Rapid preparation method and application of chimeric antigen receptor T cell

Abstract

The invention discloses a rapid preparation method and application of chimeric antigen receptor T cells, and belongs to the technical field of biological medicine. According to the method, the traditional T cell activation stage is completely avoided, and the CAR-T cell with high expression of CAR similar to the conventional T cell activation is rapidly obtained in a manner of directly co-culturing the separated T cell and nicotinamide adenine dinucleotide in vitro for a short time and then transfecting mRNA (messenger Ribonucleic Acid). The traditional T cell activation stage is skipped, the preparation time of the CAR-T cells is greatly shortened, and the cost is reduced; in-vitro co-culture with NAD < + > is utilized, so that the T cell activity and the mRNA transfection efficiency are improved; by adopting lipid nanoparticles or electrotransfection of mRNA, the potential risk of a viral vector is avoided, and the safety of the preparation process is improved; the prepared CAR-T cell has the characteristic of high expression of CAR and relatively strong in-vitro tumor cell killing activity, and is expected to improve the clinical treatment effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a rapid preparation method and application of chimeric antigen receptor T cells. Background Art

[0002] CAR-T cell therapy, also known as chimeric antigen receptor T cell therapy, is a revolutionary cancer treatment that uses genetic engineering technology to modify a patient's T cells so that they can recognize and attack specific cancer cells. The development and application of this therapy provides new treatment options for patients with a variety of cancers. CAR-T cell therapy has achieved remarkable results in the treatment of certain types of blood tumors, such as leukemia and lymphoma. For example, CAR-T cell therapy targeting CD19 and BCMA was approved by the FDA in 2017 and brought lasting clinical responses to patients. Scientists are exploring the application of CAR-T therapy in the treatment of solid tumors, but there are still many challenges in this field, although early studies have shown its potential for application. The potential of CAR-T therapy is not limited to cancer treatment. It may also play a role in areas such as autoimmune diseases, chronic infections, heart disease and aging-related diseases. CAR-T cell therapy has certain safety risks. CAR-T cell therapy may cause serious clinical complications, such as cytokine release syndrome (CRS), a systemic inflammatory response caused by the release of a large amount of cytokines, which may be accompanied by high fever, hypotension and even multiple organ failure. CAR-T cells may target healthy tissues or cells other than tumor cells, leading to severe off-target toxicity or even death.

[0003] The traditional CAR-T cell preparation method includes the steps of T cell isolation, in vitro activation, gene transduction (usually using viral vectors), in vitro expansion and preparation. The whole process is time-consuming, usually taking 9-21 days, resulting in high production costs and treatment costs of hundreds of thousands of dollars, which makes it unaffordable for many patients, making CAR-T cells poorly accessible and unable to realize the value of product application. Conventionally using CD3 and CD28 antibodies to stimulate T cells and simulate the dual signal effect of T cell activation is the most widely used method for T cell activation and expansion.

[0004] In addition, traditional preparation methods involve long-term expansion of T cells to increase the number of cells to meet clinical requirements. This step often faces challenges in cell expansion potential and quality. In terms of quality, it is mainly because the in vitro full activation and expansion time is long, T cell exhaustion increases, and early memory cells decrease, resulting in insufficient persistence of infused T cells in the body, affecting clinical effects, especially the treatment of solid tumors.

[0005] Electroporation for in vitro mRNA transfection has the advantages of simple operation and short time, but this method will cause some primary T cells to die and there are problems such as low transfection efficiency. In actual application, T cells are generally activated first. After activation, the volume of T cells increases, which can reduce the voltage during electroporation, reduce cell damage and improve transfection efficiency. However, such operation increases the process complexity of primary T cells in vitro, which is not conducive to the rapid preparation and infusion of CAR-T cells.

[0006] As mentioned above, before primary T cells are transfected with mRNA, T cells still need to be activated and expanded to obtain more cells for infusion. This CAR-T preparation method still fails to effectively shorten the preparation time, affecting the industrialization and application promotion of CAR-T.

[0007] With the continuous advancement of technology, the simple and easy CAR-T cell preparation therapy is expected to provide more effective and safe treatment options for more cancer patients and patients with non-cancer diseases. Summary of the invention

[0008] The purpose of the present invention is to provide a method for rapid in vitro preparation of chimeric antigen receptor T cells, which completely avoids the traditional T cell activation stage. By directly co-culturing isolated T cells with nicotinamide adenine dinucleotide (NAD+) or its precursor in vitro for a short time and then transfecting mRNA, CAR-T cells with high expression of CAR similar to conventional T cell activation can be quickly obtained and applied to the preparation of specific CAR-T cells.

