Isolated oncolytic adenoviruses capable of expressing exogenous genes, vectors, therapeutic agents and uses thereof

An oncolytic adenovirus and foreign gene technology, applied in the biological field, can solve problems such as reducing the oncolytic effect of oncolytic viruses

Pending Publication Date: 2022-04-01
HANGZHOU CONVERD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, host-produced neutralizing antibodies against oncolytic viruses and the restriction of viral particle diffusion by tumor tissue can reduce the oncolytic effect of oncolytic viruses.

Method used

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  • Isolated oncolytic adenoviruses capable of expressing exogenous genes, vectors, therapeutic agents and uses thereof
  • Isolated oncolytic adenoviruses capable of expressing exogenous genes, vectors, therapeutic agents and uses thereof
  • Isolated oncolytic adenoviruses capable of expressing exogenous genes, vectors, therapeutic agents and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0310] 4. Preparation of target cells expressing foreign proteins:

[0311] 293T cells or tumor cells were transfected with Lipofectamine 3000 (Thermo Fisher L3000015) according to the manufacturer's instructions. Expression plasmids include plasmids encoding the marker polypeptides, foreign HLA proteins or amino acid sequences described in this application. The method for preparing the plasmid is described below. If a plasmid containing the adenoviral genome was used to transfect 293T cells, the plasmid was predigested with PacI enzyme (New England Biolabs, R0547S) to release the adenoviral genome. Cells transiently expressing foreign proteins can be used as target cells 48-72 hours after transfection. To generate a stable SKOV3 cell line expressing NY-ESO-1 protein or a mutant KRAS protein with a G12D point mutation, 72 h after transfection with a plasmid encoding the corresponding protein, SKOV3 cells were incubated with medium supplemented with 500 μg / mL Genetin Cultiva...

Embodiment 1

[0343] This example proves that exogenous genes can be effectively expressed by the gene constructs described in this application, said constructs comprising adenoviral vectors comprising nucleic acids encoding said marker polypeptides and / or exogenous HLA class I molecules; and A lentiviral vector comprising a nucleic acid encoding a T cell receptor. Figure 1A A schematic diagram of the constructs described in this application is shown. pAdEasy-EF1α-NY-A2 is a replication-deficient adenoviral vector expressing the labeled polypeptide shown in SEQ ID NO.51, which contains NY-ESO-1 157- 165 epitope peptide, and the HLA-A2 protein shown in SEQ ID NO.12. The F2A sequence is connected between the marker polypeptide and the HLA-A2 gene. The expression unit of the marker polypeptide is flanked by an exogenous EF-1α promoter and an SV40 poly(A) signal sequence. pAd-EF1α-E1A-A2-F2A-NY is a replication-competent adenovirus vector, which contains the nucleic acid encoding the marker ...

Embodiment 2

[0347] This example proves that once the construct vector expresses the marker polypeptide and the foreign HLA class I protein, the antigenic epitope peptide can be released from the marker polypeptide and presented by the foreign source HLA class I molecule to form the antigenic peptide / HLA Class I complex, which is recognized by specific TCRs.

[0348] To assess the ability of the specific TCR expressed by JRT cells to recognize epitope peptides presented by exogenous HLA class I molecules, JRT cells were first transfected with a recombinant lentivirus co-transfected with pCDH-EF1α - 293T cell production of TCR-NY-LY, pCDH-EF1α-TCR-NY-AE or pCDH-EF1α-TCR-NY-LI and the above packaging vectors. JRT cells expressing specific TCR were co-cultured with HLA-A2 positive T2 cells, and NY-ESO-1 157-165 polypeptide in a 10-fold dilution series starting from 1 μg / ml was added to the culture wells. 16-24 hours after antigen stimulation, the percentage of CD69-positive JRT cells was ana...

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Abstract

The present invention provides isolated oncolytic adenoviruses capable of expressing exogenous genes, vectors, therapeutic agents, and uses thereof. The oncolytic adenovirus is a selective replicating type recombinant oncolytic adenovirus obtained by carrying out gene modification on an adenovirus, and the genome of the oncolytic adenovirus has the following characteristics: 1) the oncolytic adenovirus contains an E1B gene regulatory element; (2) an E1B gene coding region is deleted, an exogenous gene is inserted into the site, and the exogenous gene is positioned behind an E1B promoter and is controlled by an E1B gene regulation element; and 3) cDNA (complementary deoxyribonucleic acid) of E1A for transcribing E1A 13s mRNA (messenger Ribonucleic Acid) on the upstream of the exogenous gene, wherein the cDNA is of a wild type or an Rb protein binding region deletion type. The oncolytic adenovirus provided by the invention can more effectively introduce nucleic acid with a labeled polypeptide coding sequence into tumors and / or cancer cells, and more efficiently express exogenous labeled polypeptides in the tumors and / or cancer cells, so that the exogenous labeled polypeptides enter an MHC class I antigen presentation pathway, and the recognition sensitivity of T cell receptor modified immune cells to the tumors and / or cancer cells is enhanced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an isolated oncolytic adenovirus for expressing foreign genes, a vector, a therapeutic agent and applications thereof. Background technique [0002] As immune checkpoint inhibitors have shown significant anti-tumor efficacy in clinical trials (see the literature "Cancer Cell 27,450–461(2015)"), and CAR-T (chimeric antigen receptor (CAR) Tcells) as the representative Adoptive T cell therapy has shown long-term effective therapeutic effects on hematological tumors, and tumor immunotherapy has become one of the most promising fields. Adoptive cell therapy (ACT), including the adoptive transfer of tumor-infiltrating lymphocytes (TILs), T cells expressing exogenous tumor-specific T cell receptors (TCR-T), or CAR-T cells, has been shown to be the most effective A promising immunotherapy for cancer (see literature "N Engl J Med 2017; 377:2545-2554"). [0003] Although CAR-T the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/861A61K35/761A61K35/17A61P35/00C12R1/93
CPCA61K35/17A61K35/761A61P35/00C12N7/00C12N15/861C12N2710/00A61P35/02
Inventor 侯亚非胡放绳纪坡谭贤魁陈璨董小明
Owner HANGZHOU CONVERD CO LTD
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