Identification of Adenosine Pathway Genes Associated with Response to Therapy with the Adenosine Receptor Antagonist CPI-444

Willingham 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

ABSTRACT A2AR AGONISTS INDUCE SPECIFIC GENE SIGNATURE CPI-444CPI-444 NEUTRALIZES PHASE 1/1B THE CLINICAL ADENOSINE TRIAL DESIGNSIGNATURE SIGNATURE CORRELATES WITH PATIENT RESPONSE

Background: 1000 DMSO HUMAN PBMCs STIMULATED WITH NECA or A2AR SPECIFIC AGONIST CGS-21680 12000 Extracellular adenosine in the tumor microenvironment generates an immunosuppressive niche that promotes tumor 0.1 µm NECA CPI-444 Monotherapy 800 10000 1 µm NECA growth and metastasis by signaling through the A2A receptor (A2AR) on immune cells. CPI-444 is a selective A2AR 10 µm NECA antagonist that has demonstrated anti-tumor activity as a monotherapy and in combination with atezolizumab in an 8000 Experiment Human + NECA or + ACTIVATION NanoString 600 100 mg BID 28 days ongoing phase 1/1b trial in patients with advanced cancers. Here we analyzed gene expression profiles (GEPs) Setup PBMC CGS-21680 anti-CD3/CD28 RNASeq 1 hour 24-48 hours 600 associated with A2AR agonism to characterize a “signature” of adenosine exposure in human immune cells and 400 Renal Cell

CXCL1 (pg/ml) 400 correlated this with GEPs in tumor biopsies from patients with renal cell cancer (RCC) treated with CPI-444. CXCL10 (pg/ml) Cancer 200 200 CP-444 + Atezolizumab Dot color represents different donors and NECA concentrations Methods: Dot color represents different concentrations of NECA or GGS-21680 0 0 Human PBMCs were stimulated with NECA (a stable analog of adenosine) or a specific agonist of A2AR (CGS21680). CXCL8 100 mg BID 28 days + 15 CXCL8 17.5 Donor 1 Donor 2 Donor 1 Donor 2 SERPINB2 CXCL3 CXCL1 Eligibility Purified RNA was analyzed using the NanoString PanCancer Immune Panel in conjunction with RNASeq. Select CXCL3 CXCL1 IL-1β 840 mg, Q2W THBS1 CXCL2 15 12.5 IL-1β CXCL5 CCL2 analytes were validated in culture supernatant by ELISA. RCC tumor biopsies collected from 64 patients treated with CXCL5 Prior anti-PD-(L)1 allowed CXCL2 IL-6 CPI-444 (100 mg BID) either as a single agent (n = 32) or in combination with atezolizumab (n = 32) were analyzed using THBS1 12.5 IL-6 Progressive disease on prior therapy 10 CXCL10 IL-1α DONOR A DONOR B IL-1α IL-24 CD14 GZMB NanoString. CXCL6 1500 1500 No CPI-444 No selection for PD-L1 expression PTGS2 10 7.5 + 1 µm CPI-444

Results: 7.5 CXCL9 IFNγ 5 In vitro A2AR stimulation resulted in dose-dependent increases in CXCR2 ligands (CXCL1,2,3,5,8) and key mediators CXCL10 1000 1000 5 of neutrophil/MDSC biology (CSF3, IL-23). Increases in monocyte/macrophage inflammatory mediators such as IL-1b 2.5 2.5 CCL24 CXCL11 and CCL2,3,7,8, 20 were also observed, as were increases in SERPINB2, S100A8, PTGS2, THBS1. Expression of CXCL10 (Log2 Counts) NECA Treated 48 Hour 24 Hour 0 ADENOSINE SIGNATURE LOW ADENOSINE SIGNATURE HIGH and GZMB were decreased, consistent with a suppressed IFNg response. CPI-444 treatment inhibited these changes at 500 500 100 100 GCS-21680 or NECA (Log2 Counts)

0 CXCL5 (pg/ml) CXCL5 (pg/ml) the transcript and protein level. Preliminary biomarker analysis suggests CPI-444 anti-tumor activity in RCC was 0 2.5 5 7.5 10 12.5 15 0 2.5 5 7.5 10 12.5 15 90 CPI-444 Monotherapy 90 CPI-444 Monotherapy 80 CPI-444 + Atezolizumab 80 CPI-444 + Atezolizumab associated with increased expression of these adenosine responsive genes in pretreatment biopsies. DMSO Control (Log2 Counts) DMSO Control (Log2 Counts) 70 70 0 0 60 60 50 50 Conclusions: DMSO 0.1 µm 1 µm 10 µm DMSO 0.1 µm 1 µm 10 µm 40 40 ADENOSINE SIGNATURE - NANOSTRING NECA NECA NECA NECA NECA NECA 30 30 A2AR agonists induce a specific gene signature dominated by immunosuppressive mediators of MDSC and 20 20 ≠ monocyte/macrophage biology. Inhibition of these GEPs by CPI-444 are observed in vitro and in vivo in tumor biopsies Adjusted p Value Function Receptor 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 10 10 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 0 0 Increases angiogenesis and reduces CD8+ T-cell infiltration induction of CXCL5 by NECA as determined by ELISA (bottom panels). from treated patients. These gene signatures may be used as biomarkers for patient selection. IL23A 1.44E-04 -10 -10 SLC11A1 1.27E-03 Natural resistance-associated macrophage protein 1 -20 -20 CXCL2 1.27E-03 MIP2a: macrophage inflammatory protein 2, alpha CXCR2 -30 -30 -40 -40 CXCL7 1.27E-03 PPBP. Pro-Platelet Basic Protein CXCR2 -50 -50 CXCL6 1.40E-03 GCP2: Granulocyte chemotactic protein 2 CXCR2 + -60 -60 -70 -70 ADENOSINE INHIBITS ANTI-TUMOR IMMUNITY CXCL3 1.40E-03 Controls migration and adhesion of monocytes CXCR2 ADENOSINE SIGNATURE PRODUCED BY CD14 CELLS in SLD Maximum % Decrease -80 in SLD Maximum % Decrease -80 IL-6 1.48E-03 Pro- and anti-inflammatory cytokine -90 -90 p = 0.0075 -100 -100 IL-1α 1.73E-03 Inflammation CXCL8 1.98E-03 IL-8. Nutrophil chemotactic factor CXCR1/2 1500 250 + CD14 Monocytes 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 Tumor & TREG Cells TEFFECTOR Cell Macrophage CXCL5 1.98E-03 Attracts and activates neutrophils CXCR2 CD8+ T Cells 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. + THBS1 2.28E-03 Multiple functions. Inhibits angiogenesis & immune regulation 200 CD19 B Cells

