Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.

Gillian Houlihan,Pietro Gatti-Lafranconi,David Lowe,Florian Hollfelder

doi:10.1093/protein/gzv029

Gillian Houlihan, Pietro Gatti-Lafranconi + Show 2 more

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https://doi.org/10.1093/protein/gzv029

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Abstract

In vitro display technologies have proved to be powerful tools for obtaining high-affinity protein binders. We recently described SNAP display, an entirely in vitro DNA display system that uses the SNAP-tag to link protein with its encoding DNA in water-in-oil emulsions. Here, we apply SNAP display for the affinity maturation of a designed ankyrin repeat proteins (DARPin) that binds to the extracellular domain of HER2 previously isolated by ribosome display. After four SNAP display selection cycles, proteins that bound specifically to HER2 in vitro, with dissociation constants in the low- to sub-nanomolar range, were isolated. In vitro affinities of the panel of evolved DARPins directly correlated with the fluorescence intensities of evolved DARPins bound to HER2 on a breast cancer cell line. A stability trade-off is observed as the most improved DARPins have decreased thermostability, when compared with the parent DARPin used as a starting point for affinity maturation. Dissection of the framework mutations of the highest affinity variant, DARPin F1, shows that functionally destabilising and compensatory mutations accumulated throughout the four rounds of evolution.

Highlights

Screening and selection technologies have been fundamental for the isolation of antibodies and other binding proteins (Hoogenboom, 2005; Leemhuis et al, 2005; Bradbury et al, 2011; Colwill and Graslund, 2011)
The starting point for affinity maturation via SNAP display was designed ankyrin repeat proteins (DARPin) G3-HAVD, an anti-HER2 binder previously isolated by ribosome display (KD of 269 nM) from the N2C consensus designed ankyrin repeat library (Zahnd et al, 2007a,b)
Ligation- and transformationindependent assembly of the SNAP display construct via overlap polymerase chain reaction (PCR) (Supplementary Fig. S1) allows the construction of large libraries without introducing mutations in regulatory regions

Summary

Introduction

Screening and selection technologies have been fundamental for the isolation of antibodies and other binding proteins (Hoogenboom, 2005; Leemhuis et al, 2005; Bradbury et al, 2011; Colwill and Graslund, 2011). Binding scaffolds with superior biophysical properties have been engineered as alternatives for molecular recognition (Binz et al, 2003; Gebauer and Skerra, 2009) These alternatives include designed ankyrin repeat proteins (DARPins), designed leucine-rich repeat proteins with varying curvatures (Ramisch et al, 2014), affibodies (Nord et al, 1997; Boersma and Plückthun, 2011), monobodies (Koide et al, 1998), cystine-knot miniproteins (Kolmar, 2009) and anticalins (Beste et al, 1999). Such scaffolds consist of a characteristic framework that presents surface loops with randomised regions, setting up potential binding interfaces to interact with different target antigens

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