identification of adenosine pathway genes associated with
TRANSCRIPT
Identification of Adenosine Pathway Genes Associated with Response to Therapy with the Adenosine Receptor Antagonist CPI-444Willingham S1, Hotson A1, Laport G1, Kwei L1, Fong L2, Sznol M3, Powderly J4, Miller R1
1Corvus Pharmaceuticals, Burlingame CA, USA. 2University of California, San Francisco CA, USA 3Yale University School of Medicine, New Haven CT, USA 4Carolina BioOncology Institute, Huntersville NC, USA
0
500
1000
1500Tumor & TREG Cells
CD39CD73
ATP/ADP
AMP
Adenosine
A2AR
CD25
CD4
Macrophage
AdenosineATP A2AR
A2BRCa2+-CAM
M1 M2
GS
GS
Gq
VEGFIL-6
Angiogenesis
Fibrosis
TEFFECTOR Cell
TCR
Increased FOXP3
Promotes tumor supporting M2 phenotypeIncreased angiogenesis
Decreased IL-2 productionIncreased CTLA-4 and PD-1 expressionDecreased proliferation
A2AR
ZAP70
AP1
Activation
-
P
≠
-100-90-80-70-60-50-40-30-20-10
0102030405060708090
100
Max
imum
% D
ecre
ase
in S
LD
-100-90-80-70-60-50-40-30-20-10
0102030405060708090
100
Max
imum
% D
ecre
ase
in S
LD
ADENOSINE SIGNATURE LOW ADENOSINE SIGNATURE HIGH
CXCL
1 (p
g/m
l)
0
200
400
600
800
1000
Donor 1 Donor 2
DMSO0.1 µm NECA1 µm NECA10 µm NECA
CXCL
10 (p
g/m
l)
0
200
400
600
8000
10000
12000
Donor 1 Donor 2
0
500
1000
1500
0
500
1000
1500
CXCL
5 (p
g/m
l)
CXCL
5 (p
g/m
l)
DMSO 0.1 µmNECA
1 µmNECA
10 µmNECA
DMSO 0.1 µmNECA
1 µmNECA
10 µmNECA
No CPI-444+ 1 µm CPI-444
DONOR A DONOR B
Adjusted p Value Function ReceptorIL23A 1.44E-04 Increases angiogenesis and reduces CD8+ T-cell infiltrationSLC11A1 1.27E-03 Natural resistance-associated macrophage protein 1CXCL2 1.27E-03 MIP2a: macrophage inflammatory protein 2, alpha CXCR2CXCL7 1.27E-03 PPBP. Pro-Platelet Basic Protein CXCR2CXCL6 1.40E-03 GCP2: Granulocyte chemotactic protein 2 CXCR2CXCL3 1.40E-03 Controls migration and adhesion of monocytes CXCR2IL-6 1.48E-03 Pro- and anti-inflammatory cytokineIL-1α 1.73E-03 InflammationCXCL8 1.98E-03 IL-8. Nutrophil chemotactic factor CXCR1/2CXCL5 1.98E-03 Attracts and activates neutrophils CXCR2THBS1 2.28E-03 Multiple functions. Inhibits angiogenesis & immune regulationIL-1β 2.38E-03 InflammationPTGS2 2.65E-03 COX-2. Elevated during inflammation and cancerIL-24 2.70E-03 Cell survival and proliferation. Activates STAT1/3CXCL1 3.92E-03 Neutrophil chemotractant CXCR2CD86 5.31E-03 B7-2: Costimulatory signal for T cell activation and survivalCLEC5A 5.82E-03 Interacts with DAP-12 and may play a role in cell activationCD14 6.24E-03 Expressed by myeloid cells
Adjusted p Value Function ReceptorCXCL2 0.008 MIP2a: macrophage inflammatory protein 2, alpha CXCR2IL-23 0.008 Neutrophil attraction w/ IL-17, reduces CD8+ T cell infiltrationCSF3 0.011 G-CSF. Master regulator of neutrophil developmentCXCL3 0.011 Controls migration and adhesion of monocytes CXCR2HAS1 0.014 Hyaluronan synthase 1. ECM componentINHBA 0.016 Inhibin, beta a. Subunit of activin & inhibinCXCL5 0.019 Attracts and activates neutrophils. CXCR2PTGS2 0.019 COX-2. Elevated during inflammation and cancerPADI2 0.019 Protein-arginine deiminase type-2. NeurodegenerationCD93 0.023 C1qRp. Expressed on neutrophils. Clearance of apoptotic cellsSCL7A7 0.024 Uptake of dibasic and neutral amino acidsPID1 0.024 Phosphotyrosine Interaction Domain-Containing Protein 1ECEL1 0.024 Endothelin-converting enzyme-like 1CD300E 0.028 Expressed on myeloid cells. Interacts with TYROST6GALNAC2 0.028 Sialyltransferase 2
