CONJUGATION OF HUMAN FC-GAMMA IN CLOSED-HINGE OR OPEN-HINGE CONFIGURATION TO FAB'GAMMA AND ANALOGOUS LIGANDS

We describe a method for linking human normal Fc gamma 1, via stable t hioether bonds emerging from its hinge, to any molecule expressing a f ree sulfhydryl (SH) group. The Fc hinge may be closed by a disulfide ( SS) bond or left open. Preparation begins with reduction of the Fc hin ge to release four SH groups from its two parallel inter-gamma SS bond s. When the Fc is required in normal closed-hinge configuration, one S H group is alkylated with N-ethylmaleimide under limiting conditions, and one of the inter-gamma SS bonds is reconstituted by SS interchange . The residual SH group, to be used for linking, is left as a 4-dithio pyridyl group suitable for storage. When the Fc is required for conjug ation the 4-dithiopyridyl is replaced by a metastable maleimidyl group , which reacts rapidly with SH on the partner molecule to form a tande m thioether link. If the partner is Ab Fab'gamma, linking to cysteines in the Fab'gamma hinge yields derivatives such as FabFc and FabFc(2). Chimeric FabFc Abs (mouse Fab'gamma/human Fc gamma 1) invoked cellula r cytotoxicity in vitro, using human cell lines as targets and human l ymphocytes as effectors, whether the Fc hinge was open or closed. The same Abs could kill the same targets by activating human complement, b ut only when the Fc hinge was closed. Both effector functions were enh anced by the presence of a second Fc in FabFc(2). This method of Fc ad dition can be used to predict the performance of recombinant chimeric Abs and to provide novel molecular geometries.