC compared with normal tissue; Figure 2 D is a diagram showing the screening structure of NEPC-upregulated genes in RNA-seq data from patient-derived samples; Figure 2 E is a heatmap based on the correlation analysis of the SU2C-PCF Dream Team dataset, showing the relationship between RET and neuroendocrine characteristic genes and AR characteristic genes; Figure 2 F is a graph showing the results of quantitative analysis of RET mRNA levels in cell lines based on the Cancer Cell Line Encyclopedia (CCLE) database; Figure 2 G is Bulk tissue gene expression for RET indevelopmental Genotype-Tissue Expression (dGTEx); Figure 2 H represents a representative H&E diagram of normal human tissue;

[0026] Figure 3 This is a graph showing the experimental results of the targeting performance of the probe molecule in Experiment Example 1 of this application. Figure 3 A represents the statistical comparison of the uptake of 8 candidate peptides in NCI-H660 cells and PC3 cells (10). 5 cells); Figure 3 B is 68 Figure 1 shows the results of Ga-DOTA-RET-L7 cell uptake and self-blocking experiments; Figure 3 C is 68 Figure 1; Results of in vitro cytotoxicity assay of Ga-DOTA-RET-L7; Figure 3 D is 68 Imaging results of Ga-DOTA-RET-L1-8 in mice with NCI-H660 cell xenograft tumors (60 min); Figure 3 E represents NCI-H660 tumor in 68 Data capture results of Ga-DOTA-RET-L1-8 dynamic imaging;

[0027] Figure 4 For example 2 of this application 68 Figure of mass spectrometry analysis results for Ga-DOTA-RET-L7;

[0028] Figure 5 For example 3 of this application 68 HPLC results of radiochemical purity of Ga-DOTA-RET-L7;

[0029] Figure 6 For example 4 of this application 68 Imaging and toxicity assessment of Ga-DOTA-RET-L7 in a mouse model, where Figure 6 A and Figure 6 B is 68 120-minute dynamic imaging of Ga-DOTA-RET-L7 in NCI-H660 and PC3 cell xenograft tumors; Figure 6 C represents premature use of excessive precursors that causes self-blocking. 68 120-minute dynamic imaging of Ga-DOTA-RET-L7 on NCI-H660 cell xenograft tumors; Figure 6 D represents representative H&E and RET IHC images of NCI-H660 cells and PC3 cells xenograft tumors; Figure 6 EG represents one of the two models 68 Ga-DOTA-RET-L7 uptake in tumors and major organs at different time points. Figure 6 H is a comparison of tumor uptake and tumor-to-muscle ratio at different time points; Figure 6 I am

[0030] Results of tumor-to-non-tumor ratio at different time points in the NCI-H660 cell xenograft tumor model; Figure 6 J is 68 Biodistribution of Ga-DOTA-RET-L7 in NCI-H660 cell xenograft tumors; Figure 6 K is 68 Pharmacokinetic diagram of Ga-DOTA-RET-L7 in a non-tumor mouse model;

[0031] Figure 7 For example 4 of this application 68 H&E staining results of major organs in mice with NCI-H660 and PC3 cell xenograft tumors after Ga-DOTA-RET-L7 injection;

[0032] Figure 8 For example 5 of this application 177 The therapeutic effects and low toxicity results of Lu-DOTA-RET-L7 in cell and mouse models are shown in the figure. Figure 8 A is 177 The chemical structural formula of Lu-DOTA-RET-L7; Figure 8B is 177 Uptake of Lu-DOTA-RET-L7 in NCI-H660 and PC3 cells showed cytotoxicity (10) in NCI-H660 cells. 5 Results image (cells); Figure 8 C and Figure 8 D was the injection of different dose groups into the NCI-H660 cell xenograft model. 177 Tumor volume and mouse body weight data after Lu-DOTA-RET-L7 and saline treatment; Figure 8 E was administered at different doses in the NCI-H660 cell xenograft model. 177 Survival data of mice after Lu-DOTA-RET-L7 and saline treatment; Figure 8 F is

