Transglutaminase-Mediated Conjugations.

Abstract

Microbial transglutaminase (MTGase) catalyzes site-specific transpeptidation between a primary amine within linkers and the side chain of glutamine 295 within deglycosylated chimeric, humanized, and human IgG1s, affording homogeneous antibody-drug conjugates (ADCs). This method can be empowered by mutation of asparagine 297, insertion of a glutamine-containing peptide tag, and the use of branched linkers. Such modifications facilitate the conjugation process and provide flexibility in adjusting the conjugation site and drug-to-antibody ratio (DAR). Here, we present a protocol optimized in our group for MTGase-mediated linker incorporation and subsequent click chemistry-based payload installation. Both small linear linkers and bulky branched linkers can be incorporated into the Fc moiety within various antibodies, affording homogeneous ADCs with defined DARs. Thanks to the high homogeneity, ADCs constructed using this method can be analyzed using a single-quadrupole electrospray ionization (ESI) mass spectrometer, which many laboratories own for regular analysis of small molecules and peptides. The approach presented here allows for facile and cost-effective production of various homogeneous ADCs and other antibody conjugates for research and clinical purposes.