ATP128 Clinical Therapeutic Cancer Vaccine Activates NF-κB and IRF3 Pathways through TLR4 and TLR2 in Human Monocytes and Dendritic Cells

SIMPLE SUMMARY: Peptide-based cancer vaccines require adjuvants to increase their immunogenicity to mount an antigen-specific reliable immune response. We have previously shown that immunization with self-adjuvanting peptide-based vaccines issued from our KISIMA vaccine platform, and targeting tumor-specific antigens, resulted in potent immune responses in preclinical models. The aim of our study was to assess the molecular mechanism of action of the first human candidate vaccine derived from the KISIMA platform, ATP128. By using an in vitro (THP-1 cell line) and ex vivo model system (human monocyte-derived dendritic cells), we were able to demonstrate that ATP128 relies on both the cell-penetrating peptide and TLR agonist domain present in the construct to activate the NF-κB and IRF3 pathways, in a self-adjuvanting manner. Importantly, we demonstrated that ATP128 is the first therapeutic vaccine able to activate human DCs through TLR2 and TLR4. ABSTRACT: The use of cancer vaccines is a promising therapeutic strategy able to stimulate anti-tumor immunity by inducing both humoral and cellular immunity. In this study, antigen presenting cells play a key role by inducing a strong activation of the T cell-mediated adaptive immune response, essential for the anti-tumor potential of cancer vaccines. The first human candidate vaccine created from the KISIMA platform, ATP128, bears three tumor-associated antigens highly expressed in colorectal cancer tissues. At the N-terminus, the cell-penetrating peptide allows the antigen delivery inside the cell and, together with the TLR agonist-derived peptide at the C-terminus, ensures the activation of the monocyte-derived dendritic cells. Here, we show that ATP128 leads to both NF-κB and IRF3 pathway activation, with subsequent pro-inflammatory cytokines and type I Interferon release, as well as an increase in the expression of costimulatory molecules, alongside an upregulation of MHC class I molecules. This cellular immune response involves TLR2 and TLR4, for both membrane and intracellular signaling. We demonstrated an endocytic component in ATP128’s activity by combining the use of a variant of ATP128 lacking the cell-penetrating peptide with endocytosis inhibitors. Importantly, this internalization step is detemined essential for the activation of the IRF3 pathway. This study validates the design of the self-adjuvanting ATP128 vaccine for cancer immunotherapy.