High Level Escherichia coli Expression and Production of a Bivalent Humanized Antibody Fragment

Abstract

Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(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 F(ab′)₂ formation by chemically-directed coupling in vitro. The E. coli derived humanized F(ab′)₂ fragment is indistinguishable from F(ab′)₂ derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185^HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185^HER2. This system makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.