PD-L1 monoclonal antibody delivery system as well as preparation method and application thereof

Abstract

The invention discloses a PD-L1 monoclonal antibody delivery system as well as a preparation method and application thereof, and belongs to the technical field of medicines. The preparation method comprises the following steps: coupling amino groups of BPA-PEG9 and a PD-L1 monoclonal antibody through NHS (N-hydroxysuccinimide) esterification to form BPA6-aPDL1; the preparation method comprises the following steps: grafting a glucose derivative maltomimetic acid MA of o-benzene dihydroxyl to a lipid-like amphiphilic molecule PEG (Polyethylene Glycol) (1.5 k-DSPE) through an amido bond to form MA4-PEG (1.5 k-DSPE), namely MA4-PD; then, the BPA6-aPDL1 reacts with the MA4-PD through a boric acid ester bond, and finally, the delivery system MB-aPDL1 is formed. The delivery system not only can successfully deliver the PD-L1 to a tumor site across a blood brain barrier, but also can accurately release the PD-L1 monoclonal antibody, so that the PD-L1 monoclonal antibody is only enriched in a tumor region, the residence time of the antibody is prolonged, the tumor anchoring capability of the antibody is improved, and the occurrence of immune-related adverse events is remarkably reduced.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a PD-L1 monoclonal antibody delivery system, its preparation method, and its application. Background Technology

[0002] PD-L1 monoclonal antibody (Programmed Death-Ligand 1 monoclonal antibody) is an immunotherapy drug used to treat malignant tumors. PD-L1 is an immune checkpoint protein on the surface of tumor cells. After binding to the PD-1 protein on the surface of T cells, it can inhibit T cell activation, thereby preventing them from evading the immune system's attack. PD-L1 monoclonal antibody blocks this signaling pathway by inhibiting the binding of PD-L1 to the PD-1 ligand, thereby restoring the T cell's ability to fight tumors.

[0003] The main indications for PD-L1 monoclonal antibodies include various solid tumors, such as non-small cell lung cancer, melanoma, and squamous cell carcinoma of the head and neck. Clinical studies have shown that PD-L1 monoclonal antibodies have achieved certain efficacy in the treatment of some malignant tumors, especially in patients with tumors that have high PD-L1 expression levels, where the efficacy is more significant.

[0004] However, as an immunotherapy approach, PD-L1 monoclonal antibodies have some limitations. One significant limitation is their difficulty in crossing the blood-brain barrier (BBB). The BBB is a barrier formed by the endothelial cells of blood vessels in the brain and spinal cord, restricting the entry of many substances (including drugs) from the blood into the cerebrospinal fluid and brain tissue. Due to their large molecular structure, PD-L1 monoclonal antibodies have difficulty crossing the BBB to reach intracranial tumor lesions, thus limiting their effectiveness in treating brain tumors, including brain metastases.

[0005] To address this limitation, researchers and drug development companies have developed various PD-L1 monoclonal antibody delivery systems to improve their effectiveness in treating brain tumors. These delivery systems mainly include nanomedicine delivery systems, delivery via cerebrospinal fluid or local injection, and delivery methods relying on local brain puncture or surgery. However, current PD-L1 monoclonal antibody delivery systems still have some drawbacks. Existing delivery systems can increase the concentration of the drug at the tumor site in the brain, but the delivery efficiency is insufficient, the stability of the delivery system is poor, and problems such as the toxic side effects caused by prolonged antibody blood circulation and adverse immune reactions have not been solved. Summary of the Invention

[0006] This invention first couples BPA-PEG9 with the amino group of a PD-L1 monoclonal antibody via NHS esterification to form BPA6-aPDL1; then, it grafts the o-phenylenediol glucose derivative maltodextrin (MA) onto the lipid-like amphiphilic molecule PEG via an amide bond.1.5k -DSPE, forming MA4-PEG 1.5k -DSPE, i.e. MA4-PD; finally, BPA6-aPDL1 reacts with MA4-PD through borate ester bonds to finally form the delivery system MB-aPDL1.

[0007] The technical solution of the present invention is as follows:

[0008] This invention provides a method for preparing a PD-L1 monoclonal antibody delivery system, comprising the following steps:

[0009] (1) Synthesis of MA4-PD

[0010] NH2-PEG 1.5k -DSPE, Fmoc-lys-(Fmoc)-OH, DIC, and 6-Cl-HOBT were dissolved in DMF and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain (Fmoc)2-K-PEG. 1.5k -DSPE; Fmoc was removed using an Fmoc deprotection solution to obtain K-PEG. 1.5k -DSPE, i.e. K-PD;

[0011] K-PD, Fmoc-lys-(Fmoc)-OH, DIC, and 6-Cl-HOBT were dissolved in DMF and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain (Fmoc)2-K-PEG. 1.5k -DSPE; remove Fmoc with Fmoc deprotection solution to obtain K2-PEG. 1.5k -DSPE, which stands for K2-PD;

[0012] K2-PD, MA, NHS, and EDC.HCl were dissolved in DMF and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain MA4-PEG. 1.5k -DSPE, which stands for MA4-PD;

[0013] (2) Synthesis of BPA-PEG9-NHS

[0014] NH2-PEG9-COOtbu, BPA, NHS, and EDC.HCl were dissolved in DMF and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain BPA-PEG9-COOtbu. BPA-PEG9-COOtbu was dissolved in a TFA / DMF mixed solution and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain BPA-PEG9-COOH. Subsequently, BPA-PEG9-COOH, NHS, and EDC.HCl were dissolved in DMF and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain BPA-PEG9-NHS.

[0015] (3) Synthesis of BPA6-aPDL1

[0016] BPA-PEG9-NHS and aPDL1 were placed in PBS solution and stirred at low temperature. After the reaction was completed, the mixture was dialyzed to obtain BPA6-aPDL1.

[0017] (4) Synthesis of MB-aPDL1

[0018] MA4-PD and BPA6-aPDL1 were placed in PBS solution and reacted under low temperature with stirring. After the reaction was completed, the mixture was dialyzed to obtain MB-aPDL1.

[0019] In the above method for preparing the PD-L1 monoclonal antibody delivery system, in step (1), NH2-PEG 1.5k The molar ratio of -DSPE, Fmoc-lys-(Fmoc)-OH, DIC and 6-Cl-HOBT is selected from 1:2:2:2 to 1:6:6:6, preferably 1:4:4:4; the molar ratio of K-PD, Fmoc-lys-(Fmoc)-OH, DIC and 6-Cl-HOBT is selected from 1:2:2:2 to 1:6:6:6, preferably 1:4:4:4; the molar ratio of K2-PD, MA, NHS and EDC.HCl is selected from 1:4:4:4 to 1:12:12:12, preferably 1:8:8:8.

[0020] In the above-mentioned method for preparing the PD-L1 monoclonal antibody delivery system, in step (2), the molar ratio of NH2-PEG9-COOtbu, BPA, NHS and EDC.HCl is selected from 1:2:2:2 to 1:6:6:6, preferably 1:4:4:4.

[0021] In the above method for preparing the PD-L1 monoclonal antibody delivery system, in step (3), the molar ratio of BPA-PEG9-NHS to aPDL1 is selected from 1:1 to 10:1, preferably 6:1.

[0022] In the above method for preparing the PD-L1 monoclonal antibody delivery system, in step (4), the molar ratio of MA4-PD to BPA6-aPDL1 is selected from 2:1 to 20:1, preferably 4:1.

[0023] In the above-mentioned method for preparing the PD-L1 monoclonal antibody delivery system, the low temperature in steps (3) and (4) is selected from 1 to 16°C; preferably 4°C.

[0024] In the above-mentioned method for preparing the PD-L1 monoclonal antibody delivery system, the pH value of the PBS solution in steps (3) and (4) is selected from 7 to 8; preferably 7.4.

[0025] In the above preparation method of the PD-L1 monoclonal antibody delivery system, the reaction product is washed with diethyl ether.

[0026] In the above-mentioned method for preparing the PD-L1 monoclonal antibody delivery system, the Fmoc deprotection solution is selected from the preparation of 20% piperidine dissolved in DMF.

