Engineered Reversible Inhibition of SpyCatcher Reactivity Enables Rapid Generation of Bispecific Antibodies
bioRxiv (Cold Spring Harbor Laboratory)(2023)
摘要
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.
### Competing Interest Statement
CH, MP, FY are authors of a patent application based on this work. All authors are employees of Bio-Rad AbD Serotec GmbH.
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关键词
spycatcher reactivity,reversible inhibition
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