Toshihiko Wakabayashi MD, PhD Professor, Department of Neurosurgery, Nagoya University Graduate School of Medicine

Apr 1985-Dec 1985 Assistant in Neurosurgery, Nagoya University Hospital

Jan 1986-Jul 1987 Chief in Neurosurgery, Shizuoka-Kousei Hospital

Jul 1987-Jan 1989 Postgraduate scholarship, National Institute of Neurosurgery, Budapest, Hungary

Feb 1989 Trainee, Allgemeines Krankenhaus (AKH), Vienna, Austria

Mar 1989-Jul 1989 Registrar, Department of Neurosugery, Nagoya Daini Red Cross Hospital

July 1989- Oct 1997 Instructor in Neurosurgery, Nagoya University Hospital

Dec 1994-Feb 1995 Teaching staff of Japan International Cooperation, Center (JICA) in Sanjay Gandhi Postgraduate, Institute of Medical Sciences at Lucknow, India

Nov 1997-Dec 1997 Overseas research foundation by Japanese Ministry of Education, Department of neurosurgery, The hospital for sick children, Toronto University, Toronto, Ontario, Canada

Nov 1997- Mar 2000 Assistant Professor in Neurosurgery, Nagoya University Hospital

April 2000- May 2002 Associate Professor in Bio-Medicine, Nagoya University School of Medicine

June 2002- May 2008 Associate Professor in The Center for Genetic and Regenerative Medicine, Nagoya University Hospital

June 2008-present Professor, Department of Neurosurgery, Nagoya University Graduate School of Medicine

Epigenetic regulations and differentiation in glioma-stem cells

Development of immunetherapy using genetically engineered T cells expressing chimeric antigen receptors specific to glioma-assiciated antigens.

Purpose: Malignant glioma is one of the most lethal tumors in the malignant tumor. In this situation, immunotherapy is attracting more and more attention as one of the promising approaches. Induction of tumor-specific effecter T-cells are efficient for the eradication of bulky solid tumors, and it is the final goal of cancer immunological approaches. Tumor-specific cytotoxic T-lymphocytes (CTLs) can be genetically engineered to express CAR. CAR typically consists of a tumor antigen-specific recognition single-chain variable fragment element derived from a monoclonal antibody and components of a T cell receptors that mediate the signal transduction into CTLs. CTLs that expresses CAR bypasses the process of the tumor-antigen presenting through MHC class I. We have developed monoclonal antibody, 3C10, which recognizes EGFRvIII and then generated CAR, and also reported its ability to specifically recognize and destroy glioma cells that expressed EGFRvIII. Although the CAR that consists only scFv and CD3ζ could initiate a cytotoxic antitumor response in grafted T cells, the signals can only induce transient cell division and suboptimal cytokine secretion but cannot provide prolonged polyclonal expansion and sustained antitumor reaction in vivo. Nowadays, second and third generation CARs that contain the additional co-stimulator genes are developed to overcome these problems. The aim of this study is to develop a novel CAR and also encodes microRNA (miR)-17-92 cluster, which is known to promote cell survival, proliferation and the type-1 skewing of T-cells.

Results: 1. Generation and evaluation of new EGFRvIII-specific CAR: We created EGFRvIII-recognizing lentiviral CARs using the “third generation” CAR backbone incorporating T-cell activation domains derived from TCR/CD3ζ, CD28 as well as CD137 with or without miR17-92 (EGFR-miR-CAR and EGFR-CAR, respectively). T-cells that expressed EGFR-miR-CAR killed glioma cells that expressed EGFRvIII and conferred higher levels of IFN-γ production of transduced T-cells compared with EGFR-CAR, especially in the immuno-suppressive condition.

2. In vivo evaluation of anti-tumor activity of CAR-transfected T cells: Intracranial administration of EGFR-CAR-transduced human T-cells in NOD/scid/γc (-/-) mice bearing Day 7 EGFRvIII-positive GBM cells resulted in survival of all 5 mice over 60 days without any sign of tumor recurrence, whereas all control mice treated with mock-transduced T-cells died within 10 days following the tumor inoculation.

Iwami K, Shimato S, Ohno M, Okada H, Nakahara N, Sato Y, Yoshida J, Suzuki S, Nishikawa H, Shiku H, Natsume A, Wakabayashi T: Peptide-pulsed dendritic cell vaccination targeting interleukin-13 receptor alpha2 chain in recurrent malignant glioma patients with HLA-A*24/A*02 allele. Cytotherapy, in press (2012) Naito T, Miyachi S, Matsubara N, Isoda H, Izumi T, Haraguchi K, Takahashi I, Ishii K, Wakabayashi T: Magnetic resonance fluid dynamics for intracranial aneurysms-comparison with computed fluid dynamics. Acta Neurochir (Wien), in press (2012) Iwami K, Momota H, Natsume A, Kinjo S, Nagatani T, Wakabayashi T: A novel method of intracranial injection via the postglenoid foramen for brain tumor mouse models. J Neurosurg, 116, 630-635 (2012) Kishida Y, Natsume A, Kondo Y, Takeuchi I, An B, Okamoto Y, Shinjo K, Saito K, Ando H, Ohka F, Sekido Y, Wakabayashi T: Epigenetic subclassification of meningiomas based on genome-wide DNA methylation analyses. Carcinogenesis, 33, 436-441 (2012) Kishida Y, Natsume A, Toda H, Toi Y, Motomura K, Koyama H, Matsuda K, Nakayama O, Sato M, Suzuki M, Kondo Y, Wakabayashi T: Correlation between quantified promoter methylation and enzymatic activity of O (6)-methylguanine-DNA methyltransferase in glioblastomas. Tumour Biol, 33, 373-381 (2012) Motomura K, Natsume A, Wakabayashi T: Intravenous administration of temozolomide as a useful alternative over oral treatment with temozolomide capsules in patients with gliomas. J Neurooncol, 106, 209-211 (2012) Iwami K, Natsume A, Wakabayashi T: Cytokine networks in glioma. Neurosurg Rev, 34, 253-263; discussion 263-254 (2011) Mitsui T, Fujii M, Tsuzaka M, Hayashi Y, Asahina Y, Wakabayashi T: Skin shift and its effect on navigation accuracy in image-guided neurosurgery. Radiol Phys Technol, 4, 37-42 (2011) Momota H, Iwami K, Fujii M, Motomura K, Natsume A, Ogino J, Hasegawa T, Wakabayashi T: Rhabdoid glioblastoma in a child: case report and literature review. Brain Tumor Pathol, 28, 65-70 (2011) Motomura K, Natsume A, Fujii M, Ito M, Momota H, Wakabayashi T: Long-term survival in patients with newly diagnosed primary central nervous system lymphoma treated with dexamethasone, etoposide, ifosfamide and carboplatin chemotherapy and whole-brain radiation therapy. Leuk Lymphoma, 52, 2069-2075 (2011) Motomura K, Natsume A, Kishida Y, Higashi H, Kondo Y, Nakasu Y, Abe T, Namba H, Wakai K, Wakabayashi T: Benefits of interferon-beta and temozolomide combination therapy for newly diagnosed primary glioblastoma with the unmethylated MGMT promoter: A multicenter study. Cancer, 117, 1721-1730 (2011) Nagasaka T, Tsugeno M, Ikeda H, Okamoto T, Inao S, Wakabayashi T: A novel monoshaft bipolar cautery for use in endoscopic intracranial surgery. A short technical note. Clin Neurol Neurosurg, 113, 607-611 (2011) Natsume A, Kinjo S, Yuki K, Kato T, Ohno M, Motomura K, Iwami K, Wakabayashi T: Glioma-initiating cells and molecular pathology: implications for therapy. Brain Tumor Pathol, 28, 1-12 (2011) Wakabayashi T, Kayama T, Nishikawa R, Takahashi H, Hashimoto N, Takahashi J, Aoki T, Sugiyama K, Ogura M, Natsume A, Yoshida J: A multicenter phase I trial of combination therapy with interferon-beta and temozolomide for high-grade gliomas (INTEGRA study): the final report. J Neurooncol, 104, 573-577 (2011) Ohka F, Natsume A, Motomura K, Kishida Y, Kondo Y, Abe T, Nakasu Y, Namba H, Wakai K, Fukui T, Momota H, Iwami K, Kinjo S, Ito M, Fujii M, Wakabayashi T: The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma. PLoS One, 6, e23332 (2011) Matsubara N, Miyachi S, Nagano Y, Ohshima T, Hososhima O, Izumi T, Tsurumi A, Wakabayashi T, Sano A, Fujimoto H: Evaluation of the characteristics