Targeting Aurora Kinase with a Superior T-Cell Receptor Gene-Transfer Vector

[Background] Aurora Kinase A (AURKA) is a cancer-associated protein normally involved in the regulation of mitosis. It is over-expressed in a range of cancers, and has a ‘cancer-testis’ expression profile, making it a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor (TCR) sequences cognisant of HLA-A*0201-restricted AURKA antigen (aa207-215: YLILEYAPL) has previously been shown to transfer specific immunoreactivity against the target peptide in a HLA-restricted manner (Nagai K et al. Blood, 2012).

While TCR gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumor-reactive lymphocytes from a patient’s own cells, one hurdle is potential mispairing and competition between exogenous and endogenous TCR chains. We have used a retroviral vector design bearing siRNAs that downregulates endogenous TCR chains, without affecting expression of the transgenic TCR sequences. The TCR expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the TCR α and β chains, further enhancing formation of the desired TCR. Via a simple, modular cloning method, we have cloned the α and β chains of the anti-AURKA -reactive TCR into this ‘siTCR’ vector. In this study, we then compared the activity of this vector against the original, ‘conventional’ vector without siRNAsacross a panel of assays.

[Methods] After codon-optimization to be resistant to built-in siRNAs for endogenous TCRs, full-length of an HLA-A*0201-restricted and AURKA_207-215 nonamer -specific TCR α/β genes (Vα3 /J20 /Cα, Vβ10.3(12) /J1.1 /Cβ1, respectively) obtained from  our established epitope-specific CD8⁺ T cell clone (AUR2) linked using T2A peptide sequence were cloned into the siTCR vector carrying those siRNAs using an Infusion cloning method (Takara Bio). Either conventional retroviral AURKA-specific TCR expression vector without those siRNAs or this new retroviral AURKA-siTCR vector was transduced into CD8⁺ T cells from healthy volunteers using anti-CD3 antibody and Retronectin^TM (Takara Bio) as previously reported. The epitope-specific and leukemia specific cytotoxicity and IFN-γ production of these transfectants were examined using ⁵¹Cr-releasing assay, CD107a assay and ELISA. Introduced viral vector copy numbers of these two types of transfectants were measured using qPCR kit (Bio Line) according to manufacturer’s instruction, on an ABI Prism 7500 (Applied Biosystems/Life Technologies). To examine the potential to target leukemia stem cells mediated by these AURKA-siTCR transfectants , we employed the side population of the AURKA mRNA highly expressing HLA-A*0201⁺ leukemia cell line, GANMO-1, using the dye exclusion method with Hoechst 33342 (Sigma). Concurrently the on-target/off-tumor adverse effect against hematopoietic progenitor cells mediated by these transfectants was examined using HLA-A*0201⁺ cord blood CD34⁺cells as a target.

[Results] TCRs expressed from the AURKA-siTCR vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed TCRs demonstrated by AURKA/HLA-A*0201 tetramer labeling was superior using the siTCR design, and this was achieved at lower vector copy numbers. AURKA mRNA tended to be overexpressed by the side population of GANMO-1, and AURKA-siTCR gene-modified CD8⁺ T cells successfully recognized and killed this population, but not normal cord blood CD34⁺cells.

[Conclusion] Maintaining TCR efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of TCR gene-transfer will be crucial for clinical applications of this technology.