A car-nk cell based on a dendritic molecule and a preparation method and application thereof

Abstract

The application discloses a CAR-NK cell based on a dendritic molecule and a preparation method and application thereof. The CAR-NK cell is an assembly formed by the dendritic molecule, effectively loads nucleic acid expressing CAR through electrostatic interaction, forms a stable and uniform delivery system with nanometer size, is safely and effectively delivered to NK cells by virtue of the dendritic molecule, and prepares the CAR-NK cell with high-efficiency target gene expression, so as to solve the problems of low NK cell transduction efficiency and safety of a virus vector and improve the curative effect of the CAR-NK cell. The use of the dendritic molecule for transfection does not affect the phenotype of the NK cell and surface activation and inhibition receptors thereof. The CAR-NK cell prepared based on the dendritic molecule has a remarkable target cell killing efficiency in vitro and a remarkable anti-tumor (Raji-Luc transplanted tumor model) effect in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a CAR-NK cell based on dendritic molecules, its preparation method, and its application. Background Technology

[0002] Natural killer (NK) cells are important effector cells in the human innate immune system, accounting for about 10% of mononuclear cells in blood and lymphatic organs. They are mainly distributed in lymph nodes, bone marrow, spleen, liver, intestinal epithelium, and lung epithelium. NK cells are the body's first line of defense against viral infections and malignant cells. They can exert a cytotoxic effect by directly killing cancerous or infected cells without prior antigen sensitization. NK cells mainly exert their cytotoxic killing effect by releasing perforin and granzymes, as well as secreting cytokines. In addition, NK cells have immunomodulatory effects on dendritic cells, macrophages, neutrophils, and antigen-specific T cells and B cells.

[0003] Compared to natural NK cells, genetic modification of NK cells can enhance their targeting and anti-tumor response. Specifically, modification with chimeric antigen receptors (CARs) can improve the recognition and killing effect of NK cells on target cells. A chimeric antigen receptor is a receptor protein that specifically recognizes target proteins and induces secondary signal transduction. CAR technology refers to a novel cell therapy technique that uses genetic engineering to express an artificially constructed CAR on the surface of immune effector cells. This CAR recognizes specific tumor cell surface antigens and activates immune cells through its intracellular signal transduction activation domain, thereby exerting a killing effect. Recent studies have shown that CAR-expressing NK cells may overcome clinical problems associated with CAR-T cells, such as graft-versus-host disease (GVHD), cytokine release syndrome (CRS), and neurotoxicity. In CAR-NK cell clinical trials, this cell preparation has demonstrated significant tumor suppression and prolonged survival effects in both hematologic malignancies and solid tumors, with almost no CRS / GVHD. Therefore, introducing antigen-specific CAR genes into NK cells to develop CAR-NK cell therapy has advantages.

[0004] Due to their sensitivity to foreign genetic material and inherent defense mechanisms against viral infection, NK cells typically exhibit low transduction levels and induce apoptosis, resulting in relatively low transduction efficiency compared to T cells. Currently, CAR-NK cell preparation primarily utilizes viral vectors and electroporation for transduction. Recombinant lentiviruses show low transduction efficiency on NK cells, while retroviruses can randomly integrate into the genome but pose a potential risk of insertional mutations. Furthermore, while electroporation is a simple and cost-effective method for introducing CAR genes into NK cells, the permeabilization of the cell membrane by electrical pulses can easily lead to membrane leakage, resulting in high cell death rates. Therefore, there is an urgent need to develop safe and efficient dendritic molecule-based nucleic acid delivery systems for the preparation and application of CAR-NK cells. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a CAR gene delivery system for preparing CAR-NK cells and its application.

[0006] Another object of the present invention is to provide a CAR-NK cell based on dendritic molecules.

[0007] Another object of the present invention is to provide the application of the CAR-NK cell.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] A CAR gene delivery system for preparing CAR-NK cells, wherein the delivery system is a stable and uniform delivery system with nanoscale dimensions formed by assemblies of dendritic molecules effectively loading and expressing CAR nucleic acids through electrostatic interactions.

