August 1, 2016 (Vol. 36, No. 14)

Brandon J. Lamarche Research Scientist ACEA Biosciences
Biao Xi Research Scientist ACEA Biosciences
Fabio Cerignoli Research Scientist ACEA Biosciences

Immune Cell-Mediated Killing Kinetics and Efficacy Analysis in Real-Time without the Use of Labels

The high specificity and potent cytotoxicity of innate and adaptive immune system effector cells make them promising agents for extirpating cancer cells or, at the very least, controlling disease progression. Though the list of cell- and antibody-mediated cancer immunotherapies is growing, many of these are efficacious for only a subset of patients. 

Realizing the full therapeutic potential of this field will require: (1) continued elucidation of the mechanisms underlying cancer cell recognition and immune cell-mediated killing, and (2) an ability to screen immunotherapy constructs/conditions (CARTs, checkpoint inhibitors, etc.) using patient-derived effector and/or target cells to identify optimal treatment regimens (i.e. theranostics).

Fundamental to both of the above is the ability to quantitatively monitor the potency of immunotherapy-mediated killing of target cells under controlled conditions in vitro.  Traditional cell killing assays suffer from drawbacks that prevent them from meeting this need efficiently.

Shortcomings of Traditional Assays

Immune cell-mediated killing can be studied by measuring the activation of effector cells or their secretion of cytotoxic molecules (perforin, granzymes, etc.). While these readouts are indeed useful for reductionistic biochemical characterization, they don’t necessarily correlate with target cell killing efficiency, which is the ultimate measure of therapeutic efficacy. Assays focused on the response of target cells primarily monitor the release of either previously added labels (such as 51Cr or fluorescent dyes) or endogenous biomolecules (GAPDH, LDH, etc.) upon target cell lysis. 

Besides the potential artifacts associated with using exogenous labels, the time frame over which such labels are useful is extremely narrow (due to the perpetual leakage of label out of target cells). Moreover, release assays as a whole suffer from low sensitivity, low efficiency/throughput, and the fact that only endpoint data (mere snapshots in a cell response continuum) is produced.


Figure 1. Monitoring immune cell-mediated killing of adherent cancer cells in real-time using xCELLigence.

xCELLigence Real-Time Cell Analysis (RTCA)

ACEA’sxCELLigence® Real-Time Cell Analysis (RTCA) instruments utilize gold microelectrodes embedded in the bottom of microtiter wells to noninvasively monitor the status of adherent cells using the principle of cellular impedance. In short, adherent cells act as insulators, impeding the flow of an alternating microampere electric current between electrodes. This impedance signal is measured automatically, at a frequency defined by the user (every 10 seconds, once per hour, etc.), and provides an extremely sensitive readout of cell number, cell size/shape, and cell-substrate attachment strength. 

In contrast to adherent cancer cell targets, immune effector cells are nonadherent and therefore do not produce an impedance signal in and of themselves. Because of this, when adherent cancer cells are treated with effectors (NK cells, T cells, CARTs, oncolytic virus, checkpoint inhibitors, bispecific antibodies, BiTEs, etc.) it is possible to selectively monitor the kinetics of cancer cell destruction in real-time. Besides facile quantification of serial killing and anergy, other distinguishing features of this technology include enhanced sensitivity, the preclusion of labels, simple workflow, compatibility with orthogonal assays, and continuous kinetic measurement of cancer cell health/behavior.  The xCELLigence immunotherapy workflow is outlined in Figure 1.

Quantifying the Potency of Immunotherapies Targeting Solid Tumors

Using the assay format shown in Figure 1, target MCF7 breast cancer cells were treated with NK-92 cells at different E:T ratios (Figure 2A). Within 5 hours of effector cell addition differential responses begin to be observable in the impedance traces. The untreated breast cancer cells continue to proliferate and approach confluence (with the impedance signal beginning to plateau).

