Targeting carrier system labeled by alpha radionuclide as well as preparation method and application of targeting carrier system

Abstract

The invention discloses an alpha radionuclide labeled targeting carrier system as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention provides an alpha radionuclide labeled targeting vector system. The system comprises alpha radionuclide and a targeting vector containing primary amine which are connected through a coupling agent. The invention also provides application of the alpha radionuclide labeled targeting vector system in preparation of drugs for treating tumor diseases. According to the present invention, with the targeting vector capable of specifically binding to the PD-L1 high expression tumor cells, the in-vivo circulation time of the drug can be prolonged so as to efficiently kill the tumor cells;

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to an astatine-211 labeled PD-L1 single-domain antibody, and a preparation method and application thereof. Background Art

[0002] Melanoma is a malignant tumor derived from melanocytes, with high invasiveness and metastatic ability. Although surgical resection at an early stage can significantly improve the survival rate of patients, traditional treatment methods such as chemotherapy and radiotherapy are ineffective in controlling tumor progression in advanced or metastatic melanoma.

[0003] Specifically, chemotherapy and radiotherapy have limitations. Chemotherapy drugs often fail due to drug resistance problems and are highly toxic to healthy cells, resulting in serious side effects. Although radiotherapy can locally kill tumor cells, it also causes damage to surrounding healthy tissues and has poor efficacy in the treatment of hypoxic tumors.

[0004] In view of these deficiencies, targeted alpha-particle therapy (TAT) has attracted extensive attention as an emerging cancer treatment method. TAT precisely delivers radionuclides to tumor sites and synergistically kills tumor cells through direct ionization radiation and indirect ionization effects. However, emerging targeted radionuclide therapy still has deficiencies, such as 225 Ac, 223 Ra has problems such as too long physical half-life, complex decay chain, and unstable chemical binding, increasing the difficulty and uncertainty of treatment. In addition, traditional TAT carriers (such as monoclonal antibodies and polypeptides) also have disadvantages such as insufficient tumor penetration ability or too short in vivo circulation time, limiting their clinical application effects.

[0005] To improve the efficacy of alpha-nuclide therapy, researchers have gradually turned their attention to 211 At with a moderate physical half-life, simple decay chain, and stable chemical binding. 211 The half-life of 211 At is 7.2 hours, which is moderate and more suitable for short-term treatment. It can efficiently kill tumor cells at low doses and reduce side effects on healthy tissues. In addition, due to

[0006] In summary, the existing technical solutions have improved the treatment effect of melanoma to a certain extent, but there are still some deficiencies, such as poor tumor penetration, short residence time of drugs in the blood, and relatively high systemic toxicity during the treatment process. Summary of the Invention

[0007] In view of the problems existing in the above-mentioned prior art, in order to solve the problems of poor tumor penetration, short residence time of drugs in the blood, and relatively high systemic toxicity during the treatment process in the prior art, the object of the present invention is to design and provide a targeted α-radionuclide technical solution based on astatine-211 ( 211 At) and a single-domain antibody modified with an albumin-binding domain. Through the innovative 211 At-AuNPs-ABDMPL16 targeted vector system treatment technical solution, by utilizing 211 the α particles emitted by At, which have the characteristics of high energy and short range, to achieve efficient killing of tumor cells. The ABDMPL16 antibody targets the PD-L1 receptor, ensuring the accurate delivery of the radioisotope to melanoma cells, thereby increasing the drug concentration at the tumor site, enhancing the targeting and drug stability, significantly improving the treatment effect of melanoma, and at the same time reducing the toxic and side effects during the treatment process, providing a more effective and safe new approach for the treatment of melanoma, and being able to significantly improve the survival rate and quality of life of patients.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] On the one hand, the present invention provides a targeted vector system labeled with an α-radionuclide, comprising an α-radionuclide connected by a coupling agent and a targeting vector containing a primary amine.

