Abstract
Abstract 4713
DNA vaccination respresents a novel therapeutic strategy for cancer. Researchs have showed DNA vaccines encoding Wilms tumor gene WT1-Derived HLA *0201-restricted epitopes effectively induce epitope-specific human cytotoxic T-lymphocytes (CTLs) and kill human leukemia cells in vitro. Nevertheless, there is still a need to increase the potency of DNA vaccine and span the HLA diversity of a target population. In this study, we designed polytpoe WT1 DNA vaccine encoding multiple CTL and helper T-lymphocyte (HTL) epitopes and containing HSP70407-426 as immunoadjuvants for immunotherapy.
On the basis published data, To design a Multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted, one HLA *2402-restricted epitopes, two HTL epitopes and one universal Th Pan- DR epitope (PADRE) sequence. The construct also included IgG K chain leader sequence as signal peptide to facilitate transport into the endoplasmic reticulum and Kozak sequence at the N-terminus to support translation of the gene transcript. DNA-coding sequesces was used computer-based modeling to optimize proteasome mediated epitope processing through the introdcution of amino acid spacer sequences, then cloned into the eukaryotic vector to construct the plasmid PcDNA3.1(+). Mycobacterial HSP70 fragment including stimulatory domain P407-426 as immunotherapy was fused to the C-terminal of Multi-WT1 for enhancing CTL activity and then inserted to PcDNA3.1(+). Transfection HEK-293T cells with the Multi -WT1-HSP70 and identify the expressed product by Western bolt.
Most of Multi -WT1 epitopes can be cleaved by software Net Chop 3.1 and PAPROC± analysis. Western blot showed correct expression of target gene in HEK293T cells.
Multi-WT1-mtHSP70 fusion gene eukaryotic expression vector was successfully designed, which prepared the materials for the research of DNA vaccine.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
