THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 278, No. 48, Issue of November 28, pp. 47812–47819, 2003
Printed in U.S.A.
Intradiabodies, Bispecific, Tetravalent Antibodies for the
S
Simultaneous Functional Knockout of Two Cell Surface Receptors*□
Received for publication, July 1, 2003, and in revised form, August 5, 2003
Published, JBC Papers in Press, August 28, 2003, DOI 10.1074/jbc.M307002200
Nina Jendreyko‡, Mikhail Popkov‡, Roger R. Beerli‡, Junho Chung‡, Dorian B. McGavern§,
Christoph Rader‡¶, and Carlos F. Barbas III‡储
From the ‡Department of Molecular Biology and the Skaggs Institute for Chemical Biology and the §Department of
Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037
The specific and high affinity binding properties of
intracellular antibodies (intrabodies), combined with
their ability to be stably expressed in defined organelles, provides powerful tools with a wide range of
applications in the field of functional genomics and gene
therapy. Intrabodies have been used to specifically target intracellular proteins, manipulate biological processes, and contribute to the understanding of their
functions as well as for the generation of phenotypic
knockouts in vivo by surface depletion of extracellular
or transmembrane proteins. In order to study the biological consequences of knocking down two receptortyrosine kinases, we developed a novel intrabody-based
strategy. Here we describe the design, engineering, and
characterization of a bispecific, tetravalent endoplasmic reticulum (ER)-targeted intradiabody for simultaneous surface depletion of two endothelial transmembrane receptors, Tie-2 and vascular endothelial growth
factor receptor 2 (VEGF-R2). Comparison of the ER-targeted intradiabody with the corresponding conventional ER-targeted single-chain antibody fragment
(scFv) intrabodies demonstrated that the intradiabody
is significantly more efficient with respect to efficiency
and duration of surface depletion of Tie-2 and VEGF-R2.
In vitro endothelial cell tube formation assays suggest
that the bispecific intradiabody exhibits strong antiangiogenic activity, whereas the effect of the monospecific scFv intrabodies was weaker. These findings suggest that simultaneous interference with the VEGF and
the Tie-2 receptor pathways results in at least additive
antiangiogenic effects, which may have implications for
future drug developments. In conclusion, we have identified a highly effective ER-targeted intrabody format
for the simultaneous functional knockout of two cell
surface receptors.
* This work was supported by a Deutsche Forschungsgemeinschaft
postdoctoral fellowship (to N. J.), by National Institutes of Health
Grants CA 86258 (to C. F. B.) and CA 94966 (to C. R.), and by an
Investigator Award from the Cancer Research Institute (to C. R.). 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.
□
S The on-line version of this article (available at http://www.jbc.org)
contains two movies.
¶ To whom correspondence may be addressed: Dept. of Molecular
Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd.,
La Jolla, CA 92037. Tel.: 858-784-2738; Fax: 858-784-2583; E-mail:
raderc@mail.nih.gov.
储 To whom correspondence may be addressed: Dept. of Molecular
Biology and the Skaggs Institute for Chemical Biology, The Scripps
Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037.
Tel.: 858-784-2738; Fax: 858-784-2583; E-mail: carlos@scripps.edu.
Antibodies can bind almost any molecule with high specificity and affinity, providing powerful biotechnological tools for
diagnostic and therapeutic applications. Advances in recombinant DNA technology have facilitated the manipulation of the
antibody genes, so that design, cloning, expression, and use of
single-chain antibodies have become routine procedures in protein engineering. The potential of single-chain antibody fragments (scFv)1 for intracellular applications, termed “intrabodies,” has been exploited in a number of laboratories (1– 8). To
date, intrabodies have been utilized for targeting proteins in a
singular fashion. Bispecific and tetravalent antibody fragments
could improve and expand the inhibitory potential of intrabodies by exhibiting increased apparent affinity for their antigen
and by being more efficient at inhibiting protein function or
intracellular trafficking (9). Intrabodies present a potent alternative to methods of gene inactivation that target at the level of
DNA or mRNA, such as antisense (10), zinc finger proteins (11),
targeted gene disruption, or the relatively new RNA interference (12). Operating at the posttranslational level, intrabodies
can be directed to relevant subcellular compartments and precise epitopes on target proteins (2, 13, 14), potentially blocking
only one out of several functions of an expressed protein. Numerous studies have reported the development of engineering
antibodies that are both multispecific and multivalent (15, 16).
Applying this knowledge, the goal of our study was to develop
an ER-targeted intrabody format for the simultaneous downregulation of two independent cell surface receptors, in order to
investigate the biological consequences of knocking down two
receptor-tyrosine kinases. To accomplish this, an ER-targeted
tetravalent antibody construct, with dual specificity, was generated. Using a recombinant adenovirus as gene delivery system, we could show that this intrabody construct, termed here
an “intradiabody,” was expressed in the ER and able to trap
both targeted proteins in the same compartment. The intradiabody targets the endothelial transmembrane receptors Tie-2
and VEGF-R2, which are essential for angiogenesis. The interplay of VEGF, VEGF-R2, Tie-2, and Ang-1 and -2 has been
suggested as a key modulator in the onset of tumor angiogenesis (17). According to this model, Tie-2 is constitutively engaged with Ang-1 in quiescent blood vessels. The Tie-2/Ang-1
complex stabilizes quiescent blood vessels by promoting their
interaction with surrounding perivascular cells, smooth muscle
cells, and the extracellular matrix. The constitutive Tie-2/
1
The abbreviations used are: scFv, single-chain antibody fragment;
ER, endoplasmatic reticulum; HUVEC, human umbilical vein endothelial cells; Ang-1 or -2, angiopoietin-1 or -2; VH, heavy chain variable
domain; VL, light chain variable domain; VEGF, vascular endothelial
growth factor; ELISA, enzyme-linked immunosorbent assay; PBS,
phosphate-buffered saline; HA, hemagglutinin; MOI, multiplicity of
infection; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein.
