Application of THP in preparation of medicine for enhancing anti-tumor curative effect of V [gamma] 9V [delta] 2 T cells

Abstract

The invention relates to application of THP in preparation of a medicine for enhancing the anti-tumor curative effect of Vgamma9Vdelta2T cells, and belongs to the technical field of biological medicine. The invention finds that the surface marker MICA / B of tumor cells treated by THP is obviously enhanced, and the tumor cells induced by THP can enable Vgamma9Vdelta2T cells to highly express NKG2D. The NKG2D / MICA / B pathway is a classical pathway for playing a killing role in the Vgamma9Vdelta2T cells. THP can significantly enhance the liver cancer cell killing ability of Vgamma9Vdelta2T cells by regulating and controlling the NKG2D / MICA / B pathway, and a new therapeutic scheme is provided for TACE surgical treatment of clinical cancer patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to application of THP in the preparation of a drug for enhancing the anti-tumor efficacy of Vγ9Vδ2 T cells. Background Art

[0002] Transcatheter arterial chemoembolization (TACE) is the first choice for advanced hepatocellular carcinoma (HCC). The use of chemotherapy drugs such as pirarubicin (THP) during TACE can directly damage DNA and induce cell apoptosis. The ischemia and hypoxia caused by embolism affect the DNA synthesis of tumor cells, thereby promoting tumor cell necrosis, activating T lymphocyte responses, and changing the tumor immune microenvironment. However, many patients still have a poor prognosis after TACE. Therefore, other treatment methods are urgently needed to treat liver cancer in combination with TACE surgery.

[0003] Studies have found that allogeneic Vγ9Vδ2 T cells can improve the prognosis of some HCC patients, and a high ratio of Vγ9Vδ2 T cells in the peripheral blood of patients before TACE indicates a good prognosis. In the tumor microenvironment of HCC, it was found that γδT cells can recognize the stress-induced molecules MICA / B expressed by tumor cells through the NKG2D receptor expressed on the surface, exerting anti-tumor effects, and the Vγ9Vδ2 T cell subset in γδT cells has the potential to be used as a means of cell immunotherapy and play an important role in the treatment of HCC. However, the anti-tumor effect of Vγ9Vδ2 T cells is currently weak, and further research is urgently needed to increase the anti-tumor effect of Vγ9Vδ2 T cells and exert their function to better improve the prognosis of HCC patients. Summary of the invention

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides application of THP in preparing a medicine for enhancing the anti-tumor efficacy of Vγ9Vδ2 T cells.

[0006] Preferably, the tumor includes one or more of liver cancer, malignant lymphoma, breast cancer, ovarian cancer, uterine cancer, gastric cancer and bladder cancer.

[0007] Preferably, the effector-target ratio of Vγ9Vδ2 T cells to tumor cells is 0.5-12:1.

[0008] Preferably, the THP is used in solution.

[0009] Preferably, the THP solution comprises THP and iodized oil; the mass volume ratio of THP to iodized oil is 2 μg:1-100 μL.

[0010] The invention provides an anti-tumor drug, comprising separately packaged THP and Vγ9Vδ2 T cells.

[0011] The present invention has the following technical effects and advantages:

[0012] The present invention provides the use of THP in the preparation of a drug for enhancing the anti-tumor efficacy of Vγ9Vδ2 T cells. The present invention finds that the surface markers MICA / B of tumor cell lines treated with THP are significantly enhanced, and THP-induced tumor cells can cause Vγ9Vδ2 T cells to highly express NKG2D. The NKG2D / MIC A / B pathway is a classic pathway for Vγ9Vδ2 T cells to exert their killing effect. THP can significantly enhance the ability of Vγ9Vδ2 T cells to kill liver cancer cells by regulating the NKG2D / MICA / B pathway, providing a new treatment plan for TACE surgery in clinical cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Flow cytometry was used to detect the killing effect of Vγ9Vδ2 T cells on Huh7 tumor cells with different effector-target ratios;

[0014] Figure 2 Flow cytometry was used to detect the killing effect of Vγ9Vδ2 T cells on HepG2 tumor cells with different effector-target ratios;

[0015] Figure 3 This is a diagram showing the effect of THP enhancing Vγ9Vδ2 T cells in killing Huh7 liver cancer cells;

[0016] Figure 4 This is a diagram showing the effect of THP enhancing Vγ9Vδ2 T cells in killing HepG2 liver cancer cells;

[0017] Figure 5 This is a diagram showing the effect of THP combined with Vγ9Vδ2 T cells significantly inhibiting the proliferation of Huh7 tumor cells;

