TCR-T cells targeting a public neoantigen produced by the SF3B1 K700E mutation kill SF3B1-mutant AML/MDS Free

Myelodysplastic syndrome (MDS) is a life-threatening disease in which malignant bone marrow cells fail to differentiate, resulting in anemia, cytopenia, and 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. This is at least in part, due to the graft-vs-leukemia (GvL) effect, in which T cells within the donor graft recognize and target antigens on leukemia cells. Unfortunately, aSCT is 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 by AML/MDS are also expressed by healthy myeloid cells, and targeting them risks myeloablation. An ideal target would be 1.) exclusively expressed by leukemia cells, including leukemic stem cells (LSCs), 2.) expressed by all leukemia cells, and 3.) necessary and sufficient for leukemogenesis. 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). 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.

Here, we demonstrate that the SF3B1 K700E contains a peptide that binds HLA-A*02:01 and is bona fide leukemia-restricted T cell target. HLA binding prediction models were used to identify putative HLA binders containing the SF3B1 K700E mutation; GLVDEQQEV (wild type sequence GLVDEQQKV) was predicted to bind HLA-A*02:01, the most common HLA-A allele. We cultured CD8 T cells from healthy donor repertoires with SF3B1 K700E peptide to expand out putative SF3B1 K700E:HLA-A*02:01-specific T cells. SF3B1 K700E:A*02:01-tetramer binding cells were sorted and TCR sequenced, and TCR sequences were inserted into retroviral vectors to make TCR-T cells. Peptide dose-response expeirments with TCR-T cells identified a highly potent TCR (functional avidity < 1nM, approximating anti-viral potency) which discriminated between the mutant and wild type SF3B1 peptides with a 4 order of magnitude preference. Both CD8+ and CD4+ TCR-T cells were robustly activated in the presence of antigen, demonstrating the potency and CD8-independence of the TCR.

Co-culture cytoxocity experiments demonstrated that TCR-T cells specifically kill HLA-A*02:01, SF3B1-K700E mutant cell lines (in which SF3B1 K700E expression was either knocked-in to a single SF3B1 locus or endogenously expressed by the cell line) while sparing both HLA-A*02:01 negative and HLA-A*02:01 positive, SF3B1 WT cell lines. Sequencing experiments demonstrated that SF3B1 K700E was expressed in LSCs, and TCR-T cells were activated by culture with patient-derived SF3B1 K700E mutant primary AML/MDS (but not with SF3B1 WT AML/MDS) demonstrating that SF3B1 K700E peptide is a bona fide, patient-relevant leukemia antigen. Xscan amino acid scanning experiments tiling all possible single amino acid substitutions were performed to generate a TCR recognition motif, which was used as a ScanProsite query against the human proteome to identify possible off-target antigens for the TCR. While 18 motif matching peptides were identified in the human proteome, none activated TCR-T cells, demonstrating the exquisite specificity of the TCR.

Finally, we generated an SF3B1 K700E mutant knock-in U937 cell line, and evaluated efficacy of the TCR against the leukemia in vivo in an NSG CDX-mouse model. Mice adoptively transferred with SF3B1 K700E specific T cells demonstrated increased survival compared to mice transferred with irrelevant TCRs. In summary, we report that the SF3B1 K700E mutation yields an HLA-A*02:01 restricted AML/MDS-specific neoantigen, and that TCR-T cells targeting this mutation selectively kill leukemia.