Automatic preparation process and application of CAR-immune cells

Abstract

The invention provides an automatic preparation process and application of CAR-immune cells. Specifically, the invention provides an automatic preparation process of which all steps are carried out in a closed and sterile automatic cell processing system, automatic preparation equipment of which all corresponding modules are configured in the closed and sterile automatic cell processing system, and application of the automatic preparation process in preparation of CAR-immune cells. In the invention, a large number of CAR-immune cells meeting clinical use standards can be obtained through short-time activation transduction (about 1-4 days, and the whole process flow is less than or equal to 32 hours).

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to an automated preparation process for CAR-immune cells and its application. Background Technology

[0002] In recent years, cell therapy, especially chimeric antigen receptor (CAR) modified immune cell therapy (such as T-cell (CAR-T) therapy), has demonstrated enormous potential and significant clinical efficacy in the treatment of malignant tumors. CAR-immunotherapy, through genetic engineering, transforms a patient's immune cells into those capable of specifically recognizing and killing tumor cells, and has become an important means of treating hematological malignancies such as acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). However, despite significant breakthroughs in clinical applications, the complex manufacturing process, time-consuming nature, high cost, and limited production scale remain major bottlenecks hindering its widespread adoption and clinical application.

[0003] In the field of CAR-immune cell preparation, existing technologies often suffer from drawbacks such as long preparation cycles, small production scale, high costs, and difficulty in quality control. Therefore, optimizing the CAR-immune cell preparation process is particularly important.

[0004] Therefore, there is an urgent need in this field to develop an optimized preparation process for CAR-immune cells to obtain a larger quantity of CAR-immune cells that meet clinical use standards in a shorter time, in order to further promote the widespread adoption and clinical application of CAR-immune cell therapy and provide patients with a more efficient, convenient and economical treatment option. Summary of the Invention

[0005] The purpose of this invention is to provide an automated preparation process for CAR-immune cells and its application.

[0006] In a first aspect of the present invention, an automated preparation process for CAR-immune cells is provided, the automated preparation process comprising the following steps:

[0007] (S1) Provide peripheral blood or apheresis blood samples or PBMC samples;

[0008] (S2) The sample described in step (S1) is contacted with magnetic beads conjugated with CD3 antibody and CD28 antibody and incubated to obtain a mixture of cells and magnetic beads in the sample;

[0009] (S3) Magnetic separation and collection of T cells and / or NK cells from the mixture obtained in step (S2);

[0010] (S4) Activate the T cells and / or NK cells collected in step (S3) using the CD3 antibody and CD28 antibody conjugated with the magnetic beads;

[0011] (S5) Using the T cells and / or NK cells activated in the viral vector transduction step (S4), chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells are obtained.

[0012] (S6) Expanding the chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells obtained in step (S5) in a cell culture system; and

[0013] (S7) Collect the expanded T cells and / or NK cells from step (S6) to obtain CAR-immune cells;

[0014] All steps (S1)-(S7) are performed in a closed and sterile automated cell processing system.

[0015] In another preferred embodiment, the CAR-immune cells are CAR-T cells and / or CAR-NK cells.

[0016] In another preferred embodiment, the peripheral blood or apheresis sample is selected from the group consisting of: fresh peripheral blood or apheresis samples, frozen peripheral blood or apheresis samples, thawed peripheral blood or apheresis samples, or combinations thereof.

[0017] In another preferred embodiment, the volume of the fresh peripheral blood or apheresis sample is 10-250 mL, more preferably 50-150 mL, and even more preferably 80-120 mL.

[0018] In another preferred embodiment, the volume of the frozen peripheral blood or apheresis sample is 10-250 mL, more preferably 20-100 mL, and even more preferably 30-70 mL.

[0019] In another preferred embodiment, the volume of the resuscitated peripheral blood or apheresis sample is 10-250 mL, more preferably 20-100 mL, and even more preferably 30-70 mL.

[0020] In another preferred embodiment, the volume of the PBMC sample is 10-250 mL, more preferably 20-100 mL, and even more preferably 30-70 mL.

[0021] In another preferred embodiment, the total cell count of the peripheral blood or apheresis sample is 1 × 10⁻⁶. 7 -1×10 12 5×10 7 -1×10 10 One, better 1×10 8 -1×10 9indivual.

[0022] In another preferred embodiment, the total cell count of the PBMC sample is 1 × 10⁻⁶. 7 -1×10 12 5×10 7 -1×10 10 One, better 1×10 8 -1×10 9 indivual.

[0023] In another preferred embodiment, step (S1) further includes a sub-step:

[0024] (S1a) When the volume of peripheral blood, apheresis, or PBMC samples exceeds the maximum single processing volume of the automated cell processing system, the peripheral blood, apheresis, or PBMC samples are concentrated.

[0025] In another preferred embodiment, the concentration is performed by centrifugation and / or filtration.

[0026] In another preferred embodiment, the time for step (S1a) is ≤0.5h.

[0027] In another preferred embodiment, the automated preparation process further includes a step (Sx) between step (S1) and step (S2): isolating peripheral blood mononuclear cells (PBMCs) containing T cells and / or NK cells from the peripheral blood or apheresis sample.

[0028] In another preferred embodiment, steps (Sx) and (S1)-(S7) are performed in a closed and sterile automated cell processing system.

[0029] In another preferred embodiment, the time for step (Sx) is ≤2.5h, preferably ≤2h.

[0030] In another preferred embodiment, PBMC cells are separated using density gradient centrifugation in step (Sx).

[0031] In another preferred embodiment, the density gradient centrifugation is sucrose density gradient centrifugation.

[0032] In another preferred embodiment, the sucrose density gradient centrifugation is performed using a Ficoll solution.

[0033] In another preferred embodiment, step (Sx) further includes a sub-step:

[0034] (Sxa) dilutes the peripheral blood or apheresis sample;

[0035] (Sxb) performs density gradient centrifugation; and

[0036] (Sxc) Collect PBMC cells and resuspend them.

[0037] In another preferred embodiment, in step (Sxa), the peripheral blood or apheresis sample is diluted to 200-500 mL, preferably 200-300 mL.

[0038] In another preferred embodiment, the step (Sxa) involves dilution using a solution selected from the group consisting of physiological saline, cell culture medium, buffer solution, or a combination thereof.

[0039] In another preferred embodiment, the cell culture medium contains human serum albumin (HSA).

[0040] In another preferred embodiment, the cell culture medium contains bovine serum albumin (BSA).

[0041] In another preferred embodiment, the buffer solution is the incubation buffer solution used for incubation in step (S2).

[0042] In another preferred embodiment, the components of the buffer solution are selected from the group consisting of phosphate-buffered saline (PBS), human serum albumin (HSA), or combinations thereof.

[0043] In another preferred embodiment, the buffer solution is phosphate-buffered saline (PBS) or phosphate-buffered saline (PBS) containing human serum albumin (HSA).

[0044] In another preferred embodiment, the buffer is selected from the group consisting of: phosphate-buffered saline (PBS), phosphate-buffered saline (PBS) containing 0.5% HSA, and phosphate-buffered saline (PBS) containing 1% HSA.

[0045] In another preferred embodiment, PBMC cells are resuspended in step (Sxc) using phosphate-buffered saline (PBS).

[0046] In another preferred embodiment, the PBMC cells are resuspended in phosphate-buffered saline (PBS) containing 1% HSA in step (Sxc).

[0047] In another preferred embodiment, the resuspension volume in step (Sxc) is 10-30 mL, more preferably 12-25 mL, and even more preferably 15-20 mL.

[0048] In another preferred embodiment, the density of resuspended PBMC cells in step (Sxc) is 1 × 10⁻⁶. 5 -1×10 10 cells / mL, preferably 1×10 6 -1×10 9 cells / mL, preferably 1×10⁻⁶ 7-1×10 8 per mL.

[0049] In another preferred embodiment, step (Sx) further includes a sub-step:

[0050] (Sxd) Detection of PBMC cells after resuspending.

[0051] In another preferred embodiment, the detection includes: cell viability detection, cell count detection, cell density detection, cell purity detection, or a combination thereof.

[0052] In another preferred embodiment, the cell counting detection is performed using an automated cell counter.

[0053] In another preferred embodiment, the automated cell counter is selected from the group consisting of: benchtop automated cell counters (such as the JSY unstained fully automated cell counter, Countess...). TM Automated cell counters, Bio-Rad fully automated cell counters), digital cell imaging systems (such as...) DCI digital cell imaging system, automated bright-field / fluorescence cell counting observation station (such as Exact automated bright-field / fluorescence cell counting observation station), or a combination thereof.

[0054] In another preferred embodiment, the automated cell counter is NC-200 cell counting instrument.

[0055] In another preferred embodiment, the time for step (Sxd) is ≤0.5h.

[0056] In another preferred embodiment, when step (Sx) includes a sub-step (Sxd), the time of step (Sx) is ≤2.5h.

[0057] In another preferred embodiment, the incubation time in step (S2) is 15-40 min, more preferably 20-35 min.

[0058] In another preferred embodiment, the time for step (S2) is ≤0.5h.

[0059] In another preferred embodiment, the incubation temperature in step (S2) is room temperature.

[0060] In another preferred embodiment, the particle size of the magnetic beads in step (S2) is 1-10 μm, more preferably 2-7 μm, and even more preferably 3-5 μm.

[0061] In another preferred embodiment, the magnetic beads in step (S2) have a particle size of 4.5 μm.

[0062] In another preferred embodiment, the incubation process in step (S2) is a static-mixing dynamic incubation process, with mixing every 4-10 seconds of static incubation, preferably 5-8 seconds.

