CN113832114A - Novel oncolytic adenovirus EM/VSV-G Ad5-P and application thereof in preparation of antitumor drugs - Google Patents

Abstract

The invention relates to the field of tumor treatment, in particular to application of oncolytic adenovirus EM/VSV-G Ad5-P in preparation of antitumor drugs. The invention discloses an anti-tumor effect of oncolytic adenovirus EM/VSV-G Ad5-P in vitro and in vivo. An oncolytic adenovirus EM/VSV-G Ad5-P encapsulated by an exosome, hereinafter referred to as a novel virus, was produced using a cell expressing VSV-G and using an exosome-like technique. The novel virus realizes retargeting through VSV-G, infects CAR low expression cell line, increases gene transduction efficiency of the virus, can resist neutralization of virus antibody, and can improve virus yield. In vitro studies find that the infection efficiency, the oncolytic capacity and the expression level of PD-1 of the novel virus are remarkably improved in a CAR low-expression cell line. In vivo experiments show that the novel virus can resist the neutralization of Ad5 antibody, prolong the virus treatment window and continuously express PD-1, so the novel virus can obviously activate anti-tumor immunity and finally obviously prolong the survival of mice.

Description

Novel oncolytic adenovirus EM/VSV-G Ad5-P and application thereof in preparation of antitumor drugs

Technical Field

The invention relates to the field of tumor treatment, in particular to the field of tumor immunotherapy, and specifically relates to application of oncolytic adenovirus EM/VSV-G Ad5-P in preparation of antitumor drugs.

Background

Malignant tumor is still a major disease threatening human health worldwide¹. In recent years, the development of tumor immunotherapy against immune checkpoint receptors such as Cytotoxic T-lymphocyte-associated protein 4 (CTLA 4), apoptosis receptor 1(programmed cell-death receptor 1PD-1) and apoptosis receptor 1 ligand (programmed cell-death receptor 1PD-L1) has been a revolutionary approach to tumor therapy². In particular, anti-PD 1/PDL1 antibody treatment has been shown to have therapeutic effects on at least 15 tumors^3-6A number of PD-1/PD-L1 inhibitors have been approved worldwide for clinical therapy⁶. With the deep research and the discovery of numerous clinical trials, the treatment effect of blocking the PD-1/PD-L1 pathway is related to the T lymphocyte infiltration and the PD-L1 expression of the tumor microenvironment^2,7(ii) a The 'hot' tumor with a large number of TILs infiltrated in a tumor microenvironment and a high expression level of PD-L1 in tumor tissues has good treatment effect by using anti-PD-1/PD-L1^2,7,8(ii) a "Cold" tumors with low TILs in the tumor microenvironment, which had little effect on treatment with anti-PD-1/PD-L1 drugs^2,7,8. Therefore, increasing the number of TILs in the tumor microenvironment such that the tumor becomes "hot" is an important means to increase the effectiveness of tumor immunodetection point blockade therapy.

In addition to the PD-1/PD-L1 pathway blockade therapy limiting its effectiveness for a variety of reasons,the block of the systemic PD-1/PD-L1 pathway can cause the autoimmune damage of the self-activated T cells caused by uncontrolled, such as immune pneumonia, immune hepatitis, immune enteritis, immune myocarditis, immune islet cell damage and even nervous system damage, and the life of patients can be lost due to the autoimmune damage^1,5,6,9-11. Therefore, the tumor local blocking PD-1/PD-L1 application breaks T cell tolerance induced by tumor microenvironment, and is possibly a strategy for improving the PD-1/PD-L1 pathway blocking treatment safety¹². Numerous studies have demonstrated that oncolytic viruses can break tumor immune tolerance, induce anti-tumor immunity, and change 'cold' tumors into 'hot' tumors⁸(ii) a Simultaneously, the oncolytic virus, particularly the adenovirus can also be used as a gene therapy vector^13-16And locally expressing the target gene product in the tumor. Research proves that OV-Ad5 tumor locally expresses soluble PD-1, anti-PD-1 or anti-PD-L1 to inhibit PD-1/PD-L1 pathway to induce antitumor immunity, obviously inhibit tumor growth to prolong survival period of mice, and reduce side effect of PD-1/PD-L1 pathway blocking treatment^12,17. The treatment strategy of using the oncolytic adenovirus to locally express the PD-1/PD-L1 inhibitor in the tumor has huge clinical application prospect.

Although the strategy of locally expressing PD-1/PD-L1 inhibitor in tumor by oncolytic adenovirus has great advantages, native tropism of adenovirus limits the application of oncolytic adenovirus^18-22. Ad5 enters cells by binding of the viral fiber knob to CAR (the coxsackie and adenovirus receptor) on the target cell membrane^23,24. A large number of studies indicate that Ad5 has poor efficiency in infecting tumors with low expression of CAR on the cell surface, which also limits the clinical efficacy of oncolytic adenovirus Ad 5-mediated tumor gene therapy^25-27And also because most tumor cells express low CARs^28,29Therefore, development of a method for enhancing the infection efficiency of CAR-low expressing tumor cells is crucial. In addition, the widely-existing adenovirus neutralizing antibodies in human body are also important factors limiting the clinical efficacy of adenovirus therapy^30-32。

Methods are available to increase the efficiency of infection of CAR-low expressing cells by Ad 5. The first approach is to modify Ad with covalent bonds5 by attaching artificial polymers, including polyethylene glycol (PEG), to the Ad5 capsid^33,34,polyactic glycolic acid (PLGA)³⁵,Polyethyleneimine(PEI)⁶Or lipids^36,37. The second approach is to achieve retargeting of adenovirus by genetic engineering of Ad5, such as: ad5-RGD of Ad5 fiber knob domain with Arg-Gly-Asp (RGD) polypeptide inserted in HI loop region³⁸(ii) a Fiber chimeric Ad5/35 of Ad5 and Ad35³⁹. These approaches do increase the efficiency of viral infection to some extent, but are relatively cumbersome to operate.

In recent years, an artificial exosome-mimetic drug loading technique has been used to replace the natural collection exosome technique. The exosome-mimetic technology can overcome the problem of low yield of natural exosomes, and exosome-mimetic with different targeting properties can be manufactured by using different cell types^40-42. exosome-mimetic technology is currently used only to encapsulate drugs for targeted delivery^42-44So far, no report is found on the use of the enveloped virus. Moreover, it is widely believed that this technique is unlikely to be used to package such large therapeutic entities of viruses. Therefore, the invention hopes to realize breakthrough through our research, EM/VSV-G Ad5-P is prepared by wrapping oncolytic adenovirus capable of expressing PD-1 by the technology, VSV-G protein is also introduced on the exosome wrapping virus, the retargeting of the virus can be realized, naked virus can be infected by cells which cannot be infected or are low in infection, and not only is the EM/VSV-G Ad5-P prepared by the technology resistant to antiviral neutralizing antibody. Then the novel oncolytic virus EM/VSV-G Ad5-P did not demonstrate unexpected effects in vitro or in vivo.

Disclosure of Invention

In this study, we show an exosome-mimetic encapsulated oncolytic adenovirus expressing soluble PD-1 (Ad 5-P). Exosome-mimetic encapsulated adenovirus EM/VSV-G Ad5 was produced using exosome-mimetic technology using 293T cells expressing Vesicular stored Indiana virus G protein (VSV-G). The virus manufacturing method not only enables EM/VSV-G Ad5 to be retargeted through VSV-G, infects a CAR low expression cell line, increases the gene transduction efficiency of Ad5, can resist the neutralization effect of an Ad5 antibody, but also can improve the virus yield. In vitro studies using exosome-mimetic-encapsulated oncolytic adenovirus EM/VSV-G Ad5-P found that the infection efficiency, oncolytic capacity and expression level of soluble PD-1 of EM/VSV-G Ad5-P were all significantly improved in CAR low-expression cell lines. In vivo experiments show that EM/VSV-G Ad5-P can resist the neutralization of Ad5 antibody, prolong the virus treatment window and continuously express soluble PD-1, so that the immune activation of the tumor microenvironment of the EM/VSV-G Ad5-P treatment group is obviously higher than that of the Ad5-P control group, and finally the survival of the mice is obviously prolonged.

In the invention, an in vitro experiment shows that the novel oncolytic virus EM/VSV-G Ad5-P has stronger tumor cell oncolytic capacity on a plurality of different tumor cells, and simultaneously, a large amount of soluble PD-1 can be expressed and secreted on the different tumor cells, and the expressed PD-1 can block a PD-1/PD-L1 immunodetection point access. In vivo studies find that the novel oncolytic virus EM/VSV-G Ad5-P can remarkably prolong the survival period of tumor-bearing mice, and the antitumor immunity effect also has an immunological memory effect. The antitumor immunity of the novel oncolytic virus EM/VSV-G Ad5-P is mainly realized by CD8+ T cells and NK cells and the release of IFN-gamma. In addition, the novel oncolytic virus EM/VSV-G Ad5-P can resist the neutralizing antibody generated after the virus is used and improve the anti-tumor immune effect.

In general, the novel oncolytic virus EM/VSV-G Ad5-P prepared by the exosome-like technology has the following outstanding substantive characteristics and remarkable progress:

1. the novel oncolytic virus EM/VSV-G Ad5-P has high infection efficiency on a plurality of different tumor cells with low expression of CAR;

2. the oncolytic capacity of the novel oncolytic virus EM/VSV-G Ad5-P on various tumor cells;

3. the soluble PD-1 of the novel oncolytic virus EM/VSV-G Ad5-P in tumor cells and tissues has high expression level and is continuous and stable, and the anti-tumor treatment effect of the virus can be ensured;

4. novel oncolytic virus EM/VSV-G Ad5-P is capable of resisting neutralization of Ad5 antibody, prolonging virus treatment window, and continuously expressing soluble PD-1

5. The novel oncolytic virus EM/VSV-G Ad5-P has obvious anti-tumor immune activation effect, can obviously prolong the survival time of a mouse, and has obvious tumor treatment effect.

Therefore, the contents to be protected of the present invention include:

a novel oncolytic virus EM/VSV-G Ad5-P can express the soluble adenovirus outside PD1 extracellular region and is coated with an exosome-like nano vesicle, and VSV-G protein is arranged on the membrane of the exosome-like nano vesicle.

A novel oncolytic virus EM/VSV-G Ad5-P can express the soluble adenovirus outside PD1 extracellular region and is coated with an exosome-like nano vesicle, and VSV-G protein is arranged on the membrane of the exosome-like nano vesicle. The VSV-G protein can realize the targeting of tumor cells; and due to the existence of exosome-like nanovesicles, the novel oncolytic virus EM/VSV-G Ad5-P can not be acted by a neutralizing antibody of an anti-adenovirus, and has a remarkably prolonged administration window period; the novel oncolytic virus EM/VSV-G Ad5-P can express a soluble PD1 extracellular region more continuously and block a PD-1/PD-L1 immunodetection point path continuously.

