N o

d e p le tio n

C D 8

d e p le tio n

Tu

mo

r V

olu

me

(m

m3)

0

2 0 0

4 0 0

6 0 0

V e h ic le

* * * * * *

C B -1 1 5 8 - 1 0 0 m g /k g

* * * *

D a y s P o s t Im p la n t

Tu

mo

r V

olu

me

(m

m3)

0 2 4 6 8 1 0 1 2 1 4

0

2 0 0

4 0 0

6 0 0

* * * *

V e h ic le -c o n tr o l

C B -1 1 5 8 (1 0 0 m g /k g )

N o

d e p le tio n

N K

d e p le tio n

Tu

mo

r V

olu

me

(m

m3)

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

* * * * * * *

n s

V e h ic le

C B -1 1 5 8 - 1 0 0 m g /k g

IF N -

ng

/mL

0 .0 0

0 .0 2

0 .0 4

0 .0 6

0 .0 8

L L O Q

IL -1 2 p 7 0

0 .0 0

0 .0 1

0 .0 2

0 .0 3

0 .0 4

0 .0 5

*

L L O Q

M IP -1

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5 *

M IP -3

0 .0

0 .5

1 .0

1 .5

2 .0

*

V e h ic le C B -1 1 5 8

% o

f li

ve

ce

lls

5

1 0

1 5

* *

0

2

4

6

8

*

Abstract CB-1158 Inhibits Human Arginase and Reverses Immunosuppression of T-cells

CB-1158 has Anti-tumor Activity in Several Cancer Types

Arginase inhibitor CB-1158 is a novel immuno-oncology

agent that targets tumor-infiltrating suppressive myeloid cells

Depletion of arginine by arginase-expressing myeloid cells contributes to an

immunosuppressive tumor microenvironment that inhibits proliferation of effector

lymphocytes. Pharmacological inhibition of arginase produced by myeloid cells such as

neutrophils, macrophages and myeloid-derived suppressor cells (MDSC) is expected to

restore arginine levels and allow T-cells to proliferate, thereby leading to an immune-

mediated anti-tumor response. CB-1158 is a potent inhibitor of human arginase (IC50 = 100

nM). In a co-culture system with human neutrophils and T-cells, CB-1158 blocked arginase

activity, increased arginine levels in the media, and restored proliferation of T-cells (IC50 =

160 nM). In immune competent mice bearing Lewis Lung Carcinoma (LLC) syngeneic

tumors, BID oral dosing of 100 mg/kg CB-1158 showed good exposure in plasma and

tumor, led to pharmacodynamic increases in arginine levels in plasma (3-fold) and tumor

(4-fold), resulted in tumor growth inhibition of 54% and was well tolerated. CB-1158

treatment increased pro-inflammatory cytokine and chemokine levels in LLC tumors and

had no anti-tumor activity in immunocompromised SCID mice, consistent with an immune-

based mechanism of action. Given these findings, we investigated whether CB-1158 would

combine with checkpoint inhibitors a-CTLA-4 or a-PD-1. CB-1158 in combination with a-

CTLA-4, increased the levels of tumor-infiltrating CD8+ T-cells in LLC-tumor bearing mice.

In combination with a-CTLA-4 and a-PD-1, CB-1158 decreased tumor growth and metastatic

burden in the highly refractory 4T-1 breast cancer model. These results support the

development of CB-1158, a first-in-class arginase inhibitor, as a novel immuno-oncology

agent that targets the immunosuppressive effects of tumor-infiltrating myeloid cells.

Arginase 1 Expression in Cancer Patients

Figure 1: Proliferation of anti-CD3/anti-

CD28-stimulated human T-cells measured by

flow cytometry with anti-CD4 and anti-CD8

staining after a 4 day incubation in media

containing varying concentrations of arginine

Arginase 1 is expressed in tumor-associated myeloid cells in cancer patients

CB-1158 Increases Inflammation

in LLC Tumors

B

Conclusions

Figure 7: Levels of (A) mRNA transcripts (determined by Nanostring), (B) cytokines and chemokines (determined by

Luminex) or (C) cellular markers (determined by flow cytometry) in LLC tumors from mice treated with vehicle or oral

CB-1158 twice daily for 14 days. Arg1+ macrophages were CD68/Arg1 double positive cells. CB-1158 doses: 100

mg/kg for panel (A,C) and 200 mg/kg for panel (B).

