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http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0012031

r84, a Novel Therapeutic Antibody against Mouse and
Human VEGF with Potent Anti-Tumor Activity and
Limited Toxicity Induction
Laura A. Sullivan1, Juliet G. Carbon1, Christina L. Roland1, Jason E. Toombs1, Mari Nyquist-Andersen2,
Anita Kavlie2, Kyle Schlunegger3, James A. Richardson4, Rolf A. Brekken1,5*
1 Division of Surgical Oncology, Department of Surgery, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas,
Texas, United States of America, 2 Affitech AS, Oslo Research Park, Oslo, Norway, 3 Peregrine Pharmaceuticals, Inc., Tustin, California, United States of America,
4 Departments of Pathology and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 5 Department of
Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
Abstract


Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor
microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic
agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity
receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also
ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced
activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully
human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2
but does not interfere with VEGF:VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown
through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show
that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high
dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The
ability of r84 to specifically block VEGF:VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor
pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced
VEGFR2 signaling in tumors.


Citation: Sullivan LA, Carbon JG, Roland CL, Toombs JE, Nyquist-Andersen M, et al. (2010) r84, a Novel Therapeutic Antibody against Mouse and Human VEGF
with Potent Anti-Tumor Activity and Limited Toxicity Induction. PLoS ONE 5(8): e12031. doi:10.1371/journal.pone.0012031
Editor: Andrei L. Gartel, University of Illinois at Chicago, United States of America
Received June 1, 2010; Accepted July 13, 2010; Published August 6, 2010
Copyright:  2010 Sullivan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by a sponsored research agreement from Peregrine Pharmaceuticals (to R. A. Brekken), institutional funding from the
Simmons Comprehensive Cancer Center, the Randall Bridwell Lung Cancer Research Grant from Uniting Against Lung Cancer (to R. A. Brekken, www.
UnitingAgainstLungCancer.org), a development award from the UT SPORE in lung cancer (P50 CA070907) (to R. A. Brekken), the Effie Marie Cain Scholarship in
Angiogenesis Research (to R. A. Brekken) and Affitech AS (M. Nyquist-Andersen, A. Kavlie). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: R. A. Brekken is a consultant for, has equity interest in, and is a recipient of a sponsored research grant from Peregrine Pharmaceuticals
and Affitech AS. R. A. Brekken is also an author of a patent on technology that was used to develop the antibody r84 by Peregrine Pharmaceuticals and Affitech
AS. M. Nyquist-Andersen and A. Kavlie are employees of Affitech AS, and K. Schlunegger is an employee of Peregrine Pharmaceuticals, which supported these
studies by providing r84, mcr84, and funding. Peregrine and Affitech did not participate in the planning, execution, or interpretation of the experiments. The
authors affirm that they will adhere to the PLoS data sharing statement.
* E-mail: [e-mail]
Introduction
Angiogenesis is a tightly regulated process that is essential during
growth, wound healing and development, as well as cancer growth,
progression and metastasis [1–2]. A key stimulant of angiogenesis is
vascular endothelial growth factor-A (VEGF). VEGF induces
endothelial cell survival, proliferation, and migration its predominant
signaling receptor, VEGF receptor 2 (VEGFR2). Tumor
associated macrophages also express VEGFR2 and selective
blockade of VEGFR2 is able to decrease macrophage infiltration
into tumors [3]. VEGF signaling through VEGF receptor 1
(VEGFR1) remains unclear, although it is thought to have effects
on hematopoiesis, vascular permeability, and monocyte migration.
Importantly, there is elevated expression of VEGF, VEGFR1, and
VEGFR2 within tumors, providing a therapeutic target. In fact
targeting VEGF has lead to the development of anti-angiogenic
therapies such as sunitinib malate (SutentH, SU11248, Pfizer, Inc.),
sorafenib (NexavarH, BAY 43-9006, Bayer Pharmaceuticals Corp.),
bevacizumab (AvastinH, Genentech), IMC-1121b (ramucirumab,
ImClone), VEGF-Trap (aflibercept, Regeneron) and 2C3 [2,4–6].
Sunitinib and sorafenib are small molecule inhibitors of multiple
receptor tyrosine kinases (RTKs) including the VEGF receptors.
These drugs have been FDA-approved for the treatment of renal
cell carcinoma, gastrointestinal stromal tumors (GIST) (sunitinib),
and unresectable hepatocellular carcinoma (sorafenib) [2,7–8].
Bevacizumab is a humanized monoclonal anti-VEGF antibody
that inhibits VEGF from binding to and signaling through
VEGFR1 and VEGFR2. Bevacizumab is approved in combination
with cytotoxic chemotherapy for the treatment of colorectal
cancer, non-small cell lung cancer (NSCLC), and breast cancer, as
PLoS ONE | www.plosone.org 1 August 2010 | Volume 5 | Issue 8 | e12031
monotherapy for glioblastoma, and in combination with interferon
for renal cell carcinoma [9–11]. Treatment with bevacizumab in
these cancer types results in a delay of tumor progression and
increases in patient survival [2,9]. However, treatment with
bevacizumab, sorafenib, and sunitinib, is also associated with a
number of rare although serious toxicities including gastrointestinal
perforations, bleeding, proteinurea, and glomerulosclerosis
[9,12–13].
IMC-1121b is a high affinity, fully human IgG1 monoclonal
antibody that recognizes VEGFR2. IMC-1121b binding to
VEGFR2 inhibits ligand-induced activation of the receptor. There
are several on-going phase I, II, and III clinical trials evaluating
the efficacy of IMC-1121b in a number of tumor types [6].
VEGF-Trap is comprised of the second and third extracellular
immunoglobulin domains of VEGFR1 and VEGFR2, respectively,
joined by an IgG1 Fc region. The resulting fusion protein traps
with high affinity multiple VEGF family members including
VEGF and placental growth factor (PlGF) [14]. Currently, VEGFTrap
is being tested in phase III clinical trials in a number of
tumor types [6].
2C3 is a murine, monoclonal antibody against VEGF that
specifically blocks human VEGF binding to VEGFR2 [4]. The
selective inhibition of VEGF:VEGFR2 signaling by 2C3 reduces
vascular permeability, decreases endothelial cell growth, and
decreases tumor growth in murine xenograft models. Additionally,
2C3 reduces tumor microvessel density (MVD) and macrophage
infiltration and down-regulates VEGFR2 expression on the tumor
vasculature [3–5,15]. The desirable anti-angiogenic effects of 2C3
lead to the development of a human antibody that retains 2C3
specificity.
