Product Development for Advisory pDNA

pDNA transfected Committee (PDVAC): Advances in nucleic acid cell Humoral (-mediated) Cell-mediated immune response Low-level, persistent immunogen Engulfed by Free immunogen Immunogen on directly activates transfected cell APC directly activates delivery platforms Stimulates Stimulates Stimulates Helper CTL T cell

Memory Gives rise to Gives rise to Stimulates Helper T cell Stimulates

Memory Stimulates Memory th B cell T cell WHO PDVAC 4 Annual Meeting, 22 June 2017 Geneva Re-exposure to Immunogen Activated Plasma Memory Memory CTL cell B cell T cell Stimulates

Antibodies Cytolysis/ Apoptosis David C. Kaslow, MD Exposure to pathogen Chair, Product Development for Vaccines Advisory Committee

1 Outline

• Historical context • Current state of nucleic acid delivery platform technologies • Challenges facing next generation DNA and RNA Vaccines

Page 2 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting In the beginning (27 years ago…)

N.B. transfer = DNA or RNA

Page 3 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting The promise of nucleic acid-based technologies Disruptive technology? “typically cheaper, simpler, and more convenient-to-use...” Innovator’s Dilemma Clayton Christensen

pDNA VACCINATION

pDNA transfected cell Humoral (antibody-mediated) immune response Cell-mediated immune response Low-level, persistent immunogen Engulfed by Free immunogen Immunogen on directly activates transfected cell APC directly activates Stimulates

Stimulates Stimulates B cell Helper CTL T cell

Memory Gives rise to Stimulates Helper T cell Stimulates Gives rise to

Memory Stimulates Memory B cell T cell

Re-exposure to Immunogen Activated Plasma Memory Memory CTL cell B cell T cell Stimulates

Antibodies Cytolysis/ Apoptosis Exposure to pathogen

Page 4 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting The promise of nucleic acid-based technologies Disruptive technology? “typically cheaper, simpler, and more convenient-to-use...” Innovator’s Dilemma Clayton Christensen “when they first appear, they almost always offer lower FOREIGN OR VACCINESVACCINES performance…” SELF “always improve in NUCLEIC performance (and) ACID-BASED CANCER eventually are able to take TECNOLOGIES over older markets… …because they are able to SELF PROTEINS PROTEIN THERAPEUTICS deliver sufficient performance… and they add some new ones.”

Page 5 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting The promise of nucleic acid-based technologies Disruptive technology? “typically cheaper, simpler, and more convenient-to-use...” Innovator’s Dilemma Clayton Christensen Vaccines • Universal Infectious Diseases • Niche Infectious Diseases FOREIGN OR VACCINESVACCINES SELF ANTIGENS • Infectious Diseases, Therapeutic NUCLEIC • Self • Cancer ACID-BASED CANCER TECNOLOGIES Protein therapeutics • Cancer Immunotherapeutics SELF PROTEINS PROTEIN THERAPEUTICS • Regulated Therapeutic Proteins • Unregulated Therapeutic Proteins • Classic Gene Replacement Therapy

Page 6 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting The promise of nucleic acid-based technologies Disruptive technology? “typically cheaper, simpler, and more convenient-to-use...” Innovator’s Dilemma Clayton Christensen Vaccines • Universal Infectious Diseases • Niche Infectious Diseases FOREIGN OR VACCINESVACCINES SELF ANTIGENS • Infectious Diseases, Therapeutic NUCLEIC • Self Antigen • Cancer ACID-BASED CANCER TECNOLOGIES Protein therapeutics • Cancer Immunotherapeutics SELF PROTEINS PROTEIN THERAPEUTICS • Regulated Therapeutic Proteins • Unregulated Therapeutic Proteins • Classic Gene Replacement Therapy

Page 7 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Page 8 Slide courtesy of David Weiner WHO Meeting on Nucleic Acid Vaccines 17–18 May 1994 Geneva

World Health Organization convened a meeting in Geneva to review the early findings for DNA vaccines: • Immunology • • Efficacy • Safety • Delivery 18 papers published

(see Vaccine Volume 12 (16):1491-1568 (December 1994))

Page 9 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting WHO Meeting on Nucleic Acid Vaccines 17–18 May 1994 Geneva Historical note Second day—a vote taken to name the new technology: • Split vote between genetic , polynucleotide vaccines, and nucleic acid vaccines, with majority voting: • Nucleic acid vaccines with sub terms: • DNA vaccines • RNA vaccines “The name nucleic acid vaccines was chosen to reflect the new technology not being designed to insert DNA into the germ line of vaccinees. If this novel form of vaccination were to achieve general acceptance, the perception would need to be that this was a vaccine, and not a therapy for modifying genetic information.” Harriet L. Robinson Vaccine 15(8): 785-787, 1997

Page 10 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Intervening years—three licensed vaccines, all in veterinary applications

