Use of thp in the preparation of a drug for enhancing the antitumor efficacy of vγ9vδ2 t cells

Abstract

The application relates to application of THP in preparation of a medicine for enhancing the antitumor effect of Vgamma9VDelta2 T cells, and belongs to the technical field of biological medicine. It is found that the surface marker MICA / B of tumor cells treated by THP is significantly enhanced, and the tumor cells induced by THP can make Vgamma9VDelta2 T cells highly express NKG2D. The NKG2D / MICA / B pathway is a classic pathway for Vgamma9VDelta2 T cells to exert a killing effect. THP can significantly enhance the ability of Vgamma9VDelta2 T cells to kill hepatocarcinoma cells by regulating the NKG2D / MICA / B pathway, and provides a new treatment scheme for TACE surgical treatment of clinical cancer patients.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, and particularly relates to application of THP in preparation of a medicine for enhancing anti-tumor effect of Vgamma9Vdelta2 T cells. BACKGROUND

[0002] Transcatheter arterial chemoembolization (TACE) is a preferred treatment method for middle and late hepatocellular carcinoma (HCC), and a chemotherapeutic drug such as pirarubicin (THP) is used in TACE to directly damage DNA and induce cell apoptosis. Ischemia and hypoxia caused by embolization affect DNA synthesis of tumor cells, further promote tumor cell necrosis, activate T lymphocyte response, and change tumor immune microenvironment, but the prognosis of many patients after TACE is poor, and therefore, other treatment methods need to be combined with TACE operation for treating liver cancer.

[0003] Research finds that allogeneic Vgamma9Vdelta2 T cells can improve the prognosis of some HCC patients, and a high ratio of peripheral blood Vgamma9Vdelta2 T cells of patients before TACE indicates good prognosis. In the tumor microenvironment of HCC, it is found that gamma delta T cells can recognize stress-induced molecules MICA / B expressed by tumor cells through surface expression of NKG2D receptors, and the Vgamma9Vdelta2 T cell subgroup in gamma delta T cells has the potential to be used as a cellular immunotherapy means and plays an important role in treating HCC, but the anti-tumor effect of Vgamma9Vdelta2 T cells is weak at present, and further research is needed to increase the anti-tumor effect of Vgamma9Vdelta2 T cells and play a better function of improving the prognosis of HCC patients. SUMMARY

[0004] In order to achieve the above application purposes, the application provides the following technical scheme.

[0005] The application provides application of THP in preparation of a medicine for enhancing anti-tumor effect of Vgamma9Vdelta2 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 Vgamma9Vdelta2 T cells to tumor cells is 0.5-12:1.

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

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

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

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

[0012] The present application provides the use of THP in the preparation of a drug for enhancing the anti-tumor effect of Vγ9Vδ2 T cells. The present application found that the surface marker MICA / B of tumor cell lines treated with THP was significantly enhanced, and THP-induced tumor cells could induce high expression of NKG2D in Vγ9Vδ2 T cells. The NKG2D / MICA / B pathway is a classic pathway for Vγ9Vδ2 T cells to exert killing effect. THP can significantly enhance the ability of Vγ9Vδ2 T cells to kill hepatocarcinoma cells by regulating the NKG2D / MICA / B pathway, providing a new treatment for TACE surgical treatment of clinical cancer patients. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 For flow cytometry detection of the killing effect of Vγ9Vδ2 T cells on Huh7 tumor cells with different effector-target ratios;

[0014] Figure 2 For flow cytometry detection of the killing effect of Vγ9Vδ2 T cells on HepG2 tumor cells with different effector-target ratios;

[0015] Figure 3 For THP-enhanced Vγ9Vδ2 T cell killing effect on Huh7 hepatocarcinoma cells;

[0016] Figure 4 For THP-enhanced Vγ9Vδ2 T cell killing effect on HepG2 hepatocarcinoma cells;

[0017] Figure 5 For THP combined with Vγ9Vδ2 T cells to significantly inhibit the proliferation of Huh7 tumor cells;

[0018] Figure 6 For THP combined with Vγ9Vδ2 T cells to significantly inhibit the proliferation of HepG2 tumor cells;

[0019] Figure 7 For THP-enhanced expression of MICA and MICB in Huh7 tumor cells;

[0020] Figure 8 For THP-enhanced expression of MICA and MICB in HepG2 tumor cells;

[0021] Figure 9 Effect diagram for THP treatment of Huh7 tumor cells to promote high expression of NKG2D by Vγ9Vδ2 T cells;

[0022] Figure 10 Effect diagram for THP treatment of HepG2 tumor cells to promote high expression of NKG2D by Vγ9Vδ2 T cells;

[0023] Figure 11 Body weight change diagram during treatment of liver cancer tumor-bearing mice;

[0024] Figure 12 THP enhances the ability of Vγ9Vδ2 T cells to resist tumor immune response in the treatment of liver cancer tumor-bearing mice. DETAILED DESCRIPTION

[0025] The present application provides the use of THP in the preparation of a drug for enhancing the anti-tumor effect 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 application, 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 application, the effector-target ratio of Vγ9Vδ2 T cells to tumor cells is preferably 0.5-12:1, further preferably 1-12:1, and more preferably 10:1.

