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History and Characterization of the A3.01 Cell Line (derived from CEM) and its Tumorigenic and Oncogenic Evaluation September 19, 2012 Vaccines and Related.

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Presentation on theme: "History and Characterization of the A3.01 Cell Line (derived from CEM) and its Tumorigenic and Oncogenic Evaluation September 19, 2012 Vaccines and Related."— Presentation transcript:

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 & oncogenic evaluations Conclusion

3 Introduction A3.01 cell -T- lymphocyte originated from human -Firstly introduced for vaccine manufacture Killed-whole HIV/AIDS vaccine (SAV001-H) -Genetically modified 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 HIV

4 ChallengesSolutionsResults Safety Genetic modificationAvirulent InactivationNon infectious Production Genetic modificationHigh production yield Process developmentLarge production Immunogenicity AT-2 inactivation Minimal modification of viral protein Gamma irradiation Sumagen vaccine, SAV001-H is a genetically modified and double inactivated, safe and effective HIV vaccine. Challenges for Inactivated Vaccine Production AT-2: Aldrithiol 2

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

6 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. PBMC: Peripheral blood mononuclear cell Selection of Cell Substrate HIV-WT HIV-  Nef HIV MS SAV- HIV

7 A3.01 cell was CD4 receptor positive and sensitive for HIV infection. History of A3.01 Cell Line CategoriesA3.01 cellReference Original DonorHuman G.E.Foley et al, 1965 Tissue OriginPeripheral blood (T-Cell) Ethic/geographical OriginCaucasian/North America Age4 year old GenderFemale General Physical ConditionAcute Lymphoblastic Leukemia Cultivation History8-Azaguanine T. Folks et al, 1985 Culture MediaRPMI %; fetal bovine serum 10% Genetically ModificationHAT sensitive Gene MarkerCD4 Master Cell Bank (MCB) Under the cGMP compliance at CMO in the USA Sumagen, 2007 HAT: hypoxanthine/aminopterin/thymidine, CMO: contract manufacturing organization

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

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

10 Negative No adventitious agent was detected in Sumagen A3.01 MCB. Sterility and Mycoplasma tests In vivo adventitious 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 General Adventitious Agent Tests MRC-5 (Lung, diploid, human), HeLa (Human cervical cancer), Vero (monkey kidney epithelial)

11 No Adventitious agent was detected in Sumagen A3.01 MCB. 1.Retrovirus detection by RT assay 2.TEM assay for detection of any Virus 3.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) 4.Additional detection for human polyoma virus by PCR (BK/JC and WU/Ki) 5.Detection of adventitious agent by transcriptome analysis Target Specific Adventitious Agent Tests On Going Negative

12 Evaluation of Tumorigenicity with Intact A3.01 Cell Sumagen A3.01 MCB showed tumorigenic phenotype in high concentrated cell suspension. Concentration ( Cells/0.2mL) 1x10 3 1x10 5 1x10 7 Injection (10 animals) Adult athymic nude mice Results (4 months observation ) No tumor formation on low concentration Ten percents of tumor observed on middle concentration Ninety percents of tumors observed on high concentration

13 Concentration Lysate Equivalent of 10 7 cells/animal DNA 100  g/animal Injection (15~20 animals) - Newborn nude mice - Newborn rats - Newborn hamsters Results (4 months observation) No tumor observations in macro and histopathology examination Evaluation of Oncogenicity with Cell Lysate and DNA Sumagen A3.01 MCB has no oncogenic phenotype.

14 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. Conclusion


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