Becker A, Narasappa, N, Yin F, Zhang K, Park T, …...Becker A, Narasappa, N, Yin F, Zhang K, Park...
Transcript of Becker A, Narasappa, N, Yin F, Zhang K, Park T, …...Becker A, Narasappa, N, Yin F, Zhang K, Park...
RESEARCH POSTER PRESENTATION DESIGN © 2015
www.PosterPresentations.com
CD73i effects in a mono-therapeutic setting were assessed by
CD3/CD28/CD2 T cell stimulation and cytolytic assays.
Combinatorial settings were assessed using mixed lymphocyte
reactions (MLRs). In vivo effects of CD73i + ICI were determined
using syngeneic tumor models. Multiple structurally-related CD73
inhibitors (CD73i) used in this presentation: AB680, A000830,
A001421
INTRODUCTIONDIFFERENTIAL EXPRESSION OF CD73 AND PD-1 INTUMORS
CD73 catalyzes the extracellular generation of adenosine (ADO) from
adenosine monophosphate (AMP). ADO suppresses immune
responses, including those of T cells, NK cells and dendritic cells
through activation of A2aR and A2bR receptors. Exhausted T cells and
NK cells express high levels of several immune checkpoint proteins,
including PD-1 and TIGIT. We present here preclinical data on the
ability of CD73i to reverse effector cell suppression from exposure to
ADO even in the presence of ICI.
METHODS
AB680 Limits the Inhibitory Effect of AMP onCD4+ T Cell Activation
RESULTS AND CONCLUSIONS
CD73 is expressed across a wide range of tumor types, including
those with limited response to anti-PD-1 therapy. CD73i completely
rescued AMP-mediated inhibition of T cell proliferation and effector
function as well as NK cell cytolytic function. AMP abrogated the
enhanced allogeneic CD4+ T cell activation and IFN-γ production
mediated by blocking PD-1/PD-L1 and TIGIT, an effect that was
reversed by CD73i. Mechanistically, addition of AMP in MLRs
repressed expression of activation markers and immune checkpoint
proteins. Thus, activation of the adenosinergic pathway may limit the
efficacy of ICI. TCGA data from anti-PD-1-treated melanoma patients
identified CD73 expression as a negative prognostic factor. Finally, co-
administration of a CD73i with an anti-PD-1 mAb resulted in significant
reduction of tumor volume associated with increases in immune cell
infiltration.
CD73 inhibition, alone or in combination with anti-PD-1 and anti-TIGIT
antibodies, translates into potent enhancement of immune cell
activation in a variety of studies. These data provide a rationale for
clinical evaluation of CD73i + ICI combinations.
AB680 Reverses the Activity of AMP on a-PD-1 (AB122) and a-TIGIT (AB154) Antibodies in MLR
Figure 2: RNASeq data generated from Hugo W et al, (Cell Vol.
165 (1): 35-44, 2016 were used to plot CD73 and A2AR
expression in melanoma patients treated with nivolumab or
pembrolizumab. a-PD-1 responders (CR or PR, n = 13 patients)
and non-responders (PD, n = 13) were shown.
CD73 Inhibitors Enhance the Activity of a-PD-1in B16.F10 Melanoma Model
Figure 6: In-house generated CD73 inhibitor, A000830 or AB680,
together with a-PD-1 antibody (RMP1-14) were dosed when
tumors reached a volume of 50 mm3. Change in tumor volume
(A) associated with changes in the immune cell infiltrates (B) are
shown. Similar to A000830, AB680 also increased efficacy of a-
PD-1 antibody as measured by tumor volume (C) and survival
curves (D). Mice bearing tumors > 1000 mm3 were sacrificed as
per protocol. Pink circles indicates days when a-PD-1 antibody
dosing occurred.
Figure 1: CD73 expression were derived from RNASeq in the
TCGA database and depicted as box and whisker plots (above).
Expression of CD73 compared to PD-1 is shown (bottom)
Opposing Effects of AMP and a-PD-(L)1 on
Immune Modulatory Protein Expression inMLR
Becker A, Narasappa, N, Yin F, Zhang K, Park T, Kalisiak J, Lawson K, Jeffrey J, Powers JP, Schindler U, Walters, MJ, Tan JBL
Arcus Biosciences Inc., 3928 Point Eden Way, Hayward, CA, USA
CD73 Inhibitors (CD73i) Reverse the AMP/Adenosine-Mediated Impairment of Immune Effector Cell Activation by Immune Checkpoint Inhibitors (ICI)
0 3 6 9 1 2
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CTLA-4 LAG-3 PD-1 TIM-3 TIGIT 4-1BB ICOS CD28
AMP
a-PDL1
Figure 5: (A) Expression of indicated proteins in the presence of
AMP (top row) or a-PD-L1 antibody (bottom row) compared to
samples treated with isotype were determined. The reversibility
of AMP-mediated immune checkpoint protein expression was
shown using CD73 inhibitors (B). Gray bars represent AMP
alone; green bars represent a-PD-L1 blocking antibody alone,
red bars represent AMP + a-PD-L1 blocking antibody, and blue
bars represent AMP + a-PD-L1 blocking antibody + CD73i. (C)
Global transcriptional changes in MLR with AMP or with AMP +
AB680. Numbers indicate fold change compared to AB122
alone.
