A method for preparing genetically modified cells

Abstract

This invention relates to a method for preparing genetically modified cells. Specifically, this invention provides a method for preparing genetically modified immune cells. This cell preparation method can rapidly prepare immune cells of high quality, thereby ensuring clinical efficacy.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for preparing genetically modified cells. Background Technology

[0002] Cellular immunotherapy is a novel medical technology that collects a patient's immune cells, performs gene modification or selective amplification and culture to enhance the targeting, killing, and persistence of these immune cells. In recent years, it has shown promising results in clinical tumor immunotherapy, bringing hope for a clinical cure of cancer. However, the preparation process of immune cells is relatively complex. Deficiencies in intermediate steps such as process flow, equipment, and reagent selection can adversely affect the quality of the prepared cells, thereby impacting clinical outcomes.

[0003] Traditional immune cell preparation has disadvantages such as long cycle, complex operation and easy to cause excessive differentiation and senescence of cells during culture, which increases the preparation cost, reduces the efficacy of cell immunization and makes it difficult to meet the clinical needs of immune cell therapy.

[0004] Therefore, there is a need in this field to develop a method for preparing immune cells that improves both the efficiency and quality of immune cell preparation. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing immune cells that improves the efficiency and quality of immune cell preparation.

[0006] In a first aspect, the present invention provides a method for preparing genetically modified immune cells, the method comprising the steps of:

[0007] (a) Provide a sample containing immune cells to be genetically modified;

[0008] (b) The sample is sorted to obtain a first immune cell population rich in immune cells;

[0009] (c) The first immune cell population is activated to obtain an activated second immune cell population;

[0010] (d) The activated second immune cell population is subjected to pre-transfection culturing (also known as pre-cultivation) to obtain a pre-cultured third immune cell population;

[0011] (e) Using a viral vector, the activated third immune cell population was genetically modified by transfection to obtain a genetically modified fourth immune cell population.

[0012] (f) The genetically modified fourth immune cell population is transfected and cultured to obtain the cultured fourth immune cell population, namely the genetically modified immune cells.

[0013] The method has one or more features selected from the group consisting of:

[0014] (i) In step (c), activation is performed using magnetic beads, and the ratio of the number of magnetic beads to the number of cells is 0.5-5:1;

[0015] (ii) In step (e), the virus and the activated fourth immune cell population are mixed and incubated for a period of time. e And no centrifugation is performed after incubation;

[0016] (iii) In step (f), during culture, different perfusion methods are used based on the density of immune cells in the culture system: when the density of immune cells < 2 × 10⁻⁶ 6 Perfusion is not performed when the cell density is ≥2×10⁻⁶ / ml; perfusion is performed when the immune cell density is ≥2×10⁻⁶ / ml. 6 cells / ml and <4×10 6 When the cell density is ≥4×10⁻⁶ / ml, perfusion is performed at a rate of 0.5V / day to 1V / day, where V is the volume of the culture system; when the immune cell density is ≥4×10⁻⁶... 6 When the cell / ml ratio is 1V / day to 2V / day, perfusion is performed at a rate of 1V / day to 2V / day, where V is the volume of the culture system.

[0017] In another preferred embodiment, in step (c), the density of the first immune cell population is 0.5-10 × 10⁻⁶. 6 cells / ml.

[0018] In another preferred embodiment, the method further has one or more features selected from the group consisting of:

[0019] (t1) The total time t from step (b) to (f) (b-f) It takes 4-5 days;

[0020] (t2) The incubation time t in step (f) f It takes 1.0-3.5 days.

[0021] In another preferred embodiment, the ratio of the number of activating magnetic beads to the number of cells is 1-5:1.

[0022] In another preferred embodiment, step (b) includes: mixing the sample with sorting magnetic beads and incubating the mixture for a period of time t. b Then, the primary immune cell population rich in immune cells is selected;

[0023] Preferably, the t b The time should be 10-30 minutes, preferably 10-25 minutes.

[0024] In another preferred embodiment, step (c) includes: mixing the first immune cell population and the activating magnetic beads, and incubating the mixture for a period of time. c This allows for the acquisition of an activated second immune cell population.

[0025] In another preferred embodiment, the t in step (c) c It takes 12-24 hours.

