Anti-tumor gene nano-drug for expressing multivalent PD-L1 antibody as well as preparation method and application of anti-tumor gene nano-drug

Abstract

The invention discloses an anti-tumor gene nano-drug for expressing a multivalent PD-L1 antibody as well as a preparation method and application of the anti-tumor gene nano-drug, and relates to the technical field of immunotherapy drugs. According to the gene nano-drug, expression plasmids of a multivalent PD-L1 antibody are entrapped into polymer nano-particles through a mechanical mixing method, tumor cells can be induced to express the multivalent PD-L1 antibody through an intratumor administration mode, the effect of blocking an immune checkpoint PD-L1 is enhanced, and then the level of blocking the immune checkpoint PD-L1 in a tumor and the tumor treatment effect are improved; a tumor specific promoter is further introduced, the anti-tumor gene nano-drug can specifically express a multivalent PD-L1 antibody in melanoma through intravenous administration, expression of the multivalent PD-L1 antibody in normal tissue cells is prevented, the anti-tumor gene nano-drug has a good anti-tumor effect and in-vivo safety, and a new thought is provided for development and application of antibody drugs.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy drug technology, specifically to an anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies, its preparation method, and its application. Background Technology

[0002] Tumor immunotherapy has been a major breakthrough in cancer treatment in recent years, with immune checkpoint inhibitors (ICIs), represented by PD-1 / PD-L1 antibodies, playing a crucial role. Since the first PD-1 inhibitor was approved for the treatment of unresectable or metastatic melanoma in 2011, PD-1 / PD-L1 antibodies have been proven to bring significant long-term survival benefits to patients with various solid tumors. These antibody drugs specifically block the interaction between PD-1 and PD-L1, relieving immunosuppression in the tumor microenvironment and reactivating the anti-tumor immune function of T cells. Durvalumab, the first PD-L1-based antibody drug in China, is a fully human monoclonal antibody that blocks the interaction between PD-1 and PD-L1, thereby combating tumor immune escape mechanisms. In 2019, the National Medical Products Administration approved the PD-L1 blocking antibody Durvalumab for first-line treatment of extensive-stage small cell lung cancer. Clinical data show that, compared with placebo, adjuvant Durvalumab therapy significantly prolonged overall survival and progression-free survival in patients with extensive-stage small cell lung cancer.

[0003] Currently, the PD-1 / PD-L1 antibody drugs used in clinical practice are mainly protein drugs with specific spatial structures and biological activities. They are usually produced using mammalian cell expression systems (such as Chinese hamster ovary cells, CHO cells) to ensure correct post-translational modifications and functional activities. However, protein drugs face multiple challenges in manufacturing, storage, and delivery. For example, protein aggregation, denaturation, and degradation can occur during preparation and storage, leading to loss of activity. In addition, commercial monoclonal antibodies require therapeutic doses in grams for tumor treatment, making them expensive. In clinical treatment, antibody drugs are usually administered systemically, such as via intravenous injection. However, due to the widespread distribution of antibody drugs in the bloodstream, they may non-selectively activate T cells in non-target organs (such as the liver, intestines, and lungs), thereby triggering immune-related adverse reactions such as hepatitis, colitis, and pneumonia, affecting treatment safety. Furthermore, in systemic administration, only a small amount of antibodies can effectively accumulate in tumor tissue, resulting in insufficient drug concentration in the tumor microenvironment, making it difficult to maintain effective immune activation. Simultaneously, in some cancer patients, insufficient binding titers of antibody drugs may reduce the therapeutic effect of PD-1 / PD-L1 blockade, or even induce adaptive resistance, further limiting clinical benefit. These factors collectively affect the therapeutic efficiency of antibody drugs and may enhance tumor immune escape, ultimately leading to treatment failure.

[0004] In summary, it is essential to utilize PD-1 / PD-L1 antibodies to efficiently, specifically, and safely block the PD-1 / PD-L1 signaling pathway and enhance the blocking of the PD-L1 immune checkpoint at the tumor site. Furthermore, further breakthroughs are needed to enhance T cell-mediated anti-tumor immune responses. Summary of the Invention

[0005] To address the above problems, the present invention aims to provide an anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies, its preparation method, and its application. This invention aims to solve the technical problem in the prior art of how to efficiently and safely block the PD-1 / PD-L1 signaling pathway using PD-1 / PD-L1 antibodies, enhance the level of the PD-L1 immune checkpoint in tumors, and improve T cell-mediated anti-tumor immune effects.

[0006] To achieve the above objectives, the technical solution of this invention is as follows:

[0007] A first aspect of the present invention provides an anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies, comprising a plasmid and polymer nanoparticles; wherein the plasmid is a plasmid for expressing multivalent PD-L1 antibody-related genes; and the polymer nanoparticles are composed of ionizable lipids, cationic lipids and polymers, and can be used for in vivo and in vitro encapsulation and delivery of nucleic acid drugs.

[0008] Preferably, in conjunction with the first aspect, the plasmid is a plasmid expressing a multivalent PD-L1 antibody, and the multivalent PD-L1 antibody is a secretory IgM-like multivalent PD-L1 antibody.

[0009] Preferably, in conjunction with the first aspect, the multivalent PD-L1 antibody is a multivalent PD-L1 antibody formed by fusing a mutated Fc fragment with an IgM-expressing polymeric tail to form a polymer and linking it with at least one of an anti-PD-L1 single-chain variable region fragment and an anti-PD-L1 nanobody.

[0010] In conjunction with the first aspect, preferably, the plasmid promoter is at least one of a strong promoter and a tumor-specific promoter.

[0011] Preferably, in conjunction with the first aspect, the strong promoter is at least one of the cytomegalovirus promoter, the strong hybrid mammalian promoter, and the elongation factor-1α promoter; the tumor-specific promoter is at least one of the tyrosinase promoter and the survivin promoter.

[0012] Preferably, in conjunction with the first aspect, the plasmid is at least one of human or mouse genes.

[0013] Preferably, in conjunction with the first aspect, the ionizable lipid is at least one of Dlin-MC3-DMA, SM-102, ALC-0315, Dlin-KC2-DMA, BHEM-DBA, BHEM-APMP, BHEM-EAA, and BHEM-AEA.

