Identification of CRKII, CFL1, CNTN1, NME2, and TKT as Novel and Frequent T-cell Targets in Human IDH-Mutant Glioma

Purpose: Successful immunotherapies for IDH mut gliomas require better knowledge of T-cell target antigens. Here, we elucidated their antigen repertoire recognized by spontaneous T-cell responses using an unbiased proteomic approach. Experimental Design: Protein fractionations of tissue lysates from IDH mut gliomas (n=4) were performed. Fractions were tested by IFN-E; ELISpot assay for recognition through patient's T-cells. Proteins of immunogenic fractions were identified by mass spectrometry and validated by in silico-predicted synthetic long-peptides in patients of origin, additional IDH mut glioma patients (n=16), and healthy donors (n=13). mRNA and protein expression of immunogenic antigens was analyzed in tumor tissues and IDH mut glioma stem-like cells (GSCs). HLA-A*02-restricted T-cell epitopes were functionally determined by short peptides and numbers of antigen-specific T-cells by HLA-peptide tetramer analysis. Results: 2,897 proteins were identified in immunogenic tumor fractions. Based on a thorough filter process 79 proteins were selected as potential T-cell antigens. 26 of these were recognized by the patients' T-cells and five of them (CRKII, CFL1, CNTN1, NME2, and TKT) in up to 56% unrelated IDH mut glioma patients. Most immunogenic tumor-associated antigens (TAAs) were expressed in IDH mut gliomas and GSCs, while being almost absent in normal brain tissues. Finally, we identified HLA-A*02-restricted epitopes for CRKII, NME2, and TKT that were recognized by up to 2.82% of antigen-specific peripheral cytotoxic T-cells in IDH mut glioma patients. Conclusion: By analyzing the repertoire of T-cell target antigens in IDH mut glioma patients, we identified five novel immunogenic TAAs and confirmed their expression on IDH mut tumors and GSCs.

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