ADENOSINE SIGNATURE - NANOSTRING
ADENOSINE SIGNATURE - RNAseq
HUMAN PBMCs STIMULATED WITH NECA or A2AR SPECIFIC AGONIST CGS-21680
HumanPBMC
+ NECA orCGS-21680
+ ACTIVATIONanti-CD3/CD28
NanoStringRNASeq
1 hour 24-48 hours
ExperimentSetup
0
50
100
150
200
250 CD14+ MonocytesCD8+ T CellsCD19+ B Cells
CCL2
(% C
hang
e M
FI)
CXCL
1 (%
Cha
nge
MFI
) CD14+ MonocytesCD8+ T CellsCD19+ B Cells
CPI-444 MonotherapyCPI-444 + Atezolizumab
CPI-444 MonotherapyCPI-444 + Atezolizumab
NECA
Tre
ated
(Log
2 Co
unts
)
GCS
-216
80 o
r NEC
A (L
og2
Coun
ts)
0 152.5 5 7.5 10 12.5
• Extracellular adenosine within the tumor microenvironment create an immunosuppressive niche that promotes tumor growth and metastasis.
• CPI-444 is a potent, selective inhibitor of the adenosine 2A receptor (A2AR).
• Extracellular adenosine has a short half life and it is not feasible to routinely measure in human tumors.
• This study aims to determine genes/proteins modulated by adenosine as surrogate signature to identify patients with adenosine rich tumors
CPI-444 Monotherapy
100 mg BID 28 days
CP-444 + Atezolizumab
100 mg BID 28 days + 840 mg, Q2WEligibility
Prior anti-PD-(L)1 allowedProgressive disease on prior therapyNo selection for PD-L1 expression
Renal CellCancer
0 0.1 1 10
0
200
400
600
NECA Concentration (μM)
Purified human PBMCs from healthy donors were co-cultured with various concentrations of NECA and were stimulated with anti-human CD3 and CD28 antibodies for 1 hour. Culture supernatants were collected 48 hours later and chemokine expression levels were determined by ELISA. Adenosine signature related chemokine concentrations exhibited dose a dependent increase (CXCL1, top left panel) or decrease (CXCL10, top right panel). Addition of CPI-444 (1 µm) neutralizes the induction of CXCL5 by NECA as determined by ELISA (bottom panels).
Purified human PBMCs from healthy donors were co-cultured with various concentrations of NECA and were stimulated with anti-human CD3 and CD28 antibodies. Cells were kept in culture for 2 days. Golgi block was added 4 hours prior to collecting cells for intracellular flow cytometry analysis. CD14+ monocytic cells exhibited elevated expression ofadenosine sgnature related cytokines and chemokines as NECA concentration increased. Lymphocytes including CD8+ T cells and CD19+ B cells had minimal changes. These results indicate that the source of adenosine signature chemokines is likely to be of monocytic lineage
IL-8
(% C
hang
e M
FI)
CXCL
5 (%
Cha
nge
MFI
)
0 0.1 1 10 0 0.1 1 100 0.1 1 10NECA Concentration (μM)
0 0.1 1 10 0 0.1 1 10
0 0.1 1 10NECA Concentration (μM)
0 0.1 1 10 0 0.1 1 10 0 0.1 1 10NECA Concentration (μM)
0 0.1 1 10 0 0.1 1 10
0 152.5 5 7.5 10 12.5
Adenosine Signature levels were determined in pre-treatment biopsy tissue. In brief, RNA was extracted from tumor tissue macrodissected from patient biopsy specimens and analyzed using the NanoString PanCancer Immune Panel. Cutoffs for determining adenosine signature high and low tumors were based on the mean of the Log2 NanoString counts of select adenosine signature genes for all subjects evaluated, including screen fails. The t-test for comparing the maximum percentage decrease in SLD between the adenosine positive and negative signatures is 2-sided p-value = 0.0075, using a 5% level two-sided test.
