Alpaca single B cell interrogation and heavy-chain-only antibody discovery on an optofluidic platform

Abstract

Background 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. Methods 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. Results Anti-PSMA IgG2/3 titer was detectable by day 97 and increased until reaching a plateau on day 153. On the Beacon, rates of IgG2/3 secretion and target-specific binding likewise peaked around day 130-140. Of the 13 recombinantly expressed VHH-Fc candidates, all but one bound PSMA with a single-digit nanomolar affinity, and five 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. Conclusions 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.

A method for rapid nanobody screening with no bias of the library diversity

“…The numbers of antigen-specific immunoglobulin G (IgG)-secreting B cells in the peripheral blood range from the order of 10 1−4 per 10 6 cells after a single immunization. 19 , 20 , 21 , 22 Thus, directly following the hybridoma technology by diluting an immunized nanobody library expressing E. coli into single cell per well to perform screening for specific monoclonal nanobodies faces the problem of low throughput. 23 To solve this problem, we designed a new strategy to mix 10 n clones per well.…”