title: 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 (VH) 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 VH 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 VH domain (VH-V1a) that recognized both human and mouse VEGF with high affinity (KD=16nM or 10nM, 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 VH 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.
authors.affiliation: Department of Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Ma
authors.forename: Pierre A
authors.initials: PA
authors.lastname: Barthelemy
authors.forename: Lionel
authors.initials: L
authors.lastname: Rouge
authors.forename: Christian
authors.initials: C
authors.lastname: Wiesmann
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D000939:Epitopes / Q000378:metabolism; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D050505:Mutant Proteins; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse