Method for generating a human chimeric antigen receptor T cell population containing a high concentration of stem cell-like memory T cells

Abstract

To provide methods for preparing CAR-T cells enriched with stem cell-like memory T cells (Tscm).SOLUTION: A method provided herein comprises the steps of: (1) providing PBMCs; (2) isolating T cells from PBMCs using magnetic beads coated with T-cell specific antibodies; (3) positively selecting T cells by culturing them in a form of T cell-magnetic bead complex; (4) adding CAR-expressing lentivirus to the activated human T cell-magnetic bead complex for transducing human T cells into CAR-T cells on the human T cell-magnetic bead complex to obtain a human CAR-T cell-magnetic bead complex; (5) obtaining human CAR-T cells by dissociating the human CAR-T cells from the human CAR-T cell-magnetic bead complex; and (6) further culturing CAR-T cells to obtain CAR-T cells enriched with Tscm.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing human chimeric antigen receptor T cells (CAR-T cells), and in particular to a method for producing human CAR-T cells containing a high concentration of stem cell-like memory T cells (Tscm). [Background technology]

[0002] CAR-T cell therapy uses gene editing technology to modify T cells to express receptors that can recognize surface antigens on tumor cells. When the modified T cells are infused back into the body, they can recognize and kill tumor cells.

[0003] However, this treatment also has side effects, the most serious of which is cytokine release syndrome (CRS). CRS is described as a chain reaction that leads to excessive inflammation, microvascular leakage, and coagulation when CAR-T cells fight tumor cells. It can also lead to life-threatening events such as heart failure, lung failure, kidney failure, and brain swelling. It is generally believed that the abundance of Tscm in CAR-T cells prolongs their survival in the human body, enhances therapeutic efficacy, and reduces the incidence and severity of CRS and neurotoxicity.

[0004] However, the proportion of Tscm cells in human peripheral blood is only about 1%–5%. Conventional CAR-T cell engineering methods are unable to effectively generate CAR-T cells with a high proportion and number of Tscm cells. Because the proportion of Tscm cells in CAR-T cells prepared using conventional methods is not sufficiently high, this treatment not only carries a high risk of severe CRS and neurotoxicity, but also leads to reduced efficacy associated with a shortened in vivo lifespan. Furthermore, the in vitro generation of CAR-T cells requires a long time: it takes 9–14 days to generate enough CAR-T cells for treatment. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2020 / 047452 Summary of the Invention [Means for solving the problem]

[0006] In order to overcome the drawbacks of existing production techniques, the present invention aims to provide a method for producing sufficient CAR-T cells that also contain Tscm at high concentrations in a short production period.

[0007] In order to achieve the above-mentioned object, the present invention provides a method for producing human chimeric antigen receptor T (CAR-T) cells containing a high concentration of stem cell-like memory T cells, comprising the steps of: (1) preparing human peripheral blood mononuclear cells (PBMCs); (2) mixing magnetic beads coated with anti-CD3 antibodies and anti-CD28 antibodies with the human PBMCs, which also contain B cells, natural killer (NK) cells, and monocytes, to form human T cell-magnetic bead complexes, and positively selecting human T cells from the human PBMCs; and (3) culturing the human T cell-magnetic bead complexes for 16 to 20 hours to activate the T cells, thereby obtaining activated T cells. (4) obtaining a human T cell-magnetic bead complex; (5) obtaining a human CAR-T cell (without magnetic beads) by dissociating the human CAR-T cell from the human CAR-T cell-magnetic bead complex; and (6) further culturing the human CAR-T cell for 92 to 96 hours to obtain a human CAR-T cell containing the Tscm at a high concentration.

[0008] In the present invention, by controlling the contact time between human T cells and magnetic beads, human CAR-T cells generated by 7-day culture showed the highest proliferation rate and contained high concentrations of Tscm. The present invention provides a method for generating human CAR-T cells that have stronger immune functions than human CAR-T cells prepared using existing technologies and are therefore more suitable for CAR-T immunotherapy.

[0009] According to the present invention, human PBMCs in step (1) are isolated from either human peripheral blood or leukapheresis products, and the PBMCs include cells such as T cells, B cells, NK cells and monocytes.