47812
This paper is available on line at http://www.jbc.org
Intradiabodies for Simultaneous Knockout of VEGFR2 and Tie2
47813
TABLE I
Binding parameters of chimeric rabbit/human Fab 1S05 directed to
human Tie-2 and Fab VC06 directed to human VEGF-R2
Association (kon) and dissociation (koff) rate constants were determined using surface plasmon resonance. Human Tie-2 or human
VEGF-R2 was immobilized on the sensor chip. The dissociation constant (Kd) was calculated from koff/kon.
Fab
kon/104
M
⫺1 ⫺1
s
1S05
9.8
VC06
4.6
koff/10⫺4
s
⫺1
13.5
0.53
Kd
nM
13.8
1.2
Ang-1 complex is antagonized by Ang-2, which is up-regulated
in endothelial cells that are proximal to the tumor. By competing with Ang-1 for Tie-2 binding, Ang-2 destabilizes the interaction of endothelial cells and their microenvironment. This is
thought to sensitize the endothelial cells to VEGF signaling.
Thus, Ang-2 produced by endothelial cells promotes tumor
angiogenesis in concert with VEGF produced by the tumor.
Comparison of the effect of the intradiabody (targeting Tie-2
and VEGF-R2) with the corresponding conventional scFv intrabodies (targeting Tie-2 or VEGF-R2 alone) revealed a remarkable superiority of the intradiabody, as represented by a
complete and extended surface depletion of both Tie-2 and
VEGF-R2. This finding can be attributed to the extended halflife of our intradiabody, as determined by pulse-chase studies.
In addition, we show that the intradiabody strongly inhibits
endothelial tube formation beyond that seen with inhibitors of
either receptor-tyrosine kinase alone, thus confirming its antiangiogenic properties. Our results confirm that the inhibition
of the VEGF receptor pathway cannot be compensated by the
Tie-2 pathway, nor vice versa (18), but also demonstrates that
targeting both pathways simultaneously results in additive
antiangiogenic effects in vitro. Combining the specific and high
affinity binding properties of our intradiabody with its ability
to be stably expressed in the ER, we identified a very effective
intrabody format for the simultaneous functional knockout of
two cell surface receptors.
MATERIALS AND METHODS
Cell Culture—Human umbilical vein endothelial cells (HUVEC) (BioWhittaker, Walkersville, MD) were cultured at 37 °C in 5% CO2 in EGM
medium (BioWhittaker) supplemented with 2% bovine brain extract
(BioWhittaker). 293 cells (human embryonic kidney) (ATCC) cells were
cultured at 37 °C in 5% CO2, in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and
1% antibiotics.
Library Generation and Selection—The generation and selection of
rabbit/human chimeric Fab libraries, as well as the characterization of
positive Fabs, were done essentially as described (19, 20). In brief,
rabbit V␬-, V␭-, and VH-encoding sequences were amplified from first
strand cDNA and fused to human C␬- and CH1-encoding sequences,
respectively, followed by assembly of chimeric rabbit/human light
chain- and Fd fragment-encoding sequences and by asymmetric SfiI
cloning into phagemid vector pComb3X. Libraries were panned against
VEGF-R2/VEGF complex immobilized on Costar 3690 96-well ELISA
plates (Corning, Acton, PA). Four rounds of panning (19, 21) were
carried out using 500 ng of VEGF-R2/VEGF complex in the first round,
250 ng in the second round, and 100 ng of VEGF-R2/VEGF complex in
the third and fourth rounds. To eliminate the selection of clones that
bind to the human IgG1 Fc part of the recombinant human VEGF-R2/Fc
fusion protein, 2.5 mg/ml human IgG (Pierce) was added to the phage
preparations during selection. After the final round of panning, 10
isopropyl-1-thio-␤-D-galactopyranoside-induced clones from each library were analyzed for binding to 100 ng of immobilized VEGF-R2,
human IgG, and bovine serum albumin by ELISA using a rat anti-HA
monoclonal antibody conjugated to horseradish peroxidase for detection. Positive clones were analyzed by DNA fingerprinting and sequencing as described before (20). Soluble Fab were expressed from gene III
fragment-depleted phagemid vector pComb3X and purified using goat
anti-human F(ab⬘)2 N-hydroxysuccinimide resin columns as described
FIG. 1. A bispecific, tetravalent intradiabody. Two scFv directed
to Tie-2 and VEGF-R2, respectively, were linked through the second
and third heavy chain constant domains of human IgG1 to form a
150-kDa dimer. Through the homophilic interaction of CH3, the Nterminal and C-terminal scFv modules are displayed bivalently. The
N-terminal scFv module binds Tie-2, and the C-terminal scFv module
binds VEGF-R2. An ER retention signal (KDEL) was appended
C-terminally.