A novel oncolytic virus EM/VSV-G Ad5-P is prepared by the following method:

(1) preparing a cell in which a VSV-G protein is inserted into the outer membrane of the cell, and designating the cell as a VSV-G cell;

(2) infecting VSV-G cells by adenovirus Ad5-P, wherein the adenovirus Ad5-P is adenovirus capable of expressing a soluble PD1 extracellular region;

(3) resuspending the collected VSV-G cells after infecting adenovirus with culture medium, PBS or other buffer solution, and sequentially pressing the cell suspension through a membrane filter paper with a pore size of 10 μm 5 μm 1 μm by using a squeezer;

(4) collecting the extruded virus suspension, and enriching and collecting the novel oncolytic virus EM/VSV-G Ad5-P encapsulated by the exosome by a density gradient centrifugation method, wherein the novel oncolytic virus EM/VSV-G Ad5-P is encapsulated by the exosome, and VSV-G protein is arranged on the membrane of the encapsulated exosome, so that the targeting can be realized.

The novel oncolytic virus EM/VSV-G Ad5-P is applied to the preparation of antitumor drugs, and the tumors comprise but are not limited to tumors such as liver cancer, kidney cancer, leukemia, lung cancer, melanoma and colorectal cancer.

The novel oncolytic virus EM/VSV-G Ad5-P is applied to the preparation of tumor immunotherapy drugs, and the tumors include but are not limited to liver cancer, kidney cancer, leukemia, lung cancer, melanoma, colorectal cancer and other tumors.

The novel oncolytic virus EM/VSV-G Ad5-P can infect tumor cells, replicate and express soluble PD1, and the expressed PD1 is combined with PD-L1 so as to block the PD-1/PD-L1 immunodetection point pathway.

Drawings

FIG. 1 relationship of CAR expression to Ad5-GFP infection efficiency. Non-replicating adenovirus Ad5-GFP expressing GFP was constructed, and 293T, A549, HCC-LM3, Hepa1-6, B16-F10, CT26.WT, H22, K562 and Jurkat cells were infected with Ad5-GFP for 72 hours and then used for fluorescent microscopy analysis or flow analysis. a. The same number of 293T, A549, HCC-LM3, Hepa1-6, B16-F10, ct26.wt, H22, K562 and Jurkat cells were stained with monoclonal antibody anti-CAR-PE for flow analysis of the expression level of CAR of each cell line; isotype is a control in which cells are treated with homologous IgG-PE antibodies. b. Schematic diagram of genome structure of adenovirus Ad 5-GFP. And c, typical fluorescence photographs of Ad5-GFP virus infected 293T, A549, HCC-LM3, Hepa1-6, B16-F10 and CT26.WT cells after 72h, wherein histograms are flow statistics of GFP positive cell ratios, and the infection efficiency of the Ad5 in other cell strains in each group is calculated by taking the 293T infection efficiency as 100%. A typical scatter diagram is analyzed in a flow mode after Ad5-GFP virus infects suspension cells H22/K562/Jurkat, and a histogram is a flow result statistical chart. Data are presented as mean ± SD.

FIG. 2 preparation of exosome-mimetic Ad5 with VSV-G (EM/VSV-G Ad 5). Schematic representation of EM/VSV-G Ad5 virus preparation. Transmission electron micrographs of Ad5-GFP and EM/VSV-G Ad5-GFP viruses. The arrows in the figure indicate membrane protein protrusions on the membrane surface. Western blot analysis VSV-G content in Ad5-GFP and EM/VSV-G Ad5-GFP viruses collected by density gradient centrifugation purification. d. Virus titer determination methods different density gradients were analyzed for collection of EM/VSV-G Ad5-GFP and Ad5-GFP viruses. The left side is a picture of the distribution of the virus after EM/VSV-G Ad5-GFP is subjected to density gradient centrifugation, and the right side histogram is the proportion of the virus in different density gradients of Ad5-GFP and EM/VSV-G Ad 5-GFP. Ad5 was used to infect 293T-VSV-G cells, the cells were divided equally after 72 hours, and the virus was recovered by using exome-mimetic (new) and conventional freeze-thaw lysis method (conventional), respectively, and the virus titer was measured to calculate the amount of virus recovered, and the fold increase in the amount of virus recovered by exome-mimetic was calculated with the amount of virus in the freeze-thaw lysis method being 1. f. Ad5 and EM/VSV-G Ad5-GFP were treated with different dilutions of Ad5 neutralizing antibody, respectively, and then infected 293T cells, respectively, and the infection replication was analyzed by fluorescence microscopy after 48 hours. The left panel is representative fluorescence photograph of 3 independent experiments, and the right panel is statistical chart of the change of rate of GFP positive cells after virus infection replication by flow analysis. g. Ad5-GFP and EM/VSV-G Ad5-GFP were treated separately with different dilutions of VSV-G neutralizing serum, then infected separately into 293T cells, and the infection replication was analyzed by fluorescence microscopy after 48 hours. The left panel is representative fluorescence photograph of 3 independent experiments, and the right panel is statistical chart of the rate change of GFP positive cells after virus infection replication by flow analysis. Data were mean ± SD,. p < 0.001.

FIG. 3 infection rates of EM/VSV-G Ad5-GFP and Ad5-GFP viruses on CAR high-and low-expressing cell lines. 293T, Hepa1-6, B16/F10, CT26.WT and H22 were infected with MOI-1 Ad5-GFP and EM/VSV-G Ad5-GFP viruses, respectively, and with MOI-100 virus Jurkat and K562, and after 72 hours, the virus infection efficiency was analyzed by photographing using a fluorescence microscope and by flow analysis. a. Pictures are representative fluorescence photographs in 3 independent experiments, and histograms are flow statistics. b. The pictures are representative flow charts in 3 independent experiments, and the histogram is a flow result statistical chart. Data were mean ± SD, # p > 0.05, # p < 0.01, # p < 0.001.

FIG. 4 in vitro study of the properties of oncolytic adenovirus EM/VSV-G Ad5-P prepared by EM/VSV-G technology. Schematic structural diagram of oncolytic adenovirus Ad 5-P. EXO PD1 extracellular region, TM PD1 transmembrane region, ENDO PD1 intracellular region, 6His tag sequence. Statistics of the infection efficiency of Ad5-P and EM/VSV-G Ad5-P in 293T, Hepa1-6, B16/F10, CT26.WT, H22 cell lines. c, Ad5-P and EM/VSV-G Ad5-P are respectively infected with Hepa1-6, B16/F10, CT26.WT and H22, infected cells are collected at 12H, 24H, 36H, 48H, 60H and 72H, and virus copies changes are analyzed by using quantitative PCR, wherein the curve is that the virus copies at 12H is 1, and the virus copies changes at each time point are calculated to obtain a virus amplification curve. d. Different MOI value viruses infect different cell strains respectively, and the MTT detects the cell viability after 72 hours. e.MOI 1 Ad5-P and EM/VSV-G Ad5-P infected different cell lines, respectively, 72h collecting supernatant, ELISA detecting soluble PD1 expression level. The data are mean + -SD # # p > 0.05, p.ltoreq.0.05, p.ltoreq.0.01, p.ltoreq.0.001.

FIG. 5 in vivo study of the anti-tumor immunotherapeutic effect of EM/VSV-G Ad 5-P. 8 week old C57BL6 male mice, divided into 3 groups of 7 mice, were intraperitoneally injected with H22 cells on day 0 at 5X 10⁶cells, treatment groups were injected intraperitoneally with virus 5X 10 in each mouse on days 3,9,15, and 21⁸pfu. The control group (saline group) was injected with an equal amount of PBS buffer. All mice were tested for ascites on days 11,15, 18. Mice survival was monitored until mice died. Groups of surviving mice were injected intraperitoneally with 5X 10H 22 cells on day 80⁶cells were challenged twice and the status of the mice was monitored for 140 days. a. Schematic diagram of in vivo experiment. b. Survival curves for each group of mice. c. The survival curve of the mice after the second challenge,

the group was new 8-week-old C57/BL male mice. P < 0.01.

FIG. 6 Effect of EM/VSV-G technology on anti-tumor immune activation and anti-tumor effect of oncolytic adenovirus. The proportions of CD4+ T cells, CD8+ T cells, NK cells and PD-L1 positive cells in ascites on day 11 were analyzed by flow cytometry on total cells. a. Flow analysisThe proportion of CD4+ T cells to total cells, and the markers of CD4+ T cells are CD3-APC and CD4-FITC double positive cells; b. flow analysis of the proportion of CD8+ T cells to total cells, CD8+ T cell markers CD3-APC and CD8-PerCP-Cy^TM5.5 double positive cells; c. and analyzing the proportion of the NK cells in the total cells by flow, wherein the NK cells are marked as CD3-APC negative NK1.1-FITC positive cells. ELISA analysis of IFN-. gamma.expression in ascites of Day 11. e. The proportion of PD-L1 positive cells to total cells was analyzed by flow analysis, and PD-L1 positive cells were labeled with PD-L1-PE. Elispot assay for IFN-. gamma.expression in ascites by Day 11. The pictures are 3 photographs representing the Elispot results in each group, and the statistical chart is the statistics of all Elispot spots in each group. g. Trypan blue staining analyzes the cell viability in ascites fluid of Day 11. ELISA analysis of soluble PD1 expression in ascites fluid of Day 11,15 and 18. The results were mean. + -. SD, # p ≧ 0.05, # p < 0.01, # p < 0.001.

FIG. 7EM/VSV-G Ad5-P evading neutralizing antibody promotes sustained expression of soluble PD 1. Soluble PD1 time-courses in ascites of mice in the Ad5-P and EM/VSV-G Ad5-P treated groups. Soluble PD1 change curve in ascites of each mouse in Ad5-P treated group. c. Soluble PD1 change curves in ascites of each mouse in the EM/VSV-G Ad5-P treated group. Mice in the Ad5-P treated group and EM/VSV-G Ad5-P treated group were treated with Ad5-P and EM/VSV-G Ad5-P, respectively, in ascites of Day15, and 293T cells were infected with Ad5-P and EM/VSV-G Ad5-P after treatment, and virus infection efficiency was analyzed by flow analysis 48 hours later. d. After treating Ad5-P for Ad 5-P-treated ascites fluid, the GFP positive cell rate was flow counted. e. After treatment of EM/VSV-G Ad5-P for ascites fluid from Ad5-P treated group, GFP positive cell ratios were flow counted. f. After treating Ad5-P for ascites in the EM/VSV-G Ad5-P treated group, the GFP positive cell ratio was flow counted. g. After treatment of the ascites fluid in the EM/VSV-G Ad5-P treatment group with EM/VSV-G Ad5-P, the GFP positive cell ratio was flow counted. EM/VSV-G Ad5-P treated groups of mice were treated with ascites in Day 15: DMEM is diluted to be 1:4, the VSV-Lenti-GFP lentiviral vector is respectively treated by ascites after dilution, the 293T cell is infected by the VSV-Lenti-GFP lentiviral vector after treatment, and the ratio of GFP positive cells is analyzed in a flow mode after 48 h; the chart is a flow result statistical chart. The results were mean + -SD, # p > 0.05, # p < 0.01, # p < 0.001.

FIG. 8 EM/VSV-G Ad5-P evading neutralizing antibody promotes sustained expression of soluble PD 1.

Ad5-P encapsulated by Exosome-mimoic has significantly increased ability to infect CAR-low expressing tumor cells compared to Ad 5-P; the virus oncolytic effect is enhanced, EM/VSV-G Ad5-P coated by Exosome-mimetic can resist adenovirus neutralizing antibody, and the virus application time is prolonged; remarkably prolonging the expression and secretion time of soluble PD1, and further promoting infiltration of CD8+ T lymphocytes to tumors; finally, the anti-tumor immune response of the mouse is enhanced, and the survival period of the mouse is obviously prolonged.

