Effective immunotherapy for acute myeloid leukemia (AML) currently relies primarily on neoantigens derived from somatic mutations. However, these can sometimes cause off-target or off-tumor toxicities. Identifying additional neoantigens from alternative sources may help improve the efficacy of immunotherapy.

To discover such non-canonical neoantigens, researchers from a strong scientific community begin with RNA-seq data to screen for candidates arising from RNA missplicing. To validate whether these peptides are presented in vivo, they use LC-MS–based immunopeptidomics and compare MS2 spectra of synthetic and endogenous peptides using PEAKS software. Mirror plots of experimental versus synthetic peptides, along with the peptide scores estimated by PEAKS, clearly confirm the sequences.

In follow-up studies, the researchers isolated T cell receptors (TCRs) and demonstrated their activation by these non-canonical neoantigens. Subsequent experiments showed that immunotherapy based on these peptides could improve patient outcomes.

Tip: Non-canonical peptides like these are typically missed in conventional database searches, as standard protein databases don’t contain these sequences. In most cases, generating a custom protein database from RNA-seq data is a practical solution. Alternatively, de novo sequencing—used to deduce peptide sequences directly from fragment ions—can identify novel peptides without relying on a database.

You can find the article here: https://www.cell.com/cell/fulltext/S0092-8674%2825%2900399-X