Nano CAR T cell as well as preparation method and application thereof

Abstract

The invention discloses a nano CAR T cell as well as a preparation method and application thereof. The nano CAR T cell comprises a CAR T cell, and the surface of the CAR T cell is connected with lipid nanoparticles for activating endogenous immunity. The nano CAR T cell prepared by the invention can be rapidly homed to a tumor part by intravenous injection, the CAR T cell recognizes tumor antigen to be activated and secretes granzyme and perforin, and the lipid nanoparticles respond to CAR signal release, target in-tumor DC cells, promote maturation of the DC cells and uptake tumor-associated antigens released by CAR T cracking tumor, so that the CAR T cell can be rapidly homed to the tumor part. And a plurality of antigen-specific T cells are generated in vivo to cooperatively kill antigen-negative tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a nano CAR T cell and a preparation method and application thereof. Background Art

[0002] Currently, cell therapy has achieved remarkable results in the treatment of hematological malignancies, but the clinical recurrence rate is still very high.

[0003] For example, adoptive cell therapy (ACT) has shown great efficacy in clinical applications for the treatment of a variety of cancers. To date, the U.S. Food and Drug Administration has approved six chimeric antigen receptor T cell (CAR T) therapies and one tumor infiltrating lymphocyte (TIL) therapy, and many clinical trials of similar therapies are underway. Nevertheless, among patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL), most people experience tumor recurrence after receiving CAR T cell therapy. Current research evidence shows that this phenomenon is often closely related to the loss or downregulation of CD19 antigen on the surface of tumor cells. The loss of tumor surface antigens and the high heterogeneity of antigen expression enable tumor cells to evade the attack of immune cells, leading to resistance to cell therapy and ultimately triggering tumor recurrence.

[0004] In order to address the problem of tumor recurrence after CAR T cell therapy, bioengineering technology has been widely used to enhance the targeting of CART cells, enabling them to recognize and attack multiple tumor antigens. Specific methods include (1) developing multispecific CAR T cells and gene editing CAR T cells to secrete bispecific antibodies. (2) modifying the structure of CAR so that it can bind to adapter molecules or immunoglobulin Fc domains. Although these strategies can alleviate tumor recurrence to a certain extent, due to the high heterogeneity of tumor antigens, the strategy of increasing targets often cannot fully solve the problem. mRNA vaccines have also been introduced into CAR T cell therapy to improve its efficacy. In a clinical trial (NCT04503278), Claudin 6 (CLDN6)-specific CAR T cell therapy combined with vaccines achieved significant efficacy in the treatment of CLDN6-positive recurrent or refractory solid tumors. In addition, researchers have designed amphiphilic CAR ligand vaccines to transform dendritic cells (DCs) in lymph nodes to activate and expand CAR T cells. Although this strategy can circumvent the mechanism of antigen-negative tumor escape to a certain extent, excessive attack of CAR T cells on ligand-modified dendritic cells may also bring side effects. The problem of tumor recurrence caused by antigen loss or downregulation remains a difficult problem that needs to be solved urgently in the field of cell therapy.

[0005] Therefore, there is an urgent need to provide a method that can effectively kill antigen-negative tumor cells. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides a nano CAR T cell and a preparation method and application thereof, which combines CAR T cell therapy with in situ vaccines and activates the concept of adaptive immune response to solve the problem of tumor recurrence caused by antigen loss / downregulation during cell therapy.

[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 nano CAR T cell, comprising a CAR T cell having lipid nanoparticles that activate endogenous immunity connected to the surface of the CAR T cell.

[0009] The nano CAR T cells prepared by the present invention can be injected intravenously into the body and can quickly home to the tumor site. The CAR T cells recognize tumor antigens and are activated to secrete granzymes and perforins. Lipid nanoparticles (LNP) respond to CAR signal release, target DC cells in the tumor, promote the maturation of DC cells and the uptake and presentation of tumor-associated antigens released by CAR T lysing tumors, generate multiple antigen-specific T cells in the body, and synergistically kill antigen-negative tumor cells.

[0010] Preferably, the lipid nanoparticles comprise lipids.

[0011] Preferably, the lipid comprises any one or a combination of at least two of a cationic lipid, cholesterol, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol-mannose, distearoylphosphatidylethanolamine-polyethylene glycol or distearoylphosphatidylethanolamine-polyethylene glycol-disulfide bond-N-hydroxysuccinimide.

[0012] Preferably, the cationic lipid comprises SM102.

[0013] The present invention modifies lipid nanoparticles on the surface of CAR T cells without affecting the killing, proliferation and activation performance of CAR T cells. There are disulfide bonds in the CAR T cells and lipid nanoparticle linkers. When CAR T cells are stimulated and activated by tumor antigens, the biothiols on their surface will increase, and then the disulfide bonds will be cut to release lipid nanoparticles.

[0014] The lipid nanoparticles of the present invention are surface-modified with mannose to target DC cells, and at the same time, the lipid nanoparticles encapsulate mRNA encoding the activated STING protein. The lipid nanoparticles can translate and express the activated STING protein in DC cells, promote the activation of DC cells and the antigen presentation ability, thereby promoting the subsequent antigen-specific T cell immune response in the body.

[0015] The N-hydroxysuccinimide (NHS) on the surface of the lipid nanoparticles undergoes a cross-linking reaction with the amino groups on the surface of the CART cells, coupling the lipid nanoparticles to the surface of the CAR T cells. When the CAR T cells are activated by antigens, the disulfide bond SS is cut, and the nano-CAR T responds by releasing the lipid nanoparticles.

[0016] Preferably, the molar ratio of the cationic lipid, cholesterol, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol-mannose and distearoylphosphatidylethanolamine-polyethylene glycol is (45-50):(35-38):(9-10):(1-9):1.

[0017] Preferably, the molar ratio of the cationic lipid, cholesterol, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol-mannose and distearoylphosphatidylethanolamine-polyethylene glycol-disulfide bond-N-hydroxysuccinimide is (45-50):(35-38):(9-10):(1-9):1, for example 45:35:10:9:1, 49:38:10:2:1, 50:38:10:1:1, etc.

[0018] Preferably, the mass percentage of polyethylene glycol in the lipid is 3% to 25%.

[0019] In the present invention, by adding distearoylphosphatidylethanolamine-polyethylene glycol-mannose, DC cells can be targeted, the uptake of lipid nanomaterials by DC cells can be promoted, and the transfection efficiency can be improved. Although PEG can promote long circulation in the body, when the PEG lipid content is greater than 3%, as the PEG lipid content increases, the transfection efficiency of LNP in DC cells gradually decreases.

[0020] Preferably, the lipid nanoparticles are loaded with mRNA having an adjuvant effect, and the particle size of the lipid nanoparticles is 110-130 nm (eg, 110 nm, 120 nm, 130 nm).

[0021] Preferably, the lipid nanoparticles are loaded with mRNA encoding activated STING protein.

[0022] Preferably, the surface of the lipid nanoparticles is modified with mannose.

[0023] Preferably, the CAR T cells express the CD19 antigen.

[0024] In a second aspect, the present invention provides a method for preparing the nano CAR T cells described in the first aspect, the method comprising: preparing lipid nanoparticles that activate endogenous immunity, and connecting the lipid nanoparticles to the surface of CAR T cells to form nano CAR T cells.

[0025] In a third aspect, the present invention provides the use of the nano CAR T cells described in the first aspect in the preparation of a reagent for detecting tumor recurrence and / or a product for treating tumor recurrence.

[0026] Preferably, said tumor recurrence comprises antigen loss and / or down-regulation.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention proposes the concept of combining in situ vaccines with CAR T cells, emphasizing the importance of activating endogenous immunity in the body during CAR T cell therapy. The preparation method of nano-CAR T is simple and easy, and the use of lipid nanoparticles to assist CAR T cell therapy is a potential solution to the problem of tumor recurrence in clinical CAR T cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Transmission electron microscopy characterization of lipid nanoparticles;

[0030] Figure 2 is the particle size distribution diagram of lipid nanoparticles;

[0031] Figure 3 Statistical heat map of fluorescence expression of DC2.4 cells after treatment with lipid nanoparticles of different formulations;

[0032] Figure 4 This is a quantitative graph of the mean fluorescence intensity of intracellular STING protein in DC2.4 cells;

