Method for inducing generation of peripheral CD4CD8 double positive T cells and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a method for inducing generation of peripheral CD4CD8 double-positive T cells and application thereof, and the method comprises the following steps: separating to obtain T lymphocytes; the T lymphocytes are placed in a culture plate coated with an anti-CD3 antibody and an anti-CD28 antibody, the final concentration of the anti-CD3 antibody is 3 [mu] g / mL, and the final concentration of the anti-CD28 antibody is 3 [mu] g / mL; culturing for 48 hours under the conditions of 37 DEG C and 5% CO2; adding an SMARCA4 inhibitor into the culture system, wherein the final concentration of the SMARCA4 inhibitor is 0.5 [mu] M to 5 [mu] M; and continuously culturing for 3-7 days to obtain enriched peripheral CD4CD8 double-positive T cells, and finding that the proportion of the peripheral CD4CD8 double-positive T cells can be remarkably increased by regulating the SMARCA4 signal channel for the first time.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for inducing the generation of peripheral CD4CD8 double-positive T cells and its application. Background Technology

[0002] Malignant tumors have become a major threat to human health. In recent years, tumor immunotherapy, represented by immune checkpoint inhibitors and adoptive cell immunotherapy, has achieved significant breakthroughs, bringing new hope to cancer patients. CD8-positive T cells, as the main executors of the anti-tumor immune response, play a crucial role in tumor immunotherapy. By recognizing and killing tumor cells, CD8-positive T cells constitute an important line of defense for the body's anti-tumor immune system.

[0003] However, current tumor immunotherapy still faces many challenges. Immune cells that enter the tumor microenvironment often lose their effector function after exerting their anti-tumor effects for a period of time, becoming exhausted immune cells, or entering a resting state after completing their killing mission. This T-cell dysfunction severely limits the persistence and effectiveness of immunotherapy, especially in the treatment of solid tumors. Therefore, how to enhance T-cell function and maintain its sustained anti-tumor activity has become an urgent scientific problem to be solved.

[0004] SMARCA4, the ATP-dependent catalytic subunit of the SWI / SNF chromatin remodeling complex, plays a crucial role in cell proliferation, differentiation, and gene expression regulation. SMARCA4 participates in various biological processes by altering chromatin structure and regulating the transcriptional expression of specific genes. Studies have shown that SMARCA4 is mutated or abnormally expressed in various tumors and is closely related to tumorigenesis and development. However, the role of SMARCA4 in T cell function regulation, particularly in the formation of peripheral CD4CD8 double-positive T cells, has not yet been reported.

[0005] With the development of single-cell sequencing technology, researchers have discovered a unique subset of CD4CD8 double-positive T cells in peripheral blood. These cells simultaneously express CD4 and CD8 molecules and account for less than 2% of peripheral T cells under normal physiological conditions. Preliminary studies show that this subset of peripheral CD4CD8 double-positive T cells has stronger activation capacity and stem cell-like characteristics compared to traditional CD8 single-positive T cells. This suggests that peripheral CD4CD8 double-positive T cells may have unique advantages in anti-tumor immunity. However, due to their extremely low proportion in peripheral blood, how to effectively enrich and expand these cells has become a key obstacle to their application in clinical treatment.

[0006] Currently, there are no reported methods for inducing peripheral CD4CD8 double-positive T cells by regulating the SMARCA4 signaling pathway, and there is a lack of systematic research on the application value of these cells in tumor therapy. Therefore, there is an urgent need to develop new strategies to achieve efficient induction and enrichment of peripheral CD4CD8 double-positive T cells and explore their application potential in tumor immunotherapy. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for inducing the generation of peripheral CD4CD8 double-positive T cells. This method is simple to operate, highly efficient, and can significantly increase the proportion of peripheral CD4CD8 double-positive T cells. The present invention also provides the application of SMARCA4 inhibitors in the preparation of agents for inducing peripheral CD4CD8 double-positive T cells, and the application of the prepared peripheral CD4CD8 double-positive T cells in tumor treatment.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides a method for inducing the generation of peripheral CD4CD8 double-positive T cells, comprising the following steps: isolating and obtaining T lymphocytes; culturing the T lymphocytes in a culture plate coated with anti-CD3 antibody and anti-CD28 antibody; adding SMARCA4 inhibitor to the culture system; and continuing to culture to obtain enriched peripheral CD4CD8 double-positive T cells.

[0010] Preferably, the final concentration of the anti-CD3 antibody is 3 μg / mL, and the final concentration of the anti-CD28 antibody is 3 μg / mL. Preferably, the SMARCA4 inhibitor is added after the T lymphocytes have been cultured in the culture plate for 24 hours. Preferably, the final concentration of the SMARCA4 inhibitor is 0.5 μM to 5 μM, more preferably 1 μM. Preferably, the cells are cultured for 3 to 7 days after the addition of the SMARCA4 inhibitor, more preferably 5 days.

[0011] Preferably, the SMARCA4 inhibitor is Camibirstat or a pharmaceutically acceptable salt, solvate, prodrug, or stereoisomer thereof.

[0012] Preferably, the T lymphocytes are derived from the spleen, thymus, lymph nodes, or tumor tissue.

[0013] This invention also provides the application of SMARCA4 inhibitors in the preparation of formulations that induce peripheral CD4CD8 double-positive T cells. The formulations are used to induce the enrichment and proliferation of TCF1-positive, CD4-positive, and CD8-positive T cells, enhance the activation ability of T cells, increase the levels of cytokines IL-2, GZMB, and IFNγ secreted by T cells, promote the differentiation of TCF1-positive, CD4-positive, and CD8-positive T cells into effector T cells or memory T cells, and enhance the killing ability of T cells against tumor cells.

[0014] This invention also provides the application of peripheral CD4CD8 double-positive T cells prepared by the method of this invention in the preparation of antitumor drugs. Preferably, the antitumor drugs are used to treat breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, or lymphoma.

[0015] The present invention also provides the application of the SMARCA4 inhibitor in combination with an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] First, this invention is the first to discover that regulating the SMARCA4 signaling pathway can significantly increase the proportion of peripheral CD4CD8 double-positive T cells. In mouse T cells, treatment with a SMARCA4 inhibitor increased the proportion of peripheral CD4CD8 double-positive T cells from 0.3% to 8.48%, an increase of approximately 28-fold; in human T cells, the proportion increased from 0.16% to 3.21%, an increase of approximately 20-fold. This provides an efficient and feasible method for the in vitro enrichment and expansion of peripheral CD4CD8 double-positive T cells.

[0018] Second, the peripheral CD4CD8 double-positive T cells prepared by this invention exhibit superior functional properties. These cells not only possess stronger activation capabilities—with the proportion of CD69-positive cells increasing from 31.6% to 42.4% and the proportion of Ki67-positive proliferating cells increasing from 18.3% to 39.5%—but also secrete higher levels of effector cytokines, including IFNγ, IL-2, and Granzyme B. Simultaneously, these cells express higher levels of the stem cell-like markers TCF1 and PD-1, suggesting stronger self-renewal capacity and differentiation potential.

[0019] Third, the peripheral CD4CD8 double-positive T cells prepared in this invention exhibit significantly enhanced antitumor activity. In vitro killing experiments showed that, at an effector-to-target ratio of 1:1, the killing rate of peripheral CD4CD8 double-positive T cells against tumor cells reached 27.6%, while the killing rate of CD8 single-positive T cells was only 10.1%. In vivo experiments further confirmed that the tumor volume in mice infused with peripheral CD4CD8 double-positive T cells was significantly smaller than that in the control group infused with CD8 single-positive T cells or CD4CD8 mixed T cells, with a tumor weight reduction of approximately 40%.

[0020] Fourth, this invention found that treatment with SMARCA4 inhibitors promoted T cell differentiation into a memory phenotype. The proportion of CD44-positive and CD62L-positive central memory T cells increased from 12.8% to 24.2%, and this increase in the memory phenotype is beneficial for maintaining a long-term anti-tumor immune response.

[0021] Fifth, the method of the present invention is simple to operate and does not require complex genetic engineering modification. It can achieve efficient induction of peripheral CD4CD8 double-positive T cells simply by adding small molecule compounds, and has good clinical translation potential.

[0022] Sixth, this invention reveals a novel mechanism by which SMARCA4 regulates T cell differentiation and function through chromatin remodeling. SMARCA4 inhibitors, by altering chromatin accessibility, promote the expression of the CD4 gene and related transcription factor genes, inducing CD8 single-positive T cells to re-express CD4 molecules, forming peripheral CD4CD8 double-positive T cells with unique functional characteristics. This discovery expands our understanding of T cell plasticity and functional diversity, providing new insights for T cell engineering. Attached Figure Description

[0023] Figure 1 This image shows the effect of peripheral CD4CD8 double-positive T cells on killing tumor cells in vitro. It demonstrates that, at different effector-to-target ratios, peripheral CD4CD8 double-positive T cells have a significantly enhanced tumor cell killing ability compared to CD8 single-positive T cells.

[0024] Figure 2 The image shows the effect of peripheral CD4CD8 double-positive T cells on killing tumor cells in vivo. A is a schematic diagram of the experimental design, B is a comparison photo of tumor size in mice of different groups, C is a statistical analysis of tumor weight in each group, and D is the tumor growth curve of each group of mice.

[0025] Figure 3The images show flow cytometry analysis of peripheral CD4-CD8 double-positive T cells induced by SMARCA4 inhibitors. In Figure A, the changes in CD4 and CD8 expression in mouse CD8-positive T cells after treatment with DMSO or Camibirstat are shown. In Figure B, the changes in CD4 and CD8 expression in human CD8-positive T cells after treatment with DMSO or Camibirstat are shown.

[0026] Figure 4 The flow cytometry analysis of T cell activation promoted by the SMARCA4 inhibitor shows the expression levels of CD69 and Ki67 in CD8 single-positive T cells and peripheral CD4CD8 double-positive T cells after Camibirstat treatment.

[0027] Figure 5 The flow cytometry analysis of T cell cytokine release promoted by SMARCA4 inhibitors shows the levels of IFNγ, IL-2, and Granzyme B secreted by CD8 single-positive T cells and peripheral CD4CD8 double-positive T cells.

[0028] Figure 6 This is a flow cytometry analysis of stem cell-like T cell differentiation promoted by SMARCA4 inhibitors, showing the proportion of TCF1 and PD-1 double-positive stem cell-like T cells among CD8 single-positive T cells and peripheral CD4CD8 double-positive T cells.

[0029] Figure 7 The flow cytometry analysis of memory T cell differentiation promoted by SMARCA4 inhibitors shows the proportion of CD8 single-positive T cells and CD44 and CD62L double-positive central memory T cells in peripheral CD4 and CD8 double-positive T cells. Detailed Implementation

[0030] The present invention will be described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0031] Unless otherwise specified, all reagents and materials used in this invention are commercially available products. The anti-CD3 antibody was purchased from Thermofisher, catalog number 14-0037-82; the anti-CD28 antibody was purchased from Biolegend, catalog number 102112; the SMARCA4 inhibitor Camibirstat is a compound represented by general formula I.

[0032]

[0033] (I)

[0034] RPMI 1640 medium was purchased from Gibco; fetal bovine serum was purchased from Gibco; penicillin-streptomycin bispecific antibody was purchased from Gibco; erythrocyte lysis buffer was purchased from Beyotime Biotechnology Co., Ltd.; 40 μm cell filters were purchased from Jetech Biotechnology Co., Ltd.; anti-CD4-BV605 antibody was purchased from Biolegend; anti-CD8-PeCy5.5 antibody was purchased from Biolegend; anti-CD69-PE antibody was purchased from Biolegend (catalog number 310910); anti-Ki67-APC antibody was purchased from Biolegend. The following antibodies were purchased from Biolegend: anti-IFNγ-PE antibody, anti-IL-2-PeCy7 antibody, anti-GZMB-FITC antibody, anti-TCF1-PE antibody, anti-PD-1-APC antibody, anti-CD44-FITC antibody, anti-CD62L-PeCy7 antibody, Staining buffer (Catalog No. 420201) from Biolegend, DAPI from Sigma-Aldrich, 4% paraformaldehyde from Sangon Biotech, 0.1% Triton X-100 from Sigma-Aldrich, BFA from Sigma-Aldrich, CT26 mouse colon cancer cell line from ATCC, BALB / c mice from Shanghai Silex Laboratory Animal Co., Ltd., and flow cytometer from BDLSLRFortessa.

[0035] Example 1: Method for inducing mouse peripheral CD4CD8 double-positive T cells with SMARCA4 inhibitor

[0036] In this embodiment, mouse T cells were treated with the SMARCA4 inhibitor Camibirstat to induce the generation of peripheral CD4CD8 double-positive T cells.

[0037] Eight-week-old male BALB / c mice were used. Spleens and lymph nodes were aseptically dissected. The tissues were placed in a culture dish containing 5 mL of pre-chilled RPMI 1640 medium, and the tissues were gently abraded with a syringe needle to fully release the cells. The cell suspension was filtered through a 40 μm cell filter to remove tissue debris and cell clumps. The filtrate was collected and transferred to a 15 mL centrifuge tube, centrifuged at 500 g for 3 minutes at room temperature. The supernatant was discarded, and 3 mL of erythrocyte lysis buffer was added to the cell pellet. The cells were incubated at room temperature for 5 minutes to lyse the erythrocytes. An equal volume of complete culture medium was then added to neutralize the lysis buffer, and the cells were centrifuged at 500 g for 3 minutes. The supernatant was discarded, and the cells were resuspended in complete culture medium and washed again by centrifugation. This washing step was repeated twice. Finally, the cells were resuspended in complete culture medium, and the cell concentration was adjusted to 1 × 10⁶ cells / mL. 6The complete culture medium was RPMI 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0038] Antibody coating was performed in advance by adding coating buffer to 6-well cell culture plates. The coating buffer was prepared as follows: anti-CD3 antibody and anti-CD28 antibody were dissolved in sterile PBS to a final concentration of 3 μg / mL for both antibodies. 1000 μL of coating buffer was added to each well, and the plates were incubated overnight at 4°C or for 2 hours at room temperature. After coating, the coating buffer was discarded, and the plates were washed twice with 500 μL of sterile PBS each time.

[0039] The prepared T lymphocyte suspension was seeded into antibody-coated 6-well plates, with 2 mL of cell suspension added to each well to ensure each well contained 1 × 10⁶ cells. 6 One cell. The cell culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for 48 hours to induce initial activation of T cells.

[0040] After 24 hours of incubation, the SMARCA4 inhibitor Camibirstat was added to the culture system. Specifically, Camibirstat was dissolved in DMSO to prepare a 1 mM stock solution. Before use, it was diluted with complete culture medium to the working concentration, ensuring a final concentration of Camibirstat of 1 μM and a final concentration of DMSO not exceeding 0.1%. An equal volume of complete culture medium containing 0.1% DMSO was added to the control group. The culture was continued at 37°C and 5% CO2 for 5 days.

[0041] After culture, cells were collected for flow cytometry analysis. Cells were transferred to flow cytometry tubes, centrifuged at 500g for 5 minutes, and the supernatant was discarded. Cells were resuspended in 1 mL of PBS and centrifuged again. After discarding the supernatant, cells were resuspended in 100 μL of staining buffer, followed by the addition of anti-CD4-BV605 antibody and anti-CD8-PeCy5.5 antibody, each diluted 1:500. DAPI diluted 1:1000 was also added to distinguish dead cells. Cells were incubated at room temperature in the dark for 30 minutes, washed with 500 μL of staining buffer, and centrifuged at 500g for 5 minutes. The supernatant was discarded, and cells were resuspended in 300 μL of staining buffer for flow cytometry analysis.

[0042] like Figure 3 As shown in Figure A, the proportion of CD4-positive and CD8-positive double-positive T cells in the DMSO control group was 0.3%, while after treatment with Camibirstat, the proportion of CD4-positive and CD8-positive double-positive T cells significantly increased to 8.48%, an increase of approximately 28-fold. This result indicates that the SMARCA4 inhibitor Camibirstat can effectively induce the production of peripheral CD4-CD8 double-positive T cells in mice.

[0043] Example 2: Method for inducing human peripheral CD4CD8 double-positive T cells with SMARCA4 inhibitor

[0044] This embodiment uses SMARCA4 inhibitors to treat human T cells to verify the effectiveness of this method in human cells.

[0045] Mononuclear cells were isolated from peripheral blood of healthy volunteers. 10 mL of fresh, anticoagulated blood was collected and diluted with an equal volume of PBS. In a 15 mL centrifuge tube, 5 mL of Ficoll lymphocyte separation medium was added first. Then, the diluted blood was carefully and slowly added along the tube wall above the Ficoll, ensuring a clear interface. The tube was centrifuged at 800 g for 20 minutes at room temperature without using the brake. After centrifugation, distinct layers were observed. The middle white membrane layer, representing the mononuclear cell layer, was carefully aspirated and transferred to a new centrifuge tube. The cells were washed with 10 mL of PBS and centrifuged at 300 g for 10 minutes. This washing process was repeated twice, and the cells were resuspended in complete culture medium.

[0046] CD8-positive T cells were enriched using a CD8-positive T cell magnetic bead sorting kit. Following the kit instructions, mononuclear cells were incubated with anti-CD8 magnetic beads, and CD8-positive T cells with a purity greater than 95% were obtained after magnetic column sorting. The sorted CD8-positive T cells were resuspended in complete culture medium and the cell concentration was adjusted to 1×10⁶ cells / mL. 6 per mL.

[0047] Using the same method as in Example 1, anti-CD3 antibody and anti-CD28 antibody were coated into 6-well plates, with a final concentration of 3 μg / mL for each antibody. The prepared CD8-positive T cells were seeded into the antibody-coated 6-well plates, 1 × 10⁶ cells per well. 6 Cells were placed in a 37°C, 5% CO2 incubator and cultured for 48 hours.

[0048] After culturing for 24 hours, Camibirstat was added to the culture system to a final concentration of 1 μM. An equal volume of 0.1% DMSO was added to the control group. Cells were cultured for another 5 days, and then collected for flow cytometry analysis to detect the expression of CD4 and CD8, using the same method as in Example 1.

[0049] like Figure 3 As shown in Figure B, the proportion of human CD4-positive and CD8-positive double-positive T cells in the DMSO control group was 0.16%, while after treatment with Camibirstat, the proportion increased to 3.21%, an increase of approximately 20-fold. This result confirms that the method of inducing peripheral CD4-CD8 double-positive T cells with SMARCA4 inhibitors is also effective in human cells and has good prospects for clinical translation.

[0050] Example 3: Effect of SMARCA4 inhibitors on T cell activation capacity

[0051] This embodiment examines the effect of SMARCA4 inhibitor treatment on T cell activation capacity.

[0052] Mouse T lymphocytes were prepared according to the method in Example 1. After culturing for 48 hours with anti-CD3 and anti-CD28 antibodies, 1 μM Camibirstat or an equal volume of DMSO was added as a control, and the cells were cultured for another 5 days. After the culture was completed, CD8 single-positive T cells and CD4CD8 double-positive T cells were sorted using a CD4 and CD8 positive sorting kit, respectively.

[0053] The two types of cells obtained after sorting were tested for activation marker CD69 and proliferation marker Ki67. The CD69 staining method was as follows: cells were resuspended in staining buffer, anti-CD69-PE antibody was added at a concentration of 1:500, and DAPI was added at a concentration of 1:1000. The cells were incubated at room temperature in the dark for 30 minutes. After washing, the cells were analyzed.

[0054] The intracellular Ki67 staining method was as follows: First, stain with DAPI for 10 minutes, centrifuge at 500g for 5 minutes and discard the supernatant. Add 4% paraformaldehyde fixative and fix at room temperature for 10 minutes. Centrifuge at 500g and discard the supernatant, add 0.1% Triton X-100 permeabilization buffer and incubate at room temperature for 10 minutes. Centrifuge at 500g and discard the supernatant, add staining solution diluted 1:500 with anti-Ki67-APC antibody, and incubate at room temperature in the dark for 60 minutes. After washing, analyze the sample.

[0055] like Figure 4 As shown, in cells treated with DMSO, the CD69 positivity rate of CD8 single-positive T cells was 31.6%, and the Ki67 positivity rate was 18.3%. However, in peripheral CD4CD8 double-positive T cells generated after Camibirstat treatment, the CD69 positivity rate increased to 42.4%, and the Ki67 positivity rate increased to 39.5%. These results indicate that peripheral CD4CD8 double-positive T cells generated after treatment with the SMARCA4 inhibitor have stronger activation and proliferative capacity.

[0056] Example 4: Effects of SMARCA4 inhibitors on the ability of T cells to secrete cytokines

[0057] This embodiment examines the effect of SMARCA4 inhibitor treatment on the ability of T cells to secrete effector cytokines.

[0058] CD8 single-positive T cells and peripheral CD4CD8 double-positive T cells were prepared and sorted according to the method in Example 3. Before cytokine staining, the cells were treated with BFA for 4 hours to inhibit the secretion of cytokines from the cells into the culture medium, thereby allowing cytokines to accumulate within the cells. The concentration of BFA used was 5 μg / mL.

[0059] After treatment, the cells were first fixed and perforated, followed by intracellular cytokine staining. The specific method was as follows: Cells were collected and centrifuged at 500g for 5 minutes, then the supernatant was discarded. DAPI staining solution was added at a 1:1000 dilution, and staining was performed at room temperature for 10 minutes. After centrifugation at 500g, the supernatant was discarded, and 4% paraformaldehyde was added, with fixation at room temperature for 10 minutes. After centrifugation at 500g, the supernatant was discarded, and 0.1% Triton X-100 was added, with perforation at room temperature for 10 minutes. After centrifugation at 500g, the supernatant was discarded, and the cells were resuspended in staining buffer. Then, anti-IFNγ-PE antibody, anti-IL-2-PeCy7 antibody, and anti-GZMB-FITC antibody were added, each diluted 1:500, and incubated at room temperature in the dark for 60 minutes. After staining, the cells were washed with staining buffer and centrifuged at 500g for 5 minutes. The supernatant was discarded, and the cells were resuspended in staining buffer for analysis.

[0060] like Figure 5 As shown, in CD8 single-positive T cells, the proportion of IFNγ-positive cells was 6.56%, IL-2-positive cells was 4.48%, and Granzyme B-positive cells was 5.74%. However, in peripheral CD4CD8 double-positive T cells, the proportion of IFNγ-positive cells increased to 17.4%, IL-2-positive cells to 14.4%, and Granzyme B-positive cells to 14.8%. These results indicate that peripheral CD4CD8 double-positive T cells possess stronger effector function and can secrete higher levels of cytokines, which is significant for enhancing anti-tumor immune responses.

[0061] Example 5: Effect of SMARCA4 inhibitors on stem cell-like T cell differentiation

[0062] This embodiment examines the effect of SMARCA4 inhibitor treatment on stem cell-like T cell differentiation.

[0063] CD8 single-positive T cells and peripheral CD4CD8 double-positive T cells were prepared and sorted according to the method in Example 3. The stem cell-like markers TCF1 and PD-1 were then detected in these two cell types. Since TCF1 is an intracellular transcription factor, intracellular staining was required.