[0014] Preferably, in conjunction with the first aspect, the cationic lipid is at least one of trimethyl-2,3-diolenoyloxypropylammonium chloride (DOTMA), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), dimethyl-2-hydroxyethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-bisoctadecyloxypropylammonium bromide, N-(2-speramidyl)-N',N'-bisoctadecylglycineamide (DOGS), and 1,2-dioleoyl-3-succinyl-sn-glycerolcholine ester (DOSC); further, the cationic lipid is more preferably at least one of trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP).

[0015] Preferably, in conjunction with the first aspect, the molar ratio of the cationic lipid to the ionizable lipid is 1:1-4; and / or, the molar ratio of the cationic lipid to the nucleic acid is 1:1-4.

[0016] Preferably, in conjunction with the first aspect, the average particle size of the polymer nanoparticles is 50 nm to 350 nm; and / or, the polymer in the polymer nanoparticles is at least one of polyethylene glycol-modified poly(glycolic acid-co-lactide) and poly(glycolic acid-co-lactide); and / or, the LA / GA ratio in the polyethylene glycol-modified poly(glycolic acid-co-lactide) and the poly(glycolic acid-co-lactide) ranges from 95:5 to 50:50; and / or, the molecular weight of the polyethylene glycol ranges from 1000 to 10000 Daltons.

[0017] A second aspect of the present invention provides a method for preparing an anti-tumor gene nanomedicine expressing a multivalent PD-L1 antibody as described in any of the first aspects, comprising the following steps:

[0018] Step 1: Mix the plasmid used to express the multivalent PD-L1 antibody with DEPC water to obtain the aqueous phase;

[0019] Step 2: Mix the ionizable lipid, cationic lipid, and ethanol to obtain a lipid ethanol solution; mix the polymer and organic solvent to obtain a mixture; then mix the lipid ethanol solution and the mixture to obtain an oil phase;

[0020] Step 3: Mix the oil phase and the aqueous phase to prepare an oil-in-water emulsion; after the emulsion is left to stand at room temperature for 15-20 minutes, a gene nanomedicine for enhancing the blocking immune checkpoint PD-L1 is obtained.

[0021] The antitumor gene nanomedicine prepared in this invention for expressing multivalent PD-L1 antibodies has a nanoscale spherical structure with a polyethylene glycol shell, uniform particle size distribution, positive charge, and an average particle size of 80-150 nm. The polyethylene glycol shell provides an "invisibility" effect (reducing protein adsorption and immune clearance) and prolongs blood circulation time. The positively charged core (such as a cationic polymer / lipid) efficiently compresses negatively charged gene drugs (such as PD-L1 antibody expression plasmids) and promotes cell membrane fusion.

[0022] The third aspect of the present invention provides an anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies as described in the first aspect or a gene nanomedicine for expressing multivalent PD-L1 antibodies prepared by the method described in the second aspect. By specifically expressing multivalent PD-L1 antibodies in melanoma, the antibody titer is increased, and it can specifically recognize and act on melanomas (such as B16-F10) in vivo, thereby enhancing the anti-tumor efficacy.

[0023] Compared with the prior art, the advantages or beneficial effects of the present invention include at least the following:

[0024] The gene nanomedicine for expressing multivalent PD-L1 antibodies provided by this invention utilizes cationic lipids and ionizable lipids to assist in the delivery of plasmids via polymer nanoparticles. The plasmids are loaded by forming oil-in-water nanoparticles through hydrophilic-hydrophobic interactions, exhibiting excellent stability. The polyethylene glycol shell can achieve long-term circulation in vivo, and the nanoscale size (80-150 nm) can increase the enrichment of particles in tumors through the high permeability and retention effect (EPR effect) of solid tumors. The cationic lipids can assist the nanoparticles to be taken up by cells, and the ionizable lipids can assist the nanoparticles to escape from lysosomes.

[0025] The gene nanomedicine provided by this invention encapsulates the expression plasmid of a multivalent PD-L1 antibody into polymer nanoparticles through a mechanical mixing method. By administering the drug intratumorally, it can induce tumor cells to express the multivalent PD-L1 antibody, enhancing the effect of blocking the immune checkpoint PD-L1, thereby improving the intratumoral blocking level of the immune checkpoint PD-L1 and the therapeutic effect on tumors. Furthermore, by introducing a tumor-specific promoter, this anti-tumor gene nanomedicine can specifically express the multivalent PD-L1 antibody in melanoma via intravenous administration, preventing its expression in normal tissue cells. It exhibits good anti-tumor efficacy and in vivo safety, providing a new approach for the development and application of antibody drugs. Attached Figure Description

[0026] Figure 1 The images show the plasmids pCMV-APM, which expresses a multivalent PD-L1 antibody, and pTyr-APM, which specifically express a multivalent PD-L1 antibody, prepared in Example 1 of this invention.

[0027] Figure 2 This is a schematic diagram illustrating the preparation process of the gene nanomedicine for expressing multivalent PD-L1 antibodies provided by the present invention.

[0028] Figure 3 NP prepared in Example 2 of the present invention CMV-APM The results of particle size, potential and morphology characterization.

[0029] Figure 4 NP prepared in Example 2 of the present invention CMV-APM Figure showing the protein expression level of multivalent PD-L1 antibody after transfection into B16-F10 cells.

[0030] Figure 5 NP prepared in Example 2 of the present invention CMV-APM The results of the detection of the cytotoxic effect of multivalent PD-L1 antibody on T cells after transfection with B16-F10 cells.

[0031] Figure 6 NP prepared in Example 2 of the present invention CMV-APM The image shows the results of in vivo detection of PD-L1, which inhibits melanoma growth and enhances the blocking of immune checkpoints.

[0032] Figure 7 NP prepared in Example 2 of the present invention Tyr-APM Fluorescence flow cytometry results of tumor-specific expression of EGFP mediated by this method.

[0033] Figure 8 NP prepared in Example 2 of the present invention Tyr-APM The image shows the results of in vitro detection of tumor-specific expression of multivalent PD-L1 antibody mRNA mediated by in vitro.

