CN118165933B

CN118165933B - Composition for improving CD39-CD69-T cell ratio method

Info

Publication number: CN118165933B
Application number: CN202410594773.0A
Authority: CN
Inventors: 邓涛; 喻堃; 钟立武; 李倩; 高彦粉; 张艳
Original assignee: Chengdu Meijie Saier Biotechnology Co ltd
Current assignee: Chengdu Meijie Saier Biotechnology Co ltd
Priority date: 2024-05-14
Filing date: 2024-05-14
Publication date: 2024-07-16
Anticipated expiration: 2044-05-14
Also published as: CN118165933A

Abstract

The invention relates to the technical field of cell culture, in particular to a composition and a method for increasing the proportion of CD39 ^‑CD69^‑ T cells. The invention provides a simple and convenient culture method capable of obtaining a large number of CD39 ^‑CD69^‑ T cells. The culture method does not need to carry out gene editing or cell screening, does not have the risk of gene off-target, can greatly improve the amplification level of T cells and the proportion of CD39 ^‑CD69^‑ T cells to more than 50 percent on the premise of no purification, and also greatly improves the anti-tumor capability.

Description

Compositions and methods for increasing the proportion of CD39 -CD69- T cells

Technical Field

The invention relates to the technical field of cell culture, in particular to a composition and a method for increasing the proportion of CD39 ^-CD69^- T cells.

Background

Adoptive T cell therapy (adoptive T CELL THERAPY, ACT) refers to a tumor treatment method that enhances the anti-tumor immune function of the body by reinfusion of in vitro expanded cultured immune T cells. Such adoptive cells include Chimeric Antigen Receptor (CAR) T cells, T Cell Receptor (TCR) T cells, tumor Infiltrating Lymphocytes (TIL), and the like. The manner in which T cells are cultured not only determines the fold of cell expansion but also affects the phenotype of the cells. Both the number of T cells and the cell phenotype returned to the patient affect the anti-tumor effect. One study published in Science in 2020 shows that the number of CD8 ⁺CD39^-CD69^- cells in the feedback immune cells from melanoma patients is significantly correlated with improvement in progression-free and melanoma-specific survival and is dose-dependent. Further animal experiments prove that the CD39 ^-CD69^- T cells have stronger anti-tumor capability. Thus, how to expand in vitro to obtain a high proportion of CD39 ^-CD69^- T cells may be a break-through to improve the efficacy of adoptive T cell therapies.

The traditional T cell culture method mainly activates T cells through CD3 monoclonal antibody and/or CD28 monoclonal antibody, and then largely expands the T cells by utilizing IL-2, so that the proportion of the obtained CD39 ^-CD69^- T cells is small (< 15%). In order to obtain high-proportion CD39 ^-CD69^- T cells, gene editing methods such as Crispr/Cas, TALEN and ZFN can be adopted to knock out the CD39 and CD69 genes, but the method is complex in operation and has a risk of off-target, and the obtained T cells have a greater safety risk. In addition, the method can be used for enriching the CD39 ^-CD69^- T cells by magnetic bead sorting, flow sorting and other methods, and has the advantages of complex operation, high cost and less number of the obtained CD39 ^-CD69^- T cells.

Disclosure of Invention

In view of this, the present invention provides compositions, methods for increasing the proportion of CD39 ^-CD69^- T cells. The invention provides a simple and convenient culture method capable of obtaining a large number of CD39 ^-CD69^- T cells, and provides better possibility for durable anti-tumor effect of cell therapy.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides the use of a composition in any of the following:

(I) Culturing T cells; and/or

(II) increasing the proportion of CD39 ^-CD69^- T cells;

the compositions include IL21, IL15 and CD137L.

In some embodiments of the invention, the composition further comprises IFN-gamma, CD3 mab and CD28 mab.

The present invention also provides a composition comprising: 100-2000 ng/ml CD3 monoclonal antibody, 100-2000 ng/ml CD28 monoclonal antibody, 200-2000 IU/ml IL2, 100-2000 ng/ml IL15, 100-2000 ng/ml IL21 and CD137L cured protein.

In some embodiments of the invention, the composition comprises: 500 ng/ml CD3 mab, 500 ng/ml CD28 mab, 1000 IU/ml IL2, 500-2000 ng/ml IL15, 500-2000 ng/ml IL21, and 500-2000 ng/ml CD137L immobilized protein.

In some embodiments of the invention, the CD 137L-immobilized protein is obtained by immobilization of CD137L protein onto the surface of a carrier comprising a cell culture flask, a cell culture plate, a microsphere, or a trophoblast cell.

