Cancer Immunotherapy Targeting CD47: Wild Type SIRP Fc...
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1 Robert A. Uger, Karen Dodge, Xinli Pang and Penka S. Petrova Introduction Blocking the CD47 “Do Not Eat” Signal with SIRPα α αFc Wild Type SIRPα α αFc Binds Very Poorly to Human RBCs Compared to Commercially-Available CD47 Antibodies “Pre-clustering” CD47 on Human RBCs Leads to a Dramatic Increase in SIRPα α αFc Binding Cancer Immunotherapy Targeting CD47: Wild Type SIRPα α αFc is the Ideal CD47-Blocking Agent to Minimize Unwanted Erythrocyte Binding CD47 binds to SIRPα on the surface of macrophages and delivers a “do not eat” signal to suppress phagocytosis Cancer stem cells and bulk tumor cells in acute myeloid leukemia (AML) and other malignancies overexpress CD47 and exploit this pathway to evade macrophage-mediated destruction Blocking CD47 using its soluble ligand (SIRPαFc) or anti-CD47 antibodies has emerged as a promising strategy to neutralize the suppressive effects of CD47 and promote the eradication of tumor cells One concern with CD47-based therapies is the expression of CD47 on red blood cells (RBCs), which has the potential to act as a large antigen sink and cause hematological toxicity; anemia has been reported in animals treated with high affinity SIRPαFc variants and CD47-specific antibodies In this study we compared the RBC binding profile of wild type SIRPαFc to other CD47-specific agents The Poor Binding of Wild Type SIRPα α αFc to RBCs is Specific to Humans Trillium Therapeutics Inc./Stem Cell Therapeutics Corp., Toronto, ON, Canada The Binding Difference is RBC-Specific: Both Bind Similarly to AML and Other CD47+ Cells Fluorescence Intensity Count 10 0 10 1 10 2 10 3 10 4 0 50 100 150 200 Fluorescence Intensity Count 10 0 10 1 10 2 10 3 10 4 0 75 150 225 300 Fluorescence Intensity Count 10 0 10 1 10 2 10 3 10 4 0 100 200 300 400 Fluorescence Intensity Count 10 0 10 1 10 2 10 3 10 4 0 100 200 300 400 Fluorescence Intensity Count 10 0 10 1 10 2 10 3 10 4 0 100 200 300 400 SIRPα α αFc 2D3 B6H12 BRIC126 CC2C6 CD47 mAbs Binding to fresh human RBCs by flow cytometry at saturating concentrations Specific staining in red; secondary alone in blue; unstained in black Fluorescence Intensity Count 10 0 10 1 10 2 10 3 10 4 0 50 100 150 200 Fluorescence Intensity Count 10 0 10 1 10 2 10 3 10 4 0 50 100 150 200 Fluorescence Intensity Count 10 0 10 1 10 2 10 3 10 4 0 100 200 300 400 Human SIRPα α αFc → Human RBCs Human SIRPα α αFc → Cyno RBCs Mouse SIRPα α αFc → Mouse RBCs Binding to fresh RBCs by flow cytometry at saturating concentrations SIRPαFc staining in red; secondary alone in blue; unstained in black CD47 on tumor cells delivers a stop signal to macrophages to suppress phagocytosis Wild Type SIRPα α αFc Binds Very Poorly to Human RBCs Compared to Proprietary CD47-Specific Agents Conclusions Tumor Mφ CD47 SIRPα Tumor Cells Survive DO NOT EAT Tumor Mφ Tumor Cells Destroyed SIRPαFc The CD47 stop signal is blocked by SIRPαFc, allowing macrophages to phagocytose tumor cells EAT Wild type SIRPαFc binds very poorly to human RBCs compared to both commercial CD47 antibodies and proprietary CD47-specific agents (mAb and mutant high affinity SIRPαFc) Wild type SIRPαFc and CD47 mAbs bind similarly to other CD47+ cells (including AML cells), indicating this phenomenon is unique to RBCs The poor binding of SIRPαFc is specific to human RBCs; strong binding to monkey RBCs may overestimate the risk of hematological toxicity in non-human primate studies The mechanism underlying low RBC binding is unclear, but may relate to the lack of mobility of CD47 in the erythrocyte membrane and the inability of SIRPαFc to form high affinity clusters Our wild type SIRPαFc fusion is the ideal CD47 blocking