Table Of ContentSpringer-Verlag Berlin Heidelberg GmbH
Wayne A. Marasco, M.D., Ph.D. (Ed.)
Intrabodies
Basic Research and Clinical Gene Therapy
Applications
Springer
Wayne A. Marasco, M.D., Ph.D.
Division of Human Retrovirology
Dana-Farber Cancer Institute
Boston, Massachusetts, U.S.A.
Library of Congress Cataloging-in-Publication data
Intrabodies-Basic research and clinica! gene therapy applications / Wayne A. Marasco.
p. cm. - (Biotechnology intelligence unit)
Includes bibliographical references and index.
ISBN 978-3-662-12121-4 ISBN 978-3-662-12119-1 (eBook)
DOI 10.1007/978-3-662-12119-1
1. Immunoglobulins--Biotechnology. 2. Gene targeting. 3. Gene therapy. 4. Cellular signal
transduction. 1. Marasco, Wayne A., 1953- . II. Series.
[DNLM: 1. Antibodies--physiology. 2. Signal Transduction--physiology. 3. Gene
Expression--physiology. QW 575168519971
TP248.65.I49156 1997
616.07'98-dc21
DNLM/DLC 97-36323
for Library of Congress CIP
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DEDICATION
Dedicated to the memory of Anthony S. Marasco.
((Special Thanks" to Viola Kempf and to my girls, Je ny,
Mariah and Madison.
=====PREFACE=====
A
ntibodies have long been used in biomedical science as in vitro tools
for the identification, purification and functional manipulation of
target antigens; they have been exploited in vivo for diagnostic and
therapeutic applications as well. Recent advances in antibody engineer
ing have allowed the genes encoding antibodies to be manipulated so
that the antigen binding domain can be expressed intracellularly. The
specific and high-affinity binding properties of antibodies, combined
with their ability to be stably expressed in precise intracellular loca
tions inside mammalian cells and to unique epitopes on a target mol
ecule, has provided an extraordinary powerful new family of molecules
called intracellular antibodies or "intrabodies.,
This book is about intrabodies and contains chapters by leading
investigators in the fields of antibody engineering, signals transduc
tion as well as cancer and AIDS research. These investigators are using
intrabodies in their laboratories as tools for gene inactivation and/or
as therapeutic molecules in clinical gene transfer applications. There
are ten chapters in the book.
The first chapter is an introduction to the intrabody technology
and the use of intracellular trafficking signals to direct intrabodies to
precise subcellular locations. I have tried to briefly review published
work on intrabodies in this area as well as to discuss new vector de
signs that are currently being tested in my laboratory to broaden the
subcellular targeting potentials of these molecules. I have also discussed
where I believe this technology can go in the future.
The second chapter by Drs. Poul and Marks is an extensive review
of antibody phage display technology. Using powerful new
immunotechnological tools, the creation of large human immunoglo
bulin libraries from naive individuals has been achieved and when com
bined with phage display technology, has allowed investigators to by
pass in vivo immunization and produce high affinity human antibod
ies to human proteins. These powerful new methods have provided a
rich source of rearranged human antibody genes and, when combined
with the use of classical intracellular trafficking signals, greatly extends
and will markedly enable the further development of this new
technology.
The third chapter describes studies on the use of intrabodies to
obtain phenotypic knockout of the high affinity component of the hu
man interleukin-2 receptor a chain. These studies by Dr. Richardson
and myself demonstrate the power of this technology to obtain pheno
typic knockout of an integral membrane growth factor receptor on both
leukemic T cell lines and PBMCs. The ability to use this technology to
systematically examine different components of the IL-2 receptor path
way for their involvement in constitutive signaling and in the growth of
HTLV-I transformed cells and adult T cell leukemia cells (ATL) are dem
onstrated and discussed in detail.
In chapter four, Drs. Beerli, Graus-Porta and Hynes present their
elegant biochemical studies on the use of intrabodies as tools to study
erbB2 receptor kinases. Epidermal growth factor (EGF) receptor and
erbB-2, two receptors whose aberrant expression is frequently involved
in human cancer, were chosen as targets. Their studies withER-directed
sFv intrabodies to EGFR could compete with EGF and inhibit EGF re
ceptor function in an autocrine manner while, in contrast, the KDEL
containing sFv intrabody inactivated the EGF receptor by ER retention.
Additional studies with two KDEL-tagged sFv intrabodies against erbB-
2 have demonstrated a marked decrease in the cell-surface expression
of erbB2 in NIH3T3 fibroblasts that express an oncogenically activated
form of erbB2. Functional inactivation of the receptor is suggested by a
reduction in the phosphotyrosine content of the cells and, more impor
tantly, by the reversion of the cells to a non-transformed phenotype.