[0010] In the described α-radionuclide-labeled targeted vector system, the coupling agent is gold nanoparticles (AuNPs). In the described α-radionuclide-labeled targeted vector system, the targeting vector is one of a single-domain antibody containing a primary amine, a monoclonal antibody containing a primary amine, a bispecific antibody containing a primary amine, a polypeptide containing a primary amine, or a nucleic acid modified with a primary amine;

[0011] Preferably, the single-domain antibody containing a primary amine is a PD-L1 single-domain antibody modified with an albumin-binding domain.

[0012] The ABDMPL16 single-domain antibody can ensure that the drug specifically recognizes and binds to melanoma cells with high expression of PD-L1. The small molecule ligand has the characteristics of small volume and strong tumor penetration ability, and can quickly bind to PD-L1 on tumor cells and deliver radionuclides, thus achieving a therapeutic effect similar to that of the ABDMPL16 single-domain antibody. Bispecific antibodies can target two different antigens simultaneously, thereby further improving the specificity and efficacy of targeted therapy. For example, a bispecific antibody against PD-L1 and an anti-tumor antigen can be designed to further enhance the targeting effect on melanoma.

[0013] The described α-radionuclide-labeled targeting vector system, wherein the α-radionuclide is 211 At.

[0014] 211 At exhibits ideal physical half-life and range characteristics in the present invention.

[0015] Second, the present invention provides a preparation method of the described α-radionuclide-labeled targeting vector system, comprising the following steps:

[0016] (1) Weigh a coupling agent and perform a coupling reaction with a targeting vector containing a primary amine. After the reaction, centrifuge and wash to remove the unbound targeting vector, and obtain a targeting vector system;

[0017] (2) Label the targeting vector system obtained in the above step (1) with an α-radionuclide to obtain an α-radionuclide-labeled targeting vector system.

[0018] The described preparation method is characterized in that the labeling method is coordination binding and / or covalent binding. Among them, the radioactive covalent bonding method can introduce appropriate functional groups on the surface of AuNPs, and then covalently bond 211 At to AuNPs through a chemical reaction, further improving the stability and radiochemical yield of the labeled complex.

[0019] Third, the present invention provides the application of any one of the described α-radionuclide-labeled targeting vector systems in the preparation of drugs for treating tumor diseases;

[0020] Preferably, the tumor disease is melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, esophageal cancer, gastric cancer, bladder cancer, cervical cancer or breast cancer.

[0021] Fourth, the present invention provides a pharmaceutical composition comprising any one of the described α-radionuclide-labeled targeting vector systems;

[0022] Preferably, the administration mode of the pharmaceutical composition is intravenous injection.

[0023] It is also possible to 211 combine At-AuNPs-ABDMPL16 with other immune checkpoint inhibitors (such as PD-1 or CTLA-4 antibodies) to enhance the anti-tumor immune response. This combination therapy can act synergistically to significantly improve the effectiveness of treatment.

[0024] For the described pharmaceutical composition, the dosage of the α-radionuclide-labeled targeting vector system is 0.5 MBq - 1 MBq.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. Higher therapeutic effect: By using At with a moderate physical half-life as the α-radionuclide, the present invention can efficiently kill tumor cells in a shorter time. 211 At has the characteristics of high energy, short range, and hypoxia tolerance, making the radioactive damage more concentrated in the tumor site, thereby reducing the damage to surrounding healthy tissues. It can efficiently kill tumor cells under low-dose conditions while minimizing the radiation damage to healthy tissues, thereby improving the treatment effect of melanoma, especially being particularly prominent in the treatment of hypoxic tumors. 211 At has the characteristics of high energy, short range, and hypoxia tolerance, making the radioactive damage more concentrated in the tumor site, thereby reducing the damage to surrounding healthy tissues. It can efficiently kill tumor cells under low-dose conditions while minimizing the radiation damage to healthy tissues, thereby improving the treatment effect of melanoma, especially being particularly prominent in the treatment of hypoxic tumors.

[0027] 2. Enhanced targeting and tumor penetrability: The molecular weight of the single-domain antibody is approximately 15 kDa, which has high affinity and can better penetrate tumor tissues and specifically bind to tumor cells with high expression of PD-L1, but it is quickly cleared in the blood. In the present invention, the single-domain antibody is modified with albumin, and the single-domain antibody modified with an albumin-binding domain (ABDMPL16) is used as the 211 carrier of At, which prolongs the action time in the blood, can increase the uptake in the tumor, thereby increasing the accumulation of the drug at the tumor site, has long retention, improves the precision and efficacy of treatment, and reduces systemic toxicity, effectively solving the problem of insufficient ability of traditional monoclonal antibodies to penetrate solid tumors. It can also further increase the drug concentration and treatment effect at the tumor site by binding with gold nanoparticles.

[0028] 3. Enhanced drug stability and in vivo retention time: By combining 211 At with gold nanoparticles (AuNPs), the present invention forms a stable radioactive nanocomplex. Compared with the existing technical solutions, the application of AuNPs not only improves the 211 stability and targeting of At, but also prolongs the circulation time of the drug in the body, ensuring that 211 the radioactivity of At can fully play its role, further enhancing its cumulative effect at the tumor site. This improvement significantly reduces the systemic toxicity during the treatment process and improves the tolerance of patients. Ensure 211The efficient delivery and targeted release of At optimize the utilization rate of its half-life and achieve more precise targeted therapy. This construction method is rapid, efficient, and mild, not only optimizing 211 the utilization rate of the half-life of At, but also enabling precise targeted therapy.

[0029] 4. Enhanced immunomodulatory effect: The 211 At-AuNPs-ABDMPL16 of the present invention not only exhibits excellent effects in directly killing tumor cells, but also enhances the anti-tumor immune effect by regulating the immune response in the tumor microenvironment. The experimental results show that 211 At-AuNPs-ABDMPL16 significantly increases the proportion of CD8 + T cells in tumors and reduces the proportion of Tregs cells, further promoting the anti-tumor immune response, which cannot be achieved by traditional treatment methods.

[0030] 5. Good biosafety: In the long-term toxicity assessment of mice, the 211 At-AuNPs-ABDMPL16 of the present invention does not show significant systemic toxicity, and the main organs (such as the heart, liver, spleen, lungs, and kidneys) show no obvious damage after treatment. This result indicates that the present invention not only has significant advantages in treatment effects, but also has good safety and the potential for further clinical translation. Description of the Drawings

[0031] Figure 1 shows the binding and internalization ability of ABDMPL16. Among them, (a) analyzes the purity of ABDMPL16 by sodium dodecyl sulfate polyacrylamide gel electrophoresis; (b) the surface plasmon resonance binding curve of ABDMPL16 binding to PD-L1 at different concentrations; (c) flow cytometry analyzes the binding of AF488-ABDMPL16 to PD-L1 at low and high concentrations; (d) representative images and quantitative analysis of the binding ability of ABDMPL16 (n = 3); (e) transmission electron microscope image of AuNP-ABDMPL16; (f) the internalization ability of AuNP-ABDMPL16 is evaluated by a gold enhancement experiment. The statistical significance between groups is calculated using the student t-test, **p < 0.01;

[0032] Figure 2 is for the in vitro targeted α therapy using 211 At]-AuNP-ABDMPL16. Among them, (a) 211 preparation of 211 At]-AuNP-ABDMPL16; (b) 211Radioactive labeling stability of At]-AuNP-ABDMPL16 co-incubated in FBS or PBS over time; (d) Relative survival rate of B16F10 cells after treatment with different doses of 211 At for 24 hours; (e) Relative survival rate of B16F10 cells after treatment with different doses of 211 At]-AuNP-ABDMPL16 for 24 hours; (f) Determination of 211 At, 211 At]-AuNP-ABDMPL16 and binding after blocking of ABDMPL16; (g) Representative immunofluorescence micrographs of γ-H2AX expression in B16F10 cells after different treatments; (h) Representative immunofluorescence photographs of Fas expression in B16F10 cells after different treatments. Data are presented as mean ± standard deviation (n = 3), and G1-G4 represent the control group, AuNP-ABDMPL16, 211 At and 211 At]-AuNP-ABDMPL16 groups;

[0033] Figure 3 For 211 At]-AuNP-ABDMPL16 pharmacokinetics and biodistribution, where (a, b) 211 At]-AuNP-ABDMPL16 pharmacokinetics; (c) 211 At]-AuNP-ABDMPL16 biodistribution in healthy C57BL / 6 mice for 3 hours; (d) 211 At]-AuNP-ABDMPL16 biodistribution in healthy C57BL / 6 mice for 7 hours; (e) 211 At]-AuNP-ABDMPL16 biodistribution in healthy C57BL / 6 mice for 24 hours; (f) 211 At]-AuNP-ABDMPL16 biodistribution in melanoma for 7 hours; (g) 211 At]-AuNP-ABDMPL16 biodistribution in melanoma for 24 hours;

[0034] Figure 4In vivo anti-tumor evaluation of targeted α therapy in the B16F10 tumor model, where (a) average tumor growth curves of B16F10 tumor-bearing mice from different treatment groups (n = 5); (b) changes in mouse body weight over time after different treatments (n = 5); (c) Kaplan-Meier survival curves of mice receiving different treatments; (d) representative pictures of tumors; (e) representative H&E histopathology of tumors; (f) TUNEL staining of tumors; (g) representative H&E staining of major organs of mice in each group;

[0035] Figure 5 For in vivo anti-tumor immune response, where (a) representative flow cytometry plots of CTL cells (CD45 + CD3 + CD8 + ); (b) relative quantification of CTL cells (CD45 + CD3 + CD8 + ); (c) representative flow cytometry plots of regulatory T cells (CD45 + CD3 + CD4 + Foxp3 + ) in B16F10 tumors; (d) relative quantification of regulatory T cells (CD45 + CD3 + CD4 + Foxp3 + ) in B16F10 tumors; (e) representative flow cytometry plots of DC cells (CD45 + CD11c + CD80 + ) in B16F10 tumors; (f) relative quantification of DC cells (CD45 + CD11c + CD80 + ) in B16F10 tumors; (g) representative flow cytometry plots of DC cells (CD45 + CD11c + CD86 + ) in B16F10 tumors; (h) relative quantification of DC cells (CD45 + CD11c + CD86 + ) in B16F10 tumors, data are presented as mean ± standard deviation (n = 3), *p < 0.05, **p < 0.01;

[0036] Figure 6For the long-term in vivo safety of targeted α therapy, where (a) the body weights of healthy mice over time after different treatments (n = 5); (b) white blood cell (WBC) parameters; (c) red blood cell (RBC) parameters; (d) mean corpuscular volume (MCV) parameters; (e) mean corpuscular hemoglobin (MCH) parameters; (f) mean corpuscular hemoglobin concentration (MCHC) parameters; (g) red blood cell distribution width (RDW) parameters; (h) platelet (PLT) parameters; (i) mean platelet volume (MPV); (j) hemoglobin (HGB) parameters; (k) plateletcrit (PCT) parameters. Detailed implementation mode

[0037] The present invention will be further described below in conjunction with the drawings and embodiments.

[0038] Example 1:

[0039] The preparation method of the drug 211 of [At]-AuNP-ABDMPL16 includes the following steps:

[0040] (1) Synthesize the AuNP-ABDMPL16 complex: Dilute the ABDMPL16 single-domain antibody using the reconstitution buffer provided in the kit, and mix the diluted protein sample with the reaction buffer. Take 90 μL of the mixture and transfer it to the vial of freeze-dried NHS-activated gold nanoparticles, pipette and mix well repeatedly, and incubate at room temperature for at least 2 hours. Add 10 μL of the quenching solution to terminate the reaction, incubate for 5 minutes, and then use an Amicon ultrafiltration tube to centrifuge at 14,000 g for 30 minutes. After centrifugation, add 0.1 M PBS and repeat the centrifugation step to finally obtain the AuNP-ABDMPL16 conjugate.

[0041] (2) 211 At radioactive labeling: Mix the AuNPs-ABDMPL16 complex with 211 the At solution, and shake and react at room temperature for 5 minutes. Detect the radiochemical yield of the labeled product by thin-layer chromatography to ensure that it is higher than 90%. The labeled product is purified by centrifugation and resuspended in PBS solution for subsequent experiments and applications.

