Application of oncolytic virus and DC vaccine in preparation of antitumor drugs

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of oncolytic virus and DC vaccine in preparation of antitumor drugs. The inventor finds that the oncolytic virus can reactivate dendritic cells inhibited by tumors in vitro and can increase the infiltration proportion and tumor killing effect of T cells in a tumor microenvironment, so that the anti-tumor effect of the DC vaccine is remarkably enhanced; the invention further provides application of the oncolytic virus in preparation of DC vaccine anti-tumor synergists or drug resistance reversal agents for the first time.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of oncolytic viruses and DC vaccines in the preparation of anti-tumor drugs. Background Art

[0002] Malignant tumors are the leading cause of death and disease threatening human health. According to the Global Cancer Report, there were 19.29 million new cases of cancer and 9.95 million deaths from cancer in 2020 (SUNG H, FERLAY J, SIEGEL RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries [J]. CA Cancer J Clin, 2021, 0: 1-41.). It is estimated that by 2040, the number of new cancer cases worldwide will reach 28.4 million, an increase of 47% compared to 2020. Therefore, the general public has a very wide demand for efficient and economical anti-tumor drugs (Wanqing Chen, Rongshou Zheng, et al. Cancer Statistics in China, 2015 [J]. CA Cancer J Clin, 2016, 6 (6): 115-132.).

[0003] Traditional treatments for malignant tumors include surgery, radiotherapy, and chemotherapy. Significant progress has been made in the past few decades, but they still cannot substantially improve the long-term survival rate of patients, especially for patients with refractory tumors such as liver cancer, glioma, pancreatic cancer, and osteosarcoma. Cytotoxic drugs and molecular targeted drugs are now used as first-line clinical anti-tumor drugs, but they have the limitations of large toxic side effects and / or easy to develop drug resistance. Therefore, the treatment of malignant tumors urgently needs new treatment methods and drugs with high efficiency and low toxicity.

[0004] Dendritic cells (DCs) are the most powerful antigen-presenting cells known in the human body. They can efficiently take up and present tumor antigens and activate naive T cells, playing an indispensable role in both innate and adaptive immunity. Therefore, DCs have been developed as a new anti-tumor tool, namely DC vaccines, and have been approved by the US FDA. However, although a large number of clinical trials have confirmed the safety of DC vaccines, the objective response rate of DC vaccine treatment rarely exceeds 15%. The reasons for the poor efficacy of DC vaccines may include host tolerance, inefficient tumor antigen loading, incomplete activation of DCs, and immunosuppression mediated by the tumor microenvironment, which ultimately prevent DC vaccines from normally activating T cells and causing anti-tumor responses, thereby limiting the efficacy of DC vaccines. Therefore, how to solve these problems may be the key to improving the therapeutic effect of DC vaccines.

[0005] Oncolytic virotherapy (OV therapy) is a new type of immunotherapy for malignant tumors. This therapy uses genetically engineered or naturally occurring viruses to selectively target malignant tumor cells and replicate in tumor cells through a mechanism mediated by certain membrane receptors or intracellular factors, while having no toxic side effects on normal tissues and cells. OV drugs have multiple characteristics of targeted drugs, immunotherapy drugs and gene therapy drugs, and are highly modifiable, combinable and have the potential to bring lasting anti-tumor effects. Summary of the invention

[0006] The purpose of the first aspect of the present invention is to provide the use of oncolytic viruses in the preparation of DC vaccine anti-tumor enhancers or drug resistance reversal agents.

[0007] The purpose of the second aspect of the present invention is to provide the use of DC vaccine in the preparation of oncolytic virus anti-tumor enhancers or drug resistance reversal agents.

[0008] The third aspect of the present invention aims to provide the use of oncolytic viruses and DC vaccines in the preparation of anti-tumor drugs.

[0009] The fourth aspect of the present invention aims to provide an anti-tumor composition.

[0010] The fifth aspect of the present invention aims to provide a drug set.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides the use of oncolytic viruses in the preparation of DC vaccine anti-tumor enhancers or drug resistance reversal agents.

[0013] Preferably, the oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus; further preferably, the oncolytic virus is selected from alphavirus.

[0014] Preferably, the alpha virus comprises at least one of M1 virus and Getavirus (Getavirus is a virus with a homology of up to 97.8% (Wen et al. Virus Genes. 2007; 35(3): 597-603) with the M1 virus. The two have a high identity, and the M1 virus is also classified as a Getavirus-like virus by some literature. Therefore, it can be expected that Getavirus has similar effects to alpha virus); and further comprises M1 virus.

[0015] Preferably, the DC vaccine comprises dendritic cells loaded with specific antigens of the tumor.

[0016] Preferably, the specific antigen comprises at least one of tumor-associated antigen peptides, patient's autologous tumor antigens, tumor cell antigens, and recombinant proteins, and the specific antigen changes with the changes of the tumor; and further comprises at least one of tumor-associated antigen peptides and tumor cell antigens.

[0017] Preferably, the tumor cell antigen is obtained by lysing tumor cells.

[0018] Preferably, the lysis comprises at least one of repeated freeze-thaw lysis and oncolytic lysis; and further comprises oncolytic lysis.

[0019] Preferably, the oncolytic lysis method is: mixing oncolytic virus with tumor cells to obtain.

[0020] Preferably, the oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus; further preferably, the oncolytic virus is selected from alphavirus.

[0021] Preferably, the dendritic cells are obtained by isolating or differentiating from monocytes.

[0022] Preferably, the method of isolating or inducing differentiation of monocytes to obtain dendritic cells is a conventional method in the art: for example, the method of inducing differentiation of monocytes to obtain dendritic cells can be to mix monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to induce them.

[0023] Preferably, the monocytes include at least one of bone marrow-derived monocytes, spleen-derived monocytes, peripheral blood-derived monocytes, and umbilical cord blood-derived monocytes.

[0024] Preferably, the method for preparing the dendritic cells loaded with the tumor-specific antigen is a conventional method in the art: for example, the specific antigen is mixed with the dendritic cells and cultured.

