Research Article
Targeting Tumor Angiogenesis with Adenovirus-Delivered
Anti-Tie-2 Intrabody
1
1
2
Mikhail Popkov, Nina Jendreyko, Dorian B. McGavern, Christoph Rader,
1
and Carlos F. Barbas III
3
1
Department of Molecular Biology and Skaggs Institute for Chemical Biology and 2Department of Neuropharmacology, The Scripps
Research Institute, La Jolla, California and 3Experimental Transplantation and Immunology Branch, Center for Cancer Research, National
Cancer Institute, NIH, Bethesda, Maryland
pathway. This suggests that alternative pathways for vascular
growth exist and can drive tumor angiogenesis (4, 5). Two
structurally related tyrosine kinases with immunoglobulin
and epidermal growth factor motifs (Tie-1 and Tie-2) constitute a
second group of receptor tyrosine kinases found mainly in
endothelial cells and their progenitors (6). Whereas signaling ligands
for Tie-1 are not known, angiopoietin-1 (Ang1) and angiopoietin-2
(Ang2) are ligands of the Tie-2 receptor (7, 8). In contrast to the
VEGFR system, Tie receptors are not required for vasculogenesis.
Correct regulation of Tie-2 is required for normal vascular
development perhaps via the regulation of vascular remodeling
and endothelial cell interactions with supporting pericytes and
smooth muscle cells (8–10). Tie-2 is unique among receptor tyrosine
kinases in that its ligands, Ang1 and Ang2, apparently have opposing
actions on Tie-2 signaling and function (11). Ang1, the activating
ligand, blocks increases in vascular permeability induced by
inflammatory agents and VEGF (12). This suggests a role for Ang1
in vessel maturation and stabilization. In contrast, Ang2 is required
for VEGF-induced corneal angiogenesis (13), suggesting that Ang2
blocks the actions of Ang1 and promotes vascular destabilization and facilitates angiogenesis. This is supported by the fact that
Tie-2 / mice lack proper hierarchical organization of the
vasculature and die during embryogenesis (14). Although Tie-2
expression is down-regulated postnatally, it does persist in quiescent
adult endothelial cell, and the presence of tyrosyl phosphorylation in
the normal vasculature suggests an active role in the maintenance of
blood vessels (15). Furthermore, Tie-2 is up-regulated in capillaries
during neovascularization processes produced in skin wounds and
tumors (16, 17). Several reports have shown that interference with
the Tie-2 receptor pathway results in the inhibition of tumor
angiogenesis and growth. Such inhibition has been achieved by using
specific blocking agents that include soluble dominant-negative
receptors (18–21), antisense oligonucleotides (22), RNA aptamers
and RNA interference (23, 24), and short synthetic peptides (25).
There have been no published reports based on the use of anti-Tie-2
intrabody to inhibit tumor growth and angiogenesis.
The potential of intrabodies (i.e., antibodies for intracellular
applications) has been exploited in several laboratories (26). The
aim has been to neutralize the function of intracellular and
extracellular proteins. One widely successful strategy has been the
misdirected localization of cell membrane proteins to the
endoplasmic reticulum (ER) based on a KDEL tetrapeptide motif
(27, 28). Intrabodies present a potent alternative to methods of
gene inactivation that target at the level of DNA or mRNA, such
as antisense oligonucleotides (29), zinc finger proteins (30),
targeted gene disruption, or RNA interference (31). Operating at
the post-translational level, intrabodies can be directed to
relevant subcellular compartments and precise epitopes on target
Abstract
Inhibition of tumor angiogenesis is a promising approach
for cancer therapy. As an endothelial cell–specific receptor
kinase expressed almost exclusively on the surface of vascular
endothelium, Tie-2 has an important role in tumor angiogenesis. To explore the therapeutic potential of blocking Tie-2
receptor-interaction pathway, an adenoviral vector was used
to deliver a recombinant single-chain antibody fragment
rabbit intrabody (pAd-2S03) capable of inhibition of both
mouse and human Tie-2 surface expression. pAd-2S03 was
given to mice with well-established primary tumors, either a
human Kaposi’s sarcoma (SLK) or a human colon carcinoma
(SW1222). The intrabody significantly inhibited growth of
both tumors (75% and 63%, respectively) when compared
with pAd-GFP control-treated tumors (P < 0.01). Histopathologic analysis of cryosections taken from mice treated with
pAd-2S03 revealed a marked decrease in vessel density, which
was reduced by >87% in both tumor models when compared
with control-treated tumors (P < 0.01). In contrast, human
Tie-2-monospecific pAd-1S05 intrabody did not affect the
growth of tumors, indicating that the antitumor effect of
pAd-2S03 was due to the inhibition of tumor angiogenesis in
these murine models. Our results show that the Tie-2
receptor pathway is essential for both SLK sarcoma and
SW1222 colon carcinoma xenograft growth. The present
study shows the potential utility of antiangiogenic agents
that target the endothelium-specific receptor Tie-2 for downregulation or genetic deletion. (Cancer Res 2005; 65(3): 972-81)
Introduction
Collective experimental and clinical studies show that, in order for
a solid tumor to grow to a clinically relevant size, an adequate blood
supply is required (1). Vascular endothelial growth factor (VEGF)
receptors (VEGFR) and their ligand are currently considered the best
candidates for treatment of human cancers via angiogenesis
inhibition (2). Inhibition of angiogenesis and the resultant tumor
growth has been achieved by using specific agents that either
interrupt the VEGF/VEGFR2 interaction or block the VEGFR2 signal
transduction pathway (3). However, several studies have shown that
not all types of tumors respond to interruption of the VEGF/VEGFR
Note: Supplementary data for this are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Carlos F. Barbas III, Department of Molecular Biology,
Scripps Research Institute, BCC-550, 10550 North Torrey Pines Road, La Jolla, CA
92037. Phone: 858-784-9098; Fax: 858-784-2583; E-mail: carlos@scripps.edu.
I2005 American Association for Cancer Research.
Cancer Res 2005; 65: (3). February 1, 2005
972
www.aacrjournals.org
Intrabodies for Tie-2 Phenotypic Knockout
proteins, potentially blocking only one of several functions of an
expressed protein.
The goal of our study was to generate an ER-targeted intrabody
against Tie-2 to investigate whether a phenotypic knockout of Tie-2
could inhibit tumor cell growth in vivo. To accomplish this, an ERtargeted single-chain antibody fragment (scFv) was generated and
a replication-deficient recombinant adenovirus was used for its
local delivery in two mouse models of primary tumor growth.
was linked to the NH2-terminal VH region through a peptide linker
(SSGGGGSGGGGGGSSRSS). Control constructs 1S05 and JC7U (against
integrin avh3) were described previously (35). The scFv encoding sequences
were cloned into phagemid vector pComb3X using asymmetrical SfiI sites,
and binding activity of the expressed scFv was confirmed by ELISA.
