Quantification and functional evaluation of CD40L production from the adenovirus vector ONCOS-401.

Adaptive immunity involves activation of T cells via antigen presentation by antigen presenting cells (APCs) along with the action of co-stimulatory molecules and pattern recognition receptors. Cluster of differentiation 40 (CD40) is one such costimulatory molecule that is expressed on APCs that binds to CD40 ligand (CD40L) on T helper cells and activates a signaling cascade, subsequently resulting in a wide range of immune and inflammatory responses. Considering its important role in regulation of immune response, CD40/40 L has been used for developing antitumor vaccines. In this study, we developed methods for evaluating and quantifying the activity of CD40L expressed from an adenovirus vector ONCOS-401. Our results show that the ONCOS-401 vector produces functional CD40L, which can bind and activate a NF-κB-dependent signaling cascade, leading to secreted embryonic alkaline phosphatase reporter production in HEK293-BLUE cells. In addition, quantification of CD40L production using enzyme-linked immunosorbent assay and HEK-293 BLUE reporter cells showed reproducibly higher recovery of CD40L from ONCOS-401 than from the negative control vector or uninfected cells with consistent inter and intra-assay precision. Thus, a rapid and easy method for quantifying and assessing CD40L production and activity from adenovirus vectors would support the assessment of efficacy of the vector for gene therapy - this was the objective of our study.