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001-es BibID:	BIBFORM073405
035-os BibID:	(WoS)000413301700011 (Scopus)85030639366
Első szerző:	Tóth Gábor (általános orvos)
Cím:	Quantitating ADCC against adherent cells : impedance-based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response / Gábor Tóth, János Szöllősi, György Vereb
Dátum:	2017
ISSN:	1552-4922 1552-4930
Megjegyzések:	Monoclonal antibody-based immunotherapeutics will dominate Pharma's next generationof blockbuster drugs, and Fc-associated functions, including antibody dependent cellularcytotoxicity (ADCC) are among the highly desired activities mediated by these antibodies.Therefore, quantitative evaluation of ADCC is required during drug development. Ourobjective was to find the most suitable and reliable nonradioactive method for quantitativeanalysis of in vitro ADCC against adherent cells, which often serve as models for solidtumors. The test system was comprised the HER2 positive JIMT-1 cells targeted by thespecific therapeutic antibodies trastuzumab (HerceptinVR) 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) andcarboxyfluorescein diacetate succinimidyl ester (CFSE) release as a fluorescent alternativeto51Cr release; propidium iodide (PI) uptake revealing increased membrane permeability;the PanToxiLux assay measuring ADCC induced pro-apoptotic protease activity in flowcytometry; and an impedance-based real time cell adhesion test. We found that releaseassays are compromised by high spontaneous release of the label. PI uptake could not differentiatewell between spontaneous NK activity and specific ADCC. The PanToxiLuxassay, besides allowing for shorter assay times, offers improvement over the previousapproaches in distinguishing spontaneous and antibody mediated NK action, but, probablyowed to the prolonged detached state of adherent target cells, only at highly saturatingantibody concentrations. In the case of adherent target cells, impedance-based cell analysisattains functional information exclusively on the target cells without having to label themfor distinguishing from effectors or assay readout. It also allows continuous monitoringfor days, and specifically detects target cell detachment, as the final functional consequenceof ADCC. The sensitivity of this method even allows for quantitating the additivity andsaturability of ADCC as a function of antibody concentration. We conclude thatimpedance-based assays are the most sensitive for quantitatively assessing in vitro ADCCon adherent target cells.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Antibody Dependent Cellular Cytotoxicity nonradioactive ADCC assay adherent cell impedance-based cell analyzer PanToxiLux PI/CFSE cytometric ADCC assay
Megjelenés:	Cytometry Part A. - 91 : 10 (2017), p. 1021-1029. -
További szerzők:	Szöllősi János (1953-) (biofizikus) Vereb György (1965-) (biofizikus, orvos)
Pályázati támogatás:	K119690 OTKA TAMOP-4.2.2.A-11/1/KONV-2012-0025 TÁMOP GINOP-2.3.2-15-2016-00020 GINOP Gedeon Richter Plc. Egyéb UNKP-16-3-IV Egyéb
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