Fig. 1. (A) Schematic representation of the one-step antibody labeling approach. The payload is attached to the Fc-III peptide via a chemically reactive site “X–Z”. X in red refers to the electrophilic site, and Z in violet refers to the leaving group. Upon binding of the Fc-III reactive conjugate to IgG-Fc, the antibody lysine is poised to attack the electrophilic center of the chemically reactive site X, and the peptide vector is detached from the payload thanks to the engineered leaving group Z. This strategy allows subsequent wash out of the ligand without any additional steps. (B) Representation of the binding of Fc-III to the Fc domain of IgG based on the X-ray structure. The surface of IgG-Fc is shaded grey, and Fc-III is shown in a green ribbon structure. The antibody K317 is shown with a pink sphere. The PEG spacer is linked to a carbonate reactive site via a reactivity modulator that jointly ensures high reactivity in the presence of lysine residues and adequate stability of the reactive site towards hydrolysis. The payload (yellow star), typically a fluorescent label (fluorescein), a radiolabel, or a chelator (DOTA, DTPA, and DFO), is chemically bound to the other side of the carbonate reactive group via a short linker. (C) Chemical structure of the payload-PEGn-Fc-III reactive conjugate using the same color codes as in A and B.
