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

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-β (Aβ)-derived preparations. Immunization with Aβ _1-42 aggregate preparations generated a Fab-display library with a diversity of approximately 3.5 × 10 ⁸ . 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 (EC ₅₀ ) for Aβ _1-42 AggreSure, whereas CLAB45-h8 showed a lower apparent EC ₅₀ for pyroglutamate-modified Aβ _3-42 (Aβ _pE3-42 ). Because the preparations were not resolved into defined assembly states, these antibodies are considered Aβ-preparation-binding rather than aggregate-state-selective candidates. This workflow provides a practical route from immune-repertoire discovery to binding-positive humanized antibodies.