Tag-free, specific conjugation of glycosylated IgG1 antibodies using microbial transglutaminase. Issue 52 (22nd November 2022)

• Record Type: Journal Article
• Authors: Hadjabdelhafid-Parisien, Adem; Bitsch, Sebastian; Macarrón Palacios, Arturo; Deweid, Lukas; Kolmar, Harald; Pelletier, Joelle N.
• Is Part Of: RSC advances. Volume 12:Issue 52(2022)
• Page Start: 33510
• Page End: 33515
• Publication Date: 2022-11-22
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/d2ra05630e
• Languages: English
• ISSNs: 2046-2069

Abstract

Abstract : Substitution I253Q on a glycosylated IgG1 antibody allows microbial transglutaminase-mediated conjugation of a fluorophore or a clickable auristatin. The resulting antibody–drug conjugate showed excellent cell toxicity and no FcRn-mediated recycling. Abstract : We present an efficient approach for tag-free, site-specific conjugation of a fully glycosylated antibody using microbial transglutaminase (mTG). We created variants of trastuzumab where a single surface-exposed residue of the human crystallizable fragment had been substituted to glutamine, with the objective of enabling site-specific mTG-mediated conjugation with primary amine payloads. MTG reactivity was determined by conjugation to an amino fluorophore, demonstrating effective tag-free conjugation at the newly introduced I253Q site. The conjugation of one payload per antibody heavy chain was confirmed by mass spectrometry. We further demonstrated two-step mTG/click chemistry-based conjugation of I253Q trastuzumab with monomethyl auristatin E. Cytotoxicity and specificity of the resulting antibody–drug conjugate were indistinguishable from trastuzumab conjugated by another method although binding to the neonatal Fc receptor was impaired. The resulting fully glycosylated ADC is unique in that it results from minimal modification of the antibody sequence and offers potential for application to cellular imaging, fluorescence microscopy, western blotting or ELISA.