Background

Long-term follow-up after adoptive transfer of CD19-specific CAR+ T cells genetically modified via non-viral Sleeping Beauty system following hematopoietic stem-cell transplantation (HSCT)

Partow Kebriaei1, Helen Huls2, Simon Olivares3, Aaron Orozco3, Shihuang Su3, Sourindra Maiti3, Eleanor de Groot4, Amy Smith4, Yijun Yang4, Gabriela Rondon1, Muzaffar Qazilbash1, Stefan Ciurea1, Amin Alousi1, Yago Nieto1, David Marin1, Uday Popat1, Chitra Hosing1, Hagop Kantarjian5, Katayoun Rezvani1, Elizabeth J. Shpall1, Richard E. Champlin1, Laurence J.N. Cooper4, Harjeet Singh3

Departments of 1Stem Cell Transplantation and Cellular Therapy, 3Pediatrics and 5Leukemia, UT MD Anderson Cancer Center, Houston, TX; 2Intrexon, Germantown, MD; 4Ziopharm Oncology, Boston, MA

HSCT

Median

follow

up

PFS OS

Longest

CAR T-cell

persistence

No. pts with CAR

T-cell persistence

at 2 years**

N No. Pts

alive

Median survival

of surviving pts

Autologous 40.7 m 86% 100% 48 m 3 7 7 40.7 m

Allogeneic* 18.1 m 32% 49% 25 m 1 19 9 31.3 m

• Progression-free and overall survivals of patients receiving CD19-specific CAR+ T cells following autolo-gous and allogeneic HSCT continue to show significant benefits over historical controls of HSCT alone.2,3,4

• CAR+ T cells are detected by ddPCR and flow cytometry as long as 4+ years after infusion in autologous HSCT recipients and 2+ years in allogeneic HSCT recipients

• Persistence of T cells is noted, even as such patients have reconstituted their B-cell compartment. Further follow-up will evaluate continued persistence of CAR+ T cells and B cells.

• Patients can remain in CR for years after recovery of normal B-cell counts. • Approximately, 44 years^ of cumulative follow up in surviving recipients support the safety of infusing

CAR+ T cells modified with the SB system. • SB-modified CAR+ T cells demonstrated to be safe, further supporting non-viral gene transfer in future

clinical studies including our 2018 planned 3rd generation point-of-care trial to very rapidly manufacture (< 2 days) CD19-specific T cells.

^ The number of years of follow up were summed for all surviving patients and provided as a cumulative total

Huls: Former employee of Intrexon: Employment, Equity ownership. ZIOPHARM Oncology: Equity ownership Olivares: Intrexon Corporation: Equity Ownership. ZIOPHARM

Oncology: Equity Ownership. Su: Intrexon Corporation: Equity Ownership. ZIOPHARM Oncology: Equity Ownership Maiti: Intrexon Corporation: Equity Ownership.

ZIOPHARM Oncology: Equity Ownership. Smith: ZIOPHARM Oncology: Employment. De Groot: ZIOPHARM Oncology: Employment. Kantarjian: Pfizer: Research Funding; No-

vartis: Research Funding; Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding; Delta-Fly Pharma: Research Funding; Amgen: Research Funding. Champlin: In-

trexon Corporation: Equity Ownership. ZIOPHARM Oncology: Equity Ownership. Cooper: Miltenyi Biotec: Honoraria; Ampliphi Biosciences: Equity Ownership; Procter & Gam-

ble: Equity Ownership; Organovo Holdings: Equity Ownership; Targazyme, Inc.: Equity Ownership; Argos Therapeutics: Equity Ownership; Intrexon Corporation: Patents, Equi-

ty Ownership; Immatics: Equity Ownership, Patents & Royalties; Sangamo Biosciences: Patents & Royalties; ZIOPHARM Oncology: Employment, Equity Ownership, Research

Funding; Ferring: Consultancy. Singh: Intrexon Corporation: Equity Ownership. ZIOPHARM Oncology: Equity Ownership.

• Multi-year follow up demonstrates encouraging PFS and OS.

