****

* ** **

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

0 10 20 30 40 50 60 700

1000

2000

3000

Days post the start of treatment

Tum

or S

ize

(mm

3 )

Vehicle Anti-PD-L1DRP-104 1.4 mg/kgDRP-104 Anti-PD-L1+DRP-104 1.4 mg/kgDRP-104 0.7 mg/kgAnti-PD-L1+DRP104 0.7 mg/kg

** **

****

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

Vehicle o.5 hr 4 hr 24 hr0.00

0.05

0.10

0.15

Glu

tam

ate/

Glu

tam

ine

Glutamate/Glutamine_single dose

DRP-104 0.5 mg/kg

*

Figure 5. CT26 cells were inoculated s.c. in BALB/c mice. When tumor size reached ~400 mm3, mice were treated withDRP-104 (0.5 mg/kg) or vehicle s.c., q.d. for 5 days and/or anti-PD-1 Ab (10 mg/kg) at day1 and 4. Phenotypic analysesof tumor infiltrating immune cells and tumor cells were performed by flow cytometry. Data indicate mean+/- SEM. *:p<0.05; ***: p<0.001; ***: p<0.0001.M1 polarized TAM and decreased MDSCs were only observed in DRP-104 treated tumors.

Glutamine is a conditionally essential amino acid for rapidly proliferating cancer cells, thus depriving the same fuel from immune cells and contributing to tumor immune evasion. DRP-104 was designed as a novel prodrug of the broad acting glutamine antagonist 6-Diazo-5-oxo-L-norleucine (DON). DRP-104 is inert in its prodrug form, affords high levels of plasma and gastro-intestinal (GI) tissue stability; has high tumor cell permeability and preferential tumor versus plasma/GI tissue distribution for DON. Here we sought to (1) compare immunological modulation of DRP-104 to anti-PD-1Ab, and (2) evaluate the combination effect of DRP-104 with PD-1/PD-L1 checkpoint inhibitors.DRP-104 treatment in CT26 mouse colon carcinoma model showed broad immune cell modulation effects including increased T, NK, and macrophages; while anti-PD-1Ab affected mainly CD8+T cells. Cytokine modulation in tumor and plasma revealed that DRP-104 decreased pro-tumorigenic cytokines such as VEGF and KC(IL-8) while anti-PD-1Ab showed either no change or slight increase in these cytokines. CT26 bearing mice treated with anti-PD-1Ab alone, DRP-104, and the combination showed tumor growth inhibition at day 12 of 48%, 90%, and 94%, respectively. Median survival days were 31.5, 36, and 56 days, respectively (vehicle; 17.5 days). Notably 9 mice treated with combination of anti-PD-1 with DRP-104 were tumor free at end of the experiment (day 77) and 100% of these mice rejected a CT26 tumor re-challenge. In the H22 HCC cancer model, mice were treated with either anti-PD-L1 Ab, DRP-104, or combination. While anti-PD-L1Ab did not show tumor growth inhibition in this model, DRP-104 significantly inhibited tumor growth and the combination further enhanced efficacy, illustrated by extended survival for both DRP-104 alone (50 days) and combination (96 days) treatment groups compared to vehicle (33 days) and anti-PD-L1 alone (33 days). Combination treatment also resulted in long term durable cures in 50% of the mice.

DRP-104, A Novel Broad Acting Glutamine Antagonist, Induces Distinctive Immune Modulation Mechanisms and Synergistic Efficacy in Combination with Immune Checkpoint Blockade

Yumi Yokoyama, Ph.D., Michael Nedelcovych, Ph.D. and Robert Wild, Ph.D.Dracen Pharmaceuticals Inc., 780 Third Ave, 45th Floor, New York, NY 10017

Summary

Background

ConclusionsvBoth DRP-104 and the active form DON showed Glutamine dependent inhibition

of in vitro cancer cell growth (poster P816: Sat, Nov 9, 7:00am – 8:30pm).vDRP-104 achieved 6x tumor/plasma ratio for DON in CES-1 ko mice (poster P816:

Sat, Nov 9, 7:00am – 8:30pm).vDRP-104 treatment showed dramatic immuno-oncology and metabolism related

gene modulations.vImmuno-phenotypical analysis showed that DRP-104 induced substantial and

broad changes in various immune cell infiltrates, such as increased TIL, T, NK and NKT cells, whereas an effect of anti-PD-1 Ab was limited to CD8+T cells. DRP-104 also polarized TAMs to M1 phenotype, and decreased immunosuppressive mMDSC in the TME.

vPro-tumorigenic proteins, such as VEGF, KC (IL-8), MCP-1, LIF and IL-6 were decreased by DRP-104. Combination showed significant increase in IFN-g in TME.

vFinally DRP-104 inhibited tumor growth as a single agent in CT26 and H22 tumor models which are non-responsive to immune checkpoint inhibitors. Combination with either anti-PD-1 Ab or anti-PD-L1 Ab enhanced efficacy and achieved significantly extended survival.

vThis data support clinical development of DRP-104 in combination with anti-PD-1 Ab or anti-PD-L1 Ab immune checkpoint inhibitors including settings of non-response to IO therapies.

DRP-104 Single Administration Yields Time-Dependent Changes in Plasma Glutamine Metabolites

Figure 2. MC38 tumor bearing animals were dosed s.c. with DRP-104 0.5 mg/kg, and tumors were obtained at the indicated time after dosing. Glutamate/Glutamine in plasma was measured by Glutamine/Glutamate Glo Assay (Promega).Significant time-dependent changes in plasma Glutamate/Glutamine levels were observed linking DRP-104 treatment to target engagement. Data indicate mean+/- SEM. *: p<0.05

DRP-104 Treatment In Vivo Results in Dramatic Modulation of Multiple Relevant Tumor and Immune Cell Scores as Measured by NanostringIO360 and Metabolism Panels

Undirected Groups: differential expression in

DRB T1 vs. baseline of DRB V

Costimulatory Signaling 8.923

Myeloid Compartment 8.494

Antigen Presentation 8.473

Immune Cell Adhesion and Migration 8.326

Lymphoid Compartment 7.944

Cytokine and Chemokine Signaling 7.394

Hypoxia 7.372

JAK-STAT Signaling 7.268

Interferon Signaling 7.024

Cytotoxicity 6.699

NF-kappaB Signaling 6.577

PI3K-Akt 6.44

TGF-beta Signaling 6.272

Metabolic Stress 6.107

Matrix Remodeling and Metastasis 5.869

MAPK 5.835

Autophagy 5.6

Epigenetic Regulation 5.335

Wnt Signaling 5.001

Apoptosis 4.905

Hedgehog Signaling 4.863

Angiogenesis 4.527

Notch Signaling 4.51

Cell Proliferation 4.134

DNA Damage Repair 3.966

IO360 (Tumor) Metabolism (Tumor)

Figure 3. MC38 cells were inoculated s.c. in C57BL/6 mice. When tumor size reached ~400 mm3, mice were treatedwith DRP-104 (0.5 mg/kg) or vehicle s.c., q.d. for 5 days. mRNA was extracted from tumors and analyzed byNanostring IO360 Panel. ****: p<0.0001DRP-104 treatment was associated with dramatic modulation of multiple relevant tumor and immune cell scoressuggesting direct anti-tumor effects while stimulating immune cell function.

IO360 (Tumor)

IO360 (Tumor)

DRP-104 Treatment Shows Distinct Immuno-phenotypic Changes Compared to anti-PD-1 in the MC38 Syngeneic Model (TIL, Lymphocyte)

0 1 2 3 40

200

400

600

800

1000

1200

Days after treatment

Tu

mo

r V

olu

me (

mm

3)

Vehicle 10 ml/kg s.c. QDx5

* **

DRP-104 0.5mg/kg s.c. QDx5

DRP-104 1.4mg/kg s.c. QDx5

DRP-104+anti-PD-1

Figure 4. CT26 cells were inoculated s.c. in BALB/c mice. When tumor size reached ~400 mm3, mice were treated withDRP-104 (0.5 mg/kg) or vehicle s.c., q.d. for 5 days and/or anti-PD-1 Ab (10mg/kg) at day1 and 4. Phenotypic analysesof tumor infiltrating immune cells and tumor cells were performed by flow cytometry. *: p<0.05; ***: p<0.001; ***:p<0.0001. Distinct immunological modulation by DRP-104 and anti-PD-1 Ab was observed.

