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 V-H-C(H)1-V- H-C(H)1 cosecreted with a light chain, Functional humanized L-F(ab')(2 ) directed against p185(HER2) was secreted from Escherichia coli at hi gh titer (greater than or equal to 100 mg/l) and purified to homogenei ty, The L-F(ab')(2) binds two equivalents of antigen with an apparent affinity (K-d = 0.46 nM) that is within 3-fold of the corresponding th ioether-linked F(ab')(2) fragment, The N-terminal site binds antigen w ith an affinity (K-d = 1.2 nM) that is similar to 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) ha s greater antiproliferative activity than the thioether-linked F(ab')( 2) against the p185(HER2)-overexpressing tumor cell line BT474, Linear and thioether-linked F(ab')(2) have very similar pharmacokinetic prop erties in normal mice, and their serum permanence times are respective ly 7- and 8-fold longer than the corresponding Fab fragment, L-F(ab')( 2) offers a facile route to bivalent antibody fragments that are poten tially suitable for clinical applications, and that may have improved biological activity compared with thioether-linked F(ab')(2) fragments .