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
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.
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.
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
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
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.
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
)
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+
(%
)
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
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
G-Rex 10, 100, 100M, 100L Wilson Wolf
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
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 (%)
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 (%)
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
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
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)
CD4+ T cells
CD8+ T cells
CD4+ CM
CD4+ EM
CD8+ CM
CD8+ EM
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
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.
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)
