A polypeptide targeting LGR5, a PET molecular probe and a preparation method and application thereof

Abstract

This application relates to a peptide targeting LGR5, a PET molecular probe, its preparation method, and its application. The peptide is any one of the following: (1) its amino acid sequence is YLASRVH; (2) a derived peptide formed by substituting one or more amino acids into the amino acid sequence shown in (1), wherein the derived peptide has the same or substantially the same function as the peptide with the sequence shown in (1); (3) a derived peptide after chemical or genetic modification of the peptide in (1) or (2). The PET molecular probe targeting LGR5 includes a peptide targeting LGR5, a chelating agent, and a radionuclide. The probe of the present invention has good stability, high affinity and specificity for LGR5, and can sensitively monitor the LGR5 expression level at the tumor site; it is also rapidly metabolized in vivo and provides fast imaging. In LGR5-positive tumor models, it can reach the tumor site in a short time and obtain high-contrast PET imaging effects.

Description

Technical Field

[0001] This application belongs to the field of medical technology, and in particular relates to a peptide, PET molecular probe targeting LGR5, its preparation method and application. Background Technology

[0002] Leucine-rich G protein-coupled receptor 5 (LGR5), also known as G protein-coupled receptor 49 or 67 (GPR49 or GPR67), has been identified as a biomarker for intestinal stem cells. LGR5 is significantly overexpressed in various human malignancies, including colorectal cancer, lung cancer, gastric cancer, and breast cancer. It drives tumor progression by activating the Wnt / β-catenin signaling pathway, promoting key oncogenic processes such as cell proliferation, migration, invasion, metastasis, and angiogenesis. Given its high specificity and key role in tumor progression, LGR5 has become a promising biomarker for targeted tumor diagnosis and therapy. Currently, an increasing number of LGR5-targeted therapies are under development, including antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs), CAR-T cell therapy, and bispecific antibodies. Among these, CAR-T cell therapy has shown significant anti-tumor activity in the treatment of colorectal cancer and is currently undergoing clinical trials for colorectal cancer patients. In addition, petosemtamab, a bispecific antibody targeting LGR5 and epidermal growth factor receptor (EGFR), is currently in Phase I / II clinical trials to evaluate its efficacy in treating recurrent or metastatic head and neck squamous cell carcinoma. As LGR5-targeted therapies advance in clinical practice, developing a non-invasive, real-time, quantitative tool for detecting LGR5 expression levels is crucial.

[0003] Positron emission tomography (PET) offers advantages such as high sensitivity, deep tissue penetration, and non-invasiveness, making it a promising strategy for improving the clinical diagnosis and treatment of various cancers. Currently, only two monoclonal antibody-based LGR5-targeting PET radiotracers have been reported. 89 Zr-DFO-8F2 and 89Zr-DFO-9G5 (see the literature "Evaluation of Anti-LGR5 Antibodies by ImmunoPET for Imaging Colorectal Tumors and Development of Antibody-Drug Conjugates. Mol. Pharm. 2018, 15 (6), 2448-2454") enables sensitive, real-time, and accurate in vivo monitoring of LGR5 expression in a colorectal cancer (DLD-1) xenograft mouse model. However, due to the large molecular weight and poor tissue penetration of monoclonal antibodies, antibody-based radiotracers have poor pharmacokinetic properties and require several days to be cleared from the blood and achieve sufficient imaging contrast; this not only delays the imaging process but also hinders the timely assessment of tumor LGR5 expression. In contrast, peptide-based radiotracers exhibit unique advantages in molecular imaging, including small molecular weight, strong tissue penetration, rapid blood clearance, low immunogenicity, and ease of chemical synthesis and modification. Therefore, there is a need for a peptide-based radiotracer with high affinity and specificity for non-invasive detection of LGR5-positive tumors, thereby improving the precision and effectiveness of treatment. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, this paper provides a peptide and PET molecular probe targeting LGR5, along with their preparation method and applications.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] A polypeptide targeting LGR5, said polypeptide being any of the following:

[0007] (1) Its amino acid sequence is YLASRVH;

[0008] (2) A derivative polypeptide formed by substituting one or more amino acids into the amino acid sequence shown in (1), wherein the derivative polypeptide has the same or substantially the same function as the polypeptide of the sequence shown in (1).

[0009] (3) A derivative polypeptide derived from the polypeptide described in (1) or (2) by chemical or genetic modification.

[0010] The substitution is the replacement of one or more of tyrosine, leucine, serine, arginine, valine, and histidine with alanine.

[0011] Preferably, the substitution is that one or more of tyrosine, leucine, serine, and valine are replaced by alanine; further, the peptide targeting LGR5 is selected from any of the following amino acid sequences: YLASRVH, ALASRVH, YAASRVH, YLAARVH, YLASRAH.

[0012] The present invention also provides a PET molecular probe based on the above-mentioned polypeptide that specifically targets LGR5, comprising the above-mentioned polypeptide targeting LGR5, a chelating agent, and a radionuclide, wherein the polypeptide targeting LGR5 is covalently coupled to the chelating agent, and the radionuclide is chelated to the chelating agent.

[0013] Preferably, the radioactive nuclide is 68 Ga、 18 F, 64 Cu or 89 Zr, wherein the chelating agent is preferably DOTA, HYNIC, DTPA, NOA, or TETA.

[0014] Furthermore, R is preferably leucine or alanine, and the chelating agent is preferably NOA.

[0015] Furthermore, the PET molecular probe is preferably... or .

[0016] This invention also provides a method for preparing the above-mentioned PET molecular probe targeting LGR5, comprising: coupling and deprotecting a polypeptide compound with a chelating agent -NHS to obtain a precursor compound; radiolabeling the precursor compound with a radionuclide to obtain the PET molecular probe; wherein the polypeptide compound has the following structure:

[0017] ,

[0018] In the polypeptide compound, Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, and Trt is triphenylmethyl.

