CN114350608B - Composition for inducing T cells to be reprogrammed into NK-like cells and application thereof - Google Patents

Abstract

The invention provides a composition for inducing T cells to be reprogrammed into NK-like cells and application thereof, wherein the composition for inducing the T cells to be reprogrammed into NK-like cells comprises vitamin C and a small molecule compound; the small molecule compound comprises any one or a combination of at least two of a DNA methyltransferase inhibitor, a histone deacetylase inhibitor or a histone methyltransferase EZH2 inhibitor. The invention creatively discovers that the vitamin C can obviously promote the reprogramming of the T cells into NK-like cells induced by the small molecular compound and promote the proliferation of the cells, so that the vitamin C and the small molecular compound are combined to form a composition for inducing the reprogramming of the T cells into the NK-like cells. The composition is used for inducing the reprogramming of T cells into NK-like cells, so that the reprogramming efficiency is remarkably improved, the proliferation of the T cells is promoted, and the composition has important application value in the fields of cell immunotherapy and the like.

Description

A composition for inducing T cell reprogramming into NK-like cells and its application

Technical Field

The present invention belongs to the field of biomedicine technology and relates to a composition for inducing T cell reprogramming into NK-like cells and its application.

Background technique

Natural killer cells (NK) are important immune cells in the body. They are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity and autoimmune diseases in some cases. NK cells are an important part of the innate immune system. Their killing activity is not restricted by MHC (major histocompatibility complex) and does not rely on antibodies. NK cells are one of the most effective and active immune cells in the human body. They recognize viruses and virus-infected cells through NK cell receptors (NCR) such as NKp46, and participate in immune responses to a variety of viruses.

However, due to the limited number and regenerative capacity of NK cells naturally existing in the human body, the source of cells has become the main obstacle limiting the application of NK cells in tumor treatment. The T cell receptor (TCR) on the surface of T cells recognizes virus-infected cells or exogenous antigens and responds. NK-like cells can not only express NCR receptors such as NKp46, NKp30, NKp44 and NKG2D, but also express fully functional TCR receptors, and have the functions of both T cells and NK cells. NK-like cells have stronger killing activity and a broader spectrum of anti-tumor effects than normal NK cells. At the same time, they can proliferate in large numbers under in vitro conditions. In vitro proliferated NK-like cells can continue to survive in vivo for more than three weeks. Reprogramming T cells into NK-like cells provides a new cell source for immunotherapy of tumors and related diseases, which will greatly promote the development of tumor cell immunotherapy.

At present, T cell reprogramming methods are mainly based on transgenic means, including lentiviral transfection, PB system electroporation, CRISPR/cas9 system knockout, etc. However, these methods have many defects. For example, the lentiviral transfection method has a long processing cycle and a cumbersome process. The electroporation method will lose a large number of primary T cells and has low efficiency. There is a chance of off-target during gene overexpression or knockout. Moreover, the above three methods are difficult to achieve large-scale application and still cannot fundamentally achieve the effect of obtaining NK cells in vitro on a large scale. In addition, some researchers have used chemical small molecules to induce T cell reprogramming into NK-like cells, but the efficiency of this method is average, and chemical small molecules have certain toxicity to T cells, which limits the application of this method.

Therefore, how to develop a method to induce T cells to reprogram into NK-like cells to improve the reprogramming efficiency without affecting or promoting the growth of T cells has become an urgent problem to be solved in this field.

Summary of the invention

In view of the deficiencies of the prior art, the object of the present invention is to provide a composition for inducing T cells to reprogram into NK-like cells and its application.

In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a composition for inducing T cell reprogramming into NK-like cells, wherein the composition for inducing T cell reprogramming into NK-like cells comprises vitamin C and a small molecule compound;

The small molecule compound includes any one of a DNA methyltransferase inhibitor, a histone deacetylase inhibitor or a histone methyltransferase EZH2 inhibitor, or a combination of at least two of them.

The combination of at least two may be, for example, a combination of a DNA methyltransferase inhibitor and a histone deacetylase inhibitor, a combination of a histone deacetylase inhibitor and a histone methyltransferase EZH2 inhibitor, a combination of a DNA methyltransferase inhibitor and a histone methyltransferase EZH2 inhibitor, or any other combination.

