WO2019062250A1 - 包含分离的重组溶瘤腺病毒和nk细胞的治疗剂及应用、药盒、治疗肿瘤和/或癌症的方法 - Google Patents

包含分离的重组溶瘤腺病毒和nk细胞的治疗剂及应用、药盒、治疗肿瘤和/或癌症的方法 Download PDF

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WO2019062250A1
WO2019062250A1 PCT/CN2018/094263 CN2018094263W WO2019062250A1 WO 2019062250 A1 WO2019062250 A1 WO 2019062250A1 CN 2018094263 W CN2018094263 W CN 2018094263W WO 2019062250 A1 WO2019062250 A1 WO 2019062250A1
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cells
cancer
oncolytic adenovirus
tumor
recombinant oncolytic
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French (fr)
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绳纪坡
傅瑾
赵荣华
秦云
陈霖
康三毛
胡放
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杭州康万达医药科技有限公司
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Definitions

  • the present invention is in the field of biotechnology, and in particular, relates to therapeutic agents and applications comprising the isolated recombinant oncolytic adenovirus and NK cells, kits, methods of treating tumors and/or cancer.
  • Tumorygenesis is a multi-factor and multi-step process in which cells are stimulated by external carcinogenic factors (including physical radiation, chemicals, viruses, etc.), and the cells are damaged, causing changes in genetic material such as DNA inside the cells. It causes abnormalities and disturbances in the signal transduction pathways inside the cells. The cells appear to be madly proliferating, resisting apoptosis, stopping differentiation, and having the ability to invade tissues and migration, ultimately affecting the vital function of the human body.
  • the main treatment methods for tumors include surgery, radiation therapy, chemotherapy, biological therapy and immunotherapy. Although these treatments contribute to the control of the tumor to a certain extent, they still cannot solve the problem fundamentally.
  • Oncolytic virus therapy is also a category of biotherapeutics, and the study of oncolytic viruses dates back to the 1950s. At that time, a patient with cervical cancer was found to have resolved the tumor after infection with rabies virus. Inspired by the phenomenon of spontaneous tumor remission after infection in some cancer patients, the first wave of oncolytic virus research began.
  • Oncolytic virus refers to a type of virus that can selectively replicate in target cells after infection with tumor cells, ultimately leading to tumor cell lysis and death. These viruses rely on their own specificity to replicate in tumor cells to lyse tumor cells. The virus released after cell lysis can further infect surrounding tumor cells, and has no destructive effect on normal cells and tissues, or has less influence. .
  • Oncolytic viruses generally fall into two categories: one is a wild-type virus and an attenuated strain of natural variation, and such viruses naturally have affinity for certain tumor cells, such as reovirus, Newcastle disease virus, and small size of autonomous replication. Viruses, etc., which can multiply and lyse cells in certain tumor cells, have natural specific oncolytic activity; the other is a virus that can only be replicated in tumor cells after modification of the viral genome.
  • genetic engineering methods have been used to transform adenovirus, herpes simplex virus, influenza virus and human vaccinia virus.
  • Adenovirus is a relatively early study on the onset of oncolytic activity in oncolytic viruses. The adenovirus is more clearly studied with adenovirus type 5.
  • Adenovirus was used to treat head and neck malignant tumors shortly after the discovery. The tumors were reduced to different extents after adenovirus injection, but the tumors were easy to relapse after treatment, and the effect was difficult to last. Until 1996, Bischoff et al first reported the removal of some E1B recombination. Adenovirus Onyx-015 can cause tumor killing by selective replication of p53 abnormal tumor cells. Oncolytic adenovirus research has once again received widespread attention and rapid development, so many new oncolytic adenovirus types have emerged.
  • tumor immunotherapy is also a very important tool in the struggle against cancer. It mainly includes antibody therapy, T cell therapy and tumor vaccine.
  • the antibody is called the new target molecule "drug" of cancer, which can promote the more effective anti-tumor immunity by targeting the immune cells around the tumor, and also promote the killing of tumor cells by complement-dependent cytotoxicity, or by induction.
  • T cell therapy is a therapy in which an in vitro expanded tumor-specific autologous T cell (for example, CAR-T) is injected into a body by intravenous administration.
  • Tumor vaccine therapy is a method of producing specific antibodies and effector T cells by modulating the immune system of the motility, and is called active specific immunotherapy.
  • tumor immunotherapy plays a very positive role in the treatment of tumors, but the biggest problem in the process of tumor immunotherapy is tumor escape.
  • tumor escape There is an extremely complex relationship between the immune escape mechanism of tumors and the body's immune response to tumors.
  • Early tumor-specific CD8 + T cells are activated during tumor immunotherapy and lose their killing function as the tumor grows to a later stage.
  • a series of costimulatory molecules are required to provide a second signal, in order to allow T cells to reach physiological activation thresholds. Immune response.
  • the second signal provided by the costimulatory molecule is absent, it will lead to T cell non-reactivity or specific immune tolerance or even to apoptosis. Therefore, the regulation of positive and negative co-stimulatory signals and the balance between the two play an important regulatory role in the whole process of the body's immune response.
  • the present invention provides therapeutic agents and uses comprising the isolated recombinant oncolytic adenovirus and NK cells, kits, methods of treating tumors and/or cancer.
  • the present invention provides:
  • a therapeutic agent comprising:
  • a first pharmaceutical composition wherein the first pharmaceutical composition comprises a recombinant oncolytic adenovirus in a first pharmaceutically acceptable carrier;
  • the recombinant oncolytic adenovirus is a selective replication oncolytic adenovirus
  • the coding sequence of the exogenous shRNA capable of inhibiting PDL1 expression in tumor cells is integrated into the genome of the recombinant oncolytic adenovirus.
  • the first pharmaceutical composition comprises a dose of 5 ⁇ 10 7 to 5 ⁇ 10 12 VP / day of the recombinant oncolytic adenovirus, and the second drug said composition comprising 1 ⁇ 10 7 to 1 ⁇ 10 10 cells / day dose NK cells.
  • NK cells are selected from the group consisting of autologous NK cells and allogeneic NK cells.
  • NK cells are autologous NK cells obtained by in vitro expansion or allogeneic NK cells obtained by in vitro expansion.
  • 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 kit for a synergistic combination drug for treating tumors and/or cancer comprising: a first container containing a recombinant oncolytic adenovirus and a second container containing NK cells, wherein said a container and the second container are independent; and instructions for indicating a timing of administration and a mode of administration; wherein the recombinant oncolytic adenovirus is a selective replication oncolytic adenovirus, and the recombinant oncolytic adenovirus is A coding sequence for an exogenous shRNA capable of inhibiting PDL1 expression in tumor cells is integrated into the genome.
  • the genome of the recombinant oncolytic adenovirus comprises an E1A gene coding sequence; preferably, the E1A gene coding sequence is controlled by a CMV promoter Under.
  • NK cells are selected from the group consisting of autologous NK cells and allogeneic NK cells.
  • 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, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer.
  • a method of treating a tumor and/or cancer comprising the steps of:
  • the recombinant oncolytic adenovirus is a selective replication oncolytic adenovirus
  • the coding sequence of the exogenous shRNA capable of inhibiting PDL1 expression in tumor cells is integrated into the genome of the recombinant oncolytic adenovirus.
  • NK cells are selected from the group consisting of autologous NK cells and allogeneic NK cells.
  • 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 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 that the oncolytic adenovirus lacks the coding region of the E1B19K gene, the E1B55K gene, and the entire E3 gene, and simultaneously carries the coding sequence of the exogenous shRNA, thereby enabling the obtained recombinant oncolytic adenovirus to selectively be in the tumor.
  • the cells replicate and express shRNA capable of inhibiting PDL1 expression in tumor cells.
  • the oncolytic adenovirus provided based on this concept has strong tumoricidal ability, and its ability to replicate in normal cells is much lower than that in tumor cells, so it is less toxic to normal cells and improves safety.
  • the shRNA expressed by the virus can significantly reduce the expression level of PDL1 protein in tumor cells, thereby reducing the immunosuppression of T lymphocytes by tumor cells, thereby enhancing the anti-tumor immune killing effect of T and NK lymphocytes.
  • the present inventors have found that integration of the shRNA coding sequence in oncolytic adenovirus can produce a synergistic effect between oncolytic activity of oncolytic virus and anti-tumor immunostimulatory action of T or NK lymphocytes.
  • the recombinant oncolytic adenovirus of the present invention since the recombinant oncolytic adenovirus of the present invention selectively replicates in tumor cells and simultaneously expresses shRNA capable of inhibiting PDL1 expression in tumor cells, it enhances antitumor immunity of immune cells including T lymphocytes and NK cells.
  • the action can synergistically stimulate the anti-tumor immune response of the body, thereby enabling the recombinant oncolytic adenovirus of the present invention to be combined with NK cells.
  • the pharmaceutical composition and method provided based on the concept can fully exert the effect of the recombinant oncolytic adenovirus 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 adenovirus of the present invention selectively replicates in tumor cells, so that the tumor cells containing the recombinant oncolytic adenovirus of the present invention become NK A specific target for the cell. This ultimately results in a further enhanced synergistic killing of the tumor.
  • the present invention enables the synergistic effect of the combined application of the recombinant oncolytic adenovirus 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).
  • VP refers to the number of viral particles.
  • VP/kg refers to the number of viral particles per kilogram of patient body weight.
  • TID50 refers to a median tissue culture infective dose, which represents the viral dose at which half of the tissue culture is infected and cytopathic.
  • MOI Multiplicity of infection
  • Figure 1 shows a gel electrophoresis pattern of the E1A gene of PCR-amplified adenovirus type 5; wherein lane M is a DNA molecular weight marker and lane 1 is a PCR product using H101 genomic DNA as a template.
  • Figure 2 shows the results of PCR screening of pShuttle-E1A plasmid-positive clones; lanes M are DNA molecular weight markers and lanes 1-3 are candidate clones.
  • Figure 3 shows the construction of the pShuttle-E1A plasmid and the map of the constructed plasmid.
  • Figure 4 shows a schematic (left panel) and gel electrophoresis pattern (right panel) of PCR amplification of the E1A expression cassette from the pShuttle-E1A plasmid; wherein lane M is a DNA molecular weight marker and lanes 1-2 are PCR products.
  • Figure 5 shows the results of PCR screening of the pShuttle-MCS-E1A candidate plasmid; wherein lane M is a DNA molecular weight marker, lanes 1-13 are candidate plasmids, and lane NC is a PCR system negative control (ie, a template is water PCR product), Lane PC is a PCR system positive control (ie, the template is pShuttle-E1A plasmid DNA containing the fragment of interest).
  • Figure 6 shows the results of BglII digestion of the pShuttle-MCS-E1A candidate plasmid; wherein lane M is a DNA molecular weight marker, and samples 1-3 are candidate plasmids, two lanes of each sample, respectively: lane N is uncut The candidate plasmid, Lane B, is a candidate plasmid after BglII digestion.
  • Figure 7 shows the construction process of the pShuttle-MCS-E1A plasmid and the map of the constructed plasmid.
  • Figure 8 shows inhibition of human PDL1 mRNA in U251 and H460 cells by three shPDL1 of one embodiment of the present invention.
  • the abscissa axis represents four groups of cell samples collected at 24h and 48h after treatment of U251 and H460 cells with four shRNAs.
  • the ordinate indicates the expression level of PDL1 mRNA in cells after treatment with each shRNA and PDL1 in control cells. The ratio of the expression levels of mRNA.
  • Figure 9 shows inhibition of exogenous hPDL1 expression in 293T cells by three shPDL1 of one embodiment of the present invention.
  • the left panel shows the results of Western Blot, which indicates the expression changes of hPDL1 (containing FLAG tag) and the expression of intracellular protein ⁇ -actin ( ⁇ -actin) in cell samples after treatment with different shPDL1 cells.
  • the right panel shows the grayscale scan values of the hPDL1 bands obtained from the protein internal reference ⁇ -actin as a standardized control according to the results of Western Blot.
  • the abscissa indicates the different 293 cell sample groups after shPDL1 treatment, and the “control” refers to only transfection of pcDNA3. .3-hPDL1-FLAG
  • the control group expressing hPDL1 (containing FLAG tag), and the ordinate is the grayscale scan value of the target protein normalized by ⁇ -actin.
  • Figure 10 shows the construction process of the pShuttle-U6-shPDL1-CMV-E1A plasmid and the map of the constructed plasmid.
  • Figure 11 shows the results of restriction enzyme digestion of pShuttle-U6-shPDL1-CMV-E1A plasmid; lane M is a DNA molecular weight marker, and lane C is a KpnI/HindIII digested control plasmid (pShuttle-MCS-E1A), lane 1 -7 is a candidate plasmid after digestion with KpnI/HindIII.
  • Figure 12 is a schematic diagram showing the process of homologous recombination of pShuttle-U6-shPDL1-CMV-E1A plasmid with pAdEasy-1 in BJ5183 bacteria.
  • Figure 13 is a schematic diagram showing homologous recombination between the pShuttle-related plasmid and pAdEasy-1 during construction of the pAdEasy-U6-shPDL1-CMV-E1A plasmid.
  • Figure 14 shows the result of PacI digestion of the constructed positive pAdEasy-U6-shPDL1-CMV-E1A plasmid; wherein lane M is a DNA molecular weight marker, and lanes 1-8 are PacI digestion products of different plasmids, specifically, lanes 1 is C-4.5K PacI digestion product, lane 2 is 1-4.5K PacI digestion product, lane 3 is C-3K PacI digestion product, lane 4 is 1-3K PacI digestion product, lane 5 is a 2-4.5K PacI digestion product, lane 6 is a 3-4.5K PacI digestion product, lane 7 is a 2-3K PacI digestion product, and lane 8 is a 3-3K PacI digestion product.
  • lanes 1 is C-4.5K PacI digestion product
  • lane 2 is 1-4.5K PacI digestion product
  • lane 3 is C-3K PacI digestion product
  • lane 4 is 1-3K PacI digestion product
  • lane 5 is a 2-4.5K PacI digestion product
  • Figure 15 is a schematic diagram showing the process of viral packaging of pAdEasy-U6-shPDL1-CMV-E1A plasmid and pAdEasy-CMV-E1A control plasmid in AD293 cells, respectively.
  • Figure 16 is a schematic view showing the layout of a 12-well plate sample in one embodiment of the present invention. According to the figure, OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K), OAd-shPDL1#3-4.5K (3-4.5K) were selected. The cells were treated with the indicated oncolytic virus OAd-C-4.5K (C-4.5K) at the indicated MOI values. Wherein "NC” refers to a blank control group that has not been subjected to any virus treatment.
  • Figure 17 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed by the present invention in Example 1. Comparison of the replication ability of OAd-shPDL1#3-4.5K (3-4.5K) in different cells, wherein OAd-C-4.5K (C-4.5K) was used as a system control virus.
  • the abscissa indicates the different oncolytic virus groups, and the ordinate is the multiple of the copy number of the specific gene E1A of the oncolytic adenovirus treated with the GAPDH gene in the cells as a standardized control.
  • Figure 18 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed by the present invention in Example 2. And OAd-shPDL1#3-4.5K (3-4.5K)) and system control virus OAd-C-4.5K (C-4.5K) and control group H101 and control paclitaxel (Paclitaxel) on U251 cells.
  • the abscissa indicates the amount of virus infection (in MOI) used to treat cells, and the ordinate is the inhibition rate (%) of cell growth after virus treatment of cells.
  • the left picture shows the results of the 48-hour experiment and the right picture shows the results of the 72-hour experiment. "***" indicates p ⁇ 0.001.
  • Figure 19 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed in accordance with the present invention in Example 2. And OAd-shPDL1#3-4.5K (3-4.5K)) and system control virus OAd-C-4.5K (C-4.5K) and control group H101 and control paclitaxel on A549 cells.
  • the abscissa indicates the amount of virus infection (in MOI) used to treat cells, and the ordinate is the inhibition rate (%) of cell growth after virus treatment of cells.
  • the left picture shows the results of the 48-hour experiment and the right picture shows the results of the 72-hour experiment.
  • Figure 20 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed in accordance with the present invention in Example 2. And OAd-shPDL1#3-4.5K (3-4.5K)) and system control virus OAd-C-4.5K (C-4.5K) and control group H101 and control paclitaxel on Hela cells.
  • the abscissa indicates the amount of virus infection (in MOI) used to treat cells, and the ordinate is the inhibition rate (%) of cell growth after virus treatment of cells.
  • the left picture shows the results of the 48-hour experiment and the right picture shows the results of the 72-hour experiment.
  • FIG. 21 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed by the present invention in Example 2.
  • OAd-shPDL1#1-4.5K (1-4.5K) OAd-shPDL1#2-4.5K (2-4.5K) constructed by the present invention in Example 2.
  • the abscissa indicates different types of tumor cell groups, and the ordinate is the number of viruses (in MOI) that can kill 50% of the corresponding tumor cells when the virus is incubated for 72 hours.
  • Figure 22 shows the oncolytic adenovirus OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed by the present invention in Example 3. And OAd-shPDL1#3-4.5K (3-4.5K)) and system control virus OAd-C-4.5K (C-4.5K) and control group H101 overexpressed in A549/hPD-L1-FLAG cell line The inhibition of hPD-L1.
  • the above figure shows the results of Western blot, which indicates the expression changes of hPDL1 (including FLAG tag) and the expression of ⁇ -actin in the cell samples of cells treated with different virus treatments.
  • the "control” means that no virus treatment has been performed.
  • the blank control group; the lower panel is the grayscale scan value of the hPDL1 band obtained from the protein internal reference ⁇ -actin as a standardized control according to the results of Western blot.
  • the abscissa indicates different groups, and the ordinate is the grayscale scan value.
  • Figure 23 shows the oncolytic adenovirus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed by the present invention in Example 3. And OAd-shPDL1#3-4.5K (3-4.5K)) and control group H101 inhibited overexpression of hPD-L1 in Hela/hPD-L1-FLAG cell lines.
  • the above figure shows the results of Western blot, which indicates the expression changes of hPDL1 (including FLAG tag) and the expression of ⁇ -actin in the cell samples of cells treated with different virus treatments.
  • the "control" means that no virus treatment has been performed.
  • the blank control group; the lower panel is the grayscale scan value of the hPDL1 band obtained from the protein internal reference ⁇ -actin as a standardized control according to the results of Western blot.
  • Figure 24 shows a schematic representation of the p53 and Rb signaling pathways of cells.
  • Figure 25 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed in the present invention in Example 4. And OAd-shPDL1#3-4.5K (3-4.5K)), system control virus OAd-C-4.5K (C-4.5K), control group H101 and control group paclitaxel on HCT116 cells.
  • the abscissa indicates the amount of virus infection (in MOI) used to treat cells, and the ordinate is the inhibition rate (%) of cell growth after virus treatment of cells.
  • Panel A shows the results of the 48-hour experiment and Figure B shows the results of the 72-hour experiment.
  • Figure 26 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed in accordance with the present invention in Example 4. And OAd-shPDL1#3-4.5K (3-4.5K)), system control virus OAd-C-4.5K (C-4.5K), control group H101 and control group paclitaxel on PANC1 cells.
  • the abscissa indicates the amount of virus infection (in MOI) used to treat cells, and the ordinate is the inhibition rate (%) of cell growth after virus treatment of cells.
  • Panel A shows the results of the 48-hour experiment and Figure B shows the results of the 72-hour experiment.
  • Figure 27 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed in accordance with the present invention in Example 4. And OAd-shPDL1#3-4.5K (3-4.5K)), system control virus OAd-C-4.5K (C-4.5K), control group H101 and control group paclitaxel on HT29 cells.
  • the abscissa indicates the amount of virus infection (in MOI) used to treat cells, and the ordinate is the inhibition rate (%) of cell growth after virus treatment of cells.
  • Panel A shows the results of the 48-hour experiment and Figure B shows the results of the 72-hour experiment.
  • OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed by the present invention in Example 4.
  • system control virus OAd-C-4.5K C-4.5K
  • control group H101 and control group paclitaxel on H460 cells.
  • the abscissa indicates the amount of virus infection (in MOI) used to treat cells, and the ordinate is the inhibition rate (%) of cell growth after virus treatment of cells.
  • Panel A shows the results of the 48-hour experiment and Figure B shows the results of the 72-hour experiment.
  • FIG. 29 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed by the present invention in Example 4.
  • OAd-shPDL1 OAd-shPDL1#1-4.5K (1-4.5K)
  • OAd-shPDL1#2-4.5K 2-4.5K
  • the abscissa indicates different types of tumor cell groups, and the ordinate is the number of viruses (in MOI) that can kill 50% of the corresponding tumor cells when the virus is incubated for 72 hours.
  • Figure 30 shows the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 5.
  • OAd-shPDL1#1-4.5K (1-4.5K)
  • OAd-C-4.5K C-4.5K
  • Figure A shows the results of Western blot, showing the expression of hPDL1 in the cell samples and the expression of ⁇ -actin in the cells after treatment with different virus-treated cells.
  • control refers to the blank control group without any virus treatment
  • B is the grayscale scan value of the hPDL1 band obtained by using the protein internal reference ⁇ -actin as a standardized control according to the results of Western blot, the abscissa indicates different groups, and the ordinate is gray scale scan value.
  • Figure 31 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 5.
  • control refers to a blank control group which was not subjected to any virus treatment.
  • the abscissa indicates different groups, and the ordinate indicates the percentage (%) of cells expressing hPDL1.
  • Figure 32 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 5.
  • control refers to a blank control group which was not subjected to any virus treatment.
  • the abscissa indicates different groups, and the ordinate indicates the percentage (%) of cells expressing hPDL1.
  • Figure 33 shows the detection of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5) constructed by the present invention by Western blot in Example 5.
  • control refers to the blank control group without any virus treatment.
  • each sample has three sample wells, and the sample in each sample well comes from three different nudes in the same group. mouse.
  • Figure 34 is a graph showing the grayscale scan values of the hPDL1 bands obtained from the protein internal reference ⁇ -actin as a standardized control according to the results of Western blotting of Figure 33.
  • the abscissa indicates different groups, and the ordinate is the grayscale scan value.
  • the abscissa indicates different groups, and the ordinate indicates the percentage value of the corresponding inhibition rate.
  • the abscissa indicates different groups, and the ordinate indicates the percentage value of the corresponding inhibition rate.
  • the abscissa indicates different groups, and the ordinate indicates the percentage value of the corresponding inhibition rate.
  • Figure 38 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 7.
  • Figure 39 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 7. Changes in T/C after HCT116 tumor-bearing NOD-SCID immunodeficient mice; the gray triangle marked on the abscissa indicates the time point of administration, and the abscissa indicates the time (day) after administration, ordinate Indicates T/C (%).
  • Figure 40 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 7.
  • Figure 41 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 7. Photographs of tumors taken from mice after sacrifice in HCT116 tumor-bearing NOD-SCID immunodeficient mice.
  • Figure 42 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 7.
  • Figure 43 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 7.
  • Figure 44 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 7.
  • Figure 45 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 7. Photographs of tumors taken from mice after sacrifice in HCT116 tumor-bearing BALB/C nude mice.
  • Figure 46 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 8.
  • Figure 47 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 8.
  • Figure 48 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 8.
  • Figure 49 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 8. Photographs of tumors taken from mice after sacrifice in HCT116 tumor-bearing BALB/C nude mice (panel A) and statistical results after weighing of each group of tumors (panel B). In Fig. B, the abscissa indicates different groups, and the ordinate indicates tumor weight (g).
  • Figure 50 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 8.
  • OAd-C-4.5K system control virus
  • Figure 51 shows the treatment of the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in accordance with the present invention in Example 8.
  • the statistical results of the changes in the number of standardized T cells in the tumor, blood and spleen of each group of mice were detected by FACS.
  • Panel A shows the results in the tumor
  • Panel B shows the results in the blood
  • Panel C shows the results in the spleen.
  • the abscissa indicates the different groups set in the experiment, and the ordinate indicates the number of T cells after standardization.
  • 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 present invention proposes to utilize the oncolytic adenovirus to selectively replicate and kill tumor cells in tumor cells, while carrying a coding sequence of an exogenous shRNA capable of inhibiting PDL1 expression in tumor cells, thereby enabling recombinant oncolytic gland
  • the virus synergistically exerts a selective oncolytic effect and enhances the body's anti-tumor immune effect.
  • the inventors of the present invention discovered through experimental research and theoretical discovery that the E1B19K gene, the E1B55K gene, and the coding region of all E3 genes of the oncolytic adenovirus are simultaneously deleted, and the exogenous shRNA is integrated in the genome.
  • the coding sequence can achieve the above synergy well.
  • Adenovirus mainly interferes with the host cell cell cycle through Rb and p53 cell signaling pathways (Fig. 24) (see literature: “Chen Jianfa et al., Advances in oncolytic adenovirus research, cancer prevention research, 2004 , 31(4): 243-245.”).
  • the oncolysis function of oncolytic adenovirus is based on this principle by altering the expression of the host cell cyclin by the adenovirus.
  • the genome of the adenovirus includes four early transcription units (E1, E2, E3, and E4) with regulatory functions and one late transcription unit.
  • E1 is divided into two parts, E1A and E1B. As shown in Figure 24, E1A binds to Rb to release free E2F, and cells enter the S phase from G1 phase.
  • Adenovirus encodes both E1B55k and E1B19k proteins to inhibit p53 and Bax, respectively. The cell division and proliferation are not inhibited by the p53 cell signaling pathway.
  • the adenovirus that removes the E1A gene infects the host cell it cannot encode the E1A protein to release the free E2F, and the G1 phase cells cannot enter the S phase.
  • the E1B gene-depleted adenovirus can produce the E1A protein, the host cell enters the S phase from the G1 phase, but the cells entering the division cycle are also blocked or blocked by the p53 signaling pathway.
  • the adenovirus that removes the E1A or E1B gene cannot replicate and proliferate in host cells with normal Rb and p53 signaling pathways, and only tumor cells with abnormal Rb or p53 signaling pathways can proliferate.
  • the early oncolytic adenovirus Onyx-015 and H101 deleted the expression of E1B55K in the adenovirus (partial or total deletion of the E3 region sequence) to achieve selective replication in p53 mutant tumor cells.
  • the infected cell When such a virus infects a normal host cell, even if it can encode an E1A protein to separate the Rb-E2F conjugate and release free E2F, the infected cell enters the S phase from the G1 phase, but enters the dividing phase of the infected cell due to the inability to encode the p53 inhibitor protein E1B55K. The division of the p53 signaling pathway is blocked or apoptosis, and the intracellular adenovirus cannot be effectively replicated.
  • E1B55K expression does not inhibit the function of wild-type p53
  • the normal expression of E1B19K protein can still inhibit the function of Bax downstream of p53 and make the oncolytic gland.
  • the virus can also replicate in normal cells.
  • both E1B55K and E1B19K genes are deleted at the same time, so that the virus is more selective than the prior art oncolytic adenovirus in tumor cells. Better, the ability to replicate in normal cells is lower and the safety of normal cells is better.
  • PD-L1 (also called PDL1 or B7-H1) belongs to the B7 family and has IgV and IgC-like regions, transmembrane regions, and cytoplasmic regions.