[0018] Figure 6 This is a diagram showing the effect of THP combined with Vγ9Vδ2 T cells significantly inhibiting the proliferation of HepG2 tumor cells;

[0019] Figure 7 This is a diagram showing the effect of THP on enhancing the expression of MICA and MICB in Huh7 tumor cells;

[0020] Figure 8 This is a diagram showing the effect of THP on enhancing the expression of MICA and MICB in HepG2 tumor cells;

[0021] Fig. 9 This is a diagram showing the effect of THP treatment on Huh7 tumor cells promoting high expression of NKG2D in Vγ9Vδ2 T cells;

[0022] Fig.10 This is a diagram showing the effect of THP treatment on HepG2 tumor cells promoting high expression of NKG2D in Vγ9Vδ2 T cells;

[0023] Fig.11 This is a graph showing weight changes during treatment of liver cancer-bearing mice;

[0024] Fig.12 This is a diagram showing the ability of THP to enhance the anti-tumor immune response of Vγ9Vδ2 T cells in the treatment of liver cancer-bearing mice. DETAILED DESCRIPTION

[0025] The present invention provides the use of THP in the preparation of a drug for enhancing the anti-tumor efficacy of Vγ9Vδ2 T cells. THP can significantly enhance the ability of Vγ9Vδ2 T cells to kill liver cancer cells by regulating the NKG2D / MICA / B pathway.

[0026] In the present invention, the tumor preferably includes one or more of liver cancer, malignant lymphoma, breast cancer, ovarian cancer, uterine cancer, gastric cancer and bladder cancer.

[0027] In the present invention, the effector-target ratio of Vγ9Vδ2 T cells to tumor cells is preferably 0.5 to 12:1, more preferably 1 to 12:1, and even more preferably 10:1.

[0028] In the present invention, THP is preferably used in the form of a solution; the THP solution preferably includes THP and iodized oil; the mass volume ratio of THP to iodized oil is preferably 2 μg:1-100 μL, more preferably 2 μg:10-80 μL, and even more preferably 2 μg:50 μL; the THP solution is preferably used by injection.

[0029] The present invention provides an anti-tumor drug, preferably comprising separately packaged THP and Vγ9Vδ2 T cells, wherein tumor cells are treated with THP to enhance the surface markers MICA / B of cancer cells, and THP-induced tumor cells can cause Vγ9Vδ2 T cells to highly express NKG2D, and THP significantly enhances the ability of Vγ9Vδ2 T cells to kill liver cancer cells by regulating the NKG2D / MICA / B pathway.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Sources

[0032] Huh7 was purchased from Zhongqiao Xinzhou, with the item number ZQ 0025;

[0033] HepG2 was purchased from Servicebio with the catalog number STCC10114P;

[0034] CFSE was purchased from Sigma, catalog number 21888;

[0035] THP was purchased from Selleck, catalog number S1393;

[0036] Ficoll was purchased from cytiva, catalog number 17144003;

[0037] Zol was purchased from Sigma-Aldrich, catalog number SML0223;

[0038] rIL-2 was purchased from Beijing Sihuan Biopharmaceutical Co., Ltd.;

[0039] RPMI 1640 complete medium (containing 10% FBS) was purchased from Gibico, catalog number 6124034;

[0040] Anti-TCRVγ9Vδ2 T flow cytometry antibody was purchased from Biolegend, catalog number 331407;

[0041] Anti-CD3 flow cytometry antibody was purchased from Biolegend, catalog number 300454;

[0042] PI was purchased from Biolegend, catalog number 421301;

[0043] Ki67 flow cytometry antibody was purchased from BioLegend, catalog number 350505;

[0044] The reverse transcription kit was purchased from Servicebio, catalog number G3337-100;

[0045] SYBR Green was purchased from Servicebio, catalog number G3326-15;

[0046] Lysis buffer (RIPA:PMSF:protease inhibitor:phosphatase inhibitor=100:1:1:1) was purchased from Beyotime;

[0047] C-NKG mice (male, 4 weeks) were purchased from Saiye (Suzhou) Biotechnology Co., Ltd.;

[0048] The exposure solution was purchased from Kermei, with the product number M0101.

