Polymer lipid hybrid nano-particle with T cell activation capability and application of polymer lipid hybrid nano-particle

Abstract

The invention provides a polymer lipid hybrid nanoparticle with T cell activation ability, which is characterized in that the polymer lipid hybrid nanoparticle comprises a cationic polymer, an auxiliary lipid, cholesterol and a polyethylene glycol derived lipid, and the cationic polymer comprises a copolymer prepared from polyethyleneimine and p-toluenesulfonyl arginine. The polymer lipid hybrid nanoparticles provided by the invention have dual capabilities of T cell activation and transfection, can realize one-step activation and transfection of primary T cells, greatly shortens the preparation period, and reduces the preparation complexity. Besides, CAR-T cells generated by delivering CAR mRNA through the polymer lipid hybrid nanoparticles RT LNP have excellent cell killing ability in vitro, and can specifically secrete killer cytokines, so that specific and efficient killing of target cells is realized, and the CAR-T cells have good application prospects. It is verified that the polymer lipid hybrid nanoparticles developed in the invention are a promising platform for rapid production of mRNA CAR T cell products.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a polymer-lipid hybrid nanoparticle with T cell activation ability and its application. Background Art

[0002] Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable clinical success in the treatment of hematological malignancies. As a novel treatment modality, CAR T cell therapy endows T cells with the ability to recognize cells expressing the antigen by transferring the gene encoding the antigen receptor into T cells, thereby achieving specific killing. However, the production of these customized CAR T cells is a complex in vitro process, including leukapheresis, artificial T cell activation, and the introduction of CAR constructs. The activation step requires the participation of CD3 / CD28 antibodies, which is crucial for the transfection and differentiation of T cells [Labanieh, L., Mackall, C. L. CAR immune cells: design principles, resistance and the next generation. Nature, 2023, 614, 635 - 648.]. When constructing CAR T cells in vitro, magnetic beads conjugated with CD3 / CD28 antibodies are usually relied on to activate T cells. After a certain period of action, the magnetic beads conjugated with antibodies need to be removed and then a nucleic acid transfection reagent is added. Although effective, this artificial activation increases the complexity of CAR-T cell production because these magnetic beads must be removed before clinical use [A. E. Metzloff, M. S. Padilla, N. Gong, et al., Antigen Presenting Cell Mimetic Lipid Nanoparticles for Rapid mRNA CAR T Cell Cancer Immunotherapy. Adv. Mater. 2024, 36, 2313226].

[0003] Therefore, providing a polymer material with dual abilities of T cell activation and transfection has become a problem to be solved. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a polymer-lipid hybrid nanoparticle with T cell activation ability and its application. The polymer-lipid hybrid nanoparticle (RT LNP) with T cell activation ability provided by the present invention has dual abilities of T cell activation and transfection, can achieve one-step activation and transfection of primary T cells, greatly shortens the preparation cycle, and reduces the complexity of preparation.

[0005] The present invention provides a polymer-lipid hybrid nanoparticle with T cell activation ability, comprising a cationic polymer, a helper lipid, cholesterol, and a polyethylene glycol-derived lipid, wherein the cationic polymer comprises a copolymer prepared from polyethyleneimine and tosylarginine.

[0006] Preferably, the polyethyleneimine is linear polyethyleneimine or branched polyethyleneimine, and the number-average molecular weight of the polyethyleneimine is 300 to 80,000 Daltons;

[0007] The molar ratio of the polyethyleneimine to the tosylarginine is 1:(1 to 10,000).

[0008] Preferably, the preparation method of the cationic polymer comprises the following steps:

[0009] A) Add C 8 H 17 N 3 ·HCl and 1-hydroxybenzotriazole to a solution of tert-butoxycarbonyl and tosyl group double-protected arginine for activation to obtain an activated carboxyl solution;

[0010] B) Mix an aqueous solution of polyethyleneimine and the activated solution, and then add N,N-diisopropylethylamine for reaction to obtain a reacted solution;

[0011] C) Dialyze and lyophilize the reacted solution to obtain a lyophilized product; react the lyophilized product with trifluoroacetic acid, and then dialyze and lyophilize to obtain the cationic polymer component.

[0012] Preferably, the molar ratio of C 8 H 17 N 3 ·HCl, the 1-hydroxybenzotriazole, the N,N-diisopropylethylamine, and the tosylarginine is (1 to 5):(1 to 5):(1 to 5):1;

[0013] In step A), the activation temperature is 25 to 38 °C, and the activation time is 1 to 4 h;

[0014] In step B), the reaction temperature is 25 to 38 °C, and the reaction time is 2 to 3 days;

[0015] In step C), the molecular weight for dialysis is 500 to 5,000 Daltons; the dialysis time is 2 to 4 days; the lyophilization temperature is -50 to -80 °C.

[0016] Preferably, the polymer-lipid hybrid nanoparticle, in terms of mole fraction, comprises:

[0017] 5 to 45 parts of a cationic polymer;

[0018] 10 to 30 parts of an auxiliary lipid;

[0019] 10 to 70 parts of cholesterol;

[0020] 1 to 25 parts of a polyethylene glycol - derived lipid.

[0021] Preferably, the auxiliary lipid is selected from at least one of DSPC, DOPE, and DOTAP, and preferably DSPC;

[0022] The polyethylene glycol - derived lipid is selected from at least one of DMG - PEG, C 14 -PEG, and DSPC - PEG, and preferably DMG - PEG.

[0023] The present invention also provides an application of the above - mentioned polymer - lipid hybrid nanoparticles in the preparation of a preparation with T - cell activation ability.

[0024] Preferably, the T - cell activation ability includes up - regulating the expression of related genes and inducing the secretion of cytokines. The up - regulated genes include at least one of CD69 and CD25; the secreted cytokines include at least one of interferon - γ (IFN - γ), interleukin - 2 (IL - 2), and tumor necrosis factor - α (TNF - α).

[0025] Preferably, the T - cell activation ability also includes inducing the activation and / or proliferation of T cells.

[0026] The present invention also provides an application of the above - mentioned polymer - lipid hybrid nanoparticles in the preparation of a drug delivery carrier.

[0027] Preferably, the polymer - lipid hybrid nanoparticles are nanoparticles targeting T cells.

[0028] Preferably, the drug is selected from nucleic acid drugs, and the nucleic acid drugs are selected from non - functional mRNA and / or functional mRNA; preferably, the functional mRNA includes mRNA encoding CAR 、 mRNA encoding TCR, mRNA encoding CRISPR, mRNA encoding cytokines; more preferably, the mRNA encoding CAR includes at least one of HER2 CAR, CD19 CAR, and FAP CAR.

[0029] The present invention also provides an application of the above - mentioned polymer - lipid hybrid nanoparticles in the preparation of a drug for treating tumors.

[0030] Compared with the prior art, the present invention provides a polymer-lipid hybrid nanoparticle with T cell activation ability, which is characterized by comprising a cationic polymer, a helper lipid, cholesterol and a polyethylene glycol-derived lipid, wherein the cationic polymer comprises a copolymer prepared from polyethyleneimine (PEI) and arginine tosylate (RT). The present invention provides a polymer-lipid hybrid nanoparticle (RT LNP) with T cell activation ability, which has the dual abilities of T cell activation and transfection, can achieve one-step activation and transfection of primary T cells, greatly shortens the preparation cycle and reduces the preparation complexity. In addition, the CAR-T cells generated by delivering CAR mRNA through the polymer-lipid hybrid nanoparticle RT LNP have excellent cell killing ability in vitro and can specifically secrete cytotoxic cytokines to achieve specific and efficient killing of target cells, verifying that the polymer-lipid hybrid nanoparticle developed in the present invention is a promising platform for rapidly producing mRNA CAR T cell products. Description of the Drawings

[0031] Figure 1 Graph showing the evaluation results of the in vitro T cell activation effect of RT LNP in Example 4.

[0032] Figure 2 Graph showing the evaluation results of the in vitro T cell transfection effect of RT LNP in Example 5.

[0033] Figure 3 Graph showing the evaluation results of the in vitro promotion effect of RT LNP on T cell proliferation in Example 6.

[0034] Figure 4 Graph showing the evaluation results of the specific killing effect of in vitro constructed CAR T cells in Example 7.

[0035] Figure 5 Graph showing the evaluation results of the T cell activation effect of different polymer-lipid hybrid nanoparticles in Example 8. Detailed Description of the Invention

[0036] The present invention provides a polymer-lipid hybrid nanoparticle (RT LNP) with T cell activation ability, which comprises a cationic polymer, a helper lipid, cholesterol and a polyethylene glycol-derived lipid, wherein the cationic polymer comprises a copolymer prepared from polyethyleneimine (PEI) and arginine tosylate (RT).

[0037] Among them, the polyethyleneimine is linear polyethyleneimine or branched polyethyleneimine, and the number-average molecular weight of the polyethyleneimine is 300 to 80,000 Dalton, which can be 300, 500, 1000, 3000, 5000, 7000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, or any value or any range between 300 and 80,000 Dalton;

[0038] The molar ratio of the polyethyleneimine to tosylarginine is 1:(1 to 10,000), which can be 1:1, 1:2, 1:4, 1:5, 1:10, 1:15, 1:30, 1:50, 1:100, 1:500, 1:1000, 1:5000, 1:10,000, or any value between 1:(1 to 10,000). Preferably, it is any value between 1:(2 to 1000), more preferably any value between 1:(4 to 400), and even more preferably any value between 1:(2 to 30). In some preferred embodiments of the present invention, the molar ratio of the polyethyleneimine to tosylarginine is 1:(1 to 15).

