π-Clamp-Mediated Homo- and Heterodimerization of Single-Domain Antibodies via Site-Specific Homobifunctional Conjugation

[Image: see text] Post-translational protein–protein conjugation produces bioconjugates that are unavailable via genetic fusion approaches. A method for preparing protein–protein conjugates using π-clamp-mediated cysteine arylation with pentafluorophenyl sulfonamide functional groups is described. Two computationally designed antibodies targeting the SARS-CoV-2 receptor binding domain were produced (K(D) = 146, 581 nM) with a π-clamp sequence near the C-terminus and dimerized using this method to provide a 10–60-fold increase in binding (K(D) = 8–15 nM). When two solvent-exposed cysteine residues were present on the second protein domain, the π-clamp cysteine residue was selectively modified over an Asp-Cys-Glu cysteine residue, allowing for subsequent small-molecule conjugation. With this strategy, we build molecule–protein–protein conjugates with complete chemical control over the sites of modification.