Method for enhancing colon cancer resisting function of Vdelta1gamma deltaT cells

Abstract

The invention relates to the technical field of biological medicines, and provides a method for enhancing the colon cancer resisting function of a V delta 1 gamma delta T cell, and a CDK1 activator and the T cell are combined to prepare a colon cancer resisting medicine. According to the technical scheme, a molecular mechanism for regulating and controlling tumor antigen ectopic expression through the CDK1-NCL axis is illustrated, and the key theory blank of recognizing tumor cells through the V delta 1 gamma delta T cells is filled up; a CDK1 activator TC11 is developed to serve as an auxiliary means for enhancing the functions of the V delta 1 gamma delta T cells, and the limitation that traditional immunotherapy depends on exogenous antigen modification is broken through; in-vivo and in-vitro experiments prove that targeting CDK1 can remodel a tumor immune microenvironment, and a convertible clinical application scheme is provided for combined immunotherapy of colon cancer and other solid tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to a method for enhancing the anti-colorectal cancer function of Vδ1γδ T cells. Background Art

[0002] Cancer immunotherapy, as an important direction in cancer treatment, has made remarkable progress in recent years but still faces many challenges. Among them, Vδ1γδ T cells are regarded as highly potential effector cells due to their non-MHC-restricted antigen recognition ability and the characteristic of rapidly responding to tumor cells. Research shows that Vδ1γδ T cells play an anti-tumor role by recognizing antigens (such as nucleolin NCL) ectopically expressed on the surface of tumor cells, and their infiltration level is positively correlated with the survival rate of patients. However, the prior art has the following key limitations: (1) The regulatory mechanism of NCL membrane ectopia is unclear: NCL is usually localized in the nucleolus, and its membrane ectopia is the key for Vδ1γδ T cell recognition, but how phosphorylation modification regulates the subcellular localization of NCL has not been elucidated. (2) The role of CDK1 in tumor immunity has not been fully explored: As a cell cycle regulatory kinase, although CDK1 has been found to be involved in the regulation of PD-L1 stability, whether it affects immune recognition by regulating tumor antigen expression lacks research. (3) The limitations of existing immunotherapy strategies: Traditional methods (such as CAR-T, immune checkpoint inhibitors) rely on exogenous antigen presentation or gene modification, and have problems such as off-target toxicity, drug resistance, and insufficient efficacy against "cold tumors".

[0003] Defects of the prior art: (1) Insufficient antigen exposure: Nucleolin (NCL), as a key tumor-associated antigen recognized by Vδ1γδ T cells, is expressed on the surface of colorectal adenocarcinoma cells, but the low expression level cannot induce Vδ1γδ T cells to produce strong killing effects on colorectal cancer cells. Tumor cells often evade immune surveillance by downregulating MHC-I molecules, and the level of NCL membrane ectopia is limited by the unclear phosphorylation regulatory mechanism, resulting in the inability of Vδ1γδ T cells to efficiently recognize tumors; (2) The function of CDK1 has not been utilized: The known functions of CDK1 are mostly limited to cell cycle regulation, and its association with antigen presentation has not been explored, resulting in the failure to develop potential therapeutic targets; (3) Lack of endogenous antigen enhancement strategies: Existing therapies mostly focus on enhancing the function of effector cells, rather than improving the immune recognition efficiency by regulating tumor antigen expression, resulting in limited therapeutic effects; (4) The anti-tumor function of Vδ1γδ T cells is limited: The low response rate of existing immunotherapies in colorectal cancer is partly attributed to insufficient antigen expression or immunosuppressive characteristics in the tumor microenvironment, which limits the activation and killing efficiency of Vδ1γδ T cells. Summary of the Invention

[0004] The object of the present invention is to provide a method for enhancing the anti - colon cancer function of Vδ1γδT cells. In the field of tumor immunotherapy, Vδ1γδT cells have become a potential tool for treating solid tumors (especially colon cancer) due to their antigen recognition characteristics that do not require major histocompatibility complex (MHC) restriction. To address the above - mentioned problems, the present invention first reveals that CDK1 phosphorylated at threonine 161 can drive the ectopic expression of NCL from the cell interior to the cell membrane by phosphorylating the Thr84 site of NCL. The NCL with ectopic expression in cells mediates the recognition and killing of colon adenocarcinoma cells by Vδ1γδT cells. Further research finds that stimulating colon adenocarcinoma cells with the CDK1 activator TC11 can enhance the ectopic expression of NCL on the cell membrane and the recognition and killing functions of Vδ1γδT cells against colon cancer cells. That is, the present invention discovers that the CDK1 activator TC11 significantly improves the anti - tumor efficacy based on Vδ1γδT cells.

