Domestication of CAR-T cells by shear force treatment

Abstract

The present application relates to a method for reducing the apoptosis rate of CAR-T cells, specifically, the apoptosis rate of CAR-T cells is greater than or equal to 10%, and the shear treatment is carried out for t1 hours, t1 is 0.5-6h, and the shear stress of the shear treatment is 1-6dyn / cm 2 , so as to improve the apoptosis rate of CAR-T cells. After shear treatment, the apoptosis rate of CAR-T cells is reduced by 20%-70%, so as to improve the proliferation activity of CAR-T cells, reduce the exhaustion index, improve the memory activity, promote the short-term and long-term killing effect of tumor cells.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the acclimatization effect of shear force treatment on CAR-T cells. Background Technology

[0002] CAR-T cell (chimeric antigen receptor T cell) therapy, a revolutionary breakthrough in cancer treatment, has made significant progress in research and clinical practice in recent years. Its core lies in the genetic engineering of T cells, enabling them to specifically recognize and attack tumor cells. However, the apoptosis rate of CAR-T cells remains a significant challenge for this therapy.

[0003] First, apoptosis of CAR-T cells reduces their number, thereby decreasing their ability to recognize and attack tumor cells. This means that treatment efficacy may be weakened, and patients may not achieve the expected anti-tumor response. Second, CAR-T cell apoptosis can also trigger an immune response. Apoptotic CAR-T cells may be recognized as foreign substances by the body's immune system, triggering an immune attack. This immune response may lead to inflammation or other adverse reactions, further affecting the patient's health.

[0004] Therefore, there is an urgent need in this field for a method to reduce the apoptosis rate of CAR-T cells. Summary of the Invention

[0005] The purpose of this invention is to provide a method for reducing the apoptosis rate of CAR-T cells.

[0006] A first aspect of the present invention provides a method for reducing the apoptosis rate of CAR-T cells, comprising the steps of:

[0007] (s1) Detection of the initial apoptosis rate Zs of CAR-T cells;

[0008] (s2) When the apoptosis rate Zs of the CAR-T cells is ≥10%, the CAR-T cells are subjected to cleavage treatment for t1 hours, where t1 is 0.5–6 hours, and the shear stress of the cleavage is 1–6 dyn / cm. 2 ;

[0009] (s3) Under suitable culture conditions and without shearing, the sheared CAR-T cells were cultured for t2 days, where t2 was 2–8 days; and

[0010] (s4) Harvest the CAR-T cells.

[0011] In another preferred embodiment, the apoptosis rate (Z1) of CAR-T cells treated by the method was reduced by ≥30%, preferably ≥40%, and more preferably ≥50% compared to untreated CAR-T cells (Z0).

[0012] In another preferred embodiment, in step (s2), the initial apoptosis rate of the CAR-T cells is ≥11%, preferably ≥12%, more preferably ≥13%, and most preferably ≥14%.

[0013] In another preferred embodiment, t2 is 3 to 7 days, more preferably 4 to 6 days, for example, about 5 days.

[0014] In another preferred embodiment, in step (s2), the CAR-T cells are sheared when the apoptosis rate Zs is 5% to 50%, preferably 5% to 40%, more preferably 5% to 30%, even more preferably 10% to 30%, and most preferably 10% to 25%.

[0015] In another preferred embodiment, in step (s1), the initial apoptosis rate Zs refers to the apoptosis rate of CAR-T cells cultured for 1 to 5 days after activation and transfection or resuscitation of frozen CAR-T cells, preferably 1 to 4 days, more preferably 2 to 4 days, and most preferably 2 to 3 days.

[0016] In another preferred embodiment, in step (s2), after activation and transfection or resuscitation, the cells are sheared on day t3, where t3 is 1 to 10 days, preferably 3 to 8 days, more preferably 4 to 7 days, for example, t3 is 5 days.

[0017] In another preferred embodiment, in step (s1), the CAR-T cells are revived cryopreserved CAR-T cells.

[0018] In another preferred embodiment, the method further includes repeating step (s2) 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 (s2), 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, in step (s2), the shear stress is 2 to 6 dyn / cm. 2 Optimal ratio is 3-6 dyn / cm 2 More preferably 3-6 dyn / cm2 For example, approximately 5 dyn / cm 2 .

[0022] In another preferred embodiment, in step (s2), t1 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.

[0023] 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.

[0024] 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%.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] In another preferred embodiment, the method is in vitro.

[0029] In a second aspect, the present invention provides CAR-T cells obtained using the method described in the first aspect of the present invention.

