Therautic vaccination to improve cancer immunotherapy

Resumen

Response rates to immunecheckpoint inhibitors (ICI) are still far from satisfactory in different tumors. This poor performance is usually attributed to low lymphocyte infiltration levels, presumably due to low to moderate TMB, have been blamed for the poor performance of this therapy. Approaches aimed at promoting lymphocyte infiltration, such as vaccines, have been proposed as a possible solution. However, vaccination has not been efficient yet in achieving this purpose, which could be due to a poor design. This thesis is divided in two different projects, and each of them proposes strategies to improve one of the two main components of vaccines: the immunogen and the adjuvant. Project 1: Identification of neoAgs in HCC for future use in vaccination strategies Neoantigens, new immunogenic sequences arising from tumor mutations, have been associated with response to immunotherapy and are considered potential targets for vaccination. Hepatocellular carcinoma (HCC) is a moderately mutated tumor, where the neoantigen repertoire has not been investigated. Our aim was to analyze whether tumors in HCC patients contain immunogenic neoantigens suitable for future use in therapeutic vaccination. We recruited a group of 14 HCC patients and collected both tumor and liver tissue samples. The sequencing of these samples led to the identification of a median of 1217 missense somatic SNV per patient, narrowed to 30 when filtering by using RNAseq data. A median of 13 and 5 peptides per patient were predicted as potential binders to HLA class I and class II molecules, respectively. Considering only HLA-A*02.01- and HLA-DRB1*01-predicted binders, 70% demonstrated HLA-binding capacity and about 50% were immunogenic when tested in HHD-DR1 mice. These peptides induced polyfunctional T cells that specifically recognized the mutated but not the wild-type sequence as well as neoantigen-expressing cells. Moreover, coimmunization experiments combining CD8 and CD4 neoantigen epitopes resulted in stronger CD8 T cell responses. Finally, responses against neoantigens were also induced in vitro using human cells. These results show that mutations in HCC tumors may generate immunogenic neoantigens with potential applicability for future combinatorial therapeutic strategies. Project 2: Improvement of vaccination efficacy by blockade of vaccine-induced immunosuppressive factors. Vaccination with optimized strategies may increase tumor lymphocytic infiltration and enhance response rates to immune checkpoint inhibitors (ICI) in cold tumors. To enhance vaccine potency and improve thus responses to ICI, we analyzed the gene expression profile of an immunosuppressive dendritic cell (DC) population induced during vaccination, to identify druggable inhibitory mechanisms. RNAseq studies identified targetable genes, but their inhibition did not yield improved vaccines. However, we proved that immunosuppressive DC had a monocytic origin. Thus, monocyte depletion by gemcitabine administration reduced the generation of these DC and increased vaccine-induced immunity, which rejected about 20% tumors in LLC- OVA and B16-OVA models, non-responders to anti-PD-1. This superior efficacy was associated with higher antitumor T-cell infiltration and overexpression of PD-1/PD-L1. Therefore, combination of vaccine + gemcitabine with anti-PD-1 was superior to anti-PD-1 monotherapy in both models. B16-OVA tumors benefited from a synergistic effect, reaching 75% tumor rejection, but the higher levels in LLC-OVA tumors of exhausted T-cells co-expressing PD-1, LAG3 and TIM3 precluded similar levels of efficacy. Our results indicate that gemcitabine, by targeting vaccine-induced immunosuppressive DC, represents a suitable combinatorial partner with vaccines aimed at enhancing PD-1 therapies.