Abstract
BackgroundMonoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor are efficacious for the prevention of migraine headaches. The downstream molecular mechanisms following ligand-receptor blockade by which these antibodies prevent CGRP signaling through CGRP receptors have not been demonstrated.MethodsHere we produced tool monoclonal functional antagonist antibodies against CGRP and its canonical receptor and developed a novel cellular model using fluorogen-activated protein technology that allows detection of CGRP receptor internalization by flow cytometry and, for an extended time course, visualization by confocal microscopy.ResultsUsing this cell model we showed that these antagonist antibodies block both CGRP-induced cAMP signaling and CGRP receptor internalization. At least 10-fold higher concentrations of either antibody are necessary to block CGRP receptor internalization compared with cAMP accumulation in our cell model.ConclusionThese data reinforce our understanding of how monoclonal functional antagonist antibodies interfere with CGRP signaling.
Highlights
Monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor are efficacious for the prevention of migraine headaches
Chinese hamster ovary (CHO) cells To examine the functionality of recombinant CGPR receptors, we compared intracellular cyclic adenosine monophosphate (cAMP) concentrations in response to CGRP, CGRP8–37, anti-CGRP antibody 8E11 and anti-CGRP receptor antibody AA58 in both untagged (Fig. 1a) and calcitonin-receptor-like receptor (CLR)-fluorogenactivated protein (FAP)-tagged (Fig. 1b) CGRP receptor expressing cells
It is worth noting that CGRP is more potent in the current CHO cell model system when compared to the literature EC50 of ~ 100 pM in the Swiss3T3 or HEK293 cells [12], and in human neuroblastoma cells (SKN-MC) endogenously expressing human CGRP receptors (EC50 = 670 pM) [20]
Summary
Monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor are efficacious for the prevention of migraine headaches. Novel monoclonal antibodies that target the neuropeptide calcitonin gene-related peptide (CGRP) or its receptor have been approved for use in migraine prevention and have consistently shown positive results in clinical trials (for recent reviews, see [4, 5]). The mechanism by which these monoclonal antibodies are proposed to prevent migraines is by blocking CGRP transmission in Monoclonal antibodies that target the neuropeptide CGRP (galcanezumab, eptinezumab and fremanezumab) are proposed to bind and deactivate CGRP released by trigeminal sensory nerve fibers [7], whereas antibodies that target the CGRP receptor (erenumab) presumably act by preventing access of CGRP to its canonical receptor [8]. There are two isoforms of CGRP: CGRPα is the predominant form in the central and peripheral nervous system implicated in migraine pathology and targeted by monoclonal antagonist antibodies. Binding of CGRP to the extracellular binding pocket formed by the heterodimer causes activation of G proteins containing the Gαs subunit bound to CLR, which in turn activates
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