[0030] In another preferred embodiment, the apoptosis rate (Z1) of the CAR-T cells compared with uncut CAR-T cells (Z0) is reduced by ((Z0-Z1) / Z0) ≥30%, preferably ≥40%, and more preferably ≥50%.

[0031] A third aspect of the present invention provides a method for inhibiting the progression of cancer cells, comprising the steps of:

[0032] CAR-T cells treated as described in the first aspect of the invention or CAR-T cells as described in the second aspect of the invention are brought into contact with cancer cells to inhibit cancer cell progression.

[0033] 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.

[0034] In another preferred embodiment, the cancer is a solid tumor.

[0035] 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.

[0036] 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.

[0037] In another preferred embodiment, the method is in vitro.

[0038] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0039] In a fourth aspect, the present invention provides a method for treating cancer, comprising the steps of:

[0040] Cancer can be treated by administering CAR-T cells treated as described in the first aspect of the invention or as described in the second aspect of the invention to subjects in need.

[0041] In another preferred embodiment, the cancer is a solid tumor.

[0042] In another preferred embodiment, the cancer is selected from the group consisting of ovarian cancer, breast cancer, cervical cancer, glioma, renal clear adenocarcinoma, or a combination thereof.

[0043] 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

[0044] Figure 1 The apoptosis rate of CAR-T cells is shown, including (A) the apoptosis rate of NC-CAR-T and SS-CAR-T cells cultured to day 10; (B) the apoptosis rate of NC-CAR-T and SS-CAR-T cells after day 10, followed by a second fluid shear stress treatment for 1 hour; (C) the apoptosis rate of NC-CAR-T cells after thawing from cryopreservation, undergoing fluid shear stress treatment, and then collecting and culturing for 3 days; and (D) the apoptosis rate of CAR-T cells injected into mice with 8×10⁸ cells. 5 One hour after NC-CAR-T and SS-CAR-T, the apoptosis rate of CAR-T cells in mice was detected by blood flow cytometry.

[0045] Figure 2 This study demonstrates the antitumor effect of MSLN-CAR-T cells treated with fluid shear stress in vivo. Schematic diagram of mouse model establishment (A); flow cytometry analysis of decreased SS-MSLN-CAR-T levels relative to MSLN-CAR-T depletion levels (B), activation levels (C), and memory phenotype T in mouse blood. SCM (D) The number of tumors in the body (E) and the elevated level of INF-γ (F); mouse body weight (G); tumor volume (H); tumor photograph (I); immunohistochemical results of tumor masses obtained by euthanizing one mouse from each group on day 8, and HE staining results of other tissues (K). In all figures, *, P<0.05; **, P<0.01; ***, P<0.001. Detailed Implementation

[0046] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors unexpectedly discovered for the first time a method to reduce the apoptosis rate of CAR-T cells. Specifically, CAR-T cells with an initial apoptosis rate ≥10% are subjected to cleavage treatment for t hours, where t is 0.5–6 hours, and the shear stress of the cleavage is 1–6 dyn / cm. 2 This process increases the apoptosis rate of CAR-T cells. Experiments show that after cleavage treatment, the apoptosis rate of CAR-T cells decreases by 20% to 70%, thereby increasing CAR-T cell proliferation, reducing exhaustion index, and enhancing memory activity. Furthermore, the CAR-T cells exhibit excellent tumor cell killing activity when reinfused into mouse tumor models. Based on these findings, this invention was completed.

[0047] the term

[0048] 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.

[0049] As used herein, the term “comprising” or its variations such as “including” or “comprises” are understood to include the said element or component without excluding other elements or other components.

[0050] 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.).

[0051] 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).

[0052] 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.

[0053] As used in this article, the term "inhibit cancer cell progression" refers to inhibiting cancer cell proliferation and promoting cancer cell apoptosis.

[0054] The method of the present invention

[0055] The method of the present invention can reduce the apoptosis rate of CAR-T cells, comprising the following steps:

[0056] (s1) Detection of the initial apoptosis rate Zs of CAR-T cells;

[0057] (s2) When the initial apoptosis rate Zs of the CAR-T cells is ≥10%, the CAR-T cells are subjected to cleavage treatment for t1 hours, where t1 is 0.5–6 hours, and the shear stress of the cleavage is 1–6 dyn / cm. 2 ;

[0058] (s3) Under suitable culture conditions and without shearing, the sheared CAR-T cells were cultured for t2 days, where t2 was 2–8 days; and

[0059] (s4) Harvest the CAR-T cells.

[0060] In a preferred embodiment, the apoptosis rate (Z1) of CAR-T cells treated as described in the present invention is reduced by ((Z0-Z1) / Z0) ≥30%, preferably ≥40%, and more preferably ≥50% compared to uncut CAR-T cells (Z0).

