CHARACTERIZATION OF HUMANIZED ANTI-P185HER2 ANTIBODY FAB FRAGMENTS PRODUCED IN ESCHERICHIA-COLI

We have been using biochemical and biophysical methods to characterize chimeric and humanized variants of the murine monoclonal antibody 4D5, directed against human epidermal growth factor receptor 2 (p185HER2). These studies were performed on antibody Fab fragments produced by secretion from E. coli. Humanized Fab fragment (hu4D5-8 Fab) was expressed at very high levels (1-2 g/L), whereas chimeric Fab (ch4D5 Fab) was expressed at much lower titers (5-20 mg/L), as determined by antigen-binding ELISA of supernatants from 10 L fermentations. Hu4D5-8 Fab and ch4D5 Fab purified by using affinity chromatography on immobilized bacterial IgG-binding proteins gave identical far UV-CD spectra characteristic of the immunoglobulin fold. Thermodynamic studies of antigen binding show comparable affinities (DELTAG) for ch and hu4D5-8 Fab, but different DELTAH values suggesting slight differences in the mechanism of binding. This difference is reflected in the anti-proliferative activity of these fragments on human breast tumor cells.