Drug Discovery Stories

Cite This: ACS Pharmacol. Transl. Sci. 2019, 2, 485−490 pubs.acs.org/ptsci

Discovery of the Migraine Prevention Therapeutic Aimovig

(Erenumab), the First FDA-Approved Antibody against a G‑Protein-
Coupled Receptor
Chadwick Terence King, Colin V. Gegg, Sylvia Nai-Yu Hu, Hsieng Sen Lu, Brian M. Chan,
Kelly A. Berry, David W. Brankow, Tom J. Boone, Nebojsa Kezunovic, Matt R. Kelley, Licheng Shi,
and Cen Xu*
Amgen Research, 1 Amgen Center Drive, Thousand Oaks, California 91320-1799, United States
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

ABSTRACT: In 2018, the United States Food and Drug Administration (FDA) approved Aimovig (erenumab) for the
prevention of migraine. Erenumab is the first FDA approved antibody therapeutic against a G-protein-coupled receptor, the
canonical receptor of calcitonin gene related peptide (CGRP-R). A novel, epitope-focused antigen was created to reconstruct
Downloaded via 103.165.20.202 on March 18, 2023 at 18:04:52 (UTC).

the extracellular domains of the CGRP-R in a stable conformation. Successful inoculation of XenoMouse animals and careful
screening yielded multiple candidate molecules for high potency and exquisite selectivity toward the CGRP-R over related
receptors. These efforts led to the discovery of erenumab which has demonstrated the desired efficacy and safety profiles in
multiple clinical studies for the prevention of migraine. The innovation developed in the discovery of erenumab furthers the
ability to target G-coupled protein receptors using antibody approaches.

■ INTRODUCTION
Migraine, a leading cause of disability, affects near 15% of the
world’s population and is characterized by debilitating
headache pain, nausea, and increased sensitivity to light and
sound.1 Historically, migraine attacks have been difficult to
prevent due to poor patient adherence and a large number of
nonresponders to treatments repurposed from other indica-
tions.2,3 To answer this unmet medical need, Aimovig
(erenumab) was specifically designed and developed, leverag-
ing Amgen’s leading biotechnology expertise. Erenumab
(erenumab-aooe in the United States) is the only Food and
Drug Administration (FDA)-approved monoclonal antibody
(mAb) against the canonical receptor of calcitonin gene-
related peptide (CGRP). It is potent and specific for its target,
clinically efficacious, and well-tolerated by patients.4 In this
article, we seek to summarize the key innovations that led to
the discovery of this agent.
agonists, considered the standard of care for acute migraine for
several decades.9 In addition, intravenous infusion of CGRP in
migraineurs triggers migraine-like attacks.10 Collectively, these
data supported a substantive role for CGRP in migraine
pathology and that antagonizing the action of CGRP may be of
therapeutic benefit.
With the discovery of the major role the CGRP pathway
plays in migraine, small-molecule antagonists of the CGRP-R,
known as “gepants”, were developed. Multiple gepants were
tested in the clinic, and these compounds demonstrated
efficacy in the reversal of acute migraine attacks. This clinical
proof of concept brought further validation of CGRP
antagonism for the treatment of migraine.11−15
■ USE OF ANTIBODIES INSTEAD OF SMALL
MOLECULES
Despite their efficacy and generally good safety profile, the

■ CGRP RECEPTOR ANTAGONISM AND MIGRAINE

Migraine is a complex neurological disease involving central
and peripheral nervous systems. Studies on the interaction of
sensory nerves and cranial blood vessels in the trigeminovas-
cular system revealed that CGRP, a potent vasodilatory peptide
is involved in the headache pain occurring during a migraine
attack.5 CGRP is a 37 amino acid (AA) neuropeptide that
binds to several different G-protein-coupled receptors
(GPCRs) in the calcitonin receptor family, including CGRP,
amylin, calcitonin, and adrenomedullin receptors, with the
CGRP-R showing the highest binding affinity, making it the
canonical CGRP-R.6,7 CGRP-Rs are present in both the central
and peripheral nervous systems, as well as in smooth muscle
cells surrounding cerebral, meningeal, and dural arteries.8
CGRP plasma levels increase during migraine5 and normalize
after administration of triptans, serotonin 5-HT1B,1D receptor
first-generation gepants had limited success clinically due to
hepatoxicity concerns after extended use.16,17 In 2011, the
development of telcagepant, the most advanced small-molecule
CGRP-R antagonist, was terminated.18 The site of action of
the gepants was originally assumed to be in the central nervous
system. However, calculation of brain concentration based on
the clinically efficacious exposure and the physical-chemical
properties of these antagonists suggested that clinical efficacy is
most likely peripherally driven.14,19 Therefore, we hypothe-
sized that a peripherally restrictive antagonist antibody
targeting the CGRP-R would be therapeutically efficacious
for migraine treatment. Monoclonal antibodies provide several
potential advantages over small molecules, including high
affinity, high specificity (less off-target toxicity) through
Received: August 2, 2019
Published: September 3, 2019

