Trillium Therapeutics : AACR 2018 Abstract 2720

Introduction

•  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.

•  TTI-621 is a soluble SIRPα recombinant fusion protein with an IgG1 Fc tail that triggers macrophage phagocytosis of tumor cells in vitro and potently inhibits tumor growth in vivo.

•  TTI-621 is currently being evaluated in two clinical studies in patients with hematologic and solid cancers (NCT02663518 and NCT02890368).

•  Engagement of CD47 has been shown to directly induce apoptosis in tumor cells.

•  The objective of this study was to examine the pro-apoptotic potential of TTI-621.

Immobilized TTI-621 Induces Apoptosis in DLBCL and

T-ALL Cells but not in Normal Cells

A

NU-DUL-1

Toledo

JurkatControl Fc TTI-621

% of cells  SD

50 40 30 20

Early apoptotic (AnnexinV+)

Late apoptotic/necrotic (AnnexinV+ & N/IR+)

10

0

Untreated

Control Fc

TTI-621

B

B cells

T cellsControl Fc TTI-621

% of cells  SD

50 40 30 20 10

Early apoptotic (AnnexinV+)

Late apoptotic/necrotic (AnnexinV+ & N/IR+)

0

Untreated

Control Fc

TTI-621

Untreated

Control Fc

TTI-621

Tumor cells (NU-DUL-1, Toledo and Jurkat) (A) or normal B and T lymphocytes (B) were cultured on immobilized TTI-621 or control Fc for 48 hr (A) and 18 hr (B). Apoptosis was determined by flow cytometry. T cells are defined as the PBMCs that are singlet, live, CD14-CD16- and CD3+CD20-. B cells are defined as the PBMCs that are singlet, live, CD14-CD16- and CD3-CD20+. Representative flow cytometry histograms of Toledo cells (A) and normal B cells (B) are shown.

Cellular Stress Enables Soluble TTI-621-Mediated Caspase-Dependent and

PLCγ-1-Independent Apoptosis

A

B

C

UnstressedStressed

2.5Caspase 3/7 activity

+Caspase inhibitor

40

% of cells  SD

30

Early apoptotic (AnnexinV+)

20

Late apoptotic/necrotic (AnnexinV+ & N/IR+)

10

Luminescence fold

2.0

1.5

1.0

% of cells  SD

0.5

50 40 30 20 10

Early apoptotic (AnnexinV+)

Late apoptotic/necrotic (AnnexinV+ & N/IR+)

0

0.0

0

Control Fc TTI-621

Control Fc TTI-621

Control FcTTI-621

Control Fc TTI-621

Control Fc TTI-621

D

E

+PLC inhibitor

60

% of cells  SD

40

Early apoptotic (AnnexinV+)

P-PLCγ1

Late apoptotic/necrotic (AnnexinV+ & N/IR+)

(A) Unstressed or stressed Jurkat cells (derived from high density/low nutrient culture conditions) were treated with 1 µM of soluble TTI-621 or control Fc for 18 hr. Apoptosis was determined by flow cytometry analysis. (B) Caspase 3/7 activity was analyzed on the soluble TTI-621- or control Fc-treated stressed Jurkat cells by Caspase-Glo 3/7 assay kit and expressed as Luminescence fold relative to untreated cells.

PLCγ1

20

GAPDH

0

Control Fc TTI-621

Control Fc TTI-621

Stressed Jurkat cells were treated with 1 µM of soluble TTI-621 or control Fc in the presence of (C) 50 µM of the pan-caspase inhibitor Z-VAD-FMK or (D) 200 nM of the PLC inhibitor U73122. Apoptosis of soluble TTI-621- or control Fc-treated cells were analyzed by flow cytometry. (E) U73122 had no effect on PLCγ1 phosphorylation in soluble TTI-621-treated stressed Jurkat cells.

Fcγ Receptor Overexpressing Cells Enhance TTI-621-Induced Apoptosis in a

CD47-Dependent Manner

A

100

+CD64 cells

+CD32 cells

Cancer cell

Macrophage or Dendritic cells

CD47

Apoptosis

FcγRI (CD64) FcγRII (CD32)

FcγRIII (CD16)

% of cells  SD

75

Early apoptotic (AnnexinV+)

50

Late apoptotic/necrotic (AnnexinV+ & N/IR+)

25

SIRPαFc

Fc Receptors

0

Control Fc

TTI-621

Control Fc

TTI-621

Control Fc

B

+CD64 cells

C

% of cells  SD

50 40 30 20 10

JurkatJurkat CD47-/-JurkatJurkat CD47-/-

Early apoptotic (AnnexinV+)

Late apoptotic/necrotic (AnnexinV+ & N/IR+)

% Annexin+ cells

100 80 60 40 20

Jurkat TTI-621 Jurkat Control Fc

Jurkat + CD64 cells TTI-621 Jurkat + CD64 cells Control Fc

0

(A, B) Stressed or CD47 knockout (CD47-/-) Jurkat cells were co-cultured with cells overexpressing CD64 (Fcγ receptor I) or CD32 (Fcγ receptor IIa) in the presence of 1 µM of soluble TTI-621 or control Fc for 18 hr. Apoptosis of Jurkat cells was determined by flow cytometry. (C) Apoptosis in stressed Jurkat cells alone or in a co-culture with CD64 cells was assessed by flow cytometry in the presence of titrated amounts of TTI-621 or control Fc. EC50 values were generated by linear regression using a sigmoidal dose-response curve.

Control Fc

TTI-621

Control Fc

TTI-621

Control Fc

TTI-621

Control Fc

TTI-621

0

-4

-2

0 Log [nM]

2

4

TTI-621 Induces Tumor Regression and Apoptosis in the Toledo (DLBCL)

Xenograft Tumor Model

A

Tumor Measurements

B

Control Fc

TTI-621

Tumor Volume (mm3)

TUNEL+ cells %

10 8 6 4 2

0

10

20

30

Days Post Inoculation

Control FcTTI-621

(A) Toledo cells were implanted subcutaneously into the right flank of NOD.SCID mice on day 0. Mice were dosed (3x/week) intratumorally with TTI-621 at 10 mg/kg or control Fc when the mean tumor volumes were approximately 700 mm3. (B) Tumors were harvested 2 days following the last treatment. TUNEL assay staining was performed on formalin fixed tumor tissues and the representative histology images are shown. The percentage of TUNEL-positive cells was calculated by dividing the number of TUNEL-positive cells by the total number of cells.

Conclusions

• • In vitro, ligating CD47 with immobilized TTI-621 efficiently induced apoptosis in malignant DLBCL and T-ALL cell lines but had no effect on apoptosis in normal B and T cells.

• • Soluble TTI-621 induced apoptosis under conditions of cellular stress.

• • Anchoring the IgG1 Fc tail of TTI-621 to FcγR-expressing cells enhanced tumor cell apoptosis in a dose-dependent and CD47-dependent manner.

• • An increase in tumor cell apoptosis was observed in vivo following intratumoral injection of TTI-621 in a DLBCL xenograft model.

• • In addition to blocking the anti-phagocytic "do-not-eat" signal on tumor cells and activating FcγR on macrophages, binding of TTI-621 to FcγRs on tumor-infiltrating immune cells may provide a cross-linking scaffold to enhance TTI-621-mediated apoptosis via CD47 on cancer cells.