[0042] Stability test of the drug: Incubate the labeled 211 [At]-AuNPs-ABDMPL16 with FBS or PBS, and detect it after 0.5 hours, 3 hours, and 19 hours respectively to evaluate its stability under physiological conditions.

[0043] The molecular weight of ABDMPL16 is about 21 kDa ( Figure 1a). The binding kinetics of ABDMPL16 to PD-L1 was determined using surface plasmon resonance (SPR) technology. As the concentration of ABDMPL16 increased (from 0.78125 nM to 50 nM), the binding response increased in a concentration-dependent manner. The calculated dissociation constant (Kd) was 2.582 nM, indicating that ABDMPL16 has a high affinity for PD-L1 and can specifically bind to cells with high PD-L1 expression ( Figure 1 b). The experimental results showed that, under both high and low concentration conditions, the fluorescence intensity of AF488-ABDMPL16 was significantly increased compared to the control group, further confirming that ABDMPL16 can bind to cells with high PD-L1 expression in a concentration-dependent manner. Figure 1 c). In addition, immunofluorescence staining further verified the binding ability of ABDMPL16. Figure 1 d). Cells treated with AF488-ABDMPL16 showed strong PD-L1 signals in the images. Quantitative analysis of the positive regions showed that the PD-L1 staining signals in the AF488-ABDMPL16 treatment group were significantly enhanced compared to the control group (p < 0.01), further confirming the specific targeting effect of ABDMPL16 on PD-L1.

[0044] TEM imaging showed that the particles of AuNP-ABDMPL16 were evenly distributed, with an average diameter of approximately 10 nm. Figure 1 e), further confirming the successful conjugation of ABDMPL16 with AuNP.

[0045] The cellular internalization ability of AuNP-ABDMPL16 was further evaluated using a gold enhancement experiment. Figure 1 f). The results showed that in B16F10 cells, as the concentration of AuNP-ABDMPL16 increased, the accumulation of intracellular nanoparticles increased in a concentration-dependent manner. However, after ABDMPL16 blocked PD-L1, the uptake of AuNP-ABDMPL16 was significantly reduced. This result indicates that the internalization of nanoparticles is through a specific effect mediated by PD-L1.

[0046] 211 The synthesis schematic diagram of Figure 2 At]-AuNP-ABDMPL16 is shown in 211 a. The successful synthesis of Figure 2 At]-AuNP-ABDMPL16 was confirmed by experiment, and its radiochemical labeling rate exceeded 90%. 211 b). In an FBS or PBS environment, the radiolabeled Figure 2 At]-AuNP-ABDMPL16 remained highly stable after multiple half-lives. ​

[0047] Example 2: In vitro cytotoxicity assay

[0048] B16F10 cells were seeded in 96-well plates (3 replicates, 1×10 4 cells / well), and after adherent growth for 24 hours, they were co-cultured with RPMI 1640 medium containing 211At and [211At]-AuNP-ABDMPL16 (0, 0.125, 0.25, 0.5 MBq) for 24 hours. CCK-8 reagent (10 μL) was added for color development for 1 - 2 hours, and the absorbance (OD) at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the OD percentage of each group relative to the control group was calculated.

[0049] The in vitro cytotoxicity assay showed that 211 At could reduce cell viability (the cell survival rate was 53.82 ± 4.29% at 0.5 Mbq, Figure 2 d), while 211 treatment with [211At]-AuNP-ABDMPL16 could lead to a significant decrease in the viability of B16F10 cells, and the decrease was dose-dependent (the cell survival rate decreased to 29.98 ± 7.19% at 0.5 Mbq, Figure 2 e). In contrast, the effect of free 211 At was weaker, further confirming the enhanced effect of targeted alpha therapy in inhibiting tumor activity.

[0050] Example 3: Cellular uptake assay

[0051] B16F10 cells were seeded in 12-well plates (1×10 5 cells / mL / well), and after culturing for 24 hours, they were co-incubated with 211At, [211At]-AuNP-ABDMPL16, and AuNP-ABDMPL16 (blocking group) for 0.5, 1, and 4 hours respectively. After washing with PBS, the cells were lysed with 1 M NaOH, and the radioactivity was measured with a gamma counter.

