CN112239749B

CN112239749B - Methylated reprogramming CAR T cells with improved ratios of killing, secretion, anti-depletion and CD4, and preparation method and application thereof

Info

Publication number: CN112239749B
Application number: CN201910655957.2A
Authority: CN
Inventors: 王瑶; 韩为东; 伍志强; 佟川; 韩笑; 郭业磊; 代汉仁; 刘洋; 遆冬冬; 王晓慧
Original assignee: Chinese PLA General Hospital
Current assignee: Chinese PLA General Hospital
Priority date: 2019-07-19
Filing date: 2019-07-19
Publication date: 2022-09-02
Anticipated expiration: 2039-07-19
Also published as: CN112239749A

Abstract

The invention relates to the field of cell engineering and discloses a methylated reprogramming CAR T cell with killing, secretion, anti-exhaustion and CD4 ratio improvement, and a preparation method and application thereof. The CAR T cell modified by the method rapidly shows strong killing capacity within 12 hours, particularly under the condition of ultralow effective target ratio (E: T ═ 1:30), dCART shows remarkable killing capacity, enhances the secretion and amplification level of cytokines, has good clinical safety, considerable overall anti-tumor effect, has continuous anti-tumor capacity in vivo and has huge clinical application value compared with common CART.

Description

Methylated reprogramming CAR T cells with characteristics of killing, secretion, exhaustion resistance and increased CD4 ratio, and preparation method and application thereof

Technical Field

The invention relates to the field of cell engineering, in particular to a preparation method of CAR T cells (methylated reprogramming CAR T cells) with characteristics of killing, secretion, anti-exhaustion and improved CD4/CD8 ratio, the methylated reprogramming CAR T cells prepared by the method, and application of the cells in preparation of preparations for treating tumors.

Background

CAR-T, a chimeric antigen receptor T cell, is one of the more effective therapeutic modalities for malignant tumors at present. Chimeric Antigen Receptors (CARs) are a core component of CAR-T, conferring to T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-engineered T cells to recognize a broader range of targets than native T cell surface receptor TCRs. The basic design of a CAR includes a tumor-associated antigen (TAA) binding region (usually the scFV fragment from the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The choice of antigen of interest is a key determinant for the specificity, efficacy of the CAR and safety of the genetically engineered T cells themselves.

At present, CAR T cells have already obtained very good curative effect on hematological tumors, and more than 80% of patients obtain complete remission in B cell leukemia; in patients with B-cell lymphoma, more than 50% of patients will achieve complete remission. With the application of CAR T cells, several problems were also exposed with high remission rates. For example, 40% of patients who are in complete remission will relapse within one year, and the relapsed patients are again unresponsive to CAR T cell therapy; in addition, there remains a 20-50% of patients who respond to CAR T cell therapy and new clinical problems arising from these critical CAR T therapies need to be addressed.

Disclosure of Invention

The invention aims to reprogram CAR T cells under the action of chemical drugs, and provides a preparation method of methylated reprogrammed CAR T cells (D-CAR T cells for short) with improved killing capacity, cytokine secretion capacity, anti-exhaustion capacity and CD4 ratio, wherein the D-CAR T cells prepared by the method have improved cytokine secretion capacity, anti-exhaustion capacity and CD4/CD8 ratio, and have higher killing power on tumor cells.

The inventors of the present invention found that improving CAR T cell genome components to block or attenuate CAR T cell depleted cellular pathways, increasing CD4/CD8 cell ratio, increasing cytokine secretion capacity, enhancing T cell activation, proliferation and killer cell pathways may be effective means to effectively extend CAR T in vivo duration of action, reducing disease recurrence. Therefore, there is a need to develop a method for preparing CAR T cells with more potent killing ability, strong in vivo persistence ability, high CD4/CD8 cell ratio, and resistance to exhaustion. The inventor of the invention further finds in research that the low-dose DNA demethylation drug can reprogram CAR T cells, improve the killing capacity, cytokine secretion capacity, anti-exhaustion capacity and CD4/CD8 ratio of the CAR T cells, especially under the condition of a very low effective target ratio, the D-CAR T cells still keep high-efficiency killing capacity, and the exhaustion degree is lower than that of common CAR T, so that the D-CAR T cells can more effectively play an in-vivo anti-tumor role.

