HLA-DR TTI-621-mediated phagocytosis CD80 CD86...
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M-CSF
IFN-γ
IFN-γ + LPS
IL-4
HAGG + IL-1β
IL-10 + TGF-β
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
0
1
2
3
4
CD200R
MF
I (f
old
ch
an
ge
)
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
0
1
2
3
4
CD206
MF
I (f
old
ch
an
ge
)
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
0
2
4
6
CD163
MF
I (f
old
ch
an
ge
)
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
0.0
0.5
1.0
1.5
2.0
2.5
CD86
MF
I (f
old
ch
an
ge
)
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
0
1
2
3
4
CD80
MF
I (f
old
ch
an
ge
)
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
0.0
0.5
1.0
1.5
2.0
HLA-DR
MF
I (f
old
ch
an
ge
)
Control Fc TTI-6210
20
40
60
80
M0
% P
ha
go
cy
tos
is
***
Control Fc TTI-6210
20
40
60
80
M1
% P
ha
go
cy
tosis
***
Control Fc TTI-6210
20
40
60
80
M1 (IFN + LPS)
% P
ha
go
cy
tos
is
***
Control Fc TTI-6210
20
40
60
80
M2a (IL-4)
% P
ha
go
cy
tos
is
***
Control Fc TTI-6210
20
40
60
80
M2b (HAGG + IL-1)
% P
ha
go
cy
tos
is
***
Control Fc TTI-6210
20
40
60
80
M2c (IL-10 + TGF)
% P
ha
go
cy
tos
is
***
Gloria H.Y. Lin, Vien Chai, Vivian Lee, Karen Dodge, Tran Truong, Mark Wong, Lisa D. Johnson, Xinli Pang, Penka S. Petrova, Robert A. Uger, Natasja N. Viller
Introduction Macrophage Subsets Vary in Expression of M1 and M2 Surface Markers and Cytokine Production
Conclusions
SIRPαFc, a CD47-Blocking Cancer Immunotherapeutic, Triggers Phagocytosis of Lymphoma Cells by Both
Classically (M1) and Alternatively (M2) Activated Macrophages
CD47 binds to SIRPα on the surface of macrophages and delivers a “do not
eat” signal to suppress phagocytosis.
Tumor cells frequently overexpress CD47 and exploit this pathway to evade
macrophage-mediated destruction.
Blocking CD47 using a soluble decoy receptor (SIRPαFc) has emerged as a
promising strategy to neutralize the suppressive effects of CD47 and promote
the eradication of tumor cells.
In this study we have examined the ability of SIRPαFc to trigger phagocytosis
of lymphoma cells by six distinctly in vitro polarized macrophage populations.
TTI-621 increased phagocytosis of lymphoma tumor cells by all macrophage subsets, with M1 and M2c
MDMs being superior at phagocytosis
Macrophage subsets with slightly lower phagocytic capabilities (M0, M2a and M2b) were readily
repolarized into highly phagocytic MDMs using cytokines or TLR agonists
Macrophage expression of the high-affinity FcγRI (CD64) correlated with phagocytic activity following TTI-
621 treatment
All FcγRs (CD16, CD32, CD64) can contribute to TTI-621-mediated phagocytosis. On CD64high (M1)
macrophages, the high-affinity FcγR CD64 is the main contributor, whereas on CD64low macrophages, the
low-affinity CD16 and CD32 FcγRs play a bigger role
A Phase I clinical trial of TTI-621 in patients with advanced hematological malignancies is currently
underway (ClinicalTrials.gov # NCT02663518)
Trillium Therapeutics Inc., Toronto, ON, Canada
TTI-621 is a SIRPαFc fusion protein:
Human SIRPα linked to a human IgG1
Disrupts the interaction of CD47 with cell surface SIRPα and enables
macrophage-mediated killing of tumor cells in vitro and in vivo
Is currently in a Phase I clinical trial for lymphomas and other hematological
malignancies
AACR 2016
Abstract #2345
Macrophage Expression of the High-Affinity FcγRI (CD64) Correlates with TTI-621-Triggered Phagocytosis
TTI-621 Triggers Phagocytosis of Tumor Cells by All Macrophage Subsets
TTI-621-Mediated Phagocytosis and FcγR Expression by M0, M2a and
M2b Macrophages Can Be Further Increased by Re-Polarization with
Cytokines and Toll-Like Receptor Agonists
Expression of cell surface markers was assessed by flow cytometry and cell culture supernatants were analyzed by Cytokine Bead Array.
MDMs were co-cultured with Violet Proliferation Dye (VPD450)-labeled tumor cells for two hours in the presence of TTI-621 or isotype-matched control Fc. % Phagocytosis was determined
by flow cytometry as the % of live, single, CD14+CD11b+ MDMs that were VPD450+. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001
M1 Markers M2 Markers
Macrophage Polarization
All FcγRs (CD16, CD32, CD64) contribute to TTI-621-mediated phagocytosis
On CD64high (M1) macrophages, the high-affinity FcγR CD64 is the main contributor
On CD64low macrophages, the low-affinity CD16 and CD32 FcγRs play a bigger role
M0 or polarized M2a and M2b macrophages were left untreated (non-repolarized) or repolarized with IFN, IFNa, IL-10, Poly (I:C), LPS, R848 and CpG for
20 hours. Phagocytosis and expression of CD16, CD32 and CD64 was assessed by flow cytometry.
