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

Protein Engineering, Design & Selection : PEDS
L SchwaigerlehnerRenate Kunert

Abstract

Humanized monoclonal antibodies (mAbs) are among the most promising modern therapeutics, but defined engineering strategies are still not available. Antibody humanization often leads to a loss of affinity, as it is the case for our model antibody Ab2/3H6 (PDB entry 3BQU). Identifying appropriate back-to-mouse mutations is needed to restore binding affinity, but highly challenging. In order to get more insight, we have applied molecular dynamics simulations and correlated them to antibody binding and expression in wet lab experiments. In this study, we discuss six mAb variants and investigate a tyrosine conglomeration, an isopolar substitution and the improvement of antibody binding towards wildtype affinity. In the 3D structure of the mouse wildtype, residue R94h is surrounded by three tyrosines which form a so-called 'tyrosine cage'. We demonstrate that the tyrosine cage has a supporting function for the CDRh3 loop conformation. The isopolar substitution is not able to mimic the function appropriately. Finally, we show that additional light chain mutations can restore binding to wildtype-comparable level, and also improve the expression of the mAb significantly. We conclude that the variable light chain of Ab2/3H6 is of undere...Continue Reading

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Citations

Jul 28, 2019·Applied Microbiology and Biotechnology·Linda SchwaigerlehnerRenate Kunert
Dec 31, 2020·Cancers·Bernhard RoitherWolfgang Schreiner

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Methods Mentioned

BETA
transfection
biosensors
biosensor
X-ray
transfections
bio-layer interferometry
bio-layer

Software Mentioned

abYsis
GROMOS11
GROMOS
The Sulfinator

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