Recombinant oncolytic virus VSV-sPD1IL15RaIL15 as well as construction method and application thereof

Abstract

The invention relates to a construction method and application of a recombinant oncolytic virus VSV-sPD1IL15RaIL15. The recombinant oncolytic virus VSV-sPD1IL15RaIL15 is constructed by using homologous recombination, and the recombinant oncolytic virus VSV-sPD1IL15RaIL15 can be used for preparing the recombinant oncolytic virus. The preparation method comprises the following steps: firstly designing sPD1 and IL15RaIL15, then fusing the sPD1 and the IL15RaIL15, synthesizing a sPD1IL15RaIL15 gene, amplifying the sPD1IL15RaIL15 gene, inserting the sPD1IL15RaIL15 gene into an ATU region of a VSV whole genome cDNA cloning plasmid pT7-VSV by using a homologous recombination method to obtain a virus whole genome plasmid pT7-VSV-sPD1IL15RaIL15, and co-transfecting the virus whole genome plasmid pT7-VSV-sPD1IL15RaIL15 and an auxiliary plasmid to a 293T cell to prepare the VSV-sPD1IL15RaIL15 oncolytic virus. According to the oncolytic virus, specific anti-tumor immunity is activated, and sPD1IL15RaIL15 is locally expressed in a tumor, so that a long-acting anti-tumor immune response is generated. When the oncolytic virus is used for treating solid tumors, the functions of tumor specific immune cells in a tumor microenvironment can be improved, infiltration of CD8 + T cells and NK cells can be promoted, and the lifetime of tumor-bearing mice can be remarkably prolonged.

Description

Technical Field

[0001] This invention relates to a recombinant oncolytic virus VSV-sPD1IL15RaIL15, its construction method, and its applications, belonging to the field of viral and microbial technology. Background Technology

[0002] Ovarian cancer is a malignant tumor originating in the ovary. Ovarian cancer has diverse histological types, with epithelial ovarian cancer accounting for over 90% of cases. Among female reproductive system tumors, ovarian cancer ranks third in incidence, after cervical and uterine cancer, but its mortality rate remains the highest among female reproductive system tumors. The main treatment for ovarian cancer remains surgery plus chemotherapy, and 80% of patients are sensitive to first-line therapy based on platinum-based drugs combined with paclitaxel, which effectively improves survival. However, approximately 70%-80% of these patients eventually experience recurrence and drug resistance. In recent years, targeted therapies such as anti-angiogenic and poly(ADP-ribose) polymerase inhibitors (PARPi) have been approved for the treatment of epithelial ovarian cancer. While targeted drugs can increase progression-free survival, they do not prolong overall survival. To date, the 5-year survival rate for ovarian cancer patients is only around 40-45%, thus necessitating the development of new treatment methods and drugs.

[0003] Oncolytic viruses are a collective term for a class of viruses that selectively replicate within tumor cells and exert oncolytic activity. They replicate at low levels or not at all in normal tissue cells and do not exhibit cytotoxic effects. Common oncolytic viruses include adenovirus (Adv), herpes simplex virus type 1 (HSV-1), measles virus (MV), vesicular stomatitis virus (VSV), vaccinia virus (VV), and Newcastle disease virus (NDV). Studies have shown that oncolytic viruses infect tumor cells and replicate extensively within them, causing oncolysis, releasing tumor antigens, and stimulating antigen-presenting cells to induce specific anti-tumor immune responses. Simultaneously, oncolytic viruses can also induce immunogenic cell death in tumor cells, release damage-associated molecular patterns (DAMPs) and other danger signals, activate innate immunity, and promote the infiltration of immune cells into the tumor. In recent years, with the FDA approval of T-VEC (recombinant herpes simplex virus type 1 expressing GM-CSF) for the treatment of metastatic melanoma, the strategy of combining oncolytic viruses with immunomodulatory molecules has become a hot topic in anti-tumor development. Preclinical studies using oncolytic viruses to express PD-1 / PD-L1 inhibitory molecules or directly combining oncolytic viruses with anti-PD-1 / PD-L1 antibodies have demonstrated safety and efficacy in multiple tumor models.

[0004] Interleukin-15 (IL-15) is a pleiotropic cytokine that plays a crucial role in regulating the differentiation, activation, and proliferation of memory CD8+ T cells and NK cells. Compared to IL-2, IL-15 does not support the maintenance and proliferation of regulatory T cells (Tregs). Theoretically, the use of IL-15 could promote the activation and proliferation of tumor-specific CD8+ T cells and NK cells, kill tumor cells, enhance anti-tumor immunity, and thus have a therapeutic effect on tumors.

[0005] Clinical trials have shown that intravenous injection of recombinant IL-15 can significantly increase the proportion of γδT cells, CD8+T cells, and NK cells. However, due to the short half-life of recombinant IL-15, high doses of IL-15 have significant toxic side effects, and tumor reactivity is limited. Therefore, the development of new drugs for the treatment of solid tumors such as ovarian cancer is crucial to improve the therapeutic effect and prolong patient survival. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a recombinant oncolytic virus VSV-sPD1IL15RaIL15, its construction method, and its applications.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A recombinant oncolytic virus, VSV-sPD1IL15RaIL15, is disclosed. The recombinant oncolytic virus ligates the sPD1IL15RaIL15 fragment to a VSV viral genome plasmid to obtain the full-genome plasmid pT7-VSV-sPD1IL15RaIL15. pT7-VSV-sPD1IL15RaIL15 is co-transfected with an helper plasmid into 293T cells, and after culture, a VSV-sPD1IL15RaIL15 viral seed is obtained. The viral seed is then amplified in large quantities using 293T cells to obtain the oncolytic virus VSV-sPD1IL15RaIL15.

[0009] The method for constructing the recombinant oncolytic virus VSV-sPD1IL15RaIL15 includes the following steps:

[0010] (1) sPD1 design: The PD-1 signal peptide was removed from the extracellular region (17-170aa) of PD-1 to construct a new sPD1 sequence;

[0011] (2) IL15RaIL15 design: IL-15Ra was fused with N72D mutated IL-15, with the (GGGGS)4liner sequence used as the linker in the middle.

[0012] (3) sPD1IL15RaIL15 design: The C-terminus of sPD1 is fused with the N-terminus of IL15RaIL15, with the (GGGGS)3liner sequence used as a link in the middle. The CD33 signal peptide is inserted at the N-terminus and a 6-histidine tag is inserted at the C-terminus.

[0013] (4) Cloning of recombinant oncolytic virus VSV-sPD1IL15RaIL15: The recombinant protein sPD1IL15RaIL15 gene fragment was inserted into the ATU region of the VSV whole genome cDNA cloning plasmid pT7-VSV using homologous recombination via XhoI and MScI restriction sites to obtain the recombinant virus whole genome plasmid pT7-VSV-sPD1IL15RaIL15;

[0014] (5) Rescue of recombinant oncolytic virus VSV-sPD1IL15RaIL15: The whole genome plasmid pT7-VSV-sPD1IL15RaIL15 was co-transfected with helper plasmids pVRC-T7, pVRC-N, pVRC-P and pVRC-L into 293T cells until cytopathic effects were observed. Cell supernatant was collected, and cell debris was removed by centrifugation at 2000g for 10 min to obtain virus seeds. The virus was amplified using 293T cells to obtain recombinant virus VSV-sPD1IL15RaIL15.

[0015] The application of the recombinant oncolytic virus VSV-sPD1IL15RaIL15 in the treatment of ovarian cancer.

[0016] The application of the recombinant oncolytic virus VSV-sPD1IL15RaIL15 in the preparation of a drug for the treatment of ovarian cancer.

[0017] The application of the recombinant oncolytic virus VSV-sPD1IL15RaIL15 in the preparation of drugs for the treatment of solid tumors.

[0018] The beneficial effects of this invention are:

[0019] 1. The recombinant oncolytic virus VSV-sPD1IL15RaIL15 constructed in this invention for the treatment of solid tumors activates specific anti-tumor immunity through oncolytic virus, and simultaneously expresses sPD1IL15RaIL15 locally in the tumor. The PD-1 functional domain of sPD1IL15RaIL15 binds to PD-L1 on the surface of tumor cells, blocking the inhibition of T cells by the PD-1 / PD-L1 pathway, reversing the exhaustion of tumor-specific CD8+ T cells in the tumor microenvironment. IL15RaIL15 can maintain the activation and proliferation of tumor-specific CD8+ T cells and induce CD8+ T cells to differentiate into memory T cells, generating a long-lasting anti-tumor immune response.

[0020] 2. Cell-level studies have demonstrated that the oncolytic virus VSV-sPD1IL15RaIL15 of this invention exhibits no significant difference in oncolysis and replication compared to the control virus VSV, and the insertion of the exogenous gene sPD1IL15RaIL15 does not affect the virus's biological function. After infecting ovarian cancer cells, this oncolytic virus expresses the sPD1IL15RaIL15 fusion protein and secretes it extracellularly. Animal experiments have shown that treatment with the oncolytic virus VSV-sPD1IL15RaIL15 significantly enhances the function of tumor-specific T cells in the tumor microenvironment, promotes CD8+ T and NK cell infiltration, and significantly prolongs the survival of tumor-bearing mice.

[0021] 3. The oncolytic virus VSV-sPD1IL15RaIL15 of the present invention can improve the treatment effect of solid tumors, providing a new strategy for the development of drugs for the treatment of solid tumors, and has good application prospects.

[0022] 4. The oncolytic virus VSV-sPD1IL15RaIL15 of the present invention is not limited to being effective in the treatment of ovarian cancer, but is also applicable to the treatment of all solid tumors. Attached Figure Description

[0023] Figure 1 Schematic diagram of recombinant protein gene structure;

[0024] Figure 2 A graph showing the detection of the oncolytic ability of VSV-sPD1IL15RaIL15 virus;

[0025] Figure 3 A graph showing the detection of viral replication capacity;

[0026] Figure 4 Western blot image of viral expressed proteins;

[0027] Among them, sPD1IL15RaIL15, sPD1 and IL15RaIL15 correspond to the cell supernatant after infection with VSV-sPD1IL15RaIL15, VSV-SPD1 and VSV-IL15RaIL15, respectively.

[0028] Figure 5 Flow cytometry analysis of sPD1IL15RaIL15 combined with PD-L1;

[0029] Figure 6 T cell growth curve;

[0030] Figure 7 Mouse survival curve;

[0031] Figure 8 Image of Elispot detection of tumor-activated T cells in mouse tumor tissue;

[0032] Figure A shows typical photos of each group of Elispots, and Figure B shows the statistical results of each group of Elispot spots.

[0033] Figure 7 and Figure 8 The data is presented using Mean±SD, where ns is p>0.05, * is p<0.05, *** is p<0.001, and **** is p<0.0001.

[0034] Figure 9 Image showing the detection of infiltrating lymphocytes in mouse tumor tissue;

[0035] Figure A shows the proportion of CD8+ cells in the tumor tissue, and Figure B shows the proportion of NK1.1 positive cells in the tumor tissue.

[0036] Figure 10 Survival curves of mice after CD8+ T cells and NK cells were cleared. Detailed Implementation

[0037] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0038] Example 1: Construction of recombinant oncolytic virus sPD1IL15RaIL15

[0039] Using VSV oncolytic virus as a platform, we constructed recombinant virus VSV-sPD1IL15RaIL15 expressing sPD1IL15RaIL15, control virus VSV-sPD1 expressing sPD1 alone, and VSV-IL15RaIL15 expressing IL15RaIL15 alone.

[0040] Based on the structural diagram of the recombinant protein gene, gene fragments of recombinant proteins sPD1, IL15RaIL15, and sPD1IL15RaIL15 were obtained through gene synthesis. These three recombinant protein gene fragments were amplified using PCR. The sPD1, IL15RaIL15, and sPD1IL15RaIL15 genes were cloned into the VSV whole-genome plasmid using the In-Fusion HD Cloning Kit to obtain the recombinant viral genome. After rescue, the recombinant viruses VSV-sPD1IL15RaIL15, VSV-sPD1, and VSV-IL15RaIL15 were obtained. Specific steps:

[0041] (1) Design of sPD1

[0042] sPD1 removes the PD-1 signal peptide from the extracellular region (17-170aa) of PD-1, uses the CD33 signal peptide to increase sPD1 secretion efficiency, and adds a 6×His tag sequence to the C-terminus of sPD1 for the purification and detection of recombinant protein.

[0043] CD33 signal peptide nucleic acid sequence (SEQ ID NO.1):

[0044] ATGCCCCTATTGCTTTTGCTCCCTCTACTGTGGGCT

[0045] 6×His-tagged nucleic acid sequence (SEQ ID NO.2): CACCATCACCATCACCACPD-1

[0046] Extracellular nucleic acid sequence (SEQ ID NO.3):

[0047] CCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGCCAGGACTGCCGCTTC CGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTG.

[0048] (2) Design of IL15RaIL15

[0049] IL-15Ra (1-66aa) was fused with IL-15 (IL-15 with amino acid N mutated to D at 72 amino acid), and linked in the middle using the linker sequence (GGGGS)4 to form IL15RaIL15. The CD33 signal peptide was added to the N-terminus of IL15RaIL15, and a 6×His tag sequence was added to the C-terminus.

[0050] IL-15Ra nucleic acid sequence (SEQ ID NO.4):

[0051] ATCACCTGTCCTCCCCATGAGCGTGGAGCACGCCGACATCTGGGTTAAGAGCTACAGCCTCTACTCCAGAGAGCGCTACATCTGCAATTCTGGCTTTAAGCGGAAGGCCGGCACGAGCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCCTAGCCTGAAATGCATCAAGCCT

[0052] IL-15 nucleic acid sequence (SEQ ID NO.5):

[0053] CTGCAAAATTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGATCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTTCTGATGTGCACCCCAGCTGTAAAGTGACCGCTATGAAGTGCTTCCTGCTGGAGCTGCAAGTGATCAGCCTGGAGAGCGGGGAC GCCTCAATCCACGATACCGTGGAAAACCTCATTATCCTGGCCAACGACAGCCTTTCTAGCAACGGCAACGTCACAGAGAGCGGATGTAAAGAGTGCGAGGAACTCGAAGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATTGTGCAGATGTTCATCAACACATCT

[0054] Linker nucleic acid sequence (SEQ ID NO.6):

[0055] GGAGGTGGCGGAAGCGGAGGCGGTGGCAGTGGAGGCGGTGGCAGTGGAGGCGGCGGCAGC

[0056] (3) Design of recombinant protein sPD1IL15RaIL15

[0057] sPD1 was fused with IL15RaIL15, with the (GGGGS)3liner sequence connecting them in the middle. The CD33 signal peptide was inserted at the N-terminus and a 6-histidine tag was inserted at the C-terminus to obtain the recombinant protein sPD1IL15RaIL15.