[0025] Preferably, the culture time is 20 to 28 hours.

[0026] Preferably, the tumor comprises at least one of a solid tumor and a hematological tumor; and further comprises a solid tumor.

[0027] Preferably, the solid tumor comprises liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, adrenocortical carcinoma, pararenal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, heart tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer , Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal sinus cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumors, islet cell tumors, papillomatosis, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer and single myeloma at least one; further comprising at least one of melanoma, colon cancer, pancreatic cancer, and prostate cancer.

[0028] Preferably, the blood tumor is selected from at least one of B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia and preleukemia.

[0029] Preferably, the tumor is a tumor insensitive to DC vaccine.

[0030] The present invention is based on the inventors' discovery that oncolytic viruses can reactivate dendritic cells suppressed by tumors in vitro, and can increase the infiltration ratio of T cells and the tumor killing effect in the tumor microenvironment, thereby significantly enhancing the anti-tumor effect of DC vaccines (including some DC vaccines that have extremely poor anti-tumor effects themselves). The inventors further propose the use of oncolytic viruses in the preparation of DC vaccine anti-tumor enhancers or drug resistance reversal agents.

[0031] The second aspect of the present invention provides the use of DC vaccines in the preparation of oncolytic virus anti-tumor enhancers or drug resistance reversal agents.

[0032] Preferably, the DC vaccine comprises dendritic cells loaded with specific antigens of the tumor.

[0033] Preferably, the specific antigen comprises at least one of tumor-associated antigen peptides, patient's autologous tumor antigens, tumor cell antigens, and recombinant proteins, and the specific antigen changes with the changes of the tumor; and further comprises at least one of tumor-associated antigen peptides and tumor cell antigens.

[0034] Preferably, the tumor cell antigen is obtained by lysing tumor cells.

[0035] Preferably, the lysis comprises at least one of repeated freeze-thaw lysis and oncolytic lysis; and further comprises oncolytic lysis.

[0036] Preferably, the oncolytic lysis method is: mixing oncolytic virus with tumor cells to obtain.

[0037] Preferably, the oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus; further preferably, the oncolytic virus is selected from alphavirus.

[0038] Preferably, the dendritic cells are obtained by isolating or differentiating from monocytes.

[0039] Preferably, the method of isolating or inducing differentiation of monocytes to obtain dendritic cells is a conventional method in the art: for example, the method of inducing differentiation of monocytes to obtain dendritic cells can be to mix monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to induce them.

[0040] Preferably, the monocytes include at least one of bone marrow-derived monocytes, spleen-derived monocytes, peripheral blood-derived monocytes, and umbilical cord blood-derived monocytes.

[0041] Preferably, the method for preparing the dendritic cells loaded with the tumor-specific antigen is a conventional method in the art: for example, the specific antigen is mixed with the dendritic cells and cultured.

[0042] Preferably, the culture time is 20 to 28 hours.

[0043] Preferably, the oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus; further preferably, the oncolytic virus is selected from alphavirus.

[0044] Preferably, the alpha virus comprises at least one of M1 virus and Getavirus (Getavirus is a virus with a homology of up to 97.8% (Wen et al. Virus Genes. 2007; 35(3): 597-603) with the M1 virus. The two have a high identity, and the M1 virus is also classified as a Getavirus-like virus by some literature. Therefore, it can be expected that Getavirus has similar effects to alpha virus); and further comprises M1 virus.

[0045] Preferably, the tumor comprises at least one of a solid tumor and a hematological tumor; and further comprises a solid tumor.

[0046] Preferably, the solid tumor comprises liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, adrenal cortical carcinoma, pararenal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, heart tumor, bile duct Epithelial cancer, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal sinus cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumors, islet cell tumors, papillomatosis, paraganglioma, nasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer and single myeloma; further includes at least one of melanoma, colon cancer, pancreatic cancer, and prostate cancer; further includes at least one of colon cancer, pancreatic cancer, and prostate cancer; and further includes at least one of colon cancer and prostate cancer.

[0047] Preferably, the blood tumor is selected from at least one of B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia and preleukemia.

[0048] Preferably, the tumor is a tumor that is insensitive to oncolytic viruses.

[0049] In the first aspect and the second aspect of the present invention, the drug resistance reversal agent means that when some oncolytic viruses are used as anti-tumor drugs to treat tumors, there are some tumors that are not very sensitive to oncolytic viruses, or these tumors are resistant to oncolytic viruses. At this time, the oncolytic virus can be used in combination with a DC vaccine (as a drug resistance reversal agent) to reverse the tumor's resistance to the oncolytic virus; or, conversely, when a DC vaccine is used to treat tumors, there are some tumors that are not very sensitive to drugs, or these tumors are resistant to the DC vaccine. At this time, the oncolytic virus (as a drug resistance reversal agent) can be used in combination with the DC vaccine to reverse the tumor's resistance to the DC vaccine.

[0050] The third aspect of the present invention provides the use of oncolytic viruses and DC vaccines in the preparation of anti-tumor drugs.

[0051] The fourth aspect of the present invention provides an anti-tumor composition comprising an oncolytic virus and a DC vaccine.

[0052] Preferably, the dosage form of the composition comprises at least one of lyophilized powder injection, injection, tablet, capsule, and patch.

[0053] A fifth aspect of the present invention provides a pharmaceutical kit comprising an oncolytic virus and a DC vaccine.

[0054] Preferably, the pharmaceutical kit further comprises instructions.

[0055] Preferably, the instructions record the dosage of the drug.

[0056] In the third, fourth and fifth aspects of the present invention:

[0057] Preferably, the ratio of oncolytic virus to DC vaccine is 5×10 6 ~5×10 8 pfu:5×10 5 ~5×10 7 DC cells; further 8×10 6 ~8×10 7 pfu:8×10 5 ~3×10 6 DC cells; further 5×10 7 pfu:1×10 6 DC cells.

