Oncolytic virus VSV-aPDL1 / aCD137 expressing bispecific antibody as well as construction method and application of oncolytic virus VSV-aPDL1 / aCD137

Abstract

The invention relates to an oncolytic virus VSV-aPDL1 / aCD137 for expressing a bispecific antibody as well as a construction method and application of the oncolytic virus VSV-aPDL1 / aCD137. The preparation method comprises the following steps: firstly designing aPDL1 / aCD137, then synthesizing an aPDL1 / aCD137 gene in vitro, amplifying the aPDL1 / aCD137 gene, connecting the aPDL1 / aCD137 gene to a pT7-VSV whole genome plasmid to obtain a virus whole genome plasmid pT7-VSV-aPDL1 / aCD137, and then co-transfecting the plasmid and a helper plasmid to a 293T cell to obtain the VSV-aPDL1 / aCD137 oncolytic virus. According to the present invention, the local anti-tumor immune response of the tumor is activated through the virus, the infiltration of the lymphocyte to the tumor tissue is recruited, the anti-PD-L1 / anti-CD137 bispecific antibody is expressed, the inhibition of the PD-1 / PD-L1 pathway on the T cell is blocked, and the inhibition signal of the PD-1 / PD-L1 pathway on the tumor cell surface is reversed into the CD137 signal of the activated T cell, such that the functional failure of the T cell caused by the PD-1 / PD-L1 pathway can be blocked, and the anti-tumor immune response of the tumor can be inhibited; the function and viability of T cells can be further improved, long-term immune surveillance on tumors is realized, and long-acting anti-tumor immune response is generated. Experiments show that the oncolytic virus can significantly prolong the lifetime of tumor-bearing mice, and a new strategy is provided for development of solid tumor drugs.

Description

Technical Field

[0001] This invention relates to an oncolytic virus VSV-aPDL1 / aCD137 expressing bispecific antibodies, its construction method, and its applications, belonging to the field of viral and microbial technology. Background Technology

[0002] Malignant tumors are a major threat to human life and health. In recent years, with the continuous advancements in treatment options such as surgery, chemotherapy, radiotherapy, and targeted therapy, the survival time of cancer patients has been extended. However, a large number of patients with advanced cancer still face the predicament of having no effective treatment options. Therefore, there is an urgent clinical need to explore new and effective treatment methods and drugs for the treatment of solid tumors.

[0003] Clinically, the use of anti-PD-1 / PD-L1 antibodies to block the binding of PD-1 and PD-L1, thereby restoring tumor-specific T-cell immune function to clear tumors, has been approved for the treatment of multiple tumor types with success, such as non-small cell lung cancer, renal cell carcinoma, bladder cancer, melanoma, and Hodgkin's lymphoma. However, in ovarian cancer, the objective response rate of anti-PD-1 / PD-L1 monotherapy is only 6-15%, which can be considered almost ineffective.

[0004] The efficacy of anti-PD-1 / PD-L1 therapy depends on tumor-specific T cells in the tumor microenvironment. Tumor-specific T cell activation is highly correlated with the specific neoantigens generated by tumor gene mutations and the ability of antigen-presenting cells (DCs) to present these neoantigens. Studies have found that in most ovarian cancers, the tumor mutation burden (TMB) is generally low, resulting in fewer tumor-specific neoantigens and thus preventing tumor-specific T cells from being recognized and activated. Furthermore, ovarian cancer can induce dysfunction in antigen-presenting cells (DCs) through multiple pathways, preventing T cells from recognizing tumor neoantigens and inhibiting tumor-specific T cell activation and expansion. Due to the combined effect of these factors, ovarian cancer lacks tumor-specific T cells, leading to a low efficacy rate of anti-PD-1 / PD-L1 therapy in ovarian cancer. Therefore, promoting the recognition of tumor neoantigens by the immune system and activating and expanding tumor-specific T cells is key to solving the problem of low efficacy of anti-PD-1 / PD-L1 therapy in ovarian cancer treatment. A growing body of research has found that combining this treatment with other immunotherapies can significantly improve the efficacy of anti-PD-1 / PD-L1 therapy.

[0005] Oncolytic viruses are a collective term for a class of viruses that selectively replicate within tumor cells and exert oncolytic effects. These viruses 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 (HSV-1), measles virus (MV), vesicular stomatitis virus (VSV), vaccinia virus (VV), and Newcastle disease virus (NDV). In the past decade or so, it has been increasingly discovered that oncolytic viruses can not only directly cause tumor cell death (oncolysis) but also effectively break tumor immune tolerance and induce anti-tumor immune responses. Increasing evidence suggests that the anti-tumor effect of oncolytic viruses primarily depends on the induced immunolysis they induce, rather than the simple oncolytic action of the virus itself. Oncolytic virus-induced immunolysis not only activates immune cells by upregulating cytokines and chemokines in the tumor environment, but more importantly, it breaks down the immune tolerance of the tumor microenvironment by inducing tumor immunogenic cell death (ICD), releasing danger signals such as damage pattern-associated molecules (DAMPs), and inducing immune cells to recognize and attack tumor cells. This immunolysis by oncolytic viruses can also enhance the function of antigen-presenting cells (DCs) through multiple pathways, presenting "neoantigens" to T cells, activating and expanding tumor-specific T cells, and inducing specific anti-tumor immunity. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an oncolytic virus VSV-aPDL1 / aCD137 expressing bispecific antibodies, its construction method, and its applications.

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

[0008] An oncolytic virus expressing bispecific antibodies against PD-L1 / anti-CD137 was obtained by ligating the aPDL1 / aCD137 fragment with a VSV viral genome plasmid to obtain the full-genome plasmid pT7-VSV-aPDL1 / aCD137. This plasmid and helper plasmid were co-transfected into 293T cells, and VSV-aPDL1 / aCD137 viral seed was obtained after culture. The viral seed was then amplified in large quantities using 293T cells to obtain the oncolytic virus VSV-aPDL1 / aCD137.

[0009] The method for constructing the oncolytic virus includes the following steps:

[0010] (1) Design of aPDL1 / aCD137 fragment: Single-chain antibody aPDL1 composed of the hypervariable region of anti-PD-L1 and single-chain antibody aCD137 composed of the hypervariable region of anti-CD137 are linked by a linker sequence to fuse into bispecific antibody aPDL1 / aCD137; IgG heavy chain signal peptide is introduced at the N-terminus of aPDL1 / aCD137 and His tag is introduced at the C-terminus;

[0011] After all sequences were assembled according to the design, codon optimization was performed. The optimized nucleic acid sequence was then synthesized into a whole genome and inserted into a T vector to obtain pUC-aPDL1 / aCD137.

[0012] (2) Cloning of the oncolytic virus VSV-aPDL1 / aCD137 whole genome plasmid pT7-VSV-aPDL1 / aCD137: Using pUC-aPDL1 / aCD137 as a template, and using VSV F: TAATCAGAATTCTCGAGACCGCCACCATGGACTGGAC and VSV R: AGTTTTTTTCATATGGCCATCAGTGGTGGTGGTGGTG as primers, the aPDL1 / aCD137 fragment was amplified using a high-fidelity kit, and sequence fragments homologous to the insertion site of the whole VSV genome were introduced at both ends of the aPDL1 / aCD137 fragment, i.e., 16 homologous bases were inserted on each side;

[0013] The pT7-VSV whole genome plasmid was double-digested with MSCI and XhoI and then purified to obtain a linear pT7-VSV whole genome plasmid. The aPDL1 / aCD137 fragment was ligated with the double-digested whole genome plasmid pT7-VSV to obtain the viral whole genome plasmid pT7-VSV-aPDL1 / aCD137.

[0014] (3) Rescue of oncolytic virus VSV-aPDL1 / aCD137: pT7-VSV-aPDL1 / aCD137 was co-transfected with helper plasmid into 293T cells using lipofectamine2000 transfection reagent. The cells were then cultured in a 37°C, 5% CO2 cell culture incubator for 4-6 hours. Then, 2 mL of DMEM cell culture medium containing 2% FBS was added and the cells were cultured for another 2-3 days. Depending on the condition of the culture medium, the DMEM medium containing 2% FBS was replaced or added. The cell status was observed under a microscope every day. When cytopathic effect appeared after 4-8 days and the cytopathic effect reached 100%, the cell supernatant was collected, and cell debris was removed by centrifugation. The oncolytic virus VSV-aPDL1 / aCD137 was obtained and aliquoted and stored at -80°C.

[0015] In step (3), the amount of pT7-VSV-aPDL1 / aCD137 used is 5 μg, and the auxiliary plasmids are pVRC-T7, pVRC-N, pVRC-P and pVRC-L, each with an amount of 0.5 μg.

[0016] The application of the oncolytic virus expressing anti-PD-L1 / anti-CD137 bispecific antibodies in the preparation of a drug for the treatment of ovarian cancer.

[0017] The application of the oncolytic virus expressing anti-PD-L1 / anti-CD137 bispecific antibodies in the preparation of drugs for treating solid tumors.

[0018] The beneficial effects of this invention are:

[0019] The oncolytic virus VSV-aPDL1 / aCD137 expressing bispecific antibodies against anti-PD-L1 / anti-CD137 of the present invention has the following technical effects:

[0020] (1) The oncolytic virus VSV-aPDL1 / aCD137 of the present invention kills tumor cells through oncolysis, reduces tumor burden, and releases a variety of immune-related factors while killing tumor cells, recruits lymphocytes to infiltrate tumor tissue, and activates specific anti-tumor immunity.

[0021] (2) The oncolytic virus VSV-aPDL1 / aCD137 of the present invention selectively replicates in tumor cells and expresses aPDL1 / aCD137 only in the tumor local area, thereby achieving a high distribution of aPDL1 / aCD137 in the tumor local area, thus effectively avoiding autoimmune tissue damage caused by PD-1 / PD-L1 pathway blockade and CD137 pathway activation in normal tissues.

[0022] (3) This invention utilizes the selectivity of oncolytic viruses to tumor cells to express anti-PD-L1 / anti-CD137 bispecific antibodies locally in the tumor. While blocking the inhibitory effect of the PD-1 / PD-L1 pathway on T cells, the antibody reverses the inhibitory signal of PD-L1 on the surface of tumor cells to CD137 signal that activates T cells. It can block the functional exhaustion of tumor-specific T cells caused by the PD-1 / PD-L1 pathway, and further improve the function and survival of tumor-specific T cells, enhance the anti-tumor immune response, and promote the differentiation of T cells into memory T cells to form immune memory, thereby achieving long-term immune surveillance of tumors and generating a long-term anti-tumor immune response.

[0023] (4) The CD137 single-chain antibody in the anti-PD-L1 / anti-CD137 bispecific antibody of the present invention must be hinged to PD-L1 on the cell surface to activate the CD137 pathway. It will not induce ADCC and cause tumor-specific lymphocyte damage, and the immune activation effect of the bispecific antibody can be anchored to the tumor site. The virus is expressed locally in the tumor to form a high concentration, avoiding the toxic side effects caused by systemic administration.

[0024] (5) The novel oncolytic virus VSV-aPDL1 / aCD137 of the present invention can not only improve the efficacy of aPDL1 / aCD137 treatment, but also avoid its toxic side effects, significantly prolong the survival of mice, improve the treatment effect of solid tumors, provide a new strategy for the development of drugs for the treatment of solid tumors, and has a good application prospect.

[0025] (6) The novel oncolytic virus VSV-aPDL1 / aCD137 of the present invention is not limited to being effective against ovarian cancer, but is also applicable to the treatment of all solid tumors. Attached Figure Description

[0026] Figure 1 Schematic diagram of the aPDL1 / aCD137 gene structure;

[0027] Figure 2 Schematic diagram of the VSV-aPDL1 / aCD137 gene structure;

[0028] Figure 3 . Detection graph of viral oncolytic ability;

[0029] Figure 4 A graph showing the detection of viral replication ability;

[0030] Figure 5 Western blot identification of aPDL1 / aCD137 in cell supernatant;

[0031] Figure 6 Flow cytometry plot of aPDL1 / aCD137 combined with PD-L1;

[0032] Figure 7 Flow cytometry plot of .aPDL1 / aCD137 combined with CD137;

[0033] Figure 8 Evaluation results of the effect of aPDL1 / aCD137 on NK cell killing ability;

[0034] Figure 9 Evaluation results of the effect of aCD137 on NK cell killing ability;

[0035] Figure 10Results of ATP or HMGB1 concentration detection in cell supernatant after VSV-aPDL1 / aCD137 infection;

[0036] Figure 11 Flow cytometry results of fluorescence intensity of CALR molecules on cell surface before and after viral infection;

[0037] Figure 12 Results of IFN-γ detection in the supernatant of SKOV3 and T cell co-culture;

[0038] Figure 13 Survival curves of mice treated with VSV-aPDL1 / aCD137;

[0039] Figure 14 Elispot assay results in mice treated with VSV-aPDL1 / aCD137

[0040] Figure 15 Statistical graph of CD8+ T cells in ascites fluid by flow cytometry;

[0041] Figure 16 Statistical graph of NK cells detected by flow cytometry in ascites fluid;

[0042] Figure 17 Survival curves of mice treated with VSV-aPDL1 / aCD137 virus. Detailed Implementation

[0043] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0044] Example 1: Construction of oncolytic virus VSV-aPDL1 / aCD137 and control viruses VSV-aPDL1 and VSV-aCD137

[0045] (1) Design of aPDL1, aCD137 and aPDL1 / aCD137 gene fragments

[0046] The aPDL1, aCD137, and aPDL1 / aCD137 gene fragments were synthesized by Suzhou Genewise Biotech Co., Ltd. and inserted into the T vector as pUC-aPDL1, pUC-aCD137, and pUC-aPDL1 / aCD137, respectively.

[0047] A schematic diagram of the aPDL1 / aCD137 gene is shown below. Figure 1As shown, an IgG heavy chain signal peptide gene was added to the N-terminus of aPDL1 / aCD137. This signal peptide enables aPDL1 / aCD137 to be expressed intracellularly and then secreted extracellularly, thus realizing the secretory function of aPDL1 / aCD137. A six-His tag sequence was added to the C-terminus to facilitate the quantitative detection of aPDL1 / aCD137.

[0048] The PD-L1 binding region of the aPDL1 / aCD137 bispecific antibody is a single-chain anti-PD-L1 antibody, composed of the light chain hypervariable region (VL) and heavy chain hypervariable region (VH) of anti-PD-L1. To ensure correct folding of the anti-PD-L1 single-chain antibody, three repeating GGGGS peptide chains are used to link VL and VH. The CD137 activation region of the aPDL1 / aCD137 bispecific antibody is a single-chain anti-CD137 antibody, composed of the light chain hypervariable region (VL) and heavy chain hypervariable region (VH) of anti-CD137, linked by three repeating GGGGS peptide chains. To prevent mismatches of VL and VH between the anti-PD-L1 and anti-CD137 single-chain antibodies, GGGGS links are used between anti-PD-L1 and anti-CD137 to restrict the spatial positions of VL and VH and prevent mismatches. All sequences are assembled according to the design and codons are optimized.