[0005] To achieve the above - mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the application of the combined use of a CDK1 activator and T cells in the preparation of an anti - colon cancer drug.

[0007] Preferably, the CDK1 activator is TC11.

[0008] Preferably, the T cells are γδT cells.

[0009] Preferably, the γδT cells are Vδ1γδT cells.

[0010] The beneficial effects of the present invention are as follows:

[0011] 1. Innovative breakthroughs of the present invention: This study first reveals that CDK1 regulates its membrane ectopia by phosphorylating the Thr84 site of NCL, and develops a combined immunotherapy strategy based on the CDK1 activator (TC11), filling the following technical gaps:

[0012] (1) Mechanism innovation: It is found that CDK1 is the key kinase for NCL Thr84 phosphorylation, and the molecular pathway by which it drives the ectopia of NCL from the nucleolus to the cell membrane through phosphorylation modification is clarified. It is verified that activating CDK1 with TC11 can significantly increase the level of NCL membrane ectopia, thereby enhancing the killing efficiency of Vδ1γδT cells against colon cancer cells.

[0013] (2) Therapeutic strategy innovation: The concept of "enhancing endogenous antigens" is proposed, and the expression of tumor antigens is regulated by small - molecule activators, breaking through the MHC restriction of traditional therapies.

[0014] (3) Technical novelty and creativity: For the first time, the function of CDK1 has been extended from cell cycle regulation to the field of tumor antigen expression regulation, revealing a new mechanism of its phosphorylation modification of NCL at Thr84. The application of TC11 as a CDK1 activator in tumor immunotherapy is the first in the world. It breaks through the limitations of traditional immunotherapy that rely on exogenous antigens or genetic modification, and improves antigen visibility through endogenous regulation. By combining kinase activation and cell therapy, "synergistic enhancement of dual pathways" is achieved to solve the problem of immune suppression in the solid tumor microenvironment.

[0015] 2. Technical practicality

[0016] (1) Clinical application potential: The safety of TC11 has been verified through in vitro and animal experiments. It can be administered by injection, and the combination regimen with Vδ1γδT cells is easy to standardize. It is applicable to epithelial-derived tumors with high NCL expression (such as colorectal cancer, breast cancer), covering more than 30 types of solid tumors.

[0017] (2) Industrialization prospects:

[0018] The synthesis process of TC11 is mature and the cost is controllable; the in vitro expansion technology of Vδ1γδT cells has been optimized to a purity of >90%, and large-scale production conditions are available. Description of the drawings

[0019] Figure 1 NCL can directly activate Vδ1γδT cells. Among them, (A) sorted γδT cells were plated in a 96-well plate coated with BSA and NCL on the solid phase. After stimulation for 48 hours, the expression of activation markers CD25 and CD69 was detected by flow cytometry; (B) Quantitative analysis of the expression levels of activation-related molecules CD69 and CD25 in Vδ1γδT cells after NCL stimulation, T test, *P < 0.05, **P < 0.01;

[0020] Figure 2Ectopically expressed NCL can mediate the killing of colon cancer cells by Vδ1γδ T cells. Among them, (A) shows the detection of NCL expression on the membrane of HCT-116 cells by flow cytometry, T test, ***P < 0.001; (B) Western blot was used to detect the expression level of NCL in the total protein of HCT-116 cells transfected with blank plasmid (Control) and NCL overexpression plasmid (WT), and the gray value of the bands in different experimental groups was statistically analyzed, T test, **P < 0.01; (C) the proportion of Vδ1γδ T cells after 10 days of in vitro amplification; (D) HCT-116 cells before and after NCL overexpression were used as target cells and co-cultured with Vδ1γδ T cells respectively. Annexin V and DAPI flow cytometry double staining was used to analyze the activity of HCT-116 cells at effector-to-target ratios of 5:1 and 10:1; (E) statistical chart of three independent repeated experiments of (D), T test, ***P < 0.001, ****P < 0.0001, ns means no statistical significance;

