Site-specific modification of native IgGs with flexible drug-load.

Homogeneous, site-specifically conjugated antibodies have shown to result in antibody-drug conjugates (ADCs) with improved efficacy and tolerability compared to stochastically conjugated ADCs. However, precisely controlling the drug-load as well as attaching multiple payload moieties on the antibody remains challenging. Here, we demonstrate the simple and direct modification of native IgG-antibodies at the residue glutamine 295 (Q295) without the need for any protein engineering at flexible drug-to-antibody ratios of one or multiple payloads. The conjugation is enabled through short, positively charged lysine containing peptides and native, commercially available microbial transglutaminase. In proof-of-concept studies, HER2-targeting ADCs based on trastuzumab were generated with drug-to-antibody ratios (DARs) of 2 and 4 of the same or different payloads using orthogonal conjugation chemistries. Quantitative biodistribution studies performed with 111In-radiolabeled conjugates showed high tumour uptake and low accumulation of radioactivity in non-targeted tissues. A single dose study of trastuzumab conjugated to the highly potent payload α-Amanitin demonstrated complete and long-lasting tumour remissions and was well-tolerated at all dose levels tested.