Method for efficiently amplifying depleted precursor T cells by using rapamycin and application of depleted precursor T cells

Abstract

The invention discloses a method for efficiently amplifying depleted precursor T cells by using rapamycin and application of the depleted precursor T cells. The method comprises the following steps: adding rapamycin into an activation culture system of T cells, and culturing to obtain efficiently amplified depleted precursor T cells and inhibit the depletion degree of the CD8 + T cells, and when the rapamycin is stopped, the depleted precursor T cells can be rapidly and directionally differentiated into the CD8 + T cells. Therefore, the method provided by the invention can be applied to the in-vitro preparation process of adoptive T cell therapy so as to improve the functional state of the T cells in advance and provide technical support for clinical popularization of solid tumor infiltration lymphocyte therapy.

Description

Technical Field

[0001] This invention relates to the field of immune cell therapy, and more specifically, to a method and application for efficiently expanding exhausted precursor T cells using rapamycin. Background Technology

[0002] Tumor-infiltrating lymphocyte (TIL) therapy is one of the most promising immunotherapies for solid tumors. Its core lies in isolating, expanding, and activating CD8+ cells from the patient's tumor tissue in vitro. + T cells are then reinfused into the patient to exert an anti-tumor effect. However, it is difficult to obtain a sufficient number of well-functioning CD8 cells during in vitro culture. + T cells limit their clinical application.

[0003] Recent studies have shown that CD8 has "stem cell" characteristics. + T cells—exhausted T cell progenitor (Tpex)—are capable of self-renewal and differentiation into effector T cells. High expression of TCF1 is a marker of Tpex. Maintaining and expanding Tpex in vitro is crucial for improving the efficacy of TIL therapy.

[0004] Rapamycin is a macrolide antibiotic extracted from *Streptomyces hygroscopicus* and is an inhibitor of mammalian target of rapamycin (mTOR). Its core mechanism involves inhibiting the mTOR signaling pathway, playing a crucial role in cell growth, proliferation, metabolism, and autophagy, demonstrating superior therapeutic potential in anti-tumor, anti-aging, and tissue regeneration. Studies have shown that rapamycin plays an important role in immune regulation, enhancing antiviral immunity and regulating the reconstruction of natural killer cells. Patent CN 111494414 A states that tumor-infiltrating lymphocytes cultured in vitro in the presence of tumor antigens, antigen-presenting cells (APCs), IL-21, IL-15, and rapamycin can produce CD8+. + T cell population (including CD8) + At least 50% of the T cell population consists of CD8 cells. + T cells exhibit a central memory phenotype and are specific to tumor antigens expressed by cancer, among which IL15 and IL21 are known to drive slower homeostatic proliferation and suppress CD8+. + T cells tend to be in a central memory differentiation state. The main function of rapamycin is to increase memory T cell differentiation and enhance the anti-tumor efficacy of vaccine-induced T cells through short-term in vitro treatment. Currently, there are no publicly available reports on the effect of rapamycin in expanding exhausted precursor T cells. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a method for efficiently expanding exhausted precursor T cells.

[0006] The second objective of this invention is to provide a method for inhibiting CD8. + Methods to deplete T cells.

[0007] The third objective of this invention is to provide a highly efficient method for amplifying CD8. + T-cell approach.

[0008] The fourth objective of this invention is to provide rapamycin for the preparation and expansion of exhausted precursor T cells, the inhibition of T cell exhaustion, and the effective expansion of CD8. + Application of T-cell products.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention provides a method for efficiently expanding exhausted precursor T cells, wherein the method involves adding rapamycin to a T cell activation culture system and culturing.

[0011] This invention uses T cells from OT1 and TCF1-GFP double-positive mice (a type of CD8 cell that combines antigen specificity and TCF1 expression visualization markers). + T cells (ideal cell types for studying exhausted precursor T cells in immunological research) were cultured in activation medium for a period of time. A certain concentration of rapamycin was added to the culture system, and after 8 days of culture, flow cytometry results showed that TCF1 was present in the T cells. + TIM-3 - The significantly increased cell clustering (high TCF1 expression is a marker of Tpex) indicates that adding rapamycin to the culture system can significantly increase the number and proportion of exhausted precursor T cells (Tpex). Exhausted precursor T cells are CD8+ cells with "stem cell" characteristics. + T cells are capable of self-renewal and differentiation into effector T cells. Simultaneously, PD-1 in T cells... + TIM-3 + Clustering was significantly reduced (TIM3 is CD8) + The T cell surface exhaustion marker indicates that the addition of rapamycin to the culture system can also significantly inhibit CD8. + T cell exhaustion level. Six days after stopping drug administration, flow cytometry results showed that the number of T cells in the rapamycin-treated group was significantly higher than that in the control group, indicating that adding rapamycin to the culture system for a period of time and then stopping drug administration can significantly promote T cell proliferation. Simultaneously, the CD8+ level in the rapamycin-treated group... + T cell clusters were clearly defined and their content was as high as 97%, while the control group had CD8. +The low T-cell content indicates that the addition of rapamycin to the culture system for a period of time followed by discontinuation of drug administration can lead to the targeted expansion of CD8 cells. + T cells. Therefore, this invention provides a simple and scalable CD8 cell method. + The T-cell expansion method, by adding rapamycin, can increase the proportion of exhausted precursor T cells and reduce the level of T cell exhaustion, significantly enhancing CD8. + The number of T cells provides technical support for the clinical promotion of tumor-infiltrating lymphocyte therapy for solid tumors.

[0012] Therefore, the present invention provides a method for inhibiting CD8 + The method for T cell depletion involves adding rapamycin to a T cell activation culture system and culturing.

[0013] This invention also provides a method for efficiently amplifying CD8. + The T-cell method involves adding rapamycin to a T-cell activation culture system and culturing for 6–10 days; then culturing for 4–8 days after stopping the addition of rapamycin.

[0014] Furthermore, the final concentration of rapamycin in the activation culture system is 0.1–10 μM.

[0015] Furthermore, the activation culture system contains basal 1640 medium, 10% FBS, 1‰ IL-2, 1‰ β-mercaptoethanol and 1‰ OVA short peptide.

[0016] Furthermore, the density of the T cells is 1×10⁻⁶. 5 ~5×10 6 cells / mL.

[0017] Furthermore, the culture was incubated with rapamycin for 8 days.

[0018] Furthermore, the culture was carried out for 6 days after the addition of rapamycin was stopped.

[0019] Furthermore, when rapamycin is added, the culture medium is changed and the same concentration of rapamycin is added every 48–72 hours.

[0020] This invention also provides exhausted precursor T cells and CD8 cells obtained by the above method. + T cells.

[0021] Tumor-infiltrating lymphocyte (TIL) therapy is one of the most promising immunotherapies for solid tumors. Its core lies in isolating, expanding, and activating CD8+ cells from the patient's tumor tissue in vitro. +T cells are then reinfused into the patient to exert an anti-tumor effect. The exhausted precursor T cells obtained in this invention can be directionally and efficiently expanded by culturing them in a rapamycin-free culture system for a period of time. + T cells. The method described in this invention allows for the efficient expansion of CD8+ cells in an in vitro culture system. + T cells, to obtain a sufficient number of cells with low exhaustion and high survival rate, to overcome the bottleneck of large-scale production of TIL therapy.

[0022] Therefore, the present invention also provides the use of the above-mentioned depleted precursor T cells in the preparation of antitumor drugs.

[0023] Furthermore, the tumor is a solid tumor, including but not limited to melanoma, non-small cell lung cancer, and colorectal cancer.

[0024] This invention also provides rapamycin in the preparation of expanded exhausted precursor T cells, the inhibition of T cell exhaustion, and the expansion of CD8. + Application of T-cell products.

[0025] Furthermore, the expanded depleted precursor T cells are upregulated TCF1-positive CD8 cells. + T cell ratio.

[0026] Furthermore, the inhibition of T cell exhaustion is achieved by downregulating TIM3-positive CD8+. + T cell ratio.

[0027] Furthermore, the amplification of CD8 + T cells promote T cell proliferation and cell viability, and increase CD8+. + The proportion of T cells.

[0028] Furthermore, the product is a culture medium or a drug.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method and application for efficiently expanding exhausted precursor T cells using rapamycin. The method involves adding rapamycin to a T cell activation culture system and culturing the cells to obtain efficiently expanded exhausted precursor T cells, while simultaneously inhibiting CD8. + The degree of T cell exhaustion is such that, upon discontinuation of rapamycin, exhausted precursor T cells can rapidly differentiate into CD8 cells. + T cells. Therefore, the method provided by this invention can be applied to the in vitro preparation process of adoptive T cell therapy to improve the functional state of T cells in advance, providing technical support for the clinical promotion of solid tumor infiltrating lymphocyte therapy. Attached Figure Description

[0030] Figure 1Figure showing the results of rapamycin increasing the number and proportion of exhausted precursor T cells.

