ENGINEERING FAB FRAGMENTS FOR EFFICIENT F(AB)(2) FORMATION IN ESCHERICHIA-COLI AND FOR IMPROVED IN-VIVO STABILITY

We previously developed an efficient route to humanized F(ab') fragmen ts by high level secretion of the Fab' arms from Escherichia coli foll owed by directed chemical coupling in vitro. Here the number and type of interchain linkages in F(ab')2 molecules has been modified to simpl ify their production and improve their serum stability. All F(ab')2 va riants had comparable binding affinity for the p185HER2 Ag and antipro liferative activity against p185HER2-overexpressing tumor cells. This was anticipated since the modifications are distant from the Ag-bindin g loops. Replacement of a single disulfide bridge between Fab' arms wi th a more stable thioether bridge increased the serum permanence time in normal mice by threefold to 2.1 h. Removal of the disulfide bond be tween L and H chains in the thioether-bridged F(ab')2 did not affect t he pharmacokinetics, suggesting that the L chain remains associated wi th the H chain. An additional Fab' variant containing three repeats of the motif, CysProPro, was constructed with the aim of promoting effic ient formation of F(ab')2 in E. coli. This Fab' (CPP)3 variant was rec overed predominantly (up to 70%) as F(ab')2 directly from fermentation cell pastes, thus circumventing the need for in vitro coupling. The F (ab')2 (CPP)3 variant has a similar serum pharmacokinetics to the thio ether-bridged molecules. The improvements described here for deriving F(ab')2 fragments from E. coli should enhance the clinical potential o f these molecules.