[0039] In the present invention, the preparation method of the cationic polymer comprises the following steps:

[0040] A) Add C 8 H 17 N 3 ·HCl and 1-hydroxybenzotriazole to a solution of tert-butoxycarbonyl and tosyl group double-protected arginine for activation to obtain an activated carboxyl solution;

[0041] B) Mix an aqueous solution of polyethyleneimine and the activated solution, and then add N,N-diisopropylethylamine for reaction to obtain a reacted solution;

[0042] C) Dialyze and lyophilize the reacted solution to obtain a lyophilized product; react the lyophilized product with trifluoroacetic acid, and then dialyze and lyophilize to obtain the cationic polymer component.

[0043] The preparation method of the cationic polymer provided by the present invention first adds C 8 H 17 N 3 ·HCl and 1-hydroxybenzotriazole to a solution of tert-butoxycarbonyl and tosyl group double-protected arginine for activation to obtain an activated solution.

[0044] In the present invention, the solvent of the solution of tert-butoxycarbonyl and p-toluenesulfonyl double-protected arginine is N,N-dimethylformamide solution; the concentration of the solution is preferably 0.01 - 1 mg / mL, and can be 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, or any value between 0.01 - 1 mg / mL; more preferably 0.05 - 0.8 mg / mL; further preferably 0.1 - 0.6 mg / mL.

[0045] The activation temperature is preferably 25 - 38 °C, more preferably 25 - 35 °C; the activation time is 1 - 4 h; more preferably 2 - 2.5 h.

[0046] Then, an aqueous solution of polyethyleneimine and the activated solution are mixed, and N,N-diisopropylethylamine is added for reaction to obtain a reacted solution.

[0047] Among them, the concentration of the aqueous solution of PEI is 0.01 - 1 mg / mL, and can be 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, or any value between 0.01 - 1 mg / mL; more preferably 0.05 - 0.8 mg / mL; most preferably 0.1 - 0.7 mg / mL.

[0048] In the present invention, the C 8 H 17 N 3 The molar ratio of ·HCl, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and p-toluenesulfonyl arginine is (1 - 5):(1 - 5):(1 - 5):1, and can be 1:1:1:1, 1.5:1.5:1.5:1, 2:2:2:1, 3:3:3:1, 4:4:4:1, 5:5:5:1, or any value between (1 - 5):(1 - 5):(1 - 5):1.

[0049] The reaction temperature is preferably 25 - 35 °C; the reaction time is preferably 50 - 100 h; more preferably 50 - 90 h; most preferably 60 - 80 h.

[0050] The reacted solution is dialyzed and freeze-dried to obtain a freeze-dried product; the freeze-dried product is reacted with trifluoroacetic acid, and through dialysis and freeze-drying, the cationic polymer component is obtained.

[0051] Among them, the molecular weight of the dialysis is 500-5000 Dalton, which can be 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or any value between 500-5000 Dalton. The dialysis time is 2-4 days, which can be 2, 3, 4, or any value between 2-4 days; the freeze-drying temperature is -50 to -80 °C, which can be -50, -60, -70, -80, or any value between -50 to -80 °C.

[0052] In the polymer-lipid hybrid nanoparticles, the molar ratio of the cationic polymer is 5-45%, preferably 10-40%, more preferably 20-35%.

[0053] Except for the cationic polymer, the molar ratio of the three components of the co-lipid, cholesterol, and polyethylene glycol-derived lipid is 30-90%, preferably 40-80%, more preferably 50-70%.

[0054] In some specific embodiments of the present invention, the polymer-lipid hybrid nanoparticles provided by the present invention, in terms of molar parts, include:

[0055] 5 to 45 parts of a cationic polymer;

[0056] 10 to 30 parts of a co-lipid;

[0057] 10 to 70 parts of cholesterol;

[0058] 1 to 25 parts of a polyethylene glycol-derived lipid.

[0059] Among them, the polymer-lipid hybrid nanoparticles include 5 to 45 parts of a cationic polymer, which can be 5, 10, 15, 20, 25, 30, 35, 40, 45 parts, or any value between 5 to 45 parts, preferably 10-40 parts, more preferably 20-35 parts.

[0060] The polymer-lipid hybrid nanoparticles further include 10 to 30 parts of a co-lipid, which can be 10, 15, 20, 25, 30 parts, or any value between 10 to 30 parts. The co-lipid is selected from at least one of DSPC, DOPE, and DOTAP, preferably DSPC.

[0061] The polymer-lipid hybrid nanoparticles further include 10 to 70 parts of cholesterol, which can be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 parts, or any value between 10 to 70 parts.

[0062] The polymer-lipid hybrid nanoparticles further comprise 1 to 25 parts of polyethylene glycol-derived lipid, which can be 1 part, 3 parts, 5 parts, 7 parts, 9 parts, 10 parts, 11 parts, 13 parts, 15 parts, 17 parts, 20 parts, 23 parts, 25 parts, or any value between 1 to 25 parts. In the invention, the polyethylene glycol-derived lipid is selected from at least one of DMG-PEG, C 14 -PEG, DSPC-PEG, and is preferably DMG-PEG.

[0063] The invention also provides an application of the above polymer-lipid hybrid nanoparticles in the preparation of a preparation with T cell activation ability.

[0064] The T cell activation includes at least one of induced cytokine expression and secretion, phagocytosis, cell signal transduction, antigen processing and presentation, and target cell killing function.

[0065] In some specific embodiments of the invention, the T cell activation ability includes up-regulating the expression of related genes and inducing cytokine secretion. The up-regulated genes include at least one of CD69 and CD25; the secreted cytokines include at least one of interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α).

[0066] In the invention, if in a form without nucleic acid drugs, the T cell activation ability further includes inducing the activation and / or proliferation of T cells. Preferably, the activation and / or proliferation of T cells and the transfection of nucleic acids into T cells can be carried out simultaneously in one step using the polymer-lipid hybrid nanoparticles RT LNP to encapsulate functional mRNA.

[0067] The invention provides a method for simultaneously activating / proliferating T cells and performing T cell transfection. The polymer-lipid hybrid nanoparticles RT LNP can simultaneously activate T cells and transfect functional mRNA into T cells without the need to pre-activate T cells using CD3 / CD28 antibodies. Preferably, the polymer-lipid hybrid nanoparticles can directly deliver CAR mRNA into naive T cells, providing a simple platform for the preparation of CAR T cell products.

[0068] The invention also provides an application of the polymer-lipid hybrid nanoparticles in the preparation of a drug delivery carrier.

[0069] Among them, the polymer-lipid hybrid nanoparticles are nanoparticles targeting T cells. The invention utilizes the T cell activation performance of the polymer-lipid hybrid nanoparticles RT LNP to achieve simultaneous activation of T cells and transfection of T cells, preferably delivering CAR mRNA into T cells.

[0070] In a specific embodiment of the present invention, the drug is selected from nucleic acid drugs, and the nucleic acid drugs are selected from non-functional mRNA and / or functional mRNA; preferably, the functional mRNA includes mRNA encoding CAR. 、 mRNA encoding TCR, mRNA encoding CRISPR, mRNA encoding cytokines; more preferably, the mRNA encoding CAR includes at least one of HER2 CAR, CD19 CAR, and FAP CAR.

[0071] In the present invention, the T cells are primary mouse spleen T cells or primary human peripheral blood T cells. The polymer-lipid hybrid nanoparticles encapsulating mRNA are added to T cells, and the mRNA dose is 0.2 - 1 μg / well. The polymer-lipid hybrid nanoparticles can activate T cells, up-regulate the expression of their activation genes, and induce the secretion of cytokines, while mediating the efficient expression of mRNA in T cells. The polymer-lipid hybrid nanoparticles provided by the present invention can realize the one-step production of CAR T cells, eliminating the step of artificially activating T cells in vitro, shortening the preparation time, and reducing the preparation complexity. The mass ratio of the polymer-lipid hybrid nanoparticles RT LNP to mRNA is 1:(0.01 - 1), which can be 1:0.01, 1:0.02, 1:0.025, 1:0.05, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, or any value between 1:(0.01 - 1), preferably 1:(0.02 - 0.5).

[0072] In the present invention, the polymer-lipid hybrid nanoparticles RT LNP can deliver functional mRNA to T cells; in the present invention, the functional mRNA expresses various therapeutic proteins, including fluorescent reporter proteins, gene editing proteins, and CAR proteins. Preferably, the fluorescent reporter protein is green fluorescent protein or luciferase reporter protein; the chimeric antigen receptor protein targets include HER2 CAR, CD19 CAR, or FAP CAR.

[0073] The drug delivery carrier can activate and transfect T cells in the same step, mediate the expression of CAR mRNA in T cells, and rapidly prepare CAR T cell products by one step. Preferably, the CAR T cells of the present invention can specifically kill antigen cells in vitro and induce the secretion of killing factors.

[0074] The present invention also provides an application of the above polymer-lipid hybrid nanoparticles in the preparation of drugs for treating tumors.

[0075] The present invention uses the polymer-lipid hybrid nanoparticle RT LNP to prepare drugs related to the treatment of tumors. Preferably, a CAR T cell product is rapidly prepared by a one-step method; more preferably, the CAR T cell product is used to prepare drugs related to the treatment of cancer. The cancer includes breast cancer, pancreatic cancer, colorectal cancer or lymphoma.

[0076] The polymer-lipid hybrid nanoparticle in the present invention is an mRNA delivery vector with the ability to activate natural T cells, and can achieve efficient transfection of T cells without pre-activating T cells with antibodies during in vitro culture. The vector provided by the present invention can simultaneously activate and transfect T cells, avoiding the complex process of conventional in vitro transfection of T cells. The CAR T cell drug prepared by the delivery technology has broad market application prospects. The nanoparticle in the present invention has no ligand modification, and has the characteristics of simple composition and easy optimization. The present invention also provides the application of the activated T cells in the preparation of CAR-T cells. Using the polymer-lipid hybrid nanoparticle in the present invention in the field of drug delivery, especially in the application of T cell transfection and CAR T cell preparation, has significant technical advantages and broad prospects.

[0077] The polymer-lipid hybrid nanoparticle (RT LNP) with the ability to activate T cells provided by the present invention has the dual abilities of T cell activation and transfection, and can achieve one-step activation and transfection of primary T cells, greatly shortening the preparation cycle and reducing the complexity of preparation. In addition, the CAR-T cells generated by delivering CAR mRNA through the polymer-lipid hybrid nanoparticle RT LNP have excellent cell killing ability in vitro, and can specifically secrete cytotoxic cytokines to achieve specific and efficient killing of target cells, verifying that the polymer-lipid hybrid nanoparticle provided in the present invention is a promising platform for rapidly producing mRNA CAR T cell products. The CAR T cell drug constructed by the polymer-lipid hybrid nanoparticle RT LNP can effectively kill antigen target cells in vitro and secrete cytotoxic cytokines, and has broad application prospects in the fields of anti-tumor mRNA drugs and tumor immunotherapy.

[0078] To further understand the present invention, the following examples are used to illustrate the polymer-lipid hybrid nanoparticle with the ability to activate T cells provided by the present invention and its applications. The protection scope of the present invention is not limited by the following examples.

[0079] Example 1 Preparation of cationic polymers (LPEI1.8k-Arg(Tos)-4, LPEI1.8k-Arg-4, LPEI1.8k-Arg(NO 2 )-4, LPEI1.8k-Orn-4, LPEI1.8k-Orn(Tos)-4)

[0080] Dissolve 1 g of linear polyethyleneimine (LPEI 1.8k, 0.556 mmol) in deionized water. Dissolve 3.34 g of tert-butoxycarbonyl and p-toluenesulfonyl double-protected arginine (Boc-Arg(Tos)-OH, 7.78 mmol) in 30 mL of N,N-dimethylformamide (DMF) solution. Add 2.24 g of EDC hydrochloride (11.66 mmol) and 1.576 g of HOBT (11.66 mmol) to the Boc-Arg(Tos)-OH solution, stir at room temperature for 3 h, then gradually add the LPEI aqueous solution dropwise to the above mixed solution, and then add N,N-diisopropylethylamine (15.56 mmol). Stir and react at room temperature for 3 days. Dialyze and freeze-dry the reaction solution to obtain a white solid. Dissolve the white solid in trifluoroacetic acid, stir at room temperature for 4 h, dialyze through a dialysis bag with a molecular weight cut-off of 1000 Dalton, and freeze-dry to obtain a white powder LPEI1.8k-Arg(Tos)-4.

[0081] Dissolve 1 g of linear polyethyleneimine (LPEI 1.8k, 0.556 mmol) in deionized water. Dissolve 4.09 g of tert-butoxycarbonyl and pentamethyldihydrobenzofuran-3-sulfonyl double-protected arginine (Boc-Arg(pbf)-OH, 7.78 mmol) in 30 mL of N,N-dimethylformamide (DMF) solution. Add 2.24 g of EDC hydrochloride (11.66 mmol) and 1.576 g of HOBT (11.66 mmol) to the Boc-Arg(Tos)-OH solution, stir at room temperature for 3 h, then gradually add the LPEI aqueous solution dropwise to the above mixed solution, and then add N,N-diisopropylethylamine (15.56 mmol). Stir and react at room temperature for 3 days. Dialyze and freeze-dry the reaction solution to obtain a white solid. Dissolve the white solid in trifluoroacetic acid, stir at room temperature for 4 h, dialyze through a dialysis bag with a molecular weight cut-off of 1000 Dalton, and freeze-dry to obtain a white powder LPEI1.8k-Arg-4.

[0082] Dissolve 1 g of linear polyethyleneimine (LPEI 1.8k, 0.556 mmol) in deionized water. Dissolve 2.48 g of tert-butoxycarbonyl-protected nitroarginine (Boc-Arg(NO 2 )-OH, 7.78 mmol) in 30 mL of N,N-dimethylformamide (DMF) solution. Add 2.24 g of EDC hydrochloride (11.66 mmol) and 1.576 g of HOBT (11.66 mmol) to the Boc-Arg(NO 2)-OH solution, stirred at room temperature for 3 h. Then, an aqueous solution of LPEI was added dropwise to the above mixed solution, followed by the addition of N,N-diisopropylethylamine (15.56 mmol), and the reaction was stirred at room temperature for 3 days. The reaction solution was dialyzed and lyophilized to obtain a white solid. The white solid was dissolved in trifluoroacetic acid and stirred at room temperature for 4 h, then dialyzed through a dialysis bag with a molecular weight cut-off of 1000 Dalton and lyophilized to obtain a white powder LPEI1.8k-Arg(NO 2 )-4.

