Powering Future Oncotherapy

14th Anti-Tumor Drug Development ForumA Novel CTC-Detecting Technique Using
TelomeScan and Its Clinical Applications

Yasuo Urata
CEO and President
Oncolys BioPharma Inc.
February 16, 2013

Powering Future Oncotherapy

Telomere Length is a Limiting Factor for
Cell Replication
Cell senescence

Telomere shortening
14th Anti-Tumor Drug Development Forum

Shay JW and Wright WE, Nat Rev Mol Cell Biol. 1:72-6.

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Powering Future Oncotherapy

Telomerase Activity in Cancer Cells andBiopsies

Cell lines- Telomerase (+) in 98/100 immortal cell lines- Telomerase (-) in 22/22 mortal cell lines

Biopsies- Telomerase (+) in 90/101 human tumors- Telomerase (-) in 50/50 human normal
tissues

Telomerase is an universal marker in all
types of human cancer

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Kim NW, et al. Science. (1994)

3Telomerase and hTERTPowering Future Oncotherapy

NormalCancer

hTERT
GAPDH

Structure of Telomerase Complex
Human telomerase is a complex consisting oftemplate RNA and enzyme subunits including
hTERT, human telomerase reverse
transcriptase.

14th Anti-Tumor Drug Development Forum

Distribution of hTERT
hTERT is expressing in cancer andsome normal stem cells but not innormal somatic cells.

4Structure of TelomeScan
Powering Future Oncotherapy

Telomerase

Green Fluorescent Protein(GFP)

TelomeScan is genetically engineered GFP gene-carrying adenovirus of which
viral replication is under the control of hTERT promoter.
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Powering Future Oncotherapy

Fluorescent Detection of Cancer Cells
by TelomeScan
Normal cells(Telomerase negative)
No virus replication and GFP production

Virus infection

Fluorescence
GFP
TelomeScan

Cancer cells(Telomerase positive)
Virus replication and GFP production

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Cancer Specific GFP Expression byTelomeScan

Powering Future Oncotherapy

SW620; Human Colon Cancer Cells
2h12 h24 h48 h

NHLF; Normal Fibroblast
72 h24 h

72 h120 h

HT29; Human Colon Cancer Cells
2h

12 h

14th Anti-Tumor Drug Development Forum

24 h48 h

72 h

Kishimoto et al., Nat Med. 12:1213-1219. (2006)

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Powering Future Oncotherapy

14th Anti-Tumor Drug Development Forum

Tumor Metastasis and Circulating TumorCells (CTC)

Chaffer CL and Weinberg RA. Science, 331: 1559-1564, 2011.

8Veridex CellSearch System for CTC
detection

Powering Future Oncotherapy

CTCFerrofluid
Anti-EpCAM

WBC
Anti-CD45-APC

EpCAM
ABCD45AB

Nucleus
Nucleus

DAPI

DAPI

CK
Cytokeratin
Anti-CK 8,18,19-PE
CTC is separated by anti-EpCAM

WBC is removed by anti-CD45

The FDA-approved Veridex LLC CellSearch Circulating Tumor Cell Kit is intended for theenumeration of circulating tumor cells (CTC) of epithelia origin (CD45-, EpCAM+, andcytokeratins8, 18+, and/or 19+) in peripheral blood samples.
14th Anti-Tumor Drug Development Forum9Powering Future Oncotherapy

Correlation of Prognosis and CTC
number detected by CellSearch• The assay predicts progression-free survival (PFS) and overall survival (OS) in patientstreated for metastatic breast, colon and prostate cancer.
• A CTC count of 5 cells / 7.5mL blood or greater as determined by the assay is predictiveof shorter PFS and OS for patients with metastatic breast and/or prostate cancer.
• A CTC count of 5 cells / 7.5mL blood or greater is predictive of shorter PFS and OS forpatients with metastatic colon cancer.

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14th Anti-Tumor Drug Development Forum10Restricted Sensitivity of CellSearch
Powering Future Oncotherapy

CTC detection rate of CellSearchTM system in various
metastatic cancer patients
Prostate cancer
Ovarian cancer
Breast cancer
Stomach cancer
Colorectal cancer
Lung cancer
Pancreatic cancer
Healthy person
benign disease•••

Measured range of CTC number (mean ± S.D.); 0~23,618 cells/7.5ml (60 ± 693 cells)
Sample ratio that had more than 2 cells/7.5ml (Ratio); Mean 36%
Sample ratio that had more than 5 cells/7.5ml (Ratio); Mean 24%

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Powering Future Oncotherapy

Sensitivity of TelomeScan in NSCLC CellLines- Quantitative measurement ofGFP expression by FACS
- TelomeScan is feasible todetect CTCs in Lung Cancer
Patients.

