Construction method and anti-tumor application of multifunctional adoptive T cell

Abstract

The invention discloses a construction method and anti-tumor application of a multifunctional adoptive T cell. An active targeting peptide is fused on the surface of the multifunctional adoptive T cell through a membrane fusion liposome, and the membrane fusion liposome comprises an inner core and a shell; the inner core comprises iron oxide, a nano material and mRNA; the shell comprises phospholipid structure lipid, cationic functional lipid and active targeting peptide modified polyethylene glycol functional lipid. The multifunctional T cell provided by the invention has enhanced tumor infiltration capability, capability of overcoming immune tolerance of tumor cells and capability of reversing an immunosuppressive tumor microenvironment, and the preparation process is simple to operate, time-saving and efficient.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a construction method of multifunctional adoptive T cells and their anti-tumor application. Background Art

[0002] Adoptive cell therapy, especially CAR T cell therapy, has shown significant efficacy against certain blood tumors in clinical trials. Adoptive T cell transfer therapy mainly involves the extraction, in vitro engineering and expansion of patient T cells, and transfusion back into the patient. However, when facing solid tumors, the effect of this therapy has not yet met expectations, which is mainly attributed to the following key factors, including the insufficient tumor infiltration ability of adoptive cells, the immune tolerance of tumor cells, and the immunosuppressive tumor microenvironment. The lack of infiltration and penetration ability is mainly due to the lack of vascular endothelial barrier and tumor cell adhesion molecules, which makes it difficult for adoptively transferred T cells to penetrate into tumor tissues and infiltrate effectively. At the same time, tumor cells develop immune tolerance mechanisms by expressing immune checkpoint proteins, thereby resisting the killing effect of T cells. In addition, inhibitory cytokines and regulatory immune cells in the immunosuppressive tumor microenvironment will also hinder the activation and proliferation of reinfused T cells and other immune cells. Therefore, in order to enhance the efficacy of adoptive T cell transfer therapy for solid tumors, it is urgent to engineer T cells for multifunctionality to overcome the above challenges.

[0003] In the current research field, a variety of strategies have been reported for designing engineered T cells with specific functions to treat solid tumors. Among them, the strategy of using an external magnetic field to guide MNP-functionalized T cells has been shown to significantly enhance the infiltration and penetration of T cells into solid tumors. In addition, by modifying ligands (such as peptides, aptamers, etc.) targeting specific tumor immune checkpoints on the surface of T cells, active recognition of tumor cells can be achieved and the immune tolerance of tumor cells can be overcome. The strategy of transfecting T cells with messenger RNA expressing proinflammatory cytokines has been shown to alleviate the immunosuppressive tumor microenvironment by weakening the effects of immunosuppressive cytokines, recruiting immune cells with tumor-killing ability, and reducing the number of regulatory immune cells. Although these strategies show the potential to cope with a single challenge, they can often only achieve a single function and are not enough to fully overcome the multiple obstacles of solid tumors.

[0004] In addition, given the differences in the sites where different engineered reagents function in T cells, existing vectors make it difficult to simultaneously deliver multiple engineered reagents with different physical and chemical properties to their respective functional sites, which in turn makes the construction of multifunctional T cells dependent on complex and time-consuming engineering procedures.

[0005] Therefore, there is an urgent need to develop a T cell engineering platform that can simultaneously transform multiple functional targets at different subcellular locations to achieve multifunctional integration. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the purpose of the present invention is to provide a method for constructing multifunctional adoptive T cells and their anti-tumor applications. The first advantage of the present invention is that it can produce multifunctional T cells in a short time, making the process of engineering T cells more time-saving and efficient. The second is that the multifunctional T cells prepared by the present invention have enhanced tumor infiltration ability, the ability to overcome tumor cell immune tolerance, and the ability to reverse the immunosuppressive tumor microenvironment, so that they can overcome the three major obstacles in the treatment of solid tumors, namely, the insufficient tumor infiltration ability of adoptive cells, the immune tolerance of tumor cells, and the tumor microenvironment with immunosuppressive effects.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a membrane fusion liposome, comprising an inner core and an outer shell; the inner core comprises iron oxide, nanomaterials and mRNA; the outer shell comprises phospholipid structure lipids, cationic functional lipids and polyethylene glycol functionalized lipids modified with active targeting peptides.

[0009] The membrane fusion liposomes with a core-shell structure constructed by the present invention can fuse with the cytoplasmic membrane. This fusion not only allows the components of the lipid shell to be inserted into the cell membrane, but also effectively delivers the contents of the liposome core to the cytoplasm. Thanks to the specific distribution of the core and shell components, the fusion liposome technology facilitates the simultaneous transformation of multiple functionalized targets at different subcellular locations of the cell, thereby expanding the possibility of using a single nanoplatform to quickly construct T cells with comprehensive functions. This method indicates that in the field of cell therapy, especially in the engineering of multifunctional T cells for the treatment of solid tumors, more efficient and accurate customization of cell functions can be achieved.

[0010] The iron oxide core constructed by the present invention has paramagnetism, which can give multifunctional T cells the ability to enhance tumor infiltration under the control of an external magnetic field. The IL-12 mRNA used can enhance the IL-12 secretion level of multifunctional T cells and activate the tumor treatment effect of endogenous immune cells.

[0011] Preferably, the molar mass ratio of the phospholipid structure lipid, the cationic functional lipid and the active targeting peptide-modified polyethylene glycol functionalized lipid is (15-20):(2-5):1 (e.g., 15:2:1, 15:5:1, 76.2:20:3.8, 20:2:1, 20:5:1).

[0012] Preferably, the nanomaterial comprises any one or a combination of at least two of albumin, polycaprolactone, polyoxalate, poly-β-amino lipid, chitosan, nano-calcium carbonate or mesoporous silicon nanomaterials.

[0013] Preferably, the surface of the nanomaterial is modified with polyethyleneimine.

