Experimental Conditions for Innate Immune Stimulation of Oligonucleotide Therapeutics Using Human PBMC- Collaborative Study of the Consortium for Safety Evaluation of Oligonucleotide Therapeutics

Abstract

Oligonucleotide therapeutics are known to cause inflammatory adverse events such as influenza-like symptoms in humans as a class effect, and stimulation of the innate immunity system, including Toll-like receptors (TLRs), is suspected to be the underlying mechanism. Due to the species difference in the innate immunity, it is difficult to predict the effect in humans using animals. We investigated the experimental conditions to develop an in vitro assay using primary human peripheral blood mononuclear cells (PBMC) to detect innate immune activation potential in humans at an early stage of drug development. Freshly isolated PBMC from healthy adult volunteers were seeded in 96 well plates at 1x10^5, 2x10^5 and 4x10^5 cells/well and treated with toll-like receptor (TLR) agonists for 24 hours. Cytokine levels in the supernatant were determined. Poly(I:C) HMW (a TLR3 agonist, dsRNA) increased IL-6, IP-10, MCP-1 and IFN-α. R848 (a TLR7/8 agonist) increased IL-1β, IL-6, IL-8, IP-10, IFN-α, MCP-1 and TNF-α. ODN 2006 (a TLR9 agonist, ssDNA) increased IP-10, IFN-α, MCP-1 and IL-8, and ODN 2216 (a TLR9 agonist, ssDNA) increased IL-6, IP-10, IFN-α, MCP-1, TNF-α and IFN-β. Though the cytokine levels tended to increase in a cell density-dependent manner, the cytokine-increasing activities of the TLR agonists were detectable at almost all cell density used in this study. These results suggest that it is possible to evaluate the effect of oligonucleotide therapeutics on the activation of innate immunity by measuring the cytokines used in this study using human PBMCs.