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8th International Conference and Exhibition on:

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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˚ 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

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