Culture process and quality control method for improving stability of CAR T cells

Abstract

The invention provides a culture process and a quality control method for improving stability of CAR T cells, and relates to the technical field of cell culture.The culture process comprises the following steps that a T cell set is resuspended in a first culture medium, and an activating reagent, a first cell factor and a first costimulatory molecule inhibitor are added; putting into an incubator to obtain a first culture solution; adding polybrene into the virus supernatant, and mixing the virus supernatant and the first culture solution to obtain a mixed solution; centrifuging to obtain a first CAR T cell set; resuspending the first CAR T cell set in a second culture medium; adding a second cytokine and a second co-stimulatory molecule inhibitor; putting into an incubator to obtain a second culture solution; centrifuging the second culture solution to obtain a second CAR T cell set; resuspending the second CAR T cell set in a third culture medium; and adding a third cell factor and a third costimulatory molecule inhibitor into the third culture medium every 2-3 days, and continuously culturing for 7-14 days.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and particularly relates to a culture process and quality control method for improving the stability of CAR T cells. Background Art

[0002] Although chimeric antigen receptor (CAR) T cell therapy has achieved remarkable results in the immunotherapy of tumors, there are still problems with poor stability of CAR T cells. The specific reasons are as follows:

[0003] During in vitro culture, T cells are activated by anti-CD3 / CD28 antibodies or magnetic beads. Strong stimulation may lead to T cell exhaustion. Exhausted CAR T cells have poor persistence in vivo and weakened anti-tumor effects. During expansion, they rely on high concentrations of cytokines such as IL-2. IL-2 inhibits the formation of memory T cells to a certain extent. The lack of memory T cells will cause CAR T cells to be unable to survive and maintain their functions in vivo for a long time. Summary of the Invention

[0004] The purpose of the present invention is to provide a culture process and quality control method for improving the stability of CAR T cells, and the technical problem to be solved is how to improve the stability of CAR T cells.

[0005] The present invention is achieved through the following technical solutions:

[0006] In a first aspect, a culture process for improving the stability of CAR T cells is provided, including the following steps:

[0007] S100. Resuspend the T cell concentrate in the first culture medium, and add an activation reagent, a first cytokine, and a first co-stimulatory molecule inhibitor to the above first culture medium; when the cell density is 1×10 6 cells / mL, place the first culture medium in an incubator and culture for 24 to 48 hours to obtain a first culture solution containing activated T cells;

[0008] S200. Add polybrene to the virus supernatant, and mix the above virus supernatant and the first culture solution containing activated T cells to obtain a mixture;

[0009] S300. Centrifuge the above mixture to obtain a first CAR T cell concentrate;

[0010] S400. Resuspend the above first CAR T cell concentrate in the second culture medium; add a second cytokine and a second co-stimulatory molecule inhibitor to the above second culture medium; when the cell density is 5×10 5 cells / mL, place the second culture medium in an incubator and culture for 24 to 48 hours to obtain a second culture solution containing CAR T cells;

[0011] S500. Centrifuge the second culture solution containing the above CAR T cells to obtain a second CAR T cell aggregate;

[0012] S600. Resuspend the above second CAR T cell aggregate in a third culture medium; add a third cytokine and a third co - stimulatory molecule inhibitor to the above third culture medium every 2 - 3 days, and continuously culture for 7 - 14 days.

