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A recombinant adeno-associated virus vector carrying multi-site mutant EGFR neoantigen gene and its construction method and application

A multi-site mutation, viral vector technology, applied in the direction of virus/phage, receptor/cell surface antigen/cell surface determinant, virus, etc. The effect of high safety

Active Publication Date: 2020-03-20
COBAXER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The design of CAR relies on monoclonal antibodies targeting EGFR, but there is currently no specific antibody against EGFR point mutations, so CAR-based cellular immunotherapy is not yet suitable for tumors with EGFR point mutations

Method used

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  • A recombinant adeno-associated virus vector carrying multi-site mutant EGFR neoantigen gene and its construction method and application
  • A recombinant adeno-associated virus vector carrying multi-site mutant EGFR neoantigen gene and its construction method and application
  • A recombinant adeno-associated virus vector carrying multi-site mutant EGFR neoantigen gene and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Recombinant vector pAAV-EGFR MUT construction and identification of

[0055] 1. Materials and sources

[0056] 1. pAAV-MCS plasmid: purchased from Cell Biolabs, USA.

[0057] 2. A431 cells: purchased from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences.

[0058] 3. Gene amplification primers: designed according to the mRNA sequence of EGFR gene in NCBI database (NM_005228.3).

[0059] 4. DNA site-directed mutagenesis kit: purchased from Shanghai Saibaisheng Gene Technology Co., Ltd.

[0060] 2. Construction of recombinant adeno-associated virus vector carrying multi-site mutant EGFR neoantigen gene

[0061] The present invention uses restriction endonuclease to cut the multi-cloning site of the vector, and then uses DNA ligation technology to EGFR WT Ligation reaction between the gene and the vector to obtain pAAV-EGFR WT Recombinant vector; then get pAAV-EGFR through 9 point mutation reactions MUT Recombinant v...

Embodiment 2

[0102] Example 2 Preparation of recombinant adeno-associated virus

[0103] 1. Materials and sources

[0104] 1. The pAAV-EGFR constructed in Example 1 MUT recombinant vector.

[0105] 2. Helper vectors pAAV6-RC6 and pHelper: both were purchased from Cell Biolabs.

[0106] 3. 293AAV cells: purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences.

[0107] 4. Polyethyleneimine (PEI): purchased from Polysciences (Cat# 23966).

[0108] 5. OPTI-MEM medium: purchased from Life technology company.

[0109] 6. OptiPrep TM Density Gradient Medium (iodixanol) and Benzonase: purchased from Sigma.

[0110] 2. Preparation and identification of recombinant adeno-associated virus (AAV6)

[0111] The recombinant adeno-associated virus AAV6 of this embodiment is prepared by making the recombinant vector pAAV-EGFR MUT Co-transfected with helper vectors pAAV6-RC6 and pHelper into 293AAV cells, collected virus particles and purified and ide...

Embodiment 3

[0120] Example 3 pAAV-EGFR MUT Tumor Killing Experiment of Recombinant Vectors Introduced into Dendritic Cells

[0121] 1. Materials and sources

[0122] 1. Recombinant adeno-associated virus (AAV6): prepared according to Example 2.

[0123] 2. AIM-V cell culture medium: purchased from Lonza Company.

[0124] 3. Cytokines: GM-CSF, IL-4, IL-2 and TNFα were all purchased from Peprotech.

[0125] 4. CD3 antibody (OKT3): purchased from Life Technology Company.

[0126] 5. CD80, CD86, OX40 and 4-1BB flow detection antibodies: purchased from eBioscience.

[0127] 2. AAV6 infects dendritic cells

[0128] like Figure 5 As shown, the present embodiment pAAV-EGFR MUT The whole process of the tumor killing experiment of introducing recombinant vector into dendritic cells includes the following steps:

[0129] 1. Take 50-150mL of peripheral blood from tumor patients, use lymphocyte separation medium to obtain peripheral blood mononuclear cells (PBMC), resuspend in AIM-V medium, a...

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Abstract

The invention provides a recombinant adeno-associated virus carrier carrying a multi-site mutant EGFR (Epidermal Growth Factor Receptor) novel antigenic gene. A construction method of the recombinant adeno-associated virus carrier comprises the following steps: linking a wild type EGFR gene enzyme into a recombinant adeno-associated virus carrier; then carrying out mutation on basic groups of sites including R108K, A289V, S492R, G598V, G719S, S768I, T790M, C797S and L858R in the EGFR gene to obtain the recombinant carrier. The prepared recombinant adeno-associated virus AAV6 has the characteristics of high safety and strong infection capacity, and particularly has good preferendum on DC cells; an AAV6-DC-CTL cellular immunotherapy method of a target multi-site mutant EGFR novel antigen can be used for effectively killing positive tumor cells of the EGFR novel antigen, and targeting tumor patients of a plurality of types of mutant EGFR antigens; the applicable range is wide and the side effect is very small.

Description

technical field [0001] The invention belongs to the technical field of construction methods of recombinant adeno-associated virus vectors, and in particular relates to a recombinant adeno-associated virus vector carrying a multi-site mutant EGFR neoantigen gene, a construction method and the application of the vector in the field of anti-tumor cell immunotherapy. Background technique [0002] Tumor cells have an unlimited capacity to proliferate due to uncontrolled stimulation of growth factor signaling or aberrant expression of growth factor receptors. Epidermal growth factor receptor (EGFR) is a transmembrane protein, and its intracellular domain is a member of tyrosine kinase; when EGFR binds to its ligand epidermal growth factor (EGF), it can activate RAS-RAF-MAPK, PI3K-AKT and PLCγ signaling pathways, thereby initiating mitogenic signaling cascades. Abnormal activation of EGF / EGFR signaling pathway can also lead to tumor angiogenesis and tumor metastasis. Studies have...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66C12N15/864A61K39/00A61P35/00
CPCA61K39/0011C07K14/71C12N15/66C12N15/86C12N2750/14143C12N2800/107
Inventor 申重阳陈勇军王仲
Owner COBAXER BIOTECH
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