CN113293130B

CN113293130B - Culture method of tumor specific T cells

Info

Publication number: CN113293130B
Application number: CN202010800226.5A
Authority: CN
Inventors: 高全立; 李铁鹏; 王瑶
Original assignee: Henan Cancer Hospital
Current assignee: Henan Hualong Biotechnology Co ltd
Priority date: 2020-08-11
Filing date: 2020-08-11
Publication date: 2022-07-12
Anticipated expiration: 2040-08-11
Also published as: CN113293130A

Abstract

The invention provides a culture medium for culturing tumor specific T cells, which comprises potassium chloride with the concentration of more than 1g/L and less than or equal to 4g/L, preferably an X-VIVO culture medium, a GT-T551 culture medium and an ALYS505N-0 culture medium containing 3g/L potassium chloride. Based on the culture medium, the invention also provides a culture method of the tumor specific T cells, and potassium chloride with the concentration of more than 1g/L and less than or equal to 4g/L is added into the culture medium in the culture process. The cell product cultured by the culture medium and the culture method provided by the invention has higher proportion of tumor specific cells and improved proportion of memory T cells, and is suitable for being applied to clinically improving the adoptive immunotherapy effect.

Description

Culture method of tumor specific T cells

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to a culture method of tumor specific T cells.

Background

Malignant tumor is a common disease seriously harming human life health, and the treatment method of the malignant tumor mainly comprises surgical treatment, chemical treatment, radiation treatment and the like. However, the above-mentioned treatments have very limited therapeutic effects, and biological therapies mainly directed to immunology are increasingly advantageous, in which tumor-specific T lymphocytes (CTL) play an important role in the course of anti-tumor immunity.

The tumor specific T lymphocyte is a subgroup of T cells, and CTL is proliferated and differentiated under the premise of antigen stimulation and the assistance of cytokines to be activated into the T cells with specific killing activity. CTL is the main medium of the adoptive immunotherapy of the tumor, the adoptive immunotherapy is to direct the tumor patient to transfuse immune cells with anti-tumor activity, and directly kill the tumor or stimulate the immune response of the organism to kill the tumor cells, thereby achieving the purpose of treating the tumor.

One of the keys of the adoptive immunotherapy using CTL is to try to obtain a large amount of CTL in vitro, i.e. to screen effector cells with tumor-specific killing effect, so as to realize the features of high efficiency and low toxicity of adoptive immunotherapy. The T cell with tumor antigen specificity has the characteristics of wide tumor killing spectrum, high tumor killing activity, strong proliferation capacity and the like. How to increase the number and activity of CTL cells in vitro culture products and enhance the anti-tumor specificity of the CTL cells is the key point for improving the effect of adoptive immunotherapy.

Current research on culture methods for specific T cells has focused primarily on the induction mode.

Chinese patent 201910420697.0 discloses a method for isolating and culturing tumor-specific T cells, which comprises using peripheral blood of a tumor patient as a material, adding a PD-1 antibody to the peripheral blood, incubating, isolating mononuclear cells, and inoculating into a culture flask coated with a recombinant human fibronectin fragment and an anti-human CD3 activating antibody. The method has the advantages of convenient material selection, and high tumor specificity of the finally obtained T cells, and the T cells simultaneously comprise the T cells positive to CD4 and the T cells positive to CD 8. However, the final product obtained by the method has a low proportion of effector memory T cells contained by CD4 positive cells and CD8 positive cells, and has a further optimization space.

Chinese patent 200510038728.4 discloses an in vitro amplification method of tumor antigen specific T lymphocytes, which involves the combined use of interleukin 2, interleukin 15, an excitatory anti-CD 28 monoclonal antibody and tumor antigen loaded antigen presenting cells to induce T cells to become cytotoxic T lymphocytes.

The effect of the specific T cell medium composition on the culture effect is still under extensive study.

