Mis-splicing derived neoantigens and cognate T cell receptors in splicing factor mutant leukemias

Mutations in RNA splicing factors are prevalent across cancers and generate recurrently mis-spliced mRNA isoforms. Here we identified a series of bona fide neoantigens translated from highly stereotyped splicing alterations promoted by neomorphic, leukemia-associated somatic mutations in the splicing machinery. We utilized feature-barcoded peptide-MHC dextramers to isolate neoantigen-specific T cell receptors (TCR) from both healthy donors and patients with leukemia. While circulating neoantigen-specific CD8+ T cells were identified in patients with active disease, they were dysfunctional with reduced inflammatory response gene signatures. In contrast, donor CD8+ T cells with tumor-reactive TCRs were present following curative allogeneic hematopoietic cell transplant. T cells engineered with TCRs recognizing an SRSF2 mutant-induced neoantigen in CLK3 resulted in specific recognition and cytotoxicity of SRSF2 mutant leukemia. These data identify RNA mis-splicing derived neoantigens and neoantigen-specific TCRs across patients and provide proof-of-concept to genetically redirect T cells to public mis-splicing derived neoantigens in myeloid leukemias. Overall design: We isolated bulk T cells from patient PBMCs via negative selection and stained the T cells with a dextramer pool. We sorted live, CD3+, CD8+ dextramer+ cells and performed RNA-, TCR-, and dextramer feature barcode sequencing (Fig. S5A). For certain samples where dextramer+ cell numbers were limiting, live CD3+ CD8+ dextramer- cells were also sorted, stained with cell hashing antibodies, and doped into the dextramer+ populations. Using this approach, we single-cell profiled CD8+ T-cells from nine samples across five distinct HLA-A*02:01+ SRSF2 mutant myeloid leukemia patients (Fig. 5A, Fig.S5B, and Table S3) yielding 75,343 high-quality T cells.