Yukihiro Miyazaki

Development of a novel redirected T-cell–based adoptive immunotherapy targeting human telomerase reverse transcriptase for adult T-cell leukemia

Although adult T-cell leukemia (ATL) has a poor prognosis, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) in some cases suggests that a cellular immune-mediated strategy can be effective. So far, however, no effective target for anti-ATL immunotherapy has been defined. Here we demonstrated for the first time that human telomerase reverse transcriptase (hTERT) is a promising therapeutic target for ATL, and we developed a novel redirected T-cell-based immunotherapy targeting hTERT. hTERT messenger RNA was produced abundantly in ATL tumor cells but not in steady-state normal cells. Rearranged human leukocyte antigen-A*24:02 (HLA-A*24:02) -restricted and hTERT461-469 nonameric peptide-specific T-cell receptor (TCR) α/β genes were cloned from our previously established cytotoxic T lymphocyte clone (K3-1) and inserted into a novel retroviral TCR expression vector encoding small interfering RNAs for endogenous TCR genes in redirected T cells (hTERT-siTCR vector). Consequently, allogeneic or autologous gene-modified CD8(+) T cells prepared using the hTERT-siTCR vector successfully killed ATL tumor cells, but not normal cells including steady-state hematopoietic progenitors, in an HLA-A*24:02-restricted manner both in vitro and in vivo. Our experimental observations support the development of a novel hTERT-targeting redirected T-cell-based adoptive immunotherapy for ATL patients, especially those for whom suitable allo-HSCT donors are lacking.

The utility of positron emission tomography/computed tomography in the staging of extranodal natural killer/T‐cell lymphoma

Natural killer (NK)/T‐cell lymphoma cases are rarely discovered using positron emission tomography/computed tomography (PET/CT). We compared the utility of PET/CT and that of conventional methods (CMs; CT with IV contrast, biopsies from primary sites, and bone marrow examinations) in the staging of extranodal NK/T‐cell lymphoma. Nineteen untreated patients with extranodal NK/T‐cell lymphoma at three institutions were analyzed. PET/CT and CMs were applied for initial workups following diagnosis. PET/CT and CMs were compared and evaluated for their ability to detect tumor lesions and their influence on the staging and treatment strategies. In total, 116 lesions were detected by CM and PET/CT. Using PET/CT, 108 lesions (93%) were discovered. The number of nodal lesions was 28: all were positive by PET/CT and 26 (93%) by CMs. The number of extranodal lesions was 89: 84 (94%) and 54 (61%) lesions were positive by PET/CT and CMs, respectively. PET/CT was superior to CMs in detecting cutaneous lesions [31/31 lesions (100%) vs. 20/31 lesions (65%), respectively; P = 0.042]. Bone marrow involvement was confirmed pathologically in only seven patients; four cases (57%) were positive by PET/CT. Using CMs, ten patients (53%) were stages I–II and nine (47%) were stages III–IV. Using PET/CT, eight patients (42%) were in stages I–II and 11 (58%) were in stages III–IV. PET/CT findings altered the stage and treatment strategy in two cases (11%). Our study demonstrated that PET/CT is a useful tool for detecting extranodal lesions in NK/T‐cell lymphoma, particularly cutaneous lesions. PET/CT may therefore influence future staging and treatment strategies.

Antileukemia multifunctionality of CD4 + T cells genetically engineered by HLA class I-restricted and WT1-specific T-cell receptor gene transfer

To develop gene-modified T-cell-based antileukemia adoptive immunotherapy, concomitant administration of CD4+ and CD8+ T cells that have been gene modified using identical HLA class I-restricted leukemia antigen-specific T-cell receptor (TCR) gene transfer has not yet been fully investigated. Here, using CD4+ and CD8+ T cells that had been gene modified with a retroviral vector expressing HLA-A*24:02-restricted and Wilms’ tumor 1 (WT1)-specific TCR-α/β genes and siRNAs for endogenous TCRs (WT1-siTCR/CD4+ T cells and WT1-siTCR/CD8+ T cells), we examined the utility of this strategy. WT1-siTCR/CD4+ T cells sufficiently recognized leukemia cells in an HLA class I-restricted manner and provided target-specific Th1 help for WT1-siTCR/CD8+ T cells. By using a xenografted mouse model, we found that WT1-siTCR/CD4+ T cells migrated to leukemia sites and subsequently attracted WT1-siTCR/CD8+ T cells via chemotaxis. Therapy-oriented experiments revealed effective enhancement of leukemia suppression mediated by concomitant administration of WT1-siTCR/CD4+ T cells and WT1-siTCR/CD8+ T cells. Importantly, this augmented efficacy in the presence of WT1-siTCR/CD4+ T cells was correlated with longer survival and enhanced formation of memory T cells by WT1-siTCR/CD8+ T cells. Collectively, our experimental findings strongly suggest that this strategy would be clinically advantageous for the treatment of human leukemia.

