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 gamma 1 sequence containing 1, 2, or 4 cysteines have been produced by high level Escherichia coli peri plasmic 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 o r 4 cysteines have a high level (similar to 70%) of multiple disulphid e bonds. These F(ab')(2) molecules have an increased pharmacokinetic s tability as measured by area under the curve compared to those made by direct coupling through a single disulphide bond. One particular mole cule containing 4 hinge cysteines has a greater pharmacokinetic stabil ity 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 c hemical reduction in vitro allowing partial reduction to expose reacti ve hinge thiols. These hinge sequences provide a simple method for pro ducing robust F(ab')(2) in vitro, obviating the need to use chemical c ross-linkers, and provide a route to hinge specific chemical modificat ion with thiol-reactive conjugates. (C) 1998 Elsevier Science B.V. All rights reserved.