Engineered reversible inhibition of SpyCatcher reactivity enables rapid generation of bispecific antibodies

Christian Hentrich, Mateusz Putyrski, Hanh Hanuschka, Waldemar Preis, Sarah-Jane Kellmann, Melissa Wich, Manuel Cavada, Sarah Hanselka, Victor S. Lelyveld and Francisco Ylera ()
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Christian Hentrich: Bio-Rad AbD Serotec GmbH
Mateusz Putyrski: Bio-Rad AbD Serotec GmbH
Hanh Hanuschka: Bio-Rad AbD Serotec GmbH
Waldemar Preis: Bio-Rad AbD Serotec GmbH
Sarah-Jane Kellmann: Bio-Rad AbD Serotec GmbH
Melissa Wich: Bio-Rad AbD Serotec GmbH
Manuel Cavada: Bio-Rad AbD Serotec GmbH
Sarah Hanselka: Bio-Rad AbD Serotec GmbH
Victor S. Lelyveld: Massachusetts General Hospital
Francisco Ylera: Bio-Rad AbD Serotec GmbH

Nature Communications, 2024, vol. 15, issue 1, 1-15

Abstract: Abstract The precise regulation of protein function is essential in biological systems and a key goal in 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 perform a cysteine scan of the structured region of SpyCatcher. Except for two known reactive and catalytic residues, none of these mutations abolish reactivity. In a second screening step, we modify the cysteines with disulfide bond-forming small molecules. Here we identify 8 positions at which modifications strongly inhibit reactivity. This inhibition can be reversed by reducing agents. We call such a reversibly inhibitable SpyCatcher “SpyLock”. Using “BiLockCatcher”, a genetic fusion of wild-type SpyCatcher and SpyLock, and SpyTagged antibody fragments, we generate bispecific antibodies in a single, scalable format, facilitating the screening of a large number of antibody combinations. We demonstrate this approach by screening anti-PD-1/anti-PD-L1 bispecific antibodies using a cellular reporter assay.

Date: 2024
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DOI: 10.1038/s41467-024-50296-y

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