Liposome nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses liposome nanoparticles as well as a preparation method and application thereof, and relates to the field of biological medicines. The liposome nanoparticle comprises a lipid compound modified with a fusion protein, the fusion protein comprises apolipoprotein E or polypeptide thereof and an antibody, the modification of the fusion protein not only can actively target cells, tissues or organs of corresponding ligands, but also can effectively improve the transfection efficiency and the targeting delivery capability of the liposome nanoparticle to the cells, and the targeting delivery capability of the liposome nanoparticle to the cells can be effectively improved. The nucleic acid delivery system can be applied to preparation of anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to liposome nanoparticles and a preparation method and application thereof. Background Art

[0002] As one of the core technologies in modern biomedicine, gene therapy has become an important research direction for the treatment of various genetic diseases, cancers, and viral diseases. Nucleic acid drugs, including DNA, mRNA, and siRNA, have shown broad potential for preventing and treating diseases due to their ability to regulate or repair gene expression. However, the inherent properties of nucleic acid drugs make them extremely susceptible to degradation in the body and difficult to penetrate cell membranes independently, which greatly limits their effectiveness. Therefore, the development of safe, stable, and efficient delivery systems is a key technical challenge for the successful clinical application of nucleic acid drugs.

[0003] Currently, delivery vectors for nucleic acid drugs can be divided into two major categories: viral vectors and non-viral vectors. Viral vectors, such as adeno-associated virus (AAV) and lentivirus, have high gene transduction efficiency, but they also have several significant disadvantages: their immunogenicity may trigger a host immune response, their potential for gene integration may pose a risk of tumorigenesis, and their gene-carrying capacity is limited. Furthermore, the high production cost and complex process of viral vectors have severely limited their widespread adoption in the clinical application of nucleic acid drugs. In contrast, non-viral delivery vectors have garnered widespread attention due to their lower immunogenicity, improved biosafety, and high drug-loading capacity. Common non-viral vectors include lipid nanoparticles (LNPs), polymer nanoparticles, and cationic nanoemulsions. Among them, LNPs have demonstrated superior performance in delivering nucleic acid drugs such as mRNA and have become a hot topic of research in this field. LNPs have excellent biocompatibility, effectively protecting nucleic acid drugs from degradation by nucleases in the body, and enhancing their stability. Furthermore, LNPs have a large nucleic acid loading capacity, enabling large-scale production to achieve good batch-to-batch consistency.

[0004] While LNPs have achieved remarkable success in nucleic acid drug delivery, particularly playing a key role in the successful application of the COVID-19 mRNA vaccine, most LNPs currently face a significant technical bottleneck: insufficient specificity in targeted delivery. Studies have shown that LNPs are primarily absorbed by liver cells after entry into the body, with low delivery efficiency to other tissues or targeted cells. This nonspecific distribution significantly limits the application of LNPs in the treatment of various diseases, particularly in areas such as tumors and neurological disorders that require precise targeting of specific cells.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a liposome nanoparticle and a preparation method and application thereof.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides a liposome nanoparticle, which includes a lipid complex modified with a fusion protein; the fusion protein includes apolipoprotein E or its polypeptide, and an antibody or its antigen-binding fragment; the antibody or its antigen-binding fragment is connected to the C-terminus of the apolipoprotein E or its polypeptide.

[0009] In a second aspect, an embodiment of the present invention provides a composition comprising: the components of the lipid complex described in the aforementioned embodiment.

[0010] In a third aspect, embodiments of the present invention provide the use of the fusion protein described in the aforementioned embodiments in the preparation of liposome nanoparticles.

[0011] In a fourth aspect, an embodiment of the present invention provides a method for preparing the liposome nanoparticles as described in the aforementioned embodiment, comprising: preparing the liposome nanoparticles using the fusion protein described in the aforementioned embodiment.

[0012] In a fifth aspect, embodiments of the present invention provide uses of the fusion proteins described in the preceding embodiments or the liposome nanoparticles described in the preceding embodiments in the preparation of CAR-T cells, CAR-T transfection reagents, or products for producing in vivo CAR-T cells.

[0013] In a sixth aspect, embodiments of the present invention provide the use of the fusion protein described in the preceding embodiments or the liposome nanoparticles described in the preceding embodiments in the preparation of nucleic acid drug targeted delivery preparations or drugs for the prevention, treatment or auxiliary treatment of tumors.

[0014] The present invention has the following beneficial effects:

[0015] By coupling apolipoprotein E or its polypeptide with an antibody to obtain a fusion protein, which is then used to modify liposome nanoparticles, the modification of the fusion protein can not only actively target the cells, tissues or organs of the corresponding ligand, but also effectively improve the transfection efficiency and targeted delivery ability of the liposome nanoparticles to cells, achieve efficient delivery of nucleic acid drugs, and thus enhance the therapeutic effect of the drugs;

[0016] The liposome nanoparticles modified with fusion proteins provided in the embodiments of the present invention have the advantages of mild preparation conditions, simple route, wide applicability, and the like, and can be produced on a large scale with high quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 Schematic diagram of the structure of lipid nanoparticles modified with targeted antibody-APOE fusion protein;