[0033] Figure 5 It is the expression statistics of CD80, CD86 and CD40 on the surface of DC2.4 cells;

[0034] Figure 6 This is a quantitative graph of the mean fluorescence intensity of lipid nanoparticles on the surface of nano-CART cells;

[0035] Figure 7 This is a statistical chart of the killing results of nano-CART cells;

[0036] Figure 8 This is a diagram showing the tumor treatment effect of nano-CART;

[0037] Fig. 9 This is a graph showing the results of in vivo antigen-specific T cells after nano-CART treatment. DETAILED DESCRIPTION

[0038] 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.

[0039] 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.

[0040] Reagents used and their sources:

[0041] Cationic lipid (SM102) was purchased from Avanti.

[0042] Cholesterol was purchased from Sigma-Aldrich.

[0043] Distearoylphosphatidylcholine (DSPC) was purchased from Avanti.

[0044] Distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) was purchased from Avanti.

[0045] Distearoylphosphatidylethanolamine-polyethylene glycol-mannose (DSPE-PEG-Mannose) was purchased from Tanshtech.

[0046] Distearoylphosphatidylethanolamine-polyethylene glycol-disulfide bond-N-hydroxysuccinimide (DSPE-PEG-SS-NHS) was purchased from Ruixibio.

[0047] The mRNA encoding STING protein was purchased from VectorBuilder.

[0048] CAR plasmid was purchased from Addgene.

[0049] Ultracentrifuge, instrument model OPTIMAXPN-100, purchased from BECKMAN.

[0050] Transmission electron microscope, instrument model HT-7700, was purchased from Hitachi, Japan.

[0051] Laser particle size analyzer, instrument model Nano-ZS, was purchased from Malvern.

[0052] Flow cytometer, instrument model Accuri C6, was purchased from BD.

[0053] Example 1

[0054] This embodiment provides a method for preparing lipid nanoparticles and a prescription screening method.

[0055] (1) SM102, cholesterol, DSPC, DSPE-PEG-Mannose and DSPE-PEG were dissolved in ethanol. Orthogonal screening experiments were performed by adjusting the ratio of PEG lipids in total lipids (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) and the ratio of DSPE-PEG-Mannose to DSPE-PEG (e.g., 1:9, 2:8, 3:7, 4:6, 5:5, 7:3, 6:4, 8:2, 9:1) to optimize the transfection performance of the lipid formulation in DC cells.

[0056] (2) The mRNA encoding STING protein was diluted in sodium citrate buffer (10 mM, pH 4.0).

[0057] (3) The two solutions prepared in step (1) and step (2) were quickly mixed at a volume ratio of 1:3 between the ethanol phase and the aqueous phase, and then diluted with PBS (pH 7.4), and centrifuged using a Millipore 30 kD ultrafiltration tube to synthesize LNPs encapsulating luciferase mRNA.

[0058] (4) Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to evaluate the morphology of lipid nanoparticles. Figure 1 ), and the hydrodynamic diameter is about 120nm ( Figure 2 ).

[0059] (5) The preparation was stored at 4°C for further use.

[0060] (6) DC2.4 cells were seeded at a density of 10,000 cells per well in a 96-well plate.

[0061] (7) Add the LNP prepared in step (5) above to the DC2.4 cell culture medium, with the mRNA concentration of 200 ng per well.

[0062] (8) After culturing for 24 h, DC2.4 cells were collected, cell lysis buffer was added, and then luciferase substrate was added. The fluorescence signal was measured using a microplate reader to evaluate the transfection efficiency of lipid nanoparticles.

[0063] (9) The optimal transfection formula of lipid nanoparticles was screened out by changing the ratio of PEG lipids in the total lipid content and the ratio of DSPE-PEG-Mannose to DSPE-PEG. Figure 3As shown, when the proportion of PEG lipids in the total lipids is greater than 3%, the transfection efficiency of DC2.4 cells gradually decreases with the increase of PEG lipids. When the PEG lipid content accounts for 3% of the total lipids and the ratio of DSPE-PEG-Mannose to DSPE-PEG is 7:3, the fluorescence expression is the strongest. According to the order described in (1), the optimal molar percentages are 49.2%, 37.9%, 9.8%, 2.1% and 0.9%, respectively.