  • the molecule has a broad tissue expression profile and is highly expressed on some tumor cell lines, and many studies have shown that it is associated with the immune escape mechanism of tumors.
  • the microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells, and the expression is broad, and the expressed PD-L1 is beneficial to the occurrence and growth of tumors.
  • PD-L1 expressed by APCs in tumor cells and tumor microenvironment interacts with receptor PD1 on T cells.
  • the PD-1/PD-L1 signaling pathway inhibits the activation of tumor antigen-specific T cells and down-regulates T cell-mediated Tumor immune response.
  • blocking PD-L1/PD-1 signaling pathway can promote the proliferation of tumor antigen-specific T cells, up-regulate the secretion of infiltrating CD8 + T cells IFN- ⁇ and effectively inhibit tumor growth, indicating PD-1/PD Blocking of the -L1 signaling pathway plays an important role in tumor immune responses for the purpose of inducing an immune response.
  • experiments have shown that the selection of anti-PD-L1 monoclonal antibody combined with tumor vaccine for tumor immunotherapy can effectively enhance the immune activation of tumor vaccine and reduce the impact of tumor microenvironment on efficacy.
  • the oncolytic adenovirus of the present invention has a higher oncolytic activity than the modified oncolytic virus genome, and also adds a shRNA that can express shPDL1 (inhibiting PDL1 expression).
  • shPDL1 can efficiently degrade the mRNA of PDL1 in cells to achieve gene silencing, thereby reducing the expression of PDL1 in tumor cells, attenuating the transmission of PD1/PDL1 signaling pathway to T cells, and enhancing the killing effect of T cells on tumors.
  • the oncolytic virus of the present invention can be used as an oncolytic agent alone or as an effective carrier of the coding frame of shPDL1, so that shPDL1 is abundantly expressed along with viral replication, and at the same time exerts the dual functions of viral treatment and gene therapy.
  • the present invention provides an isolated recombinant oncolytic adenovirus, wherein the recombinant oncolytic adenovirus is a selective replication oncolytic adenovirus, and the recombinant oncolytic adenovirus is integrated into a genome capable of being in a tumor cell A coding sequence for an exogenous shRNA that inhibits PDL1 expression.
  • the coding sequence of the exogenous shRNA is as set forth in any one of SEQ ID NOs. 16, 19 and 22.
  • the E1B19K gene, the E1B55K gene, and all E3 region genes are deleted from the genome of the recombinant oncolytic adenovirus.
  • the possible mechanism of action of oncolytic virus into tumor cells leading to tumor cell lysis is: (1) direct cytotoxicity of viral proteins: both death proteins and late proteins produced by adenovirus can effectively mediate tumor cell lysis. (2) Producing an anti-tumor immune response: On the one hand, the virus can play a tumoricidal effect by enhancing the sensitivity of tumor cells to various cytokines, such as adenovirus, which is enhanced by replicating and expressing E1A protein in infected tumor cells.
  • Tumor necrosis factor-mediated tumoricidal effect when tumor cells are infected by virus, the viral antigen on the surface of tumor cells forms a complex with the major histocompatibility complex class I antigen, which is easily cytotoxic T lymphocyte The cells are recognized to mediate specific attacks on virally infected tumor cells.
  • Enhance the sensitivity of tumor cells to chemoradiotherapy The product of adenovirus E1A gene expression is a powerful chemical sensitizer, and the expression product of E1A gene in tumor cells can induce high level expression of p53 protein, and In order to enhance the damage of DNA by chemotherapy and radiotherapy.
  • the genome of the recombinant oncolytic adenovirus comprises an E1A gene coding sequence.
  • the E1A gene coding sequence is under the control of a CMV promoter, thereby enhancing its oncolytic killing effect on tumor cells by increasing the expression of E1A.
  • the recombinant oncolytic adenovirus is obtained by genetically modifying an adenovirus type 5.
  • An example of an adenovirus type 5 is H101.
  • the oncolytic adenovirus genome integrates a coding cassette comprising a U6 promoter and human PDL1 shRNA (shPDL1) after the ES sequence, and includes a CMV promoter, an E1A coding region and a portion thereof 3' end E1A expression cassettes in the UTR region and SV40 polyA.
  • a coding cassette comprising a U6 promoter and human PDL1 shRNA (shPDL1) after the ES sequence, and includes a CMV promoter, an E1A coding region and a portion thereof 3' end E1A expression cassettes in the UTR region and SV40 polyA.
  • the recombinant oncolytic adenovirus of the invention is applicable to various human tumor cells (for example, human glioma cell line U251, human lung cancer cell A549, human cervical cancer cell line Hela, human large cell lung cancer H460, human colorectal cancer cell HCT116, human pancreas Cancer cells PANC1, human colon cancer cells HT29, etc.) have strong killing ability.
  • human tumor cells for example, human glioma cell line U251, human lung cancer cell A549, human cervical cancer cell line Hela, human large cell lung cancer H460, human colorectal cancer cell HCT116, human pancreas Cancer cells PANC1, human colon cancer cells HT29, etc.
  • human shPDL1 expressed by this virus can significantly reduce the level of highly expressed PDL1 protein in human tumor cells.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the recombinant oncolytic adenovirus according to the present invention as an active ingredient, and a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition comprises a therapeutically effective amount of the recombinant oncolytic adenovirus. More preferably, the recombinant oncolytic adenovirus therapeutically effective amount of 5 ⁇ 10 7 -5 ⁇ 10 12 vp / day dose.
  • the oncolytic virus 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 adenovirus should be present independently in the pharmaceutical composition
  • compositions of the present invention may also comprise suitable pharmaceutically acceptable excipients.
  • the present invention provides a vector for the preparation of the recombinant oncolytic adenovirus of the present invention, wherein the vector comprises an exogenous shRNA coding sequence under the control of a promoter, and the shRNA coding sequence is SEQ ID Shown as any of NOs. 16, 19 and 22.
  • the vector employs pShuttle as a basic backbone, and in turn comprises, in the basic backbone, a promoter operably linked to control expression of the exogenous shRNA coding sequence, the exogenous shRNA coding sequence a promoter that controls expression of the E1A gene coding sequence, and the E1A gene coding sequence.
  • Another aspect of the invention also provides a host cell comprising the vector of the invention.
  • the host cell stably expresses the vector.
  • the present invention provides an isolated shRNA, wherein the coding sequence of the shRNA is as shown in any one of SEQ ID NOs. 16, 19 and 22, and the shRNA is capable of inhibiting the expression of PDL1 in tumor cells .
  • Another aspect of the invention also provides the use of a recombinant oncolytic adenovirus of the invention for 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 to a tumor and/or cancer patient a recombinant oncolytic adenovirus according to the invention.
  • 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 adenovirus is administered in a therapeutically effective amount, 1-2 times a day for 1-7 days (including continuous administration for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days).
  • the therapeutically effective amount is preferably a dose of 5 x 10 7 to 5 x 10 12 vp per day (e.g., a dose of 5 x 10 7 to 5 x 10 12 vp per day, a dose of 5 x 10 7 to 1.5 x 10 12 VP per day) 5 x 10 8 to 1 x 10 12 VP/day dose, 1 x 10 9 to 5 x 10 11 VP/day dose, 3 x 10 10 to 3 x 10 11 VP/day dose).
  • a dose of 5 x 10 7 to 5 x 10 12 vp per day e.g., a dose of 5 x 10 7 to 5 x 10 12 vp per day, a dose of 5 x 10 7 to 1.5 x 10 12 VP per day
  • 5 x 10 8 to 1 x 10 12 VP/day dose e.g., a dose of 5 x 10 7 to 5 x 10 12 vp per day, a dose of 5 x 10 7 to 1.5 x 10 12
  • the recombinant oncolytic adenovirus 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 oncolytic virus can be administered by administration in a manner generally employed in the art, for example, by intratumoral injection or intravenous administration.
  • the present invention provides an isolated recombinant oncolytic adenovirus, wherein the recombinant oncolytic adenovirus is a selective replication oncolytic adenovirus, and the E1B19K gene, E1B55K is deleted from the genome of the recombinant oncolytic adenovirus Gene, and all E3 region genes; preferably, the genome of the recombinant oncolytic adenovirus comprises an E1A gene coding sequence; further preferably, the E1A gene coding sequence is under the control of a CMV promoter.
  • the oncolytic adenovirus has strong tumoricidal ability, and its ability to replicate in normal cells is much lower than its ability to replicate in tumor cells, and thus has low toxicity to normal cells and improves safety.
  • the inventors of the present invention have also proposed new combination therapies based on the oncolytic adenovirus described above in combination with systematic thinking.
  • many non-cytotoxic anti-tumor drugs when combined with chemotherapy to treat tumors, patients' long-term survival results are not satisfactory, the reason is the lack of systematic thinking.
  • traditional chemotherapy mainly interferes with RNA or DNA synthesis and mitosis. It is mainly for fast-growing cells. It also attacks the normal immune system of the human body while removing tumor cells. As the body's immunity is destroyed, tumor cells are bound to " look up”.
  • Systematic thinking is based on a holistic view, and comprehensively examines the interactions and interactions between drugs, diseases, systems, and humans.
  • the present invention believes that other methods for improving immunity can be employed, and various treatments can be combined by system to maximize the overall therapeutic effect while minimizing the damage to the immune system.
  • the present invention proposes a novel combination therapy that combines the recombinant oncolytic adenovirus with NK cells to treat tumors and/or cancer.
  • the present invention can produce a synergistic effect only by using the recombinant oncolytic adenovirus in combination with NK cells.
  • the present invention also provides a therapeutic agent comprising:
  • a first pharmaceutical composition wherein the first pharmaceutical composition comprises the recombinant oncolytic adenovirus of the present invention in a first pharmaceutically acceptable carrier;
  • the first pharmaceutical composition and the second pharmaceutical composition are each independently present in the pharmaceutical composition without mixing with each other.
  • 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 active ingredient of the first pharmaceutical composition is the recombinant oncolytic adenovirus, and wherein the active ingredient of the second pharmaceutical composition is the NK cell.
  • the first pharmaceutical composition comprises a therapeutically effective amount of the recombinant oncolytic adenovirus (preferably, the first pharmaceutical composition comprises 5 ⁇ 10 7 -5 ⁇ 10 12 vp / day dose).
  • the recombinant oncolytic adenovirus more preferably comprising a recombinant oncolytic adenovirus at a dose of 5 x 10 7 to 1.5 x 10 12 VP/day, more preferably comprising a dose of 5 x 10 8 to 1 x 10 12 VP per day
  • the recombinant oncolytic adenovirus more preferably comprising 1 x 10 9 to 5 x 10 11 VP/day of the recombinant oncolytic adenovirus, still more preferably comprising 3 x 10 10 to 3 x 10 11 VP/day
  • the present invention also provides a pharmaceutical composition wherein the active ingredient of the pharmaceutical composition comprises the recombinant oncolytic adenovirus and NK cells of the present invention.
  • the active ingredient of the pharmaceutical composition consists of the recombinant oncolytic adenovirus and NK cells.
  • the recombinant oncolytic adenovirus and the NK cells are each independently present in the pharmaceutical composition without mixing with each other.
  • 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 skillfully combines them.
  • the antiviral mechanism of NK cells is equally applicable to tumor cells infected with oncolytic viruses and complements their anti-tumor mechanisms.
  • the combination also makes tumor cells containing oncolytic viruses a specific target for 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 NK cells of the present invention include autologous NK cells and allogeneic NK cells.
  • the NK cells may be 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. Expansion of NK Cells Using Genetically Engineered K562 Feeder Cells. Methods Mol Biol. 2016;1441:167-74 .”). Clinical data confirmed that autologous NK cells, haploidentical NK cells (which belong to allogeneic NK cells), and umbilical cord blood were not toxic and side effects after NK cells were returned to humans, and were not long-term dependent, safe and
  • 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 dose, administration sequence and administration interval of the recombinant oncolytic adenovirus and NK cells, which are crucial, and the determination is crucial.
  • the pharmaceutical composition or therapeutic agent comprises a therapeutically effective amount of the recombinant oncolytic adenovirus (preferably, the pharmaceutical composition or therapeutic agent comprises 5 x 10 7 - 5 x 10 12 vp / day
  • the dose of the recombinant oncolytic adenovirus more preferably comprising 5 x 10 7 to 1.5 x 10 12 VP/day of the recombinant oncolytic adenovirus, more preferably 5 x 10 8 to 1 x 10 12 VP / day
  • the dose of the recombinant oncolytic adenovirus more preferably comprising a dose of 1 x 10 9 to 5 x 10 11 VP per day of the recombinant oncolytic adenovirus, still more preferably comprising 3 x 10 10 to 3 x 10 11 VP / a daily dose of the recombinant oncolytic adenovirus
  • the pharmaceutical composition or therapeutic agent comprises 1 x 10 7 - 1 x 10 10 cells
  • the recombinant oncolytic adenovirus can be administered by administration 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 active ingredients of the pharmaceutical composition or therapeutic agent of the present invention include 5 ⁇ 10 7 to 5 ⁇ 10 12 VP / day dose of the oncolytic adenovirus recombinant (e.g., for 1.5 to 5 ⁇ 10 7 ⁇ 10 12 VP/day dose of the recombinant oncolytic adenovirus, 5 ⁇ 10 8 to 1 ⁇ 10 12 VP/day dose of the recombinant oncolytic adenovirus, 1 ⁇ 10 9 to 5 ⁇ 10 11 VP/day a dose of the recombinant oncolytic adenovirus, a 3 x 10 10 to 3 x 10 11 VP/day dose of the recombinant oncolytic adenovirus, etc.) and a dose of 1 x 107 to 1 x 10 10 cells per day NK cells (for example, 1 ⁇ 10 8 to 5 ⁇ 10 9 cells/day dose of the NK cells, 1 ⁇ 10 9 to 4 ⁇ 10 9 cells/day dose of the NK cells,
  • the pharmaceutical or therapeutic agents of the present invention may also comprise suitable pharmaceutically acceptable excipients.
  • the pharmaceutical composition or therapeutic of the present invention may further comprise other active ingredients known in the art, such as interleukin-2 (IL-2), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon. - ⁇ (IFN- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ) and the like.
  • IL-2 interleukin-2
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • IFN- ⁇ interferon.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • a pharmaceutical or 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 pharmaceutical or therapeutic agents of the present invention can be found in the scientific literature and in the patent literature, for
  • the pharmaceutical composition or therapeutic of the present invention can be used for the treatment of various tumors and/or cancers including, but 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, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer.
  • lung cancer eg, non-small cell lung cancer
  • melanoma melanoma
  • head and neck cancer liver cancer
  • brain cancer colorectal Cancer
  • bladder cancer breast cancer
  • ovarian cancer uterine cancer
  • cervical cancer cervical cancer
  • lymphoma stomach cancer
  • esophageal cancer esophageal cancer
  • kidney cancer esophageal cancer
  • prostate cancer pancreatic cancer
  • leukemia e.g., testicular cancer.
  • the pharmaceutical composition or therapeutic agent of the present invention is administered by first administering the recombinant oncolytic adenovirus to a tumor and/or cancer patient, and then, 18-72 hours after administration of the recombinant oncolytic adenovirus ( For example, 20-70 hours, 22-48 hours, 24-48 hours, 30-48 hours, etc., the NK cells are administered to the tumor and/or cancer patient.
  • 18-72 hours after administration of the recombinant oncolytic adenovirus 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 administration of the first recombinant oncolytic adenovirus 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 adenovirus 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 adenovirus that is most adjacent to it before 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 adenovirus administered immediately before it is 24-48 hours.
  • the recombinant oncolytic adenovirus is administered in a therapeutically effective amount (for example, 5 ⁇ 10 7 - 5 ⁇ 10 12 vp / day, 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP /day, 5 ⁇ 10 8 to 1 ⁇ 10 12 VP / day, 1 ⁇ 10 9 to 5 ⁇ 10 11 VP / day, 3 ⁇ 10 10 to 3 ⁇ 10 11 VP / day), 1-2 times a day, continuous Administration for 1-7 days (eg, once a day, continuous administration for 1-6 days); 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 a day, continuous administration of 1-6 day.
  • a therapeutically effective amount for example, 5 ⁇ 10 7 - 5 ⁇
  • the recombinant oncolytic adenovirus is administered in a therapeutically effective amount (for example, 5 ⁇ 10 7 - 5 ⁇ 10 12 vp / day, 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP/day, 5 ⁇ 10 8 to 1 ⁇ 10 12 VP/day, 1 ⁇ 10 9 to 5 ⁇ 10 11 VP/day, 3 ⁇ 10 10 to 3 ⁇ 10 11 VP/day), once every 2 days, Continuous administration for 2-6 days; 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 every 2 days, continuous administration for 2-6 days.
  • a therapeutically effective amount for example, 5 ⁇ 10 7 - 5 ⁇ 10 12 vp / day, 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP/day, 5 ⁇ 10 8 to
  • administration of the recombinant oncolytic adenovirus and administration of the NK cells may be by intermittent administration (for example, administration of recombinant oncolytic adenovirus on day 1, administration of NK cells on day 2, and administration of recombinant oncolytic adenovirus on day 3, Administration of NK cells for 4 days...
  • sequential administration for example, administration of recombinant oncolytic adenovirus on day 1, administration of recombinant oncolytic adenovirus and NK cells on day 2, and sequential administration on day 3
  • Recombinant oncolytic adenovirus and NK cells sequential administration of recombinant oncolytic adenovirus and NK cells on day 4, etc.
  • other modes of administration eg, first administration of recombinant oncolytic adenovirus, 1-2 times per day
  • Continuous administration for 1-7 days for example, once a day, continuous administration for 1-6 days
  • administration of NK cells at intervals of 18-72 hours, once a day for 1-6 days.
  • the recombinant oncolytic adenovirus is administered first, and the NK cells are administered 18 to 72 hours after the total administration of the recombinant oncolytic adenovirus.
  • the recombinant oncolytic adenovirus is first administered to a tumor and/or cancer patient, the recombinant oncolytic adenovirus being administered at a therapeutically effective amount (eg, 5 x 10 7 -5 ⁇ 10 12 vp, 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP, 5 ⁇ 10 8 to 1 ⁇ 10 12 VP, 1 ⁇ 10 9 to 5 ⁇ 10 11 VP, 3 ⁇ 10 10 to 3 ⁇ 10 11 VP), Administration 1 time; and administering the NK cells to the tumor and/or cancer patient from 18 hours to 72 hours after administration of the recombinant oncolytic adenovirus, the NK cells are administered at a dose of 1 ⁇ 10 7 Up to 1 ⁇ 10 10 cells (for
  • the recombinant oncolytic adenovirus is capable of selective replication in tumor or cancer cells and peaks over time.
  • the inventors of the present invention have found that recombinant retinal adenovirus in tumor cells promotes killing of tumor cells by NK cells after a period of replication. Therefore, the administration interval of the recombinant oncolytic adenovirus and NK cells proposed by the present invention achieves a bimodal overlap of the peaks of action of both.
  • 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.
  • 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.
  • the invention also provides a kit for synergistic combination therapy for treating tumors and/or cancer, comprising a first container containing the recombinant oncolytic adenovirus of the invention and comprising the NK cell of the invention a second container, 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 adenovirus of the invention and the NK cells of the 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.
  • 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.
  • the first container containing the recombinant oncolytic adenovirus comprises a therapeutically effective amount (for example, a dose of 5 x 10 7 - 5 x 10 12 vp / day, a dose of 5 x 10 7 to 1.5 x 10 12 VP / day) , 5 ⁇ 10 8 to 1 ⁇ 10 12 VP / day dose, 1 ⁇ 10 9 to 5 ⁇ 10 11 VP / day dose, 3 ⁇ 10 10 to 3 ⁇ 10 11 VP / day dose) of the recombinant oncolytic gland
  • the virus, and the second container containing the NK cells comprises the NK cells sufficient to provide a dose of 1 x 10 7 - 1 x 10 10 cells per day (e.g., 1 x 10 8 to 5 x 10 9 cells per day dose)
  • the NK cells may be selected from autologous NK cells and allogeneic NK cells.
  • the NK cells may be autologous NK cells obtained by in vitro expansion or allogeneic NK cells obtained by in vitro expansion.
  • the recombinant oncolytic adenovirus can be administered by the respective modes of administration conventionally employed in the art, such as by intratumoral injection or intravenous administration.
  • the NK cells can be administered by administration generally employed in the art, for example, by intravenous administration.
  • 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.
  • 18-72 hours after administration of the recombinant oncolytic adenovirus 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 administration of the first recombinant oncolytic adenovirus 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 adenovirus 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 adenovirus that is most adjacent to it before 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 adenovirus 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.
  • 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.
  • the recombinant oncolytic adenovirus is administered in a therapeutically effective amount (for example, 5 ⁇ 10 7 - 5 ⁇ 10 12 vp / day, 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP /day, 5 ⁇ 10 8 to 1 ⁇ 10 12 VP / day, 1 ⁇ 10 9 to 5 ⁇ 10 11 VP / day, 3 ⁇ 10 10 to 3 ⁇ 10 11 VP / day), 1-2 times a day, continuous Administration for 1-7 days (eg, once a day, continuous administration for 1-6 days); 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 a day, continuous administration of 1-6 day.
  • a therapeutically effective amount for example, 5 ⁇ 10 7 - 5 ⁇
  • the recombinant oncolytic adenovirus is administered in a therapeutically effective amount (for example, 5 ⁇ 10 7 - 5 ⁇ 10 12 vp / day, 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP/day, 5 ⁇ 10 8 to 1 ⁇ 10 12 VP/day, 1 ⁇ 10 9 to 5 ⁇ 10 11 VP/day, 3 ⁇ 10 10 to 3 ⁇ 10 11 VP/day), once every 2 days, Continuous administration for 2-6 days; 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 every 2 days, continuous administration for 2-6 days.
  • a therapeutically effective amount for example, 5 ⁇ 10 7 - 5 ⁇ 10 12 vp / day, 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP/day, 5 ⁇ 10 8 to
  • administration of the recombinant oncolytic adenovirus and administration of the NK cells may be by intermittent administration (for example, administration of recombinant oncolytic adenovirus on day 1, administration of NK cells on day 2, and administration of recombinant oncolytic adenovirus on day 3, Administration of NK cells for 4 days...
  • sequential administration for example, administration of recombinant oncolytic adenovirus on day 1, administration of recombinant oncolytic adenovirus and NK cells on day 2, and sequential administration on day 3
  • Recombinant oncolytic adenovirus and NK cells sequential administration of recombinant oncolytic adenovirus and NK cells on day 4, etc.
  • other modes of administration eg, first administration of recombinant oncolytic adenovirus, 1-2 times per day
  • Continuous administration for 1-7 days for example, once a day, continuous administration for 1-6 days
  • administration of NK cells at intervals of 18-72 hours, once a day for 1-6 days.
  • the recombinant oncolytic adenovirus is administered first, and the NK cells are administered 18 to 72 hours after the total administration of the recombinant oncolytic adenovirus.
  • the recombinant oncolytic adenovirus is first administered to a tumor and/or cancer patient, the recombinant oncolytic adenovirus being administered at a therapeutically effective amount (eg, 5 x 10 7 -5 ⁇ 10 12 vp, 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP, 5 ⁇ 10 8 to 1 ⁇ 10 12 VP, 1 ⁇ 10 9 to 5 ⁇ 10 11 VP, 3 ⁇ 10 10 to 3 ⁇ 10 11 VP), Administration 1 time; and administering the NK cells to the tumor and/or cancer patient from 18 hours to 72 hours after administration of the recombinant oncolytic adenovirus, the NK cells are administered at a dose of 1 ⁇ 10 7 Up to 1 ⁇ 10 10 cells (for
  • 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.
  • the NK cells may be autologous NK cells obtained by in vitro expansion or allogeneic NK cells obtained by in vitro expansion.
  • the recombinant oncolytic adenovirus can be administered by the respective modes of administration conventionally employed in the art, such as by intratumoral injection or intravenous administration.
  • the 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.
  • Cells AD293, MRC-5, Hela, A549, U251, HCT116, PANC1, HT29, H460, MDA-MB-231 were purchased from ATCC; HUVEC was purchased from Aussell Biotechnology (Shanghai) Co., Ltd.
  • Oncolytic adenovirus H101 was purchased from Shanghai 3D Biotechnology Co., Ltd.
  • 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 mouse was purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • 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.
  • 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 light blue, living cells refused to stain. The number of viable cells was taken as the final data.
  • 6-well cell culture plates (2 ml per well) in each case, 12-well cell culture plates (1 ml per well), 24-well cell culture plates (500 ⁇ l per well), 96-well cell culture Plates (100 ⁇ l per well culture volume) were obtained from Corning.
  • the segment sequence includes the coding region of the E1A gene (excluding the E1A promoter sequence) and a portion of the 3' UTR region.
  • the obtained PCR product was digested with BglII and cloned between the BglII and EcoRV sites in the multiple cloning site (MCS) of the vector pShuttle-CMV (purchased from Agilent) to obtain the intermediate vector pShuttle-E1A.
  • pShuttle-E1A positive clones were confirmed by PCR screening using P1 and P2, and the results are shown in Fig. 2, and the construction process is shown in Fig. 3. Positive clones were sequenced and the sequencing results were identical to the corresponding sequences on the AD5 genomic DNA.
  • PCR primers P3 and P4 (P3: CGC GTCGAC TACTGTAATAGTAATCAATTACGG ( SEQ ID No.3) , and P4: GAC GTCGAC TAAGATACATTGATGAGTTTGGAC (SEQ ID No.4); NOTE: 5 'end of the two primers are added to the SalI restriction site Point, underlined, and obtained the pShuttle-E1A positive clone as a template for high-fidelity PCR amplification.
  • the PCR product contains the C1 promoter, E1A gene fragment and SV40polyA, and the PCR product size is 2017bp (Fig. 4).
  • the obtained PCR product of the E1A expression cassette was subjected to SalI digestion and cloned into the SalI site in the MCS region of the pShuttle vector (purchased from Agilent), and the positive clones inserted into the E1A expression cassette were screened by PCR using P3 and P4 primers.
  • Figure 5 and confirmed by BglII digestion, the clone inserted into the E1A expression cassette will produce 7200 bp and 1400 bp fragments after BglII digestion, and the reverse insertion of the E1A expression cassette will be 7970 bp after BglII digestion.
  • two fragments of 630 bp (Fig. 6)
  • the #2 plasmid of Fig. 6 was selected for subsequent experiments.
  • the intermediate vector pShuttle-MCS-E1A was finally obtained, and the construction process is shown in Fig. 7.
  • the obtained pShuttle-MCS-E1A positive clones were sequenced and the results
  • shPDL1-1 Three shRNA sequences (shPDL1-1) were designed based on the human PDL1 variant1 sequence in Genbank on the NCBI website (ACCESSION: NM_014143), targeting the 168-190, 430-452 and 589-611 regions of their coding region mRNA, respectively.
  • Said shPDL1-#1), shPDL1-2 (or shPDL1-#2) and shPDL1-3 (or shPDL1-#3) respectively SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO.