Chinese patent 201810955224.6 discloses an immune cell culture medium and its application, which explores the influence of various inorganic salts such as anhydrous calcium chloride, various amino acids such as L-leucine, interleukin 2, interleukin 4 and other components on immune cell culture. However, the culture medium provided by the invention has complicated and variable component types and large component concentration range, and the specific component concentration of the culture medium with the optimal culture effect cannot be determined, thereby bringing great difficulty in culture selection.

The general concentration of potassium chloride in a culture medium of general specific T cells or immune cells is lower (Chinese patent 201810955224.6: 310.5 mg/L-311.3 mg/L; Chinese patent 201711319524.7: 10-1000 mg/L; Chinese patent 201711321217.2: 10-1000 mg/L; Chinese patent 201710797665.3: 250-500mg/L), the highest concentration of potassium chloride conventionally used in the field is 1g/L, and the proportion of total specific T cells and memory T cells of a final product of cell culture is improved by optimizing the concentration of potassium chloride in the culture medium.

Disclosure of Invention

The invention provides a culture medium and a culture method for culturing tumor-specific T cells. The content of potassium chloride in the conventional tumor specific T cells is up to 1g/L, and the invention unexpectedly discovers that the proportion of the tumor specific T cells is higher and the proportion of the memory T cells is also improved in cell products cultured by a culture medium containing high-concentration potassium chloride.

In one aspect, the invention provides a culture medium for tumor-specific T cell culture.

The concentration of potassium chloride in the culture medium is more than 1g/L and less than or equal to 4 g/L.

Preferably, the culture medium comprises potassium chloride with the concentration of 3 g/L.

The culture medium includes but is not limited to serum-free culture medium; the culture medium includes but is not limited to KBM581 culture medium, GT-T551 culture medium, X-VIVO culture medium, GT-T505 culture medium, StemXVivo culture medium, TexMACS GMP culture medium, EX-CELL culture medium, ImmunoCult culture medium, ALYS505N-0 culture medium.

The components of the culture medium include but are not limited to: anhydrous calcium chloride, lipoic acid, sodium pyruvate, magnesium chloride, vitamins, ethanolamine, sodium chloride, nicotinamide, amino acids and antibiotics.

In some embodiments, the medium is X-VIVO medium supplemented with 3g/L potassium chloride.

In some embodiments, the medium is GT-T551 medium supplemented with 3g/L potassium chloride.

In some embodiments, the medium is ALYS505N-0 supplemented with 3g/L potassium chloride.

Optionally, the culture medium further comprises one or more of human IL-2, human IL-7, human IL-15 or human IL-21, preferably at a final concentration of 0.1X 10⁶-7×10⁶U/L human IL-2.

In another aspect, the invention provides a method of culturing tumor-specific T cells.

The culture method comprises the following steps:

(1) peripheral blood PD-1 pretreatment of tumor patients;

(2) obtaining mononuclear cells from the peripheral blood in the step (1) by adopting a Ficoll density gradient method;

(3) incubating the mononuclear cells separated in the step (2) with biotinylated anti-human IgG4 antibody at 25 ℃ for 20min, adding magnetic beads coated with anti-biotin antibody, incubating at 25 ℃ for 20min, and performing magnetic separation to obtain PD-1 positive mononuclear cells;

(4) and (3) resuspending the PD-1 positive mononuclear cells separated in the step (3) by using a serum-free culture solution, wherein the serum-free culture solution comprises potassium chloride with the concentration of more than 1g/L and less than or equal to 4g/L, and performing amplification culture on the resuspended mononuclear cells to obtain a product of the tumor specific T cells.

The tumor patient in the step (1) is one of lung cancer, kidney cancer, intestinal cancer, esophageal cancer, malignant melanoma or cholangiocarcinoma patients.

The pretreatment method in the step (1) is to add the PD-1 antibody into peripheral blood after a tumor patient receives the PD-1 antibody treatment or takes out the peripheral blood of the tumor patient, and then incubate.

Further, in the step (1), after peripheral blood of a tumor patient is taken out during pretreatment, PD-1 antibody is added into the peripheral blood to achieve the final concentration of 0.2-0.8 mu g/mL, the incubation temperature is 0-6 ℃, and the incubation time is 0.5-24 h.

The addition amount of potassium chloride in the serum-free culture solution in the step (4) is preferably 3 g/L.

The serum-free culture solution in the step (4) also comprises one or more of human IL-2, human IL-7, human IL-15 or human IL-21, and the final concentration is preferably 0.1X 10⁶-7×10⁶U/L human IL-2.

The amplification culture method in the step (4) is to seed the strain into a pre-coated bottle, the pre-coated bottle is treated by fibronectin, and the pre-coated bottle is also treated by anti-human CD3 antibody or OKT 3; wherein the concentration of fibronectin is 0.5-10 μ g/mL, preferably 6 μ g/mL; the concentration of the anti-human CD3 antibody is 0.1-4 mug/mL, preferably 1.5 mug/mL; the number of days of culture is 8 to 15 days, preferably 10 days.

In yet another aspect, the invention also provides a product comprising tumor specific T cells.

The product contains the tumor specific T cell final product obtained by the culture method.

The product types include, but are not limited to, pharmaceuticals and vaccines.

Compared with the culture method of specific T cells disclosed at present, the final cell product of the culture method provided by the invention has higher proportion of tumor specific cells, and the proportion of memory T cells is also improved.

Drawings

FIG. 1 shows that the specific T cell culture end product in the presence of high concentration of potassium chloride stimulates the secretion of IFN-. gamma.cells occupying CD8 in tumor cells derived from tumor patients (not treated with anti-PD-1 antibody)⁺And CD4⁺The proportion of cells.

FIG. 2 shows the secretion of IFN-. gamma.cells occupying CD8 by specific T cell culture final product in the presence of high concentration of potassium chloride under stimulation by tumor cells derived from tumor patients (treated with anti-PD-1 antibody)⁺And CD4⁺The proportion of cells.

FIG. 3 shows the ratio of effective memory T cells in the final product of specific T cell culture (L-TIL cell product) under high concentration of potassium chloride.

FIG. 4 is a graph showing the proportion of potent memory T cells in the final product of specific T cell culture (CD19-CART cell product) under high potassium chloride concentration conditions.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

EXAMPLE 1 Effect of high concentrations of Potassium chloride on the proportion of (tumor) antigen-specific T cells in the end product of specific T cell cultures (patients not receiving anti-PD-1 antibody treatment)

(1) Taking peripheral blood of a malignant melanoma patient, adding a PD-1 antibody for incubation (pretreatment);

(3) and (3) incubating the mononuclear cells obtained in the step (2) with biotinylated anti-human IgG4 antibody for 20min at 25 ℃, adding magnetic beads coated with anti-biotin antibody for incubating for 20min at 25 ℃, and performing magnetic separation to obtain the mononuclear cells positive to PD-1.

(4) Respectively resuspending the cells in the step (3) by using potassium chloride-added serum-free culture solution (the serum-free culture solution is purchased from semer fly) with the concentration of 3g/L and serum-free culture solution without potassium chloride, respectively inoculating 6 mu g/mL fibronectin and 1.5 mu g/mL anti-human CD3 antibody into the resuspended mononuclear cells, then respectively carrying out amplification on the mononuclear cells in a pre-coated culture flask, obtaining the amplified T cells after 14 days, and counting the CD8 occupation of IFN-gamma cells secreted by the IFN-gamma cells under the stimulation of tumor cells⁺And CD4⁺The proportion of cells. The specific method comprises the following steps:

obtaining autologous tumor cells (derived from tumor tissue) of a tumor patient: the tumor tissue obtained by operation is cut into pieces, added with collagenase, hyaluronidase and DNA enzyme and digested for 0.5 to 2 hours in a constant temperature incubator at 37 ℃, and then washed for 2 times by phosphate buffer. Tumor cells were obtained by Ficoll density gradient method and frozen in liquid nitrogen (as target cells for specific T lymphocytes).

