Search

Adenosine signaling through A2A receptors (A2AR) expressed on immune cells suppresses antitumor immunity. CPI-444 is a potent, selective, oral A2AR antagonist. Blockade of A2AR with CPI-444 restored T-cell signaling, IL2, and IFNγ production that were suppressed by adenosine analogues in vitro. CPI-444 treatment led to dose-dependent inhibition of tumor growth in multiple syngeneic mouse tumor models. Concentrations of extracellular adenosine in the tumor microenvironment, measured using microdialysis, were approximately 100–150 nmol/L and were higher than corresponding subcutaneous tissue. Combining CPI-444 with anti–PD-L1 or anti–CTLA-4 treatment eliminated tumors in up to 90% of treated mice, including restoration of immune responses in models that incompletely responded to anti–PD-L1 or anti–CTLA-4 monotherapy. Tumor growth was fully inhibited when mice with cleared tumors were later rechallenged, indicating that CPI-444 induced systemic antitumor immune memory. CD8+ T-cell depletion abrogated the efficacy of CPI-444 with and without anti–PD-L1 treatment, demonstrating a role for CD8+ T cells in mediating primary and secondary immune responses. The antitumor efficacy of CPI-444 with and without anti–PD-L1 was associated with increased T-cell activation, a compensatory increase in CD73 expression, and induction of a Th1 gene expression signature consistent with immune activation. These results suggest a broad role for adenosine-mediated immunosuppression in tumors and justify the further evaluation of CPI-444 as a therapeutic agent in patients with solid tumors. Cancer Immunol Res; 6(10); 1136–49. ©2018 AACR.

Supplementary Data

Adenosine mediates immunosuppression within the tumor microenvironment through triggering adenosine 2A receptors (A2AR) on immune cells. To determine whether this pathway could be targeted as an immunotherapy, we performed a phase I clinical trial with a small-molecule A2AR antagonist. We find that this molecule can safely block adenosine signaling in vivo. In a cohort of 68 patients with renal cell cancer (RCC), we also observe clinical responses alone and in combination with an anti–PD-L1 antibody, including subjects who had progressed on PD-1/PD-L1 inhibitors. Durable clinical benefit is associated with increased recruitment of CD8+ T cells into the tumor. Treatment can also broaden the circulating T-cell repertoire. Clinical responses are associated with an adenosine-regulated gene-expression signature in pretreatment tumor biopsies. A2AR signaling, therefore, represents a targetable immune checkpoint distinct from PD-1/PD-L1 that restricts antitumor immunity.Significance:This first-in-human study of an A2AR antagonist for cancer treatment establishes the safety and feasibility of targeting this pathway by demonstrating antitumor activity with single-agent and anti–PD-L1 combination therapy in patients with refractory RCC. Responding patients possess an adenosine-regulated gene-expression signature in pretreatment tumor biopsies.See related commentary by Sitkovsky, p. 16.This article is highlighted in the In This Issue feature, p. 1

Supplemental Appendix

PDF - 35K, Circulating biomarker distribution across arms.

PDF file - 22K, Supplemental Fig. S3. Effect of cytoplasmic MET immunohistochemistry (IHC) staining on objective response rate in patients treated with panitumumab (pmab) plus rilotumumab (AMG 102) versus pmab plus placebo.

PDF - 43K

PDF file - 37K, Supplemental Fig. S4. The effect of baseline IGF and IGF-related protein expression on overall survival.

PDF - 74K

PDF file - 76K, Supplemental Fig. S1. (A) Randomized phase Ib/II trial of panitumumab plus rilotumumab (AMG 102) or ganitumab (AMG 479). (B) CONSORT diagram for part 2. *Panitumumab 6 mg/kg; rilotumumab 10 mg/kg with dose de-escalation to 5 mg/kg as necessary. �Panitumumab 6 mg/kg; rilotumumab 10 mg/kg; ganitumab 12 mg/kg. �Rilotumumab 10 mg/kg; ganitumab 12 mg/kg.

PDF file - 38K, Supplemental Fig. S5. The effect of baseline IGF and IGF-related protein expression on objective response rate.

Purpose: This phase 1b dose-escalation study assessed safety, tolerability, and pharmacokinetics of ganitumab, a fully human monoclonal antibody against the insulin-like growth factor 1 (IGF1) receptor, combined with targeted agents or cytotoxic chemotherapy in patients with advanced solid tumors.Experimental Design: Patients with treatment-refractory advanced solid tumors were sequentially enrolled at 2 ganitumab dose levels (6 or 12 mg/kg i.v. every 2 weeks) combined with either sorafenib 400 mg twice daily, panitumumab 6 mg/kg every 2 weeks, erlotinib 150 mg once daily, or gemcitabine 1,000 mg/m2 on days 1, 8, and 15 of each 4-week cycle. The primary end points were safety and pharmacokinetics of ganitumab.Results: Ganitumab up to 12 mg/kg appeared well tolerated combined with sorafenib, panitumumab, erlotinib, or gemcitabine. Treatment-emergent adverse events were generally mild and included fatigue, nausea, vomiting, and chills. Three patients had dose-limiting toxicities: grade 3 hyperglycemia (ganitumab 6 mg/kg and panitumumab), grade 4 neutropenia (ganitumab 6 mg/kg and gemcitabine), and grade 4 thrombocytopenia (ganitumab 12 mg/kg and erlotinib). Ganitumab-binding and panitumumab-binding antibodies were detected in 5 and 2 patients, respectively; neutralizing antibodies were not detected. The pharmacokinetics of ganitumab and each cotherapy did not appear affected by coadministration. Circulating total IGF1 and IGF binding protein 3 increased from baseline following treatment. Four patients (9%) had partial responses.Conclusions: Ganitumab up to 12 mg/kg was well tolerated, without adverse effects on pharmacokinetics in combination with either sorafenib, panitumumab, erlotinib, or gemcitabine. Ganitumab is currently under investigation in combination with some of these and other agents. Clin Cancer Res; 18(12); 3414–27. ©2012 AACR.

PDF file - 38K, Supplemental Fig. S2. Best percent change of the sum of longest diameters of target lesions from baseline to post-baseline. (A) Panitumumab plus rilotumumab.(B) Panitumumab plus ganitumab. (C) Panitumumab alone.

Purpose: Panitumumab, a fully human anti-epidermal growth factor receptor monoclonal antibody (mAb), has demonstrated efficacy in patients with wild-type KRAS metastatic colorectal cancer (mCRC). Rilotumumab and ganitumab are investigational, fully human mAbs against hepatocyte growth factor (HGF)/scatter factor and IGF1R, respectively. Here we evaluate combining rilotumumab or ganitumab with panitumumab in previously treated patients with wild-type KRAS mCRC.Experimental Design: Part 1 was a phase Ib dose-finding study of panitumumab plus rilotumumab. The primary endpoint was the incidence of dose-limiting toxicities (DLT). Part 2 was a randomized phase II trial of panitumumab in combination with rilotumumab, ganitumab, or placebo. The primary endpoint was objective response rate (ORR); safety, progression-free survival (PFS), and overall survival (OS) were secondary endpoints. Archival tissue specimens were collected for exploratory correlative work.Results: In part 1, no DLTs were reported. A recommended phase II dose of 10 mg/kg rilotumumab was selected. In part 2, for the panitumumab plus rilotumumab (n = 48), panitumumab plus ganitumab (n = 46), and panitumumab plus placebo arms (n = 48), the ORRs were 31%, 22%, and 21%, respectively. The median PFS was 5.2, 5.3, and 3.7 months and median OS 13.8, 10.6, and 11.6 months, respectively. Adverse events were tolerable. Exploratory biomarker analyses, including MET and IGF-related protein expression, failed to indicate conclusive predictive evidence on efficacy endpoints.Conclusions: Panitumumab plus rilotumumab met the prespecified criterion for improvement in ORR whereas ganitumab did not. This is the first study to suggest a benefit for combining an HGF inhibitor (rilotumumab) with panitumumab in previously treated patients with wild-type KRAS mCRC. Clin Cancer Res; 20(16); 4240–50. ©2014 AACR.

