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TitleIntrabodies: Basic Research and Clinical Gene Therapy Applications
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Springer-Verlag Berlin Heidelberg GmbH

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Wayne A. Marasco, M.D., Ph.D. (Ed.)

Basic Research and Clinical Gene Therapy


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98 Intra bodies: Basic Research and Clinical Gene Therapy Applications

is replacement of the deficient function with a wild-type tumor suppressor gene
counterpart. This general strategy has been shown to allow phenotypic correction
in vitro. For instance, Vogelstein et al have demonstrated that delivery of the wild-
type p53 gene to transformed p53-deficient colonic carcinoma cells can abrogate
the malignant phenotype.9 In addition, similar studies carried out for other tumor
suppressor genes and other tumor targets have further demonstrated the poten-
tial therapeutic effects achievable with re-establishment of wild-type tumor-sup-
pressor gene function.10-12 The concept has also been establishment in in vivo
models. Roth et al have shown that delivery of the wild-type P53 gene via recombi-
nant retrovirus or recombinant adenovirus, by direct in vivo delivery, can have a
therapeutic effect in a murine model employing human lung cancer xenografts.13
Importantly, it has been shown that despite the presence of multiple genetic le-
sions, the targeted rectification of only one of these is, in many instances, sufficient
to revert the neoplastic phenotype. 10'11 This work has established the rationale for
human clinical gene therapy trials designed to achieve mutation compensation in
epithelial carcinomas of multiple sites, including lung, liver, and the head and neck.

For dominant oncogenes, it is the aberrant expression of the corresponding
gene product which elicits the associated neoplastic transformation. In this con-
text, molecular therapeutic interventions are designed to ablate expression of the
dominant oncogene. The most universally employed methodology to achieve this
is the utilization of"antisense" molecules (DNA or RNA oligonucleotides).14-17 These
molecules are designed to specifically target sequences to achieve blockade of the
encoded genetic informational flow. Approaches have included the use of"triplex"
DNA to achieve functional ablation of transcriptional activation through block-
ade of transcription factor binding sites. This has been used in in vitro model sys-
tems for targeting the c-myc/8 ras/9 and erbB-2 oncogenes.20 Targeting has also
been achieved at levels of gene expression distal to transcription. Specific anti-
sense binding to transcribed RNA sequences may interrupt the flow of genetic
information through several mechanisms, including degradation, impaired trans-
port, and translational arrest. These interventions may be accomplished by simple
antisense oligonucleotides, as well as by antisense molecules which possess cata-
lytic activity to accomplish cleavage of target "sense" sequences.19'21'22 A variety of
experimental models have demonstrated the utility of the antisense approach as
an anti-cancer therapeutic. Importantly, several studies have shown the ability to
selectively ablate a dominant oncogene with reversion of the malignant pheno-
type.18'19'22 In selected instances, the in vivo demonstration of this effect could also
be accomplished by direct, in vivo delivery of the antisense molecules.23'24 Thus,
the antisense approach offers the potential to achieve targeted disruption of spe-
cific genes in anti-cancer therapy models.

Despite the potentially novel therapeutic strategies offered by the antisense
approach, this methodology has in practice been associated with severe limita-
tions. These practical constraints have limited wide employment for this technol-
ogy in human gene therapy anti-cancer protocols. In this regard, there do not exist
universal rules dictating the efficacy of a given antisense oligonucleotide for achiev-
ing specific gene inhibition.14-16 Thus, despite the utility of antisense inhibition,
there are a great many cancer-related genes which have resisted attempts to achieve
their antisense ablation. In addition, delivery of antisense molecules has been highly
problematic.14-16 It is often difficult to achieve effective sustained intracellular lev-
els of the antisense molecules sufficient for a therapeutic effect. To circumvent this

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Intracellular Antibody-Mediated Knockout of the ErbB-2 Oncoprotein 99

problem, a number of design modifications have been developed to enhance their
stability.14-16•25 In addition, a number of vector approaches have been explored for
effective cellular delivery.24'26'27 Despite these maneuvers, the overriding limita-
tions to the employment of this therapeutic modality remains the idiosyncratic
efficacy of specific antisense for a given target gene and the sub-optimal delivery
of antisense molecules.

Based upon these recognized limitations of antisense methodologies, we en-
deavored to develop alternative approaches, to achieve functional "knock-out' of
oncoproteins. To this end, a large number of specific monoclonal antibodies (mAbs)
have been developed to target these oncoproteins. Further, techniques have been
developed to allow the derivation of recombinant molecules which possess anti-
gen binding specificities expropriated from immunoglobins. In this regard, single-
chain immunoglobin (scFv) molecules retain the antigen-binding specificity of
the immunoglobulin from which they were derived; however, they lack other func-
tional domains characterizing the parent molecule. The basis of constructing scFvs
has been established. Pastan et al have developed methods to derive cDNAs which
encode the variable regions of specific immunoglobins.28'29 Specifically, a single-
chain antibody gene is derived which contains the coding sequences for variable
regions from the heavy chain (V H) and the light chain (V L) of the immunoglobulin
separated by a short linker (L) of hydrophilic amino acids. The resultant recombi-
nant molecule, when expressed in prokaryotic systems, is a single-chain antibody
which retains the antigen recognition and binding profile of the parent. The devel-
opment of recombinant immunotoxins employing scFv moieties achieves cell-spe-
cific binding of the toxin to the exterior of the target cell, allowing receptor-medi-
ated endocytosis to accomplish toxin internalization. A variety of strategies
employing the recombinant scFv-directed immunotoxins have been developed by
a number of investigators.28-33 In addition, it has recently been shown that scFv
molecules may be expressed intracellularly in eucaryotic cells by gene transfer of
scFv cDNAs. The encoded scFv may be expressed in the target cell and localized to
specific, targeted subcellular compartments by appropriate signal molecules. Im-
portantly, these intracellular scFvs may recognize and bind antigen within the tar-
get cell. Marasco et al have shown that intracellular antibodies against HIV can
abrogate production of progeny virion in HIV-infected cells.34•35 Thus, intracellu-
lar scFvs appeared to offer the means to achieve intracellular "knock-out" of spe-
cific oncoproteins for therapeutic purposes.

As an initial proof of principle, we endeavored to achieve functional knock -out
of the erbB-2 oncoprotein. In this regard, erbB-2 is a 185-kDa transmembrane pro-
tein kinase receptor with extensive homology to the family of epithelial growth
factor receptors. 36 Several lines of evidence suggest that aberrant expression of the
erbB-2 gene may play an important role in neoplastic transformation and progres-
sion. Specifically, ectopic expression of erbB-2 has been shown to be capable of
transforming rodent fibroblasts in vitro.37 In addition, transgenic mice carrying
either normal or mutant erbB-2 develop a variety of tumors, including neoplasms
of mammary origin.38 Importantly, it has been shown that amplification and/or
overexpression of the erbB-2 gene occurs in a variety of human epithelial carcino-
mas, including malignancies of the ovary, breast, gastrointestinal tract, salivary
gland, and lung.39 In the instance of ovarian carcinoma, a direct correlation has
been noted between overexpression of erbB-2 and aggressive tumor growth with

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210 Intrabodies: Basic Research and Clinical Gene Therapy Applications

Helper phage 32-33, 35, 198
High-density screening array 15
HIV-1 2, 4-5, 9-10,14,35, 89,147-150,

153-158,163-177,183-191,194-198, 200-
201, 203-206

HIV-1 gp120 5
HIV-1 Tat 14
HTLV-I 48-49,52-54,57,59
Human sFv libraries 2
Humanization 155
HUT102 49, 52-53
Hybridoma 2, 4, 23, 25, 29-31,35-36,39, so,

61, 64, 72, 75, 86, 88,133,148,152, 167,
174, 187, 188, 191, 194


IL-2 47-54,56-57, 153
IL-7 54
Immunoglobulin 1-2, 4, 9, 15, 23, 25, 27, 31,

36, 75, 79, 83, 99> 124,157,165,174,185,

In vitro affmity maturation 2
Inducible knockout 53, 56
Infection of tumor cells 69, 141
Inhibitory potential139
Integral membrane proteins 7
Intracellular immunization 1, 39, 148,

163-164, 177, 184-187, 200, 205-206
Intracellular targeting 2, 75, 133
Intracellular trafficking 23

Jak3 48-49, 57
Joining segment 24, 28
Jun kinases 11
Junctional diversity 24
Jurkat 5, 50-52,165-172,175


K-Ras 12
Kabat 4, 25, 196
KDEL ER retention signal4, 56, 64
Killer gene 143
Kit225 54, 55


