Quantitating ADCC against adherent cells: Impedance‐based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response

Abstract

Running headline (45 chars. incl. space) Impedance-based cell analyzer for ADCC Conflicts of interest:None of the authors have conflicts interest. AbstractMonoclonal antibody-based immunotherapeutics will dominate Pharma's next generation of blockbuster drugs, and Fc-associated functions, including antibody dependent cellular cytotoxicity (ADCC) are among the highly desired activities mediated by these antibodies. Therefore, quantitative evaluation of ADCC is required during drug development. Our objective was to find the most suitable and reliable non-radioactive method for quantitative analysis of in vitro ADCC against adherent cells, which often serve as models for solid tumors. The test system was comprised the HER2 positive JIMT-1 cells targeted by the specific therapeutic antibodies trastuzumab (Herceptin ® ) and pertuzumab (Perjeta ® ). These cells are resistant to the direct biological effects of these antibodies, and therefore allow the isolated assessment of ADCC. We compared fluorescein diacetate (FDA) and carboxyfluorescein diacetate succinimidyl ester (CFSE) release as a fluorescent alternative to 51 Cr release; propidium iodide (PI) uptake revealing increased membrane permeability; the PanToxiLux assay measuring ADCC induced proapoptotic protease activity in flow cytometry; and an impedance-based real time cell adhesion test. We found that release assays are compromised by high spontaneous release of the label. PI uptake could not differentiate well between spontaneous NK activity and specific ADCC. The PanToxiLux assay, besides allowing for shorter assay times, offers improvement over the previous approaches in distinguishing spontaneous and antibody mediated NK action, but, probably owed to the prolonged detached state of adherent target cells, only at highly saturating antibody concentrations. In the case of adherent target cells, impedance-based cell analysis attains functional information exclusively on the target cells without having to label them for distinguishing from effectors or assay readout. It also allows continuous monitoring for days, and specifically detects target cell detachment, as the final functional consequence of ADCC. The sensitivity of this method even allows for quantitating the additivity and saturability of ADCC as a function of antibody concentration. We conclude that impedance-based assays are the most sensitive for quantitatively assessing in vitro ADCC on adherent target cells.

The multitargeted receptor tyrosine kinase inhibitor sunitinib induces resistance of HER2 positive breast cancer cells to trastuzumab-mediated ADCC

“…This cancer cell line was selected as it is known to be resistant to trastuzumab thus representing a clinically challenging cellular model. ECIS proved to be superior to some alternative methods for the quantitation of ADCC in this model [ 14 ]. Therefore, the first optimization assays were run on the ECIS platform (Fig.…”

Improvements in Flow Cytometry‐Based Cytotoxicity Assay

“…In early studies (8,9), CFSE was shown to be released spontaneously from CFSE‐labeled cells, especially during early time points within 3 or 4 h following initial labeling. In accord with this finding, our data show a significant decrease in CFSE intensity after 4‐h culture and when the target killed by effector cells.…”

“…In the current study, carboxyfluorescein diacetate succinimidyl ester (CFSE), a dye frequently utilized in both in vitro cell proliferation and in vivo cell tracking analyses was used to label the target cells and Hoechst 33258 was used to stain the cell membrane‐compromised cells. As with other cell trace dyes, CFSE has been reported with problems of spontaneous release and leakage from the initial labeling (8,9). A clear discrimination between CFSE labeled target cells and unstained effector cells was shown by analyzing the fluorescence intensity of CFSE‐labeled K562 cells cocultured with PBMNCs (8).…”

Enhancing antibody-dependent cell-mediated cytotoxicity: a strategy for improving antibody-based immunotherapy

“…A potential drawback of relying on reporter molecules is that they may spontaneously release even from live cells. Impedance-based analysis avoids this issue and has shown promise as a quantitative measure of cell-mediated cytotoxicity that can continuously measure ADCC over time [40]. More recently, scientists have recognized the need for a sensitive, reliable, and accurate cytotoxicity assay that is compliant with regulatory requirements.…”