[0010] According to the present invention, the source of human PBMCs described herein can be from healthy donors or cancer patients.

[0011] According to the present invention, magnetic beads are capable of activating human T cells. Preferably, the magnetic beads are Dynabeads. TM Human T-Expander CD3 / CD28 (Gibco, Cat. No. 11141D) or CTS TM (Cell Therapy Systems)Dynabeads TM CD3 / CD28 (Gibco, CAT. No. 40203D).

[0012] Preferably, the number of magnetic beads coated with anti-CD3 and anti-CD28 antibodies in step (2) is 2 to 4 times the number of human T cells in human PBMCs, and more preferably, the number of magnetic beads is 3 times the number of human T cells in human PBMCs.

[0013] Preferably, the culture area (cm ) of the culture flask in steps (3) and (4) 2 ) for the complete X-VIVO TM The ratio of the total volume of 15 medium (mL) is 0.12 mL / cm 2 ~0.3mL / cm 2 That is, when the medium is filled into the culture flask, it is 0.12 mL / cm2 ~0.3mL / cm 2 More preferably, the volume to surface area ratio of the complete x-vivo culture flask in steps (3) and (4) is 1 / 1000. TM The ratio of the total volume of 15 medium is 0.2 mL / cm 2 ~0.27mL / cm 2 For example, if the culture area of a culture flask is 75 cm 2 In case of X-VIVO TM 15 The total volume of the medium is 9 mL to 22.5 mL. More preferably, the culture area of the culture flask is 75 cm. 2 If so, apply to X-VIVO TM 15 medium is 15 mL to 20.25 mL.

[0014] Preferably, the number of magnetic beads in step (2) is controlled to be 2 to 4 times the number of human T cells in the human PBMC, and at the same time, the complete x-vivo culture relative to the culture area of the flask in steps (3) and (4) is performed. TM The ratio of the total volume of 15 medium is 0.12 mL / cm 2 ~0.3mL / cm 2 That is, the contact time between the human T cells and the magnetic beads in step (2), the ratio of the magnetic beads to the human T cells, and the complete x-vivo TM 15 By simultaneously controlling the ratio of the medium volume to the culture surface area of the culture flask, T cells can be expanded many-fold in a short period of time, and a large population can be generated as Tscm.

[0015] Preferably, the human T cell-magnetic bead complexes in step (3) are cultured at 36.5°C to 37.5°C for 16 to 20 hours. For example, the culture period can be 16 to 19 hours, or 17 to 19 hours. More preferably, the human T cell-magnetic bead complexes in step (3) are incubated at 37°C for 18 hours in a humidified environment containing 5% CO2.

[0016] Preferably, the lentiviral transduction in step (4) is carried out for 40 to 56 hours at 36.5°C to 37.5°C. More preferably, the lentiviral transfection in step (4) is carried out for 48 hours at 37°C in a humidified environment containing 5% CO2.

[0017] Preferably, in step (6), the human CAR-T cells are cultured for 92 to 96 hours at 36.5°C to 37.5°C. More preferably, in step (6), the human T cells are cultured for 96 hours at 37°C in a humidified environment containing 5% CO2.

[0018] Preferably, the culturing of the human T cell-magnetic bead complex in step (3), the lentiviral transduction in step (4), and the culturing of the human CAR-T cells in step (6) are carried out using complete x-vivo immunoprecipitation containing IL-2. TM 15 (Lonza, Cat. No. 04-418Q) medium, and complete x-vivo TM The IL-2 concentration in the medium is 200 IU / mL.

[0019] According to the present invention, complete X-VIVO TM 15 medium is X-VIVO medium containing 200 IU / mL of recombinant human IL-2 (R&D Systems, Cat No. 202-IL-500). TM 15 medium.

[0020] Preferably, the lentiviral transduction in step (4) involves transducing human T cells with a gene comprising an anti-CD19, anti-CD22, anti-B-cell maturation antigen (BCMA), or anti-mesothelin receptor sequence.

