Characterizing the mechanism of action of a HER2xCD3 bispecific T-cell engager (BiTE) antibody in 2D and 3D immune cell killing assays using a combined live-cell imaging and flow cytometry workflow

Abstract

Abstract Solid cancers have proved to be a challenge for immunotherapies and targeted approaches are needed. Evaluation of these therapies using relevant models plays a large part in their development. A workflow that combines live-cell imaging and flow cytometry was used to characterize immune cell killing induced by a BiTE antibody. Immune cell co-culture assays were set up in 2D or 3D formats with green nuclear labeled AU565 HER2 positive breast cancer cells and various effector cell types. Immune cell activation was induced using HER2xCD3 BiTE or CD3/CD28 Dynabeads. Images were captured every 4 h with IncuCyte and quantified for numerous cellular parameters. Cytokine analysis of supernatant samples was performed using a multiplex QBead panel alongside assessment of immune cell activation markers using the iQue3. Quantification of PBMC mediated cytotoxicity showed a comparable 70 ± 2% and 62 ± 1% reduction in spheroid growth with BiTE (10 ng mL−1) and Dynabead (1:1 bead-to-cell ratio) activation. In contrast to Dynabeads, BiTE activation did not induce immune cell shape change or upregulation of T cell activation markers; 1 ± 0.2% CD69 expression was observed on CD3+CD8+ cells, compared to 53 ± 0.6%. Further evaluation found BiTE activated isolated CD56+ cells induced less target cell death compared to PBMCs and granzyme B secretion was not detected, suggesting a lack of involvement of NK cells. Release of IL-6 and CCL2 cytokines indicate immune cell mediated cytotoxicity via an inflammatory pathway. These data suggest HER2xCD3 BiTE activity may not be mediated through T cell or NK cell subtypes. At present the mechanism of target cell killing is unclear, however further studies combining imaging and flow cytometry may provide the required insight.