[0027] In the above-mentioned method for preparing the PD-L1 monoclonal antibody delivery system, the reaction time of each stirring reaction is preferably 6 to 12 hours, based on the sufficient reaction of the substrate.

[0028] The present invention provides a PD-L1 monoclonal antibody delivery system prepared by the above method.

[0029] This invention provides the application of the above-described PD-L1 monoclonal antibody delivery system in the preparation of drugs for treating brain metastases.

[0030] The beneficial effects of this invention are as follows:

[0031] As a large molecule drug, PD-L1 monoclonal antibody delivery across the blood-brain barrier is extremely challenging. The delivery system MB-aPDL1 developed in this invention not only successfully delivers PD-L1 across the blood-brain barrier to the tumor site but also precisely releases the PD-L1 monoclonal antibody, causing it to accumulate only in the tumor region and prolonging the antibody's retention time, thus increasing its ability to anchor to the tumor. Furthermore, the MB-aPDL1 delivery system avoids leakage during long-term blood circulation, thereby extending the blood circulation time. Simultaneously, MB-aPDL1 flowing into other organs maintains a compact structure and does not trigger an immune response in normal tissues. In summary, the MB-aPDL1 delivery system not only improves the therapeutic effect of brain metastases but also significantly reduces irreversible adverse events (irAEs), demonstrating significant application value. Attached Figure Description

[0032] Figure 1 For MA4-PD 1 H-NMR spectrum;

[0033] Figure 2MALDI-TOF mass spectrometry of MA4-PD;

[0034] Figure 3 For BPA-PEG9-NHS 1 H-NMR spectrum;

[0035] Figure 4 MALDI-TOF mass spectrometry of BPA-PEG9-NHS;

[0036] Figure 5 Transmission electron microscopy (TEM) image of MB-aPDL1 and the quantification results under TEM;

[0037] Figure 6 This is a graph showing the ability of MB-aPDL1 to bind to PDL1 on the cell surface before and after release;

[0038] Figure 7 Figure showing the effect of different concentrations of MB-aPDL1 on cell viability;

[0039] Figure 8 The figure shows the statistical results of the delivery efficiency of the MB-aPDL1 in vitro BBB model.

[0040] Figure 9 The fluorescence and quantification of tumors in Lewis-GFP-Luc brain-bearing mice under different treatment groups;

[0041] Figure 10 Fluorescence signal images and quantification results of MB-PDL1 treatment of tumors at different time points;

[0042] Figure 11 Image showing HE staining and quantification results of brain tissue after MB-PDL1 treatment;

[0043] Figure 12 Survival statistics for a mouse model of NSCLC brain metastases treated with MB-PDL1;

[0044] Figure 13 MB-PDL1 activates tumor CD8 + • T cell count results;

[0045] Figure 14 The pharmacokinetic diagram of MB-PDL1 in blood circulation;

[0046] Figure 15 The results of the antibody anchoring ability of MB-aPDL1 are shown in the figure.

[0047] Figure 16 Liver function was measured in mice after MB-PDL1 treatment.

[0048] Figure 17The results show the number of CD8+ T cells in the liver of mice after MB-PDL1 treatment. Detailed Implementation

[0049] The materials used in this invention are as follows:

[0050] Phospholipid-polyethylene glycol-amino (NH2-PEG) 1.5k -DSPE), Xi'an Ruixi Company; N,N'-bisfluorenemethoxycarbonyl-L-lysine [Fmoc-lys-(Fmoc)-OH], N,N'-diisopropylcarbodiimide (DIC), Maclean's; 6-chloro-1-hydroxybenzotriazole (6-Cl-HOBT), N,N-dimethylformamide (DMF), maltobionic acid (MA), bis-N-hydroxysuccinimide (NHS), NH2-PEG9-COOtbu, 2-carboxypyridine-5-boronic acid (BPA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl), N,N-dimethylformamide (DMF), trifluoroacetic acid (TFA), ethyl-3-(3-dimethylammoniumpropyl)carboammonium (EDC), Aladdin; PD-L1 monoclonal antibody (aPDL1), Bio X Cell.

