HLA-DR TTI-621-mediated phagocytosis CD80 CD86...

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Page 1: HLA-DR TTI-621-mediated phagocytosis CD80 CD86 …s2.q4cdn.com/.../2016/Poster-AACR-2016-final-jpeg-images-10APR16.… · L P S ) M 2 a 2 b M c 0 1 2 3 4 CD200R M F I (f o l d 100000

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)