[0049] The nucleic acid sequence of the aPDL1 / aCD137 gene (SEQ ID NO.1):

[0050] ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCCGCCGCCACCGGGGCCCACAGC TATCCTTACGA

[0051] CGTGCCCGACTACGCCGACATTCAGATGACACAGAGCCCTAGCTCGCTTTCTGCTTCCGTGGGCGATAG

[0052] AGTGACAATCACATGCAGAGCCTCCCAGGACGTGAGCACCGCCGTGGCCTGGTATCAGCAAAAGCCC

[0053] GGAAAAGCCCCTAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACTCTGGCGTGCCTAGCAGGTTCAG

[0054] CGGCTCTGGCTCCGGCACAGATTTCACCCTGACCATCTCTAGCCTGCAACCTGAGGATTTCGCCACATA

[0055] TTACTGCCAGCAGTACCTCTACCACCCCGCCACCTTCGGCCAGGGCACAAAGGTGGAAATCAAGCGGA

[0056] CCGTGGCAGGCGGCGGCGGAAGTGGAGGCGGTGGTTCTGGCGGCGGCGGCAGCGAGGTGCAGCTGG

[0057] TGGAAAGCGGAGGAGGCCTGGTGCAGCCTGGAGGCTCTCTGAGACTGAGCTGTGCTGCCAGCGGCTT

[0058] TACATTCTCTGATAGTTGGATTCACTGGGTGCGGCAGGCCCCTGGCAAAGGCCTGGAATGGGTCGCCTG

[0059] GATCTCCCCATACGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCGCCG

[0060] ATACCAGCAAGAATACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACAGCCGTGTACTAC

[0061] TGTGCCCGCAGACACTGGCCTGGCGGCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTGAGCTC

[0062] CGCCAGCACAAAAGGAGGCGGCGGCAGCGAAGTGCAGCTGGTGCAGAGCGGTGCTGAGGTGAAGAA

[0063] GCCTGGCGAGAGCCTGAGAATTAGCTGCAAGGGCAGCGGCTACAGCTTCAGCACATACTGGATCAGCT

[0064] GGGTTAGACAGATGCCTGGAAAGGGCCTGGAATGGATGGGCAAAATCTACCCCGGCGACTCCTACACA

[0065] AACTACAGTCCATCATTTCAGGGCCAAGTGACCATCAGCGCTGATAAGAGCATCAGCACTGCCTACCTG

[0066] CAGTGGTCCAGCCTGAAGGCCTCAGATACCGCCATGTACTACTGCGCCAGAGGATATGGCATCTTCGAC

[0067] TATTGGGGCCAGGGCACCCTGGTCACAGTGTCTTCTGCCTCTACCAAGGGCGGCGGAGGCGGCAGCGG

[0068] AGGCGGCGGCTCCGGAGGGGGAGGCTTCTAGCTACGAGCTGACCCAGCCCCCCAGCGTGAGCGTTAGC

[0069] CCGGGCCAGACCGCCTCTATCACCTGCAGCGGCGACAACATCGGCGACCAGTACGCCCACTGGTATCA

[0070] GCAGAAACCTGGCCAAAGCCCTGTGCTGGTGATTCTATCAGGACAAGAACAGACCTAGCGGAATCCCCG

[0071] AGCGGTTCTCCGGCTCTAATAGCGGCAACACCGCTACCCTGACCATCCTCGGAACACAAGCTATGGAC

[0072] GAGGCCGACTACTACTGCGCTACCTACACCGGCTTCGGATCTCTGGCCGTGTTCGGCGGCGGCACAAAGCTGACCGTGCTGGGCCAGCCTAAGGCTCAC CATCACCACCACCATCACTGA .

[0073] The underlined portion represents the IgG heavy chain signal peptide sequence and the His tag sequence (SEQ ID NO.2-3 is the same).

[0074] The aPDL1 gene of the control virus VSV-aPDL1 is an anti-PD-L1 single-chain antibody sequence with an IgG heavy chain signal peptide added to the N-terminus and a 6-His tag sequence added to the C-terminus.

[0075] aPDL1 gene sequence (SEQ ID NO.2):

[0076] ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCCGCCGCCACCGGGGCCCACAGC TATCCTTACGA

[0077] CGTGCCCGACTACGCCGACATTCAGATGACACAGAGCCCTAGCTCGCTTTCTGCTTCCGTGGGCGATAG

[0078] AGTGACAATCACATGCAGAGCCTCCCAGGACGTGAGCACCGCCGTGGCCTGGTATCAGCAAAAGCCC

[0079] GGAAAAGCCCCTAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACTCTGGCGTGCCTAGCAGGTTCAG

[0080] CGGCTCTGGCTCCGGCACAGATTTCACCCTGACCATCTCTAGCCTGCAACCTGAGGATTTCGCCACATA

[0081] TTACTGCCAGCAGTACCTCTACCACCCCGCCACCTTCGGCCAGGGCACAAAGGTGGAAATCAAGCGGA

[0082] CCGTGGCAGGCGGCGGCGGAAGTGGAGGCGGTGGTTCTGGCGGCGGCGGCAGCGAGGTGCAGCTGG

[0083] TGGAAAGCGGAGGAGGCCTGGTGCAGCCTGGAGGCTCTCTGAGACTGAGCTGTGCTGCCAGCGGCTT

[0084] TACATTCTCTGATAGTTGGATTCACTGGGTGCGGCAGGCCCCTGGCAAAGGCCTGGAATGGGTCGCCTG

[0085] GATCTCCCCATACGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCGCCG

[0086] ATACCAGCAAGAATACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACAGCCGTGTACTAC

[0087] TGTGCCCGCAGACACTGGCCTGGCGGCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTGAGCTCCGCCAGCACAAAA CATCACCACCACCATCACTGA .

[0088] The aCD137 gene of the control virus VSV-aCD137 is an anti-CD137 single-chain antibody sequence with an IgG heavy chain signal peptide added to the N-terminus and a 6-His tag sequence added to the C-terminus.

[0089] aCD137 gene nucleic acid sequence (SEQ ID NO.3):

[0090] ATGGACTGGACCTGGAGAATCCTGTTCCTGGTGGCCGCCGCCACCGGGGCCCACAGC GAAGTGCAGC

[0091] TGGTGCAGAGCGGTGCTGAGGTGAAGAAGCCTGGCGAGAGCCTGAGAATTAGCTGCAAGGGCAGCG

[0092] GCTACAGCTTCAGCACATACTGGATCAGCTGGGTTAGACAGATGCCTGGAAAGGGCCTGGAATGGATG

[0093] GGCAAAATCTACCCCGGCGACTCCTACACAAACTACAGTCCATCATTTCAGGGCCAAGTGACCATCAG

[0094] CGCTGATAAGAGCATCAGCACTGCCTACCTGCAGTGGTCCAGCCTGAAGGCCTCAGATACCGCCATGTA

[0095] CTACTGCGCCAGAGGATATGGCATCTTCGACTATTGGGGCCAGGGCACCCTGGTCACAGTGTCTTCTGC

[0096] CTCTACCAAGGGCGGCGGAGGCGGCAGCGGAGGCGGCGGCCTCCGGAGGGGGAGGCTCTAGCTACGA

[0097] GCTGACCCAGCCCCCCAGCGTGAGCGTTAGCCCGGGCCAGACCGCCTCTATCACCTGCAGCGGCGACA

[0098] ACATCGGCGACCAGTACGCCCACTGGTATCAGCAGAAACCTGGCCAAAGCCCTGTGCTGGTGATCTAT

[0099] CAGGACAAGAACAGACCTAGCGGAATCCCCGAGCGGTTCTCCGGCTCTAATAGCGGCAACACCGCTAC

[0100] CCTGACCATCTCCGGAACACAAGCTATGGACGAGGCGACTACTACTGCGCTACCTACACCGGCTTCG

[0101] GATCTCTGGCCGTGTTCGGCGGCGGCACAAAGCTGACCGTGCTGGGCCAGCCTAAGGCT CATCACCAC CACCATCAC TGA.

[0102] (2) The design diagram of the clonal oncolytic virus VSV-aPDL1 / aCD137 of the viral whole genome plasmids pT7-VSV-aPDL1 / aCD137, pT7-VSV-aPDL1, and pT7-VSV-aCD137 is shown in the figure below. Figure 2 As shown, N is the nucleocapsid protein gene, P is the phosphoprotein gene, M is the basic protein gene, G is the VSV membrane protein gene, and L is the viral polymerase gene.

[0103] Using pUC-aPDL1 / aCD137, pUC-aPDL1, and pUC-aCD137 as templates, the aPDL1 / aCD137, aPDL1, and aCD137 gene fragments were amplified, respectively.

[0104] Using VSV-F: taatcagaattctcgagaccgccaccatggactggac (SEQ ID NO.4) and VSV-R: agttttttttcatatggccatcagtggtg gtggtggtg (SEQ ID NO.5) as primers, amplification was performed using the HiFimix high-fidelity PCR kit from Shanghai Micro-Chemistry. Sequence fragments homologous to the insertion site in the entire VSV genome were introduced at both ends of the amplified fragment, i.e., 16 homologous bases were inserted at each end.

[0105] Amplification reaction conditions: 94℃ pre-denaturation for 15 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min, storage at 4℃. aPDL1 / aCD137, aPDL1, and aCD137 fragments were obtained by PCR amplification; the PCR reaction system is shown in Table 1.

[0106] Table 1 PCR reaction system

[0107] Component Name Added amount 2×HiFi mix 25μl KDS 1μl (aPDL1 / aCD137) / aPDL1 / aCD137 100ng VSV-F (10μM) 1μl VSV-R (10μM) 1μl <![CDATA[Make up to with ddH2O]]> 50μl

[0108] The pT7-VSV whole genome plasmid was digested with XhoI and MSCI under the following conditions: 37℃ for 4 h. Then, it was purified using a gel purification kit (Tiangen Biotech) to obtain the linear pT7-VSV whole genome.

[0109] The VSV full genome sequence is based on vesicular stomatitis Indiana virus as a template, with a new transcription unit (ATU) inserted before its N gene containing restriction sites XhoI and MScI, transcription start and termination sites, to facilitate the above-mentioned restriction and gene expression.

[0110] Table 2 Double enzyme digestion reaction system

[0111] 10×cutsmart buffer 10μl MscI-HF 5μl XhoI-HF 5μl pT7-VSV 10μg <![CDATA[Make up to with ddH2O]]> 100μl

[0112] The aPDL1 / aCD137, aPDL1, and aCD137 fragments were cloned into the ATU region of pT7-VSV (ATU is an additional transcription unit) 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-aPDL1 / aCD137, pT7-VSV-aPDL1, and pT7-VSV-aCD137.

[0113] Table 3 Homologous recombination reaction system

[0114] Infusion MIX 5μl Linearized pT7-VSV 300ng (aPDL1 / aCD137) / aPDL1 / aCD137 80ng <![CDATA[Make up to with ddH2O]]> 10μl

[0115] The full genome sequences of pT7-VSV, pT7-VSV-aPDL1 / a CD137, pT7-VSV-aPDL1, and pT7-VSV-a CD137 are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively (end of text).

[0116] (3) Rescue of oncolytic virus VSV-aPDL1 / aCD137

[0117] Preparation of 293T cells: Culture 293T cells, digest them with 0.25% trypsin during the logarithmic growth phase, resuspend them in complete culture medium (DMEM + 10% FBS + 1% PS) to prepare a cell suspension of 400,000 cells / mL, and seed 2 mL (containing 800,000 cells) into 6-well plates. Incubate the 6-well plates overnight in a CO2 incubator (37℃, 5% CO2) until the cell density reaches 70-80%, then set aside for use.

[0118] Transfection: Following the instructions for the Lipofectmine 2000 transfection reagent, the helper plasmid and transfection reagent were diluted with Opti-MEM, thoroughly mixed, and allowed to stand at room temperature for 5 minutes to obtain plasmid dilution A and Lipofectmine 2000 reagent dilution B. The composition of plasmid dilution A and transfection reagent dilution B is shown in Table 4. Table 4. Preparation of Transfection Reagents

[0119]

[0120] Add diluent A to diluent B, mix thoroughly, let stand at room temperature for 10 minutes, add 600 μL of opti-MEM to obtain a diluted mixture of A and B, and gently mix by pipetting.

[0121] Remove the prepared 293T cells (6-well plate), discard the supernatant, and wash once with PBS. Add all of the diluted A and B mixture to the 293T cells and incubate in a CO2 incubator (37℃, 5% CO2) for 4-6 hours. Add 2 mL of DMEM cell culture medium containing 2% FBS and continue culturing for 2-3 days. Replace or supplement the DMEM with 2% FBS as needed. Observe the cell status under a microscope daily. Cytopathic effects will begin to appear around 4-8 days. When cytopathic effects reach 100%, collect the cell supernatant, centrifuge at 4000×g for 20 min to remove cell debris, aliquot into 500 μL / tube, and store at -80℃.

[0122] The rescue methods for the viruses VSV-aPDL1 and VSV-aCD137 are the same as those for VSV-aPDL1 / aCD137.

[0123] Example 2: Functional testing of VSV-aPDL1 / aCD137

[0124] 1. Viral oncolytic ability detection

[0125] Ovarian cancer cell lines SKOV3, ES2, OVCAR3, and ID8 were cultured using RPMI 1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (cell culture reagents were purchased from Thermo Fisher Scientific (China) Co., Ltd.). Cells were placed in T75 culture flasks and cultured until the cell density reached approximately 90%. Cells were then digested with 0.25% trypsin-EDTA to prepare single-cell suspensions. After cell counting, 10,000 cells per well (100 μL total volume) were seeded into 96-well plates, with 5 rows of each cell type and 6 replicates per row. The 96-well plates were incubated at 37°C in a cell culture incubator containing 5% carbon dioxide for 6 hours until cell adhesion.

[0126] Cells were divided into five groups: Mock, VSV, VSV-aPDL1, VSV-aCD137, and VSV-aPDL1 / aCD137, with one group labeled per column. The Mock group was added to 5 μL of RPMI 1640 medium, while the other groups were each added to 5 μL of virus until the final MOI was 0.01. Cells were cultured 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 formaldehyde. The cells were then washed twice 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 rinsed with water, and the cells were air-dried at room temperature before photographing and analysis.

[0127] Cell viability was analyzed using ImageJ2 software. The gray values ​​of cells in each group and the blank well were measured. The gray values ​​of the blank wells were used as the baseline, and the gray values ​​of the other wells were subtracted from the gray values ​​of the blank wells. The cell viability of the mock blank group was taken as 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.

[0128] The results are as follows Figure 3 As shown, the virus treatment groups VSV, VSV-aPDL1, VSV-aCD137, and VSV-aPDL1 / aCD137, using viruses with an MOI of 0.01, all caused approximately 80% cell death within 48 hours, with no significant differences among the viruses. This indicates that the insertion of exogenous genes into aPDL1, aCD137, and aPDL1 / aCD137 does not affect the oncolytic ability of the viruses.

[0129] 2. Virus replication ability detection

[0130] SKOV3 cell lines were cultured, digested with 0.25% trypsin-EDTA to prepare single-cell suspensions, and 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 wells in total, 1 cell per well) were placed in a 37°C, 5% CO2 incubator and cultured for 4 hours until the cells were completely adhered to the well.

[0131] VSV, VSV-aPDL1, VSV-aCD137, and VSV-aPDL1 / aCD137 viruses were diluted to a final concentration of 1×10 using RPMI 1640 medium. 4 PFUs / mL, 24-well plates inoculated with SKOV3 cell line were divided into four groups: VSV, VSV-aPDL1, VSV-aCD137, and VSV-aPDL1 / aCD137, with three wells in each group and labeled. 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, and RPMI 1640 complete medium was added. 293T cells were infected with VSV, VSV-aPDL1, VSV-aCD137, and VSV-aPDL1 / aCD137 viruses with a multiplicity of infection (MOI) of 0.01, respectively. Cell supernatant was collected after 48 hours, and the virus titer in each well was determined using a dilution method.

[0132] Virus titer assay: 10,000 Vero cells per well were seeded into 96-well plates and cultured overnight. The virus stock solution was diluted 1:10 to 10⁻¹⁰ using DMEM medium. -10 Add 100 μL of Vero cells to each well of a 96-well plate, with 10 wells per dilution. Incubate at 37°C for 4 hours, remove the virus solution, and continue culturing in complete medium containing 2% fetal bovine serum. Observe cytopathic effects after 1 week and calculate the viral titer. Observe under a microscope; wells showing cytopathic effects are defined as positive, with each positive result recorded as 0.1. Sum all positive wells across all dilutions to obtain S.

[0133] Virus TCID50 = 10 2+(S-0.5) / mL, pfu / mL=0.7×TCID50 / mL, virus titer per well (PFUs / well)=PFUs / mL×virus volume per well (ml).

[0134] The results are as follows Figure 4 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 PFUs, VSV, VSV-aPDL1, VSV-aCD137, and VSV-aPDL1 / aCD137. These results indicate that the insertion of exogenous genes does not affect viral replication capacity.

[0135] 4. Detection of recombinant protein expression and secretion

[0136] SKOV3 cells were infected with VSV-aPDL1 / aCD137 with a multiplicity of infection (MOI) of 0.01. After 72 hours, the supernatant was collected and analyzed by 8-20% polyacrylamide gel electrophoresis. Western blotting was used to detect the expression of aPDL1 / aCD137, with anti-His-Tag as the primary antibody. The predicted molecular size was 57 kDa.

[0137] The results are as follows Figure 5 As shown, a single band between 55-70 kDa was observed in the cell supernatant after VSV-aPDL1 / aCD137 infection, with a size of approximately 60 kDa, which is close to the predicted molecular weight of aPDL1 / aCD137. This result indicates that VSV-aPDL1 / aCD137 infection of SKOV3 ovarian cancer cells enables the expression of the target protein aPDL1 / aCD137 in the ovarian cancer cells and its secretion into the cell supernatant.

