# 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**

|  | Ligand category | Ligand | EC50(μM) | IC50(μM) |
| --- | --- | --- | --- | --- |
| Endogenous | Neuropeptides | Cortistatin-14 | 0.02 | 0.06 |
| Endogenous | Neuropeptides | Substance P | 0.05 | 0.10 |
| Endogenous | Neuropeptides | PACAP 1-27 | 0.08 | 0.10 |
| Endogenous | Neuropeptides | PACAP 1-38 | 0.33 | 0.25 |
| Endogenous | Neuropeptides | Dynorphin A | 2.05 | 0.13 |
| Endogenous | Neuropeptides | Neuropeptide FF | 6.18 | 0.10 |
| Endogenous | Antimicrobial peptides | LL-37 | 0.18 | 0.17 |
| Endogenous | Antimicrobial peptides | Catestatin | 3.29 | 0.10 |
| Endogenous | Antimicrobial peptides | β-defensin 2 | 3.82 | 0.04 |
| Endogenous | Peptide hormones | PAMP-12 | 0.04 | 0.15 |
| Endogenous | Peptide hormones | VIP | 0.45 | 0.07 |
| Endogenous | Peptide hormones | Vasopressin | 6.04 | 0.09 |
| Endogenous | Protein fragments | Eosinophil cationic protein | 9.32 | 0.15 |
| Hemokinin-1 | Protein fragments | 1.10 | 0.04 |
|  |  |  |  |  |
| Exogenous |  | Dextromethorphan | 2.31 | 0.12 |
| Exogenous |  | C48/80 | 3.72 | 0.08 |
| Exogenous |  | Icatibant | 6.18 | 0.05 |
| Exogenous |  | Chlorpromazine | 13.53 | 0.20 |
| Exogenous |  | Ciprofloxacin | 29.95 | 0.04 |

**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 Fc**e**R1**  
**mediated mast cell activation**

**A Synergy between MRGPRX2 and Fc**e**R1 pathways of mast cell activation**

| 50.21 | 39.35 | 29.56 | 17.33 | -4.54 |
| --- | --- | --- | --- | --- |
| 38.36 | 44.29 | 29.53 | 10.57 | -5.06 |
| 21.08 | 14.53 | 7.82 | -2.58 | -5.51 |
| -4.45 | -4.40 | -5.06 | -5.50 | -5.84 |
| 200 | 40 | 8 | 1.6 | 0 |

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.