In-vitro culture method of CD8 < + > dry memory T cells, product and application of CD8 < + > dry memory T cells

Abstract

The invention belongs to the field of bioengineering, and particularly relates to an in-vitro culture method of CD8 < + > dry memory T cells, a product and application of the product. The method comprises the following specific steps: (1) coating, stimulating and activating T cells by using an anti-CD3 antibody and RetroNectin; (2) performing resting culture on the activated T cells in a resting culture solution for 0-60 hours; and (3) adding cell factors into the resting culture solution to perform large-scale multiplication culture on the T cells after resting. According to the method, the efficiency of obtaining the CD8 + TSCM cells through in-vitro culture is improved, the culture process is simplified, and the risk of microbial contamination in the culture process and the cost of obtaining the CD8 + TSCM cells are reduced.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering and specifically relates to a CD8 + In vitro culture method, product and application of stem memory T cells. Background Art

[0002] T cell immunotherapy has become a new type of tumor treatment method. It is a method of infusing T cells activated and expanded in vitro to induce the body's immune response to achieve the purpose of treating or alleviating the disease. It is considered to be a very promising tumor treatment strategy. However, the infusing effector T cells have poor long-term anti-tumor effects in vivo, which may be related to their terminal differentiation and exhaustion phenotypes during in vitro expansion. Studies have shown that the use of T cells with a lower degree of differentiation and a wide range of differentiation and expansion potential can achieve a more lasting anti-tumor effect. Cytotoxic T cells (Cytotoxic T Lymphocytes), also known as CD8 + T cells are an important part of T lymphocytes in the human immune system and play a key role in antiviral and anti-tumor immune responses. + T cells can recognize and kill tumor cells in the human body, and at the same time secrete a variety of cytokines to enhance the anti-tumor effects of other cells. + When T cells are terminally differentiated, CD8 + T SCM The superior expansion of cells in vitro is of great significance to improving the effect of adoptive cell therapy.

[0003] Chinese patent CN201610281300.0 provides a method for constructing human stem memory T cells. First, mononuclear cells are extracted from human peripheral blood, and then the initial T cells are sorted using immunomagnetic beads (CD45RA positive sorting, CD45RO and CD95 secondary negative sorting). The initial T cells are activated by CD3 / CD28 magnetic beads and TWS119, IL-2, IL-7, IL-15, etc. induce the production and proliferation of stem memory T cells. Prior art CN114958737B discloses an in vitro expansion method of stem memory T cells and its application, which mainly adopts the method of adjusting the cell culture conditions to acidic culture conditions to obtain a lymphocyte population dominated by stem memory T cells, and the lymphocyte population includes CD4, CD8T cells, γδT, TCRT, CART cells and other in vitro amplified T cells, and the stem memory T cells obtained by this method can be used in clinical treatment, especially anti-tumor and anti-infection immunotherapy.

[0004] The existing method of obtaining stem memory T cells relies on flow cytometer sorting technology, which has high requirements for instruments and operating environment, is not suitable for large-scale cell production processes, and has a high risk of microbial contamination. In addition, in vitro optimization of culture medium and inducing or maintaining stem memory T cell subsets by adding cytokines have become the mainstream method for preparing stem memory T cells. For example, interleukin 21 or interleukin 7 and interleukin 15 are used to induce amplification or maintain stem memory T cells. However, in large-scale production processes, the amount of cytokine drugs used is large, which will inevitably increase production costs. Therefore, it is of great application value to explore a method for preparing stem memory T cells that is fast, efficient, low-cost, and has a high safety factor. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a CD8 + The in vitro culture method, product and application of stem memory T cells, and the specific technical solutions are as follows:

[0006] In one aspect, the present invention provides a CD8 + The in vitro culture method of stem memory T cells comprises the following steps:

[0007] (1) Anti-CD3 antibodies and recombinant human fibronectin (RetroNectin) were used to coat and stimulate activated T cells;

[0008] (2) Activated T cells were cultured in resting medium for 0-60 h;

[0009] (3) Add cytokines to the resting culture medium to perform large-scale expansion culture of resting T cells.

[0010] Specifically, the concentration of the anti-CD3 antibody in step (1) is 0.8-1.5 μg / mL.