[0013] Activate T cells in the above first culture medium through an activation reagent, enabling T cells to enter a proliferative state to prepare for subsequent CAR gene transduction; promote the survival and proliferation of T cells through a first cytokine and enhance the activity of T cells; block the inhibitory signal of T cells through a first co - stimulatory molecule inhibitor to prevent T cells from prematurely entering an exhausted state, thereby maintaining the long - term activity of T cells, and then culture in an incubator for 24 - 48 hours to ensure sufficient activation of T cells while avoiding cell exhaustion caused by over - activation, providing a healthy T cell population for subsequent CAR gene transduction. Add polybrene to the viral supernatant to enhance the interaction between viral particles and the T cell membrane, improve the transduction efficiency of the CAR gene, and ensure that more T cells successfully express the CAR molecule; separate T cells through centrifugation; complete the amplification of CAR T cells in the second culture medium, promote the amplification and survival of CAR T cells through a second cytokine, while maintaining the memory phenotype of T cells and enhancing their long - term survival ability; the second co - stimulatory molecule inhibitor further blocks the inhibitory signal to prevent CAR T cells from entering an exhausted state during the amplification process and maintain their functional stability, and then culture in an incubator for 24 - 48 hours to ensure that CAR T cells maintain high activity and function during the amplification process and avoid a decline in cell function caused by over - amplification. Complete the long - term culture of CAR T cells in the third culture medium, maintain the long - term survival and function of CAR T cells through a third cytokine, promote their differentiation into memory T cells, and enhance persistence; continuously block the inhibitory signal through a third co - stimulatory molecule inhibitor to prevent CAR T cells from entering an exhausted state during the long - term culture process and maintain their anti - tumor activity; continuously culture for 7 - 14 days to ensure that CAR T cells obtain sufficient amplification and functional maturation in vitro while maintaining their stability and persistence, providing high - quality CAR T cell products for subsequent clinical applications.

[0014] Furthermore, the above activation reagent includes one or more of an anti - CD3 antibody, an anti - CD28 antibody, and an anti - CD2 antibody.

[0015] Using a low - concentration activation reagent reduces the exhaustion caused by strong activation of T cells, which helps maintain the long - term activity and function of CAR T cells.

[0016] Furthermore, the first cytokine includes interleukin-7 and interleukin-15;

[0017] After adding the activation agent to the above first culture medium, 10 to 20 parts of interleukin-7 and 10 to 20 parts of interleukin-15 are added.

[0018] The above-mentioned interleukin-7 and interleukin-15 can improve the survival rate of T cells and ensure that more T cells enter the subsequent CAR gene transduction and amplification stage. By promoting the proliferation and functional maintenance of T cells, interleukin-7 and interleukin-15 can ensure that CAR T cells maintain high activity and functional stability during in vitro culture. The combined action of interleukin-7 and interleukin-15 helps CAR T cells differentiate into memory T cells and enhances the long-term survival ability and anti-tumor effect of CAR T cells in the body.

[0019] Furthermore, the first co-stimulatory molecule inhibitor includes a CTLA-4 inhibitor; and 500 to 1000 parts of an activation reagent and 1000 to 3000 parts of a CTLA-4 inhibitor are added to the first culture medium at the same time.

[0020] CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is an inhibitory receptor on the surface of T cells, which can transmit inhibitory signals by binding to CD80 / CD86, limiting the activation and function of T cells. The above-mentioned CTLA-4 inhibitor blocks the inhibitory signals mediated by CTLA-4, enhances the activity of T cells, and delays the exhaustion of T cells; adding activation reagents and CTLA-4 inhibitors to the first culture medium at the same time ensures that T cells can release inhibitory signals while obtaining sufficient activation signals, thereby maintaining the long-term activity and function of T cells.

[0021] Furthermore, the second cytokine includes interleukin-7, interleukin-15 and interleukin-21;

[0022] The above-mentioned second co-stimulatory molecule inhibitor includes a PD-1 inhibitor.

[0023] Furthermore, the specific steps of adding the second cytokine and the second co-stimulatory molecule inhibitor to the second culture medium include:

[0024] Adding 10 to 20 parts of interleukin-7, 10 to 20 parts of interleukin-15 and 10 to 50 parts of interleukin-21 to the second culture medium;

[0025] adding the first CAR T cell set to the second culture medium;

[0026] 1000 to 3000 portions of PD-1 inhibitor are added to the second culture medium.

[0027] By adding the above-mentioned interleukin-7, interleukin-15 and interleukin-21, the survival rate and expansion efficiency of CAR T cells are improved, the differentiation of CAR T cells is promoted, and the number of cells is rapidly increased; the use of PD-1 inhibitors can prevent CAR T cells from entering a state of exhaustion during the expansion process and maintain their long-term activity and function; by promoting the functional differentiation of CAR T cells and relieving inhibitory signals, the anti-tumor activity of CAR T cells is enhanced.

[0028] Furthermore, the third cytokine includes interleukin-7, interleukin-15 and interleukin-21;

[0029] The above-mentioned third co-stimulatory molecule inhibitors include CTLA-4 inhibitors and PD-1 inhibitors.

