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.