Epitope Mapping of Anti-TNFα Monoclonal Antibodies Using Mutational Analysis

GABRIELA CANZIANI, SHENG-JIUN SAM WU, DEIDRA BETHEA, YAN CHEN, AUDREY BAKER, MICHAEL BRIGHAM-BURKE, AND GEORGE A. HEAVNER

Philadelphia, PA, USA

Simposio; DiPIA (Development in Protein Interaction Analysis); 2005

GE HEALTH SCIENCE

Surface plasmon resonance (SPR, Biacore) and ELISA were used to screen the binding of TNFα constructs to a panel of anti-TNFα human and murine monoclonal antibodies. Individual substitutions of 24 residues by alanine on the human TNFα molecule surface were used to map the contact surface of five potential therapeutic antibodies of high binding affinity. The 24 muteins were expressed as soluble proteins by in vitro translation and purified using an affinity column prepared by immobilizing an anti-human TNFα polyclonal antibody. Size and purity of both wild type TNFα and the muteins were assessed by SDS-PAGE and the protein secondary structure was evaluated by circular dichroism. This approach allowed defining with single-residue resolution five different interfaces corresponding to the conformational epitopes of these anti-TNF α antibodies.