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.
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