WO2019062234A1 - 分离的重组溶瘤痘病毒、药物组合物及其在***和/或癌症的药物中的用途 - Google Patents

分离的重组溶瘤痘病毒、药物组合物及其在***和/或癌症的药物中的用途 Download PDF

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WO2019062234A1
WO2019062234A1 PCT/CN2018/093404 CN2018093404W WO2019062234A1 WO 2019062234 A1 WO2019062234 A1 WO 2019062234A1 CN 2018093404 W CN2018093404 W CN 2018093404W WO 2019062234 A1 WO2019062234 A1 WO 2019062234A1
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cancer
cells
gene
tumor
poxvirus
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PCT/CN2018/093404
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English (en)
French (fr)
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傅瑾
赵荣华
张蓉
王婷婷
陈霖
胡放
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杭州康万达医药科技有限公司
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Priority to JP2020518664A priority Critical patent/JP7248325B2/ja
Priority to EP18861531.4A priority patent/EP3690034A4/en
Priority to CN202410133935.0A priority patent/CN118105415A/zh
Priority to US16/650,076 priority patent/US20200289591A1/en
Priority to CN201880062059.XA priority patent/CN111315873B/zh
Publication of WO2019062234A1 publication Critical patent/WO2019062234A1/zh
Priority to JP2023035979A priority patent/JP2023071953A/ja

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Definitions

  • the present invention is in the field of biotechnology and, in particular, relates to isolated recombinant oncolytic poxviruses, pharmaceutical compositions and their use in the treatment of tumors and/or cancer.
  • Oncolytic virus is a type of virus that selectively replicates and kills tumor cells in tumor cells without harming normal cells. After the virus replicates in the infected tumor cells, it releases new virus particles, which in turn infect the surrounding tumor cells, further inducing oncolytic effects. In addition to this direct oncolytic effect, the oncolytic virus can also be effective. Stimulates the body to produce an immune response against the virus itself and against infected tumor cells. Thus, the anti-tumor effect of oncolytic viruses is achieved by selectively killing tumor cells and inducing the body to produce a systemic anti-tumor immune response.
  • viruses can alleviate the progress of tumor development, suggesting the potential of the virus in the field of cancer treatment.
  • gene technology With the development of gene technology, it can be selectively replicated in tumor cells by changing the viral genome structure to enhance its oncolytic targeting.
  • viruses such as adenovirus, herpes virus, picornavirus, and poxvirus by techniques such as gene recombination, gene transfer, and gene knockout.
  • Oncolytic virus products To date, more than 20 products have entered different stages of clinical research. Among them, in 2005, China CFDA approved the genetic modification of oncolytic adenovirus H101 of Shanghai 3D Biotechnology Co., Ltd.
  • the present invention provides isolated recombinant oncolytic poxviruses, pharmaceutical compositions and their use in the treatment of tumors and/or cancers.
  • the present invention provides:
  • a pharmaceutical composition wherein the pharmaceutical composition comprises, as an active ingredient, the recombinant oncolytic poxvirus according to any one of (1) to (7), and a pharmaceutically acceptable excipient.
  • tumor and/or cancer comprises lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterus Cancer, cervical cancer, lymphoma, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer.
  • a method of treating a tumor and/or a cancer comprising administering to a tumor and/or a cancer patient a recombinant oncolytic poxvirus according to any one of (1) to (7).
  • the tumor and/or cancer comprises lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterus Cancer, cervical cancer, lymphoma, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer.
  • the invention has the following advantages and positive effects:
  • the present invention firstly proposes a functional defect of the TK gene and the VGF gene of the oncolytic poxvirus, and at the same time carries the gene of the immunoregulatory factor IL-21, thereby enabling the resulting recombinant oncolytic poxvirus to selectively replicate in tumor cells. And express the immunoregulatory factor IL-21.
  • the oncolytic pox virus, pharmaceutical composition and method provided based on the concept can fully exert the effect that the oncolytic virus selectively replicates in tumor cells and kills tumor cells, and further causes subsequent immune responses, and is capable of Give full play to the anti-tumor immunity of exogenous IL-21.
  • the present inventors have found that the integration of the IL-21 gene in the oncolytic poxvirus can synergistically effect the oncolytic killing effect of the oncolytic virus and the anti-tumor immunostimulatory effect of IL-21.
  • TK and VGF double gene function defects of poxvirus effectively enhance their tumor targeting, thereby improving safety.
  • the recombinant oncolytic poxvirus of the present invention expresses exogenous IL-21 while selectively replicating in tumor cells, it can synergistically stimulate the anti-tumor immune response of the organism, thereby enabling the recombinant oncolytic poxvirus of the present invention to be combined with NK.
  • the cells are used in combination.
  • the pharmaceutical composition and method provided based on the concept can fully exert the effect of the recombinant oncolytic poxvirus of the present invention selectively replicating and killing tumor cells in tumor cells, and further causing a subsequent immune response, and also capable of Fully exerting the function of NK cells to kill tumor cells, and skillfully utilizing the feature that the recombinant oncolytic poxvirus of the present invention selectively replicates in tumor cells, so that the tumor cells containing the recombinant oncolytic poxvirus of the present invention become NK
  • the specific target of the cell, and the expressed exogenous IL-21 can also enhance the killing power of NK cells, thereby further enhancing the tumor killing effect of NK cells. This ultimately results in a further enhanced synergistic killing of the tumor.
  • the present invention enables the synergistic effect of the combined administration of the two of the recombinant oncolytic poxvirus and NK cells of the present invention to achieve maximum efficiency by research, while avoiding both of them. Inter-restriction to achieve effective treatment of tumors and/or cancer.
  • oncolytic virus refers to a virus that is capable of selectively replicating and lysing tumor cells in tumor cells.
  • terapéuticaally effective amount refers to an amount of a functional agent or pharmaceutical composition capable of exhibiting a detectable therapeutic or inhibitory effect, or an amount that exerts an anti-tumor effect. The effect can be detected by any test method known in the art.
  • administering refers to providing a compound, complex or composition (including viruses and cells) to a subject.
  • patient refers to a human or non-human organism.
  • the methods and compositions described herein are suitable for use in human diseases and veterinary diseases.
  • the patient has a tumor.
  • the patient has one or more types of cancer at the same time.
  • the term "synergistic effect” refers to the effect of two or more agents acting together, which is greater than the sum of the individual effects of the agents therein.
  • plaque forming unit means that the amount of virus that produces an plaque is referred to as a plaque forming unit (pfu).
  • MOI Multiplicity of infection
  • Figure 1 shows a map of a recombinant plasmid constructed for insertion of an IL-21 gene into the TK gene of poxvirus VSC20 (i.e., "Dvv-VSC20/VGF-" shown in the figure), constructed in one embodiment of the present invention, And a schematic diagram of the recombination mechanism by which a recombinant double gene defective poxvirus (recombinant DDvv) of one embodiment of the present invention is obtained.
  • DDvv recombinant double gene defective poxvirus
  • Figure 2 shows a flow diagram of one embodiment of obtaining a recombinant oncolytic poxvirus of the invention.
  • Figure 3 shows a map of the plasmid constructed in Example 1 of the present invention.
  • Figure 4 shows the results of PCR and ELISA to identify the P0 generation recombinant virus DDvv-IL21, wherein A is PCR detection of P0 virus DDvv-mIL21, B is PCR detection of P0 virus DDvv-hIL21, in the figure Lane M: D000 marker, Lane 1: Negative control (ie, PCR reaction), Lane 2: VSC20, Lane 3: CV1 cells, Lane 4: P0 generation virus; C is the identification of IL21 in the culture supernatant of the P0 virus by ELISA The level of expression.
  • A is PCR detection of P0 virus DDvv-mIL21
  • B PCR detection of P0 virus DDvv-hIL21
  • Lane M D000 marker
  • Lane 1 Negative control (ie, PCR reaction)
  • Lane 2 VSC20
  • Lane 3 CV1 cells
  • Lane 4 P0 generation virus
  • C is the identification of IL21 in the culture supernatant of the P0 virus by ELISA The level
  • the abscissa is the group, the ordinate is the IL21 concentration (pg/mL), the "NC” shown in the abscissa is the CV1 cell, and the "plasmid” is the pCB-mIL21.
  • Figure 5 shows the results of identification of recombinant virus after plaque screening; wherein A and B respectively identified the oncolytic poxvirus DDvv-mIL21, DDvv-hIL21 by PCR, lane M: D000 marker, lane 1: negative control ( Namely, PCR reaction solution), Lane 2: P0 generation virus, Lane 3: DDvv-mIL21 (Fig. 5A) or DDvv-hIL21 (Fig.
  • C and D are respectively identified by PCR method for TK region, in which lane M :D000 marker, Lane 1: Negative control (ie, PCR reaction), Lane 2: VSC20, Lane 3: P0 generation virus, Lane 4: DDvv-mIL21 ( Figure 5C) or DDvv-hIL21 ( Figure 5D); E and G was used to detect the expression of IL21 protein in the oncolytic virus DDvv-mIL21 and DDvv-hIL21 using Western Blot, respectively.
  • the indicated "NC” refers to CV1 cells; F and H are ELISA kits, respectively. The content of IL21 in the culture solution and cell lysate of the cells infected with the oncolytic virus DDvv-mIL21 and DDvv-hIL21 was detected.
  • Figure 6 shows the killing effect of recombinant poxvirus DDvv-mIL21 carrying mouse IL-21 fragment on five kinds of mouse cancer cells, wherein A is the result of B16 cell experiment, B is the result of 4T1 cell experiment, and C is the result of LLC cell experiment. D is the result of GL261 cell experiment, and E is the result of CT26 cell experiment. 6A-E is the time (hour) and the ordinate is the cell killing rate (%).
  • Figure 7 shows the killing effect (AE) and IC 50 value (F) of recombinant poxvirus DDvv-mIL21 carrying murine IL-21 fragment against five mouse cancer cells, wherein A is the result of B16 cell experiment and B is CT26. Cell experiment results, C is the result of 4T1 cell experiment, D is the result of LLC cell experiment, and E is the result of GL261 cell experiment. 7A-E is the log value of the MOI, and the ordinate is the cell killing rate (%). The abscissa of Fig. 7F is the cell group, and the ordinate is the IC 50 value (MOI).
  • Figure 8 shows the killing effect of recombinant poxvirus DDvv-hIL21 carrying human IL-21 fragment on four human cancer cells, wherein A is the result of A549 cell experiment, B is the result of Hela cell experiment, and C is the result of SKOV3 cell experiment. D is the result of U251 cell experiment. 8A-D is the time (hour) and the ordinate is the cell killing rate (%).
  • Figure 9 shows the killing effect (AH) and IC 50 value (I) of recombinant poxvirus DDvv-hIL21 carrying human IL-21 fragment against 8 human cancer cells, wherein A is the result of A549 cell experiment and B is HepG2 Cell test results, C is the result of Hela cell experiment, D is the result of HT29 cell experiment, E is the result of SKOV3 cell experiment, F is the result of PANC1 cell experiment, G is the result of SK-HEP-1 cell experiment, and H is the result of FaDu cell experiment.
  • 9A-H is the log value of the MOI, and the ordinate is the cell killing rate (%).
  • Figure 9I shows the cell group on the abscissa and the IC 50 value (MOI) on the ordinate.
  • Figure 10 shows the antitumor effect of recombinant poxvirus DDvv-mIL21 carrying a murine IL-21 fragment on B16 tumor-bearing mice.
  • Fig. 10A is a graph showing changes in tumor volume with time, the abscissa is the administration time (days), and the ordinate is the tumor volume (mm 3 ).
  • Fig. 10B shows a plot of T/C as a function of time, the abscissa is the administration time (days), and the ordinate is T/C (%).
  • Figure 10C shows the comparison of tumor weights for different dose groups, with the abscissa being the group and the ordinate as the tumor weight (g).
  • Figure 11 shows the flow cytometric analysis of DDvv-mIL21 on B16 tumor-bearing mice; A and B for spleen extraction of PBMC for flow cytometry to detect changes in CD4 + cells (A) and CD8 + cells (B) ; C and D respectively for the flow analysis of tumor tissue extracted tumor cells to detect changes in CD4 + cells (C) and changes in CD8 + cells (D).
  • 11A-D is the dosing group, and the ordinate is the percentage (%) of CD4 + T or CD8 + T cells.
  • Figure 12 shows the tumor suppressor effect of recombinant poxvirus DDvv-mIL21 carrying a murine IL-21 fragment on LLC tumor-bearing mice.
  • Fig. 12A is a graph showing changes in tumor volume with time, the abscissa is the administration time (days), the ordinate is the tumor volume (mm 3 );
  • Fig. 12B is the curve of T/C with time, and the abscissa is the administration time ( Day), the ordinate is T/C (%).
  • Figure 13 shows the killing effect of DDvv-hIL21 poxvirus and NK cells carrying human IL-21 fragment on human tumor cell SK-HEP-1.
  • the abscissa is the group and the ordinate is the percentage value of the corresponding inhibition rate.
  • Figure 14 shows the anti-tumor effect of DDvv-mIL21 poxvirus carrying a murine IL-21 fragment on drug-induced immunocompromised B16 tumor-bearing mice.
  • Figure 14A shows the change in tumor volume after treatment, the abscissa is the B16 tumor cell inoculation time (days), the arrow on the abscissa indicates the time point of administration of DDvv-mIL21, and the ordinate is the tumor volume (mm 3 );
  • Figure 14B shows The T/C curve after treatment, the abscissa is B16 tumor cell inoculation time (days), and the ordinate is T/C (%).
  • Figure 15 shows a comparison of the anti-tumor effect of DDvv-mIL21 poxvirus carrying a murine IL-21 fragment with a control poxvirus on drug-induced immunocompromised B16 tumor-bearing mice.
  • Figure 15A shows the change in tumor volume after treatment, the abscissa is the time after administration (days), the ordinate is the tumor volume (mm 3 );
  • Figure 15B shows the change curve of T/C after treatment, and the abscissa is given The post-drug time (days), the ordinate is T/C (%);
  • Figure 15C shows the tumor weight after treatment, the abscissa is the group, and the ordinate is the tumor weight (g).
  • Figure 16 shows a flow cytometric comparison of the immunological effects of DDvv-mIL21 poxvirus carrying a murine IL-21 fragment and control poxvirus on drug-induced immunocompromised B16 tumor-bearing mice.
  • Figures 16A-C show flow analysis to detect changes in CD4 + cells (A), changes in CD8 + cells (B), and changes in NK cells (C), respectively.
  • FIGS 16A-C, the abscissa represents the administration group, respectively ordinate percentage of the total number of CD4 + T cells in the immune cells and tumor tissue (%) (A) in tumor tissue, CD8 + T cells and tumor tissues Total Percentage (%) of immunocytes (B) or percentage (%) of total NK cells to total immune cells in tumor tissues (C).
  • Figure 17 is a graph showing the antitumor effect of DDvv-hIL21 poxvirus-conjugated human NK cells carrying human IL-21 fragment and control poxvirus combined with human NK cells on HCT116 tumor-bearing severe immunodeficient mice.
  • Fig. 17A shows the change curve of the tumor volume after treatment, the abscissa is the HCT116 tumor cell inoculation time (days), the arrow on the abscissa indicates the time point of administration of DDvv-hIL21, and the ordinate is the tumor volume (mm 3 );
  • Fig. 17B shows The T/C curve after treatment, the abscissa is the time of inoculation of HCT116 tumor cells (days), and the ordinate is T/C (%).
  • Figure 18A-D shows comparison of tumor tissue analysis after treatment of HCT116 tumor-bearing severe immunodeficient mice with DDvv-hIL21 poxvirus-bearing human NK cells carrying human IL-21 fragments and control poxvirus combined with human NK cells (using quantification) PCR method, including the relative expression level of the poxvirus A46R gene in the tumor tissue (A), the relative expression level of the IL-21 gene (B), the relative expression level of the NKG2D gene of the NK cell (C), and the IFN- ⁇ gene. Relative expression level (D).
  • 18A-D is the dosing group, and the ordinate is the target gene in the tumor tissue (A46R gene (A) of the poxvirus in the tumor, IL-21 gene (B) in the tumor, and NK cells in the tumor.
  • Figure 19 shows the distribution of intravenously administered DDvv-mIL21 poxvirus in B16 tumor mice.
  • the abscissa is the tissue and organ type, and the ordinate is the comparison of the expression level of the poxvirus A46R gene in each tissue organ and the expression level of the poxvirus A46R gene in the kidney organs.
  • Figure 20 shows the killing effect of DDvv-hIL21 poxvirus carrying human IL-21 fragment and human NK cells on human tumor cell HepG2.
  • the abscissa is the group and the ordinate is the percentage value of the corresponding inhibition rate.
  • Figure 21 shows a comparison of the killing effect of DDVV-hIL21 poxvirus in combination with human NK cells carrying human IL-21 fragment and control poxvirus in combination with human NK cells on human tumor cell HCT116.
  • 21A shows a comparison of the NK group, the DDvv-hIL21 group, and the DDvv-hIL21+NK group;
  • FIG. 21B shows a comparison of each experimental group.
  • the abscissa is a group, and the ordinate is a percentage value of the corresponding inhibition rate.
  • Figure 22 shows a comparison of the killing effect of DDVV-hIL21 poxvirus in combination with human NK cells carrying human IL-21 fragments and control poxvirus in combination with human NK cells on human tumor cells FaDu.
  • Figure 22A shows a comparison of the NK group, the DDvv-hIL21 group, and the DDvv-hIL21+NK group;
  • Figure 22B shows a comparison of each experimental group.
  • the abscissa is a group, and the ordinate is a percentage value of the corresponding inhibition rate.
  • the human body is a complex system consisting of ten systems, including breathing, circulation, and digestion. These systems work together to make various complex life activities in the human body work normally.
  • the anti-tumor mechanism of the body includes both cellular immunity and humoral immunity. They are closely related and interact with each other and involve a variety of immune effector molecules and effector cells. It is generally believed that cellular immunity plays a leading role in the anti-tumor process, and humoral immunity plays a synergistic role in some cases.
  • the invention proposes to utilize the oncolytic pox virus to selectively replicate and kill tumor cells in tumor cells, and at the same time, to carry the immunoregulatory factor IL-21, so that the recombinant oncolytic pox virus synergistically exerts selective oncolytic and enhancement.
  • the role of the body against tumor immune effects Based on this concept, the inventors of the present invention discovered through experimental research and theoretical exploration that the functional defects of the TK gene and the VGF gene of the oncolytic virus are simultaneously integrated, and the IL-21 gene is integrated in the genome, which can achieve the above synergy well. effect.
  • Vaccinia virus is one of the largest and most complex viruses ever discovered. It is a vaccine virus against smallpox. The special biological properties of this virus have made it more and more concerned in the field of tumor immunotherapy/gene therapy: (1) Safety: Poxvirus is a DNA virus that replicates in the cytoplasm of cells and does not integrate into the host cell genome. To reduce the variation induced by foreign genes (see literature: "Hruby, DEVaccinia Virus Vectors: New Strategies for Producing Recombinant Vaccines. Clin. Microbiol. Rev.
  • Poxvirus can be cultured to very high titers (>10 9 pfu/ml), usually infected for 1-3 hours, allowing more than 90% of infected cells to express the target gene product (see literature: "Pfleiderer, M., Falkner, FG & Dorner, FA novel vaccinia virus expression system allows construction of recombinants without the need for selection markers, plasmids and bacterial hosts. J. Gen. Virol.
  • infected cells Wide range almost infects all types of mammalian cells, including dividing and non-dividing cells (see the literature: "Hruby, DEVaccinia Virus Vectors: New Strategies for Producing Rec Ombinant Vaccines. Clin. Microbiol. Rev. 3, 153-170 (1990).”); (4) Large genomic capacity: at least 25 kb of foreign gene can be inserted without affecting its genetic stability (see literature: "Hruby, DEVaccinia Virus Vectors: New Strategies for Producing Recombinant Vaccines. Clin. Microbiol. Rev.
  • TK thymidine kinase gene of poxvirus
  • the thymidine kinase (TK) gene of poxvirus is one of the genes that the poxvirus replication process depends on. If the poxvirus lacks the TK gene, it needs to provide TK protein replenishment through the host cell, while the TK protein can only be transiently expressed in the normal cell cycle, but the TK protein is persistently highly expressed in most tumor cells. Therefore, the expression characteristics of TK protein in tumor tissues are limited to the replication of TK-deficient poxvirus in tumor tissues, but not in normal cells, thereby improving tumor targeting. Since the introduction of the poxvirus genetic engineering system in 1982, the TK gene region has been used for the insertion region of foreign genes (see the literature: "Byrd, C. & Hruby, D. Construction of Recombinant Vaccinia Virus. in Vaccinia Virus and Poxvirology :Methods and Protocols 269, 31-40 (2004).”).
  • VGF virus growth factor
  • IL-21 (interleukin-21) is a multi-directional type I cytokine produced primarily by T cells that regulates innate and acquired immune responses and plays a role in anti-tumor immune responses. Important role. The role of IL-21 in various immune cells and signal transduction has been reported in the literature (see the literature: "Leonard, WJ & Wan, C. IL-21 Signaling in Immunity.
  • the immune cells mainly include: 1) CD4 + T cells: promote proliferation, produce cytokines; T fh cells: promote differentiation, improve developmental center function; Th17 cells: promote differentiation and proliferation; Treg cells: inhibit their production and survival; NKT cells: proliferation, enhance cytotoxicity; 3) CD8 + T cells: enhance cytotoxicity, proliferation and / or survival, anti-tumor effect; 4) NK cells: promote cell maturation, proliferation, increase cytotoxicity, enhance anti-tumor Activity; 5) DC cells: inhibit antigen presentation function, induce apoptosis; 6) macrophages: enhance phagocytosis; 7) B cells: promote proliferation and / or apoptosis, promote plasma cell differentiation and immunoglobulin production 8) Additional IL-21 can activate a variety of tumor-associated signaling pathways, including JAK/STAT, MARK/PI3K signaling pathways, and regulate tumor progression.
  • T fh cells promote differentiation, improve developmental center function
  • Th17 cells promote differentiation and proliferation
  • Treg cells inhibit their production
  • IL-21 plays an important role in this procedure.
  • IL-21 promotes maturation of NK cells to produce IFN- ⁇ and perforin, and induces NK cell-mediated anti-tumor cytotoxicity targeting NKG2D ligands on the surface of tumor cells via antibody-dependent cell-mediated cytotoxicity (ADCC) Increasing the lethality of NK cells (see the literature: "Spolski, R. & Leonard, WJ Interleukin-21: a double-edged sword with therapeutic potential. Nat. Rev. Drug Discov. 13, 379-395 (2014).”).
