Cancer Gene Therapy https://doi.org/10.1038/s41417-018-0038-x BRIEF COMMUNICATION Quantification and functional evaluation of CD40L production from the adenovirus vector ONCOS-401 Lukasz Kuryk 1,2 ● Anne-Sophie W. Møller 3 ● Magnus Jaderberg 3 Received: 7 May 2018 / Revised: 18 May 2018 / Accepted: 24 May 2018 © Springer Nature America, Inc. 2018 Abstract 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. Antigen presentation Antigen presentation, which involves the display of intra and extracellular antigens complexed with major histo- compatibility complexes (MHCs) on the surfaces of antigen presenting cells (APCs) such as dendritic cells (DCs), macrophages, and B cells, to effector cells such as T cells (helper and cytotoxic), is key to the development of adap- tive immunity and defense against tumors. APCs are clas- sified as professional and non-professional. Those that express MHC class II molecules along with co-stimulatory molecules and pattern recognition receptors are called pro- fessional antigen-presenting cells [1], which efficiently internalize antigens either by phagocytosis (macrophages and dendritic cells) or receptor-mediated endocytosis (B cells), process the antigens into peptide fragments, and display them as MHC-bound peptides on their surfaces. The T cell recognizes and interacts with the antigen-class II MHC molecule complex via T cell receptors (TCRs) [2]. Subsequently, an additional co-stimulatory signal is pro- duced by the APCs, leading to activation of the T cell. CD40 Cluster of differentiation 40 (CD40) is a co-stimulatory pro- tein found on APCs that is required for their activation. The binding of CD40L (CD154) on T H cells to CD40 activates APCs and induces a variety of downstream effects. CD40 is a member of the tumor necrosis factor (TNF) receptor super- family and is essential in mediating a wide range of immune and inflammatory responses, including T cell-dependent immunoglobulin class switching, memory B cell develop- ment, and germinal center formation [3]. The TNFR-receptor associated factor adaptor proteins TRAF1, TRAF2, TRAF6, and possibly TRAF5 interact with this receptor and act as mediators of the signal transduction [4–8]. * Lukasz Kuryk lukasz.kuryk@targovax.com 1 Targovax Oy, Clinical Science, Helsinki, Finland 2 Department of Virology, National Institute of Public Health–National Institute of Hygiene, Warsaw, Poland 3 Targovax ASA, Clinical Science, Oslo, Norway 1234567890();,: 1234567890();,: