Pro region engineering of nerve growth factor by deep mutational scanning enables a yeast platform for conformational epitope mapping of anti‐NGF monoclonal antibodies

• Published in: Biotechnology and bioengineering 2018-08, Vol.115 (8), p.1925-1937
• Main Authors: Medina‐Cucurella, Angélica V., Zhu, Yaqi, Bowen, Scott J., Bergeron, Lisa M., Whitehead, Timothy A.
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - immunology; Antibodies, Neutralizing - immunology; Cell Surface Display Techniques - methods; Chronic pain; conformational epitope mapping; Crystallography; deep mutational scanning; Drug development; Epitope Mapping; Flow cytometry; Fluorescence; Growth factors; Monoclonal antibodies; Mutagenesis; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutants; Mutation; Nerve growth factor; Nerve Growth Factor - genetics; Nerve Growth Factor - metabolism; Neutralizing; Opioids; Pain; pro region; Protein Binding; Protein engineering; Proteins; Residues; tanezumab; Yeast; yeast display; Yeasts - genetics; Yeasts - metabolism
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.26706

Nerve growth factor (NGF) plays a central role in multiple chronic pain conditions. As such, anti‐NGF monoclonal antibodies (mAbs) that function by antagonizing NGF downstream signaling are leading drug candidates for non‐opioid pain relief. To evaluate anti‐canine NGF (cNGF) mAbs we sought a yeast surface display platform of cNGF. Both mature cNGF and pro‐cNGF displayed on the yeast surface but bound conformationally sensitive mAbs at most 2.5‐fold in mean fluorescence intensity above background, suggesting that cNGF was mostly misfolded. To improve the amount of folded, displayed cNGF, we used comprehensive mutagenesis, FACS, and deep sequencing to identify point mutants in the pro‐region of canine NGF that properly enhance the folded protein displayed on the yeast surface. Out of 1,737 tested single point mutants in the pro region, 49 increased the amount of NGF recognized by conformationally sensitive mAbs. These gain‐of‐function mutations cluster around residues A‐61–P‐26. Gain‐of‐function mutants were additive, and a construct containing three mutations increased amount of folded cNGF to 23‐fold above background. Using this new cNGF construct, fine conformational epitopes for tanezumab and three anti‐cNGF mAbs were evaluated. The epitope revealed by the yeast experiments largely overlapped with the tanezumab epitope previously determined by X‐ray crystallography. The other mAbs showed site‐specific differences with tanezumab. As the number of binding epitopes of functionally neutralizing anti‐NGF mAbs on NGF are limited, subtle differences in the individual interacting residues on NGF that bind each mAb contribute to the understanding of each antibody and variations in its neutralizing activity. These results demonstrate the potential of deep sequencing‐guided protein engineering to improve the production of folded surface‐displayed protein, and the resulting cNGF construct provides a platform to map conformational epitopes for other anti‐neurotrophin mAbs. A deep sequencing‐guided protein engineering workflow combined with yeast display was developed to increase the production of folded canine nerve growth factor (NGF). Dozens of mutations within the pro‐region of NGF enhanced the display of mature NGF on the yeast surface. Combining a subset of these beneficial mutations enabled a NGF construct that was used for conformational epitope mapping of different anti‐NGF monoclonal antibodies.