[0021] Preferably, the number of human CAR-T cells in the human CAR-T cell population containing Tscm at a high concentration obtained in step (6) is more than 10 times the number of human T cells in the human T cell-magnetic bead complexes in step (2), i.e., the number of human T cells isolated using magnetic beads in step (2). That is, the number of human CAR-T cells obtained by culture expansion in step (6) is more than 10 times the number of human T cells in the human T cell-magnetic bead complexes in step (2). More preferably, the number of human CAR-T cells containing Tscm at a high concentration obtained in step (6) is more than 20 times the number of human T cells in the human T cell-magnetic bead complexes in step (2). Even more preferably, the number of human CAR-T cells containing Tscm at a high concentration obtained in step (6) is more than 30 times the number of human T cells in the human T cell-magnetic bead complexes in step (2).

[0022] Preferably, the culture of human CAR-T in step (6) is carried out using a culture medium mixture, and the culture medium mixture is fresh complete x-vivo culture medium. TM The mixture contains the conditioned medium of the human CAR-T cells obtained in step (5) and the conditioned medium of the human CAR-T cells obtained in step (1). That is, the conditioned medium obtained by culturing the human CAR-T cells through steps (1) to (5) is added with fresh complete X-VIVO TM Add 15 medium, preferably fresh complete X-VIVO TM The volume ratio of the 15 medium to the conditioned medium by human CAR-T cells obtained in step (5) is 1:1 to 3:1.

[0023] Preferably, the proportion of Tscm in the Tscm-enriched human CAR-T cells obtained in step (6) containing Tscm at a high concentration exceeds 55%. That is, more than 55% of all human CAR-T cells obtained after the culture expansion in step (6) are Tscm. More preferably, the proportion of Tscm in the Tscm-enriched human CAR-T cells obtained in step (6) exceeds 60%. More preferably, the proportion of Tscm in the Tscm-enriched human CAR-T cells obtained in step (6) exceeds 70%. Even more preferably, the proportion of Tscm in the Tscm-enriched human CAR-T cells obtained in step (6) exceeds 75%.

[0024] Preferably, the present invention further comprises the step of (7) cryopreserving the human CAR-T cells containing the above-mentioned Tscm at a high concentration.

[0025] The advantages of the present invention lie in the CAR-T manufacturing procedure, which results in sufficient T cell proliferation in a short period of time and generates human CAR-T cells enriched in Tscm, which can be further differentiated, have a long lifespan, and are less likely to cause side effects such as CRS and neurotoxicity. Therefore, the human CAR-T cells generated by the method of the present invention are more suitable for CAR-T cell therapy. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 shows the fold expansion of human CAR-T cells on day 7 compared to day 0 for the Examples and Comparative Examples. [Figure 2] FIG. 2 shows the fold expansion of human T cells on day 7 of the Example and day 9 of the Comparative Example, compared to day 0. [Figure 3] FIG. 3 shows human T cell subpopulations on day 7 of the example and day 9 of the comparative example. [Figure 4A] FIG. 4A is an example flow cytometry of human T cell subpopulations on day 7. [Figure 4B] FIG. 4B is flow cytometry of human T cell subpopulations on day 9 of a comparative example. DETAILED DESCRIPTION OF THE INVENTION

[0027] The technical means adopted to achieve the intended purpose of the present invention will be further explained through the accompanying drawings and the following preparation examples and examples of the present invention. It should be understood that the descriptions proposed in this specification are merely preferred examples for illustrative purposes only and are not intended to limit the scope of the present disclosure. Various modifications and changes can be made to implement or apply the present disclosure without departing from the spirit and scope of the present disclosure.