Specific T lymphocyte evaluation (co-incubation method): taking the two groups of cultured T cells (a potassium chloride group with the concentration of 3g/L and a potassium chloride-free group), washing for two times by serum-free culture solution, then re-suspending by the serum-free culture solution which is added with IFN-gamma secretion blocker Brefeldin A (the concentration is 5ng/mL) and does not contain any cell factor, mixing with autologous tumor cells according to the proportion of 1:1, and incubating in a constant-temperature incubator at 37 ℃ for 6h and then collecting the cells. Labeling CD3, CD4 and CD8 fluorescent antibodies on the cell surface, adding a fixing solution for fixation, then breaking the membrane by using an intracellular staining kit, labeling IFN-gamma fluorescent antibodies, and finally detecting the IFN-gamma cell secretion in CD8 by using a flow cytometer⁺And CD4⁺Proportion of cells (CD8+ IFN-. gamma.)⁺％，CD4+IFN-γ⁺％)。

The results of the relevant experiments are shown in FIG. 1, which shows that the specific T cell ratio of the tumor antigen in the final product of the specific T cell cultured by adding the culture medium with high concentration (3g/L) of potassium chloride is higher.

EXAMPLE 2 Effect of high concentrations of Potassium chloride on the proportion of (tumor) antigen-specific T cells in the final product of a specific T cell culture (patients treated with anti-PD-1 antibody)

(1) Taking peripheral blood of a patient with malignant melanoma;

obtaining autologous tumor cells (derived from tumor tissue) of a tumor patient: cutting the tumor tissue obtained by operation, adding collagenase, hyaluronidase and DNase, digesting for 0.5-2h in a constant temperature incubator at 37 ℃, and then washing for 2 times by using phosphate buffer solution. Tumor cells were obtained by Ficoll density gradient method and frozen in liquid nitrogen (as target cells for specific T lymphocytes).

Specific T lymphocyte evaluation (co-incubation method): taking the two groups of cultured T cells (a potassium chloride group with the concentration of 3g/L and a potassium chloride-free group), washing for two times by serum-free culture solution, then re-suspending by the serum-free culture solution which is added with IFN-gamma secretion blocker Brefeldin A (the concentration is 5ng/mL) and does not contain any cell factor, mixing with autologous tumor cells according to the proportion of 1:1, and incubating in a constant-temperature incubator at 37 ℃ for 6h and then collecting the cells. Marking fluorescent antibodies of CD3, CD4 and CD8 on the cell surface, adding a fixing solution for fixation, breaking a membrane by using an intracellular staining kit, marking the fluorescent antibodies of IFN-gamma, and finally utilizing a flow cytometerDetection of IFN-gamma cell secreting CD8⁺And CD4⁺Proportion of cells (CD8+ IFN-. gamma.)⁺％，CD4+IFN-γ⁺％)。

The results of the relevant experiments are shown in FIG. 2, which shows that the specific T cell ratio of the tumor antigen in the final product of the specific T cell cultured by the medium with high concentration (3g/L) of potassium chloride is higher.

EXAMPLE 3 Effect of high concentrations of Potassium chloride on the proportion of effector memory T cells in the final tumor-specific T cell culture product (L-TIL cell product)

(1) Peripheral blood was taken from cancer patients treated with the PD-1 antibody within 3 weeks, 10 patients were involved in the present example, and tumor types and number of patients were as follows: 2 patients with renal cancer, 2 patients with malignant melanoma, 1 patient with lung cancer, 2 patients with liver cancer, 1 patient with stomach cancer, 1 patient with esophageal cancer and 1 patient with intestinal cancer. The tumor patients are selected from tumor hospitals in Henan province and have obtained patient permission.

