Engineering Antibodies with C-Terminal Sortase-Mediated Modification for Targeted Nanomedicine

Rania A. Hashad, Jaclyn L. Lange, Natasha C.W. Tan, Karen Alt, Christoph E. Hagemeyer

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The current advances in nanoengineered materials coupled with the precise targeting capability of recombinant antibodies can create nanoscale diagnostics and therapeutics which show enhanced accumulation and extended retention at a target tissue. Smaller antibodies such as single-chain variable fragments (scFv) preserve the selective and strong binding of their parent antibody to their antigen with the benefits of low immunogenicity, more efficient tissue penetration and easy introduction of functional residues suitable for site-specific conjugation. This is of high importance as nonspecific antibody modification often involves attachment to free cysteine or lysine amino acids which may reside in the active site, leading to reduced antigen binding. In this chapter, we outline a facile and versatile chemoenzymatic approach for production of targeted nanocarrier scFv conjugates using the bacterial trans-peptidase Sortase A (Srt A). Srt A efficiently mediates sequence-specific peptide ligation under mild conditions and has few undesirable side reactions. We first describe the production, purification and characterization of Srt A enzyme and a scFv construct which targets activated platelets, called scFvanti-GPIIb/IIIa. Following this, our protocol illustrates the chemoenzymatic modification of the antibody at the C-terminus with an orthogonal click chemistry linker. This avoids any random attachment to the biologically active antigen binding site of the antibody. Finally, we describe the modification of a nanoparticle surface with scFv attachment via two methods: (1) direct Sortase-mediated conjugation; or (2) a two-step system which consists of scFv Sortase-mediated conjugation followed by strain promoted azide-alkyne cycloaddition. Finally, methodology is described to assess the successful assembly of targeted particles.

Original languageEnglish
Title of host publicationBioconjugation
Subtitle of host publicationMethods in Molecular Biology
EditorsSam Massa, Nick Devoogdt
Place of PublicationNew York, NY
PublisherHumana Press
Number of pages14
ISBN (Electronic)9781493996544
ISBN (Print)9781493996537
Publication statusPublished - 2019

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Chemoenzymatic
  • Nanomedicine
  • Recombinant antibodies
  • Site-specific bioconjugation
  • Sortase A
  • Targeting

Cite this

Hashad, R. A., Lange, J. L., Tan, N. C. W., Alt, K., & Hagemeyer, C. E. (2019). Engineering Antibodies with C-Terminal Sortase-Mediated Modification for Targeted Nanomedicine. In S. Massa, & N. Devoogdt (Eds.), Bioconjugation : Methods in Molecular Biology (pp. 67-80). (Methods in Molecular Biology; Vol. 2033). Humana Press.