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

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 (KD = 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 (KD = 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.