[0011] In some embodiments, the anti-CD3 antibody is at a concentration of 1.5 μg / mL.

[0012] Specifically, the concentration of RetroNectin in step (1) is 5-10 μg / mL.

[0013] In some embodiments, the RetroNectin concentration is 6 μg / mL.

[0014] Specifically, the resting culture medium in step (2) is selected from: X-VIVO TM 15 Serum-free culture medium, any one of GT-T551, GT-T55H3, ALyS505N, and RPMI-1640.

[0015] In some embodiments, the resting culture medium is selected from: X-VIVO TM 15 Serum-free culture medium.

[0016] Specifically, the static culture time in step (2) is 0-48h.

[0017] Preferably, the static culture time is 24 hours.

[0018] Specifically, the cytokine in step (3) is selected from any one of rhIL-2, H9T, IL-7+IL-15+IL-21 and IL-2.

[0019] Here, IL-7+IL-15+IL-21 refers to the cytokines IL-7, IL-15 and IL-21 being mixed together at a certain concentration and added to the culture medium.

[0020] Specifically, the cytokine in step (3) is rhIL-2, and the concentration of the rhIL-2 is 800-1000 U / mL.

[0021] In some embodiments, the concentration of the cytokine is 1000 U / mL.

[0022] Specifically, the T cells in step (1) are selected from PBMCs derived from human peripheral blood or initial T cells sorted from PBMCs.

[0023] In another aspect, the present invention provides a lymphocyte population prepared by the above method.

[0024] In yet another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned lymphocyte population.

[0025] In another aspect, the present invention discloses CD8 + An in vitro culture method for stem memory T cells or the use of the lymphocyte population or the pharmaceutical composition in the preparation of adoptive immunotherapy drugs.

[0026] Preferably, the adoptive immunotherapy is anti-tumor immunotherapy, anti-infection immunotherapy and autoimmune disease treatment.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a CD8 + In vitro culture method, product and application of stem memory T cells. When culturing T cells, this patent does not require the initial T cells to be sorted from PBMCs by flow cytometry or magnetic beads. Only a certain period of resting culture is performed after the T cells are activated, and then conventional cell expansion culture is performed to obtain a high proportion of CD8 +T SCM This patent improves the in vitro culture of CD8 + T SCM The efficiency of cells is improved, the culture process is simplified, the risk of microbial contamination during the culture process is reduced, and the CD8 + T SCM The cost of cells.

[0029] When culturing T cells, this patent can obtain a high proportion of CD8 + T SCM Cell products. Since the market price of clinical application-level rhIL-2 is much lower than IL-7, IL-15, IL-21, H9T and other cytokines, this patent can significantly reduce the preparation of clinical application-level CD8 + T SCM Cost of cell products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Flow chart of cell culture for the control group and the resting culture group.

[0031] Figure 2 CD8 in the final products of T cell culture obtained from different culture systems + T cells and CD4 + The proportion of T cells.

[0032] Figure 3 T cells in the T cell products obtained from the control group and the resting culture group SCM CD8 + The proportion of T cells (T SCM The phenotype is CD95 + CD45Ra + CCR7 + ).

[0033] Figure 4 T cells in the T cell products obtained from the control group and the resting culture group SCM CD4 + The proportion of T cells (T SCM The phenotype is CD95 + CD45Ra + CCR7 + ).

[0034] Figure 5 A shows the expression of different immunosuppressive molecules (TIM-3, LAG-3, PD-1) and stem cell factor (TCF-1) in the final T cell products obtained from the control group and the resting culture group in CD8 +B is the expression of different immunosuppressive molecules (TIM-3, LAG-3, PD-1) and stem cell factor (TCF-1) in the final T cell products obtained from the control group and the resting culture group on CD4 + Expression on T cells. "ns" in the figure indicates no statistical difference between the two groups, P>0.05; "*" indicates a statistical difference between the two groups, where "*" means P<0.05, "**" means P<0.01, and "***" means P<0.001.

[0035] Figure 6 CD8 + Comparison of differences in cytokine secretion (IL-2 and IFN-γ) by T cells. The "*" in the figure indicates that there is a statistical difference between the two groups, where "*" means P < 0.05.