[0064] The staining method was as follows: Collect cells, centrifuge at 500g for 5 minutes and discard the supernatant. Add DAPI staining solution diluted 1:1000 and stain at room temperature for 10 minutes. Centrifuge at 500g and discard the supernatant, add 4% paraformaldehyde and fix at room temperature for 10 minutes. Centrifuge at 500g and discard the supernatant, add 0.1% Triton X-100 and rupture the membrane at room temperature for 10 minutes. Centrifuge at 500g and discard the supernatant, resuspend the cells in staining buffer, then add anti-TCF1-PE antibody and anti-PD-1-APC antibody, each diluted 1:500, and incubate at room temperature in the dark for 60 minutes. After staining, wash and analyze.

[0065] like Figure 6 As shown, the proportion of TCF1-positive and PD-1-positive stem cell-like T cells in CD8 single-positive T cells was 3.75%. However, in peripheral CD4 / CD8 double-positive T cells, the proportion of TCF1-positive and PD-1-positive stem cell-like T cells significantly increased to 8.97%, an increase of approximately 2.4-fold. Stem cell-like T cells possess self-renewal capacity and multi-lineage differentiation potential, characteristics crucial for maintaining long-term anti-tumor immune responses. This result indicates that SMARCA4 inhibitor treatment not only induces the generation of peripheral CD4 / CD8 double-positive T cells but also promotes their differentiation into stem cell-like phenotypes, providing a new strategy for improving the durability of adoptive cell therapy.

[0066] Example 6: Effects of SMARCA4 inhibitors on memory T cell differentiation

[0067] This embodiment examines the effect of SMARCA4 inhibitor treatment on memory T cell differentiation.

[0068] CD8 single-positive T cells and peripheral CD4CD8 double-positive T cells were prepared and sorted according to the method in Example 3. The memory markers CD44 and CD62L were then detected in these two cell types. Both CD44 and CD62L are cell surface molecules and can be directly stained.

[0069] The staining method was as follows: Collect cells, centrifuge at 500g for 5 minutes and discard the supernatant. Resuspend the cells in staining buffer, add anti-CD44-FITC antibody and anti-CD62L-PeCy7 antibody (each diluted 1:500), and add DAPI diluted 1:1000. Incubate at room temperature in the dark for 30 minutes, wash with staining buffer, centrifuge at 500g for 5 minutes. Discard the supernatant, resuspend the cells in staining buffer, and perform analysis.

[0070] like Figure 7As shown, in CD8 single-positive T cells, the proportion of CD44-positive and CD62L-positive central memory T cells was 12.8%. However, in peripheral CD4-CD8 double-positive T cells, the proportion of CD44-positive and CD62L-positive central memory T cells increased to 24.2%, an increase of approximately 1.9-fold. Central memory T cells possess long-term survival and rapid response capabilities, and can rapidly proliferate and differentiate into effector cells upon re-stimulation by antigens. This result indicates that SMARCA4 inhibitor treatment promotes T cell differentiation into a memory phenotype, which is of significant value for establishing long-term anti-tumor immune memory.

[0071] Example 7: In vitro tumor cell killing experiment using peripheral CD4CD8 double-positive T cells

[0072] This embodiment evaluates the in vitro killing ability of peripheral CD4CD8 double-positive OT1 cells against tumor cells.

[0073] OT1 mouse T cells were prepared according to the method in Example 1 and treated with Camibirstat for 5 days. CD8-positive OT1 cells and peripheral CD4 / CD8-positive OT1 cells were then sorted using a CD4 and CD8-positive sorting kit. CT26-OVA mouse colon cancer cells were cultured as target cells, and CT26-GFP cells were used as control cells. CT26 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the logarithmic growth phase.

[0074] Flow cytometry was used to detect the cytotoxic activity of T cells against tumor cells. CT26-GFP and CT26-OVA cells were digested and collected, resuspended in complete culture medium, counted, and the cell concentration was adjusted to 1×10⁶. 5 Target cells / mL. After mixing target cells and control cells at a 1:1 ratio, the mixture was seeded into 96-well plates at 100 μL per well, i.e., 1 × 10⁶ cells per well. 4 Target cells were selected. CD8 single-positive OT1 cells and peripheral CD4CD8 double-positive OT1 cells were used as effector cells and added to the corresponding wells according to different effector-target ratios. The effector-target ratios were set to six gradients: 0:1, 1:8, 1:4, 1:1, 4:1, and 8:1.

[0075] After co-culturing the cells for 48 hours, the supernatant was collected, and the ratio of CT26-OVA to CT26-GFP cells was determined by flow cytometry.

[0076] The formula for calculating the percentage of cytotoxicity is: Cytotoxicity% = (1 - number of CT26-OVA cells / number of CT26-GFP cells) × 100%.

[0077] like Figure 1As shown, at an effector-to-target ratio of 0:1, the background killing rate was close to 0. With increasing effector-to-target ratio, the killing rate of tumor cells by both groups of T cells gradually increased, exhibiting a dose-dependent effect. However, under all effector-to-target ratio conditions, the killing ability of peripheral CD4CD8 double-positive T cells was significantly higher than that of CD8 single-positive T cells. At an effector-to-target ratio of 1:8, the killing rate of CD8 single-positive T cells was 2.8%, and that of peripheral CD4 / CD8 double-positive T cells was 6.5%; at an effector-to-target ratio of 1:4, the killing rate of CD8 single-positive T cells was 10.1%, and that of peripheral CD4 / CD8 double-positive T cells was 27.6%; at an effector-to-target ratio of 1:1, the killing rate of CD8 single-positive T cells was 21.3%, and that of peripheral CD4 / CD8 double-positive T cells was 40.8%; at an effector-to-target ratio of 4:1, the killing rate of CD8 single-positive T cells was 28.6%, and that of peripheral CD4 / CD8 double-positive T cells was 45.2%; and at an effector-to-target ratio of 8:1, the killing rate of CD8 single-positive T cells was 30.1%, and that of peripheral CD4 / CD8 double-positive T cells was 47.8%. These results fully demonstrate that peripheral CD4 / CD8 double-positive T cells have a significantly enhanced tumor cell killing ability.

[0078] Example 8: In vivo antitumor experiment of peripheral CD4CD8 double-positive T cells

[0079] This embodiment evaluates the inhibitory effect of peripheral CD4CD8 double-positive OT1 cells on tumor growth in vivo.

[0080] A subcutaneous xenograft tumor model was used in BALB / c mice. CT26-OVA colon cancer cells were cultured in vitro to the logarithmic growth phase from 8-week-old female BALB / c mice. After digestion and collection, the cells were resuspended in serum-free RPMI 1640 medium and the cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 6 Cells / mL. 100 μL of cell suspension was subcutaneously injected into the right axilla of each mouse, i.e., inoculated with 5 × 10⁶ cells / mL. 5 One CT26-OVA cell.

[0081] On the 4th day after tumor inoculation, when the tumor volume reached approximately 50 mm 3 Mice were randomly divided into four groups of five each: a blank control group, a CD4 single-positive T cell group, a CD8 single-positive OT1 cell group, a peripheral CD4-CD8 double-positive OT1 cell group, and a mixed CD4 single-positive and CD8 single-positive T cell group.

[0082] T cells were prepared according to the method in Example 1 and treated with Camibirstat. CD4-positive T cells, CD8-positive OT1 cells, and peripheral CD4 / CD8 double-positive OT1 cells were obtained using a CD4 and CD8 positive sorting kit. The CD4 / CD8 mixed T cell group consisted of CD4-positive T cells and CD8-positive OT1 cells mixed at a 1:1 ratio. All cell groups were washed twice with serum-free RPMI 1640 medium, and the cell concentration was adjusted to 5 × 10⁶ cells / mL. 6 per mL.

[0083] The drug was administered via tail vein injection to mice, with each mouse receiving 100 μL of cell suspension, or 5 × 10⁶ cells. 5 100 T cells. The blank control group was injected with an equal volume of serum-free culture medium. Tumor size and mouse weight were measured every 2 days after administration. The major and minor diameters of the tumor were measured using calipers, and the tumor volume was calculated according to the formula V = (major diameter × minor diameter²) / 2.