[0138] Example 3: Functional evaluation of the bispecific antibody aPDL1 / aCD137

[0139] 1. Binding ability of aPDL1 / aCD137 to PD-L1 and CD137

[0140] 1.1 aPDL1 / aCD137 binding assay with PD-L1: 293T cell lines were transfected with the PD-L1 expression plasmid pCMV-PDL1. After 48 h, the cells were digested with 0.25% trypsin and neutralized with DMEM complete medium. The cells were centrifuged at 300×g for 4 min to remove the supernatant, and resuspended in PBS to prepare a single-cell suspension with a concentration of 1 million cells per mL. 100 μL of cell suspension was added to each of the following: anti-PD-L1-PE antibody (2 μL), anti-His-FITC antibody (2 μL), and aPD-L1 / aCD137 (2 μL) + anti-His-FITC (2 μL). The mixture was incubated at room temperature for 30 min (gently vortexed every 10 min to mix), and then washed twice with 1 mL of PBS to remove excess antibody. The fluorescence intensity of PE or FITC was detected by flow cytometry.

[0141] The results are as follows Figure 6 As shown, approximately 50% of 293T cells transfected with pCMV-PDL1 showed significantly enhanced fluorescence signal after anti-PD-L1-PE labeling, while no change was observed in 293T cells. Neither 293T nor PD-L1-293T cells showed enhanced fluorescence signal when labeled with Anti-his-FITC alone. When both 293T and PD-L1-293T cells were co-labeled with Anti-his-FITC and aPDL1 / aCD137, the fluorescence intensity of 293T cells showed no significant change, while some PD-L1-293T cells showed significantly enhanced fluorescence.

[0142] These results indicate that some 293T cells transfected with pCMV-PDL1 highly express PD-L1. Neither 293T nor PD-L1-293T cells have His-tag protein molecules on their surface, and anti-his-FITC labeling showed no fluorescent signal. When 293T and PD-L1-293T cells were co-labeled with Anti-his-FITC and aPDL1 / aCD137, 293T cells did not express PD-L1 molecules, and aPDL1 / aCD137 could not bind to 293T cells, therefore no fluorescent signal change was observed in 293T cells. Only about 50% of PD-L1-293T cells expressed PD-L1. aPDL1 / aCD137 bound to PD-L1-positive cells, and anti-his-FITC bound to aPDL1 / aCD137. Therefore, approximately 50% of PD-L1-293T cells co-labeled with anti-his-FITC and aPDL1 / aCD137 showed enhanced fluorescence signal. This result indicates that aPDL1 / aCD137 can bind to PD-L1.

[0143] 1.2 Binding experiment of aPDL1 / aCD137 to CD137: A stable CD137-expressing cell line, 293T-CD137, was constructed using a lentiviral vector. Cells were cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated in a cell culture incubator at 37°C with 5% CO2. Single-cell suspensions of 293T-CD137 cells were prepared using PBS to a final concentration of 1 million cells per milliliter. 100 μL of this suspension was then added to anti-CD137-FITC, anti-His-FITC, or aPDL1 / aCD137+anti-His-FITC as required by the experimental protocol.

[0144] FITC was used to incubate 293T-CD137 cells. After 30 min, the cells were washed twice with PBS to remove excess antibody. The FITC fluorescence intensity was then measured by flow cytometry.

[0145] The results are as follows Figure 7As shown, anti-CD137-FITC labeling significantly enhanced cell fluorescence intensity, while anti-His-FITC labeling alone resulted in fluorescence intensity similar to that of Isotype labeling. Simultaneous labeling with aPDL1 / aCD137 and anti-His-FITC significantly enhanced cell fluorescence intensity, approaching that of anti-CD137-FITC labeled cells. This indicates that anti-His-FITC can bridge with 293T-CD137 via aPDL1 / aCD137, enabling 293T-CD137 cells to be labeled with FITC fluorescence signals. aPDL1 / aCD137 can bind to CD137 molecules.

[0146] 2. αPDL1 / αCD137 activation of the CD137 pathway depends on the binding of PD-L1 positive cells.

[0147] Further investigation is needed to determine whether the aPDL1 / aCD137 activation of the CD137 pathway depends on the hinge of PD-L1-positive cells.

[0148] First, a stable PD-L1-expressing cell line, SKOV3-PDL1, was constructed using a lentiviral vector. Then, NK cells were co-cultured with either SKOV3-PDL1 or SKOV3 cells. The cytotoxicity of NK cells against SKOV3 and SKOV3-PDL1 was evaluated in the co-culture system with or without the addition of aPDL1 / aCD137 bispecific antibody. aPDL1 / aCD137 activation of the CD137 pathway in NK cells significantly enhanced their cytotoxicity. However, aPDL1 / aCD137 activation of the CD137 pathway depends on its hinge with PD-L1-positive cells. Therefore, only the addition of aPDL1 / aCD137 to a mixture of NK cells and SKOV3-PDL1-positive cells significantly improved the cytotoxicity of NK cells.

[0149] This experiment consisted of 5 groups: NK+SKOV3 group, NK+aPDL1 / aCD137+SKOV3 group, NK+SKOV3-PDL1 group, NK+SKOV3-PDL1+aPDL1 / aCD137 group, and Mock group (Mock group was untreated SKOV3 cells). NK cells were mixed with SKOV3 or SKOV3-PDL1 at a ratio of 10:1 and cultured. Purified aPDL1 / aCD137 protein at a final concentration of 200 ng / ml was added or not added according to the group. Cell viability was detected after culturing at 37℃ for 12 h.

[0150] Simultaneously, to evaluate whether the aCD137 single-chain antibody could activate the CD137 pathway, the co-culture method of NK cells and SKOV3 was also used. The experiment consisted of three groups: the SKOV3 group, the SKOV3+NK group, and the SKOV3+NK+aCD137 group. NK cells and SKOV3 were mixed at a ratio of 10:1, and purified aCD137 protein at a final concentration of 200 ng / ml was added or not added according to the group. Cell viability was then assessed after incubation at 37°C for 12 h.

[0151] The results are as follows Figure 8 As shown, there was no difference in SKOV3 cell viability among the NK+SKOV3 group, NK+aPDL1 / aCD137+SKOV3 group, and NK+SKOV3-PDL1 group, all of which were significantly lower than the Mock group. The SKOV3-PDL1 cell viability in the NK+aPDL1 / aCD137+SKOV3-PDL1 group was significantly lower than that in the other groups.

[0152] Figure 9 As shown, after adding aCD137 single-chain antibody to the NK and SKOV3 co-culture system, there was no significant difference in SKOV3 cell viability between the NK+SKOV3 and NK+aCD137+SKOV3 groups, both of which were significantly lower than the SKOV3 culture alone group (data in the figure are presented as Mean±SD, ns P>0.05).

[0153] The results above indicate that aPDL1 / aCD137 can enhance the cytotoxic effect of NK cells against PD-L1-positive cells, but cannot improve the cytotoxic effect of NK cells against PD-L1-negative cells; aCD137 itself cannot enhance the cytotoxic effect of NK cells. Therefore, the activation of CD137 molecules by aPDL1 / aCD137 to enhance NK cell function depends on the expression of PD-L1 on the cell surface. aCD137 alone cannot activate CD137 molecules and cannot enhance the cytotoxic function of NK cells.

[0154] The above evidence suggests that aPDL1 / aCD137 activation of the CD137 pathway depends on the binding of PD-L1 positive cells.

[0155] Example 4: Study on the activation of VSV-aPDL1 / aCD137 for anti-tumor immunity

[0156] 1. VSV-aPDL1 / aCD137 induces immunogenic cell death in ovarian cancer cells.

[0157] SKOV3 cells were infected with VSV-aPDL1 / aCD137 at an MOI of 0.01. Supernatants were collected at 24 and 48 hours, and the concentrations of ATP and HMGB1 in the supernatants were measured. Cells were collected at 24 hours, labeled with anti-CALR-PE for 30 minutes, and CALR molecules on the cell surface were detected by flow cytometry. The study aimed to analyze whether VSV-aPDL1 / aCD137 could induce immunogenic cell death in ovarian cancer cells.

[0158] like Figure 10 As shown, the concentrations of ATP and HMGB1 in the supernatant after VSV-aPDL1 / aCD137 treatment were significantly increased, and the concentrations of ATP and HMGB1 at 48 h were significantly higher than those at 24 h.

[0159] in Figure 10 A represents the result of ATP concentration detection in cell supernatant after viral infection. Figure 10 B represents the concentration of HMGB1 in the cell supernatant after viral infection;

[0160] Figure 11 The flow cytometry results of the fluorescence intensity of CALR molecules on the cell surface before and after viral infection show that, 24 h after viral infection, the fluorescence signal of anti-CALR-PE labeled SKOV3 cells was significantly enhanced compared with the Mock untreated group, indicating a significant increase in CALR molecules on the cell surface.

[0161] The above results indicate that VSV-aPDL1 / aCD137 treatment can induce immunogenic cell death in SKOV3 cells.

[0162] 2. Study on the enhancement of DC cell function by VSV-aPDL1 / aCD137

[0163] SKOV3 cells were infected with VSV-aPDL1 / aCD137 at an MOI of 0.01. The supernatant was collected after 48 hours, and the virus was inactivated by UV irradiation for 30 minutes, serving as a tumor antigen. Peripheral blood PBMCs were isolated, and peripheral blood dendritic cells (DCs) were isolated using the adherent method. These cells were cultured in 1640 medium containing GM-CSF 10 ng / ml and IL-4 10 ng / ml at 37°C in a 5% CO2 cell culture incubator. On day 5, supernatant from virus-infected SKOV3 cells (pre-prepared tumor antigen) was added. For the control group, freeze-thawed lysed SKOV3 cells were added as the tumor antigen. Cells were cultured for another 2 days, and then collected. T cells and DCs were added at a 1:10 ratio and cultured for another 7 days. T cells and SKOV3 cells were cultured overnight at a 5:1 ratio, and the supernatant was collected. The IFN-γ level in the supernatant was detected using ELISA. The study investigated whether VSV-aPDL1 / aCD137 could enhance DC antigen presentation and induce more tumor-specific T cells.

[0164] The results are as follows Figure 12 After treatment with VSV-aPDL1 / aCD137, the supernatant from the cells activated by dendritic cells (DCs) showed significantly higher interferon release upon re-stimulation with SKOV3 compared to the control group. This indicates that the number of SKOV3-specific T cells induced by the virus group was significantly higher than that in the control group, and that VSV-aPDL1 / aCD137 infection significantly enhanced the antigen presentation capacity of dendritic cells (data presented in the figure are presented using Mean ± SD, ****P < 0.0001). Application Example: Study on the antitumor effect of VSV-aPDL1 / aCD137

[0165] 1. VSV-aPDL1 / aCD137 can significantly prolong the survival of mice with ovarian cancer.

[0166] Using 8-10 week old C57BL6 mice, on day 1, each mouse was injected intraperitoneally with 1×10 7 On day 10, in vivo imaging confirmed intraperitoneal tumor growth. Mice were randomly divided into 5 groups: VSV, VSV-aPDL1, VSV-aCD137, and VSV-aPDL1 / aCD137 virus treatment groups, and an untreated mock group. Ten mice were injected intraperitoneally with 1 × 10⁶ cells of the virus on days 10, 13, and 16. 7 PFUs / mouse / dose. Cells were isolated from ascites fluid collected from mice on day 22. The number of tumor-specific T cells in the ascites fluid was detected using EliSpot technology, and CD8+ cells in the ascites fluid were analyzed by flow cytometry. + The infiltration status of T and NK cells.

[0167] Mice survival curves were plotted by monitoring their condition. The results are as follows: Figure 13 As shown, the survival time of mice treated with VSV and VSV-aCD137 viruses was not significantly different from that of the MOCK groups. However, the survival time of mice treated with VSV-aPDL1 was significantly prolonged compared to the Mock, VSV, and VSV-aCD137 groups. The median survival time of mice in the VSV-aPDL1 / aCD137 treatment group was 75 days, significantly longer than the other four groups, indicating that the antitumor effect of VSV-aPDL1 / aCD137 was stronger than that of the other groups.

[0168] Elispot test results are as follows: Figure 14 After viral treatment, the number of tumor-activated T cells increased significantly compared to the Mock group. The number of tumor-activated T cells in the VSV-aPDL1 group was higher than that in the VSV and VSV-aCD137 groups, and the number of tumor-activated T cells in the VSV-aPDL1 / aCD137 group was significantly higher than that in the other four groups.

[0169] Figure 15 , Figure 16 The graphs show the flow cytometry results of CD8+ T cells and NK cells in the ascites fluid. It can be seen that CD8+ T cells are present in the ascites fluid. + All virus treatment groups for T cells and NK cells were significantly higher than the Mock group. The VSV-aPDL1 / aCD137 group was significantly higher than the other four groups. The VSV-aPDL1 group was higher than the VSV and VSV-aCD137 groups. There was no significant difference between the VSV and VSV-aCD137 groups.

[0170] The above results indicate that VSV-aPDL1 / aCD137 significantly prolongs mouse survival by activating anti-tumor immunity in the tumor microenvironment.

[0171] 2. Research on the anti-tumor mechanism of the novel oncolytic virus VSV-aPDL1 / aCD137

[0172] An ascites tumor model was established using 8-10 week old female C57BL6 mice. Each mouse was inoculated with 1×10 7 ID8-Luc cells. On day 10, mice were randomly divided into 4 groups: A. untreated MOCK group, B. VSV-aPDL1 / aCD137 group, C. CD8 cell depletion + VSV-aPDL1 / aCD137 group, and D. NK cell depletion + VSV-aPDL1 / aCD137 group.

[0173] Group C received intraperitoneal injection of anti-CD8 antibody, and Group D received intraperitoneal injection of anti-NK1.1 antibody, administered every 5 days until the end of the experiment. Intratumoral injection of 1×10⁻⁶ virus was also administered according to group assignments. 7 PFUs / mouse / dose, 4 days apart, for a total of 3 doses. Mouse survival status was monitored until death or the end of the experiment, and mouse survival curves were plotted. The role of immune cells in VSV-aPDL1 / aCD137 anti-tumor immunity was investigated.

[0174] The results are as follows Figure 17 As shown, VSV-aPDL1 / aCD137 treatment significantly prolonged the survival of mice. The use of anti-NK1.1 to clear mouse NK cells did not affect the survival of mice treated with VSV-aPDL1 / aCD137, and the survival rate was the same as that of the VSV-aPDL1 / aCD137-only treatment group. When anti-CD8 antibodies were used to clear mouse CD8 cells... + Following T cell therapy, the therapeutic effect of VSV-aPDL1 / aCD137 was significantly inhibited, and the survival time of mice was significantly shortened, similar to the Mock group. These results indicate that the antitumor effect of VSV-aPDL1 / aCD137 mainly relies on CD8+ T cells.

