Use of overexpression of cdca5 in preparation of a drug for enhancing the anti-tumor function of t cells

Abstract

The application provides application of overexpressed CDCA5 in preparation of a drug for enhancing antitumor function of T cells, and relates to the technical field of cellular immunotherapy. The method for overexpressing CDCA5 is to use a CDCA5 overexpression agent, and the CDCA5 overexpression agent is a CDCA5 overexpression vector, and the CDCA5 overexpression vector is any one of a lentivirus and a retrovirus. The application overcomes the defects of the prior art, verifies that CAR-T with overexpressed CDCA5 can significantly enhance antitumor function in vivo and in vitro, and provides a new technical direction for antitumor treatment.

Description

Technical Field

[0001] This invention relates to the field of cell immunotherapy technology, specifically to the application of overexpression of CDCA5 in the preparation of anti-tumor drugs that enhance the function of T cells. Background Technology

[0002] The high incidence and mortality rates of malignant tumors have become a major challenge in global public health. While traditional treatments (such as surgery, chemotherapy, and radiotherapy) can control tumor progression in the short term, they are limited by toxicity to normal tissues, tumor heterogeneity, and drug resistance, making it difficult for most late-stage patients to achieve long-term survival benefits. In recent years, anti-tumor immunotherapy strategies, represented by immune checkpoint inhibitors (ICIs) and adoptive cell immunotherapy (ACT), have fundamentally changed the landscape of cancer treatment by "relieving immunosuppression" or "enhancing effector T cell function." The former is currently the most mature immunotherapy applicable to the widest range of cancer types, but it is only effective in 20%-40% of patients, and its efficacy is related to indicators such as tumor PD-L1 expression and mutation burden. The latter can be genetically modified (e.g., by adding TCR or CAR receptors) or massively expanded in vitro to enable precise recognition of tumor antigens before being reinfused into the patient to kill tumors.

[0003] As the core of the body's adaptive immune system, T cells not only specifically recognize and kill tumor cells, but their functional state also directly determines whether tumors can be effectively controlled. However, tumor cells actively remodel the tumor microenvironment (TME), forming multiple mechanisms that hinder effective anti-tumor immune responses. On the one hand, continuous antigen stimulation within the TME induces T cells into a "dysregulated state"—manifested as a decline in proliferative capacity, difficulty in forming a sufficient number of effector cell populations, reduced secretion of effector molecules such as IFN-γ and granzymes, high expression of inhibitory receptors such as PD-1 and TIM-3, and abnormal expression of transcription factors such as TOX, leading to irreversible loss of T cell killing ability. On the other hand, metabolic disorders in the TME further exacerbate immunosuppression—tumor cells competitively take up glucose through the "Warberg effect," while releasing metabolites such as lactic acid, leading to TME acidification and nutrient deprivation, inhibiting T cell glycolysis for energy supply, and ultimately causing T cells to fall into an "energy crisis" and "functional paralysis." Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides the application of overexpressing CDCA5 in the preparation of anti-tumor drugs that enhance the function of T cells. It verifies that CAR-T cells overexpressing CDCA5 significantly enhance anti-tumor function in vitro and in vivo, providing a new technical direction for anti-tumor treatment.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The application of overexpression of CDCA5 in the preparation of anti-tumor drugs that enhance the function of T cells, wherein the method of overexpressing CDCA5 is to use a CDCA5 overexpression agent, and the CDCA5 overexpression agent is a vector for overexpressing CDCA5.

[0007] Preferably, the vector overexpressing CDCA5 is any one of lentivirus and retrovirus.

[0008] A pharmaceutical composition for enhancing the anti-tumor effect of T cells, the pharmaceutical composition comprising a CDCA5 overexpression agent, wherein the CDCA5 overexpression agent is a vector for overexpressing CDCA5; the vector for overexpressing CDCA5 is any one of lentivirus and retrovirus.

[0009] This invention provides the application of overexpression of CDCA5 in the preparation of antitumor drugs that enhance the function of T cells, and its advantages compared with the prior art are:

[0010] Existing studies have shown that CDCA5 overexpression primarily affects tumors by promoting tumor cell proliferation, migration, and invasion, and is associated with poor patient prognosis. The CDCA5 gene is highly expressed in cervical cancer tissues, and its overexpression enhances the proliferative capacity of human cervical cancer cells. Furthermore, CDCA5 may promote liver cancer development by activating tumor cell proliferation. However, in this invention, due to T cell dysfunction in the tumor microenvironment, CDCA5 overexpression in T cells leads to enhanced T cell proliferation. This, in turn, activates the PI3K / AKT / mTOR pathway, promoting T cell effector function and enhancing the in vitro anti-tumor activity of T cells. Ultimately, this demonstrates that CAR-T cells overexpressing CDCA5 significantly enhance anti-tumor function both in vivo and in vitro. Attached Figure Description

[0011] Figure 1 This is a schematic diagram illustrating the proliferation and survival of CDCA5-overexpressing T cells in Example 1 of this invention; where A represents the expression of the specified protein in Tn, Teff, and Tex cells and the ratios of Teff / Tn and Teff / Tex; B represents verification of the overexpression effect at the protein level; C represents verification of the overexpression effect at the mRNA level; D represents cell counts after 6 days of culture in complete medium containing 50 U / mL interleukin-2 (IL-2); E represents the T cell proliferation after 4 days of culture in complete medium containing 50 U / mL IL-2 following Cell Trace Violet (CTV) staining; and F represents the proliferation of control-T cells and CDCA5-overexpressing cells. OE Ki67 protein expression in -T cells; GI is the expression of CD3 by flow cytometry. + CD4 + and CD8 +Ki67 expression in T cells; J represents cell cycle analysis by flow cytometry; KL represents cell survival and apoptosis analysis by flow cytometry. M represents T cells in the control group and CDCA5. OE Expression of Bcl-2 and Bax proteins in -T cells;