[0033] 177 Biodistribution map of Lu-DOTA-RET-L7 in mice; Figure 8 G represents the H&E and γ-H2AX, Ki67 and RET IHC images of mouse tumors after treatment in each group;

[0034] Figure 9 Different doses were injected in Experimental Example 5 of this application. 177 Representative images of major organs in the NCI-H660 cell xenograft model were obtained after H&E staining with Lu-DOTA-RET-L7 and saline. Detailed Implementation

[0035] To make the technical problems, technical solutions and advantages of this application clearer, a detailed description will be provided below in conjunction with the accompanying drawings and specific embodiments.

[0036] The following embodiments are used to illustrate this application, but are not intended to limit the scope of this application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.

[0037] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0038] Example 1

[0039] Target screening and identification for the diagnosis and treatment of neuroendocrine prostate cancer

[0040] To identify potential targets for the diagnosis and treatment of neuroendocrine prostate cancer (NEPC), five patients pathologically diagnosed with NEPC (all patients gave informed consent) were selected. FFPE samples from these NEPC patients were subjected to bulk RNA sequencing (BRNA-Seq) along with paired adenocarcinoma and normal tissue samples. t-SNE dimensionality reduction analysis was performed based on the RNA-seq data from the samples. The results are as follows: Figure 2 As shown in A: Figure 2 A showed aggregation of normal tissue while NEPC tissue and paired adenocarcinoma tissue were mixed. Genes upregulated in NEPC were screened using Log2FC > 1 and P < 0.05 as criteria, and the results are as follows: Figure 2 B~ Figure 2 As shown in D, many differentially expressed genes, either upregulated or downregulated, were identified. These upregulated gene sets were intersected with the sets of genes highly expressed in NEPC from previous gene expression datasets SU2C-PCF Dream Team (AbidaW, Cyrta J, Heller G, et al. Genomic correlates of clinical outcome in advanced prostate cancer. Proc Natl Acad Sci US A. 2019; 116:11428–36.) and Beltran (AbidaW, Cyrta J, Heller G, et al. Genomic correlates of clinical outcome in advanced prostate cancer. Proc Natl Acad Sci US A. 2019; 116:11428–36.), yielding candidate genes RET (such as...). Figure 1 As shown in Figure A). In normal tissues, RET expression is elevated in the parathyroid glands, brain tissue, and adrenal glands (e.g., Figure 2 Correlation analysis showed that RET expression was positively correlated with the expression of neuroendocrine characteristic genes NEO2, ASCL1, FOXA2, CHGA, and SYP, and negatively correlated with AR-related genes and FOLH1, the gene encoding PSMA, suggesting the potential of RET for the diagnosis and treatment of PSMA-negative patients (e.g., Figure 1 B Figure 2 As shown in E). Quantitative analysis of these genes in the SU2C-PCF Dream Team dataset showed the same results (e.g., Figure 1 As shown in C). Prostate cell atlas data based on patients showed that RET expression was significantly elevated in NEPC patients (p < 0.0001) compared to patients without neuroendocrine features (e.g., ...). Figure 1(as shown in D).

