gene therapy of cancer, methods and protocols

Methods and Protocols
Methods and Protocols
Gene
Therapy
of Cancer
M E T H O D S I N M O L E C U L A R M E D I C I N E
TM
Humana Press
Edited by
Wolfgang Walther
Ulrike Stein
Gene
Therapy
of Cancer
Edited by
Wolfgang Walther
Ulrike Stein
Humana Press
Gene Therapy of Cancer
M E T H O D S I N M O L E C U L A R M E D I C I N E
TM
John M. Walker, S
ERIES
E
DITOR
40. Diagnostic and Therapeutic
Antibodies, edited by Andrew J. T.
George and Catherine E. Urch, 2000
39. Ovarian Cancer: Methods and
Protocols, edited by John M. S.
Bartlett, 2000

38. Aging Methods and Protocols, edited
by Yvonne A. Barnett and Christopher
P. Barnett, 2000
37. Electrically Mediated Delivery of
Molecules to Cells, edited by Mark J.
Jaroszeski, Richard Heller, and
Richard Gilbert, 2000
36. Septic Shock Methods and Protocols,
edited by Thomas J. Evans, 2000
35. Gene Therapy of Cancer: Methods
and Protocols, edited by Wolfgang
Walther and Ulrike Stein, 2000
34. Rotavirus Methods and Protocols,
edited by James Gray and Ulrich
Desselberger, 2000
33. Cytomegalovirus Protocols, edited by
John Sinclair, 2000
32. Alzheimer’s Disease: Methods and
Protocols, edited by Nigel M. Hooper, 1999
31. Hemostasis and Thrombosis
Protocols: Methods in Molecular
Medicine, edited by David J. Perry
and K. John Pasi, 1999
30. Vascular Disease: Molecular Biology
and Gene Therapy Protocols, edited
by Andrew H. Baker, 1999
29. DNA Vaccines: Methods and
Protocols, edited by Douglas B.
Lowrie and Robert Whalen, 1999
28. Cytotoxic Drug Resistance

Mechanisms, edited by Robert Brown
and Uta Böger-Brown, 1999
27. Clinical Applications of Capillary
Electrophoresis, edited by Stephen M.
Palfrey, 1999
26. Quantitative PCR Protocols, edited
by Bernd Kochanowski and Udo
Reischl, 1999
25. Drug Targeting, edited by G. E.
Francis and Cristina Delgado, 1999
24. Antiviral Methods and Protocols,
edited by Derek Kinchington
and Raymond F. Schinazi, 1999
23. Peptidomimetics Protocols, edited by
Wieslaw M. Kazmierski, 1999
22. Neurodegeneration Methods and
Protocols, edited by Jean Harry
and Hugh A. Tilson, 1999
21. Adenovirus Methods and Protocols,
edited by William S. M. Wold, 1998
20. Sexually Transmitted Diseases:
Methods and Protocols, edited by
Rosanna Peeling and P. Frederick
Sparling, 1999
19. Hepatitis C Protocols, edited by
Johnson Yiu-Nam Lau, 1998
18. Tissue Engineering, edited by
Jeffrey R. Morgan and Martin L.
Yarmush, 1999
17. HIV Protocols, edited by Nelson

Michael and Jerome H. Kim, 1999
16. Clinical Applications of PCR, edited
by Y. M. Dennis Lo, 1998
Humana Press Totowa, New Jersey
Edited by
Wolfgang Walther
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
and
Ulrike Stein
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
M E T H O D S I N M O L E C U L A R M E D I C I N E
TM
Gene Therapy
of Cancer
Methods and Protocols
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1. Cancer Gene therapy Laboratory manuals. I. Walther,
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[DNLM: 1. Neoplasms therapy. 2. Gene Therapy methods. QZ 266
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Preface
v
Since the discovery of the molecular structure of genes and the unveiling
of the molecular basis of numerous human diseases, scientists have been fasci-
nated with the possibility of treating certain diseases by transducing foreign
DNA into the affected cells. Initially, it was proposed that the foreign DNA
could either replace defective nonfunctional genes, or code for therapeutic
proteins. This concept has evolved into the rapidly growing field of gene
therapy.
Even though surgery, radiotherapy, and chemotherapy are widely avail-
able and routinely used for cancer treatment, these therapies fail to cure
approximately 50 percent of cancer patients. Therefore, since it is a disease
characterized by aberrant gene expression, cancer has been a target of gene
therapy research since the inception of this treatment modality. Numerous
cancer gene therapy strategies are currently being investigated, including gene
replacement therapy, the regulation of gene expression to modulate immuno-
logical responses to tumors, the direct killing of tumor cells, and direct inter-
ference with tumor growth. In this context, gene transfer systems, tumor-specific
expression vectors, and novel therapeutic genes have been extensively stud-
ied. All these strategies aim for the selective destruction of human malignant
disease while circumventing the destruction of nonmalignant cells and tissues
thereby minimizing toxicity to the patient.
Rapid progress in the field of cancer gene therapy, exemplified by the
vast number of publications in this area, creates a challenging situation for
scientists and clinicians who need to be cognizant of the most recent advances
in gene transfer techniques. This volume of Gene Therapy of Cancer: Meth-
ods and Protocols in the Methods in Molecular Medicine series will provide
researchers with a broad array of methods used to study cancer gene therapy
in both the laboratory and clinical trials. Moreover, several chapters are included
to provide short overviews of specialized gene therapy strategies for the treat-

ment of particular malignancies.
Gene Therapy of Cancer: Methods and Protocols does not provide com-
prehensive reviews of all methodologies currently used for gene therapy of
cancer. Rather the topics we have selected consist of approved procedures,
vi Preface
Wolfgang Walther
Ulrike Stein
current trends, and representative strategies in cancer gene therapy using dif-
ferent classes of therapeutic genes, suppressor genes, antisense oligonucle-
otides, ribozymes, viral- and nonviral-vector systems, and tumor targeting
approaches at the preclinical and, more importantly, at the clinical level. For
cancer gene therapy to be successful in the treatment of human cancers, exten-
sive preclinical evaluation is essential. Therefore, the first part of this book
discusses relevant experiments from preclinical studies followed by clinical
gene therapy protocols in the second part.
Gene Therapy of Cancer: Methods and Protocols should provide practi-
cal guidance for basic and clinical researchers, as well as graduate and post-
graduate students working in the exciting and emerging field of gene therapy.
Contents
Preface
v
Contributors
xi
PART I. EXPERIMENTAL APPROACES TO CANCER GENE THERAPY
A:
Immunotherapy/Tumor Vaccination
1 Immunizing Potential of Cytokine-Transduced Tumor Cells
Mario P. Colombo and Monica Rodolfo 3
2 Particle-Mediated Gene Transfer into Dendritic Cells:
A Novel

Strategy for the Induction of Immune Responses against
Tumor Antigens
Thomas Tüting and Andreas Albers 27
3Cancer Gene Therapy with Heat Shock Protein-65 Gene
Katalin V. Lukacs and Artit Nakakes 49
4Recombinant Vaccinia Virus MVA for Generation and Analysis
of T Cell Responses Against Tumor Associated Antigens
Ingo Drexler, Karl Heller, Marion Ohlmann, Volker Erfle,
and Gerd Sutter 57
B:
Suicide Gene Therapy
5 Selection of Cytochrome
P450
Genes for Use in Prodrug
Activation-Based Cancer Gene Therapy
Jodi E. D. Hecht and David J. Waxman 77
6Construction of P450-Expressing Tumor Cell Lines
Using Retroviruses
Jodi E. D. Hecht, Youssef Jounaidi,
and David J. Waxman 85
7 In Vitro Methods for Evaluation of
P450
-Based
Anticancer Gene Therapy
Jodi E. D. Hecht and David J. Waxman 95
8 Tumor Models for Evaluation of
P450
Gene Therapy In Vivo
Jodi E. D. Hecht, Pamela Schreiber Schwartz,
and David J. Waxman 107

vii
C:
Anti-Oncogene and Suppressor Gene Therapy
9 Intracellular Single-Chain Antibodies for Gene Therapy
Guadalupe Bilbao, Jesus Gomez-Navarro, Keizo Kazano,
Juan Luis Contreras, and David T. Curiel 121
10 Combined Adenoviral Transfer of Tumor Suppressor
and Cell-Cycle Genes for Tumor-Cell Apoptosis
Karsten Brand, Volker Sandig, and Michael Strauss 151
D:
Antisense Gene Therapy
11 Inhibition of Cell Growth by Antisense Oligonucleotides
Targeting the Growth-Related Protein Kinase c-raf
Doriano Fabbro, B. P. Monia, K H. Altmann,
and Thomas Geiger 167
12 IGF-1 Antisense Strategies for Cancer Treatment
Yue Xin Pan and Donald D. Anthony 189
E:
Ribozyme Gene Therapy
13 Anti-
MDR1
Ribozyme Gene Therapy
Takao Ohnuma, Hiroyuki Kobayashi, and Fu-Sheng Wang 207
14 Anti-c-
erb
B2 Ribozyme for Gene Therapy of Breast Cancer
Toshiya Suzuki, Masami Bessho, and Kevin J. Scanlon 247
15 Anti-K-
ras
Ribozyme Adenoviral Vector for Gene Therapy

of Non-Small Cell Lung Cancer
Yu-An Zhang, John Nemunaitis, and Alex W. Tong 261
F:
Delivery Systems and Tumor Targeting
16 Green Fluorescent Protein Retroviral Vector:
Generation
of High-Titer Producer Cells and Virus Supernatant
Wolfgang Uckert, Lene Pedersen, and Walter Günzburg 275
17 HSV-1 Vectors for Gene Therapy of Experimental CNS Tumors
Ulrich Herrlinger, Andreas Jacobs, Manish Aghi,
Deborah E. Schuback, and Xandra O. Breakefield 287
18 Intratumoral Injection of Naked DNA
Jingping Yang 313
19 Cationic Liposome Gene Transfer
Kyonghee Kay Son 323
20 In Vivo Particle-Mediated Gene Transfer for Cancer Therapy
Alexander L. Rakhmilevich and Ning-Sun Yang 331
viii Contents
Contents ix
21 Gene Targeting to Hepatomas (AFP)
Shotaro Tsuruta, Akio Ido, and Shigenobu Nagataki 345
22 Adenovirus-Mediated Targeted Gene Therapy for Breast Cancer
and for Purging Hematopoietic Stem-Cell Sources
Ling Chen 361
23 Chemotherapy-Inducible Vector for Gene Therapy of Cancer
Wolfgang Walther, Ulrike Stein, Robert H. Shoemaker,
and Peter M. Schlag 371
G:
Alternative Approaches in Cancer Gene Therapy
24 Oncolytic Adenoviral Vectors

Ramon Alemany and Wei-Wei Zhang 395
25 Genetically Modified Clostridium for Gene Therapy of Tumors
Mary E. Fox, Marilyn J. Lemmon, Amato J. Giaccia,
Nigel P. Minton, and J. Martin Brown 413
26 Tumor-Targeted
Salmonella
:
Strain Development
and Expression of the
HSV-tK
Effector Gene
David Bermudes, Brooks Low, and John M. Pawelek 419
Part II. Clinical Protocols for Cancer Gene Therapy
A:
Immunotherapy/Tumor Vaccination
27 Ex Vivo Cytokine Gene Transfer in Melanomas
by Using Particle Bombardment
Dirk Schadendorf 439
28 Intratumoral Gene Transfer of the
HLA-B7
Gene
Into Colon Carcinoma Metastases
Evanthia Galanis and Joseph Rubin 453
29 Hybrid Cell Vaccination in Patients with Metastatic Melanoma
Uwe Trefzer, Guido Weingart, Wolfram Sterry,
and Pete Walden 469
B:
Suicide Gene Therapy
30 Retroviral Transfer of the Herpes Simplex Virus-Thymidine
Kinase (

