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RESEARCH POSTER PRESENTATION DESIGN © 2015
www.PosterPresentations.com
• Robust assays have been developed to measure the peripheral levels and activity of the
adenosine-generating enzyme, CD73.
• Immunohistochemical methods have been developed to measure and quantify adenosine-
generating enzymes CD73 and TNAP in human tumor tissue.
• These assays are being implemented to assess the adenosine generating potential of subjects
in clinical studies with AB928.
• AB928 is currently in clinical trials in combination with standard of care chemotherapeutics and
an anti-PD-1 immunotherapeutic.
DiRenzo D, Ashok D, Anderson AE, Udyavar A, Park A, Tan JBL, Luu I, Zhang K, Jeffrey JL, Seitz L, Leleti MR, Young SW, Powers JP, Walters MJ
Arcus Biosciences, Inc.; 3928 Point Eden Way, Hayward, CA 94545 (USA)
Methods for Assessment of the “Adenosine Fingerprint”
in Clinical Trials of AB928
pCREB Assay: Whole blood from healthy volunteers or cancer subjects was stimulated ex vivo
with the adenosine agonist NECA. Levels of CREB phosphorylation (pCREB) were assessed by
flow cytometry and AB928 plasma concentrations were determined using LC-MS/MS.
CD73 ELISA: Circulating levels of CD73 were quantified in matched serum and plasma samples
from healthy donors and cancer patient samples with an in-house ELISA. Validation studies were
performed to correlate CD73 levels between serum and plasma and to ensure acceptable assay
parallelism and inter-assay variation.
AMPase Activity: AMP hydrolytic activity in serum from healthy volunteers or patients with solid
tissue tumors was assessed using the AMP GloTM (Promega) assay. CD73 and TNAP inhibitor
cocktails were used to assess the relative contribution of each enzyme to AMPase activity.
Gene Expression: Expression of CD73 (NT5E) and TNAP (ALPL) on select tumor types were
derived from RNASeq in The Cancer Genome Atlas (TCGA) database. Data is displayed as log2
transformed expression of counts per million.
Histology: Immunohistochemistry (IHC) was performed using CD73 (Cell Signaling, D7F9A)
and TNAP (Sino Biological, R034) on formalin fixed paraffin embedded (FFPE) human tissue.
QuPath software was used for quantification and H-score = 3x(high intensity staining area) +
2x(medium intensity staining area) + (low intensity staining area). .
Introduction
Figure 1. (A.) AB928 suppresses NECA-induced pCREB activation in CD8+ T cells of healthy
volunteers. (B.) Healthy volunteers and cancer patients exhibit a similar pattern of pCREB
inhibition and an overlapping PK/PD relationship.
Results
Materials and Methods
Conclusions
AACR 2019 National Meeting; Atlanta
Abstract # 3168
The tumor microenvironment (TME) contains high levels of immunosuppressive adenosine
(ADO), which activates the A2aR and A2bR receptors on immune cells, leading to an ineffective
anti-tumor response. Ecto-5’-nucleotidase (CD73) and tissue non-specific alkaline phosphatase
(TNAP) are primarily responsible for the conversion of extracellular adenosine mono-phosphate
(AMP) to ADO and exhibit both membrane-bound
and secreted forms. We have previously shown
that AB928, a dual A2aR/A2bR antagonist,
rescues the immuno-suppressive effects of ADO
in experimental tumor models. Herein, we
describe the development of assays to measure
the expression and activity of adenosine-
generating enzymes in human tumor samples
and peripheral blood. These assays are being
used to define an “adenosine fingerprint” to
identify tumor types and patients most sensitive
to adenosine inhibition by AB928.
0
50
100
% pC
RE
B S
ignal
Pre-dose Day 4Pre-dose
Day 42 hours
Day 424 hours
Placebo 10 mg
75 mg 150 mg
25 mg
200 mg
1000 2000 3000 4000
0
50
100
AB928 measured in plasma (nM)
%
Inhib
itio
n fro
m P
redose
Cancer patientsHealthy volunteersA. B.