[0175] The following is a partial sequence from the text:

[0176] PT7-VSV nucleic acid sequence (SEQ ID NO.6)

[0177] acgaagacaaacaaaccattattatcattaaaaggctcaggagaaactttaacagtaatcagaattctcgagaccgccacctggccatatgaaaaaaactaacagtaatc

[0178] aaaatgtctgttacagtcaagagaatcattgacaacacagtcatagttccaaaacttcctgcaaatgaggatccagtggaatacccggcagattacttcagaaaatcaaag

[0179] gagattcctctttacatcaatactacaaaaagtttgtcagatctaagaggatatgtctaccaaggcctcaaatccggaaatgtatcaatcatacatgtcaacagctacttgtat

[0180] ggagcattaaaggacatccggggtaagttggataaagattggtcaagtttcggaataaacatcgggaaagcaggggatacaatcggaatatttgaccttgtatccttgaa

[0181] agccctggacggcgtacttccagatggagtatcggatgcttccagaaccagcgcagatgacaaatggttgcctttgtatctacttggcttatacagagtgggcagaacac

[0182] aaatgcctgaatacagaaaaaagctcatggatgggctgacaaatcaatgcaaaatgatcaatgaacagtttgaacctcttgtgccagaaggtcgtgacatttttgatgtgtg

[0183] gggaaatgacagtaattacacaaaaattgtcgctgcagtggacatgttcttccacatgttcaaaaaacatgaatgtgcctcgttcagatacggaactattgtttccagattca

[0184] aagattgtgctgcattggcaacatttggacaccctgcaaaataaccggaatgtctacagaagatgtaacgacctggatctgaaccgagaagttgcagatgaaatggtc

[0185] caaatgatgcttccaggccaagaaattgacaaggccgattcatacatgccttatttcgactttggattgtcttctaagtctccatattctctcgtcaaaaaccctgccttcc

[0186] acttctgggggcaattgacagctctctgctcagatccaccagagcaaggaatgcccgagcctgacattgagtatacatctcttactacagcaggttttgttgtacgc

[0187] tttgcagtaggatcctgccgacttggcacaacagttttgtgttggagataaaatacactccagatgatagtaccggaggattgacgactaatgcaccgccacaag

[0188] gcagagatgtggtcgaatggctcggatggttgaagatcaaaacagaaaccgactcctgatgatgcagtatgcgaaaagagcagtcatgtcactgcaaggcctaa

[0189] gagagacaattggcaagtatgctaagtcagaatttgacaaatgaccctataattctcagatcacctattatatattatgctacatatgaaaaaaactaacagatatcatg

[0190] gataatctcacaaaagttcgtgagtatctcaagtcctactctcgtctagatcaggcggtaggagagatgagatcgaagcaacgagctgaaaagtccaattatga

[0191] gttgttccaagaggacggagtggaagagcatactaggccctcttattttcaggcagcagatgattctgacacagaatctgaaccagaaattgaagacaatcaaggcttgt

[0192] atgtaccagatccggaagctgagcaagttgaaggctttatacaggggcctttagatgactatgcagatgaggacgtggatgttgtattcacttcggactggaaacagcct

[0193] gagcttgaatccgacgagcatggaaagaccttacggttgacattgccagagggtttaagtggagagcagaaatcccagtggcttttgacgattaaagcagtcgttcaaa

[0194] gtgccaaacactggaatctggcagagtgcacatttgaagcatcgggagaaggggtcatcataaaaaagcgccagataactccggatgtatataaggtcactccagtgat

[0195] gaacacacatccgtaccaatcagaagccgtatcagatgtttggtctctctcaaagacatccatgactttccaacccaagaaagcaagtcttcagcctctcaccatatccttg

[0196] gatgaattgttctcatctagaggagaattcatctctgtcggaggtaacggacgaatgtctcataaagaggccatcctgctcggtctgaggtacaaaaagttgtacaatcag

[0197] gcgagagtcaaatattctctgtagactatgaaaaaaagtaacagatatcacaatctaagtgttatcccaatccattcatcatgagttccttaaagaagattctcggtctgaagg

[0198] ggaaaggtaagaaatctaagaaattagggatcgcaccacccccttatgaagaggacactagcatggagtatgctccgagcgctccaattgacaaatcctattttggagtt

[0199] gacgagatggacacctatgatccgaatcaattaagatatgagaaattcttctttacagtgaaaatgacggttagatctaatcgtccgttcagaacatactcagatgtggcag

[0200] ccgctgtatcccattgggatcacatgtacatcggaatggcagggaaacgtcccttctacaaaatcttggcttttttgggttcttctaatctaaaggccactccagcggtattg

[0201] gcagatcaaggtcaaccagagtatcacgctcactgcgaaggcagggcttatttgccacataggatggggaagacccctcccatgctcaatgtaccagagcacttcaga

[0202] agaccattcaatataggtctttacaagggaacgattgagctcacaatgaccatctacgatgatgagtcactggaagcagctcctatgatctgggatcatttcaattcttccaa

[0203] attttctgatttcagagagaaggccttaatgtttggcctgattgtcgagaaaaaggcatctggagcgtgggtcctggattctatcagccacttcaaatgagctagtctagctt

[0204] ccagcttctgaacaatccccggtttactcagtctctcctaattccagcctttcgaacaactaatatcctgtcttttctatccctatgaaaaaaactaacagagatcgatctgtttc

[0205] cttgacacgcgttacgatgcggccgatgaagtgccttttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacacaaccaaaaaggaaactg

[0206] gaaaaatgttccttctaattaccattattgcccgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaaggcta

[0207] ttcaagcagacggttggatgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccgatccttcactccatctgtag

[0208] aacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcctcaaagttgtggatatgcaactgtgacggatgccgaagcagtgat

[0209] tgtccaggtgactcctcaccatgtgctggttgatgaatacacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccataa

[0210] ctctacaacctggcattctgactataaggtcaaagggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaag

[0211] gagggcacagggttcagaagtaactactttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtctggt

[0212] tcgagatggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgtaagtctaattcagga

[0213] cgttgagaggatcttggattattccctctgccaagaaacctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaacccagg

[0214] aaccggtcctgctttcaccataatcaatggtaccctaaaatactttgagaccagatacatcagagtcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtg

[0215] gaactaccacagaaagggaactgtgggatgactgggcaccatatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatac

[0216] atgattggacatggtatgttggactccgatcttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagttta

[0217] ttttttggtgatactgggctatccaaaaatccaatcgagcttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatagggttaatcattggactat

[0218] tcttggttctccgagttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaagtaagcggccgccct

[0219] gcacacagattcttcatgtttgaaccaaatcaacttgtgatatcatgctcaaagaggccttaattatattttaatttttaatttttatgaaaaaaactaacagcaatcatggaagtc

[0220] cacgattttgagaccgacgagttcaatgatttcaatgaagatgactatgccacaagagaattcctgaatcccgatgagcgcatgacgtacttgaatcatgctgattacaattt

[0221] gaattctcctctaattagtgatgatattgacaatttgatcaggaaattcaattctcttccgattccctcgatgtgggatagtaagaactgggatggagttcttgagatgttaacat

[0222] catgtcaagccaatcccatctcaacatctcagatgcataaatggatgggaagttggttaatgtctgataatcatgatgccagtcaagggtatagttttttacatgaagtggac

[0223] aaagaggcagaaataacatttgacgtggtggagaccttcatccgcggctggggcaacaaaccaattgaatacatcaaaaaggaaagatggactgactcattcaaaattc

[0224] tcgcttatttgtgtcaaaagtttttggacttacacaagttgacattaatcttaaatgctgtctctgaggtggaattgctcaacttggcgaggactttcaaaggcaaagtcagaag

[0225] aagttctcatggaacgaacatatgcaggattagggttcccagcttgggtcctacttttatttcagaaggatgggcttacttcaagaaacttgatattctaatggaccgaaactt

[0226] tctgttaatggtcaaagatgtgattatagggaggatgcaaacggtgctatccatggtatgtagaatagacaacctgttctcagagcaagacatcttctcccttctaaatatcta

[0227] cagaattggagataaaattgtggagaggcagggaaatttttcttatgacttgattaaaatggtggaaccgatatgcaacttgaagctgatgaaattagcaagagaatcaag

[0228] gcctttagtcccacaattccctcattttgaaaatcatatcaagacttctgttgatgaaggggcaaaaattgaccgaggtataagattcctccatgatcagataatgagtgtgaa

[0229] aacagtggatctcacactggtgatttatggatcgttcagacattggggtcatccttttatagattattacactggactagaaaaattacattcccaagtaaccatgaagaaaga

[0230] tattgatgtgtcatatgcaaaagcacttgcaagtgatttagctcggattgttctatttcaacagttcaatgatcataaaaagtggttcgtgaatggagacttgctccctcatgatc

[0231] atccctttaaaagtcatgttaaagaaaatacatggcccacagctgctcaagttcaagattttggagataaatggcatgaacttccgctgattaaatgttttgaaatacccgact

[0232] tactagacccatcgataatatactctgacaaaagtcattcaatgaataggtcagaggtgttgaaacatgtccgaatgaatccgaacactcctatccctagtaaaaaggtgtt

[0233] gcagactatgttggacacaaaggctaccaattggaaagaatttcttaaagagattgatgagaagggcttagatgatgatgatgatctaattattggtcttaaaggaaaggagag

[0234] ggaactgaagttggcaggtagatttttctccctaatgtcttggaaattgcgagaatactttgtaattaccgaatatttgataaagactcatttcgtccctatgtttaaaggcctga

[0235] caatggcggacgatctaactgcagtcattaaaaagatgttagattcctcatccggccaaggattgaagtcatatgaggcaatttgcatagccaatcacattgattacgaaaa

[0236] atggaataaccaccaaaggaagttatcaaacggcccagtgttccgagttatgggccagttcttaggttatccatccttaatcgagagaactcatgaattttttgagaaaagtc

[0237] ttatatactacaatggaagaccagacttgatgcgtgttcacaacaacacactgatcaattcaacctcccaacgagtttgttggcaaggacaagagggtggactggaaggt

[0238] ctacggcaaaaaggatggagtatcctcaatctactggttattcaaagagaggctaaaatcagaaacactgctgtcaaagtcttggcacaaggtgataatcaagttatttgc

[0239] acacagtataaaacgaagaaatcgagaaacgttgtagaattacagggtgctctcaatcaaatggtttctaataatgagaaaattatgactgcaatcaaaagggacagg

[0240] gaagttaggacttttgataaatgacgatgagactatgcaatctgcagattacttgaattatggaaaaataccgattttccgtggagtgattagagggttagagaccaagaga

[0241] tggtcacgagtgacttgtgtcaccaatgaccaaatacccacttgtgctaatataatgagctcagtttccacaaatgctctcaccgtagctcattttgctgagaacccaatcaat

[0242] gccatgatacagtacaattattttgggacatttgctagactcttgttgatgatgcatgatcctgctcttcgtcaatcattgtatgaagttcaagataagataccgggcttgcaca

[0243] gttctactttcaaatacgccatgttgtatttggacccttccattggaggagtgtcgggcatgtctttgtccaggtttttgattagagccttcccagatcccgtaacagaaagtct

[0244] ctcattctggagattcatccatgtacatgctcgaagtgagcatctgaaggagatgagtgcagtatttggaaaccccgagatagccaagtttcgaataactcacatagacaa

[0245] gctagtagaagatccaacctctctgaacatcgctatgggaatgagtccagcgaacttgttaaagactgaggttaaaaaatgcttaatcgaatcaagacaaaccatcagga

[0246] accaggtgattaaggatgcaaccatatatttgtatcatgaagaggatcggctcagaagtttcttatggtcaataaatcctctgttccctagatttttaagtgaattcaaatcagg

[0247] cactttttgggagtcgcagacgggctcatcagtctatttcaaaattctcgtactattcggaactcctttaagaaaaaagtatcatagggaattggatgatttgattgtgaggagt

[0248] gaggtatcctctttgacacatttagggaaacttcatttgagaaggggatcatgtaaaatgtggacatgttcagctactcatgctgacacattaagatacaaatcctggggcc

[0249] gtacagttatgggacaactgtaccccatccattagaaatgttgggtccacaacatcgaaaagagactccttgtgcaccatgtaacacatcagggttcaattatgtttctgtt

[0250] cattgtccagacgggatccatgacgtctttagttcacggggaccattgcctgcttatctagggtctaaaacatctgaatctacattctattttgcagccttgggaaagggaaag

[0251] caaagtcccactgattaaaagagctacacgtcttagagatgctatctcttggtttgttgaacccgactctaaactagcaatgactatactttctaacatccactctttaacaggc

[0252] gaagaatggaccaaaaggcagcatgggttcaaaagaacagggtctgcccttcataggttttcgacatctcggatgagccatggtgggttcgcatctcagagcactgcag

[0253] cattgaccaggttgatggcaactacagacaccatgagggatctgggagatcagaatttcgacttttattccaagcaacgttgctctatgctcaaattaccaccactgttgca

[0254] agagacggatggatcaccagttgtacagatcattatcatattgcctgtaagtcctgtttgagacccatagaagagatcaccctggactcaagtatggactacacgccccca

[0255] gatgtatcccatgtgctgaagacatggaggaatggggaaggttcgtggggacaagagataaaacagatctatcctttagaagggaattggaagaatttagcacctgctg

[0256] agcaatcctatcaagtcggcagatgtataggttttctatatggagacttggcgtatagaaaatctactcatgccgaggacagttctctatttcctctatctatacaaggtcgtat

[0257] tagaggtcgaggtttcttaaaagggttgctagacggattaatgagagcaagttgctgccaagtaatacaccggagaagtctggctcatttgaagaggccggccaacgca

[0258] gtgtacggaggtttgatttacttgattgataaattgagtgtatcacctccattcctttctcttactagatcaggacctattagagacgaattagaaacgattccccacaagatcc

[0259] caacctcctatccgacaagcaaccgtgatatgggggtgattgtcagaaattacttcaaataccaatgccgtctaattgaaaagggaaaatacagatcacattattcacaatt

[0260] atggttattctcagatgtcttatccatagacttcattggaccattctctatttccaccaccctcttgcaaatcctatacaagccatttttatctgggaaagataagaatgagttgag

[0261] agagctggcaaatctttcttcattgctaagatcaggagaggggtgggaagacatacatgtgaaattcttcaccaaggacatattattgtgtccagaggaaatcagacatgc

[0262] ttgcaagttcgggattgctaaggataataataaagacatgagctatcccccttggggaagggaatccagagggacaattacaacaatccctgtttattatacgaccacccc

[0263] ttacccaaagatgctagagatgcctccaagaatccaaaatcccctgctgtccggaatcaggttgggccaattaccaactggcgctcattataaaattcggagtatattacat

[0264] ggaatgggaatccattacagggacttcttgagttgtggagacggctccggagggatgactgctgcattactacgagaaaatgtgcatagcagaggaatattcaatagtct

[0265] gttagaattatcagggtcagtcatgcgaggcgcctctcctgagccccccagtgccctagaaactttaggaggagataaatcgagatgtgtaaatggtgaaacatgttggg

[0266] aatatccatctgacttatgtgacccaaggacttgggactatttcctccgactcaaagcaggcttggggcttcaaattgatttaattgtaatggatatggaagttcgggattcttc

[0267] tactagcctgaaaattgagacgaatgttagaaattatgtgcaccggattttggatgagcaaggagttttaatctacaagacttatggaacatatatttgtgagagcgaaaaga

[0268] atgcagtaacaatccttggtcccatgttcaagacggtcgacttagttcaaacagaatttagtagttctcaaacgtctgaagtatatatggtatgtaaaggtttgaagaaattaat

[0269] cgatgaacccaatcccgattggtcttccatcaatgaatcctggaaaaacctgtacgcattccagtcatcagaacaggaatttgccagagcaaagaaggttagtacatacttt

[0270] accttgacaggtattccctcccaattcattcctgatccttttgtaaacattgagactatgctacaaatattcggagtacccacgggtgtgtctcatgcggctgccttaaaatcat

[0271] ctgatagacctgcagatttattgaccattagccttttttatatggcgattatatcgtattataacatcaatcatatcagagtaggaccgatacctccgaaccccccatcagatgg

[0272] aattgcacaaaatgtggggatcgctataactggtataagcttttggctgagtttgatggagaaagacattccactatatcaacagtgtttggcagttatccagcaatcatttcc

[0273] gattaggtgggaggctatttcagtaaaaggaggatacaagcagaagtggagtactagaggtgatgggctcccaaaagatacccgaatttcagactccttggccccaatc

[0274] gggaactggatcagatctttggaattggtccgaaaccaagttcgtctaaatccattcaataagatcttgttcaatcagctatgtcgtacagtggataatcatttgaagtggtca

[0275] aatttgcgaaaaaacacaggaatgattgaatggatcaatgggcgaatttcaaaagaagaccggtctatactgatgttgaagagtgacctacatgaggaaaactcttggag

[0276] agattaaaaaatcaggaggagactccaaactttaagtatgaaaaaaactttgatccttaagaccctcttgtggtttttatttttttatctggttttgtggtcttcgtgggtcggcat

[0277] ggcatctccacctcctcgcggtccgacctgggcatccgaaggaggacgtcgtccactcggatggctaagggagagctcggatccggctgctaacaaagcccgaaag

[0278] gaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggatc

[0279] gagatcctctagagtcgacctgcaggcatgcaagcttgtattctatagtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgttctaacgac

[0280] aatatgtacaagcctaattgtgtagcatctggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctggt

[0281] aacgccgtcccgcacccggaaatggtcagcgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccggcgccaca

[0282] ggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggcccc

[0283] gtggccgggggactgttgggcgccatctccttgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataa

[0284] gggagagcgtcgaatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgt

[0285] ctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtg

[0286] atacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaa

[0287] atatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatttt

[0288] gccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc

[0289] cttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgcc

[0290] gcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtg

[0291] ataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccg

[0292] gagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttccc

[0293] ggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgt

[0294] gggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacag

[0295] atcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaag

[0296] atcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgt

[0297] aatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagat

[0298] accaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtg

[0299] gcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcga

[0300] acgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcgga

[0301] acaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagg

[0302] ggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataa

[0303] ccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaacc

[0304] gcctctccccgcgcgttggccgattcattaatgcagggggatctcgatcccgcgaaattaatacgactcactatagg

[0305] pT7-VSV-aPDL1 / aCD137(SEQ ID NO.7)

[0306] acgaagacaaacaaaccattattatcattaaaaggctcaggagaaactttaacagtaatcagaattctcgaggccaccatggactggacctggagaatcctgttcctggtg

[0307] gccgccgccaccggggcccacagctatccttacgacgtgcccgactacgccgacattcagatgacacagagccctagctcgctttctgcttccgtgggcgatagagtg

[0308] acaatcacatgcagagcctcccaggacgtgagcaccgccgtggcctggtatcagcaaaagcccggaaaagcccctaagctgctgatctacagcgccagcttcctgta

[0309] ctctggcgtgcctagcaggttcagcggctctggctccggcacagatttcaccctgaccatctctagcctgcaacctgaggatttcgccacatattactgccagcagtacct

[0310] ctaccaccccgccaccttcggccagggcacaaaggtggaaatcaagcggaccgtggcaggcggcggcggaagtggaggcggtggttctggcggcggcggcagc

[0311] gaggtgcagctggtggaaagcggaggaggcctggtgcagcctggaggctctctgagactgagctgtgctgccagcggctttacattctctgatagttggattcactggg

[0312] tgcggcaggcccctggcaaaggcctggaatgggtcgcctggatctccccatacggcggcagcacctactacgccgacagcgtgaagggcagattcaccatcagcg

[0313] ccgataccagcaagaataccgcctacctgcagatgaacagcctgcgggccgaggacacagccgtgtactactgtgcccgcagacactggcctggcggctttgactac

[0314] tggggccagggaaccctggtcaccgtgagctccgccagcacaaaaggaggcggcggcagcgaagtgcagctggtgcagagcggtgctgaggtgaagaagcctg

[0315] gcgagagcctgagaattagctgcaagggcagcggctacagcttcagcacatactggatcagctgggttagacagatgcctggaaagggcctggaatggatgggcaa

[0316] aatctaccccggcgactcctacacaaactacagtccatcatttcagggccaagtgaccatcagcgctgataagagcatcagcactgcctacctgcagtggtccagcctg

[0317] aaggcctcagataccgccatgtactactgcgccagaggatatggcatcttcgactattggggccagggcaccctggtcacagtgtcttctgcctctaccaagggcggcg

[0318] gaggcggcagcggagggcggcggctccggaggggaggctctagctacgagctgacccagccccccagcgtgagcgttagcccgggccagaccgcccttatcac

[0319] ctgcagcggcgaacaatcggcgaccagtacgccactggtatcagcagaaacctggccaaagccctgtgctggtgatctatcagcaagaagaacctagcgga

[0320] atccccgagcggttctccggctctaaatagcggcaacaccgctaccctgaccatctccggaacacaagctatggacgaggccgactactactgcgctacctacaccggc

[0321] ttcggatctctggccgtgttcggcggcggcacaaagctgaccgtgctggggccagcctaaggctcaccatcaccaccaccatcactgatggccatatgaaaaaactaa

[0322] footatcaaaaatgtctgttacagtcaagagaatcattgacaacacagtcatagttccaaaacttcctgcaaatgaggatccagtggaatacccggcagattacttcagaa

[0323] aatcaaaggagattcctctttacatcaatactacaaaaagtttgtcagatctaagaggatatgtctaccaaggcctcaaatccggaaatgtatcaatcatacatgtcaacagc

[0324] tacttgttggagcattaaaaggacatccggggtaagttggataaagattggtcaagtttcgggaataaacatcgggaaagcaggggatacaatcggaatatttgaccttgta

[0325] tccttgaaagccctggacggcgtacttccagatggagtatcggatgcttccagaaccagcgcagatgacaaatggttgcctttgtatctacttggcttatacagagtgggc

[0326] agaacacaaatgcctgaatacagaaaaaagctcatggatgggctgacaaatcaatgcaaaatgatcaatgaacagtttgaacctcttgtgccagaaggtcgtgacattttt

[0327] gatgtgtggggaaatgacagtaattacacaaaaattgtcgctgcagtggacatgttcttccacatgttcaaaaaacatgaatgtgcctcgttcagatacggaactattgtttc

[0328] cagattcaaagattgtgctgcattggcaacatttggacacctctgcaaaataaccggaatgtctacagaagatgtaacgacctggatcttgaaccgagaagttgcagatg

[0329] aaatggtccaaatgatgcttccaggccaagaaattgacaaggccgattcatacatgccttatttgatcgactttggattgtcttctaagtctccatattcttccgtcaaaaaccc

[0330] tgccttccacttctgggggcaattgacagctcttctgctcagatccaccagagcaaggaatgcccgacagcctgatgacattgagtatacatctcttactacagcaggtttg

[0331] ttgtacgcttatgcagtaggatcctctgccgacttggcacaacagttttgtgttggagataacaaatacactccagatgatagtaccggaggattgacgactaatgcaccg

[0332] ccacaaggcagagatgtggtcgaatggctcggatggtttgaagatcaaaacagaaaaccgactcctgatatgatgcagtatgcgaaaagagcagtcatgtcactgcaa

[0333] ggcctaagagagaagacaattggcaagtatgctaagtcagaatttgacaaatgaccctataattctcagatcacctattatatattatgctacatatgaaaaaaactaacaga

[0334] tatcatggataatctcacaaaagttcgtgagtatctcaagtcctactctcgtctagatcaggcggtaggagagatagatgagatcgaagcacaacgagctgaaaagtcca

[0335] attatgagttgttccaagaggacggagtggaagagcatactaggccctcttattttcaggcagcagatgattctgacacagaatctgaaccagaaattgaagacaatcaa

[0336] ggcttgtatgtaccagatccggaagctgagcaagttgaaggctttatacaggggcctttagatgactatgcagatgaggacgtggatgttgtattcacttcggactggaaa

[0337] cagcctgagcttgaatccgacgagcatggaaagaccttacggttgacattgccagagggtttaagtggagagcagaaatcccagtggcttttgacgattaaagcagtcg

[0338] ttcaaagtgccaaacactggaatctggcagagtgcacatttgaagcatcgggagaaggggtcatcataaaaaagcgccagataactccggatgtatataaggtcactcc

[0339] agtgatgaacacacatccgtaccaatcagaagccgtatcagatgtttggtctctctcaaagacatccatgactttccaacccaagaaagcaagtcttcagcctctcaccata

[0340] tccttggatgaattgttctcatctagaggagaattcatctctgtcggaggtaacggacgaatgtctcataaagaggccatcctgctcggtctgaggtacaaaaagttgtaca

[0341] atcaggcgagagtcaaatattctctgtagactatgaaaaaaagtaacagatatcacaatctaagtgttatcccaatccattcatcatgagttccttaaagaagattctcggtct

[0342] gaaggggaaaggtaagaaatctaagaaattagggatcgcaccacccccttatgaagaggacactagcatggagtatgctccgagcgctccaattgacaaatcctatttt

[0343] ggagttgacgagatggacacctatgatccgaatcaattaagatatgagaaattcttctttacagtgaaaatgacggttagatctaatcgtccgttcagaacatactcagatgt

[0344] ggcagccgctgtatcccattgggatcacatgtacatcggaatggcagggaaacgtcccttctacaaaatcttggcttttttgggttcttctaatctaaaggccactccagcg

[0345] gtattggcagatcaaggtcaaccagagtatcacgctcactgcgaaggcagggcttatttgccacataggatggggaagacccctcccatgctcaatgtaccagagcact

[0346] tcagaagaccattcaatataggtctttacaagggaacgattgagctcacaatgaccatctacgatgatgagtcactggaagcagctcctatgatctgggatcatttcaattct

[0347] tccaaattttctgatttcagagagaaggccttaatgtttggcctgattgtcgagaaaaaggcatctggagcgtgggtcctggattctatcagccacttcaaatgagctagtct

[0348] agcttccagcttctgaacaatccccggtttactcagtctctcctaattccagcctttcgaacaactaatatcctcttttctatccctatgaaaaaaactaacagagatcgatct

[0349] gtttccttgacacgcgttacgatgcggccgatgaagtgcctttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacaacaccaaaaaggaa

[0350] actggaaaaatgttccttctaattaccattatgcccgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaag

[0351] gctattcaagcagacggttggatgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccgatccttcactccatct

[0352] gtagaacaatgcaaggaaagcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcctcaagttgtggatatgcaactgtgacggatgccgaagca

[0353] gtgattgtccaggtgactcctcaccatgtgctggttgaatgaatacacaggagaatgggttgattcacagttcatcaacggagaaaatgcagcaattacatatgccccactgtcc

[0354] ataactctacaacctggcattctgactataaggtcaaagggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctggga

[0355] aagggcacagggttcagaagtaactactttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtct

[0356] ggttcgagatgggctgataaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgtaagtctaattca

[0357] ggacgttgagaggatcttggattatccctctgccaagaaaacctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaaaccc

[0358] aggaaccggtcctgctttcaccataatcaatggtaccctaaaatactttgagaccagatacatcagagtcgatatgctgctccaatcctctcaagaatggtcggaatgatc

[0359] agtggaactaccacagaaagggaactgtgggatgactgggcaccatatgaagcgtgggaaattggacccaatgggagttctgaggaccagttcaggatataaggtttcctt

[0360] tatacatgattggacatggtatgttggactccgatcttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgaga

[0361] gtttattttttggtgatactgggctatccaaaaatccaatcgagcttgtagaaggttggttcagtagttggaaaagctctctattgcctcttttttctttatcatagggttaatcattgg

[0362] actattcttggttctccgagttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaagtaagcggcc

[0363] gccctgcacacagattcttcatgtttgaaccaaatcaacttgtgatatcatgctcaaagaggccttaattatattttaatttttaatttttatgaaaaaaactaacagcaatcatgg

[0364] aagtccacgattttgagaccgacgagttcaatgatttcaatgaagatgactatgccacaagagaattcctgaatcccgatgagcgcatgacgtacttgaatcatgctgatta

[0365] caatttgaattctcctctaattagtgatgatattgacaatttgatcaggaaattcaattctcttccgattccctcgatgtgggatagtaagaactgggatggagttcttgagatgtt

[0366] aacatcatgtcaagccaatcccatctcaacatctcagatgcataaatggatgggaagttggttaatgtctgataatcatgatgccagtcaagggtatagttttttacatgaagt

[0367] ggaaagaggcagaaataatttgacgtggtggagaccttcatccgcggctggggcaacaaaccaattgaatacatcaaaaaaggaaagatggactgactcattcaa

[0368] aattctcgcttatttgtgtcaaaagtttttggacttacacaagttgacattaatcttaaatgctgtctctgaggtggaattgctcaacttggcgaggactttcaaaggcaaagtca

[0369] gaagaagttctcatggaacgaacatatgcaggattaggttcccagcttgggtcctacttttattcagaaggatgggctacttcaagaaacttgatattctaatggaccga

[0370] aactttctgttaatggtcaaagatgtgattatagggaggatgcaaacggtgctatccatggtatgtagaatagacaacctgttctcagagcaagacatcttctcccttctaaat

[0371] atctacagaattggagataaaattgtggagaggcagggaaatttttcttatgacttgattaaaatggtggaaccgatatgcaacttgaagctgatgaaattagcaagagaat

[0372] caaggcctttagtcccacaattccctcattttgaaaatcatatcaagacttctgttgatgaaggggcaaaaattgaccgaggtataagattcctccatgatcagataatgagt

[0373] gtgaaaacagtggatctcacactggtgatttatggatcgttcagacattggggtcatccttttatagattattacactggactagaaaaattacattcccaagtaaccatgaag

[0374] aaagatattgatgtgtcatatgcaaaagcacttgcaagtgatttagctcggattgttctatttcaacagttcaatgatcataaaaagtggttcgtgaatggagacttgctccctc

[0375] atgatcatccctttaaaagtcatgttaaagaaaatacatggcccacagctgctcaagttcaagattttggagataaatggcatgaacttccgctgattaaatgttttgaaatac

[0376] ccgacttactagacccatcgataatatactctgacaaaagtcattcaatgaataggtcagaggtgttgaaacatgtccgaatgaatccgaacactcctatccctagtaaaaa

[0377] ggtgttgcagactatgttggacacaaaggctaccaattggaaagaatttcttaaagagattgatgagaagggcttagatgatgatgatctaattattggtcttaaaggaaag

[0378] gagagggaactgaagttggcaggtagatttttctccctaatgtcttggaaattgcgagaatactttgtaattaccgaatatttgataaagactcatttcgtccctatgtttaaag

[0379] gcctgacaatggcggacgatctaactgcagtcattaaaaagatgttagattcctcatccggccaaggattgaagtcatatgaggcaatttgcatagccaatcacattgatta

[0380] cgaaaaatggaataaccaccaaaggaagttatcaaacggcccagtgttccgagttatgggccagttcttaggttatccatccttaatcgagagaactcatgaattttttgag

[0381] aaaagtcttatatactacaatggaagaccagacttgatgcgtgttcacaacaacacactgatcaattcaacctcccaacgagtttgttggcaaggacaagagggtggact

[0382] ggaaggtctacggcaaaaaggatggagtatcctcaatctactggttattcaaagagaggctaaaatcagaaacactgctgtcaaagtcttggcacaaggtgataatcaag

[0383] ttatttgcacacagtataaaacgaagaaatcgagaaacgttgtagaattacagggtgctctcaatcaaatggtttctaataatgagaaaattatgactgcaatcaaaatagg

[0384] gacagggaagttaggacttttgataaatgacgatgagactatgcaatctgcagattacttgaattatggaaaaataccgattttccgtggagtgattagagggttagagacc

[0385] aagagatggtcacgagtgacttgtgtcaccaatgaccaaatacccacttgtgctaatataatgagctcagtttccacaaatgctctcaccgtagctcattttgctgagaaccc

[0386] aatcaatgccatgatacagtacaattattttgggacatttgctagactcttgttgatgatgcatgatcctgctcttcgtcaatcattgtatgaagttcaagataagataccgggct

[0387] tgcacagttctactttcaaatacgccatgttgtatttggacccttccattggaggagtgtcgggcatgtctttgtccaggtttttgattagagccttcccagatcccgtaacaga

[0388] aagtctctcattctggagattcatccatgtacatgctcgaagtgagcatctgaaggagatgagtgcagtatttggaaaccccgagatagccaagtttcgaataactcacata

[0389] gacaagctagtagaagatccaacctctctgaacatcgctatgggaatgagtccagcgaacttgttaaagactgaggttaaaaaatgcttaatcgaatcaagacaaaccat

[0390] caggaaccaggtgattaaggatgcaaccatatatttgtatcatgaagaggatcggctcagaagtttcttatggtcaataaatcctctgttccctagatttttaagtgaattcaaa

[0391] tcaggcacttttttgggagtcgcagacgggctcatcagtctatttcaaaattctcgtactattcggaactcctttaagaaaaagtatcatagggaattggatgatttgattgtga

[0392] ggagtgaggtatcctctttgacacatttagggaaacttcatttgagaaggggatcatgtaaaatgtggacatgttcagctactcatgctgacacattaagatacaaatcctgg

[0393] ggccgtacagttattgggacaactgtaccccatccattagaaatgttgggtccacaacatcgaaaagagactccttgtgcaccatgtaacacatcagggttcaattatgttt

[0394] ctgtgcattgtccagacgggatccatgacgtctttagttcacggggaccattgcctgcttatctagggtctaaaacatctgaatctacatctattttgcagccttgggaaagg

[0395] gaaagcaaagtcccactgattaaaagagctacacgtcttagagatgctatctcttggtttgttgaacccgactctaaactagcaatgactatactttctaacatccactctttaa

[0396] caggcgaagaatggaccaaaaggcagcatgggttcaaaagaacagggtctgcccttcataggttttcgacatctcggatgagccatggtgggttcgcatctcagagca

[0397] ctgcagcattgaccaggttgatggcaactacagacaccatgagggatctgggagatcagaatttcgactttttattccaagcaacgttgctctatgctcaaattaccaccac

[0398] tgttgcaagagacggatggatcaccagttgtacagatcattatcatattgcctgtaagtcctgtttgagacccatagaagagatcaccctggactcaagtatggactacacg

[0399] cccccagatgtatcccatgtgctgaagacatggaggaatggggaaggttcgtggggacaagagataaaacagatctatcctttagaagggaattggaagaatttagcac

[0400] ctgctgagcaatcctatcaagtcggcagatgtataggttttctatatggagacttggcgtatagaaaatctactcatgccgaggacagttctctatttcctctatctatacaag

[0401] gtcgtattagaggtcgaggtttcttaaaagggttgctagacggattaatgagagcaagttgctgccaagtaatacaccggagaagtctggctcatttgaagaggccggcc

[0402] aacgcagtgtacggaggtttgatttacttgattgataaattgagtgtatcacctccattcctttctcttactagatcaggacctattagagacgaattagaaacgattccccaca