[0034] Figure 9 NP prepared in Example 2 of the present invention Tyr-APM Image showing the results of in vivo detection of tumor-specific expression of multivalent PD-L1 antibodies.

[0035] Figure 10 NP prepared in Example 2 of the present invention Tyr-APM The image shows the results of in vivo detection of PD-L1, which inhibits melanoma growth and enhances the blocking of immune checkpoints. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on this invention. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0037] In the following description, references to "some embodiments" refer to a subset of all possible embodiments; however, it is understood that "some embodiments" may be the same or different subsets of all possible embodiments and may be combined with each other without conflict. Unless otherwise defined, all technical and scientific terms used in the embodiments of the invention have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of the invention pertain. The terminology used in the embodiments of the invention is for the purpose of describing the embodiments of the invention only and is not intended to limit the invention.

[0038] In the following description of this embodiment, the terms "including", "comprising", "having", and "containing" are all open-ended terms, meaning that they include but are not limited to.

[0039] It should be noted that all raw materials / reagents in the embodiments of the present invention can be purchased on the market or prepared according to conventional methods known to those skilled in the art; the term "and / or" in the embodiments of the present invention is only used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B means three cases: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural. The character " / " generally indicates that the related objects before and after are in an "or" relationship.

[0040] In the following description of this embodiment, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0041] Those skilled in the art should understand that, in the following description of the embodiments of the present invention, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0042] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0043] Those skilled in the art will understand that the numerical ranges in the embodiments of this invention should be understood as each intermediate value between the upper and lower limits of the specifically disclosed range. Each smaller range between any stated value and an intermediate value within the stated range, as well as any other stated value or an intermediate value within the stated range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein. While only preferred methods and materials are described herein, any methods and materials similar to or equivalent to those described herein may be used in embodiments or test cases of this invention. All references to this specification are generally incorporated herein by reference to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the contents of this specification shall prevail.

[0044] It should be noted that all raw materials and / or reagents in the embodiments of the present invention were purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0045] In a first aspect, embodiments of the present invention provide a gene nanomedicine for expressing multivalent PD-L1 antibodies, comprising a plasmid and polymer nanoparticles; the plasmid is a plasmid expressing a gene related to multivalent PD-L1 antibodies; the polymer nanoparticles are composed of ionizable lipids, cationic lipids and polymers, and can be used for in vivo and in vitro encapsulation and delivery of nucleic acid drugs.

[0046] In a specific embodiment, the plasmid in this embodiment of the invention is a plasmid expressing a multivalent PD-L1 antibody, and the multivalent PD-L1 antibody is a secretory IgM-like multivalent PD-L1 antibody.

[0047] In a specific embodiment, the multivalent PD-L1 antibody in this invention is formed by linking a multimer formed by fusing the mutated Fc fragment with the IgM-expressing multimer tail to at least one of an anti-PD-L1 single-chain variable region fragment and an anti-PD-L1 nanobody.

[0048] In a specific embodiment, the plasmid promoter in this invention is at least one of a strong promoter and a tumor-specific promoter.

[0049] In specific embodiments, the strong promoter in the present invention is at least one of the cytomegalovirus promoter, the strong hybrid mammalian promoter, and the elongation factor-1α promoter; the tumor-specific promoter is at least one of the tyrosinase promoter and the survivin promoter.

[0050] It should be noted that tumor-specific promoters can selectively drive gene transcription and expression in tumor cells, while having no effect on other normal cells. Tyrosinase promoters can achieve melanoma-specific gene expression.

[0051] In a specific embodiment, the plasmid in the present invention is at least one of human or mouse genes.

[0052] In a specific embodiment, the ionizable lipid in the present invention is at least one of Dlin-MC3-DMA, SM-102, ALC-0315, Dlin-KC2-DMA, BHEM-DBA, BHEM-APMP, BHEM-EAA, and BHEM-AEA.

[0053] It should be noted that these ionizable lipids are electrically neutral at physiological pH when forming gene nanomedicines. After being taken up by endocytosis, they become positively charged in the acidic endosome environment and interact with negatively charged phospholipids on the endosome membrane, thereby disrupting the endosome membrane and enabling endosome escape. This helps to promote the release of delivered nucleic acid drugs and improve transfection efficiency.

[0054] In specific embodiments, the cationic lipids in the embodiments of the present invention are at least one of trimethyl-2,3-diolenoyloxypropylammonium chloride (DOTMA), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), dimethyl-2-hydroxyethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-bisoctadecyloxypropylammonium bromide, N-(2-speramidyl)-N',N'-bisoctadecylglycineamide (DOGS), and 1,2-dioleoyl-3-succinyl-sn-glycerolcholine ester (DOSC); further, the cationic lipid is more preferably trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP).

[0055] It should be noted that these cationic lipids play the following roles in the formation of gene nanomedicines: 1) They can form stable complexes with negatively charged nucleic acids (such as DNA or RNA). These complexes easily cross the cell membrane and enter the cell, improving gene transfection of gene nanomedicines. These cationic liposomes bind to nucleic acids through charge interactions, greatly compressing the volume of nucleic acid molecules and promoting their transfection and expression within cells; 2) After gene transfer mediated by cationic liposomes, the liposomes are degraded by intracellular enzymes, exhibiting no toxic side effects on cells and demonstrating good biocompatibility. Compared with viral vectors, cationic liposomes as gene carriers have lower immunogenicity and toxicity, reducing immune responses and cytotoxicity; 3) The complexes formed by cationic liposomes and nucleic acids can promote endocytosis, effectively delivering nucleic acids into cells. Inside the cell, the complexes can release nucleic acids, enabling them to be transcribed and expressed, thereby achieving the purpose of gene therapy; 4) Cationic liposomes such as DOTAP and DOTMA have hydrophobic tails and hydrophilic positively charged heads, enabling them to form stable interfaces between the aqueous and organic phases, which is beneficial for binding with nucleic acids and forming complexes. Cationic liposomes such as DOGS have specific chemical structures, such as hydrophobic tails and hydrophilic positively charged heads, which give them unique biological properties and application potential.

[0056] In a specific embodiment, the molar ratio of the cationic lipid to the ionizable lipid in the present invention is 1:1-4.