[0052] The time-dependent cellular uptake assay showed that compared with free 211 At, 211 the uptake of [211At]-AuNP-ABDMPL16 was significantly higher at 1 hour. The blocking experiment showed a decrease in the uptake amount ( Figure 2 f), further confirming receptor-mediated endocytosis.

[0053] Example 4: γ-H2AX and FAS expression assay

[0054] B16F10 cells were seeded in a glass-bottomed culture dish. After 24 hours of adherence, they were co-cultured with RPMI 1640 medium containing [211At]-AuNP-ABDMPL16 (0, 0.01, 0.1, 0.5, 1 MBq / mL) for 24 hours. They were fixed with 4% paraformaldehyde for 10 minutes, blocked with a permeabilization solution and a rapid blocking solution at room temperature for 10 minutes. Anti-γ-H2AX antibody was added and incubated overnight at 4°C. The fluorescent secondary antibody was incubated for 1 hour in the dark. The cell nuclei were stained with Hoechst 33342, and photographs were taken with a fluorescence microscope. The FAS detection method was similar.

[0055] The results of immunofluorescence staining showed that 211 the expression of γ-H2AX increased significantly after treatment with [211At]-AuNP-ABDMPL16, indicating aggravated DNA damage ( Figure 2 g). In addition, 211 [211At] and 211 after irradiation with [211At]-AuNP-ABDMPL16, the expression level of Fas increased, while the Fas level in the AuNP-ABDMPL16 treatment group showed no obvious change ( Figure 2 h), indicating that targeted α therapy could effectively induce apoptosis of tumor cells.

[0056] Example 5: Pharmacokinetics and tissue distribution experiments

[0057] Normal C57BL / 6 mice were injected via the tail vein with 211 [211At]-AuNP-ABDMPL16 (200 KBq, n = 3 / group). At 10, 30, 60, 210, 390, 510, 1050, and 1740 minutes, 10 μL of tail vein blood was collected, and the radioactivity (MBq / L) was measured with a γ counter. The Phoenix 8.1 software was used to fit a non-linear regression curve to calculate the pharmacokinetic parameters. Another injection of 211 [211At]-AuNP-ABDMPL16 (200 KBq, n = 4 / group) was performed for biodistribution studies. At 3, 7, and 24 hours, the tissues were weighed, and the radioactive uptake rate per gram of tissue (%ID / g) was calculated. 1 × 10 6 B16F10 cells were subcutaneously inoculated into C57BL / 6 mice. When the tumor size reached approximately 100 mm 3 , 211 [211At]-AuNP-ABDMPL16 (100 Kbq) was injected via the tail vein. The mice were euthanized at 7 hours and 24 hours after injection for collecting tumors and organs. The tissues were collected, weighed, and their radioactive levels were measured.

[0058] 211 The curve of the blood drug concentration of [211At]-AuNP-ABDMPL16 changing with time indicated that it conformed to a biphasic clearance pattern ( Figure 3 ​a). Pharmacokinetic analysis showed that its alpha-phase half-life (Alpha_HL) was 6.34 minutes, beta-phase half-life (Beta_HL) was 694 minutes, and the total area under the curve (AUC) was 3.76×10 9 Bq·min / L, indicating that the radioactive tracer had a high exposure level in the body.

[0059] The biodistribution results in normal mice showed that at 3 h, there was moderate uptake in the liver, spleen, and lungs; after 7 h, the liver uptake increased, suggesting that clearance mainly occurred through liver metabolism; after 24 h, radioactivity still mainly remained in the liver and spleen ( Figure 3 c-3e).

[0060] In tumor model mice, at 7 h 211 the uptake of [211At]-AuNP-ABDMPL16 in tumor tissues was the highest (20.64±4.71% ID / g), significantly higher than that in most normal tissues; after 24 h, the uptake in tumor tissues decreased slightly ( Figure 3 f, 3g). These results indicated that 211 [211At]-AuNP-ABDMPL16 could effectively target and accumulate in tumor tissues, and was mainly cleared through the liver and spleen.