[0026] In another preferred embodiment, the method has one or more features selected from the group consisting of:

[0027] In step (d), the pre-incubation time t d The optimal time is 1.5-3 days, with 1.5-2.5 days being the best.

[0028] In step (e), the transfection culture time t e The time is 0.5-2.5 days, preferably 1-2 days; and / or

[0029] In step (f), the incubation time t after transfection f The time is 1-3.5 days, with 1.5-3 days being ideal.

[0030] In another preferred embodiment, the method further includes:

[0031] (f) When the number or density of the cultured fourth immune cell population reaches a predetermined value, the cultured fourth immune cell population is harvested.

[0032] In another preferred embodiment, the predetermined value is 2 × 10⁻⁶. 6 cells / ml ~ 20 × 10 6 Cells / ml.

[0033] In another preferred embodiment, in step (e), the virus and the activated fourth immune cell population are mixed and incubated for a period of time. e The incubation solution is obtained by diluting the incubation solution with a culture medium by 0.5-2 times (preferably 0.75-1.5 times) by volume to obtain the diluted incubation solution.

[0034] In another preferred embodiment, in step (f), the diluted incubation solution is incubated for 0.5-1.5 days and then inoculated into a bioreactor such as Xuri Wave for further culture for 1-7 days.

[0035] In another preferred embodiment, the sample is selected from the group consisting of blood, cells, fresh apheresis, frozen apheresis, PBMC collections, or combinations thereof.

[0036] In another preferred embodiment, the immune cells are selected from the group consisting of T cells, NK cells, or combinations thereof.

[0037] In another preferred embodiment, the sample is washed before being sorted.

[0038] In another preferred embodiment, the washing process includes the steps of: adding washing solution to the cell sample and mixing, centrifuging, removing the supernatant, and obtaining a precipitate.

[0039] In another preferred embodiment, the washing is performed on a Sepax 2, Sepax C-pro, Sefia, Lovo, CS 5+, CSElite, or Prodigy device.

[0040] In another preferred embodiment, the washing is performed on a Sepax C-pro device.

[0041] In another preferred embodiment, sample washing and magnetic bead incubation are performed using the Sepax C-pro "Beadwash" program, with Beadwash parameters including one or more parameters selected from Table A below:

[0042] Table A

[0043]

[0044]

[0045] In another preferred embodiment, in step (b), the sorting includes positive sorting or negative sorting.

[0046] In another preferred embodiment, in step (b), the sorting includes sorting by adding sorting magnetic beads containing traps that bind to immune cell surface markers (such as CD4 and / or CD8). The sorting magnetic beads bind to the immune cell surface markers through the traps to form a sorting magnetic bead-cell complex, thereby obtaining a first immune cell population rich in immune cells.

[0047] In another preferred embodiment, the trapping body is an antibody.

[0048] In another preferred embodiment, the antibody is a specific antibody.

[0049] In another preferred embodiment, the antibody is selected from the group consisting of: CD4 + Antibodies, CD8 + Antibodies, or combinations thereof.

[0050] In another preferred embodiment, the cell surface marker is selected from the group consisting of: CD4 + CD8 + , or a combination thereof.

[0051] In another preferred embodiment, the trapping body specifically binds to the cell surface marker.

[0052] In another preferred embodiment, the sorting beads are CD4-containing + Antibodies and CD8 + Antibody sorting magnetic beads.

[0053] In another preferred embodiment, the washing solution is a buffer solution.

[0054] In another preferred embodiment, the sorting solution is a buffer solution containing sorting magnetic beads.

[0055] In another preferred embodiment, the buffer solution is a pH 6.8-7.4 PBS buffer.

[0056] In another preferred embodiment, in step (c), the activating magnetic bead is a CD3. + CD28 + Or a combination of them, to activate magnetic beads.

[0057] In another preferred embodiment, the activating magnetic beads are Dynabeads.

[0058] In another preferred embodiment, in step (c), the cell density in the second immune cell population is 0.5-10 × 10⁻⁶. 6 cells / ml.

[0059] In another preferred embodiment, in step (e), the virus is a lentivirus.

[0060] In another preferred embodiment, the gene is a tumor-killing gene.