[0036] Figure 7 A is a T cell product obtained by resting culture and combining with multiple γ chain cytokines. SCM CD8 + B is the ratio of immunosuppressive molecule TIM-3 in CD8 T cells in the final product of T cells obtained by resting culture and combining with various γ chain cytokines. + Expression on T cells. The "*" in the figure indicates that there is a statistical difference between the two groups, where "*" means P < 0.05 and "**" means P < 0.01.

[0037] Figure 8 A is the T cell products obtained after resting culture for 0h, 10h, 24h and 48h. SCM CD8 + B is the ratio of T cells in T cell products obtained after resting culture at 0h, 10h, 24h and 48h. SCM CD8 + C is the percentage of immunosuppressive molecules TIM-3 in the final T cell products obtained at 0h, 10h, 24h and 48h of resting culture in CD8 + Expression on T cells. Among them, "*" means P < 0.05, "**" means P < 0.01, "***" means P < 0.001, and "****" means P < 0.0001. DETAILED DESCRIPTION

[0038] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0039] Experimental Materials:

[0040] Peripheral blood was obtained from healthy donors, and the peripheral blood collection process, experimental methods, and uses were approved by the Ethics Committee of Henan Cancer Hospital, approval number 2022-KY-0150-001.

[0041] Experimental reagents / instruments:

[0042] Table 1

[0043]

[0044]

[0045] Example 1CD8 + In vitro expansion method of stem memory T cells

[0046] (1) Human peripheral blood collection: Peripheral blood from healthy donors is collected manually and anticoagulated using sodium heparin, EDTA salt or citrate anticoagulant;

[0047] (2) Extraction of mononuclear cells (PBMCs): density gradient centrifugation;

[0048] The collected human peripheral blood was centrifuged at 800g for 10min at 4℃, and the upper plasma was aspirated and stored at 4℃. The remaining blood cells were diluted with 2 volumes of normal saline and added to the upper layer of 0.5 volumes of human lymphocyte separation solution, centrifuged at 800g for 22min at 20℃, with an acceleration of 3, and the middle white film layer was aspirated as mononuclear cells. The mononuclear cells were resuspended in normal saline at 350g and centrifuged for 5min to wash twice;

[0049] (3) Stimulation and activation of PBMCs using anti-CD3 antibody and RetroNectin coated plates:

[0050] Preparation before the experiment: Prepare the coating solution (anti-CD3 antibody: 1.5 μg / mL; RetroNectin: 6 μg / mL) with sterile PBS and add 156 μL / cm 2 Add the coating solution to the well plate (48-well plate: add 126 μL of coating solution to each well), seal with sealing film, and place in a 4°C refrigerator away from light for 24 h;

[0051] With X-VIVOTM 15 Serum-free culture medium (containing 3% fetal bovine serum) was used to resuspend PBMCs to 2×10 6 / mL, according to 250μL / cm 2 Add the cell suspension to the wells coated with anti-CD3 antibody and RetroNectin (discard the coating solution before adding the cell suspension to the wells, and then use an equal volume of X-VIVO TM 15 serum-free culture medium, saline or PBS, incubate for 10 min at room temperature and discard), 37°C, 5% CO 2 Culture for 48 hours;

[0052] (4) Using resting culture medium (X-VIVO TM 15 serum-free culture medium) to culture the activated T cells in a static manner:

[0053] T cells activated for 48 h were washed twice with resting culture medium or saline, and the washing conditions were centrifugation at 300 g for 5 min. 6 Resuspend the cells at a density of 1 / mL and inoculate into new wells / flasks and incubate at 37°C with 5% CO 2 Incubate in a saturated humidity incubator for 24 h;

[0054] (5) Use expansion culture medium (containing 1000U / mL rhIL-2X-VIVO TM 15 serum-free culture medium) to perform large-scale expansion culture of resting T cells:

[0055] After resting culture of T cells, they were washed once with saline and then resuspended in expansion medium to 2×10 5 / mL-5×10 5 / mL, at 37°C, 5% CO 2 Culture in a saturated humidity incubator. Replace the culture medium every 1-2 days and maintain the cell density at 2×10 5 / mL-5×10 5 / mL, cultured for 7-14 days, can obtain CD8-rich cells of clinical application magnitude + T SCM of cell products.