[0010] Preferably, the dendritic molecule is an amphiphilic dendritic molecule with different chain lengths and different end modifications, consisting of a hydrophilic end and a hydrophobic end; the hydrophobic end of the amphiphilic dendritic molecule is a hydrophobic alkyl chain, and the hydrophilic end is a dendritic structure, including but not limited to polyamide and polyurethane backbones, and its end is modified with functional groups with different functions, including but not limited to guanidinium, N,N-dimethyl, N,N-diethyl, pyrrole, piperidine, morpholine, and piperazine.

[0011] Preferably, the amphiphilic dendritic molecule is selected from AD type amphiphilic dendritic molecules, AB2 type amphiphilic dendritic molecules, and AB3 type amphiphilic dendritic molecules.

[0012] The AD-type amphiphilic dendritic molecule is selected from any one of the following (I), (II), and (III):

[0013]

[0014] In the formula,

[0015] R1 is C 6-22 Alkyl, C 4-22 Fluorinated alkyl or C 6-22 alkenyl;

[0016] R2 is S;

[0017] R3 is C 4-10 Alkylene;

[0018] R4, R5, R6, R7, R8, and R9 are all C. 2-6 Alkylene;

[0019] R is C 1-3 Alkoxy, amino, guanidinyl or -NH-R 10 ;

[0020] R 10 For R 11 Replacement C 1-6 alkyl;

[0021] R 11 For amino, , C 1-6 Alkyl monosubstituted amino, or ;

[0022] R 12 C 1-6 Alkylene.

[0023] The AB2 type amphiphilic dendritic molecule is selected from the following:

[0024]

[0025] In the formula,

[0026] R 13 It is a methyl, methoxy, or halogen group;

[0027] n = 4 to 12;

[0028] X can be represented independently in the following two structures: (Ⅳ), (Ⅴ), and (Ⅵ).

[0029]

[0030] in,

[0031] R 14 R 17 or R 19 C independently 1-5 alkylene groups;

[0032] R15 R 18 or R 20 Independently, they are covalent bonds and C 1-4 alkylene groups;

[0033] R 16 It is an amino, carboxyl, or -NHR group. 21 ;

[0034] R 21 It is tert-butyloxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyl, trifluoroacetyl, , , , , , , , .

[0035] The AB3 type amphiphilic dendritic molecule is selected from the following:

[0036]

[0037] In the formula,

[0038] R 22 C 1-3 Alkyl, C 1-3 alkoxy, halogen or ;

[0039] M is , , or ;

[0040] n is an integer from 2 to 16;

[0041] Y can be represented independently in three structures: (VII), (VIII), or (IX).

[0042]

[0043] in,

[0044] R 23 R 25 R 26 R 27 or R 28 C independently 2-6 alkylene groups;

[0045] R 24 It is a hydroxyl, hydrazine, substituted or unsubstituted amino group, substituted or unsubstituted C group. 1-6 Alkylamino or substituted or unsubstituted C1-6 Alkoxy groups; their substituents are halogens, amino groups, C... 1-4 Alkylamino, C 1-5 Alkyl, benzyl, phenyl, carboxyl, C 2-5 Ester group, benzyl ester group, , or ;

[0046] R 29 It is hydrogen or substituted or unsubstituted C 1-6 Alkyl groups; whose substituents are halogen, amino, carboxyl, C 1-4 Alkylamino or C 1-4 Alkoxy;

[0047] R 30 C 1-6 Alkyl, C 1-6 Alkoxy or C 1-6 Alkylamino.

[0048] Preferably, the CAR nucleic acid is selected from nucleic acids in the form of plasmid DNA or mRNA containing the CAR gene (including but not limited to transient transposon plasmids or transposon plasmids).

[0049] The application of the CAR gene delivery system in the preparation of CAR-NK cells.

[0050] A CAR-NK cell based on a dendritic molecule, wherein the CAR-NK cell is an NK cell transfected with the CAR gene delivery system of claim 5.

[0051] The method for preparing CAR-NK cells includes the following steps:

[0052] (1) Preparation of dendritic molecules: Under aseptic conditions, the amphiphilic dendritic molecules were dissolved in sterile water, sonicated, and allowed to stand to prepare a stock solution;

[0053] (2) Preparation of dendritic molecule delivery system: The stock solution of dendritic molecule is rapidly mixed with the aqueous solution of CAR nucleic acid to prepare a solution with a certain N / P ratio, where N / P is the ratio of amino groups in dendritic molecule to phosphate groups in nucleotide. After standing, the solution of dendritic molecule delivery system is obtained.