S.M. Steggerda, M. Bennett, J. Chen, E. Emberley, J. Janes, W. Li, A. MacKinnon, G. Marguier, A. Pan, F. Parlati, M. Rodriguez, T. Wang,

M. Works, J. Zhang, W. Zhang, and M. Gross; Calithera Biosciences, Inc., South San Francisco, California

↓ expression of TCRz

↓ production of IFN

↓ proliferation

↓ Arginine

MDSC/

neutrophil T-cell

NK-cell

= Arginase

Nutrient sensor

pathways

Arginine Depletion Blocks T-cell and NK-cell Activation

Arginase plays a major immunosuppressive role in human cancer:

• Arginase-expressing MDSCs/neutrophils are found in many tumor types and are

associated with poor prognosis

• Arginase depletes arginine, required for the activation/proliferation of T- and NK-cells

• Arginase inhibition is a novel strategy to target myeloid-mediated immunosuppression

[A rg in in e ] (M )

Ce

ll C

ou

nt

(% m

ax

ima

l p

ro

life

ra

tio

n)

1 0 1 0 0 1 0 0 0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

C D 8+

C D 4+

0

u n s timu la

ted

no

rm

al

pla

sm

a l

ev

el

• Normal plasma arginine levels

are 80-160 µM in humans

• Arginine levels <40 µM suppress

T-cell proliferation

• A small decrease in arginine is

immunosuppressive

Human Arginase Source IC50

Arginase 1 (recombinant) 98 nM

Arginase 2 (recombinant) 274 nM

Neutrophil lysate 162 nM

Red blood cell lysate 116 nM

Hepatocyte lysate 139 nM

RCC patient plasma #1 127 nM

RCC patient plasma #2 174 nM

• CB-1158 is a potent inhibitor of arginase 1 and 2

• CB-1158 inhibits extracellular arginase in plasma and cell lysates

• CB-1158 is not cytotoxic to cancer cell lines or primary T-cells (up to 1 mM)

• CB-1158 is a selective arginase inhibitor (minimal off-target activity at 50 M)

Arginase Reaction

CB-1158 reverses human T-cell immunosuppression by neutrophils or cancer patient-derived MDSCs ex vivo

• Purified neutrophils were mixed with T-cells a from healthy volunteer

• Neutrophils suppress T-cell proliferation

• CB-1158 relieves T-cell suppression by neutrophils (IC50 = 162 nM)

• CB-1158 maintains arginine levels with an (IC50 = 240 nM)

1 01 0 0

1 0 0 0

1 0 0 0 0

3 0

4 0

5 0

6 0

7 0

[C B -1 1 5 8 ] n M

% C

ell

Div

isio

n (

CF

SE

)

F u ll

R e c o v e r y

3 0

4 0

5 0

6 0

7 0

% C

ell

Div

isio

n (

CF

SE

)

T -c e lls T -c e lls +

n eu tro p h ils

1 01 0 0

1 0 0 0

1 0 0 0 0

3 0

4 0

5 0

6 0

7 0

8 0

0

1 0

2 0

3 0

4 0

[C B -1 1 5 8 ] n M

Arg

inin

e (

M

)

Orn

ithin

e (

M)

• Cancer patient MDSCs from PBMCs purified with a-CD66b antibodies

• Media was conditioned with MDSCs for 48 hours

• MDSC-conditioned media suppress T-cell proliferation

• CB-1158 relieves T-cell suppression by MDSCs (IC50 = 177 nM)

• CB-1158 maintains arginine levels (IC50 = 288 nM)

1 1 01 0 0

1 0 0 0

1 0 0 0 0

2 0

3 0

4 0

5 0

6 0

[C B -1 1 5 8 ] n M

% C

ell

Div

isio

n (

CF

SE

)

F u ll

R e c o v e r y

2 0

3 0

4 0

5 0

% C

ell

Div

isio

n (

CF

SE

)

T -c e lls T -c e lls

+ M D S C s

1 01 0 0

1 0 0 0

1 0 0 0 0

2 0

3 0

4 0

5 0

6 0

0

2 0

4 0

6 0

8 0

[C B -1 1 5 8 ] n M

Arg

inin

e (

M

)

Orn

ithin

e (

M)

• Increasing oral doses of CB-1158 increases drug exposures in plasma and tumor

• Elevated exposures of CB-1158 increases plasma and tumor arginine levels

Plasma CB-1158

CB

-1158 (

M

)

0

50

100

150

200

Vehicle 25 mg/kg 250 mg/kg

Tumor CB-1158

CB

-1158 (

nm

ol/g

tis

su

e)

0

20

40

60

Plasma ArginineA

rgin

ine (

M

)

