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Adoptive Cell Therapy for the Treatment
of Cancer: The CAR-T Cell Experience
Steven A. Feldman, Ph.D.
Surgery Branch, NCI
MEASUREMENT CHALLENGES FOR CAR-T
BIOMANUFACTURING
January 29, 2016
1
Disclosures
No financial disclosures
This work is supported, in part,
through a CRADA with KITE Pharma
2
Outline
•
•
•
•
Background
CD19 CAR-T process development
CD19 CAR-T clinical experience
CAR-T therapy for solid cancers
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Three Main Approaches to Cancer Immunotherapy
1. Non-specific stimulation of immune reactions
• Stimulate effector cells (IL-2, IL-12)
• Inhibit regulatory factors (PD-1, CTLA-4)
2. Active immunizations to enhance anti-tumor reactions
• Cancer vaccines
3. Passively transfer activated immune cells with anti-tumor activity
• Adoptive cell transfer
4
Gene Therapy Approach for Adoptive Cell Transfer
Tumor-specific TCR/CAR
Clinical
retroviral vector
Morgan RA, et al. Science 2006; 314(5796): 68-9.
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Advantages of Cell Transfer Therapy Using Genetically
Engineered Cells
1. High avidity anti-tumor T cell receptors (TCR) and/or chimeric
antigen receptors (CAR) can be identified and cloned using in
vitro assays.
2. Peripheral blood lymphocytes can be genetically modified to
express these high avidity TCRs/CARs.
3. Large numbers of tumor-specific lymphocytes can be grown in
vitro.
4. The host can be manipulated to provide a favorable tumor
microenvironment prior to administering the cells.
5. ACT can mediate tumor regressions.
6
A Critical Challenge Confronting the Development of
Human Cancer Immunotherapy is the Identification of
Antigens to Target
1. Differentiation antigens overexpressed on cancers compared
to normal tissue (MART-1, gp100, CEA, Her-2, Mesothelin)
2. Antigens expressed on cancers and on non-essential normal
tissues (CD19, thyroglobulin)
3. Shared antigens unique to cancer (cancer-testes antigens,
NY-ESO-1, MAGE-A)
4. Critical components of the tumor stroma (VEGFR2, FAP)
5. Mutations unique to each cancer (EGFRvIII)
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Chimeric Antigen Receptors (CARs)
Step 2
Step 1
Step 3
Ig scFv
Linker/TM
T cell signaling
Antibody Producing Hybridoma
Ig Genes
Chimeric Antigen Receptor (CAR)
sd
Step 5

Step 4
sa
LTR
CD28
Anti-tumor Ag-scFv
CD3 zeta
LTR
Transduce PBL
sd
LTR

sa
Anti-tumor Ag-scFv
CD8
CD28
4-1BB
CD3 zeta
LTR
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CAR-T Program for the Treatment of CD19+ Hematologic
Malignancies
• More than 20,000 people die of B-cell malignancies annually
in the U.S.
• CD19 is expressed by more than 90% of B-cell malignancies.
• CD19 is expressed by mature B cells, B-cell precursors and
plasma cells but not any other normal tissues.
• B cells are considered a “non-essential” tissue.
Anti-CD19 CAR
5’ LTR

FMC63 scFv
CD28
CD3-zeta
3’ LTR
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A Rapid Cell Expansion Process for the Production of CD19
CAR-T Cells
Process Development Objectives:
• Develop a closed process
• Production time of 6 days
• Cryopreserved cell product
• GMP-compliant
Stimulation
Approx 1E9
Cell #
Days
0
<1E9
1
Transduction
2E8
2E8
2
3
Expansion
2.5E8
5E8
4
5
1E9
6
10
Development of a CAR-T Cell Closed Production Process
Current NCI Process
•
•
Manual Ficoll Separation
of PMBC
•
•
Stim in T175 Flask (open)
AIM V + 5% Hu serum
supplemented with anti-CD3 Ab
and rIL2
Wash cells after Stim (open)
Days
Transduction in 6 - well plates
by Spinoculation (open
process)
•
•
•
•
•
Expansion in T-175 flasks
(open process)
7 day expansion
Cell concentration and wash
(open process)
Administer fresh cells
Improved Process
Apheresis product
Enrich for lymphocytes
•
•
Ficoll Separation of PBMC by
Sepax 2 (closed process)
Stim in Culture bags (closed)
Serum-free medium with anti-CD3
Ab and rIL-2
Wash cells after Stim
(Sepax 2, closed process)
T Cell
Days
0-2
Activation
0-2
•
Days
Retroviral
Days
•
2-3
Transduction
2-3
Transduction in Culture bags
(closed process)
•
Expansion in bags (closed
process) without antibiotics
3 day expansion
Days
3-10
T Cell
Expansion
Days
3-6
•
•
Harvest
Cell concentration and wash
(closed process)
Cryopreserve product
11
Optimization Parameters
Process Steps:
• Cell culture medium
• Serum replacement
• Bag comparison
• Cell density at OKT3 stimulation
• Post-stimulation wash
• Transduction





Retronectin concentration
Vector dilution
1 vs 2 transductions
Bag flip
Cell density
Analytical Assays:
• FACS
– CAR+ cells
– Phenotype
• Co-culture
• ELISA
• qPCR
– Copy number
– Persistence
• Sepax
 Apheresis
 OKT3 wash
 Final product wash
• Cryopreservation
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Sepax 2 can Efficiently Process Apheresis Products
Sepax Technologies, Inc.
13
Optimizer-TCSR Medium can Support T Cell Growth
Cell Number
1.5×108
OpTmizer CTS
1.0×108
AIM V medium
X-VIVO 20 Medium
5.0×107
TexMACS GMP medium
AIM V 5%
0.0
0
2
4
6
8
10
1 .5  1 0
8
1 .0  1 0
8
5 .0  1 0
7
p = 0 .7 7 8 1
iz
e
IM
r
V
C
T
5
S
%
0
O
p
T
m
A
C e ll N u m b e r
Day
14
Optimization of Cell Concentration During Transduction
6 well (9.5 cm2):
• 2e6 cell/well
• 10 ng/ml RN
• 4ml (1:1 vector)
• 0.5e6 cell/ml
• 1e5 cell/cm2
PL240 (540 cm2):
2e8 cell/bag
10 ng/ml RN
400ml (1:1 vector)
0.5e6 cell/ml
3.7e5 cell/cm2
100
% T r a n s d u c t io n
80
60
40
20
c
u
h
s
d
8
t
ra
n
a
p
T
li
2
n
d
F
g
0
.5
e
6
/m
l
B
a
o
ti
u
o
6
e
.5
0
.2
0
n
U
n
rs
l
/m
/m
6
e
5
e
.1
0
tr
a
n
s
d
u
6
c
e
/m
d
l
l
0
15
6 Day Closed Production Process – Engineering Runs
• 5 engineering runs at scale and compared to standard open
process
• Evaluated
•
•
•
•
Transduction efficiency
Function (IFNg secretion)
Cell expansion
Phenotype
16
A Comparison of the Standard Open and 6 Day Closed
Production Process
50000
Bag
PL240 Bag
P la te
40000
IF N g ( p g /m L )
***
80
60
40
30000
20000
10000
20
M
-N
C
E
R
F
G
L
A
2
6
5
K
* * * p < 0 .0 0 1
M
o
d
T
o
le
o
C e ll P r o d u c t io n P r o c e s s
N
P la t e
e
B ag
n
UT
6
0
0
N
% T r a n s d u c t io n
100
C e ll L in e
17
A Comparison of the Standard Open and 6 Day Closed
Production Process
Bag
P la te
80
60
40
20
ra
m
T
e
T
e
m
m
c
T
a
iv
e
0
N
P e r c e n t a g e o f T o t a l C e lls
100
C e ll P h e n o t y p e
Naïve:
Tcm:
Tem:
Temra:
CD45RA+/CCR7+
CD45RA-/CCR7+
CD45RA-/CCR7CD45RA+/CCR7-
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Development of a CAR-T Cell Closed Production Process
Current NCI Process
•
•
Manual Ficoll Separation
of PMBC
•
•
Stim in T175 Flask (open)
AIM V + 5% Hu serum
supplemented with anti-CD3 Ab
and rIL2
Wash cells after Stim (open)
Days
Transduction in 6 - well plates
by Spinoculation (open
process)
•
•
•
•
•
Expansion in T-175 flasks
(open process)
7 day expansion
Cell concentration and wash
(open process)
Administer fresh cells
Improved Process
Apheresis product
Enrich for lymphocytes
•
•
FicollSeparation of PBMC by
Sepax 2 (closed process)
Stim in Culture bags (closed)
Serum-free medium with anti-CD3
Ab and rIL-2
Wash cells after Stim
(Sepax 2, closed process)
T Cell
Days
0-2
Activation
0-2
•
Days
Retroviral
Days
•
2-3
Transduction
2-3
Transduction in Culture bags
(closed process)
•
Expansion in bags (closed
process) without antibiotics
3 day expansion
Days
3-10
T Cell
Expansion
Days
3-6
•
•
Harvest
Cell concentration and wash
(closed process)
Cryopreserve product
19
Efficacy of CD19 CAR-T Cells in CLL
Before treatment
3 months after treatment
CD19
CD19
CD20
CD20
20
Kochenderfer et al. Blood 2012
CD19 CAR-T cells can Mediate Tumor regression
21
CD19 CAR-T Cells Eliminate normal B cells and Show
Minimal Persistence in Vivo
22
CAR-T Cell Production Process for Solid Cancers
REP:
6000 IU/ml IL-2
200:1 irradiated feeder cells
OKT3
Gene
Modification
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CAR-T Program for the Treatment of Solid Cancers
Receptor
Generation
Cancers
Status
ERBB2 (Her2/neu)
3rd
All Her2+ Cancers
Closed
VEGFR2
3rd
All cancers
Closed
EGFRvIII
3rd
Glioblastoma
Accruing
Mesothelin
2nd
Pancreatic/Mesothelioma/Ovarian
Accruing
Receptor
Patients
Treated
Cells Infused
ORR
ERBB2 (Her2/neu)
1
1e10
0/1
VEGFR2
23
1e6 – 3e10
1/23 (4%)
EGFRvIII
16
1e7 – 3e10
0/16
Mesothelin
14
1e6 – 1e8
0/13
PR (2 months)
(Pt. 14, too early)
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Summary
• CD19 CAR-T cell therapy effectively treats a variety of B cell malignancies
• We have developed a closed cell production process sufficient for current
CD19 CAR-T cell demands
–
–
–
–
Closed system
Serum-free
6 days
Cryopreserved cell product
• Currently no effective CAR-T therapies for the treatment of solid cancers
• Considerations
–
–
–
–
–
–
How to handle increased cell numbers
Increase transduction efficiency
RCR testing beyond 6 days
Direct detection of CD19 CAR+ T cells (other CARs ???)
Patient variability
How to extend CAR T therapies to solid cancers
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