P-01.10 Promoter methylation facilitates escape from specific immune responses against tumor-associated antigens across cancer types and can be overcome by demethylating agents

<h3>Background</h3> Targeting tumor-associated antigens (TAAs) holds promise for cancer immunotherapy; however, antigen loss remains a major obstacle, leading to immune evasion. The synergistic effect of targeting multiple TAAs and their shared expression across cancer types has renewed interest in TAAs. In contrast to abundant single-antigen studies, comprehensive combined assessments of expression, humoral and cellular immune responses against multiple TAAs are rare. <h3>Materials and Methods</h3> NanoString, FluoroSpot, and protein-bound bead assays were used to explore the immunologic landscape of TAAs across five cancer types, while DNA promoter methylation of these tumor antigens was assessed using the MethylationEPIC v2.0 BeadChip assay. <h3>Results</h3> We identified frequent and tumor-specific (co-)expression patterns of TAAs, with notably high frequencies of endogenous cellular and humoral anti-TAA immune responses. Interestingly, co-occurrence between TAA expression and a corresponding anti-TAA immune response was rarely detectable in the same patient, suggesting that immune evasion, rather than a lack of immunogenicity, as the reason for tumor persistence despite frequent endogenous anti-TAA immune responses. T cell to B-cell clusters within the tumor microenvironment correlate with the presence of tertiary lymphoid structures (TLS) and tumor-specific immune responses, suggesting TLS as local sites for inducing anti-TAA immune responses. We found DNA promoter methylation to be inversely correlated with TAA expression. Our data identify methylation as an important regulator of TAA expression, leading to antigen escape in various cancer types. DNA methyltransferase inhibitors induced re-expression of TAAs and enhanced T-cell-mediated tumor cell killing in vitro. <h3>Conclusions</h3> Collectively, these data provide a foundation for optimizing multi-targeted immunotherapies. Our results suggest that therapies combining enhancement of anti-tumor immune responses with drugs antagonizing antigen escape may overcome immune evasion and improve responsiveness to immunotherapies such as immune checkpoint inhibition. M. Thelen: None. E. Preugszat: None. M. Garcia-Marquez: None. K. Wennhold: None. J. Lehmann: None. E. Hagen: None. M. Werner: None. P. Volkmar: None. K. Siepmann: None. H. Eckel: None. L.S. Rudeck: None. F. Thangarajah: None. N. Zaburannyi: None. C.J. Bruns: None. A. Quaas: None. M. von Bergwelt-Baildon: None. A.M. Hillmer: None. H.A. Schlößer: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Astrazeneca.