The heavy chain variable (VH) domain is the smallest functional antigen-binding fragment of antibodies. The VH domain contains only three of the six complementarity determining regions (CDRs) of a conventional antibody, which are the hypervariable loops that mediate antigen recognition. Our objective is to design an autonomous human VH domain that can be used as a scaffold for the rapid design of synthetic phage-displayed VH libraries. Because of their smaller size, VH domains should be highly expressed in bacterial hosts, and due to their monomeric nature, are better suited for in vitro selection technologies like phage display. However, most isolated human VH domains are usually difficult to express in E. coli and tend to aggregate, properties mainly attributed to the exposure to the solvent of a large hydrophobic region following the removal of the light chain. In this study, we describe the discovery and characterization of a stable, monomeric human VH domain derived from a humanized antibody. Using rational design, naïve phage-displayed libraries and biophysical characterization of purified proteins, we were able to obtain variants which are 5 to 10 times better expressed that the wild-type 4D5 VH domain, are essentially monomeric, resistant to aggregation, and refold reversibly upon thermodenaturation. The results emphasize the importance of engineering the VH / VL interface for solubilizing an isolated human VH domain and may prove useful for the construction of functional synthetic phage-displayed VH libraries.

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