Dimer bicyclic peptide nuclide ligand and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a dimer bicyclic peptide nuclide ligand and application thereof. The dimer bicyclic peptide nuclide ligand disclosed by the invention has a structure as shown in a formula I: # imgabs0 # formula I. The dimer bicyclic peptide nuclide ligand disclosed by the invention can be prepared into a nuclide probe targeting Nectin-4 after being labeled by radionuclide, the dimer bicyclic peptide nuclide ligand is good in radiation stability and ideal in pharmacokinetics, a tumor overexpressed by the Nectin-4 is high in uptake of the dimer bicyclic peptide nuclide ligand, and the tumor and muscle uptake ratio and in-vivo metabolic performance are good.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more particularly to a dimer bicyclic peptide radionuclide ligand and its application. Background Art

[0002] Breast cancer is one of the common malignant tumors, and its molecular classification is of great significance for clinical diagnosis and treatment. According to immunohistochemical characteristics, breast cancer can be divided into luminal A type, luminal B type, human epidermal growth factor receptor 2 (HER2)-positive type, and triple-negative breast cancer (TNBC). Triple-negative breast cancer has limited clinical treatment options due to the lack of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression, and traditional chemotherapy remains the main treatment option. However, this type of breast cancer shows high invasiveness and recurrence rate, and some patients are resistant to chemotherapy, with a short median overall survival and poor long-term prognosis. Therefore, finding specific biomarkers and new targeted treatment strategies is crucial for improving the treatment effect of triple-negative breast cancer patients.

[0003] In recent years, the role of adhesion molecules in tumorigenesis and development has received extensive attention. The nectin protein belongs to the adhesion receptor of the immunoglobulin superfamily (IgSF), including four members from nectin-1 to nectin-4. Among them, nectin-4 (PVRL4) is a type I transmembrane protein that is highly expressed in various malignant tumors. Existing studies have shown that the expression level of nectin-4 increases in tumor tissues such as breast cancer, bladder cancer, ovarian cancer, pancreatic cancer, and lung cancer, and is related to tumor cell proliferation, invasion, and drug resistance. Especially in triple-negative breast cancer and basal-like breast cancer, the expression rate of nectin-4 is relatively high, while it is low or not expressed in normal breast tissue, making it a potential diagnostic and therapeutic target.

[0004] Currently, the research on targeted therapy for nectin-4 mainly focuses on the field of antibody-drug conjugates (ADCs), such as Enfortumab Vedotin (PADCEV ®)(It) has been approved for the treatment of advanced or metastatic bladder cancer. In addition, preclinical studies have also explored the use of radionuclide conjugates of Nectin-4 monoclonal antibodies for molecular imaging. However, antibodies have a relatively large molecular weight, a long circulation time in the body, and a high non-specific tissue uptake, which limits their application in rapid diagnosis and treatment evaluation. In contrast, small molecule probes, such as peptide molecules, have the advantages of small molecular weight, good tissue permeability, fast clearance, and low immunogenicity, and show good application prospects in tumor-targeted imaging and radionuclide therapy. Therefore, the development of a radioactive molecular probe based on Nectin-4-targeted peptides will contribute to the accurate diagnosis and efficacy evaluation of Nectin-4-positive tumors, and provide an imaging basis for individualized treatment. Summary of the Invention

[0005] One of the objectives of the present invention is to provide a radionuclide ligand that can specifically target Nectin-4 and has ideal pharmacokinetics.

[0006] The present invention provides a dimer bicyclic peptide radionuclide ligand having a structure shown in Formula I: Formula I.

[0007] The present invention constructs a dimer bicyclic peptide radionuclide ligand that can target Nectin-4. Through a specific amino acid sequence and cyclization method, its parent nucleus can achieve high affinity for the Nectin-4 target. The radionuclide chelating group can bind to a variety of radionuclides to achieve the labeling of the bicyclic peptide targeting Nectin-4. When two bicyclic peptide radionuclide ligands of the present invention are linked in a specific structure to form a dimer and then radionuclide-labeled, the obtained dimer probe can improve the enrichment level in tumor tissues through multivalent effects, while reducing non-specific binding and increasing the tumor / normal tissue ratio. In addition, the dimer probe of the present invention also exhibits a higher apparent affinity, reduces the dissociation rate ( K d ), enhances the imaging contrast, and thus improves the diagnostic accuracy. Due to the increased binding time to the target protein, the use of the dimer bicyclic peptide radionuclide ligand of the present invention can enhance the uptake and retention time of tumors. The dimer bicyclic peptide molecular probe targeting Nectin-4 can be used for preclinical verification of the in vivo identification of Nectin-4 receptor-positive expression tumor models through PET whole-body imaging (which can obtain high resolution and clearly distinguish tumor regions). It is of great significance for the early detection of triple-negative breast cancer with high Nectin-4 expression, the screening of populations benefited from Nectin-4-targeted therapeutic drugs, the efficacy monitoring and evaluation of anti-tumor drugs, and prognosis evaluation, and has great potential for clinical translation.