[0057] In a specific embodiment, the molar ratio of cationic lipid to nucleic acid in this invention is 1:1-4.

[0058] In a specific embodiment, the average particle size of the polymer nanoparticles in the present invention is 50nm-350nm.

[0059] In a specific embodiment, the polymer in the polymer nanoparticles of the present invention is at least one of polyethylene glycol-modified poly(glycolic acid-co-lactide) (PEG-b-PLGA) and poly(glycolic acid-co-lactide) (PLGA).

[0060] It should be noted that, in the embodiments of the present invention, the preferred ratio range of LA / GA in the polyethylene glycol-modified poly(glycolic acid-co-lactide) and the poly(glycolic acid-co-lactide) is 95:5-50:50.

[0061] In a specific embodiment, the molecular weight range of the polyethylene glycol in the present invention is 1000-10000 Daltons.

[0062] In a second aspect, embodiments of the present invention provide a method for preparing the anti-tumor gene nanomedicine expressing multivalent PD-L1 antibodies as described in the first aspect, comprising the following steps:

[0063] Step 1: Mix the plasmid used to express the multivalent PD-L1 antibody with DEPC water to obtain the aqueous phase;

[0064] Step 2: Mix the ionizable lipid, cationic lipid, and ethanol to obtain a lipid ethanol solution; mix the polymer and organic solvent to obtain a mixture; then mix the lipid ethanol solution and the mixture to obtain an oil phase;

[0065] Step 3: Mix the oil phase and the aqueous phase, and pipette to mix them 20-40 times to prepare an oil-in-water emulsion; after the emulsion is left to stand at room temperature for 15-20 minutes, the gene nanomedicine for enhancing the blocking immune checkpoint PD-L1 is obtained.

[0066] Thirdly, embodiments of the present invention provide a gene nanomedicine for expressing multivalent PD-L1 antibodies as described in the first aspect, or a gene nanomedicine for expressing multivalent PD-L1 antibodies prepared by the method described in the second aspect. Through melanoma-specific expression of multivalent PD-L1 antibodies, sustained antibody secretion is achieved locally in the tumor. The multivalent PD-L1 antibodies, through spatial arrangement, allow multiple antigen-binding sites to simultaneously occupy PD-L1 molecules on the surface of tumor cells, enhancing the efficiency of blocking PD-1 / PD-L1 signaling. This significantly improves affinity for PD-L1, completely blocking PD-1 / PD-L1 immune checkpoint signals and relieving T cell exhaustion; simultaneously, local antibody expression remodels the immune microenvironment and activates CD8. +T cell killing function and induction of immune memory. Compared with monovalent antibodies, multivalent PD-L1 antibodies have a stronger affinity for PD-L1, which can enhance the effect of blocking the immune checkpoint PD-L1. This strategy increases PD-L1 occupancy and enhances T cell activity, significantly improving tumor suppression efficiency in melanoma models.

[0067] The technical method of the present invention will be further described below with reference to specific embodiments.

[0068] Sources of raw materials used in the examples:

[0069] DOTAP and Dlin-MC3-DMA were purchased from Aivito Pharmaceutical Technology Co., Ltd.

[0070] The polyester material was purchased from Guangzhou Xinheng Biotechnology Co., Ltd.

[0071] Dimethyl sulfoxide was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0072] B16-F10, MC38, Panc02, 4T1, C2C12, NIH-3T3, DC2.4, and RAW264.7 cells were purchased from ATCC;

[0073] CD8 sorting magnetic beads were purchased from Miltenyi Biotec in Germany.

[0074] The anti-Histag antibody was purchased from Sangon Biotech in China.

[0075] Recombinant mouse IFN-γ cytokines were purchased from PeproTech, USA.

[0076] Flow cytometry antibodies: anti-mouse CD16 / 32 (Catalog No. 101302), PE anti-mouse PD-L1 (Catalog No. 124308), AF647 anti-mouse 6×Histag (Catalog No. 652513), BV510 anti-mouse CD45 (Catalog No. 157219), BV650 anti-mouse CD80 (Catalog No. 104731), BV785 anti-mouse CD8 (Catalog No. 100742), FITC anti-mouse CD3 (Catalog No. 100204), APC / Cy7 anti-mouse CD4 (Catalog No. 100414), BV605 anti-mouse CD69 (Catalog No. 104529), FITC anti-mouse CD11b (Catalog No. 101206), BV650 anti-mouse... IFN-γ (part number 505832), BV421 anti-mouse F4 / 80 (part number 123131), PE anti-mouse CD206 (part number 141706), and APC anti-mouse Ki-67 (part number 652406) were purchased from Biolegend.

[0077] Instrument models and manufacturers used in the examples:

[0078] Nanoparticle size and Zeta potential meter: Model NanoZSE, Malvern, UK;

[0079] Analytical flow cytometer: Model FACSCelesta, BD Biosciences, USA;

[0080] Chemiluminescence imager: Model ChemiDocMP, Bio-RAD, USA;

[0081] Benchtop micro-refrigerated centrifuge: Model Microfuge20R, Beckman Coulter, USA;

[0082] Microplate reader: Model 800TS, BioTek, USA.

[0083] Example 1

[0084] The multimeric IgG Fc-TP and anti-PD-L1 scFv (αPD-L1) gene sequences were ligated and inserted into the pCMV or pTyr plasmid vector to construct a multivalent PD-L1 antibody expressing secretory IgM, resulting in the multivalent PD-L1 antibody plasmid pCMV-APM and the tumor-specific multivalent PD-L1 antibody plasmid pTyr-APM. A similar plasmid carrying the EGFP reporter gene, pTyr-APM-EGFP, was constructed. The resulting plasmid map P... CMV-APM and P Tyr-APM like Figure 1 As shown. The main components of this expression plasmid include the anti-PD-L1 scFv (αPD-L1) gene element and the multimeric IgG Fc-TP gene element, which are linked using a linker to achieve co-expression.