[0012] Figure 2 This is a schematic diagram illustrating the activation and effector function of T cells overexpressing CDCA5 in Example 2 of the present invention; where A represents the analysis of CD3 by flow cytometry. + CD4 + and CD8 + Changes in T cell activation; B represents CD3. + CD4 + and CD8 + Changes in the T cell population: Tn (CD44-CD62L+), Teff (CD44+CD62L-). CE represents CD3+. + CD4 + and CD8 + Changes in T cell function; F represents changes in OT-1 cell function;

[0013] Figure 3 In Example 3 of this invention, CDCA5 activates the PI3K-AKT-mTOR signaling pathway; where A is a volcano plot showing differentially expressed genes, with red indicating upregulated genes and blue indicating downregulated genes; B is a heatmap showing the expression of specified genes; C is a KEGG pathway analysis of the 10 most significantly upregulated pathways; DE is GSEA analysis of the PI3K-AKT and mTOR signaling pathways; F is the expression of CDCA5 in control-T cells and CDCA5. OE Expression of mTOR, p-mTOR, PI3K, p-PI3K, AKT, and p-AKT proteins in -T cells; GH represents the expression of p-mTOR and p-4EBP1 analyzed by flow cytometry.

[0014] Figure 4 In Example 4 of this invention, CDCA5 enhances T cell proliferation and effector function through the mTOR signaling pathway; where A is a heatmap showing the expression of the specified gene; B shows the measurement and statistical results of extracellular acidification rate (ECAR) using a Seahorse XF-96 Cell Energy Metabolism Analyzer (Seahorse Bioscience); CF shows the control group T cells and CDCA5. OE -T cells were treated with 50 nM rapamycin for 2 days or not; changes in cell function were analyzed by flow cytometry.

[0015] Figure 5To demonstrate that T cells overexpressing CDCA5 in Example 5 of this invention have a stronger anti-tumor effect in vivo; where A is the flowchart of the in vivo experiment; BC is the tumor growth curve, tumor image and tumor weight; DH is the proportion, number and function of tumor-infiltrating lymphocytes analyzed by flow cytometry; I is the flowchart of the in vivo experiment; JK is the tumor growth curve, tumor image and tumor weight; LO is the proportion, number and function of tumor-infiltrating lymphocytes analyzed by flow cytometry.

[0016] Figure 6 This is a schematic diagram illustrating the anti-tumor ability of CAR-T cells overexpressing CDCA5 in Example 6 of the present invention; where A represents tNGFR. OE -CAR-T cells and CDCA5 OE - Schematic diagram of the CAR-T cell experimental procedure; B shows a schematic diagram of a second-generation CAR expressing human tNGFR (a truncated nerve growth factor receptor molecule) targeting hCD19 (tNGFR CD19 CAR) as a control and a second-generation CAR expressing human CDCA5 targeting hCD19 (CDCA5 CD19 CAR), both modified and inserted after the P2A element; C shows cell counting after 8 days of culture in complete medium containing 200 U / mL IL-2; D shows the proliferation of CAR-T cells after 4 days of culture in complete medium containing 200 U / mL IL-2 following Cell CTV staining; E shows the Ki67 expression of CAR-T cells analyzed by flow cytometry.

[0017] F represents the apoptosis of CAR-T cells analyzed by flow cytometry; GH represents the activation of CAR-T cells analyzed by flow cytometry; IL represents the function of CAR-T cells analyzed by flow cytometry; M represents the secretion of key immune cytokines such as TNF-α and IFN-γ in the cytotoxic supernatant detected by ELISA; NO represents the cytotoxicity of CAR-T cells against Raji or Su-4 cells at a specified effector cell to target cell ratio (E:T ratio).

[0018] Figure 7 To demonstrate that CAR-T cells overexpressing CDCA5 in Example 7 of this invention exhibit stronger anti-tumor activity in vivo; where A is a flowchart of the in vivo experiment; B is a tumor bioluminescence image; C is a survival curve; D is a quantitative value of tumor bioluminescence; E shows changes in mouse body weight; F shows the detection of the number of CAR-T cells in peripheral blood; G shows changes in the CAR-T cell population (Teff(CD45RO)). - CD62L - H represents the survival time of B-cell malignant tumor patients who received anti-CD19 CAR-T therapy after stratification based on the mean expression of CDCA5 in T cells. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1:

[0021] CDCA5 promotes T cell proliferation and survival.

[0022] 1. Mouse Cdca5 The gene sequence is shown in SEQ ID NO.1 in the sequence listing.

[0023] 2. Cdca5 Gene sequence bacterial culture preparation:

[0024] Using seamless cloning technology Cdca5 Inserted into the reverse transcription vector psir-hu6-GFP, and selected bacterial cultures with the correct constructed sequence were identified by sequencing:

[0025] Seamless cloning: using homologous recombinase, Cdca5 Gene sequence fragments, double-distilled water, and linearized psir-hu6-GFP vector digested with Not I restriction endonuclease were used in a 10 μL reaction system: 2 μL homologous recombinase, Cdca5 50 ng of gene sequence fragment, 100 ng of linearized psir-hu6-GFP vector, and other components were added to make up 10 μL of double-distilled water. The temperature was controlled at 50℃ and the reaction time was 60 min to obtain seamless cloning products.

[0026] Transformation and plating: Remove competent cells (DH5α) from -80℃ and immediately place them on ice to thaw. Take 5 μL of seamless cloning product and add it to the pre-thawed competent cells. Mix gently and place on ice for 30 min. Heat shock at 42℃ for 90 s without shaking the test tube, and immediately stop the ice bath for 5 min.

