Presented at the American Academy of Allergy, Asthma & Immunology (AAAAI), Philadelphia, PA, March 13th – 16th, 2020

METHODS

• Human skin mast cells express the inhibitory receptor Siglec-8, and activation of mast cells cells via FcεRI is inhibited with antolimab

• Mast cells are elevated in number and are basally activated in AD biopsies with high levels of surface-bound IgE

• Antolimab inhibits IL-33/TSLP-mediated mast cell activation in AD skin biopsies, suggesting antolimab can broadly inhibit multiple modes of mast cell stimulation including, IgE, IL-33, and TSLP

• Mast cells appear to be important in AD, and targeting mast cells via Siglec-8 with antolimab may represent a novel therapeutic approach to the treatment of AD and other allergic diseases

CONCLUSIONS

Figure 8. Resting Mast Cells are Activated in AD Skin Biopsies

RESULTS

Figure 5. Non-Diseased Human Skin Mast Cells Express Siglec-8 and Multiple Types of Activating Receptors

Figure 6. Antolimab (AK002) Inhibits IgE-Dependent Human Skin Mast Cell Activation

Figure 9. Antolimab Inhibits IL-33/TSLP-mediated MC Activation in AD Skin Biopsies

• Single-cell suspensions were prepared by enzymatic & mechanical digestion of fresh biopsies from patients clinically diagnosed with AD (n=6) or non-disease control skin tissue (n=10)

• Multi-color flow cytometry was performed to quantify immune cells and evaluate the activation state of eosinophils & mast cells as shown in Figure 4

• Mast cells were FACS-sorted from AD biopsies or non-diseased skin tissues followed by overnight incubation with or without PMA/Ionomycin

• Cell-free supernatants were collected the following day and cytokines were quantified using meso scale discovery (MSD) system

• The following cytokines were analyzed: IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-18, IL-33, GM-CSF, INF𝞬, TNF⍺, CCL2, CCL3, CCL4, and VEGF

7AAD

CD

45

FcεRI

CD

117

CD16

SSC

CCR3

SSC

Mast Cells Eosinophils

1. BiopsyAcquisition

2. TissueDigestion

3. FluorescentAntibodyStaining

4. FlowCytometry

5. Immune CellCharacterization

6. Evaluateantolimab

Activity

Figure 3. Study Design

Figure 4. Flow Cytometry Gating Strategy for Mast Cells and Eosinophils in AD Biopsy Tissue

Figure 5: (Left) Representative contour plot of human mast cells from skin tissue. (Right) Expression of Siglec-8 (blue), Substance P receptor (red), ST2L (black), and TSLP receptor (yellow) on human skin mast cells compared to a fluorescence minus one (FMO) control (gray).

Figure 7. Mast Cells and Eosinophils are Elevated in AD Skin Biopsies

Figure 8: The expression of Siglec-8 and the known mast cell activation markers, CD63, CD203c, and IgE were determined by flow cytometry in non-diseased normal tissue (black) and AD skin tissue (gray). Data are shown as mean +/- SEM for n=10 non-diseased and n=6 for AD donors; ** p<0.01 * p<0.05; *** p<0.001

Figure 9: (Top Left) Schematic of IL-33+TSLP mediated human AD skin mast cell activation assay to evaluate the inhibitory activity of AK002. (Top Right) Mast cell activation as assessed by CD63 expression on mast cells for AD skin mast cells activated with IL-33 + TSLP (10 ng/mL) in the presence of an isotype control mAb(gray) or AK002 (blue). (Bottom) Cytokine and chemokine production in cell-free supernatant induced by IL-33 + TSLP from AD skin mast cells treated with an isotype control mAb (gray) or AK002 (blue). Data are shown as mean +/- SEM for n=3 AD donors; ** p<0.01

Atopic Dermatitis Skin Biopsies Have High Numbers of Activated Mast Cells that Are Inhibited by Antolimab (AK002) After Stimulation Ex Vivo

Bradford A. Youngblood, Simon Gebremeskel, Alan Wong, Julia Schanin, Amol P. Kamboj, and Nenad TomasevicAllakos Inc. Redwood City, CA

• Loss of epithelial barrier integrity is a critical step in the development of atopic dermatitis (AD) whereby the alarmin cytokines IL-33 and TSLP activate inflammatory cells such as mast cells (MCs) (Figure 1)

• While MCs have been shown to be elevated AD, there is need for further characterization of their pathogenic role

• Siglec-8 is an inhibitory receptor expressed on mast cells and eosinophils and represents a new potential therapeutic target for AD given the pathogenic role of MCs