[0030] Furthermore, 10 to 20 parts of interleukin-7, 10 to 20 parts of interleukin-15, 10 to 50 parts of interleukin-21, 1000 to 3000 parts of CTLA-4 inhibitor and 1000 to 3000 parts of PD-1 inhibitor are added to the third culture medium at the same time.

[0031] During the long-term culture process, interleukin-7, interleukin-15, interleukin-21, CTLA-4 inhibitors and PD-1 inhibitors are added regularly; the above interleukin-7, interleukin-15 and interleukin-21 act synergistically through different signaling pathways to improve the survival rate, functional activity and memory phenotype formation of CAR T cells; the above CTLA-4 inhibitors and PD-1 inhibitors block different inhibitory signaling pathways to synergistically prevent CAR T cells from entering a state of exhaustion during long-term culture, enhance the functional activity and persistence of CAR T cells, and ensure their long-term survival in the body and exert anti-tumor effects.

[0032] A second aspect provides a quality control method for detecting the quality of CAR T cells cultured by the above-mentioned culture process; the quality control method comprises the following steps:

[0033] Preset quality control standards;

[0034] The proportion of CAR-positive cells, stem cell-like memory T cells, and central memory T cells were detected by flow cytometry;

[0035] co-culturing CAR-T cells and target cells to determine the killing rate of the above CAR-T cells;

[0036] The IFN-γ and interleukin-2 secretions of the above CAR-T cells were detected by ELISA;

[0037] Determine whether the above-mentioned CAR-positive cell ratio, stem cell-like memory T cell ratio, central memory T cell ratio, CAR-T cell killing rate, IFN-γ secretion amount, and interleukin-2 secretion amount meet the quality control standards;

[0038] When the above-mentioned CAR-positive cell ratio, stem cell-like memory T cell ratio, central memory T cell ratio, CAR-T cell killing rate, IFN-γ secretion amount, and interleukin-2 secretion amount all meet the quality control standards, qualified CAR T cells are obtained.

[0039] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0040] Activate T cells in the above-mentioned first culture medium, activate T cells through an activation reagent to make T cells enter a proliferative state, and prepare for subsequent CAR gene transduction; promote the survival and proliferation of T cells through the first cytokine and enhance the activity of T cells; block the inhibitory signal of T cells through the first co-stimulatory molecule inhibitor to prevent T cells from prematurely entering an exhausted state, thereby maintaining the long-term activity of T cells, and then culture in an incubator for 24 to 48 hours to ensure sufficient activation of T cells and avoid cell exhaustion caused by over-activation, providing a healthy T cell population for subsequent CAR gene transduction.

[0041] Add polybrene to the virus supernatant to enhance the interaction between virus particles and the T cell membrane, improve the transduction efficiency of the CAR gene, and ensure that more T cells successfully express the CAR molecule.

[0042] Amplify CAR T cells in the second culture medium, promote the amplification and survival of CAR T cells through the second cytokine, while maintaining the memory phenotype of T cells and enhancing their long-term survival ability; further block the inhibitory signal through the second co-stimulatory molecule inhibitor to prevent CAR T cells from entering an exhausted state during the amplification process and maintain their functional stability, and then put them back into the incubator for culture for 24 to 48 hours to ensure that CAR T cells maintain high activity and function during the amplification process and avoid a decrease in cell function caused by over-amplification.

[0043] Perform long-term culture of CAR T cells in the third culture medium, maintain the long-term survival and function of CAR T cells through the third cytokine, promote their differentiation into memory T cells, and enhance persistence; continuously block the inhibitory signal through the third co-stimulatory molecule inhibitor to prevent CAR T cells from entering an exhausted state during long-term culture and maintain their anti-tumor activity; continuously culture for 7 - 14 days to ensure sufficient amplification and functional maturation of CAR T cells in vitro, while maintaining their stability and persistence, and providing high-quality CAR T cell products for subsequent clinical applications. Description of the Drawings

[0044] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the drawings required for the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can be obtained based on these drawings. In the drawings:

[0045] Figure 1 It is a culture process flow chart. Specific embodiments

[0046] To make the purpose, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with the embodiments and the drawings. The illustrative embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0047] First embodiment:

