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.

Feline Interleukin-31 Shares Overlapping Epitopes with the Oncostatin M Receptor and IL-31RA

“…fIL-31 immobilization on the CM5 sensor and the binding measurements were conducted as previously described. 24 Data were analyzed with Biacore T200 Evaluation software by using the method of double referencing. The resulting curve was fit with the 1:1 binding model.…”

“…Surface plasmon resonance was performed on a Biacore T200 instrument (GE Healthcare, Pittsburgh, PA) to measure binding affinities of fOSMR-ECD and mAb#1 for fIL-31. fIL-31 immobilization on the CM5 sensor and the binding measurements were conducted as previously described . Data were analyzed with Biacore T200 Evaluation software by using the method of double referencing.…”

Understanding and Modulating Antibody Fine Specificity: Lessons from Combinatorial Biology

“…Cys, Pro and Gly are excluded as substituents, as the first one can cause structural changes due to the formation of non-natural disulfide bonds, and the two others can distort the backbone. Recent advances in epitope mapping by yeast display have taken advantage of a very efficient way of constructing single-pot saturation mutagenesis libraries containing all the single-mutated alanine replacement variants derived from a given antigen [ 68 , 69 , 70 , 71 ]. Yeast display has been extremely useful to delineate the whole landscape of potential escape mutations of SARS-CoV-2 emerging receptor binding domain (RBD) variants [ 21 , 22 , 72 , 73 , 74 , 75 ].…”

“…Furthermore, NGS analysis of selected multi-clonal populations is powerful enough to underscore a plethora of significant selection-driven molecular patterns beyond the few molecules that typically dominate the enrichment procedure and can be identified through limited clonal screening. Deep sequencing of libraries and library products is becoming a common practice in many laboratories [ 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 116 ].…”

An antibody targeting type III secretion system induces broad protection against Salmonella and Shigella infections

“…To obtain high-resolution conformational epitope mapping, we used deep mutational scanning (DMS) associated with YSD to identify key IpaD amino acid positions necessary for IpaD-318 binding [74][75][76][77][78][79]. This method consists of the functional assessment of every possible amino acid change at each position in a protein.…”