Engineering of Human T-Cells with a Novel Aurora-A Kinase-Specific T-Cell Receptor Gene Transfer Confers Anti-Leukemia Reactivity

Recently we have identified a novel HLA-A*0201-restricted antigenic 9mer epitope (aa207-215: YLILEYAPL) derived from Aurora-A kinase (Aurora-A) which is capable of generating anti-leukemia cytotoxic T-lymphocytes (CTL).(Blood, 2009) To improve the feasibility of Aurora-A targeting cellular immunotherapy against leukemia, we have established a Aurora _A207-215-specific CTL clone, and have obtained the full-length T-cell receptor (TCR) α/β genes for TCR gene transfer. In this study, using human leukemia cell lines and patients' leukemia cells, we examined the anti-leukemia reactivity of engineered T-cells with Aurora-A-specific TCR gene transfer.

Full-length of an HLA-A*0201-restricted and Aurora-A_207-215-specific TCR α/β genes (Vα3/J20/Cα, Vβ10.3(12)/J1.1/Cβ1, respectively) were cloned into bicistronic GaLV-pseudotyped retroviral vector. Using Retronectin (Takara Bio. Japan)-coated plates, Aurora-A-specific TCR α/β genes were inserted into lymphocytes. Whether this vector was capable of generating a functional Aurora-A_207-215-specific TCR heterodimer was examined using Jurkat/MA cells (kindly gifted from Prof. Erik Hooijberg, Netherlands). The epitope-specific and leukemia specific cytotoxicity and IFN-γ production of gene-modified normal CD8⁺ and CD4⁺ T-cells were examined by ⁵¹Cr-releasing assay and ELISA. HLA-A*0201-restriction of engineered T-cell responses was examined by inhibition assay with antibodies, and HLA-A*0201 transduced human leukemia cell line: MEG01 which abundantly expresses Aurora-A. In vivo anti-leukemia effect of gene-modified CD8⁺T-cells was examined using NOD/SCID/γ_c^null (NSG) mice. Eventually, the on-target adverse effect of these Aurora-A-specific TCR-gene transferred CD8⁺T-cells against autologous hematopoietic progenitor cells was examined using cord blood CD34⁺ cells.

The Aurora-A specific-TCR expressing retroviral vector was capable of generating a functional TCR in Jurkat/MA cells which could produce luciferase in response to Aurora-A peptide on C1R-A2 cells in a dose dependent manner. Aurora-A-specific TCR-transduced CD8⁺ T-cells produced IFN-γ and exerted cytotoxicity against Aurora-A peptide-loaded C1R-A2 cells in an HLA-A*0201 restricted fashion. These engineered CD8⁺ T-cells also killed HLA-A*0201⁺ leukemia cell line and patient leukemia cells, but not HLA-A*0201⁺ normal PBMC and normal mitotic PHA-stimulated lymphoblasts. The anti-leukemia effect of These engineered CD8⁺ T cells was significantly abrogated by the anti-HLA-class I monoclonal antibody (MoAb), but not by anti-HLA-DR-MoAb. These engineered CD8⁺ T-cells killed HLA-A*0201-transduced MEG01 cells which were abundantly expressing Aurora-A, but not parent HLA-A*0201-negative MEG01 cells. Aurora-A-specific TCR gene transduced CD4⁺ T-cells produced IFN-γ in response to the epitope recognition, which was also in an HLA-A*0201-restricted fashion. Furthermore, Aurora-A-specific TCR-transduced CD8⁺ T cells did not damage the viability of autologous cord blood CD34⁺ cells in vitro. Finally, These engineered CD8⁺ T-cells successfully inhibited the engraftment and growth of inoculated leukemia cell line cells in the NOD/SCIDγ/_c^null mice.

In this study, Aurora-A kinase-specific TCR gene transferred T-cells successfully recognized the target epitope and exerted the target-specific cytotoxicity. Additionally these engineered CD8⁺ T-cells exerted anti-leukemia effect both in vitro and in vivo. While those these transfectants did not damage autologous hematopoietic progenitor cells in vitro. Collectively, the novel anti-leukemia adoptive therapy using Aurora-A-specific TCR-gene transferred T-cells appears promising, and further investigations are warranted for the clinical application.

No relevant conflicts of interest to declare.