Development of PD-1 gene-disrupted EGFRvIII-specific CAR-T cell-based immunotherapy against glioblastoma

• Project/Area Number: 18K16567
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 56010:Neurosurgery-related
• Research Institution: Nara Medical University
• Principal Investigator: Nakazawa Tsutomu 奈良県立医科大学, 医学部, 研究員 (00772500)
• Project Period (FY): 2018-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2019: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2018: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: CAR / PD-1 / CRISPR/Cas / EGFRvIII / CRISPR/Cas9 / EGFRｖIII / CAR-T / Glioma

Outline of Final Research Achievements

The present study showed that PD-1-disrupted EGFRvIII-specific CAR-T cells were established using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The sgRNA/Cas9 expression vectors designed precisely disrupted the target region of PD-1 and inhibited the expression of PD-1 in EvCAR-T cells. The PD-1-disrupted EvCAR-T cells had an in vitro growth inhibitory effect on EGFRvIII-expressing glioblastoma cells without altering the T-cell phenotype and the expression of other checkpoint receptors.

Academic Significance and Societal Importance of the Research Achievements

将来的に、PD-1破壊EGFRvIII特異的CAR-T細胞のin vivoにおける抗がん効果解析や3次元培養系を用いた抗がん効果解析を実施し、膠芽腫に対する抗がん効果が認められれば、GBMに対するEGFRvIII特異的vCAR-T細胞を用いた膠芽腫に対するがん免疫治療の有効性を向上させることが可能と考えられる。

Research Products

• [Journal Article] Effect of CRISPR/Cas9-Mediated PD-1-Disrupted Primary Human Third-Generation CAR-T Cells Targeting EGFRvIII on in vitro Human Glioblastoma Cell Growth (2020)
  • Author(s): Nakazawa T, Nishimura F, Matsuda R, Nakamura M, Nakase H et al.
  • Journal Title: Cells Volume: 9 Issue: 4 Pages: 998-1017
  • DOI: 10.3390/cells9040998
  • Peer Reviewed / Open Access
• [Journal Article] Ex vivo-expanded highly purified natural killer cells in combination with temozolomide induce antitumor effects in human glioblastoma cells in vitro. (2019)
  • Author(s): Tanaka Y, Nakazawa T(corresponding author), Nakamura M, Nishimura F, Matsuda R, Omoto K, Shida Y, Murakami T, Nakagawa I, Motoyama Y, Morita H, Tsujimura T, Nakase H.
  • Journal Title: PLOS ONE Volume: 14 Issue: 3 Pages: e0212455-e0212455
  • DOI: 10.1371/journal.pone.0212455
  • Peer Reviewed / Open Access
• [Journal Article] Expression of peptide transporter 1 has a positive correlation in protoporphyrin IX accumulation induced by 5-aminolevulinic acid with photodynamic detection of non-small cell lung cancer and metastatic brain tumor specimens originating from non-small cell lung cancer. (2019)
  • Author(s): Omoto K, Matsuda R, Nakai Y, Tatsumi Y, Nakazawa T, Tanaka Y, Shida Y, Murakami T, Nishimura F, Nakagawa I, Motoyama Y, Nakamura M, Fujimoto K, Hiroyuki N.
  • Journal Title: Photodiagnosis Photodyn Ther Volume: 25 Pages: 309-316
  • DOI: 10.1016/j.pdpdt.2019.01.009
  • Peer Reviewed / Open Access
• [Journal Article] Risk of brain herniation after craniotomy with lumbar spinal drainage: a propensity score analysis. (2019)
  • Author(s): Yasushi Motoyama
  • Journal Title: Journal of Neurosurgery Volume: 130 Issue: 5 Pages: 1710-1720
  • DOI: 10.3171/2017.12.jns172215
  • Peer Reviewed
• [Journal Article] Novel Human NK Cell Line Carrying CAR Targeting EGFRvIII Induces Antitumor Effects in Glioblastoma Cells. (2018)
  • Author(s): Murakami T, Nakazawa T(equally contribution), Natsume A, Nishimura F, Nakamura M, Matsuda R, Omoto K, Tanaka Y, Shida Y, Park YS, Motoyama Y, Nakagawa I, Yamada S, Tamura K, Takeshima Y, Takamura Y,
  • Journal Title: Anticancer Res Volume: 38 Issue: 9 Pages: 5049-5056
  • DOI: 10.21873/anticanres.12824
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Characterization of a novel type NK cell line KHYG-1 carrying EGFRvIII-specific CAR in glioblastoma cells (2020)
  • Author(s): Morimoto T, Nakazawa T, Natsume A, Nishimura F, Matsuda R, Murakami T, Nakagawa I, Motoyama Y, Park YS, Tsujimura T, Wakabayashi T, Nakase H
  • Organizer: American Association of Immunology (AAI) 2020
  • Int'l Joint Research
• [Presentation] Effect of CRISPR/Cas9-mediated PD-1-disrupted primary human third-generation CAR-T cells targeting EGFRvIII on human glioblastoma cell growth (2020)
  • Author(s): Nakazawa T, Natsume A, Nishimura F, Matsuda R, Murakami T, Nakagawa I, Motoyama Y, Park YS, Tsujimura T, Wakabayashi T, Nakase H
  • Organizer: American Association of Immunology (AAI) 2020
  • Int'l Joint Research
• [Presentation] 膠芽腫に対する高純度NK細胞と免疫チェックポイント阻害薬併用による抗腫瘍効果の検討 (2019)
  • Author(s): 至田洋一、中澤務、中村光利、辻村貴弘、森本尭之、田中祥貴、村上敏春、尾本幸治、松田良介、中川一郎、西村文彦、本山靖、朴永銖、中瀬裕之
  • Organizer: 第20回日本分子脳神経外科学会
• [Presentation] Establishment of highly purified human natural killer cells and evaluation of their antitumor activity on glioblastoma (2019)
  • Author(s): 松田良介、中澤務、田中祥貴、至田洋一、中村光利、西村文彦、村上敏春、中川一郎、本山靖、辻村貴弘、中瀬裕之
  • Organizer: 第23回日本がん免疫学会総会
• [Presentation] Establishment of an efficient ex-vivo expansion method for highly purified human natural killer cells and evaluation of their antitumor activity on glioblastoma. (2019)
  • Author(s): Nakazawa T, Tanaka Y, Shida Y, Nakamura M, Nishimura F, Matsuda R, Murakami T, Nakagawa I, Motoyama Y, Tsujimura T, Nakase H.
  • Organizer: Keystone Symposia: Innate and Non-Classical Immune Cells in Cancer Immunotherapy
  • Int'l Joint Research
• [Presentation] Capability of dendritic cells loaded with induced-pluripotent stem cells to induce cancer-responsive T cells from a donor with HLA class I-A33 alleles in vitro. (2019)
  • Author(s): Nakazawa T, Nishimura F, Matsuda R, Yamashita Y, Nakamura M, Nakagawa I, Motoyama Y, Tsujimura T, Nakase H.
  • Organizer: European Association for Cancer Research: Defence is the Best Attack: Immuno-Oncology Breakthroughs
  • Int'l Joint Research
• [Presentation] Novel human NK cell line carrying CAR Targeting EGFRvIII Induces antitumor effects in glioblastoma cells (2019)
  • Author(s): Murakami T, Nakazawa T, Nishimira F, Natsume A, Wakabayashi T, Nakase H
  • Organizer: European Association for Cancer Research: Defence is the Best Attack: Immuno-Oncology Breakthroughs
  • Int'l Joint Research
• [Presentation] Novel human NK cell line carrying CAR targeting EGFRvIII induces antitumor effects in glioblastoma cells in vitro (2018)
  • Author(s): 村上敏春、中澤 務、夏目敦至、西村文彦、中村光利、松田良介、至田洋一、朴永銖、本山 靖、中川一郎、山田修一、田村健太郎、竹島靖浩、高村慶旭、若林俊彦、中瀬裕之
  • Organizer: 第19回日本分子脳神経外科学会