[0041] Analysis of cell lines showed that RET mRNA levels were significantly elevated in the NCI-H660 cell line, representing neuroendocrine prostate cancer, compared to cell lines representing normal tissue, benign prostatic hyperplasia (BPH), and NEPC (p < 0.0001). Quantitative analysis of RET mRNA levels in the cell lines was performed using the Cancer Cell Line Encyclopedia (CCLE) database. Transcriptome data from the cell lines also showed similar results (e.g., ...). Figure 1 E, Figure 2 (As shown in F). Protein level detection showed that the RET-encoded protein was specifically expressed in NCI-H660, while PSMA protein expression was negative (e.g., Figure 1 (As shown in F). Transcriptome sequencing data of genetically engineered mice showed that RET expression was increased in DKO and TKO mice (GSE90891) compared to wild-type and SKO mice (p < 0.0001), while FOLH1 was highly expressed in SKO mice. SKO mice are similar to human ADPC, while DKO and TKO mice are considered to have NEPC characteristics (e.g., Figure 1 F, Figure 1 (As shown in G). Next, RET protein expression was further evaluated in patient-derived samples, and the IHC results for a total of 134 patient-derived FFPE samples are as follows. Figure 1 H, Figure 1 As shown in Figure I: the RET positivity rate was 45% (9 / 20) in normal prostate tissue, 50% (11 / 22) in BPH tissue, 64.2% (50 / 78) in adenocarcinoma tissue, and 85.7% (12 / 14) in NEPC tissue. Notably, the histochemical score (H-score) of RET in NEPC tissue was significantly higher than that in other tissue types (p < 0.0001). Figure 1 H, Figure 1 (As shown in Figure I). Furthermore, no significant RET staining was observed in normal human organs and tissues, suggesting that RET expression is specific in tumor tissues (e.g., ...). Figure 1 J、 Figure 2 (As shown in H). Finally, immunofluorescence staining in the PC3 and NCI-H660 cell lines showed that RET was expressed on the cell membrane (as shown in H). Figure 1Transcriptomic analysis based on patient FFPE samples revealed aberrant RET expression in NEPC. Extensive online data and IHC analysis of patient samples confirmed elevated RET expression in NEPC, but not in adenocarcinoma or normal tissues. RET expression remained strong even in PSMA-negative models. These results indicate increased RET expression in NEPC-characteristic cell lines, animal models, and NEPC patient tissues, validating that the RET-encoded tyrosine kinase receptor is an important imaging and therapeutic target.

[0042] Example 2

[0043] Phage peptide library screening for peptides targeting RET protein

[0044] Five rounds of biological screening were performed on recombinant human RET protein using two random phage display peptide library kits containing the linear peptide Ph.D.-C7C and the cyclic peptide Ph.D.-12. After identification by phage ELISA and DNA sequencing, eight candidate peptide sequences that could be developed as radiotracers were obtained, as shown in Table 2 below. The eight peptides were then successfully synthesized using the F-moc solid-phase synthesis method.

[0045] Table 2 Candidate peptides and their encoding genes

[0046]

[0047] Example 3

[0048] Preparation of molecular imaging probes

[0049] Preparation of candidate molecular imaging probes specifically targeting RET-encoded tyrosine kinase receptors 68 The specific process for Ga-DOTA-RET-L1---L8 is as follows:

[0050] (1) L1 to L8 peptides were synthesized using the F-moc solid-phase synthesis method, and precursors were prepared by chelating candidate peptides L1 to L8 with the bifunctional chelating agent DOTA.

[0051] (2) Take 10-30 nmol of the precursor and add it to 1.3 mL of HEPPS (solution, 500 mg / mL). After dissolving, transfer it to a 5 or 10 mL reaction flask.

[0052] (3) Take 4 mL of 0.6 M HCl for rinsing. 68 Ge / 68 In the Ga generator, discard the first and fourth mL portions, and add the second and third mL portions to the reaction flask. React at 90-100℃ for 10 min.

[0053] (4) Add 5 mL of deionized water to quench the reaction, use a 10 mL syringe to extract the reaction system and pass it through C18. After the reaction is complete, wash the C18 with 10 mL of water to further remove free 68Ga and inorganic salts.

[0054] (5) Elute the C18 column with 1 mL of ethanol, collect the eluent in a product bottle, and wash the C18 column with 5 mL of physiological saline. Filter the eluent through a sterile membrane into the product bottle to obtain the product solution, which contains the molecular imaging probes corresponding to the L1 to L8 peptides. 68 Ga-DOTA-RET-L1---L8.

[0055] Experimental Example 1

[0056] Investigating the targeting performance of the prepared molecular imaging probe

[0057] To verify the specificity of the molecular imaging probes prepared in Example 3 for RET-encoded tyrosine kinase receptors, the uptake of eight molecular imaging probes in human neuroendocrine prostate cancer cell line (NCI-H660) and human prostate cancer cell line (PC3) was investigated.

