IMPROVING IN-VIVO FOLDING AND STABILITY OF A SINGLE-CHAIN FV ANTIBODYFRAGMENT BY LOOP GRAFTING

The complementary determining regions (CDRs) from the fluorescein-bind ing antibody 4-4-20, which yields almost no soluble protein in peripla smic expression in Escherichia coli, were transplanted to the framewor k of the humanized antibody 4D5. The resulting single-chain Fv fragmen t (scFv) 4D5Flu showed both a dramatic improvement in soluble expressi on, even at 37 degrees C, and an improved thermodynamic stability. Ant igen affinity was maintained upon this engineering by paying attention to crucial framework-CDR contacts. This demonstrates that the use of superior frameworks is a robust strategy to improve the physical prope rties of scFv fragments, We also report that the grafted version was s elected in phage display over several competing variants of the same a ntibody, with identical binding constant but poorer folding or stabili ty properties. The selection required four panning rounds and a temper ature of 37 degrees C and we show that the underlying reason for this selection is a higher fraction of phages carrying functional scFv mole cules.