1. Electroporation mediated DNA vaccination results in improved immune responses
8th International Conference and Exhibition on:
Pharmaceutics & Novel Drug Delivery Systems
Madrid, Spain
March 9, 2016
Paul Fisher, Ph.D.
Inovio Pharmaceuticals

2. Attractiveness of DNA Vaccines
Safer than live virus vaccines
Cannot cause disease
No significant side effects
Prevent and treat
Vastly expands market size
Generate T cell & Ab responses
Faster development
Easier to manufacture
Combination vaccines possible
Key limitation of delivery being recently overcome with electroporation
Electroporation is a key enabling technology

3. Better DNA Vaccine Delivery: Electroporation
Inovio Confidental

4. INOVIO: Fulfilling the promise of DNA Vaccine
No vector induced responses – repeat boosts; multiple/combination vaccines
Greater potency than viral vectors in primates and in humans
Manufacturing advantages
Inovio Confidental

5. Muscle EP Device – In the clinic: CELLECTRA®-5P
Phase II Device
Phase III Device
IM-Electroporation
Prophylactic Phase I
FLU-001 US
FLU-001 Korea
HIV-080
RV-262
EBOV (Ebola)
(HIV)
(Flu)
Therapeutic Phase II
HPV-003 (HPV-assoc cervical dysplasia)
EORTC-1411 (HPV-associated cervical cancer)
How can this possibly be applied in vivo, in a clinical setting? Electroporation has actually been used in the clinic for many years. Inovio has immunized nearly 1000 patients.
Question we always get: Needles and electric pulses in your muscle? How do people tolerate it? Answer: only a small number of patients have elected not to return for addtl tx due to pain. Most cite the injection itself as more painful than the insertion of the needle electrodes or the electroporation event.
Therapeutic Phase I
HIV (HIV)
HPV-001
HPV-002
HPV-004
HPV-005
HPV-006
TRT-001 (breast, lung, pancreatic cancers)
PCa-001 (prostate cancer)
VGX (chonic HepC)
HBV-001 (chronic HepB)
(HPV-associated CIN)
(HPV-associated cancers)
Number of total patients treated
Number of total immunizations
Inovio Confidental

6. VGX-3100 Phase II: Robust HPV-16 and HPV-18 T Cell Responses
So the question is: does it work? Here we can see that patients receiving VGX-3100, our HPV vaccine used in a recently-completed ph II trial, had very significant cellular immune responses.
Keep in mind that this clinical trial was a therapeutic vaccine treating patients already infected with HPV, who exhibited precancerous lesions.
So, our ability to promote a powerful T cell response specific to the antigens of interest is very important if we are seeking to actively clear the disease, not just prevent it.
Trimble et al, Lancet 2015
Inovio Confidental

7. VGX-3100 Phase II: Clinical Efficacy in the Treatment of Cervical Dysplasia
Post hoc
Any way we look at it, we hit our primary endpoint, which is regression of the disease to a less severe state. Nearly half of patients receiving VGX-3100 saw their lesions improve, compared to less than one third of the placebo group.
Furthermore, almost all of our vaccinated patients who regressed also hit our secondary endpoint, which is clearance of the virus itself. Compare that to the placebo group, where about 14% of patients hit both endpoints.
Finally, post hoc analysis showed that around 40% of patients receiving VGX-3100 had their disease regress to a normal, healthy state, while only 17% of placebo patients had this occur.
Evidence is clear: EP-enabled DNA vaccine can promote specific, T-cell mediated clearance of an active viral infection 1˚
1˚ and 2 ˚
Trimble et al, Lancet 2015
First demonstration of clinical efficacy from a DNA vaccine using electroporation
Inovio Confidental

8. GFP expression on skin surface
Surface EP Device – Transfection localization
x20
+ EP
x20
- EP
IM: incredibly successful proof of concept. There are limitations
Pediatric / prophylactic vaccinations? IM not the best fit. Focus: tolerability, user experience. Think RSV vaccination for young children, or annual flu vaccination
Principle: E-field shallower than the depth of the subdermal nerves
Epidermis is richly populated with immune cells.
+ EP
x40
x40
+ EP
- EP
GFP expression on skin surface
Broderick et al, Gene Ther Mar;18(3):
CONFIDENTIAL
Inovio Confidential
Inovio Confidental

9. Evolution of Surface EP to a Multi-head Device
GFP only
Dose limitations, multivalent vaccines (no need to test interference with a co-formulation, more ‘plug and play’. For example, in response to bioterrorism event or a pandemic)
Skin injection limited to about 100 uL due to delamination of the tissue layers (Dose sparing effect may not overcome this)
Increase dosage from a single treatment. Also, some evidence that there is interference between multiple DNA treatments at the same location.
Injection only (No EP)
RFP only
RFP and GFP
McCoy et al, 2014
Inovio Confidental

10. Monocyte/Granulocyte Infiltration
Expression Kinetics in Skin
1 Hr
2 Hr
4 Hr
6 Hr
8 Hr
24 Hr
48 Hr
Day 3
Day 7
Day 12
Day 14
Day 21
No EP
1 Hr
1 Hr
1 Hr
2 Hr
2 Hr
2 Hr
4 Hr
4 Hr
4 Hr
6 Hr
6 Hr
6 Hr
8 Hr
8 Hr
8 Hr
24 Hr
24 Hr
24 Hr
48 Hr
48 Hr
48 Hr
Day 3
Day 3
Day 3
Day 7
Day 7
Day 7
Day 12
Day 12
Day 12
Day 14
Day 14
Day 14
Day 21
Day 21
Day 21
Days
Muscle cells have a very long life, but skin cells have higher turnover rate.
Expression begins within 1h after administration, peak around 24-48h.
Signs of infiltration, but no clear cases of inflammation or damage following tx
Hours
Surface Expression
Cellular Expression
Monocyte/Granulocyte Infiltration
Mendoza et al, 2013
Inovio Confidental