[0031] Figure 2 Rapamycin inhibits CD8 + The result of T cell depletion.

[0032] Figure 3 This is a diagram showing the results of rapamycin promoting T cell proliferation.

[0033] Figure 4 CD8 was directionally amplified for rapamycin. + The results of T cell analysis. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] Rapamycin (Sirolimus): is a potent and specific mTOR inhibitor.

[0037]

[0038] Example 1: Rapamycin increases the number and proportion of exhausted precursor T cells (Tpex). I. Experimental Methods 1. Acquisition and activation of mouse spleen T cells This embodiment uses T cells from OT1 and TCF1-GFP double-positive mice (mouses that are double-positive by hybridization of OT1 and TCF1 mice). To obtain mouse spleen T cells, the spleen was first placed in a culture dish containing 2% FBS solution in a sterile operating table, then transferred to a 70 μm filter screen, 2 mL of 2% FBS solution was added, and the cells were gently ground with the bottom of a syringe plunger. The cell suspension was collected into a 15 mL centrifuge tube, and another 3 mL of 2% FBS was added, followed by repeated grinding. The suspensions were combined and the volume was adjusted to 5 mL. After centrifugation at 4°C and 1500 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in 1 mL of erythrocyte lysis buffer and lysed at room temperature for 5 min. 2 mL of 2% FBS was added to stop lysis, and the cells were centrifuged again and the supernatant was discarded. The cells were resuspended in 1 mL of 2% FBS, centrifuged, and the supernatant was discarded. The supernatant was replaced with 1 mL of activation medium (basal 1640 medium containing 10% FBS and 1‰ IL-1). 2.1‰ β Resuspend the cells in mercaptoethanol and 1‰ OVA short peptide. Filter the suspension through a 40 μm filter into a 10 cm culture dish, add 9 mL of activation medium, and incubate. Change the medium every other day. On day 4, collect the cells, centrifuge, resuspend in 1 mL of activation medium, count the cells, and adjust the cell concentration to 1×10⁻⁶. 6 / mL.

[0039] 2. Effects of rapamycin on T cells Press 5×10 5 Cells were seeded into culture plates and treated with drugs according to their respective groups (control group, 0.1 μM rapamycin, 1 μM rapamycin, 10 μM rapamycin (final concentration)); the medium was changed and the drugs were administered again every other day. On day 8, the number and proportion of exhausted precursor T cells (Tpex) were detected by flow cytometry.

[0040] II. Experimental Results After adding rapamycin to the culture medium, the flow cytometry results were as follows: Figure 1 As shown, TCF1 in T cells + TIM-3 - The significant increase in cell clusters (high TCF1 expression is a marker of Tpex) indicates that adding rapamycin to the culture system can reduce the expression of exhaustion markers, improve cell function and survival, and significantly increase the number and proportion of exhausted precursor T cells (Tpex), with the proportion of exhausted precursor T cells increasing sixfold. Exhausted precursor T cells are CD8+ cells with "stem cell" characteristics. + T cells are capable of self-renewal and differentiation into effector T cells.

[0041] Example 2: Rapamycin inhibits CD8 + T cell exhaustion I. Experimental Methods 1. The acquisition and activation of mouse spleen T cells are the same as the steps described above.

[0042] 2. Effects of rapamycin on T cells Press 5×10 5 Cells were seeded into culture plates and treated with the appropriate drugs according to their respective groups (control group, 0.1 μM rapamycin, 1 μM rapamycin, 10 μM rapamycin (final concentration)); the medium was changed and the drugs were administered again every other day. On day 8 of culture, CD8+ was detected by flow cytometry. + The number and proportion of T cells.

[0043] II. Experimental Results After adding rapamycin to the culture medium, the flow cytometry results were as follows: Figure 2 As shown, PD-1 in T cells + TIM-3+ Clustering was significantly reduced (TIM3 is CD8) + T cell surface exhaustion markers indicate that the addition of rapamycin to the culture system can significantly inhibit CD8. + The degree of T cell exhaustion is defined as a decrease in the proportion of exhausted T cells ≥95%.