  • a negative control sequence shPDL1-NC not related to human PDL1 mRNA was designed. The sequence is as follows:
  • Synthetic justice sequence (SEQ ID No. 14):
  • Synthetic justice sequence (SEQ ID No. 17):
  • Synthetic justice sequence (SEQ ID No. 20):
  • the four-sequence sequence was ligated between the BbsI and HindIII sites on the DSGU6/GFP/Neo vector (purchased from Shanghai Shenggong Biotechnology Co., Ltd.) using the sticky ends complementary to BbsI and HindIII reserved at both ends of the shRNA sequence.
  • Four vectors that can express shPDL1 pSGU6/GFP/Neo-shPDL1-NC, pSGU6/GFP/Neo-shPDL1-1, pSGU6/GFP/Neo-shPDL1-2, and pSGU6/GFP/Neo-shPDL1-3).
  • the inhibitory effect of shPDL1 on hPDL1 mRNA was examined in U251 and H460 cells.
  • U251 and H460 were first inoculated in a 12-well plate at 2 ⁇ 10 5 cells per well for 12 hours, and each well was transfected with U251 and H460 in a ratio of 4 ⁇ l of lipofectamin 2000:1.6 ⁇ g shRNA expression vector DNA, respectively, at 24 hours.
  • Two cell samples were taken at 48 hours, total RNA was extracted and reverse transcription was performed, and the expression level of human PDL1 mRNA in the cells was detected by Real-time PCR using the mRNA level of GAPDH gene as a control.
  • the results showed that shPDL1-# was compared with the control. 1, 2, and 3 all produced inhibition of hPDL1 mRNA in a certain period of time, and shPDL1-#1 had the most significant inhibitory effect on hPDL1 mRNA (Fig. 8).
  • the above pcDNA3.3-hPDL1-3 ⁇ FLAG plasmid was constructed as follows: First, two primers (P11: CGCGTCGACATGAGGATATTTGCTGTCTTTAT (SEQ ID No. 11), P12:CCGCTCGAGCGTCTCCTCCAAATGTATCAC (SEQ ID No.) were designed based on the mRNA sequence of the human PDL1 gene in NCBI.
  • RT-PCR was used to obtain hPDL1 cDNA and cloned into pShuttle-IRES-hrGFP-1 vector (purchased from Agilent) to make hPDL1 and downstream
  • the FLAG tag was fused to express the intermediate vector pShuttle-HPDL1-IRES-hrGFP-1.
  • a primer (P15: CGCCTATTACACCCACTCGTGCAG (SEQ ID No.
  • the coding cassette including the entire sequence of the U6 promoter and shPDL1 was cloned into pShuttle-MCS-E1A.
  • the pSGU6/GFP/Neo-shPDL1 vector was digested with SacI, and then the sticky end formed by SacI digestion was digested with T4 DNA polymerase, then subjected to ethanol/ammonium acetate precipitation recovery, and finally recovered by KpnI digestion.
  • BJ5183 (transfected into pAdEasy-1 plasmid) stored in our laboratory at -80 °C was inoculated into LB/Amp medium, and cultured at 37 ° C, 200 RPM overnight.
  • BJ5183 competent bacteria were prepared using Supercompetent Cell Preparation Kit (B529303-0040) of Shenggong Bioengineering Co., Ltd., and 100 ⁇ l of each tube was dispensed and stored at -80 °C until use.
  • the digested product was directly added to 100 ⁇ l of BJ5183 competent bacteria for routine transformation experiments, and finally the transformed bacterial solution was spread on Kana-resistant LB plates and cultured overnight at 37 °C. On the next day, the colonies appearing on the plate were picked and inoculated into LB/Kana. After overnight culture, the plasmid DNA was extracted and subjected to conventional PacI digestion, and the digested product was subjected to electrophoresis analysis. Three different ways of homologous recombination may occur depending on the location at which homologous recombination may occur (Fig. 13).
  • Plasmids that can produce 4.5Kb or 3Kb fragments after PacI digestion are plasmids in which correct homologous recombination occurs.
  • the electrophoresis results of the digested products proved: pShuttle-MCS-E1A, pShuttle-U6-shPDL1-1-CMV-E1A, pShuttle-U6-shPDL1-2-CMV-E1A and pShuttle-U6-shPDL1-3-
  • the correct homologous recombination between CMV-E1A and pAdEasy-1 plasmids successfully obtained the genomic DNA of packaged oncolytic adenovirus (collectively referred to as OAd-shPDL1) and its control virus (pAdEasy-U6-shPDL1#1-CMV- E1A, pAdEasy-U6-shPDL1#2-CMV-E1A, p
  • AD293 cells in good growth condition were inoculated into a 6-well plate one day in advance, and the cell coverage was preferably 60-70% in the transfection experiment on the second day.
  • the cytopathic condition was observed every 2-3 days.
  • the small cells become “beaded” the range is gradually expanded until the cells are largely detached, and the cells can be gently beaten to recover the cells and the culture supernatant, and stored in a -80 ° C refrigerator or directly expanded further, as shown in FIG.
  • the AD293 cells in good growth state were also inoculated into a 6 cm cell culture dish one day earlier, and the cell coverage was preferably 70-80% in the transfection experiment on the second day.
  • Each 6 cm cell culture dish was added to the previously collected virus supernatant 800-1000 ul, and the mixture was also cross-mixed and returned to the cell culture medium to continue the culture.
  • the cells are largely rounded and detached, and at this time, the cells and the culture supernatant can be collected.
  • the cell density is also about 70%.
  • Each 10cm cell culture dish is added to the previously collected virus supernatant 1200-1500 ⁇ l, and the cells are visible after 48 hours. A large number of lesions fall off and the cells and supernatant are recovered. Finally, the virus was amplified in a 15 cm culture dish, and when the cell density was about 70%, 2 ml of the virus culture supernatant collected in a 10 cm culture dish was added, and after mixing, the cells were cultured for 48 hours to collect the cells and the culture supernatant. The virus can then be cycled in a 15 cm culture dish to the desired amount of virus.
  • the methods for determining the titer of adenovirus include VP method, GTU/BFU method, plaque method, TCID50 method and Hexon staining counting (kit) method.
  • the more accurate and high repetition rate methods are the TCID50 method and the Hexon staining method.
  • the number of active virus particles (unit: PFU/ml) in the obtained virus supernatant was measured by Hexon staining counting method. The result is as follows.
  • the three pShuttle-U6-shPDL1-CMV-E1A plasmids described in the present invention and one control plasmid pShuttle-MCS-E1A were homologously recombined with the pAdEasy-1 plasmid to generate six pAdEasy-U6-shPDL1-CMV-E1A plasmids and 2 pAdEasy-CMV-E1A (see Figure 13 for each plasmid to generate two correct ways of homologous recombination, and to obtain two correct plasmids, which can be cleaved by PacI to produce 4.5K or 3K bands, respectively).
  • the virus was packaged in AD293, and 8 oncolytic viruses were obtained: OAd-C-4.5K, OAd-C-3K, OAd-shPDL1#1-4.5K, OAd-shPDL1#1 -3K, OAd-shPDL2#1-4.5K, OAd-shPDL1#2-3K, OAd-shPDL1#3-4.5K and OAd-shPDL1#3-3K, 8 viruses are respectively written in short: C-4.5K, C-3K, 1-4.5K, 1-3K, 2-4.5K, 2-3K, 3-4.5K and 3-3K.
  • C-4.5K and C-3K are the same virus with the same sequence
  • 1-4.5K and 1-3K are the same virus with the same sequence
  • 2-4.5K and 2-3K are the same virus with the same sequence
  • 3 -4.5K and 3-3K are the same virus with the same sequence.
  • the titer of the virus has been obtained:
  • Example 1 Replication ability of oncolytic adenovirus (OAd-shPDL1) in cells (tumor cells and normal cells)
  • the cells (HUVEC, MRC-5, Hela, A549, and U251) were seeded in a 12-well plate at a dose of 1.5 ⁇ 10 5 cells per well.
  • the medium (HUVEC cells were treated with Allcells-specific medium). Cells and media were purchased from Aussell Biotechnology (Shanghai) Co., Ltd.; MRC-5 cells were cultured using MEM+10% FBS medium; Hela cells were cultured using RPMI1640+10% FBS medium; A549 cells were cultured using DMEM. /F12+10% FBS medium; U251 cells were cultured using MEM + 10% FBS, all of which were purchased from Gibco) in a volume of 1 ml.
  • the medium was aspirated and the cells were rinsed once with PBS, and 500 ⁇ l of the virus suspension (the virus was prepared in Preparation Example 4) was added as shown in Fig. 16 to have a viral infection number (MOI) of 10.
  • MOI viral infection number
  • the virus suspension was aspirated, and the cells were rinsed twice with PBS at this time for 0 hr, one cell sample was taken by trypsinization, and a second cell sample was taken after 48 hr.
  • the results shown in Figure 17 show that the four types of oncolytic adenoviruses (control virus C-4.5K, OAd-shPDL1 viruses 1-4.5K, 2-4.5K and 3-4.5K) constructed in the present invention are among the cells tested.
  • the replication capabilities vary widely.
  • the oncolytic virus of the present invention has a very strong ability to replicate in the detected tumor cells, and also exhibits strong replication ability in the immortalized human embryonic lung fibroblast cell line, but in the human primary cell HUVEC.
  • the ability to replicate is very low, and the ability to replicate in human normal cells is less than that in tumor cell lines or cell lines with tumorigenic tendency (eg, immortalized human embryonic lung fibroblast cell line MRC5). 444 times. Therefore, it can be considered that the oncolytic adenovirus of the present invention has a strong tumor cell bias in selective replication, has higher safety in the future clinical oncolytic virus application, and has a larger virus use amount. space.
  • Example 2 Killing ability of oncolytic adenovirus (OAd-shPDL1) on tumor cells
  • This example detects the killing ability of the oncolytic adenovirus (OAd-shPDL1) of the present invention by CCK8 assay.
  • the cells (U251, Hela and A549) were seeded in 96-well plates in an amount of 1.5 ⁇ 10 3 cells per well.
  • the medium was per well (U251 cell culture was MEM+10% FBS; Hela cell culture was RPMI1640+10%).
  • FBS medium; A549 cells were cultured using DMEM/F12 + 10% FBS medium; all media were purchased from Gibco) and the volume was 100 ⁇ l.
  • the damage of the cells by different viruses was judged according to the obtained light absorption value. ability.
  • the commercial oncolytic adenovirus H101 was used as a control, and the same cells were treated with the same MOI virus number in the experiment, and the light absorption values were simultaneously detected at the same time point.
  • a 1 ⁇ M paclitaxel solution was used as a system positive control.
  • the dose killing effect and half-killing dose (IC 50 ) results of oncolytic adenovirus (OAd-shPDL1) against three tumor cells are shown in Figures 18-21.
  • the oncolytic adenovirus of the present invention (control virus C-4.5K, OAd-shPDL1 virus 1-4.5K, 2-4.5K and 3-4.5K) had significant doses for killing U251, A549 and Hela.
  • the virus of the present invention has a similar killing effect, and the killing of U251 cells is even better than that of H101, and the statistical analysis of the killing effect shows that there is a significant difference.
  • this example deliberately constructed two cell lines that stably express the FLAG-tagged hPDL1: A549/hPDL1-FLAG and Hela /hPDL1-FLAG.
  • the construction process is briefly described as follows:
  • the pcDNA3.3-hPDL1-FLAG vector also referred to as pcDNA3.3-hPDL1-FLAG-IRES-hrGFP vector) obtained according to the method of Preparation Example 2 was transfected into A549 and Hela using lipofectamin 2000.
  • the above treatment was also carried out using H101 and the four oncolytic adenoviruses described in the present invention (the control virus C-4.5K prepared in Preparation Example 4, OAd-shPDL1 virus 1-4.5K, 2-4.5K and 3-4.5K).
  • the control virus C-4.5K prepared in Preparation Example 4 OAd-shPDL1 virus 1-4.5K, 2-4.5K and 3-4.5K.
  • Two cell lines, the viral multiplicity (MOI) was 10, and the cell samples were collected 48 hours after virus infection.
  • Western blot analysis was performed, and the expression level of the FLAG-tagged hPDL1 was detected by Anti-FLAG antibody. The results are shown in Figures 22 and 23.
  • the results of Western blot showed that the expression level of hPDL1 was increased in different degrees after treatment of A549 and Hela cells with oncolytic adenovirus, but it was elevated after treatment of the same cells with three OAd-shPDL1 oncolytic viruses.
  • the expression of hPDL1 was inhibited to varying degrees, especially the inhibition ability of OAd-shPDL1-1 (1-4.5K) was the most obvious, which was confirmed by the previous Q-PCR results and Western blot results after co-transfection.
  • CPE cytopathic effect
  • the amount of virus purified by one ultracentrifugation is about 60-80 15 CM culture dishes (including medium supernatant and infected 293 cells).
  • the collected 293 cells containing the oncolytic virus and the culture supernatant were centrifuged at 3,000 RPM for 30 minutes at 4 ° C, and the subsequent experiments were carried out separately for the supernatant and the precipitate.
  • the supernatant after centrifugation is transferred to another new bottle and can be temporarily stored at 4 ° C for subsequent infection of cells to amplify the virus.
  • the freeze-thaw process was repeated 3-5 times to completely destroy the cell membrane, and the virus was released from the cells to obtain a virus solution. If it is not immediately purified, it can be stored at -20 °C. Before ultracentrifugation purification, the virus solution obtained in this step was thawed in a 37 ° C water bath and centrifuged at room temperature for 10 minutes at a rotational speed of 16000 RPM. The virus-containing supernatant was collected, and the virus supernatant was temporarily stored on ice for cryopreservation.
  • Barium chloride density gradient centrifugation is still the most common method for separating and purifying various viruses. It is mainly based on different types of viruses with different buoyant densities, which are separated from other components in the cell lysate in CsCl solution. After the specific virus-specific band was collected, the cesium chloride was removed by a PD-10 desalting column to finally obtain a purified virus. Very high purity viruses can be obtained using this method.
  • the specific purification procedures are as follows:
  • the virus solution collected by the second ultracentrifugation is added to the empty PD-10 column, and the filtered inside the column is collected by the prepared centrifuge tube according to the volume difference (1.2-1.5 mi/ tube).
  • an adenovirus titration titration kit (Shanghai Yuxiang) is used. Biotechnology Co., Ltd.) detects the precise titer of the virus.
  • the high-quality virus solution purified by ultracentrifugation was dispensed into different clean centrifuge tubes according to the concentration and the experiment, and the date and virus name were marked and stored at -80 ° C for use.
  • Example 4 Killing ability of oncolytic adenovirus (OAd-shPDL1) on tumor cells (HCT116, PANC1, HT29 and H460)
  • OAd-shPDL1 oncolytic adenovirus
  • HCT116 Human tumor cells
  • PANC1 Human tumor cells
  • HT29 Human tumor cells
  • H460 human tumor cells
  • HCT116 cell culture was performed using McCoy's 5A+10% FBS medium
  • PANC-1 cells were cultured in DMEM + 10% FBS medium
  • HT29 cells were cultured in DMEM/F12 + 10% FBS medium
  • H460 cells were cultured in RPMI 1640 + 10% FBS, all media were purchased from Gibco
  • the volume was 100 ⁇ l. .
  • the virus used was the control virus C-4.5K prepared by the method described in Preparation Example 5, OAd-shPDL1 virus 1-4.5K, 2-4.5K and 3, respectively.
  • -4.5K Mixture with serum-free medium (time point is recorded as 0 hr), and the multiplicity of infection (MOI) is 1, 3, 10, 30, 100 and 300, respectively. There are 3 duplicate wells for each infection.
  • MTT solution (purchased from Solite Biotech Co., Ltd.) (5 mg/ml, 0.5% MTT) was added to each well at 48 hr and 72 hr, respectively, and incubation was continued for 4 hr; Culture medium, try to avoid contact and aspirate cells. 150 ⁇ l of DMSO was added to each well, and shaken at a low speed for 10 minutes on a shaker to sufficiently dissolve the crystals, and the absorbance at 490 nm was measured on a microplate reader.
  • the commercial oncolytic adenovirus H101 was used as a control in this experiment. In the experiment, the same MOI virus was used to treat the same cell, and the light absorption value was simultaneously detected at the same time point.
  • a 1 ⁇ M paclitaxel solution was used as a system positive control.
  • Using GraphPad Prism 5.04 for data analysis and generate a dose - response curves, and calculate the IC 50. Inhibition rate calculation formula: percentage inhibition of cell proliferation (IR%) 1 - (OD test - OD blank) ⁇ 100%.
  • IC50 dose killing effect and half-killing dose (IC50) results of oncolytic adenovirus (OAd-shPDL1) on four tumor cells are shown in Figure 25-29.
  • the oncolytic adenovirus prepared by the present invention (control virus C-4.5K, OAd-shPDL1 virus 1-4.5K, 2-4.5K and 3-4.5K) has the killing effect on HCT116, PANC1, HT29 and H460. Significant dose dependence and strong killing ability.
  • the virus of the present invention has a similar killing effect as compared to the commercial oncolytic adenovirus H101. It is expected that the oncolytic adenovirus of the present invention can be used for the treatment of the above-mentioned types of tumors in future clinical applications.
  • This example includes in vitro functional assays performed in cells and in vivo functional assays performed in tumor-bearing mouse models.
  • the oncolytic adenovirus used in the experiment was a control virus C-4.5K and an OAd-shPDL1 virus 1-4.5K (the virus used was prepared by the method described in Preparation Example 5).
  • the cell line used in this part of the experiment was human breast cancer cell line MDA-MB-231 with a high level of human PDL1 expression.
  • cell samples were recovered after trypsinization of the cells.
  • the recovered cell sample was rinsed twice with clean PBS, and the recovered cell pellet was suspended in RIPA buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% deoxygenated with protease inhibitor.
  • RIPA buffer 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% deoxygenated with protease inhibitor.
  • sodium cholate, 0.1% SDS and 1/20 Cocktail protease inhibitors the cells were lysed on ice for 30 minutes, and the cell samples were mixed by shaking or shaking. After centrifugation at 12000 RPM for 5 minutes, the lysed supernatant and cell pellet were separately recovered and stored at -20. °C.
  • the protein level of hPDL1 in the supernatant of the recovered MDA-MB-231 cells was detected by a conventional Western blotting method, and the primary antibody was a rabbit-derived PDL1 antibody (No. NBP2-15791) from Novusbio. Each set of experiments was repeated three times. Western results and grayscale scan values are analyzed in Figure 30.
  • the expression level of hDPL1 in the cells can be significantly changed after treating the MDA-MB-231 cells treated with the OAd-shPDL1 oncolytic virus of the present invention, and the OAd-shPDL1 treatment group (1)
  • the expression level of hPDL1 in MDA-MB-231 cells in the 4.5K group was reduced by about 72.4% compared with the control group, while the expression level of hPDL1 in the cells of the oncolytic virus control group (C-4.5K group) was not. Significant changes have occurred.
  • the experimental results showed that the percentage of cells detecting hPDL1 on the cell membrane decreased by about 5 percentage points compared with the blank control group without any treatment after treatment of MDA-MB-231 cells with the OAd-shPDL1 oncolytic virus of the present invention.
  • the percentage of cells that can detect hPDL1 in the cells of the oncolytic virus control group (C-4.5K group) increased by about 9 percentage points compared with the blank control group without any treatment, which is related to the oncolytic virus in the related literature. Stimulation of tumor cells is consistent with an increase in the expression level of hPDL1 on the tumor surface (see, for example, the following scientific literature: "Dmitriy Zamarin, et al.
  • OAd-shPDL1 oncolytic virus treatment of MDA-MB-231 cells can reduce the percentage of cells in which the oncolytic virus stimulates tumor cells and increases hPDL1 expression to 95% of the percentage of unstimulated cells.
  • shPDL1 expressed in OAd-shPDL1 oncolytic virus does knock down the expression level of hPDL1 in MDA-MB-231 tumor cells and decreases the number of cells expressing hPDL1 on the surface of MDA-MB-231 tumor cells.
  • the results of the experiment showed that the percentage of cells detecting hPDL1 on the cell membrane decreased by about 19.1% compared with the blank control group without any treatment after treatment of HCT116 cells with the OAd-shPDL1 oncolytic virus of the present invention, and the oncolytic virus In the control group (C-4.5K group), the percentage of cells that can detect hPDL1 increased by about 5.3% compared with the blank control group without any treatment, which can be caused by the oncolytic virus stimulating tumor cells in related literature.
  • the expression level of hPDL1 on the tumor surface is increased (see, for example, the following scientific literature: "Dmitriy Zamarin, et al. PD-L1 in tumor microenvironment mediates resistance to oncolytic immunotherapy. J Clin Invest.
  • results shown indicate that the OAd-shPDL1 oncolytic virus treatment of HCT116 cells reduced the percentage of cells in which the oncolytic virus stimulated tumor cells and increased hPDL1 expression to 80.9% of the percentage of unstimulated cells.
  • shPDL1 expressed in OAd-shPDL1 oncolytic virus does knock down the expression level of hPDL1 in HCT116 tumor cells and reduces the number of cells expressing hPDL1 on the surface of HCT116 tumor cells.
  • human colon cancer cells HCT116 were first inoculated subcutaneously in the back of 9 BALB/C nude mice, and the number of cells inoculated into each nude mouse was 5 ⁇ 10 6 . After about 9 days, the cells were randomly divided into 3 groups after subcutaneous tumor formation in nude mice. The first group was treated as a blank control group without any treatment, and the second group was injected with oncolytic tumor in the subcutaneous tumor by intratumoral injection.
  • the control virus C-4.5K, the virus injection amount per mouse was 1 ⁇ 10 9 PFU, the injection volume was 100 ⁇ l, and the third group also injected intratumoral injection of the oncolytic adenovirus 1-4.5K in the subcutaneous tumor.
  • the amount of virus injected per nude mouse was 1 ⁇ 10 9 PFU, and the injection volume was 100 ⁇ l.
  • the injection was performed once a day for 3 days, and the treatment was continued for 3 days on the 4th day.
  • the nude mice were sacrificed to recover the tumor tissue.
  • a portion of all tissue samples were added to the RIPA buffer containing the protease inhibitor (formulation as above), and the tissue protein was extracted by homogenization.
  • the protein expression level of hPDL1 in the tumor tissues was detected by Western blotting. The results are shown in the figure. 33 and Figure 34.
  • the strip scan of the Western result shown in Fig. 33 is converted into a gray value and normalized according to the gray value of the respective housekeeping gene ( ⁇ -actin) to obtain the scatter map of Fig. 34, according to the median value of each It can be seen that after treatment of HCT116 cells subcutaneously inoculated with BALB/C by oncolytic adenovirus control virus (C-4.5), the expression of hPDL1 in this group was up-regulated by 20% compared with the control group without any treatment.
  • hPDL1 in the group treated with the oncolytic adenovirus OAd-shPDL1 treated with BALB/C subcutaneously inoculated HCT116 cells was 25% lower than that of the control virus-treated group, and the expression level was also lower than that without any treatment.
  • the expression of hPDL1 in the group therefore, this result demonstrates that shPDL1 expressed in the oncolytic adenovirus OAd-shPDL1 (1-4.5) does reduce the expression of hPDL1 in human tumor cell transplants in vivo.
  • Example 6 Joint killing experiment of oncolytic adenovirus OAd-shPDL1 and human NK cells on human tumor cell lines
  • the oncolytic adenovirus OAd-shPDL1 of the present invention comprises a shPDL1 expression cassette, which can express shPDL1 in the infected tumor cells, thereby knocking down the expression level of hPDL1 in the tumor cells, thereby reducing the presence of hPDL1 on the surface of the tumor cells.
  • the immunosuppressive effect on immune cell activation caused by the binding of hPDL1 on the surface of tumor cells to PD1 of immune cells (including T cells or NK cells) is eventually attenuated or eliminated.
  • the human tumor is detected by detecting the oncolytic adenovirus OAd-shPDL1 (1-4.5K) (prepared by the method described in Preparation 5) and human NK cells simultaneously at the cellular level Cell killing has a synergistic effect on killing.
  • the synergistic effect of oncolytic adenovirus OAd-shPDL1 (1-4.5K) and human NK cells in the joint killing process was detected by trypan blue staining counting method for human colon cancer cell HCT116 and human lung cancer cell A549, respectively.
  • Trypan blue staining is a classic method of dead/live cell counting. When cells are damaged or die, trypan blue penetrates the denatured cell membrane, binds to and stains the disintegrated DNA, and living cells prevent the dye from entering the cell, thus identifying dead and living cells.
  • the killing dose of human NK cells to HCT116 cells is about 5:1 than NK:HCT116, the killing rate is between 10% and 20%.
  • the killing doses are suitable respectively, which is suitable for joint killing experiments.
  • HCT116 cells were seeded in 24-well plates, and 2 ⁇ 10 4 cells were seeded per well.
  • the CO 2 incubator was further cultured for 6 hr, and then the supernatant containing the virus was aspirated, and washed gently with clean PBS once, and then fresh complete medium (McCoy's 5A + 10% FBS) was added. .
  • the experimental group was 1-4.5K+NK group.
  • the number of viable cells was counted after 24 hr of collection of cells after addition of NK cells by trypan blue staining.
  • a group of HCT116 cells were preserved in the experiment, without the oncolytic adenovirus OAd-shPDL1 (1-4.5K), without NK, as a blank control group; one group added oncolytic adenovirus OAd-shPDL1 at the corresponding time point (1- 4.5K), but no NK was added as a 1-4.5K group; one group added NK at the corresponding time point, but did not add oncolytic adenovirus OAd-shPDL1 (1-4.5K) as the NK group.
  • the control group was subjected to the corresponding liquid exchange operation at the corresponding time. Each group of experiments was repeated three times or more, and the average was taken for statistical analysis.
  • the MOI of the oncolytic adenovirus OAd-shPDL1 (1-4.5K) is 1 and the NK cell-to-target ratio (E:T) is 5:1.
  • the results are shown in Figure 35 (where the X-axis is different, Y The axis is the percentage of the corresponding inhibition rate), which shows a significant synergistic effect of the combination of oncolytic adenovirus OAd-shPDL1 (1-4.5K) and NK cells on HCT116 killing, and the synergistic inhibition rate is about 83%. .
  • the MOI of the oncolytic adenovirus OAd-shPDL1 (1-4.5K) was 3, and the NK cell-to-target ratio (E:T) was 5:1.
  • the results are shown in Figure 36 (where the X-axis is different, Y The axis is the percentage of the corresponding inhibition rate), which shows a significant synergistic effect of the combination of oncolytic adenovirus OAd-shPDL1 (1-4.5K) and NK cells on HCT116 killing, and the synergistic inhibition rate is about 91%. .
  • A549 cells were inoculated into 24-well plates, and 2 ⁇ 10 4 cells were seeded per well.
  • the incubator was further cultured for 6 hr, and then the supernatant containing the virus was aspirated, and washed gently with clean PBS once, and then fresh complete medium (DMEM/F12 + 10% FBS) was added.
  • the experimental group was 1-4.5K+NK group.
  • the number of viable cells was counted after 24 hr of collection of cells after addition of NK cells by trypan blue staining.