[0085] Example 2

[0086] NP, a gene nanomedicine for expressing multivalent PD-L1 antibodies CMV-APM or NP Tyr-APM Preparation method and characterization of particle size, potential and morphology

[0087] Reference Figure 2 Prepare ethanol solutions of Dlin-MC3-DMA and DOTAP respectively, with a concentration of 20-30 mg / mL for both; prepare a dimethyl sulfoxide solution of polyethylene glycol-modified poly(glycolic acid-co-lactide) (PEG2k-b-PLGA2k) with a concentration of 150 mg / mL. Dlin-MC3-DMA ethanol solution, DOTAP ethanol solution, PEG2k-b-PLGA2k dimethyl sulfoxide solution, and anhydrous ethanol were added to an enzyme-free EP tube and mixed by pipetting to obtain the oil phase. pCMV-APM and pTyr-APM prepared in Example 1 were dissolved in enzyme-free DEPC water and mixed by pipetting to obtain the aqueous phase. The aqueous phase was added to the oil phase, and after gentle pipetting for 15 seconds, it was allowed to stand at room temperature for 10 minutes to obtain the nanomedicine. This was then mixed with enzyme-free DEPC water and transferred to a 100 kDa ultrafiltration tube. The tube was centrifuged at 3000g for 5 minutes, and this process was repeated three times to remove the organic solvent, yielding the gene nanomedicine NP for enhancing the blocking immune checkpoint PD-L1. CMV-APM or NP Tyr-APM .

[0088] To verify the gene nanomedicine NP prepared in Example 2 for expressing multivalent PD-L1 antibodies CMV-APM The relevant properties were characterized, and the prepared NP was taken. CMV-APMIn the sample cell, the nanoparticles were characterized using a nanoparticle size analyzer and a zeta potential analyzer to determine their hydration diameter, corresponding particle size, and zeta potential distribution. Figure 3 As shown in Figure A, this gene nanomedicine for enhancing the blocking immune checkpoint PD-L1 is nanoscale in size, with a uniform particle size distribution and positive charge. The average particle size is 144.80 nm, and the zeta potential is 32.60 mV.

[0089] Take the prepared NP CMV-APM The particles were diluted 50 times with ultrapure water and then ultrasonically dispersed. 10 μL of the particle solution was dropped onto a copper grid and allowed to stand at room temperature for 5-10 minutes. The moisture was then blotted dry with filter paper. Under light-protected conditions, 10 μL of 2% phosphotungstic acid solution was dropped into a clean 60 mm petri dish. The copper grid was gently held at the edge with tweezers to contact the droplet. After negative staining for 30 seconds, the dish was placed on filter paper and allowed to dry. The morphology of the nanomedicine was observed using a field emission transmission electron microscope at 200 kV. Figure 3 As shown in Figure B, the gene nanomedicine used to enhance the blocking immune checkpoint PD-L1 is a nanoscale spherical structure with a polyethylene glycol shell. The polyethylene glycol shell is approximately 80-150 nm thick.

[0090] Test Example 1

[0091] Gene nanomedicines for expressing multivalent PD-L1 antibodies NP CMV-APM In vitro expression of multivalent PD-L1 antibody protein

[0092] In this invention, B16-F10 cells in the logarithmic growth phase are digested, and 5.0 × 10⁶ cells are taken. 4 Cells were seeded at a density of 1000 μg / mL in 24-well plates; after 12 hours, the old culture medium was removed, and serum-free culture medium containing particles was added. The plasmid concentration was 1 μg / mL. The experimental groups were PBS, NP, and NP. CMV-AP NP CMV-APMAfter 6 hours, the culture medium was replaced with Opti-MEM medium. After 72 hours, the supernatant of transfected B16-F10 cells was collected, and the B16-F10 cells were lysed with RIPA lysis buffer. The total protein in the supernatant and cell lysate was quantified using a BCA protein quantification kit. The samples were adjusted to the same protein concentration using PBS. Denaturing and reducing protein loading buffer was added to one sample of cell supernatant, and denaturing and reducing protein loading buffer was added to the cell lysate. The samples were heated at 95°C for 20 minutes. The other sample of cell supernatant was added to non-denaturing and non-reducing protein loading buffer and subjected to SDS-PAGE electrophoresis. After SDS-PAGE, the samples were transferred to a membrane and then blocked with 5% skim milk powder. The supernatant and cell lysate samples were labeled with anti-His antibody and GAPDH antibody. The protein expression of PD-L1 antibody after transfection of B16-F10 cells was characterized by Western blotting. The results are as follows. Figure 4 As shown, NP CMV-APM Multivalent PD-L1 antibody protein is expressed in B16-F10 cells.

[0093] Test Example 2

[0094] Gene nanomedicines for expressing multivalent PD-L1 antibodies NP CMV-APM It expresses multivalent PD-L1 antibodies, has a stronger affinity for PD-L1, and promotes enhanced T cell killing of tumors in vitro.

[0095] CD8 extracted from OT-1 mice + T cells, and collection of PBS, NP, NP CMV-AP NP CMV-APM Supernatant after transfection of B16-F10 cells. B16-F10-OVA cells were stimulated with IFN-γ for 24 h, and 1.0 × 10⁶ cells were collected. 4 5.0 × 10 B16-F10-OVA cells and 5.0 × 10 4 Activated CD8+ T cells were added to 96-well plates, and the original culture medium was replaced with PBS, NP, and NP. CMV-AP NP CMV-APM The concentrated supernatant after transfection of B16-F10 cells was cultured for 24 hours in a cell culture incubator at 37°C and 5% CO2. After 24 hours, the supernatant was discarded, and 100 μL of CCK8 working solution (1640 basal medium and CCK8 stock solution mixed at a volume ratio of 9:1) was added. The cells were then incubated in the dark for 3 hours. After incubation, cell viability in each well was detected using a microplate reader. Figure 5 As shown, NP CMV-APMThe multivalent PD-L1 antibody secreted after transfection of B16-F10 cells promoted the killing effect of T cells on tumor cells. Under conditions of T cell co-culture, the tumor cell survival rate in the PBS group was close to 100%, indicating that the killing effect of T cells was limited without antibody blockade. (NP group and NP...) CMV-AP Cell viability decreased to approximately 80% and 65% in the two groups, respectively, indicating that delivery of a monovalent PD-L1 antibody could partially block the PD-1 / PD-L1 pathway, thereby enhancing the cytotoxicity of T cells. In contrast, NP... CMV-APM The survival rate of tumor cells in the treatment group further decreased significantly to approximately 50%, indicating that the multivalent PD-L1 antibody can simultaneously bind multiple PD-L1 molecules in its spatial structure, achieving more efficient immune checkpoint blockade. Notably, the survival rate of the PBS control group, which was administered T cells alone without any delivery system, was significantly higher than that of all nanomedicine-treated groups, further demonstrating the promoting effect of nanomedicine-mediated antibody expression on immune killing. In summary, NP CMV-APM It effectively enhances T-cell-mediated tumor cell killing, and its immune activation effect is superior to that of the monovalent antibody delivery group.

