1. No category

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