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

10 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

Funding

K.J.M. acknowledges support from the NIH/NCI training grant T32 CA186897 and an American Cancer Society–2017 Seattle Gala Paddle Raise Postdoctoral Fellowship, PF-18-118-01-CDD. B.R.K. and K.A.P. acknowledge support from the Ryan Fellowship, the International Institute for Nanotechnology at Northwestern University, and the National Science Foundation Graduate Research Fellowship under Grant DGE-1842165. Research reported in this publication was supported by AFRL FA8650-15-2-5518, ARO MURI FA9550-16-1-0150, and from the National Cancer Institute of the National Institutes of Health under Award Numbers U54CA199091and U01CA199315. This work made use of the BioCryo facility of Northwestern University’s NUANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205); and made use of facilities partially supported by the MRSEC program (NSF DMR-1720139) at the Materials Research Center.

ASJC Scopus subject areas

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

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