[0058] Preferably, the dosage of the oncolytic virus is 5×10 6 ~5×10 8 pfu / mouse (weight based on 20 g), the dosage of the DC vaccine was 5×105 ~5×10 7 DC cells / mouse (weight 20 g); further preferably, the dosage of the oncolytic virus is 8×10 6 ~8×10 7 pfu / mouse (weight based on 20 g), the dosage of the DC vaccine was 5×10 5 ~5×10 7 DC cells / mouse (weight 20 g); more preferably, the dosage of the oncolytic virus is 5×10 7 pfu / mouse (weight based on 20 g), the dosage of the DC vaccine was 1×10 6 DC cells / mouse (body weight 20g).

[0059] Preferably, the dosage of the oncolytic virus is 1.5×10 10 ~1.5×10 12 pfu / person (weight 60kg), the dosage of the DC vaccine is 1.5×10 9 ~1.5×10 11 DC cells / person (weight 60 kg); further preferably, the dosage of the oncolytic virus is 2.4×10 10 ~2.4×10 11 pfu / person (weight 60kg), the dosage of the DC vaccine is 1.5×10 9 ~1.5×10 11 DC cells / person (weight 60 kg); more preferably, the dosage of the oncolytic virus is 1.5×10 11 pfu / person (weight 60kg), the dosage of the DC vaccine is 3×10 9 DC cells / person (body weight 60kg).

[0060] Preferably, the oncolytic virus and DC vaccine exist independently.

[0061] Preferably, the oncolytic virus and DC vaccine can be administered simultaneously or in any order.

[0062] Preferably, the drug, composition, or drug kit is intended for use in mammals; further, humans.

[0063] Preferably, the oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus; further preferably, the oncolytic virus is selected from alphavirus.

[0064] Preferably, the alpha virus comprises at least one of M1 virus and Getavirus (Getavirus is a virus with a homology of up to 97.8% (Wen et al. Virus Genes. 2007; 35(3): 597-603) with the M1 virus. The two have a high identity, and the M1 virus is also classified as a Getavirus-like virus by some literature. Therefore, it can be expected that Getavirus has similar effects to alpha virus); and further comprises M1 virus.

[0065] Preferably, the DC vaccine comprises dendritic cells loaded with specific antigens of the tumor.

[0066] Preferably, the specific antigen comprises at least one of tumor-associated antigen peptides, patient's autologous tumor antigens, tumor cell antigens, and recombinant proteins, and the specific antigen changes with the changes of the tumor; and further comprises at least one of tumor-associated antigen peptides and tumor cell antigens.

[0067] Preferably, the tumor cell antigen is obtained by lysing tumor cells.

[0068] Preferably, the lysis comprises at least one of repeated freeze-thaw lysis and oncolytic lysis; and further comprises oncolytic lysis.

[0069] Preferably, the oncolytic lysis method is: mixing oncolytic virus with tumor cells to obtain.

[0070] Preferably, the oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus; further preferably, the oncolytic virus is selected from alphavirus.

[0071] Preferably, the dendritic cells are obtained by isolating or differentiating from monocytes.

[0072] Preferably, the method of isolating or inducing differentiation of monocytes to obtain dendritic cells is a conventional method in the art: for example, the method of inducing differentiation of monocytes to obtain dendritic cells can be to mix monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to induce them.

[0073] Preferably, the monocytes include at least one of bone marrow-derived monocytes, spleen-derived monocytes, peripheral blood-derived monocytes, and umbilical cord blood-derived monocytes.

[0074] Preferably, the method for preparing the dendritic cells loaded with the tumor-specific antigen is a conventional method in the art: for example, the specific antigen is mixed with the dendritic cells and cultured.

[0075] Preferably, the culture time is 20 to 28 hours.

[0076] Preferably, the tumor comprises at least one of a solid tumor and a hematological tumor; and further comprises a solid tumor.

[0077] Preferably, the solid tumor comprises liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, adrenocortical carcinoma, pararenal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, heart tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer , Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal sinus cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumors, islet cell tumors, papillomatosis, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer and single myeloma at least one; further comprising at least one of melanoma, colon cancer, pancreatic cancer, and prostate cancer.

[0078] Preferably, the blood tumor is selected from at least one of B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom macroglobulinemia and preleukemia.

[0079] In the present invention, the inventors found that the anti-tumor effect of the combination of oncolytic virus and DC vaccine is better than the sum of the anti-tumor effects of oncolytic virus and DC vaccine alone, that is, the combination of oncolytic virus and DC vaccine has a synergistic effect in anti-tumor.

[0080] The beneficial effects of the present invention are:

[0081] The present invention is based on the inventors' discovery that oncolytic viruses can reactivate dendritic cells suppressed by tumors in vitro, and can increase the infiltration ratio of T cells and the tumor killing effect in the tumor microenvironment, thereby significantly enhancing the anti-tumor effect of DC vaccines. Furthermore, the inventors proposed for the first time the use of oncolytic viruses in the preparation of DC vaccine anti-tumor enhancers or drug resistance reversal agents.

[0082] The present invention discloses for the first time the use of DC vaccines in the preparation of oncolytic virus anti-tumor enhancers or drug resistance reversal agents.

[0083] The present invention is based on the inventors' discovery that the anti-tumor effect of the combination of oncolytic virus and DC vaccine is better than the sum of the anti-tumor effects of oncolytic virus and DC vaccine alone, that is, the combination of oncolytic virus and DC vaccine has a synergistic effect in anti-tumor. Furthermore, the inventors proposed for the first time the use of oncolytic virus and DC vaccine in the preparation of anti-tumor drugs, and a composition and drug kit containing oncolytic virus and DC vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 The results of oncolytic virus M1 reactivating tumor-suppressed dendritic cells in vitro: A is the expression level of CD86, CD83 and MHC II in BMDCs co-cultured with B16-F10 after LPS (positive control) and M1 virus treatment; B is the concentration of TNF-α, IL-12p70 and IFN-β in the supernatant of BMDCs co-cultured with B16-F10 after M1 virus treatment; C is the concentration of CD4+ / -1 / -2 in BMDCs co-cultured with B16-F10 after M1 virus treatment and co-cultured with T cells. + T cells and CD8 + The expression levels of CD44 and CD69 on the surface of T cells are shown in the figure; D is the concentration of IFN-γ, granzyme, and perforin in the supernatant of BMDCs co-cultured with B16-F10 and then co-cultured with T cells after being treated with M1 virus; the number of repetitions for each treatment is n=3; "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, and "ns" indicates P > 0.05.

