第4回がん新薬開発合同シンポジウム がん免疫細胞療法開発への企業側の取り組み がん免疫遺伝子治療への取り組み 峰野純一 タカラバイオ株式会社 バイオ産業支援事業部門 Takara Bio, Gene therapy supporting history TCR gene therapy clinical research (JP) Center for Cell & Gene Therapy (GMP) HSV-TK ex vivo gene therapy clinical trial (JP) RetroNectin 1990 2000 Center for Gene & Cell Processing (GMP) 2010 HF10(oncolytic virus) clinical trial (USA) MazF gene therapy clinical trial (USA) RetroNectin manufacturing (GMP) 2 Nov. 28, 2014 CPCs (GMP) がん免疫細胞療法開発への企業側の取り組み TCR gene therapy clinical trial (JP) Gene Therapy in vivo Gene Therapy ex vivo Gene Therapy Target cells Viral vector with therapeutic gene Cell expansion Gene transfer Viral vector with therapeutic gene Gene modified cells 3 Nov. 28, 2014 がん免疫細胞療法開発への企業側の取り組み Schedule for Clinical Development of Gene Medicine of Takara Bio Preclinical trials HF10 (oncolytic virus) anticancer therapy Phase I clinical trials Phase II clinical trials Phase III clinical trials Phase II clinical trials in the U.S. Commerci alization FY2018 Phase I clinical trials in Japan （commence in FY 2014） MAGE-A4 TCR gene therapy for cancer Phase I clinical trials in Japan FY2021 MazF gene therapy for AIDS Phase I clinical trials in the U.S. FY2022 NY-ESO-1 TCR gene therapy for cancer 4 Nov. 28, 2014 Phase I clinical trials in Japan （commence in FY 2014） がん免疫細胞療法開発への企業側の取り組み TCR Gene Therapy TCR gene-modified CD8+ T cell recognition of cancer antigen Cancer cell TCR transduced T cell CD8+ T cell CD8 TCR MHC-1 Cancer antigen recognition Transduced TCR TCR α β Cancer specific TCR gene recombinant Retroviral vector 5 Nov. 28, 2014 がん免疫細胞療法開発への企業側の取り組み δ ε εγ ζ ζ CD3 What we have been doing 6 Vector development Development of cell expansion Development of gene transfer method Closed system cell processing GMP manufacturing of MCB and vector Development of QC method and validation Construction of GMP facility Nov. 28, 2014 がん免疫細胞療法開発への企業側の取り組み Vector development: siTCR - silencing of endogenous TCR by siRNAs TCR Gamma-retroviral siTCR Gamma-retroviral Vector Vector TCRTCR α β1LTR LTR PGKp PGKp si-α2 si-β2 si-β1 si-α1 Therapeutic TCR α β Endogenous TCR α β ε εγ ζζ ε εγ ζζ δ α β Knockdown of endogenous TCRs could prevent TCR mispairing and lead to efficient expression of the introduced TCRs ε εγ ζζ ε εγ ζ ζ CD3 ε εγ ζζ δ α β δ α β Endogenous TCR δ codon-modified(optimized）TCR α: TCR α1 codon-modified(optimized） TCR β: TCR β1 siRNAs for TCR α :si-α1, si-α2 siRNAs for TCR β :si-β1, si-β2 δ TCR α1 β LTR LTR TCR siTCR vector can express 6 kinds of genes TCR mispairng Okamoto et al., Cancer Res 2009 7 Nov. 28, 2014 がん免疫細胞療法開発への企業側の取り組み Vector development: siTCR - Higher expression and cytotoxic activity Tetramer Staining WT CO 3.1 copies /cell 2.0 copies /cell 11.3 % tetramer siTCR 2.7 copies /cell 20.9 % WT: wt TCR vector CO: codon-optimized TCR vector siTCR: siTCR vector 51.5 % Higher Expression CD8 CTL assay CTL % 50 siTCR/11-18(MAGE-A4+/HLA-A24+) 40 30 CO/11-18(MAGE-A4+/HLA-A24+) Higher Cytotoxic Activity 20 siTCR/QG56(MAGE-A4+/HLA-A24-) CO/QG56(MAGE-A4+/HLA-A24-) 10 0 0 10 20 E/T ratio 8 Nov. 28, 2014 30 siTCR : 4.0 copies/cell co-control: 4.2 copies/cell がん免疫細胞療法開発への企業側の取り組み Okamoto et al., Cancer Res 2009 2nd Generation siTCR Vector - Enhancement of TCR specific lysis SD Ψ SA 1st gen. siTCR (internal promoter) Ψ SD 2nd gen. siTCR (2A peptide) PGK p TCR α LTR TCR β SA TCR β LTR 2A TCR α HLA-A*0201 restricted MART-1 specific TCR α β genes Tetramer Staining MFI of tetramer+ 2nd gen. siTCR 60 1st gen. siTCR 40 20 0 0 1 2 3 copies/cell 4 MFI of tetramer in tet+CD8+ % of tetramer+ cells in CD8+ 80 LTR CTL assay 40 40000 35000 30000 25000 20000 15000 10000 5000 0 2nd gen. siTCR CTL % % of tetramer+ LTR 2nd gen. siTCR 30 20 10 1st gen. siTCR 1st gen. siTCR 0 10 3 1 E/T ratio 0 1 2 3 copies/cell 4 Target cell: MART-1 specific peptide pulsed T2 cells Okamoto et al., Mol Ther Nucleic Acids. 2012 9 Nov. 28, 2014 がん免疫細胞療法開発への企業側の取り組み Development of cell expansion - RetroNectin Expansion Method day0 day4 αCD3mAb±costimulation mol. +IL-2 day10 day7 transfer to new medium +IL-2 add medium +IL-2 day14 add medium +IL-2 RetroNectin, αCD28, α4-1BB Expansion fold 1200 * 1400 1400 day10 1200 * 1000 1000 800 800 600 600 400 400 200 200 0 0 αCD3 αCD3 /RN αCD3 /28 day14 αCD3 /4-1BB αCD3 αCD3 /RN αCD3 /28 αCD3 /4-1BB error bar: means±SEM (5 donors) *p<0.05, Tukey-kramer test Yu et al., Cancer Gene Ther 2008 10 Nov. 28, 2014 がん免疫細胞療法開発への企業側の取り組み Development of gene transfer method & Closed system cell processing Stimulation PBMC recovery Stimulation by TExpander CD3/CD28 Transduction (RBV-LTS) Vector pre-load Transduction GaLV retroviral vector Vector pre-load Expansion Expansion (CultiLife Eva) Harvest Transduction GaLV retroviral vector CultiLife Eva （640cm2） 11 Nov. 28, 2014 がん免疫細胞療法開発への企業側の取り組み 12 Nov. 28, 2014 がん免疫細胞療法開発への企業側の取り組み Center for Gene & Cell Processing (Kusatsu, Shiga, Japan) Total floor space: 6,500 m2, 1st floor: Cell banking (e.g. E. coli) Plasmid vector manufacturing E. coli culture for protein production QC test (sterility, Mycoplasma) Cell bank storage 2nd floor: Viral vector production gamma retrovirus, lentivirus, HSV, adenovirus, AAV, HVJ , etc. Cell culture, Media preparation Protein purification Aseptic filling 3rd floor: Cell processing QC test (test for cells & viruses, qPCR, bio assay, etc.) 13 Nov. 28, 2014 がん免疫細胞療法開発への企業側の取り組み Center for Gene & Cell Processing 14 Nov. 28, 2014 がん免疫細胞療法開発への企業側の取り組み
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