A549 : A Novel Cell Substrate Enabling Production of New Vaccine Candidates VRBPAC Silver Spring, MD September 19, 20

1. A549: A Novel Cell Substrate Enabling Production of New Vaccine CandidatesVRBPACSilver Spring, MDSeptember 19, 2012

2. Presentation Overview • PaxVax Vision • Rationale for Selection of the A549 Cell Substrate for adenovirus based vaccines production • The origin and regulatory status of the A549 cell line • Evaluation of A549 for Vaccine Production • Absence of adventitious agents • Absence of oncogenic agents • Clearance of intact cells and residual DNA • Conclusions VRBPAC, 19th Sept 2012

3. PaxVax Vision Potential Novel Vaccine Candidates: • H5, H7 pandemic flu • HIV • Anthrax • HSV • HPV • CMV • Malaria • Tuberculosis Unique Live Oral Ad4- and Ad7-Based Replication Competent Vaccines Insert gene of relevant antigen(s) Grow in high yield cell substrate A549 VRBPAC, 19th Sept 2012

4. A549 is an Ideal Cell Substrate for Ad4-Based Vaccine Production • PaxVax reviewed available cell substrates for suitable Ad4 productivity • Ruled out non-human cells due to species specificity • Evaluated human diploid cell lines that are used for licensed vaccines: WI-38, which is used for the Ad4/Ad7 vaccine, and MRC-5 • Typically, PaxVax vaccines have a lower yield compared to wild type Ad4 • Ruled out human diploid cells for our use due to low productivity • After screening human continuous cell lines, A549 identified as the optimum candidate for production purposes VRBPAC, 19th Sept 2012

5. A549 Cell Substrate Essential for Current Vaccine Development Work • Initial Phase 1 H5 pandemic influenza vaccine candidate produced in MRC-5 cells • However, yield in MRC-5 is suboptimal to supply large-scale clinical trials and insufficient to support potential pandemic needs • Two Anthrax vaccine candidates in development with NIH/DMID • Yield in MRC-5 very low; insufficient for even Phase 1 studies VRBPAC, 19th Sept 2012

6. A549 Cell Substrate Essential for Current Vaccine Development Work • HIV vaccine candidates based on gag and truncated env gp145 • Phase 1 material produced in MRC-5 but scale insufficient for further vaccine development • HIV vaccine candidate based on full length native gp160 • Production only possible in A549 cell substrate; gp160 reduces vaccine yield from MRC-5 cells • gp160 antigen is of interest to HIV vaccine community • Further clinical development of this important antigen in jeopardy if no suitable cell substrate available for production VRBPAC, 19th Sept 2012

7. A549 Shows 10-40x Higher Yields for Several PaxVax Ad4 Vaccines Summary: • Anthrax and HIV (gp160) dependent on A549 for development • A549 essential for Pan Flu and HIV (gp145) to reach needed production scale VRBPAC, 19th Sept 2012

8. Origin and Regulatory Status of the A549 Cell Substrate • Human lung adenocarcinoma-derived (source: 58 yr old Caucasian male, 1972) • Deposited at ATCC in 1976 • PaxVax prepared Master Cell Bank; undertook comprehensive characterization with support of Wellcome Trust and NIH, with guidance from CBER • Biologics Master File submitted to CBER (Oct 2011) • Condition of W. Trust funding is that PaxVax A549 cell substrate be made available for licensure to other parties VRBPAC, 19th Sept 2012

9. Evaluation of Use of A549 Cell Substrate for Vaccine Production • Extensive Characterization of A549 Cell Substrate • Adventitious Agents Testing • General screening methods and specific agent PCR tests • Chemical induction studies for latent or occult viruses • Massively parallel sequencing • Oncogenicity testing of cell lysates and DNA • Animal studies in mice, rats and hamsters • Tumorigenicity profile assessment in mice • Review of production process for ability to clear intact viable cells, cellular DNA and proteins VRBPAC, 19th Sept 2012

10. Adventitious Agent Studies: All Tests NegativeGeneral Screens for Microbial and Viral Agents VRBPAC, 19th Sept 2012

11. Adventitious Agent Studies:All Tests Negative for Specific Agents VRBPAC, 19th Sept 2012

12. Adventitious Agent Studies:All Tests Negative for Specific Agents VRBPAC, 19th Sept 2012

13. Chemical Induction Studies for Latent or Occult Viruses: None Detected • Viral induction conditions optimized with guidance from Dr. Arifa Khan, CBER* • TPA & NaB for DNA viruses • IdU or AzaC for RNA viruses • No viruses detected by extensive analyses • TEM and PERT: Negative for retroviruses • Coculture by serial passage with indicator cells: Negative for latent RNA viruses • Degenerative PCR: Negative for DNA viruses *Khan et al, Proposed Algorithm to Investigate Latent and Occult Viruses in Vaccine Cell Substrates by Chemical Induction. Biologicals 2009 37:196-201 VRBPAC, 19th Sept 2012

14. Massively Parallel Sequencing (MPS)For Detection of Adventitious Agents Cellular transcriptome analysis (mRNA Shotgun MPS) [Latent/Integrated virus detection] DNA Transcription RNA Extracellular nucleic acids & Culture media components (Amplicon MPS) [Encapsidated/Productive virus detection] A549 DNA RNA VRBPAC, 19th Sept 2012

15. Adventitious Agent Studies: Massively Parallel Sequencing (MPS) • A549 MPS Data • 2009 Analysis (database of 300,000 entries) • 2012 Reanalysis of data (database expanded to >1,000,000); same result • No viral sequences detected by analysis of cellular transcriptome • Extracellular medium: bovine viral sequences present • Bovine viral sequences traced to preparatory serum (MPS and qPCR) • A549 MCB negative for the bovine viral sequences (qPCR) • Conclusion: • No  transcripts associated with replicating, latent or transforming viruses detected in A549 cells (or MRC-5 control) • MPS sensitive enough to detect viral sequences • Future production serum (γ­irradiated) lots now screened for specific bovine sequences VRBPAC, 19th Sept 2012

16. Conclusion:Adventitious Agents Not Detected in A549 • None detected in general screens (broad detection) • No viruses detected by extensive type-specific assays • 30 standard plus 11 human specific viruses per FDA suggestion • Rare bovine, porcine, rodent, simian • Human retroviruses, adeno-associated viruses • General antibody production: hamster, mouse & rat • Latent or occult DNA and RNA viruses not detected by chemical induction tests • Massively Parallel Sequencing did not detect viral signatures in A549 cells • Also, PRNP gene sequencing found no mutations that predispose to transmissible spongiform encephalopathies (TSEs) VRBPAC, 19th Sept 2012

17. A549 DNA or Cell Lysate is Not Oncogenic • Oncogenicity: Ability of DNA or acellular materials to induce tumor formation in animals • PaxVax studies showed A549 cell lysates and A549 DNA did not induce tumors in nude newborn mice, rats or hamsters • The lack of tumor formation by high molecular weight A549 cellular DNA result is consistent with publicly available data, including that in Newborn CD3 Epsilon mice (Dr Keith Peden, CBER) VRBPAC, 19th Sept 2012

18. A549 Cell Substrate Tumorigenicity Profile • A549 cells, known to be tumorigenic, had a TPD50 value of log10 4.4 in a study in athymic nude mice conducted by PaxVax VRBPAC, 19th Sept 2012

19. Clearance of A549 Cells and DNA • Concern is about potential presence of transforming genes from residual cell substrate DNA and presence of intact viable cells in final vaccine product • The clearance of intact viable A549 cells, DNA and cellular protein is achieved by multiple purification steps in the PaxVax manufacturing process VRBPAC, 19th Sept 2012

20. PaxVax Manufacturing Process RemovesA549 Components at Multiple Steps A549 MCB/WCB Clarified Virus Benzonase A549 DNA Digestion Virus Infected A549 Intermediate #1 Detergent Lysis Intact Cell Destruction Anion Exchange Chromatography A549 DNA & Protein Removal Intermediate #2 Virus Lysate #1 Freeze-Thaw Intact Cell Destruction Diafiltration A549 DNA & Protein Removal Intermediate #3 Virus Lysate #2 Filtration #1 Depth 0.8/0.22 µm Intact Cell Removal Filtration #2 0.22 µm Intact Cell Removal Clarified Virus Bulk Drug Substance Oral Dosage Form VRBPAC, 19th Sept 2012

21. Residual Cellular DNA Below Limit of Detection • Several process steps remove or degrade DNA • No residual cellular DNA detected in 5 small-scale Ad4 wild type BDS lots made using the PaxVax process • High sensitivity qPCR (non-validated) • All results below the level of detection (LOD <0.13 ng/mL) • Equivalent to <0.013 ng/dose (1010vp vaccine dose) • Absence of residual DNA makes DNA fragment size determination impractical • Any DNA fragments are likely to be <200 bp due to Benzonase treatment in the PaxVax process • Benzonase able to reduce DNA to 2-5 nucleotides in length • PaxVax process uses 25,000 U/L for 3 hours (exhaustive digestion) VRBPAC, 19th Sept 2012

22. The A549 Cell Substrate is Suitable for Production of Human Vaccines • A549 cell substrate is uniquely suited for Ad4-based vaccine production at a scale to meet potential global medical needs • 10 to 40 fold higher yield than MRC-5 • Extensive characterization failed to identify any adventitious agents in the A549 MCB • General microbial and viral screening tests and specific virus PCR tests • Chemical induction for latent/occult viruses • Massively parallel sequencing • Lack of oncogenicity of A549 DNA or cell lysates in standard animal models • Robust manufacturing process includes orthogonal clearance steps which remove A549 DNA and intact viable cells PaxVax recommends that the A549 cell substrate be accepted for use for the production of human vaccines VRBPAC, 19th Sept 2012