Scheme 1

Conjugation of trastuzumab and PE24-AzF. Trastuzumab, Her2-targeting IgG, was engineered with an unpaired Cys, and an unnatural amino acid having the azido group was incorporated into PE24 to install the bioorthogonal reactive moiety at the toxin protein. The two proteins were conjugated via a linker having bifunctional groups of maleimide and dibenzocyclooctyne (DBCO), which are reactive with the thiol and azido groups, respectively. The introduced Cys residues are shown in the heavy chain for simplicity even though various positions in the both chains were tested in this study.