Conventional dendritic cell (DC) vaccine strategies, in which DCs are loaded with antigens , suffer biological issues such as impaired DC migration capacity and laborious GMP production procedures. In a promising alternative, antigens are targeted to DC-associated endocytic receptors with antibody-antigen conjugates co-administered with toll-like receptor (TLR) agonists as adjuvants. To combine the potential advantages of targeting of DCs with those of conjugated TLR agonists, we generated a multifunctional antibody construct integrating the DC-specific delivery of viral- or tumor-associated antigens and DC activation by TLR ligation in one molecule. We validated its functionality and determined if TLR ligation might improve the efficacy of such a molecule. In proof-of-principle studies, an αCD40 antibody containing a CMV pp65-derived peptide as an antigen domain (αCD40) was genetically fused to the TLR5-binding D0/D1 domain of bacterial flagellin (αCD40.Flg). The analysis of surface maturation markers on immature DCs revealed that fusion of flagellin to αCD40 highly increased DC maturation (3.4-fold elevation of CD80 expression compared to αCD40 alone) by specifically interacting with TLR5. Immature DCs loaded with αCD40.Flg induced significantly higher CMV-specific T cell activation and proliferation compared to αCD40 in co-culture experiments with allogeneic and autologous T cells (1.8-fold increase in % IFN-γ/TNF-α CD8 T cells and 3.9-fold increase in % CMV-specific dextramer CD8 T cells). More importantly, we confirmed the beneficial effects of flagellin-dependent DC stimulation using a tumor-specific neoantigen as the antigen domain. Specifically, the acute myeloid leukemia (AML)-specific mutated NPM1 (mNPM1)-derived neoantigen CLAVEEVSL was delivered to DCs in the form of αCD40 and αCD40.Flg antibody constructs, making this study the first to investigate mNPM1 in a DC vaccination context. Again, αCD40.Flg-loaded DCs more potently activated allogeneic mNPM1-specific T cells compared to αCD40. These results confirmed the functionality of our multifunctional antibody construct and demonstrated that TLR5 ligation improved the efficacy of the molecule. Future mouse studies are required to examine the T cell-activating potential of αCD40.Flg after targeting of dendritic cells using AML xenograft models.