[0027] Add 200 μL of antibiotic-free LB liquid medium to each tube and incubate at 37°C and 150 rpm for 30 min with shaking. Spread 100 μL of bacterial culture onto the surface of an LB agar plate containing the appropriate antibiotic (Amp) using sterile glass beads, incubate at 37°C upright for 30 min, and then invert overnight.

[0028] Picking and Sequencing: Randomly pick several plaques from LB agar plates and place them into 5 mL shaker tubes containing ampicillin-resistant LB liquid medium. Shake at 37°C and 200 rpm for 10-12 hours until the bacterial culture becomes turbid. Take 100 μL of each tube for sequencing. The sequencing results and... Cdca5 Gene sequences were compared, and bacterial cultures that matched correctly yielded... Cdca5 Gene sequence bacterial culture.

[0029] 3. Preparation containing Cdca5 Gene retroviruses:

[0030] After plasmid extraction, it is packaged with retroviruses and contains... Cdca5 Viral particles were obtained by co-transfecting plat-E cells with the psir-hu6-GFP vector and the helper plasmid PCL-ECO.

[0031] Plasmid extraction: Take 100 μL of bacterial culture containing the target sequence and put it into 200 mL of LB liquid medium. Control the temperature at 37℃ and 200 rpm and shake the culture on a shaker. After 12 h, extract the psir-hu6-GFP vector containing the target sequence, including PCL-ECO.

[0032] Virus Packaging: One day before transfection, passage plat-E cells 2:5 into 10cm dishes and incubate at 37°C with 5% CO2. Transfection can be performed when the cell density reaches 80%–90%. 1–2 hours before transfection, replace the cell culture medium with 6 mL of pre-warmed complete medium. Add 300 μL of jetPRIME buffer and the corresponding weight of plasmid (PCL-ECO: psir-hu6-GFP = 1:2, total 12 μg) to a sterile 1.5 mL EP tube A. Mix well, then add 25 μL of jetPRIME and gently shake. Incubate at room temperature for 10 min. Transfer the mixture to the plat-E cell culture medium, mix well, and incubate at 37°C with 5% CO2. After 8 hours, replace with 10 mL of pre-warmed complete medium. Collect the viral supernatant at 48 h and 72 h. The collected supernatant was filtered through a 0.45 μm (PES) membrane filter to remove cell residues.

[0033] 4. Preparation of mouse T cells:

[0034] Antibody plating: Anti-mouse CD3 / CD28 antibody plating. Add antibody to PBS to a final concentration of 5 μg / mL anti-mouse CD3 and 2.5 μg / mL anti-mouse CD28. Add 500 μL to each well of a 12-well plate, seal with tape, and incubate overnight at 4°C.

[0035] T cell isolation: Mouse spleen T cells were isolated, washed with 1×PBS, and the cell count was determined using a Countstar automated cell counter (Shanghai, China). T cells (1×10⁶) were... 6 Cells were seeded (1 cell / well) and transduced after 24 hours.

[0036] T cell infection and culture: Activated T cells were co-transduced with 8 μg / mL polybrene retrovirus. To improve transduction efficiency, plates were centrifuged at 1000×g for 1.5 h at 37°C, and the culture medium was changed 6-8 h post-infection. Transduced lymphocytes were maintained in culture medium supplemented with IL-2 (50 IU). On day 4 post-transduction, GFP+ was used as a reporter gene for quantification by flow cytometry. Cdca5 Express.

[0037] 5. Western blotting (WB) detection of related protein expression levels

[0038] Preparing the adhesive: Prepare the lower layer of adhesive (10%), pour it into the glass plate, press it down with isopropyl alcohol, and allow it to solidify. Prepare the upper layer of adhesive, pour it onto the solidified lower layer of adhesive, install the comb, and allow it to solidify naturally.

[0039] Electrophoresis: Fix the gel plate, add the electrophoresis solution, remove the comb, and add the standard marker and sample. Set the electrophoresis voltage to 90V for 30 minutes and 120V for 60 minutes.

[0040] Transfer: Prepare the transfer clamp, construct the sandwich structure, add the moistened PVDF membrane, and install the transfer tank. Set the voltage to 200mA, transfer for 60 minutes, and then cool to an ice bath.

[0041] Sealing: Clean the PVDF membrane after transfer, place it in the sealing solution, and seal it on a shaker for 20 minutes.

[0042] Primary antibody incubation: After washing the membrane, add preheated primary antibody solution and incubate overnight at 4°C. Mouse anti-CDCA5 (Cat#67418-1-Ig, Proteintech), rabbit anti-Ki67 (28074-1-AP, Proteintech), rabbit anti-Bcl2 (Cat#82469-6-RR, Proteintech), rabbit anti-Bax (Cat#50599-2-Ig, Proteintech)

[0043] Secondary antibody incubation: After washing the membrane, add the secondary antibody solution and incubate on a shaker for 1 hour. HRP goat anti-mouse IgG (Cat#LF101, Epizyme), HRP goat anti-rabbit IgG (Cat#LF102, Epizyme)

[0044] Development: Mix the developer solution, apply it to the membrane, and develop until the bands appear.

[0045] 6. Flow cytometry detection of T cell proliferation and survival:

[0046] Cells were collected, washed with 1 mL of PBS, and then incubated with a dead / live cell dye (Zombie Dye) to remove dead cells. Following this step, cells were stained with all relevant antibodies (anti-mouse CD3-APC-Cy7 (Clone17A2; Cat#100222), anti-mouse CD4-PerCP-CY5.5 (Clone GK1.5; Cat#100434), anti-mouse CD4-APC-Cy7 (Clone GK1.5; Cat#100414), anti-mouse CD8α-APC-CY7 (Clone 53-6.7; Cat#557654), anti-mouse Ki67-PerCP-CY5.5 (Clone 11F6; Cat#50-237-5907)) at room temperature for 0.5 h. Cells were then washed with PBS and finally remixed with 200 μL of PBS. The data was analyzed using FlowJo software.