(19). Library generation and selection for Tie-2 clones is described
elsewhere (20). For a control construct, designated T2V2, one clone was
selected from each generated library (VEGF-R2 and Tie-2) prior to
selection. ELISA was done the same way as for detection of binders
after panning, determining no binding to VEGF-R2 or Tie-2.
Surface Plasmon Resonance—Surface plasmon resonance for the determination of association (kon) and dissociation (koff) rate constants for
the interaction of chimeric rabbit/human Fab with Tie-2 was performed
on a Biacore instrument (Biacore AB, Uppsala, Sweden). A CM5 sensor
chip (Biacore AB) was activated for immobilization with N-hydroxysuccinimide and N-ethyl-N⬘-(3-dimethylaminopropyl)carbodiimide according to the methods outlined by the supplier. Recombinant human
VEGF-R2/Fc fusion protein was coupled at a low density (500 –1000
resonance units) to the surface by injection of 10 –20 ␮l of a 10 ng/␮l
sample in 20 mM sodium acetate (pH 3.5). Subsequently, the sensor chip
was deactivated with 1 M ethanolamine hydrochloride (pH 8.5). Binding
of chimeric rabbit/human Fab to immobilized human VEGF-R2 was
studied by injection of Fab at five different concentrations ranging from
50 to 150 nM. PBS was used as the running buffer. The sensor chip was
regenerated with 20 mM HCl and remained active for at least 20
measurements. The kon and koff values were calculated using Biacore
AB evaluation software. The equilibrium dissociation constant Kd was
calculated from koff/kon.
Conversion of a VEGF-R2 and a Tie-2-specific Fab into a Singlechain Antibody Fragment (scFv)—Specific oligonucleotide primers (19,
22) were used to amplify VH and VL gene segments from purified
phagemid DNA of Fab VC06 and Fab 1S05. VL of VC06 and 1S05 were
amplified with extompseq (5⬘-GCG GAG GAG CTT GCT AGC TGC
GAG AAG ACA GCT ATC GCG ATT GCA TGT) and RJ␭O-BL. VH of
VC06 and 1S05 were amplified with RSCVH4 or RSCVH3, respectively,
and HSCG1234-B. Overlap extension PCR was done using primers ext
and RSC-B. The resulting overlap-PCR product encodes an scFv in
which the C-terminal VL region is linked to the N-terminal VH region
through a peptide linker (SSGGGGSGGGGGGSSRSS). Control construct JC7U (against integrin ␣v␤3) was constructed as described (22).
The scFv encoding sequences were cloned into phagemid vector
pComb3X using asymmetric SfiI sites and binding activity of the expressed scFv was confirmed by ELISA.
Cloning of Diabodies and Corresponding scFv—The scFv 1S05 and
VC06 genes were linked through the second and third heavy chain
constant domains of human IgG1 to generate a bispecific diabody. ScFv
1S05 was PCR-amplified using extompseq and Tie-2-B (5⬘-GCC AGA
CCC ACC GCC TCT AGA TGA GGA GAC GGT GAC CAG GGT G-3⬘).
CH2-CH3 was amplified using CH2-F (5⬘-TCT AGA GGC GGT GGG
TCT GGC GGG GGC TCG-3⬘) and CH3-B (5⬘-CGA CTG AGT CAG CAC
GAG CTC GGC CGC CTG TGC CGA GCC ACC CCC AGA ACC-3⬘).
ScFv VC06 was amplified using VEGF-R2-F (5⬘-TCT CCG GGT GGC
GCG CCT GGT GGC GGT TCT GGC GGT GGT TCT GGG GGT GGC
TCG GCA CAG GCG GCC GAG CTC GTG CTG ACT CAG TCG CCC
TC-3⬘) and dpseq (19). ScFv 1S05 and CH2-CH3 were combined by
overlap extension PCR using primer ext (22) and CH3-B. ScFv VC06
47814
Intradiabodies for Simultaneous Knockout of VEGFR2 and Tie2
FIG. 2. Intrabody and Tie-2/VEGF-R2
colocalization on HUVEC. Maximal projections of three-dimensional data sets show
the total cellular distribution of Tie-2 (a–f)
and VEGF-R2 (g–l). HUVEC cells were infected with an MOI of 50 with the intradiabody against Tie-2 (a–c) and VEGF-R2 (g–i)
and a control intrabody against integrin
␣v␤3 (d–f, j–l). The left panels show the target
antigen in red; middle panels show intrabodies in blue; right panels show overlapping
antigen (red) and intrabody (blue) signals in
purple and nuclei in green. Antigen/intrabody colocalization was observed in intradiabody-infected cells (c and i, purple). By contrast, a homogenous antigen distribution
was observed in control intrabody-infected
HUVEC (f and l, red and blue).
was SacI/SpeI-cloned into phagemid vector pComb3X. Finally, both
constructs were combined by SacI cloning and confirmed by DNA sequence analysis. The T2 scFv was amplified using primers extompseq
and T2-B (5⬘-GCC AGA CCC ACC GCC TCT AGA TGA GGA GAC GGT
GAC CAG-3⬘), CH2-CH3 was amplified with primers CH2/T2-F (5⬘-TCT
AGA GGC GGT GGG TCT GGC GGG GGC TCG-3⬘) and CH3/V2-B
(5⬘-AGT CTG GGT CAT CAC GAG CTC GGC CGC CTG TGC CGA GCC
ACC CCC AGA ACC-3⬘), and the scFv for V2 was amplified using dpseq
and primer V2-F (5⬘-TCT GGG GGT GGC TCG GCA CAG GCG GCC
GAG CTC GTG ATG ACC CAG ACT-3⬘). scFv T2 and CH2-CH3 were
combined by overlap extension PCR using ext and CH3/V2-B. ScFv V2
was SacI/SpeI cloned into phagemid vector pComb3X. Both constructs
were then combined in the same manner as the Tie-2/VEGF-R2
intrabody.
Assembly of Intrabody Constructs in pAdTrackCMV and Generation
of Adenoviral Plasmids by Homologous Recombination—Intrabody coding regions were initially assembled in pBabePuro essentially as described (23). In these constructs, the scFv coding regions are flanked by
a human ␬ light chain leader sequence at the 5⬘-end and a sequence
encoding the HA tag (YPYDVPDYA) and the ER retention signal
(KDEL) at the 3⬘-end. The intrabody coding regions were then excised
by digestion with BamHI and SalI and ligated into pAdTrackCMV (24)
digested with BglII and SalI. This adapter fragment contains also
compatible SfiI sites, which were used for cloning the different intrabodies (intradiabody against Tie-2/VEGF-R2, control intradiabody
T2V2, scFv intrabody against Tie-2, and scFv intrabody against VEGFR2) into the adenovirus vector. The generation of recombinant adenoviruses was done essentially as described (24). High titer viral stocks
were produced and purified by CsCl banding. All virus preparations
were GFP-corrected (25).
Infection of HUVEC with Intrabodies Using Recombinant Adenovi-
ruses—1.5 ⫻ 106 HUVEC were infected with 10 MOI (⬃80% of the cells
were infected) of adenovirus encoding Tie-2/VEGF-R2-specific intradiabody, Tie-2-specific intrabody, VEGF-R2-specific intrabody, control intrabody, or no insert (mock). Tie-2 and VEGF-R2 expression of HUVEC
cells was monitored for 15 days by flow cytometry.
Flow Cytometry—HUVEC were washed once with Hepes-buffered
saline solution, trypsinized, washed once with PBS, and resuspended at
6
10 cells/ml in FACS buffer (1% (w/v) bovine serum albumin, 0.03%
(w/v) NaN3, 25 mM Hepes in PBS, pH 7.4). 105 cells were stained with
biotinylated Tie-2 or VEFG-R2 polyclonal antibodies (R&D Systems,
Minneapolis, MN) (1:200) in FACS buffer, followed by streptavidin-APC
(1:100). Incubation times were 30 min at room temperature. Flow
cytometry was performed using a FACSort instrument from BectonDickinson measuring at FL4. For control purposes, cells were also
stained with LM609 (␣v␤3) and P1F6 (␣v␤5) (Chemicon, Temecula, CA)
and detected with biotinylated donkey anti-mouse antibody followed by
streptavidin-APC. The formula for calculations of surface inhibition,
after subtracting the background of normal goat IgG-stained cells was
as follows: VEGF-R2GFP or Tie-2GFP minus VEGF-R2intrabody or
Tie-2intrabody, divided by VEGF-R2GFP or Tie-2GFP.
Immunocytochemical Analysis of Antigen/Intrabody Colocalization—
For analysis of Tie-2, VEGF-R2, and intrabodies on GFP-positive HUVEC, cells were seeded on collagen-coated Lab-Tek coverglasses and
infected with 50 MOI of adenovirus encoding intradiabody or control
intrabody (JC7U). Forty-eight hours postinfection, HUVEC were
washed with copious amounts of PBS, followed by incubation in a
humidifying chamber at room temperature for 1 h with a primary
antibody mixture of rat anti-HA monoclonal antibody (5 ␮g/ml; Roche
Applied Science) and biotinylated goat anti-human Tie-2 or anti-human
VEGF-R2 polyclonal antibodies (R&D Systems). The cells were then
stained for 1 h at room temperature with the mixture of Cy5-conjugated
Intradiabodies for Simultaneous Knockout of VEGFR2 and Tie2
47815
FIG. 3. A, kinetics of surface expression of Tie-2 and VEGF-R2 after gene transfer of the intradiabody and conventional scFv intrabodies on
HUVEC using recombinant adenoviruses with an MOI of 10. The surface expression of Tie-2 and VEGF-R2 is specifically blocked over a period of
more than 15 days to the extent of 97.5 and 96% inhibition, respectively, with the intradiabody. The corresponding scFv intrabodies against Tie-2
(1S05) and VEGF-R2 are much less efficient in terms of duration of surface depletion. Open circle and open triangle, Tie-2. Filled circle and filled
triangle, VEGF-R2. Circles, intradiabody; triangles, the two corresponding scFv intrabodies. B, gene transfer of intrabodies directed to human
Tie-2, VEGF-R2, or Tie-2/VEGF-R2 by recombinant adenoviruses on day 6 after infection. HUVEC were infected with an MOI of 10 for delivery
of the intradiabody (DIA) and conventional scFv intrabodies (1S05, VC06). Controls consisted of an anti-integrin ␣v␤3 scFv intrabody and
mock-infected cells (data not shown). The flow cytometry histograms show the binding of anti-VEGF-R2 (thin line) and anti-Tie-2 (boldface line)
biotinylated polyclonal antibodies to HUVEC. Normal goat IgG was used as primary control antibody (dotted black line). Streptavidin-APC was
used for detection. The y axis gives the number of events in linear scale, the x axis shows the fluorescence intensity in logarithmic scale.
