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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 Products FDA Advisory Committee Meeting
Sumagen Co., Ltd
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
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 stud-
ies
- 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 ongoingSAV001-H: Sumagen AIDS Vaccine 001 - HIV
Challenges Solutions Results
SafetyGenetic modification Avirulent
Inactivation Non infectious
ProductionGenetic modification High production yield
Process development Large production
Immunogeni-city
AT-2 inactivation Minimal modification of viral proteinGamma irradiation
Sumagen vaccine, SAV001-H is a genetically modified and dou-ble inactivated, safe and effective HIV vaccine.
Challenges for Inactivated Vaccine Production
AT-2: Aldrithiol 2
Li, Luo, Thomas & Kang, Virology, 204, 266-278, 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
Genetically modified HIV was infected 4 dif-ferent 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- DNef HIV MS SAV- HIV
A3.01 cell was CD4 receptor positive and sensitive for HIV infection.
History of A3.01 Cell Line
Categories A3.01 cell Reference
Original Donor Human
G.E.Foley et al, 1965
Tissue Origin Peripheral blood (T-Cell)
Ethic/geographical Origin Caucasian/North America
Age 4 year old
Gender Female
General Physical Condition Acute Lymphoblastic Leukemia
Cultivation History 8-Azaguanine
T. Folks et al,1985
Culture Media RPMI 1640 90%; fetal bovine serum 10%
Genetically Modification HAT sensitive
Gene Marker CD4
Master Cell Bank (MCB) Under the cGMP compliance at CMO in the USA Sumagen, 2007
HAT: hypoxanthine/aminopterin/thymidine, CMO: contract manufacturing organization
Unknown characteristics
No information of adventi-tious agents
Tumorigenic and oncogenic properties
Potential Risks of A3.01 as a New Cell Substrate
Potential Risks
Assessments
Characterization studies
General and target specific adven-
titious agent tests
In vivo tumorigenicity tests with
live cell and oncogenicity tests
with cell lysate/DNA
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
AnalysisCharacteristics
PrP: Prion Protein
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 detec-tions
General Adventitious Agent Tests
MRC-5 (Lung, diploid, human), HeLa (Human cervical cancer), Vero (monkey kidney epithelial)
No Adventitious agent was detected in Sumagen A3.01 MCB.
1. Retrovirus detection by RT assay2. TEM assay for detection of any Virus3. 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 tran-scriptome analysis
Target Specific Adventitious Agent Tests
On Going
Negative
Evaluation of Tumorigenicity with Intact A3.01 Cell
Sumagen A3.01 MCB showed tumorigenic phenotype in high concentrated cell suspension.
Concentration
(Cells/0.2mL)
1x103
1x105
1x107
Injection(10 animals)
Adult athymic
nude mice
Results (4 months observation)
No tumor formation on low concentration
Ten percents of tumor ob-served on middle concentra-
tion
Ninety percents of tumors observed on high concentra-
tion
Concentration
Lysate
Equivalent of 107
cells/animal
DNA
100mg/animal
Injection (15~20 animals)
- Newborn nude mice
- Newborn rats
- Newborn hamsters
Results (4 months observation)
No tumor observa-tions in macro and histopathology ex-
amination
Evaluation of Oncogenicity with Cell Lysate and DNA
Sumagen A3.01 MCB has no oncogenic phenotype.
•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