A Public Neoantigen Produced By the SF3B1 K700E Mutation Is a Bona Fide T Cell Target in AML/MDS

Myelodysplastic syndrome (MDS) is a life-threatening disease in which malignant bone marrow cells fail to differentiate, resulting in anemia, cytopenia, and increased risk of progression to acute myeloid leukemia (AML). T cell-based therapies have great potential in treating AML/MDS, given that allogeneic stem cell transplantation (aSCT) can be curative in such settings. aSCT is associated with the graft-vs-leukemia (GvL) effect, in which T cells within the donor graft recognize and target antigens expressed by leukemia cells. Unfortunately, aSCT is also associated with considerable morbidity and mortality, as donor T cells may also recognize antigens expressed by non-malignant tissues and kill them, leading to graft-vs-host disease (GvHD). A T cell-based therapy that targets an AML/MDS-exclusive antigen has the potential to eliminate leukemic cells while sparing healthy tissues.

Most known antigens expressed on AML/MDS are also expressed by healthy myeloid cells. An ideal T cell target would be 1.) only expressed by leukemia cells, including leukemic stem cells (LSCs), 2.) expressed by all leukemia cells, and 3.) required for continued leukemia persistence. Somatic mutations in genes encoding spliceosomal machinery are common in MDS and have the potential to generate such neoantigens. In particular, a conserved missense mutation encoding a K700E substitution in SF3B1 is present in about 20% of patients with MDS, and approximately 80% of patients with MDS with ringed sideroblasts (MDS-RS). Moreover, SF3B1 K700E-mutant MDS has been proposed to be a distinct disease entity in which the SF3B1 mutation is a necessary, disease-initiating event. Further, SF3B1 K700E mutations are broadly detected across the spectrum of myeloid neoplasms from clonal hematopoiesis (CH) to relapsed/refractory AML and are associated with worse overall survival (OS) in the latter.

We demonstrate that the SF3B1 K700E mutant protein produces a leukemia-restricted T cell target. NetMHCpan4.1 binding algorithm predicted that a nonameric SF3B1 K700E-derived peptide could bind HLA-A*02:01, the most common HLA-A allele. A similar peptide derived from the wild type (WT) SF3B1 sequence was also predicted to bind HLA-A*02:01 with similar affinity. We identify potent TCRs from the T cell repertoires of healthy HLA-A*02:01 positive individuals, which effectively discriminate between WT and K700E peptides. We further show that the SF3B1 K700E: HLA-A*02:01 is a bona fide T cell antigen, as it is naturally processed by the proteasome and presented on the surface of SF3B1 K700E mutant cell lines and primary patient samples. Primary human CD8 T cells that were retrovirally engineered to express an SF3B1 K700E:HLA-A2 specific TCR are activated by, and kill SF3B1 K700E mutant cancer cells, while sparing SF3B1 WT cells.

Antigen dose response experiments demonstrate that this TCR has an approximately 4 order of magnitude specificity for the K700E peptide over the WT peptide, and amino acid substitution scanning experiments tiling the mutant peptide suggested no other naturally occurring HLA-A*02:01 restricted peptides from the human proteome that could activate the TCR. The EC ₅₀ of the K700E:A0201-specific TCR in peptide pulsing experiments was <1nM, which is comparable to the potency observed in viral antigen-specific TCRs.

In summary, we report that the SF3B1 K700E mutation yields an HLA-A*02:01 restricted AML/MDS-specific neoantigen which can be effectively targeted with TCR-T cells. We identified a highly potent TCR that eliminates cancer cells while sparing mutation negative cells. These data support further development of this TCR as a potential therapeutic for MDS and AML.