[0021] Figure 3 It is about the relationship between the ectopic expression level of NCL on the cell membrane and the phosphorylation level of Thr84. Among them, (A) Membrane, cytoplasm and nuclear proteins were extracted from HCT-116 cells, and Western blot was used to detect the expression levels of phosphorylated NCL and total NCL. Three independent repeated experiments were carried out, and after analyzing the gray values of the bands, one-way ANOVA was performed, *P < 0.05, ns means no statistical significance; (B) HCT-116 cells before and after STS treatment were used to extract total protein, membrane, cytoplasm and nuclear proteins respectively, and Western blot was used to detect the expression levels of phosphorylated NCL and total NCL; Three independent repeated experiments were carried out, and after analyzing the gray values of the bands, the expression level of phosphorylated NCL at Thr84 was analyzed, T test, *P < 0.05, ns means no statistical significance. (C) Before and after STS treatment, flow cytometry was used to detect the ectopic expression level of NCL on the membrane, and the mean fluorescence intensity was analyzed. Five independent repeated experiments were carried out, T test, *P < 0.05;

[0022] Figure 4Effect of phosphorylation of Thr84 on the subcellular localization of NCL. (A) HCT-116 cells infected with different viruses were used to extract membrane, cytoplasmic, and nuclear proteins, and Western blot was performed to detect the expression levels of phosphorylated NCL at Thr84 and total NCL. The gray values of the phosphorylated NCL at Thr84 and total NCL bands were analyzed, and the ratio of the gray values of the phosphorylated NCL at Thr84 and total NCL bands was used to represent the level of phosphorylated NCL at Thr84. One-way ANOVA was performed after three independent repeated experiments, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no statistical significance. (B) Flow cytometry was used to detect the expression level of NCL on the cell membrane infected with different viruses, and the mean fluorescence intensity was analyzed. One-way ANOVA was performed on the three independent repeated experiments, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no statistical significance;

[0023] Figure 5 Effect of knocking down the expression of CDK1 in colon cancer cells on the ectopic expression of NCL on the membrane. (A) Western Blot was used to detect the expression of CDK1 and phosphorylated CDK1 at Thr161 in the control group and siRNA interference group cells; one-way ANOVA was used for the analysis of differences between groups, *P < 0.05, **P < 0.01, ***P < 0.001; (B) Flow cytometry was used to detect the ectopic expression of NCL on the membrane in the control group and siRNA interference group cells; the statistical chart of the mean fluorescence intensity of three experiments, one-way ANOVA was used for the analysis of differences between groups, *P < 0.05;

[0024] Figure 6 Effect of activating CDK1 on the ectopic expression of NCL on the membrane. (A) Western blot was used to detect the expression of CDK1 and phosphorylated CDK1 (Thr161) before and after treatment with TC11; (for three independent repeated experiments, T-test was used for the analysis of the gray value statistical chart, ****P < 0.0001, ns indicates no statistical significance; (B) Flow histograms of NCL membrane expression after treatment with different concentrations of TC11 for different times; the statistical chart of the mean fluorescence intensity of NCL in three independent repeated experiments, T-test was used, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0025] Figure 7 Effect of CDK1-regulated phosphorylation of Thr84 on the ectopic expression of NCL on the membrane ((A) HCT-116 cells were seeded at 5×10 4Cells were seeded in 96-well plates at a density of

[0026] Figure 8 To investigate the effect of CDK1-regulated Thr84 phosphorylation on the cellular distribution of NCL, (A) HCT-116 cells infected with different viruses were treated with 2.5 μM TC11 for 12 h. Membrane proteins, cytoplasmic proteins, and nuclear proteins were extracted before and after TC11 treatment, and the expression levels of phosphorylated NCL and total NCL were detected by Western blot. (B) The expression level of phosphorylated NCL protein before and after TC11 treatment was analyzed. The gray values of the Thr84-phosphorylated NCL and total NCL bands were analyzed, and the ratio of the gray values of the Thr84-phosphorylated NCL and total NCL bands was used to represent the level of Thr84-phosphorylated NCL. After three independent repeated experiments, a T-test was performed, with *P < 0.05, **P < 0.01, and ns indicating no statistical significance. (C) The expression level of total NCL protein before and after TC11 treatment was analyzed. After three independent repeated experiments, a T-test was performed on the gray values of the bands, with *P < 0.05, **P < 0.01, and ns indicating no statistical significance.

