| Abstract | An attractive feature of human Vₕs over camelid VₕHs as immunotherapeutics is their perceived lower risk of immunogenicity. While human Vₕs can readily be obtained from synthetic phage display libraries, they often suffer from low affinity and poor solubility compared to VₕHs derived from immune libraries. Using SARS-CoV-2 spike protein as a model antigen, we screened a synthetic human Vₕ phage display library and identified a diverse set of antigen-specific Vₕs. However, the Vₕs 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 Vₕ to in vitro affinity maturation. We created a yeast surface display library of Vₕ 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 Vₕs as potentially improved candidates. Characterization of these candidates revealed several Vₕs with improved affinities of up to 100-fold (KDs 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 Vₕ (a process referred to as camelization), we were able to completely solubilize the Vₕ 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 Vₕs from synthetic libraries through a combination of in vitro affinity maturation and minimal camelization. |
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