[0096] Test Example 3

[0097] Gene nanomedicines for expressing multivalent PD-L1 antibodies NP CMV-APM Blocking the immune checkpoint PD-L1 in melanoma and its tumor growth inhibitory effect

[0098] After hair removal, C57BL / 6J mice were subcutaneously implanted with B16-F10 melanoma tumors. The tumor volume in the mice was approximately 100 mm². 3 Mice were randomly divided into four groups: PBS, NP, NP, and NP. CMV-AP NP CMV-APM The experimental group received intratumoral administration every other day, for a total of three times, with each dose being 0.5 mg / kg. The length and width of the mouse tumors were measured daily using electronic calipers, and the mice were weighed daily using an electronic balance. This was to evaluate the NP... CMV-APM To assess the antitumor activity in vivo, we established a B16-F10 melanoma-bearing mouse model and followed the experimental protocol ( Figure 6 A) When the tumor grows to approximately 80-100 mm 3 Intratumoral administration began at a certain time, with two-day intervals between doses, for a total of three doses. Tumor tissue was collected for analysis on day 14. Tumor growth curve ( Figure 6 (B, left) shows that the tumor volume in the PBS group and the NP empty vector group continued to grow rapidly during the drug administration period, reaching approximately 1900 mm on day 14. 3 and 1750mm 3 NP CMV-AP The tumor growth in this group slowed significantly, with a final volume of approximately 600 mm.3 The levels were significantly lower in the NP group compared to the PBS group, suggesting that delivery of a monovalent PD-L1 antibody can inhibit tumor progression. CMV-APM The group exhibited the most significant tumor-suppressing effect, with a tumor volume of only about 400 mm. 3 Compared to NP CMV-AP The group showed a further decrease, indicating that multivalent PD-L1 antibody delivery can more effectively block the PD-1 / PD-L1 pathway and enhance the immune response. Mouse body weight changes ( Figure 6 The results for B (right) showed no significant differences among the groups, and the curves were stable, suggesting that the administration process did not cause significant systemic toxicity.

[0099] Tumor tissue was collected to detect T cell activation. Digestion solution was added to the minced tumor tissue, and PBS was added to terminate digestion after 30 minutes. The tissue was centrifuged, the supernatant was discarded, and the cell pellet was resuspended in 40% Percoll solution. The pellet was centrifuged at 800g for 20 minutes at room temperature (increase speed 6, decrease speed 2). The supernatant was aspirated, and erythrocyte lysis buffer was added to resuspend the cell pellet. Lysis was performed at room temperature for 3 minutes, followed by PBS to terminate lysis. The pellet was centrifuged at 3000rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in PBS. An appropriate amount of cells was passed through a 200-mesh nylon mesh into an EP tube, and anti-mouse CD16 / 32 antibody was added. The tube was blocked on ice for 15 minutes. Flow cytometry antibodies BV510 anti-mouse CD45, FITC anti-mouse CD3, APC / Cy7 anti-mouse CD4, BV785 anti-mouse CD8, BV605 anti-mouse CD69, FITC anti-mouse CD11b, and BV421 anti-mouse were added. F4 / 80, BV650 anti-mouse CD80, PE anti-mouse CD206, and BV650 anti-mouse IFN-γ were used to detect T cell activation in tumors using flow cytometry. To investigate the tumor suppression mechanism, we also measured tumor-infiltrating CD8+. + The percentage of activated T cells (CD69 positive percentage), Figure 6 (C, left). The results showed that the activation rates in the PBS and NP groups were approximately 14%, with no significant difference; NP CMV-AP The group rose to about 25%, while NP CMV-APM The percentage of the group significantly increased to approximately 40%, indicating that multivalent antibodies can more effectively activate CD8. + The cytotoxic function of T cells. Simultaneously, we assessed the ratio of M1 pro-inflammatory macrophages to M2 immunosuppressive macrophages in tumor tissue. Figure 6 (C, right). The M1 / M2 ratio in both the PBS and NP groups was less than 2, and the NP group... CMV-APNo significant improvement was observed in the NP group, while... CMV-APM The proportion of this group increased to 3, suggesting that multivalent antibodies not only enhanced T cell immune activity but also reshaped the tumor immune microenvironment, transforming it towards pro-inflammatory and anti-tumor effects.

[0100] In summary, NP CMV-APM It significantly inhibited tumor growth in a mouse melanoma model and enhanced CD8. + T cell activation and promotion of M1 macrophage polarization improved the tumor immune microenvironment, with better results than the monovalent antibody delivery group.

[0101] Test Example 4

[0102] Gene nanomedicines for expressing multivalent PD-L1 antibodies NP Tyr-APM In vitro EGFP fluorescence detection

[0103] Five 5.0 × 10⁻⁶ samples were taken from four types of tumor cell lines (B16-F10, CT-26, Pan02, 4T1) and four types of non-tumor cell lines (NIH / 3T3, RAW264.7, DC2.4, C2C12) that were in good growth condition. 4 Cells were seeded at a density of 1000 μg / mL in 24-well plates. After 12 hours, the old culture medium was removed, and serum-free culture medium containing granules was added. The plasmid concentration was 1 μg / mL. The experimental groups were PBS, NP, and NP. Tyr-APM-EGFP After 6 hours, the culture medium was replaced with complete culture medium, and the cells were cultured for another 48 hours. One sample was stained with Hoechst 33342 to detect EGFP expression in different cell types using a fluorescence microscope. The other sample was digested with trypsin, cells were collected, washed once with PBS, centrifuged at 200g for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 300 μL of PBS. The pellet was then transferred through a 200-mesh nylon mesh to a flow cytometer to detect EGFP expression in different cell types. Results are as follows: Figure 7 As shown, EGFP fluorescence was expressed only in B16-F10 cells, and the EGFP positivity rate, as detected by flow cytometry, was high in B16-F10 cells, while it was low or absent in other cells.

[0104] Test Example 5

[0105] Gene nanomedicines for expressing multivalent PD-L1 antibodies NP Tyr-APM Detection of in vitro multivalent PD-L1 antibody mRNA

[0106] Four types of tumor cell lines (B16-F10, CT-26, Pan02, 4T1) and four types of non-tumor cell lines (NIH / 3T3, RAW264.7, DC2.4, C2C12) with good growth were digested with trypsin and counted. 5.0 × 10⁻⁶ cells were collected. 4Cells were seeded at a density of 1000 cells / well in 24-well plates and cultured overnight in a cell culture incubator at 37°C and 5% CO2. After 12 hours, cell adhesion and cell status were observed under a microscope. The old culture medium was removed, and serum-free culture medium containing particles was added. The plasmid concentration was 1 μg / mL. The experimental groups were PBS, NP, and NP. Tyr-APM After 6 hours, the culture medium was replaced with complete medium, and the cells were cultured for another 48 hours. Cells were then collected, and RNA was extracted using RNAiso Plus and reverse transcribed. qRT-PCR was then used to detect the transfection of NP cells. Tyr-APM The expression of the PD-L1 antibody gene in different cell types was then analyzed. The results are as follows: Figure 8 As shown, the multivalent PD-L1 antibody mRNA is expressed only in B16-F10. NP Tyr-APM It can specifically identify and act on melanoma B16-F10.

[0107] Test Example 6

[0108] Gene nanomedicines for expressing multivalent PD-L1 antibodies NP Tyr-APM Detection of multivalent PD-L1 antibodies in vivo

[0109] Mice were sacrificed after treatment, and their tumors and organs were removed and rapidly cryopreserved in liquid nitrogen. The tumors and organs were ground into powder using a mortar containing liquid nitrogen, and then RIPA lysis buffer was added to extract total protein from the tumors. The mixture was lysed on ice for 30 min. The mixture was then centrifuged at 10,000 g for 15 min, and the supernatant was collected. The total protein was quantified using a BCA protein quantification kit. The sample was adjusted to the same protein concentration using PBS, and protein loading buffer was added. The sample was heated at 95°C for 20 min.

[0110] After SDS-PAGE gel running, the sample was transferred to a membrane and then blocked with 5% skim milk powder. The sample was labeled with anti-His antibody and GAPDH antibody. It was incubated with secondary antibody goat anti-mouse IgG-HRP, developed with ELC chemiluminescence imaging solution, and imaged using a multi-functional imaging system. NP was detected by Western blotting. Tyr-APM Protein expression levels of PD-L1 antibodies in tumors and various organs after intravenous administration. Results are as follows: Figure 9 As shown, the protein of the multivalent PD-L1 antibody is expressed only in B16-F10.

[0111] Test Example 7

[0112] Gene nanomedicines for expressing multivalent PD-L1 antibodies NP Tyr-APM Expressing multivalent PD-L1 antibodies in melanoma, which have a stronger affinity for PD-L1, can enhance T cell activation and tumor growth inhibition.

[0113] After hair removal, B16-F10 melanomas were subcutaneously implanted into C57BL / 6J mice. The tumor volume in the mice was approximately 50 mm². 3 Mice were randomly divided into four groups: PBS, NP, NP, and NP. Tyr-AP NP Tyr-APM The experimental group received intratumoral administration every other day for a total of three times, with each dose being 0.5 mg / kg. The length and width of the mouse tumors were measured daily using electronic calipers, and the mice were weighed daily using an electronic balance. The results are as follows: Figure 10 As shown, NP Tyr-APM It inhibited the growth of melanoma in mice. To evaluate NP... Tyr-APM To investigate its antitumor effects in vivo and its regulatory role on the tumor immune microenvironment, we established a B16-F10 melanoma-bearing model in C57BL / 6 mice and followed the experimental protocol ( Figure 10 A) When the tumor volume reaches approximately 80-100 mm 3 Intravenous administration began at [time], once every other day for a total of five times. Samples were collected for analysis on day 18. Tumor volume change curve ( Figure 10 (B, left) shows that the tumors in both the PBS and NP groups exhibited a sustained and rapid growth trend during drug administration, with the volume exceeding 1500 mm² at the end of day 18. 3 This indicates that the empty nanoparticles have no significant antitumor activity. Tyr-AP The tumor growth in this group slowed significantly, with a final volume of approximately 600 mm. 3 The levels were significantly lower in the PD-L1 group compared to the PBS group, suggesting that the monovalent PD-L1 antibody can inhibit tumor progression to some extent. Tyr-APM The group exhibited the most significant tumor-suppressing effect, with a terminal tumor volume of only about 250 mm. 3 Compared to NP Tyr-AP The group showed a further decrease, indicating that multivalent PD-L1 antibody delivery is more efficient in blocking immune checkpoint signaling and inhibiting tumor growth. Mouse body weight monitoring results ( Figure 10 As shown in Figure B (right), the body weight of each group did not fluctuate significantly and there was no statistical difference throughout the entire treatment period, indicating that the treatment regimen has good biosafety.