Supplementary Table S1 (Multivariate analysis for circulating biomarkers.) Supplementary Table S2 (Summary of KRAS mutational status in the ganitumab arm.) Supplementary Table S3 (Association of KRAS mutational status with OS in the ganitumab arm.) Supplementary Table S4 (Summary of PTEN expression in the ganitumab arm.) Supplementary Table S5 (Association of PTEN expression with OS in the ganitumab arm.)

PDF - 26K, Receiver Operator Characteristic Analysis for circulating biomarkers.

PDF file, 93KB, Supplemental Table 1. Study Drug Administration; Supplemental Table 2. Summary of Somatic Mutation Status and Tumor PTEN Expression Versus Best Result Change in Tumor Dimensions.

Purpose: This planned exploratory analysis assessed the predictive nature of baseline circulating factors of the insulin-like growth factor (IGF) axis on the treatment effect of ganitumab (monoclonal antibody inhibitor of IGF-1 receptor) plus gemcitabine in a randomized phase II study in metastatic pancreatic adenocarcinoma.Experimental Design: Baseline levels of IGFs/IGF binding proteins (IGFBP) were analyzed in serum or plasma. Mutations and gene expression were analyzed in archival samples. Treatment effects between biomarker subgroups were compared for overall survival (OS). Associations of tumor markers with OS were evaluated.Results: For patients with evaluable samples, ganitumab was associated with improved OS versus placebo (HR, 0.49; 95% CI: 0.28–0.87). The treatment effect on improved OS was strong in the patient subset with higher levels of IGF-1, IGF-2, or IGFBP-3, or lower levels of IGFBP-2, but not so on the other corresponding subset. Median OS of ganitumab versus placebo in patients with higher levels of IGF-1, IGF-2, and IGFBP-3 was 16 versus 6.8 months (HR, 0.25; 95% CI: 0.09–0.67), 16 versus 5.9 months (HR, 0.24; 95% CI: 0.09–0.68), and 16 versus 6.8 months (HR, 0.28; 95% CI: 0.11–0.73), and in patients with lower IGFBP-2 levels was 12.7 versus 6.6 months (HR, 0.19; 95% CI: 0.07–0.55). Interaction between treatment and IGFs/IGFBPs in multivariate analyses suggested predictive potential for IGF-2 (P = 0.002) and IGFBP-2 (P = 0.02). KRAS mutation status and PTEN expression were not associated with OS.Conclusions: Baseline circulating factors of the IGF axis may predict OS benefit from ganitumab plus gemcitabine in metastatic pancreatic adenocarcinoma. Clin Cancer Res; 19(15); 4282–9. ©2013 AACR.

PDF file - 86K

PDF file, 86KB.

Supplemental Digital Content is available in the text., Cancer immunotherapies, such as atezolizumab, are proving to be a valuable therapeutic strategy across indications, including non–small cell lung cancer (NSCLC) and urothelial cancer (UC). Here, we describe a diagnostic assay that measures programmed-death ligand 1 (PD-L1) expression, via immunohistochemistry, to identify patients who will derive the most benefit from treatment with atezolizumab, a humanized monoclonal anti-PD-L1 antibody. We describe the performance of the VENTANA PD-L1 (SP142) Assay in terms of specificity, sensitivity, and the ability to stain both tumor cells (TC) and tumor-infiltrating immune cells (IC), in NSCLC and UC tissues. The reader precision, repeatability and intermediate precision, interlaboratory reproducibility, and the effectiveness of pathologist training on the assessment of PD-L1 staining on both TC and IC were evaluated. We detail the analytical validation of the VENTANA PD-L1 (SP142) Assay for PD-L1 expression in NSCLC and UC tissues and show that the assay reliably evaluated staining on both TC and IC across multiple expression levels/clinical cut-offs. The reader precision showed high overall agreement when compared with consensus scores. In addition, pathologists met the predefined training criteria (≥85.0% overall percent agreement) for the assessment of PD-L1 expression in NSCLC and UC tissues with an average overall percent agreement ≥95.0%. The assay evaluates PD-L1 staining on both cell types and is robust and precise. In addition, it can help to identify those patients who may benefit the most from treatment with atezolizumab, although treatment benefit has been demonstrated in an all-comer NSCLC and UC patient population.

Adenosine signaling through A2A receptors (A2AR) expressed on immune cells suppresses antitumor immunity. CPI-444 is a potent, selective, oral A2AR antagonist. Blockade of A2AR with CPI-444 restored T-cell signaling, IL2, and IFNγ production that were suppressed by adenosine analogues in vitro. CPI-444 treatment led to dose-dependent inhibition of tumor growth in multiple syngeneic mouse tumor models. Concentrations of extracellular adenosine in the tumor microenvironment, measured using microdialysis, were approximately 100–150 nmol/L and were higher than corresponding subcutaneous tissue. Combining CPI-444 with anti–PD-L1 or anti–CTLA-4 treatment eliminated tumors in up to 90% of treated mice, including restoration of immune responses in models that incompletely responded to anti–PD-L1 or anti–CTLA-4 monotherapy. Tumor growth was fully inhibited when mice with cleared tumors were later rechallenged, indicating that CPI-444 induced systemic antitumor immune memory. CD8+ T-cell depletion abrogated the efficacy of CPI-444 with and without anti–PD-L1 treatment, demonstrating a role for CD8+ T cells in mediating primary and secondary immune responses. The antitumor efficacy of CPI-444 with and without anti–PD-L1 was associated with increased T-cell activation, a compensatory increase in CD73 expression, and induction of a Th1 gene expression signature consistent with immune activation. These results suggest a broad role for adenosine-mediated immunosuppression in tumors and justify the further evaluation of CPI-444 as a therapeutic agent in patients with solid tumors. Cancer Immunol Res; 6(10); 1136–49. ©2018 AACR.

Adenosine mediates immunosuppression within the tumor microenvironment through triggering adenosine 2A receptors (A2AR) on immune cells. To determine whether this pathway could be targeted as an immunotherapy, we performed a phase I clinical trial with a small-molecule A2AR antagonist. We find that this molecule can safely block adenosine signaling in vivo. In a cohort of 68 patients with renal cell cancer (RCC), we also observe clinical responses alone and in combination with an anti–PD-L1 antibody, including subjects who had progressed on PD-1/PD-L1 inhibitors. Durable clinical benefit is associated with increased recruitment of CD8+ T cells into the tumor. Treatment can also broaden the circulating T-cell repertoire. Clinical responses are associated with an adenosine-regulated gene-expression signature in pretreatment tumor biopsies. A2AR signaling, therefore, represents a targetable immune checkpoint distinct from PD-1/PD-L1 that restricts antitumor immunity. Significance: This first-in-human study of an A2AR antagonist for cancer treatment establishes the safety and feasibility of targeting this pathway by demonstrating antitumor activity with single-agent and anti–PD-L1 combination therapy in patients with refractory RCC. Responding patients possess an adenosine-regulated gene-expression signature in pretreatment tumor biopsies. See related commentary by Sitkovsky, p. 16. This article is highlighted in the In This Issue feature, p. 1

Introduction The Blueprint Programmed Death Ligand 1 (PD-L1) Immunohistochemistry (IHC) Assay Comparison Project is an industrial-academic collaborative partnership to provide information on the analytical and clinical comparability of four PD-L1 IHC assays used in clinical trials. Methods A total of 39 NSCLC tumors were stained with four PD-L1 IHC assays (22C3, 28-8, SP142, and SP263), as used in the clinical trials. Three experts in interpreting their respective assays independently evaluated the percentages of tumor and immune cells staining positive at any intensity. Clinical diagnostic performance was assessed through comparisons of patient classification above and below a selected expression cutoff and by agreement using various combinations of assays and cutoffs. Results Analytical comparison demonstrated that the percentage of PD-L1–stained tumor cells was comparable when the 22C3, 28-8, and SP263 assays were used, whereas the SP142 assay exhibited fewer stained tumor cells overall. The variability of immune cell staining across the four assays appears to be higher than for tumor cell staining. Of the 38 cases, 19 (50.0%) were classified above and five (13%) were classified below the selected cutoffs of all assays. For 14 of the 38 cases (37%), a different PD-L1 classification would be made depending on which assay/scoring system was used. Conclusions The Blueprint PD-L1 IHC Assay Comparison Project revealed that three of the four assays were closely aligned on tumor cell staining whereas the fourth showed consistently fewer tumor cells stained. All of the assays demonstrated immune cell staining, but with greater variability than with tumor cell staining. By comparing assays and cutoffs, the study indicated that despite similar analytical performance of PD-L1 expression for three assays, interchanging assays and cutoffs would lead to "misclassification" of PD-L1 status for some patients. More data are required to inform on the use of alternative staining assays upon which to read different specific therapy-related PD-L1 cutoffs.