  • IL-21 enhances the killing of CD8 + T cells by promoting the proliferation of CD8 + T cells, inducing the generation of memory T cells, and promoting the secretion of IFN ⁇ /granzyme, thereby facilitating the memory of recurrent tumor cells.
  • Immune response Importantly, unlike IL-2, IL-21 does not induce expansion of Treg cells, and further enhances the immune function response of CD8 + T cells (see literature: "Spolski, R. & Leonard, WJ Interleukin-21: a double -edged sword with therapeutic potential. Nat. Rev. Drug Discov. 13, 379-395 (2014).”). Based on the diversification of immune cells by IL-21, IL-21 can “reactivate” multiple effector cells in the tumor microenvironment. Several clinical studies have been used alone or in combination with other drugs. tumor treatment.
  • the present invention provides an isolated recombinant oncolytic poxvirus, wherein the recombinant oncolytic poxvirus is functionally deficient in the TK gene and the VGF gene, and the exogenous IL-integrated in the genome of the recombinant oncolytic poxvirus 21 gene, and the IL-21 gene can be expressed in tumor cells.
  • the term "functional deficiency" as used in reference to the gene of oncolytic virus of the present invention means that the oncolytic virus is unable to exert its intended function, ie, loss of function, which can be inserted, for example, by insertion into a gene.
  • the source fragment is obtained by knocking out the gene.
  • an exogenous nucleotide sequence can be inserted into the TK gene, thereby rendering its function defective.
  • An exogenous nucleotide sequence may also be inserted into the VGF gene to render it functionally defective, but the VGF gene is preferably knocked out.
  • the exogenous IL-21 gene is inserted into the TK gene, which enables the TK gene to be functionally deficient and capable of expressing the IL-21 gene after infection of the tumor cell.
  • the poxvirus which can be used includes Wyeth strain or WR strain, and an example of the WR strain is VSC20.
  • the recombinant oncolytic poxvirus is obtained by genetic engineering of a VSC20 poxvirus.
  • VSC20 poxvirus is a VGF gene-deficient poxvirus, in which the LacZ gene is inserted at the C11R site.
  • the genetic modification involves insertion of an exogenous IL-21 gene into the TK gene of the VSC20 poxvirus, thereby rendering the TK gene functionally defective.
  • a map of a plasmid for inserting the IL-21 gene into the TK gene of poxvirus VSC20 and a schematic diagram for achieving insertion by a recombination mechanism are shown in FIG.
  • the exogenous screening gene may also be integrated into the genome of the recombinant oncolytic poxvirus, and the exogenous screening gene includes a gpt (guanine phosphoribosyltransferase) gene and/or a LacZ gene, but does not include a fluorescent protein gene. Avoid the safety hazards caused by the expression of fluorescent proteins in patients.
  • gpt guanine phosphoribosyltransferase
  • the exogenous screening gene may also not be integrated into the genome of the recombinant oncolytic poxvirus.
  • the invention controls the gpt gene by using the poxvirus early/late promoter p7.5, respectively, and the artificially synthesized poxvirus early promoter pSEL to control the exogenous IL-21 gene, using in vitro intracellular recombination techniques to gpt
  • the IL-21 gene was inserted into the TK gene region of the poxvirus VSC20 strain to construct an oncolytic virus, and the two promoters respectively activated the expression of the respective regulatory genes in a back-to-back manner.
  • the present invention further finds that only the exogenous IL-21 gene is inserted into the TK gene, thereby causing functional defects, and the function of the VGF gene of the oncolytic poxvirus is deficient, thereby enabling the oncolytic virus to selectively act on the tumor.
  • the cells replicate and kill tumor cells, causing subsequent immune responses, and the exogenous IL-21 gene carried by the oncolytic pox virus directly induces anti-tumor immunity.
  • the exogenous IL-21 gene is derived from a mouse or a human.
  • the recombinant oncolytic poxvirus of the present invention can be obtained by a related known method in the field of bioengineering, and the flow of one embodiment is shown in Fig. 2.
  • the invention takes into consideration the respective characteristics of the oncolytic poxvirus and IL-21, and skillfully integrates them to work together, wherein the oncolytic pox virus can be amplified by the function of replicating in tumor cells while oncolytic IL-21 is expressed in tumor cells and then excreted to the outside of the cells, thereby further inducing tumor immunity in the body, and the combined effect of the two has a better therapeutic effect.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant oncolytic poxvirus according to the present invention as an active ingredient, and a pharmaceutically acceptable excipient, based on the recombinant oncolytic poxvirus developed by the present invention.
  • the pharmaceutical composition comprises a therapeutically effective amount of the recombinant oncolytic poxvirus.
  • the active ingredient of the pharmaceutical composition comprises a 1 x 10 5 to 5 x 10 9 pfu/day dose of a recombinant oncolytic poxvirus according to the invention (eg, 1 x 10 5 to 3) ⁇ 10 9 pfu/day dose of recombinant oncolytic poxvirus according to the invention, 1 x 10 5 to 1 x 10 8 pfu/day dose of recombinant oncolytic pox virus according to the invention, etc.).
  • the recombinant oncolytic poxvirus can be administered by administration in a manner generally employed in the art, for example, by intratumoral injection or intravenous administration.
  • compositions of the invention may also comprise other active ingredients known in the art, such as interleukin-2 (IL-2), IL-15, IL-18, granulocyte-macrophage colony stimulating factor (GM- CSF), interferon- ⁇ (IFN- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ), and the like, can be administered in a conventional manner in the manner of administration and administration.
  • active ingredients such as interleukin-2 (IL-2), IL-15, IL-18, granulocyte-macrophage colony stimulating factor (GM- CSF), interferon- ⁇ (IFN- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ), and the like
  • IL-2 interleukin-2
  • IL-15 IL-15
  • IL-18 granulocyte-macrophage colony stimulating factor
  • IFN- ⁇ interferon- ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • the recombinant oncolytic poxvirus should be present independently in the pharmaceutical composition without inter
  • compositions of the present invention may also comprise suitable pharmaceutically acceptable excipients.
  • the pharmaceutical compositions of the invention comprise one or more pharmaceutically acceptable carriers.
  • Pharmaceutical formulations can be prepared by methods known in the art.
  • an active ingredient such as a compound can be formulated with a common excipient, a diluent (for example, phosphate buffer or physiological saline), a tissue culture medium, and a carrier (for example, autologous plasma or human serum albumin) as a suspension.
  • a diluent for example, phosphate buffer or physiological saline
  • tissue culture medium for example, autologous plasma or human serum albumin
  • carriers for example, autologous plasma or human serum albumin
  • Other carriers may include liposomes, micelles, nanocapsules, polymeric nanoparticles, solid lipid particles (see, for example, the literature "E. Koren and V. Torchilin, Life, 63:586-595, 2011").
  • Specific methods of formulating the pharmaceutical compositions of the present invention can be found in the scientific literature and in the patent literature, for example, see
  • Another aspect of the invention also provides a vector for the preparation of the recombinant oncolytic poxvirus of the invention.
  • the vector can cause functional defects in the TK gene and the VGF gene in the poxvirus by a recombinant mechanism.
  • the recombinant vector comprises a TK homologous fragment TK-L, TK-R, and an expression cassette that initiates the IL-21 gene and the exogenous screening gene gpt.
  • the poxvirus is a WR strain
  • the sequence of the TK gene is the poxvirus gene numbered NC_006998 in GenBank of NCBI (ie, National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov). In the sequence shown in No.
  • the sequence of TK-L may be, for example, a sequence fragment shown in No. 80724-80961bp
  • the sequence of TK-R may be, for example, a sequence fragment shown in No. 81033-81257bp.
  • the vector can insert the IL-21 gene expression cassette and the gpt gene expression cassette into the TK gene region of the poxvirus by an intracellular recombination mechanism (such that, for example, the 80962-81032 bp in the poxvirus gene numbered NC_006998 in GenBank) The sequence fragment shown is deleted), thereby causing the recombinant poxvirus to lose its TK gene function.
  • Another aspect of the invention also provides a host cell comprising the vector of the invention.
  • Another aspect of the invention also provides the use of a recombinant oncolytic poxvirus of the invention in the manufacture of a medicament for the treatment of a tumor and/or cancer.
  • the tumor and/or cancer includes, but is not limited to, lung cancer (eg, non-small cell lung cancer), melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma head and neck cancer
  • liver cancer brain cancer
  • colorectal cancer bladder cancer
  • breast cancer breast cancer
  • ovarian cancer uterine cancer
  • Cervical cancer lymphoma
  • gastric cancer esophageal cancer
  • kidney cancer esophageal cancer
  • prostate cancer pancreatic cancer
  • leukemia e.g., testicular cancer, etc.
  • Another aspect of the invention also provides a method of treating a tumor and/or cancer comprising administering a recombinant oncolytic poxvirus according to the invention to a tumor and/or cancer patient.
  • the tumor and/or cancer includes, but is not limited to, lung cancer (eg, non-small cell lung cancer), melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma head and neck cancer
  • liver cancer brain cancer
  • colorectal cancer bladder cancer
  • breast cancer breast cancer
  • ovarian cancer uterine cancer
  • Cervical cancer lymphoma
  • gastric cancer esophageal cancer
  • kidney cancer esophageal cancer
  • prostate cancer pancreatic cancer
  • leukemia e.g., testicular cancer, etc.
  • the recombinant oncolytic poxvirus is administered in a therapeutically effective amount once a day for 1-6 days (including continuous administration for 1 day, 2 days, 3 days, 4 days). , 5 or 6 days).
  • the therapeutically effective amount is preferably from 1 x 10 5 to 5 x 10 9 pfu per day (e.g., 1 x 10 5 to 3 x 10 9 pfu per day dose, 1 x 10 5 to 1 x 10 8 pfu per day dose) Wait).
  • the recombinant oncolytic poxvirus of the present invention can also be used in combination with other drugs, such as interleukin-2 (IL-2), IL-15, IL-18, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon- ⁇ (IFN- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ), and the like, the administration dose and administration manner can be carried out in a conventional manner.
  • IL-2 interleukin-2
  • IL-15 IL-15
  • IL-18 granulocyte-macrophage colony-stimulating factor
  • IFN- ⁇ interferon- ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • the method of treating tumors and/or cancer of the present invention may be performed one or more times on the patient according to actual conditions and needs.
  • the recombinant oncolytic poxvirus can be administered by administration in a manner generally employed in the art, for example, by intratumoral injection or intravenous administration.
  • the invention also provides a therapeutic agent comprising:
  • a first pharmaceutical composition wherein the first pharmaceutical composition comprises the recombinant oncolytic poxvirus of the present invention in a first pharmaceutically acceptable carrier;
  • the first pharmaceutically acceptable carrier and the second pharmaceutically acceptable carrier are the same. In other embodiments, the first pharmaceutically acceptable carrier and the second pharmaceutically acceptable carrier are different.
  • the therapeutic agent can also be understood as a combination of drugs.
  • the oncolytic virus contacts the tumor cells by intratumoral or intravenous administration and the infection enters the tumor cells. Since the oncolytic virus is characterized in that it mainly replicates and proliferates in tumor cells, but does not replicate or replicate in normal cells, a large number of oncolytic viruses appear in infected tumor cells, causing tumor cell lysis and death. . The dissolution of tumor cells releases a large number of tumor antigens and proliferating oncolytic viruses. The antigen further activates the immune system in the body, stimulating NK cells and T cells in the body to continue to attack tumor cells that have not yet died, and the new oncolytic virus will Continue to infect tumor cells that have not yet been infected.
  • NK cells are broad-spectrum immune cells that kill tumor cells, and NK cells can distinguish between tumor cells and normal cells. NK contacts and recognizes tumor cells, recognizes it as an abnormal cell, and then kills it through receptor recognition, antibody-targeted recognition (ADCC), granzyme secretion, perforin secretion, and indirect killing of interferon. The effect of dead tumor cells. In vitro experiments have shown that a healthy NK cell can kill 27 tumor cells in a row during its lifetime.
  • ADCC antibody-targeted recognition
  • NK cells also have antiviral functions.
  • a normal cell is infected with a virus, as the virus replicates a lot, the cell exhibits an aging lesion, and the composition of the protein cluster reflected on the cell membrane changes.
  • the NK cell can recognize the infected patient sharply and efficiently.
  • the cells by the means described above similar to killing tumor cells, kill the infected cells, thereby achieving the purpose of inhibiting viral replication and proliferation. Subsequently, under the action of factors such as antigen stimulation and interferon, other immune cells will continue to act against the virus.
  • the present invention takes into consideration the respective characteristics of oncolytic viruses and NK cells, and uses them in combination.
  • the antiviral mechanism of NK cells is equally applicable to tumor cells infected with oncolytic viruses, and is complementary to its anti-tumor mechanism.
  • the combination also makes tumor cells containing oncolytic virus a specific target of NK cells, thereby enhancing the tumor killing effect of NK cells.
  • the oncolytic virus selectively proliferates in cancer cells, plays a role in killing cancer cells in the cell, and can cause changes in protein receptor clusters on the cancer cell membrane, enhancing the recognition of cancer cells by NK cells, and NK cells outside the cancer cells. Attack, the two together to kill cancer cells, have a better therapeutic effect.
  • the recombinant oncolytic poxvirus of the present invention simultaneously expresses exogenous IL-21, and the expressed exogenous IL-21 can enhance the killing power of NK cells to further enhance the killing effect of NK cells, so that the present invention
  • the combination of recombinant oncolytic poxvirus and NK cells has a surprising effect on the killing effect of tumors.
  • the active ingredient of the first pharmaceutical composition is a recombinant oncolytic poxvirus of the invention
  • the active ingredient of the second pharmaceutical composition is the NK cell.
  • the first pharmaceutical composition and the second pharmaceutical composition are each independently present in the therapeutic agent without mixing with each other.
  • the NK cells may be selected from autologous NK cells and allogeneic NK cells; preferably, the NK cells are autologous NK cells obtained by in vitro expansion or allogeneic NK cells obtained by in vitro expansion.
  • Large-scale in vitro expansion culture techniques for NK cells are known and have been largely mature (see, for example, the following scientific literature: "Somanchi SS, Lee DA. Ex Vivo Expansion of Human NK Cells Using K562 Engineered to Express Membrane Bound IL21 .Methods Mol Biol.2016;1441:175-93.” or "Phan MT, Lee SH, Kim SK, Cho D.
  • the purity of the NK cells which can be used for treatment may be: the purity of the autologous NK cells may be 85% or more, and the purity of the allogeneic NK cells may be 90% or more; the impurity cells may be NK-T and/or ⁇ T cells. .
  • the NK cell activity (survival rate) is 90% or more, and the NK cell killing activity is 80% or more.
  • the present invention further explores the optimization of the respective administration doses of oncolytic virus and NK cells, which is critical.
  • the first pharmaceutical composition comprises a dose of 1 ⁇ 10 5 - 5 ⁇ 10 9 pfu / day of the recombinant oncolytic pox virus (for example, a dose of 1 ⁇ 10 5 - 3 ⁇ 10 9 pfu / day) Recombinant oncolytic poxvirus, 1 ⁇ 10 5 -1 ⁇ 10 8 pfu/day dose of the recombinant oncolytic poxvirus, etc.), and the second pharmaceutical composition comprises 1 ⁇ 10 7 -1 ⁇ 10 10 a cell/day dose of said NK cells (preferably, said second pharmaceutical composition comprises from 1 x 10 8 to 5 x 10 9 cells per day of said NK cells; further preferably said second drug
  • the composition comprises 1 x 10 9 to 4 x 10 9 cells per day of the NK cells; more preferably, the second pharmaceutical composition comprises 1 x 10 9 to 3
  • the active ingredient of the therapeutic agent is from 1 x 10 5 to 5 x 10 9 pfu per day of the recombinant oncolytic poxvirus (e.g., 1 x 10 5 to 3 x 10 9 pfu per day dose) Recombinant oncolytic poxvirus, 1 ⁇ 10 5 to 1 ⁇ 10 8 pfu/day dose of the recombinant oncolytic poxvirus, etc.) and 1 ⁇ 10 7 to 1 ⁇ 10 10 cells/day dose of the NK cells (For example, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells/day of the NK cells, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells/day of the NK cells, 1 ⁇ 10 9 to 3 ⁇ 10 9 cells/day dose of the NK cells, etc.).
  • the recombinant oncolytic poxvirus e.g., 1 x 10 5 to 3 x 10 9 pfu per day dose
  • Recombinant oncolytic poxvirus e.g., 1 x
  • the recombinant oncolytic poxvirus can be administered by administration in a manner generally employed in the art, for example, by intratumoral injection or intravenous administration.
  • NK cells can be administered by administration generally employed in the art, for example, by intravenous administration.
  • therapeutic agents of the present invention may also comprise suitable pharmaceutically acceptable excipients.
  • the therapeutic agents of the invention may also comprise other active ingredients known in the art, such as interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN). - ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ) and the like.
  • IL-2 interleukin-2
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • IFN interferon-gamma
  • TNF- ⁇ tumor necrosis factor- ⁇
  • a therapeutic agent of the invention comprises one or more pharmaceutically acceptable carriers.
  • Pharmaceutical formulations can be prepared by methods known in the art.
  • an active ingredient such as a compound can be formulated with a common excipient, a diluent (for example, phosphate buffer or physiological saline), a tissue culture medium, and a carrier (for example, autologous plasma or human serum albumin) as a suspension.
  • a diluent for example, phosphate buffer or physiological saline
  • tissue culture medium for example, autologous plasma or human serum albumin
  • carrier for example, autologous plasma or human serum albumin
  • Other carriers may include liposomes, micelles, nanocapsules, polymeric nanoparticles, solid lipid particles (see, for example, the literature "E. Koren and V. Torchilin, Life, 63:586-595, 2011").
  • Specific methods of formulating the therapeutic agents of the present invention can be found in the scientific literature and in the patent literature, for example, see the
  • the therapeutic agents of the present invention can treat a variety of tumors and/or cancers including, but not limited to, lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer , cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer.
  • the therapeutic agent of the present invention is administered by first administering the recombinant oncolytic poxvirus to a tumor and/or cancer patient, and then, 18-72 hours after administration of the recombinant oncolytic poxvirus (for example, the 20th
  • the NK cells are administered to the tumor and/or cancer patient at -70 hours, 22-48 hours, 24-48 hours, 30-48 hours, and the like.
  • the 18th to 72th hours after administration of the recombinant oncolytic poxvirus eg, 20-70 hours, 22-48 hours, 24-48 hours, 30-48 hours, etc.
  • the tumor And/or administration of the NK cells to a cancer patient means that the time interval between administration of the first NK cells and the first recombinant oncolytic pox virus administration is 18-72 hours (eg, 20-70 hours, 22-48 hours, 24-48) Hours, 30-48 hours, etc., or the time interval between administration of the first NK cells and the recombinant oncolytic pox virus administered immediately before it is 18-72 hours (eg, 20-70 hours, 22- 48 hours, 24-48 hours, 30-48 hours, etc.).
  • the time interval between administration of the first NK cell and the recombinant oncolytic pox virus administered immediately before it is 18-72 hours (eg, 20-70 hours, 22-48 hours, 24-48 hours) , 30-48 hours, etc.). Also preferably, the time interval between administration of the first NK cells and the recombinant oncolytic pox virus administered immediately before it is 24-48 hours.
  • the recombinant oncolytic poxvirus is administered in a therapeutically effective amount once a day for 1-6 days; and the NK cells are administered at a dose of 1 ⁇ 10 7 to 1 ⁇ 10 10 cells/day dose (for example, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 3 ⁇ 10 9 cells/day dose), once a day, for 1-6 days.
  • the recombinant oncolytic poxvirus is administered in a therapeutically effective amount once every two days for 2-6 days; and the NK cells are administered at a dose of 1 ⁇ 10 7 to 1 ⁇ 10 10 cells/day dose (for example, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 3 ⁇ 10 9 cells / day dose), once every 2 days, continuous administration for 2-6 days.
  • the NK cells are administered at a dose of 1 ⁇ 10 7 to 1 ⁇ 10 10 cells/day dose (for example, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 3 ⁇ 10 9 cells / day dose), once every 2 days, continuous administration for 2-6 days.
  • administration of the recombinant oncolytic poxvirus and administration of NK cells may be by intermittent administration (for example, administration of the recombinant oncolytic poxvirus on day 1, administration of NK cells on day 2, administration of the recombination on day 3) Oncolytic pox virus, administration of NK cells on day 4, etc.) or sequential administration (for example, administration of the recombinant oncolytic poxvirus on day 1 and sequential administration of the recombinant oncolytic poxvirus on day 2 and NK cells, the recombinant oncolytic poxvirus and NK cells are sequentially administered on day 3, the recombinant oncolytic poxvirus and NK cells are sequentially administered on day 4, and so on; or other modes of administration (eg, first administration)
  • the recombinant oncolytic poxvirus is administered once a day for 1-6 days, followed by administration of NK cells at intervals of 18-72 hours, once a day for 1-6 days.
  • the recombinant oncolytic pox virus is administered first, and NK cells are administered at intervals of 18-72 hours after all administration of the recombinant oncolytic pox virus.
  • the recombinant oncolytic poxvirus is first administered to a tumor and/or cancer patient, the recombinant oncolytic pox virus is administered in a therapeutically effective amount, administered once; and at the administration site
  • the NK cells are administered to the tumor and/or cancer patient from 18 hours to 72 hours after the recombinant oncolytic pox virus, and the NK cells are administered at a dose of 1 ⁇ 10 7 to 1 ⁇ 10 10 cells / Day dose (for example, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 3 ⁇ 10 9 cells / day dose) , applied once.
  • the therapeutically effective amount of the recombinant oncolytic poxvirus is preferably from 1 x 10 5 to 5 x 10 9 pfu per day (e.g., 1 x 10 5 to 3 x 10 9 pfu per day dose, 1 x 10 5 to 1 x). 10 8 pfu / day dose, etc.).
  • the invention also provides the use of a therapeutic agent according to the invention in the manufacture of a medicament for the treatment of tumors and/or cancer.
  • the tumor and/or cancer includes, but is not limited to, lung cancer (eg, non-small cell lung cancer), melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma head and neck cancer
  • liver cancer brain cancer
  • colorectal cancer bladder cancer
  • breast cancer breast cancer
  • ovarian cancer uterine cancer
  • Cervical cancer lymphoma
  • gastric cancer esophageal cancer
  • kidney cancer esophageal cancer
  • prostate cancer pancreatic cancer
  • leukemia e.g., testicular cancer, etc.