Detailed Description

All experimental materials and methods referred to in the examples section

1. Cell lines and cell culture methods

Human and murine hepatocellular carcinoma cell lines HCC-LM3 and H22 were purchased from the cell bank of the culture collection committee of the chinese academy of sciences and were identified by STR and detected by mycoplasma. The mouse liver cell cancer cell line Hepa1-6 human embryonic kidney cell line 293T, human T cell leukemia cell line Jurkat, human chronic myelocytic leukemia cell line K562, human alveolar adenocarcinoma cell line A549, mouse melanoma B16-F10, and Lewis colon cancer cell line CT26.WT were from ATCC company of America. 293T-VSV-G is an engineered 293T cell line expressing VSV-G. Jurkat K562 and H22 were cultured using RPMI 1640 medium containing 10% fetal bovine serum 2mM glutamine, 100units/mL penicillin, and 0.1mg/mL streptomycin. HCC-LM3 Hepa 1-6A 549B 16-F10 and 293 were cultured using DMEM medium containing 10% fetal bovine serum 2mM glutamine, 100units/mL penicillin, and 0.1mg/mL streptomycin. All cell cultures were incubated in a thermostatted cell incubator containing 5% carbon dioxide at 37 ℃.

Construction of 293T-VSV-G cell line

293T cells were co-transfected with eukaryotic expression plasmid pCMV-VSV-G expressing VSV-G, eukaryotic expression vector pCMV-REV expressing REV, eukaryotic plasmid pCMV-gag-pol expressing gag-pol lentiviral backbone protein, and eukaryotic expression plasmid pCMV-CAG-VSV-G-2A-puro expressing lentiviral genome carrying the VSV-G gene, and the supernatant was collected after 72 hours and filtered using a 0.45 micron syringe filter. 293T cells were infected with viral supernatant and maintained in culture after 48 hours using puromycin containing 1 microgram per ml. The sequence of CAG-VSV-G-2A-PURO is shown in SEQ ID NO 13.

2. Construction of recombinant adenovirus

Construction of the Adenoviral vector Ad5 and oncolytic adenovirus Ad5-P ViraPower was used^TMAdenoviral Expression System and

pENTR^TMvectors (all from Life Technologies, Grand Island, NY). Virus construction was according to manufacturer's instructions. E1A, CMV, polyA, GFP, and PD-1 extracellular regions were first amplified using PCR techniques, E1A PCR template using the 293T genome, PD-1cDNA purchased from Sino Biological Inc. CMV, GFP and poly A PCR templates using the lenti-GFP plasmid. The DNA sequence of the PD-1 extracellular region is shown in SEQ ID NO. 1, the DNA sequence of the CMV promoter is shown in SEQ ID NO. 3, and the DNA sequence of E1A is shown in SEQ ID NO. 5. Obtaining CMV-GFP-POLYA and CMV-GFP-CMV-sPD1-POLYA gene fragments by PCR amplification and ligation technology, and cloning the obtained gene fragments to a penetrator adenovirus plasmid pENTR by using TOPO-clone technology^TMVectors. The cloned shuttle plasmid is recombined with an adenovirus skeleton pAd/PL-DEST to obtain recombinant adenovirus vectors pAd5-GFP and pAd 5-P. Rescue of recombinant viruses: the recombinant adenovirus vectors pAd5 and pAd5-P restriction enzyme PacI are linearized and purified. 293T cells were prepared one day in advance and seeded with 6-well plates 50 million cells/well, and 1ug of linearized adenovirus vector was transfected into the prepared 293T cells using Lipofectamine 2000. Continuously culturing for 10-14 days, and replacing complete culture medium containing 5% FBS once for 2-3 days until 70-80% of cells have cytopathic effect, collecting cells and supernatant, freezing and thawing for 3 times, centrifuging at 4000 Xg for 20min, and collecting virus seeds. Using 293T-VSV cells for Ad5 and Ad5-P adenovirus amplification and purification, infecting the cells for 72 hours by using viruses with MOI of 5, repeatedly freezing and thawing the cells, centrifuging the cells for 20min at 4000 Xg to remove cell debris, and collecting supernatant to obtain virus suspension; purification of the virus was performed using iodixanol density gradient centrifugation. Virus titer was determined by inoculating 96-Wells plates with 293T cells, 10000 cells/well; diluting the virus by 10 times; 100ul of the water is added to the water,wells with green fluorescent cells were defined as positive by 4 days of culture, using a fluorescence microscope, counting 0.1 per positive well, and all positive wells were summed to S. Virus TCID50 ═ 10^2+(S/10-0.5)/ml, pfu/ml＝0.7×TCID50/ml。

3. Preparation of Exosome-mimetic adenoviruses EM/VSV-G Ad5 and EM/VSV-G Ad5-P

exosome-mimetic adenovirus was prepared by inoculating 6cells of 10cm in size with 293T-VSV-G cells²Culture dish (1X 10)⁷cells/plate), after overnight incubation, inoculated at 5X 10⁷pfu of Ad5-GFP or Ad5-P virus, culturing for a further 72h, collecting the cells, removing the cell culture supernatant, resuspending in 30ml of DMEM, pressing the cell suspension in succession through a 10 μm 5 μm 1 μm polycarbonate membrane filter (whatman) using a mini-extruder (avanti polar lipids), collecting the pressed virus suspension, adding the virus suspension to a centrifuge tube with a 15% 20% and 40% iodixanol (iodoxanol) cushion, centrifuging at 100000 Xg for 90min, and collecting the zones between 15% and 20% and between 20% and 40% iodoxanol, respectively. Dialyzing with 5% glycerol, 1mM MgCl2, 150mM NaCl, 10mM Tris-HCl (pH7.6) for 2 times, determining virus titer, and storing in-80 deg.C refrigerator.

4. Detection of viral infection efficiency

The virus infection efficiency test firstly uses 1 × 10⁵cells/well to be detected cells are inoculated on a 24-well plate, corresponding viruses are added according to the specified virus amount, the cells are cultured for 48 hours at 37 ℃ under 5 percent CO2, and the cells are observed and photographed by a fluorescence microscope; the cells were collected and the rate of GFP positive cells was counted by flow counting, and the infection efficiency of other cell viruses was calculated with the rate of 293T GFP positive cells infected with Ad5-GFP or Ad5-P virus being 1.

5. Transmission electron microscope scanning

EM/VSV-G Ad5-GFP and Ad5-GFP virus suspensions were added to a copper magnetic film mesh (400mesh, Agar Scientific) projection electron microscope and allowed to air dry at room temperature. Staining with 1% phosphotungstic acid, and observing using JEM-200CX projection electron microscope (JEOL, Japan).

6. Neutralization test

Adenovirus neutralizing antibody is presented to gland by Wang Shi (Zhejiang university of science and technology)Virus immune rabbit serum. VSV-G neutralizing antibodies were from verified volunteer sera. Animal Experimental ascites sample treatment cells and cell debris were removed by centrifugation for 10min at 400 Xg and 12000 Xg respectively. Neutralization test serum and ascites were both inactivated at 56 ℃ for 30 min. 293T inoculation with 24well-plate (1X 10)⁵cells/well), cultured at 37 ℃ for 4 hours, and then used in 100ul of 1X 10-containing medium⁵DMEM of pfu virus was incubated with the indicated dilution of neutralizing serum or ascites fluid at 37 ℃ for 30min, and the virus antibody mixture was added to pre-inoculated 293T cells (1X 10)⁵cells/well) at 37 ℃ for 1 hour, removing the virus supernatant, adding complete medium for another 48 hours, observing fluorescence, performing flow statistics on the ratio of GFP positive cells, and calculating the ratio of antibody neutralization in each treatment group with the ratio of GFP positive cells in the group not treated with the antibody as 100%.

7. MTT cell viability assay

Inoculating a cell strain to be detected to 96-well plate, culturing 1 ten thousand cells in each hole at 37 ℃ for 4 hours, adding corresponding viruses according to the specified virus amount, removing virus suspension after 4 hours, replacing complete culture medium, and continuously culturing for 72 hours. 100ul of MTT dilution (1mg/ml) per well was incubated for an additional 4 hours. The supernatant was removed, 150ul isopropanol was added, shaking was carried out for 15min, and absorbance was measured using 570 wavelengths. The cell force of the other treatment groups was calculated by taking the absorbance of the untreated cell culture wells as 100% of the cell viability.

8. Q-PCR detection of replication function of virus

Cells were harvested at 6H, 24H, 36H, 48H, 60H, 72H after treatment with 5 MOI virus Ad5-P or EM/VSV-G Ad5-P inoculated with Hepa1-6, B16/F10, CT26.WT and H22 cells, respectively, and total DNA was extracted using a genome extraction kit (Tiangen Biochemical). Using PowerUp^TM SYBR^TMAnd (3) Green Master Mix, taking the quantified shuttle-Ad5-P plasmid as a standard substance and Q-E1A F and Q-EIA R as primers, and carrying out quantitative PCR detection on the genome copies of the virus at each time point. The number of 6h virus copies was 1 for each treatment group, and the fold increase of virus copies at each time was calculated.

9. Western Blot detection of target protein expression in supernatant

Purified Ad5-GFP and EM/VSV-G Ad5-GFP virus suspensions were density gradient centrifuged, and 80ul of protein concentration was measured using BCA followed by addition of 5 Xloading buffer (containing. beta. mercaptoethanol) (Beyotime, Hangzhou)20 ul. After treatment at 100 ℃ for 5min, the cells were electrophoresed to polyvinyidene fluoride membranes (# 03010040001; Roche) using SDS-PAGE at a concentration of 12%, and then blocked for 30min using 10% skim milk TBS blocking solution. The results were obtained by imaging the primary antibody with Anti-VSV-G tag antibody (ab1874 abcam 1:2000), the secondary antibody with rabbit Anti-mouse monoclonal antibody labeled with horseradish peroxidase (A00160 GenScript Biotech Corp.1:5000), with chemiluminescent reagent (WBKLS 0500; Millipore, Billerica, MA, USA), and with a chemiluminescence imager (ChampChemi 610; Sage Creation Science, Beijing, China).

10. Enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay ELISA)

Quantitative detection of soluble protein SPD1

ELISA 96-well plates were coated with 2. mu.g/ml Anti-His-tag monoclonal antibody (purchased from Nanjing King Shirui Biotech Co., Ltd., Nanjing, China). Adding 100 mul of cell supernatant or ascites, and incubating at 37 ℃ for 2 hours; washing away unbound soluble protein with phosphate buffered saline (PBS-T) containing 0.1% Tween 20, adding rabbit anti-mouse PD-1 monoclonal antibody (purchased from Beijing Yiqianzhou Biotech limited, Beijing, China) as detection antibody, and incubating at 37 deg.C for 1 hr; PBS-T washing for 5 times, adding 100 μ l biotin-labeled goat anti-rabbit IgG antibody and HRP-labeled avidin diluted solution, and incubating for 1 hour at room temperature. After washing with PBS-T for 10 times, substrate TMB was added thereto and reacted at room temperature for 30 minutes, 50. mu.l of stop solution (2N sulfuric acid) was added thereto, and OD450 was detected using a microplate reader.

