title: High level secretion of a humanized bispecific diabody from Escherichia coli.
abstract: 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.
authors.affiliation: Department of Molecular Oncology, Genentech Inc., South San Francisco, CA 94080, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Zhu
authors.forename: G
authors.initials: G
authors.lastname: Zapata
authors.forename: R
authors.initials: R
authors.lastname: Shalaby
authors.forename: B
authors.initials: B
authors.lastname: Snedecor
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: P
authors.initials: P
authors.lastname: Carter
mesh_terms: D000595:Amino Acid Sequence; D018033:Antibodies, Bispecific / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000276:immunology*; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D012636:Secretory Rate; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D017186:Titrimetry; D014407:Tumor Cells, Cultured
chemical_list: D018033:Antibodies, Bispecific; D007140:Immunoglobulin Fab Fragments