Application of histone deacetylase inhibitor and CAR-T cell targeting DR5 in preparation of medicine for treating tumors

Abstract

The invention discloses application of a histone deacetylase inhibitor combined with a CAR-T cell targeting DR5 in preparation of a medicine for treating tumors, and further provides a combined medicine for treating tumors, and the combined medicine comprises the histone deacetylase inhibitor and the CAR-T cell targeting DR5. The CAR-T cells targeting DR5 and the histone deacetylase inhibitor are combined for use, and the CAR-T cells and the histone deacetylase inhibitor can generate a synergistic effect, can generate an excellent curative effect on malignant tumors, and can reduce the dosage of the CAR-T cells, reduce the depletion of the CAR-T cells, reduce the level of cell factors, increase the safety of CAR-T treatment and reduce the treatment cost; moreover, tumor cells are more sensitive to DR5CAR-T, and tumor escape is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the use of histone deacetylase inhibitors combined with DR5-targeted CAR-T cells in the preparation of drugs for treating tumors. Background Art

[0002] CAR-T cell therapy expresses chimeric antigen receptor (CAR) molecules on the surface of T cells. CAR-T cells can not only specifically recognize and bind to tumor antigens, but also possess the self-renewal and killing abilities of T cells, and specifically kill tumor cells in a non-MHC-dependent manner. CAR-T cell therapy is a new type of precision targeted therapy for treating tumors. Currently, 10 CAR-T cell products have been approved for marketing globally, with the treatment targets concentrated on CD19 and BCMA, and the treated diseases concentrated in the fields of B-cell leukemia, lymphoma, and multiple myeloma. However, in the face of solid tumors accounting for more than 90%, there is currently no successful CAR-T cell product on the market, and there is an urgent need to develop new immunocyte therapy methods.

[0003] Death receptor 5 (DR5) is highly expressed in human cancers, including liver cancer, colon cancer, gastric cancer, pancreatic cancer, ovarian cancer, breast cancer, non-small cell lung cancer, etc., and is not expressed or is lowly expressed in normal cells. Therefore, DR5 is considered to be a target for various tumors. The applicant has previously developed a murine monoclonal antibody 3E7 against DR5 (ZL201711166864.0), obtained a humanized monoclonal antibody against DR5 and CAR-T cells using the antibody scFv sequence, and proved that DR5 CAR-T cells can target DR5-positive tumors and exert an anti-tumor effect (patent application number: 202210932327.7).

[0004] Although DR5 CAR-T has shown significant anti-tumor effects in immunodeficient mice, in clinical treatment, low-dose DR5 CAR-T is not strong enough in killing tumor cells. The reason may be that the abundance of DR5 expressed by human tumor cells is lower than that of tumor cell lines cultured in vitro, making the in vivo tumor cells less sensitive to the killing of DR5 CAR-T. In addition, the immune system of tumor patients is often damaged, the T cell function is weak or in a depleted state, or more inhibitory molecules such as PD1 and Tim3 are expressed. Therefore, it is necessary to study how to enhance the killing of tumor cells by CAR-T cell therapy to improve the anti-tumor efficacy.

[0005] Histone deacetylase inhibitors (HDACi) are a class of compounds that interfere with the function of histone deacetylases. By increasing the degree of histone acetylation in cells, they inhibit the proliferation of tumor cells and induce cell differentiation and / or apoptosis. According to the structure of HDACi, they can be divided into: benzamides (such as MS-275), hydroxamic acids (such as Trichostatin (TSA), Vorinostat (SAHA)), fatty acids (such as valproic acid, butyrate, phenylbutyrate), and cyclic peptides (such as the natural product depsipeptide FK-228, Trapoxin A (TPA), Apicidin). TSA and SAHA are non-selective and can inhibit both class I and class II HDACs simultaneously. Currently, four HDACi have been approved by the FDA for clinical use, including romidepsin, suberoylanilide hydroxamic acid (SAHA), and belinostat, which are approved for T-cell lymphoma, and panobinostat, which is used to treat multiple myeloma. Currently, there are no reports on the combined use of HDACi and DR5 CAR-T cells in the treatment of tumors. Summary of the Invention

[0006] In previous studies by the applicant, it was found that in clinical treatment with DR5 CAR-T products, low-dose DR5 CAR-T had poor killing effects on tumor cells, while high-dose CAR-T might increase the risks in terms of safety such as cytokine storm or neurotoxicity. To address the deficiencies in the prior art, the purpose of the present invention is to provide the use of histone deacetylase inhibitors in combination with DR5-targeted CAR-T cells in the preparation of drugs for treating tumors.

[0007] The specific technical solution of the present invention is as follows:

[0008] On the one hand, the present invention provides the use of histone deacetylase inhibitors in combination with DR5-targeted CAR-T cells in the preparation of drugs for treating tumors.

[0009] Further, the histone deacetylase inhibitor is selected from one or more of hydroxamic acid histone deacetylase inhibitors.

[0010] Further, the histone deacetylase inhibitor is selected from SAHA and / or TSA.

[0011] Further, the DR5-targeted CAR-T cell is a T cell expressing a DR5-targeted CAR;

[0012] Preferably, the T cells are derived from autologous T cells, allogeneic T cells, or iPSC-induced T cells.

[0013] Furthermore, the DR5 CAR comprises, from the amino terminus to the carboxyl terminus: a DR5 antigen recognition domain, a hinge region, a transmembrane region, a co-stimulatory region, and a signal transduction region;

[0014] Preferably, the DR5 antigen recognition domain comprises a heavy chain, a linker peptide, and a light chain, the amino acid sequence of the heavy chain is selected from one of SEQ ID NO: 1-6, and the amino acid sequence of the light chain is selected from one of SEQ ID NO: 7-12;

[0015] Preferably, the amino acid sequence of the linker peptide comprises (GGGGS)n, where n is a positive integer from 2 to 50;

[0016] Preferably, the DR5 antigen recognition domain comprises, from the amino terminus to the carboxyl terminus, a light chain, a linker peptide, and a heavy chain, the amino acid sequence of the light chain is as shown in SEQ ID NO: 11, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 4 or SEQ ID NO: 6, and the amino acid sequence of the linker peptide comprises (GGGGS)n, where n is 3;

[0017] Preferably, the hinge region is selected from CD8α;

[0018] Preferably, the transmembrane region is selected from CD8 or CD28;

[0019] Preferably, one or two co-stimulatory regions are selected;

[0020] Preferably, the co-stimulatory region is selected from CD28 and / or 4-1BB;

[0021] Preferably, the signal transduction region is selected from CD3ζ.

[0022] Furthermore, the tumor is a tumor expressing DR5;

[0023] Preferably, the tumor is selected from one or more of malignant lymphoma, liver cancer, colon cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, cervical cancer, and prostate cancer.

[0024] The present invention also provides a combined drug for treating tumors, which comprises a histone deacetylase inhibitor and CAR-T cells targeting DR5;

[0025] Preferably, the histone deacetylase inhibitor is selected from the above-mentioned histone deacetylase inhibitors;

[0026] Preferably, the CAR-T cells targeting DR5 are selected from the above-mentioned CAR-T cells targeting DR5.

[0027] Furthermore, the combination drug also includes other tumor treatment drugs;

[0028] Preferably, the other tumor therapeutic drugs include immune checkpoint inhibitors;

[0029] Preferably, the immune checkpoint inhibitor is selected from one or more of anti-PD-1 antibodies, anti-PDL1 antibodies, anti-TIM3 antibodies, and anti-CTLA-4 antibodies.

[0030] Furthermore, the combination drug is administered sequentially or simultaneously by injection;

[0031] Preferably, the histone deacetylase inhibitor is administered by intraperitoneal injection, and the CAR-T cells targeting DR5 are administered by intravenous injection.

[0032] Furthermore, the histone deacetylase inhibitor is administered first, and the CAR-T cells targeting DR5 are administered later, so that the histone deacetylase inhibitor can first stimulate the tumor cells in the patient's body to produce more DR5 expression, thereby enhancing the recognition and treatment effect of the CAR-T cells targeting DR5 on malignant tumor cells; and it will not affect the cell activity of the CAR-T cells targeting DR5 themselves.

