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M2-defective poxvirus

A pox virus and virus technology, applied in the field of oncolytic virus, can solve problems such as difficulty in implementation

Pending Publication Date: 2021-09-28
TRANSGENE SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, preclinical and even more clinical studies may be difficult to implement due to the complex nature of these immune interacting molecules and viral vectors and the risk of triggering a cascade of events

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0259] Example 1: Identification of the ability of the vaccinia virus m2 protein to interfere with the B7-mediated co-stimulatory pathway and characterization of its binding properties

[0260] Supernatants of vaccinia virus-infected cells inhibit the interaction of CTLA4 with CD80 or CD86

[0261] Two assays were established to quantitatively monitor the CD80 / CTLA4 and CD86 / CTLA4 blocking activity provided by different candidate viruses. In these assays, human CTLA4 (hCTLA4) was immobilized on ELISA plates and soluble labeled hCD80 or hCD86 was added. In this setup, any competing molecule bound to the immobilized or soluble partner will induce a decrease in signal (competition assay). Anti-hCTLA4 antibody ipilimumab (Yervoy) and uninfected DF1 (chicken cell line available; e.g. from CRL-12208 TM ) were used as positive and negative controls, respectively. Surprisingly, all supernatants of cells infected with vaccinia virus (Copenhagen, Wyeth and Western Reserve strains) ...

Embodiment 2

[0275] Example 2: m2-deficient poxviruses no longer produce IF

[0276] Construction of M2L-deleted poxvirus

[0277] The involvement of m2 in IF was further investigated by deleting the M2L gene in the vaccinia virus genome. Specifically, in a double deletion (DD) vaccinia virus expressing luciferase (i.e. tk as described in WO2009 / 065546 - , rr- - , and named VVTG18277) to disrupt the M2L locus, resulting in a recombinant triple deletion (TD) virus expressing luciferase as described above (i.e. tk - rr - , m2 - ) (COPTG19289). Deletion of the M2L portion extending from 64 nucleotides upstream of the m2 ORF to the first 169 codons resulted in repression of expression of the m2 protein (m2-) and did not have any significant effect on viral replication in CEFs compared to the parent (data not shown). show).

[0278] M2L-deleted viruses no longer produce IF

[0279] Supernatants obtained after infection of human HeLa and avian DF1 cells with DD and TD viruses were studie...

Embodiment 3

[0281] Example 3: m2-deficient recombinant poxvirus

[0282] The luciferase-expressing tk-rr-m2-oncolytic vaccinia virus (gene inserted into the J2R locus) was constructed as described above.

[0283] Oncolytic activity

[0284] will LOVO ( CCL-229 TM ) and HT116 ( CCL-247 TM ) colon cancer cells with 8.10 5 Cells / well were seeded in 96-well plates. Before infection, at 37°C, 5% CO 2 Plates were incubated for 4 hours. Use tk-rr-m2-COPTG19289 virus or tk-rr-VVTG18277 virus expressing luciferase in 10 -1 to 10 -4 Cells were infected at an MOI range of particles / cell. Cell viability was determined 96 hours post-infection (D4) by trypan blue exclusion using a cell counter (Vi-Cell, Beckman coulter). LOVO ( Figure 7A ) and HCT116 ( Figure 7B) showed that the oncolytic potency conferred by the m2-deficient COPTG19289 virus was comparable to that obtained with m2-positive VVTG18277 in both LOVO cells and HCT116. Specifically, at 10 -1 and 10 -2 LOVO cells were lys...

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Abstract

The present invention is in the field of oncolytic viruses. The invention provides new poxviruses which are engineered to be defective for the function encoded by the M2L locus (i.e., m2 function). Such poxviruses lack a functional m2 binding activity to at least one or both of CD80 and CD86 co-stimulatory antigens. Said oncolytic poxviruses are preferably vaccinia virus having a total or partial deletion of the M2L locus. The present invention also relates to cells and compositions comprising such poxviruses and their use for treating proliferative diseases such as cancers and for preventing diseases (vaccination, especially in veterinary field). More precisely, the invention provides an alternative to the existing oncolytic viruses which are largely used in virotherapy. The m2-defective poxviruses are particularly useful for the expression of immunomodulatory polypeptides such as anti-CTLA-4 antibodies with the purposes of stimulating or improve immune response.

Description

technical field [0001] The present invention belongs to the field of oncolytic viruses. The present invention provides novel poxviruses engineered to be deficient in the function encoded by the M2L locus (ie m2 function). Such poxviruses lack functional m2-binding activity for at least one or both of the CD80 and CD86 costimulatory antigens. The oncolytic poxvirus is preferably a vaccinia virus in which all or part of the M2L locus has been deleted. The invention also relates to cells and compositions comprising such poxviruses, and their use for the treatment of proliferative diseases such as cancer and for the prevention of diseases (vaccination, especially in the veterinary field). More precisely, the present invention provides an alternative to existing oncolytic viruses primarily used in virotherapy. m2-deficient poxviruses are particularly useful for expressing immunomodulatory polypeptides, such as anti-CTLA-4 antibodies, for the purpose of stimulating or improving a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/275C12N7/00C12N15/863
CPCA61K35/768C12N15/86C12N2710/24121C12N2710/24122C12N2710/24132C12N2710/24143A01K67/0278A01K2207/12A01K2227/105A01K2267/0331A61K2039/54A61K2039/5256A61K39/0011A61P35/00A61P19/02A61P9/00C07K16/2818
Inventor P·克莱因彼得J-B·马尔尚C·雷米D·施密特
Owner TRANSGENE SA