The present invention creatively discovered that vitamin C can significantly promote the induction of T cell reprogramming into NK-like cells by small molecule compounds, and can promote cell proliferation. (Other anti-apoptotic drugs such as Q-VD-Oph have no promoting effect on the process of small molecule compounds inducing T cell reprogramming into NK-like cells) Therefore, vitamin C is combined with small molecule compounds to form a composition that induces T cell reprogramming into NK-like cells. The composition is used to induce T cell reprogramming into NK-like cells, which significantly improves the reprogramming efficiency and promotes T cell proliferation, and has important application value in the fields of cell immunotherapy.

Preferably, the DNA methyltransferase inhibitor comprises decitabine and/or GSK-3484862.

Preferably, the histone deacetylase inhibitor includes any one of Mocetinostat, Givinostat or Entinostat, or a combination of at least two of them. The combination of at least two of them can be, for example, a combination of Mocetinostat and Givinostat, a combination of Givinostat and Entinostat, a combination of Mocetinostat and Entinostat, or the like. Any other combination is also acceptable.

Preferably, the histone methyltransferase EZH2 inhibitor includes Tazemetostat and/or GSK126.

In a second aspect, the present invention provides a method for inducing T cell reprogramming into NK-like cells, the method comprising co-culturing activated T cells with the composition for inducing T cell reprogramming into NK-like cells as described in the first aspect to obtain NK-like cells.

Preferably, the final concentration of vitamin C is 40-60 ng/mL, for example, 40 ng/mL, 42 ng/mL, 45 ng/mL, 48 ng/mL, 50 ng/mL, 52 ng/mL, 55 ng/mL, 58 ng/mL, 60 ng/mL, etc.

Preferably, the final concentration of decitabine is 0.05-0.5 μM, for example, 0.05 μM, 0.07 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, etc.

Preferably, the final concentration of GSK-3484862 is 0.5-8 μM, for example, 0.5 μM, 0.7 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, etc.

Preferably, the final concentration of Mocetinostat is 0.1-0.5 μM, such as 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, etc.

Preferably, the final concentration of Givinostat is 0.05-1 μM, for example, 0.05 μM, 0.07 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 0.95 μM, 1 μM, etc.

Preferably, the final concentration of Entinostat is 0.05-1 μM, for example, 0.05 μM, 0.07 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 0.95 μM, 1 μM, etc.

Preferably, the final concentration of Tazemetostat is 0.1-5 μM, for example, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.7 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, etc.

Preferably, the final concentration of GSK126 is 0.05-1 μM, for example, 0.05 μM, 0.07 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 0.95 μM, 1 μM, etc.

Preferably, the co-cultivation period is 3-10 days.

Preferably, during the co-cultivation process, half of the medium is replaced every day.

In a third aspect, the present invention provides a NK-like cell prepared by the method described in the second aspect, wherein the NK-like cell expresses an NK cell receptor and a T cell receptor.

Preferably, the NK cell receptor includes any one of NKp46, NKp30, NKp44 or NKG2D, or a combination of at least two of them. The combination of at least two may be, for example, a combination of NKp46 and NKp30, a combination of NKp44 and NKG2D, a combination of NKp30 and NKp44, or any other combination.

Preferably, the NK cell receptor comprises NKp46 and/or NKp30.

In a fourth aspect, the present invention provides a composition for inducing T cell reprogramming into NK-like cells as described in the first aspect, a method for inducing T cell reprogramming into NK-like cells as described in the second aspect, or the use of NK-like cells as described in the third aspect in the preparation of cellular immunotherapy drugs.

In a fifth aspect, the present invention provides the use of vitamin C in promoting small molecule compounds to induce T cells to reprogram into NK-like cells;

The small molecule compound comprises: any one of a DNA methyltransferase inhibitor, a histone deacetylase inhibitor or a histone methyltransferase EZH2 inhibitor or a combination of at least two thereof.

Preferably, the DNA methyltransferase inhibitor comprises decitabine and/or GSK-3484862;

Preferably, the histone deacetylase inhibitor comprises any one of Mocetinostat, Givinostat or Entinostat, or a combination of at least two thereof.

Preferably, the histone methyltransferase inhibitor EZH2 includes Tazemetostat and/or GSK126.

Preferably, the NK-like cells express NK cell receptors and T cell receptors.