[0014] The polyethyleneimine-modified nanomaterial constructed by the present invention can effectively compress mRNA through electrostatic interaction to form a stable nanostructure, which is beneficial to the intracellular delivery of mRNA.

[0015] Preferably, the mRNA includes IL-12 mRNA and / or enhanced green fluorescent protein mRNA.

[0016] Preferably, the phospholipid structure lipid includes any one of dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or dioleoylphosphatidylethanolamine, or a combination of at least two thereof.

[0017] Preferably, the cationic functional lipid comprises trimethyl-2,3-dioleyloxypropylammonium bromide and / or trimethyl-2,3-dioleyloxypropylammonium chloride.

[0018] Preferably, the polyethylene glycol functionalized lipid comprises distearoylphosphatidylethanolamine-polyethylene glycol.

[0019] Preferably, the active targeting peptide comprises DPPA peptide.

[0020] The DPPA peptide constructed by the present invention has the ability to target the PD-L1 receptor highly expressed on the surface of tumor cells, and endows multifunctional T cells with the ability to actively recognize and kill tumor cells.

[0021] In a second aspect, the present invention provides a method for preparing the membrane fusion liposome according to the first aspect, the method comprising the following steps:

[0022] (1) mixing iron oxide, nanomaterials, mRNA and a solvent to obtain a suspension of polymer nanocores loaded with mRNA;

[0023] (2) taking phospholipid structure lipids, cationic functional lipids and polyethylene glycol functionalized lipids, and using a thin film hydration method to prepare a suspension of a preliminary membrane fusion liposome shell;

[0024] (3) mixing the suspension of the polymer nanocore loaded with mRNA with the suspension of the membrane fusion liposome shell obtained in step (2) to obtain preliminary membrane fusion liposomes;

[0025] (4) The preliminary membrane fusion liposomes obtained in step (3) are mixed with a solution containing an active targeting peptide, and the active targeting peptide is modified on the surface of the preliminary membrane fusion liposomes to obtain membrane fusion liposomes.

[0026] In a third aspect, the present invention provides a multifunctional adoptive T cell, wherein an active targeting peptide is fused to the surface of the multifunctional adoptive T cell via a membrane fusion liposome; and mRNA is delivered to the interior of the multifunctional adoptive T cell via a membrane fusion liposome.

[0027] Preferably, the membrane fusion liposome comprises the membrane fusion liposome described in the first aspect.

[0028] The preparation and principle diagram of membrane fusion liposome engineered T cells for solid tumor treatment in the present invention are as follows: Figure 1 shown.

[0029] In a fourth aspect, the present invention provides a method for preparing the multifunctional adoptive T cells according to the third aspect, the method comprising:

[0030] The activated T cells are co-incubated with the membrane fusion liposomes described in the first aspect to obtain multifunctional adoptive T cells.

[0031] In a fifth aspect, the present invention provides the use of the membrane fusion liposome described in the first aspect and / or the multifunctional adoptive T cells described in the third aspect in the preparation of drugs for anti-tumor and / or reversal of immunosuppressive tumor microenvironment.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The membrane fusion liposomes of the present invention are composed of a lipid shell modified with a targeting peptide and having a membrane fusion effect, and an inner core containing magnetic nanoparticles and IL-12 mRNA. They are uniform in size and can efficiently engineer human T cells and mouse T cells;

[0034] (2) The adoptive T cells prepared by the present invention have enhanced tumor infiltration ability, the ability to overcome tumor cell immune tolerance and the ability to reverse the immunosuppressive tumor microenvironment, and can overcome the three major obstacles in the treatment of solid tumors (i.e., insufficient tumor infiltration ability of adoptive cells, immune tolerance of tumor cells and tumor microenvironment with immunosuppressive effects), thereby achieving the purpose of enhancing the treatment effect of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the preparation and principle of immune encapsulated T cell membrane fusion liposome engineered T cells for solid tumor treatment;

[0036] Figure 2AThe 10-nanometer nuclear magnetic resonance (NMR) and mass spectrometry spectra of DPPA peptide (CNYSKPTDRQYHF);

[0037] Figure 2B DSPE-PEG-Mal is a hydrogen nuclear magnetic resonance spectrum and a mass spectrum;

[0038] Figure 2C It is the nuclear magnetic resonance hydrogen spectrum and mass spectrum of DSPE-PEG-DPPA;

[0039] Figure 3 Transmission electron microscopy images of MNP-PEI and MNP-PEI / IL-12mRNA;

[0040] Figure 4A Gel retardation electrophoresis diagrams of different MNP-PEI / mRNA;

[0041] Figure 4B is the particle size diagram of different MNP-PEI / mRNA;

[0042] Figure 4C Surface potential diagrams of different MNP-PEI / mRNA;

[0043] Figure 5A The particle size diagrams of MNP-PEI, MNP-PEI / IL-12mRNA and membrane fusion liposomes;

[0044] Figure 5B Potential diagrams of MNP-PEI, MNP-PEI / IL-12mRNA and membrane fusion liposomes;

[0045] Figure 5C Transmission electron microscopy images of membrane fusion liposomes;

[0046] Fig. 6A Schematic diagram of the experimental principle of fluorescence resonance energy transfer (FRET) between membrane fusion liposomes and non-membrane fusion liposomes;

[0047] Figure 6B The diagram shows the membrane fusion efficiency of liposomes with different DOTAP contents quantified based on FRET experiments;

[0048] Figure 6C FRET curves of membrane fusion liposomes and non-membrane fusion liposomes;

[0049] Fig. 7A Confocal microscopic images of T cells treated with membrane fusogenic liposomes and non-membrane fusogenic liposomes;

[0050] Figure 7B for Fig. 7A The distribution of YOYO-1 and DiD signals along the white arrows;

[0051] Figure 7C for Fig. 7A Quantitative results of the co-localization level of YOYO-1 and DiD signals in the figure;