[0058] The sPD1IL15RaIL15 nucleic acid sequence (SEQ ID NO.7), with the underlined sequences being the CD33 signal peptide sequence and the His tag sequence:

[0059] ATGCCCCTATTGCTTTTGCTCCCTCTACTGTGGGCTCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTG GGAGGTGGCGGAAGCGGAGGCGGTGGCAGTGGAGGCGG CGGCAGCATCACCTGTCCTCCCCCCATGAGCGTGGAGCACGCCGACATCTGGGTTAAGAGCTACAGCCTCTACTCCAGAGAGCGCTACATCTGCAATTCTGGCTTTAAGCGGAAGGCCGGCACGAGCAGCCTGACAGAGTGCGTGCTGAACAAGGCCACCAACGTGGCCCACTGGACCACCCCTAGCCTGAAATGCATCAAGCCTTCTGGCGGCAGCGGAGGTGGCGGAAGCGGCGGTGGCAGTGGAGGCGGCGGCAGCCTGCAAAATTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGATCTGATCCAGAGCATGCACATCGACGCCACACTGTACACCGAGTCTGATGTGCACCCCAGCTGTAAAGTGACCGCTATGAAGTGCTTCCTGCTGGAGCTGCAAGTGATCAGCCTGGAGAGCGGGGACGCCTCAATCCACGATACCGTGGAAAACCTCATTATCCTGGCCAACGACAGCCTTTCTAGCAACGGCAACGTCACAGAGAGCGGATGTAAAGAGTGCGAGGAACTCGAAGAGAAGAACATCAAGGAGTTCCTGCAGAGCTTCGTGCACATTGTGCAGATGTTCATCAACACATCT CACCATCACCATCACCAC TGA.

[0060] (4) Cloning of viral whole genome plasmids pT7-VSV-sPD1IL15RaIL15, pT7-VSV-sPD1 and pT7-VSV-IL15RaIL15

[0061] Using the synthetic genes sPD1IL15RaIL15, sPD1 and IL15RaIL15 as templates, and

[0062] VSV-F: TAATCAGAATTCTCGAGACCGCCACCATGGACTGGAC (SEQ ID NO.8) and

[0063] VSV-R: AGTTTTTTTCATATGGCCATCAGTGGTGGTGGTGGT (SEQ ID NO.9)G was used as primers. The Shanghai Micro-Chemistry HiFi mix high-fidelity PCR kit was used with the following reaction conditions: 94℃ pre-denaturation for 15 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, repeated 35 times; 72℃ extension for 5 min; and storage at 4℃. The sPD1IL15RaIL15, sPD1, and IL15RaIL15 fragments were amplified by PCR.

[0064] Table 1 PCR reaction system

[0065] Components Dosage 2×HiFi mix 25μl KDS 1μl sPD1IL15RaIL15 / sPD1 / IL15RaIL15 100ng VSV-F (10μM) 1μl VSV-R (10μM) 1μl <![CDATA[ddH2O]]> Add to a total volume of 50 μl

[0066] The pT7-VSV whole genome plasmid was digested with XhoI and MSCI under the following conditions: 37℃ for 4 hours. Then, it was purified using a gel purification kit (Tiangen Biotech) to obtain the linear pT7-VSV whole genome.

[0067] The VSV full genome sequence is based on vesicular stomatitis Indiana virus, with a new transcription unit (ATU) inserted before its N gene. This ATU contains the restriction enzyme sites XhoI and MScI, as well as transcription start and termination sites, facilitating enzyme digestion and gene expression.

[0068] Table 2 Enzyme digestion reaction system

[0069] Components Dosage 10×CutSmart buffer 10μl MscI 5μl XhoI 5μl pT7-VSV 5μg <![CDATA[ddH2O]]> Add to total 100 μl

[0070] The sPD1IL15RaIL15, sPD1, and IL15RaIL15 fragments were cloned into the ATU region (ATU is an additional transcription unit) of pT7-VSV using the In-Fusion kit. The reaction conditions were 37℃ for 15 min and 50℃ for 15 min to obtain the recombinant viral whole genome plasmids pT7-VSV-sPD1IL15RaIL15, pT7-VSV-sPD1, and pT7-VSV-IL15RaIL15.

[0071] Table 3. Cloning system of the In-Fusion reagent kit

[0072] Component Name Added amount In-Fusion mix 10μl sPD1IL15RaIL15 / sPD1 / IL15RaIL15 80ng pT7-VSV (linearization) 160ng <![CDATA[ddH2O]]> Add to a total volume of 20 μl

[0073] The full genome sequences of pT7-VSV, pT7-VSV-sPD1IL15RaIL15, pT7-VSV-sPD1, and pT7-VSV-IL15RaIL15 are shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13, respectively.

[0074] (5) Rescue of recombinant oncolytic viruses VSV-sPD1IL15RaIL15, VSV-sPD1 and VSV-IL15RaIL15

[0075] Seed 293T cells at a ratio of 800,000 cells per well into 6-well cell culture plates and cultured overnight at 37°C with 5% CO2 in complete medium (high glucose DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin). The cells reached a cell density of 70-80% at the bottom of the culture plate. Prepare solutions A and B according to the transfection reagent preparation table. Add solution A to solution B, mix thoroughly, and incubate at room temperature for 10 minutes. Add 600 μl of opti-MEM and mix gently.

[0076] Table 4. Preparation of Transfection Reagents

[0077]

[0078] Take 293T cells cultured overnight, remove the cell supernatant, add a mixture of A and B solutions, and incubate at 37℃ in a 5% CO2 cell culture incubator for 4 hours. Add 3 ml of complete culture medium and continue culturing. After approximately 60-96 hours until cytopathic effect reaches 90-100%, collect the cell supernatant, centrifuge at 2000g for 10 min to remove cell debris, collect the supernatant to obtain VSV-sPD1IL15RaIL15 seed virus, aliquot 500 μl / tube, and store at -80℃.

[0079] VSV-sPD1IL15RaIL15 amplification: 293T cells were cultured in large quantities and seeded into 10 petri dishes with a diameter of 10 cm. When the cell density reached about 70-80%, 100 μL of seed virus was added per dish. The cells were cultured in a 37℃, 5% CO2 cell culture incubator for 48-72 hours until all cells showed cytopathic effects. The supernatant was collected, centrifuged at 4000 rpm for 20 minutes to remove cell debris, and the supernatant was collected again to obtain the oncolytic virus VSV-sPD1IL15RaIL15. After aliquoting, the virus was stored at -80℃.

[0080] The rescue and amplification of VSV-sPD1 and VSV-IL15RaIL15 are performed using the same steps as VSV-sPD1IL15RaIL15.

[0081] (6) Virus titer determination

[0082] Vero cells were seeded into 96-well plates at a concentration of 1 × 10⁶. 4 Cells / well were cultured overnight at 37°C in a 5% CO2 cell incubator; the viral stock solution was serially diluted to 10^10 using DMEM culture medium at a 1:10 ratio. -9The concentration gradients are 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 and 10 -9 Seed Vero cells at 100 μl per well in 96-well plates, with 10 wells per dilution. Incubate at 37°C in a 5% CO2 incubator for 4 hours. Remove the virus solution and continue culturing in complete culture medium containing 2% fetal bovine serum. Observe cytopathic effects after 96 hours, record and calculate the viral titer using the formula: TCID50 = 10-1. 2+(S / 10-0.5) / mL, PFUs / mL=TCID50 / mL×0.7, S is the total number of cytopathic wells.

[0083] Example 2: Testing of sPD1IL15RaIL15 Function

[0084] 1. Viral oncolytic ability detection

[0085] Human ovarian cancer cell lines SKOV3, ES2, OVCAR3 and mouse ovarian cancer cell line ID8 were amplified in T75 culture flasks until the cell density reached approximately 90%. After digestion with 0.25% trypsin-EDTA digestion solution, single-cell suspensions were prepared. After cell counting, 10,000 cells per well (total volume 100 μL) were seeded into 96-well plates, with 5 rows of each cell type and 6 replicate wells per row. The 96-well plates were placed in a cell culture incubator at 37°C with 5% CO2 and cultured for 6 hours until the cells adhered.

[0086] VSV-sPD1 and VSV-IL15RaIL15 viral plasmids were constructed according to the method in Example 1. The cells were divided into five groups: blank Mock group, VSV, VSV-sPD1, VSV-IL15RaIL15, and VSV-sPD1IL15RaIL15, with one group labeled per column. 5 μL of virus was added to each group, with a multiplicity of infection (MOI) of 0.01. The blank control Mock group was incubated with 5 μL of RPMI 1640 medium for 48 hours. The cell supernatant was removed, and the cells were gently washed twice with PBS. 200 μL of 4% paraformaldehyde was added, and the cells were incubated at room temperature for 10 minutes to remove the paraformaldehyde. The cells were then washed twice more with PBS, and 100 μL of 0.1% crystal violet staining solution was added. The cells were incubated at room temperature for 10 minutes to remove the stain. Excess stain was washed away with water, and the cells were air-dried at room temperature before photographing and analysis.

[0087] Cell viability was analyzed using ImageJ2 software. The gray values ​​of cells in each well and the gray values ​​of blank wells were measured. The gray values ​​of blank wells were used as the baseline. The gray values ​​of each well were subtracted from the gray values ​​of the blank wells. The cell viability of the blank mock group was set at 100%. The proportion of live cells in each virus treatment group was calculated. Data analysis was performed using Graphpad Prism 6.0 software and the One-way ANOVA method was used for statistical analysis.

[0088] The results are as follows Figure 2 As shown, after using the virus with an MOI of 0.01, the virus treatment groups VSV, VSV-sPD1, VSV-IL15RaIL15 and VSV-sPD1IL15RaIL15 all resulted in approximately 80% cell death after 48 hours, with no significant difference between the groups. This indicates that the insertion of exogenous genes into sPD1, IL15RaIL15 and sPD1IL15RaIL15 does not affect the oncolytic ability of the virus.

[0089] 2. Virus replication ability detection

[0090] Human ovarian cancer cell line SKOV3 was inoculated into T75 cell culture flasks and cultured overnight until the cell density reached approximately 90% the next day. Cells were then digested with 0.25% trypsin-EDTA to prepare a single-cell suspension. After cell counting, the cells were diluted to a final concentration of 2 × 10⁶ cells / mL. 5 500 μL (1 × 10⁶ cells) of SKOV3 were seeded into 24-well plates. 5 (12 cells) were collected and cultured in 12 wells at 37°C with 5% CO2 for 4 hours until the cells were fully adhered to the well.

[0091] VSV, VSV-sPD1, VSV-IL15RaIL15, and VSV-sPD1IL15RaIL15 viruses were diluted to a final concentration of 1×10 using RPMI 1640 complete culture medium. 4 PFUs / ml, the prepared 24-well SKOV3 plates were divided into four groups: VSV, VSV-sPD1, VSV-IL15RaIL15, and VSV-sPD1IL15RaIL15, with 3 wells in each group and labeled. The cell supernatant was removed, and 500 μL of diluted virus suspension was added to each well according to the group. The plates were incubated at 37°C for 30 minutes, the virus suspension was removed, RPMI-1640 complete medium was added, and the plates were cultured for another 48 hours. The cell supernatant was collected, and the virus titer in each well was detected using the dilution method.

[0092] Virus titer assay: Vero cells were inoculated into 96-well plates with 10,000 cells per well, arranged in 8 rows of 10 wells each, and incubated in a CO2 incubator for 4 hours until cell adhesion. The collected virus stock solution with an MOI of 0.01 was serially diluted 10-fold to a final concentration of 10.-10 , 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 and 10 -10 Add the diluted virus to the prepared Vero cells, adding 100 μL of diluted virus to each well, and continue culturing for 5 days. Observe the cell lesions in each well, record the number of lesion wells, and calculate the virus titer.

[0093] like Figure 3 As shown, after 48 hours, each virus group could obtain 4×10 7 -9×10 7 There were no significant differences in viral load among the PFUs groups VSV, VSV-sPD1, VSV-IL15RaIL15, and VSV-sPD1IL15RaIL15.

[0094] The above results indicate that the insertion of exogenous genes does not affect the virus's replication ability.

[0095] 3. Detection of recombinant protein expression and secretion

[0096] Human ovarian cancer cell line SKOV3 was infected with viral loads of VSV-sPD1, VSV-IL15RaIL15 and VSV-sPD1IL15RaIL15 at MOI=0.01, respectively. After 72 h, the cell culture supernatant was collected, and protein expression was detected by Western blot using His-tagged antibody as the primary antibody.

[0097] like Figure 4 As shown, bands of approximately 40-55 kDa were observed in the VSV-sPD1IL15RaIL15 lane, while the bands of VSV-sPD1 and VSV-IL15RaIL15 were approximately 25 kDa, consistent with the predicted band sizes. This indicates that the VSV-sPD1, VSV-IL15RaIL15, and VSV-sPD1IL15RaIL15 groups can infect ovarian cancer cells and express the target proteins sPD1, IL15RaIL15, and sPD1IL15RaIL15, which are secreted into the cell supernatant.

[0098] 4. Functional assay of recombinant protein sPD1IL15RaIL15 binding to PD-L1

[0099] 293T cells were infected with VSV-sPD1IL15RaIL15, and the cell culture supernatant was collected. The sPD1IL15RaIL15 protein was purified by nickel column chromatography. During SKOV3 cell culture, IFN-γ was added to a final concentration of 2000 IU / ml and the cells were cultured at 37℃ for 12 hours. After digestion with 0.25% trypsin, the resulting solution was prepared as 1×10⁻⁶ cells / mL. 6 For a single-cell suspension of cells / ml, add 100ul of purified sPD1IL15RaIL15 protein to a final concentration of 300ng / ml, incubate at room temperature for 1 hour, then stain with fluorescently labeled anti-His-FITC and detect by flow cytometry.

[0100] The results are as follows Figure 5 After stimulation with IFN-γ at a final concentration of 2000 IU / ml, SKOV3 cells highly expressed PDL1, and the fluorescence signal of SKOV3 cells was significantly enhanced after staining with ANTI-PDL1-PE antibody. This indicates that SKOV3 cells highly express PD-L1 molecules on their surface after IFN-γ stimulation (see...). Figure 5 A).

[0101] When SKOV3 cells were co-incubated with sPD1IL15RaIL15 and IFN-γ after stimulation, followed by anti-His-FITC staining, flow cytometry analysis revealed that SKOV3 cells incubated with sPD1IL15RaIL15 showed a stronger fluorescence signal. However, when anti-His-FITC staining was performed without sPD1IL15RaIL15 incubation, the fluorescence signal of SKOV3 cells was not significantly enhanced and was the same as that of SKOV3 cells without IFN-γ stimulation (see [link to relevant documentation]). Figure 5 (B) These results indicate that IFN-γ stimulation can induce SKOV3 cells to overexpress PD-L1 molecules. sPD1IL15RaIL15 can bind to PD-L1, and anti-His-FITC recognition of sPD1IL15RaIL15 significantly enhances the fluorescence signal in PD-L1-positive SKOV3 cells.