© 2019 American Chemical Society 485 DOI: 10.1021/acsptsci.9b00061

ACS Pharmacol. Transl. Sci. 2019, 2, 485−490
ACS Pharmacology & Translational Science
Figure 1. CGRP and related receptors of the calcitonin family. The CGRP receptor is similar in composition to several other members of the
calcitonin family of receptors (receptor name listed above diagrams). These receptors are formed by a complex of the calcitonin-like receptor
(CLR) or calcitonin receptor (CTR) with receptor-activity-modifying proteins (RAMPs 1−3), predominantly binding ligands CGRP,
adrenomedullin (AM), and amylin (AMY). Due to the close similarity between receptor complexes, these agonists cross-react with other receptors
of the family.
binding to a unique epitope, and prolonged plasma half-life
enabling less frequent dosing. In addition, limited CNS
exposure can reduce potential liability associated with
CGRP-R blockade behind the blood brain barrier. This
approach was later supported by positron-emission tomog-
raphy (PET) imaging biodistribution data of an efficacious
dose of telcagepant in migraineurs, which demonstrated that
CGRP-R antagonism in the periphery is sufficient for migraine
pain relief.20
■
CHALLENGES OF TARGETING THE CGRP-R WITH
AN ANTIBODY
GPCRs represent about 35% of targets of prescription drugs
approved by the FDA.21 Despite the significant therapeutic
opportunities in this target class, none of these drugs were
antibody therapeutics prior to the approval of erenumab. This
scarcity of antibody therapeutics is due to the technical
challenges associated with generating functional antibodies to
GPCRs. Generally, the success rates of antibody discovery
campaigns increased with the availability of the soluble forms
of antigens, and maintaining the structural and functional
accuracy of the soluble antigen is critical. This is difficult in the
case of GPCRs, as they contain multiple transmembrane
domains, causing their structural stability to depend on the
lipid layers of the cell membrane. Full-length membrane-
associated forms of GPCRs can also be used for antibody
discovery, but historically these had lower success rates due to
the inherently poor immunogenic and antigenic characteristics.
For the CGRP-R, there are added challenges in that the
receptor is a heterodimeric complex of the calcitonin receptor-
like receptor (CLR), a class B GPCR, and the receptor activity-
modifying protein 1 (RAMP1), a type 1 transmembrane
protein; both proteins are required for CGRP binding and
subsequent complexing with the Gs-protein heterotrimer, the
main transducer protein for this receptor. The CGRP-R is
closely related to adrenomedullin (AM) and amylin (AMY)
receptors of the calcitonin receptor family, shown in Figure 1.
The two components of the CGRP-R, CLR and RAMP1, are
also individually found in AM1 (CLR + RAMP2), AM2 (CLR
+ RAMP3), and AMY1 (CTR + RAMP1) receptors.22 These
receptors demonstrate sensitivity to CGRP;6 however, neither
AM1 nor AMY1 have been linked to migraine pathophysiol-
ogy. In contrast, circulating levels of amylin are raised in
response to meal ingestion, and the peptide potently inhibits
gastric emptying and gastric acid secretion,22,23 while
adrenomedullin has been shown to have a remarkable range
of actions, from regulating cellular growth and differentiation,
through modulating hormone secretion, to antimicrobial
486
Drug Discovery Stories
effects.24 These functional indications call for an exquisite
selectivity to the CGRP-R. However, components shared
between these related receptors present additional challenges
where asymmetric binding of an antagonist antibody to either
component alone would result in lack of selectivity.
■
DESIGN CRITERIA OF AN ANTIBODY AGAINST
THE CGRP-R
At the time of designing our strategy, it was known that CGRP
interacted with the CGRP-R through two distinct regions: the
CGRP C-terminal domain, conferring high-affinity binding,
and the CGRP N-terminal domain, driving agonist activities.25
The CGRP C-terminal domain interacts with a high-affinity
binding region formed at the distal end of the CLR-RAMP1
extracellular domains. The CGRP N-terminal domain drives
agonist activity, through an interaction with a “classical”
agonist binding region formed in the CGRP-R by the
transmembrane α-helices of the CLR heterodimer, with no
direct interaction with RAMP1.26,27 The range of interaction
between CGRP and its receptor presents alternate strategies
for designing antibody antagonists: (1) Blocking the agonist
binding region from interacting with the CGRP N-terminus.
This strategy may result in less-selective inhibitors as it does
not involve RAMP1, as demonstrated by small-molecule
antagonists that interact within the transmembrane region.26
(2) Directly blocking the high-affinity binding area so that the
CGRP is blocked from associating with the receptor. We
decided that such blocking of the high-affinity binding region
was likely the most effective strategy and the best epitope
would be an extracellular region of CGRP-R that includes both
RAMP1 and CLR, as this unique combination in the epitope
would likely provide selectivity over the AM and AMY
receptors.
An important additional consideration in our design strategy
is that CGRP has a very high affinity for the CGRP-R with a
dissociation constant (Kd) of ∼15 pM.28 Therefore, we
expected that for an antibody to be effective in vivo it should
bind to the receptor in the low picomolar range.
The design criteria for an anti-CGRP-R antagonist antibody
were therefore strict, requiring innovative solutions. The
antibody would need to block a very specific binding region
on the receptor with selectivity for CGRP-R over related
receptor family members, along with high potency and affinity
necessary to be therapeutically relevant. To tackle this
formidable challenge, we focused on developing novel and
specific immunogens to conduct large-scale antibody discovery
campaigns.
DOI: 10.1021/acsptsci.9b00061
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ACS Pharmacology & Translational Science Drug Discovery Stories

Figure 2. Design, purification, and competition binding assay of the soluble CGRPR-ECD Fc fusion protein immunogen. (A) Space-filling model
of the theoretical structure of the CGRP-R ECD-Fc immunogen. This protein was constructed through fusing the N-terminal extracellular domains
of RAMP1 and CLR (yellow and purple) to human IgG1 Fc domain (green). A 5× glycine linker was inserted between the extracellular domains
and Fc region to confer additional flexibility and rotational freedom (orange). Coexpression of the two receptor components will generate a
heterodimeric protein of 81.5 kDa. (B) MALDI-TOF mass data for native sample of CGRPR ECD-Fc immunogen. Peak 1 (76.1 kDa) represents
the homodimer of RAMP1 ECD-Fc, peak 2 (86.3 kDa) represents the heterodimer of CGRP-R ECD-Fc (CLR + RAMP1), and peak 3 (99.1 kDa)
represents CLR ECD-Fc. (C) Results of a fluorescence activated cell sorting (FACS)-based inhibition of ligand binding study, plotted as percent
inhibition. Measured as percent reduction in binding of ALEXA-647 CGRP after 1 h of preincubation at room temperature with increasing
concentrations of purified CGRP-R (ECD)-Fc. An Fc-fusion protein OPG-Fc was used as a negative control.

