EMEA/ICH WORKSHOP ON VIRAL/VECTOR SHEDDINGin conjunction with:

The XVth Annual Congress of the European Society of Gene and Cell Therapy

Vector Shedding and Biodistribution Following Intratumoral Delivery of Adenoviral p53 Gene (ADVEXIN) in Patients with

Advanced Non-small Cell Lung Cancer

Toshiyoshi Fujiwara, Noriaki Tanaka

Center for Gene and Cell TherapyDepartment of Surgery

Okayama University Graduate School

October 30, 2007 Rotterdam, The Netherlands

A Phase I Study of Adenovirus-Mediated Wild-Type p53 Gene Transfer in Patients with Advanced

Non-Small Cell Lung Cancer

Okayama University Hospital

Human Gene Therapy Protocol

Chromosome 17

DNA

Transcriptional Factor

Cho Y, et al.: Science 265, 346-355 (1994)

S-46 P

S-15 P

S-20 P

SG1/S

G2/M

G1M

G2

Cell Cycle Control Apoptosis

DNA Repair

GADD45

p53R2

Angiogenesis

p53

Tumor Suppressor p53 Gene

Accura Biopsy SystemTM : 19G co-axial introducer needle21G injection needle

CT-guided Injection

Ad5CMV-p53Advexin®

BronchoscopicInjection

Advexin(3 ml)

23G OLYMPUSInjection needle

0 10 20 30 40 50 60 70 80 90 100 mu

Human wild-type p53 expression cassette

CMVpromoter

wt-p53 SV40 polyA

AdE1 AdE3

Adenoviral p53 Gene Therapy for Non-Small Cell Lung Cancer

Institutions

Okayama Univ. Hosp.Dept. of Surgery

Tohoku Univ.Institute of Development, Aging, and CancerRespiratory Oncology and Molecular Medicine

Jikei Univ. Sch. of Med.Institute of DNA Med.Dept. of Internal Med. (IV)Dept. of Surgery (I)

Tokyo Med. Univ.First Dept. of Surgery

1010 PFU

1011 PFU

109 PFU

Clinical Trial

ADVEXIN ADVEXIN+ CDDP

Total Pt. No. 15 cases

Multicenter Phase I Study

Characteristics of patients that received injections of Advexin®

PT.#

01

02

03

04

05

06

07

08

09

10

11

12

13

14

15

Sex

Male

Male

Male

Female

Male

Male

Male

Male

Male

Male

Male

Male

Male

Male

Male

Age

58

58

66

46

56

54

71

52

66

51

55

61

62

52

62

Histology

squamous

squamous

squamous

adenocar-cinoma

squamous

squamous

squamous

squamous

squamous

adenocar-cinoma

adenocar-cinoma

squamous

squamous

squamous

squamous

Location oftumor

carina

left lowerlobe

right mainstembronchus

left upperlobe

right lowerlobe

left upper lobe

left upper lobe

right lowerlobe

left upperlobe

right lowerlobe

right upper lobe

right upper lobe

left mainstembronchus

left upper lobe

right upper lobe

Prior therapy

chemotherapyradiotherapy

chemotherapyradiotherapy

surgery, laserchemotherapy

chemotherapy

chemotherapyradiotherapy

chemotherapyradiotherapy

chemotherapyradiotherapy

chemotherapyradiotherapy

surgery, laserchemotherapy

chemotherapy

chemotherapy

chemotherapyradiotherapy

chemotherapyradiotherapy

chemotherapy

chemotherapyradiotherapy

Method ofinjection

BF

BF/CT

BF

CT

BF/CT

CT

BF

BF

CT

CT

BF/CT

CT

BF

BF

BF

Viral dose+/- CDDP

109 PFU

109 PFU

109 PFU

109 PFU+ CDDP

109 PFU+ CDDP

109 PFU+ CDDP

1010PFU

1010 PFU

1010 PFU

1010 PFU+ CDDP

1010 PFU+ CDDP

1010 PFU+ CDDP

1011PFU

1011 PFU

1011 PFU

Number ofcourses

14(485)

9(340)

4(109)

10(619)

3(165)

2(207)

2(178)

1(115)

4(590)

1(150)

2(3y9m)

2(390)

1(88)

4(2y4m)

4(94)

TNM Staging

cT4N0M0 St IIIB

cT4N2M0 St IIIB

cT2N0M0 St IB(pre-operative)

cT2N3M1 St IV

cT4N1M0 St IIIB

cT3N2M0 St IIIB

cT4N1M0 St IIIB

cT2N2M0 St IIB

cT2N0M0 St IB(pre-operative)

cT2N2M1 St IV

cT4N3M0 St IIIB

cT4N2M0 St IIIB

cT4N3M0 St IIIB

cT2N2M0 St IIIA

cT4N2M0 St IIIB

(Tohoku Univ.)

(Okayama Univ.)

(Okayama Univ.)

(Tokyo Jikei Univ.)

(Tokyo Med. Univ.)

(Tokyo Med. Univ.)

(Okayama Univ.)

(Tohoku Univ.)

(Tokyo Med. Univ.)

Adverse events associated with Advexin® gene therapy in patients with non-small cell lung cancer

mild moderate severe

gradesadverse events

Total (100%) n = 12

fever 1 8 2 11（91.7%）hemoptysis 7 1 0 8（66.7%）dyspnea 6 0 0 6（50.0%）nausea 2 3 0 5（41.7%）vomiting 3 2 0 5（41.7%）cough 4 0 0 4（33.3%）constipation 3 0 0 3（25.0%）back pain 2 1 0 3（25.0%）obst. pneumo. 0 2 1 3（25.0%）chest pain 3 0 0 3（25.0%）diarrhea 3 0 0 3（25.0%）stomatitis 2 0 0 2（16.7%）shoulder pain 2 0 0 2（16.7%）infection 1 1 0 2（16.7%）anemia 0 0 2 2（16.6%）pneumonia 0 1 0 1（8.3%）atelectasis 0 0 1 1（8.3%）abd. fullness 1 0 0 1（8.3%）dysesthesia 1 0 0 1（8.3%）peumothorax 1 0 0 1（8.3%）hematoma 1 0 0 1（8.3%）fatigue 1 0 0 1（8.3%）

pituita 1 0 0 1（8.3%）arm pain 0 1 0 1（8.3%）pleu. effusion 1 0 0 1（8.3%）anal bleeding 1 0 0 1（8.3%）dullness 1 0 0 1（8.3%）tooth pain 1 0 0 1（8.3%）lumbsgo 1 0 0 1（8.3%）bronchitis 0 1 0 1（8.3%）dysthymia 1 0 0 1（8.3%）paracusis 0 2 0 1（8.3%）inj. site pain 1 0 0 1（8.3%）finger abscess 1 0 0 1（8.3%）weight loss 0 0 1 1（8.3%）

gradesadverse events

Total (100%) n = 12mild moderate severe

Biodistribution Data of Advexin®: gargle(DNA-PCR analysis)

Pt #1

Pt #2

Pt #3

Pt #4

Pt #5

Pt #7

Pt #6

Pt #8

Pt #9

Pt #10

Pt #11

Pt #12

Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

123456

123456

1234

123456

123

12

12

1234

12

12

(Tohoku)

(Jikei Med.)

(Tokyo Med.)

(Tokyo Med.)

(Okayama)

(Okayama)

(Okayama)

(Okayama)

(Okayama)

(Okayama)

(Okayama)

(Okayama)

positivenegative

Pt #1

Pt #2

Pt #3

Pt #4

Pt #5

Pt #7

Pt #6

Pt #8

Pt #9

Pt #10

Pt #11

Pt #12

Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

123456

123456

1234

123456

123

12

12

1234

12

12

(Tohoku)

(Jikei Med.)

(Tokyo Med.)

(Tokyo Med.)

(Okayama)

(Okayama)

(Okayama)

(Okayama)

(Okayama)

(Okayama)

(Okayama)

(Okayama)

Biodistribution Data of Advexin®: urine(DNA-PCR analysis)

positivenegative

Pt #3

Pt #2

Pt #1

Pt #4

Pt #8

Pt #6

Pt #5

Pt #9

+ +

+ + + +

+ + + +

+ + + + +

+ +

+ + +

Biodistribution Data of Advexin®: plasma(DNA-PCR analysis & CPE assay)

0 306090 0 306090 0 306090 0 306090 0 306090 0 3060901

10

100

1000

10000Ti

ter (

PFU

)

Time after Ad5CMV-p53 Injection (min)0 306090 0 306090 0 306090

1

10

100

1000

10000

Tite

r (P

FU)

0 306090 0 306090 0 306090 0 306090 0 306090 0 3060901

10

100

1000

10000

Tite

r (P

FU)

0 306090 0 3060901

10

100

1000

10000

Tite

r (P

FU)

0 306090 0 306090 0 306090 0 3060901

10

100

1000

1000020000

Tite

r (P

FU)

0 3060 900 3060 901

10

100

1000

10000

1

10

100

1000

10000

Tite

r (PF

U)

Tite

r (PF

U)

0 306090 0 306090 0 306090 0 306090 0 306090 0 3060901

10

100

1000

10000

Tite

r (P

FU)

0 306090 0 306090 0 306090 0 3060901

10

100

1000

10000

Tite

r (P

FU)

0 306090 0 3060901

10

100

1000

10000

Tite

r (P

FU)

0 306090 0 3060901

10

100

1000

10000

100000

Tite

r (P

FU)

0 30 60 901

10

100

1000

10000Ti

ter (

PFU

)

Time after ADVEXIN Injection (min)

TF-03-36

Pt #7

Pt #10

+

Pt #11

+ + + + + +

Pt #12

+

(Tohoku)

(Jikei Med.)

(Tokyo Med.)

Biodistribution Data of Advexin® : plasma(DNA-PCR analysis & CPE assay)

0 306090 0 3060901

10

100

1000

10000

100000

Tite

r (P

FU)

Patient #3　(Okayama)

Autopsy samples (9/25/99) : 25 days after the 4th Advexin injection (8/31/99)

・Tumor (+) ・Liver (-)・Distant LN (-) ・Kidney (-)・Testis (-)

Patient #7　(Tohoku)

Autopsy samples (12/9/00) : 151 days after the 2nd Advexin injection (7/12/00)

・Tumor (+) ・Liver (-)・Proximal LN (+) ・Kidney (-)・Testis (-)

Biodistribution Data of Advexin®: autopsy samples (DNA-PCR analysis)

Antibodies in the Serum after Advexin® Administration

Pt No. / Initial

Pt #1 / F-A

Pt #2 / T-M

Pt #3 / S-I

Pt #4 / N-M

Cycle

pre1234567

finalpre123456

finalpre123

pre123456

final

Anti-p53

( - )( - )( - )( - )( - )( - )( - )( - )( - )( - )( - )( - )( - )( - )( - )( + )( - )( - )( - )( - )( - )( - )( + )( + )( + )( - )( + )( + )( + )

Anti-Ad(U/ml) x 1000

17.7126.4629.6535.2470.6351.3556.46

62.4712.1631.0343.5365.8356.8276.4755.03

10.3622.8223.8624.6313.1448.6814.9818.10

30.1148.94

Ad5 Neutralisation

Assay20

12801280256012801280256025605120< 20320320640320160160340< 20204020320160128012801280128051202560

Enzyme Immuno AssayPt No. / Initial

Pt #5 / Y-T

Pt #6 / M-H

Pt #7 / I-S

Pt #8 / Y-K

Pt #9 / I-H

Pt #10 / T-Y

Pt #11 / K-K

Pt #12 / K-K

Cycle

pre12

pre1

pre1

pre1

pre123

pre1

pre12

pre123

Anti-p53

( - )( - )( - )

( + )( + )

( - )( - )( - )( - )( - )( - )

( - )( - )

( + )( + )( + )( - )( - )( - )( - )

Anti-Ad(U/ml) x 1000

118.378.49

89.815

5.1955.85

3.149114.3114.37827.770142.68177.93

4.78468.02

8.839370.69391.760.8859.49566.8481.59

Ad5 Neutralisation

Assay40

> 204802560< 202560

2020480

2051203205120

1024010240

40>20480

<2080

5120<2016025601280

Enzyme Immuno Assay

(Jikei Med.)

(Tokyo Med.)

(Okayama)

(Tohoku)

Pt # 01

Pt # 02

Pt # 03

Pt # 04

Pt # 05

Pt # 06

Pt # 07

Pt # 08

Pt # 09

Pt # 10

Pt # 11

Pt # 12

cycle01 02 03 04 05 06 07 08 09 10 11 12 13

48 hr

109 PFU +CDDP

109 PFU

1010PFU +CDDP

1010 PFU

(Tohoku)

(Okayama)

(Okayama)

(Jikei)

(Tokyo Med.)

(Tokyo Med.)

p53 mRNA Expression in Tumor Tissues Following Advexin® Administration (RT-PCR Analysis)

positivenegative

Best Response of Indicated Lesion Assessment of Advexin®

for Non-Small Cell Lung Cancer

109PFU 109

PFU+CDDP 1010 1010 Total

3 3 3 3 15PR 1 1SD 1 3 1 3 10ResponsePD 1 1 2

Pretreatment5 months

After 4 cycles

Case#01

Pretreatment5 months

After 4 cycles

Case#04

12 monthsAfter 11 cycles

Case#10

Before chemotherapy

BeforeADVEXIN®

After ADVEXIN®

(March，2001)

After ADVEXIN®

15 monthsAfter radiotherapy

13 months(June, 2002)

(Tokyo Med. Sch.)

PFU PFU+CDDP

Evaluable PatientsTreatments 1011

3

2

PFU

* *

*One patient was not evaluable because their tumor sizes could not be measured due to obstructive pneumonia.

Lessons Learned from Phase I Study of Advexin® Gene Therapy

Safety- Vector-related adverse events were minimal. Repeated intratumoral injections of Advexin up to a dose of 1011 PFU could be well tolerated.

