A culture medium for improving CAR-T cell function and its application

Abstract

The present invention relates to a culture medium, and in particular to a culture medium for improving the function of CAR-T cells and its application. The culture medium for improving the function of CAR-T cells is composed of a basal culture medium and the following components added to the basal culture medium: ergobenzyl ester, IL-2, IL-15, basic fibroblast growth factor, ethinyl estradiol, transferrin, and vitamin C. The addition of ergobenzyl ester and ethinyl estradiol can effectively improve the survival rate and proliferation rate of CAR-T cells; it can also effectively increase the proportion of central memory T cells of CAR-T cells after culture, thereby improving the efficiency and durability of CAR-T cell therapy. In addition, after culturing CAR-T cells with the culture medium of the present invention, they have a strong specific killing effect on tumor cells in vitro.

Description

Technical Field

[0001] The present invention relates to a culture medium, and in particular to a culture medium for improving the function of CAR-T cells and an application thereof. Background Art

[0002] CAR-T cell therapy is a chimeric antigen receptor T cell immunotherapy. It is considered to be a new tumor immunotherapy method for curing cancer because of its advantages such as specific recognition of tumor antigens, strong specificity, non-MHC restriction, high efficacy, CAR-T cell replication and expansion, and strong killing effect.

[0003] The CAR-T cell killing process of tumors mainly includes two pathways: one is that after CAR-T cells bind to tumor cells through immune synapses, they secrete substances such as cytolysin and granzyme B. After cytolysin forms holes in the membrane of tumor cells, CAR-T cells transport granzyme B into the tumor cells through the holes, causing a chain reaction of cysteine ​​aspartic acid proteases, causing tumor cells to lyse; the other is that TNF ligands exist on the surface of CAR-T cells, which can induce apoptosis of target cells after binding to target cells. In addition, CAR-T cells can also promote their own activity through autocrine cytokines to further enhance their ability to kill tumors.

[0004] CAR-T cells are cells that integrate the DNA construct of CAR into T cells isolated from the patient's own peripheral blood through methods such as lentiviral transfection in vitro, and then amplify in vitro before returning to the patient's body after lymphocyte clearance pretreatment to exert anti-tumor effects. Existing in vitro amplification culture media have technical problems such as low CAR-T cell survival rate and proliferation rate, as well as low tumoricidal activity of CAR-T cells after culture. Therefore, it is urgent to provide a new type of CAR-T cell culture medium. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a culture medium for improving the function of CAR-T cells, which can effectively improve the survival rate and proliferation rate of CAR-T cells; and can effectively increase the proportion of central memory T cells of CAR-T cells after culture.

[0006] The second purpose of the present invention is to provide a culture medium for improving the function of CAR-T cells and its application in CAR-T cell culture. After culturing with the culture medium of the present invention, CAR-T cells have a strong specific killing effect on tumor cells in vitro.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] A culture medium for improving the function of CAR-T cells, comprising a basic culture medium and the following components added to the basic culture medium: ergobenzyl ester, IL-2, IL-15, basic fibroblast growth factor, ethinyl estradiol, transferrin, and vitamin C.

[0009] Furthermore, in terms of final concentration, the concentrations of the added components in the basal culture medium are: ergobenzyl ester 110-130 ng / mL, IL-2 60-80 ng / mL, IL-15 3-7 ng / mL, basic fibroblast growth factor 10-20 ng / mL, ethinyl estradiol 1-6 μg / mL, transferrin 5-12 μg / mL, and vitamin C 35-50 μg / mL.

[0010] Furthermore, the basal culture medium is DMEM serum-free culture medium.

[0011] The second object of the present invention is achieved by adopting the following technical solution:

[0012] The use of the above-mentioned culture medium for improving the function of CAR-T cells in CAR-T cell culture comprises the following steps: inoculating CAR-T cells in the culture medium and culturing them for 13-15 days.

[0013] Furthermore, the concentration of the CAR-T cells in the culture medium is 4×10 5 -6×10 5 Pieces / mL.

[0014] Furthermore, the culture conditions are 37°C and 5% CO2.

[0015] Furthermore, the culture medium needs to be replaced every 2-3 days during the culture.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. Improvement of culture medium formulation

[0018] The present invention provides a novel culture medium for improving the function of CAR-T cells, wherein ingredients such as ergobenzyl ester and ethinyl estradiol are added to the culture medium formula.

[0019] 2. Achieved unexpected technical effects

[0020] The test results of the present invention show that:

[0021] (1) The addition of ergosterol and ethinyl estradiol can effectively improve the survival rate and proliferation rate of CAR-T cells.

[0022] (2) The addition of ergosterol and ethinyl estradiol can also effectively increase the proportion of central memory T cells in CAR-T cells after culture, thereby improving the efficiency and durability of CAR-T cell therapy.

[0023] (3) CAR-T cells cultured with the culture medium of the present invention have a strong specific killing effect on tumor cells in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a morphological diagram of CAR-T obtained in Example 1 of the present invention;

[0025] Figure 2 This is a result analysis diagram of the percentage of central memory T cells of CAR-T cells after culture in the culture medium of Examples 1-3 and Comparative Examples 1-6 of the present invention;

[0026] Figure 3 This is a result analysis diagram of the specific killing effect of CAR-T cells on tumor cells in vitro after being cultured in the culture medium of Examples 1-3 and Comparative Examples 1-6 of the present invention. DETAILED DESCRIPTION

[0027] The technical scheme of the present invention is further explained below in conjunction with specific embodiments, comparative examples and test examples. In the following embodiments, comparative examples and test examples, the raw materials and preparation methods used are conventional materials and techniques in the art unless otherwise specified.

[0028] Example 1

[0029] Example 1 provides a culture medium for improving the function of CAR-T cells, the culture medium consisting of DMEM serum-free medium and the following components added to the DMEM serum-free medium: ergobenzyl ester, IL-2, IL-15, basic fibroblast growth factor, ethinyl estradiol, transferrin, and vitamin C. In terms of final concentration, the concentrations of the above-mentioned added components in the DMEM serum-free medium are: ergobenzyl ester 120ng / mL, IL-2 75ng / mL, IL-15 6ng / mL, basic fibroblast growth factor 12ng / mL, ethinyl estradiol 3μg / mL, transferrin 7μg / mL, and vitamin C 41μg / mL.

[0030] Example 1 also provides the use of the above-mentioned culture medium for improving the function of CAR-T cells in CAR-T cell culture, and the specific process is as follows:

[0031] 1) Preparation of CAR-T cells:

[0032] Umbilical cord blood was obtained and CAR-T cells were separated. After centrifugation, 2 mg / mL trypsin EDTA was added for digestion. Then 2 mL DMEM complete medium was added to terminate trypsin digestion. D-Hanks solution was added and centrifuged for 5 min at a centrifugal force of 320 g and a centrifugal speed of 1600 r / min. CAR-T cells were obtained. The cell morphology is shown in Figure 2. Figure 1 .

[0033] 2) The CAR-T cells from step 1) were inoculated into the above-mentioned culture medium for improving the function of CAR-T cells. The concentration of CAR-T cells in the culture medium was 5×10 5 The cells were cultured at 37°C and 5% CO2 for 14 days, and the culture medium was replaced every 2 days during the culture process.

[0034] Example 2

[0035] Example 2 provides a culture medium for improving the function of CAR-T cells, the culture medium consisting of DMEM serum-free medium and the following components added to the DMEM serum-free medium: ergobenzyl ester, IL-2, IL-15, basic fibroblast growth factor, ethinyl estradiol, transferrin, and vitamin C. In terms of final concentration, the concentrations of the above-mentioned added components in the DMEM serum-free medium are: ergobenzyl ester 130ng / mL, IL-2 80ng / mL, IL-15 3ng / mL, basic fibroblast growth factor 20ng / mL, ethinyl estradiol 1μg / mL, transferrin 12μg / mL, and vitamin C 35μg / mL.

[0036] Example 2 also provides the use of the above-mentioned culture medium for improving the function of CAR-T cells in CAR-T cell culture, and the specific process is as follows:

[0037] 1) Preparation of CAR-T cells:

[0038] Same as Example 1.

[0039] 2) The CAR-T cells from step 1) were inoculated into the above-mentioned culture medium for improving the function of CAR-T cells. The concentration of CAR-T cells in the culture medium was 6×10 5 The cells were cultured at 37°C and 5% CO2 for 13 days, and the culture medium was replaced every 2 days during the culture process.

[0040] Example 3

[0041] Example 3 provides a culture medium for improving the function of CAR-T cells, the culture medium consisting of DMEM serum-free medium and the following components added to the DMEM serum-free medium: ergobenzyl ester, IL-2, IL-15, basic fibroblast growth factor, ethinyl estradiol, transferrin, and vitamin C. In terms of final concentration, the concentrations of the above-mentioned added components in the DMEM serum-free medium are: ergobenzyl ester 110 ng / mL, IL-2 60 ng / mL, IL-15 7 ng / mL, basic fibroblast growth factor 10 ng / mL, ethinyl estradiol 6 μg / mL, transferrin 5 μg / mL, and vitamin C 50 μg / mL.

[0042] Example 3 also provides the use of the above-mentioned culture medium for improving the function of CAR-T cells in CAR-T cell culture, and the specific process is as follows:

[0043] 1) Preparation of CAR-T cells:

[0044] Same as Example 1.

[0045] 2) The CAR-T cells from step 1) were inoculated into the above-mentioned culture medium for improving the function of CAR-T cells. The concentration of CAR-T cells in the culture medium was 4×10 5 The cells were cultured at 37°C and 5% CO2 for 15 days, and the culture medium was replaced every 3 days during the culture process.

[0046] Comparative Example 1

[0047] Comparative Example 1 provides a culture medium, which consists of a DMEM serum-free culture medium and the following components added to the DMEM serum-free culture medium: IL-2, IL-15, basic fibroblast growth factor, ethinyl estradiol, transferrin, and vitamin C. In terms of final concentration, the concentrations of the above-mentioned added components in the DMEM serum-free culture medium are: IL-2 75 ng / mL, IL-15 6 ng / mL, basic fibroblast growth factor 12 ng / mL, ethinyl estradiol 3 μg / mL, transferrin 7 μg / mL, and vitamin C 41 μg / mL, and the rest are the same as in Example 1.

[0048] Comparative Example 2

[0049] Comparative Example 2 provides a culture medium, which consists of a DMEM serum-free culture medium and the following components added to the DMEM serum-free culture medium: ergobenzyl ester, IL-2, IL-15, basic fibroblast growth factor, ethinyl estradiol, transferrin, and vitamin C. In terms of final concentration, the concentrations of the above-mentioned added components in the DMEM serum-free culture medium are: ergobenzyl ester 30 ng / mL, IL-2 75 ng / mL, IL-15 6 ng / mL, basic fibroblast growth factor 12 ng / mL, ethinyl estradiol 3 μg / mL, transferrin 7 μg / mL, and vitamin C 41 μg / mL, and the rest are the same as in Example 1.

[0050] Comparative Example 3

[0051] Comparative Example 3 provides a culture medium, which is composed of a DMEM serum-free culture medium and the following components added to the DMEM serum-free culture medium: ergobenzyl ester, IL-2, IL-15, basic fibroblast growth factor, ethinyl estradiol, transferrin, and vitamin C. In terms of final concentration, the concentrations of the above-mentioned added components in the DMEM serum-free culture medium are: ergobenzyl ester 60 ng / mL, IL-2 75 ng / mL, IL-15 6 ng / mL, basic fibroblast growth factor 12 ng / mL, ethinyl estradiol 3 μg / mL, transferrin 7 μg / mL, and vitamin C 41 μg / mL, and the rest are the same as in Example 1.

[0052] Comparative Example 4

[0053] Comparative Example 4 provides a culture medium, which is composed of a DMEM serum-free culture medium and the following components added to the DMEM serum-free culture medium: ergobenzyl ester, IL-2, IL-15, basic fibroblast growth factor, ethinyl estradiol, transferrin, and vitamin C. In terms of final concentration, the concentrations of the above-mentioned added components in the DMEM serum-free culture medium are: ergobenzyl ester 90 ng / mL, IL-2 75 ng / mL, IL-15 6 ng / mL, basic fibroblast growth factor 12 ng / mL, ethinyl estradiol 3 μg / mL, transferrin 7 μg / mL, and vitamin C 41 μg / mL, and the rest are the same as in Example 1.