0

500

1000

****

****

Tumor Arginine

Arg

inin

e (

nm

ol/g

tis

su

e)

0

200

400

600

800

1000

***

****

100 mg/kg50 mg/kg

CB-1158 Elevates Arginine in Tumors

Single agent CB-1158 efficacy CD8 depletion

CB-1158 Anti-tumor Efficacy is Immune Based

• Single agent CB-1158 reduces the growth of LLC tumors in immuno-competent mice

• CB-1158 has no anti-tumor activity in immuno-compromised mice

• NK-cell or CD8 depletion partially reverses the anti-tumor effects of CB-1158

Th1 Cytokines M1 Macrophage

Chemokines Arg1+ Macrophage

TCRz+ Lymphocytes

Single agent CB-1158 Combination with checkpoint inhibitors T

um

or V

olu

me

(m

m3)

0 7 1 4 2 1 2 8

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

4 9 % T G I

*

M a d is o n 1 0 9 (L u n g )

D a y s P o s t Im p la n t

Tu

mo

r V

olu

me

(m

m3)

0 5 1 0 1 5

0

5 0 0

1 0 0 0

1 5 0 0

V e h ic le C B -1 1 5 8 (1 0 0 m g /k g )

* * *

4 1 % T G I

B 1 6 (M e la n o m a )

D a y P o s t Im p la n t

Tu

mo

r V

olu

me

(m

m3)

0 5 1 0 1 5 2 0 2 5

0

5 0 0

1 0 0 0

1 5 0 0

V e h ic le

C B -1 1 5 8

(1 0 0 m g /k g )

C B -1 1 5 8

+ a P D -1

+ a C T L A -4

a P D -1

+ a C T L A -4

* * *

P rim a ry T u m o r

4 T 1 (B re a s t)

Nu

mb

er o

f L

un

g M

eta

ste

se

s

0

2 5

5 0

7 5

1 0 0

1 2 5

* *

L u n g M e ta s ta s is

= arginase 1 positive neutrophil/granulocyte

Liver Tonsil Bone marrow Kidney

Normal Human Tissues

HCC_1

Renal NSCLC (squam) NSCLC (adeno) TNBC

Colorectal Prostate

Pancreas

Ovarian Bladder Gastric

Hepatocellular Cancer

Tu

mo

r T

issu

e M

icro

arr

ay

Normal Tissues Cancer Tissue

No rm

a l

He a d &

Ne c k

Me s o th

e liom

a

Co lo

n

L u n g (NS C

)

L u n g (SC

)

Kid

n e y

Bre a s t

1

1 0

1 0 0

Pla

sm

a A

rg

ina

se

1 (

ng

/ml)

Arg

ina

se

1 P

os

itiv

e C

ell

s (

nu

mb

er/m

m2)

L u n g

S tom

a c h

Co lo

n

Bla

d d e r

P a n c re a s

Ov a ry

Kid

n e y

P ro s tate

Bre a s t

1

1 0

1 0 0

1 0 0 0

0 -

• HCC: Moderate expression

• Other tumor types:

– No expression in tumor cells

– High expression in granulocytic myeloid cells

• Liver: High expression

• Other Tissue: no expression

• Bone Marrow: Expression

confined to neutrophils

A

B Arginase 1 in cancer

patient plasma

Arginase 1 expressing

myeloid cells in tumors C

Figure 2: (A) Immunohistochemistry staining for arginase 1 in sections of normal human tissues (N = 33 tissues

analyzed) and human tumor tissues (N = 12 tumor histologies analyzed). Representative images are shown

(red arrows point to arginase-expressing myeloid cells). (B) Quantitation by digital histopathology of arginase 1-

expressing myeloid cells in human tumor tissues. (C) ELISA determination of arginase 1 levels in plasma

samples from cancer patients.

Figure 3: (A) Schema of arginase reaction. (B) IC50 values for inhibition

of arginase reaction by CB-1158 using various sources of arginase as

indicated. Activity was measured by urea and/or ornithine production

using a dose titration of CB-1158.