  • a group of A549 cells were preserved in the experiment, without the oncolytic adenovirus OAd-shPDL1 (1-4.5K), without NK, as a blank control group; one group added oncolytic adenovirus OAd-shPDL1 at the corresponding time point (1- 4.5K), but no NK was added as a 1-4.5K group; one group added NK at the corresponding time point, but did not add oncolytic adenovirus OAd-shPDL1 (1-4.5K) as the NK group.
  • the control group was subjected to the corresponding liquid exchange operation at the corresponding time. Each group of experiments was repeated three times or more, and the average was taken for statistical analysis.
  • Example 7 In vivo growth inhibition assay of oncolytic adenovirus OAd-shPDL1 on human tumor cells subcutaneously inoculated into immunodeficient mice
  • the NOD-SCID immunodeficient mice were first inoculated subcutaneously (the number of cells subcutaneously inoculated into each mouse was 5 ⁇ 10 6 cells).
  • the human colon cancer cell HCT116 was used to prepare a tumor-bearing mouse animal model for detecting the tumor-bearing gland.
  • the growth inhibitory effect of the virus OAd-shPDL1 (1-4.5K) on HCT116, the oncolytic control virus C-4.5K was used as a negative control virus.
  • the control virus C-4.5K and OAd-shPDL1 virus 1-4.5K used in the experiment were prepared by the method described in Preparation Example 5.
  • mice with subcutaneously inoculated tumor volume were randomly divided into 4 groups, 3 mice in each group.
  • the first group was a control group (blank control), and each mouse was injected with 100 ⁇ l of adenovirus stock solution (ie, 10 mM Tris solution (pH 7.4) containing 1 mM MgCl 2 and 10% glycerol);
  • the second group was Medium dose oncolytic control virus group (C-4.5K (middle)), each mouse was injected with 100 ⁇ l each containing 1 ⁇ 10 8 PFU of C-4.5K virus suspension;
  • the third group was medium dose oncolytic virus group (1-4.5K (middle)), each mouse was injected 100 ⁇ l each containing 1 ⁇ 10 8 PFU of 1-4.5K virus suspension;
  • the fourth group was high dose oncolytic virus group (1-4.5K (high) ))
  • Each mouse was injected with 100 ⁇ l of 1 ⁇ 10 9 PFU of 1-4.5K virus suspension per injection.
  • HCT116 Human colon cancer cells
  • HCT116 were prepared for tumor-bearing mouse model for verification of oncolytic adenovirus OAd-
  • the growth inhibitory effect of shPDL1 (1-4.5K) on HCT116, the oncolytic control virus C-4.5K was used as a negative control virus.
  • Twenty-five tumor-bearing mice with subcutaneously inoculated tumor volume (tumor volume of 90-120 mm 3 ) were randomly divided into 5 groups, 5 mice in each group.
  • the first group was the control group (Control), each mouse was injected with 100 ⁇ l of adenovirus preservation solution each time; the second group was the medium dose oncolytic control virus group (C-4.5K (middle)), each Each injection of 100 ⁇ l of mouse contains 1 ⁇ 10 8 PFU of C-4.5K virus suspension; the third group is low dose oncolytic virus group (1-4.5K (low)), each mouse contains 100 ⁇ l per injection.
  • the oncolytic adenovirus OAd-shPDL1 (1-4.5K) treated the HCT116 tumor cells inoculated subcutaneously in BALB/C nude mice. Both the high-dose group and the middle-dose group showed very good anti-tumor effect. (See Figure 42), in which the relative tumor proliferation rate (T/C%) of the high-dose group reached below 40%, and the relative tumor growth rate (T/C%) of the middle-dose group also reached below 40% in the experiment. (See Figure 43). More importantly, the tumor on the back of the nude mice in the high-dose group showed obvious "oncolytic" phenomenon - the surface was ruptured, and the tumor tissue at the original inoculation site was basically removed after the rupture of the rupture site.
  • Example 8 Verification of growth inhibition of human tumor cell HCT116 subcutaneously inoculated into BALB/C nude mice by oncolytic adenovirus OAd-shPDL1
  • OAd-shPDL1 (1-4.5K) for the growth inhibition of HCT116 cells subcutaneously inoculated into BALB/C nude mice
  • this example expanded the sample size of the animal model to verify OAd-shPDL1 again (1-4.5K). Effective dose.
  • the human colon cancer cell HCT116 was first inoculated into the BALB/C nude mice to prepare a tumor-bearing mouse model. The cell inoculation amount of each nude mouse was 5 ⁇ 10 6 cells.
  • the oncolytic virus C-4.5K was also selected as a negative control virus.
  • mice with subcutaneously inoculated tumor volume were randomly divided into 5 groups, 7 mice in each group.
  • the first group was the control group (Control group), and each mouse was injected with 100 ⁇ l of adenovirus preservation solution (ie, 10 mM Tris solution (pH 7.4) containing 1 mM MgCl 2 and 10% glycerol); the second group was dissolved.
  • adenovirus preservation solution ie, 10 mM Tris solution (pH 7.4) containing 1 mM MgCl 2 and 10% glycerol
  • Tumor control virus medium dose group (C-4.5K (1 ⁇ 10 8 )
  • each mouse was injected with 100 ⁇ l each containing 1 ⁇ 10 8 PFU of C-4.5K virus suspension
  • the third group was oncolytic control virus High dose group (C-4.5K (1 ⁇ 10 9 ))
  • each mouse was injected with 100 ⁇ l of C-4.5K virus suspension containing 1 ⁇ 10 9 PFU each time
  • the fourth group was oncolytic virus 1-4.5K.
  • each mouse was injected with 100 ⁇ l of 1 ⁇ 10 8 PFU of 1-4.5K virus suspension each time; the fifth group was the oncolytic virus high dose group ( 1-4.5K (1 ⁇ 10 9 )), each mouse was injected with 100 ⁇ l of 1 ⁇ 10 9 PFU of 1-4.5K virus suspension per injection.
  • a total of five doses were administered throughout the experiment, administered every other day, and the first dose was started on the day of grouping (denoted as day 0).
  • the tumor diameter was measured twice a week and the body weight of the mice was weighed.
  • the nude mice in the control group and the oncolytic control virus group developed a tumor to reach the end of the experiment.
  • mice All 35 mice were sacrificed and the tumor was weighed. And photographing, together with the blood and spleen of the same mouse, preparing a cell suspension and adding a flow antibody (anti-mouse CD49b antibody and anti-mouse CD3 antibody) for detecting BALB/C nude mouse NK cells and T cells, both All were purchased from ebioscience) and subjected to FACS after staining to analyze changes in the ratio of NK and T cells of tumor cells in tumor, blood and spleen. The experimental results are shown in Fig. 46, Fig. 47, Fig. 48, Fig. 49, Fig. 50 and Fig. 51, respectively.
  • a flow antibody anti-mouse CD49b antibody and anti-mouse CD3 antibody
  • Preparation of blood cell suspension After taking the eyeball of the mouse, the blood was taken and added to the anticoagulation tube and shaken and mixed, and then placed on ice. After centrifugation at 500 ⁇ g for 5 minutes, the supernatant was discarded and red blood cell lysate (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., item number: #122-02) was added and the precipitated cells were thoroughly mixed and reacted at room temperature for 10-15 minutes. After centrifugation at 500 ⁇ g for 5 minutes, the supernatant was discarded and the cell pellet was retained.
  • red blood cell lysate purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., item number: #122-02
  • the cells were again resuspended in PBS containing 1% BSA and centrifuged at 500 ⁇ g for 5 minutes, and then the supernatant was discarded to retain the cell pellet, and the cell pellet was divided into two. One part was not treated as a control, and the other was added to the anti-BALB/C mouse CD3 and CD49b flow-type antibody, mixed and allowed to react at room temperature for 30 minutes in the dark, and then subjected to FACS.
  • mice were dissected and the spleens of the mice were taken and placed in a 1.5 ml centrifuge tube and temporarily stored on ice. 5 ml of a PBS solution containing 1% BSA was added to a clean 6 cm culture dish, and the spleen was wrapped with a nylon mesh to squeeze the spleen in a culture dish to fully release the spleen cells. Centrifuge the cell suspension at 500 ⁇ g for 5 minutes, discard the supernatant, add the red blood cell lysate, mix the precipitated cells thoroughly and react at room temperature for 10-15 minutes, then centrifuge again at 500 ⁇ g for 5 minutes, discard the supernatant and retain the cell pellet.
  • mice were dissected and the subcutaneous tumors were placed and placed in a 1.5 ml centrifuge tube and temporarily stored on ice. Each tumor tissue was cut into an equal volume and placed in another set of centrifuge tubes, and then 500 ⁇ l of collagenase was added. The tumor was cut into small pieces by ophthalmic scissors and reacted at 37 ° C for 30 minutes to add a cell suspension of 500 ⁇ g. After centrifugation for 5 minutes, the supernatant was discarded, and the precipitated cells were thoroughly mixed by erythrocyte lysate and reacted at room temperature for 10-15 minutes.
  • the oncolytic adenovirus OAd-shPDL1 (1-4.5K) treated the HCT116 tumor cells inoculated subcutaneously in BALB/C nude mice. Both the high-dose group and the middle-dose group showed very good anti-tumor effect. (See Figure 46), where the relative tumor proliferation rate (T/C%) of the middle dose group reached 40% and the high dose group reached 40% or less (see Figure 47).
  • T/C% tumor proliferation rate
  • the inhibition of HCT116 growth was observed, and the oncolytic virus 1-4.5K showed stronger.
  • the growth inhibition effect was statistically analyzed for the tumor volume of the two groups, and there was a very significant difference between the two groups.
  • the inhibition of HCT116 growth was compared between the control virus C-4.5K (1 ⁇ 10 9 ) group and the target oncolytic virus 1-4.5K (1 ⁇ 10 9 ) group. Both viruses were subcutaneously inoculated at the initial stage of administration. HCT116 showed very strong growth inhibition, but on the 10th day of the experiment, C-4.5K could not continue to inhibit tumor growth like 1-4.5K, and the nude mice in the C-4.5K (1 ⁇ 10 9 ) group were subcutaneous.
  • the NK cells in the blood of the 1-4.5K oncolytic virus group were twice as high as the NK cells in the blood of the control group, and the NK cells in the tumor tissue were increased by 5 compared with the control group. At times, there was no significant change in NK cells in the spleens of each group of animals (see Figure 50). Analysis of T cells revealed that the proportion of T cells in the blood of each group did not change significantly.
  • the T cells in the spleen and tumor tissues of the 1-4.5K (1 ⁇ 10 8 ) experimental group were C-4.5K (1 ⁇ ). 10 8 ) Significant increases in T cells in the same tissues (see Figure 51).
  • HCT116 subcutaneously inoculated into BALB/C nude mice at the same concentration (1 ⁇ 10 9 ) backbone control virus (C-4.5K) that did not contain the shPDL1 expression cassette showed OAd-shPDL1 only at the initial stage of administration.
  • C-4.5K backbone control virus
  • Oncolytic virus has similar tumor suppressing effects, but does not continue to inhibit tumor growth like OAd-shPDL1 (1-4.5K) after stopping administration.
  • tumor cells will highly express PDL1 or PDL2 ligands on their own surface. The binding of the above ligands to PD-1 leads to the intracellular domain of PD-1.
  • NK, macrophages or dendritic cells may play an important joint killing effect in inhibiting tumor growth (see, for example, the following scientific literature: "Kevin C. Barry, et al.A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments. Nat Med. 2018 Jun 25.doi: 10.1038/s41591-018-0085-8.”).
  • the oncolytic adenovirus OAd-shPDL1 (1-4.5K) differs from its backbone control virus C-4.5K in that it contains an expression cassette that expresses shPDL1, which can express shPDL1 in invading tumor cells, and shPDL1 reduces human tumor cells.
  • shPDL1 The level of hPDL1 in the interior, which in turn attenuates or relieves the immunosuppressive state in the peritumoral environment, ultimately leads to a longer inhibition of tumor growth.
  • hPDL1 expression level in tumor cells caused by shPDL1 expressed by oncolytic adenovirus OAd-shPDL1 (1-4.5K) in human tumor cell HCT116 attenuates and relieves tumor-to-tumor immunity in BALB/C nude mice.
  • the inhibition of cells NK, macrophages or dendritic cells, thus showing a sustained inhibition of the growth of HCT116.

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Abstract

一种包含分离的重组溶瘤腺病毒和NK细胞的治疗剂及应用、药盒、治疗肿瘤和/或癌症的方法。治疗剂包含第一药物组合物，其中该第一药物组合物包含位于第一可药用载体中的重组溶瘤腺病毒；和第二药物组合物，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。重组溶瘤腺病毒为选择复制型溶瘤腺病毒，并且该重组溶瘤腺病毒的基因组中整合有能够在肿瘤细胞中抑制PDL1表达的外源shRNA的编码序列。重组溶瘤腺病毒使其溶瘤杀伤作用和T淋巴细胞的抗肿瘤免疫刺激作用产生协同效果。重组溶瘤腺病毒与NK细胞联用可产生进一步协同杀伤肿瘤的作用。

Description

包含分离的重组溶瘤腺病毒和NK细胞的治疗剂及应用、药盒、治疗肿瘤和/或癌症的方法 技术领域
本发明属于生物技术领域，具体而言，涉及包含分离的重组溶瘤腺病毒和NK细胞的治疗剂及应用、药盒、治疗肿瘤和/或癌症的方法。
背景技术
根据2013年1月世界卫生组织网站公布的人口死亡病因最新报告，2008年全世界约有760万人死于癌症，占全世界死亡总数的13％，而且这个数字每年还在迅猛增长，预计到2030年，这个数字将超过1310万。在我国，恶性肿瘤发病率正以年均3％～5％的速度递增，2002年全国每年恶性肿瘤新发病例达219万，到2020年，每年新发病人数量将会超过300万人。癌症已经成为世界上的头号死因之一。而且肿瘤的治疗已经成为当今医学领域的重点和难点，也越来越受到人们的重视。
肿瘤发生(Tumorigenesis)是一个多因素和多步骤的过程，主要为细胞受到外界致癌因子(包括物理辐射、化学物质和病毒等)的刺激，细胞受到损伤导致细胞内部DNA等遗传物质发生改变，进而引起细胞内部信号转导通路的异常与紊乱，细胞表现为疯狂增殖、抵抗凋亡、停止分化和具备侵袭组织与迁移的能力，最终影响人体重要脏器功能危及生命。目前肿瘤的主要治疗方式包括手术治疗、放射治疗、化学治疗、生物治疗和免疫治疗等。虽然这些治疗手段在一定程度上有助于肿瘤的控制，但还是不能从根本上解决问题。
溶瘤病毒疗法也属于生物治疗的范畴，溶瘤病毒的研究最早可以追溯到上个世纪五十年代。当时发现一名宫颈癌患者在感染狂犬病病毒后肿瘤随之消退。受某些肿瘤患者在感染病毒后出现自发性肿瘤 缓解这一现象的启发，第一波溶瘤病毒研究热潮自此开始。溶瘤病毒是指感染肿瘤细胞后能够选择性地在靶细胞内复制，最终导致肿瘤细胞裂解和死亡的一类病毒。这类病毒依靠其本身的特异性在肿瘤细胞中复制来裂解肿瘤细胞，细胞裂解后释放出来的病毒又可以进一步感染周围的肿瘤细胞，同时对正常细胞和组织则没有破坏作用，或影响较小。溶瘤病毒一般分为二类：一类为野生型病毒和自然变异的弱毒病毒株，这类病毒天然就对某些肿瘤细胞有亲和力，如呼肠孤病毒、新城疫病毒以及自主复制的细小病毒等，这些病毒能够在某些肿瘤细胞中繁殖并裂解细胞，具有天然的特异性溶瘤活性；另一类是对病毒基因组进行改造后，只能在肿瘤细胞内复制的病毒。目前人们已经通过基因工程方法改造了腺病毒、单纯疱疹病毒、流感病毒和人牛痘病毒等。其中腺病毒是溶瘤病毒研究开展相对较早溶瘤机理研究相对较为清楚的，腺病毒中又以5型腺病毒研究得更为清楚。腺病毒发现后不久曾被用来治疗头颈部恶性肿瘤，注射腺病毒后肿瘤有不同程度缩小，但治疗后肿瘤易复发，效果难以持久；直到1996年，Bischoff等首次报道去除部分E1B的重组腺病毒Onyx-015能在p53异常的肿瘤细胞选择性复制引起肿瘤杀伤作用，溶瘤腺病毒研究才再次受到广泛关注并且发展迅速，因此出现了许多新型溶瘤腺病毒种类。我国在2006年就批准上市了上海三维生物技术有限公司研发的一种具有溶瘤作用的重组人5型腺病毒H101(一种删除了E1B55K和E3基因的溶瘤腺病毒)(NDA号为：国药准字S20060027)，可以实现在p53突变型的肿瘤细胞中特异性复制并溶解肿瘤细胞。溶瘤腺病毒日益成为恶性肿瘤治疗的一种新手段。