Large T antigen 12
Leader sequences 4, 165, 196
Lentiviral vector 52, 54, 89
Localization sequence 133, 149, 156

LTR 111,148-150,158,164,169,189, 196,

Lysosomes 7-8


Matrix 11, 15, 34, 164, 206
Matrix protein 4, 11
Mitochondria 9, 11, 15
Mitogen-activated protein kinases

(MAPKs) 11
Murine IgG3 hinge 7
Mutator strain 37


N-segment addition 24
NDF 62-63, 67,70-71
Neu differentiation 63
NF-KB 154
Non-immunogenic 16
Non-toxic 16
Nuclear export signal14
Nuclear localization signal12, 14, 164, 196
Nucleocapsid 164,173, 174
Nucleus 12, 14, 23, 156, 164, 169, 172, 184,



Oligoligation PCR 31
Oncogene 48, 89,97-98, 108,111, 124,

Oncogenic Raf 139
Ovary 69, 99-102,105-106,108-109,



P34CdC2 135, 139
P53 98,129,142,143-144,149
pBabe-puromycin retrovirus 69
PBMCs 54, 153-155,157, 167
PCR 4, 25, 29, 31-32,35,37,133-135,148-149,


Peroxide metabolism 9
Peroxisomal targeting signal (PTS) 11
Peroxisomes 9, 11
Phage antibody library 35-38
Phage display 2, 15,31-32,34-37,39,

Phage vector 32-33, 35

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Phagemid vector 32, 33, 198
Phenotypic knockout 4, 5, 37, 39, 52, 54
Phenotypic reversion 5, 130, 139
pill 32-33.35. 38, 157
Primary human T cells 54,56
Primary human tumors 69
Primary lymphocytes 158, 169
Proopiomelanocortin (POMC) 9
Proteasome 5, 52
Protein disulfide isomerase 4


Rabbit anti-scFv antibodies 135
Ras 12-14,98, 129-131,133,135-137,139,141,

Ras proteins 11, 130, 133
Ras-responsive promoter 139
Receptor heterodimer 62-63
Receptor homodimer 63
Receptor-ligand interaction 61, 70
Redox state 4
Regulated secretory pathway 9, 86
Repertoire cloning 2, 157
Replication 115,147,149,153-156,163-164,

175.184-185,187, 189,191,194.196-198,
200-201, 204-205

Retroviral vector 65-66,194,196, 201,

Retrovirus 48, 53, 68-69,71, 89, 98, 129,
163, 164, 173-174. 187,189, 194. 196

Rev 14, 16-17,148, 150,154-158, 164, 184, 191,
196-198, 200, 204-205

Reverse transcriptase 147, 158, 164,173
Reversion of the transformed phenotype

Ribozymes 1, 148,156,163-164,200-201
RNA decoys 1, 156, 163-164
Rough endoplasmic reticulum 2, 4
RRE 150, 155-158, 184, 195, 200-201,

RTK 61-62,64

scFv-CAAX 132,136,139, 141-143
scFv-CAAX construct 133
scFv-CK 132, 134, 135, 136, 139, 141
Semisynthetic libraries 2
Serum stimulation 139
sFv intrabody 1, 4-5, 9,11-12,14-15

sFvTac 5, 50-53,55-56
sFvTacKDEL 5, 50-54,56-57
Single-chain antibodies 1, 129, 187
Somatic hypermutation 31, 36-37
Surface plasmon resonance 38-39
SV 40 virus 12
Syntaxin 12


Tac (CD25, IL-zRa) 4
TAR 148, 150,156,158, 184
Targeted gene disruption 1
Targeting protein 164
Tat 14,147-150,152-154,156-158,164,166,

174-175.184,198, 207
Tat 148
Tat protein 166
Tetracycline 33, 54-55,57, 77
TGF-a 63
TIM system 15
TOM system 15
Trabodies 143
Trans-Golgi 2, 7-8
Transactivator 54, 148-149, 164
Transcriptional activity 142-143, 203
Transgenic mice 2, 75-76, 78-83, 86, 99
Transmembrane spanning region 7


Tumor cel167-71, 91,100,104-105,108-117,
120, 123, 128-130, 139. 141, 143

Thmor regression 129, 141


Variable fragment 24, 27, 183, 187, 205


Xenopus laevis 135


YQRL motif, 7

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