[0033] The beneficial effects of the present invention are:

[0034] The present invention provides a new use of a histone deacetylase inhibitor combined with DR5-targeted CAR-T cells in the preparation of a drug for treating tumors. Compared with the use of DR5 CAR-T alone, the combination of histone deacetylase inhibitor and DR5 CAR-T can produce a synergistic effect and can significantly enhance the killing effect on malignant tumor cells.

[0035] The present invention found that HDACi can enhance the sensitivity of tumor cells to DR5 CAR-T killing, and then DR5 CAR-T cells can kill tumor cells faster and more. The combination of the two shows a synergistic effect, and the anti-tumor effect is significantly enhanced. It can also reduce the dosage of DR5 CAR-T cells and reduce the level of cytokine secretion by CAR-T cells, thereby reducing the side effects caused by CAR-T cell therapy. And HDACi can inhibit the expression of CAR-T cell immunosuppressive molecules PD1 and Tim3, reduce CAR-T cell exhaustion, and thus enhance the anti-tumor function of CAR-T. In addition, HDACi is a small molecule compound drug, which is cheap and can reduce the cost of CAR-T cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1Flow cytometry graph for detecting the positive rate of CAR in DR5 CAR-T, where DR5 CAR-T uses the TND5 sequence.

[0037] Figure 2 RTCA detection results of SAHA or TSA combined with DR5 CAR-T killing Huh7 cells, where DR5 CAR-T uses the TND5 sequence. Medium represents the normal growth of tumor cells in the culture medium; DR5 CAR-T 1:1 means that DR5 CAR-T cells are added to the tumor cells with an effector-to-target ratio of 1:1; SAHA or TSA + DR5 CAR-T means that both CAR-T cells and SAHA or TSA are added to the tumor cells; SAHA-0.25 μM or TSA-0.05 μM means that only SAHA or TSA is added to the tumor cells.

[0038] Figure 3 RTCA detection results of DR5 CAR-T killing Huh7 cells (effector-to-target ratio is 5:1), where DR5 CAR-T uses the TND5 sequence.

[0039] Figure 4 Graph of cytokine release after co-culturing SAHA combined with DR5 CAR-T and Huh7 for 33 h (effector-to-target ratio 10:1), where DR5 CAR-T uses the TND5 sequence.

[0040] Figure 5 Graph of cytokine release after co-culturing SAHA combined with DR5 CAR-T and HepG2 for 66 h (effector-to-target ratio 5:1), where DR5 CAR-T uses the TND6 sequence.

[0041] Figure 6 RTCA detection results of SAHA combined with DR5 CAR-T killing PANC1 cells, where DR5 CAR-T uses the TND4 sequence.

[0042] Figure 7 Results of fluorescence quantitative PCR for detecting the mRNA levels of PD1 and Tim3 after treating DR5 CAR-T with SAHA for 24 hours, where DR5 CAR-T uses the TND5 sequence.

[0043] Figure 8 Results of anti-PANC1 tumor in mice with SAHA combined with DR5 CAR-T, where DR5 CAR-T uses the TND5 sequence. Detailed implementation methods

[0044] To better understand the present invention, the present invention will be further described with reference to the following embodiments and accompanying drawings. The embodiments are only for explanation and do not limit the present invention in any way. In the embodiments, all original reagent materials are commercially available. The experimental methods without specific conditions are conventional methods and conventional conditions well-known in the art, or the conditions recommended by the instrument manufacturer.

