Abstract 2008: The oncogenic fusion protein DNAJB1-PRKACA can be actively targeted by peptide-based immunotherapy in fibrolamellar hepatocellular carcinoma
Authors: Bauer Jens , Köhler Natalie , Maringer Yacine , Bucher Philip , Bilich Tatjana , Zwick Melissa , Dicks Severin , Nelde Annika , Dubbelaar Marissa , Scheid Jonas , Wacker Marcel , Heitmann Jonas J. , Schroeder Sarah , Rieth Jonas , Denk Monika , Richter Marion , Klein Reinhild , Bonzheim Irina , Luibrand Julia , Holzer Ursula , Ebinger Martin , Brecht Ines B. , Bitzer Michael , Boerries Melanie , Salih Helmut R. , Rammensee Hans-Georg , Hailfinger Stephan , Walz Juliane S. 
Published: June 2022

Abstract
Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare tumor disease, which affects children and adolescents without history of primary liver disease. Beside surgical resection established treatment options are lacking for FL-HCC. Recently, the DNAJB1-PRKACA fusion transcript was identified as the oncogenic driver of tumor pathogenesis in 100% of FL-HCC patients. Here, we investigated the role of the DNAJB1-PRKACA fusion protein as a source for immunogenic neoepitopes and showed first immunotherapeutic application of these antigens in a FL-HCC patient.HLA class I- and class II-presented neoantigens derived from the DNAJB1-PRKACA fusion protein were predicted in silico using NetMHCpan 4.1 and SYFPEITHI 1.0, or NetMHCIIpan 4.0, respectively. With this workflow nine binding cores of nine amino acid length for a total of 1290 different HLA class II alleles, as well as 13 HLA class I ligands for the 20 most frequent HLA class I allotypes (European population, iedb.org) were identified. Cellular processing and HLA presentation of DNAJB1-PRKACA-derived peptides was proven by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) of DNAJB1-PRKACA-transduced HCC cell lines. Immunogenicity of DNAJB1-PRKACA-derived peptides was assessed for the HLA class II peptide (PII-1) and the HLA-A*24 peptide (PA*24) by in vitro priming experiments which showed an induction of multifunctional peptide-specific CD4+ and CD8+ T cells, respectively, with expression of CD107a, IFNγ, and TNF upon peptide-pulsing. Furthermore, PA*24-specific T cells showed antigen-specific lysis of autologous peptide-loaded target cells and single-cell next-generation sequencing (10x Genomics) of PA*24-specific CD8+ T cells further enabled the identification of DNAJB1-PRKACA-reactive T cell receptors. Based on these preclinical data we applied a peptide vaccine, consisting of three HLA class I ligands (PA*02, PB*44, and PC*05) and PII-1 spanning the DNAJB1-PRKACA fusion region, to a 15-year old patient with histologically confirmed FL-HCC, who experienced multiple tumor relapses after early liver transplant due to unresectable FL-HCC not responsive to chemotherapy. After two vaccinations in vivo induction of multifunctional CD4+ T cells targeting PII-1 and PB*44 was observed by IFNγ ELISPOT. Single-cell RNA sequencing of vaccine-induced CD4+ T cells revealed distinct gene expression clusters of T cell activation and high TCR clonality. DNAJB1-PRKACA-specific T cells persisted in peripheral blood and were accompanied by relapse free survival of the patient until now, more than one year post vaccination. These findings identified the DNAJB1-PRKACA fusion transcript as novel prime source for broadly applicable neoepitopes and corresponding TCRs and provide first evidence for their application in cancer immunotherapy of FL-HCC.
Citation Format: Jens Bauer, Natalie Köhler, Yacine Maringer, Philip Bucher, Tatjana Bilich, Melissa Zwick, Severin Dicks, Annika Nelde, Marissa Dubbelaar, Jonas Scheid, Marcel Wacker, Jonas J. Heitmann, Sarah Schroeder, Jonas Rieth, Monika Denk, Marion Richter, Reinhild Klein, Irina Bonzheim, Julia Luibrand, Ursula Holzer, Martin Ebinger, Ines B. Brecht, Michael Bitzer, Melanie Boerries, Helmut R. Salih, Hans-Georg Rammensee, Stephan Hailfinger, Juliane S. Walz. The oncogenic fusion protein DNAJB1-PRKACA can be actively targeted by peptide-based immunotherapy in fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2008.