[0403] agatcccaacctcctatccgacaagcaaccgtgatatgggggtgattgtcagaaattacttcaaataccaatgccgtctaattgaaaagggaaaatacagatcacattattc

[0404] acaattatggttattctcagatgtcttatccatagacttcattggaccattctctatttccaccaccctcttgcaaatcctatacaagccatttttatctgggaaagataagaatga

[0405] gttgagagagctggcaaatctttcttcattgctaagatcaggagaggggtgggaagacatacatgtgaaattcttcaccaaggacatattattgtgtccagaggaaatcag

[0406] acatgcttgcaagttcgggattgctaaggataataataaagacatgagctatcccccttggggaagggaatccagagggacaattacaacaatccctgtttattatacgac

[0407] caccccttacccaaagatgctagagatgcctccaagaatccaaaatcccctgctgtccggaatcaggttgggccaattaccaactggcgctcattataaaattcggagtat

[0408] attacatggaatgggaatccattacagggacttcttgagttgtggagacggctccggagggatgactgctgcattactacgagaaaatgtgcatagcagaggaatattca

[0409] atagtctgttagaattatcagggtcagtcatgcgaggcgcctctcctgagccccccagtgccctagaaactttaggaggataaatcgagatgtgtaaatggtgaaaca

[0410] tgttgggaatatccatctgacttatgtgacccaaggacttgggactatttcctccgactcaaagcaggcttggggcttcaaattgatttaattgtaatggatatggaagttcgg

[0411] gattcttctactagcctgaaaattgagacgaatgttagaaattatgtgcaccggattttggatgagcaaggagttttaatctacaagacttatggaacatatatttgtgagagc

[0412] gaaaagaatgcagtaacaatccttggtcccatgttcaagacggtcgacttagttcaaacagaatttagtagttctcaaacgtctgaagtatatatggtatgtaaaggtttgaa

[0413] gaaattaatcgatgaacccaatcccgattggtcttccatcaatgaatcctggaaaaacctgtacgcattccagtcatcagaacaggaatttgccagagcaaagaaggttag

[0414] tacatactttaccttgacaggtattccctcccaattcattcctgatccttttgtaaacattgagactatgctacaaatattcggagtacccacgggtgtgtctcatgcggctgcct

[0415] taaaatcatctgatagacctgcagatttattgaccattagccttttttatggcgattatatcgtattataacatcaatcatatcagagtaggaccgatacctccgaacccccca

[0416] tcagatggaattgcacaaaatgtggggatcgctataactggtataagcttttggctgagtttgatggagaaagacattccactatatcaacagtgtttggcagttatccagca

[0417] atcatttccgattaggtgggaggctatttcagtaaaaggaggatacaagcagaagtggagtactagaggtgatgggctcccaaaagatacccgaatttcagactccttgg

[0418] ccccaatcgggaactggatcagatctttggaattggtccgaaaccaagttcgtctaaatccattcaataagatcttgttcaatcagctatgtcgtacagtggataatcatttga

[0419] agtggtcaaatttgcgaaaaaacacaggaatgattgaatggatcaatgggcgaatttcaaaagaagaccggtctatactgatgttgaagagtgacctacatgaggaaaa

[0420] ctcttggagagattaaaaaatcaggaggagactccaaactttaagtatgaaaaaaactttgatccttaagaccctcttgtggtttttatttttttatctggttttgtggtcttcgtgg

[0421] gtcggcatggcatctccacctcctcgcggtccgacctgggcatccgaaggaggacgtcgtccactcggatggctaagggagagctcggatccggctgctaacaaag

[0422] cccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactat

[0423] atccggatcgagatcctctagagtcgacctgcaggcatgcaagcttgtattctatagtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgtt

[0424] ctaacgacaatatgtacaagcctaattgtgtagcatctggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctga

[0425] gtttctggtaacgccgtcccgcacccggaaatggtcagcgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccgg

[0426] cgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggc

[0427] aggccccgtggccgggggactgttgggcgccatctccttgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagt

[0428] cgcataagggagagcgtcgaatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacg

[0429] ggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaaggg

[0430] cctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaata

[0431] cattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgc

[0432] ggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcgg

[0433] taagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactc

[0434] ggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataacc

[0435] atgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttg

[0436] ggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactct

[0437] agcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccg

[0438] gtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaa

[0439] atagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatct

[0440] aggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttt

[0441] ctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga

[0442] gcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctg

[0443] ctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagc

[0444] ttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggc

[0445] agggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatg

[0446] ctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgatt

[0447] ctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaat

[0448] acgcaaaccgcctctccccgcgcgttggccgattcattaatgcagggggatctcgatcccgcgaaattaatacgactcactatagg

[0449] Nucleic acid sequence of pT7-VSV-aPDL1 (SEQ ID NO.8)

[0450] acgaagacaaacaaaccattattatcattaaaaggctcaggagaaactttaacagtaatcagaattctcgaggccaccatggactggacctggagaatcctgttcctggtg

[0451] gccgccgccaccggggcccacagctatccttacgacgtgcccgactacgccgacattcagatgacacagagccctagctcgctttctgcttccgtgggcgatagagtg

[0452] acaatcacatgcagagcctcccaggacgtgagcaccgccgtggcctggtatcagcaaaagcccggaaaagcccctaagctgctgatctacagcgccagcttcctgta

[0453] ctctggcgtgcctagcaggttcagcggctctggctccggcacagatttcaccctgaccatctctagcctgcaacctgaggatttcgccacatattactgccagcagtacct

[0454] ctaccaccccgccaccttcggccagggcacaaaggtggaaatcaagcggaccgtggcaggcggcggcggaagtggaggcggtggttctggcggcggcggcagc

[0455] gaggtgcagctggtggaaagcggaggaggcctggtgcagcctggaggctctctgagactgagctgtgctgccagcggctttacattctctgatagttggattcactggg

[0456] tgcggcaggcccctggcaaaggcctggaatgggtcgcctggatctccccatacggcggcagcacctactacgccgacagcgtgaagggcagattcaccatcagcg

[0457] ccgataccagcaagaataccgcctacctgcagatgaacagcctgcgggccgaggacacagccgtgtactactgtgcccgcagacactggcctggcggctttgactac

[0458] tggggccagggaaccctggtcaccgtgagctccgccagcacaaaacatcaccaccaccatcactgatggccatatgaaaaaaactaacagtaatcaaaatgtctgttac

[0459] agtcaagagaatcattgacaacacagtcatagttccaaaacttcctgcaaatgaggatccagtggaatacccggcagattacttcagaaaatcaaaggagattcctcttta

[0460] catcaatactacaaaaagtttgtcagatctaagaggatatgtctaccaaggcctcaaatccggaaatgtatcaatcatacatgtcaacagctacttgtatggagcattaaag

[0461] gacatccggggtaagttggataaagattggtcaagtttcggaataaacatcgggaaagcaggggatacaatcggaatatttgaccttgtatccttgaaagccctggacgg

[0462] cgtacttccagatggagtatcggatgcttccagaaccagcgcagatgacaaatggttgcctttgtatctacttggcttatacagagtgggcagaacacaaatgcctgaata

[0463] cagaaaaaagctcatggatgggctgacaaatcaatgcaaaatgatcaatgaacagtttgaacctcttgtgccagaaggtcgtgacatttttgatgtgtggggaaatgaca

[0464] gtaattacacaaaaattgtcgctgcagtggacatgttcttccacatgttcaaaaaacatgaatgtgcctcgttcagatacggaactattgtttccagattcaaagattgtgctg

[0465] cattggcaacatttggacacctctgcaaaataaccggaatgtctacagaagatgtaacgacctggatcttgaaccgagaagttgcagatgaaatggtccaaatgatgcttc

[0466] caggccaagaaattgacaaggccgattcatacatgccttatttgatcgactttggattgtcttctaagtctccatattcttccgtcaaaaaccctgccttccacttctgggggc

[0467] aattgacagctcttctgctcagatccaccagagcaaggaatgcccgacagcctgatgacattgagtatacatctcttactacagcaggtttgttgtacgcttatgcagtagg

[0468] atcctctgccgacttggcacaacagttttgtgttggagataacaaatacactccagatgatagtaccggaggattgacgactaatgcaccgccacaaggcagagatgtg

[0469] gtcgaatggctcggatggtttgaagatcaaaacagaaaaccgactcctgatatgatgcagtatgcgaaaagagcagtcatgtcactgcaaggcctaagagagaagaca

[0470] attggcaagtatgctaagtcagaatttgacaaatgaccctataattctcagatcacctattatatattatgctacatatgaaaaaaactaacagatatcatggataatctcacaa

[0471] aagttcgtgagtatctcaagtcctactctcgtctagatcaggcggtaggagagatagatgagatcgaagcacaacgagctgaaaagtccaattatgagttgttccaagag

[0472] gacggagtggaagagcatactaggccctcttattttcaggcagcagatgattctgacacagaatctgaaccagaaattgaagacaatcaaggcttgtatgtaccagatcc

[0473] ggaagctgagcaagttgaaggctttatacaggggcctttagatgactatgcagatgaggacgtggatgttgtattcacttcggactggaaacagcctgagcttgaatccg

[0474] acgagcatggaaagaccttacggttgacattgccagagggtttaagtggagagcagaaatcccagtggcttttgacgattaaagcagtcgttcaaagtgccaaacactg

[0475] gaatctggcagagtgcacatttgaagcatcgggagaaggggtcatcataaaaaagcgccagataactccggatgtatataaggtcactccagtgatgaacacacatcc

[0476] gtaccaatcagaagccgtatcagatgtttggtctctctcaaagacatccatgactttccaacccaagaaagcaagtcttcagcctctcaccatatccttggatgaattgttctc

[0477] atctagaggagaattcatctctgtcggaggtaacggacgaatgtctcataaagaggccatcctgctcggtctgaggtacaaaaagttgtacaatcaggcgagagtcaaat

[0478] attctctgtagactatgaaaaaaagtaacagatatcacaatctaagtgttatcccaatccattcatcatgagttccttaaagaagattctcggtctgaaggggaaaggtaaga

[0479] aatctaagaaattagggatcgcaccacccccttatgaagaggacactagcatggagtatgctccgagcgctccaattgacaaatcctattttggagttgacgagatggac

[0480] acctatgatccgaatcaattaagatatgagaaattcttctttacagtgaaaatgacggttagatctaatcgtccgttcagaacatactcagatgtggcagccgctgtatcccat

[0481] tgggatcacatgtacatcggaatggcagggaaacgtcccttctacaaaatcttggcttttttgggttcttctaatctaaaggccactccagcggtattggcagatcaaggtca

[0482] accagagtatcacgctcactgcgaaggcagggcttatttgccacataggatggggaagacccctcccatgctcaatgtaccagagcacttcagaagaccattcaatata

[0483] ggtctttacaagggaacgattgagctcacaatgaccatctacgatgatgagtcactggaagcagctcctatgatctgggatcatttcaattcttccaaattttctgatttcaga

[0484] gagaaggccttaatgtttggcctgattgtcgagaaaaaggcatctggagcgtgggtcctggattctatcagccacttcaaatgagctagtctagcttccagcttctgaacaa

[0485] tccccggtttactcagtctctcctaattccagcctttcgaacaactaatatcctgtcttttctatccctatgaaaaaaactaacagagatcgatctgtttccttgacacgcgttac

[0486] gatgcggccgatgaagtgccttttgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacacaaccaaaaaggaaactggaaaaatgttccttct

[0487] aattaccattattgcccgtcaagctcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaaggctattcaagcagacggtt

[0488] ggatgtgtcatgcttccaaatgggtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccgatccttcactccatctgtagaacaatgcaaggaa

[0489] agcattgaacaaacgaaacaaggaacttggctgaatccaggcttccctcctcaaagttgtggatatgcaactgtgacggatgccgaagcagtgattgtccaggtgactc

[0490] ctcaccatgtgctggttgatgaatacacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccataactctacaacctggc

[0491] attctgactataaggtcaaagggctatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaaggagggcacagggt

[0492] tcagaagtaactactttgcttatgaaactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtctggttcgagatggctgat

[0493] aaggatctctttgctgcagccagattccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgtaagtctaattcaggacgttgagaggatct

[0494] tggattattccctctgccaagaaacctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaacccaggaaccggtcctgcttt

[0495] caccataatcaatggtaccctaaaatactttgagaccagatacatcagagtcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtggaactaccacaga

[0496] aagggaactgtgggatgactgggcaccatatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatacatgattggacatg

[0497] gtatgttggactccgatcttcatcttagctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagtttattttttggtgatact

[0498] gggctatccaaaaatccaatcgagcttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatagggttaatcattggactattcttggttctccga

[0499] gttggtatccatctttgcattaaattaaagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaagtaagcggccgccctgcacacagattct

[0500] tcatgtttgaaccaaatcaacttgtgatatcatgctcaaagaggccttaattatattttaatttttaatttttatgaaaaaaactaacagcaatcatggaagtccacgattttgaga

[0501] ccgacgagttcaatgatttcaatgaagatgactatgccacaagagaattcctgaatcccgatgagcgcatgacgtacttgaatcatgctgattacaatttgaattctcctcta

[0502] attagtgatgatattgacaatttgatcaggaaattcaattctcttccgattccctcgatgtgggatagtaagaactgggatggagttcttgagatgttaacatcatgtcaagcca

[0503] atcccatctcaacatctcagatgcataaatggatgggaagttggttaatgtctgataatcatgatgccagtcaagggtatagttttttacatgaagtggacaaagaggcaga

[0504] aataacatttgacgtggtggagaccttcatccgcggctggggcaacaaaccaattgaatacatcaaaaaggaaagatggactgactcattcaaaattctcgcttatttgtgt

[0505] caaaagtttttggacttacacaagttgacattaatcttaaatgctgtctctgaggtggaattgctcaacttggcgaggactttcaaaggcaaagtcagaagaagttctcatgg

[0506] aacgaacatatgcaggattagggttcccagcttgggtcctacttttatttcagaaggatgggcttacttcaagaaacttgatattctaatggaccgaaactttctgttaatggtc

[0507] aaagatgtgattatagggaggatgcaaacggtgctatccatggtatgtagaatagacaacctgttctcagagcaagacatcttctcccttctaaatatctacagaattggag

[0508] ataaaattgtggagaggcagggaaatttttcttatgacttgattaaaatggtggaaccgatatgcaacttgaagctgatgaaattagcaagagaatcaaggcctttagtccc

[0509] acaattccctcattttgaaaatcatatcaagacttctgttgatgaaggggcaaaaattgaccgaggtataagattcctccatgatcagataatgagtgtgaaaacagtggatc

[0510] tcacactggtgatttatggatcgttcagacattggggtcatccttttatagattattacactggactagaaaaattacattcccaagtaaccatgaagaaagatattgatgtgtc

[0511] atatgcaaaagcacttgcaagtgatttagctcggattgttctatttcaacagttcaatgatcataaaaagtggttcgtgaatggagacttgctccctcatgatcatccctttaaa

[0512] agtcatgttaaagaaaatacatggcccacagctgctcaagttcaagattttggagataaatggcatgaacttccgctgattaaatgttttgaaatacccgacttactagaccc

[0513] atcgataatatactctgacaaaagtcattcaatgaataggtcagaggtgttgaaacatgtccgaatgaatccgaacactcctatccctagtaaaaaggtgttgcagactatg

[0514] ttggacacaaaggctaccaattggaaagaatttcttaaagagattgatgagaagggcttagatgatgatgatctaattattggtcttaaaggaaaggagagggaactgaag

[0515] ttggcaggtagatttttctccctaatgtcttggaaattgcgagaatactttgtaattaccgaatatttgataaagactcatttcgtccctatgtttaaaggcctgacaatggcgga

[0516] cgatctaactgcagtcattaaaaagatgttagattcctcatccggccaaggattgaagtcatatgaggcaatttgcatagccaatcacattgattacgaaaaatggaataac

[0517] caccaaaggaagttatcaaacggcccagtgttccgagttatgggccagttcttaggttatccatccttaatcgagagaactcatgaattttttgagaaaagtcttatatactac

[0518] aatggaagaccagacttgatgcgtgttcacaacaacacactgatcaattcaacctcccaacgagtttgttggcaaggacaagagggtggactggaaggtctacggcaa

[0519] aaaggatggagtatcctcaatctactggttattcaaagagaggctaaaatcagaaacactgctgtcaaagtcttggcacaaggtgataatcaagttatttgcacacagtata

