A method for improving the killing ability of CAR-T cells by shear treatment

Abstract

The present application relates to a method for enhancing the performance of CAR-T cells, specifically, comprising the step of: performing a shear treatment on the stimulated CAR-T cells. The CAR-T cells treated by the method of the present application have high proliferation activity, low exhaustion index, good memory activity, and excellent short-term and long-term tumor cell killing effect. In practical applications, especially in the treatment of solid tumors, the CAR-T cells treated by the method of the present application have excellent effects.

Description

Technical Field

[0001] This invention relates to cell immunotherapy, and more specifically to a method for enhancing the killing ability of CAR-T cells through cleavage processing. Background Technology

[0002] CAR-T cells (chimeric antigen receptor T cells), as an innovative immunotherapy, have achieved significant results in cancer treatment in recent years. However, CAR-T cells may face several challenges in the human bloodstream. First, due to the complexity of the blood environment, CAR-T cells may be attacked by the immune system, leading to decreased cell survival. Second, the immunosuppressive microenvironment within solid tumors may inhibit CAR-T cell migration and infiltration, reducing their anti-tumor effects. Furthermore, continuous antigen stimulation may lead to CAR-T cell depletion, manifested as decreased cell proliferation and weakened cytotoxic activity. Simultaneously, CAR-T cell activation may release large amounts of cytokines, triggering side effects such as cytokine release syndrome. Finally, the preparation process of CAR-T cells is complex, and the infusion method may also affect its efficacy and safety.

[0003] Shear force is a mechanical force exerted on cells during blood flow, originating from the interaction between blood flow, the blood vessel wall, and cells. For CAR-T cells, the effects of shear force are mainly manifested in two aspects: physical damage to cells and alterations in cell function.

[0004] Therefore, there is an urgent need in the field for a method to enhance the performance of CAR-T cells, improve their stability and anti-tumor effects, and reduce the risk of side effects in complex environments such as blood. Summary of the Invention

[0005] The purpose of this invention is to provide a method for enhancing the killing ability of CAR-T cells by treating them with shear force.

[0006] A first aspect of the present invention provides a method for enhancing CAR-T cell performance, comprising the steps of:

[0007] (s1) Provide T cells, culture the T cells under suitable culture conditions for a period of time t1, and activate and transfect the T cells during the t1 culture period to obtain activated and transfected CAR-T cells, where t1 is 1 to 3 days;

[0008] (s2) The activated and transfected CAR-T cells were cultured for t2 days under suitable culture conditions and without shearing, where t2 was 2 to 8 days.

[0009] (s3) The CAR-T cells are subjected to shearing treatment for t3 hours, where t3 is 0.5–6 hours, and the shearing stress is 1–6 dyn / cm.2 CAR-T cells were obtained after cleavage treatment;

[0010] (s4) The cleaved CAR-T cells were cultured for t4 days under suitable culture conditions without shearing, where t4 was 2–8 days; and

[0011] (s5) Harvest the CAR-T cells.

[0012] In another preferred embodiment, step (s1) further includes removing activating factors and viruses from activated and transfected CAR-T cells.

[0013] In another preferred embodiment, the activation and transfection processes are performed simultaneously or separately.

[0014] In another preferred embodiment, in step (s1), the activated and transfected CAR-T cells are free of activating factors and viruses.

[0015] In another preferred embodiment, in step (s3), t3 is 0.5 to 5 hours, more preferably 0.5 to 3 hours, more preferably 0.5 to 2 hours, for example, about 1 hour.

[0016] In another preferred embodiment, in step (s2), t2 is 3 to 7 days, more preferably 4 to 6 days, for example, about 5 days.

[0017] In another preferred embodiment, in step (s3), the shear stress is 2–6 dyn / cm. 2 Optimal ratio is 3-6 dyn / cm 2 More preferably 3-6 dyn / cm 2 For example, approximately 5 dyn / cm 2 .

[0018] In another preferred embodiment, the method further includes step (s3) being repeated 0 to 3 times, more preferably 0 to 2 times, and even more preferably 0 to 1 times.