[0061] Example 6: In vivo anti-tumor efficacy experiment

[0062] 1×106 B16F10 cells were subcutaneously inoculated into the right back of C57BL / 6 mice, and the tumor volume was measured with a vernier caliper (formula: volume = length × width2 / 2). When the tumor reached approximately 100 mm 3 , the mice were randomly divided into 4 groups: G1 (control group), G2 (AuNP-ABDMPL16 group), G3 ( 211 [211At] group), G4 ([[211At]-AuNP-ABDMPL16 group), and the therapeutic dose was injected via the tail vein (n = 5 / group). The body weight and tumor volume were measured every 2 days, and the experiment was terminated when the tumor volume > 1000 mm 3 or the body weight loss > 20%.

[0063] HE staining: Paraffin sections were dewaxed and hydrated, stained with hematoxylin for 5 - 10 minutes, differentiated with hydrochloric acid ethanol, stained with eosin for 1 - 2 minutes, dehydrated and cleared, and then sealed for observation under an optical microscope.

[0064] TUNEL staining: Antigens were repaired by microwave in EDTA buffer (pH 8.0), blocked with BSA for 30 minutes, reacted with TUNEL reaction solution overnight at 4 °C, incubated with HRP-labeled secondary antibody at 37 °C for 50 minutes, counterstained with DAPI, and observed under a fluorescence microscope after anti-quenching sealing.

[0065] The results were as Figure 4showed that, compared with the control group, 211 At]-AuNP-ABDMPL16 was able to significantly inhibit tumor growth ( Figure 4 a). As the tumors grew in the control group mice, their body weights increased slightly, while 211 in the At group and 211 the At]-AuNP-ABDMPL16 group, the mice's body weights remained relatively stable ( Figure 4 b). In addition, the survival time of the mice in this treatment group was significantly prolonged ( Figure 4 c). On the 10th day of treatment, 211 the tumor volume of the mice in the At]-AuNP-ABDMPL16 group increased slowly, while 211 the tumors in the At group continued to grow, further demonstrating the effectiveness of the combination therapy (see Figure 4 d).

[0066] The HE staining results showed that the tumor cells in the control group were dense and there were a large number of mitotic structures, while 211 in the At]-AuNP-ABDMPL16-treated group, the tumor cells were significantly reduced and accompanied by an enlarged necrotic area ( Figure 4 e). TUNEL staining further confirmed that this treatment could induce apoptosis of tumor cells ( Figure 4 f).

[0067] Example 7: In vivo anti-tumor immune response experiment

[0068] Ten days after the end of the treatment, the tumor tissues were taken, minced, passed through a 200-mesh cell sieve, and the cell suspension was collected by centrifugation. CTL and Treg were labeled with APC anti-mouse CD45, FITC anti-mouse CD3, BV421 anti-mouse CD4, APC / Cy7 anti-mouse CD8a, and PE anti-mouse FOXP3 antibodies respectively; DC was labeled with FITC anti-mouse CD11c, PE anti-mouse CD86, APC anti-mouse CD80, and PE / Cy7 anti-mouse CD45 antibodies. Incubate in the dark at 4 °C for 40 minutes and analyze by flow cytometry.

[0069] The results showed no differences in CD45 211 CD3 211 CD8 + T cells, regulatory T cells (Tregs), and dendritic cells (DCs) expressing co-stimulatory molecules CD80 and CD86 among the control group, free + CD8 + T cells, regulatory T cells (Tregs), and dendritic cells (DCs) expressing co-stimulatory molecules CD80 and CD86 among the control group, free Figure 5 a showed that, compared with the control group, 211 in the At]-AuNP-ABDMPL16 treatment group, CD3 + CD8+ The proportion of T cells increased significantly. Statistical analysis showed that 211 After treatment with At]-AuNP-ABDMPL16, this proportion increased from 29.95 ± 4.34% to 46.69 ± 1.19% (p < 0.01)( Figure 5 b). This finding indicates that the treatment may have promoted the activation or recruitment of T cells. In the analysis of regulatory T cells, 211 In the At]-AuNP-ABDMPL16 treatment group, the proportion of Foxp3+ cells decreased significantly, from 3.16 ± 1.43% in the control group to 0.7 ± 0.18% (p < 0.05)( Figure 5 c, 5d). This may reflect the attenuation of the immunosuppressive environment in the TME, thereby enhancing the anti-tumor immune response.