Detection of interferon gamma: for detection of interferon gamma in mouse ascites and serum, mouse IFN-. gamma.ELISA kit (BD Biosciences, Franklin Lakes, NJ, USA) from BD Co was used, and the detection procedure was performed according to the instruction.

11. Mouse tumor model

Mouse hepatocellular carcinoma ascites tumor model using SPF grade 8 week old C57BL/6 male mice (purchased from the university of Nanjing model animal research center). Each mouse was injected intraperitoneally with 5 x 106H22 cells. Mice were each injected intraperitoneally with 5X 108PFUs of virus and controls with equal amounts of viral solutes on days 3,9,15 and 21, respectively. Ascites fluid was collected for examination on days 11,15 and 18 after tumor injection. The cured mice were re-injected intraperitoneally with 5X 106H22 cells on day 80, and untreated mice were used as the negative control group. Mice survival was monitored and recorded. .

12. Flow cytometry analysis

Immune cell subpopulation detection in ascites

Ascites from mice were extracted according to the protocol, centrifuged at 400 Xg for 5 minutes to collect cells, and washed twice with PBS. The antibodies were incubated for 30min at room temperature in the dark according to the cell marker combination. PBS wash twice using 500 u l PBS heavy suspension, using flow cytometry detection analysis. T cells were transfected with CD3-APC, CD4-FITC, and CD8-PerCP-Cy^TM5.5 two T cell subsets of labeled CD3+ CD4+ or CD3+ CD8 +. NK cells CD3-APC and NK1.1-FITC labeled CD3-NK1.1+ cells were used. PD-L1 positive cells were labeled with CD274 PE. CD3 APC, CD8a PerCP-Cy^TM5.5, CD4 FITC, NK1.1 FITC and CD274 PE (available from BD Biosciences, Franklin Lakes, NJ, USA).

Expression detection of CAR on tumor cell surface

100 million cells were collected from each cell line by centrifugation and washed 2 times with PBS. Incubate with anti-CAR-PE antibody for 30min at room temperature (protected from light). Washed 2 times with PBS and resuspended in 500. mu.l of PBS. Analysis was detected using a flow cytometer.

13. IFN-gamma enzyme linked spot assay (IFN-gamma EILSpot assay)

Activated lymphocyte detection in ascites

Preparation of IFN- γ EILSpot assay plate: IFN-. gamma.EILSpot detection plates were prepared as per the instructions of the Mouse IFN-. gamma.EILSpot PLUS kit (available from 3321-2 AW-PLUS; Mabtech, Nacka Strand, Sweden). Ascites of the mice are extracted at the appointed time point according to the experimental scheme, and cells in the ascites are collected by removing the ascites through a centrifugal method. After washing twice with PBS, the cells were stained with placental blue and counted. A cell suspension of 1X 106cells/ml was prepared. The cell suspension was added to a previously prepared IFN- γ EILSpot assay plate in a volume of 200. mu.l per well. The cells were incubated overnight at 37 ℃ in a cell incubator containing 5% carbon dioxide for 20 hours. The cell suspension was removed, treated as per the instructions and developed. Detection assays were performed using an ELISpot plate reader.

Example 1 adenovirus Ad5 infection of cells with low expression of CAR was inefficient

CAR (the coxsackie and adenovirus receptor) is the main receptor for adenovirus Ad5 infected cells. Early studies have demonstrated that adenovirus Ad5 infects cells with low expression of CAR with low efficiency.^23,29We found by flow analysis that 293T, A549, HCC-LM3 and Hepa1-6 cells stained with anti-CAR-PE showed a significant increase in fluorescence intensity compared to the isotype treated group, indicating high expression of 293T, A549, HCC-LM3 and Hepa1-6 cell surface CARs (fig. 1 a); and after the surfaces of B16-F10, CT26.WT and H22 cells are stained by using the CAR antibody, the fluorescence intensity is not obviously changed compared with that of an isotype treatment group; indicating that these cell lines do not express CAR; whereas, the fluorescence intensity of K562 and Jurkat cells stained with the CAR antibody was only slightly increased compared to the isotype treated group, much lower than that of 293T cell lines, indicating that these cell lines underexpress CAR (FIG. 1 a). Next, to verify the relationship between the expression of CAR and the infection efficiency of Ad5, we constructed non-replicative adenovirus Ad5-GFP expressing GFP (fig. 1B), 293T, A549, HCC-LM3, Hepa1-6, B16-F10, ct26.wt and H22 cells treated with Ad5-GFP with MOI of 1, and found that 50% -60% of the 293T, A549, HCC-LM3, and Hepa1-6 cells with high expression of CAR were infected with Ad5-GFP and express GFP after 72H, and the infection efficiency of Ad5-GFP was not significantly different among these several cells; however, the efficiency of infection of adherent cells B16-F10, CT26.WT, H22 with Ad5-GFP, which are low in expression of CAR, is less than 5%, and is very significantly lower than 293T and other CAR-highly expressing cell strains (P < 0.001) (FIG. 1c, d). Suspension cell lines K562 and Jurkat infected only 8.26% + -0.64% and 12.08% + -0.81% of cells with Ad5-GFP virus at MOI of 100 for 72h, respectively, and very significantly less than the infection efficiency (52.3% + -2.61%) of Ad5-GFP virus at MOI of 1 for 293T (P < 0.001) (FIG. 1 d). The results prove that the infection efficiency of the adenovirus Ad5 on cells is related to the amount of the CAR expressed on the cell surface, the infection efficiency of the adenovirus Ad5 on the cells with high expression of the CAR is high, and the infection efficiency of the adenovirus Ad5 on the cells with low expression of the CAR is low.

Example 3 EM/VSV-G Ad5-GFP can counteract the effect of anti-Ad5 neutralizing antibodies and the entrained VSV-G mediates its entry into cells

To further demonstrate that EM/VSV-G Ad5-GFP is encapsulated in vesicles, and that such encapsulation brings new attributes, we treated EM/VSV-G Ad5-GFP with anti-Ad5 neutralizing antibody and VSV-G neutralizing serum, respectively, and followed analyses. After treatment of EM/VSV-G Ad5-GFP and Ad5-GFP viruses with anti-Ad5 neutralizing antibodies at 1/32, 1/64 and 1/128 dilution times, respectively, the infection efficiencies of the Ad5-GFP viruses were only 5.66 + -2.1%, 9.3 + -2.02% and 12.6 + -3.84% of the PBS-treated control group, while the infection efficiencies of EM/VSV-G Ad5-GFP were 61.0 + -3.10%, 71.3 + -3.7% and 81.6 + -1.2%, respectively (FIG. 2 f); therefore, the Ad5-GFP virus almost loses the infection capacity after the anti-Ad5 neutralizing antibody treatment, and the infection capacity of the EM/VSV-G Ad5-GFP can be remarkably rescued due to the encapsulation of an exosome-like outer membrane and the existence of VSV-G. After treatment of the two viruses with 1/4, 1/8, and 1/16 fold dilutions of VSV-G neutralizing serum, respectively, the infection efficiency of Ad5 was 97.66 + -4.97%, 97.33 + -1.76%, and 97 + -3.78% of the PBS control, while the infection efficiency of EM/VSV-G Ad5-GFP was 24.66 + -2.6%, 26.33 + -2.6%, and 67.33 + -5.45%, respectively (FIG. 2G); thus, it can be seen that the EM/VSV-G Ad5 virus entry into the infected cells is mediated by VSV-G. The above results indicate that EM/VSV-G Ad5-GFP can resist the action of anti-Ad5 neutralizing antibody and the carried VSV-G can help it obtain the infection ability of uninfected or low infected cells to naked virus.

Example 4 EM/VSV-G Ad5-GFP has high infection efficiency on CAR-low expressing cell lines

Previous studies have found that adenovirus Ad5 has low infection efficiency on cells with low expression of CAR, and we wanted to experimentally observe whether EM/VSV-G Ad5-GFP virus has high infection efficiency on cell lines with low expression of CAR, unlike Ad5-GFP virus. Here we examined the infection efficiency of EM/VSV-G Ad5-GFP and Ad5-GFP viruses on CAR-overexpressing cells. Surprisingly, the infection efficiency of EM/VSV-G Ad5-GFP was increased extremely significantly in cell lines B16/F10, CT26.WT, H22, K562 and Jurkat with low expression of CAR, to 8.00. + -. 0.54-fold, 2.83. + -. 0.04-fold, 14.99. + -. 1.09-fold, 11.35. + -. 0.37-fold and 6.25. + -. 0.58-fold, respectively, compared to Ad5-GFP (FIGS. 3a, B). In addition to the detection of the infection efficiency of EM/VSV-G Ad5-GFP and Ad5-GFP viruses on CAR-low expressing cells, we also detected the infection efficiency of EM/VSV-G Ad5-GFP and Ad-GFP viruses on CAR-high expressing cells, and the results showed that there was no significant difference in the infection efficiency between the CAR-high expressing cell strain 293T, A549 (data not shown) and LM3-HCC (data not shown); however, surprisingly, the infection efficiency of EM/VSV-G Ad5-GFP on Hepa1-6 cells was increased 1.253. + -. 0.028 fold significantly compared to Ad5-GFP virus (FIG. 3 a). The results show that the EM/VSV-G technology completely changes the property of low infection of Ad5-GFP virus to the CAR low-expression cell line, the EM/VSV-G Ad5-GFP virus prepared by the EM/VSV-G technology has extremely high infection efficiency to the CAR low-expression cell line, and the EM/VSV-G technology can also improve the infection efficiency of AD5-GFP to the individual CAR high-expression cell line, so that the possible application range of the EM/VSV-G technology is expanded to a certain extent. .

Example 5 EM/VSV-G technology EM/VSV-G Ad5-P has higher infection efficiency, ability to express soluble PD1 and oncolytic ability in vitro

The oncolytic adenovirus Ad5-P is an oncolytic adenovirus Ad5 (named Ad5-P) expressing soluble PD1, and the genome structure schematic diagram of the oncolytic adenovirus Ad5-P is shown in FIG. 4 a. The low infection rate of oncolytic adenovirus Ad5-P to CAR-low expressing tumor types severely limits the transformation applications of Ad 5-P. On the basis that the EM/VSV-G technology can completely change the low infectivity of Ad5 virus to a CAR low expression cell line, the EM/VSV-G technology is applied to oncolytic adenovirus Ad5-P, the infection efficiency of the oncolytic adenovirus Ad5-P to the CAR low expression cell line is expected to be remarkably increased through the EM/VSV-G technology, the treatment effect of the oncolytic adenovirus Ad5-P is improved, and the application range of the oncolytic adenovirus Ad5-P is expanded. The virus obtained by preparing oncolytic adenovirus Ad5-P by EM/VSV-G technology is named as EM/VSV-G Ad 5-P.

Compared with Ad5-P virus, the infection efficiency of EM/VSV-G Ad5-P to CAR low expression tumor cell line is remarkably improved (FIG. 4B), and the infection efficiency is respectively improved to 8.7 +/-0.62, 2.948 +/-0.039 and 14.596 +/-1.56 on B16/F10, CT26.WT and H22 cell strains.

We compared the replication efficiencies of EM/VSV-G Ad5-P and Ad5-P in different cell lines, and found that there was no significant difference in the replication efficiencies of EM/VSV-G Ad5-P and Ad5-P between the CAR high expression cell line Hepa1-6 and the CAR low expression cell line B16/F10, CT26.WT and H22 (FIG. 4 c). The results show that EM/VSV-G technology only increases the efficiency of viral infection and does not affect viral replication.