[0049] Example 1: Cytotoxic effect of Vγ9Vδ2 T cells on HCC (Huh7, HepG2) tumor cells

[0050] 1) In vitro expansion of Vγ9Vδ2 T cells

[0051] Peripheral blood was obtained from healthy donors with a body mass index (BMI) of 18.5 to 24.99, and PBMCs were isolated by Ficoll density gradient centrifugation. Vγ9Vδ2 T cells were then expanded and cultured: on day 0, 3×10 6 Vγ9Vδ2 T cells were added with 50μM Zol and 100IU / mL rIL-2, and cultured in RPMI 1640 complete medium (containing 10% FBS), 37°C, 5% CO2, and saturated humidity incubator. Thereafter, on days 3, 5, 7, and 9, the medium containing 100IU / mL rIL-2 was replaced. After 9 days of induction, immunofluorescence double staining was performed with anti-TCRVγ9Vδ2 T and anti-CD3 flow cytometry antibodies, and the purity of anti-TCRVγ9Vδ2 T cells was analyzed by flow cytometry. The ratio of more than 80% can be used for experiments.

[0052] 2) Flow cytometry detection of the killing effect of Vγ9Vδ2 T cells on HCC (Huh7, HepG2) tumor cells

[0053] Vγ9Vδ2 T cells (effector cells, E) were incubated with HepG2 and Huh7 cells (target cells, T) at an effector-target ratio of E:T = 0:1 (Vγ9Vδ2 T was 0, tumor cells were 2×10 5 )、1:1(Vγ9Vδ2T is 0.2×10 6 , tumor cells are 2×10 5 )、5:1(Vγ9Vδ2 T is 1×10 6 , tumor cells are 2×10 5 ) and 10:1(Vγ9Vδ2 T is 2×10 6 , tumor cells are 2×10 5 ), placed in a round-bottom flow tube for co-culture, cultured with RPMI 1640 medium (serum-free), with a culture volume of 150 μL, placed in a 37°C incubator, and incubated in the dark for 5-6 h.

[0054] After the incubation of the above experimental groups, add 1 mL PBS to resuspend, centrifuge, discard the supernatant, add 100 μL PBS and 2 μL PI to each tube, stain at 4 ° C in the dark for 10 min, add 400 μL PBS and resuspend, and use flow cytometry to detect the killing effect of Vγ9Vδ2 T cells on tumor cells (HepG2, Huh7) with different effector-target ratios. Figure 1 and Figure 2 .

[0055] Figure 1 and Figure 2 This indicates that the killing effect of the effect-target ratio of 10:1 is significantly enhanced compared to 0:1, 1:1, and 5:1.

[0056] Example 2 The killing effect of Vγ9Vδ2 T cells on HCC (Huh7, HepG2) tumor cells treated with THP and its effect on tumor cell proliferation

[0057] 1) Experimental Grouping

[0058] Blank control group: tumor cells were plated at 3×10 5 Plate into 12-well plates and incubate in a 37°C, 5% CO2 incubator for 30 h;

[0059] THP group: Tumor cells were plated at 3×10 5 The cells were plated in 12-well plates and incubated in a 37°C, 5% CO2 incubator for 18 h. After the cells adhered to the wall, 2 μM THP was added to treat the cells for 12 h.

[0060] Vγ9Vδ2 T group: Tumor cells were plated at 3×10 5 The cells were plated in 12-well plates and incubated in a 37°C, 5% CO2 incubator for 24 h. 3 × 10 5 tumor cells and 3×10 6 Vγ9Vδ2 T cells were co-cultured with tumor cells (HepG2 and Huh7) in round-bottom flow tubes at an effector-target ratio of 10:1. The culture volume was 150 μL and the cells were placed in a 37°C incubator in the dark for 6 h.

[0061] THP+Vγ9Vδ2 T group: Tumor cells were plated at 3×10 5 The cells were plated in 12-well plates and incubated in a 37°C, 5% CO2 incubator for 12 h. After the cells adhered to the wall, 2 μM THP was added to treat the cells for 12 h. 3×10 5 tumor cells and 3×10 6 Vγ9Vδ2 T cells were co-cultured with THP-treated HCC (HepG2 and Huh7) cells in round-bottom flow tubes at an effector-target ratio of 10:1 in a culture volume of 150 μL. The cells were placed in a 37°C incubator and incubated in the dark for 6 h.

[0062] 2) Cytotoxic effect of Vγ9Vδ2 T cells on HCC (Huh7, HepG2) tumor cells treated with THP

[0063] After the incubation of the above experimental groups, add 1 mL PBS to resuspend, centrifuge, discard the supernatant, add 100 μL PBS and 2 μL PI to each tube, stain at 4 ° C in the dark for 10 min, add 400 μL PBS and resuspend, and use flow cytometry to detect the killing effect of Vγ9Vδ2 T cells on THP-treated tumor cells (HepG2, Huh7). Figure 3 and Figure 4 .