[0028] In the present application, 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 and iodized oil is preferably 2 μg:1-100 μL, further preferably 2 μg:10-80 μL, and more preferably 2 μg:50 μL; and the THP solution is preferably used by injection.

[0029] The present application provides an anti-tumor drug, preferably including THP and Vγ9Vδ2 T cells packaged separately, tumor cells treated by THP, enhanced surface markers MICA / B of cancer cells, and THP-induced tumor cells capable of high expression of NKG2D by Vγ9Vδ2 T cells, THP significantly enhancing 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 application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0031] Material sources

[0032] Huh7 was purchased from Zhijiaoxinzhou with the item number ZQ 0025;

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

[0034] CFSE was purchased from Sigma with the item number 21888;

[0035] THP was purchased from Selleck with the item number S1393;

[0036] Ficoll was purchased from cytiva with the item number 17144003;

[0037] Zol was purchased from Sigma-Aldrich with the item 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 with the item number 6124034;

[0040] Anti-TCRVγ9Vδ2 T flow antibody was purchased from Biolegend with the item number 331407;

[0041] Anti-CD3 flow antibody was purchased from Biolegend with the item number 300454;

[0042] PI was purchased from Biolegend with the item number 421301;

[0043] Ki67 flow antibody was purchased from BioLegend with the item number 350505;

[0044] Reverse transcription kit was purchased from Servicebio with the item number G3337-100;

[0045] SYBR Green was purchased from Servicebio with the item number G3326-15;

[0046] Lysis solution (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] Exposure solution was purchased from Kelmee with the item number M0101.

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

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

[0051] Peripheral blood was taken from healthy blood donors with a body mass index (BMI) of 18.5-24.99, and PBMCs were isolated by Ficoll liquid density gradient centrifugation. Subsequently, Vγ9Vδ2 T cell expansion culture was performed: on day 0, 3x10 6 Vγ9Vδ2 T cells were added with 50 μM Zol and 100 IU / mL rIL-2, and cultured in RPMI 1640 complete medium (containing 10% FBS) in a 37°C, 5% CO2, saturated humidity incubator. On days 3, 5, 7, and 9, the medium containing 100 IU / mL rIL-2 was replaced, and after 9 days of induction, immunofluorescence double staining was performed using anti-TCRVγ9Vδ2 T and anti-CD3 flow cytometry antibodies, and the purity of anti-TCRVγ9Vδ2 T cells was analyzed by flow cytometry. When the proportion reached more than 80%, the cells were used for experiments.

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

[0053] Vγ9Vδ2 T cells (effector cells, E) were mixed with HepG2 and Huh7 cells (target cells, T) at effector-to-target ratios E:T = 0:1 (0 Vγ9Vδ2 T and 2x10 5 ), 1:1 (0.2x10 6 Vγ9Vδ2 T and 2x10 5 ), 5:1 (1x10 6 Vγ9Vδ2 T and 2x10 5 ), and 10:1 (2x10 6 Vγ9Vδ2 T and 2x10 5 ), respectively, in round-bottom flow tubes, and cultured in RPMI 1640 medium (without serum) with a culture volume of 150 μL in a 37°C incubator, avoiding light for 5-6 h.

[0054] After incubation of the above experimental groups, 1 mL of PBS was added for resuspension, centrifugation was performed, the supernatant was discarded, 100 μL of PBS and 2 μL of PI were added to each tube, 4°C staining was performed for 10 min, 400 μL of PBS was added for resuspension, and flow cytometry was used to detect the killing effect of Vγ9Vδ2 T cells on tumor cells (HepG2, Huh7) at different effector-to-target ratios. The results are shown in detail in Figure 1 and Figure 2 .

[0055] Figure 1 and Figure 2 The killing effect of 10:1 effector target ratio is significantly enhanced compared to 0:1, 1:1 and 5:1 killing effect.

[0056] Example 2 Killing effect of Vγ9Vδ2 T cells on HCC (Huh7, HepG2) tumor cells treated by THP and the effect on tumor cell proliferation

[0057] 1) Experimental grouping

[0058] Blank control group: tumor cells were plated at 3x10 5 into 12-well plates and incubated in a 37°C, 5% CO2 incubator for 30h;

[0059] THP group: tumor cells were plated at 3x10 5 into 12-well plates and incubated in a 37°C, 5% CO2 incubator for 18h, and then 2μM THP was added to treat the cells for 12h;

[0060] Vγ9Vδ2 T group: tumor cells were plated at 3x10 5 into 12-well plates and incubated in a 37°C, 5% CO2 incubator for 24h, and then 3x10 5 tumor cells and 3x10 6 Vγ9Vδ2 T cells were collected, and Vγ9Vδ2 T cells and tumor cells (HepG2 and Huh7) were co-cultured in round-bottom flow tubes at an effector target ratio of 10:1, with a culture volume of 150μL, in a 37°C incubator, and incubated for 6h in the dark.

