Development of a Novel Anti-Leukemia Gene Immunotherapy Using Aurora-A Kinase-Specific T-Cell Receptor Gene Transfer.

Aurora-A kinase (Aurora-A) is the member of therine/threonine kinase family, which regulates proper cell division process. Aurora-A currently has become the focus of public attention in the field of cancer therapy, because dysregulation of Aurora-A has been shown to be implicated in carcinogenesis, and its overexpression has been demonstrated in various kinds of cancer. Recently we have identified the HLA-A*0201-restricted 9-mer immunogenic epitope derived from Aurora-A (aa207-215: YLILEYAPL) which is well recognized by leukemia-reactive cytotoxic T lymphocytes (CTLs) (Blood, 2009). The clinical effect of peptide vaccination trials against leukemia reported previously is not satisfactory, therefore, the adoptive transfer of T cells genetically modified to express target antigen-specific T-cell receptor (TCR) has currently been highlighted. In the present study, we investigated the feasibility of anti-leukemia gene immunotherapy using Aurora-A-specific TCR gene transfer.

We established the Aurora-A_207-215-specific CD8⁺ CTL clone (AUR-2) from an HLA-A*0201⁺ healthy individual. The full length TCR alpha and beta genes were cloned from AUR-2, and the bicistronic GALV retroviral vector carrying these TCR genes was developed. At first, we evaluated the antigen specificity of TCR gene-transduced T cells by using Jurkat/MA cells (kindly gifted from Prof. Erik Hooijberg, Vrije Universiteit Medisch Centrum, Netherlands). Jurkat/MA is the modified Jurkat cell line devoid of intrinsic TCR, and engineered to express both CD8alpha molecule and NFAT-luciferase construct for detecting signaling via transferred TCR. Aurora-A-specific TCR-transduced Jurkat/MA cells were cocultured with various concentrations of Aurora-A peptide-loaded CIR-A2 cells, and then the expression level of luciferase was measured. Next, we transduced Aurora-A specific TCR gene into peripheral blood CD8⁺ T cells, and evaluated their Aurora-A-specific response against peptide-loaded C1R-A2 cells by detecting IFN-gamma production and cytotoxicity. Finally, we examined the cytotoxicity of Aurora-A-specific TCR-transduced T cells against various leukemia cells.

DNA sequencing revealed that TCR genes of AUR-2 are Valpha3/J20 for alpha-chain and Vbeta10.3/J1.1 for beta-chain. Mean transfection efficacy determined as the percentage of cells positively labeled with Vbeta12 antibody (identical to Vbeta10.3) by flow cytometry was about 20% in Jurkat/MA and more than 50% in normal CD8+ T cells. TCR-transduced Jurkat/MA produced luciferase in response to stimulation with Aurora-A peptide-loaded C1R-A2 cells in the dose dependent manner. Aurora-A-specific TCR-transduced CD8⁺ cells appeared to exert cytotoxicity against Aurora-A peptide-loaded C1R-A2 cells but not against peptide-unloaded C1R-A2 cells. Aurora-A TCR-transduced T cells produced IFN-gamma abundantly in response to stimulation with Aurora-A peptide-loaded but not with peptide-unloaded C1R-A2 cells. These TCR gene-transfectants lysed Aurora-A-expressing and HLA-A*0201⁺ leukemia cell line, GANMO-1. The cytotoxicity of Aurora-A-specific TCR gene-transduced T cells against GANMO-1 was significantly attenuated by anti-HLA-class I framework monoclonal antibody (MoAb), but not by anti-HLA-DR-MoAb.

In this study, we demonstrated for the first time that anti-leukemia effect can be mediated by Aurora-A _208-215-epitope specific TCR gene transfer into normal peripheral blood CD8⁺ T cells. The clinical trials using small molecule inhibitors for Aurora-A are currently being conducted. Our findings may develop a comprehensive strategy for cancer treatment using the combination of immunotherapy and small molecule inhibitors, both are targeting the identical molecule which plays an important role in carcinogenesis and progression of cancer.

No relevant conflicts of interest to declare.