Human Telomerase Reverse Transcriptase–specific T-Cell Receptor Gene Transfer Redirects T Cells to Display an Effective Antitumor Reactivity Against Adult T-Cell Leukemia
Authors: Fujiwara Hiroshi , Ochi Fumihiro , Miyazaki Yukihiro , Asai Hiroaki , Ishida Takashi , Okamoto Sachiko , Mineno Junichi , Kuzushima Kiyotaka , Shiku Hiroshi , Yasukawa Masaki 
Published: October 2019

Abstract
Abstract 1902

Background and Purpose:
Adult T-cell leukemia (ATL) is one of the most refractory forms of T-cell malignancy that preferentially affects aged population that constrains clinical efficacy of chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, objective responses in a small part of patients who received allo-HSCT evidently emphasize the therapeutic potential of immune-mediated approaches. While, some previous studies described that activation of human telomerase reverse transcriptase (hTERT), one of tumor antigens was detected in ATL tumor cells. Collectively, in this study, we examined the feasibility of redirected T-cell based immunotherapy targeting hTERT to explore a novel treatment strategy for ATL.

Methods:
Approval for this study was obtained from our institutional review board. Written informed consent was obtained from all patients, healthy volunteers, and parents of cord blood donors in accordance with the Declaration of Helsinki. HLA-A*2402-restricted and hTERT461–469 epitope (VYGFVRACL)-specific TCR-α/β genes were newly cloned into a novel retroviral vector encoding siRNAs for endogenous TCR-α/β genes (hTERT-siTCR vector). Using hTERT-siTCR vector, hTERT-siTCRR-α/β genes were transduced into normal peripheral CD8+ T cells (hTERT-siTCR/CD8+), employed as effectors. HLA-A*2402+ or HLA-A*2402− ATL cell lines, freshly isolated patients' ATL cells, normal peripheral blood mononuclear cells (PBMCs) from healthy volunteers, and CD34+ cells isolated from cord blood mononuclear cells (CBMCs) were employed as target cells. Anti-tumor reactivity mediated by hTERT-siTCR/CD8+ was assessed using standard 51Cr release assay. Expressions of hTERT mRNA and hTERT protein in ATL cell lines, freshly isolated ATL cells and normal CD4+ T cells were assessed by quantitative real-time PCR (QRT-PCR) and Western blotting. Frequencies of hTERT461–469–specific cytotoxic T-lymphocyte (CTL) precursors in periphery of ATL patients or healthy donors were measured by hTERT461–469/HLA-A*2402 tetramer assay. Finally, NOD/scid/γcnull (NOG) mice subcutaneously inoculated with luciferase-gene-modified HLA-A*2402+ ATL cell line, ATN-1 cells were treated with intravenous infusion of hTERT-siTCR/CD8+or non-gene-modified (NGM) ones, respectively. Thereafter, these mice were serially monitored for tumor growth using bioluminescence imaging and survival.

Results:
QRT-PCR and Western blotting revealed that hTERT was markedly overexpressed in both ATL cell lines and freshly isolated ATL cells, while less than undetectable in normal cells. hTERT461–469-specific CTL precursors were variably detectable in periphery of HLA-A*2402+ ATL patients, but not HLA-A*2402− ATL patients or HLA-A*2402+ healthy individuals. hTERT-siTCR/CD8+ were 30–50% positive for hTERT461–469/HLA-A*2402 tetramer and displayed HLA-A*2402-restricted and hTERT461–469–specific cytocidal activity against peptide-loaded K562-A24 cells. Those hTERT-siTCR/CD8+ successfully displayed tumoricidal activity against HLA-A*2402+ ATL cell line cells and freshly isolated HLA-A*2402+ ATL cells, but not HLA-A*2402− ones or HLA-A*2402+ normal cells including CD34+ CBMCs. Moreover, hTERT-siTCR/CD8+ generated from HLA-A*2402+ ATL patients also lysed autologous ATL tumor cells. Finally, using xenografted NOG mouse models, therapeutically infused hTERT-siTCR/CD8+, but not NGM ones from the identical donor successfully suppressed growth of inoculated ATL cells in vivo. Furthermore, NOG mice bearing advanced ATL tumor masses were also successfully treated with hTERT-siTCR/CD8+ in combination with bortezomib.

Summary:
In this study, we newly demonstrated that hTERT is a promising therapeutic tumor antigen for ATL. Moreover, redirected CD8+ T cells to express hTERT-specific TCR displayed antitumor activity against ATL tumor cells both in vitro and in vivo. Furthermore, our preliminary data represented antitumor activity against advanced ATL, in combination with bortezomib. Collectively, redirected T cell-based immunotherapy targeting hTERT seems clinically feasible and promising for the treatment of almost all ATL patients who are ineligible for allo-HSCT.

Disclosures:
No relevant conflicts of interest to declare.