Maturation of Monocyte-Derived Dendritic Cells Induced by Dual Targeting of Endocytosis Receptor and CD40

Abstract 4293

Dendritic cells (DC) are professional antigen presenting cells, which can induce and regulate adaptive immune responses. Hence, DC are attractive candidates for anti-tumor vaccination strategies. The aim of this study was to create recombinant bispecific Fab to target and activate DC, by using derivates of monovalent agonistic CD40 scFv and endocytosis receptors FcγRI (CD64), FcαR (CD89), DEC205 (CD205), as well as DC-SIGN (CD209), respectively. The recombinant bispecific molecules were expressed in the eukaryotic cell line Hek 293T, enriched to homogeneity by affinity chromatography and analyzed for specific binding to the targeted receptors. Functional analysis of these molecules indicated, that concurrent engagement of CD40 and endocytosis receptors expressed by immature IL-4/GM-CSF cultured monocytes-derived DC induced maturation of these DC. This was suggested by changes of DC phenotype, especially enhanced surface expression of co-stimulatory molecules like CD80 and CD86, and gain of CD83 expression as measured by flow cytometry. To particularly analyze the T cell activation properties of these DC, MACS enriched T cells of autologous donors were co-cultivated with antigen-loaded and subsequently maturated DC for seven days. After restimulation of T cells, flow cytometric detection of intracellular IFN-γ levels and surface expression of CD69 used as markers for T cell activation implies, that these DC exhibit elevated properties to activate antigen specific T cells. In contrast, a bispecific [CD64scFv × CD40scFv] Fab used as control failed to induce maturation of DC lacking CD64 expression, which may be due to abrogation of bivalent binding of this molecule and therefore CD40 cross-linking. Moreover, dual targeting with these novel immunoconstructs when linked to peptides of a tumor-associated antigen may allow direct antigen delivery to DC in combination with DC activation, and thus anti-tumor vaccination in vivo.