Here we describe a fully human monoclonal antibody, r84
(AT001, Affitech AS) that binds to mouse and human VEGF and
specifically inhibits VEGF binding to VEGFR2, while leaving
intact VEGF interaction with VEGFR1. Through blockade of
VEGFR2 signaling, r84 inhibits the migration of VEGFR2
positive endothelial cells, and blocks VEGFR2 phosphorylation
and downstream signaling. In addition, treatment of mice bearing
tumor xenografts with r84 delays tumor take resulting in tumor
vascular changes, including reductions in tumor MVD and in
tumor lymphatic vessel density (LVD). Furthermore, extended
treatment with r84 does not induce significant systemic toxicity in
mice.

Materials and Methods
Construction of human IgG anti-VEGF antibodies
Human anti-VEGF single chain variable fragments (scFvs) were
created by Affitech AS (Olso, Norway) and Peregrine Pharmaceuticals,
Inc. (Tustin, CA) and screened for specific VEGF
binding characteristics. The most desirable scFvs were cloned into
full length antibody expression vectors containing the glutamine
synthetase gene, transfected into CHO K1SV cells, and selected in
a glutamine free cell culture media. The cells were plated into flat
bottom 96 well culture plates, and wells with antibody production
were diluted and the cells were subcloned. Once subcloned, the
high production cells were grown to 500 mL cultures and the
antibody was purified by Protein-A affinity chromatography and
size-exclusion chromatography for purities of greater than 90%
monomer.
ELISA analysis of r84
To evaluate the binding specificities of r84, a series of ELISAs
were performed.
Determination of r84 specificity. Relative binding affinity
of r84 for mouse and human VEGF was determined by ELISA.
Recombinant human VEGF (R&D SystemsH, Minneapolis, MN)
or mouse VEGF (Sigma-AldrichH, St. Louis, MO) was coated onto
the bottom of 96-well plates at 0.5 mg/mL. Wells were blocked
and then incubated with r84 starting at 2 mg/mL with a serial
dilution factor of four. Antibody bound to the wells was detected
by incubation with anti-human Fc HRP-conjugated antibody
followed by development with HRP substrate.
r84 specificity within VEGF family. Human VEGF-A,
mouse VEGF-A, human VEGF-B, human VEGF-C, human
VEGF-D, and human PlGF (R&D SystemsH) were coated onto
96-well ELISA plates at 0.5 mg/mL. Wells were blocked and then
incubated with human r84 at 1 mg/mL. Antibody bound to the
wells was detected as described above.
r84 receptor blocking ELISAs. Recombinant human
VEGFR1/Fc or VEGFR2/Fc (R&D SystemsH) was coated onto
the bottom of 96-well plates at 1 mg/mL. Wells were blocked and
then incubated with 2.38 nM or 4.76 nM biotinylated VEGF for
VEGFR1 and VEGFR2, respectively, +/2 fold the indicated
molar excess of antibody. Labelled VEGF bound to the wells was
detected by incubation with strepavidin HRP-conjugated
antibody, developed as described above, and displayed as a
percentage of VEGF binding alone in the absence of antibody.
Endothelial cell in vitro assays
The effect of r84 on endothelial cell function and signaling in
vitro was assessed using HDMEC (ScienCellTM Research Laboratories,
Carlsbad, CA), PAE-KDR [16], PAE-Flt-1 [16] endothelial
cell lines.
Migration assays. A modified Boyden chamber assay was
used. 20,000 endothelial cells (ECs) (HDMEC (ScienCellTM
Research Laboratories, Carlsbad, CA), PAE-KDR [16], PAEFlt-
1 [16]) were plated in serum free media on 8.0 mm pore size
cell culture inserts (BD FalconTM, San Jose, CA) and allowed to
migrate overnight at 37uC. Recombinant human or mouse VEGF
(Sigma-AldrichH) was used as a chemo-attractant at 100 ng/mL,
with antibodies added at a 500-fold molar excess. Insert
membranes were isolated following migration and stained with
DAPI to allow for quantification of migrated cells (total
magnification, 1006).
Stimulation assays. HDMEC and PAE-KDR, -Flt-1 cell
lines were maintained in 100 mm2 tissue culture dishes in MCDB
131 (GibcoH, Carlsbad, CA) media supplemented with 0.4 mg/mL
ECGF and 20% fetal bovine serum (FBS). Following 24 hour
serum starvation, cells were stimulated for two minutes with
100 ng recombinant human VEGF or mouse (R&D SystemsH) +/
2 500-fold molar excess antibody. Cell lysates of stimulated cells
were prepared and analyzed by Western blot using commerciallyavailable
antibodies specific for targets of interest ( total and
phospho- VEGFR2, p38, PLCc, Erk1/2 (Cell Signaling
TechnologyH, Danvers, MA), and VEGFR1 (AbcamH,
Cambridge, MA)).
Animal studies
4–6-week-old NOD/SCID mice were purchased from the
breeding core at the University of Texas Southwestern Medical
Center. Animals were housed in a pathogen-free facility and all
procedures were performed in accordance with a protocol (APN
0974-07-05-1) approved by the IACUC of the University of Texas
Southwestern Medical Center.
Tumor models and treatment. All tumor cells (H460,
H1299, A549, PANC-1) were grown in culture in RPMI-1640
medium (HyCloneH, Waltham, MA) supplemented with 5% FBS.
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Cell lines were confirmed to be pathogen free and were
authenticated to confirm origin prior to use.
Subcutaneous xenograft therapy study. 2.5 million H460,
H1299, A549 NSCLC cells (provided by Dr. John Minna) were
injected (in PBS) subcutaneously into the right flank of NOD/
SCID mice. Mice were treated with 50 mg/kg/week r84 and
25 mg/kg/week bevacizumab/AvastinH and palivizumab/
SynagisH (anti-respiratory syncytial virus) via intraperitoneal (IP)
injection starting one day post tumor cell injection (TCI) (n =8–9/
group). Mice were monitored twice a week, recording weights,
taking perpendicular tumor measurements, and observing for signs
of distress such as weight loss and inactivity. Therapy continued
until average control-treated tumor volume reached 1500 mm3 or
until day 60 post TCI, at which point animals were sacrificed.
Toxicity studies. 5 million PANC-1 human pancreatic
cancer cells (ATCC, Manassas, VA) (in PBS) were injected
subcutaneously into the right flank of NOD/SCID mice. An equal
number of NOD/SCID mice were not injected with tumor cells.