[0061] In another preferred embodiment, in step (e), the ratio of virus to cells in the transfection step is 1-10:1.

[0062] In another preferred embodiment, in step (f), the culture is carried out in a Wave device.

[0063] In another preferred embodiment, the culture is carried out in a wave culture bag.

[0064] In another preferred embodiment, the wave culture bag has a size of 2L-10L.

[0065] In another preferred embodiment, the wave parameters are: temperature 35-39℃, gas flow 0.08-0.15L / min, CO2 4-6%, rocking speed 10-18rpm, and rocking angle 6-10°.

[0066] In another preferred embodiment, in step (f), after cultivation, the activating magnetic beads are removed using Dynamag CTS.

[0067] In another preferred embodiment, step (f) further includes concentrating the cultured fourth immune cell population, the concentration being performed on a Sepax C-pro device;

[0068] In another preferred embodiment, concentration is performed using the "culturewash" program of Sepax C-pro, wherein the culturewash parameters include one or more selected from the group consisting of:

[0069]

[0070] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0071] Through extensive and in-depth research, the inventors have unexpectedly developed a method for preparing genetically modified immune cells for the first time. This cell preparation method can rapidly prepare immune cells of high quality, thereby ensuring clinical efficacy.

[0072] the term

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0074] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only open-ended definitions but also semi-closed and closed definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0075] Preparation method

[0076] This invention provides a method for preparing gene-modified immune cells, the method comprising the following steps:

[0077] (a) Provide a sample containing immune cells to be genetically modified;

[0078] (b) The sample is sorted to obtain a first immune cell population rich in immune cells;

[0079] (c) The first immune cell population is activated to obtain an activated second immune cell population;

[0080] (d) The activated second immune cell population is subjected to pre-transfection culturing (also known as pre-cultivation) to obtain a pre-cultured third immune cell population;

[0081] (e) Using a viral vector, the activated third immune cell population was genetically modified by transfection to obtain a genetically modified fourth immune cell population.

[0082] (f) The genetically modified fourth immune cell population is transfected and cultured to obtain the cultured fourth immune cell population, namely the genetically modified immune cells.

[0083] The method has one or more features selected from the group consisting of:

[0084] (i) In step (c), activation is performed using magnetic beads, and the ratio of the number of magnetic beads to the number of cells is 0.5-5:1;

[0085] (ii) In step (e), the virus and the activated fourth immune cell population are mixed and incubated for a period of time. e And no centrifugation is performed after incubation;

[0086] (iii) In step (f), during culture, different perfusion methods are used based on the density of immune cells in the culture system: when the density of immune cells < 2 × 10⁻⁶ 6 Perfusion is not performed when the cell density is ≥2×10⁻⁶ / ml; perfusion is performed when the immune cell density is ≥2×10⁻⁶ / ml. 6 cells / ml and <4×10 6 When the cell density is ≥4×10⁻⁶ / ml, perfusion is performed at a rate of 0.5V / day to 1V / day, where V is the volume of the culture system; when the immune cell density is ≥4×10⁻⁶... 6 When the cell / ml ratio is 1V / day to 2V / day, perfusion is performed at a rate of 1V / day to 2V / day, where V is the volume of the culture system.

[0087] In another preferred embodiment, in step (c), the density of the first immune cell population is 0.5-10 × 10⁻⁶. 6 cells / ml.

[0088] The method described in this invention can rapidly prepare cells in a short time, which is beneficial for industrial production.

[0089] Preferably, the method further comprises one or more features selected from the group consisting of:

[0090] (t1) The total time t from step (b) to (f) (b-f) It takes 4-5 days;

[0091] (t2) The incubation time t in step (f) f It takes 1.0-3.5 days.

[0092] In another preferred embodiment of the present invention, the ratio of the number of activating magnetic beads to the number of cells is 1-5:1;

[0093] In another preferred embodiment of the invention, step (b) includes: mixing the sample with sorting magnetic beads and incubating the mixture for a period of time t. b Then, the primary immune cell population rich in immune cells is selected;

[0094] Preferably, the t b The time should be 10-30 minutes, preferably 10-25 minutes.

