Engineering linear F(ab')2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity

: 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.