Therapy began one day post TCI. Tumor bearing (TB) and nontumor
bearing (NTB) mice were treated with 50 mg/kg/week r84
and palivizumab via IP injection. Each group consisted of five
mice. Mice were monitored as previously described. All mice were
sacrificed following 12 weeks of continuous therapy and evaluated
for r84-induced toxicity. Blood was collected from animals at
sacrifice; serum was isolated following centrifugation and analyzed
by the mouse metabolic phenotyping core at the University of
Texas Southwestern Medical Center. A second toxicity study was
performed in immunocompetent mice harbouring spontaneous
pancreatic cancer (p48cre/KrasG12D/INK4a) [17]. Mice were treated
with saline (n =4) or 25 mg/kg/week mouse chimeric r84 (mcr84,
n =3) via IP injection or with 50 mg/kg/week sunitinib (n= 4) by
daily oral gavage five days per week. Sunitinib was purchased from
LC laboratories (Woburn, MA). Therapy began when mice
reached eight weeks old. Mice were monitored for weight gain as
previously described. At weeks two and seven of therapy, tail vein
cuff blood pressures of all mice were measured using the Visitech
Systems BP-2000 Series II Blood Pressure Analysis SystemTM
through the O’Brien Kidney Research Core Center at the
University of Texas Southwestern Medical Center. To
familiarize mice to the procedure, tail cuff blood pressures were
measured for five consecutive days, with data collection on the fifth
day. Average systolic pressures were calculated from data collected
on the last day of measurement (day five). At week six of therapy
metabolic cages obtained through the O’Brien Kidney Research
Core Center at the University of Texas Southwestern Medical
Center were used to collect urine from all animals over a 24-hour
collection period. Fresh urine samples were then submitted to the
University of Texas Southwestern Medical Center mouse
metabolic phenotyping core for analysis of total levels of urine
protein and creatine. All mice were sacrificed following eight
weeks of continuous therapy and evaluated for mcr84- and
sunitinib-induced toxicity. Blood was collected from animals at
sacrifice; serum was isolated following centrifugation and analyzed
by the mouse metabolic phenotyping core at the University of
Texas Southwestern Medical Center.
Therapy dose titration. 2.5 million A549 NSCLC cells (in
PBS) were injected subcutaneously into the right flank of NOD/
SCID mice. Mice were treated with 5, 15, or 50 mg/kg/week r84
and bevacizumab and 15 mg/kg/week control IgG (Peregrine
Pharmaceuticals, Inc.) via IP injection starting one day post tumor
cell injection (TCI). Each group consisted of eight mice and were
monitored as above. Therapy continued until average controltreated
tumor volume reached 1200 mm3, at which point animals
were sacrificed.
Histology and Immunohistochemical Studies
Formalin-fixed, paraffin-embedded tissues were sectioned and
stained with hematoxylin and eosin by the molecular pathology core
laboratory at the University of Texas Southwestern Medical Center.
Snap frozen tumors were sectioned, blocked with 20% Aquablock
(East Coast Biologics, North Berwick, ME) and stained for markers
of interest. Primary antibodies used include MECA-32 (DSHB;
University of Iowa), endomucin (Santa Cruz BiotechnologyH, Inc.,
Santa Cruz, CA), NG2 (MilliporeH, Billerica, MA), smooth muscle
actin (NeoMarkers, Fremont, CA), Lyve1, VEGFR2 (55B11) (Cell
Signaling TechnologyH, Danvers, MA), rabbit anti-VEGFR2 T014
(purified in our laboratory) [4,18], rat anti-VEGFR2 RAFL-2 [19],
and insulin (Dako, Glostrup, Denmark).
Statistics
Data were analyzed using GraphPad software (GraphPad Prism
version 5.00 for Windows, GraphPad Software). Results are
expressed as mean 6 SE. Differences are analyzed by t test or
ANOVA, and results are considered significant at a p value of
,0.05.
Results
Generation of a fully human monoclonal antibody
against VEGF
The success of 2C3 in preclinical models led to the development
of a fully human monoclonal antibody that recognizes VEGF and
retains many of the characteristics of 2C3. A number of anti-
VEGF human single chain variable fragments (scFv) were
screened for several characteristics such as a competition with
2C3 for binding to VEGF, the ability to block VEGF:VEGFR2
binding, and the ability of the scFv to bind to different VEGF
isoforms such as VEGF165 and VEGF121.
r84 binds human and mouse VEGF-A and specifically
blocks VEGF from binding to VEGFR2
To determine the binding specificity of r84, a series of ELISAs
were performed. A titration of r84 against recombinant human or
mouse VEGF demonstrated that r84 binds with equal affinity to
both species (Figure 1A). This established r84 as an important tool
in evaluating the contribution of both tumor cell- and host-derived
VEGF in tumor progression using xenograft models. The binding
specificity of r84 differs from other anti-VEGF antibodies, such as
bevacizumab and 2C3 that recognize only human VEGF. Next,
the specificity of r84 within the VEGF family was determined. r84
only bound wells coated with recombinant human and mouse
VEGF-A (Figure 1B).
The effect of r84 on VEGF binding to VEGFR1 and VEGFR2
was determined using ligand-receptor ELISAs. 2C3 and r84 at
increasing fold molar excess significantly reduced biotinylated-
VEGF binding to VEGFR2, compared to binding of biotinylated-
VEGF alone or in the presence of a non-specific control IgG
(Figure 1C). In contrast, neither 2C3 nor r84 inhibited binding of
biotinylated-VEGF to VEGFR1 (Figure 1D, left panel). However,
at a 500 fold molar excess of antibody to biotinylated-VEGF,
bevacizumab decreased VEGF binding to VEGFR1 by approximately
80% (Figure 1D, right panel). These blocking ELISAs
demonstrate the precise binding of r84 to VEGF to selectively
inhibit the VEGF:VEGFR2 interaction.
r84 effects VEGFR2-mediated endothelial cell function
The effect of r84 on endothelial cells was determined using
several in vitro assays. First, a transwell assay was used to test the
r84 Inhibits VEGFR2
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effects of anti-VEGF antibodies on VEGF-induced endothelial cell
migration. Three different endothelial cell lines, selected for their
VEGF receptor expression, were used for the migration assays.
