a-real-time-potency-assay-for-chimeric-antigen-receptor-t-cells

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

ACEA Biosciences, Inc

Immunoglobulin E (IgE)-mediated mast cell activation is involved in the immediate phase of allergic reactions and plays a central role in the onslaught and persistence of allergic diseases. IgE-mediated mast cell activation includes two important events: cell sensitization resulting from IgE binding to Fc (FcepsilonRI) receptor and cell activation triggered by allergen-mediated oligomerization of membrane-bound IgE. Real-time monitoring of these events is needed to dissect the molecular mechanisms underlying IgE-mediated mast cell activation. Existing technologies are limited to label-based end-point assay formats, which detect either early signaling or final phase of mast cell activation. We describe a microelectronic cell sensor-based technology allowing dynamic monitoring of IgE-mediated mast cell sensitization and activation in real-time without any labeling steps. RBL-2H3 mast cells were cultured onto the surface of microelectronic cell sensor arrays integrated into the bottom of microtiter plates, which record electric properties, such as impedance between cell membrane and sensor surface. In the presence of the allergen, dinitrophenyl (DNP)-bovine serum albumin (BSA), anti-DNP IgE-sensitized cells were activated within 5 min and the entire activation process was quantitatively and continuously recorded. Impedance measurements correlate with morphological dynamics and mediator release as measured by beta-hexosaminidase activity, and can be blocked by pharmacological agents, inhibiting IgE-mediated signaling. The assay on microelectronic cell sensor arrays can be scaled up for high-throughput screening of pharmacological inhibitors of IgE-mediated mast cell activation and other cell-based receptor-ligand assays.

Label-free, real-time monitoring of IgE-mediated mast cell activation on microelectronic cell sensor arrays.

In this paper we have explored the utility of the real-time cell electronic sensing (RTCES, ACEA Biosciences Inc., San Diego, CA) system for monitoring the quality of live cells in cell-based assays as well as for assay development. We have demonstrated that each cell type displays unique growth kinetic profiles that provide a quantitative account of cell behavior and can be used as a diagnostic tool for cellular quality control. The utility of the specific signature patterns was shown by demonstrating the significant differences in primary cell behavior depending on the supplier. In addition, the RT-CES system was able to differentiate cell behavior depending on the passage stage of the cells. The utility of the RT-CES system as an assay development tool was demonstrated in cytotoxicity assays. The RT-CES system not only provides information regarding the potency of cytotoxic compounds, but in addition relates potency to the rate of the response for each concentration of the compound tested, which is important for understanding the mechanism of compound action. Moreover, real-time display of cytotoxicity data by the RT-CES system allows for calculation of real-time 50% inhibitory concentration (IC50) values or determination of optimal IC(50) value. In summary, the RT-CES system provides high content and information-rich data that are beyond the scope of single-point assays.

Live cell quality control and utility of real-time cell electronic sensing for assay development.

Cell-based assays have become an integral part of the preclinical drug development process. Recently, noninvasive label-free cell-based assay technologies have taken center stage, offering important and distinct advantages over and in addition to traditional label-based endpoint assays. Dynamic monitoring of live cells, the preclusion of label, and kinetics are some of the fundamental features of cell-based label-free technologies. In this article we will discuss the real-time cell electronic sensing (RT-CES, ACEA Biosciences Inc., San Diego, CA) system and some of its key applications for cell-based assays such as cell proliferation and cytotoxicity, functional assays for receptor-ligand analysis, cell adhesion and spreading assays, dynamic monitoring of endothelial barrier function, and dynamic monitoring of cell migration and invasion. Also, where appropriate we will briefly discuss other label-free technologies in an application-specific manner.

Dynamic and label-free cell-based assays using the real-time cell electronic sensing system.

Cell-based assays are an important part of the drug discovery process allowing for interrogation of targets and pathways in a more physiological setting compared to biochemical assays. One of the main hurdles in the cell-based assay field is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the pathway or target being investigated. Conventional label and reporter-based cell assays may be more prone to artifacts due to considerable manipulation of the cell either by the label or over-expression of targets or reporter proteins. Cell-based label-free technologies preclude the need for cellular labeling or over-expression of reporter proteins, utilizing the inherent morphological and adhesive characteristics of the cell as a physiologically relevant and quantitative readout for various cellular assays. Furthermore, these technologies utilize non-invasive measurements allowing for time resolution and kinetics in the assay. In this article, we have reviewed the various label-free technologies that are being used in drug discovery settings and have focused our discussion on impedance-based label-free technologies and its main applications in drug discovery.

