Linker-dependent modulation of anti-CD22 scFv antibody stability and avidity: Combined structural and experimental insights.

Monoclonal antibodies targeting CD22, a B-cell surface antigen overexpressed in hematologic malignancies such as acute lymphoblastic leukemia (ALL), have shown promise for targeted immunotherapy. Single-chain variable fragments (scFvs) offer enhanced modularity for CAR-T platforms, and linker length between VH and VL domains is known to influence their conformation and binding. Here, we systematically compared two anti-CD22 scFvs with short (GGGGS) or long ((GGGGS)₄) linkers. Using molecular dynamics simulations (in aqueous and membrane environments), bio-layer interferometry (BLI), and flow cytometry, we found that membrane proximity influences binding, and the short-linker scFv exhibited higher affinity (Kd = 5.1 nM vs. 42.1 nM), slower dissociation, and greater avidity for CD22. Binding free energy decomposition and RMSD analyses revealed a more stable interface for the short-linker construct, especially near the membrane. SEC analysis showed a modestly higher dimeric fraction for the short-linker scFv, although flow cytometry indicated comparable CD22 binding across monomeric and dimeric forms. These findings support linker engineering as a strategy to optimize scFv performance and inform the rational design of CAR-T receptors with improved therapeutic potential.