Alaunos Therapeutics (TCRT) 8-K ZIOPHARM Announces Oral Presentation Highlighting Ad-RTS-IL-12

INTRATUMORAL REGULATED

EXPRESSION OF IL-12 AS A

GENE THERAPY APPROACH

TO IMMUNOTHERAPY

John

Nemunitis

1

,

John

A.

Barrett

2

,

Francois

Lebel

2

,

Thomas

D.

Reed

3

,

E.

Antonio

Chiocca

4

,

Antonio

M.

Omuro

5

,

Larry

Norton

5

,

Jonathan

Lewis

2

1

Mary

Crowley

Cancer

Research

Centers,

Dallas,

TX,

United

States,

75201

2

ZIOPHARM Oncology Inc., Boston, MA, United States, 02129

3

Intrexon Corporation, Germantown, MD, United States, 20876

4

Brigham and Woman’s Hospital, Harvard Medical School, Boston, MA, United States, 02115

5

Memorial Sloan-Kettering Cancer Center, New York, NY, United States, 10065

Exhibit 99.2

Background & Rationale IL-12 in Glioma

•

Tumors escape the immune system through

the process of immunoediting. Thus,

restoration of the immune system’s ability to

detect the tumor should result in improved

treatment outcomes.

•

Localized IL-12 administration has been

shown to have antitumor activity that is

mediated by direct tumor cell cytotoxicity, and

enhancement of immuno-regulatory activities

including activation of anti-tumor natural killer

(NK) cells, CD4

+

T cells and CD8

+

T cells.

Background & Rationale IL-12 in Glioma

•

Roy

&

Kranz

(University

of

Illinois)

IL-12.

Journal

of

Immunology,

2000,

165:

7293–7299.

Model

SV11

Transgenic

mouse

administered

recombinant

mIL-12

i.c.

Findings

Increased

survival

infiltration

of

activated

CD8

and

CD4

T

cells.

•

Vom

Berg

&

Becher

(University

of

Zurich)

J

Exp

Med,

2013,

210:

2803-2811.

Model:

GL261

transduced

to

constitutively

express

IL-12

i.c.

Findings:

Increased

survival

combination of IL-12 + CTLA4 elicits decrease Tregs while increasing Teff.

•

Dimeco

&

Olivi

Johns

Hopkins

School

of

Medicine

&

Istituto

Nazionale

Tumori,

Milan,

Italy)

J

Neurosurg

2000,

92:419–427,

Model:

Rat

9L

gliosarcoma

cells

expressing

IL-12.

Findings

Local

delivery

of

IL-12

in

rat

brain

prolongs

survival

in

animals

challenged

i.c.

with

a

malignant

glioma

cells.

•

Sonabend

&

Lesniak

(University

of

Chicago).

Anti-Cancer

Drugs

2008,

19:133–142.

Model:

GL-261

orthotopic

glioma

model.

Findings:

Synergy

in

survival

with

locally

administered

pmIL-12/PPC

+

biodegradable

carmustine

•

Markert

&

Whitley

(University

of

Alabama)

Journal

of

Virology

2012,86:

5304–5313

Model:

4C8

glioma

cells

orthotopic

B6D2F1

mouse.

Findings:

Hsv

mutant

M002

expressing

IL-12

demonstrated

prolonged

survival

vs.

control

•

Liu

&

Yu

(Cedars

Sinai

Medical

Center)

Cancer

Gene

Therapy

(2002)

9,

9–15

Model:

GL-26

orthotopic

mouse.

Ad5-mIL-12

Findings:

Survival

was

significantly

prolonged

in

Ad-mIL-12–treated

animals

with

increased

CD4+

and

CD8+

T-

cell

infiltration

(

:

:

:

1.

The

Switch

Components:

The

RTS®

gene

program

includes

2

receptor

protein

fusions:

VP16-RXR and Gal4-EcR.  They form unstable and unproductive heterodimers in the

absence of any ligand.

2.

The

Inducible

Promoter:

A

customizable

promoter

to

which

basal

transcription

proteins

are recruited and the target gene is transcribed.

3.

The

Activator

Ligand

(veledimex):

An

ecdysone

analog,

diacylhydrazine-based

small

molecule functions as an activator. In the presence of the ligand, the protein

heterodimer changes to a stable conformation and binds to the inducible promoter.

EcR

RXR

VP16

Gal4

Basal

Transcription

Proteins

Activator

Ligand

RXR

VP16

Gal4

EcR

Inducible Gene Program

Inducible Gene Program

RheoSwitch Therapeutic System®

(RTS®) is a 3-component transcriptional regulator

Inducible Gene Regulation: RheoSwitch Therapeutic System

®

IL-12 Production is Modulated by Activator Ligand in

HT 1080 Cells

5

On

Off

On

AL=75nM

5

Dose-Dependent Increase in Expression of Tumor IL-12

mRNA & IL-12 Protein in Response to Veledimex

10

2

10

10

4

10

5

10

6

0

2

4

6

8

10

12

14

RTS gDNA

Tumor Veledimex Level

10

0

10

1

10

2

10

3

0

2

4

6

8

10

12

14

Time (days)

Time (days)

Vehicle/Vehicle

Vehicle/Veledimex

150

mg/m

Ad (1e10) + Veledimex 15 mg/m

Ad (1e10) + Veledimex 30 mg/m

Ad (1e10) + Veledimex 75 mg/m

Ad

(1e10)