[0019] In another preferred embodiment, in step (s3), the density of CAR-T cells during the cleavage treatment is 1 × 10⁻⁶. 4 ~1×10 8 cells / mL, preferably 1×10 5 ~1×10 7 cells / mL, preferably 5 × 10⁻⁶ 5 ~5×10 6 cells / mL, for example, about 1×10 6 per mL.

[0020] In another preferred embodiment, the CAR-T cells are derived from peripheral blood, preferably peripheral blood mononuclear cells.

[0021] In another preferred embodiment, step (s1) includes:

[0022] (s1a) Provide T cells, add activating factors to the T cells under suitable culture conditions to activate the T cells, and culture the activated T cells for 0.5 to 1 day;

[0023] (s1b) CAR-T cells were obtained by transfecting the activated T cells with the virus and culturing them for 0.5–1 day; and

[0024] (s1c) Remove the activating factors and virus from the CAR-T cells to obtain activated and transfected CAR-T cells.

[0025] In another preferred embodiment, in step (s1), the T cells are activated using an activating factor, preferably CD3 / CD28.

[0026] In another preferred embodiment, in step (s1), the T cells are transfected with a virus selected from the group consisting of lentiviruses, adenoviruses, adeno-associated viruses, retroviruses, or combinations thereof, more preferably MSLN virus.

[0027] In another preferred embodiment, a step of changing the CAR-T cell culture medium is included between steps (s1b) and (s1c).

[0028] In another preferred embodiment, t4 is 3 to 7 days, more preferably 4 to 6 days, for example, about 5 days.

[0029] In another preferred embodiment, the suitable culture conditions include a CAR-T basal medium, preferably an X-VIVO medium comprising Gibco fetal bovine serum and IL-2.

[0030] In another preferred embodiment, the Gibco fetal bovine serum content in the X-VIVO medium is 1-10% (w / w), more preferably 3-8%, more preferably 4-6%, for example, about 5%.

[0031] In another preferred embodiment, the amount of IL-2 in the X-VIVO medium is 50 to 500 IU, more preferably 70 to 300 IU, more preferably 90 to 150 IU, for example, about 100 IU.

[0032] In another preferred embodiment, the suitable conditions for cultivation include 25–45°C, more preferably 28–40°C, and even more preferably 30–40°C, for example, about 37°C.

[0033] In another preferred embodiment, the suitable conditions for cultivation include 1% to 10% CO2, more preferably 3% to 8% CO2, and even more preferably 4% to 6% CO2.

[0034] In another preferred embodiment, the method is in vitro.

[0035] In a second aspect, the present invention provides CAR-T cells obtained using the method described in the first aspect of the present invention.

[0036] In another preferred embodiment, the ratio (Z1 / Z0) of the activation rate (Z1) of the CAR-T cells to that of CAR-T cells (Z0) not treated by the method of the present invention is ≥1.2, preferably ≥1.5, and more preferably ≥2.0.

[0037] In another preferred embodiment, the ratio (X1 / X0) of the exhaustion rate (X1) of the CAR-T cells to that of CAR-T cells (X0) not treated by the method of the present invention is ≤0.8, preferably ≤0.5, and more preferably ≤0.4.

[0038] In another preferred embodiment, the ratio (Y1 / Y0) of the central memory phenotype (Y1) of the CAR-T cells to that of CAR-T cells (Y0) not treated by the method of the present invention is ≥1.1, preferably ≥1.3, and more preferably ≥1.5.

[0039] In another preferred embodiment, the ratio (N1 / N0) of the CAR-T cell lysis rate (N1) to that of CAR-T cells not treated by the method of the present invention is ≥1.5, preferably ≥1.8, and more preferably ≥2.0.

[0040] A third aspect of the present invention provides a method for inhibiting the progression of cancer cells, comprising the steps of:

[0041] By contacting CAR-T cells as described in the second aspect of the present invention with cancer cells, cancer cell progression can be inhibited.

[0042] In another preferred embodiment, the ratio of CAR-T cells to cancer cells is 1:1 to 5, for example, about 1:2 or 1:4.

[0043] In another preferred embodiment, the cancer is a solid tumor.

