Arginase Inhibitor CB-1158 Alleviates Immunosuppression And Enhances Anti-Tumor Responses As A Single Agent
And In Combination With Other Immunotherapies
Amani Makkouk, Mark Bennett, Jason Chen, Ethan Emberley, Matt Gross, Tony Huang, Weiqun Li, Andy Mackinnon, Gisele Marguier, Silinda Neou,
Alison Pan, Susanne Steggerda, Tracy Wang, Jing Zhang, Winter Zhang, and Francesco Parlati
Calithera Biosciences Inc., South San Francisco, CA
Abstract
CB-1158 Synergizes with Adoptive Cell Therapy
Arginase 1 is Expressed in Human Cancer
Background. T cells and natural killer (NK) cells require L-arginine for proliferation. Arginine depletion by arginase in
the tumor microenvironment induces immunosuppression and is associated with tumor immune evasion. Arginase is
expressed by myeloid-derived suppressor cells (MDSCs) and granulocytes, and its pharmacological inhibition is
expected to restore arginine levels and relieve immunosuppression, leading to anti-tumor immune responses.
CB-1158 Synergizes with Adoptively-Transferred Antigen-Specific T cells to Inhibit Tumor Growth
Arginase 1 is Expressed in Tumor-Infiltrating Leukocytes in Human Tumors
C B -1 1 5 8
100
900
3
T u m o r V o lu m e (m m
3
T u m o r V o lu m e (m m
In vivo, CB-1158 has high oral bioavailability and is very well tolerated. In tumor-bearing mice, twice daily dosing of CB1158 causes dose-dependent pharmacodynamic increases in plasma and tumor arginine levels associated with single
agent anti-tumor efficacy in multiple syngeneic models. The anti-tumor efficacy of CB-1158 is abrogated in
immunocompromised mice or via depletion of either CD8 T cells or NK cells, confirming an immune-mediated
mechanism of action. Moreover, CB-1158 enhances CD8 T cell infiltration into tumors and increases expression of Th1
cytokines, T cell and NK cell activation markers, and interferon-inducible genes in the tumor.
600
****
400
200
600
80
P e r c e n t s u r v iv a l
)
)
P m e l- 1 T c e lls
C B - 1 1 5 8 + P m e l- 1 T c e lls
300
0
1200
C B -1 1 5 8 + P m e l-1 T c e lls
900
5
10
15
20
0
0
0
D a y s P o s t Im p la n t
5
10
15
20
15
20
L
25
A
3000
α -P D -L 1
1500
V e h ic le
Arginase 1
C B -1 1 5 8 + α -P D -L 1
6 /1 0 C R
2000
40
V e h ic le
30
0
5
10
15
20
30 %
C B -1 1 5 8
**
20
20 %
α -P D -L 1
10
C B -1 1 5 8 + α -P D -L 1
0
25
0
10
20
30
+
A rg 1
C D 68
/
+
25
40
0
10
20
30
D a y s P o s t Im p la n t
40
B
H ead & N eck
E so p h ag eal
(N = 3 3 )
(N = 4 2 )
(N = 3 8 )
(N = 4 0 )
A
B
N=
4T1 (breast)
C o lo n
Higher Arginase and Lower Arginine Levels are Observed in Cancer Patient Plasma
CB-1158 Combines with anti-PD-1, anti-CTLA-4 and IDO Inhibitors to Inhibit Tumor Growth
A
L u n g
3 1
5
4
6
1 7
8
2 2
2 7
3 2
2 8
****
****
**
****
***
****
****
****
****
N=
2 0
3
•
Efficacy of CB-1158 correlates with increases in tumor arginine levels
•
CB-1158 has no anti-tumor activity in SCID mice or in mice depleted of CD8 T cells or NK cells (LLC model)
V e h ic le
*
D
4
50
0 .5
Figure 6: (A) 4T1 cells were implanted orthotopically into female Balb/c mice. CB-1158 was dosed at 100 mg/kg PO BID starting on day
2. α-CTLA-4 (9H10) was dosed at 5 mg/kg IP on days 2, 5 and 8. α-PD-1 (RPMI-14) was dosed at 5 mg/kg IP on days 3, 6 and 9. Lung
metastases were counted on day 26. (B) B16F10 cells were implanted in C57.Bl/6 mice. CB-1158 and Epacadostat were both dosed at
100 mg/kg PO BID starting on day 2. (N = 10 per group; **** P < 0.0001; ** P < 0.01; * P < 0.05).
200
8
1
-1
200
200
100
4
6
8
10
12
14
0
0
2
4
6
8
10
12
0
5
10
15
20
D a y s P o s t Im p la n t
C
D a y s P o s t Im p la n t
G e m c ita b in e
600
C B -1 1 5 8
****
+ G e m c ita b in e
400
*
200
G e m c ita b in e
C B -1 1 5 8 +
1000
G e m c ita b in e
****
0
e
M
500
0
5
10
15
A v g a r g in in e
50
v o lu n te e rs
D a y s P o s t Im p la n t
0
V e h ic le
*
150
**
1600
D
V e h ic le
15
10
5
30
N K C e lls
+
****
+
G e m c ita b in e
of C D 45
C B -1 1 5 8 +
800
***
400
30
***
75
25
70
20
65
15
10
5
0
60
V e h ic le
C B -1 1 5 8
10
5
10
15
20
C B -1 1 5 8
Figure 3: (left) Levels of immune cell subsets (determined by flow cytometry, N = 10 per group), or (right) levels of mRNA
transcripts (determined by Nanostring, N = 6 per group) in LLC tumors from mice treated with vehicle or CB-1158 at 100 mg/kg
BID starting on day 2 for 14 days. (** P < 0.01; * P < 0.05).
1 2
t
s
re
a
C
N
B
S
R
A
C
L
M
C
L
C
R
C
P a t ie n t 3
1
8
15
22
29
36
1
8
S tu d y D a y
15
22
29
36
S tu d y D a y
G e m c ita b in e
•
55
•
CB-1158 increases inflammation and lymphocyte activation in the tumor microenvironment
•
Addition of CB-1158 to adoptive cell therapy, checkpoint blockade, or chemotherapy results
in further tumor growth inhibition
25
Figure 7: (A) LLC cells were implanted in C57.Bl/6 mice. CB-1158 was dosed at 100 mg/kg PO BID starting on day 2. Gemcitabine was
dosed at 60 mg/kg IP on days 6 and 10. (B) 4T1 cells were implanted orthotopically into female Balb/c mice. CB-1158 was dosed at 100
mg/kg PO BID starting on day 2. Gemcitabine was dosed at 30 mg/kg IP on day 5. (C) CT26 cells were implanted in Balb/c mice.
CB-1158 was dosed at 100 mg/kg PO BID starting on day 2. Gemcitabine was dosed at 50 mg/kg IP on days 10 and 16. (D) Levels of
immune cell subsets determined by flow cytometry in CT26 tumors from mice treated as in (C) for 13 days.
(N = 10 per group; **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05).
SITC Conference, November 9-13, 2016, National Harbor, MD
CB-1158 increases tumor and plasma arginine levels and has single agent efficacy in
multiple syngeneic mouse models
C B -1 1 5 8 +
G e m c ita b in e
CB-1158 potently inhibits arginase and reverses MDSC/granulocyte-induced suppression of
T cell proliferation
C B -1 1 5 8
10
V e h ic le
•
0
0
6
P a t ie n t 2
Conclusions
)
3
**
G e m c ita b in e
1200
D a y s P o s t Im p la n t
0
0
P a t ie n t 1
80
35
30
50
18
2
Figure 11: (A) Plasma levels of CB-1158 in the first dose cohort (50 mg BID) of clinical trial CX-1158-101
(ClinicalTrials.gov #NCT02903914) evaluated after the first dose (Day 1) and at steady state (Day 15). (B) Fasted
plasma arginine levels (absolute concentration or normalized to total plasma amino acid levels) in samples collected
pre-dose (trough) on the indicated days. Analytes were quantitated by LC/MS/MS.
