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Abstract:
De novo protein design has evolved into a powerful tool providing a new generation of
potential therapeutics. Recently, we have designed novel proteins which act as granulocyte-colony stimulating factor receptor (G-CSFR) agonists utilizing a topological rescaffolding (1) and refactoring strategy (2). Unlike the native receptor ligand (G-CSF), which represents an important immunotherapeutic, these proteins are small, highly stable, and can be produced with high yields in Escherichia coli. In order to optimize such promising molecules, we developed a pipeline that is able to quickly and efficiently affinity-maturate de novo designed proteins. In essence, a bacterial display is used in combination with computationally-designed
libraries, utilizing the new design software Damietta. Affinity enhanced variants were
investigated as tandem fusions in a cell-based assay, from which the most active variant nearly reached the activity of recombinant human G-CSF itself. Additionally, high affinity variants were utilized to design competitive G-CSFR antagonists which can serve as a protein-
based drug to treat G-CSF dependent types of cancer.
