Immune cells with enhanced anti-tumor effect and preparation method and application thereof

Abstract

This invention discloses an immune cell with enhanced anti-tumor effect, its preparation method, and its application. Experiments using this invention show that... SERTAD1 CD8 gene overexpression + T cells can enhance their function in anti-tumor immunotherapy. SERTAD1 Gene overexpression can downregulate CD8 + The expression of immune checkpoint molecules on T cells upregulates the expression of mRNAs of tumor-killing cytokines. In the MC38-OVA subcutaneous tumor model, Naïve CD8 cells were extracted from OT-I mice. + T cells were infected with a virus to overexpress SERTAD1, followed by adoptive transfer to mice. The results showed that SERTAD1 overexpression enhanced CD8 expression. + T cells kill MC38-OVA cells, inhibit the growth of MC38-OVA tumor cells, and overexpress SERTAD1 CD8. + The tumor volume and weight in the T-cell adoptive group were significantly smaller than those in the control group.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an immune cell with enhanced anti-tumor effects, its preparation method, and its application. Background Technology

[0002] Malignant tumors are caused by the malignant proliferation of cells, and their incidence and mortality rates are rising year by year, becoming a major public health problem that seriously threatens human life and health. Traditional treatment methods directly target tumor cells, including surgery, radiotherapy / chemotherapy, and targeted therapy. Among these, surgery is mainly suitable for early-stage patients, while radiotherapy / chemotherapy, due to its lack of specific targeting, often causes significant damage to normal tissues such as bone marrow hematopoietic system, digestive tract mucosa, and hair follicles while killing tumor cells, seriously affecting patients' quality of life. In addition, although targeted therapy has specific targeting, it faces significant bottlenecks in drug resistance, has a narrow applicable population, and cannot activate a systemic anti-tumor immune response. In contrast, as a novel treatment method, immunotherapy utilizes the patient's own immune system to kill tumor cells, which not only has advantages such as specific targeting, low tissue toxicity, and broad indications, but also long-lasting effects and synergistic potential.

[0003] The core mechanism of immunotherapy lies in activating tumor-specific T cell function (such as enhancing CD8). + Immunotherapy aims to restore the body's immune surveillance function against tumors by addressing the cytotoxicity of effector T cells and relieving the immunosuppressive microenvironment (e.g., by blocking the immunosuppressive effects of regulatory T cells). However, current clinical applications of immunotherapy still face challenges: 1) Immune cells possess multiple immune checkpoints, while most existing immune checkpoint inhibitors target only a single checkpoint; 2) Significant immune heterogeneity exists among different cancers, leading to varying therapeutic effects of the same drug in different cancers; 3) Tumor cells can suppress T cell function by upregulating multiple immune checkpoint molecules (such as TIM-3 and LAG-3). Therefore, effectively reducing the expression of immune checkpoint molecules in T cells and preventing the inhibitory effect of tumor cells on T cell function is a key issue that urgently needs to be addressed in the field of immunotherapy.

[0004] To address the aforementioned issues, a 2015 study by Ho PC, Bihuniak JD, and others in Cell found that increasing phosphoenolpyruvate (PEP) in T cells by overexpressing phosphoenolpyruvate carboxykinase 1 (PCK1) can improve T cell exhaustion. Furthermore, adoptive T cell therapy, which involves reinfusing PCK1-overexpressing T cells into mice, can achieve significant anti-tumor effects. [Ho PC, Bihuniak JD, Macintyre AN, et al. Phosphoenolpyruvate Is a Metabolic Checkpoint of Anti-tumor T Cell Responses.] Cell .2015;162(6):1217-1228.】. Existing research has found SERTAD1 The gene has a role in promoting cancer in prostate cancer [Hu B, Hu H, Yin M, et al. Sertad1 promotes prostate cancer progression through binding androgen receptor ligandbinding domain. Int J Cancer. 2019 Feb 1;144(3):558-568.] and breast cancer [Nguyen HA, Vu SH, Jung S, et al. SERTAD1 Sensitizes Breast Cancer Cells to Doxorubicin and Promotes Lysosomal Protein Biosynthesis. Biomedicines. 2022 May 17;10(5):1148.]. However, regarding its role in prostate cancer [Hu B, Hu H, Yin M, et al. Sertad1 promotes prostate cancer progression through binding androgen receptor ligandbinding domain. Int J Cancer. 2019 Feb 1;144(3):558-568.], the role of SERTAD1 in promoting cancer in breast cancer [Nguyen HA, Vu SH, Jung S, et al. SERTAD1 Sensitizes Breast Cancer Cells to Doxorubicin and Promotes Lysosomal Protein Biosynthesis. Biomedicines. 2022 May 17;10(5):114 ...]. SERTAD1 Whether genes can solve the above problems in immune cells and achieve the dual function of inhibiting tumor progression and activating anti-tumor immunity has not yet been reported. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an immune cell with enhanced anti-tumor effects, its preparation method, and its application, specifically an immune cell overexpressing SERTAD1, its preparation method, and its application. This invention constructs a SERTAD1-overexpressing CD8... + T cells revealed the dual function of SERTAD1 in inhibiting tumor progression and activating anti-tumor immunity, achieving significant results.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] In a first aspect, the present invention provides an immune cell that overexpresses SERTAD1.

