Background
Immune system primary effector cells, such as macrophage and natural killer cells, are crucial in combating cancer through mechanisms such as antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC). Both ADCP and ADCC are key mechanisms in antibody-based cancer immunotherapies, beginning when antibodies bind to Fc gamma receptors (FcRs) on the immune cells, triggering a cascade of events that lead to the downstream cellular response.
Methods
We have developed stable luminescent plate-based reporter bioassays to measure the binding affinity and functional potency of antibodies in response to various antigenic targets. For each assay, an 8-point dose response curve was completed to identify EC50 values of relevant biologics. All luminescent readings were measured on a GloMax Discover Luminescent Reader. Biologics tested are highlighted in each figure.
Results
Binding affinities of anti-CD20 rituximab IgG variants were tested in the presence of CD20+ target cells (Raji) against single FcgR based reporter bioassays. Rank order binding affinities of Cetuximab, Panitumumab and Pembrolizumab were also rank ordered using a Lumit based FcgR binding immunoassays. A physiologically relevant FcgR bioassay was also qualified using Cetuximab and Rituximab against A549 and Raji target cells. Fold inductions greater than 10 were achieved for all FcgR bioassays. Potency assays using primary human PBMCs and macrophages were developed using our HiBiT Targeted Cell Killing platform to further define the physiologically relevant EC50 values of indicated biologics (figure 5). Differentiated macrophages were used in a thaw-and-use format against Ramos or SK-BR-3 target cells to measure ADCP activity of ritixumab and trastuzumab respectively. Similarly, primary human PBMCs were Ramos and SK-BR-3 cells to compare the EC50 values of the same biologic across different effector cell types. Trastuzumab demonstrated an approximate 100-fold higher potency for ADCP activity in primary macrophages compared to PBMC activity.
Conclusions
These bioassays are both robust and user-friendly and adhere to ICH guidelines with pre-qualification. Collectively, these innovative reporter bioassays furnish a sturdy, high-throughput toolkit to expedite the exploration and advancement of immunotherapies targeting macrophage effector functions.