Quantifying <i>in vitro</i>complement-dependent cytotoxicity (CDC) using advanced flow cytometry

Abstract Complement dependent cytotoxicity (CDC) is a key Fc mediated function of monoclonal antibody (mAb) therapeutics. mAb binding to antigen triggers complex molecular cascade with sequential recruitment serum proteins, eventuating in target cell lysis. Here we demonstrate CDC quantification streamlined, vitro, advanced flow cytometry assay. Anti-CD20 mAbs were incubated lines 96- or 384-well plates. Human (15%) was then added induce CDC. Cells labeled iQue® Cell Membrane Integrity (R/Red) Dye enable assessment death using the Advanced Flow Cytometry Platform. Induction by anti-CD20 correlated CD20 expression, most observed high-CD20 expressing Ramos cells. Maximal induced Rituximab and Truxima® 68% 70%, respectively. Comparatively, mid-CD20 Raji cells lower at 34% 24%. No negative Jurkat Another anti-CD20-IgG1 compared against two isotype mutants: IgG1fut (non-fucosylated) IgG1NQ (non-glycosylated). Their activity profiled towards three previously described effector functions: antibody-dependent cellular (ADCC); phagocytosis (ADCP) induction comparable between mAbs, however their ADCC ADCP differed. The did not exert activity, whilst showed more less than native. These data exemplify use quantify range mechanisms, highlighting potential profile libraries novel therapeutics minimal time.