[0009] The present invention is achieved through the following technical solutions:

[0010] A method for rapidly preparing chimeric antigen receptor T cells comprises the following steps:

[0011] S1. Isolation of T cells:

[0012] An in vitro peripheral blood sample was selected, and peripheral blood mononuclear cells were separated using a leukocyte separation method, and then T cells were separated using a magnetic bead separation method;

[0013] S2. In vitro cell co-culture:

[0014] The T cells obtained in S1 are selected to be co-cultured with a metabolic regulator in vitro to obtain co-cultured T cells;

[0015] S3, transfection of mRNA:

[0016] The mRNA encoding the chimeric antigen receptor is introduced into the co-cultured T cells by lipid nanoparticle encapsulated mRNA transfection or electroporation, and after the treatment is completed, the transfected T cells are obtained;

[0017] S4, finished product:

[0018] The transfected T cells are cultured in vitro and collected to obtain chimeric antigen receptor T cells.

[0019] Preferably, the metabolic regulator is nicotinamide adenine dinucleotide or a precursor thereof.

[0020] Preferably, the nicotinamide adenine dinucleotide precursor is nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN).

[0021] Precursors of nicotinamide adenine dinucleotide (NAD+), such as nicotinamide riboside (NR) or β-nicotinamide mononucleotide (NMN), can also play a similar role. NR can be converted into NMN by nicotinamide riboside kinase (NRK) in cells, and then NAD+ is synthesized from NMN by intracellular nicotinamide mononucleotide adenosyltransferase (NMNAT).

[0022] Preferably, the mass concentration of the metabolic regulator is 0.1-1.5 mM.

[0023] Preferably, the mass concentration of the metabolic regulator is 0.5 mM.

[0024] Preferably, in S4, the in vitro culture time is 45 to 48 hours.

[0025] A chimeric antigen receptor T cell is prepared by the rapid preparation method.

[0026] A use of a chimeric antigen receptor T cell in the preparation of a drug for treating cancer.

[0027] A use of a chimeric antigen receptor T cell in the preparation of an in vitro killing preparation.

[0028] Compared with the prior art, the present invention has at least the following technical effects:

[0029] The present invention provides a method for rapidly preparing chimeric antigen receptor T cells. The method does not adopt a traditional T cell activation method, but directly co-cultures the separated T cells with nicotinamide adenine dinucleotide (NAD+) in vitro for a short time, and then introduces mRNA into the cultured T cells by lipid nanoparticle encapsulation mRNA transfection or electroporation technology, thereby rapidly obtaining CAR-T cells with high expression of CAR similar to conventional T cell activation and applying them to the preparation of specific CAR-T cells.

[0030] Experimental verification shows that the CAR-T cells obtained through this rapid preparation method have strong in vitro killing ability.

[0031] This preparation method has the following advantages:

[0032] (1) It provides a new method for preparing CAR-T cells, which does not go through the traditional T cell activation stage and does not cause T cell exhaustion. This method helps to shorten the preparation time of CAR-T cells and reduce costs, and has industrial significance;

[0033] Traditional T cell activation requires 24 hours, while this technology only requires 6 hours of co-culture time;

[0034] At the same time, this preparation technology can achieve significant tumor cell killing effects by omitting the amplification process, while the traditional preparation time takes 9-21 days;

[0035] (2) Achieve high mRNA transfection efficiency by using simple NAD+ in vitro co-culture;

[0036] (3) The introduction of mRNA through electroporation or lipid nanoparticles avoids the potential risks of viral vectors and improves the safety of the preparation process;

[0037] (4) The CAR-T cells prepared by this method have similar tumor cell killing activity as those prepared by traditional methods, and are expected to achieve better clinical treatment effects. DETAILED DESCRIPTION

[0038] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0039] Example 1: Primary human T cells transfected with mRNA

[0040] 1. Experimental Materials

[0041] Fresh anticoagulated peripheral blood from healthy subjects, human peripheral blood mononuclear cell (PBMC) isolation kit, human pan T cell isolation reagent (Pan T Cell Isolation Kit, human), 6-well culture plates, supplemented TexMACS medium (TexMACS Medium (GMP grade): containing IL-2, IL-7, IL-15); 50 mL centrifuge tubes, liquid nitrogen and other reagents and consumables. mRNA encoding enhanced green fluorescent protein (eGFP), catalog number SC2346, purchased from GenScript Biotech Co., Ltd. Coenzyme I (NAD+) for injection, purchased from Conno Biopharmaceutical Co., Ltd. Electroporator MaxCyte, ATx system, electroporation cup OC-25x3, electroporation solution EPB-1. Keyence fluorescence microscope system (Japan), flow cytometer (CANTO II, BD).

