THE DEVELOPMENT, CHARACTERIZATION AND APPLICATION OF TCR-LIKE MONOCLONAL ANTIBODIES WITH SPECIFICITY FOR EPSTEIN-BARR VIRUS LATENT EPITOPES

Abstract

In this study, we detail the generation and thorough characterization of three monoclonal antibodies that recognizes a peptide epitope associated with MHC class I. This type of antibody is known as a TCR-like monoclonal antibody (TCR-like mAb) in that it has the same specificity as a cytotoxic T-cell receptor. The three antibodies characterizd target epitopes derived from the EBV latent gene products LMP-1, LMP-2A, and EBNA-1. We demonstrate that these monoclonal antibodies exhibit specific high affinity binding to their respective HLA-A0201/peptide complexes and determined sites within peptides that affect antibody binding. We also investigate the effects of HLA-A02 subtypes and clinical peptide variants for antibody binding. These antibodies are used to visualize EBV latent epitopes in infected humanized NOD-scid IL2Rgammanull mice and NPC biopsies. In addition, we describe the quantity and expression hierarchy of these EBV latent epitopes in EBV infected HLA-A0201 positive tumor cell lines (RPMI-6666, CCRF-SB and C666-1A2). Finally, with our anti-HLA-A0201/LMP-2A antibody, we investigate the dynamics of epitope processing and MHC class I presentation using a Proteotuner¿ system (Clontech).