Fusion of Bacterial Flagellin to a Dendritic Cell-Targeting αCD40 Antibody Construct Coupled With Viral or Leukemia-Specific Antigens Enhances Dendritic Cell Maturation and Activates Peptide-Responsive T Cells

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.