[0042] 2.NAD+ in vitro co-culture:

[0043] The isolated T cells were co-cultured with NAD+ in vitro for 3h, 6h, 12h, and 24h.

[0044] In this step, NAD+ acts as a key metabolic regulator, promoting the metabolic activity and stability of T cells and providing a better cell state for subsequent mRNA electroporation.

[0045] The mass concentration gradients of NAD+ were 0.1mM, 0.5mM, 1mM, and 1.5mM, which were used for electro-transfer mRNA group 1, electro-transfer mRNA group 2, electro-transfer mRNA group 3, and electro-transfer mRNA group 4, respectively.

[0046] 3. Electrotransfer of mRNA:

[0047] The mRNA encoding eGFP (5 μg) was electroporated into the co-cultured T cells, and the number of T cells was 1×10 6 The electric pulse condition was 500V / cm, duration 10ms, and electroporation was performed once. The electroporation efficiency reached 50%, and the target gene mRNA could be expressed in T cells.

[0048] 4. Collection and preparation of transfected T cells:

[0049] The electroporated T cells were cultured in vitro for 18 h, and the GFP expression level of the T cells was detected by fluorescence microscopy to ensure that the expression level reached the standard required for treatment. The excitation wavelength was 488 nm and the emission wavelength was 507 nm.

[0050] The results combined with Table 1 show that when the NAD+ mass concentration is at a medium concentration of 0.5mM and co-cultured for 6h, the expression of the tool protein eGFP can reach a high level. Compared with relatively high concentrations of NAD+ (1mM, 1.5mM) and co-cultured for 12h and 24h, there is no significant difference.

[0051] Table 1 Electroporation efficiency detection (eGFP expression positive rate) (average of 3 times)

[0052] Group 3h 6h 12h 24h Electroporation control 1.0±0.2 1.3±0.1 0.9±0.2 1.3±0.5 Electroporation of mRNA Group 1 32.0±5.1 41.1±6.3 42.9±6.3 48.0±2.9 Electroporation of mRNA Group 2 58.2±6.5 74.3±8.1 73.9±7.6 73.5±6.5 Electroporation of mRNA Group 3 59.3±7.2 72.1±7.2 78.0±5.7 74.1±6.1 Electroporation of mRNA Group 4 60.2±7.0 71.0±7.9 69.3±4.3 63.0±9.0

[0053] Example 2: Preparation and application of CAR-T cells targeting Claudin 18.2 (using the CAR-T cells prepared in Example 1 to verify the anti-tumor effect in vivo)

[0054] 1. Experimental Materials

[0055] Peripheral blood mononuclear cells (PBMC) were collected from the peripheral blood of healthy volunteers by leukocyte separation and T cells were sorted in the same manner as in Example 1.

[0056] 2.CAR design and coding mRNA synthesis

[0057] In the CAR structure, the antigen recognition domain is derived from the single-chain variable fragment (scFv) of the monoclonal antibody zolbetuximab (IMAB362) targeting Claudin 18.2. The hinge region and transmembrane region are derived from human CD8α, the co-stimulatory signaling domain is CD28, and the intracellular signaling domain is the intracellular part of the CD3ζ chain. It was commissioned to be synthesized by GenScript Biotech Co., Ltd.

[0058] 3. T cell culture and electroporation

[0059] NAD+ was co-cultured with T cells in vitro, and the method was the preferred method of Example 1, and the isolated T cells were co-cultured with NAD+ at a mass concentration of 0.5 mM in vitro for 6 hours.

[0060] Electroporation of mRNA: The mRNA encoding CAR targeting Claudin 18.2 was electroporated into the co-cultured T cells using electroporation technology. The electric pulse conditions were 1500V / cm, the duration was 10ms, and the electroporation was repeated once.

[0061] 4. Collection and preparation of CAR-T cells:

[0062] The electroporated T cells were cultured in vitro for 48 hours, and the expression level of CAR was detected by flow cytometry. The reagent for detecting CAR expression was fluorescein-labeled human Claudin-18.2 full-length protein, catalog number / price CL2-HF218-20μg. CAR-T cell preparations were collected and prepared for in vitro killing experiments.