CT265x105cells/mouse

Tumor Size(~400mm3)

Harvest 1hr after the last dosing• Immuno-phenotyping (tumor)• Plasma for Luminex

Anti-PD-1DRP-104

Tumor Growth Inhibition

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

10

20

30

40

TIL

(CD4

5+%

in L

ive

cells

) ***

*

TIL CD8+T

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

2

4

6

8

10

CD8+ T

cel

l (%

in C

D45+ c

ells

)

** **** **

NK Cell

CD4+T

Vehicl

e

DRP-104_

0.5

anti-P

D-1

DRP-104+

anti-P

D-10

2

4

6

8

10

CD4+ T

cel

l (%

in C

D45+ c

ells

)

** **

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

10000

20000

30000

40000

50000

Ki67

in C

D8+

cell

(MFI

)

**

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

10000

20000

30000

Ki67

in C

D4+

cell

(MFI

)

******

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

1

2

3

4

5

NK c

ell (

% in

CD4

5+ cel

ls)

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

10000

20000

30000

40000

Ki67

in N

K cel

l (M

FI)

**

**

Proliferation

CD8+T CD4+T NK Cell

NKT Cell

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10.0

0.2

0.4

0.6

0.8

NKT

cell

(% in

CD4

5+ cel

l)

**

***

TAM M1 Macrophage

gMDSC+Neutrophil mMDSC

Vehicl

e

DRP-104

_0.5

anti-

PD-1

DRP-104

+anti-

PD-10

20

40

60

80

100

Mac

roph

age

(% in

CD

45+

cells

)

** *

**

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

50

100

M1

(MH

C II

+ CD

206- )

Mac

roph

age

(%)

******* ***

*

Vehicl

e

DRP-104

_0.5

anti-

PD-1

DRP-104

+anti-

PD-10

2

4

6

8

gMD

SC

+Neu

trop

hil

(% in

CD

45+

cells

)

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

10

20

30

40

mM

DSC

(% in

CD

45+ c

ells

)

** *****

DRP-104 Showed Distinctive Modulation of In Vivo Cytokines in TME Compared to Anti-PD-1 Ab

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

2

4

6

IFN

-g (p

g/m

g)

IFN-g*

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

5

10

15

IL6

(pg/

mg)

IL6

*

Vehicl

e

DRP-104_

0.5

anti-P

D-1

DRP-104+

anti-P

D-10

50

100

150

200

KC

(pg/

mg)

KC*

Vehicl

e

DRP-104_

0.5

anti-P

D-1

DRP-104+

anti-P

D-10

50

100

150

200

250

MC

P-1

(pg/

mg)

MCP-1**

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10.0

0.5

1.0

1.5

2.03.54.0

IL1b

(pg/

ml)

IL-1b

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10.0

0.5

1.0

1.5

2.0

IL2

(pg/

mg)

IL2

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

2

4

6

8

LIF

(pg/

mg)

LIF

**

Vehicl

e

DRP-104

anti-P

D-1

DRP-104+

anti-P

D-10

5

10

15

20

25

VEG

F (p

g/m

g)

VEGF*

Figure 6. Cytokines in tumor lysates from CT26 study were measured by Luminex. Data indicate mean+/- SEM. *:p<0.05; **: p<0.01.Distinct immunological modulation by DRP-104 and anti-PD-1 Ab and enhanced IFN-g production in combination wasobserved.

Combination of DRP-104 with Anti-PD-1 Ab Enhanced Anti-Tumor Efficacy and Survival in CT26 Colon Cancer Model

***

*

**

**0 5 10 15 20 25 30 35 40 45

0

500

1000

1500

2000

Days post the start of treatment

Tum

or v

olum

e (m

m3 )

Vehicle q.d.x28, s.c.

Group2:anti-PD-1Ab 10 mg/kg, q4dx8, i.p.

Group3: DRP-104 1.4mg/kg 5-days ON 2-days OFF x 4 cycles, s.c.

Group4:DRP-104 1.4mg/kg 5-days ON 2-days OFF x 4 cycles, s.c. + anti-PD-1Ab 10 mpk q4d x 8, i.p.

