Abstract 3168: Methods for assessment of the “adenosine fingerprint” in clinical trials of AB928
Authors: DiRenzo Daniel , Ashok Devika , Anderson Amy E. , Udyavar Akshata , Tan Joanne B. , Luu Irene M. , Zhang Kristen , Jeffrey Jenna L. , Seitz Lisa , Leleti Manmohan R. , Young Stephen W. , Powers Jay P. , Walters Matthew J. 
Published: August 2019

Abstract
BACKGROUND: The tumor microenvironment (TME) contains high levels of immunosuppressive adenosine (ADO), which activates the A2aR and A2bR receptors on immune cells, leading to an ineffective anti-tumor response. Ecto-5’-nucleotidase (CD73) and tissue non-specific alkaline phosphatase (TNAP) are primarily responsible for the conversion of extracellular adenosine monophosphate (AMP) to ADO and exhibit both membrane-bound and secreted forms. We have previously shown that AB928, a dual A2aR/A2bR antagonist, rescues the immunosuppressive effects of ADO in experimental tumor models. Herein, we describe the development of assays to measure the expression and activity of adenosine-generating enzymes in human tumor samples and peripheral blood. These assays are being used to define an “adenosine fingerprint” that identifies tumor types and patients most sensitive to adenosine inhibition by AB928.
METHODS: CD73 and TNAP immuno-histochemistry (IHC) and mRNA analysis were performed on sections of formalin fixed paraffin embedded (FFPE) tumor tissue. Circulating levels of CD73 were quantified with an in-house developed ELISA, and AMP-ase enzymatic activity in serum was determined using an AMP-GloTM(Promega) assay. Gene expression data were extracted from The Cancer Genome Atlas (TCGA) and expressed as a ratio of log2 counts per million per sample.
RESULTS: TCGA data identified non-small cell lung (NSCLC), renal clear cell, triple-negative breast, ovarian, colorectal, and gastro-esophageal cancers as tumors that highly express the adenosine producing enzymes CD73 or TNAP. To confirm these gene expression patterns, IHC assays for both CD73 and TNAP were developed using normal and tumor human tissue. IHC for CD73 was strongest in NSCLC (54.3 +/- 11.2 µm2) and colorectal (22.5 +/- 8.1 µm2) adenocarcinomas, whereas prostate (1.0 +/- 0.3 µm2) cancer exhibited the weakest staining. In contrast, TNAP staining was strongest in ovarian cancer and NSCLC adenocarcinoma, whereas gastric and colorectal adenocarcinomas showed very little TNAP staining. Therefore, CD73 and TNAP IHC broadly recapitulate the gene expression patterns found in TCGA. A CD73-specific ELISA was developed using human cancer patient blood which established a range of circulating CD73 protein levels (2-8 ng/mL) and showed a strong correlation between plasma and serum levels (r2 = 0.94). To measure adenosine-generating enzyme activity in peripheral blood, the AMP-Glo biochemical assay was performed and showed strong concordance with the CD73 ELISA in cancer patient serum (r2 = 0.72).
CONCLUSIONS: These assays provide a detailed picture of the adenosine-generating capacity in the local TME as well as the peripheral activity and levels of CD73/TNAP to better identify patients that may benefit from adenosine inhibition.
Citation Format: Daniel DiRenzo, Devika Ashok, Amy E. Anderson, Akshata Udyavar, Joanne B. Tan, Irene M. Luu, Kristen Zhang, Jenna L. Jeffrey, Lisa Seitz, Manmohan R. Leleti, Stephen W. Young, Jay P. Powers, Matthew J. Walters. Methods for assessment of the “adenosine fingerprint” in clinical trials of AB928 [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 3168.