Abstract
The elucidation of proteins’ structure and function is of great importance for the understanding of disease triggering mechanisms and for the development of new drugs. In this thesis, different biophysical and biochemical techniques were employed to gain insights into the structural and functional features of three proteins belonging to two
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important cell surface receptor families: low density lipoprotein (LDL) receptor-related protein 1 (LRP1) and human epidermal growth factor receptor 2 (HER2) and 3 (HER3). LRP1 is the largest member of the low density lipoprotein receptor family and it is known to interact with numerous binding partners, fulfilling many biological tasks besides its main role as a scavenger. Small-angle X-ray scattering (SAXS) and negative stain electron microscopy (EM) were combined with surface plasmon resonance (SPR), native and crosslink mass spectrometry (MS) to elucidate the binding behavior and structural features of LRP1. This synergistic approach made it possible to obtain the first structural characterization of the entire extracellular region of LRP1 at acidic and neutral pH. Moreover, LRP1’s binding properties were investigated using the chaperone RAP as model ligand. Another chapter of this thesis describes the identification of a novel bispecific antibody that targets the receptor tyrosine kinases HER2 and HER3. These receptors normally regulate cell proliferation and survival by binding to growth factors, which trigger the activation of different signaling pathways. However, when their activity is dysregulated they are involved in tumor growth. Bispecific antibodies are a novel class of bio-pharmaceuticals that, by combining two different antigen binding sites in a single molecule, have the potential to exert a more potent and durable action compared to monospecific antibodies. By combining in vitro and in vivo assays with X-ray crystallography, SAXS and statistical modeling we could elucidate the mechanism of action of this novel therapeutic molecule. MCLA-128 specifically and potently inhibits ligand dependent HER2:HER3 signaling in HER2-overexpressing breast cancer cells resulting in suppression of tumor growth. Finally, the engineering efforts behind the development of MCLA128 are described. The heterodimerization of two different antibody heavy chains was promoted by introducing asymmetric mutations in the CH3 domains of the Fc region of the IgG. The most promising mutations were investigated by in vitro assays, computational docking tools and X-ray crystallography. Furthermore, a common light chain strategy was adopted to overcome the problem related to the mispairing of the heavy and light chains. By combining these approaches we could develop a very stable bispecific antibody based on the architecture of a natural IgG with optimal biophysical properties.
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