Neoantigen-specific T-cell receptors targeting recurrent mutations in acute myeloid leukemia
Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy with poor prognosis. Clinical trials investigating novel therapies for AML increasingly involve immunotherapy, such as Chimeric Antigen Receptor-engineered T cells. However, their use is often limited by severe myelotoxicity, emphasizing the need for effective and safer alternatives. Neoantigens are attractive targets for immunotherapy as they are exclusively expressed on malignant cells. Neoantigens can be targeted by T-cell receptor-engineered T cells (TCR-T cells), as we previously demonstrated for neoantigens encoded by mutated NPM1, occurring in ~30% of AML. Here, we searched for TCRs targeting neoantigens encoded by other prevalent mutations in AML.
Immunopeptidomics was performed on EBV-B cells expressing common HLA-I alleles transduced with retroviral constructs encoding recurrent mutations in AML. pHLA-tetramers were produced for 19 neopeptides to search for specific T cells in 42 healthy donors. Tetramer-positive CD8 T cells were single-cell sorted, and T-cell clones were sequentially tested against peptide-pulsed cells, cell lines transduced with the mutated gene, cell lines expressing the mutation naturally or after CRISPR/Cas9 editing, and finally against patient-derived AML.
T-cell clones for 4 neoantigens were reactive against patient-derived AML. These antigens include an HLA-A*01:01 neoantigen encoded by DNMT3A-R882H (DNMT3A-A1), HLA-A*02:01 and HLA-B*08:01 neoantigens encoded by FLT3-D835Y (FLT3-A2 & FLT3-B8), and an HLA-B*07:02 neoantigen encoded by frameshift mutations in RUNX1 (RUNX1-B7). Despite high avidity in peptide-titration assays (EC50 ~1 nM), the clone for DNMT3A-A1 displayed weak reactivity against AML. The other clones were more reactive against AML. In peptide-titration experiments, T cells for FLT3 neoantigens (2-5 nM) had higher avidities than T cells for RUNX1-B7 and NPM1 neoantigens (~30 nM). Next, the TCRs of the clones were used to engineer T cells. TCR-T cells targeting FLT3-A2, RUNX1-B7, and NPM1 neoantigens were able to kill AML, but no killing was observed for TCR-T cells targeting FLT3-B8. Moreover, killing by FLT3-A2-specific TCR-T cells was relatively weak as compared to RUNX1-B7- and NPM1-specific TCR-T cells, highlighting the importance of neoantigen surface expression levels on tumor cells for efficient T-cell killing.
In conclusion, we identified various neoantigens and TCRs to develop effective and safe immunotherapies for patients with AML.