JNJ-4528 is a chimeric antigen receptor T cell (CAR-T) therapy containing two B-cell maturation antigen (BCMA)-targeting single-domain antibodies designed to confer avidity. We present translational data from the phase 1b cohort of an ongoing phase 1b/2 study conducted in the US to explore the safety and efficacy of JNJ-4528 in patients with R/R MM (NCT03548207). Apheresis samples were collected from 25 patients, and T cells were selected and transduced with a lentivirus encoding the BCMA CAR construct. Immune cell composition of both the apheresis samples and transduced cells following expansion ("drug product" [DP]) was evaluated by multiparametric flow cytometry. The median proportion of CAR+ T cells in the DP was 16% (range 6-28%) of total cells, with a median proportion of 12% (range 4-22%) CD4+ CAR+ and 7% (range 3-20%) CD8+ CAR+ T cells. The CD4:CD8 ratio of CAR+ T cells in the DP was variable between patients, with a median of 1.5 (range 0.62-4.40). Significant variability between patients was observed in the composition of the DP regarding T cell subsets (i.e., naïve, Tscm, Tcm, Tem, Teff, Temra) although this profile was comparable in the CAR- and CAR+ T cell subpopulations within each patient. Patients were administered a single infusion of the JNJ-4528 DP at a target dose of 0.75x106 CAR+ T cells/kg (target range 0.5-1.0x106). Of the 21 patients with a postbaseline disease evaluation, the overall response rate was 91% at a median follow-up of 3 months (range 1-10). Among the 15 patients with post-infusion day 28 evaluable bone marrow (BM) samples by next generation flow cytometry and/or next generation sequencing, 10 were minimal residual disease negative at the 10-5 level of sensitivity, 2 at 10-4 level of sensitivity, and 3 had unidentified clones. All patients expressed BCMA in BM tumor cells at baseline, as assessed by flow cytometry, although levels varied among patients. Clinical responses appear independent of BM BCMA expression. Following infusion, CAR+ T cells expanded reaching a peak between 20-87% of the total T cells in blood between days 10-14 post-infusion. The CD4:CD8 ratio and the proportion of T cell memory subsets in the final DP did not correlate with peak CAR+ T cell expansion. Peak CAR+ T cell expansion did not correlate with response. Ten patients had a follow-up of enumeration of CAR+ T cells in blood of at least 8 weeks. In 5 patients, CAR+ T cells decreased to below the limit of quantification (BLOQ) of the flow cytometry assay (5 cells/µl) within 8 weeks (range 2-7) post-infusion. Although preliminary, no difference was observed in the response rate between these patients compared with patients with measurable CAR+ T cells after 8 weeks. A similar trend was observed when expansion and persistence were assessed by measuring transgene levels. While both CD4+ and CD8+ CAR+ T cells expanded in vivo, the CD4:CD8 CAR+ ratio decreased at peak expansion compared with the final DP (from a median of 1.35 to 0.35), indicating a preferential expansion of CD8+ CAR+ T cells in blood. At peak expansion, CD8+ CAR+ T cells showed predominantly a central memory (Tcm) phenotype (CCR7+ CD45RO+; median of 90% [range 29.3-98.5%]). In contrast, CD4+ CAR+ T cells were enriched in effector memory (Tem) cells (CCR7- CD45RO+; median of 87% [range 69.5-98.1%]) at peak expansion. A similar trend in the CD4:CD8 ratio, as well as the T cell memory subset composition, was observed in BM of all 11 patients with evaluable samples at day 28. CD8+ CAR- T cells showed an approximate 50:50 ratio of stem memory (Tscm):Tcm subsets while CD4+ CAR- T cells showed an approximate 50:50 ratio of Tcm:Tem subsets, indicating a differential T cell maturation course for CD4+ and CD8+ CAR+ and CAR- T cells. Expansion of CAR+ T cells correlated with increases in serum cytokines levels (i.e., IL-6, IFNγ, IL-10) which peaked around day 10, coinciding with maximal expansion of CAR+ T cells. Generally, increases in some proinflammatory cytokines (i.e., IL-6) correlated with onset of cytokine release syndrome symptoms (median time to onset of 7 days [range 2-12]). Findings from the ongoing CARTITUDE-1 trial suggest that JNJ-4528 is a differentiated CAR-T cell therapy that is highly active at a relatively low dose, potentially related to a preferential and consistent in vivo expansion of CD8+ CAR+ T cells displaying a central memory phenotype. Disclosures Zudaire: Janssen R&D: Employment, Equity Ownership. Madduri:Foundation Medicine: Consultancy; Takeda: Consultancy; AbbVie: Consultancy; Celgene: Consultancy. Usmani:Bristol-Myers Squibb: Consultancy, Research Funding; Pharmacyclics: Patents & Royalties, Research Funding; Celgene: Consultancy, Patents & Royalties, Research Funding, Speakers Bureau; Janssen: Consultancy, Patents & Royalties, Research Funding, Speakers Bureau; Merck: Consultancy, Research Funding; Skyline DX: Consultancy; Amgen: Consultancy, Patents & Royalties, Research Funding, Speakers Bureau; Array Biopharma: Patents & Royalties, Research Funding; Sanofi: Patents & Royalties, Research Funding, Speakers Bureau; Takeda: Consultancy, Patents & Royalties, Research Funding, Speakers Bureau. Jakubowiak:Adaptive Biotechnologies: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; SkyLineDx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; KaryoPharm Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Juno: Consultancy, Honoraria. Berdeja:AbbVie Inc, Amgen Inc, Acetylon Pharmaceuticals Inc, Bluebird Bio, Bristol-Myers Squibb Company, Celgene Corporation, Constellation Pharma, Curis Inc, Genentech, Glenmark Pharmaceuticals, Janssen Biotech Inc, Kesios Therapeutics, Lilly, Novartis, Poseida: Research Funding; Poseida: Research Funding; Amgen Inc, BioClinica, Celgene Corporation, CRISPR Therapeutics, Bristol-Myers Squibb Company, Janssen Biotech Inc, Karyopharm Therapeutics, Kite Pharma Inc, Prothena, Servier, Takeda Oncology: Consultancy. Geng:Legend Biotech: Employment, Equity Ownership. Rizvi:Legend Biotech: Employment, Equity Ownership. Nesheiwat:Legend Biotech: Employment, Equity Ownership. Schecter:Janssen R&D, LLC: Employment, Equity Ownership. Goldberg:Janssen R&D: Employment, Equity Ownership. Banerjee:Janssen R&D: Employment, Equity Ownership. Allred:Janssen R&D: Employment, Equity Ownership. Singh:Janssen R&D: Employment, Equity Ownership. Verona:Janssen R&D: Employment, Equity Ownership. McCaffery:Janssen R&D: Employment, Equity Ownership. Jagannath:Celgene Corporation: Consultancy; AbbVie: Consultancy; Bristol-Myers Squibb: Consultancy; Karyopharm Therapeutics: Consultancy; Janssen Pharmaceuticals: Consultancy; Merck & Co.: Consultancy.

Background: The androgen receptor (AR) is a clinically important driver in prostate cancer. In metastatic castration resistant prostate cancer (mCRPC), increased expression of the ligand-independent AR variant 7 (AR-v7) is a biomarker of hormonal therapy resistance. However, the prevalence and clinical importance of AR-v7 in non-metastatic CRPC (nmCRPC) is not yet known. Low circulating tumor cell (CTC) frequencies in these patients make emergence of AR-v7 during anti-androgen therapy difficult to study. We report blood-based detection of AR-v7 using digital droplet PCR (ddPCR) in nmCRPC patients enrolled in the SPARTAN trial, a randomized phase 3 study testing ADT vs apalutamide (APA)+ADT. Method: To investigate simultaneous and quantitative expression of AR-v7 and AR, we utilized ddPCR to measure individual mRNA transcripts in blood samples collected in PAXgene tubes. AR-v7 positivity was calculated as the normalized fraction of AR-v7 vs total AR transcripts (AR-v7/AR). Normalized AR-v7/AR frequency in healthy volunteers (HV) and mCRPC was measured to determine an expression cutoff to separate normal and prostate cancer blood samples. The ddPCR AR-v7 biomarker assay was then used to measure AR-v7 expression in ADT (N=47) and APA+ADT (N=53) SPARTAN samples taken at time of study initiation and correlated with clinical outcome. Results: By setting a cutoff at 0.3 AR-v7/AR normalized fraction we could differentiate HV from mCRPC patients. In nmCRPC, mean AR-v7 and AR-FL expression were calculated as 1.2 and 349.3 transcripts, respectively. The 0.3 AR-v7/AR normalized fraction cutoff could not differentiate AR-v7 expression between HV and nmCRPC. Using this assay, we detected AR-v7 transcripts in 47% of nmCRPC SPARTAN patients analyzed. However, results of AR-v7 expression as a continuous and discretized variable were inconclusive when correlated with clinical outcome. Conclusion: This study reports ddPCR-based detection of whole blood mRNA as a sensitive assay to detect simultaneously low and high expressing AR transcripts in nmCRPC. Our technical analysis demonstrates that unlike in mCRPC, low level transcript counts of AR-v7 in nmCRPC may not distinguish expression from baseline in healthy patients. Data from this limited cohort suggest that while AR-v7 is detected in 47% of patients, a higher threshold of expression may be biologically important for driving treatment resistance. Further analysis of this assay in mCRPC and APA refractory samples sequenced with other therapies are needed to confirm the clinical and biological utility of AR-v7 detection by ddPCR assay and inform disease continuum management. Citation Format: Mel Pilar Espaillat, Yashoda Rajpurohit, Mike Gormley, Denis Smirnov, Ian McCaffery, Angela Lopez-Gitlitz, Deborah Ricci, Shibu Thomas. Digital droplet PCR (ddPCR)-based detection of androgen receptor splice variant 7 (AR-v7) in non-metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1335.

420 Background: Plasma samples from a Phase 2 study of the pan-FGFR inhibitor erdafitinib in advanced UC patients (pts) with FGFR mutations (mut) or fusions, were tested using next generation sequencing (NGS) for circulating tumor DNA (ctDNA), and results compared to central FGFR status determination from tissue. Methods: FGFR alterations were measured in archival tissue using an RNA-based RT-PCR test and compared with FGFR alterations identified in pre-treatment plasma specimens using the Guardant360 ctDNA test. Sensitivity of the ctDNA test to detect the FGFR alterations identified by RT-PCR from tissue, the proportion of pts with a study-eligible FGFR alteration in ctDNA, and the ability to detect a tissue FGFR alteration in ctDNA in relation to clinical response were assessed. Results: Samples from 155 pts with detectable baseline ctDNA were included. Overall, concordance between ctDNA and tissue-based FGFR results was 56% (87/155): 63% (77/122) for tissue mut-positive pts vs 24% (7/29) in fusion-positive pts. 61% of pts (94/155) were positive for a study-eligible FGFR alteration from blood-based testing in the tissue-positive population. The response rate was 47% (36/77) for patients for which FGFR mutations could be detected in blood (ctDNA- FGFR-positive) compared with 32% (14/44) in patients for which mutations were not detected in blood (ctDNA- FGFR-negative), with an estimated odds ratio of 1.882 (95% CI: 0.866; 4.090) Conclusions: The 63% concordance rate for detecting FGFR mut in temporally unmatched blood and tissue supports potential for patient selection with blood-based testing, while ctDNA FGFR fusion detection may require further optimization. Although differences in clinical response rate were observed between ctDNA- FGFR-positive and negative patients, the results were not statistically significant. Importantly, a proportion of ctDNA-negative pts responded to erdafitinib, indicating that tissue-based testing may be warranted for pts negative for FGFR alterations via blood-based testing. (BLC2001, NCT02365597). Clinical trial information: NCT02365597.

Ganitumab is an investigational, fully human monoclonal antibody antagonist of the insulin-like growth factor-1 receptor (IGF1R) that has shown trends towards improved progression-free survival and overall survival in a phase 2 pancreatic cancer clinical trial. To characterize ganitumab pharmacokinetics (PK) and identify factors affecting PK, ganitumab serum concentration data from three clinical trials were analyzed. The PK of ganitumab as monotherapy and in combination with gemcitabine in patients with pancreatic or non-pancreatic cancer were assessed with a non-linear mixed-effect model. We found that ganitumab exhibited linear and time-invariant kinetics. A two-compartment model adequately described data over a dose range of 1-20 mg/kg with good predictive capability. Typical clearance and central volume of distribution values were 1.7- and 1.3-fold higher, respectively, in patients with pancreatic cancer than in patients with other advanced solid cancers, resulting in lower ganitumab exposure. Covariate analysis was used to evaluate effects of cancer type, gemcitabine coadministration, clinical study, demographics, and laboratory values on ganitumab PK. Pancreatic cancer type was the most significant covariate on clearance along with weight, albumin, and serum creatinine. Gemcitabine coadministration did not affect ganitumab clearance. Thus, disease state can significantly affect PK and should be considered when selecting the clinically effective dose.

Summary Background Insulin-like growth factors (IGF-1 and IGF-2) bind to the IGF-1 receptor (IGF-1R), increasing cell proliferation and survival. Ganitumab is a monoclonal IgG1 antibody that blocks IGF-1R. We tested the efficacy and safety of adding ganitumab to endocrine treatment for patients with hormone-receptor-positive breast cancer. Methods We did this phase 2 trial in outpatient clinics and hospitals. We enrolled postmenopausal women with hormone-receptor-positive, locally advanced or metastatic breast cancer previously treated with endocrine treatment. They were randomly assigned (2:1) with a central randomisation schedule to receive intravenous ganitumab 12 mg per kg bodyweight or placebo in combination with open-label intramuscular fulvestrant (500 mg on day 1, then 250 mg on days 15, 29, and every 28 days) or oral exemestane (25 mg once daily) on a 28-day cycle. Patients, investigators, study monitors, and the sponsor staff were masked to treatment allocation. Response was assessed every 8 weeks. The primary endpoint was median progression-free survival in the intention-to-treat population. We analysed overall survival as one of our secondary endpoints. The study is registered at ClinicalTrials.gov, number NCT00626106. Findings We screened 189 patients and enrolled 156 (106 in the ganitumab group and 50 in the placebo group). Median progression-free survival did not differ significantly between the ganitumab and placebo groups (3·9 months, 80% CI 3·6–5·3 vs 5·7 months, 4·4–7·4; hazard ratio [HR] 1·17, 80% CI 0·91–1·50; p=0·44). However, overall survival was worse in the the ganitumab group than in the placebo group (HR 1·78, 80% CI 1·27–2·50; p=0·025). With the exception of hyperglycaemia, adverse events were generally similar between groups. The most common grade 3 or higher adverse event was neutropenia—reported by six of 106 (6%) patients in the ganitumab group and one of 49 (2%) in the placebo group. Hyperglycaemia was reported by 12 of 106 (11%) patients in the ganitumab group (with six patients having grade 3 or 4 hyperglycaemia) and none of 49 in the placebo group. Serious adverse events were reported by 27 of 106 (25%) patients in the ganitumab group and nine of 49 (18%) patients in the placebo group. Interpretation Addition of ganitumab to endocrine treatment in women with previously treated hormone-receptor-positive locally advanced or metastatic breast cancer did not improve outcomes. Our results do not support further study of ganitumab in this subgroup of patients. Funding Amgen.