[0063] In another preferred embodiment, the dynamic incubation in step (S2) is performed by mixing at a speed of 100-300 rpm / min, preferably 125-200 rpm / min.

[0064] In another preferred embodiment, the time for step (S3) is ≤1h, preferably ≤0.5h.

[0065] In another preferred embodiment, step (S3) further includes a sub-step:

[0066] (S3a) Detect the collected T cells and / or NK cells.

[0067] In another preferred embodiment, the detection includes: cell viability detection, cell count detection, cell density detection, cell purity detection, or a combination thereof.

[0068] In another preferred embodiment, the cell counting detection is performed using an automated cell counter.

[0069] In another preferred embodiment, the yield of T cells and / or NK cells in step (S3) is ≥70%, more preferably ≥75%, and even more preferably ≥80%.

[0070] In another preferred embodiment, the purity of T cells and / or NK cells in step (S3) is detected using flow cytometry.

[0071] In another preferred embodiment, the time for step (S3a) is ≤0.5h.

[0072] In another preferred embodiment, when step (S3) includes a sub-step (S3a), the time of step (S3) is ≤1h.

[0073] In another preferred embodiment, the time for steps (S4)-(S6) is ≤24h.

[0074] In another preferred embodiment, the viral vector in step (S5) is a lentiviral vector.

[0075] In another preferred embodiment, the cell culture system in step (S6) includes components selected from the group consisting of serum-free culture medium, supplemental culture medium, cytokines, gentamicin sulfate, or combinations thereof.

[0076] In another preferred embodiment, the cell seeding density used during amplification in step (S6) is 1×10⁻⁶. 3 -1×10 9 cells / mL, preferably 1×104 -1×10 8 cells / mL, preferably 1×10⁻⁶ 5 -1×10 7 per mL.

[0077] In another preferred embodiment, the cell seeding density used during amplification in step (S6) is 1×10⁻⁶. 6 per mL.

[0078] In another preferred embodiment, the number of cells seeded during amplification in step (S6) is 1 × 10⁻⁶. 5 -1×10 15 One, preferably 1×10 6 -1×10 12 One, better 1×10 7 -1×10 10 indivual.

[0079] In another preferred embodiment, the number of cells seeded during amplification in step (S6) is 1.8 × 10⁶. 8 indivual.

[0080] In another preferred embodiment, step (S7) further includes a sub-step:

[0081] (S7a) Remove the magnetic beads from the T cells and / or NK cells; and

[0082] (S7b) Clean the T cells and / or NK cells obtained in step (S7a) after removing the magnetic beads to remove impurities.

[0083] In another preferred embodiment, step (S7) further includes a sub-step:

[0084] (S7c) The collected T cells and / or NK cells were tested.

[0085] In another preferred embodiment, the detection includes: cell viability detection, cell count detection, cell density detection, cell purity detection, or a combination thereof.

[0086] In another preferred embodiment, the cell counting detection is performed using an automated cell counter.

[0087] In another preferred embodiment, the time for step (S7a) is ≤1.5h.

[0088] In another preferred embodiment, the time for step (S7b) is ≤1 hour.

[0089] In another preferred embodiment, the time for step (S7c) is ≤0.5h.

[0090] In another preferred embodiment, the automated preparation process further includes the step of:

[0091] (S8) The CAR-immune cells obtained in step (S7) are cryopreserved to obtain a CAR-immune cell cryopreservation preparation.

[0092] In another preferred embodiment, steps (S8) and (S1)-(S7) are performed in a closed and sterile automated cell processing system.

[0093] In another preferred embodiment, steps (Sx), (S8) and (S1)-(S7) are all performed in a closed and sterile automated cell processing system.

[0094] In another preferred embodiment, one or more of steps (Sx), (S8), and (S1)-(S7) are performed in the same or different chambers of the automated cell processing system.

[0095] In another preferred embodiment, one or more of steps (Sx), (S8), and (S1)-(S7) are performed within the same chamber of the automated cell processing system.

[0096] In another preferred embodiment, the time for step (S8) is ≤0.5h.

[0097] In another preferred embodiment, the total time of the automated preparation process is ≤32h, preferably ≤31.5h, ≤31h, ≤30.5h, ≤30h, ≤29.5h, ≤29h, ≤28.5h, ≤28h, ≤27.5h or ≤27h, etc.

[0098] In a second aspect of the invention, an automated preparation device for CAR-immune cells is provided, the automated preparation device comprising:

[0099] (M1) Sample loading module, which is configured to provide peripheral blood or apheresis blood samples or PBMC samples;

[0100] (M2) Magnetic bead incubation module, which is configured to: contact the sample in module (M1) with magnetic beads conjugated with CD3 antibody and CD28 antibody and incubate them to obtain a mixture of cells and magnetic beads in the sample;

[0101] (M3) Magnetic bead sorting module, which is configured to magnetically separate and collect T cells and / or NK cells from the mixture obtained from module (M2);

[0102] (M4) Activation module, the activation module being configured to: activate T cells and / or NK cells collected in module (M3) using CD3 and CD28 antibodies conjugated to the magnetic beads;

[0103] (M5) Transduction module, the transduction module being configured to: obtain chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells by using activated T cells and / or NK cells in the viral vector transduction module (M4);

[0104] (M6) Amplification module, configured to: chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells obtained in a cell culture system from amplification module (M5); and

[0105] (M7) Harvesting module, which is configured to collect expanded T cells and / or NK cells in module (M6) to obtain CAR-immune cells;

[0106] The modules (M1)-(M7) are all configured in a closed and sterile automated cell processing system.

[0107] In another preferred embodiment, the CAR-immune cells are CAR-T cells and / or CAR-NK cells.

[0108] In another preferred embodiment, the peripheral blood or apheresis sample is selected from the group consisting of: fresh peripheral blood or apheresis samples, frozen peripheral blood or apheresis samples, thawed peripheral blood or apheresis samples, or combinations thereof.

[0109] In another preferred embodiment, the volume of the fresh peripheral blood or apheresis sample is 10-250 mL, more preferably 50-150 mL, and even more preferably 80-120 mL.

[0110] In another preferred embodiment, the volume of the frozen peripheral blood or apheresis sample is 10-250 mL, more preferably 20-100 mL, and even more preferably 30-70 mL.

[0111] In another preferred embodiment, the volume of the resuscitated peripheral blood or apheresis sample is 10-250 mL, more preferably 20-100 mL, and even more preferably 30-70 mL.

[0112] In another preferred embodiment, the volume of the PBMC sample is 10-250 mL, more preferably 20-100 mL, and even more preferably 30-70 mL.

[0113] In another preferred embodiment, the total cell count of the peripheral blood or apheresis sample is 1 × 10⁻⁶. 7 -1×10 12 5×10 7 -1×10 10 One, better 1×10 8 -1×10 9 indivual.

[0114] In another preferred embodiment, the total cell count of the PBMC sample is 1 × 10⁻⁶. 7 -1×10 12 5×10 7 -1×10 10 One, better 1×10 8 -1×10 9 indivual.

[0115] In another preferred embodiment, the module (M1) further includes a submodule:

[0116] (M1a) Sample Concentration Module, which is configured to concentrate peripheral blood or apheresis samples or PBMC samples when the volume of the peripheral blood or apheresis sample or PBMC sample exceeds the maximum single processing volume of the automated cell processing system.

[0117] In another preferred embodiment, the concentration is performed by centrifugation and / or filtration.

[0118] In another preferred embodiment, the unit working time of the module (M1a) is ≤0.5h.

[0119] In another preferred embodiment, the automated preparation equipment further includes a module:

[0120] (Mx)PBMC separation module, the PBMC separation module being configured to: separate peripheral blood mononuclear cells (PBMCs) containing T cells and / or NK cells from the peripheral blood or apheresis sample.

[0121] In another preferred embodiment, modules (Mx) and (M1)-(M7) are all configured in a closed and sterile automated cell processing system.

[0122] In another preferred embodiment, the unit working time of the module (Mx) is ≤2.5h, preferably ≤2h.

[0123] In another preferred embodiment, the module (Mx) uses density gradient centrifugation to separate PBMC cells.

[0124] In another preferred embodiment, the density gradient centrifugation is sucrose density gradient centrifugation.

[0125] In another preferred embodiment, the sucrose density gradient centrifugation is performed using a Ficoll solution.

[0126] In another preferred embodiment, the module (Mx) further includes a submodule:

[0127] (Mxa) Dilution module, the dilution module being configured to dilute the peripheral blood or apheresis sample;

[0128] (Mxb) centrifugation module, the centrifugation module being configured to: perform density gradient centrifugation; and

[0129] (Mxc) resuspension module, the resuspension module being configured to collect PBMC cells and resuspend them.

[0130] In another preferred embodiment, the peripheral blood or apheresis sample in the module (Mxa) is diluted to 200-500 mL, preferably 200-300 mL.

[0131] In another preferred embodiment, the module (Mxa) is diluted using a solution selected from the group consisting of physiological saline, cell culture medium, buffer solution, or a combination thereof.

[0132] In another preferred embodiment, the cell culture medium contains human serum albumin (HSA).

[0133] In another preferred embodiment, the cell culture medium contains bovine serum albumin (BSA).

[0134] In another preferred embodiment, the buffer solution is an incubation buffer solution used for incubation in module (M2).

[0135] In another preferred embodiment, the module (Mxc) uses phosphate-buffered saline (PBS) to resuspend PBMC cells.

[0136] In another preferred embodiment, the PBMC cells are resuspended in phosphate-buffered saline (PBS) containing 1% HSA in the module (Mxc).