BACKGROUND

• Siglec-8 is an inhibitory receptor selectively expressed on human eosinophils and mast cells, and therefore represents a novel target for the treatment of debilitating allergic, inflammatory, and proliferative diseases

• Antolimab is a novel, humanized, non-fucosylated IgG1 monoclonal antibody to Siglec-8

• Engagement of Siglec-8 receptor by antolimab triggers:– Antibody dependent cell mediated cytotoxicity (ADCC) against blood

eosinophils and apoptosis of tissue eosinophils– Inhibition of mast cells

• Here we immunophenotype mast cells and examine the ex vivo activity of antolimab in AD biopsies

Figure 2. Antolimab (AK002) Mechanism of ActionActivatingReceptors

Activation

Siglec-8

Mast CellEosinophil

InflammatoryResponse

Antolimab

InhibitionMast CellEosinophil

InhibitionADCC/Apoptosis

Antolimab

Figure 1. Mast Cells and Eosinophils are Key Drivers of Acute and Chronic Inflammation

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

IL-33 TSLP

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

SENSITIZATION ACUTE AND CHRONIC INFLAMMATION

Sub P

Human Skin Mast Cells

CD

117

FcɛRI Expression

Siglec-8Substance P

Receptor IL-33 Receptor TSLP Receptor

Normal AD0

20000

40000

60000

80000

Δ MFI

CD203c

**

Normal AD0

50000

100000

150000

Δ MFI

IgE

**

0

20

40

60

80

VEGF

% C

hang

e fro

m U

nstim

ulat

ed

**

0

10

20

30

40

50

% C

hang

e fro

m U

nstim

ulat

ed

CXCL1/KC

**

0

20

40

60

80

100MIP-1α

% C

hang

e fro

m U

nstim

ulat

ed

**

0

10

20

30

% C

D63

+ M

ast C

ells

(N

orm

aliz

ed to

Con

trol)

Mast Cell Activation

**

ISO + IL-33/TSLPAK002 + IL-33/TSLP

• Mast cells in AD skin biopsies are activated by IL-33/TSLP suggesting they are important target cells for alarmin cytokines released by epithelial cells

• Treatment with AK002 significantly reduces IL-33/TSLP mast cell activation as evidenced by decreased surface markers of activation and cytokine production

Normal AD0

2000

4000

6000

8000

10000

Δ MFI

Siglec-8

Normal AD0

1000

2000

3000

Δ MFI

CD63

*

Figure 6: (Left) Schematic of IgE-mediated human skin mast cell activation assay to evaluate the inhibitory activity of AK002. (Right) Markers of mast cell degranulation (CD107a) and activation (CD63) as assessed by flow cytometry for human skin mast cells. Activation was normalized to an unstimulated control. Data are shown as mean +/- SEM for n=5 donors; * p<0.05; ** p<0.01

ISO + anti-FcɛRIAK002 + anti-FcɛRI

0

5

10

15

20

% C

D63

+ M

ast C

ells

(N

orm

aliz

ed to

Con

trol)

Mast Cell Activation

**

0

4

8

12

% C

D10

7a+

Mas

t Cel

ls

(Nor

mal

ized

to C

ontro

l)

Mast Cell Degranulation

**

Normal AD0

1

2

3

4

% o

f CD

45+

Cel

ls

Skin Eosinophils

**

Normal AD0

5

10

15

20

25

% o

f CD

45+

Cel

ls

Skin Mast Cells

** Figure 7: Percentage of eosinophils (left) and mast cells (right) among CD45+ immune cells in non-diseased (black) or AD (gray) skin. Eosinophils and mast cells were gated on viable CD45+ cells in processed skin tissue as shown in figure 4. Data are shown as mean +/- SEM for n=10 non-diseased and n=6 for AD donors; ** p<0.01

• Siglec-8 expression remains high on human skin mast cells independent of disease state

• Resting mast cells in AD skin tissue display an activated atopic phenotype compared to non-diseased skin mast cells

• AK002 inhibits FcεRI activation of skin mast cells, suggesting an anti-Siglec-8 approach may be effective in MC-driven skin diseases, such as AD

Cou

nts

AK002 or Isotype Control

0h

Activating anti-FcεRIantibody

16hProcess human skin tissue into

single cells

Measure mast cell activation by flow

cytometry

AK002 or Isotype Control

0h

IL-33 + TSLP

16hProcess human skin tissue into

single cells

Measure mast cell activation by flow cytometry and

cytokines in supernatant