We next analyzed EM/VSV-G Ad5-P and Ad5-P for different viral titers for oncolytic effects in Hepa1-6, B16/F10, CT26.WT and H22 cells. There was no significant difference in the oncolytic effect of the virus in the CAR high expressing cell line Hepa1-6, perhaps because there was no significant difference in the infection and replication efficiencies of the two viruses; in the B16/F10, CT26.WT and H22 cell lines, the oncolytic effect of EM/VSV-G Ad5-P was significantly increased at high viral load (FIG. 4d), perhaps because the infection efficiency of EM/VSV-G Ad5-P was significantly higher than that of Ad5-P, and the amount of virus entering the cells was significantly increased although there was no increase in the virus replication efficiency.

Next, we infected cells with the same virus titer of EM/VSV-G Ad5-P and Ad5-P, and then examined the content of soluble PD1 secreted in the supernatant of the virus-infected cells, and the results showed that soluble PD1 expression in the supernatant of the culture medium of B16/F10, CT26.WT and H22 cells showed a very significant increase after EM/VSV-G Ad5-P infection, compared to Ad5-P (FIG. 4 e).

In general, EM/VSV-G Ad5-P prepared by using EM/VSV-G technology can improve the infection efficiency of oncolytic adenovirus Ad5-P in a low CAR expression cell strain, increase the oncolytic effect and the expression level of soluble PD1, and the results of in vitro researches indicate that the EM/VSV-G technology can possibly improve the anti-tumor effect of oncolytic adenovirus Ad5-P in vivo.

Example 6EM/VSV-G Ad5-P induced a stronger anti-tumor effect and immune memory, with a very significant extension of mouse survival.

We established a mouse ascites tumor model using the H22 cell line to study the anti-tumor effect of EM/VSV-G Ad5-P in vivo. The experimental protocol is shown in figure 5 a. Animal results found that the survival of mice was significantly prolonged in the EM/VSV-G Ad5-P treated group and Ad5-P treated group compared to the saline control group, and 5 and 2 mice were cured in the EM/VSV-G Ad5-P and Ad5-P groups, respectively, accounting for 71.4% and 28.5% (FIG. 5 b); the survival of mice was also significantly prolonged in the EM/VSV-G Ad5-P treated group compared to the Ad5-P treated group (FIG. 5 b). And when 2 attacks were performed on the cured mice, the Naive group mice all died on day 26; mice cured with EM/VSV-G Ad5-P and Ad5-P groups were observed for 60 days for no tumor growth (FIG. 5 c). These results indicate that EM/VSV-G Ad5-P obtained after the oncolytic virus Ad5-P is processed by EM/VSV-G technology has obviously enhanced anti-tumor effect; the secondary challenge experiment proves that EM/VSV-G Ad5-P induced tumor elimination has immunological memory.

To explore the mechanism of enhancement of the antitumor effect of EM/VSV-G Ad5-P, we examined the immune status in ascites on day 11 of the H22 ascites carcinoma model. Flow analysis found that CD4+ T, CD8+ T and NK cells were both significantly elevated in ascites in the EM/VSV-G Ad5-P virus treated group compared to the control group (salt group) (P ═ 0.009, P ═ 0.003, P ═ 0.0002); furthermore, the EM/VSV-G Ad5-P treated group was unable to elevate CD4+ T cells in ascites, and was able to extremely significantly elevate CD8+ T and NK cells (P0.012, P0.0042) compared to the Ad5-P virus treated group (fig. 6a, b, c). The IFN-gamma elispot method detects activated lymphocytes in ascites, and the IFN-gamma positive cells in the ascites of the EM/VSV-G Ad5-P group are found to be remarkably higher than those in the Ad5-P group (P ═ 0.0128) and very remarkably higher than those in the saline group (P ═ 0.0007) (FIG. 6f), while the Ad5-P group has no remarkable difference compared with the saline group. The ELISA method detects IFN-gamma concentration in ascites, the IFN-gamma concentration in the ascites of EM/VSV-G Ad5-P group is remarkably higher than that in Ad5-P group (P is 0.004) and saline group (P is 0.0016) (figure 6d), and the Ad5-P group has no remarkable difference compared with the saline group. The above results indicate that EM/VSV-G Ad5-P is able to induce stronger anti-tumor immunity than Ad 5-P.

EM/VSV-G Ad5-P was able to induce a stronger immune response than Ad5-P, meaning that a stronger immune negative regulatory response might be induced, and we examined immunosuppressive molecules PD-L1 positive cells in ascites. The results showed that PD-L1 positive cells in ascites in EM/VSV-G Ad5-P group were significantly higher than in Ad5-P group (P0.036) and very significantly higher than in salene group (P0.0023) (fig. 6 e). Therefore, EM/VSV-G Ad5-P can induce stronger immune response than Ad5-P, so that more tumor cells express PD-L1, and therefore the anti-tumor effect of EM/VSV-G Ad5-P can be ensured only by blocking the PD-1/PD-L1 pathway.

Oncolytic virus-induced anti-tumor immune response associated with its induced tumor cell death¹²In vitro studies, the EM/VSV-G Ad5-P is found to remarkably enhance the virus oncolytic effect after enhancing the virus infection efficiency to H22, and whether the EM/VSV-G Ad5-P has the same effect in vivo or not is researched. We analyzed the cell viability in day 11 ascites using Trypan blue staining and found that the viable cell ratio in the ascites of EM/VSV-G Ad5-P group was 65.9 + -5.36% very significantly lower than 74.45 + -3.38% of the Ad5-P group (P ═ 0.0032) and 90.15 + -5.71% of the salene group (P < 0.001) (FIG. 6G). ELISA detects the expression level of soluble PD1 in abdominal water on days 11,15 and 18, and finds that the expression level of soluble PD1 in the group EM/VSV-G Ad5-P is obviously higher than that in the group Ad 5-P; while the amount of soluble PD1 in Day15/18 ascites of some mice in Ad5-P group was lower than the test line. The results show that the enhancement of the anti-tumor effect of EM/VSV-G Ad5-P and the enhancement of the oncolytic effect of EM/VSV-G Ad5-P can induce ICD of a large number of tumor cells and stimulate the immune system of a mouse to generate stronger anti-tumor immune response; meanwhile, the increase of the infection efficiency of EM/VSV-G Ad5-P causes the increase of the expression level of soluble PD1, more soluble PD1 can be utilized to block the PD1/PD-L1 immune suppression pathway so as to inhibit the PD1/PD-L1 immune suppression pathway to cause T cell failure, and the combined action increases the anti-tumor effect of EM/VSV-G Ad 5-P.

Example 7EM/VSV-G Ad5-P anti-Ad 5-P neutralizing antibodies to extend viral treatment time

Oncolytic viruses can induce the body to produce antiviral immunity, produce neutralizing antibodies, and eliminate viruses. Neutralizing antibodies not only block the spread of the virus in tumor tissue but also limit the reuse of the virus. The concentration of soluble PD1 at day 18 was 27.59. + -. 27.59pg/ml, and the concentration of soluble PD1 at day15 was 58.68. + -. 20.75pg/ml, significantly lower than 130.2. + -. 7.77pg/ml at day 11 in the Ad5-P treated group (FIG. 7 a); although one virus injection was performed on day15, only 1 mouse soluble PD1 was 110.36 pg/ml on day 18, and soluble PD1 was lower than the test line in the remaining mouse ascites (FIG. 7 b). In sharp contrast, the concentrations of soluble PD1 at days 11,15 and 18 in the EM/VSV-G Ad5-P group were 276.4 ± 28.34 pg/ml, 132 ± 19.55pg/ml and 166.4 ± 26.66pg/ml, respectively, although the concentrations of soluble PD1 were significantly reduced at days 15(P ═ 0.0018) and 18 (P ═ 0.023) compared to day 11, but increased at day 18 compared to day15, and secretion was able to be expressed continuously (P ═ 0.641) (fig. 7 a). The time-varying curves of soluble PD1 were analyzed independently for each mouse in group EM/VSV-G Ad5-P, and after virus injection on day15, the expression level of soluble PD1 in ascites in group EM/VSV-G Ad5-P was significantly increased in 1 mouse, and was not significantly changed in the remaining 3 mice (FIG. 7 c). The above results suggest that the virus in Ad5-P group ascites fluid may induce the mouse to produce neutralizing antibodies, resulting in the virus being unable to infect tumor cells any more.

To ascertain whether neutralizing antibodies were produced in ascites from the Ad5-P treated group, we found by a neutralization test on day15 ascites test that ascites from all mice from the Ad5-P treated group significantly blocked infection of 293T cells by Ad5-P virus (P < 0.001), but had essentially no blocking effect on infection of 293T cells by EM/VSV-G Ad5-P (FIG. 7 d); the results indicate that group Ad5-P induced the production of neutralizing antibodies against Ad5-P, which were able to neutralize Ad5-P but not EM/VSV-G Ad 5-P. We also used the EM/VSV-G Ad 5-P4 mice in the ascites were neutralized, and showed that 4 mice in the EM/VSV-G Ad5-P in the ascites blocked the infection of 293T cells by Ad5-P virus, but did not show a certain blocking effect but did not show significant effect on the infection of 293T cells by EM/VSV-G Ad5-P (FIG. 7 e); the results indicate that the EM/VSV-G Ad5-P group also induced the production of neutralizing antibodies against Ad5-P, which were able to neutralize Ad5-P but not EM/VSV-G Ad 5-P.

We constructed VSV-lenti-GFP virus, neutralized the VSV-lenti-GFP virus using the ascites of 4 mice in the EM/VSV-G Ad5-P group, and then observed the infectivity of the VSV-lenti-GFP virus, and the results showed that NO 4 and 5 mouse ascites significantly blocked the VSV-lenti-GFP infection, but the blocking effect was much weaker than that of the preceding blocking Ad 5-P; NO2 and 7 mouse ascites had a blocking effect on VSV-lenti-GFP virus infection, but were not significant (FIG. 7 f). The above results indicate that EM/VSV-G Ad5-P can induce the production of neutralizing antibodies to Ad5-P, and only a small amount of VSV-G neutralizing antibodies. The reason for this may be that after the cells are infected with EM/VSV-G Ad5-P, the cells can be replicated and amplified, the virus can be used as a heterologous antigen to induce the mouse to produce neutralizing antibody, while the VSV-G protein is a heterologous protein but only exists in a small amount on the exosome and enters the mouse along with the virus injection, and the low protein amount causes the mouse to be induced to produce anti-VSV-G neutralizing antibody, so the EM/VSV-G Ad5-P ascites VSV-G neutralizing antibody is low.

In conclusion, after the Ad5-P treatment, a large amount of neutralizing antibodies against Ad5-P are generated, so that the infection of Ad5-P is blocked, and the treatment effect of Ad5-P is influenced; after EM/VSV-G Ad5-P treatment, although a large amount of neutralizing antibodies against Ad5-P can be generated, the treatment effect is remarkably improved compared with that of Ad5-P because EM/VSV-G Ad5-P is wrapped in exosome-like bodies and the infection of EM/VSV-G Ad5-P cannot be effectively blocked by the neutralizing antibodies. Although the EM/VSV-G Ad5-P carries the heterologous protein VSV-G, the induction of mice produces less neutralizing antibody against VSV-G due to the smaller amount of VSV-G, and thus has less effect on EM/VSV-G Ad5-P, and EM/VSV-G Ad5-P exhibits a significantly enhanced therapeutic effect than Ad 5-P.