[0095] In another preferred embodiment, step (c) includes: mixing the first immune cell population and the activating magnetic beads, and incubating the mixture for a period of time. c This allows for the acquisition of an activated second immune cell population;

[0096] In another preferred embodiment, the t in step (c) c It takes 12-24 hours.

[0097] In another preferred embodiment of the invention, the method has one or more features selected from the group consisting of:

[0098] In step (d), the pre-incubation time t d The optimal time is 1.5-3 days, with 1.5-2.5 days being the best.

[0099] In step (e), the transfection culture time t e The time is 0.5-2.5 days, preferably 1-2 days; and / or

[0100] In step (f), the incubation time t after transfection f The time is 1-3.5 days, with 1.5-3 days being ideal.

[0101] In another preferred embodiment of the invention, the method further includes:

[0102] (f) When the number or density of the cultured fourth immune cell population reaches a predetermined value, the cultured fourth immune cell population is harvested.

[0103] In another preferred embodiment, the predetermined value is 2 × 10⁻⁶. 6 cells / ml ~ 20 × 10 6 Cells / ml.

[0104] In another preferred embodiment, in step (e), the virus and the activated fourth immune cell population are mixed and incubated for a period of time. e The incubation solution is obtained by diluting the incubation solution with a culture medium by 0.5-2 times (preferably 0.75-1.5 times) by volume to obtain the diluted incubation solution.

[0105] In another preferred embodiment, in step (f), the diluted incubation solution is incubated for 0.5-1.5 days and then inoculated into a bioreactor such as Xuri Wave for further culture for 1-7 days.

[0106] In the method described in this invention, the sample is not particularly limited. Preferably, the sample includes (but is not limited to): blood, cells, fresh aspirate, frozen aspirate, PBMC aspirate, or a combination thereof.

[0107] In another preferred embodiment, the immune cells include (but are not limited to): T cells, NK cells, or combinations thereof.

[0108] In another preferred embodiment of the invention, the sample is washed before being sorted.

[0109] In another preferred embodiment, the washing process includes the steps of adding washing solution to the cell sample, mixing, centrifuging, removing the supernatant, and obtaining a precipitate. The washing process described in this invention can be performed on Sepax 2, Sepax C-pro, Sefia, Lovo, CS 5+, CS Elite, or Prodigy devices.

[0110] In another preferred embodiment, the washing is performed on a Sepax C-pro device.

[0111] Sepax C-Pro is a fully automated, closed-loop cell processing system. It is an automated, functional, closed-loop technology product used to process cells in the production of cell therapy products. When used in conjunction with software solutions and kits, it can achieve a multifunctional combination of various processing steps, including but not limited to the enrichment, separation, washing, concentration, dilution, and bagging of cells from various sources (umbilical cord blood, bone marrow, peripheral blood, fat, cultured cells, etc.).

[0112] Typically, sample washing and magnetic bead incubation were performed using the Sepax C-pro's "Beadwash" program, with Beadwash parameters including one or more parameters selected from Table A above.

[0113] In another preferred embodiment, in step (b), the sorting includes positive sorting or negative sorting.

[0114] In another preferred embodiment, in step (b), the sorting includes sorting by adding sorting magnetic beads containing traps that bind to immune cell surface markers (such as CD4 and / or CD8). The sorting magnetic beads bind to the immune cell surface markers through the traps to form a sorting magnetic bead-cell complex, thereby obtaining a first immune cell population rich in immune cells.

[0115] In a preferred embodiment, the trapping agent is an antibody. The antibody may be a specific antibody. Typically, the antibody is selected from the group consisting of CD4. + Antibodies, CD8 + Antibodies, or combinations thereof.

[0116] In another preferred embodiment, the cell surface marker is selected from the group consisting of: CD4 + CD8 + or combinations thereof. The capture body described in this invention can specifically bind to the cell surface markers described herein.

[0117] In another preferred embodiment of the present invention, the sorting magnetic beads are CD4-containing + Antibodies and CD8 + Antibody sorting magnetic beads.

[0118] In another preferred embodiment of the present invention, in step (c), the activating magnetic bead is a CD3. + CD28 + Or a combination of them, to activate magnetic beads.

[0119] Typically, the activated magnetic beads are Dynabeads.