Preferably, the NK cell receptor comprises any one of NKp46, NKp30, NKp44 or NKG2D, or a combination of at least two thereof.

Preferably, the NK cell receptor comprises NKp46 and/or NKp30.

The numerical range described in the present invention not only includes the point values listed above, but also includes any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.

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

The present invention creatively discovered that vitamin C can significantly promote the induction of T cell reprogramming into NK-like cells by small molecule compounds and can promote cell proliferation, so vitamin C is combined with small molecule compounds to form a composition that induces T cell reprogramming into NK-like cells. The composition is used to induce T cell reprogramming into NK-like cells, significantly improves the reprogramming efficiency and promotes T cell proliferation, and has important application value in the fields of cell immunotherapy and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the expression results of NKp30 and NKp46 in CD4 T cells without induction of small molecule drugs.

FIG1B is a graph showing the expression results of NKp30 and NKp46 in CD4 T cells without small molecule drug induction after the addition of Vc.

FIG. 1C is a graph showing the expression of NKp30 and NKp46 in CD4 T cells induced by DAC drugs.

FIG. 1D is a graph showing the expression of NKp30 and NKp46 in CD4 T cells induced by Vc+DAC drugs.

FIG. 1E is a graph showing the expression of NKp30 and NKp46 in CD4 T cells induced by GSK3484862.

FIG1F is a graph showing the expression results of NKp30 and NKp46 in CD4 T cells induced by Vc+GSK3484862.

FIG1G is a graph showing the expression of NKp30 and NKp46 in CD4 T cells induced by the combination of DAC+GSK3484862 drugs.

FIG1H is a graph showing the expression of NKp30 and NKp46 in CD4 T cells induced by the combination of Vc+DAC+GSK3484862.

FIG. 2A is a graph showing the expression results of NKp30 and NKp46 in CD8 T cells without induction of small molecule drugs.

FIG2B is a graph showing the expression results of NKp30 and NKp46 in CD8 T cells without small molecule drug induction after the addition of Vc.

FIG2C is a graph showing the expression of NKp30 and NKp46 in CD8 T cells induced by DAC drugs.

FIG2D is a graph showing the expression results of NKp30 and NKp46 in CD8 T cells induced by Vc+DAC drugs.

FIG. 2E is a graph showing the expression results of NKp30 and NKp46 in CD8 T cells induced by GSK3484862.

FIG2F is a graph showing the expression results of NKp30 and NKp46 in CD8 T cells induced by Vc+GSK3484862.

Figure 2G is a graph showing the expression results of NKp30 and NKp46 in CD8 T cells induced by the combination of DAC+GSK3484862 drugs.

Figure 2H is a graph showing the expression results of NKp30 and NKp46 in CD8 T cells induced by the combination of Vc+DAC+GSK3484862 drugs.

FIG. 3A is a graph showing the results of Vc promoting the proliferation of GSK-3484862-induced T cells reprogrammed into NK-like cells.

FIG. 3B is a graph showing the results of Vc promoting the proliferation of DAC-induced T cells reprogrammed into NK-like cells.

FIG3C is a graph showing the results of Vc promoting the proliferation of T cells co-induced by GSK-3484862+Mocetinostat to reprogram T cells into NK-like cells.

Detailed ways

The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

The sources of materials involved in the following examples are as follows:

Umbilical cord blood was obtained from the Guangdong Province Umbilical Cord Blood Hematopoietic Stem Cell Bank;

Vitamin C (Vc) was purchased from Sigma-Aldrich (USA).

Pan T cell isolation kit was purchased from STEMCELL Technologies (Canada);

Transact was purchased from Miltenyi Biotec GmbH, Germany;

Decitabine (DAC) was purchased from Selleck (Shanghai Lanmu Chemical Co., Ltd., China);

GSK-3484862 and Mocetinostat were purchased from MCE (MedChemExpress);

CD3 PE-Cy7, CD4 APC-Cy7, CD8 FITC, NKp30 PE and NKp46 APC were purchased from BioLegend (USA).