[0102] 5. Detection of T cell amplification capacity of sPD1IL15RaIL15

[0103] 20 ml of venous blood was drawn from a healthy volunteer using a heparin anticoagulant tube, centrifuged at 1000 g for 20 min, and the plasma was removed. The blood cells were then resuspended in 40 ml of normal saline. 15 ml of lymphocyte separation medium was added to a 50 ml centrifuge tube, and the resuspended cell suspension was slowly added to the upper layer of lymphocytes. The cells were centrifuged at 2000 rpm for 20 min to separate peripheral blood mononuclear cells (PBMCs). PBMCs were resuspended in RPMI-1640 medium containing 10% fetal bovine serum and 100 ng / ml anti-CD3. They were randomly divided into three groups with three replicates each: sPD1IL15RaIL15, IL-2, and I-L15. PBMCs were added to each group at a final concentration of 50 ng / ml for sPD1IL15RaIL15, 1000 IU / ml for IL-2, or 50 ng / ml for IL-15, respectively. The cells were incubated at 37°C in a 5% CO2 incubator for 10 days. Depending on the cell count, complete RPMI-1640 medium (containing 10% fetal bovine serum, supplemented with the corresponding cytokines sPD1IL15RaIL15 50 ng / ml, IL-2 1000 IU / ml, or IL-15 50 ng / ml) was added every 3-4 days. Cell counts were recorded on days 4 and 10, and cell growth curves were plotted.

[0104] The results are as follows Figure 6 As shown, the number of T cells counted on day 10 of the sPD1IL15RaIL15, IL-2, and IL-15 groups were 8.6 × 10⁻⁶. 7 1.1×10 8 and 1.8×10 7 There was no significant difference between the sPD1IL15RaIL15 group and the IL-2 group, and the sPD1IL15RaIL15 group was higher than the IL-15 group. The results indicate that the ability of sPD1IL15RaIL15 to expand T cells is no different from that of IL-2, and is higher than that of IL-15. The ability of sPD1IL15RaIL15 to activate T cells is stronger than that of IL-15.

[0105] Application example: Antitumor immunotherapy of recombinant viruses in a mouse ovarian cancer model.

[0106] 1. Study on the antitumor effect of recombinant virus in mouse ovarian cancer ascites tumor model.

[0107] Using 8-10 week old C57BL6 mice, on day 1, each mouse was injected intraperitoneally with 5×10⁻⁶ mg / L. 6ID8-Luc cells. On day 10, tumor formation in mice was assessed using in vivo imaging. Tumor-forming mice were randomly divided into 5 groups: a VSV, VSV-sPD1, VSV-IL15RaIL15, VSV-sPD1IL15RaIL15 virus treatment group, and an untreated mock group, with 7 mice in each group. The virus was injected intraperitoneally into mice on days 10, 14, and 18 after grouping, at a dose of 5 × 10⁶ cells. 8 PFUs / mouse / time were used to monitor the survival status of mice until their death, and the lifespan of the mice was calculated.

[0108] The results are as follows Figure 7 As shown, the median survival time of mice in each group was: 27 days in the Mock group, 18 days in the VSV group, 42 days in the VSV-sPD1 group, 52 days in the VSV-IL15RaIL15 group, and 75 days in the VSV-sPD1IL15RaIL15 group. It is evident that the survival time of mice in the VSV-sPD1IL15RaIL15 group was significantly longer than that of the other control groups.

[0109] 2. Study on the activation of anti-tumor immunity by VSV-sPD1IL15RaIL15

[0110] 8-10 week old C57BL6 mice were subcutaneously injected with 1×10⁻⁶ ozontally into the right axillary region. 7 ID8-Luc cells. Tumor size was monitored, and when the tumor volume reached 500 mm². 3 At the tumor size, mice were randomly divided into 5 groups: a VSV, VSV-sPD1, VSV-IL15RaIL15, and VSV-sPD1IL15RaIL15 virus treatment group and an untreated control group (Mock group), with 5 mice in each group. According to the grouping, 5 × 10⁶ viruses were injected intratumorally into the mice. 8 PFUs / mouse / dose, each mouse was injected with the virus 3 times, with an interval of 3 days between each injection. The mice were sacrificed on the 4th day after the virus injection, the tumor was isolated and made into a single cell suspension, and the tumor-activated lymphocytes, tumor-infiltrating CD8+ T cells and NK cells in the microenvironment were detected by Elispot and flow cytometry.

[0111] Tumor-specific lymphocytes, activated by tumor cells, release IFN-γ. Single-cell suspensions of isolated tumor tissue were seeded at a rate of 200,000 cells per well into an Elispot assay plate and incubated at 37°C with 5% CO2 for 12 hours. The tumor-specific lymphocytes, activated by tumor cells, release IFN-γ, which is then captured by antibodies at the bottom of the culture plate. Enzyme-linked immunosorbent assay (ELISA) is then used to display the captured IFN-γ as brown spots at the bottom of the plate. Each spot represents one or a cluster of tumor-activated lymphocytes.

[0112] The results are as follows Figure 8As shown in Figure A, typical Elispot images of each group are presented, and Figure B shows the statistical results of Elispot spots in each group. The number of brown spots in all virus groups was significantly higher than in the untreated control group. The number of spots in the VSV-sPD1IL15RaIL15 treatment group was significantly higher than in other treatment groups. The VSV-IL15RaIL15 group had more spots than the VSV-sPD1 group, and the VSV-sPD1 group had more spots than the VSV group. These results indicate that the proportion of tumor-specific lymphocytes in the tumor tissue of the VSV-sPD1IL15RaIL15 treatment group was significantly higher than in other groups, and VSV-sPD1IL15RaIL15 can induce more tumor-specific lymphocytes.

[0113] CD8+ T cells and NK cells infiltrating tumor tissue, such as Figure 9 As shown, the levels of VSV-sPD1IL15RaIL15 were significantly higher than in other groups, while there was no significant difference in the number of infiltrating lymphocytes between the VSV-sPD1 and VSV-IL15RaIL15 groups. This indicates that VSV-sPD1IL15RaIL15 treatment can significantly increase the level of tumor-specific lymphocytes in the tumor microenvironment. Figure 9 Figure A shows the proportion of CD8+ cells in the tumor tissue, and Figure B shows the proportion of NK1.1 positive cells in the tumor tissue.

[0114] 3. The role of recombinant viruses in different types of immune cells in anti-tumor responses

[0115] The experiment was conducted using 8-10 week old female C57BL6 mice. On day 1, each mouse was injected intraperitoneally with 5×10 6 ID8-Luc cells were collected. On day 10, mice were randomly divided into four groups: Mock group, anti-CD8a+VSV-sPD1IL5RaIL5 group, anti-NK1.1+VSV-sPD1IL5RaIL5 group, and VSV-sPD1IL5RaIL5 group. Each mouse received an intraperitoneal injection of either anti-CD8a 200 ng / mouse or anti-NK1.1 200 ng / mouse, once weekly, to deplete CD8+ T cells or NK cells. Simultaneously, on days 10, 14, and 18, mice were injected intraperitoneally with 5 × 10⁶ recombinant virus cells. 8 PFUs / mouse / dose. Monitor mouse survival status until death and calculate mouse lifespan. Investigate the impact of lymphocyte clearance on viral therapy to determine which lymphocytes play a major role in treatment.

[0116] The results are as follows Figure 10As shown, the use of NK antibodies to deplete mouse NK cells did not affect the therapeutic effect of VSV-sPD1IL15RaIL15, and there was no significant difference in survival time compared with the VSV-sPD1IL15RaIL15 alone treatment group; however, the use of anti-CD8a antibodies to deplete mouse CD8+ T cells significantly inhibited the therapeutic effect of VSV-sPD1IL15RaIL15, and the median survival time of mice was significantly shortened, which was not different from the control group.

[0117] The results showed that the anti-tumor effect of VSV-sPD1IL15RaIL15 depends on CD8+ T cells.

[0118] Partial sequence from the instruction manual:

[0119] PT7-VSV nucleic acid sequence (SEQ ID NO.10)

[0120] acgaagacaaacaaaccattattatcattaaaaggctcaggagaaactttaacagtaatcagaattctcgagaccgccacctggccatatgaaaaaaactaacagtaatc

[0121] aaaatgtctgttacagtcaagagaatcattgacaacacagtcatagttccaaaacttcctgcaaatgaggatccagtggaatacccggcagattacttcagaaaatcaaag

[0122] gagattcctctttacatcaatactacaaaaagtttgtcagatctaagaggatatgtctaccaaggcctcaaatccggaaatgtatcaatcatacatgtcaacagctacttgtat

[0123] ggagcattaaaggacatccggggtaagttggataaagattggtcaagtttcggaataaacatcgggaaagcaggggatacaatcggaatatttgaccttgtatccttgaa

[0124] agccctggacggcgtacttccagatggagtatcggatgcttccagaaccagcgcagatgacaaatggttgcctttgtatctacttggcttatacagagtgggcagaacac

[0125] aaatgcctgaatacagaaaaaagctcatggatgggctgacaaatcaatgcaaaatgatcaatgaacagtttgaacctcttgtgccagaaggtcgtgacatttttgatgtgtg

[0126] gggaaatgacagtaattacacaaaaattgtcgctgcagtggacatgttcttccacatgttcaaaaaacatgaatgtgcctcgttcagatacggaactattgtttccagattca

[0127] aagattgtgctgcattggcaacatttggacacctctgcaaaataaccggaatgtctacagaagatgtaacgacctggatcttgaaccgagaagttgcagatgaaatggtc

[0128] caaatgatgcttccaggccaagaaattgacaaggccgattcatacatgccttatttgatcgactttggattgtcttctaagtctccatattcttccgtcaaaaaccctgccttcc

[0129] acttctgggggcaattgacagctcttctgctcagatccaccagagcaaggaatgcccgacagcctgatgacattgagtatacatctcttactacagcaggtttgttgtacgc

[0130] ttatgcagtaggatcctctgccgacttggcacaacagttttgtgttggagataacaaatacactccagatgatagtaccggaggattgacgactaatgcaccgccacaag

[0131] gcagagatgtggtcgaatggctcggatggtttgaagatcaaaacagaaaaccgactcctgatatgatgcagtatgcgaaaagagcagtcatgtcactgcaaggcctaa

[0132] gagagacaattggcaagtatgctaagtcagaatttgacaaatgaccctataattctcagatcacctattatatattatgctacatatgaaaaaaactaacagatatcatg

[0133] gataatctcacaaaagttcgtgagtatctcaagtcctactctcgtctagatcaggcggtaggagagatgagatcgaagcaacgagctgaaaagtccaattatga

[0134] gttgttccaagacggagtggagagcatactaggccctttatttcaggcagcagatgattctgacacagaatctgaaccagaaattgaagacaatcaaggcttgt

[0135] atgtaccagatccggaagctgagcaagttgaaggctttatacaggggccttagatgactatgcagatgagacgtggatgttgtattcacttcggactggaaacagcct

[0136] gagcttgaatccgacgagcatggaaagaccttacggttgacattgccagagggtttaagtggagagcagaaatcccagtggctttgacgacgattaaagcagtcgttcaaa

[0137] gtgccaaacactggaatctggcagagtgcacatttgaagcatcgggagaaggggtcatcataaaaagcgccagataactccggatgtataaggtcactccagtgat

[0138] gaacacacatccgtaccaatcagaagccgtatcagattgtggtctctcaagacatccatgacttccaacccaagaaagcaagtcttcagcctctcaccatatccttg

[0139] gatgaattgttctcatctagaggagaattcatctctgtcggaggtaacggacgaatgtctcataaagaggccatcctgctcggtctgaggtacaaaagttgtacaatcag

[0140] gcgagagtcaaatattctctgtagactatgaaaaaaagtaacagatatcacaatctaagtgttatcccaatccattcatcatgagttccttaaagaagattctcggtctgaagg

[0141] ggaaaggtaagaaatctaagaaattagggatcgcaccacccccttatgaagaggacactagcatggagtatgctccgagcgctccaattgacaaatcctattttggagtt

[0142] gacgagatggacacctatgatccgaatcaattaagatatgagaaattcttctttacagtgaaaatgacggttagatctaatcgtccgttcagaacatactcagatgtggcag

[0143] ccgctgtatcccattgggatcacatgtacatcggaatggcagggaaacgtcccttctacaaaatcttggcttttttgggttcttctaatctaaaggccactccagcggtattg

[0144] gcagatcaaggtcaaccagagtatcacgctcactgcgaaggcagggcttatttgccacataggatggggaagacccctcccatgctcaatgtaccagagcacttcaga

[0145] agaccattcaatataggtctttacaagggaacgattgagctcacaatgaccatctacgatgatgagtcactggaagcagctcctatgatctgggatcatttcaattcttccaa

[0146] attttctgatttcagagagaaggccttaatgtttggcctgattgtcgagaaaaaggcatctggagcgtgggtcctggattctatcagccacttcaaatgagctagtctagctt

[0147] ccagcttctgaacaatccccggtttactcagtctctcctaattccagcctttcgaacaactaatatcctgtcttttctatccctatgaaaaaaactaacagagatcgatctgtttc

[0148] cttgacacgcgttacgatgcggccgatgaagtgccttttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacacaaccaaaaaggaaactg

[0149] gaaaaatgttccttctaattaccattattgcccgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaaggcta

[0150] ttcaagcagacggttggatgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccgatccttcactccatctgtag

[0151] aacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcctcaaagttgtggatatgcaactgtgacggatgccgaagcagtgat

[0152] tgtccaggtgactcctcaccatgtgctggttgatgaatacacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccataa

[0153] ctctacaacctggcattctgactataaggtcaaagggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaag

[0154] gagggcacagggttcagaagtaactactttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtctggt

[0155] tcgagatggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgtaagtctaattcagga

[0156] cgttgagaggatcttggattattccctctgccaagaaacctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaacccagg

[0157] aaccggtcctgctttcaccataatcaatggtaccctaaaatactttgagaccagatacatcagagtcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtg

[0158] gaactaccacagaaagggaactgtgggatgactgggcaccatatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatac

[0159] atgattggacatggtatgttggactccgatcttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagttta

[0160] ttttttggtgatactgggctatccaaaaatccaatcgagcttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatagggttaatcattggactat

[0161] tcttggttctccgagttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaagtaagcggccgccct

[0162] gcacacagattcttcatgtttgaaccaaatcaacttgtgatatcatgctcaaagaggccttaattatattttaatttttaatttttatgaaaaaaactaacagcaatcatggaagtc

