Optimization of synthetic human VH affinity and solubility through in vitro affinity maturation and minimal camelization.

Abstract

An attractive feature of human V_Hs over camelid V_HHs as immunotherapeutics is their perceived lower risk of immunogenicity. While human V_Hs can readily be obtained from synthetic phage display libraries, they often suffer from low affinity and poor solubility compared to V_HHs 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_Hs. However, the V_Hs 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_Hs as potentially improved candidates. Characterization of these candidates revealed several V_Hs with improved affinities of up to 100-fold (K_Ds 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_Hs from synthetic libraries through a combination of in vitro affinity maturation and minimal camelization.