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