F(ab′) 2 molecules made from Escherichia coli produced Fab′ with hinge sequences conferring increased serum survival in an animal model

Fab′s with hinges based on the human γ1 sequence containing 1, 2, or 4 cysteines have been produced by high level Escherichia coli periplasmic secretion, and coupled in vitro by reduction/oxidation to form F(ab′) 2. We find that the F(ab′) 2 made with hinges containing 2 or 4 cysteines have a high level (∼70%) of multiple disulphide bonds. These F(ab′) 2 molecules have an increased pharmacokinetic stability as measured by area under the curve compared to those made by direct coupling through a single disulphide bond. One particular molecule containing 4 hinge cysteines has a greater pharmacokinetic stability than a F(ab′) 2 formed by chemical cross-linking. F(ab′) 2 made from the Fab′ with 4 hinge cysteines is also relatively resistant to chemical reduction in vitro allowing partial reduction to expose reactive hinge thiols. These hinge sequences provide a simple method for producing robust F(ab′) 2 in vitro, obviating the need to use chemical cross-linkers, and provide a route to hinge specific chemical modification with thiol-reactive conjugates.