[0048] Combined with Figure 1 , a culture process for improving the stability of CAR T cells includes the following steps:

[0049] Activating T cells: First, resuspend the T cell concentrate in 1 L of the first culture medium (the first culture medium can use LONZA X-VIVO 15 culture medium); then, add 1000 μg of anti-CD3 antibody and 2000 μg of anti-CTLA-4 antibody (i.e., CTLA-4 inhibitor) to the above-mentioned first culture medium; then, add 15 μg of interleukin-7 (IL-7) and 15 μg of interleukin-15 (IL-15); then, adjust the cell density to 1×10 6 cells / mL; finally, place the first culture medium in an incubator and culture for 36 hours to obtain the first culture solution containing activated T cells; wherein, the temperature of the incubator is 37°C and the CO2 concentration is 5%.

[0050] CAR gene transduction: Add polybrene to the virus supernatant and mix it with the activated T cells at a ratio of 1:1, and centrifuge to obtain the first CAR T cell concentrate.

[0051] Expansion of CAR T cells: First, add 15 μg of interleukin-7 (IL-7), 15 μg of interleukin-15 (IL-15), and 30 μg of interleukin-21 (IL-21) to the second culture medium; then, take a portion of the CAR T cells from the first CAR T cell pool and resuspend them in 1 L of the second culture medium (the second culture medium can be LONZA X-VIVO 15 medium); then, add 2000 μg of anti-PD-1 antibody (i.e., PD-1 inhibitor); then, detect the cell density in the second culture medium. When the cell density is 1×10 5 cells / mL, place the second culture medium in an incubator and culture for 36 hours to obtain a second culture solution containing CAR T cells; wherein, the temperature of the above incubator is 37 °C and the CO2 concentration is 5%.

[0052] Long-term culture of CAR T cells: First, take a portion of the CAR T cells from the second CAR T cell pool and resuspend them in 1 L of the third culture medium (the third culture medium can be LONZA X-VIVO 15 medium); then, add 15 μg of interleukin-7 (IL-7), 15 μg of interleukin-15 (IL-15), 30 μg of interleukin-21 (IL-21), 2000 μg of anti-PD-1 antibody, and 2000 μg of anti-CTLA-4 antibody to the third culture medium every 2 days; continuously culture for 10 days.

[0053] Second Embodiment:

[0054] A culture process for improving the stability of CAR T cells, comprising the following steps:

[0055] Activation of T cells: First, resuspend the T cell pool in 1 L of the first culture medium (the first culture medium can be LONZA X-VIVO 15 medium); then, add 500 μg of anti-CD3 antibody, 500 μg of anti-CD28 antibody, and 3000 μg of anti-CTLA-4 antibody (i.e., CTLA-4 inhibitor) to the above first culture medium; then, add 20 μg of interleukin-7 (IL-7) and 20 μg of interleukin-15 (IL-15); then, adjust the cell density to 1×10 6 cells / mL; finally, place the first culture medium in an incubator and culture for 48 hours to obtain a first culture solution containing activated T cells; wherein, the temperature of the incubator is 37 °C and the CO2 concentration is 5%.

[0056] CAR gene transduction: Add polybrene to the viral supernatant and mix it with the activated T cells at a ratio of 1:1, and centrifuge to obtain the first CAR T cell pool.

[0057] Expansion of CAR T cells: First, add 20 μg of interleukin-7 (IL-7), 20 μg of interleukin-15 (IL-15), and 50 μg of interleukin-21 (IL-21) to the second culture medium; then, resuspend a portion of the CAR T cells from the first CAR T cell pool in 1 L of the second culture medium (the second culture medium can be LONZA X-VIVO 15 medium); then, add 3000 μg of anti-PD-1 antibody (i.e., PD-1 inhibitor); then, detect the cell density in the second culture medium. When the cell density is 1×10 5 cells / mL, place the second culture medium in an incubator and culture for 48 hours to obtain a second culture solution containing CAR T cells; wherein, the temperature of the incubator is 37 °C and the CO2 concentration is 5%.