donkey anti-rat IgG polyclonal antibodies and streptavidin/rhodamine
red-X (both from Jackson Immunoresearch, West Grove, PA) diluted to
1:100 in FACS buffer, 0.1% saponin. Finally, the cells were covered with
SlowFade Antifade reagent. Three-color (GFP, rhodamine red-X, and
Cy5) three-dimensional data sets were collected with a DeltaVision
system (Applied Precision, Issaquah, WA); this consisted of an Olympus
IX-70 fluorescence microscope, a motorized high precision xyz stage, a
100-watt mercury lamp, and a KAF1400 chip-based cooled chargecoupled device camera. Exposure times were 0.2– 0.5 s (2-binning), and
images were obtained with a ⫻60 oil objective. Three-dimensional reconstructions were generated by capturing 150-nm serial sections along
the z axis. Images were deconvolved (based on the Agard-Sadat inverse
matrix algorithm) and analyzed with softWorX version 2.5.
Pulse-chase for Determination of Half-life—HUVEC cells were
seeded at a density of 1 ⫻ 106 cells in T175 flasks and infected 24 h later
with recombinant adenoviruses using an MOI of 10. After infection for
24 h, cells were washed with Hepes-buffered saline solution and
trypsinized. Cells were starved for 2 h in 10 ml of serum-free, methionine-free, cysteine-free minimal essential medium at 37 °C and
swirled periodically. Samples were then labeled with Tran35S-label
medium (50 ␮Ci/ml; ICN, Aurora, OH) for 2 h at 37 °C and subsequently
chased with EGM medium, containing a 40-fold excess of methionine
and 20-fold excess of cysteine for various time points (0, 4, 8, 16, 24, and
48 h). At each time point, cells were washed once with ice-cold PBS
containing 1 mM phenylmethylsulfonyl fluoride and lysed (Promega
lysis buffer, containing complete protease inhibitor mixture). Supernatants were collected and stored at ⫺80 °C. Samples were precipitated
using protein G and monoclonal anti-HA antibody (Covance) for scFv or
protein G alone for the diabody. Immunocomplexes were washed twice
with lysis buffer and twice with TBS, boiled in 1.5⫻ SDS loading buffer,
and separated by a 4 –20% gradient SDS-PAGE gel under reducing
conditions. The gels were stained, dried, and exposed to autoradiography as well as quantitatively analyzed with a PhosphorImager.
Endothelial Cell Tube Formation Assay—6 ⫻ 104 HUVEC cells were
infected with recombinant adenovirus using an MOI of 20 in an Eppendorf tube for 45 min and were then transferred to 6-well plates. Three
days after infection, cells were washed with Hepes-buffered saline
solution, trypsinized, and counted. 2 ⫻ 104 cells/well (volume 50 ␮l) in
complete EGM medium were seeded in triplicates for each virus in a
96-well plate coated with Matrigel Basement Membrane Matrix (BD
Bioscience, Bedford, MA) and incubated for 15.5 h. Cells were then
stained and fixed with the Diff-Quik® staining set (DADE BEHRING
Inc., Newark, DE). For this, cells were fixed with Diff-Quik Fixative,
followed by Diff-Quik Solution I and II, each with 100 ␮l for 2–3 min.
Cells were then washed four times with distilled H2O, and pictures
were taken under an inverted light microscope at ⫻2 magnification.
The number of tube branches for each virus was counted in triplicates
to calculate the average ⫾ S.D.
Cell Proliferation Assay—HUVEC cells were seeded (5 ⫻ 104) in
96-well plates in complete EGM medium and infected with different
recombinant adenoviral constructs with an MOI of 10 and 50. Three
days later, the cells were trypsinized, stained with trypan blue, and
counted in triplicate by a hemacytometer. Cell proliferation was also
measured by adding [3H]thymidine (ICN Radiochemicals) in a concentration of 0.5 ␮Ci/well (1 Ci ⫽ 37 GBq) during the last 24 h of incubation. The cells were frozen at ⫺80 °C overnight and subsequently processed on a multichannel automated cell harvester (Cambridge
Technology, Cambridge, MA) and counted in a liquid scintillation
47816
Intradiabodies for Simultaneous Knockout of VEGFR2 and Tie2
FIG. 4. A, pulse-chase experiment. [35S]Methionine/cysteine was used to determine the half-life of the intradiabody (left) and the scFv intrabody
against Tie-2 (right). HUVEC were infected with an MOI of 10 for each virus for 24 h. After 2-h starvation in methionine/cysteine-free medium,
cells were incubated for 2 h with medium containing 50 ␮Ci/ml [35S]methionine/cysteine, for protein labeling. HUVEC were lysed following
different time points of chase. Precipitation was done with an anti-HA monoclonal antibody followed by protein G (scFv intrabody) or protein G
alone (intradiabody). The precipitated proteins were separated on a 4 –20% gradient SDS-PAGE gel under reducing conditions. The intradiabody
showed the expected size of 78 kDa, and the scFv intrabody was 28 kDa. The intradiabody showed an expected size of 150 kDa under nonreducing
conditions (data not shown). Numbers on the left indicate molecular masses (kDa). Chase times in hours are labeled at the bottom. Lane 1 contains
the nonspecific control. B, determination of half-life of the intradiabody and scFv intrabody 1S05. The average half-life of the intradiabody and scFv
intrabody 1S05 were calculated (t ⫽ ln2/k, k ⫽ first order rate constant) after quantification using a PhosphorImager. The intradiabody reveals
a half-life beyond 48 h (t 227 h), whereas the half-life of the scFv intrabody 1S05 is ⬃22 h.