[0058] NCI-H660 or PC-3 cells were used at a concentration of 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of 10⁵ cells / well in 24-well plates and cultured at 37°C and 5% CO₂ for 24 hours. Eight different molecular imaging probes, 68Ga-DOTA-RET-L1-L8 (74 kBq per well), were then added, and the plates were cultured under the same conditions. At 30, 60, and 90 minutes, the culture medium was removed, the plates were washed with ice-cold PBS, and the cells were lysed with 1M NaOH. The lysates were collected and counted using a gamma counter. Simultaneously, three aliquots of standards were measured for radioactivity using a gamma counter as the injected dose (ID). Results are expressed as the percentage of injected dose taken up per 10⁵ cells (%ID). Experimental results are as follows: Figure 3 As shown in Figure A, after 90 minutes of co-incubation, the probe... 68 Ga-DOTA-RET-L7 showed the highest uptake of all probes in the NCI-H660 cell line (3.612±0.6425% ID), which was significantly higher than that in PC3 cells (0.5526±0.2358% ID; P=0.0015).

[0059] Further investigation 68 The uptake of Ga-DOTA-RET-L7 in NCI-H660 and PC3 cell lines was as follows: Figure 3 As shown in B, 68Ga-DOTA-RET-L7 uptake in the NCI-H660 cell line showed a significant time-dependent effect, reaching 4.163 ± 0.188855025% ID at 120 min. In contrast, uptake in the PC3 cell line was significantly lower than in NCI-H660 cells (0.8080 ± 0.6487; p < 0.0001) and did not show a time-dependent effect. Self-blocking evidence was demonstrated. 68 Ga-DOTA-RET-L7 exhibits good targeting of the RET-encoded tyrosine kinase receptor. In the blockade assay, cells were pre-incubated with 10 μg of unlabeled DOTA-RET-L7, followed by the same procedure as in the cell uptake assay. Results were expressed as per 1 × 10⁻⁶ cells. 5 The percentage of the injected dose in cells is expressed. Following injection of the unlabeled precursor peptide 1 hour prior to injection for inhibition, the NCI-H660 cell line showed efficacy at 120 min. 68 The uptake of Ga-DOTA-RET-L7 decreased significantly to 1.414 ± 0.07672% ID (p < 0.0001). 68 Ga-DOTA-RET-L7 did not exhibit significant cytotoxicity in PC3 and NCI-H660 cell lines (e.g., Figure 3 (as shown in C).

[0060] Meanwhile, to clarify the differences in cell line and in vivo uptake, separate methods were used. 68 Ga-DOTA-RET-L1~L8 were used to perform dynamic imaging on a mouse subcutaneous tumor model loaded with NCI-H660 cells. The results are as follows: Figure 3 D、 Figure 3 As shown in E, among all 8 molecular probes 68 The uptake of Ga-DOTA-RET-L7 increased significantly over time and had the highest uptake value among all probes.

[0061] The peptide-protein docking binding model showed that the binding site of DOTA-RET-L7 and the RET-encoded tyrosine kinase receptor protein exhibited suitable spatial complementarity; the binding energy between DOTA-RET-L7 and the RET-encoded tyrosine kinase receptor protein was the highest among all peptides, at -19.40 kcal / mol. Furthermore, hydrogen bond interactions were also formed between RET and the peptide.

[0062] Experiment Example 2

[0063] 68 Ga-DOTA-RET-L7 Structure and Stability Analysis

[0064] The product obtained in Example 3 68 Mass spectrometry analysis was performed on the Ga-DOTA-RET-L7, and the mass spectrum is shown below. Figure 4As shown, it has a radiochemical yield of 80.8 ± 4.9%.

[0065] Radio-HPLC purity analysis: Mobile phase A was acetonitrile containing 0.1% TFA, mobile phase B was distilled water containing 0.1% TFA, the chromatographic column was Symmetrix ODS-R (4.6*250mm, 5μm), and the elution mode was gradient elution. 68 The radiochemical purity HPLC results of Ga-DOTA-RET-L7 are as follows: Figure 5 As shown, 68 The peak elution time of Ga-DOTA-RET-L7 was 13.288 min, the radiochemical purity was greater than 99%, and the specific activity of radioactivity was 59.6 ± 6.1 GBq / μmol.

[0066] 68 The stability of Ga-DOTA-RET-L7 in physiological saline or serum was determined using radiochemical purity analysis methods, which were performed by Radio-HPLC. The prepared... 68 Ga-DOTA-RET-L7 was incubated with serum or physiological saline at 37°C. Samples were taken at 0.5, 1, 2, and 4 hours, and the radiochemical purity was determined using Radio-HPLC to evaluate the in vitro stability of the label in physiological saline / serum. Radio-HPLC analysis showed that within 4 hours... 68 Ga-DOTA-RET-L7 exhibits good stability (>95%) in physiological saline and serum.

[0067] Experimental Example 3

[0068] 68 Imaging and toxicity assessment of Ga-DOTA-RET-L7 in mouse models

[0069] All animal experiments in this study strictly followed the experimental protocol approved by the laboratory's animal ethics committee (Approval No.: 202409121832000585636). Six- to eight-week-old male BALB / c nude mice (Slikejingda, Changsha, China) were used in the experiments. These animals were housed in a specific pathogen-free (SPF) environment with a 12-hour light-dark cycle and provided with free access to water and food. After a one-week acclimatization period, the mice contained at least 5 × 10⁻⁶ mmol / L of X-rays. 6 100 μL of phosphate-buffered saline (PBS) suspension of NCI-H660 and PC3 cells was mixed with an equal volume of Matrigel basement membrane matrix (Corning, NY, USA) to prepare a 200 μL mixture. This mixture was subcutaneously injected into the right axilla or ventral side of mice to establish tumors. Tumors were allowed to grow for 3 to 6 weeks. When the tumor volume reached approximately 100 mm², the tumor was considered established.3 Treatment research began when the tumor reached approximately 1500 mm². 3 At that time, small animal PET / CT imaging or biodistribution studies were conducted. Assuming the tumor volume is spherical, it was measured using calipers and the result was calculated according to the formula V = [length × (width)]. 2 ] / 2 Calculate the tumor volume.

[0070] Further evaluation was conducted in NCI-H660 and PC3 xenograft mouse models. 68 The targeting specificity and imaging capabilities of Ga-DOTA-RET-L7. Mice were anesthetized with 1–2% isoflurane and then injected with approximately 7.4 MBq via the tail vein. 68 Ga-DOTA-RET-L7. Animals were placed in the gantry of a Siemens Inveon PET / CT scanner, prone on a 37°C heated pad, for 60 minutes of static scanning or 120 minutes of dynamic scanning. Image reconstruction was performed using the 3D Ordered Subset Expectation-Maximization (OSEM3D) algorithm implemented in Inveon Research Workplace 4.0 (IRW 4.0). Based on the registered CT images, regions of interest (ROIs) were manually delineated on the tumor and major organs, and the mean signal intensity within the ROIs was measured as a percentage of the injected dose per gram of tissue (%ID / g). Figure 6 A, Figure 6 As shown in F, 120 minutes of dynamic PET imaging revealed... 68 The uptake of Ga-DOTA-RET-L7 in NCI-H660 subcutaneous xenografts increased over time, with tumor uptake at 60, 90 and 120 minutes being 3.215±0.1131%ID / g, 3.933±0.2326%ID / g and 5.684±0.2334%ID / g, respectively.

[0071] In contrast to the NCI-H660 mouse model, 68 The uptake and metabolism of Ga-DOTA-RET-L7 in tumor tissues and non-tumor tissues and organs (such as heart, liver, kidney, muscle, and brain tissue) are as follows: Figure 6 As shown in E, 68 Ga-DOTA-RET-L7 is rapidly metabolized in lung tumor tissue, and its uptake gradually decreases over time; during 120 minutes of imaging, the ratio of uptake in tumor tissue to non-tumor tissue gradually increases over time (e.g., ...). Figure 6 (as shown in G); different time points 68 Tumor uptake and tumor-to-muscle ratio of Ga-DOTA-RET-L7 in NCI-H660, PC3, and blocked peptide model mice are as follows: Figure 6 As shown in E~6H, 68In the NCI-H660 mouse model, the tumor-to-muscle uptake ratio of Ga-DOTA-RET-L7 was highest at 120 minutes, at 10.98 ± 0.1671.

[0072] Meanwhile, another group of NCI-H660 xenograft mice were pretreated with an excessive amount of unlabeled peptide (Selpercatinib, a RET targeting inhibitor) to block the probe's specificity (Blocked group). The results were as follows: Figure 6 As shown in Figure C, no significant tumor uptake was observed during the 120-minute PET imaging period. Tumor uptake at 60, 90, and 120 minutes was 1.955±0.06845, 2.081±0.06343, and 2.424±0.0934%ID / g, respectively. The tumor uptake values ​​and tumor-to-muscle ratios are shown in Figure C. Figure 6 As shown in Figure I, compared to the unblocked group, both tumor uptake and tumor-to-muscle ratio were significantly reduced. The tumor-to-muscle ratio at 120 minutes was significantly lower than that in the unblocked group, decreasing to 3.719 ± 0.08326 (p < 0.0001), confirming the effectiveness of the molecular probe. 68 Ga-DOTA-RET-L7 was specifically taken up in NCI-H660 model mice and showed high uptake capacity.

[0073] As a comparison 68 No tumor imaging was observed during the 120-minute imaging period in a RET-negative PC3 xenograft mouse model using Ga-DOTA-RET-L7 (e.g., Figure 6 (As shown in B). Tumor uptake was weak and gradually decreased over time, reaching 0.8375 ± 0.03832% ID / g at 120 minutes, significantly lower than that of NCI-H660 xenografts (p < 0.0001). The tumor-to-muscle ratio did not change significantly over time, reaching 3.411 ± 0.03075 at 120 minutes, comparable to the ratio after NCI-H660 xenograft blockade (p = 0.4947), both significantly lower than that of unblocked NCI-H660 xenografts (p < 0.0001). Figure 6 E, Figure 6 Representative images of H&E and RET quantitative immunohistochemical (IHC) data from NCI-H660 cell and PC3 cell xenograft tumors are shown in Figure F. Figure 6 As shown in Figure D, immunohistochemical staining of NCI-H660 xenografts revealed strong RET expression on the cell membrane, while it was negative in PC xenografts.

[0074] Research 68 The biodistribution of Ga-DOTA-RET-L7 in NCI-H660 cell xenograft tumors is as follows: Figure 6 As shown in J, the results indicate that...68 Ga-DOTA-RET-L7 is widely distributed in vivo, rapidly accumulating in the blood and kidneys and being rapidly excreted through the urinary system. Tumor uptake of the radiotracer increased rapidly within 120 minutes, while uptake in non-tumor tissues was relatively low and gradually decreased over time. At 120 minutes, tumor uptake was 6.763 ± 0.234% ID / g. After blocking with an excessive amount of unlabeled peptide (Blocked group), tumor uptake significantly decreased to 0.446 ± 0.1337% ID / g (p < 0.0001). The biodistribution results validated the aforementioned observations in PET imaging. Furthermore, pharmacokinetic analyses, such as... Figure 6 As shown in I, 68 Ga-DOTA-RET-L7 is rapidly eliminated from the bloodstream, with a half-life of 14.22 minutes. 68 Hematological examinations of non-tumor mice injected with Ga-DOTA-RET-L7 and saline showed no significant differences in the levels of major blood cells (leukocytes, lymphocytes, monocytes, neutrophils, erythrocytes, hemoglobin, etc.), and were generally within the normal range. The above-mentioned injections... 68 After obtaining Ga-DOTA-RET-L7 and saline, H&E staining was performed on the major organs of the NCI-H660 cell xenograft model. The results are as follows: Figure 7 As shown, regardless 68 No significant toxicity was observed in Ga-DOTA-RET-L7 compared to physiological saline, further confirming the effectiveness of the molecular probe. 68 Ga-DOTA-RET-L7 has high biocompatibility.