[0044] In another preferred embodiment, the cancer cells are selected from the group consisting of ovarian cancer cells, breast cancer cells, cervical cancer cells, glioma cells, clear cell renal adenocarcinoma cells, or combinations thereof; preferably ovcar3-luc cells, Hela-luc cells, 1806-luc cells, 786-0-luc cells, u87-luc cells, or combinations thereof.

[0045] In another preferred embodiment, the CAR-T cells are exposed to cancer cells for 5 to 40 hours, more preferably 10 to 30 hours, more preferably 20 to 30 hours, for example, about 24 hours.

[0046] In another preferred embodiment, the method is in vitro.

[0047] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0048] 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

[0049] Figure 1 A schematic diagram of the process for cutting and processing CAR-T cells is shown.

[0050] Figure 2 The performance of CAR-T cells after shear stress treatment is shown. A: Cell viability (%); B-C: Activation rate; D-E: Exhaustion rate; F: T SCM (%); G:T EM (%); H~J: lysis rate of tumor cells (%).

[0051] Figure 3 The effect of shear stress treatment time on the performance of CAR-T cells is shown. A: Effect of different treatment days on CAR-T cells; B-J: Performance of CAR-T cells treated with shear stress on day 7, where B-C: activation rate; D-E: exhaustion rate; F: T SCM (%); G:T EM (%); H~J: lysis rate of tumor cells (%).

[0052] Figure 4 The effects of the method of the present invention on CAR-T cells are shown in comparison with existing technologies. A: Cell viability (%); B-C: Activation rate; D-E: Exhaustion rate; F: T SCM (%); G:T EM (%); H~J: lysis rate of tumor cells (%). Detailed Implementation

[0053] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors have unexpectedly discovered for the first time a method to enhance the performance of CAR-T cells. This method includes the steps of: removing activating factors and viruses from activated and transfected CAR-T cells, and then subjecting them to a shearing treatment with a shear stress of 1–6 dyn / cm. 2The shear stress in the method of this invention is similar to the shear stress in venous blood in vivo. Experiments show that CAR-T cells treated by the method of this invention have high proliferative activity, low exhaustion index, good memory activity, and excellent short-term and long-term killing effects on tumor cells. Based on this, this invention was completed.

[0054] the term

[0055] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0056] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0057] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0058] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0059] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0060] The method of the present invention

[0061] The method of the present invention can enhance the performance of CAR-T cells, comprising the following steps:

[0062] (s1) Provide T cells, culture the T cells under suitable culture conditions for a period of time t1, and activate and transfect the T cells during the t1 culture period to obtain activated and transfected CAR-T cells, where t1 is 1 to 3 days;

[0063] (s2) The activated and transfected CAR-T cells were cultured for t2 days under suitable culture conditions and without shearing, where t2 was 2 to 8 days.

[0064] (s3) The CAR-T cells are subjected to shearing treatment for t3 hours, where t3 is 0.5–6 hours, and the shearing stress is 1–6 dyn / cm. 2 CAR-T cells were obtained after cleavage treatment;

[0065] (s4) The cleaved CAR-T cells were cultured for t4 days under suitable culture conditions without shearing, where t4 was 2–8 days; and

[0066] (s5) Harvest the CAR-T cells.

[0067] In a preferred embodiment, in step (s3), t3 is 0.5 to 5 hours, more preferably 0.5 to 3 hours, and even more preferably 0.5 to 2 hours, for example, about 1 hour. In a preferred embodiment, in step (s2), t2 is 3 to 7 days, more preferably 4 to 6 days, for example, about 5 days.

[0068] In a preferred embodiment, in step (s3), the shear stress is 2–6 dyn / cm. 2 Optimal ratio is 3-6 dyn / cm 2 More preferably 3-6 dyn / cm 2 For example, approximately 5 dyn / cm 2 .

[0069] In a preferred embodiment, in step (s3), the density of CAR-T cells during the cleavage treatment is 1×10⁻⁶. 4 ~1×10 8 cells / mL, preferably 1×10 5 ~1×10 7 cells / mL, preferably 5 × 10⁻⁶ 5 ~5×10 6 cells / mL, for example, about 1×10 6 per mL.

[0070] In a preferred embodiment, the CAR-T cells are derived from peripheral blood, preferably peripheral blood mononuclear cells.