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EpCAM and Cytokeratin Independent
GFP expression in NSCLC Cell Lines

Powering Future Oncotherapy

Cytokeratin

EpCAM

HCC827

TelomeScan(GFP)

Cytokeratin

A549

EpCAM

TelomeScan(GFP)
EpCAM
Negative

H358

H460

H2170

H1299

H1975

H522

H322

H661

H1650

H1703

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EpCAM/CKNegative

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High Sensitivity of TelomeScan
Powering Future Oncotherapy

GFP

Number of spiked cells

Bright14th Anti-Tumor Drug Development Forum

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Clinical Application of TelomeScan for
CTC detection and Analyses

Powering Future Oncotherapy

Sample Preparation

CTC Enumeration

T-CAS
TelomeScan
CTCAnalysis
SystemTelomeScan kit

Super early detectionof cancer
Prognosis definition
Evaluation ofmalignancy

CTC isolation and its gene analysis
Lysis

Infection Immunostaining
24 hrsWhole blood

PBMC

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T-GEN
TelomeScan
Genotyping
SystemGene analysis

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Phenotyping of CTC using TelomeScan
amd Immunostaining

Powering Future Oncotherapy

TelomeScan（GFP）

CD45

VimentinE-cadhelin（E-cad）

CTC detection

Elimination of WBC

Mesenchymal marker

Epithelial marker

Bright Field

H661 (NSLCS)

Merge: GFP+CD45+Vim GFP+CD45+E-cad
EMT: Epithelial to Mesenchymal
Transition

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Powering Future Oncotherapy

Multiple Immunostaining Identifies
Characteristics of NSCLC Cell Lines
GFP

CD45

Vimentin

E-Cadherin

MergedBright field

Epithelial

H322A549

Mesenchymal (EMT)

H661H1299

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CTC Detection from NSCLC Patients
Powering Future Oncotherapy

TelomeScan
GFP

PBMC
CD45

Phenotyping marker
VimMerge

Bright field

E-CadPatient#1

CD14Patient#2

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CTC Detection from Patients of Various
Types of Cancer

Powering Future Oncotherapy

Cancer type

TelomeScan
GFP

PBMC

Phenotyping marker

Merge

Bright field

CD45

Pancreas#1
Vim

Pancreas#2

ECadColon

CD14

StomachBreastBone
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CTC Isolation Using Single-Cell Isolator
Powering Future Oncotherapy

Nano-litter scale
manipulator equippedwith Microscope

Define a cell

X-Y-Z coordinates werecalculated and cell collection isachieved automaticallyCapture

A cell is collected and drainedout on PCR tube or glass slide

Deposit

Cells are collected on
cap of PCR tube anddirectly processed forgenetic mutational
analysis
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Genetic Mutational Analysis Test for
EGFR in NSCLC Cell Lines

Powering Future Oncotherapy

Virus infection does not influence CTC
mutational analysis
Cells

PC-9(EGFR exon 19deletionmutation)

Virus MOI（MOI）Result0Exon 19del10

Exon 19del100

Exon 19del

1000

Exon 19del

EGFR mutation was detected fromNSCLC cells
Cells
H358
EGFR
Wild Type
HCC827EGFR
E746A750del# of cell

Result1PCR failed

3WT10WT1

PCR failed

3E746-A750del

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E746-A750del

EGFR mutation was detected from 3 NSCLC cells with PBMC contaminantCells

HCC827
EGFR E746-A750del14th Anti-Tumor Drug Development Forum# of cell

Result3 cancer cells + 12 PBMC

E746-A750del detected

3 cancer cells + 17 PBMC

E746-A750del detected

3 cancer cells + 17 PBMCE746-A750del detected21

Specification of TelomeScan Systems
Powering Future Oncotherapy

T-CAS (TelomeScan CTC Analysis System) is;
1. To detect telomerase-positive tumor cells
2. Not depends on EpCAM enrichment
3. Biological tool to detect only living CTC (L-CTC)

T-GEN (TelomeScan Genotyping System) is;
1. To isolate a single L-CTC
2. To identify allele specific gene mutation of EGFR
3. Gene analysis system without biopsy

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