Presentation Title

1. Are HeLa Cells an Acceptable Vaccine Cell Substrate? The answer might surprise you. September 19, 2012 Vaccines & Related Biological Products Advisory Committee meeting White Oak FDA Campus Conference Name Presentation Title Date CAPT Rebecca Sheets, Ph.D., USPHS USNIH/NIAID

2. Outline • The need for cell substrates • Evolving cell substrate policy • Why HeLa cells? • HeLa cell bank characterization • Are there differences between HeLa cells & other cell substrates & what do those differences mean in terms of product safety? • Conclusion

3. The Need for Cell Substrates • Over the past 60+ years, cells propagated in culture have been safely & successfully used to produce viral vaccines • Without cell culture, we would not have the following vaccines: • Poliovirus • Measles, Mumps, Rubella • Varicella (chickenpox), Zoster (shingles) • Rotavirus • And more

4. Evolution of Cell Substrate Policy Over this time, primary cells, while still in use, have fallen out of favor, because they cannot be banked and well-characterized prior to use in production. They must be sourced carefully each time and characterized as they are used in production. If they fail characterization, the batch or lot in production must be discarded.

5. What has driven evolution? • Better understanding of cell biology & molecular basis of cancer • Better tools to characterize cells & products • Greater experience using various cells, including in the veterinary field, biotech therapeutics field • Need to support development of HIV/AIDS vaccines and improved influenza virus vaccines (e.g., pandemic)

6. What We Know Now That We Didn’t Then • Tumors arise from genetic changes that result in inappropriate expression or knock-out of proto-oncogenes turning them into oncogenes (i.e., it’s in the DNA) • Viruses that cause cancer express proteins that interact with cellular oncoproteins or are themselves oncoproteins • How to carefully measure cell residuals in products, e.g., host cell nucleic acids, host cell proteins • Improved methods for detection of viruses & viral nucleic acids • Improved purification processes to remove or destroy cell residuals & viruses that might have been present

7. What about HeLa cells? • HeLa cells derive from a woman with cervical cancer in the 1950’s • The cause of the cancer was HPV18 • The nucleic acid remains of HPV18 in HeLa cells are the cause for the cellular transformation in culture (i.e., immortalized growth) – E6, E7 genes • The cell transformation can be reversed in culture by silencing these genes (Lea et al.) • HeLa cells grow readily in culture & are easily engineered

8. Adeno-Associated Virus Vectors • AAV vectors can be engineered by entirely removing the 2 viral protein genes (cap, rep) and simply using the empty backbone to insert foreign genes • The vector can be encapsidated only if the viral capsid gene is provided in trans in the cell line – i.e., requires a packaging cell line • The vector can only replicate to amplify itself if the viral replicase& other needed genes are provided in trans from the cell line

9. Alternatives to Using HeLa Cells • Efforts were undertaken to establish packaging cell lines based on Vero cells instead of HeLa cells • Requires change from human helper virus to simian helper virus • Use of simian virus for helper – are there unique safety concerns introduced? • Yield & manufacturability of AAV on Vero cells not been established • Robustness of Vero packaging cell line for commercial or clinical trial manufacture undetermined • (293 cells won’t work because of E1 gene already in the cell line & its interaction with AAV rep gene product)

10. Characterization of Packaging Cell Line • HeLa cells were engineered to permit packaging of AAV vectors expressing HIV antigen genes • These engineered clones were amplified to establish Master Cell Banks • These banks and “end-of-production (EOP)” passage level cells were thoroughly characterized prior to use in production

11. Identity Testing • Isoenzyme testing to demonstrate human origin of cells (as would be done for all cell substrates) • Southern blot to demonstrate cells were HeLa cells • Restriction digest & southern blot to distinguish between different packaging cell lines based on HeLa used by manufacturer • Sequencing of engineered inserted gene (capsid gene)

12. Tests for Adventitious Agents • All the usual tests were done & were negative • Bacterial & Fungal Sterility • Mycoplasmas (cultivatable & non-cultivatable) • in vitro in Vero, MRC-5, HeLa • in vivo in adult & suckling mice, guinea pigs, embryonated hens’ eggs • Bovine viruses, porcine viruses • Specific PCRs for rcAAV, human viruses panel (retroviruses, herpesviruses, hepatitis viruses, B19), bovine polyoma • Product-enhanced reverse transcriptase (PERT) assay for retroviruses • Transmission electron microscopy for broad general screening

13. Tests for Adventitious Agents 2 • In addition to the usual tests, additional tests were done for: • Product-enhanced reverse transcriptase (PERT) assay for retroviruses under induction conditions • Bovine & porcine circoviruses • Mouse antibody production test (usual test for rodent cell lines commonly used for monoclonal antibody production) • These were also negative

14. Tests for Tumorigenicity • 2 MCB and an EOP cell bank were tested to determine a 50% tumor producing dose (TPD50) • Culture media – negative control • HT1080 (another human tumor line) – positive control • Doses from 10^0 to 10^7 cells/nude mouse into 10 mice/group

15. TPD50 values determined Reported TPD50 value for HeLa cells from FDA: 10^4.9

16. Tests for Oncogenicity (Lysates) • Cell lysates (to release theoretical oncogenic or latent agents present in cells) – equivalent of >10^7 cells injected into newborn rats or hamsters, which were followed for 150 days • No tumors were observed in any group