[0056] Example 2 Effect of resting culture on the preparation of T cell final product

[0057] (1) Human peripheral blood collection: same as in Example 1;

[0058] (2) Extraction of mononuclear cells (PBMCs): same as in Example 1;

[0059] (3) Stimulation and activation of PBMCs using anti-CD3 antibody and RetroNectin coated plates:

[0060] Quiescent culture group: X-VIVO TM 15 Serum-free culture medium (containing 3% fetal bovine serum) was used to resuspend PBMCs to 2×10 6 / mL, according to 250μL / cm 2 Add the cell suspension to the wells coated with anti-CD3 antibody and RetroNectin (discard the coating solution before adding the cell suspension to the wells, and then use an equal volume of X-VIVO TM 15 serum-free culture medium, physiological saline or PBS, incubate at room temperature for 10 min and discard);

[0061] Control group: X-VIVO TM 15 Serum-free culture medium (containing 3% fetal bovine serum and 1000 U / mL rhIL-2) was used to resuspend PBMCs to the same density and inoculated into the coated wells in the same manner;

[0062] Both groups of cells were placed at 37°C with 5% CO 2 Culture in an incubator for 48 h.

[0063] (4) Using resting culture medium to rest culture the activated T cells:

[0064] Resting culture group: T cells activated for 48 hours were washed twice with resting culture medium or physiological saline, and the washing condition was centrifugation at 300g for 5 minutes. 6 Resuspend the cells at a density of 1 / mL and inoculate into new wells / flasks and incubate at 37°C with 5% CO 2 Incubate in a saturated humidity incubator for 24 h;

[0065] Control group: Synchronously add an equal volume of culture medium and continue culturing.

[0066] (5) Use expansion culture medium (containing 1000U / mL rhIL-2X-VIVO TM 15 serum-free culture medium) to perform large-scale expansion culture of resting T cells:

[0067] Resting culture group: After resting culture of T cells, wash them with physiological saline and then resuspend the cells in expansion medium to 2×10 5 / mL-5×10 5 / mL, at 37°C, 5% CO 2 Culture in a saturated humidity incubator. Replace the culture medium every 1-2 days and maintain the cell density at 2×10 5 / mL-5×105 / mL, cultured to day 10;

[0068] Control group: cells were cultured in expansion medium until day 10 and then harvested for subsequent experiments. + The experimental process of stem memory T cells is as follows Figure 1 shown.

[0069] Take 1×10 in step (5) respectively 6 The cells obtained from the control group and the resting culture group were analyzed by flow cytometry for the stem memory T cells (CD95 + CD45Ra + CCR7 + ) ratio for testing, the results are as follows Figure 2-4 As shown, the T cell products in the resting culture group contained a higher proportion of CD8 + T cells, a lower percentage of CD4 + T cells, resting culture group CD8 + T cells SCM The phenotype ratio is higher.

[0070] The expression of two groups of cellular immunosuppressive molecules (TIM-3, LAG-3, PD-1) and stem cell transcription factor (TCF-1) was detected. Figure 5 As shown, CD8 + TCF-1 was higher in T cells, while the immunosuppressive molecule TIM-3 was lower.

[0071] The difference in cytokine secretion function of the final products of the two groups of cells obtained in step (5) was detected by flow cytometry. 1×10 6 The cells obtained from the control group and the resting culture group were washed twice with resting culture medium or saline. The cells were stimulated with PMA (30 ng / mL), ionomycin (500 ng / mL), and BFA (1×) for 2 h. The cells were then collected again and flow cytometry was performed according to the instructions of the intracellular cytokine detection kit. The results are shown in Figure 2. Figure 6 As shown, CD8 + IL-2 in T cells + IFN-γ - The percentage of phenotype was significantly higher than that of the control group, while the percentage of CD8 + IL-2 in T cells + IFN-γ + and IL-2 - IFN-γ + The percentage of the phenotype was significantly lower than that of the control group.

[0072] In summary, the technical solution of this application can cultivate a large number of CD8 + T SCM cell.