[0061] THP+Vγ9Vδ2 T group: tumor cells were plated at 3x10 5 into 12-well plates and incubated in a 37°C, 5% CO2 incubator for 12h, and then 2μM THP was added to treat the cells for 12h, and 3x10 5 tumor cells and 3x10 6 Vγ9Vδ2 T cells were collected, and Vγ9Vδ2 T cells and THP-treated HCC (HepG2 and Huh7) cells were co-cultured in round-bottom flow tubes at an effector target ratio of 10:1, with a culture volume of 150μL, in a 37°C incubator, and incubated for 6h in the dark.

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

[0063] After the incubation of the above experimental groups, 1 mL of PBS was added for resuspension, centrifugation, and discarding of the supernatant. 100 μL of PBS and 2 μL of PI were added to each tube, and staining was performed at 4°C in the dark for 10 min. After the addition of 400 μL of PBS for resuspension, the killing effect of Vγ9Vδ2 T cells on THP-treated tumor cells (HepG2, Huh7) was detected by flow cytometry. The results are shown in detail in Figure 3 and Figure 4 .

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

[0065] 3) Flow cytometry detection of the effect of THP combined with Vγ9Vδ2 T cells on HCC (HepG2 and Huh7) cell proliferation

[0066] After the incubation of the above blank control and experimental groups, 1 mL of PBS was added for resuspension, centrifugation, and discarding of the supernatant. 100 μL of PBS and 2.5 μL of ki67 were added to each tube, and staining was performed at 4°C in the dark for 10 min. After the addition of 400 μL of PBS for resuspension, the samples were detected by flow cytometry. The results are shown in detail in Figure 5 and Figure 6 .

[0067] The results show 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 of HCC (Huh7, HepG2) tumor cells

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

[0070] Experimental grouping

[0071] Control group: tumor cells were plated at 5 × 10 5 per well in a 12-well plate and incubated at 37°C in a 5% CO2 incubator for 30 h;

[0072] THP group: tumor cells were plated at 5 × 10 5 per well in a 12-well plate and incubated at 37°C in a 5% CO2 incubator for 18 h. After the cells adhered, 2 μM THP was used to treat the cells for 12 h.

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

[0074] 2) The effect of THP on the expression of MICA and MICB in HCC (Huh7, HepG2) tumor cells was detected by Western Blot method

[0075] Experimental grouping

[0076] Control group: the cells were plated at 6x10 5 per well in a 12-well plate and incubated at 37°C in a 5% CO2 incubator for 30h;

[0077] THP group: the cells were plated at 6x10 5 per well in a 12-well plate and incubated at 37°C in a 5% CO2 incubator for 18h, and after the cells adhered, the cells were treated with 2μM THP for 12h.

[0078] The supernatant was discarded, the cells were washed twice with 1mL PBS per well, 200μL of prepared lysis solution (RIPA:PMSF:protease inhibitor:phosphatase inhibitor = 100:1:1:1) was added per well, the cells were lysed on ice for 5min, and then the cells were scraped from the well wall with a cell scraper to obtain a cell suspension, which was transferred to an EP tube and lysed on a 4°C rotating disc for 20min. Centrifugation was performed at 12000rpm for 20min, 170μL of cell supernatant was taken, 85μL of 3xLoading Buffer (Beyotime) was added, and the metal bath was boiled at 100°C for 10min. After SDS-PAGE of the protein, electrophoresis was performed at a constant current of 300mA for 2h, and the PVDF membrane after electrophoresis was placed in 5% skim milk and blocked on a horizontal shaker at room temperature for 2h. After blocking, 15mL of TBST was added and washed for 10min x 3 times. After incubation with the corresponding MICA / B primary antibody overnight, TBST was washed for 10min x 3 times, and after incubation with the corresponding secondary antibody, it was washed again for 10min x 3 times. According to the size of the protein molecular weight, exposure solution (A solution:B solution = 1:1) was added evenly on the membrane for exposure.

[0079] The results of the effect of THP on the expression of MICA and MICB in HCC (Huh7, HepG2) tumor cells were detected by qPCR and Western Blot method, which are shown in Figure 7 and Figure 8 .

[0080] The results showed that after THP treatment, the gene level of NKG2D ligand-MICA / MICB on the surface of Huh7 and HepG2 hepatoma cell lines was significantly increased.

