A replicating LCMV-based vaccine for the treatment of solid tumors.

Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of lymphocytic choriomeningitis virus (LCMV)-based vectors to break tolerance and induce effective antigen-specific CD8+ T cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T cell priming, which renders previously "cold" tumors open to immune infiltration and reprograms the tumor microenvironment to "hot." Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.

Viral vector-mediated reprogramming of the fibroblastic tumor stroma sustains curative melanoma treatment.

The tumor microenvironment (TME) is a complex amalgam of tumor cells, immune cells, endothelial cells and fibroblastic stromal cells (FSC). Cancer-associated fibroblasts are generally seen as tumor-promoting entity. However, it is conceivable that particular FSC populations within the TME contribute to immune-mediated tumor control. Here, we show that intratumoral treatment of mice with a recombinant lymphocytic choriomeningitis virus-based vaccine vector expressing a melanocyte differentiation antigen resulted in T cell-dependent long-term control of melanomas. Using single-cell RNA-seq analysis, we demonstrate that viral vector-mediated transduction reprogrammed and activated a Cxcl13-expressing FSC subset that show a pronounced immunostimulatory signature and increased expression of the inflammatory cytokine IL-33. Ablation of Il33 gene expression in Cxcl13-Cre-positive FSCs reduces the functionality of intratumoral T cells and unleashes tumor growth. Thus, reprogramming of FSCs by a self-antigen-expressing viral vector in the TME is critical for curative melanoma treatment by locally sustaining the activity of tumor-specific T cells.

Adenovirus vector vaccination reprograms pulmonary fibroblastic niches to support protective inflating memory CD8+ T cells.

Pathogens and vaccines that produce persisting antigens can generate expanded pools of effector memory CD8+ T cells, described as memory inflation. While properties of inflating memory CD8+ T cells have been characterized, the specific cell types and tissue factors responsible for their maintenance remain elusive. Here, we show that clinically applied adenovirus vectors preferentially target fibroblastic stromal cells in cultured human tissues. Moreover, we used cell-type-specific antigen targeting to define critical cells and molecules that sustain long-term antigen presentation and T cell activity after adenovirus vector immunization in mice. While antigen targeting to myeloid cells was insufficient to activate antigen-specific CD8+ T cells, genetic activation of antigen expression in Ccl19-cre-expressing fibroblastic stromal cells induced inflating CD8+ T cells. Local ablation of vector-targeted cells revealed that lung fibroblasts support the protective function and metabolic fitness of inflating memory CD8+ T cells in an interleukin (IL)-33-dependent manner. Collectively, these data define a critical fibroblastic niche that underpins robust protective immunity operating in a clinically important vaccine platform.

Heterologous Prime Boost Vaccination Induces Protective Melanoma-Specific CD8+ T Cell Responses.

Cancer vaccination aims at inducing an adaptive immune response against tumor-derived antigens. In this study, we utilize recombinant human adenovirus serotype 5 (rAd5) and recombinant lymphocytic choriomeningitis virus (rLCMV)-based vectors expressing the melanocyte differentiation antigen gp100. In contrast to single or homologous vaccination, a heterologous prime boost vaccination starting with a rAd5-gp100 prime immunization followed by a rLCMV-gp100 boost injection induces a high magnitude of polyfunctional gp100-specific CD8+ T cells. Our data indicate that an optimal T cell induction is dependent on the order and interval of the vaccinations. A prophylactic prime boost vaccination with rAd5- and rLCMV-gp100 protects mice from a B16.F10 melanoma challenge. In the therapeutic setting, combination of the vaccination with low-dose cyclophosphamide showed a synergistic effect and significantly delayed tumor growth. Our findings suggest that heterologous viral vector prime boost immunizations can mediate tumor control in a mouse melanoma model.

Type I interferon signaling in fibroblastic reticular cells prevents exhaustive activation of antiviral CD8+ T cells.

Fibroblastic reticular cells (FRCs) are stromal cells that actively promote the induction of immune responses by coordinating the interaction of innate and adaptive immune cells. However, whether and to which extent immune cell activation is determined by lymph node FRC reprogramming during acute viral infection has remained unexplored. Here, we genetically ablated expression of the type I interferon-α receptor (Ifnar) in Ccl19-Cre+ cells and found that sensing of type I interferon imprints an antiviral state in FRCs and thereby preserves myeloid cell composition in lymph nodes of naive mice. During localized lymphocytic choriomeningitis virus infection, IFNAR signaling precipitated profound phenotypic adaptation of all FRC subsets enhancing antigen presentation, chemokine-driven immune cell recruitment, and immune regulation. The IFNAR-dependent shift of all FRC subsets toward an immunostimulatory state reduced exhaustive CD8+ T cell activation. In sum, these results unveil intricate circuits underlying type I IFN sensing in lymph node FRCs that enable protective antiviral immunity.

Molecular, clinicopathological, and immune correlates of LAG3 promoter DNA methylation in melanoma.

BACKGROUND: The co-receptor lymphocyte-activation gene-3 (LAG3, LAG-3, CD223) is a potential target for immune checkpoint inhibition immunotherapies. However, little is known about the biological and clinical significance of LAG3 DNA methylation in melanoma and its microenvironment. METHODS: We evaluated LAG3 promoter and gene body methylation in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas (TCGA cohort), an independent cohort of N = 120 patients from the University Hospital Bonn, and in subsets of peripheral blood leukocytes, melanocytes, and melanoma cell lines. We validated the association of LAG3 methylation with mRNA expression in vitro in the melanoma cell line A375 treated with the hypomethylating agent 5-azacytidine and stimulated with interferon-γ. Finally, we investigated correlations between LAG3 methylation and progression-free survival in patients treated with immune checkpoint blockade (ICB cohort, N = 118). FINDINGS: Depending on the analysed locus (promoter, gene body) we found region-dependent significant LAG3 methylation differences between monocytes, B cells, CD8+ and CD4+ T cells, regulatory T cells, melanocytes, and melanoma cell lines. In tumor tissues, methylation correlated significantly with LAG3 mRNA expression, immune cell infiltrates (histopathologic lymphocyte score and RNA-Seq signatures of distinct immune infiltrates), and an interferon-γ signature. Finally, LAG3 methylation was associated with overall survival in the TCGA cohort and progression-free survival in the ICB cohort. We detected basal LAG3 mRNA expression in the melanoma cell A375 and an interferon-γ inducible expression after demethylation with 5-azacytidine. INTERPRETATION: Our study points towards an epigenetic regulation of LAG3 via promoter methylation and suggests a prognostic and predictive significance of LAG3 methylation in melanoma. Our results give insight in the tumor cell-intrinsic transcriptional regulation of LAG3 in melanoma. In perspective, our results might pave the way for investigating LAG3 methylation as a predictive biomarker for response to anti-LAG3 immune checkpoint blockage. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Prognostic and predictive value of PD-L2 DNA methylation and mRNA expression in melanoma.

BACKGROUND: PD-L1 (programmed cell death 1 ligand 1) expression in melanoma has been associated with a better response to anti-PD-1 (programmed cell death 1) therapy. However, patients with PD-L1-negative melanomas can respond to anti-PD-1 blockade, suggesting that the other PD-1 ligand, PD-L2 (programmed cell death 1 ligand 2), might also be relevant for efficacy of PD-1 inhibition. We investigated PD-L2 expression and methylation as a prognostic and predictive biomarker in melanoma. METHODS: DNA methylation at five CpG loci and gene expression of PD-L2 were evaluated with regard to survival in 470 melanomas from The Cancer Genome Atlas. PD-L2 promoter methylation in correlation with PD-L2 mRNA and protein expression was analyzed in human melanoma cell lines. Prognostic and predictive value of PD-L2 methylation was validated using quantitative methylation-specific PCR in a multicenter cohort of 129 melanoma patients receiving anti-PD-1 therapy. mRNA sequencing data of 121 melanoma patients receiving anti-PD-1 therapy provided by Liu et al. were analyzed for PD-L2 mRNA expression. RESULTS: We found significant correlations between PD-L2 methylation and mRNA expression levels in melanoma tissues and cell lines. Interferon-γ inducible PD-L2 protein expression correlated with PD-L2 promoter methylation in melanoma cells. PD-L2 DNA promoter hypomethylation and high mRNA expression were found to be strong predictors of prolonged overall survival. In pre-treatment melanoma samples from patients receiving anti-PD-1 therapy, low PD-L2 DNA methylation and high PD-L2 mRNA expression predicted longer progression-free survival. CONCLUSION: PD-L2 expression seems to be regulated via DNA promoter methylation. PD-L2 DNA methylation and mRNA expression may predict progression-free survival in melanoma patients receiving anti-PD-1 immunotherapy. Assessment of PD-L2 should be included in further clinical trials with anti-PD-1 antibodies.

BP180-specific IgG is associated with skin adverse events, therapy response, and overall survival in non-small cell lung cancer patients treated with checkpoint inhibitors.

BACKGROUND: Anti-programmed cell death protein 1 (PD1)/programmed death-ligand 1(PD-L1) therapy frequently entails immune-related adverse events (irAEs), and biomarkers to predict irAEs are lacking. Although checkpoint inhibitors have been found to reinvigorate T cells, the relevance of autoantibodies remains elusive. OBJECTIVE: Our aim was to explore whether IgG autoantibodies directed against coexpressed antigens by tumor tissue and healthy skin correlate with skin irAEs and therapy outcome. METHODS: We measured skin-specific IgG via enzyme-linked immunosorbent assay in patients with non-small cell lung cancer (NSCLC) who received anti-PD1/PD-L1 treatment between July 2015 and September 2017 at the Kantonsspital St. Gallen. Sera were sampled at baseline and during therapy after 8 weeks. RESULTS: Analysis of publicly available tumor expression data revealed that NSCLC and skin coexpress BP180, BP230, and type VII collagen. A skin irAE developed in 16 of 40 recruited patients (40%). Only elevated anti-BP180 IgG at baseline significantly correlated with the development of skin irAEs (P = .04), therapy response (P = .01), and overall survival (P = .04). LIMITATIONS: The patients were recruited in a single tertiary care center. CONCLUSIONS: Our data suggest that the level of anti-BP180 IgG of NSCLC patients at baseline is associated with better therapy response and overall survival and with a higher probability to develop skin irAEs during anti-PD1/PD-L1 treatment.