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Apr 5, 2017 - BET (bromodomain and extraterminal) family proteins (BRD2, BRD3, BRD4, and BRDT) are epigenetic readers that bind to acetylated-lysine ...

Abstract LB:113

Immune-mediated anti-tumor activity with a clinical stage BET bromodomain inhibitor ODM-207 in pre-clinical models Pratima Deshpande2, Ravi Krishna Babu2, Prashant Yallappa Vadnal2, Mahaboobi Jaleel2, Chandrasekhar Abbineni 2, Anu Moilanen1, Pekka Kallio1,, Murali Ramachandra2, Susanta Samajdar2 1Orion Corporation Orion Pharma, Espoo, Finland, 2Aurigene Discovery Technologies Limited, Bangalore, India

Results

BET (bromodomain and extraterminal) family proteins (BRD2, BRD3, BRD4, and BRDT) are epigenetic readers that bind to acetylated-lysine residues in histones and recruit protein complexes to promote transcription elongation. In many cancers, BET proteins have been shown to regulate expression of MYC and other oncogenic drivers that are important for cell proliferation and survival. Pharmacologic inhibition of the BET–histone interaction has been shown to result in transcriptional downregulation of a number of oncogenes and inhibition of tumor growth providing a novel strategy for treatment of cancer.

1. ODM-207 activates mouse and human immune system in in

carcinoma and human H1975 lung carcinoma cell lines

vitro cultures

ODM-207 treatment

Inhibition of the cell surface expression of PD-L1

Day -10

IF N  - s t im u la t e d C T 2 6 c e ll lin e (4 8 h )

ODM-207 enhances cytotoxic nature of cultured mouse CD8+ T-cells

Frequency of either live tumor cells or immune infiltrates unchanged (except CD8 T cells) 7 - A A D n e g a t iv e c e lls

200

8

90

0 C on.

0 .0 1

0 .0 3

0 .1

0 .3

1

3

H 1 9 7 5 c e ll lin e ( 7 2 h )

In view of the recent publications implicating a role for BET protein BRD4 in the suppression of PD-L1 expression, an immune checkpoint ligand for PD-1, we sought to evaluate ODM207 for its effect on immune-mediated anti-tumor efficacy in pre-clinical models.

80

70

60

100

95

90

4

2

85

50 V e h ic le c o n t r o l

P D - L 1 ( M F I)

300

80

100

200

60

90

C D 4 + T c e lls 25

p = 0 .0 3 7 4

100

40

% CD4+

20

80

20

0 .0 1

0 .0 3

0 .1

0 .3

1

3

0

5

0

60

O D M -2 0 7

V e h ic le c o n t r o l

O D M -2 0 7

+ 10

ODM-207 decreases frequency of FOXP3+ cells and expression level in mouse splenocytes and LN cell cultures

O D M -2 0 7 ( M )

Reduction in MDSCs, decrease in PD-L1 positivity, increase in DCs and Granzyme B M -M D S C s

Lack of direct cytotoxicity of ODM-207 in CT26 cell line C y t o t o x ic it y X T T a s s a y ( 7 2 h )

110

15

10

80

150

100

50

70

V e h ic le c o n t r o l

0 V e h ic le c o n t r o l

O D M -2 0 7

O D M -2 0 7

V e h ic le c o n t r o l

O D M -2 0 7

(3 0 m p k )

(3 0 m p k )

T o t a l G r a n z y m e B c e lls p = 0 .0 4 3 3

30

0 .5

p = 0 .0 0 2 1

250

20

10

0

200

200

100

150

100

50

1

1

0

.0

.0

0

p = 0 .0 4 0 9

300

(M F I)

1 .0

G r a n z y m e B le v e ls ( C D 4 )

G r a n z y m e B le v e ls (C D 8 )

G ran zym e B

1

.1

90

0

% G ran zym e B +

2

0

100

5

1 .5

.0

p = 0 .0 5 9 9

(3 0 m p k )

3

0

200

p = 0 .0 0 2 9

% A n n e x in V + a n d / o r P I+ T c e lls

O .D . 4 5 0 n M

( n o r m a liz e d t o s t im .)

