Synthetic Tuning of Domain Stoichiometry in Nanobody–Enzyme Megamolecules
| Autor: | Eric Berens, Daniel J Sykora, Milan Mrksich, Justin A. Modica, Vinayak P. Dravid, Zena Werb, Blaise R. Kimmel, Kelly Parker, Kevin J. Metcalf, Raymond Dai |
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| Rok vydání: | 2020 |
| Předmět: |
Protein domain
Biomedical Engineering Pharmaceutical Science Antineoplastic Agents Bioengineering 02 engineering and technology Proof of Concept Study 01 natural sciences Article Structure-Activity Relationship Cell Line Tumor Humans Prodrugs Avidity Cytotoxicity Pharmacology chemistry.chemical_classification biology 010405 organic chemistry Chemistry Organic Chemistry Single-Domain Antibodies Prodrug 021001 nanoscience & nanotechnology Enzyme assay Enzymes 0104 chemical sciences Enzyme Covalent bond biology.protein Biophysics 0210 nano-technology Biotechnology Conjugate |
| Zdroj: | Bioconjug Chem |
| ISSN: | 1520-4812 1043-1802 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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