[0073] Example 3 Resting culture combined with multiple γ-chain cytokines enhances PBMCs to generate CD8 + T SCM cell

[0074] The experimental method of this example refers to that of Example 1, with the following differences: ① the raw materials used in this example are derived from the peripheral blood of tumor patients; ② the cytokines added to the amplification culture medium for amplifying T cells are IL-2 (1000U / mL), H9T (10nM) or IL-7 (10ng / mL) + IL-15 (50U / mL) + IL-21 (10ng / mL).

[0075] Take 1×10 6 The cells obtained from the control group and the resting culture group were tested by flow cytometry. The results are as follows: Figure 7 As shown in the figure, it shows that resting culture and different γ-chain cytokine and cytokine combinations can enhance the production of CD8 by PBMCs compared with the corresponding control groups. + T SCM cells, and reduced CD8 + The results of the expression of TIM-3 (immunosuppressive molecule) on T cells showed statistically significant differences.

[0076] The T cell product obtained by the technical solution of the present invention is T SCM CD8 + The proportion of T cells is about 42%. The cytokine rhIL-2 in the technical solution is replaced with other cytokines on the market, such as IL-2 (1000U / mL), H9T (10nM) or IL-7 (10ng / mL) + IL-15 (50U / mL) + IL-21 (10ng / mL) for experiments. The T cells in the obtained cell products are about 42%. SCM CD8 + The proportion of T cells is about 24%-30%. In addition, the market price of rhIL-2 is relatively low, which shows that this technical solution can achieve better technical effects.

[0077] Comparative Example

[0078] The experimental method of this comparative example refers to that of Example 1, except that: when PBMCs are activated and then cultured, the time for culture is set to 0 h, 10 h, 24 h and 48 h, respectively, and the subsequent cell expansion scheme is the same as that of Example 1.

[0079] Take 1×10 6The cells obtained from different groups were cultured and tested by flow cytometry. The results are as follows: Figure 8 As shown in the figure, it shows that as the resting culture time increases, PBMCs generate CD8 + T SCM Cell enhancement and reduction of CD8 in the final product + The expression of TIM-3 (immunosuppressive molecule) on T cells was statistically significant. In the prior art CN118726254A, human stem memory T cells were obtained after 12 days of culture, accounting for CD8 + The proportion of T cells is 27%, which shows that the technical effect of the present invention is better than the effect disclosed in the prior art.

Claims

1. A CD8 + The in vitro culture method of stem memory T cells is characterized by: The following steps are involved: (1) Stimulate and activate T cells by coating with anti-CD3 antibodies and recombinant human fibronectin; (2) Activated T cells were cultured in resting medium for 0-60 h; (3) Add cytokines to the resting culture medium to expand and culture the resting T cells.

2. The in vitro culture method according to claim 1, characterized in that: In step (1), the concentration of the anti-CD3 antibody is 0.8-1.5 μg / mL, and the concentration of RetroNectin is 5-10 μg / mL.

3. The in vitro culture method according to claim 1, characterized in that: The resting culture medium in step (2) is selected from: X-VIVO TM 15 Serum-free culture medium, any one of GT-T551, GT-T55H3, ALyS505N, and RPMI-1640.

4. The in vitro culture method according to claim 1, characterized in that: The static culture time in step (2) is 0-48h.

5. The in vitro culture method according to claim 1, characterized in that: The cytokine in step (3) is selected from any one of rhIL-2, H9T, IL-7+IL-15+IL-21 and IL-2.

6. The in vitro culture method according to claim 5, characterized in that: The cytokine in step (3) is rhIL-2, and the concentration of the rhIL-2 is 800-1000U / mL.

7. The in vitro culture method according to claim 1, characterized in that: The T cells in step (1) are selected from PBMCs derived from human peripheral blood or initial T cells sorted from PBMCs.

8. A lymphocyte population, characterized in that: Prepared by the in vitro culture method according to any one of claims 1 to 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the lymphocyte population according to claim 8.

10. Use of the in vitro culture method according to any one of claims 1 to 7, the lymphocyte population according to claim 8, or the pharmaceutical composition according to claim 9 in the preparation of an adoptive immunotherapy drug.

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