[0008] The optimization of dimeric probes involves multiple key factors, including steric hindrance, target binding efficiency, in vivo distribution, and pharmacokinetic properties. Existing studies have shown that not all dimerizations of probes can bring about ideal performance improvements, and may even lead to a decrease in affinity or a decline in tumor uptake rate. For example, the literature "Krasniqi A et al. Pharmacokinetics of radiolabeled dimeric sdAbs constructs targeting human CD20. New biotechnology, 2018, 45: 69-79" reported that although the dimerization of CD20 single-domain antibody (sdAb) improved endocytosis, its apparent affinity decreased, indicating that the optimization of dimeric probes requires precise design for specific targets. Another example is the literature "Garousi J et al. Comparative evaluation of dimeric and monomeric forms of ADAPT scaffold protein for targeting of HER2-expressing tumours. European journal of pharmaceutics and biopharmaceutics, 2019, 134: 37-48", which reported that the affinity of the HER2-targeting ADAPT6 dimer was significantly improved, but its tumor uptake rate was lower than that of the monomeric probe, indicating that an increase in molecular size may have an adverse effect on tissue permeability. Therefore, the optimization of dimeric probes does not solely rely on increasing the binding sites. Different ligation strategies may lead to a decrease in targeting ability, deterioration of pharmacokinetic properties, or molecular conformational constraints, thus affecting the target binding affinity. Therefore, factors such as spatial structure, binding kinetics, and in vivo metabolism need to be comprehensively considered to ensure that the optimized probe exhibits good targeting performance in vivo.

[0009] The optimization of dimeric probes requires precise design for specific targets, molecular conformations, and in vivo distribution characteristics, rather than simply achieving performance improvement through structural repetition. In the process of optimizing the dimeric radionuclide ligand of the present invention, systematic screening was carried out for different ligation methods, and some failed construction schemes were verified. Specifically, in the preliminary experiment, the present invention attempted to directly connect the two-terminal cyclic peptides with an amide bond, but this scheme may have changed the conformation of the cyclic peptide, thereby affecting the target binding ability. The present invention also attempted to directly couple a polyethylene glycol linker with a bicyclic peptide, but this scheme failed during the synthesis process. The possible reason is that the direct coupling of polyethylene glycol with the bicyclic peptide has poor chemical stability, resulting in low coupling efficiency.

[0010] To overcome the above problems, the present invention adopts the maleimide-cysteine conjugation strategy, which not only improves the coupling efficiency of the dimer linker but also ensures the spatial arrangement of the polyethylene glycol linker, thereby reducing the influence of steric hindrance on target binding. Compared with the failed scheme, the optimized dimer probe of the present invention shows significant improvements in terms of Nectin-4 target affinity ( K D decreased from 23.7 nM to 0.37 nM), tumor uptake rate, etc., successfully avoiding the common affinity loss and poor pharmacokinetics problems caused by dimerization. In addition, the hydrophilicity of polyethylene glycol reduces the liver uptake of the dimer probe caused by the increase in molecular weight, improves the tumor tissue targeting, and makes the probe of the present invention more widely used in molecular imaging and radiotherapy.

[0011] The dimer probe of the present invention successfully overcomes the influence of steric hindrance on target binding through the optimization of specific connection methods, peptide chain lengths, and dimerization strategies, achieving higher affinity and better pharmacokinetic properties, and overcoming the deficiencies in tumor uptake and in vivo distribution. After dimerization, the probe of the present invention not only shows a significantly higher affinity improvement than the traditional dimerization strategy ( K D decreased from 23.7 nM to 0.37 nM), but also shows a significantly increased tumor uptake rate compared with the control monomer probe in animal experiments, fully demonstrating its application value in molecular imaging and radiotherapy. The present invention also optimizes the dimer connection strategy to maintain high affinity while avoiding the problem of reduced tumor tissue permeability caused by the increase in dimer size.

[0012] In summary, the Nectin-4-targeted dimer polypeptide probe of the present invention not only overcomes the defects of existing dimerization probes but also realizes the comprehensive optimization of affinity, tumor uptake rate, and pharmacokinetics, solving the key technical challenges in the dimerization modification process. Compared with the traditional dimerization strategy, the present invention significantly improves the imaging and therapeutic effects by precisely regulating the molecular structure of the probe, showing higher clinical application potential.

[0013] In addition, the optimization process of the present invention involves the regulation of multiple key parameters, which has high technical challenges, and this technical solution is not only applicable to Nectin-4-targeted probes but also can be extended to the design of other bicyclic peptide-targeted probes, providing important technical support for molecular imaging and radiotherapy of similar targets.