[0120] In another preferred embodiment, in step (c), the cell density in the second immune cell population is 0.5-10 × 10⁻⁶. 6 cells / ml.

[0121] In another preferred embodiment of the invention, in step (e), the virus is a lentivirus.

[0122] In another preferred embodiment, the gene is a tumor-killing gene.

[0123] In another preferred embodiment, in step (e), the ratio of virus to cells in the transfection step is 1-10:1.

[0124] In another preferred embodiment of the invention, in step (f), the culture is carried out in a Wave device.

[0125] In another preferred embodiment, the culture is carried out in a wave culture bag.

[0126] In another preferred embodiment, the wave culture bag has a size of 2L-10L.

[0127] In another preferred embodiment, the wave parameters are: temperature 35-39℃, gas flow 0.08-0.15L / min, CO2 4-6%, rocking speed 10-18rpm, and rocking angle 6-10°.

[0128] In another preferred embodiment, in step (f), after cultivation, the activating magnetic beads are removed using Dynamag CTS.

[0129] In another preferred embodiment, step (f) further includes concentrating the cultured fourth immune cell population, the concentration being performed on a Sepax C-pro device;

[0130] Preferably, the concentration is performed using the "culturewash" program of Sepax C-pro, wherein the culturewash parameters include one or more selected from the group consisting of:

[0131]

[0132]

[0133] The main advantages of this invention include:

[0134] The cell preparation method of the present invention can rapidly prepare immune cells, reduce enterprise costs, increase production capacity, and is suitable for industrial production. At the same time, the immune cells prepared by the cell preparation method of the present invention are of high quality and can guarantee clinical efficacy.

[0135] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0136] Example 1: Cell preparation method for cell immunotherapy

[0137] 1. Cell culture:

[0138] All cell cultures were performed under normal cell culture conditions.

[0139] 2. Preparation method

[0140] (1) Provide cell samples:

[0141] Resuscitate 10-440 ml of cryopreserved CD4 expression + and CD8 + The cells (blood sample) are used as cell samples.

[0142] (2) Washing and sorting steps:

[0143] Cell washing was performed using the Sepax C-Pro “beadWash” program and with CD4-containing... + Antibodies and CD8 + The parameters for antibody sorting magnetic bead incubation are shown in Table 1 below:

[0144] Table 1 Sepax Pro Beadwash Parameter Settings

[0145]

[0146]

[0147] The Sepax Pro “beadWash” program works as follows:

[0148] After adding washing solution (pH 7.2) and PBS buffer to the cell sample, mix, centrifuge, remove the supernatant, and obtain the precipitate.

[0149] Add sorting solution to the precipitate and incubate for 10-30 min to obtain an incubation mixture. The sorting solution is a pH 7.2 PBS buffer containing sorting magnetic beads. The volume of sorting magnetic beads is calculated as: CD4 / CD8 lymphocyte count / [(200-800) × 10⁻⁶]. 6 / ml], the sorting magnetic beads contain CD4 + Antibodies and CD8 + Antibodies act as trapping agents, and the sorting magnetic beads are processed via CD4. + Antibodies and CD8 + Antibodies and CD4 on the cell surface + and CD8 + They bind specifically to form sorting magnetic beads-cell complexes.

[0150] In step (2), it was found that too short an incubation time would affect the binding of target cells. Too short an incubation time between cells and magnetic beads would prevent some target cells from binding to the sorting beads, affecting sorting efficiency. Conversely, when the incubation time was too long, the cells did not grow well. When the volume of sorting magnetic beads was too low, some target cells could not be labeled, affecting sorting efficiency. When the volume of magnetic beads was too high, the amount of magnetic beads remaining after incubation and washing would increase, occupying the binding sites on the sorting column and affecting the sorting effect.

[0151] The sorted magnetic bead-cell complex was isolated from the incubation mixture described using the CliniMacs device in pH 7.2 PBS buffer.

[0152] CliniMacs devices function as follows:

[0153] First, the sorting magnetic beads-cell complex is adsorbed using a magnetic field to remove liquid from the incubation mixture. Then, the magnetic field is removed, and the sorting magnetic beads-cell complex is washed with pH 7.2 PBS buffer to obtain sorting magnetic beads-cell complex pH 7.2 PBS buffer.

[0154] (3) Cell activation steps:

[0155] The sorting magnetic bead-cell complex was centrifuged in pH 7.2 PBS buffer, and the supernatant was removed to obtain a precipitate containing the sorting magnetic bead-cell complex. The precipitate was resuspended in culture medium and then added to cell culture medium containing Dynabeads for cell activation, resulting in a cell mixture. After incubation for 12-24 hours, further processing was performed. In the cell mixture, the ratio of Dynabeads to cells was 0.5-5:1, and the density of activated cells was 0.5-10 × 10⁶ cells / mL. 6 cells / ml.

[0156] In step (4), it was found that using too high a proportion of activating magnetic beads may cause overactivation of lymphocytes and an increase in the amount of residual magnetic beads during removal. Overactivation of cells can lead to apoptosis and differentiation, and too much magnetic bead can overload the magnet's adsorption capacity, resulting in excessive magnetic bead residue in the final cell product, which poses a high risk. When the activation density is too high or too low, it will affect the probability of cell contact with the activating magnetic beads, thereby affecting the activation effect of the magnetic beads on cells and thus affecting cell proliferation.

[0157] (4) Transfection steps:

[0158] After culturing the cell mixture for 2 days, the cells were transfected with a virus carrying the target gene at a virus-to-cell ratio (MOI) of 1-10:1. After incubation for 2 days, an incubation solution was obtained. The virus was diluted 1-fold by adding an equal volume of cell culture medium to the incubation solution and cultured for another day. The cells were then seeded into a Xuri Wave for further culture. After culturing in the Wave for 1-2 days, the culture was terminated, and a cell mixture carrying the target gene was obtained. The Wave cell culture parameters were: temperature 37℃, gas flow 0.1L / min, CO2 5%, rocking speed 10-18rpm, and rocking angle 6-10°.

[0159] In step (5), it was found that excessively low or high Wave culture temperature would affect the metabolic growth rate of cells; excessively low or high CO2 ratio, gas flow rate, rocking angle and rate would affect the dissolved oxygen rate and other culture conditions, inhibiting cell growth. Wave culture can strictly control the above parameters.

[0160] (5) Steps to remove the activating magnetic beads:

[0161] The activation magnetic beads in the cell mixture carrying the target gene were removed using the Dynamag CTS device to obtain a cell mixture without the activation magnetic beads.

[0162] (6) Cell perfusion

[0163] The cell mixture after removing the activating magnetic beads was cultured, and perfusion (flowing supplemental medium to maintain a cell culture volume of 500 ml) was performed during the culture process to obtain the cultured cell solution. The perfusion process is shown in Table 2 below:

[0164] Table 2 Cell perfusion culture

[0165] Irrigation rate Cell density No irrigation <![CDATA[Cell density < 2 × 10 6 cell / ml]]> 0.25~0.5L / day <![CDATA[2×10 6 cell / ml ≤ cell density < 4×10 6 cell / ml]]> 0.5~1L / day <![CDATA[4×10 6 cell / ml ≤ cell density]]>

[0166] (7) Cell concentration and freeze-drying:

[0167] After washing and concentrating the cell slurry obtained in step (7), a freeze-drying protective solution was added, and the cells were dispensed and freeze-dried to obtain the gene-modified cells.

[0168] The cell washing and concentration step was performed using the "culturewash" program of Sepax C-pro, and the culturewash parameters are shown in Table 3 below:

[0169] Table 3 Culturewash parameter settings

[0170]

[0171] 3. Experimental Results

[0172] Multiple parallel experiments were conducted using the method described above to investigate different process parameters. The results are as follows:

[0173] 3.1 In step (2) washing and incubation, after the washing and incubation step was performed using the Sepax Pro “beadWash” program, the recovery rates of monocytes and lymphocytes are shown in Table 4.

[0174] Table 4 Recovery rates of monocytes and lymphocytes

[0175]

[0176] As can be seen from Table 4, after washing and incubating cell samples with Sepax Pro “beadWash”, the cell recovery rate is as high as 90% or more, indicating that washing and incubating cell samples with Sepax Pro “beadWash” will basically not cause cell loss.

