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History and Characterization of the A3.01 Cell Line ( derived from CEM) and its Tumorigenic and Oncogenic Evalua PowerPoint Presentation
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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

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slide1

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
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 & oncogenicevaluations

  • Conclusion
introduction
Introduction
  • A3.01 cell
    • T- lymphocyte originated from human
    • Firstly introduced for vaccine manufacture
  • Killed-whole HIV/AIDS vaccine (SAV001-H)
    • Geneticallymodified 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 001 - HIV

slide4

Challenges for Inactivated Vaccine Production

Sumagen vaccine, SAV001-H is a genetically modified and double inactivated, safe and effective HIV vaccine.

AT-2: Aldrithiol 2

slide5

Replacement of envSignal Sequence

Replacement of envsignal sequence to melittin signal sequence caused the secretion of gp120 increase dramatically.

Li, Luo, Thomas & Kang, Virology, 204, 266-278, 1994

NSS: Natural signal sequence, MSS: Melittin signal sequence

slide6

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

slide7

History of A3.01Cell Line

A3.01 cell was CD4 receptor positive and sensitive for HIV infection.

HAT: hypoxanthine/aminopterin/thymidine, CMO: contract manufacturing organization

slide8

Potential Risks of A3.01 as a New Cell Substrate

Assessments

Potential Risks

  • Characterization studies
  • General and target specific adventitious agent tests
  • In vivo tumorigenicitytests with live cell and oncogenicitytests with cell lysate/DNA
  • Unknown characteristics
  • No information of adventitious agents
  • Tumorigenicand oncogenic properties
slide9

Characteristics of A3.01 Cell

Characteristics

Analysis

  • Human origin
  • Karyotypicallyabnormal
  • Normal PrP gene
  • Lymphoblast-like morphology
  • 28 hours doubling times
  • Isoenzyme analysis
  • Karyotyping
  • PrP genomic sequencing
  • Cellular morphology
  • Growth characteristics

PrP: Prion Protein

slide10

General Adventitious Agent Tests

  • Sterility and Mycoplasma tests
  • In vivoadventitious 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)

slide11

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 transcriptomeanalysis

On Going

slide12

Evaluation of Tumorigenicity with Intact A3.01 Cell

Sumagen A3.01 MCB showed tumorigenic phenotype in high concentrated cell suspension.

slide13

Evaluation of Oncogenicity with Cell Lysate and DNA

Sumagen A3.01 MCB has no oncogenicphenotype.

slide14

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.