Peptide‐Linked Bivalent Nanobody Constructs as Positive Allosteric Modulators of the Calcium‐Sensing Receptor

ABSTRACT We previously developed nanobodies acting as positive allosteric modulators (PAMs) of the calcium‐sensing receptor (CaSR). Here, we sought to develop more potent CaSR modulators by creating bivalent nanobody constructs using ‘tail‐to‐head’ peptide‐linker conjugation. The constructs were functionally characterized at CaSR in HEK293 cells in a Ca 2+ mobilization assay. Homobivalent Nb4‐Nb4 and Nb10‐Nb10 constructs with linkers comprising 3, 5, 6 or 9 GGGG(S/T) repeats all displayed CaSR PAM potencies comparable with those exhibited by their monovalent counterparts. Likewise, five heterobivalent nanobodies combining Nb4, Nb10, Nb11, Nb36 or Nb37 through [GGGG(S/T)] 9 linkers exhibited PAM potencies similar to that of the monovalent version of the first (N‐terminal) nanobody in the respective constructs. This prompted us to examine the functional properties of [GGGG(S/T)] 9 ‐linked constructs combining Nb4 and the mGluR5‐selective nanobody PAM Nb43 mGlu5 . Nb4‐L9‐Nb43 mGlu5 displayed slightly increased CaSR PAM potency relative to Nb4, whereas Nb43 mGlu5 ‐L9‐Nb4 exhibited ~40‐ and ~10‐fold reduced potency compared with Nb4‐L9‐Nb43 mGlu5 and Nb4, respectively. The findings indicate that ‘tail‐to‐head’ conjugation markedly reduces CaSR binding affinity of the second (C‐terminal) nanobody and likely explains the lack of synergistic enhancement of PAM potency in the bivalent constructs. Thus, the suitability of tail‐to‐head linker conjugation of nanobodies is context dependent, governed by how the nanobody engages its target and by whether the linker perturbs these interactions.