Vaccine immunotherapy with ARNAX induces tumor‐specific memory T cells and durable anti‐tumor immunity in mouse models

Immunological checkpoint blockade therapies benefit a limited population of cancer patients. We have previously shown that vaccine immunotherapy with Toll‐like receptor (TLR)3‐adjuvant and tumor antigen overcomes anti‐programmed death ligand‐1 (PD‐L1) resistance in mouse tumor models. In the present study, 4 different ovalbumin (OVA)‐expressing tumor cell lines were implanted into syngeneic mice and subjected to anti‐tumor immunotherapy using ARNAX and whole OVA protein. ARNAX is a TLR3‐specific agonist that does not activate the mitochondrial antiviral‐signaling protein (MAVS) pathway, and thus does not induce systemic inflammation. Dendritic cell priming and proliferative CTL were induced by ARNAX + OVA, but complete remission was achieved only in a PD‐L1‐low cell line of EG7. Addition of anti‐PD‐L1 antibody to the ARNAX + OVA therapy brought complete remission to another PD‐L1‐high subline of EG7. Tumor shrinkage but not remission was observed in MO5 in that regimen. We analyzed tumor cells and tumor‐infiltrating immune cells to identify factors associated with successful ARNAX vaccine therapy. Tumors that responded to ARNAX therapy expressed high levels of MHC class I and low levels of PD‐L1. The tumor‐infiltrating immune cells in ARNAX‐susceptible tumors contained fewer immunosuppressive myeloid cells with low PD‐L1 expression. Combination with anti‐PD‐L1 antibody functioned not only within tumor sites but also within lymphoid tissues, augmenting the therapeutic efficacy of the ARNAX vaccine. Notably, ARNAX therapy induced memory CD8(+) T cells and rejection of reimplanted tumors. Thus, ARNAX vaccine + anti‐PD‐L1 therapy enabled permanent remission against some tumors that stably present antigens.