[0019] The present invention also provides a precursor compound for the above-mentioned LGR5-targeting PET molecular probe, which is composed of an LGR5-targeting peptide and a chelating agent. The LGR5-targeting peptide and the chelating agent are covalently coupled, and the chelating agent is preferably DOTA, HYNIC, DTPA, NOA, or TETA.

[0020] Preferably, the precursor compound of the PET molecular probe targeting LGR5 has the following structure:

[0021] or

[0022] .

[0023] The present invention also provides the application of the above-mentioned LGR5-targeting PET molecular probe in the preparation of LGR5-targeting imaging agents, wherein the LGR5-targeting imaging agent is preferably an LGR5-targeting imaging agent or an imaging drug targeting LGR5-positive tumors for non-disease diagnosis or treatment.

[0024] The present invention also provides an imaging agent targeting LGR5, the imaging agent containing the above-mentioned PET molecular probe targeting LGR5, wherein the imaging agent targeting LGR5 is preferably an imaging agent targeting LGR5 for non-disease diagnosis or treatment or an imaging drug targeting LGR5-positive tumors.

[0025] Preferably, the LGR5-positive tumor includes at least one of colorectal cancer, non-small cell lung cancer, gastric cancer, liver cancer, and breast cancer.

[0026] The beneficial effects of this invention are:

[0027] This invention provides a peptide-based PET molecular probe targeting LGR5. This peptide-based molecular probe is based on a specific peptide targeting LGR5, obtained by structural modification through the introduction of a chelating agent and subsequent radionuclide labeling. This peptide-based molecular probe has the following advantages:

[0028] First, it has good stability and good hydrophilicity, and has a high affinity and specificity for LGR5. It can sensitively monitor the expression level of LGR5 in tumor sites, and its uptake in LGR5-positive tumors is significantly higher than that in LGR5-negative tumors.

[0029] Second, it is rapidly metabolized in vivo and has fast imaging capabilities. In LGR5 positive tumor models, it can reach the tumor site in a short time and obtain high-contrast PET imaging results.

[0030] Third, the precursor synthesis steps are simple, the production cost is low, and the labeling method is simple and fast, requiring only 20 minutes to complete;

[0031] Fourth, radiolabeled nuclides can be used. 68 Ga is a short-lived radioactive nuclide that emits less radiation per unit time, further enhancing its safety.

[0032] Therefore, this polypeptide molecular probe can monitor the LGR5 expression level in tumors in real time and dynamically at the molecular level through PET imaging, and has great application prospects in the preparation of tumor imaging drugs. Attached Figure Description

[0033] The technical solution of this application will be further described below with reference to the accompanying drawings and embodiments.

[0034] Figure 1 Binding mode and affinity prediction of peptide P01 to LGR5.

[0035] Figure 2 Binding mode and affinity prediction of peptide PO2 and LGR5.

[0036] Figure 3 Binding modes and affinity prediction of LGR5 for peptides YLA, P01, and their alanine scanning analogs.

[0037] Figure 4 HPLC analysis chromatogram of precursor compound LTP-01.

[0038] Figure 5 ESI-MS analysis chromatogram of precursor compound LTP-01.

[0039] Figure 6 Precursor compound LTP-01 and probe [ 68 HPLC chromatogram of Ga]Ga-LTP-01.

[0040] Figure 7 HPLC analysis chromatogram of precursor compound LTP-02.

[0041] Figure 8 ESI-MS analysis chromatogram of precursor compound LTP-02.

[0042] Figure 9 Precursor compound LTP-02 and probe [ 68 HPLC chromatogram of Ga]Ga-LTP-02.

[0043] Figure 10 : probe[ 68 HPLC analysis of the stability of Ga]Ga-LTP-01 after incubation in PBS for 0.5, 1, and 2 hours.

[0044] Figure 11 : probe[ 68 HPLC analysis of the stability of Ga]Ga-LTP-01 after incubation in Mouse serum for 0.5, 1, and 2 hours.

[0045] Figure 12 : probe[ 68 HPLC analysis of the stability of Ga]Ga-LTP-02 after incubation in PBS for 0.5, 1, and 2 hours.

[0046] Figure 13 : probe[ 68HPLC analysis of the stability of Ga]Ga-LTP-02 after incubation in Mouse serum for 0.5, 1, and 2 hours.

[0047] Figure 14 : probe[ 68 Figure 1 shows the results of the affinity test between Ga]Ga-LTP-01 and LGR5.

[0048] Figure 15 : probe[ 68 Figure 1 shows the results of the affinity test between Ga]Ga-LTP-02 and LGR5.

[0049] Figure 16 : probe[ 68 Uptake of Ga]Ga-LTP-01 in A549, HCT116 and NCI-H1299 cells.

[0050] Figure 17 : probe[ 68 Uptake of Ga]Ga-LTP-02 in A549, HCT116 and NCI-H1299 cells.

[0051] Figure 18 : probe[ 68 Pharmacokinetic study of Ga]Ga-LTP-01 in normal ICR mice.

[0052] Figure 19 : probe[ 68 Pharmacokinetic study of Ga]Ga-LTP-02 in normal ICR mice.

[0053] Figure 20 : probe[ 68 1-hour dynamic PET imaging of Ga]Ga-LTP-01 in A549, HCT116 and NCI-H1299 tumor-bearing mice.

[0054] Figure 21 : probe[ 68 Changes in tumor and muscle uptake of Ga]Ga-LTP-01 over time in A549 tumor-bearing mice.

