manufacturing of genetically-engineered t cells expressing a … · manufacturing of...
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Manufacturing of genetically-engineered T cells expressing a thymidlate kinase (Tmpk)- based suicide transgene for the treatment
of graft-versus-host disease follow cellular therapy
Fenlu Zhu, Ph.D. BMT Laboratories
Division of Hematology and Oncology Medical College of Wisconsin
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Figure 3
Donor derived T lymphocytes
Donor derived T lymphocytes: Mediate graft-versus-tumor (GVT) effects.
The curative potential of AlloSCT and augmentation of anti-tumor activity by DLI strongly rely on donor derived T lymphocytes.
Attack immunocompromised recipient-lead to graft-
versus-host disease (GVHD). Donor derived T lymphocytes play an important role in pathogenesis of both acute and chronic GVHD.
Dissociate GvT from GVHD is difficult.
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Rationale of Suicide Gene Therapy to Treat and Prevent GVHD
The rationale of suicide gene therapy is to genetically modify T cells to express a suicide gene that allows T cells to be harnessed for their graft-versus-tumor effects when infused, and safely eliminated in the event of severe GVHD.
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Suicide gene therapy for GVHD treatment has been proved feasible and effective
• Proof-of-concept studies on mouse models using T cells expressing Herpes Simplex Thymidine Kinase (HSV1-TK) suicide gene combined with GCV treatment were able to control GVHD.
• Clinical study using T cells modified with retroviral vector expressing HSV1-TK gene to treat patients with hematological malignancies has demonstrated feasibility and effectiveness.
• Limitations of using HSV1-TK: HSV1-TK transgene immunogenic
Prophylactic use of GCV limits the use HSV1-TK schema.
Ciceri F et al, Blood. 2007; 109: 4698-4707
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Vector features: 1. The Tmpk/Prodrug Azidoxythymidine (AZT) combination induces apoptosis of
the cells. 2. Not immunogenic, no sequence in the vector longer than 5aa other than
transgene. 3. Self-inactivating with a deletion in the 3’ LTR resulting in non-functional LTRs. 4. The inclusion of low affinity nerve growth factor receptor (LNGFR) in the
expression cassette allows enrichment of the Tmpk expressing T cells.
LTR5’Ψ
SD
ΔGAGRRE
SA EF1-α
cPPT
WPRE SIN/LTR 3’Transgene
Tmpk LNGFR
EMCV-IRES
Lentiviral Vector From MCW Vector Facility
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Factors in the manufacturing process that need to be optimized
• Lentiviral vector: TMPK- LNGFR • Culture medium: GIBCO OpTmizer™ and Miltenyi Tex MACS
medium • Activation: Dynabeads® Human T-Activator CD3/CD28 and TransAct
CD3/28 reagent. • Transduction • Culture devices: G-Rex 10, 100 • Enrichment: Miltenyi CD271 Microbeads and CD271-PE/APC
Microbeads • Final cell analysis, functional study.
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Day
0
Day
3
Day
5
Day
7
Day
11
Day
14
0
2
4
6
82%HSOpt+Prometh IL-2+Dynabeads
2%HSOpt+Miltenyi IL-2+Dynabead
Days in culture
Expensio
n (
fold
)
Day
0
Day
3
Day
5
Day
7
Day
11
Day
14
0
2
4
6
8
10
2%HSOpt+Prometh IL-2+Dynabeads2%HSOpt+Prometh IL-2+TransAct3%HSTex+Prometh IL-2+Dynabeads2%HSOpt+Milten IL-2+Dynabeads3%HSTex+Milten IL-2+TransAct3%HSTex+Milten IL-2+Dynabeads
Days in cultureE
xpensio
n (
fold
)
Day
0
Day
3
Day
5
Day
7
Day
11
Day
14
0
2
4
6
8
2%HSOpt+Prometh IL-2+Dynabeads
3%HSTex+Prometh IL-2+Dynabeads
Days in culture
Expensio
n (
fold
)
Day
0
Day
3
Day
5
Day
7
Day
11
Day
14
0
2
4
6
8 2%HSOpt+Prometh IL-2+Dynabeads
2%HSOpt+Prometh IL-2+TransAct
