The well-characterized binding site of the therapeutic antibody Avastin was selected for an initial proof of concept experiment. A 267 clone Fab library was expressed on the surface of phage. This library was separated into bound and expressed fractions in a 72hr. equilibrium binding reaction. Both fractions were then sequenced using the MiSeq system. 267 mutations were quantitatively assessed in a single experiment. The results of this study can conveniently be presented as an engineering heatmap. MapEng™ identified all higher, lower, and neutral mutations

Avastin and VEGF

Consistency of the method can be seen by comparing MapEng™ values between synonymous codons 

MapEng™ simultaneously provided the relative binding affinities of all single amino acid variants for the entire region analyzed:
   •Most of the mutations negatively impacted binding
   •Many mutations adjacent to the core binding site were well tolerated
   •A smaller number of novel up mutations were identified in the CDR region.
 

The accuracy of the binding data from multiple MapEng™ experiments was assessed against the Ala scan results originally presented by Muller et. al., Structure 6: 1153-67 (1998).   The MapEng™ data show good agreement with the original Ala scan data.  The MapEng™ data also show good reproducibility across different samples (A,B), different sequencing runs (A,C) and different sampling techniques (A,D).

The plasticity of individual sites in VH CDR3, as defined by the available number of useful substitutions, was mapped onto the co-crystal structure of VEGF (sticks ± shading) bound to Avastin (spheres).  These data show a decrease in residue plasticity as the proximity to the binding site increases.