Article Text
PDF
Abstract
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.
References
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.
Alternative splicing resulting in targetable peptides. Alternatively spliced RNA that is translated into a protein and processed by the proteasome may produce mutation-specific peptides. When loaded into an HLA and presented on the surface, these peptide-HLA (pHLA) complexes can be recognized by TCR-T cell therapies
Single chain dimer peptide discovery pipeline. Single chain dimers (SCDs) for each HLA of interest are transduced into SRSF2 WT and P95H K562s. Transduced cells are sorted and cultured, usually with the addition of IFNy and TNFα. During the culture period, SCDs are secreted into the supernatant which is collected at the end of the culture. Peptides are then eluted from the SCDs and identified using mass spectometry
T cell line discovery pipeline. Dendritic cells (DCs) are isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and matured using a cytokine treatment. DCs are loaded with peptides of interest and cultured with donor-matched CD8+ T cells for two weeks. This cycle is repeated 1–2 more times using peptide-loaded PBMCs. After overnight stimulation with peptides, CD137+ (41BB) cells are sorted and expanded. These T cell lines are then tested for their ability to respond to SRSF2 wild type and mutant (P95H) K562 lines. This protocol aims to identify lines that kill mutant cells while sparing wild type cells
Venn diagram comparisons of eluted peptides. The three HLAs selected have distinct binding preferences (left). Combining peptides from the three HLAs, approximately 11% of peptides are exclusive to the SRSF2 mutant (P95H) cells (right). These peptides are potential candidates for the development of TCR-T cells. Results from 2x A2, 3x All, and 3x A24 biological replicates
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.