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Isolated recombinant oncolytic poxvirus, pharmaceutical composition and use thereof in the treatment of tumors and/or cancer

A poxvirus and composition technology, applied in the field of drugs for the treatment of tumors and/or cancers, to achieve the effects of improving safety and enhancing tumor targeting

Active Publication Date: 2019-04-02
HANGZHOU CONVERD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No genetically engineered poxvirus is currently marketed as a drug

Method used

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  • Isolated recombinant oncolytic poxvirus, pharmaceutical composition and use thereof in the treatment of tumors and/or cancer
  • Isolated recombinant oncolytic poxvirus, pharmaceutical composition and use thereof in the treatment of tumors and/or cancer
  • Isolated recombinant oncolytic poxvirus, pharmaceutical composition and use thereof in the treatment of tumors and/or cancer

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

[0113] Preparation Example 1: Construction of DDvv-IL21 Oncolytic Poxvirus

[0114] The following process can be found in figure 2 .

[0115] (1) Plasmid construction

[0116] Two strains of oncolytic poxviruses were constructed to carry human IL-21 and mouse IL-21 genes respectively, which were marked as DDvv-hIL21 and DDvv-mIL21 respectively.

[0117] 1) According to the NCBI gene bank sequences NM_001291041 and NM_021803, a total of two mouse IL21 DNA fragments (mIL-21) and human IL21 DNA fragments (hIL-21) were synthesized by gene synthesis, specifically:

[0118] mIL-21 comprises a murine IL21 fragment of normal sequence (i.e. a fragment of 72-560bp in the nucleotide sequence of Genbank No. NM_001291041), and its sequence (SEQ ID NO.1) is as follows:

[0119] AGATCTATGGAGAGGACCCTTGTCTGTCTGGTAGTCATCTTCTTGGGGACAGTGGCCCATAAATCAAGCCCCCAAGGGCCAGATCGCCTCCTGATTAGACTTCGTCACCTTATTGACATTGTTGAACAGCTGAAAATCTATGAAAATGACTTGGATCCTGAACTTCTATCAGCTCCACAAGATGTAAAGGGGCACTGTGAGCATGCAGCT...

preparation example 2

[0142] Preparation Example 2: Production and Purification of Oncolytic Poxvirus

[0143] (1) Production and purification

[0144] 1) Amplify the P5-1 sample of DDvv-mIL21 and the P4-3 sample of DDvv-hIL21 respectively, using 50 150mm culture dishes. When the Hu-143B cells grow to about 80%, each culture dish adds 50 μL small amplified virus about 2×10 4 Infect cells with PFU at 37°C, 5% CO 2nourish. After about 2 to 3 days, observe under the microscope that the cells become round and beaded, and some cells are detached from the culture dish. Use a cell scraper to collect the culture medium / cells and store them in a -80°C refrigerator.

[0145] 2) The collected virus liquid was repeatedly frozen and thawed three times with liquid nitrogen and autoclaved water, centrifuged at 2000 rpm for 3 minutes, and the supernatant was collected.

[0146] 3) Centrifuge the collected supernatant at 12,000 rpm for 10 minutes, discard the supernatant, suspend the precipitate with 5 mL of PB...

Embodiment 1

[0161] Example 1: Killing effect of DDvv-mIL21 poxvirus carrying mouse IL-21 on different mouse tumor cells

[0162] Place mouse tumor cells in 96-well plates, including B16 cells (mouse melanoma cells), GL261 cells (mouse glioma cells), LLC cells (mouse Lewis lung cancer cells), CT-26 cells (mouse colon Cancer cells), 4T-1 cells (mouse breast cancer cells), 5000 cells per hole, cultivated overnight to make it adhere to the wall, and then used 1MOI of DDvv-mIL21 virus prepared by the above method to infect the cells, respectively in 24 hours, 48 ​​hours After 1 hour and 60 hours (tests for LLC cells, GL261 cells), or after 24 hours, 48 ​​hours, and 72 hours (tests for B16 cells, CT-26 cells, 4T-1 cells), tumor cells were detected using the MTT assay Apoptosis (n=3, 2-3 repeated experiments). The control groups of this experiment were: negative control group (no virus infection), recombinant mouse IL-21 protein (rmIL-21, purchased from R&D Systems) and positive control group (...

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Abstract

The invention provides isolated recombinant oncolytic poxvirus, a pharmaceutical composition and use thereof in the treatment of tumors and / or cancer. The isolated recombinant oncolytic poxvirus is TKgene and the VGF gene functionally deficient type, an exogenous IL-21 gene is integrated in the genome of the recombinant oncolytic poxvirus, and the IL-21 gene can be expressed in tumor cells. The recombinant oncolytic poxvirus can be selectively replicated in tumor cells, and can fully exert the anti-tumor immunity of the exogenous IL-21, so that the oncolytic killing effect of the oncolytic virus and the anti-tumor immune stimulation effect of IL-21 can produce a synergistic effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an isolated recombinant oncolytic poxvirus, a pharmaceutical composition and its use in drugs for treating tumors and / or cancers. Background technique [0002] Oncolytic virus is a kind of virus that can selectively replicate in tumor cells and kill tumor cells, but not harm normal cells. After the virus replicates in the infected tumor cells, it releases new virus particles, and then infects the surrounding tumor cells, further playing an oncolytic effect. In addition to this direct oncolytic effect, the oncolytic virus can also effectively Stimulate the body to generate an immune response against the virus itself and against infected tumor cells. It can be seen that the anti-tumor effect of oncolytic viruses is achieved through the dual effects of selectively killing tumor cells and inducing a systemic anti-tumor immune response in the body. [0003] As early as the e...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/863A61P35/00A61P35/02C12R1/93
CPCA61K35/17A61K35/768C12N7/00C12N15/86C07K14/55C12N2710/24021C12N2710/24043A61K2300/00A61K39/285A61P35/00A61P35/02C07K14/54C12N2710/24132C12N2710/24143A61K38/00A61K9/0019C12N2710/24121C12N2710/24122
Inventor 傅瑾赵荣华张蓉王婷婷陈霖胡放
Owner HANGZHOU CONVERD CO LTD
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