[0058] Long-term culture of CAR T cells: First, resuspend a portion of the CAR T cells from the second CAR T cell pool in 1 L of the third culture medium (the third culture medium can be LONZA X-VIVO 15 medium); then, add 20 μg of interleukin-7 (IL-7), 20 μg of interleukin-15 (IL-15), 50 μg of interleukin-21 (IL-21), 3000 μg of anti-PD-1 antibody, and 3000 μg of anti-CTLA-4 antibody to the third culture medium every 3 days; continuously culture for 14 days.

[0059] The first comparative example:

[0060] Activation of T cells: First, resuspend the T cell pool in 1 L of the first culture medium (the first culture medium can be LONZA X-VIVO 15 medium); then, add 500 μg of anti-CD3 antibody and 500 μg of anti-CD28 antibody to the above first culture medium; then, add 5 μg of interleukin-7 (IL-7) and 5 μg of interleukin-15 (IL-15); then, adjust the cell density to 1×10 6 cells / mL; finally, place the first culture medium in an incubator and culture for 24 hours to obtain a first culture solution containing activated T cells; wherein, the temperature of the incubator is 37 °C and the CO2 concentration is 5%.

[0061] CAR gene transduction: Add polybrene to the virus supernatant and mix it with the activated T cells at a ratio of 1:1. After centrifugation, obtain the first CAR T cell pool.

[0062] Expansion of CAR T cells: First, add 5 μg of interleukin-7 (IL-7), 5 μg of interleukin-15 (IL-15), and 5 μg of interleukin-21 (IL-21) to the second culture medium; then, resuspend a portion of the CAR T cells from the first CAR T cell pool in 1 L of the second culture medium (the second culture medium can be LONZA X-VIVO 15 medium); then, detect the cell density in the second culture medium. When the cell density is 1×10 5 cells / mL, place the second culture medium in an incubator and culture for 24 hours to obtain a second culture solution containing CAR T cells; wherein, the temperature of the incubator is 37 °C and the CO2 concentration is 5%.

[0063] Long-term culture of CAR T cells: First, resuspend a portion of the CAR T cells from the second CAR T cell pool in 1 L of the third culture medium (the third culture medium can be LONZA X-VIVO 15 medium); then, add 5 μg of interleukin-7 (IL-7), 5 μg of interleukin-15 (IL-15), and 5 μg of interleukin-21 (IL-21) to the third culture medium every 2 days; continuously culture for 7 days.

[0064] Second comparative example:

[0065] Activation of T cells: First, resuspend the T cell pool in 1 L of the first culture medium (the first culture medium can be LONZA X-VIVO 15 medium); then, add 500 μg of anti-CD3 antibody, 500 μg of anti-CD28 antibody, and 1000 μg of anti-CTLA-4 antibody to the above first culture medium; then, add 5 μg of interleukin-7 (IL-7) and 5 μg of interleukin-15 (IL-15); then, adjust the cell density to 1×10 6 cells / mL; finally, place the first culture medium in an incubator and culture for 24 hours to obtain a first culture solution containing activated T cells; wherein, the temperature of the incubator is 37 °C and the CO2 concentration is 5%.

[0066] CAR gene transduction: Add polybrene to the viral supernatant and mix it with the activated T cells at a ratio of 1:1. After centrifugation, obtain the first CAR T cell pool.

[0067] Expanding CAR T cells: First, add 5 μg of interleukin-7 (IL-7), 5 μg of interleukin-15 (IL-15), and 5 μg of interleukin-21 (IL-21) to the second culture medium; then, resuspend a portion of the CAR T cells from the first CAR T cell pool in 1 L of the second culture medium (the second culture medium can be LONZA X-VIVO 15 medium); then, add 1000 μg of anti-PD-1 antibody; then, detect the cell density in the second culture medium, and when the cell density is 1×10 5 cells / mL, place the second culture medium in an incubator and culture for 24 hours to obtain a second culture solution containing CAR T cells; wherein, the temperature of the incubator is 37°C and the CO2 concentration is 5%.

