Search

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.

The ongoing COVID-19 pandemic has prompted intense research on the role of antibodies in characterizing the emergence and sustenance of the immune response. Antibody isotype information is important in understanding disease mechanism, seroconversion, and vaccine response. While several methods provide qualitative information on SARS-CoV-2 antibodies, there is a need for quick methods with high detection sensitivity that provide relative response levels against multiple SARS-CoV-2 antigens. In this study, we show results from the development of a high-sensitivity bead-based immunoassay for flow cytometry that enables detection of IgG, IgM, or IgA antibodies in serum or plasma samples against multiple SARS-CoV-2 antigens. Antigenic targets include SARS-CoV-2 nucleoprotein, S1-subunit of the spike protein, and receptor-binding domain of the spike protein. The assay allows for quick processing, displays clear visual shifts, and provides fluorescent intensity information allowing for high detection sensitivity. Application of the assay to 30 PCR positive SARS-CoV-2 samples and 30 pre-pandemic samples provides high sensitivity and specificity for IgG analysis. IgG detection was observed in SARS-CoV-2 samples ranging from 6–75 days post-onset of symptoms in the study. IgM and IgA antibodies were also detected in several samples. The assay demonstrates good precision, with low %CVs of For Research Use Only. Not for use in diagnostic procedures.

Mitochondria are critical cellular organelles that play a fundamental role in cellular metabolism and oxidative stress and are well known to trigger multiple cell death pathways. The study of sequence of mitochondrial events as it relates to apoptotic/cell death events can provide critical insights into mechanism of cellular homeostasis, stress and death. Availability of rapid and simplified cytometric testing methods for evaluating mitochondrial changes, apoptosis and cell death in parallel can greatly enhance our understanding of mechanism of compound action. In this study, we investigated a series of compounds to evaluate apoptotic/cell death effects in context of mitochondrial changes using plate-based assays on Guava® easyCyte systems. Studies utilized multiplexed assays for mitochondrial membrane potential changes and apoptosis/cell death markers and allowed for easy identification of hit compounds. Dose and time response studies with Niclosamide, an anti-helmintic drug and comparison of effects with Gambogic acid and celastrol demonstrated early and significant mitochondrial impacts for niclosamide and gambogic acid. No apoptotic or cell death impacts were observed in parallel at low doses/short times of incubation for niclosamide, while increased time with niclosamide caused increase in mitochondrial, apoptotic and cell death response. The method demonstrates great power in being able to distinguish between potency of compounds and conditions in modulating mitochondrial/apoptotic changes. The simplicity of the assays described coupled with the ease of use of plate based microcapillary cytometry can provide researchers valuable tools to obtain a more comprehensive insight into how compounds modulate mitochondria and its relationship with subsequent apoptosis/cell death pathways.

Drug-based screening, identification of ‘hit’ compounds, and evaluation of mode of action require the parallel understanding of how the drug modulates critical cellular parameters which eventually lead to cytotoxicity or disrupt cellular proliferation. Given the increased need for assays with high informational content and mechanism-based data, researchers turn to flow cytometry with its potential for quantifiable comparisons between cell populations.

The degree of apoptosis in a cell population is an important parameter of cell health and is characterized by distinct morphological changes. Current methods of accurate detection and measurement of cellular apoptosis require expensive and complicated instrument platforms and expertise. The Muse Cell Analyzer is a unique instrument that enables multidimensional cell health analysis on a single platform. In this study, we used the Muse Cell Analyzer for apoptosis studies using the Muse Annexin VDead Cell Assay. The assay is based on the detection of phosphatidylserine (PS) on the surface of apoptotic cells. The results obtained from Muse Cell Analyzer were compared with traditional methods for apoptosis analysis. Our results indicate that Muse Annexin VDead Cell Assay and software module enabled the acquisition of accurate and highly precise measurements of cellular apoptosis. The assay is versatile and works with both suspension and adherent cell lines and multiple treatment conditions.

Multiplexing cell health parameters with phenotyping markers provides greater understanding of time and sequence for activation and cellular stress pathways in immune cells. Sample heterogeneity often makes this type of assessment complex to perform and evaluate. The development of the easyCyte™ 12 instruments, which combines 3 lasers and 12 parameters, has greatly facilitated 6-9 color multiparametric analysis of immune cell populations using small amounts of sample without compensation. In this study, we evaluated the impact of activation agents and cytotoxic treatment on PBMCs using a multiparametric assay which identifies lymphocyte subsets in combination with cell health or activation parameters. Our studies demonstrate that overnight treatment with anisomycin resulted in more sensitive impacts on mitochondrial depolarization on all lymphocyte subsets with lower apoptotic and cell death effects being observed. Overnight treatment with celastrol demonstrated almost parallel changes in mitochondrial potential and annexin V based apoptosis effects in lymphocyte subpopulations. The methodology was also utilized to study dose and time course treatments of activation, cellular stress, and cell death caused by PHA or PMA/ionomycin with PBMCs. Which allowed for dissection of sequence of changes in multiple subpopulations. Such studies can greatly enhance our understanding on response of immune cell subpopulations to compounds that cause activation or cytotoxicity.

Cellular health enumeration and assessment of heterogeneous cellular sample such as whole blood and PBMCs often require many colors to accurately characterize subpopulations and are critical in defining immune cell handling as well as in functional studies ranging from cytokine secretion to proliferation. Traditional multi-color flow cytometers have posed challenges in multi-parametric experiments, managing compensation, and analyzing results. Existing solutions are expensive, cumbersome to run and require extensive training or dedicated personnel. In this study, we will present data from simplified methods allowing identification of multiple subsets including T,B, NK, naïve and memory, monocytes, and granulocytes while simultaneously assessing cell health for studies with activation agents, cytotoxic treatments or culture conditions on a novel instrument platform, the guava easyCyte™ 12. The guava easyCyte™ 12 is a novel small footprint cytometer, with 3 lasers including blue, red and violet. This allows for data on up to 12 parameters in a microplate or tube-based format. The system is based on principles of microcapillary cytometery, provides absolute count and percentage data without external beads, uses low sample volume and uses flexible and intuitive software. The combination of multicolor antibody panel and cell health markers with the powerful instrument platform enables obtaining critical sub-population level information for PBMC and whole blood samples.

A comprehensive mechanistic understanding of cytotoxic compounds action requires knowledge of their impact on different immune sub-populations such as CD4 and CD8 T cells. In this study, we explored the mechanism of action of Gambogic acid, a natural product with potent anti-tumor activity and Anisomycin, an antibiotic that inhibits protein synthesis on peripheral blood mononuclear cells (PBMCs) using assays that combine CD4 and CD8 T cell identification with cell health parameters followed by microcapilllary cytometry. Our studies demonstrate that gambogic acid results in significant apoptosis in both CD4 and CD8 T cells. Further, mitochondrial depolarization plays a significant role in gambogic acid induced cytotoxicity of both CD4 and CD8 T cells with a greater percentage of cells demonstrating loss of mitochondrial membrane potential at lower concentration compared to other apoptosis markers. Anisomycin also results in significant mitochondrial membrane potential loss in T cells. Significant Caspase activation was observed in PBMC’s on treatment with anisomycin; with Caspase 3/7, Caspase 8 and Caspase 9 activity being observed in CD4 and CD8 T cells. Data on dose and time based responses in the sequence of cell health impacts will be further presented for these compounds on PBMC. Evaluating mechanistic impact of compounds on T lymphocytes can provide a more enriched understanding of complete activity of compounds that induce cytotoxicity.

Activation of lymphocytes leads to expression of cytokine receptors, cytokine secretion and the expression of specific cell surface molecules resulting in divergent immune responses. Correlation of cell surface activation with cytokine secretion levels can provide valuable information to evaluate secretion levels. In this study, we demonstrate the kinetics of CD69 and CD25 expression on human peripheral blood lymphocytes from multiple donors following activation with polyclonal activator, phytohaemagglutinin or phorbol myristate acetate and Ionomycin. The studies were performed using novel no-wash assays on the Muse™ Cell analyzer to assess CD69 and CD25 levels on total lymphocytes. Our data shows that the kinetics of CD69 and CD25 expression demonstrates that a high level of lymphocytes depict CD69 expression as early as 6 hours of stimulation, peaked at 24 hours and down regulated after 48 hours. The observed CD25 expression on lymphocytes was significantly less than that of CD69 at 6 hours, increased at 24 hours and peaked at 48 hours with differences between PHA and PMA-ionomycin activation with some variability in extent of activation between donors. Comparison of the level of cytokines secreted and lymphocyte activation will be presented. The combined measurements of activation markers at the cellular level and the secreted cytokines can provide a comprehensive analysis in monitoring lymphocyte response to polyclonal stimuli.

Characterization and enumeration of T and B cell lymphocyte sub-populations is crucial in immunological research applications for understanding of immune status and response. This analysis has typically been performed using sophisticated single platform flow cytometry or double platforms, to which many researchers may have limited access. In this study, we evaluated performance of a novel, low-cost, small footprint platform, the Muse™ Cell Analyzer which is based on microcapillary cytometry and utilizes guided touchscreens, for immunophenotyping analysis. Whole blood and peripheral mononuclear blood cell samples a were analyzed using simplified, no wash, novel two color strategies and results for CD4 T cells, CD8 T Cells and B cells count and percentage of lymphocytes were compared against well-established predicate methods. Comparison of results demonstrates good correlations and low bias for each strategy. Precise results were obtained for all assays tested as shown by the low coefficient of variation (CV

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

Immunological studies evaluating phenotypic, mechanistic and activation characteristics of peripheral blood mononuclear cells (PBMCs) involve multiple samples, long treatment times, and laborious methods. The initial screening and assessment of PBMC quality for parameters such as count, viability and apoptosis is critical in determining the success of downstream immunological studies. Traditionally, such screening has been limited since it has required access to complicated and expensive platforms. In this study, we present results from a simplified method for the characterization of fresh or cryopreserved PBMC’s on the novel Muse Cell Analyzer. The methods utilize simple mix and read assays, provide quick assessment of count and viability measurements and the apoptotic status of PBMC’s. Our results indicate that good accuracy and linearity data on count and viability can be obtained for PBMC’s in a wide concentration range can be obtained using sample sizes as low as 20 µL. Precise results were obtained for all assays tested as shown by the low coefficient of variation (CV

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

Caspase activation is critical to immune cell apoptosis, with different caspases activated in different pathways of cell death. Thus, Caspase 8 is implicated in CD95-mediated apoptosis, Caspase 9 is crucial to the intrinsic pathway of apoptosis and Caspase 3,7 activation, is observed in multiple pathways of cell death. Caspases also play a role in immune cell inflammation and proliferation. Simplified methods that allow identification of immune cells and specific caspase activity can provide important mechanistic insights in immune cell apoptosis. We present here results from a simplified strategy to assess caspase activity in CD4 and CD8 T cells using T cell specific cocktails and fluorescently labeled caspase inhibitors. Using this approach, caspase activation with multiple compounds such as anisomycin and staurosporine were studied. Our results indicate that anisomycin causes early activation of both Caspase 9 and Caspase 8 followed by slower activation of Caspase 3,7 activity in both CD4 and CD8 T cells. Differential dose response curves were obtained for Caspase levels in CD4 and CD8 T cells on treatment with staurosporine. In some cases, donor specific differences were observed in the timing and relative activation of the different caspases. Simultaneous assessment of caspase activites in different immune sub-populations allows for a deeper understanding of mechanistic pathways of apoptosis, which is important in drug development and understanding of diseases.

The study of mitochondrial membrane potential (ΔΨm) changes in immune cells is important in understanding immune cell apoptosis, mechanism of diseases, and evaluating the action of compounds and drugs that cause mitochondrial stress. Simultaneous identification of T cells along with ΔΨm changes is challenging due to the broad fluorescence of traditional mitochondrial dyes, such as JC-1, in multiple channels. Here we demonstrate results from a novel approach for the simultaneous evaluation of ΔΨm in CD4 & CD8 T cells using simplified cocktails and a mitochondrial responsive dye followed by microcapillary flow cytometry. PBMC’s were treated with a range of compounds including staurosporine and gambogic acid and evaluated for mitochondrial membrane potential changes and annexin V binding on T cells. Our results demonstrate that with most treatments, a greater % of cells exhibit earlier and more sensitive changes in ΔΨm, than Annexin V binding. Further, some donors treated with staurosporine, a significantly greater % of CD8 T cells demonstrated changes in ΔΨm vs. CD4 T cells. Gambogic acid demonstrated a dramatic reduction of ΔΨm and showed increased apoptosis on both CD4 and CD8 T cells simultaneously. The ability to measure mitochondrial membrane potential changes in CD4 and CD8 T cell simultaneously can allow more sensitive identification of immune cell perturbation and provide deeper insights into the mechanism of immune cell apoptosis in disease and drug discovery.

Simultaneous assessment of cell surface phenotypic markers, activation markers such as CD69 and apoptosis related markers such as CD95/FAS can provide deeper understanding of mechanism and impacts of in vitro mitogenic stimulation on immune cell populations.. Kinetic and dose response studies of PBMC’s with different stimulants was performed and followed with novel simplified assays that could assess levels of CD69 and CD95 on T and B cells followed by plate-basedmicrocapillary flow cytometry. Information on the CD4, CD8, CD3,CD19, CD45 expression levels was also obtained. Our results demonstrate that while CD4 expression on CD4 T cells was rapidly downregulated with phorbol myristate acetate or combinations with ionomycin, but was not impacted by PHA treatment. Tracking of CD95 expression levels demonstrate the elevation of CD95 on T and B cells with PHA treatment with no significant loss in CD4 expression levels. Correlation of T celll activation markers and apoptotic markers and expression levels of phenotypic markers would facilitate further elucidation of the mechanism of T cell activation.