[0075] Molecular probe 68 Ga-DOTA-RET-L7 exhibited significantly higher uptake in RET-positive cell models compared to RET-negative models. It provided clear and persistent visualization of NEPC tumors in small animal PET imaging. Tumor uptake and the tumor-to-non-tumor ratio were positively correlated with RET expression. (RET-positive tumor model)

[0076] NCI-H660 exhibits higher performance 68 Ga-DOTA-RET-L7 uptake, in contrast, the RET-negative model showed no uptake. This demonstrates 68 Ga-DOTA-RET-L7 exhibits good targeting of NEPC and can be effectively visualized, achieving the goal of diagnosing NEPC and possessing the potential for clinical translation.

[0077] Experiment Example 4

[0078] Molecular probe 177Therapeutic effects and biosafety of Lu-DOTA-RET-L7 in cell and mouse models

[0079] Following the preparation method of Example 3, the radioactive isotope was replaced with 177 Lu, prepared molecular probes 177 Lu-DOTA-RET-L7, chemical formula as follows Figure 8 As shown in Figure A.

[0080] Investigation 177 The uptake of Lu-DOTA-RET-L7 in NCI-H660 and PC3 cell lines was as follows: Figure 8 As shown in B, the cellular uptake experiment demonstrated... 177 Lu-DOTA-RET-L7 bound to the NCI-H660 cell line but not PC3 cells, and interfered with cell growth in a dose-dependent manner. Meanwhile, PC3 cells showed only a weak marginal effect, which preliminarily demonstrates... 177 Lu-DOTA-RET-L7 exhibits specific killing effect on RET-positive cells.

[0081] Mice carrying subcutaneous xenografts of NCI-H660 were randomly divided into four groups. The experimental group received a single dose of the drug. 177 Lu-DOTA-RET-L7 (3.7 MBq, 7.4 MBq, or 18.5 MBq). The control group received a single dose of normal saline. To balance... 177 The therapeutic efficacy and toxicity of Lu-DOTA-RET-L7 were assessed. The highest injection dose was set at 18.5 MBq, the intermediate dose at 7.4 MBq (approximately half that of the high-dose group), and the lowest dose at 3.7 MBq (a 50% reduction from 7.4 MBq). This was used to determine the minimum effective dose and to reduce off-target organ radiotoxicity. Tumor volume, body weight, and survival data were measured in four groups of mice over 60 consecutive days. The results are as follows: Figure 8 C~ Figure 8 As shown in E, compared to the saline group... 177 Lu-DOTA-RET-L7 effectively delayed tumor growth in a dose-dependent manner during the observation period. The median survival in the saline group was 37.5 days. Two mice (D98, D89) died for unknown reasons on days 15 and 24, respectively. The remaining mice died due to tumor volume (>1500 mm). 3Mice with unexplained weight loss (>20%) exceeding ethical standards were euthanized during the observation period. The median survival in the low-dose group (3.7 MBq) was 46.5 days, not significantly different from the saline group (p = 0.1121). Weight loss gradually decreased over time but did not exceed 20%. One mouse in the low-dose group died for unknown reasons on day 30, and three were euthanized during the observation period. The median survival in the medium-dose group (7.4 MBq) was 60 days, superior to the saline group (p = 0.0161). One mouse died for unknown reasons, two were euthanized during the observation period, and the remaining three did not meet ethical standards or died during the observation period. The high-dose group (18.5 MBq) inhibited tumor growth rate for approximately 35 days, but tumor growth eventually resumed. One mouse experienced rapid weight loss during treatment but recovered quickly. No mice met ethical standards or died during the observation period, significantly superior to the saline group (p = 0.0005).