[0068] Long-term culture of CAR T cells: First, resuspend a portion of the CAR T cells from the second CAR T cell pool in 1 L of the third culture medium (the third culture medium can be LONZA X-VIVO 15 medium); then, add 5 μg of interleukin-7 (IL-7), 5 μg of interleukin-15 (IL-15), 5 μg of interleukin-21 (IL-21), 1000 μg of anti-PD-1 antibody, and 1000 μg of anti-CTLA-4 antibody to the third culture medium every 2 days; continuously culture for 7 days.

[0069] The third comparative example:

[0070] Activating T cells: First, resuspend the T cell pool in 1 L of the first culture medium (the first culture medium can be LONZA X-VIVO 15 medium); then, add 500 μg of anti-CD3 antibody and 500 μg of anti-CD3 antibody to the above first culture medium; then, add 30 μg of interleukin-7 (IL-7) and 30 μg of interleukin-15 (IL-15); then, adjust the cell density to 1×10 6 cells / mL; finally, place the first culture medium in an incubator and culture for 48 hours to obtain a first culture solution containing activated T cells; wherein, the temperature of the incubator is 37°C and the CO2 concentration is 5%.

[0071] CAR gene transduction: Add polybrene to the viral supernatant and mix it with the activated T cells at a ratio of 1:1, and centrifuge to obtain the first CAR T cell pool.

[0072] Expansion of CAR T cells: First, add 30 μg of interleukin-7 (IL-7), 30 μg of interleukin-15 (IL-15), and 30 μg of interleukin-21 (IL-21) to the second culture medium; then, take a portion of the CAR T cells from the first CAR T cell pool and resuspend them in 1 L of the second culture medium (the second culture medium can be LONZA X-VIVO 15 medium); then, detect the cell density in the second culture medium. When the cell density is 1×10 5 cells / mL, place the second culture medium in an incubator and culture for 48 hours to obtain a second culture solution containing CAR T cells; wherein, the temperature of the above incubator is 37 °C and the CO2 concentration is 5%.

[0073] Long-term culture of CAR T cells: First, take a portion of the CAR T cells from the second CAR T cell pool and resuspend them in 1 L of the third culture medium (the third culture medium can be LONZA X-VIVO 15 medium); then, add 30 μg of interleukin-7 (IL-7), 30 μg of interleukin-15 (IL-15), and 30 μg of interleukin-21 (IL-21) to the third culture medium every 3 days; continuously culture for 14 days.

[0074] The results comparison between the examples and the comparative examples is shown in the following table:

[0075]

[0076]

[0077] Among them, the baseline of the cytokine secretion level refers to the cytokine secretion level under certain conditions. Here, the cytokine secretion amount of the control group is used as the 100% baseline; the cytokine secretion level of the experimental group is expressed as a percentage by comparing with the baseline; the baseline of the inhibitory molecule expression refers to the expression level of the inhibitory molecule under certain conditions. Here, the inhibitory molecule expression amount of the control group is used as the 100% baseline; the inhibitory molecule expression level of the experimental group is expressed as a percentage by comparing with the baseline.

[0078] The first embodiment and the second embodiment improve the amplification multiple, survival rate, and stability of CAR T cells by optimizing the concentrations and combinations of activation reagents, cytokines, and costimulatory molecule inhibitors. Among them, in the first comparative example, due to the lack of costimulatory molecule inhibitors and low cytokine concentration, the amplification multiple of CAR T cells is low, the survival rate is poor, and the stability is insufficient. In the second comparative example, although CTLA-4 inhibitor and PD-1 inhibitor are added, due to the low cytokine concentration, the amplification multiple and survival rate of CAR T cells are still lower than those of the embodiments. In the third comparative example, although high-concentration cytokines are used, due to the lack of costimulatory molecule inhibitors, the inhibitory molecule expression of CAR T cells is high, and the apoptosis rate is high (i.e., the stability is poor). In the first embodiment and the second embodiment, the use of CTLA-4 inhibitor and PD-1 inhibitor effectively reduces the expression of inhibitory molecules and further enhances the function and stability of CAR T cells.

