EAACI June 6th-8th

A Siglec-8 Antibody Reduces Substance P-induced Inflammation by Inhibiting MRGPR-mediated

Mast Cell Activation

Simon Gebremeskel, Alan Wong, Tina Davis, Emily C. Brock, John Leung, Julia Schanin, Bradford A. Youngblood

Allakos, Inc., Redwood City, CA

EAACI 2020London, UK June 6th-8th 2020

EAACI June 6th-8th

Disclosures

• Authors are employees of Allakos, Inc.

• AK002 is an investigational drug in clinical development as is not FDA or EMA approved

2

EAACI June 6th-8th

Mast Cells Are Key Drivers of Inflammatory Disease

3

Tissue damage, fibrosisBronchoconstriction, increased GI motility, pain, itch

T Cell B Cell

ActivatedB Cell

IgE

ACTIVATION AND RECRUITMENT OF OTHERIMMUNE CELLS AND TISSUE INFLAMMATION

Smooth Muscle

Histamine, LTC4, PGD2 and proteases

IL- 4 IL-13

IL- 4 IL-13

HistamineNGF, Histamine

MacrophageEosinophil Neutrophil

IL-5 IL-8 IL-6, TNFa

Allergens

Epithelium

Mast CellNeuron

Sub P

IL-33 TSLP

ECP, MBP, elastase, MMP, TNFa , IL-1b, TGFb

Sub P

ACUTE AND CHRONIC INFLAMMATIONSENSITIZATION

EAACI June 6th-8th

Mast Cell

Substance P Drives Neurogenic Inflammation and Pain through Mast Cell Activation

Adapted from Green et al. 2019. Neuron

Neutrophil

Tissue Damage

Substance P

Neuron

• Substance P is a neuropeptide that activates mast cells through the MrgprX2/B2 receptor, leading to cytokine release, pain, itch, and recruitment of immune cells1,2

• Multiple diseases have been shown to be associated with Substance P-mediated mast cell activation, including chronic urticaria, psoriasis, and atopic dermatitis2,3

• Elevated Substance P levels as well as increased mast cell numbers and activation have also been reported in the mucosa of patients with gastrointestinal disorders4

• Based on numerous studies implicating Substance P/MrgprX2-mediated mast cell activation in the pathogenesis of multiple diseases there is a significant need to identify potential therapies that can modulate this pathway

Epithelium

NGF, TNF, Histamine

CXCL1, IL-8

MrgprX2/B2

1Green, DP et al. Neuron (2019); 2Varricchi, G et al Frontiers (2019); 3Vena, GA et al. Clin Mol Allergy (2018); Sohn, W et al. Scand J Gastroenterol (2014)

EAACI June 6th-8th

AK002 Broadly Inhibits Mast Cells and Depletes Eosinophils

5

ActivatingReceptors

Activation

Siglec-8

Mast cellEosinophil

Inflammatoryresponse

AK002

Inhibition

Mast cellEosinophil

InhibitionADCC/Apoptosis

AK002

Youngblood, BA et al IAAI 2019

EAACI June 6th-8th

Substance P Selectively Activates Human and Mouse Mast Cells through MrgprX2/B2

6

MrgprX2

CD

117

Mast Cells

Human Skin

0 0.025 0.250 2.50

10

20

30

40

50

% o

f MC

s

CD107a (Degranulation)

SubP (µg/mL):

MrgprX2/B2 is selectively expressed on mast cells and stimulation with Substance P induces degranulation of human and mouse mast cells

0 0.05 0.1 0.5 10

20

40

60

80

% o

f MC

s

CD107a (Degranulation)

SubP (mg/mL):

Human Skin Mast Cells Murine Peritoneal Mast Cells

Degranulation (CD107a) Degranulation (CD107a)

EAACI June 6th-8th

Mouse Model to Examine the Role of Mast Cells in Substance P-Induced Inflammation

7

0h

Substance PIP Injection

3h Number of immune cells in peritoneal

cavity by flow cytometry

AssessmentsWild-type

or MC deficient KitW-sh mice

• Substance P and other MrgprB2 agonists have been shown to recruit neutrophils into local tissues, including the mouse footpad and peritoneal cavity1,2

1Green, DP et al. Neuron (2019); 2Arifuzzaman, M et al Sci. Advances (2019)

Evaluate Inflammation

EAACI June 6th-8th

Mast Cells Play an Important Role in Substance P-Mediated Neutrophil Recruitment

8

Substance P induces mast cell-dependent recruitment of neutrophils into the peritoneal cavity

Vehicle Sub P0

10

20

30

40

% o

f CD

45+

Via

ble

Cel

ls

Neutrophils

*

Vehicle Sub P0

3

6

9

Abs

olut

e C

ount

s (x

105 )

Neutrophils

WT

KitW-sh

* WTKitW-sh

Substance PWild-type KitW-shWild-type

0.18%

Neutrophils14%

Neutrophils

4.89%

Neutrophils

Ly6G

Ly6C

* p<0.05

EAACI June 6th-8th

Anti-Siglec-8 mAb Reduces Sub P-Mediated Neutrophil Infiltration

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Anti-Siglec-8 mAb treatment significantly reduces Substance P-mediated neutrophil recruitment into the peritoneal cavity

0h

Substance PIP Injection

4h1h

Isotype or anti-Siglec-8 mAb

0

10

20

30

% o

f CD

45+

Via

ble

Cel

ls

Neutrophils

**** ****

0

2

4

6

Abso

lute

Cou

nts

(x10

5 )

Neutrophils

Vehicle

ISO + Sub. P

Anti-S8 + Sub. P

**** ***Evaluate

Inflammation

Vehicle ISO + Sub P Anti-Siglec-8 mAb + Sub P

*** p<0.001 **** p<0.0001

EAACI June 6th-8th 10

Anti-Siglec-8 mAb Inhibits Substance P-Mediated Inflammation and Mast Cell Activation

0

4

8

12

pg/mL

CCL3

**** ***

0

10

20

30

pg/mL

CCL4

**** **

0

10

20

30

40

pg/mL

KC/CXCL1

**** *

0

20

40

60

80

% C

hang

e in

CD

63

from

Veh

icle

Mast Cell Activation

**

*** p<0.001**** p<0.0001

* p<0.05

** p<0.01

Inflammatory MediatorsMast Cell Activation

ISO + Sub P Anti-Siglec-8 mAb + Sub P Vehicle ISO + Sub P Anti-Siglec-8 mAb + Sub P

Anti-Siglec-8 mAb decreases Substance P-induced chemokine production and mast cell activation, indicative of direct mast cell inhibition

EAACI June 6th-8th

Conclusions

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• Studies using mast cell deficient mice show reduced Substance P-mediated inflammation, implicating mast cells as key effector cells in Substance P-mediated inflammation

• Treatment with an anti-Siglec-8 mAb significantly inhibits Substance P-mediated mast cell degranulation, chemokine production, and neutrophil infiltration

• Targeting mast cells with an anti-Siglec-8 mAb may have the potential to treat diseases associated with Substance P/MrgprX2-mediated mast cell activation, such as irritable bowel syndrome, functional dyspepsia, atopic dermatitis, and chronic urticaria