␤-counter (Beckman Coulter). The background was defined by uninfected cells. The inhibition was calculated according to the following
formula: (background ⫺ infected cell count)/background ⫻ 100%.
RESULTS
Selection of Rabbit Fabs Binding to VEGF-R2—Using phage
display, several New Zealand White rabbit Fabs against
VEGF-R2 were selected by panning (19) chimeric rabbit/human antibody libraries (20, 21) on a human VEGF-R2䡠VEGF
complex. For further analysis, chimeric rabbit/human Fab
VC06 was selected and produced as soluble Fab in Escherichia
coli and purified by affinity chromatography using goat antihuman F(ab⬘)2 N-hydroxysuccinimide resin columns. Fab VC06
demonstrated a strong binding to human VEGF-R2 in ELISA
and to HUVEC in flow cytometry (not shown). Thus, the
VEGF-R2 epitope recognized by VC06 is displayed by native
VEGF-R2 expressed on the cell surface and is an accessible
target for antiangiogenic therapy. Surface plasmon resonance
studies of VC06 revealed that it possessed a monovalent dissociation constant of ⬃1 nM to human VEGF-R2, whereas Fab
1S05, which we described earlier (20), bound with a dissociation constant of 14 nM to human Tie-2 (Table I).
Generation of an Intradiabody against Tie-2 and VEGF-R2
and scFv Intrabodies against Tie-2 or VEGF-R2 Alone—Fabs
were converted into scFv, in which the VL and VH fragments
were covalently linked with a peptide linker consisting of 18
amino acids. Preserved binding to their respective antigens
was confirmed for both scFv VC06 (VEGF-R2) and scFv 1S05
(Tie-2) by ELISA (not shown). Next, scFv 1S05 and scFv VC06
were linked through the second and third heavy chain constant
domains of human IgG1, resulting in a scFv-CH2-CH3-scFv
expression cassette. As a key feature, the scFv-CH2-CH3-scFv
expression cassette provides for the production of a bifunctional tetravalent antibody construct from a single polypeptide.
Through the homophilic interaction of CH3, two scFv-CH2CH3-scFv molecules associate to form a 150-kDa dimer, which
displays both the N-terminal and C-terminal scFv module
bivalently as the intradiabody (Fig. 1). The scFv against Tie-2
or VEGF-R2 and the scFv-CH2-CH3-scFv genes were cloned
into a modified adenovirus shuttle vector pAdTrackCMV (24)
using two asymmetric SfiI sites. In these constructs, the scFv
coding regions are flanked by a human ␬ light chain leader
sequence at the 5⬘-end, and a sequence encoding the HA tag
(YPYDVPDYA) and the ER retention signal (KDEL) at the
3⬘-end. The generation of replication-deficient recombinant adenoviruses was done essentially as described (24). The resulting recombinant adenoviruses were purified by CsCl banding,
and final yields were between 4.3 ⫻ 1011 particles/ml and 1.2 ⫻
1012 particles/ml.
Colocalization of Intradiabody and Targeted Proteins in the
ER—To investigate whether our intrabody constructs were
expressed in the ER and able to trap the targeted proteins in
the same compartment, we infected HUVEC with an MOI of 50
and verified the endoplasmatic reticulum localization of intrabodies and proteins by deconvolution microscopy on day 3 after
infection (Fig. 2). As a control, HUVEC were infected with an
intrabody against integrin ␣v␤3 (scFv JC7U). To visualize the
intrabodies in the ER, intracellular staining was carried out
with saponin and rat anti-HA monoclonal antibody, followed by
donkey anti-rat Cy5 polyclonal antibodies. Both intrabody constructs were found to be expressed in the ER, as indicated by a
staining of the characteristic tubular network. To confirm that
the targeted proteins were trapped in the ER, another staining
protocol using biotinylated Tie-2 and VEGF-R2 polyclonal antibodies, followed by streptavidin-rhodamine red-X polyclonal
antibodies was performed. As shown in Fig. 2 and in threedimensional movies (see Supplemental Material), the intradia-
Intradiabodies for Simultaneous Knockout of VEGFR2 and Tie2
47817
FIG. 5. Inhibition of capillary tube
formation in vitro. A, HUVEC cells
were infected with different recombinant
adenoviruses for 3 days and then seeded
in 96-well plates coated with Matrigel.
After incubation for 15.5 h, cells were
stained and fixed, and pictures were
taken. Uninfected cells (a), mock-infected
cells (b), and cells infected with the control diabody construct T2V2 (c) formed
capillary tubes. By contrast, cells infected
with the intradiabody (f) were strongly
inhibited in their capability to form capillary tubes. B, the intradiabody revealed
an inhibitory effect of 90 ⫾ 4%, whereas
scFv intrabodies showed inhibitory effects
to 44 ⫾ 4.3% (A, d, VC06 against VEGFR2) and 12 ⫾ 5.8% (A, e, 1S05 against
Tie-2). The number of tube branches in
three independent wells per sample were
counted and averaged with S.D. values.
body against Tie-2/VEGF-R2 was found to be co-localized with
both targeted proteins in the ER, indicated by the purple color
in the merged picture. On the other hand, the control intrabody
(JC7U), although expressed in the ER, did not retain either
Tie-2 or VEGF-R2.