[0052] Figure 8 Figure 2 shows the results of flow cytometry analysis of liposome uptake under low temperature or pretreatment with different inhibitors;

[0053] Fig.9A The graph shows the uptake results of YOYO-1 and DiD by T cells under different concentrations;

[0054] Fig. 9B The results of T cell uptake of YOYO-1 and DiD after treatment for different time periods;

[0055] Fig. 10A Confocal microscopy images of non-engineered or engineered T cells co-incubated with B16F10 tumor cells;

[0056] Fig. 10B The figure shows the flow cytometry results of co-incubation of non-engineered or engineered T cells with B16F10 tumor cells;

[0057] Fig. 10C for Fig. 10B Quantitative result diagram of

[0058] Fig.11A The results of cell activity of B16F10 tumor cells after treatment with different doses of T cells;

[0059] Fig. 11B This is a graph showing the cell activity results of RM-1 tumor cells after T cell treatment;

[0060] Fig. 12A The result graph is the IFNγ cytokine level in the supernatant of T cells and tumor cells co-cultured;

[0061] Fig. 12B The result graph is the TNFα cytokine level in the supernatant of co-culture of T cells and tumor cells;

[0062] Fig.13A This is the result diagram of the hysteresis loop of membrane fusion liposomes and engineered T cells;

[0063] Fig. 13B This is a graph showing the migration rate of engineered T cells under the action of an external magnetic field;

[0064] Fig. 13C This is a diagram of the migration direction of engineered T cells under the action of external magnetic fields in different directions;

[0065] Fig.14AFlow chart of the in vivo biodistribution experiment of engineered T cells;

[0066] Fig. 14B This is an in vivo imaging image of the biodistribution of engineered T cells;

[0067] Fig. 14C This is a distribution diagram of engineered T cells in tumor tissue sections;

[0068] Fig.15A The figure is the ratio of endogenous T cells and engineered T cells based on flow cytometry;

[0069] Fig. 15B Graph showing the percentage of injected dose quantified based on flow cytometry. DETAILED DESCRIPTION

[0070] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0071] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0072] In order to more easily understand the present invention, some terms are first defined. As used in the present invention, unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. All patents and publications mentioned in the present invention are incorporated into the present invention in their entirety by reference.

[0073] The term "DMPC" refers to dimyristoylphosphatidylcholine.

[0074] The term "DOTAP" refers to trimethyl-2,3-dioleyloxypropylammonium bromide.

[0075] The term "DSPE-PEG-Mal" refers to distearoylphosphatidylethanolamine-polyethylene glycol-maleimide.

[0076] The term "DSPE-PEG-DPPA" refers to distearoylphosphatidylethanolamine-polyethylene glycol-DPPA.

[0077] The term "EGFP" refers to enhanced green fluorescent protein.

[0078] The term "IL-12" refers to interleukin 12.

[0079] The term "mRNA" refers to messenger RNA.

[0080] The term "PEI" refers to polyethyleneimine.

[0081] The term "MNP-PEI" refers to polyethyleneimine-modified magnetic nanoparticles.

[0082] The term "PCR" refers to polymerase chain reaction.

[0083] The term "DEPC" refers to diethyl pyrocarbonate.

[0084] The term "FRET" refers to Fluorescence Resonance Energy Transfer.

[0085] The term "DiO" refers to 3,3'-dioctadecyloxycarbocyanine perchlorate.

[0086] The term "DiI" refers to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate.

[0087] The term "DiD" refers to 1,1'-tetracosyl-4,4'-dipyridylacridinium iodide.

[0088] The term "DiR" refers to 1,1'-dioctadecyl-3,3,3',3'-tetramethylindole tricarbene iodide.

[0089] The term "DAPI" refers to 4',6-diamidino-2-phenylindole.

[0090] The term "Cal-AM" refers to Calcein AM.

[0091] The term "CLSM" refers to confocal laser scanning microscopy.

[0092] The term "FACS" refers to fluorescence activated cell sorting.

[0093] The term "ELISA" refers to enzyme-linked immunosorbent assay.

[0094] The term "FBS" refers to fetal bovine serum.

[0095] The term "BSA" refers to bovine serum albumin.

[0096] The term "PFA" refers to paraformaldehyde.

[0097] The term "PBS" refers to phosphate buffered saline.

[0098] The term "H&E" refers to hematoxylin and eosin.

[0099] Preparation Example 1

[0100] This preparation example provides a method for preparing a polypeptide-modified lipid molecule for engineering T cells, and the specific method includes:

[0101] The synthesis of DSPE-PEG-DPPA is based on the Michael addition reaction between the thiol group of the cysteine ​​residue in the DPPA peptide (CNYSKPTDRQYHF) and the maleimide group in DSPE-PEG-Mal. First, 3.53 mg of DPPA peptide was dissolved in 2 mL of deionized water. At the same time, 10 mg of DSPE-PEG-Mal was dissolved in 0.5 mL of dimethylformamide. Under vigorous stirring, the DPPA peptide solution was slowly added dropwise to the DMF solution of DSPE-PEG-Mal. To ensure that the two reactants can fully contact and promote the Michael addition reaction. The mixture was stirred continuously at 25 ° C for 24 h. After the reaction was completed, the product was dialyzed with deionized water, and the molecular weight cutoff of the dialysis bag was 3000 Da to remove unreacted raw materials and by-products. The dialyzed product was freeze-dried to remove moisture to obtain dry DSPE-PEG-DPPA powder for easy storage and subsequent use. The synthesized DSPE-PEG-DPPA was characterized by 600 MHz nuclear magnetic resonance hydrogen spectrum and mass spectrometry to confirm its chemical structure. Figure 2A-2C shown.

