High level secretion of a humanized bispecific diabody from Escherichia coli.

Clinical development of bispecific antibodies (BsAb) has been effectively stymied by the lack of efficient production methods. We therefore attempted to produce a humanized BsAb fragment using an expression system that has proved very successful for secretion of monospecific Ab fragments from E. coli. An anti-p185HER2/anti-CD3 BsF(ab')2 was first recast into the diabody format and then periplasmically secreted from E. coli grown to high cell density in a fermentor. The diabody was recovered in very high yield (up to 935 mg/l) after protein A purification and predominantly (> or = 80%) as a dimer as judged by size exclusion chromatography. Diabody dimers were found to be mainly functional heterodimers (approximately 75%) by titration with p185HER2 extracellular domain. The diabody binds p185HER2 extracellular domain and human T lymphocytes with affinities close to those of the parent BsF(ab')2. Furthermore, the diabody is capable of simultaneous binding to tumor cells overexpressing p185HER2 and CD3 on T cells as shown by cellular rosetting. The diabody is equally potent as the parent BsF(ab')2 in retargeting IL-2 activated T-enriched peripheral blood lymphocytes to lyse tumor cells overexpressing p185HER2.