+

Veledimex

150

mg/m

2

Tumor IL-12 Protein Level

IL-12 RNA

0.1

1

10

0

2

4

6

8

10

12

14

Time (days)

10

0

10

1

10

2

10

3

10

4

0

2

4

6

8

10

12

14

Time (days)

2

2

2

2

3

Ad-RTS-mIL-12 + Veledimex Increases Tumor

CD8

+

& CD4

+

While Decreasing CD4

+

Fox P3

+

TILs

in the 4T1 Syngeneic Mouse

Vehicle

Ad-RTS-mIL-12

1 x 10

10

vp

+ Veledimex

150 mg/m

7

CD8

+

CD4

+

CD4

+

Fox P3

+

2

Dose-Dependent Anti-Tumor Activity of Ad-RTS-

mIL-12 + Veledimex (AL) in Murine 4T1 Model 

Start of treatment

Tumor

volume

reached

100-200

mm

3

0

4

8

12

16

20

24

28

32

0

5

10

15

Vehicle/Vehicle

Vehicle/Ad-RTS-mIL12

Vehicle/Veledimex

15

mg/m

2

Ad-RTS-mIL12

+

AL

15

mg/m

2

Ad-RTS-mIL12

+

AL

30

mg/m

2

Ad-RTS-mIL12

+

AL

75

mg/m

2

Ad-RTS-mIL12

+

AL

150mg/m

2

Time (Days)

•

High expression of IL-12 mRNA in tumors, tightly controlled by

veledimex dose

•

Tumor

biopsies

show

increased

tumor

infiltrating

lymphocytes

in

both

injected and systemic non-injected lesions

•

Serious adverse events are mechanism-based and consistent with

immunotherapy (Fever, N&V, leukopenia, increased LFTs,

hyponatremia, cytokine release response)

•Serious adverse events reversed within days after stopping veledimex

dosing

•Subjects

who

have

had

IL-12

expression

turned

“off”

have

been

redosed, and IL-12 turned “on”

again

Clinical Observations to Date

9

We can control gene expression to achieve a systemic immune

response

We have seen systemic and fully reversible toxicity

Higher Veledimex Levels Normal and in GL261

Orthotopic Glioma Mouse Brains

Veledimex levels at 24 hr posttreatment

0

1000

2000

3000

4000

5000

6000

450 mg/m

2

/day

1200 mg/m

2

/day

Effects of Ad-RTS-mIL-12 + Veledimex (AL)

in the Orthotopic GL261 Mouse

Normal Mouse

Vehicle

BID x 14

Ad-RTS-mIL-12 1x10

10

vp

+

AL

450

mg/m

/day

BID x14

Treatment For 14 days

Day 74 (end of study)

Control Day 20

2

Ad-RTS-mIL-12 + Veledimex (in Chow)

Results in Increased Survival in the GL261

Orthotopic Glioma Mouse Model

Veledimex administered ad lib in chow from Day 4 to EOS at ~ 675 mg/m

/day

Ad-RTS-mIL12 administered on Day 5

0

20

40

60

80

0

20

40

60

80

100

Vehicle/Vehicle

Vehicle/AD (5e9)

Vehicle/Activator chow (1000)

Activator chow + AD (5e9)

Activator chow + AD (1e9)

Activator chow + AD (1e8)

Time (Days)

2

Ad-RTS-mIL-12 + Veledimex (AL) Results in

Increased Survival When Compared to Control

in the GL261 Orthotopic Glioma Mouse Model

Ad-RTS-mIL12

administered

on

Day

5

;

Veledimex

(mg/m

2

)

administered

BID

for

14

days

from

Day 5;

0

20

40

60

80

0

10

20

30

40

50

60

70

80

90

100

maisine/saline

maisine/vector

Veledimex (AL)/saline

AL 150/day + AD 1e10

AL 300/day + AD 1e10

AL 450/day + AD 1e10

AL 675/day + AD 1e10

AL 1200/day + AD 1e10

dexamethasone 6 bidx14

bevacizumab 30 biwkx3

temozolamide 300Qdx5

Time (Days)

Ad-RTS-mIL-12 + veledimex significantly

reduces brain cancer stem cells

in GL-261

Orthotopic Glioma Model

Nestin levels (marker for cancer stem cells)

inverse correlation with survival (Pearson r= 0.92)

0

10

20

30

Vehicle/Vehicle

Vehicle/Ad-RTS-mIL-12

Vehicle/Veledimex

Veledime

x 450 mg/m

/day

Veledime

x 600 mg/m

/day

Veledime

x 1200 mg/m

/day

Nestin

2

2

2

15

Conclusions

•

Ad-RTS-mIL-12 + veledimex PO exhibits controllable

systemic immune activation in human subjects with

melanoma and breast cancer. 

•

Veledimex exhibits dose-related increases in plasma and

brain tissue exposure with no accumulation in brain.

•

Ad-RTS-mIL-12 (1x10

10

vp) + veledimex PO improves

survival over temozolomide, dexamethasone and

bevacizumab.

•

Ad-RTS-mIL-12 + veledimex significantly reduces brain

cancer stem cells

•

These findings support the utility of localized, regulatable IL-

12 production as an approach for the treatment of malignant

glioma in human subjects.