[0102] Preparation Example 2

[0103] This preparation example provides a method for preparing polyethyleneimine-modified magnetic nanoparticles for engineering T cells, and the specific method includes:

[0104] First, in a round-bottom flask, 0.25 g of ferric chloride tetrahydrate (FeCl2·4H2O) and 0.05 g of 25k branched PEI were mixed. The above solid mixture was dissolved in 12.75 mL of deionized water to ensure complete dissolution to form a uniform solution. Under vigorous stirring, 1.25 mL of ammonia water was quickly added to the solution, and a black precipitate was observed, indicating the formation of Fe3O4. Subsequently, the reaction mixture solution was transferred to a hydrothermal autoclave reactor and treated at 134 ° C and 2 bar for 3 h to promote the formation of polyethyleneimine-modified magnetic nanoparticles (MNP-PEI). After the reaction was completed, the reaction mixture was cooled to 25 ° C, and the MNP-PEI product was collected by magnetic separation. The MNP-PEI was washed and purified with a large amount of deionized water to remove unreacted raw materials and by-products. The purified MNP-PEI was lyophilized to obtain a dry powder. Then, it was concentrated to 50 mg mL -1The concentration of MNP-PEI was redissolved in sterile deionized water for further use. Figure 3 shown.

[0105] Preparation Example 3

[0106] This preparation example provides a mRNA design and preparation method for improving the immune function of engineered T cells, and the specific method includes:

[0107] The mRNA of IL-12 and EGFP was synthesized by in vitro transcription (IVT). Specifically, the upstream primer (5'-TAATACGACTCACTATAGGG-primer) and downstream primer containing the T7 promoter were designed using PCR technology to amplify the coding sequences of EGFP (derived from the pEGFP plasmid, Addgene ID#165830) and IL-12 (derived from the mIL12-N1 plasmid, Addgene ID#123139) (Table 1). The resulting PCR product was purified by a PCR product purification kit and used as a transcription template for the standard IVT protocol. The synthesized mRNA sequence contains an anti-reverse transcription cap structure analog and a polyA tail at the 5' end. The purification of IVT mRNA was completed by lithium chloride precipitation and 1μgμL -1 The final samples were stored at -80 °C for further use.

[0108] Table 1

[0109] name Forward primer (5'-3') Reverse primer (5'-3') Amplification length template IL-12 SEQ ID NO.1 SEQ ID NO.2 1773bp mIL12-N1 Plasmid EGFP SEQ ID NO.3 SEQ ID NO.4 717bp pEGFP Plasmid

[0110] SEQ ID NO.1:

[0111] TAATACGACTCACTATAGGGATGTGTCCTCAGAAGCTAACC.

[0112] SEQ ID NO. 2: TCAGGCGGAGCTCAGATAGCCCAT.

[0113] SEQ ID NO.3:

[0114] TAATACGACTCACTATAGGGGTGAGCAAGGGCGAGGAGCT.

[0115] SEQ ID NO. 4: TTACTTGTACAGCTCGTCCA.

[0116] Preparation Example 4

[0117] This preparation example provides a method for preparing a MNP / IL-12 nanocore, and the specific method includes:

[0118] First, 50 mg mL -1 The MNP-PEI solution (5 μL) with a concentration of 1 μg / mL was diluted with DEPC water (495 μL) to obtain a final volume of 500 μL of solution A. Next, a 1 μg / mL solution containing -1 The IL-12 mRNA solution (10 μL) was mixed with DEPC water (490 μL) to prepare 500 μL of solution B. Subsequently, under continuous stirring, solution B was slowly added dropwise to an equal volume of solution A. After vigorous vortex mixing for 5 min and incubation at 25°C for 15 min, a mixture containing MNP / IL-12 core (total volume of 1 mL) was formed for subsequent experiments.

[0119] Preparation Example 5

[0120] This preparation example provides a method for preparing a MNP / EGFP nanocore, and the specific method includes:

[0121] First, 50 mg mL -1 The concentration of MNP-PEI solution (5 μL) was diluted with DEPC water (495 μL) to obtain a final volume of 500 μL of solution A. Then, a commercial 1 μg μL -1 The EGFP mRNA solution (10 μL) was mixed with DEPC water (490 μL) to prepare 500 μL of solution B. Subsequently, under continuous stirring, solution B was slowly added dropwise to an equal volume of solution A. After vigorous vortex mixing for 5 min and incubation at 25°C for 15 min, a mixture containing MNP / EGFP core (total volume of 1 mL) was formed for subsequent experiments.

[0122] Preparation Example 6

[0123] This preparation example provides a method for preparing membrane fusion liposomes for engineering T cells, and the specific method includes:

[0124] The core-shell membrane fusion liposomes were prepared by thin film hydration method. Specifically, DMPC, DOTAP and DSPE-PEG-DPPA lipids were dissolved in chloroform at a concentration of 1 mg mL -1. In order to prepare 1 mg of liposomes, DMPC (582 μL), DOTAP (148 μL) and DSPE-PEG-DPPA (270 μL) were mixed in a 25 mL round-bottom flask at a molar ratio of 76.2:20:3.8. The solvent was removed by a rotary evaporator, and then further dried in a vacuum oven to form a lipid film. Afterwards, the MNP / IL-12 core solution (500 μL) prepared according to the method in Preparation Example 4 was added to the flask containing the lipid film and ultrasonically hydrated to form a core-shell structure of membrane fusion liposomes. Finally, the membrane fusion liposome mixed solution was repeatedly extruded using a polycarbonate nanoporous membrane with a diameter of 880 nm to ensure the uniformity and stability of the liposomes. The synthesis and ratio optimization results of the MNP / IL-12 core are shown in Figure 2. Figure 4A-4C As shown in the figure, it shows that when MNP-PEI and IL-12 mRNA are mixed at a mass ratio of 1:25, mRNA can be completely loaded on MNP-PEI to form a nanocore with uniform particle size and positive surface charge. Figure 5A-Figure 5C As shown, it indicates that more stable core-shell nanostructures can be formed through lipid membranes.

