Pro region engineering of nerve growth factor by deep mutational scanning enables a yeast platform for conformational epitope mapping of anti‐NGF monoclonal antibodies
Abstract 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.
유료 다운로드의 경우 해당 사이트의 정책에 따라 신규 회원가입, 로그인, 유료 구매 등이 필요할 수 있습니다. 해당 사이트에서 발생하는 귀하의 모든 정보활동은 NDSL의 서비스 정책과 무관합니다.
원문복사신청을 하시면, 일부 해외 인쇄학술지의 경우 외국학술지지원센터(FRIC)에서
무료 원문복사 서비스를 제공합니다.
NDSL에서는 해당 원문을 복사서비스하고 있습니다. 위의 원문복사신청 또는 장바구니 담기를 통하여 원문복사서비스 이용이 가능합니다.
- 이 논문과 함께 출판된 논문 + 더보기