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.

Submission Details

ID: 8vj9yRoA

Submitter: Shu-Ching Ou

Submission Date: July 11, 2018, 11:41 a.m.

Version: 1

Publication Details
Medina-Cucurella AV;Zhu Y;Bowen SJ;Bergeron LM;Whitehead TA,Biotechnol Bioeng (2018) Pro region engineering of nerve growth factor by deep mutational scanning enables a yeast platform for conformational epitope mapping of anti-NGF monoclonal antibodies. PMID:29663315
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1WWW 1999-09-15 2.2 NGF IN COMPLEX WITH DOMAIN 5 OF THE TRKA RECEPTOR
1SG1 2004-06-01 2.4 Crystal Structure of the Receptor-Ligand Complex between Nerve Growth Factor and the Common Neurotrophin Receptor p75
4ZBN 2015-06-10 2.45 Non-helical DNA Triplex Forms a Unique Aptamer Scaffold for High Affinity Recognition of Nerve Growth Factor
4EDW 2014-04-02 2.48 Nerve Growth Factor in Complex with Fab from humanized version of mouse mAb 911 (tanezumab)
4EDX 2014-04-02 2.5 Nerve Growth Factor in Complex with Fab from mouse mAb 911
5JZ7 2017-01-11 3.4 NGF IN COMPLEX WITH MEDI578 scFv
2IFG 2007-02-13 3.4 Structure of the extracellular segment of human TRKA in complex with nerve growth factor

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.0 Canine Nerve growth factor (cNGF) Q9N2E9 NGF_PONPY
91.5 Canine Nerve growth factor (cNGF) P01138 NGF_HUMAN
92.0 Canine Nerve growth factor (cNGF) Q9N2F1 NGF_PANTR
92.0 Canine Nerve growth factor (cNGF) Q9N2F0 NGF_GORGO
91.0 Canine Nerve growth factor (cNGF) P13600 NGF_BOVIN
92.5 Canine Nerve growth factor (cNGF) Q5ISB0 NGF_SAIBB
93.5 Canine Nerve growth factor (cNGF) Q29074 NGF_PIG