Abstract 7276: Accurate prediction of clinical immunotoxicity of oligonucleotide therapeutics using ex vivo circulating fresh human whole blood test system

Abstract Few oligonucleotide (ONDs) therapeutics are currently approved for cancer treatment due to their ability to modulate gene expression and protein function and their reduced toxicity compared to traditional chemotherapeutics. Existing preclinical animal models demonstrate varied sensitivities to ONDs for detecting clinical immunotoxicity. Non-Human Primates (NHPs) show a higher sensitivity to phosphorothioate oligonucleotides and tend to overpredict the risk of complement activation. Additionally, NHPs were unable to predict antisense oligonucleotide (ASO) induced cytokine release prior to first-in-human (FIH) trials. A notable example is ISIS 353512 (a 2′ methoxyethyl ASO, 2’ MOE ASO), which unexpectedly induced IL-6 release in human volunteers. Here, we describe the use of a physiologically relevant ex vivo model based on circulating fresh human whole blood, to accurately predict the clinical immunotoxicity of two FDA-approved ONDs: imetelstat (OND) and inotersen (a 2’ MOE ASO). Both ONDs were tested in six donors at three concentrations each 1, 5 and 25 uM), covering the clinical Cmax for both inotersen (at the lowest concentration) and imetelstat (at the highest concentration). This ex vivo test system enables the study of early human immune responses by preserving all blood immune cells, immunoglobulins, and active coagulation and complement cascade systems. The system has previously been validated for predicting infusion reactions of antibody-based drugs prior to FIH trials.Both inotersen and imetelstat are known to cause clinical side effects such as flu-like symptoms. The model system reflects these symptoms with a significant increase in pro-inflammatory cytokine release (IL-8, and TNF-α) observed at the highest dose of both ONDs. Imetelstat demonstrated significant activation of granulocytes and monocytes, correlating with the observed high release of IL-6 and IL-8 cytokines in this model system and its intended action against myeloid tumors. In contrast, inotersen only triggered B cell activation, correlating with the IL-6 release caused by Toll-like receptor (TLR) stimuli in B cells, a response previously observed in patients treated with ISIS 353512. Previous studies indicate that both the investigated ONDs result in complement activation in NHPs, while imetelstat alone caused significant complement activation in the clinic. In line with clinical observations, the high dose of imetelstat (but not inotersen) in our test system showed significantly increased activation of complement factors (C3a, and C5a). Additionally, imetelstat caused a decreasing trend with platelet counts, which correlates with its clinical manifestations of thrombocytopenia. Taken together, the data show that our ex vivo circulating whole blood test system has the potential to predict human immunotoxicity responses to ONDs before FIH trials. Citation Format: Erika A. Fletcher, Gunilla Törnqvist, Helena Harlin Laven, Sakthi Srinivasan. Accurate prediction of clinical immunotoxicity of oligonucleotide therapeutics using ex vivo circulating fresh human whole blood test system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7276.