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.
Zhu
Z
Z
Department of Molecular Oncology, Genentech Inc., South San Francisco, CA 94080, USA.
Zapata
G
G
Shalaby
R
R
Snedecor
B
B
Chen
H
H
Carter
P
P
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
D018033:Antibodies, Bispecific; D007140:Immunoglobulin Fab Fragments