A combination drug for preventing and / or treating prostate cancer, use thereof, and a pharmaceutical composition

Abstract

The present application belongs to the field of chemical medicine, and particularly relates to a combined drug for preventing and / or treating prostate cancer, use and pharmaceutical composition thereof. The combined drug of the present application is an immune cell with enhanced infiltration degree and a galectin-1 inhibitor; the immune cell with enhanced infiltration degree is a natural killer (NK) cell with TTTY15-USP9Y chimera RNA knocked out, which can increase the infiltration degree and anti-tumor activity of the NK cell in the prostate cancer microenvironment, and the activated NK cell has stronger killing effect on tumor cells in vitro. The present application combines the immune cell with enhanced infiltration degree and the galectin-1 inhibitor, and the combination shows synergistic effect in treating prostate cancer, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical medicine, and particularly relates to a combined drug for preventing and / or treating prostate cancer, use thereof and a pharmaceutical composition. BACKGROUND

[0002] Prostate cancer (PCa) is the most common malignant tumor of the male urinary system, causing more than 370,000 deaths worldwide each year. The incidence of prostate cancer in China is increasing year by year, and more than 50% of patients are in the advanced stage at the first diagnosis, missing the best opportunity for surgical treatment. Compared with traditional therapies such as radiotherapy and chemotherapy, tumor immunotherapy has the characteristics of less side effects and lower drug resistance, and has made progress in the clinical application of various advanced tumors. However, the effective rate of existing immunotherapy strategies in patients with advanced prostate cancer is still less than 15%, and it does not bring long-term survival advantage to patients.

[0003] Chimeric RNA refers to a fusion RNA sequence composed of exons or exon fragments of different genes, that is, an RNA molecule containing multiple gene sequence fragments. In recent years, with the continuous progress of sequencing technology, more than two million chimeric RNAs have been found in human cells. It has been reported in the literature that the expression of TTTY15-USP9Y chimeric RNA is closely related to the proliferation ability of prostate cancer cells, which can accelerate the cells into the G2 phase and enhance the migration and invasion ability of prostate cancer cells. It can be seen that intervention of the expression of TTTY15-USP9Y chimeric RNA may become a potential strategy for treating prostate cancer.

[0004] Galectin-1 (LGALS1, Gal-1) is a known immunosuppressive factor, and the inhibitor OTX008 of Gal-1 can inhibit the proliferation of some types of tumors in vivo and in vitro.

[0005] Prostate cancer is considered an immune "cold tumor" with insufficient immune cell infiltration and immunosuppressive microenvironment, and if the prostate cancer can be converted from an immune "cold tumor" to an immune "hot tumor", the efficacy of immunotherapy in prostate cancer is expected to be improved. However, there is still a lack of effective means for regulating the immune microenvironment of prostate cancer. Therefore, it is of great significance to develop new drugs for treating prostate cancer to provide more treatment options and improve the prognosis for patients. SUMMARY

[0006] In view of the problems of the prior art, the present application provides a combined drug for preventing and / or treating prostate cancer, use thereof and a pharmaceutical composition.

[0007] The present application provides a combination drug for preventing and / or treating prostate cancer, wherein the combination drug is immune cells with enhanced infiltration and a galectin-1 inhibitor.

[0008] Preferably, the immune cells with enhanced infiltration and the galectin-1 inhibitor are used in a ratio of 2×10 5 immune cells with enhanced infiltration: (0.001-0.125) mg.

[0009] Preferably, the immune cells are selected from natural killer cells.

[0010] Preferably, the immune cells with enhanced infiltration are selected from immune cells with knockout or knockdown of TTTY15-USP9Y chimera RNA.

[0011] Preferably, the immune cells with knockout or knockdown of TTTY15-USP9Y chimera RNA are achieved by gene knockout or knockdown through a gene editing tool; and the gene editing tool is a CRISPR / Cas9 gene editing system.

[0012] Preferably, the nucleotide sequence of the TTTY15-USP9Y chimera RNA is shown in SEQ ID NO. 3.

[0013] Preferably, the galectin-1 inhibitor is selected from a small molecule compound.