In some embodiments of the invention, the concentration of the CD137L immobilized protein comprises 100-2000 ng/ml; preferably, the concentration of the CD137L immobilized protein comprises 500-2000 ng/ml.

The invention also provides the use of said composition in any of the following:

(I) Culturing T cells; and/or

(II) increasing the proportion of CD39 ^-CD69^- T cells.

The invention also provides a method for culturing T cells and/or a method for increasing the proportion of CD39 ^-CD69^- T cells, and the composition is used for culturing mononuclear cells to obtain target cells.

In some embodiments of the invention, the method comprises the steps of:

step 1, taking mononuclear cells, and culturing in an A0 culture medium; the A0 culture medium comprises 5% -10% (v/v) of blood plasma and/or blood serum, 2000 IU/ml of IFN-gamma;

Step 2, culturing in an A1 culture medium; the A1 culture medium comprises 5% -10% (v/v) plasma and/or serum, 500 ng/ml CD3 monoclonal antibody, 500 ng/ml CD28 monoclonal antibody and 1000 IU/ml IL2;

step 3, activating and culturing in an A2 culture medium; the A2 culture medium comprises 5% -10% (v/v) plasma and/or serum, 100% -2000 ng/ml IL15 and 100% -2000 ng/ml IL21;

The activation culture comprises a carrier culture comprising CD137L immobilized protein;

step 4, culturing in an amplification culture medium to obtain target cells; the amplification medium included 1000 IU/ml IL2.

In some embodiments of the invention, the source of the mononuclear cells comprises peripheral blood and/or cord blood; or (b)

The culture medium comprises an immune cell culture medium; or (b)

The serum and/or plasma comprises one or more of autologous plasma, umbilical plasma, AB serum, fetal bovine serum or serum substitutes; or (b)

The preparation method of the carrier containing the CD137L immobilized protein comprises the following steps: and (3) taking protein coating liquid containing 100-2000 ng/ml CD137L-Fc, and incubating for 1-24 h at 4-37 ℃ in a culture bottle to obtain the carrier containing the CD137L immobilized protein.

In some embodiments of the invention, the A2 medium comprises 5% -10% (v/v) plasma and/or serum, 1000 IU/ml IL2, 500-2000 ng/ml IL15 and 500-2000 ng/ml IL21;

The preparation method of the carrier containing the CD137L immobilized protein comprises the following steps: and (3) taking a protein coating solution containing 500-2000 ng/ml of CD137L-Fc, placing the protein coating solution into a culture flask according to the usage amount of 50-300 mu L/cm ², and incubating at 4-37 ℃ for 1-24 hours to obtain the carrier containing the CD137L immobilized protein.

In some embodiments of the invention, the culturing period of step1 comprises 1 day; or (b)

The culturing time in the step 2 comprises 3 days; or (b)

The culturing time in the step 3 comprises 3 days; or (b)

And (3) culturing the amplification culture medium in the step (4) for 7-21 days.

In the preparation method of the carrier containing the CD137L immobilized protein, the incubation condition comprises standing overnight at 4 ℃, standing at normal temperature for 2-3 hours or standing at 37 ℃ for 1 hour.

The invention also provides cells obtained by the culture method.

The invention also provides application of the cell in preparing antitumor drugs and/or drug combinations.

The invention also provides a medicament and/or a pharmaceutical combination comprising said cells.

The invention also provides a method of preventing and/or treating a tumor, comprising administering to a subject any of:

(I) -said cells;

(II), the medicament and/or the pharmaceutical combination.

The present invention includes, but is not limited to, providing the following benefits:

the invention provides a simple and convenient culture method capable of obtaining a large number of CD39 ^-CD69^- T cells. The culture method does not need to carry out gene editing or cell screening, does not have the risk of gene off-target, can greatly improve the amplification level of T cells and the proportion of CD39 ^-CD69^- T cells to more than 50 percent on the premise of no purification, and also greatly improves the anti-tumor capability.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows cell expansion curves for 21 days of different combinations of cultures;

FIG. 2 shows a flow chart of the ratio of different combinations of cultured CD3 ⁺、CD4⁺ and CD8 ⁺ T cells;

FIG. 3 shows a bar graph of CD3 ⁺、CD4⁺ and CD8 ⁺ T cell ratios for each group; wherein, the difference is significant; * Representing the difference was very significant;

FIG. 4 shows a proportional flow diagram of different combinations of cultured CD39 ^-CD69^- T cells (gated on CD3 ⁺ T cells);

FIG. 5 shows a CD39 ^-CD69^- T cell scale bar graph; * Indicating that the difference is significant; * Representing the difference was very significant;

FIG. 6 shows in vitro anti-tumor experiments with different combinations of cultured T cells; wherein, the difference is significant; * Representing the difference was very significant;

FIG. 7 shows graphs of orthogonal experimental trends in IL15, IL21 protein and CD137L-Fc concentration;

FIG. 8 shows orthogonal experimental bar graphs of IL15, IL21 protein and CD137L-Fc concentration; wherein, the difference is significant.