除此之外，在与肿瘤的斗争过程中肿瘤免疫治疗也是一种非常重要的手段。它主要包括抗体疗法、T细胞疗法和肿瘤疫苗等。抗体被称为癌症的新靶分子“药物”，可通过打靶肿瘤周围的免疫细胞辅助激活效应细胞，促进更有效的抗肿瘤免疫；也可通过补体依赖细胞毒导致肿瘤细胞的杀伤，或通过诱导肿瘤细胞凋亡。T细胞疗法是通过静脉给药，把体外扩增的肿瘤特异性自体T细胞(例如：CAR-T)注入体内的一种疗法。肿瘤疫苗治疗是通过调动机体的免疫系统产生 特异性抗体及效应性T细胞的方法，称为主动特异性免疫治疗。大量的临床实验证明肿瘤免疫治疗在治疗肿瘤的过程中发挥了非常积极的作用，但是肿瘤免疫治疗过程中的最大问题还是肿瘤逃逸。肿瘤的免疫逃逸机制与机体对肿瘤的免疫应答之间存在着极为复杂的关系。肿瘤免疫治疗的过程中早期肿瘤特异性的CD8 +T细胞是激活的，随着肿瘤生长到后期失去了杀伤的功能。通常T细胞的活化除了需要通过APC递呈MHC-抗原肽给抗原特异性T细胞提供第一信号外，还需要一系列协同刺激分子提供第二信号，进而才能使T细胞达到生理活化阈值产生正常的免疫应答。如果缺少共刺激分子提供的第二信号，将会导致T细胞的无反应性或特异性免疫耐受甚至进入凋亡。因此，正性和负性协同刺激信号的调节及两者之间的平衡在机体免疫应答的整个过程中起着重要的调节作用。
目前在肿瘤和/或癌症的免疫治疗中，仍然需要更加有效的治疗方案和由此开发出的药物。
发明内容
为解决上述现有技术中所存在的问题，本发明提供了包含分离的重组溶瘤腺病毒和NK细胞的治疗剂及应用、药盒、治疗肿瘤和/或癌症的方法。
具体而言，本发明提供了：
(1)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重组溶瘤腺病毒；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；
其中所述重组溶瘤腺病毒为选择复制型溶瘤腺病毒，并且该重组溶瘤腺病毒的基因组中整合有能够在肿瘤细胞中抑制PDL1表达的外源shRNA的编码序列。
(2)根据(1)所述的治疗剂，其中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。
(3)根据(1)所述的治疗剂，其中所述第一药物组合物的活性成分为所述重组溶瘤腺病毒，并且其中所述第二药物组合物的活性成分为所述NK细胞。
(4)根据(1)所述的治疗剂，其中所述第一药物组合物包含5×10 7至5×10 12VP/天剂量的所述重组溶瘤腺病毒，并且所述第二药物组合物包含1×10 7至1×10 10个细胞/天剂量的所述NK细胞。
(5)根据(1)所述的治疗剂，其中所述外源shRNA的编码序列如SEQ ID NOs.16、19和22中的任一者所示。
(6)根据(1)所述的治疗剂，其中所述重组溶瘤腺病毒的基因组中缺失了E1B19K基因、E1B55K基因、和全部E3区基因。
(7)根据(1)或(6)所述的治疗剂，其中所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列；优选的是，所述E1A基因编码序列是在CMV启动子控制下的。
(8)根据(1)所述的治疗剂，其中所述重组溶瘤腺病毒是对5型腺病毒进行基因改造而得到的。
(9)根据(1)所述的治疗剂，其中所述NK细胞选自自体NK细胞和异体NK细胞。
(10)根据(9)所述的治疗剂，其中所述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。
(11)根据(1)所述的治疗剂，其中所述重组溶瘤腺病毒配制成通过瘤内注射给药或静脉给药；并且其中所述NK细胞配制成通过静脉给药。
(12)根据(1)所述的治疗剂，其中所述治疗剂由所述第一药物组合物和所述第二药物组合物组成。
(13)根据(1)-(12)中任一项所述的治疗剂在制备用于治疗肿瘤和/或癌症的药物中的应用。
(14)根据(13)所述的应用，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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌、白血病、骨癌、睾丸癌。
(15)一种用于治疗肿瘤和/或癌症的具有协同作用的联合药物的药盒，包括：装有重组溶瘤腺病毒的第一容器和装有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；其中所述重组溶瘤腺病毒为选择复制型溶瘤腺病毒，并且该重组溶瘤腺病毒的基因组中整合有能够在肿瘤细胞中抑制PDL1表达的外源shRNA的编码序列。
(16)根据(15)所述的药盒，其中所述第一容器包含5×10 7至5×10 12VP/天剂量的所述重组溶瘤腺病毒，并且所述第二容器包含1×10 7至1×10 10个细胞/天剂量的所述NK细胞。
(17)根据(15)所述的药盒，其中所述外源shRNA的编码序列如SEQ ID NOs.16、19和22中的任一者所示。
(18)根据(15)所述的药盒，其中所述重组溶瘤腺病毒的基因组中缺失了E1B19K基因、E1B55K基因、和全部E3区基因。
(19)根据(15)或(18)所述的药盒，其中所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列；优选的是，所述E1A基因编码序列是在CMV启动子控制下的。
(20)根据(15)所述的药盒，其中所述重组溶瘤腺病毒是对5型腺病毒进行基因改造而得到的。
(21)根据(15)所述的药盒，其中所述NK细胞选自自体NK细胞和异体NK细胞。
(22)根据(21)所述的药盒，其中所述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。
(23)根据(15)所述的药盒，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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌、白血病、骨癌、睾丸癌。
(24)根据(15)所述的药盒，其中所述重组溶瘤腺病毒配制成通过瘤内注射给药或静脉给药；所述NK细胞配制成通过静脉给药。
(25)一种治疗肿瘤和/或癌症的方法，包括以下依次进行的步骤：
1)对肿瘤和/或癌症患者施用重组溶瘤腺病毒，该重组溶瘤腺病毒能够选择性地在肿瘤细胞中复制；
2)在施用所述重组溶瘤腺病毒之后的第18小时至72小时，对所述肿瘤和/或癌症患者施用NK细胞；
其中所述重组溶瘤腺病毒为选择复制型溶瘤腺病毒，并且该重组溶瘤腺病毒的基因组中整合有能够在肿瘤细胞中抑制PDL1表达的外源shRNA的编码序列。
(26)根据(25)所述的方法，其中所述外源shRNA的编码序列如SEQ ID NOs.16、19和22中的任一者所示。
(27)根据(25)所述的方法，其中所述重组溶瘤腺病毒的基因组中缺失了E1B19K基因、E1B55K基因、和全部E3区基因。
(28)根据(25)或(27)所述的方法，其中所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列；优选的是，所述E1A基因编码序列是在CMV启动子控制下的。
(29)根据(25)所述的方法，其中所述重组溶瘤腺病毒是对5型腺病毒进行基因改造而得到的。
(30)根据(25)所述的方法，其中所述NK细胞选自自体NK细胞和异体NK细胞。
(31)根据(30)所述的方法，其中所述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。
(32)根据(25)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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌、白血病、骨癌、睾丸癌。
(33)根据(25)所述的方法，其中所述重组溶瘤腺病毒的施用剂量为5×10 7至5×10 12VP/天，每天1-2次，连续施用1-7天。
(34)根据(25)所述的方法，其中所述NK细胞的施用剂量为1×10 7至1×10 10个细胞/天剂量，每天1次，连续施用1-6天。
(35)根据(25)所述的方法，其中所述重组溶瘤腺病毒的施用剂量为5×10 7至5×10 12VP/天，每2天1次，连续施用2-6天。
(36)根据(25)所述的方法，其中所述NK细胞的施用剂量为1×10 7至1×10 10个细胞/天剂量，每2天1次，连续施用2-6天。
(37)根据(25)所述的方法，其中所述重组溶瘤腺病毒通过瘤内注射给药或静脉给药；所述NK细胞通过静脉给药。
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腺病毒缺失E1B19K基因、E1B55K基因、和全部E3基因的编码区，同时使其携带外源shRNA的编码序列，从而使所得的重组溶瘤腺病毒能够选择性地在肿瘤细胞中复制，并且表达能够在肿瘤细胞中抑制PDL1表达的shRNA。基于该构思而提供的溶瘤腺病毒具有较强的杀瘤能力，并且在正常细胞中的复制能力远远低于其在肿瘤细胞中的复制能力，因此对正常细胞的毒性小，提高了安全性；并且该病毒所表达的shRNA能够显著降低肿瘤细胞内的PDL1蛋白表达水平，从而降低了肿瘤细胞对T淋巴细胞的免疫抑制，因此增强了T和NK淋巴细胞的抗肿瘤免疫杀伤作用。本发明发现在溶瘤腺病毒中整合所述shRNA编码序列，能够使溶瘤病毒的溶瘤杀伤作用和T或NK淋巴细胞的抗肿瘤免疫刺激作用产生协同效果。
此外，由于本发明的重组溶瘤腺病毒在肿瘤细胞中选择性复制的同时表达能够在肿瘤细胞中抑制PDL1表达的shRNA，因此增强了免疫细胞(包括T淋巴细胞和NK细胞)的抗肿瘤免疫作用，从而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腺病毒能够与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腺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腺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腺病毒的肿瘤细胞成为了NK细胞的特异性靶标。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腺病毒和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，从而达到有效地治疗肿瘤和/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“VP”是指病毒颗粒的个数。
本文所用的术语“VP/kg”是指病毒颗粒数/千克患者体重。
本文所用的术语“TCID50”是指半数组织培养感染剂量(median tissue culture infective dose)，表示使半数组织培养物遭受感染，而发生细胞病变的病毒剂量。
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PCR扩增5型腺病毒的E1A基因的凝胶电泳图；其中泳道M为DNA分子量标记，泳道1为以H101基因组DNA为模板的PCR产物。
图2示出pShuttle-E1A质粒阳性克隆的PCR筛选结果；其中泳道M为DNA分子量标记，泳道1-3为候选克隆。
图3示出pShuttle-E1A质粒的构建过程和所构建的质粒的图谱。
图4示出从pShuttle-E1A质粒PCR扩增E1A表达框的示意图(左图)和凝胶电泳图(右图)；其中泳道M为DNA分子量标记，泳道1-2为PCR产物。
图5示出pShuttle-MCS-E1A候选质粒的PCR筛选结果；其中泳道M为DNA分子量标记，泳道1-13为候选质粒，泳道NC为PCR系统阴性对照(即，模板为水的PCR产物)，泳道PC为PCR系统阳性对照(即，模板为包含目的片段的pShuttle-E1A质粒DNA)。
图6示出pShuttle-MCS-E1A候选质粒的BglII酶切鉴定结果；其中泳道M为DNA分子量标记，样品1-3为候选质粒，每种样品两条泳道，分别为：泳道N为未酶切的候选质粒，泳道B为BglII酶切后的候选质粒。
图7示出pShuttle-MCS-E1A质粒的构建过程和所构建的质粒的图谱。
图8示出本发明的一个实施方案的三种shPDL1对U251和H460细胞中人PDL1 mRNA的抑制情况。横坐标轴表示利用4种shRNA处理U251和H460细胞后分别在24h和48h收取的4组细胞样品，纵坐标表示每种shRNA处理细胞后细胞中PDL1 mRNA的表达水平与对照shRNA处理细胞后其中PDL1 mRNA的表达水平的比率。
图9示出本发明的一个实施方案的三种shPDL1对293T细胞中外源hPDL1表达的抑制情况。左图为蛋白质印迹法(Western Blot)结果，表示不同shPDL1处理细胞后细胞样品中hPDL1(含FLAG标签)的表达变化情况和细胞内的蛋白内参β-actin(β肌动蛋白)的表达情况；右图为根据Western Blot结果，以蛋白内参β-actin作为标准化对照获得的hPDL1条带的灰度扫描值，横坐标表示不同shPDL1 处理后的293细胞样品组，“对照”是指仅转染pcDNA3.3-hPDL1-FLAG表达hPDL1(含FLAG标签)的对照组，纵坐标为利用β-actin标准化后的目的蛋白的灰度扫描值。
图10示出pShuttle-U6-shPDL1-CMV-E1A质粒的构建过程和所构建的质粒的图谱。
图11示出pShuttle-U6-shPDL1-CMV-E1A质粒的酶切鉴定结果；其中泳道M为DNA分子量标记，泳道C为KpnI/HindIII酶切后的对照质粒(pShuttle-MCS-E1A)，泳道1-7为KpnI/HindIII酶切后的候选质粒。
图12示出pShuttle-U6-shPDL1-CMV-E1A质粒在BJ5183细菌中与pAdEasy-1进行同源重组的过程示意图。
图13示出pAdEasy-U6-shPDL1-CMV-E1A质粒构建过程中pShuttle相关质粒与pAdEasy-1之间的同源重组的示意图。
图14示出所构建的阳性pAdEasy-U6-shPDL1-CMV-E1A质粒的PacI酶切鉴定结果；其中泳道M为DNA分子量标记，泳道1-8为不同质粒的PacI酶切产物，具体而言，泳道1为C-4.5K的PacI酶切产物，泳道2为1-4.5K的PacI酶切产物，泳道3为C-3K的PacI酶切产物，泳道4为1-3K的PacI酶切产物，泳道5为2-4.5K的PacI酶切产物，泳道6为3-4.5K的PacI酶切产物，泳道7为2-3K的PacI酶切产物，泳道8为3-3K的PacI酶切产物。
图15示出pAdEasy-U6-shPDL1-CMV-E1A质粒和pAdEasy-CMV-E1A对照质粒分别在AD293细胞中完成病毒包装的过程示意图。
图16示出本发明的一个实施方案中12孔板样品布局示意图。按照图中所示分别选用OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K)、OAd-shPDL1#3-4.5K(3-4.5K)和对照溶瘤病毒OAd-C-4.5K(C-4.5K)以所示MOI值处理细胞。其中“NC”是指未进行任何病毒处理的空白对照组。
图17示出在实施例1中本发明所构建的溶瘤病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K) 和OAd-shPDL1#3-4.5K(3-4.5K))在不同细胞中复制能力的比较结果，其中OAd-C-4.5K(C-4.5K)作为系统对照病毒。横坐标表示不同溶瘤病毒组，纵坐标为利用细胞中GAPDH基因作为标准化对照处理后的溶瘤腺病毒的特异性基因E1A的拷贝数的倍数。
图18示出在实施例2中本发明所构建的溶瘤病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K)和OAd-shPDL1#3-4.5K(3-4.5K))和系统对照病毒OAd-C-4.5K(C-4.5K)以及对照组H101和对照组紫杉醇(Paclitaxel)对U251细胞的杀伤效果。横坐标表示处理细胞所用的不同的病毒感染量(单位为MOI)，纵坐标为病毒处理细胞后对细胞生长的抑制率(％)。左图为48小时实验结果，右图为72小时实验结果。“***”表示p＜0.001。
图19示出在实施例2中本发明所构建的溶瘤病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K)和OAd-shPDL1#3-4.5K(3-4.5K))和系统对照病毒OAd-C-4.5K(C-4.5K)以及对照组H101和对照组紫杉醇对A549细胞的杀伤效果。横坐标表示处理细胞所用的不同的病毒感染量(单位为MOI)，纵坐标为病毒处理细胞后对细胞生长的抑制率(％)。左图为48小时实验结果，右图为72小时实验结果。
图20示出在实施例2中本发明所构建的溶瘤病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K)和OAd-shPDL1#3-4.5K(3-4.5K))和系统对照病毒OAd-C-4.5K(C-4.5K)以及对照组H101和对照组紫杉醇对Hela细胞的杀伤效果。横坐标表示处理细胞所用的不同的病毒感染量(单位为MOI)，纵坐标为病毒处理细胞后对细胞生长的抑制率(％)。左图为48小时实验结果，右图为72小时实验结果。
图21示出在实施例2中本发明所构建的溶瘤病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K)和OAd-shPDL1#3-4.5K(3-4.5K))和系统对照病毒OAd-C-4.5K(C-4.5K)以及对照组H101对不同细胞的IC 50(72h)剂量的比较结果。横坐标表示不同种类的肿瘤细胞组，纵坐标为病毒孵育细胞 72h时能够杀伤50％对应肿瘤细胞所用的病毒数量(单位为MOI)。
图22示出在实施例3中本发明所构建的溶瘤腺病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K)和OAd-shPDL1#3-4.5K(3-4.5K))和系统对照病毒OAd-C-4.5K(C-4.5K)以及对照组H101对A549/hPD-L1-FLAG细胞株中过表达的hPD-L1的抑制情况。上图为Western blot结果，表示不同病毒处理细胞后细胞样品中hPDL1(含FLAG标签)的表达变化情况和细胞内的蛋白内参β-actin的表达情况，“对照”是指未进行任何病毒处理的空白对照组；下图为根据Western blot结果，以蛋白内参β-actin作为标准化对照获得的hPDL1条带的灰度扫描值。横坐标表示不同组别，纵坐标为灰度扫描值。
图23示出在实施例3中本发明所构建的溶瘤腺病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K)和OAd-shPDL1#3-4.5K(3-4.5K))以及对照组H101对Hela/hPD-L1-FLAG细胞株中过表达的hPD-L1的抑制情况。上图为Western blot结果，表示不同病毒处理细胞后细胞样品中hPDL1(含FLAG标签)的表达变化情况和细胞内的蛋白内参β-actin的表达情况，“对照”是指未进行任何病毒处理的空白对照组；下图为根据Western blot结果，以蛋白内参β-actin作为标准化对照获得的hPDL1条带的灰度扫描值。
图24示出细胞的p53和Rb信号通路示意图。
图25示出在实施例4中本发明所构建的溶瘤病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K)和OAd-shPDL1#3-4.5K(3-4.5K))、系统对照病毒OAd-C-4.5K(C-4.5K)、对照组H101和对照组紫杉醇对HCT116细胞的杀伤效果。横坐标表示处理细胞所用的不同的病毒感染量(单位为MOI)，纵坐标为病毒处理细胞后对细胞生长的抑制率(％)。图A为48小时实验结果，图B为72小时实验结果。
图26示出在实施例4中本发明所构建的溶瘤病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K) 和OAd-shPDL1#3-4.5K(3-4.5K))、系统对照病毒OAd-C-4.5K(C-4.5K)、对照组H101和对照组紫杉醇对PANC1细胞的杀伤效果。横坐标表示处理细胞所用的不同的病毒感染量(单位为MOI)，纵坐标为病毒处理细胞后对细胞生长的抑制率(％)。图A为48小时实验结果，图B为72小时实验结果。
图27示出在实施例4中本发明所构建的溶瘤病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K)和OAd-shPDL1#3-4.5K(3-4.5K))、系统对照病毒OAd-C-4.5K(C-4.5K)、对照组H101和对照组紫杉醇对HT29细胞的杀伤效果。横坐标表示处理细胞所用的不同的病毒感染量(单位为MOI)，纵坐标为病毒处理细胞后对细胞生长的抑制率(％)。图A为48小时实验结果，图B为72小时实验结果。
图28示出在实施例4中本发明所构建的溶瘤病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K)和OAd-shPDL1#3-4.5K(3-4.5K))、系统对照病毒OAd-C-4.5K(C-4.5K)、对照组H101和对照组紫杉醇对H460细胞的杀伤效果。横坐标表示处理细胞所用的不同的病毒感染量(单位为MOI)，纵坐标为病毒处理细胞后对细胞生长的抑制率(％)。图A为48小时实验结果，图B为72小时实验结果。
图29示出在实施例4中本发明所构建的溶瘤病毒OAd-shPDL1(OAd-shPDL1#1-4.5K(1-4.5K)、OAd-shPDL1#2-4.5K(2-4.5K)和OAd-shPDL1#3-4.5K(3-4.5K))和系统对照病毒OAd-C-4.5K(C-4.5K)以及对照组H101对不同细胞的IC 50(72h)剂量的比较结果。横坐标表示不同种类的肿瘤细胞组，纵坐标为病毒孵育细胞72h时能够杀伤50％对应肿瘤细胞所用的病毒数量(单位为MOI)。
图30示出在实施例5中本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)对人乳腺癌细胞MDA-MB-231中表达的hPDL1的抑制情况。图A为Western blot结果，表示不同病毒处理细胞后细胞样品中hPDL1的表达变化情况和细胞内的蛋白内参β-actin的表达情况，“对照” 是指未进行任何病毒处理的空白对照组；图B为根据Western blot结果，以蛋白内参β-actin作为标准化对照获得的hPDL1条带的灰度扫描值，横坐标表示不同组别，纵坐标为灰度扫描值。
图31示出在实施例5中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理MDA-MB-231细胞后，利用FACS检测肿瘤细胞膜上表达hPDL1的细胞的百分率变化；其中“对照”是指未进行任何病毒处理的空白对照组。横坐标表示不同组别，纵坐标表示表达hPDL1的细胞百分率(％)。
图32示出在实施例5中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116细胞后，利用FACS检测细胞膜上表达hPDL1的细胞的百分率变化；其中“对照”是指未进行任何病毒处理的空白对照组。横坐标表示不同组别，纵坐标表示表达hPDL1的细胞百分率(％)。
图33示出在实施例5中用Western blot检测本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)对BALB/C裸鼠背部皮下接种的HCT116细胞中hPDL1表达的hPDL1的抑制情况；图中示出Western blot结果，表示不同病毒处理细胞后细胞样品中hPDL1的表达变化情况和细胞内的蛋白内参β-actin的表达情况，“对照”是指未进行任何病毒处理的空白对照组，图中每组三个上样孔，每个上样孔中的样品来自同一组中三个不同的裸鼠。
图34示出根据图33的Western blot结果，以蛋白内参β-actin作为标准化对照获得的hPDL1条带的灰度扫描值。横坐标表示不同组别，纵坐标为灰度扫描值。
图35示出在实施例6中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)(MOI＝1)和NK细胞(E∶T＝5∶1)联用对HCT116细胞的协同杀伤作用。横坐标表示不同组别，纵坐标表示相应的抑制率的百分比数值。
图36示出在实施例6中用本发明所构建的溶瘤腺病毒 OAd-shPDL1#1-4.5K(1-4.5K)(MOI＝3)和NK细胞(E∶T＝5∶1)联用对HCT116细胞的协同杀伤作用。横坐标表示不同组别，纵坐标表示相应的抑制率的百分比数值。
图37示出在实施例6中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)(MOI＝30)和NK细胞(E∶T＝5∶1)联用对A549细胞的协同杀伤作用。横坐标表示不同组别，纵坐标表示相应的抑制率的百分比数值。
图38示出在实施例7中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤NOD-SCID免疫缺陷鼠后，肿瘤体积的变化情况；图中横坐标上标出的灰色三角形指示给药处理的时间点，横坐标表示给药处理后时间(天)，纵坐标表示肿瘤体积(mm 3)。
图39示出在实施例7中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤NOD-SCID免疫缺陷鼠后，T/C的变化情况；图中横坐标上标出的灰色三角形指示给药处理的时间点，横坐标表示给药处理后时间(天)，纵坐标表示T/C(％)。
图40示出在实施例7中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤NOD-SCID免疫缺陷鼠后，小鼠体重的变化情况；图中横坐标上标出的灰色三角形指示给药处理的时间点，横坐标表示给药处理后时间(天)，纵坐标表示体重(g)。
图41示出在实施例7中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤NOD-SCID免疫缺陷鼠后，从处死后的小鼠体内取出的肿瘤的照片。
图42示出在实施例7中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤BALB/C裸鼠后，肿瘤体积的变化情况；图中横坐标上标出的灰色三角形指示给药处理的时间点，横坐标表示给药处理 后时间(天)，纵坐标表示肿瘤体积(mm 3)。
图43示出在实施例7中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤BALB/C裸鼠后肿瘤相对增殖率(T/C)的变化情况；图中横坐标上标出的灰色三角形指示给药处理的时间点，横坐标表示给药处理后时间(天)，纵坐标表示T/C(％)。
图44示出在实施例7中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤BALB/C裸鼠后，小鼠体重的变化情况；图中横坐标上标出的灰色三角形指示给药处理的时间点，横坐标表示给药处理后时间(天)，纵坐标表示体重(g)。
图45示出在实施例7中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤BALB/C裸鼠后，从处死后的小鼠体内取出的肿瘤的照片。
图46示出在实施例8中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤BALB/C裸鼠后肿瘤体积的变化情况；图中横坐标上标出的灰色三角形指示给药处理的时间点，横坐标表示给药处理后时间(天)，纵坐标表示肿瘤体积(mm 3)。
图47示出在实施例8中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤BALB/C裸鼠后肿瘤相对增殖率(T/C)的变化情况；图中横坐标上标出的灰色三角形指示给药处理的时间点，横坐标表示给药处理后时间(天)，纵坐标表示T/C(％)。
图48示出在实施例8中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤BALB/C裸鼠后小鼠体重的变化情况；图中横坐标上标出的灰色三角形指示给药处理的时间点，横坐标表示给药处理后时间(天)，纵坐标表示体重(g)。
图49示出在实施例8中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤BALB/C裸鼠后，从处死后的小鼠体内取出的肿瘤的照片(图A)和各组肿瘤称重后的统计结果(图B)。图B中横坐标表示不同组别，纵坐标表示肿瘤重量(g)。
图50示出在实施例8中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤BALB/C裸鼠后，利用FACS检测各组小鼠的肿瘤、血液和脾脏中的标准化处理后NK细胞数目变化的统计结果。图A表示肿瘤中的结果，图B表示血液中的结果，图C表示脾脏中的结果。各图中横坐标表示实验中设置的不同组别，纵坐标表示标准化处理后的NK细胞的数目。
图51示出在实施例8中用本发明所构建的溶瘤腺病毒OAd-shPDL1#1-4.5K(1-4.5K)和系统对照病毒OAd-C-4.5K(C-4.5K)处理HCT116荷瘤BALB/C裸鼠后，利用FACS检测各组小鼠的肿瘤、血液和脾脏中的标准化处理后T细胞数目变化的统计结果。图A表示肿瘤中的结果，图B表示血液中的结果，图C表示脾脏中的结果。各图中横坐标表示实验中设置的不同组别，纵坐标表示标准化处理后的T细胞的数目。
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。
人体是一个复杂的系统，它是由呼吸、循环、消化等十大系统组成，这些系统协调配合，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腺病毒选择性地在肿瘤细胞中复制并杀伤肿瘤细胞的特点，同时使其携带能够在肿瘤细胞中抑制PDL1表达的外源shRNA的编码序列，从而使重组的溶瘤腺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，发现同时使溶瘤腺病毒的E1B19K基因、E1B55K基因、和全部E3基因的编码区缺失，并在基因组中整合所述外源shRNA的编码序列，能够很好地实现上述协同作用。
另外，许多种DNA病毒，如腺病毒等都可以改变宿主细胞细胞周期，其原因主要是病毒产生一些蛋白作用于宿主细胞细胞周期调节蛋白，使静止期细胞进入细胞周期以利于病毒DNA的复制。不同病毒影响细胞周期的机制不一，腺病毒主要通过Rb和p53细胞信号通路干扰宿主细胞细胞周期(图24)(参见文献：“陈建发等，溶瘤腺病毒的研究进展，肿瘤防治研究，2004，31(4)：243-245.”)。溶瘤腺病毒的溶瘤功能便是基于此原理通过改变腺病毒的调节宿主细胞细胞周期蛋白的表达来实现的。腺病毒的基因组包括4个具有调节功能的早期转录单位(early transcription unit，即E1、E2、E3和E4)和1个晚期转录单位(late transcription unit)。E1分为E1A和E1B两部分，如图24所示，E1A与Rb结合释放游离E2F，细胞由G1期进入S期；腺病毒同时编码产生E1B55k和E1B19k两种蛋白分别抑制p53和Bax，使宿主细胞的分裂增殖不受p53细胞信号通路所抑制，大量宿主细胞由静止期进入分裂期，腺病毒得以大量复制繁殖。而去除E1A基因的腺病毒感染宿主细胞时不能编码产生E1A蛋白使游离E2F释放，G1期细胞不能进入S期。同样，去除E1B基因的腺病毒即使可以产生E1A蛋白使宿主细胞由G1期进入S期，但进入分裂周期的细胞也会通过p53信号通路发生凋亡或分裂受阻。因此，去除E1A或E1B基因的腺病毒在Rb和p53细胞信号通路正常的宿主细胞无法进行复制增殖，只有在Rb或p53信号通路异常的肿瘤细胞才能 增殖。早期的溶瘤腺病毒Onyx-015和H101都是删除了腺病毒中的E1B55K的表达(E3区序列部分或全部删除)来实现其在p53突变型的肿瘤细胞内选择性复制的。此类病毒感染正常宿主细胞时即使可以编码产生E1A蛋白使Rb-E2F结合物分离释放游离E2F，感染细胞由G1期进入S期，但由于不能编码产生p53抑制蛋白E1B55K，进入分裂期的感染细胞通过p53信号通路发生分裂受阻或细胞凋亡，细胞内的腺病毒不能得到有效复制。在p53信号通路功能异常的细胞中，进入分裂期的感染细胞不会通过p53信号通路发生分裂受阻或细胞凋亡，细胞大量增殖，细胞内腺病毒便大量复制从而使细胞溶解。但是后面的实验也证明了上述两类病毒在正常细胞中的选择性复制并没有预期的那样理想，分析原因可能是因为它们只是删除了E1B55K，但是仍旧保留了E1B19K的正常表达，而根据图24，E1B55K和E1B19K在p53信号通路中发挥着相似的作用，虽然删除了E1B55K的表达不能抑制野生型p53的功能的发挥，但是E1B19K蛋白的正常表达仍能抑制p53下游Bax的功能从而使溶瘤腺病毒在正常细胞中也能完成复制。在本发明描述的溶瘤腺病毒中，除了删除E3区外，也同时删除了E1B55K和E1B19K两个基因，因此该类病毒比现有技术中的溶瘤腺病毒在肿瘤细胞中的选择复制性更好，在正常细胞中的复制能力更低，对正常细胞的安全性更好。
另一方面，PD-L1(也称PDL1或B7-H1)属于B7家族，具有IgV和IgC样区、跨膜区及胞浆区。该分子具有广泛的组织表达谱，在一些肿瘤细胞系上有较高的表达，许多研究均表明其与肿瘤的免疫逃逸机制相关。肿瘤部位的微环境可诱导肿瘤细胞上的PD-L1的表达，且表达广泛，表达的PD-L1有利于肿瘤的发生和生长。肿瘤细胞以及肿瘤微环境中的APCs表达的PD-L1与T细胞上的受体PD1相互作用经PD-1/PD-L1信号通路抑制肿瘤抗原特异性T细胞的活化，下调T细胞介导的肿瘤免疫应答。另外也有研究表明通过阻断PD-L1/PD-1信号通路可以促进肿瘤抗原特异性T细胞的增殖、上调浸润CD8 +T细胞IFN-γ的分泌和有效抑制肿瘤生长，表明PD-1/PD-L1信号通路的阻断在以诱导免疫应答为目的的肿瘤免疫应 答中发挥重要的作用。而且还有实验证明选择抗PD-L1单抗配合肿瘤疫苗进行肿瘤免疫治疗可有效加强肿瘤疫苗的免疫激活作用，减弱肿瘤微环境对疗效的影响。
基于以上的理论研究和探索，本发明的溶瘤腺病毒除了改构溶瘤病毒基因组构成使其具备更高的溶瘤杀伤能力以外，还加入了一个可以表达shPDL1(抑制PDL1表达的shRNA)的编码框，期望借助shPDL1可以高效降解细胞内PDL1的mRNA实现其基因沉默，进而降低肿瘤细胞内PDL1的表达，减弱PD1/PDL1信号通路向T细胞抑制信号的传递，加强T细胞对肿瘤的杀伤作用。因此，本发明的溶瘤病毒既可以单独作为溶瘤剂，又可以作为shPDL1的编码框的有效载体，使shPDL1伴随病毒复制而大量表达，同时发挥病毒治疗和基因治疗的双重功能。
由此，本发明提供了一种分离的重组溶瘤腺病毒，其中该重组溶瘤腺病毒为选择复制型溶瘤腺病毒，并且该重组溶瘤腺病毒的基因组中整合有能够在肿瘤细胞中抑制PDL1表达的外源shRNA的编码序列。
优选地，所述外源shRNA的编码序列如SEQ ID NOs.16、19和22中的任一者所示。
优选地，所述重组溶瘤腺病毒的基因组中缺失了E1B19K基因、E1B55K基因、和全部E3区基因。
溶瘤病毒进入肿瘤细胞后导致肿瘤细胞裂解的可能作用机制还有：(1)病毒蛋白的直接细胞毒作用：如腺病毒产生的死亡蛋白和晚期蛋白都能有效地介导肿瘤细胞裂解。(2)产生抗肿瘤免疫反应：一方面，病毒可通过增强肿瘤细胞对多种细胞因子的敏感性而起到杀瘤作用，如腺病毒通过在感染的肿瘤细胞内复制和表达E1A蛋白，增强肿瘤坏死因子所介导的杀瘤作用；另一方面当肿瘤细胞被病毒感染后，肿瘤细胞表面的病毒抗原与主要组织相容性复合体I类抗原形成复合物，很容易被细胞毒性T淋巴细胞所识别，从而介导对病毒感染的肿瘤细胞的特异性攻击。(3)增强肿瘤细胞对放化疗的敏感性：腺病毒E1A基因表达的产物就是一种强有力的化学增敏剂，在肿瘤细胞中E1A基因的表达产物可诱导p53蛋白的高水平表达，并 以此增强化疗和放疗对DNA的损伤作用。
因此，优选地，所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列。还优选地，所述E1A基因编码序列是在CMV启动子控制下的，从而通过增加E1A的表达来增强其对肿瘤细胞的溶瘤杀伤效果。
优选地，所述重组溶瘤腺病毒是对5型腺病毒进行基因改造而得到的。5型腺病毒的一个例子为H101。
在一个优选实施方案中，所述溶瘤腺病毒基因组在ES序列后整合了包括U6启动子和人PDL1 shRNA(shPDL1)的编码框，以及包括CMV启动子、E1A编码区及其部分3’端UTR区和SV40polyA在内的E1A表达框。
本发明的重组溶瘤腺病毒对多种人肿瘤细胞(例如人神经胶质细胞瘤细胞U251、人肺癌细胞A549、人宫颈癌细胞Hela、人大细胞肺癌H460、人结直肠癌细胞HCT116、人胰腺癌细胞PANC1、人结肠癌细胞HT29等)具有较强的杀伤能力。该病毒在人正常原代细胞中的复制能力远远低于其在人肿瘤细胞中的复制能力(相差约2个数量级)。该病毒所表达的人shPDL1能够显著降低人肿瘤细胞内高表达的PDL1蛋白水平。
基于本发明开发的重组溶瘤腺病毒，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腺病毒，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腺病毒。更优选地，所述重组溶瘤腺病毒的治疗有效量为5×10 7-5×10 12vp/天剂量。
所述溶瘤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，那么所述重组溶瘤腺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，将重组溶瘤腺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。
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腺病毒的载体，其中所述载体包含在启动子控制下的外源shRNA编码序列，该shRNA编码序列如SEQ ID NOs.16、19和22中的任一者所示。
在一个具体实施方案中，所述载体采用pShuttle作为基本骨架，并且在该基本骨架中依次包含可操作地连接的、控制所述外源shRNA编码序列表达的启动子、所述外源shRNA编码序列、控制所述E1A基因编码序列表达的启动子、和所述E1A基因编码序列。
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。优选地，所述宿主细胞稳定表达所述载体。
本发明另一方面还提供了一种分离的shRNA，其中该shRNA的编码序列如SEQ ID NOs.16、19和22中的任一者所示，并且该shRNA能够在肿瘤细胞中抑制PDL1的表达。
本发明另一方面还提供了本发明所述的重组溶瘤腺病毒在制备用于治疗肿瘤和/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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、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治疗肿瘤和/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腺病毒。
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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、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，所述重组溶瘤腺病毒的施用剂量为治疗有效量，每天1-2次，连续施用1-7天(包括连续施用1天、2天、3天、4天、5天、6天或7天)。所述治疗有效量优选为5×10 7-5×10 12vp/天剂量(例如，5×10 7-5×10 12vp/天剂量、5×10 7至1.5×10 12VP/天剂量、5×10 8至1×10 12VP/天剂量、1×10 9至5×10 11VP/天剂量、3×10 10至3×10 11VP/天剂量)。
如果需要，还可以将本发明的重组溶瘤腺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治疗肿瘤和/或癌症的方法。
所述溶瘤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。
本发明另一方面还提供了一种分离的重组溶瘤腺病毒，其中该重组溶瘤腺病毒为选择复制型溶瘤腺病毒，并且该重组溶瘤腺病毒的基因组中缺失了E1B19K基因、E1B55K基因、和全部E3区基因；优选的是，该重组溶瘤腺病毒的基因组中包含E1A基因编码序列；进一步优选的是，所述E1A基因编码序列是在CMV启动子控制下的。
该溶瘤腺病毒具有较强的杀瘤能力，并且在正常细胞中的复制能力远远低于其在肿瘤细胞中的复制能力，因此对正常细胞的毒性小，提高了安全性。
在本发明的另一个方面中，本发明的发明人还在上述溶瘤腺病毒的基础上，结合系统化思维提出了新的联合疗法。当前，很多非细胞毒性抗肿瘤药物，在联合化疗治疗肿瘤时，患者的长期生存结果都不甚理想，其原因是缺少了系统思维的考虑。例如，传统化疗主要是干扰RNA或DNA合成以及有丝分裂等，主要针对生长快速的细胞，在清除肿瘤细胞的同时也打击了人体正常的免疫系统，随着机体免疫力被摧跨，肿瘤细胞势必“抬头”。系统化思维是从整体观出发，把药物作用、疾病、系统和人体的相互联系、相互作用进行综合考察。 在该系统化思维的基础上，本发明认为可以采用其它提高机体免疫的方法，通过系统组合各种治疗手段，以最大可能地提高综合疗效，同时使对免疫系统的伤害最小化。由此，本发明提出了新的联合疗法，将所述重组溶瘤腺病毒与NK细胞联用以治疗肿瘤和/或癌症。特别是，本发明仅将所述重组溶瘤腺病毒与NK细胞联用就能够产生协同效果。
因此，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腺病毒；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。
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药物组合物中而互不混合。
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。
在一些实施方案中，所述第一药物组合物的活性成分为所述重组溶瘤腺病毒，并且其中所述第二药物组合物的活性成分为所述NK细胞。在一些实施方案中，所述第一药物组合物包含治疗有效量的所述重组溶瘤腺病毒(优选地，所述第一药物组合物包含5×10 7-5×10 12vp/天剂量的所述重组溶瘤腺病毒，更优选包含5×10 7至1.5×10 12VP/天剂量的所述重组溶瘤腺病毒，更优选包含5×10 8至1×10 12VP/天剂量的所述重组溶瘤腺病毒，更优选包含1×10 9至5×10 11VP/天剂量的所述重组溶瘤腺病毒，还更优选包含3×10 10至3×10 11VP/天剂量的所述重组溶瘤腺病毒)，并且所述第二药物组合物包含1×10 7至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)。
本发明还提供了一种药物组合物，其中该药物组合物的活性成分包括本发明所述的重组溶瘤腺病毒和NK细胞。优选的是，该药物组合物的活性成分由所述重组溶瘤腺病毒和NK细胞组成。
优选地，所述重组溶瘤腺病毒和所述NK细胞各自独立地存在于所述药物组合物中而互不混合。
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系统，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。
本发明所述的NK细胞包括自体NK细胞和异体NK细胞。所述NK细胞可以为经体外扩增得到的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所述重组溶瘤腺病毒和NK细胞各自的施用剂量、施用顺序和施用间隔，这几点是至关重要的，其决定了所述重组溶瘤腺病毒的抗肿瘤疗效、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。
因此，优选地，所述药物组合物或治疗剂包含治疗有效量的所述重组溶瘤腺病毒(优选地，所述药物组合物或治疗剂包含5×10 7-5×10 12vp/天剂量的所述重组溶瘤腺病毒，更优选包含5×10 7至1.5×10 12VP/天剂量的所述重组溶瘤腺病毒，更优选包含5×10 8至1×10 12VP/天剂量的所述重组溶瘤腺病毒，更优选包含1×10 9至5×10 11VP/天剂量的所述重组溶瘤腺病毒，还更优选包含3×10 10至3×10 11VP/天剂量的所述重组溶瘤腺病毒)，并且所述药物组合物或治疗剂包含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)。
所述重组溶瘤腺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。
在特定的实施方案中，本发明的药物组合物或治疗剂的活性成分包括5×10 7至5×10 12VP/天剂量的所述重组溶瘤腺病毒(例如，5×10 7至1.5×10 12VP/天剂量的所述重组溶瘤腺病毒、5×10 8至1×10 12VP/天剂量的所述重组溶瘤腺病毒、1×10 9至5×10 11VP/天剂量的所述重组溶瘤腺病毒、3×10 10至3×10 11VP/天剂量的所述重组溶瘤腺病毒等)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)；优选的是，该药物组合物或治疗剂的活性成分由5×10 7至5×10 12VP/天剂量的所述重组溶瘤腺病毒(例如，5×10 7至1.5×10 12VP/天剂量的所述重组溶瘤腺病毒、5×10 8至1×10 12VP/天剂量的所述重组溶瘤腺病毒、1×10 9至5×10 11VP/天剂量的所述重组溶瘤腺病毒、3×10 10至3×10 11VP/天剂量的所述重组溶瘤腺病毒等)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。
本领域的技术人员可以理解，本发明的药物组合物或治疗剂还可包含合适的可药用的辅料。
本发明的药物组合物或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，本发明的药物组合物或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”)。本发明的药物组合物或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″)。
本发明的药物组合物或治疗剂可以用于治疗多种肿瘤和/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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌、白血病、骨癌、睾丸癌。
本发明的药物组合物或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腺病毒，然后，在施用所述重组溶瘤腺病毒之后的第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腺病毒之后的第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腺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腺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腺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腺病毒施用的时间间 隔为24-48小时。
在本发明的一个优选实施方案中，所述重组溶瘤腺病毒的施用剂量为治疗有效量(例如，5×10 7-5×10 12vp/天、5×10 7至1.5×10 12VP/天、5×10 8至1×10 12VP/天、1×10 9至5×10 11VP/天、3×10 10至3×10 11VP/天)，每天1-2次，连续施用1-7天(例如每天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，所述重组溶瘤腺病毒的施用剂量为治疗有效量(例如，5×10 7-5×10 12vp/天、5×10 7至1.5×10 12VP/天、5×10 8至1×10 12VP/天、1×10 9至5×10 11VP/天、3×10 10至3×10 11VP/天)，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腺病毒之后的第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。其中重组溶瘤腺病毒的施用和NK细胞的施用可以是间隔给药方式(例如，第1天施用重组溶瘤腺病毒，第2天施用NK细胞，第3天施用重组溶瘤腺病毒，第4天施用NK细胞...以此类推)；或依次给药方式(例如，第1天施用重组溶瘤腺病毒，第2天依次施用重组溶瘤腺病毒和NK细胞，第3天依次施用重组溶瘤腺病毒和NK细胞，第4天依次施用重组溶瘤腺病毒和NK细胞...以此类推)；或其它给药方式(例如首先施用重组溶瘤腺病毒，每天1-2次，连续施用1-7天(例如每天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，首先施用重组溶瘤腺病毒，在重组溶瘤腺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腺病毒，所述重组溶瘤腺病毒的施用剂量为治疗有效量(例如，5×10 7-5×10 12vp、5×10 7 至1.5×10 12VP、5×10 8至1×10 12VP、1×10 9至5×10 11VP、3×10 10至3×10 11VP)，施用1次；并且在施用所述重组溶瘤腺病毒之后的第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(例如，1×10 8至5×10 9个细胞、1×10 9至4×10 9个细胞、1×10 9至3×10 9个细胞)，施用1次。
所述重组溶瘤腺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，肿瘤细胞里的重组溶瘤腺病毒会促进NK细胞对肿瘤细胞的杀伤。因此，本发明提出的所述重组溶瘤腺病毒和NK细胞的施用间隔实现了两者作用峰值的双峰重叠。
本发明还提供了本发明所述的治疗剂在制备用于治疗肿瘤和/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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌、白血病、骨癌、睾丸癌。
本发明还提供了一种用于治疗肿瘤和/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腺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腺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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌、白血病、骨癌、睾丸癌。
优选地，所述装有重组溶瘤腺病毒的第一容器包含治疗有效量(例如，5×10 7-5×10 12vp/天剂量、5×10 7至1.5×10 12VP/天剂量、5 ×10 8至1×10 12VP/天剂量、1×10 9至5×10 11VP/天剂量、3×10 10至3×10 11VP/天剂量)的所述重组溶瘤腺病毒，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NK细胞可选自自体NK细胞和异体NK细胞。所述NK细胞可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。
所述重组溶瘤腺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。
所述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。
本发明另一方面还提供了一种治疗肿瘤和/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腺病毒；
2)在施用所述重组溶瘤腺病毒之后的第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腺病毒之后的第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腺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腺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腺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腺病毒施用的 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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌、白血病、骨癌、睾丸癌。
在本发明的一个优选实施方案中，所述重组溶瘤腺病毒的施用剂量为治疗有效量(例如，5×10 7-5×10 12vp/天、5×10 7至1.5×10 12VP/天、5×10 8至1×10 12VP/天、1×10 9至5×10 11VP/天、3×10 10至3×10 11VP/天)，每天1-2次，连续施用1-7天(例如每天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，所述重组溶瘤腺病毒的施用剂量为治疗有效量(例如，5×10 7-5×10 12vp/天、5×10 7至1.5×10 12VP/天、5×10 8至1×10 12VP/天、1×10 9至5×10 11VP/天、3×10 10至3×10 11VP/天)，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腺病毒之后的第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。其中重组溶瘤腺病毒的施用和NK细胞的施用可以是间隔给药方式(例如，第1天施用重组溶瘤腺病毒，第2天施用NK细胞，第3天施用重组溶瘤腺病毒，第4天施用NK细胞...以此类推)；或依次给药方式(例如，第1天施用重组溶瘤腺病毒，第2天依次施用重组溶瘤腺病毒和NK细胞，第3天依次施用重组溶瘤腺病毒和NK细胞，第4天依次施用重组溶瘤腺病毒和NK细胞...以此类推)；或其它给药方式(例如首先施用重组溶瘤腺病毒，每天1-2次，连续施用1-7天(例如每天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，首先施用重组溶瘤腺病毒，在重组溶瘤腺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腺病毒，所述重组溶瘤腺病毒的施用剂量为治疗有效量(例如，5×10 7-5×10 12vp、5×10 7至1.5×10 12VP、5×10 8至1×10 12VP、1×10 9至5×10 11VP、3×10 10至3×10 11VP)，施用1次；并且在施用所述重组溶瘤腺病毒之后的第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(例如，1×10 8至5×10 9个细胞、1×10 9至4×10 9个细胞、1×10 9至3×10 9个细胞)，施用1次。
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治疗肿瘤和/或癌症的方法。
所述NK细胞可选自自体NK细胞和异体NK细胞。所述NK细胞可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。
所述重组溶瘤腺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。
所述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))。
以下例子中所用材料来源如下：
1.细胞AD293、MRC-5、Hela、A549、U251、HCT116、PANC1、 HT29、H460、MDA-MB-231购自ATCC；HUVEC购自澳赛尔斯生物技术(上海)有限公司。
2.溶瘤腺病毒H101购自上海三维生物技术有限公司。
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.小鼠购自北京维通利华实验动物技术有限公司。
5.PBS配方：8mM Na 2HPO 4、136mM NaCl、2mM KH 2PO 4、2.6mM KCl，pH7.2-7.4。
6.以下例子中所用的细胞计数方法如下：
CCK8法：每孔细胞中加入10μl CCK8溶液，在37℃培养箱培养1～4小时，置摇床上低速震荡5分钟，使结晶物充分溶解并混匀，使用酶标仪检测其在450nm处的吸光值(OD 450)。抑制率计算公式：细胞增殖抑制百分率(IR％)＝1-(OD 450供试品-OD 450空白)/(OD 450阴性对照-OD 450空白)×100％。
MTT法：每孔细胞中加入10μl MTT溶液(5mg/ml)，在37℃培养箱培养4小时，吸弃培养液，每孔加入150μl DMSO，置摇床上低速震荡10分钟，使结晶物充分溶解并混匀，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，死细胞会染成浅蓝色，活细胞拒染。取活细胞数为最终数据。
7.培养板
各例子中采用的6孔细胞培养板(每孔培养基体积为2ml)、12孔细胞培养板(每孔培养体积1ml)、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100μl)均得自Corning公司。
制备例1：E1A基因表达载体构建
根据NCBI(即，美国国立生物技术信息中心，网址：https：//www.ncbi.nlm.nih.gov)的Genbank中的人5型腺病毒(AD5)基因组DNA序列(ACCESSION：AC_000008)设计两条PCR引物(P1：GGA AGATCTGGACTGAAAATGAG(SEQ ID No.1)，P2：TGAGGTCAGATGTAACCAAGATTA(SEQ ID No.2)；注：引物P1的5’端加入了BglII酶切位点，下划线标出)；提取上海三维生物技术有限公司生产的溶瘤病毒(H101)基因组DNA做为模板，进行高保真PCR扩增AD5基因组DNA上551-1714之间1164bp的序列，实际大小为1173bp(见图1)，该段序列包括E1A基因的编码区(不包括E1A启动子序列)和部分的3’UTR区。利用BglII酶切获得的PCR产物，并将其克隆至载体pShuttle-CMV(购自Agilent公司)上多克隆位点区(MCS)中的BglII和EcoRV位点之间，获得中间载体pShuttle-E1A，所得pShuttle-E1A阳性克隆用P1和P2进行PCR筛选确认，结果见图2，构建过程见图3。对获得阳性克隆进行测序，测序结果与AD5基因组DNA上的对应序列完全一致。
再次设计PCR引物P3和P4(P3：CGC GTCGACTACTGTAATAGTAATCAATTACGG(SEQ ID No.3)和P4：GAC GTCGACTAAGATACATTGATGAGTTTGGAC(SEQ ID No.4)；注：两条引物的5’端均加入了SalI酶切位点，下划线标出)，以获得的pShuttle-E1A阳性克隆为模板进行高保真PCR扩增，PCR产物包含CMV启动子、E1A基因片段和SV40polyA在内的E1A表达框，PCR产物大小为2017bp(图4)。
将获得的E1A表达框的PCR产物进行SalI酶切后克隆到pShuttle 载体(购自Agilent公司)上MCS区中的SalI位点中，利用P3和P4引物进行PCR筛选插入E1A表达框的阳性克隆(图5)，并用BglII进行酶切进行确认，E1A表达框正向插入的克隆经BglII酶切后将产生7200bp和1400bp两条片段，E1A表达框反向插入的克隆经BglII酶切后将产生7970bp和630bp两条片段(图6)，选择图6中#2质粒进行后续试验。最终获得中间载体pShuttle-MCS-E1A，构建过程见图7。对获得的pShuttle-MCS-E1A阳性克隆进行测序，结果与预期序列完全一致。
制备例2：shRNA表达载体构建
根据NCBI网站上Genbank中的人PDL1 variant1序列(ACCESSION：NM_014143)设计了分别靶向其编码区mRNA的168-190、430-452和589-611三个区域的三条shRNA序列(shPDL1-1(或称shPDL1-#1)、shPDL1-2(或称shPDL1-#2)和shPDL1-3(或称shPDL1-#3)，分别为SEQ ID NO.16、SEQ ID NO.19、SEQ ID NO.22)，除此之外，还设计了一个与人PDL1mRNA无关的一个阴性对照序列shPDL1-NC。序列如下：
(1)shPDL1-1
合成正义序列(SEQ ID No.14)：
Figure PCTCN2018094263-appb-000001
合成反义序列(SEQ ID No.15)：
Figure PCTCN2018094263-appb-000002
shRNADNA(SEQ ID No.16)：
Figure PCTCN2018094263-appb-000003
(2)shPDL1-2
合成正义序列(SEQ ID No.17)：
Figure PCTCN2018094263-appb-000004
合成反义序列(SEQ ID No.18)：
Figure PCTCN2018094263-appb-000005
shRNA DNA(SEQ ID No.19)：
Figure PCTCN2018094263-appb-000006
合成正义序列(SEQ ID No.20)：