[0054] (3) Preparation of CAR-NK cells based on dendritic molecules: NK cells were transfected with a solution of a well-established dendritic molecule delivery system, and the expression of the target gene was measured to determine the CAR-NK cells obtained.

[0055] Preferably, the dissolution temperature of the dendritic molecule in step (1) is 20-30°C, and the sonication time is 30 min; the CAR nucleic acid in step (2) is selected from nucleic acid in the form of plasmid DNA or mRNA containing the CAR gene, and the N / P ratio of the dendritic molecule forming the nanodelivery system to the CAR nucleic acid is 1:10-20:1, preferably 1:5-10:1, and the standing time is 10-40 min.

[0056] The application of the CAR-NK cells in the preparation of gene therapy drugs; preferably in the preparation of gene therapy drugs for cancer or autoimmune diseases.

[0057] Beneficial effects:

[0058] In this invention, the preferred dendritic molecule has advantages such as stronger nuclear penetration and higher transfection efficiency, which can further improve the efficiency of exogenous gene transfection into NK cells. Its killing efficiency against specific target cells (Raji-Luc cells) is significantly higher than that of untransfected NK cells, demonstrating that anti-CD19-CAR-NK cells, after expressing the CD19 receptor, enhance their targeting ability and killing activity. After co-incubating anti-CD19-CAR-NK cells and Raji-Luc cells for 24 hours, the expression levels of cytokines IFN-γ and TNF-α, as well as perforin and granzyme, were significantly higher than those of NK cells, indicating that anti-CD19-CAR-NK cells have an enhanced anti-tumor effect on specific target cells. Simultaneously, the CAR-NK cells prepared using the dendritic molecule provided by this invention exhibit significant anti-tumor activity in vivo, providing a new non-viral vector-based tool and method for CAR-NK cell therapy. Attached Figure Description

[0059] Figure 1 To investigate the cytotoxicity of amphiphilic dendritic molecules with different configurations on NK-92 cells using the CCK-8 assay.

[0060] Figure 2 To screen for pEGFP delivery activity of different conformations of amphiphilic dendritic molecular nucleic acid delivery systems on NK-92 cells using flow cytometry

[0061] Figure 3 For the optimization of AD type TK-AmD C for agarose gel electrophoresis 8-14 8. The binding affinity of Gua amphiphilic dendritic molecules to nucleic acids

[0062] Figure 4 For flow cytometry analysis, the AD type TK-AmD C is preferred. 8-14 Screening of pEGFP delivery activity in NK-92 cells using the 8Gua amphiphilic dendritic molecular nucleic acid delivery system

[0063] Figure 5 To investigate pDNA / TK-AmD C by flow cytometry 8-14 8Gua uptake capacity in NK-92 cells

[0064] Figure 6 To investigate pDNA / TK-AmDC using laser confocal microscopy 8-14 8. Uptake and endosome escape ability of Gua in NK-92 cells

[0065] Figure 7 TK-AmD C is the preferred choice for flow cytometry analysis. 8-14 Transfection activity of 8Gua amphiphilic dendritic molecules against anti-pCD19 CAR plasmids in NK-92 cells

[0066] Figure 8 To investigate TK-AmD C by flow cytometry 8-14 Effects of Gua transfection on NK-92 cell viability

[0067] Figure 9 To investigate TK-AmD C by flow cytometry 8-14 Effects of Gua transfection on NK-92 cell phenotype

[0068] Figure 10 To investigate TK-AmD C by flow cytometry 8-14 Effects of 8Gua transfection on the activation and inhibition of receptors on the surface of NK-92 cells

[0069] Figure 11 To investigate TK-AmD C using the ELISA method 8-14 Figure 8 shows the secretion of cytokines from CAR-NK cells prepared with Gua after co-incubation with target cells.

[0070] Figure 12 To investigate TK-AmD C for LDH detection method 8-14 The killing ability of CAR-NK cells prepared by 8Gua and untransfected NK cells against Raji-Luc cells and K562 cells.

[0071] Figure 13 For TK-AmD C 8-14 8. In vivo antitumor effects of CAR-NK cells prepared by Gua and untransfected NK cells on the Raji-Luc xenograft model. Detailed Implementation

[0072] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to embodiments. The described embodiments are only some embodiments of this application, and not all embodiments.

[0073] The dendritic molecular structure used in the examples is shown below:

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] Example 1: Toxicity of amphiphilic dendritic molecules with different configurations on NK-92 cells

[0097] NK-92 cells were cultured using commercially available serum-free NK cell culture medium and incubated at 37°C with 5% carbon dioxide. Subsequently, well-grown NK-92 cells were seeded into 96-well plates at 1 × 10⁶ cells per well. 5 Cells. After 24 hours, solutions of amphiphilic dendritic molecules with different configurations (including AB2 type AB2 C) were prepared under the condition of N / P=2 (N / P is the ratio of amino groups in the dendritic molecule to phosphate groups in the nucleotide). 11 4A, AB3 type AB3 C 11 4A, AD type TK-AmD C 8-10 2A), and let stand for 10 min. Add different concentrations of dendritic molecular solutions to each well, and continue incubation at 37℃ for 48 h. Then add 10 µL of CCK-8 solution to each well and continue incubation. After 4 h, detect the absorbance with a microplate reader, and calculate the corresponding cell viability based on the absorbance. AB2 type AB2 C 11 4A, AB3 type AB3 C 11 4A, AD type TK-AmD C 8-10 Cell viability of NK-92 cells under the influence of 2A dendritic molecules, such as Figure 1 As shown.

[0098] Example 2: Screening of pEGFP plasmid delivery activity in NK-92 cells using amphiphilic dendritic molecular nucleic acid delivery systems with different configurations.

[0099] Dendritic molecular compounds and their preparation methods with pDNA (plasmid DNA):

[0100] Step 1) Preparation of dendritic molecule stock solution: Under aseptic conditions, amphiphilic dendritic molecules of different configurations (including AD type, AB2 type, AB3 type) are dissolved in sterile water, sonicated for 30 min, and then stored at -20℃ after standing overnight at 4℃ for later use.

[0101] Step 2) Preparation of dendritic molecule-pDNA complex: Under sterile conditions, according to a specific N / P ratio (N / P is the ratio of amino groups in the dendritic molecule to phosphate groups in the nucleotide), the stock solution of the amphiphilic dendritic molecule and the aqueous solution of the plasmid are mixed and allowed to stand at room temperature for 10 to 40 min to obtain the nanocomplex solution.

[0102] Cell plating and transfection methods:

[0103] Well-grown NK-92 cells were seeded into 24-well plates, with 2 × 10⁶ cells per well. 5Cells were cultured. After 24 hours, following the above-described preparation method for the dendritic compound and its pDNA complex, a nanocomplex solution with a specific N / P ratio was prepared and added to 24-well plates. The plates were then incubated at 37°C for 48 hours. The transduction efficiency was calculated by detecting the GFP positivity rate using flow cytometry. The pEGFP delivery activity of different conformations of amphiphilic dendritic molecules in NK-92 cells is shown in [reference needed]. Figure 2 Experimental results show that the AD-type amphiphilic dendritic molecule has the best delivery activity. Furthermore, we preferentially selected the AD-type G3 generation TK-AmD C... 8-14 8. Research continues on the amphiphilic dendritic molecules of Gua.

[0104] Example 3: Preferred AD type TK-AmD C 8-14 8. The binding affinity of Gua amphiphilic dendritic molecules to pDNA

[0105] Preparation of dendritic molecular compounds and their complexes with pDNA (pEGFP plasmid):

[0106] Step 1) Preparation of dendritic stock solution: Under aseptic conditions, TK-AmD C 8-14 8Gua amphiphilic dendritic molecules were dissolved in sterile water, sonicated for 30 min, and then left to stand overnight at 4°C before being stored in a -20°C refrigerator for later use.

[0107] Step 2) Preparation of the dendritic molecule-pDNA complex: Under sterile conditions, according to a specific N / P ratio (N / P is the ratio of amino groups in the dendritic molecule to phosphate groups in the nucleotide), TK-AmD C 8-14 Mix the stock solution of 8Gua amphiphilic dendritic molecules with the aqueous solution of plasmid, and incubate at room temperature for 10 to 40 minutes to obtain pDNA / TK-AmDC. 8-14 8Gua complex solution.

[0108] The binding affinity of dendritic molecules to pDNA:

[0109] TK-AmD C was investigated using agarose gel electrophoresis. 8-14 The binding ability of 8Gua to plasmids was assessed using naked plasmids as a positive control, and agarose gel electrophoresis was performed as follows: Figure 3 As shown. The results show that TK-AmD C 8-14 The fact that the effective payload plasmid of 8Gua did not leak indicates that the dendritic molecule has good nucleic acid loading stability.

[0110] Example 4: Preferred AD type TK-AmD C 8-14 Screening for pEGFP delivery activity of 8Gua dendritic molecules in NK cells.

[0111] Well-grown NK-92 cells were seeded into 24-well plates, with 2 × 10⁶ cells per well. 5 Cells. After 24 hours, pEGFP / TK-AmD C cells with different N / P ratios were prepared according to the method in Example 2. 8-14 8Gua complex solution was added to 24-well plates and incubated at 37°C for 48 hours. Transduction efficiency was calculated by detecting the GFP positivity rate using flow cytometry. (TK-AmD C) 8-14 pEGFP delivery activity of 8Gua in NK-92 cells is shown in the figure. Figure 4 .

[0112] Example 5: pDNA / TK-AmD C 8-14 Intracellular transport of 8Gua in NK-92 cells.

[0113] To examine the preferred TK-AmD C of this invention 8-14 8Gua dendritic molecules exhibit high transfection efficiency in NK-92 cells. This example also investigated their intracellular transport process. Using YOYO-1 fluorescently labeled pEGFP as a model plasmid, a certain amount of pEGFP plasmid was incubated with YOYO-1 fluorescent dye at room temperature in the dark for 10 min (30 μg DNA was labeled with 10 μL of 80 μM YOYO-1 dye). YOYO-1-pDNA / TK-AmDC was prepared according to the method in Example 2. 8-14 8Gua complex solution.

[0114] First, the effects of NK-92 cells on YOYO-1-pDNA / TK-AmD C were investigated. 8-14 8. Gua uptake capacity. NK-92 cells were transfected according to the method in Example 2, and the uptake capacity of NK cells in each group for YOYO-1-pDNA / TK-AmD C was detected by flow cytometry. 8-14 The uptake rate of the 8Gua complex and the mean intracellular YOYO-1 fluorescence intensity were compared using untransfected NK cells as a negative control. Figure 5 The results showed that NK cells responded to YOYO-1-pDNA / TK-AmD C 8-14 The uptake rate of the 8Gua complex increased over time, and the average fluorescence intensity of intracellular YOYO-1 also increased over time.

[0115] Subsequently, laser confocal microscopy was used to investigate YOYO-1-pDNA / TK-AmD C 8-14Uptake and endosome escape of the 8Gua complex in NK-92 cells. NK-92 cells were transfected as described in Example 2 and cultured in a cell culture incubator. After reaching specific time points, NK cells from each group were collected. After washing with PBS, 1 mL of NK cell culture medium containing lysosomal green fluorescent probe (Lyso-Tracker green) and nuclear blue fluorescent dye (Hoechst 33342) was added. The cells were incubated at 37°C for 30 min. After washing with PBS, 1 mL of PBS was added, and the intracellular transport process was immediately observed and photographed using a confocal microscope. Figure 6 The results showed that the red fluorescence signal of YOYO-1 in NK cells gradually increased with time, indicating that NK cells respond to pDNA / TK-AmDC. 8-14 The uptake of the 8Gua complex gradually increased. At 1 hour, a yellow signal appeared intracellularly, indicating colocalization of the red signal of YOYO-1 and the green signal of lysosomes. After 4 hours, the red signal of YOYO-1 and the green signal of lysosomes began to separate, indicating that the pDNA / TK-AmD C... 8-14 The 8Gua complex can effectively escape from the endosomes of NK-92 cells into the nucleus, which is beneficial for subsequent plasmid expression.

[0116] Example 6: Preferred TK-AmD C 8-14 Transfection activity of 8Gua dendritic molecules in NK-92 cells with anti-pCD19 CAR plasmid (Miaoling Plasmid Company, catalog number: P44108).

[0117] Well-grown NK-92 cells were seeded into 24-well plates, with 2 × 10⁶ cells per well. 5 Cells. After 24 hours, prepare an anti-pCD19 CAR / TK-AmD C formulation with a specific N / P ratio. 8-14 The 8Gua complex solution was added to 24-well plates after standing, with lentivirus transfection as a control. The plates were incubated at 37°C for 48 hours. Cells from each group were then collected, and CD19 protein expression was detected by flow cytometry. Figure 7 The results showed that TK-AmD C 8-14 The transfection efficiency of the anti-pCD19 CAR plasmid of 8Gua in NK-92 cells was higher than that of lentivirus.

[0118] Example 7: Based on TK-AmD C 8-14 Physiological activity study of CAR-NK prepared from 8Gua dendritic molecules.

[0119] To evaluate the use of TK-AmD C 8-14The CAR-NK cells prepared by 8Gua still maintain normal physiological activity. This invention studies the cell viability, cell phenotype, changes in cell surface activating and inhibiting receptors, and cytokine secretion of CAR-NK cells.

[0120] First, CD19 CAR-NK cells were prepared according to Example 6. We used the Annexin V-PE / 7-AAD apoptosis assay kit to verify TK-AmD C 8-14 Cell viability of CAR-NK cells prepared by 8Gua dendritic molecules was assessed, with CAR-NK cells prepared by electroporation serving as a control. 48 h after NK cell transfection, CAR-NK cells from each group were collected, washed twice with PBS, resuspended in 100 μL x Binding Buffer, and then 5 μL Annexin V-PE and 5 μL 7-AAD staining solution were added. The cells were incubated at room temperature in the dark for 10 min, and then 400 μL x Binding Buffer was added. After gentle mixing, the ratio of Annexin V-PE / 7-AAD double positivity was assessed using flow cytometry. Figure 8 The results showed that TK-AmD C 8-14 NK cells transfected with 8Gua dendritic molecules showed no significant difference in cell viability compared to untreated NK cells, but CAR-NK cells prepared by electroporation exhibited significant apoptosis.

[0121] To avoid TK-AmD C 8-14 8Gua transfection affects the phenotype of NK cells and their surface functional receptors. This study investigated the effects of TK-AmD C transfection on NK cell phenotype. 8-14 The phenotype of CAR-NK cells prepared using 8Gua dendritic molecules, and the activation and inhibition receptors on the surface of CAR-NK cells. Untransfected NK cells were used as a blank control. 48 h after NK cell transfection, CAR-NK cells were collected, washed twice with PBS, counted, and incubated with a solution containing fluorescently labeled flow cytometry antibodies CD3 and CD56. After 30 min, the cells were washed twice with PBS, and the CD3 / CD56 ratio of CAR-NK cells was examined using flow cytometry. Untransfected NK cells were used as a blank control group. Figure 9 The results showed that TK-AmD C 8-14 Transfection with 8Gua dendritic molecules did not affect the phenotype of NK cells. CAR-NK cells were collected using the same method and incubated with flow cytometry antibodies containing fluorescently labeled NKG2D, NKp30, NKp40, NKp46, TIGIT, PD-1, and CD96 to test the effect of dendritic molecule transfection on their surface activating and inhibiting receptors. Figure 10 The results show that based on TK-AmD C8-14 CAR-NK cells prepared by 8Gua dendritic molecules showed no significant difference from untransfected NK cells.

[0122] Finally, we examined TK-AmD C 8-14 Cytokine secretion after co-incubation of CAR-NK cells prepared with 8Gua and target cells. Human lymphoma Raji cells were incubated at 1×10⁸ cells / cells. 4 The substrate was laid at a density of 1×10⁶ cells / well in a 96-well plate, with 1×10⁶ cells / well added to each well. 5 CAR-NK cells were incubated at 37°C for 24 hours, and supernatants from all groups were collected. Cytokine secretion from CAR-NK cells was measured using an ELISA kit, with untransfected NK cells serving as a control. Figure 11 The results showed that, under stimulation by target cell antigens, TK-AmD C... 8-14 CAR-NK cells constructed from 8Gua dendritic molecules secreted significantly more cytokines than untransfected NK cells, indicating that these CAR-NK cells have an enhanced anti-tumor effect on specific target cells.

[0123] Example 8: Based on TK-AmD C 8-14 CAR-NK cells prepared from 8Gua dendritic molecules specifically kill target cells.