D e a d C D 3 T c e ll f r a c t io n

CD8

SSC

P D - L 1 in C T 2 6 T u m o r c e lls

p = 0 .0 0 2 6

2 .0 4

M a t u r e m y e lo id c e lls / D C

25

20

No cytotoxicity up to 1 μM

O D M -2 0 7 (3 0 m p k )

P D - L 1 ( M F I)

+ 0.1

% Ly6C- Ly6G -

+ -

( M F I)

-

V e h ic le c o n t r o l

(3 0 m p k )

G ra n zy m e B

+ 10

% Ly6C+

+ 0.1

10

0

(3 0 m p k )

+ -

15

70

V e h ic le c o n t r o l

-

O D M -2 0 7 (3 0 m p k )

C D 8 + T c e lls

C D 3 + T c e lls

C on.

Anti-CD3/CD28 ODM-207 (μM)

V e h ic le c o n t r o l

(3 0 m p k )

% CD8+

Granzyme B levels

O D M -2 0 7

(3 0 m p k )

% CD3+

IFNγ levels

0

V e h ic le c o n t r o l

O D M -2 0 7

400

Methods

Regulatory T cell (Treg) assay: Spleens and lymph nodes (LNs) were harvested from an 8 week old male C57BL/6 mouse. After homogenization, the cells were counted and plated in a 96-well plate pre-coated with 2.5 µg/mL of anti-CD3 antibody. Soluble anti-CD28 was added at 1 µg/mL, and IL-2 and TGFβ at 2 ng/mL along with requisite concentrations of ODM-207. Following incubation period, cells were stained with anti-FOXP3 PE, anti-CD3 APC, anti-CD4 PerCP-Cy5.5 antibodies, and the data was acquired on a BD FACSCaliburTM.

6

500

ODM-207 (3µM) DMSO ODM-207 (1µM) No stain

Effector cell phenotype assay: CD8+ T-cells were isolated using commercially available isolation kit and stimulated for 2.5 d with coated anti-CD3 (2.5 µg/ml) and anti-CD28 (0.5 µg/ml). Following 3.5 d of incubation the cells were restimulated with PMA, Ionomycin with brefeldin A and stained with anti-CD8 PerCP-Cy5.5, anti-granzyme B FITC and anti-IFNγ PE antibodies.

C D 4 5 + c e lls (T IL s )

C D 4 5 - c e lls ( T u m o r c e lls ) 105

% C D 4 5 - c e lls

Granzyme B

Granzyme B

CD8

ODM-207 (3µM) + IFNγ DMSO + IFNγ DMSO No stain

Biomarker modulation in tumor

400

% L iv e c e lls

CD8+ Granzyme B-

Tumor, whole blood & serum sample collection for bio-marker analysis

1 X 106 CT26cells s.c. injection/mice

600

P D - L 1 ( M F I)

CD8+ Granzyme B+

SSC

SSC

CD8+ gate

Day 5

Day 1

% C D 4 5 + c e lls

Background

ODM-207 is a potent and selective BET inhibitor that is structurally unrelated to the benzodiazepine-based inhibitors including JQ1, I-BET762, and OTX015. Phase I clinical trial is ongoing with this agent based on its potent anti-tumor activity in several in vitro and in vivo models of hematologic malignancies and solid tumors.

4. Antitumor activity of ODM-207 correlates with immune pharmacodynamic effect in the tumor

2. ODM-207 downregulates PD-L1 in mouse CT26 colon

O D M -2 0 7 ( (M) M) ODM-207

Poster presented at AACR Symposium 2017 April 1 - 5, 2017. Washington, D.C., USA

3

1

.3

V e h ic le c o n t r o l

O D M -2 0 7 (3 0 m p k )

(3 0 m p k )

0

o C

10 µM ODM-207

Biomarker modulation in blood Reduction in the immuno-suppressive subsets C D 4 7 + P D - L 1 + c e lls in L y 6 C - L y 6 G -

C D 4 7 + P D L - 1 + c e lls in L y 6 C + ( m M D S C s )

( m a t u r e m y e lo id c e lls / D C ) 100 100

% C D 4 7 + P D L-1 +

% C D 4 7 + P D -L 1 +

3. ODM-207 inhibits tumor growth in CT26 syngeneic modeI

p = 0 .0 1 5 9

7000

P D - L 1 ( M F I)

CD3

p = 0 .0 1 5 9

6000

5000

4000

90

80

70

60

V e h ic le c o n t r o l

O D M -2 0 7

V e h ic le c o n t r o l

(3 0 m p k )

p = 0 .0 3 1 7 90

80

70

60

O D M -2 0 7

V e h ic le c o n t r o l

(3 0 m p k )

O D M -2 0 7 (3 0 m p k )

FOXP3 expression level 25

V e h ic le C o n t r o l

20

O D M -2 0 7 3 m p k q d p .o

5

(m e a n  S E M )

