Biomarkers measuring the activity of Toll-like receptor ligands in clinical development programs

Efforts to develop therapeutic approaches based on stimulation of Toll-like receptor (TLR) pathways have increased in recent years (Nat Med 13:552-559). The effectiveness of TLR agonists is currently being tested in diseases such as cancer, asthma, allergic rhinitis, and viral infections (J Clin Invest 117: 1184-1194; Blood 105: 489-495; Proc Am Thorac Soc 4:289-294; N Engl J Med 355:1445-1455; Am J Respir Crit Care Med 174:15-20). For a successful clinical trial program, it is important to know whether the therapeutic agent under development is both pharmacologically active and activating the intended pathway in humans. A biomarker reflecting this in an accurate and sensitive manner greatly facilitates dose/regimen-finding and is a "must-have." In this chapter, we describe a polymerase chain reaction (PCR)-based method that quantifies gene expression levels indicative of TLR-stimulation in human samples. We focus on genes specifically induced in an IFN-alpha-dependent manner, as this pathway is activated after stimulation of both TLR-7 and TLR-9. We demonstrate that IFN-alpha-inducible gene expression levels can be successfully applied in a clinical trial setting as a marker of drug activity in a variety of human samples, including peripheral blood mononuclear cells (PBMC), cells derived from the airways, as well as cells from induced-sputum.