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N o
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L L O Q
IL -1 2 p 7 0
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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