[0084] like Figure 2 As shown, the tumors in the blank control group mice grew the fastest, reaching an average tumor volume of approximately 1200 mm² by day 21. 3 The CD4 single-positive T cell group showed a weak inhibitory effect on tumor growth, with an average tumor volume of approximately 1100 mmHg on day 21. 3 The CD8 single-positive OT1 cell group and the CD4CD8 mixed T cell group showed certain anti-tumor activity, with an average tumor volume of approximately 900 mm on day 21. 3 and 850mm 3 The peripheral CD4CD8 double-positive OT1 cell group showed the most significant inhibitory effect on tumor growth, with an average tumor volume of only about 500 mm on day 21. 3 .

[0085] Mice were sacrificed on day 21 of the experiment, and tumor tissue was removed and weighed. The average tumor weight was 1.12g in the blank control group, 1.05g in the CD4 single-positive T cell group, 0.84g in the CD8 single-positive OT1 cell group, 0.78g in the CD4CD8 mixed T cell group, and only 0.45g in the peripheral CD4CD8 double-positive OT1 cell group. Statistical analysis showed that the tumor weight in the peripheral CD4CD8 double-positive OT1 cell group was significantly lower than that in the other groups.

[0086] Comparative Example 1: T cells not treated with SMARCA4 inhibitors

[0087] This comparative study investigated the proportion and functional characteristics of peripheral CD4CD8 double-positive T cells in T cells that were not treated with SMARCA4 inhibitors.

[0088] Mouse T lymphocytes were prepared according to the method in Example 1. The cells were seeded into 24-well plates coated with anti-CD3 and anti-CD28 antibodies and cultured at 37°C in a 5% CO2 incubator for 48 hours. After 48 hours of culture, an equal volume of complete culture medium containing 0.1% DMSO was added as a control; no SMARCA4 inhibitor was added. Cells were collected after another 5 days of culture and analyzed by flow cytometry.

[0089] The results showed that in the control group without SMARCA4 inhibitor treatment, the proportion of CD4-CD8-positive double-positive T cells was only 0.32%, which was significantly lower than the 8.48% achieved after Camibirstat treatment in Example 1. This result indicates that under standard culture conditions, even after stimulation and activation by anti-CD3 and anti-CD28 antibodies, the natural production rate of peripheral CD4-CD8 double-positive T cells remains extremely low and cannot meet the needs of clinical applications.

[0090] Further analysis of the functional characteristics of these cells revealed that the CD69 positivity rate of T cells in the control group was 28.3%, and the Ki67 positivity rate was 15.7%, both significantly lower than those of peripheral CD4CD8 double-positive T cells generated after treatment with the SMARCA4 inhibitor. This indicates that treatment with the SMARCA4 inhibitor not only increases the proportion of peripheral CD4CD8 double-positive T cells but also significantly enhances their activation and proliferation capabilities.

[0091] Comparative Example 2: Stimulation with CD3 antibody alone without co-stimulation with CD28 antibody

[0092] This comparative study investigated the effect of stimulation with anti-CD3 antibody alone, without co-stimulation with anti-CD28 antibody, on the induction efficiency of peripheral CD4CD8 double-positive T cells.

[0093] Mouse T lymphocytes were prepared according to the method in Example 1, but only anti-CD3 antibody was used for antibody coating at a concentration of 3 μg / mL; anti-CD28 antibody was not added. T cells were seeded into 24-well plates coated with anti-CD3 antibody, cultured for 48 hours, and then 1 μM Camibirstat was added for further 5 days of culture.

[0094] The results showed that in the experimental group stimulated only with anti-CD3 antibody, even with the addition of SMARCA4 inhibitor, the proportion of CD4-positive and CD8-positive double-positive T cells was only 3.21%, far lower than the 8.48% achieved in Example 1 when both anti-CD3 and anti-CD28 antibodies were used for stimulation. This result indicates that adequate T cell activation is a necessary prerequisite for the effective induction of peripheral CD4-CD8 double-positive T cell production by SMARCA4 inhibitor. Anti-CD3 antibody provides the first signal by binding to the T cell receptor complex, while anti-CD28 antibody provides the co-stimulatory signal by binding to the CD28 molecule. Their synergistic effect enables T cells to reach an optimal activation state, thus allowing the SMARCA4 inhibitor to exert its full effect. In the absence of the co-stimulatory signal, although T cells can receive the antigen signal, the degree of activation is insufficient, limiting the efficiency of SMARCA4 inhibitor in inducing CD4 gene re-expression.

[0095] Comparative Example 3: Effects of excessively high concentrations of SMARCA4 inhibitors

[0096] This comparative study investigated the effects of high concentrations of SMARCA4 inhibitors on cell viability and induction efficiency.

[0097] Mouse T lymphocytes were prepared and cultured according to the method in Example 1. After 48 hours of culture with anti-CD3 and anti-CD28 antibodies, 10 μM of Camibirstat was added and cultured for another 5 days.

[0098] The results showed that treatment with a high concentration of 10 μM Camibirstat significantly decreased T cell viability, with the proportion of DAPI-positive dead cells increasing from 5.2% in the control group to 32.8%. Although the proportion of CD4-positive and CD8-positive double-positive T cells among surviving cells still increased to some extent to 5.67%, the actual absolute number of double-positive T cells obtained was lower than that obtained in the treatment groups using appropriate concentrations such as 1 μM or 2 μM due to the high cell death rate. This result indicates that the concentration of SMARCA4 inhibitors needs to be strictly controlled; excessively high concentrations can have cytotoxic effects and are not conducive to the enrichment of peripheral CD4-CD8 double-positive T cells.

[0099] Comparative Example 4: Insufficient processing time

[0100] This comparative study investigated the effect of excessively short treatment time with SMARCA4 inhibitors on induction efficiency.

[0101] Mouse T lymphocytes were prepared and cultured according to the method in Example 1. After 48 hours of culture with anti-CD3 and anti-CD28 antibodies, 1 μM of Camibirstat was added, but the culture was continued for only 1 day.

[0102] The results showed that the proportion of CD4- and CD8-positive double-positive T cells was only 2.15% after one day of treatment, far lower than the 8.48% achieved after five days of treatment. This indicates that the induction of peripheral CD4- and CD8-positive T cells is a time-consuming process involving multiple steps such as chromatin remodeling, altered gene expression, and cell phenotype conversion. Insufficient treatment time prevents these changes from being fully completed, thus affecting the induction efficiency.

[0103] Comparative Example 5: Comparison of T cells from different sources

[0104] This comparative study compares the induction efficiency of peripheral CD4CD8 double-positive T cells from T cells of different origins after treatment with SMARCA4 inhibitor.

[0105] T cells were isolated from the spleen, lymph nodes, thymus, and tumor tissue of mice, and processed according to the method in Example 1.

[0106] The results showed that the proportion of double-positive T cells after treatment was 8.35% from the spleen, 7.89% from lymph nodes, 6.12% from the thymus, and 5.45% from tumor tissue. These results indicate that although there are some differences in induction efficiency among T cells from different sources, treatment with the SMARCA4 inhibitor effectively induced the production of peripheral CD4CD8 double-positive T cells. The relatively high induction efficiency of T cells from the spleen and lymph nodes may be related to the presence of more resting or nascent T cells in these tissues. The relatively low induction efficiency of T cells from tumor tissue may be related to the inhibitory effect of the tumor microenvironment leading to impaired cell function.

[0107] Test Example 1: Combined application of peripheral CD4CD8 double-positive T cells and immune checkpoint inhibitors

[0108] This test case evaluates the synergistic antitumor effect of peripheral CD4CD8 double-positive T cells combined with anti-PD-1 antibody.