[0044] Example 3: Increased CD8 count after rapamycin culture + Number and proportion of T cells I. Experimental Methods 1. Acquisition and activation of mouse spleen T cells This invention uses T cells from OT1 and TCF1-GFP double-positive mice for experiments. To obtain mouse spleen T cells, the spleen was first placed in a culture dish containing 2% FBS solution in a sterile operating table, then transferred to a 70 μm filter screen, and 2 mL of 2% FBS solution was added and gently ground with the bottom of a syringe plunger. The cell suspension was collected into a 15 mL centrifuge tube, and 3 mL of 2% FBS was added and the grinding was repeated once. The suspensions were combined and the volume was adjusted to 5 mL. After centrifugation at 4°C and 1500 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in 1 mL of erythrocyte lysis buffer and lysed at room temperature for 5 min. 2 mL of 2% FBS was added to stop lysis, and the cells were centrifuged again and the supernatant was discarded. The cells were resuspended in 1 mL of 2% FBS, centrifuged, and the supernatant was discarded. The supernatant was replaced with 1 mL of activation medium (basal 1640 medium containing 10% FBS and 1‰ IL-1). 2.1‰ β Resuspend the cells in mercaptoethanol and 1‰ OVA short peptide. Filter the suspension through a 40 μm filter into a 10 cm culture dish, add 9 mL of activation medium, and incubate. Change the medium every other day. On day 4, collect the cells, centrifuge, resuspend in 1 mL of activation medium, count the cells, and adjust the cell concentration to 1×10⁻⁶. 6 / mL.

[0045] 2. 5×10 5 Cells were seeded into culture plates and treated according to their respective groups (control group, 0.1 μM rapamycin, 1 μM rapamycin, 10 μM rapamycin (final concentration)). The medium was changed, cells were counted, and the treatment repeated every other day. On day 8, cells were collected, centrifuged, and treated again without further administration. The cells were resuspended in 1 mL of activation medium, counted using trypan blue, and seeded into culture plates. The medium was changed and cells were counted every other day for a total of 14 days. CD8+ was detected by flow cytometry. + The number and proportion of T cells.

[0046] II. Experimental Results Flow cytometry results as follows Figure 3As shown, the number of T cells in the treatment group was significantly higher than that in the control group, indicating that adding rapamycin to the culture system for a period of time and then stopping the administration can significantly promote T cell proliferation, increasing the total cell number by ≥400%. Further flow cytometry results are shown below. Figure 4 As shown, CD8 in the drug-treated group + T cell clusters were clearly defined, with a content as high as 97%, significantly increasing the total number of CD8+ T cells (proliferation rate >10-fold), compared to the control group's CD8+ T cells. + The low T-cell content indicates that the addition of rapamycin to the culture system for a period of time followed by discontinuation of drug administration can lead to the targeted expansion of CD8 cells. + T cells.

[0047] In summary, this invention provides a simple and scalable method for TIL amplification. By adding rapamycin to the T cell activation culture system, the proportion of exhausted precursor T cells can be increased and the level of T cell exhaustion can be reduced, significantly enhancing CD8+. + The number of T cells provides technical support for the clinical promotion of tumor-infiltrating lymphocyte therapy for solid tumors.

Claims

1. A method for efficiently expanding exhausted precursor T cells, characterized in that, The method involves adding rapamycin to a T-cell activation culture system and then culturing.

2. A method for inhibiting CD8 + The method for T cell exhaustion is characterized by... The method involves adding rapamycin to a T-cell activation culture system and then culturing.

3. A highly efficient method for amplifying CD8 + The T-cell method, characterized in that... The method involves adding rapamycin to the T cell activation culture system and culturing for 6–10 days; then culturing for 4–8 days after stopping the addition of rapamycin.

4. The method according to any one of claims 1 to 3, characterized in that, The final concentration of rapamycin in the activated culture system is 0.1–10 μM.

5. The method according to any one of claims 1 to 3, characterized in that, The activation culture system contains basic 1640 medium, 10% FBS, 1‰ IL-2, 1‰ β-mercaptoethanol and 1‰ OVA short peptide.

6. The method according to any one of claims 1 to 3, characterized in that, The density of the T cells was 1×10⁻⁶. 5 ~5×10 6 cells / mL.

7. The method according to any one of claims 1 to 3, characterized in that, When rapamycin is added, the culture medium should be changed and the same concentration of rapamycin should be added every 48–72 hours.

8. Rapamycin in the preparation of exhausted precursor T cells, inhibition of T cell exhaustion, and expansion of CD8+. + Application of T-cell products.

9. The application according to claim 8, characterized in that, The expanded depleted precursor T cells were upregulated TCF1-positive CD8 cells. + T cell ratio.

10. The application according to claim 8, characterized in that, The inhibition of T cell exhaustion is achieved by downregulating TIM3-positive CD8+ cells. + T cell ratio.

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