[0079] Third embodiment:

[0080] A quality control method for detecting the quality of CAR T cells cultured by the above-mentioned culture process; the quality control method includes the following steps:

[0081] Preset quality control standards; among them, the above-mentioned quality control standards include the CAR-positive cell ratio threshold, stem cell-like memory T cell ratio threshold, central memory T cell ratio threshold, CAR-T cell killing rate threshold, IFN-γ secretion amount threshold, and interleukin-2 secretion amount threshold;

[0082] Detect the CAR-positive cell ratio, stem cell-like memory T cell ratio, and central memory T cell ratio by flow cytometry;

[0083] Co-culture CAR-T cells and target cells to determine the killing rate of the above-mentioned CAR-T cells;

[0084] Detect the IFN-γ secretion amount and interleukin-2 secretion amount of the above-mentioned CAR-T cells by ELISA;

[0085] Judge whether the CAR-positive cell ratio, stem cell-like memory T cell ratio, central memory T cell ratio, CAR-T cell killing rate, IFN-γ secretion amount, and interleukin-2 secretion amount meet the quality control standards;

[0086] When the CAR-positive cell ratio, stem cell-like memory T cell ratio, central memory T cell ratio, CAR-T cell killing rate, IFN-γ secretion amount, and interleukin-2 secretion amount all meet the quality control standards, qualified CAR T cells are obtained.

[0087] The values of each index can be determined by existing methods as follows:

[0088] The specific method for detecting the proportion of CAR-positive cells is as follows: Collect CAR-T cells after long-term culture, place the CAR-T cells in phosphate-buffered saline, and adjust the cell density to 1×106 cells / mL; Take 100 μL of the cell suspension and add it to a flow tube; Add an anti-CAR antibody (such as anti-CD19-CAR antibody) to the above flow tube, and incubate in the dark for 30 minutes; Wash twice with phosphate-buffered saline to remove unbound antibodies; Resuspend the cells in 200 μL of phosphate-buffered saline and perform on-machine detection; Use a flow cytometer to analyze the proportion of CAR-positive cells. When the proportion of CAR-positive cells > 70%, it meets the quality control standard.

[0089] The specific method for detecting the killing rate of CAR-T cells is as follows: Prepare target cells (such as NALM-6 or Raji cells) and adjust the cell density to 1×105 cells / mL; Co-culture CAR-T cells and target cells at an effector-to-target ratio of 10:1 in a 96-well plate, and add 100 μL of culture medium to each well; Set up a group with only target cells (negative control) and a group with only CAR-T cells (background control). Place the 96-well plate in an incubator with 5% CO2 at 37°C for 4 - 6 hours; Use a lactate dehydrogenase (LDH) release kit or a fluorescent dye (such as Calcein-AM) to detect the death of target cells; Read the data through an enzyme-linked immunosorbent assay (ELISA) reader or a fluorescence microscope and calculate the killing rate of CAR-T cells; When the killing rate > 50%, it meets the quality control standard.

[0090] The specific method for detecting the IFN-γ secretion and interleukin-2 secretion of CAR-T cells is as follows: Collect the supernatant of the co-culture of CAR-T cells and target cells, and centrifuge to remove cell debris; Use IFN-γ and IL-2 ELISA kits, add the supernatant to the pre-coated ELISA plate, and incubate for 2 hours; Add the detection antibody and the enzyme-labeled secondary antibody, and incubate for 1 hour; Add the substrate for color development, use an ELISA reader to read the absorbance, and obtain the secretion of IFN-γ and IL-2; When the IFN-γ secretion > 1000 pg / mL, it meets the quality control standard; When the IL-2 secretion > 500 pg / mL, it meets the quality control standard.

[0091] The specific methods for detecting the proportions of stem cell-like memory T cells and central memory T cells in CAR-T cells are as follows: Collect CAR-T cells and adjust the cell density to 1×106 cells / mL; Take 100 μL of the cell suspension (about 1×105 cells) and add it to a flow tube; Add anti-CD45RA-FITC and anti-CD62L-PE antibodies (1 μg / mL), and incubate in the dark for 30 minutes. Wash twice with phosphate buffered saline to remove unbound antibodies; Resuspend the cells in 200 μL of phosphate buffered saline and perform on-machine detection; Analyze the proportions of stem cell-like memory T cells (CD45RA+CD62L+(Tscm)) and central memory T cells (CD45RO+CD62L+(Tcm)). When the proportion of CD45RA+CD62L+(Tscm) > 10%, it meets the quality control standard; When the proportion of CD45RO+CD62L+(Tcm) > 20%, it meets the quality control standard.