[0028] Example first, In step (1), On Day 0 Human PBMCs were prepared Specifically, whole blood was collected from a healthy donor and mixed evenly with an equal volume of 2% albumin / phosphate-buffered saline (Alb / PBS) buffer to obtain a diluted whole blood sample. TM Human PBMCs were isolated from diluted whole blood samples by density gradient centrifugation using SepMate-50 tubes. The detailed procedure is shown below. TM Add 15 mL of Ficoll®-Paque Premium density gradient medium (GE Healthcare) to the SepMate™ by carefully pipetting it through the center hole of the insert. TM The diluted whole blood sample was then added to a SepMate 50 tube. TM Add the -50 solution slowly by pipetting it up and down the side of the tube. TM The -50 tube was centrifuged at 1200 × g for 20 minutes at room temperature with the brake on. After centrifugation, the upper layer, which is thought to contain human PBMCs, was poured into a new tube and centrifuged at 300 × g for 8 minutes at room temperature. The supernatant was then discarded, and the human PBMC cell pellet was resuspended in an appropriate amount of 2% Alb / PBS buffer to adjust the human PBMC concentration to 2–5 × 10. 7 Adjusted to cells / mL.

[0029] The cell number was measured by first sampling 7.5 μL of the cell suspension and mixing it with 6 μL of PBS buffer and 1.5 μL of acridine orange / propidium iodide staining solution by pipetting. TM The density, viability, and size of the cell samples were measured using an automated fluorescent cell counter. The total number of cells in the suspension was calculated by multiplying the viable cell density by the total volume of the cell suspension.

[0030] Furthermore, before carrying out step (2), CD3 in human PBMC was analyzed to calculate the number of magnetic beads required for step (2). + We needed to know the percentage of T cells. To do this, we co-incubated 10 μL of MACS 8-color immunophenotyping cocktail and 1 μL of 7-AAD Staining Solutions for 10 minutes at 4°C in the dark to obtain 1 × 10 5 PBMCs were stained. Cells were washed with 2 mL of 2% hAlb / saline buffer and then centrifuged at 350 × g for 5 minutes at room temperature. The supernatant was then discarded, and the cell pellet was gently tapped and resuspended in 200 μL of 2% Alb / saline buffer. PBMC subpopulations were analyzed using flow cytometry (CytoFLEX). CD3 + The proportion of T cells was approximately 40% to 70%.

[0031] CD3 surface antigen in human PBMCs + The number of cells showing this, i.e., the number of human T cells, was calculated using the following formula. All PBMC×CD3 + T cell%=CD3 + T cell number

[0032] next, In step (2), T cells were positively selected from human PBMCs, which also contained B cells, natural killer (NK) cells, and monocytes, by mixing antibody-coated magnetic beads with human PBMCs to form human T cell-magnetic bead complexes, where the magnetic beads were coated with anti-CD3 and anti-CD28 antibodies. Specifically, 2% hAlb / PBS buffer was added to the tube to resuspend human PBMCs, resulting in a maximum total nucleated cell (PBMC) count of 2 × 10 per mL. 8 Maintain a human T cell density of 2-5 x 10 without exceeding 1 x 10 7The concentration was adjusted to 1000 cells / mL. Before mixing with PBMCs, magnetic beads coated with anti-CD3 / CD28 antibodies (Dynabeads Human T-Expander CD3 / CD28: Gibco, Cat. No. 11141D) were washed twice with 2% Alb / PBS buffer. The magnetic beads were added to the human PBMC suspension to obtain a mixture of human PBMCs and magnetic beads, with a bead-to-human T cell ratio of 3:1. The human PBMCs and magnetic beads were mixed by rotating the tube at 6 rpm at room temperature for 30 minutes. After mixing, the tube containing the human PBMCs and magnetic beads was placed in a magnet for 1–2 minutes, after which the supernatant, containing unbound cells such as B cells, NK cells, and monocytes, was discarded. Most human T cells expressing CD3 bind to the magnetic beads via antigen-antibody interactions, forming the resulting T cell-magnetic bead complex.

[0033] Before step (3), the number of human T cells bound to the magnetic beads was calculated. TM 15 medium (X-VIVO containing 200 IU / mL of recombinant human IL-2 (R&D Systems, Cat. No. 202-IL-500) TM The density of the human T cell-magnetic bead complexes was readjusted by adding 5 times the volume of the human T cell-magnetic bead complex suspension (15 medium). 7.5 μL of the suspension containing the human T cell-magnetic bead complexes was sampled and mixed with 6 μL of PBS buffer and 1.5 μL of acridine orange / propidium iodide staining solution. The number of human T cells bound to the magnetic beads was then measured using an automated fluorescent cell counter.

