Table 6.
Advanced in vitro antitumor efficacy assays
| Assay | Investigation of focus | Description | Recommended assay(s) |
|---|---|---|---|
| Antigen stress test | Adaptive resistance, cytotoxicity following functional exhaustion | Repeated antigen stimulation by multiple rounds of target cell addition to investigate effector cell cytotoxicity under high antigen stress | 51Cr, BLI, impedance, or flow cytometry assay* |
| Differential cytotoxicity on target cells with heterogeneous antigen expression | Cytotoxicity on target cells with antigen heterogeneity (low and high), on-target/off-tumor toxicity on normal cells with very low antigen expression | Cytotoxicity on target cells with variable antigen expression to investigate the correlation between antigen density and cytotoxicity and any unanticipated cytotoxicity of activated effector cells on normal cells with very low antigen expression | If target cells with different antigen expression levels are assessed individually (one cell type per well): 51Cr, BLI, impedance, or flow cytometry assay. If target cells are plated as mixture: flow cytometry assay. |
| Multi-CAR cytotoxicity | Antigen heterogeneity, antigen escape | Cytotoxicity assessment of effector cells with multiple CAR constructs targeting different antigens simultaneously | 51Cr, BLI, impedance, or flow cytometry assay |
| Cytotoxicity in the presence of soluble factors | Immunosuppression by soluble factors known to be present in the tumor environment | Cytotoxicity in the presence of various doses of single or multiple inhibitory or excitatory cytokines and/or soluble factors | 51Cr, BLI, impedance†, or flow cytometry assay |
| Cytotoxicity in the presence of immune cells | Immunosuppression by immune cells known to be present in the tumor immune environment | Cytotoxicity in the presence of various ratios of single or multiple immunomodulatory immune cells | Flow cytometry assay to investigate cytotoxicity against heterogenous (target) cell populations |
It is imperative that the target cells are completely lysed before the effector cells are pooled from the coculture condition and exposed to freshly plated target cells. Transfer of viable target cells to the next round of antigen stimulation might change the E:T ratio, potentially confounding comparison across different effector constructs.
Addition of soluble factors to the impedance assay may impact tumor cell morphology and cell index. It is critical to include a control to account for soluble factor–induced changes in cell index in absence of effector cells. As an example, treatment of target cells with soluble factors such as TGFβ or TNFα can alter the size, adherence, and cell surface receptor expression on target as well as T cells, and it is important to include controls at different concentrations and durations of incubation.
CAR, chimeric antigen receptor.