Invention summary knot

In the present invention, we present an exosome-mimetic encapsulated oncolytic adenovirus expressing soluble PD-1 (Ad 5-P). Exosome-mimetic encapsulated adenovirus EM/VSV-G Ad5 was produced using 293T cells stably expressing Vesicular storage Indiana virus G protein (VSV-G) using exosome-mimetic technology. The virus manufacturing method not only enables EM/VSV-G Ad5 to be retargeted through VSV-G, infects a CAR low expression cell line, increases the gene transduction efficiency of Ad5, can resist the neutralization effect of an Ad5 antibody, but also can improve the virus yield. In vitro studies using exosome-mimetic-encapsulated oncolytic adenovirus EM/VSV-G Ad5-P found that the infection efficiency, oncolytic capacity and expression level of soluble PD-1 of EM/VSV-G Ad5-P were all significantly improved in CAR low-expression cell lines. In vivo experiments show that EM/VSV-G Ad5-P can resist the neutralization of Ad5 antibody, prolong the virus treatment window and continuously express soluble PD-1, so that the immune activation of the tumor microenvironment of the EM/VSV-G Ad5-P treatment group is obviously higher than that of the Ad5-P control group, and finally the survival of the mice is obviously prolonged. Detailed effects please refer to fig. 8. It is seen that the claims of the present invention are supported by the results.

Reference documents:

1.Walter,O,Treiner,E,Bonneville,F,Mengelle,C,Vergez,F,Lerebours,F, et al.(2019).Treatment of Progressive Multifocal Leukoencephalopathy with Nivolumab.New England Journal of Medicine 380:1674-1676.

2.Smyth,MJ,Ngiow,SF,Ribas,A,and Teng,MW(2016).Combination cancer immunotherapies tailored to the tumour microenvironment.Nature reviews Clinical oncology 13:143-158.

3.Hellmann,MD,Friedman,CF,and Wolchok,JD(2016).Combinatorial Cancer Immunotherapies.Advances in immunology 130:251-277.

4.Sharma,P,and Allison,JP(2015).The future of immune checkpoint therapy. Science 348:56-61.

5.Topalian,SL,Hodi,FS,Brahmer,JR,Gettinger,SN,Smith,DC,McDermott, DF,et al.(2012).Safety,activity,and immune correlates of anti-PD-1antibody in cancer.The New England journal of medicine 366:2443-2454.

6.Moslehi,JJ,Salem,JE,Sosman,JA,Lebrun-Vignes,B,and Johnson,DB (2018).Increased reporting of fatal immune checkpoint inhibitor-associated myocarditis.Lancet 391:933.

7.Teng,MW,Ngiow,SF,Ribas,A,and Smyth,MJ(2015).Classifying Cancers Based on T-cell Infiltration and PD-L1.Cancer research 75:2139-2145.

8.Gujar,S,Pol,JG,Kim,Y,Lee,PW,and Kroemer,G(2018).Antitumor Benefits of Antiviral Immunity:An Underappreciated Aspect of Oncolytic Virotherapies.Trends in immunology 39:209-221.

9.Garon,EB,Rizvi,NA,Hui,R,Leighl,N,Balmanoukian,AS,Eder,JP,et al. (2015).Pembrolizumab for the treatment of non-small-cell lung cancer.The New England journal of medicine 372:2018-2028.

10.Rini,BI,Plimack,ER,Stus,V,Gafanov,R,Hawkins,R,Nosov,D,et al. (2019).Pembrolizumab plus Axitinib versus Sunitinib for Advanced Renal-Cell Carcinoma.New England Journal of Medicine 380:1116-1127.

11.Esfahani,K,Buhlaiga,N,Thébault,P,Lapointe,R,Johnson,NA,and Miller, WH(2019).Alemtuzumab for Immune-Related Myocarditis Due to PD-1 Therapy. New England Journal of Medicine 380:2375-2376.

12.Shin,SP,Seo,HH,Shin,JH,Park,HB,Lim,DP,Eom,HS,et al.(2013). Adenovirus expressing both thymidine kinase and soluble PD1 enhances antitumor immunity by strengthening CD8 T-cell response.Molecular therapy:the journal of the American Society of Gene Therapy 21:688-695.

13.Choi,IK,Lee,JS,Zhang,SN,Park,J,Sonn,CH,Lee,KM,et al.(2011). Oncolytic adenovirus co-expressing IL-12 and IL-18 improves tumor-specific immunity via differentiation of T cells expressing IL-12Rbeta2 or IL-18Ralpha.Gene therapy 18:898-909.

14.Choi,IK,Li,Y,Oh,E,Kim,J,and Yun,CO(2013).Oncolytic adenovirus expressing IL-23 and p35 elicits IFN-gamma-and TNF-alpha-co-producing T cell-mediated antitumor immunity.PloS one 8:e67512.

15.Freytag,SO,Barton,KN,and Zhang,Y(2013).Efficacy of oncolytic adenovirus expressing suicide genes and interleukin-12 in preclinical model of prostate cancer.Gene therapy 20:1131-1139.

16.Volpers,C,and Kochanek,S(2004).Adenoviral vectors for gene transfer and therapy.The journal of gene medicine 6 Suppl 1:S164-171.

17.Tanoue,K,Rosewell Shaw,A,Watanabe,N,Porter,C,Rana,B,Gottschalk, S,et al.(2017).Armed Oncolytic Adenovirus-Expressing PD-L1 Mini-Body Enhances Antitumor Effects of Chimeric Antigen Receptor T Cells in Solid Tumors. Cancer research 77:2040-2051.

18.Gall,J,Kass-Eisler,A,Leinwand,L,and Falck-Pedersen,E(1996). Adenovirus type 5 and 7 capsid chimera:fiber replacement alters receptor tropism without affecting primary immune neutralization epitopes.Journal of virology 70: 2116-2123.

19.Gall,JG,Crystal,RG,and Falck-Pedersen,E(1998).Construction and characterization of hexon-chimeric adenoviruses:specification of adenovirus serotype. Journal of virology 72:10260-10264.

20.Molinier-Frenkel,V,Lengagne,R,Gaden,F,Hong,SS,Choppin,J, Gahery-Segard,H,et al.(2002).Adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response.Journal of virology 76:127-135.

21.Nigatu,AS,Vupputuri,S,Flynn,N,Neely,BJ,and Ramsey,JD(2013). Evaluation of cell-penetrating peptide/adenovirus particles for transduction of CAR-negative cells.Journal of pharmaceutical sciences 102:1981-1993.

22.Dmitriev,I,Krasnykh,V,Miller,CR,Wang,M,Kashentseva,E,Mikheeva, G,et al.(1998).An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism.Journal of virology 72:9706-9713.

23.Roy,S,Shirley,PS,McClelland,A,and Kaleko,M(1998).Circumvention of immunity to the adenovirus major coat protein hexon.Journal of virology 72: 6875-6879.

24.Bergelson,JM,Cunningham,JA,Droguett,G,Kurt-Jones,EA,Krithivas,A, Hong,JS,et al.(1997).Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5.Science 275:1320-1323.

25.Kuroki,LM,Jin,X,Dmitriev,IP,Kashentseva,EA,Powell,MA,Mutch,DG, et al.(2017).Adenovirus platform enhances transduction efficiency of human mesenchymal stem cells:An opportunity for cellular carriers of targeted TRAIL-based TR3 biologics in ovarian cancer.PloS one 12:e0190125.

26.Cody,JJ,and Douglas,JT(2009).Armed replicating adenoviruses for cancer virotherapy.Cancer gene therapy 16:473-488.

27.Aldhamen,YA,Seregin,SS,and Amalfitano,A(2011).Immune recognition of gene transfer vectors:focus on adenovirus as a paradigm.Frontiers in immunology 2:40.

28.Douglas,JT,Kim,M,Sumerel,LA,Carey,DE,and Curiel,DT(2001). Efficient Oncolysis by a Replicating Adenovirus(Ad)<em>in Vivo</em>Is Critically Dependent on Tumor Expression of Primary Ad Receptors.Cancer research 61:813-817.

29.Li,Y,Pong,R-C,Bergelson,JM,Hall,MC,Sagalowsky,AI,Tseng,C-P,et al.(1999).Loss of Adenoviral Receptor Expression in Human Bladder Cancer Cells: A Potential Impact on the Efficacy of Gene Therapy.Cancer research 59:325-330.

30.Sumida,SM,Truitt,DM,Kishko,MG,Arthur,JC,Jackson,SS,Gorgone, DA,et al.(2004).Neutralizing antibodies and CD8+T lymphocytes both contribute to immunity to adenovirus serotype 5 vaccine vectors.Journal of virology 78: 2666-2673.

31.Bottermann,M,Foss,S,van Tienen,LM,Vaysburd,M,Cruickshank,J, O'Connell,K,et al.(2018).TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination.Proceedings of the National Academy of Sciences of the United States of America 115:10440-10445.

32.Nwanegbo,E,Vardas,E,Gao,W,Whittle,H,Sun,H,Rowe,D,et al.(2004). Prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of The Gambia,South Africa,and the United States.Clinical and diagnostic laboratory immunology 11:351-357.

33.Cheng,X,Ming,X,and Croyle,MA(2003).PEGylated adenoviruses for gene delivery to the intestinal epithelium by the oral route.Pharmaceutical research 20:1444-1451.

34.Croyle,MA,Chirmule,N,Zhang,Y,and Wilson,JM(2002).PEGylation of E1-Deleted Adenovirus Vectors Allows Significant Gene Expression on Readministration to Liver.Human Gene Therapy 13:1887-1900.

35.Matthews,C,Jenkins,G,Hilfinger,J,and Davidson,B(1999).Poly-L-lysine improves gene transfer with adenovirus formulated in PLGA microspheres.Gene therapy 6:1558-1564.

36.Lee,SG,Yoon,SJ,Kim,CD,Kim,K,Lim,DS,Yeom,YI,et al.(2000). Enhancement of adenoviral transduction with polycationic liposomes in vivo.Cancer gene therapy 7:1329-1335.

37.Wonganan,P,and Croyle,MA(2010).PEGylated Adenoviruses:From Mice to Monkeys.Viruses 2:468-502.

38.Martínez-Vélez,N,Garcia-Moure,M,Marigil,M,González-Huarriz,M, Puigdelloses,M,Gallego Pérez-Larraya,J,et al.(2019).The oncolytic virus Delta-24-RGD elicits an antitumor effect in pediatric glioma and DIPG mouse models. Nature Communications 10:2235.

39.Kuryk,L,

A-SW,and Jaderberg,M(2019).Combination of immunogenic oncolytic adenovirus ONCOS-102 with anti-PD-1 pembrolizumab exhibits synergistic antitumor effect in humanized A2058 melanoma huNOG mouse model.OncoImmunology 8:e1532763.

40.Jang,SC,Kim,OY,Yoon,CM,Choi,DS,Roh,TY,Park,J,et al.(2013). Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors.ACS nano 7:7698-7710.

