Document resource
Background The tumor suppressor genes encoded in the CDKN2A locus, p16INK4A and p14ARF, are among the most frequently mutated genes in cancers. Functional loss of either gene product contributes to tumorigenesis. However, when their downstream effectors get inactivated, both genes can often be aberrantly overexpressed by a multitude of cancers. Here we assess whether peptide antigens derived from overexpressed p16INK4A and p14ARF can be processed/presented by tumors and recognized by T cells with the goal of identifying T cell receptors (TCRs) for the development of TCR-T cell therapies to eliminate p16INK4A- and p14ARF-expressing cancer cells.Methods We used the NetMHCpan4.1 program to predict HLA-A*02:01-associated peptide antigens derived from p16INK4A and p14ARF and tested those peptides for their ability to elicit potent and specific T cell responses. For each peptide, antigen-specific T cell lines were generated from the peripheral repertoire of healthy donors, and peptide/HLA tetramers were employed to identify and enrich peptide specific CD8 + T cells. T cell lines were tested for recognition of p6INK4A/p14ARF+ target cell lines and TRA/TRB genes were cloned and used to engineer TCR-T cells to evaluate cytotoxic potential and target specificity in vitro.Results Antigen-specific T cell lines targeting twelve p16INK4A peptides and four p14ARF peptides were generated, sort-purified and tested for recognition of p16INK4A and p14ARF-expressing target cells, respectively. For p16INK4A, only one peptide was presented by HLA-A*02:01 and recognized by T cells, albeit with low efficiency. However, this epitope was not specific for p16INK4A, and shared with the broadly expressed cell cycle inhibitor p15. In contrast, we identified a p14ARF-derived peptide efficiently presented by p14ARF + targets resulting in highly potent T cell responses. Specificity was further confirmed with a p14ARF knockout cell line, which failed to elicit a response from p14ARF-specific T cells. Eight healthy donor repertoires were then screened for high affinity p14ARF-specific TCRs. Seventeen TCRs were synthesized, of which two proved particularly potent when engineered into TCR-T cells, with efficient recognition of multiple p14ARF+ tumor cell lines and robust killing of p14ARF-expressing tumors at a low 1:4 effector-to-target ratio. No alloreactivity was observed for either TCR, and X-scan analysis revealed limited cross-reactivity.Conclusions Human TCRs specific for HLA-A*02:01/p14ARF can specifically and potently lyse p14ARF-expressing cancer cells. Given the clinical progress and promising anti-tumor activity of TCR-T cell therapies targeting MAGEA4, NY-ESO1, WT1, and PRAME, p14ARF-specific TCR-T cell therapies hold promise for treating a variety of cancers overexpressing this tumor suppressor.