[0137] In another preferred embodiment, the resuspension volume in the module (Mxc) is 10-30 mL, more preferably 12-25 mL, and even more preferably 15-20 mL.

[0138] In another preferred embodiment, the resuspended PBMC cell density in the module (Mxc) is 1 × 10⁻⁶. 5 -1×10 10 cells / mL, preferably 1×10 6 -1×10 9 cells / mL, preferably 1×10⁻⁶ 7 -1×10 8 per mL.

[0139] In another preferred embodiment, the module (Mx) further includes a submodule:

[0140] (Mxd) detection module, the detection module being configured to detect resuspended PBMC cells.

[0141] In another preferred embodiment, the detection includes: cell viability detection, cell count detection, cell density detection, cell purity detection, or a combination thereof.

[0142] In another preferred embodiment, the cell counting detection is performed using an automated cell counter.

[0143] In another preferred embodiment, the automated cell counter is selected from the group consisting of: benchtop automated cell counters (such as the JSY unstained fully automated cell counter, Countess...). TM Automated cell counters, Bio-Rad fully automated cell counters), digital cell imaging systems (such as...) DCI digital cell imaging system, automated bright-field / fluorescence cell counting observation station (such as Exact automated bright-field / fluorescence cell counting observation station), or a combination thereof.

[0144] In another preferred embodiment, the automated cell counter is NC-200 cell counting instrument.

[0145] In another preferred embodiment, the unit working time of the module (Mxd) is ≤0.5h.

[0146] In another preferred embodiment, when the module (Mx) includes a submodule (Mxd), the unit working time of the module (Mx) is ≤2.5h.

[0147] In another preferred embodiment, the incubation time in the module (M2) is 15-40 min, more preferably 20-35 min.

[0148] In another preferred embodiment, the time of the module (M2) is ≤0.5h.

[0149] In another preferred embodiment, the incubation temperature in the module (M2) is room temperature.

[0150] In another preferred embodiment, the magnetic beads in the module (M2) have a particle size of 1-10 μm, more preferably 2-7 μm, and even more preferably 3-5 μm.

[0151] In another preferred embodiment, the magnetic beads in the module (M2) have a particle size of 4.5 μm.

[0152] In another preferred embodiment, the incubation process in the module (M2) is a static-mixing dynamic incubation process, with mixing occurring every 4-10 seconds of static time, preferably 5-8 seconds.

[0153] In another preferred embodiment, the dynamic incubation in the module (M2) is performed by mixing at a speed of 100-300 rpm / min, preferably 125-200 rpm / min.

[0154] In another preferred embodiment, the unit working time of the module (M3) is ≤1h, preferably ≤0.5h.

[0155] In another preferred embodiment, the module (M3) is equipped with a magnetic field.

[0156] In another preferred embodiment, the module (M3) further includes a submodule:

[0157] (M3a) Detection module, which is configured to detect collected T cells and / or NK cells.

[0158] In another preferred embodiment, the detection includes: cell viability detection, cell count detection, cell density detection, cell purity detection, or a combination thereof.

[0159] In another preferred embodiment, the cell counting detection is performed using an automated cell counter.

[0160] In another preferred embodiment, the yield of T cells and / or NK cells in the module (M3) is ≥70%, more preferably ≥75%, and even more preferably ≥80%.

[0161] In another preferred embodiment, the purity of T cells and / or NK cells in the module (M3) is detected using flow cytometry.

[0162] In another preferred embodiment, the time of the module (M3a) is ≤0.5h.

[0163] In another preferred embodiment, when module (M3) includes submodule (M3a), the unit working time of module (M3) is ≤1h.

[0164] In another preferred embodiment, the unit working time of the modules (M4)-(M6) is ≤24h.

[0165] In another preferred embodiment, the viral vector in the module (M5) is a lentiviral vector.

[0166] In another preferred embodiment, the cell culture system in the module (M6) includes components selected from the group consisting of serum-free culture medium, supplemental culture medium, cytokines, gentamicin sulfate, or combinations thereof.

[0167] In another preferred embodiment, the cell seeding density used during amplification in the module (M6) is 1 × 10⁻⁶. 3 -1×10 9 cells / mL, preferably 1×104 -1×10 8 cells / mL, preferably 1×10⁻⁶ 5 -1×10 7 per mL.

[0168] In another preferred embodiment, the cell seeding density used during amplification in the module (M6) is 1 × 10⁻⁶. 6 per mL.

[0169] In another preferred embodiment, the number of cells seeded during amplification in the module (M6) is 1 × 10⁻⁶. 5 -1×10 15 One, preferably 1×10 6 -1×10 12 One, better 1×10 7 -1×10 10 indivual.

[0170] In another preferred embodiment, the number of cells seeded during amplification in the module (M6) is 1.8 × 10⁶. 8 indivual.

[0171] In another preferred embodiment, the module (M7) further includes a submodule:

[0172] (M7a) Magnetic bead removal module, the magnetic bead removal module being configured to: remove magnetic beads from the T cells and / or NK cells; and

[0173] (M7b) Cleaning module, the cleaning module being configured to remove impurities from T cells and / or NK cells obtained in cleaning module (M7a) after the removal of magnetic beads.

[0174] In another preferred embodiment, the module (M7) further includes a submodule:

[0175] (M7c) Detection module, which is configured to detect collected T cells and / or NK cells.

[0176] In another preferred embodiment, the detection includes: cell viability detection, cell count detection, cell density detection, cell purity detection, or a combination thereof.

[0177] In another preferred embodiment, the cell counting detection is performed using an automated cell counter.

[0178] In another preferred embodiment, the unit working time of the module (M7a) is ≤1.5h.

[0179] In another preferred embodiment, the unit working time of the module (M7b) is ≤1h.

[0180] In another preferred embodiment, the unit working time of the module (M7c) is ≤0.5h.

[0181] In another preferred embodiment, the device further includes a module:

[0182] (M8) Cryopreservation module, which is configured to: cryopreserve the CAR-immune cells obtained in module (M7) to obtain a CAR-immune cell cryopreservation preparation.

[0183] In another preferred embodiment, modules (M8) and (M1)-(M7) are all configured in a closed and sterile automated cell processing system.

[0184] In another preferred embodiment, the modules (Mx), (M8) and (M1)-(M7) are all configured in a closed and sterile automated cell processing system.

[0185] In another preferred embodiment, one or more of the modules (Mx), (M8), and (M1)-(M7) are configured to operate in the same or different chambers of the automated cell processing system.

[0186] In another preferred embodiment, one or more of the modules (Mx), (M8), and (M1)-(M7) are configured to operate within the same chamber of the automated cell processing system.

[0187] In another preferred embodiment, the unit working time of the module (M8) is ≤0.5h.

[0188] In another preferred embodiment, the unit working time of the device is ≤32h, preferably ≤31.5h, ≤31h, ≤30.5h, ≤30h, ≤29.5h, ≤29h, ≤28.5h, ≤28h, ≤27.5h or ≤27h, etc.

[0189] In another preferred embodiment, the device further includes a control module (M9) configured to control the operation of modules (M1)-(M7), or modules (M1)-(M8), or modules (M1), (Mx) and (M2)-(M7), or modules (M1), (Mx) and (M2)-(M8).

[0190] In a third aspect of the invention, the use of an automated CAR-immune cell preparation apparatus as described in the second aspect of the invention is provided for preparing CAR-immune cells.

[0191] In another preferred embodiment, the purpose is for non-disease diagnosis purposes or non-disease treatment purposes.

[0192] In another preferred embodiment, the CAR-immune cells are CAR-T cells and / or CAR-NK cells.

[0193] In another preferred embodiment, the CAR-immune cell is a CAR-T cell.

[0194] 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. Attached Figure Description

[0195] Figure 1 The calculation results show the cell yield after magnetic bead sorting: Total Cell yield is the total number of cells after sorting divided by the total number of cells before sorting; CD3+ Cell yield is the total number of cells after sorting multiplied by the percentage of CD3+ cells divided by the total number of cells before sorting multiplied by the percentage of CD3+ cells.

[0196] Figure 2 The figure shows the cell yields after removing magnetic beads at different time points. In the legend, 9BK364-A0 D19 represents experimental group 9, BK364 is the Donor number; A0 indicates simultaneous activation and transduction, A24 indicates activation for 24 hours; D1 indicates magnetic bead removal on Day 1, D2 on Day 2, and D3 on Day 3. Other groups follow the same pattern. In groups 9-16, BC represents cells obtained after bead sorting.

[0197] Figure 3 The amplification folds of antibody groups 1 through 8 are displayed. The vertical axis represents the amplification fold; the horizontal axis D1 / D0 represents the number of cells in D1 divided by the number of cells in D0, D2 / D0 represents the number of cells in D2 divided by the number of cells in D0, and D3 / D0 represents the number of cells in D3 divided by the number of cells in D0.

[0198] Figure 4 The display shows the amplification folds of Beads sorting groups 9 through 16. The vertical axis represents the amplification fold; the horizontal axis D1 / D0 represents the amplification fold of D1 by dividing the number of cells in D1 by the number of cells in D0, D2 / D0 represents the amplification fold of D2 by dividing the number of cells in D0, and D3 / D0 represents the amplification fold of D3 by dividing the number of cells in D0.

[0199] Figure 5 The percentage of CD3+CAR+ cells in groups 1-16 is displayed. The vertical axis represents the percentage of CD3+CAR+ cells, and the horizontal axis represents different culture days; D1 represents day 1 of culture.

[0200] Figure 6The data shows the fold increase of CAR+ cells in groups 1-16. The vertical axis represents the fold increase of CAR+ cells, and the horizontal axis represents the ratio of the number of cells at different culture days to the starting number of cells at D0. D1 / D0 represents the ratio of the number of CAR+ cells on Day 1 to the starting number of cells at D0.