[0051] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0052] Example 1

[0053] The PD-L1 monoclonal antibody delivery system is prepared as follows:

[0054] (1) Synthesis of MA4-PD

[0055] NH2-PEG 1.5k DSPE (2000 mg), Fmoc-lys-(Fmoc)-OH (1523.8 mg), N,N'-diisopropylcarbodiimide [(DIC) 399.6 μL] and 6-chloro-1-hydroxybenzotriazole [(6-Cl-HOBT) 437.4 mg] were dissolved in 20 mL of N,N-dimethylformamide (DMF) and stirred at room temperature for 12 h. The reaction endpoint was determined by Kaiser assay. The resulting mixture was then precipitated in diethyl ether, washed with diethyl ether, and dried under vacuum to give the product (Fmoc)2-K-PEG. 1.5k-DSPE. Fmoc was removed using Fmoc deprotection solution (20% piperidine dissolved in DMF, 5 mL) to obtain K-PEG. 1.5k -DSPE (abbreviated as K-PD) was washed three times with ether.

[0056] K-PD (2000 mg), Fmoc-lys-(Fmoc)-OH (2285.7 mg), N,N'-diisopropylcarbodiimide [(DIC) 599.4 μL] and 6-chloro-1-hydroxybenzotriazole [(6-Cl-HOBT) 656.1 mg] were dissolved in 20 mL of N,N-dimethylformamide (DMF) and stirred at room temperature for 12 h. The reaction endpoint was determined by Kaiser assay. The resulting mixture was then precipitated in diethyl ether, washed with diethyl ether, and dried under vacuum to obtain the product (Fmoc)4-K2-PEG. 1.5k -DSPE. Fmoc was removed using Fmoc deprotection solution (20% piperidine dissolved in DMF, 5 mL). The reaction product was precipitated in 100 mL of ice-cold diethyl ether, centrifuged at 4°C and 12000 rpm, and the precipitate was washed three times with cold diethyl ether, followed by vacuum drying to obtain the product K2-PEG. 1.5k -DSPE (abbreviated as K2-PD).

[0057] K2-PEG 1.5k DSPE (1000 mg), MA (1602.4 mg), NHS (515.4 mg), and EDC.HCl (858.4 mg) were dissolved in 20 mL of DMF and stirred at room temperature for 12 h. The resulting mixture was precipitated in diethyl ether, washed with diethyl ether, and dried under vacuum to obtain crude MA4-PEG. 1.5k -DSPE (abbreviated as MA4-PD). The crude MA4-PD is then dialyzed and finally lyophilized to obtain pure MA4-PD.

[0058] MA4-PD 1 H-NMR spectrum and MALDI-TOF mass spectrometry are as follows: Figure 1 and Figure 2 As shown.

[0059] (2) Synthesis of BPA-PEG9-NHS

[0060] NH2-PEG9-COOtbu (2000 mg), BPA (2443.2 mg), bis-N-hydroxysuccinimide [(NHS) 1684.8 mg] and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride [(EDC.HCl) 2805.6 mg] were dissolved in 20 mL of N,N-dimethylformamide (DMF) and stirred at room temperature for 12 h. The resulting mixture was precipitated in diethyl ether, and after washing with cold ether and vacuum drying, crude BPA-PEG9-COOtbu was obtained. The crude BPA-PEG9-COOtbu was dissolved in 10 mL of trifluoroacetic acid (TFA) / DMF (v / v: 1 / 1) and stirred at room temperature for 2 h. Then it was washed with cold ether and vacuum dried to obtain crude BPA-PEG9-COOH. Subsequently, BPA-PEG9-COOH (1000 mg), NHS (545.62 mg), and EDC.HCl (908.54 mg) were dissolved in 10 mL of DMF and stirred at room temperature for 6 h. The resulting mixture was precipitated in cold ether, washed with cold ether, and vacuum dried to obtain crude BPA-PEG9-NHS. Finally, the crude BPA-PEG9-NHS was dialyzed and lyophilized to obtain pure BPA-PEG9-NHS.

[0061] BPA-PEG9-NHS 1 H-NMR spectrum and MALDI-TOF mass spectrometry are as follows: Figure 3 and Figure 4 As shown.

[0062] (3) Synthesis of BPA6-aPDL1

[0063] BPA-PEG9-NHS (4.39 g) and aPDL1 (13 g) were placed in PBS solution (pH 7.4) and stirred at 4 °C for 8 h. Then, they were dialyzed using a dialysis bag with a molecular weight of 3000 to obtain BPA6-aPDL1.

[0064] (4) Synthesis of MB-aPDL1

[0065] MA4-PD (16g) and BPA6-aPDL1 (17.2g) were placed in PBS solution (pH 7.4) and stirred at 4°C for 8 hours. Then, they were dialyzed using a dialysis bag with a molecular weight of 3000 to obtain MB-aPDL1.

[0066] I. Morphological Structure of MB-aPDL1

[0067] 5 mg of MB-aPDL1 was dissolved in 1 mL of PBS solution at pH 7.4, 6.8 and 6.5, respectively. The reaction solution was then dropped onto a supporting carbon membrane. After vacuum drying, the morphology and structure of the delivery system MB-aPDL1 at different pH values ​​were observed under a 120 kV transmission electron microscope.

[0068] Observation results as follows Figure 5 As shown:

[0069] MB-aPDL1 exhibits different structures at different pH levels, responding to pH and disintegrating into smaller structures with a particle size of 25 nm at pH 6.5. This indicates that MB-aPDL1 has pH-responsive characteristics, and the smaller, dispersed structures are more conducive to penetrating deeper into tumors.

[0070] II. The blocking effect of MB-aPDL1

[0071] HeLa cells were seeded in 6-well plates, with 10 cells per well. 5 Cells were cultured for 16 hours. The transient overexpression plasmid mPDL1-GFP was transfected into HeLa cells at 500 ng / well using Lipo6000. 24 hours after transfection, different components of aPDL1 were added to different cell culture wells at 1 mg / mL. After co-culturing for 24 hours, fluorescent secondary antibody was added to the culture medium, and after 1 hour of co-culturing, the location of the fluorescence signal was observed under a laser confocal microscope.

[0072] Observation results as follows Figure 6 As shown:

[0073] At pH 7.4, MB-aPDL1 can block the binding of antibodies to antigens, while at pH 6.5, the antibodies regain their biological function and can co-localize with the PDL1 protein. This indicates that MB-aPDL1 has a good blocking effect before depolymerization, and that depolymerization does not affect the antibody's function.

[0074] III. Cytotoxicity of MB-aPDL1

[0075] Cell viability was assessed using CCK8 assay. 4000 cells were seeded into each well of a 96-well plate, and different concentrations of MB-aPDL1 were added. After co-culturing for 24 hours, 10 μL of CCK8 was added to each well, and the cells were co-cultured for another hour. The absorbance was then read using a microplate reader at a wavelength of 450 nm.

[0076] The test results are as follows Figure 7 As shown:

[0077] At different concentrations, MB-aPDL1 had very little effect on cell viability; at a concentration of 1 g / L, cells could still maintain more than 80% viability. This indicates that MB-aPDL1 has excellent biocompatibility.

[0078] IV. In vitro cross-BBB efficiency of MB-aPDL1

[0079] 1. Delivery efficiency of in vitro BBB model

[0080] In vitro, using a Transwell cell apparatus, bEnd.3 was seeded in the upper chamber to simulate the BBB, and the concentration of aPDL1 was detected by ELISA.

[0081] The test results are as follows Figure 8 As shown:

[0082] MB-aPDL1 showed 40% antibody detection in the lower chamber, compared to only 10% for the pure antibody Native aPDL1.

[0083] 2. Cross-BBB enrichment

[0084] MB-aPDL1 was injected via the tail vein in mice with brain metastases from non-small cell lung cancer. Red Alexa 647 indicated the location of the antibody, and green GFP indicated the location of the tumor.