[0008] In this invention, SERTAD1 is an oncogene (also known as SEI1 or TRIP-Br1), a cell cycle regulatory gene located at 19q13.1 to 19q13.2.

[0009] In this invention, SERTAD1 is treated / overexpressed / induced to be overexpressed in immune cells, which reduces the expression of immune checkpoint molecules on T cells and prevents tumor cells from inhibiting T cell function.

[0010] Preferably, the amino acid sequence of SERTAD1 is shown in SEQ ID NO.1.

[0011] Preferably, the nucleotide sequence of SERTAD1 is shown in SEQ ID NO.2.

[0012] Preferably, the immune cells are T cells.

[0013] Furthermore, the T cells are CD8. + T cells.

[0014] Secondly, the present invention also provides a method for preparing the above-mentioned immune cells, the method comprising overexpressing SERTAD1 in immune cells.

[0015] Preferably, the overexpression is performed via viral transduction.

[0016] Furthermore, the overexpression is performed using a viral vector; the viral vector is a lentiviral vector; the lentiviral vector is the lentiviral vector pMSCV-IRES-GFP II (addgene: #52107).

[0017] Thirdly, the present invention provides a pharmaceutical composition comprising the aforementioned immune cells.

[0018] Preferably, the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

[0019] Fourthly, the present invention provides the use of the above-described immune cells and / or the above-described pharmaceutical compositions in the preparation of the following products;

[0020] (a) Products used to treat tumors;

[0021] (b) Products that inhibit the proliferation and / or growth of tumor cells.

[0022] Preferably, the tumor is a solid tumor.

[0023] Preferably, the solid tumor is colon cancer.

[0024] Furthermore, the product is a medicine;

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention constructs a SERTAD1-overexpressing CD8 expression system. + T cells revealed the dual function of SERTAD1 in inhibiting tumor progression and activating anti-tumor immunity, achieving significant results. Experimental data showed that SERTAD1 overexpression of CD8... + T cells significantly inhibited the growth of MC38 tumor cells. In in vivo experiments, overexpression of SERTAD1 CD8 + The tumor volume and weight in the T-cell adoptive group were significantly smaller than those in the control group.

[0027] Meanwhile, SERTAD1 activates CD8 by downregulating immune checkpoint molecules. + The anti-tumor immune response of T cells and the expression of mRNA of tumor-killing cytokines were significantly upregulated.

[0028] Compared with existing technologies, this invention has significant advantages in elucidating the SERTAD1 mechanism and supporting the development of treatment strategies. By optimizing experimental procedures and shortening the experimental cycle, this invention reduces the number of experimental animals used and experimental costs, making it more environmentally friendly and economical.

[0029] In summary, this invention provides efficient and precise tools and methods for the research and treatment of anti-tumor therapies, and has significant scientific value and clinical application potential. Attached Figure Description

[0030] Figure 1 After SERTAD1 overexpression, CD8 + Protein expression levels of immune checkpoint molecules on T cells. Figure A shows the expression levels of Tight protein in CD8. + Flow cytometry analysis results of T cells; Figure B shows the Lag3 protein in CD8. + Flow cytometry analysis results of T cells; Figure C shows PD-1 protein in CD8. + Flow cytometry analysis results of T cells; Figure D shows Tim3 protein in CD8. + Flow cytometry analysis results of T cells.

[0031] Figure 2Figure A shows the expression of immune checkpoint molecules and tumor-killing cytokines mRNA after SERTAD1 overexpression. Figure B shows the expression of immune checkpoint molecule mRNA after 5 days of in vitro differentiation and the expression of tumor-killing cytokines mRNA after 8 days of in vitro differentiation.

