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History and Characterization of the A3.01 Cell Line ( derived from CEM) and its Tumorigenic and Oncogenic Evalua

History and Characterization of the A3.01 Cell Line ( derived from CEM) and its Tumorigenic and Oncogenic Evaluation. September 19, 2012 Vaccines and Related Products FDA Advisory Committee Meeting Sumagen Co., Ltd. Contents. Introduction - A3.01 cell

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History and Characterization of the A3.01 Cell Line ( derived from CEM) and its Tumorigenic and Oncogenic Evalua

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  1. History and Characterization of the A3.01 Cell Line (derived from CEM)and its Tumorigenic and Oncogenic Evaluation September 19, 2012 Vaccines and Related Products FDA Advisory Committee Meeting Sumagen Co., Ltd

  2. Contents • Introduction - A3.01 cell - Killed-whole HIV vaccine - Selection of a cell substrate • Characterization of A3.01 cells - History - Risk assessment - General and target specific adventitious agent tests - Tumorigenic & oncogenicevaluations • Conclusion

  3. Introduction • A3.01 cell • T- lymphocyte originated from human • Firstly introduced for vaccine manufacture • Killed-whole HIV/AIDS vaccine (SAV001-H) • Geneticallymodified killed whole HIV/AIDS vaccine and double inactivated by chemical and physical methods • Induce strong humoral and cellular immunity in non-human primate studies • Invented at the University of Western Ontario in Canada and developed by Sumagen Co., Ltd. • Approved for phase I clinical trial from US FDA and the study is ongoing SAV001-H: Sumagen AIDS Vaccine 001 - HIV

  4. Challenges for Inactivated Vaccine Production Sumagen vaccine, SAV001-H is a genetically modified and double inactivated, safe and effective HIV vaccine. AT-2: Aldrithiol 2

  5. Replacement of envSignal Sequence Replacement of envsignal sequence to melittin signal sequence caused the secretion of gp120 increase dramatically. Li, Luo, Thomas & Kang, Virology, 204, 266-278, 1994 NSS: Natural signal sequence, MSS: Melittin signal sequence

  6. Selection of Cell Substrate Genetically modified HIV was infected 4 different T-cell lines to evaluate their ability to produce HIV and A3.01 was found the most reliable cell to produce genetically modified Sumagen-HIV. HIV-WT HIV- DNef HIV MS SAV- HIV PBMC: Peripheral blood mononuclear cell

  7. History of A3.01Cell Line A3.01 cell was CD4 receptor positive and sensitive for HIV infection. HAT: hypoxanthine/aminopterin/thymidine, CMO: contract manufacturing organization

  8. Potential Risks of A3.01 as a New Cell Substrate Assessments Potential Risks • Characterization studies • General and target specific adventitious agent tests • In vivo tumorigenicitytests with live cell and oncogenicitytests with cell lysate/DNA • Unknown characteristics • No information of adventitious agents • Tumorigenicand oncogenic properties

  9. Characteristics of A3.01 Cell Characteristics Analysis • Human origin • Karyotypicallyabnormal • Normal PrP gene • Lymphoblast-like morphology • 28 hours doubling times • Isoenzyme analysis • Karyotyping • PrP genomic sequencing • Cellular morphology • Growth characteristics PrP: Prion Protein

  10. General Adventitious Agent Tests • Sterility and Mycoplasma tests • In vivoadventitious virus detections with cell and supernatant • (Newborn and adult mice and embryonated chicken eggs) • In vitro adventitious virus detection with cell and supernatant • (MRC-5, HeLa, Vero and CEM-A cell lines) • In vitro bovine adventitious virus detections Negative No adventitious agent was detected in Sumagen A3.01 MCB. MRC-5 (Lung, diploid, human), HeLa (Human cervical cancer), Vero (monkey kidney epithelial)

  11. Target Specific Adventitious Agent Tests Retrovirus detection by RT assay TEM assay for detection of any Virus Detection of viruses by PCR (CMV, EBV, HAV, HBV, HCV, HHV-6, HHV-7, HHV-8, HIV-2, HTLV-1, HTLV-2, AAV, and HIV-1) Additional detection for human polyoma virus by PCR (BK/JC and WU/Ki) Negative No Adventitious agent was detected in Sumagen A3.01 MCB. Detection of adventitious agent by transcriptomeanalysis On Going

  12. Evaluation of Tumorigenicity with Intact A3.01 Cell Sumagen A3.01 MCB showed tumorigenic phenotype in high concentrated cell suspension.

  13. Evaluation of Oncogenicity with Cell Lysate and DNA Sumagen A3.01 MCB has no oncogenicphenotype.

  14. Conclusion • The A3.01 cell line is human T-cell line and the best cell line for manufacturing of Sumagen HIV/AIDS vaccine. • No adventitious agent was detected in Sumagen A3.01 MCB. • Sumagen A3.01 MCB has tumorigenic phenotype at high cell concentration; however, there was no oncogenic phenotype in various animal species.

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