CAR-T cells in Leukemia. Prof. Keiya Ozawa

1. Keiya Ozawa
Director, IMSUT Hospital
Professor, Division of Genetic Therapeutics
The Advanced Clinical Research Center
The Institute of Medical Science
The University of Tokyo
Visiting Professor
Division of Immuno-Gene & Cell Therapy (Takara Bio)
Jichi Medical University
CD19-targeted CAR (chimeric antigen
receptor)-expressing T-cell gene therapy
for B-cell lymphoma

2. Engineered T cell therapy
☞ TCR (T cell receptor) gene therapy
☞ CAR (chimeric antigen receptor)-T gene therapy
Cancer Gene TherapyCancer Gene Therapy

3. Chimeric Antigen Receptor (CAR)
CARs are hybrid proteins consisting of an extracelluar single chain fragment
of variable region (scFv) fused to co-stimulatory signaling domains CD28 or
4-1BB (CD137), coupled with CD3ζ to mediate T-cell activation.

4. Tumor antigen specific gene therapy by a
gene-modified T lymphocytes
Nature Reviews Cancer 13,525–
541(2013)
TCR CAR
1. Sensitive signal amplification derived by
evolution
2. Low avidity
3. Targets intracellular proteome
4. Requires MHC class I expression and HLA
matching on tumor cell
5. Possible mispairing with endogenous TCR
1. Signal amplification derived by
synthetic biology
2. Avidity controllable
3. Targets only surface structures
4. HLA independent antigen recognition,
universal application
5. No mispairing with endogenous TCR

5. Cytotoxicity of CD19-specific CAR-expressing
T Lymphocytes against B Cell Lymphoma
T cells
TCR
MHC
class I
cytotoxicity
scFv(CD19)CD28CD3ζ
CAR
CD19
B lymphoma cell
CD19-CAR T cells, which are engineered to express extracellular single-
chain immunoglobulin variable fragments to CD19, linked to cytoplasmic
T cell activation domains including CD3-ζ, showed remarkable
therapeutic benefits toward CD19+
B cell malignancies.

6. Adoptive Immuno-Gene Therapy using
CAR-T-cells for Refractory B Cell Non-Hodgkin Lymphoma

7. Ex vivo expansion using 3T3/CD19+
feeder cells
Day0
Week2 Week3 Week4Week1
Gene transduction
19-28ζ retroviral vector* Harvest
PBMC activation
(α-CD3/Retronectin)
Preparation and Ex Vivo Expansion
of CD19-CAR-T Lymphocytes
*19-28ζ retroviral vector was provided by Dr. Brentjens (MSKCC).

8. IFN-γ(pg/ml)
NIH3T3
NIH3T3/CD19
CD19CAR+
T-cell
+
--
- +
+
-
-
+
+
+ -
*
IFN-γ Production by CD19-CAR-T
Lymphocytes (ELISA)

9. Cytotoxic Activity of CD19-CAR-T
Lymphocytes
Raji Daudi
E/T ratio
Specificlysis(%)
CD19-CAR
Control
CD19-CAR
Control

10. Tumor alone Tumor + CAR-T Tumor + control T
Raji-bearing
mice
Raji-luc tumors (s.c.)
CAR+
T or control T
(i.v.)
IHC with CD3 Ab (α day
1)
Accumulation of CD19-CAR-T-cells at Raji-
subcutaneous tumors in Rag2-/-
γc-/-
mice
CD3 staining

11. CD19-CAR-T-cell therapy in Raji-bearing mice
Raji-Luc Control
day1 4 15 21 29
Raji-Luc
5×104
(i.v.)
CAR-T cells
1×107
(i.v.)
Treatment protocol
Bioluminescence
imaging

12. day15
day2
1
day29
No treatment CD19-CAR-T
Efficacy of CD19-CAR-T-cell Therapy
T. Tsukahara et al., BBRC 2013
control T

13. Enhanced survival of CD19-CAR-T-cell-treated Raji-bearing mice
Immunohistochemical staining for human CD3 (brown) in spleen lesions
from Raji-bearing mice

14. The ability of CD19-CAR-T cells to accumulate
at tumor lesions may be pivotal for their anti-
tumor effects, and therefore may enhance the
clinical efficacy of adoptive T-cell therapy for
relapsed/refractory B-cell lymphoma.

15. Clinical Research of Gene Therapy Using
CD19-specific CAR-expressing T-cells for
Refractory B-cell Malignant Lymphoma
PI: Keiya Ozawa (Jichi Medical University)
Objective
To evaluate the safety, efficacy and kinetics of autologous
T cells genetically modified to express anti-CD19 chimeric
antigen receptor (CAR) in patients with B-NHL.
CD19-CAR-T Clinical Study at
Jichi Medical University

16. Characteristics of CD19-CAR-T-cell Therapy
Target disease: Relapsed/Refractory B-cell NHL
(Follicular lymphoma, Mantle cell lymphoma, DLBCL)
Timing of Tx: After the reduction of tumor mass by chemo-Tx.
Target molecule:
CD19 --- different from the target of rituximab (CD20)
Strategy: Infusion of engineered T cells
☞ Effective in immuno-deficient Pts after long-term chemo.
Therapeutic efficacy persists over a long period of time.