IL-1β 2.38E-03 Inflammation 1000 PTGS2 2.65E-03 COX-2. Elevated during inflammation and cancer 150 CD39 IL-24 2.70E-03 Cell survival and proliferation. Activates STAT1/3 AMP CD73 A2AR Adenosine Neutrophil chemotractant CXCR2 100 ATP/ TCR ATP CXCL1 3.92E-03 500 CONCLUSIONS ADP CD86 5.31E-03 B7-2: Costimulatory signal for T cell activation and survival G S A2BR 50 GS CLEC5A 5.82E-03 Interacts with DAP-12 and may play a role in cell activation 2+ CCL2 (% Change MFI) P ZAP70 Ca -CAM G CXCL5 (% Change MFI) q CD14 6.24E-03 Expressed by myeloid cells 0 0 ● A2AR agonists induce a specific gene signature in human immune cells. This “Adenosine Signature” is dominated A2AR - 0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 M1 M2 VEGF by inflammatory myeloid cytokines and chemokines that signal through CXCR2. Adenosine IL-6 NECA Concentration (μM) NECA Concentration (μM) AP1 ADENOSINE SIGNATURE - RNAseq 600 500 ● Adenosine induction of these genes dampens T cell immunity and shifts the balance away from T effector responses A2AR Fibrosis Adjusted p Value Function Receptor CD14+ Monocytes Angiogenesis and toward myeloid suppressor functions. Activation CXCL2 0.008 MIP2a: macrophage inflammatory protein 2, alpha CXCR2 CD8+ T Cells 400 + CD25 IL-23 0.008 Neutrophil attraction w/ IL-17, reduces CD8+ T cell infiltration CD19 B Cells CSF3 0.011 G-CSF. Master regulator of neutrophil development 400 ● CPI-444 blocks the induction of the Adenosine Signature by A2AR agonists in vitro. Decreased IL-2 production 300 CD4 Promotes tumor supporting M2 phenotype CXCL3 0.011 Controls migration and adhesion of monocytes CXCR2 Increased CTLA-4 and PD-1 expression ● Expression of the “Adenosine Signature” correlates with tumor regression in Corvus’ ongoing Ph 1/1b trial with Decreased proliferation Increased angiogenesis HAS1 0.014 Hyaluronan synthase 1. ECM component Increased FOXP3 200 CPI-444 treatment in RCC. Patients with high expression of the Adenosine Signature were more likely to have tumor INHBA 0.016 Inhibin, beta a. Subunit of activin & inhibin 200 CXCL5 0.019 Attracts and activates neutrophils. CXCR2 regression than those patients with low expression 100 IL-8 (% Change MFI) • Extracellular adenosine within the tumor microenvironment create an immunosuppressive niche that promotes PTGS2 0.019 COX-2. Elevated during inflammation and cancer CXCL1 (% Change MFI) tumor growth and metastasis. PADI2 0.019 Protein-arginine deiminase type-2. Neurodegeneration ● The Adenosine Signature may be used as a predictive biomarker that can be used to select patients most likely to 0 0 CD93 0.023 C1qRp. Expressed on neutrophils. Clearance of apoptotic cells 0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 0 0.1 1 10 respond to therapy with agents that antagonize adenosine production or signaling. • CPI-444 is a potent, selective inhibitor of the adenosine 2A receptor (A2AR). SCL7A7 0.024 Uptake of dibasic and neutral amino acids NECA Concentration (μM) NECA Concentration (μM) PID1 0.024 Phosphotyrosine Interaction Domain-Containing Protein 1 • Extracellular adenosine has a short half life and it is not feasible to routinely measure in human tumors. ● Neutralization of the Adenosine Signature confirms the mechanism of action of CPI-444 as an A2AR antagonist. ECEL1 0.024 Endothelin-converting enzyme-like 1 CD300E 0.028 Expressed on myeloid cells. Interacts with TYRO • This study aims to determine genes/proteins modulated by adenosine as surrogate signature to identify patients 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 ● Further data along with an update on clinical results will be presented at the Society of Immunotherapy of Cancer 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+ with adenosine rich tumors ST6GALNAC2 0.028 Sialyltransferase 2 B cells had minimal changes. These results indicate that the source of adenosine signature chemokines is likely to be of monocytic lineage (SITC) meeting in November 2018. Identification of Adenosine Pathway Genes Associated with Response to Therapy with the Adenosine Receptor Antagonist CPI-444

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.

• 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