[0085] Figure 2The results of the release of tumor-associated antigens after M1 virus infection of tumor cells: A is the BMDCs treated with OVA (positive control), M1 virus and B16-F10-OVA oncolytic lysate and OT-I CD8 + Schematic diagram of co-culture of T cells; B is the co-culture of BMDCs treated with OVA (positive control), M1 virus, and B16-F10-OVA oncolytic lysate with OT-ICD8 + Flow cytometry of CD69 and CD44 expression on T cells after co-culture with T cells; C is a flow cytometric analysis of BMDCs treated with OVA (positive control), M1 virus, and B16-F10-OVA oncolytic lysate and OT-I CD8 + The mean fluorescence intensity (MFI) of CD69 and CD44 of T cells was detected after co-culture with T cells; D is the result of BMDCs and OT-I CD8 + Schematic diagram of co-culture of T cells; E is a diagram of BMDCs and OT-I CD8 + Flow cytometric analysis of the expression of CD69 and CD44 on T cells after co-culture with T cells; F is a flow cytometric analysis of BMDCs and OT-I CD8 + The mean fluorescence intensity (MFI) of CD69 and CD44 on T cells was detected after T cell co-culture; the number of repetitions for each treatment was n=3; "*" indicates P < 0.05, "**" indicates P < 0.01, "***" indicates P < 0.001, and "ns" indicates P > 0.05.

[0086] Figure 3 The figures are the results of M1 virus enhancing the anti-tumor effect of DC vaccine in different tumor-bearing mouse models: A is the tumor growth curve, T / C%, and survival curve of the melanoma mouse model after different treatments; B is the tumor growth curve, T / C%, and survival curve of the colon cancer mouse model after different treatments; C is the tumor growth curve, T / C%, and survival curve of the prostate cancer mouse model after different treatments; D is the tumor growth curve, T / C%, and survival curve of the pancreatic cancer mouse model after different treatments; the specific number of treatment repetitions is as follows: B16-F10 (n=9), CT-26 (n=7), RM-1 (n=8), Pan02 (n=7), T / C%=tumor volume after each treatment (OVM, DC, OVM+DC) / tumor volume of the control group (Vehicle).

[0087] Figure 4 The results of M1 virus-enhanced DC vaccine activation of anti-tumor T cell response: A is the CD3 in T cells in the tumor microenvironment of melanoma mouse model after different treatments + CD45 + B is a flow cytometric graph of the content of T cells in the tumor microenvironment of a melanoma mouse model after different treatments. + CD4 + Flow cytometry of T cell content; C is a flow cytometer showing CD3 in T cells in the tumor microenvironment of a melanoma mouse model after different treatments. + CD45 + The statistical results of the content of T cells; D is the CD4 + CD3 in T cells + CD45 + CD4 + The statistical results of the content of T cells; E is the CD8 + CD3 in T cells + CD45 + CD8 + The statistical results of T cell content; F is the CD4 + T cells and CD3 + The statistical results of the proportion of T cells; G is the CD8 + T cells and CD3 + The statistical results of the proportion of T cells; H is the CD4 + The mean fluorescence intensity (MFI) of CD44 T cells in the tumor microenvironment of the melanoma mouse model after different treatments. + The mean fluorescence intensity (MFI) of T cell CD44 is shown in Figure 2; J is the CD4 in the tumor microenvironment of the melanoma mouse model after different treatments. + Central memory T cells (T CM ) + CD44 + CD62L + T CM The statistical results of the content of CD8 in the tumor microenvironment of the melanoma mouse model after different treatments. + T CM CD8 + CD44 + CD62L +T CM The statistical results of the content of CD4 in the tumor microenvironment of the melanoma mouse model after different treatments. + Effector memory T cells (T EM ) + CD44 + CD62L + T EM M is the CD8 in the tumor microenvironment of the melanoma mouse model after different treatments. + T EM CD8 + CD44 + CD62L + T EM Statistical results of the content of; N is the CD4 in the tumor microenvironment of the melanoma mouse model after different treatments + Exhausted T cells (T ex ) + T ex The statistical results of the content of CD8 in the tumor microenvironment of the melanoma mouse model after different treatments. + T ex PD-1 + T ex ; P is the result of the killing effect of tumor cells after separation of lymphocytes from the tumor microenvironment of the melanoma mouse model after different treatments and co-culture with tumor cells; Q is the concentration of IFN-γ in the supernatant after co-culture with tumor cells after separation of lymphocytes from the tumor microenvironment of the melanoma mouse model after different treatments; the specific number of treatment repetitions is as follows: in Figure CG: Vehicle (n=8), M1 (n=8), DC (n=9), M1+DC (n=9), in Figure HO: Vehicle (n=6), M1 (n=5), DC (n=7), M1+DC (n=10), in Figure PQ: n=8; "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "ns" indicates P>0.05. DETAILED DESCRIPTION

[0088] The present invention is further described in detail below through specific examples.

[0089] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0090] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0091] The oncolytic virus M1 in the following examples is the M1 virus with a deposit number of CCTCCV201423.

[0092] Example 1 Oncolytic virus M1 reactivates tumor-suppressed dendritic cells (DC) in vitro

[0093] 1) Induction of mouse bone marrow-derived dendritic cells (BMDCs)

[0094] The femur and tibia of the mouse were obtained, and the bone marrow cavity was repeatedly flushed with DPBS using a syringe to collect bone marrow single cell suspension. After lysing the red blood cells, the mononuclear cells were induced with RPMI 1640 complete medium containing 20 ng / mL mGM-CSF and 20 ng / mL mIL-4. Half of the medium was changed on the 3rd and 5th days, and fresh RPMI 1640 complete medium containing GM-CSF and IL-4 was supplemented. The suspended immature BMDCs could be collected on the 7th day.