The application of cell-based label-free technology in drug discovery.

We describe a cell-based kinetic profiling approach using impedance readout for monitoring the effect of small molecule compounds. This noninvasive readout allows continuous sampling of cellular responses to biologically active compounds and the ensuing kinetic profile provides information regarding the temporal interaction of compounds with cells. The utility of this approach was tested by screening a library containing FDA approved drugs, experimental compounds, and nature compounds. Compounds with similar activity produced similar impedance-based time-dependent cell response profiles (TCRPs). The compounds were clustered based on TCRP similarity. We identified novel mechanisms for existing drugs, confirmed previously reported calcium modulating activity for COX-2 inhibitor celecoxib, and identified an additional mechanism for the experimental compound monastrol. We also identified and characterized a new antimitotic agent. Our findings indicate that the TCRP approach provides predictive mechanistic information for small molecule compounds.

Kinetic cell-based morphological screening: prediction of mechanism of compound action and off-target effects.

Strict quality control of cells is required for the standardization and interpretation of results in all areas of cell-based research, especially in drug discovery. Real-time cellular analysis using electrical impedance as a readout offers a rapid and highly reproducible method for quality control as it provides a quantitative measure of overall cell morphology and growth. In a case study, the authors demonstrate that samples of a single cell line obtained from several different labs show clear differences in their impedance profiles when compared with the corresponding standard cell line. A number of kinetic parameters were derived from the impedance profiles and used to quantify the differences among these cell lines. Our findings indicate that this methodology can detect cell line differences including mix-ups or contaminations, genetic alterations, and potential epigenetic changes occurring during passaging, all of which can occur in the time scale of a screening campaign. Finally, we provide evidence that these impedance profile differences can be predictive of different outcomes in cell-based functional assays for the effects of small molecules on otherwise seemingly identical cell lines.

Rapid and quantitative assessment of cell quality, identity, and functionality for cell-based assays using real-time cellular analysis.

We describe here the use of the xCELLigence system for label-free and real-time monitoring of cell -viability. The xCELLigence system uses specially designed microtiter plates containing interdigitated gold microelectrodes to noninvasively monitor the viability of cultured cells using electrical impedance as the readout. The continuous monitoring of cell viability by the xCELLigence system makes it possible to distinguish between different perturbations of cell viability, such as senescence, cell toxicity (cell death), and reduced proliferation (cell cycle arrest). In addition, the time resolution of the xCELLigence system allows for the determination of optimal time points to perform standard cell viability assays as well as other end-point assays to understand the mode of action. We have used the WST-1 assay (end-point viability readout), the cell index determination (continuous monitoring of viability by xCELLigence), and the DNA fragmentation assay (end-point apoptosis assay) to systematically examine cytotoxic effects triggered by two cytotoxic compounds with different cell-killing kinetics. Good correlation was observed for viability readouts between WST-1 and cell index. The significance of time resolution by xCELLigence readout is exemplified by its ability to pinpoint the optimal time points for conducting end point viability and apoptosis assays.

The xCELLigence system for real-time and label-free monitoring of cell viability.

Cardiac toxicity is a major concern in drug development and it is imperative that clinical candidates are thoroughly tested for adverse effects earlier in the drug discovery process. In this report, we investigate the utility of an impedance-based microelectronic detection system in conjunction with mouse embryonic stem cell-derived cardiomyocytes for assessment of compound risk in the drug discovery process.

Dynamic monitoring of beating periodicity of stem cell-derived cardiomyocytes as a predictive tool for preclinical safety assessment.

Cardiac safety testing of lead drug candidates is an important part of the drug discovery and development process. All new chemical entities need to be subjected to extensive preclinical assessment for cardiac liability, especially for a potentially fatal form of ventricular arrhythmia referred to as Torsades de Pointes. We have developed an innovative label-free, real-time system, the xCELLigence RTCA Cardio System, which is designed to monitor contractility of cardiomyocytes based on impedance measurement. The assay is performed using specially designed microtiter plates that are integrated with gold microelectrodes. The system was validated using mouse embryonic stem cell-derived cardiomyocytes, human-induced pluripotent stem cell-derived cardiomyocytes, and rat neonatal primary cardiomyocytes by applying a variety of tool compounds and drugs with known mechanisms of action. Our data show that the time resolution in the assay can provide important information about compound action. Furthermore, the impedance-based beating profile in response to compound treatment can provide mechanistic toxicity information regarding the target being modulated and may be able to flag pro-arrhythmic compounds. We believe the real-time and kinetic aspect of this technology combined with beat-to-beat measurement of cardiomyocyte contraction would make this instrument an important part of preclinical cardiac safety assessment.