agent to reduce the potential RBC sink effect and minimize hematological toxicity Binds to CD47 with nanomolar affinity Disrupts the interaction of CD47 with cell surface SIRPα Enables macrophage-mediated killing of tumor cells in vitro Exhibits potent in vivo anti-leukemic activity in AML xenograft models Is in pre-clinical development as a therapy for AML SIRPα α αFc: 2D3 B6H12 BRIC126 C C2C6 S I R P aFc C o ntro l Fc 0 1000 2000 3000 4000 CD47 mAbs FI (Geo Mean) 2 D3 B6H12 BRI C1 2 6 CC2C6 S I RP a Fc Control Fc 0 50 100 150 200 250 300 350 400 450 CD47 mAbs FI (Geo Mean) 2 D 3 B6H12 BRIC126 CC2C6 S I R P aFc C o ntro l Fc 0 50 100 150 200 250 300 350 CD47 mAbs FI (Geo mean) 2 D 3 B6H12 BRIC126 C C2C6 S I R P aFc C o ntro l Fc 0 2500 5000 7500 10000 12500 15000 17500 CD47 mAbs FI (Median) 2D3 B 6 H12 BRIC126 C C2C6 S I R P aFc Co nt r o l Fc 0 500 1000 1500 2000 2500 CD47 mAbs FI (Geo Mean) RBCs (n=14) Primary AML AML-2 Cell Line TEX Cell Line Jurkat Cell Line 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 0 0 10000 20000 30000 40000 50000 60000 SIRPα α αFc or CD47 mAb (μ μ μM) RBC Binding (Median FI) Wild type SIRPα α αFc vs. anti-CD47 mAb 5F9* Wild type SIRPα α αFc Clone 5F9* 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 0 0 1000 2000 3000 4000 5000 6000 7000 8000 SIRPα α αFc ( μ μ μM) RBC Binding (Mean FI) Wild type SIRPα α αFc vs. high affinity SIRPα α αFc** Wild type SIRPα α αFc High affinity SIRPα α αFc** **Engineered high affinity SIRPα (FD6) described in Weiskopf et al. 2013 Science 341:88, linked to human IgG4 Fc *Humanized anti-CD47 mAb (5F9) described in WO2011/143624, linked to human IgG4 Fc Fluorescence Intensity Count 10 0 10 1 10 2 10 3 10 4 0 75 150 225 300 1 1 2 2 Wild type SIRPαFc binds poorly to human RBCs Wild type SIRPαFc binds strongly to human RBCs that were pre-treated with non- blocking anti-CD47 (2D3)
Transcript of Cancer Immunotherapy Targeting CD47: Wild Type SIRP Fc...
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Robert A. Uger, Karen Dodge, Xinli Pang and Penka S. Petrova
Introduction
Blocking the CD47 “Do Not Eat” Signal with SIRP ααααFc
Wild Type SIRP ααααFc Binds Very Poorly to Human RBCs Compared to Commercially-Available CD47 Antibodies
“Pre-clustering” CD47 on Human RBCs Leads to a Dramatic Increase in SIRP ααααFc Binding
Cancer Immunotherapy Targeting CD47: Wild Type SIRP ααααFc is the Ideal CD47-Blocking Agent to Minimize Unwanted Eryt hrocyte Binding
� CD47 binds to SIRPα on the surface of macrophages and delivers a “do not eat” signal to suppress phagocytosis
� Cancer stem cells and bulk tumor cells in acute myeloid leukemia (AML) and other malignancies overexpress CD47 and exploit this pathway to evade macrophage-mediated destruction
� Blocking CD47 using its soluble ligand (SIRPαFc) or anti-CD47 antibodies has emerged as a promising strategy to neutralize the suppressive effects of CD47 and promote the eradication of tumor cells
� One concern with CD47-based therapies is the expression of CD47 on red blood cells (RBCs), which has the potential to act as a large antigen sink and cause hematological toxicity; anemia has been reported in animals treated with high affinity SIRPαFc variants and CD47-specific antibodies
� In this study we compared the RBC binding profile of wild type SIRPαFc to other CD47-specific agents
The Poor Binding of Wild Type SIRP ααααFc to RBCs is Specific to Humans
Trillium Therapeutics Inc./Stem Cell Therapeutics Corp., Toronto, ON, Canada
The Binding Difference is RBC-Specific: Both Bind Similarly to AML and Other CD47+ Cells
Fluorescence Intensity
Cou
nt
100
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103
104
0
50
100
150
200
Fluorescence Intensity
Cou
nt
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103