[0014] Preferably, the small molecule compound is selected from OTX008.

[0015] The present application also provides use of the above-mentioned combination drug in the preparation of a drug for preventing and / or treating prostate cancer.

[0016] The present application also provides a pharmaceutical composition for preventing and / or treating prostate cancer, which is prepared by adding a pharmaceutically acceptable adjuvant to immune cells with enhanced infiltration and a galectin-1 inhibitor as active ingredients; the immune cells with enhanced infiltration are selected from immune cells with knockout or knockdown of TTTY15-USP9Y chimera RNA; and the immune cells with enhanced infiltration and the galectin-1 inhibitor are used in a ratio of 2×10 5 immune cells with enhanced infiltration: (0.001-0.125) mg.

[0017] The application combines the infiltration-enhanced immune cells and the galectin-1 inhibitor; the infiltration-enhanced immune cells are natural killer (NK) cells with knockout TTTY15-USP9Y chimera RNA, which can increase the infiltration degree and anti-tumor activity of NK cells in the prostate cancer microenvironment, and the activated NK cells have stronger killing effect on tumor cells in vitro. The application combines the infiltration-enhanced immune cells and the galectin-1 inhibitor, which can efficiently kill prostate cancer cells and has a synergistic effect in the treatment of prostate cancer, and has a wide application prospect.

[0018] Obviously, according to the above content of the application, according to the ordinary technical knowledge and means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the application.

[0019] The above content of the application will be further described in detail through the specific embodiments in the form of examples. However, it should not be understood that the scope of the above subject matter of the application is limited to the following examples. Any technology achieved based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Schematic diagram for gene editing knockout TTTY15-USP9Y chimera RNA and construction of knockout cell line. (A) TTTY15 gene transcribes TTTY15 and USP9Y on one RNA chain through transcription read-through, forming TTTY15-USP9Y chimera RNA; (B) using gRNA to guide Cas9 protein to the region between TTTY15 and USP9Y genes, Cas9 cuts the genome, at this time, the DNA fragment containing the transcription terminator and the puromycin gene is inserted into the cell genome, and by using puromycin to screen cells, two stable transfection monoclonal cell strains (TUKO) are obtained; (C) TUKO-6 and TUKO-8 only contain separated TTTY15 and USP9Y gene transcripts, and do not contain TTTY15-USP9Y chimera RNA, which has the effect of knocking out TTTY15-USP9Y chimera RNA.

[0021] Figure 2 Knocking out TTTY15-USP9Y chimera RNA can increase the infiltration degree and anti-tumor activity of NK cells in the prostate cancer microenvironment. (A) In vivo imaging shows the tumor growth of DU145 and TUKO cells in nude mice; (B) immunohistochemical analysis shows the infiltration of NK cells in tumor tissues.

[0022] Figure 3 TUKO-activated NK cells have a killing effect on tumors in vitro.

[0023] Figure 4 Effect of different concentrations of OTX008 on cell viability of prostate cancer cells.

[0024] Figure 5 OTX008 can improve the killing ability of TUKO-NK cells on tumors.

[0025] Figure 6 TUKO-activated NK cells have therapeutic effects on prostate cancer in vivo.

[0026] Figure 7 Effect of OTX008 on DU145 prostate cancer cells in vivo.

[0027] Figure 8 Therapeutic effect of TUKO-NK cells combined with OTX008 on prostate cancer. DETAILED DESCRIPTION

[0028] In the following examples and experimental examples, the reagents and raw materials not specifically explained are commercially available.

[0029] Example 1 Combination drug for preventing and / or treating prostate cancer

[0030] This example provides a combination drug for preventing and / or treating prostate cancer, which is a combination drug of 3000 NK cells knocking out TTTY15-USP9Y chimera RNA in 200 μL of OTX008 solution of Gal-1 inhibitor at a concentration of 0.1 μM. The combination drug has a synergistic effect.

[0031] Example 2 Combination drug for preventing and / or treating prostate cancer

[0032] This example provides a combination drug for preventing and / or treating prostate cancer, which is a combination drug of 3000 NK cells knocking out TTTY15-USP9Y chimera RNA in 200 μL of OTX008 solution of Gal-1 inhibitor at a concentration of 10 μM. The combination drug has a synergistic effect.

