Single-residue engineering of lambda (λ) antibody light chains reduces conformational flexibility and enhances thermal stability.

Monoclonal antibodies with lambda (λ) light chains are less commonly used in therapeutics due to their lower biophysical stability compared to kappa (κ) variants. Here, we identify a conserved glycine residue (Gly111) in the λ light chain hinge as a driver of large-scale Fab elbow-angle transitions. Using microsecond-scale molecular dynamics simulations of the EBV-neutralizing Fab AMMO1, we show that substituting Gly111 with threonine (G111T) increases the free energy barrier between conformational states, effectively arresting these transitions. Structural and biophysical analyses-including crystallography, differential scanning fluorimetry, and surface plasmon resonance-confirm that the mutation maintains Fab architecture and antigen binding while increasing thermal stability by up to 2.5 °C. The same mutation applied to a second λ-Fab yielded similar stabilization, and simulations of three clinical λ-Fabs revealed consistent reductions in elbow-angle flexibility. These results demonstrate a generalizable, single-residue engineering strategy to enhance the stability of λ-based Fabs without compromising function, with direct implications for therapeutic antibody development and manufacturability.