Microproteins encoded by noncanonical ORFs are a major source of tumor-specific antigens in a liver cancer patient meta-cohort.

The expression of tumor-specific antigens during cancer progression can trigger an immune response against the tumor. Here, we investigate if microproteins encoded by noncanonical open reading frames (ncORFs) are a relevant source of tumor-specific antigens. We analyze RNA sequencing data from 117 hepatocellular carcinoma (HCC) tumors and matched healthy tissue together with ribosome profiling and immunopeptidomics data. Combining human leukocyte antigen-epitope binding predictions and experimental validation experiments, we conclude that around 40% of the tumor-specific antigens in HCC are likely to be derived from ncORFs, including two peptides that can trigger an immune response in humanized mice. We identify a subset of 33 tumor-specific long noncoding RNAs expressing novel cancer antigens shared by more than 10% of the HCC samples analyzed, which, when combined, cover a large proportion of the patients. The results of the study open avenues for extending the range of anticancer vaccines.

Relevance of mutation-derived neoantigens and non-classical antigens for anticancer therapies.

Efficacy of cancer immunotherapies relies on correct recognition of tumor antigens by lymphocytes, eliciting thus functional responses capable of eliminating tumor cells. Therefore, important efforts have been carried out in antigen identification, with the aim of understanding mechanisms of response to immunotherapy and to design safer and more efficient strategies. In addition to classical tumor-associated antigens identified during the last decades, implementation of next-generation sequencing methodologies is enabling the identification of neoantigens (neoAgs) arising from mutations, leading to the development of new neoAg-directed therapies. Moreover, there are numerous non-classical tumor antigens originated from other sources and identified by new methodologies. Here, we review the relevance of neoAgs in different immunotherapies and the results obtained by applying neoAg-based strategies. In addition, the different types of non-classical tumor antigens and the best approaches for their identification are described. This will help to increase the spectrum of targetable molecules useful in cancer immunotherapies.

Identification of HLA class I-restricted immunogenic neoantigens in triple negative breast cancer.

Immune checkpoint inhibitor (ICI)-based immunotherapy in triple negative breast cancer (TNBC) is achieving limited therapeutic results, requiring the development of more potent strategies. Combination of ICI with vaccination strategies would enhance antitumor immunity and response rates to ICI in patients having poorly infiltrated tumors. In heavily mutated tumors, neoantigens (neoAgs) resulting from tumor mutations have induced potent responses when used as vaccines. Thus, our aim was the identification of immunogenic neoAgs suitable as vaccines in TNBC patients. By using whole exome sequencing, RNAseq and HLA binding algorithms of tumor samples from a cohort of eight TNBC patients, we identified a median of 60 mutations/patient, which originated a putative median number of 98 HLA class I-restricted neoAgs. Considering a group of 27 predicted neoAgs presented by HLA-A*02:01 allele in two patients, peptide binding to HLA was experimentally confirmed in 63% of them, whereas 55% were immunogenic in vivo in HLA-A*02:01+ transgenic mice, inducing T-cells against the mutated but not the wild-type peptide sequence. Vaccination with peptide pools or DNA plasmids expressing these neoAgs induced polyepitopic T-cell responses, which recognized neoAg-expressing tumor cells. These results suggest that TNBC tumors harbor neoAgs potentially useful in therapeutic vaccines, opening the way for new combined immunotherapies.