Levels and enzyme activity of CD73 in primary samples from cancer patients

Abstract

CD73 is an ectonucleotidase that converts extracellular AMP to Adenosine. Within the tumor microenvironment, adenosine produced by CD73 is believed to have an immunosuppressive effect upon anti-tumor immunity. CD73 is found on lymphoid and myeloid cells, and blockade of this enzyme has been shown to alter the suppressive capacity of cells. Expression of surface CD73 as well as soluble CD73(sCD73) levels were measured in serum, peripheral blood mononuclear cells, and tumor samples from healthy and cancer patient donors. Enzymatic activity of both surface CD73 and sCD73 was determined and differences in levels across varying tumor types and disease status were quantified. In addition, CD73 levels and activity were measured after incubation or treatment with anti-CD73 antibody, MEDI9447. These studies suggest a potential role of surface and sCD73 as a pharmacodynamic and/ or predictive biomarker of anti-CD73 immunotherapy.

Full text

Modified fromZhang, 2012.
Levels and Enzyme Activity of CD73 in Primary Samples
From Cancer Patients
Qihui Huang*, Nicholas Durham*, Erin Sult, Jia Li, Yuling Wu, Nicholas Holoweckyj, Robert Hollingsworth,
Brett Hall, Ronald Herbst, Mary Antonysamy, Ching Ching Leow, and Kris F. Sachsenmeier
AACR Abstract # 1538, 20April2015
CD73 is an ecto-nucleotidase that converts extracellular AMP to
adenosine. Within the tumor microenvironment, adenosine produced
by CD73 is believed to have an immunosuppressive effect upon anti-
tumor immunity. CD73 is found on lymphoid and myeloid cells, and
blockade of this enzyme has been shown to alter the suppressive
capacity of cells. Expression of surface CD73 as well as soluble
CD73 (sCD73) levels were measured in serum, peripheral blood
mononuclear cells, and tumor samples from healthy and cancer
patient donors. Enzymatic activity of both surface CD73 and sCD73
was determined and differences in levels across varying tumor types
and disease status were quantified. In addition, CD73 levels and
activity were measured after incubation or treatment with anti-CD73
antibody, MEDI9447. These studies suggest a potential role of
surface and sCD73 as pharmacodynamic biomarkers for anti-CD73
therapy.
Abstract
Background
•CD73, an ecto-5’-nucleotidease, is expressed on many cells types
including tumor and immune cells and, together with CD39, converts
extracellular adenosine tri-phosphate (ATP) to adenosine [ 2]. CD73
can also be found in soluble form (sCD73) in the blood.
•Adenosine enhances polarization of myeloid and T cell subsets to
immuno-suppressive phenotypes, promoting tumor growth and
survival [ 1].
•Targeting CD73 relieves adenosine-mediated immunosuppression
within the tumor microenvironment [ 1].
•MEDI9447 is a monoclonal antibody that inhibits CD73 enzyme
activity. Figure 4. Balb/C mice with CT26 tumors were treated with MEDI9447. MEDI9447
significantly reduced levels of CD73 on Tregs, CD8s, and CD11b+ cells in the
peripheral blood as well as in the tumor microenvironment
Conclusions
•MEDI9447 treatment significantly decreases the level
of cell surface CD73 on Tregs, CD8s, and CD11b+
cells. Correspondingly, a significant decrease in CD73
enzyme activity was observed on these cells following
treatment with MEDI9447.
•In mice and humans with tumors, elevated serum
levels of sCD73 were observed.
•MEDI9447 is blocks the enzymatic activity of both cell
surface and serum CD73.
•Measuring CD73 levels and enzyme activity could be
a valuable tool in determining pharmacodynamic
effects of MEDI9447 and for identifying patient
populations that may benefit from MEDI9447
treatment.
Figure 7. Two groups of cancer patient serum samples (BioreclamationIVT), f rom a variety of
indications, were tested for CD73 enzyme activity and compared with samples from normal
healthy donors (Panels A and B). The patient sample with highest CD73 enzyme levels was
incubated with the increasing amounts of MEDI9447 (C). The activi ty was calibrated using