[0045] Example 1

[0046] 1. Preparation of lentivirus expressing DR5 CAR

[0047] The structure of DR5 CAR used in this example is shown in Table 1, where L6 represents the light chain, H5 and H7 represent the heavy chains, and linker1 represents the linker peptide. CD8h represents the CD8α hinge region, CD8Tm represents the CD8 transmembrane region, CD28 represents the co-stimulatory region, 4-1BB represents the co-stimulatory region, and CD3ζ represents the signal transduction region. The amino acid sequence of L6 is shown in SEQ ID NO:11, and the encoding nucleotide sequence is shown in SEQ ID NO:14. The amino acid sequences of H5 and H7 are shown in SEQ ID NO:4 and SEQ ID NO:6 respectively, and the encoding nucleotide sequences are shown in SEQ ID NO:15 and SEQ ID NO:16 respectively. The amino acid sequence of linker1 is shown in SEQ ID NO:13. The amino acid sequence of CD8h is shown in SEQ ID NO:17, the amino acid sequence of CD8Tm is shown in SEQ ID NO:18, the amino acid sequence of CD28Tm is shown in SEQ ID NO:19, the amino acid sequence of CD28 is shown in SEQ ID NO:20, the amino acid sequence of 4-1BB is shown in SEQ ID NO:21, and the amino acid sequence of CD3ζ is shown in SEQ ID NO:22. Synthesize the encoding nucleotide sequences of the CARs shown in Table 1 and construct them into the expression plasmid pCDH respectively to obtain the recombinant expression plasmids TND4, TND5, and TND6 (as shown in Table 1 below). Co-transfect the recombinant expression plasmid with the packaging plasmids (pMDLg / pRRE and pRSV-REV) and the envelope plasmid (pMD2.G) into the cultured HEK293T cells. After 48 hours, harvest the cell culture supernatant, centrifuge, and harvest the recombinant lentivirus, which is the lentivirus expressing DR5 CAR.

[0048] Table 1 List of CAR-DR5 structures

[0049] CAR-DR5 structure DR5 CAR recombinant expression plasmid number L6-linker1-H7-CD8h-CD8Tm-41BB-CD3ζ TND4 L6-linker1-H7-CD8h-CD28Tm-CD28-41BB-CD3ζ TND5 L6-linker1-H5-CD8h-CD8Tm-41BB-CD3ζ TND6

[0050] 2. Preparation of CAR-T cells

[0051] Peripheral blood was collected, and the buffy coat cells were taken after Ficoll density gradient centrifugation to obtain mononuclear cells PBMC. According to the ratio of magnetic beads to cells of 3:1, anti-CD3 / anti-CD28 immunomagnetic beads were added for incubation, and CD3-positive T lymphocytes were screened out. The cells were resuspended with the medium, and after 1 day of culture, lentivirus expressing DR5 CAR with a multiplicity of infection (MOI) of 3 - 5 was added for co-culture. After the cells were cultured for 8 - 14 days, the magnetic beads were removed by a magnet, and centrifuged at 500g for 8 min to obtain chimeric antigen receptor T cells targeting DR5 (DR5 CAR-T cells).

[0052] Take 1×10 6 The DR5 CAR-T cells were first incubated with 1 μL of DR5 protein in the dark at 4°C for 30 min, then 1 mL of Dulbecco's phosphate buffered saline (DPBS) was added for washing, centrifuged at 300g for 5 min, the supernatant was removed, and resuspended with 100 μL of DPBS + 2% fetal bovine serum (FBS). 1 μL of CD3 antibody and 0.5 μL of phycoerythrin-labeled streptavidin (PE-streptavidin) were added to each tube, mixed well and incubated in the dark at 4°C for 30 min. 1 mL of DPBS was added for washing, centrifuged to remove the supernatant, and after resuspending with 200 μL of DPBS, the positive rate of the obtained DR5 CAR-T cells was detected by flow cytometry, using T cells only stimulated by magnetic beads without virus infection (UTD) as a control. The results are as Figure 1 shown.

[0053] Example 2

[0054] 1. Detection of in vitro killing function

[0055] The real-time cell analyzer (xCElligence RTCA SP) was used to perform the tumor cell killing experiment. First, 50 μL of complete medium containing 10,000 Huh7 or HepG2 or PANC1 cells was added to the wells of the E-Plate supporting this instrument for culture. After about 5 - 24 h, the corresponding number of DR5 CAR-T cells and different concentrations of SAHA or TSA were added according to different effector-to-target ratios (E:T), and then co-cultured for a period of time (>24 h). The cell killing effect was analyzed according to the CI value of the real-time cell analyzer. The cell culture supernatant was taken, and the cytokine levels were detected using the BD CBA detection kit.

[0056] (1) Analysis of tumor cell killing effect

[0057] The RTCA detection results of SAHA or TSA combined with DR5 CAR-T killing Huh7 cells are as Figure 2 shown, and from Figure 2It can be seen that DR5 CAR-T cells have an obvious killing effect on DR5-positive tumor cells Huh7. Treatment of tumor cells with low concentrations of SAHA or TSA for 24 hours has almost no effect on killing tumor cells, while the combination of DR5 CAR-T cells with SAHA or TSA can significantly enhance the effect of killing tumor cells at 24 hours.

[0058] The RTCA detection results of DR5 CAR-T killing Huh7 cells are as Figure 3 shown. From Figure 2 and 3 it can be seen that the anti-tumor effect of 0.25 μM SAHA combined with DR5 CAR-T cells with an effector-to-target ratio of 1:1 on Huh7 can reach the anti-tumor effect of DR5 CAR-T cells with an effector-to-target ratio of 5:1 on Huh7.

[0059] The RTCA detection results of SAHA combined with DR5 CAR-T killing PANC1 cells are as Figure 6 shown. From Figure 6 it can be seen that under the same effector-to-target ratio condition (0.5:1), the combination of SAHA and DR5 CAR-T cells has a significantly enhanced killing effect on DR5-positive tumor cells PANC1 compared with DR5 CAR-T cells alone, while the use of the same concentration of SAHA alone has almost no effect on killing tumor cells at 24 hours.

[0060] According to the analysis of the combination index (CI)-compusyn software, the calculation results of the combination index of SAHA or TSA and DR5 CAR-T cells killing tumors are shown in Table 2-4 below:

[0061] Table 2 Calculation table of the anti-tumor combination index of SAHA and DR5 CAR-T cells

[0062] SAHA (μM) DR5 CAR-T (E:T) Tumor cell killing (%) CI 0.125 1:1 65.89 0.59939 0.25 1:1 76.57 0.42206 0.5 1:1 79.35 0.39007

[0063] Table 3 Calculation table of the anti-tumor combination index of TSA and DR5 CAR-T cells

[0064]

[0065]

[0066] Table 4 Definition of the combination index

[0067] CI Description <0.1 Very strong synergism 0.1～0.3 Strong synergism 0.3～0.7 Synergism 0.7～0.85 Moderate synergism 0.85～0.90 Slight synergism 0.90～1.10 Nearly additive

[0068] As can be seen from the above table, when SAHA at 0.125 - 0.5 μM or TSA at 0.05 - 0.1 μM acts in combination with DR5 CAR-T, the combination index is between 0.3 and 0.7. Therefore, there is an obvious synergistic effect in killing tumors. When 0.2 μM TSA acts in combination with DR5 CAR-T, the combination index is between 0.1 and 0.3. Therefore, there is a strong synergistic effect in killing tumors.

[0069] (2) Results of cytokine level detection

[0070] The figure showing the cytokines released after co-culturing SAHA combined with DR5 CAR-T and Huh7 for 33 h (effector-to-target ratio 10:1) is as Figure 4 shown. As Figure 4 can be seen, after co-culturing DR5 CAR-T cells and DR5-positive tumor cells Huh7, a large amount of cytokines can be secreted, such as IL-2, IFN-γ, and TNF-α. When SAHA is added simultaneously, the level of cytokines secreted by CAR-T cells shows a dose-dependent decrease.

[0071] The figure showing the cytokines released after co-culturing SAHA combined with DR5 CAR-T and HepG2 for 66 h (effector-to-target ratio 5:1) is as Figure 5 shown. As Figure 5 can be seen, after co-culturing DR5 CAR-T cells and DR5-positive tumor cells HepG2, IL-6 and TNF-α can be secreted. When SAHA is added simultaneously, the levels of IL-6 and TNF-α secreted by CAR-T cells show a dose-dependent decrease.

[0072] 2. Effects of SAHA on immunosuppressive molecules of DR5 CAR-T cells

[0073] After treating DR5 CAR-T with SAHA for 24 hours, the levels of PD1 and Tim3 mRNA were detected by fluorescence quantitative PCR. The results are as Figure 7 , as Figure 7 can be seen, low-concentration SAHA can reduce the expression of immunosuppressive molecules of CAR-T cells and reduce T cell exhaustion.

[0074] 3. Antitumor experiments in vivo

[0075] Twenty 6-week-old female B-NDG mice were subcutaneously inoculated with 0.2 mL / mouse of PANC1 cells (containing 5×10 6 cells). When the average tumor size grew to ~100 mm 3 , the animals were divided into 4 groups and were respectively given SAHA (2 mg / kg, i.p., injected 5 times in total, once every other day), DR5 CAR-T (1.32×10 6CAR-T cells / only, i.v., CAR positive rate 39.9%, injected only once), SAHA combined with DR5 CAR-T and solvent (model group), where i.p. is intraperitoneal injection and i.v. is intravenous injection. Tumor size was measured with vernier calipers, weighed, and the survival of mice was observed. 37 days after DR5 CAR-T injection, the mice were euthanized, and the tumors were isolated and weighed. Tumor volume = 1 / 2 × (long diameter) × (short diameter) × (short diameter).

[0076] Anti-PANC1 tumor in SAHA combined with DR5 CAR-T mice in vivo was as Figure 8 shown, and it can be seen from Figure 8 that DR5 CAR-T cells have significant anti-tumor effects in vivo. In terms of tumor weight, CAR-T group vs model group, p < 0.0001; CAR-T group vs SAHA+CAR-T group, p < 0.05; SAHA group vs model group, p > 0.5. In addition, the body weights of the mice in the CAR-T injection group, SAHA group, and combined treatment group were all stable, with no significant difference from the body weights of the mice in the model group, indicating that the combined treatment of SAHA and CAR-T cells has good safety in vivo.

[0077] According to Appendix Figure 2 , Table 2 and Table 3, it can be seen that when histone deacetylase inhibitor SAHA or TSA acts on tumor cells in combination with DR5 CAR-T cells, it can significantly synergistically kill tumors and significantly enhance the anti-tumor effect. According to Appendix Figure 4 and 5 , it can be seen that when histone deacetylase inhibitor SAHA acts on tumor cells in combination with DR5 CAR-T cells, SAHA can dose-dependently reduce the levels of cytokines secreted by CAR-T cells, such as IL-2, IFN-γ, TNF-α, and IL-6. According to Appendix Figure 2 and 6 , it can be seen that the synergistic anti-tumor effect produced by the combination of histone deacetylase inhibitor and DR5 CAR-T cells exists in a variety of DR5-positive tumors, such as liver cancer and pancreatic cancer. According to Appendix Figure 7 , it can be seen that histone deacetylase inhibitor SAHA can reduce the expression of immunosuppressive molecules of CAR-T cells and reduce T cell exhaustion. According to Appendix Figure 8 , it can be seen that histone deacetylase inhibitor SAHA can synergize with DR5 CAR-T in vivo to enhance the anti-tumor efficacy and has good safety. In summary, the combination of histone deacetylase inhibitor and DR5 CAR-T can produce a synergistic effect and can significantly enhance the killing effect on malignant tumor cells.

[0078] SEQ ID NO:1

[0079] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTCWMNWVRQAPGKGLVWIGEINPDSSRINYMPSLKERFTISRDNAKNTLYLQMNSLRAEDTAVYYCARGGTFYAMDYWGQGTTVTVSS

[0080] SEQ ID NO:2

[0081] EVQLVESGGGLVQPGGSLRLSCAASGFTFSTCWMNWVRQAPGKGLEWIGEINPDSSRINYMPSLKERFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGGTFYAMDYWGQGTTVTVSS

[0082] SEQ ID NO:3

[0083] EVQLLESGGGLVKPGGSLRLSCAASGFTFSTCWMNWVRQAPGKGLEWIGEINPDSSRINYMPSLKEKFTISRDNSKNTLYLQMSSVRAEDTALYYCARGGTFYAMDYWGQGTTVTVSS