• Survival appears longer than data reported for autologous (compared with 49% 3 year PFS)2,

allogeneic (compared with 1-year 20-34% OS)3, and haploidentical (haplo) allogeneic (compared

with 3 year 37% OS and 31% DFS)4 HSCT without infusion of CAR+ T cells.

Autologous HSCT

followed by T-cell

infusion

26 patients w/

advanced CD19+ non-

Hodgkin lymphoma (NHL)

or acute lymphoblastic

leukemia (ALL)

Patients subsequently enrolled onto Long-Term

Follow Up (LTFU) study. During this time, patients

could be tested annually for the persistence of

SB-modified T cells using PCR and flow cytometry.

Patients from two clinical trials

at MD Anderson Cancer Center

(NCT00968760, NCT01497184)

15 Year Long Term Follow Up

(NCT01492036, IRB: 2006-0676, Ongoing)

2015: Initial data reported1

2017: LTFU update

B-cells

(normal range: 7.2—11.2%)

CAR

Patient Summary

P471

Autologous

NHL

4 year

Post-infusion

• Pt remains in remission

• PFS and OS: 3.8 years

• No disease in marrow

• IgG: Normal

• No Toxicity

P458

Autologous

NHL

4 year

Post-infusion

• Pt remains in remission

• PFS and OS: 4.2 years

• No disease in marrow

• IgG: Normal

• No Toxicity

P708

Autologous

NHL

2 year

Post-infusion

• Pt remains in remission

• PFS and OS: 2.2 years

• No disease in marrow

• IgG: Normal

• No Toxicity

T-cell Dose

Level/ BSA

No. Allogeneic

pts dosed

(haploidentical

subset)

No. Autologous

pts dosed

106/m2 5 (2) -

107/m2 6 (3) -

5x107/m2 5 (1) -

108/m2 3 (2) -

5x108/m2 - 5

109/m2 - 2

Implementing Sleeping Beauty-Modified Cells, a Non-viral Approach

Study Design

CD19 Specific CAR Structure and Patient Dose Levels

Patient Summary

Summary and Conclusions

Patients in remission show long-term T Cell persistence

References

Overall Survival (OS) and Progression-Free Survival (PFS)

CAR Structure: Depicted as homodimer of CD19-specific

CAR CD19RCD28. CD19 scfv is appended from T-cell

surface by IgG4 Fc region and signals through chimeric

CD28 and CD3 endodomains.

Long-term persistence of CAR+ T cells: Autologous and allogeneic HSCT patients receiving genetically modified T cells from the 1st generation SB trial (NCT00968760, NCT01497184) were enrolled in LTFU study (NCT01492036).

PBMC were isolated from peripheral blood collected from these patients at their yearly visits and analyzed for the presence of CAR+ T cells using ddPCR and flow cytometry. Presence of CAR by ddPCR is shown through out the

follow up period. Lower limit of quantification is 32 copies/g of DNA. Grayed area represents previously published data.1 Dot-plots depict presence of CAR and B-cells (CD19, CD20) in viable (CD45+) lymphocytes at the last

time point for each patient circled in red. Results from representative patients are shown.

P788

Allogeneic

ALL

2 year

Post-infusion

• Pt remains in remission

• PFS and OS: 2.2 years

• No disease in marrow

• IgG: Normal

• No Toxicity

Table 1: T-cell dose levels/BSA for patients dosed on

NCT00968760 and NCT01497184.

Table 3: Median follow up, OS, PFS, and T-cell persistence data for patients dosed on NCT00968760 and NCT01497184 from T-cell infusion. *Haploidentical subset data show OS of 63% and PFS of 50% with a median follow up of 20.8 months. **Persistence was determined using CAR-specific ddPCR.

