1. Vaccines and Related Products FDA Advisory Committee Meeting
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 Killed-whole HIV/AIDS vaccine (SAV001-H)
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. Minimal modification of viral protein
Challenges for Inactivated Vaccine Production
Challenges
Solutions
Results
Safety
Genetic modification
Avirulent
Inactivation
Non infectious
Production
High production yield
Process development
Large 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.
AT-2: Aldrithiol 2 4

5. Replacement 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, , 1994
NSS: Natural signal sequence, MSS: Melittin signal sequence 5

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 A3.01 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 %; 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
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 tumorigenicity tests with live cell and oncogenicity tests with cell lysate/DNA
Unknown characteristics
No information of adventitious agents
Tumorigenic and oncogenic properties

9. Characteristics of A3.01 Cell
Analysis
Human origin
Karyotypically abnormal
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 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
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 transcriptome analysis
On Going

12. Evaluation of Tumorigenicity with Intact A3.01 Cell
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 observed on middle concentration
Ninety percents of tumors observed on high concentration
Sumagen A3.01 MCB showed tumorigenic phenotype in high concentrated cell suspension.

13. No tumor observations in macro and histopathology examination
Evaluation of Oncogenicity with Cell Lysate and DNA
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 observations in macro and histopathology examination
Sumagen A3.01 MCB has no oncogenic phenotype.

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.