[0083] Dissolve 1 g of linear polyethyleneimine (LPEI1.8k, 0.556 mmol) in deionized water, and dissolve 1.80 g of tert-butoxycarbonyl-protected ornithine (Boc-Orn-OH, 7.78 mmol) in 30 mL of N,N-dimethylformamide (DMF) solution. Add 2.24 g of EDC hydrochloride (11.66 mmol) and 1.576 g of HOBT (11.66 mmol) to the Boc-Orn-OH solution, stir at room temperature for 3 h, then add the aqueous solution of LPEI dropwise to the above mixed solution, followed by the addition of N,N-diisopropylethylamine (15.56 mmol), and the reaction was stirred at room temperature for 3 days. The reaction solution was dialyzed and lyophilized to obtain a white solid. The white solid was dissolved in trifluoroacetic acid and stirred at room temperature for 4 h, then dialyzed through a dialysis bag with a molecular weight cut-off of 1000 Dalton and lyophilized to obtain a white powder LPEI1.8k-Orn-4.

[0084] Dissolve 1 g of linear polyethyleneimine (LPEI1.8k, 0.556 mmol) in deionized water, and dissolve 3.00 g of tert-butoxycarbonyl and p-toluenesulfonyl double-protected ornithine (Boc-Orn(Tos)-OH, 7.78 mmol) in 30 mL of N,N-dimethylformamide (DMF) solution. Add 2.24 g of EDC hydrochloride (11.66 mmol) and 1.576 g of HOBT (11.66 mmol) to the Boc-Orn(Tos)-OH solution, stir at room temperature for 3 h, then add the aqueous solution of LPEI dropwise to the above mixed solution, followed by the addition of N,N-diisopropylethylamine (15.56 mmol), and the reaction was stirred at room temperature for 3 days. The reaction solution was dialyzed and lyophilized to obtain a white solid. The white solid was dissolved in trifluoroacetic acid and stirred at room temperature for 4 h, then dialyzed through a dialysis bag with a molecular weight cut-off of 1000 Dalton and lyophilized to obtain a white powder LPEI1.8k-Orn(Tos)-4.

[0085] Example 2 Polymer-lipid hybrid nanoparticles RT LNP, Arg LNP, Arg(NO 2)Preparation of LNP, Orn LNP, Orn(Tos)LNP

[0086] Dissolve the above cationic polymer, cholesterol, DMG-PEG, and DSPC in ethanol at a concentration of 10 mg / mL respectively, and mix them in a molar ratio of 35:10:24:10. Dissolve mRNA in 10 mM citrate buffer (pH 3.0) (0.25 mg / mL). Subsequently, quickly mix the two solutions under shaking at a ratio of aqueous phase:ethanol phase volume of 3:1, and then dialyze in neutral phosphate buffer for 4 hours for collection and use.

[0087] Example 3 Extraction and Culture of Mouse Primary T Cells

[0088] In this experiment, female C57 / 6J mice aged 6 - 8 weeks were prepared. After sacrificing the mice, soak them in 75% alcohol for sterilization, and then place the mice in a laminar flow hood for dissection to remove the spleen, which was soaked in phosphate buffer. Grind the spleen into a single cell suspension, filter and collect single cells using a cell strainer, and sort out CD3 + T cells. Culture the sorted CD3 + T cells in RPMI 1640 medium containing 10% (v / v) fetal bovine serum and 0.1% (v / v) interleukin-2 factor in an incubator set at 37°C and 5% (v / v) CO 2 incubator.

[0089] Example 4 Evaluation of the T Cell Activation Effect of RT LNP

[0090] The mRNA used in this experiment was non-functional mRNA. Prepare RT LNP / NC mRNA according to the steps of Example 2, and the mass ratio of RTLNP to NC mRNA was 40:1. Culture mouse primary T cells in RPMI 1640 medium containing 10% (v / v) fetal bovine serum and 0.1% (v / v) interleukin-2 factor, seed 1×10 6 cells per well in a 96-well plate, add RT LNP / mRNA nanoparticles to the 96-well cell plate at a dosage of 0.2 μg mRNA per well, and continue to culture for 24 h. After incubation, detect the expression of CD69 or CD25 by flow cytometry to evaluate the in vitro T cell activation effect. The results are shown in Figure 1 , Figure 1 which is the result graph of the evaluation of the in vitro T cell activation effect of RT LNP in Example 4. As Figure 1 can be seen, RT LNP can significantly up-regulate the expression of the T surface marker CD69, while the untreated control group did not show the expression of CD69. CD69 is a surface marker up-regulated in the early stage of T cell activation, and its significant up-regulation represents the activation state of T cells.