41.Hwang,DW,Choi,H,Jang,SC,Yoo,MY,Park,JY,Choi,NE,et al.(2015). Noninvasive imaging of radiolabeled exosome-mimetic nanovesicle using (99m)Tc-HMPAO.Scientific reports 5:15636.

42.Lunavat,TR,Jang,SC,Nilsson,L,Park,HT,Repiska,G,Lasser,C,et al. (2016).RNAi delivery by exosome-mimetic nanovesicles-Implications for targeting c-Myc in cancer.Biomaterials 102:231-238.

43.Wu,JY,Ji,AL,Wang,ZX,Qiang,GH,Qu,Z,Wu,JH,et al.(2018). Exosome-Mimetic Nanovesicles from Hepatocytes promote hepatocyte proliferation in vitro and liver regeneration in vivo.Scientific reports 8:2471.

44.Tao,SC,Rui,BY,Wang,QY,Zhou,D,Zhang,Y,and Guo,SC(2018). Extracellular vesicle-mimetic nanovesicles transport LncRNA-H19 as competing endogenous RNA for the treatment of diabetic wounds.Drug delivery 25:241-255.

sequence listing

<110> Nanjing university

<120> a novel oncolytic adenovirus EM/VSV-G Ad5-P and application thereof in preparing antitumor drugs

<130> 20200623

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 510

<212> DNA

<213> Artificial sequence (.)

<400> 1

atgtgggtcc ggcaggtacc ctggtcattc acttgggctg tgctgcagtt gagctggcaa 60

tcagggtggc ttctagaggt ccccaatggg ccctggaggt ccctcacctt ctacccagcc 120

tggctcacag tgtcagaggg agcaaatgcc accttcacct gcagcttgtc caactggtcg 180

gaggatctta tgctgaactg gaaccgcctg agtcccagca accagactga aaaacaggcc 240

gccttctgta atggtttgag ccaacccgtc caggatgccc gcttccagat catacagctg 300

cccaacaggc atgacttcca catgaacatc cttgacacac ggcgcaatga cagtggcatc 360

tacctctgtg gggccatctc cctgcacccc aaggcaaaaa tcgaggagag ccctggagca 420

gagctcgtgg taacagagag aatcctggag acctcaacaa gatatcccag cccctcgccc 480

aaaccagaag gccggtttca aggcatggtc 510

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence (.)

<400> 2

caccaccacc accaccac 18

<210> 3

<211> 538

<212> DNA

<213> Artificial sequence (.)

<400> 3

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 60

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 120

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 180

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 240

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 300

cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 360

ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 420

aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 480

taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatc 538

<210> 4

<211> 720

<212> DNA

<213> Artificial sequence (.)

<400> 4

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga 720

<210> 5

<211> 870

<212> DNA

<213> Artificial sequence (.)

<400> 5

atgagacata ttatctgcca cggaggtgtt attaccgaag aaatggccgc cagtcttttg 60

gaccagctga tcgaagaggt actggctgat aatcttccac ctcctagcca ttttgaacca 120

cctacccttc acgaactgta tgatttagac gtgacggccc ccgaagatcc caacgaggag 180

gcggtttcgc agatttttcc cgactctgta atgttggcgg tgcaggaagg gattgactta 240

ctcacttttc cgccggcgcc cggttctccg gagccgcctc acctttcccg gcagcccgag 300

cagccggagc agagagcctt gggtccggtt tctatgccaa accttgtacc ggaggtgatc 360

gatcttacct gccacgaggc tggctttcca cccagtgacg acgaggatga agagggtgag 420

gagtttgtgt tagattatgt ggagcacccc gggcacggtt gcaggtcttg tcattatcac 480

cggaggaata cgggggaccc agatattatg tgttcgcttt gctatatgag gacctgtggc 540

atgtttgtct acagtcctgt gtctgaacct gagcctgagc ccgagccaga accggagcct 600

gcaagaccta cccgccgtcc taaaatggcg cctgctatcc tgagacgccc gacatcacct 660

gtgtctagag aatgcaatag tagtacggat agctgtgact ccggtccttc taacacacct 720

cctgagatac acccggtggt cccgctgtgc cccattaaac cagttgccgt gagagttggt 780

gggcgtcgcc aggctgtgga atgtatcgag gacttgctta acgagcctgg gcaacctttg 840

gacttgagct gtaaacgccc caggccataa 870

<210> 6

<211> 54

<212> DNA

<213> Artificial sequence (.)

<400> 6

gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54

<210> 7

<211> 297

<212> DNA

<213> Artificial sequence (.)

<400> 7

ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc 60

cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 120

actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 180

attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 240

catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctgccac 297

<210> 8

<211> 3739

<212> DNA

<213> Artificial sequence (.)

<400> 8

cacctatcga taagcttggg agttccgcgt tacataactt acggtaaatg gcccgcctgg 60

ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 120

gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 180

ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 240

atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 300

catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 360

gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 420

gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 480

attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt 540

agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca 600

ccgactctag aggatccgcc accatggtga gcaagggcga ggagctgttc accggggtgg 660

tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 720

agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 780

agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca 840

gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 900

acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 960

tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 1020

aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata 1080

tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg 1140

aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 1200

ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca 1260

acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg 1320

gcatggacga gctgtacaag gctagcgagg gcagaggaag tcttctaaca tgcggtgacg 1380

tggaggagaa tcccggccct accggaatga gacatattat ctgccacgga ggtgttatta 1440

ccgaagaaat ggccgccagt cttttggacc agctgatcga agaggtactg gctgataatc 1500

ttccacctcc tagccatttt gaaccaccta cccttcacga actgtatgat ttagacgtga 1560

cggcccccga agatcccaac gaggaggcgg tttcgcagat ttttcccgac tctgtaatgt 1620

tggcggtgca ggaagggatt gacttactca cttttccgcc ggcgcccggt tctccggagc 1680

cgcctcacct ttcccggcag cccgagcagc cggagcagag agccttgggt ccggtttcta 1740

tgccaaacct tgtaccggag gtgatcgatc ttacctgcca cgaggctggc tttccaccca 1800

gtgacgacga ggatgaagag ggtgaggagt ttgtgttaga ttatgtggag caccccgggc 1860

acggttgcag gtcttgtcat tatcaccgga ggaatacggg ggacccagat attatgtgtt 1920

cgctttgcta tatgaggacc tgtggcatgt ttgtctacag tcctgtgtct gaacctgagc 1980

ctgagcccga gccagaaccg gagcctgcaa gacctacccg ccgtcctaaa atggcgcctg 2040

ctatcctgag acgcccgaca tcacctgtgt ctagagaatg caatagtagt acggatagct 2100

gtgactccgg tccttctaac acacctcctg agatacaccc ggtggtcccg ctgtgcccca 2160

ttaaaccagt tgccgtgaga gttggtgggc gtcgccaggc tgtggaatgt atcgaggact 2220

tgcttaacga gcctgggcaa cctttggact tgagctgtaa acgccccagg ccataacacc 2280

tatcgataag cttgggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 2340

ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 2400

atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 2460

gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 2520

cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 2580

tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt 2640

ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt 2700

ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg 2760

acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg 2820

aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccga 2880

ctctagagga tccgccacca tgaccggtat gtgggtccgg caggtaccct ggtcattcac 2940

ttgggctgtg ctgcagttga gctggcaatc agggtggctt ctagaggtcc ccaatgggcc 3000

ctggaggtcc ctcaccttct acccagcctg gctcacagtg tcagagggag caaatgccac 3060

cttcacctgc agcttgtcca actggtcgga ggatcttatg ctgaactgga accgcctgag 3120

tcccagcaac cagactgaaa aacaggccgc cttctgtaat ggtttgagcc aacccgtcca 3180

ggatgcccgc ttccagatca tacagctgcc caacaggcat gacttccaca tgaacatcct 3240

tgacacacgg cgcaatgaca gtggcatcta cctctgtggg gccatctccc tgcaccccaa 3300

ggcaaaaatc gaggagagcc ctggagcaga gctcgtggta acagagagaa tcctggagac 3360

ctcaacaaga tatcccagcc cctcgcccaa accagaaggc cggtttcaag gcatggtcca 3420

ccaccaccac caccaccact aactcgagtc tagagggccc gtttaaaccc gctgatcagc 3480

ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3540

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 3600

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 3660

ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc 3720

ggaaagaacc agctgccac 3739

<210> 9

<211> 1687

<212> DNA

<213> Artificial sequence (.)

<400> 9

ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 60

ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 120

ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 180

tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 240

tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 300

cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 360

ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 420

aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 480

taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc 540

ctggagacgc catccacgct gttttgacct ccatagaaga caccgactct agaggatccg 600

ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc 660

tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca 720

cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc 780

ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca 840

tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca 900

tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca 960

ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg 1020

ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga 1080

agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc 1140

tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca 1200

accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca 1260

tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca 1320

agtaatggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc agatatccag 1380

cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 1440

tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 1500

aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 1560

gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 1620

aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 1680

ccagctg 1687

<210> 10

<211> 1536

<212> DNA

<213> Artificial sequence (.)

<400> 10

atgaagtgcc ttttgtactt agccttttta ttcattgggg tgaattgcaa gttcaccata 60

gtttttccac acaaccaaaa aggaaactgg aaaaatgttc cttctaatta ccattattgc 120

ccgtcaagct cagatttaaa ttggcataat gacttaatag gcacagcctt acaagtcaaa 180

atgcccaaga gtcacaaggc tattcaagca gacggttgga tgtgtcatgc ttccaaatgg 240

gtcactactt gtgatttccg ctggtatgga ccgaagtata taacacattc catccgatcc 300

ttcactccat ctgtagaaca atgcaaggaa agcattgaac aaacgaaaca aggaacttgg 360

ctgaatccag gcttccctcc tcaaagttgt ggatatgcaa ctgtgacgga tgccgaagca 420

gtgattgtcc aggtgactcc tcaccatgtg ctggttgatg aatacacagg agaatgggtt 480

gattcacagt tcatcaacgg aaaatgcagc aattacatat gccccactgt ccataactct 540

acaacctggc attctgacta taaggtcaaa gggctatgtg attctaacct catttccatg 600

gacatcacct tcttctcaga ggacggagag ctatcatccc tgggaaagga gggcacaggg 660

ttcagaagta actactttgc ttatgaaact ggaggcaagg cctgcaaaat gcaatactgc 720

aagcattggg gagtcagact cccatcaggt gtctggttcg agatggctga taaggatctc 780

tttgctgcag ccagattccc tgaatgccca gaagggtcaa gtatctctgc tccatctcag 840

acctcagtgg atgtaagtct aattcaggac gttgagagga tcttggatta ttccctctgc 900

caagaaacct ggagcaaaat cagagcgggt cttccaatct ctccagtgga tctcagctat 960

cttgctccta aaaacccagg aaccggtcct gctttcacca taatcaatgg taccctaaaa 1020

tactttgaga ccagatacat cagagtcgat attgctgctc caatcctctc aagaatggtc 1080

ggaatgatca gtggaactac cacagaaagg gaactgtggg atgactgggc accatatgaa 1140

gacgtggaaa ttggacccaa tggagttctg aggaccagtt caggatataa gtttccttta 1200

tacatgattg gacatggtat gttggactcc gatcttcatc ttagctcaaa ggctcaggtg 1260

ttcgaacatc ctcacattca agacgctgct tcgcaacttc ctgatgatga gagtttattt 1320

tttggtgata ctgggctatc caaaaatcca atcgagcttg tagaaggttg gttcagtagt 1380

tggaaaagct ctattgcctc ttttttcttt atcatagggt taatcattgg actattcttg 1440

gttctccgag ttggtatcca tctttgcatt aaattaaagc acaccaagaa aagacagatt 1500

tatacagaca tagagatgaa ccgacttgga aagtaa 1536

<210> 11

<211> 934

<212> DNA

<213> Artificial sequence (.)

