ADENOVIRAL-BASED VACCINE PROMOTES NEOANTIGEN-SPECIFIC CD8+ T CELL STEMNESS AND TUMOR REJECTION

Upon chronic antigen exposure, CD8+ T cells become exhausted, acquiring a dysfunctional state correlated with the inability to control infection or tumor progression. In contrast, stem- like CD8+ T progenitors maintain the ability to promote and sustain effective immunity. Adenovirus (Ad)–vectored vaccines encoding tumor neoantigens have been shown to eradicate large tumors when combined with anti–programmed cell death protein 1 (PD-1) in murine models; however, the mechanisms and translational potential have not yet been elucidated. Here, we show that gorilla Ad vaccine targeting tumor neoepitopes from colon rectal cancer (CRC) enhances responses to PD-1 therapy by improving immunogenicity and antitumor efficacy. Single-cell RNA sequencing (scRNA-seq) demonstrated that the combination of Ad vaccine and PD-1 increased the number of murine polyfunctional neoantigen-specific CD8+ T cells over PD-1 monotherapy, with an accumulation of Tcf1+ stem-like progenitors in draining lymph nodes and effector CD8+ T cells in tumors. Combined T cell receptor (TCR) sequencing analysis highlighted a broader spectrum of neoantigen-specific CD8+ T cells upon vaccination compared to PD-1 monotherapy. The translational relevance of these data is supported by results obtained in the first 12 patients with metastatic deficient mismatch repair (dMMR) tumors were treated with a combination of immune check-point blockade antibody (Pembrolizumab) and a heterologous prime boost protocol of Modified Vaccinia Ankara (MVA) and Ad vaccine both encoding shared neoantigens across three different tumor types (gastric, colorectal and gastroesophageal junction cancers). Expansion and diversification of TCRs were observed in post-treatment biopsies of patients with clinical response, as well as an increase in tumor-infiltrating T cells with an effector memory signature. As proof of concept, we validated these findings by single-cell RNA sequencing of a partial responder, the analysis revealed an increase of transitory memory neoantigen-specific CD8+ T cells following Ad vaccination. In particular, TCR sequences of neoantigen-specific clones detected in peripheral blood mononuclear cells (PBMCs) after PD-1 treatment were expanded following Ad vaccination. Additionally, the analysis identified the same TCR clones both in peripheral blood and tumor biopsies. Taken together all these findings indicate a new vaccination protocol to overcome resistance to PD- 1 blockade by promoting immunogenicity and broadening the spectrum and magnitude of neoantigen-specific T cells infiltrating tumors.