[0201] Figure 7 Representative group 5 is shown. Figure 7 A), group 6( Figure 7 B), Group 13 / 14 Figure 7 C) and group 15 / 16 ( Figure 7 D) Cell typing. The horizontal axis represents different culture days, and the vertical axis represents the proportion of different cell types measured by flow cytometry. In the legend, T represents CD3+ T cells, NKT represents CD3+CD56+ NKT cells, Monocyte represents CD14+ cells, B represents B cells with CD19+ surface markers, and NK cells with CD3-CD56+ surface markers.

[0202] Figure 8 Representative group 3 is shown. Figure 8 A), group 4( Figure 8 B), Group 5 Figure 8 C), Group 6 Figure 8 D), Group 9 / 10 Figure 8 E), Group 11 / 12 ( Figure 8 F), Group 13 / 14 Figure 8 G) and group 15 / 16 ( Figure 8 The ratio of CD4+ / CD8+ cells in CD3+ T cells (H). The horizontal axis represents different culture days, and the vertical axis represents the percentage of CD4+ / CD8+ cells in CD3+ T cells.

[0203] Figure 9 The percentage of CD69+ cells, an activation marker, in CD3+ T cells is shown in groups 1-16. The x-axis represents different culture days, and the y-axis represents the percentage of CD69+ cells among CD3+ T cells.

[0204] Figure 10 The CAR+% plots for the MANUAL manual test group and the MACS full-scale experimental instrument test group are shown.

[0205] Figure 11 The flow cytometry plots for the MANUAL manual test group and the MACS full-scale experimental instrument test group are shown. Figure 11 A represents the MACS full-scale experimental instrumentation test group. Figure 11 B represents the MANUAL manual test group, which shows the ratio of CD4+ cells to CD8+ cells in all CD45+ leukocytes.

[0206] Figure 12 This displays a graph showing cell viability versus diameter. The vertical axis represents cell viability (…). Figure 12 A) and diameter ( Figure 12 B), with the horizontal axis representing D0 PBMC, Day 1 MANUAL manual test group, Day 1 MACS full-scale experimental instrument group, Day 1 MANUAL after BEAD removal (the viability and diameter of Day 1 MANUAL manual test group after BEAD removal), and Day 1 MACS after BEAD removal (the viability and diameter of Day 1 MACS full-scale experimental instrument group after BEAD removal).

[0207] Figure 13 The proportion of CD69+ cells, the activation marker, in CD3+ T cells is shown in each test group. Z0624PBMC represents Day 0 PBMCs from Donor Z0624; Manual BC represents T cells after Day 0 sorting in the Manual group; Manual Supernatant represents negative cells after Day 0 sorting in the Manual group; Manual CAR-T D1 represents Day 1 CAR-T cells in the Manual group; MACS BC represents T cells after Day 0 sorting in the MACS full-scale experimental instrument group; MACS Supernatant represents negative cells after Day 0 sorting in the MACS full-scale experimental instrument group; MACSD1 CAR-T represents Day 1 CAR-T cells in the MACS full-scale experimental instrument group.

[0208] Figure 14 The proportion of CD25+ cells, the activation marker, in CD3+ T cells was shown in each test group. The definitions of each test group were the same as before.

[0209] Figure 15 The proportion of TIM3+ cells (a marker of exhaustion) in each test group is shown. The definitions of each test group are the same as above.

[0210] Figure 16 The proportion of PD-1+TIM3+ cells, a marker of exhaustion, is shown in each test group. The definitions of each test group are the same as above. Detailed Implementation

[0211] Through extensive and in-depth research and optimization of the process flow, the inventors have, for the first time, creatively designed and optimized an automated preparation process and corresponding automated preparation equipment in a closed and sterile automated cell processing system. The automated preparation process and equipment of this invention use fresh, frozen, or resuscitated apheresis blood, peripheral blood, or PBMC samples as starting cells. After a short period of activation and transduction (approximately 1-4 days, with the entire process ≤32 hours, preferably ≤31.5 hours, ≤31 hours, ≤30.5 hours, ≤30 hours, ≤29.5 hours, ≤29 hours, ≤28.5 hours, ≤28 hours, ≤27.5 hours, or ≤27 hours), a large number of CAR-immune cells (preferably CAR-T cells) meeting clinical use standards can be obtained. This invention was completed based on this.

[0212] the term

[0213] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0214] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0215] CD3 / CD28 sorting activation beads

[0216] In this invention, CD3 / CD28 sorting and activating magnetic beads can be used to sort human CD3+ T cells, and based on this, a simple method for activating and expanding T cells without the need for antigen-presenting cells or antigens is provided. By conjugating anti-CD3 and anti-CD28 monoclonal antibodies to the magnetic beads, primary and co-stimulatory signals required for T cell activation and expansion are provided, thereby achieving the sorting, activation, and expansion of T cells.

[0217] The automated preparation process of the present invention

[0218] This invention provides an automated preparation process for CAR-immune cells, the automated preparation process comprising the following steps:

[0219] (S1) Provide peripheral blood or apheresis blood samples or PBMC samples;

[0220] (S2) The sample described in step (S1) is contacted with magnetic beads conjugated with CD3 antibody and CD28 antibody and incubated to obtain a mixture of cells and magnetic beads in the sample;

[0221] (S3) Magnetic separation and collection of T cells and / or NK cells from the mixture obtained in step (S2);

[0222] (S4) Activate the T cells and / or NK cells collected in step (S3) using the CD3 antibody and CD28 antibody conjugated with the magnetic beads;

[0223] (S5) Using the T cells and / or NK cells activated in the viral vector transduction step (S4), chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells are obtained.

[0224] (S6) Expanding the chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells obtained in step (S5) in a cell culture system; and

[0225] (S7) Collect the expanded T cells and / or NK cells from step (S6) to obtain CAR-immune cells;

[0226] All steps (S1)-(S7) are performed in a closed and sterile automated cell processing system.

[0227] A closed system is a cell processing system that is largely or completely closed off from the environment in which cells are processed, cultured, and / or transported. This environment may include the room environment, or even the fume hood of the system, the tubing (such as test tubes), and the environment outside the chamber. One of the greatest risks to safety and control in cell processing procedures is the risk of contamination through frequent exposure to the environment, as found in traditional open cell culture systems. To mitigate this risk, especially in the absence of antibiotics, several commercial methods have been developed that utilize single-use (disposable) equipment. However, even under aseptic conditions, there is always a risk of contamination when opening flasks to sample or add additional growth medium. To overcome this problem, the methods presented herein (typically in vitro methods) are typically performed within a closed system. This method is designed and operable so that the product is not exposed to the external environment. Material transfer is performed via aseptic connections, such as aseptic tubing or aseptic welded connections. Air for gas exchange is provided via a breathable membrane through a 0.2 μm filter to prevent environmental exposure. Such closed system methods can be performed using commercially available devices. Different closed-system devices can be used at different steps within the method, and cells can be transferred between such devices using tubes and connections (such as welds, Luer, nails, or ports) to prevent cell or culture medium exposure to the environment. The method can be performed on any device or combination of devices suitable for closed-system T cell and / or NK cell production. Non-limiting examples of such devices include G-Rex devices (Wilson Wolf), GatheRex (WilsonWolf), Sepax 2 (Biosafe), WAVE Bioreactors (General Electric), CultiLife CellCulture bags (Takara), PermaLife bags (OriGen), CliniMACS Prodigy (Miltenyi Biotec), and VueLife bags (Saint-Gobain). In illustrative embodiments, activation, transduction, and amplification are performed within the same chamber or container within the closed system. For example, in an illustrative embodiment, the chamber may be a chamber of a G-Rex device, and PBMCs may be transferred to the chamber of the G-Rex device after enrichment and separation and may remain in the same chamber of the G-Rex device until harvest. The sealing system typically includes a step of coating some or all of the device surface in contact with the cells. For example, the surface may be coated with recombinant fibronectin or fibronectin fragments (such as RetroNectin (Takara)), without theoretical limitation, which can promote the transduction of T cells and / or NK cells using non-replicating competent recombinant retroviral particles. Washing must be performed before cells can be introduced into the coated device. The steps of coating and washing the device introduce additional risks of contamination.Therefore, in any of the embodiments provided herein, T cells and / or NK cells can be advantageously introduced into the closed system device without coating the surface. In some embodiments, the method can be performed in the absence of recombinant fibronectin or RetroNectin.

[0228] Peripheral blood or apheresis samples may be fresh peripheral blood or apheresis samples, frozen peripheral blood or apheresis samples, or thawed peripheral blood or apheresis samples. The volume of fresh peripheral blood or apheresis samples is preferably 10-250 mL, more preferably 50-150 mL, and even more preferably 80-120 mL. The volume of frozen peripheral blood or apheresis samples is preferably 10-250 mL, more preferably 20-100 mL, and even more preferably 30-70 mL. The volume of thawed peripheral blood or apheresis samples is preferably 10-250 mL, more preferably 20-100 mL, and even more preferably 30-70 mL. The volume of PBMC samples is preferably 10-250 mL, more preferably 20-100 mL, and even more preferably 30-70 mL. The total cell count of the above samples is preferably 1×10⁻⁶. 7 -1×10 12 5×10 7 -1×10 10 One, better 1×10 8 -1×10 9 indivual.

