Figure 3
Mapping MEDI1912 self-interactions.
Site-localised protection against hydrogen-exchange in MEDI1912 heavy (a) and light (b) chain variable domains. For concentrations at which MEDI1912 is predominantly monomeric (M) and dimeric (D), the difference in uptake of deuterium label was calculated as ΔHDX(M-D). This was normalised per protein, with the region that showed maximum difference set to 100%. Protection due to self-association shows as a positive value on the y-axis (solid line – measured value; dashed line – 99% confidence limit). Amino acids altered in affinity maturation from MEDI-578 are marked in red circles. CDR sequences are listed for those that were affinity matured: red – mutated residue; underlined – significant HDX protection (p < 0.01). A spatial aggregation propensity (SAP) algorithm was applied to structural models of MEDI-578 (c) and MEDI1912 (d) revealing surface exposed hydrophobic patches (red)15. Three amino acids coincident with significant HDX protection in MEDI1912 dimer and SAP prediction: W30, F31 and L56 (blue circles) were chosen for reversion to the corresponding MEDI-578 amino acids: S30, T31, and T56 (red circles). The crystal structure of NGF (coloured green) in complex with the Fab of the MEDI1912 parental antibody, MEDI-578 (blue), (e) reveals that while the VHCDR3 (cyan lines) forms a loop that extends into a cavity within NGF, the amino acid residues 30 and 31 in the VHCDR1 and 56 in the VHCDR2 (blue sticks) are located at the periphery of the binding interface and do not make specific interactions with epitope residues.