[0032] Figure 3 Figure A shows the experimental results of the SERTAD1 mouse tumor adoptive cell therapy model. The upper part of Figure A represents the overall tumor change, while the lower part represents the tumor size change in each mouse. The dashed line is a simulated change line based on the tumor change data. Figure B is a direct camera image of the mouse tumor tissue. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the embodiments. Unless otherwise specified, all reagents or instruments used are considered to be conventional products that can be purchased on the market.

[0034] MC38-OVA cells were a cell line constructed in the laboratory through lentiviral infection. The cell line was cultured in RPMI 1640 medium (C3010-0500, Viva Cell) containing 10% fetal bovine serum (C29100500) and 1% penicillin and streptomycin. All cells were maintained in an incubator at 37°C and 5% CO2.

[0035] Six- to eight-week-old SPF-grade C57BL / 6J mice and six- to eight-week-old SPF-grade OT-I C57BL / 6J mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., and housed in the SPF-grade barrier facility of the Experimental Animal Center of Zhejiang University. A 12-hour light / dark cycle was maintained (light from 07:30 to 19:30, 5 mice per group, housed in individual ventilated cages; darkness from 19:30 to 07:30), with controlled room temperature (23±2℃) and relative humidity (40%~50%). Mice were marked before grouping and then randomly assigned to different groups by an independent person. Six mice were used in each group. The experimental groups were ensured to be balanced in terms of animal age and weight. All animal handling complied with the standards of the Experimental Animal Welfare and Ethics Review Committee of Zhejiang University.

[0036] Example 1: After SERTAD1 overexpression, CD8 + Protein expression levels of immune checkpoint molecules on T cells

[0037] Spleens and lymph nodes were extracted from C57BL / 6J mice and placed in a 70μM filter. Cells were then ground using a syringe tip to obtain a cell suspension. The suspension was centrifuged at 500×g for 5 minutes, the supernatant was removed, and 1 ml of erythrocyte lysis buffer was added to resuspend the cells. Erythrocytes were lysed at room temperature for 1 minute, followed by the addition of 9 ml of 1× PBS. The suspension was centrifuged at 500×g for 5 minutes, the supernatant was removed, and the cells were washed once with 10 ml of PBS while counting. The cells were then centrifuged at 500×g for 5 minutes, the supernatant was removed, and sorting buffer (PBS containing 0.5% bovine serum albumin and 2 mM EDTA) corresponding to the cell count was added. Cells were then sorted according to the Miltenyi mouse Naive CD8... + Following the instructions of the T Cell sorting kit (130-096-543), Naive CD8 was obtained through sorting. + T cells were resuspended in T cell culture medium and adjusted to a concentration of 0.5E6 / ml (basal medium was RIPM-1640, with the following additives and amounts: fetal bovine serum (FBS, FBS to RIPM-1640 basal medium volume ratio 10:90), 100 μM non-essential amino acids, 14 μM folic acid, 100 U / ml penicillin, 0.1 mg / ml streptomycin, 50 μM β-mercaptoethanol, vitamins, 2 mM L-glutamine, 0.66 mM aspartate / 0.24 mM arginine, 1 mM pyruvate, and 100 mM 4-hydroxyethylpiperazine ethanesulfonic acid). 200 μl of cells were placed in 96-well plates pre-coated with 2 μg / ml CD3 and CD28 antibodies and activated for one day. The next day, the cells were used with packaged pMSCV-IRES-GFP II (addgene: #52107)-SERTAD1 (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.1). (As shown in NO.2) Infect the cells with the virus. Remove 100 μl of culture medium, add 100 μl of virus solution, and centrifuge at 1000g at 37℃ for 1 hour. Then remove 100 μl of culture medium, add 100 μl of T cell culture medium, and transfer to a 96-well U-shaped plate. Add cytokines 2 ng / ml IL2, 20 ng / ml IL7, and 20 ng / ml IL5. Let the cells rest for two days. Then transfer the cells to a 96-well plate pre-coated with 2 μg / ml CD3 and CD28 antibodies for reactivation and differentiation for two days. After that, perform flow cytometry staining and flow cytometry sorting to detect immune checkpoint proteins on CD8. + T cell expression.

[0038] Flow cytometry results showed that CD8+ cells overexpressing the SERTAD1 gene... + The expression of PD-1, Tight, Lag3, and Tim3 immune checkpoint molecules on T cells was downregulated. Figure 1 ).

