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