17. Cancer Research 71(9): 3175-3181, 2011
Mitigating the potential for early toxicity
 Dose-escalation
 Splitting the T-cell dose
 (Co-expression of conditional suicide genes)
Mitigating the potential for late toxicity
 Depletion of B cells ----- Ig transfer

18. Design
This study is a phase I/II clinical trial in which dose-escalation of anti-
CD19 CAR expressing T cells (CD19-CAR-T) will be performed to
determine the maximum tolerated dose (MTD).
The maximum dosage of cohorts in which DLT occurred in less than 33%
of patients will be set as the MTD.
Dose levels
Dose Levels
Cell numbers
(CAR-positive cells)
Population
-1 1×106
/kg 0-6
1 (starting dose) 3×106
/kg 3-6
2 1×107
/kg 3-6
3 3×107
/kg 3-6
CD19-CAR-T Clinical Study
N = 6-18 ; As dose limiting toxicity (DLT) evaluation subjects

19. Peripheral blood
up to 600 mL
2nd
CD19-CAR-T infusion
Split dose (optional)
For 15years
Long-Term
Follow-up
Cell processing
Day 0 & Day 1
1st
CD19-CAR-T infusion
Split Dose
(Day 0 ： 1/3, Day 1 ：
2/3)
Day 84
End of study
Day 28
DLT evaluation
CD19-CAR-T
Clinical Study - Schedule -
Preconditioning regimen ：
Day -2
Cyclophosphamide
(1.5 g/m2
)
Day -3 & Day -2
Bendamustine
(120 mg/m2
x 2 days)
or
Day -4 or Day -3
2nd
registration
Informed
consent &
1st
registration
HospitalizationHospitalization

24. Collaborator
s
Jichi Medical University
Department of Medicine
Division of Hematology
Chihiro Yamamoto Iekuni Oh Ken Ohmine
Takahiro Suzuki Tadashi Nagai Yoshinobu Kanda
Division of Cell Transplantation and Transfusion
Koji Kishino Kazuo Muroi
Center for Molecular Medicine
Division of Genetic Therapeutics
Tomonori Tsukahara Masashi Urabe Akihiro Kume
Hiroaki Mizukami
Division of Immuno- Gene & Cell therapy (Takara Bio)
Takeshi Teruya Hiroyuki Ido Ryosuke Uchibori
Takara Bio Inc.
Asuka Okazaki Hideto Chono Jun-ichi Mineno
Kazuto Takesako
Memorial Sloan Kettering Cancer Center
Isabelle RiviereIsabelle Riviere Renier BrentjensRenier BrentjensMichel SadelainMichel Sadelain

25. Jinshanling Great Wall

27. Structure of Chimeric Antigen Receptors
(CARs)
Sadelain M et al. Cancer Discovery 2013;3:388-398
First-generation CAR Second-generation CAR Third-generation CAR

28. TCR CAR (T
body)
Engineered T Cell Therapy
 HLA restriction (-)
 Applicable to HLA-deficient cancer
cells
 Peptide processing is not required
 No mispairing with endogenous TCR
 Target: cell surface antigens

29. T lymphocytes
Expansion
Y Y Y
Gene transfer
YYYYYY
YYYYYY
YYYYYY
Tumor specificity
Tumor
Adoptive Immuno-Gene Therapy using CAR
(chimeric antigen receptor)-Expressing T lymphocytes

30. CD3
CD19CAR
Day7
Day22
98%
97%
92%
90%
isotype mIg
0%
0.3%
0.9%
0.5%
Expression of CD19-CAR on T Lymphocytes
(Flow Cytometry)

31. Protocol of CD19-CAR-T Cell Therapy for B-cell Lymphoma
Cell Preparation
QC
2nd
Enrollment
Day-4 or -3
Day-3, -2
or Day-2
Day
0, 1
Day
28 〜
Day
84
Infusion Level
-1
1 (Initial Dose)
2
3
No. of Subjects
0-6
3-6
0-6
0-6
No. of CAR-T Cells
3×105
/kg
1×106
/kg
3×106
/kg
1×107
/kg
Eligibility/IC
Eligibility/IC
1st
Enrollment
PBcollection
Cyclophosphamide
or
Bendamustine
CAR-TInfusion
CAR-TInfusion
(option)
Follow-up Survey
Hospitalization
Number of subjects;
N = 6-18 ; As dose limiting toxicity (DLT) evaluation subjects

32. CAR CD19+
disease
Pre-
conditioning
Additional
therapy
Institute
ScFv-CD28-
CD3ζ
CLL, ALL Yes No SKCC
ScFv-41BB-
CD3ζ
CLL, ALL Yes No U Penn
ScFv-CD28-
CD3ζ
B-NHL Yes IL-2 i.v. NCI
ScFv-CD3ζ
vs ScFv-
CD28-CD3ζ
B-NHL or CLL No No BCM
Cooper, Blood 2012
Clinical Trials of CD19-CAR-T-cell Therapy