Figure 4: AB122 (A) or AB122 and AB154 (B) were
simultaneously added to CD4+ T cells and monocytic DC
cultures containing 100 mM AMP. Addition of AB680
completely reversed the inhibitory effects of AMP.
Ig G 4 AB 1 2 2 0 0 .4 1 .2 3 .7 1 1 .1 3 3 .3 1 0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
IFN
-g(p
g/m
l)
A B 6 8 0 (n M )
A B 1 2 2 + A M P
A
B
B
C T L A -4 L A G -3
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(%
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P D -1 T IM -3 T IG IT
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gG
(%
)
AMP AMP + AB680CD39 6.29 1.76
CD73 0.51 1.18
ADA 0.22 1.22
CD26 1.15 1.03
IL2 0.15 0.85
IFNG 0.52 0.69
TNF 1.13 0.69
IL10 2.91 0.96
TGFB 3.66 0.74
TBX21 0.39 0.91
GATA3 1.35 1.13
FOXP3 1.53 1.26
CTLA-4 0.96 1.2
LAG-3 0.72 0.93
PD-1 0.19 0.73
TIM-3 1.63 0.82
TIGIT 1.02 1.08
PDL-1 2 1.08
PDL-2 2.67 1.63
LGALS3 1.93 0.73
IDO-1 1.92 1.14
CSF1 1.19 0.51
Immune
Modulation
AB122
Adenosine
Pathway
Cytokines
Transcription
Factors
Ig G 4 AB 6 8 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
3 0 0 0
IFN
-g (
pg
/ml)
+ A B 122
+ A M P
+ A B 154
Ig G 1 +
Ig G 4
CD73 AND A2AR Expression in a-PD-1 Respondersand Non-responders
C
AB680 (nM)
+
Act AMP
0.3
1
3
30
10
60
200
A B
Figure 3: Proliferation (A) and cytokine secretion (B) in the
presence of AMP and varying concentrations of AB680 tested.
+ +
A c t 0 1 3 1 0 3 0 6 0 2 0 0 4 0 0
0
5 0 0
1 0 0 0
1 5 0 0
IL-2
(p
g/m
L)
A B 6 8 0 (n M )
A M P + E H N A
Re
stin
g
N .D .
A c t 0 1 3 1 0 3 0 6 0 2 0 0 4 0 0
0
2 0 0
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IFN
-g (
pg
/mL
)
A B 6 8 0 (n M )
A M P + E H N A
Re
stin
g
# 3501
1 0
1 0 0
1 0 0 0
1 0 0 0 0
T u m o r T y p e s
RP
KM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
11. Sarcoma
12. Kidney (papillary)
13. Head and Neck
14. Esophageal
15. Pancreatic
16. Stomach
17. Colorectal
Ovarian1.
Kidney (Clear Cell)2.
Adenocortical3.
Lung (squamous)4.
Bladder5.
Breast6.
Liver7.
Lung 8.(adenocarcinoma)
Prostate9.
Melanoma10.
C R /P R P D
1 0
1 0 0
1 0 0 0
1 0 0 0 0
1 0 0 0 0 0
CD
73
FP
KM
C R /P R P D
1
1 0
A2
aR
FP
KM
CD73 Inhibitors Enhance the Activity ofLymphokine Activated Killer Cells
0 .0 0
0 .0 5
0 .1 0
0 .1 5
0 .2 0
0 .2 5
No
rm
ali
ze
d t
o H
PR
T
N .D
A 1 R A 2 a R A 2 b R A 3 R
LLC
only 0
AM
P0.3
1
3
10
30
100
300
0
2 0
4 0
6 0
8 0
Ca
sp
as
eh
i (%
)
A M P + A 0 0 1 4 2 1 (n M )
A B
Figure 7: Lymphokine activated killer cells (LAKs) were
generated from C57BL/6 splenocytes cultured in IL-2.
Expression of adenosine receptors were assessed by qPCR
(A). LAK cells pre-incubated with A001421 and AMP were co-
cultured with LLC target cells loaded with caspase detection
reagent (B). Cytolytic activity of LAKs were depicted as percent
caspase-positive LLCs.
CTLA-4 LAG-3 PD-1 TIM-3 TIGIT 4-1BB ICOS CD28
CD73 PD-1Sarcoma 1077 19.88
Liver 975.9 16.46
Pancreatic 973.5 32.1
Colorectal 963.5 21.62
Stomach 935.2 51.78
Adeno (lung) 908.9 54.8
Esophageal 821.8 28.28
Head and Neck 664.1 39.91
Clear cell (kidney) 649.1 46.11
Papillary Cell (kidney) 557.1 19.53
Ovarian 342.9 22.94
Breast 299.4 17.94
Bladder 286.9 19.48
Melanoma 280.1 53.49
Squamous (lung) 253.6 43.43
Adenocortical 105.5 3.353