836. Changes in Maturation Profiles of Dendritic Cells Transduced with a CD40-Targeted Adenoviral Vector
Authors: Disha A. Mody, Alexander V. Pereboev, Don A. Sibley, David T. Curiel, J. Michael Mathis
Journal: Molecular Therapy (2006)

Introduction: Adenovirus (Ad)-mediated transduction of dendritic cells (DCs) is inefficient because of the lack of the primary Ad receptor, CAR. CD40 is a surface marker expressed by DCs that plays a crucial role in their maturation and subsequent stimulation of T cells. DC infection with Ad targeted to the CD40 results in increased gene transfer. Recently, we characterized a new model using a mouse ovarian carcinoma cell line (MOVCAR) that expresses the SV40 large T-Ag and forms tumors in syngeneic immunocompetent B6C3F1 mice. The SV40 large T-Ag is highly immunogenic, inducing both antibody and cytotoxic T lymphocyte (CTL) responses. Since this antigen is synthesized in MOVCAR cells, the SV40 large T-Ag is an attractive candidate as a model system for the development of a DC-targeted cancer vaccine. We describe the further characterization of the CD40-targeting approach using an adapter molecule that bridges the fiber of the Ad5 to CD40 on mouse DC. This adapter molecule, CFm40L, consists of the ectodomain of CAR genetically linked via a trimerization motif to the extracellular domain of mouse CD40 ligand. We have demonstrated that DCs transduced with a CD40-targeted Ad vector expressing SV40 T-Ag (Ad5-SV40-TAg) showed an increased expression of cell surface activation markers and were effective in inducing an antigen-specific CTL response. To examine CD maturation induced by transduction with the CD40-targeted Ad vector, we examined changes in a panel of cytokines and chemokines. In addition, we examined the kinase signalling pathways involved in cytokine regulation. Methods: We used a Bio-Plex assay was performed on supernatants from untransduced DCs and DCs transduced with untargeted Ad and CD40-targeted Ad5 to determine changes in production of a panel of 23 cytokines and chemokines at 24, 48, and 72 hours post infection. We uses a Kinexus phospho-antibody screening system on cell lysates from untransduced DCs and DCs transduced with untargeted Ad and CD40-targeted Ad to examine changes in phosphorylation / activation of 34 different protein phospho-kinases. Results: DCs transduced with a CD40-targeted Ad showed increased secretion of the cytokines IL-1A, IL-1B, IL-6, and IL-12, as well as chemokines MIP-1A and RANTES compared to untransduced DCs or DCs transduced with untargeted Ad. Similarly, DCs transduced with a CD40-targeted Ad showed a decreased secretion of cytokine KC. DCs transduced with a CD40-targeted Ad showed increased phosphorylation of MEK1, Erk1, Erk, and STAT1(T385), and decreased phosphorylation of Akt1, compared to untransduced DCs or DCs transduced with untargeted Ad. In contrast, DCs transduced with a CD40-targeted Ad showed an inhibition of Src, Gsk3a, Gsk3b, and STAT1(S727) phosphorylation compared to DCs transduced with untargeted Ad. Conclusions: We demonstrated Ad-mediated CD40-targeted gene transfer to murine DCs using an adapter molecule CFm40L promotes DC maturation with induction of a complex signaling cascade accompanied by characteristic changes in cytokine production.