[0163] cacgattttgagaccgacgagttcaatgatttcaatgaagatgactatgccacaagagaattcctgaatcccgatgagcgcatgacgtacttgaatcatgctgattacaattt

[0164] gaattctcctctaattagtgatgatattgacaatttgatcaggaaattcaattctcttccgattccctcgatgtgggatagtaagaactgggatggagttcttgagatgttaacat

[0165] catgtcaagccaatcccatctcaacatctcagatgcataaatggatgggaagttggttaatgtctgataatcatgatgccagtcaagggtatagttttttacatgaagtggac

[0166] aaagaggcagaaataacatttgacgtggtggagaccttcatccgcggctggggcaacaaaccaattgaatacatcaaaaaggaaagatggactgactcattcaaaattc

[0167] tcgcttatttgtgtcaaaagtttttggacttacacaagttgacattaatcttaaatgctgtctctgaggtggaattgctcaacttggcgaggactttcaaaggcaaagtcagaag

[0168] aagttctcatggaacgaacatatgcaggattagggttcccagcttgggtcctacttttatttcagaaggatgggcttacttcaagaaacttgatattctaatggaccgaaactt

[0169] tctgttaatggtcaaagatgtgattatagggaggatgcaaacggtgctatccatggtatgtagaatagacaacctgttctcagagcaagacatcttctcccttctaaatatcta

[0170] cagaattggagataaaattgtggagaggcagggaaatttttcttatgacttgattaaaatggtggaaccgatatgcaacttgaagctgatgaaattagcaagagaatcaag

[0171] gcctttagtcccacaattccctcattttgaaaatcatatcaagacttctgttgatgaaggggcaaaaattgaccgaggtataagattcctccatgatcagataatgagtgtgaa

[0172] aacagtggatctcacactggtgatttatggatcgttcagacattggggtcatccttttatagattattacactggactagaaaaattacattcccaagtaaccatgaagaaaga

[0173] tattgatgtgtcatatgcaaaagcacttgcaagtgatttagctcggattgttctatttcaacagttcaatgatcataaaaagtggttcgtgaatggagacttgctccctcatgatc

[0174] atccctttaaaagtcatgttaaagaaaatacatggcccacagctgctcaagttcaagattttggagataaatggcatgaacttccgctgattaaatgttttgaaatacccgact

[0175] tactagacccatcgataatatactctgacaaaagtcattcaatgaataggtcagaggtgttgaaacatgtccgaatgaatccgaacactcctatccctagtaaaaaggtgtt

[0176] gcagactatgttggacacaaaggctaccaattggaaagaatttcttaaagagattgatgagaagggcttagatgatgatgatctaattattggtcttaaaggaaaggagag

[0177] ggaactgaagttggcaggtagatttttctccctaatgtcttggaaattgcgagaatactttgtaattaccgaatatttgataaagactcatttcgtccctatgtttaaaggcctga

[0178] caatggcggacgatctaactgcagtcattaaaaagatgttagattcctcatccggccaaggattgaagtcatatgaggcaatttgcatagccaatcacattgattacgaaaa

[0179] atggaataaccaccaaaggaagttatcaaacggcccagtgttccgagttatgggccagttcttaggttatccatccttaatcgagagaactcatgaattttttgagaaaagtc

[0180] ttatatactacaatggaagaccagacttgatgcgtgttcacaacaacacactgatcaattcaacctcccaacgagtttgttggcaaggacaagagggtggactggaaggt

[0181] ctacggcaaaaaggatggagtatcctcaatctactggttattcaaagagaggctaaaatcagaaacactgctgtcaaagtcttggcacaaggtgataatcaagttatttgc

[0182] acacagtataaaacgaagaaatcgagaaacgttgtagaattacagggtgctctcaatcaaatggtttctaataatgagaaaattatgactgcaatcaaaatagggacagg

[0183] gaagttaggacttttgataaatgacgatgagactatgcaatctgcagattacttgaattatggaaaaataccgattttccgtggagtgattagagggttagagaccaagaga

[0184] tggtcacgagtgacttgtgtcaccaatgaccaaatacccacttgtgctaatataatgagctcagtttccacaaatgctctcaccgtagctcattttgctgagaacccaatcaat

[0185] gccatgatacagtacaattattttgggacatttgctagactcttgttgatgatgcatgatcctgctcttcgtcaatcattgtatgaagttcaagataagataccgggcttgcaca

[0186] gttctactttcaaatacgccatgttgtatttggacccttccattggaggagtgtcgggcatgtctttgtccaggtttttgattagagccttcccagatcccgtaacagaaagtct

[0187] ctcattctggagattcatccatgtacatgctcgaagtgagcatctgaaggagatgagtgcagtatttggaaaccccgagatagccaagtttcgaataactcacatagacaa

[0188] gctagtagaagatccaacctctctgaacatcgctatgggaatgagtccagcgaacttgttaaagactgaggttaaaaaatgcttaatcgaatcaagacaaaccatcagga

[0189] accaggtgattaaggatgcaaccatatttgtatcatgaagaggatcggctcagaagtttcttatggtcaataaatcctctgttccctagatttttaagtgaattcaaatcagg

[0190] cacttttttgggagtcgcagacgggctcatcagtctatttcaaaattctcgtactattcggaactcctttaagaaaaagtatcatagggaattggatgatttgattgtgaggagt

[0191] gaggtatcctctttgacacatttagggaaacttcatttgagaaggggatcatgtaaaatgtggacatgttcagctactcatgctgacacattaagatacaaatcctggggcc

[0192] gtacagttattgggacaactgtaccccatccattagaaatgttgggtccacaacatcgaaaagagactccttgtgcaccatgtaacacatcagggttcaattatgtttctgtg

[0193] cattgtccagacgggatccatgacgtctttagttcacggggaccattgcctgcttatctagggtctaaaacatctgaatctacatctattttgcagccttgggaaagggaaag

[0194] caaagtcccactgattaaaagagctacacgtcttagagatgctatctcttggtttgttgaacccgactctaaactagcaatgactatactttctaacatccactctttaacaggc

[0195] gaagaatggaccaaaaggcagcatgggttcaaaagaacagggtctgcccttcataggttttcgacatctcggatgagccatggtgggttcgcatctcagagcactgcag

[0196] cattgaccaggttgatggcaactacagacaccatgagggatctgggagatcagaatttcgacttttattccaagcaacgttgctctatgctcaaattaccaccactgttgca

[0197] agagacggatggatcaccagttgtacagatcattatcatattgcctgtaagtcctgtttgagacccataagagatcaccctggactcaagttggactacacgcccccc

[0198] gatgtatcccatgtgctgaagacatggaggaatgggggaaggttcgtggggcaagagagataaaacagatctatcctttagaagggaattggaagaatttagcacctgctg

[0199] agcaatcctatcaagtcggcagatgtataggttttctatatggagaacttggcgtatagaaaatctactcatgccgaggacagttctctatttccttatctatacaaggtcgtat

[0200] tagaggtcgaggtttcttaaaagggttgctagacggattaatgagagcaagttgctgccaagtaataacaccggagaagtctggctcatttgaagaggccggccaacgca

[0201] gtgtacggaggtttgatttacttgattgataaattgagtgtatcacctccattcctttctcttactagatcaggacctattagagacgaattagaaacgattccccacaagatcc

[0202] caacctcctatccgacaagcaaccgtgatatgggggtgattgtcagaaattacttcaaataccaatgccgtctaattgaaaagggaaaatacagatcacattattcacaatt

[0203] atggttattctcagatgtcttatccatagacttcattggaccattctctatttccaccaccctcttgcaaatcctatacaagccatttttatctgggaaagataagaatgagttgag

[0204] agagctggcaaatctttcttcattgctaagatcaggagaggggtgggaagacatacatgtgaaattcttcaccaaggacatattattgtgtccagaggaaatcagacatgc

[0205] ttgcaagttcgggattgctaaggataataataaagacatgagctatcccccttggggaagggaatccagagggacaattacaacaatccctgtttattatacgaccacccc

[0206] ttacccaaagatgctagagatgcctccaagaatccaaaatcccctgctgtccggaatcaggttgggccaattaccaactggcgctcattataaaattcggagtatattacat

[0207] ggaatgggaatccattacagggacttcttgagttgtggagacggctccggagggatgactgctgcattactacgagaaaatgtgcatagcagaggaatattcaatagtct

[0208] gttagaattatcagggtcagtcatgcgaggcgcctctcctgagccccccagtgccctagaaactttaggaggagataaatcgagatgtgtaaatggtgaaacatgttggg

[0209] aatatccatctgacttatgtgacccaaggacttgggactatttcctccgactcaaagcaggcttggggcttcaaattgatttaattgtaatggatatggaagttcgggattcttc

[0210] tactagcctgaaaattgagacgaatgttagaaattatgtgcaccggattttggatgagcaaggagttttaatctacaagacttatggaacatatatttgtgagagcgaaaaga

[0211] atgcagtaacaatccttggtcccatgttcaagacggtcgacttagttcaaacagaatttagtagttctcaaacgtctgaagtatatatggtatgtaaaggtttgaagaaattaat

[0212] cgatgaacccaatcccgattggtcttccatcaatgaatcctggaaaaacctgtacgcattccagtcatcagaacaggaatttgccagagcaaagaaggttagtacatacttt

[0213] accttgacaggtattccctcccaattcattcctgatccttttgtaaacattgagactatgctacaaatattcggagtacccacgggtgtgtctcatgcggctgccttaaaatcat

[0214] ctgatagacctgcagatttattgaccattagccttttttatatggcgattatatcgtattataacatcaatcatatcagagtaggaccgatacctccgaaccccccatcagatgg

[0215] aattgcacaaaatgtggggatcgctataactggtataagcttttggctgagtttgatggagaaagacattccactatatcaacagtgtttggcagttatccagcaatcatttcc

[0216] gattaggtgggaggctatttcagtaaaaggaggatacaagcagaagtggagtactagaggtgatgggctcccaaaagatacccgaatttcagactccttggccccaatc

[0217] gggaactggatcagatctttggaattggtccgaaaccaagttcgtctaaatccattcaataagatcttgttcaatcagctatgtcgtacagtggataatcatttgaagtggtca

[0218] aatttgcgaaaaaacacaggaatgattgaatggatcaatgggcgaatttcaaaagaagaccggtctatactgatgttgaagagtgacctacatgaggaaaactcttggag

[0219] agattaaaaaatcaggaggagactccaaactttaagtatgaaaaaaactttgatccttaagaccctcttgtggtttttatttttttatctggttttgtggtcttcgtgggtcggcat

[0220] ggcatctccacctcctcgcggtccgacctgggcatccgaaggaggacgtcgtccactcggatggctaagggagagctcggatccggctgctaacaaagcccgaaag

[0221] gaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggatc

[0222] gagatcctctagagtcgacctgcaggcatgcaagcttgtattctatagtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgttctaacgac

[0223] aatatgtacaagcctaattgtgtagcatctggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctggt

[0224] aacgccgtcccgcacccggaaatggtcagcgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccggcgccaca

[0225] ggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggcccc

[0226] gtggccgggggactgttgggcgccatctccttgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataa

[0227] gggagagcgtcgaatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgt

[0228] ctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtg

[0229] atacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaa

[0230] atatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatttt

[0231] gccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc

[0232] cttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgcc

[0233] gcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtg

[0234] ataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccg

[0235] gagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttccc

[0236] ggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgt

[0237] gggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacag

[0238] atcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaag

[0239] atcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgt

[0240] aatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagat

[0241] accaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtg

[0242] gcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcga

[0243] acgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcgga

[0244] acaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagg

[0245] ggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataa

[0246] ccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaacc

[0247] gcctctccccgcgcgttggccgattcattaatgcagggggatctcgatcccgcgaaattaatacgactcactatagg

[0248] PT7-VSV-sPD1IL15RaIL15 nucleic acid sequence (SEQ ID NO.11)

[0249] acgaagacaaacaaaccattattatcattaaaaggctcaggagaaactttaacagtaatcagaattctcgagaccgccaccatgcccctattgcttttgctccctctactgtg

[0250] ggctccaggatggttcttagactccccagacaggccctggaacccccccaccttctccccagccctgctcgtggtgaccgaaggggacaacgccaccttcacctgcag

[0251] cttctccaacacatcggagagcttcgtgctaaactggtaccgcatgagccccagcaaccagacggacaagctggccgccttccccgaggaccgcagccagcccggc

[0252] caggactgccgcttccgtgtcacacaactgcccaacgggcgtgacttccacatgagcgtggtcagggcccggcgcaatgacagcggcacctacctctgtggggccat

[0253] ctccctggcccccaaggcgcagatcaaagagagcctgcgggcagagctcagggtgacagagagaagggcagaagtgcccacagcccaccccagcccctcaccc

[0254] aggccagccggccagttccaaaccctggtgggaggtggcggaagcggaggcggtggcagtggaggcggcggcagcatcacctgtcctccccccatgagcgtgga

[0255] gcacgccgacatctgggttaagagctacagcctctactccagagagcgctacatctgcaattctggctttaagcggaaggccggcacgagcagcctgacagagtgcgt

[0256] gctgaacaaggccaccaacgtggcccactggaccacccctagcctgaaatgcatcaagccttctggcggcagcggaggtggcggaagcggcggtggcagtggag

[0257] gcggcggcagcctgcaaaattgggtgaacgtgatcagcgacctgaagaagatcgaggatctgatccagagcatgcacatcgacgccacactgtacaccgagtctgat

[0258] gtgcaccccagctgtaaagtgaccgctatgaagtgcttcctgctggagctgcaagtgatcagcctggagagcggggacgcctcaatccacgataccgtggaaaacctc

[0259] attatcctggccaacgacagcctttctagcaacggcaacgtcacagagagcggatgtaaagagtgcgaggaactcgaagagaagaacatcaaggagttcctgcagag

[0260] cttcgtgcacattgtgcagatgttcatcaacacatctcaccatcaccatcaccactgatggccatatgaaaaaaactaacagtaatcaaaatgtctgttacagtcaagagaat

[0261] cattgacaacacagtcatagttccaaaacttcctgcaaatgaggatccagtggaatacccggcagattacttcagaaaatcaaaggagattcctctttacatcaatactaca

[0262] aaaagtttgtcagatctaagaggatatgtctaccaaggcctcaaatccggaaatgtatcaatcatacatgtcaacagctacttgtatggagcattaaaggacatccggggt

[0263] aagttggataaagattggtcaagtttcggaataaacatcgggaaagcaggggatacaatcggaatatttgaccttgtatccttgaaagccctggacggcgtacttccagat

[0264] ggagtatcggatgcttccagaaccagcgcagatgacaaatggttgcctttgtatctacttggcttatacagagtgggcagaacacaaatgcctgaatacagaaaaaagct

[0265] catggatgggctgacaaatcaatgcaaaatgatcaatgaacagtttgaacctcttgtgccagaaggtcgtgacatttttgatgtgtggggaaatgacagtaattacacaaa

[0266] aattgtcgctgcagtggacatgttcttccacatgttcaaaaaacatgaatgtgcctcgttcagatacggaactattgtttccagattcaaagattgtgctgcattggcaacattt

[0267] ggacacctctgcaaaataaccggaatgtctacagaagatgtaacgacctggatcttgaaccgagaagttgcagatgaaatggtccaaatgatgcttccaggccaagaaa

[0268] ttgacaaggccgattcatacatgccttatttgatcgactttggattgtcttctaagtctccatattcttccgtcaaaaaccctgccttccacttctgggggcaattgacagctctt

[0269] ctgctcagatccaccagagcaaggaatgcccgacagcctgatgacattgagtatacatctcttactacagcaggtttgttgtacgcttatgcagtaggatcctctgccgact

[0270] tggcacaacagttttgtgttggagataacaaatacactccagatgatagtaccggaggattgacgactaatgcaccgccacaaggcagagatgtggtcgaatggctcgg

[0271] atggtttgaagatcaaaacagaaaaccgactcctgatatgatgcagtatgcgaaaagagcagtcatgtcactgcaaggcctaagagagaagacaattggcaagtatgct

[0272] aagtcagaatttgacaaatgaccctataattctcagatcacctattatatattatgctacatatgaaaaaaactaacagatatcatggataatctcacaaaagttcgtgagtatc

[0273] tcaagtcctactctcgtctagatcaggcggtaggagagatagatgagatcgaagcacaacgagctgaaaagtccaattatgagttgttccaagaggacggagtggaag

[0274] agcatactaggccctcttattttcaggcagcagatgattctgacacagaatctgaaccagaaattgaagacaatcaaggcttgtatgtaccagatccggaagctgagcaa

[0275] gttgaaggctttatacaggggcctttagatgactatgcagatgaggacgtggatgttgtattcacttcggactggaaacagcctgagcttgaatccgacgagcatggaaa

[0276] gaccttacggttgacattgccagagggtttaagtggagagcagaaatcccagtggcttttgacgattaaagcagtcgttcaaagtgccaaacactggaatctggcagagt

[0277] gcacatttgaagcatcgggagaaggggtcatcataaaaaagcgccagataactccggatgtatataaggtcactccagtgatgaacacacatccgtaccaatcagaag

[0278] ccgtatcagatgtttggtctctctcaaagacatccatgactttccaacccaagaaagcaagtcttcagcctctcaccatatccttggatgaattgttctcatctagaggagaat

[0279] tcatctctgtcggaggtaacggacgaatgtctcataaagaggccatcctgctcggtctgaggtacaaaaagttgtacaatcaggcgagagtcaaatattctctgtagacta

[0280] tgaaaaaaagtaacagatatcacaatctaagtgttatcccaatccattcatcatgagttccttaaagaagattctcggtctgaaggggaaaggtaagaaatctaagaaatta

[0281] gggatcgcaccacccccttatgaagaggacactagcatggagtatgctccgagcgctccaattgacaaatcctattttggagttgacgagatggacacctatgatccgaa

[0282] tcaattaagatatgagaaattcttctttacagtgaaaatgacggttagatctaatcgtccgttcagaacatactcagatgtggcagccgctgtatcccattgggatcacatgta

[0283] catcggaatggcagggaaacgtcccttctacaaaatcttggcttttttgggttcttctaatctaaaggccactccagcggtattggcagatcaaggtcaaccagagtatcac

[0284] gctcactgcgaaggcagggcttatttgccacataggatggggaagacccctcccatgctcaatgtaccagagcacttcagaagaccattcaatataggtctttacaaggg

[0285] aacgattgagctcacaatgaccatctacgatgatgagtcactggaagcagctcctatgatctgggatcatttcaattcttccaaattttctgatttcagagagaaggccttaat

[0286] gtttggcctgattgtcgagaaaaaggcatctggagcgtgggtcctggattctatcagccacttcaaatgagctagtctagcttccagcttctgaacaatccccggtttactca

[0287] gtctctcctaattccagcctttcgaacaactaatatcctgtcttttctatccctatgaaaaaaactaacagagatcgatctgtttccttgacacgcgttacgatgcggccgatga

[0288] agtgccttttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacacaaccaaaaaggaaactggaaaaatgttccttctaattaccattattgcc

[0289] cgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaaggctattcaagcagacggttggatgtgtcatgctt

[0290] ccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccgatccttcactccatctgtagaacaatgcaaggaaagcattgaacaaac

[0291] gaaacaaggaacttggctgaatccaggcttccctcctcaaagttgtggatatgcaactgtgacggatgccgaagcagtgattgtccaggtgactcctcaccatgtgctgg

[0292] ttgatgaatacacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccataactctacaacctggcattctgactataaggt

[0293] caaagggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaaggagggcacagggttcagaagtaactactt

[0294] tgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtctggttcgagatggctgataaggatctctttgctg

[0295] cagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgtaagtctaattcaggacgttgagaggatcttggattattccctctg

[0296] ccaagaaacctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaacccaggaaccggtcctgctttcaccataatcaatgg

[0297] taccctaaaatactttgagaccagatacatcagagtcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtggaactaccacagaaagggaactgtggg

[0298] atgactgggcaccatatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatacatgattggacatggtatgttggactccga

[0299] tcttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagtttattttttggtgatactgggctatccaaaaat

[0300] ccaatcgagcttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatagggttaatcattggactattcttggttctccgagttggtatccatctttg

[0301] cattaaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaagtaagcggccgccctgcacacagattcttcatgtttgaaccaaa

[0302] tcaacttgtgatatcatgctcaaagaggccttaattatattttaatttttaatttttatgaaaaaaactaacagcaatcatggaagtccacgattttgagaccgacgagttcaatg

[0303] atttcaatgaagatgactatgccacaagagaattcctgaatcccgatgagcgcatgacgtacttgaatcatgctgattacaatttgaattctcctctaattagtgatgatattga

[0304] caatttgatcaggaaattcaattctcttccgattccctcgatgtgggatagtaagaactgggatggagttcttgagatgttaacatcatgtcaagccaatcccatctcaacatc

[0305] tcagatgcataaatggatgggaagttggttaatgtctgataatcatgatgccagtcaagggtatagttttttacatgaagtggacaaagaggcagaaataacatttgacgtg

[0306] gtggagaccttcatccgcggctggggcaacaaaccaattgaatacatcaaaaaggaaagatggactgactcattcaaaattctcgcttatttgtgtcaaaagtttttggactt

[0307] acacaagttgacattaatcttaaatgctgtctctgaggtggaattgctcaacttggcgaggactttcaaaggcaaagtcagaagaagttctcatggaacgaacatatgcag

[0308] gattagggtcccagcttgggtcctacttttatttcagaaggatgggcttacttcaagaaacttgatattctaatggaccgaaactttctgttaatggtcaaagatgtgattatag

[0309] ggaggatgcaaacggtgctatccatggtatgtagaatagacaacctgttctcagagcaagacatctctcccttctaaatatctacagaattggataaaattgtggagag

[0310] gcagggaaatttttcttatgacttgattaaaatggtggaaccgatatgcaacttgaagctgatgaaattagcaagagaatcaaggcctttagtcccacaattccctcatttga

[0311] aaatcatatcaagacttgttgatgaaggggcaaaattgaggtataagattcctccatgatcagataatgagtgtgaaaacagtggatctcacactggtgattat

[0312] ggatcgttcagacattggggtcatccttttagattattacactggactagaaaatacattcccaagtaaccatgaagaaagatattgatgtgtcattgcaaaagcactt

[0313] gcaagtgatttagctcggattgttcttatttcaacagttcaatgatcatcataaaaagtggttcgtgaatggagacttgctccctcatcatcatccctttaaaagtcatgttaaagaaa

[0314] atacatggcccacagctgctcaagttcaagattttggataaatggcatgaacttccgctgattaaatgttttgaaatacccgacttactagacccatcgataatactctg

[0315] acaaaagtcattcaatgaataggtcagaggtgttgaaacatgtccgaatgaatccgaacactcctatccctagtaaaaaggtgttgcagactatgttggacacaaaggcta

[0316] ccaattggaaagaatttcttaaagagattgatgagaagggcttagatgatgatgatgatctaattattggtcttaaaggaaaggagagggaactgaagttggcaggtagattttt

[0317] ctccctaatgtcttggaaattgcgagaatactttgtaattaccgaatatttgataaagactcatttcgtccctatgtttaaaggcctgacaatggcggacgatctaactgcagtc

[0318] attaaaaagatgttagattcctcatccggccaaggattgaagtcatatgaggcaatttgcatagccaatcacattgattacgaaaaatggaataaccaccaaaggaagttat

[0319] caaacggcccagtgttccgagttatgggccagttcttaggttatccatccttaatcgagagaactcatgaattttttgagaaaagtcttatatactacaatggaagaccagac

[0320] ttgatgcgtgttcacaacaacacactgatcaattcaacctcccaacgagtttgttggcaaggacaagagggtggactggaaggtctacggcaaaaaggatggagtatcc

[0321] tcaatctactggttattcaaagagaggctaaaatcagaaacactgctgtcaaagtcttggcacaaggtgataatcaagttatttgcacacagtataaaacgaagaaatcga

[0322] gaaacgttgtaagattacagggtgctctcaatcaaatggtttctaataatgagaaaaattatgactgcaatcaaaagggacagggaagttaggacttttgataaatgacga

[0323] tgagactatgcaatctgcagattacttgaattatggaaaaataccgattttccgtggagtgattagagggttagagaccaagagatggtcacgagtgacttgtgtcaccaat

[0324] gaccaaatacccacttgtgctaatataatgagctcagtttccacaaatgctctcaccgtagctcattttgctgagaacccaatcaatgccatgatacagtacaattattttggg

[0325] acatttgctagactcttgttgatgatgcatgatcctgctcttcgtcaatcattgtatgaagttcaagataaagataccgggcttgcacagttctactttcaaatacgccatgttgtat

[0326] ttggacccttccattggaggagtgtcgggcatgtctttgtccaggtttttgattagagccttcccagatcccgtaacagaaagtctctcattctggagattcatccatgtacat

[0327] gctcgaagtgagcatctgaaggagatgagtgcagtatttggaaaccccgagatagccaagtttcgaataactcacatagacaagctagtagaagatccaacctctctga

[0328] acatcgctatgggaatgagtccagcgaacttgttaaagactgaggttaaaaaatgcttaatcgaatcaagacaaaccatcaggaaccaggtgattaaggatgcaaccata

[0329] tatttgtatcatgaagaggatcggctcagaagtttcttatggtcaataaatcctctgttccctagatttttaagtgaattcaaatcaggcacttttttgggagtcgcagacgggct

[0330] catcagtctatttcaaaattctcgtactattcggaactcctttaagaaaaagtatcatagggaattggatgatttgattgtgaggagtgaggtatcctctttgacacatttaggg

[0331] aaacttcatttgagaaggggatcatgtaaaatgtggacatgttcagctactcatgctgacacattaagatacaaatcctggggccgtacagttattgggacaactgtacccc

[0332] atccattagaaatgttgggtccacaacatcgaaaagagactccttgtgcaccatgtaacacatcagggttcaattatgtttctgtgcattgtccagacgggatccatgacgtc

[0333] tttagttcacggggaccattgcctgcttatctagggtctaaaacatctgaatctacatctattttgcagccttgggaaagggaaagcaaagtcccactgattaaaagagctac

[0334] acgtcttagagatgctatctcttggtttgttgaacccgactctaaactagcaatgactatactttctaacatccactctttaacaggcgaagaatggaccaaaaggcagcatg

[0335] ggttcaaaagaacagggtctgcccttcataggttttcgacatctcggatgagccatggtgggttcgcatctcagagcactgcagcattgaccaggttgatggcaactaca

[0336] gacaccatgagggatctgggagatcagaatttcgactttttattccaagcaacgttgctctatgctcaaattaccaccactgttgcaagagacggatggatcaccagttgta

[0337] cagatcattatcatattgcctgtaagtcctgtttgagacccatagaagagatcaccctggactcaagtatggactacacgcccccagatgtatcccatgtgctgaagacatg

[0338] gaggaatggggaaggttcgtggggacaagagataaaacagatctatcctttagaagggaattggaagaatttagcacctgctgagcaatcctatcaagtcggcagatgt

[0339] ataggttttctatatggagacttggcgtatagaaaatctactcatgccgaggacagttctctatttcctctatctatacaaggtcgtattagaggtcgaggtttcttaaaagggtt

[0340] gctagacggattaatgagagcaagttgctgccaagtaatacaccggagaagtctggctcatttgaagaggccggccaacgcagtgtacggaggtttgatttacttgattg

[0341] ataaattgagtgtatcacctccattcctttctcttactagatcaggacctattagagacgaattagaaacgattccccacaagatcccaacctcctatccgacaagcaaccgt

[0342] gatatgggggtgattgtcagaaattacttcaaataccaatgccgtctaattgaaaagggaaaatacagatcacattattcacaattatggttattctcagatgtcttatccatag

[0343] acttcattggaccattctctatttccaccaccctcttgcaaatcctatacaagccatttttatctgggaaaagaaaatgagttgagagagctggcaaatctttcttcattgcta

[0344] agatcaggagaggggtgggaagacatacatgtgaaattcttcaccaaggacatattattgtgtccagaggaaatcagacatgcttgcaagttcgggattgctaaggataa

[0345] taataaagacatgagctatcccccttggggaagggaatccagagggacaattacaacaatccctgtttattatacgaccaccccttaacccaaagatgctagagatgcctc

[0346] caagaatccaaaatcccctgctgtccggaatcaggttgggccaattaccaactggcgctcattataaaattcggagtatattacatggaatgggaatccattacagggact

[0347] tcttgagttgtggagacggctccggagggatgactgctgcattactacgagaaaatgtgcatagcagaggaatattcaatagtctgttagaattatcagggtcagtcatgc

[0348] gaggcgcctctcctgagccccccagtgccctagaaactttaggaggagataaatcgagatgtgtaaatggtgaaacatgttgggaatatccatctgacttatgtgaccca

[0349] aggacttgggactatttcctccgactcaaagcaggcttggggcttcaaattgatttaattgtaatggatatggaagttcgggattcttctactagcctgaaaattgagacgaat

[0350] gttagaaattatgtgcaccggattttggatgagcaaggagttttaatctacaagacttatggaacatatatttgtgagagcgaaaagaatgcagtaacaatccttggtcccat

[0351] gttcaagacggtcgacttagttcaaacagaatttagtagttctcaaacgtctgaagtatatatggtatgtaaaggtttgaagaaattaatcgatgaacccaatcccgattggtc