Construction of pAd-2S03 Adenoviral Vector and Production of
Recombinant Virus. 2S03 scFv coding regions were initially assembled in
pBabePuro essentially as described (36). In these construct, the scFv coding
regions were flanked by a human n light-chain leader sequence at the 5V end
and a sequence encoding the HA-tag (YPYDVPDYA) and the ER retention
signal (KDEL) at the 3V end. The scFv coding regions were excised by
digestion with BamHI and SalI and ligated into pAd-TrackCMV (37). Control
adenovirus vectors, pAd-1S05 and pAd-JC7U, encoding 1S05 and JC7U scFv
intrabodies and empty adenovirus vector, pAd-GFP, were generated as
described previously (35). The generation of recombinant adenoviruses was
done essentially as described (37). High-titer viral stocks were produced and
purified by CsCl banding. Final yields were between 5.3 1011 and 1.5 1012
particles/mL. All virus preparations were green fluorescent protein (GFP)
corrected (38).
Flow Cytometry. Analysis of Tie-2 binding on HUVECs and MAECs was
done as described previously (32). Briefly, 100 AL of F(ab) 1S05, 2S03, or
control mAb solution at 5 Ag/mL in fluorescence-activated cell sorting
(FACS) buffer were added to the 105 cells and incubated for 40 minutes at
room temperature. Cells were washed once with 200 AL FACS buffer and
incubated for 40 minutes at room temperature with 100 AL FITCconjugated donkey anti-human or anti-mouse IgG antibody diluted to
1:100 in FACS buffer. Cells were washed twice, resuspended in 200 AL FACS
buffer, and transferred to FACS tube for analysis in a FACScan flow
cytometer (Becton Dickinson, San Jose, CA).
For Tie-2 detection on adenovirus-infected cells, 1.5 106 HUVECs were
infected with 10 multiplicities of infection (MOI) of adenovirus (f80% of
the cells were infected) encoding Tie-2-specific intrabody (pAd-1S05 or pAd2S03) or with control adenovirus (pAd-JC7U or pAd-GFP), and Tie-2
expression was monitored for 7 days by flow cytometry as described
previously (35). The formula used for calculation of surface inhibition, after
subtraction of the background of biotinylated normal-goat IgG-stained
cells, was (Tie-2GFP Tie-2intrabody) / Tie-2GFP.
Analysis of Tie-2/Intrabody Colocalization by Immunofluorescence.
For analysis of Tie-2, 2.5 103 HUVECs were seeded on collagen-coated LabTek 8-chambered slide (Nalge Nunc International, Naperville, IL) and infected
with 50 MOI of adenovirus encoding Tie-2-specific intrabody (pAd-1S05
or pAd-2S03) or with control adenovirus (pAd-JC7U). Forty-eight hours
postinfection, HUVECs were fixed and double stained with biotinylated goat
human anti-Tie-2 polyclonal antibody and rat anti-HA mAb as described
previously (35). Three-color (GFP, rhodamine red-X, and Cy5) threedimensional data sets were collected with a DeltaVision system (Applied
Precision, Issaquah, WA), and images were deconvolved (based on the AgardSadat inverse matrix algorithm) and analyzed with softWorX version 2.5.
Ultrastructural Characterization. For transmission electron microscopy (TEM), adenovirus-infected cells were cultured in 35-mm dishes to 90%
confluence and fixed in 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer.
After a brief rinse, cells were postfixed in 1% osmium tetroxide, rinsed,
incubated in 0.5% tannic acid followed by 1% sodium sulfate, and
subsequently dehydrated in graded ethanol series. Cells were embedded
in LX112 resin (Ladd Research Industries, Williston, VT), each disc of
embedded cells was cut into small pieces and mounted on blank resin
blocks, and ultrathin sections were cut en face. Sections were mounted on
parlodion-coated copper slot grids, stained with uranyl acetate and lead
citrate, and viewed in a Philips CM100 TEM (FEI, Hillsbrough, OR) at 80 kV.
Images were documented on Kodak SO-163 film for later analysis.
Immunoelectron microscopy on ultrathin cryostat sections was done as
described (39) with minor variations. Cell were cultured as described above,
fixed in 4% paraformaldehyde-0.025% glutaraldehyde in 0.1 mol/L
Materials and Methods
Cell Lines. Human umbilical vein endothelial cells (HUVEC) were
purchased from BioWhittaker (Walkersville, MD) and maintained in
complete endothelial growth medium supplemented with bovine brain
extract (BioWhittaker). The mouse endothelial cell line MAEC was provided
by W.B. Stallcup (Burnham Institute, La Jolla, CA) and maintained in
DMEM supplemented with 4 mmol/L L-glutamine, 1.5 g/L sodium
bicarbonate, 4.5 g/L glucose, 1 mmol/L sodium pyruvate, 10% FCS, and
antibiotics. Human Kaposi’s sarcoma cell line (SLK) was provided by R.
Pasqualini (University of Texas M.D. Anderson Cancer Center, Houston, TX)
with permission from S. Levinton-Kriss (Tel Aviv, Israel). Human colon
cancer cell line (SW1222) was provided by L.J. Old (Ludwig Institute for
Cancer Research, New York, NY). All human cell lines were maintained in
RPMI 1640 containing 10% FCS and antibiotics.
Antibodies and Other Proteins. The lyophilized recombinant human
Tie-2/Fc chimera (330 kDa) and mouse Tie-2/Fc chimera (270 kDa), which
contain the extracellular domain of human or murine Tie-2 receptors fused to
human IgG1 Fc via a polypeptide linker, were purchased from R&D Systems
(Minneapolis, MN). Biotinylated goat anti-human Tie-2 polyclonal antibodies
and biotinylated normal goat IgG were also purchased from R&D Systems.
Mouse anti-human/mouse Tie-2 monoclonal antibody (mAb), rat anti-mouse
CD31 (platelet/endothelial cell adhesion molecule 1) mAb, APC-conjugated
streptavidin, and biotinylated rabbit anti-rat IgG polyclonal antibodies were
purchased from PharMingen (San Diego, CA). FITC-conjugated donkey antimouse and anti-human IgG polyclonal antibodies, Cy5-conjugated donkey
anti-rat IgG polyclonal antibodies, horseradish peroxidase–conjugated goat
anti-mouse IgG polyclonal antibodies, and rhodamine red-X–conjugated
streptavidin were from Jackson ImmunoResearch Laboratories (West Grove,
PA). High-affinity rat anti-hemagglutinin (HA) mAb 3F10 was from Roche
Diagnostics (Indianapolis, IN). Mouse anti-HA mAb was from Covance
Research Products (Denver, PA). Horseradish peroxidase–conjugated goat
anti-human n light-chain polyclonal antibodies and mouse IgG1 mAb were
from Southern Biotechnology Associates (Birmingham, AL). Protein A tagged
with 10-nm gold was provided by J. Slot (Department of Cell Biology,
University of Utrecht, Utrecht, the Netherlands).
Chimeric rabbit/human F(ab) 1S05 and 2S03 against the extracellular
domain of Tie-2 were generated as described previously (32).
Analysis of Tie-2 Binding in ELISA. Costar 96-well ELISA plates
(Corning, Acton, MA) were coated with 100 ng antigen (human or mouse
Tie-2 or bovine serum albumin) in 25 AL PBS and incubated overnight at
4jC. After blocking with 150 AL TBS/3% bovine serum albumin for 1 hour at
37jC, 50 AL of 2 Ag/mL F(ab) 1S05, 2S03, or control mAb solution was added
into each well, and the plates were incubated for 2 hours at 37jC. Washing
and detection were done essentially as described (33) using horseradish
peroxidase–conjugated goat anti-human n or anti-mouse IgG antibody
(diluted 1:2,500 in TBS/1% bovine serum albumin).
Conversion of a Tie-2-Specific F(ab) 2S03 into a scFv. Specific
oligonucleotide primers were used to amplify VH and VL gene segments from
purified phagemid DNA of F(ab) 2S03 (34). VL was amplified with extompseq
(5V-GCGGAGGAGCTTGCTAGCTGCGAGAAGACAGCTATCGCGATTGCATGT-3V) and RJEO-BL. VH was amplified with RSCVH3 and HSCG1234-B.
Overlap extension PCR was done using primers ext and RSC-B. The resulting
overlap-PCR product encoded a scFv in which the COOH-terminal VL region
www.aacrjournals.org
973
Cancer Res 2005; 65: (3). February 1, 2005
Cancer Research
experiment was done with SW1222 tumor cells (2 106 cells per mouse),
except three injections of 108 plaque-forming units per mouse were
administrated on days 7, 14, and 21, and the animals were sacrificed on
day 24. Results are reported as means F SD for each group.
Immunohistochemistry and Microvessel Density Determination.
Immunohistochemical localization was done with rat anti-mouse CD31
using a Vectastain Elite Peroxidase kit (Vector Laboratories, Inc.,
Burlingame, CA). Tumors represented each group of treated animals were
freshly frozen in OCT solution and stored at 80jC. Cryostat sections (6 Am)
were cut, fixed in 3.7% paraformaldehyde, and blocked with 0.3% H2O2, 5%
goat serum, and avidin-biotin blocking reagents (Vector Laboratories). With
PBS washes between all steps, mouse endothelial cells were detected with rat
anti-mouse CD31 (platelet/endothelial cell adhesion molecule 1) mAb at 0.5
Ag/mL (incubation overnight at 4jC) followed by mouse-adsorbed
biotinylated goat anti-rat IgG polyclonal antibodies at 5 Ag/mL (incubation
for 1 hour at room temperature) and visualized using Avidin-BiotinPeroxidase Complex kit from Vector Laboratories. Peroxidase activity was
revealed by the 3,3V-diaminobenzidine (Vector Laboratories) cytochemical
reaction. Sections were then weakly counterstained with hematoxylin
(Vector Laboratories) and mounted with VectaMount (Vector Laboratories).
The microvessel density was determined by digitally imaging five fields of
the tumor stroma that showed the highest vascularity [hotspots; at 100
magnification with a CCD1317 camera (Princeton Instruments, Inc.,
Trenton, NJ) mounted on a Axiovert 100 microscope (Carl Zeiss, Inc.,
Thornwood, NY)] and processing them with Abode Photoshop 7.0 software
(Adobe Systems, Mountain View, CA) as follows: (CD31-stained pixels per
field) / (total pixels per field) 100%. The final count per group represents
the average (mean F SD) of five fields.
Statistics. Tumor volume, tumor weight, and percentage of CD31positive cells were compared by using two-tailed Student’s t test.
Differences were considered statistically significant at P < 0.05.
phosphate buffer, and embedded in gelatin. Cryosections (f100 nm) were
cut and mounted on nickel mesh grids, and each grid was processed on
individual droplets of the solutions [50 mmol/L glycine, 10% FCS in PBS,
and primary mouse anti-HA mAb diluted in 5% FCS (controls grids were
incubated in an irrelevant mouse IgG1 mAb)] at room temperature, washed
in 0.2% FCS, and incubated in protein A tagged with 10-nm gold. Grids were
then washed in PBS, fixed in 1% glutaraldehyde in PBS, washed in dH2O,
and contrasted in uranyl oxalate (pH 7). Each individual grid was then
picked up in ice-cold uranyl acetate/methyl cellulose mixture (pH 4). Once
dry, the grids were examined on the CM100 microscope and documented as
described above.
In vitro Cell Proliferation Assay. A total of 1 103 (SLK), 2 103
(HUVEC), 2.5 103 (SW1222), or 5 103 (MAEC) cells per well in a 96-well
tissue culture plate (type I collagen-coated 96-well plate for HUVEC) were
infected with various amount of different adenoviruses ranging from 0.5 to
500 MOI, and plate was incubated at 37jC for 64 hours (72 hours for
HUVEC) in a humidified CO2 incubator. [3H]thymidine (ICN Radiochemicals, Irvine, CA) was added to 0.5 ACi/well (1 Ci = 37 GBq) during
the last 16 hours of incubation (24 hours for HUVEC). The cells were frozen
at 80jC overnight and subsequently processed on a multichannel
automated cell harvester (Cambridge Technology, Cambridge, MA), and
the [3H]thymidine incorporated into DNA was determined by in a liquid
scintillation h counter (Beckman Coulter, Fullerton, CA). The background
was defined by running the same assay in the absence of adenovirus. The
inhibition in experiment E was calculated according to the formula:
(background
E) / background 100%. All experiments were done in
triplicate.
In vivo Tumor Growth. Confluent cultured tumor cells (SLK and
SW1222) were harvested by incubation with 5 mL trypsin solution (0.25%).