p = 0.0075
ADENOSINE INHIBITS ANTI-TUMOR IMMUNITY
ABSTRACT
Background:Extracellular adenosine in the tumor microenvironment generates an immunosuppressive niche that promotes tumor growth and metastasis by signaling through the A2A receptor (A2AR) on immune cells. CPI-444 is a selective A2AR antagonist that has demonstrated anti-tumor activity as a monotherapy and in combination with atezolizumab in an ongoing phase 1/1b trial in patients with advanced cancers. Here we analyzed gene expression profiles (GEPs) associated with A2AR agonism to characterize a “signature” of adenosine exposure in human immune cells and correlated this with GEPs in tumor biopsies from patients with renal cell cancer (RCC) treated with CPI-444.
Methods:Human PBMCs were stimulated with NECA (a stable analog of adenosine) or a specific agonist of A2AR (CGS21680). Purified RNA was analyzed using the NanoString PanCancer Immune Panel in conjunction with RNASeq. Select analytes were validated in culture supernatant by ELISA. RCC tumor biopsies collected from 64 patients treated with CPI-444 (100 mg BID) either as a single agent (n = 32) or in combination with atezolizumab (n = 32) were analyzed using NanoString.
Results:In vitro A2AR stimulation resulted in dose-dependent increases in CXCR2 ligands (CXCL1,2,3,5,8) and key mediators of neutrophil/MDSC biology (CSF3, IL-23). Increases in monocyte/macrophage inflammatory mediators such as IL-1b and CCL2,3,7,8, 20 were also observed, as were increases in SERPINB2, S100A8, PTGS2, THBS1. Expression of CXCL10 and GZMB were decreased, consistent with a suppressed IFNg response. CPI-444 treatment inhibited these changes at the transcript and protein level. Preliminary biomarker analysis suggests CPI-444 anti-tumor activity in RCC was associated with increased expression of these adenosine responsive genes in pretreatment biopsies.
Conclusions:A2AR agonists induce a specific gene signature dominated by immunosuppressive mediators of MDSC and monocyte/macrophage biology. Inhibition of these GEPs by CPI-444 are observed in vitro and in vivo in tumor biopsies from treated patients. These gene signatures may be used as biomarkers for patient selection.
A2AR AGONISTS INDUCE SPECIFIC GENE SIGNATURE
0
100
200
300
400
500
CPI-444 PHASE 1/1B CLINICAL TRIAL DESIGN
ADENOSINE SIGNATURE PRODUCED BY CD14+ CELLS
CPI-444 NEUTRALIZES THE ADENOSINE SIGNATURE SIGNATURE CORRELATES WITH PATIENT RESPONSE
CONCLUSIONS
● A2AR agonists induce a specific gene signature in human immune cells. This “Adenosine Signature” is dominated by inflammatory myeloid cytokines and chemokines that signal through CXCR2.
● Adenosine induction of these genes dampens T cell immunity and shifts the balance away from T effector responses and toward myeloid suppressor functions.
● CPI-444 blocks the induction of the Adenosine Signature by A2AR agonists in vitro.
● Expression of the “Adenosine Signature” correlates with tumor regression in Corvus’ ongoing Ph 1/1b trial with CPI-444 treatment in RCC. Patients with high expression of the Adenosine Signature were more likely to have tumor regression than those patients with low expression
● The Adenosine Signature may be used as a predictive biomarker that can be used to select patients most likely to respond to therapy with agents that antagonize adenosine production or signaling.
