AACR TTI-622 (SIRPα-IgG4 Fc), a CD47- Blocking Innate Immune … · 2018-08-03 · TTI-622...

TTI-622 (SIRPα-IgG4 Fc), a CD47-Blocking Innate Immune Checkpoint Inhibitor, Suppresses Tumor Growth and Demonstrates Enhanced Efficacy in Combination with Anti-Tumor Antibodies in Both Hematological and Solid Tumor Models

1Trillium Therapeutics Inc., Mississauga, ON, Canada, 2London Regional Cancer Program, London Health Sciences Centre, Lawson Health Research Institute, London, ON, Canada, 3Department of Oncology, University of Western Ontario, London, ON, Canada

TTI-622 Potently Induces Macrophage-Mediated Phagocytosis of Malignant Human Cell Lines

TTI-622 Potentiates the Efficacy of Daratumumab(anti-CD38 mAb) in Burkitt Lymphoma

and Multiple Myeloma Xenograft Models

FaDu cells were implanted subcutaneously into the right flank of NOD.SCID mice (n=8 mice per group) on day 0. Micewere randomized on day 7 and received IP injections of TTI-622 10 mg/kg and/or cetuximab 3 mg/kg as indicated by theinverted triangles (left). Statistical analysis of the tumor growth curves was performed using one-way ANOVA withTukey’s multiple comparison test comparing tumor volumes at day 29 and survival curves were analyzed by Log-ranktest (corrected for multiple comparisons). Where indicated, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Both Early and Delayed Administration of TTI-622 Decrease Tumor Growth and Improve Survival in a DLBCL

Xenograft Tumor Model

TTI-622 Enhances the Efficacy of Cetuximab(anti-EGFR mAb) in a Head and Neck

Squamous Cell Carcinoma Xenograft Model

Toledo cells were implanted subcutaneously into the right flank of NOD.SCID mice (n=9 mice per group) on day 0. Micewere dosed intraperitoneally (IP) with TTI-622 at 10 mg/kg or control Fc on day 10 (A) or day 19 (B). Mice were sacrificedwhen at least one tumor dimension exceeded 15 mm. The study terminated on day 63. Statistical analysis of the tumorgrowth curves was performed using t-test comparing tumor volumes at day 35 (A) and day 40 (B). Survival curves wereanalyzed by Log-rank test. Where indicated, *** p ≤ 0.001, **** p ≤ 0.0001.

Macrophage-mediated phagocytosis of primary B-ALL, T-ALL, MDS and AML patient samples was assessed by flow cytometry in the presence of 1 μM TTI-622 or control Fc.

Macrophage-mediated phagocytosisof human tumor cell lines wasassessed by flow cytometry in thepresence of titrated amounts of TTI-622 or control Fc. EC50 values weregenerated by linear regressionusing a sigmoidal dose-responsecurve.

TTI-622

Control Fc

Macrophage-mediated phagocytosis ofplatelets and tumor cells (SU-DHL-6)(alone or in competition) in the presenceof 1 μM TTI-622 or control Fc.

The ratio of macrophages to targets was1:40 for platelets and 1:2.5 for tumorcells.

(A) Representative confocal microscopy images. Macrophages were stained red, platelets green and tumor cells blue. Non-phagocytosed target cells were removed during washing.

(B) The phagocytic index was determined as the average of the total number of engulfed target cells divided by the total number of macrophages in 3-5 fields. Platelets outnumbered tumor cells by 16-fold in competition conditions. Where indicated, *** p ≤ 0.001, NS = not significant.

Daudi cells (Burkitt lymphoma) wereimplanted subcutaneously into the rightflank of NOD.SCID mice (n=9-11 miceper group). Mice were randomized onday 3 and received IP injections of TTI-622 10 mg/kg and/or daratumumab 10mg/kg as indicated by the invertedtriangles (left). Statistical analysis ofthe tumor growth curves wasperformed using one-way ANOVA withTukey’s multiple comparison testcomparing tumor volumes at day 32and survival curves were analyzed byLog-rank test (corrected for multiplecomparisons). Where indicated, * p ≤0.05, *** p ≤ 0.001

MM.1S cells (multiple myeloma) wereimplanted subcutaneously into the rightflank of NOD.SCID mice (n=9-10 miceper group). Mice were randomized onday 11 and received IP injections ofTTI-622 10mg/kg and/or daratumumab10 mg/kg as indicated by the invertedtriangles (left). Statistical analysis of thetumor growth curves was performedusing one-way ANOVA with Tukey’smultiple comparison test comparingtumor volumes at day 26 and survivalcurves were analyzed by Log-rank test(corrected for multiple comparisons).Where indicated, * p ≤ 0.05, ** p ≤ 0.01,*** p ≤ 0.001

Conclusions TTI-622 potently induces phagocytosis of a broad panel of tumor cells

derived from patients with both hematological and solid tumors.

Unlike CD47-blocking antibodies, TTI-622 binds minimally to humanerythrocytes and does not induce hemagglutination in vitro.

TTI-622 preferentially induces phagocytosis of tumor cells over platelets ina competitive phagocytosis assay.

In a DLBCL xenograft tumor model, both early and delayed treatmentsresulted in statistically significant decreases in tumor growth, and improvedsurvival relative to treatment with control Fc.

TTI-622 potentiates the efficacy of cetuximab and daratumumab in solidand hematological xenograft tumor models, respectively.

Gloria H. Y. Lin1, Natasja Nielsen Viller1, Marilyse Charbonneau1, Laura Brinen1, Tapfuma Mutukura1, Karen Dodge1, Simone Helke1, Vien Chai1, Violetta House1, Vivian Lee1, Hui Chen1, Alison O’Connor1, Debbie Jin1, Rene Figueredo2,3, Saman Maleki Vareki2,3, Mark Wong1, Emma Linderoth1, Lisa D. S. Johnson1, Xinli Pang1, James Koropatnick2,3, Jeff Winston1, Penka S. Petrova1, Robert A. Uger1

TTI-622 (SIRPα-IgG4 Fc): A Novel Biologic that Blocks the CD47 “Do Not Eat” Signal

CD47 binds to SIRPα on the surface of macrophages and delivers a “do not eat” signalto suppress phagocytosis.

Tumor cells frequently overexpress CD47 and exploit this pathway to evademacrophage-mediated destruction.

TTI-622 (human SIRPα linked to a human IgG4) is a soluble decoy receptor thatneutralizes the suppressive effects of CD47 and promotes macrophage-mediatedphagocytosis of tumor cells.

Previous studies have shown that blockade of the CD47-SIRPα pathway using TTI-621,a soluble SIRPα-IgG1 Fc fusion protein, triggers macrophage phagocytosis of tumor cellsin vitro, and potently inhibits tumor growth in vivo.

In this study, the in vitro and in vivo efficacy of TTI-622, a soluble SIRPα-Fc variantprotein containing an IgG4 Fc tail, was evaluated in multiple model systems.

TTI-622 Preferentially Induces Phagocytosis of Tumor Cells over Platelets

TTI-622 Potently Induces Macrophage-Mediated Phagocytosis of Primary Malignant Human Cells

Binding of TTI-622 or anti-CD47 mAbs to fresh human RBCs at saturating concentrations. Data were analyzed by flow cytometry, graph represents mean fluorescence intensity for 43 human donors.

Agglutination of human RBCs by TTI-622 and anti-CD47mAbs. PBS washed RBCs from healthy donors were treatedwith TTI-622 or anti-CD47 mAbs overnight at 37oC, 5% CO2.The extent of hemagglutination was assessed by scoringeach well on a scale of 1 to 6, with 1 representing theabsence of hemagglutination and 6 representing completehemagglutination.

0.01 0.1 1 10 100 1000 100000

1

2

3

4

5

62D3B6H12BRIC1265F9

TTI-622

Concentration (nM)

Hem

aggl

utin

atio

n Sc

ore

(mea

n±

SE)

TTI-622

contro

l Fc

BRIC126

2D3

CC2C6

B6H12 5F

9

mIgG1

mIgG2b100

101

102

103

104

105

106

Mea

n F

luor

esce

nce

Inte

nsity

CD47 mAbs ControlmAbs

TTI-622 Binds Minimally to Human RBCs and Does Not Induce Hemagglutination

Daudi (Burkitt Lymphoma)

MM.1S (Multiple Myeloma)

Platelets only Tumor cells only Tumor cells + Platelets

0

20

40

60

80

100

% P

hago

cyto

sis

140979 140606 4354 150110 130089 120779 130667 120093

B-ALL T-ALL MDS AML Control FcTTI-622

Patient ID

-4 -3 -2 -1 0 1 2 3 40

20

40

60

80

Toledo(Diffuse Large B Cell Lymphoma)

Concentration (Log nM)

% P

hago

cyto

sis Control Fc

TTI-622

EC50 = 1.8 nM

-4 -3 -2 -1 0 1 2 3 40

20

40

60

80

SU-DHL-6(Diffuse Large B Cell Lymphoma)

Concentration (Log nM)

% P

hago

cyto

sis Control Fc

TTI-622

EC50 = 25.9 nM

-4 -3 -2 -1 0 1 2 3 40

10

20

30

40

NU-DUL-1(Diffuse Large B Cell Lymphoma)

Concentration (Log nM)

% P

hago

cyto

sis Control Fc

TTI-622

EC50 = 9.2 nM

-4 -3 -2 -1 0 1 2 3 40

2

4

6

HCT-15(Colorectal Adenocarcinoma)

Concentration (Log nM)

% P

hago

cyto

sis Control Fc

TTI-622

EC50 = 3.5 nM

-4 -3 -2 -1 0 1 2 3 40

10

20

30

40

50

NCI-H446(Small Cell Lung Cancer)

Concentration (Log nM)

% P

hago

cyto

sis Control Fc

TTI-622

EC50 = 6.7 nM

-4 -3 -2 -1 0 1 2 3 40

10

20

30

40

NCI-1568(Non-Small Cell Lung Cancer)

Concentration (Log nM)

% P

h ag o

c yto

sis Control Fc

TTI-622

EC50 = 12.4 nM

0 20 40 600

400

800

1200

1600Tumor Measurements

Days post inoculation

Tum

or V

olum

e (m

m3 )

VehicleTTI-622

Daratumumab + TTI-622

Daratumumab

TTI-622 Dosing scheduleDaratumumab Dosing schedule

**

0 20 40 60 80 1000

50

100

Survival

Days post inoculation

Perc

ent s

urvi

val

VehicleTTI-622

Daratumumab + TTI-622Daratumumab

****

0 20 40 60 80 1000

50

100

Survival

Days post inoculation

Perc

ent s

urvi

v al

TTI-622

Daratumumab + TTI-622Daratumumab

Vehicle*

*****

0 20 40 600

500

1000

1500Tumor measurements

Days post inoculation

Tum

or V

olum

e (m

m3 )

TTI-622

Daratumumab + TTI-622Daratumumab

Vehicle

**

Daratumumab Dosing scheduleTTI-622 Dosing schedule

0

100

200

300

400

Platelet Phagocytosis

Phag

ocyt

osis

Inde

x( P

l ate

lets

)

TTI-622Control Fc

***

Platelets Platelets +Tumor Cells

0

25

50

75

100

125

Tumor Cell Phagocytosis

Phag

ocyt

osis

Inde

x(T

umor

Cel

ls)

TTI-622Control Fc

NS

Tumor Cells Tumor Cells +Platelets

A

B

0 20 40 60 800

500

1000

1500

2000

Tumor Measurements (early dosing)

Days post inoculation

Tum

or V

olum

e (m

m3 ) TTI-622

Control FcDosing schedule

****

0 20 40 600

50

100

Survival (early dosing)

Days post inoculation

Perc

ent s

urvi

val

TTI-622Control Fc ****

0 20 40 600

500

1000

1500

2000

Tumor Measurements (delayed dosing)

Days post inoculation

Tum

or V

olum

e (m

m3 )

Control FcTTI-622

Dosing schedule

****

0 20 40 600

50

100

Survival (delayed dosing)

Days post inoculation

Perc

ent s

urvi

v al Control Fc

TTI-622***

A

B

AACR # 2709

Based on these data, a clinical study of TTI-622 in patients withadvanced lymphoma or myeloma is being initiated.

0 20 40 600

500

1000

1500

2000

Tumor Measurements

Days post inoculation

Tum

or V

olum

e (m

m3 )

VehicleTTI-622CetuximabCetuximab + TTI-622TTI-622 Dosing scheduleCetuximab Dosing schedule

******** ********

*

0 20 40 600

50

100

Survival

Days post inoculation

Perc

ent s

urvi

val

VehicleTTI-622CetuximabCetuximab + TTI-622

****** *** ** ***