[0019] Figure 2 Statistical graph of the in vitro Hela cell transfection percentage of GFP mRNA@LNP bound / unbound to APOE-B7H3VHH and B7H3VHH-APOE in Example 3;

[0020] Figure 3 Statistical graph of the in vitro Hela cell targeting of GFP mRNA@LNP bound / unbound to APOE-B7H3VHH and B7H3VHH-APOE in Example 3;

[0021] Figure 4 Statistical graph of the in vitro T cell transfection percentage of GFP mRNA@LNP bound / unbound to APOE-anti-CD3 antibody and anti-CD3 antibody-APOE in Example 4;

[0022] Figure 5 Statistical graph of in vitro T cell targeting of GFP mRNA@LNP bound / unbound to APOE-anti-CD3 antibody and anti-CD3 antibody-APOE in Example 4;

[0023] Figure 6 Statistical graph of the relative mean fluorescence intensity of in vitro T cell transfection of GFP mRNA@LNP bound / unbound to APOE-anti-CD3 antibody and anti-CD3 antibody-APOE in Example 4;

[0024] Figure 7 The distribution of Luc mRNA@LNP bound / unbound to APOE-B7H3VHH and B7H3VHH-APOE in various organs and tumors in mice after intravenous injection in Example 5;

[0025] Figure 8 The in vivo T cell targeted delivery of GFP mRNA@LNP bound / unbound to APOE-anti-CD3 antibody and anti-CD3 antibody-APOE in Example 6;

[0026] Figure 9Statistical graph of in vitro CAR-T cell preparation efficiency of B7H3.CARmRNA@LNP bound / unbound to APOE-anti-CD3 antibody and anti-CD3 antibody-APOE in Example 7;

[0027] Figure 10 The tumor killing activity of CAR-T cells prepared in vitro by nucleic acid lipid nanoparticles modified with antibody-APOE fusion protein in Example 8;

[0028] Figure 11 Preparation of in vivo mCD19 CAR-T cell CAR using mCD19 CAR mRNA@LNP modified with APOE (SEQ ID NO.1)-GGGGS-mCD3scFv in Example 9 + T cell positive rate;

[0029] Figure 12 Detection of tumor volume changes after intravenous injection of mCD19 CAR mRNA@LNP modified with APOE (SEQ ID NO. 1)-GGGGS-mCD3scFv in Example 9;

[0030] Figure 13 Monitoring the survival of tumor-bearing mice after intravenous injection of mCD19 CAR mRNA@LNP modified with APOE (SEQ ID NO. 1)-GGGGS-mCD3scFv in Example 9;

[0031] Figure 14 This is a statistical diagram of the in vitro targeting of GFP mRNA@LNP modified with different target antibodies-APOE fusion proteins in Example 10;

[0032] Figure 15 This is a statistical comparison of the in vitro targeting properties of GFP mRNA@LNP modified with APOE-DLL3scFv fusion protein of different APOE sequences (full length, polypeptide or fragment thereof) in Example 11; wherein SEQ1 to SEQ 13 are abbreviations of SEQ ID NO: 1 to SEQ ID NO: 13, respectively. DETAILED DESCRIPTION

[0033] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0034] On the one hand, an embodiment of the present invention provides a liposome nanoparticle, which includes a lipid complex modified with a fusion protein; the fusion protein includes apolipoprotein E or its polypeptide, and an antibody or its antigen-binding fragment; the antibody or its antigen-binding fragment is connected to the C-terminus of the apolipoprotein E or its polypeptide.

[0035] Liposome nanoparticles modified with fusion proteins can actively target and deliver drugs to target antigen-positive cells, tissues or organs, deliver the encapsulated drugs to the lesions, and thus increase the therapeutic effect of the drugs. They can be used for in vitro or in vivo targeted gene delivery, preparation of CAR-T cells (in vitro preparation), CAR-T transfection preparations and in vivo CAR-T cells, and preparation of anti-tumor drugs. Compared with unmodified fusion proteins or connecting antibodies or their antigen-binding fragments to the N-terminus of apolipoprotein E or its polypeptide, connecting the antibody or its antigen-binding fragment to the C-terminus of apolipoprotein E or its polypeptide can significantly improve the infection efficiency of liposome nanoparticles to cells, so that liposome nanoparticles have better ability to target target-positive cells.

[0036] In some embodiments, the apolipoprotein E includes any one of APOE2, APOE3, and APOE4.

[0037] In some embodiments, the apolipoprotein E comprises APOE4.

[0038] In some embodiments, the amino acid sequence of the apolipoprotein E or its polypeptide comprises a sequence that is at least 80% identical to the sequence shown in any one of SEQ ID NOs: 1 to 13.

[0039] As used herein, "having at least 80% identity" may mean: having an identity of any one of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100%, or a range between any two of the above.

[0040] In some embodiments, the amino acid sequence of the apolipoprotein E or its polypeptide is as shown in any one of SEQ ID NOs: 1 to 13.

[0041] In some embodiments, the antigen is selected from any one or more of B7H3, HER-2 / 3, EGFR, EPCAM, GPC3, CLDN18.2, NECTIN4, TROP2, DLL3, cMET, PSMA, PSCA, VEGFR, CD117, PD-1, PD-L1, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD52, CD38, BCMA, and CD45.

[0042] In some embodiments, the antibody comprises a monoclonal antibody;

[0043] In some embodiments, the antibody or antigen-binding fragment thereof comprises any one of VHH, scFv, F(ab')2, Fab', Fab, scFab, and dsFv.

[0044] In some embodiments, the apolipoprotein E polypeptide or fragment thereof and the antibody or antigen-binding fragment thereof are directly linked or linked via a linker.

[0045] In some embodiments, the linker includes (GGGGS)n, (GS)4, (Gly)m and Cys-X-Cys; wherein n is a positive integer selected from 1 to 3, specifically 1, 2 or 3; m is a positive integer selected from 2 to 4, specifically 2, 3 or 4, and X is selected from amino acids other than Cys.

[0046] In some embodiments, the components of the lipoplex include at least one of the fusion protein and a polyethylene glycol lipid modified with the fusion protein.

[0047] In some embodiments, the components further include at least one or more of: ionizable lipids, auxiliary phospholipids, cholesterol, and polyethylene glycol lipids.

[0048] In some embodiments, the ionizable lipid is at least one of DLin-MC3-DMA, SM-102, 5A2-SC8, and C12-200.

[0049] In some embodiments, the auxiliary phospholipid includes at least one of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).

[0050] In some embodiments, the polyethylene glycol lipid includes at least one of 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG), distearoyl-rac-glycerol-polyethylene glycol (DSG-PEG), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), dimyristoylphosphatidylethanolamine-polyethylene glycol (DMPE-PEG), and dioleoylphosphatidylethanolamine-polyethylene glycol (DOPE-PEG), and the PEG molecular weight is 500-5000; optionally, the polyethylene glycol lipid is DMG-PEG2000.

[0051] In some embodiments, the molar percentage of the fusion protein or the polyethylene glycol lipid modified by the fusion protein is 0.1% to 5%, specifically any one of 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or a range between any two of them.

[0052] In some embodiments, the molar percentage of the ionizable lipid is 35% to 65%, specifically any one of 35%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 65%, or a range between any two of them.

[0053] In some embodiments, the molar percentage of the auxiliary phospholipid is 10% to 20%, specifically any one of 10%, 12%, 14%, 16%, 18%, 20%, or a range between any two of them.

[0054] In some embodiments, the molar percentage of cholesterol is 20% to 50%, specifically any one of 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, or a range between any two of them.

[0055] In some embodiments, the molar percentage of the polyethylene glycol lipid is 1% to 10%, specifically any one of 1%, 2%, 4%, 6%, 8%, 10%, or a range between any two of them.

[0056] There are many ways to modify the lipid complex with a fusion protein. The fusion protein can be pre-modified on the components of the lipid complex. After all components are assembled, liposome nanoparticles modified with the fusion protein are obtained. Alternatively, a lipid complex that is not modified with a fusion protein can be prepared first and then mixed with the fusion protein. The apolipoprotein E or the C-terminus of its polypeptide in the fusion protein has a natural affinity for the lipid complex, and after mixing, it can self-assemble to obtain liposome nanoparticles modified with the fusion protein. The specific preparation process is described in the subsequent examples.

[0057] In some embodiments, the method for preparing the fusion protein-modified polyethylene glycol lipid comprises: modifying the fusion protein on the polyethylene glycol lipid by covalent coupling based on amino groups and / or thiol groups.

[0058] In some embodiments, the fusion protein modified with polyethylene glycol lipid is prepared from a polyethylene glycol lipid containing a maleimide terminus and a fusion protein containing a cysteine ​​terminus.

[0059] In some embodiments, the maleimide-terminated polyethylene glycol lipid comprises at least one of: distearoylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-Mal), dipalmitoylphosphatidylethanolamine-polyethylene glycol-maleimide (DPPE-PEG-Mal), dimyristoylphosphatidylethanolamine-polyethylene glycol-maleimide (DMPE-PEG-Mal), and dioleoylphosphatidylethanolamine-polyethylene glycol-maleimide (DOPE-PEG-Mal); the PEG molecular weight is 500-5000; optionally, the maleimide-terminated polyethylene glycol lipid is DSPE-PEG2000-Mal.

[0060] In some embodiments, the polyethylene glycol lipid modified by the fusion protein is DSPE-PEG2000-antibody or its antigen-binding fragment-APOE protein or its polypeptide.

[0061] In some embodiments, the lipid complex further encapsulates a nucleic acid drug. All nucleic acid drugs may be nucleic acid drugs composed of natural bases, nucleic acid drugs containing non-natural bases or base modifications, or nucleic acid analogs.

[0062] In some embodiments, the nucleic acid drug includes at least one of an RNA drug, an siRNA drug, an miRNA drug, a dsRNA drug, an antisense nucleic acid drug, an aptamer drug, and a DNA drug.

[0063] In some embodiments, the mass ratio of the nucleic acid drug to the lipid complex is 1:5 to 30, specifically any one of 1:5, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:22, 1:24, 1:26, 1:28, and 1:30, or a range between any two of them.

[0064] In some embodiments, the structural diagram of the liposome nanoparticles provided by the present invention is as follows Figure 1 shown.

