Influence of water-structures and interface conformations on GIST free energy calculations of Format Chain Exchange systems

Despite their wide-ranging applications and undeniable presence on the pharmaceutical market, monoclonal antibodies (mAB) are limited by the nature of their bivalent monospecific format. While mABs can bind a single target with high affinity, the extensive crosstalk fount in many cellular signalling pathways, impairs their therapeutic usefulness in many relevant indications. Bispecific antibodies (bsAB) have been subject to extensive research and development in the recent years, rectifying these issues by being able to bind multiple different antigens while retaining their ability to be directed against a virtually infinite number of targets. Especially obligate asymmetric bispecific antibodies have recently started to pass clinical trials, mimicking the natural shape of antibodies, and thus facing minimal immune response. However, they are held back primarily by the difficulty one faces in their production. Numerous different platforms have been developed with the vast majority of them relying on heterodimerisation of the antibody heavy chain, mediated by the intramolecular interaction of the CH3-CH3 domains in the crystallizable fragment (Fc) of the antibody. Understanding the complexity of this interface is therefore fundamental to inform rational design, streamline optimisation and improve bsAB production. By enabling the study of the dynamics of the CH3 domains and the interactions between them at atomic resolution, molecular dynamics simulations are ideally suited to elucidate their intricate interplay. Furthermore, free energy calculations can be used to give a qualitative understanding of the impact of mutations and thus gauge their stability. Herin, we present a thorough investigation of various CH3-CH3 interfaces employed by the Format Chain Exchange (FORCE) bsAB production platform developed by Roche with the goal to understand their interactions and ability to enable half-molecule exchange. The systems exhibit numerous variations of the Knob-into-Hole (K
by Robert Franz Wild
Masterarbeit University of Innsbruck 2023