A method for efficiently transfecting exogenous substances into immune cells

Abstract

The application provides an electrotransfection device which adopts titanium plating platinum as an electrotransfection electrode material, and can effectively overcome the technical problem of aluminum ion precipitation during electrotransfection of a conventional aluminum electrode; meanwhile, the application provides an electrotransfection system which can efficiently electrotransfect a to-be-electrotransfected substance into immune cells; meanwhile, the electrotransfection method of the application can be used for preparation of CAR-T cells; meanwhile, the electrotransfection method of the application can also be combined with a CRISPR / Cas9 editing system to prepare universal CAR-T cells, and provides a solution for immunotherapy.

Description

Technical Field

[0002] This invention relates to the field of electroporation, to an electroporation apparatus, and more specifically, to a method for efficiently electroporating exogenous substances into immune cells using an electroporation apparatus. Background Technology

[0004] A fast-paced lifestyle, immense work pressure, unhealthy eating habits, and poor environmental conditions all contribute to the development of cancer (malignant tumors), leading to a significant increase in cancer incidence and a trend towards affecting younger people. Cancer has now become the leading cause of death worldwide. Current traditional treatments have reached their limits, necessitating the exploration of more effective treatment methods to improve the survival rate and quality of life for cancer patients. Leukemia, commonly known as "blood cancer," is a type of malignant clonal disease originating from hematopoietic stem cells. Clonal leukemia cells proliferate continuously, infiltrating other non-hematopoietic tissues and organs, and suppressing normal hematopoietic function. In China, approximately tens of thousands of patients are diagnosed with B-cell acute lymphoblastic leukemia (B-ALL) each year. In the United States, about 80% of acute leukemia cases are B-ALL, with 80% of B-ALL patients achieving remission with first-line chemotherapy, but 60% of B-ALL patients relapse. Because leukemia is classified and its prognosis is complex—for example, it can be divided into acute and chronic leukemia based on the speed of onset, and into myeloid and lymphoid leukemia based on the type of cells involved—there is no one-size-fits-all treatment method.

[0005] Immunotherapy for malignant tumors has developed rapidly in recent years, achieving remarkable clinical efficacy. In 2011, Nature and JCO, the top clinical oncology journal, published commentaries with the same title, "The Era of Tumor Immunotherapy Has Arrived" (Nature. 2011; 480(7378):480; J Clin Oncol. 2011; 29(36):4828), respectively. Tumor immunotherapy has ushered in a new wave of research and may occupy a very important position in tumor treatment in the future. One method for treating leukemia patients is to genetically modify T cells to target and bind to antigens expressed on tumor cells through the expression of chimeric antigen receptors (CARs), transmitting signals into the cells, causing T cell proliferation and activation, thereby targeting and killing tumor cells.

[0006] "Immune function" can be divided into cellular immunity and humoral immunity, which have distinctly different ways of killing tumors or viruses. Cellular immunity involves cells directly exerting their effects to eliminate foreign substances; it is a participant in the body's immune response and an executor of immune function. Currently, effector immune cells used in clinical treatment, namely cytotoxic immune cells, mainly include natural killer (NK) cells, lymphokine-activated killer cells (LAK), cytokine-induced killer cells (CIK), dendritic cell-stimulated cytokine-induced killer cells (DC-CIK), cytotoxic T lymphocytes (CTL), γδT cells, tumor-infiltrating lymphocytes (TIL), and CD3AK cells (cytotoxic cells with anti-CD3 monoclonal antibodies). These cells do not express antibodies; they directly kill or act through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0007] Humoral immunity is mediated by antibodies, which are produced by B lymphocytes in the human body. In 1997, the first monoclonal antibody used in clinical cancer treatment—anti-human CD20 (rituximab)—was approved by the FDA. With the development of antibody technology, more than 100,000 antibodies have been reported globally to date, including more than 1,000 genetically engineered antibodies and more than 200 humanized antibodies. As of March 2016, the FDA had approved 50 antibodies for marketing, 26 of which are used for cancer treatment. These antibodies exert their effects either directly on tumor cells or indirectly on immune cells.

[0008] However, while effector immune cells possess certain anti-tumor effects, their clinical therapeutic efficacy is limited. Furthermore, large-molecule antibodies have insufficient penetration into solid tumors, and systemic administration may cause systemic adverse reactions. For example, HER2 monoclonal antibodies have cardiotoxicity, while PD1 antibodies disrupt the body's immune balance, potentially causing autoimmune diseases. Moreover, antibody drugs involve complex in vitro production and preparation processes, requiring high purity and large dosages, leading to high treatment costs. Therefore, if effector cells can efficiently express antibodies with anti-tumor activity while maintaining cytotoxicity, it would simultaneously overcome the limitations of insufficient cellular immunotherapy and the difficulty of large-molecule antibodies penetrating solid tumors, while also reducing treatment costs. These cells, possessing both cytotoxicity and high antibody expression levels, can actively enter tumor tissue through cell deformation under the influence of chemokines, thereby expressing antibodies at high levels locally within the tumor tissue, avoiding the side effects of systemic administration. Meanwhile, antibodies that act on immune cells themselves (such as the HER2 antibody Herceptin) can produce strong ADCC and CDC effects due to the dual presence of antibodies and cytotoxic immune cells, effectively killing tumor cells; while antibodies that act on T cells themselves (such as the PD1 antibody Keytruda) can avoid the inhibitory effect of the tumor microenvironment on the reinfused effector T cells, allowing them to continue to exert their therapeutic effects.

[0009] Although there are reports of introducing exogenous genes into NK cells or T cells, commonly used gene transfection vector systems have low transfection rates on effector immune cells with cytotoxic effects, or fail to achieve high-level expression of exogenous genes within these cells. Adenoviral vectors (non-integrating) can mediate relatively efficient short-term expression of exogenous genes in NK cells or T cells, but activated NK cells or T cells proliferate very rapidly, and the exogenous gene expression cassettes they carry are quickly lost during cell passage, making sustained expression difficult. Retroviruses or lentiviruses can mediate the integration of exogenous genes into the genome of NK cells or T cells, theoretically achieving stable expression. However, antibodies contain light and heavy chains, have long coding sequences, and large molecular weights, making the packaging and preparation of retroviruses or lentiviruses carrying full-length antibody expression cassettes difficult, hindering efficient antibody expression. They can only be used to express structurally simple single-chain antibodies (lacking Fc fragments, functionally incomplete, and with short half-lives). Chemical transfection methods are generally ineffective at transfecting exogenous substances into immune cells.

[0010] CAR-T cells targeting CD19 molecules have achieved clinical efficacy in the treatment of refractory and relapsed B-cell-derived hematologic malignancies that traditional therapies cannot reach, with complete remission (CR) rates generally reaching 80%, and some clinical trials even reaching 100%, making it the most attention-grabbing area in current tumor immunotherapy. However, it has also exposed problems such as poor CAR-T cell persistence, antigen escape leading to tumor recurrence, and safety risks: (1) Short survival time. The length of survival time has been proven to be closely related to efficacy, and ensuring the survival time of CAR-T cells after reinfusion in the patient is crucial; (2) Prone to relapse. Insufficient survival time of CAR-T cells in vivo leads to antigen loss and immune escape mechanisms, ultimately resulting in relapse; (3) Low preparation success rate. Retroviral systems are commonly used to prepare CAR-T cells, which can effectively infect host cells, but their loading capacity is limited, and the preparation process of recombinant viral particles is relatively complex. In addition, viral systems may pose safety risks. Furthermore, patients who have undergone multiple treatments may have low T-cell activity, and in vitro culture cannot guarantee the amount of CAR-T cells to be reinfused.

[0011] CN201710539385 discloses the use of an electrotransfection reagent from a commercially available Amaxa electrotransfection kit, which is mixed with a CD19CAR plasmid and its transposase plasmid, an sgRNA plasmid for knocking out the PD-1 gene, and a CAS9 plasmid to form a mixture; and the electrotransfection is performed by receiving PBMC cells from a PBMC cell preparation system and adding the mixture thereto.

[0012] However, due to the extensive radiotherapy and chemotherapy treatments received by hematological malignancy patients, there is significant variability among patient samples. Using Amaxa electroporation, the transfection efficiency and survival rate vary considerably between different samples, with the lower limit of transfection efficiency potentially falling below 20%. Secondly, commercially available electroporation cups are all made of aluminum alloy, and during the electroporation process, some Al reacts with the ions, releasing... 3+ Precipitation can damage cells. In immunotherapy, cell samples after electroporation need to be reinfused into the body. How can residual Al be removed in this process? 3+ It is a very complicated process. Summary of the Invention

[0014] The present invention addresses the technical problems existing in the prior art by providing an electro-dyeing apparatus to overcome the problem of aluminum ion precipitation during the electro-dyeing process.

[0015] The present invention also provides an electrotransfection system, providing a solution for efficiently transfecting exogenous substances into immune cells.

[0016] This invention also provides an electroporation method that can efficiently electroporate exogenous substances into immune cells.

[0017] To achieve the above objectives, the technical solution of the electroporation apparatus provided by this invention is summarized as follows:

[0018] The electrotransfer dyeing apparatus of the present invention uses titanium-plated platinum as the electrode material, and the electrode is a planar electrode or a needle electrode, preferably a planar electrode; the planar spacing of the planar electrodes is 1-5 mm; preferably, the planar spacing of the planar electrodes is 2 mm.

[0019] The preferred electroporation apparatus is an electroporation cup, comprising a planar electrode composed of a pair of titanium-plated platinum electrode sheets and a tube with an opening at only one end. The planar electrode is embedded parallel to the non-conductive sidewall of the tube, forming a cavity with a volume of (5-15 mm) × (10-30 mm) × (1-5 mm); preferably 10.3 mm × 20 mm × 2 mm.

[0020] The electroporation device of the present invention is used in combination with the Yida EBXP-H1 electroporation host, the wireless PAD control system and the dedicated base for electroporation cups to form an electroporation system.

[0021] The electrotransfer parameters of this invention are as follows: the pulse voltage is 400-500V, preferably 420-450V; the pulse width is 300-1000μs, preferably 500-800μs; the number of pulses is 1-8 times, preferably 2-3 times; the pulse interval is 10-2000ms, preferably 50-2000ms; and the pulse waveform is preferably a square wave.

[0022] This invention provides a method for electroporating exogenous substances into immune cells, wherein the exogenous substance to be transfected is used in combination with an electroporation buffer, preferably the electroporation buffer is EBEL electroporation buffer (product number H10305) independently developed by Yida.

[0023] The immune cells described in this invention are subjected to adhesion treatment for 8-20 hours before electroporation.

[0024] After electroporation of the immune cells described in this invention, they are transferred to the culture medium at a volume ratio of 1:8.

[0025] The exogenous substances described in this invention include DNA, RNA, proteins, therapeutic agents that can express nucleic acid sequences encoding therapeutic peptides or nucleic acids, or derivatives thereof.

[0026] The immune cells described in this invention include natural killer cells (NK cells), dendritic cells (DC cells), macrophages, neutrophils, eosinophils, T lymphocytes, or combinations thereof; preferably, the immune cells include NK cells, DC cells, and T lymphocytes. The immune cells described in this invention also include mammalian cell CD8+ cytotoxic T cells, CD4+ helper T cells, naive T cells, memory T cells, central memory T cells, effector memory T cells, stem cell-like memory T cells, homeostatic T cells, NKT cells, cytokine-induced cytotoxic T cells, g / d T cells, B lymphocytes, natural killer cells, monocytes, macrophages, dendritic cells, or granulocytes.

[0027] The present invention provides a method for electrotransfection of immune cells, comprising the step of electrotransfection using the electrotransfection system of the present invention, wherein the electrotransfection is performed on the immune cells and the plasmid to be transfected at a voltage of 400-500V, and the pulse width is 50-2000μs.

[0028] This invention provides a method for preparing T cells for immunotherapy, characterized by using the electrotransfection system of this invention.

[0029] This invention provides a method for preparing chimeric antigen receptor T cell (CAR-T) drugs for treating diseases using the electrotransfection system of this invention and unactivated or activated T cells.

[0030] This invention provides the use of isolated T cells or cell lines or their secondary cultures, which can be obtained by a method for preparing T cells for immunotherapy, in the preparation of drugs for treating diseases.

[0031] Preferably, the T cells or cell lines or their secondary cultures include exogenous polynucleotide sequences encoding chimeric antigen receptors.

[0032] This invention provides a method for preparing chimeric antigen receptor cell drugs for treating diseases using the electrotransfection system of this invention and human NK cells, DC cells, and macrophages.

[0033] Preferably, the chimeric antigen receptor is a multi-chain chimeric antigen receptor, and preferably the multi-chain chimeric antigen receptor is specific for CD19, CD20, CD22, CD33, CD30, CDS, CD38, CD37, CD52, CD70, CD44v6, CD123, CD135, EpCAM, EGFR, EGFR variants, GD2, ROR1, ROR2, CD269, CD319, CD38, BCMA or CD138, CD139, CLDN6, CEA, CSPG4, C-MET, FR, TRBC1, Het2, WT1, CLL-1, GFR alpha4, IL13Rα2, meso, TEM8, B7-H4, SSEA4, MUC1, PSMA, and PSA.

[0034] The disease is preferably cancer (malignant tumor), viral infection or autoimmune disorder.

[0035] The preferred diseases are lymphoma, leukemia, myeloma, glioma, ovarian tumor, colon cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, melanoma, or liver cancer.

[0036] The beneficial effects of the technical solution of this invention are as follows:

[0037] The electroporation scheme of this invention adopts a relatively low-pressure, short-pulse-width opening method, an independently developed electroporation buffer, and optimized pre- and post-electroporation processing of PBMC, as well as optimized electroporation parameters and electroporation cup materials, which significantly improves the transfection efficiency to 70-75%.

[0038] The electrotransfection method of the present invention can be used for the preparation of CAR-T cells, and can transfect the cutting enzyme protein and guide RNA, which are CRISPER gene editing tools, into the cells to perform gene editing.

[0039] The electrotransfection material of this invention is novel and groundbreaking, and can ensure high transfection efficiency while improving cell survival rate. Attached Figure Description

[0041] Figure 1 PBMC transfection efficiency at 250-600V voltage;

[0042] Figure 2 Transfection efficiency of PBMCs at pulse widths of 100-2000 μs;

[0043] Figure 3 The difference in transfection efficiency between direct centrifugation after electroporation and centrifugation after incubation at 37°C for 20 min.

[0044] Figure 4The difference in transfection efficiency between direct centrifugation after electroporation and dilution at a volume ratio of 1:8.

[0045] Figure 5 Flow cytometry analysis of GFP transfection 24 h later; Implementation

[0046] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0047] Example 1

[0048] Electroporation parameter optimization

[0049] PBMC cell preparation

[0050] Collect peripheral blood from healthy individuals using an anticoagulant tube (10ml BD K2D (EDTA) anticoagulant tube, 367525). Collect the appropriate volume of whole blood from a healthy individual at the designated blood collection point. Invert the tube approximately 5 times to prevent clotting. Label the tube, wrap it with 2-3 layers of gauze or paper towels, and place it in an ice pack for transport. Slowly add peripheral blood diluent to a 50ml centrifuge tube containing an equal volume of Ficoll lymphocyte separation medium. Centrifuge at 400g for 30 minutes using a slow ascending and descending motion without stopping. After centrifugation, carefully aspirate the white membrane cells from the top of the lymphocyte separation medium and transfer them to a new 50ml centrifuge tube. Add 1×PBS, centrifuge at 300g for 10 minutes using a slow ascending and descending motion, discard the supernatant, and retain the cell pellet at the bottom of the centrifuge tube. Add 1×PBS again, centrifuge at 1100rpm for 10 minutes using a slow ascending and descending motion, and discard the supernatant. Finally, add 1×PBS, centrifuge at 800rpm for 10 minutes using a slow ascending and descending motion, and discard the supernatant to obtain PBMC cells.

[0051] Electroporation

[0052] The specific steps are as follows: Centrifuge the overnight adherent PBMC cells to discard the culture medium, wash with 1×PBS, add EBEL electroporation buffer, then add 5μg of pMax-GFP plasmid DNA, 5×10⁻⁶ 6Cells were used to obtain 100 μl of electroporation mixture containing plasmids. Electroporation parameters were then determined using three frozen samples and two fresh samples (both from healthy individuals). Voltages ranged from 250-600 V, and pulse widths from 100 μs-2000 μs. Five rounds of experiments were conducted with 1-3 different electroporation cycles, yielding 118 data points. A 100 μl electroporation mixture containing plasmids was used as a control for electroporation. After electroporation, cells were transferred to 6-well plates containing 2 ml of culture medium. The GFP positivity rate was detected by flow cytometry 24 h post-transfection. Figure 1 , 2 As shown: For transfection efficiencies above 10% and relatively stable data distribution, the voltage range is 350-450V. Under high voltage, the data is more significantly affected by cell condition, resulting in relatively large fluctuations. For transfection efficiencies above 10% and relatively stable data distribution, the pulse width is 500-1000μs. For good repeatability and transfection efficiencies above 15%, the parameters are: voltage 400-500V, pulse width 500-1000μs, and 2-3 transfections.

[0053] Example 2

[0054] Electro-rotor material optimization

[0055] PBMC cell preparation was performed as described in Example 1.

[0056] Electroporation

[0057] The specific steps are as follows: Centrifuge the overnight adherent PBMC cells to discard the culture medium, wash with 1×PBS, add EBEL electroporation buffer, and then perform 5×10⁻⁶ ppm electroporation. 6 5 μg of pmax-GFP plasmid DNA was added to cells to obtain 100 μl of electroporation mixture containing the plasmid. This mixture was then transferred to three electroporation cuvettes: a Bio-Rad aluminum alloy cuvette, a Lonza aluminum alloy cuvette, and a Yida titanium-plated platinum cuvette. The parameters were set as follows: voltage 400-500 V, pulse width 500-1000 μs, and 2-3 cycles. After electroporation, the cells were transferred to 6-well plates containing 2 ml of culture medium. The GFP positivity rate was detected by flow cytometry 24 h post-transfection. The results are shown in Table 1.

[0058] Table 1: Differences in electroporation efficiency among different electroporation cups NC 0.06% No flocculent matter Aluminum alloy electro-rotator (Bio-Rad) 17.7% Large amounts of cellular flocculent material Lonza aluminum alloy electric rotary cup 25.5% Large amounts of cellular flocculent material Titanium-plated platinum electrorotating cup 30.1% No flocculent matter

[0059] Example 3

[0060] Optimization of pre- and post-processing of PBMC electro-polarization

[0061] PBMC cell preparation was performed as described in Example 1.

[0062] Pre- and post-electroplasty treatments:

[0063] The collected PBMC cells were processed at a ratio of 5 × 10⁻⁶. 6 Cells were cultured at a density of 1 / ml for adherence at 3, 8, 12, 20, and 26 h. Cells were then harvested, centrifuged, and the culture medium was discarded. After washing with 1×PBS, EBEL electroporation buffer was added, followed by 5 μg of pmax-GFP plasmid to obtain 100 μl of electroporation mixture containing the plasmid. The electroporation parameters were set as follows: voltage 400V, pulse width 800μs, 2 electroporations, and 784ms interval. Electroporation was performed using EBXP-H1, with the 100 μl electroporation mixture containing the plasmid serving as a control. After electroporation, cells were transferred to 6-well plates containing 2 ml of culture medium and cultured. The GFP positivity rate was detected by flow cytometry after 24 h. Post-electroporation treatment methods: (1) After electroporation, cells were collected, centrifuged, and incubated with culture medium for 20 min before centrifugation; (2) Centrifugation; (3) 1:8 direct dilution. The results are shown in Table 2. The optimal electroporation efficiency was achieved with a wall adhesion time of 8-20 hours. Both excessively short and long wall adhesion times decreased the transfection efficiency. Results of different post-electroporation treatments are shown in (see...). Figure 3 and Figure 4 Centrifugation after electroporation is significantly more efficient than incubation in culture medium for 20 minutes after electroporation, thus shortening the overall electroporation time. Furthermore, direct dilution at a 1:8 ratio after electroporation results in a 5-10% higher transfection efficiency than direct centrifugation, further simplifying the procedure and reducing the risk of contamination. Therefore, PBMCs can be allowed to adhere to culture media for 8-20 hours, followed by electroporation, and then directly transferred to culture medium at a 1:8 ratio, which will significantly improve transfection efficiency.

[0064] Table 2: Differences in PBMC staining efficiency at different times before electroporation NC 0.5% 3 hours before electroplating 16.6% 8 hours before electroplating 26.5% 12 hours before electroplating 26.8% 20 hours before electroplating 25.3% 26 hours before electroplating 19.9%

[0065] Example 4

[0066] Compared with existing products on the market

[0067] PBMC cell preparation was performed as described in Example 1.

[0068] Preparation of activated T cells

[0069] PBMCs were cultured in X-VIVO medium (without IL-2 cytokine) for 2-20 hours to adhere to the culture medium. Unadhered cells were then harvested and cultured at 2×10⁻⁶ cells per culture medium. 6 Transfer / ml to wells coated with OKT3 and CD28 antibodies for incubation. Perform electroporation 72-120 hours after cell activation.

[0070] Electroporation

[0071] The specific steps are as follows: activate 5×10 6 Activated T cells were centrifuged and the culture medium was discarded. After washing with 1×PBS, electroporation buffer developed by Yida was added, followed by 2 μg of pmax-GFP plasmid, resulting in 100 μl of electroporation mixture containing the plasmid. The electroporation parameters were set to 450V, 800μs, 2 times with a 784ms interval. Electroporation was performed using EBXP-H1, and simultaneously, electroporation was performed according to Lonza's standard operating procedure (SOP). Activated T cells (5×10⁶ cells) were centrifuged and the culture medium was discarded. After washing with 1×PBS, electroporation buffer developed by Yida was added, followed by 2 μg of pmax-GFP plasmid, resulting in 100 μl of electroporation mixture containing the plasmid, which served as a control. After electroporation, the cells were transferred to 12-well plates containing 1.5 ml of culture medium. The GFP positivity rate was detected by flow cytometry 24 h after transfection.

[0072] like Figure 5 As shown in Table 3: with the same plasmid and cell quantity, the optimal electroporation parameters and electroporation system for each cell type, the EBXP-H1 electroporation instrument achieved an efficiency of 74.14%, which is much higher than the Lonza's transfection efficiency of 42.12%.

[0073] Table 3: Flow cytometry results 24 h after GFP transfection NC 3.03% EBXP-H1 74.14% Lonza 42.12%

[0074] While the invention has been described in detail, modifications within the spirit and scope of the invention will be readily apparent to those skilled in the art. It should be understood that aspects and various embodiments of the invention described above and / or in the appended claims, as well as various features, can be combined or interchanged in whole or in part. As those skilled in the art will appreciate, those embodiments referred to as one in the foregoing description of various embodiments may be suitably combined with other embodiments. Furthermore, those skilled in the art will recognize that the foregoing description is merely illustrative and is not intended to limit the invention.