[0125] Preparation Example 7

[0126] This preparation example provides a method for preparing non-membrane fusion liposomes for engineering T cells, and the specific method includes:

[0127] The core-shell membrane fusion liposomes were prepared by thin film hydration method. Specifically, DMPC and DSPE-PEG-DPPA lipids were dissolved in chloroform at a concentration of 1 mg mL -1 . To prepare 1 mg of liposomes, DMPC (730 μL) and DSPE-PEG-DPPA (270 μL) were mixed in a 25 mL round-bottom flask at a molar ratio of 96.2:3.8. The solvent was removed by a rotary evaporator and then further dried in a vacuum oven to form a lipid film. Afterwards, the MNP / IL-12 inner core solution (500 μL) prepared by the method in Preparation Example 4 was added to the flask containing the lipid film and ultrasonically hydrated to form a core-shell structure of non-membrane fusion liposomes. Finally, the non-membrane fusion liposome mixed solution was repeatedly extruded using a polycarbonate nanoporous membrane with a diameter of 880 nm to ensure the uniformity and stability of the liposomes.

[0128] Preparation Example 8

[0129] This preparation example provides a method for preparing a membrane fusion liposome that does not carry a DPPA targeting peptide and is used for engineering T cells. The specific method includes:

[0130] The core-shell membrane fusion liposomes were prepared by thin film hydration method. Specifically, DMPC, DOTAP and DSPE-PEG-Mal lipids were dissolved in chloroform at a concentration of 1 mg mL -1 . To prepare 1 mg of liposomes, DMPC (582 μL), DOTAP (148 μL) and DSPE-PEG-Mal (270 μL) were mixed in a 25 mL round-bottom flask at a molar ratio of 76.2:20:3.8. The solvent was removed by a rotary evaporator and then further dried in a vacuum oven to form a lipid film. Afterwards, the pre-prepared MNP / IL-12 core solution (500 μL) was added to the flask containing the lipid film and hydrated by ultrasound to form the core-shell structure of membrane fusion liposomes. Finally, the membrane fusion liposome mixed solution was repeatedly extruded using a polycarbonate nanoporous membrane with a diameter of 880 nm to ensure the uniformity and stability of the liposomes.

[0131] Preparation Example 9

[0132] This preparation example provides a method for preparing membrane fusion liposomes not loaded with IL-12 mRNA for engineering T cells, and the specific method includes:

[0133] The core-shell membrane fusion liposomes were prepared by thin film hydration method. Specifically, DMPC, DOTAP and DSPE-PEG-DPPA lipids were dissolved in chloroform at a concentration of 1 mg mL -1 . To prepare 1 mg of liposomes, DMPC (582 μL), DOTAP (148 μL) and DSPE-PEG-DPPA (270 μL) were mixed in a 25 mL round-bottom flask at a molar ratio of 76.2:20:3.8. The solvent was removed by a rotary evaporator and then further dried in a vacuum oven to form a lipid film. Afterwards, the MNP / EGFP core solution (500 μL) prepared by the method in Preparation Example 5 was added to the flask containing the lipid film and ultrasonically hydrated to form a core-shell structure of membrane fusion liposomes. Finally, the membrane fusion liposome mixed solution was repeatedly extruded using a polycarbonate nanoporous membrane with a diameter of 880 nm to ensure the uniformity and stability of the liposomes.

[0134] Preparation Example 10

[0135] This preparation example provides a method for preparing multifunctional engineered T cells based on liposomes, and the specific method includes:

[0136] For the standard T cell engineering procedure, activated mouse T cells were first collected by centrifugation at 3000 rpm for 5 min and then resuspended in serum-free RPMI 1640 medium to a concentration of 1 × 10 per ml.6 According to the general procedure, add 100 μL of liposome solution to 1 mL of T cell suspension to make the liposome concentration reach 400 μg mL -1 , and then co-cultured with T cells at 37°C for 6 hours for cell engineering. After the culture was completed, the engineered T cells were collected by centrifugation at 3000 rpm for 5 minutes, and washed with pre-cooled 1× PBS to remove unbound liposomes, and the cells were collected for further experiments.

[0137] Example 1

[0138] This example provides a method for quantifying and optimizing membrane fusion efficiency based on fluorescence resonance energy transfer (FRET) technology, and the specific method includes:

[0139] Fluorescence resonance energy transfer technology was used to explore the efficiency of membrane fusion effect. First, a series of dual-fluorescence labeled liposomes with different DOTAP contents were prepared, and the molar ratios were DMPC / DOTAP / DSPE-PEG-DPPA = 96.0:0:3.8, 91.2:5:3.8, 86.2:10:3.8, 76.2:20:3.8, 66.2:30:3.8, 46.2:50:3.8. In the 1 mg liposome preparation system, 5 μL of DiO and DiI chloroform solutions (1 mg mL -1 ) to label the lipid membrane. As expected, when the dual fluorescent labeled liposomes fuse with the T cell membrane, the relative distance between the markers DiO and DiI will increase, significantly reducing the absorbance of the FRET absorption peak.

[0140] Next, the freshly prepared liposomes (10 μL) were added to a 96-well plate containing primary mouse T cells from the spleen of C57BL / 6 mice (100 μL of RPMI 1640 medium without FBS was added to each well at a density of 1 × 10 5 After incubation at 37°C for 6 h, the fluorescence intensity of DiO in each well was measured using a microplate reader (excitation / emission wavelength = 483 / 506 nm). Subsequently, the membrane fusion efficiency was calculated according to the following formula:

[0141]

[0142] The initial DiO fluorescence intensity of the liposomes before incubation with T cells was defined as F0, and the fluorescence intensity measured after adding 0.1% (v / v) Triton X-100 solution was defined as F 100 The DiO fluorescence intensity of each well is F DiO .