[0064] Example 2

[0065] This embodiment provides a method for preparing CART cells by a retroviral method.

[0066] (1) Mix 20 μg of CAR plasmid with 60 μL of Lipofectamine 2000 to form a transfection complex.

[0067] (2) The above complex was added to Platinum-E cells in a T150 culture flask. After 24 hours of transfection, the old culture medium was replaced with preheated fresh DMEM culture medium.

[0068] (3) After 24 h, the viral supernatant was harvested and centrifuged at 800 g for 5 min and filtered through a 0.45 μm filter to remove cell debris.

[0069] (4) Store the produced retroviral supernatant at -80°C for subsequent use.

[0070] (5) CD3 T cells were isolated and purified from the spleen of female C57BL / 6 mice aged 6-8 weeks using a mouse CD3 T cell isolation kit.

[0071] (6) Use mouse T cell activator CD3 / CD28 magnetic beads to culture and activate T cells at a ratio of 1:1 between cells and magnetic beads.

[0072] (7) Add the Retronectin solution to a non-tissue culture treated 24-well plate to coat the plate, and add the retroviral supernatant produced in the above step (4) to the Retronectin-coated 24-well plate.

[0073] (8) After centrifugation at 2000 g for 1 h, the T cells pre-activated in step (6) were added. After 24 h, transfection was completed and CAR T cells were obtained.

[0074] Example 3

[0075] This embodiment provides a method for preparing nano CAR T.

[0076] (1) 1×10 6CAR T cells were resuspended in 1 mL PBS, and then LNP (6 μg mRNA) was added and incubated at 37°C for 2 h.

[0077] (2) NanoCAR T cells were centrifuged twice (500 g, 5 min) to remove free lipid nanoparticles and then resuspended in PBS for in vitro or in vivo studies.

[0078] Test Example 1

[0079] This test example tests the DC cell transfection by lipid nanoparticles prepared in Example 2.

[0080] (1) DC2.4 cells were plated in a confocal microscopy dish at a density of 20,000 cells per well.

[0081] (2) DC2.4 cells were treated with lipid nanoparticles encapsulating STING protein mRNA.

[0082] (3) After 12 hours, the supernatant was removed, the cells were washed twice with PBS, and then 4% paraformaldehyde was added to fix the cells. Immunofluorescence experiments were performed to observe the lipid nanoparticles. The test results are shown in Figure 4 .

[0083] from Figure 4 It can be seen that the average fluorescence intensity of STING protein is significantly increased.

[0084] Test Example 2

[0085] This test example tests the lipid nanoparticles prepared in Example 2 on the maturation and antigen presentation of DC cells.

[0086] (1) Primary cells were isolated from the femurs and tibias of 6-8 week old male C57BL / 6 mice.

[0087] (2) Add 20 ng / mL GM-CSF and 20 ng / mL IL-4 to RPMI 1640 complete medium to induce differentiation into BMDC cells.

[0088] (3) On the seventh day, lipid nanoparticles were added for treatment. BMDC cells and supernatant were collected 48 hours later. Flow cytometry was used to detect the expression of BMDC cell surface activation factors CD80, CD86, and CD40. The test results are shown in Figure 5 .

[0089] from Figure 5 It can be seen that the expression levels of CD80, CD86 and CD40 markers on the surface of BMDC cells were significantly increased, and LNP has a good DC cell activation effect.

[0090] Test Example 3

[0091] This test example verifies the modification of lipid nanoparticles in the nano-CART prepared in Example 3.

[0092] DiD-labeled lipid nanoparticles were coupled to Hoechst 33342-stained CAR T cells, and the mean fluorescence intensity ( Figure 6 ) to verify the modification of lipid nanoparticles on the surface of CART cells.

[0093] Test Example 4

[0094] This test example tests the biological function of the NanoCAR T cells prepared in Example 3.

[0095] (1) Untransfected T cells, CAR T cells and NanoCART cells were collected and incubated with pancreatic cancer functional cell lines overexpressing CD19 (CD19 + The cells and supernatant were collected after 48 h.