[0114] Tumor tissue was collected to detect T cell activation. Digestion solution was added to the minced tumor tissue, and PBS was added to terminate the digestion after 30 minutes. The tissue was centrifuged, the supernatant was discarded, and the cell pellet was resuspended in 40% Percoll solution. The pellet was centrifuged at 800g for 20 minutes at room temperature (increase speed 6, decrease speed 2). The supernatant was aspirated, and erythrocyte lysis buffer was added to resuspend the cell pellet. Lysis was performed at room temperature for 3 minutes, followed by PBS to terminate the lysis. The pellet was centrifuged at 3000rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in PBS. An appropriate amount of cells was passed through a 200-mesh nylon mesh into an EP tube, and anti-mouse CD16 / 32 antibody was added. The tube was blocked on ice for 15 minutes. Flow cytometry was then performed using BV510 anti-mouse CD45, FITC anti-mouse CD3, APC / Cy7 anti-mouse CD4, BV785 anti-mouse CD8, BV605 anti-mouse CD69, FITC anti-mouse CD11b, and BV421 anti-mouse antibodies. F4 / 80, BV650 anti-mouse CD80, and PE anti-mouse CD206 were used to detect T cell activation in tumors using flow cytometry. Results are as follows: Figure 10 As shown, NP Tyr-APM Enhanced blocking of the immune checkpoint PD-L1 promoted T cell activation and inhibited tumor growth. Flow cytometry results ( Figure 10 (C, left) shows the CD3 infiltration of tumors in the PBS group and the NP group. + The proportion of T cells was less than 15% in both cases, with no significant difference. NP Tyr-AP The group significantly increased to about 20%, while NP Tyr-APM The percentage further increased to approximately 25%, suggesting that multivalent antibody delivery can more effectively promote T cell infiltration into tumor tissue. (In CD8...) + The expression ratio of the activation marker CD69 in T cells ( Figure 10 C (in the PBS and NP groups) was below 10% in both the PBS and NP groups; NP Tyr-AP The group rose to about 15%, while NP Tyr-APM The proportion of M1 / M2 macrophages increased to approximately 24%, indicating that multivalent antibody delivery not only promoted T cell infiltration but also enhanced T cell activation levels, contributing to improved cytotoxic function. Figure 10 The right (C) reflects the pro-inflammatory / anti-inflammatory state of the tumor immune microenvironment. The M1 / M2 ratio in both the PBS and NP groups was approximately 0.8-1.2, indicating an immunosuppressive tendency; NP... Tyr-AP The group slightly increased to about 1.7, but the difference was not significant. Meanwhile, NP... Tyr-APMThe significant increase in the group to approximately 2.0 suggests that multivalent antibody delivery can significantly promote macrophage polarization towards pro-inflammatory M1, thereby improving the tumor immune microenvironment and enhancing the anti-tumor immune response.

[0115] In summary, NP Tyr-APM It exhibited superior tumor-suppressive effects compared to monovalent antibody delivery in the B16-F10 melanoma model, and its mechanism may be related to promoting CD3. + Cell infiltration, enhanced CD8 + T cell activation is closely related to the regulation of tumor-associated macrophages toward pro-inflammatory polarization, and it has good in vivo safety.

[0116] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A tumor gene nanomedicine for expressing multivalent PD-L1 antibodies, characterized in that, It includes plasmids and polymer nanoparticles; the plasmid is a gene expression plasmid for a multivalent PD-L1 antibody; the polymer nanoparticles are composed of ionizable lipids, cationic lipids and polymers, and can be used for in vivo and in vitro encapsulation and delivery of nucleic acid drugs.

2. The anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies according to claim 1, characterized in that, The plasmid is a plasmid expressing a multivalent PD-L1 antibody, and the multivalent PD-L1 antibody is a secretory IgM-like multivalent PD-L1 antibody.

3. The anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies according to claim 2, characterized in that, The multivalent PD-L1 antibody is formed by linking a multimer formed by fusing the mutated Fc fragment with the IgM-expressing multimer tail to at least one of an anti-PD-L1 single-chain variable region fragment and an anti-PD-L1 nanobody.

4. The anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies according to claim 1, characterized in that, The plasmid is at least one of human and mouse genes; the plasmid promoter is at least one of a strong promoter and a tumor-specific promoter; the strong promoter is at least one of a cytomegalovirus promoter, a strong hybrid mammalian promoter, and an elongation factor-1α promoter; the tumor-specific promoter is at least one of a tyrosinase promoter and a survivin promoter.

5. The anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies according to claim 1, characterized in that, The ionizable lipid is at least one of Dlin-MC3-DMA, SM-102, ALC-0315, Dlin-KC2-DMA, BHEM-DBA, BHEM-APMP, BHEM-EAA, and BHEM-AEA.

6. The anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies according to claim 1, characterized in that, The cationic lipid is at least one of trimethyl-2,3-diolenooxypropylammonium chloride, trimethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-bisoctadecyloxypropylammonium bromide, N-(2-speramido)-N',N'-bisoctadecylglycineamide, and 1,2-dioleoyl-3-succinoyl-sn-glycerolcholine ester; further, the cationic lipid is more preferably at least one of trimethyl-2,3-dioleoyloxypropylammonium bromide.

7. The anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies according to claim 1, characterized in that, The molar ratio of the cationic lipid to the ionizable lipid is 1:1-4; and / or the molar ratio of the cationic lipid to the nucleic acid is 1:1-4.

8. The anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies according to claim 1, characterized in that, The average particle size of the polymer nanoparticles is 50 nm to 350 nm; and / or, the polymer in the polymer nanoparticles is at least one of polyethylene glycol-modified poly(glycolic acid-co-lactide) and poly(glycolic acid-co-lactide); and / or, the LA / GA ratio in the polyethylene glycol-modified poly(glycolic acid-co-lactide) and the poly(glycolic acid-co-lactide) ranges from 95:5 to 50:50; and / or, the molecular weight of the polyethylene glycol ranges from 1000 to 10000 Daltons.

9. The method for preparing antitumor gene nanomedicine expressing multivalent PD-L1 antibodies according to any one of claims 1-8, characterized in that, include: Step 1: Mix the plasmid used to express the multivalent PD-L1 antibody with DEPC water to obtain the aqueous phase; Step 2: Mix the ionizable lipid, cationic lipid, and ethanol to obtain a lipid ethanol solution; mix the polymer and organic solvent to obtain a mixture; then mix the lipid ethanol solution and the mixture to obtain an oil phase; Step 3: Gently mix the oil phase and the aqueous phase to prepare a water-in-oil emulsion; after the emulsion is left to stand at room temperature, an anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies is obtained.

10. The anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies according to any one of claims 1-8, or the anti-tumor gene nanomedicine for expressing multivalent PD-L1 antibodies prepared by the method of claim 9, kills tumor cells by tail vein injection.

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