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MHC CLASS I PEPTIDE ANALYSIS FROM A BREAST CANCER CELL LINE TRANSFECTED WITH sHLA.
Oriana Hawkins 1, Melva Gonzalez Ph.D. 1, Angela Gilb 1, Tamara Potapova 1 and William Hildebrand Ph.D. 1. 1 Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA .
The human immune system represents a powerful mechanism for detecting and destroying cancer cells. Immune recognition is mediated by the major histocompatibility complex class I (MHC I) molecules, which scan the cell
s proteome and carry small peptides of intracellular origin to the cell surface. Effector T-lymphocytes (CTL) survey MHC-peptide complexes and target cells displaying cancer-specific peptides. However, our understanding of the epitopes that differentiate cancer cells from normal cells is incomplete. Determining the MHC-peptide phenotype of cancer cells is a critical step in the development of CTL-activating vaccines. The process of epitope discovery from cancer cells requires large amounts of HLA to extract and identify the peptides. Breast cancer cell lines, MCF-7 and T47D, were transfected with sHLA-A*0201 and sHLA-B*0702. sHLA constructs lacking the intracellular and transmembrane domains of the a-chain were modified at the 3 end by addition of 10 amino acids from the rat VLDL receptor (VLDLr). sHLA-VLDLr were cloned into the mammalian expression vector pcDNA3.1(-) Geneticin (Invitrogen) using the FuGENE 6 Transfection Reagent (Roche). Transfected cell lines were selected with Geneticin and assayed for sHLA production by ELISA. High producing transfectants obtained from each cell line were subcloned. Stable MCF-7 clones producing sHLA >50 ng/ml were inoculated into Bioreactors and peptides have been eluted from purified sHLA from transfected MCF-7 cells and analyzed by mass spectroscopy. MCF7 and T47D breast cancer cell lines have been successfully transfected with sHLA and MHC Class I peptide discovery from MCF-7 cancer cells is in process.