Background T cells genetically modified to express a high affinity T cell receptor (TCR) have considerable potential for the treatment of cancer, however identifying immunogenic, tumor-specific targets remains a challenge. Recent transcriptomic analysis has shown that splicing mutations are frequently found across many different cancer types and can be drivers of tumorigenesis. Heterozygous mutations in SRSF2 (Serine and arginine Rich Splicing Factor 2), a protein involved in pre-mRNA splicing are prevalent across multiple types of high-risk myeloid neoplasms.1 The P95H mutation results in altered binding affinity of SRSF2 for cognate RNA site in target transcripts, producing abnormal proteins. Indeed, cell lines modified to express a SFSR2-P95H/+ mutation showed mis-regulation of hundreds of splicing events potentially exposing a large pool of unique, tumor-associated peptides that could be used to develop effective treatments (figure 1).
Methods To identify presented peptides, HLA-specific single chain dimers, which have the transmembrane domain replaced with a His-Tag allowing for secretion of the peptide/HLA in the supernatant, are transduced into SRSF2 wild type and mutant cells lines for downstream mass-spectrometry analysis (figure 2).2 We used this versatile platform to discover HLA-specific peptides resulting from alternative splicing events induced by a mutation in SRSF2. We then used recently published computational analysis to select promising mutant-derived peptides by aligning mass spectrometry-derived peptides to RNAseq data from our cell lines as well as broader tumor transcriptome databases.3 Finally, we tested our candidate peptides for their ability to induce tumor-specific killing by growing out T cell lines from healthy donors through repeated cycles of peptide stimulation (figure 3).
Results Using eluted single chain dimers, we identified thousands of peptides exclusive to mutant P95H/+ K562s cells for three different HLAs (HLA- A2, A11, and A24) that together cover 71% of phenotype of the US. We found that 11% of peptides are exclusive to the mutant cells (figure 4). Next, computational analysis of RNA sequencing data from wild type and mutant cells narrowed the list of peptides to those which resulted from alternate splicing events, against which T cell lines could be developed. Validating the identified peptides, we found multiple peptides which were able to trigger T cell activation and proliferation.
Conclusions This work demonstrates a robust method to discover novel HLA/peptide pairs and extend the reach of engineered TCR-T cell therapies. These techniques can easily be adapted for other mutations associated with RNA processing including SF3B1, U2AF1 and DDX41.
Acknowledgements This work was funded by the Fred Hutch Immunotherapy Integrated Research Center (IIRC) and the MPN Research Foundation. The authors wish to thank Kathryn Finton for developing the ARTEMIS system, Deborah Baker for writing and editing support, and the staff of the Fred Hutch flow core for making sure experiments run smoothly.
Kim E, et al. SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition. Cancer Cell. 2015;27:617–630.
Finton K, et al. ARTEMIS: A Novel Mass-Spec Platform for HLA-Restricted Self and Disease-Associated Peptide Discovery. Front Immunol. 2021;12:658372.
Pan Y, et al. IRIS: Discovery of cancer immunotherapy targets arising from pre-mRNA alternative splicing. Proc National Acad Sci. 2023;120:e2221116120.
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