  • kits for synergistic combination therapy for treating tumors and/or cancer comprising a first container containing the recombinant oncolytic poxvirus of the present invention and containing the present invention A second container of NK cells, wherein the first container and the second container are independent; and instructions for administering the timing and mode of administration.
  • the kit consists of separate containers each containing the recombinant oncolytic poxvirus of the present invention and the NK cells of the present invention, respectively, together with instructions for the timing and mode of administration.
  • the tumor and/or cancer includes, but is not limited to, lung cancer (eg, non-small cell lung cancer), melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma head and neck cancer
  • liver cancer brain cancer
  • colorectal cancer bladder cancer
  • breast cancer breast cancer
  • ovarian cancer uterine cancer
  • Cervical cancer lymphoma
  • gastric cancer esophageal cancer
  • kidney cancer esophageal cancer
  • prostate cancer pancreatic cancer
  • leukemia e.g., testicular cancer, etc.
  • the first container containing the recombinant oncolytic poxvirus of the present invention comprises a therapeutically effective amount of the recombinant oncolytic poxvirus
  • the second container containing the NK cells comprises sufficient to provide 1 x 10 7 -1 x 10 10 cells/day dose of said NK cells (for example, 1 x 10 8 to 5 x 10 9 cells/day dose of said NK cells, 1 x 10 9 to 4 x 10 9 cells/day dose The NK cells, 1 x 10 9 to 3 x 10 9 cells/day of the NK cells, etc.).
  • the therapeutically effective amount of the recombinant oncolytic poxvirus is preferably from 1 x 10 5 to 5 x 10 9 pfu per day (e.g., 1 x 10 5 to 3 x 10 9 pfu per day dose, 1 x 10 5 to 1 x). 10 8 pfu / day dose, etc.).
  • the first container containing the recombinant oncolytic poxvirus comprises a dose of 1 ⁇ 10 5 -1 ⁇ 10 8 pfu/day of the recombinant oncolytic poxvirus, and the NK cell-containing fraction
  • the second container contains 1 x 10 9 to 3 x 10 9 cells per day of the NK cells.
  • the NK cells may be selected from autologous NK cells and allogeneic NK cells; preferably, the NK cells are autologous NK cells obtained by in vitro expansion or allogeneic NK cells obtained by in vitro expansion.
  • the recombinant oncolytic poxvirus can be administered by its respective mode of administration conventionally employed in the art, for example, by intratumoral injection or intravenous administration.
  • NK cells can be administered by administration generally employed in the art, for example, by intravenous administration.
  • the tumor and/or cancer includes, but is not limited to, lung cancer (eg, non-small cell lung cancer), melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma head and neck cancer
  • liver cancer brain cancer
  • colorectal cancer bladder cancer
  • breast cancer breast cancer
  • ovarian cancer uterine cancer
  • Cervical cancer lymphoma
  • gastric cancer esophageal cancer
  • kidney cancer esophageal cancer
  • prostate cancer pancreatic cancer
  • leukemia e.g., testicular cancer, etc.
  • Another aspect of the invention also provides a method of treating a tumor and/or cancer, comprising the steps of:
  • the NK cells of the invention are administered to a tumor and/or cancer patient.
  • the 18th to 72th hours after administration of the recombinant oncolytic poxvirus eg, 20-70 hours, 22-48 hours, 24-48 hours, 30-48 hours, etc.
  • the tumor And/or administration of a NK cell of the invention to a cancer patient means that the time interval between administration of the first NK cell and the first recombinant oncolytic pox virus administration is 18-72 hours (eg, 20-70 hours, 22-48 hours, 24-48 hours, 30-48 hours, etc.), or the time interval between administration of the first NK cells and the recombinant oncolytic pox virus administered immediately before it is 18-72 hours (eg, 20-70 hours) , 22-48 hours, 24-48 hours, 30-48 hours, etc.).
  • the time interval between administration of the first NK cell and the recombinant oncolytic pox virus administered immediately before it is 18-72 hours (eg, 20-70 hours, 22-48 hours, 24-48 hours) , 30-48 hours, etc.). Also preferably, the time interval between administration of the first NK cells and the recombinant oncolytic pox virus administered immediately before it is 24-48 hours.
  • the tumor and/or cancer includes, but is not limited to, lung cancer (eg, non-small cell lung cancer), melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, Cervical cancer, lymphoma, gastric cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer, etc.
  • lung cancer eg, non-small cell lung cancer
  • melanoma head and neck cancer
  • liver cancer brain cancer
  • colorectal cancer bladder cancer
  • breast cancer breast cancer
  • ovarian cancer uterine cancer
  • Cervical cancer lymphoma
  • gastric cancer esophageal cancer
  • kidney cancer esophageal cancer
  • prostate cancer pancreatic cancer
  • leukemia e.g., testicular cancer, etc.
  • Oncolytic viruses are capable of selective replication in tumor or cancer cells and peak over time.
  • the inventors of the present invention found that after a period of replication, the oncolytic virus in the tumor cells promotes killing of tumor cells by NK cells. Therefore, the application sequence and interval of the recombinant oncolytic poxvirus and NK cells proposed by the present invention achieve a bimodal overlap of the peaks of action of both.
  • the present invention further explores that the respective administration doses of the recombinant oncolytic poxvirus and NK cells are optimized, and the cooperation with the above administration sequence and administration interval is crucial, which determines the anti-tumor of the recombinant oncolytic poxvirus. Efficacy, anti-tumor efficacy of NK cells, and optimal synergistic killing of both tumor cells.
  • the recombinant oncolytic poxvirus is administered in a therapeutically effective amount once a day for 1-6 days; and the NK cells are administered at a dose of 1 ⁇ 10 7 to 1 ⁇ 10 10 cells/day dose (for example, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 3 ⁇ 10 9 cells/day dose), once a day, for 1-6 days.
  • the recombinant oncolytic poxvirus is administered in a therapeutically effective amount once every two days for 2-6 days; and the NK cells are administered at a dose of 1 ⁇ 10 7 to 1 ⁇ 10 10 cells/day dose (for example, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 3 ⁇ 10 9 cells / day dose), once every 2 days, continuous administration for 2-6 days.
  • the NK cells are administered at a dose of 1 ⁇ 10 7 to 1 ⁇ 10 10 cells/day dose (for example, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 3 ⁇ 10 9 cells / day dose), once every 2 days, continuous administration for 2-6 days.
  • administration of recombinant oncolytic poxvirus and administration of NK cells may be by intermittent administration (for example, administration of recombinant oncolytic poxvirus on day 1, administration of NK cells on day 2, and administration of recombinant oncolytic poxvirus on day 3, 4 days of administration of NK cells, etc.) or sequential administration (eg, administration of recombinant oncolytic poxvirus on day 1, sequential administration of recombinant oncolytic poxvirus and NK cells on day 2, and sequential reconstitution on day 3) Tumor pox virus and NK cells, on the 4th day, the recombinant oncolytic poxvirus and NK cells are administered sequentially...
  • intermittent administration for example, administration of recombinant oncolytic poxvirus on day 1, administration of NK cells on day 2, and administration of recombinant oncolytic poxvirus on day 3, 4 days of administration of NK cells, etc.
  • sequential administration eg, administration of recombinant oncolytic poxvirus on day 1, sequential
  • NK cells were administered again at intervals of 18-72 hours, once a day for 1-6 days.
  • the recombinant oncolytic pox virus is administered first, and the NK cells are administered 18 to 72 hours after the full administration of the recombinant oncolytic pox virus.
  • the recombinant oncolytic poxvirus is first administered to a tumor and/or cancer patient, the oncolytic virus is administered in a therapeutically effective amount, administered once; and in the administration of the dissolution
  • the NK cells are administered to the tumor and/or cancer patient from 18 hours to 72 hours after the tumor virus, and the NK cells are administered at a dose of 1 ⁇ 10 7 to 1 ⁇ 10 10 cells/day (for example) , 1 ⁇ 10 8 to 5 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells / day dose, 1 ⁇ 10 9 to 3 ⁇ 10 9 cells / day dose), once administered .
  • the therapeutically effective amount of the recombinant oncolytic poxvirus is preferably from 1 x 10 5 to 5 x 10 9 pfu per day (e.g., 1 x 10 5 to 3 x 10 9 pfu per day dose, 1 x 10 5 to 1 x). 10 8 pfu / day dose, etc.).
  • the method of treating tumors and/or cancer of the present invention may be performed one or more times on the patient according to actual conditions and needs.
  • the NK cells may be selected from autologous NK cells and allogeneic NK cells; preferably, the NK cells are autologous NK cells obtained by in vitro expansion or allogeneic NK cells obtained by in vitro expansion.
  • the tumor and/or cancer includes lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, lymphoma, gastric cancer, esophageal cancer, Kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer, etc.
  • the recombinant oncolytic poxvirus can be administered by administration in a manner generally employed in the art, for example, by intratumoral injection or intravenous administration.
  • NK cells can be administered by administration generally employed in the art, for example, by intravenous administration.
  • the percent concentration (%) of each reagent refers to the volume percent concentration (% (v/v)) of the reagent.
  • CV1 cells and Hu-143B cells are derived from the China Center for Type Culture Collection of Wuhan University.
  • the remaining tumor cells are derived from the China Center for Type Culture Collection and ATCC.
  • the cells were cultured in the normal culture environment McCoy's 5A + 10% FBS and MEM + 10% FBS. McCoy's 5A and MEM were purchased from GIBCO. Fetal bovine serum FBS was purchased from SIGMA.
  • the sources of NK cells used in the experiments are as follows:
  • the preparation method of the human NK cells is as follows: the peripheral blood of a healthy person is taken by a blood collection needle by puncture the elbow vein by a conventional method in the art, and the whole immune cell PBMC is extracted.
  • the irradiated K562 trophoblast cells purchased from Hangzhou Dingyun Biotechnology Co., Ltd.
  • the purity of NK cells was 90%, and the survival rate of NK cells was 90%.
  • the kill rate reached 85%.
  • the oncolytic poxvirus DDVV-RFP which is a control virus, is known to belong to the oncolytic poxvirus WR strain (see, for example, the scientific literature: "X Song, et al. T-cell Engager-armed Oncolytic Vaccinia Virus Significantly Enhances Antitumor Therapy Molecular Therapy. (2014); 22 1,102-111")
  • the TK gene and VGF gene of the virus are both functionally defective and carry the exogenous red fluorescent protein (RFP) gene. Since the RFP gene only plays a screening/reporting role, the anti-tumor function of the oncolytic poxvirus DDVV-RFP is substantially equivalent to the oncolytic poxvirus with a defective TK gene and VGF gene function.
  • the oncolytic poxvirus DDvv-RFP can also be obtained by genetic modification of the VSC20 poxvirus using conventional techniques in the art.
  • VSC20 poxvirus is a VGF gene-deficient poxvirus.
  • For the preparation method please refer to the scientific literature: "McCart, JA, et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Res (2001 ) 61:8751-8757.”.
  • the genetic modification involves the use of a synthetic vaccinia early/late promoter pSEL to regulate the exogenous DsRed gene (ie, the RFP gene), and the DsRed gene is inserted into the TK gene region of the poxvirus VSC20 strain using an in vitro intracellular recombination technique to construct
  • the oncolytic poxvirus DDVV-RFP was obtained. Its production and purification can be carried out in accordance with Preparation Example 2 described below.
  • C57BL/6 mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Severe immunodeficient NCG mice were obtained from the Zhejiang Animal Center.
  • a 6-well cell culture plate (2 ml per well), a 24-well cell culture plate (500 ⁇ l per well), and a 96-well cell culture plate (200 ⁇ l per well) were obtained from Corning.
  • gpt screening drug preparation method using 0.1M NaOH to prepare 10mg/ml mycophenolic acid (400 ⁇ ), 10mg/ml 40 ⁇ astragalus (40 ⁇ ), 10mg/ml hypoxanthine (670 ⁇ ), protected from light Store at -20 ° C; prepare 1 ⁇ working solution at the time of use: add 25 ⁇ l of mycophenolic acid + 250 ⁇ l of xanthine + 15 ⁇ l of jaundice in 10 ml of DMEM.
  • PBS formulation 8 mM Na 2 HPO 4 , 136 mM NaCl, 2 mM KH 2 PO 4 , 2.6 mM KCl, pH 7.2-7.4.
  • STE buffer formulation 10 mM Tris-Cl, 0.1 M NaCl, 1 mM EDTA, pH 8.0.
  • Trypan blue staining method After washing the cells with PBS, trypsin digestion, the cells were suspended in PBS, and a final concentration of 0.04% (w/v) of trypan blue dye solution was added, and the cells were counted under a microscope. Dyeed in blue, living cells are transparent and colorless. The number of viable cells was taken as the final data.
  • FBS fetal bovine serum
  • PSG penicillin-streptomycin-glutamine
  • PBMC peripheral blood mononuclear cells
  • RFP red fluorescent protein
  • TIL tumor infiltrating lymphocytes
  • mIL-21 contains a murine IL21 fragment of the normal sequence (ie, a fragment of 72-560 bp in the nucleotide sequence of Genbank No. NM_001291041), the sequence of which (SEQ ID NO. 1) is as follows:
  • AGATCT is a BglII recognition sequence
  • hIL-21 contains a human IL21 fragment of the normal sequence (ie, a 47-535 bp fragment of the nucleotide sequence of Genbank number NM_021803), the sequence of which (SEQ ID NO. 2) is as follows:
  • AGATCT ATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAAAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTGA Where AGATCT is a BglII recognition sequence; Identify
  • CV1 cells with good growth conditions were selected and plated in 6-well plates at about 4 ⁇ 10 5 cells/well, so that the next day cell density reached 80%-90%.
  • plasmid/Lipo2000 mixture 300 ⁇ l was added to one of the wells infected with the virus, and a control well transfected with the plasmid was further set up, and cultured at 37 ° C for 4 hours, and 2 ml of DMEM medium containing 10% FBS + 1% PSG was added to each well.
  • Primer pair Authenticated virus Strip size (bp) P1/P2 VSC20 622 P15/P11 DDvv-mIL21 1893 P15/zP1 DDvv-hIL21 1792
  • IL-21 gene sequence was integrated into the viral TK region, ie, the virus was successfully recombined (Fig. 4A-B); the ELISA results showed that the recombinant virus supernatant containing IL21 (shown as "DDvv-mIL-21” in the figure) There was expression of IL-21 protein, and the remaining cells (shown as "NC” in the figure), the transfected plasmid (pCB-mIL21), and the VSC20 control showed no expression of IL-21 (Fig. 4C).
  • CV1 cells were cultured in a 60 mm culture dish, and 1 ml of virus dilution solution was added at 80% of full time, containing 150 ⁇ l of P0 virus. After 2 hours of infection, 3 ml of 1 ⁇ gpt screening drug was added for screening, cell lesions were observed, and virus labeling was collected. P1 virus.
  • the viral gene was extracted using a viral genome extraction kit for PCR verification.
  • step 2) Repeat step 2) to obtain the P3 virus and verify it by PCR.
  • CV1 cells were cultured in a 10 cm culture dish to a density of 80%. The medium was discarded, and 3 ml of a gradient dilution of P3 virus was added for 2 hours to remove the culture solution, and 8 ml of a solution containing 0.8 ⁇ gpt of the screening drug was added (w/v).
  • cultured at 37 ° C for 48 hours pick a single plaque to 300 ⁇ l of sterile PBS, repeated freeze-thaw three times
  • the virus was released, and 150 ⁇ l of virus solution was taken from each plaque in a 24-well plate using CV1 cells for small expansion. After about 48 hours, after the virus was completely damaged, the virus was collected, and 200 ⁇ l of virus solution was taken to extract the genome, and verified by PCR.
  • the remaining viral fluid was labeled P4 and stored at -80 °C.
  • PCR confirmed that the recombinant virus does not contain the skeleton virus VSC20, and simultaneously use ELISA and Western-blot method (primary antibody: rabbit anti-mouse IL21, PEpro Tech.INC., #500-P278 ; rabbit anti-IL21 antibody, abcam, ab5978; secondary antibody: Goat anti-Rb, Abcam, ab97051) confirmed the expression of IL21 protein.
  • Fig. 5A-D Western blot results also showed that DDvv-IL21 virus can effectively express IL-21 protein with the correct size (Fig. 5E, G).
  • the ELISA results showed that the recombinant virus DDvv-IL21 can detect IL-21 protein in cell culture supernatant and cell lysis (shown as "cell” in the figure), and further demonstrates the characteristics of IL-21 protein as exogenous protein.
  • Fig. 5F, H The results showed that the virus obtained from the above step 4) was identified by PCR and showed no banding of the PCR results of P1/P2, indicating that the recombinant virus is a purebred virus carrying the exogenous target gene IL
  • sucrose gradient Add 2 mL of 60% (w/v), 50% (w/v), 40% (w/v) sucrose solution in the ultracentrifuge tube from bottom to top, and add 30% to the top layer. % (w/v) sucrose resuspended virus solution 4 mL.
  • the virus After sucrose gradient was applied, after 39 minutes of ultracentrifugation at 39,000 g, the virus showed a milky white band distributed in the 50% (w/v) sucrose layer, and the virus strip was aspirated using a 1 mL flat/large mouth. The strip was placed in a new centrifuge tube (the volume of the strip taken from the virus strip was recorded as V1).
  • each well was inoculated with about 4 ⁇ 10 5 HuTK-143B cells, and the cells were brought to near 80%-90% in the 5% CO 2 incubator at 37 ° C overnight.
  • virus titer average number of viral plaques / (virus dilution x added virus volume).
  • Example: If there are 30 viral plaques in a 10 -5 dilution well, the virus titer is 30 / (10 -5 ⁇ 0.5) 6 ⁇ 10 6 pfu / ml.
  • the titer of each batch of DDvv-mIL21 virus and DDvv-hIL21 virus after amplification/purification was 2 x 10 8 to 4 x 10 8 PFU/ml.
  • Example 1 Killing effect of DDvv-mIL21 poxvirus carrying murine IL-21 fragment on different murine tumor cells
  • Mouse tumor cells in 96-well plates including B16 cells (mouse melanoma cells), GL261 cells (mouse glioma cells), LLC cells (mouse Lewis lung cancer cells), CT-26 cells (mouse colon) Cancer cells), 4T-1 cells (mouse breast cancer cells), 5000 cells per well, cultured overnight to adhere to cells, and infected cells with 1 MOI of DDvv-mIL21 virus prepared by the above method, respectively, at 24 hours, 48 After hours and 60 hours (test against LLC cells, GL261 cells), or after 24 hours, 48 hours, and 72 hours (tests against B16 cells, CT-26 cells, 4T-1 cells), tumor cells were detected using the MTT assay.
  • the control group of this experiment was: negative control group (no virus infection), recombinant mouse IL-21 protein (rmIL-21, purchased from R&D Systems) and positive control group (1 ⁇ M paclitaxel), purchased from Beijing Shuangluo 3)
  • rmIL-21 recombinant mouse IL-21 protein
  • positive control group (1 ⁇ M paclitaxel
  • Each of the treatment groups and the control group were set with 3 duplicate wells, and the cell killing rate was the percentage of the experimental group and the negative control group.
  • the results showed that DDvv-mIL21 virus reached a killing rate of more than 60% for each tumor cell after 60 hours or 72 hours, wherein DDvv-mIL21 virus reached 70% killing of CT-26 and 4T1 cells after 24 hours of infection. Rate (see Figure 6).
  • Example 2 IL-21 fragment carrying the murine DDvv-mIL21 poxvirus different murine tumor cell killing IC 50
  • the IC 50 of recombinant poxvirus against B16 cells, CT26 cells, 4T1 cells, LLC cells, and GL261 cells were 0.232 MOI, 0.216 MOI, 0.07 MOI, 0.304 MOI, and 0.227 MOI, respectively (see Figure 7). It can be seen that the recombinant poxvirus DDvv-mIL21 of the present invention has a low IC 50 value (less than or about equal to 0.3 MOI) for different tumor cells, and has a good promising prospect.
  • Example 3 Killing effect of DDvv-hIL21 poxvirus carrying human IL-21 fragment on different human tumor cells
  • A549 human non-small cell lung cancer cells
  • SKOV3 human ovarian adenocarcinoma cells
  • Hela human cervical cancer cells
  • U251 human glioma cells
  • the control group of this experiment was: negative control group (no virus infection), recombinant human IL-21 protein (rhIL-21, purchased from R&D Systems) and positive control group (1 ⁇ M paclitaxel), and each treatment group and control group Three replicate wells were set, and the cell killing rate was the percentage of the experimental group and the negative control group.
  • the results showed that the killing effect of recombinant virus on Hela, A549, SKVO3, U251 cells was time-dependent and strengthened with time (see Figure 8).
  • the results also showed that the DDvv-hIL21 virus reached a killing rate of more than 60% for each tumor cell after 48 hours, and reached a killing rate of more than 70% after 72 hours.
  • Example 4 carrying the human IL-21 fragment DDvv-hIL21 poxvirus different human tumor cell killing IC 50
  • A549, HepG2 (human liver cancer cells), Hela, HT29 (human colorectal cancer cells), SKOV3 (human ovarian adenocarcinoma cells), PANC1 (human pancreatic cancer cells), SKHEP-1 (human liver cancer cells), FaDu (human pharynx) Squamous cell carcinoma cells were plated in 96-well plates at 5000 cells per well.
  • the DDvv-hIL21 virus-infected cells prepared by the above method were diluted with a 3-fold dilution of MOI, and the MOI concentrations of DDvv-hIL21 virus were 0.003, 0.01, respectively.
  • Example 5 Anti-tumor effect of DDvv-mIL21 poxvirus carrying murine IL-21 fragment on B16 tumor mice
  • Fig. 10A The results showed that the recombinant virus DDvv-mIL21 was able to effectively inhibit tumor growth in a dose-dependent manner.
  • the T/C of each administration group the tumor inhibition efficiency, that is, the percentage of the tumor size of the administration group and the tumor size of the control group, the value of ⁇ 40% indicates that the drug is effective
  • Fig. 10B Mice were sacrificed on day 12 and tumors were removed for tumor weight and similar results were shown (Fig. 10C).
  • Example 6 Anti-tumor immune activation of DDvv-mIL21 poxvirus carrying murine IL-21 fragment in B16 tumor mice
  • CD8 + T cells (CD3 + CD8 + ) were detected by flow cytometry (antibody: anti-mouse CD3, Anti-mouse CD8a, eBioscience) and CD4 + T cells (CD3 + CD4 + ) (antibody: anti-mouse CD4, eBioscience).