[0033] The technical solutions of the present application are further illustrated by experiments below.

[0034] Experimental Example 1: Killing effect of TUKO-NK cells on prostate cancer cells in vitro

[0035] I. Experimental method

[0036] 1. Gene editing to knock out TTTY15-USP9Y chimera RNA and construct a knockout cell line

[0037] (1) Using specially designed primers, PCR is performed in cancer tissues of prostate cancer patients and prostate cancer cell lines, and the sequence of TTTY15-USP9Y chimeric RNA (TU) is determined by Sanger sequencing of the PCR products. A DNA fragment containing a transcription terminator and a puromycin resistance gene is inserted into the intergenic region between TTTY15 gene and USP9Y, thereby blocking the production of TU chimeric RNA. The composition of the DNA fragment is as follows: 5'-transcription terminator (transcription terminator sequence repeated 5 times)-puromycin (puromycin resistance gene repeated once)-3'.

[0038] The sequence of TTTY15-USP9Y chimeric RNA (TU) is (SEQ ID NO. 3):

[0039]

[0040] (2) Synthesis of gRNA expression lentivirus, gRNA sequence is shown in Table 1.

[0041] (3) The above gene fragments, gRNA and Cas9 protein are transcribed into prostate cancer cell line (DU145) by lentivirus vector (pLenti-CMV-GFP plasmid), and stable cell strain is obtained by puromycin (Puro) screening. Figure 1 ).

[0042] The nucleotide sequences involved in the above method are shown in Table 1.

[0043] Table 1

[0044]

[0045] 2, Immunohistochemical method

[0046] 1x10 6 DU145 cells and tumor cells (TUKO) knocked out TTTY15-USP9Y chimera RNA were injected subcutaneously into 4-week-old Balbc nude mice weighing 20 g, and photographed using a live imaging instrument after one month. The tumor was peeled off, fixed with 4% paraformaldehyde for 24 hours, paraffin-embedded and sectioned, and immunohistochemical analysis was performed using a mouse-derived CD49b antibody.

[0047] 3, Acquisition of DU145 tumor-bearing mouse-derived NK cells (DU145-NK) and TUKO tumor-bearing mouse-derived NK cells (TUKO-NK)

[0048] (1) DU145 and TUKO cells were injected subcutaneously into CB-17 SCID mice (1x10 6 cells per mouse). Note: In order to exclude the interference of T and B lymphocytes, CB-17 SCID mice with simple immune microenvironment were used in this experiment. The immune system of this mouse only has NK cells.

[0049] (2) The mice were sacrificed after 10 days, the spleen was taken, and the spleen was ground in a 70 μm filter screen. Red blood cells were broken by using red cell lysing solution, and other cells in the spleen were collected. Phosphate buffered saline (PBS) was washed for 15 seconds, and centrifuged at 300g for 5 minutes.

[0050] (3) Discard the supernatant, resuspend the cells with MACS Running Buffer (Miltenyi, Cat# 130-091-221), and incubate with CD49b magnetic bead antibody (Miltenyi, Cat# 130-052-501) and perform magnetic sorting (LS column, Miltenyi, Cat# 130-042-401) according to the instructions, to obtain DU145 tumor-bearing mouse-derived NK cells (DU145-NK) and TUKO tumor-bearing mouse-derived NK cells (TUKO-NK). The above steps need to be performed in a clean bench and under sterile conditions.

[0051] 4. CCK8 method

[0052] Mix the two kinds of NK cells sorted above with DU145 cells at a ratio of 1:1 in 1640 medium, to obtain two groups of DU145-NK vs DU145 and TUKO-NK vs DU145 cells, and plate the mixed cells in a 96-well plate at 200 microliters per well and 6000 cells per well, with 5 replicate wells per group. After 48 hours, discard the culture medium and suspended cells, and perform CCK-8 detection.

[0053] II. Experimental results

[0054] As shown in Figure 2 Compared with the DU145 group, the TUKO group of nude mice had smaller tumor volume Figure 2 A), and more NK cell (CD49b antigen positive cell) infiltration in the tumor Figure 2 B).

