title: Engineering linear F(ab')2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity.
abstract: We developed a novel bivalent antibody fragment, the linear (L-) F(ab')2, comprising tandem repeats of a heavy chain fragment VH-CH1-VH-CH1 cosecreted with a light chain. Functional humanized L-F(ab')2 directed against p185HER2 was secreted from Escherichia coli at high titer (> or = 100 mg/l) and purified to homogeneity. The L-F(ab')2 binds two equivalents of antigen with an apparent affinity (Kd = 0.46 nM) that is within 3-fold of the corresponding thioether-linked F(ab')2 fragment. The N-terminal site binds antigen with an affinity (Kd = 1.2 nM) that is approximately 4-fold greater than that for the C-terminal site, as shown by the comparison of L-F(ab')2 variants containing a single functional binding site. L-F(ab')2 has greater antiproliferative activity than the thioether-linked F(ab')2 against the p185HER2-overexpressing tumor cell line BT474. Linear and thioether-linked F(ab')2 have very similar pharmacokinetic properties in normal mice, and their serum permanence times are respectively 7- and 8-fold longer than the corresponding Fab fragment. L-F(ab')2 offers a facile route to bivalent antibody fragments that are potentially suitable for clinical applications, and that may have improved biological activity compared with thioether-linked F(ab')2 fragments.
authors.affiliation: Department of Process Sciences, Genentech Inc., South San Francisco, CA 94080-4990, USA.
authors.forename: G
authors.initials: G
authors.lastname: Zapata
authors.forename: J B
authors.initials: JB
authors.lastname: Ridgway
authors.forename: J
authors.initials: J
authors.lastname: Mordenti
authors.forename: G
authors.initials: G
authors.lastname: Osaka
authors.forename: W L
authors.initials: WL
authors.lastname: Wong
authors.forename: G L
authors.initials: GL
authors.lastname: Bennett
authors.forename: P
authors.initials: P
authors.lastname: Carter
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003602:Cytotoxicity, Immunologic; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D015202:Protein Engineering; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases