Immune infiltration of advanced human gliomas has been shown, but it is doubtful whether these immune cells affect tumor progression. It could be hypothesized that this infiltrate reflects recently recruited immune cells that are immediately overwhelmed by a high tumor burden. Alternatively, if there is earlier immune detection and infiltration of the tumor, the question arises as to when antitumor competency is lost. To address these issues, we analyzed a transgenic mouse model of spontaneous astrocytoma (GFAP-V¹²HA-ras mice), which allows the study of immune interactions with developing glioma, even at early asymptomatic stages. T cells, including a significant proportion of Tregs, are already present in the brain before symptoms develop, followed later by macrophages, natural killer cells, and dendritic cells. The effector potential of CD8 T-cells is defective, with the absence of granzyme B expression and low expression of IFN-γ, tumor necrosis factor, and interleukin 2. Overall, our results show an early defective endogenous immune response to gliomas, and local accumulation of immunosuppressive cells at the tumor site. Thus, the antiglioma response is not simply overwhelmed at advanced stages of tumor growth, but is counterbalanced by an inhibitory microenvironment from the outset. Nevertheless, we determined that effector molecule expression (granzyme B, IFN-γ) by brain-infiltrating CD8 T-cells could be enhanced, despite this unfavorable milieu, by strong immune stimuli. This potential to modulate the strong imbalance in local antiglioma immunity is encouraging for the development and optimization of future glioma immunotherapies. Cancer Res; 70(12); 4829–39. ©2010 AACR.

Functional tumor-specific cytotoxic T cells elicited by therapeutic cancer vaccination in combination with oncolytic viruses offer opportunities to address resistance to checkpoint blockade therapy. Two cancer vaccines, the self-adjuvanting protein vaccine KISIMA, and the recombinant oncolytic vesicular stomatitis virus pseudotyped with LCMV-GP expressing tumor-associated antigens, termed VSV-GP-TAA, both show promise as a single agent. Here we find that, when given in a heterologous prime-boost regimen with an optimized schedule and route of administration, combining KISIMA and VSV-GP-TAA vaccinations induces better cancer immunity than individually. Using several mouse tumor models with varying degrees of susceptibility for viral replication, we find that priming with KISIMA-TAA followed by VSV-GP-TAA boost causes profound changes in the tumor microenvironment, and induces a large pool of poly-functional and persistent antigen-specific cytotoxic T cells in the periphery. Combining this heterologous vaccination with checkpoint blockade further improves therapeutic efficacy with long-term survival in the spectrum. Overall, heterologous vaccination with KISIMA and VSV-GP-TAA could sensitize non-inflamed tumors to checkpoint blockade therapy.

Combining different immunotherapy approaches is currently building the future of immunotherapy, with the view to maximize anti-tumoral efficacy for larger patient population. The KISIMA™ platform allows the development of protein-based cancer vaccines able to induce tumor-specific T cell response resulting in anti-tumoral efficacy in various mouse models. Intra-tumoral administration of stimulator of interferon gene agonists (STINGa) was shown to induce a potent inflammatory response leading to the development of tumor-specific immunity. Here, we explored the efficacy and mechanisms of action of subcutaneous STINGa treatment combined with therapeutic vaccination in various mouse tumor models. This combinatory treatment highly enhanced frequency and effector function of both peripheral and intra-tumoral antigen-specific CD8 T cells, promoting potent IFNγ and TNFα production along with increased cytotoxicity. Moreover, combination therapy favorably modulated the tumor microenvironment by dampening immune-suppressive cells and increasing CD4 T cell infiltration together with their polarization toward Th1 phenotype. Combination with STINGa treatment improved the effect of therapeutic vaccination, resulting in a prolonged control and slower growth of B16-OVA and TC-1 tumors. Altogether, the results presented here highlight the potential of combining STINGa with a therapeutic protein vaccine for cancer treatment.

Novel immunopreventive strategies are emerging that show great promise for conferring long-term protection to individuals at high risk of developing colorectal cancer. The KISIMA vaccine platform utilizes a chimeric protein comprising: (1) a selected tumor antigen; (2) a cell-penetrating peptide to improve antigen delivery and epitope presentation, and (3) a TLR2/4 agonist to serve as a self-adjuvant. This study examines the ability of a KISIMA vaccine against achaete-scute family bHLH transcription factor 2 (Ascl2), an early colon cancer antigen, to reduce colon tumor formation by stimulating an anti-tumor immune response. Vaccine administrations were well-tolerated and led to circulating antibodies and antigen-specific T cells in a mouse model of colorectal cancer. To assess preventive efficacy, the vaccine was administered to mice either alone or in combination with the immune checkpoint inhibitor anti-PD-1. When delivered to animals prior to colon tumor formation, the combination strategy significantly reduced the development of colon microadenomas and adenomas, as compared to vehicle-treated controls. This response was accompanied by an increase in the intraepithelial density of CD3+ T lymphocytes. Together, these data indicate that the KISIMA-Ascl2 vaccine shows great potential to be a safe and potent immunopreventive intervention for individuals at high risk of developing colorectal cancer.

Heterologous prime-boost settings with a protein vaccine and the viral vector vesicular stomatitis virus, both expressing tumor-associated antigens (KISIMA-TAA and VSV-GP-TAA), have been previously shown to generate potent antitumor immunity. In the cold TC-1 model (HPV antigen) and the immune-infiltrate MC-38 model (Adpgk, Reps1 and Rpl18 neo-antigens), we further investigated pivotal immune cells that educate CD8+ T cells. Heterologous prime-boost vaccination induced a superior antitumor response characterized by the increase in number and functionality of antigen-specific CD8+ T cells, recruitment of cross-presenting dendritic cells, and polarization of CD4+ T cells towards an antitumor Th1 phenotype within the tumor and tumor-draining lymph nodes, turning the cold TC-1 tumor into a hot, inflamed tumor. In the inflamed MC-38 tumor model, treatment combination markedly prolonged the overall survival of mice. Treatment with multi-epitope vaccines also induced high frequencies of multiple antigen specificities in the periphery and in the tumor. Prime-boost treatment reduced tumor-infiltrating regulatory CD4+ T cells whilst increasing cross-presenting dendritic cells in tumor-draining lymph nodes. In conclusion, heterologous prime-boost vaccination possesses the ability to induce a potent anti-tumor response in both immune-excluded and immune-infiltrated mouse tumor models. Additionally, this study highlights the design of a multi-epitope vaccine for cancer immunotherapy.

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.