Few actionable tumour-specific antigens (TSAs) successfully used in therapeutic cancer vaccines have been discovered so far. Most efforts to date have employed the approach of reverse immunology to identify mutations from whole-exome sequencing of tumour cells, and major histocompatibility complex (MHC)-binding prediction software algorithms to assess the binding affinities of mutated peptides. Yet, this method has drawbacks: 90% of TSA candidates are false positives, as the software tools fail to predict steps other than binding necessary for the processing of MHC peptides, and using only protein-coding exons as a source of TSAs is limiting. To address this, Laumont, Vincent et al. developed a proteogenomic strategy for high-throughput mass spectrometry (MS) discovery of aberrantly expressed TSAs (aeTSAs) from non-coding sequences as well as mutated TSAs (mTSAs) encoded by all genomic regions.

Most MS software tools rely on a user-defined reference proteome that does not include TSAs, and so the authors built a tailored database from tumour RNA sequencing data. This global cancer database comprises a canonical cancer proteome of proteins encoded by normal or single base-mutated exonic sequences, and a cancer-specific proteome of peptides encoded by any reading frame of any genomic region that are absent from normal MHC IIhi medullary thymic epithelial cells (mTECs, which are responsible for T cell selection).

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Utilizing the database with MS sequencing of two mouse tumour cell lines, colorectal carcinoma CT26 and T lymphoblastic lymphoma EL4, followed by further validation identified 6 mTSAs and 11 aeTSAs. Interestingly, most of the TSAs came from atypical translation events: translation of an out-of-frame coding exon or non-coding regions. Furthermore, these TSAs derived from various non-coding regions, such as intergenic and intronic sequences, non-coding exons, untranslated region (UTR)–exon junctions, endogenous retroelements and even structural variants.

Double immunization of mice with either unpulsed or individual TSA-pulsed dendritic cells before challenge with live EL4 cells demonstrated that the identified TSAs displayed differential but overall long-lasting protection. Crucially, the immunogenicity of the TSAs was determined by both TSA-responsive T cell expansions upon vaccination and TSA expression levels on tumour cells.

Last, applying this strategy to human primary tumour samples — specifically four B cell acute lymphoblastic leukaemias (B-ALLs) and three lung cancers — by subtracting mRNA sequences of TECs and mTECs from unrelated donors led to the identification of 2 mTSAs and 20 aeTSAs. One of the B-ALL aeTSAs originates from the 3' UTR of TCL1A, a gene associated with lymphoid malignancies. The next step would be to validate the immunogenicity of these TSAs in mice.

targetable antigens that can be used for cancer vaccine development

By identifying TSAs from non-coding regions, this study expands the number of targetable antigens that can be used for cancer vaccine development. Even more appealing as preferred targets, aeTSAs can be widely shared by multiple tumours (including those with low mutational burdens), meaning a single vaccine could be generated, unlike neoantigen-directed immunotherapy, which is likely to be personalized.