Abstract
Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal expansion of myeloid progenitor cells in the bone marrow. Mutations in the nucleophosmin-1 (NPM1) gene are essential for leukemogenesis in 30% of AML. The mutations consist of 4 base pair insertions in exon 12, resulting in an elongated protein and translation of the C-terminal 11 amino acids in an alternative reading frame (CLAVEEVSLRK). We previously identified various HLA binding mutated NPM1 (dNPM1) derived peptides on the cell surface of AML by tandem mass spectrometry. These peptides include CLAVEEVSL, which binds to HLA-A*02:01 and AVEEVSLRK and CLAVEEVSLRK both binding to HLA-A*03:01 as well as HLA-A*11:01. For CLAVEEVSL, we previously isolated a T cell clone from an HLA-A*02:01-positive healthy donor that was able to react against dNPM1 AML. The dNPM1-A2 specific T-cell receptor (TCR) of this clone has been sequenced and cloned and can be used to engineer and redirect patient T-cells to target AML in vitro and in vivo in immunodeficient mice engrafted with the HLA-A*02:01- and dNPM1-positive OCI-AML3 cell line. The safety and preliminary efficacy of the dNPM1-A2 TCR-T cells will be evaluated in a phase I/II clinical trial.
Here, we searched for new dNPM1 specific T cells in peripheral blood mononuclear cells of healthy donors using peptide-HLA (pHLA)-tetramers for AVEEVSLRK in HLA-A*03:01 and HLA-A*11:01. We isolated one dNPM1-A3 tetramer positive T cell clone (31.3.F1) from an HLA-A*03:01-positive donor, which specifically released IFN-γ upon stimulation with HLA-A*03:01-tranduced OCI-AML3 (dNPM1), but not HLA-A*03:01-transduced OCI-AML2 (wtNPM1) cells. The T-cell clone also recognized three HLA-A*03:01- and dNPM1-positive AML cases in vitro (Figure 1A). In addition to the dNPM1-A3 T cell clone, two dNPM1-A11 tetramer positive T cell clones were isolated from an HLA-A*11:01-positive and HLA-A*11:01-negative healthy individual. Both T-cell clones specifically reacted against HLA-A*11:01-transduced OCI-AML3 cells, but not against OCI-AML2 cells transduced with HLA-A*11:01. Both T-cell clones also recognized three HLA-A*11:01- and dNPM1-positive AML cases (Figure 1A). In all experiments, the dNPM1-A11 T cell clone from the HLA-A*11:01 negative donor (clone 26.2.D6) outperformed the dNPM1-A11 T cell clone from the HLA-A*11:01 positive donor (clone 6F11) in its reactivity against AML, suggesting expression of a higher affinity TCR. A preliminary safety analysis did not reveal any reactivity of clone 26.2.D6 against primary HLA-A*11:01-positive hematopoietic cell types including mature dendritic cells.
To investigate whether the TCRs of the T-cell clones may be useful to produce dNPM1-A3 and dNPM1-A11 TCR-T cells to treat patients with dNPM1 positive AML, the TCRs were sequenced, cloned and used to engineer CD8+ T cells from healthy individuals. HLA-A*03:01 or HLA-A*11:01 restricted TCRs for EBNA3A or EBNA3B, respectively, were introduced as controls. The dNPM1-A3 and dNPM1-A11 TCR-T cells showed specific binding to the corresponding pHLA tetramer and reactivity against HLA-A*03:01- and A*11:01-positive EBV-B cells pulsed with a mix of dNPM1 and EBV peptides. The dNPM1-A3 and dNPM1-A11 TCR-T cells were also reactive against OCI-AML3 cells transduced with the relevant HLA allele, while no reactivity was observed against irrelevant targets (Figure 1B). Similar as the parental T-cell clones, T cells with the dNPM1-A11 TCR from clone 26.2.D6 outperformed T cells with the dNPM1-A11 TCR from clone 6F11. Currently, dNPM1-A3 and dNPM1-A11 TCR-T cells are analyzed for their potential to target AML in vitro and in OCI-AML3-engrafted immunodeficient mice. These data show that AVEEVSLRK is a neoantigen on HLA-A*03:01 and A*11:01 dNPM1 AML that could serve as a target for TCR gene therapy.
Disclosures
Koutsoumpli:Miltenyi Biotec B.V & Co. KG: Research Funding. Lock:Miltenyi Biotec B.V & Co. KG: Current Employment. Johnston:Miltenyi Biotec B.V & Co. KG: Current Employment. Griffioen:Miltenyi Biotec B.V & Co. KG: Research Funding.