Human dermal microvascular endothelial cells (HDMEC) express
VEGFR1 and VEGFR2, porcine aortic endothelial cells (PAE)-
KDR and PAE-Flt-1 express high levels of VEGFR2 or VEGFR1,
respectively [16]. Human VEGF significantly induced migration
of all three cell types compared to serum free media (SFM) alone
(p,0.05 for HDMEC, p,0.001 for PAE-KDR, -Flt-1), and a nonspecific
control IgG did not affect VEGF-induced migration
(Figure 2A). Both r84 and bevacizumab significantly inhibited
VEGF-induced migration of VEGFR2-expressing endothelial cells
(p,0.001, Figure 2A, HDMEC, PAE-KDR). However, only
bevacizumab was able to decrease the migration of PAE-Flt-1 cells
Figure 1. r84 binds human and mouse VEGF-A and specifically blocks VEGF-A binding to VEGFR2, not VEGFR1. A, Recombinant
human VEGF coated at 0.5 mg/mL was detected with a titration of fully human monoclonal antibody r84. r84 bound to VEGF was detected with an
anti-human Fc HRP-conjugated antibody, demonstrating r84 binds both human and mouse VEGF-A (open squares and circles, respectively). B,
Recombinant human and mouse VEGF-A, and human VEGF-B, -C, -D, and PlGF coated at 0.5 mg/mL was detected with r84 at 1 mg/mL. Binding of r84
to VEGF family member was detected as in A, demonstrating r84 binds only human and mouse VEGF-A and not other VEGF family members. VEGFR2
(C) and VEGFR1 (D, left panel) coated at 1 mg/mL were incubated with 4.76 nM or 2.38 nM biotinylated VEGF, respectively, +/2 the indicated fold
excesses of antibody (Control IgG, 2C3, r84). r84, 2C3 specifically block biotinylated-VEGF binding to VEGFR2 (C), but not VEGFR1 (D, left panel). In
contrast, a 500-fold molar excess bevacizumab (bev) reduces biotinylated-VEGF binding to VEGFR1, compared to biotinylated-VEGF alone or plus r84
(D, right panel).
doi:10.1371/journal.pone.0012031.g001
r84 Inhibits VEGFR2
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towards VEGF (Figure 2A, PAE-Flt-1). To further evaluate the
specificity of r84 to mouse VEGF, migration assays were
performed with PAE-KDR cells using mouse VEGF as the
chemotactic agent. As seen with human VEGF, mouse VEGF
significantly induced the migration of PAE-KDR cells as
compared to SFM alone (Figure S1A). Only r84 was able to
significantly inhibit this migration, while bevacizumab and a
control IgG had no effect on cell migration (p,0.001, Figure S1A).
The ability of r84 to specifically block both human and mouse
VEGF-induced migration of VEGFR2-expressing endothelial cells
(HDMEC, PAE-KDR) but not VEGFR1-expressing endothelial
cells (PAE-Flt-1) demonstrates the selectivity of r84 to inhibit
VEGF-induced VEGFR2 activity.
VEGF binding to VEGFR2 initiates receptor phosphorylation
and subsequent phosphorylation of downstream pathway components
such as phospholipase C gamma (PLCc), p38, and the MAP
kinase extracellular signal-regulated kinase (ERK1/2). PAE-KDR
cells stimulated in vitro with human VEGF (100 ng, two minutes)
induced phosphorylation of VEGFR2, PLCy, p38, and ERK
(Figure 2B). Human VEGF stimulation of HDMECs induced
phosphorylation of PLCc and ERK (Figure 2C). Stimulation of
PAE-KDR and HDMEC cells with human VEGF plus 500-fold
molar excesses r84 or bevacizumab inhibited phosphorylation of
VEGFR2 and downstream targets (Figure 2B, 2C). However, only
bevacizumab blocked human VEGF-induced phosphorylation of
VEGFR1 in PAE-Flt-1 (Figure 2D). Further, stimulation of PAEKDR
cells with mouse VEGF induced phosphorylation of
VEGF2, PLCy, and ERK that was only inhibited by r84 and
not by bevacizumab or a control IgG (Figure S1B). This data
shows that r84 selectively inhibits human and mouse VEGF
Figure 2. r84 reduces endothelial cell migration and signaling in vitro. A, A modified Boyden chamber migration assay was used to assess
the effect of r84, bevacizumab (bev) on VEGF-induced endothelial cell (EC) migration. 20,000 HDMEC, PAE-KDR, PAE-Flt-1 cells were plated on 8.0 mm
cell culture inserts and allowed to migrate overnight towards SFM or human VEGF (100 ng/mL) +/2 500-fold molar excess antibody (bev, r84, control
IgG). r84, bev block the VEGF-induced migration of VEGFR2-expressing ECs (A, HDMEC, PAE-KDR). Bev blocks VEGF-induced migration of endothelial
cells expressing VEGR1, but r84 does not (A, PAE-Flt-1). Western blots of VEGF-induced signaling in PAE-KDR (B), HDMEC (C), and PAE-Flt-1 (D) lysates
following stimulation of cells with 50 ng/mL human VEGF +/2 500-fold molar excess antibody (bev, r84, control IgG). r84 and bev block p-VEGFR2
and downstream phosphorylation (p38, PLCc, ERK1/2) (B, C), but only bev blocks VEGF-induced VEGFR1 phosphorylation in PAE-Flt-1 stimulated cells
(D). *p,0.05, **p,0.01, ***p,0.001, statistical differences in A compared to VEGF alone, unless otherwise indicated.
doi:10.1371/journal.pone.0012031.g002
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binding and signaling through VEGFR2 without interrupting
VEGFR1 signaling. The ability of r84 to bind human and mouse
VEGF (Figure 1A, B) and block VEGF from binding and signaling
through VEGFR2 (Figure 1C, Figure 2A–D, Figure S1A, B)
makes r84 a unique tool for studying VEGF inhibition in tumor
xenograft models, assessing possible toxicity induction and
analyzing the importance of VEGFR1 signaling in these processes.
r84 delays take of human xenograft tumors
Previous studies in our lab have demonstrated the ability of r84
to control tumor growth and decrease tumor angiogenesis in
established models of breast cancer [20–21]. The efficacy of r84 as
a cancer therapeutic was further assessed in tumor xenograft
models in NOD/SCID mice. Briefly, four- to six-week old female
NOD/SCID mice were implanted subcutaneously (s.c.) with 2.5
million human non-small cell lung cancer (NSCLC) cell lines
H460, H1299, or A549. Treatment began one day post tumor cell
injection (TCI) and continued until the average tumor volume in
control IgG treated tumors reached 1500 mm3, at which time all
animals were sacrificed. Tumor-bearing animals were treated with
50 mg/kg/week r84 and 25 mg/kg/week bevacizumab and
control IgG (palivizumab/SynagisH). r84 and bevacizumab
similarly delayed tumor take, thereby controlling H460 and
H1299 tumor growth compared to control IgG therapy by both
tumor volume and final tumor weights at sacrifice (Figure 3A, B).
In A549 xenografts, r84 delayed tumor take better than
bevacizumab, and the mean final tumor weight at sacrifice of
animals treated with r84 was significantly smaller than animals
treated with bevacizumab (Figure 3C, p,0.05).
Bevacizumab has a half life in mice of approximately two weeks
[22]. Pharmacokinetic studies (data not shown) determined the
half life of r84 in mice to be approximately five days. This
difference, along with the fact that r84 binds both human and
mouse VEGF and thus has more target to bind in tumor xenograft
models than bevacizumab, led to the differences in antibody doses
used in tumor studies. Consequently, this increase in dose could
lead to better control of tumor growth as was seen in the A459
model (Figure 3C). To evaluate the effect of antibody dose, A549
tumor cells were implanted into mice as previously described. One
day post TCI, animals began therapy, receiving 5, 15, or 50 mg/
kg/week of r84 or bevacizumab, or 15 mg/kg/week of a nonspecific
control human IgG. The different doses of bevacizumab
had the same effect on tumor growth and final tumor weight
(Figure 3D). In contrast, there was an observable titration of tumor
growth and final tumor weight with r84 therapy, with tighter
control seen at higher doses of antibody. In addition, treatment of
A549 tumor-bearing animals with 15 and 50 mg/kg/week r84
resulted in smaller tumors as compared to the same dose of
bevacizumab (Figure 3D, p,0.001 and p,0.01, respectively).
Therefore, these results indicate that r84 may be more effective at
controlling tumor take and growth than bevacizumab independent
of dose in certain models. We propose that the appropriate
therapeutic antibody dose should be determined independently for
different tumor types to maximize therapeutic benefit with
minimal induction of toxicity [23–25].
r84 effects the tumor microenvironment
The phenotypic effects of r84 therapy within NSCLC tumors
were assessed by immunohistochemistry (IHC). As was expected
for anti-angiogenic therapies, treatment with r84 and bevacizumab
resulted in a significant decrease in tumor MVD as
demonstrated using two endothelial cell markers, MECA-32 (data
not shown) and endomucin (Figure 4A). There was a trend
towards an increase in the number of pericyte-(PC) associated
blood vessels in r84- and bevacizumab-treated tumors as
compared to control IgG, although this increase only reached
significance in the H460 model (Figure 4A). Treatment of H460
and H1299 xenograft tumors with r84 or bevacizumab also
reduced the number of VEGFR2 positive cells, as analyzed by
IHC (Figure 4B). Interestingly, VEGFR2 expression in A549
tumors was only decreased following r84 and not bevacizumab
(Figure 4B, p,0.05) therapy, perhaps reflecting the difference in
the efficacy of these two drugs in controlling tumor growth in this
model. Additionally, inhibition of VEGF with r84 or bevacizumab
decreased tumor LVD as compared to control IgG therapy in both
H460 and H1299 models (Figure 4C). However, bevacizumab
therapy failed to reduce LVD in the A549 model. These results
suggest that in a model-dependent manner, r84 and bevacizumab
may be able to disrupt lymphatic vessel-mediated tumor
metastasis.
Extended r84 therapy does not induce toxicity
The use of bevacizumab and other anti-angiogenic therapies in
the clinic is associated with a number of rare although serious
toxicities. Toxicity associated with r84 can be evaluated in
preclinical mouse xenograft models because of its ability of r84
to bind both human and mouse VEGF. To assess the potential of
r84 to induce toxicities, NOD/SCID mice were injected with five
million PANC-1 tumor cells (a slow-growing human pancreatic
cancer line) s.c. Treatment began one day post TCI, with 50 mg/
kg/week r84 or a non-specific control IgG (palivizumab/
SynagisH). An equal number of non-tumor-bearing (NTB) animals
received antibody treatment as well. Therapy continued for 12
weeks, at which point animals were sacrificed and tumor, organs
and blood were collected for toxicity assessment. As was seen in
the NSCLC models, r84 therapy significantly reduced PANC-1
tumor growth and final tumor weight, as compared to control
(Figure S2A, p,0.05). In addition, r84 treatment resulted in
decreased tumor MVD (Figure S2B, p,0.001). r84 did not induce
histological changes (as assessed by a pathologist) within the kidney
or liver of tumor-bearing (TB) or NTB mice as compared to agematched
nai¨ve animals (Figure 5A, nai¨ve and TB r84 displayed).
Blood was collected from all animals at the time of sacrifice, and a
serum analysis of 20 metabolic markers was performed at UT
Southwestern Medical Center’s mouse metabolic phenotyping
core (Table S1). There were no significant changes in any of these
analytes between treated animals and age-matched nai¨ve animals.
This analysis included no observable change in alanine aminotransferase,
aspartate aminotransferase, and blood urea nitrogen
levels (Figure 5B), markers of liver and kidney function,
respectively. These three markers are elevated in correlation with
toxicity in animals treated for 12 weeks with bevacizumab and
high-affinity anti-VEGF antibodies [12].
It has been reported that anti-VEGF treatment can reduce
pancreatic islet vascular density in adult mice, leaving the
supporting pericytes behind [9]. In this study, the pancreatic islets
of TB and NTB animals treated for 12 weeks with r84 showed a
reduction in MVD as compared to control IgG-treated TB and
NTB animals (p,0.01), but there was no significant change when
compared to nai¨ve animals (Figure S3A). Additionally, there was
no observable change in the percentage of pericytes without
endothelial cell association (Figure S3A). Furthermore, there was
no change in serum glucose levels, nor was there a change in
insulin staining in pancreatic islets of experimental animals (Figure
S3B, C). Taken together, long-term therapy with r84 produced no
observable toxicity in TB or NTB animals.