[0352] ttccatcaatgaatcctggaaaaacctgtacgcattccagtcatcagaacaggaatttgccagagcaaagaaggttagtacatactttaccttgacaggtattccctcccaat

[0353] tcattcctgatccttttgtaaacattgagactatgctacaaatattcggagtacccacgggtgtgtctcatgcggctgccttaaaatcatctgatagacctgcagatttattgac

[0354] cattagccttttttatatggcgattatatcgtattataacatcaatcatatcagagtaggaccgatacctccgaaccccccatcagatggaattgcacaaaatgtggggatcg

[0355] ctataactggtataagcttttggctgagtttgatggagaaagacattccactatatcaacagtgtttggcagttatccagcaatcatttccgattaggtgggaggctatttcagt

[0356] aaaaggaggatacaagcagaagtggagtactagaggtgatgggctcccaaaagatacccgaatttcagactccttggccccaatcgggaactggatcagatctttgga

[0357] attggtccgaaaccaagttcgtctaaatccattcaataagatcttgttcaatcagctatgtcgtacagtggataatcatttgaagtggtcaaatttgcgaaaaaacacaggaat

[0358] gattgaatggatcaatgggcgaatttcaaaagaagaccggtctatactgatgttgaagagtgacctacatgaggaaaactcttggagagattaaaaaatcaggaggaga

[0359] ctccaaactttaagtatgaaaaaaactttgatccttaagaccctcttgtggtttttatttttttatctggttttgtggtcttcgtgggtcggcatggcatctccacctcctcgcggtc

[0360] cgacctgggcatccgaaggaggacgtcgtccactcggatggctaagggagagctcggatccggctgctaacaaagcccgaaaggaagctgagttggctgctgcca

[0361] ccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggatcgagatcctctagagtcgacctg

[0362] caggcatgcaagcttgtattctatagtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgttctaacgacaatatgtacaagcctaattgtgta

[0363] gcatctggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctggtaacgccgtcccgcacccggaa

[0364] atggtcagcgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatat

[0365] cgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggc

[0366] gccatctccttgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgaatggtgca

[0367] ctctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacag

[0368] acaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgt

[0369] catgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaat

[0370] aaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccag

[0371] aaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaaga

[0372] acgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgact

[0373] tggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttc

[0374] tgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaa

[0375] acgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatg

[0376] gaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagc

[0377] actggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcact

[0378] gattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgacca

[0379] aaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaa

[0380] aaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgta

[0381] gccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgg

[0382] gttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgag

[0383] atacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagg

[0384] gagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaa

[0385] aacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtg

[0386] agctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagggggatctcgatcccgcgaaattaatacgactcactatagg.

[0387] PT7-VSV-sPD1 sequence (SEQ ID NO.12)

[0388] acgaagacaaacaaaccattattatcattaaaaggctcaggagaaactttaacagtaatcagaattctcgagaccgccaccatgcccctattgcttttgctccctctactgtg

[0389] ggctccaggatggttcttagactccccagacaggccctggaacccccccaccttctccccagccctgctcgtggtgaccgaaggggacaacgccaccttcacctgcag

[0390] cttctccaacacatcggagagcttcgtgctaaactggtaccgcatgagccccagcaaccagacggacaagctggccgccttccccgaggaccgcagccagcccggc

[0391] caggactgccgcttccgtgtcacacaactgcccaacgggcgtgacttccacatgagcgtggtcagggcccggcgcaatgacagcggcacctacctctgtggggccat

[0392] ctccctggcccccaaggcgcagatcaaagagagcctgcgggcagagctcagggtgacagagagaagggcagaagtgcccacagcccaccccagcccctcaccc

[0393] aggccagccggccagttccaaaccctggtgcaccatcaccatcaccactgatggccatatgaaaaaaactaacagtaatcaaaatgtctgttacagtcaagagaatcatt

[0394] gacaacacagtcatagttccaaaacttcctgcaaatgaggatccagtggaatacccggcagattacttcagaaaatcaaaggagattcctctttacatcaatactacaaaa

[0395] agtttgtcagatctaagaggatatgtctaccaaggcctcaaatccggaaatgtatcaatcatacatgtcaacagctacttgtatggagcattaaaggacatccggggtaagt

[0396] tggataaagattggtcaagtttcggaataaacatcgggaaagcaggggatacaatcggaatatttgaccttgtatccttgaaagccctggacggcgtacttccagatgga

[0397] gtatcggatgcttccagaaccagcgcagatgacaaatggttgcctttgtatctacttggcttatacagagtgggcagaacacaaatgcctgaatacagaaaaaagctcat

[0398] ggatgggctgacaaatcaatgcaaaatgatcaatgaacagtttgaacctcttgtgccagaaggtcgtgacatttttgatgtgtggggaaatgacagtaattacacaaaaatt

[0399] gtcgctgcagtggacatgttcttccacatgttcaaaaaacatgaatgtgcctcgttcagatacggaactattgtttccagattcaaagattgtgctgcattggcaacatttgga

[0400] cacctctgcaaaataaccggaatgtctacagaagatgtaacgacctggatcttgaaccgagaagttgcagatgaaatggtccaaatgatgcttccaggccaagaaattga

[0401] caaggccgattcatacatgccttatttgatcgactttggattgtcttctaagtctccatattcttccgtcaaaaaccctgccttccacttctgggggcaattgacagctcttctgc

[0402] tcagatccaccagagcaaggaatgcccgacagcctgatgacattgagtatacatctcttactacagcaggtttgttgtacgcttatgcagtaggatcctctgccgacttggc

[0403] acaacagttttgtgttggagataacaaatacactccagatgatagtaccggaggattgacgactaatgcaccgccacaaggcagagatgtggtcgaatggctcggatgg

[0404] tttgaagatcaaaacagaaaaccgactcctgatatgatgcagtatgcgaaaagagcagtcatgtcactgcaaggcctaagagagaagacaattggcaagtatgctaagt

[0405] cagaatttgacaaatgaccctataattctcagatcacctattatatattatgctacatatgaaaaaaactaacagatatcatggataatctcacaaaagttcgtgagtatctcaa

[0406] gtcctactctcgtctagatcaggcggtaggagagatagatgagatcgaagcacaacgagctgaaaagtccaattatgagttgttccaagaggacggagtggaagagca

[0407] tactaggccctcttattttcaggcagcagatgattctgacacagaatctgaaccagaaattgaagacaatcaaggcttgtatgtaccagatccggaagctgagcaagttga

[0408] aggctttatacaggggcctttagatgactatgcagatgaggacgtggatgttgtattcacttcggactggaaacagcctgagcttgaatccgacgagcatggaaagacct

[0409] tacggttgacattgccagagggtttaagtggagagcagaaatcccagtggcttttgacgattaaagcagtcgttcaaagtgccaaacactggaatctggcagagtgcac

[0410] atttgaagcatcgggagaaggggtcatcataaaaaagcgccagataactccggatgtatataaggtcactccagtgatgaacacacatccgtaccaatcagaagccgta

[0411] tcagatgtttggtctctctcaaagacatccatgactttccaacccaagaaagcaagtcttcagcctctcaccatatccttggatgaattgttctcatctagaggagaattcatct

[0412] ctgtcggaggtaacggacgaatgtctcataaagaggccatcctgctcggtctgaggtacaaaaagttgtacaatcaggcgagagtcaaatattctctgtagactatgaaa

[0413] aaaagtaacagatatcacaatctaagtgttatcccaatccattcatcatgagttccttaaagaagattctcggtctgaaggggaaaggtaagaaatctaagaaattagggat

[0414] cgcaccacccccttatgaagaggacactagcatggagtatgctccgagcgctccaattgacaaatcctattttggagttgacgagatggacacctatgatccgaatcaatt

[0415] aagatatgagaaattcttctttacagtgaaaatgacggttagatctaatcgtccgttcagaacatactcagatgtggcagccgctgtatcccattgggatcacatgtacatcg

[0416] gaatggcagggaaacgtcccttctacaaaatcttggcttttttgggttcttctaatctaaaggccactccagcggtattggcagatcaaggtcaaccagagtatcacgctca

[0417] ctgcgaaggcagggcttatttgccacataggatggggaagacccctcccatgctcaatgtaccagagcacttcagaagaccattcaatataggtctttacaagggaacg

[0418] attgagctcacaatgaccatctacgatgatgagtcactggaagcagctcctatgatctgggatcatttcaattcttccaaattttctgatttcagagagaaggccttaatgtttg

[0419] gcctgattgtcgagaaaaaggcatctggagcgtgggtcctggattctatcagccacttcaaatgagctagtctagcttccagcttctgaacaatccccggtttactcagtct

[0420] ctcctaattccagcctttcgaacaactaatatcctgtcttttctatccctatgaaaaaaactaacagagatcgatctgtttccttgacacgcgttacgatgcggccgatgaagt

[0421] gccttttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacacaaccaaaaaggaaactggaaaaatgttccttctaattaccattattgcccgt

[0422] caagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaaggctattcaagcagacggttggatgtgtcatgcttcca

[0423] aatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccgatccttcactccatctgtagaacaatgcaaggaaagcattgaacaaacgaa

[0424] acaaggaacttggctgaatccaggcttccctcctcaaagttgtggatatgcaactgtgacggatgccgaagcagtgattgtccaggtgactcctcaccatgtgctggttga

[0425] tgaatacacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccataactctacaacctggcattctgactataaggtcaaa

[0426] gggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaaggagggcacagggttcagaagtaactactttgctt

[0427] atgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtctggttcgagatggctgataaggatctctttgctgcagc

[0428] cagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgtaagtctaattcaggacgttgagaggatcttggattattccctctgccaa

[0429] gaaacctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaacccaggaaccggtcctgctttcaccataatcaatggtacc

[0430] ctaaaatactttgagaccagatacatcagagtcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtggaactaccacagaaagggaactgtgggatga

[0431] ctgggcaccatatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatacatgattggacatggtatgttggactccgatcttc

[0432] atcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagtttattttttggtgatactgggctatccaaaaatccaat

[0433] cgagcttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatagggttaatcattggactattcttggttctccgagttggtatccatctttgcatta

[0434] attaaagcacaccaagaaagacagatttatacacacagagatgaacgacttggaaagtaagcggccgccctgcacacagattctcatgtttgaaccaaatcaa

[0435] cttgtgatatcatgctcaaagaggccttaattatattttaatttttaatttgaaaaaaactaacagcaatcatggaagtccacgatttgagaccgacgagttcaatgatttc

[0436] aatgaagatgactatgccacaagagaattcctgaatcccgatgagcgcatgacgtacttgaatcatgctgattacaatttgaattctctctattagtgatgatattgacaat

[0437] ttgatcaggaaattcaattcttccgattccctcgatgtgggatagtaagactgggatggtcttgagatgttaacatcatgtcaagccaatcccatctcaacatctcag

[0438] atgcataaatggatgggaagttggttaatgtctgataatcatgatgccagtcaagggtatagttttacatgaagtggacaaagaggcagaaataacatttgacgtggtgg

[0439] agaccttcatcccgggctggggcaaaaccaattgaatacatcaaaaaggaagatggactgactcattcaaaattcgcttatttgtgtcaaaagtttttggacttacac

[0440] aagttgacattaattaaatgctgtctctgaggtggaattgctcaacttggcgaggactttcaaggcaaagtcagaagaagttctcatggaacgaacatatgcaggatta

[0441] gggttcccagcttgggtcctacttttatttcagaaggatgggcttacttcaagaaacttgatattctaatggaccgaaactttctgttaatggtcaaagatgtgattatagggag

[0442] gatgcaaacggtgctatccatggtatgtagaatagacaacctgttctcagagcaagacatcttctcccttctaaatatctacagaattggagataaaattgtggagaggcag

[0443] ggaaatttttcttatgacttgattaaaatggtggaaccgatatgcaacttgaagctgatgaaattagcaagagaatcaaggcctttagtcccacaattccctcattttgaaaat

[0444] catatcaagacttctgttgatgaaggggcaaaaattgaccgaggtataagattcctccatgatcagataatgagtgtgaaaacagtggatctcacactggtgatttatggat

[0445] cgttcagacattggggtcatccttttatagattattacactggactagaaaaattacattcccaagtaaccatgaagaaagatattgatgtgtcatatgcaaaagcacttgcaa

[0446] gtgatttagctcggattgttctatttcaacagttcaatgatcataaaaagtggttcgtgaatggagacttgctccctcatgatcatccctttaaaagtcatgttaaagaaaataca

[0447] tggcccacagctgctcaagttcaagattttggagataaatggcatgaacttccgctgattaaatgttttgaaatacccgacttactagacccatcgataatatactctgacaaa

[0448] agtcattcaatgaataggtcagaggtgttgaaacatgtccgaatgaatccgaacactcctatccctagtaaaaaggtgttgcagactatgttggacacaaaggctaccaat

[0449] tggaaagaatttcttaaagagattgatgagaagggcttagatgatgatgatgatctaattattggtcttaaaggaaaggagagggaactgaagttggcaggtagatttttctccct

[0450] aatgtcttggaaattgcgagaatactttgtaattaccgaatatttgataaagactcatttcgtccctatgtttaaaggcctgacaatggcggacgatctaactgcagtcattaaa

[0451] aagatgttagattcctcatccggccaaggattgaagtcatatgaggcaatttgcatagccaatcacattgattacgaaaaatggaataaccaccaaaggaagttatcaaac

[0452] ggcccagtgttccgagttatgggccagttcttaggttatccatccttaatcgagagaactcatgaattttttgagaaaagtcttatatactacaatggaagaccagacttgatg

[0453] cgtgttcacaacaacacactgatcaattcaacctcccaacgagtttgttggcaaggacaagagggtggactggaaggtctacggcaaaaaggatggagtatcctcaatc

[0454] tactggttattcaaagagaggctaaaatcagaaacactgctgtcaaagtcttggcacaaggtgataatcaagttatttgcacacagtataaaacgaagaaatcgagaaacg

[0455] ttgtagaattacagggtgctctcaatcaaatggtttctaataatgagaaaattatgactgcaatcaaaatagggacagggaagttaggacttttgataaatgacgatgagact

[0456] atgcaatctgcagattacttgaattatggaaaaataccgattttccgtggagtgattagagggttagagaccaagagatggtcacgagtgacttgtgtcaccaatgaccaa

[0457] atacccacttgtgctaatataatgagctcagtttccacaaatgctctcaccgtagctcattttgctgagaacccaatcaatgccatgatacagtacaattattttgggacatttg

[0458] ctagactcttgttgatgatgcatgatcctgctcttcgtcaatcattgtatgaagttcaagataagataccgggcttgcacagttctactttcaaatacgccatgttgtatttggac

[0459] ccttccattggaggagtgtcgggcatgtctttgtccaggtttttgattagagccttcccagatcccgtaacagaaagtctctcattctggagattcatccatgtacatgctcga

