SF3B1K700E Neoantigen Is a CD8+ T-cell Target Shared across Human Myeloid Neoplasms

Abstract

Acquired mutations in spliceosome genes in early hematopoietic stem/progenitor cells are common events in myelodysplastic neoplasms (MDS) and related myeloid malignancies. Mutations in the spliceosome factor subunit B1 (SF3B1) gene occur in ≥20% of MDS cases at conserved hotspots and in early neoplastic clones as driver events. Neoantigens from aberrant SF3B1 proteins could serve as shared T-cell therapy targets for SF3B1-mutated myeloid neoplasms. We identified a candidate neoantigen from the prevalent SF3B1K700E variant using in silico predictions of epitope processing and presentation and then validated presentation and immunogenicity in vitro. CD8+ T cells recognizing SF3B1K700E demonstrated high functional avidity and killed neoplastic myeloid cell lines and primary cells in an antigen-specific manner. We then sequenced, cloned, and transduced an SF3B1K700E-specific T-cell receptor into third-party T cells and confirmed that T-cell receptor transfer conferred antigen specificity and killing of neoplastic myeloid cells in vitro and in vivo. The data indicate that the SF3B1K700E neoantigen represents a promising T-cell target for patients with SF3B1-mutated MDS and acute myeloid leukemia.

Figure 1.

Predicted binding affinity and eluted ligand ranking of SF3B1^K700E peptides to 20 prevalent HLA class I alleles. Heatmaps of A, the predicted binding affinity or B, the eluted ligand rank as an output from a netMHCpan 4.1 analysis of the SF3B1^K700E-spanning 18-mer amino acid sequence for the 20 most prevalent HLA class I alleles in the U.S. population. For both heatmaps, data is −log₁₀ transformed and shown for mutant (gray) and wild-type (black) equivalent peptides (indicated at the top of heatmaps). Created in BioRender. Lok, J. (2025) https://BioRender.com/4i4i1dy

Figure 2.

The SF3B1^K700E neoantigen epitope is immunogenic and primes a high-avidity epitope-specific CD8⁺ T cell clone. A, Schematic of altered SF3B1^K700E protein and neoantigen epitope peptide. B, A clone was identified after primary in vitro stimulation of CD8⁺ T cells from an HLA-B*40:01-positive donor with QEVRTISAL peptide-pulsed DC and tested in peptide titration CRA against autologous LCL pulsed with varying peptide concentrations (3 technical replicate experiments). C, HLA restriction of D1.C24 was confirmed by testing in CRA against a panel of HLA-typed LCL with single HLA overlap with the original T cell donor. LCL were pulsed with QEVRTISAL peptide at 1000 ng/mL prior to coculture (≥5 biological replicates per HLA). D, D1.C24 was tested for recognition of known immunogenic HLA-B*40:01-presented peptides in CRA using autologous LCL pulsed with 1000 ng/mL of each peptide. QEVRTISAL peptide and the wild-type equivalent QKVRTISAL were included as controls (3 technical replicate experiments). E, Alanine scanning for D1.C24 was performed using autologous LCL pulsed with a panel of peptides (1000 ng/mL) with alanine residues substituted at each position, along with two peptides with either a glycine or valine substitution at position 8, a natural alanine residue in the QEVRTISAL peptide (3 technical replicate experiments). These data were used to identify critical residues for HLA and TCR binding and define the core motif xExRTIxxL. F, Four peptides derived from wild-type human proteins and sharing the xExRTIxxL motif were identified using the Scan ProSite tool. To evaluate for cross-recognition of these peptides by D1.C24, autologous LCL were pulsed with each peptide (1000 ng/mL) and used as targets for D1.C24 in CRA (3 technical replicate experiments). For all experiments, mean and SEM are shown. Created in BioRender. Lok, J. (2025) https://BioRender.com/pijsqsm

Figure 3.

The SF3B1^K700E neoantigen is naturally processed and presented on neoplastic myeloid cells. A, Percent survival of HNT-34/B*40:01 cells cocultured with either D1.C24 (blue) or irrelevant neoantigen-specific clone (gray) in a flow cytometry cytotoxicity assay. Mean and SEM from ≥3 technical replicates shown. B, Percent survival of SF3B1^K700E minigene transduced NB-4 cells cocultured with either clone D1.C24 (blue) or irrelevant neoantigen-specific clone (gray) in flow cytometry cytotoxicity assay. Mean and SEM from ≥3 technical replicates shown. C, Representative flow plots from CD107a assay demonstrating D1.C24 degranulation in response to genotypically SF3B1^K700E-positive HLA-B*40:01-positive primary neoplastic myeloid cells (MDS or sAML) but not controls lacking either the mutation or HLA. D, Summary data from CD107a assays of D1.C24 response to primary neoplastic myeloid cells (≥1 biological replicates, ≥2 technical replicates). E, Representative flow plots from CD107a assay demonstrating D1.C24 degranulation with genotypically SF3B1^K700E-positive HLA-B*40:01-positive MPP-5F line but not SF3B1^K700E-negative HLA-B*40:01-positive isogenic control. MPP-5F lines were pre-cultured with IFNγ for at least 24 hours. F, Summary data from CD107a assays of D1.C24 response to MPP-5F lines under various conditions (≥3 biological replicates). For summary figures, mean and SEM are shown. Created in BioRender. Lok, J. (2025) https://BioRender.com/6rz6fds

Figure 4.

Transfer of the SF3B1^K700E/B*40:01-specific TCR confers specificity and function. A, Representative flow plots demonstrating expression of the D1.C24 SF3B1^K700E/B*40:01-specific TCR (TCR24) transduced (TD) into primary human CD8⁺ T cells after CRISPR/Cas9-mediated knock-out of endogenous TCR alpha and beta chains (middle) showing staining for the RQR8 transduction marker and the transgenic TCR with SF3B1^K700E/B*40:01-pHLA tetramer. B, Cytolytic activity of TCR24 TD T cells and corresponding T cell clone in CRA (technical triplicates). C, Percent survival of HNT-34 cells without and with transduction of HLA-B*40:01 cocultured with TCR24 TD T cells, parental clone, or controls in flow cytometry cytotoxicity assay (≥3 technical replicates). D, Percent survival of NB-4 cells without and with transduction of SF3B1^K700E minigene cocultured with TCR24 TD T cells, parental clone, or controls in flow cytometry cytotoxicity assay (≥3 technical replicates). Created in BioRender. Lok, J. (2025) https://BioRender.com/xtc8n5z