Scheme 1
Cysteine hinge specific Click chemistry for generation of dbBiTES. Blue = reduced antibody 1 with DBCO. Red = reduced antibody 2 with PEGn-azide. Click = the two derivatized antibodies were mixed 1:1. The cross-linking between two heavy chains is likely the favored result, including the possibility of two heavy chain cross-links per dbBiTE. Evidence for cross-linking between two light chains (side to side) is shown in the EM studies as a rare event