Expression of mucin (MUC-1) from a mini-Epstein-Barr virus in immortalizedB-cells to generate tumor antigen specific cytotoxic T cells

Background EBV immortalized B-cells can be used as antigen presenting cells (APC) to stimulate specific T-cell responses. Mini-Epstein-Barr virus (min i-EBV) plasmids contain all functional elements of Epstein-Barr virus (EBV) necessary to immortalize B-cells in vitro. These immortalized B-cells are incapable of releasing infectious virus in contrast to cells immortalized b y wildtype EBV. In addition, mini-EBVs can be modified in E. coli to alter their genetic composition or adopt new genes. Methods We constructed a mini-EBV plasmid carrying an expression cassette f or the human tumor antigen mucin encoded by the gene MUC-1. Primary human B -cells were infected with the MUC-1 carrying mini-EBV plasmid packaged into an EBV coat and immortalized B-cell clones were expanded in vitro. These B -cells were analyzed by FAGS analyses for the expression of mucin and co-st imulatory molecules and were subsequently used as antigen presenting cells (APC) to stimulate peripheral blood mononuclear cells from healthy donors. Results Several B-cell lines were established that were shown to be free of helper virus or wildtype EBV. These B-cells expressed the relevant tumor-s pecific epitopes of mucin and the co-stimulatory ligands B7.1 and B7.2 nece ssary for efficient T-cell activation. Using the mucin expressing B-cells a s antigen presenting cells (APC) mucin-epitope specific cytotoxic T-cells w ere established. Conclusions Virus-free B-cell lines expressing tumor-associated epitopes su ch as mucin or other antigens of interest provide an unlimited and safe sou rce of APC to generate antigen specific T-cells which could be used for cli nical trials in adoptive immune therapy or cancer vaccines. Copyright (C) 1 999 John Wiley & Sons, Ltd.