Detailed Description

The present invention discloses compositions and methods for increasing the proportion of CD39 ^-CD69^- T cells, and one skilled in the art can, based on the teachings herein, suitably modify the process parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

CD39 ^-CD69^- T cells are defined as stem cell-like T cells, which have a stronger and more durable anti-tumor effect, and the proportion of CD39 ^-CD69^- T cells in cell therapy has a strong correlation with the efficacy of tumor patients. The traditional T cell culture method has low CD39 ^-CD69^- T cell proportion (less than 15 percent), and the invention provides a culture method which can expand a large amount of T cells on the premise of no purification, and the CD39 ^-CD69^- T cell proportion exceeds 50 percent, thereby providing better possibility for durable anti-tumor effect of cell therapy.

The culture method disclosed by the invention is added with the combination of IL21, IL15 and solidified CD137L protein, so that the expansion level of T cells can be greatly increased, the proportion of CD39 ^-CD69^- T cells is increased to more than 50%, and the anti-tumor capability is also greatly improved.

The compositions, materials and reagents used in the methods of increasing the proportion of CD39 ^-CD69^- T cells provided by the present invention are commercially available unless otherwise specified.

The invention is further illustrated by the following examples:

Examples

1. Experimental method

1.1 PBMC isolation and autologous plasma preparation

(1) Peripheral blood was collected from 3 healthy persons, 40 ml each.

(2) The peripheral blood of healthy persons was placed in a sterile 50 ml centrifuge tube, 600g, and centrifuged for 10 minutes.

(3) Autologous plasma preparation: sucking upper plasma, inactivating at 56 deg.C for 30min, centrifuging at 800 g min to obtain autologous plasma, and preserving at 4 deg.C;

(4) The same volume of PBS was added to the lower blood sediment of step (3) for dilution.

(5) Add 20 ml Ficoll to a new 50 ml centrifuge tube and gently spread the PBS-diluted blood from step (4) over Ficoll, 20 per tube ml.

(6) 400 G was centrifuged for 30min, the supernatant was discarded, and the middle buffy coat cells were transferred to a new centrifuge tube.

(7) Cells were washed with more than 1 volume of PBS, centrifuged at 400 g for 10 min, and the supernatant was discarded.

(8) Cells were washed 2 times with 40 ml PBS and the supernatant was discarded to obtain a PBMC cell pellet.

1.2 T cell in vitro activation of different culture combinations

(1) The PBMC cell pellet obtained by Ficoll isolation is resuspended by adding an appropriate amount of immunocyte medium containing 10% (v/v) autologous plasma, the cell density being 1-2×10 ⁶ cells/ml. The plasma used was autologous plasma obtained after 1.1 (3) inactivation, and the immune cell culture medium used was GT-T551H 3 of Takara.

(2) Activation cultures of Day0 to Day7 were performed according to the experimental group of table 1:

TABLE 1 grouping of experiments

(3) The coating method of the culture bottle of the group D and the group E is as follows:

① CD137L-Fc fusion protein (Sigma-Aldrich, cat# SRP 0556) was diluted to 2000 ng/ml with D-PBS to prepare a coating solution.

② The 5 ml coating solution was pipetted into a T25 flask and placed at 4℃overnight.

③ The coating was removed and the T25 flask was gently washed once with 5ml PBS.

④ Used immediately after washing.

1.3 T cell in vitro expansion culture

The activated T cells are amplified by using an amplification culture medium. Counting every 2-3 days of expansion culture, and regulating the density of living cells to 1-2X 10 ⁶/ml by using an expansion culture medium, and continuing to culture for 21 days. The amplification medium used was Takara GT-T551H 3 medium (cat# WK 593S) +10% autologous plasma (v/v) +1000 IU/ml IL2.