[0055] As shown in Figure 3 The killing ability of the NK cells sorted from the spleen of DU145 tumor-bearing mice on DU145 cells was weak, while the killing ability of the NK cells sorted from the spleen of TUKO tumor-bearing mice on DU145 cells was enhanced, and the tumor cell activity decreased to 81.06% of the previous group.

[0056] The above results show that knocking out TTTY15-USP9Y chimera RNA in tumor cells can increase the degree of NK cell infiltration and anti-tumor activity in the microenvironment of prostate cancer, and TUKO-activated NK cells have stronger killing effect on tumors in vitro.

[0057] Effect of Gal-1 inhibitor OTX008 on the cell activity of prostate cancer cells

[0058] I. Experimental method

[0059] CCK8 method:

[0060] (1) DU145 cells were cultured in 10cm dishes using 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin). When the cell density reached 80%, the cells were digested with trypsin, centrifuged at 150g, the supernatant was discarded, and the cells were washed and resuspended with PBS for cell counting. 1.2 × 10⁻⁶ cells were collected. 4 Each cell was placed in a 15 ml centrifuge tube, for a total of 6 centrifuge tubes, centrifuged at 150 g, and the supernatant was discarded.

[0061] (2) Dissolve 1 mg of OTX008 in 106.7 μL of ethanol to obtain a stock solution with a molar concentration of 10 mM; add 10 μL of the stock solution to 990 μL of 1640 complete culture medium to obtain 1 mL of a 100 μM diluent; add 100 μL of the 100 μM diluent to 900 μL of 1640 complete culture medium to obtain a 10 μM OTX008 diluent; add 100 μL of the 10 μM diluent to 900 μL of 1640 complete culture medium to obtain a 10 μM OTX008 diluent; The diluent was added to 900 μL of 1640 complete medium to obtain a 1 μM OTX008 dilution; 100 μL of the 1 μM diluent was added to 900 μL of 1640 complete medium to obtain a 0.1 μM OTX008 dilution; 100 μL of the 0.1 μM diluent was added to 900 μL of 1640 complete medium to obtain a 0.01 μM OTX008 dilution. Separately, 1 mL of 1640 complete medium was mixed with 1 μL of ethanol to serve as a control medium.

[0062] (3) Use 600 μL of the above-mentioned 1640 complete medium containing OTX008 (OTX008 concentrations of 0, 0.01, 0.1, 1, 10, and 100 (μM)) to resuspend the 6 tubes of cells in (1), and seed the cells in 96-well plates with 100 μL per well and 5 replicates for each concentration.

[0063] (4) Place the cells in a 37°C carbon dioxide cell culture incubator and culture for 46 hours. Add 10 μL of CCK-8 reagent to each well and continue culturing for 2 hours. Then place the cells in an ELISA reader for absorbance detection.

[0064] II. Experimental Results

[0065] The results are as follows Figure 4 As shown, different concentrations of OTX008 had no significant effect on the cell viability of prostate cancer DU145 cells compared to the control group. This indicates that OTX008 has no killing effect on prostate cancer cells.

[0066] Experimental Example 3: The therapeutic effect of TUKO-NK cells combined with OTX008 on prostate cancer in vitro.

[0067] I. Experimental Methods

[0068] (1) The sorted TUKO-NK cells are mixed with DU145 cells in a ratio of 1:1 in 1640 medium to obtain a mixed cell solution of TUKO-NK mixed DU145, and the mixed cells are plated in a 96-well plate at 200 microliters per well and 6000 cells per well, for a total of 20 wells. The 20 wells are divided into 4 groups, 5 replicates per group. The control group is added with 0.2 microliters of ethanol, and the experimental groups are added with 0.2 microliters of OTX008 solution with concentrations of 0.1 mM, 1 mM and 10 mM respectively (prepared in advance: 1 milligram of OTX008 is dissolved in 106.7 microliters of ethanol to obtain a stock solution with a molar concentration of 10 mM; 10 microliters of the stock solution is added to 90 microliters of PBS to obtain a dilution solution with a concentration of 1 mM; 10 microliters of the dilution solution with a concentration of 1 mM is added to 90 microliters of PBS to obtain an OTX008 dilution solution with a concentration of 0.1 mM), so that the final concentrations of the drugs in each group are 0.1 μM, 1 μM and 10 μM.