2005 2005 2007 → 2010 MERIAL Receives Full License Approval for ONCEPT™ Canine Melanoma Vaccine ONCEPT is the First and Only USDA-Approved, Therapeutic Vaccine for the Treatment of Cancer IHNV DNA vaccine (Apex-IHN, Novartis), containing a CMV February 16, 2010 Duluth, GA — Merial, a world-leading driving expression of animal health company, has gained full-licensure from the IHNV G gene, approved for the U.S. Department of Agriculture for ONCEPT™ Canine commercialization in July 2005 Melanoma Vaccine, DNA. ONCEPT is a breakthrough by the Canadian Food WEST NILE-INNOVATOR vaccines contain the adjuvant vaccine indicated for aiding in extending survival of dogs ® Inspection Agency, Veterinary MetaStim , presentation which is designed for safe, enhanced of with stage II or stage III oral canine melanoma, a Biologist and Biotechnology antigens to the horse’s . MetaStim features a common yet deadly form of cancer in dogs. Division. dual-phase formulation shown to stimulate both cell-mediated and humoral immunity to West Nile virus.

Side note: LifeTideSW5, an -delivered DNA expressing porcine releasing hormone (GHRH) licensed as a product for use in 2007.

Page 11 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting And huge amount of R&D on DNA vaccines

pubmed - (DNA vaccine) NOT cancer count ClinicalTrials.gov N=22,957 1400 Infectious Disease | DNA Vaccine 1200 N= 159 (July 2014) 1000 (N=173 (June 2017)) 800 US EU 600

400 Phase 1 121 3 200 Phase 2 38 9 0

Phase 3 0 0

1949 1961 1963 1965 1967 1969 1971 1973 1975 1977 1979 1981 1983 1985 1987 1989 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015 2017

N.B. >55,000 articles in PubMed+/-Google Scholar HIV 66 Racz R et al. BMC Bioinformatics 15:S2 2014 15

See also: DNAVaxDB ( http://www.violinet.org/dnavaxdb), Tregoning JS, Kinnear E. 2014. Microbiol Spectrum 2(6):PLAS- 0028-2014. a Web-based database and analysis resource of doi:10.1128/microbiolspec.PLAS-0028-2014 experimentally verified DNA vaccines

Page 12 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Evolution of Regulatory Environment (US FDA) for Plasmid DNA Vaccines for Preventive Infectious Diseases 1996 2007

See also: Klinman DM et al. FDA guidance on prophylactic DNA vaccines: analysis and recommendations. Vaccine 28: 2801-2805, 2010.

Page 13 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Circa 1996 Guidance

“Until such time that CBER has accrued sufficient information regarding the safety of plasmid DNA preventive vaccines, a new plasmid construct using a common vector and differing only in the antigen to be expressed may be considered a new product and subject to preclinical safety evaluation as delineated in later sections. Once CBER has more experience in the review of plasmid DNA vaccine products, abbreviated preclinical development programs may be proposed for common plasmid vectors.”

Page 14 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Circa 1996 Guidance

Circa 1996 concerns: • Measures of potency: In vivo required • Autoimmunity: IgG anti-DNA autoantibodies • Persistent expression: At site of injection or distally • Tolerance: Increased disease risk upon subsequent • Integration: Enhanced risk of malignant transformation

Page 15 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Updated Guidance 2007: Autoimmunity

Published preclinical studies indicate that DNA vaccination can activate autoreactive B cells to secrete IgG anti-DNA autoantibodies (See Section VI, References). However, the magnitude and duration of this response appears to be insufficient to cause disease in normal animals or accelerate disease in autoimmune-prone mice. These preclinical studies helped to establish that systemic autoimmunity is unlikely to result from DNA vaccination. Similarly, the absence of an immune response against cells expressing the vaccine- encoded antigen (including muscle cells and dendritic cells) suggests that an autoimmune response directed against tissues in which such cells reside is unlikely. Based on these findings, we will no longer expect that you perform preclinical studies to specifically assess whether vaccination causes .

Page 16 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Updated Guidance 2007: Tolerance

Published studies to address whether DNA vaccines could induce neonatal tolerance yielded divergent results (see Section VI, References). Most DNA vaccines did not induce tolerance in neonatal animals, but idiosyncratic examples of neonatal tolerance have been observed (see Section VI, References). Tolerance has never been observed following vaccination of mature animals. Taken together, these studies suggest that the capacity of a DNA vaccine to induce tolerance may depend on the nature of the encoded antigen and the age at which, and frequency with which, the vaccine is administered. Based on these findings and other considerations, we recommend that prior to use of a DNA vaccine in children or newborns that: i) you first test the vaccine for safety and immunogenicity in adults, and ii) you utilize appropriate preclinical models to evaluate the potential of such vaccines to induce neonatal tolerance.

