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

Alpaca single B cell interrogation and heavy-chain-only antibody discovery on an optofluidic... In vivo VHH discovery approaches have been limited by the lack of methodologies for camelid B cell interrogation. Here, we report a novel application of the Beacon® optofluidic platform to the discovery of heavy-chain-only antibodies by screening alpaca B cells. Methods for alpaca B cell enrichment, culture, IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH from an alpaca immunized with prostate-specific membrane antigen (PSMA) or a second target. PSMA-specific hits were expressed as VHH-Fc and characterized using label-free techniques. Anti-PSMA IgG2/3 titer plateaued on day 153, when on-Beacon IgG2/3 secretion and target binding rates peaked. Of 13 recombinantly expressed VHH-Fc, all but one bound with nanomolar affinity, and five were successfully humanized. Repertoire sequencing uncovered additional variants within the clonal lineages of the validated hits. The establishment of this workflow extends the powerful Beacon technology to enable rapid VHH discovery directly from natural camelid immune repertoires. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Antibody Therapeutics Oxford University Press

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

13 pages

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References (30)

Publisher
Oxford University Press
Copyright
© The Author(s) 2023. Published by Oxford University Press on behalf of Antibody Therapeutics. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
eISSN
2516-4236
DOI
10.1093/abt/tbad018
Publisher site
See Article on Publisher Site

Abstract

In vivo VHH discovery approaches have been limited by the lack of methodologies for camelid B cell interrogation. Here, we report a novel application of the Beacon® optofluidic platform to the discovery of heavy-chain-only antibodies by screening alpaca B cells. Methods for alpaca B cell enrichment, culture, IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH from an alpaca immunized with prostate-specific membrane antigen (PSMA) or a second target. PSMA-specific hits were expressed as VHH-Fc and characterized using label-free techniques. Anti-PSMA IgG2/3 titer plateaued on day 153, when on-Beacon IgG2/3 secretion and target binding rates peaked. Of 13 recombinantly expressed VHH-Fc, all but one bound with nanomolar affinity, and five were successfully humanized. Repertoire sequencing uncovered additional variants within the clonal lineages of the validated hits. The establishment of this workflow extends the powerful Beacon technology to enable rapid VHH discovery directly from natural camelid immune repertoires.

Journal

Antibody TherapeuticsOxford University Press

Published: Aug 17, 2023

Keywords: VHH; alpaca; single domain antibody; Beacon; single B cell; antibody discovery; humanization; PSMA

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