EVO756 potently blocks MRGPRX2-mediated mast cell activation as well as MRGPRX2 and FceR1- dependent synergistic responses

Sreya Bagchi, Claudia Montllor Albalate , Alexandra Pavel , Josh Leung , Jeegar Patel , Lorena Riol Blanco , Jamie L. Harden
Evommune, Inc. Palo Alto, CA 94304, presenting author

EVO756 inhibits synergistic responses A B

Abstract

Mas-related G-protein coupled receptor X2 (MRGPRX2) is a
GPCR expressed predominantly by mast cells and has been
implicated in playing an important role in diseases such as
chronic spontaneous urticaria, atopic dermatitis, asthma and
ulcerative colitis. Cationic ligands, either endogenous or
exogenous, can activate the receptor and induce IgEindependent mast cell activation, resulting in allergic responses
and non-histaminergic itch. Therefore, blocking MRGPRX2
represents a promising therapeutic modality for a plethora of
autoimmune conditions. Here we show that EVO756, a novel
small molecule antagonist of MRGPRX2, demonstrates potent,
concentration dependent inhibition of MRGPRX2 mediated
activation in MRGPRX2 transfectants and LAD2 cells.
Furthermore, EVO756 inhibits primary humans mast cell activity
at the transcriptional and proteomic levels, as determined by the
release of tryptase and surface expression of mast cell granules.
We also demonstrate synergism between MRGPRX2-mediated
and IgE-dependent primary human mast cell activation, which is
inhibited by EVO756. These studies establish EVO756 as a
potent inhibitor of MRGPRX2-mediated mast cell activation and
therefore, a promising therapeutic for the treatment of mast cell
driven diseases.

Background

Figure 1. MRGPRX2 mediated activation of mast cells.

EVO756 blocks MRGPRX2 mediated
signaling in the presence of various
agonists

Table 1. IC50 values (µM) for EVO756 in the presence of various MRGPRX2 agonists

Figure 2. Concentration dependent inhibition
of MRGPRX2 signaling by EVO756 in CHO-
MRGPRX2 transfectant. CHO cells expressing
MRGPRX2 were loaded with the FLIPR calcium
dye and incubated for 30 minutes with EVO756
at varying doses. Subsequently, various classes
of both endogenous and exogenous agonists
were added by the FLIPR Penta instrument and
calcium flux measured over time. Percent
inhibition was calculated based on CHO-
MRGPRX2 cells treated with only EVO756 or
only agonist.

EVO756 blocks MRGPRX2 mediated
mast cell degranulation in the
presence of various agonists

Skin-derived primary human mast cells (skphMC)

Figure 3. Concentration dependent inhibition of
MRGPRX2-mediated mast cell degranulation by
EVO756. LAD2 and skphMCs were treated with
EVO756 at varying concentrations or at 1 mm (for
tryptase release) for 5 minutes. Cells were then
incubated with MRGPRX2 agonists at greater than
ec80 concentrations. For assays assessing CD63
surface expression, cells were incubated for 1 hour
with agonists before flow cytometry-based analysis of
CD63 expression was performed. For the bhexosaminidase assay, released versus cellular
content of the enzyme was detected in the
supernatant and cell pellets after 1 hour of incubation
with agonists. For the tryptase assay, the enzyme
was measured in the supernatant by ELISA after 24
hours of incubation with agonists.

EVO756 inhibits synergistic responses
elicited by MRGPRX2 and FceR1
mediated mast cell activation

A Synergy between MRGPRX2 and FceR1 pathways of mast cell activation

Anti-IgE (ng/ml)

C Synergy and its inhibition
by EVO756 demonstrated by
CD107a MFI

Figure 4. EVO756 blocks MRGPRX2 and FceR1 mediated synergy in skphMCs. skphMCs
were treated with different concentrations of SP and/or IgE/anti-IgE in the presence or absence of
EVO756 (5 mM). For CD63 and CD107a analysis,
cells were incubated for 1 hour before flow
cytometry was performed. Percent synergy was calculated using the Bliss model of independence.

Conclusions

• EVO756 potently inhibits MRGPRX2 responses to various
endogenous and exogenous ligands in vitro

• EVO756 effectively reduced LAD2 and donor-derived mast cell
activation upon treatment with different MRGPRX2 ligands

• EVO756 inhibited synergistic responses upon activation of
mast cells through concomitant activation through MRGPRX2
and FceR1.

Acknowledgements

We would like to acknowledge and thank the Maurer at Charité
(Institute of Allergology) and the Galli lab at Stanford University for
their invaluable inputs to the project and testing the activity of
EVO756 in primary human mast cells and ROSA cells,
respectively. Lastly, we would like to thank Raine Osterling for her
help and support for isolating primary human mast cells from skin
tissues. Illustrations were created with BioRender.com

Disclosures

SB, CMA, AP, JL, JP, LRB and JLH are employees of, and hold
stock in, Evommune.