Importance Dysregulation of the mesenchymal-epithelial transition (MET) signaling pathway is associated with poor prognosis in gastroesophageal adenocarcinoma (GEC). We report results of METGastric, a phase 3 trial of the MET inhibitor onartuzumab plus standard first-line chemotherapy for human epidermal growth factor receptor 2 (HER2)-negative, MET-positive, advanced GEC. Objective To determine whether the addition of onartuzumab to first-line fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) improves efficacy compared with mFOLFOX6 plus placebo in HER2-negative, MET-positive GEC. Design, Setting, and Participants Randomized, double-blind, multicenter trial conducted from November 2012 to March 2014. Patients were 18 years or older with an adenocarcinoma of the stomach or gastroesophageal junction with metastatic disease not amenable for curative therapy. Tumor samples were centrally tested for MET expression using Ventana anti-Total c-MET (SP44) rabbit monoclonal antibody, HER2 status, and Lauren histologic subtype. MET-positive tumors were defined as at least 50% of tumor cells showing weak, moderate, and/or strong staining intensity (MET 1+/2+/3+, respectively) by immunohistochemistry. Interventions Patients with HER2-negative, MET-positive GEC were enrolled and randomized 1:1 to receive mFOLFOX6 with or without onartuzumab (10 mg/kg). Main Outcomes and Measures Co–primary end points: overall survival in the intent-to-treat (ITT) population and in patients with MET 2+/3+ GEC. Secondary end points: progression-free survival (PFS), overall response rate (ORR), and safety. Results Enrollment was stopped early due to sponsor decision, which was agreed with an independent data monitoring committee. At the data cutoff (April 25, 2014) there were 562 patients in the ITT population (n = 283 placebo plus mFOLFOX6 [median age, 58 y; 65% male]; n = 279 onartuzumab plus mFOLFOX6 [median age, 60 y; 67% male]); 109 (38.5%) and 105 (37.6%) of the ITT population were MET 2+/3+, respectively. Addition of onartuzumab to mFOLFOX6 did not significantly improve OS, PFS, or ORR vs placebo plus mFOLFOX6 in the ITT (OS hazard ratio [HR], 0.82; 95% CI, 0.59-1.15; P = .24; PFS HR, 0.90; 95% CI, 0.71-1.16; P = .43; ORR, 46.1% vs 40.6%) or MET 2+/3+ populations (OS HR, 0.64; 95% CI, 0.40-1.03; P = .06; PFS HR, 0.79; 95% CI, 0.54-1.15; P = .22; ORR, 53.8% vs 44.6%). Safety was as expected for onartuzumab. Conclusions and Relevance Addition of onartuzumab to first-line mFOLFOX6 did not significantly improve clinical benefits in the ITT or MET 2+/3+ populations. Trial Registration clinicaltrials.gov Identifier:NCT01662869

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Purpose Ganitumab is a fully human monoclonal antibody against type-1 insulin-like growth factor receptor (IGF1R). An open-label phase II study was conducted to evaluate the efficacy and safety of ganitumab monotherapy in patients with metastatic Ewing family tumors (EFT) or desmoplastic small round cell tumors (DSRCT). Patients and Methods Patients ≥16 years of age with relapsed or refractory EFT or DSRCT received 12 mg/kg of ganitumab every 2 weeks. Objective response rate (ORR) was the primary end point. Secondary end points included clinical benefit rate (CBR = complete + partial responses + stable disease [SD] ≥ 24 weeks) and safety and pharmacokinetic profiles of ganitumab. The relationship between tumor response and EWS gene translocation status and IGF-1 levels was evaluated. Results Thirty-eight patients (22 with EFT; 16 with DSRCT) received one or more doses of ganitumab. Twenty-four patients (63%) experienced ganitumab-related adverse events. Grade 3 related events included hyperglycemia (n = 2), thrombocytopenia (n = 5), neutropenia (n = 2), leukopenia (n = 1), and transient ischemic attack (n = 1). There were no grade 4 or 5 treatment-related events. Of 35 patients assessed for response, two had partial responses (ORR, 6%) and 17 (49%) had SD. Four patients had SD ≥ 24 weeks, contributing to a CBR of 17%. The pharmacokinetic profile of ganitumab was similar to that observed in the first-in-human trial. Elevation of IGF-1 levels was observed postdose. EWS-Fli1 translocations were analyzed by RNA sequencing and fluorescent in situ hybridization, and novel translocations were observed in EFT and DSCRT. No apparent relationship between tumor response and IGF-1 levels or EWS gene translocations was observed. Conclusion Ganitumab was well tolerated and demonstrated antitumor activity in patients with advanced recurrent EFT or DSRCT.

Erythropoiesis stimulating agents (ESAs) have been reported to activate erythropoietin receptors (EpoR) on cell types, including endothelial, neuronal, renal tubule, and cardiac cells. ESAs have also been reported to promote angiogenesis. However, those findings are controversial and confounded by methodologic issues. We show that EpoR mRNA was detected in essentially all cell types examined, including primary human endothelial, renal, cardiac, and neuronal cells but 10- to 100-fold lower than Epo-responsive cells using quantitative reverse-transcribed polymerase chain reaction. Total endothelial EpoR protein examined using a new monoclonal antibody was low to undetectable. Surface EpoR on endothelial cells was not detected using [125I]-rHuEpo surface-binding studies. There was no evidence of ESA-induced intracellular signaling in endothelial cells. There was a similar lack of EpoR expression and signaling in other cell types examined. Experiments were performed examining ESA function on these cells. An in vivo rat corneal angiogenesis assay demonstrated neo-vessel formation in response to recombinant human vascular endothelial growth factor (rHuVEGF). However, recombinant mouse Epo did not induce vessel formation. Similarly, ESAs did not reproducibly provide cytoprotection to neuronal, renal, or cardiac cells. Taken together, our data challenge the notion of presence or function of EpoR on nonhematopoietic cells, and call into question the preclinical basis for clinical studies exploring direct, “pleiotropic” actions of ESAs.

Certain oncology trials showed worse clinical outcomes in the erythropoiesis-stimulating agent (ESA) arm. A potential explanation was that ESA-activated erythropoietin (Epo) receptors (EpoRs) promoted tumor cell growth. Although there were supportive data from preclinical studies, those findings often used invalidated reagents and methodologies and were in conflict with other studies. Here, we further investigate the expression and function of EpoR in tumor cell lines. EpoR mRNA levels in 209 human cell lines representing 16 tumor types were low compared with ESA-responsive positive controls. EpoR protein production was evaluated in a subset of 66 cell lines using a novel anti-EpoR antibody. EpoR+ control cells had an estimated 10 000 to 100 000 EpoR dimers/cell. In contrast, 54 of 61 lines had EpoR protein levels lower than 100 dimers/cell. Cell lines with the highest EpoR protein levels (400-3200 dimers/cell) were studied further, and, although one line, NCI-H661, bound detectable levels of [125I]–recombinant human Epo (rHuEpo), none showed evidence of ESA-induced EpoR activation. There was no increased phosphorylation of STAT5, AKT, ERK, or S6RP with rHuEpo. In addition, EpoR knockdown with siRNAs did not affect viability in 2 cell lines previously reported to express functional EpoR (A2780 and SK-OV-3). These results conflict with the hypothesis that EpoR is functionally expressed in tumors.

Background: Tumor biopsies obtained from patients are often limited in size and availability, and the ability to perform multiple diagnostic assays depends on the quantity and quality of the tissue. Here we describe and evaluate a method for performing DNA-based mutational analyses after immunohistochemistry analysis has been performed, using a single tissue section. Method: Immunohistochemistry analysis was performed on 4-5 μm formalin-fixed paraffin-embedded tumor tissue sections and immunohistochemistry-stained sections were stored for subsequent genomic analysis. DNA was isolated from these immunohistochemistry-stained sections and DNA quality was assessed using a multiplexpolymerase chain reaction method as well as real time quantitative polymerase chain reaction of commonly used reference genes. Subsequently, genomic DNA was pre-amplified and mutations in KRAS, BRAF, NRAS and PIK3CA were detected by validated Taqman assays. Comparisons were made with results from unstained formalinfixed paraffin-embedded sections obtained from the same paraffin block. Results: Our results demonstrate that genomic DNA isolated from immunohistochemistry-stained and unstained formalin-fixed paraffin-embedded tissue sections are comparable in quality and are suitable for down-stream analysis using polymerase chain reaction based assays. We also found that the sensitivity and specificity in detecting hotspot mutations are comparable in both sources of genomic DNA. This study reports 100% concordance in detecting hotspot mutations in KRAS, BRAF, NRAS and PIK3CA using quantitative real-time polymerase chain reaction between stained and unstained formalin-fixed paraffin-embedded sections. Conclusion: We conclude that by using our novel approach, it is possible to perform immunohistochemistry staining followed by genomic analysis using a single 4-5 μm section of formalin-fixed paraffin-embedded tissue.