[0084] SEQ ID NO:4

[0085] EVQLVESGGGVVQPGGSLRLSCAASGFTFDTCWMNWVRQAPGKGLEWIGEINPDSSRINYMPSLKEKFTISRDNSKNTLYLQMSSVRTEDTALYYCARGGTFYAMDYWGQGTTVTVSS

[0086] SEQ ID NO:5

[0087] QVQLVESGGGLVQPGGSLRLSCAASGFTFSTCWMNWVRQAPGKGLVWIGEINPDSSRINYMPSLKEKFTISRDNAKNTLYLQMSSVRAEDTALYYCARGGTFYAMDYWGQGTTVTVSS

[0088] SEQ ID NO:6

[0089] EVQLVESGGGLVKPGGSLRLSCAASGFDFSTCWMNWVRQAPGKGLEWIGEINPDSSRINYMPSLKEKFTISRDNAKNTLYLQMSSVRAEDTALYYCARGGTFYAMDYWGQGTTVTVSS

[0090] SEQ ID NO:7

[0091] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLPFTFGQGTKLEIK

[0092] SEQ ID NO:8

[0093] DIVMTQTPLSLSVTPGQPASISCKSSQSLLNSRTRKNYLAWYLQKPGQSPQLLIYWASTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCKQSYNLPFTFGQGTKLEIK

[0094] SEQ ID NO:9

[0095] DIVMTQSPDSLAVSLGERVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLPFTFGQGTKLEIK

[0096] SEQ ID NO:10

[0097] DIVMTQSPLSLAVSPGQPVSMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPQLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLPFTFGQGTKLEIK

[0098] SEQ ID NO:11

[0099] EIVMTQSPPSLAVSPGERVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQAPRLLIYWASTRESGVPARFSGSGSGTDFTLTISSVQPEDLAVYYCKQSYNLPFTFGQGTKLEIK

[0100] SEQ ID NO:12

[0101] DIQMTQSPSSLAVSVGDRVTMSCKSSQSLLNSRTRKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSVQPEDLAVYYCKQSYNLPFTFGQGTKLEIK

[0102] SEQ ID NO:13

[0103] GGGGSGGGGSGGGGS

[0104] SEQ ID NO:14

[0105] GAGATCGTGATGACCCAGAGCCCTCCCAGCCTTGCTGTGTCTCCTGGCGAGAGAGTGACCATGAGCTGCAAGAGCAGCCAGAGCCTGCTGAACAGCAGGACCAGGAAGAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTAGGCTGTTGATCTACTGGGCCAGCACAAGGGAGAGCGGCGTGCCTGCTAGATTCTCTGGAAGCGGATCCGGAACCGACTTCACCCTGACCATCAGCAGCGTGCAGCCCGAAGACCTTGCCGTGTACTACTGCAAGCAGAGCTACAACCTGCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG

[0106] SEQ ID NO:15

[0107] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGAGTTGTTCAACCTGGAGGAAGCTTGAGGCTGAGCTGCGCTGCCAGCGGCTTCACCTTCGACACCTGTTGGATGAACTGGGTGAGGCAGGCCCCCGGAAAAGGCCTGGAGTGGATTGGCGAGATCAACCCTGACAGCAGCAGGATCAACTACATGCCCAGCCTGAAGGAGAAGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAGCAGCGTGAGGACCGAGGACACCGCCCTGTACTATTGTGCCAGGGGAGGAACCTTCTACGCCATGGACTACTGGGGCCAGGGCACAACCGTGACCGTCAGCAGC

[0108] SEQ ID NO:16

[0109] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCTTAGTTAAACCTGGCGGAAGCTTGAGGCTGAGCTGCGCTGCCAGCGGCTTCGATTTCAGCACCTGCTGGATGAACTGGGTGAGGCAGGCCCCCGGCAAGGGATTAGAGTGGATCGGCGAGATCAATCCCGACAGCAGCAGGATCAACTACATGCCCAGCCTGAAGGAGAAGTTCACCATCAGCAGGGACAACGCCAAGAACACCCTGTACCTGCAGATGAGCAGCGTGAGGGCCGAGGACACCGCTCTGTATTACTGCGCTAGGGGAGGAACCTTCTACGCCATGGACTACTGGGGCCAGGGAACCACCGTGACCGTCAGCAGC