Wherein, before each fluid infusion, the AO/PI counting method is adopted for cell counting, and the specific method is as follows:

① After the cells are blown and evenly mixed, 10 microliter of cell suspension is sucked, and 10 microliter of AO/PI reagent is added;

② Transferring all 20 mu l of uniformly mixed cells and AO/PI mixed solution into a counting plate;

③ Detecting density and activity rate by adopting a full-automatic fluorescent cell analyzer;

④ Cell number = viable cell density (individual/ml) x culture volume (ml).

1.4 Flow cytometry detection of T cell surface markers

The ratio of the surface markers of CD3 ⁺、CD4⁺、CD8⁺ and CD39 ^-CD69^- was measured by flow cytometry from cells cultured for 21 days, and the specific method was as follows:

① Placing 1×10 ⁶ cells into a centrifuge tube, centrifuging for 5min at 500g, discarding supernatant, adding 1: 1ml PBS to wash the cells, centrifuging for 5min at 500g, and discarding supernatant;

② Adding 150 μl of PBS to resuspend the cells, and blowing and uniformly mixing; split charging into 32 ml flow-through loading tubes (50 μl/tube), labeled 1#,2# and 3#, respectively; tubes 1# and 2# were supplemented with flow-through fluorescent antibodies in the amounts indicated in table 2, tube 3# was supplemented with 200 μl PBS alone as a negative control;

TABLE 2 flow antibody dosage

③ After incubating the 1# and 2# tubes for 15 minutes at room temperature in the dark, adding 2ml PBS vortex to wash for 5 seconds, centrifuging 500g for 5 minutes, removing the supernatant, and re-suspending the cell pellet by 200 μl PBS;

④ And (5) detecting by a flow cytometer.

2. Experimental results:

(1) Cell expansion fold

According to the above experimental method, PBMCs were activated for 7 days in five combined culture methods, and after further culturing in an amplification medium (medium+10% autologous plasma+1000 IU/ml IL 2) for 21 days, amplification curves were plotted and the differences in amplification factors of each group were counted (n=3), and the results are shown in table 3 and fig. 1. The results show that the amplification factor of group D is highest, reaching 1055.95 +/-93.06 times, and is extremely remarkably higher than that of A, B and group C (P value < 0.01) and is remarkably higher than that of group E (P value < 0.05). The balance of the group C culture 21 is subjected to expansion multiple of 703.00 +/-90.63, which is obviously higher than that of the group A and the group B (P value is less than 0.05). The results show that the addition of IL15 and IL21 simultaneously in the culture medium can improve the expansion capacity of T cells, and the expansion capacity of T cells can be further improved by using a culture flask coated with the CD137L-Fc fusion protein for culture.

TABLE 3 total cell expansion for 21 days in each group

(2) T cell phenotype assay

The cell phenotypes of each group were flow tested and the results are shown in fig. 2,3 and table 4. The results in Table 4 and FIG. 3 show that A, B, C and D groups have a ratio of CD3 ⁺ T cells greater than 95% and that the sum of the ratios of CD8 ⁺ and CD4 ⁺ T cells is greater than 85%, indicating that the immune cells obtained from the groups containing CD3 and CD28 antibodies are predominantly T cells with no significant difference between the groups (P value > 0.05), whereas the NK cell ratio in the cells obtained from the group E protocol culture is higher and the ratio of CD3 ⁺ T is only 45.70% + -5.16% with very significant differences from the other groups (P value < 0.01). The results indicate that the combination of IL15, IL21 and CD137L-Fc requires the cooperation of CD3 and CD28 monoclonal antibodies to ensure the massive expansion of T cells in PBMC but not NK cells.

Table 4 ratios of samples CD3 ⁺、CD4⁺ and CD8 ⁺

(3) CD39 ^-CD69^- T cell ratio

The flow-through assay for CD39 ^-CD69^- stem cell-like T cell fraction and the results are shown in FIGS. 4, 5 and Table 5. The results show that the proportion of CD39 ^-CD69^- T cells in group D is significantly higher than that in groups A and B (P value < 0.01), and significantly higher than that in groups C and E (P value < 0.05), reaching 55.26% + -8.31%. The result shows that the combined solidification of the cytokines IL15 and IL21 can greatly improve the proportion of the CD39 ^-CD69^- stem cell-like T cells.

TABLE 5 sample CD39 ^-CD69^- T cell fraction (Gated on CD3 ⁺)

(4) Tumor killing experiment

To further verify the antitumor activity of different ratios of CD39 ^-CD69^- stem cell-like T cells, we performed tumor killing experiments using the a549 tumor cell line as target cells. Since the NK cells of the cells cultured in the E group are too high in proportion and are non-target cells, killing experiments are carried out by adopting A, B, C and D groups of cells only.