[0061] In a preferred embodiment, in step (s2), the initial apoptosis rate of the CAR-T cells is ≥11%, preferably ≥12%, more preferably ≥13%, and most preferably ≥14%.

[0062] In a preferred embodiment, in step (s1), the initial apoptosis rate Zs refers to the apoptosis rate of CAR-T cells cultured for 1-5 days after activation and transfection or revival of frozen CAR-T cells, preferably 1-4 days, more preferably 2-4 days, and most preferably 2-3 days. The apoptosis rate of activated or transfected CAR-T cells or revival of frozen CAR-T cells increases significantly after 1-5 days of culture, and then tends to level off. CAR-T cells treated by the method of the present invention can reduce their apoptosis rate.

[0063] In a preferred embodiment, in step (s2), a shearing process is performed on day t3 after activation and transfection or resuscitation, where t3 is 1 to 10 days, preferably 3 to 8 days, more preferably 4 to 7 days, for example, t3 is 5 days.

[0064] In a preferred embodiment, in step (s2), 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.

[0065] The main advantages of this invention include:

[0066] The method of the present invention can increase the apoptosis rate of CAR-T cells. Compared with CAR-T cells not treated by the method of the present invention, the apoptosis rate is reduced by 20% to 70%, thereby improving the proliferative activity of CAR-T cells, reducing the exhaustion index, improving memory activity, and promoting the short-term and long-term killing effects of tumor cells.

[0067] 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.

[0068] Example 1: Preparation of CAR-T cells

[0069] 1.1 T cell sorting experiment:

[0070] 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.

[0071] 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. Repeat the incubation three times. After incubation, culture the cells in a T25 flask at a ratio of 2E6 cells / ml of culture medium (marked as day 1, D1).

[0072] The T cells cultured overnight should be used promptly for lentivirus transfection the next day to construct CAR-T cells.

[0073] 1.2 Construction of CAR-T cells:

[0074] 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.

[0075] 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.

[0076] Table 1

[0077]

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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 (referred to as day 2).

[0082] 1.3 Cell Culture Experiments:

[0083] 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.

[0084] Example 2 Shear Force Treatment

[0085] 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 .

[0086] 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.

[0087] 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.

[0088] Example 3 Detection Method

[0089] 3.1 Cell viability

[0090] 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.

[0091] 3.2 Phenotypic Changes of CAR-T Cells

[0092] 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 each sample was labeled with a specific antibody, such as activating cell markers (CD25, CD69), exhausted cell 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.

[0093] 3.3 Detection of CAR-T cell-mediated tumor cell killing

[0094] 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.

[0095] 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%.

[0096] 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%.

[0097] Example 4

[0098] Apoptosis detection: Divide the cells to be tested into multiple samples, each containing 5E5 cells. Centrifuge to remove the supernatant, wash with pre-chilled PBS, centrifuge again, and add 100 μl of 1x Binding Buffer, gently mixing until a single-cell suspension is formed. Add 5 μl of Annexin V-PE and 5 μl of 7-AAD Staining Solution, gently mixing; incubate in the dark at room temperature (20–25°C) for 10 min; add 400 μl of 1x Binding Buffer, gently mixing. Analyze the stained samples using flow cytometry.

[0099] After 1 hour of SS treatment on CAR-T cells from day 3 to day 10, apoptosis was detected in CAR-T cells and SS-CAR-T cells. Based on the detection results in Table 1, we chose to perform the subsequent SS treatment on day 7.

[0100] Table 1 Apoptosis rate

[0101] D3 D4 D5 D6 D7 D8 D9 D10 NC-CAR-T 9.99％ 10.41％ 14.25％ 14.16％ 14.55％ 13.98％ 14.24％ 14.74％ SS-CAR-T 6.17％ 6.50％ 8.70％ 8.34％ 7.11％ 7.65％ 9.64％ 9.22％

[0102] Example 5

[0103] Apoptosis rates of CAR-T cells treated with and without SS were measured, as shown in Table 1. Figure 1 As shown.

[0104] Group A: NC-CAR-T cells were subjected to fluid shear stress treatment for 1 hour on day 7 and then collected and cultured. NC-CAR-T and SS-CAR-T cells were cultured to day 10 and apoptosis was detected. SS-CAR-T cells showed relatively lower apoptosis.

[0105] Group B: After NC-CAR-T and SS-CAR-T were subjected to fluid shear stress treatment again for 1 hour on day 10, SS-CAR-T had relatively lower apoptosis.