[0085] The test results are as follows Figure 9 As shown:

[0086] Only in the MB-aPDL1 administration group were large amounts of antibodies observed around cells, 5.56 times that of native aPDL1. This indicates that MB-aPDL1 can effectively cross the BBB and accumulate around tumors in vivo.

[0087] 3. Tumor growth inhibition

[0088] Because Lewis cells with stable luciferase transduction were constructed in advance, tumor size can be detected through in vivo animal imaging.

[0089] Test results as follows Figure 10 and Figure 11 As shown:

[0090] Depend on Figure 10 It can be seen that in mice with non-small cell lung cancer brain metastases treated with MB-aPDL1, the fluorescence signal of the tumor was weakened, indicating that tumor growth was inhibited. Figure 11 It can visually show the size of the tumor. Dark HE staining indicates the tumor, and it is clear that the tumor growth has been inhibited, accounting for only 5% of the brain.

[0091] V. Therapeutic effects of MB-aPDL1

[0092] 1. Survival time of mice in NSCLC brain metastasis model

[0093] A brain metastasis model of NSCLC was established using 8-week-old C57 mice. Treatment began on day 6 after tumor transplantation. After four treatments, the survival of the mice was observed at a dose of 10 mg / kg. Mice were considered dead when their body weight decreased by 20% and they showed no vital signs.

[0094] The test results are as follows Figure 12 As shown:

[0095] Statistical analysis of survival revealed that MB-aPDL1 not only prolonged the survival time of mice with brain metastases from non-small cell lung cancer, but also achieved a cure rate of up to 20%. Brain tissue sections from three cured mice showed no residual tumor.

[0096] 2. CD8 + T cell activation

[0097] After treatment, the tumor was removed, and the CD8+ of the brain tumor was analyzed by flow cytometry. + • T cell count, collected 10 cells 5 Each one was tested.

[0098] The test results are as follows Figure 13 As shown:

[0099] MB-aPDL1 activated a large number of CD8s. + T cells, these CD8 + T cells attack tumor cells in large numbers, inhibiting tumor growth.

[0100] VI. Pharmacokinetics of MB-aPDL1

[0101] Different components of aPDL1 were injected into normal individuals, serum was collected, and antibody concentrations at different time points were detected by ELISA.

[0102] The test results are as follows Figure 14 As shown:

[0103] Pharmacokinetic studies show that MB-aPDL1 can prolong the retention time of aPDL1 in the blood, with a drug half-life of 14.73 h, which means it can have a sustained effect on the body.

[0104] VII. Antibody Anchoring Ability of MB-aPDL1

[0105] A mouse subcutaneous tumor model was constructed, and different components of Cy5.5-labeled aPDL1 were injected into the tumor.

[0106] Imaging results as follows Figure 15 As shown:

[0107] MB-aPDL1 prolongs the retention time of antibodies within tumors and makes them less likely to diffuse outside the tumor, indicating an increased antibody anchoring ability.

[0108] 8. MB-aPDL1 reduces adverse immune responses

[0109] Mouse serum was collected, and liver function was assessed using a mouse blood biochemistry analyzer. Mouse liver was collected, ground into a cell suspension, and then CD8+ levels in the liver were detected by flow cytometry. + T cell percentage, 10 cells collected 5 Each one was tested.

[0110] The test results are as follows Figure 16 and Figure 17 As shown:

[0111] Depend on Figure 16 It can be seen that, after the treatment, liver function tests on mice showed that the MB-aPDL1 treatment group did not exhibit abnormally elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), indicating normal liver function. Figure 17 It can be seen that CD8 in the liver was further detected by flow cytometry. + The results showed that the MB-aPDL1 treatment group did not activate hepatic CD8 cells. + T cells, and randomly activated CD8 + T cells can attack normal cells without provocation, causing liver inflammation.

[0112] In summary, the MB-aPDL1 delivery system developed in this invention not only successfully delivers PD-L1 across the blood-brain barrier to the tumor site, but also precisely releases the PD-L1 monoclonal antibody, causing it to accumulate only in the tumor region and prolonging the antibody's retention time, thus increasing its ability to anchor to the tumor. Furthermore, the MB-aPDL1 delivery system avoids leakage during long-term blood circulation, thereby extending the blood circulation time. Simultaneously, MB-aPDL1 flowing into other organs maintains a compact structure and does not trigger an immune response in normal tissues.