100
20
12
4
25
M y e lo id C e lls
%
20
20
-
50
15
D a y s P o s t Im p la n t
C D 3 N K p46
*
T r a n s c r ip t C o u n t
of C D 45
+
% C D 68
10
40
*
+
20
/A r g 1
****
25
10
CT26 (colon)
T u m o r V o lu m e (m m
IL12RB
+
+
30
+
of C D 45
40
+
/C D 2 0 6
CD69
5
+
% C D 68
CD3-δ
6
6
0
0
T im e (h r )
0
C
g
u
L
ll
100
P a t ie n t 3
of C D 45
+
150
c o n c . in h e a lth y
+
of C D 45
e
S
P a t ie n t 1
P a t ie n t 2
C D 11b
+
CB-1158 Increases T cell Gene Expression
in LLC Tumors
M2 Macrophages
CD8 T Cells
% C D 8
c o n c e n tr a tio n
8
%
CB-1158 Increases CD8 T cells and
Decreases M2 Macrophages in LLC Tumors
0
90
0 .1
0
C B -1 1 5 8
1
C
s
a
e
H
e
H
IC
0 .0 1
C
CB-1158 Increases Tumor Inflammation
2
m
N
&
d
y
h
lt
a
ll
a
m
1
***
D a y s P o s t Im p la n t
Figure 2: (A) Concentration of arginine in plasma or LLC tumor lysates from C57.Bl/6 mice dosed orally with four doses of CB1158 at 100 mg/kg BID (samples collected 2 h after the last dose; N = 5 per group). LLC (B) and B16F10 (C) cells were
implanted in C57.Bl/6 mice and CT26 cells (D) were implanted in Balb/c mice (N =10 per group). CB-1158 was dosed at 100
mg/kg BID starting on day 2 and continued throughout each study. (N = 10 per group; **** P < 0.0001; *** P < 0.001).
**
o
e
n
o
D
B
N
e
C
o
200
0
3
250
1500
3
400
****
2
B
D ay 15
B
h
****
0
D ay 1
C B -1 1 5 8
300
0
10
V e h ic le
400
0
5
le
ic
5
e
V
B
C
V
e
h
-1
1
ic
le
8
0
A
4T1 (breast)
C B -1 1 5 8
600
****
B
V e h ic le
C B -1 1 5 8
400
200
CB-1158 Elevates Plasma Arginine in Patients
at the Lowest Dose Level in Trial CX-1158-101
A r g in in e ( µ M )
400
C B -1 1 5 8
Figure 10: (A) Arginase 1 protein levels in plasma from healthy donors and cancer patients as determined by ELISA.
(B) Plasma arginine concentrations in healthy donor and cancer patient samples as determined by LC/MS.
(**** P < 0.0001; *** P < 0.001; ** P < 0.01; vs. healthy).
CB-1158 Combines with Standard-of-Care Chemotherapy
LLC (lung)
n
r
o
t
a
re
R
A
C
s
C
L
M
C
L
C
S
C
L
ll
e
R
n
u
m
o
li
d
la
C
g
a
r
d
c
e
N
s
D a y s P o s t Im p la n t
th
12
e
8
e
k
r
o
n
o
4
a
0
li
200
0
)
***
****
25
0
V e h ic le
C B -1 1 5 8
****
1
0
T u m o r V o lu m e (m m
****
500
****
75
25
3
3
600
****
2
+ α -P D -1
800
V e h ic le
V e h ic le
)
Tum or
T u m o r V o lu m e (m m
(µ M o r n m o l/g tis s u e )
A r g in in e
P la s m a
****
ll
+ α -C T L A -4
400
8
CT26 (colon)
800
**
a
C B -1 1 5 8
*
16
k
50
E p a c a d o s ta t
C B -1 1 5 8 (µ M )
600
**
e
+ α P D -1
C B -1 1 5 8 +
CB-1158 Combines with MDSC-depleting Gemcitabine to Inhibit Tumor Growth
B16F10 (melanoma)
2 6
m
α -C T L A 4
75
600
32
c
C B -1 1 5 8
*
E p a c a d o s ta t
)
C
LLC (lung)
2 3
100
th
100
T u m o r V o lu m e (m m
B
Arginine
1 8
64
o
T u m o r V o lu m e (m m
3
)
V e h ic le
A
A
2 0
A r g in in e (% o f T o ta l A m in o A c id s )
Single agent CB-1158 reduces the growth of LLC, B16F10 and CT26 tumor models
9
128
800
A r g in in e ( µ M )
**
S
•
3
125
Figure 1: (A) Proliferation (CFSE)
of
α-CD3/α-CD28-stimulated
human T cells after a 4-day
incubation in the presence or
absence of neutrophils or CB-1158
(1.6 µM). (B) Human T cells from a
healthy donor were cultured in
media pre-conditioned for 2 days
with granulocytes isolated from a
patient with head & neck cancer.
The media was collected and
analyzed for the indicated analytes
by Cytometric Bead Array.
CB-1158 Increases Arginine Levels and Has
Single-Agent Anti-Tumor Efficacy in Animal Models
6
B16F10 (melanoma)
M
C B -1 1 5 8 (n M )
50
&
+
/
+
C D 15
d
-
+
A rg 1
D
-
75
a
C B -1 1 5 8
1000
Figure 9: (A) Immunofluorescent
staining (MultiOmyx) of Arginase 1
and CD15 in a tumor section from a
patient with head & neck cancer. (B)
Percentage of arginase 1 positive
cells that co-express the granulocyte
marker CD15 or macrophage marker
CD68 in tumor tissue microarrays as
determined by quantitation of
MultiOmyx immunofluorescence.
50
e
+
100
Overlay (95% overlap)
100
c e lls
3000
B
60
+
500
P e rc e n t s u rv iv a l
****
0
y
+
10
CD15 (granulocytes)
)
****
h
-
0
1
t
as
e
r
70
lt
N e u t r o p h ils
0
ro
P
)
ry
as
ey
C
n
B
va
re
d
N
c
i
O
K
-T
an
n
P
o
(n
1000
a
+
B
te
a
st
80
3
1000
T u m o r V o lu m e (m m
C B -1 1 5 8 + α -P D -L 1
e
+
d
t
as
e
r
Arginase 1 and CD15 Staining of Tumor Tissue Microarray
H
+
d
ea
an
N
k
ec
90
α -P D -L 1
N u m b e r o f L u n g M e ta s te s e s
)
G r a n z y m e -B (p g /m L
% C e ll D iv is io n (C F S E )
T c e lls
20000
)
30
1000
In te r fe r o n - γ (p g /m L
40
h
S
n
ki
B
A
C B -1 1 5 8
40000
p
s
2000
125
50
so
u
ag
100%
100
3 /1 0 C R
CB-1158 Reverses Myeloid Cell Mediated T Cell Suppression
2000
E
n
H
B
CB-1158 is not cytotoxic to cancer cell lines or primary T cells at concentrations up to 1 mM
60000
C
o
lo
Arginase 1 Co-localizes with Tumor-Infiltrating Granulocytes
Figure 5: (A) CT26 cells were implanted in Balb/c mice. CB-1158 was dosed at 100 mg/kg PO BID starting on day 2. α-PD-L1 (10F.9G2)
was dosed at 5 mg/kg IP on days 5, 7, 9, 11, 13 and 15. Mice were sacrificed when tumor volumes reached 2000 mm3. CR: complete
regression. (N = 10 per group; **** P < 0.0001; ** P < 0.01). (B) Survival curves. P < 0.0001 by Mantel-Cox test (experiment is ongoing).
•
60
B
er
0
CB-1158 is a potent and selective inhibitor of arginase 1 (IC50 98 nM) and arginase 2 (IC50 274 nM)
3000
to
d
la
d
D a y s P o s t Im p la n t
•
80000
n
u
L
S
m
h
ac
B
0
CB-1158 Potently Inhibits Arginase
B
n
(S
d
(A
g
)
o
CB-1158 Combines with anti-PD-L1 to Inhibit Tumor Growth
0
70
am
u
en
CB-1158 Combines with Checkpoint Blockade
D a y s P o s t Im p la n t
A
u
g
q
o
s)
u
Figure 8: Human tumor tissue microarrays were stained with anti-Arginase 1 antibody by immunohistochemistry.
Arginase 1-positive infiltrating granulocytes were quantified by digital histopathology.
Figure 4: (A) B16F10 cells were implanted in C57.Bl/6 mice. Non-myeloablative chemotherapy (Cyclophosphamide 250 mg/kg and
Fludarabine 50 mg/kg) was dosed IP on day 7 (orange arrow) to all groups. Pmel-1 CD8 T cells (1 x 106) were adoptively transferred IV
on day 9 (blue arrow). Recombinant human IL-2 (200,000 UI) was dosed IP BID on days 9, 10 and 11 in mice receiving T cells (no effect
on tumor growth; data not shown). CB-1158 was dosed at 100 mg/kg PO BID starting on day 2. (N = 9-10 per group; **** P < 0.0001).
(B) Survival curves. P = 0.0137 by Mantel-Cox test.
1000
T cell
NK cell
10
D a y s P o s t Im p la n t
↓ Production of IFN-γ
= Arginase
5
D a y s P o s t Im p la n t
↓ Proliferation
MDSC/
Neutrophil
0
H
↓ Arginine
1
25
A r g in a s e 1 (n g /m L )
Nutrient sensor
pathways
10
10%
% o f A rg 1
↓ Expression of TCR-ζ
100
P m e l- 1 T c e lls
0
0
1000
P m e l- 1 T c e lls
0
3
Arginase depletes arginine, which is required for the proliferation of T cells and NK cells
C B -1 1 5 8
300
)
•
V e h ic le
C B -1 1 5 8 +
T u m o r V o lu m e (m m
Arginase-expressing MDSCs/neutrophils are present in multiple tumor types & are associated with poor prognosis
44%
40
20
C B -1 1 5 8
•
78%
60
600
To assess the clinical potential of CB-1158, the abundance of arginase in tumors and plasma from cancer patients
across multiple cancer histotypes was surveyed. Arginase-expressing granulocytic infiltrates are abundant in multiple
tumor types. Plasma arginase levels are elevated in cancer patients compared to healthy controls, and are associated
with decreased plasma arginine. In an ongoing Phase I clinical trial (NCT02903914) for patients with solid tumors, CB1158 shows significant PK/PD effects as a monotherapy at the first dose of 50 mg BID.
Arginase is an Immunosuppressive Enzyme
500 0
A r g in a s e 1 P o s itiv e c e lls /m m
800
Results. In a co-culture system of T cells with granulocytes or MDSCs, CB-1158 reverses granulocyte- or MDSCmediated immunosuppression by blocking arginine depletion, thereby allowing T cells to proliferate. T cells activated in
the presence of granulocyte-conditioned media show suppressed production of cytokines involved in Th1-type adaptive
immunity, and this effect is reversed by the addition of CB-1158.
Conclusions. These results support the clinical development of CB-1158, a first-in-class arginase inhibitor, as a novel
immunomodulatory agent antagonizing myeloid-mediated immunosuppression alone or in combination with other
immunotherapies.
P m e l-1 T c e lls
V e h ic le
Materials and Methods. We developed CB-1158, a potent and selective small molecule inhibitor of arginase (IC50 = 98
nM). The activity of CB-1158 was examined ex vivo using immune cells isolated from healthy volunteers or cancer
patients, and in vivo using murine syngeneic tumor models. Arginase abundance in cancer patient plasma and in tumor
tissue microarrays was also examined.
The immunomodulatory activity of CB-1158 supports the potential of its combination with other immunotherapies and/or
standard-of-care therapies. CB-1158 enhances the anti-tumor efficacy of adoptive cell therapies such as T cell therapy
in the B16F10 model. CB-1158 also enhances checkpoint inhibitors, including anti-PD-L1 in the B16F10 model.
Moreover, CB-1158 enhances the anti-tumor efficacy of chemotherapies with immunomodulatory activity such as
Gemcitabine in several models.
B
1200
2
A
•
Cancer patients have arginase-containing tumor immune cell infiltrates, increased plasma
arginase, and decreased plasma arginine compared to healthy individuals
•
CB-1158 is currently in a Phase I clinical trial in solid tumor patients (CX-1158-101)
•
CB-1158 shows significant pharmacodynamic effects in patients at the first dose level
(For more details on the design of clinical study CX-1158-101, see Poster # 155)