[0091] Example 5 Evaluation of the in vitro transfection effect of RT LNP on primary mouse T cells

[0092] The mRNA used in this experiment was mGFP encoding green fluorescent protein (GFP), and RT LNP / mGFP was prepared according to the above steps ， The mass ratio of RT LNP to mGFP was 40:1. Primary mouse T cells were cultured in RPMI 1640 medium containing 10% (v / v) fetal bovine serum and 0.1% (v / v) interleukin-2 factor, and seeded at 1×10 6 cells / well in a 96-well plate. At the time of transfection, RT LNP / mRNA nanoparticles were added to the 96-well cell plate at a dosage of 0.2 μg mRNA / well, and the cells were further cultured for 24 h. After transfection, the GFP expression was detected by flow cytometry to evaluate the in vitro T cell transfection effect. The results are shown in Figure 2 , Figure 2 which is the result graph of the evaluation of the in vitro T cell transfection effect of RT LNP in Example 5. It can be seen from Figure 2 that RT LNP can achieve a 25% positive rate of T cell transfection in vitro

[0093] Example 6 Evaluation of the effect of RT LNP on the proliferation of primary mouse T cells

[0094] The mRNA used in this experiment was non-functional mRNA, and RT LNP / NC mRNA was prepared according to the above steps. The mass ratio of RT LNP to mRNA was 40:1. Primary mouse T cells labeled with CFSE were cultured in RPMI 1640 medium containing 10% (v / v) fetal bovine serum and 0.1% (v / v) interleukin-2 factor, and seeded at 1×10 6 cells / well in a 96-well plate. RT LNP / mRNA nanoparticles were added to the 96-well cell plate at a dosage of 0.2 μg mRNA / well, and the cells were further cultured for different times. After the experiment, the fluorescence intensity of CFSE was detected by flow cytometry to evaluate the in vitro T cell proliferation effect. The results are shown in Figure 3 , Figure 3 which is the result graph of the evaluation of the effect of RT LNP in promoting T cell proliferation in vitro in Example 6. It can be seen from Figure 3 that as T cells divide and proliferate, the fluorescence intensity of CFSE will decrease. Compared with the control group, the average fluorescence intensity of CFSE in the RT LNP treatment group was significantly reduced, indicating that RT LNP promoted the proliferation of T cells, and with the prolongation of time, the effect of RT LNP in promoting T cell proliferation was more significant

[0095] Example 7 Evaluation of the in vitro killing effect of CAR T cell drugs prepared by RT LNP

[0096] The functional mRNA used in this in vivo treatment experiment was FAP CAR mRNA. RT LNP / FAP CAR mRNA was prepared according to the above steps, and the mass ratio of RT LNP to mRNA was 40:1. The FAP-GFP plasmid was transfected into 293T cells to obtain FAP + cells constructed in vitro. The transfected FAP CAR T cells were co-cultured and incubated with FAP-293T cells, and the in vitro killing effect of CAR T cells was evaluated by detecting the viability of FAP-293T cells and the secreted cytotoxic cytokines. The results are shown in Figure 4 , Figure 4 Figure showing the evaluation results of the specific killing effect of CAR T cells constructed in vitro in Example 7. As Figure 4 can be seen, as the ratio of CAR T cells to FAP-target cells increased, the killing power of CAR T cells also increased. When the ratio of effector CAR T cells to target cells increased to 8:1, CAR T cells were able to kill 60% of the target cells, showing a strong killing ability.

[0097] Comparison of T cell activation effects of different polymer-lipid hybrid nanoparticles in Example 8

[0098] The mRNA used in this experiment was non-functional mRNA. Different polymer-lipid hybrid nanoparticles were prepared according to the steps of Example 2, including RT LNP, Arg LNP, Arg(NO 2 )LNP, Orn LNP, Orn(Tos)LNP. The mass ratio of polymer-lipid hybrid nanoparticles to mRNA was 40:1. Mouse primary T cells were cultured in RPMI 1640 medium containing 10% (v / v) fetal bovine serum and 0.1% (v / v) interleukin-2 factor, and seeded in 96-well plates at a density of 1×10 6 cells / well. Different polymer-lipid hybrid nanoparticles were added to the 96-well cell plates at a dosage of 0.2 μg mRNA / well and cultured for another 24 h. After incubation, the expression of CD69 or CD25 was detected by flow cytometry to evaluate the in vitro T cell activation effect. The results are shown in Figure 5 , Figure 5 Figure showing the evaluation results of the in vitro T cell activation effect of different polymer-lipid hybrid nanoparticles in Example 8. As Figure 5 can be seen, when the composition of the hybrid nanoparticles was changed, specifically, when the polymer component was changed, the activation ability of T cells also showed different effects. Specifically, the upregulation rate of the T cell surface marker CD69 in the Arg LNP treatment group was 4%, and Arg(NO 2) The upregulation rate of CD69 in the treatment group was 2%, the upregulation rate of CD69 in the Orn LNP treatment group was 5%, and the upregulation rate of CD69 in the Orn(Tos)LNP treatment group was 11%. Compared with these four polymer-lipid hybrid nanoparticles, RT LNP could induce a significant upregulation of CD69 (68%) and had the strongest T cell activation ability. It further indicated that this T cell activation ability was unique to RT LNP.