[0124] CD19 CAR-NK cells were prepared according to Example 6. Human Raji lymphoma cells expressing CD19 were used as antigen-positive target cells, and human chronic myeloid leukemia K562 cells not expressing CD19 were used as antigen-negative target cells. Untransfected NK cells were used as controls. The target cells were prepared at a concentration of 1 × 10⁻⁶ cells / cells. 4 CAR-NK cells were seeded at a density of 1 cell / well in 96-well plates, with the appropriate number of cells added to each well according to the effector-to-target ratio. The plates were then incubated in a cell culture incubator for 24 hours. Sample control wells containing only tumor cells and maximum enzyme activity control wells for subsequent lysis were also included. One hour before the predetermined detection time, the 96-well plates were removed from the cell culture incubator. LDH release reagent was added to the sample control wells at 10% of the original culture medium volume. After adding the LDH release reagent, the plates were mixed thoroughly by pipetting several times, and then incubated in the cell culture incubator. After the predetermined time, the cell suspension from each well was collected and centrifuged. 100 μL of the supernatant was added to a new 96-well plate, and 60 μL of LDH detection working solution was added to each well. The plates were mixed and incubated at room temperature in the dark for 30 minutes. The absorbance was then measured at 490 nm using a microplate reader. Cell mortality rate (%) = (Absorbance of treated sample - Absorbance of sample control well) / (Absorbance of maximum enzyme activity of cells - Absorbance of sample control well) × 100%. Figure 12As shown, based on TK-AmD C 8-14 CAR-NK cells prepared by 8Gua dendritic molecules have good specific killing ability against CD19-positive target cells, while their killing ability against target cells that do not express CD19 is comparable to that of untransfected NK cells.

[0125] Example 9: Based on TK-AmD C 8-14 The antitumor efficacy of CAR-NK cells prepared by 8Gua dendritic molecules in an in vivo tumor model of Raji-Luc transplantation.

[0126] Using NOD-SCID IL-2 receptor γ-deficient (NSG) mice, 1×10⁻⁶ mmol / L was injected via tail vein. 6 Raji-Luc cells were used to establish an acute lymphoblastic leukemia model. After observing luciferase-induced fluorescence (approximately 5 days later), tumor-bearing mice were randomly divided into three groups of five mice each. Each group was administered PBS and TK-AmD C via tail vein injection. 8-14 CAR-NK cells constructed from 8Gua (2×10) 6 CAR-NK cells constructed from lentiviruses (2×10⁻⁶ cells) 6 Cells were used as a control. Tumor burden was monitored every 5 days by quantitative fluorescence. After intraperitoneal injection of 0.15 mg D-fluorescein per gram of body weight, bioluminescence imaging was performed on a small animal in vivo imaging system (IVIS), and the fluorescence data were analyzed using imaging system software. Figure 13 The therapeutic effect was demonstrated in immunodeficient tumor-bearing mice inoculated with human Raji-Luc tumor cells. Results showed that, compared to the control group, TK-AmD C... 8-14 The CAR-NK cell group constructed by 8Gua has a significant anti-tumor effect, comparable to the therapeutic effect of CAR-NK cells constructed by lentivirus.

Claims

1. A CAR gene delivery system for preparing CAR-NK cells, characterized in that, The delivery system is a stable and uniform nanoscale delivery system formed by the electrostatic interaction of assemblies of dendritic molecules to effectively load and express CAR nucleic acids.

2. The CAR gene delivery system according to claim 1, characterized in that, The aforementioned dendritic molecules are amphiphilic dendritic molecules with different chain lengths and different end modifications, consisting of hydrophilic and hydrophobic ends; the hydrophobic end of the amphiphilic dendritic molecule is a hydrophobic alkyl chain, and the hydrophilic end is a dendritic structure, including but not limited to polyamide and polyurethane backbones, and its ends are modified with functional groups with different functions, including but not limited to guanidinyl, N,N-dimethyl, N,N-diethyl, pyrrole, piperidine, morpholine, and piperazine.

3. The CAR gene delivery system according to claim 1, characterized in that, The amphiphilic dendritic molecules are selected from AD type amphiphilic dendritic molecules, AB2 type amphiphilic dendritic molecules, and AB3 type amphiphilic dendritic molecules.