[0014] The present invention also provides a method for preparing the above-mentioned dimer bicyclic peptide radionuclide ligand, which includes: (1) By solid-phase synthesis method, the amino acids contained in Formula I are coupled to obtain Compound 1; (2) Compound 1 and Compound 2 are subjected to a cyclization reaction in a mixed solvent of ammonium carbonate buffer solution and ACN / H2O (volume ratio 1:1) to obtain Compound 3; Preferably, the volume ratio of the ACN / H2O mixed solvent to the ammonium carbonate buffer solution is 3:1, and the concentration of NH4HCO3 in the ammonium carbonate buffer solution is 1 M; The molar ratio of Compound 1 to Compound 2 is 1:1.5; (3) Compound 4 is dissolved in DMF, activated in the presence of DCC, HOSu and DIPEA, and then subjected to acid amide condensation with Compound 3; after the reaction is completed, DMF is removed, Solution E is added for reaction, and then ether is added and dried to obtain Compound 5; Solution E refers to a mixed solution of 90% TFA + 5% anisole sulfide + 2.5% phenol + 2.5% 1,2-ethanedithiol; Preferably, the molar ratio of Compound 3 to Compound 4 is 1:2; The molar ratio of DCC, HOSu and DIPEA is 2:2:5; the molar ratio of Compound 3 to DIPEA is 1:5; (4) Compound 7 is dissolved in DMF, activated in the presence of DCC, HOSu, DIPEA, and then subjected to acid amide condensation with Compound 6. After the reaction is completed, DMF is removed to obtain Compound 8; Preferably, the molar ratio of Compound 6 to Compound 7 is 1:1.2; The molar ratio of DCC, HOSu, DIPEA is 1.2:1.2:5; the molar ratio of Compound 6 to DIPEA is 1:5; (5) Compound 8 is dissolved in DMF, and acid amide condensation is carried out with Compound 9 under the catalysis of EDC·HCl, HOBt and NMM. After the reaction is complete, DMF is removed, the tert-butyl protecting group is removed with TFA solution, ether is added and dried to obtain Compound 10; Preferably, the molar ratio of Compound 8 to Compound 9 is 1:5; The molar ratio of EDC·HCl, HOBt and NMM is 1:1:1; the molar ratio of Compound 8 to NMM is 1:5; (6) Compound 10 and Compound 5 are dissolved in ACN / H2O (1:1), and then co-react in a phosphate buffer solution (pH = 7.2 0.2 M) to obtain the dimeric bicyclic peptide radionuclide ligand; Preferably, the molar ratio of Compound 5 to Compound 10 is 2.5:1; The structural formula of Compound 1 is as follows: ; Compound 2 is 1,3,5-triacryloyl-1,3,5-triazine; The structural formula of Compound 3 is as follows: ; The structural formula of Compound 4 is as follows: ; The structural formula of Compound 5 is as follows: ; The structural formula of Compound 6 is as follows: ; The structural formula of Compound 7 is as follows: ; The structural formula of Compound 8 is as follows: ; The structural formula of Compound 9 is as follows: ; The structural formula of Compound 10 is as follows: 。

[0015] The present invention also provides a radionuclide probe targeting Nectin-4, which is a radionuclide-labeled above-mentioned dimeric bicyclic peptide radionuclide ligand or the dimeric bicyclic peptide radionuclide ligand prepared by the above method.

[0016] In the radionuclide probe targeting Nectin-4 of the present invention, the radionuclide includes a diagnostic radionuclide or a therapeutic radionuclide, and the diagnostic radionuclide is 68 Ga, 64 Cu, 18 F, 86 Y, 90 Y, 89 Zr, 111 In, 99m Tc, 11 C, 123 I, 125 I and 124 I at least one of them; the therapeutic radionuclide includes 177 Lu, 125 I, 131 I, 211 At, 111 In, 153 Sm, 186 Re, 188 Re, 67 Cu, 212 Pb, 225Ac, 213 Bi, 212 Bi and 212 at least one of Pb; preferably, the radionuclide is 68 Ga.

[0017] Compared with monoclonal antibodies, the radionuclide probe of the present invention has a reasonable half-life, can penetrate tumor tissues more effectively, and can provide accurate evaluation results in a shorter time, thus achieving more precise diagnosis. In addition, the radionuclide probe targeting Nectin-4 of the present invention has the characteristics of rapid labeling and high labeling rate, its purity exceeds 95%, it is mainly excreted through the kidneys, the uptake by other non-target organs is low, and it shows an ideal uptake amount in tumor tissues, with high sensitivity and specificity.

[0018] The present invention also provides a method for preparing the above-mentioned radionuclide probe targeting Nectin-4, which includes the step of labeling the above-mentioned dimeric bicyclic peptide radionuclide ligand or the dimeric bicyclic peptide radionuclide ligand prepared by the above method with a radionuclide.