[0229] Preferably, step (S1) may include sub-steps:

[0230] (S1a) When the volume of peripheral blood, apheresis, or PBMC samples exceeds the maximum processing volume of the automated cell processing system, the peripheral blood, apheresis, or PBMC samples are concentrated. Concentration can be performed by centrifugation and / or filtration to discard excess plasma, ensuring a suitable volume for the next stage of cell processing. This step (S1a) should take ≤0.5 hours. It should be understood that this step (S1a) is not mandatory and is only required when the sample volume exceeds the maximum processing volume.

[0231] For example, density gradient centrifugation for PBMC separation is a commonly used cell separation technique, primarily used to separate peripheral blood mononuclear cells (PBMCs), including lymphocytes, monocytes, and dendritic cells (DCs), from peripheral blood. Ficoll density gradient centrifugation utilizes the difference in specific gravity between a Ficoll (a polymer of sucrose) solution and different cellular components in the blood. Ficoll solution is neutral, highly hydrophilic, with an average molecular weight of 400,000. At a density of 1.2 g / mL, it does not exceed normal physiological osmotic pressure and does not permeate biological membranes. Red blood cells and granulocytes, being denser, sink to the bottom of the tube after centrifugation; lymphocytes and monocytes, with a specific gravity less than or equal to that of the separating solution, float on the surface of the separating solution after centrifugation, although a small number of cells may remain suspended in the separating solution. By aspirating cells from the surface of the separating solution, mononuclear cells can be separated from peripheral blood. The blood sample is slowly layered onto the Ficoll solution along the tube wall, maintaining a clear interface between the two liquids. Note that you should set a slow acceleration and deceleration to avoid disrupting the separation of the layers. After centrifugation, the liquid in the tube will separate into three layers: a first layer of plasma and PBS, a middle layer of Ficoll solution, and a third layer of erythrocytes and granulocytes. At the interface between the first and middle layers, there is a narrow band of white, cloudy layer dominated by mononuclear cells; this is the target PBMC. Subsequently, aspirate the target cells, wash, centrifuge, and resuspend them in a suitable solution before proceeding to the next step.

[0232] It should be understood that the PBMC separation step (i.e., step (Sx)) is not necessary in the automated preparation process of the present invention. For example, when the peripheral blood or apheresis sample is a frozen peripheral blood or apheresis sample, a thawed peripheral blood or apheresis sample, or a PBMC sample, the automated preparation process can directly use the thawed and washed frozen peripheral blood and frozen apheresis sample, the thawed peripheral blood or apheresis sample, or the PBMC sample as the starting cells, without the need for the PBMC separation step. When the peripheral blood or apheresis sample is a fresh peripheral blood or apheresis sample, the automated preparation process may additionally include step (Sx) between step (S1) and step (S2): separating peripheral blood mononuclear cells (PBMCs) containing T cells and / or NK cells from the peripheral blood or apheresis sample. This step (Sx) can be performed together with other steps of the automated preparation process in a closed and sterile automated cell processing system, or it can be performed independently outside the system and then added to the automated cell processing system as the initial sample. The time for this step (Sx) is ≤2.5h, preferably ≤2h.

[0233] Preferably, this step (Sx) may further include sub-steps:

[0234] (Sxa) dilutes the peripheral blood or apheresis sample;

[0235] (Sxb) performs density gradient centrifugation; and

[0236] (Sxc) Collect PBMC cells and resuspend them.

[0237] Furthermore, this step (Sx) may also include sub-steps:

[0238] (Sxd) Detection of PBMC cells after resuspending.

[0239] The time for step (Sxd) is ≤0.5h. When step (Sx) includes a sub-step (Sxd), the time for step (Sx) is ≤2.5h.

[0240] The detections in step (Sxd) may include: cell viability detection, cell count detection, cell density detection, cell purity detection, or a combination thereof. Preferably, an automated cell counter is used for cell counting. Typically, the automated cell counter may be a benchtop automated cell counter (such as the JSY unstained fully automated cell counter, Countess...). TM Automated cell counters, Bio-Rad fully automated cell counters), digital cell imaging systems (such as...) Digital cell imaging (DCI) systems, automated bright-field / fluorescent cell counting stations (such as the Exact automated bright-field / fluorescent cell counting station), etc.

[0241] In a preferred embodiment, using The NC-200 cell counting instrument is used for cell counting. The NC-200 cell counting instrument is a device that uses disposable Via-Cassette cartridges. TM A counting chamber is an automated cell counter that combines sample loading, staining, and counting into one step. Via1-Cassette TM The counting chamber uses nuclear fluorescent dyes to automatically stain cell nuclei, enabling the counter to accurately detect cells in various states: live cells, dead cells, and cell clusters.

[0242] For step (S2), the cells and magnetic beads are placed in the same container and dynamically shaken to ensure full contact between the cells and magnetic beads. The surface of the magnetic beads is coupled with corresponding antibodies through appropriate surface chemical modifications. These antibodies can bind to CD3 surface markers expressed on the surface of CD3 cells in PBMCs. After sufficient incubation, the magnetic beads bind to the cells through antigen-antibody binding. The incubation time is 15-40 min, preferably 20-35 min; the incubation temperature is room temperature; the particle size of the magnetic beads is 1-10 μm, preferably 2-7 μm, more preferably 3-5 μm, for example 4.5 μm; the incubation process is a static-mixing dynamic incubation process, mixing every 4-10 seconds, preferably 5-8 seconds; the dynamic incubation is performed at a mixing speed of 100-300 rpm / min, preferably 125-200 rpm / min; the time for step (S2) is ≤0.5 h.

[0243] For step (S3), the principle of magnetic bead sorting is mainly based on the principle of antigen-antibody specific recognition. Highly specific monoclonal antibodies coupled with magnetized microparticles are used to specifically magnetically label target cells, and a high-intensity, gradient magnetic field is used to achieve magnetic separation of cells. A high-intensity, gradient magnetic field is applied, causing the magnetic beads to produce a magnetic adsorption effect under the influence of magnetic force. This adsorption effect concentrates the magnetic beads and their bound target substances (target cells) in a specific area. The incubated mixture of cells and magnetic beads is passed through a corresponding magnetic field. Under the influence of the magnetic field, the magnetic beads coupled with the antibody and capturing the corresponding cells are adsorbed by the magnet, while other impurity cells and liquid flow out. Finally, the magnetic field is removed, and the captured cells are collected along with the magnetic beads. The time for this step (S3) is ≤1 hour, preferably ≤0.5 hours. Preferably, it may also include a sub-step: (S3a) detecting the collected T cells and / or NK cells. The time for step (S3a) is ≤0.5 hours. When step (S3) includes a sub-step (S3a), the time for step (S3) is ≤1 hour.

[0244] The detection in step (S3a) is similar to the detection in (Sxd) and may include: cell viability detection, cell count detection, cell density detection, cell purity detection, or a combination thereof. Preferably, an automated cell counter is used for cell counting. In a preferred embodiment, an automated cell counter is used. The NC-200 cell counting instrument is used for cell counting.

[0245] In step (S3), the yield of T cells and / or NK cells is ≥70%, preferably ≥75%, and more preferably ≥80%. The purity of T cells and / or NK cells in step (S3) is detected using flow cytometry.

[0246] For steps (S4)-(S6), the time for steps (S4)-(S6) is ≤24h.

[0247] For step (S4), the cells collected after magnetic bead sorting, coupled with CD3 / CD28 activation antibodies, are mixed and directly proceed to the activation step. CD3 is part of the T cell receptor (TCR) complex. It transmits a specific antigenic stimulation signal, the first signal for T cell activation, by binding to a specific MHC molecule antigen peptide complex on the surface of antigen-presenting cells (APCs). CD28 is a co-stimulatory molecule, mainly expressed on the surface of T cells. It transmits a non-specific co-stimulatory signal, the second signal for T cell activation, by binding to B7 molecules (CD80 / CD86) on the surface of APCs. The second signal is crucial for the complete activation of T cells; it enhances T cell proliferation, promotes the production of cytokines (such as IL-2), and prevents T cells from entering an unresponsive state or undergoing apoptosis. In vitro, the combined use of CD3 and CD28 antibodies to stimulate T cells mimics the dual-signal action of in vivo T cell activation, effectively activating T cells.

[0248] In step (S5), a chimeric antigen receptor (CAR) gene fragment is introduced into T cells using genetic engineering techniques, causing them to express CAR. Lentiviral vectors, with their strong infectivity, can effectively infect T cells and are a commonly used tool. They can integrate foreign genes into the host cell genome, thereby achieving long-term stable gene expression. Viral vectors with CAR structures integrate CAR fragments into T cells, which are then expressed on the cell membrane surface, targeting and recognizing antigens on the surface of target cells (such as tumor cells). Through antigen-antibody interaction, the target cells are cleared. In step (S5), the preferred viral vector is a lentiviral vector. Of course, other vectors can also be used in this invention, as long as they can transduce or transfect T cells and / or NK cells to obtain chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells. The illustrative methods (typically in vitro methods) provided herein include the following steps: transducing activated T cells and / or NK cells with recombinant lentivirus or recombinant lentiviral particles within a closed system, typically in a single chamber of the closed system (also referred to as a reactor, vessel, container, compartment, or receiving container), to produce genetically modified T cells and / or NK cells. The chamber may be flexible or rigid. In the illustrative embodiment, the chamber may be rigid.

