ALX148 Enhances the Depth and Durability ofResponse to Multiple AML Therapies(Abstract #1965)
Amy Chen, Ons Harrabi, Abraham P. Fong, Katherine L. Ruffner, Alison J. Forgie, Janet Sim,Sophia S. Randolph, Hong I. Wan, Jaume Pons, and Tracy C. Kuo
ALX Oncology Inc., Burlingame, CA

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Disclosures

Chen: ALX Oncology Inc: Consultancy, Equity Ownership. Harrabi: ALX Oncology Inc: Consultancy, Equity Ownership. Fong:
ALX Oncology Inc: Employment, Equity Ownership. Ruffner: ALX Oncology Inc: Employment, Equity Ownership. Forgie: ALX
Oncology Inc: Employment, Equity Ownership. Sim: ALX Oncology Inc: Consultancy, Equity Ownership. Randolph: ALX
Oncology Inc: Employment, Equity Ownership. Wan: ALX Oncology Inc: Consultancy, Equity Ownership. Pons: ALX Oncology
Inc: Employment, Equity Ownership; Membership on an entity's Board of Directors or advisory committees. Kuo: ALX
Oncology Inc: Consultancy, Equity Ownership.

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Background
High Affinity CD47
Binding Domains of SIRP⍺• CD47, a myeloid checkpoint and marker of self, signals themacrophage to ignore the cell on which CD47 is expressed bybinding its receptor, SIRPα.1 Tumors upregulate CD47 to evadethe immune response.

Optimized, picomolar binding affinity
Fc domain mutated to eliminate Fcɣ receptor
binding and minimize associated toxicity• Fc domain enables antibody-like PK• Molecular weight half the size oftypical antibody

CD47/SIRP⍺
Macrophage
SIRPα

CD47

CRTCancer Cell

ALX148LRP

AZA or VEN

Macrophage
SIRPα

ALX148
CD47

CRT• ALX148 is a high affinity CD47 blocker fusion protein with aninactive human immunoglobulin Fc region designed to enhance
the activity of anti-cancer targeted antibodies and checkpoint
inhibitors with minimal hematologic toxicity.2

LRP• Calreticulin is a pro-phagocytic multifunctional protein that istranslocated to the cell surface under conditions of cellular stressand apoptosis, marking the cell for phagocytic clearance.3

AZA or VEN• Azacitidine (AZA) is a hypomethylating chemotherapeutic agent
used in acute myeloid leukemia (AML) and myelodysplastic
syndrome (MDS) treatments that induces upregulation of both
CD47 and calreticulin.

Cancer Cell• Venetoclax (VEN) is a specific B-cell lymphoma-2 (BCL2) inhibitorthat can restore apoptosis in malignant cells and has been
recently granted approval in combination with AZA for treatmentof newly diagnosed AML.4

CRT – Calreticulin; LRP – Low density lipoprotein receptor–related protein; AZA – Azacitidine; VEN – Venetoclax.
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Azacitidine or Venetoclax Treatments Increase Surface Levels of Calreticulin and CD47 in
AML Cell Lines and Primary AML Blasts
Calreticulin
Primary AML Don 3

Primary AML Don 1

80004002503002002001501001006000

NA
2.
5CD47
Primary AML Don 11000

Primary AML Don 2

1500

3000

600

1000

2000

400

500

100000• AZA and VEN increase expression of thepro-phagocytic signal calreticulin in both
AML cell lines and primary AML blasts.
• AZA and VEN also increase expression ofthe CD47 anti-phagocytic signal.