[0520] aaacgaagaaatcgagaaacgttgtaagattacagggtgctctcaatcaaatggtttctaataatgagaaaattatgactgcaatcaaaagggacagggaagttagga

[0521] ctttgataaatgacgatgagactatgcaatctgcagattacttgaattatggaaaaataccgattttccgtggagtgattagagggttagagaccaagagatggtcacgag

[0522] tgacttgtgtcaccaatgaccaaatacccacttgtgctaatataatgagctcagtttccacaaatgctctcaccgtagctcattttgctgagaacccaatcaatgccatgatac

[0523] agtacaattattttgggacatttgctagactcttgttgatgatgcatgatcctgctcttcgtcaatcattgtatgaagttcaagataagataccgggcttgcacagttctactttca

[0524] aatacgccatgttgtatttggacccttccattggaggagtgtcgggcatgtctttgtccaggtttttgattagagccttcccagatcccgtaacagaaagtctctcattctgga

[0525] gattcatccatgtacatgctcgaagtgagcatctgaaggagatgagtgcagtatttggaaaccccgagatagccaagtttcgaataactcacatagacaagctagtagaa

[0526] gatccaacctctctgaacatcgctatgggaatgagtccagcgaacttgttaaagactgaggttaaaaaatgcttaatcgaatcaagacaaaccatcaggaaccaggtgatt

[0527] aaggatgcaaccatatatttgtatcatgaagaggatcggctcagaagtttcttatggtcaataaatcctctgttccctagatttttaagtgaattcaaatcaggcacttttttggg

[0528] agtcgcagacgggctcatcagtctatttcaaaattctcgtactattcggaactcctttaagaaaaagtatcatagggaattggatgatttgattgtgaggagtgaggtatcctc

[0529] tttgacacatttagggaaacttcatttgagaaggggatcatgtaaaatgtggacatgttcagctactcatgctgacacattaagatacaaatcctggggccgtacagttattg

[0530] ggacaactgtaccccatccattagaaatgttgggtccacaacatcgaaaagagactccttgtgcaccatgtaacacatcagggttcaattatgtttctgtgcattgtccaga

[0531] cgggatccatgacgtctttagttcacggggaccattgcctgcttatctagggtctaaaacatctgaatctacatctattttgcagccttgggaaagggaaagcaaagtccca

[0532] ctgattaaaagagctacacgtcttagagatgctatctcttggtttgttgaacccgactctaaactagcaatgactatactttctaacatccactctttaacaggcgaagaatgg

[0533] accaaaaggcagcatgggttcaaaagaacagggtctgcccttcataggttttcgacatctcggatgagccatggtgggttcgcatctcagagcactgcagcattgacca

[0534] ggttgatggcaactacagacaccatgagggatctgggagatcagaatttcgactttttattccaagcaacgttgctctatgctcaaattaccaccactgttgcaagagacgg

[0535] atggatcaccagttgtacagatcattatcatattgcctgtaagtcctgtttgagacccatagaagagatcaccctggactcaagtatggactacacgcccccagatgtatcc

[0536] catgtgctgaagacatggaggaatggggaaggttcgtggggacaagagataaaacagatctatcctttagaagggaattggaagaatttagcacctgctgagcaatcct

[0537] atcaagtcggcagatgtataggttttctatatggagacttggcgtatagaaaatctactcatgccgaggacagttctctatttcctctatctatacaaggtcgtattagaggtcg

[0538] aggtttcttaaaagggttgctagacggattaatgagagcaagttgctgccaagtaatacaccggagaagtctggctcatttgaagaggccggccaacgcagtgtacgga

[0539] ggtttgatttacttgattgataaattgagtgtatcacctccattcctttctcttactagatcaggacctattagagacgaattagaaacgattccccacaagatcccaacctccta

[0540] tccgacaagcaaccgtgatatgggggtgattgtcagaaattacttcaaataccaatgccgtctaattgaaaagggaaaatacagatcacattattcacaattatggttattct

[0541] cagatgtcttatccatagacttcattggaccattctctatttccaccaccctcttgcaaatcctatacaagccatttttatctgggaaagataagaatgagttgagagagctggc

[0542] aaatctttcttcattgctaagatcaggagaggggtgggaagacatacatgtgaaattcttcaccaaggacatattattgtgtccagaggaaatcagacatgcttgcaagttc

[0543] gggattgctaaggataataataaagacatgagctatcccccttggggaagggaatccagagggacaattacaacaatccctgtttattatacgaccaccccttacccaaa

[0544] gatgctagagatgcctccaagaatccaaaatcccctgctgtccggaatcaggttgggccaattaccaactggcgctcattataaaattcggagtatattacatggaatggg

[0545] aatccattacagggacttcttgagttgtggagacggctccggagggatgactgctgcattactacgagaaaatgtgcatagcagaggaatattcaatagtctgttagaatt

[0546] atcagggtcagtcatgcgaggcgcctctcctgagccccccagtgccctagaaactttaggaggagataaatcgagatgtgtaaatggtgaaacatgttgggaatatccat

[0547] ctgacttatgtgacccaaggacttgggactatttcctccgactcaaagcaggcttggggcttcaaattgatttaattgtaatggatatggaagttcgggattcttctactagcc

[0548] tgaaaattgagacgaatgttagaaattatgtgcaccggattttggatgagcaaggagttttaatctacaagacttatggaacatatatttgtgagagcgaaaagaatgcagt

[0549] aacaatccttggtcccatgttcaagacggtcgacttagttcaaacagaatttagtagttctcaaacgtctgaagtatatatggtatgtaaaggtttgaagaaattaatcgatga

[0550] acccaatcccgattggtcttccatcaatgaatcctggaaaaacctgtacgcattccagtcatcagaacaggaatttgccagagcaaagaaggttagtacatactttaccttg

[0551] acaggtattccctcccaattcattcctgatccttttgtaaacattgagactatgctacaaatattcggagtacccacgggtgtgtctcatgcggctgccttaaaatcatctgata

[0552] gacctgcagatttattgaccattagccttttttatatggcgattatatcgtattataacatcaatcatatcagagtaggaccgatacctccgaaccccccatcagatggaattgc

[0553] acaaaatgtggggatcgctataactggtataagcttttggctgagtttgatggagaaagacattccactatatcaacagtgtttggcagttatccagcaatcatttccgattag

[0554] gtgggaggctatttcagtaaaaggaggatacaagcagaagtggagtactagaggtgatgggctcccaaaagatacccgaatttcagactccttggccccaatcgggaa

[0555] ctggatcagatctttggaattggtccgaaaccaagttcgtctaaatccattcaataagatcttgttcaatcagctatgtcgtacagtggataatcatttgaagtggtcaaatttg

[0556] cgaaaaaacacaggaatgattgaatggatcaatgggcgaatttcaaaagaagaccggtctatactgatgttgaagagtgacctacatgaggaaaactcttggagagatt

[0557] aaaaaatcaggaggagactccaaactttaagtatgaaaaaaactttgatccttaagaccctcttgtggtttttatttttttatctggttttgtggtcttcgtgggtcggcatggcat

[0558] ctccacctcctcgcggtccgacctgggcatccgaaggaggacgtcgtccactcggatggctaagggagagctcggatccggctgctaacaaagcccgaaaggaag

[0559] ctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggatcgaga

[0560] tcctctagagtcgacctgcaggcatgcaagcttgtattctatagtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgttctaacgacaatat

[0561] gtacaagcctaattgtgtagcatctggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctggtaacg

[0562] ccgtcccgcacccggaaatggtcagcgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgc

[0563] ggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggc

[0564] cgggggactgttgggcgccatctccttgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggag

[0565] agcgtcgaatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcc

[0566] cggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcc

[0567] tatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtat

[0568] ccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcc

[0569] tgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgaga

[0570] gttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcataca

[0571] ctattctcagaatgacttggttgagtactcaccagtcacagaaaagcattacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacac

[0572] tgcggccaacttacttgacaacgatcggaggaccgaaggagctaaccgctttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctg

[0573] aatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatgcaaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaac

[0574] attaatagactggatggaggcggataaagttgcaggaccactctgcgctcggccctcggctggctttgctgataaatctggagccggtgagcgtgggtct

[0575] cgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatcgacggggagtcaggcaactatggatgaacgaatagacagatcgct

[0576] gagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctt

[0577] ttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatctcttgagatccttttttctgcgcgtaatctg

[0578] ctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaa

[0579] atactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgat

[0580] aagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacga

[0581] cctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacag

[0582] gagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagggggg

[0583] cggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgta

[0584] ttaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctc

[0585] tccccgcgcgttggccgattcattaatgcagggggatctcgatcccgcgaaattaatacgactcactatagg

[0586] Nucleic acid sequence of pT7-VSV-aCD137 (SEQ ID NO.9)

[0587] acgaagacaaacaaaccattattatcattaaaaggctcaggagaaactttaacagtaatcagaattctcgaggccaccatggactggacctggagaatcctgttcctggtg

[0588] gccgccgccaccggggcccacagcgaagtgcagctggtgcagagcggtgctgaggtgaagaagcctggcgagagcctgagaattagctgcaagggcagcggct

[0589] acagcttcagcacatactggatcagctgggttagacagatgcctggaaagggcctggaatggatgggcaaaatctaccccggcgactcctacacaaactacagtccat

[0590] catttcagggccaagtgaccatcagcgctgataagagcatcagcactgcctacctgcagtggtccagcctgaaggcctcagataccgccatgtactactgcgccagag

[0591] gatatggcatcttcgactattggggccagggcaccctggtcacagtgtcttctgcctctaccaagggcggcggaggcggcagcggaggcggcggctccggagggg

[0592] gaggctctagctacgagctgacccagccccccagcgtgagcgttagcccgggccagaccgcctctatcacctgcagcggcgacaacatcggcgaccagtacgccc

[0593] actggtatcagcagaaacctggccaaagccctgtgctggtgatctatcaggacaagaacagacctagcggaatccccgagcggttctccggctctaatagcggcaaca

[0594] ccgctaccctgaccatctccggaacacaagctatggacgaggccgactactactgcgctacctacaccggcttcggatctctggccgtgttcggcggcggcacaaagc

[0595] tgaccgtgctgggccagcctaaggctcatcaccaccaccatcactgatggccatatgaaaaaaactaacagtaatcaaaatgtctgttacagtcaagagaatcattgaca

[0596] acacagtcatagttccaaaacttcctgcaaatgaggatccagtggaatacccggcagattacttcagaaaatcaaaggagattcctctttacatcaatactacaaaaagttt

[0597] gtcagatctaagaggatatgtctaccaaggcctcaaatccggaaatgtatcaatcatacatgtcaacagctacttgtatggagcattaaaggacatccggggtaagttgga

[0598] taaagattggtcaagtttcggaataaacatcgggaaagcaggggatacaatcggaatatttgaccttgtatccttgaaagccctggacggcgtacttccagatggagtatc

[0599] ggatgcttccagaaccagcgcagatgacaaatggttgcctttgtatctacttggcttatacagagtgggcagaacacaaatgcctgaatacagaaaaaagctcatggatg

[0600] ggctgacaaatcaatgcaaaatgatcaatgaacagtttgaacctcttgtgccagaaggtcgtgacatttttgatgtgtggggaaatgacagtaattacacaaaaattgtcgc

[0601] tgcagtggacatgttcttccacatgttcaaaaaacatgaatgtgcctcgttcagatacggaactattgtttccagattcaaagattgtgctgcattggcaacatttggacacct

[0602] ctgcaaaataaccggaatgtctacagaagatgtaacgacctggatcttgaaccgagaagttgcagatgaaatggtccaaatgatgcttccaggccaagaaattgacaag

[0603] gccgattcatacatgccttatttgatcgactttggattgtcttctaagtctccatattcttccgtcaaaaaccctgccttccacttctgggggcaattgacagctcttctgctcag

[0604] atccaccagagcaaggaatgcccgacagcctgatgacattgagtatacatctcttactacagcaggtttgttgtacgcttatgcagtaggatcctctgccgacttggcaca

[0605] acagttttgtgttggagataacaaatacactccagatgatagtaccggaggattgacgactaatgcaccgccacaaggcagagatgtggtcgaatggctcggatggtttg

[0606] aagatcaaaacagaaaaccgactcctgatatgatgcagtatgcgaaaagagcagtcatgtcactgcaaggcctaagagagaagacaattggcaagtatgctaagtcag

[0607] aatttgacaaatgaccctataattctcagatcacctattatatattatgctacatatgaaaaaaactaacagatatcatggataatctcacaaaagttcgtgagtatctcaagtcc

[0608] tactctcgtctagatcaggcggtaggagagatagatgagatcgaagcacaacgagctgaaaagtccaattatgagttgttccaagaggacggagtggaagagcatact

[0609] aggccctcttattttcaggcagcagatgattctgacacagaatctgaaccagaaattgaagacaatcaaggcttgtatgtaccagatccggaagctgagcaagttgaagg

[0610] ctttatacaggggcctttagatgactatgcagatgaggacgtggatgttgtattcacttcggactggaaacagcctgagcttgaatccgacgagcatggaaagaccttacg

[0611] gttgacattgccagagggtttaagtggagagcagaaatcccagtggcttttgacgattaaagcagtcgttcaaagtgccaaacactggaatctggcagagtgcacatttg

[0612] aagcatcgggagaaggggtcatcataaaaaagcgccagataactccggatgtatataaggtcactccagtgatgaacacacatccgtaccaatcagaagccgtatcag

[0613] atgtttggtctctctcaaagacatccatgactttccaacccaagaaagcaagtcttcagcctctcaccatatccttggatgaattgttctcatctagaggagaattcatctctgt

[0614] cggaggtaacggacgaatgtctcataaagaggccatcctgctcggtctgaggtacaaaaagttgtacaatcaggcgagagtcaaatattctctgtagactatgaaaaaaa

[0615] gtaacagatatcacaatctaagtgttatcccaatccattcatcatgagttccttaaagaagattctcggtctgaaggggaaaggtaagaaatctaagaaattagggatcgca

[0616] ccacccccttatgaagaggacactagcatggagtatgctccgagcgctccaattgacaaatcctattttggagttgacgagatggacacctatgatccgaatcaattaaga

[0617] tatgagaaattcttctttacagtgaaaatgacggttagatctaatcgtccgttcagaacatactcagatgtggcagccgctgtatcccattgggatcacatgtacatcggaat

[0618] ggcagggaaacgtcccttctacaaaatcttggcttttttgggttcttctaatctaaaggccactccagcggtattggcagatcaaggtcaaccagagtatcacgctcactgc

[0619] gaaggcagggcttatttgccacataggatggggaagacccctcccatgctcaatgtaccagagcacttcagaagaccattcaatataggtctttacaagggaacgattga

[0620] gctcacaatgaccatctacgatgatgagtcactggaagcagctcctatgatctgggatcatttcaattcttccaaattttctgatttcagagagaaggccttaatgtttggcct

[0621] gattgtcgagaaaaaggcatctggagcgtgggtcctggattctatcagccacttcaaatgagctagtctagcttccagcttctgaacaatccccggtttactcagtctctcct

[0622] aattccagcctttcgaacaactaatatcctgtcttttctatccctatgaaaaaaactaacagagatcgatctgtttccttgacacgcgttacgatgcggccgatgaagtgccttt

[0623] tgtacttagcctttttattcattggggtgaattgcaagttcaccatagtttttccacacaaccaaaaaggaaactggaaaaatgttccttctaattaccattattgcccgtcaagc

[0624] tcagatttaaattggcataatgacttaataggcacagccttacaagtcaaaatgcccaagagtcacaaggctattcaagcagacggttggatgtgtcatgcttccaaatgg

[0625] gtcactacttgtgatttccgctggtatggaccgaagtatataacacattccatccgatccttcactccatctgtagaacaatgcaaggaaagcattgaacaaacgaaacaag

[0626] gaacttggctgaatccaggcttccctcctcaaagttgtggatatgcaactgtgacggatgccgaagcagtgattgtccaggtgactcctcaccatgtgctggttgatgaat

[0627] acacaggagaatgggttgattcacagttcatcaacggaaaatgcagcaattacatatgccccactgtccataactctacaacctggcattctgactataaggtcaaagggc

[0628] tatgtgattctaacctcatttccatggacatcaccttcttctcagaggacggagagctatcatccctgggaaaggagggcacagggttcagaagtaactactttgcttatga

[0629] aactggaggcaaggcctgcaaaatgcaatactgcaagcattggggagtcagactcccatcaggtgtctggttcgagatggctgataaggatctctttgctgcagccaga

[0630] ttccctgaatgcccagaagggtcaagtatctctgctccatctcagacctcagtggatgtaagtctaattcaggacgttgagaggatcttggattattccctctgccaagaaa