Viable cells were counted by trypan blue exclusion. Fractions containing
>95% of viable cells were used in this study. SLK cells were suspended in
PBS, and 0.2 mL (3 106 cells per mouse) of the suspension was
inoculated s.c. into right flanks of 20 nude mice on day 0. Tumors were
allowed to establish for 6 days. On day 7, four groups of five mice were
formed, and each mouse received 1.5 108 plaque-forming units per
mouse (50 MOI per initial tumor load) of adenovirus-encoded intrabody
(pAd-2S03, pAd-1S05, or pAd-JC7U) or control adenovirus (pAd-GFP)
administrated peritumorally in 70 AL PBS on days 7, 14, 21, and 28. The
resulting tumor was measured over the skin in two dimensions using
a slide caliper, and the tumor volume was calculated according to the
formula: 0.5(width)2 length. All animals were sacrificed after 45 days,
and the tumors were completely dissected and then weighed. The same
Results
Characterization of Rabbit Antibody Fragments and Binding
to Tie-2. F(ab) 1S05 and 2S03 were subcloned and overexpressed in
Escherichia coli with subsequent purification to yield nearhomogenous protein solutions (data not shown). ELISA was used
to check for cross-reactivity of rabbit F(ab) 1S05 and 2S03 with
human and mouse Tie-2. F(ab) 1S05 bound only to human Tie-2
and did not reveal any cross-reactivity with mouse Tie-2. In
contrast to F(ab) 1S05, F(ab) 2S03 was found to bind both human
Figure 1. Analysis of F(ab) 1S05 and 2S03 binding to Tie-2 receptors. A, signal intensity of Tie-2 receptor-specific binders. Specific binding of selected F(ab) to
human and mouse receptors was measured by ELISA as described in MATERIALS AND METHODS. B, HUVECs and MAECs were stained with the anti-Tie-2
antibody (bold line ), irrelevant F(ab) (thin line ), and secondary antibody alone (dashed line ). Bound antibodies were detected with FITC-conjugated donkey
anti-human (1S05 and 2S03 ) or anti-human/mouse (m/h Tie-2 ) IgG.
Cancer Res 2005; 65: (3). February 1, 2005
974
www.aacrjournals.org
Intrabodies for Tie-2 Phenotypic Knockout
and mouse Tie-2 (Fig. 1A). Flow cytometry analysis further revealed
that F(ab) 2S03 bound to both HUVECs, which express native
human Tie-2 on their surface, and to MAECs, which express a very
low level of mouse Tie-2 (Fig. 1B). In contrast, F(ab) 1S05 was able
to bind HUVECs but not to MAECs (Fig. 1B), thus confirming
previous ELISA results demonstrating cross-reactivity within the
Tie-2. It should be emphasized that this anti-Tie-2 cross-reacting
rabbit F(ab) 2S03 was generated and selected using human Tie-2
for both immunization and panning (32). Despite using human Tie2 for immunization, rabbit F(ab) 2S03 binds to the mouse antigen
as well. The affinity of F(ab) 2S03 for the human or mouse Tie-2
was determined by surface plasmon resonance. The rabbit F(ab)
2S03 was found to bind human and mouse Tie-2 with a monovalent
affinity of 6.8 and 9.6 nmol/L (Supplementary Table S1). The K d for
F(ab) 1S05 for human Tie-2 was described previously and equal to
15 nmol/L (32). The relative ease with which cross-reacting
antibodies can be selected points at the potential of the rabbit
antibody repertoire as a source for therapeutic antibodies.
Tie-2 Surface Depletion with Intrabodies. F(ab) 2S03 was
converted into scFv in which the VL and VH fragments were
covalently linked with a peptide linker consisting of 18 amino acids.
Antigen binding was confirmed by ELISA (data not shown). Next,
the 2S03 scFv against Tie-2 was cloned into a modified adenovirus
shuttle vector, pAd-TrackCMV.
HUVECs were infected with 10 MOI of adenovirus encoding Tie2-specific intrabody (pAd-1S05 or pAd-2S03) or, as a control, with
an intrabody against integrin avh3 (pAd-JC7U). Surface Tie-2
expression was monitored for 7 days by flow cytometry as
described in MATERIALS AND METHODS (Fig. 2A). More than
90% of the infected HUVECs showed GFP expression, suggesting
that virtually all infected cells expressed the vector-delivered
intrabody (data not shown). At 24 hours postinfection, the surface
expression of human Tie-2 on HUVECs infected with the 1S05
intrabody was blocked specifically up to 62% (Fig. 2A). On day 2
after infection, Tie-2 expression was reduced by 95%. Seven days
after infection, surface expression remained efficiently blocked up
to 92%. In comparison, the surface expression of human Tie-2 on
HUVECs infected with the 2S03 intrabody was only blocked with
25% efficiency on day 1. However, on day 3, Tie-2 surface depletion
reached a maximum of 82% and remained >65% inhibition through
day 7 postinfection (Fig. 2). No inhibition of Tie-2 expression was
observed for the cells infected with JC7U intrabody (Fig. 2). These
results underscore the ability of ER-targeted intrabodies 1S05 and
2S03 to down-regulate the HUVEC surface receptor Tie-2.
Colocalization of Intrabody and Tie-2 Protein in the ER. To
investigate whether the intrabody constructs were expressed in the
ER and able to trap the targeted proteins in the same compartment,
HUVECs were infected with the adenovirus-encoding intrabodies
(pAd-1S05 and pAd-2S03; 50 MOI). As a control, HUVECs were
infected with pAd-JC7U. Representative data from deconvolution
microscopy for 1S05, 2S03, and JC7U scFv staining on day 3 after
infection are shown in Fig. 3A. To visualize the intrabodies,
intracellular staining was carried out with rat anti-HA-tag mAb
followed by donkey anti-rat Cy5 polyclonal antibodies. As expected,
all scFv showed significant intracellular accumulation in the ER as
indicated by a staining of the characteristic tubular network (Fig. 3A,
b, e, and h). To confirm that the targeted proteins (Tie-2) were
trapped in the ER, cells were costained with biotinylated anti-Tie-2
polyclonal antibodies followed by streptavidin-rhodamine red-X.
As shown in Fig. 3A and in the three-dimensional movies (see
Supplementary Data), the intrabody against Tie-2 was found to be
www.aacrjournals.org
Figure 2. Kinetics of surface expression of Tie-2. A, kinetics assay for different
days after gene transfer of the intrabodies on HUVEC using recombinant
adenoviruses with a MOI of 10. Surface expression of Tie-2 was specifically
blocked over a period of >7 days by intrabodies against Tie-2 (1S05 and 2S03)
but not by anti-integrin avh3 intrabody JC7U. B, Tie-2 down-regulation on day 3
after infection. HUVECs were infected with a MOI of 10 with two anti-Tie-2
intrabodies, 1S05 and 2S03. Controls consisted of an anti-integrin avh3 intrabody
JC7U and mock-infected cells (data not shown). Flow cytometry histograms
show the binding of anti-Tie-2 (bold line ) biotinylated polyclonal antibodies to
HUVECs in intrabody-infected cells. Normal goat IgG was used as primary
control antibody (thin line ). Dashed line, Tie-2 expression by mock
adenovirus-infected HUVEC. Streptavidin-APC was used for detection.
colocalized with targeted proteins in the ER. The control intrabody
(JC7U), although expressed in the ER, did not retain Tie-2. These
data show that an intrabody with an ER localization signal is
sufficient to retain a membrane protein.
Intracellular Localization of Intrabodies. To confirm that our
intrabody constructs were expressed in the ER, HUVECs were
infected with the adenovirus-encoding intrabodies (pAd-1S05, pAd2S03, and pAd-JC7U; 50 MOI). As a control, HUVECs were infected
with a vector alone (pAd-GFP). Representative data from immunogold electron microscopy, which is the technique of choice for
precise subcellular localization, revealed that on day 3 after
infection most of the 10-nm gold particles were present in the
ERs of 1S05, 2S03, and JC7U pAd-infected HUVECs (Fig. 3B). No
signal was detected in the nucleus (data not shown). On the other
hand, the control infection with the vector alone (pAd-GFP) did not
reveal any ER staining.
Intrabodies Effect on Tumor Cell Proliferation. We next
sought to evaluate general cytotoxicity from adenoviral infection
and the cytotoxicity related to scFv expression in ER. For this
analysis, HUVEC, MAEC, SW1222, and SLK cells were treated
in vitro with adenovirus encoding the ER-directed anti-Tie-2 scFv
(pAd-1S05 and pAd-2S03), anti-avh3 scFv (pAd-JC7U), or an
adenovirus encoding the GFP reporter gene (pAd-GFP). Adenoviral
vector-infected cells were examined for viability using the
proliferation assay described in MATERIALS AND METHODS.
Using this assay, it was observed that scFv accumulation in the ER
results in an elevated inhibition of proliferation in all the cell lines
tested when compared with control pAd-GFP (Fig. 4A). The most
sensitive cell line was HUVEC, where a MOI of 5 resulted in a >20%
975
Cancer Res 2005; 65: (3). February 1, 2005
Cancer Research
Figure 3. Localization of adenovirus-delivered intrabodies. A,
maximal projections of three-dimensional data sets show the intrabody
and Tie-2 colocalization in HUVEC. Cells were infected with a MOI
of 50 with the intrabodies against Tie-2, 1S05 (d-f ), and 2S03 (g-i) and
a control intrabody against integrin avh3, JC7U (a-c ). Left, distribution
of Tie-2 in red; middle, intrabodies in blue ; right, overlapping signal
between Tie-2 (red ) and intrabody (blue ) in pink and cell nuclei in
green . Tie-2/intrabody colocalization was observed in cells expressing
1S05 and 2S03 (f and i, pink ). Note the complete overlap in signal.
By contrast, a homogenous distribution of Tie-2 in control
intrabody-infected HUVECs and a minimal overlap in signal were
observed (c ). For three-dimensional images of c , f , and i, see
Supplementary Movies S1-S3. B, immunolocalization of intrabodies in
HUVEC by electron microscopy. Cells were cultured in 35-mm dishes,
fixed, and processed for immunoelectron microscopy as described in
MATERIALS AND METHODS. Intrabodies were detected with
anti-HA mAb and 10-nm colloidal gold-conjugated protein A.
Localization of intrabodies can be seen in ER of HUVEC infected with
scFv-containing adenovirus (JC7U, 1S05, and 2S03) and not in
HUVEC infected with empty vector (GFP). Bar, 200 nm.
observation suggests that the elevated cytotoxicity of adenoviral
vectors encoding intrabodies could be attributed to extensive scFv
accumulations in the ER.
In vivo Localization of the scFv Gene after Peritumoral
Vector Delivery. We next sought to establish the localization of
transduced cells following peritumoral administration of the
adenoviral vector. Nude mice bearing SLK tumors were injected
peritumorally with 1.5 108 viral particles of pAd-2S03, pAd-1S05,
pAd-JC7U, or pAd-GFP. Six days later, the animals were sacrificed
and DNA was extracted from lung, heart, liver, spleen, stomach,
small intestine, kidney, and brain. PCR analysis was used to detect
the presence of the scFv and GFP-encoding sequences. A previous
study had shown that target sequences are detectable at a level
of one gene copy per 10 pg of total DNA (40). Identical levels of the
b-actin gene were detected in all mouse normal and tumor tissues,
inhibition of proliferation. Colon carcinoma cell line SW1222 was
less affected by intrabody accumulation in the ER, with 32% to 58%
inhibition observed at a MOI of 50 (Fig. 4A). However, a MOI of 50
did only slightly inhibit the proliferation of MAEC and human
tumor SLK cells (<20% and <10%, respectively). Because low
cytotoxicity of intrabodies was usually observed at MOI 50, we
selected this concentration for antiangiogenic studies in vivo.
To further investigate the nature of pAd-mediated cytotoxicity,
HUVEC treated with 50 MOI of pAd-1S05, 2S03, JC7U, and GFP
in vitro were analyzed by TEM. Image analysis of high-magnification
TEM micrographs ( five for each cell culture conditions) allowed us
to identify a pronounced swelling of ERs in 10% to 15% of the cells
treated with pAd-1S05, 2S03, and JC7U (Fig. 4B, arrows). The
ER in GFP-treated cells as well as the nuclear and mitochondrial morphology in all cells remained unaffected (Fig. 4B). This
Cancer Res 2005; 65: (3). February 1, 2005
976
www.aacrjournals.org
Intrabodies for Tie-2 Phenotypic Knockout
to be within normal limits with no pathologic changes in the
vasculature of these organs (Supplementary Fig. S2).
Antiangiogenic Activity of 2S03 Intrabody In vivo. The ability
of F(ab) 2S03 to recognize mouse endothelial cell receptor Tie-2
enabled us to use this adenovirus-encoded scFv intrabody for an
experimental antiangiogenic therapy in xenograft tumor models.
To determine whether administration of pAd-2S03 could inhibit the
growth rate of well-established primary tumors, two human tumor
cell lines, SLK Kaposi sarcoma and SW1222 colon carcinoma, were
used. SLK, an avh3 integrin-positive tumor cell line, is of interest
because of its endothelial origin. In this analysis, tumor cells (3 106 in 100 AL PBS) were implanted s.c. into the right flank of female
nude mice on day 0 followed by the peritumoral administrations of
the control adenovirus pAd-GFP or the adenovirus encoding the
anti-Tie-2 scFv pAd-1S05 and pAd-2S03 as well as the anti-avh3
scFv pAd-JC7U adenovirus. Four injections of 1.5 108 plaqueforming units each were given on days 7, 14, 21, and 28 post-tumor
verifying the quality of DNA in these samples. The presence of scFv
and/or GFP genes was restricted to the tumor tissue as shown by
gel electrophoresis (Supplementary Fig. S1). All tested mouse
organs failed to show the presence of scFv/GFP sequences at the
limits of detection.
Toxicity Study with Adenoviral Vector Encoding scFv Intrabody. A toxicity study was carried out with the adenoviral vector to
determine any potential toxicity associated with vector delivery of
intrabody genes. For this study, SLK tumor-bearing nude mice, ages
8 weeks, were injected peritumorally four times with 1.5 108
plaque-forming units per mouse of pAd-2S03, pAd-1S05, pAd-JC7U,
or pAd-GFP on days 7, 14, 21, and 28. On day 34, animals were
sacrificed and underwent autopsy. On pathologic examination,
none of the animals showed any detectable pathologic changes,
and all organs were appropriately placed without evidence of
abscess formation, necrosis zones, or infarction. In addition, the
major organs were retained for histologic examination and found
Figure 4. Cytotoxicity of adenovirus-delivered intrabodies.
