Sortase A Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are highly potent targeted anticancer therapies. They rely on the linking of a selectively targeting antibody moiety with potent cytotoxic payloads to effect antitumoral activity. In recent years, one focus in the ADC field was to create novel methods for site-specifically conjugating payloads to antibodies. The method presented here is based on the S. aureus sortase A-mediated transpeptidation reaction. This method requires antibodies to be engineered in such a way that they possess the sortase recognition pentapeptide motif LPETG on the C-terminus of the immunoglobulin heavy and/or light chains. In addition, the toxin must contain an oligoglycine motif in order to make it a suitable substrate for sortase A. Here we describe a detailed method to conjugate a pentaglycine-modified toxin to the C-termini of LPETG-tagged antibody heavy and light chains using sortase-mediated antibody conjugation (SMAC-Technology™). Highly homogenous, site-specifically conjugated ADCs with controlled drug to antibody ratio and improved overall properties can be obtained with this method.