Site-Specific Conjugation of Thiol-Reactive Cytotoxic Agents to Nonnative Cysteines of Engineered Monoclonal Antibodies.

Antibody-drug conjugates (ADCs) have been proven to be a successful therapeutic concept, allowing targeted delivery of highly potent active pharmaceutical ingredients (HPAPIs) selectively to tumor tissue. So far, HPAPIs have been mainly attached to the antibody via a chemical reaction of the payload with lysine or cysteine side chains of the antibody backbone. However, these conventional conjugation technologies result in formation of rather heterogeneous products with undesired properties. To overcome the limitations of heterogeneous ADC mixtures, several site-specific conjugation technologies have been developed over the last years. Originally pioneered by scientist from Genentech with their work on THIOMABs, several engineered cysteine mAb ADCs (ECM-ADCs) are now investigated in clinical trials. Here, we describe in detail how to engineer additional cysteines into antibodies and efficiently use them as highly site-specific conjugation sites for HPAPIs.