[0631] cctggagcaaaatcagagcgggtcttccaatctctccagtggatctcagctatcttgctcctaaaaacccaggaaccggtcctgctttcaccataatcaatggtaccctaaa

[0632] atactttgagaccagatacatcagagtcgatattgctgctccaatcctctcaagaatggtcggaatgatcagtggaactaccacagaaagggaactgtgggatgactggg

[0633] caccatatgaagacgtggaaattggacccaatggagttctgaggaccagttcaggatataagtttcctttatacatgattggacatggtatgttggactccgatcttcatctta

[0634] gctcaaaggctcaggtgttcgaacatcctcacattcaagacgctgcttcgcaacttcctgatgatgagagtttattttttggtgatactgggctatccaaaaatccaatcgag

[0635] cttgtagaaggttggttcagtagttggaaaagctctattgcctcttttttctttatcatagggttaatcattggactattcttggttctccgagttggtatccatctttgcattaaatta

[0636] aagcacaccaagaaaagacagatttatacagacatagagatgaaccgacttggaaagtaagcggccgccctgcacacagattcttcatgtttgaaccaaatcaacttgtg

[0637] atatcatgctcaaagaggccttaattatattttaatttttaatttttatgaaaaaaactaacagcaatcatggaagtccacgattttgagaccgacgagttcaatgatttcaatga

[0638] agatgactatgccacaagagaattcctgaatcccgatgagcgcatgacgtacttgaatcatgctgattacaatttgaattctcctctaattagtgatgatattgacaatttgatc

[0639] aggaaattcaattctcttccgattccctcgatgtgggatagtaagaactgggatggagttcttgagatgttaacatcatgtcaagccaatcccatctcaacatctcagatgca

[0640] taaatggatgggaagttggttaatgtctgataatcatgatgccagtcaagggtatagttttttacatgaagtggacaaagaggcagaaataacatttgacgtggtggagacc

[0641] ttcatccgcggctggggcaacaaaccaattgaatacatcaaaaaggaaagatggactgactcattcaaaattctcgcttatttgtgtcaaaagtttttggacttacacaagtt

[0642] gacattaatcttaaatgctgtctctgaggtggaattgctcaacttggcgaggactttcaaaggcaaagtcagaagaagttctcatggaacgaacatatgcaggattagggtt

[0643] cccagcttgggtcctacttttatttcagaaggatgggcttacttcaagaaacttgatattctaatggaccgaaactttctgttaatggtcaaagatgtgattatagggaggatg

[0644] caaacggtgctatccatggtatgtagaatagacaacctgttctcagagcaagacatcttctcccttctaaatatctacagaattggagataaaattgtggagaggcaggga

[0645] aatttttcttatgacttgattaaaatggtggaaccgatatgcaacttgaagctgatgaaattagcaagagaatcaaggcctttagtcccacaattccctcattttgaaaatcata

[0646] tcaagacttctgttgatgaaggggcaaaaattgaccgaggtataagattcctccatgatcagataatgagtgtgaaaacagtggatctcacactggtgatttatggatcgtt

[0647] cagacattggggtcatccttttatagattattacactggactagaaaaattacattcccaagtaaccatgaagaaagatattgatgtgtcatatgcaaaagcacttgcaagtg

[0648] atttagctcggattgttctatttcaacagttcaatgatcataaaaagtggttcgtgaatggagacttgctccctcatgatcatccctttaaaagtcatgttaaagaaaatacatgg

[0649] cccacagctgctcaagttcaagattttggagataaatggcatgaacttccgctgattaaatgttttgaaatacccgacttactagacccatcgataatatactctgacaaaagt

[0650] cattcaatgaataggtcagaggtgttgaaacatgtccgaatgaatccgaacactcctatccctagtaaaaaggtgttgcagactatgttggacacaaaggctaccaattgg

[0651] aaagaatttcttaaagagattgatgagaagggcttagatgatgatgatctaattattggtcttaaaggaaaggagagggaactgaagttggcaggtagatttttctccctaat

[0652] gtcttggaaattgcgagaatactttgtaattaccgaatatttgataaagactcatttcgtccctatgtttaaaggcctgacaatggcggacgatctaactgcagtcattaaaaa

[0653] gatgttagattcctcatccggccaaggattgaagtcatatgaggcaatttgcatagccaatcacattgattacgaaaaatggaataaccaccaaaggaagttatcaaacgg

[0654] cccagtgttccgagttatgggccagttcttaggttatccatccttaatcgagagaactcatgaattttttgagaaaagtcttatatactacaatggaagaccagacttgatgcg

[0655] tgttcacaacaacacactgatcaattcaacctcccaacgagtttgttggcaaggacaagagggtggactggaaggtctacggcaaaaaggatggagtatcctcaatcta

[0656] ctggttattcaaagagaggctaaaatcagaaacactgctgtcaaagtcttggcacaaggtgataatcaagttattgcacacagtataaaacgaagaaatcgagaaacgtt

[0657] gtagaattacagggtgctctcaatcaaatggtttctaataatgagaaaattatgactgcaatcaaaatagggacagggaagttaggacttttgataaatgacgatgagacta

[0658] tgcaatctgcagattacttgaattatggaaaaataccgattttccgtggagtgattagagggttagagaccaagagatggtcacgagtgacttgtgtcaccaatgaccaaat

[0659] acccacttgtgctaatataatgagctcagtttccacaaatgctctcaccgtagctcattttgctgagaacccaatcaatgccatgatacagtacaattattttgggacatttgct

[0660] agactcttgttgatgatgcatgatcctgctcttcgtcaatcattgtatgaagttcaagataagataccgggcttgcacagttctactttcaaatacgccatgttgtatttggaccc

[0661] ttccattggaggagtgtcgggcatgtctttgtccaggtttttgattagagccttcccagatcccgtaacagaaagtctctcattctggagattcatccatgtacatgctcgaag

[0662] tgagcatctgaaggagatgagtgcagtatttggaaccccgatagccaagtttcgaataactcacatagacaagctagtagagaatccaacctctctgaacatcgcta

[0663] tgggaatgagtccagcgaacttgttaaagactgaggttaaaaaatgcttaatcgaatcaagacaaaccatcaggaaccaggtgattaaggatgcaaccatatatttgtatc

[0664] atgaagaggatcggctcagaagtttcttatggtcaataaatcctctgttccctagatttttaagtgaattcaaatcaggcacttttttgggagtcgcagacgggctcatcagtct

[0665] atttcaaaattctcgtactattcggaactcctttaagaaaaagtatcatagggaattggatgatttgattgtgaggagtgaggtatcctctttgacacatttagggaaacttcatt

[0666] tgagaaggggatcatgtaaaatgtggacatgttcagctactcatgctgacacattaagatacaaatcctggggccgtacagttattgggacaactgtaccccatccattag

[0667] aaatgttgggtccacaacatcgaaaagagactccttgtgcaccatgtaacacatcagggttcaattatgtttctgtgcattgtccagacgggatccatgacgtctttagttca

[0668] cggggaccattgcctgcttatctagggtctaaaacatctgaatctacatctattttgcagccttgggaaagggaaagcaaagtcccactgttaaaagagctacacgtctta

[0669] gagatgctatctcttggtttgttgaacccgactctaaactagcaatgactatactttctaacatccactctttaacaggcgaagaatggaccaaaaggcagcatgggttcaa

[0670] aagaacagggtctgcccttcataggttttcgacatctcggatgagccatggtgggttcgcatctcagagcactgcagcattgaccaggttgatggcaactacagacacca

[0671] tgagggatctgggagatcagaatttcgactttttattccaagcaacgttgctctatgctcaaattaccaccactgttgcaagagacggatggatcaccagttgtacagatcat

[0672] tatcatattgcctgtaagtcctgtttgagacccatagaagagatcaccctggactcaagtatggactacacgcccccagatgtatcccatgtgctgaagacatggaggaat

[0673] ggggaaggttcgtggggacaagagataaaacagatctatcctttagaagggaattggaagaatttagcacctgctgagcaatcctatcaagtcggcagatgtataggttt

[0674] tctatatggagacttggcgtatagaaaatctactcatgccgaggacagttctctatttcctctatctatacaaggtcgtattagaggtcgaggtttcttaaaagggttgctagac

[0675] ggattaatgagagcaagttgctgccaagtaatacaccggagaagtctggctcatttgaagaggccggccaacgcagtgtacggaggtttgatttacttgattgataaattg

[0676] agtgtatcacctccattcctttctcttactagatcaggacctattagagacgaattagaaacgattccccacaagatcccaacctcctatccgacaagcaaccgtgatatgg

[0677] gggtgattgtcagaaattacttcaaataccaatgccgtctaattgaaaagggaaaatacagatcacattattcacaattatggttattctcagatgtcttatccatagacttcatt

[0678] ggaccattctctatttccaccaccctcttgcaaatcctatacaagccatttttatctgggaaagataagaatgagttgagagagctggcaaatctttcttcattgctaagatcag

[0679] gagaggggtgggaagacatacatgtgaaattcttcaccaaggacatattattgtgtccagaggaaatcagacatgcttgcaagttcgggattgctaaggataataataaa

[0680] gacatgagctatcccccttggggaagggaatccagagggacaattacaacaatccctgtttattatacgaccaccccttaacccaaagatgctagagatgcctccaaaat

[0681] ccaaaatcccctgctgtccggaatcaggttgggccaattaccaactggcgctcattataaaattcggagtatattacatggaatgggaatccattacagggacttcttgagt

[0682] tgtggagacggctccggagggatgactgctgcattactacgaaaatgtgcatagcagaggaatattcaatagtctgttagaattatcagggtcagtcatgcgaggcg

[0683] cctctcctgagccccccagtgccctagaaactttaggaggagataaatcgagatgtgtaaatggtgaaacatgttgggaatatccatctgacttatgtgacccaaggactt

[0684] gggactatttcctccgactcaaagcaggcttggggcttcaaattgatttaattgtaatggatatggaagttcgggattcttctactagcctgaaaattgagacgaatgttagaa

[0685] attatgtgcaccggattttggatgagcaaggagttttaatctacaagacttatggaacatatatttgtgagagcgaaaagaatgcagtaacaatccttggtcccatgttcaag

[0686] acggtcgacttagttcaaacagaatttagtagttctcaaacgtctgaagtatatatggtatgtaaaggtttgaagaaattaatcgatgaacccaatcccgattggtcttccatc

[0687] aatgaatcctggaaaaacctgtacgcattccagtcatcagaacaggaatttgccagagcaaagaaggttagtacatactttaccttgacaggtattccctcccaattcattcc

[0688] tgatccttttgtaaacattgagactatgctacaaatattcggagtacccacgggtgtgtctcatgcggctgccttaaaatcatctgatagacctgcagatttattgaccattagc

[0689] cttttttatatggcgattatatcgtattataacatcaatcatatcagagtaggaccgatacctccgaaccccccatcagatggaattgcacaaaatgtggggatcgctataact

[0690] ggtataagcttttggctgagtttgatggagaaagacattccactatatcaacagtgtttggcagttatccagcaatcatttccgattaggtgggaggctatttcagtaaaagg

[0691] aggatacaagcagaagtggagtactagaggtgatgggctcccaaaagatacccgaatttcagactccttggccccaatcgggaactggatcagatctttggaattggtc

[0692] cgaaaccaagttcgtctaaatccattcaataagatcttgttcaatcagctatgtcgtacagtggataatcatttgaagtggtcaaatttgcgaaaaaacacaggaatgattga

[0693] atggatcaatgggcgaatttcaaaagaagaccggtctatactgatgttgaagagtgacctacatgaggaaaactcttggagagattaaaaaatcaggaggagactccaa

[0694] actttaagtatgaaaaaactttgatccttaagaccctcttgtggtttttattttttatctggttttgtggtcttcgtgggtcggcatggcatctccacctcctcgcggtccgacct

[0695] gggcatccgaaggaggacgtcgtccactcggatggctaagggagagctcggatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctg

[0696] agcaataactagcataaccccttggggcctctaaacgggtcttgaggggtttttgctgaaaggaggaactatatccggatcgagatcctctagagtcgacctgcaggca

[0697] tgcaagcttgtattctatagtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgttctaacgacaatatgtacaagcctaattgtgtagcatct

[0698] ggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctggtaacgccgtcccgcacccggaaatggtc

[0699] agcgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccg

[0700] acatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatc

[0701] tccttgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgaatggtgcactctca

[0702] gtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaag

[0703] ctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatga

[0704] taataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccc

[0705] tgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacg

[0706] ctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttt

[0707] tccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggtt

[0708] gagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgac

[0709] aacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacga

[0710] cgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggag

[0711] gcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcact

[0712] ggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgatt

[0713] aagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaa

[0714] tcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaac

[0715] caccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagcc

[0716] gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttg

[0717] gactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatac

[0718] ctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagc

[0719] ttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacg

[0720] ccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagct

[0721] gataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgat

[0722] tcattaatgcagggggatctcgatcccgcgaaattaatacgactcactatagg。

Claims

1. An oncolytic virus expressing an anti-PD-L1 / anti-CD137 bispecific antibody, characterized in that: The aPDL1 / aCD137 fragment was connected to the VSV viral genome plasmid to obtain the viral full genome plasmid pT7-VSV-aPDL1 / aCD137; the plasmid was co-transfected with the auxiliary plasmid into 293T cells, and VSV-aPDL1 / aCD137 viral seeds were obtained after culture; the viral seeds were amplified in large quantities using 293T cells to obtain the oncolytic virus VSV-aPDL1 / aCD137.

2. A method for constructing an oncolytic virus according to claim 1, characterized in that: The following steps are involved: (1) Design of aPDL1 / aCD137 fragments: The single-chain antibody aPDL1 composed of the hypervariable region of anti-PD-L1 and the single-chain antibody aCD137 composed of the hypervariable region of anti-CD137 were connected through a linker sequence to fuse into a bispecific antibody aPDL1 / aCD137; an IgG heavy chain signal peptide was introduced at the N-terminus of aPDL1 / aCD137, and a His tag was introduced at the C-terminus; All sequences were spliced ​​according to the design and then codon optimized. The optimized nucleic acid sequence was fully synthesized and inserted into the T vector to obtain pUC-aPDL1 / aCD137; (2) Cloning of the oncolytic virus VSV-aPDL1 / aCD137 full genome plasmid pT7-VSV-aPDL1 / aCD137: Using pUC-aPDL1 / aCD137 as a template and VSV F: TAATCAGAATTTCTCGAGACCGCCACCATGGACTGGAC and VSV R: AGTTTTTTTCATATGGCCATCAGTGGTGGTGGTGGTG as primers, a high-fidelity kit was used to amplify the aPDL1 / aCD137 fragment, and sequence fragments homologous to the insertion position of the VSV full genome were introduced at both ends of the aPDL1 / aCD137 fragment, that is, 16 homologous bases were inserted on each side; The pT7-VSV whole genome plasmid was double-digested with MscI and XhoI, and then purified to obtain a linear pT7-VSV whole genome plasmid, and the aPDL1 / aCD137 fragment was connected to the double-digested whole genome plasmid pT7-VSV to obtain the virus whole genome plasmid pT7-VSV-aPDL1 / aCD137; (3) Rescue of oncolytic virus VSV-aPDL1 / aCD137: pT7-VSV-aPDL1 / aCD137 and auxiliary plasmid were co-transfected into 293T cells using lipofectamine2000 transfection reagent, and then cultured in a 37°C, 5% CO2 cell culture incubator for 4-6 hours. Then, 2 mL of DMEM cell culture medium containing 2% FBS was added and cultured for 2-3 days. Depending on the culture medium, DMEM with 2% FBS was replaced or supplemented. The cell state was observed under a microscope every day. When cytopathic effect appeared in 4-8 days and reached 100% cytopathic effect, the cell supernatant was collected and centrifuged to remove cell debris to obtain the oncolytic virus VSV-aPDL1 / aCD137, which was aliquoted and stored at -80°C.

3. The construction method according to claim 2, characterized in that: In the step (3), the dosage of pT7-VSV-aPDL1 / aCD137 is 5 μg, and the auxiliary plasmids are pVRC-T7, pVRC-N, pVRC-P and pVRC-L, and the dosages thereof are all 0.5 μg.

4. Use of the oncolytic virus expressing the anti-PD-L1 / anti-CD137 bispecific antibody according to claim 1 in the preparation of a drug for the treatment of ovarian cancer.

5. Use of the oncolytic virus expressing the anti-PD-L1 / anti-CD137 bispecific antibody according to claim 1 in the preparation of a drug for treating solid tumors.

Images