● Neutralization of the Adenosine Signature confirms the mechanism of action of CPI-444 as an A2AR antagonist.
● Further data along with an update on clinical results will be presented at the Society of Immunotherapy of Cancer (SITC) meeting in November 2018.
Dot color represents different donors and NECA concentrations Dot color represents different concentrations of NECA or GGS-21680
CCL2
15
12.5
10
7.5
5
2.5
0
17.5
15
12.5
10
7.5
5
2.5
0
CXCL8
IL-1β
CXCL2
CXCL1THBS1
CXCL5
IL-6
CXCL3
GZMBIL-1αCD14IL-24
IFNγ
CXCL10
CXCL9
CXCL11CCL24
24 Hour
CXCL1
CXCL8
IL-1βCXCL3
SERPINB2
CXCL5CXCL2
IL-6
IL-1αTHBS1
PTGS2CXCL6
CXCL10
48 Hour
DMSO Control (Log2 Counts) DMSO Control (Log2 Counts)
Identification of Adenosine Pathway Genes Associated with Response to Therapy with the Adenosine Receptor Antagonist CPI-444Willingham S1, Hotson A1, Laport G1, Kwei L1, Fong L2, Sznol M3, Powderly J4, Miller R1
1Corvus Pharmaceuticals, Burlingame CA, USA. 2University of California, San Francisco CA, USA 3Yale University School of Medicine, New Haven CT, USA 4Carolina BioOncology Institute, Huntersville NC, USA
• Extracellular adenosine within the tumor microenvironment create an immunosuppressive niche that promotes tumor growth and metastasis.
• CPI-444 is a potent, selective inhibitor of the adenosine 2A receptor (A2AR).
• Extracellular adenosine has a short half life and it is not feasible to routinely measure in human tumors.
• This study aims to determine genes/proteins modulated by adenosine as surrogate signature to identify patients with adenosine rich tumors
Background:Extracellular adenosine in the tumor microenvironment generates an immunosuppressive niche that promotes tumor growth and metastasis by signaling through the A2A receptor (A2AR) on immune cells. CPI-444 is a selective A2AR antagonist that has demonstrated anti-tumor activity as a monotherapy and in combination with atezolizumab in an ongoing phase 1/1b trial in patients with advanced cancers. Here we analyzed gene expression profiles (GEPs) associated with A2AR agonism to characterize a “signature” of adenosine exposure in human immune cells and correlated this with GEPs in tumor biopsies from patients with renal cell cancer (RCC) treated with CPI-444.
Methods:Human PBMCs were stimulated with NECA (a stable analog of adenosine) or a specific agonist of A2AR (CGS21680). Purified RNA was analyzed using the NanoString PanCancer Immune Panel in conjunction with RNASeq. Select analytes were validated in culture supernatant by ELISA. RCC tumor biopsies collected from 64 patients treated with CPI-444 (100 mg BID) either as a single agent (n = 32) or in combination with atezolizumab (n = 32) were analyzed using NanoString.
Results:In vitro A2AR stimulation resulted in dose-dependent increases in CXCR2 ligands (CXCL1,2,3,5,8) and key mediators of neutrophil/MDSC biology (CSF3, IL-23). Increases in monocyte/macrophage inflammatory mediators such as IL-1b and CCL2,3,7,8, 20 were also observed, as were increases in SERPINB2, S100A8, PTGS2, THBS1. Expression of CXCL10 and GZMB were decreased, consistent with a suppressed IFNg response. CPI-444 treatment inhibited these changes at the transcript and protein level. Preliminary biomarker analysis suggests CPI-444 anti-tumor activity in RCC was associated with increased expression of these adenosine responsive genes in pretreatment biopsies.
Conclusions:A2AR agonists induce a specific gene signature dominated by immunosuppressive mediators of MDSC and monocyte/macrophage biology. Inhibition of these GEPs by CPI-444 are observed in vitro and in vivo in tumor biopsies from treated patients. These gene signatures may be used as biomarkers for patient selection.