Human monocyte-derived macrophages (MDMs) were
differentiated from peripheral blood CD14+ monocytes
using M-CSF and polarized into subsets as shown.
Isotype Control
Non-repolarized
IFN
IL-10
LPS
CpG
IFNa
Poly (I:C)
• Additional cytokine/chemokine data not shown: M1(+LPS) and M2b specific: IL-6. M1 and M1(+LPS) specific: CXCL-10 (IP-10), CXCL-11 (I-TAC).
M1, M1(+LPS) and M2b specific: CCL-3 (MIP-1a), CCL-4 (MIP-1), CCL-20 (MIP-3a), CCL-5 (Rantes), CCL-2 (MCP-1), CXCL-8 (IL-8).
TTI-621 (SIRPαFc): A Novel Biologic that
Blocks the CD47 “Do Not Eat” Signal
Cytokines Chemokines
0 2000 4000 6000 80000
20
40
60
80
100
CD64 expression on M
% P
ha
go
cyto
sis
Correlation between CD64 levels on M and
TTI-621-mediated phagocytosis
M0 (M-CSF or GM-CSF)
M1 (+IFN)
M1 (+IFN+LPS)
M2a (+IL-4)
M2b (+HAGG + IL-1)
M2c (+L-10 +TGF)
r2=0.53
TTI-621
+ aCD16
+ aCD32
+ aCD64
+ aCD16
/CD32
/CD64
0
50
100
150
M0
% P
ha
go
cyto
sis
TTI-621
+ aCD16
+ aCD32
+ aCD64
+ aCD16
/CD32
/CD64
0
50
100
150
M1
% P
ha
go
cyto
sis
TTI-621
+ aCD16
+ aCD32
+ aCD64
+ aCD16
/CD32
/CD64
0
50
100
150
M1 (+LPS)
% P
ha
go
cyto
sis
TTI-621
+ aCD16
+ aCD32
+ aCD64
+ aCD16
/CD32
/CD64
0
50
100
150
M2a
% P
ha
go
cyto
sis
TTI-621
+ aCD16
+ aCD32
+ aCD64
+ aCD16
/CD32
/CD64
0
50
100
150
M2b
% P
ha
go
cyto
sis
TTI-621
+ aCD16
+ aCD32
+ aCD64
+ aCD16
/CD32
/CD64
0
50
100
150
M2c
% P
ha
go
cyto
sis
non-rep
olarize
dIF
N
IFNa
IL-1
0
Poly
(I:C)
LPS
R84
8CpG
0
20
40
60
80
M0 Macrophages
% P
ha
go
cy
tos
is
**
Control Fc
TTI-621
**
non-rep
olarize
dIF
N
IFNa
IL-1
0
Poly
(I:C)
LPS
R84
8CpG
0
20
40
60
M2a Macrophages
% P
ha
go
cy
tos
is
*
**
**
**
TTI-621
Control Fc
non-rep
olarize
dIF
N
IFNa
IL-1
0
Poly
(I:C)
LPS
R84
8CpG
0
20
40
60
M2b Macrophages
% P
ha
go
cy
tos
is
Control Fc
TTI-621
***
***
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
10
100
1000
10000
100000
1000000
CXCL-9 (MIG)
CX
CL
-9 (
pg
/mL
)
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
10
100
1000
10000
100000
CXCL-1 (GROa)
CX
CL
-1 (
pg
/mL
)
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
0.1
1
10
100
1000
10000
CCL-17 (TARC)
CC
L-1
7 (
pg
/mL
)
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
1
10
100
1000
10000
IL-10
IL-1
0 (
pg
/mL
)
n.d.
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
0.01
0.1
1
10
100
1000
10000
100000
TNFa
TN
Fa
(p
g/m
L)
M0
M1
M1
(+LP
S)
M2a
M2b
M2c
0.1
1
10
100
1000
10000
100000
IL-12p70
IL-1
2p
70 (
pg
/mL
)
R848
Isotype Control
Non-repolarized
IFN
IL-10
LPS
CpG
IFNa
Poly (I:C)
R848
Isotype Control
Non-repolarized
IFN
IL-10
LPS
CpG
IFNa
Poly (I:C)
R848
FITC CD16
FITC CD16
FITC CD16
FITC CD32
FITC CD32
V450 CD64
V450 CD64
FITC CD32 V450 CD64
Blood
monocytes
Macrophages
M-CSF
10-14 days
Priming
24 hours
M0 Mϕ
M1 Mϕ
M1 Mϕ
M2a Mϕ
M2b Mϕ
M2c Mϕ
Unpolarized
M1
(classical)
M2
(alternative)