[0065] On the other hand, an embodiment of the present invention provides a composition comprising: the components of the lipid complex described in any of the aforementioned embodiments.

[0066] In some embodiments, the composition is a pharmaceutical composition.

[0067] In some embodiments, the composition further comprises: a pharmaceutically acceptable carrier.

[0068] In some embodiments, the pharmaceutically acceptable carrier includes any one or more of a pH adjuster, an isotonicity adjuster, and a lyoprotectant.

[0069] In some embodiments, the pH adjuster includes but is not limited to one or more of Tris, HEPES, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and dipotassium hydrogen phosphate; the isotonicity adjuster includes but is not limited to one or more of sucrose, sodium chloride, and potassium chloride; the lyoprotectant includes but is not limited to one or more of sucrose, trehalose, lactose, and mannitol.

[0070] In some embodiments, the pharmaceutical composition is an injection or an inhalation liquid preparation, and the injection and inhalation liquid preparation can be prepared by conventional preparation methods in the art.

[0071] On the other hand, embodiments of the present invention provide use of the fusion protein as described in any of the aforementioned embodiments in preparing liposome nanoparticles.

[0072] On the other hand, an embodiment of the present invention provides a method for preparing liposome nanoparticles as described in any of the aforementioned embodiments, comprising: preparing the fusion protein described in any of the aforementioned embodiments.

[0073] In some embodiments, when the components of the lipid complex include fusion protein-modified polyethylene glycol lipids, the preparation method includes the following steps (1) or (2):

[0074] (1) Dissolving the components of the lipid complex (e.g., ionizable lipids, auxiliary phospholipids, cholesterol, polyethylene glycol lipids, and fusion protein-modified polyethylene glycol lipids) in an organic solvent to obtain an organic phase, and dissolving the nucleic acid drug in a buffer solution to obtain an aqueous phase; mixing the organic phase and the aqueous phase to obtain a receiving solution 1.

[0075] (2) Dissolving the other components of the lipid complex except the fusion protein-modified polyethylene glycol lipid (e.g., ionizable lipid, auxiliary phospholipid, cholesterol, and polyethylene glycol lipid) in an organic solvent to obtain an organic phase, and dissolving the nucleic acid drug solvent in a buffer to obtain an aqueous phase; mixing the organic phase and the aqueous phase and then mixing them with the fusion protein-modified polyethylene glycol lipid to obtain a receiving solution 2.

[0076] In some embodiments, after mixing the organic phase and the aqueous phase, the preparation method further comprises diluting the receiving solution 1 or the receiving solution 2 with a phosphate or Tris buffer, and purifying the solution by dialysis or ultrafiltration to obtain liposome nanoparticles modified with the fusion protein and loaded with the nucleic acid drug. Optionally, the pH of the Tris buffer is 7.4; optionally, the ultrafiltration step is performed using a microporous filter membrane (e.g., 0.22 μm) for sterile filtration.

[0077] (3) When the components of the lipid complex do not include polyethylene glycol lipid modified with a fusion protein, the preparation method includes the following steps: dissolving the components of the lipid complex in an organic solvent to obtain an organic phase, and dissolving the nucleic acid drug in a buffer solution to obtain an aqueous phase; mixing the organic phase and the aqueous phase to obtain a receiving solution 3, and mixing the receiving solution 3 with the fusion protein.

[0078] In some embodiments, before mixing the receiving solution 3 with the fusion protein, the preparation method further comprises: diluting the receiving solution 3 with a phosphate or Tris buffer, dialyzing to remove ethanol, and then mixing with the fusion protein.

[0079] In some embodiments, the flow rate ratio of the aqueous phase to the organic phase is 2 to 5:1, and the total flow rate is 2 to 20 mL / min. The flow rate ratio can be any one of 2:1, 3:1, 4:1, and 5:1, or a range between any two thereof; the total flow rate can be any one of 2 mL / min, 4 mL / min, 5 mL / min, 6 mL / min, 8 mL / min, 10 mL / min, 15 mL / min, and 20 mL / min, or a range between any two thereof.

[0080] In some embodiments, the organic solvent comprises anhydrous ethanol.

[0081] In some embodiments, the buffer comprises a citric acid buffer. Optionally, the citric acid buffer has a concentration of 10 to 100 mM and a pH of 3 to 5; the concentration can be any one of 10, 20, 40, 50, 60, 80, and 100 mM, or a range between any two thereof; and the pH can be any one of 3, 3.5, 3.8, 4, 4.2, 4.5, and 5, or a range between any two thereof.

[0082] On the other hand, embodiments of the present invention provide the use of the fusion protein as described in any of the foregoing embodiments or the liposome nanoparticles as described in any of the foregoing embodiments in the preparation of CAR-T cells, CAR-T transfection reagents, or products for producing in vivo CAR-T cells.

[0083] In addition, embodiments of the present invention also provide the use of the fusion protein described in any of the foregoing embodiments or the liposome nanoparticles described in any of the foregoing embodiments in the preparation of nucleic acid drug targeted delivery preparations or drugs for the prevention, treatment or auxiliary treatment of tumors.

[0084] In some embodiments, the formulation is used to deliver nucleic acid drugs to cells, tissues, or organs that are positive for target antigen expression.

