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

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2012; p. 1
• Main Authors: Gébleux, Rémy, Briendl, Manfred, Grawunder, Ulf, Beerli, Roger R
• Format: Journal Article
• Language: English
• Published: United States 01.01.2019
• Subjects: Aminoacyltransferases - chemistry; Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - isolation & purification; Antibodies, Monoclonal - pharmacology; Bacterial Proteins - chemistry; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Cysteine Endopeptidases - chemistry; Drug Compounding; Humans; Immunoconjugates - chemistry; Immunoconjugates - immunology; Immunoconjugates - isolation & purification; Immunoconjugates - pharmacology; Molecular Structure; ADC; Anthracycline; Antibody–drug conjugate; Maytansine; Auristatin; SMAC; Site-specific conjugation; Sortase A-mediated ligation; Sortase-mediated antibody conjugation; Transpeptidation
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-9546-2_1

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.