Molecular Recognition of Antibodies studied with High Speed AFM

The medical application of nanotechnology, i.e. nanomedicine, has enormous potential to improve health care. Aided by nanomedicine, pathomechanisms of disease are being discovered and molecular diagnostics refined. This novel research area has also led to the discovery, development, and new ways of delivery of drugs. A variety of macromolecular and nanoparticulate pharmacological compounds, as well as polymeric nanomedicines and polymer-drug conjugates endowed with receptor-specific recognition sites are already being utilized. Amongst them are antibodies, which may be considered as superior natural nanomedicines. The high specificity for their cognate antigens constitutes a paradigm for molecular recognition. It is exploited in many applications throughout biochemistry, molecular biology, medical research, and increasingly in the therapy of cancer, autoimmunity, and infectious diseases. They play a key role in opsonisation of pathogens by assembling into clusters that attract phagocytes and thus promote binding of the complement system resulting in clearance of the invader. These important recent developments point the way towards a more effective treatment of disease. It is thus essential to fundamentally advance our understanding of the nanomechanical properties of IgG molecules in the process of antigen recognition. Novel antibody formats and paratope-carrying scaffolds with improved activity need to be developed. To this end, a detailed description of the dynamic process of antibody-antigen binding and dissociation on the molecular level is required. Atomic force microscopy is the method of choice for such measurements under physiological conditions with respect to biological imaging, recognition, and localization of specific binding sites, as well as for measuring the strength of receptor-ligand bonds and other nanomechanical properties of bio-molecules at the single-molecule level. We thus aim to use the atomic force microscope for characterizing the dynamic and nanomechanical molecular properties of therapeutic antibodies and new antibody structures to identify features of optimal clinical trial candidates that facilitate drug development.

The project focused on two highly medically relevant systems: (a) on comparing the dynamic and nanomechanical properties of different IgG subclasses that bind agonistically to the receptor CD40, and (b) on antibody binding to the broadly expressed microbial antigen polysaccharide poly-N-acetyl glucosamine (PNAG) that is found on the surface of the most important causative agents of several severe diseases. Antibodies can bind to bacterial cell wall components (i.e. PNAG) and block initial attachment to the target surface and prevent biofilm formation (Raafat et al., 2019). Therefore, complete understanding of dynamics and kinetics of PNAG binding and dissociation mechanism to surface-bound antibodies (most prominently IgG) at the single molecule level is important. Human monoclonal antibodies (MAbs) used for the research of PNAG show different activities, with respect to their complement-depositing activity. They share, however, identical H and L constant regions, as 2 different pairs of V and H variable regions were cloned into the same vector. MAb F598 binds to PNAG and deposits complement on the bacterial surface effectively, thereby providing protection against infections in animals. In contrast, MAb F628 shows low binding to the PNAG and ineffcient binding of complement to the bacterial surface (Casie Kelly-Quintos et al. and Gerald B. Pier, 2006). Although MAb F598 is superior when compared to MAb F628 with respect to antigen binding, the molecular forces involved are not known. IgG subclasses and isoforms used in the research of the CD40 system differ by their amino acid sequence in their hinge region, e.g., disulphide bonds and hinge length. The project's goal was to investigate the impact of these hinge region differences on the antigen-antibody binding dynamics and kinetics, and gain a deeper understanding on their influence on the binding mechanism of the antibody coupling to the antigen.