[0085] In some embodiments, the tumor comprises a malignant tumor.

[0086] In some embodiments, the malignant tumor includes but is not limited to squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading Melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phakomatoses, edema (such as that associated with brain tumors) and Meigs syndrome, brain tumors and brain cancers, and head and neck cancers, and related metastases.

[0087] In some embodiments, adjuvant therapy refers to a clinical setting in which an individual has a history of cancer and is generally (but not necessarily) responsive to therapy, but is considered at risk for developing the disease because of the individual's history of cancer. The treatment or medication in "adjuvant therapy" refers to a subsequent treatment mode.

[0088] In some embodiments, the treatment refers to contacting (e.g., administering) the targeted lipid nanoparticles, nucleic acid-lipid nanoparticle compositions, and / or pharmaceutical formulations described herein after a subject has contracted a disease, thereby alleviating the symptoms of the disease compared to the absence of such contact, and does not necessarily mean that the symptoms of the disease are completely suppressed. Suffering from a disease means that symptoms of the disease appear in the body.

[0089] In some embodiments, the prevention refers to: before a subject develops a disease, by exposing the subject to the targeted lipid nanoparticles, nucleic acid lipid nanoparticle compositions and / or pharmaceutical preparations of the present invention, thereby reducing the probability of developing the disease and / or alleviating the symptoms after developing the disease compared to when the subject does not develop the disease, and does not mean that the disease must be completely suppressed.

[0090] In this article, the use of "may" means both performing a certain process and not performing a certain process.

[0091] In this document, “nanoparticle” is the same as “nanoparticle”; lipid nanoparticle is the same as “liposome nanoparticle”; “nucleic acid lipid nanoparticle” may refer to liposome nanoparticle encapsulated with nucleic acid.

[0092] As used herein, "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0093] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0094] Example 1: Preparation of APOE (SEQ ID NO. 1)-GGGGS-B7H3VHH and B7H3VHH-GGGGS-APOE (SEQ ID NO. 1) fusion proteins

[0095] The designed and synthesized APOE (SEQ ID NO. 1)-GGGGS-B7H3VHH sequence was amplified by PCR and cloned into the pcDNA3.1 expression vector (Invitrogen). The amino acid sequence of B7H3VHH is shown in SEQ ID NO. 14. The cells were transiently transfected into 293FT cells and cultured in shake flasks using FreeStyle™ serum-free medium (Life Technologies) for 5-7 days. The supernatant was collected, ultrafiltered by centrifugation, and then purified over a nickel column to obtain the His-tagged APOE (SEQ ID NO. 1)-GGGGS-B7H3VHH fusion protein.

[0096] The fusion proteins B7H3VHH-GGGGS-APOE (SEQ ID NO.1), APOE (SEQ ID NO.1)-GGGGS-CD3scFv and CD3scFv-GGGGS-APOE (SEQ ID NO.1) were prepared in the same manner, wherein the amino acid sequence of CD3scFv is shown in SEQ ID NO.15.

[0097] Example 2: Synthesis of Antibody-APOE Fusion Protein Modified Lipid Nanoparticles

[0098] DLin-MC3-DMA (ionizable lipid), cholesterol, DSPE (auxiliary phospholipid), and PEG lipid (PEG2000-DSPE, polyethylene glycol lipid) were dissolved in anhydrous ethanol at a molar ratio of 35:46.5:16:2.2 to obtain an organic phase. The mRNA encoding GFP or B7H3.CAR was dissolved in 50 mM citric acid buffered saline solution at pH 4.0 to obtain an aqueous phase. The mass ratio of lipid complex (ionizable lipid, cholesterol, auxiliary phospholipid and polyethylene glycol lipid) to mRNA was 10:1 and the volume ratio was 1:3. The two phases were quickly mixed using a nanoparticle preparation instrument and dialyzed using a 7 kDa dialysis bag for 24 hours. The buffer environment was replaced with PBS at pH 7.4 to remove ethanol to prepare GFP mRNA@LNP or B7H3.CAR mRNA@LNP lipid nanosolutions.

[0099] Then, the APOE (SEQ ID NO.1)-GGGGS-B7H3VHH, B7H3VHH-GGGGS-APOE (SEQ ID NO.1), APOE (SEQ ID NO.1)-GGGGS-CD3scFv or CD3scFv-GGGGS-APOE (SEQ ID NO.1) fusion protein (1 mg / mL) obtained in Example 1 was dissolved in PBS at pH 7.4 and added to the GFP mRNA@LNP or B7H3.CAR mRNA@LNP lipid nanosolution (1 mg / mL) and incubated at 4°C for 6 hours to obtain antibody-APOE fusion protein-modified GFP mRNA@LNP or B7H3.CAR mRNA@LNP.

