Work

52 Immunotoxicity assessment of biotherapeutics using the NAM ID.Flow®: an ex vivo whole blood assay under continuous flow

Year: 2026

Type: conference-abstract

Abstract: Background Assessing the immunotoxicity of advanced biotherapeutics and translational data currently available from in vitro and in vivo Methods to the clinic can be challenging. In particular, the limited physiological relevance of many models makes it difficult to assess the risks of tested drug candidates. Here, we used the Novel approach methodology (NAM), ID.Flow®, an ex vivo test system mimicking human blood in circulation.Methods In this study, we selected two antibodies (alemtuzumab and cetuximab) for further investigation, aiming to identify the relevance and sensitivity of our NAM test system, ID.Flow® with regard to cytokine release and complement activation. Fresh whole blood was collected from healthy volunteers using an open system, with a minimal addition of anti-coagulant. The blood was transferred to the ID.Flow system where it was kept in constant circulation. We assessed the immunotoxicity of these therapeutic antibodies in a larger cohort and compared our test system to other commonly used in vitro-based assays.Results We measured cytokine release in plasma for IFN-γ, IL-2, IL-6, IL-8, and TNF-α. After 4 hours, no differences in cytokines were seen when comparing vehicle control (PBS) to cetuximab. Alemtuzumab increased the levels of IFN-γ, IL-6, IL-8, and TNF-α compared to PBS, confirming clinical data. In ID.Flow®, there was a greater separation of IFN-γ and IL-8 release between low- and high-risk antibodies when compared to the plate-based whole blood assays. ID.Flow® also exhibited less Background and less variability. In these same runs we saw that alemtuzumab but not cetuximab induced C3a (2.1-fold vs. PBS) and C5a (2.5-fold vs. PBS) in ID.Flow®.Conclusions In conclusion, we show that the NAM ID.Flow® is a sensitive assay that can be used to detect even low to moderate clinically relevant unwanted immunotoxic responses of biotherapeutics with higher sensitivity over conventional in vitro models. Therapeutic antibodies, such as alemtuzumab and cetuximab exhibited a similar toxicity profile within ID.Flow® to the observed clinical findings. What makes this system unique is the ability to simultaneously measure multiple immune-toxicological readouts and provide comprehensive human specific data for candidate selection and dose selection before IND.

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0052