Lessons learned from merging wet lab experiments with molecular simulation to improve mAb humanization.

Area of Science:

• Biochemistry
• Immunology
• Structural Biology

Background:

• Humanized monoclonal antibodies (mAbs) are vital therapeutics, yet engineering strategies to maintain affinity are lacking.
• Antibody humanization frequently causes affinity loss, exemplified by the model antibody Ab2/3H6.
• Restoring binding affinity requires identifying specific back-to-mouse mutations, a complex challenge.

Purpose of the Study:

• To investigate strategies for restoring binding affinity in humanized monoclonal antibodies.
• To understand the role of specific structural features, like tyrosine cages, in antibody function.
• To evaluate the impact of light chain mutations on antibody binding and expression.

Main Methods:

• Utilized molecular dynamics simulations to analyze antibody behavior.
• Correlated simulation data with experimental wet lab results on antibody binding and expression.
• Generated and characterized six distinct mAb variants, including those with tyrosine conglomerations and isopolar substitutions.

Main Results:

• Identified a 'tyrosine cage' in the mouse wildtype antibody supporting CDRh3 loop conformation.
• Demonstrated that isopolar substitutions do not effectively mimic the tyrosine cage's function.
• Showed that light chain mutations successfully restored wildtype-comparable binding affinity and enhanced mAb expression.

Conclusions:

• The variable light chain of Ab2/3H6 plays a critical, underestimated role in antigen interaction (mAb 2F5).
• Targeting light chain modifications offers a viable strategy for improving humanized mAb therapeutics.
• Structural insights from simulations can guide experimental engineering of antibodies.