■ NOVEL ANTIGEN DESIGN: RECONSTRUCTING THE

EXTRACELLULAR DOMAIN OF THE CGRP-R
In addition to the conventional cellular and cell-membrane
immunogen preparations, we specifically designed and
produced a novel soluble protein immunogen containing
only the extracellular domains (ECDs) of the CGRP-R,
seeking to recapitulate the unique heterodimeric structure of
this receptor by coexpressing the extracellular regions of both
CLR and RAMP1. Achieving a structurally stable heterodimer
was essential to represent the unique heterodimeric epitope for
immunization. Our approach was to express each individual
ECD component as a fusion protein to an antibody Fc domain.
Fc domains are expected to form dimers when coexpressed to
promote the reassembly of CLR and RAMP1. In addition, a
linker region was inserted between the ECD and Fc domains
to decrease the rotational constraint on the ECDs and promote
native reassembly of the CLR and RAMP-1 domains (Figure
2A).
The two components, CLR ECD-Fc and RAMP1 ECD-Fc,
were coexpressed to generate heterodimer Fc proteins. A
complicating factor in producing this immunogen was the
uncontrolled dimerization of the Fc domains. This process
produced three species: a CLR ECD homodimer, a RAMP-1
ECD homodimer, and the CGRP-R ECD-heterodimer.
Applying conventional chromatography purification strategies
allowed separation of the smaller RAMP1-ECD-Fc homo-
dimers, but the CLR ECD-Fc homodimers and CGRP-R ECD-
Fc heterodimers could not be completely separated due to
their size similarity (Figure 2B). Overall, we estimated the final
purified sample to be 40% CGRP-R ECD-Fc and 60%
homodimeric species.
To assess whether the partially purified CGRP-R ECD-Fc
heterodimer structurally recapitulated ligand binding, it was
tested for activity in a fluorescence activated cell sorting
(FACS)-based CGRP ligand binding assay. The CGRPR ECD-
Fc sample was incubated with a labeled form of CGRP
(ALEXA-647 CGRP) and added to cells expressing CGRP-R.
A reduction in the amount of ALEXA-647 CGRP binding to
the cells was then calculated as the percent inhibition. ECD
complex demonstrated concentration-dependent inhibition of
the binding of CGRP to the CGRP-R (Figure 2C). These data
487
provided confidence that the heterodimer was likely replicating
the structure of the native CGRP-R ligand binding sites
between CLR and RAMP1.
■ ANTIBODY DISCOVERY
XenoMouse animals, a transgenic mouse strain that has fully
human immunoglobulin genes,29 were used to generate human
CGRP-R antibodies. The advantage of fully human antibodies
as therapeutic molecules is the relatively lower rates of
immunogenicity.30
To ensure maximal diversity and identification of the highest
quality candidates, two antibody generation campaigns were
run using XenoMouse animals: the first inoculating with the
soluble CGRP-R ECD-Fc immunogen and the second with a
combination of CGRP-R expressing cells and their cell-
membrane preparations as immunogens.
The first campaign focused on the soluble protein
immunogen, with a pool of immune repertoires from the
best-responding animals used for hybridoma generation.
Approximately 100 000 of these hybridoma clones were
screened for binding using Chinese hamster ovary (CHO)
cells stably expressing CGRP-R and counter-screened on CHO
cells stably expressing the AM1 receptor. A total of 1092
hybridomas were identified with selective binding to CGRP-R.
These 1092 hybridomas were then screened for the ability to
block CGRP-induced signaling in a cell-based cAMP assay, the
key second messenger of CGRP-R action. Antagonist activity
of each hybridoma supernatant was expressed as a percent
inhibition of cAMP production induced by 1 nM of CGRP. Of
the panel of CGRP-R specific binders, 28% achieved >50%
inhibition in this assay.
The campaign with cells and cell-membrane immunogens
was far less productive for generating specific binders and
antagonists compared to results with the CGRP-R ECD-Fc
soluble immunogen. In this case, approximately 103 000
hybridomas were screened and a total of 119 binders of
CGRP-R were identified. This panel of binders was then
screened in the CGRP signaling assay using the same
conditions as the campaign above, with only 2.5% of the
panel achieving >50% inhibition of CGRP signaling.
DOI: 10.1021/acsptsci.9b00061
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The immunization of XenoMouse animals with the soluble Table 1. Potency and Selectivity of Representative
CGRP-R ECD-Fc protein resulted in robust target-specific Antibodies
immune responses compared to results with the native cell-
based immunogens. The dramatically different outcome of
these two screening campaigns clearly indicates the benefit of
the epitope-focused soluble immunogen strategy. The soluble
CGRP-R ECD-Fc heterodimer immunogen generated signifi-
cantly stronger immune responses in XenoMouse animals,
allowing identification of a 10-fold larger specific binder panel
than a comparably scaled campaign with the cellular
immunogens. The soluble immunogen also gave rise to a
much higher frequency of antibody antagonists specific to
CGRP-R, critically important for campaign success. This larger
panel enabled stringent refinement of the lead candidates to
only those highly selective antagonists. Figure 3 summarizes

human CGRP-R over the other members of this receptor

family. Taken together, these results indicated the design goal
had been achieved.

■ CONFIRMATION OF HETEROMETRIC
INTERACTIONS
We inferred that the antibody drug candidates identified must
bind to a heterodimeric region on the CGRP-R formed by the
CLR and RAMP1 components. To test this hypothesis, we
conducted protease protection assay to map the binding
regions. After generating a disulfide peptide map of the CGRP-
R ECD-Fc protein (method described in ref 31), peptides
produced through proteolytic digestion using AspN (which
cleaves after aspartic acid and some glutamic acid residues)
were analyzed by LC-MS to determine the sequences of
peptide fragments of CLR and RAMP1. Protection assays were
then performed with the presence of an anti-CGRP-R
Figure 3. Functional selectivity screening of most potent CGRP-R
antagonists. Large panel of 167 of the most potent CGRP-R antibody. The difference between the peptide maps indicated
antagonist hybridomas (>70% inhibition) were tested for functional the region(s) of the CGRP-R ECD Fc protein that were
activity in AMY1 (CTR+RAMP1) and AM1 (CLR+RAMP2) cell- protected from proteolytic digestion by AspN when bound to
based assays. The majority of samples tested showed little to no the anti-CGRP-R antibody (method described in ref 32). A
activity. A subset of the most selective hybridomas were then representative result of these studies is shown in Table 2. The
recombinantly expressed for potency determination.
Table 2. Result of AspN Proteolytic Digestion of the CGRP-
R ECD-Fc Fusion Proteina
the functional selectivity of this panel. In these screens the
most potent set of 167 CGRP-R antagonists (>70% inhibition) sequence protection assay − HPLC
were tested for selectivity against AMY1 and AM1 receptors. peptide origin location chromatogram peak height
The majority of candidates were functionally inactive at these C5 CLR D55-P67/D86- decreased
related receptors, indicating a high degree of selectivity, and H110
the advantage of using an epitope focused immunization C6 CLR D8-Y24 decreased
strategy with the soluble antigen. Immunization with CGRP-R C7 CLR E25-Q32/D48- decreased
N54
ECD-Fc protein yielded almost the entirety of the lead panel of R1 RAMP1 D32-A44 decreased
candidate antibodies. R2 RAMP1 E12-V20/D45- decreased
High-throughput screening performed with hybridoma A51
supernatants identified a lead candidate panel. From this a
Change to peptide peak height as measured by HPLC chromato-
subset, we generated clonal and purified antibody samples for gram are reported in the last column. Reduction in peak height
potency determination. Potency and selectivity data for a indicates a protection.
representative set of four sequence-unique and functionally
selective CGRP-R antagonist antibodies is shown in Table 1,
which summarizes the activity profiles. These four candidates study identified peptide fragments from both CLR and
were shown to have dissociation constants (Kd) in the double- RAMP1 that are protected by the binding of anti-CGRP-R
digit pM range (compared to a Kd of CGRP at 20 pM when Ab from AspN digestion. Our analysis indicated that on CLR
tested side by side in these studies, data not shown) and there were multiple cleavage sites protected by the bound
antagonist IC50’s in the single-digit nM range in cell-based antibody; these included regions between D55-P67/D86-
cAMP signaling assay. Furthermore, the binding and functional H110, D8-Y24, and E25-Q32/D48-N54. On RAMP1, the
antagonistic activities were shown to be exquisitely selective to results indicated that the antibody protected multiple cleavage
488 DOI: 10.1021/acsptsci.9b00061
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ACS Pharmacology & Translational Science
sites, including regions between D32-A44 and E12-V20/D45-
A51. The data, although with limited resolution, provided
direct evidence that regions on both CLR and RAMP1 are
closely associated with the anti-CGRP-R antibody when
bound. The binding of the antibody to the unique complex
of CGRP-R ECD-Fc likely contributes to the specificity over
other related receptors in the receptor family.
■
CONCLUSION
In this article, we highlight the key innovations that led to the
discovery of erenumab, a preventive therapy for migraine. With
the aim of developing a first-in-class molecule, we generated a
fully human, highly potent monoclonal antibody antagonist of
the human CGRP-R, a complex GPCR formally only targeted
by small-molecule approaches. Epitope-focused design of the
specific antigen led to a panel of candidate antibodies with
desired potency and specificity. Clinical trials for erenumab
demonstrated safety and efficacy for the prevention of episodic
and chronic migraine.33−36 The high potency and exquisite
specificity of erenumab were key in this successful develop-
ment in the clinic as the first FDA approved antibody therapy
for the prevention of migraine. The innovation implemented in
the discovery of erenumab furthers the ability to target GPCRs
therapeutically using antibody technology. We look forward to
the application of this strategy to the development of many
agents in the near future.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: cenx@amgen.com.
Notes
The authors declare the following competing financial
interest(s): All authors are or were employed by Amgen Inc.
■
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490 DOI: 10.1021/acsptsci.9b00061

ACS Pharmacol. Transl. Sci. 2019, 2, 485−490