FDG-PET can evaluate the treatment for fungal liver abscess much earlier than other imagings

Dear Editor, Fungal liver abscesses (FLA), mainly caused by Candida, are among the most serious complications in patients with acute leukemia [1–4]. Diagnosing FLA by imaging such as ultrasound (US) [5] or computed tomography (CT) [6] is easily performed when multiple nodular lesions in the patients liver can be seen. However, these nodular lesions can still be found by imaging for a long time after clinical symptoms begin to improve with anti-fungal treatments. Therefore, it is very difficult to evaluate the early effectiveness of treatments. We first report here a case in which the use of 18F-Fluorodeoxyglucose (FDG) positron emission tomography (PET) imaging was more beneficial than US or CT in evaluating FLA in the early stage of treatment. A 32-year-old woman with relapsed acute myeloid leukemia was given re-induction chemotherapy and achieved second complete remission. Allogeneic bone marrow transplantation from an HLA-identical unrelated donor was planned, but low-grade fever (37–38°C) appeared before the transplantation. At that time, her C-reactive protein (CRP) level was about 3 mg/dL, but her blood cultures and beta-D-glucan were negative. Abdominal US and CT revealed multiple low-density areas in her liver, which suggested the FLA or leukemic infiltration. FDG-PET imaging examination was performed, and FDG uptake was shown to be increased at these lesions (Fig. 1 (1)). She was diagnosed with FLA when the periodic acid-Schiff staining of the liver biopsy specimens showed granuloma and yeast-like fungus, although the strain of the fungus could not be identified. Liposomal amphotericin B and Micafungin were administered daily, but the low-grade fever continued, and her CRP gradually increased. Consequently, the scheduled transplantation was postponed. After a month, the antifungal treatment was changed to oral Voriconazole, then her fever lowered promptly and her CRP level decreased gradually. At 3 months after the anti-fungal treatment was started, she had no clinical symptoms, and her CRP level was negative. While the US and CT tests at that time Y. Miyazaki (*) :M. Yasukawa Department of Bioregulatory Medicine, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan e-mail: miya2ymy@yahoo.co.jp

Gene-Modified Human α/β-T Cells Expressing a Chimeric CD16-CD3ζ Receptor as Adoptively Transferable Effector Cells for Anticancer Monoclonal Antibody Therapy

Ochi and colleagues engineered T cells to express chimeric CD16 V158-CD3ζ receptors that mediate antibody-dependent tumoricidal activity; upon stimulation these T cells proliferate and differentiate into effector memory T cells, which could combine with and enhance clinical responses by anticancer monoclonal antibodies. The central tumoricidal activity of anticancer monoclonal antibodies (mAb) is exerted by FcγR IIIa (CD16)–expressing effector cells in vivo via antibody-dependent cell-mediated cytotoxicity (ADCC), as observed for natural killer (NK) cells. In practice, chemotherapy-induced leukopenia and exhaustion of NK cells resulting from ADCC often hamper the clinical efficacy of cancer treatment. To circumvent this drawback, we examined in vivo the feasibility of T cells, gene-modified to express a newly generated affinity-matured (158V/V) chimeric CD16-CD3ζ receptor (cCD16ζ-T cells), as a transferable alternative effector for cancer mAb therapy. cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human interleukin-2 (rhIL-2), and they successfully displayed ADCC-mediated tumoricidal activity in vitro. During ADCC, ligation of opsonized cancer cells to the introduced cCD16ζ-T cells stimulated the effector cells to produce proinflammatory cytokines and release toxic granules through the activation of the Nuclear factor of activated T cells (NFAT) pathway after phosphorylation of the CD3ζ chain. In parallel, these stimulated cCD16ζ-T cells transiently proliferated and differentiated into effector memory T cells. In contrast, NK cells activated by rhIL-2 displayed similar ADCC activity, but failed to proliferate. Human cCD16ζ-T cells infused concomitantly with anti-CD20 mAb synergistically inhibited the growth of disseminated Raji cells, a CD20+ lymphoma cell line, in immunodeficient mice, whereas similarly infused rhIL-2–treated NK cells survived for a shorter time and displayed less effective tumor suppression. Our findings strongly suggest the clinical feasibility of cCD16ζ-T cells as adoptively transferable ADCC effector cells that could potentially enhance the clinical responses mediated by currently available anticancer mAbs. Cancer Immunol Res; 2(3); 249–62. ©2014 AACR.

Adoptive transfer of genetically engineered WT1-specific cytotoxic T lymphocytes does not induce renal injury

Because WT1 is expressed in leukemia cells, the development of cancer immunotherapy targeting WT1 has been an attractive translational research topic. However, concern of this therapy still remains, since WT1 is abundantly expressed in renal glomerular podocytes. In the present study, we clearly showed that WT1-specific cytotoxic T lymphocytes (CTLs) certainly exerted cytotoxicity against podocytes in vitro; however, they did not damage podocytes in vivo. This might be due to the anatomical localization of podocytes, being structurally separated from circulating CTLs in glomerular capillaries by an exceptionally thick basement membrane.