In contrast, at the highest E:T ratios the target cells are killed within the first few hours, resulting in an impedance signal that approaches zero, signifying complete cytolysis of the target cells. At intermediate E:T ratios (1.25:1 and 2.5:1) the cancer cells are effectively killed but with significantly delayed killing kinetics. In a second example, killing of PC3 prostate cancer cells by PBMCs was evaluated in the presence of a bispecific T cell engager (BiTE) with specificity for both CD3 on T cells and EpCAM on PC3 cells. Under these conditions, in the absence of the BiTE PBMCs have little if any effect on the target cells. In contrast, the BiTE stimulates PC3 cell killing in a dose dependent manner.  


Figure 2. Using xCELLigence to quantify solid tumor cell killing. (A) NK-92 cell-mediated killing of MCF7 breast cancer cells at different E:T ratios. (B) Killing of PC3 cells by PBMCs in the presence of an EpCAM-specific BiTE.

A Novel Killing Assay for B Cell Cancers

With dozens of peer-reviewed studies published over the past decade, the utility of xCELLigence RTCA for probing the efficacy of immunotherapies targeting solid/adherent cancers is now firmly established.  However, ~10% of all cancers are liquid in nature, are therefore nonadherent, and cannot be directly monitored by the standard impedance assay. 

Moreover, because they are readily accessible within the bloodstream and aren’t confounded by the microenvironment complexities/heterogeneities associated with solid tumors, liquid cancers (B cell cancers in particular) are prominent immunotherapy targets at present. To help accelerate research in this area, ACEA recently developed an xCELLigence Immunotherapy Kit focused on B cell killing.

For this purpose, the wells of ACEA’s electronic microtiter plate are precoated with a B cell-specific antibody, enabling B cells to be immobilized on the plate bottom (Figure 3A). Whereas antibody immobilized B cells generate a robust impedance signal and proliferate to the point of confluence (resulting in a plateaued impedance signal), the growth of untethered B cells is essentially undetectable (Figure 3B). Importantly, with or without antibody coating of the wells effector cells, such as the NK-92 cells used here, produce minimal signal on their own (Figure 3B).

Addition of NK-92 cells on top of immobilized B cells results in target cell death in a dose-dependent manner (Figure 3C).  The tethering and killing behaviors seen in Figures 3B & 3C have been observed in all three of the B cell lines tested (Daudi, Raji, and Ramos), for multiple effector cell types (NK, T, CART), and for combination therapies (CART + checkpoint inhibitors, etc.). 

An important question is whether the physical immobilization of B cells via antibody tethering affects the efficiency with which they are killed. To assess this, side-by-side four hour assays were performed for NK-92 cell-mediated killing of Raji B cells that were either immobilized (analyzed by xCELLigence) or in suspension (analyzed by flow cytometry). As seen in Figure 3D, the killing trends observed by these two methods correlate perfectly, with the magnitude of % cytolysis varying minimally. This is consistent with a large number of publications showing that xCELLigence data consistently recapitulates data obtained by traditional assays. 


Figure 3. The xCELLigence Immunotherapy Kit for monitoring B cell killing. (A) Antibody-mediated tethering of B cells to electrodes. (B) Selective proliferation of Daudi B cells on electrodes coated with B cell-specific antibody. Nonadherent NK-92 effector cells produce minimal signal. (C) Raji B cell killing by NK-92 cells. (D) The impact of B cell immobilization on killing efficiency. Raji B cells, either immobilized by antibody or in suspension, were treated with different numbers of NK-92 cells. % cytolysis was determined by xCELLigence (tethered) or flow cytometry (in suspension).

Conclusion

In a label-free manner, xCELLigence instruments provide a quantitative kinetic assessment of immune cell-mediated killing of both solid and liquid tumor cells. This functional assay is currently being used for evaluating/optimizing combination therapies, and for the development of adoptive cell therapies and engineered antibodies. Beyond the arena of R&D, we envision xCELLigence being utilized as a quality control assay for manufactured immuno-oncology therapies.

Brandon J. Lamarche (blamarche@aceabio.com), Biao Xi, and Fabio Cerignoli are
research scientists at ACEA Biosciences.

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