11. Transfection in the Epidermis
Reporter gene expression spatially matches, almost exactly, the section of the skin in contact with our surface electroporation device (4.5mm)
Keratinocyte marker shows outermost layer of skin, and merged image shows essentially all RFP+ cells in the epidermis.
Stratum corneum
Epidermis
Dermis
Inovio Confidental

12. Directly Transfecting Keratinocytes
Note keratinocytes, with their rounded, “fried egg” morphology, are indeed being transfected.
x40
Mendoza et al, Hum Vacc Imm 2013
Inovio Confidental

13. Directly Transfecting Dendritic Cells
4h post treatment
RFP positive dendritic cell
x40
GFP positive dendritic cell
6h post treatment
x 60
x 60
DCs, with their very distinctive projections, are also transfected (very exciting). Majority of DCs in this layer of skin are LCs.
This particular cell is traversing the membrane between dermis and epidermis, showing motility, UNLIKE keratinocytes
We have also observed transfected cells in dermis, but EP does not extend to dermis  suggest migration of transfected cells.
Dendrites themselves seem to be interacting with other cells
IMARIS-3D Rendered
Amante, Hum Gene Ther Methods 2015
Inovio Confidental

14. DC migration into the dermis
Answering question of DC migration.
Gradually move through epidermis, dermis, and hypodermis, eventually interacting w/ lymphatic system
Inovio Confidental

15. GFP positive cells in the draining lymph nodes
Kinetics of GFP+ cells in T cell zone in draining LNs correspond to expression levels in the skin.
More cells expressing GFP in skin  more GFP+ cells in draining LNs
GFP positive cell in the T cell zone of the cortex in the inguinal lymph node
Smith, Mol Ther Methods Clin Dev 2014
Inovio Confidental

16. Minimally invasive dermal electroporation – CELLECTRA®-3P
Tethered 3P System
Next Gen Tethered 3PSystem
Portable Cordless 3P System
No EP P-EP
Skin surface
Dermal EP (as opposed to surface EP)
This device has been in the clinic for our flu and EBOLA trials, and is currently being evaluated in an HIV trial
Electrodes do penetrate, unlike surface EP, so the E-field covers the dermis and subdermal regions (as opposed to epidermis for surface EP)
Skin underside
Inovio Confidental

17. CELLECTRA®-3P targets a wide range of cell types in the skin
Keratinocytes after 1 hour
Fibroblasts
Langerhans Cell
25um
25um
25um
Keratinocytes after 24 hours
Adipocytes
Myocytes
Speaks for itself. The dermal electroporation device transfects a wide range of cells.
25um
50um
25um
Amante, Hum Gene Ther Methods 2015
Inovio Confidental

18. Minimally invasive EP produces comparable immunology to IM-EP
Central dogma has historically been: IM to drive T cell response, and skin to drive humoral response. Here, we can see that the lines are a bit blurred and the difference is not clear cut
ID drives comparable cellular response (left, HIV vaccine), while IM is also capable of driving antibody response (flu, right)
Inovio Confidental

19. + +++++ +++ - - +++++ 1:0 5:0 3:5 + + +++++ - ++++ +++ To be adde
DNA Injection Only (No EP)
DNA Injection Only (No EP)
SEP
CELLECTRA-3P
SEP
CELLECTRA-3P
Skin Surface
Epidermal Transfection +
+++++
+++
Skin Underside
Dermal/ Subdermal Transfection - -
+++++
Epidermis :Dermis Transfection Ratio
Skin Section (DAPI stained)
1:0
5:0
3:5
Comparing surface and intradermal 3P devices. Each has pros and cons, in particular the balance between tissue layers transfected and damage
Design of the device and parameters result in very distinct biological responses.
Inovio has established a fairly comprehensive platform of devices, and we want to think about each treatment individually: what device would be the best fit?
Electroporation may not be a “one size fits all” system
Skin Section (H&E stained)
Necrosis + +
+++++
Skin Section (TUNEL stained)
Apoptosis -
++++
+++
To be adde
To be adde
To be adde
Inovio Confidental

20. University of Pennsylvania
Acknowledgments
University of Pennsylvania
Muthumani Karuppiah
David Weiner
Inovio
Kate Broderick
Laurent Humeau
Jay McCoy
Trevor Smith
Janess Mendoza
Dinah Amante
Katherine Schultheis
Stephanie Ramos
Bernadette Ferraro
Maria Yang
Lauren Jann
Steve Kemmerrer
Jingjing Jiang
Niranjan Sardesai
Mark Bagarazzi
J. Joseph Kim
Amir Khan
Michael Dallas
Anna Slager
Kim Kraynyak
Matthew Morrow
Jian Yan
Lindsay Sakata
JC Stone
Jessica Lee
USAMRIID
Connie Schmaljohn
Kat Cashman
Scripps
Bill Kiosses
VGXI, Inc.
Dorothy Peterson
This work was supported in part by US Army grant W23RYX-8141-N604: # and US Army SBIR W81XWH-11-C-0051:#
Additional funding support from DTRA and NIH/NIAID