<400> 11

attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 60

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 120

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 180

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 240

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 300

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 360

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 420

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 480

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg 540

ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 600

gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 660

gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct 720

ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc 780

ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag 840

cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg 900

ttattgtgct gtctcatcat tttggcaaag aatt 934

<210> 12

<211> 600

<212> DNA

<213> Artificial sequence (.)

<400> 12

atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta 60

cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac 120

cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 180

atcggcaagg tgtgggtcgc ggacgacggc gcggccgtgg cggtctggac cacgccggag 240

agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 300

tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 360

cccgcgtggt tcctggccac cgtcggagtc tcgcccgacc accagggcaa gggtctgggc 420

agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 480

gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 540

gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga 600

<210> 13

<211> 3136

<212> DNA

<213> Artificial sequence (.)

<400> 13

attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg 60

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 120

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca 180

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 240

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 300

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 360

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 420

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 480

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg 540

ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 600

gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 660

gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct 720

ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc 780

ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag 840

cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg 900

ttattgtgct gtctcatcat tttggcaaag aattgccacc atgaagtgcc ttttgtactt 960

agccttttta ttcattgggg tgaattgcaa gttcaccata gtttttccac acaaccaaaa 1020

aggaaactgg aaaaatgttc cttctaatta ccattattgc ccgtcaagct cagatttaaa 1080

ttggcataat gacttaatag gcacagcctt acaagtcaaa atgcccaaga gtcacaaggc 1140

tattcaagca gacggttgga tgtgtcatgc ttccaaatgg gtcactactt gtgatttccg 1200

ctggtatgga ccgaagtata taacacattc catccgatcc ttcactccat ctgtagaaca 1260

atgcaaggaa agcattgaac aaacgaaaca aggaacttgg ctgaatccag gcttccctcc 1320

tcaaagttgt ggatatgcaa ctgtgacgga tgccgaagca gtgattgtcc aggtgactcc 1380

tcaccatgtg ctggttgatg aatacacagg agaatgggtt gattcacagt tcatcaacgg 1440

aaaatgcagc aattacatat gccccactgt ccataactct acaacctggc attctgacta 1500

taaggtcaaa gggctatgtg attctaacct catttccatg gacatcacct tcttctcaga 1560

ggacggagag ctatcatccc tgggaaagga gggcacaggg ttcagaagta actactttgc 1620

ttatgaaact ggaggcaagg cctgcaaaat gcaatactgc aagcattggg gagtcagact 1680

cccatcaggt gtctggttcg agatggctga taaggatctc tttgctgcag ccagattccc 1740

tgaatgccca gaagggtcaa gtatctctgc tccatctcag acctcagtgg atgtaagtct 1800

aattcaggac gttgagagga tcttggatta ttccctctgc caagaaacct ggagcaaaat 1860

cagagcgggt cttccaatct ctccagtgga tctcagctat cttgctccta aaaacccagg 1920

aaccggtcct gctttcacca taatcaatgg taccctaaaa tactttgaga ccagatacat 1980

cagagtcgat attgctgctc caatcctctc aagaatggtc ggaatgatca gtggaactac 2040

cacagaaagg gaactgtggg atgactgggc accatatgaa gacgtggaaa ttggacccaa 2100

tggagttctg aggaccagtt caggatataa gtttccttta tacatgattg gacatggtat 2160

gttggactcc gatcttcatc ttagctcaaa ggctcaggtg ttcgaacatc ctcacattca 2220

agacgctgct tcgcaacttc ctgatgatga gagtttattt tttggtgata ctgggctatc 2280

caaaaatcca atcgagcttg tagaaggttg gttcagtagt tggaaaagct ctattgcctc 2340

ttttttcttt atcatagggt taatcattgg actattcttg gttctccgag ttggtatcca 2400

tctttgcatt aaattaaagc acaccaagaa aagacagatt tatacagaca tagagatgaa 2460

ccgacttgga aagagcggcg ccaccaactt cagcctgctg aagcaggccg gcgacgtgga 2520

ggagaacccc ggccccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga 2580

cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca 2640

caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac 2700

gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgcgg ccgtggcggt 2760

ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat 2820

ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc 2880

gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca 2940

gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg 3000

ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg 3060

cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg 3120

caagcccggt gcctga 3136

<210> 14

<211> 511

<212> PRT

<213> Artificial sequence (.)

<400> 14

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys

1 5 10 15

Lys Phe Thr Ile Val Phe Pro His Asn Gln Lys Gly Asn Trp Lys Asn

20 25 30

Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp Leu Asn Trp

35 40 45

His Asn Asp Leu Ile Gly Thr Ala Leu Gln Val Lys Met Pro Lys Ser

50 55 60

His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys His Ala Ser Lys Trp

65 70 75 80

Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr His

85 90 95

Ser Ile Arg Ser Phe Thr Pro Ser Val Glu Gln Cys Lys Glu Ser Ile

100 105 110

Glu Gln Thr Lys Gln Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln

115 120 125

Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Ala Val Ile Val Gln

130 135 140

Val Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp Val

145 150 155 160

Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Tyr Ile Cys Pro Thr

165 170 175

Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val Lys Gly Leu

180 185 190

Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr Phe Phe Ser Glu Asp

195 200 205

Gly Glu Leu Ser Ser Leu Gly Lys Glu Gly Thr Gly Phe Arg Ser Asn

210 215 220

Tyr Phe Ala Tyr Glu Thr Gly Gly Lys Ala Cys Lys Met Gln Tyr Cys

225 230 235 240

Lys His Trp Gly Val Arg Leu Pro Ser Gly Val Trp Phe Glu Met Ala

245 250 255

Asp Lys Asp Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys Pro Glu Gly

260 265 270

Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val Ser Leu Ile

275 280 285

Gln Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr Trp

290 295 300

Ser Lys Ile Arg Ala Gly Leu Pro Ile Ser Pro Val Asp Leu Ser Tyr

305 310 315 320

Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro Ala Phe Thr Ile Ile Asn

325 330 335

Gly Thr Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Val Asp Ile Ala

340 345 350

Ala Pro Ile Leu Ser Arg Met Val Gly Met Ile Ser Gly Thr Thr Thr

355 360 365

Glu Arg Glu Leu Trp Asp Asp Trp Ala Pro Tyr Glu Asp Val Glu Ile

370 375 380

Gly Pro Asn Gly Val Leu Arg Thr Ser Ser Gly Tyr Lys Phe Pro Leu

385 390 395 400

Tyr Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Leu Ser Ser

405 410 415

Lys Ala Gln Val Phe Glu His Pro His Ile Gln Asp Ala Ala Ser Gln

420 425 430

Leu Pro Asp Asp Glu Ser Leu Phe Phe Gly Asp Thr Gly Leu Ser Lys

435 440 445

Asn Pro Ile Glu Leu Val Glu Gly Trp Phe Ser Ser Trp Lys Ser Ser

450 455 460

Ile Ala Ser Phe Phe Phe Ile Ile Gly Leu Ile Ile Gly Leu Phe Leu

465 470 475 480

Val Leu Arg Val Gly Ile His Leu Cys Ile Lys Leu Lys His Thr Lys

485 490 495

Lys Arg Gln Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys

500 505 510

<210> 15

<211> 170

<212> PRT

<213> Artificial sequence (.)

<400> 15

Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln

1 5 10 15

Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp

20 25 30

Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala

35 40 45

Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met

50 55 60

Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala

65 70 75 80

Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln

85 90 95

Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp

100 105 110

Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val

130 135 140

Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro

145 150 155 160

Lys Pro Glu Gly Arg Phe Gln Gly Met Val

165 170

Claims (6)

1. A novel oncolytic virus EM/VSV-G Ad5-P, characterized by: the novel oncolytic virus EM/VSV-GAd5-P is an exosome-like nano vesicle which is wrapped outside an adenovirus capable of expressing a soluble PD1 extracellular region, and VSV-G protein is arranged on the membrane of the exosome-like nano vesicle; the DNA sequence of the VSV-G protein is shown in SEQ ID NO 10, and the amino acid sequence of the VSV-G protein is shown in SEQ ID NO 14; the DNA sequence of the soluble PD1 extracellular region is shown in SEQ ID NO. 1, and the protein sequence of the soluble PD1 extracellular region is shown in SEQ ID NO. 15.

2. The novel oncolytic virus EM/VSV-G Ad5-P of claim 1, characterized by: the VSV-G protein can realize the targeting of tumor cells; and due to the existence of exosome-like nanovesicles, the novel oncolytic virus EM/VSV-G Ad5-P can not be acted by a neutralizing antibody of an anti-adenovirus, and has a remarkably prolonged administration window period; the novel oncolytic virus EM/VSV-G Ad5-P can express a soluble PD1 extracellular region more continuously and block a PD-1/PD-L1 immunodetection point path continuously.

3. The novel oncolytic virus EM/VSV-G Ad5-P of claim 1, prepared by the following method:

(1) preparing a cell in which a VSV-G protein is inserted into the outer membrane of the cell, and designating the cell as a VSV-G cell;

(2) infecting VSV-G cells by adenovirus Ad5-P, wherein the adenovirus Ad5-P is adenovirus capable of expressing a soluble PD1 extracellular region;

(3) resuspending the collected VSV-G cells after infecting adenovirus with culture medium, PBS or other buffer solution, and sequentially pressing the cell suspension through a membrane filter paper with a pore size of 10 μm 5 μm 1 μm by using a squeezer;

(4) collecting the extruded virus suspension, and enriching and collecting the novel oncolytic virus EM/VSV-G Ad5-P encapsulated by the exosome by a density gradient centrifugation method, wherein the novel oncolytic virus EM/VSV-G Ad5-P is encapsulated by the exosome, and VSV-G protein is arranged on the membrane of the encapsulated exosome, so that the targeting can be realized.

4. The use of the novel oncolytic virus EM/VSV-G Ad5-P of claim 1 for the preparation of anti-tumor drugs including but not limited to liver cancer, kidney cancer, leukemia, lung cancer, melanoma and colorectal cancer.

5. The use of a novel oncolytic virus EM/VSV-G Ad5-P according to claim 1 for the manufacture of a medicament for immunotherapy of tumors including but not limited to liver, kidney, leukemia, lung, melanoma and colorectal cancer.

6. The use of the novel oncolytic virus EM/VSV-G Ad5-P of claim 1 for the preparation of a medicament for blocking the immunodetection site pathway of tumor PD-1/PD-L1, wherein the tumor includes but is not limited to liver cancer, kidney cancer, leukemia, lung cancer, melanoma and colorectal cancer, the novel oncolytic virus EM/VSV-G Ad5-P can infect tumor cells and replicate and express soluble PD1, and the expressed PD1 binds to PD-L1 to block the immunodetection site pathway of PD-1/PD-L1.

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