Design of Synthetic Autonomous VH Domain Libraries and Structural Analysis of a VH Domain Bound to Vascular Endothelial Growth Factor

Abstract We compared the capacity of an autonomous heavy chain variable (V H ) domain (VH-B1a) to support diversity within its antigen-binding site relative to the conventional antigen-binding fragment (Fab) from which it was derived. We find that VH-B1a can tolerate significant diversity within all three complementarity-determining regions (CDRs) and also within framework 3, and thus, VH-B1a and the Fab are similar in terms of the regions of the antigen-binding site that can tolerate diversity without compromising stability. We constructed libraries of synthetic V H domains and isolated binders with moderate affinity for vascular endothelial growth factor (VEGF) from a library in which only CDR3 was randomized. One binder was subjected to affinity maturation to derive an autonomous V H domain (VH-V1a) that recognized both human and mouse VEGF with high affinity ( K D  = 16 nM or 10 nM, respectively). Structural analysis revealed that VH-V1a binds to an epitope that is distinct from the epitopes of a natural VEGF receptor and six different anti-VEGF Fabs. Moreover, VH-V1a recognizes VEGF by using an unusual paratope consisting predominantly of CDR3 but with significant contributions from framework residues within the former light chain interface. These results suggest that VH-B1a and other autonomous V H domains may be useful scaffolds to support both conventional libraries with antigen-binding sites built from the three CDR loops and, also, nonconventional libraries with antigen-binding sites built from CDR3 and the former light chain interface.