Mutational and biophysical robustness in a prestabilized monobody
| Autor: | Colin J. Jackson, Li Lynn Tan, James S. Green, Sebastian S. Broendum, Robert J. Falconer, Peter G. Chandler, Benjamin T. Porebski, Blake T. Riley, David E. Hoke, Trent P. Munro, Ashley M. Buckle, Malcolm Buckle |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Scaffold VEGF receptors Fibronectin Type III Domain LTS long-term stability non-antibody scaffold Complementarity determining region FN3 fibronectin type III Biochemistry mini proteins Antibodies loop grafting 03 medical and health sciences consensus design fibronectin CDR complementarity determining region Humans stability–function trade-off adnectin Molecular Biology Thermostability 030102 biochemistry & molecular biology biology Chemistry Robustness (evolution) Cell Biology monobodies Directed evolution Matrix Attachment Regions Vascular Endothelial Growth Factor Receptor-2 Peptide Fragments Monobody Fibronectins 030104 developmental biology Mutation Biophysics biology.protein Genetic Engineering Function (biology) Protein Binding Research Article |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability–function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics. |
| Databáze: | OpenAIRE |
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