Accelerating ADCP screening using the iQue® Advanced Flow Cytometry Platform

Abstract

Abstract Antibody-dependent cellular phagocytosis (ADCP) is an immune process stimulated by monoclonal antibodies (mAbs) that promotes specific recruitment of phagocytes to eliminate targeted cancer cells. In vitro functional assays are used extensively in drug development to characterize the effects of mAbs on a range of mechanisms of action, including ADCP. We describe a high throughput assay for ADCP quantification via co-localization analysis of labeled target cells with CD14 positive effector cells. Target cells were incubated in 96 or 384 well plates with clinically relevant mAbs prior to addition of PBMCs or isolated immune cells (e.g. monocytes or macrophages). Cells were analyzed using the iQue®3 Advanced Flow Cytometer and inbuilt software. Various effector:target (E:T) cell ratios were used to validate the assay. Maximal ADCP of non-adherent Ramos target cells by PBMCs in the presence of Truxima was 43 ± 2% with an E:T ratio of 20:1, reducing to 19 ± 6% with a 5:1 ratio. Further pharmacological data was obtained for adherent AU565 cells, comparing the response between anti-HER2 mAb isotypes. The potency of response to anti-HER2-IgG1 mAb (Trastuzumab) was five times greater than to another native human isotype, anti-HER2-IgA2, with EC50 values of 6.6 ng/mL and 35 ng/mL, respectively. These data are in line with previous characterizations of isotype effector functions which showed increased ADCP functionality with IgG1 compared to IgA2 mAbs. The iQue® platform provides a solution for rapid profiling of ADCP effector function in both adherent and suspension target cell models. This accelerates antibody discovery by highlighting potential efficacy and toxicity of antibody drug candidates, leading to greater productivity and insight.