Example 1

Isolation and in vitro culture of primary T cells from human umbilical cord blood

Ficoll-hypaque density gradient centrifugation was used to separate mononuclear cells (UCBMC) from umbilical cord blood. 4×10 ⁷ CD3-positive UCBMC were cultured overnight with a cell density of 2×10 ⁶ /mL, and the remaining UCBMC were frozen. T cells were sorted using the Pan T cell isolation kit, and ≥4×10 ⁷ CD3-positive T cells were obtained after culture for a period of time with a viability of ≥70%, without contamination by exogenous microorganisms such as bacteria, fungi, and mycoplasma.

Example 2

Small molecule drugs induce T cell reprogramming

(1) Take the Transact activated T cells cultured for 36 hours, centrifuge at 300 g for 5 min, resuspend the T cells in T551 + 5% FBS + 1% double antibody (100× penicillin-streptomycin mixed solution) + IL2 (300 U) and adjust the T cell density to (2-3) × 10 ⁵ cells/mL;

(2) Each group of small molecule drugs was added to the resuspended T cells, and the final concentration of each drug was shown in Table 1. Half of the medium was changed every day, and new drugs were added according to the volume after the change of medium; the first addition of drugs was recorded as Day 0 (Day 0), and the drug was continued until Day 5 (Day 5). The medium was changed by centrifugation at 300g for 5 minutes, and then T551+5% FBS+1% double antibody (100× penicillin-streptomycin mixed solution) was used for continued culture.

Table 1

Example 3

Flow cytometry was used to detect the effect of Vc on T cell reprogramming into NK cell-like phenotype

(1) Take 200 μL of each of the drug groups in Table 1 in Example 2 (i.e., after induction with GSK-3484862 without Vc, after induction with DAC without Vc, after co-induction with GSK-3484862 without Vc and Mocetinostat, after induction with DAC with Vc, after induction with GSK-3484862 with Vc, after co-induction with GSK-3484862 with Vc and Mocetinostat) on Day 6, uninduced T cells, and uninduced T cells with 50 ng/mL Vc added, centrifuge at 400 g for 4 min, discard the supernatant, add 50 μL of phosphate buffered saline (PBS) to resuspend the cells, then add 0.5 μL of CD3 PE-Cy7, CD4 APC-Cy7, CD8 FITC, NKp30 PE, and NKp46 APC, respectively, and incubate at 4° C. in the dark for 30 min; add 500 μL of Dilute the antibody with PBS, centrifuge at 400 g for 4 min, carefully discard the supernatant, add 300 μL PBS to resuspend the cells, transfer to the flow tube, and put on the machine;

(2) BD-flowcyto analysis software was used for data analysis. 10,000 cells were collected from each tube and the percentages of NKp46-positive T cells and NKp30-positive T cells were calculated.

As shown in Figures 1A and 1B, the expression rate of NKp30 in CD4 T cells of the uninduced treatment group was similar to that of the uninduced treatment group with Vc added; as shown in Figures 1C and 1D, the expression rate of NKp30 in CD4 T cells in the DAC-induced group was approximately 15.1%, while the expression rate of NKp30 in CD4 T cells in the DAC+Vc group was approximately 22.8%; as shown in Figures 1E and 1F, the expression rate of NKp30 in CD4 T cells in the GSK3484862-induced group was approximately 12.6%, while the expression rate of NKp30 in CD4 T cells in the GSK3484862+Vc group was approximately 22.9%; as shown in Figures 1G and 1H, the expression rate of NKp30 in CD4 T cells in the GSK-3484862+Mocetinostat co-induced group was approximately 57.3%, while the expression rate of NKp30 in CD4 T cells in the GSK-3484862+Mocetinostat+Vc co-induced group was approximately 57.3%. The expression rate of NKp30 in T cells was about 67.6%. In summary, after adding Vc to small molecule drugs, the expression rate of NKp30 in CD4 T cells in each group increased significantly, which fully proved the significant promotion effect of Vc on small molecule drugs-induced T cell reprogramming into NK cells, especially the phenotype of NKp30 in CD4 T cells.

As shown in Figures 2A and 2B, the expression rate of NKp30 in CD8 T cells in the untreated group was about 14.7%, while the expression rate of NKp30 in CD8 T cells in the untreated group with Vc added was about 10.3%; as shown in Figures 2C and 2D, the expression rate of NKp30 in CD8 T cells in the DAC-induced group was about 23.2%, while the expression rate of NKp30 in CD8 T cells in the DAC+Vc group was about 47.8%, and the phenotype of NKp46 began to appear (increased from 0.62% to 3.3%); as shown in Figures 2E and 2F, the expression rate of NKp30 in CD8 T cells in the GSK3484862-induced group was about 24.6%, while the expression rate of NKp30 in CD8 T cells in the GSK3484862+Vc group was about 5.8%. The expression rate of NKp30 in T cells was about 36.7%, and at the same time, the phenotype of NKp46 began to appear obviously (increased from 0.56% to 5.6%); as shown in Figure 2G and Figure 2H, the expression rate of NKp30 in CD8 T cells in the GSK-3484862+Mocetinostat co-induction group was about 49.6%, while the expression rate of NKp30 in CD8 T cells in the GSK-3484862 and Mocetinostat+Vc co-induction group was about 46.0%. The addition of Vc slightly reduced the phenotype expression of NKp30, but the phenotype of NKp46 began to appear (increased from 2.51% to 6.63%). The above results show that in CD8 T cells, the addition of Vc can not only affect the phenotype of NKp30, but also increase the expression of NKp46.

The above results show that Vc itself cannot induce T cells to reprogram into NK-like cells, but the addition of Vc significantly promotes the small molecule compounds to induce T cells to reprogram into NK-like cells.

Example 4

Plotting cell proliferation curves

10 μL of T cells induced by DAC, GSK-3484862, GSK-3484862+Mocetinostat co-induced, DAC+Vc induced, GSK-3484862+Vc induced, and GSK-3484862+Mocetinostat+Vc co-induced for 2, 4, 6, and 10 days, as well as untreated T cells, were mixed with 10 μL of trypan blue in a 1:1 ratio for counting and cell proliferation curves were drawn.

The results are shown in Figures 3A, 3B, and 3C. After the addition of Vc, the cell proliferation rate was significantly accelerated, indicating that Vc significantly promoted the proliferation of small molecule compounds-induced T cell reprogramming into NK-like cells.

The applicant declares that the present invention illustrates a composition for inducing T cell reprogramming into NK-like cells and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims (12)

1. A method for inducing T cell reprogramming into NK-like cells, the method comprising co-culturing activated T cells with a composition that induces T cell reprogramming into NK-like cells to obtain NK-like cells, wherein the composition that induces T cell reprogramming into NK-like cells comprises vitamin C and a small molecule compound; The small molecule compound comprises any one or a combination of at least two of a DNA methyltransferase inhibitor, a histone deacetylase inhibitor or a histone methyltransferase EZH2 inhibitor; The DNA methyltransferase inhibitors include decitabine and/or GSK-3484862; The histone methyltransferase EZH2 inhibitor includes Tazemetostat and/or GSK126; The histone deacetylase inhibitor includes any one of Mocetinostat, Givinostat or Entinostat or a combination of at least two thereof. 2. The method according to claim 1, wherein the final concentration of vitamin C is 40-60 ng/mL. 3. The method according to claim 1, wherein the final concentration of decitabine is 0.05-0.5 μM. 4. The method according to claim 1, wherein the final concentration of GSK-3484862 is 0.5-8 μM. 5. The method according to claim 1, wherein the final concentration of Mocetinostat is 0.1-0.5 μM. 6. The method according to claim 1, wherein the final concentration of Givinostat is 0.05-1 μM. 7. The method of claim 1, wherein the final concentration of Entinostat is 0.05-1 μM. 8. The method of claim 1, wherein the final concentration of Tazemetostat is 0.1-5 μM. 9. The method according to claim 1, wherein the final concentration of GSK126 is 0.05-1 μM. 10. The method according to claim 1, wherein the co-cultivation period is 3-10 days. The method according to claim 1 , wherein during the co-cultivation process, half of the medium is replaced every day. 12. Application of vitamin C in promoting small molecule compounds to induce T cell reprogramming into NK-like cells; The small molecule compound comprises: any one or a combination of at least two of a DNA methyltransferase inhibitor, a histone deacetylase inhibitor or a histone methyltransferase EZH2 inhibitor; The DNA methyltransferase inhibitor is decitabine and/or GSK-3484862; The histone methyltransferase EZH2 inhibitor is Tazemetostat and/or GSK126; The histone deacetylase inhibitor is any one of Mocetinostat, Givinostat or Entinostat, or a combination of at least two of them.