800

NA

VE
uM

1.
25

1.
25uM

AZ

only

VE

med
ia

A
AZ

uM

uM
2.
5

2.
5med
ia

N0M
ed
ia
on0.
ly
62
5uM
AZ
A
2.
5uM
VE
N200only

CD47

Median Fluorescent Intensity (MFI)MV411• AML cells were incubated for 24-48 hours
with AZA or VEN, and calreticulin andCD47 expression were measured by flowcytometry.

uM

VE

AZ

uM

VEmed
ia

2.
5NA
uM
2.
5med
ia

AZ

only

VE

2.
5uM

AAZ

uM

uM
2.
5med
ia

N

0

1.
25

2000

only

4000

only

Calreticulin

Median Fluorescent Intensity (MFI)

MV411

Range of AZA and VEN concentration tested, representative Concentration with maximal effect shown
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4ALX148 Enhances Phagocytosis of AML Cell Lines and Primary AML Blasts in Combination
with Azacitidine or Venetoclax
Azacitidine
HL60

3152

10

150.000MediaonM nly
AL
X148
1uM
AZAZ
A
A+
AL
X148

M
ed
iaonM nly
AL
X148
16nM
AZ
AZ
A
A+
AL
X148

M
ediaonM nly
AL
X1
1.
48
25uM
AZ
AZ
A
A+
AL
X1
48

0.5

• AML cells were incubated with AZA or VEN
for 24-48 hours and then co-cultured for
2 hours with human monocyte derived
macrophages in the presence or absenceof ALX148.
25

1.04

204Phagocytosis(fold over media only)

Primary AML Don 1

OCIAML3
4

1.5

Venetoclax

2.0

2.0

40

1.5

1.530

1.0

1.0

20

0.5

0.5

100.0

0.00252020• ALX148 in combination with AZA or VEN
enhanced phagocytotic elimination of AML
cells by human macrophages compared toeither treatment alone.

M
ediaonM nly
AL
X1
1.
48
25uM
VE
VE
N
N+
AL
X1
4850M
ed
iaonM nly
AL
X1
48
1uM
VE
VE
NN+
AL
X148

2.5M
ed
iaonM nly
AL
X112485nMVEVENN+AL
X148

Phagocytosis(fold over media only)

Primary AML Don 1

OCIAML3

HL60
2.5• Phagocytosis of AML cells was determinedby flow cytometry as the percentage ofmacrophages that have engulfed AML(CFSE+) cells compared to total number ofmacrophages.

Range of AZA and VEN concentration tested, representative results shown
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Combination of ALX148 with Azacitidine Demonstrated Significant and Durable Anti-Leukemic
Activity and Prolonged Survival in Aggressive AML Tumor Model
HL60 LUC

HL60 Survival

10 1210080

10 10
109PBS

108AZA (5 mg/kg)

107ALX148 (30 mg/kg)
AZA + ALX148 (30)10 6
10 5Probability of Survival

Total Flux (photons/sec)

10 11PBS60

ALX148 (30 mg/kg)
AZA (5mg/kg)

40

AZA + ALX148

**

**

20

10 4

**P value <0.002

10 3003ÝÝÝÝÝÝÝÝÝÝÝÝÝÝ50

100

Day Post Innoculation

150

050100150

Day Post Innoculation• Utilizing an aggressive AML tumor model, HL60 cells were transplanted by intravenous injection in NOD SCID mice withengraftment and tumor burden measured by bioluminescent imaging until end of study. AZA and ALX148 therapies wereinitiated on day 4 post engraftment and dosed intraperitoneally every 3 days for a total of 14 doses.
• AZA and ALX148 monotherapies produced moderate tumor growth inhibition with all mice succumbed to disease by day 85.
• Combination of AZA and ALX148 completely eliminated tumor growth with 100% animal survival up to study termination onday 147.
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6ALX148 Maintenance Therapy Controlled Disease and Extended Survival
HL60 LUC

HL60 LUC
10010 1280

10 1010 9AZA (5 mg/kg)

10 8

AZA + ALX148 (10 mg/kg)

10 7

ALX148 continued

10 6
10 5

Probability of Survival

Total Flux (photons/sec)

101160AZA (5mg/kg)
AZA + ALX148 (10 mg/kg)

40

ALX148 continued

20

10 40

10 3050100

Day Post Innoculation

150050100

150

Day Post Innoculation• AZA and ALX148 therapies were initiated day 4 post engraftment and dosed intraperitoneally every 3 days for a total of14 doses.
• ALX148 maintenance monotherapy was continued in half the combination cohort for an additional 16 doses.
• After 14 total doses of AZA and ALX148 combination dosing, tumor inhibition was observed until day 105.
• ALX148 maintenance therapy extended tumor inhibition with 3 out of 4 mice surviving until study termination on day 147.