[0099] In summary, the present invention provides an mRNA delivery platform with T cell activation ability, which can simultaneously activate and transfect T cells in one step. Using the said delivery platform, CAR mRNA can be delivered into T cells without pre-activating T cells with antibodies, realizing the rapid preparation of CAR T cell drugs in one step. In the present invention, the cytotoxicity of CART cell drugs was verified in vitro, realizing a proof-of-concept of the therapeutic potential of rapidly preparing CAR T cell products. This technical solution omits the step of artificially activating T cells in vitro, shortens the preparation time, reduces the preparation complexity, and has good application prospects and transformation potential.

[0100] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A polymer lipid hybrid nanoparticle having T cell activation ability, characterized in that: The invention comprises a cationic polymer, an auxiliary lipid, cholesterol and a polyethylene glycol-derivatized lipid, wherein the cationic polymer comprises a copolymer prepared from polyethyleneimine and p-toluenesulfonylarginine.

2. The polymer lipid hybrid nanoparticle according to claim 1, characterized in that The polyethyleneimine is linear polyethyleneimine or branched polyethyleneimine, and the number average molecular weight of the polyethyleneimine is 300 to 80,000 Dalton; The molar ratio of the polyethyleneimine to p-toluenesulfonylarginine is 1:(1-10000).

3. The polymer lipid hybrid nanoparticle according to claim 1, characterized in that The preparation method of the cationic polymer comprises the following steps: A) C8H 17 N3·HCl and 1-hydroxybenzotriazole are added to a solution of arginine double-protected by tert-butyloxycarbonyl and p-toluenesulfonyl to activate the solution, thereby obtaining a solution after carboxyl activation; B) mixing the aqueous solution of polyethyleneimine and the activated solution, and then adding N,N-diisopropylethylamine to react to obtain a reacted solution; C) dialyzing and freeze-drying the solution after the reaction to obtain a freeze-dried product; reacting the freeze-dried product with trifluoroacetic acid, dialyzing and freeze-drying to obtain a cationic polymer component.

4. The polymer lipid hybrid nanoparticle according to claim 3, characterized in that: The C8H 17 The molar ratio of N3·HCl, the 1-hydroxybenzotriazole, the N,N-diisopropylethylamine and the p-toluenesulfonylarginine is (1-5): (1-5): (1-5): 1; In step A), the activation temperature is 25-38°C and the activation time is 1-4h; In step B), the reaction temperature is 25-38°C and the reaction time is 2-3 days; In step C), the molecular weight of the dialyzed solution is 500 to 5000 Dalton; the dialysis time is 2 to 4 days; and the freeze-drying temperature is -50 to -80°C.

5. The polymer lipid hybrid nanoparticle according to claim 1, characterized in that: In terms of molar fractions, it includes: 5 to 45 parts of a cationic polymer; 10 to 30 parts of auxiliary lipids; 10 to 70 parts of cholesterol; 1 to 25 parts of polyethylene glycol-derivatized lipids.

6. The polymer lipid hybrid nanoparticle according to claim 1, characterized in that: The auxiliary lipid is selected from at least one of DSPC, DOPE, and DOTAP, preferably DSPC; The polyethylene glycol-derived lipid is selected from DMG-PEG, C 14 -PEG, at least one of DSPC-PEG, preferably DMG-PEG.

7. Use of the polymer lipid hybrid nanoparticles according to any one of claims 1 to 6 in the preparation of a preparation having T cell activation ability.

8. The use according to claim 7, characterized in that: The T cell activation ability includes up-regulating the expression of related genes and inducing cytokine secretion, wherein the up-regulated genes include at least one of CD69 and CD25; and the secreted cytokines include at least one of interferon-γ, interleukin-2 and tumor necrosis factor-α.

9. The use according to claim 7, characterized in that: The T cell activation ability also includes inducing the activation and / or proliferation of T cells.

10. Use of the polymer lipid hybrid nanoparticles according to any one of claims 1 to 6 in the preparation of drug delivery carriers.

11. The use according to claim 10, characterized in that: The polymer lipid hybrid nanoparticles are T cell-targeted nanoparticles.

12. The use according to claim 10, characterized in that: The drug is selected from a nucleic acid drug, and the nucleic acid drug is selected from a non-functional mRNA and / or a functional mRNA; preferably, the functional mRNA includes an mRNA encoding CAR 、 mRNA encoding TCR, mRNA encoding CRISPR, mRNA encoding cytokine; more preferably, the mRNA encoding CAR includes at least one of HER2 CAR, CD19 CAR, and FAP CAR.

13. Use of the polymer lipid hybrid nanoparticles according to any one of claims 1 to 6 in the preparation of drugs for treating tumors.

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