Site-Specific Antibody Labeling Using Phosphopantetheinyl Transferase-Catalyzed Ligation

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2012; p. 237
• Main Authors: Grünewald, Jan, Brock, Ansgar, Geierstanger, Bernhard H
• Format: Journal Article
• Language: English
• Published: United States 01-01-2019
• Subjects: Antibodies - chemistry; Antineoplastic Agents - chemistry; Bacterial Proteins - chemistry; Catalysis; Immunoconjugates - chemistry; Molecular Structure; Recombinant Proteins; Structure-Activity Relationship; Transferases (Other Substituted Phosphate Groups) - chemistry; Enzymatic ligation; Antibody–drug conjugates; 4′-Phosphopantetheinyl transferase; Site-specific conjugation; Bioorthogonal chemistry; Enzymatic modification; Protein tags
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-9546-2_13

4'-Phosphopantetheinyl transferases (PPTases) have been employed by researchers as versatile biocatalysts for the site-specific modification of numerous protein targets with structurally diverse molecules. Here we describe the use of these enzymes for the production of homogeneous antibody-drug conjugates (ADCs), which have garnered much attention as innovative anticancer drugs. The exceptionally broad substrate tolerance of PPTases allows for one-step and two-step conjugation strategies for site-specific ADC synthesis. While one-step conjugation involves direct coupling of a drug molecule to an antibody, two-step conjugation provides increased flexibility and efficiency of the conjugation process by first attaching a bioorthogonal chemical handle that is then used for drug molecule attachment in a second step. The aim of this chapter is to outline detailed protocols for both labeling procedures, as well as to provide guidance on enzyme and substrate preparation.