Charakterisierung immunogener Neoantigene des DNAJB1-PRKACA-Fusionsproteins als Zielstrukturen für T-Zell-basierte Immuntherapien

01.01.2023 bis 31.12.2025

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare but lethal tumor disease, which affects children and adolescents without history of primary liver disease. Beside surgical resection established treatment options are lacking. Recently, the DNAJB1-PRKACA fusion transcript was identified as the oncogenic driver of tumor pathogenesis in 100% of FL-HCC patients representing an attractive target for the development of novel therapies for this devastating tumor disease. Moreover, the recent identification of other cancer entities that express the DNAJB1-PRKACA fusion transcript give the prospect that targeting the DNAJB1-PRKACA fusion might improve treatment options in multiple cancer entities. We postulate that (i) the DNAJB1-PRKACA fusion transcript is a source for immunogenic neoepitopes and thus (ii) can be actively targeted by T cell-based immunotherapy. These hypotheses are based on recent reports on T-cell responses against neoepitopes derived from gene fusion transcripts in patients receiving immune checkpoint inhibitors and their correlation with treatment response as well as on own preliminary work applying a DNAJB1-PRKACA-derived peptide vaccine to a FL-HCC patient suffering from recurrent disease relapses that induced persistent DNAJB1-PRKACA-specific T-cell responses accompanied by relapse-free survival. Thus, in the proposed project we aim to characterize the DNAJB1-PRKACA fusion transcript as a source for broadly applicable neoepitopes together with their corresponding T-cell receptors (TCRs) and to provide first evidence for their application in cancer immunotherapy. Therefore, we will establish DNAJB1-PRKACA-expressing tumor cell lines and evaluate the natural processing and HLA presentation of in silico predicted DNAJB1-PRKACA-derived peptides by mass spectrometry-based immunopeptidome analyses of these cell lines and primary tumor samples. The immunogenicity of DNAJB1-PRKACA-derived HLA ligands will further be evaluated by in vitro priming of CD4+ and CD8+ T cells followed by the characterization of functionality and cytotoxicity of DNAJB1-PRKACA-specific T cells. In addition, preexisting DNAJB1-PRKACA-specific memory T-cell responses in patients with DNAJB1-PRKACA-expressing tumors will be analyzed. In vitro and in vivo induced DNAJB1-PRKACA-specifc T cells will be assessed by single-cell RNA sequencing for in-depth characterization of T-cell phenotypes and functionality and isolation of TCR sequences with subsequent analysis of TCR signaling, affinity and conferrable effector functions. Together, successful conduct of this project will enable the development of T-cell based immunotherapies actively targeting the DNAJB1-PRKACA fusion transcript to optimize treatment of FL-HCC patients and further tumor entities carrying this oncogenic driver fusion.