[0069] (2) After 48 hours of culture, the supernatant is discarded (NK cells and dead tumor cells float in the supernatant), and CCK-8 detection is performed on the remaining cells.

[0070] II. Experimental results

[0071] The results are shown in Table 1. Figure 5 As shown in Table 1, the addition of OTX008 to the mixed cell solution of TUKO-NK and DU145 can significantly reduce the activity of tumor cells; when the concentration of OTX008 is 0.1 μM, 1 μM and 10 μM, the activity of DU145 cells can be reduced to 65.75%, 26.79% and 4.87% respectively by TUKO-NK cells, and the reduction amplitude is significantly higher than the sum of the effects of TUKO-NK cells and OTX008 used alone, which has a synergistic effect and can be used for the treatment of prostate cancer.

[0072] Experimental Example 4: Inhibition of TUKO-NK cells on the growth of prostate cancer in mice in vivo

[0073] I. Experimental method

[0074] (1) Prostate cancer DU145 cells are injected subcutaneously into CB-17 SCID nude mice (1 x 10 6 cells per mouse), to construct 6 tumor-bearing mice.

[0075] (2) At the same time, 1 x 10 6 DU145 cells and TUKO cells are injected subcutaneously into CB-17 SCID mice, for a total of 2 groups, 4 NK donor mice per group.

[0076] (3) Ten days later, following the experimental method in Example 1, DU145-NK and TUKO-NK cells were isolated from the spleens of the two groups of NK donor mice. Two × 10⁶ cells of each DU145-NK and TUKO-NK cell line were collected. 5 Each mouse was resuspended in 200 μL of PBS and injected via the tail vein into the tumor-bearing mice of step (1) (2 mice per group, 100 μL per mouse). The remaining 2 mice from step (1) were injected via the tail vein with 100 μL of PBS as a control group.

[0077] (4) After continuing to feed for 3 weeks, the size of the tumor was detected using a live imaging system.

[0078] II. Experimental Results

[0079] The results are as follows Figure 6 As shown, compared with the control group, the tumors in mice infused with DU145-NK cells did not shrink significantly, while the tumors in mice infused with TUKO-NK cells shrank. This indicates that TUKO-activated NK cells have an inhibitory effect on prostate cancer in vivo.

[0080] Experimental Example 5: The Gal-1 inhibitor OTX008 had no significant inhibitory effect on prostate cancer tumor growth in mice in vivo.

[0081] I. Experimental Methods

[0082] (1) DU145 cells were subcutaneously injected into the groin of four CB-17 SCID nude mice, with each mouse receiving 1×10⁻⁶ cells. 5 One tumor cell.

[0083] (2) Preparation of OTX008 solution: Take 1 mg of OTX008, add 200 μL of DMSO, and sonicate to dissolve to obtain a 5 mg / ml OTX008 stock solution. Take 16 μL of the stock solution and add it to 384 μL of PBS to obtain an OTX008 injection solution with a concentration of 0.2 mg / ml.

[0084] (3) Ten days after the mice were born with tumors, 100 μL of the above OTX008 injection solution was injected into the mice via the tail vein.

[0085] (4) Continue feeding the mice for 3 weeks and use an in vivo imaging system to detect the size of the tumor.

[0086] II. Experimental Results

[0087] The results are as follows Figure 7 As shown, the size of the tumors in the mice treated with OTX008 was not significantly different from that in the control group, indicating that OTX008 treatment did not significantly inhibit tumor growth.

[0088] Experimental Example 6 In vivo, the therapeutic effect of TUKO-NK cells combined with OTX008 on prostate cancer

[0089] I. Experimental Methods

[0090] (1) 8 CB-17 SCID nude mice were subcutaneously injected at the root of the thigh with DU145 cells, 1 x 10 5 tumor cells per mouse.