It has been demonstrated that a higher CD8+ to CD4+FoxP3+ T regulatory cells (Tregs) ratio coincides with improved therapeutic outcomes for patients receiving immunotherapies in the clinic. The last several years of research have drawn attention to many such therapeutics and strategies. Adenosine is an abundant extracellular signaling molecule in the tumor microenvironment (TME) of many cancers. Signal transduction through the GPCR A2AR enhances the immunosuppressive activity of Tregs while simultaneously attenuating tumor-specific CD4+/CD8+ T cells. We have evaluated the appropriate coordination and dosing for a small-molecule inhibitor of A2AR in a Her-2/neu expressing murine model of breast cancer. We have demonstrated its capacity to augment existing therapeutic strategies in this tolerant model. To test our hypotheses on the ability of the small molecule to work in concert with existing therapeutics, we tested the role of CPI-444 when given with a T cell-inducing vaccine. Neu-expressing mammary tumor bearing HER-2/neu transgenic (Neu-N) mice were administered combinations of low-dose cyclophosphamide (Cy) to deplete Tregs, followed one day later with a granulocyte-macrophage colony-stimulating factor (GM-CSF) and neu-expressing whole cell vaccine (GVAX). The day following administration of GVAX, mice received adoptively transferred high-avidity naïve neu-specific CD8+ T cells intravenously one day after vaccination. Administration of specific components in this strategy (i.e. Cy, GVAX, adoptive transfer) were altered to gain insight into the mechanistic effects of CPI-444 in vivo through analysis of tumor progression, tumor clearance, and flow cytometric analysis of the TIL. Mice treated with the A2AR inhibitor or the vehicle control were administered each by oral gavage daily for 14 days (survival) or until 4 days post-adoptive transfer (TIL) at the peak infiltrate time. Of the strategies tested, Cy, followed by concomitant administration of GVAX with 100mg/kg of CPI-444 for 14 days, and a single adoptive therapy treatment provided a 55% overall survival (OS) compared with 0%-20% OS in vehicle controls (P Citation Format: Blake A. Scott, Todd Armstrong, Stephen Willingham, Ian McCaffery, Elizabeth M. Jaffee. Strategic inhibition of adenosine A2A receptor (A2AR) by CPI-444 improves CD8+:Treg ratios and enhances T-cell killing of a HER-2/neu expressing murine tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5579. doi:10.1158/1538-7445.AM2017-5579

Adenosine is present at high concentrations in the tumor microenvironment and is immunosuppressive acting on multiple cell types, including suppression of effector T cells. CD73, an ectonucleotidase that converts AMP to adenosine, is expressed on a subset of B and T cells and is a major source of extracellular adenosine. Elevated CD73 expression has been observed in multiple tumor types and is prognostic in triple negative breast cancer supporting a role for CD73 in tumor progression1. Inhibiting catalytic activity of CD73 is an attractive therapeutic strategy to reduce adenosine-mediated suppression of tumor immunity. We developed two types of humanized monoclonal anti-CD73 antibodies. CPX-006 inhibits CD73 catalytic activity by competing directly with AMP for the active site with an affinity of 0.64 nM. CPX-016 is similar to anti-CD73 antibodies described by others2, and inhibits CD73 activity allosterically by binding to a distal site. This was demonstrated using CD73 expressing cells incubated with APCP, a non-hydrolyzable analog of AMP. APCP competes with CPX-006 for binding to CD73 in contrast to CPX-016. The CPX-016 mechanism requires higher order complexes and results in loss of inhibition at high CPX-016/CD73 ratios. In contrast, CPX-006 reduces catalytic activity completely in cell based assays to levels seen when CD73 gene is deleted using CRISPR technology and inhibition was unaffected at high CPX-006/CD73 ratios. In a functional assay, CPX-006 inhibited immune suppression of T cell function induced by exposure of human PBMCs to AMP through direct inhibition of catalytic activity with a mean EC50 of 137 nM (interferon gamma production) and 189 nM (T cell proliferation) (n=12 donors). We analyzed the prevalence of CD73 expression across tumor histologies by immunohistochemistry including renal cell carcinoma (RCC, n=62), non small cell lung cancer (NSCLC, n=68), melanoma (n= 68) and breast cancer (n=94). CD73 was found to be heterogeneously expressed on tumor cells, immune cells and other stromal elements within each of the histologies examined. Expression on tumor cell only was found in 15% of melanoma and 14% of squamous NSCLC cases. In contrast, tumor cell staining was found in a significant fraction of the adenocarcinoma sub-type of NSCLC (55%). Stromal cell staining to varying degrees was seen in all tumor tissues. In summary, we have a generated a therapeutic antibody, CPX-006, that utilizes a novel mechanism of binding to the CD73 active site to completely inhibit CD73 enzymatic activity and restore T cell function. The finding of CD73 expression on tumor cells and stromal cells in a variety of tumors suggest this as a potential new target for immunotherapy of these malignancies. 1. Loi et al., Proc Natl Acad Sci USA 2013; 110: 11091-11096. 2. Geoghegan et al., mAbs 2016; 8: 454-467. Citation Format: Emily C. Piccione, Glen Mikesell, Barbara Daine-Matsuoka, Kimberly Walter, Richard Miller, Ian McCaffery. A novel CD73-blocking antibody reduces production of immunosuppressive adenosine and restores T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5577. doi:10.1158/1538-7445.AM2017-5577

Elevated levels of extracellular adenosine within the tumor microenvironment create an immunosuppressive niche that promotes tumor growth and metastasis. Adenosine signaling via the A2A receptor (A2AR) on immune cells suppresses anti-tumor immunity and has been shown to limit the efficacy of immunotherapies such as anti-PD-L1 or anti-PD-1 monoclonal antibodies (mAbs). CPI-444 is a potent and selective oral A2AR antagonist that is currently being investigated in Phase 1/1b clinical trials alone and in combination with an anti-PD-L1 antibody (Atezolizumab) in selected solid tumors. New preclinical data suggests that combining CPI-444 with anti-CTLA-4 mAbs as well as chemotherapy treatment are also promising therapeutic strategies in solid tumors, suggesting a broad role for adenosine as an immune suppressive mechanism. The efficacy of CPI-444 + anti-CTLA-4 mAb treatment was evaluated in MC38 and CT26 syngeneic mouse tumor models. In CT26, combination treatment eliminated established tumors in up to 90% of mice approximately 2 weeks after treatment was initiated. In MC38, combination CPI-444 and anti-CTLA-4 mAb treatment prolonged survival of 80% of mice compared to only 40% of mice that received CPI-444 or anti-CTLA-4 mAbs alone. The effect of CPI-444 + anti-CTLA-4 treatment on T-cell proliferation, T cell activation, and TREG function will be discussed. Chemotherapy releases adenosine and ATP into the tumor microenvironment (TME). Multiple chemotherapies have also been shown to up-regulate the ecto-enzymes CD39 and CD73 that produce adenosine and further suppress immune function. In the MC38 model, CPI-444 treatment synergized with doxorubicin and eliminated established tumors 80% of treated mice. CPI-444 treatment was also synergistic with cyclophosphamide, inhibiting the growth of RENCA tumors, a model that is considered resistant to chemotherapy. Ongoing studies are evaluating the effect of CPI-444 + chemotherapy on tumor infiltrating lymphocyte localization, activation, and expression of CD73 and CD39. These results suggest that blockade of the adenosine signaling pathway may be vital for enhancing anti-tumor responses in solid tumors that show an incomplete response to anti-CTLA4 therapy or chemotherapy. Citation Format: Po Ho, Meng-Yin Hsieh, Andrew Hotson, Richard Miller, Ian McCaffery, Stephen Willingham. Adenosine signaling through A2AR limits the efficacy of anti-CTLA4 and chemotherapy in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5598. doi:10.1158/1538-7445.AM2017-5598