[0177] 3.2 In step (3), the cell sorting was performed using CliniMacs, and the cell recovery rate is shown in Table 5:

[0178] Table 5 Cell recovery rate

[0179] <![CDATA[Number of CD4 + +CD8 + T cells in the cell sample]]> <![CDATA[1832.7×10 6 ]]> <![CDATA[CD4 + + CD8 + T cell count]]> <![CDATA[1500×10 6 ]]> <![CDATA[Sort CD4 + + CD8 + T cell recovery rate]]> 82％

[0180] As can be seen from Table 5, after using CliniMacs to sort cell samples, the cell recovery rate is as high as 80% or more, indicating that CliniMacs can collect most of the target cells after sorting cell samples.

[0181] 3.3 In the transfection step (5), the cell activation rate was detected after culturing in the cell mixture for 1 day. Cells were incubated for another 2 days after virus transfection. The cell activation rate on day 1, the cell count on days 3 and 4, and the cell positivity rate are shown in Table 6.

[0182] Table 6 shows the activation effects in step (4) of the cell activation process.

[0183]

[0184]

[0185] As can be seen from Table 6, after one day of culture, up to 85% of the cells in the cell mixture of step (4) were in an activated state, indicating that the cell activation effect was good. Continued culture of the cells can continuously expand and efficiently express transgenic expression products.

[0186] 3.4 In the cell preparation method of this embodiment, the cell subpopulation information of the cell samples in step (1) (the initial blood drug sample), after sorting in step (3), and after incubation for 2 days after virus transfection in step (5) is shown in Table 7:

[0187] Table 7 Cell Subpopulation Information

[0188]

[0189] Remarks: Tnaive: naive T cell; Tcm: Central Memory T cell.

[0190] As can be seen from Table 7, the proportion of Tnaive and Tcm cells continuously increased during the cell preparation process, indicating that the cell viability was continuously enhanced during the preparation process.

[0191] 3.5 The cell recovery rate and viability before and after removal of dynabeads using Dynamag CTS are shown in Table 8:

[0192] Table 8. Cell recovery rate and viability before and after dynabead removal in Dynamag CTS.

[0193]

[0194] As can be seen from Table 8, Dynamag CTS can ensure high cell recovery and viability when removing dynabeads.

[0195] 3.6 Cell washing and concentration process experiment

[0196] Table 9 shows the cell recovery rate and viability before and after washing and concentrating cell samples using Sepax Pro:

[0197] Table 9. Cell recovery rate and viability before and after cell washing and concentration.

[0198]

[0199] As can be seen from Table 9, the cell samples washed and concentrated using Sepax Pro exhibited excellent cell recovery and viability.

[0200] 3.7 Cell dispensing process experiment

[0201] Using Cell Connect dispensing tubing or Sefia to dispense cell samples ensures accurate output volume and consistent cell density during dispensing, as shown in Table 10.

[0202] Table 10

[0203] Cell Connect dispensing tubing Set value / theoretical value Actual volume The first bag of sample volume was then repackaged. 88ml 86ml The first bag of sample concentration was then dispensed. <![CDATA[16×10 6 / ml]]> <![CDATA[16.4×10 6 / ml]]> The second bag of sample volume was then repackaged. 880ml 86ml The second bag of sample concentration was dispensed. <![CDATA[16×10 6 / ml]]> <![CDATA[16.4×10 6 / ml]]> Sefia Set value / theoretical value Actual volume The first bag of sample volume was then repackaged. 88ml 86.2ml The first bag of sample concentration was then dispensed. <![CDATA[12×10 6 / ml]]> <![CDATA[12×10 6 / ml]]> The second bag of sample volume was then repackaged. 88ml 82.4ml The second bag of sample concentration was dispensed. <![CDATA[12×10 6 / ml]]> <![CDATA[11.7×10 6 / ml]]>