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7Combination of ALX148 with Venetoclax Abrogated Tumor Growth and Prolonged Survival
in AML Tumor Model
HL60 LUC

HL60 LUC

10 12

10080

10 10
10 9PBS10 8

ALX148 (30 mg/kg)

10 7

VEN (100 mg/kg)

10 6

VEN + ALX148

10 5

Probability of Survival

Total Flux (photons/sec)

10 11PBS60

ALX148 (30 mg/kg)
VEN (100 mg/kg)

40

VEN+ALX148

20****P value <0.002

10 410 3**

00 Ý Ý Ý Ý Ý 2040
Day post Inoculation

6080

02040

60

80

Day Post Innoculation• VEN and ALX148 therapies were initiated day 4 post engraftment with ALX148 given intraperitoneally every 3 days for atotal of 5 doses and for 14 consecutive days by oral gavage for VEN.
• VEN and ALX148 monotherapies produced moderate tumor growth inhibition but did not maintain durable responses withall mice succumbing to disease by day 40.
• In contrast, combination of VEN+ALX148 completely eliminated tumor growth in 6 out 8 mice within an 80 day evaluation
period. Study is ongoing.
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8Combining ALX148 with both Venetoclax and Azacitidine Inhibited Tumor Growth andProlonged Survival in AML Tumor Model
HL60 LUC

HL60 LUC
10010 12PBS

10 10

ALX148 (30 mg/kg)

10 9

AZA (5mg/kg)

10 8

VEN (100 mg/kg)

10 7

AZA+VEN

10 6

AZA+VEN+ALX148

10 580

Probability of Survival

Total Flux (photons/sec)

1011PBS60

ALX148AZA40VEN
AZA+VEN

20AZA+VEN+ALX148

10 4010 302040Day Post Innoculation

6080

020406080

Day Post Innoculation• AZA, VEN and ALX148 therapies were initiated day 4 post engraftment with AZA and ALX148 given intraperitoneally every
3 days for a total of 5 doses and for 14 consecutive days by oral gavage for VEN.
• AZA and VEN combination therapies yielded 4 out of 8 mice with progressive disease.
• In contrast, the combination of AZA+VEN+ALX148 completely eliminated tumor growth for 7 out of 9 mice within an 80
day evaluation period. Study is ongoing.

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9Conclusion
ALX148 in combination with VEN/AZA demonstrates superior tumor control and significant
prolongation of survival compared to VEN, AZA, or VEN/AZA in aggressive murine models of AML• AZA and VEN increase the cell surface expression of calreticulin and CD47 in AML cell lines and primary AML blasts in vitro.
• Co-treatment of ALX148 with AZA as well as VEN leads to increased clearance of AML cell lines and primary AML blasts byhuman macrophages in vitro.
• Combination treatment of ALX148 with AZA, VEN or AZA+VEN leads to tumor elimination and prolonged survival in AMLxenograft models.
• ALX148 maintenance therapy significantly prolonged duration of tumor remission and overall survival after discontinuationof AZA treatment in AML xenograft models.
• These results support evaluation of ALX148 in combination with VEN/AZA in patients with AML.
• The ASPEN-02 clinical trial evaluates ALX148 in combination with AZA for patients with 1L MDS and is ongoing(NCT04417517), ASPEN-05 evaluating ALX148 in patients with AML is planned.

1. Weiskopf, K., Eur J Cancer. 2017 May;76:100-109; 2. Kauder, S.E. et al., PLoS ONE. 2018 August;13(8): e0201832; 3. Chao MP. et al., Sci Transl Med. 2010 Dec 22;2(63):63ra94;
4. https://www.fda.gov/drugs/fda-approves-venetoclax-combination-aml-adults
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