In vivo discovery approaches for single domain antibodies such as VHH have been limited by the lack of methodologies available for camelid B cell interrogation. Here, we report a novel application of the Berkeley Lights Beacon® optofluidic platform to the discovery of heavy-chain-only antibodies by screening single B cells from alpacas immunized with two different targets. Custom methods for alpaca B cell enrichment, culture, on-Beacon IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH candidates from an alpaca immunized with either human prostate specific membrane antigen (PSMA) or a second blinded target, in a proof-of-concept study. PSMA-specific VHH hits discovered on the Beacon were recombinantly expressed as VHH-Fc, purified, and characterized using label-free techniques. All but one VHH-Fc bound PSMA with a single-digit nanomolar affinity, and four candidates were successfully humanized in silico using a rapid bulk humanization approach. In addition, next-generation repertoire sequencing was performed at longitudinal timepoints after immunization, uncovering additional variants within the clonal lineages of the validated hits discovered on the Beacon platform. The establishment of this single B cell VHH discovery workflow extends the powerful Beacon technology to enable rapid discovery of VHH directly from natural camelid immune repertoires.

Competing Interest Statement
The authors have declared no competing interest.