[0095] 2) Inhibitory effect of tumor cells B16-F10 (mouse skin melanoma cells) on BMDCs

[0096] Tumor cells in the logarithmic growth phase were digested and counted in advance, and the cell density was adjusted to be inoculated into a 6-well plate and cultured overnight. The induced BMDCs in 1) were collected and added to a 6-well plate pre-plated with tumor cells (NC / Co-DC, the control group added an equal amount of BMDCs to a blank 6-well plate, iDC) at a ratio of tumor cells: BMDCs = 1:3 (15w:45w), and 2 mL of complete PRMI 1640 medium was added to each well for a total of 48 hours of culture. After culture, the suspended BMDCs were collected and CD11c was analyzed by flow cytometry. + CD86, CD83, and MHC II on the surface of BMDCs were detected, and the co-culture supernatants were collected to detect the secretion of cytokines, including TNF-α, IL-12p70, and IFN-β.

[0097] The magnetic bead sorting process of BMDCs after co-culture with tumor cells is as follows: all cells in the co-culture system of tumor cells and BMDCs (the culture method is the same as the above co-culture, the only difference is that this co-culture is carried out in a large dish, and the ratio of tumor cells: BMDCs = 1:3 (100w:300w) are collected and sorted using EasySep TMResuspend in cell sorting buffer and transfer to flow cytometry tubes. The volume of each tube should be between 0.5 and 2 mL. + The cell positive selection kit was used to sort BMDCs in the co-culture system. According to the volume of each tube of cell suspension, rat serum was added at a ratio of 50 μL / mL. Solution A and solution B in the kit were mixed at a ratio of 1:1 and left for 5 minutes (prepared according to the volume). Then, the cocktail obtained by mixing solution A and solution B was added to the cell suspension at a volume of 100 μL / mL, gently blown, incubated at room temperature for 5 minutes, and violently shaken RapidSpheresTM until the particles in the tube were evenly dispersed. RapidSpheresTM was added at a ratio of 80 μL / mL, mixed and incubated at room temperature for 3 minutes. The total volume was filled to 2.5 mL with EasySepTM cell sorting buffer, inserted into the magnetic pole, left for 5 minutes, and the supernatant was discarded. The flow tube was removed, and the cells were resuspended with 2.5 mL of buffer, inserted into the magnetic pole again, and left for 5 minutes, and the supernatant was discarded. The flow tube was removed, and the cells were resuspended with RPMI 1640 complete medium, centrifuged at 500×g at 4℃ for 5 minutes, and the supernatant was discarded. Then used for co-culture with T cells.

[0098] 3) BMDCs and spleen origin Co-culture of T cells

[0099] Take the mouse spleen and put it in a 70μm nylon mesh sieve, place it in a 60mm culture dish, and add 5mL of mouse lymphocyte separation solution. Use a 2.5mL syringe piston rod to gently grind the spleen until only a small amount of white tissue is left in the cell sieve. Discard the sieve, and the liquid in the culture dish is the spleen cell suspension. Transfer the spleen cell suspension to a 15mL centrifuge tube, and gently add 1mL of RPMI 1640 culture medium along the wall to keep the liquid surface boundary clear. Set the acceleration and deceleration of the high-speed centrifuge to third gear, and centrifuge at room temperature at 800×g for 30 minutes. The cell layer between the RPMI 1640 culture medium cover layer and the separation liquid layer after the centrifugation is the lymphocyte layer. The lymphocyte layer was aspirated into a new 15 mL centrifuge tube, and 10 mL of RPMI 1640 medium was added, and the tube was inverted for washing. The tube was centrifuged at 300 × g for 10 min at room temperature, and the supernatant was discarded. The cells were resuspended in RPMI 1640 complete medium containing 10 ng / mL of mIL-2, and co-cultured with BMDCs at a ratio of BMDCs: lymphocytes = 1:5 for 96 hours. The culture supernatant was collected for the detection of cytokines (including IFN-γ, granzyme B and perforin), and the cells were collected by centrifugation, and the expression of CD69 and CD44 on the surface of T cells was detected by flow cytometry.

[0100] 4) M1 virus treatment of BMDCs suppressed by tumor cells B16-F10

[0101] According to the method in 2), BMDCs were co-cultured with tumor cells for 24 hours to obtain BMDCs inhibited by tumor cells, and then M1 virus (M1 / Co-DC) with a final concentration of 1 MOI and LPS (positive control, LPS / Co-DC) with a final concentration of 1 μg / mL were added and treated for 24 hours. After culture, the suspended BMDCs were collected, and CD86, CD83 and MHC II on the surface of CD11c+BMDCs were detected by flow cytometry, and the co-culture supernatant was collected to detect the secretion of cytokines, including TNF-α, IL-12p70 and IFN-β.

[0102] The magnetic bead sorting process of BMDCs treated with M1 virus and inhibited by tumor cells B16-F10 is the same as that in 2), except that the BMDCs are BMDCs treated with M1 virus and inhibited by tumor cells B16-F10: BMDCs and tumor cells are co-cultured for 24 hours according to the method in 2) to obtain BMDCs inhibited by tumor cells, and then M1 virus (M1 / Co-DC) with a final concentration of 1 MOI is added for 24 hours, and the sorted BMDCs are compared with spleen-derived T cells were co-cultured, and the levels of CD69 and CD44 on the T cell surface were detected by flow cytometry, and IFN-γ, granzyme B and perforin in the supernatant were detected by ELISA (the method is the same as 3)).

[0103] The results of the above experiments are as follows Figure 1 As shown in Figure 2, after BMDCs were co-cultured with tumor cells B16-F10, the expression levels of maturation markers CD86, CD83, and MHC II on the surface of BMDCs and the concentrations of inflammatory cytokines TNF-α, IL-12p70, and IFN-β in the supernatant were significantly downregulated. The activation capacity of T cells (the concentrations of IFN-γ, granzyme B, and perforin in the supernatant, and the expression levels of CD69 and CD44 on the surface of T cells) was significantly reduced, indicating that tumor cells inhibited the maturation and function of DCs; after treatment with M1 virus, the expression levels of maturation markers CD86, CD83, and MHC II on the surface of BMDCs, as well as the concentrations of TNF-α, IL-12p70, and IFN-β were significantly increased, and the activation capacity of T cells (the concentrations of IFN-γ, granzyme B, and perforin in the supernatant, and the expression levels of CD69 and CD44 on the surface of T cells) was significantly reduced, indicating that tumor cells inhibited the maturation and function of DCs; after treatment with M1 virus, the expression levels of maturation markers CD86, CD83, and MHC II on the surface of BMDCs, as well as The activation ability of T cells (the concentrations of IFN-γ, granzyme B, and perforin in the supernatant, and the expression levels of CD69 and CD44 on the surface of T cells) was significantly improved, indicating that the M1 virus can promote the maturation of DCs inhibited by tumor cells, and at the same time, improve the function of DCs inhibited by tumor cells (activate T cells); that is, the M1 virus has the potential to enhance the anti-tumor effect of DC vaccines.

[0104] Example 2 Oncolytic virus M1 enhances the anti-tumor effect of DC vaccine in a tumor-bearing mouse model

[0105] 1) Activation of antigen-specific T cells by BMDCs

[0106] BMDCs were seeded into 6-well plates and treated in the following four ways: (1) control group (NC), without any treatment; (2) positive control (OVA), stimulated with 1 μg / mL OVA protein for 24 h; (3) 500 μL M1 (1×10 6 (4) Add 500 μL of oncolytic lysate of B16-F10-OVA infected with M1 virus and stimulate for 24 hours (Oncolysate (B16-F10-OVA)).

[0107] To further prove that M1 virus infection releases tumor antigens and completely exclude the effect of M1 virus, BMDCs were pre-inoculated into 6-well plates and treated as follows: (1) control group ( T cells) without any treatment; (2) adding 500 μL of oncolytic lysate of B16-F10-OVA inactivated by UV cross-linking to stimulate for 24 hours (Oncolysate (UV) (B16-F10-OVA)); (3) adding 500 μL of oncolytic lysate of B16-F10 inactivated by UV cross-linking to stimulate for 24 hours (Oncolysate (UV) (B16-F10)).

[0108] The preparation method of B16-F10-OVA and B16-F10 oncolytic lysate is as follows: B16-F10-OVA or B16-F10 cells are pre-cultured at 3×10 6 The number of cells / dish was inoculated into a 100 mm cell culture dish. After the cells adhered, the culture medium was replaced with 10 mL of RPMI 1640 complete culture medium containing 20 ng / mL mGM-CSF and 10 ng / mL mIL-4, and then the M1 virus with a final concentration of 0.1 MOI was added. After 24 hours of infection, the supernatant culture fluid was collected and filtered through 0.22 μm to remove cell debris to obtain the oncolytic lysate of B16-F10-OVA or B16-F10. The oncolytic lysate was frozen in a 4 °C refrigerator for later use.

[0109] The preparation method of the oncolytic lysate of B16-F10-OVA or B16-F10 inactivated by UV crosslinking is as follows: 3 mL of B16-F10-OVA or B16-F10 oncolytic lysate was placed in a 100 mm cell culture dish, placed in the HL-2000 HybriLinker hybridization oven, and after opening the lid of the dish, 1000 × 100 μJ / cm 2 After irradiating the oncolytic lysis solution with ultraviolet light for 15 seconds, gently shake the culture dish and irradiate it twice under the same conditions. After irradiation, filter through 0.22 μm to remove bacteria. After aliquoting, freeze in a 4°C refrigerator for later use.

[0110] BMDCs to OT-I CD8 + Stimulation of T cells: BMDCs treated with different methods were combined with CD8 + T cells were co-cultured at a ratio of BMDCs:T = 1:2 for 48 hours. CD8 + Expression of CD69 and CD44 on T cell populations.

[0111] The results are as follows Figure 2 As shown: Compared with the control group, the activation level of T cells by BMDCs treated with OVM remained almost unchanged, while the activation level of T cells by BMDCs treated with OVA and Oncolysate (B16-F10-OVA) was significantly enhanced, and the activation level of T cells by BMDCs treated with Oncolysate (UV) (B16-F10-OVA) was significantly better than that by BMDCs treated with Oncolysate (UV) (B16-F10); that is, the infection of tumor cells by M1 virus can release tumor-associated antigens, which can be used for the preparation of DC vaccines.

[0112] 2) Construction and treatment of tumor-bearing mice

[0113] Tumor cells (B16-F10 (mouse skin melanoma cells), CT-26 (mouse colon cancer cells), RM-1 (mouse prostate cancer cells), Pan02 (mouse pancreatic cancer cells)) in the logarithmic growth phase were taken respectively. After trypsin digestion, digestion was terminated with complete medium, centrifuged at 4°C 500×g for 5 minutes, the supernatant was discarded, and the cells were resuspended with DPBS, pipetted evenly, centrifuged at 4°C 500×g for 5 minutes, the supernatant was discarded, and the cells were resuspended with DPBS again. The cells were counted and the concentration of the cell suspension was adjusted to 1-3×10 7 cells / mL (specifically as follows: B16-F10 / CT-26 / RM-1: 1×10 7 cells / mL, Pan02: 3×10 7Cells / mL) were placed on ice. The right posterior skin of the mouse was depilated in advance, and 0.1 mL of cell suspension was injected subcutaneously into the depilated area with a 1 mL syringe. The sixth day after tumor bearing was recorded as day 0. When the tumor volume of the tumor-bearing mouse grew to an appropriate size (50-100 mm 3 ), the mice were randomly divided into groups and given corresponding medications (specifically, Figure 3 (1) Vehicle control group: PBS solution was injected into the tail vein for 5 consecutive days, 300 μL each time; (2) M1 group (OVM): M1 virus was injected into the tail vein for 5 consecutive days, 300 μL each time (1×10 7 pfu); (3) DC group: single intratumoral injection of PBS solution containing DC vaccine, 100 μL each time (1×10 6 (3) M1+DC group (OVM+DC): single intratumoral injection of PBS solution containing DC vaccine, 100 μL each time (1×10 6 The M1 virus was injected into the tail vein for 5 consecutive days, 300 μL each time (1×10 7 pfu).