[0113] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a PD-L1 monoclonal antibody delivery system, characterized in that, The steps are as follows: (1) Synthesis of MA4-PD NH2-PEG 1.5k -DSPE, Fmoc-lys-(Fmoc)-OH, DIC, and 6-Cl-HOBT were dissolved in DMF and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain (Fmoc)2-K-PEG. 1.5k -DSPE; Fmoc was removed using an Fmoc deprotection solution to obtain K-PEG. 1.5k -DSPE, i.e. K-PD; K-PD, Fmoc-lys-(Fmoc)-OH, DIC, and 6-Cl-HOBT were dissolved in DMF and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain (Fmoc)2-K-PEG. 1.5k -DSPE; remove Fmoc with Fmoc deprotection solution to obtain K2-PEG. 1.5k -DSPE, which stands for K2-PD; K2-PD, MA, NHS, and EDC.HCl were dissolved in DMF and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain MA4-PEG. 1.5k -DSPE, which stands for MA4-PD; (2) Synthesis of BPA-PEG9-NHS NH2-PEG9-COOtbu, BPA, NHS, and EDC.HCl were dissolved in DMF and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain BPA-PEG9-COOtbu. BPA-PEG9-COOtbu was dissolved in a TFA / DMF mixed solution and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain BPA-PEG9-COOH. Subsequently, BPA-PEG9-COOH, NHS and EDC.HCl were dissolved in DMF and reacted with stirring at room temperature. After the reaction was completed, the reaction product was washed to obtain BPA-PEG9-NHS. (3) Synthesis of BPA6-aPDL1 BPA-PEG9-NHS and aPDL1 were placed in PBS solution and reacted under low temperature with stirring. After the reaction was completed, the mixture was dialyzed to obtain BPA6-aPDL1. (4) Synthesis of MB-aPDL1 MA4-PD and BPA6-aPDL1 were placed in PBS solution and reacted under low temperature with stirring. After the reaction was completed, the mixture was dialyzed to obtain MB-aPDL1.

2. The preparation method according to claim 1, characterized in that, In step (1), NH2-PEG 1.5k The molar ratios of -DSPE, Fmoc-lys-(Fmoc)-OH, DIC, and 6-Cl-HOBT are selected from 1:2:2:2 to 1:6:6:6; the molar ratios of K-PD, Fmoc-lys-(Fmoc)-OH, DIC, and 6-Cl-HOBT are selected from 1:2:2:2 to 1:6:6:6; and the molar ratios of K2-PD, MA, NHS, and EDC.HCl are selected from 1:4:4:4 to 1:12:12:

12.

3. The preparation method according to claim 1, characterized in that, In step (2), the molar ratio of NH2-PEG9-COOtbu, BPA, NHS and EDC.HCl is selected from 1:2:2:2 to 1:6:6:

6.

4. The preparation method according to claim 1, characterized in that, In step (3), the molar ratio of BPA-PEG9-NHS to aPDL1 is selected from 1:1 to 10:

1.

5. The preparation method according to claim 1, characterized in that, In step (4), the molar ratio of MA4-PD to BPA6-aPDL1 is selected from 2:1 to 20:

1.

6. The preparation method according to claim 1, characterized in that, The low temperature in steps (3) and (4) is selected from 1 to 16°C.

7. The preparation method according to claim 1, characterized in that, The pH value of the PBS solution in steps (3) and (4) is selected from 7 to 8.

8. The preparation method according to claim 1, characterized in that, The reaction time for each stirring reaction should be based on the complete reaction of the substrate, and can preferably be 6 to 12 hours.

9. The PD-L1 monoclonal antibody delivery system prepared by the method according to any one of claims 1 to 8.

10. The use of the PD-L1 monoclonal antibody delivery system of claim 9 in the preparation of a medicament for treating brain metastases.

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