  • the results showed that the proportion of CD8 + T cells and CD4 + T cells in the spleen PBMC of the recombinant virus DDvv-mIL21 was significantly increased relative to the PBS group (Fig. 11A-B); at the same time, the immune cell invasion analysis in the tumor tissue was found to be administered.
  • the proportion of CD8 + T cells and CD4 + T cells (TIL) in the high dose group of DDvv-mIL2 was increased relative to the PBS group (Fig. 11C-D).
  • Example 7 Anti-tumor effect of DDvv-mIL21 poxvirus carrying murine IL-21 fragment on LLC tumor mice
  • each mouse was given 1 million cells inoculated into the back of the hind legs of C57BL/6 mice, and the tumor growth was observed.
  • the tumor volume was about 100-200 mm 3
  • the intratumoral injection was performed.
  • the administration method was as follows: 100 ⁇ l of PBS containing 1 ⁇ 10 6 pfu (low dose), 1 ⁇ 10 7 pfu (high dose) of DDvv-mIL21 prepared by the above method and 100 ⁇ l of PBS as a control group. 5 mice per group were tested for tumor size and body weight every 3 days. The results showed that the recombinant virus DDvv-mIL21 was able to effectively inhibit the growth of LLC tumors in a dose-dependent manner (Fig. 12A), and on the 9th day after administration, the T/C of the high dose administration group was less than 40% (Fig. 12B).
  • Example 8 Killing effect of DDvv-hIL21 poxvirus and NK cells carrying human IL-21 fragment on human tumor cell SK-HEP-1
  • SK-HEP-1 cells were seeded into 24-well culture plates and confluent in 30%. Incubate in MEM + 10% FBS environment for 24 hours at 37 ° C, 5% CO 2 , and add MOI in the serum-free environment of DMEM. 0.15 of the DDvv-hIL21 virus prepared by the above method was infected for 6 hours, and the medium was changed to MEM + 10% FBS, and cultured at 37 ° C, 5% CO 2 for 24 hours.
  • the cells were counted by trypan blue staining, and the experimental group was the DDvv-hIL21+NK group.
  • a group of SK-HEP-1 cells were additionally preserved in the experiment, no virus, no NK, as a blank group; one group was added with DDvv-hIL21 virus at the corresponding time point, but no NK was added as DDvv-hIL21 virus.
  • Example 9 Anti-tumor effect of DDvv-mIL21 poxvirus carrying murine IL-21 fragment on drug-induced immunocompromised B16 tumor-bearing mice
  • the next day (ie, the 10th day after inoculation) was administered intramuscularly in 100 ⁇ l PBS containing 1 ⁇ 10 5 pfu (low dose (low) Dose)), 1 ⁇ 10 6 pfu (middle dose), 1 ⁇ 10 7 pfu (high dose) of DDvv-mIL21 prepared by the above method and 100 ⁇ l of PBS, 5 mice per group
  • the tumor size and body weight were measured once every 3 days.
  • the results showed that the recombinant virus DDvv-mIL21 was able to effectively inhibit tumor growth and was dose-dependent (Fig. 14A).
  • T/C in the middle and high dose groups (effective tumor inhibition rate, ie the percentage of tumor size in the drug-administered group and tumor size in the control group, which is lower than 40%)
  • the time indicates that the drug is effective is less than 40% (Fig. 14B).
  • Example 10 Comparison of anti-tumor effect of DDvv-mIL21 poxvirus carrying mouse IL-21 fragment and control poxvirus on drug-induced immunocompromised B16 tumor-bearing mice
  • each mouse was inoculated with 200,000 cells in the back of the hind legs of C57BL/6 mice, and the tumor growth was observed.
  • the tumor volume was measured at 80-150 mm 3 and the daily intraperitoneal injection was 30 mg/kg.
  • Cyclosporine was administered to 100 ⁇ l of PBS containing 1 ⁇ 10 6 pfu of DDvv-mIL21 prepared by the above method, 1 ⁇ 10 6 pfu of DDvv-RFP and 100 ⁇ l of PBS, respectively, until the end of the experiment.
  • Three mice in each group were tested for tumor size and body weight every 3 days.
  • DDvv-mIL21 was effective in inhibiting tumor growth compared to DDvv-RFP (Fig. 15A), and the DDvv-mIL21 group had a T/C of less than 40% from day 6 after administration (Fig. 15B).
  • the mice were sacrificed and the tumor tissues were weighed, and the tumor weight of the DDvv-mIL21 group was significantly reduced (Fig. 15C).
  • TIL in tumor tissues was detected by flow cytometry (including CD8 + T cells (CD3 + CD8 + ) (antibody: anti-mouse CD3, anti-mouse CD8a, eBioscience) and CD4 + T cells (CD3 + CD4 + ) ( Antibody: anti-mouse CD3, anti-mouse CD4, eBioscience) and NK cells (CD3 - NK1.1 + ) (antibody: anti-mouse NK1.1, eBioscience), the results showed that DDvv-RFP and DDvv-mIL21 can be significantly Increasing the TIL content in tumor tissues showed a significant increase in CD4 + T cells in the tumor TIL compared with the DDvv-mP21 group (Fig. 16A-C).
  • Example 11 Comparison of anti-tumor effect of DDvv-hIL21 poxvirus carrying human IL-21 combined with human NK cells and control poxvirus combined with human NK cells on HCT116 tumor-bearing severe immunodeficiency mice
  • NCG mice obtained from Zhejiang Animal Center
  • 5 million HCT116 cells were inoculated subcutaneously in the back of the hind legs of the mice to observe the tumor growth.
  • the tumor volume was measured at about 80-150 mm 3 (about after inoculation).
  • Day 7 100 ⁇ l of PBS containing 5 ⁇ 10 6 pfu of DDvv-hIL21 virus, 5 ⁇ 10 6 pfu of control poxvirus DDvv-RFP and PBS, 5 mice per group, were administered intratumorally.
  • 5 ⁇ 10 7 NK cells/day were administered to the tail vein, and the cells were continuously administered for 3 days and stopped for 3 days.
  • a cycle a total of 3 cycles of NK cells were administered. Tumor size and body weight were measured every 2-5 days from the start of virus administration.
  • the results showed that both the oncolytic virus DDvv-RFP and DDvv-hIL21 were able to effectively inhibit tumor growth, and DDvv-hIL21 showed better antitumor effect than DDvv-RFP (Fig. 17A), starting on the 12th day after administration.
  • the T/C of the DDvv-RFP and DDvv-hIL21 groups were both less than 40% (Fig. 17B). At the end of the experiment, the mice were sacrificed and the tumor tissues were collected.
  • the expression level of the poxvirus A46R gene in the tumor tissues was detected by quantitative PCR (the primer sequence of the target gene was: 5'-CAGGGAAACGGATGTATA-3' (SEQ ID NO. 8) and 5'. -TGTGTTACAGAATCATATAAGG-3' (SEQ ID NO. 9)), IL-21 gene expression level (primer gene primer sequence: 5'-CCAACTAAAGTCAGCAAATACAGG-3' (SEQ ID NO. 10) and 5'-CTTTCTAGGAATTCTTTGGGTGG-3' (SEQ ID NO. 11)), NK cell NKG2D gene expression level (primer gene primer sequence: 5'-GGCTTTTATCCACAAGAATCAAGATC-3' (SEQ ID NO.
  • the housekeeping gene GAPDH was used as an internal reference (the primer sequences of the target gene were: 5'-GGTCTCCTCTGACTTCAACA-3' (SEQ ID NO. 16) and 5'-AGCCAAATTCGTTGTCATAC-3' (SEQ ID NO. 17))
  • the expression level of the target gene was pg/ ⁇ g GAPDH.
  • Example 12 Distribution of intravenously administered DDvv-mIL21 poxvirus in B16 tumor mice
  • each mouse was inoculated with 200,000 cells in the back of the hind legs of C57/B16 female mice, and the tumor growth was observed.
  • the tumor volume was measured at 100-200 mm 3 to start the administration.
  • Administration method 5 ⁇ 10 9 pfu of DDvv-mIL21 oncolytic virus prepared by the above method was administered to 100 ⁇ l of PBS, and 4 mice were sacrificed. After 1 week, the mice were sacrificed to collect heart, liver, spleen, lung, kidney, brain.
  • ovary, tumor tissue, PCR method was used to detect the distribution of poxvirus in various organs, specifically, the expression level of poxvirus A46R gene in each tissue and organ was compared with the expression level of poxvirus A46R gene in kidney organs.
  • the primer sequences of the gene were: 5'-CAGGGAAACGGATGTATA-3' (SEQ ID NO. 8) and 5'-TGTGTTACAGAATCATATAAGG-3' (SEQ ID NO. 9), and the results showed that the administered DDvv-mIL21 oncolytic virus was mainly distributed in In the tumor tissue, the virus was detected in the ovarian tissue of one of the mice, and the DDvv-mIL21 oncolytic virus was not detected in other organs (Fig. 19).
  • Example 13 Killing effect of DDvv-hIL21 poxvirus carrying human IL-21 and NK cells on human tumor cell HepG2
  • the DDvv-hIL21 virus prepared by the method was infected for 6 hours, and then changed to DMEM + 10% FBS, and incubation was continued for 18 hours at 37 ° C, 5% CO 2 .
  • the experimental group was the DDvv-hIL21+NK group.
  • a group of HepG2 cells were additionally preserved in the experiment, no virus was added, no NK was added, as a blank (BKK) group; a group of DDvv-hIL21 virus was added at the corresponding time point, but no NK was added as the DDvv-hIL21 virus group.
  • Example 14 Comparison of the killing effect of DDVV-hIL21 poxvirus combined with human NK cells carrying human IL-21 fragment and control poxvirus combined with human NK cells on human tumor cell HCT116
  • the experimental group was DDVV-RFP+NK group and DDVV-hIL21+NK group, respectively.
  • a group of HCT116 cells were preserved in the experiment, no virus, no NK, as a blank group; one group did not add virus, no NK, but added to the final concentration of 50 ng/ml at the time corresponding to the above NK addition time point.
  • the IL21 recombinant protein was used as a blank + IL21 group.
  • One group added DDVV-RFP and NK at the corresponding time points, and added human IL21 recombinant protein with a final concentration of 50 ng/ml at the same time as NK, as DDVV-RFP+NK+IL21 group; the control group did at the corresponding time. The corresponding liquid exchange operation. Each group of experiments was repeated three times or more, and the average was taken for statistical analysis.
  • DDvv-hIL21 virus and NK cells are combined (the oncolytic virus is administered first, and the NK cells are administered later).
  • the killing of HCT116 cells showed a significant synergistic effect, and the synergistic inhibition rate was about 86%.
  • the inhibition rate of DDvv-hIL21 virus alone was about 43%, and the inhibition rate of NK cells alone was about 10%, and the superimposed values of the two were shown by dashed lines in the figure.
  • the blank group inhibition rate is about 0 (not shown in Fig. 21A or B).
  • the killing effect of DDVV-hIL21 and NK cells on HCT116 cells was combined (inhibition rate was about 86%).
  • the same dose of DDVV-RFP combined with NK cells inhibition rate of about 67%), also significantly higher than the same dose of DDVV-RFP and NK cells and human IL21 recombinant protein combined killing effect ( The inhibition rate is about 64%).
  • the inhibition rate of DDVV-RFP alone was about 37%, the inhibition rate of blank + IL21 group was not detected, and the inhibition rate of NK+IL21 group was about 12%.
  • the data of other groups were the same as above.
  • exogenous IL-21 can be expressed by DDVV-hIL21 selectively replicating in HCT116 cells while killing HCT116 cells, and the expressed exogenous IL-21 can enhance the killing power of NK cells, thereby further enhancing HCT116 of NK cells.
  • Cell killing effect the combination of DDVV-hIL21 and NK cells has a surprising effect on the killing effect of HCT116 cells.
  • Example 15 Comparison of the killing effect of DDVV-hIL21 poxvirus combined with human NK cells carrying human IL-21 fragment and control poxvirus combined with human NK cells on human tumor cell FaDu
  • the DDVV-hIL21 or DDVV-RFP prepared by the above method was infected for 6 hours, and then incubated for 18 hours at 37 ° C, 5% CO 2 in a MEM + 10% FBS environment.
  • the experimental group was DDVV-RFP+NK group and DDVV-hIL21+NK group, respectively.
  • a group of FaDu cells were kept, no virus was added, no NK was added, as a blank group; one group was not added with virus, no NK was added, but a person with a final concentration of 50 ng/ml was added at the time corresponding to the above-mentioned NK addition time point.
  • the IL21 recombinant protein was used as a blank + IL21 group.
  • DDvv-hIL21 virus and NK cells are combined (the oncolytic virus is administered first, and the NK cells are administered later).
  • the killing of FaDu cells has a significant synergistic effect, and the synergistic inhibition rate is about 73%.
  • the inhibition rate of DDvv-hIL21 virus alone was about 37%, and the inhibition rate of NK cells alone was about 13%.
  • the superimposed values of the two were shown by dashed lines in the figure.
  • the blank group inhibition rate is about 0 (not shown in Fig. 22A or B).
  • the killing effect of DDVV-hIL21 and NK cells on FaDu cells was combined (inhibition rate was about 73%).
  • the same dose of DDVV-RFP combined with NK cells inhibition rate of about 66%), also significantly higher than the same dose of DDVV-RFP and NK cells and human IL21 recombinant protein combined killing effect ( The inhibition rate is about 65%).
  • the inhibition rate of DDVV-RFP alone was about 39%, the inhibition rate of blank + IL21 group was not detected, and the inhibition rate of NK+IL21 group was about 14%.
  • the data of other groups were the same as above.
  • exogenous IL-21 can be expressed by DDVV-hIL21 selectively replicating in FaDu cells while killing FaDu cells, and the expressed exogenous IL-21 can enhance the killing power of NK cells, thereby further enhancing the FaDu of NK cells.
  • Cell killing effect the combination of DDVV-hIL21 and NK cells has a surprising effect on the killing effect of FaDu cells.

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Abstract

分离的重组溶瘤痘病毒、药物组合物及其在***和/或癌症的药物中的用途。该分离的重组溶瘤痘病毒是TK基因和VGF基因功能缺陷型的，并且该重组溶瘤痘病毒的基因组中整合有外源IL-21基因，该IL-21基因能够在肿瘤细胞中表达。

Description

分离的重组溶瘤痘病毒、药物组合物及其在***和/或癌症的药物中的用途 技术领域
本发明属于生物技术领域，具体而言，涉及分离的重组溶瘤痘病毒、药物组合物及其在***和/或癌症的药物中的用途。
背景技术
溶瘤病毒是一种能够在肿瘤细胞中选择性复制并杀伤肿瘤细胞，但不伤害正常细胞的一类病毒。病毒在受感染的肿瘤细胞内进行复制后，释放出新的病毒颗粒，进而感染其周围的肿瘤细胞，进一步起了溶瘤作用，除了这一直接的溶瘤作用外，溶瘤病毒还可有效刺激机体产生针对病毒自身及针对被感染肿瘤细胞的免疫应答。由此可见，溶瘤病毒的抗肿瘤作用是通过选择性杀伤肿瘤细胞和诱导机体产生***性抗肿瘤免疫反应的双重作用来实现的。
早在19世纪末人们就发现多种病毒可缓解肿瘤发展的进程，提示了病毒在肿瘤治疗领域的潜力。随着基因技术的发展，可通过改变病毒基因组结构使其可选择性地在肿瘤细胞内进行复制，提升其溶瘤靶向性。近十年来，研究者们以基因重组、基因转入、基因敲除等技术开始对腺病毒、疱疹病毒、小核糖核酸病毒、痘病毒等病毒进行了基因改构，为此开发出一系列的溶瘤病毒产品。迄今为止大约有20多个产品进入临床研究的不同阶段。其中，2005年中国CFDA批准了上海三维生物技术有限公司的基因改构溶瘤腺病毒H101上市，用于治疗头颈部肿瘤，这是世界范围内首个上市的溶瘤病毒药物。10年之后的2015年，第二个溶瘤病毒药物，即Amgen公司的基因改构溶瘤单纯疱疹病毒T-Vec才被美国FDA以及欧盟EMA批准上市，用于治疗晚期恶性黑素瘤。目前尚未有基因改构的溶瘤痘病毒作为药物上市。
目前在肿瘤和/或癌症的溶瘤病毒免疫治疗中，仍然需要开发出效果更好的药物。
发明内容
为解决上述现有技术中所存在的问题，本发明提供了分离的重组溶瘤痘病毒、药物组合物及其在***和/或癌症的药物中的应用。
具体而言，本发明提供了：
(1)一种分离的重组溶瘤痘病毒，其中该重组溶瘤痘病毒是TK基因和VGF基因功能缺陷型的，并且该重组溶瘤痘病毒的基因组中整合有外源IL-21基因，并且该IL-21基因能够在肿瘤细胞中表达。
(2)根据(1)所述的重组溶瘤痘病毒，其中所述TK基因通过***外源核苷酸序列而使该TK基因功能缺陷。
(3)根据(1)所述的重组溶瘤痘病毒，其中所述外源IL-21基因***在所述TK基因中，从而使该TK基因功能缺陷。
(4)根据(1)所述的重组溶瘤痘病毒，其中所述VGF基因通过基因敲除或***外源核苷酸序列而使该VGF基因功能缺陷。
(5)根据(1)所述的重组溶瘤痘病毒，其中所述重组溶瘤痘病毒是惠氏株或WR株。
(6)根据(1)所述的重组溶瘤痘病毒，其中所述重组溶瘤痘病毒的基因组中还整合有外源筛选基因，所述外源筛选基因包括gpt基因和/或LacZ基因，但不包括荧光蛋白基因。
(7)根据(1)或(5)所述的重组溶瘤痘病毒，其中所述外源IL-21基因来自于小鼠或人。
(8)一种药物组合物，其中该药物组合物包括作为活性成分的根据(1)-(7)中任一项所述的重组溶瘤痘病毒，及可药用辅料。
(9)根据(8)所述的药物组合物，其中所述药物组合物包含1×10 5-5×10 9pfu/天剂量的所述重组溶瘤痘病毒。
(10)根据(8)所述的药物组合物，其中所述重组溶瘤痘病毒通过瘤内注射给药或静脉给药。
(11)一种用于制备(1)-(7)中任一项所述的重组溶瘤痘病毒的载体，其中所述载体包含在启动子控制下的外源IL-21基因。
(12)一种含有(11)所述的载体的宿主细胞。
(13)根据(1)-(7)中任一项所述的重组溶瘤痘病毒在制备用于***和/或癌症的药物中的用途。
(14)根据(13)所述的用途，其中所述肿瘤和/或癌症包括肺癌、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌。
(15)一种***和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据(1)-(7)中任一项所述的重组溶瘤痘病毒。
(16)根据(15)所述的方法，其中所述重组溶瘤痘病毒的施用剂量为1×10 5-5×10 9pfu/天，每天1次，连续施用1-6天。
(17)根据(15)所述的方法，其中所述重组溶瘤痘病毒通过瘤内注射给药或静脉给药。
(18)根据(15)所述的方法，其中所述肿瘤和/或癌症包括肺癌、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌。
本发明与现有技术相比具有以下优点和积极效果：
本发明首次提出使溶瘤痘病毒的TK基因和VGF基因的功能缺陷，同时使其携带免疫调控因子IL-21的基因，从而使所得的重组溶瘤痘病毒能够选择性地在肿瘤细胞中复制，并且表达免疫调控因子IL-21。基于该构思而提供的溶瘤痘病毒、药物组合物和方法能够充分发挥溶瘤病毒选择性地在肿瘤细胞中复制并杀死肿瘤细胞、以及进一步引起随后的机体免疫反应的作用，同时还能够充分发挥外源IL-21的抗肿瘤免疫作用。本发明发现在溶瘤痘病毒中整合IL-21基因，能够使溶瘤病毒的溶瘤杀伤作用和IL-21的抗肿瘤免疫刺激作用产生协同效果。
此外，使痘病毒的TK和VGF双基因功能缺陷有效增强了其肿瘤靶向性，从而提高了安全性。
此外，由于本发明的重组溶瘤痘病毒在肿瘤细胞中选择性复制的同时表达外源IL-21，能够协同刺激机体的抗肿瘤免疫反应，进而使得本发明的重组溶瘤痘病毒能够与NK细胞得以联用。基于该构思而提供的药物组合物和方法能够充分发挥本发明的重组溶瘤痘病毒选择性地在肿瘤细胞中复制并杀死肿瘤细胞、以及进一步引起随后的机体免疫反应的作用，同时还能够充分发挥NK细胞杀伤肿瘤细胞的功能，并且巧妙地利用了本发明的重组溶瘤痘病毒选择性地在肿瘤细胞中复制的特点，使得含有本发明的重组溶瘤痘病毒的肿瘤细胞成为了NK细胞的特异性靶标，而表达的外源IL-21也能提升NK细胞的杀伤力从而进一步增强了NK细胞的肿瘤杀伤作用。这样最终产生进一步加强的协同杀伤肿瘤的作用。
进一步地，本发明通过研究，使得本发明的重组溶瘤痘病毒和NK细胞各自的施用剂量、施用顺序和施用间隔能够使两者的联合施用达到最大效率的协同作用，同时避免了两者之间的相互制约，从而达到有效地***和/或癌症的效果。
定义
在本发明中，词语“肿瘤”、“癌症”、“肿瘤细胞”、“癌细胞”涵盖本领域通常认为的含义。
本文所用的词语“溶瘤病毒”是指能够选择性地在肿瘤细胞中复制并裂解肿瘤细胞的病毒。
本文所用的词语“治疗有效量”是指功能药剂或药物组合物能够表现出可检测的治疗效果或抑制效果的量，或者起到抗肿瘤效果的量。所述效果可以通过本领域任何已知的检验方法检测。
本文所用的词语“给药”或“施用”是指向受试者提供化合物、复合物或组合物(包括病毒和细胞)。
本文所用的词语“患者”是指人或非人类生物。因此，本文所述的方法和组合物适用于人类疾病和兽类疾病。在一些实施方案中， 患者患有肿瘤。在一些例子中，患者同时患有一种或多种类型的癌症。
本文所用的词语“协同效果”是指两种或多种药剂共同起到的效果，该效果大于其中各药剂的单独效果的总和。
本文所用的术语“pfu”或“蚀斑形成单位”(plague forming unit)是指：产生一个蚀斑的病毒量称为一个蚀斑形成单位(pfu)。
本文所用的术语“MOI”或“感染复数”(Multiplicity of infection)也即，病毒与细胞个数比，是指用以起始病毒感染的每个细胞感染病毒颗粒的粒数。MOI＝pfu/细胞，即细胞个数×MOI＝总PFU。
附图说明
图1示出在本发明一个实施方案中所构建的用于在痘病毒VSC20(即图中所示“Dvv-VSC20/VGF-”)的TK基因中***IL-21基因的重组质粒的图谱，以及重组机制的示意图，通过所示重组机制得到本发明的一个实施方案的重组双基因缺陷痘病毒(重组DDvv)。
图2示出获得本发明的重组溶瘤痘病毒的一个实施方案的流程图。
图3示出本发明实施例1中所构建的质粒的图谱。
图4示出分别用PCR和ELISA法鉴定P0代重组病毒DDvv-IL21的结果；其中A为用PCR法检测P0代病毒DDvv-mIL21，B为用PCR法检测P0代病毒DDvv-hIL21，图中泳道M：D000 marker，泳道1：阴性对照(即，PCR反应液)，泳道2：VSC20，泳道3：CV1细胞，泳道4：P0代病毒；C为用ELISA鉴定P0代病毒培养上清中IL21的表达水平。图4C中横坐标为组别，纵坐标为IL21浓度(pg/mL)，横坐标中所示“NC”是指CV1细胞，“质粒”是指pCB-mIL21。
图5示出空斑筛选后重组病毒的鉴定结果；其中A和B分别为用PCR法鉴定溶瘤痘病毒DDvv-mIL21、DDvv-hIL21，图中泳道M：D000 marker，泳道1：阴性对照(即，PCR反应液)，泳道2：P0代病毒，泳道3：DDvv-mIL21(图5A)或DDvv-hIL21(图5B)；C和D分别为用PCR法进行TK区鉴定，图中泳道M：D000 marker，泳道1：阴性对照(即，PCR反应液)，泳道2：VSC20，泳道3： P0代病毒，泳道4：DDvv-mIL21(图5C)或DDvv-hIL21(图5D)；E和G分别为用Western Blot使用抗IL-21抗体检测溶瘤痘病毒DDvv-mIL21和DDvv-hIL21内IL21蛋白的表达，其中所示“NC”是指CV1细胞；F和H分别为用ELISA试剂盒检测溶瘤痘病毒DDvv-mIL21和DDvv-hIL21感染细胞后的培养液及细胞裂解液内IL21的含量。
图6示出携带鼠IL-21片段的重组痘病毒DDvv-mIL21对5种鼠源癌细胞的杀伤效果，其中A为B16细胞实验结果，B为4T1细胞实验结果，C为LLC细胞实验结果，D为GL261细胞实验结果，E为CT26细胞实验结果。图6A-E横坐标为时间(小时)，纵坐标为细胞杀伤率(％)。
图7示出携带鼠IL-21片段的重组痘病毒DDvv-mIL21对5种鼠源癌细胞的杀伤效果曲线(A-E)和IC 50值(F)，其中A为B16细胞实验结果，B为CT26细胞实验结果，C为4T1细胞实验结果，D为LLC细胞实验结果，E为GL261细胞实验结果。图7A-E横坐标为MOI的log值，纵坐标为细胞杀伤率(％)。图7F横坐标为细胞组别，纵坐标为IC 50值(MOI)。
图8示出携带人IL-21片段的重组痘病毒DDvv-hIL21对4种人源癌细胞的杀伤效果，其中A为A549细胞实验结果，B为Hela细胞实验结果，C为SKOV3细胞实验结果，D为U251细胞实验结果。图8A-D横坐标为时间(小时)，纵坐标为细胞杀伤率(％)。
图9示出携带人IL-21片段的重组痘病毒DDvv-hIL21对8种人源癌细胞的杀伤效果曲线(A-H)和IC 50值(I)，其中A为A549细胞实验结果，B为HepG2细胞实验结果，C为Hela细胞实验结果，D为HT29细胞实验结果，E为SKOV3细胞实验结果，F为PANC1细胞实验结果，G为SK-HEP-1细胞实验结果，H为FaDu细胞实验结果。图9A-H横坐标为MOI的log值，纵坐标为细胞杀伤率(％)。图9I横坐标为细胞组别，纵坐标为IC 50值(MOI)。
图10示出携带鼠IL-21片段的重组痘病毒DDvv-mIL21对B16荷瘤小鼠的抑瘤作用。图10A表示肿瘤体积随时间变化的曲线，横 坐标为给药时间(天)，纵坐标为肿瘤体积(mm 3)。图10B表示T/C随时间变化的曲线，横坐标为给药时间(天)，纵坐标为T/C(％)。图10C表示不同剂量组的肿瘤重量的比较结果，横坐标为组别，纵坐标为肿瘤重量(g)。
图11示出流式分析DDvv-mIL21对B16荷瘤小鼠的免疫作用；A和B分别为脾脏提取PBMC的流式分析检测CD4 +细胞的变化(A)和CD8 +细胞的变化(B)；C和D分别为肿瘤组织提取肿瘤细胞的流式分析检测CD4 +细胞的变化(C)和CD8 +细胞的变化(D)。图11A-D横坐标为给药组别，纵坐标为CD4 +T或CD8 +T细胞的百分比(％)。
图12示出携带鼠IL-21片段的重组痘病毒DDvv-mIL21对LLC荷瘤小鼠的抑制瘤作用。图12A表示肿瘤体积随时间变化的曲线，横坐标为给药时间(天)，纵坐标为肿瘤体积(mm 3)；图12B表示T/C随时间变化的曲线，横坐标为给药时间(天)，纵坐标为T/C(％)。
图13示出携带人IL-21片段的DDvv-hIL21痘病毒和NK细胞联合对人肿瘤细胞SK-HEP-1的杀伤作用。横坐标为组别，纵坐标为相应的抑制率的百分比数值。
图14示出携带鼠IL-21片段的DDvv-mIL21痘病毒对药物诱导免疫低下B16荷瘤小鼠的抑瘤作用。图14A示出处理后肿瘤体积的变化曲线，横坐标为B16肿瘤细胞接种时间(天)，横坐标上的箭头表示给予DDvv-mIL21时间点，纵坐标为肿瘤体积(mm 3)；图14B示出处理后T/C的变化曲线，横坐标为B16肿瘤细胞接种时间(天)，纵坐标为T/C(％)。
图15示出携带鼠IL-21片段的DDvv-mIL21痘病毒与对照痘病毒对药物诱导免疫低下B16荷瘤小鼠的抑瘤作用的比较。图15A示出处理后肿瘤体积的变化曲线，横坐标为给药后时间(天)，纵坐标为肿瘤体积(mm 3)；图15B示出处理后T/C的变化曲线，横坐标为给药后时间(天)，纵坐标为T/C(％)；图15C示出处理后的肿瘤重量，横坐标为组别，纵坐标为肿瘤重量(g)。
图16示出携带鼠IL-21片段的DDvv-mIL21痘病毒与对照痘病 毒对药物诱导免疫低下B16荷瘤小鼠的免疫作用的流式分析比较。图16A-C分别示出流式分析检测CD4 +细胞的变化(A)、CD8 +细胞的变化(B)和NK细胞的变化(C)。图16A-C中横坐标为给药组别，纵坐标分别为肿瘤组织内CD4 +T细胞与肿瘤组织内总免疫细胞的数量百分比(％)(A)、CD8 +T细胞与肿瘤组织内总免疫细胞的数量百分比(％)(B)或NK细胞与肿瘤组织内总免疫细胞的数量百分比(％)(C)。
图17示出携带人IL-21片段的DDvv-hIL21痘病毒联合人NK细胞与对照痘病毒联合人NK细胞对HCT116荷瘤的重度免疫缺陷型小鼠的抑瘤作用比较。图17A示出处理后肿瘤体积的变化曲线，横坐标为HCT116肿瘤细胞接种时间(天)，横坐标上的箭头表示给予DDvv-hIL21时间点，纵坐标为肿瘤体积(mm 3)；图17B示出处理后T/C的变化曲线，横坐标为HCT116肿瘤细胞接种时间(天)，纵坐标为T/C(％)。
图18A-D示出携带人IL-21片段的DDvv-hIL21痘病毒联合人NK细胞与对照痘病毒联合人NK细胞对HCT116荷瘤的重度免疫缺陷型小鼠处理后肿瘤组织分析比较(使用定量PCR法)，包括肿瘤组织内痘病毒A46R基因的相对表达水平(A)、IL-21基因的相对表达水平(B)、NK细胞的NKG2D基因的相对表达水平(C)、IFN-γ基因的相对表达水平(D)。图18A-D横坐标为给药组别，纵坐标分别为肿瘤组织内各靶点基因(肿瘤内痘病毒的A46R基因(A)、肿瘤内IL-21基因(B)、肿瘤内NK细胞的NKG2D基因(C)、肿瘤内IFN-γ基因(D))的表达水平(单位pg)相对于GAPDH基因的表达水平(单位μg)的比值(pg/μg)。
图19示出静脉给予的DDvv-mIL21痘病毒在B16肿瘤小鼠中的分布。横坐标为组织器官类别，纵坐标为各组织器官内痘病毒A46R基因的表达量分别与肾脏器官内痘病毒A46R基因的表达量的比较倍数。
图20示出携带人IL-21片段的DDvv-hIL21痘病毒和人NK细胞联合对人肿瘤细胞HepG2的杀伤作用。横坐标为组别，纵坐标为 相应的抑制率的百分比数值。
图21示出携带人IL-21片段的DDVV-hIL21痘病毒联合人NK细胞与对照痘病毒联合人NK细胞对人肿瘤细胞HCT116的杀伤作用比较。图21A示出NK组、DDvv-hIL21组和DDvv-hIL21+NK组的比较；图21B示出各实验组的比较。图21A和B中横坐标为组别，纵坐标为相应的抑制率的百分比数值。
图22示出携带人IL-21片段的DDVV-hIL21痘病毒联合人NK细胞与对照痘病毒联合人NK细胞对人肿瘤细胞FaDu的杀伤作用比较。图22A示出NK组、DDvv-hIL21组和DDvv-hIL21+NK组的比较；图22B示出各实验组的比较。图22A和B中横坐标为组别，纵坐标为相应的抑制率的百分比数值。
各图中，如果显示的话，*表示p＜0.05，**表示p＜0.01，***表示p＜0.001，****表示p＜0.0001(与所示的对照组比较，使用One-way ANOVA统计分析法得到)。
具体实施方式
以下通过具体实施方式的描述并参照附图对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。
在本发明中，词语“肿瘤”、“癌症”、“肿瘤细胞”、“癌细胞”涵盖本领域通常认为的含义。
人体是一个复杂的***，它是由呼吸、循环、消化等十大***组成，这些***协调配合，使人体内各种复杂的生命活动能够正常进行。当肿瘤发生后，机体可通过多种免疫效应机制发挥抗肿瘤作用，机体的抗肿瘤机制包括细胞免疫和体液免疫两个方面。它们联系密切，相互影响，涉及多种免疫效应分子和效应细胞。一般认为，细胞免疫在抗肿瘤过程中起到主导作用，体液免疫在某些情况下起协同作用。本发明提出利用溶瘤痘病毒选择性地在肿瘤细胞中复制并杀伤肿瘤细胞的特点，同时使其携带免疫调控因子IL-21，从而使重组的溶瘤 痘病毒协同发挥选择性溶瘤和增强机体抗肿瘤免疫效应的作用。基于该构思，本发明的发明人通过实验研究和理论摸索，发现同时使溶瘤痘病毒的TK基因和VGF基因的功能缺陷，并在基因组中整合IL-21基因，能够很好地实现上述协同作用。