A, cells seeded in 96-well plate were infected with different
recombinant adenoviruses. Three days after infection,
the inhibition of cell proliferation was examined by a
[3H]thymidine incorporation assay. Points, mean; bars,
SD. B, differences in ER morphology of HUVEC were
examined by TEM. Nuclear and mitochondrial
morphologies are normal in all cells. Swollen ERs
(arrows ) can be seen in HUVEC infected with
scFv-containing adenovirus (JC7U, 1S05, and 2S03)
but not in HUVEC treated with GFP-encoded
adenovirus (GFP). Bar, 200 nm.
www.aacrjournals.org
977
Cancer Res 2005; 65: (3). February 1, 2005
Cancer Research
Figure 5. pAd-2S03 inhibition of
the growth of primary tumors.
Cultured SLK or SW1222 cells
were inoculated s.c. into nude mice
on day 0. After a palpable tumor
was observed (day 6), the mice
were divided randomly into four
groups of five mice each.
Adenovirus encoding 1S05, 2S03,
and JC7U scFv or control
adenovirus (GFP) was
administrated peritumorally
according to a schedule described
in MATERIALS AND METHODS.
A, tumor volume from five growing
tumors per group measured at
3-day intervals from 6 to 45 days
postgrafting for SLK xenografts
or from 6 to 24 days postgrafting
for SW1222 xenografts. Points,
mean; bars, SD. B, tumor weight.
Columns, mean; bars, SD. *,
P < 0.001, compared with GFP;
**, P < 0.01, compared with GFP.
human Tie-2 or for avh3-specific pAd-JC7U intrabody, confirming
that the antitumor effect of the cross-reactive pAd-2S03 intrabody
could be attributed to the surface depletion of Tie-2 from mouse
endothelial cells infiltrating the human tumor.
In view of our findings and work showing that inhibition of Tie-2
signaling prevents tumor growth (23), it is tempting to speculate
that reduced expression of Tie-2 may contribute to growth
inhibition in angiogenesis-dependent tumors. These results suggest
that inhibition of tumor angiogenesis by pAd-2S03 limited tumor
growth.
Determination of Tumor Microvessel Density and Tumor
Cell Apoptosis. To establish the mechanism underlying the
inhibition in tumor growth, the effect of Tie-2 receptor downregulation on tumor vascularization was evaluated by the
determination of microvessel density. Cryosections were stained
for murine CD31, and the number of discrete microvessels was
compared in areas showing the highest vascularity (hotspots) of the
tumor stroma. Strong CD31 expression was observed at the edge of
the SLK tumors as well as in neovessels penetrating the substance
of the tumor (Fig. 6A). A similar pattern of CD31 localization was
also observed in human SW1222 tumor, in which the strongest
CD31 expression was at the tumor boundary in regions of extensive
tumor neovascularization (Fig. 6B). These results are consistent
with capillary sprouting from surrounding mouse normal tissues,
tumor vessel elongation, and/or formation of connections between
tumor vessels and host vessels. CD31 expression was remarkably
inhibited in both tumors when treated with 2S03 intrabody,
whereas 1S05 and JC7U intrabody treatments did not affect
angiogenesis in surrounding mouse normal tissues. The tumor
vascularization index was measured from photomicrographs of
cell inoculation. The mean tumor size of all treated mice was
monitored from days 6 to 45. As depicted in Fig. 5, all injected
animals developed palpable tumors within 6 days. Beginning at day
18, a reduced tumor growth rate was observed in the pAd-2S03treated group when compared with the control group of mice
treated with pAd-GFP. On average, 69% reduction in tumor growth
was observed by day 45 in the pAd-2S03-treated group relative to
the pAd-GFP-treated group of mice. Repeated inoculation of
human Tie-2-specific intrabody pAd-1S05 in mice bearing SLK
tumors did not result in lower tumor sizes compared with the
control group. The observed antitumor activity of pAd-JC7U
intrabody (f30%) could be due to specific action of the avh3
down-regulation on tumor cells. No side effects, such as loss of
body weight, were observed in mice treated with any of the
antibodies. Tumor weights were determined at sacrifice in all
animals on day 45 post-tumor injection and confirmed the
reduction in tumor growth during intrabody treatment.
The ability of the pAd-2S03 vector to modulate tumor-induced
angiogenesis was also evaluated using avh3-negative tumor cell line
SW1222 (Fig. 5). For this study, nude mice were xenografted s.c. on
day 0 with 2 106 SW1222 cells into the right flank. After the
establishment of tumor, the animals were peritumorally treated
with the same intrabodies as above, except three injections of 108
plaque-forming units were given on day 7, 14, and 21 post-tumor
cell inoculation. Xenografts raised from SW1222 tumor cells grew
significantly faster than SLK tumors (Fig. 5). The treatment with
recombinant adenovirus encoding the cross-reactive pAd-2S03
intrabody significantly inhibited tumor growth when compared
with the control adenovirus pAd-GFP. In addition, no inhibition of
tumor growth was observed for the pAd-1S05 intrabody targeting
Cancer Res 2005; 65: (3). February 1, 2005
978
www.aacrjournals.org
Intrabodies for Tie-2 Phenotypic Knockout
angiogenesis, the ability to measure any decrement in tumor
vascular density strongly suggests that the primary action of the
2S03 intrabody was to inhibit tumor neovascularization. Thus,
intracellular expression of 2S03 led to reduced tumor vascularization through reduced Tie-2 surface expression.
CD31-stained tumors. Quantification of microvessel density
revealed a statistically significant (P < 0.01) 8- to 8.5-fold reduction
of microvessel density in pAd-2S03-treated tumors, whereas a
similar reduction was not observed in either pAd-JC7U- or pAd1S05-treated tumors (Fig. 6, top right). These results indicate that
the Tie-2 pathway is essential for the vascularization and tumor
growth of both SLK and SW1222 xenografts.
TUNEL staining of tumor sections from the four groups of SLK
tumor-bearing animals revealed no difference in apoptosis and
tumor necrosis among the four groups (Supplementary Fig. S3).
Therefore, apoptosis was not a factor in tumor growth inhibition.
Considering the strong link between tumor growth and tumor
Discussion
Because angiogenesis is required for the growth and metastasis
of solid tumors, we hypothesized that an ER-targeted intrabody
with Tie-2 specificity could be beneficial to the inhibition of
tumor angiogenesis and should provide a practical approach to
Figure 6. Effect of Tie-2 down-regulation
on the vascularization of transplanted
tumors. Cryostat sections of tumor grown
from SLK (A ) or SW1222 (B) xenografted
mice were stained for the mouse
endothelial marker CD31. Control (GFP,
JC7U, and 1S05) treated tumors
developed a substantial intratumor
vasculature and an obvious
peritumor-hypervasculature border zone.
In contrast, 2S03-treated tumors
possessed a sparse vasculature with no
obvious hypervascular border zone.