[0054] Comparative Example 5

[0055] Comparative Example 5 provides a culture medium, which is composed of a DMEM serum-free culture medium and the following components added to the DMEM serum-free culture medium: ergobenzyl ester, IL-2, IL-15, basic fibroblast growth factor, ethinyl estradiol, transferrin, and vitamin C. In terms of final concentration, the concentrations of the above-mentioned added components in the DMEM serum-free culture medium are: ergobenzyl ester 150 ng / mL, IL-2 75 ng / mL, IL-15 6 ng / mL, basic fibroblast growth factor 12 ng / mL, ethinyl estradiol 3 μg / mL, transferrin 7 μg / mL, and vitamin C 41 μg / mL, and the rest are the same as in Example 1.

[0056] Comparative Example 6

[0057] Comparative Example 6 provides a culture medium, which consists of a DMEM serum-free medium and the following components added to the DMEM serum-free medium: ergobenzyl ester, IL-2, IL-15, basic fibroblast growth factor, estradiol, transferrin, and vitamin C. In terms of final concentration, the concentrations of the above-mentioned added components in the DMEM serum-free medium are: ergobenzyl ester 120 ng / mL, IL-2 75 ng / mL, IL-15 6 ng / mL, basic fibroblast growth factor 12 ng / mL, estradiol 3 μg / mL, transferrin 7 μg / mL, and vitamin C 41 μg / mL.

[0058] Test Example 1

[0059] Experimental Example 1 The survival rate and proliferation rate of CAR-T cells after culture in the culture medium of Examples 1-3 and Comparative Examples 1-6 were investigated, and the detection method was as follows:

[0060] ① Survival rate: The concentration of cell suspension in each test group was adjusted to 1×10 5 / mL, the cell suspensions of each group were mixed with 0.4% trypan blue dye at a ratio of 9:1, and observed under an optical microscope after 2 minutes. Among them, living cells were not stained, and dead cells were stained. Cell survival rate = (total cell number - dead cell number) / total cell number × 100%, the results are shown in Table 1.

[0061] ② Proliferation rate: The number of CAR-T cells in Example 1-3 and Comparative Example 1-6 groups after the culture was completed was counted using a cell counter. Proliferation rate = (total number of cells after culture - number of cells inoculated) / number of cells inoculated × 100%. The results are shown in Table 1.

[0062] Table 1

[0063]

[0064] As can be seen from Table 1, compared with Example 1-3, the survival rate and proliferation rate of CAR-T cells after culture in Comparative Example 1-4 culture medium are significantly reduced. The formula of the culture medium of Example 1-3 and Comparative Example 1-4 is compared and analyzed, wherein no ergobenzyl ester is added to the formula of the culture medium of Comparative Example 1. The dosage of ergobenzyl ester is reduced in the formula of the culture medium of Comparative Example 2-4. Compared with Example 1-3, there is no significant difference in the survival rate and proliferation rate of CAR-T cells after culture in the culture medium of Comparative Example 5. The dosage of ergobenzyl ester is increased in the formula of the culture medium of Comparative Example 5. It can be seen from the above test results that the addition of ergobenzyl ester can effectively improve the survival rate and proliferation rate of CAR-T cells, and when the concentration of ergobenzyl ester is 110-130ng / mL, the effect is better; when the concentration of ergobenzyl ester is 120ng / mL, the effect is best.

[0065] Compared with Examples 1-3, the survival rate and proliferation rate of CAR-T cells after culture in the culture medium of Comparative Example 6 were reduced. The formula of the culture medium of Examples 1-3 and Comparative Example 6 was compared and analyzed, and Comparative Example 6 used estradiol instead of ethinyl estradiol. It can be seen from the above test results that changing the ingredients in the culture medium formula of the present invention will reduce the survival rate and proliferation rate of CAR-T cells after culture.

[0066] Test Example 2

[0067] The proportion of central memory T cells of CAR-T cells after culture in the culture medium of Example 1-3 and Comparative Example 1-6 was investigated, and the detection method was as follows:

[0068] After culture in the culture medium of Example 1-3 and Comparative Example 1-6, the CAR-T cells were counted and placed in flow cytometry tubes, with 2×10 cells per tube. 4 After centrifugation, discard the supernatant, wash the cell pellet with PBS, centrifuge again, discard the supernatant, add 200μL PBS and 1μL anti-human CD62L and 1μL anti-human CD45RO antibodies to each tube, and incubate in the dark for 30min. Wash the antibody with PBS, discard the supernatant after centrifugation, resuspend the cell pellet in PBS, and detect it by flow cytometry. The present invention uses CD45RO and CD62L as the phenotypic standards of central memory T cells, and the results are shown in Figure 2 .

[0069] Figure 2 This is a result analysis chart of the percentage of central memory T cells of CAR-T cells after culture in the culture medium of Examples 1-3 and Comparative Examples 1-6 of the present invention. Figure 2It is known that, compared with comparative examples 1-6, the use of the culture medium of Example 1-3 can effectively increase the central memory T cell ratio of CAR-T cells after culture. It can be seen from the above test results that the culture medium of the present invention can effectively increase the central memory T cell ratio of CAR-T cells, thereby improving the efficiency and durability of CAR-T cell therapy.

[0070] Test Example 3

[0071] Experimental Example 3 explores the in vitro specific killing effect of CAR-T cells on tumor cells and the in vivo anti-tumor activity after culture in the culture medium of Examples 1-3 and Comparative Examples 1-6. The detection method is as follows:

[0072] After culture in the culture medium of Example 1-3 and Comparative Example 1-6, CAR-T cells were added to K562 at an effector-target ratio of 20:1 and inoculated into a 96-well plate. After co-culture for 6 hours, the cells were centrifuged, the supernatant was discarded, 100 mL of DMSO was added to each well and vibrated for dissolution for 10 minutes. The OD value of each test group at 570 nm was detected by a microplate reader. At the same time, a blank control group, a target cell control group, and an effector cell control group were set up. The in vitro specific killing activity = [OD value of the target cell control group - (OD value of the test group - OD value of the effector cell control group)] / OD value of the target cell control group × 100%. The results are shown in Figure 3 .

[0073] Figure 3 This is a result analysis diagram of the specific killing effect of CAR-T cells on tumor cells in vitro after culture in the culture medium of Examples 1-3 and Comparative Examples 1-6 of the present invention. Figure 3 It can be seen that compared with Comparative Examples 1-6, the CAR-T cells cultured with the culture medium of Example 1-3 have a stronger specific killing effect on tumor cells in vitro.

[0074] In summary, CAR-T cells cultured with the culture medium of the present invention have a strong specific killing effect on tumor cells in vitro.

[0075] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A culture medium for improving the function of CAR-T cells, characterized in that: It consists of a basal medium and the following components added to the basal medium: ergobenzyl ester, IL-2, IL-15, basic fibroblast growth factor, ethinyl estradiol, transferrin, and vitamin C; In terms of final concentration, the concentrations of the added components in the basal culture medium are: ergobenzyl ester 110-130 ng / mL, IL-2 60-80 ng / mL, IL-15 3-7 ng / mL, basic fibroblast growth factor 10-20 ng / mL, ethinyl estradiol 1-6 μg / mL, transferrin 5-12 μg / mL, and vitamin C 35-50 μg / mL; the basal culture medium is DMEM serum-free culture medium.

2. The use of the culture medium for improving the function of CAR-T cells according to claim 1 in CAR-T cell culture, characterized in that: The following steps are involved: The CAR-T cells were inoculated into the culture medium and cultured for 13-15 days.

3. Use of the culture medium for improving CAR-T cell function according to claim 2 in CAR-T cell culture, characterized in that: The concentration of the CAR-T cells in the culture medium was 4×10 5 -6×10 5 Pieces / mL.

4. The use of the culture medium for improving the function of CAR-T cells in CAR-T cell culture according to claim 2, characterized in that: The culture conditions were 37° C. and 5% CO 2 .

5. Use of the culture medium for improving CAR-T cell function according to claim 2 in CAR-T cell culture, characterized in that: During the culture, the culture medium needs to be replaced every 2-3 days.

Images