Therefore, in order to achieve the above objects, in a first aspect, the present invention provides a method of producing a methylated reprogrammed CAR T cell, the method comprising culturing a CAR T cell in the presence of a DNA demethylating drug, or infecting a T cell with a lentiviral expression vector expressing a CAR in the presence of a DNA demethylating drug, to obtain a methylated reprogrammed CAR T cell having improved killing ability, cytokine secretion ability, anti-depletion ability, and CD4/CD8 ratio.

In a second aspect, the invention provides a D-CAR T cell made by the method described above.

In a third aspect, the invention provides the use of a D-CAR T cell as described above in the preparation of an agent for the treatment of a tumour.

In a fourth aspect, the invention provides the use of a DNA demethylating drug in the preparation of a methylated reprogrammed CAR T cell having improved killing ability, cytokine secretion ability, anti-exhaustion ability and CD4/CD8 ratio.

The invention can significantly increase the ratio of CD4/CD8 in CAR T cells without affecting the expression of the corresponding CAR by reprogramming the CAR T cells with a DNA demethylating drug; and has more efficient and rapid killing capability on target cells, especially under the condition of ultra-low effective target ratio; after contacting with target cells, the secretion amount of cytokines (such as TNF-alpha, IL-2 and IFN-gamma) is obviously increased, the killing activity to tumor cells is obviously enhanced, and in addition, the reprogrammed CAR T cells block or weaken a CAR T cell exhausted cell pathway and enhance the T cell activation, proliferation and cell killing pathway through genomics analysis. The D-CAR T cell provided by the invention has good clinical safety, no toxic or side effect, considerable overall anti-tumor effect and great clinical application value. In particular, the method comprises the following steps of,

1) as shown in example 2, DNA demethylating drugs did not substantially affect CAR expression in CAR 19T cells;

2) as shown in example 3, the ratio of CD4/CD8 in CAR 19T cells was significantly increased and was close to 1;

3) as shown in example 4, at an effective target ratio of 0.2:1, 83% of the target cells were killed within 20 hours, significantly higher than CAR 19T cells and NT groups that were not treated with DNA demethylating drugs;

4) as shown in example 5, the ability of demethylating drug-enhanced CAR 19T cells to secrete cytokines was significantly higher than that of the plain type CAR 19T cells;

5) as shown in example 6, under the condition of an ultra-low effective target ratio (E: T ═ 1:30), the D-CAR 19T cell provided by the invention rapidly shows a strong killing capability within 12 hours, and compared with the ordinary CAR 19T, the D-CAR 19T cell shows a remarkable killing capability, and the 96-hour killing efficiency can reach more than 90%;

6) as shown in example 7, the memory phenotype cells of D-CAR 19T obtained by the method are increased, which shows that the D-CAR T prepared by the method provided by the invention has more efficient and rapid killing capability on target cells;

7) as shown in example 8, it was shown by genomic analysis that reprogrammed CAR 19T cells blocked or attenuated the CAR 19T cell-depleted cellular pathway, enhancing T cell activation, proliferation and killing cellular pathways.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 shows the effect of CAR19 expression in DNA demethylating drug treated CAR 19T cells (FIG. 1-1) and untreated CAR 19T cells (FIG. 1-2);

FIG. 2 is a graph of the CD4/CD8 ratio increase in CAR 19T cells after DNA demethylation drug treatment;

FIG. 3 is a graph of the killing effect of DNA demethylating drug treated CAR 19T cells on CD19 positive Raji cells at an effective target ratio of 0.2: 1;

FIG. 4 shows the secretion of cytokines (IL-2 (FIG. 4-1), IFN- γ (FIG. 4-2), TNF- α (FIG. 4-3)) in the culture supernatant after DNA demethylating drug treated CAR 19T cells were exposed to the effective target ratio of 1:1 for 24 hours;

FIG. 5 shows killing of Naml6 cells by D-CAR T cells prepared by the method of the invention in the case of ultra-low target ratios (E: T ═ 1: 30);

FIG. 6 is the memory phenotype cell count of D-CAR T cells obtained by the methods of the invention;

FIG. 7 is a diagram of a genomics analysis of D-CAR T cells obtained by the method of the invention.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are given by way of illustration and explanation only, not limitation.

