Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies

Human V H single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human V H domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (V H H) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human V H single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human V H domains and the selection of binders against a diverse set of antigens. Unlike "camelized" human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the V H /V L interface. Structure determination in complex with hen egg white lysozyme revealed an extended V H binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human V H domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies. Background: Camelid antibody domains are naturally stable and capable of cleft binding. Results: Protein engineering can endow human antibody domains with such properties. Conclusion: Our strategy does not require undesirable antibody framework changes. Significance: Robust building blocks for human therapeutic applications.