Beyond Canonical Neoantigens: Emerging Technologies for Identification of Noncanonical Antigens and Implications for Personalized Cancer Vaccines

Over the past decade, advances in sequencing technologies and computational pipelines enabled the development of personalized cancer vaccines (PCVs). Current PCV strategies primarily target cancer neoantigens generated by non-synonymous DNA mutations, which can result in altered amino acid sequences capable of eliciting tumor-specific immune responses. More recently, a distinct class of tumor-specific antigens (TSA), termed noncanonical or cryptic antigens, has emerged as an additional source of immunogenic targets. Unlike canonical neoantigens, noncanonical antigens typically cannot be identified by tumor/normal whole-exome sequencing, as they do not arise from classical DNA mutations. Instead, they are often associated with less well recognized and/or aberrant processes in the pathways from DNA to human leukocyte antigen (HLA)-presented peptides. Examples include transposable elements, circular RNA, translation of alternative open reading frames and/or long non-coding RNA, among others. Emerging evidence suggests that noncanonical antigens represent a substantial portion of the tumor-specific immunopeptidome and, similar to canonical neoantigens, are absent during thymic selection and can evade central tolerance and elicit T cell responses. Technological advances have increasingly facilitated the identification of noncanonical antigens. Long-read RNA sequencing reveals noncanonical transcripts by improving transcriptome assembly, while ribosome profiling provides genome-wide maps of actively translated regions, facilitating the discovery of peptides from aberrant translation events. Specialized molecular approaches enable enrichment and sequencing of circular RNAs, and immunopeptidomics using mass spectrometry allows for direct characterization of HLA-presented peptides. Together, these technological advances have led to an increasing interest in prioritizing and targeting noncanonical antigens in the next generation of PCVs. This review provides an overview of the diverse origins of TSAs beyond classical neoantigens and discusses emerging approaches that may enable the integration of these antigens in future clinical trials.