104
0
75
150
225
300
Fluorescence Intensity
Cou
nt
100
101
102
103
104
0
100
200
300
400
Fluorescence Intensity
Cou
nt
100
101
102
103
104
0
100
200
300
400
Fluorescence Intensity
Cou
nt
100
101
102
103
104
0
100
200
300
400
SIRPααααFc 2D3 B6H12 BRIC126 CC2C6
CD47 mAbs
Binding to fresh human RBCs by flow cytometry at saturating concentrations Specific staining in red; secondary alone in blue; unstained in black
Fluorescence Intensity
Cou
nt
100
101
102
103
104
0
50
100
150
200
Fluorescence Intensity
Cou
nt
100
101
102
103
104
0
50
100
150
200
Fluorescence Intensity
Cou
nt
100
101
102
103
104
0
100
200
300
400
Human SIRP ααααFc →Human RBCs
Human SIRP ααααFc →Cyno RBCs
Mouse SIRP ααααFc →Mouse RBCs
Binding to fresh RBCs by flow cytometry at saturating concentrations SIRPαFc staining in red; secondary alone in blue; unstained in black
CD47 on tumor cells delivers a stop signal to macrophages to suppress phagocytosis
Wild Type SIRP ααααFc Binds Very Poorly to Human RBCs Compared to Proprietary CD47-Specific Agents
Conclusions
Tumor
Mφ
CD47
SIRPα
Tumor CellsSurvive
DO NOT EAT
Tumor
Mφ
Tumor CellsDestroyed
SIRPαFc
The CD47 stop signal is blocked by SIRPαFc, allowing macrophages to phagocytose tumor cells
EAT
� Wild type SIRPαFc binds very poorly to human RBCs compared to both commercial CD47 antibodies and proprietary CD47-specific agents (mAb and mutant high affinity SIRPαFc)
� Wild type SIRPαFc and CD47 mAbs bind similarly to other CD47+ cells (including AML cells), indicating this phenomenon is unique to RBCs
� The poor binding of SIRPαFc is specific to human RBCs; strong binding to monkey RBCs may overestimate the risk of hematological toxicity in non-human primate studies
� The mechanism underlying low RBC binding is unclear, but may relate to the lack of mobility of CD47 in the erythrocyte membrane and the inability of SIRPαFc to form high affinity clusters
� Our wild type SIRPαFc fusion is the ideal CD47 blocking agent to reduce the potential RBC sink effect and minimize hematological toxicity
� Binds to CD47 with nanomolar affinity
� Disrupts the interaction of CD47 with cell surface SIRPα
� Enables macrophage-mediated killing of tumor cells in vitro
� Exhibits potent in vivo anti-leukemic activity in AML xenograft models
� Is in pre-clinical development as a therapy for AML
SIRPααααFc:2D3
B6H12BRIC
126
CC2C6SIR
PaFcContr
o l Fc
0
1000
2000
3000
4000
CD47 mAbs
FI (
Geo
Mea
n)
2D3
B6H12
BRIC126
CC2C6SIR
PaFc
Contro
l Fc
050
100150
200250300350400
450
CD47 mAbs
FI (
Geo
Mea
n)
2D3
B6H12BRIC
126
CC2C6SIR
PaFcContr
o l Fc
0
50
100
150
200
250
300
350
CD47 mAbs
FI (
Geo
mea
n)
2D3
B6H12BRIC
126
CC2C6SIR
PaFcContr
o l Fc
0
2500
5000
7500
10000
12500
15000
17500
CD47 mAbs
FI (
Med
ian)
2D3B6H
12BRIC
126
CC2C6SIR
PaFcCont
rol F
c
0
500
1000
1500
2000
2500
CD47 mAbs
FI (
Geo
Mea
n)
RBCs (n=14) Primary AML AML-2 Cell Line TEX Cell Line J urkat Cell Line
10-6 10-5 10-4 10-3 10 -2 10 -1 100 10100
10000
20000
30000
40000
50000
60000
SIRPααααFc or CD47 mAb ( µµµµM)
RB
C B
indi
ng (
Med
ian
FI)
Wild type SIRP ααααFc vs. anti-CD47 mAb 5F9*
Wild type SIRPααααFc
Clone 5F9*
10 -6 10-5 10-4 10 -3 10-2 10-1 100 10100
1000
2000
3000
4000
5000
6000
7000
8000
SIRPααααFc ( µµµµM)
RB
C B
indi
ng (
Mea
n FI
)
Wild type SIRP ααααFc vs. high affinity SIRP ααααFc**
Wild type SIRPααααFc
High affinity SIRPααααFc**
**Engineered high affinity SIRPα (FD6) described in Weiskopf et al. 2013 Science 341:88, linked to human IgG4 Fc
*Humanized anti-CD47 mAb (5F9) described in WO2011/143624, linked to human IgG4 Fc
Fluorescence Intensity
Co
unt
100
101
102
103
104
0
75
150
225
300
1
1
2
2
Wild type SIRPαFc binds poorly to human RBCs
Wild type SIRPαFc binds strongly to human RBCs that were pre-treated with non-blocking anti-CD47 (2D3)
