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)Center for Gene &Cell Processing
(GMP)

2000

2010

RetroNectin
1990

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
3Nov. 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
Preclinicaltrials
HF10 (oncolyticvirus) anticancer therapy

Phase I
clinicaltrialsPhase II
clinicaltrialsPhase III
clinicaltrialsPhase II clinical trials
in the U.S.

Commerci
alization
FY2018Phase I clinical trials in Japan（commence in FY 2014）

MAGE-A4 TCR
gene therapy
for cancer

Phase I clinical trials in Japan

FY2021MazF genetherapy forAIDS

Phase I clinical trials in the U.S.

FY2022

NY-ESO-1 TCR
gene therapy
for cancer
4Nov. 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 transducedT cellCD8+ T cell
CD8TCR

MHC-1
Cancer antigen

recognition
Transduced TCR
TCR
α βCancer specific TCR generecombinant Retroviral vector
5Nov. 28, 2014

がん免疫細胞療法開発への企業側の取り組み

δε εγζ ζ CD3What we have been doing 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
6Nov. 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-retroviralsiTCR
Gamma-retroviral
VectorVectorTCRTCR
α β1LTR LTRPGKp
PGKp

si-α2
si-β2
si-β1
si-α1

TherapeuticTCR

α βEndogenousTCR

α βε εγζζε εγζζδα βKnockdown of endogenous TCRs could prevent
TCR mispairing and lead to efficient expression ofthe introduced TCRsε εγ
ζζε εγζ ζ CD3ε εγζζδα βδα βEndogenousTCR

δcodon-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-β2δTCR
α1 βLTRLTR TCR

siTCR vector can express 6 kinds of genes

TCRmispairng

Okamoto et al., Cancer Res 2009
7Nov. 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
3.1 copies /celltetramer

11.3 %

COsiTCR

2.0 copies /cell

2.7 copies /cell20.9 %

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+)

4030

CO/11-18(MAGE-A4+/HLA-A24+)

Higher Cytotoxic Activity

20

siTCR/QG56(MAGE-A4+/HLA-A24-)
CO/QG56(MAGE-A4+/HLA-A24-)

10001020

E/T ratio
8Nov. 28, 201430

siTCR : 4.0 copies/cellco-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)ΨSD

2nd gen. siTCR(2A peptide)

PGKpTCR αLTR

TCR βSATCR βLTR

2ATCR αHLA-A*0201 restricted MART-1 specific TCR α β genes

Tetramer Staining
MFI of tetramer+

2nd gen. siTCR

60

1st gen. siTCR

402000123copies/cell4

MFI of tetramer
in tet+CD8+% of tetramer+cells in CD8+

80LTR

CTL assay

4040000
35000
30000
25000
20000
15000
10000
50000

2nd gen. siTCR

CTL %% of tetramer+LTR

2nd gen. siTCR

30
20
10

1st gen. siTCR

1st gen. siTCR

010

3

1E/T ratio
01

2

3copies/cell

4

Target cell: MART-1 specificpeptide pulsed T2 cells

Okamoto et al., Mol Ther Nucleic Acids. 2012
9Nov. 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 day0day4

αCD3mAb±costimulation mol.
+IL-2transfer to new medium+IL-2

day7

day10

add medium+IL-2

add medium
+IL-2

day14

RetroNectin, αCD28, α4-1BBExpansion fold1200

*1400

1400

day101200

*1000

1000

800

800600600

400

400200200

00

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
10Nov. 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/CD28Transduction (RBV-LTS)

Vectorpre-load

TransductionGaLV retroviral vector
Vectorpre-load

Expansion

Expansion(CultiLife Eva)

HarvestTransduction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 productiongamma 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.)
13Nov. 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