Group5: DRP-104 0.5mg/kg 5-days ON 2-days OFF x 4 cycles, s.c.

Group6: DRP-104 0.5mg/kg 5-days ON 2-days OFF x 4 cycles, s.c. + anti-PD-1Ab 10 mpk q4d x 8, i.p.

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

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

*****

9 Cures and 8 naïve mice re-

challenged with CT26

without additional treatment

Re-challenge

Combination of DRP-104 with Anti-PD-L1 Ab Enhanced Anti-Tumor Efficacy and Survival in the H22 HCC Cancer Model

Figure 8. H22 mouse hepatocellular carcinoma cells were inoculated s.c. in BALB/c mice. When tumor size reached 50-100 mm3, mice were randomized and treated with DRP-104, anti-PD-L1Ab, or DRP-104/anti-PD-L1Ab combination. Anti-PD-1Ab did not show any effect in this model, while DRP-104 alone showed potent single agent efficacy and combination with anti-PD-L1 Ab further inhibited tumor growth and significantly improved OS and long term durable cures.(Mean+/- SEM. n=10, *: p<0.05, **: p<0.01, ***: p<0.001)

DRP-104 Treatment Shows Distinct Immuno-Phenotypic Changes Compared to Anti-PD-1 Ab in the CT26 Syngeneic Model (TIL, Lymphocyte)

DRP-104 Treatment Shows Distinct Immuno-Phenotypic Changes Compared to Anti-PD-1 Ab in the CT26 Syngeneic Model (Macrophage and Tumor)

0 20 40 60 800

20

40

60

80

100

Days post the start of treatment

Per

cent

sur

viva

l

**********

****

****

497

Dracen’s Glutamine Antagonist, DRP-104, Preferentially Delivers DON to Tumors Leading to Broad Glutamine Pathway Inhibition

Figure 1. A. DRP-104 is a prodrug of the broad acting glutamine antagonist DON (6-Diazo-5-oxo-L-norleucine). DRP-104 is inactive in its prodrug form with high plasma and GI tissue stability. DRP-104 is preferentially distributed in tumors where it is biotransformed and activated to the active moiety DON. B. Glutamine dependent pathways inhibited by DON. Enzymes targeted by DON are shown in blue bold letters.

Figure 7. CT26 mouse colon carcinoma cells were inoculated s.c. in BALB/c mice. When tumor size reached 50-100 mm3, mice were randomized and treated with DRP-104, anti-PD-1Ab, or DRP-104/anti-PD-1Ab combination. Anti-PD-1Ab showed marginal effect, while DRP-104 alone showed potent single agent efficacy and combination with anti-PD-1 Ab further inhibit tumor growth and significantly improved OS and long term durable cures (p<0.001 vs monotherapy).(Mean+/- SEM. n=10, **: p<0.01; ***: p<0.001; ****: p<0.0001). Cured animals from the combination group were re-challenged with CT26 tumor cells. 100% of the mice rejected the tumor implant suggesting potential long term immune memory response elicited by DRP-104 and PD-1 combination treatment.

A B

Vehicle DRP-104-4

-2

0

2

4

Cytotoxicity

Cyt

otox

icity

Sco

re

Vehicle DRP-104-6

-4

-2

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Met

abol

ic S

tres

s S

core

Vehicle DRP-104-4

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ptos

is S

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Vehicle DRP-104-3

-2

-1

0

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l Pro

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Vehicle DRP-104-6

-4

-2

0

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6

Antigen Presentation

Ant

igen

Pre

sent

atio

n S

core

********

****

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

Vehicle DRP-104-10

-5

0

5

10

Cytokine and Chemokine Signaling

Cyt

okin

e an

d C

hem

okin

e S

core

Treatment (Vehicle or DRP-104, 0.5 mg/kg, q.d.x5)

MC382x105cells/mouse

Tumor Size(~400mm3)

Harvest 1hr after the last dosing• Nanostring analysis (tumor)

0 20 40 60 80 1000

20

40

60

80

100

Days

Perc

ent s

urvi

val

***

***

****

VehicleDRP-104, 0.5 mg/kg, s.c. q.d.x5Anti-PD-1 Ab 10 mg/kg, i.p., q4dx2DRP-104+Anti-PD-1 Ab