Abstract
Random conjugation of chemical linkers to endogenous lysines or cysteines within an antibody yields a heterogeneous mixture of conjugates with various drug-to-antibody ratios. One approach for generating homogeneous antibody conjugates utilizes enzymatic transfer of payloads onto a specific glycan or amino acid residue. Microbial transglutaminase (MTG) is an enzyme that catalyzes the formation of a stable isopeptide bond between a glutamine and a lysine. We have previously identified and reported several sites throughout the antibody structure where an engineered lysine is sufficient for transfer of a glutamine-based substrate onto the antibody. Whereas other enzymatic transfer strategies typically require significant antibody engineering to either modify the N-glycans or introduce a multi-amino acid enzyme recognition site, the lower contextual specificity of MTG for lysines allows just a single lysine point mutation in an antibody to be efficiently transamidated. Here we describe the molecular positioning of these single engineered lysine residues and the conjugation conditions for producing homogeneous antibody conjugates exemplified using azido- and auristatin F-based acyl donor substrates.
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Acknowledgments
We thank Ben Vaessen for his work optimizing the MTG reaction conditions, Dr. Xin Cheng for ESI-MS analysis, and Sara Jacob for antibody purification.
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Spidel, J.L., Albone, E.F. (2019). Efficient Production of Homogeneous Lysine-Based Antibody Conjugates Using Microbial Transglutaminase. In: Massa, S., Devoogdt, N. (eds) Bioconjugation. Methods in Molecular Biology, vol 2033. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9654-4_5
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DOI: https://doi.org/10.1007/978-1-4939-9654-4_5
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