[0071] In the method of the present invention, the device that provides shear force to CAR-T cells is not limited and can be a peristaltic pump, such as the peristaltic pump shown in CN 2024306153043.

[0072] CAR-T cells obtained using the method of this invention exhibit high proliferation activity, low exhaustion index, good memory activity, and good short- and long-term killing performance. The activation rate (Z1) of CAR-T cells obtained using the method of this invention, compared to CAR-T cells (Z0) not treated by this invention, has a ratio (Z1 / Z0) ≥ 1.2, preferably ≥ 1.5, and more preferably ≥ 2.0. The exhaustion rate (X1) of CAR-T cells obtained using the method of this invention, compared to CAR-T cells (X0) not treated by this invention, has a ratio (X1 / X0) ≤ 0.8, preferably ≤ 0.5, and more preferably ≤ 0.4. The central memory phenotype (Y1) of CAR-T cells obtained using the method of this invention, compared to CAR-T cells (Y0) not treated by this invention, has a ratio (Y1 / Y0) ≥ 1.1, preferably ≥ 1.3, and more preferably ≥ 1.5. The lysis rate (N1) of CAR-T cells obtained by the method of the present invention is ≥1.5, preferably ≥1.8, and more preferably ≥2.0, compared with that of CAR-T cells (N0) not treated by the method of the present invention.

[0073] The main advantages of this invention include:

[0074] 1. CAR-T cells treated using the method of this invention exhibit high proliferative activity, low exhaustion index, good memory activity, and good short- and long-term killing performance.

[0075] 2. The method of the present invention simulates the shear force in venous blood in the human body, thereby enhancing the effect of CAR-T cells in practical applications.

[0076] 3. In practical applications, especially in the treatment of solid tumors, CAR-T cells treated by the method of this invention have excellent effects.

[0077] 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 by weight.

[0078] Example 1: Preparation of CAR-T cells

[0079] 1.1 T cell sorting experiment:

[0080] Remove a cryovial containing 5E7 PBMC cells from the liquid nitrogen tank and thaw it in a 37°C water bath. Place a new sterile cryovial in a magnetic rack and add 1 ml of X-VIVO medium (containing 5% Gibco fetal bovine serum and 100 IU IL-2; the X-VIVO medium is the same throughout). Then, take a 15 ml centrifuge tube, add 4 ml of X-VIVO medium, transfer the thawed PBMC cells to the 15 ml centrifuge tube, centrifuge (500×g, 5 min), discard the supernatant, add 50 μl of Stemcell easysep separation antibody and 800 μl of X-VIVO medium, incubate for 5 min, add 50 μl of magnetic separation beads, resuspend the cells, and transfer them to the prepared cryovial. Mix well and incubate for 3 min.

[0081] After incubation, take a 15ml centrifuge tube, add 7ml of culture medium, and transfer the cells from the cryopreservation tube to the centrifuge tube. Centrifuge at 500×g for 5min and count the cells. After counting, centrifuge again (500×g, 5min), discard the supernatant, and add 100μl of activating magnetic beads and 50μl of culture medium per 1E7 cells, according to the counting results. Resuspend the cells several times by pipetting, and incubate in an incubator for 7min. After incubation, repeat the incubation three times. After incubation, culture the cells in a T25 flask at a ratio of 2E6 cells / ml of culture medium.

[0082] The T cells cultured overnight should be used promptly for lentivirus transfection the next day to construct CAR-T cells.

[0083] 1.2 Construction of CAR-T cells:

[0084] 293T cells intended for virus packaging were cultured in a cell culture incubator at 37°C and 5% CO2. The culture medium used was DMEM containing 10% Gibco fetal bovine serum. The day before the actual virus packaging, the cultured 293T cells were passaged three times in T75 cell flasks at a cell density of 1E7. Lentiviral packaging began when the 293T cells reached 70-80% confluence and were evenly distributed in the culture flasks.

[0085] Prepare plasmid and transfection reagent dilution solutions by vortexing the PEI 40K transfection reagent. Prepare two centrifuge tubes and prepare plasmid and transfection reagent dilution solutions in the following order.

