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Novel vesicle-like oncolytic virus and application thereof in preparation of antitumor drugs

An oncolytic virus and vesicle technology, applied in the field of tumor immunotherapy and tumor treatment, can solve the problems of low yield of natural exosomes, and achieve the effects of reduced preparation cost, high infection efficiency, and extended window period.

Pending Publication Date: 2021-06-11
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Artificial vesicle technology can overcome the problem of low yield of natural exosomes. At present, artificial vesicle technology is only used to encapsulate drugs to achieve targeted delivery. So far, there has been no report on the use of its encapsulated virus
Moreover, it is widely believed that the technique is unlikely to be useful for encapsulating therapeutic entities as large as viruses

Method used

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  • Novel vesicle-like oncolytic virus and application thereof in preparation of antitumor drugs
  • Novel vesicle-like oncolytic virus and application thereof in preparation of antitumor drugs
  • Novel vesicle-like oncolytic virus and application thereof in preparation of antitumor drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Infection efficiency of adenovirus Ad5 to cells with low CAR expression is low

[0076] CAR (the coxsackie and adenovirus receptor) is the main receptor of adenovirus Ad5 infected cells. Early studies have demonstrated that adenovirus Ad5 has low infection efficiency for cells with low CAR expression. 13,19 We found that the fluorescence intensity of 293T, A549, HCC-LM3 and Hepa1-6 cells stained with anti-CAR-PE was significantly increased compared with the isotype treatment group by flow cytometric analysis, indicating that 293T, A549, HCC-LM3 and Hepa1-6 High expression of CAR on the cell surface ( figure 1 a); while the surface of B16-F10, CT26.WT and H22 cells were stained with CAR antibody, there was no significant change in fluorescence intensity compared with the isotype treatment group; indicating that these cell lines do not express CAR; while K562 and Jurkat cells were stained with CAR antibody After that, compared with the isotype treatment group,...

Embodiment 2

[0078] Example 2 Vesicle-like technology is used to prepare a new type of vesicle-like oncolytic virus EM / VSV-GAd5sPVRCD137L with VSV-G

[0079] Vesicular stomatitis Indiana virus G protein (VSV-G) is capable of mediating viral entry into all cell types tested so far and is widely used in gene transduction and gene therapy 26 . This study hopes to use vesicle-like technology to take advantage of the broad tropism of VSV-G on cells to realize the retargeting of Ad5 and increase the infection efficiency of Ad5 on cell lines with low CAR expression.

[0080] First, we constructed 293T cells expressing VSV-G (293T-VSV-G), infected 293T-VSV-G cells with Ad5sPVRCD137L at an MOI of 5, and collected cells after culturing at 37°C for 72 hours. The collected cells were divided into two parts, and one part was used After freezing and thawing 3 times in the traditional protocol, the virus was purified by density gradient centrifugation with iodixanol; the other part produced a new type o...

Embodiment 3

[0086] Example 3 EM / VSV-G Ad5sPVRCD137L prepared by EM / VSV-G technology has higher infection efficiency, ability to express and secrete soluble PVRCD137L and oncolytic ability in vitro

[0087] The oncolytic adenovirus Ad5sPVRCD137L is an oncolytic adenovirus Ad5 expressing soluble PVRCD137L (named Ad5sPVRCD137L), and the genome structure of the oncolytic adenovirus Ad5sPVRCD137L is shown in image 3 shown. The low infection rate of oncolytic adenovirus Ad5sPVRCD137L to tumor types with low CAR expression severely limits the transformational application of Ad5sPVRCD137L. We applied EM / VSV-G technology to oncolytic adenovirus Ad5sPVRCD137L, expecting to significantly increase the infection efficiency of CAR low-expressing tumor cell lines through EM / VSV-G technology, and improve the therapeutic effect and expansion of oncolytic adenovirus Ad5sPVRCD137L its scope of application. The virus prepared by the oncolytic adenovirus Ad5sPVRCD137L through EM / VSV-G technology is named E...

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Abstract

The invention belongs to the field of tumor treatment and relates to application of a novel vesicle-like oncolytic virus in preparation of antitumor drugs. The virus Ad5sPVRCD137L infects cells capable of expressing specific membrane protein VSV-G, then a centrifugal extrusion / shearing method is utilized to enable the cells to pass through membrane holes so as to form vesicles, the oncolytic virus wrapped by the vesicles is manufactured, and the membrane protein VSV-G on the surfaces of the cells is reserved on the outer surfaces of the vesicles in the preparation process. The prepared novel vesicle-like oncolytic virus can achieve heavy targeting through the membrane protein VSV-G, and therefore, the gene transduction efficiency of the virus is improved, the neutralizing effect of an antiviral antibody can be avoided, and the yield of the virus can be improved; and a soluble fusion protein sPVRCD137L expressed by the virus can achieve the dual functions of blocking an immune detection point and activating immune costimulation. The novel vesicle-like oncolytic virus can significantly activate anti-tumor immunity and finally significantly prolong survival of mice.

Description

technical field [0001] The invention relates to the field of tumor treatment, especially the field of tumor immunotherapy, and in particular to the application of a novel vesicle-like oncolytic virus EM / VSV-G Ad5sPVRCD137L in the preparation of antitumor drugs. Background technique [0002] Cancer is one of the most harmful diseases to human life and health, and millions of people die of cancer every year. Many patients are already in the middle and late stages at the time of diagnosis and thus lose the opportunity for surgical treatment. Traditional radiotherapy and chemotherapy have not brought breakthrough progress to the tumor. Even small molecule targeted drugs are facing a huge challenge of short-term recurrence . [0003] In the past ten years, exciting results have emerged in the clinical research of anti-tumor immunotherapy, bringing hope to cancer patients again. Simultaneously blocking immune negative regulation and activating co-stimulatory pathways is proven t...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/62C12N15/861A61K35/761A61K38/17A61K47/62A61K47/69A61P35/00
CPCC12N7/00C12N15/86C07K14/70596C07K14/47A61K35/761A61K38/1709A61K47/62A61K47/6917A61P35/00C12N2710/10021C07K2319/00C12N2710/10043A61K2300/00
Inventor 吴俊华魏继武张海林张永辉吴俊艺刘淑雯马丁
Owner NANJING UNIV
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