17. Tests for Oncogenicity (DNA) • Cellular DNA (>200 mcg)/nude mouse was injected into 30 animals/group and they were followed for 150+3 days • 4 groups received either vehicle (Tris buffer, negative control), MRC-5 cell DNA (negative control), HeLa S3 MCB DNA, or T3B12-5B EOPC DNA • No tumors were observed in any group

18. Risk Assessment for Transmissible Spongiform Encephalopathy Agents • Cells grown in the presence of bovine serum could theoretically have been exposed to the TSE agent of cattle (bovine spongiform encephalopathy – BSE) • Calculated dilution factor from theoretical exposure to contaminated lot of serum in legacy • Cells could theoretically express the cellular gene (PrPC) that can convert to the human TSE agent • Sequenced cellular gene looking for familial mutations associated with disease • Western blot for protease-resistant form of PrP

19. Results (TSE risk) • Calculated risk is less than 1 infectious dose of BSE agent per 10^19 doses of vaccine had there actually been BSE exposure in legacy • No protease-resistant PrP protein detected (human TSE or BSE) • limit of detection 1 ng/4.7x10^5 cells HeLa S3 MCB, 5.8x10^5 cells T3B12-5B MCB, 5.8x10^5 cells T3B12-5B EOPC, or 1.2X10^11 DRP product • Sequence normal at known mutation points, heterozygous gene (normal full-length, truncated at known octapeptide repeat region – R3-R4)

20. Product Characterization • Residual cells • Residual host cell DNA • Residual host cell proteins • E6, E7 gene DNA • Residual helper virus • Biosafety (bacteria, fungi, mycoplasmas, viruses)

21. Host Cell DNA in Product • Residual host cell DNA from a preparation of AAV vaccine was assessed by PCR for the E6 gene sequences (LLOD – 24.6 copies/mL) • Total cellular DNA was measured as 60 pg/human dose of vaccine (3x10^11 DNase-Resistant Particles) below limit of 10 ng/dose limit recommended originally by WHO • E6 gene fragment residuals unmeasurable (below limit of detection of assay) • Product is treated with Benzonase, which cuts up the DNA to sizes less than an intact gene size (potentially less than 100 bp, which was the size of the PCR product sought)

22. Cell Clearance in Product • Manufacturing process included 6 filter steps – calculated removal clearance of 10^22 (10^8 cells to make a dose of product – giving 10^14-fold margin-of-safety per dose)

23. Viral Clearance in Product • Ad5 – Relevant Virus – Medium Non-Enveloped Virus • BVDV – Model Virus – Small Enveloped Virus • SV40 – Model Virus – Small Non-Enveloped Virus – Moderately resistant to Inactivation

24. How This Might Differ From Cell Substrates Already in Use? • Cells do cause tumors in animals at doses less than 10^7 cells/animal • Residual Cell DNA could contain unmeasurable amounts of oncogene DNA (HPV genes), although this DNA would be degraded to sizes less than that of an intact gene

25. Why Should This Be Safe or Suitable? • Product purification processes remove intact cells with large margin of safety • Processes degrade residual cellular nucleic acids and purify away cellular residuals (NAs and proteins) • Processes remove model viruses with significant margin of safety (were there undetected viruses in the cell line, the processes used have the potential to clear these as they do model viruses) • Final product does not contain HeLa cells & the majority of cellular residuals are processed away, similar to other biologicals & significantly more thoroughly than some traditional licensed vaccines made in primary cells or diploid cells

26. Conclusion • AAV vectored vaccines can be successfully made in engineered packaging cell lines based on HeLa cells, which have been banked & thoroughly characterized • These products can be thoroughly characterized for cellular residuals • Such vaccine candidates may be safely given to humans • Clinical experience with preventive vaccines to date is limited to ~150 healthy adults

27. Summary • Cell lines derived from human tumors can be banked & well characterized prior to use in product manufacture • Such cell lines propagate well in culture & often are readily genetically engineered to support growth of viral vectors that require genes for complementation or packaging • This capability can mean the difference between successful production of or inability to produce vaccines that have the potential to fill unmet public health needs • Such well characterized cell lines, like other non-tumor-derived cell lines, can be safe & suitable cell substrates for the production of preventive vaccines

28. Other NIAID-supported projects using tumor cell lines • NIAID also supports a project for bio-defense & a potential project for HIV using A549 cells from PaxVax • NIAID is supporting and/or collaborating on two separate AAV vector projects to use gene transfer to “passively” immunize individuals with HIV-specific antibody genes that express broadly cross-neutralizing Ab

29. Acknowledgements • Michael Pensiero, team lead & program officer for project • Phil Johnson, Childrens Hospital of Philadelphia, vaccine champion • PervinAnklesaria, formerly Targeted Genetics, Inc. Regulatory Affairs (now at B&M Gates Foundation) • International AIDS Vaccine Initiative (funded original HeLa cell line characterization) • Funded by NIH HIV Vaccine Design & Development Team HHSN266200500008C

30. References • Tatalick LM et al., 2005 “Safety Characterization of HeLa-based cell substrates used in the manufacture of a recombinant adeno-associated virus-HIV vaccine” Vaccine 23:2628-38 • Lea JS et al., 2007 “Silencing of HPV18 Oncoproteins with RNA Interference Causes Growth Inhibition of Cervical Cancer Cells” Reproductive Sciences 14:20-28