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bioRxiv · 10.64898/2026.08.14.744767

Focused framework sampling recovers binding-positive humanized anti-amyloid-β antibodies in a single sorting round

Kim, Y.·Kwon, H.·Song, J.·Lee, Y.·Park, M.·Lee, C.-H.
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Abstract

Therapeutic antibody development requires workflows that integrate antigen-reactive clone discovery with efficient humanization and early developability assessment. Here, we combined immune yeast fragment antigen-binding (Fab) display with single-round focused humanization and applied the workflow to antibodies against amyloid-{beta} (A{beta})-derived preparations. Immunization with A{beta}1-42 aggregate preparations generated a Fab-display library with a diversity of approximately 3.5 x 108. Magnetic enrichment followed by fluorescence-activated cell sorting (FACS) identified three sequence-distinct immunoglobulin G (IgG)-format candidates, of which CLAB17 and CLAB45 were advanced to humanization. Structure-guided libraries sampled framework positions predicted to support complementarity-determining regions (CDRs) or heavy-and light-chain variable-domain packing, and a single FACS round recovered binding-positive variants CLAB17-h2 and CLAB45-h8. Both retained the parental CDRs and showed increased predicted humanness, favorable computational developability triage profiles, and high purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). By enzyme-linked immunosorbent assay (ELISA), CLAB17-h2 showed a lower apparent half-maximal effective concentration (EC50) for A{beta}1-42AggreSure, whereas CLAB45-h8 showed a lower apparent EC50 for pyroglutamate-modified A{beta}3-42 (A{beta}pE3-42). Because the preparations were not resolved into defined assembly states, these antibodies are considered A{beta}-preparation-binding rather than aggregate-state-selective candidates. This workflow provides a practical route from immune-repertoire discovery to binding-positive humanized antibodies.

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Kim, Y., Kwon, H., Song, J., Lee, Y., Park, M., Lee, C.-H.. 2026-08-18. Focused framework sampling recovers binding-positive humanized anti-amyloid-β antibodies in a single sorting round. https://doi.org/10.64898/2026.08.14.744767

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