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

[0082] Experimental grouping:

[0083] Control group: tumor cells were plated at 6x10 5 per well in a 6-well plate and incubated in a 37°C, 5% CO2 incubator for 24 h, and 6x10 5 Vγ9Vδ2 T cells were co-cultured with HCC (HepG2 and Huh7) tumor cells at an effector-to-target ratio of 1:2 in round-bottom flow tubes.

[0084] THP group: tumor cells were plated at 6x10 5 per well in a 6-well plate and incubated in a 37°C, 5% CO2 incubator for 12 h, and after the cells adhered, 2 μM THP was added to treat the cells for 12 h, and 6x10 5 Vγ9Vδ2 T cells were co-cultured with THP-treated HCC (HepG2 and Huh7) tumor cells at an effector-to-target ratio of 1:2 in round-bottom flow tubes.

[0085] The co-culture volume was 150 μL, and the co-culture was incubated in a 37°C incubator in the dark for 6 h. After co-culture, 1 mL PBS was added to resuspend the cells, and the cells were centrifuged and the supernatant was discarded. 100 μL PBS, 2 μL PI, and 2 μL NKG2D were added to each tube, and the cells were stained in the dark at 4°C for 25 min. After the addition of 400 μL PBS, the cells were resuspended and detected by flow cytometry. The results are shown in Figure 9 and Figure 10 .

[0086] The results show that after THP treatment of hepatoma cells, Vγ9Vδ2 T cells are promoted to highly express NKG2D.

[0087] Example 5 Animal experiment

[0088] 1) Construction of humanized mice

[0089] C-NKG mice weighing 23 ± 2 g were taken and raised in a clean environment at a temperature of 25 ± 2°C. Each mouse was injected with 5x10 6 PBMCs in the tail vein, and the mice were taken for eye vein blood every other week. The proportion of CD45 was detected by flow cytometry, and the proportion of CD45 reached more than 10% in the second week, indicating that the construction was successful.

[0090] 2) Construction of hepatoma mouse model

[0091] Huh7 cells (5x10 6The right hind leg of the humanized mouse which has been constructed is inoculated with 100 μL / each of subcutaneous tumor inoculation. The tumor growth is observed within 12-14 days after the subcutaneous tumor inoculation, and if there is a significant bulge at the injection site, the construction is successful. The successfully constructed mice are randomly divided into four groups: a control group, a Vγ9Vδ2 T group, a THP group, and a Vγ9Vδ2 T group combined with the THP group. The number of mice in each group is 5.

[0092] 3) Mouse treatment

[0093] The corresponding treatment is performed on the next day after the successful construction of the liver cancer mouse. The control group is intratumorally injected with 100 μL of PBS solution per mouse, the THP group is intratumorally injected with 2 μg of THP + 50 μL of iodized oil per mouse, the Vγ9Vδ2 T group is tail vein injected with 5 × 10 6 Vγ9Vδ2 T cells per mouse, and the THP + Vγ9Vδ2 T group is intratumorally injected with 2 μg of THP + 50 μL of iodized oil and tail vein injected with 5 × 10 6 Vγ9Vδ2 T cells per mouse. The injection method of the THP + Vγ9Vδ2 T group is intratumorally injected with 2 μg of THP + 50 μL of iodized oil for two days, and then tail vein injected with Vγ9Vδ2 T cells once every three days, for a total of five times. The weight of the mouse is recorded every other day during the treatment (see Table 1 and Figure 11 ), and all the mice in each group are sacrificed seven days after the last injection of the THP + Vγ9Vδ2 T group. The mouse spleen and tumor tissue are separated, weighed, and placed in order from large to small for photographing. The mouse tumor is fixed overnight and embedded in paraffin. All animal studies are performed in accordance with the approved protocol of the Ethics Committee of Xinxiang Medical College. The tumor sizes of the mice in different groups are shown in Figure 12 .

[0094] Table 1 Weight change data table of mice during treatment

[0095]

[0096]

[0097] Table 1 Weight change data table of mice during treatment (continued)

[0098]

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

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

[0101] The above merely preferred embodiments of the present application, it should be noted that for those of ordinary skill in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the scope of protection of the present application.

Claims

1. Use of pirarubicin in the preparation of a medicament for enhancing the efficacy of Vγ9Vδ2 T cells against liver cancer.

2. Use according to claim 1, characterized in that, The pirarubicin is used in the form of a solution.

3. Use according to claim 2, characterized in that, The pirarubicin solution comprises pirarubicin and iodized oil; the mass / volume ratio of the pirarubicin and the iodized oil is 2 μg: 1 ~ 100 μL.

4. An anti-liver cancer drug, characterized by, The pirarubicin and the Vγ9Vδ2 T cells used in the application of any one of claims 1 to 3 are packaged separately.

Images