vector shedding and biodistribution following intratumoral ...€¦ · 19g co-axial introducer...
TRANSCRIPT
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