Figure PCTCN2018094263-appb-000007
合成反义序列(SEQ ID No.21)：
Figure PCTCN2018094263-appb-000008
shRNADNA(SEQ ID No.22)：
Figure PCTCN2018094263-appb-000009
(4)shPDL1-NC
合成NC正义序列(SEQ ID No.23)：
Figure PCTCN2018094263-appb-000010
合成NC反义序列(SEQ ID No.24)：
Figure PCTCN2018094263-appb-000011
shNC DNA(SEQ ID No.25)：
Figure PCTCN2018094263-appb-000012
利用shRNA序列两端预留的可以与BbsI和HindIII互补的粘性末端将四段序列连接到DSGU6/GFP/Neo载体(购自上海生工生物技术有限公司)上的BbsI和HindIII位点之间获得四个可以表达shPDL1的载本(pSGU6/GFP/Neo-shPDL1-NC、pSGU6/GFP/Neo-shPDL1-1、pSGU6/GFP/Neo-shPDL1-2和pSGU6/GFP/Neo-shPDL1-3)。
试验例1：shPDL1的抑制效果检测
在U251和H460细胞中检测shPDL1对hPDL1 mRNA(人PDL1mRNA)的抑制效果。首先在12孔板中以每孔2×10 5个细胞提前12小时接种U251和H460，每个孔以4μl lipofectamin 2000∶1.6μg shRNA表达载体DNA的比例关系转染U251和H460，分别在24小时和48小时收取两份细胞样品，提取总RNA并进行反转录后以GAPDH基因的mRNA水平作为对照进行Real-time PCR检测细胞中人PDL1 mRNA的表达水平，结果显示与对照相比shPDL1-#1、2、3均在一定时间内产生对hPDL1 mRNA的抑制作用，其中shPDL1-#1对hPDL1 mRNA的抑制效果最显著(图8)。
另外也在蛋白水平上对三个shPDL1的抑制效果进行了检测。将4个质粒pSGU6/GFP/Neo-shPDL1-NC、pSGU6/GFP/Neo-shPDL1-1、pSGU6/GFP/Neo-shPDL1-2和pSGU6/GFP/Neo-shPDL1-3分别与pcDNA3.3-hPDL1-3×FLAG等摩尔比(1∶1)瞬间转染293T细胞，其中质粒pcDNA3.3-hPDL1-3×FLAG可以表达融合了3×FLAG标签的 人PDL1蛋白。48小时后收取细胞样品，裂解后进行Western blot分析，结果(见图9)证明shPDL1-#1能够显著降低过表达的hPDL1。
上述pcDNA3.3-hPDL1-3×FLAG质粒的构建过程如下：首先根据NCBI中人PDL1基因的mRNA序列设计两条引物(P11：CGCGTCGACATGAGGATATTTGCTGTCTTTAT(SEQ ID No.11)，P12：CCGCTCGAGCGTCTCCTCCAAATGTGTATCAC(SEQ ID No.12))，利用Trizol提取U251细胞的总RNA，以该RNA为模板进行RT-PCR获得hPDL1cDNA并将其克隆至pShuttle-IRES-hrGFP-1载体(购自Agilent公司)中，使hPDL1与下游的FLAG标签融合表达，获得中间载体pShuttle-HPDL1-IRES-hrGFP-1。再次设计一条引物(P15：CGCCTATTACACCCACTCGTGCAG(SEQ ID No.13))与P11联合扩增pShuttle-hPDL1-IRES-hrGFP-1上的一段包括hPDL1cDNA-FLAG-IRES-hrGFP的序列，将该段片段克隆至pcDNA3.3-TOPO载体(购自Invitrogen公司)上，最后获得pcDNA3.3-hPDL1-FLAG载体。插入的片段经过测序确认序列完全正确。
制备例3：溶瘤腺病毒(OAd-shPDL1)的基因组DNA的制备
1.在确定了shPDL1的抑制效果后，将包括U6启动子和shPDL1全部序列在内的编码框克隆到pShuttle-MCS-E1A中。首先利用SacI酶切pSGU6/GFP/Neo-shPDL1载体，然后利用T4DNA聚合酶将SacI酶切后形成的粘性末端进行切平处理后进行乙醇/乙酸铵沉淀回收，最后再用KpnI酶切后回收包括U6启动子和shPDL1序列在内的编码框序列；同时利用KpnI和EcoRV双酶切pShuttle-MCS-E1A载体并回收，最后将3个shPDL1编码框分别连接到pShuttle-MCS-E1A载体上得到最终的载体pShuttle-U6-shPDL1-CMV-E1A，过程见图10。选取几个菌落提取质粒后进行KpnI/HindIII酶切鉴定，正确克隆将产生一条大小为370bp的条带(见图11)。选取酶切正确的pShuttle-U6-shPDL1-CMV-E1A质粒进行测序，测序结果完全正确。
2.将获得的pShuttle-MCS-E1A(对照质粒)、pShuttle-U6-shPDL1-1-CMV-E1A、pShuttle-U6-shPDL1-2-CMV-E1A和 pShuttle-U6-shPDL1-3-CMV-E1A四个质粒利用PmeI酶切将其线性化后转入BJ5183菌中，与其中包含AD5腺病毒基因组DNA(缺失E1和E3区)的pAdEasy-1质粒(购自Agilent公司)进行同源重组，借此将CMV-E1A-SV40pA表达框和/或U6-shPDL1编码框整合到AD5腺病毒的基因组DNA中，从而获得可以表达目的基因的并能进行复制的溶瘤腺病毒(OAd-shPDL1)的基因组DNA(图12)。
具体实验过程如下：
(1)BJ5183感受态细胞的制备。
将本实验室-80℃保存的BJ5183(已转入pAdEasy-1质粒)接种于LB/Amp培养基中，37℃、200RPM过夜培养活化。利用生工生物工程股份有限公司的超级感受态细胞制备试剂盒(B529303-0040)制备BJ5183感受态细菌，每管分装100μl，保存于-80℃备用。
(2)pShuttle相关质粒与pAdEasy-1质粒之间的同源重组。
分别取pShuttle-MCS-E1A、pShuttle-U6-shPDL1-1-CMV-E1A、pShuttle-U6-shPDL1-2-CMV-E1A和pShuttle-U6-shPDL1-3-CMV-E1A质粒DNA各1μg，利用1μl的PmeI对其进行酶切，37℃反应1.5小时后加入1μl碱性磷酸酶对线性化的DNA片段进行脱磷处理。而后将酶切产物直接加入到100μl BJ5183感受态细菌中进行常规的转化实验，最后将转化后的菌液涂布于Kana抗性的LB平板上，37℃培养过夜。第二天挑取平板上出现的菌落接种于LB/Kana中过夜培养后提取质粒DNA并进行常规的PacI酶切，酶切产物进行电泳分析。根据可能发生同源重组的位置不同，可能发生三种不同方式的同源重组(图13)，经PacI酶切后可以产生4.5Kb或3Kb片段的质粒均为发生正确同源重组的质粒。酶切产物的电泳结果(图14)证明：pShuttle-MCS-E1A，pShuttle-U6-shPDL1-1-CMV-E1A，pShuttle-U6-shPDL1-2-CMV-E1A和pShuttle-U6-shPDL1-3-CMV-E1A与pAdEasy-1质粒间发生了正确的同源重组，成功获得了包装溶瘤腺病毒(统称为OAd-shPDL1)和其对照病毒的基因组DNA(pAdEasy-U6-shPDL1#1-CMV-E1A、pAdEasy-U6-shPDL1#2-CMV-E1A、pAdEasy-U6-shPDL1#3-CMV-E1A和pAdEasy-CMV-E1A)。对获得的阳性克隆中插入的shPDL1编码框 和E1A表达框进行测序，序列完全正确。
制备例4：溶瘤腺病毒(OAd-shPDL1)的包装与扩增
(1)包装溶瘤腺病毒(OAd-shPDL1)的基因组DNA的准备
分别取2μg获得的pAdEasy-CMV-E1A、pAdEasy-U6-shPDL1#1-CMV-E1A、pAdEasy-U6-shPDL1#2-CMV-E1A和pAdEasy-U6-shPDL1#3-CMV-E1A质粒DNA，各加入2μl PacI酶，37℃反应2小时后进行乙醇乙酸铵沉淀DNA，70％乙醇漂洗后将DNA沉淀溶解于10μl洁净的ddH 2O中，然后将其转染进入AD293进行病毒的包装。
(2)溶瘤腺病毒(OAd-shPDL1)的包装
提前一天将生长状态良好的AD293细胞接种于6孔板中，细胞接种数量以第二天进行转染实验时细胞覆盖率60-70％为宜。将准备好的约2μg线性化DNA与QIAGEN公司的6μl Attractene转染试剂混匀后加入AD293细胞中，十字混匀后放入细胞培养箱(37℃，5％CO 2)中继续培养约10-14天，中间每隔2-3天观察细胞病变情况。待出现小片细胞变为“念珠状”，范围逐渐扩大直至细胞大量脱落即可轻轻吹打细胞回收细胞和培养上清，保存于-80℃冰箱或直接进行进一步扩增，过程见图15。
(3)溶瘤腺病毒(OAd-shPDL1)的扩增
同样提前一天将生长状态良好的AD293细胞接种于6cm细胞培养皿中，细胞接种数量以第二天进行转染实验时细胞覆盖率70-80％为宜。每个6cm细胞培养皿加入前面收集的病毒上清800-1000ul，同样十字混匀后放回细胞培养基继续培养。通常48小时后可见细胞大量变圆并脱落，此时可收集细胞和培养上清。接下来继续放大在10cm细胞培养皿中扩增病毒，接种病毒时细胞密度同样以约70％为宜，每个10cm细胞培养皿加入前面收集的病毒上清1200-1500μl，同样48小时后可见细胞大量病变脱落，回收细胞和上清。最终放大到15cm培养皿中扩增病毒，细胞密度约为70％时加入10cm培养皿收集的病毒培养上清2ml，混匀后继续培养48小时收集细胞和培养上清。后面可在15cm培 养皿中循环扩增病毒至所需病毒量。
(4)溶瘤腺病毒(OAd-shPDL1)滴度的测定
目前腺病毒滴度的测定方法有VP法、GTU/BFU法、空斑法、TCID50法和Hexon染色计数(试剂盒)法。较为准确且重复率高的方法为TCID50法和Hexon染色计数法。本实施例采用Hexon染色计数法测定了获得的病毒上清中的活性病毒颗粒数(单位：PFU/ml)。结果如下所示。本发明描述的3个pShuttle-U6-shPDL1-CMV-E1A质粒和1个对照质粒pShuttle-MCS-E1A与pAdEasy-1质粒同源重组后产生6个pAdEasy-U6-shPDL1-CMV-E1A质粒和2个pAdEasy-CMV-E1A(参见图13中每个质粒可产生2种正确方式的同源重组，并获得两种正确的质粒，经PacI酶切后可分别产生4.5K或3K的条带)，利用获得的8个同源重组质粒在AD293中包装病毒，共获得8个溶瘤病毒：OAd-C-4.5K、OAd-C-3K、OAd-shPDL1#1-4.5K、OAd-shPDL1#1-3K、OAd-shPDL2#1-4.5K、OAd-shPDL1#2-3K、OAd-shPDL1#3-4.5K和OAd-shPDL1#3-3K，8个病毒依次分别简写作：C-4.5K、C-3K、1-4.5K、1-3K、2-4.5K、2-3K、3-4.5K和3-3K。其中C-4.5K和C-3K是序列相同的同一种病毒、1-4.5K和1-3K是序列相同的同一种病毒、2-4.5K和2-3K是序列相同的同一种病毒、3-4.5K和3-3K是序列相同的同一种病毒。
已获得病毒的滴度：
Figure PCTCN2018094263-appb-000013
实施例1：溶瘤腺病毒(OAd-shPDL1)在细胞(肿瘤细胞和正常细胞)中的复制能力
接种细胞(HUVEC、MRC-5、Hela、A549和U251)于12孔板 中，接种数量为每孔1.5×10 5个细胞，培养基(HUVEC细胞使用澳赛尔斯(Allcells)专用培养基，细胞和培养基均购自澳赛尔斯生物技术(上海)有限公司；MRC-5细胞培养使用MEM+10％FBS培养基；Hela细胞培养使用RPMI1640+10％FBS培养基；A549细胞培养使用DMEM/F12+10％FBS培养基；U251细胞培养使用MEM+10％FBS，全部培养基均购自Gibco公司)体积为1ml。12hr后吸去培养基并以PBS漂洗细胞1次后按图16所示方式加入500μl病毒悬液(病毒为制备例4制备所得)，病毒感染复数(MOI)为10。病毒与细胞共孵育90分钟后吸去病毒悬液，以PBS漂洗细胞2次，以此时为0hr，胰酶消化收取一份细胞样品，48hr后收取第二份细胞样品。分别提取0hr和48hr收取的细胞样品的基因组DNA，利用针对5型腺病毒E1A(P5：TCCGGTTTCTATGCCAAACCT(SEQ ID No.5)和P6：TCCTCCGGTGATAATGACAAGA(SEQ ID No.6))、Hexon基因(P7：CCATTACCTTTGACTCTTGTGT(SEQ ID No.7)和P8：GGTAGTCCTTGTATTTAGTATC(SEQ ID No.8))、以及人GAPDH(P9：CATGCCTTCTTGCCTCTTGTCTCTTAGAT(SEQ ID No.9)和P10：CCATGGGTGGAATCATATTGGAACATGTAA(SEQ ID No.10))基因的特异性引物进行Q-PCR，根据Q-PCR结果分析病毒感染细胞48hr后在细胞中的复制情况。溶瘤腺病毒(OAd-shPDL1)在不同细胞中复制能力的比较见图17。
如图17结果显示：本发明中构建的四类溶瘤腺病毒(对照病毒C-4.5K、OAd-shPDL1病毒1-4.5K、2-4.5K和3-4.5K)在所检测的细胞中的复制能力差别较大。本发明的溶瘤病毒在所检测的肿瘤细胞中的复制能力非常强，包括在永生化的人胚肺成纤维细胞系中也表现出了较强的复制能力，但是在人原代细胞HUVEC中的复制能力非常低，在人正常细胞中的复制能力比在肿瘤细胞系或者有成瘤倾向的细胞系(例如：永生化的人胚肺成纤维细胞系MRC5)中的复制能力低约42-444倍。因此，可以认为本发明的溶瘤腺病毒在选择性复制方面有着很强的肿瘤细胞偏向性，在未来的临床溶瘤病毒应用方面具有更高的安全性，在病毒使用量方面具有更大的空间。
实施例2：溶瘤腺病毒(OAd-shPDL1)对肿瘤细胞的杀伤能力
本实施例通过CCK8实验检测本发明的溶瘤腺病毒(OAd-shPDL1)的杀伤能力。接种细胞(U251、Hela和A549)于96孔板中，接种数量为每孔1.5×10 3个细胞，每孔培养基(U251细胞培养使用MEM+10％FBS；Hela细胞培养使用RPMI1640+10％FBS培养基；A549细胞培养使用DMEM/F12+10％FBS培养基；全部培养基均购自Gibco公司)体积为100μl。12hr后吸去50μl培养基，并加入50μl的病毒(分别为制备例4制备所得对照病毒C-4.5K、OAd-shPDL1病毒1-4.5K、2-4.5K和3-4.5K)与单纯培养基的混合物(时间点记为0hr)，感染复数(MOI)分别为1、3、10、30、100和300。每个感染复数设3个复孔。分别在48hr和72hr 2个时间点加入10μl CCK8(购自东仁化学科技有限公司)继续孵育1hr后检测培养物在450nm处的光吸收值，根据获得的光吸收值判断不同病毒对细胞的杀伤能力。商业化的溶瘤腺病毒H101被用来作为对照，实验中使用相同MOI的病毒数量处理相同的细胞，在相同的时间点同时检测光吸收值。另外，1μM的紫杉醇溶液用来作为系统阳性对照。溶瘤腺病毒(OAd-shPDL1)对三种肿瘤细胞的剂量杀伤效果以及半杀伤剂量(IC 50)结果如图18-21所示。
结果显示：本发明的溶瘤腺病毒(对照病毒C-4.5K、OAd-shPDL1病毒1-4.5K、2-4.5K和3-4.5K)对U251、A549和Hela的杀伤存在着明显的剂量依赖关系，与商业化溶瘤腺病毒H101相比，本发明所述病毒有着相似的杀伤效果，对U251细胞的杀伤甚至优于H101，杀伤效果的统计学分析显示存在显著性差异。另外，对本发明的溶瘤病毒针对不同细胞在72h的半杀伤剂量(IC 50)进行比较后发现，该类病毒对人脑胶质瘤细胞U251的IC 50最低，提示其可能在对人脑胶质瘤的治疗方面具有更加重要的临床价值。
实施例3：溶瘤腺病毒(OAd-shPDL1)所表达的shPDL1的功效性
为了方便检测本发明的溶瘤腺病毒(OAd-shPDL1)所表达的 shPDL1的功效，本实施例特意构建了两个可以稳定表达带有FLAG标签的hPDL1的细胞系：A549/hPDL1-FLAG和Hela/hPDL1-FLAG。构建过程简述如下：将按照制备例2所示方法得到的pcDNA3.3-hPDL1-FLAG载体(也称为pcDNA3.3-hPDL1-FLAG-IRES-hrGFP载体)利用lipofectamin 2000转染至A549和Hela细胞中，因为该载体携带Neomycin基因，随后加入G418进行三轮筛选，最终获得可以稳定表达带FLAG标签的hPDL1和GFP蛋白的A549/hPDL1-FLAG和Hela/hPDL1-FLAG细胞株。
同样使用H101和本发明所述的四种溶瘤腺病毒(为制备例4制备所得对照病毒C-4.5K、OAd-shPDL1病毒1-4.5K、2-4.5K和3-4.5K)处理上述两个细胞系，病毒感染复数(MOI)为10，病毒感染48hr后收取细胞样品，裂解后进行Western blot分析，利用Anti-FLAG抗体检测带有FLAG标签的hPDL1的表达水平的变化。结果见图22和23。Western blot结果表明：在使用溶瘤腺病毒处理A549和Hela细胞后，hPDL1的表达水平都有了不同程度的提高，但是在使用3种OAd-shPDL1溶瘤病毒处理相同的细胞后，原本升高的hPDL1的表达都得到了不同程度的抑制，尤其OAd-shPDL1-1(1-4.5K)的抑制能力最为明显，与前面的Q-PCR结果和共转染后的Western blot结果相互印证。
制备例5：溶瘤腺病毒超速离心纯化程序
1)超速离心前溶瘤腺病毒溶液的制备。
将制备例4获得的腺病毒培养上清在15CM培养皿中按病毒上清与细胞培养液体积比例(AD∶DMEM＝2∶20)感染293细胞，37℃和5％CO 2孵箱里继续培养约36小时，镜下观察约50％细胞发生致细胞病变效应(CPE)且细胞悬浮后，通过吹打或细胞刮刀等方式收集培养皿中的细胞和培养液，短暂可保存于4℃，长期需要保存于-20℃或-80℃；通常情况下一次超速离心纯化病毒的量约为60-80个15CM培养皿所产出的病毒量(包括培养基上清和感染的293细胞)。将收集的包含溶瘤病毒的293细胞和培养基上清4℃，3000RPM，离心 30分钟，后面的实验分上清和沉淀两部分分别进行。A)离心后的上清转移至另一新瓶中，可暂时保存于4℃，用于后面感染细胞扩增病毒用。到病毒扩增结束时可将全部上清与PEG8000溶液(含20％PEG8000的2.5M NaCl的水溶液)按2∶1的比例混合，冰上沉淀1小时或过夜。而后12000RPM离心20分钟后弃上清并保留病毒沉淀，最后利用适量的10mM Tris·Cl(pH8.0)溶液重悬病毒沉淀至全部吹散，7000RPM离心5分钟后保留含病毒上清，直接将含病毒上清上样至梯度氯化铯离心管中进行超速离心。B)离心后的细胞沉淀用少量10mM Tris·Cl(pH8.0)溶液轻洗(切勿吹打)，清洗细胞表面的培养基，吸弃。每10-12个15CM的培养皿的细胞沉淀用3ml Tris·Cl重悬，分装细胞悬液，-20℃保存。集中纯化前保存时间不超过2个月。待样品收齐后，将细胞悬液置于37℃水浴锅中融解，剧烈震荡30秒后将样品放回-80℃冰箱或置于干冰/纯乙醇混合物中快速冰冻细胞悬液。重复该冻融过程3-5次，彻底破坏细胞膜，将病毒从细胞中释放出来，获得病毒液。若不立即进行提纯，可保存于-20℃。超速离心纯化前将本步中获得的病毒液在37℃水浴锅中融解后常温离心10分钟，转速为16000RPM，收集含病毒上清，病毒上清可暂放冰上低温保存。
2)氯化铯密度梯度离心纯化AD病毒
氯化铯密度梯度离心法至今仍是分离纯化各种病毒的最常用方法。主要是依据不同类型病毒具有不同的浮力密度，使其与细胞裂解液中的其它成分在CsCl溶液中分离开来。收集到目标病毒特异的条带后，再用PD-10脱盐柱除去其中的氯化铯，最终获得纯化的病毒。使用这种方法可获得极高纯度的病毒。具体纯化程序如下：
1.制备第一次超速离心的氯化铯密度梯度：在Beckman超速离心管中先加入1.6ml轻CsCl(1.2g/ml)，然后在底部慢慢加入1.05毫升重CsCl(1.45g/ml)；也可根据超速离心管的体积改变加入的轻重CsCl的体积，分别加入2.5毫升轻CsCl和2.5毫升重CsCl(注意：此步切勿将轻重CsCl混匀，使之间保持清晰的分界线)。
2.将3-7ml含病毒上清轻轻加至轻CsCl液面上，然后将超离 管放入离心套管中，称重并配平相对的两个离心管后分别挂到离心转子上相对应的两个挂钩上。(注意：整个过程动作务必要轻柔。)
3.将离心管完全挂好后，20℃、40000RPM离心1小时15分钟。
4.离心结束后，将离心管牢固地固定在生物安全柜中铁架台上的万能夹上。小心地使用装有18G针头的5ml注射器从最下面的完整病毒颗粒条带下方(～1cm)刺入离心管，仅吸出完整病毒颗粒条带，可短暂保存于冰上。
5.制备第二次超速离心的CsCl溶液：在超离管中加入6ml等体积均匀混合的CsCl溶液(轻重CsCl各3ml)，将上一步收集的病毒液小心地灌注在制备好的CsCl液面上。同样小心地将灌注好的离心管放入到超速离心管套管中，而后将欲放置到离心转子上相对应的两个离心管称重并配平。小心地将配平的离心管挂到离心转子上(注意：整个过程动作务必要轻柔)。
6.设置离心参数：20℃，40000RPM，离心18小时。
7.离心结束后小心取出离心管，将离心管牢固地固定在生物安全柜中的铁架台上的万能夹上。小心地使用装有18G针头的5ml注射器从完整病毒颗粒条带下方刺入离心管，吸出完整病毒颗粒条带，可短暂保存于冰上。
8.在生物安全柜中，取出一支PD-10脱盐柱(柱体积为5ml)并剪去底部末端，将其固定在铁架台上使其内部的保养液在重力作用下自然流出。加3-4倍柱体积(15-20ml)的透析液(含1mM MgCl 2和10％甘油的10mM Tris溶液(pH7.4)，过滤除菌后保存于4℃)彻底清洗脱盐柱，完全置换保养液。
9.准备10个1.5ml做好编号的离心管用于收集病毒。
10.待加入的透析液完全流出后将第二次超速离心收集的病毒液加入空的PD-10柱内，根据体积差利用准备好的离心管收集柱内的滤过夜(1.2-1.5mi/管)。
11.继续加透析液滤过，每次加入量为2.5ml，持续收集到准备好的离心管内，直至收集完10个离心管。
12.另备11个1.5ml离心管，每管先加入90μl 0.1％(w/v)SDS。 从10个病毒收集管中分别取出10μl病毒加入到提前加入SDS的离心管中并做相应的编号。另取10μl透析液同样加入到加有SDS的离心管中作为系统对照。充分混匀病毒和SDS溶液后，室温放置15-30分钟，期间间或震荡。后测量其在260nm和280nm处的光吸收值，并根据公式(1OD 260＝1×10 12VP)计算病毒溶液中的病毒光学颗粒数浓度。OD 260数值反映收集液中的病毒DNA浓度，OD 280数值反映收集液中的蛋白质浓度，OD 260/OD 280比值约为1.2-1.3。
13.将获得病毒进行相应稀释后，选取两个合适稀释度的稀释液100μl，感染已经接种到24孔板中合适浓度的AD293细胞中，48小时后利用腺病毒滴度滴定试剂盒(上海翱翔生物技术有限公司)检测病毒的精确滴度。
将超速离心纯化的优质病毒液根据浓度大小和实验需要按照不同体积分装到不同的洁净离心管中，标记日期和病毒名称后保存于-80℃备用。
实施例4：溶瘤腺病毒(OAd-shPDL1)对肿瘤细胞(HCT116、PANC1、HT29和H460)的杀伤能力
本实施例通过MTT实验检测溶瘤腺病毒(OAd-shPDL1)的杀伤能力。接种人肿瘤细胞(HCT116、PANC1、HT29和H460)于96孔板中，每孔的细胞接种数量为3×10 3个，每孔培养基(HCT116细胞培养使用McCoy’s 5A+10％FBS培养基；PANC-1细胞培养使用DMEM+10％FBS培养基；HT29细胞培养使用DMEM/F12+10％FBS培养基；H460细胞培养使用RPMI1640+10％FBS，全部培养基均购自Gibco公司)体积为100μl。12小时后吸去50μl培养基并加入50μl的病毒(所用病毒分别为通过制备例5所述方法制备得到的对照病毒C-4.5K、OAd-shPDL1病毒1-4.5K、2-4.5K和3-4.5K)与无血清培养基的混合物(时间点记为0hr)，感染复数(MOI)分别为1、3、10、30、100和300。每个感染复数设3个复孔。分别在48hr和72hr这2个时间点在每孔中分别加入10μl MTT溶液(购自索莱宝生物科技有限公司)(5mg/ml，即0.5％MTT)，继续培养4hr；之后小心吸弃 全部培养液，尽量避免接触和吸弃细胞。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，在酶标仪上检测其在490nm处的吸光值。本实验中商业化的溶瘤腺病毒H101被用来作为对照，实验中使用相同MOI的病毒处理同一种细胞，在相同的时间点同时检测光吸收值。另外，1μM的紫杉醇溶液用来作为系统阳性对照。使用GraphPad Prism 5.04对数据进行分析和绘制剂量-效应曲线，并计算IC 50。抑制率计算公式：细胞增殖抑制百分率(IR％)＝1-(OD供试品-OD空白)×100％。溶瘤腺病毒(OAd-shPDL1)对四种肿瘤细胞的剂量杀伤效果以及半杀伤剂量(IC50)结果如图25-29所示。
结果显示：本发明制备的溶瘤腺病毒(对照病毒C-4.5K、OAd-shPDL1病毒1-4.5K、2-4.5K和3-4.5K)对HCT116、PANC1、HT29和H460的杀伤存在着明显的剂量依赖关系，并均有着较强的杀伤能力。与商业化溶瘤腺病毒H101相比，本发明所述病毒有着相似的杀伤效果。可以期待本发明的溶瘤腺病毒在未来的临床应用上可用于上述种类肿瘤的治疗。
实施例5：溶瘤腺病毒OAd-shPDL1所表达的shPDL1的功效性
本实施例包括在细胞中进行的体外功能检测和在荷瘤小鼠模型中进行的体内功能检测。实验中所用溶瘤腺病毒为对照病毒C-4.5K和OAd-shPDL1病毒1-4.5K(所用病毒通过制备例5所述方法制备得到)。
1.溶瘤腺病毒OAd-shPDL1所表达的shPDL1功能检测的体外细胞实验
(1)针对人乳腺癌细胞MDA-MB-231的细胞实验
本部分实验中使用的细胞株为人PDL1表达水平较高的人乳腺癌细胞MDA-MB-231。在6孔板的3个孔中接种MDA-MB-231细胞，每孔的接种数量为1×10 6个细胞。12hr后分别在其中的2个孔中分别加入包含1×10 7PFU(MOI＝10)的C-4.5K和1-4.5K溶瘤腺病毒的100μl病毒无血清L15培养基(L15培养基购自Gibco公司)混合物，剩余1个孔加入100μl无血清培养基作为空白对照，轻轻地“十”字 晃动6孔板使病毒在培养孔中尽量均匀分布。37℃和5％CO 2培养箱中培养24小时后，使用胰酶消化细胞后回收细胞样品。使用洁净PBS漂洗回收的细胞样品两次，将回收的细胞沉淀悬浮于加有蛋白酶抑制剂的RIPA缓冲液(50mM Tris-Cl(pH7.4)，150mM NaCl，1％NP-40，0.5％脱氧胆酸钠，0.1％SDS和1/20Cocktail蛋白酶抑制剂)中，放置于冰上裂解细胞30分钟，间或震荡混匀细胞样品，12000RPM离心5分钟后分别回收裂解上清和细胞沉淀并保存于-20℃。进行常规Western印迹方法检测回收的三份MDA-MB-231细胞裂解上清中的hPDL1的蛋白水平，其中一抗为Novusbio公司的兔源PDL1抗体(货号：NBP2-15791)。每组实验重复三次。Western结果与灰度扫描值分析见图30。
根据上面的Western实验结果，可以用看到本发明的OAd-shPDL1溶瘤病毒处理MDA-MB-231细胞后，细胞中的hDPL1的表达水平发生了明显的变化，OAd-shPDL1处理组(1-4.5K组)的MDA-MB-231细胞中的hPDL1的表达水平与对照组相比降低了约72.4％，而溶瘤病毒对照组(C-4.5K组)的细胞中的hPDL1的表达水平未发生明显变化。
接下来，在12孔板的3个孔中分别接种MDA-MB-231细胞，每孔接种1×10 4个细胞，12hr后在其中的2个孔中分别加入1×10 5PFU(MOI＝10)的C-4.5K和1-4.5K溶瘤腺病毒与无血清培养基混合液，剩余1个孔加入等体积的无血清培养基作为空白对照，轻轻地“十”字晃动12孔板使病毒在培养孔中尽量均匀分布。同样37℃和5％CO 2培养箱中培养24小时后，回收细胞样品，利用CD274的流式抗体(购自ebioscience公司)进行FACS，检测细胞样品中表达hPDL1的细胞百分率。每组实验重复三次。流式结果见图31。
实验结果表明：用本发明的OAd-shPDL1溶瘤病毒处理MDA-MB-231细胞后，细胞膜上检测到hPDL1的细胞数的百分率与未进行任何处理的空白对照组相比下降了约5个百分点，而溶瘤病毒对照组(C-4.5K组)的细胞中可以检测到hPDL1的细胞百分率与未进行任何处理的空白对照组相比上升了约9个百分点，这与相关文献 中溶瘤病毒刺激肿瘤细胞后能够引起肿瘤表面hPDL1的表达水平升高相符(参见(例如)以下科技文献：“Dmitriy Zamarin，et al.PD-L1in tumor microenvironment mediates resistance to oncolytic immunotherapy.J Clin Invest.2018；128(4)：1413-1428.”；“Zuqiang Liu，et al.Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy.Nat Commun.2017 Mar27；8：14754-14765.”；或“Praveen K.Bommareddy，et al.Integrating oncolytic viruses in combination cancer immunotherapy.Nat Rev Immunol.2018 May 9.doi：10.1038/s41577-018-0014-6.”)。所示结果表明OAd-shPDL1溶瘤病毒处理MDA-MB-231细胞后能够将溶瘤病毒刺激肿瘤细胞并使hPDL1表达升高的细胞百分率降低至未刺激前细胞百分率的95％。这也充分证明了OAd-shPDL1溶瘤病毒中表达的shPDL1的确敲低了MDA-MB-231肿瘤细胞中hPDL1的表达水平并且降低了MDA-MB-231肿瘤细胞表面表达hPDL1的细胞数量。
(2)针对人结肠癌细胞HCT116的细胞实验
在12孔板的3个孔中分别接种人结肠癌细胞HCT116细胞，每孔接种1×10 4个细胞，12hr后在其中的2个孔中分别加入1×10 5PFU(MOI＝10)的C-4.5K和1-4.5K溶瘤腺病毒与无血清培养基混合液，剩余1个孔加入等体积的无血清培养基McCoy′s 5A作为空白对照，轻轻地“十”字晃动12孔板使病毒在培养孔中尽量均匀分布。同样37℃和5％CO 2培养箱中培养24小时后，使用胰酶消化细胞后回收细胞样品，利用CD274的流式抗体进行FACS，检测细胞样品中表达hPDL1的细胞百分率。每组实验重复三次。流式结果见图32。