[0027] Figure 9 To investigate the effect of CDK1 activator TC11-regulated NCL expression on the killing of colon cancer cells by Vδ1γδT cells, (A) Flow cytometry was used to detect the proportion of apoptotic cells after TC11 treatment. Three independent repeated experiments on cell apoptosis after TC11 treatment were performed, and a T-test was performed, with ns indicating no statistical significance. (B) HCT-116 cells before and after TC11 treatment were used as target cells and co-cultured with Vδ1γδT cells at an effector-to-target ratio of 5:1 and 10:1. Flow cytometry scatter plots were obtained. (C) A T-test was performed on three independent repeated experiments, with *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns indicating no statistical significance.

[0028] Figure 10 To study the effect of CDK1 activator TC11 on the anti-tumor effect of Vδ1γδ T cells, where (A) is a schematic diagram of the experimental procedure. When the tumor volume reached 50 mm 3 Grouping was performed (D0). In the experimental group, TC11 with a concentration of 20 mg / mL was subcutaneously injected from the fourth day. Within 24 hours after the fourth injection of TC11, 1×10 7 Vδ1γδ T cells were intratumorally injected. (B) Representative images of tumors in mice in different treatment groups. (C) Line graph showing the change in tumor volume over time in different treatment groups. It shows the dynamic changes in tumor volume of the four groups from day 4 to day 18. Two-way ANOVA was performed, *P<0.05, ****P<0.0001. Detailed implementation manners

[0029] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1 NCL can directly activate Vδ1γδ T cells, and ectopically expressed NCL can mediate the killing of colon cancer cells by Vδ1γδ T cells

[0031] Previous studies have confirmed that nucleolin (NCL) is mainly localized in the nucleolus in normal cells, while it shows abnormal high expression and ectopic membrane surface localization characteristics in malignant tumor cells. To verify the activation effect of NCL on Vδ1γδ T cells, in this study, solid-phase NCL recombinant protein was used to stimulate γδ T cells isolated from the peripheral blood of healthy donors. Flow cytometry analysis showed that NCL stimulation significantly upregulated the expression levels of activation markers such as CD69( Figure 1 (A)) and CD25( Figure 1 (B)) in the Vδ1γδ T cell subset (p<0.01), and this result was consistent with the previously reported antigen characteristics of NCL.

[0032] It was confirmed by flow cytometry of membrane surface proteins that there was a significant phenomenon of ectopic membrane expression of NCL in HCT-116 cells( Figure 2 (A)). To clarify whether membrane-localized NCL mediates the specific killing of Vδ1γδ T cells, we constructed an HCT-116 cell line overexpressing NCL (WT group, Figure 2 (B)), and used cells transfected with empty plasmid as a control (Control group). Using highly purified in vitro-expanded Vδ1γδ T cells( Figure 2 (C)) for killing experiments, it was found that at effector-to-target ratios of 5:1 and 10:1, the proportions of early apoptosis (AnnexinV+PI-) and late apoptosis (AnnexinV+PI+) of target cells in the WT group were significantly higher than those in the Control group (p<0.05,Figure 2 D - E). These data indicate that the ectopic expression level of NCL on the surface of colon cancer cell membranes is positively correlated with its cytotoxic sensitivity mediated by Vδ1γδT cells.

[0033] Example 2 Thr84 Phosphorylation Mediates Ectopic Expression of NCL on Colon Cancer Cell Membranes

[0034] Post - translational phosphorylation modification plays an important role in the translocation of intracellular NCL to the cell membrane. Previous studies have found that NCL with threonine 84 phosphorylation modification exists in colon cancer, but the relationship between Thr84 phosphorylation modification and NCL membrane translocation has not been studied. In this study, the total NCL and its Thr84 phosphorylation modification in membrane, cytoplasm, and nuclear proteins were analyzed by Western blot in HCT - 116 cells. The results showed that the NCL phosphorylated at Thr84 was most highly expressed in the cell membrane ( Figure 3 A). Staurosporine (STS) is a very effective protein kinase inhibitor that can inhibit the phosphorylation modification of NCL at Thr84. In this study, cells were treated with STS and the total NCL and its Thr84 phosphorylation modification in whole protein, membrane, cytoplasm, and nuclear proteins were analyzed. It was found that the phosphorylation modification level of Thr84 was significantly down - regulated in whole protein and membrane proteins ( Figure 3 B). Before and after STS treatment, the ectopic expression level of cellular NCL was detected by flow cytometry. The results showed that the expression of NCL on the cell membrane surface decreased significantly after STS treatment ( Figure 3 C). The above results indicate that the ectopic expression level of NCL on the cell membrane decreases with the down - regulation of Thr84 phosphorylation level.

