Deciphering Binding Patterns of Therapeutic Antibodies with Immune Cells : From Method Development to Application

Abstract: Reversible binding, for example between signaling molecules and receptors on the cell surface, is one of the main means to communicate information in cellular systems. Knowledge about how molecules interact is crucial for both understanding biological function and for therapeutic intervention. The cellular environment often makes ligand-receptor interactions complex with the membrane providing structural support and containing other components that interfere with the interaction. One of the fastest growing drug classes for targeting cellular receptors are monoclonal antibodies (mAb), in particular within oncology. Therapeutic mAbs can have direct effects on target cells mediated via the Fab-domain and immune-related effects that are mediated via the Fc-domain. An example of the latter is activation of the complement system by binding of its first component C1q to Fc-domains. Furthermore, immune cells can recognize Fc-domains via Fc-receptors and cause target cell death by a process called antibody-dependent cellular cytotoxicity (ADCC).Increased understanding about structure-binding-function relationships facilitates rational drug design, as has been demonstrated with the development of next-generation mAbs that harbor a structural modification on their Fc-domain that strengthens the interaction with immune cells thereby increasing ADCC efficacy. In this thesis, assays for characterizing mAb binding and mAb mediated interactions on live cells were developed and applied to illustrate how detailed knowledge about binding processes helps to understand the relation between binding and biological function.Paper I describes a protocol for real-time interaction analysis of antibodies with live immune cells enabling binding measurements in a relevant cellular context with the data resolution needed to study complex binding processes.Paper II presents a novel real-time proximity assay that allows to study binding kinetics in connection with receptor dimerization and clustering thereby aiding in decipher complex interactions.In paper III, binding patterns of the CD20 mAbs rituximab, ofatumumab and obinituzumab were established on cells revealing that the fraction of bivalently bound mAbs differed resulting in dose-dependent affinities for rituximab and obinituzumab.In paper IV, a C1q binding assay to mAb opsonized cells was developed and it was shown that a higher degree of bivalent binding correlated with stronger C1q binding for the CD20 mAbs evaluated in paper III.In paper V, an assay to study mAb mediated cell-cell interactions was set-up and it was found that neutrophil engagement with target cells was similar for antibodies of IgG and IgA isotype.