Pre-clinical Combination of AO-176, a Highly Differentiated Clinical Stage CD47 Antibody, with Either Azacitidine or Venetoclax Significantly Enhances DAMP Induction and Phagocytosis of Acute Myeloid Leukemia

ASH POSTER PRESENTATIONDecember 2020

Michael J. Donio, W. Casey Wilson, Isra Darwech, Gabriela Andrejeva, Benjamin J. Capoccia, Robyn J. Puro, Arun K. Kashyap and Daniel S. Pereira

Arch Oncology, 4340 Duncan Avenue, St. Louis, MO 63110 and 2000 Sierra Point Parkway, Brisbane, CA 94005

Disclosures

• All authors are employees of Arch Oncology, Inc.

AO-176: Clearly Differentiated in the CD47 LandscapeHumanized IgG2 anti-CD47 Antibody with Multiple Mechanisms of Action

Differentiated Best-in-Class Antibody | Blocking and Direct Killing | Unique Among the Anti-CD47 Field

CONVENTIONALANTI-CD47 APPROACH AO-176

POTENTIAL ADVANTAGES

Blocking ✓ ✓

Preferential Binding to Tumor Cells vs. Normal Cells X ✓ SAFETY

Better Binding in Tumor Environment (Low pH) X ✓ SAFETY + EFFICACY

Direct Killing & DAMP Induction X ✓ EFFICACY

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3 Chakraborty P et al, Cancer Res 2019;79(13 Suppl):Abstract nr 540; Wilson WC et al, Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B100, Puro R et al, Cancer Res 2018;78(13 Suppl):Abstract nr 1765; Capoccia BJ et al, J ImmunoTherapy of Cancer (2018) 6 (Suppl 1) 115

Azacitidine Enhances AO-176-Mediated Phagocytosis of HL-60 Cells

HL-60 Cells

AO-176 Increases Phagocytosis WhenCombined with Azacitidine. Humanmonocyte derived macrophages wereplated at a concentration of 5 x 104

cells per well in a 96 well plate. 8 x 104

CFSE (1μM) labeled human HL-60 acutemyeloid leukemia cells were treatedwith 3 or 10µM azacitidine overnightprior to being incubated with increasingconcentrations of AO-176 and added tothe macrophage cultures at 37°C fortwo hours. Non-phagocytosed targettumor cells were removed, andmacrophage cultures were washedextensively. Macrophages weretrypsinized and stained for CD14 priorto analysis by flow cytometry. Percent(%) phagocytosis is calculated from theratio of CFSE+/CD14+ to total CD14+macrophages. Figures show singleconcentrations of each agent alone, orin combination, as optimized per eachcell line. Azacitidine also increasedsurface exposure of calreticulin. Twoother AML cell lines, MV4-11 and KG-1showed similar effects.

% P

hago

cyto

sis

60

40

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0IgG2 3 10

AO-176 (µg/mL)

0 µM Azacitidine3 µM Azacitidine10 µM Azacitidine

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% P

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IgG2 3 µg/mLAO-176

3 µM Aza

AO-176 +Aza

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% C

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/SYT

OX-

0 3Azacitidine (µM)

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Venetoclax Enhances AO-176-Mediated Phagocytosis of HL-60 Cells

HL-60 Cells

AO-176 Increases Phagocytosis WhenCombined with Venetoclax. Humanmonocyte derived macrophages wereplated at a concentration of 5 x 104 cellsper well in a 96 well plate. 8 x 104 CFSE(1μM) labeled human HL-60 acutemyeloid leukemia cells were treatedwith 3 or 10nM venetoclax overnightprior to being incubated with increasingconcentrations of AO-176 and added tothe macrophage cultures at 37°C fortwo hours. Non-phagocytosed targettumor cells were removed, andmacrophage cultures were washedextensively. Macrophages weretrypsinized and stained for CD14 priorto analysis by flow cytometry. Percent(%) phagocytosis is calculated from theratio of CFSE+/CD14+ to total CD14+macrophages. Figures show singleconcentrations of each agent alone, orin combination, as optimized per eachcell line. Venetoclax also increasedsurface exposure of calreticulin. Twoother AML cell lines, MV4-11 and KG-1showed similar effects.