[0047] 7. T proliferation detection experimental procedure:

[0048] Cell staining: T cells were suspended in PBS and the cell concentration was adjusted to 1×10⁶. 6 Cells / mL. Prepare CTV working solution according to experimental needs. Typically, the final working concentration of CTV is 5 μM, dissolved in sterile PBS. Add the CTV working solution to the cell suspension, ensuring uniform dye distribution. Stain cells with 5 μM CTV dye and incubate for 25 min. After staining, add cold PBS or culture medium and centrifuge to remove the CTV solution. Wash cells 2-3 times to remove unbound CTV. Finally, culture in complete medium containing 50 U / mL interleukin-2 (IL-2) for 4 days.

[0049] 8. Experimental Results:

[0050] Mouse spleen T cells were isolated and encoded with Cdca5Gene (CDCA5) OE The expression of CDCA5 was transduced. Successful overexpression of CDCA5 was confirmed at both the protein and mRNA levels. When co-cultured with IL-2 in vitro, CDCA5... OE -T cells exhibited enhanced proliferative capacity, manifested as increased cell count, a higher proportion of cells with low CellTrace Violet (CTV) expression, and upregulated Ki67 expression. Specifically, compared to the control group, CD4+... + CDCA5 OE -T cells and CD8 + CDCA5 OE The proportion of Ki67-positive cells was significantly increased in all T cells. Cell cycle analysis further showed that CDCA5 OE The proportion of T cells in the G2 / M phase increased. These results collectively indicate that CDCA5 promotes T cell proliferation. To further investigate the effect of CDCA5 on T cell survival, we performed live / dead cell staining experiments and found that CDCA5-overexpressing T cells exhibited a higher survival rate compared to control T cells. Annexin V and propidium iodide (PI) staining showed that CDCA5... OE - The apoptosis rate of T cells decreased. Consistent with this, overexpression of CDCA5 inhibited the expression of the pro-apoptotic protein Bax while upregulating the expression of the anti-apoptotic protein BCL-2. These results suggest that CDCA5 can enhance the proliferation and survival of T cells.

[0051] Example 2:

[0052] CDCA5 promotes T cell activation and effector function:

[0053] 1. Flow cytometry detection of T cell activation and function:

[0054] Collect cells, wash them with 1 mL of PBS, and then incubate them with a dead / live cell dye (Zombie Dye) to remove dead cells. Following this step, the cells were treated with all relevant antibodies (anti-mouse CD3-APC (Clone17A2; Cat#100235), anti-mouse CD3-APC-Cy7 (Clone 17A2; Cat#100222), anti-mouse CD4-PerCP-CY5.5 (Clone GK1.5; Cat#100434), anti-mouse CD4-APC-Cy7 (Clone GK1.5; Cat#100414), anti-mouse CD8-PE (Clone 53-6.7; Cat#100708), anti-mouse CD8α-APC-CY7 (Clone 53-6.7; Cat#557654), anti-mouse Ki67-PerCP-CY5.5 (Clone 11F6; Cat#50-237-5907), anti-mouse CD62L-PE (Clone 17A2; Cat#100235), anti-mouse CD3-APC-Cy7 (Clone 17A2; Cat#100222), anti-mouse CD4-PerCP-CY5.5 (Clone GK1.5; Cat#100434), anti-mouse CD4-APC-Cy7 (Clone GK1.5; Cat#100414), anti-mouse CD8-PE (Clone 53-6.7; Cat#557654), anti-mouse Ki67-PerCP-CY5.5 (Clone 11F6; Cat#50-237-5907), anti-mouse CD62L-PE (Clone 17A2; Cat#100235), anti-mouse CD3-APC-Cy7 (Clone 17A2; Cat#100232), anti-mouse CD62L-PE (Clone 17A2; Cat#100232), anti-mouse Cells were stained with the following anti-mouse / human CD44-APC (Clone IM7; Cat#103012), anti-mouse IFN-γ-APC (Clone XMG1.2; Cat#505810), anti-mouse IFN-γ-PE (Clone XMG1.2; Cat#05808), anti-mouse / human GZMB-BV421 (Clone GB11; Cat#563389), anti-mouse CD25-PerCP-CY5.5 (Clone 3C7; Cat#101912), and anti-mouse TNF-α-PE-CY7 (Clone MP6-XT22; Cat#506324) at room temperature for 0.5 h. Cells were then washed with PBS and remixed with 200 μL of PBS. Data were analyzed using FlowJo software.

[0055] 2. Experimental Results:

[0056] In total CDCA5 OE Increased expression of the activation marker CD25 was detected in T cells, and this phenomenon was also observed in the CD4⁺ and CD8⁺ subsets. Furthermore, CDCA5 overexpression significantly reduced expression in Tn cells (CD62). + CD44- The proportion of ) increased, while Teff cells (CD62) were also increased. - CD44 + The proportions of CDCA5 in total T cells, CD4⁺ T cells, and CD8⁺ T cells indicate that CDCA5 promotes T cell activation and differentiation into effector phenotypes. Results showed that, compared to the control group, CDCA5... OE -T cells significantly increased the production of key antitumor effector molecules TNF-α, IFN-γ, and GZMB. To further evaluate antigen-specific responses, CDCA5 was generated from ovalbumin (OVA)-specific TCR transgenic OT-1 mice. OE -T cells, CDCA5 OE The levels of TNF-α, IFN-γ, and GZMB produced by OT-1 cells were higher than those in the control group.

[0057] Example 3:

[0058] CDCA5 enhances T cell proliferation and effector function through the PI3K / AKT / mTOR pathway:

[0059] 1. RNA-seq

[0060] T cells isolated from the spleen were stimulated for 2 days with anti-mouse CD3 antibody (5 μg / mL), anti-mouse CD28 antibody (2.5 μg / mL), and recombinant mouse IL-2 (50 U / mL), followed by the use of encoding... Cdca5 Gene (CDCA5) OE Cells were transduced using either a retrovirus or an empty vector. Cells were collected on day 4, and total RNA was extracted using TRIzol reagent. RNA-Seq data analysis was performed by Beijing Qingke Biotechnology Co., Ltd.

[0061] 2. Western blotting (WB) was used to detect the expression levels of relevant proteins.

[0062] Preparing the adhesive: Prepare the lower layer of adhesive (10%), pour it into the glass plate, press it down with isopropyl alcohol, and allow it to solidify. Prepare the upper layer of adhesive, pour it onto the solidified lower layer of adhesive, install the comb, and allow it to solidify naturally.

[0063] Electrophoresis: Fix the gel plate, add the electrophoresis solution, remove the comb, and add the standard marker and sample. Set the electrophoresis voltage to 90V for 30 minutes and 120V for 60 minutes.

[0064] Transfer: Prepare the transfer clamp, construct the sandwich structure, add the moistened PVDF membrane, and install the transfer tank. Set the voltage to 200mA, transfer for 60 minutes, and then cool to an ice bath.

[0065] Sealing: Clean the PVDF membrane after transfer, place it in the sealing solution, and seal it on a shaker for 20 minutes.

[0066] Primary antibody incubation: After washing the membrane, add preheated primary antibody solution and incubate overnight at 4°C. Rabbit anti-phospho-PI3 Kinase (Cat#17366, CST), rabbit anti-Akt (Cat#9272, CST), rabbit anti-phospho-Akt (Cat#4060, CST), rabbit anti-mTOR (Cat#F0169, Selleck), rabbit anti-phospho-mTOR (Cat#F2584, Selleck), mouse anti-b-actin (Cat#4970, CST).

[0067] Secondary antibody incubation: After washing the membrane, add the secondary antibody solution and incubate on a shaker for 1 hour. HRP goat anti-mouse IgG (Cat#LF101, Epizyme), HRP goat anti-rabbit IgG (Cat#LF102, Epizyme)

[0068] Development: Mix the developer solution, apply it to the membrane, and develop until the bands appear.

[0069] 3. Flow cytometry detection of T cell proliferation and survival:

[0070] Cells were collected, washed with 1 mL of PBS, and then incubated with a dead / live cell dye (Zombie Dye) to remove dead cells. Following this step, cells were stained with all relevant antibodies (anti-mouse CD3-APC-Cy7 (Clone17A2; Cat#100222), anti-human / mouse Phospho-mTOR (Cat#25-9718-42, eBioscience), anti-human / mouse Phospho-4EBP1 (Cat#740048M, ThermoFisher)) at room temperature for 0.5 h. Cells were then washed with PBS and finally remixed with 200 μL of PBS. Data were analyzed using FlowJo software.

[0071] 4. Experimental Results:

[0072] Control group T cells and CDCA5 OE RNA sequencing analysis was performed on -T cells. CDCA5 OEThe transcriptional profile of T cells differed significantly from that of control T cells. Differential expression analysis showed that CDCA5... OE - T cells activate related genes (including Cd69 and Cd44 , Il2ra and Il2rg) and effect-related genes ( Gzmb and Ifng The expression levels of CDCA5 were elevated. In summary, in addition to its classic role in cell cycle regulation, this study revealed a novel function of CDCA5 in promoting T cell activation and effector function. To further elucidate the molecular mechanisms of CDCA5-mediated T cell proliferation and enhanced effector function, we performed KEGG pathway analysis, and the results showed that CDCA5... OE -T cells show upregulated expression of the PI3K-AKT signaling pathway. It is known that activation of the PI3K-AKT pathway further triggers mTOR kinase activity. Gene set enrichment analysis (GSEA) results further confirm this. OE In -T cells, both the PI3K-AKT and mTOR signaling pathways were significantly enriched. Western blotting and flow cytometry analysis revealed that CDCA5 overexpression increased the phosphorylation levels of PI3K, AKT, and mTOR, but did not alter the total protein expression. Furthermore, CDCA5... OE The phosphorylation level of 4EBP1, a key substrate of mTORC1, was also significantly increased in -T cells. These results collectively indicate that CDCA5 can enhance the activity of the PI3K / AKT / mTOR signaling pathway.

[0073] Example 4:

[0074] CDCA5 enhances mTOR-mediated metabolic processes:

[0075] 1. ECAR

[0076] ECAR was measured using a Seahorse XF96 extracellular throughput analyzer (Agilent Technologies). In short, cells were collected and resuspended in unbuffered assay medium at pH 7.4, then seeded onto poly-L-lysine-pretreated XF96 microplates and incubated in a non-CO2 incubator for 30 minutes before measurement. ECAR was measured using the XF Glycolysis Stress Assay Kit (catalog number #103020-100, Agilent Technologies, USA). After baseline recording, 10 mM glucose, 1 μM oligomycin A, and 50 mM 2-DG were added sequentially to the microplates.

[0077] 2. Flow cytometry for T cell function detection:

[0078] Control group T cells and CDCA5OE -T cells were treated for 2 days in the presence or absence of 50 nM rapamycin. Cells were collected, washed with 1 mL PBS, and then incubated with a dead / live cell dye (Zombie Dye) to remove dead cells. Following this step, cells were stained with all relevant antibodies (anti-mouse CD3-APC (Clone 17A2; Cat#100235), anti-mouse Ki67-PerCP-CY5.5 (Clone 11F6; Cat#50-237-5907), anti-mouse IFN-γ-APC (Clone XMG1.2; Cat#505810), anti-mouse IFN-γ-PE (CloneXMG1.2; Cat#05808), anti-mouse / human GZMB-BV421 (Clone GB11; Cat#563389), and anti-mouse TNF-α-PE-CY7 (Clone MP6-XT22; Cat#506324)) at room temperature for 0.5 h. Cells were then washed with PBS and finally remixed with 200 μL of PBS. Data were analyzed using FlowJo software.

[0079] 3. Experimental Results:

[0080] RNA sequencing analysis showed that glycolysis-related genes (such as...) in CDCA5OE-T cells were present. Hk2 , Ldha and Pkm The expression level of CDCA5 was significantly increased. Seahorse analysis consistently showed that CDCA5 overexpression significantly enhanced glycolysis, manifested as an increased extracellular acidification rate (ECAR). To investigate whether the enhanced PI3K / AKT / mTOR signaling pathway mediates CDCA5-driven T cell proliferation and effector function, we treated CDCA5-transduced or empty vector-derived T cells with the mTOR inhibitor rapamycin. The results showed that rapamycin treatment significantly inhibited the upregulation of Ki67, TNF-α, IFN-γ, and GZMB expression induced by CDCA5. These results indicate that CDCA5 enhances T cell proliferation and effector function by activating the PI3K / AKT / mTOR signaling pathway.

[0081] Example 5:

[0082] CDCA5 enhances the efficacy of ACT:

[0083] 1. Adoptive transfer experiment

[0084] MC38-OVA cells (1×10⁻⁶) 6OT-1 (cells / mouse) was subcutaneously injected into the right axilla of 6-week-old C57BL / 6J mice. On day 6 post-injection, tumor-bearing mice were randomly divided into two groups to receive OT-1 treatment (1.5 × 10⁻⁶ cells / mouse). 6 Cells / mouse). Cells were preactivated with OVA peptide for 24 hours, then treated with recombinant mouse IL-2 (50 U / mL), followed by transduction of the encoding... Cdca5 Gene retrovirus (CDCA5) OE (or empty vector) were used for amplification over 3 days. Tumor size was measured every 3 days using calipers, calculated as (length × width) 2 ) / 2. On day 21 post-tumor inoculation, Tetramer-positive T cells were isolated from the tumor tissue and analyzed by flow cytometry.

[0085] 2. Flow cytometry for T cell function detection:

[0086] Collect cells, wash them with 1 mL of PBS, and then incubate them with a dead / live cell dye (Zombie Dye) to remove dead cells. Following this step, the cells were treated with all relevant antibodies (anti-mouse CD45.2-APC-CY7 (Clone 104; Cat#109824), anti-mouse CD3-APC (Clone 17A2; Cat#100235), anti-mouse CD3-APC-Cy7 (Clone 17A2; Cat#100222), anti-mouse Ki67-PerCP-CY5.5 (Clone 11F6; Cat#50-237-5907), anti-mouse IFN-γ-APC (Clone XMG1.2; Cat#505810), anti-mouse IFN-γ-PE (Clone XMG1.2; Cat#05808), anti-mouse / human GZMB-BV421 (Clone GB11; Cat#563389), anti-mouse TNF-α-PE-CY7 (Clone 104; Cat#563389), and anti-mouse TNF-α-PE-CY7 (Clone 104; Cat#109824). MP6-XT22 (Cat#506324) cells were stained at room temperature for 0.5 h. Cells were then washed with PBS and finally remixed with 200 μL of PBS. Data were analyzed using FlowJo software.

[0087] 3. Experimental Results:

[0088] Cells isolated from the spleen of OT-1 mice were activated with initial OVA peptides and then transduced into CDCA5 or empty vectors, respectively, and co-cultured with IL-2 for 3 days. These cells were then adoptively transferred into mice carrying B16-OVA. Compared with control OT-1 cells, CDCA5 cells showed significantly higher levels of activity. OE -OT-1 cell treatment significantly delayed B16-OVA tumor growth. Consistently, we observed this in patients receiving CDCA5. OE An increased number of OVA-specific T cells and elevated Ki67 expression were detected in the mouse tumor microenvironment of -OT-1 cells, indicating that CDCA5 enhances the persistence and proliferation of T cells in vivo. Furthermore, tumor-infiltrating OVA-specific CDCA5... OE -T cells exhibit a stronger ability to produce cytokines. Similarly, CDCA5 OE -OT-1 cells more effectively controlled MC38-OVA tumor growth, accompanied by enhanced proliferation and effector function of tumor-infiltrating T cells. These results collectively demonstrate that CDCA5 can enhance the anti-tumor immune response of T cells in vivo.

[0089] Example 6:

[0090] CDCA5 overexpression enhances the anti-tumor ability of CAR-T cells in vitro:

[0091] 1. Design of CARs overexpressing CDCA5 and tNGFR to resist human CD19:

[0092] The CDCA5 and tNGFR gene sequences were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0093] The CDCA5 gene sequence is shown in SEQ ID NO.2 of the sequence listing;

[0094] The tNGFR gene sequence is shown in SEQ ID NO.3 of the sequence listing;

[0095] 2. Preparation of bacterial culture containing CAR gene sequence:

[0096] Seamless cloning technology was used to insert CDCA5 and tNGFR gene sequence fragments into the lentiviral vector pCDH-CD19-CAR, and bacterial cultures with the correct CAR sequences were selected by sequencing.

[0097] Seamless cloning: Using homologous recombinase, CAR gene sequence fragment, and double-distilled water, the linearized pCDH-CD19-CAR vector was digested by Not I restriction endonuclease. The reaction system was 10 μL: 2 μL homologous recombinase, 50 ng CAR gene sequence fragment, 100 ng linearized pCDH-CD19-CAR vector, and the rest was made up to 10 μL with double-distilled water. The temperature was controlled at 50℃ and the reaction time was 60 min to obtain the seamless cloning product.