Since hypertension and proteinuria are among the most
common toxicity-related side effects associated with anti-VEGF
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therapy and given recent data suggesting a role for VEGFR2 in
controlling blood pressure [13,26], we investigated the effects of
r84 therapy on hypertension and proteinuria in a spontaneous,
immunocompetent model of pancreatic cancer. Mice (p48-
Cre:LSL-KrasG12D:p16ink4a/arf+/lox) expressing a pancreas-specific
Cre recombinanse activating a constitutively active Kras allele
(KrasG12D) and inactivating a single copy of Ink4a/Arf that
spontaneously develop pancreatic ductal adenocarcinoma (PDAC)
[17] were separated into three groups receiving either saline,
mouse chimeric r84 (mcr84 [21]), or sunitinib. Therapy began
when mice when mice were eight weeks old, with weekly IP
injections of saline 25 mg/kg/week or mcr84, or daily oral gavage
of 50 mg/kg/week sunitinib. Therapy continued for a total of
eight weeks, at which time all animals were sacrificed. Tumor
burden, as assessed by final pancreas weight at sacrifice, was
reduced in mcr84-treated animals as compared to control- and
sunitinib-treated animals, although this trend failed to reach
significance (Figure S2C). However, treatment with mcr84 or
sunitinib resulted in decreased tumor MVD (Figure S2D, p,0.01
or p,0.05, respectively). The effects of r84 in PDAC and its
possible therapeutic benefit in animal models of PDAC are active
areas of research in our lab. Tail cuff blood pressure measurements
were gathered during weeks two and seven of therapy. At
week two, animals in the mcr84 and sunitinib groups displayed
elevated systolic blood pressure as compared to control-treated
animals (Figure 5C, left panel, p,0.001). However, at week seven
neither mcr84- nor sunitinib-treated animals displayed elevated
systolic blood pressures as compared to control-treated animals
although blood pressures were significantly higher in sunitinibtreated
animals than in those receiving mcr84 (Figure 5C, right
panel, p,0.05). Thus in this model, inhibiting VEGFR2 with
mcr84 or multiple receptor tyrosine kinases including both
VEGFR1 and VEGFR2 with sunitinib increased systolic blood
pressure after acute but not chronic therapy. During week six of
therapy, metabolic cages were used to collect urine samples from
all mice, which were subsequently analyzed for urine protein
(Upro) and creatine levels by UT Southwestern Medical Center’s
mouse metabolic phenotyping core. The Upro:creatine ratio did
not differ between the three treatment groups, suggesting that long
term treatment with mcr84 and sunitinib does not induce kidney
damage (Figure 5D). Similar to the initial toxicity study in NOD/
SCID mice, blood was collected from all animals at sacrifice for
analysis by the mouse metabolic phenotyping core, which again
yielded no significant changes in any of the 18 tested analytes
between mcr84-, sunitinib-, or control-treated animals (Table S2).
Therefore, in a spontaneous tumor model in immunocompetent
animals, chronic treatment with mcr84 failed to produce
observable, lasting toxicity.
Discussion
Angiogenesis is a crucial process during embryonic development
and normal physiology, and during tumor development, growth,
and progression [2]. Anti-angiogenic therapy therefore presents an
exciting and rational approach for tumor therapy. However, the
clinical efficacy of anti-angiogenic therapies have been mostly
Figure 3. r84 controls tumor growth in vivo. 2.5 million human
NSCLC cells were injected subcutaneously into the right flank of NOD/
SCID mice. Therapy began one day post tumor cell injection (TCI), and
continued for 4–8 weeks. Tumor volumes were measured twice/week
and final tumor weights were recorded at sacrifice. r84 and
bevacizumab (bev) similarly control tumor growth and final weight
compared to control IgG treatment in H460, H1299 models (A, B). C, In
A549 NSCLC tumor bearing animals, r84’s control of tumor growth was
significantly different from bev (p,0.05). D, Titration of antibody dosing
in A549 tumor xenografts showed no change in tumor growth and final
tumor weight with increasing doses of bev, however there was
increasing control of tumor growth with increasing doses of r84, with
doses of r84 controlling growth better than bev (D). A–C, n = 8 mice per
group; D, n = 6 mice per group. *p,0.05, **p,0.01, ***p,0.001,
statistical differences compared to Ctrl treatment, unless otherwise
indicated.
doi:10.1371/journal.pone.0012031.g003
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disappointing, with modest increases in patient overall survival
[27]. Therefore, there is still much to learn about angiogenic
signaling and angiogenesis dependence within tumors, which can
be aided through the development and use of new investigative
tools.
Here we describe r84, a fully human monoclonal antibody
specific for VEGF, a key mediator of angiogenesis. r84 binds to
human and mouse VEGF-A, but not other VEGF family members
(VEGF-B, -C, -D, PlGF), and specifically blocks subsequent
binding of VEGF to VEGFR2, leaving intact VEGF:VEGFR1
interaction. Through its unique VEGF binding properties, r84
blocked VEGFR2-mediated endothelial cell migration and
signaling. In vivo, r84 controlled tumor growth in NOD/SCID
mice similarly to bevacizumab. r84-treated tumors had reduced
MVD, VEGFR2 expression, and LVD as compared to controltreated
tumors, and showed a trend towards increased pericyteassociated
blood vessels. Importantly, chronic exposure to r84 in
tumor bearing and non-tumor bearing NOD/SCID mice and in a
spontaneous, immunocompetent model of pancreatic cancer did
not induce toxicity.
The discriminating specificity of r84 in that it recognizes one
ligand (VEGF) and inhibits binding only to VEGFR2 establishes
r84 as a beneficial tool for elucidating VEGFR1 signaling
pathways and functional contributions of VEGFR1 and VEGFR2
in vitro and in vivo. r84 binds both human and mouse VEGF
(Figure 1A, B), and a mouse chimeric version of r84 (mcr84) has
been developed, thereby obviating the need for complex mouse
model systems genetically engineered to express human VEGF
[12] to study contributions of host- and tumor-derived VEGF in
human xenograft or syngeneic tumor models. Previous work has
directly compared the efficacy of r84 with other anti-angiogenic
agents in established human tumor xenografts and syngeneic
tumor models [20–21]. In these studies, r84 has been shown to be
more effective than bevacizumab, sunitinib, an anti-VEGFR2
antibody (RAFL-2), and a peptoid against VEGFR1 and
VEGFR2 (GU81) in controlling tumor growth and infiltration of
immune suppressor cell populations [19,21]. Functionally, r84
inhibits VEGFR2 activity by specifically blocking only VEGF.
This distinguishes this r84 from anti-VEGFR2 antibodies such as
DC101 that block the activity of all VEGFR2 ligands [28]. The
importance of r84’s specificity is best observed through direct
comparisons where r84 has been shown to outperform less specific
anti-VEGFR2 strategies [21]. The present study supports the
previous investigations, highlighting that selective inhibition of
VEGFR2 with r84 can delay tumor take and control tumor
growth similar to blockade of both VEGFR1 and VEGFR2
(Figure 3), bringing to question the function of VEGFR1 in tumor
angiogenesis and in physiological homeostasis. A caveat to the
specificity of r84 is that we have been unable to determine
conclusively the effect of r84 on VEGF binding to neuropilin-1 or -
2, which might impact the biological effect of r84.