[0034] In step (3), the human T cell-magnetic bead complex was cultured for 16 to 20 hours to activate the T cells, thereby obtaining activated human T cell-magnetic bead complexes. Specifically, a moderate amount of complete X-VIVO TM Using 15 medium, human T cells were grown in a T-flask at a density of 1 x 10 6 The human T cell-magnetic bead complexes were cultured to a concentration of cells / mL and incubated at 37°C for 20 hours in a humidified environment containing 5% CO2 to obtain activated human T cell-magnetic bead complexes.

[0035] Next, on the first day of this example, In step (4), a CAR-expressing lentivirus was added to the culture of activated human T cell-magnetic bead complexes to transduce the human T cells into CAR-T cells on the cell-magnetic bead complexes. Specifically, lentivirus was added to the culture of activated human T cell-magnetic bead complexes obtained in step (3) at a multiplicity of infection (MOI) of 0.5 to 3, with gentle mixing at a gentle speed to prevent dissociation of the human T cells and magnetic beads. After mixing for 30 seconds, the culture was again incubated at 37°C in a humidified atmosphere containing 5% CO2 for an additional 48 hours to transduce the lentivirus and generate human CAR-T cell-magnetic bead complexes.

[0036] On the third day of this example, In step (5), human CAR-T cells (without magnetic beads) were obtained from the human CAR-T cell-magnetic bead complexes by dissociation. First, the total volume of the human CAR-T cell-magnetic bead complex culture was measured. Next, 7.5 μL of the human CAR-T cell-magnetic bead complex culture was sampled and mixed with 6 μL of PBS buffer and 1.5 μL of acridine orange / propidium iodide staining solution. LUNA-FL TM The density, viability, and size of the cell samples were measured using an automated fluorescent cell counter. The total number of human CAR-T cells in suspension (in human CAR-T cell-magnetic bead complexes) was calculated by multiplying the viable cell density by the total volume of the human CAR-T cell-magnetic bead complex culture. The tube containing the cell suspension containing the human CAR-T cell-magnetic bead complexes was gently pipetted to dissociate the CAR-T cells from the magnetic beads, then placed in a magnet for 1-2 minutes to allow the magnetic beads to be fixed and removed from the cell sample. This process was repeated three times to sufficiently remove the magnetic beads from the cell sample and obtain a cell suspension consisting solely of human CAR-T cells.

[0037] Prior to step (6), the number and recovery rate of human CAR-T cells obtained from the CAR-T cell-magnetic bead complex culture were calculated. Specifically, the total volume of the human CAR-T cell suspension was calculated. 7.5 μL of the suspension containing human CAR-T cells was sampled and mixed with 6 μL of PBS buffer and 1.5 μL of acridine orange / propidium iodide staining solution. LUNA-FL was then used. TMThe density, viability, and size of the cell samples were measured using an automated fluorescent cell counter. The total number of CAR-T cells in the suspension was calculated by multiplying the viable cell density by the total volume of the human CAR-T cell suspension. The percentage of cell recovery from the magnetic bead depletion procedure was also calculated.

[0038] lastly, In step (6), the human CAR-T cells were further cultured to obtain human CAR-T cells containing high concentrations of Tscm. Specifically, the original conditioned medium of human CAR-T cells containing human CAR-T cells in suspension was added to fresh complete x-vivo culture. TM 15 medium was added to bring the cell density in the T flask to 7 x 10 5 The T-flask was incubated at 37°C in a humidified atmosphere containing 5% CO2 for a total of 96 hours (4 days). During this process, not only did the human CAR-T cells further proliferate, but the Tscm population also became highly enriched. The product of this process was human CAR-T cells containing a high concentration of Tscm. The CAR-T cell density in the same flask was also adjusted to 7 x 10 5 Add an appropriate amount of fresh complete x-vivo conditioned medium every 2-3 days to maintain the cells / mL. TM 15 medium was added. Now, fresh complete X-VIVO TM When adjusting the cell density by adding 15 medium, it is preferable to dilute the medium less than three-fold.

