Discovery and pharmacological characterization of nanobodies acting as potent positive allosteric modulators of the calcium-sensing receptor

The calcium-sensing receptor (CaSR) is responsible for sustaining a stable blood calcium concentration. Consequently, genetic and acquired changes in this G protein-coupled receptor can give rise to various calcium homeostasis disorders. Synthetic positive allosteric modulators targeting CaSR are currently used to treat hypercalcemia, but their usage is highly limited due to the high risk of severe hypocalcemia and gastrointestinal intolerance. In this study, we aimed to generate pharmacologically active CaSR-specific nanobodies that could be employed as a new generation of pharmacological tools to investigate the receptor function and potentially serve as a new drug modality for effective treatment of CaSR-related disorders. Nanobodies were generated by immunization of a llama with CHO cells recombinantly overexpressing a myc-epitope-tagged human CaSR. Following construction of a phage display library representing the repertoire of nanobody genes, nanobodies binding to the CaSR were isolated by FACS of whole HEK293 cells recombinantly overexpressing HA-epitope-tagged human CaSR. Based on sequence comparison, 37 nanobodies from 25 different sequence families were purified and subsequent characterized in vitro for modulation of CaSR signaling. The nanobodies were screened for agonist, as well as positive and negative allosteric modulators activity in in vitro cellular assays downstream of CaSR activation. We identified eight pharmacologically active nanobodies acting as positive allosteric modulators that could be divided into five main families based on their sequence identity. The most potent nanobody (Nb4) binding to the extracellular domain of CaSR was slightly more potent than the reference small molecule PAM NPS R-568. This study describes the discovery and pharmacological characterization of nanobodies acting as potent CaSR positive allosteric modulators. These nanobodies are a new class of pharmacological research tools for the CaSR, which potentially can be developed into new therapeutics in the treatment of CaSR-related disorders.