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

Abstract

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.

An HLA-A*11:01-Binding Neoantigen from Mutated NPM1 as Target for TCR Gene Therapy in AML

“…Therefore, identification of an HLA-A11 TCR with enhanced affinity for dNPM1 for TCR gene therapy seems advisable. In addition to TCR gene therapy, AVEEVSLRK may also be a promising target for other immunotherapeutic strategies, such as neoantigen vaccination [ 44 ]. These dNPM1-directed immunotherapies could provide new and effective treatment options for patients with relapsed or refractory AML.…”

Harnessing Engineered Immune Cells and Bacteria as Drug Carriers for Cancer Immunotherapy

“…Schmitt et al modified flagellin on anti-CD40 antibody with leukemia-specific antigen, respectively. The hybrid antibody carried the flagellin and targeted to DCs, which enhanced DCs’ maturation and promoted antigen-specific T-cell responses owing to the addition of flagellin . In consideration of the excellent T-cell priming capacity, flagellin tends to be a great choice to combine with adoptive T-cell therapy.…”

“…The hybrid antibody carried the flagellin and targeted to DCs, which enhanced DCs' maturation and promoted antigen-specific T-cell responses owing to the addition of flagellin. 224 In consideration of the excellent Tcell priming capacity, flagellin tends to be a great choice to combine with adoptive T-cell therapy. Song et al employed an adenoviral vector to deliver antigen, flagellin, and an inhibitor of the antigen-presenting attenuator A20.…”

Engineered Bacteriophage-Based In Situ Vaccine Remodels a Tumor Microenvironment and Elicits Potent Antitumor Immunity

“…CD40 is a member of the tumor necrosis factor receptor superfamily (TNFRSF), which is expressed at a relatively high level on the cell surface of both immature and mature DCs. 21 In addition, CD40 plays a central role in DC maturation and activation as a costimulatory molecule. CD40 engagement on the surface of DCs promotes cytokine secretion and helps to bridge DCs with adaptive immunity for enhanced tumor regression.…”