Although the function and signaling pathways of VEGFR1
remain elusive, there is data supporting the concept that VEGFR1
is a negative regulator of VEGFR2 signaling. VEGFR1 deficient
mice die in utero due to an over abundance of endothelial cells
[29–30], whereas mice expressing only the extracellular domain of
VEGFR1 are viable [31]. These studies established that VEGFR1
does not need to signal through its cytoplasmic domain and
functions during development as a decoy receptor for VEGF,
sequestering the ligand and regulating VEGFR2-mediated
angiogenesis. Roberts et al., [32] demonstrated that the VEGFR1
mutant phenotype in embryonic stem cell-derived blood vessels
could be rescued by incubation with small molecule inhibitors of
VEGFR2. These data further supports that VEGFR1 controls
blood vessel development by negatively regulating VEGFR2
signaling. In addition, work by Nozaki et al., [33] demonstrated
that VEGF binding to VEGFR1 induced the activity of SHP-1
phosphatase that in turn reduced levels of VEGFR2 phosphorylation.
Therefore, active VEGF binding and signaling through
VEGFR1 could potentially negatively regulate tumor angiogenesis,
an interesting concept that warrants further investigation.
Hypertension is likely caused by decreased levels of nitric oxide
(NO) resulting from blockade of VEGF signaling through
VEGFR2 and VEGFR1 by current anti-angiogenic strategies.
VEGF activation of VEGFR1 has been demonstrated to induce
NO production [34–35]. Therefore, it is possible that hypertension
may be reduced or eliminated following r84 therapy.
Additionally, studies have demonstrated the importance of
VEGFR1 function in tumor cell survival. Neutralizing antibodies
against VEGFR1 [36–37] and PlGF [38], a VEGFR1 specific
ligand, have successfully controlled tumor growth in preclinical
models. Adding to the complexity of this pathway, PlGF over
expression has also been shown to inhibit tumor growth and
angiogenesis through increased levels of functionally inactive
VEGF:PlGF heterodimers [39–40]. Further, Bais et al. recently
demonstrated that although anti-PlGF antibodies were able to
inhibit wound healing and cancer cell extravasation, these
antibodies only inhibited tumor growth in tumors that over
expressed VEGFR1 [41]. These papers question the importance of
directly blocking PlGF or VEGFR1 therapeutically and highlight
the potential benefit of anti-angiogenic agents such as r84 that
allow for PlGF and VEGFR1 interactions. VEGFR1 has also been
linked to tumor metastasis [42–43]. However, selective blockade of
VEGFR2 in our models was sufficient to control tumor growth as
compared to simultaneous inhibition of VEGFR1 and VEGFR2
(Figure 3A–D). Increased metastasis was not observed from tumor
xenografts treated with r84 or the phenotypic precursor of r84,
2C3, in subcutaneous or orthotopic models [3,20]. Nevertheless,
the effects of anti-angiogenic therapy on tumor progression and
metastasis are still being elucidated [44–45] and could benefit from
selective tools, such as r84, to delineate important pathways and
mechanisms of action in these processes.
In the present study, delayed tumor take through selective
inhibition of VEGFR2 with r84 was associated with several
histological changes. r84 reduced tumor MVD similar to
bevacizumab treatment (Figure 4A). Consistent with the concept
of anti-angiogenic therapies functioning by pruning nascent tumor
vasculature, we observed a trend of increased pericyte association
with endothelial cells in r84 and bevacizumab treated animals,
though this only reached statistical significance in the H460 model
Figure 4. r84 therapy induces vascular changes within tumors. Frozen sections of A549, H460, H1299 tumors treated with control IgG (Ctrl),
r84, or bevacizumab (bev) were analyzed by immunofluorescence for endothelial, pericyte (PC), and lymphatic markers, as well as for VEGFR2
expression. Number of positive-staining entities per high powered field was evaluated using Nikon Elements software. A, r84, bev treatment
significantly decreases tumor MVD, shown by a reduction in tumor endomucin positive endothelial cells (red). r84, bev treatment induced a trend
towards increased NG2 positive (green) PC coverage of vessels as compared to Ctrl. B, r84, bev treatment significantly reduces the number of VEGFR2
positive cells in H460, H1299 tumors as shown by RAFL-2 staining (red). Only r84 treatment significantly reduced VEGFR2 staining in A549 tumors. C,
r84, bev treatment significantly decreased H460, H1299 tumor LVD, as indicated by a reduction in lyve1 positive cells (red). Only r84 treatment
significantly reduced A549 tumor LVD. *p,0.05, **p,0.01, ***p,0.001, statistical differences compared to Ctrl, unless otherwise indicated.
doi:10.1371/journal.pone.0012031.g004
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Figure 5. Extended r84 therapy controls tumor growth without induction of toxicity. 5 million PANC-1 tumor cells were injected
subcutaneously into NOD/SCID mice. Tumor bearing (TB) and non tumor bearing (NTB) mice received long-term 12-week therapy with 50 mg/kg/
week r84 or a control IgG. Following 12-weeks of therapy, animals were sacrificed and organs and blood were collected for toxicity analysis. n = 5
animals per group. A, Hematoxylin and eosin staining of formalin-fixed, paraffin-embedded kidney and liver sections demonstrated that control of
tumor growth is achieved without induction of kidney or liver histopathologic changes. B, Blood chemistry analysis of serum samples collected from
mice at sacrifice indicated that r84 treatment does not induce changes in serum levels of alanine aminotransferase (ALT), aspartate aminotransferase
(AST), or blood urea nitrogen (BUN) as compared to control IgG therapy and to age-matched Nai¨ve animals that did not have tumor and never
received antibody therapy. Immunocompetent Kras/INK4a mice that spontaneously develop pancreatic cancer were treated for eight weeks with
r84 Inhibits VEGFR2
PLoS ONE | www.plosone.org 10 August 2010 | Volume 5 | Issue 8 | e12031
(Figure 4A). NSCLC tumors treated with r84 and bevacizumab
showed a reduction in VEGFR2 staining (Figure 4B), with the
exception of A549 tumors where bevacizumab had no effect,
suggesting specific inhibition of VEGF:VEGFR2 binding by r84
can down regulate receptor expression. As VEGFR2 is considered
the predominant angiogenic signaling receptor, decreasing its
expression within tumors could promote the anti-angiogenic
effects of r84. Tumor LVD was also decreased in mice treated
with r84 and bevacizumab, with the exception of A549 tumors
where bevacizumab had no effect. In several tumor types,
including lung cancer, lymphatic vasculature participates in tumor
metastasis [46]. Although predominately mediated by VEGF-C
and -D interaction with VEGFR3, recent data demonstrated
elevated expression of tumor-derived VEGF-A contributes to
pathological lymphangiogenesis [47–48]. In a corneal injury
model, Cursiefen et al., [47] demonstrated that elevated levels of
VEGF-A recruits macrophages and inflammatory cells secreting
VEGF-C and -D to the site of injury, thereby inducing
lymphangiogenesis. This mechanism may explain the decrease in
LVD seen in treated tumors in our studies. Therefore, reduced
LVD observed with r84 and bevacizumab therapy is perhaps
mechanistically similar to the reduction in LVD observed in 2C3-
treated breast cancer xenografts, which correlated with a
VEGFR2-mediated down regulation of VEGFR3 in lymphatic
endothelial cells and a decrease in Ang-2 expression in endothelial
cells and tumor cells [49].