[0098] Transformation and plating: Remove competent cells (DH5α) from -80℃ and immediately place them on ice to thaw. Take 5 μL of seamless cloning product and add it to the pre-thawed competent cells. Mix gently and place on ice for 30 min. Heat shock at 42℃ for 90 s without shaking the test tube, and immediately stop the ice bath for 5 min.

[0099] Add 200 μL of antibiotic-free LB liquid medium to each tube and incubate at 37°C and 150 rpm for 30 min with shaking. Spread 100 μL of bacterial culture onto the surface of an LB agar plate containing the appropriate antibiotic (Amp) using sterile glass beads, incubate at 37°C upright for 30 min, and then invert overnight.

[0100] Picking and sequencing: Randomly pick several plaques from LB plates and place them into 5 mL of ampicillin-resistant LB liquid medium in a shaker. Shake the plates at 37°C and 200 rpm for 10-12 h until the bacterial culture becomes turbid. Take 100 μL of each plate for sequencing. Compare the sequencing results with the CAR gene sequence. The bacterial culture with the correct sequence is the CAR gene sequence bacterial culture.

[0101] 3. Preparation of lentiviruses containing the CDCA5 and tNGFR genes:

[0102] After plasmid extraction, lentiviruses were used for packaging, and the pCDH-CD19-CAR vector containing the CDCA5 and tNGFR genes was co-transfected with helper plasmids pMD2.G and pSPAX2 to obtain viral particles.

[0103] Plasmid extraction: Take 100 μL of bacterial culture containing the target sequence and put it into 200 mL of LB liquid medium. Control the temperature at 37℃ and 200 rpm and shake the culture on a shaker. After 12 h, extract the pCDH-CD19-CAR vector containing the target sequence, including pMD2.G and pSPAX2.

[0104] Virus Packaging: One day before transfection, passage 293T cells 2:5 into 10cm dishes and incubate at 37°C with 5% CO2. Transfection can be performed when the cell density reaches 80%–90%. 1–2 hours before transfection, replace the cell culture medium with 6 mL of pre-warmed complete medium. Add 300 μL of jetPRIME buffer and the corresponding mass of plasmid (pMD2g:psPAX2:PCDH-CAR-CD19 = 1:3:4, total 12 μg) to a sterile 1.5 mL EP tube A. Mix well, then add 25 μL of jetPRIME and gently shake. Incubate at room temperature for 10 min. Transfer the mixture to the 293T cell culture medium, mix well, and incubate at 37°C with 5% CO2. After 8 hours, replace with 10 mL of pre-warmed complete medium. Collect the viral supernatant at 48 h and 72 h. The collected supernatant was filtered through a 0.45 μm (PES) membrane filter to remove cell residues.

[0105] 4. Preparation of human anti-CD19 CAR-T cells:

[0106] Antibody plating: Anti-human CD3 / CD28 antibody plating. Add antibody to PBS to a final concentration of 5 μg / mL anti-human CD3 and 2.5 μg / mL anti-human CD28. Add 500 μL to each well of a 12-well plate, seal with tape, and incubate overnight at 4°C.

[0107] PBMC isolation and plating: Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using density gradient centrifugation with Ficoll. Fresh blood was diluted with phosphate-buffered saline (PBS), plated onto Ficoll plates, and centrifuged at 500×g for 30 min at room temperature. The mononuclear cell layer was collected, washed with 1×PBS, and cell counts were determined using a Countstar automated cell counter (Shanghai, China). PBMCs (1×10⁶ cells / well) were seeded and transduced after 24 h.

[0108] PBMC Infection and Culture: Activated T cells were co-transduced with lentivirus in a medium containing 8 μg / mL polybrene and an MOI of 10. To improve transduction efficiency, plates were centrifuged at 800 × g for 1.5 h at 37°C, and the medium was changed 6–8 h post-infection. Transduced lymphocytes were maintained in medium supplemented with IL-2 (200 IU). On day 4 post-transduction, GFP was used for cell culture by flow cytometry. + Quantitative CAR expression as a reporter gene.

[0109] 5. Raji stimulation of CAR-T cells by tumor cells:

[0110] CAR-T cells (2×10) 4 ) with Raji (2×10 4 They were cultured in 96-well U-shaped plates for 12 hours at an E:T ratio of 1:1.

[0111] 6. Flow cytometry detection of CAR-T cell effector function:

[0112] Cells were collected, washed with 1 mL of PBS, and then incubated with a dead / live cell dye (Zombie Dye) to remove dead cells. Following this step, cells were stained with all relevant antibodies (APC / Cy7 anti-human CD3 (clone UCHT1; 300470); PE / Cyanine7 anti-human CD8 (clone SK1; 344712); APC anti-human IFN-γ (clone 4S.B3; 502512); PE anti-human TNF-α (clone MAb11; 502909)) at room temperature for 0.5 h. Cells were then washed with PBS and finally remixed with 200 μL of PBS. Data were analyzed using FlowJo software.

[0113] 7. Cytotoxicity of CAR-T cells:

[0114] Cell staining and plating: Raji cells, Daudi cells, and SU-DHL-4 cells were suspended in PBS and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells / mL. Prepare CTV working solution according to experimental needs. The final working concentration of CTV is 5 μM, dissolved in sterile PBS. Add the CTV working solution to the cell suspension, ensuring uniform dye distribution. Stain cells with 5 μM CTV dye and incubate for 20 min. After staining, add cold PBS or culture medium and centrifuge to remove the CTV solution. Wash cells 2-3 times to remove unbound CTV. Co-culture for 24 h using different effector-target ratios (0.25:1, 0.5:1, 1:1).