4. The CAR gene delivery system according to claim 1, characterized in that, The AD-type amphiphilic dendritic molecule is selected from any one of the following (I), (II), and (III):

5. In the formula, R1 is C 6-22 Alkyl, C 4-22 Fluorinated alkyl or C 6-22 alkenyl; R2 is S; R3 is C 4-10 Alkylene; R4, R5, R6, R7, R8, and R9 are all C. 2-6 Alkylene; R is C 1-3 Alkoxy, amino, guanidinyl or -NH-R 10 ; R 10 For R 11 Replacement C 1-6 alkyl; R 11 For amino, , C 1-6 Alkyl monosubstituted amino, or ; R 12 C 1-6 Alkylene.

6. The AB2 type amphiphilic dendritic molecule is selected from the following:

7. In the formula, R 13 It is a methyl, methoxy, or halogen group; n＝4～12； X can be represented independently in the following two structures: (Ⅳ), (Ⅴ), and (Ⅵ).

8. Among them, R 14 R 17 or R 19 C, each independently 1-5 alkylene groups; R 15 R 18 or R 20 Independently, they are covalent bonds and C 1-4 alkylene groups; R 16 It is an amino, carboxyl, or -NHR group. 21 ; R 21 It is tert-butyloxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyl, trifluoroacetyl, , , , , , , , .

9. The AB3 type amphiphilic dendritic molecule is selected from the following:

10. In the formula, R 22 C 1-3 Alkyl, C 1-3 alkoxy, halogen or ; M is , , or ; n is an integer from 2 to 16; Y can be represented independently in three structures: (VII), (VIII), or (IX).

11. Among them, R 23 R 25 R 26 R 27 or R 28 C, each independently 2-6 alkylene groups; R 24 It is a hydroxyl, hydrazine, substituted or unsubstituted amino group, substituted or unsubstituted C group. 1-6 Alkylamino or substituted or unsubstituted C 1-6 Alkoxy groups; their substituents are halogens, amino groups, C... 1-4 Alkylamino, C 1-5 Alkyl, benzyl, phenyl, carboxyl, C 2-5 Ester group, benzyl ester group, , or ; R 29 It is hydrogen or substituted or unsubstituted C 1-6 Alkyl groups; whose substituents are halogen, amino, carboxyl, C 1-4 Alkylamino or C 1-4 Alkoxy; R 30 C 1-6 Alkyl, C 1-6 Alkoxy or C 1-6 Alkylamino.

12. The CAR gene delivery system according to any one of claims 1-4, characterized in that, The CAR nucleic acid is selected from nucleic acids in the form of plasmid DNA or mRNA containing the CAR gene, wherein the plasmid DNA includes, but is not limited to, transient transgenic plasmids or transposon plasmids.

13. The application of the CAR gene delivery system of claim 5 in the preparation of CAR-NK cells.

14. A CAR-NK cell based on dendritic molecules, characterized in that, The CAR-NK cells are NK cells transfected with the CAR gene delivery system of claim 5.

15. The method for preparing CAR-NK cells according to claim 7, characterized in that, Includes the following steps: (1) Preparation of dendritic molecules: Under aseptic conditions, the amphiphilic dendritic molecules were dissolved in sterile water, sonicated, and allowed to stand to prepare a stock solution. (2) Preparation of dendritic molecule delivery system: The stock solution of dendritic molecule is rapidly mixed with the aqueous solution of CAR nucleic acid to prepare a solution with a certain N / P ratio, where N / P is the ratio of amino groups in dendritic molecule to phosphate groups in nucleotide. After standing, the solution of dendritic molecule delivery system is obtained. (3) Preparation of CAR-NK cells based on dendritic molecules: NK cells were transfected with a solution from a well-established dendritic molecule delivery system, and the expression of the target gene was measured to determine the availability of CAR-NK cells.

16. The preparation method according to claim 8, characterized in that, The dissolution temperature of the dendritic molecule in step (1) is 20-30℃, and the sonication time is 30 min; the CAR nucleic acid in step (2) is selected from plasmid DNA or mRNA containing the CAR gene, and the N / P ratio of the dendritic molecule forming the nanodelivery system to the CAR nucleic acid is 1:10-20:1, preferably 1:5-10:1, and the standing time is 10-40 min.

17. The use of the CAR-NK cells of claim 7 in the preparation of gene therapy drugs; preferably in the preparation of gene therapy drugs for cancer or autoimmune diseases.

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