In the present invention, unless otherwise specified, the steps such as culturing are carried out in vitro.

In a first aspect, the invention provides a method of making a methylated reprogrammed CAR T cell, the method comprising culturing a CAR T cell in the presence of a DNA demethylating drug, or infecting a T cell with a lentiviral expression vector expressing a CAR in the presence of a DNA demethylating drug, to obtain a methylated reprogrammed CAR T cell having improved killing ability, cytokine secretion ability, anti-exhaustion ability, and a CD4/CD8 ratio.

According to the present invention, the timing of the addition of the DNA demethylating agent can be selected within a wide range, for example, it can be added immediately after the CAR T cell culture (i.e., the first day of culture) or after a certain period of culture. Preferably, the DNA demethylating agent is added on days 1-7 of CAR T cell culture. More preferably, the DNA demethylating agent is added at 12-72 hours, e.g., 12 hours, 15 hours, 18 hours, 20 hours, 25 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, 72 hours of CAR T cell culture.

According to the invention, the time for which the CAR T cells are cultured in DNA demethylating drugs can also be selected within a wide range. Preferably, the CAR T cells are cultured in the presence of the DNA demethylating agent for a time period of 24-72 hours (e.g., 24 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, 72 hours, or a range between any two of the foregoing values), within which the CAR T cells can more efficiently undergo DNA reprogramming, thereby achieving the objects of the invention.

According to the present invention, in the case where a DNA demethylating agent is added during infection of a T cell by a CAR-expressing lentiviral expression vector, the method further comprises culturing the infected T cell (i.e., the resulting CAR T cell) in the DNA demethylating agent for a further 24-72 hours (e.g., 24 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, 72 hours, or a range between any two of the foregoing values) after the infection is complete.

According to the invention, there is no particular requirement for the amount of the DNA demethylating agent, but preferably the amount of the DNA demethylating agent is such that it is present in the cell culture medium in a concentration of 5-2000nM, preferably 10-1000nM (e.g.10 nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1000nM or a range between any of the two).

According to the invention, the medium used for the culture may be a medium conventional in the art for CAR T cells, and may be, for example, X-VIVO 15 ^TM Serum-free cell culture media.

According to the present invention, the DNA demethylating agent may be a conventional agent for DNA demethylation, such as decitabine and/or azacytidine, and the like. Preferably, the DNA demethylating agent is decitabine.

According to the invention, the CAR T cell to be reprogrammed may be any CAR T cell in the art, which may be a single CAR T cell and/or a multiple CAR T cell. Wherein the term "single CAR T cell" refers to a single targeted CAR T cell, i.e., the CAR portion expresses one receptor, and the term "multi CAR T cell" refers to a multi-targeted CAR T cell, i.e., the CAR portion expresses at least two receptors.

Preferably, the CAR T cell is selected from the group consisting of a CAR 19T cell, a CAR 20T cell, a CAR 22T cell, a CAR 30T cell, a CAR 133T cell, a carmln T cell, a carmer 1T cell, a carmer 2T cell, a CAR 138T cell, a CAR 20-19T cell, and a CAR 22-19T cell; more preferably, the CAR T cells are CAR 19T cells, CAR 20T cells, CAR 22T cells, CAR 20-19T cells, and CAR 22-19T cells.

Where it is noted that the numbers followed by CAR in CAR T cells as above represent the type of antigen targeted by the CAR T cells, for example CAR 19T cells represent CAR T cells targeting CD19, and the like.