[0064] Figure 1 and Figure 2 The results showed that the killing effect of Vγ9Vδ2 T cells on THP-treated tumor cells (HepG2, Huh7) was significantly increased compared with the blank control, THP and Vγ9Vδ2 T cells, proving that there is a synergistic effect between the two.

[0065] 3) Flow cytometry was used to detect the effect of THP combined with Vγ9Vδ2 T cells on the proliferation of HCC (HepG2 and Huh7) cells

[0066] After the blank control group and experimental group were incubated, 1 mL of PBS was added for re-suspending, centrifuged, and the supernatant was discarded. 100 μL of PBS and 2.5 μL of ki67 were added to each tube, and 400 μL of PBS was added for re-suspending after staining at 4°C in the dark for 10 min. The samples were detected by flow cytometry. Figure 5 and Figure 6 .

[0067] The results showed that THP combined with Vγ9Vδ2 T cells significantly inhibited the proliferation of tumor cells.

[0068] Example 3 Effect of THP on the expression of MICA and MICB in HCC (Huh7, HepG2) tumor cells

[0069] 1) Detection of the effect of THP on the expression of MICA and MICB in HCC (Huh7, HepG2) tumor cells using qPCR

[0070] Experimental Grouping

[0071] Control group: Tumor cells were plated at 5×10 5 Plate into 12-well plates and incubate in a 37°C, 5% CO2 incubator for 30 h;

[0072] THP group: tumor cells were plated at 5×10 5 The cells were plated into 12-well plates and incubated in a 37°C, 5% CO2 incubator for 18 h. After the cells adhered to the wall, the cells were treated with 2 μM THP for 12 h.

[0073] RNA was extracted from the tumor cells of the Control group and THP group after treatment, and reverse transcription was performed using a reverse transcription kit, followed by cDNA quantification using SYBR Green.

[0074] 2) Western Blot was used to detect the effect of THP on the expression of MICA and MICB in HCC (Huh7, HepG2) tumor cells

[0075] Experimental Grouping

[0076] Control group: cells were plated at 6×10 5 Plate into 12-well plates and incubate in a 37°C, 5% CO2 incubator for 30 h;

[0077] THP group: cells were plated at 6×10 5 The cells were plated into 12-well plates and incubated in a 37°C, 5% CO2 incubator for 18 h. After the cells adhered to the wall, the cells were treated with 2 μM THP for 12 h.

[0078] Discard the supernatant, wash each well twice with 1 mL PBS, add 200 μL of prepared lysis buffer (RIPA: PMSF: protease inhibitor: phosphatase inhibitor = 100: 1: 1: 1) to each well, lyse on ice for 5 min, scrape the cells from the well wall with a cell scraper to obtain a cell suspension, transfer the cell suspension to an EP tube, lyse on a 4°C turntable for 20 min, centrifuge at 12000 rpm for 20 min, take 170 μL of cell supernatant, add 85 μL of 3× Loading Buffer (Beyotime), and boil in a metal bath at 100°C for 10 min. After SDS-PAGE of the protein, perform electrotransfer with a constant current of 300 mA for 2 h, place the electrotransferred PVDF membrane in 5% skim milk and block it at room temperature on a horizontal shaker for 2 h. After blocking, add 15 mL of TBST to wash for 10 min × 3 times. After incubation with the corresponding MICA / B primary antibody overnight, wash with TBST for 10 min × 3 times, incubate with the corresponding secondary antibody, and then wash again for 10 min × 3 times. According to the molecular weight of the protein, evenly drop the exposure solution (A solution: B solution = 1:1) on the membrane for exposure.

[0079] The results of the qPCR and Western Blot assays to examine the effects of THP on the expression of MICA and MICB in HCC (Huh7, HepG2) tumor cells are detailed in Figure 7 and Figure 8 .

[0080] The results showed that after THP treatment, the gene levels of NKG2D ligands-MICA / MICB on the surface of Huh7 and HepG2 liver cancer cell lines were significantly increased.

[0081] Example 4 Effect of THP treatment of HCC (Huh7, HepG2) tumor cells on NKG2D on the surface of Vγ9Vδ2 T cells

[0082] Experimental groups:

[0083] Control group: Tumor cells were plated at 6×10 5 The cells were plated in a 6-well plate and incubated in a 37°C, 5% CO2 incubator for 24 h. 6 × 10 cells of each tumor were collected. 5 Vγ9Vδ2 T cells were co-cultured with HCC (HepG2 and Huh7) tumor cells in round-bottom flow tubes at an effector-target ratio of 1:2.