Background: Accumulation of extracellular adenosine in the tumor microenvironment activates A2aR suppressing anti-tumor immunity. CPI-444 is an oral, selective A2aR antagonist with single agent (SA) antitumor efficacy in mouse models, and the addition of anti-PD-1/PD-L1 antibodies leads to synergistic anti-tumor activity. CPI-444 was well-tolerated in previous clinical trials in the non-oncology setting. This phase 1/1b open-label clinical trial uses a 2-step adaptive design to evaluate CPI-444 alone and in combination with atezolizumab (atezo, Tecentriq®) in patients(pts) with advanced cancers. Preliminary efficacy and safety results in the dose selection phase (Step 1) are reported. This is the first report of adenosine inhibition as a treatment for cancer. Methods: Adult pts who have failed up to 5 standard therapies with advanced solid tumors were enrolled. In Step 1, pts were randomized 1:1:1:1 to 1 of 4 dose cohorts (28 days/cycle) including 3 SA cohorts (100mg BID x 14 days(d), 100mg BID x 28d and 200 mg QD x 14d) or combined with atezo (CPI-444 50mg or 100 mg BID x 14d + atezo 840mg IV q2 weeks). Primary objectives: safety and efficacy and defining the optimal dose and schedule of CPI-444. Results: 48 pts were enrolled in Step 1; 47 received treatment. Median age was 65 years (range, 36-84).Tumor types enrolled were: non- small cell lung cancer(NSCLC, n=10), triple negative breast cancer (TNBC,n=10),melanoma(MEL,n=7), urothelial bladder cancer(UBC,n=6), renal cell cancer(RCC,n=5), MSI-H colorectal cancer(CRC,n=4), head and neck cancer (SCCHN, n=3)and prostate cancer (n=2). Median number of prior systemic regimens was 4(range, 1-5). Twenty-four pts received prior anti PD-1/PD-L1 therapy. One pt in the combination cohort had reversible grade 3 autoimmune hemolytic anemia which was the only DLT observed. Biomarker analyses defined the optimal dose of CPI-444 as 100 mg bid x 28 days. With a median follow-up time of 12 weeks(range, 2-32), the overall disease control rate (DC defined as CR, PR or SD) was 45% per RECIST. Nineteen pts had SD;15 pts received SA CPI-444. Two pts had a PR; both received SA. Of 5 RCC pts enrolled, 4 had DC (1 PR, 3 SD). Of these 4 RCC pts, 3 are receiving SA CPI-444 including the patient with PR. These 4 RCC pts remain on treatment with 2 pts on treatment for > 30 weeks. Of 7 MEL pts, 1 pt receiving SA had a PR and 2 had SD. SD was observed in 5/10 NSCLC pts, 4/10 TNBC pts, as well as 2/6 UBC pts and 1 pt each with CRC, SCCHN and prostate cancer. The proportion of pts with DC were similar for pts treated with CPI-444 alone and for pts treated with CPI-444 combined with atezo. 9/18(50%) pts who were naïve to anti-PD-1/PD-L1 treatment achieved DC as well as 10/24(42%) of pts who were resistant/ refractory to anti-PD1/PDL1 therapy. The most common (≥ 10% of pts) adverse events related to study drug were Grade 1 or 2 nausea (13%) and fatigue (19%). Conclusion: Inhibition of adenosine signaling through the A2aR results in anti-tumor activity in pts with advanced solid tumors. Responses are observed in multiple histologies both as a SA and in combination with atezo and in pts naïve or resistant/refractory to anti-PD-1/PD-L1 therapy. The optimal dose is 100 mg bid continuous. Enrollment in Step 2, evaluating disease-specific cohorts, is ongoing. Citation Format: Leisha Emens, John Powderly, Lawrence Fong, Joshua Brody, Patrick Forde, Matthew Hellmann, Brett Hughes, Shivaani Kummar, Sherene Loi, Jason Luke, Daruka Mahadevan, Benjamin Markman, Ian McCaffery, Richard Miller, Ginna Laport. CPI-444, an oral adenosine A2a receptor (A2aR) antagonist, demonstrates clinical activity in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT119. doi:10.1158/1538-7445.AM2017-CT119

Adenosine signaling via A2A receptor (A2AR) on immune cells suppresses anti-tumor immunity and limits the efficacy of immunotherapy, chemotherapy, CAR-T, and vaccines. CPI-444 is a potent and selective oral A2AR antagonist. Daily treatment of mice with CPI-444 led to dose-dependent inhibition of tumor growth in multiple syngeneic tumor models. Combining CPI-444 with anti-PD-L1 treatment synergistically eliminated tumors in up to 90% of treated mice, including restoration of immune responses in models that are poorly responsive to anti-PD-1 or anti-PD-L1 monotherapy. We have initiated a Phase 1/1b clinical trial to examine safety, tolerability, biomarkers, and preliminary efficacy of CPI-444 as a single agent and in combination with the anti-PD-L1 antibody, atezolizumab, in patients with non-small cell lung, melanoma, renal, triple negative breast, and other (bladder, prostate, head and neck, colorectal) tumors. Step 1 of the trial focused on determining the optimal dose and schedule for CPI-444; Step 2 is currently evaluating the efficacy of optimal CPI-444 dosing alone and with atezolizumab. In 48 patients treated in Step 1, CPI-444 was well tolerated with 1 Grade 3 or 4 treatment related adverse events. Preliminary evidence of clinical activity was observed in patients treated with single agent CPI-444, including patients who previously failed anti-PD-1 therapy. A pCREB-based pharmacodyamic assay showed that 100 mg, BID dose of CPI-444 resulted in a complete, sustained inhibition of A2AR on circulating immune cells. CPI-444 treatment alone or in combination with atezolizumab resulted in increased frequency of circulating CD8+PD-1+ cells and memory/effector subsets of CD4+ and CD8+ T cells. Substantial changes in TCR repertoire (Morisita Index Citation Format: Stephen Willingham, Andrew Hotson, Po Ho, Carmen Choy, Kim Walter, Erik Yusko, Sharon Benzeno, Ginna Laport, Richard Miller, Ian McCaffery. Inhibition of A2AR induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical and clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5593. doi:10.1158/1538-7445.AM2017-5593

3004 Background: Adenosine production in the tumor leads to immunosuppression through A2aR on infiltrating immune cells. CPI-444 is an oral A2aR antagonist with single agent(SA) anti-tumor activity in pre-clinical models. This phase 1/1b clinical trial uses a 2-step adaptive design to evaluate CPI-444 as a SA and in combination (combo) with the anti-PDL1 antibody, atezolizumab (atezo). We report results of RCC and NSCLC cohorts. Methods: Primary objectives: safety, efficacy and to identify optimal dose/schedule. Step 1 utilized 3 SA and 1 combo cohort to select dose/schedule. Step 2 included disease-specific expansion cohorts including RCC and NSCLC. Eligible pts had selected advanced cancers and failed standard therapies including checkpoint inhibitors. Results: 34 pts have enrolled and 25 pts were evaluable for response (Table 1). Median prior regimens: 3 (range,1-5) and most pts were resistant/refractory to anti PD1/PDL1 therapy (R/R). Most common AEs were Gr 1 nausea (n = 3) and pyrexia (n = 3); Gr 3 tachycardia was the only possibly related SAE. The selected Step 2 doses were CPI-444 100mg BID as a SA and in combo with atezo 840mg IV q2 weeks. The disease control rate (DCR, CR+PR+SD; duration 2 mo to > 8 mo) for pts with RCC and NSCLC cohorts were 86% and 50%, (100% and 43% for R/R pts), respectively. DCRs were similar in the SA and combo cohorts. Of 7 evaluable RCC pts, 1 pt has an ongoing PR (SA cohort, > 4 mo) and 5 have ongoing SD, duration 3 mo to > 8 mo (2 SA, 3 combo). Biopsy of the PR pt showed no detectable tumor and infiltration with CD8+ lymphocytes. In 18 evaluable NSCLC pts, 1 PR (PDL1 negative pt) and 8 SD were seen. PRs and SDs were seen in R/R pts and in PDL1 negative pts in both diseases. Conclusions: CPI-444 is well tolerated and shows anti-tumor activity in RCC and NSCLC pts as a SA and in combo. Pts who are R/R to anti PD1/PDL1 therapy and who are PDL1 negative can also benefit. Clinical trial information: NCT02655822. [Table: see text]