Functional cardiotoxicity profiling and screening using the xCELLigence RTCA Cardio System.

Cardiovascular toxicity, a prominent reason for late-stage failures in drug development, has resulted in a demand for in vitro assays that can predict this liability in early drug discovery. Current in vitro cardiovascular safety testing primarily focuses on ion channel modulation and low throughput cardiomyocyte (CM) contractility measurements. We evaluated both human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and rat neonatal CMs (rat CMs) on the xCELLigence Cardio system which uses impedance technology to quantify CM beating properties in a 96-well format. Forty-nine compounds were tested in concentration-response mode to determine potency for modulation of CM beating, a surrogate biomarker for contractility. These compounds had previously been tested in vivo and in a low throughput in vitro optical-based contractility assay that measures sarcomere shortening in electrically paced dog CMs. In comparison with in vivo contractility effects, hiPSC-CM impedance had assay sensitivity, specificity, and accuracy values of 90%, 74%, and 82%, respectively. These values compared favorably to values reported for the dog CM optical assay (83%, 84%, and 82%) and were slightly better than impedance using rat CMs (77%, 74%, and 74%). The potency values from the hiPSC-CM and rat CM assays spanned four orders of magnitude and correlated with values from the dog CM optical assay (r(2 )= 0.76 and 0.70, respectively). The Cardio system assay has >5× higher throughput than the optical assay. Thus, hiPSC-CM impedance testing can help detect the human cardiotoxic potential of novel therapeutics early in drug discovery, and if a hazard is identified, has sufficient throughput to support the design-make-test-analyze cycle to mitigate this liability. 

An impedance-based cellular assay using human iPSC-derived cardiomyocytes to quantify modulators of cardiac contractility.

The ICH S7B guidelines recommend that all new chemical entities should be subjected to hERG repolarization screening due to its association with life-threatening "Torsades de Pointes" (TdP) arrhythmia. However, it has become evident that not all hERG channel inhibitors result in TdP and not all compounds that induce QT prolongation and TdP necessarily inhibit hERG. In order to address the limitations of the S7B/E14 guidelines, the FDA through a public/private partnership initiated the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative to examine the possible modification and refinement of the ICH E14/S7B guidelines. One of the main components of the CiPA initiative is to utilize a predictive assay system together with human cardiomyocytes for risk assessment of arrhythmia.

Multi-parametric assessment of cardiomyocyte excitation-contraction coupling using impedance and field potential recording: A tool for cardiac safety assessment.

Recent in vitro cardiac safety studies demonstrate the ability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to detect electrophysiologic effects of drugs. However, variability contributed by unique approaches, procedures, cell lines, and reagents across laboratories makes comparisons of results difficult, leading to uncertainty about the role of hiPSC-CMs in defining proarrhythmic risk in drug discovery and regulatory submissions. A blinded pilot study was conducted to evaluate the electrophysiologic effects of 8 well-characterized drugs on 4 cardiomyocyte lines using a standardized protocol across 3 microelectrode array platforms (18 individual studies). Drugs were selected to define assay sensitivity of prominent repolarizing currents (E-4031 for IKr, JNJ303 for IKs) and depolarizing currents (nifedipine for ICaL, mexiletine for INa) as well as drugs affecting multichannel block (flecainide, moxifloxacin, quinidine, and ranolazine). Inclusion criteria for final analysis was based on demonstrated sensitivity to IKr block (20% prolongation with E-4031) and L-type calcium current block (20% shortening with nifedipine). Despite differences in baseline characteristics across cardiomyocyte lines, multiple sites, and instrument platforms, 10 of 18 studies demonstrated adequate sensitivity to IKr block with E-4031 and ICaL block with nifedipine for inclusion in the final analysis. Concentration-dependent effects on repolarization were observed with this qualified data set consistent with known ionic mechanisms of single and multichannel blocking drugs. hiPSC-CMs can detect repolarization effects elicited by single and multichannel blocking drugs after defining pharmacologic sensitivity to IKr and ICaL block, supporting further validation efforts using hiPSC-CMs for cardiac safety studies.

Cross-Site Reliability of Human Induced Pluripotent stem cell-derived Cardiomyocyte Based Safety Assays Using Microelectrode Arrays: Results from a Blinded CiPA Pilot Study.

Yama A. Abassi

ACEA Biosciences, Inc

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells