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AB598, an IgG1 Fc-silent antibody that blocks CD39 (ENTPD1) enzymatic activity, is being developed as a novel cancer immunotherapy. CD39 is highly expressed on immune and stromal cells within the tumor microenvironment (TME) and is responsible for the conversion of adenosine triphosphate (ATP) into adenosine monophosphate (AMP). AB598 can bind and inhibit CD39 activity on primary human immune cells, leading to an increase in local levels of immunostimulatory ATP. In the TME, elevated ATP exerts its effects by signaling through the purinergic family of P2X and P2Y receptors. Using a combined approach of nanostring profiling of myeloid cell subsets derived from healthy donors, flow cytometry on healthy human peripheral blood, and bioinformatic analysis of the MET500 and TCGA databases, we define a profile of relevant receptors that can act as effectors to promote anti-tumor responses upon CD39 inhibition. Nanostring and flow cytometry show CD39 and P2X7 are ubiquitously expressed across the myeloid cell lineage. P2RX7, CASP1, and GSDMD gene expression is highest in M1 macrophages, a finding corroborated by the boost in inflammasome activation seen on in vitro-derived macrophages with AB598 treatment. P2RY11 gene expression shows a bimodal distribution with the highest expression in monocyte-derived dendritic cells (moDCs) and M2 macrophages, a finding that supports previously published work identifying P2Y11 as the receptor responsible for ATP-induced maturation of moDCs. Using CD83 and CD86 as markers, we show that AB598 enhances ATP-induced moDC activation in a concentration-dependent manner. Supportive of the broad expression of CD39 on myeloid cell subsets, analysis of TCGA data shows an inverse correlation of ENTPD1 gene expression and tumor purity. ENTPD1 expression is positively correlated with P2RX7, NLRP3, ITGAM (CD11b), and ITGAX (CD11c) expression, establishing the presence of the machinery for AB598 inhibition of CD39 to promote myeloid cell activation in the TME. Clinically, immunostimulation in the TME can be achieved by the administration of an immunogenic cell death (ICD)-inducing chemotherapy capable of releasing ATP; however, the ATP can be degraded by CD39. Using continuous ATP monitoring, we show that AB598 treatment increases and maintains the pool of extracellular ATP from human cancer cell lines treated with ICD-inducing agents. Our results demonstrate that the TME has the machinery to respond to an environment rich in immunostimulatory ATP, common chemotherapeutic agents can cause tumor cells to release ATP extracellularly, and AB598 can effect immunogenic change through inflammasome activation and dendritic cell maturation when ATP is present in the TME. Overall, the findings presented here establish AB598 as a promising agent targeting CD39 for cancer immunotherapy and provide a rationale for the combination of AB598 with ICD-inducing chemotherapy in the clinic. Citation Format: Kaustubh Parashar, Julie Clor, Dana Piovesan, Casey Mitchell, LC Stetson, Ke Jin, Gonzalo Barajas, Sean Cho, Janine Kline, Stephen W. Young, Nigel P. Walker, Matthew J. Walters, Ester Fernandez-Salas, Christine E. Bowman. AB598, a therapeutic anti-CD39 antibody, elevates ATP and increases immunogenicity in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B08.

CD39 (ENTPD1) is an ecto-nucleoside triphosphate diphosphohydrolase expressed widely in the tumor microenvironment (TME) responsible for catalyzing the conversion of ATP to AMP. Inhibition of CD39 enzymatic activity can promote anti-tumor immune responses by increasing the immunostimulatory substrate ATP and decreasing the formation of the product AMP, a precursor to immunoinhibitory adenosine. CD39 inhibition has been shown to have an anti-tumor effect by activating dendritic cells, macrophages, and NK cells in the TME to promote antigen presentation and inflammatory cytokine release. AB598 is a novel antibody, highly specific for human and cynomolgus CD39. AB598 potently binds to CD39 expressed on human primary myeloid cells and tumor cells and potently inhibits both soluble and membrane-bound CD39 enzymatic activity. CD39 inhibition results in increased extracellular ATP (eATP) in the TME and subsequent activation of P2X and P2Y receptors. Myeloid cells highly express both CD39 and P2X7, the P2X receptor with the weakest ligand affinity for ATP (greater than 100 μM), and most likely to be specifically activated by very high levels of eATP resulting from CD39 inhibition. Thus, the myeloid compartment has emerged as a key target associated with the therapeutic effects of CD39 inhibition. AB598 retains the ability to bind and inhibit membrane bound CD39 in the presence of high ATP (400 μM). AB598 promotes maturation of human dendritic cells in the presence of ATP, as determined by increased expression of CD83 and CD86 and decreased expression of CD14. In human macrophages treated with ATP, AB598 activates the inflammasome, resulting in the secretion of pro-inflammatory IL-18 and IL-1β. The ATP-dependent effects of AB598 support a therapeutic rationale of combining it with immunogenic therapies that induce release of ATP, such as chemotherapy and radiation, which might represent a new paradigm in the treatment of advanced solid tumors. Citation Format: Christine E. Bowman, Ada Chen, Damie Juat, Kaustubh Parashar, Julie Clor, Hema Singh, Ritu Kushwaha, Bryan Handlos, Suan Liu, Janine Kline, Xiaoning Zhao, Hyock Joo Kwon, David Green, Stephen W. Young, Ester Fernandez-Salas, Matthew J. Walters, Nigel P. Walker. Inhibition of CD39 results in elevated ATP and activation of myeloid cells to promote anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 321.

Drug discovery and toxicity studies require access to methods and technologies that can provide comprehensive evaluation of the types of cellular impacts caused by anti-cancer compounds. In particular, there is growing emphasis on the study of mitochondrial toxicity at an early phase of compound evaluation and its relations to multiple cell death processes that include apoptotic cell death, caspase-independent death and autophagic processes. The study of oxidative stress and its relation to cell death pathways also provides insights into mechanism of cellular stress. In this study we have used multiparametric assays that correlate changes in mitochondria with different cell health parameters using plate-based microcapillary cytometry on guava easyCyte platforms. Compound screening studies were performed on Jurkats (suspension) and HeLa adherent cell lines using an array of over 80 compounds to study inter-relationships between mitochondrial perturbations and different cell health parameters. Compounds were evaluated for mitochondrial depolarization and relationship to apoptosis and cell death; mitochondrial perturbations with regard to caspase levels were also evaluated; and the parallel connection between mitochondrial superoxide stress and apoptosis was also studied. Our studies identified several hit compounds, examples of which included compounds like gambogic acid, thimerosal, camtothecin, antimycin A, staurosporine, celastrol and juglone which caused significant mitochondrial perturbation and apoptosis. In addition, compounds such as antimycin A and staurosporine caused significant oxidative stress. Simplified and rapid screening assays that provide information on multiple parameters of cellular stress and apoptosis can greatly benefit the identification of hit compounds and provide a more comprehensive understanding of the mechanism of compound toxicity. Citation Format: Katherine Gillis, Julie Clor, Kimvan Tran, Asima Khan, Kamala Tyagarajan. Multiparametric approaches to evaluate mitochondrial toxicity, oxidative stress and apoptosis in drug discovery studies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1738. doi:10.1158/1538-7445.AM2013-1738

The study of anti-cancer compounds and cancerous cell lines requires access to cell health information that includes parameters such as cellular vitality, cell cycle and apoptosis and provides a complete picture of cell health. Traditionally, expensive and complicated instrumentation has been required for comprehensive cell health analysis. Here we present data from a novel, low-cost, easy to use, compact, high performance instrument, the Muse Cell Analyzer which provides multi-dimensional cell health information. The instrument utilizes a combination of highly simplified mix and read assay protocols with a touch-screen interface which greatly simplifies data acquisition and analysis. The objective of this study was to evaluate performance of Muse Count and Viability Assays when compared to traditional predicate methods including hemacytometers and automated image based systems. The mix and read format of the Muse™ Count & Viability assay provides quantitative detection of multiple suspension cell lines (e.g. Jurkats, Hl60s, K562s) and adherent cell lines (e.g. CHOs, PC3s, MCF-7s) across a wide range of concentrations and viabilities. Comparison of count and viability data from multiple platforms indicate that count and viability information on a vast variety of cancer cell lines can be robustly and accurately obtained on the Muse Cell Analyzer and the system depicts good linearity over a wide concentration range. Superior precision was obtained on the Muse Cell Analyzer for a variety of sample types with %CV ranging from 1-8% compared to %CV of 5-20% for predicate methods on the same samples. Cytotoxicity effects of anti-cancer compounds such as staurosporine, gambogic acid etc.on multiple cell types could be reliabily obtained on the instrument platform. The mix and read format of the Annexin V & Dead Cell assay and simplified Cell Cycle protocol on the instrument further allows for the easy and reliable study of impacts of anti-cancer compounds. The combination of three critical assays for cell count and viability, apoptosis and cell cycle status on a simple low cost instrument makes the Muse Cell Analyzer a versatile and accurate solution for comprehensive cell health analysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4073. doi:1538-7445.AM2012-4073