dilutions of recombinant human CD73. Data in panel C represent the mean and standard
deviation of duplicate samples.
Figure 8. Sera of different cancer indications show differing levels of sCD73 enzyme activity.
Cancer patient serum samples ( from BioreclamationIVT) and normal healthy donors were
tested for CD73 enzyme activity. The activity was calibrated using dilutions of recombinant
human CD73. Some samples showed no detectable enzyme activity.
Figure 6. Serum from Balb/C m ice with or without syngeneic CT26 colorectal tumors was
analyzed for CD73 enzyme activity.The activity was calibrated using dilutions of
recombinant mouse CD73.
Results
Figure 1: Figure 1. CD73 is
expressed on tumorcells, in
the vasculature, in peripheral
blood cells as well as tumor-
infiltrating leukocytes. Within
the tumor microenvironment,
adenosine supports tumor
growth and
immunosuppression and
impacts T, B, & NK cells as
well as monocytes.
Figure 2. Tumor-free CD1 m ice were injected with a single intravenous dose of anti-CD73
antibody MEDI9447 at 3 and 10 mg per kg (mpk) mouse. At the indicated time points,
mice were bled and levels of antibody (A) and antibody-complexed soluble CD73 (B) were
determined by ELISA. The mean and standard deviation of 3 indivi dual m ice ar e shown.
C) Normal Balb/C mice were treated with MEDI9447. Whole bl ood cell s were harvested
CD73 levels were evaluated by flow cytometry. MEDI9447 significantly (p<0.05) reduced
CD73 surface expression levels.
Cellular CD73 Levels Cellular CD73 Activity
A
Tumor
B
ng/ml sCD73
ng/ml MEDI9447
MEDI9447 Levels and Pharmacodynamics in Tumor-Free Mice
A. B.
The Role of CD73 in Tumor Biology
CD73 Activity (RLU)
Methods
CD73 Enzyme Activity:
The Promega CellTiter-Glo (CTG) kit was used to measure the
conversion of AMP to Ado by ecto-5
’-nucleotidase by detection of
ATP levels through the luciferase-linked emission of light
accompanying catabolism of ATP to AMP and free diphosphate as
described by Sachsenmeier et al[3].
CT26 Flow Cytometry:
1x10^5 CT26 cells were injected sub-cutaneously into the right flank
of a BALB/c mice. Mice were treated on Day 12 and 16 with
MEDI9447. On day 17 mice were sacrificed and blood and tumor
analyzed. To detect total CD73, a non-competing antibody clone
TY/23 was used for flow cytometry.
References:
1. Antonioli L, Blandizzi C, Pacher P, Haskó G. Immunity, inflammation and cancer:
a leading role for adenosine. Nat Rev Cancer. 2013 Dec;13(12):842-57.
2. Linden J, Cekic C. Regulation of lymphocyte function by adenosine. Art T hromb
Vasc Biol. 2012 Sep;32(9):2097-10\3.
3. Sachsenmeier KF, Hay C, Brand E, Clarke L, Rosenthal K, Guillard S, Rust S,
Minter R, Hollingsworth R. Development of a novel ectonucleotidase assay
suitable for high-throughput screening. J Biomol Screen. 2012 Aug;17(7):993-8.
For further information please visit posters for abstracts 285 and 272.
Tumor-Bearing Mice Show Elevated Serum CD73 Enzyme Activity
C.
Cancer Patients Show Elevated Serum CD73 Enzyme Activity
A. B. C.
MEDI9447 Reduces CD73 Levels in Different Immune Cell Subsets
Of CT26 Tumor-bearing Mice
Figure 3. Balb/C mice with CT26 tumors were treated with MEDI9447. Cell surface
CD73 levels were evaluated by flow cytometry and CD73 enzyme activity was
measured from dissociated whole blood (A) and tumor cell s (B). MEDI9447
significantly (p<0.05) reduced CD73 surface levels and enzyme activity in the tumor
and the periphery.
% CD73+ Cells
% CD73+ Cells
Whole
Blood
Cells
MEDI9447 Reduces CD73 Levels and Activity in Tumor and Whole Blood
Cells Of CT26 Tumor-bearing Mice
Correlation Between Peripheral and Tumor cCD73 Enzyme Activity
Figure 5. CT26/CRC tumor-bearing im munocompetent syngeneic were tr eated with
MEDI9447 before tumor cel ls or whole blood cells were isolated and assayed for CD73
enzyme activity. There is significant correlation in cell surface CD73 activities between
whole blood cells and tumor cells.
*These authors contributed equally to this work