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-p185(HER2)/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 fermenter. The diabody was recovered in very high yield (up to 935 mg/I) after protein A purification and predominantly (greater than or equal to 80%) as a dimer as judged by size exclusion chromatography. Diabody dimers were found to be mainly functional heterodimers (similar to 75%) by titration with p185(HER2) extracellular domain. The diabody binds p185(HER2) 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 p185(HER2) and CD3 on T cells as shown by cellular resetting. 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 p185(HER2).