实验结果表明：用本发明的OAd-shPDL1溶瘤病毒处理HCT116细胞后，细胞膜上检测到hPDL1的细胞数的百分率与未进行任何处理的空白对照组相比下降了约19.1％，而溶瘤病毒对照组(C-4.5K组)的细胞中可以检测到hPDL1的细胞百分率与未进行任何处理的空白对照组相比上升了约5.3％，这与相关文献中溶瘤病毒刺激肿瘤细胞后能够引起肿瘤表面hPDL1的表达水平升高相符(参见(例如)以下科技文献：“Dmitriy Zamarin，et al.PD-L1 in tumor  microenvironment mediates resistance to oncolytic immunotherapy.J Clin Invest.2018；128(4)：1413-1428.”；“Zuqiang Liu，et al.Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy.Nat Commun.2017 Mar 27；8：14754-14765.”；或“Praveen K.Bommareddy，et al.Integrating oncolytic viruses in combination cancer immunotherapy.Nat Rev Immunol.2018 May 9.doi：10.1038/s41577-018-0014-6.”)。所示结果表明OAd-shPDL1溶瘤病毒处理HCT116细胞后能够将溶瘤病毒刺激肿瘤细胞并使hPDL1表达升高的细胞百分率降低至未刺激前细胞百分率的80.9％。这也充分证明了OAd-shPDL1溶瘤病毒中表达的shPDL1的确敲低了HCT116肿瘤细胞中hPDL1的表达水平并且降低了HCT116肿瘤细胞表面表达hPDL1的细胞数量。
2.对溶瘤腺病毒OAd-shPDL1所表达的shPDL1进行功能检测的体内荷瘤小鼠模型实验
本部分实验首先在9只BALB/C裸鼠的背部皮下接种人结肠癌细胞HCT116，每只裸鼠的细胞接种数量为5×10 6个。约9天细胞在裸鼠皮下成瘤后将其随机分成3组，其中的第1组不做任何处理作为空白对照组，第2组采用瘤内注射的方式在皮下肿瘤中注射溶瘤腺病毒对照病毒C-4.5K，每只裸鼠的病毒注射量为1×10 9PFU，注射体积为100μl，第3组同样采用瘤内注射的方式在皮下肿瘤中注射溶瘤腺病毒1-4.5K，每只裸鼠的病毒注射量为1×10 9PFU，注射体积为100μl。按照这种注射剂量和方式每天注射一次，连续注射3天，第4天间隔1天不做任何处理，第5天处死裸鼠回收肿瘤组织。从全部组织样品中分别选取一部分加入包含蛋白酶抑制剂的RIPA缓冲液(配方同上)后进行匀浆处理提取组织蛋白，进行Western印迹实验检测不同处理后肿瘤组织中hPDL1的蛋白表达水平，结果见图33和图34。
把图33所示Western结果的条带扫描转换为灰度值并根据各自的看家基因(β-actin)的灰度值进行标准化后得到图34的散点分布图，根据各自的中位值可以看到：在利用溶瘤腺病毒对照病毒(C-4.5) 处理BALB/C皮下接种的HCT116细胞后，该组hPDL1的表达较无任何处理的空白对照组中hPDL1的表达上调了20％，而利用溶瘤腺病毒OAd-shPDL1处理BALB/C皮下接种的HCT116细胞的一组中的hPDL1的蛋白表达水平较对照病毒处理组下降了25％，而且表达水平也低于无任何处理的空白对照组中的hPDL1的表达，因此该结果证明了溶瘤腺病毒OAd-shPDL1(1-4.5)中表达的shPDL1的确降低了体内人肿瘤细胞移植物中hPDL1的表达。
实施例6：溶瘤腺病毒OAd-shPDL1与人NK细胞对人肿瘤细胞系的联合杀伤实验
本发明的溶瘤腺病毒OAd-shPDL1包含一个shPDL1的表达框，可以在其感染的肿瘤细胞内表达shPDL1，并由此可以敲低肿瘤细胞内hPDL1的表达水平，从而减少肿瘤细胞表面hPDL1的存在，最终减弱或消除因为肿瘤细胞表面的hPDL1与免疫细胞(包括T细胞或NK细胞)PD1的结合而导致的对免疫细胞激活的免疫抑制作用。因此在本实施例中主要通过在细胞水平上检测溶瘤腺病毒OAd-shPDL1(1-4.5K)(由制备例5所述方法制备得到)与人NK细胞同时存在的情况下是否对人肿瘤细胞的杀伤存在杀伤协同效应。其中，采用台盼蓝染色计数法分别针对人结肠癌细胞HCT116和人肺癌细胞A549检测了溶瘤腺病毒OAd-shPDL1(1-4.5K)与人NK细胞在联合杀伤过程中是否存在协同效应。
台盼蓝染色计数法，是一种经典的死/活细胞计数方法。细胞损伤或死亡时，台盼蓝可穿透变性的细胞膜，与解体的DNA结合并使其着色，而活细胞能阻止染料进入细胞内，因此可以鉴别死细胞和活细胞。
1.针对人结肠癌细胞HCT116的细胞实验
经由台盼蓝染色计数法试验确定，当溶瘤腺病毒OAd-shPDL1(1-4.5K)对HCT116细胞的杀伤剂量为约MOI＝1～3时，杀伤率在40～60％之间；当人NK细胞对HCT116细胞的杀伤剂量为效靶比NK∶HCT116为约5∶1时，杀伤率在10～20％之间；杀伤剂量分别较为 合适，适宜进行联合杀伤实验。
首先在24孔板中接种HCT116细胞，每孔接种2×10 4个细胞，12hr后按照不同的MOI(MOI＝1、3)加入溶瘤腺病毒OAd-shPDL1(1-4.5K)，混合均匀后放回37℃、CO 2培养箱继续培养6hr，而后吸去包含病毒的培养基上清，并用洁净的PBS轻洗1次后加入新鲜的完全培养基(McCoy′s 5A+10％FBS)。去除病毒18hr后按照设定的效靶比(E∶T＝5∶1)在每个孔中加入已经活化的NK细胞(冻存NK复苏后培养)。该实验组为1-4.5K+NK组。在加入NK细胞后的24hr收集细胞加入台盼蓝染色后计数活细胞的数目。实验中保留一组HCT116细胞，不加溶瘤腺病毒OAd-shPDL1(1-4.5K)，不加NK，作为空白对照组；一组在相应时间点加溶瘤腺病毒OAd-shPDL1(1-4.5K)，但不加NK，作为1-4.5K组；一组在相应时间点加NK，但不加溶瘤腺病毒OAd-shPDL1(1-4.5K)，作为NK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。
溶瘤腺病毒OAd-shPDL1(1-4.5K)的MOI为1、NK细胞效靶比(E∶T)为5∶1时的结果如图35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)，其中显示出联合使用溶瘤腺病毒OAd-shPDL1(1-4.5K)和NK细胞对HCT116的杀伤有显著的协同作用，并且协同抑制率为约83％。而本实验中单用溶瘤腺病毒OAd-shPDL1(1-4.5K)的抑制率为约51％，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，空白组抑制率约为0(未示出在该图中)。
溶瘤腺病毒OAd-shPDL1(1-4.5K)的MOI为3、NK细胞效靶比(E∶T)为5∶1时的结果如图36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)，其中显示出联合使用溶瘤腺病毒OAd-shPDL1(1-4.5K)和NK细胞对HCT116的杀伤有显著的协同作用，并且协同抑制率为约91％。而本实验中单用溶瘤腺病毒OAd-shPDL1(1-4.5K)的抑制率为约58％，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，空白组抑制率约为 0(未示出在该图中)。
2.针对人肺癌细胞A549的细胞实验
经由台盼蓝染色计数法实验确定，当溶瘤腺病毒OAd-shPDL1(1-4.5K)对A549细胞的杀伤剂量为约MOI＝30时，杀伤率在40～60％之间；当人NK细胞对A549细胞的杀伤剂量为效靶比为约5∶1时，杀伤率在10～20％之间；杀伤剂量分别较为合适，适宜进行联合杀伤实验。
首先在24孔板中接种A549细胞，每孔接种2×10 4个细胞，12hr后按照MOI＝30加入溶瘤腺病毒OAd-shPDL1(1-4.5K)，混合均匀后放回37℃、CO 2培养箱继续培养6hr，而后吸去包含病毒的培养基上清，并用洁净的PBS轻洗1次后加入新鲜的完全培养基(DMEM/F12+10％FBS)。去除病毒18hr后按照设定的效靶比(E∶T＝5∶1)在每个孔中加入已经活化的NK细胞(冻存NK细胞复苏后培养)。该实验组为1-4.5K+NK组。在加入NK细胞后的24hr收集细胞加入台盼蓝染色后计数活细胞的数目。实验中保留一组A549细胞，不加溶瘤腺病毒OAd-shPDL1(1-4.5K)，不加NK，作为空白对照组；一组在相应时间点加溶瘤腺病毒OAd-shPDL1(1-4.5K)，但不加NK，作为1-4.5K组；一组在相应时间点加NK，但不加溶瘤腺病毒OAd-shPDL1(1-4.5K)，作为NK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37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)，其中显示出联合使用溶瘤腺病毒OAd-shPDL1(1-4.5K)和NK细胞对A549的杀伤有显著的协同作用，并且协同抑制率为约83％。而本实验中单用溶瘤腺病毒OAd-shPDL1(1-4.5K)的抑制率为约45％，单用NK细胞的抑制率为约16.9％，二者的叠加值在图中用虚线示出。此外，空白组抑制率约为0(未示出在该图中)。
实施例7：溶瘤腺病毒OAd-shPDL1对免疫缺陷小鼠皮下接种的人肿瘤细胞的体内生长抑制实验
本实施例中首先对NOD-SCID免疫缺陷鼠皮下接种(每只小鼠 皮下接种细胞的数量为5×10 6个细胞)人结肠癌细胞HCT116制备荷瘤鼠动物模型以用于检测溶瘤腺病毒OAd-shPDL1(1-4.5K)对HCT116的生长抑制作用，溶瘤对照病毒C-4.5K作为阴性对照病毒。实验中所用对照病毒C-4.5K和OAd-shPDL1病毒1-4.5K通过制备例5所述方法制备得到。选取皮下接种的肿瘤体积达到要求(肿瘤体积为90-120mm 3)的12只荷瘤鼠按照区组随机分组的方式分成4组，每组3只小鼠。第一组为对照组(空白对照Control)，每只小鼠每次注射100μl腺病毒保存液(即，含1mM MgCl 2和10％甘油的10mM Tris溶液(pH7.4))；第二组为中剂量溶瘤对照病毒组(C-4.5K(中))，每只小鼠每次注射100μl包含1×10 8PFU的C-4.5K病毒悬液；第三组为中剂量溶瘤病毒组(1-4.5K(中))，每只小鼠每次注射100μl包含1×10 8PFU的1-4.5K病毒悬液；第四组为高剂量溶瘤病毒组(1-4.5K(高))，每只小鼠每次注射100μl包含1×10 9PFU的1-4.5K病毒悬液。整个实验过程中一共给药三次，隔天给药，第一次给药从分组当天开始(记为第0天)。每周两次测量肿瘤直径和称量小鼠体重，第21天后处死小鼠取肿瘤进行拍照。实验结果分别见图38、图39、图40和图41。
综合上述结果：溶瘤腺病毒OAd-shPDL1(1-4.5K)处理NOD-SCID免疫缺陷鼠皮下接种的HCT116肿瘤细胞，在一定程度上可以抑制肿瘤细胞的生长，高剂量组(1-4.5K(高))较中剂量组(1-4.5K(中))和对照病毒组(C-4.5K(中))表现出更强的生长抑制作用，呈现出明显的剂量依赖关系(参见图38)，但是相对肿瘤增殖率(T/C％)均未达到40％以下(参见图39)。同等剂量的OAd-shPDL1(1-4.5K)和骨架对照病毒C-4.5K在抑制肿瘤生长方面并未因为OAd-shPDL1(1-4.5K)病毒所表达的shPDL1导致肿瘤细胞内hPDL1的表达水平的降低而呈现出更好的抑瘤效果(参见图38、39、41)。全部组小鼠的体重未发生明显的变化，表明溶瘤腺病毒OAd-shPDL1(1-4.5K)和对照病毒组(C-4.5K)对NOD-SCID缺陷鼠未表现出明显的毒性(参见图40)。
接下来对BALB/C裸鼠皮下接种(每只小鼠皮下接种5×10 6个细 胞接种多少个细胞？)人结肠癌细胞HCT116制备荷瘤鼠动物模型以用于验证溶瘤腺病毒OAd-shPDL1(1-4.5K)对HCT116的生长抑制作用，溶瘤对照病毒C-4.5K作为阴性对照病毒。选取皮下接种的肿瘤体积达到要求(肿瘤体积为90-120mm 3)的25只荷瘤鼠按照区组随机分组的方式分成5组，每组5只小鼠。第一组为对照组(空白对照(Control))，每只小鼠每次注射100μl腺病毒保存液；第二组为中剂量溶瘤对照病毒组(C-4.5K(中))，每只小鼠每次注射100μl包含1×10 8PFU的C-4.5K病毒悬液；第三组为低剂量溶瘤病毒组(1-4.5K(低))，每只小鼠每次注射100μl包含1×10 7PFU的1-4.5K病毒悬液；第四组为中剂量溶瘤病毒组(1-4.5K(中))，每只小鼠每次注射100μl包含1×10 8PFU的1-4.5K病毒悬液；第五组为高剂量溶瘤病毒组(1-4.5K(高))，每只小鼠每次注射100μl包含1×10 9PFU的1-4.5K病毒悬液。整个实验过程中一共给药五次，隔天给药，第一次给药从分组当天开始(记为第0天)。每周两次测量肿瘤直径和称量小鼠体重，第28天后处死小鼠取肿瘤进行拍照。实验结果分别见图42、图43、图44和图45。
综合上述结果：溶瘤腺病毒OAd-shPDL1(1-4.5K)处理BALB/C裸鼠皮下接种的HCT116肿瘤细胞的实验中，高剂量组和中剂量组均表现出了非常好的抑瘤效果(参见图42)，其中高剂量组的相对肿瘤增殖率(T/C％)达到了40％以下，中剂量组的相对肿瘤增殖率(T/C％)在实验中也达到过40％以下(参见图43)。更为重要的是高剂量组中裸鼠背上的肿块出现了明显的“溶瘤”现象-表面破溃，破溃部位结痂脱落后原有接种部位的肿瘤组织基本上被清除。从动物的身体状况和体重情况来看，高剂量组小鼠除1只裸鼠身上的肿瘤细胞附着于背部脊柱的肌肉上，使得OAd-shPDL1(1-4.5K)对其抑制效果不是十分明显外，剩余4只背部接种的肿瘤细胞的生长得到了有效的抑制，该4只裸鼠的的身体较其它组的裸鼠圆润和强壮，其中2只裸鼠背部接种的肿瘤细胞经过OAd-shPDL1(1-4.5K)处理后肉眼已不可见(参见图44、45)。同种剂量的溶瘤病毒骨架对照病毒组(C-4.5K)和OAd-shPDL1(1-4.5K)中剂量组相比，OAd-shPDL1 (1-4.5K)中剂量组中裸鼠皮下接种的肿瘤的生长也呈现出了较为显著的抑制效果(参见图42、43、45)，由此可以认为OAd-shPDL1(1-4.5K)中剂量组中shPDL1表达框所表达的shPDL1所导致的肿瘤细胞内HPDL1表达水平的降低在抑制肿瘤生长的过程中发挥了关键的作用。病毒保存液组的裸鼠体重减轻明显，多表现出肿瘤生长相关的恶性消耗病体质。
实施例8：溶瘤腺病毒OAd-shPDL1对BALB/C裸鼠皮下接种的人肿瘤细胞HCT116的生长抑制的验证
为了进一步确认OAd-shPDL1(1-4.5K)对BALB/C裸鼠皮下接种的HCT116细胞生长抑制的有效剂量，本实施例扩大了动物模型的样本量再次验证了OAd-shPDL1(1-4.5K)的有效剂量。首先将人结肠癌细胞HCT116接种到BALB/C裸鼠的皮下制备荷瘤鼠动物模型，每只裸鼠的细胞接种量为5×10 6个细胞。同样选取溶瘤病毒C-4.5K作为阴性对照病毒。选取皮下接种的肿瘤体积达到要求(肿瘤体积为80-130mm 3)的35只荷瘤鼠按照区组随机分组的方式分成5组，每组7只小鼠。第一组为对照组(Control组)，每只小鼠每次注射100μl腺病毒保存液(即，含1mM MgCl 2和10％甘油的10mM Tris溶液(pH7.4))；第二组为溶瘤对照病毒中剂量组(C-4.5K(1×10 8))，每只小鼠每次注射100μl包含1×10 8PFU的C-4.5K病毒悬液；第三组为溶瘤对照病毒高剂量组(C-4.5K(1×10 9))，每只小鼠每次注射100μl包含1×10 9PFU的C-4.5K病毒悬液；第四组为溶瘤病毒1-4.5K中剂量组(1-4.5K(1×10 8))，每只小鼠每次注射100μl包含1×10 8PFU的1-4.5K病毒悬液；第五组为溶瘤病毒高剂量组(1-4.5K(1×10 9))，每只小鼠每次注射100μl包含1×10 9PFU的1-4.5K病毒悬液。整个实验过程中一共给药五次，隔天给药，第一次给药从分组当天开始(记为第0天)。每周两次测量肿瘤直径和称量小鼠体重，第25天因为对照组和溶瘤对照病毒组中裸鼠荷瘤出现了破溃到达实验终点，处死全部35只小鼠取肿瘤进行称重和拍照，并连同同一只小鼠的血和脾，制备细胞悬液后加入检测BALB/C裸鼠NK细胞和T细胞的流式抗 体(抗小鼠CD49b抗体和抗小鼠CD3抗体，两者均购自ebioscience公司)染色后进行FACS，分析肿瘤、血和脾中肿瘤细胞的NK和T细胞比例的改变。实验结果分别见图46、图47、图48、图49、图50和图51。
血细胞悬液的制备：摘取小鼠眼球后取血并加入抗凝管中震荡混匀后放置于冰上。500×g离心5分钟后弃去上清后加入红细胞裂解液(购自天根生化科技(北京)有限公司，货号：#122-02)充分混匀沉淀细胞并于室温反应10-15分钟后再次500×g离心5分钟后弃去上清保留细胞沉淀，再次利用包含1％BSA的PBS溶液重新悬浮细胞并500×g离心5分钟后弃去上清保留细胞沉淀，将细胞沉淀分为两份，一份不进行任何处理作为对照，另外一份加入抗BALB/C鼠的CD3和CD49b的流式抗体，混匀后室温避光反应30分钟后上机进行FACS。
脾脏细胞悬液的制备：解剖小鼠取小鼠脾脏后放置于1.5ml离心管中暂时保存于冰上。在一个洁净的6cm培养皿中加入5ml包含1％BSA的PBS溶液，利用尼龙网包裹脾脏在培养皿中挤压研磨脾脏充分释放脾脏细胞。将细胞悬液500×g离心5分钟后弃去上清，加入红细胞裂解液充分混匀沉淀细胞并于室温反应10-15分钟后，再次500×g离心5分钟，弃去上清保留细胞沉淀，再次利用包含1％BSA的PBS溶液重新悬浮细胞并500×g离心5分钟，弃去上清保留细胞沉淀，将细胞沉淀分为两份，一份不进行任何处理作为对照，另外一份加入抗BALB/C鼠的CD3和CD49b的流式抗体，混匀后室温避光反应30分钟后上机进行FACS。
肿瘤组织细胞悬液的制备：解剖小鼠取皮下肿瘤后放置于1.5ml离心管中暂时保存于冰上。每个肿瘤组织剪取等体积一小块后放入另一套离心管中后加入500μl胶原酶，利用眼科剪刀将肿瘤剪成细小碎块后37℃反应30分钟，将细胞悬液500×g离心5分钟后弃去上清后加入红细胞裂解液充分混匀沉淀细胞并于室温反应10-15分钟，再次500×g离心5分钟后弃去上清保留细胞沉淀，再次利用包含1％BSA的PBS溶液重新悬浮细胞并500×g离心5分钟后弃去上清保留细胞沉淀，将细胞沉淀分为两份，一份不进行任何处理作为对照，另外一 份加入抗BALB/C鼠的CD3和CD49b的流式抗体，混匀后室温避光反应30分钟后上机进行FACS。
综合上述结果：溶瘤腺病毒OAd-shPDL1(1-4.5K)处理BALB/C裸鼠皮下接种的HCT116肿瘤细胞的实验中，高剂量组和中剂量组均表现出了非常好的抑瘤效果(参见图46)，其中中剂量组的相对肿瘤增殖率(T/C％)达到了40％，并且高剂量组达到40％以下(参见图47)。对比比较对照病毒C-4.5K(1×10 8)组和目的溶瘤病毒1-4.5K(1×10 8)组对HCT116生长的抑制情况，溶瘤病毒1-4.5K表现出了较强的生长抑制作用，对两组肿瘤体积进行统计学分析，两组之间存在非常显著的差异。对比比较对照病毒C-4.5K(1×10 9)组和目的溶瘤病毒1-4.5K(1×10 9)组对HCT116生长的抑制情况，在给药初期，两种病毒均对皮下接种的HCT116表现出了非常强烈的生长抑制作用，但在实验第10天C-4.5K不能像1-4.5K那样持续抑制肿瘤的生长，C-4.5K(1×10 9)组中裸鼠皮下的肿瘤开始生长，而1-4.5K(1×10 9)组中裸鼠皮下的肿瘤仍持续得到抑制直至实验第17天，两组的肿瘤体积同样存在非常显著的差异。综合分析上述结果，认为C-4.5K和1-4.5K溶瘤病毒充分表现出了溶瘤病毒的特性，另外1-4.5K因为可以表达shPDL1，较C-4.5K表现出了对HCT116细胞的生长抑制作用的更长的持续性。本实施例完全体现出溶瘤病毒1-4.5K中所表达的shPDL1由于改变了人肿瘤细胞内hPDL1的表达水平，可能减弱或解除了瘤周的免疫抑制作用而活化了周围的免疫细胞，因此实现了对肿瘤的联合杀伤作用。全部5组小鼠的体重均未发生明显变化，说明两种溶瘤腺病毒对BALB/C裸鼠均未发现明显毒性(参见图48)。对各组小鼠的肿瘤进行拍照和称重后(参见图49)，发现1-4.5K实验组与C-4.5K实验组的瘤重之间存在着显著的差异。对肿瘤、血和脾脏中的NK和T细胞进行FACS分析后发现，施加1-4.5K溶瘤病毒的小鼠的肿瘤组织和血中的NK细胞均较对照组(Control组)和C-4.5K对照溶瘤病毒组得到了很大的提高，其中1-4.5K溶瘤病毒组血中的NK细胞是对照组血中NK细胞的2倍，肿瘤组织中的NK细胞较对照组提高了5倍，各组动物脾中的NK细胞未发生明显变化(参 见图50)。对T细胞的分析发现，各组动物的血中的T细胞比例未发生明显改变，1-4.5K(1×10 8)实验组的脾脏和肿瘤组织中T细胞较C-4.5K(1×10 8)同样组织中的T细胞发生了显著的提高(参见图51)。
综合分析上面的实验结果(参见实施例7和8)，利用相同的溶瘤腺病毒OAd-shPDL1(1-4.5K)处理两种不同遗传背景的免疫缺陷小鼠皮下接种的人结肠癌细胞HCT116，获得了差别非常明显的抑瘤效果。OAd-shPDL1(1-4.5K)对BALB/C裸鼠皮下接种的HCT116表现出了非常好的抑制肿瘤生长和清除肿瘤的作用，而同剂量(1×10 9)的OAd-shPDL1(1-4.5K)溶瘤病毒对NOD-SCID免疫缺陷鼠皮下接种的同种细胞的抑瘤效果则不明显。另外一种不包含shPDL1表达框的同浓度(1×10 9)骨架对照病毒(C-4.5K)对BALB/C裸鼠皮下接种的HCT116仅在给药初期表现出了与OAd-shPDL1(1-4.5K)溶瘤病毒相似的抑瘤效果，但在停止给药后即不能像OAd-shPDL1(1-4.5K)一样持续抑制肿瘤的生长。通常情况下肿瘤细胞为了逃避机体免疫系统对其杀伤与清除会在其自身表面高表达PDL1或PDL2等配体，上述配体与PD-1的结合会导致PD-1的胞内结构域的酪氨酸磷酸化，并招募酪氨酸磷酸酶SHP-2，从而减少TCR信号通路的磷酸化，降低了TCR通路下游的激活信号以及T细胞的激活和细胞因子的生成，从而在肿瘤周围形成一种免疫抑制的微环境，处于该环境中的免疫细胞处于一种非活化状态。另外从免疫缺陷小鼠品系来分析，NOD-SCID免疫缺陷小鼠免疫缺陷程度较高，体内缺失了T细胞，B细胞虽然存在但功能缺失，NK细胞功能极度低下；而BALB/C裸鼠的免疫缺陷程度较低，体内同样缺失了T细胞，B细胞虽然存在但功能缺失，但保留有完整功能的NK细胞、巨噬细胞和以树突状细胞为主的抗原提呈细胞。已有文献报告小鼠体内的NK、巨噬细胞或树突状细胞等可能在抑制肿瘤生长过程中可能发挥了重要的联合杀伤作用(参见(例如)以下科技文献：“Kevin C.Barry，et al.A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumormicroenvironments.Nat  Med.2018 Jun 25.doi：10.1038/s41591-018-0085-8.”)。溶瘤腺病毒OAd-shPDL1(1-4.5K)与其骨架对照病毒C-4.5K的区别在于前者包含有可以表达shPDL1的表达框，可以在侵入的肿瘤细胞内部表达shPDL1，shPDL1减少了人肿瘤细胞内的hPDL1水平，继而减弱或解除了瘤周环境中免疫抑制状态，最终使其对肿瘤的生长保持一个较长的抑制状态。另外也有文献报道，小鼠免疫细胞表面的PD1与人细胞表面上的PDL1可以相互结合并传递免疫抑制信号；反过来人肿瘤细胞内因为shPDL1的表达降低了hPDL1的表达水平(参见(例如)以下科技文献：“David Yin-wei Lin，et al.The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors.Proc Natl Acad Sci U S A.2008 Feb 26；105(8)：3011-6.”；“Clement Viricel，et al.Human PD-1binds differently to its human ligands：A comprehensive modeling study.J Mol Graph Model.2015 Apr；57：131-42.”；或“Aaron T.Mayer，et al.Practical ImmunoPET radiotracer design considerations for human immune checkpoint imaging.J Nucl Med.2017 Apr；58(4)：538-546.”)。由此认为溶瘤腺病毒OAd-shPDL1(1-4.5K)在人肿瘤细胞HCT116内表达的shPDL1所导致的肿瘤细胞内hPDL1表达水平的下降在BALB/C裸鼠上减弱和解除了肿瘤周围对免疫细胞(NK、巨噬细胞或树突状细胞)的抑制作用，从而对HCT116的生长表现出持续的抑制能力。
该部分结果也与上述溶瘤病毒OAd-shPDL1(1-4.5K)与人NK细胞在体外对肿瘤细胞的联合杀伤过程中表现出来的协同效果相吻合。

Claims (37)

  1. 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重组溶瘤腺病毒；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；
    其中所述重组溶瘤腺病毒为选择复制型溶瘤腺病毒，并且该重组溶瘤腺病毒的基因组中整合有能够在肿瘤细胞中抑制PDL1表达的外源shRNA的编码序列。
  2. 根据权利要求1所述的治疗剂，其中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。
  3. 根据权利要求1所述的治疗剂，其中所述第一药物组合物的活性成分为所述重组溶瘤腺病毒，并且其中所述第二药物组合物的活性成分为所述NK细胞。
  4. 根据权利要求1所述的治疗剂，其中所述第一药物组合物包含5×10 7至5×10 12VP/天剂量的所述重组溶瘤腺病毒，并且所述第二药物组合物包含1×10 7至1×10 10个细胞/天剂量的所述NK细胞。
  5. 根据权利要求1所述的治疗剂，其中所述外源shRNA的编码序列如SEQ ID NOs.16、19和22中的任一者所示。
  6. 根据权利要求1所述的治疗剂，其中所述重组溶瘤腺病毒的基因组中缺失了E1B19K基因、E1B55K基因、和全部E3区基因。
  7. 根据权利要求1或6所述的治疗剂，其中所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列；优选的是，所述E1A基因编 码序列是在CMV启动子控制下的。
  8. 根据权利要求1所述的治疗剂，其中所述重组溶瘤腺病毒是对5型腺病毒进行基因改造而得到的。
  9. 根据权利要求1所述的治疗剂，其中所述NK细胞选自自体NK细胞和异体NK细胞。
  10. 根据权利要求9所述的治疗剂，其中所述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。
  11. 根据权利要求1所述的治疗剂，其中所述重组溶瘤腺病毒配制成通过瘤内注射给药或静脉给药；并且其中所述NK细胞配制成通过静脉给药。
  12. 根据权利要求1所述的治疗剂，其中所述治疗剂由所述第一药物组合物和所述第二药物组合物组成。
  13. 根据权利要求1-12中任一项所述的治疗剂在制备用于治疗肿瘤和/或癌症的药物中的应用。
  14. 根据权利要求13所述的应用，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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌、白血病、骨癌、睾丸癌。
  15. 一种用于治疗肿瘤和/或癌症的具有协同作用的联合药物的药盒，包括：装有重组溶瘤腺病毒的第一容器和装有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；其中所述重组溶瘤腺病毒为选择复制型溶瘤 腺病毒，并且该重组溶瘤腺病毒的基因组中整合有能够在肿瘤细胞中抑制PDL1表达的外源shRNA的编码序列。
  16. 根据权利要求15所述的药盒，其中所述第一容器包含5×10 7至5×10 12VP/天剂量的所述重组溶瘤腺病毒，并且所述第二容器包含1×10 7至1×10 10个细胞/天剂量的所述NK细胞。
  17. 根据权利要求15所述的药盒，其中所述外源shRNA的编码序列如SEQ ID NOs.16、19和22中的任一者所示。
  18. 根据权利要求15所述的药盒，其中所述重组溶瘤腺病毒的基因组中缺失了E1B19K基因、E1B55K基因、和全部E3区基因。
  19. 根据权利要求15或18所述的药盒，其中所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列；优选的是，所述E1A基因编码序列是在CMV启动子控制下的。
  20. 根据权利要求15所述的药盒，其中所述重组溶瘤腺病毒是对5型腺病毒进行基因改造而得到的。
  21. 根据权利要求15所述的药盒，其中所述NK细胞选自自体NK细胞和异体NK细胞。
  22. 根据权利要求21所述的药盒，其中所述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。
  23. 根据权利要求15所述的药盒，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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌、白血病、骨癌、睾丸癌。
  24. 根据权利要求15所述的药盒，其中所述重组溶瘤腺病毒配制成通过瘤内注射给药或静脉给药；所述NK细胞配制成通过静脉给药。
  25. 一种治疗肿瘤和/或癌症的方法，包括以下依次进行的步骤：
    1)对肿瘤和/或癌症患者施用重组溶瘤腺病毒，该重组溶瘤腺病毒能够选择性地在肿瘤细胞中复制；
    2)在施用所述重组溶瘤腺病毒之后的第18小时至72小时，对所述肿瘤和/或癌症患者施用NK细胞；
    其中所述重组溶瘤腺病毒为选择复制型溶瘤腺病毒，并且该重组溶瘤腺病毒的基因组中整合有能够在肿瘤细胞中抑制PDL1表达的外源shRNA的编码序列。
  26. 根据权利要求25所述的方法，其中所述外源shRNA的编码序列如SEQ ID NOs.16、19和22中的任一者所示。
  27. 根据权利要求25所述的方法，其中所述重组溶瘤腺病毒的基因组中缺失了E1B19K基因、E1B55K基因、和全部E3区基因。
  28. 根据权利要求25或27所述的方法，其中所述重组溶瘤腺病毒的基因组中包含E1A基因编码序列；优选的是，所述E1A基因编码序列是在CMV启动子控制下的。
  29. 根据权利要求25所述的方法，其中所述重组溶瘤腺病毒是对5型腺病毒进行基因改造而得到的。
  30. 根据权利要求25所述的方法，其中所述NK细胞选自自体NK细胞和异体NK细胞。
  31. 根据权利要求30所述的方法，其中所述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。
  32. 根据权利要求25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、宫颈癌、淋巴癌、胃癌、食道癌、肾癌、前列腺癌、胰腺癌、白血病、骨癌、睾丸癌。
  33. 根据权利要求25所述的方法，其中所述重组溶瘤腺病毒的施用剂量为5×10 7至5×10 12VP/天，每天1-2次，连续施用1-7天。
  34. 根据权利要求25所述的方法，其中所述NK细胞的施用剂量为1×10 7至1×10 10个细胞/天剂量，每天1次，连续施用1-6天。
  35. 根据权利要求25所述的方法，其中所述重组溶瘤腺病毒的施用剂量为5×10 7至5×10 12VP/天，每2天1次，连续施用2-6天。
  36. 根据权利要求25所述的方法，其中所述NK细胞的施用剂量为1×10 7至1×10 10个细胞/天剂量，每2天1次，连续施用2-6天。
  37. 根据权利要求25所述的方法，其中所述重组溶瘤腺病毒通过瘤内注射给药或静脉给药；所述NK细胞通过静脉给药。
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