[0086] Table 1

[0087]

[0088] Mix thoroughly. Add the transfection reagent dilution (centrifuge tube 2) dropwise to the plasmid DNA solution (centrifuge tube 1), mixing thoroughly after each addition. The order of addition is crucial. Incubate the transfection mixture at room temperature for 15–20 minutes.

[0089] Discard the old culture medium in the culture flask containing the 293T cells, wash once with PBS, and add 9 ml of fresh DMEM medium. Then add 1 ml of the incubated transfection mixture (carefully and slowly add dropwise from the opposite side of the cells to prevent them from floating). Incubate the cells at 37°C and 5% CO2 for 6 hours, then discard the culture medium in the flask and add 15 ml of fresh culture medium to continue culturing.

[0090] Forty-eight hours post-transfection, the cell culture supernatant was collected for the first time, and 15 ml of fresh culture medium was added for continued culturing. A second collection of cell culture supernatant was conducted 72 hours post-transfection, yielding a total of 30 ml. The supernatant was centrifuged at 1000×g for 8 min, then filtered through a 0.45 nm pore size filter to remove cell debris, and transferred to ultracentrifuge tubes. After centrifugation at 22,000 rpm for 2 h at 4°C, the virus location was immediately marked on the tube, and the supernatant was aspirated and discarded. The virus pellet was resuspended in 200 μl of X-VIVO medium, transferred to EP tubes, aliquoted, and stored overnight at 4°C. Storage at 4°C is recommended for one week; for longer storage, storage at 4°C overnight followed by storage at -80°C is recommended.

[0091] Remove 1E6 T cells isolated the previous day (as described above) from the incubator, add 50 μl of virus resuspended solution, and incubate at 37°C for 24 h. After 24 h, aspirate all cells, centrifuge, change the medium, and continue culturing. The cultured cells are the desired CAR-T cells.

[0092] 1.3 Cell Culture Experiments:

[0093] CAR-T cells were cultured normally in X-VIVO medium containing 5% Gibco fetal bovine serum and 100 IU of IL-2 for subsequent confirmatory experiments.

[0094] Example 2 Shear Force Treatment

[0095] Place the peristaltic pump in a sterile operating table. First, sterilize the peristaltic pump with UV light for 10 minutes. Start the peristaltic pump and add 75% alcohol to each of the four 1ml nozzles for 5 minutes of circulation sterilization. Then collect and discard the alcohol. Next, add sterile deionized water for 5 minutes of circulation rinsing. Collect and discard the rinse water. Set the shear stress to 5 dyn / cm. 2 .

[0096] Dilute the cells to be processed with the appropriate culture medium to a density of 1E6 / ml. Take 4ml of cells and add them to four 1ml pipette tips. Then remove air bubbles. After removing the air bubbles, keep the four tips at the same height and immerse them in the liquid. Then plug the tips with sterile cotton. Then transfer the peristaltic pump to a cell culture incubator at 37°C and 5% CO2 and start the cycle timing.

[0097] After circulation, transfer the peristaltic pump back to a sterile operating table, discard the cotton, collect the circulating cells, centrifuge them at 500g for 5 minutes, and then replace with fresh culture medium for further culture. Next, add 75% alcohol to the four 1ml pipette tips of the peristaltic pump for 5 minutes of circulation sterilization, then collect and discard the alcohol. Then add sterile deionized water for 5 minutes of circulation rinsing, then collect and discard the rinse water, and finally turn off the peristaltic pump.

[0098] Example 3 Detection Method

[0099] 3.1 Cell viability

[0100] Trypan blue is a cell viability dye commonly used for cell counting and cell viability assays. It can cross the cell membrane to enter the cell and bind to intracellular nucleic acids, turning the cell blue. For cells that have lost viability or have incomplete cell membranes, the cell membrane permeability increases, allowing trypan blue dye to enter and stain the cells blue. The cell concentration can be determined by observing the number of blue-stained cells. To test cell viability, take 10 μl of cells, add 10 μl of trypan blue dye (0.2%), and gently mix with a pipette. Add 20 μl of the mixture to a counting chamber and use a Countstar cell counter to measure cell viability according to the cell counter's operating instructions.