[0019] The present invention also provides the application of the radionuclide probe targeting Nectin-4 in the preparation of a Nectin-4 imaging agent or a PET imaging probe.

[0020] The present invention also provides the application of the radionuclide probe targeting Nectin-4 in the preparation of a reagent or kit for diagnosing, treating and / or preventing diseases; the diseases are characterized by overexpression of Nectin-4.

[0021] The present invention also provides a detection reagent or kit, which includes the above-mentioned radionuclide probe targeting Nectin-4.

[0022] The present invention also provides a drug, which includes the above-mentioned dimeric bicyclic peptide radionuclide ligand or the radionuclide probe targeting Nectin-4 and pharmaceutically acceptable excipients.

[0023] In the present invention, "pharmaceutically acceptable" means that a compound or composition is chemically and / or toxicologically compatible with other components constituting the preparation and with humans or mammals for which it is used to prevent, diagnose and treat diseases or disorders.

[0024] In the drug of the present invention, the dimeric bicyclic peptide radionuclide ligand or the radionuclide probe targeting Nectin-4 is also conjugated with a therapeutic agent that can prevent, inhibit and / or treat diseases characterized by overexpression of Nectin-4.

[0025] The dimeric bicyclic peptide radionuclide ligand of the present invention has excellent targeting effect, and it can be combined with a substance with drug activity to accurately deliver the therapeutic agent to the lesion site, thereby improving the targeting of the drug, reducing side effects and enhancing the therapeutic effect.

[0026] The beneficial effects of the present invention are at least as follows: The present invention provides a new dimer bicyclic peptide radionuclide ligand, which can target Nectin-4. Compared with the existing bicyclic peptide radionuclide probes targeting Nectin-4, the dimer bicyclic peptide radionuclide ligand and probe of the present invention have good stability, ideal pharmacokinetics, strong uptake by tumors with overexpression of Nectin-4, good tumor-to-muscle uptake ratio and in vivo metabolism performance, and have significant clinical transformation potential, providing a new technical means for the precise diagnosis of solid tumors. Brief Description of the Drawings

[0027] Figure 1 It is one of the schematic diagrams of the synthesis route of the bicyclic peptide radionuclide ligand DOTA-HTA-DM of the present invention; Figure 2 It is another schematic diagram of the synthesis route of the bicyclic peptide radionuclide ligand DOTA-HTA-DM of the present invention; Figure 3 It is the third schematic diagram of the synthesis route of the bicyclic peptide radionuclide ligand DOTA-HTA-DM of the present invention; Figure 4 It is the fourth schematic diagram of the synthesis route of the bicyclic peptide radionuclide ligand DOTA-HTA-DM of the present invention; Figure 5 It is the mass spectrum of DOTA-HTA-DM; Figure 6 It is the binding affinity graph of DOTA-HTA-DM and human Nectin-4 detected by SPR; Figure 7 It is 68 The HPLC chromatogram of the Figure 8 It is the Western blot detection graph of tumor cells MDA-MB-468; Figure 9 It is for the breast cancer MDA-MB-468 tumor model mice injected with 200 μCi of 68 Ga-DOTA-HTA-DM, 68 Ga-DOTA-HTA-DM and blocking dose of DOTA-HTA-DM (blocking) and 68 Ga-DOTA-HTA (monomer) and then the whole body Micro-PET / CT MIP imaging graphs at 30 min, 1 h, and 2 h respectively; the arrow indicates the tumor location; Figure 10 It is for the breast cancer MDA-MB-468 tumor model mice injected with 200 μCi of 68Ga-DOTA-HTA-DM and 68 The ratio of the tumor / muscle uptake values ​​of Ga-DOTA-HTA-DM and blocking dose of DOTA-HTA-DM at 30 min, 1 h, and 2 h after ROI delineation; represents P < 0.05, represents P < 0.01, ns represents no significant difference; Figure 11 MDA-MB-468 breast cancer model mice were injected with 50 μCi of 68 Uptake values ​​of Ga-DOTA-HTA-DM in tumors and other tissues and organs at 30 min, 1 h, and 2 h (%ID / g) (mean±SD, n=3); Figure 12 is the mass spectrum of DOTA-HTA; Figure 13 for 68 The structural formula of Ga-DOTA-HTA; Figure 14 is the mass spectrum of DOTA-HTA-DM intermediate compound 1; Figure 15 is the mass spectrum of DOTA-HTA-DM intermediate compound 3; Figure 16 is the mass spectrum of DOTA-HTA-DM intermediate compound 5; Figure 17 is the mass spectrum of DOTA-HTA-DM intermediate compound 8; Figure 18 This is the mass spectrum of DOTA-HTA-DM intermediate compound 10. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0029] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0030] Example 1 This example discloses a method for synthesizing a bicyclic peptide DOTA-HTA-DM targeting nectin-4 dimer (see the schematic diagram of the synthetic route). Figures 1 to 4 ), specifically: Synthesis of Compound 1: First, Fmoc-Cys(Trt)-OH was coupled to Rink Amide MBHA Resin, and then Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-HomoArg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Cys(Trt)-OH, Fmoc-DAsp(OtBu)-OH, Fmoc-1Nal-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH were coupled in sequence. The coupling of all amino acids (1 eq) was carried out in DMF using DIC (3 eq) and HOBt (3 eq) as coupling reagents. The Fmoc protecting group was cleaved with 20% Pip / DMF solution. Finally, the crude peptide compound 1 was cleaved from the resin with TFA solution at room temperature for 2 h. The crude peptide was purified by HPLC on a C18 preparative column to obtain pure compound 1 (Yield: 23%). Its structure was identified by mass spectrometry, and the mass spectrum is as shown in Figure 14 shown, which is consistent with the synthesis target.

