Journal: PLoS Computational Biology

Article Title: Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces

doi: 10.1371/journal.pcbi.1007207

Figure Lengend Snippet: ( a ) G6 and 18 low-energy designs, each encoding 4–10 mutations relative to G6 (number of mutations is indicated above the bars) were tested for binding using yeast display at 8 nM VEGF concentration, resulting in seven designs that showed comparable or higher binding signal compared to G6. G6 des1 and G6 des13 were chosen for further characterization (colored in blue and orange, respectively). ( b ) SPR kinetic analysis of VEGF binding with twofold dilutions from a maximal concentration of 100 nM by G6, G6 des1 , and G6 des13 Fabs demonstrated faster binding on-rate in the designs ( k a = 2.3 * 10 5 M -1 s -1 , 3.27 * 10 5 M -1 s -1 and 5.3 * 10 5 M -1 s -1 , respectively). G6 des13 also improved binding off-rate ( k d = 3.2 * 10 −5 s -1 compared to 6 * 10 −5 s -1 in G6), resulting in an improved dissociation constant ( K D = 60 pM compared to 270 pM in G6). ( c & d ) Thermal denaturation and temperature of aggregation onset experiments, respectively, using microscale thermophoresis indicated substantially higher apparent stability in the designs. ( e ) Computational mutation-tolerance mapping indicated 11 positions at the vL-vH interface of the anti-VEGF antibody G6 (spheres) with potentially tolerated mutations. Thumbnails indicate selected mutations in a model structure of G6 des13 relative to G6 (gray). ( f ) Expression levels in HEK293 cells of G6 and the designs formatted as IgG were measured using Western blot analysis showing approximately an order of magnitude improvement in IgG expression levels for the designs. (g) Native mass-spectrometry analysis exhibited higher tolerance to applied collision energy in G6 des13 compared to G6, both formatted as IgG. The error bars represent standard deviations inferred from three repeats.

Article Snippet: The wild-type and designed antibodies were tested for binding by flow cytometry with 8 nM biotinylated VEGF (Recombinant Human VEGF 165, Biotinylated Protein R&D systems).

Techniques: Binding Assay, Concentration Assay, Microscale Thermophoresis, Mutagenesis, Expressing, Western Blot, Mass Spectrometry