[0114] The preparation method of the DC vaccine for treating melanoma is as follows: BMDCs are induced by the method 1) in Example 1, OVA protein is added at a concentration of 1 μg / mL on the 7th day of induction, and the DC vaccine is collected after 24 hours of stimulation;

[0115] The preparation method of DC vaccine for the treatment of colon cancer, prostate cancer and pancreatic cancer is as follows: tumor cells (CT-26, RM-1 or Pan02) are pre-cultured at 3×10 6 The number of cells / dish was inoculated into a 100 mm cell culture dish, and after the cells adhered, 10 mL of RPMI 1640 complete medium containing 20 ng / mL mGM-CSF and 10 ng / mL mIL-4 was replaced, and M1 virus with a final concentration of 0.1 MOI was added to the cells for infection for 24 hours. The supernatant was collected in a 15 mL centrifuge tube and filtered through 0.22 μm to remove cell debris. Then, the oncolytic lysis buffer was used at a rate of 1×10 6 BMDCs induced by the method of 1) in Example 1 were cultured at a concentration of 10 cells / mL, and the DC vaccine was collected after 24 hours.

[0116] The results are as follows Figure 3As shown in Tables 1 to 5: M1 virus can significantly enhance the therapeutic effect of DC vaccine and prolong the survival of tumor-bearing mice; and DC vaccine can also significantly enhance the therapeutic effect of M1 virus and prolong the survival of tumor-bearing mice; in particular, in different tumor mouse models, the survival time extended by OVM+DC compared with Vehicle is greater than the sum of the survival time extended by OVM compared with Vehicle and the survival time extended by DC compared with Vehicle, indicating that the combination of oncolytic virus and DC vaccine has a synergistic effect in anti-tumor.

[0117] Table 1 Tumor volume (mm) of melanoma mouse model after different treatments at different times 3 )

[0118] Day 6 Day 8 Day 10 Day 12 Day 14 Vehicle 63.2±41.2 173.3±97.2 657.9±201.3 1229.8±2268.3 1970.9±264.9 OVM 64.2±27.4 172.9±101.7 457.0±148.0 850.4±292.2 1318.2±231.0 DC 65.7±23.0 135.5±84.5 390.4±155.5 866.2±393.4 1186.0±343.7 OVM+DC 63.8±39.3 100.5±123.5 185.9±87.2 279.1±67.2 398.3±93.0

[0119] Table 2 Tumor volume (mm) of colon cancer mouse model after different treatments at different times 3 )

[0120] Day 10 Day 14 Day 18 Day 20 Day 23 Day 27 Vehicle 61.5±17.1 292.8±117.6 436.0±132.8 902.5±146.5 1350.9±329.0 2677.7±539.6 OVM 49.0±14.7 128.3±70.3 221.2±92.7 579.8±295.7 1036.75±611.8 2024.3±645.9 DC 50.9±17.3 121.6±63.7 242.0±98.8 566.4±343.8 814.7±397.4 1679.2±825.4 OVM+DC 55.4±15.0 53.6±12.7 93.0±38.3 230.2±155.5 398.1±389.2 664.4±501.7

[0121] Table 3 Tumor volume (mm) of prostate cancer mouse model after different treatments at different times 3 )

[0122]

[0123]

[0124] Table 4 Tumor volume (mm) of pancreatic cancer mouse model after different treatments at different times 3 )

[0125] Day 7 Day 14 Day 21 Day 27 Day 35 Day 42 Vehicle 42.8±17.9 68.9±13.4 76.4±21.5 110.8±33.9 205.9±81.3 373.4±80.2 OVM 50.1±20.3 73.7±24.0 76.6±29.4 97.3±26.0 228.9±58.0 407.4±40.9 DC 51.3±10.2 56.7±27.0 73.5±31.5 103.6±22.8 169.3±60.9 364.4±151.9 OVM+DC 50.7±10.3 45.3±22.8 40.5±17.7 53.5±23.4 105.6±57.0 212.7±79.1

[0126] Table 5 Survival days of different tumor mouse models after different treatments

[0127] Melanoma Mouse Model Colon cancer mouse model Prostate cancer mouse model Pancreatic cancer mouse model Vehicle 16 30 20 90 OVM 18 32 23 90 DC 20 41 23 90 OVM+DC 27 ＞45 37 111

[0128] Example 3 The combination of M1 virus and DC vaccine significantly increased the infiltration ratio of T cells in the tumor microenvironment and the tumor killing effect

[0129] 1) Preparation of tumor single cell suspension and flow cytometry detection

[0130] On the second day after the last administration, the tumor tissue of the melanoma mouse was peeled off, placed in a 70μm nylon mesh, and placed in a culture dish. An appropriate amount of DPBS was added, and the piston rod of the syringe gently ground the tumor block until only a small amount of tissue remained in the cell mesh. The mesh was discarded, the cell suspension in the culture dish was collected, and filtered again with a nylon mesh, and the filtrate was collected to obtain a single cell suspension of tumor tissue, which was counted and set aside. Tumor cells were collected from the above single cell suspension of tumor tissue in a flow tube, and dead and live cells were stained and blocked using FVS 510 and CD16 / CD32 respectively according to the instructions, and finally the pre-mixed flow antibody was added for staining and put on the machine.