[0106] Group C: After 7 days of NC-CAR-T culture, the samples were frozen in a liquid nitrogen tank at -80℃ for 10 days. After the frozen NC-CAR-T was thawed, it was subjected to fluid shear stress treatment and then collected for culture. After 3 days of culture, apoptosis detection was performed on NC-CAR-T and SS-CAR-T. SS-CAR-T had relatively lower apoptosis.

[0107] Group D: NC-CAR-T cells were subjected to fluid shear stress treatment for 1 hour on day 7 and then collected and cultured. NC-CAR-T and SS-CAR-T cells were cultured until day 10. Mice were injected with 8 x 10⁸ cells. 5Blood flow cytometry was performed on mice 1 hour after NC-CAR-T and SS-CAR-T treatment. CAR-T cells were labeled with CD3 antibody to detect CAR-T cell apoptosis. It was found that the apoptosis level of SS-MSLN-CAR-T cells treated with fluid shear force was reduced in vivo.

[0108] Table 2 Apoptosis rate of CAR-T cells

[0109]

[0110] The results showed that apoptosis of CAR-T cells was significantly reduced after shear stress treatment.

[0111] Example 6

[0112] Tumor 1806 cells were subcutaneously implanted in NSG mice. Ten days after tumor formation, the mice were divided into three groups. The first group was injected with 200 μL of PBS, and the second group was injected with 200 μL of 8x10... 5 MSLN-CAR-T, third group, 200ul injection (8x10) 5 SS-MSLN-CAR-T.

[0113] Blood flow cytometry analysis performed on mice on day 12 showed a decrease in SS-MSLN-CAR-T depletion levels relative to MSLN-CAR-T in vivo. Figure 2 C), activation level increased ( Figure 2 (D), memory phenotype T SCM Increase ( Figure 2 The amount of D in the body increases ( Figure 2 The level of INF-γ increased (in F). Figure 2 (G).

[0114] Mice were weighed every two days from the time of CAR-T injection (day 0). No significant difference in weight was observed among the three groups of mice. Figure 2 (H).

[0115] Tumor size was measured in mice every two days, and it was observed that tumors in the SS group eliminated faster. Figure 2 Middle I).

[0116] Mice were sacrificed on day 25 and their tumors were dissected. It was observed that the tumors in the SS group had completely disappeared by day 25. Figure 2 (JK).

[0117] On day 8, one mouse from each group was randomly selected and sacrificed to collect its tissue. Immunohistochemistry was performed on the tumor mass, and it was observed that the SS group had better infiltration. Figure 2 In the case of H&E staining of other tissues, it can be seen that CAR-T cells in the SS group did not cause damage to other tissues. Figure 2 (Middle K).

[0118] 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 reducing the apoptosis rate of CAR-T cells, characterized in that, Including the following steps: (s1) Detection of the initial apoptosis rate Zs of CAR-T cells; (s2) When the initial apoptosis rate Zs of the CAR-T cells is ≥10%, the CAR-T cells are subjected to cleavage treatment for t1 hours, where t1 is 0.5–6 hours, and the shear stress of the cleavage is 1–6 dyn / cm. 2 ; (s3) Under suitable culture conditions and without shearing, the sheared CAR-T cells were cultured for t2 days, where t2 was 2–8 days; and (s4) Harvest the CAR-T cells.

2. The method as described in claim 1, characterized in that, Compared with uncut CAR-T cells (Z0), the apoptosis rate (Z1) of CAR-T cells treated with the method of claim 1 was reduced by ((Z0-Z1) / Z0) ≥30%, preferably ≥40%, more preferably ≥50%.

3. The method as described in claim 1, characterized in that, In step (s2), the initial apoptosis rate of the CAR-T cells is ≥11%, preferably ≥12%, more preferably ≥13%, and most preferably ≥14%.

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

5. The method as described in claim 1, characterized in that, In step (s1), the initial apoptosis rate Zs refers to the apoptosis rate of activated and transfected CAR-T cells or revived frozen CAR-T cells cultured for 1 to 5 days, preferably 1 to 4 days, more preferably 2 to 4 days, and most preferably 2 to 3 days.

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

7. The method as described in claim 1, characterized in that, In step (s2), 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 .

8. A CAR-T cell obtained using the method of claim 1.

9. The CAR-T cells as described in claim 8, characterized in that, Compared with uncut CAR-T cells (Z0), the apoptosis rate of the CAR-T cells (Z1) was reduced by ((Z0-Z1) / Z0) ≥30%, preferably ≥40%, and more preferably ≥50%.

10. A method for inhibiting the progression of cancer cells, characterized in that, Including the following steps: By contacting cancer cells with CAR-T cells treated as described in claim 1 or CAR-T cells as described in claim 8, cancer cell progression can be inhibited.

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