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

Localized Regulated Expression of IL12 as a Gene Therapy Approach to
Cancer Immunotherapy
John A. Barrett Ph.D.
Ziopharm Oncology, Boston MA
02129
Background & Rationale IL-12 in Oncology
• Tumors grow & 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.
Inducible Gene Regulation: RheoSwitch Therapeutic System®
RheoSwitch Therapeutic System® (RTS®) is a 3-component transcriptional regulator
EcR
VP16
Activator
Ligand
RXR
Basal
Transcription
Proteins
Gal4
RXR
VP16
Gal4
EcR
Inducible Gene Program
Inducible Gene Program
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.
Regulated intratumoral expression of IL-12
promotes activation of TIL’s to drive a cytotoxic
immune response against distant tumors
IL-12 Production is Modulated by Activator Ligand in
HT 1080 Cells
Off
On
AL=75nM
On
5
Dose-Dependent Increase in Expression of Tumor IL-12
mRNA & IL-12 Protein in Response to Veledimex
Tumor Veledimex (ng/g)
10 6
10 3
Excipient
INXN-1001 150mg/m2
Ad10
(1e10)+INXN
15mg/m2
2
Ad (1e10)+INXN 30mg/m2
Ad(1e10)+INXN 75mg/m2
Ad(1e10)+INXN
150mg/m2
10 1
10 5
10 4
10 3
10 2
0
Relative Expression
Tumor Veledimex Level
2
4
6
8
10
12
14
Vehicle/Vehicle
Vehicle/Veledimex 150 mg/m2
Ad (1e10) + Veledimex 15 mg/m2
Ad (1e10) + Veledimex 30 mg/m2
Ad (1e10) + Veledimex 75 mg/m2
Ad (1e10) + Veledimex 150 mg/m2
10 0
0
2
4
6
8
10
12
Time (days)
Time (days)
IL-12 RNA
Tumor IL-12 Protein Level
Tumor IL-12 (pg/mg protein)
RTS Copy Number/g DNA
RTS gDNA
14
1000
Excipient
INXN 150mg/m2
Ad(1e10)+INXN 15mg/m2
100
Ad(1e10)+INXN 30mg/m2
Ad(1e10)+INXN 75mg/m2
Ad(1e10)+INXN
150mg/m2
10
10
1
0.1
0
2
4
6
8
Time (days)
10
12
14
Vehicle
AL 150 mg/m2
AL 15 mg/m2 + Ad 1e
AL 30 mg/m2 + Ad 1e
AL 75 mg/m2 + Ad 1e
AL 150 mg/m2 + Ad 1
1
0
2
4
6
8
10
Days post Ad injection
12
14
Ad-RTS-mIL-12 + Veledimex Increases Tumor
CD8+ & CD4+ While Decreasing CD4+ Fox P3+ TILs
in the 4T1 Syngeneic Mouse
CD8+
CD4+
CD4+ Fox P3+
Vehicle
Ad-RTS-mIL-12
1 x 1010 vp
+ Veledimex 150
mg/m2
7
Dose-Dependent Anti-Tumor Activity of Ad-RTSmIL-12 + Veledimex (AL) in Murine 4T1 Model
Tumor Volume V/V0
15
Vehicle/Vehicle
Vehicle/Ad-RTS-mIL12
Vehicle/Veledimex 15 mg/m2
10
Ad-RTS-mIL12 + AL 15 mg/m2
5
Ad-RTS-mIL12 + AL 30 mg/m2
Start of treatment
Ad-RTS-mIL12 + AL 75 mg/m2
Ad-RTS-mIL12 + AL 150mg/m2
0
0
4
8
12
16
20
Time (Days)
Tumor volume reached 100-200 mm3
24
28
32
RIGHT Flank
LEFT Flank
9
Clinical Observations to Date
We can control gene expression to achieve a systemic immune
response
• 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
We have seen systemic and fully reversible toxicity
• 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
10
Increase in Patient Serum Cytokine Expression
Cycle 1
IL-12
80
pg/mL
60
40
20
0
0
0
0
0
0
0
0
0
0
0
16 20 24 28 30 42 56 70 98
Veledimex (mg)
Effects of Ad-RTS-hIL-12 +
Veledimex (AL) in Melanoma Patient
C1D1
C1D15
Day 30
•
•
Initial increase in lesion size due to
inflammatory response seen at Cycle
1 Day 15
Lesion was undetectable at Day 30
12
Veledimex (ng/g)
Higher Veledimex Levels Normal and in GL261
Orthotopic Glioma Mouse Brains
6000
450 mg/m2 /day
5000
1200 mg/m2 /day
4000
3000
2000
1000
0
r
No
,
ce
i
lM
a
m
D
2
y
a
2
GL
M
1
6
,
ice
D
2
y
a
61
2
GL
,D
e
ic
a
M
Veledimex levels at 24 hr posttreatment
3
1
y
Effects of Ad-RTS-mIL-12 + Veledimex (AL)
in the Orthotopic GL261 Mouse
Normal Mouse
Control Day 20
Vehicle
BID x 14
Treatment For 14 days
Day 74 (end of study)
Ad-RTS-mIL-12 1x1010vp
+ AL 450 mg/m2/day
BID x14
Increased Expression of Tumor IL-12 mRNA &
IL-12 Protein in Response to Veledimex
Tumor & Plasma INXN-1001 Ctrough Day 4
200
150
100
50
1.010 6
1.010 4
1.010 2
1.010 0
Day 4
a
sm
Pl
a
Tumor IL-12 protein
Tumor IL-12 mRNA
Ad1e10+AL 450mg/m2
500
Ad1e10+AL 450mg/m2
400
100
pg/mg
Relative Expression
150
Ad1e10+AL 450mg/m2
IN
IN
XN
or
Tu
m
1.010 8
XN
-1
00
-1
00
1
1
0
RTS copy number/µg DNA
INXN-1001, ng/mL
250
RTS gDNA
50
300
200
100
0
0
Day 4
Day 4
Ad-RTS-mIL-12 + Veledimex (AL) Results in
Increased Survival When Compared to Control
in the GL261 Orthotopic Glioma Mouse Model
100
90
80
maisine/saline
% Survival
70
maisine/vector
Veledimex (AL)/saline
60
AL 150/day + AD 1e10
50
AL 300/day + AD 1e10
AL 450/day + AD 1e10
40
AL 675/day + AD 1e10
30
AL 1200/day + AD 1e10
dexamethasone 6 bidx14
20
bevacizumab 30 biwkx3
10
temozolamide 300Qdx5
0
0
20
40
60
80
Time (Days)
Ad-RTS-mIL12 administered on Day 5 ; Veledimex (mg/m2) administered BID for 14 days from
Day 5;
Ad-RTS-mIL-12 + veledimex significantly
reduces brain cancer stem cells in GL-261
Orthotopic Glioma Model
Sum Score
30
Vehicle/Vehicle
20
Vehicle/Ad-RTS-mIL-12
Vehicle/Veledimex
Veledimex 450 mg/m2 /day
10
Veledimex 600 mg/m2 /day
Veledimex 1200 mg/m2 /day
0
Nestin
Nestin levels (marker for cancer stem cells)
inverse correlation with survival (Pearson r= 0.92)
Conclusions
• Ad-RTS-hIL-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 (1x1010 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 IL12 production as an approach for the treatment of malignant
glioma in human subjects.
18