[0039] Comparative Example The method of the comparative example was similar to that of the example, except for steps (4) through (6). In the example, removal of the magnetic beads in step (5) was performed after 48 hours of lentiviral transduction, as shown in step (4). In contrast, in the comparative example, the magnetic beads were not removed until after 96 hours of lentiviral transduction. That is, the method of the comparative example was (4') adding lentivirus to the culture of activated human T cell-magnetic bead complexes for 96 hours to transduce the human T cells into CAR-T cells on the cell-magnetic bead complexes. This step was carried out on the first day of the comparative example. Additional medium was also supplied 48 hours after the start of lentiviral transduction. (5') A step of removing the magnetic beads from the human CAR-T cell-magnetic bead complex to obtain human CAR-T cells. was carried out on the fifth day of the preparation of the comparative example. (6') A step of culturing the human CAR-T cells at 37°C for 96 hours to obtain expanded human CAR-T cells. was carried out.

[0040] Since magnetic beads can activate, stimulate and proliferate T cells, the T cells in the comparative example were indeed activated for a longer period of time by magnetic beads compared to the example.

[0041] Test Example 1 Determination of T cell proliferation fold 7.5 μL of the cell suspension containing expanded human CAR-T cells obtained in step (6) of the Example and step (6') of the Comparative Example were sampled and mixed with 6 μL of PBS buffer and 1.5 μL of acridine orange / propidium iodide staining solution, respectively. TM The density, viability, and size of the cell samples were measured using an automated fluorescent cell counter. The total number of human CAR-T cells in the Examples and Comparative Examples was calculated by multiplying the viable cell density by the total volume of the cell suspension and compared to the total number of human T cells measured on day 0. Figure 1 shows the results of the expansion fold on day 7 of culture for the Examples and Comparative Examples. Figure 2 shows the results of the expansion fold of expanded human CAR-T cells obtained after 96 hours (4 days) of culture after magnetic bead removal for the Examples (day 7) and Comparative Examples (day 9). The total number of human CAR-T cells for the Examples on days 0, 3, 6, and 7, and the total number of human CAR-T cells for the Comparative Examples on days 0, 5, 6, 7, 8, and 9 are shown in Table 1 below.

[0042] [Table 1]

[0043] According to the results of Figures 1, 2 and Table 1, the total number of expanded human CAR-T cells obtained on day 7 in the Example was an expansion fold of 12.17 relative to the total number of human T cells on day 0. Although the total cell number and expansion fold obtained on day 9 in the Comparative Example were higher than those on day 7 in the Example, the total cell number on day 7 in the Comparative Example was 1.97 x 10 7 In the example (3.65 × 10 7) is much lower than that of the comparative example. In addition, the expansion ratio of 6.99 on day 7 relative to day 0 in the comparative example is only about half that of the example. Therefore, it has been demonstrated that the present invention enables T cells to grow more rapidly and proliferate significantly.

[0044] Test Example 2: Characterization of human T cell subpopulations The expanded human CAR-T cells obtained in step (6) of the Example and step (6') of the Comparative Example were fluorescently labeled with surface markers CD3, CD4, CD8, CD95, CD45RA, and CCR7, and then analyzed by flow cytometry to identify Tscm, central memory T cells (T CM ), effector memory T cells (T EM ) and terminally differentiated effector memory T cells (T EMRA We characterized human T cell subpopulations, including the IL-1 ...

[0045] [Table 2]