These latter studies also show the importance of receptor
transmodulation among the type I RTKs.
Neuroantibodies or recombinant antibody expression in the cen
tral nervous system is the topic of chapter five. Here, Drs. Cattaneo,
Piccioli and Ruberti present their pioneering work on expressing re
combinant antibodies in the central nervous system of transgenic mice.
They describe their studies with glial and neuronal cell expression of
anti-neurotrophic factor (NGF) and anti-substance P (SP) recombinant
antibodies and their neutralizing activity of the corresponding anti
gens. They also discuss the potential applications of this approach of
local production of antibodies within the CNS as a promising thera
peutic strategy.
Chapters six and seven contain very valuable contributions from
two leading laboratories on the use of intrabodies for the gene therapy
of cancer. In chapter six, Dr. Curiel presents a number of studies from
his laboratory, complementary to those presented by Drs. Beerli, Graus
Porta and Hynes, on an ER-directed sFv intrabody that has been used
to downregulate erbB2 in the human ovarian-carcinoma cell line SKOV3.
They demonstrate that constitutive downregulation of erbB2 is incom
patible with the long-term survival of these cells which are killed by
apoptosis. When ex vivo transduced SKOV3 cells were introduced sub
cutaneously into nude mice, no tumors grew and complete tumor eradi
cation at necropsy was observed. Tumor cell eradication in transduced
primary cultures of human ovarian carcinoma cells could also be dem
onstrated. Using a replicative-defective adenovirus to in vivo trans
duce the anti-erbB2 sFv intrabody gene into i.p. transplanted SKOV3
cells, these investigators further demonstrated in vivo tumor cell kill
ing and a significantly prolonged survival of animals compared to con
trol groups. These preclinical studies form the basis of a clinical gene
therapy protocol to treat a metastatic ovarian cell carcinoma in patients
that have failed conventional chemotherapy.
Chapter seven describes in vitro and in vivo studies by Dr. Cochet
and colleagues on an anti-ras sFv intrab ody. They demonstrate first in
Xenopus laevis oocytes that the anti-ras sFv intrabody can inhibit mei
otic maturation in response to insulin which activates the Ras signaling
pathway. Germinal vesical breakdown ( GVBD ), a hallmark of matura
tion and activation of maturation promoting factor or P34cdc• were also
inhibited. In mammalian cells, the anti-Ras sFv could also inhibit the
activation of the transcriptional AP-1 complex by oncogenic ras. Cell
death by apoptosis was observed in human lung carcinoma H460 cells
which express the mutant Ki-ras gene. When direct intratumor injec
tion of an adenovirus expressing the anti-ras sFv intrabody was per
formed in nude mice, pronounced tumor regression was observed. Fi
nally, they discuss their proposed studies to determine if selected anti
P53 sFv intrabodies can restore wild-type p53-associated properties such
as specific DNA binding and transcriptional activation, which may re
sult in cell cycle arrest and/or apoptosis of certain tumor cells that con
tain point mutations in one of the P53 alleles.
Chapters eight through ten detail studies from both my labora
tory and the laboratories of Drs. Duan and Pomerantz on the use of
anti-HIV- 1 intrabodies for the gene therapy ofHIV- 1-infection and AIDS.
In chapter eight, Dr. Mhashilkar and I describe studies that evaluate
anti-tat and anti-rev sFv intrabodies for "intracellular immunization''
against HIV-1 infection. In these studies we present experimental data
that demonstrate that the epitope of the target protein is critical for
inhibition of HIV-1 replication, both for anti-tat and anti-rev sFv
intrabodies.
In chapter nine, Dr. Rondon and I describe studies on intrabodies
that are directed against the HIV-1 gag proteins, p17 (matrix (MA)) and
p7 (nucleocapsid (NC) ). A Fab intrabody against MA was used to block
both afferent and efferent arms of the viral life cycle since the matrix
protein is both required for nuclear import of the viral preintegration
complex and for particle assembly. In these studies we show that a
marked inhibition of proviral gene expression occurred in stably trans
fected T cell lines when single-round HIV-1 CAT virus was used for
infections. In challenge experiments using both laboratory strains and
syncytium -inducing primary isolates of HIV -1, a substantial reduction
in the infectivity of virions released from the cells was also observed.
We also present preliminary data with an anti-NC sFv intrabodywhich
suggests that the anti-viral activity that is observed is due to a blockade
at both the afferent and efferent arms of the life cycle.