[0031] Synthesis of Compound 3: Compound 1 (1 eq) was dissolved in a mixed solvent of 75 mL ACN and 75 mL H2O, and then compound 2 - 1,3,5-triacryloyl-1,3,5-triazine (1.5 eq) and 50 mL 1M NH4HCO3 were added. The reaction was carried out at room temperature for 1 h, and the reaction was monitored by LC-MS until completion. Then it was directly purified by reversed-phase preparative liquid chromatography to obtain compound 3 (Yield: 28%). Its structure was identified by mass spectrometry, and the mass spectrum is as shown in Figure 15 shown.

[0032] Synthesis of Compound 5: Compound 4 (2 eq) was dissolved in 10 ml DMF, and then DCC (2 eq) and HOSu (2 eq) were added. The reaction was carried out at room temperature for 6 h, and then compound 3 (1 eq) and DIPEA (5 eq) were added. The reaction was carried out at room temperature for 1 h, and the reaction was monitored by LC-MS until completion. The DMF was removed by rotary evaporation, and then 10 ml of solution E (90% TFA + 5% thioanisole + 2.5% phenol + 2.5% 1,2-ethanedithiol) was added. The reaction was carried out at room temperature for 2 h, 100 ml of ether was added, a large amount of solid precipitated, and it was centrifuged and dried. After purification by reversed-phase preparative liquid chromatography, compound 5 was obtained (Yield: 28.8%). The structure of compound 5 was identified by mass spectrometry, and the mass spectrum is as shown in Figure 16 shown.

[0033] Synthesis of Compound 8: Compound 7 (1.2 eq) was dissolved in 10 ml of DMF, and then DCC (1.2 eq) and HOSu (1.2 eq) were added. The reaction was carried out at room temperature for 6 hours. Then, Compound 6 (1 eq) and DIPEA (5 eq) were added, and the reaction was continued at room temperature for 1 hour. The reaction was monitored by LC-MS until completion. DMF was removed by rotary evaporation, and the residue was purified by reverse-phase preparative liquid chromatography to obtain Compound 8 (Yield: 63%). Compound 8 was identified by mass spectrometry, and the mass spectrum is shown as Figure 17 shown.

[0034] Synthesis of Compound 10: Compound 8 (1 eq) was dissolved in 10 ml of DMF, HOBt (5 eq) and EDC·HCl (5 eq) were added, and the reaction was carried out at 0 °C for 10 minutes. Then, Compound 9 (5 eq) and NMM (5 eq) were added, and the reaction mixture was transferred to room temperature and reacted for 12 hours. The reaction was monitored by LC-MS until completion. DMF was evaporated to dryness, 10 mL of TFA solution was added, and the reaction was carried out at room temperature for 2.5 hours. 100 ml of ether was added, and a large amount of solid precipitated. The solid was centrifuged and dried, and then purified by reverse-phase preparative liquid chromatography to obtain Compound 10 (yield: 16%). Compound 10 was identified by mass spectrometry, and the mass spectrum is shown as Figure 18 shown.

[0035] The structural formula of Compound 1 is as follows: ; Compound 2 is 1,3,5-triacryloyl-1,3,5-triazine; The structural formula of Compound 3 is as follows: ; The structural formula of Compound 4 is as follows: ; The structural formula of Compound 5 is as follows: ; The structural formula of Compound 6 is as follows: ; The structural formula of Compound 7 is as follows: ; The structural formula of Compound 8 is as follows: ; The structural formula of Compound 9 is as follows: ; The structural formula of Compound 10 is as follows: .