[0115] Flow cytometry was used to detect the survival and mortality rates of target cells.

[0116] After co-culture, 7-AAD (420404, BioLegend, USA) was added, and the samples were analyzed by flow cytometry. Cytotoxicity was calculated using the following formula:

[0117] (Percentage of 7-AAD+ in the experimental group - Percentage of 7-AAD+ in the control group) / (100% - Percentage of 7-AAD+ in the control group).

[0118] 8. ELISA detection of CAR-T cell cytokines:

[0119] Sample preparation: Collection of supernatant: After 24 hours of cell culture, the culture supernatant is collected for ELISA analysis.

[0120] Add sample: Add 100 µL of the collected CAR-T cell supernatant to each well of the ELISA plate. Choose an appropriate dilution factor based on the expected concentration range.

[0121] Incubation: Incubate at room temperature for 1-2 hours to allow cytokines to bind to the capture antibody.

[0122] Washing: After incubation, wash the plate with PBST to remove unbound samples.

[0123] Add detection antibody: Add 100 µL of detection antibody that binds to the target cytokine, i.e., HRP (horseradish peroxidase) labeled antibody, to each well. Incubate for 1 h.

[0124] Washing: Wash the plate again with PBST to remove unbound detection antibodies.

[0125] Substrate reaction: Add substrate solution (TMB, tetramethylbenzidine). The substrate will bind with HRP to produce a visible color reaction. Incubate at room temperature for 20 minutes.

[0126] Stop the reaction: Add a stopping solution (sulfuric acid) to terminate the reaction.

[0127] Absorbance measurement: The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0128] Data analysis: Calculate cytokine concentrations based on a standard curve. The standard curve is prepared using standards of known concentrations.

[0129] 9. Experimental Results:

[0130] With tNGFR OE Compared to CAR-T cells, CDCA5 OE CAR-T cells exhibited stronger proliferative capacity, specifically manifested in increased cell count, a higher proportion of CTVlow cells, and an increased proportion of Ki67-positive cells. Furthermore, CDCA5... OE -The apoptosis rate of CAR-T cells was also reduced.

[0131] When exposed to Raji cells expressing CD19, CDCA5 OECAR-T cells showed increased expression of activation markers CD69 and CD25, and produced higher levels of effector molecules TNF-α, IFN-γ, IL-2, and GZMB, along with tNGFR. OE Compared to CAR-T cells, ELISA analysis further confirmed the role of these effector molecules in CDCA5. OE - Increased secretion in the supernatant of CAR-T co-culture. Consistently, when co-cultured with Raji cells, CDCA5... OE -CAR-T cells exhibited enhanced cytotoxicity. Overall, these results indicate that CDCA5 overexpression improves CAR-T cell proliferation, survival, and effector function.

[0132] Example 7:

[0133] CDCA5 overexpression enhances the anti-tumor function of CAR-T cells in vivo:

[0134] 1. Mouse experiment:

[0135] The experiment used the same amount of 6- to 8-week-old NOD / ShiLtJGpt- Prkdc em26Cd52 Il2rg em26Cd22 / Gpt(NCG) male mice, with an initial weight of 20-25 grams, were obtained from the Shanghai Model Biology Center (Shanghai, China). All animal experiments strictly followed the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Ethics Committee of Anhui Medical University (Approval No.: LLSC20211220).

[0136] 2. In vivo experimental model:

[0137] On day 0, Raji-Luc cells (5 × 10⁻⁶) were... 5 (By intravenously injecting NCG into NCG mice) to establish a tumor model. On day 7, CAR-T cells (1×10⁻⁶) were injected into the mice. 6 The drug was administered intravenously to assess its therapeutic effect. Tumor burden and survival in mice were monitored every 3-4 days using bioluminescence imaging (BLI) until the end of the experiment. Tumor growth and mouse survival data were recorded at each time point.

[0138] 3. Experimental Results:

[0139] We then evaluated CDCA5. OE- The antitumor efficacy of CAR-T cells in vivo. In short, Raji cells expressing luciferase (Raji-luc) were intravenously injected into immunodeficient NCG mice, followed by adoptive transfer using a suboptimal dose of CAR-T cells. Encouragingly, the efficacy of CAR-T cells received from CDCA5... OE Mice receiving CAR-T cells showed significantly improved survival and superior tumor control, which was validated by bioluminescent radiation. The clinical application of CAR-T therapy is often limited by toxic side effects. Interestingly, overexpression of CDCA5 appears to alleviate these toxicities; compared to the control group, mice receiving CDCA5 showed improved survival rates. OE Mouse CAR-T cells showed minimal weight loss. Consistent with the enhanced therapeutic effect, CDCA5 overexpression also promoted CAR-T cell persistence and increased CD62L levels in peripheral blood. - CD45RO - The proportion of effector subsets. To further explore the clinical significance of CDCA5 expression, we analyzed single-cell RNA sequencing data from B-cell malignancies treated with anti-CD19 CAR-T therapy. This analysis showed that higher CDCA5 expression levels in pretreated T cells were associated with improved overall survival after CAR-T therapy. In summary, these results indicate that CDCA5 overexpression can enhance the antitumor efficacy of CAR-T cells in vivo, potentially representing a promising strategy for improving the efficacy of CAR-T cell therapy.

[0140] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of overexpression of CDCA5 in the preparation of antitumor drugs that enhance the function of T cells, characterized in that, The method for overexpressing CDCA5 involves using a CDCA5 overexpression agent, which is a vector for overexpressing CDCA5, and the vector for overexpressing CDCA5 is used to transfect T cells.

2. The application according to claim 1, characterized in that: The vector overexpressing CDCA5 can be any one of lentivirus or retrovirus.

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