of various types of coils for the embolization of intracranial aneurysms with an optical pressure sensor system. Neuroradiology, 53, 169-175 (2011) Matsubara N, Miyachi S, Tsukamoto N, Izumi T, Naito T, Haraguchi K, Wakabayashi T: Endovascular coil embolization for saccular-shaped blood blister-like aneurysms of the internal carotid artery. Acta Neurochir (Wien), 153, 287-294 (2011) Iwami K, Natsume A, Wakabayashi T: Gene therapy for high-grade glioma. Neurol Med Chir (Tokyo), 50, 727-736 (2010) Ohno M, Natsume A, Ichiro Iwami K, Iwamizu H, Noritake K, Ito D, Toi Y, Ito M, Motomura K, Yoshida J, Yoshikawa K, Wakabayashi T: Retrovirally engineered T-cell-based immunotherapy targeting type III variant epidermal growth factor receptor, a glioma-associated antigen. Cancer Sci, 101, 2518-2524 (2010) Motomura K, Ogura M, Natsume A, Yokoyama H, Wakabayashi T: A free-radical scavenger protects the neural progenitor cells in the dentate subgranular zone of the hippocampus from cell death after X-irradiation. Neurosci Lett, 485, 65-70 (2010) Maesawa S, Fujii M, Nakahara N, Watanabe T, Wakabayashi T, Yoshida J: Intraoperative tractography and motor evoked potential (MEP) monitoring in surgery for gliomas around the corticospinal tract. World Neurosurg, 74, 153-161 (2010) Miyachi S, Izumi T, Matsubara N, Naito T, Haraguchi KI, Wakabayashi T: The mechanism of catheter kickback in the final stage of coil embolization for aneurysms: the straightening phenomenon. Interv Neuroradiol, 16, 353-60 (2010) Kato T, Natsume A, Toda H, Iwazumi H, Sugita T, Hachisu R, Watanabe R, Yuki K, Motomura K, Bankiewicz K, Wakabayashi T. Efficient delivery of liposome-mediated MGMT-siRNA reinforces the cytotoxity of temozolomide in GBM-initiating cells. Gene Therapy, 17, 1363-1371 (2010) Natsume A, Kondo Y, Ito M, Motomura M, Wakabayashi T, Yoshida J. Epigenetic aberrations and therapeutic implications in gliomas. Cancer Sci, 101, 1331-1336 (2010) Ito S, Natsume A, Shimato S, Ohno M, Kato T, Chansakul P, Wakabayashi T, Kim S U. Human neural stem cells transduced with IFN-beta and cytosine deaminase genes intensify bystander effect in experimental glioma. Cancer Gene Ther, 17, 299-306 (2010) Yuki K, Natsume A, Yokoyama H, Kondo Y, Ohno M, Kato T, Chansakul P, Ito M, Kim SU, Wakabayashi T. Induction of oligodendrogenesis in glioblastoma-initiating cells by IFN-mediated activation of STAT3 signaling. Cancer Lett, 284, 71-79 (2009) Wakabayashi T, Natsume A, Hatano H, Fujii M, Shimato S, Ito M, Ohno M, Ito S, Ogura M, Yoshida J. p16 promoter methylation in the serum as a basis for the molecular diagnosis of gliomas. Neurosurgery,64, 455-461 (2009) Oi S, Natsume A, Ito M, Kondo Y, Shimato S, Maeda Y, Saito K, Wakabayashi T. Synergistic induction of NY-ESO-1 antigen expression by a novel histone deacetylase inhibitor, valproic acid, with 5-aza-2'-deoxycytidine in glioma cells. J Neurooncol, 92, 15-22 (2009) Ohno M, Natsume A, Kondo Y, Iwamizu H, Motomura K. Toda H, Ito M, Kato T, Wakabayashi T. The modulation of microRNAs by type I IFN through the activation of signal transducers and activators of transcription 3 in human glioma. Mol Cancer Res, 7, 2022-2030 (2009) Natsume A, Kondo Y, Wakabayashi T. Epigenetic analyses of brain tumor stem cells. Brain Nerve, 61, 791-798 (2009) Ito M, Natsume A, Takeuchi H, Shimato S, Ohno M, Wakabayashi T, Yoshida J. Type I interferon inhibits astrocytic gliosis and promotes functional recovery after spinal cord injury by deactivation of the MEK/ERK pathway. J Neurotrauma, 26, 41-53 (2009) Fujii M,Wakabayashi T. Image-guided neurosurgery using intraoperative MRI. Brain Nerve, 61, 823-834 (2009) Wakabayashi T, Natsume A, Hashizume