[0143] In order to further distinguish the behavior of membrane fusion liposomes and non-membrane fusion liposomes through FRET, the prepared membrane fusion liposomes and non-membrane fusion liposomes were incubated with mouse T cells for 6 hours, and the incubation temperature was set to 37°C. The incubation process is designed to simulate the interaction between liposomes and cell membranes, thereby triggering possible membrane fusion events. After the incubation is completed, the mixture of liposomes and T cells is subjected to fluorescence spectral analysis using a fluorescence spectrophotometer. During this analysis, the sample was excited at a wavelength of 483nm, and the fluorescence spectrum was recorded in the range of 505nm to 600nm. This range covers the emission peaks of DiO and DiI fluorescent dyes, so that the occurrence of the FRET effect can be evaluated by observing changes in fluorescence intensity at specific wavelengths, thereby indirectly reflecting the fusion properties between liposomes and T cell membranes. The results of quantification and optimization of membrane fusion efficiency based on fluorescence resonance energy transfer are shown in Figure 2. Figure 6A-6C As shown, it indicates that membrane fusion liposomes can fuse with T cell membranes.

[0144] Example 2

[0145] This example provides an experimental method for verifying the internalization pathway of the multifunctional T cell engineering vector in Preparation Examples 6-9, and the specific method includes:

[0146] In order to intuitively study the membrane fusion process between membrane fusion liposomes and T cells, CLSM was used for high-resolution imaging. In this study, YOYO-1 dye was used to label IL-12 mRNA during the preparation of membrane fusion liposomes, and DiD dye was used to label the lipid layer. The specific experimental steps are as follows: First, mouse T cells were rotated at 3000 rpm min. -1 The cells were centrifuged at a speed of 500 μg for 5 min and then resuspended in RPMI 1640 medium without FBS to adjust the cell concentration to 1 × 10 cells per ml. 6 cells. Afterwards, 100 μL of dual fluorescent labeled membrane fusion liposomes were co-incubated with 1 mL of activated mouse T cell suspension for 2 h to promote membrane fusion events. After incubation, the cells were rinsed three times with pre-cooled 1× PBS to remove unfused liposomes, and then fixed with 4% PFA. T cell nuclei were stained with DAPI and rinsed again with 1× PBS three times. Subsequently, the cells were centrifuged at 3000 rpm min -1 T cells were collected by centrifugation at a speed of 5 min, and the cells were resuspended in an anti-fade mounting medium containing glycerol and sliced. When microscopic observation was performed under a 63x oil immersion objective, different fluorescent dyes were excited using excitation wavelengths of 305 nm (DAPI), 488 nm (YOYO-1), and 645 nm (DiD).

[0147] To investigate the effects of different cellular uptake mechanisms on plasmid delivery efficiency, T cells were first grown at 1 × 106 The cells were divided into centrifuge tubes at a density of 100 cells. Subsequently, in order to evaluate the effects of different cellular uptake pathways on the internalization of membrane fusion liposomes, the cells were pretreated with four different cellular uptake inhibitors: Z-Phe-Phe-Phe-OH (membrane fusion inhibitor), chlorpromazine (clathrin-mediated endocytosis inhibitor), amiloride (macropinocytosis inhibitor), and nystatin (caveolin-mediated endocytosis inhibitor). These inhibitors were respectively added at 50 μg mL -1 , 10 μg mL -1 , 100μgmL -1 and 15 μg mL -1 The concentration of 100 μg / mL was added to the culture medium and the cells were pretreated for 30 min to specifically inhibit different substance uptake pathways in the cells. For the low-temperature treatment group, the cells were placed in a 4°C refrigerator for 30 min to evaluate whether the uptake pathway was energy-dependent.

[0148] After pretreatment, the -1 The medium containing the inhibitor was removed by centrifugation at a speed of 5000 rpm for 5 min, and the cells were resuspended in 1 mL of serum-free RPMI 1640 medium containing 100 μL of YOYO-1 and DiD dual fluorescent labeled membrane fusion liposomes. After incubation at 37°C for 2 h, the cells were incubated at 3000 rpm for 5 min. -1 The transfection reagent solution was removed by centrifugation for 5 min, and the cells were washed three times with pre-cooled 1× PBS. The cells were then fixed with 4% PFA and subjected to FACS analysis. Representative CLSM images of membrane fusion liposomes and non-membrane fusion liposomes are shown in Figure 7. The cell nucleus, IL-12 mRNA, and lipid molecules of the lipid layer were labeled with DAPI (blue), YOYO-1 (green), and DiD (red), respectively. The gray value curves of the green (IL-12 mRNA labeled with YOYO-1) and red (lipid labeled with DiD) channels were analyzed by ImageJ software, and the statistical results of the co-localization of the red and green channels are shown in Figure 7. Figure 7A-7C As shown in Figure 2, mRNA in the nucleus can be directly delivered to the cytoplasm and the lipid components can fuse with the cell membrane. The FACS results of liposome uptake under low temperature or different inhibitor pretreatment are shown in Figure 2. Figure 8 As shown, it indicates that membrane fusion liposomes fuse with T cell membrane mainly through membrane fusion.

[0149] Example 3

[0150] This example provides an experimental method for engineering T cells using the multifunctional T cell engineering vector in Preparation Examples 6-9. The specific method includes:

[0151] For the standard T cell engineering procedure, first pass the 3000 rpm min-1 The activated mouse T cells were collected by centrifugation at high speed for 5 min, and then the cells were resuspended in serum-free RPMI 1640 medium to a concentration of 1 × 10 per ml. 6 According to the general procedure, add 100 μL of membrane fusion liposome solution to 1 mL of T cell suspension to make the concentration of membrane fusion liposome reach 400 μg mL -1 , and then co-cultured with T cells at 37°C for 6 h to perform membrane fusion liposome@T cell engineering. In the control group experiment, the added concentration of liposomes (400 μg mL -1 ) and culture time (6 h) were the same as those of the membrane fusion liposome treatment group. -1 The engineered T cells were collected by centrifugation at a speed of 5 min, and washed with pre-cooled 1× PBS to remove unbound liposomes. The cells were collected and used for further experimental studies.