30

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0

% P

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IgG2 3 µg/mLAO-176

3 nM Veneto

AO-176 +Veneto

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% C

RT+

/SYT

OX-

0 3Venetoclax (nM)

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% P

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0IgG2 3 10

AO-176 (µg/mL)

0 nM Venetoclax3 nM Venetoclax10 nM Venetoclax

AO-176 Combines with Azacitidine to Increase AML Cell Killing

HL-60 MV4-11

AO-176 Enhances Cell Killing in Combination with Azacitidine. HL-60 (left panel) or MV4-11 (right panel) acute myeloid leukemia cells were incubated with 100µg/mL AO-176 alone, 5 µM azacitidine alone, or a combination of AO-176 and azacitidine in RPMI media at 37oC for 24 hours. Cells were washed and then stainedwith Annexin V PE and SYTOX Blue followed by flow cytometry analysis.

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% T

otal

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UntreatedCells

100 µg/mLAO-176

5 µM Aza

AO-176 +Aza

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+/SY

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UntreatedCells

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5 µM Aza

AO-176 +Aza

AO-176 Combines with Venetoclax to Increase AML Cell Killing

MV4-11 KG-1

AO-176 Enhances Cell Killing in Combination with Venetoclax. MV4-11 or KG-1 acute myeloid leukemia cells were incubated with 100 µg/mL (MV4-11, left panel)or 10 µg/mL (KG-1, right panel) AO-176 alone, 0.3 µM (MV4-11) or 2.5 µM (KG-1) venetoclax alone and their respective combinations with AO-176 in RPMI mediaat 37oC for 24 hours. Cells were washed and then stained with Annexin V PE and SYTOX Blue followed by flow cytometry analysis.

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100 µg/mLAO-176

2.5 µM Veneto

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in V

+/SY

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3 nM Veneto

AO-176 +Veneto

AO-176 Increases HL-60 Surface DAMP Exposure, Alone and In Combination with Azacitidine

PDIA3Calreticulin

AO-176 Enhances DAMP Induction. Left panel-HL-60 acute myeloid leukemia cells were incubated with 10, 30 or 100 µg/mL AO-176 alone in RPMI media at 37oCfor 24 hours. Cells were washed and then stained for calreticulin and viability (SYTOX Blue) followed by flow cytometry analysis. Cell surface exposure ofcalreticulin was increased by treatment with AO-176 in a concentration-dependent manner. Right Panel-AO-176 Enhances DAMP Induction in Combination with 5-Azacitidine. HL-60 acute myeloid leukemia cells were incubated with 100 µg/mL AO-176 alone, 5 µM azacitidine alone, or a combination of AO-176 and azacitidinein RPMI media at 37oC for 24 hours. Cells were washed and then stained for PDIA3 and viability (SYTOX Blue) followed by flow cytometry analysis. PDIA3 cellsurface exposure was increased by AO-176 treatment and further enhanced in combination with azacitidine.

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AO-176 Treatment of HL-60 Xenograft Mice Results in Significant Increases in Survival, Alone and In Combination with Azacitidine

Treatment with AO-176 Extends Survival in a Human Acute Myeloid Leukemia Model. Human HL-60 acute myeloid leukemia cells were implanted intravenouslyinto NSG mice (N=10/group) and treatment was initiated four days after implantation. Human IgG2 (hIgG2), 2.5% DMSO:PBS solution, azacitidine, AO-176, 5F9, Azaand AO-176 or Aza and 5F9 in combination were administered by intraperitoneal (IP) injection and survival was monitored and plotted versus days following tumorinoculation. **** p<0.0001

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Dosing ScheduleAO-176:

5F9:Aza:

IgG2 + IgG4 control 10 MPK

DMSO Control

AO-176 10 MPK

5F9 10 MPKAza 7.5 MPK

AO-176 10 MPK + Aza 7.5 MPK

5F9 10 MPK + Aza 7.5 MPK

0 20Days Post Inoculation

40 60 80 100

AO-176 is Efficacious in Pre-Clinical AML Models

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Azacitidine and venetoclax enhance AO-176-mediate phagocytosis of AML cells

AO-176 combines with azacitidine and venetoclax to increase AML cell killing

AO-176, alone and in combination with azacitidine, significantly increases survival in an HL-60 xenograft model. AO-176 activity is comparable to that of a magrolimab benchmark antibody.

AO-176 induces cell surface DAMP exposure, alone and in combination, with azacitidine on HL-60 cells

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AO-176 is currently being evaluated in two phase 1/2 studies for the treatment of solid tumors (NCT03834948) and multiple myeloma (NCT044445701).

Taken together, these data highlight the strong therapeutic potential of AO-176 for AML patients.

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