T-cell engineering for adoptive immunotherapy using TAL-

effector nucleases (TALEN®)

Sophie Derniame PhD

Project Leader, CellectisTherapeutics

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statements concerning Cellectis SA and its business.

Such statements involve certain known and unknown risks, uncertainties and

other factors, which could cause the actual results, financial condition,

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future results, performance or achievements expressed or implied by such

forward-looking statements.

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Not to be copied, distributed or used without Cellectis’s prior written consent.

FORWARD LOOKING STATEMENT

September 10th, 2013 2

3 September 10th, 2013

Retargeting T-cells to tumors with CARs

Tumor cell

T-cell

Tumor antigen

binding:

mAb scFv

Chimeric Antigen Receptors (CARs)

• CARs = cell membrane bound mAbs

• Allows MHC-independent antigen

recognition

• T-cells replicate in response to contact

with antigen in vivo

Adoptive autologous T-cell immunotherapy using CARs directed at CD19 has shown

impressive results with complete remissions of late stage leukemias

4 September 10th, 2013

“Universal” anti-CD19+ adoptive T-cell immunotherapy

• CAR expression to redirect T-cells to

tumor antigens

• Suicide gene for safety

• Off the shelf (TCR disruption*)

Minimal potential for GvHD

• For CLL - CD52 disruption* to prevent

destruction by high risk CLL mAb

Alemtuzumab therapy

(*): knock-out by using TALEN® mRNA

Engineered

SG SG

Allogeneic T-cell therapy using cells derived from healthy donors

5 September 10th, 2013

Platform development: combining multiple technologies

Apheresis (Frozen PBMC)

T-cell

activation Gene KO (transient

nuclease

expression)

Stable

gene

transfer

Product

storage

T-cell

expansion

and purification

CAMPATH

conditioning

(non-cytotoxic)

Patients

17 Days

6 September 10th, 2013

An

ti-F

ab

(scF

v)

CAR transduction

-10

0

10

20

30

40

50

60

70

80

30:1 15:1 8:1 3:1

NT

supeMP13622

supeMP13624

% c

ell

lys

is

Effector:Target

Benchmark CAR

Ctx CAR

SUPT1-CD19

-10

0

10

20

30

40

50

60

70

80

30:1 15:1 8:1 3:1

NT

supeMP13622

supeMP13624

% c

ell

lys

is

Benchmark CAR

Ctx CAR

Effector:Target

SUPT1 CD19 negative cells CD19 expressing cells

UCART19 CAR exhibits

comparable/higher cytolytic capability

than benchmark CAR on target cells

In collaboration with Dr. Martin Pule, UCL, UK

7 September 10th, 2013

CTx proprietary technology for TALEN® electroporation

Large volume chamber

(1.5 -12 ml)

Electroporation buffer

• A series of high voltage, short duration pulses

followed by lower voltage, longer duration

pulses

• High transfection efficacy

• High cell viability

AgilePulseTM Electroporation system

SS

C

FSC GFP

Via

bili

ty

Cell

co

un

t

GFP

93% GFP+

8 September 10th, 2013

TALEN®-mediated knock-outs

TALEN® mRNA: transient gene transfer

CD52

TC

R/C

D3

42.5%

72.9 %

TCR

FCS

51.1%

CD52

FCS

Functional tests: TCR KO cells are unresponsive to TCR stimulation

CD52 KO cells are resistant to Campath depletion

9 September 10th, 2013

End product

Cell expansion around 80 fold in 11 days (5 x 106 to 378 x 106 ± 54 cells)

Depletion of TCRαβ+ cells (Miltenyi)

Final phenotype (day 17):

After purification Before purification

Starting population

(CD3+)

Final population

(CD3-)

CD

45

RA

CD62L

CD

4

CD8

TC

R/C

D3

CD52

10 September 10th, 2013

CAR19 TCR- cells eradicate lymphoma in an orthotopic model

No CAR CAR19 T-cell CAR19 TCR- T-cell

Day 2

Day 7

Day 12

Day 1:

iv infusion with 250k Raji

Day 2:

iv infusion with 4x106 T-cells

In collaboration with Dr. Martin Pule, UCL, UK

TCR-deficient cells

engraft as WT T-cells

TCR-deficient cells

eradicate Raji lymphoma

11 September 10th, 2013

Cellectis’ established manufacturing process platform

A robust process designed for cGMP compatibility

Healthy

Donor

Apheresis

Frozen PBMC

Patients

109 T-cells

Efficiency >50%

2x 1011 T-cells

Double KO Efficiency >40%

1 x 1010 CAR+ TCR- T-cells

>40% CD52 KO

“Off the shelf”

cell therapy product

Activation

T-cells

D0

Transduction

D3

Electroporation

TALEN® mRNA

D5

Cell Separation

D17

12 September 10th, 2013

Conclusion

Development of an off-the-shelf therapeutic T-cell platform

• Efficient T-cell engineering for exogenous CAR expression

• TALENs® permit efficient and simultaneous elimination of

Endogenous T-cell receptor → significant decrease of GvHD risk

Cellular target for a lymphodepleting treatment → for

proliferation & activity of final T-cells versus immunosuppressive

drugs

A robust process designed for cGMP compatibility

Generation of non alloreactive T-cells for cancer therapy

13 September 10th, 2013

Acknowledgments

Cellectis therapeutics

Collaborators:

University College, London

Martin Pule

- Brian Philip

- Gordon Weng-Kit Cheung

- Leila Mekkaoui

Karl Peggs

Waseem Qasim

Cellectis Platform Philippe Duchateau

Jean-Charles Epinat Julianne

Smith

Andrew

Scharenberg

Isabelle

Chion

Diane

LeClerre

Céline

Lebuhotel

Justin

Eyquem

Laetitia

Lemaire

Cécile

Bas

Pierrick

Potrel

Cécile

Schiffer-

Mannioui

Laurent

Poirot

Roman

Galetto

Agnès

Gouble

Stéphanie

Grosse

Sylvain

Arnould

THANK YOU