[0046] In the whole blood of healthy donors, Tscm usually account for 2% to 3% of the total T cell population (Gattinoni, Luca, et al. "A human memory T cell subset with stem cell-like properties." Nature Medicine 17.10(2011):1290-1297). According to the results in Table 2, Figure 3, Figure 4A, and Figure 4B, Tscm accounted for 79.68% of the expanded human CAR-T cells in the example, which is much higher than the Tscm percentage of 34.14% in the comparative example. In addition, the total number of Tscm in the example was 2.91 × 10 7 pieces (3.65×10 7 ×79.68%=2.91×10 7 pcs), whereas the total number of Tscm in the comparative example is 2.09 × 10 7 pieces (6.13×10 7 ×34.14%=2.09×10 7It can be seen that there is a 39% difference in the ratio of Tscm between the Example and the Comparative Example. EMRA The proportion of T cells in the example was 8.35%, which is significantly lower than that of the comparative example (47.81%). This is because, conceptually, the majority of T cells in the example are less differentiated and are likely not senescent, and therefore T CM and T EM They retain the ability to further differentiate into T CM , T EM and T EMRA This means that human CAR-T cells containing a high concentration of Tscm populations, such as those prepared in the Examples of the present invention, are more suitable for CAR-T cell therapy than those prepared in the Comparative Examples. Furthermore, human CAR-T cells prepared using the present invention may be able to reduce the side effects of CRS and neurotoxicity induced by CAR-T cell therapy.

[0047] In summary, the method of the present invention enables the generation of human CAR-T cells containing a high concentration of Tscm in a shorter period of time. Because the generated human CAR-T cells have a high proportion of Tscm, they have a higher ability to further differentiate, a longer lifespan, and may be able to reduce the side effects of CRS and neurotoxicity induced by CAR-T cell therapy. Therefore, human CAR-T cells generated by the method of the present invention are more suitable for CAR-T cell therapy.

[0048] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. The scope of the rights claimed by the present invention shall be determined by the claims.

Claims

1. 1. A method for producing human chimeric antigen receptor T (CAR-T) cells enriched in stem cell-like memory T cells (Tscm), comprising: (1) mixing isolated human peripheral blood mononuclear cells (PBMCs) with magnetic beads coated with anti-CD3 and anti-CD28 antibodies to form a human T cell-magnetic bead complex, and positively selecting human T cells from the isolated human PBMCs; (2) culturing the human T cell-magnetic bead complex for 16 to 20 hours to activate the T cells, thereby obtaining activated human T cell-magnetic bead complexes; (3) adding a CAR-expressing lentivirus to the activated human T cell-magnetic bead complex for 40 to 48 hours to transduce the activated human T cells with the activated human T cell-magnetic bead complex, thereby obtaining a human CAR-T cell-magnetic bead complex; (4) obtaining human CAR-T cells by dissociating the human CAR-T cells from the human CAR-T cell-magnetic bead complex; and (5) further culturing the human CAR-T cells for 92 to 96 hours to obtain human CAR-T cells containing Tscm at a high concentration, wherein the human CAR-T cells containing Tscm at a high concentration exhibit a proportion of Tscm higher than 55% based on the total number of the human CAR-T cells. A method comprising:

2. 10. The method of claim 1, wherein the isolated human PBMCs are obtained by isolation from human whole blood or leukapheresis products.

3. 2. The method of claim 1, wherein the number of magnetic beads coated with the anti-CD3 antibody and the anti-CD28 antibody in step (1) is 2 to 4 times the number of the human T cells in the isolated human PBMC.

4. The method of claim 1, wherein the human T cell-magnetic bead complex in step (2) is cultured at 36.5°C to 37.5°C for 16 to 20 hours.

5. 2. The method of claim 1, wherein the transduction in step (3) is carried out at 36.5°C to 37.5°C for 40 to 48 hours.

6. The method of claim 1, wherein the transduction in step (3) is transduction of a gene encoding anti-CD19, anti-CD22, anti-B-cell maturation antigen (BCMA), or anti-mesothelin CAR into the activated human T cells of the activated human T cell-magnetic bead complex.

7. The method of claim 1, wherein in step (5), the human CAR-T cells are cultured at 36.5°C to 37.5°C for 92 to 96 hours.

8. The method of claim 1, wherein the culturing of the human T cell-magnetic bead complex in step (2), the transduction of the activated human T cell with the activated human T cell-magnetic bead complex in step (3), and the further culturing of the human CAR-T cells in step (5) are carried out in a medium containing 200 IU / mL of IL-2.

9. The method according to any one of claims 1 to 8, wherein the number of human CAR-T cells containing Tscm at a high concentration obtained in step (5) is more than 10 times the number of human T cells positively selected using magnetic beads in step (1).

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