Hentrich, C.; Putyrski, M.; Hanuschka, H.; Preis, W.; Kellmann, S.-J.; Wich, M.; Cavada, M.; Hanselka, S.; Ylera, F.

The precise regulation of protein function is essential in biological systems, and achieving such control is a fundamental objective in the fields of chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we performed a cysteine scan of SpyCatcher, exchanging each amino acid in the structured region against cysteine. Except for the two known reactive and catalytic residues, none of these mutations abolished reactivity. In a second screening step, we modified the cysteines with disulfide bond-forming small molecules and screened for reactivity again. Here we found 8 positions that, when modified, strongly inhibited reactivity. This inhibition could be reversed by treatment with reducing agents. We call such a reversibly inhibitable SpyCatcher SpyLock. We then used BiLock, a fusion of SpyLock and wildtype SpyCatcher, in combination with SpyTagged antibody fragments to generate bispecific antibodies. A first antibody was reacted with the regular SpyCatcher moiety, followed by unlocking of the SpyLock through reduction and its reaction with a second antibody. This method to generate bispecific antibodies requires only a single antibody format and is readily scalable, facilitating the screening of a large number of antibody combinations. We demonstrate the utility of this approach to screen anti-PD-1/anti-PD L1 bispecific antibodies using a cellular reporter assay.