MDSCs/ Neutrophils

Impaired T-cell Proliferation Arginase Arginine

T-cells

A

Figure 4: (A) Schema of MDSC/neutrophil mediated suppression of T-cell proliferation via arginase. (B) Proliferation of anti-CD3/anti-CD28-stimulated human T-cells measured by flow cytometry

after a 4 day incubation in the presence or absence of neutrophils. Neutrophils were pre-incubated with media for 2 days prior to T-cell addition and stimulation. T-cell proliferation in the presence of

neutrophils (left panel), increasing doses of CB-1158 (middle panel) and arginine/ornithine concentrations in the media from neutrophils incubated for 48 h with a dose titration of CB-1158 (left panel)

are shown. (C) Proliferation of anti-CD3/anti-CD28-stimulated human T-cells measured by flow cytometry after a 4 day incubation in the presence or absence of MDSC-conditioned media. MDSCs

were pre-incubated with media for 2 days prior to transferring the media to stimulated T-cells. T-cell proliferation in MDSC-conditioned media (left panel), increasing doses of CB-1158 (middle panel)

and arginine/ornithine concentrations in the media from MDSCs incubated for 48 hr with a dose titration of CB-1158 (left panel) are shown. Dotted line represents minimal CB-1158 concentration to

achieve full recovery of T-cell proliferation.

Figure 5: (A-B) Concentration of CB-1158 in (A) plasma or (B) Lewis Lung Carcinoma (LLC) tumor lysates from

C57.Bl/6 mice dosed orally with 5 doses of CB-1158 on a BID schedule. Samples were collected 2 h after the last

dose of CB-1158 (N = 5 per group). (C-D) Arginine concentration in (C) plasma or (D) tumor of samples in A-B.

C D 3

Tra

ns

crip

t C

ou

nt

2 0

3 0

4 0

*

T C R z

0

2 0

4 0

*

C D 6 9

1 0

3 0

5 0

*

IL 2 R B

5 0

1 0 0

1 5 0

* *

C D 9 4

4 0

8 0

1 2 0

*

N C R 1

0

2 0

4 0

*

IN F A 1

Tra

ns

crip

t C

ou

nt

2 0

3 0

4 0

* *

IS G 1 5

2 0 0

4 0 0

6 0 0

*

U S P 1 8

4 0

8 0

1 2 0

*

IR F 5

1 0 0

2 0 0

3 0 0

*

IR F 7

1 0 0

2 0 0

3 0 0

*

D d x 6 0

4 0

8 0

1 2 0

* *

V e h ic le C B -1 1 5 8

T-cell markers NK-cell markers

Interferon response genes

C

• CB-1158 increases T-cell/NK-cell markers and interferon-stimulated genes

• Cytokine/chemokine changes suggest skewing toward a pro-inflammatory Th1/M1 phenotype

• CB-1158 decreases immuno-suppressive M2 macrophages

• CB-1158 increases TCRz positive cells suggesting lymphocyte activation

• Single agent CB-1158 inhibits the growth of checkpoint-refractory models Madison 109 and B16

• 4T1 breast tumors are resistant to combined treatment with anti-CTLA-4 and anti-PD-1

• The addition of CB-1158 inhibits the growth rate of the primary tumors and reduces the number of lung metastases

Figure 8: (A) Madison109 cells were implanted in balb/c mice and B16 cells were implanted in C57.Bl/6 mice and mice were dosed orally with vehicle or CB-

1158 BID (N=10 per group). (B) Growth of 4T1 mammary carcinoma cells implanted orthotopically into female balb/c mice and treated with either vehicle, CB-

1158 (100 mg/kg PO BID), anti-CTLA-4 (5 mg/kg IP on Days 2, 5, 8) plus anti-PD-1 (5 mg/kg IP on days 3, 6, and 9), or the combination of CB-1158 with anti-

CTLA-4 and anti-PD-1 (N = 10 per group; *P < 0.05; ***P < 0.001, **** P < 0.0001 vs vehicle).

• CB-1158 potently inhibits arginase and reverses MDSC/neutrophil induced suppression of T-cell proliferation

• CB-1158 increases tumor and plasma arginine levels and has single agent efficacy in syngeneic mouse models

• CB-1158 efficacy is immune mediated and creates a pro-inflammatory tumor microenvironment

• In the refractory 4T-1 model, addition of CB-1158 to anti-PD-1 and anti-CTLA-4 results in tumor growth inhibition

• CB-1158 is currently in IND-enabling studies

Figure 6: (A) Lewis Lung Carcinoma

cells were implanted in C57.Bl/6

mice and mice were dosed orally

with vehicle or CB-1158 twice daily at

100 mg/kg. (B) An additional group of

mice in (A) were treated with anti-

CD8 prior to the start of CB-1158

dosing. (C) In a separate experiment,

tumor-bearing mice were treated with

vehicle or CB-1158 in the presence

or absence of anti-NK1.1 (N = 10 per

group; **** P < 0.0001; *** P < 0.001;

** P < 0.01; ns, not significant).

A

B

C B

B

A B C D

A

A B

A

NK cell depletion C