[0039] Example 2: Expression of mRNAs of immune checkpoint molecules and tumor-killing cytokines after SERTAD1 overexpression.

[0040] Naive CD8 was obtained by magnetic bead sorting according to the experimental scheme of Example 1. + T cells were infected with pMSCV-IRES-GFPII-SERTAD1 virus, allowed to rest for two days, and then activated for differentiation for two days. Cultured cells were collected, and RNA was extracted using the Ultrapure RNA Kit (Kangwei Century, Cat# CW0581M). DNA was then removed from the RNA using the FastKing cDNA First-Strand Synthesis Kit (Tiangen Biotech, Cat# KR116), followed by reverse transcription. Real-time quantitative PCR was then performed using Magic SYBR Green qPCR Mix (Maibo Biotech, Cat# M223) to detect the expression of immune checkpoint molecules' mRNA.

[0041] Naive CD8 was obtained by magnetic bead sorting according to the experimental scheme of Example 1. + T cells were infected with pMSCV-IRES-GFPII-SERTAD1 virus, rested for five days, and then activated to differentiate for two days to obtain the corresponding cells. mRNA was extracted from the cells, and cDNA was obtained by reverse transcription. The expression of mRNA of tumor killing-related cytokines was detected by qPCR.

[0042] qPCR results showed that ( Figure 2 ), SERTAD1 CD8 gene overexpression + The expression of mRNAs of T cell immune checkpoint molecules was downregulated, while the expression of mRNAs of tumor-killing cytokines was upregulated. These findings suggest that SERTAD1 may exert its inhibitory effect by downregulating CD8+. + T cell immune checkpoint molecules, increase the expression of tumor-killing cytokines, and promote CD8. + The anti-tumor immune response of T cells. This series of findings provides a theoretical basis and experimental support for further research on SERTAD1 as a gene with potential important value in cancer treatment, and also reveals new perspectives on its mechanism of action and therapeutic potential.

[0043] Example 3: Experimental results of a mouse adoptive cell therapy model of SERTAD1 for tumors

[0044] In C57BL / 6J mice, 1 million MC38-OVA cells were subcutaneously implanted into tumors, which were then allowed to grow to 4×4 mm. 2At that time, 1 million CD8 were performed. + T-cell adoption.

[0045] Adoption CD8 + T cells were obtained by extracting spleens and lymph nodes from OT-I mice and sorting them with magnetic beads according to the experimental protocol in Example 1 to obtain Naive CD8 cells. + T cells were infected with pMSCV-IRES-GFP II-SERTAD1 virus and pMSCV-IRES-GFP II control virus, and the cells rested for two days. Mice were continuously observed, and their physiological status and tumor progression were recorded. Fifteen days after inoculation with MC38-OVA cells, mice were euthanized, and tumor tissue was collected to further evaluate SERTAD1 overexpression of CD8. + The anti-tumor immune effect of T cells in the body.

[0046] By observing and recording the growth curve of the tumor ( Figure 3 A) and images after the tumor tissue was removed ( Figure 3 B), found that SERTAD1 overexpression of CD8 + T cells significantly inhibited tumor growth in the MC38-OVA subcutaneous tumor model. This finding strongly suggests that SERTAD1 exerts its anti-tumor immune function by regulating the expression of immune checkpoint molecules and enhancing the expression of tumor-killing cytokines.

[0047] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. An immune cell, characterized in that, The immune cell is a CD8 + T cell obtained by overexpressing SERTAD1; the amino acid sequence of the SERTAD1 is shown as SEQ ID NO.

1.

2. The immune cell of claim 1, wherein, The nucleotide sequence of SERTAD1 is shown in SEQ ID NO.

2.

3. A method of producing the immune cell according to claim 1 or 2, characterized in that, The method comprises overexpressing SERTAD1 in CD8 + T cells.

4. The method for preparing immune cells according to claim 3, characterized in that, The overexpression is achieved through viral transduction.

5. The method for preparing immune cells according to claim 4, characterized in that, The overexpression was performed using a viral vector. The viral vector is a lentiviral vector; The lentiviral vector is the lentiviral vector pMSCV-IRES-GFP II.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the immune cells as described in claim 1 or 2.

7. The use of the immune cells of claim 1 or 2 or the pharmaceutical composition of claim 6 in the preparation of a medicament for treating a tumor, wherein the tumor is colon cancer.

Images