High-throughput developability assessment of 181 untagged V H H single-domain antibodies

Antibodies composed of only heavy chains are naturally produced in camelids and recognize their target through a single variable domain. Single‑domain antibodies (VHHs) are derived from this camelid variable domain and have multiple advantages over monoclonal antibodies (mAbs), such as their small size (12–15 kDa), better tissue penetration in vivo, thermal and chemical stability, simplicity, and the ability to be fused to Fc-domains as well as other VHHs. As such, VHHs offer an exciting opportunity to develop new therapeutic modalities. The first VHH-based drug was approved in 2018, and to date, a total of five VHH-derived drugs have been approved for therapeutic use. However, in comparison to the 160+ mAb-based drugs that have been approved since 1986, VHHs are relatively new and consequently less studied. To gain insights into VHH stability and developability, we expressed, purified, and characterized a diverse library of VHHs. From a broad sequence space of VHHs, we selected and produced 521 representatives. Of these, we characterized 181 untagged VHHs for key developability endpoints, including titer, purity, aggregation, thermal stability, and hydrophobicity. We compared these VHH characterization data to internal mAb developability acceptance criteria and VHHs from clinically approved drugs, used statistical analyses to connect sequence and structural features to key characterization endpoints, identified optimally developable VHHs and their associated sequence features, and developed predictive machine‑learning models. This work establishes practical guidelines for ranking VHHs by their developability profile and designing VHHs with optimal stability and developability that promise to accelerate the VHH drug development pipeline.