[0055] Figure 22 : probe[ 68 Changes in tumor and muscle uptake of Ga]Ga-LTP-01 over time in HCT116 tumor-bearing mice.

[0056] Figure 23 : probe[ 68 Changes in tumor and muscle uptake of Ga]Ga-LTP-01 over time in NCI-H1299 tumor-bearing mice.

[0057] Figure 24 : probe[ 68 Tumor-muscle uptake ratio of Ga]Ga-LTP-01 in A549, HCT116 and NCI-H1299 tumor-bearing mice.

[0058] Figure 25 : probe[ 68 1-hour dynamic PET imaging of Ga]Ga-LTP-02 in A549, HCT116 and NCI-H1299 tumor-bearing mice.

[0059] Figure 26 : probe[ 68 Changes in tumor and muscle uptake of Ga]Ga-LTP-02 over time in A549 tumor-bearing mice.

[0060] Figure 27 : probe[ 68 Changes in tumor and muscle uptake of Ga-LTP-02 over time in HCT116 tumor-bearing mice.

[0061] Figure 28 : probe[ 68 Changes in tumor and muscle uptake of Ga]Ga-LTP-02 over time in NCI-H1299 tumor-bearing mice.

[0062] Figure 29 : probe[ 68 Tumor-muscle uptake ratio of Ga]Ga-LTP-02 in A549, HCT116 and NCI-H1299 tumor-bearing mice.

[0063] Figure 30 : probe[ 68 In vivo biodistribution of Ga]Ga-LTP-01 in A549, HCT116 and NCI-H1299 tumor-bearing mice.

[0064] Figure 31 : probe[ 68 In vivo biodistribution of Ga]Ga-LTP-02 in A549, HCT116 and NCI-H1299 tumor-bearing mice.

[0065] Figure 32 : NOTA-YLA AND [ 68 Structure of Ga]Ga-NOTA-YLA.

[0066] Figure 33 : probe[ 68 Cellular uptake of Ga]Ga-NOTA-YLA.

[0067] Figure 34 : probe[ 681-hour dynamic PET imaging of Ga]Ga-NOTA-YLA in A549 and HCT116 tumor-bearing mice.

[0068] Figure 35 : probe[ 68 Changes in tumor and muscle uptake over time in A549 tumor-bearing mice using Ga-NOTA-YLA.

[0069] Figure 36 : probe[ 68 Changes in tumor and muscle uptake over time in HCT116 tumor-bearing mice using Ga-NOTA-YLA.

[0070] Figure 37 : probe[ 68 Tumor-muscle uptake ratio of Ga-NOTA-YLA in A549 and HCT116 tumor-bearing mice. Detailed Implementation

[0071] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0072] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0073] Example 1

[0074] This embodiment provides an LGR5-targeted molecular probe. 68 Preparation method of Ga]Ga-LTP-01

[0075] The molecular probe [ 68 Ga]Ga-LTP-01 has the following structure:

[0076] ;

[0077] The molecular probe [ 68 The preparation method of Ga]Ga-LTP-01 is as follows:

[0078] Step 1: Synthesize polypeptide compound 1 using the Fmoc solid-phase polypeptide synthesis method.

[0079] (1) After adding 2-chlorotriphenylmethyl chloride resin (262 mg, 0.3 mmol) and dichloromethane (DCM) (ultra-dry type, 10 mL) to the polypeptide synthesis tube, place it on a shaker and shake for 10 min. After the resin is fully swollen, filter it with a vacuum pump to remove DCM.

[0080] (2) Add N-Fmoc-N'-triphenylmethyl-L-histidine (232 mg, 0.375 mmol) and N,N-dimethylformamide (DMF) (ultra-dry type, 10 mL) to the polypeptide synthesis tube, and add N,N-diisopropylethylamine (DIPEA) to adjust the pH of the reaction system to 8. Then, place the tube on a shaker and shake for 3 h. After the reaction is completed, filter the solution using a vacuum pump to remove the reaction solution.

[0081] (3) After adding DMF mixture (DMF / MeOH / DIPEA=17:2:1, v / v / v, 10 mL) to the polypeptide synthesis tube, first place it on a shaker and shake for 10 min, then filter it. Repeat this operation 3 times. After adding N,N-dimethylformamide (DMF) (HPLC type, 10 mL) to the polypeptide synthesis tube, first shake and wash manually, then filter it with a vacuum pump to remove the reaction solution. Repeat this operation 3 times.

[0082] (4) Take a sample (0.5 mg) from the polypeptide synthesis tube, add Kaiser reagent (100 μL), and incubate at 110℃ for 2 min. The solution turns pale yellow, indicating that the condensation reaction is complete. Add DMF (10 mL) containing 20% ​​(v / v) piperidine to the polypeptide synthesis tube, shake on a shaker for 10 min, and then filter. Repeat this operation 3 times. Add DMF (HPLC type, 10 mL) to the polypeptide synthesis tube, wash manually, and then filter with a vacuum pump to remove the reaction solution. Repeat this operation 3 times.

[0083] (5) Add Fmoc-L-valine (127 mg, 0.375 mmol), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) (164 mg, 0.345 mmol) and DMF (ultra-dry type, 10 mL) to the polypeptide synthesis tube, and add DIPEA to adjust the pH of the reaction system to 8. Then, place the tube on a shaker and shake for 3 h. After the reaction is completed, filter the solution using a vacuum pump to remove the reaction solution.

[0084] (6) Repeat steps 3 to 5, and connect the following amino acids in sequence: Fmoc-Pbf-L-arginine (243 mg, 0.375 mmol), FMOC-O-tert-butyl-L-serine (144 mg, 0.375 mmol), Fmoc-L-alanine (117 mg, 0.375 mmol), Fmoc-L-leucine (133 mg, 0.375 mmol), and Fmoc-O-tert-butyl-L-tyrosine (173 mg, 0.375 mmol).

