Keiya OzawaDirector, IMSUT HospitalProfessor, Division of Genetic Therapeutics The Advanced Clinical Research CenterThe Institute of Medical ScienceThe University of Tokyo

Visiting ProfessorDivision of Immuno-Gene & Cell Therapy (Takara Bio)Jichi Medical University

CD19-targeted CAR (chimeric antigen receptor)-expressing T-cell gene therapyfor B-cell lymphoma

Engineered T cell therapy

☞ TCR (T cell receptor) gene therapy

☞ CAR (chimeric antigen receptor)-T gene therapy

Cancer Gene TherapyCancer Gene Therapy

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.

Tumor antigen specific gene therapy by agene-modified T lymphocytes

Nature Reviews Cancer 13,525–541(2013)

TCR CAR

1. Sensitive signal amplification derived by evolution

2. Low avidity3. Targets intracellular proteome4. Requires MHC class I expression and HLA

matching on tumor cell5. Possible mispairing with endogenous TCR

1. Signal amplification derived by synthetic biology

2. Avidity controllable3. Targets only surface structures4. HLA independent antigen recognition,

universal application5. No mispairing with endogenous TCR

Cytotoxicity of CD19-specific CAR-expressingT 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.

Adoptive Immuno-Gene Therapy usingCAR-T-cells for Refractory B Cell Non-Hodgkin Lymphoma

Ex vivo expansion using 3T3/CD19+ feeder cells

Day0

Week2 Week3 Week4Week1

Gene transduction19-28ζ retroviral vector* Harvest

PBMC activation(α-CD3/Retronectin)

Preparation and Ex Vivo Expansionof CD19-CAR-T Lymphocytes

*19-28ζ retroviral vector was provided by Dr. Brentjens (MSKCC).

IFN

-γ (

pg

/ml)

NIH3T3

NIH3T3/CD19

CD19CAR+T-cell

+

--

- +

+

-

-

+

+

+ -

*

IFN-γ Production by CD19-CAR-T Lymphocytes (ELISA)

Cytotoxic Activity of CD19-CAR-T Lymphocytes

Raji Daudi

E/T ratio

Sp

eci

fic

lysi

s(%

)

CD19-CAR

Control

CD19-CAR

Control

Tumor alone Tumor + CAR-T Tumor + control T

Raji-bearingmice

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

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

day15

day21

day29

No treatment CD19-CAR-T

Efficacy of CD19-CAR-T-cell Therapy

T. Tsukahara et al., BBRC 2013

control T

Enhanced survival of CD19-CAR-T-cell-treated Raji-bearing mice

Immunohistochemical staining for human CD3 (brown) in spleen lesions from Raji-bearing mice

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.

Clinical Research of Gene Therapy Using

CD19-specific CAR-expressing T-cells for

Refractory B-cell Malignant Lymphoma

PI: Keiya Ozawa (Jichi Medical University)

ObjectiveTo 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

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.

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

DesignThis 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 LevelsCell 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

Peripheral bloodup to 600 mL

2nd CD19-CAR-T infusionSplit dose (optional)

For 15yearsLong-TermFollow-up

Cell processing

Day 0 & Day 11st CD19-CAR-T infusion

Split Dose(Day 0 ： 1/3, Day 1 ：

2/3)

Day 84End of study

Day 28DLT evaluation

CD19-CAR-TClinical Study - Schedule -

Preconditioning regimen ：

Day -2Cyclophosphamide

(1.5 g/m2)

Day -3 & Day -2Bendamustine

(120 mg/m2 x 2 days)

or

Day -4 or Day -32nd registration

Informed consent &

1st registration

HospitalizationHospitalization

Collaborators

Jichi Medical University

Department of Medicine Division of Hematology

Chihiro Yamamoto Iekuni Oh Ken OhmineTakahiro 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 MinenoKazuto Takesako

Memorial Sloan Kettering Cancer CenterIsabelle RiviereIsabelle Riviere Renier BrentjensRenier BrentjensMichel SadelainMichel Sadelain

Jinshanling Great Wall

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

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

T lymphocytes

Expansion

Y Y Y

Gene transfer

Y Y Y Y Y Y

Y Y Y Y Y Y

Y Y Y Y Y Y

Tumor specificity

Tumor

Adoptive Immuno-Gene Therapy using CAR(chimeric antigen receptor)-Expressing T lymphocytes

CD3

CD

19C

AR

Day7

Day22

98%

97%

92%

90%

isotype mIg

0%

0.3%

0.9%

0.5%

Expression of CD19-CAR on T Lymphocytes(Flow Cytometry)

Protocol of CD19-CAR-T Cell Therapy for B-cell Lymphoma

Cell PreparationQC

2nd EnrollmentDay-4 or -3

Day-3, -2or 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

Elig

ibili

ty /

IC

Elig

ibili

ty /

IC

1st E

nrol

lmen

t

PB

col

lect

ion

Cyc

loph

osph

amid

eor

Ben

dam

ustin

e

CA

R-T

Inf

usio

n

CA

R-T

Inf

usio

n

(option)

Follow-up Survey

Hospitalization

Number of subjects;N = 6-18 ; As dose limiting toxicity (DLT) evaluation subjects

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-CD3ζvs ScFv-

CD28-CD3ζB-NHL or CLL No No BCM

Cooper, Blood 2012

Clinical Trials of CD19-CAR-T-cell Therapy