Survival for recipients of allogeneic CAR+ T cells

HSCT N

Histology Prior lines of Rx

(Mean)

Age (years)

NHL ALL Median Min Max

Autologous 7 7 0 52 36 61 3

Allogeneic

(Haploidentical subset)

19

(8)

2

(1)

17

(7)

31

(34)

21

(21)

56

(52)

3

(3)

1Kebriaei, P. et al, .J. Clin. Invest., 126, pp. 3363-3376 (2016) 2Sauter, C.S. et al., Blood 125, 2579-2581 (2015) 3Kebriaei, P. & Poon, L.M. Curr Hematol Malig Rep 7, 144-152 (2012) 4 Srour et al, BBMT, Feb; 23(2):318-324 (2017)

Disclosures

• Non-viral gene transfer – Sleeping Beauty (SB) transposon/transposase system – used to stably express a CD19-specific chimeric antigen receptor (CAR, designated CD19RCD28) that signals via chimeric CD28 and CD3-zeta

• T cells from peripheral blood mononuclear cells (PBMC) were manufactured by electro-transfer of SB-derived DNA plasmids coding for transposon (CAR) and transposase (SB11) and propagation on irradiated feeder cells derived from K-562 genetically modified to co-express CD19 and co-stimulatory molecules and thus serve as activating and propagating cells (AaPC)

• First-generation clinical trials using SB modified T cells as an adjuvant therapy to hematopoietic stem-cell transplantation (HSCT) have been reported1

• Twenty-six patients with advanced non-Hodgkin lymphoma (n=9) or acute lymphoblastic leukemia (n=17) underwent autologous (n=7) or allogeneic (n=19) HSCT. Patients were subsequently enrolled onto a LTFU study, to be followed for up to 15 years. During this time, patients could be tested annually for the persistence of genetically modified T cells using droplet digital polymerase chain reaction (ddPCR) and flow cytometry.

Table 2: Patient summary characteristics for patients dosed on NCT00968760 and NCT01497184.

Allogeneic HSCT

followed by T-cell

infusion

0 10 20 30 40

Survival Time Since Infusion (Months)

0.0

0.2

0.4

0.6

0.8

1.0

Pro

ba

bili

ty o

f S

urv

iva

l

PFS_HaploOS_HaploPFS_AlloOS_Allo

0 10 20 30 40 50

Survival Time Since Infusion (Months)

0.0

0.2

0.4

0.6

0.8

1.0

Pro

ba

bili

ty o

f S

urv

iva

l

PFSOS

Survival for recipients of autologous CAR+ T cells

PCR

0 .0 0 .3 0 .6 0 .9

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

6 1 2 1 8 2 4 3 0 3 6 4 2 4 8

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

4 0 0 0 0

5 0 0 0 0

6 0 0 0 0

P 7 0 8 (2 0 0 7 -0 6 3 5 )

M o n th s a fte r C A R + T -c e ll in fu s io n

Tra

ns

ge

ne

co

pie

s b

y d

dP

CR

(no

./u

g o

f g

DN

A)

Months after CAR T cell infusion

0 .0 0 .3 0 .6 0 .9

0

2 0 0

4 0 0

6 0 0

8 0 0

2 0 0 0

3 0 0 0

4 0 0 0

6 1 2 1 8 2 4 3 0 3 6 4 2 4 8

P 4 5 8 (2 0 0 7 -0 6 3 5 )

M o n th s a fte r C A R + T -c e ll in fu s io n

Tra

ns

ge

ne

co

pie

s b

y d

dP

CR

(no

./u

g o

f g

DN

A)

L e g e n d

Tran

sgen

e c

op

ies

by

dd

PC

R (

no

./

g o

f gD

NA

)

0 .0 0 .3 0 .6 0 .9

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

1 0 0 0

1 5 0 0

6 1 2 1 8 2 4

P 7 8 8 (2 0 0 9 -0 5 2 5 )

D a y s a fte r C A R + T -c e ll in fu s io n

Tra

ns

ge

ne

co

pie

s B

Y d

dP

CR

(no

./u

g o

f g

DN

A)

L e g e n d

0 .0 0 .3 0 .6 0 .9

0

7 5 0

1 5 0 0

2 2 5 0

3 0 0 0

3 7 5 0

4 5 0 0

6 1 2 1 8 2 4 3 0 3 6 4 2 4 8

7 5 0 0

1 0 0 0 0

1 2 5 0 0

1 5 0 0 0

P 4 7 1 (2 0 0 7 -0 6 3 5 )

M o n th s a fte r C A R+

T -c e ll in fu s io n

Tra

ns

ge

ne

co

pie

s b

y d

dP

CR

(no

./u

g o

f g

DN

A)