Extended Surface Depletion of Tie-2 and VEGF-R2 with Intradiabody—To compare the effect of the intradiabody with
that of the scFv intrabodies on the surface expression of Tie-2
and VEGF-R2, HUVEC were infected with an MOI of 10 and
analyzed by flow cytometry over a period of 15 days. The
surface expression of human Tie-2 and VEGF-R2 on HUVEC
infected with the intradiabody, was specifically blocked up to
57% (Tie-2) and 78% (VEGF-R2), respectively, on day one. On
day three after infection, blockade of Tie-2 and VEGF-R2 was
98% complete. Fifteen days after infection, surface expression
remained efficiently blocked, 97.5 and 96% for Tie-2 and
VEGF-R2, respectively. In comparison, the surface expression
of HUVEC infected with the scFv intrabodies, were blocked
with 80% (Tie-2) and 83% (VEGF-R2) efficiency on day one and
84% (Tie-2) and 90% (VEGF-R2) on day three postinfection.
However, on day 15, surface depletion was only 68 and 5% for
Tie-2 and VEGF-R2, respectively (Fig. 3, a and b). No downregulation of integrins ␣v␤3 and ␣v␤5 was observed during 15
days of the experiment (data not shown).
Extended Half-life of the Intradiabody—To determine
whether the remarkable superiority of the intradiabody with
respect to effectiveness and duration of surface depletion of the
targeted proteins can be attributed to an extended half-life,
pulse-chase studies were performed. In these experiments, we
compared the intradiabody with the 1S05 scFv intrabody,
which had been found to be more efficient than the VC06 scFv
intrabody. In brief, 106 HUVEC cells were infected with an
MOI of 10 with each virus for 24 h. Proteins were labeled with
[35S]methionine/cysteine, and HUVEC were lysed following different time points of chase. Following immunoprecipitation,
proteins were separated on a 4 –20% gradient SDS-PAGE gel
under reducing conditions, visualized by autoradiography, and
quantified using a PhosphorImager. Pulse-chase experiments
revealed a half-life of the intradiabody beyond 48 h (t ⬃230 h),
whereas the half-life of the scFv intrabody 1S05 was ⬃22 h
(Fig. 4, a and b). The half-life of the scFv intrabody VC06 was
even shorter than 22 h (data not shown).
Antiangiogenic Effect of Intradiabody—To test whether our
intradiabody can inhibit angiogenesis in vitro, we used a threedimensional capillary tube formation assay. This in vitro assay
has been used as a model of the early organization of new blood
vessels and is consistently found to recapitulate to a large
extent the process of angiogenesis in vivo (26, 27). HUVEC
were infected with an MOI of 20 of each virus. Cells were
transferred to the Matrigel-coated 96-well plate 3 days after
infection to achieve maximum surface depletion of the targeted
proteins. All cells were incubated in the presence of exogenous
growth factors and serum (complete EGM medium). Controls
consisted of mock-infected cells (empty vector) and a control
intradiabody without specificity for any known target (T2V2).
These controls were used to determine possible toxic or growth
inhibition (antiproliferative) effects of either the vector or the
intradiabody format. As shown in Fig. 5, formation of capillaries in Matrigel was inhibited 90 ⫾ 3% with the intradiabody
(VEG-R2/Tie-2), 44 ⫾ 4.3% with the scFv intrabody VC06
(VEGF-R2), and 11 ⫾ 5.8% with the scFv intrabody 1S05 (Tie2). Significantly, cells infected with the empty vector and the
47818
Intradiabodies for Simultaneous Knockout of VEGFR2 and Tie2
FIG. 6. Cell viability assay on HUVEC. To determine the effect of
the intradiabody on the proliferation rate of HUVEC, cells (5 ⫻ 104)
were seeded in 96-well plates and infected with different recombinant
adenoviruses. 3 days after infection the extent of cell survival was
examined by trypan blue staining under a light microscope (A) or via a
[3H]thymidine incorporation assay (B). Cells infected with the intradiabody (DIA) showed the same survival rates as cells infected with the
control construct (T2V2) and the scFv intrabodies (1S05 and VC06).
Results are expressed as mean ⫾ S.D.
control intradiabody (T2V2) were able to form capillaries to the
same extent as uninfected cells.
Intradiabody Does Not Affect Cell Viability—In order to determine that the antiangiogenic effects of the intradiabody are
not related to cytotoxic effects on HUVEC, cell proliferation
and survival were assessed via [3H]thymidine incorporation
assay and trypan blue staining. HUVEC were infected with an
MOI of 10 and 50 of each virus. Cell viability was assessed 3
days after infection. All cells were incubated in complete EGM
medium. As shown in Fig. 6, cell survival and proliferation of
cells infected with the intradiabody are similar as for the scFv
intrabodies or the control intradiabody. We demonstrate
hereby that accumulation of the bispecific intradiabody in the
ER does not affect cell viability to a greater extent than scFv
intrabodies or the control intradiabody and exclude that the
antiangiogenic effects are related to cytotoxic effects caused by
ER stress.