Purpose: Panitumumab, a fully human anti-epidermal growth factor receptor monoclonal antibody (mAb), has demonstrated efficacy in patients with wild-type KRAS metastatic colorectal cancer (mCRC). Rilotumumab and ganitumab are investigational, fully human mAbs against hepatocyte growth factor (HGF)/scatter factor and IGF1R, respectively. Here we evaluate combining rilotumumab or ganitumab with panitumumab in previously treated patients with wild-type KRAS mCRC. Experimental Design: Part 1 was a phase Ib dose-finding study of panitumumab plus rilotumumab. The primary endpoint was the incidence of dose-limiting toxicities (DLT). Part 2 was a randomized phase II trial of panitumumab in combination with rilotumumab, ganitumab, or placebo. The primary endpoint was objective response rate (ORR); safety, progression-free survival (PFS), and overall survival (OS) were secondary endpoints. Archival tissue specimens were collected for exploratory correlative work. Results: In part 1, no DLTs were reported. A recommended phase II dose of 10 mg/kg rilotumumab was selected. In part 2, for the panitumumab plus rilotumumab (n = 48), panitumumab plus ganitumab (n = 46), and panitumumab plus placebo arms (n = 48), the ORRs were 31%, 22%, and 21%, respectively. The median PFS was 5.2, 5.3, and 3.7 months and median OS 13.8, 10.6, and 11.6 months, respectively. Adverse events were tolerable. Exploratory biomarker analyses, including MET and IGF-related protein expression, failed to indicate conclusive predictive evidence on efficacy endpoints. Conclusions: Panitumumab plus rilotumumab met the prespecified criterion for improvement in ORR whereas ganitumab did not. This is the first study to suggest a benefit for combining an HGF inhibitor (rilotumumab) with panitumumab in previously treated patients with wild-type KRAS mCRC. Clin Cancer Res; 20(16); 4240–50. ©2014 AACR.

Purpose: This planned exploratory analysis assessed the predictive nature of baseline circulating factors of the insulin-like growth factor (IGF) axis on the treatment effect of ganitumab (monoclonal antibody inhibitor of IGF-1 receptor) plus gemcitabine in a randomized phase II study in metastatic pancreatic adenocarcinoma. Experimental Design: Baseline levels of IGFs/IGF binding proteins (IGFBP) were analyzed in serum or plasma. Mutations and gene expression were analyzed in archival samples. Treatment effects between biomarker subgroups were compared for overall survival (OS). Associations of tumor markers with OS were evaluated. Results: For patients with evaluable samples, ganitumab was associated with improved OS versus placebo (HR, 0.49; 95% CI: 0.28–0.87). The treatment effect on improved OS was strong in the patient subset with higher levels of IGF-1, IGF-2, or IGFBP-3, or lower levels of IGFBP-2, but not so on the other corresponding subset. Median OS of ganitumab versus placebo in patients with higher levels of IGF-1, IGF-2, and IGFBP-3 was 16 versus 6.8 months (HR, 0.25; 95% CI: 0.09–0.67), 16 versus 5.9 months (HR, 0.24; 95% CI: 0.09–0.68), and 16 versus 6.8 months (HR, 0.28; 95% CI: 0.11–0.73), and in patients with lower IGFBP-2 levels was 12.7 versus 6.6 months (HR, 0.19; 95% CI: 0.07–0.55). Interaction between treatment and IGFs/IGFBPs in multivariate analyses suggested predictive potential for IGF-2 (P = 0.002) and IGFBP-2 (P = 0.02). KRAS mutation status and PTEN expression were not associated with OS. Conclusions: Baseline circulating factors of the IGF axis may predict OS benefit from ganitumab plus gemcitabine in metastatic pancreatic adenocarcinoma. Clin Cancer Res; 19(15); 4282–9. ©2013 AACR.

Background Targeted agents presently available for mutant KRAS metastatic colorectal cancer (mCRC) are bevacizumab and aflibercept. We evaluated the efficacy and safety of conatumumab (an agonistic monoclonal antibody against human death receptor 5) and ganitumab (a monoclonal antibody against the type 1 insulin-like growth factor receptor) combined with standard FOLFIRI chemotherapy as a second-line treatment in patients with mutant KRAS mCRC. Patients and methods Patients with mutant KRAS metastatic adenocarcinoma of the colon or rectum refractory to fluoropyrimidine- and oxaliplatin-based chemotherapy were randomized 1 : 1 : 1 to receive intravenous FOLFIRI plus conatumumab 10 mg/kg (Arm A), ganitumab 12 mg/kg (Arm B), or placebo (Arm C) Q2W. The primary end point was progression-free survival (PFS). Results In total, 155 patients were randomized. Median PFS in Arms A, B, and C was 6.5 months (HR, 0.69; P = 0.147), 4.5 months (HR, 1.01; P = 0.998), and 4.6 months, respectively; median overall survival was 12.3 months (HR, 0.89; P = 0.650), 12.4 months (HR, 1.27; P = 0.357), and 12.0 months; and objective response rate was 14%, 8%, and 2%. The most common grade ≥3 adverse events in Arms A/B/C included neutropenia (30%/25%/18%) and diarrhea (18%/2%/10%). Conclusions Conatumumab, but not ganitumab, plus FOLFIRI was associated with a trend toward improved PFS. Both combinations had acceptable toxicity.

Elevated extracellular adenosine in the tumor microenvironment generates an immunosuppressive niche that promotes tumor growth and metastasis that is mediated via A2A receptor (A2AR) on immune cells. CPI-444 is a potent, oral, selective A2AR antagonist that has been well tolerated in Ph 1/1b studies in non-oncology indications. CPI-444 is being investigated in a Ph 1b study in solid tumors as a single agent and in combination with the anti-PD-L1 antibody atezolizumab. In preclinical studies, daily treatment of the syngeneic mouse model MC38 with CPI-444 (1, 10, 100 mg/kg) led to dose-dependent inhibition of tumor growth, leading to tumor elimination in ∼30% of treated mice. Combining CPI-444 (100 mg/kg, qd, 14 days) with anti-PD-L1 (200 μg, 3qw, 4 doses) treatment in MC38 models synergistically inhibited tumor growth and eliminated tumors in 90% of treated mice. When cured mice were later re-challenged with MC38 cells, tumor growth was rejected in 100% of challenged mice, indicating that CPI-444 induced systemic anti-tumor immune memory. CD8+ T cell depletion abrogated the efficacy of CPI-444 treatment as a single agent as well as in combination with anti-PD-L1, demonstrating a role for CD8+ T cells in mediating primary and secondary immune responses. Anti-tumor efficacy of CPI-444 ± anti-PD-L1 was associated with increased CD8+ cell infiltration and activation in MC38 tumor tissues, and a corresponding rise in PD-1 expression on CD8+ T cells in the spleen. Additionally, levels of immune checkpoints were modulated by treatment with CPI-444, including GITR, OX40, and LAG3 on tumor infiltrating lymphocytes and circulating T cells, suggesting a broad role for adenosine mediated immunosuppression. Based on these results and others, we have initiated a Phase 1b clinical trial to examine safety, tolerability, biomarkers, and preliminary efficacy of CPI-444 as a single agent and in combination with the anti-PD-L1 antibody, atezolizumab, in patients with non-small cell lung, melanoma, renal, triple negative breast, and other (head and neck, colorectal [MSI-H], bladder) tumors. Peripheral blood and tumor biopsies are collected pre- and post-treatment for biomarker analysis of adenosine pathway expression and activity, immune cell activation and tumor infiltration, and mutational burden. To demonstrate CPI-444 blockade of A2AR, a functional signaling assay was developed. Peripheral blood samples from treated patients were stimulated ex vivo with the adenosine analog NECA and CREB phosphorylation was quantified using flow cytometry in B and T cells. Following 14 days of CPI-444 treatment, NECA signaling was robustly inhibited at the doses selected for the Ph1b study in both the CPI-444 single agent and combination cohorts. In summary, this shows that CPI-444 induces anti-tumor immunity in mouse tumor models and inhibits adenosine signaling in lymphocytes of treated humans. This is the first demonstration of immune modulation in cancer patients receiving an adenosine antagonist. Evaluation of patient clinical responses and additional biomarkers are ongoing. Citation Format: Stephen Willingham, Andrew Hotson, Po Ho, Carmen Choy, Ginna Laport, Ian McCaffery, Richard Miller. CPI-444: A potent and selective inhibitor of A2AR induces antitumor responses alone and in combination with anti-PD-L1 in preclinical and clinical studies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR04.