[0249] For step (S6), the genetically modified T cells / NK cells are expanded in vitro to obtain a sufficient number of CAR-T / CAR-NK cells for treatment. The expansion process is typically carried out in a cell culture system, including providing suitable growth factors, nutrients, and cytokines. In illustrative embodiments, such methods typically include expanding the genetically modified T cells and / or NK cells in a cell expansion medium within a closed system, typically in a single chamber of the closed system, after transducing T cells and / or NK cells. Activation and transduction are performed within the same closed system without washing the cells between activation and transduction. In illustrative embodiments, activation, transduction, and expansion are performed in the same chamber of the same closed system. Preferably, the cell culture system in step (S6) comprises components selected from the group consisting of serum-free medium, supplemental medium, cytokines, gentamicin sulfate, or combinations thereof. The cell seeding density used during expansion in step (S6) can be, for example, 1 × 10⁻⁶ cells / year. 6 cells / mL, but not limited to this, for example 1×10 3 -1×10 9 cells / mL, 1×10 4 -1×10 8 cells / mL, 1×10 5 -1×10 7 The cell seeding density, such as cells / mL, can also be adjusted according to actual conditions and needs. For example, the number of cells seeded during amplification in step (S6) can be 1.8 × 10⁶. 8 However, this is not the only one; for example, 1×10 5 -1×10 15 1×10 6 -1×10 12 1×10 7 -1×10 10 The number of cells seeded can also be adjusted according to the actual situation and needs.

[0250] For step (S7), it may preferably include a sub-step:

[0251] (S7a) Remove the magnetic beads from the T cells and / or NK cells; and

[0252] (S7b) Clean the T cells and / or NK cells obtained in step (S7a) after removing the magnetic beads to remove impurities. The time for step (S7a) is ≤1.5h. The time for step (S7b) is ≤1h.

[0253] Under the influence of a magnetic field, magnetic beads bound to the target substance are concentrated in a specific area, while unbound impurities can be removed through washing. Finally, by changing or removing the magnetic field, the magnetic beads bound to the target substance can be separated from the solution, thus achieving the removal of the target substance. Cell washing and impurity removal is crucial in cell culture and biopharmaceutical fields, as it relates to the purity and quality of the final product. Cells are washed and liquid replaced through centrifugation, filtration, or washing to remove impurities, minimizing the impurity content of the final product and ensuring its safety.

[0254] Preferably, step (S7) may further include a sub-step: (S7c) detecting the collected T cells and / or NK cells. The detection in step (S7) is similar to the detection in (Sxd) and may include: cell viability detection, cell count detection, cell density detection, cell purity detection, or a combination thereof. Preferably, an automated cell counter is used for cell counting. In a preferred embodiment, an automated cell counter is used... Cell counting is performed using an NC-200 cell counting instrument. The time for step (S7c) is ≤0.5h.

[0255] Additionally, the automated manufacturing process may, as needed, include the following steps:

[0256] (S8) The CAR-immune cells obtained in step (S7) are cryopreserved to obtain a CAR-immune cell cryopreservation formulation. A cell cryopreservation formulation is a preparation used to preserve cells at low temperatures to maintain their activity and function. Cell cryopreservation formulations play a crucial role in cell cryopreservation, protecting cells from ice crystal damage at low temperatures and maintaining cell viability, thereby ensuring successful thawing and continued growth and differentiation when needed. The CAR-ART cells, after washing and liquid replacement, are encapsulated according to the cryopreservation solution formulation ratio and clinical infusion dose, minimizing the contact time between cells and the cryopreservation solution. After encapsulation, the cells are rapidly cooled to -80°C using a programmed cooling device and then transferred to liquid nitrogen for storage. Steps (S8) and (S1)-(S7) are all performed in a closed and sterile automated cell processing system. If the automated preparation process also includes step (Sx), then steps (Sx), (S8), and (S1)-(S7) are all performed in a closed and sterile automated cell processing system. The time for step (S8) is ≤0.5h. One or more of steps (Sx), (S8), and (S1)-(S7) are performed in the same or different chambers of the automated cell processing system. Preferably, one or more of steps (Sx), (S8), and (S1)-(S7) are performed in the same chamber of the automated cell processing system.

[0257] When the automated preparation process includes steps (Sx) and (S8), the total time of the automated preparation process of the present invention is ≤32h. If the automated preparation process does not include step (Sx) but includes step (S8), the total time of the automated preparation process of the present invention is ≤29.5h or ≤30h. If the automated preparation process includes step (Sx) but does not include step (S8), the total time of the automated preparation process of the present invention is ≤31.5h. If the automated preparation process does not include either step (Sx) or step (S8), the total time of the automated preparation process of the present invention is ≤29h or ≤29.5h. Furthermore, step (S1a) is not mandatory, and each detection step can be selectively performed; therefore, the total time of the automated preparation process of the present invention can also be ≤28.5h, ≤28h, ≤27.5h, or ≤27h.

[0258] The automated preparation equipment of the present invention

[0259] This invention provides an automated preparation device for CAR-immune cells, the automated preparation device comprising:

[0260] (M1) Sample loading module, which is configured to provide peripheral blood or apheresis blood samples or PBMC samples;

[0261] (M2) Magnetic bead incubation module, which is configured to: contact the sample in module (M1) with magnetic beads conjugated with CD3 antibody and CD28 antibody and incubate them to obtain a mixture of cells and magnetic beads in the sample;

[0262] (M3) Magnetic bead sorting module, which is configured to magnetically separate and collect T cells and / or NK cells from the mixture obtained from module (M2);

[0263] (M4) Activation module, the activation module being configured to: activate T cells and / or NK cells collected in module (M3) using CD3 and CD28 antibodies conjugated to the magnetic beads;

[0264] (M5) Transduction module, the transduction module being configured to: obtain chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells by using activated T cells and / or NK cells in the viral vector transduction module (M4);

[0265] (M6) Amplification module, configured to: chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells obtained in a cell culture system from amplification module (M5); and

[0266] (M7) Harvesting module, which is configured to collect expanded T cells and / or NK cells in module (M6) to obtain CAR-immune cells;

[0267] The modules (M1)-(M7) are all configured in a closed and sterile automated cell processing system.

[0268] The automated preparation equipment of the present invention is configured to be consistent with the automated preparation process of the present invention and can be used to implement the automated preparation process of the present invention.

[0269] As needed, module (M1) may further include a sub-module: (M1a) a sample concentration module, which is configured to concentrate the peripheral blood, apheresis, or PBMC samples when the volume exceeds the maximum single-processing volume of the automated cell processing system. The unit working time of module (M1a) is ≤0.5h.

[0270] As needed, the automated preparation equipment may further include a module: a (Mx) PBMC separation module, configured to separate peripheral blood mononuclear cells (PBMCs) containing T cells and / or NK cells from the peripheral blood or apheresis sample. Modules (Mx) and (M1)-(M7) are all configured in a closed and sterile automated cell processing system. The unit working time of module (Mx) is ≤2.5h, preferably ≤2h.

[0271] Preferably, module (Mx) may further include sub-modules: (Mxa) a dilution module configured to dilute the peripheral blood or apheresis sample; (Mxb) a centrifugation module configured to perform density gradient centrifugation; and (Mxc) a resuspension module configured to collect and resuspend PBMC cells. Further, module (Mx) may also include a sub-module: (Mxd) a detection module configured to detect the resuspended PBMC cells. The unit working time of module (Mxd) is ≤0.5h. When module (Mx) includes sub-module (Mxd), the unit working time of module (Mx) is ≤2.5h.

[0272] Preferably, the incubation time in module (M2) is 15-40 min, more preferably 20-35 min. The incubation time in module (M2) is ≤0.5 h. The incubation temperature in module (M2) is room temperature. The particle size of the magnetic beads in module (M2) is 1-10 μm, more preferably 2-7 μm, more preferably 3-5 μm, for example 4.5 μm. The incubation process in module (M2) is a dynamic incubation process of static-mixing, with mixing every 4-10 s, more preferably 5-8 s. The dynamic incubation in module (M2) is carried out at a mixing speed of 100-300 rpm / min, more preferably 125-200 rpm / min.

[0273] Preferably, the unit working time of module (M3) is ≤1 hour, more preferably ≤0.5 hours. Module (M3) is generally equipped with a magnetic field for magnetic separation and collection of T cells / NK cells. Module (M3) may also include a sub-module: (M3a) a detection module configured to detect the collected T cells and / or NK cells. The time of module (M3a) is ≤0.5 hours. When module (M3) includes sub-module (M3a), the unit working time of module (M3) is ≤1 hour.

[0274] Preferably, the unit working time of modules (M4)-(M6) is ≤24h. The viral vector in module (M5) is a lentiviral vector. The cell culture system in module (M6) includes components selected from the group consisting of: serum-free culture medium, supplemented culture medium, cytokines, gentamicin sulfate, or combinations thereof. The cell seeding density used for amplification in module (M6) is 1×10⁻⁶. 6 cells / mL, but not limited to this, for example 1×10 3 -1×10 9 cells / mL, 1×10 4 -1×10 8 cells / mL, 1×10 5 -1×10 7 The cell seeding density, such as cells / mL, can also be adjusted according to actual conditions and needs. The cell seeding density used for amplification in module (M6) is 1.8 × 10⁶ cells / mL. 8 However, this is not the only one; for example, 1×10 5 -1×10 15 1×10 6 -1×10 12 1×10 7 -1×10 10 The number of cells seeded can also be adjusted according to the actual situation and needs.

[0275] Preferably, module (M7) may further include sub-modules: (M7a) a magnetic bead removal module configured to remove magnetic beads from the T cells and / or NK cells; and (M7b) a washing module configured to wash the T cells and / or NK cells obtained in module (M7a) after magnetic bead removal to remove impurities. Further, module (M7) may further include a sub-module: (M7c) a detection module configured to detect the collected T cells and / or NK cells. The unit working time of module (M7a) is ≤1.5h. The unit working time of module (M7b) is ≤1h. The unit working time of module (M7c) is ≤0.5h.