[0085] (7) After adding 10 mL of DCM containing 1% (v / v) trifluoroacetic acid (TFA) to the polypeptide synthesis tube, the tube was shaken on a shaker for 10 min, then filtered. This operation was repeated 3 times. The filtrate was evaporated to dryness, and 5 mL of cold diethyl ether at 4°C was added to precipitate the filtrate. The precipitate was collected and dried in a vacuum drying oven to obtain a yellow solid polypeptide compound 1 (100 mg, yield 22.97%). The polypeptide compound 1 has the following structure:

[0086] .

[0087] Step 2: Synthesis of precursor compound LTP-01

[0088] In a reaction flask, polypeptide compound 1 (10 mg, 0.0069 mmol), 2,2'-(7-(2-((2,5-dioxopyrrolidone-1-yl)oxy)-2-oxoethyl)-1,4,7-triazane-1,4-diyl)diacetic acid (NOTA-NHS ester) (4.13 mg, 0.010 mmol), and DMF (ultra-dry type, 500 μL) were added. After adjusting the pH of the reaction system to 8 with DIPEA, the mixture was stirred at 150 rpm for 3 h under nitrogen protection at room temperature (25 °C). After the reaction was complete, the reaction solution was evaporated to dryness. Acetonitrile (ACN) (0.2 mL), trifluoroacetic acid (TFA) (1.8 mL), and triisopropylsilane (TIPS) (40 μL) were added to the reaction flask, and the mixture was stirred at 150 rpm for 1 h at room temperature (25 °C). After the reaction was complete, the reaction solution was evaporated to dryness, the precipitate was collected, and the product was separated and purified by semi-preparative HPLC to obtain the purified product. The purified product was then evaporated to dryness and freeze-dried to obtain a white solid precursor compound LTP-01 (1.68 mg, yield 21.53%). The precursor compound LTP-01 has the following structure:

[0089] .

[0090] The HPLC results of the precursor compound LTP-01 are as follows: Figure 4ESI-MS results are as follows Figure 5 .

[0091] Step 3: Probe [ 68 Preparation of Ga]Ga-LTP-01

[0092] Rinse with hydrochloric acid (HCl) solution (4 mL, 0.05 M). 68 Ge / 68 The Ga generator, using the section with the highest radioactive dose in the middle. 68 GaCl3 solution (1.0 mL) was used for subsequent radiolabeling. Add [the following to the reaction tube]... 68 GaCl3 solution (500 μL) and 10 μg of the probe precursor compound LTP-01 were added, and the pH of the reaction system was adjusted to 4 by adding sodium acetate solution (0.25 M). The mixture was then incubated at 37°C for 15 min to obtain the probe. 68 Ga]Ga-LTP-01. A sample was taken, and the radiochemical purity of the probe was analyzed using Radio-HPLC. The results are as follows: Figure 6 As shown, its radiochemical purity is >98%, and its retention time is 15.8 min, which is basically consistent with the retention time of the precursor compound, indicating that its structure is correct. After attenuation correction, the molar activity of the probe is 19.96 ± 0.89 GBq / μmol.

[0093] Example 2

[0094] This embodiment provides an LGR5-targeted molecular probe. 68 The preparation method of Ga]Ga-LTP-02, wherein the molecular probe [ 68 Ga]Ga-LTP-02 has the following structure:

[0095] ;

[0096] The molecular probe [ 68 The preparation method of Ga]Ga-LTP-02 is as follows: replace Fmoc-L-leucine in step one of Example 1 with Fmoc-L-alanine, and the remaining steps are the same as in Example 1. The precursor compound LTP-02 has the following structure: ;

[0097] HPLC analysis of the precursor compound LTP-02 as follows Figure 7 As shown, the ESI-MS results are as follows: Figure 8 As shown. Probe [ 68 The HPLC results of Ga]Ga-LTP-02 are as follows Figure 9As shown, the radiochemical purity is >98%, and the retention time is 12.35 min, which is basically consistent with the retention time of the precursor compound LTP-02 in HPLC, indicating that the labeled product structure is correct. After attenuation correction, the calculated [ 68 The molar activity of Ga]Ga-LTP-02 is 20.64 ± 1.29 GBq / μmol.

[0098] Example 3: Design of LGR5-targeting peptides

[0099] This experimental example demonstrates the molecular docking of a peptide with the LGR5 protein. The specific procedure is as follows:

[0100] The crystal structure of the LGR5 extracellular domain complex with R-spondin-2 (PDB ID: 4UFR) was used for computer simulation studies. Molecular docking was performed using Autodock Vina to predict the binding mode and interaction between the peptide and LGR5, and alanine scans were used to assess the contribution of each amino acid residue to the binding affinity. Subsequently, molecular dynamics (MD) simulations were performed using GROMACS software to investigate the interaction between the peptide and LGR5. The results are as follows: Figure 1 , Figure 2 and Figure 3 As shown, the docking fraction of control peptide YLA (the polypeptide containing the amino acid sequence of SEQ ID NO:1 in patent WO2024210395A1) with LGR5 is -7.23 kcal / mol, and the docking fraction of peptide P01 (tyrosine-leucine-alanine-serine-arginine-valine-histidine) with LGR5 is -7.55 kcal / mol. Among all alanine analogues of peptide P01, peptide P02 (where leucine in P01 is mutated to alanine) has the best affinity for LGR5 (-10.14 kcal / mol).