[0036] Synthesis of DOTA-HTA-DM: Compound 10 (1 eq) and Compound 5 (2.5 eq) were dissolved in 10 mL of ACN / H2O (1:1), and then 10 mL of 0.2 M phosphate buffer solution with pH = 7.2 was added. The reaction was carried out at room temperature for 1 hour. The reaction was monitored by LC-MS until completion, and then directly purified by reverse-phase preparative liquid chromatography to obtain DOTA-HTA-DM (Yield: 18.6%). Its structural formula is shown in Formula I.

[0037] Formula I.

[0038] The structure was confirmed by negative-ion mode mass spectrometry method, and the purity was greater than 95% by HPLC quantitative analysis. The mass spectrometry results are shown in Table 1. As can be seen from the mass spectrometry Figure 5 that the peak at 1186.6854 is the [M + 5H] + / 5 peak, and the peak at 1483.1050 is the [M + 4H] + / 4 peak, and its molecular weight is actually 5928.82. The structure was confirmed to be correct according to the mass spectrometry structure.

[0039] Table 1 Mass spectrometry results of compound DOTA-HTA-DM

[0040] Example 2 This example discloses the determination of the binding affinity between DOTA-HTA-DM and human Nectin-4 protein, specifically as follows: Surface plasmon resonance affinity assay (SPR) experiment was carried out on Biacore 8K to determine the k a (1 / Ms), k d (1 / s) and K D(M) value. First, the sensor chip was activated with freshly mixed 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) for 420 s. Subsequently, the Nectin-4 protein was diluted to 20 μg / mL with 10 mM sodium acetate (pH 4.5). After the Nectin-4 protein coupling level reached approximately 800 RU, EA was flowed over the chip surface to block the excess sites. After the reference channel was activated, it was directly blocked. The diluted polypeptide DOTA-HTA-DM prepared in Example 1 (concentrations were 100, 50, 25, 12.5, 6.25, 3.125 nM respectively) was injected onto the chip at a flow rate of 30 μL / min at 25 °C. The binding time was 60 s and the dissociation time was 600 s. Detection was performed using the single-cycle kinetic mode. All data were processed using Biacore 8K evaluation software version 4.0. Appropriate consecutive concentrations (at least 5 concentrations) were selected for kinetic 1:1 binding and steady-state analysis. The results are as Figure 6 shown in Table 2.

[0041] Table 2 Binding affinity of DOTA-HTA-DM to human Nectin-4 protein

[0042] The results showed that DOTA-HTA-DM had a very high binding affinity for human Nectin-4 protein, and its K D value was 0.37 nM.

[0043] Example 3 68 Radioactive labeling of Ga: A DMSO solution (concentration 10 mg / ml) of the Nectin-4-targeted dimeric bicyclic peptide radionuclide ligand prepared in Example 1 was prepared as the precursor solution. 20 μL of the precursor solution was placed in a reaction flask, 65 μL of 1 M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly rinsed 68 Ga eluate ( 68 Ga elution was carried out in 0.1 M hydrochloric acid solution) were added to the reaction flask, and then 0.5 mL of deionized water was added. The pH value of the reaction solution was approximately 4.0. After mixing, the mixture was placed at 95 °C for reaction for 10 min to obtain 68 Ga-labeled complex ( 68 Ga-DOTA-HTA-DM bicyclic peptide radionuclide probe).

[0044] Quality control of Nectin-4 targeting probe: 68 The radiochemical purity of the Ga-DOTA-HTA-DM bicyclic peptide radionuclide probe was determined by radio-high performance liquid chromatography (Radio-HPLC). Chromatographic method: Mobile phase: water containing 0.1% TFA and acetonitrile solution containing 0.1% TFA, 0 - 20 min: 30 - 50% acetonitrile phase. The radiochemical purity of the product detected by Radio-HPLC was greater than 95%, and the detection results are as Figure 7 shown.

[0045] Example 4 Western blot detection of Nectin-4 expression in human breast cancer MDA-MB-468 cell line: Sample lysis: Appropriate lysis buffer (containing protease inhibitor) was added to the tissue, and it was ground with a tissue grinding cryo-homogenizer. After taking it out, it was left standing on ice for 30 min, and shaken every 10 min. Centrifuge at 12000 rpm at 4°C for 30 min, and collect the supernatant, which is the total protein solution. Protein concentration determination: The protein concentration was detected using a BCA protein concentration determination kit. Protein sample preparation: The treated protein solution, RAPI lysis buffer and SDS-PAGE protein loading buffer were mixed in proportion, and denatured in a metal bath at 95°C for 5 min, and stored at -20°C in the refrigerator for later use. SDS-PAGE electrophoresis: Protein samples and protein markers (Marker) were added in sequence according to the sample layout, constant voltage 80V / 15 min, 120V / 1 hr. Blotting: The blotting tank was placed in an ice-water bath, and transferred at a constant current of 300 mA for 1 hr. Blocking: Incubate with 5% skim milk on a shaker at room temperature for 1 hr. Primary antibody incubation: The PVDF membrane was incubated with the primary antibody on a shaker at a constant temperature of 4°C overnight. Secondary antibody incubation: The PVDF membrane was incubated with the secondary antibody on a shaker at room temperature for 1 hr. ECL color development: The color developing solution was added dropwise, and automatic exposure was performed using a chemiluminescence imaging system. The Western blot detection results are as Figure 8 shown. Using GAPDH protein as an internal standard, the molecular weight of Nectin-4 protein is about 60 - 70 kDa. The relative expression of Nectin-4 to GAPDH in MDA-MB-468 tumor tissue is relatively high, while the expression of Nectin-4 was not detected in the control MDA-MB-231 tumor tissue. It is proved that MDA-MB-468 is a high-expression model of Nectin-4, while MDA-MB-231 is a low-expression model of Nectin-4. T-47D has been reported to express Nectin-4, which is used as a positive control to prove the effectiveness of the antibody and the reliability of the Western blot experiment operation.