HSV-tK
) Gene for the Treatment of Cancer
Rajagopal Ramesh, Anupama Munshi, Aizen J. Marrogi,
and Scott M. Freeman 479
31 Gene Therapy for Treatment of Brain Tumors
(
HSV-tK
In Vivo Gene Transfer):
A Case Study
Friedrich Weber, Frank Floeth, and Hans Bojar 499
x Contents
32 Gene Therapy of Glioblastoma Multiforme with a Bicistronic
Retroviral Vector Expressing Human IL-2 and
HSV-tk
Giorgio Palù, Massimo Pizzato, Roberta Bonaguro,
and Frederico Colombo 511
33 Intratumoral Gene Transfer of the Cytosine Deaminase Gene
for the Treatment of Breast Cancer
Hardev S. Pandha and Nicholas R. Lemoine 523
C:
Anti-Oncogene and Suppressor Gene Therapy
34 Adenovirus-Mediated Wild-Type
p53
Gene Transfer into Head
and Neck Cancers
Gary L. Clayman, Douglas K. Frank,
and Patricia A. Bruso 537
35 Direct DNA Injection (
p53
) into HCC Tumors

Ragai R. Mitry and Nagy A. Habib 545
36 A Phase II Trial of Intratumoral Injection with Selectively Replicating
Adenovirus (ONYX-015) in Patients with Recurrent, Refractory
Squamous Cell Carcinoma of the Head and Neck
David H. Kirn 559
D:
Antisense Gene Therapy
37 c
-myb
Antisense Oligonucleotide Therapeutics
for Hematologic Malignancies
Selina Luger 577
38 Ovarian Cancer Gene Therapy with
BRCA1
— An Overview
Patrice S. Obermiller and Jeffrey T. Holt 593
39 Methods for Chemoprotection and Chemosensitization:
MDR-1
For Chemoprotection Using Retroviruses to Modify Hematopoietic
Cells and Cytosine Deaminase for Chemosensitization Using
Adenoviral Vectors to Modify Epithelian Neoplastic Cells
Shrinavassan Shrimdkandada, Si Qing Fu, Lian Hua Yin,
Xiang Yang David Guo, Thong Nanakorn, Xue Yan Peng,
Don Dizon, Debbie Lin, Matthew Cooperberg,
Jong Ho Won, and Albert Deisseroth 609
Index
617
x Contents
Contributors
MANISH AGHI • Molecular Neurogenetics Unit, Massachusetts General

Hospital East, Charlestown, MA
A
NDREAS ALBERS • Centrum Somatische Gentherapie at Freie Universität
Berlin, Berlin, Germany
R
AMON ALEMANY • Wallace Tumor Institute, Department of Medicine–
Pulmonary and Critical Care, University of Alabama, Birmingham, AL
K H. A
LTMAN • Oncology Research, Novartis Pharma, Basel, Switzerland
D
ONALD D. ANTHONY • Department of Pharmacology, Case Western Reserve
University, Cleveland, OH
D
AVID BERMUDES • VION Pharmaceuticals, New Haven, CT
M
ASAMI BESSHO • First Department of Internal Medicine, Saitama Medical
School, Saitama, Japan
G
UADALUPE BILBAO • Gene Therapy Program, University of Alabama at
Birmingham, Birmingham, AL
H
ANS BOJAR • Institut für Onkologische Chemie, Heinrich Heine Universität
Düsseldorf, Düsseldorf, German
R
OBERTA BONAGURO • Institute of Microbiology, University of Padova
Medical School, Padova, Italy
K
ARSTEN BRAND • Max-Delbrück-Center for Molecular Medicine, Berlin,
Germany
X

ANDRA O. BREAKEFIELD • Molecular Neurogenetics Unit, Massachusetts
General Hospital East, Charlestown, MA
J. M
ARTIN BROWN • Cancer Biology Research Laboratories, Department of
Radiation Oncology, Stanford University School of Medicine, Stanford, CA
P
ATRICIA A. BRUSO • Department of Head and Neck Surgery, The University
of Texas M.D. Anderson Cancer Center, Houston, TX
L
ING CHEN • Department of Human Genetics, Merck Research Laboratories,
West Point, PA
G
ARY L. CLAYMAN • Department of Head and Neck Surgery, M.D. Anderson
Cancer Center, University of Texas, Houston, TX
xi
xii Contributors
FREDERICO COLOMBO • Institute of Microbiology, University of Padova
Medical School, Padova, Italy
MARIO P. COLOMBO • Experimental Oncology Department, Istituto Nazionale
Tumori, Milano, Italy
J
UAN LUIS CONTRERAS • Gene Therapy Program, Lurleen B. Wallace Tumor
Institute, Comprehensive Cancer Center, University of Alabama at
Birmingham, Birmingham, AL
M
ATTHEW COOPERBERG • Genetic Therapy Program of the Yale Cancer
Center, and the Medical Oncology Section, Department of Internal
Medicine, Yale University School of Medicine, New Haven, CT
D
AVID T. CURIEL • Gene Therapy Program, University of Alabama at

Birmingham, Birmingham, AL
A
LBERT DEISSEROTH • Department of Internal Medicine, Yale University
School of Medicine, New Haven, CT
D
ON DIZON • Genetic Therapy Program of the Yale Cancer Center and the
Medical Oncology Section, Department of Internal Medicine, Yale
University School of Medicine, New Haven, CT
I
NGO DREXLER • Institut für Molekulare Medizin, Bavarian Nordic
Research Institute, Neuherberg, Germany
V
OLKER ERFLE • Institut für Molekulare Medizin, Bavarian Nordic
Research Institute, Neuherberg, Germany
D
ORIANO FABBRO • Oncology Research, Novartis Pharma, Basel, Switzerland
F
RANK FLOETH • Neurochirurgische Klinik, Heinrich Heine Universität
Dusseldorf, Germany
M
ARY E. FOX • Division of Basic Sciences, Fred Hutchinson Cancer
Research Center, Seattle, WA
D
OUGLAS K. FRANK • Department of Head and Neck Surgery, M.D. Anderson
Cancer Center, University of Texas, Houston, TX
S
COTT M. FREEMAN • Clinical Research Oncology, Schering–Plough
Research Institute, Kenilworth, NJ
S
I QING FU • Genetic Therapy Program of the Yale Cancer Center and the

Medical Oncology Section, Department of Internal Medicine, Yale
University School of Medicine, New Haven, CT
E
VANTHIA GALANIS • Division of Medical Oncology, Mayo Clinic, Rochester, MN
T
HOMAS GEIGER • Oncology Research, Novartis Pharma, Basel, Switzerland
A
MATO J. GIACCIA • Cancer Biology Research Laboratories, Department of
Radiation Oncology, Stanford University School of Medicine, Stanford, CA
Contributors xiii
JESUS GOMEZ-NAVARRO • Gene Therapy Program, University of Alabama at
Birmingham, Birmingham, AL
WALTER GÜNZBURG • Institute of Virology, University of Veterinary Sciences,
Vienna, Austria
X
IANG YANG DAVID GUO • Genetic Therapy Program of the Yale Cancer
Center and the Medical Oncology Section, Department of Internal
Medicine, Yale University School of Medicine, New Haven, CT
N
AGY B. HABIB • Liver Surgery Section, Imperial College School of
Medicine Hammersmith Hospital Campus, London, UK
J
ODI E. D. HECHT • Department of Biology, Boston University, Boston, MA
K
ARL HELLER • Institut für Molekulare Medizin, Bavarian Nordic Research
Institute, Neuherberg, Germany
U
LRICH HERRLINGER • Neurologische Universitätsklinik Tübingen,
Tübingen, Germany
J

EFFREY T. HOLT • Department of Cell Biology,Vanderbilt University,
Nashville, TN
A
KIO IDO • Second Department of Internal Medicine, Miyazaki Medical
College, Miyazaki, Japan
A
NDREAS JACOBS • Molecular Neurogenetics Unit, Massachusetts General,
Hospital East, Charlestown, MA
Y
OUSSEF JOUNAIDI • Department of Biology, Boston University, Boston, MA
K
EIZO
K
AZANO
• Gene Therapy Program, University of Alabama
at Birmingham,
Birmingham, AL
D
AVID H. KIRN • Onyx Pharmaceuticals, Richmond, CA
H
IROYUKI
K
OBAYASHI
• Division of Neoplastic Diseases, Department of Medicine,
Mount Sinai School of Medicine, New York, NY
W
EN
-H
WA
L

EE
•
Department of Molecular Medicine, Institute of Biotechnology,
The University of Texas Health Science Center, San Antonio, TX
MARILYN J. LEMMON • Cancer Biology
Research Laboratories,
Department of
Radiation Oncology,
Stanford University School of Medicine, Stanford, CA
NICHOLAS R. LEMOINE • Imperial Cancer Research Fund Oncology Group,
Imperial College School of Medicine at Hammersmith, Hammersmith
Hospital, London, UK
D
EBBIE LIN • Genetic Therapy Program of the Yale Cancer Center and the
Medical Oncology Section, Department of Internal Medicine, Yale
University School of Medicine, New Haven, CT
B
ROOKS LOW • Therapeutic Radiology, Yale University School of Medicine,
New Haven, CT
xiv Contributors
SELINA LUGER • Bone Marrow Transplant Program, Division of Hematology
and Oncology, Hospital of the University of Pennsylvania, Philadelphia, PA
KATALIN V. LUKACS • National Heart and Lung Institute, Imperial College,
London, UK
A
IZEN J. MARROGI • Department of Surgery, Louisiana State University
School of Medicine, New Orleans, LA
N
IGEL P. MINTON • Centre for Applied Microbiology and Research, Porton
Down, Salisbury, Wiltshire, UK

R
AGAI R. MITRY • Liver Surgery Section, Imperial College School
of Medicine, Hammersmith Hospital Campus, London, UK
B. P. M
ONIA • Department of Molecular Pharmacology, Carlsbad Research
Center, ISIS Pharmaceuticals, Carlsbad, CA
A
NUPAMA MUNSHI • Department of Surgery, Louisiana State University
School of Medicine, New Orleans, LA
S
HIGENOBU NAGATAKI • Radiation Effects Research Foundation,
Hiroshima, Japan
A
RTIT
N
AKAKES
• National Heart and Lung Institute, Imperial College,
London, UK
THONG NANAKORN • Genetic Therapy Program of the Yale Cancer Center
and the Medical Oncology Section, Department of Internal Medicine,
Yale University School of Medicine, New Haven, CT
J
OHN NEMUNAITIS • Mary C. Crowley Cancer Research Program, Baylor
Research Institute, Baylor University Medical Center, Dallas, TX
P
ATRICE S. OBERMILLER • Department of Cell Biology, Vanderbilt University,
Nashville, TN
M
ARION OHLMANN • Institut für Molekulare Medizin, Bavarian Nordic
Research Institute, Neuherberg, Germany

T
AKAO OHNUMA • Division of Neoplastic Diseases, Department of Medicine,
Mount Sinai School of Medicine, New York, NY
G
IORGIO PALÙ • Institute of Microbiology, University of Padova Medical
School, Padova, Italy
Y
UE XIN PAN • Department of Pharmacology, School of Medicine, Case
Western Reserve University, Cleveland, OH
H
ARDEV
S. P
ANDHA
• Department of Medicine, Royal Marsden Hospital,
Surrey, UK
JOHN M. PAWELEK • Departments of Dermatology and Pharmacology, Yale
University School of Medicine, New Haven, CT
L
ENE PEDERSEN • Department of Molecular and Structural Biology,
University of Aarhus, Aarhus, Denmark
Contributors xv
XUE YAN PENG • Genetic Therapy Program of the Yale Cancer Center and
the Medical Oncology Section, Department of Internal Medicine, Yale
University School of Medicine, New Haven, CT
M
ASSIMO PIZZATO • Institute of Microbiology, University of Padova Medical
School, Padova, Italy
A
LEXANDER L. RAKHMILEVICH • Department of Human Oncology, University
of Wisconsin Medical School, Madison, WI

R
AJAGOPAL RAMESH • Department of Thoracic and Cardiovascular Surgery,
M.D. Anderson Cancer Center, Houston, TX
D
ANIEL J. RILEY • Department of Medicine, Division of Nephrology, University
of Texas Health Center, San Antonio, TX
MONICA RODOLFO • Department of Experimental Oncology, Istituto Nazionale
Tumori, Milano, Italy
J
OSEPH RUBIN • Division of Medical Oncology, Mayo Clinic, Rochester, MN
V
OLKER SANDIG • Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
K
EVIN J. SCANLON • Berlex Biosciences, Richmond, CA
D
IRK SCHADENDORF • Clinical Cooperation Unit for Dermatology at the
Department of Dermatology Clinics, Mannheim Universität Heidelberg,
Mannheim, Germany
P
AMELA SCHREIBER SCHWARTZ • Department of Biology, Boston University,
Boston, MA
P
ETER M. SCHLAG • Departments of Surgery and Surgical Oncology,
Robert-Rössle-Clinic, Charité at the Humboldt University, Berlin Germany
D
EBORAH E. SCHUBACK • Molecular Neurogenetics Unit, Massachusetts
General Hospital East, Charlestown, MA
R
OBERT H. SHOEMAKER • Antiviral Evaluations Branch, Developmental
Therapeutics Program, National Cancer Institute, Rockville, MD

S
HRINAVASSAN SHRIMDKANDADA • Genetic Therapy Program of the Yale
Cancer Center and the Medical Oncology Section, Department of
Internal Medicine, Yale University School of Medicine, New Haven, CT
K
YONGHEE KAY SON • Department of Pharmaceutics, College of Pharmacy,
Rutgers, The State University of New Jersey, Piscataway, NJ
U
LRIKE STEIN • Max-Delbrück-Center of Molecular Medicine, Robert-Rössle
Strasse, Berlin, Germany
W
OLFRAM STERRY • Department of Dermatology Charité, Humboldt University
Berlin, Berlin, Germany
M
ICHAE
L
S
TRAUSS
•
Max
-
Delbrück
-Center for
Molecular Medicine
, Berlin,
Germany
GERD SUTTER • Institut für Molkulare Medizin, Bavarian Nordic Research
Institute, Neuherberg, Germany
xvi Contributors
TOSHIYA SUZUKI • First Department of Internal Medicine, Saitama Medical

School, Saitama, Japan
A
LEX W. TONG • Mary C. Crowley Cancer Research Program, Baylor
Research Institute, Baylor University Medical Center, Dallas, TX
U
WE TREFZER • Department of Dermatology, Medical Faculty Charité,
Humboldt-University Berlin, Berlin, Germany
S
HOTARO TSURUTA • The First Department of Internal Medicine, Nagasaki
University School of Medicine, Nakgasaki, Japan
T
HOMAS TÜTING • Department of Dermatology, University of Mainz,
Mainz, Germany
W
OLFGANG UCKERT • Max-Delbrück-Center for Molecular Medicine,
Berlin, Germany
P
ETER WALDEN • Department of Dermatology, Medical Faculty Charité,
Humboldt-University Berlin, Berlin, Germany
W
OLFGANG WALTHER • Max-Delbrück-Center of Molecular Medicine,
Robert-Rössle-Strasse, Berlin, Germany
F
U-SHENG WANG • Department of Medicine, Division of Neoplastic Diseases,
Mount Sinai School of Medicine, New York, NY
D
AVID J. WAXMAN • Department of Biology, Boston University, Boston, MA
FRIEDRICH WEBER • Neurochirurgische Klinik, Heinrich Heine Universität
Düsseldorf, Düsseldorf, Germany
G

UIDO WEINGART • Department of Dermatology, Medical Faculty Charité,
Humboldt-University Berlin, Berlin, Germany
J
ONG HO WON • Genetic Therapy Program of the Yale Cancer Center and
the Medical Oncology Section, Department of Internal Medicine, Yale
University School of Medicine, New Haven, CT
L
IAN HUA YIN • Genetic Therapy Program of the Yale Cancer Center and
the Medical Oncology Section, Department of Internal Medicine, Yale
University School of Medicine, New Haven, CT
J
INGPING YANG • Genetic Therapy, Gaithersburg, MD
N
ING-SUN YANG • Comprehensive Cancer Center, University of Wisconsin
Medical School, Madison, WI
W
EI-WEI ZHANG • GenStar Therapeutics, San Diego, CA
Y
U-AN ZHANG • Mary C. Crowley Cancer Research Program, Baylor
Research Institute, Baylor University Medical Center, Dallas, TX
Cytokine-Transduced Tumor Cells 1
I
E
XPERIMENTAL APPROACHES
IN CANCER GENE THERAPY
A: Immunotherapy/Tumor Vaccination
Cytokine-Transduced Tumor Cells 3
3
From:
Methods in Molecular Medicine, Vol. 35: Gene Therapy: Methods and Protocols

