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

Area of Science:

• Biochemistry
• Immunology
• Structural Biology

Background:

• Monoclonal antibodies with lambda (λ) light chains are less utilized therapeutically due to lower biophysical stability than kappa (κ) variants.
• The conformational flexibility of the Fab fragment, particularly the elbow angle, contributes to reduced stability in λ light chains.

Purpose of the Study:

• To identify key residues influencing Fab elbow-angle transitions in λ light chains.
• To develop a strategy for enhancing the biophysical stability of λ-based therapeutic antibodies.

Main Methods:

• Microsecond-scale molecular dynamics simulations of the EBV-neutralizing Fab AMMO1.
• Site-directed mutagenesis (Gly111 to Threonine).
• Crystallography, differential scanning fluorimetry, and surface plasmon resonance for structural and biophysical characterization.

Main Results:

• A conserved glycine at position 111 (Gly111) in the λ light chain hinge was identified as critical for large-scale Fab elbow-angle transitions.
• The G111T mutation increased the free energy barrier, reducing conformational transitions and enhancing thermal stability by up to 2.5°C.
• The mutation was validated in a second λ-Fab and shown to reduce flexibility in clinical λ-Fabs via simulations, without compromising antigen binding.

Conclusions:

• A generalizable single-residue engineering strategy can enhance the stability of λ-based Fabs.
• This approach improves thermal stability and reduces conformational flexibility without sacrificing antibody function.
• The findings have significant implications for the development and manufacturability of λ-based therapeutic antibodies.