Days in culture
Expensio
n (
fold
)
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Dynabeads CD3/28 , three days, MOI 5 TransAct CD3/28 reagents , one day, MOI 5
54.9 15.3
CD271
CD
45
Pre-activation conditions
% G
FP
expre
ssio
n
Day
-3 D
ynab
eads
Day
-2 D
ynab
eads
Day
0 D
ynab
eads
No
activ
ation
0
20
40
60
80 Day -3 Dynabeads
Day -2 Dynabeads
Day 0 Dynabeads
No activation
Day
-4 T
rans
Act
Day
-3 T
rans
Act
Day
-2 T
rans
Act
Day
-1 T
rans
Act
Day
0 T
rans
Act
Day
-3 D
ynab
eads
0
20
40
60Day-4 TransAct
Day-3 TransAct
Day-2 TransAct
Day-1 TransAct
Day 0 TransActDay-3 Dynabeads
Pre-activation with different beads at various days
CD
45+
LN
GF
R+
(%
)
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73.0 61.2 51.5 45.5
34.6 61.0
CD271
CD
45
Variations between different batches of vector
CD271
CD
45
MOI 5 MOI 20
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82.3 83.0
Without Protamine Sulfate With Protamine Sulfate
CD271
CD
45
A
23.4 23.0
Without Retronectin With Retronectin
CD271
CD
45
B
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G-Rex 10, 100, 100M, 100L Wilson Wolf
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Culture Device
Fo
ld o
f cell e
xp
an
sio
n
T75
Flas
k
G-R
ex0
20
40
60
80
T75 Flask
G-Rex
P=0.0131
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CD271
CD
45
75.7 83.1
200 mL culture medium 400 mL culture medium
200
mL
400
mL
0
10
20
60
80
100
Volume of Culture Medium
Perc
en
tag
e (
%)
Expansion (Fold)Viability (%)LNGFR Expression (%)
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CD271
CD
45
71.4 70.0 64.9 59.2
6.25e4/cm2 1.25e5/cm2 2.5e5/cm2 5e5/cm2
6.25
e4/cm2
1.25
e5/cm2
2.5e
5/cm
2
5e5/
cm2
0
10
20
30
40
60
80
100
Cell Seeding Density in G-Rex
Perc
enta
ge (
%)
Expansion (Fold)
Viability (%)LNGFR EXpression (%)
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94.5 73.2
After 1st enrichment Cultured for one week after 1st enrichment
After 2nd enrichment
95.3
Cultured for one week after 2nd enrichment
83.5
CD271
CD
45
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Day -1 PBL activation
Day 0 Transduction with
LV-LNGFRtmpk
Day 4 First Enrichment
Day 18 Harvest
Transduction and expansion protocol
Day 11 Second Enrichment
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95.3
CD271
CD
45
95.9
82.9
Days in culture
Cell
num
ber
(10
6)
Day
0
Day
4
Day
11
Day
18
0
200
400
600
Experiment 1 (106)Experiment 2 (106)Experiment 3 (106)
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CD4+ T cells
CD8+ T cells
CD4+ CM
CD4+ EM
CD8+ CM
CD8+ EM
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AZT Concentration
Liv
e C
ell P
erc
enta
ge (%
)
0 (u
M)
1 (u
M)
10 (u
M)
20 (u
M)
50 (u
M)
100
(uM
)
200
(uM
)0
50
100
150
Untransduced
Transduced
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Conclusion A feasible manufacturing protocol that starting with less than
10x106 cells, low MOI (MOI 5), provides sufficient clinical dose of genetically modified T cells with the majority of the cells expressing LNGFR was established.
Phenotypic analysis revealed that most of the cells were T cells, with both CD4 and CD8 exist at the same time. Central memory and effector memory T cells account for most of the cells.
The transduced T cells could be eliminated by culturing with AZT. This protocol can also be useful for manufacturing all the genetically
modified T cells like CAR T cells.
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Acknowledgements
• Carolyn A. Keever-Taylor, Ph.D.
• Huiqing Xu, MD
• William R Drobyski, MD
• Jeffrey A. Medin, PhD
• W. Monty McKillop, PhD
• Funding Support:
Midwest Athletes Against Childhood Cancer (MACC Fund)