[0101] 3.2 Phenotypic Changes of CAR-T Cells

[0102] The cells to be tested were divided into multiple samples, each containing 5E5 cells. After centrifugation, the supernatant was discarded, and the cells were washed with PBS, centrifuged again, and resuspended in 100 μl of PBS. 2 μl of specific antibodies were then labeled for each sample, such as cell activation markers (CD25, CD69), cell exhaustion markers (PD-1, LAG-3), and memory cell markers (CD45RO, CD62L). The labeled cells were incubated in the dark for 20 min, then washed again with PBS to remove unbound antibodies and resuspended in 200 μl of PBS. The fluorescence intensity of each marker was detected using flow cytometry, and the data were recorded and analyzed to assess changes in CAR-T cell activation, exhaustion, and memory phenotypes.

[0103] 3.3 Detection of CAR-T cell-mediated tumor cell killing

[0104] Tumor cells 1806-luc were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) to the logarithmic growth phase. Cells were then digested with 0.05% trypsin and counted to ensure cell viability was above 90%. 1806-luc cells were added to 96-well plates according to the required sample volume, 2E4 cells per well (100 μl volume), and incubated in a normoxic incubator for 24 h. After discarding the original medium, the suspension concentration of CAR-T cells and fluid shear force-treated CAR-T cells was adjusted according to different positive rates at effector-to-target ratios of 1:2 and 1:4. The total volume should always be 100 μl. The co-incubated cells were then placed in a cell culture incubator (37℃, 5% CO2) for 24 h.

[0105] A 1:4 effector-to-target ratio was used for short-acting cell killing. After 24 hours of stimulation, the supernatant was discarded. 100 μl of reagent E605A from the ONE-Glo Luciferase Assay System was added to each well, mixed thoroughly, and incubated at room temperature in the dark for 10 minutes. 100 μl of the solution from the cell culture plate was then transferred horizontally into 96-well white flat-bottomed plates. A multi-mode microplate reader and software were turned on, and the Luminescence mode was selected for plate layout. The 96-well white flat-bottomed plates were placed in the microplate reader to read the data, which was then exported and saved for calculating the cell killing rate. Cell killing rate = (sample luminescence value - background luminescence value) / (control group luminescence value - background luminescence value) * 100%.

[0106] A 1:2 effector-to-target ratio was used for long-lasting cell killing. Stimulation was repeated three times, with each round lasting 24 hours. After each round, cells from each well were transferred to the next round of tumor cells for a second round of co-incubation. 100 μl of the E605A reagent from the ONE-Glo Luciferase Assay System was added to each well of the plate, mixed thoroughly by pipetting, and incubated at room temperature in the dark for 10 minutes. 100 μl of the solution from the cell culture plate was then transferred horizontally into a 96-well white plate. A multi-mode microplate reader and software were turned on, and the Luminescence mode was selected for plate layout. The 96-well white plate was placed in the microplate reader to read the data, which was then exported and saved for calculating the cell killing rate. Cell killing rate = (sample luminescence value - background luminescence value) / (control group luminescence value - background luminescence value) * 100%.

[0107] Example 4: Effects of different rounds of shear force treatment on CAR-T cells

[0108] The flowchart of the shearing process in this embodiment is as follows: Figure 1 As shown.

[0109] T cells were isolated from PBMCs as Day 0. CD3 / CD28 activation magnetic beads were added during isolation. On Day 1, the T cells were transfected with MSLN virus to enable the T cells to carry the MSLN sequence CAR structure. On Day 2, the CAR-T cells were centrifuged to replace the culture medium and remove the virus and CD3 / CD28 activation magnetic beads.

[0110] C1: When CAR-T cells are cultured to Day 4, 4E6 CAR-T cells are harvested and diluted to a density of 1E6 / ml with a shear stress of 5 dyn / cm. 2 After undergoing fluid shear stress treatment for 1 hour, the samples were collected by centrifugation at 500g for 5 minutes, and then resuspended in fresh culture medium for further culture.

[0111] C2: Take 4E6 cells from C1, dilute to a density of 1E6 / ml, and apply a shear stress of 5 dyn / cm. 2 After undergoing a second fluid shear stress treatment for 1 hour, the sample was collected by centrifugation at 500g for 5 minutes, and then resuspended in fresh culture medium for further culture.