The final chapter prepared by Drs. Duan and Pomerantz details
their studies on sFv intrabodies against HIV-1 integrase and reverse
transcriptase. These two targets are critical for the afferent arm of the
HIV-1life cycle and it is thought that intracellular immunization which
results in preventing the establishment of infection may be more effi
cient than strategies aimed at inhibiting gene expression after integra
tion of the provirus has occurred. These investigators present data to
demonstrate that the anti-IN and anti-RT sFv intrab ody transduced cells
are protected against HIV-1 challenge. They also discuss their studies
on combining sFv intrabodies with ribozymes to increase the anti-viral
activity of their retroviral transduced cells.
In summary, this book will broaden the interest and knowledge of
scientists already familiar with specific aspects of antibody engineer
ing and gene therapy, for whom the extensive and up-to-date data list
will prove particularly useful. It will also be useful to students of cell
biology, biochemistry, microbiology, molecular biology and gene
therapy. Finally, this book will grab the attention of scientists that are
involved in the human genome project and in identifying small mol
ecule drugs for the treatment of human diseases who may wish to uti
lize intrabodies as a means to achieve their goals. They will surely learn
a great deal about intracellular protein trafficking along the way.
CONTENTS
1. Designing Intrabodies: Structural Features and the Use
of Intracellular Trafficking Signals ............................................ 1
Wayne A. Marasco
Introduction ................................................................................. 1
Starting Materials For lntrabody Construction ....................... 2
Intracellular Trafficking ............................................................. 2
Summary and Future Prospects ............................................... 15
2. Phage Libraries for Generation of Single Chain Fv Antibodies
for Intracellular Immunization ................................................. 23
Marie Alix Poul and ]ames D. Marks
Introduction ............................................................................... 23
3· Phenotypic Knockout of the Human Interleukin-2
Receptor a Chain On Primary and HTLV -I
Transformed T Cells .................................................................... 47
Jennifer H. Richardson and Wayne A. Marasco
Introduction ............................................................................... 47
Functional Studies ..................................................................... 50
Conclusions ............................................................................... 56
4· Intracellular Antibodies as Tools to Study ErbB
Receptor Tyrosine Kinases ......................................................... 61
Roger R. Beerli, Diana Graus-Porta and Nancy E. Hynes
Introduction ............................................................................... 61
5· Neuroantibodies: The Use of Recombinant Antibody
Expression in the Central Nervous System .............................. 75
Antonino Cattaneo, Patrizia Piccioli and Francesca Ruberti
Introduction ............................................................................... 75
Refining the Mode of Action of Ne uroantibodies .................. 86
Bypassing the Blood-Brain Barrier: Grafting
of Engineered Cells or Direct Gene Transfer ...................... 87
Antibody Expression in the CNS
for Intercellular Immunization ............................................ 91
Conclusions ............................................................................... 92
6. Intracellular Antibody-Mediated Knockout of the ErbB-2
Oncoprotein as a Cancer Gene Therapy Approach ................ 97
David T. Curiel
Introduction ............................................................................... 97
Summary .................................................................................. 124
7· Intracellular Targeting of Oncogenes: A Novel Approach
for Cancer Therapy ................................................................... 129
Olivier Cochet, Isabelle Delumeau, Mireille Kenigsberg,
Nadege Grue~ Fabien Schweighoffer, Laurent Bracco,
Jean Luc Teillaud and Bruno Tocque
Introduction ............................................................................. 129
Future Directions .................................................................... 142
8. Intrabodies Against the HIV -1 Regulatory Proteins:
Tat and Rev as Targets for Gene Therapy .............................. 147
Abner M. Mhashilkar and Wayne A. Marasco
Introduction .............................................................................. 147
9· Gene Therapy for HIV-1 Using Intracellular Antibodies
Against HIV -1 Gag Proteins ..................................................... 163
Isaac]. Rondon and Wayne A. Marasco
Introduction .............................................................................. 163
Genetic-Based Strategies Used to Inhibit
HIV-1 Replication ................................................................. 163
Intracellular Antibodies Against HIV -1 ................................ 164
Matrix (p17) .............................................................................. 164
Nucleocapsid (p7) .................................................................... 173
Conclusion ............................................................................... 176
10. Single Chain Variable Fragment-Based Strategies
for Anti-HIV-1 Gene Therapy: Targeting the Viral
Preintegration Complex and Combination
Molecular Approaches .............................................................. 183
Roger]. Pomerantz and Lingxun Duan
Introduction .............................................................................. 183
Conclusion ............................................................................... 205
Index ..................................................................................................... 209