[0131] 2) Isolation of tumor lymphocytes and lymphocyte killing experiments

[0132] Separation of tumor tissue infiltrating lymphocytes: Take a 15mL centrifuge tube and add 5mL separation solution. Carefully draw 5mL of tumor tissue single cell suspension and add it to the surface of the separation solution, and pay attention to keep the interface between the two liquid surfaces clear. (You can use a Pasteur pipette to draw the single cell suspension, and then carefully spread it on the separation solution. Because of the density difference between the two, a clear stratified interface will be formed) Centrifuge at room temperature and 800×g for 25 minutes. After centrifugation, the cells in the centrifuge tube are divided into four layers from top to bottom. The first layer is the diluent layer; the second layer is the ring-shaped milky white lymphocyte layer; the third layer is the transparent separation liquid layer; and the fourth layer is the red blood cell layer. Carefully draw the second layer of ring-shaped milky white lymphocytes into another clean 15mL centrifuge tube, add 10mL of cell washing solution to the centrifuge tube to wash the white film layer cells, and centrifuge at 4℃250×g for 10 minutes. Discard the supernatant, resuspend the cells with 5mL of cell washing solution, and centrifuge at 4℃250×g for 10 minutes. Discard the supernatant, resuspend the cells, count, and set aside.

[0133] Lymphocyte killing experiment: Tumor cells B16-F10-OVA were pre-incubated at 1×10 4Cells / well were inoculated into 96-well plates. After the tumor cells adhered to the wall, tumor-infiltrating lymphocytes were added at a ratio of Tumor:T=3:1. After 72 hours of co-culture with tumor cells, the co-culture supernatant of tumor cells and lymphocytes was collected, centrifuged at 1000×g for 5 minutes at 4℃, and the supernatant was transferred to a 1.5mL EP tube for subsequent ELISA experiments. The remaining lymphocytes were removed by rinsing with PBS twice, and the viability of adherent tumor cells was detected by MTT method: the MTT mother solution was diluted in RPMI 1640 culture medium at a ratio of 10% (V / V), and then the culture medium containing MTT was added to the tumor cells at a volume of 200μL / well, and incubated in a 30℃ incubator for 3-4 hours. Since the succinate dehydrogenase in the living cells can oxidize MTT to formazan, blue-purple crystals can be observed on the bottom of the 96-well plate. Carefully remove the supernatant from the well plate, add 100 μL DMSO to each well to dissolve the formazan crystals, and shake on a microporous oscillator for 5 minutes to completely dissolve the crystals. After mixing, use an enzyme marker to detect the OD value at 570 nm. The worse the vitality of the tumor cells at the bottom, the stronger the lymphocytotoxicity. The calculation formula for lymphocytotoxicity is: Lymphocytotoxicity = (A Tumor cells -A Tumor cells+lymphocytes ) / (A Tumor cells -A blank )x100%.

[0134] The above experimental results are as follows Figure 4 As shown: The results of flow cytometry analysis of T cells in the tumor microenvironment using lymphocyte killing experiments showed that CD3 + T cells, especially CD3 + CD8 + The proportion of T cells was significantly upregulated ( Figure 4 A~I); through further phenotypic analysis, lymphocyte killing experiments and ELISA experiments, it was found that in the combined treatment group, the effector T cells and memory T cells increased significantly, while the proportion of exhausted T cells decreased, the specific killing of tumor cells was enhanced, and the secretion of inflammatory cytokines increased; the above results indicate that M1 virus enhances the anti-tumor T cell response activated by DC vaccine.

[0135] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of oncolytic viruses in the preparation of DC vaccine anti-tumor enhancers or drug resistance reversal agents.

2. Application of DC vaccines in the preparation of oncolytic virus anti-tumor enhancers or drug resistance reversal agents.

3. Application of oncolytic viruses and DC vaccines in the preparation of anti-tumor drugs.

4. An anti-tumor composition comprising an oncolytic virus and a DC vaccine.

5. A pharmaceutical kit comprising an oncolytic virus and a DC vaccine.

6. The use, composition or pharmaceutical kit according to any one of claims 1 to 5, characterized in that: The oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus; Preferably, the oncolytic virus is selected from alphavirus; Preferably, the alphavirus comprises at least one of M1 virus and Geta virus.

7. The use, composition or pharmaceutical kit according to any one of claims 1 to 5, characterized in that: The DC vaccine comprises dendritic cells loaded with tumor-specific antigens; Preferably, the specific antigen comprises at least one of tumor-associated antigen peptides, patient autologous tumor antigens, tumor cell antigens, and recombinant proteins; Preferably, the dendritic cells are obtained by isolating or differentiating from monocytes; Preferably, the monocytes include at least one of bone marrow-derived monocytes, spleen-derived monocytes, peripheral blood-derived monocytes, and umbilical cord blood-derived monocytes.

8. The use, composition or pharmaceutical kit according to any one of claims 1 to 5, characterized in that: The tumor comprises at least one of a solid tumor and a blood tumor; Preferably, the solid tumor comprises liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, adrenocortical carcinoma, pararenal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, heart tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, embryonic tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, renal cancer, Langerhans cell tissue cell At least one of: leukocyte proliferation, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, sinonasal cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumor, islet cell tumor, papillomatosis, paraganglioma, sinonasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer and single myeloma; Preferably, the blood tumor is selected from at least one of B-cell acute lymphoid leukemia, T-cell acute lymphoid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia and preleukemia.

9. The use, composition or pharmaceutical kit according to any one of claims 3 to 5, characterized in that: The ratio of oncolytic virus and DC vaccine is 5×10 6 ~5×10 8 pfu:5×10 5 ~5×10 7 DC cells; Preferably, the dosage form of the composition comprises at least one of lyophilized powder injection, injection, tablet, capsule, and patch.

10. The use, composition or pharmaceutical kit according to any one of claims 3 to 5, characterized in that: The oncolytic virus and DC vaccine are administered simultaneously or in any order; Preferably, the pharmaceutical kit further comprises instructions; Preferably, the instructions record the dosage of the drug.

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