[0063] 5.CAR-T cell killing experiment in vitro.

[0064] 1) Target cells were digested and counted, and the density was adjusted to 2×10 in culture medium (Myco5A or 1640 + 10% FBS). 5 cells / ml of target cell suspension. The target cells are NUGC4 expressing CLDN18.2.

[0065] 2) Add 50 μl of CAR-T cell culture medium (MEM) to the Eplate plate, place the Eplate in the DP monitoring tank, and prepare to measure the baseline.

[0066] 3) After the baseline measurement, remove the Eplate and add 1×10 4 (50 μl) of target cells and place at room temperature for 30 min.

[0067] 4) Place the E plate with the cell suspension back into the corresponding monitoring tank and start monitoring.

[0068] 5) After 18 hours, count the CAR-T and NTD cells, and adjust the effector cells to the corresponding density according to the effector-target ratio (E:T) in the table below.

[0069] Effect-target ratio Number of effector cells Cell density and volume per well 0:1 0 50μl 1:1 <![CDATA[1×10 4 ]]> <![CDATA[2×10 5 cells / ml, 50μl]]> 5:1 <![CDATA[5×10 4 ]]> <![CDATA[1×10 6 cells / ml, 50μl]]> 10:1 <![CDATA[1×10 6 ]]> <![CDATA[2×10 6 cells / ml, 50μl]]>

[0070] 6) Pause data collection of all detection slots;

[0071] 7) Take out the E-plate that has been placed overnight and add 50 μl of the corresponding effector cells to each well;

[0072] 8) Place the E-plate back into the corresponding monitoring slot and continue data acquisition using the real-time label-free dynamic cell analysis (RTCA) system;

[0073] 9) After the effector cells are added, the monitoring time should not exceed 48 hours.

[0074] 10) Detection of the killing ability of CAR-T cells

[0075] 11) Test results: NTD had no killing effect on target cells; when the effector-target ratio (E:T) was 0:1, 1:1, 5:1, and 10:1, CAR-T cells showed obvious killing effects at low efficiency-target ratios of 1:1 and 5:1.

[0076] Effect-target ratio Maximum lethality (%) 0:1 2.1±0.3 1:1 60.2±12.3 5:1 88.1±10.7 10:1 90.6±14.9

[0077] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for rapidly preparing chimeric antigen receptor T cells, characterized in that: The steps include: S1. Isolation of T cells: An in vitro peripheral blood sample was selected, and peripheral blood mononuclear cells were separated using a leukocyte separation method, and then T cells were separated using a magnetic bead separation method; S2. In vitro cell co-culture: The T cells obtained in S1 are selected to be co-cultured with a metabolic regulator in vitro to obtain co-cultured T cells; S3. Transfection of mRNA: The mRNA encoding the chimeric antigen receptor is introduced into the co-cultured T cells by lipid nanoparticle encapsulated mRNA transfection or electroporation, and after the treatment is completed, the transfected T cells are obtained; S4, finished product: The transfected T cells are cultured in vitro and collected to obtain chimeric antigen receptor T cells.

2. The method for rapidly preparing chimeric antigen receptor T cells according to claim 1, characterized in that: The metabolic regulator is nicotinamide adenine dinucleotide or a precursor thereof.

3. The method for rapidly preparing chimeric antigen receptor T cells according to claim 2, characterized in that: The nicotinamide adenine dinucleotide precursor is nicotinamide riboside or nicotinamide mononucleotide.

4. The method for rapidly preparing chimeric antigen receptor T cells according to claim 1, characterized in that: The mass concentration of the metabolic regulator is 0.1-1.5 mM.

5. The method for rapidly preparing chimeric antigen receptor T cells according to claim 4, characterized in that: The mass concentration of the metabolic regulator is 0.5 mM.

6. The method for rapidly preparing chimeric antigen receptor T cells according to claim 1, characterized in that: In S2, the in vitro cell co-culture time is 5 to 6 hours.

7. The method for rapidly preparing chimeric antigen receptor T cells according to claim 1, characterized in that: In the S4, the in vitro culture time is 45 to 48 hours.

8. A chimeric antigen receptor T cell, characterized in that The product is prepared by the rapid preparation method according to any one of claims 1 to 7.

9. Use of the chimeric antigen receptor T cell according to claim 8 in the preparation of a drug for treating cancer.

10. Use of the chimeric antigen receptor T cell according to claim 8 in preparing an in vitro killing preparation.