Wherein the preparation of each CAR T cell is well known to those skilled in the art, for example, CAR 19T cells, CAR 22T cells, CAR 22-19T cells can be prepared according to the methods disclosed in 201811190780.5; CAR 20T cells, preparation of CAR 20-19T cells can be according to the method disclosed in 201811062721. X; CAR 30T cells can be prepared according to the methods disclosed in 2015106705068; CAR 133T cells can be prepared according to the method disclosed in 201648750. X; the preparation of CAR 138T cells can be according to the method disclosed in 201510531218.4, which patent applications are incorporated herein by reference in their entirety.

According to the present invention, the CAR T cell culture conditions may be those known in the art for culturing T cells, which are described in the above-cited patent applications, and are not repeated here in order to avoid unnecessary repetition.

According to the present invention, preferably, to further facilitate reprogramming of CAR T cells, the culturing is performed in the presence of cytokines; more preferably, the cytokine is interleukin 2, and still more preferably, the cytokine is recombinant human interleukin 2.

In a second aspect, the invention discloses methylated reprogrammed CAR T cells made as described above.

According to the invention, compared with CAR T cells which are not co-cultured by DNA demethylation drugs, the CAR T cells which are reprogrammed by DNA demethylation drugs have obviously improved CD4/CD8 ratio which is close to 1, the cytokine secretion capacity is also obviously improved, the killing efficiency can reach more than 90% in 96 hours under the condition of 1:30 effective target ratio, and the anti-exhaustion capacity and the activation and proliferation capacity are also enhanced through genomics analysis.

In a third aspect, the invention provides the use of a methylated reprogrammed CAR T cell as described above in the preparation of a formulation for the treatment of a tumour.

Wherein the type of tumor to be treated can be selected according to different CAR T cell types, which are well known to those skilled in the art and will not be described in detail here.

In a fourth aspect, the present invention provides the use of a DNA demethylating drug in the preparation of a methylated reprogrammed CAR T cell, as described above, the inventors of the present invention have found that treatment of a CAR T cell with a DNA demethylating drug enables reprogramming of a CAR T cell, resulting in a methylated reprogrammed CAR T cell having improved killing ability, cytokine secretion ability, anti-exhaustion ability and CD4/CD8 ratio.

The present invention will be described in detail below by way of examples.

The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used in the following examples were all obtained commercially, unless otherwise specified.

The CD8 monoclonal antibody, the CD3 monoclonal antibody, the CD4 monoclonal antibody, the Fab monoclonal antibody, the CD62L monoclonal antibody and the CD45RA monoclonal antibody are all purchased from BD company in the United states.

T cell culture solution X-VIVO 15 ^TM Serum-free cell culture medium was purchased from Lonza corporation, usa.

ELISA kits for IL-2, IFN γ and TNF- α were purchased from R & D, USA.

Raji cells expressing CD19 were purchased from ATCC, usa.

Naml6 cells expressing both CD19+ and CD20+ were purchased from ATCC in the USA.

K562 cells expressing CD19 were purchased from ATCC in usa.

pLenti-CMV-luc2-IRES-Puro virus was purchased from Shanghai and Yuan.

Example 1

This example illustrates the preparation of reprogrammed CAR 19T cells (D-CAR 19T)

Lentivirus (lentivirus expression vector pRRLsin-CAR19) infected T cells were prepared as disclosed in 201811190780.5, and the cells infected for 12 hours and discarded culture were transferred to a 6-well plate without any coating, and fresh T cell culture broth, 300U/mL of recombinant human interleukin 2 and Decitabine (DAC) were added at final concentrations of 0, 10nM, 100nM, 1000nM, 2000nM, respectively, to give a concentration of 1X 10T cells ⁶ CO at 37 ℃ and 5% saturation humidity/ml ₂ And (3) continuously culturing in the incubator for 3 days to obtain decitabine enhanced CAR 19T cells (namely reprogrammed CAR 19T cells), namely D-CAR 19T for short.

Example 2

This example serves to illustrate the effect of CAR19 expression in D-CAR 19T of the present invention.