The specific experimental steps are as follows:

① The test adopts a 96-hole cell plate, A549 tumor cells are attached for 24 hours in advance, and the inoculation cell density is 3000 cells/hole;

② Adding each group of T cells cultured for 21 days into tumor cell holes according to different target ratios (E: T) of 1:1, 5:1 and 10:1, and setting a control group without T cells;

③ After the co-culture is continued for 72 hours, the killing rate of the tumor cells is detected by adopting a CCK8 method.

As shown in FIG. 6, the killing rate of the tumor cells in the group D is extremely higher than that in the groups A, B and C (P value is less than 0.01) when the effective target ratio (E: T) is 5:1 and 10:1; when the effective target ratio (E: T) is 1:1, the killing rate of the tumor cells of the group D is extremely higher than that of the tumor cells of the group A and the group B (the P value is less than 0.01), the killing rate of the tumor cells of the group C is remarkably higher than that of the tumor cells of the group C (the P value is less than 0.05), and the killing rate of the tumor cells of the group C is also improved relative to that of the tumor cells of the group A and the tumor cells of the group B. The results indicate that a high proportion of CD39 ^-CD69^- T cells can enhance antitumor activity.

3. Optimum combined concentration screening

The experimental method comprises the following steps:

Taking group A cells cultured until 4 days in 1.2 experiments, performing orthogonal experiments in a 24-well plate, adding cytokines with different concentrations or coating CD137L-Fc fusion proteins with different concentrations according to the following table, and performing expansion culture according to 1.3 experiments; flow cytometry was used to detect the proportion of CD39 ^-CD69^- stem cell-like T cells after each group of cells was cultured until 21 days.

The results of the detection are shown in FIG. 7, FIG. 8 and Table 6. As the concentrations of IL15 and IL21 factors increased, the proportion of CD39 ^-CD69^- T cells increased gradually, with significant differences in the remaining concentrations compared to 0 ng/ml (P value < 0.05); the proportion of CD39 ^-CD69^- T cells was comparable after 500 ng/ml concentration, without significant differences (P value > 0.05). As the concentration of CD137L-Fc coating increased, the proportion of CD39 ^-CD69^- T cells also increased; there were significant differences (P value < 0.05) in concentrations of 500 ng/ml, 1000 ng/ml and 2000 ng/ml compared to 0 ng/ml; there was no significant difference between 1000 ng/ml and 2000 ng/ml (P value > 0.05). The results show that increasing the concentration of IL15 and IL21 factors and the concentration of coated CD137L-Fc can increase the proportion of CD39 ^-CD69^- T cells, but the increasing trend of the concentration range of 500 ng/ml to 2000 ng/ml is gentle, namely, the concentration range of 500 ng/ml to 2000 ng/ml is the optimal concentration range.

TABLE 6 influence of the differences in the amount of factor added and the protein coating concentration on the proportion of CD39 ^-CD69^- T cells

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

A method of T cell culture and/or a method of increasing the proportion of CD39 ^-CD69^- T cells comprising the steps of:

Step 1, taking mononuclear cells, and culturing in an A0 culture medium; the A0 culture medium comprises 5% -10% of plasma and 2000 IU/ml IFN-gamma in a volume fraction ratio;

Step 2, culturing in an A1 culture medium; the A1 culture medium comprises 5% -10% of plasma, 500 ng/ml of CD3 monoclonal antibody, 500 ng/ml of CD28 monoclonal antibody and 1000 IU/ml of IL2 in a volume fraction ratio;

step 3, activating and culturing in an A2 culture medium; the A2 culture medium comprises 5% -10% of plasma, 500% -2000 ng/ml IL15 and 500% -2000 ng/ml IL21 in a volume fraction ratio;

The activation culture comprises a carrier culture comprising CD137L immobilized protein;

the concentration of the CD137L cured protein is 500-2000 ng/ml;

step 4, culturing in an amplification culture medium to obtain target cells; the amplification medium included 1000 IU/ml IL2.
2. The method of claim 1, wherein the source of mononuclear cells comprises peripheral blood and/or cord blood; or (b)

The culture medium comprises an immune cell culture medium; or (b)

The plasma comprises autologous plasma or umbilical plasma; or (b)

The preparation method of the carrier containing the CD137L immobilized protein comprises the following steps: and (3) taking protein coating liquid containing 500-2000 ng/ml CD137L-Fc, and incubating for 1-24 h at 4-37 ℃ in a culture bottle to obtain the carrier containing the CD137L immobilized protein.