Downsizing the downsized: Nanobody CDR peptidomimicry

Compared to conventional antibodies, Nanobodies (Nbs), antigen-binding fragments derived from heavy-chain only antibodies, are smaller, show favorable in vivo pharmacokinetics, and have the ability to bind highly specifically and efficiently to antigens. Even though Nbs are being fully validated in preclinical settings with the ultimate goal to translate them to clinical trials, their translation is cumbersome from a regulatory perspective. Besides immunogenicity issues, the requirements for production implicate extremely high costs, and these limitations advocate the development of smaller non-protein alternatives. In this proposal, we aim to identify the minimal structural components needed to mimic the high efficiency binding that is characteristic of Nbs. The most important subdomains in the antigen-recognition event are the complementarity determining region (CDR) loops, and hence cyclic peptidomimetics of the CDR loops will be constructed. Importantly, a thorough structural analysis by circular dichroism,2D-NMR and
molecular modeling will allow to narrow down the conformational space of the designed mimetics. Next to the synthesis of individual CDR mimetics, the preparation of CDR strings is proposed to determine the individual contribution of each loop in the binding event. Two receptors, the β2- adrenergic receptor (β2AR) and the human epidermal growth factor receptor 2 (HER2), will be
used in this project to verify the peptidomimetics’ ability to bind their antigens.