500. mu.l each of D0-CAR 19T (indicating a final concentration of decitabine of 0) and D100-CAR 19T (indicating a final concentration of decitabine of 100nM) in example 1 were taken, centrifuged and the supernatant discarded; cells were washed 2 times with PBS, centrifuged to discard the supernatant, 100 μ l PBS was added, CAR19 expression detection antibody (Fab antibody) was added, and the effect of CAR19 expression was examined by flow cytometry. Wherein, the expression of CAR19 in D0-CAR 19T is shown in FIG. 1-2 (54.9%), and the expression of CAR19 in D100-CAR 19T is shown in FIG. 1-1 (53.23%).

The results show that CAR expression in D-CAR 19T is similar to the non-enhanced CART results with no significant difference, i.e. addition of DAC did not substantially affect CAR19 expression in CAR 19T cells.

Example 3

This example serves to illustrate the increase in the cellular proportion of CD4/CD8 in D-CAR 19T of the invention.

Taking 500. mu.l each of the T cells (abbreviated as NT) prepared according to the method disclosed in 201811190780.5, D0-CAR 19T (indicating that the final concentration of decitabine is 0) and D100-CAR 19T (indicating that the final concentration of decitabine is 100nM) in example 1, centrifuging and discarding the supernatant; the cells were washed 2 times with PBS, centrifuged to discard the supernatant, 100. mu.l of PBS was added, and phenotypically detected flow antibodies (CD4, CD8) were added, respectively, and cell phenotype was detected by flow cytometry, and CD4/CD8 was calculated, and the results are shown in FIG. 2.

As can be seen from fig. 2, CD4/CD8 was significantly improved in CAR 19T cells treated with decitabine (D100-CAR 19T) compared to T cells (NT), CAR 19T cells not treated with decitabine (D0-CAR 19T), and was close to 1.

Example 4

This example illustrates the dynamic assay of killing activity of D-CAR 19T by RTCA

The T cell-mediated killing activity of infected T cells (abbreviated NT) was examined using T cells prepared according to the method disclosed in 201811190780.5, D0-CAR 19T (indicating a final concentration of decitabine of 0) and D100-CAR 19T (indicating a final concentration of decitabine of 100nM) in example 1, and Raji cells expressing CD 19. Each well of the 96-well E-Plates was inoculated with 5,000 Raji cells expressing CD19 as target cells and cultured with effector cells for 7 days, while Raji cells expressing CD19 were cultured with medium without effector cells for 7 days as positive controls (i.e., showing the growth of target cells in the absence of effector cells), CAR 19T cells were cultured alone for 7 days as negative controls, and the RTCA system recorded the growth of Raji cells expressing CD19 per hour. The NT is added at a potent target ratio (effector cell: target cell) of 1:1, the D0-CAR 19T, D100-CAR 19T is added at a potent target ratio (effector cell: target cell) of 0.2:1, the NT, D0-CAR 19T and D100-CAR 19T are used as effector cells, and the killing process of the effector cells on the target cells is dynamically detected, and the result is shown in FIG. 3.

As can be seen from FIG. 3, D100-CAR 19T killed 83% of the Raji cells positive for CD19 within 20 hours at an effective target ratio of 0.2:1, which is significantly higher than those in the D0-CAR 19T and NT groups.

Example 5

This example illustrates the cytokine release of D-CAR 19T

(1) The effector cells and target cells (Raji cells expressing CD19 and K562 cells, respectively) of D0-CAR 19T (indicating a final concentration of decitabine of 0), D5-CAR 19T (indicating a final concentration of decitabine of 5nM), D10-CAR 19T (indicating a final concentration of 10Nm), and D2000-CAR 19T (indicating a final concentration of 2000nM) of example 1 were added to a 96-well plate at an effective-to-target ratio of 1:1 at 37 ℃ with 5% CO ₂ After 24 hours of interaction in the cell culture chamber, the supernatant was collected and centrifuged. Cytokine levels in the supernatants were measured individually according to the ELISA kit instructions for IL2, IFN-. gamma.and TNF. alpha. (labeled D-CAR 19T in the figure). As shown in FIG. 4 (IL2 is shown in FIG. 4-1, IFN-. gamma.is shown in FIG. 4-2, TNF. alpha. is shown in FIG. 4-3).