Angiogenic index of tumor vascularization
(top right ) was calculated as described
in MATERIALS AND METHODS and
expressed as the percentage of
CD31-stained area. Tumor
neovascularization was significantly
inhibited by 2S03 intrabody (P < 0.001
versus GFP). Mean F SD (n = 5). Bar,
50 Am for all sections.
www.aacrjournals.org
979
Cancer Res 2005; 65: (3). February 1, 2005
Cancer Research
lack the support of underlying periendothelial mesenchymal cells,
which implicates a predominant role for Tie-2 in vessel maturation
and maintenance. This results in collapse of the endothelial cells
into the lumen of vessels that were preformed by tissue fold and
vessel occlusion (45).
Disruption of angiogenesis via Tie-2 down-regulation may be
globally useful in diseases that result in pathologic neovascularization. Interestingly, both tumor cell lines used in this study
produce VEGF.4 The ability of pAd-2S03 to inhibit angiogenesis
despite the presence of VEGF suggests that disrupting the
angiogenic program at stages distal to endothelial activation will
provide effective inhibition of neovascularization. One potential
limitation of this study is that only partial tumor growth
inhibition was achieved. This result indicates that blocking Tie-2
activation alone may not be sufficient to completely halt tumor
angiogenesis. Because the VEGF pathway also is critical for
tumor angiogenesis, future work will test the efficacy of a
combined delivery of the Tie-2 intrabody and the VEGFR2
intrabody to knockout the cell surface expression of both
proteins simultaneously (35).
long-term control of disease. A gene therapy strategy was
designed to deliver a Tie-2-specific intrabody, pAd-2S03, and
the importance of the Tie-2 receptor tyrosine kinase pathways for
tumor angiogenesis in vivo was analyzed using human Kaposi’s
sarcoma and colon carcinoma xenograft models. Treatment with
adenovirus-delivered anti-mouse Tie-2 intrabody 2S03 resulted in
a 75% reduction in Kaposi’s sarcoma tumor growth over a period
of 45 days and a 63% reduction in colon carcinoma tumor
growth over a period of 24 days. The inhibition of tumor growth
was likely secondary to a blockade in tumor angiogenesis. This
notion is supported by the decreased vascular density in the
pAd-2S03-treated tumors and the ability of 2S03 antibody to
recognize mouse Tie-2. These findings provide the first
demonstration of the anti-Tie-2 intrabody effect on tumor
angiogenesis and suggest that therapeutic approaches similarly
designed to inhibit the Tie-2 pathway could be clinically useful.
For example, other transcriptional or post-transcriptional
approaches, such as RNA interference and zinc finger transcription factors, may be promising alternative strategies to our
intrabody-based approach. It is important to note that the
phenotypic outcome of receptor antagonism and down-regulation/knockout need not be the same as studies of the integrins
avh3 and avh5 have revealed (41, 42). Significantly, we did not
find any evidence for adverse effects of Tie-2 down-regulation
after peritumoral delivery of the 2S03 intrabody, suggesting that
safe therapeutic regimens are possible despite the expression of
Tie-2 in normal vasculature.
The molecular mechanism by which pAd-2S03 blocks tumor
angiogenesis is not yet clear. However, based on several previously
published results, it is likely that pAd-2S03 blocks tumor
angiogenesis at a step distal to endothelial cell activation (18, 43,
44). This is supported by the results of gene-targeting experiments
of Tie-2 (18, 43) that revealed indispensable but distinct functions
of the receptor pathway. In Tie-2-deficient mice, endothelial cells
Acknowledgments
Received 6/15/2004; revised 11/15/2004; accepted 11/16/2004.
Grant support: NIH grant RO1-CA086258.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Jody D. Berry for discussion. The TEM and immunoelectron
microscopy experiments were carried out by Malcolm R. Wood and Theresa Fassel in
the TSRI Core Microscopy Facility.
4
N. Jendreyko and M. Popkov, unpublished data.
1. Folkman J. What is the evidence that tumors are
angiogenesis dependent? J Natl Cancer Inst 1990;82:4–6.
2. Ferrara N, Gerber H-P, LeCouter J. The biology of
VEGF and its receptors. Nat Med 2003;9:669–76.
3. Zogakis TG, Libutti SK. General aspects of antiangiogenesis and cancer therapy. Expert Opin Biol Ther
2001;1:253–75.
4. Millauer B, Longhi MP, Plate KH, et al. Dominantnegative inhibition of Flk-1 suppresses the growth of
many tumor types in vivo . Cancer Res 1996;56:1615–20.
5. Goldman CK, Kendall RL, Cabrera G, et al. Paracrine
expression of a native soluble vascular endothelial
growth factor receptor inhibits tumor growth, metastasis, and mortality rate. Proc Natl Acad Sci U S A
1998;95:8795–800.
6. Partanen J, Dumont DJ. Functions of Tie1 and Tie2
receptor tyrosine kinases in vascular development. Curr
Top Microbiol Immunol 1999;237:159–72.
7. Davis S, Aldrich TH, Jones PF, et al. Isolation of
angiopoietin-1, a ligand for the TIE2 receptor, by
secretion-trap expression cloning. Cell 1996;87:1161–9.
8. Maisonpierre PC, Suri C, Jones PF, et al. Angiopoietin2, a natural antagonist for Tie2 that disrupts in vivo
angiogenesis. Science 1997;277:55–60.
9. Hanahan D. Signaling vascular morphogenesis and
maintenance. Science 1997;277:48–50.
10. Davis S, Yancopoulos GD. The angiopoietins: Ying
and Yang in angiogenesis. Curr Top Microbiol Immunol
1999;237:173–85.
11. Holash J, Wiegand SJ, Yancopoulos GD. New model of
tumor angiogenesis: dynamic balance between vessel
regression and growth mediated by angiopoietins and
VEGF. Oncogene 1999;18:5356–62.
12. Thurston G, Rudge JS, Ioffe E, et al. Angiopoietin-1
protects the adult vasculature against plasma leakage.
Nat Med 2000;6:460–3.
13. Yancopoulos GD, Davis S, Gale NW, Rudge JS,
Wiegand SJ, Holash J. Vascular-specific growth factors
and blood vessel formation. Nature 2000;407:242–8.
14. Sato TN, Tozawa Y, Deutsch U, et al. Distinct roles of
the receptor tyrosine kinase Tie-1 and Tie-2 in blood
vessel formation. Nature 1995;376:70–4.
15. Wong AL, Haroon ZA, Werner S, Dewhirst MW,
Greenberg CS, Peters KG. Tie2 expression and phosphorylation in angiogenic and quiescent adult tissues.
Circ Res 1997;81:567–74.
16. Peters KG, Coogan A, Berry D, et al. Expression of
Tie2/Tek in breast tumour vasculature provides a new
marker for evaluation of tumour angiogenesis. Br J
Cancer 1998;77:51–6.
