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

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 Ca²⁺ 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.