[0096] (2) After washing the cells twice with PBS, the cell killing function was determined by flow cytometry. The test results are shown in Figure 7 .

[0097] from Figure 7 It can be seen that the modification of lipid nanoparticles does not affect the biological function of CART cells.

[0098] Test Example 5

[0099] This test example tests the effects of the nano-CART prepared in Example 3 on tumor inhibition and antigen-specific immunity in vivo.

[0100] (1) On day 0, 5×10 5 CD19 + B16F10-OVA cells.

[0101] (2) Lymphodepletion was performed using cyclophosphamide (2 mg / kg) and fludarabine (2 mg / kg).

[0102] (3) On day 7, PBS, lipid nanoparticles, CAR T cells, a direct mixture of lipid nanoparticles and CART cells (lipid nanoparticles + CART), or nano-CART cells were intravenously injected.

[0103] (4) Regularly record the size of mouse tumors. The test results are shown in Figure 8 .

[0104] (5) After 14 days, the mice were anesthetized and their peripheral blood was collected through orbital sampling.

[0105] (6) After adding red blood cell lysis buffer, it was used for in vivo flow cytometry analysis. The test results are shown in Fig. 9 .

[0106] from Figure 8 and Fig. 9 It can be seen that nano-CAR T has a significant anti-tumor effect, and the level of OVA antigen-specific T cells in mice was significantly increased after nano-CAR T treatment.

[0107] In summary, the nano-CART cells prepared by the present invention can quickly home to the tumor site through intravenous injection into the body, the CAR T cells recognize tumor antigens and are activated, secrete granzymes and perforins, and the lipid nanoparticles are released in response to the CAR signal, targeting the DC cells in the tumor, promoting the maturation of DC cells and the uptake and presentation of tumor-associated antigens released by CAR T lysing the tumor, generating a variety of antigen-specific T cells in the body, and synergistically killing antigen-negative tumor cells.

[0108] 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 nano CAR T cell, characterized in that: The nano CAR T cells include CAR T cells, and lipid nanoparticles that activate endogenous immunity are connected to the surface of the CAR T cells.

2. The nano CAR T cell according to claim 1, characterized in that The lipid nanoparticles include lipids; Preferably, the lipid comprises any one of a cationic lipid, cholesterol, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol-mannose, distearoylphosphatidylethanolamine-polyethylene glycol or distearoylphosphatidylethanolamine-polyethylene glycol-disulfide bond-N-hydroxysuccinimide or a combination of at least two thereof; Preferably, the cationic lipid comprises SM102.

3. The nano CAR T cell according to claim 2, characterized in that The molar ratio of the cationic lipid, cholesterol, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol-mannose and distearoylphosphatidylethanolamine-polyethylene glycol is (45-50):(35-38):(9-10):(1-9):1; Preferably, the molar ratio of the cationic lipid, cholesterol, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol-mannose and distearoylphosphatidylethanolamine-polyethylene glycol-disulfide bond-N-hydroxysuccinimide is (45-50):(35-38):(9-10):(1-9):1; Preferably, the mass percentage of polyethylene glycol in the lipid is 3% to 25%.

4. The nano CAR T cell according to any one of claims 1-3, characterized in that The lipid nanoparticles are loaded with mRNA with adjuvant effect, and the particle size of the lipid nanoparticles is 110-130 nm.

5. The nano CAR T cell according to any one of claims 1-4, characterized in that The lipid nanoparticles are loaded with mRNA encoding the activated STING protein.

6. The nano CAR T cell according to any one of claims 1 to 5, characterized in that: The surface of the lipid nanoparticles is modified with mannose.

7. The nano CAR T cell according to any one of claims 1-6, characterized in that The CAR T cells express the CD19 antigen.

8. A method for preparing the nano CAR T cell according to any one of claims 1 to 7, characterized in that: The method comprises: preparing lipid nanoparticles for activating endogenous immunity, and connecting the lipid nanoparticles to the surface of CAR T cells to form nano CAR T cells.

9. Use of the nano CAR T cells described in any one of claims 1 to 7 in the preparation of reagents for detecting tumor recurrence and / or products for treating tumor recurrence.

10. The use according to claim 9, characterized in that: The tumor recurrence includes antigen loss and / or down-regulation.

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