Y, Fujii M, Mizuno M, Yoshida J. A phase I clinical trial of interferon-beta gene therapy for high-grade glioma: novel findings from gene expression profiling and autopsy. J Gene Med, 10, 329-339 (2008) Wakabayashi T, Kayama T, Nishikawa R, Takahashi H, Yoshimine T, Hashimoto N, Aoki T, Kurisu K, Natsume A, Ogura M, Yoshida J. A multicenter phase I trial of interferon-beta and temozolomide combination therapy for high-grade gliomas (INTEGRA Study). Jpn J Clin Oncol, 38, 715-718 (2008) Shimato S, Natsume A, Wakabayashi T, Tsujimura K, Nakahara N, Ishii J, Ito M, Akatsuka Y, Kuzushima K, Yoshida J. Identification of a human leukocyte antigen-A24-restricted T-cell epitope derived from interleukin-13 receptor alpha2 chain, a glioma-associated antigen. J Neurosurg, 109, 117-122 (2008) Natsume A, Wakabayashi T, Tsujimura K, Shimato S, Ito M, Kuzushima K, Kondo Y, Sekido Y, Kawatsura H, Narita Y, Yoshida J. The DNA demethylating agent 5-aza-2'-deoxycytidine activates NY-ESO-1 antigenicity in orthotopic human glioma. Int J Cancer, 122, 2542-2553 (2008) Natsume A, Wakabayashi T, Ishii D, Maruta H, Fujii M, Shimato S, Ito M, Yoshida J. A combination of IFN-beta and temozolomide in human glioma xenograft models: implication of p53-mediated MGMT downregulation. Cancer Chemother Pharmacol, 61, 653-659 (2008) Ishii D et al. Efficacy of temozolomide is correlated with 1p loss and methylation of the deoxyribonucleic acid repair gene MGMT in malignant gliomas. Neurologia medico-chirurgica 47:341-349 (2007) Ishii J et al. The free-radical scavenger edaravone restores the differentiation of human neural precursor cells after radiation-induced oxidative stress. Neurosci lett. 423:225-230 (2007) Shimato S et al. Human neural stem cells target and deliver therapeutic gene to experimental leptomeningeal medulloblastoma. Gene Ther 14:1132-1142 (2007) Takeuchi H et al. Intravenously transplanted human neural stem cells migrate to the injured spinal cord in adult mice in an SDF-1- and HGF-dependent manner. Neurosci lett 426:69-74 (2007) Tsuno T et al. Inhibition of Aurora-B function increases formation of multinucleated cells in p53 gene deficient cells and enhances anti-tumor effect of temozolomide in human glioma cells. J Neuro-oncol 2007:83(3):249-258. Natsume A et al. IFN-beta down-regulates the expression of DNA repair gene MGMT and sensitizes resistant glioma cells to temozolomide. Cancer Res. 65:7573-9 (2005) Shimato S, et al. EGFR mutation in patients with brain metastases from lung cancer: correlation with the efficacy of gefitinib. Neuro-Oncol., 8, 137-44 (2006) Yoshida J et al. Human gene therapy for malignant gliomas (Glioblastoma multiforme and anaplastic astrocytoma) by in vivo transduction with human interferon beta gene using cationic liposomes. Human Gene Ther 15: 77-86 (2004) Hatano N et al. Efficacy of post operative adjuvant therapy with human interferon beta, MCNU and radiation (IMR) for malignant glioma: comparison among three protocols. Acta Neurochir (Wien) 142: 633-639, (2000) Okada H et al. Suppression of CD44 expression decreases migration and invasion of human glioma cells. Int J Cancer. 10:255-60 (1996) Okada H et al. Anti-(glioma surface antigen) monoclonal antibody G-22 recognizes overexpressed CD44 in glioma cells. Cancer Immunol Immunother. 39:313-7 (1994) Wakabayashi T et al. Characterization of neuroectodermal antigen by a monoclonal antibody and its application in CSF diagnosis of human glioma. J Neurosurg. 68:449-55 (1988)

2001 Outstanding paper award of 2001 Journal of chemical engineering of Japan 2007 Research Award of Japan Society of Gene Therapy 2008 Best Simulation Awards of Japan Virtual Reality Medical Society