[0109] A BALB / c mouse CT26 subcutaneous xenograft model was established using the same method as in Example 10. On day 7 after tumor inoculation, mice were randomly divided into 5 groups of 6 mice each: blank control group, anti-PD-1 antibody alone group, CD8 single-positive T cell alone group, peripheral CD4CD8 double-positive T cell alone group, and peripheral CD4CD8 double-positive T cell combined with anti-PD-1 antibody group.

[0110] The preparation and administration of T cells were the same as in Example 10, with 2 × 10⁶ cells injected into the tail vein of each mouse. 6Each mouse received 100 μg of anti-PD-1 antibody via intraperitoneal injection, every 3 days for a total of 4 doses. The combination therapy group received both T-cell infusion and anti-PD-1 antibody therapy.

[0111] Tumor growth and changes in mouse body weight were monitored. Results showed that by day 21, the average tumor volume in the control group was 1180 mmHg. 3 The anti-PD-1 antibody alone group had a concentration of 820 mm. 3 The CD8 single-positive T cell group alone had a count of 890 mm. 3 The peripheral CD4-CD8 double-positive T cell group alone had a cell count of 510 mm. 3 The combined treatment group had only 280mm. 3 Statistical analysis showed that the combined treatment group had significantly better anti-tumor effects than any single treatment group, demonstrating a clear synergistic effect.

[0112] Further analysis of immune cell infiltration in tumor tissue was conducted. Flow cytometry analysis showed that the combined treatment group had the highest proportion of CD3-positive T cells infiltrating the tumor tissue, reaching 28.5%, and these T cells expressed a relatively low proportion of PD-1, suggesting that the anti-PD-1 antibody effectively blocked PD-1-mediated immunosuppressive signals. Simultaneously, the IFNγ and Granzyme B secretion levels of tumor-infiltrating T cells in the combined treatment group were significantly higher than those in the single treatment group. These results indicate that the combined application of peripheral CD4CD8 double-positive T cells and immune checkpoint inhibitors has a significant synergistic anti-tumor effect, providing a new strategy for improving the efficacy of tumor immunotherapy.

[0113] Test Example 2: Comprehensive Comparison of Cell Characteristics of Various Examples and Comparative Examples

[0114] This test case systematically compares and analyzes the functional characteristics of T cells obtained in each embodiment and comparative example.

[0115] T cell samples prepared in Examples 1-8 and Comparative Examples 1-5 were collected and subjected to the following standardized tests: proportion of CD4-positive and CD8-positive double-positive T cells, CD69 positivity rate, Ki67 positivity rate, proportion of IFNγ-secreting cells, proportion of IL-2-secreting cells, proportion of Granzyme B-secreting cells, proportion of TCF1-positive and PD-1-positive stem cell-like T cells, and proportion of CD44-positive and CD62L-positive central memory T cells. The detection methods were the same as those in the aforementioned examples.

[0116] The data from each group are summarized in Table 1:

[0117] Table 1. Comparison of functional characteristics of T cells under different treatment conditions

[0118] Note: * indicates that the cell mortality rate in this group is as high as 32.8%, and the data shown is the proportion of surviving cells.

[0119] Table 1 clearly shows that under optimized treatment conditions, namely treatment with 1 μM Camibirstat for 5 days, the best peripheral CD4CD8 double-positive T cell induction efficiency and functional characteristics can be obtained. These cells are significantly superior to the control group and other treatment groups in all functional indices, fully demonstrating the superiority of the method of the present invention.

[0120] Further statistical analysis revealed a significant positive correlation between the proportion of CD4-positive and CD8-positive double-positive T cells and various functional indicators. Correlation analysis showed that the correlation coefficient (r) between the proportion of double-positive T cells and the CD69 positivity rate was 0.92, the correlation coefficient (r) with the Ki67 positivity rate was 0.89, and the correlation coefficient (r) with the proportion of IFNγ-secreting cells was 0.94. These results suggest that peripheral CD4-CD8 double-positive T cells induced by SMARCA4 inhibitors do indeed possess unique functional advantages, which are closely related to the double-positive phenotype.

[0121] This invention, through systematic research, has discovered that SMARCA4 inhibitors induce the production and enhance the function of peripheral CD4CD8 double-positive T cells through the following mechanism:

[0122] First, SMARCA4, as the core ATPase subunit of the SWI / SNF chromatin remodeling complex, plays a crucial role in T cell differentiation and functional regulation. SMARCA4 uses energy generated from ATP hydrolysis to drive the sliding or repositioning of nucleosomes along the DNA, altering chromatin accessibility and thus regulating the transcriptional expression of specific genes. Under normal conditions, the chromatin at the CD4 gene locus in CD8 single-positive T cells is relatively dense, making it difficult for transcription factors to access this region, resulting in CD4 gene silencing.

[0123] When treated with SMARCA4 inhibitors, the function of the SWI / SNF complex is suppressed, leading to reduced chromatin remodeling activity. This change causes chromatin opening at some previously silenced gene loci, particularly the CD4 gene locus, allowing transcription factors to enter and initiate gene transcription. Simultaneously, T cells receive dual stimulation signals from anti-CD3 and anti-CD28 antibodies, entering a highly activated state. Intracellular signal transduction pathways are activated, and the expression and activity of related transcription factors such as RUNX3 and ThPOK are altered. Under these conditions, the CD4 gene is re-expressed, transforming previously CD8 single-positive T cells into CD4CD8 double-positive T cells.

[0124] Secondly, the effects of SMARCA4 inhibitors extend beyond inducing CD4 gene expression; they broadly influence the functional state of T cells. By altering chromatin accessibility, a series of genes associated with T cell activation, effector function, and stem cell-like characteristics are synergistically regulated. Experiments have shown that peripheral CD4CD8 double-positive T cells treated with SMARCA4 inhibitors exhibit multifaceted functional enhancements, including higher activation levels manifested as upregulated CD69 expression, stronger proliferative capacity manifested as increased Ki67 expression, higher effector cytokine secretion, and a higher proportion of stem cell-like and memory phenotypes. These synergistic enhancements in functional characteristics are not simply additive effects, but rather epigenetic reprogramming at the chromatin level, endowing these cells with unique functional advantages.

[0125] Third, the superior antitumor activity exhibited by peripheral CD4CD8 double-positive T cells may be closely related to their unique phenotype and functional characteristics. Co-expression of CD4 and CD8 molecules endows these cells with certain characteristics of both helper T cells and cytotoxic T cells. CD4 molecules can recognize antigens presented by MHC class II molecules, while CD8 molecules can recognize antigens presented by MHC class I molecules. This dual recognition capability may give peripheral CD4CD8 double-positive T cells a broader antigen recognition spectrum. Simultaneously, these cells secrete higher levels of cytokines such as IFNγ and IL-2, which not only directly kill tumor cells but also recruit and activate other immune cells, forming a stronger antitumor immune microenvironment.

[0126] Fourth, increasing the proportion of stem cell-like T cells among peripheral CD4CD8 double-positive T cells is significant. TCF1-positive and PD-1-positive stem cell-like T cells possess self-renewal capacity and multi-lineage differentiation potential, making them a key cell population for maintaining long-term anti-tumor immune responses. These cells can rapidly proliferate and differentiate into effector T cells after antigen stimulation, while retaining some stem cell-like cells to maintain the cell pool. In the tumor microenvironment, stem cell-like T cells are the primary responder cell population for immune checkpoint inhibitors. Therefore, enriching peripheral CD4CD8 double-positive T cells with a high proportion of stem cell-like characteristics is valuable for improving the durability of adoptive cell therapy and its synergistic effect with immune checkpoint inhibitors.

[0127] Fifth, the differentiation of peripheral CD4-CD8 double-positive T cells into memory phenotypes enhances their long-term anti-tumor effects. Central memory T cells possess long-term survival capabilities, can reside in lymphoid tissues, and upon re-stimulation by antigens, can rapidly proliferate and differentiate into effector cells, achieving continuous monitoring of tumors. The establishment of this memory characteristic provides an immunological basis for preventing tumor recurrence and metastasis.

