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Site-Specific Antibody Labeling Using Phosphopantetheinyl Transferase-Catalyzed Ligation

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Enzyme-Mediated Ligation Methods

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2012))

Abstract

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.

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Acknowledgments

The authors acknowledge the support from Tetsuo Uno, Yunho Jin, Hsien-Po Chiu, Xing Wang, and Yongqin Wan, who synthesized analogs of pantoate, pantothenate, and coenzyme A, as well as auristatins. Julie Vance’s efforts were instrumental in the production of CoA biosynthetic enzymes and PPTases. The authors further thank Badry Bursulaya for conjugation site modelling, as well as Heath E. Klock, Susan E. Cellitti, and Paula Patterson for generating a library of peptide-tagged antibodies that enabled the subsequent profiling of conjugation sites. We are also indebted to Daniel McMullan, Hung Tong, David Jones, and Jessica Read for purification and biophysical characterization of the immunoconjugates.

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Authors and Affiliations

  1. Biotherapeutics, Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA

    Jan Grünewald, Ansgar Brock & Bernhard H. Geierstanger

Authors
  1. Jan Grünewald
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  2. Ansgar Brock
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  3. Bernhard H. Geierstanger
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Correspondence to Jan Grünewald .

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  1. EnzyPep B.V., Geleen, The Netherlands

    Timo Nuijens  & Marcel Schmidt  & 

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Grünewald, J., Brock, A., Geierstanger, B.H. (2019). Site-Specific Antibody Labeling Using Phosphopantetheinyl Transferase-Catalyzed Ligation. In: Nuijens, T., Schmidt, M. (eds) Enzyme-Mediated Ligation Methods. Methods in Molecular Biology, vol 2012. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9546-2_13

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  • DOI: https://doi.org/10.1007/978-1-4939-9546-2_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9545-5

  • Online ISBN: 978-1-4939-9546-2

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