痘病毒(Vaccinia virus，VV)是迄今发现最大、最复杂的病毒之一，它是预防天花的疫苗病毒。该病毒的特殊生物属性使其在肿瘤免疫治疗/基因治疗领域获得越来越大的关注：(1)安全性：痘病毒是在细胞胞浆内复制的DNA病毒，不会整合进宿主细胞基因组，减少由于外源基因诱发的变异(参见文献：“Hruby，D.E.Vaccinia Virus Vectors：New Strategies for Producing Recombinant Vaccines.Clin.Microbiol.Rev.3，153-170(1990).”)；(2)表达效率高：痘病毒可培养到很高滴度(＞10 9pfu/ml)，通常感染1-3小时，可使90％以上的受感染细胞表达目的基因产物(参见文献：“Pfleiderer，M.，Falkner，F.G.&Dorner，F.A novel vaccinia virus expression system allowing construction of recombinants without the need for selection markers，plasmids and bacterial hosts.J.Gen.Virol.76，2957-2962(1995).”)；(3)感染的细胞范围广：几乎可感染所有类型的哺乳动物细胞，包括***和非***细胞(参见文献：“Hruby，D.E.Vaccinia Virus Vectors：New Strategies for Producing Recombinant Vaccines.Clin.Microbiol.Rev.3，153-170(1990).”)；(4)基因组容量大：至少可***25kb的外源基因而不影响其遗传稳定性(参见文献：“Hruby，D.E.Vaccinia Virus Vectors：New Strategies for Producing Recombinant Vaccines.Clin.Microbiol.Rev.3，153-170(1990).”)；(5)稳定性：对温度不敏感，成药性好，方便使用和运输(参见文献：“Hruby，D.E.Vaccinia Virus Vectors：New Strategies for Producing Recombinant Vaccines.Clin.Microbiol.Rev.3，153-170(1990).”)；(6)表达产物具有天然结构：它表达的产物可具有正确的糖基化和翻译后加工，接近天然构型(参见文献：“Hruby，D.E.Vaccinia Virus Vectors：New Strategies for Producing Recombinant Vaccines.Clin.Microbiol.Rev.3，153-170(1990).”)，这对于病毒携带免疫调控基因用以激发机体免疫反应是至关重要的。
痘病毒的胸苷激酶(thymidine kinase(TK))基因是痘病毒复制过程需依赖的基因之一。如果痘病毒缺失了TK基因，则需通过宿主细胞提供TK蛋白补给，而TK蛋白在正常细胞周期中只能短暂表达，但在大多数肿瘤细胞中TK蛋白呈持续性高表达。因此利用TK蛋白在肿瘤组织的表达特性使得TK缺失痘病毒的复制局限在肿瘤组织内，而不在正常细胞内复制，从而提高了肿瘤靶向性。自从1982年开始使用痘病毒基因改造体系以来，TK基因区一直是被用于外源基因的***区(参见文献：“Byrd，C.&Hruby，D.Construction of Recombinant Vaccinia Virus.in Vaccinia Virus and Poxvirology：Methods and Protocols 269，31-40(2004).”)。
其次，痘病毒在细胞内的复制和在细胞间的播散与宿主细胞表皮生长因子受体(EGFR)信号通路的活化密切相关。痘病毒感染细胞后分泌病毒生长因子(virus growth factor(VGF))，其与已感染的或邻近的未感染细胞表面的EGFR结合并激活EGFR/Ras信号通路，为痘病毒的复制及感染邻近细胞提供有利的环境。因此VGF缺失的痘病毒在正常细胞内无法启动EGFR/Ras通道，使其感染正常细胞受限。而肿瘤细胞中EGFR信号通路处于活化状态，故缺失VGF基因的痘病毒在肿瘤细胞内的复制、感染不受影响，亦即相对提高了对肿瘤作用的特异性(参见文献：“Autio，K.et al.Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory Beagles.Mol.Ther.-Oncolytics 1，1-8(2014).”)。
IL-21(即白细胞介素-21(interleukin-21))是一种多向性I型细胞因子，主要由T细胞产生，调节先天免疫和获得性免疫应答，在抗肿瘤免疫反应中发挥了重要作用。已有文献报道IL-21对各类免疫细胞及信号转导的作用(参见文献：“Leonard，W.J.&Wan，C.IL-21Signaling in Immunity.F1000Research 5，1-10(2016).”)，各类免疫细胞主要包括：1)CD4 +T细胞：促进增殖、产生细胞因子；T fh细胞：促进分化、提高发育中心功能；T h17细胞：促进分化、增殖；Treg细胞：抑制其产生与存活；2)NKT细胞：增殖、增强细胞毒性；3)CD8 +T细胞：提升细胞毒性、增殖和/或存活、抗肿瘤作用；4)NK 细胞：促进细胞成熟、增殖、提高细胞毒性作用、增强抗肿瘤活性；5)DC细胞：抑制抗原递呈功能、诱导凋亡；6)巨噬细胞：提升吞噬作用；7)B细胞：促进增殖和/或凋亡、促进浆细胞分化和免疫球蛋白的生成；8)另外IL-21可激活多种与肿瘤相关信号通道，包括JAK/STAT、MARK/PI3K等信号通道，调控肿瘤的发展过程。在肿瘤免疫治疗中，激活NK细胞和CD8 +T细胞的细胞毒性是关键，许多研究已充分表明IL-21在这一程序中起着重要作用。IL-21促进NK细胞成熟使其产生IFN-γ和穿孔素，诱发NK细胞介导的抗肿瘤细胞毒性作用靶向于肿瘤细胞表面的NKG2D配体，通过抗体依赖细胞介导的细胞毒性(ADCC)提升NK细胞的杀伤力(参见文献：“Spolski，R.&Leonard，W.J.Interleukin-21：a double-edged sword with therapeutic potential.Nat.Rev.Drug Discov.13，379-395(2014).”)。其次，IL-21通过诱导CD8 +T细胞的增殖、诱导记忆T细胞的生成、促进IFNγ/颗粒酶的分泌等，以增强CD8 +T细胞对肿瘤的杀伤和有利于对复发肿瘤细胞产生记忆性免疫应答。重要的是不同于IL-2，IL-21不会诱导Treg细胞的扩增，也进一步增强了CD8 +T细胞的免疫功能应答(参见文献：“Spolski，R.&Leonard，W.J.Interleukin-21：a double-edged sword with therapeutic potential.Nat.Rev.Drug Discov.13，379-395(2014).”)。基于IL-21对免疫细胞的多样化作用，体现了IL-21可在肿瘤微环境内“重新活化”多种效应细胞，已有多个临床研究使用IL-21单独或与其它药物联合用于肿瘤治疗。
因此，本发明提供了一种分离的重组溶瘤痘病毒，其中该重组溶瘤痘病毒是TK基因和VGF基因功能缺陷型的，并且该重组溶瘤痘病毒的基因组中整合有外源IL-21基因，并且该IL-21基因能够在肿瘤细胞中表达。
本发明在提及溶瘤病毒的基因时所使用的术语“功能缺陷”是指该溶瘤病毒无法发挥该基因应有的功能，即功能丧失，该目的可通过(例如)在基因中***外源片段或敲除该基因而实现。
因此，可以在所述TK基因中***外源核苷酸序列，从而使其功能缺陷。也可以在所述VGF基因中***外源核苷酸序列，从而使其 功能缺陷，但优选将所述VGF基因敲除。
优选地，将外源IL-21基因***到TK基因中，这样能够使该TK基因发生功能缺陷，并且在感染肿瘤细胞之后能够表达IL-21基因。
在本发明中，可使用的痘病毒包括惠氏株或WR株，WR株的一个例子为VSC20。
在一个优选的实施方案中，所述重组溶瘤痘病毒是对VSC20痘病毒进行基因改造而得到的。VSC20痘病毒是VGF基因缺失的痘病毒，其中在C11R位点***LacZ基因，制备方法可参见科技文献：“McCart，JA，et al.Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes.Cancer Res(2001)61：8751-8757.”。所述基因改造包括将外源IL-21基因***该VSC20痘病毒的TK基因中，从而使该TK基因功能缺陷。具体地，用于在痘病毒VSC20的TK基因中***IL-21基因的质粒的图谱，以及通过重组机制实现***的示意图如图1所示。
所述重组溶瘤痘病毒的基因组中还可以整合有外源筛选基因，所述外源筛选基因包括gpt(鸟嘌呤磷酸核糖转移酶)基因和/或LacZ基因，但不包括荧光蛋白基因，以避免荧光蛋白的表达在患者体内带来的安全隐患。
所述重组溶瘤痘病毒的基因组中也可以不整合有外源筛选基因。
在一些实施方案中，本发明通过分别使用痘病毒早/晚启动子p7.5控制gpt基因，以及人工合成痘病毒早启动子pSEL控制外源IL-21基因，使用体外细胞内重组技术将gpt和IL-21基因***到痘病毒VSC20株的TK基因区中，以此构建溶瘤病毒，两个启动子以背靠背的方式分别启动各自调控基因的表达。
本发明进一步发现，仅将外源IL-21基因***TK基因中，从而使其发生功能缺陷，并且使该溶瘤痘病毒的VGF基因的功能缺陷，即可发挥溶瘤病毒选择性地在肿瘤细胞中复制、杀死肿瘤细胞、引起随后的机体免疫反应的作用，并且溶瘤痘病毒携带的外源IL-21基因可直接诱发抗肿瘤免疫作用。
优选地，所述外源IL-21基因来自于小鼠或人。
本发明的重组溶瘤痘病毒可通过生物工程领域的相关已知方法获得，一个具体实施方案的流程如图2所示。
本发明考虑了溶瘤痘病毒和IL-21各自的特点，巧妙地使其整合在一起而联合发挥作用，其中溶瘤痘病毒在溶瘤的同时，通过其在肿瘤细胞内复制的功能可放大IL-21在肿瘤细胞内表达，随后外泌到细胞外，以此进一步诱发机体的肿瘤免疫作用，两者联合作用具有更好的治疗效果。
基于本发明开发的重组溶瘤痘病毒，本发明还提供了一种药物组合物，其中该药物组合物包括作为活性成分的根据本发明所述的重组溶瘤痘病毒，及可药用辅料。
优选地，所述药物组合物包含治疗有效量的所述重组溶瘤痘病毒。在某些实施方案中，所述药物组合物的活性成分包括1×10 5至5×10 9pfu/天剂量的根据本发明所述的重组溶瘤痘病毒(例如，1×10 5至3×10 9pfu/天剂量的根据本发明所述的重组溶瘤痘病毒、1×10 5至1×10 8pfu/天剂量的根据本发明所述的重组溶瘤痘病毒等)。
所述重组溶瘤痘病毒可采用本领域通常所采用的给药方式给药，例如通过瘤内注射给药或静脉给药。
本发明的药物组合物还可以包含本领域已知的其它活性成分，例如白细胞介素-2(IL-2)、IL-15、IL-18、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)等，其施用剂量和施用方式可以按照各自常规的方式进行。如果包含其它活性成分，那么所述重组溶瘤痘病毒应独立地存在于所述药物组合物中而不与其它活性成分相互混合。例如，将重组溶瘤痘病毒独立地装在独立容器中。
本领域的技术人员可以理解，本发明的药物组合物还可包含合适的可药用的辅料。
在一些实施方案中，本发明的药物组合物包含一种或多种可药用载体。可以通过本领域已知的方法制备药物制剂。例如，可以将化合物等活性成分与常见的赋形剂、稀释剂(例如磷酸盐缓冲液或生理 盐水)、组织培养基、和载体(例如自体血浆或人血清白蛋白)进行配制，并作为悬浮剂施用。其它的载体可以包括脂质体、胶团、纳米胶囊、聚合纳米颗粒、固体脂颗粒(例如参见文献“E.Koren and V.Torchilin，Life，63：586-595，2011”)。本发明的药物组合物的具体配制方法可参见科学文献和专利文献中的描述，例如参见最新版雷明登氏药物科学，Maack出版公司，Easton PA(″Remington′s″)。
本发明另一方面还提供了一种用于制备本发明所述的重组溶瘤痘病毒的载体。
所述载体可通过重组机制使痘病毒内的TK基因和VGF基因发生功能缺陷。例如，在一个具体实施方案中，如图1所示，重组载体包含TK同源片段TK-L、TK-R、及启动IL-21基因和外源筛选基因gpt的表达框。当痘病毒为WR株时，TK基因的序列为NCBI(即，美国国立生物技术信息中心，网址：https：//www.ncbi.nlm.nih.gov)的GenBank中编号为NC_006998的痘病毒基因中第80724-81257bp所示的序列，则TK-L的序列可为例如第80724-80961bp所示序列片段，TK-R的序列可为例如第81033-81257bp所示序列片段。该载体可通过细胞内重组机制使IL-21基因表达框和gpt基因表达框***到痘病毒的TK基因区中(从而使(例如)GenBank中编号为NC_006998的痘病毒基因中第80962-81032bp所示的序列片段缺失)，从而使重组痘病毒丧失TK基因功能。本发明另一方面还提供了一种含有本发明所述的载体的宿主细胞。
本发明另一方面还提供了本发明所述的重组溶瘤痘病毒在制备用于***和/或癌症的药物中的用途。
所述肿瘤和/或癌症包括但不限于：肺癌(例如非小细胞肺癌)、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌等。
本发明另一方面还提供了一种***和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据本发明所述的重组溶瘤痘病毒。
所述肿瘤和/或癌症包括但不限于：肺癌(例如非小细胞肺癌)、 黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌等。
在本发明的一个优选实施方案中，所述重组溶瘤痘病毒的施用剂量为治疗有效量，每天1次，连续施用1-6天(包括连续施用1天、2天、3天、4天、5天或6天)。所述治疗有效量优选为1×10 5至5×10 9pfu/天剂量(例如，1×10 5至3×10 9pfu/天剂量、1×10 5至1×10 8pfu/天剂量等)。
如果需要，还可以将本发明的重组溶瘤痘病毒与其它药物联合使用，例如白细胞介素-2(IL-2)、IL-15、IL-18、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)等，其施用剂量和施用方式可以按照各自常规的方式进行。
可以根据实际情况和需要对患者进行一次或多次本发明的***和/或癌症的方法。
所述重组溶瘤痘病毒可采用本领域通常所采用的给药方式给药，例如通过瘤内注射给药或静脉给药。
本发明还提供了一种治疗剂，包含：
(a)第一药物组合物，其中该第一药物组合物包含位于第一可药用载体中的本发明所述的重组溶瘤痘病毒；和
(b)第二药物组合物，其中该第二药物组合物包含位于第二可药用载体中的NK细胞。
在一些实施方案中，所述第一可药用载体和第二可药用载体是相同的。在另一些实施方案中，所述第一可药用载体和第二可药用载体是不同的。
在一些情况下，所述治疗剂也可以理解为药物的组合。
所有溶瘤病毒杀伤肿瘤细胞的机制都大体相似。在不同实施方案中，通过瘤内注射或静脉给药的方式，溶瘤病毒与肿瘤细胞接触，感染进入肿瘤细胞内。由于溶瘤病毒的特性是，其主要在肿瘤细胞内复制增殖，而在正常细胞内低复制或不复制，因此被感染的肿瘤细胞中会出现大量的溶瘤病毒，造成肿瘤细胞的溶解和死亡。肿瘤细胞的 溶解会释放出大量的肿瘤抗原和增殖的溶瘤病毒，抗原会进一步激活体内的免疫***，刺激体内的NK细胞和T细胞继续攻击尚未死亡的肿瘤细胞，同时新的溶瘤病毒会继续感染尚未被感染的肿瘤细胞。
NK细胞是广谱型杀伤肿瘤细胞的免疫细胞，NK细胞可以辨别肿瘤细胞与正常细胞的区别。NK通过与肿瘤细胞接触，识别确认其为非正常细胞，然后通过受体识别、抗体靶向识别(ADCC)、颗粒酶分泌、穿孔素分泌和分泌干扰素间接杀伤等多种协同手段，达到杀死肿瘤细胞的效果。体外实验显示，一个健康的NK细胞在生命期内可以连续杀死27个肿瘤细胞。
NK细胞还具有抗病毒的功能。当正常细胞感染了病毒后，随着病毒的大量复制，细胞体现出衰老病变，体现在细胞膜上的蛋白簇的组成发生变化，在这个过程中，NK细胞就可以敏锐而高效地识别被感染的细胞，通过类似于杀伤肿瘤细胞的上述手段，杀死被感染的细胞，从而达到抑制病毒复制增殖的目的。随后在抗原刺激和干扰素等因子的作用下，其它免疫细胞会持续作用，抵抗病毒。
本发明考虑了溶瘤病毒和NK细胞各自的特点，巧妙地将其联用，在联用时，NK细胞的抗病毒机制对于被溶瘤病毒感染的肿瘤细胞同样适用，并且与其抗肿瘤机制互补。此外，联用还使得含有溶瘤病毒的肿瘤细胞成为了NK细胞的特异性靶标，从而增强了NK细胞的肿瘤杀伤作用。溶瘤病毒选择性地在癌细胞内增殖，在胞内起作用杀伤癌细胞，同时能够导致癌细胞膜上的蛋白受体簇发生变化，增强NK细胞对癌细胞的识别，NK细胞在癌细胞外攻击，两者联合起来协同杀伤癌细胞，具有更好的治疗效果。进一步地，本发明所述的重组溶瘤痘病毒还同时表达外源IL-21，而表达的外源IL-21能够提升NK细胞的杀伤力从而进一步增强NK细胞的杀伤作用，使得本发明所述的重组溶瘤痘病毒与NK细胞的联用能够对肿瘤的杀伤效果产生令人惊奇的效果。
优选的是，所述第一药物组合物的活性成分为本发明所述的重组溶瘤痘病毒，并且所述第二药物组合物的活性成分为所述NK细胞。
优选地，所述第一药物组合物和所述第二药物组合物各自独立地 存在于所述治疗剂中而互不混合。
在本发明中，所述NK细胞可以选自自体NK细胞和异体NK细胞；优选地，所述NK细胞为经体外扩增得到的自体NK细胞或经体外扩增得到的异体NK细胞。NK细胞的大规模体外扩增培养技术是已知的，并且已经基本成熟(参见(例如)以下科技文献：“Somanchi SS，Lee DA.Ex Vivo Expansion of Human NK Cells Using K562 Engineered to Express Membrane Bound IL21.Methods Mol Biol.2016；1441：175-93.”或“Phan MT，Lee SH，Kim SK，Cho D.Expansion of NK Cells Using Genetically Engineered K562 Feeder Cells.Methods Mol Biol.2016；1441：167-74.”)。临床数据证实自体NK细胞、半相合异体NK细胞(属于异体NK细胞)、以及脐血制备NK细胞回输人体后均无毒副作用，无长期依赖性，安全有效。
可用于治疗的NK细胞的纯度范围可以是：自体NK细胞的纯度可为大于等于85％，异体NK细胞的纯度可为大于等于90％；其中的杂质细胞可为NK-T和/或γδT细胞。优选地，NK细胞活性(存活率)大于等于90％，NK细胞杀伤力活性大于等于80％。
在本发明的所述联用治疗方案中，本发明进一步探索优化了溶瘤病毒和NK细胞各自的施用剂量，这是至关重要的。优选地，所述第一药物组合物包含1×10 5-5×10 9pfu/天剂量的所述重组溶瘤痘病毒(例如，1×10 5-3×10 9pfu/天剂量的所述重组溶瘤痘病毒、1×10 5-1×10 8pfu/天剂量的所述重组溶瘤痘病毒等)，并且所述第二药物组合物包含1×10 7-1×10 10个细胞/天剂量的所述NK细胞(优选地，所述第二药物组合物包含1×10 8至5×10 9个细胞/天剂量的所述NK细胞；还优选地，所述第二药物组合物包含1×10 9至4×10 9个细胞/天剂量的所述NK细胞；更优选地，所述第二药物组合物包含1×10 9至3×10 9个细胞/天剂量的所述NK细胞)。优选的是，该治疗剂的活性成分由1×10 5至5×10 9pfu/天剂量的所述重组溶瘤痘病毒(例如，1×10 5至3×10 9pfu/天剂量的所述重组溶瘤痘病毒、1×10 5至1×10 8pfu/天剂量的所述重组溶瘤痘病毒等)和1×10 7至1×10 10个细胞/天剂量的所述NK细胞(例如，1×10 8至5×10 9个细胞/天剂量的 所述NK细胞、1×10 9至4×10 9个细胞/天剂量的所述NK细胞、1×10 9至3×10 9个细胞/天剂量的所述NK细胞等)组成。
所述重组溶瘤痘病毒可采用本领域通常所采用的给药方式给药，例如通过瘤内注射给药或静脉给药。
NK细胞可采用本领域通常所采用的给药方式给药，例如可通过静脉给药。
本领域的技术人员可以理解，本发明的治疗剂还可包含合适的可药用的辅料。
本发明的治疗剂还可以包含本领域已知的其它活性成分，例如白细胞介素-2(IL-2)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)等。
在一些实施方案中，本发明的治疗剂包含一种或多种可药用载体。可以通过本领域已知的方法制备药物制剂。例如，可以将化合物等活性成分与常见的赋形剂、稀释剂(例如磷酸盐缓冲液或生理盐水)、组织培养基、和载体(例如自体血浆或人血清白蛋白)进行配制，并作为悬浮剂施用。其它的载体可以包括脂质体、胶团、纳米胶囊、聚合纳米颗粒、固体脂颗粒(例如参见文献“E.Koren and V.Torchilin，Life，63：586-595，2011”)。本发明的治疗剂的具体配制方法可参见科学文献和专利文献中的描述，例如参见最新版雷明登氏药物科学，Maack出版公司，Easton PA(″Remington′s″)。
本发明的治疗剂可以治疗多种肿瘤和/或癌症，包括但不限于：肺癌、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌等。
本发明的治疗剂的施用方法为，对肿瘤和/或癌症患者首先施用所述重组溶瘤痘病毒，然后，在施用所述重组溶瘤痘病毒之后的第18-72小时(例如，第20-70小时、第22-48小时、第24-48小时、第30-48小时等)，对所述肿瘤和/或癌症患者施用所述NK细胞。“在施用所述重组溶瘤痘病毒之后的第18-72小时(例如，第20-70小时、第22-48小时、第24-48小时、第30-48小时等)，对所述肿 瘤和/或癌症患者施用所述NK细胞”是指首次NK细胞的施用与首次重组溶瘤痘病毒施用的时间间隔为18-72小时(例如，20-70小时、22-48小时、24-48小时、30-48小时等)，或首次NK细胞的施用与在其之前最相邻一次的所述重组溶瘤痘病毒施用的时间间隔为18-72小时(例如，20-70小时、22-48小时、24-48小时、30-48小时等)。优选地，首次NK细胞的施用与在其之前最相邻一次的所述重组溶瘤痘病毒施用的时间间隔为18-72小时(例如，20-70小时、22-48小时、24-48小时、30-48小时等)。还优选地，首次NK细胞的施用与在其之前最相邻一次的所述重组溶瘤痘病毒施用的时间间隔为24-48小时。
在本发明的一个优选实施方案中，所述重组溶瘤痘病毒的施用剂量为治疗有效量，每天1次，连续施用1-6天；并且所述NK细胞的施用剂量为1×10 7至1×10 10个细胞/天剂量(例如，1×10 8至5×10 9个细胞/天剂量、1×10 9至4×10 9个细胞/天剂量、1×10 9至3×10 9个细胞/天剂量)，每天1次，连续施用1-6天。在本发明的另一个优选实施方案中，所述重组溶瘤痘病毒的施用剂量为治疗有效量，每2天1次，连续施用2-6天；并且所述NK细胞的施用剂量为1×10 7至1×10 10个细胞/天剂量(例如，1×10 8至5×10 9个细胞/天剂量、1×10 9至4×10 9个细胞/天剂量、1×10 9至3×10 9个细胞/天剂量)，每2天1次，连续施用2-6天。无论本发明采用上述何种实施方案或其它实施方案，只要满足在施用所述重组溶瘤痘病毒之后的第18小时至72小时，对所述肿瘤和/或癌症患者施用NK细胞的条件即可。其中所述重组溶瘤痘病毒的施用和NK细胞的施用可以是间隔给药方式(例如，第1天施用所述重组溶瘤痘病毒，第2天施用NK细胞，第3天施用所述重组溶瘤痘病毒，第4天施用NK细胞…以此类推)；或依次给药方式(例如，第1天施用所述重组溶瘤痘病毒，第2天依次施用所述重组溶瘤痘病毒和NK细胞，第3天依次施用所述重组溶瘤痘病毒和NK细胞，第4天依次施用所述重组溶瘤痘病毒和NK细胞…以此类推)；或其它给药方式(例如首先施用所述重组溶瘤痘病毒，每天1次，连续施用1-6天，之后间隔18-72小时再施用NK细 胞，每天1次，连续施用1-6天)。优选的是，首先施用所述重组溶瘤痘病毒，在所述重组溶瘤痘病毒全部施用之后间隔18-72小时再施用NK细胞。在本发明的一个优选实施方案中，对肿瘤和/或癌症患者首先施用所述重组溶瘤痘病毒，所述重组溶瘤痘病毒的施用剂量为治疗有效量，施用1次；并且在施用所述重组溶瘤痘病毒之后的第18小时至72小时，对所述肿瘤和/或癌症患者施用所述NK细胞，所述NK细胞的施用剂量为1×10 7至1×10 10个细胞/天剂量(例如，1×10 8至5×10 9个细胞/天剂量、1×10 9至4×10 9个细胞/天剂量、1×10 9至3×10 9个细胞/天剂量)，施用1次。所述重组溶瘤痘病毒的治疗有效量优选为1×10 5至5×10 9pfu/天剂量(例如，1×10 5至3×10 9pfu/天剂量、1×10 5至1×10 8pfu/天剂量等)。
本发明还提供了本发明所述的治疗剂在制备用于***和/或癌症的药物中的应用。
所述肿瘤和/或癌症包括但不限于：肺癌(例如非小细胞肺癌)、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌等。
本发明另一方面还提供了一种用于***和/或癌症的具有协同作用的联合药物的药盒，包括装有本发明所述重组溶瘤痘病毒的第一容器和装有本发明所述NK细胞的第二容器，其中所述第一容器和所述第二容器是独立的；以及载明给药时机和给药方式的说明书。优选的是，该药盒由分别独立地装有本发明所述的重组溶瘤痘病毒和本发明所述的NK细胞的独立容器组成，以及载明给药时机和给药方式的说明书。
所述肿瘤和/或癌症包括但不限于：肺癌(例如非小细胞肺癌)、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌等。
优选地，装有本发明所述重组溶瘤痘病毒的第一容器包含治疗有效量的所述重组溶瘤痘病毒，并且装有NK细胞的第二容器包含足 够提供1×10 7-1×10 10个细胞/天剂量的所述NK细胞(例如，1×10 8至5×10 9个细胞/天剂量的所述NK细胞、1×10 9至4×10 9个细胞/天剂量的所述NK细胞、1×10 9至3×10 9个细胞/天剂量的所述NK细胞等)。所述重组溶瘤痘病毒的治疗有效量优选为1×10 5至5×10 9pfu/天剂量(例如，1×10 5至3×10 9pfu/天剂量、1×10 5至1×10 8pfu/天剂量等)。
优选地，所述装有所述重组溶瘤痘病毒的第一容器包含1×10 5-1×10 8pfu/天剂量的所述重组溶瘤痘病毒，并且所述装有NK细胞的第二容器包含1×10 9至3×10 9个细胞/天剂量的所述NK细胞。
在本发明中，所述NK细胞可以选自自体NK细胞和异体NK细胞；优选地，所述NK细胞为经体外扩增得到的自体NK细胞或经体外扩增得到的异体NK细胞。
所述重组溶瘤痘病毒可采用其各自的本领域通常所采用的给药方式给药，例如通过瘤内注射给药或静脉给药。
NK细胞可采用本领域通常所采用的给药方式给药，例如可通过静脉给药。
所述肿瘤和/或癌症包括但不限于：肺癌(例如非小细胞肺癌)、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌等。
本发明另一方面还提供了一种***和/或癌症的方法，包括以下依次进行的步骤：
1)对肿瘤和/或癌症患者施用根据本发明所述的重组溶瘤痘病毒；
2)在施用所述重组溶瘤痘病毒之后的第18-72小时(例如，第20-70小时、第22-48小时、第24-48小时、第30-48小时等)，对所述肿瘤和/或癌症患者施用本发明所述的NK细胞。
“在施用所述重组溶瘤痘病毒之后的第18-72小时(例如，第20-70小时、第22-48小时、第24-48小时、第30-48小时等)，对所述肿瘤和/或癌症患者施用本发明所述的NK细胞”是指首次NK细胞的施用与首次重组溶瘤痘病毒施用的时间间隔为18-72小时(例 如，20-70小时、22-48小时、24-48小时、30-48小时等)，或首次NK细胞的施用与在其之前最相邻一次的所述重组溶瘤痘病毒施用的时间间隔为18-72小时(例如，20-70小时、22-48小时、24-48小时、30-48小时等)。优选地，首次NK细胞的施用与在其之前最相邻一次的所述重组溶瘤痘病毒施用的时间间隔为18-72小时(例如，20-70小时、22-48小时、24-48小时、30-48小时等)。还优选地，首次NK细胞的施用与在其之前最相邻一次的所述重组溶瘤痘病毒施用的时间间隔为24-48小时。
所述肿瘤和/或癌症包括但不限于：肺癌(例如非小细胞肺癌)、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌等。
溶瘤病毒能够在肿瘤或癌症细胞中选择性复制，经过一定时间达到峰值。本发明的发明人发现，在经过一段时间的复制之后，肿瘤细胞里的溶瘤病毒会促进NK细胞对肿瘤细胞的杀伤。因此，本发明提出的重组溶瘤痘病毒和NK细胞的施用顺序和间隔实现了两者作用峰值的双峰重叠。
本发明进一步探索优化了所述重组溶瘤痘病毒和NK细胞各自的施用剂量，其与上述施用顺序和施用间隔的配合是至关重要的，其决定了所述重组溶瘤痘病毒的抗肿瘤疗效、NK细胞的抗肿瘤疗效、以及两者对肿瘤细胞的最佳协同杀伤。
在本发明的一个优选实施方案中，所述重组溶瘤痘病毒的施用剂量为治疗有效量，每天1次，连续施用1-6天；并且所述NK细胞的施用剂量为1×10 7至1×10 10个细胞/天剂量(例如，1×10 8至5×10 9个细胞/天剂量、1×10 9至4×10 9个细胞/天剂量、1×10 9至3×10 9个细胞/天剂量)，每天1次，连续施用1-6天。在本发明的另一个优选实施方案中，所述重组溶瘤痘病毒的施用剂量为治疗有效量，每2天1次，连续施用2-6天；并且所述NK细胞的施用剂量为1×10 7至1×10 10个细胞/天剂量(例如，1×10 8至5×10 9个细胞/天剂量、1×10 9至4×10 9个细胞/天剂量、1×10 9至3×10 9个细胞/天剂量)， 每2天1次，连续施用2-6天。无论本发明采用上述何种实施方案或其它实施方案，只要满足在施用所述重组溶瘤痘病毒之后的第18小时至72小时，对所述肿瘤和/或癌症患者施用NK细胞的条件即可。其中重组溶瘤痘病毒的施用和NK细胞的施用可以是间隔给药方式(例如，第1天施用重组溶瘤痘病毒，第2天施用NK细胞，第3天施用重组溶瘤痘病毒，第4天施用NK细胞…以此类推)；或依次给药方式(例如，第1天施用重组溶瘤痘病毒，第2天依次施用重组溶瘤痘病毒和NK细胞，第3天依次施用重组溶瘤痘病毒和NK细胞，第4天依次施用重组溶瘤痘病毒和NK细胞…以此类推)；或其它给药方式(例如首先施用重组溶瘤痘病毒，每天1次，连续施用1-6天，之后间隔18-72小时再施用NK细胞，每天1次，连续施用1-6天)。优选的是，首先施用重组溶瘤痘病毒，在重组溶瘤痘病毒全部施用之后间隔18-72小时再施用NK细胞。在本发明的一个优选实施方案中，对肿瘤和/或癌症患者首先施用所述重组溶瘤痘病毒，所述溶瘤病毒的施用剂量为治疗有效量，施用1次；并且在施用所述溶瘤病毒之后的第18小时至72小时，对所述肿瘤和/或癌症患者施用所述NK细胞，所述NK细胞的施用剂量为1×10 7至1×10 10个细胞/天剂量(例如，1×10 8至5×10 9个细胞/天剂量、1×10 9至4×10 9个细胞/天剂量、1×10 9至3×10 9个细胞/天剂量)，施用1次。所述重组溶瘤痘病毒的治疗有效量优选为1×10 5至5×10 9pfu/天剂量(例如，1×10 5至3×10 9pfu/天剂量、1×10 5至1×10 8pfu/天剂量等)。
可以根据实际情况和需要对患者进行一次或多次本发明的***和/或癌症的方法。
在本发明中，所述NK细胞可以选自自体NK细胞和异体NK细胞；优选地，所述NK细胞为经体外扩增得到的自体NK细胞或经体外扩增得到的异体NK细胞。
所述肿瘤和/或癌症包括肺癌、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌等。
所述重组溶瘤痘病毒可采用本领域通常所采用的给药方式给药，例如通过瘤内注射给药或静脉给药。
NK细胞可采用本领域通常所采用的给药方式给药，例如可通过静脉给药。
以下通过例子的方式进一步解释或说明本发明的内容，但这些例子不应被理解为对本发明的保护范围的限制。
例子
以下除非特别说明，否则以下例子中所用实验方法均使用生物工程领域的常规实验流程、操作、材料和条件进行。
以下除非特别说明，否则各试剂的百分浓度(％)均指该试剂的体积百分浓度(％(v/v))。
以下例子中所用的材料如下：
1.CV1细胞和Hu-143B细胞来源于武汉大学中国典型培养物保藏中心。
2.肿瘤细胞
除FaDu人头颈癌细胞来源于杭州市第一人民医院，其余各肿瘤细胞来源于中国典型培养物保藏中心和ATCC。细胞培养为正常培养环境McCoy′s 5A+10％FBS和MEM+10％FBS。McCoy′s 5A和MEM购自GIBCO公司。胎牛血清FBS购自SIGMA公司。
3.NK细胞
实验中采用的NK细胞来源如下：
各例子中采用杭州康万达医药科技有限公司培养冻存的人NK细胞。该人NK细胞的制备方法如下：采用本领域常规的方法，用采血针穿刺肘部静脉，取健康人的外周静脉血，提取全免疫细胞PBMC。采用经辐照后的K562滋养细胞(购自杭州鼎云生物技术有限公司)，以自体血浆培养法扩增NK细胞，最终NK细胞纯度达到90％，NK细胞存活活性达到90％，NK细胞体外杀伤率达到85％。
4.对照病毒
作为对照病毒的溶瘤痘病毒DDVV-RFP是已知的，属于溶瘤痘病毒WR株(可参见(例如)科技文献：“X Song，et al.T-cell Engager-armed Oncolytic Vaccinia Virus Significantly Enhances Antitumor TherapyMolecular Therapy.(2014)；22 1，102-111”)，该病毒的TK基因和VGF基因均为功能缺陷型，且携带有外源红色荧光蛋白(RFP)基因。由于RFP基因仅起到筛选/报告作用，因此溶瘤痘病毒DDVV-RFP的抗肿瘤功能基本等同TK基因和VGF基因功能缺陷型的溶瘤痘病毒。溶瘤痘病毒DDvv-RFP也可采用本领域常规技术对VSC20痘病毒进行基因改造而得到。VSC20痘病毒是VGF基因缺失的痘病毒，制备方法可参见科技文献：“McCart，JA，et al.Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes.Cancer Res(2001)61：8751-8757.”。所述基因改造包括使用人工合成痘病毒早/晚期启动子pSEL调控外源DsRed基因(即RFP基因)，使用体外细胞内重组技术将DsRed基因***到痘病毒VSC20株的TK基因区中，从而构建得到溶瘤痘病毒DDVV-RFP。其生产和纯化可按照下文所述制备例2进行。
5.C57BL/6小鼠购自北京维通利华实验动物技术有限公司。重度免疫缺陷型NCG小鼠得自浙江省动物中心。
6.培养板
6孔细胞培养板(每孔培养体积2ml)、24孔细胞培养板(每孔培养体积500μl)、96孔细胞培养板(每孔培养体积200μl)均得自：Corning(康宁)公司。
7.gpt筛选药物配制方法：使用0.1M NaOH分别配制10mg/ml霉酚酸(400×)、10mg/ml 40×黄嘌呤(40×)、10mg/ml次黄嘌呤(670×)，避光-20℃储存；使用时配制1×工作液：在10ml DMEM内加入25μl霉酚酸+250μl黄嘌呤+15μl次黄嘌呤。
8.PBS配方：8mM Na 2HPO 4、136mM NaCl、2mM KH 2PO 4、2.6mM KCl，pH7.2-7.4。
9.STE缓冲液配方：10mM Tris-Cl、0.1M NaCl、1mM EDTA， pH8.0。
10.以下例子中所用的细胞计数方法如下：
MTT法：每孔细胞中加入10μl MTT溶液(5mg/ml)，在37℃培养箱培养4～6小时，吸弃培养液，每孔加入150μl DMSO，置摇床上低速震荡10分钟，使结晶物充分溶解，使用酶标仪检测其在490nm处的吸光值(OD 490)。抑制率计算公式：细胞增殖抑制百分率(IR％)＝1-(OD 490供试品-OD 490空白)/(OD 490阴性对照-OD 490空白)×100％。
台盼蓝染色法计数：将细胞用PBS洗后，用胰蛋白酶消化，细胞悬浮在PBS中，加入终浓度为0.04％(w/v)的台盼蓝染液，显微镜下计数，死细胞会染成蓝色，活细胞为透明无色。取活细胞数为最终数据。
以下例子中所用的缩写说明如下：
FBS：胎牛血清
PSG：青霉素-链霉素-谷氨酰胺
PBMC：外周血单个核细胞
RFP：红色荧光蛋白
TIL：肿瘤浸润淋巴细胞
DMSO：二甲亚砜
制备例1：DDvv-IL21溶瘤痘病毒的构建
以下过程可参见图2。
(1)质粒构建
构建两株溶瘤痘病毒使其分别携带人IL-21、鼠IL-21基因，分别标注为DDvv-hIL21、DDvv-mIL21。
1)根据NCBI基因库序列NM_001291041和NM_021803，用基因合成方法合成鼠IL21 DNA片段(mIL-21)和人IL21 DNA片段(hIL-21)共2条，具体而言：
mIL-21包含正常序列的鼠源IL21片段(即Genbank编号NM_001291041的核苷酸序列中72-560bp的片段)，其序列(SEQ ID NO.1)如下所示：
AGATCTATGGAGAGGACCCTTGTCTGTCTGGTAGTCATCTTCTTGGGGACAGTGGCCCATAAATCAAGCCCCCAAGGGCCAGATCGCCTCCTGATTAGACTTCGTCACCTTATTGACATTGTTGAACAGCTGAAAATCTATGAAAATGACTTGGATCCTGAACTTCTATCAGCTCCACAAGATGTAAAGGGGCACTGTGAGCATGCAGCTTTTGCCTGTTTTCAGAAGGCCAAACTCAAGCCATCAAACCCTGGAAACAATAAGACATTCATCATTGACCTCGTGGCCCAGCTCAGGAGGAGGCTGCCTGCCAGGAGGGGAGGAAAGAAACAGAAGCACATAGCTAAATGCCCTTCCTGTGATTCGTATGAGAAAAGGACACCCAAAGAATTCCTAGAAAGACTAAAATGGCTCCTTCAAAAGGTATGCACCTTAAATGCATTTCTTTCACTTCCATGTTGTGTCCGGGTACCTCCTGTGCCCAGTGACTCATAG
Figure PCTCN2018093404-appb-000001
其中 AGATCT为BglII识别序列；
Figure PCTCN2018093404-appb-000002
为SpeI识别序列；以及
hIL-21包含正常序列的人源IL21片段(即Genbank编号为NM_021803的核苷酸序列中47-535bp的片段)，其序列(SEQ ID NO.2)如下所示：
AGATCTATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTGA
Figure PCTCN2018093404-appb-000003
其中 AGATCT为BglII识别序列；
Figure PCTCN2018093404-appb-000004
为SpeI识别序列。
使用BglII内切酶与SpeI内切酶进行剪切，把IL21基因***pCB 质粒(来源可参见文献“房有容等，Zeocin和GFP双筛选标记重组痘苗病毒载体的构建.《国际流行病学传染病学杂志》，2012.39(3)：148-152.”)内，使用大肠杆菌DH5α转化提取质粒pCB-mIL21、pCB-hIL21(图3)。
2)T7通用引物测序检测质粒pCB-mIL21、pCB-hIL21，序列正确。
3)使用TIANprep Rapid Mini Plasmid试剂盒(TIANGEN，DP105-03)进行中等量质粒DNA提取。
(2)重组溶瘤痘病毒的包装及鉴定
1)选取生长状态良好的CV1细胞铺于6孔板中，约4×10 5个细胞/孔，使得次日细胞密度达到80％-90％。
2)弃掉6孔板中的培养基，两个孔分别加入1ml无血清无抗生素的DMEM培养基，其中分别含有9×10 3pfu的Dvv-VSC20病毒(病毒来源可参见文献“McCart，JA，et al.Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes.Cancer Res(2001)61：8751-8757.”)使其浓度为0.03pfu/细胞，十字混匀，37℃培养2小时，期间每20分钟混匀一次，同时设置一个不加病毒液的对照孔。
3)配制质粒/Lipo2000(
Figure PCTCN2018093404-appb-000005
转染试剂，Life Technologies，11668-019)混合液：溶液A-吸取5μg的质粒加入到300μl的Opti-MEM I(Gibco，1802679)中混匀，室温孵育5分钟；溶液B-吸取12μl的Lipo2000，加入至300μl的Opti-MEM I中混匀，室温孵育5分钟；将配置好的溶液A及溶液B混合，温和混匀，室温孵育15分钟，得到质粒/Lipo2000混合液。向感染病毒的其中一个孔加入300μl质粒/Lipo2000混合液，另设置一个只转染质粒的对照孔，37℃培养4小时，每孔补加2ml含10％FBS+1％PSG的DMEM培养基。
4)24小时后，弃旧的培养基，换含5％FBS+1％PSG的DMEM培养基，继续培养约48小时，显微镜下观察见细胞完全病变后，收 集上清及细胞，反复冻融3次，释放病毒。
5)取出200μl的病毒液，使用TIANamp Virus DNA/RNA试剂盒(TIANGEN，DP315)抽提病毒基因组，用于PCR法检测外源***的IL-21基因是否整合到骨架病毒内；同时取50μl的病毒液离心取上清用于ELISA检测(Mouse IL-21 DuoSet ELISA，R&D，DY594-05；Human IL-21 Uncoated ELISA，Invitrogen，88-8218)，进一步确定重组痘病毒是否表达IL-21蛋白，剩余病毒液保存于-80℃，标示为P0。PCR鉴定所用引物序列如表1，PCR检测获得阳性病毒的鉴定条带大小如表2所示。
表1.PCR引物序列
Figure PCTCN2018093404-appb-000006
表2.PCR结果条带大小
引物对 鉴定的病毒 条带大小(bp)
P1/P2 VSC20 622
P15/P11 DDvv-mIL21 1893
P15/zP1 DDvv-hIL21 1792
PCR结果显示，IL-21基因序列整合到病毒TK区，即病毒重组成功(图4A-B)；ELISA结果显示含有IL21的重组病毒上清(图中示为“DDvv-mIL-21”)中有IL-21蛋白的表达，其余细胞(图中示为“NC”)、转染质粒(pCB-mIL21)及VSC20对照未见IL-21的表达(图4C)。
(3)重组溶瘤痘病毒的筛选
1)使用60mm培养皿培养CV1细胞，达到80％满时加入1ml病毒稀释液，含150μl P0代病毒，感染2小时后，加3ml含1×gpt筛选药物进行筛选，观察细胞病变，收集病毒标注P1病毒。使用病毒基因组抽提试剂盒抽提病毒基因进行PCR验证。
2)重复2次步骤1)获得P3病毒，使用PCR法进行验证。
3)采用10cm培养皿培养CV1细胞待其密度达到80％，弃培养基，加入3ml梯度稀释的P3病毒感染2小时，去除培养液，加入8ml含有0.8×gpt筛选药物的1％(w/v)琼脂糖凝胶(V 5％的琼脂糖凝胶∶V 1×gpt工作 液＝1∶4)，37℃培养48小时，挑取单个空斑至300μl的无菌PBS中，反复冻融三次释放病毒，每个空斑分别取150μl的病毒液在24孔板中采用CV1细胞进行小扩，约48小时待病毒完全病变后，收集病毒，吸取200μl病毒液抽提基因组，使用PCR法验证，其余病毒液标记为P4，保存于-80℃。
4)重复2-3次步骤3)，PCR法确认重组病毒中不含骨架病毒VSC20，同时使用ELISA和Western-blot方法(一抗：兔抗鼠IL21，PEpro Tech.INC.，#500-P278；兔抗IL21抗体，abcam，ab5978；二抗：Goat anti-Rb，Abcam，ab97051)确认IL21蛋白的表达。
结果显示：从上述4)步骤获得的病毒经PCR法鉴定显示P1/P2的PCR结果无条带，表明重组病毒为携带外源目的基因IL-21的纯种病毒，不含其骨架背景病毒VSC20(图5A-D)。Western blot结果同样显示DDvv-IL21病毒可有效表达IL-21蛋白，且大小正确(图5E、G)。ELISA结果显示重组病毒DDvv-IL21在细胞培养上清及细胞裂解(图中示为“细胞”)中均可检测到IL-21蛋白，也进一步体现了IL-21蛋白为外泌型蛋白的特性(图5F、H)。
制备例2：溶瘤痘病毒的生产和纯化
(1)生产和纯化
1)分别对DDvv-mIL21的P5-1样本和DDvv-hIL21的P4-3样本进行大量扩增，使用50个150mm培养皿，当Hu-143B细胞长至80％左右时，每个培养皿加50μL小扩后的病毒约2×10 4PFU感染细胞，37℃、5％CO 2培养。约2～3天后，显微镜下观察细胞变圆呈串珠状，部分细胞脱离培养皿，使用细胞刮刀收集培养液/细胞，保存于-80℃冰箱。
2)使用液氮、高压灭菌水将所收集的病毒液反复冻融三次，2000rpm离心3分钟，收集上清。
3)将收集的上清在12,000rpm离心10分钟，弃上清，沉淀物用5mL PBS悬浮，12,000rpm离心10分钟，弃上清，沉淀物用4mL30％(w/v)蔗糖重悬。
4)蔗糖梯度的制备：在超速离心管中从下至上依次加入60％(w/v)、50％(w/v)、40％(w/v)的蔗糖溶液各2mL，最上层加30％(w/v)蔗糖重悬的病毒液4mL。
5)铺好蔗糖梯度后，39,000g超速离心20分钟后，肉眼可看到病毒呈乳白色条带，分布在50％(w/v)蔗糖层，使用1mL剪平/大口的 枪头吸取病毒条带，置于新的离心管中(记录吸取病毒条带的体积为V1)。
6)使用2倍V1体积的STE缓冲液清洗病毒去除残余的蔗糖，离心35,000×g、1小时，弃上清，保留沉淀。
7)用V1体积的STE重悬沉淀，离心35,000×g，30分钟，保留沉淀，重复此操作2次，随后用PBS洗3次。
8)最后用预冷的PBS重悬沉淀，混匀后分装在无菌的EP管中，储存于-80℃，预留1管放置4℃进行病毒滴度检测。
(2)病毒滴度检测
1)使用2个6-well培养皿，每孔接种约4×10 5HuTK-143B细胞，在37℃、5％CO 2培养箱过夜，使其次日细胞量接近80％-90％。
2)将待测病毒液200μl用无血清DMEM 1800μl作10倍梯度稀释，使其稀释度在10 -4-10 -8范围。
3)吸去6孔板内细胞培养液，加入0.5ml稀释的病毒液，标注各孔稀释度，同时设立两个空白对照孔，置37℃感染2小时，期间每20分钟摇动6孔板一次，以保证孔内均匀湿润和避免局部干燥。
4)每孔加2ml含5％FBS的DMEM，37℃培养48小时。
5)吸去培养液，每孔加0.5ml含0.1％结晶紫染色液，室温染色10分钟，吸去染色液，PBS清洗3次，倒扣自然风干，拍照。
6)计数各孔内的病毒噬斑数，病毒滴度＝平均病毒噬斑数/(病毒稀释度×加入的病毒体积)。
举例：假如在10 -5稀释度孔内有30个病毒噬斑，病毒滴度为30/(10 -5×0.5)＝6×10 6pfu/ml。
放大/纯化后各个批号的DDvv-mIL21病毒和DDvv-hIL21病毒的滴度在2×10 8～4×10 8PFU/ml。
实施例1：携带鼠IL-21片段的DDvv-mIL21痘病毒对不同鼠肿瘤细胞的杀伤作用
在96孔板铺鼠肿瘤细胞，包括B16细胞(小鼠黑色素瘤细胞)、 GL261细胞(小鼠脑胶质瘤细胞)、LLC细胞(小鼠Lewis肺癌细胞)、CT-26细胞(小鼠结肠癌细胞)、4T-1细胞(小鼠乳腺癌细胞)，每孔5000个细胞，培养过夜使其贴壁后使用1MOI的通过上述方法制备的DDvv-mIL21病毒感染细胞，分别于24小时、48小时和60小时后(针对LLC细胞、GL261细胞的试验)，或者24小时、48小时和72小时后(针对B16细胞、CT-26细胞、4T-1细胞的试验)，使用MTT法检测肿瘤细胞凋亡情况(n＝3，2-3次重复实验)。本试验的对照组为：阴性对照组(无病毒感染)、重组鼠源IL-21蛋白(rmIL-21，购自R&D Systems)及阳性对照组(1μM紫杉醇(Paclitaxel)，购自北京双鹭药业)，每个处理组及对照组均设置3个复孔，细胞杀伤率为实验组与阴性对照组百分比值。结果显示DDvv-mIL21病毒对各肿瘤细胞在60小时或72小时后均达到超过60％的杀伤率，其中，DDvv-mIL21病毒对CT-26和4T1细胞在感染24小时后就达到70％的杀伤率(参见图6)。
实施例2：携带鼠IL-21片段的DDvv-mIL21痘病毒对不同鼠肿瘤细胞杀伤的IC 50
B16细胞、CT26细胞、4T1细胞、LLC细胞、GL261细胞铺于96孔板，每孔5000个细胞，使用0.002MOI、0.02MOI、0.2MOI、1MOI、2MOI的通过上述方法制备的DDvv-mIL21病毒感染细胞72小时后使用MTT试剂检测细胞杀伤，并计算DDvv-mIL21重组痘病毒对不同肿瘤细胞杀伤的半数致死量(IC 50值)(n＝3，1次实验)。结果显示，重组痘病毒对B16细胞、CT26细胞、4T1细胞、LLC细胞、GL261细胞杀伤的IC 50分别为0.232MOI、0.216MOI、0.07MOI、0.304MOI、0.227MOI(参见图7)。由此可见，本发明的重组痘病毒DDvv-mIL21对不同肿瘤细胞的IC 50值均较低(小于或约等于0.3MOI)，具有较好的成药前景。
实施例3：携带人IL-21片段的DDvv-hIL21痘病毒对不同人肿瘤细胞的杀伤作用
A549(人非小细胞肺癌细胞)、SKOV3(人卵巢腺癌细胞)、Hela(人***细胞)、U251(人神经胶质细胞瘤细胞)铺于96孔板，每孔5000个细胞，培养过夜使其贴壁后使用1MOI的通过上述方法制备的DDvv-hIL21病毒感染细胞，分别于24小时、48小时和72小时后，使用MTT法检测肿瘤细胞凋亡情况(n＝3，3次重复实验)。本试验的对照组为：阴性对照组(无病毒感染)、重组人源IL-21蛋白(rhIL-21，购自R&D Systems)及阳性对照组(1μM紫杉醇)，每个处理组及对照组均设置3个复孔，细胞杀伤率为实验组与阴性对照组百分比值。结果显示重组病毒对Hela、A549、SKVO3、U251细胞杀伤作用呈时间依赖性，随时间而加强(参见图8)。此外，结果还显示DDvv-hIL21病毒对各肿瘤细胞在48小时后均达到超过60％的杀伤率，在72小时后均达到超过70％的杀伤率。
实施例4：携带人IL-21片段的DDvv-hIL21痘病毒对不同人肿瘤细胞杀伤的IC 50
A549、HepG2(人肝癌细胞)、Hela、HT29(人结直肠癌细胞)、SKOV3(人卵巢腺癌细胞)、PANC1(人胰腺癌细胞)、SKHEP-1(人肝癌细胞)、FaDu(人咽鳞癌细胞)铺于96孔板，每孔5000个细胞，以3倍稀释MOI梯度的通过上述方法制备的DDvv-hIL21病毒感染细胞，使DDvv-hIL21病毒的MOI浓度分别为：0.003、0.01、0.03、0.1、0.3、1、3、10，感染48小时后使用MTT试剂检测细胞杀伤，并计算病毒对不同肿瘤细胞杀伤的IC 50值(n＝3，1次实验)，本研究每个处理组及对照组均设置3个复孔。结果显示，重组痘病毒对各株肿瘤细胞杀伤的IC 50在0.05-0.4MOI之间，均较低(参见图9)。由此可见，本发明的重组痘病毒DDvv-hIL21具有较好的成药前景。
实施例5：携带鼠IL-21片段的DDvv-mIL21痘病毒对B16肿瘤小鼠的抑瘤作用
构建免疫健全荷瘤小鼠：使用对数生长期B16细胞，每只小鼠给予20万个细胞接种于C57BL/6小鼠(北京维通力华实验动物中心) 后腿背部皮下，观察肿瘤生长情况，测量肿瘤体积在100-200mm 3左右开始给药，以瘤内给药方式分别给予100μl PBS内包含5×10 6pfu(低剂量(low dose))、1×107pfu(高剂量(high dose))的通过上述方法制备的DDvv-mIL21及PBS，每组3只小鼠，每3天测定1次瘤体大小及体重。结果显示重组病毒DDvv-mIL21能够有效的抑制肿瘤的生长，并且呈剂量依赖性(图10A)。截至给药后第9天，各给药组T/C(抑瘤有效率，即给药组肿瘤大小与对照组肿瘤大小的百分比率，该值＜40％时表示药物有效)都小于40％(图10B)。第12天处死小鼠，取出肿瘤检测瘤重，也显示出相似的结果(图10C)。
实施例6：携带鼠IL-21片段的DDvv-mIL21痘病毒对B16肿瘤小鼠的抗肿瘤免疫激活作用
使用对数生长期B16细胞，每只小鼠给予20万个细胞接种于C57/B16小鼠后腿背部皮下，观察肿瘤生长情况，测量肿瘤体积在100-200mm 3左右开始给药，以瘤内给药方式分别给予100μl PBS内包含5×10 6pfu(低剂量(low dose))、1×10 7pfu(高剂量(high dose))的通过上述方法制备的DDvv-mIL21及PBS，每组3只小鼠，2周后处死小鼠收取脾脏、肿瘤组织，提取脾脏PBMC和肿瘤组织内肿瘤细胞，采用流式细胞术检测CD8 +T细胞(CD3 +CD8 +)(抗体：抗鼠CD3、抗鼠CD8a，eBioscience)和CD4 +T细胞(CD3 +CD4 +)(抗体：抗鼠CD4，eBioscience)。结果显示给重组病毒DDvv-mIL21的小鼠脾脏PBMC内的CD8 +T细胞和CD4 +T细胞比例相对于PBS组有显著增加(图11A-B)；同时肿瘤组织内免疫细胞侵润分析发现给予DDvv-mIL2高剂量组的CD8 +T细胞和CD4 +T细胞(TIL)比例相对于PBS组增加(图11C-D)。
实施例7：携带鼠IL-21片段的DDvv-mIL21痘病毒对LLC肿瘤小鼠的抑瘤作用
使用对数生长期LLC细胞，每只小鼠给予100万个细胞接种于C57BL/6小鼠后腿背部皮下，观察肿瘤生长情况，当肿瘤体积在 100-200mm 3左右开始给药，以瘤内给药方式分别给予100μl PBS内包含1×10 6pfu(低剂量(low dose))、1×10 7pfu(高剂量(high dose))的通过上述方法制备的DDvv-mIL21和100μlPBS作为对照组，每组5只小鼠，每3天测定1次瘤体大小及体重。结果显示重组病毒DDvv-mIL21能够有效地抑制LLC肿瘤的生长，并且呈剂量依赖性(图12A)，给药后第9天，高剂量给药组T/C小于40％(图12B)。
实施例8：携带人IL-21片段的DDvv-hIL21痘病毒和NK细胞联合对人肿瘤细胞SK-HEP-1的杀伤作用
取SK-HEP-1细胞种入24孔培养板，铺满30％，在MEM+10％FBS环境中，在37℃、5％CO 2下孵育24小时，在DMEM无血清环境下加入MOI＝0.15的通过上述方法制备的DDvv-hIL21病毒，感染6小时，换液为MEM+10％FBS，在37℃、5％CO 2下培养24小时。之后换新鲜MEM+10％FBS，加入NK细胞(效靶比NK∶SK-HEP-1＝5∶1)，继续孵育48小时，洗去死细胞和碎片，对残留的活SK-HEP-1细胞进行台盼蓝染色计数，该实验组为DDvv-hIL21+NK组。实验中另外保留一组SK-HEP-1细胞，不加病毒，不加NK，作为空白(Blank)组；一组在相应时间点加DDvv-hIL21病毒，但不加NK，作为DDvv-hIL21病毒组；一组在相应时间点加NK，但不加DDvv-hIL21病毒，作为NK组；一组在相应时间点加NK，并同时加入终浓度为50ng/ml的人IL21重组蛋白，但不加DDvv-hIL21病毒，作为NK+IL21组；一组不加病毒，不加NK，但在相应的时间加入终浓度为50ng/ml的人IL21重组蛋白作为空白+IL21组。每组实验重复三次以上，取平均值做统计学分析。
结果如图13所示(其中横坐标为不同组别，纵坐标为相应的抑制率的百分比数值)，联合使用DDvv-hIL21病毒和NK细胞(溶瘤病毒在先施用，NK细胞在后施用)对SK-HEP-1杀伤有显著的协同作用，并且协同抑制率为约87％。而本实验中单用DDvv-hIL21病毒的抑制率为约47％，单用NK细胞的抑制率为约11％，NK+IL21组的抑制率为约22％，空白+IL21组的抑制率为约1％。而空白组抑制 率约为0(未示出在图13中)。
结果显示联合使用DDvv-hIL21病毒和NK细胞(溶瘤病毒在先施用，NK细胞在后施用)对SK-HEP-1细胞杀伤有显著的协同作用，并且协同抑制率为约87％。
实施例9：携带鼠IL-21片段的DDvv-mIL21痘病毒对药物诱导免疫低下B16荷瘤小鼠的抑瘤作用
构建药物诱导免疫低下荷瘤小鼠：使用对数生长期B16细胞，每只小鼠使用20万个细胞接种于C57BL/6小鼠后腿背部皮下，观察肿瘤生长情况，测量肿瘤体积在80-150mm 3左右开始每天腹腔注射30mg/kg环孢素直到实验结束，第二天(即接种后第10天)以瘤内给药方式分别给予100μl PBS内包含1×10 5pfu(低剂量(low dose))、1×10 6pfu(中剂量(middle dose))、1×10 7pfu(高剂量(high dose))的通过上述方法制备的DDvv-mIL21及100μl PBS，每组5只小鼠，每3天测定1次瘤体大小及体重。结果显示重组病毒DDvv-mIL21能够有效的抑制肿瘤的生长，并且呈剂量依耐性(图14A)。截至给药后第9天(即接种后第19天)，中、高剂量组T/C(有效抑瘤率，即给药组肿瘤大小与对照组肿瘤大小的百分比，该值低于40％时表示药物有效)都小于40％(图14B)。
实施例10：携带鼠IL-21片段的DDvv-mIL21痘病毒与对照痘病毒对药物诱导免疫低下B16荷瘤小鼠的抑瘤作用的比较
使用对数生长期B16细胞，每只小鼠使用20万个细胞接种于C57BL/6小鼠后腿背部皮下，观察肿瘤生长情况，测量肿瘤体积在80-150mm 3左右开始每天腹腔注射30mg/kg环孢素直到实验结束，第二天以瘤内给药方式分别给予100μl PBS内包含1×10 6pfu的通过上述方法制备的DDvv-mIL21、1×10 6pfu的DDvv-RFP和100μl PBS，每组3只小鼠，每3天测定1次瘤体大小及体重。结果显示与DDvv-RFP相比，DDvv-mIL21可有效抑制肿瘤的生长(图15A)，给药后第6天开始，DDvv-mIL21组T/C都小于40％(图15B)。实 验结束后处死小鼠收取肿瘤组织称重，DDvv-mIL21组肿瘤重量明显降低(图15C)。同时采用流式细胞仪检测肿瘤组织内TIL的含量(包括CD8 +T细胞(CD3 +CD8 +)(抗体：抗鼠CD3、抗鼠CD8a，eBioscience)和CD4 +T细胞(CD3 +CD4 +)(抗体：抗鼠CD3、抗鼠CD4，eBioscience))及NK细胞(CD3 -NK1.1 +)(抗体：抗鼠NK1.1，eBioscience)的含量，结果显示给予DDvv-RFP和DDvv-mIL21可明显提高肿瘤组织内TIL含量，与DDvv-RFP组比较，DDvv-mIL21组可见肿瘤TIL内CD4 +T细胞显著增高(图16A-C)。
实施例11：携带人IL-21的DDvv-hIL21痘病毒联合人NK细胞与对照痘病毒联合人NK细胞对HCT116荷瘤的重度免疫缺陷型小鼠的抑瘤作用比较
使用重度免疫缺陷型NCG小鼠(得自浙江省动物中心)，在小鼠后腿背部皮下接种500万个HCT116细胞，观察肿瘤生长情况，测量肿瘤体积在80-150mm 3左右开始(约接种后第7天)以瘤内给药方式分别给予100μl PBS内包含5×10 6pfu的DDvv-hIL21病毒、5×10 6pfu的对照痘病毒DDvv-RFP和PBS，每组5只小鼠。次日开始尾静脉给予5×10 7NK细胞/天，连续给予3天，停止3天，以此为一个周期，共给予3个周期的NK细胞。在开始给予病毒起每2-5天测定1次瘤体大小及体重。结果显示溶瘤痘病毒DDvv-RFP和DDvv-hIL21都能够有效的抑制肿瘤的生长，DDvv-hIL21呈现出比DDvv-RFP更好的抑瘤作用(图17A)，给药后第12天开始，DDvv-RFP和DDvv-hIL21组T/C都小于40％(图17B)。实验结束后处死小鼠收取肿瘤组织，采用定量PCR法检测肿瘤组织内痘病毒A46R基因的表达水平(目标基因的引物序列为：5’-CAGGGAAACGGATGTATA-3’(SEQ ID NO.8)和5’-TGTGTTACAGAATCATATAAGG-3’(SEQ ID NO.9))、IL-21基因表达水平(目标基因的引物序列为：5’-CCAACTAAAGTCAGCAAATACAGG-3’(SEQ ID NO.10)和5’-CTTTCTAGGAATTCTTTGGGTGG-3’(SEQ ID NO.11))、NK 细胞NKG2D基因的表达水平(目标基因的引物序列为：5’-GGCTTTTATCCACAAGAATCAAGATC-3’(SEQ ID NO.12)和5’-GTGCACGTCTACCGCAGAGA-3’(SEQ ID NO.13))、IFN-γ基因表达水平(目标基因的引物序列为：5’-AGTGTGGAGACCATCAAGGAAG-3’(SEQ ID NO.14)和5’-GTATTGCTTTGCGTTGGACAT-3’(SEQ ID NO.15))，以看家基因(House Keeping Gene)GAPDH为内参(目标基因的引物序列为：5’-GGTCTCCTCTGACTTCAACA-3’(SEQ ID NO.16)和5’-AGCCAAATTCGTTGTCATAC-3’(SEQ ID NO.17))，靶点基因的表达量为pg/μg GAPDH。结果显示DDvv-hIL21溶瘤病毒在肿瘤内的复制与对照痘病毒DDvv-RFP无显著区别(图18A)，同时IL21的表达进一步验证了，DDvv-hIL21溶瘤病毒在肿瘤组织中的存在(图18B)。与给予DDvv-RFP病毒的对照组比较，发现给予DDvv-hIL21溶瘤病毒组的肿瘤内NK含量明显增高(图18C)，同时伴有免疫因子INF-γ的表达增高(图18D)。
实施例12：静脉给予的DDvv-mIL21痘病毒在B16肿瘤小鼠中的分布
使用对数生长期B16细胞，每只小鼠使用20万个细胞接种于C57/B16雌小鼠后腿背部皮下，观察肿瘤生长情况，测量肿瘤体积在100-200mm 3左右开始给药，以静脉给药方式给予100μl PBS内包含5×10 9pfu的通过上述方法制备的DDvv-mIL21溶瘤病毒，共4只小鼠，1周后处死小鼠收取心脏、肝脏、脾脏、肺、肾脏、脑、卵巢、肿瘤组织，使用PCR法检测痘病毒在各脏器的分布，具体而言将各组织器官内痘病毒A46R基因的表达量分别与肾脏器官内痘病毒A46R基因的表达量进行比较(目标基因的引物序列为：5’-CAGGGAAACGGATGTATA-3’(SEQ ID NO.8)和5’-TGTGTTACAGAATCATATAAGG-3’(SEQ ID NO.9))，结果显示给予的DDvv-mIL21溶瘤病毒主要分布在肿瘤组织内，其中有一只小鼠的卵巢组织也可检测到病毒，其他脏器都未检测到DDvv-mIL21 溶瘤病毒(图19)。
实施例13：携带人IL-21的DDvv-hIL21痘病毒和NK细胞联合对人肿瘤细胞HepG2的杀伤作用
取HepG2细胞种入24孔培养板，铺满30％，在DMEM+10％FBS环境中，在37℃、5％CO 2下孵育24小时，在DMEM无血清环境下加入MOI＝0.027的通过上述方法制备的DDvv-hIL21病毒，感染6小时，之后换液为DMEM+10％FBS，在37℃、5％CO 2下继续孵育18小时。之后换新鲜DMEM+10％FBS，加入NK细胞(效靶比NK∶HepG2＝3∶1)，继续孵育48小时，洗去死细胞和碎片，对残留的活HepG2细胞进行台盼蓝染色计数，该实验组为DDvv-hIL21+NK组。实验中另外保留一组HepG2细胞，不加病毒，不加NK，作为空白(Blank，简称BK)组；一组在相应时间点加DDvv-hIL21病毒，但不加NK，作为DDvv-hIL21病毒组；一组在相应时间点加NK，但不加DDvv-hIL21病毒，作为NK组；一组在相应时间点加NK，并同时加入终浓度为50ng/ml的人IL21重组蛋白，但不加DDvv-hIL21病毒，作为NK+IL21组；一组不加病毒，不加NK，但在与上述NK加入时间点相应的时间加入终浓度为50ng/ml的人IL21重组蛋白作为空白+IL21组；对照组均在相应时间做相应换液操作。每组实验重复三次以上，取平均值做统计学分析。
结果如图20所示(其中横坐标为不同组别，纵坐标为相应的抑制率的百分比数值)，联合使用DDvv-hIL21病毒和NK细胞(溶瘤病毒在先施用，NK细胞在后施用)对HepG2杀伤有显著的协同作用，并且协同抑制率为约71％。而本实验中单用DDvv-hIL21病毒的抑制率为约34％，单用NK细胞的抑制率为约14％，二者的叠加值在图中用虚线示出。此外，NK+IL21组的抑制率为约25％，空白+IL21组的抑制率为约2％。而空白组抑制率约为0(未示出在图20中)。
结果显示联合使用DDvv-hIL21病毒和NK细胞(溶瘤病毒在先施用，NK细胞在后施用)对HepG2细胞杀伤有显著的协同作用，并且协同抑制率为约71％。
实施例14：携带人IL-21片段的DDVV-hIL21痘病毒联合人NK细胞与对照痘病毒联合人NK细胞对人肿瘤细胞HCT116的杀伤作用比较
由试验确定，DDVV-RFP对HCT116细胞的杀伤剂量适宜为约MOI＝0.7；NK细胞对HCT116细胞的杀伤剂量适宜为效靶比NK∶HCT116为约5∶1。
取HCT116细胞种入24孔培养板，铺满30％，在McCoy′s5A+10％FBS环境中，在37℃、5％CO 2下孵育24小时，在McCoy′s 5A的无血清环境下加入MOI＝0.7的通过上述方法制备的DDVV-hIL21或DDVV-RFP，感染6小时，之后在McCoy′s 5A+10％FBS环境中，在37℃、5％CO 2下继续孵育18小时。随后(未换液)加入NK细胞(效靶比NK∶HCT116＝5∶1)，继续孵育48小时，洗去死细胞和碎片，对残留的活HCT116细胞进行台盼蓝染色计数。该实验组分别为DDVV-RFP+NK组和DDVV-hIL21+NK组。实验中保留一组HCT116细胞，不加病毒，不加NK，作为空白组；一组不加病毒，不加NK，但在与上述NK加入时间点相应的时间加入终浓度为50ng/ml的人IL21重组蛋白作为空白+IL21组。一组在相应时间点只加DDVV-RFP，但不加NK，作为DDVV-RFP组；一组在相应时间点只加DDVV-hIL21，但不加NK，作为DDVV-hIL21组；一组在相应时间点加NK，但不加病毒，作为NK组；一组在相应时间点加NK，并同时加入终浓度为50ng/ml的人IL21重组蛋白，但不加病毒作为NK+IL21组。一组在相应时间点分别加DDVV-RFP、NK，并在加NK的同时加入终浓度为50ng/ml的人IL21重组蛋白，作为DDVV-RFP+NK+IL21组；对照组均在相应时间做相应换液操作。每组实验重复三次以上，取平均值做统计学分析。
如图21A所示(其中X轴为不同组别，Y轴为相应的抑制率的百分比数值)，联合使用DDvv-hIL21病毒和NK细胞(溶瘤病毒在先施用，NK细胞在后施用)对HCT116细胞的杀伤有显著的协同作用，并且协同抑制率为约86％。而本实验中单用DDvv-hIL21病毒的 抑制率为约43％，单用NK细胞的抑制率为约10％，二者的叠加值在图中用虚线示出。而空白组抑制率约为0(未示出在图21A或B中)。
此外，如图21B所示(其中X轴为不同组别，Y轴为相应的抑制率的百分比数值)，联合使用DDVV-hIL21与NK细胞对HCT116细胞的杀伤效果(抑制率为约86％)显著高于同样剂量的DDVV-RFP和NK细胞联用的杀伤效果(抑制率为约67％)，也显著高于同样剂量的DDVV-RFP和NK细胞以及人IL21重组蛋白联用的杀伤效果(抑制率为约64％)。而单用DDVV-RFP的抑制率为约37％，空白+IL21组的抑制率为未测出，NK+IL21组的抑制率为约12％，其它组数据同上。
上述结果表明由于DDVV-hIL21在HCT116细胞中选择性复制从而杀伤HCT116细胞的同时表达外源IL-21，而表达的外源IL-21能够提升NK细胞的杀伤力从而进一步增强了NK细胞的HCT116细胞杀伤作用，使得DDVV-hIL21与NK细胞的联用对HCT116细胞的杀伤效果产生了令人惊奇的效果。
实施例15：携带人IL-21片段的DDVV-hIL21痘病毒联合人NK细胞与对照痘病毒联合人NK细胞对人肿瘤细胞FaDu的杀伤作用比较
取FaDu细胞种入24孔培养板，铺满30％，在MEM+10％FBS环境中，在37℃、5％CO 2下孵育24小时，在MEM的无血清环境下加入MOI＝0.2的通过上述方法制备的DDVV-hIL21或DDVV-RFP，感染6小时，之后在MEM+10％FBS环境中，在37℃、5％CO 2下继续孵育18小时。随后(未换液)加入NK细胞(效靶比NK∶FaDu＝5∶1)，继续孵育48小时，洗去死细胞和碎片，对残留的活FaDu细胞进行台盼蓝染色计数。该实验组分别为DDVV-RFP+NK组和DDVV-hIL21+NK组。实验中保留一组FaDu细胞，不加病毒，不加NK，作为空白组；一组不加病毒，不加NK，但在与上述NK加入时间点相应的时间加入终浓度为50ng/ml的人IL21重组蛋白作为空白+IL21组。一组在相应时间点只加DDVV-RFP，但不加NK，作为 DDVV-RFP组；一组在相应时间点只加DDVV-hIL21，但不加NK，作为DDVV-hIL21组；一组在相应时间点加NK，但不加病毒，作为NK组；一组在相应时间点加NK，并同时加入终浓度为50ng/ml的人IL21重组蛋白，但不加病毒，作为NK+IL21组；一组在相应时间点加DDVV-RFP，加NK的同时加入终浓度为50ng/ml的人IL21重组蛋白，作为DDVV-RFP+NK+IL21组；对照组均在相应时间做相应换液操作。每组实验重复三次以上，取平均值做统计学分析。
如图22A所示(其中X轴为不同组别，Y轴为相应的抑制率的百分比数值)，联合使用DDvv-hIL21病毒和NK细胞(溶瘤病毒在先施用，NK细胞在后施用)对FaDu细胞的杀伤有显著的协同作用，并且协同抑制率为约73％。而本实验中单用DDvv-hIL21病毒的抑制率为约37％，单用NK细胞的抑制率为约13％，二者的叠加值在图中用虚线示出。而空白组抑制率约为0(未示出在图22A或B中)。
此外，如图22B所示(其中X轴为不同组别，Y轴为相应的抑制率的百分比数值)，联合使用DDVV-hIL21与NK细胞对FaDu细胞的杀伤效果(抑制率为约73％)显著高于同样剂量的DDVV-RFP和NK细胞联用的杀伤效果(抑制率为约66％)，也显著高于同样剂量的DDVV-RFP和NK细胞以及人IL21重组蛋白联用的杀伤效果(抑制率为约65％)。而单用DDVV-RFP的抑制率为约39％，空白+IL21组的抑制率为未测出，NK+IL21组的抑制率为约14％，其它组数据同上。
上述结果表明由于DDVV-hIL21在FaDu细胞中选择性复制从而杀伤FaDu细胞的同时表达外源IL-21，而表达的外源IL-21能够提升NK细胞的杀伤力从而进一步增强了NK细胞的FaDu细胞杀伤作用，使得DDVV-hIL21与NK细胞的联用对FaDu细胞的杀伤效果产生了令人惊奇的效果。

Claims (18)

  1. 一种分离的重组溶瘤痘病毒，其中该重组溶瘤痘病毒是TK基因和VGF基因功能缺陷型的，并且该重组溶瘤痘病毒的基因组中整合有外源IL-21基因，并且该IL-21基因能够在肿瘤细胞中表达。
  2. 根据权利要求1所述的重组溶瘤痘病毒，其中所述TK基因通过***外源核苷酸序列而使该TK基因功能缺陷。
  3. 根据权利要求1所述的重组溶瘤痘病毒，其中所述外源IL-21基因***在所述TK基因中，从而使该TK基因功能缺陷。
  4. 根据权利要求1所述的重组溶瘤痘病毒，其中所述VGF基因通过基因敲除或***外源核苷酸序列而使该VGF基因功能缺陷。
  5. 根据权利要求1所述的重组溶瘤痘病毒，其中所述重组溶瘤痘病毒是惠氏株或WR株。
  6. 根据权利要求1所述的重组溶瘤痘病毒，其中所述重组溶瘤痘病毒的基因组中还整合有外源筛选基因，所述外源筛选基因包括gpt基因和/或LacZ基因，但不包括荧光蛋白基因。
  7. 根据权利要求1或5所述的重组溶瘤痘病毒，其中所述外源IL-21基因来自于小鼠或人。
  8. 一种药物组合物，其中该药物组合物包括作为活性成分的根据权利要求1-7中任一项所述的重组溶瘤痘病毒，及可药用辅料。
  9. 根据权利要求8所述的药物组合物，其中所述药物组合物包含1×10 5-5×10 9pfu/天剂量的所述重组溶瘤痘病毒。
  10. 根据权利要求8所述的药物组合物，其中所述重组溶瘤痘病毒配制成通过瘤内注射给药或静脉给药。
  11. 一种用于制备权利要求1-7中任一项所述的重组溶瘤痘病毒的载体，其中所述载体包含在启动子控制下的外源IL-21基因。
  12. 一种含有权利要求11所述的载体的宿主细胞。
  13. 根据权利要求1-7中任一项所述的重组溶瘤痘病毒在制备用于***和/或癌症的药物中的用途。
  14. 根据权利要求13所述的用途，其中所述肿瘤和/或癌症包括肺癌、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌。
  15. 一种***和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据权利要求1-7中任一项所述的重组溶瘤痘病毒。
  16. 根据权利要求15所述的方法，其中所述重组溶瘤痘病毒的施用剂量为1×10 5-5×10 9pfu/天，每天1次，连续施用1-6天。
  17. 根据权利要求15所述的方法，其中所述重组溶瘤痘病毒通过瘤内注射给药或静脉给药。
  18. 根据权利要求15所述的方法，其中所述肿瘤和/或癌症包括肺癌、黑色素瘤、头颈部癌症、肝癌、脑癌、结直肠癌、膀胱癌、乳腺癌、卵巢癌、子宫癌、***、淋巴癌、胃癌、食道癌、肾癌、***癌、胰腺癌、白血病、骨癌、睾丸癌。
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