[0084] THP group: Tumor cells were plated at 6×10 5 The cells were plated in 6-well plates and incubated in a 37°C, 5% CO2 incubator for 12 h. After the cells adhered to the wall, 2 μM THP was added to treat the cells for 12 h. 6×10 cells of each tumor type were collected. 5 In this study, Vγ9Vδ2 T cells were co-cultured with THP-treated HCC (HepG2 and Huh7) tumor cells in round-bottom flow tubes at an effector-target ratio of 1:2.

[0085] The co-culture volume was 150 μL, placed in a 37°C incubator, and incubated for 6 hours in the dark. After co-culture, add 1 mL of PBS to resuspend, centrifuge, discard the supernatant, add 100 μL of PBS, 2 μL of PI, and 2 μL of NKG2D to each tube, stain at 4°C in the dark for 25 minutes, add 400 μL of PBS and resuspend, and detect by flow cytometry. The results are detailed in Fig. 9 and Fig.10 .

[0086] The results showed that THP treatment of liver cancer cells promoted high expression of NKG2D in Vγ9Vδ2 T cells.

[0087] Example 5 Animal Experiment

[0088] 1) Construction of humanized mice

[0089] C-NKG mice weighing 23±2g were raised in a clean environment at 25±2℃. Each mouse was injected with 5×10 6 PBMCs were cultured and ocular venous blood was collected from mice every other week. The CD45 ratio was detected by flow cytometry. If the CD45 ratio reached more than 10% in the second week, it indicated that the construction was successful.

[0090] 2) Construction of liver cancer mouse model

[0091] Huh7 cells (5×10 6 / 100μL / mouse) was inoculated into the right hind leg of the constructed humanized mice for subcutaneous tumor inoculation. The tumor growth was observed within 12-14 days after the inoculation of the subcutaneous tumor. If there was an obvious bulge at the injection site, the construction was successful. The mice with successful construction were randomly divided into four groups: control group, Vγ9Vδ2 T group, THP group, and Vγ9Vδ2 T group combined with THP group. The number of mice in each group was 5.

[0092] 3) Mouse treatment

[0093] The next day after the liver cancer mice were successfully constructed, the corresponding treatments were carried out. Each mouse in the control group was injected with 100 μL PBS solution into the tumor, each mouse in the THP group was injected with 2 μg THP + 50 μL iodized oil into the tumor, and each mouse in the Vγ9Vδ2T group was injected with 5×10 6 Each mouse in the THP+Vγ9Vδ2 T group was injected with 2 μg THP+50 μL iodized oil intratumorally and 5×10 6 Vγ9Vδ2 T cells were injected into the THP+Vγ9Vδ2 T group by intratumoral injection of 2μg THP+50μL iodized oil for two days, and Vγ9Vδ2 T cells were injected into the tail vein once every 3 days, for a total of five injections of Vγ9Vδ2 T cells. The weight of the mice was recorded every other day during the treatment period (see Table 1 and Fig.11 ), seven days after the last injection of the THP+Vγ9Vδ2 T group, mice in all groups were killed at the same time, spleens and tumor tissues of mice were separated, weighed, and photographed according to the size of the tumor tissues. The tumors of mice were fixed overnight and embedded in paraffin. All animal studies were performed in accordance with the protocols approved by the Ethics Committee of Xinxiang Medical College. The tumor sizes of mice in different groups are detailed in Fig.12 .

[0094] Table 1. Weight changes of mice during treatment

[0095]

[0096]

[0097] Table 1: Weight changes of mice during treatment

[0098]

[0099] The results showed that THP significantly synergized with Vγ9Vδ2 T to inhibit tumor progression.

[0100] It can be seen from the above examples that THP can increase the killing ability of Vγ9Vδ2 T by regulating the NKG2D / MIC A / B pathway, thereby improving the anti-tumor immune response ability of liver cancer-bearing mice.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of THP in the preparation of drugs for enhancing the anti-tumor efficacy of Vγ9Vδ2T cells.

2. The application according to claim 1, characterized in that: The tumor includes one or more of liver cancer, malignant lymphoma, breast cancer, ovarian cancer, uterine cancer, gastric cancer and bladder cancer.

3. The use according to claim 2, characterized in that: The effector-target ratio of Vγ9Vδ2T to tumor cells is 0.5-12:

1.

4. The use according to claim 3, characterized in that: The THP is used in solution.

5. The use according to claim 4, characterized in that: The THP solution includes THP and iodized oil; the mass volume ratio of the THP to the iodized oil is 2 μg:1-100 μL.

6. An anti-tumor drug, characterized in that: The invention comprises the THP and Vγ9Vδ2T cells for use according to any one of claims 1 to 5 which are packaged separately.

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