[0276] As needed, the device may further include a module: a cryopreservation module (M8), configured to cryopreserve the CAR-immune cells obtained in module (M7) to obtain a CAR-immune cell cryopreservation formulation. Modules (M8) and (M1)-(M7) are all configured in a closed and sterile automated cell processing system. If the device further includes a module (Mx), then modules (Mx), (M8), and (M1)-(M7) are all configured in a closed and sterile automated cell processing system. The unit operating time of module (M8) is ≤0.5h. One or more of modules (Mx), (M8), and (M1)-(M7) are configured to operate in the same or different chambers of the automated cell processing system. Preferably, one or more of modules (Mx), (M8), and (M1)-(M7) are configured to operate in the same chamber of the automated cell processing system.

[0277] The unit working time of the equipment is ≤32h, preferably ≤31.5h, ≤31h, ≤30.5h, ≤30h, ≤29.5h, ≤29h, ≤28.5h, ≤28h, ≤27.5h or ≤27h, etc.

[0278] Typically, the device of the present invention further includes a control module (M9) configured to control the operation of modules (M1)-(M7), or modules (M1)-(M8), or modules (M1), (Mx) and (M2)-(M7), or modules (M1), (Mx) and (M2)-(M8).

[0279] Main advantages of the invention

[0280] This invention provides a method, equipment, and application for obtaining large quantities of CAR-immune cells (preferably CAR-T cells) that meet clinical use standards in a closed and sterile automated cell processing system, using fresh, frozen, or resuscitated apheresis, peripheral blood, or PBMC samples as starting cells, after a short period of activation and transduction (approximately 1-4 days, with the entire process ≤32 hours).

[0281] 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, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0282] Example 1: Small-scale testing and optimization of CAR-T cell preparation process

[0283] Day 0: Frozen apheresis cells were thawed, centrifuged, and washed. Some groups underwent cell sorting and activation, while others were activated directly without sorting. Different cell types were used as starting cells, different activators were used, and cell transduction was performed at different times. Days 1-3: Cell expansion and culture were conducted, with daily samples taken for counting, flow cytometry analysis, etc. See the table below for detailed grouping information:

[0284] Table 1 Group Description

[0285] Group Donor Activator type / brand Transduction time Bead removal time 1BK364-M-A0 BK364 Antibody M Activate synchronous transduction NA 2BK364-M-A24 BK364 Antibody M Transduction after 24 hours of activation NA 3BK364-ACRO-A0 BK364 Antibody ACRO Activate synchronous transduction NA 4BK364-ACRO-A24 BK364 Antibody ACRO Transduction after 24 hours of activation NA 5Z0628-M-A0 Z0628 Antibody M Activate synchronous transduction NA 6Z0628-M-A24 Z0628 Antibody M Transduction after 24 hours of activation NA 7Z0628-ACRO-A0 Z0628 Antibody ACRO Activate synchronous transduction NA 8Z0628-ACRO-A24 Z0628 Antibody ACRO Transduction after 24 hours of activation NA 9BK364-BC-A0 BK364 CD3 / CD28 sorting activation beads Activate synchronous transduction Day 1 10BK364-BC-A0 BK364 CD3 / CD28 sorting activation beads Activate synchronous transduction Day 2 11BK364-BC-A24 BK364 CD3 / CD28 sorting activation beads Transduction after 24 hours of activation Day 2 12BK364-BC-A24 BK364 CD3 / CD28 sorting activation beads Transduction after 24 hours of activation Day 3 13Z0628-A0 Z0628 CD3 / CD28 sorting activation beads Activate synchronous transduction Day 1 14Z0628-A0 Z0628 CD3 / CD28 sorting activation beads Activate synchronous transduction Day 2 15Z0628-A24 Z0628 CD3 / CD28 sorting activation beads Transduction after 24 hours of activation Day 2 16Z0628-BC-A24 Z0628 CD3 / CD28 sorting activation beads Transduction after 24 hours of activation Day 3

[0286] As shown in the table, groups 9 and 10 are equivalent to the same experimental group, with the only difference being the magnetic bead removal time.

[0287] Groups 11 and 12 are equivalent to the same experimental group, differing only in the time required to remove the magnetic beads.

[0288] Groups 13 and 14 are equivalent to the same experimental group, differing only in the time required to remove the magnetic beads.

[0289] Groups 15 and 16 are equivalent to the same experimental group, differing only in the time required to remove the magnetic beads.

[0290] Figures 1-16 Z0628 and BK364 represent two different Donors.

[0291] Figure 1The calculation of cell yield after magnetic bead sorting is as follows: Total Cell yield is the total number of cells after sorting divided by the total number of cells before sorting; CD3+ Cell yield is the total number of cells after sorting multiplied by the percentage of CD3+ cells divided by the total number of cells before sorting multiplied by the percentage of CD3+ cells. The results show that effective sorting can be achieved for different donors, especially for CD3+ cells.

[0292] Figure 2 This method was used to compare cell yield after removing magnetic beads at different time points. The calculation method was: cell density after magnetic bead removal divided by cell count density before magnetic bead removal. In the legend, 9BK364-A0 D1 represents experimental group 9, BK364 is the Donor number; A0 indicates simultaneous activation and transduction, A24 represents activation for 24 hours; D1 represents magnetic bead removal on Day 1, D2 on Day 2, and D3 on Day 3. Other groups follow the same pattern. In groups 9-16, BC represents cells obtained after bead sorting. The results showed that the magnetic bead removal time on Day 1 and Day 2 had no significant impact on cell yield; therefore, magnetic beads can be removed on Day 1 to shorten the process. Furthermore, simultaneous activation and transduction versus transduction after 24 hours of activation also had no significant impact on cell yield; therefore, simultaneous activation and transduction can be performed to shorten the process.

[0293] Figure 3 This represents the amplification fold of antibody groups 1 through 8. The vertical axis represents the amplification fold; the horizontal axis D1 / D0 represents the number of cells in D1 divided by the number of cells in D0, D2 / D0 represents the number of cells in D2 divided by the number of cells in D0, and D3 / D0 represents the number of cells in D3 divided by the number of cells in D0. See Table 1 for the meaning of the symbols in the legend.

[0294] Figure 4 The amplification folds for groups 9-16 sorted by Beads are shown. The vertical axis represents the amplification fold; the horizontal axis D1 / D0 represents the amplification fold of D1 by dividing the number of cells in D1 by the number of cells in D0, D2 / D0 represents the amplification fold of D2 by dividing the number of cells in D0, and D3 / D0 represents the amplification fold of D3 by dividing the number of cells in D0. See Table 1 for the meaning of the symbols in the legend. Comparing the amplification folds of antibody groups 1-8 and Beads sorted groups 9-16, it can be seen that the amplification folds of the Beads sorted groups are higher. The amplification folds of the antibody groups did not exceed 1.0-fold in D1-D3, while the amplification folds of the Beads sorted groups were significantly higher than those of the antibody groups in D1-D3, reaching a maximum of approximately 2.3-2.4-fold above D0. Therefore, Beads are the preferred method for sorting.

[0295] Figure 5The graph shows the percentage of CD3+CAR+ cells in groups 1-16. The vertical axis represents the percentage of CD3+CAR+ cells, and the horizontal axis represents different culture days, with D1 representing day 1 of culture. See Table 1 for the meaning of the symbols in the legend. The results show that the percentage of CD3+CAR+ cells in the Beads sorting groups was superior to that in the antibody groups. Furthermore, group 13 / 14 (Z0628-A0) had the highest percentage of CD3+CAR+ cells. Compared to group 15 / 16 (Z0628-A24) which was transduced 24 hours after activation, group 13 / 14 achieved a higher percentage of CD3+CAR+ cells with simultaneous activation transduction. Therefore, using Beads and employing simultaneous activation transduction are preferred to obtain a higher percentage of CD3+CAR+ cells. The same results were observed in another study.

[0296] Figure 6 This represents the fold increase of CAR+ cells in groups 1-16. The vertical axis represents the fold increase of CAR+ cells, and the horizontal axis represents the ratio of the number of cells at different culture days to the starting number of cells at D0. D1 / D0 represents the ratio of the number of CAR+ cells on Day 1 to the starting number of cells at D0. See Table 1 for the meaning of the symbols in the legend. Figure 5 The results were similar, indicating that the preferred use of Beads and the adoption of activation-synchronized transduction resulted in a higher fold increase in CAR+ cells.

[0297] Figure 7 For representative group 5 ( Figure 7 A), group 6( Figure 7 B), Group 13 / 14 Figure 7 C) and group 15 / 16 ( Figure 7 D) Cell typing (not fully shown due to space limitations). The horizontal axis represents different culture days, and the vertical axis represents the proportion of different cell types measured by flow cytometry. In the legend, T represents CD3+ T cells, NKT represents CD3+CD56+ NKT cells, Monocyte represents CD14+ cells, B represents B cells with CD19+ surface markers, and NK cells with CD3-CD56+ surface markers. The titles represent grouping information, see Table 1 for details. The results show that the Beads group obtained a higher proportion of T cells compared to the antibody group; therefore, Beads are preferred over antibodies in the process flow.

[0298] Figure 8 For representative group 3 ( Figure 8 A), group 4( Figure 8 B), Group 5 Figure 8 C), Group 6 Figure 8 D), Group 9 / 10 Figure 8 E), Group 11 / 12 ( Figure 8 F), Group 13 / 14 Figure 8G) and group 15 / 16 ( Figure 8 The ratio of CD4+ / CD8+ cells in CD3+ T cells (H) is not fully shown due to space limitations. The horizontal axis represents different culture days, and the vertical axis represents the percentage of CD4+ / CD8+ cells in CD3+ T cells. The title is group information, see Table 1 for details. The results show that: Comparison Figure 8 C, 8D and Figure 8 As shown in G and 8H, the Beads group achieved a higher proportion of CD3+CD8+ cells compared to the antibody group in this treatment. However, the CD4+ / CD8+ cell ratio varied considerably depending on the treatment, for example, when comparing... Figure 8 A, 8B and Figure 8 As shown in E and 8F, there was no significant difference in the CD4+ / CD8+ cell ratio between the Beads group and the antibody group in this study.