[0070] The expression of co-stimulatory molecules CD80 and CD86 on dendritic cells was further analyzed ( Figure 5 e and 5g). The results showed that in 211 the At]-AuNP-ABDMPL16 treatment group, the proportion of CD11c + CD80 + DCs increased significantly, reaching 19.97 ± 2.33% (p < 0.05)( Figure 5 f), while the proportion of CD11c + CD86 + DCs rose to 18.05 ± 1.69% (p < 0.01)( Figure 5 h). These data indicate that the treatment may have enhanced the antigen-presenting ability of dendritic cells, thus promoting the T cell-mediated immune response. In summary, these results indicate 211 the regulatory effect of At]-AuNP-ABDMPL16 on immune cells in the tumor microenvironment, highlighting its promising strategy as radioimmunotherapy.

[0071] Example 8: Long-term in vivo safety of targeted α therapy

[0072] The pathological changes of the heart, liver, spleen, lung, and kidney of mice treated with At]-AuNP-ABDMPL16 were observed by H&E staining. In addition, normal mice were injected with At]-AuNP-ABDMPL16 (1 MBq, n = 5 / group) via the tail vein. After continuously observing the vital signs for 63 days, the mice were sacrificed for blood routine detection. 211 At]-AuNP-ABDMPL16(1MBq, n = 5 / group) via the tail vein of normal mice, and the vital signs were continuously observed for 63 days before sacrificing for blood routine detection. 211 At]-AuNP-ABDMPL16(1MBq, n = 5 / group), and the mice were sacrificed for blood routine detection after continuously observing the vital signs for 63 days.

[0073] The long-term safety study showed that there was no significant difference in the body weight of mice during the treatment process ( Figure 6a). Hematological index analysis showed that there were no significant changes in various blood parameters of the treatment group and the control group, further proving 211 At]-AuNP-ABDMPL16 had good biosafety ( Figure 6 b-6k).

Claims

1. An α-radionuclide-labeled targeting vector system, characterized in that, Comprising an α-radionuclide linked by a coupling agent and a targeting vector containing a primary amine.

2. The α-radionuclide-labeled targeting vector system according to claim 1, characterized in that, The coupling agent is a gold nanoparticle.

3. The α-radionuclide-labeled targeting vector system according to claim 1, characterized in that, The targeting vector is one of a single-domain antibody containing a primary amine, a monoclonal antibody containing a primary amine, a bispecific antibody containing a primary amine, a polypeptide containing a primary amine, or a nucleic acid modified with a primary amine; Preferably, the single-domain antibody containing a primary amine is a PD-L1 single-domain antibody modified with an albumin-binding domain.

4. The α-radionuclide-labeled targeting vector system according to claim 1, wherein, The α-radionuclide is 211 At.

5. The preparation method of a targeted vector system labeled with an α-radionuclide according to claim 1, characterized in that, Comprising the following steps: (1) Weigh a coupling agent and a targeting vector containing a primary amine for a coupling reaction. After the reaction, centrifuge and wash to remove the unbound targeting vector, and obtain a targeting vector system; (2) Label the targeting vector system obtained in the above step (1) with an α-radionuclide to obtain an α-radionuclide-labeled targeting vector system.

6. The preparation method according to claim 5, characterized in that, The labeling method is coordination binding and / or covalent binding.

7. Use of an α-radionuclide-labeled targeting vector system according to any one of claims 1-4 in the preparation of a medicament for treating tumor diseases.

8. The application according to claim 7, characterized in that, The tumor diseases are melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, esophageal cancer, gastric cancer, bladder cancer, cervical cancer, or breast cancer.

9. A pharmaceutical composition, characterized in that, Comprising an α-radionuclide-labeled targeting vector system according to any one of claims 1-4; Preferably, the administration method of the pharmaceutical composition is intravenous injection.

10. A pharmaceutical composition according to claim 9, wherein, The dosage of the α-radionuclide-labeled targeting vector system is 0.5 MBq - 1 MBq.

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