[0046] Example 5 Micro-PET / CT Imaging of Human Breast Cancer MDA-MB-468 Model Mice: This example discloses the in vivo imaging experiment of the dimer bicyclic peptide radionuclide ligand targeting Nectin-4 prepared in Example 1. Nude mice with MDA-MB-468 xenograft tumors were given tail vein injection (200 μCi / mouse) when the tumor tissue reached 100 - 300 mm 3 . Static scans were performed for 10 minutes at different time points (30, 60, and 120 min) after injection, and the tumors were clearly visualized. After the scan, the scan data were iteratively reconstructed (OSEM 3D), and the QD software was used to delineate two tumors and other tissues as regions of interest (ROI) to calculate the radioactive uptake %ID / g of each ROI. Among them, breast cancer MDA-MB-468 tumor model mice were respectively injected with 200 μCi of 68 68Ga-DOTA-HTA-DM, 68 68Ga-DOTA-HTA-DM with a blocking dose (1000-fold excess) of DOTA-HTA-DM, and 68 68Ga-DOTA-HTA (monomer), and then whole-body Micro-PET / CT MIP was performed at 30 min, 1 h, and 2 h later. The imaging diagrams are as shown in Figure 9 . Breast cancer MDA-MB-468 tumor model mice were respectively injected with 200 μCi of 68 68Ga-DOTA-HTA-DM and 68 68Ga-DOTA-HTA-DM with a blocking dose of DOTA-HTA-DM, and then the ratios of the uptake values of the tumors / muscles delineated by ROI at 30 min, 1 h, and 2 h later are as shown in Figure 10 (the data are presented in the form of mean ± standard deviation (n = 3).

[0047] 68 The preparation method of 68Ga-DOTA-HTA (monomer) is as follows: (1) Preparation of DOTA-HTA (monomer): Dissolve the compound 1 (1 eq) prepared in Example 1 in a mixed solvent of 15 mL ACN and 15 mL H2O, then add compound 2-1,3,5-triacryloyl-1,3,5-triazine (1.5 eq) and 10 mL 1M NH4HCO3. React at room temperature for 1 hour, monitor the reaction completion by LC-MS, and directly purify by reverse-phase preparative liquid chromatography to obtain DOTA-HTA (Yield: 34.5%). The mass spectrometry diagram is shown in Figure 12 . The structural formula is shown in Figure 13 .

[0048] (2) 68Radioactive labeling of Ga: Refer to the description in Example 3 to prepare and obtain 68 Ga-DOTA-HTA.

[0049] As Figure 9 and Figure 10 shown, 68 Ga-DOTA-HTA-DM accumulates significantly in the tumor region, with good sensitivity, and is mainly excreted from the body through the kidneys. As time prolongs, the uptake ratio of the complex in the tumor to muscle increases. At 30 min, 1 h, and 2 h after injection, the tumor uptake values are 2.18 ± 0.34, 2.01 ± 0.33, and 1.98 ± 0.22 %ID / g respectively, and the uptake ratios of the tumor to muscle are 4.52 ± 1.55, 9.41 ± 1.41, and 6.83 ± 1.75 respectively. In the blocking imaging experiment ( 68 Ga-DOTA-HTA-DM + blocking), it can be seen that there is still relatively high uptake in the kidneys and bladder, less uptake in other organs, and no significant uptake at the tumor location. The tumor uptake of the probe in the blocking group is significantly lower than that in the positive group, showing obvious statistical differences. The above research shows that 68 Ga-DOTA-HTA-DM has good prospects for clinical research.

