Human Telomerase Reverse Transcriptase-Specific T-Cell Receptor Gene Transfer Redirects T-Lymphocytes to Exert Effective Antitumor Reactivity Against Adult T-Cell Leukemia,

Currently adult T-cell leukemia (ATL) is one of the most chemotherapy-resistant T cell malignancies and the prognosis of ATL patients still remains very poor. Although several novel treatment options, for example anti-chemokine receptor 4 (CCR4) monoclonal antibody, look promise, development of other novel treatment options still remains warranted. Currently activated status of human telomerase reverse transcriptase (hTERT) in ATL cells has been underscored. Thus, in this study, in order to develop a novel T cell-based immunotherapy for the treatment of ATL, we investigated the feasibility of redirected T-cell using hTERT-specific T-cell receptor (TCR) gene transfer.

Approval for this study was obtained from the Institutional Review Board of Ehime University Hospital. HLA-A*24:02-restricted and hTERT_461–469epitope (residues: VYGFVRACL)-specific TCR -a/b genes (Tajima K et al. Int J Cancer 2004) cloned from our established hTERT-specific CTL clone were inserted into a novel GaLV-pseudotyped retroviral vector encoding built-in siRNAs for constant regions of endogenous TCR a /b genes (hTERT-siTCR vector). hTERT-siTCR vector was transfected into normal CD8⁺ T cells in RetroNectin (Takara Bio Inc.) coated-plates and these redirected CD8⁺ T cells (hTERT-siTCR CD8) were used as effector cells. HLA-A*24:02⁺ or A*24:02⁻ ATL cell lines, HTLV-1 infected T-cell lines and freshly isolated ATL cells from patients were examined as target cells. All patients gave written informed consents in accordance with the declaration of Helsinki. Normal HLA-A*24:02⁺CD4⁺ T cells andHLA-A*24:02⁺cord blood CD34⁺ mononuclear cells (CB-CD34⁺) were similarly obtained from healthy volunteers. Expression of hTERT mRNA and hTERT protein in ATL cell lines, freshly isolated ATL cells, and HTLV-1 infected T cells were evaluated by quantitative real time PCR (QRT-PCR) using DCt method and Western blotting. Antileukemia reactivity mediated by hTERT-siTCR CD8 against ATL cells were examined by standard ⁵¹chromium release assay and flow-based CD107a assay. For the safety assessment, effector cells-mediated cytotoxicity against CB-CD34⁺ cells as normal hematopoietic progenitors was similarly examined. Peripheral blood mononuclear cells (PBMCs) from ATL patients were obtained on admission and in stable disease or remission after receiving treatments, and hTERT_461–469 epitope responsive CD8⁺ T cells were detected using hTERT_461–469peptide/ HLA-A*2402 tetramer or ELISPOT assay.

QRT-PCR revealed that both freshly isolated ATL cells and ATL cell lines markedly overexpressed hTERT mRNA, while that in normal CD4⁺ cells and CB-CD34⁺ cells was less than undetectable. hTERT-siTCR CD8 cells were more than 40% positive for hTERT/HLA-A*2402 tetramer and successfully displayed HLA-A*2402-restricted and hTERT_461–469-specific cytocidal effect against target-loaded C1R-A24 cells. Additionally, hTERT-siTCR CD8 cells successfully discriminated A*2402⁺ ATL cell lines (ATN-1, TL-Su) from A*2402⁻ ones (TLO-m1, HUT-102); those all cell lines similarly overexpressed hTERT. Furthermore, those effector cells successfully killed freshly isolated HLA-A*2402⁺ ATL cells, but not A*2402⁻ ones. Finally, hTERT_461–469-specific cytotoxic T-lymphocyte precursors were detected at variable levels in PBMCs obtained from HLA-A*2402⁺ ATL patients, which shows hTERT overexpressed by ATL cells may be naturally processed in vivo.

Although much further invetigations are warranted, in our preliminary study, we have shown for the first time that hTERT could be a potential therapeutic target of redirected T cell-based immunotherapy for the treatment of ATL. We are now in detail examining efficacy and safety of those redirected T cells using xenograft mouse model.

No relevant conflicts of interest to declare.