[0082] Images of tumors in mice after treatment were obtained by quantitative immunohistochemistry (IHC) for H&E, γ-H2AX, K167, and RET, as shown below. Figure 8 As shown in G, IHC of subcutaneous tumors in mice after treatment showed a significant positivity for γ-H2A.X. 177 Compared to saline, Lu-DOTA-RET-L7 promoted damage to tumor cell nuclear DNA, and KI67 levels decreased with increasing injection dose. Furthermore, biodistribution studies showed that injection into nude mice with NCI-H660 xenograft tumors... 177 Tumor uptake after Lu-DOTA-RET-L7 treatment peaked at 5.019 ± 0.5876% ID / g at 6 hours, and then gradually decreased over time, reaching 0.471 ± 0.1419% ID / g at 48 hours (e.g., ...). Figure 8 (as shown in F), while non-tumor tissue is rapidly metabolized after a brief uptake within 1 hour.

[0083] Using different concentrations 177 Lu-DOTA-RET-L7 (3.7MBq, 7.4MBq, or 18.5MBq) and saline were injected separately into non-tumor mice. After reaching the treatment endpoint, blood samples were collected via retro-orbital hemorrhage for hematological examination. The results showed no significant differences in the levels of major blood cells (leukocytes, lymphocytes, monocytes, neutrophils, erythrocytes, hemoglobin, etc.), and were generally within the normal range. The animals were subsequently euthanized, and major organs were dissected for macroscopic and histopathological examination. H&E staining results are shown below. Figure 9 As shown, regardless of the dosage 177 No significant toxicity was observed in Lu-DOTA-RET-L7 compared to physiological saline.

[0084] In summary, the above experiments confirmed the effectiveness of molecular probes. 177 Lu-DOTA-RET-L7 effectively delayed the growth of NCI-H660 tumors without significant toxicity, demonstrating high biocompatibility.

[0085] The embodiments described above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the technical scope of this application, based on the technical solution and concept of this application, should be covered within the scope of protection of this application.

Claims

1. A molecular probe targeting a tyrosine kinase receptor, characterized in that, The device comprises a radioactive isotope, a targeting peptide, and a chelating group. The targeting peptide can specifically target a tyrosine kinase receptor. The chelating group connects the targeting peptide to form a targeting precursor. The radioactive isotope labels the targeting precursor to form a molecular probe. The sequence of the targeting peptide is shown in any one of SEQ ID No. 1 to SEQ ID No.

8. The tyrosine kinase receptor is a membrane protein encoded by the RET gene.

2. The molecular probe targeting tyrosine kinase receptor according to claim 1, characterized in that, The chelating group is one of DOTA or NOTA.

3. The molecular probe targeting tyrosine kinase receptor according to claim 1, characterized in that, The radioactive isotope is 68 Ga、 177 Lu、 18 F, 161 Tb, 223 Ra、 89 Sr、 99m Tc, 64 Cu、 89 Zr、 225 Any of Ac.

4. The molecular probe targeting tyrosine kinase receptor according to claim 1, characterized in that, The structure of the targeting precursor is shown in Formula I:

5. The molecular probe targeting tyrosine kinase receptor according to claim 1, characterized in that, The structure of the molecular probe is shown in Formula II below: Where X is the radioactive isotope.

6. A method for preparing a molecular probe targeting a tyrosine kinase receptor as described in any one of claims 1 to 5, characterized in that, The process includes the following steps: combining a targeting peptide with a chelating group to form a precursor compound, then mixing the precursor compound with a radioactive isotope, reacting under acidic conditions, and purifying to obtain the molecular probe.

7. The use of a molecular probe targeting a tyrosine kinase receptor as described in any one of claims 1 to 5, or a molecular probe targeting a tyrosine kinase receptor prepared by the method described in claim 6, in the preparation of a PET imaging agent for neuroendocrine prostate cancer.

8. The use of a molecular probe targeting a tyrosine kinase receptor as described in any one of claims 1 to 5, or a molecular probe targeting a tyrosine kinase receptor prepared by the method described in claim 6, in the preparation of a neuroendocrine radiopharmaceutical for prostate cancer.

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