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Sci Rep. 2016 Dec 16;6:39291. doi: 10.1038/srep39291.

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates.

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Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, 64287 Darmstadt, Germany.
Merck KGaA, Frankfurter Straße 250, 64293 Darmstadt, Germany.
EMD Serono Research &Development Institute, Inc., 45A Middlesex Turnpike, Billerica, MA 01821, USA.


Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation.

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