[0101] Example 4: In vitro stability experiment of LGR5-targeted molecular probe

[0102] This embodiment provides an in vitro stability experiment of the LGR5-targeted molecular probe, the specific process of which is as follows:

[0103] The probes prepared in Examples 1 and 2 [ 68 Ga]Ga-LTP-01 and [ 68Ga]Ga-LTP-02 (148 MBq) was mixed with PBS buffer (200 μL, pH = 7.4) or mouse serum (purchased from Nanjing Senbeiga Biotechnology Co., Ltd.) (200 μL) and incubated at room temperature (25°C). Samples were taken after incubation for 30, 60, and 120 min for HPLC analysis. The results are as follows: Figure 10 , Figure 11 , Figure 12 and Figure 13 As shown, after incubation for 120 min, the radiochemical purity of the probes was >90%, indicating that the probes have good in vitro stability.

[0104] Example 5: Determination of the lipid-water partition coefficient of an LGR5-targeted molecular probe

[0105] This experiment provides a method for determining the lipid-water partition coefficient (log P) of an LGR5-targeted molecular probe. The specific procedure is as follows:

[0106] The probes prepared in Examples 1 and 2 [ 68 Ga]Ga-LTP-01 and [ 68 Ga]Ga-LTP-02 (~1.85 MBq) was placed in separate centrifuge tubes, and 1 mL of n-octanol and 1 mL of ultrapure water were added. The mixture was shaken to ensure thorough dispersion of the probe in both phases. The tubes were centrifuged at 5000 rpm for 5 min to separate the phases. 500 μL of each phase was taken and placed in separate radioimmunoassay tubes. The radioactivity of the n-octanol phase and the aqueous phase was measured using a gamma counter. This was repeated three times. According to the formula log P = log(C o / C w Calculate the log P of the probe, where C o Indicates the radioactivity of the probe in the n-octanol phase, C w The results represent the radioactivity of the probe in the aqueous phase, expressed as mean ± standard deviation.

[0107] probe [ 68 Ga]Ga-LTP-01 and [ 68 The log P values ​​of Ga]Ga-LTP-02 were -2.92 ± 0.08 and -3.33 ± 0.28, respectively, indicating that the probe has good hydrophilicity.

[0108] Example 6: Affinity determination of LGR5-targeted molecular probes

[0109] This experiment provides an affinity determination procedure for LGR5-targeted molecular probes, and the specific steps are as follows:

[0110] A549 cells with high LGR5 expression (purchased from the Cell Bank of the Chinese Academy of Sciences) were selected for saturation binding assays to study the uptake of probes by cells at different concentrations. Total binding assay procedure: A549 cells (5 × 10⁻⁶) were... 4 (1 cell / well) was inoculated into 48-well plates and cultured for 24 hours, followed by the addition of different concentrations of […]. 68 Ga]Ga-LTP-01 (12.5, 25, 50, 100, 150, 200, 300, 400 nM) or [ 68 Incubation with Ga-LTP-02 (12.5, 25, 50, 100, 150, 200, 300 nM) for 60 minutes (n=3). The non-specific binding group was pretreated with a 500-fold molar excess of peptide PO1 or PO2 for 60 minutes. After incubation, cells were washed twice with PBS (500 μL), collected with NaOH (500 μL, 1 M), and radioactivity was measured using a gamma counter. The specific binding of the radiotracer to LGR5 was determined by subtracting the non-specific binding group radioactivity from the total binding group radioactivity. The dissociation constant (K0) of the probe was calculated using nonlinear regression "One site Specific binding". d The results were fitted to the mean ± standard deviation and expressed as mean ± standard deviation.

[0111] The probe was measured. 68 Ga]Ga-LTP-01 and [ 68 Ga]Ga-LTP-02 of K d like Figure 14 and Figure 15 As shown, the values ​​were 155.20 ± 17.94 nM and 74.24 ± 14.41 nM, respectively, indicating that the probe has a good binding affinity for LGR5.

[0112] Example 7: Cellular uptake assay of LGR5-targeted molecular probes

[0113] This experiment provides a cellular uptake assay of an LGR5-targeting molecular probe, the specific procedure of which is as follows:

[0114] Cell uptake experiments were performed using A549, HCT116, and NCI-H1299 cells (purchased from the Cell Bank of the Chinese Academy of Sciences). In the cell uptake experiments, A549, HCT116, and NCI-H1299 cells were cultured at a density of 3 × 10⁻⁶ cells / cells. 5Cells were seeded at a density of 10 cells / well in 12-well plates and cultured overnight. The culture supernatant was then removed, and 500 μL of serum-free medium containing 0.37 MBq of radiotracer was added. Cells were cultured at 37°C for 30, 60, 90, and 120 minutes. At each time point, cells were washed with cold PBS and collected with 1 M NaOH solution. Radioactivity was measured using a gamma counter, and uptake was quantified as a percentage of the additive amount (%AD).

[0115] The probe of Example 1 [ 68 The cellular uptake results of Ga]Ga-LTP-01 are as follows Figure 16 As shown, throughout the incubation process, the uptake of the probe by A549 and HCT116 cells with high LGR5 expression was consistently significantly higher than that by NCI-H1299 cells with low LGR5 expression; within 120 min of incubation, the uptake of the probe by A549 and HCT116 cells was significantly higher than that by […]. 68 The uptake of Ga]Ga-LTP-01 increased in a time-dependent manner, reaching its peak at 120 minutes, at 0.30 ± 0.01 %AD and 0.26 ± 0.03 %AD, respectively, while the maximum uptake of the probe by NCI-H1299 was only 0.08 ± 0.01 %AD. These results indicate that the probe has good targeting specificity for both A549 and HCT116 cells with high LGR5 expression.