[0128] Based on the above mechanistic analysis, this invention, through the regulation of chromatin remodeling by SMARCA4 inhibitors, achieves multi-level reprogramming of T cell phenotype and function, generating peripheral CD4CD8 double-positive T cells with unique advantages. These cells are not only enriched in number, but more importantly, exhibit significantly enhanced function, including activation capacity, effector function, stem cell-like characteristics, and memory properties. This comprehensive functional enhancement makes peripheral CD4CD8 double-positive T cells a highly promising adoptive cell therapy product, providing a new strategy and direction for tumor immunotherapy.

[0129] Example 9: Preparation and application of an inducer containing a SMARCA4 inhibitor

[0130] This embodiment provides an inducing agent containing a SMARCA4 inhibitor for inducing the enrichment and proliferation of peripheral CD4CD8 double-positive T cells.

[0131] Preparation of the formulation:

[0132] Dissolve 10 mg of the SMARCA4 inhibitor Camibirstat in 1 mL of anhydrous dimethyl sulfoxide to prepare a 10 mM stock solution. Aliquot the stock solution into sterile EP tubes, 100 μL per tube, and store at -20°C protected from light. When using, remove one tube of stock solution and dilute it to the required working concentration with serum-free RPMI 1640 medium. For in vitro T cell culture applications, the working concentration range is 0.5 μM to 5 μM, preferably 1 μM.

[0133] To improve the stability and ease of use of formulations, lyophilized powder dosage forms can be prepared. The specific method is as follows: Camibirstat is dissolved in an aqueous solution containing 5% trehalose and 2% mannitol to prepare a 5mM concentration, which is then dispensed into 1mL lyophilization vials. The lyophilization process is completed through three stages: pre-freezing, sublimation drying, and desorption drying. The lyophilized powder is stable at room temperature and can be reconstituted with the appropriate volume of sterile water for injection before use.

[0134] Application of the formulation in inducing peripheral CD4CD8 double-positive T cells:

[0135] Mouse T lymphocytes were isolated and prepared according to the method in Example 1. The cells were seeded into 6-well plates coated with anti-CD3 and anti-CD28 antibodies, 2 × 10⁶ cells per well. 6 Cells were cultured at 37°C and 5% CO2 for 24 hours. The prepared inducing agent was then added to the culture system to achieve a final Camibirstat concentration of 1 μM. Cells were collected after culturing for another 5 days.

[0136] Flow cytometry analysis showed that after treatment with the inducing agent, the proportion of TCF1-positive, CD4-positive, and CD8-positive T cells increased from 0.3% before treatment to 8.52%, an increase of approximately 28-fold. Further analysis revealed that these induced peripheral CD4-CD8 double-positive T cells exhibited significantly enhanced functional properties. The positive rate of CD69, a T cell activation marker, increased from 28.1% in the untreated group to 42.8%, indicating that the agent effectively enhanced T cell activation. The positive rate of Ki67, a cell proliferation marker, increased from 15.4% to 39.8%, confirming that the agent promoted T cell proliferation.

[0137] The results of effector cytokine secretion assays showed that among the peripheral CD4CD8 double-positive T cells generated after treatment with the formulation, the proportions of IFNγ-positive cells were 17.6%, IL-2-positive cells were 14.6%, and Granzyme B-positive cells were 15.1%, all significantly higher than those in the untreated control group. The proportion of cells double-positive for stem cell-like T cell markers TCF1 and PD-1 reached 9.05%, and the proportion of cells double-positive for central memory T cell markers CD44 and CD62L reached 24.5%. These results fully demonstrate that the inducing agent containing a SMARCA4 inhibitor prepared in this invention can effectively induce the enrichment and proliferation of TCF1-positive, CD4-positive, and CD8-positive T cells, while enhancing T cell activation, increasing effector cytokine secretion levels, and promoting the differentiation of stem cell-like T cells and memory T cells.

[0138] Example 10: Application of peripheral CD4CD8 double-positive T cells in the treatment of different types of tumors

[0139] This embodiment evaluates the application effect of peripheral CD4CD8 double-positive T cells in the treatment of various types of tumors and verifies their broad-spectrum anti-tumor activity.

[0140] 10.1 Application in the treatment of colon cancer

[0141] A subcutaneous xenograft model of colon cancer (CT26) was established in BALB / c mice. Eight-week-old female BALB / c mice were subcutaneously injected with 5 × 10⁻⁶ spores into the right axilla. 5 CT26-OVA cells. On day 7 post-inoculation, when the tumor volume reached approximately 50 mm... 3 Mice were randomly divided into a control group and a treatment group, with 8 mice in each group. Peripheral CD4CD8 double-positive OT1 cells were prepared according to the method in Example 1, and the drug was administered via tail vein injection, with each mouse receiving 2 × 10⁻⁶ cells. 6 Cells. The control group was injected with an equal volume of PBS. Dosage was administered every 3 days for a total of 3 doses.

[0142] Tumor growth curve monitoring showed that by day 24, the average tumor volume in the control group was 1350 mm. 3 The average tumor volume in the treatment group was only 480 mm. 3 The tumor growth inhibition rate reached 64.4%. At the end of the experiment, the tumors were removed and weighed. The average tumor weight in the control group was 1.28g, while that in the treatment group was 0.52g, a statistically significant difference. Histological analysis showed that the tumor tissue in the treatment group had extensive T-cell infiltration, and the tumor cells exhibited obvious apoptosis and necrosis.

[0143] Example 11: Preparation of antitumor drugs for peripheral CD4CD8 double-positive T cells

[0144] This embodiment provides a complete preparation method for an anti-tumor drug based on peripheral CD4CD8 double-positive T cells.

[0145] 11.1 Collection and processing of donor T cells

[0146] For clinical applications, patient-derived peripheral blood or tumor-infiltrating lymphocytes can be collected as starting materials. Collect 100-200 mL of peripheral blood and separate mononuclear cells using Ficoll density gradient centrifugation with lymphocyte separation medium. Specifically, mix anticoagulated blood with an equal volume of PBS, carefully add this mixture to the Ficoll separation medium, and centrifuge at 800g for 20 minutes at room temperature. Aspirate the cells from the middle white membrane layer, wash twice with PBS, and resuspend in complete culture medium. For tumor-infiltrating lymphocytes, mince freshly removed tumor tissue, add a digestion solution containing collagenase and DNase, digest at 37°C for 60 minutes, and filter through a cell filter to obtain a single-cell suspension.

[0147] 11.2 In vitro culture and induction of T cells

[0148] The isolated mononuclear cells were seeded into cell culture flasks pre-coated with anti-CD3 and anti-CD28 antibodies. The antibody coating method was as follows: anti-CD3 and anti-CD28 antibodies were dissolved in PBS at a concentration of 3 μg / mL, and added to the culture flask at a volume of 0.5 mL per square centimeter of culture area. Coating was carried out overnight at 4°C or for 2 hours at room temperature. After coating, the cells were washed twice with PBS.

[0149] Cells were loaded at a rate of 1×10 6Inoculated at a density of 100 cells / mL into coated culture flasks, the culture medium was RPMI 1640 complete medium containing 10% human AB serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 50 U / mL recombinant human IL-2. After culturing at 37°C and 5% CO2 for 24 hours, the SMARCA4 inhibitor Camibirstat was added to the culture system to achieve a final concentration of 1 μM. Culture continued for 5 days, with fresh medium added every 2 days to maintain an IL-2 concentration of 50 U / mL.

[0150] 11.3 Sorting and expansion of peripheral CD4-CD8 double-positive T cells

[0151] After culture, peripheral CD4 and CD8 positive T cells were enriched using a CD4 and CD8 positive cell magnetic bead sorting kit. After cell collection, following the kit instructions, CD4 positive cells were first labeled and sorted using anti-CD4 magnetic beads, and then CD4 and CD8 double-positive T cells were further sorted from the CD4 positive cells using anti-CD8 magnetic beads. The purity of the sorted peripheral CD4 and CD8 double-positive T cells reached over 90%.

[0152] To obtain a sufficient number of cells for clinical treatment, the sorted peripheral CD4CD8 double-positive T cells need to be expanded and cultured. Cells are seeded into new culture flasks and cultured using a rapid expansion protocol. The culture medium includes anti-CD3 antibody, irradiated inactivated allogeneic peripheral blood mononuclear cells as feeder cells, and 50 U / mL IL-2. Fresh culture medium and IL-2 are added every 3 days. After 7 to 14 days of expansion culture, the cell number can increase 100 to 1000-fold.

[0153] 11.4 Quality Control and Testing

[0154] Strict quality control is required during cell preparation. Key testing indicators include: cell viability should be greater than 85%, detected using trypan blue staining; cell purity, detected by flow cytometry, should have a CD3-positive T cell ratio greater than 95% and a CD4-positive / CD8-positive double-positive T cell ratio greater than 80%; sterility testing, with no bacterial or fungal growth in the culture supernatant; endotoxin testing, with an endotoxin content below 5 EU / mL; and a negative mycoplasma test.

[0155] Simultaneously, it is necessary to test the functional characteristics of the cells, including the expression levels of CD69 and Ki67, the secretion capacity of IFNγ, IL-2, and Granzyme B, and their in vitro tumor cell killing activity. Only cell products that pass all quality control indicators can be used for clinical treatment.

[0156] 11.5 Preparation and Preservation of Cell Preparations

[0157] Peripheral CD4CD8 double-positive T cells that passed quality inspection were collected and washed three times with physiological saline or Ringer's solution to remove culture medium components. Finally, the cells were resuspended in physiological saline containing 5% human serum albumin and the cell concentration was adjusted to 1×10⁻⁶. 7 From 1×10⁻⁶ cells / mL to 1×10⁻⁶ cells / mL 8 Cells / mL. The cell suspension is dispensed into sterile infusion bags, with the amount per bag determined according to the patient's treatment dose, typically 50 mL to 100 mL.

[0158] Freshly prepared cell preparations should be stored at 4°C and used within 24 hours. For long-term storage, cryopreservation can be used. Resuspend the cells in a cryopreservation solution containing 10% dimethyl sulfoxide and 90% fetal bovine serum to a cell concentration of 1×10⁻⁶. 7 The samples were dispensed at a density of 1 / mL into cryovials, cooled to a programmed temperature, and then transferred to a liquid nitrogen tank for storage. Before use, the samples were rapidly thawed and washed with culture medium to remove the cryoprotectant.

[0159] 11.6 Clinical Dosing Regimen

[0160] The prepared peripheral CD4CD8 double-positive T-cell preparation was administered to the patient via intravenous infusion. Pre-treatment of the patient, such as lymphocyte-depleting chemotherapy with cyclophosphamide and fludarabine, was performed before infusion to enhance the efficacy of adoptive cell therapy. The infused cell dose was determined based on the patient's weight and disease condition, typically 1 × 10⁻⁶ cells / day. 8 Up to 1×10¹ 0 Cells / infusion. Infusion rate controlled at 1 mL to 2 mL per minute, with patient vital signs monitored throughout. IL-2 maintenance therapy may be administered post-infusion as needed to promote in vivo cell survival and proliferation.

[0161] The peripheral CD4CD8 double-positive T-cell antitumor drug prepared in this embodiment has the following characteristics: high cell purity, excellent functional properties, controllable quality, standardized preparation process, and is suitable for clinical treatment of various tumor types.

[0162] To gain a deeper understanding of the synergistic mechanism between peripheral CD4CD8 double-positive T cells and immune checkpoint inhibitors, tumor tissues, spleens, and lymph nodes were collected and analyzed at different time points in the combination therapy experiment.

[0163] Studies have found that peripheral CD4CD8 double-positive T cells, after infusion, can effectively infiltrate tumor tissue and maintain high activity in the tumor microenvironment. These cells express certain levels of immune checkpoint molecules such as PD-1 and CTLA-4, but due to their inherent potent functional properties, they can still exert anti-tumor effects. When used in combination with immune checkpoint inhibitors, these inhibitors can block the PD-1 / PD-L1 or CTLA-4 / CD80 / CD86 signaling pathways, relieving the suppression of T cells and further enhancing the function of peripheral CD4CD8 double-positive T cells.

[0164] Meanwhile, peripheral CD4CD8 double-positive T cells show a high proportion of stem cell-like T cells, which are the main responder cell population for immune checkpoint inhibitors. Under the influence of immune checkpoint inhibitors, these stem cell-like T cells can rapidly proliferate and differentiate into effector T cells, producing a stronger anti-tumor effect. Furthermore, cytokines such as IFNγ secreted by peripheral CD4CD8 double-positive T cells can upregulate the expression of PD-L1 on the surface of tumor cells, increasing the target sites for anti-PD-1 / PD-L1 antibodies and thus enhancing the efficacy of immune checkpoint inhibitors.

[0165] These results indicate that the combined use of peripheral CD4CD8 double-positive T cells with immune checkpoint inhibitors, including anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, has a significant synergistic anti-tumor effect, providing a new combined strategy for clinical tumor immunotherapy.

[0166] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for inducing the generation of peripheral CD4CD8 double-positive T cells, characterized in that... This includes the following steps: T lymphocytes were isolated and obtained; The T lymphocytes were placed in a culture plate coated with anti-CD3 antibody and anti-CD28 antibody, with a final concentration of 3 μg / mL for both the anti-CD3 antibody and the anti-CD28 antibody. Incubate at 37℃ and 5% CO2 for 48 hours; Add SMARCA4 inhibitor to the culture system, wherein the final concentration of SMARCA4 inhibitor is 0.5 μM to 5 μM; Continue culturing for 3 to 7 days to obtain enriched peripheral CD4CD8 double-positive T cells.

2. The method according to claim 1, characterized in that... The final concentration of the SMARCA4 inhibitor is 1 μM.

3. The method according to claim 1, characterized in that... The SMARCA4 inhibitor is Camibirstat, which is a small molecule compound of general formula I or a pharmaceutically acceptable salt, solvate, prodrug, or stereoisomer thereof.

4. The method according to claim 1, characterized in that... The T lymphocytes are derived from the spleen, thymus, lymph nodes, or tumor tissue.

5. The method according to claim 1, characterized in that... The peripheral CD4CD8 double-positive T cells exhibit at least one of the following characteristics: The proportion of TCF1-positive, CD4-positive, and CD8-positive T cells increased to 5% to 15%; The expression level of CD69 on the membrane surface is increased; The levels of secreted cytokines IL-2, GZMB, and IFNγ were elevated; The expression levels of stem cell-like T cell markers TCF1 and PD-1 were elevated; The expression levels of central memory T cell markers CD44 and CD62L were elevated.

6. Use of SMARCA4 inhibitors in the preparation of formulations that induce peripheral CD4CD8 double-positive T cells, said formulations being used for at least one of the following purposes: Induces enrichment and proliferation of TCF1-positive, CD4-positive, and CD8-positive T cells; Enhance the activation ability of T cells; Increase the levels of cytokines IL-2, GZMB, and IFNγ secreted by T cells; Promote the differentiation of TCF1-positive, CD4-positive, and CD8-positive T cells into effector T cells or memory T cells; Enhance the ability of T cells to kill tumor cells.

7. The application according to claim 6, characterized in that... The SMARCA4 inhibitor is Camibirstat, which is a small molecule compound of general formula I or a pharmaceutically acceptable salt, solvate, prodrug, or stereoisomer thereof.

8. The use of peripheral CD4CD8 double-positive T cells prepared by the method according to any one of claims 1 to 5 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that... The antitumor drug is used to treat at least one of the following diseases: breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, and lymphoma.

10. The application according to claim 6 or 7, characterized in that... The SMARCA4 inhibitor is used in combination with an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

Images