[0204] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for improving the efficiency and quality of preparing gene-modified immune cells, characterized in that, The method includes the following steps: (a) Provide a sample containing immune cells to be genetically modified, wherein the sample is a blood sample; (b) The blood sample was washed and incubated with sorting magnetic beads using the Sepax C-Pro "beadWash" program to perform specific cell sorting, thereby obtaining a first immune cell population rich in immune cells; (c) The first immune cell population is activated to obtain an activated second immune cell population; (d) The activated second immune cell population is cultured before transfection to obtain a pre-cultured third immune cell population; (e) Genetically modify the activated third immune cell population by transfection to obtain a genetically modified fourth immune cell population. (f) The genetically modified fourth immune cell population is transfected and cultured to obtain the cultured fourth immune cell population, namely the genetically modified immune cells. wherein the total time t of step (b) to step (f) (b-f) was 4-5 days, wherein the positive rate of transduction of the cell population reached 56.80% 2 days after the virus transfection of step (e), and the cell proportion of naive T cells (Tnaive) and central memory T cells (Tcm) increased relative to both the cell sample and after sorting; The sample washing and magnetic bead incubation were performed using the "Beadwash" program of Sepax C-pro. The sorting magnetic beads contained CD4+ and CD8+ antibodies. The Beadwash parameters included:

2. The method of claim 1, wherein, In step (c), the first immune cell population is activated using activating magnetic beads, and the ratio of the number of activating magnetic beads to the number of cells is 0.5-5:

1.

3. The method of claim 1, wherein, In step (c), the activation magnetic bead is a CD3+, CD28+, or a combination thereof.

4. The method of claim 1, wherein, In step (c), the cell density in the second population of immune cells is 0.5-10 x 10 6 cells / ml.

5. The method of claim 1, wherein, Step (b) includes: mixing the sample with sorting magnetic beads and incubating the mixture for a period of time t. b Then, the first immune cell population, which is rich in immune cells, is selected.

6. The method of claim 1, wherein, In step (e), the virus and the activated fourth immune cell population are mixed and incubated for a period of time. e Furthermore, no centrifugation is performed after incubation.

7. The method of claim 1, wherein, In step (f), during culture, different perfusion methods are used based on the density of immune cells in the culture system: when the density of immune cells < 2 × 10⁻⁶ 6 Perfusion is not performed when the cell density is ≥2×10⁻⁶ / ml; perfusion is performed when the immune cell density is ≥2×10⁻⁶ / ml. 6 cells / ml and <4×10 6 When the cell density is ≥4×10⁻⁶ / ml, perfusion is performed at a rate of 0.5V / day to 1V / day, where V is the volume of the culture system; when the immune cell density is ≥4×10⁻⁶... 6 When the cell / ml ratio is 1V / day to 2V / day, perfusion is performed at a rate of 1V / day to 2V / day, where V is the volume of the culture system.

8. The method of claim 1, wherein, The incubation time t in step (f) f It takes 1.0-3.5 days.

9. The method of claim 1, wherein, In step (d), the pre-incubation time t d It takes 1.5-3 days.

10. The method of claim 9, wherein, The pre-culture time t d It takes 1.5-2.5 days.

11. The method of claim 1, wherein, In step (e), the transfection culture time t e It takes 0.5-2.5 days.

12. The method of claim 11, wherein, The transfection culture time t e It takes 1-2 days.

13. The method of claim 8, wherein, In step (f), the incubation time t after transfection f It takes 1.5-3 days.

14. The method of claim 1, wherein, The immune cells mentioned are T cells.

15. The method of claim 1, wherein, In step (e), a viral vector is used for transfection.

16. The method of claim 15, wherein, The viral vector is a lentiviral vector.

17. The method of claim 1, wherein, The method further includes: (f) When the number or density of the cultured fourth immune cell population reaches a predetermined value, the cultured fourth immune cell population is harvested.

18. The method as described in claim 1, characterized in that, In step (e), the virus and the activated fourth immune cell population are mixed and incubated for a period of time. e To obtain the incubation solution; The incubation solution is diluted with culture medium by 0.5-2 times by volume to obtain a diluted incubation solution.

19. The method of claim 18, wherein the incubation solution is diluted with a culture medium by 0.75-1.5 times by volume to obtain a diluted incubation solution.

20. The method according to any one of claims 1-19, wherein, The samples are selected from the following group: fresh single samples, frozen single samples, or combinations thereof.

21. The method as described in claim 1, characterized in that, The activation is performed using Dynabeads.

Images