HLA class I loss in metachronous metastases prevents continuous T cell recognition of mutated neoantigens in a human melanoma model

// Barbara Schrors 1 , Silke Lubcke 1 , Volker Lennerz 1 , Martina Fatho 1 , Anne Bicker 2 , Catherine Wolfel 1 , Patrick Derigs 1 , Thomas Hankeln 2 , Dirk Schadendorf 3 , Annette Paschen 3 , Thomas Wolfel 1 1 Internal Medicine III, University Cancer Center (UCT) and Research Center for Immunotherapy (FZI), University Medical Center (UMC) of the Johannes Gutenberg University and German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Mainz, Germany 2 Institute for Molecular Genetics, Johannes Gutenberg University, Mainz, Germany 3 Department of Dermatology, University Hospital, University Duisburg-Essen and German Cancer Consortium (DKTK), Partner Site Essen/Dusseldorf, Essen, Germany Correspondence to: Thomas Wolfel, email: thomas.woelfel@unimedizin-mainz.de Keywords: melanoma, mutated neoantigens, high-throughput sequencing, immune escape, HLA class I loss Received: July 05, 2016      Accepted: February 27, 2017      Published: March 09, 2017 ABSTRACT T lymphocytes against tumor-specific mutated neoantigens can induce tumor regression. Also, the size of the immunogenic cancer mutanome is supposed to correlate with the clinical efficacy of checkpoint inhibition. Herein, we studied the susceptibility of tumor cell lines from lymph node metastases occurring in a melanoma patient over several years towards blood-derived, neoantigen-specific CD8 + T cells. In contrast to a cell line established during early stage III disease, all cell lines generated at later time points from stage IV metastases exhibited partial or complete loss of HLA class I expression. Whole exome and transcriptome sequencing of the four tumor lines and a germline control were applied to identify expressed somatic single nucleotide substitutions (SNS), insertions and deletions (indels). Candidate peptides encoded by these variants and predicted to bind to the patient’s HLA class I alleles were synthesized and tested for recognition by autologous mixed lymphocyte-tumor cell cultures (MLTCs). Peptides from four mutated proteins, HERPUD1 G161S , INSIG1 S238F , MMS22L S437F and PRDM10 S1050F , were recognized by MLTC responders and MLTC-derived T cell clones restricted by HLA-A*24:02 or HLA-B*15:01. Intracellular peptide processing was verified with transfectants. All four neoantigens could only be targeted on the cell line generated during early stage III disease. HLA loss variants of any kind were uniformly resistant. These findings corroborate that, although neoantigens represent attractive therapeutic targets, they also contribute to the process of cancer immunoediting as a serious limitation to specific T cell immunotherapy.