Presentation on theme: "Vaccines and Related Products FDA Advisory Committee Meeting"— Presentation transcript:
1Vaccines and Related Products FDA Advisory Committee Meeting History and Characterization of the A3.01 Cell Line (derived from CEM) and its Tumorigenic and Oncogenic EvaluationSeptember 19, 2012Vaccines and Related ProductsFDA Advisory Committee MeetingSumagen Co., Ltd
2Contents Introduction - A3.01 cell - Killed-whole HIV vaccine - Selection of a cell substrateCharacterization of A3.01 cells- History- Risk assessment- General and target specific adventitious agent tests- Tumorigenic & oncogenic evaluationsConclusion
3Introduction A3.01 cell Killed-whole HIV/AIDS vaccine (SAV001-H) T- lymphocyte originated from humanFirstly introduced for vaccine manufactureKilled-whole HIV/AIDS vaccine (SAV001-H)Genetically modified killed whole HIV/AIDS vaccine and double inactivated by chemical and physical methodsInduce strong humoral and cellular immunity in non-human primate studiesInvented 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 ongoingSAV001-H: Sumagen AIDS Vaccine HIV
4Minimal modification of viral protein Challenges for Inactivated Vaccine ProductionChallengesSolutionsResultsSafetyGenetic modificationAvirulentInactivationNon infectiousProductionHigh production yieldProcess developmentLarge productionImmunogenicityAT-2 inactivationMinimal modification of viral proteinGamma irradiationSumagen vaccine, SAV001-H is a genetically modified and double inactivated, safe and effective HIV vaccine.AT-2: Aldrithiol 24
5Replacement of env Signal Sequence Replacement of env signal sequence to melittin signal sequence caused the secretion of gp120 increase dramatically.Li, Luo, Thomas & Kang, Virology, 204, , 1994NSS: Natural signal sequence, MSS: Melittin signal sequence5
6Selection 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-WTHIV- DNefHIV MSSAV- HIVPBMC: Peripheral blood mononuclear cell
7History of A3.01 Cell Line Categories A3.01 cell Reference Original DonorHumanG.E.Foley et al, 1965Tissue OriginPeripheral blood (T-Cell)Ethic/geographical OriginCaucasian/North AmericaAge4 year oldGenderFemaleGeneral Physical ConditionAcute Lymphoblastic LeukemiaCultivation History8-AzaguanineT. Folks et al,1985Culture MediaRPMI %; fetal bovine serum 10%Genetically ModificationHAT sensitiveGene MarkerCD4Master Cell Bank (MCB)Under the cGMP compliance at CMO inthe USASumagen, 2007A3.01 cell was CD4 receptor positive and sensitive for HIV infection.HAT: hypoxanthine/aminopterin/thymidine, CMO: contract manufacturing organization
8Potential Risks of A3.01 as a New Cell Substrate AssessmentsPotential RisksCharacterization studiesGeneral and target specific adventitious agent testsIn vivo tumorigenicity tests with live cell and oncogenicity tests with cell lysate/DNAUnknown characteristicsNo information of adventitious agentsTumorigenic and oncogenic properties
9Characteristics of A3.01 Cell AnalysisHuman originKaryotypically abnormalNormal PrP geneLymphoblast-like morphology28 hours doubling timesIsoenzyme analysisKaryotypingPrP genomic sequencingCellular morphologyGrowth characteristicsPrP: Prion Protein
10General Adventitious Agent Tests Sterility and Mycoplasma testsIn 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 detectionsNegativeNo adventitious agent was detected in Sumagen A3.01 MCB.MRC-5 (Lung, diploid, human), HeLa (Human cervical cancer), Vero (monkey kidney epithelial)
11Target Specific Adventitious Agent Tests Retrovirus detection by RT assayTEM assay for detection of any VirusDetection 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)NegativeNo Adventitious agent was detected in Sumagen A3.01 MCB.Detection of adventitious agent by transcriptome analysisOn Going
12Evaluation of Tumorigenicity with Intact A3.01 Cell Concentration(Cells/0.2mL)1x1031x1051x107Injection(10 animals)Adult athymic nude miceResults (4 months observation)No tumor formation on low concentrationTen percents of tumor observed on middle concentrationNinety percents of tumors observed on high concentrationSumagen A3.01 MCB showed tumorigenic phenotype in high concentrated cell suspension.
13No tumor observations in macro and histopathology examination Evaluation of Oncogenicity with Cell Lysate and DNAConcentrationLysateEquivalent of 107 cells/animalDNA100mg/animalInjection(15~20 animals)- Newborn nude mice- Newborn rats- Newborn hamstersResults (4 months observation)No tumor observations in macro and histopathology examinationSumagen A3.01 MCB has no oncogenic phenotype.
14ConclusionThe 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.