Edited by: W. Walther and U. Stein © Humana Press, Inc., Totowa, NJ
1
Immunizing Potential of Cytokine-Transduced
Tumor Cells
Mario P. Colombo and Monica Rodolfo
1. Introduction
1.1. Cytokine-Gene-Modified Tumor Cell Vaccines for Cancer
Immunotherapy: Concepts, Rationale, and Prospective
The molecular definition of tumor antigens, costimulatory signals, and the
possibility to genetically engineer tumor cells as well as simple protocols for
efficient isolation and preparation of dendritic cells (DC) renew the interest in
tumor immunotherapy and vaccination, in particular. Engineering of tumor
cells with the gene of a particular cytokine is a way of releasing that cytokine at
the tumor site. In contrast to bolus administration, it provides a constant supply
of cytokine. If live-engineered tumor cells are injected, their proliferation
results in both the provision of antigen and an increase of cytokine concentra-
tion until a physiological or a pharmacological threshold is reached, and its
biological activity begins. The following inflammatory reaction is then respon-
sible for tumor destruction, thus, turning off the initial trigger. The efficacy of
this feedback action is determined by the type of cytokine, its quantity and
activity, the histotype of the tumor and the molecules it releases, and its extra-
cellular matrix (1). However, the relevant point is that a cascade of events
other than tumor debulking are initiated by the transduced cytokines. Infiltra-
tion of different leukocyte types, including antigen-presenting cells (APC), and
the release of secondary cytokines contribute to the induction of a systemic
and memory response. Also, injection of replication in competent cells because
of irradiation can exert the same effect, in this case the amount of cytokine to
be released in situ to trigger the system, should be predetermined.
4 Colombo and Rodolfo
The events caused by the injection of cytokine-transduced tumors include

both the early infiltration of granulocytes and then of macrophages and lym-
phocytes, and the release of secondary cytokines. Tumor-cell debris derived
from tumor destruction may represent the source of tumor antigens that APC
process and bring to draining lymph-nodes, which appear often enlarged with
expanded cortical and paracortical area and rich of tingible-body macrophages
(2). Although cellular debris captured by phagocytic cells contain antigens that
are presented through the Class II pathway, CD8
+
cytotoxic T lymphocyte
(CTL) are often induced in this setting because systemic immunity and genera-
tion of cytotoxic T lymphocytes generally follow tumor destruction (2).The
finding of GM-CSF-dependent DC Class I presentation of soluble proteins for
CTL induction (3,4) as well as of the role of bone marrow-derived APC (5) in
mediating in vivo cross-priming (6) indicate that intratumoral DC can induce
protective immunity by uptaking and processing antigen for presentation within
their own major histocompatibility complex (MHC) (5).
The efficacy of such vaccines largely depends on their ability to provide all
the repertoire of relevant antigens and the cytokines and cosignals that favor
DC recruitment and function for both T-cell priming and T-T help. The per-
spective should consider the possibility of favoring the interaction between
tumor-cell vaccine and DC, rather than genetically manipulate tumor cells to
transform them into APC-like cells that, by interacting directly with T cells,
may bypass the need of DC.
1.2. Inhibition of Tumor Take, Induction of Systemic
Immunity, and Curative Effects Involve Different
Mechanisms and Different Players
The main criterion in the assessment of cytokine-transduced tumor-cell vac-
cines is the therapeutic efficacy. Tumor inhibition and/or the induction of sys-
temic immunity are not in themselves sufficient for evaluation of treatment
efficacy and curative potential. In fact, tumor inhibition has been studied by

injecting tumor-cell suspensions, although it is known that solid tumor frag-
ments often grow progressively, whereas even 10-fold-higher numbers of the
identical tumor-cell type injected as a suspension are rejected (7). The tumor
stroma, which consists of vessels, sessile and migratory cells, and extracellular
matrix, plays an important role in tumor growth and progression. In addition,
tumor cells modified to produce cytokines are likely to have additional regula-
tory signals resulting from cytokine-extracellular matrix crosstalk. Thus, the
interaction of the tumor with host immune cells and the features of the effector
cells mediating destruction of a tumor injected as a cell suspension differ from
those required to reject an established tumor nodule, as shown in the case
of C-26 colon carcinoma transduced with the G-CSF gene (8). Moreover,
Cytokine-Transduced Tumor Cells 5
induction of systemic immunity with activation of CTL might not be sufficient
to destroy existing tumor nodules although tumor-bearing mice still retain the
ability to recognize the same antigen present within the tumor when presented
on a normal tissue and outside the tumor environment (9). This suggests that
whereas a tumor patient could be immunized against that tumor, the induced
immunity is insufficient to fight an established tumor growing within its own
stroma. Tumor escape from T-cell cytotoxicity may also result from loss of
MHC Class-I antigens or from impaired migration of CTL at the tumor site.
The latter occurs in C-26 carcinoma cells transduced with IL-12 genes, and
CD4
+
cell depletion can replenish CD8
+
T-cell tumor infiltration (10).
1.3. Tumor and Animal Models for Preclinical Studies:
Working With Known Target Antigen(s)
Other crucial points underscore the differences between experiments per-
formed in the mouse system and ongoing clinical investigations. For some

human tumors, the target antigen(s) of an antitumor immune response has been
identified at the molecular level (11), whereas for most murine neoplasms, the
target tumor antigen remains unknown. Moreover, in the case of melanomas
most of the dominant antigens cloned come from specific differentiation anti-
gens of the melanocytic lineage whereas few are from mutated gene products
(11). This implies that vaccines directed against antigens common to different
tumors in patients sharing the relevant HLA haplotype are possible in humans,
whereas similar evidence in the mouse is lacking. Exceptions are viral-tumor
antigens such as Friend-Moloney and Rauscher gag/env proteins, which are
common to some lymphomas and leukemias and lung tumor-associated anti-
gen connexin-1 (12). Other antigens that are frequently expressed on different
tumors are those derived from mutated oncogenes, such as ras or from fusion
proteins derived by chromosomal translocations, such as PML/RAR_, or from
genes overexpressed in tumors, such as HER-2/neu. The immunogenicity of
peptides deriving from these gene products has been better demonstrated in
humans than in mice. Several ras mutations that are commonly associated with
carcinogen-induced tumors are reportedly immunogenic based on the use of
peptides or recombinant proteins; however, their role as tumor-transplantation
antigens (13) is not clear in the absence of experiments with tumors carrying
such mutations. By contrast, tumor antigens cloned from mouse tumors appear
to be unique and tumor specific.
In all mouse experiments that require a well-characterized target antigen,
proteins not classifiable as tumor antigens have been used. In fact, to test
whether a recombinant vaccine carrying the gene encoding an antigen recog-
nized by CTL can treat established metastasis, Restifo and co-workers (14)
used the `-galactosidase (`-gal) gene of Escherichia coli. We have also used
6 Colombo and Rodolfo
(`-gal by to demonstrate Class I-restricted CTL priming by dendritic cells, and
cytolysis and in vivo protection against a `-gal -transduced tumor (3). As a
model, `-gal offers many advantages; `-gal soluble protein, its peptide, as well

as a retroviral vector able to transduce the gene into tumor cells are all avail-
able. In addition, `-gal, as a soluble protein, is unable to enter the cell outside
the endosomal compartment and is unable to stimulate CTL in vivo. Finally,
the animal model utilizing transplantable tumors are of limited preclinical
value. New models employing transgenic mice carrying oncogenes under
tissue-specific promoters mimic more closely the clinical setting.
2. Materials
2.1. Solutions and Materials
2.1.1. Cell Culture
1. RPMI-1640 (Life Technologies, Bethesda, MD) supplemented shortly before use
with 5–10% fetal calf serum (FCS) heat inactivated 1 h at 56°C, 2 mM
L-glutamine, 25 mM HEPES buffer, 1% nonessential amino acids, 1% Na
piruvate, 50 mM 2-mercaptoethanol (2-ME), 100 U/mL penicillin, 100 µg/mL
streptomycin sulfate.
2. ACK lysing buffer: add to 1 L distilled sterile H
2
0: 8.29 g NH
4
Cl (0.15M), 1 g
KHCO
3
(1 mM), 37.2 mg Na
2
EDTA (0.1 mM) and bring to pH 7.2–7.4 with HCl
1M; filter 0.2 µm, store at 4°C.
3. Low tox-M rabbit complement: Cederlane (Hornby, Ontario, Canada), cod
CL3051.
4. 1 mCi/mL Na
2
51

CrO
4
in isotonic saline solution (ICN 620152).
2.1.2. In Vivo Reduction of Metastases
1. India Ink 15% in distilled H
2
O, add 2 drops ammonia water.
2. Fekete solution (to bleach white tumor nodules against blacked lung parenchima):
100 mL 70% ethanol, 100 mL formaldehyde, 5 mL CH
3
COOH.
2.1.3.
In situ
Hybridization
1. OCT compound (Tissue Tek II, Miles cod 4583).
2. SSC (sodium chloride/sodium citrate), 20X: 3M NaCl (175 g/L), 0.3M Na
3
citrate
2H
2
O (88 g/L), adjust pH to 7.0 with 1M HCl.
3. 100x Denhardt’s solution: 10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g
bovine serum albumin (Pentax Fraction V, Miles Lab. Rexdale, Ont., Canada).
4. Ammonium acetate (Sigma A-1542).
5. Dextran sulfate (Sigma D-8906).
6. Dithiothreitolo (DTT) (BRL 5527UA).
7. Formamide (Sigma F-7508).
8. Glycine (USB 16407).
Cytokine-Transduced Tumor Cells 7
9. Triethanolamine (Sigma T-1502).

10. EDTA (Sigma E-5134).
11. Salmon Sperm DNA (SSDNA) (Sigma D-9156).
2.1.4. Immunohistochemical Staining of Tissue Sections
1. Poly-L-lysine (Sigma, MW> 150,000).
2. Tris-buffered saline (TBS): 10 mM Tris Cl, pH 7.6, 1 mM EDTA.
2.2. Mice (Short Note on Animal Care and Handling)
Ethical use of experimental animals include the concepts outlined by Russel
and Burch in 1959 (15), replacement, refinement, and reduction of the use of
laboratory animals. Besides the ethical considerations, experiments with ani-
mal models is strictly regulated by local law, submitted for approval to Institu-
tional Committee and eventually regulated by Institutional Guidelines for
Animal Experimentation. It is important to know that health conditions may
affect results, because common mycoplasma and viral infection determine
immunosuppression. Thus, health monitoring is recommended and a stable
environment for maintenance and quarantine of newly arrived animals should
be followed. Immunocompromised mice, because of irradiation or other
immunosuppressive treatment or because of genetic defects, cannot survive in
conventional animal facilities and should be maintained in conditions aimed to
prevent adventitious infections. Handling requires knowledge of the proper
methods for avoiding injury to the handler and to the animal. Pain and distress
should be avoided as far as possible, anesthesia should be used for all surgical
procedures, and euthanasia by acceptable methods that minimize pain should
be used, following fixed GLP standards (16–18).
2.3. Hybridomas
Most, if not all, the hybridomas listed below can be obtained from American
Type Culture Collection (ATCC). On-line database of ATCC http://www.
atcc.org gives information on monoclonal antibodies (MAbs).
2.3.1. Antibodies for Immunocytochemistry
1. hamster antimouse CD3¡ (154-2C11 clone)
2. rat antimouse CD8 (53.6.72 hybridoma)

3. rat antimouse CD4 (GK1.5 hybridoma)
4. rat antimouse CD28 (37.51 hybridoma)
5. rat antimouse CD31/PECAM-1 (MEC 13.3 hybridoma)
6. rat antimouse CD34 (14.7 MEC hybridoma)
7. rat antimouse CD45 (M1/9.3.4.HL2 hybridoma)
8. rat antimouse CD51/_v integrin (H9.2B8 hybridoma)
8 Colombo and Rodolfo
9. rat antimouse CD54 (3C2 hybridoma)
10. rat antimouse CD61/`
3
integrin (2C9.G2 hybridoma )
11. rat antimouse CD86 (GL-1 hybridoma)
12. rat antimouse Mac-3 (M3/84,6,34 hybridoma)
13. rat antimouse MHC-II (B21-2 hybridoma)
14. rat antimouse GR-1 (RB6-8C5 hybridoma)
15. rat antimouse DEC205 (NDLC-145 hybridoma)
16. rabbit antiasialo GM1 serum (Wako, Osaka, Japan)
2.3.2. Antibodies for In Vivo Leukocyte Depletion
1. rat antimouse CD4 (GK1.5 hybridoma)
2. rat antimouse CD8 (2.43 hybridoma)
3. rat antimouse NK1.1 (PK136 hybridoma)
4. rat antimouse granulocytes (RB6.8C5 hybridoma)
5. rabbit antiasialo GM1 serum (Wako, Osaka, Japan)
2.3.3. Antibodies for TCR V
`
Usage
1. rat antimouse V` 2 (B20.6 hybridoma)
2. hamster antimouse V` 3 (KJ-25 hybridoma)
3. rat antimouse V` 4 (KT4 hybridoma)
4. mouse antimouse V` 5 (MR 9.4 hybridoma)

5. rat antimouse V` 6 (44.22.1 hybridoma)
6. rat antimouse V` 7 (TR310 hybridoma)
7. mouse antimouse V` 8 (F23.1 hybridoma)
8. mouse antimouse V` 9 (MR 10.2 hybridoma)
9. rat antimouse V` 10 (B21.5 hybridoma)
10. rat antimouse V` 11 (RR3-15 hybridoma)
11. rat antimouse V` 12 (MR 11-1 hybridoma)
12. rat antimouse V` 13 (MR 12.4 hybridoma)
13. rat antimouse V` 14 (14.2 hybridoma)
14. mouse antimouse V` 17 (KJ 23 hybridoma)
3. Methods
3.1. In Vivo Tumorigenicity and Challenge-Protection Assay
(Tumor Growth Curves: Tumor Take, Onset, and Survival)
The induction of an in vivo measurable antitumor response in mice follow-
ing immunization with genetically modified tumor cells has a major relevance
for perspectives of clinical application. In fact, although in vitro methods have
been developed to measure different specific effector cells or molecules
involved in tumor regression, a correlation between in vitro detected antitumor
responses and clinical response is still undetermined. In addition, there are
Cytokine-Transduced Tumor Cells 9
instances in which the positive demonstration of antitumor reactivity in vitro
bears no correlation with the extent of the antitumor response in vivo. Thus,
although several aspects of the mechanisms at the basis of immune tumor
destruction have been described, the related immunological parameters are
likely to vary in the different tumor models and treatments tested.
In vivo tumorigenicity of engineered tumor cells can be primarily used to
determine the mechanisms of tumor regression as far as lymphoid subpopula-
tions involved by depletion experiments (see Subheading 3.3.1.) or by histo-
logical techniques. Challenge-protection assays are used to measure
immunogenicity of cytokine-transduced tumors as their ability to induce a

tumor rejection response. They are intended to measure a secondary or memory
response rather then a primary response. Here, parental tumor cells are injected
in mice that have been previously immunized with irradiated engineered cells,
or that have rejected an injection of live engineered cells. Both assays require
tumor-cell injection in mice and monitor of tumor growth and can follow this
scheme:
1. Inject groups of syngeneic mice with engineered tumor cells and parental or
mock-engineered parental cells at doses that have been predetermined in prelimi-
nary experiments. Generally, TD 50/100 tumor-cell inoculum-producing tumor
growth in 50 and 100% of injected animals are used for the first experiments.
Subcutaneous implant in the flank is used because the tumor growth is most
easily observed. Use at least 5 mice per group. For protection assays, the number
of parental tumor cells producing tumor growth in 100% of untreated animals is
injected in immunized mice and control group.
2. Evaluate and record incidence and tumor growth twice weekly in a blinded
fashion. Check tumor appearance by palpation and measure tumor nodules in the
two perpendicular diameter by caliper.
3. To evaluate data by analyzing differences in:
a. Tumor takes or incidence of tumor-free animals.
b. Tumor growth curves constructed by calculation of tumor volume, in mm
3
or
in mg, as: (minor diameter)
2
× (major diameter)/2 (19), or /r
3
. Tumor area
can be alternatively used, calculated by the measures of the longest and short-
est tumor diameters. Tumor growth curves are constructed by plotting the
tumor measures, mg or mm

3
or mm
2
, against time in days or weeks.
c. Tumor onset, defining latency as the time period between challenge and
growth of neoplastic mass.
d. Survival, by observing tumor growth until mice die of tumors or until they
become moribund or until tumor masses become excessively large. Use
r-square test to determine significance in the difference of tumor takes, and
survivors, student’s t- or Fisher test for differences in tumor growth.