Elucidation of Critical Amino Acid Residues for Antibody and Transmembrane Antigen Interactions via Cell‐Free Protein Expression‐Based Approach

The generation of antibodies against integral membrane proteins (IMPs) presents unique challenges due to IMPs' low natural abundance, reduced biosynthesis rate, limited level of extraction, and low purification yield. That stems from their intricate structure defining the conformal epitope location. This study introduces a novel approach to identify crucial amino acid residues involved in the interaction between antibodies and tumor-associated transmembrane antigens based on utilizing a prokaryotic cell-free expression (CFPE) system. We considered human transmembrane prostate androgen-induced protein 1 (hTMEPA1) as the target antigen. It is a single-pass α-helical protein possessing hallmarks of a promising cancer biomarker. A several-step procedure was implemented to determine key residues of hTMEPA1. We created mimotope mAbs based on in silico analysis of immunogenic regions followed by target protein antigen production in the CFPE system and further antibody characterization. The results demonstrated an exceptional way for robust and high-throughput target protein production combined with in-depth biophysical and immunochemical characterization of therapeutic and diagnostic antibody-based molecules.