Synthetic Tuning of Domain Stoichiometry in Nanobody-Enzyme Megamolecules

Kevin J. Metcalf, Blaise R. Kimmel, Daniel J. Sykora, Justin A. Modica, Kelly A. Parker, Eric Berens, Raymond Dai, Vinayak P. Dravid, Zena Werb, Milan Mrksich*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

This paper presents a method to synthetically tune atomically precise megamolecule nanobody-enzyme conjugates for prodrug cancer therapy. Previous efforts to create heterobifunctional protein conjugates suffered from heterogeneity in domain stoichiometry, which in part led to the failure of antibody-enzyme conjugates in clinical trials. We used the megamolecule approach to synthesize anti-HER2 nanobody-cytosine deaminase conjugates with tunable numbers of nanobody and enzyme domains in a single, covalent molecule. Linking two nanobody domains to one enzyme domain improved avidity to a human cancer cell line by 4-fold but did not increase cytotoxicity significantly due to lowered enzyme activity. In contrast, a megamolecule composed of one nanobody and two enzyme domains resulted in an 8-fold improvement in the catalytic efficiency and increased the cytotoxic effect by over 5-fold in spheroid culture, indicating that the multimeric structure allowed for an increase in local drug activation. Our work demonstrates that the megamolecule strategy can be used to study structure-function relationships of protein conjugate therapeutics with synthetic control of protein domain stoichiometry.

Original languageEnglish (US)
Pages (from-to)143-152
Number of pages10
JournalBioconjugate Chemistry
Volume32
Issue number1
DOIs
StatePublished - Jan 20 2021

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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