[0152] To optimize the conditions for engineered T cells, other conditions were kept constant and different culture times (0.5 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h) or liposome concentrations (100 μg mL -1 , 200 μg mL -1 , 400 μg mL -1 , 600 μg mL -1 , 800 μg mL -1 , 1000 μg mL -1 ), YOYO-1 and DiD dual fluorescent labeled membrane fusion liposomes were used to treat mouse T cells. After incubation at 37°C for a corresponding period of time, 3000 rpm min -1 The transfection reagent solution was removed by centrifugation for 5 min and washed three times with pre-cooled 1× PBS. The cells were then fixed with 4% PFA and subjected to FACS analysis. Fig.9A and Fig. 9B As shown, it is indicated that at 400 μg mL -1 The best transfection efficiency can be achieved by treating with liposome concentration for 6 hours.

[0153] Example 4

[0154] This example provides an experimental method for evaluating the tumor recognition ability of the engineered T cells in Example 3, and the specific method includes:

[0155] The DPPA-mediated tumor recognition effect was verified by CLSM imaging and FACS analysis. In the CLSM analysis, B16F10 melanoma cells were first stained with Hoechst 33342, and then mouse T cells were labeled with Cal-AM. The specific operation process is as follows: First, B16F10 were stained with 5×10 4 The cells were seeded in a confocal culture dish at a density of 10 cells and cultured for 24 h at 37 ° C and 5% CO2 to facilitate cell attachment. For the membrane fusion liposome @ T plus PD-L1 antibody (aPD-L1) treatment group, B16F10 cells were pre-treated with aPD-L1 for 20 min at 37 ° C. Subsequently, these cells were co-cultured with PBS, non-engineered T cells, membrane fusion liposome engineered T cells without IL-12 mRNA loading, membrane fusion liposome engineered T cells without DPPA, non-membrane fusion liposome engineered T cells, or membrane fusion liposome engineered T cells at 37 ° C. Wash with pre-cooled 1× PBS to remove T cells that are not attached to the surface of tumor cells, then fix the attached cells with 4% PFA, and image Hoechst 33342 (excitation wavelength 305nm) and Cal-AM (excitation wavelength 488nm) by CLSM.

[0156] In FACS analysis, the steps were similar to those above, but B16F10 melanoma cells were prestained with DiD, while T cells were labeled with Cal-AM. After incubation, the co-cultured cells were treated with 0.25% trypsin for 2 min and then fixed with 4% PFA. Afterwards, the clustering of B16F10 cells and T cells was analyzed in detail. The specific recognition results of engineered T cells on tumor cells are shown in Figure 2. Figure 10A-10C As shown, it is shown that the ability of T cells to specifically recognize tumor cells can be significantly improved by DPPA targeting peptide modification based on membrane fusion liposomes.

[0157] Example 5

[0158] The present application example provides an experimental method for evaluating the tumor killing ability of the engineered T cells in Example 3, and the specific method includes:

[0159] In order to evaluate the effect of different types of T cells on the proliferation of B16F10 melanoma cells, B16F10 cells were seeded in 96-well plates at 2 × 10 3Cells were cultured at 37°C and 5% CO2 for 24 h to facilitate cell attachment. After continued culture, PBS, non-engineered T cells, negative control fusion liposome engineered T cells without IL-12 mRNA, fusion liposome engineered T cells without DPPA, non-fusion liposome engineered T cells or fusion liposome engineered T cells were added to B16F10 cells and cultured for 48 h to evaluate their inhibitory effects. After the culture was completed, MTT solution (5 mg mL -1 , 150 μL) and continued to culture for 4 h so that MTT can be reduced by living cells. Subsequently, 150 μL DMSO was added to each well to dissolve the formed formazan crystals. The absorbance was measured at a wavelength of 570 nm by an ELISA reader to quantitatively evaluate cell activity. In addition, the supernatant of B16F10 cells co-cultured with T cells was collected to detect the levels of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) using ELISA technology to evaluate T cell-mediated immune responses.

[0160] Similarly, to evaluate the effect of engineered T cells on the RM-1-luc prostate cancer cell line with luciferase activity, RM-1-luc cells were seeded in black 96-well plates at 2 × 10 cells per well. 3 The cells were cultured at 37°C and 5% CO2 for 24 h. Afterwards, non-engineered or engineered T cells were treated for 48 h. Subsequently, the culture medium was replaced with D-luciferin potassium salt (150 μg mL -1 ) preheated culture medium, and after 10 minutes, the bioluminescent signal was measured using an ELISA instrument to evaluate the inhibitory effect of T cells on the proliferation of RM-1-luc cells. The results of the killing effect of engineered T cells on tumor cells are shown in Fig.11A and Fig. 11B As shown, it shows that membrane fusion liposome engineered T cells can significantly kill various types of tumor cells. The expression levels of IFNγ (A) and TNFα (B) in the supernatant of B16F10 cells and T cells co-cultured are shown in Fig. 12A and Fig. 12B As shown, it indicates that membrane fusion liposome engineered T can increase the secretion level of anti-tumor cytokines to enhance the ability to kill tumors.

[0161] Example 6

[0162] This example provides an experimental method for evaluating the magnetic properties of the engineered T cells in Example 3, and the specific method includes:

[0163] In order to evaluate the superparamagnetic properties of membrane fusion liposomes and their engineered T cells, the MPMS XL7 SQUID magnetic measurement system was used to measure the magnetization curve and hysteresis loop of the freeze-dried samples at 25°C. Furthermore, in order to achieve live cell tracking, 1×10 5 Non-engineered T cells, non-membrane fusion liposome engineered T cells, and membrane fusion liposome engineered T cells labeled with Cal-AM were seeded in 6-well plates at different cell concentrations. A Zeiss inverted fluorescence microscope was used to record real-time images of labeled T cells at a rate of 1 frame per second, and the excitation wavelength was set to 488nm. During live cell imaging, a 50×25×10mm NdFeB N52 magnet was placed at a predetermined time point and position to apply an external magnetic field to simulate the migration behavior of T cells under the action of a magnetic field. The recorded time-lapse images were processed using ImageJ software, and cell tracking analysis was performed using the TrackMate plug-in. The results of the in vitro evaluation of the magnetic properties of engineered T cells are shown in Figure 2. Figure 13A-13C As shown, it is shown that membrane fusion liposome engineered T cells have superparamagnetism and can migrate rapidly and directionally under the action of an external magnetic field.