[0112] C3: Take 4E6 cells from C2, dilute to a density of 1E6 / ml, and apply a shear stress of 5 dyn / cm. 2 After being subjected to a third fluid shear stress treatment for 1 hour, it was collected by centrifugation at 500g for 5 minutes, and then resuspended in fresh culture medium for further culture.

[0113] When C1, C2, and C3 cells were cultured to Day 12, their cell viability, activation, exhaustion, memory phenotype, and killing ability were detected according to the method described in Example 3.

[0114] The results are as follows Figure 2 As shown, CAR-T cells were treated with fluid shear force for 1 hour per cycle, which yielded the best results.

[0115] Example 5: Effects of fluid shear stress treatment at different time points on CAR-T cells

[0116] T cells were isolated from PBMCs as Day 0. CD3 / CD28 activation magnetic beads were added during isolation. On Day 1, the T cells were transfected with MSLN virus to enable the T cells to carry the MSLN sequence CAR structure. On Day 2, the CAR-T cells were centrifuged to replace the culture medium and remove the virus and CD3 / CD28 activation magnetic beads.

[0117] D4 CAR-T: When CAR-T cells are cultured to Day 4, 4E6 CAR-T cells are harvested and diluted to a density of 1E6 / ml with a shear stress of 5 dyn / cm. 2After undergoing fluid shear stress treatment for 1 hour, the samples were collected by centrifugation at 500g for 5 minutes, and then resuspended in fresh culture medium for further culture.

[0118] D7 CAR-T: On Day 7, 4E6 untreated CAR-T cells were collected and diluted to a density of 1E6 / ml with a shear stress of 5 dyn / cm. 2 After being subjected to fluid shear stress for 1 hour, the sample was collected by centrifugation at 500g for 5 minutes, and then resuspended in fresh culture medium for further culture.

[0119] D10 CAR-T: On Day 10, 4E6 untreated CAR-T cells were collected and diluted to a density of 1E6 / ml with a shear stress of 5 dyn / cm. 2 After being subjected to fluid shear stress for 1 hour, the sample was collected by centrifugation at 500g for 5 minutes, and then resuspended in fresh culture medium for further culture.

[0120] When D4 CAR-T, D7 CAR-T, and D10 CAR-T cells were cultured to Day 12, their cell viability, activation, exhaustion, memory phenotype, and killing ability were detected according to the method described in Example 3.

[0121] Table 2. Activation rate, exhaustion, and memory phenotype of CART-T cells

[0122] NC D4 D7 D10 CD25+ 27.97 39.38 49.24 36.50 CD69+ 15.16 18.14 43.79 15.42 PD-1+ 22.76 54.82 24.72 57.14 LAG-3+ 29.17 22.09 13.07 28.73 <![CDATA[T SCM ]]> 17.87 24.74 29.30 20.41 <![CDATA[T EM ]]> 17.78 18.12 20.49 31.27

[0123] Table 3. Lysis rate of CAR-T cells against cancer cells

[0124]

[0125] The results are as follows Figure 3 As shown in Tables 2 and 3, the best results were obtained by subjecting CAR-T cells to fluid shear force for 1 hour on Day 7.

[0126] Comparative Example

[0127] NC group: T cells were isolated from PBMCs as Day 0, with CD3 / CD28 activation magnetic beads added during isolation. On Day 1, the T cells were transfected with MSLN virus, enabling the T cells to carry the MSLN sequence CAR structure. On Day 2, the CAR-T cells were centrifuged to replace the culture medium, removing the virus and CD3 / CD28 activation magnetic beads.

[0128] SS group: NC group cells were cultured to Day 7, and 4E6 CAR-T cells were harvested, diluted to a density of 1E6 / ml, and subjected to a shear stress of 5 dyn / cm. 2After undergoing fluid shear stress treatment for 1 hour, the samples were collected by centrifugation at 500g for 5 minutes, and then resuspended in fresh culture medium for further culture.

[0129] CD3 / CD28-SS group: T cells were isolated from PBMCs as Day 0. The T cells were activated using CD3 / CD28 magnetic beads and incubated at 37°C for 15 minutes, followed by incubation at 5 dyn / cm². 2 After 1 hour of shear stress, the cells were collected by centrifugation at 500g for 5 minutes, resuspended in fresh medium, and cultured. On Day 1, the T cells were transfected with MSLN virus, enabling them to carry the MSLN sequence CAR structure. On Day 2, the CAR-T cells were centrifuged and the culture medium was changed to remove the virus.

[0130] When the cells in each group were cultured to Day 12, their cell viability, activation, exhaustion, memory phenotype, and killing ability were tested according to the method described in Example 3.

[0131] Table 4. Activation rate, exhaustion, and memory phenotype of CART-T cells

[0132] NC CD3 / CD28-SS SS CD25+ 19.56 32.11 44.47 CD69+ 21.46 26.24 31.39 PD-1+ 28.38 25.93 12.50 LAG-3+ 60.19 60.26 29.70 TSCM 31.57 35.68 37.94 TEM 26.41 19.48 17.35

[0133] Table 5. Lysis rate of CAR-T cells against cancer cells

[0134]

[0135] The results are as follows Figure 4 As shown in Tables 4 and 5, the best results were obtained by culturing without CD3 / CD28 magnetic beads and subjecting the culture to fluid shear stress treatment for 1 hour on day 7.

[0136] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for enhancing the performance of CAR-T cells, characterized in that, Including the following steps: (s1) Provide T cells, culture the T cells under suitable culture conditions for a period of time t1, and activate and transfect the T cells during the t1 culture period to obtain activated and transfected CAR-T cells, where t1 is 1 to 3 days; (s2) The activated and transfected CAR-T cells were cultured for t2 days under suitable culture conditions and without shearing, where t2 was 2 to 8 days. (s3) The CAR-T cells are subjected to shearing treatment for t3 hours, where t3 is 0.5–6 hours, and the shearing stress is 1–6 dyn / cm. 2 CAR-T cells were obtained after cleavage treatment; (s4) The cleaved CAR-T cells were cultured for t4 days under suitable culture conditions without shearing, where t4 was 2–8 days; and (s5) Harvest the CAR-T cells.

2. The method as described in claim 1, characterized in that, In step (s3), t3 is 0.5 to 5 hours, preferably 0.5 to 3 hours, more preferably 0.5 to 2 hours, for example, about 1 hour.

3. The method as described in claim 1, characterized in that, In step (s2), t2 is 3 to 7 days, preferably 4 to 6 days, for example, about 5 days.

4. The method as described in claim 1, characterized in that, In step (s3), the shear stress is 2–6 dyn / cm. 2 The optimal ratio is 3–6 dyn / cm. 2 More preferably 3-6 dyn / cm 2 For example, approximately 5 dyn / cm 2 .

5. The method as described in claim 1, characterized in that, The method further includes step (s3) being repeated 0 to 3 times, preferably 0 to 2 times, and more preferably 0 to 1 time.

6. The method as described in claim 1, characterized in that, In step (s3), the density of CAR-T cells during the cleavage treatment is 1×10⁻⁶. 4 ~1×10 8 cells / mL, preferably 1×10 5 ~1×10 7 cells / mL, preferably 5 × 10⁻⁶ 5 ~5×10 6 cells / mL, for example, about 1×10 6 per mL.

7. The method as described in claim 1, characterized in that, The CAR-T cells are derived from peripheral blood, preferably peripheral blood mononuclear cells.

8. The method as described in claim 1, characterized in that, Step (s1) includes: (s1a) Provide T cells, add activating factors to the T cells under suitable culture conditions to activate the T cells, and culture the activated T cells for 0.5 to 1 day; (s1b) CAR-T cells were obtained by transfecting the activated T cells with the virus and culturing them for 0.5–1 day; and (s1c) Remove the activating factors and virus from the CAR-T cells to obtain activated and transfected CAR-T cells.

9. Using the CAR-T cells obtained by the method as described in claim 1.

10. A method for inhibiting the progression of cancer cells, characterized in that, Including the following steps: By contacting the CAR-T cells as described in claim 9 with cancer cells, the progression of cancer cells can be inhibited.

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