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 gamma1, via stable thioether bonds emerging from its hinge, to any molecule expressing a free sulfhydryl (SH) group. The Fc hinge may be closed by a disulfide (SS) bond or left open. Preparation begins with reduction of the Fc hinge to release four SH groups from its two parallel inter-gamma SS bonds. When the Fc is required in normal closed-hinge configuration, one SH 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-dithiopyridyl group suitable for storage. When the Fc is required for conjugation the 4-dithiopyridyl is replaced by a metastable maleimidyl group, which reacts rapidly with SH on the partner molecule to form a tandem thioether link. If the partner is Ab Fab'gamma, linking to cysteines in the Fab'gamma hinge yields derivatives such as FabFc and FabFc2. Chimeric FabFc Abs (mouse Fab'gamma/human Fc gamma1) invoked cellular cytotoxicity in vitro, using human cell lines as targets and human lymphocytes as effectors, whether the Fc hinge was open or closed. The same Abs could kill the same targets by activating human complement, but only when the Fc hinge was closed. Both effector functions were enhanced by the presence of a second Fc in FabFc2. This method of Fc addition can be used to predict the performance of recombinant chimeric Abs and to provide novel molecular geometries.