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