Optimization of synthetic human V(H) affinity and solubility through in vitro affinity maturation and minimal camelization.

An attractive feature of human V(H)s over camelid V(H)Hs as immunotherapeutics is their perceived lower risk of immunogenicity. While human V(H)s can readily be obtained from synthetic phage display libraries, they often suffer from low affinity and poor solubility compared to V(H)Hs derived from immune libraries. Using SARS-CoV-2 spike protein as a model antigen, we screened a synthetic human V(H) phage display library and identified a diverse set of antigen-specific V(H)s. However, the V(H)s exhibited low affinity, and many had low solubility; that is, they were prone to aggregation. To explore the feasibility of improving the affinity, we subjected a representative V(H) to in vitro affinity maturation. We created a yeast surface display library of V(H) variants employing a site-saturated mutagenesis approach targeting complementarity-determining regions and selected against the target antigen. Next-generation sequencing of the selected variants, combined with structural modeling, identified a set of V(H)s as potentially improved candidates. Characterization of these candidates revealed several V(H)s with improved affinities of up to 100-fold (K(D)s as low as 3 nM) and potent neutralization capabilities; however, they still showed significant aggregation. By introducing as few as two camelid residues into the framework region 2 of a high-affinity V(H) (a process referred to as camelization), we were able to completely solubilize the V(H) without compromising its affinity and other important attributes, including thermostability and protein A binding. This study demonstrates the feasibility of generating high-affinity, -solubility, and -stability human V(H)s from synthetic libraries through a combination of in vitro affinity maturation and minimal camelization.