[0164] Example 7

[0165] This example provides an experimental method for evaluating the tumor infiltration ability of the engineered T cells in Example 3, and the specific method includes:

[0166] To accurately track the distribution of T cells in vivo, 100 μL of 1×10 6 RM-1 prostate cancer cells. Non-engineered and membrane fusion liposome-engineered mouse T cells were labeled with DiR dye and 1×10 7 The cells were resuspended in 1× PBS. Seven days after tumor implantation, DiR-labeled T cells (single dose per mouse: 1×10 6 cells, total volume 100 μL) were injected into tumor-bearing mice. For the group of mice treated with magnetic field, a 10×10×5 mm NdFeB N52 magnet was placed at the tumor site of each mouse under anesthesia. Timed in vivo imaging was performed over the next 48 h using a live imaging system to monitor the distribution of labeled T cells.

[0167] After the experiment, the tumor tissue was collected and weighed. Some tumor samples were taken to prepare frozen sections, fixed with 4% PFA, and the cell nuclei were stained with DAPI. The sections were observed using CLSM, where the excitation wavelength of DAPI was 305nm and the excitation wavelength of DiR was 750nm. The CLSM images were analyzed using ImageJ software.

[0168] In addition, tumor samples were weighed, ground, and filtered through a 100 μm cell sieve. Tumor cells were fixed with 1% PFA and 1% BSA in PBS and stained with APC anti-mouse CD3 antibody. CD3 was evaluated by FACS analysis. + DiR + Cell populations and DiR + The number of cells. And the injection dose percentage (Injection dose percentage, ID% g) is calculated by formula (3-2) -1 ).

[0169]

[0170] The in vivo distribution of engineered T cells Figure 14A-Figure 14C As shown. Engineered T cells in tumor tissue are more Fig.15A and Fig. 15B As shown, it is shown that engineered T cells can aggregate and reside in solid tumor sites under the action of an external magnetic field.

[0171] In summary, the multifunctional T cells prepared by the present invention have enhanced tumor infiltration ability, the ability to overcome tumor cell immune tolerance and the ability to reverse the immunosuppressive tumor microenvironment, and are simple to operate, time-saving and efficient.

[0172] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A membrane fusion liposome, characterized in that: The membrane fusion liposome comprises an inner core and an outer shell; the inner core comprises iron oxide, nanomaterials and mRNA; and the outer shell comprises phospholipid structural lipids, cationic functional lipids and polyethylene glycol functional lipids modified with active targeting peptides.

2. The membrane fusion liposome according to claim 1, characterized in that: The molar mass ratio of the phospholipid structure lipid, the cationic functional lipid and the active targeting peptide modified polyethylene glycol functional lipid is (15-20):(2-5):

1.

3. The membrane fusion liposome according to claim 1 or 2, characterized in that: The nanomaterial includes any one or a combination of at least two of albumin, polycaprolactone, polyoxalate, poly-β-amino lipid, chitosan, nano-calcium carbonate or mesoporous silicon nanomaterials; Preferably, the surface of the nanomaterial is modified with polyethyleneimine.

4. The membrane fusion liposome according to any one of claims 1 to 3, characterized in that: The mRNA includes IL-12 mRNA and / or enhanced green fluorescent protein mRNA.

5. The membrane fusion liposome according to any one of claims 1 to 4, characterized in that: The phospholipid structure lipids include any one of dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or dioleoylphosphatidylethanolamine or a combination of at least two thereof; Preferably, the cationic functional lipid comprises trimethyl-2,3-dioleyloxypropylammonium bromide and / or trimethyl-2,3-dioleyloxypropylammonium chloride; Preferably, the polyethylene glycol functionalized lipid comprises distearoylphosphatidylethanolamine-polyethylene glycol; Preferably, the active targeting peptide comprises DPPA peptide.

6. A method for preparing the membrane fusion liposome according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) mixing iron oxide, nanomaterials, mRNA and a solvent to obtain a suspension of polymer nanocores loaded with mRNA; (2) taking phospholipid structure lipids, cationic functional lipids and polyethylene glycol functionalized lipids, and using a thin film hydration method to prepare a suspension of a preliminary membrane fusion liposome shell; (3) mixing the suspension of the polymer nanocore loaded with mRNA with the suspension of the membrane fusion liposome shell obtained in step (2) to obtain preliminary membrane fusion liposomes; (4) The preliminary membrane fusion liposomes obtained in step (3) are mixed with a solution containing an active targeting peptide, and the active targeting peptide is modified on the surface of the preliminary membrane fusion liposomes to obtain membrane fusion liposomes.

7. A multifunctional adoptive T cell, characterized in that: The surface of the multifunctional adoptive T cells is fused with active targeting peptides via membrane fusion liposomes; and the interior of the multifunctional adoptive T cells is delivered with mRNA via membrane fusion liposomes.

8. The multifunctional adoptive T cell according to claim 7, characterized in that The membrane fusion liposome comprises the membrane fusion liposome according to any one of claims 1 to 5.

9. A method for preparing the multifunctional adoptive T cells according to claim 7 or 8, characterized in that: The method comprises: The activated T cells are co-cultured with the membrane fusion liposomes according to any one of claims 1 to 5 to obtain multifunctional adoptive T cells.

10. Use of the membrane fusion liposome according to any one of claims 1 to 5 and / or the multifunctional adoptive T cells according to claim 7 or 8 in the preparation of drugs for anti-tumor and / or reversal of immunosuppressive tumor microenvironment.

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