[0299] In addition, the proportions of cell subtypes Tn, Tscm, Tcm, Tem, and Teff in CD3+ T cells were also detected (not fully shown due to space limitations).

[0300] Figure 9 The graph shows the proportion of CD69+ cells, an activation marker, in CD3+ T cells from groups 1-16. The horizontal axis represents different culture days, and the vertical axis represents the percentage of CD69+ cells among CD3+ T cells. See Table 1 for the legend. The results indicate that the Beads group achieved a higher proportion of CD69+ cells compared to the antibody group, suggesting a stronger activation effect. Therefore, Beads are the preferred choice over antibodies in the process.

[0301] Example 2: Full-scale testing and optimization of CAR-T cell preparation process

[0302] D0 involves thawing one bag of cryopreserved PBMCs, washing the cells on the instrument, and then incubating them with micron-sized magnetic beads. Following the instrument's instructions, different liquid bags are soldered to different tubing. After incubation, magnetic separation is performed to remove negative cells, and positive cells are counted, resuspended, and cultured. Simultaneously, a small-scale experiment is conducted manually while the instrument performs full-scale experiments, and the results of the small-scale and full-scale experiments are compared.

[0303] Figures 10-16 In the text, MANUAL represents the manual testing group, and MACS represents the full-scale experimental instrument testing group.

[0304] Figure 10 The CAR+% graph shows that the vertical axis represents the percentage of CD3+CAR+ cells, and the horizontal axis represents different culture days, with D1 representing day 1 of culture. The comparison shows that the CAR+% in the MACS full-scale experimental instrument testing group was higher than that in the MANUAL manual testing group.

[0305] Figure 11 Flow cytometry for the MANUAL manual test group and the MACS full-scale experimental instrument test group. Figure 11 A represents the MACS full-scale experimental instrumentation test group. Figure 11 B represents the MANUAL manual testing group, which shows the ratio of CD4+ cells to CD8+ cells among all CD45+ leukocytes. The results showed that the proportion of CD8+ cells in the MACS full-scale experimental instrument testing group was slightly higher than that in the MANUAL manual testing group, but the difference was not significant.

[0306] Figure 12 This is a graph showing cell viability versus diameter. The vertical axis represents cell viability (…). Figure 12 A) and diameter ( Figure 12 B), the x-axis represents D0 PBMC, Day 1 MANUAL manual testing group, Day 1 MACS full-scale experimental instrument group, Day 1 MANUAL after BEAD removal (for the Day 1 MANUAL manual testing group), and Day 1 MACS after BEAD removal (for the Day 1 MACS full-scale experimental instrument group). The results show that there was no significant difference in cell viability and diameter between the MACS full-scale experimental instrument group and the MANUAL manual testing group, indicating that the MACS full-scale experimental instrument does not adversely affect cell viability and diameter.

[0307] In addition, the proportions of different cell types in each test group (Z0624PBMC represents Day 0 PBMC of Donor Z0624; ManualBC represents T cells after Day 0 sorting in the Manual group; Manual Supernatant represents negative cells after Day 0 sorting in the Manual group; Manual CAR-T D1 represents CAR-T cells after Day 1 sorting in the Manual group; MACS BC represents T cells after Day 0 sorting in the MACS full-scale experimental instrument group; MACS Supernatant represents negative cells after Day 0 sorting in the MACS full-scale experimental instrument group; MACSD1 CAR-T represents CAR-T cells after Day 1 sorting in the MACS full-scale experimental instrument group) were also analyzed (not fully shown due to space limitations). The proportion of T cells after Day 0 sorting in the MACS full-scale experimental instrument group was slightly higher than that in the Manual group. The proportion of Day 1 CAR-T cells in the MACS full-scale experimental instrument group was slightly higher than that in the Manual group. The percentages of different subtypes of cells (Tn, Tscm, Tcm, Tem, and Teff) in CD3+ T cells were also measured (not fully shown due to space limitations). The CD4+ / CD8+ ratio in each test group was also measured, and there was no significant difference between the results of the MACS full-scale experimental instrument test group and the MANUAL manual test group (not fully shown due to space limitations).

[0308] like Figure 13 , Figure 14 As shown, the proportion of CD25+ / CD69+ cells, which are activation markers, in CD3+ T cells was detected in each test group. Figure 13 The vertical axis represents the percentage of CD69+ cells out of CD3+ T cells. Figure 14 The vertical axis represents the percentage of CD25+ cells among CD3+ T cells. The results showed that the CD25+ / CD69+ ratio in the full-scale experimental instrument group was higher than that in the manual testing group, indicating a higher proportion of T cell activation in the full-scale experimental instrument group.

[0309] like Figure 15 , Figure 16 As shown, the proportions of CD3+ T cells (TIM3+ and PD-1+TIM3+ cells) were detected in each test group. Figure 15 The vertical axis represents the proportion of TIM3+ cells to CD3+ T cells. Figure 16The vertical axis represents the proportion of PD-1+TIM3+ cells to CD3+ T cells. The results showed that the proportions of exhaustion markers TIM3+ and PD-1+TIM3+ cells in the full-scale experimental instrument test group were lower than those in the manual test group, indicating that the full-scale experimental instrument test group had lower T cell exhaustion.

[0310] 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. An automated process for preparing CAR-immune cells, characterized in that, The automated preparation process includes the following steps: (S1) Provide peripheral blood or apheresis blood samples or PBMC samples; (S2) The sample described in step (S1) is contacted with magnetic beads conjugated with CD3 antibody and CD28 antibody and incubated to obtain a mixture of cells and magnetic beads in the sample; (S3) Magnetic separation and collection of T cells and / or NK cells from the mixture obtained in step (S2); (S4) Activate the T cells and / or NK cells collected in step (S3) using the CD3 antibody and CD28 antibody conjugated with the magnetic beads; (S5) Using the T cells and / or NK cells activated in the viral vector transduction step (S4), chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells are obtained. (S6) Expanding the chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells obtained in step (S5) in a cell culture system; and (S7) Collect the expanded T cells and / or NK cells from step (S6) to obtain CAR-immune cells; All steps (S1)-(S7) are performed in a closed and sterile automated cell processing system.

2. The automated preparation process as described in claim 1, characterized in that, The CAR-immune cells are CAR-T cells and / or CAR-NK cells.

3. The automated preparation process as described in claim 1, characterized in that, Step (S1) further includes sub-steps: (S1a) When the volume of peripheral blood, apheresis, or PBMC samples exceeds the maximum single processing volume of the automated cell processing system, the peripheral blood, apheresis, or PBMC samples are concentrated.

4. The automated preparation process as described in claim 1, characterized in that, The automated preparation process further includes a step (Sx) between step (S1) and step (S2): separating peripheral blood mononuclear cells (PBMCs) containing T cells and / or NK cells from the peripheral blood or apheresis sample.

5. The automated preparation process as described in claim 1, characterized in that, The automated preparation process also includes the following steps: (S8) The CAR-immune cells obtained in step (S7) are cryopreserved to obtain a CAR-immune cell cryopreservation preparation.

6. The automated preparation process as described in claim 1, characterized in that, The total time of the automated preparation process is ≤32h, preferably ≤31.5h, ≤31h, ≤30.5h, ≤30h, ≤29.5h, ≤29h, ≤28.5h, ≤28h, ≤27.5h or ≤27h.

7. An automated preparation device for CAR-immune cells, characterized in that, The automated preparation equipment includes: (M1) Sample loading module, which is configured to provide peripheral blood or apheresis blood samples or PBMC samples; (M2) Magnetic bead incubation module, which is configured to: contact the sample in module (M1) with magnetic beads conjugated with CD3 antibody and CD28 antibody and incubate them to obtain a mixture of cells and magnetic beads in the sample; (M3) Magnetic bead sorting module, which is configured to magnetically separate and collect T cells and / or NK cells from the mixture obtained from module (M2); (M4) Activation module, the activation module being configured to: activate T cells and / or NK cells collected in module (M3) using CD3 and CD28 antibodies conjugated to the magnetic beads; (M5) Transduction module, the transduction module being configured to: obtain chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells by using activated T cells and / or NK cells in the viral vector transduction module (M4); (M6) Amplification module, configured to: chimeric antigen receptor (CAR) genetically modified T cells and / or NK cells obtained in a cell culture system from amplification module (M5); and (M7) Harvesting module, which is configured to collect expanded T cells and / or NK cells in module (M6) to obtain CAR-immune cells; The modules (M1)-(M7) are all configured in a closed and sterile automated cell processing system.

8. The automated preparation equipment as described in claim 7, characterized in that, The automated preparation equipment also includes modules: (Mx)PBMC separation module, the PBMC separation module being configured to: separate peripheral blood mononuclear cells (PBMCs) containing T cells and / or NK cells from the peripheral blood or apheresis sample.

9. The automated preparation equipment as described in claim 7, characterized in that, The device also includes modules: (M8) Cryopreservation module, which is configured to: cryopreserve the CAR-immune cells obtained in module (M7) to obtain a CAR-immune cell cryopreservation preparation.

10. Use of an automated CAR-immune cell preparation device as described in any one of claims 7-9, characterized in that, Used to prepare CAR-immune cells.

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