[0460] agtgagcatctgaaggagatgagtgcagtatttggaaaccccgagatagccaagtttcgaataactcacatagacaagctagtagaagatccaacctctctgaacatcgc

[0461] tatgggaatgagtccagcgaacttgttaaagactgaggttaaaaaatgcttaatcgaatcaagacaaaccatcaggaaccaggtgattaaggatgcaaccatatatttgta

[0462] tcatgaagaggatcggctcagaagtttcttatggtcaataaatcctctgttccctagatttttaagtgaattcaaatcaggcacttttttgggagtcgcagacgggctcatcagt

[0463] ctatttcaaaattctcgtactattcggaactcctttaagaaaaagtatcatagggaattggatgatttgattgtgaggagtgaggtatcctctttgacacatttagggaaacttca

[0464] tttgagaaggggatcatgtaaaatgtggacatgttcagctactcatgctgacacattaagatacaaatcctggggccgtacagttattgggacaactgtaccccatccatta

[0465] gaaatgttgggtccacaacatcgaaaagagactccttgtgcaccatgtaacacatcagggttcaattatgtttctgtgcattgtccagacgggatccatgacgtctttagttc

[0466] acggggaccattgcctgcttatctagggtctaaaacatctgaatctacatctattttgcagccttgggaaagggaaagcaaagtcccactgattaaaagagctacacgtctt

[0467] agagatgctatctcttggtttgttgaacccgactctaaactagcaatgactatactttctaacatccactctttaacaggcgaagaatggaccaaaaggcagcatgggttca

[0468] aaagaacagggtctgcccttcataggttttcgacatctcggatgagccatggtgggttcgcatctcagagcactgcagcattgaccaggttgatggcaactacagacacc

[0469] atgagggatctgggagatcagaatttcgactttttattccaagcaacgttgctctatgctcaaattaccaccactgttgcaagagacggatggatcaccagttgtacagatc

[0470] attatcatattgcctgtaagtcctgtttgagacccatagaagagatcaccctggactcaagtatggactacacgcccccagatgtatcccatgtgctgaagacatggagga

[0471] atggggaaggttcgtggggacaagagataaaacagatctatcctttagaagggaattggaagaatttagcacctgctgagcaatcctatcaagtcggcagatgtataggt

[0472] tttctatatggagacttggcgtatagaaaatctactcatgccgaggacagttctctatttcctctatctatacaaggtcgtattagaggtcgaggtttcttaaaagggttgctag

[0473] acggattaatgagagcaagttgctgccaagtaatacaccggagaagtctggctcatttgaagaggccggccaacgcagtgtacggaggtttgatttacttgattgataaat

[0474] tgagtgtatcacctccattcctttctcttactagatcaggacctattagagacgaattagaaacgattccccacaagatcccaacctcctatccgacaagcaaccgtgatatg

[0475] ggggtgattgtcagaaattacttcaaataccaatgccgtctaattgaaaagggaaaatacagatcacattattcacaattatggttattctcagatgtcttatccatagacttca

[0476] ttggaccattctctatttccaccaccctcttgcaaatcctatacaagccatttttatctgggaaagataagaatgagttgagagagctggcaaatctttcttcattgctaagatc

[0477] aggagaggggtggagaacatacatgtgaaattcttcaccaaggacatattattgtgtccagaggaaatcagacatgcttgcaagttcgggattgctaaggataataata

[0478] aagacatgagctatcccccttggggaagggaatccagagggacaattacaacaatccctgtttattatacgaccaccccttacccaaagatgctagagatgcctccaaga

[0479] atccaaaatcccctgctgtccggaatcaggttgggccaattaccaactggcgctcattataaaaaattcggagtatattacatggaatgggaatccattacagggacttcttga

[0480] gttgtggagacggctccggagggatgactgctgcattactacgagaaaatgtgcatagcagaggaatattcaatagtctgttagaattatcagggtcagtcatgcgaggc

[0481] gcctctcctgagccccccagtgccctagaaactttaggaggagataaatcgagatgtgtaaatggtgaaacatgttgggaatatccatctgacttatgtgacccaaggact

[0482] tgggactatttcctccgactcaaagcaggcttggggcttcaaattgatttaattgtaatggatatggaagttcgggattcttctactagcctgaaattgagacgaatgttaga

[0483] aattatgtgcaccggattttggatgagcaaggagttttaatctacaagacttatggaacatatatttgtgagagcgaaaagaatgcagtaacaatccttggtcccatgttcaa

[0484] gacggtcgacttagttcaaacagaatttagtagttctcaaacgtctgaagtatattggtatgtaaaggtttgaagaaattaatcgatgaacccaatcccgattggtcttccat

[0485] caatgaatcctggaaaaacctgtacgcattccagtcatcagaacaggaatttgccagagcaaagaaggttagtacatactttaccttgacaggtattccctcccaattcattc

[0486] ctgatccttttgtaaacattgagactatgctacaaatattcggagtacccacgggtgtgtctcatgcggctgccttaaaatcatctgatagacctgcagatttattgaccattag

[0487] ccttttttattggcgattatatcgtattaataacatcaatcatatcagagtaggaccgatacctccgaaccccccatcagatggaattgcacaaaatgtggggatcgctataac

[0488] tggtataagcttttggctgagtttgatggagaaagacattccactatatcaacagtgtttggcagttatccagcaatcatttccgattaggtgggaggctatttcagtaaagg

[0489] aggatacaagcagaagtggagtactagaggtgatgggctcccaaaagatacccgaatttcagactccttggccccaatcgggaactggatcagatctttggaattggtc

[0490] cgaaaccaagttcgtctaaatccattcaataagatcttgttcaatcagctatgtcgtacagtggataatcatttgaagtggtcaaatttgcgaaaaaacacaggaatgattga

[0491] atggatcaatgggcgaatttcaaaagaagaccggtctatactgatgttgaagagtgacctacatgaggaaaactcttggagagattaaaaaatcaggaggagactccaa

[0492] actttaagtatgaaaaaaactttgatccttaagaccctcttgtggtttttatttttttatctggttttgtggtcttcgtgggtcggcatggcatctccacctcctcgcggtccgacct

[0493] gggcatccgaaggaggacgtcgtccactcggatggctaagggagagctcggatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctg

[0494] agcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggatcgagatcctctagagtcgacctgcaggca

[0495] tgcaagcttgtattctatagtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgttctaacgacaatatgtacaagcctaattgtgtagcatct

[0496] ggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctggtaacgccgtcccgcacccggaaatggtc

[0497] agcgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccg

[0498] acatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatc

[0499] tccttgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgaatggtgcactctca

[0500] gtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaag

[0501] ctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatga

[0502] taataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccc

[0503] tgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacg

[0504] ctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttt

[0505] tccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggtt

[0506] gagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgac

[0507] aacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacga

[0508] cgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggag

[0509] gcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcact

[0510] ggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgatt

[0511] aagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaa

[0512] tcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaac

[0513] caccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagcc

[0514] gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttg

[0515] gactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatac

[0516] ctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagc

[0517] ttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacg

[0518] ccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagct

[0519] gataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagggggatctcgatcccgcgaaattaatacgactcactatagg.

[0520] PT7-VSV-IL15Ra IL15 sequence (SEQ ID NO.13)

[0521] acgaagacaaacaaaccattattatcattaaaaggctcaggagaaactttaacagtaatcagaattctcgagaccgccaccatgcccctattgcttttgctccctctactgtg

[0522] ggctatcacctgtcctccccccatgagcgtggagcacgccgacatctgggttaagagctacagcctctactccagagagcgctacatctgcaattctggctttaagcgga

[0523] aggccggcacgagcagcctgacagagtgcgtgctgaacaaggccaccaacgtggcccactggaccacccctagcctgaaatgcatcaagccttctggcggcagcg

[0524] gaggtggcggaagcggcggtggcagtggaggcggcggcagcctgcaaaattgggtgaacgtgatcagcgacctgaagaagatcgaggatctgatccagagcatg

[0525] cacatcgacgccacactgtacaccgagtctgatgtgcaccccagctgtaaagtgaccgctatgaagtgcttcctgctggagctgcaagtgatcagcctggagagcggg

[0526] gacgcctcaatccacgataccgtggaaaacctcattatcctggccaacgacagcctttctagcaacggcaacgtcacagagagcggatgtaaagagtgcgaggaactc

[0527] gaagagaagaacatcaaggagttcctgcagagcttcgtgcacattgtgcagatgttcatcaacacatctcaccatcaccatcaccactgatggccatatgaaaaaaacta

[0528] acagtaatcaaaatgtctgttacagtcaagagaatcattgacaacacagtcatagttccaaaacttcctgcaaatgaggatccagtggaatacccggcagattacttcaga

[0529] aaatcaaaggagattcctctttacatcaatactacaaaaagtttgtcagatctaagaggatatgtctaccaaggcctcaaatccggaaatgtatcaatcatacatgtcaacag

[0530] ctacttgtatggagcattaaaggacatccggggtaagttggataaagattggtcaagtttcggaataaacatcgggaaagcaggggatacaatcggaatatttgaccttgt

[0531] atccttgaaagccctggacggcgtacttccagatggagtatcggatgcttccagaaccagcgcagatgacaaatggttgcctttgtatctacttggcttatacagagtggg

[0532] cagaacacaaatgcctgaatacagaaaaaagctcatggatgggctgacaaatcaatgcaaaatgatcaatgaacagtttgaacctcttgtgccagaaggtcgtgacatttt

[0533] tgatgtgtggggaaatgacagtaattacacaaaaattgtcgctgcagtggacatgttcttccacatgttcaaaaaacatgaatgtgcctcgttcagatacggaactattgtttc

[0534] cagattcaaagattgtgctgcattggcaacatttggacacctctgcaaaataaccggaatgtctacagaagatgtaacgacctggatcttgaaccgagaagttgcagatg

[0535] aaatggtccaaatgatgcttccaggccaagaaattgacaaggccgattcatacatgccttatttgatcgactttggattgtcttctaagtctccatattcttccgtcaaaaaccc

[0536] tgccttccacttctgggggcaattgacagctcttctgctcagatccaccagagcaaggaatgcccgacagcctgatgacattgagtatacatctcttactacagcaggtttg

[0537] ttgtacgcttatgcagtaggatcctctgccgacttggcacaacagttttgtgttggagataacaaatacactccagatgatagtaccggaggattgacgactaatgcaccg

[0538] ccacaaggcagagatgtggtcgaatggctcggatggtttgaagatcaaaacagaaaaccgactcctgatatgatgcagtatgcgaaaagagcagtcatgtcactgcaa

[0539] ggcctaagagagaagacaattggcaagtatgctaagtcagaatttgacaaatgaccctataattctcagatcacctattatatattatgctacatatgaaaaaaactaacaga

[0540] tatcatggataatctcacaaaagttcgtgagtatctcaagtcctactctcgtctagatcaggcggtaggagagatagatgagatcgaagcacaacgagctgaaaagtcca

[0541] attatgagttgttccaagaggacggagtggaagagcatactaggccctcttattttcaggcagcagatgattctgacacagaatctgaaccagaaattgaagacaatcaa

[0542] ggcttgtatgtaccagatccggaagctgagcaagttgaaggctttatacaggggcctttagatgactatgcagatgaggacgtggatgttgtattcacttcggactggaaa

[0543] cagcctgagcttgaatccgacgagcatggaaagaccttacggttgacattgccagagggtttaagtggagagcagaaatcccagtggcttttgacgattaaagcagtcg

[0544] ttcaaagtgccaaacactggaatctggcagagtgcacatttgaagcatcgggagaaggggtcatcataaaaaagcgccagataactccggatgtatataaggtcactcc

[0545] agtgatgaacacacatccgtaccaatcagaagccgtatcagatgtttggtctctctcaaagacatccatgactttccaacccaagaaagcaagtcttcagcctctcaccata

[0546] tccttggatgaattgttctcatctagaggagaattcatctctgtcggaggtaacggacgaatgtctcataaagaggccatcctgctcggtctgaggtacaaaaagttgtaca

[0547] atcaggcgagagtcaaatattctctgtagactatgaaaaaaagtaacagatatcacaatctaagtgttatcccaatccattcatcatgagttccttaaagaagattctcggtct

[0548] gaaggggaaaggtaagaaatctaagaaattagggatcgcaccacccccttatgaagaggacactagcatggagtatgctccgagcgctccaattgacaaatcctatttt

[0549] ggagttgacgagatggacacctatgatccgaatcaattaagatatgagaaattcttctttacagtgaaaatgacggttagatctaatcgtccgttcagaacatactcagatgt

[0550] ggcagccgctgtatcccattgggatcacatgtacatcggaatggcagggaaacgtcccttctacaaaatcttggcttttttgggttcttctaatctaaaggccactccagcg

[0551] gtattggcagatcaaggtcaaccagagtatcacgctcactgcgaaggcagggcttatttgccacataggatggggaagacccctcccatgctcaatgtaccagagcact

[0552] tcagaagaccattcaatataggtctttacaagggaacgattgagctcacaatgaccatctacgatgatgagtcactggaagcagctcctatgatctgggatcatttcaattct

[0553] tccaaattttctgatttcagagagaaggccttaatgtttggcctgattgtcgagaaaaaggcatctggagcgtgggtcctggattctatcagccacttcaaatgagctagtct

[0554] agcttccagcttctgaacaatccccggtttactcagtctctcctaattccagcctttcgaacaactaatatcctcttttctatccctatgaaaaaaactaacagagatcgatct

[0555] gtttccttgacacgcgttacgatgcggccgatgaagtgcctttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacaacaccaaaaaggaa

[0556] actggaaaaatgttccttctaattaccattatgcccgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaag

[0557] gctattcaagcagacggttggatgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccgatccttcactccatct

[0558] gtagaacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcctcaagttgtggatatgcaactgtgacggatgccgaagca

[0559] gtgattgtccaggtgactcctcaccatgtgctggttgatgaatacacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtcc

[0560] ataactctacaacctggcattctgactataaggtcaaagggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctggga

[0561] aagggcacagggttcagaagtaactactttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtct

[0562] ggttcgagatgggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgtaagtctaattca

[0563] ggacgttgagaggatcttggattatccctctgccaagaaaacctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaaaccc

[0564] aggaaccggtcctgctttcaccataatcaatggtaccctaaaatactttgagaccagatacatcagagtcgatatgctgctccaatcctctcaagaatggtcggaatgatc

[0565] agtggaactaccacagaaagggaactgtgggatgactgggcaccatatgaagcgtgggaaattggacccaatgggagttctgaggaccagttcaggatataaggtttcctt

[0566] tatacatgattggacatggtatgttggactccgatcttcatcttagctcaaaggctcaggtgttcgaaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgaga

[0567] gtttattttttggtgatactgggctatccaaaaatccaatcgagcttgtagaaggttggttcagtagttggaaaagctctattgcctctttttttctttatcatagggttaatcattgg

[0568] actattcttggttctccgagttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaagtaagcggcc

[0569] gccctgcacacagattcttcatgtttgaaccaaatcaacttgtgatatcatgctcaaagaggccttaattatattttaatttttaatttttatgaaaaaaactaacagcaatcatgg

[0570] aagtccacgattttgagaccgacgagttcaatgatttcaatgaagatgactatgccacaagagaattcctgaatcccgatgagcgcatgacgtacttgaatcatgctgatta

[0571] caatttgaattctcctctaattagtgatgatattgacaatttgatcaggaaattcaattctcttccgattccctcgatgtgggatagtaagaactgggatggagttcttgagatgtt

[0572] aacatcatgtcaagccaatcccatctcaacatctcagatgcataaatggatgggaagttggttaatgtctgataatcatgatgccagtcaagggtatagttttttacatgaagt

[0573] ggacaaagaggcagaaataacatttgacgtggtggagaccttcatccgcggctggggcaacaaaccaattgaatacatcaaaaaggaaagatggactgactcattcaa

[0574] aattctcgcttatttgtgtcaaaagtttttggacttacacaagttgacattaatcttaaatgctgtctctgaggtggaattgctcaacttggcgaggactttcaaaggcaaagtca

[0575] gaagaagttctcatggaacgaacatatgcaggattaggttcccagcttgggtcctacttttattcagaaggatgggctacttcaagaaacttgatattctaatggaccga

[0576] aactttctgttaatggtcaaagatgtgattatagggaggatgcaaacggtgctatccatggtatgtagaatagacaacctgttctcagagcaagacatcttctcccttctaaat

[0577] atctacagaattggagataaaattgtggagaggcagggaaatttttcttatgacttgattaaaatggtggaaccgatatgcaacttgaagctgatgaaattagcaagagaat

[0578] caaggcctttagtcccacaattccctcattttgaaaatcatatcaagacttctgttgatgaaggggcaaaaattgaccgaggtataagattcctccatgatcagataatgagt

[0579] gtgaaaacagtggatctcacactggtgatttatggatcgttcagacattggggtcatccttttatagattattacactggactagaaaaattacattcccaagtaaccatgaag

[0580] aaagatattgatgtgtcatatgcaaaagcacttgcaagtgatttagctcggattgttctatttcaacagttcaatgatcataaaaagtggttcgtgaatggagacttgctccctc

[0581] atgatcatccctttaaaagtcatgttaaagaaaatacatggcccacagctgctcaagttcaagattttggagataaatggcatgaacttccgctgattaaatgttttgaaatac

[0582] ccgacttactagacccatcgataatatactctgacaaaagtcattcaatgaataggtcagaggtgttgaaacatgtccgaatgaatccgaacactcctatccctagtaaaaa

[0583] ggtgttgcagactatgttggacacaaaggctaccaattggaaagaatttcttaaagagattgatgagaagggcttagatgatgatgatctaattattggtcttaaaggaaag

[0584] gagagggaactgaagttggcaggtagatttttctccctaatgtcttggaaattgcgagaatactttgtaattaccgaatatttgataaagactcatttcgtccctatgtttaaag

[0585] gcctgacaatggcggacgatctaactgcagtcattaaaaagatgttagattcctcatccggccaaggattgaagtcatatgaggcaatttgcatagccaatcacattgatta

[0586] cgaaaaatggaataaccaccaaaggaagttatcaaacggcccagtgttccgagttatgggccagttcttaggttatccatccttaatcgagagaactcatgaattttttgag

[0587] aaaagtcttatatactacaatggaagaccagacttgatgcgtgttcacaacaacacactgatcaattcaacctcccaacgagtttgttggcaaggacaagagggtggact

[0588] ggaaggtctacggcaaaaaggatggagtatcctcaatctactggttattcaaagagaggctaaaatcagaaacactgctgtcaaagtcttggcacaaggtgataatcaag

[0589] ttatttgcacacagtataaaacgaagaaatcgagaaacgttgtagaattacagggtgctctcaatcaaatggtttctaataatgagaaaattatgactgcaatcaaaatagg

[0590] gacagggaagttaggacttttgataaatgacgatgagactatgcaatctgcagattacttgaattatggaaaaataccgattttccgtggagtgattagagggttagagacc

[0591] aagagatggtcacgagtgacttgtgtcaccaatgaccaaatacccacttgtgctaatataatgagctcagtttccacaaatgctctcaccgtagctcattttgctgagaaccc

[0592] aatcaatgccatgatacagtacaattattttgggacatttgctagactcttgttgatgatgcatgatcctgctcttcgtcaatcattgtatgaagttcaagataagataccgggct

[0593] tgcacagttctactttcaaatacgccatgttgtatttggacccttccattggaggagtgtcgggcatgtctttgtccaggtttttgattagagccttcccagatcccgtaacaga

[0594] aagtctctcattctggagattcatccatgtacatgctcgaagtgagcatctgaaggagatgagtgcagtatttggaaaccccgagatagccaagtttcgaataactcacata

[0595] gacaagctagtagaagatccaacctctctgaacatcgctatgggaatgagtccagcgaacttgttaaagactgaggttaaaaaatgcttaatcgaatcaagacaaaccat

[0596] caggaaccaggtgattaaggatgcaaccatatatttgtatcatgaagaggatcggctcagaagtttcttatggtcaataaatcctctgttccctagatttttaagtgaattcaaa

[0597] tcaggcacttttttgggagtcgcagacgggctcatcagtctatttcaaaattctcgtactattcggaactcctttaagaaaaagtatcatagggaattggatgatttgattgtga

[0598] ggagtgaggtatcctctttgacacatttagggaaacttcatttgagaaggggatcatgtaaaatgtggacatgttcagctactcatgctgacacattaagatacaaatcctgg

[0599] ggccgtacagttattgggacaactgtaccccatccattagaaatgttgggtccacaacatcgaaaagagactccttgtgcaccatgttaacacatcagggttcaattatgttt

[0600] ctgtgcattgtccagacgggatccatgacgtctttagttcacggggaccattgcctgcttatctagggtctaaaacatctgaatctacatctatttttgcagccttgggaaagg

[0601] gaaagcaaagtcccactgattaaaagagctacacgtcttagagatgctatctcttggtttgttgaacccgactctaaactagcaatgactatactttctaacatccactctttaa

[0602] caggcgaagaatggaccaaaaggcagcatgggttcaaaagaacagggtctgcccttcataggttttcgacatctcggatgagccatggtgggttcgcatctcagagca

[0603] ctgcagcattgaccaggttgatggcaactacagacaccatgagggatctgggagatcagaatttcgactttttattccaagcaacgttgctctatgctcaaattaccaccac

[0604] tgttgcaagagacggatggatcaccagttgtacagatcattatcatattgcctgtaagtcctgtttgagacccatagaagagatcaccctggactcaagtatggactacacg

[0605] cccccagatgtatcccatgtgctgaagacatggaggaatggggaaggttcgtggggacaagagataaaacagatctatcctttagaagggaattggaagaatttagcac

[0606] ctgctgagcaatcctatcaagtcggcagatgtataggttttctatatggagacttggcgtatagaaaatctactcatgccgaggacagttctctatttcctctatctatacaag

[0607] gtcgtattagaggtcgaggtttcttaaaagggttgctagacggattaatgagagcaagttgctgccaagtaatacaccggagaagtctggctcatttgaagaggccggcc

[0608] aacgcagtgtacggaggtttgatttacttgattgataaattgagtgtatcacctccattcctttctcttactagatcaggacctattagagacgaattagaaacgattccccaca

[0609] agatcccaacctcctatccgacaagcaaccgtgatatgggggtgattgtcagaaattacttcaaataccaatgccgtctaattgaaaagggaaaatacagatcacattattc

[0610] acaattatggttattctcagatgtcttatccatagacttcattggaccattctctatttccaccaccctcttgcaaatcctatacaagccatttttatctgggaaagataagaatga

[0611] gttgagagagctggcaaatctttcttcattgctaagatcaggagaggggtgggaagacatacatgtgaaattcttcaccaaggacatattattgtgtccagaggaaatcag

[0612] acatgcttgcaagttcgggattgctaaggataataataaagacatgagctatcccccttggggaagggaatccagagggacaattacaacaatccctgtttattatacgac

[0613] caccccttacccaaagatgctagagatgcctccaagaatccaaaatcccctgctgtccggaatcaggttgggccaattaccaactggcgctcattataaaattcggagtat

[0614] attacatggaatgggaatccattacagggacttcttgagttgtggagacggctccggagggatgactgctgcattactacgagaaaatgtgcatagcagaggaatattca

[0615] atagtctgttagaattatcagggtcagtcatgcgaggcgcctctcctgagccccccagtgccctagaaactttaggaggagataaatcgagatgtgtaaatggtgaaaca

[0616] tgttgggaatatccatctgacttatgtgacccaaggacttgggactatttcctccgactcaaagcaggcttggggcttcaaattgatttaattgtaatggatatggaagttcgg

[0617] gattcttctactagcctgaaaattgagacgaatgttagaaattatgtgcaccggattttggatgagcaaggagttttaatctacaagacttatggaacatatatttgtgagagc

[0618] gaaaagaatgcagtaacaatccttggtcccatgttcaagacggtcgacttagttcaaacagaatttagtagttctcaaacgtctgaagtatatatggtatgtaaaggtttgaa

[0619] gaaattaatcgatgaacccaatcccgattggtcttccatcaatgaatcctggaaaaacctgtacgcattccagtcatcagaacaggaatttgccagagcaaagaaggttag

[0620] tacatactttaccttgacaggtattccctcccaattcattcctgatccttttgtaaacattgagactatgctacaaatattcggagtacccacgggtgtgtctcatgcggctgcct

[0621] taaaatcatctgatagacctgcagatttattgaccattagccttttttatggcgattatatcgtattataacatcaatcatatcagagtaggaccgatacctccgaacccccca

[0622] tcagatggaattgcacaaaatgtggggatcgctataactggtataagcttttggctgagtttgatggagaaagacattccactatatcaacagtgtttggcagttatccagca

[0623] atcatttccgattaggtgggaggctatttcagtaaaaggaggatacaagcagaagtggagtactagaggtgatgggctcccaaaagatacccgaatttcagactccttgg

[0624] ccccaatcgggaactggatcagatctttggaattggtccgaaaccaagttcgtctaaatccattcaataagatcttgttcaatcagctatgtcgtacagtggataatcatttga

[0625] agtggtcaaatttgcgaaaaaacacaggaatgattgaatggatcaatgggcgaatttcaaaagaagaccggtctatactgatgttgaagagtgacctacatgaggaaaa

[0626] ctcttggagagattaaaaaatcaggaggagactccaaactttaagtatgaaaaaaactttgatccttaagaccctcttgtggtttttatttttttatctggttttgtggtcttcgtgg

[0627] gtcggcatggcatctccacctcctcgcggtccgacctgggcatccgaaggaggacgtcgtccactcggatggctaagggagagctcggatccggctgctaacaaag

[0628] cccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactat

[0629] atccggatcgagatcctctagagtcgacctgcaggcatgcaagcttgtattctatagtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgtt

[0630] ctaacgacaatatgtacaagcctaattgtgtagcatctggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctga

[0631] gtttctggtaacgccgtcccgcacccggaaatggtcagcgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccgg

[0632] cgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggc

[0633] aggccccgtggccgggggactgttgggcgccatctccttgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagt

[0634] cgcataagggagagcgtcgaatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacg

[0635] ggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaaggg

[0636] cctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaata

[0637] cattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgc

[0638] ggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcgg

[0639] taagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactc

[0640] ggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataacc

[0641] atgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttg

[0642] ggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactct

[0643] agcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccg

[0644] gtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaa

[0645] atagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatct

[0646] aggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttt

[0647] ctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga

[0648] gcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctg

[0649] ctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagc

[0650] ttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggc

[0651] agggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatg

[0652] ctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgatt

[0653] ctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagggggatctcgatcccgcgaaattaatacgactcactatagg.

Claims

1. A recombinant oncolytic virus VSV-sPD1IL15RaIL15, characterized in that The recombinant oncolytic virus connects the sPD1IL15RaIL15 fragment with the VSV viral genome plasmid to obtain the viral full genome plasmid pT7-VSV-sPD1IL15RaIL15; pT7-VSV-sPD1IL15RaIL15 and the auxiliary plasmid are co-transfected into 293T cells, and VSV-sPD1IL15RaIL15 virus seeds are obtained after culture; the virus seeds are amplified in large quantities using 293T cells to obtain the oncolytic virus VSV-sPD1IL15RaIL15.

2. A method for constructing the recombinant oncolytic virus VSV-sPD1IL15RaIL15 according to claim 1, characterized in that: The method comprises the following steps: (1) Design of sPD1: The PD-1 signal peptide was removed from the extracellular region of PD-1 (17-170aa) to construct a new sPD1 sequence; (2) Design of IL15RaIL15: IL-15Ra was fused with N72D mutated IL-15 and connected with (GGGGS) 4liner sequence; (3) Design of sPD1IL15RaIL15: The C-terminus of sPD1 was fused to the N-terminus of IL15RaIL15, connected by a (GGGGS) 3-liner sequence, with a CD33 signal peptide inserted at the N-terminus and a 6-histidine tag inserted at the C-terminus; (4) Cloning of the recombinant oncolytic virus VSV-sPD1IL15RaIL15: The recombinant protein sPD1IL15RaIL15 gene fragment was inserted into the ATU region of the VSV full genome cDNA cloning plasmid pT7-VSV through the XhoI and MscI restriction sites using homologous recombination to obtain the recombinant virus full genome plasmid pT7-VSV-sPD1IL15RaIL15; (5) Rescue of recombinant oncolytic virus VSV-sPD1IL15RaIL15: The whole genome plasmid pT7-VSV-sPD1IL15RaIL15 was co-transfected with auxiliary plasmids pVRC-T7, pVRC-N, pVRC-P and pVRC-L into 293T cells until cytopathic effect was observed. The cell supernatant was collected and centrifuged at 2000 g for 10 min to remove cell debris to obtain virus seeds. The virus was amplified using 293T cells to obtain the recombinant virus VSV-sPD1IL15RaIL15.

3. Use of the recombinant oncolytic virus VSV-sPD1IL15RaIL15 according to claim 1 in the treatment of ovarian cancer.

4. Use of the recombinant oncolytic virus VSV-sPD1IL15RaIL15 according to claim 1 in the preparation of a drug for the treatment of ovarian cancer.

5. Use of the recombinant oncolytic virus VSV-sPD1IL15RaIL15 according to claim 1 in the preparation of drugs for the treatment of solid tumors.

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