Claims

1. A method for electroporating exogenous substances into immune cells, characterized in that, The invention includes an electroporation system for immune cells, comprising an electroporation device and a power source for providing electrical pulses. The electrodes of the electroporation device are made of titanium plated with platinum, and are planar electrodes with a planar spacing of 1-5 mm. The pulse voltage is 400-500 V, the pulse width is 300-1000 μs, the number of pulses is 1-8, and the pulse interval is 10-2000 ms. The immune cells include one or more combinations of NK cells, macrophages, dendritic cells (DCs), neutrophils, eosinophils, and T lymphocytes.

2. The method according to claim 1, characterized in that, The immune cells were treated for adhesion for 8-20 hours before electroporation.

3. The method according to claim 1, characterized in that, After electroporation, the immune cells were transferred to the culture medium at a volume ratio of 1:

8.

4. The method according to claim 1, characterized in that, The exogenous substance includes one or more complexes of DNA, RNA, protein, therapeutic forms of expressing nucleic acid sequences encoding therapeutic peptides or nucleic acids, or derivatives thereof.

5. The method according to claim 1, characterized in that, The planar spacing between the planar electrodes is 2 mm.

6. The method according to claim 1, characterized in that, The pulse voltage is 420-450V.

7. The method according to claim 1, characterized in that, The pulse width is 500-800µs.

8. The method according to claim 1, characterized in that, The number of pulses is 2-3.

9. The method according to claim 1, characterized in that, The pulse waveform is a square wave.

10. The method according to claim 1, characterized in that, The pulse interval is 50-2000ms.

11. A method for electroporation of immune cells, characterized in that, The procedure includes the step of electrotransfection using an electrotransfection system for immune cells, wherein a liquid containing immune cells and plasmids to be transfected is electrotransfected with a pulse voltage of 400-500V, and the pulse width is 300-1000µs; the electrotransfection system includes an electrotransfection device and a power supply for providing the electrical pulses, wherein the electrodes of the electrotransfection device are made of titanium plated with platinum, the electrodes are planar electrodes, the planar electrode spacing is 1-5mm, the number of pulses is 1-8, the pulse interval is 10-2000ms, and the immune cells include one or more combinations of NK cells, macrophages, DC cells, neutrophils, eosinophils, and T lymphocytes.

12. The method according to claim 11, characterized in that, The immune cells were treated for adhesion for 8-20 hours before electroporation.

13. The method according to claim 11, characterized in that, After electroporation, the immune cells were transferred to the culture medium at a volume ratio of 1:

8.

14. The method according to claim 11, characterized in that, The plasmid to be transfected includes one or more complexes of DNA, RNA, protein, a therapeutic form of a nucleic acid sequence encoding a therapeutic polypeptide or nucleic acid, or a derivative thereof.

15. The method according to claim 11, characterized in that, The planar spacing between the planar electrodes is 2 mm.

16. The method according to claim 11, characterized in that, The pulse voltage is 420-450V.

17. The method according to claim 11, characterized in that, The pulse width is 500-800µs.

18. The method according to claim 11, characterized in that, The number of pulses is 2-3.

19. The method according to claim 11, characterized in that, The pulse waveform is a square wave.

20. The method according to claim 11, characterized in that, The pulse interval is 50-2000ms.

21. A method for preparing a cell-based drug for immunotherapy, characterized in that, The method includes using an electroporation system for immune cells; the electroporation system includes an electroporation device and a power supply for providing electrical pulses, wherein the electrodes of the electroporation device are made of titanium plated with platinum, the electrodes are planar electrodes, the planar electrode spacing is 1-5 mm, the pulse voltage is 400-500 V, the pulse width is 300-1000 μs, the number of pulses is 1-8, and the pulse interval is 10-2000 ms; the immune cells include one or more combinations of NK cells, macrophages, DC cells, neutrophils, eosinophils, and T lymphocytes; the immune cells are pretreated for adhesion for 8-20 h before electroporation.

22. A method for preparing a chimeric antigen receptor T-cell drug for treating diseases according to claim 21, characterized in that, Use either inactive or activated T cells.

23. A method for preparing a cell drug for immunotherapy according to claim 21, characterized in that, Human NK cells, DC cells, and macrophages are used.

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