17. Stratmann A, Risau W, Plate KH. Cell type-specific
expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis. Am J Pathol
1998;153:1459–66.
18. Lin P, Polverini P, Dewhirst M, Shan S, Rao PS, Peters
K. Inhibition of tumor angiogenesis using a soluble
receptor establishes a role for Tie2 in pathologic
vascular growth. J Clin Invest 1997;100:2072–8.
19. Lin P, Buxton JA, Acheson A, et al. Antiangiogenic
gene therapy targeting the endothelium-specific recep-
Cancer Res 2005; 65: (3). February 1, 2005
980
References
tor tyrosine kinase Tie2. Proc Natl Acad Sci U S A
1998;95:8829–34.
20. Siemeister G, Schirner M, Weindel K, et al. Two
independent mechanisms essential for tumor angiogenesis: inhibition of human melanoma xenograft
growth by interfering with either the vascular endothelial growth factor receptor pathway or the Tie-2
pathway. Cancer Res 1999;59:3185–91.
21. Stratmann A, Acker T, Burger AM, Amann K, Risau
W, Plate KH. Differential inhibition of tumor angiogenesis by tie2 and vascular endothelial growth factor
receptor-2 dominant-negative receptor mutants. Int J
Cancer 2001;91:273–82.
22. Hayes AJ, Huang WQ, Mallah J, Yang D, Lippman
ME, Li LY. Angiopoietin-1 and its receptor Tie-2
participate in the regulation of capillary-like tubule
formation and survival of endothelial cells. Microvasc
Res 1999;58:224–37.
23. White RR, Shan S, Rusconi CP, et al. Inhibition of rat
corneal angiogenesis by a nuclease-resistant RNA
aptamer specific for angiopoietin. Proc Natl Acad Sci
U S A 2003;100:5028–33.
24. Niu Q, Perruzzi C, Voskas D, Lawler J, Dumont DJ,
Benjamin LE. Inhibition of Tie-2 signaling induces
endothelial cell apoptosis, decreases Akt signaling, and
induces endothelial cell expression of the endogenous
anti-angiogenic molecule, thrombospondin-1. Cancer
Biol Ther 2004;3:402–5.
25. Tournaire R, Simon M-P, le Noble F, Eichmann A,
England P, Pouyssegur J. A short synthetic peptide inhibits signal transduction, migration and
www.aacrjournals.org
Intrabodies for Tie-2 Phenotypic Knockout
angiogenesis mediated by Tie2 receptor. EMBO
Reports 2004;5:1–6.
26. Marasco WA. Intrabodies: turning the humoral
immune system outside in for intracellular immunization. Gene Ther 1997;4:11–5.
27. Marasco WA, Haseltine WA, Chen SY. Design,
intracellular expression, and activity of a human
anti-human immunodeficiency virus type 1 gp120
single-chain antibody. Proc Natl Acad Sci U S A
1993;90:7889–93.
28. Beerli RR, Wels W, Hynes NE. Intracellular expression
of single chain antibodies reverts ErbB-2 transformation. J Biol Chem 1994;269:23931–6.
29. Wagner RW, Flanagan WM. Antisense technology
and prospects for therapy of viral infections and cancer.
Mol Med Today 1997;3:31–8.
30. Beerli RR, Barbas III CF. Engineering polydactyl
zinc-finger transcription factors. Nat Biotechnol
2002;20:135–41.
31. Hannon GJ. RNA interference. Nature 2002;418:
244–51.
32. Popkov M, Mage RG, Alexander CB, Thundivalappil S, Barbas III CF, Rader C. Rabbit immune
repertoires as sources for therapeutic monoclonal
antibodies: the impact of n allotype-correlated
variation in cysteine content on antibody libraries
selected by phage display. J Mol Biol 2003;325:
325–35.
www.aacrjournals.org
33. Steinberger P, Sutton JK, Rader C, Elia M, Barbas III
CF. Generation and characterization of a recombinant
human CCR5-specific antibody. A phage display approach for rabbit antibody humanization. J Biol Chem
2000;275:36073–8.
34. Barbas III CF, Burton DR, Scott JK, Silverman GJ.
Phage display: a laboratory manual. Cold Spring
Harbor (NY): Cold Spring Harbor Laboratory Press;
2001.
35. Jendreyko N, Popkov M, Beerli RR, et al. Intradiabodies, bispecific, tetravalent antibodies for the simultaneous functional knockout of two cell surface
receptors. J Biol Chem 2003;278:47812–9.
36. Steinberger P, Andris-Widhopf J, Buehler B, Torbett
BE, Barbas CF III. Functional deletion of the CCR5
receptor by intracellular immunization produces cells
that are refractory to CCR5-dependent HIV-1 infection
and cell fusion. Proc Natl Acad Sci U S A 2000;97:
805–10.
37. He T, Zhou S, da Costa LT, Yu J, Kinzler KW,
Vogelstein B. A simplified system for generating
recombinant adenoviruses. Proc Natl Acad Sci U S A
1998;95:2509–14.
38. Hitt DC, Booth JL, Dandpani V, Pennington LR,
Gimble JM, Metcalf J. A flow cytometric protocol for
titering recombinant adenoviral vectors containing
the green fluorescent protein. Mol Biotechnol 2000;
14:197–203.
39. Raposo G, Kleijmeer MJ, Posthuma G, Slot JW, Geuze
HJ. Immunogold labeling of ultrathin cryosections:
application in immunology. In: Weir DM, Blackwell
LA, Herzenberg C, editors. Weir’s handbook of experimental immunology. Ames, IA: Blackwell Publishers,
1996. p. 1–11.
40. Deshane J, Siegal GP, Wang M, et al. Transductional
efficacy and safety of an intraperitoneally delivered
adenovirus encoding an anti-erbB-2 intracellular singlechain antibody for ovarian cancer gene therapy.
Gynecol Oncol 1997;64:378–85.
41. Cheresh DA, Stupack DG. Integrin-mediated death:
an explanation of the integrin-knockout phenotype?
Nat Med 2002;8:193–4.
42. Hynes RO. A reevaluation of integrins as regulators of
angiogenesis. Nat Med 2002;8:918–21.
43. Dumont DJ, Gradwohl G, Fong GH, et al.
Dominant-negative and targeted null mutations in
the endothelial receptor tyrosine kinase, tek, reveal a
critical role in vasculogenesis of the embryo. Genes
Dev 1994;8:1897–909.
44. Suri C, Jones PF, Patan S, et al. Requisite role of
angiopoietin-1, a ligand for the Tie2 receptor, during
embryonic angiogenesis. Cell 1996;87:1171–80.
45. Patan S. TIE1 and TIE2 receptor tyrosine kinases
inversely regulate embryonic angiogenesis by the
mechanism of intussusceptive microvascular growth.
Microvasc Res 1998;56:1–21.
981
Cancer Res 2005; 65: (3). February 1, 2005