[0110] SEQ ID NO:17

[0111] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI

[0112] SEQ ID NO:18

[0113] YIWAPLAGTCGVLLLSLVITLYCKRG

[0114] SEQ ID NO:19

[0115] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0116] SEQ ID NO:20

[0117] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0118] SEQ ID NO:21

[0119] RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0120] SEQ ID NO:22

[0121] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0122] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description.

Claims

1. Use of histone deacetylase inhibitor combined with DR5-targeted CAR-T cells in the preparation of a medicament for treating tumors.

2. The use according to claim 1, wherein The histone deacetylase inhibitor is selected from one or more of hydroxamic acid-based histone deacetylase inhibitors.

3. The use according to claim 1, characterized in that, The histone deacetylase inhibitor is selected from SAHA and / or TSA.

4. The use according to claim 1, characterized in that, The DR5-targeted CAR-T cells are T cells expressing DR5-targeted CAR; Preferably, the T cells are derived from autologous T cells, allogeneic T cells or iPSC-induced T cells.

5. The use according to claim 4, characterized in that, The DR5 CAR comprises, from the amino terminus to the carboxyl terminus: a DR5 antigen recognition domain, a hinge region, a transmembrane region, a co-stimulatory region and a signal transduction region; Preferably, the DR5 antigen recognition domain comprises a heavy chain, a linker peptide and a light chain, the amino acid sequence of the heavy chain is selected from one of SEQ ID NO:1-6, and the amino acid sequence of the light chain is selected from one of SEQ ID NO:7-12; Preferably, the amino acid sequence of the linker peptide comprises (GGGGS)n, where n is a positive integer from 2 to 50; Preferably, the DR5 antigen recognition domain comprises, from the amino terminus to the carboxyl terminus, a light chain, a linker peptide and a heavy chain, the amino acid sequence of the light chain is as shown in SEQ ID NO:11, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:4 or SEQ ID NO:6, and the amino acid sequence of the linker peptide comprises (GGGGS)n, where n is 3; Preferably, the hinge region is selected from CD8α; Preferably, the transmembrane region is selected from CD8 or CD28; Preferably, one or two co-stimulatory regions are selected; Preferably, the co-stimulatory region is selected from CD28 and / or 4-1BB; Preferably, the signal transduction region is selected from CD3ζ.

6. The use according to claim 1, wherein The tumor is a tumor expressing DR5; Preferably, the tumor is selected from one or more of malignant lymphoma, liver cancer, colon cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, cervical cancer and prostate cancer.

7. A combined drug for treating tumors, characterized in that, The combined medicament comprises a histone deacetylase inhibitor and DR5-targeted CAR-T cells; Preferably, the histone deacetylase inhibitor is selected from the histone deacetylase inhibitors described in claim 2 or 3; Preferably, the DR5-targeted CAR-T cells are selected from the DR5-targeted CAR-T cells described in claim 4 or 5.

8. The combined drug according to claim 7, wherein, The combined medicament further comprises other anti-tumor drugs; Preferably, the other anti-tumor drugs comprise immune checkpoint inhibitors; Preferably, the immune checkpoint inhibitors are selected from one or more of anti-PD-1 antibody, anti-PDL1 antibody, anti-TIM3 antibody, anti-CTLA-4 antibody.

9. The combination drug according to claim 7, wherein, The administration mode of the combined medicament is sequential administration or simultaneous administration by injection; Preferably, the histone deacetylase inhibitor is administered by intraperitoneal injection, and the DR5-targeted CAR-T cells are administered by intravenous injection.

10. The combination drug according to claim 7, characterized in that, The histone deacetylase inhibitor is administered first, and the DR5-targeted CAR-T cells are administered later.

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