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An attenuated strain of oncolytic baculovirus and its application in tumor therapy

An oncolytic virus and virus technology, applied in the direction of viruses, applications, viral peptides, etc., can solve the problems of poor treatment of solid tumors, poor oncolytic effects, and safety risks, and achieve the induction and promotion of anti-tumor immune responses , Eliminate immunosuppression in the microenvironment of tumor tissue, and low toxicity

Active Publication Date: 2021-01-29
FANTASIA BIOPHARMA ZHEJIANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the genetically recombinant viruses based on VSV known in the prior art either have certain toxicity to normal somatic cells, resulting in safety risks; or have poor oncolytic effects, resulting in poor efficacy in treating solid tumors

Method used

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  • An attenuated strain of oncolytic baculovirus and its application in tumor therapy
  • An attenuated strain of oncolytic baculovirus and its application in tumor therapy
  • An attenuated strain of oncolytic baculovirus and its application in tumor therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Virus rescue of the attenuated mutant strain (RV-Mut) constructed based on VSV rod-shaped oncolytic virus

[0086] Based on the efficient rescue system of VSV virus, different attenuated strain systems were constructed, and the virus rescue situation of single point and multiple points was randomly mutated. Then confirm whether the M protein of VSV virus can be mutated arbitrarily, and virions can be obtained (that is, whether any mutation can be performed, and virions can be rescued).

[0087] The concrete steps of above-mentioned virus rescue system construction are as follows:

[0088] 1. Spread BSR-T7 cells in a 6-well plate to make the cell volume reach 4×10 5 1 / well, add vT7 14-16 hours after plating, and transfect after 4 hours of virus infection.

[0089]2. Use opti-MEM medium to dilute the plasmid. Among them, the total amount of plasmid was 5 μg, and then 7.5 μl PLUS Reagent was added. Dilute 10 μl of Lipofectamine LTX with culture medium.

...

Embodiment 2

[0097] Embodiment 2: the virus titer detection of different RV-Mut virus strains

[0098] In the MEF / LLC cell culture medium, add the following viruses: VSV-GFP-WT, RV-GFP-M51R, RV-GFP-M51R-V221F-S226R (RV-3Mut), RV-GFP-G21E-M51A-L111F -V221F (RV-4Mut) each 200PFU, detect the titer of the virus that virus strain produces (TCID 50 ).

[0099] The concrete steps of detecting the titer of above-mentioned virus are as follows:

[0100] 1. Add 3 mL of Vero (LLC / Hela) cell suspension to each well of a 6-well culture plate to make the cell volume reach 4×10 5 pcs / well, 6 holes in total, 37°C, 5% CO 2 Cultivate for 16h.

[0101] 2. Add 200 PFU each of viruses VSV-GFP-WT, RV-GFP-M51R, RV-GFP-M51R-V221F-S226R, RV-GFP-G21E-M51A-L111F-V221F to each well, and set 2 normal cells as controls hole. Harvest 100 μl of cell supernatant at each time point of 12h, 24h, 48h, 72h, 80h, and 96h.

[0102] 3. Add 100 μl of Vero cell suspension to each well of the 96-well culture plate to make ...

Embodiment 3

[0110] Example 3: Comparison of the in vitro killing of different tumor cells by detecting RV-4Mut with different viral loads

[0111] By MTT detection method, the in vitro killing effect of RV-4Mut attenuated strains with different viral loads on different tumor cells was detected.

[0112] The specific steps of the above detection method are as follows:

[0113] 1. Add 100 μl of Vero (LLC / Hela / MEF / MC38) cell suspension to each well of a 96-well culture plate to make the cell volume reach 1×10 4 pc / well, 37°C, 5% CO 2 Cultivate for 16h.

[0114] 2. Dilute viruses VSV-GFP-WT, RV-GFP-M51R, RV-GFP-M51R-V221F-S226R (RV-3Mut), RV-GFP-G21E-M51A-L111F-V221F (RV-4Mut) to MOI (multiplicity of infection) were 0.001, 0.01, 0.1, 1.0, inoculate 4 wells for each dilution gradient, 100 μl per well, 37°C, 5% CO 2 Cultivate for 40h.

[0115] 3. Discard the supernatant in the 96-well culture plate, add fresh medium, and add MTT solution, 20 μL / well. 37°C, 5% CO 2 Cultivate for 4h.

...

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PUM

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Abstract

The present disclosure relates to a modified matrix protein of recombinant oncolytic baculovirus, an attenuated strain of oncolytic baculovirus with the aforementioned modified matrix protein, a composition containing the aforementioned attenuated strain and its preparation for killing abnormal proliferative cells, Applications in drugs that induce and promote anti-tumor immune responses or eliminate immunosuppression in the tumor tissue microenvironment. The attenuated strain of oncolytic baculovirus involved in the present disclosure has the characteristics of continuous replication expression, high titer, stimulation of immune response in the local microenvironment of the tumor, and low toxicity to normal cells while maintaining high selectivity for infecting tumor cells. It is of great significance in the clinical treatment of tumors.

Description

technical field [0001] The present disclosure relates primarily to the field of biotechnology. In particular, the present disclosure relates to an attenuated virus strain and its use in the treatment of diseases. More specifically, the present disclosure relates to a mutated attenuated oncolytic baculovirus strain, particularly the VSV-MuddSummer strain, and its therapeutic methods and applications in cancer. Background technique [0002] At present, small molecule drugs and monoclonal antibodies have been developed and applied to new tumor treatments, but the cure rate is not high, and more research is needed. In addition, treatment with only a single drug may lead to drug resistance in tumor cells, so the development of effective biological therapies is urgently needed. Oncolytic virus is a virus that has the ability to replicate through genetic changes. The highly diluted attenuated virus can take advantage of the inactivation or defect of tumor suppressor genes in tumo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/145C12N15/47C12N7/01A61K35/766A61K45/06A61P35/00A61P35/04
CPCC07K14/005C12N7/00A61K35/766A61K45/06A61P35/00A61P35/04C12N2760/20222C12N2760/20221C12N2760/20232C12N2760/20271A61K39/00A61K39/4611A61K2239/57A61K2239/50A61K2239/38A61K39/46449A61K2300/00C07K14/145A61K39/205
Inventor 秦晓峰吴飞夏菁
Owner FANTASIA BIOPHARMA ZHEJIANG CO LTD