Page 17 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Updated Guidance 2007: Biodistribution and Integration Analysis Plasmid biodistribution, persistence and integration studies were initially recommended by CBER to determine whether subjects in DNA vaccine trials were at heightened risk from i) the long-term expression of the encoded antigen either at the site of injection or an ectopic site, and/or ii) integration of the plasmid that might increase susceptibility to malignant transformation. Publications resulting from the use of DNA vaccines in clinical studies under IND indicate that intramuscular, subcutaneous, intradermal, or particle-mediated delivery does not result in long-term persistence of plasmid at ectopic sites, and that <30 copies of plasmid per 105 host cells persist at the site of injection after 60 days (see Section VI, References). Before conducting biodistribution/persistence studies, you should contact FDA for advice concerning the need for these studies in particular, when: i) new or significantly modified are proposed for clinical use, and/or ii) the formulation of the DNA vaccine and/or its method/route of delivery may significantly increase cellular uptake or alter plasmid distribution Page 18 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting WHO TRS Guidelines on DNA Vaccines

• Measures of potency: in vitro assay should be shown to correlate with immunogenic activity or efficacy in an animal model • Autoimmunity: IgG anti-DNA autoantibodies analysis in nonclinical programmes not generally warranted • Persistent expression: Biodistribution and persistence studies are required, unless substantial experience has already been gained with an almost identical or similar product • Tolerance: Tolerance has not been observed in adult animals and humans and the initial concern may have been overstated; the possibility of tolerance may remain a concern, particularly in neonates • Integration: Integration studies may not be necessary for a plasmid DNA vaccine if prior information on a similar plasmid, with the same mode of administration already exists. • Risks of encoding or co- stimulatory molecules: Studies should continue to address the possibility that some cytokines may produce local or systemic toxic effects Page 19 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Naked Plasmid DNA: Advantages, limitations and alternatives

Advantages Limitations Electroporation Jet injection Simple design; low complexity Weak to moderate production immunogenicity in humans Easy to scale up; large scale Limited to protein immunogens production methods available delivery Potential for atypical processing Safety of bacterial and parasite proteins Noninfectious, non-replicative; unable to revert to virulent forms Stable formulation Self-amplifying RNA vaccines Easy storage and shipping; no cold chain requirement for transport Cost effective, depending on dose

Immunogenic in animals Humoral and cellular specific immune responses (including CTL)

Page 20 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Next Gen DNA Vaccines: Growing pipeline in infectious diseases

HIV (B)

HIV (ACD) Electroporation Delivery HIV (ABCD) 7 of 15 current programs in clinical development in infectious Flu Universal disease indications: All Phase 1 Ebola ≈500 subjects in trials MERS ≈1500 safety database Zika

http://www.inovio.com/products/pipeline/ accessed 20 June 2017

Page 21 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting What about RNA Vaccines?

DNA Vaccines RNA Vaccines pubmed - (DNA vaccine) NOT cancer count N=22,957 1400

1200 1000 pubmed - (RNA vaccine) NOT cancer count N=9,970 800 800

600 600

400 400

200 200

0 0

1949 1961 1963 1965 1967 1969 1971 1973 1975 1977 1979 1981 1983 1985 1987 1989 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015 2017 1951 1954 1959 1963 1965 1967 1969 1971 1973 1975 1977 1979 1981 1983 1985 1987 1989 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015 2017

1990 2003 1990 2003

Page 22 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Early barriers to RNA Vaccines

• Instability • Hydrolysis • RNases • Billion year old barriers • Dowdy Nature Biotechnology 35: 222–229 (2017) • Escape from endosomes • Innate immune responses (e.g., Toll-like receptors, RNA helicase RIG-I and protein kinase R) • Production requires “clean” enzymes • Unmodified mRNA → Low expression levels in vivo

Page 23 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting RNA Vaccine enhancements

• ↑ Stability and/or expression levels • 5′ cap (m7GpppG) • 3′ poly(A) tail to the end, • Optimizing codon usage and GC content • 5′ and 3′ untranslated elements • 2o structure to increase ribosome engagement • Traversing the billion year barrier • nanoparticle (LNP) • Cationic—delivery and protection against RNAses • Anionic—targeting dendritic cells Nature Biotechnology 35, 193–197 (2017) • Modified bases (e.g., pseudouridine, 5-methylcytidine) • Self-amplification • Encoding viral replicases (e.g., )

Page 24 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting RNA Vaccines in Clinical Development

Adapted from Nature Biotechnology 35, 193–197 (2017)

10 of 19 in clinical development in infectious disease indications: 4 Pre-clinical; 4.5 Phase 1; 1.5 Phase 2

Page 25 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Challenges facing next generation Nucleic Acid Vaccines

• “New products”, from a regulatory perspective • Moving from full to abbreviated preclinical studies? • “Disruptive technology”* without bridges to immunological surrogates/correlates of protection for conventional vaccines • Modernizing legacy vaccines? • Well-characterized biologics v conventional vaccine • mRNA platform v gene of interest (product-specific) quality control and lot release testing? • Scale-up synthetic manufacturing • Enzymes and raw materials (COGs) • Distributed manufacturing (self-contained modules) • Acceptability

*Trans R Soc Trop Med Hyg 98 (10): 593-601 (2004).

Page 26 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting

Progress is being made…

Courtesy of Dr. Frank Bähner, CureVac

Page 27 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting Next Gen Nucleic Acid Vaccines Outstanding Questions

• Efficacy in adequately powered studies in humans • Duration of Immunity • Long Term Safety • Demonstrating scalability of Manufacturing and Formulation presentations for commercial scale

Courtesy of Dr. Mike Watson,

Page 28 Advances in nucleic acid delivery vaccine platforms 4th Annual (2017) PDVAC meeting