[0035] To investigate the effect of Thr84 phosphorylation on the subcellular localization of NCL, we constructed plasmids of NCL with Thr84 mutation and wild-type NCL and transfected them into HCT-116 cells. The cell membrane, cytoplasm, and nuclear proteins of cells in the blank group (Blank), vector group (Vector), NCL wild-type group (WT), and Thr84 mutant group (Mutant) were extracted, and the phosphorylation modification of NCL in each group of cells was analyzed by Western blot. The results showed that the Thr84 phosphorylation level was the highest in the membrane protein fraction in the Blank, Vector, and WT groups, while in the Mutant group, the Thr84 phosphorylation level was the highest in the nuclear protein fraction, indicating that the mutation changed the cellular distribution of Thr84 phosphorylated NCL. Comparing the Thr84 phosphorylation levels of NCL in the Blank, Vector, WT, and Mutant groups in different cell fractions, it was found that in the membrane protein, the Thr84 phosphorylated NCL level in the WT group was significantly higher than that in the Blank and Vector groups, indicating that overexpression of NCL increased the Thr84 phosphorylated NCL level in the membrane protein, while after Thr84 mutation, the Thr84 phosphorylated NCL level decreased significantly. In the cytoplasmic protein, the Thr84 phosphorylated NCL level in the WT group decreased significantly; in the nuclear protein, the Thr84 phosphorylation level in the Mutant group was significantly higher than that in the Blank, Vector, and WT groups; the phosphorylated NCL level in the WT group was significantly lower than that in the Blank and Vector groups ( Figure 4 A). The ectopic expression levels of NCL on the cell membrane in different groups were detected by flow cytometry, and the results showed that the ectopic expression level of NCL in the WT group was significantly higher than that in the Blank and Vector groups; after Thr84 mutation, the ectopic expression level of NCL was significantly lower than that in the WT group ( Figure 4 B). Overall, the change trend of the NCL membrane ectopic expression level in the Blank, Vector, WT, and Mutant groups was consistent with the change in the Thr84 phosphorylation modification level in the membrane protein fraction, suggesting that the phosphorylation modification of Thr84 mediates the ectopic localization of NCL from the nucleus and cytoplasm to the cell membrane.

[0036] Example 3 CDK1 can regulate the ectopic expression of NCL

[0037] Previous studies have shown that CDK1 is the upstream kinase for phosphorylating NCL, but the effect of CDK1 phosphorylating NCL on its ectopic expression has not been studied. To clarify whether CDK1 phosphorylating NCL affects the membrane ectopic expression of NCL, this application used siRNA to knockdown the expression of CDK1 in colon cancer cells and constructed a CDK1 knockdown cell model. Western Blot was used to detect the expression of total CDK1 and phosphorylated CDK1 at the Thr161 site. The results showed that compared with the control group, the levels of total CDK1 and Thr161 phosphorylated CDK1 in the siCDK1 group showed an obvious downward trend ( Figure 5 A). Flow cytometry was used to detect the ectopic expression of NCL on the cell membrane. The results showed that compared with the control group, the expression of NCL in siCDK1 decreased ( Figure 5 B). The above results prove that interfering with the phosphorylation modification of the 161st threonine of CDK1 downregulates the level of NCL membrane ectopia, suggesting that the phosphorylation level of CDK1 is closely related to the membrane ectopic expression of NCL.

[0038] As an activator of CDK1, TC11 can phosphorylate CDK1 at Thr161. To clarify the effect of CDK1 activation on the membrane ectopic expression of NCL in colon cancer cells, this study treated HCT-116 cells with TC11. Western blot showed that TC11 (2.5 μM, 12 h) could significantly upregulate the phosphorylation level of CDK1 at Thr161 ( Figure 6 A). Further, different concentrations (0, 2.5, 5 μM) of TC11 were added to the HCT-116 cell culture system. Cells were collected after culturing for 12, 24, 36, and 48 hours, respectively. Flow cytometry was used to detect the level of NCL membrane ectopic expression in colon cancer cells. Flow cytometry detection showed that the membrane ectopic expression in the TC11 treatment group reached the peak MFI at 2.5 μM TC11 treatment for 12 hours ( Figure 6 B). The above results prove that upregulating the phosphorylation level of CDK1 at Thr161 promotes the membrane ectopic expression of NCL, suggesting that CDK1 can regulate the membrane ectopic expression of NCL.

[0039] Example 4 TC11 promotes the phosphorylation modification of NCL by CDK1 at Thr84 and affects its subcellular localization

[0040] In colon cancer cell lines, it is not clear whether CDK1, as an upstream kinase, can phosphorylate NCL at Thr84. To further verify this, this study stimulated HCT-116 cells with TC11. Western blot results showed that after TC11 treatment, the expression level of total NCL did not change significantly, while the phosphorylation modification level of Thr84 showed a significant upregulation, suggesting that TC11 can upregulate the phosphorylation modification level of NCL at Thr84 ( Figure 7A). To investigate whether CDK1 regulates the membrane ectopic expression of NCL through phosphorylation modification at Thr84, in this study, the activator TC11 of CDK1 was used to treat HCT-116 cells in the Blank, Vector, WT, and Mutant groups, and Western blot was performed to analyze the expression levels of total NCL and phosphorylated NCL ( Figure 7 B). The results showed that there were no significant differences in the expression levels of total NCL and phosphorylated NCL among the cells in each group without TC11 treatment. After TC11 treatment, the expression level of phosphorylated NCL increased in the Blank, Vector, and WT groups, while there was no significant difference in the Mutant group before and after TC11 treatment ( Figure 7 B). The above results indicated that the mutation at the Thr84 site led to the inability of CDK1 activated by TC11 to phosphorylate and modify NCL at this site. To further analyze the effect of Thr84 mutation on the membrane ectopic expression of NCL, flow cytometry was used to analyze the changes in the membrane ectopic expression level of NCL ( Figure 7 C). The experimental results suggested that after TC11 treatment, the membrane ectopic expression level of NCL in the Blank, Vector, and WT groups increased significantly, while there was no statistically significant difference in the change of the membrane ectopic expression level of NCL in the Mutant group before and after TC11 treatment ( Figure 7 C). It was suggested that after Thr84 of NCL was mutated to alanine, the membrane ectopic expression level of NCL was not regulated by the activation of CDK1. The above results indicated that CDK1 could regulate the membrane surface ectopic expression of NCL through Thr84.

[0041] To explore the effect of phosphorylation modification of CDK1 at the Thr84 site on the subcellular localization of NCL, in this study, Western blot was used to analyze the expression levels of total NCL and Thr84 phosphorylated NCL in different components (nucleus, cytoplasm, cell membrane) of HCT-116 cells before and after TC11 treatment ( Figure 8 A). The results showed that in membrane proteins, after TC11 treatment, the Thr84 phosphorylated NCL in the Blank, Vector, and WT groups increased significantly, while there was no significant change in the Mutant group. In cytoplasmic proteins, the Thr84 phosphorylation level increased in the Blank, Vector, and WT groups, and there was no change in the Mutant group. In nuclear proteins, the level of Thr84 phosphorylated NCL did not change in the Blank, Vector, WT, and Mutant groups ( Figure 8B). The above results indicate that after treatment with TC11, the upregulation of Thr84 phosphorylation modification levels in the cell membrane and cytoplasm is the main change. Further analysis of the changes in the cellular distribution of total NCL revealed that compared with before TC11 stimulation, the distribution of NCL in the cell nucleus or (and) cytoplasm decreased in the Blank, Vector, and WT groups, while the distribution of NCL in the cell membrane increased, shifting from the cell nucleus or (and) cytoplasm to the cell membrane. However, no such changes occurred in the Mutant group ( Figure 8 C). The above results indicate that TC11 promotes the ectopic translocation of NCL from the cell nucleus or (and) cytoplasm to the cell membrane through CDK1-mediated Thr84 phosphorylation modification.

[0042] Example 5: The effect of the CDK1 activator TC11 on regulating NCL expression and influencing the anti-tumor effect of Vδ1γδ T cells

