Generation of a high-fidelity antibody against nerve growth factor using library scanning mutagenesis and validation with structures of the initial and optimized Fab-antigen complexes

Porte, Sherry L. La; Ultsch, Mark; Ho, Wei-Hsien; Foletti, Davide; Forgie, Alison; Lindquist, Kevin C.; Shelton, David L.; Pons, Jaume

doi:10.4161/mabs.28677

Cited by 8 publications

(8 citation statements)

References 37 publications

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“…Thus, both tanezumab and mAb#1 recognize a conformational epitope on cNGF and all mAbs function as cNGF antagonists. This biochemical data are corroborated with a previously published co‐structure of tanezumab with hNGF (La Porte et al, ) that reveals binding mainly at the homodimer interface between subunits in a conformation that requires properly folded hNGF. In the remainder of this work we use the term “folded” to describe cNGF that is recognized by these conformationally sensitive mAbs.…”

Section: Resultssupporting

confidence: 87%

“…Tanezumab, a humanized anti‐NGF mAb, was expressed recombinantly in CHO cells based upon the published sequences (La Porte et al, ) on hIgG2/kappa constant regions. This antibody was purified using Protein A resin, dialyzed into PBS, and sterile filtered.…”

Section: Methodsmentioning

confidence: 99%

“…These mAbs function by interfering with binding to p75NTR and/or TrkA. Humanized Tanezumab (La Porte et al, ) is furthest along in a phase III clinical trial. Comprehensive reviews of clinical studies with tanezumab and others mAbs are found elsewhere (Chang, Hsu, Hottinger, & Cohen, ; Kumar & Mahal, ).…”

Section: Introductionmentioning

confidence: 99%

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

Medina-Cucurella

Zhu

Bowen

et al. 2018

Biotech & Bioengineering

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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.

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Section: Resultssupporting

confidence: 87%

Section: Methodsmentioning

confidence: 99%

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

Medina-Cucurella

Zhu

Bowen

et al. 2018

Biotech & Bioengineering

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“…Also available are the structures of NGF bound to its receptors (Wiesmann et al, 1999; He and Garcia, 2004; Wehrman et al, 2007; Feng et al, 2010), neutralizing antibodies (Covaceuszach et al, 2008; La Porte et al, 2014), and small ligands, such as lysophospholipids (Tong et al, 2012; Sun and Jiang, 2015) and DNA-aptamers (Jarvis et al, 2015). These studies have led to an emerging picture of the binding determinants on NGF to its key receptors.…”

Section: Introductionmentioning

confidence: 99%

Conformational Rigidity within Plasticity Promotes Differential Target Recognition of Nerve Growth Factor

Paoletti

Chiara

Kelly

et al. 2016

Front. Mol. Biosci.

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Nerve Growth Factor (NGF), the prototype of the neurotrophin family, is essential for maintenance and growth of different neuronal populations. The X-ray crystal structure of NGF has been known since the early '90s and shows a β-sandwich fold with extensive loops that are involved in the interaction with its binding partners. Understanding the dynamical properties of these loops is thus important for molecular recognition. We present here a combined solution NMR/molecular dynamics study which addresses the question of whether and how much the long loops of NGF are flexible and describes the N-terminal intrinsic conformational tendency of the unbound NGF molecule. NMR titration experiments allowed identification of a previously undetected epitope of the anti-NGF antagonist antibody αD11 which will be of crucial importance for future drug lead discovery. The present study thus recapitulates all the available structural information and unveils the conformational versatility of the relatively rigid NGF loops upon functional ligand binding.

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“…In both cases, a standard mAb crystal structure was used as the mAb moiety (PDB ID: 1IGT, Harris et al 1997). A ligand-bound anti-NGF Fab co-crystal structure (PDB ID: 4EDX 34 ) was used to generate the remainder of the structure for the mAb-dAb incorporating an anti-NGF dAb; similarly, a ligand-bound anti-VEGF dAb co-crystal structure (generated in-house) was used to recreate the remainder of the proposed structure for the mAb-dAb incorporating an anti-VEGF mAb-dAb. In both cases, the schematic structural model took into account whether the target was an anti-parallel (VEGF) or parallel (NGF) dimer.…”

Section: Discussionmentioning

confidence: 99%

‘In-Format’ screening of a novel bispecific antibody format reveals significant potency improvements relative to unformatted molecules

Scott

Lee

Wake

et al. 2016

mAbs

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Bispecific antibodies (BsAbs) are emerging as an important class of biopharmaceutical. The majority of BsAbs are created from conventional antibodies or fragments engineered into more complex configurations. A recurring challenge in their development, however, is the identification of components that are optimised for inclusion in the final format in order to deliver both efficacy and robust biophysical properties. Using a modular BsAb format, the mAb-dAb, we assessed whether an 'in-format' screening approach, designed to select format-compatible domain antibodies, could expedite lead discovery. Human nerve growth factor (NGF) was selected as an antigen to validate the approach; domain antibody (dAb) libraries were screened, panels of binders identified, and binding affinities and potencies compared for selected dAbs and corresponding mAb-dAbs. A number of dAbs that exhibited high potency (IC 50 ) when assessed in-format were identified. In contrast, the corresponding dAb monomers had »1000-fold lower potency than the formatted dAbs; such dAb monomers would therefore have been omitted from further characterization. Subsequent stoichiometric analyses of mAb-dAbs bound to NGF, or an additional target antigen (vascular endothelial growth factor), suggested different target binding modes; this indicates that the observed potency improvements cannot be attributed simply to an avidity effect offered by the mAb-dAb format. We conclude that, for certain antigens, screening na€ ıve selection outputs directly in-format enables the identification of a subset of format-compatible dAbs, and that this offers substantial benefits in terms of molecular properties and development time.

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Generation of a high-fidelity antibody against nerve growth factor using library scanning mutagenesis and validation with structures of the initial and optimized Fab-antigen complexes

Cited by 8 publications

References 37 publications

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

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

Conformational Rigidity within Plasticity Promotes Differential Target Recognition of Nerve Growth Factor

‘In-Format’ screening of a novel bispecific antibody format reveals significant potency improvements relative to unformatted molecules

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