Abstract
Antibodies have enormous therapeutic and biotechnology potential. G protein-coupled receptors (GPCRs), the main targets in drug development, are of major interest in antibody development programs. Metabotropic glutamate receptors are dimeric GPCRs that can control synaptic activity in a multitude of ways. Here we identify llama nanobodies that specifically recognize mGlu2 receptors, among the eight subtypes of mGluR subunits. Among these nanobodies, DN10 and 13 are positive allosteric modulators (PAM) on homodimeric mGlu2, while DN10 displays also a significant partial agonist activity. DN10 and DN13 have no effect on mGlu2-3 and mGlu2-4 heterodimers. These PAMs enhance the inhibitory action of the orthosteric mGlu2/mGlu3 agonist, DCG-IV, at mossy fiber terminals in the CA3 region of hippocampal slices. DN13 also impairs contextual fear memory when injected in the CA3 region of hippocampal region. These data highlight the potential of developing antibodies with allosteric actions on GPCRs to better define their roles in vivo.
Highlights
Antibodies have enormous therapeutic and biotechnology potential
Whereas DN1 displays the same affinity for the active and inactive forms of rat mGlu[2] (Fig. 1d, Supplementary Table 1), DN10 and DN13 bind to the active form stabilized by the orthosteric agonist, LY379268 (Fig. 1e, f, Supplementary Table 1)
Because the epitope recognized by DN13 likely involves both subunits in the mGlu dimer, and DN10 binds to an overlapping area, we examined whether the nanobodies could bind to the mGlu[2,3] and mGlu[2,3,4] heterodimers by transfer measured in a time resolved manner (TR-FRET) using d2 labeled secondary anti-c-Myc antibodies and Lumi4-Tb labeled SNAP-mGlu[3] or SNAP-mGlu[4], in cells co-transfected with a mGlu[2] subunit (Fig. 4a)
Summary
Antibodies have enormous therapeutic and biotechnology potential. G protein-coupled receptors (GPCRs), the main targets in drug development, are of major interest in antibody development programs. This led us to identify two nanobodies that bind to the active form of the mGlu[2] These nanobodies act as PAMs, enhancing the agonist action at mGlu[2] receptors in transfected cells and in brain slices. When injected in the hippocampus, one of these nanobodies enhances the effect of a group-II mGluR agonist in the fear-conditioning test, demonstrating their possible use to decipher the physiological role of mGlu[2] receptors in the brain. These data nicely illustrate novel possibilities to develop mGlu allosteric modulators for numerous therapeutic actions, and exemplify the use of nanobodies to allosterically modulate GPCRs
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