Oncolytic virus vaccine and adoptive immune cell combination therapy

An oncolytic virus and oncolytic technology, applied in the direction of viruses, viral peptides, viruses/bacteriophages, etc., can solve a lot of manpower and material resources and other problems

Inactive Publication Date: 2020-06-23
SUZHOU ULTRAIMMUNE CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A method for Tcm culture for the production of TAA-specific T cells from PBMC samples has been published [21], but this protocol requires a clinical-grade cell sorter, and for the enrichment of antigen-specific cells, the described preparation protocol requires a large amount of Human and material resources (see U.S. Patent Application Publication No.US2015 / 0023938)

Method used

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  • Oncolytic virus vaccine and adoptive immune cell combination therapy
  • Oncolytic virus vaccine and adoptive immune cell combination therapy
  • Oncolytic virus vaccine and adoptive immune cell combination therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0224] Example 1: Virus rescue of the attenuated mutant strain (RV-Mut) constructed based on VSV rod-shaped oncolytic virus

[0225] 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. It is further confirmed whether the M protein of the VSV virus can be mutated arbitrarily, and virions can be obtained (that is, whether any mutation can be performed, and the virions can be rescued).

[0226] The concrete steps of above-mentioned virus rescue system construction are as follows:

[0227] 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.

[0228] 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 c...

Embodiment 2

[0236] Example 2: Virus titer detection of different RV-Mut virus strains, virus replication of different attenuated strains including RV-4Mut at different time points in normal cell MEF and tumor cell LLC.

[0237] In the MEF / LLC cell culture medium, add the following viruses: VSV-GFP-WT, RV-GFP-M51R, RV-GFP-M51R-V221F-G226R (RV-3Mut), RV-GFP-G21E-M51A-L111F - V221F (RV-4Mut) each 200PFU, detect the titer (TCID50) of the virus produced by the virus strain.

[0238] The concrete steps of detecting the titer of above-mentioned virus are as follows:

[0239] 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.

[0240]2. Add 200 PFU each of viruses RV-WT, RV-M51R, RV-M51R-V221F-G226R, RV-G21E-M51A-L111F-V221F to each well, and set 2 wells for normal cell control. Harvest 100 μl of cell supernatant at each time point of 12h, 24h, 48h, 72h, 80h, an...

Embodiment 3

[0249] Example 3: Detection of the comparison of the in vitro killing of various tumor cells by RV-4Mut with different viral loads

[0250] By MTT detection method, the in vitro killing effect of RV-4Mut attenuated strains with different viral loads on different tumor cells was detected.

[0251] The specific steps of the above detection method are as follows:

[0252] 1. Add 100 μl of (LLC / Hela / MEF) 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.

[0253] 2. Dilute the viruses RV-WT, RV-M51R, RV-M51R-V221F-G226R (RV-3Mut), RV-G21E-M51A-L111F-V221F (RV-4Mut) to an MOI (multiplicity of infection) of 0.001, respectively , 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.

[0254] 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.

[025...

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Abstract

The invention relates to an oncolytic virus vaccine and adoptive immune cell combination therapy. Specifically, the present disclosure relates to a modified matrix protein (M) of recombinant oncolyticrod-like virus, an oncolytic virus, an oncolytic virus vaccine, and combination therapy of the oncolytic virus vaccine with endogenous and adoptive immune cells. The present disclosure also providesantineoplastic preparations, including independent administration for oncolytic virus, oncolytic virus vaccine, and multiple-route and combination administration therapies. The present disclosure alsoprovides methods and strategies of the combination therapy for treating cancers, as well as a method for preparing an oncolytic virus vaccine and a tumor antigen specific central memory CD8+T cell population. According to the combination therapy provided by the invention, all potentials of adoptive immune cells and endogenous T cells aiming at various tumor-associated antigens can be developed, so that primary and secondary metastatic primary tumors are effectively eliminated, immune escape is prevented, long-time memory protection is generated, and the effect of radically treating tumors isachieved.

Description

technical field [0001] The present disclosure relates to immunotherapeutic methods for the treatment of cancer, including oncolytic virus therapy, oncolytic virus vaccines, and combination therapy of oncolytic virus vaccines and T cells. Background technique [0002] Cancer and conventional cancer therapeutics currently impose a significant socioeconomic burden in terms of emotional / physical distress, loss of life, and increased healthcare costs. Conventional therapies have shown some beneficial clinical effects but are accompanied by toxic side effects that reduce the quality of life of patients [1]. There is a need for more effective and less toxic cancer therapy in clinical treatment. [0003] It is well understood that neoplastic malignancy can be effectively eliminated by the immune system, and that immunotherapy is the most promising alternative in cancer treatment compared to other treatment modalities [2]. [0004] For example, it is generally believed that immune ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/145C12N15/47C12N7/00A61K35/766A61K39/00A61K45/06A61P35/00C12R1/93
CPCA61K35/766A61K39/0011A61K45/06A61K2039/5156A61K2039/5256A61P35/00C07K14/005C12N7/00C12N2760/20221C12N2760/20222C12N2760/20232
Inventor 秦晓峰万永红斯科特·沃尔什吴飞陈岚
Owner SUZHOU ULTRAIMMUNE CO LTD
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