The procedure of reference example 2 was followed to examine the effect of high concentrations of potassium chloride on the proportion of effector memory T cells in the final tumor-specific T cell culture product.

For effector memory T cell CD3 contained in final product⁺CD4⁺CCR7^-CD45RA^-(CD4⁺)，CD3⁺CD8⁺CCR7^-CD45RA^-(CD8⁺) The results are shown in FIG. 3. The result shows that the high concentration of potassium chloride (3g/L) can obviously improve the expansion effect of the T cells, and has statistical difference.

Example 4 Effect of high Potassium chloride concentrations on the proportion of potent memory T cells in the final product of specific T cell cultures (CD19-CART cell product)

CD19-CART cells were cultured in a medium containing 3g/L potassium chloride (LONZA, X-VIVO medium) and a medium containing no potassium chloride (LONZA, X-VIVO medium), and 4 groups of parallel CD19-CART cells were prepared and cultured in the same manner as described in patent 201810462786.7 (paragraph 0050-0062).

After the culture is finished, detecting the CD19-CART cell product, wherein the specific detection method comprises the following steps: the collected cells were washed 2 times with PBS and their expression of surface markers CD4, CD8, CD45RA, CCR7 and CD19CAR was detected by flow cytometry.

The results are shown in FIG. 4, the ratio of the T cells with memory effect in the final product of T cells cultured in the medium containing high concentration (3g/L) of potassium chloride is obviously increased, which indicates that the high concentration of potassium chloride can improve the expansion effect of T cells.

Comparative example 1

Compared with the method disclosed in the Chinese patent 201910420697.0, the method disclosed by the application is adopted to carry out a comparison experiment, only the concentration of potassium chloride in the culture medium is different in the experimental setting, and peripheral blood is taken from a renal cancer patient treated by PD-1 in the comparison experiment. The results are as follows:

it should be noted that: 3g/L of potassium chloride in the culture medium is added with KCl with the final concentration of 3g/L on the basis of the original commercial culture solution, so that when the culture medium is compared with the original commercial culture medium, the relative potassium chloride content of the comparative example is 0 g/L.

Claims

1. The application of a culture medium in improving the proportion of memory cells of tumor-specific T cells is characterized in that the concentration of potassium chloride in the culture medium is 3g/L, and the culture medium is an X-VIVO culture medium.

2. A method of increasing the proportion of memory cells in tumor specific T cells comprising the steps of:

(1) peripheral blood PD-1 pretreatment of a tumor patient;

(4) and (3) resuspending the PD-1 positive mononuclear cells separated in the step (3) by using a serum-free culture medium, wherein the serum-free culture medium is an X-VIVO culture medium, the serum-free culture medium comprises potassium chloride with the concentration of 3g/L, and the resuspended mononuclear cells are subjected to amplification culture to obtain a product of the tumor specific T cells.

3. The method of claim 2, wherein the expanding culture in step (4) comprises inoculating into a pre-coated flask, treating with fibronectin, and treating with anti-human CD3 antibody or OKT 3; wherein the concentration of fibronectin is 0.5-10 μ g/mL; the concentration of the anti-human CD3 antibody is 0.1-4 mug/mL; the culture days are 8-15 days.

4. The method according to claim 2, wherein the serum-free medium in step (4) further comprises one or more of human IL-2, human IL-7, human IL-15 or human IL-21.

5. The method according to claim 4, wherein the serum-free medium in step (4) further comprises a final concentration of 0.1X 10⁶-7×10⁶U/L human IL-2.

6. The method of claim 5, wherein the PD-1 antibody is added to the peripheral blood of the tumor patient at a final concentration of 0.2-0.8 μ g/mL after the peripheral blood is taken out from the tumor patient in the pretreatment in the step (1), the temperature of the incubation is 0-6 ℃, and the time of the incubation is 0.5-24 h.