[0100] Example 3: In vitro tumor cell targeted delivery of antibody-APOE fusion protein modified nucleic acid lipid nanoparticles

[0101] B7H3 + After HeLa cells (overexpressing human B7H3 molecules / mCherry fluorescent protein) were recovered and cultured, they were mixed at a ratio of 1:1 and then 1.0×10 5 The inoculum size of cells / mL was inoculated into a 12-well plate. After the cells adhered, the APOE (SEQ ID NO.1)-GGGGS-B7H3VHH modified GFP mRNA@LNP or B7H3VHH-GGGGS-APOE (SEQ ID NO.1) modified GFP mRNA@LNP prepared in Example 2 was added, and the unmodified GFP mRNA@LNP was used as a control. After culturing for 24 hours, the cells were collected and the expression of mCherry and GFP in the cells was detected by flow cytometry for analysis. The results are shown in Figure 2. Figure 2 , Figure 3 Compared with the unmodified GFP mRNA@LNP control group, the antibody-APOE fusion protein modified GFP mRNA@LNP can significantly improve the infection efficiency of cells and has a better ability to target target-positive cells.

[0102] Example 4: In vitro T cell-targeted delivery of antibody-APOE fusion protein modified nucleic acid lipid nanoparticles

[0103] CD3 - After culture, T cells (CD3 knockout) were mixed with T cells at a ratio of 1:1, and then 1.0×10 5The inoculation amount of cells / mL was inoculated into a 12-well plate. APOE (SEQ ID NO.1)-GGGGS-CD3scFv modified GFP mRNA@LNP or CD3scFv-GGGGS-APOE (SEQ ID NO.1) modified GFP mRNA@LNP prepared in Example 2 was added, and unmodified GFP mRNA@LNP was used as a control. After culturing for 24 hours, the cells were collected and the expression of cell CD3 molecules and GFP in the cells was detected by flow cytometry for analysis. The results are shown in Figure 2. Figures 4-6 Compared with the unmodified GFPmRNA@LNP control group, the antibody-APOE fusion protein modified GFP mRNA@LNP can significantly improve the infection efficiency of T cells and has better T cell targeting ability.

[0104] Example 5: In vivo cell-targeted delivery of antibody-APOE fusion protein modified nucleic acid lipid nanoparticles

[0105] The GFP mRNA in Example 2 was replaced with Luciferase mRNA (Luc mRNA) to prepare APOE (SEQ ID NO. 1)-GGGGS-B7H3VHH-modified Luc mRNA@LNP or B7H3VHH-GGGGS-APOE (SEQ ID NO. 1)-modified Luc mRNA@LNP, and unmodified Luc mRNA@LNP was used as a control.

[0106] The Luc mRNA@LNP solution (mRNA 1.0 mg / kg) obtained above was injected into the B7H3-implanted + In C57BL / 6 mice with a B16F10 (overexpressing human B7H3) subcutaneous melanoma model, the fluorescence intensity and organ distribution in the mice were imaged and analyzed using a live imaging device 6 hours later. Figure 7 As shown. Unmodified Luc mRNA@LNP is mainly distributed in the liver (about 50%), and only about 5% is distributed in tumor tissue. In comparison, APOE (SEQ ID NO.1)-GGGGS-B7H3VHH modified Luc mRNA@LNP or B7H3VHH-GGGGS-APOE (SEQ ID NO.1) modified Luc mRNA@LNP can effectively target tumor tissue and increase the distribution in tumor tissue, which is 68.2% and 49.8%, respectively. This result shows that antibody-APOE fusion protein modified nucleic acid lipid nanoparticles can be used for in vivo cell targeted delivery and have good targeted delivery effect.

[0107] Example 6: In vivo T cell-targeted delivery of antibody-APOE fusion protein modified nucleic acid lipid nanoparticles

[0108] According to Example 2, APOE (SEQ ID NO. 1)-GGGGS-CD3scFv-modified GFP mRNA@LNP or CD3scFv-GGGGS-APOE (SEQ ID NO. 1)-modified GFP mRNA@LNP was prepared, and unmodified GFP mRNA@LNP was used as a control.

[0109] The GFP mRNA@LNP solution (mRNA 1.0 mg / kg) obtained above was injected into C57BL / 6 mice via the tail vein. After 24 hours, the spleen was collected and cells were isolated. After antibody incubation, the expression of GFP in spleen T cells was detected by flow cytometry. Figure 8 As shown, GFP expression in spleen + The proportion of T cells. Compared with the unmodified GFP mRNA@LNP control group, the antibody-APOE fusion protein-modified GFP mRNA@LNP can more effectively target T cells in vivo.

[0110] Example 7: In vitro CAR-T cell preparation using nucleic acid lipid nanoparticles modified with antibody-APOE fusion protein

[0111] T cells were plated at 1.0 × 10 5 The inoculation amount of cells / mL was inoculated into a 12-well plate. APOE (SEQ ID NO.1)-GGGGS-CD3scFv modified B7H3.CAR mRNA@LNP or CD3scFv-GGGGS-APOE (SEQ ID NO.1) modified B7H3.CAR mRNA@LNP prepared in Example 2 was added, and the unmodified B7H3.CAR mRNA@LNP was used as a control. After 48 hours of culture, the cells were collected and the expression of cell CD3 molecules and B7H3.CAR was detected by flow cytometry for analysis. The results are shown in Figure 2. Figure 9 Compared with the unmodified B7H3.CAR mRNA@LNP control group, the antibody-APOE fusion protein modified B7H3.CAR mRNA@LNP can significantly improve the efficiency of CAR-T cell preparation.