In addition, cytokine levels in the supernatants of each effector cell not contacted with target cells (labeled D-CAR 19T in the figure) were measured separately according to ELISA kit instructions for IL2, IFN-. gamma., TNF. alpha., as shown in FIG. 4 (IL2 in FIG. 4-1, IFN-. gamma., FIG. 4-2, TNF. alpha., FIG. 4-3).

Wherein, in the figure, A represents D0-CAR 19T, B represents D5-CAR 19T, C represents D10-CAR 19T, and D represents D2000-CAR 19T, and in each group, the leftmost column represents Raji cells, the middle column represents K562 cells, and the rightmost column represents effector cells not in contact with target cells.

(2) Manufactured by the method disclosed in 1201811190780.5The cells from which the culture solution was discarded were transferred to 6-well plates without any coating, and fresh T cell culture solution, 300U/mL of recombinant human interleukin 2 and Decitabine (DAC) were added to the plates at final concentrations of 0, 5nM, 10nM, 100nM, 1000nM and 2000nM, respectively, to give a T cell concentration of 1X 10 ⁶ CO at 37 ℃ and 5% saturation humidity/ml ₂ And continuously culturing for 3 days in the incubator to obtain decitabine enhanced T cells, D-T for short.

Respectively taking D0-T (representing the final concentration of decitabine to be 0), D5-T (representing the final concentration of decitabine to be 5nM), D10-T (representing the final concentration of decitabine to be 10nM) and D2000-T (representing the final concentration of decitabine to be 2000nM) and adding effector cells and target cells (Raji cells expressing CD19 and K562 cells) into a 96-well plate according to an effective target ratio of 1:1, and adding 5% CO at 37 ℃ to the 96-well plate ₂ After 24 hours of interaction in the cell culture chamber, the supernatant was collected and centrifuged. The cytokine levels in the supernatants were measured (labeled NT in the figure) according to the ELISA kit instructions for IL2, IFN-. gamma.and TNF. alpha. As shown in FIG. 4 (IL2 is shown in FIG. 4-1, IFN-. gamma.is shown in FIG. 4-2, TNF. alpha. is shown in FIG. 4-3).

In addition, cytokine levels (labeled NT in the figure) were measured in the supernatants of each effector cell not contacted with the target cells according to the ELISA kit instructions for IL2, IFN-. gamma.and TNF-. alpha.as shown in FIG. 4 (IL2 is shown in FIG. 4-1, IFN-. gamma.is shown in FIG. 4-2, and TNF. alpha. is shown in FIG. 4-3).

In the figure, A represents D0-T, B represents D5-T, C represents D10-T, and D represents D2000-T, and in each group, the leftmost column represents Raji cells, the middle column represents K562 cells, and the rightmost column represents effector cells not in contact with target cells.

As can be seen from fig. 4, upon contact with the target cells, DAC-enhanced CAR 19T cells secreted cytokines significantly higher than the normal CAR 19T, whereas for T cells, DAC did not enhance their ability to secrete cytokines.

Example 6

This example illustrates the in vitro detection of killing Activity of D-CAR 19T

Target cell: naml6 cells expressing both CD19+ and CD20+ were infected with pLenti-CMV-luc2-IRES-Puro virus, allowing Naml6 cells to express luciferase. Naml6 cells stably expressing luciferase were established by puromycin selection.

To test the lethality of effector cells against target cells in vitro, effector cells (NT cells, D0-CAR T cells, and D10-CAR T cells) were compared to target cells in a 96-well plate at an effective-to-target ratio E: t is 1: co-culturing at a ratio of 30. Mu.l of 2 XD-fluorescein solution (300. mu.g/ml) was added to each well and the signal was measured after 2-5 minutes by Varioskan TM LUX (Thermo Fisher). The signal values are the target cell viability rates and the results are shown in FIG. 5.

As can be seen from fig. 5, under the condition of ultra-low effective target ratio (E: T ═ 1:30), the D10-CAR 19T cell provided by the invention rapidly shows strong killing capability within 12 hours, and compared with the ordinary D0-CAR 19T cell, the killing capability is remarkably shown, and the killing efficiency at 96 hours can reach more than 90%.

Example 7

This example illustrates memory phenotype cells of D-CAR 19T

Lentivirus (lentivirus expression vector pRRLsin-CAR19) infected T cells (derived from the same patient) were prepared as disclosed in 201811190780.5, and the cells infected for 12 hours and discarded culture were transferred to a 6-well plate without any coating, and fresh T cell culture, 300U/mL of recombinant human interleukin 2 and Decitabine (DAC) were added at final concentrations of 0, 10nM, 100nM, 1000nM, and 2000nM, respectively, to give a concentration of 1X 10T cells ⁶ CO at 37 ℃ and 5% saturation humidity/ml ₂ The incubator was continued for 14 days and the memory phenotype cells were detected using flow antibodies (CD3, CD62L, CD45RA) on days 0, 3, 5, 7, 14 of culture, with the results shown in fig. 6.

As can be seen in FIG. 6, compared with NT cells and D0-CAR 19T cells (DAC concentration is 0nM), the memory phenotype cells of D10-CAR 19T (DAC concentration is 10nM) obtained by the method are increased, which indicates that the D-CAR 19T prepared by the method provided by the invention has more efficient killing capability. The remaining concentration results are not shown.

Example 8

This example serves to illustrate the genomics analysis of D-CAR 19T

Lentivirus (lentivirus expression vector pRRLsin-CAR19) -infected T cells (from the same patient) were prepared as disclosed in 201811190780.5, and the cells infected for 12 hours and discarded were transferred to 6-well plates without any coating, and fresh T cell culture solution, 300U/mL of recombinant human interleukin 2 and Decitabine (DAC) were added at final concentrations of 0, 10nM, 100nM, 1000nM and 2000nM, respectively, to give a T cell concentration of 1X 10 ⁶ CO at 37 ℃ and 5% saturation humidity/ml ₂ The incubation in the incubator was continued for 3 days, after which time the RNA was extracted and subjected to eukaryotic transcriptome sequencing analysis, and the results of D10-CAR 19T (DAC concentration 10nM) are shown in FIG. 7, the remainder not shown.

From fig. 7, it can be seen that, in genomics, the genome components of the D-CAR 19T cell are changed under the action of DAC, wherein, the expression of genes related to the anti-exhaustion, proliferation and activation abilities is improved, further demonstrating that the method of the present invention can improve the anti-exhaustion ability, proliferation activity and target cell killing activity of the CAR 19T cell.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims

1. The use of decitabine to increase the CD4/CD8 cell ratio in CAR T cells, increase the cytokine secretion, killing and CAR T cell memory of CAR T cells;

wherein decitabine is added at 12-72 hours of CAR T cell culture.

2. A method of increasing the CD4/CD8 cell ratio in CAR T cells, increasing the cytokine secretion capacity, killing capacity, and CAR T cell memory of CAR T cells, comprising culturing CAR T cells in the presence of a DNA demethylating agent, or infecting T cells with a lentiviral expression vector expressing a CAR in the presence of a DNA demethylating agent, to give methylated reprogrammed CAR T cells;

wherein the CAR T cells are cultured in the presence of a DNA demethylating agent for 24-72 hours; or a lentiviral expression vector expressing the CAR is cultured for 24-72 hours after infection of T cells in the presence of a DNA demethylating agent;

the concentration of the DNA demethylating agent in the cell culture medium is 10-1000 nM;

the DNA demethylating drug is decitabine;

wherein the DNA demethylating agent is added at 12-72 hours of CAR T cell culture.

3. The method of claim 2, wherein the CAR is a single CAR and/or a multiple CAR.

4. The method of claim 3, wherein said CAR is selected from CAR19, CAR20, CAR22, CAR30, CAR133, CARMSLN, carmer 1, carmer 2, CAR138, CAR20-19, and CAR 22-19.

5. The method of any one of claims 2-4, wherein the culturing is performed in the presence of a cytokine.

6. The method of claim 5, wherein the cytokine is interleukin 2.