Biodistribution- Results of plasma and aerodigestive secretion analysis confirmed transient systemic spread of the vector; however, no vector dissemination-related toxicity was observed. Transgene expression was detected in the presence of neutralizing anti-adenovirus antibody.

Clinical Benefit- Evidence of clinical antitumor activity, including tumor regression, tumor growth stabilization, and decrease of tumor markers, was observed in some patients. Note the Advexin-mediated radiosensitization, suggesting the clinical feasibility of Advexin gene therapy.

Phase I/II Adenoviral p53 Gene Therapy for Chemoradiation Resistant Advanced Esophageal

Squamous Cell Carcinoma

Chiba University Graduate School of Medicine

Shimada H, Matsubara H, Shiratori T, Shimizu T, Myazaki S, Okazumi S, Nabeya Y, Shuto K, Hayashi H, Tanizawa T, Nakatani Y, Nakase H, Kitada M, Ochiai T

Human Gene Therapy Protocol

1 64 M CR llB E 5 15 x 1011 2

PatientNo.

AgeGender

LocalResponse

to CRT

UICCStage

InjectionArea

Exon Site 0f P53

mutation

Viral particles

Treatmentcycles

2 71 M PR lll E 7 10 x 1011 53 62 M SD lVA E negative 15 x 1011 34 78 M CR lVA E 8 10 x 1011 25 66 M CR llA E 7 10 x 1011 36 60 M SD lVB E 7 20 x 1011 17 67 F SD lVA E 5 10 x 1011 28 58 M SD llA E 6 10 x 1011 29 48 M PR lVA E 7 25 x 1011 410 77 M NA lVA N negative 25 x 1011 2

Characteristics of Patients with Esophageal SCC with Advexin®

CR; complete response, PR; partial response, SD; stable disease, NA; not assessedE; Esophagus, N; lymph nodes

Pt #1

1 3 1 3

FecesGargleUrinePlasma

Day 1 7 14 21 28 7 14 21 28

28Tumor biopsy

1 3 1 3 1 3 1 3

Day 1 7 14 21 28 7 14 21 28 7 14 21 28

1 3

7 14 21 28 7 14 21 28

FecesGargleUrinePlasma

Tumor biopsy 28

1 3 1 3 1 3

Day 1 7 14 21 28 7 14 21 28

FecesGargleUrinePlasma

Tumor biopsy

7 14 21 28

Pt #2

Pt #3

1 2 courses

1 2

1 2

3

3 courses

4 5 courses

Biodistribution Data of Advexin®

Day 0 5 7 12 5 7 12 5 7

Pt #4

Pt #7

Pt #10

Pt #5

Pt #6

Pt #8

Pt #9

12 5 7 12

1 2 3 4クール

FecesGargle

FecesGargle

FecesGargle

FecesGargle

FecesGargle

FecesGargle

FecesGargle

Biodistribution Data of Advexin®

Lessons Learned from Phase I/II Study of Advexin® Gene Therapy for Esophageal Cancer

-Transient shedding of the virus into feces and gargle could be detected in some of the treated patients; however, urine rarely contained the virus.

-Systemic spread of the virus could not be detected in plasma 24 hours after virus injection, although the lung study demonstrated a transient dissemination of the virus just after virus injection.

-Most of biodistribution data in the esophageal study are consistent with those in the lung study.

Okayama University

Toshiyoshi Fujiwara, M. D., Ph. D.

Noriaki Tanaka, M. D., Ph. D.

Shunsuke Kagawa, M. D., Ph. D. Futoshi Uno, M. D., Ph. D.

Center for Gene and Cell Therapy

Toru Kojima, M. D.Shinji Kuroda, M. D.Shuya Yano, M. D.

(Oncolys BioPharma, Inc.)Yuichi Watanabe, Ph. D.Hideki Onimatsu, Ph. D.Yuri Hashimoto Masaaki Ouchi

Kanazawa University

Satoru Kyo, M. D., Ph. D.Masaki Inoue, M. D., Ph. D.

National Institute of Biomedical Innovation

Hiroyuki Mizuguchi, Ph. D.

Oncolys BioPharma, Inc.

Yasuo UrataKatsuyuki NagaiHitoshi Kawamura, Ph. D.Daiju Ichimaru

Acknowledgements

The University of TexasM. D. Anderson Cancer Center

Jack A. Roth, M. D.Stephen G. Swisher, M. D.

Bingliang Fang, M. D., Ph. D.

Ryo Sakai, M. D.

Elizabeth A. Grimm, Ph. D.

Introgen Therapeutics, Inc.

Sunil Chada, Ph. D.Peter M. Clarke, Ph. D.David G. Nance