10

0

* ## *** ****

600

O D M -2 0 7 3 0 m p k q d p .o Is o t y p e c o n t r o l 1 0 0 µ g i . p

+ 0.1

D ay 4

D ay 7

D ay 11

1500 20000

5000

0

+ 0.1

1

8

+ 0.3

9

7

6

5

4

3

2

1

0

+ 1

+ 0.03

No cytotoxicity seen in PBMCs with ODM-207 up to 1.0 μM

+ 0.01

(m e a n  S E M )

10000

C u b ic m i l l i m e t e r

IF N  (p g /m l)

O D M -2 0 7 3 m p k q d p .o

+ 3

20

50

40 10

30

0 O D M -2 0 7

40

20

0

V e h ic le c o n t r o l

O D M -2 0 7

V e h ic le c o n t r o l

O D M -2 0 7

(3 0 m p k )

(3 0 m p k )



ODM-207 activates mouse and human immune system in the in vitro cultures as demonstrated by the activation of effector function of mouse CD8+ T cells, reduction in the level and frequency of mouse FoxP3+ Treg cells and secretion of IFNγ cytokine in human whole blood assay



ODM-207 downregulates PD-L1 in mouse (CT26, colon carcinoma) and human (H1975, lung carcinoma) cell lines



ODM-207 shows the anti-tumor activity in a syngeneic model of colon carcinoma in the absence of a direct anti-proliferative activity on tumor cells



Observed tumor growth inhibition correlated with the in vitro activation of cytotoxic CD8+ T cells supporting an immune-mediated effect leading to tumor growth inhibition



In view of the remarkable success with the immune-based therapeutic approaches, these findings are relevant in devising appropriate strategies for the continued clinical development of ODM-207

V e h ic le C o n tro l 15000

+ 10

30

(3 0 m p k )

Tumor ona l final T u m o r vvolume o lu m e o n fin d a y day

IFN-Υ levels in whole blood

+ -

60

D ay 14

D a y s o f d o s in g

ODM-207 modulates effector cytokine production in whole blood from healthy humans

-

40

0 D ay 0

Anti-CD3/CD28 ODM-207 (μM)

p = 0 .0 0 7 9

60

Conclusions

M .1

+ 1.0

FO XP3+)

J 4 3 1 0 0 µ g i.p o n c e w e e k ly

u

M

1

+ 10

0

1

G

0

+ -

µ

µ

ß

M

+ IL

-2

+

T

S

ti

F

m

m

ti n

U

-

(g a te d o n F O X P 3 + ) 70

50

V e h ic le c o n t r o l

o n c e w e e k ly

300 s

Anti-CD3/CD28 ODM-207 (μM)

O D M -2 0 7 1 0 m p k q d p .o

900

15 C u b ic m illim e t e r

(m e d ia n )

F O X P 3 le v e ls

1200

F r e q u e n c y o f P D - 1 e x p r e s s in g c e lls

F r e q u e n c y o f L A G 3 e x p r e s s in g c e lls ( g a t e d o n

% L A G 3 + ( t o t a l)

T u m o r v ovolume lu m e Tumor

F O X P 3 e x p r e s s io n le v e l

p = 0 .0 9 5 2

% P D -1 +

% F O X P 3 + P D -1 + L A G 3 +

F r e q u e n c y o f F O X P 3 + P D - 1 + L A G 3 + c e lls

O D M -2 0 7

Measurement of immune PD in CT26 syngeneic tumor model: Tumors were established by subcutaneous injection of CT26 cells into male Balb/C mice. After initial tumor growth, when the average tumor volume reached 122 mm3, oral treatment with ODM-207 30 mg/kg qd or vehicle was initiated on day-1 and continued for 5 days. Samples (tumor, whole blood & plasma) were collected 24 h after the last dose of compound administration for bio-marker analysis by FACS. Statistical differences between vehicle control and treatment group were tabulated by Mann-Whitney U test.

O D M -2 0 7

(3 0 m p k )

P D - L 1 le v e ls in L y 6 C + ( M - M D S C s )

106

Subcutaneous CT26 syngeneic tumor model: Tumors were established by subcutaneous injection of CT26 cells into female BALB/c mice. After initial tumor growth, when the average tumor volume reached ~40 mm3, oral treatment with ODM-207 at 3, 10 and 30 mg/kg qd, or vehicle was initiated and continued for 14 days. Tumor volume was measured with a caliper during the course of the study twice weekly. Statistical analysis: *p