HIGH-LEVEL ESCHERICHIA-COLI EXPRESSION AND PRODUCTION OF A BIVALENT HUMANIZED ANTIBODY FRAGMENT

Many clinical uses of antibodies will re quire large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized FA(ab')2 fragments by secretion from E. coli. Titers of 1-2 g l-1 of soluble and functional Fab' fragments have been routinely achieved as judged by antigen-binding ELISA. Surprisingly, this high expression level of Fab' in the periplasmic space of E. coli does not drive dimerization. However, we have developed a protocol to directly and efficiently recover Fab' with the single hinge cysteine in the free thiol state, allowing FA(ab')2 formation by chemically-directed coupling in vitro. The E. coli derived humanized FA(ab')2 fragment is indistinguishable from FA(ab')2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2. This system makes EB. coli expression of bivalent antibody fragments for human therapy (or ther uses) practical.