[0050] Example 6 Tissue distribution in human breast cancer MDA-MB-468 model mice: When the MDA-MB-468 xenograft tumor model nude mice reached 100 - 300 mm in tumor tissue 3 respectively, they were given tail vein injection of 68 Ga-DOTA-HTA-DM (50 μCi / mouse), and blood was collected by dissection at 30, 60, and 120 min after injection. Tumor tissues and tissue organs such as the heart, liver, spleen, lung, stomach, pancreas, small intestine, large intestine, kidney, muscle, and bone were taken from each group of mice, weighed, and their radioactivity (CPM) was measured. The results were converted to ID% / g and referred to Figure 11 .

[0051] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A dimeric bicyclic peptide radionuclide ligand, characterized in that, It has the structure shown in Formula I: 。 2. A method for preparing the dimer bicyclic peptide radionuclide ligand according to claim 1, characterized in that, It includes: (1) By solid-phase synthesis method, the amino acids contained in Formula I are coupled to obtain Compound 1; (2) Compound 1 and Compound 2 are subjected to a cyclization reaction in an ammonium carbonate buffer solution and a mixed solvent of ACN / H2O to obtain Compound 3; (3) Compound 4 is dissolved in DMF, activated in the presence of DCC, HOSu and DIPEA, and then subjected to acid amide condensation with Compound 3; after the reaction is completed, DMF is removed, Liquid E is added for reaction, and then ether is added and dried to obtain Compound 5; Liquid E refers to a mixed solution of 90% TFA + 5% thioanisole + 2.5% phenol + 2.5% 1,2-ethanedithiol; (4) Compound 7 is dissolved in DMF, activated in the presence of DCC, HOSu, DIPEA, and then subjected to acid amide condensation with Compound 6; after the reaction is completed, DMF is removed to obtain Compound 8; (5) Compound 8 is dissolved in DMF, and acid amide condensation is carried out with Compound 9 under the catalysis of EDC·HCl, HOBt and NMM; after the reaction is complete, DMF is removed, the tert-butyl protecting group is removed with a TFA solution, and ether is added and dried to obtain Compound 10; (6) Compound 10 and Compound 5 are dissolved in ACN / H2O, and co-reacted in a phosphate buffer solution to obtain the dimeric bicyclic peptide radionuclide ligand; The structural formula of Compound 1 is as follows: ； Compound 2 is 1,3,5-triacryloyl-1,3,5-triazine; The structural formula of Compound 3 is as follows: ； The structural formula of Compound 4 is as follows: ； The structural formula of Compound 5 is as follows: ； The structural formula of Compound 6 is as follows: ； The structural formula of Compound 7 is as follows: ； The structural formula of Compound 8 is as follows: ； The structural formula of Compound 9 is as follows: ； The structural formula of Compound 10 is as follows: 。 3. A radionuclide probe targeting Nectin-4, characterized in that, It is the dimeric bicyclic peptide radionuclide ligand as claimed in claim 1 radiolabeled with a radionuclide or the dimeric bicyclic peptide radionuclide ligand prepared by the method of claim 2.

4. The radionuclide probe targeting Nectin-4 according to claim 3, wherein The radioactive nuclides include diagnostic radioactive nuclides or therapeutic radioactive nuclides. The diagnostic radioactive nuclides are 68 Ga, 64 Cu, 18 F, 86 Y, 90 Y, 89 Zr, 111 In, 99m Tc, 11 C, 123 I, 125 I and 124 I, and at least one of 177 Lu, 125 I, 131 I, 211 At, 111 In, 153 Sm, 186 Re, 188 Re, 67 Cu, 212 Pb, 225 Ac, 213 Bi, 212 Bi and 212 Pb, and at least one of 5. A method for preparing the radionuclide probe targeting Nectin-4 as claimed in claim 3 or 4, characterized in that, It includes the step of radiolabeling the dimeric bicyclic peptide radionuclide ligand as claimed in claim 1 or the dimeric bicyclic peptide radionuclide ligand prepared by the method of claim 2 with a radionuclide.

6. Use of the radionuclide probe targeting Nectin-4 as claimed in claim 3 or 4 in the preparation of a Nectin-4 imaging agent or a PET imaging probe.

7. Use of the radionuclide probe targeting Nectin-4 as claimed in claim 3 or 4 in the preparation of a reagent or kit for diagnosing, treating and / or preventing a disease; the disease is characterized by overexpression of Nectin-4.

8. A detection reagent or kit, characterized in that, It includes the radionuclide probe targeting Nectin-4 as claimed in claim 3 or 4.

9. A drug, characterized in that, It includes the dimeric bicyclic peptide radionuclide ligand as claimed in claim 1 or the radionuclide probe targeting Nectin-4 as claimed in claim 3 or 4 and a pharmaceutically acceptable excipient.

10. The drug according to claim 9, characterized in that, The dimeric bicyclic peptide radionuclide ligand or the radionuclide probe targeting Nectin-4 is also conjugated with a therapeutic agent for preventing, inhibiting and / or treating a disease characterized by overexpression of Nectin-4.

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