Development of a Validated ELISA Assay to Quantify Soluble
CD73 in Peripheral Blood
Figure 3. (A.) Healthy volunteer serum was tested in the presence of CD73 and/or TNAP
inhibitors. (B.) Average CVs across multiple conditions show the assay is robust for the
determination of AMP hydrolysis in human serum. (C.) Healthy volunteer and cancer patient
serum display a strong correlation between CD73 protein concentration and AMP hydrolysis.
Determination of AMP Hydrolysis by CD73 and TNAP in Serum
Using an AMP GloTM Assay
Figure 2. (A.) Serum and sodium heparin plasma from healthy donors were evaluated in an
ELISA to measure soluble CD73 and were highly correlated. (B.) Parallelism assessment was
performed to identify the quantitative range of the assay. (C.) Soluble CD73 levels in serum are
elevated in cancer patients compared to healthy controls. *p<0.05
1 2 3 4 51
2
3
4
5
Plasma vs. Serum
Serum CD73 [ng/ml]
Pla
sm
a C
D73 [ng/m
l]
R2 = 0.9597
0 2 4 6 8 10 12 14 16 18 2040
60
80
100
120
140
160
180
Parallelism Assessment
Sample Dilution Factor
Adju
ste
d %
Recovery
Below Detection
2
4
5
8
10
MRD
Healthy Cancer0
2
4
6
8
10
Seru
m C
D73 [ng/m
l]
*A. B. C.
TCGA Analysis Reveals Unique Expression Patterns of CD73 and
TNAP in Human Tumors
Figure 4. CD73 (left) and TNAP (right) expression from RNAseq data retrieved from The
Cancer Genome Atlas (TCGA) samples. Numbers indicate a ratio of log2 counts per million per
sample. Samples are ordered according to their expression with higher expressing tumors on
the left and lower expressing tumors on the right.
TNAP Expression is Heterogeneous Across Human Tumors from
Different Indications
Figure 5. (A.) Representative images of immunostaining for CD73 (brown) on human FFPE
tumor samples. Nuclei are stained with hematoxylin (blue) (B.) Quantification of CD73 staining
area as a percentage of total tumor area. (C.) A strong correlation is seen when comparing an
H-score to % staining area.
CD73 is Detected and Quantifiable in Human Tumors via IHC
NSCLC NSCLC TNBC CRC
NSCLCadeno
CRC HNSCC NSCLCsquam
Gastric Esoph Breast0
10
20
30
40
% C
D73
+ a
rea
0 20 40 60 80 100
0
20
40
60
80
100
% CD73+ Area
H s
co
re
A.
B. C.
Ovarian NSCLC(adeno)
Breast Liver Stomach CRC0
25
50
75
100
% T
NA
P+ A
rea
Figure 6. (A.) Representative images of immunostaining for TNAP (brown) on human FFPE
tumor samples. Strong staining can be seen in ovarian and NSCLC tumors whereas liver and
CRC exhibit much less staining. (C.) Quantification of TNAP staining area as a percentage of
total tumor area.
A.
C.
AB928 Inhibits A2aR Activation Following Dosing with ≥75 mg in
Healthy Volunteers and Cancer Subjects
0
3×105
6×105
9×105
1.2×106
AMP Hydrolysis in Healthy Serum
Lum
inescence (
RLU
)
CD73 Inhibitor
TNAP Inhibitor +
+ +
+
--
- -0 2 4 6 8
0
20
40
60
80
100
CD73 Activity vs. Protein Correlation
CD73 [ng/ml]
% H
ydro
lytic A
ctivity
R2 = 0.7151
TNBC
R2 = 0.9532
Healthy
NSCLC
CRC
A.
B. C.
Ovarian NSCLC Breast Liver
CRCStomachB.
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CD73 TNAP
Teff
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