[0112] Example 8: In vitro preparation of nucleic acid lipid nanoparticles modified with antibody-APOE fusion protein and tumor killing activity of CAR-T cells

[0113] The B7H3.CAR-T cells prepared based on APOE (SEQ ID NO. 1)-GGGGS-CD3scFv modified B7H3.CAR mRNA@LNP in Example 6 were mixed with B7H3 + Hela cells were co-cultured for killing experiments, and CD19.CAR-T cells were used as a control. Different effector-target ratios (CAR-T cells: Hela cell numbers) were used, and the cell killing was detected after 12 hours of co-culture. The results are shown in Figure 2. Figure 10 Compared with the CD19.CAR-T cell control group, the B7H3.CAR-T cells prepared in vitro by nucleic acid lipid nanoparticles modified with antibody-APOE fusion protein showed good tumor killing activity.

[0114] Example 9: In vivo preparation of CAR-T cells and their anti-tumor activity using nucleic acid lipid nanoparticles modified with antibody-APOE fusion protein

[0115] Construction of mouse B cell lymphoma model: Fifteen normal healthy 6-8 week old Balb / c mice were selected and inoculated subcutaneously with A20 cells (1.0×10 6 cells / mouse), and there were 5 mice in each group.

[0116] According to the method in Example 2, APOE (SEQ ID NO. 1)-GGGGS-mCD3scFv modified mCD19 CAR mRNA@LNP was prepared, and unmodified mCD19 CAR mRNA@LNP was used as a control. On the 7th day after Balb / c mice were inoculated with mouse B cell lymphoma cells, the modified or unmodified mCD19 CAR mRNA@LNP was intravenously infused into the tumor-bearing mice at different doses (0.5 mg / kg or 2 mg / kg). After 48 hours, the expression of CAR in peripheral blood of different treatment groups was evaluated. + The positive rate of T cells was measured. At the same time, the tumor size of the mice was measured regularly with a vernier caliper, and the survival time of the tumor-bearing mice was observed and recorded.

[0117] The results are as attached Figure 11 , Figure 12 and Figure 13 Compared with unmodified mCD19 CAR mRNA@LNP, APOE (SEQID NO.1)-GGGGS-mCD3scFv modified mCD19 CAR mRNA@LNP can significantly increase the mCD19 CAR expression in peripheral blood. +The ratio of T cells in the tumor tissues was 247.3% and 29.7% respectively, indicating that the antibody-APOE fusion protein modified nucleic acid lipid nanoparticles can be used to prepare in vivo CAR-T cells. At the same time, compared with the unmodified mCD19 CAR mRNA@LNP, APOE (SEQ ID NO.1)-GGGGS-mCD3scFv modified mCD19 CAR mRNA@LNP can effectively inhibit tumor growth and prolong the survival of tumor-bearing mice, indicating that the antibody-APOE fusion protein modified nucleic acid lipid nanoparticles can be used to prepare in vivo CAR-T cells and thus exert their anti-tumor activity.

[0118] Example 10: In vitro cell-targeted delivery of nucleic acid lipid nanoparticles modified with different target antibodies-APOE fusion proteins

[0119] HeLa cells overexpressing different antigen targets (co-expressing mCherry) were revived and cultured with HeLa cells, mixed at a ratio of 1:1, and then 1.0×10 5 The inoculum size of cells / mL was inoculated into a 12-well plate. After the cells adhered to the wall, the prepared APOE (SEQ ID NO.1)-GGGGS-antigen target scFv modified GFP mRNA@LNP or antigen target scFv-GGGGS-APOE (SEQ ID NO.1) modified GFP mRNA@LNP was added, and the unmodified GFP mRNA@LNP was used as a control. After culturing for 24 hours, the cells were collected and the expression of mCherry and GFP in the cells was detected by flow cytometry for analysis. The results are shown in Figure 2. Figure 14 Compared with the unmodified GFP mRNA@LNP control group, GFP mRNA@LNP modified with different target antibodies-APOE fusion proteins can significantly improve the targeting of antigen target positive cells.

[0120] Example 11: Comparison of in vitro cell targeting of nucleic acid lipid nanoparticles modified with APOE-DLL3 scFv fusion proteins of different APOE sequences (full length, polypeptide or fragment thereof)

[0121] DLL3 + After HeLa cells (overexpressing human DLL3 molecules / mCherry fluorescent protein) were recovered and cultured, they were mixed at a ratio of 1:1 and then 1.0×10 5The cells were inoculated into 12-well plates at a seeding rate of 10 cells / mL. After the cells adhered to the wall, the prepared APOE (SEQ ID NO.1-13)-GGGGS-DLL3scFv modified GFP mRNA@LNP was added, and the unmodified GFP mRNA@LNP was used as a control. After culturing for 24 hours, the cells were collected and the expression of mCherry and GFP in the cells was detected by flow cytometry for analysis. The results are shown in Figure 2. Figure 15 As shown in the figure. Compared with the unmodified GFP mRNA@LNP control group, GFP mRNA@LNP modified with APOE-DLL3scFv fusion proteins of different APOE sequences (full-length, polypeptide, or fragment) can significantly improve the targeting of antigen-positive cells. Compared with APOE-DLL3scFv with a functional LDLR binding domain, APOE-DLL3scFv modified with a functional LDLR binding domain can further improve the targeting effect on antigen-positive cells.