[0092] The specific embodiments described above further elaborate on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A culture process for improving the stability of CAR T cells, characterized in that: The following steps are involved: S100, resuspending the T cell set in a first culture medium, adding an activation reagent, a first cytokine and a first co-stimulatory molecule inhibitor to the first culture medium; when the cell density is 1×10 6 cells / mL, placing the first culture medium in an incubator and culturing for 24 to 48 hours to obtain a first culture fluid containing activated T cells; S200, adding polybrene to the viral supernatant, mixing the viral supernatant and the first culture medium containing activated T cells to obtain a mixed solution; S300, centrifuging the mixed solution to obtain a first CAR T cell set; S400, resuspending the first CAR T cell set in a second culture medium; adding a second cytokine and a second co-stimulatory molecule inhibitor to the second culture medium; when the cell density is 5×10 5 cells / mL, placing the second culture medium in an incubator and culturing for 24 to 48 hours to obtain a second culture fluid containing CAR T cells; S500, centrifuging the second culture solution containing the CAR T cells to obtain a second CAR T cell set; S600, resuspending the second CAR T cell set in a third culture medium; adding a third cytokine and a third co-stimulatory molecule inhibitor to the third culture medium every 2 to 3 days, and continuing the culture for 7 to 14 days.

2. The culture process according to claim 1, characterized in that: The activation reagent includes one or more of anti-CD3 antibody, anti-CD28 antibody and anti-CD2 antibody.

3. The culture process according to claim 1, characterized in that: The first cytokines include interleukin-7 and interleukin-15; After adding the activation agent to the first culture medium, 10 to 20 parts of interleukin-7 and 10 to 20 parts of interleukin-15 are added.

4. The culture process according to claim 1, characterized in that: The first co-stimulatory molecule inhibitor includes a CTLA-4 inhibitor; and 500 to 1000 parts of an activation reagent and 1000 to 3000 parts of a CTLA-4 inhibitor are added to the first culture medium at the same time.

5. The culture process according to claim 1, characterized in that: The second cytokines include interleukin-7, interleukin-15 and interleukin-21; The second co-stimulatory molecule inhibitor includes a PD-1 inhibitor.

6. The culture process according to claim 5, characterized in that: The specific steps of adding the second cytokine and the second co-stimulatory molecule inhibitor to the second culture medium include: adding 10 to 20 parts of interleukin-7, 10 to 20 parts of interleukin-15 and 10 to 50 parts of interleukin-21 to the second culture medium; adding a first CAR T cell set to the second culture medium; 1000 to 3000 portions of PD-1 inhibitor are added to the second culture medium.

7. The culture process according to claim 1, characterized in that: The third cytokine includes interleukin-7, interleukin-15 and interleukin-21; The third co-stimulatory molecule inhibitor includes a CTLA-4 inhibitor and a PD-1 inhibitor.

8. The culture process according to claim 7, characterized in that: At the same time, 10 to 20 parts of interleukin-7, 10 to 20 parts of interleukin-15, 10 to 50 parts of interleukin-21, 1000 to 3000 parts of CTLA-4 inhibitor and 1000 to 3000 parts of PD-1 inhibitor are added to the third culture medium.

9. A quality control method, characterized in that: The quality control method is used to detect the quality of CAR T cells cultured by the culture process according to any one of claims 1 to 8; the quality control method comprises the following steps: Preset quality control standards; The proportion of CAR-positive cells, stem cell-like memory T cells, and central memory T cells were detected by flow cytometry; co-culturing CAR-T cells and target cells, and determining the killing rate of the CAR-T cells; The IFN-γ secretion and interleukin-2 secretion of the CAR-T cells were detected by ELISA; Determine whether the CAR-positive cell ratio, stem cell-like memory T cell ratio, central memory T cell ratio, CAR-T cell killing rate, IFN-γ secretion, and interleukin-2 secretion meet the quality control standards; When the CAR-positive cell ratio, stem cell-like memory T cell ratio, central memory T cell ratio, CAR-T cell killing rate, IFN-γ secretion and interleukin-2 secretion all meet the quality control standards, qualified CAR T cells are obtained.

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