Conventional dendritic cell (DC) vaccine strategies, in which DCs are loaded with antigens
ex vivo
, 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
in vivo
with antibody–antigen conjugates co-administered with toll-like receptor (TLR) agonists as adjuvants. To combine the potential advantages of
in vivo
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
in vitro
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
CMV
) was genetically fused to the TLR5-binding D0/D1 domain of bacterial flagellin (αCD40.Flg
CMV
). The analysis of surface maturation markers on immature DCs revealed that fusion of flagellin to αCD40
CMV
highly increased DC maturation (3.4-fold elevation of CD80 expression compared to αCD40
CMV
alone) by specifically interacting with TLR5. Immature DCs loaded with αCD40.Flg
CMV
induced significantly higher CMV
NLV
-specific T cell activation and proliferation compared to αCD40
CMV
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
NLV
-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
mNPM1
and αCD40.Flg
mNPM1
antibody constructs, making this study the first to investigate mNPM1 in a DC vaccination context. Again, αCD40.Flg
mNPM1
-loaded DCs more potently activated allogeneic mNPM1
CLA
-specific T cells compared to αCD40
mNPM1
. These
in vitro
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
mNPM1
after targeting of dendritic cells
in vivo
using AML xenograft models.
Helicobacter pylori
is one of the most prevalent bacterial pathogens, inflicting hundreds of thousands of peptic ulcers and gastric cancers to patients every year. Antibacterial treatment of
H. pylori
is complicated due to the need of combining multiple antibiotics, entailing serious side effects and increasing selection for antibiotic resistance.
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