[0043] To evaluate the effect of the CDK1 activator TC11 on promoting the ectopic expression of NCL in the cell membrane on the anti-tumor effect of Vδ1γδ T cells, we first analyzed the differences in the sensitivity of HCT-116 cells in the Blank, Vector, WT, and Mutant groups to TC11. The results showed that there were no significant differences in the sensitivity of the four groups of cells to TC11 ( Figure 9 A). We further co-cultured HCT-116 cells before and after treatment with TC11 as target cells with Vδ1γδ T cells respectively ( Figure 9 B). The effector-to-target ratio was set at 5:1 and 10:1. The HCT-116 cells were divided into 4 groups: blank group (Blank), empty vector group (Vector), wild-type NCL overexpression group (WT), and Thr84 mutant group (Mutant). Each group of HCT-116 cells was treated with 2.5 μM TC11 for 12 hours and then co-incubated with Vδ1γδ T cells for 4 hours. The results showed that compared with the group of Vδ1γδ T cells alone, after combining with TC11, the proportion of viable cells in the Blank, Vector, and WT groups decreased significantly, and the proportions of early and late apoptotic cells increased significantly. The trend was the same at effector-to-target ratios of 5:1 and 10:1, indicating that the CDK1 activator TC11 promoting the ectopic expression of NCL in the cell membrane enhanced the anti-tumor effect of Vδ1γδ T cells. The proportions of viable and apoptotic cells in the Mutant group after treatment with TC11 did not show significant changes at the two effector-to-target ratios. Figure 9 C). It shows that the mutation of NCL at the Thr84 site abolishes the enhancing effect of TC11 on the anti-tumor effect of Vδ1γδ T cells. The above results suggest that promoting the ectopic expression of NCL in the cell membrane by the CDK1 activator can enhance the killing of tumor cells by Vδ1γδ T cells.

[0044] To further verify in in vivo experiments that CDK1-mediated phosphorylation modification promotes the ectopic expression of NCL in the membrane, thereby enhancing the anti-tumor effect of Vδ1γδ T cells. We established a xenograft tumor model of immunodeficient mice with HCT-116 cells, subcutaneously injected TC11, and within 24 hours after every four injections of TC11, injected Vδ1γδ T cells into the tumor Figure 10 A). During the treatment process, the tumor volume was measured regularly to analyze the differences in tumor growth rates and the survival periods of mice in each group. The results showed that compared with the control group, the tumor volume and weight in each experimental group decreased. Compared with the group treated with Vδ1γδ T cells alone, the tumor significantly decreased after treatment with TC11 combined with Vδ1γδ T cells Figure 10 B-C), indicating that the CDK1 activator TC11 can enhance the anti-tumor effect of Vδ1γδ T cells.

[0045] As can be seen from the above Examples 1 to 5,

[0046] 1. NCL can directly activate Vδ1γδ T cells, and the ectopically expressed NCL can mediate the killing of colon cancer cells by Vδ1γδ T cells;

[0047] 2. Thr84 phosphorylation mediates the ectopic expression of NCL in the membrane of colon cancer cells;

[0048] 3. The present invention for the first time reveals that CDK1 phosphorylated at the 161st threonine can drive the ectopic expression of NCL from the cell interior to the cell membrane by phosphorylating the Thr84 site of NCL, and the NCL ectopically expressed in the cell mediates the recognition and killing of colon adenocarcinoma cells by Vδ1γδ T cells;

[0049] 4. Stimulating colon adenocarcinoma cells with the CDK1 activator TC11 can enhance the ectopic expression of NCL in the cell membrane and enhance the recognition and killing functions of Vδ1γδ T cells against colon cancer cells. That is, the present invention discovers that the CDK1 activator TC11 significantly improves the anti-tumor efficacy based on Vδ1γδ T cells.

[0050] As can be seen from the above examples, the present invention provides a method for enhancing the anti-colon cancer function of Vδ1γδ T cells, and combines a CDK1 activator and T cells to prepare an anti-colon cancer drug. The technical solution of the present invention clarifies the molecular mechanism of the CDK1-NCL axis regulating the ectopic expression of tumor antigens, filling the key theoretical gap in the recognition of tumor cells by Vδ1γδ T cells; developing the CDK1 activator TC11 as an auxiliary means to enhance the function of Vδ1γδ T cells, breaking through the limitation of traditional immunotherapy relying on the modification of exogenous antigens; and confirming through in vitro and in vivo experiments that targeting CDK1 can reshape the tumor immune microenvironment, providing a translatable clinical application plan for the combined immunotherapy of colon cancer and other solid tumors.

[0051] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a CDK1 activator in combination with T cells in the preparation of an anti-colorectal cancer drug.

2. The application according to claim 1, characterized in that The CDK1 activator is TC11.

3. The application according to claim 1, characterized in that, The T cells are γδ T cells.

4. The application according to claim 3, characterized in that, The γδ T cells are Vδ1γδ T cells.

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