[0091] (2) At the same time, 1 x 10 6 TUKO cells were subcutaneously inoculated in CB-17 SCID mice, a total of 8 NK donor mice.

[0092] (3) After 10 days, the TUKO-NK cells in the spleen of the CB-17 SCID mice in (2) above were sorted according to the method of Experimental Example 1; 4 x 10 5 TUKO-NK cells were taken, washed and centrifuged with PBS, and the supernatant was discarded.

[0093] (4) Preparation of OTX008 solution: Take 1 mg OTX008, add 200 μL DMSO, ultrasonic dissolution, get 5 mg / ml OTX008 stock solution. Take 32 μL stock solution and add to 768 μL PBS to get 800 μL of 0.2 mg / ml OTX008 injection.

[0094] (5) Take 200 μL OTX008 injection, transfer into a centrifuge tube containing 2 x 10 5 TUKO-NK cells, resuspend the cells to get a mixed treatment solution.

[0095] (6) TUKO-NK + OTX008 combined treatment group: inject the mixed treatment solution into the tumor-bearing mice in (1) through the tail vein, 100 μL per mouse;

[0096] OTX008 treatment group: inject OTX008 injection into the tumor-bearing mice in (1) through the tail vein, 100 μL per mouse;

[0097] TUKO-NK reinfusion treatment group: take 2 x 10 5 TUKO-NK cells, resuspend in 200 μL PBS, and inject into the tumor-bearing mice in (1) through the tail vein, 100 μL per mouse, a total of 2 mice;

[0098] PBS control group: inject 100 μL of PBS into the tail vein of the remaining 2 mice in step (1).

[0099] (7) Continue to raise the mice for 3 weeks, and observe the tumor size by live imaging instrument.

[0100] II. Experimental results

[0101] Results as shown in Figure 8 Compared with the control group, TUKO-NK combined with OTX008 can significantly inhibit the growth of prostate cancer in mice, and the inhibitory effect on tumors is better than the sum of the effects of the two alone, has a synergistic effect, and shows a significant therapeutic effect.

[0102] In summary, knocking out TTTY15-USP9Y chimera RNA in tumor cells can increase the infiltration and anti-tumor activity of natural killer (NK) cells in the microenvironment of prostate cancer, and NK cells activated by TUKO have stronger killing effect on tumor cells in vitro, however, Gal-1 inhibitor OTX008 alone has no obvious killing effect on prostate cancer cells; the present application combines Gal-1 inhibitor with activated NK cells, which can efficiently kill prostate cancer cells, showing a synergistic effect in the treatment of prostate cancer, and has a broad application prospect.

Claims

1. A combination drug for treating prostate cancer, characterized in that: The combination drug is infiltrating immune cells with enhanced degree and galectin-1 inhibitor, the infiltrating immune cells with enhanced degree are selected from NK cells of spleen of tumor cell TUKO of DU145 tumor cell TTTY15-USP9Y chimera RNA knockout mice, and the galectin-1 inhibitor is selected from OTX008.

2. The combination of claim 1, wherein: The use amount ratio of the immune cells with enhanced infiltration and the galectin-1 inhibitor is: 2 x 10 5 immune cells with enhanced infiltration: (0.001-0.125) mg.

3. The combination of claim 1, wherein: The nucleotide sequence of the TTTY15-USP9Y chimera RNA is shown as SEQ ID NO.

3.

4. Use of the combination drug according to any one of claims 1-3 in the preparation of a drug for treating prostate cancer.

5. A pharmaceutical composition for treating prostate cancer, characterized by: It is an infiltrating degree enhanced immune cell and a galectin-1 inhibitor as active ingredients, adding a pharmaceutically acceptable adjuvant to form; the infiltrating degree enhanced immune cell is selected from the NK cells of the spleen of the tumor cell TUKO of the DU145 tumor cell TTTY15-USP9Y chimera RNA knockout of the tumor-bearing mouse, and the galectin-1 inhibitor is selected from OTX008; the use amount ratio of the infiltrating degree enhanced immune cell and the galectin-1 inhibitor is: 2×10 5 Infiltrating degree enhanced immune cells: (0.001-0.125) mg.

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