Abstract
Monoclonal 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 nonradioactive 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 pro-apoptotic 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. © 2017 International Society for Advancement of Cytometry.
Highlights
Immunotherapy of cancer and other diseases has become one of the most promising and popular research areas in medicine [1]
The same label released from the target cells by cytolysis during antibody dependent cell-mediated cytotoxicity (ADCC) can be quantitated in the supernatant
Given the time needed to implement ADCC resulting in measurable membrane damage, first we tested the spontaneous release of the label from the target cells
Summary
Immunotherapy of cancer and other diseases has become one of the most promising and popular research areas in medicine [1]. One focus of these therapeutic approaches is how we can modify or improve the patient’s cellular immune system to cope with a specific disease. When antibody therapy using humanized monoclonal antibodies against tumors was first coined, parent antibodies were selected based on their in vitro antitumor effect. It became apparent that in addition to direct cell biological effects, antibody dependent cell-mediated cytotoxicity (ADCC), and other Fc fragment-associated functions such as complement-dependent cytotoxicity (CDC) and complement activation are if not more important in the antitumor therapeutic effect of these antibodies, when applied in the clinical setting
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