[0116] Probe of Example 2 68 The cellular uptake results of Ga]Ga-LTP-02 are as follows Figure 17 As shown, throughout the incubation process, the uptake of the probe by A549 and HCT116 cells with high LGR5 expression was consistently significantly higher than that by NCI-H1299 cells with low LGR5 expression; within 120 min of incubation, the uptake of the probe by A549 and HCT116 cells was significantly higher than that by […]. 68 The uptake of Ga-LTP-O2 increased in a time-dependent manner, reaching its highest value at 120 minutes, at 0.78 ± 0.08 %AD and 0.62 ± 0.03 %AD, respectively, while the maximum uptake of the probe by NCI-H1299 was only 0.12 ± 0.01 %AD. This result indicates that the probe [ 68 Ga]Ga-LTP-02 can distinguish tumors with different LGR5 expression levels at the cellular level.

[0117] Example 8: Pharmacokinetics of an LGR5-targeted molecular probe

[0118] This embodiment provides a pharmacokinetic experiment of an LGR5-targeting molecular probe, the specific process of which is as follows:

[0119] Female ICR mice (purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) were injected with a probe via the tail vein. 68 Ga]Ga-LTP-01 and [ 68 After administering Ga-LTP-02 (~7.4 MBq / animal, the probe was diluted with 150 μL of physiological saline before injection), blood was collected from the tail vein at different time points (1, 3, 5, 7, 10, 15, 20, 30, 45, 60, 90 and 120 min) and weighed. The radioactivity of the samples was determined using a gamma counter, and the pharmacokinetic parameters of the probe were analyzed using DAS software.

[0120] The probe of Example 1 [ 68 The metabolism of Ga]Ga-LTP-01 in normal ICR mice is as follows: Figure 18 As shown, the drug delivery curve conforms to the characteristics of a two-compartment model, with a distribution half-life of 2.21 min and an elimination half-life of 24.08 min. These results indicate that the probe can rapidly distribute throughout the mouse body, reach the tumor site, and then be rapidly cleared, consistent with the metabolic characteristics of peptide probes in vivo.

[0121] Probe of Example 2 68 The metabolism of Ga]Ga-LTP-O2 in normal ICR mice is as follows: Figure 19 As shown, with probe [ 68 Similar to Ga]Ga-LTP-01, the probe [ 68 The pharmacokinetic curve of Ga]Ga-LTP-02 also conforms to the characteristics of a two-compartment model; the distribution half-life of the probe is 0.94 min, and the elimination half-life of the probe is 21.93 min. These results indicate that the probe can be rapidly distributed throughout the mouse body and reach the tumor site, and then rapidly metabolized and cleared.

[0122] Example 9: PET imaging experiment of LGR5-targeted molecular probe

[0123] This experiment provides a PET imaging experiment using an LGR5-targeted molecular probe. The specific procedure is as follows:

[0124] Female BALB / c nude mice aged 4-6 weeks (purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) were selected and housed in the SPF laboratory animal facility for one week to acclimatize, thus establishing a tumor-bearing mouse model. Logarithmically growing A549, HCT116, and NCI-H1299 cells were subcutaneously inoculated into the right axilla of the mice (5 × 10⁻⁶ cells per cell line). 6 / mouse, cells suspended in PBS buffer before injection). The tumor-bearing mice were then housed in the SPF animal facility. Subsequent animal experiments were conducted when the tumor diameter reached 0.6 cm.

[0125] To investigate the distribution and metabolism of the probe in tumor-bearing mice, A549, HCT116, and NCI-H1299 tumor-bearing mouse models were selected for PET imaging studies. The tumor-bearing mice were anesthetized with isoflurane / O2 (2%, v / v, flow rate 2 mL / min), and placed prone on the PET scanning table. The probe was injected into the tail vein of each tumor-bearing mouse. 68 Ga]Ga-LTP-01, [ 68 Ga]Ga-LTP-02 (~5.5 MBq / mouse, probe diluted with 150 μL of physiological saline before injection); immediately after injection, a micro-PET dynamic scan was performed for 60 min to obtain whole-body PET images of tumor-bearing mice. The images were reconstructed using 3D-OSEM, and regions of interest (ROIs) were manually delineated. The uptake of the probe in the tumor and muscle of the tumor-bearing mice was quantitatively analyzed, and the results are expressed as %ID / mL.

[0126] The probe of Example 1 [ 68 PET imaging results of Ga-LTP-01 in A549, HCT116, and NCI-H1299 tumor-bearing mice are as follows: Figure 20 As shown, the probe can rapidly distribute throughout the mouse body and reach the tumor site, consistent with the characteristics of peptide probes: strong tissue penetration, rapid diffusion in the tumor microenvironment, uniform distribution, and easy access to deep solid tumors. Ten minutes after probe injection, significant radioactive signals were observed at the tumor sites of A549 and HCT116 tumor-bearing mice, and radioactive signals accumulated at the tumor sites within 1 hour of imaging. Conversely, no radioactive signals were observed at the tumor sites of NCI-H1299 tumor-bearing mice during imaging. Quantitative analysis results indicate that ( Figure 21 , Figure 22 and Figure 23 Ten minutes after injection, the uptake at the tumor site in A549 and HCT116 tumor-bearing mice reached its maximum, at 3.86 ± 0.60 %ID / mL and 2.60 ± 0.15 %ID / mL, respectively, significantly higher than the probe uptake in NCI-H1299 tumor-bearing mice (1.15 ± 0.25 %ID / mL). Figure 21 , Figure 22 and Figure 23 middle,[ 68 Ga]Ga-LTP-01-T probe[ 68 Ga]Ga-LTP-01 uptake at the tumor site in tumor-bearing mice, [ 68 Ga]Ga-LTP-01-M finger probe[ 68Ga-LTP-01 uptake in the muscle of tumor-bearing mice. The maximum tumor-to-muscle ratio of the probe in A549 and HCT116 tumor-bearing mice was 3.50 ± 0.67 and 2.45 ± 0.26, respectively, significantly higher than that in NCI-H1299 tumor-bearing mice (0.87 ± 0.20). Figure 24 The above results indicate that the probe [ 68 Ga]Ga-LTP-01 exhibits good targeting of LGR5 at the animal level, and can rapidly identify tumors with high LGR5 expression and quickly clear them from non-target organs.

[0127] Probe of Example 2 68 PET imaging results of Ga-LTP-02 in A549, HCT116, and NCI-H1299 tumor-bearing mice are as follows: Figure 25 As shown, the probe can rapidly distribute throughout the mouse body and reach the tumor site. Quantitative analysis results indicate that ( Figure 26 , Figure 27 and Figure 28 Ten minutes after the probe was injected into mice, the tumor outlines of A549 and HCT116 tumor-bearing mice were clearly visible, with maximum uptake values ​​of 4.74 ± 0.44 %ID / mL and 3.71 ± 0.30 %ID / mL, respectively, significantly higher than the probe uptake in NCI-H1299 tumor-bearing mice (0.98 ± 0.08 %ID / mL). Figure 26 , Figure 27 and Figure 28 middle,[ 68 Ga]Ga-LTP-02-T probe[ 68 Ga]Ga-LTP-02 uptake at the tumor site in tumor-bearing mice, [ 68 Ga]Ga-LTP-02-M finger probe[ 68 Ga]Ga-LTP-02 uptake in the muscle of tumor-bearing mice. The maximum tumor-to-muscle ratio of the probe in A549 and HCT116 tumor-bearing mice was 4.07 ± 0.07 and 3.39 ± 0.27, respectively, which was significantly higher than that in NCI-H1299 tumor-bearing mice (0.76 ± 0.01). Figure 29 The above results indicate that replacing leucine with alanine improved the probe […]. 68 Ga]Ga-LTP-02 showed increased targeting in tumor-bearing mice.

[0128] Example 10: Biodistribution experiment of LGR5-targeted molecular probe

[0129] This experiment provides a biodistribution assay for LGR5-targeted molecular probes, and the specific procedure is as follows:

[0130] Female BALB / c nude mice aged 4-6 weeks (purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) were selected and housed in the SPF laboratory animal facility for one week to acclimatize, thus establishing a tumor-bearing mouse model. Logarithmically growing A549, HCT116, and NCI-H1299 cells were subcutaneously inoculated into the right axilla of the mice (5 × 10⁻⁶ cells per cell line). 6 (Cells were suspended in PBS buffer and injected into each mouse to obtain A549, HCT116, and NCI-H1299 tumor-bearing mice). These tumor-bearing mice were then housed in the SPF animal facility. Subsequent animal experiments were conducted when the tumor diameter reached 0.6 cm.

[0131] Probes were injected into the tail veins of A549, HCT116, and NCI-H1299 tumor-bearing mice, respectively. 68 Ga]Ga-LTP-01 or [ 68 Ga-LTP-02 (~7.4 MBq / mouse, probe diluted with 150 μL physiological saline before injection); mice were euthanized 30 min after injection, and the mice were dissected. Major organs and tissues (heart, lungs, liver, spleen, kidneys, stomach, intestines, bones, brain, tumors, and muscles) were collected and weighed. The radioactivity of the samples was detected using a gamma counter. The results are expressed as a percentage of the uptake per gram of organ and tissue (%ID / g).

[0132] The probe of Example 1 [ 68 The biodistribution of Ga-LTP-01 in A549, HCT116, and NCI-H1299 tumor-bearing mice is as follows: Figure 30 As shown, the uptake of the probe at the tumor site in A549 and HCT116 tumor-bearing mice was significantly higher than that in NCI-H1299 tumor-bearing mice, at 3.04 ± 0.31 %ID / g, 2.58 ± 0.36 %ID / g, and 0.96 ± 0.36 %ID / g, respectively, consistent with the PET results, indicating that the probe […]. 68 Ga]Ga-LTP-01 can distinguish tumors with different LGR5 expression levels. The radioactive signal in the kidneys of A549, HCT116, and NCI-H1299 tumor-bearing mice was significantly higher than that in other organs, indicating that the probe is mainly metabolized through the kidneys.

[0133] Probe of Example 2 68 The biomass of Ga-LTP-02 in A549, HCT116, and NCI-H1299 tumor-bearing mice is as follows: Figure 31As shown, the uptake of the probe at the tumor site in A549 and HCT116 tumor-bearing mice was significantly higher than that in NCI-H1299 tumor-bearing mice, at 4.76 ± 0.65 %ID / g, 3.58 ± 0.08 %ID / g, and 1.49 ± 0.23 %ID / g, respectively, consistent with the PET results, indicating that the probe […]. 68 Ga]Ga-LTP-02 can distinguish tumors with different LGR5 expression levels. The radioactive signal in the kidneys of A549, HCT116, and NCI-H1299 tumor-bearing mice was significantly higher than that in other organs, indicating that the probe is mainly metabolized through the kidneys.

[0134] Comparative Example 1: Probe [ 68 Cellular uptake experiment of Ga-NOTA-YLA

[0135] NOTA-YLA and [ 68 The structure of Ga]Ga-NOTA-YLA is as follows: Figure 32 As shown.

[0136] Cell uptake experiments were performed using A549, HCT116, and NCI-H1299 cells (purchased from the Cell Bank of the Chinese Academy of Sciences). In the cell uptake experiments, A549, HCT116, and NCI-H1299 cells were cultured at a density of 3 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 10 cells / well in 12-well plates and cultured overnight. The culture supernatant was then removed, and 500 μL of serum-free medium containing 0.37 MBq of radiotracer was added. Cells were cultured at 37°C for 30, 60, 90, and 120 minutes. At each time point, cells were washed with cold PBS and collected with 1 M NaOH solution. Radioactivity was measured using a gamma counter, and uptake was quantified as a percentage of the additive amount (%AD).

[0137] The probe of Comparative Example 1 [ 68 The cellular uptake results of Ga]Ga-NOTA-YLA are as follows Figure 33 As shown, throughout the incubation process, A549 and HCT116 cells showed […]. 68 The uptake of Ga-NOTA-YLA increased in a time-dependent manner, reaching its highest value at 120 minutes, which was 0.21 ± 0.01 %AD and 0.16 ± 0.03 %AD, respectively. At all incubation time points, its uptake value was significantly lower than that of the probes in Examples 1 and 2. The maximum uptake of the probe by NCI-H1299 was 0.06 ± 0.01 %AD.

[0138] Comparative Example 2: Probe [ 68 Ga]Ga-NOTA-YLA PET Imaging Experiment

[0139] [ 68 PET imaging results of Ga-NOTA-YLA in A549 and HCT116 tumor-bearing mice are as follows: Figure 34 As shown. Quantitative analysis results indicate that ( Figure 35 and Figure 36 Ten minutes after the probe was injected into mice, the uptake of the probe by A549 and HCT116 tumors reached its maximum, at 3.27 ± 0.11 %ID / mL and 1.55 ± 0.06 %ID / mL, respectively. Figure 35 and Figure 36 middle,[ 68 Ga]Ga-NOTA-YLA-T probe[ 68 Ga]Ga-NOTA-YLA uptake at the tumor site in tumor-bearing mice, [ 68 Ga]Ga-NOTA-YLA-M finger probe[ 68 Uptake of Ga-NOTA-YLA in the muscle of tumor-bearing mice. Probe [ 68 The maximum tumor-to-muscle ratios of Ga-NOTA-YLA in A549 and HCT116 tumor-bearing mice were 2.17 ± 0.07 and 1.25 ± 0.40, respectively. Figure 37 The above results indicate that the control peptide has poor specificity for detecting LGR5 in vivo.

[0140] Based on the above-described preferred embodiments according to this application, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical concept of this application. The technical scope of this application is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A polypeptide targeting LGR5, characterized in that, The polypeptide is any one of the following: (1) Its amino acid sequence is YLASRVH; (2) A derivative polypeptide formed by substituting one or more amino acids into the amino acid sequence shown in (1), wherein the derivative polypeptide has the same or substantially the same function as the polypeptide of the sequence shown in (1). (3) A derivative polypeptide derived from the polypeptide described in (1) or (2) by chemical or genetic modification. The substitution is the replacement of one or more of tyrosine, leucine, serine, arginine, valine, and histidine with alanine.

2. The LGR5-targeting polypeptide of claim 1, wherein, The substitution is the replacement of one or more of tyrosine, leucine, serine, and valine with alanine; preferably, the peptide targeting LGR5 is selected from any of the following amino acid sequences: YLASRVH, ALASRVH, YAASRVH, YLAARVH, YLASRAH.

3. A PET molecular probe targeting LGR5, characterized in that, The invention comprises the LGR5-targeting peptide, chelating agent, and radionuclide as described in claim 1 or 2, wherein the LGR5-targeting peptide is covalently coupled to the chelating agent, and the radionuclide is chelated to the chelating agent.

4. The PET molecular probe targeting LGR5 according to claim 3, characterized in that, The radionuclide is 68 Ga, 18 F, 64 Cu or 89 Zr, the chelator is preferably DOTA, HYNIC, DTPA, NOTA or TETA; further, R is preferably leucine or alanine, the chelator is preferably NOTA, further, the PET molecular probe is preferably or .

5. A method for preparing the PET molecular probe targeting LGR5 as described in claim 3 or 4, characterized in that, include: The polypeptide compound was coupled with the chelating agent -NHS and deprotected to obtain the precursor compound; the precursor compound was radiolabeled to obtain the PET molecular probe. The polypeptide compound has the following structure: ， In the polypeptide compound, Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, and Trt is triphenylmethyl.

6. A precursor compound for a PET molecular probe targeting LGR5, characterized in that, By the claims The LGR5-targeting peptide and chelating agent described in 1 or 2 are covalently coupled, wherein the LGR5-targeting peptide and the chelating agent are preferably DOTA, HYNIC, DTPA, NOA, or TETA.

7. The precursor compound of the PET molecular probe targeting LGR5 according to claim 6, characterized in that, The precursor compound of the PET molecular probe targeting LGR5 has the following structure: or 。 8. The application of a PET molecular probe targeting LGR5 in the preparation of an imaging agent targeting LGR5, characterized in that, The imaging agent targeting LGR5 is preferably an imaging agent targeting LGR5 for non-disease diagnosis or treatment, or an imaging drug targeting LGR5-positive tumors.

9. An imaging agent targeting LGR5, characterized in that, The imaging agent contains the PET molecular probe targeting LGR5 as described in claim 3 or 4, and the imaging agent targeting LGR5 is preferably an imaging agent targeting LGR5 for non-disease diagnosis or treatment or an imaging drug targeting LGR5-positive tumors.

10. The application of the LGR5-targeting PET molecular probe according to claim 8 in the preparation of an LGR5-targeting imaging agent or the LGR5-targeting imaging agent according to claim 9, characterized in that, The LGR5-positive tumors include at least one of colorectal cancer, non-small cell lung cancer, gastric cancer, liver cancer, and breast cancer.

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