[0122] The sequences involved in this application are shown in the table below.

[0123]

[0124]

[0125]

[0126] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A liposome nanoparticle, characterized in that: The invention comprises a lipid complex which is modified with a fusion protein; the fusion protein comprises apolipoprotein E or its polypeptide, and an antibody or its antigen-binding fragment; the antibody or its antigen-binding fragment is connected to the C-terminus of the apolipoprotein E or its polypeptide.

2. The liposome nanoparticle according to claim 1, characterized in that The apolipoprotein E includes any one of APOE2, APOE3 and APOE4; Optionally, the apolipoprotein E comprises APOE4; Optionally, the amino acid sequence of apolipoprotein E or a polypeptide thereof comprises a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NOs: 1 to 13; Optionally, the amino acid sequence of the apolipoprotein E or its polypeptide is as shown in any one of SEQ ID NOs: 1 to 13.

3. The liposome nanoparticle according to claim 1, characterized in that The antigen is selected from any one or more of B7H3, HER-2 / 3, EGFR, EPCAM, GPC3, CLDN18.2, NECTIN4, TROP2, DLL3, cMET, PSMA, PSCA, VEGFR, CD117, PD-1, PD-L1, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD52, CD38, BCMA and CD45; Optionally, the antibody comprises a monoclonal antibody; Optionally, the antibody or antigen-binding fragment thereof includes any one of VHH, scFv, F(ab')2, Fab', Fab, scFab, and dsFv.

4. The liposome nanoparticle according to claim 1, characterized in that The apolipoprotein E polypeptide or fragment thereof and the antibody or antigen-binding fragment thereof are directly connected or connected through a linker; Optionally, the linker includes (GGGGS)n, (GS)4, (Gly)m and Cys-X-Cys; wherein n is a positive integer selected from 1 to 3, m is a positive integer selected from 2 to 4, and X is selected from amino acids other than Cys.

5. The liposome nanoparticle according to any one of claims 1 to 4, characterized in that The components of the lipid complex include: at least one of the fusion protein and a polyethylene glycol lipid modified with the fusion protein; Optionally, the components further include: at least one or more of ionizable lipids, auxiliary phospholipids, cholesterol, and polyethylene glycol lipids; Optionally, the molar percentage of the fusion protein or the polyethylene glycol lipid modified by the fusion protein is 0.1% to 5%; Optionally, the molar percentage of the ionizable lipid is 35% to 65%; Optionally, the molar percentage of the auxiliary phospholipid is 10% to 20%; Optionally, the molar percentage of cholesterol is 20% to 50%; Optionally, the molar percentage of the polyethylene glycol lipid is 1% to 10%; Optionally, the preparation method of the fusion protein modified polyethylene glycol lipid comprises: modifying the fusion protein on the polyethylene glycol lipid by covalent coupling based on amino or sulfhydryl groups; Optionally, the lipid complex further encapsulates a nucleic acid drug; Optionally, the nucleic acid drug includes at least one of: RNA drug, siRNA drug, miRNA drug, dsRNA drug, antisense nucleic acid drug, aptamer drug and DNA drug.

6. A composition, characterized in that It includes: A component of the lipid complex according to any one of claims 1 to 5.

7. Use of the fusion protein according to any one of claims 1 to 5 in the preparation of liposome nanoparticles.

8. The method for preparing liposome nanoparticles according to any one of claims 1 to 5, wherein: The method comprises: preparing the fusion protein according to any one of claims 1 to 5; Optionally, when the components of the lipid complex include fusion protein-modified polyethylene glycol lipids, the preparation method includes the following steps (1) or (2): (1) dissolving the components of the lipid complex in an organic solvent to obtain an organic phase, dissolving the nucleic acid drug in a buffer to obtain an aqueous phase; and mixing the organic phase and the aqueous phase; (2) dissolving the components of the lipid complex except the fusion protein-modified polyethylene glycol lipid in an organic solvent to obtain an organic phase, and dissolving the nucleic acid drug solvent in a buffer to obtain an aqueous phase; mixing the organic phase and the aqueous phase and then mixing with the fusion protein-modified polyethylene glycol lipid; When the components of the lipid complex do not include a polyethylene glycol lipid modified with a fusion protein, the preparation method comprises the following steps: dissolving the components of the lipid complex in an organic solvent to obtain an organic phase, dissolving the nucleic acid drug in a buffer to obtain an aqueous phase; mixing the organic phase and the aqueous phase, and then mixing with the fusion protein; Optionally, the organic solvent includes anhydrous ethanol: Optionally, the buffer comprises a citrate buffer.

9. Use of the fusion protein according to any one of claims 1 to 5 or the liposome nanoparticles according to any one of claims 1 to 5 in the preparation of CAR-T cells, CAR-T transfection reagents, or products for producing in vivo CAR-T cells.

10. Use of the fusion protein according to any one of claims 1 to 5 or the liposome nanoparticles according to any one of claims 1 to 5 in preparing a nucleic acid drug targeted delivery preparation or a drug for preventing, treating or assisting in the treatment of tumors.

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