DISCUSSION
The use of scFv fragments as intrabodies has received considerable attention over the past 10 years. Intrabodies have
been utilized to neutralize the function of endogenous target
proteins using several different strategies (13, 28, 29). Among
these, the misdirected localization of the target to another
subcellular region features as the most popular methodology
employed. We hypothesized that an ER-targeted intrabody
with dual specificity and valency could be generated by fusing
two scFv modules with unique specificity to the N terminus and
C terminus of an Fc domain, respectively. Although there are
reports of presenting functional bispecific antibodies in scFv-Fc
formats (30, 31), none of those antibody constructs have been
studied as functional intrabodies targeted to the ER. Furthermore, these antibody constructs bind in a monovalent fashion
to each of the target proteins. As a prototype and proof of
concept, we demonstrate here the development of an ER-targeted intradiabody format for the simultaneous down-regulation of two independent cell surface receptors using an adenovirus-mediated gene delivery system. Our prototypical
intradiabody targets the endothelial transmembrane tyrosine
kinase receptors Tie-2 and VEGF-R2. Both of these receptors
have been shown to be key in angiogenesis.
The endothelial cell receptor-tyrosine kinases, VEGF-R2 and
Tie-2, as well as their ligands, VEGF and angiopoietin-1 and -2,
respectively, play key roles in tumor angiogenesis. A model for
the interplay of VEGF, VEGF-R2, Tie-2, and Ang-1 and -2 in
tumor angiogenesis is discussed by Holash et al. (17). From a
therapeutic perspective, the Tie-2/Ang-2 complex and the
VEGF/VEGF-R2 complex, which are formed on the surface of
proliferating endothelial cells, are attractive targets for antiangiogenic agents. Several reports (32, 33) have been published
showing the inhibition of tumor growth by interference with
the VEGF or Tie-2 receptor pathway by means of antibodies
and soluble or dominant-negative receptor domains (34, 35). As
of yet, no reports have been published based on the use of an
ER-targeted intrabody to inhibit expression of the two endothelial transmembrane receptors Tie-2 and VEGF-R2, either
separately or simultaneously. Our results demonstrate the feasibility of expressing functional, bispecific, and tetravalent antibodies intracellularly and the possibility of inhibiting the
transit of two integral membrane proteins simultaneously
through the ER by means of diabody expression. The potential
for inhibiting angiogenesis by this approach should have implications for the treatment of cancer, since VEGF-R2 and
Tie-2 are essential for tumor angiogenesis. In vitro angiogenesis assays using Matrigel showed that the intradiabody significantly reduces the number of capillary tubes formed, demonstrating its potential as an antiangiogenic drug. Interestingly,
monospecific scFv intrabodies against VEGF-R2 or Tie-2 inhibited capillary tube formation to a significantly lesser extent.
Moreover, the toxicity of the bispecific intradiabody and the
control intradiabody (T2V2) are comparable. The relative efficiency of the intradiabody over the traditional intrabody-based
approach could be explained by several reasons: first, the
bispecificity of the intradiabody versus the monospecificity of
the scFv intrabodies; second, simply their monovalency versus
the bivalency of the intradiabody. A third possibility is that the
relative stability of the proteins could also account for these
findings. Fourth, the bivalent display of the ER retention signal may lead to better retention of the targeted proteins. We
found that the superiority of the intradiabody with respect to
effectiveness and duration of surface depletion of the targeted
proteins can be attributed to an extended half-life. This was
demonstrated in pulse-chase studies, which revealed a half-life
of the intradiabody beyond 48 h (t ⬃230 h), whereas the halflife of the scFv intrabody 1S05 was ⬃22 h and the half-life of
scFv intrabody VC06 was even shorter (data not shown). The
extended half-life of the intradiabody correlates well with its
more efficient and durable surface depletion of the targeted
proteins compared with the scFv intrabody. A plausible explanation for the extended half-life of the intradiabody format is
the bivalent display of the ER retention signal (KDEL) through
the homodimerization of the constant domains (Fig. 1). This
Intradiabodies for Simultaneous Knockout of VEGFR2 and Tie2
may increase the efficiency of recovery of the intradiabody from
the Golgi by the KDEL receptor (36). In addition, fusion to
constant domains per se has been suggested as a means of
increasing the stability of intrabodies (37). Although the increased valency of the intradiabody, which results in a higher
apparent affinity (avidity) for the antigen, is probably an important factor for the efficient surface depletion we observed,
other factors might contribute to the superiority of the intradiabody. In particular, it is known that intrabodies with extended half-lives achieve higher steady-state expression levels,
which increases effectiveness in target molecule inactivation.
In fact, intracellular stability can be more important than
affinity for the antigen (28, 38, 39).
A major application for intrabodies lies in functional genomics (3, 5, 6). Since the number of proteins with unknown function and their interactions with other proteins steadily increases, bispecific and tetravalent intrabodies could provide an
attractive proteomic tool to analyze these networks. This novel
tool provides for the simultaneous surface depletion of two
independent receptors while improving both the efficiency and
duration of the resulting phenotypic knockout. Intradiabodies
may be used to investigate linked regulatory pathways or block
redundant pathways. In addition, intradiabodies may extend
the therapeutic applicability of intrabodies. For example, our
prototype developed in this study provides a precise tool for the
simultaneous silencing of two independent signaling pathways
essential for angiogenesis (18).
Acknowledgment—We thank Roberta Fuller for assistance.
9.
10.
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12.
13.
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25.
26.
27.
28.
29.
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