Identifying the potential for cytokine release is a critical aspect of preclinical safety assessment. Immuneed’s cytokine release assay (CRA) offers unmatched quality in evaluating the potential for adverse pro-inflammatory reactions following clinical infusion. Given the severe dangers posed by cytokine release syndrome (CRS), as evidenced clearly in the disastrous clinical trial of TGN1412, the value of predicting even slight responses cannot be underestimated.
Regulatory authorities demand an assessment of cytokine release potential, emphasizing the importance of human-based methods in determining potential immunotoxicity. However, even human-based methods differ greatly in their sensitivity. Traditional methods based on microwell plates are unable to detect low-to-moderate cytokine responses, and they have been shown to miss several key control compounds known to trigger cytokine release in humans.
Our CRA reliably detects the responses plate-based assays miss because our platform provides a low-background, physiologically relevant environment that is far closer to that of the actual human body. Based on circulating, fresh human blood, our platform allows your drug to be introduced within minutes rather than hours – and avoids the interference of anticoagulants. The latter, which are required with any amount of blood storage, increase the risk of a missed response by inactivating complement and cascade systems.
Finally, our CRA provides readouts within four hours. This perfectly mirrors the clinical situation, where reactions develop swiftly after drug administration and not following a lengthy incubation period. Between the high sensitivity and speed of our method, you can effectively assess the hazards of cell activation and cytokine release posed by your drug candidate.
Shown above is a direct comparison of Immuneed’s platform with two plate-based assays where either PBMCs with coated antibodies or whole blood with soluble antibodies were used for cytokine release assessment. It involves antibodies associated with a low risk of causing CRS, namely anti-integrin alpha 4 (1 µg/ml) and anti-EGFR antibody (200 µg/ml), as well antibodies associated with a high risk of CRS, namely anti-CD52 antibody (3 µg/ml) and anti-CD28 (aCD28). The level of cytokines was compared to vehicle and zero (blood directly after the acquisition). In the Immuneed assay, cytokine release was measured at a time point of 4 hours, while the plate-based assays required a 24-hour incubation time before readouts could be taken.
Therapeutic antibodies known to be associated with low and high risk of cytokine release-mediated clinical toxicity have been used to correlate the type and number of cytokines induced with the frequency of donor responses, creating a comprehensive profile of cytokine release. When Immuneed’s platform was used to test a TGN1412-like antibody,* it was shown that this antibody – but not the isotype-matched control – induced a clear cytokine release (n=33-38). The threshold for a positive antibody response was set at above the 95th percentile of the calculated ratio of vehicle/baseline value. Read more about the study in Fletcher Int Immunopharmacol. 2017 Oct 27;54:1-11.
*TGN1412 is an anti-CD28 monoclonal antibody that caused catastrophic systemic organ failure due to cytokine release in healthy volunteers in a clinical trial in 2006. (REF: Suntharalingham et. al. N Engl J Med. 2006 Sep 7;355(10):1018-28) https://www.nejm.org/doi/full/10.1056/NEJMoa063842