Extended therapy with r84 in tumor bearing and non-tumor
bearing mice did not induce toxicity, as measured by weight
maintenance, blood pressure levels, proteinuria analysis, and
preservation of renal, hepatic, and pancreatic structure and
function. Previous studies assessing the safety of anti-VEGF
antibodies, including bevacizumab, demonstrated increased hepatic
and renal damage with antibodies of increasing affinity to
VEGF. Hepatic and renal toxicity produced elevated serum levels
of ALT, AST, and BUN as well as glomerulosclerosis and loss of
structural integrity seen by H&E staining [12]. These toxicityinducing
antibodies were first characterized in 2006 as crossreactive
antibodies that recognized human and mouse VEGF and
highlighted the importance of blocking stromal-derived VEGF in
some tumor models [50]. Our current work with r84 in the A549
xenograft model (Figure 3C–D) highlights the importance of host
VEGF in the progression of some tumors. However, in our studies,
long-term therapy with r84 does not induce the renal or hepatic
toxicities (Figure 5, Table S1). This separates r84 from previously
developed cross-reactive antibodies as a unique therapeutic tool
with the potential to answer key questions on the function of
stromal VEGF in tumor progression and the importance of
VEGFR1 activity in avoiding anti-VEGF induced toxicity. The
endocrine pancreas is especially sensitive to VEGF inhibition
[9,51]. However, extended therapy with r84 did not result in
changes in pancreatic islet structure or function (Figure S3A-C). In
an immunocompetent model of spontaneous pancreatic cancer,
extended therapy with mcr84 did not induce renal or hepatic
toxicities as indicated by urine analysis and serum metabolic
markers (Figure 5D, Table S2) and acute increases in systolic
blood pressure were resolved over time without cessation of
therapy (Figure 5C). Thus, we conclude that r84 and mcr84 do not
induce significant toxicities in mice perhaps due to the lower
affinity of r84 for VEGF as compared to other anti-VEGF
antibodies or from a protective function of VEGFR1. Overall, the
in vitro and in vivo characteristics of r84 establish this antibody as an
important tool to further elucidate the importance of VEGF
signaling through VEGFR1 and VEGFR2 within tumors and
during normal physiology and as a potential adjuvant therapy. At
the present time the production of clinical grade r84 is being
evaluated and we anticipate that initial safety trials in humans will
begin in the near future.
Supporting Information
Table S1 Extended r84 therapy does not induce significant
changes in blood serum chemistry. NOD/SCID mice bearing
subcutaneous PANC-1 tumors received long-term 12-week
therapy with 50 mg/kg/week r84 or a control IgG. Blood
chemistry analysis of serum samples collected from mice at
sacrifice indicated that extended r84 treatment does not induce
changes in serum levels of 20 different markers, as compared to
control-treated (Ctrl) or Nai¨ve animals.
Found at: doi:10.1371/journal.pone.0012031.s001 (0.03 MB
DOC)
Table S2 Extended mcr84 therapy does not induce significant
changes in blood serum chemistry. Immunocompetent mice
heterozygous for a spontaneous model of pancreatic cancer
received extended 8-week therapy with saline, 25 mg/kg/week
mouse chimeric r84 (mcr84), or 50 mg/kg/week sunitinib. Blood
chemistry analysis of serum samples collected from mice at
sacrifice indicated that extended mcr84 and sunitinib treatment
does not induce changes in serum levels of 18 different markers, as
compared to saline-treated animals in this model.
Found at: doi:10.1371/journal.pone.0012031.s002 (0.03 MB
DOC)
Figure S1 r84 reduces mouse VEGF-induced endothelial cell
migration and signaling in vitro. A, A modified Boyden chamber
migration assay was used to assess the effect of r84, bevacizumab
(bev) on mouse VEGF-induced endothelial cell (EC) migration.
20,000 PAE-KDR cells were plated on 8.0 mm cell culture inserts
and allowed to migrate overnight towards SFM or mouse VEGF
(100 ng/mL)+/2500-fold molar excess antibody (bev, r84, control
IgG). Only r84 blocks mouse VEGF-induced migration of
VEGFR2-expressing PAE-KDR ECs. B, Western blots of mouse
VEGF-induced signaling in PAE-KDR lysates following stimulation
of cells with 50 ng/mL mouse VEGF+/2500-fold molar
excess antibody (bev, r84, control IgG). Only r84 blocks p-
VEGFR2 and downstream phosphorylation (PLC-c, ERK1/2) in
mouse VEGF-stimulated cells. ***p,0.001, statistical differences
in A compared to mouse VEGF alone, unless otherwise indicated.
Found at: doi:10.1371/journal.pone.0012031.s003 (0.68 MB TIF)
Figure S2 Efficacy of long-term anti-VEGF therapy. r84 and
mcr84 were able to control tumor growth in two extended therapy
models. A–B, NOD/SCID mice bearing subcutaneous PANC-1
tumors received long-term 12-week therapy with 50 mg/kg/week
r84 or a control IgG. r84 therapy significantly controls tumor
growth and final tumor weight compared to control IgG (A,
*p,0.05). B, r84 significantly decreases PANC-1 tumor microvessel
density as compared to control IgG (Ctrl) treatment as
saline, 25 mg/kg/week mcr84, or 50 mg/kg/week sunitinib. After 2 weeks of therapy, mcr84 and sunitinib significantly increased mean systolic blood
pressure (C, left panel, ***p,0.001), but this effect was lost by week 7 of continuous therapy (C, right panel). D, Urine samples collected during week
6 of therapy and assayed for total levels of urine protein and creatine (Upro/Creatine ratio displayed) showed no significant difference between
treated animals as compared to control, indicating that extended therapy with mcr84 and sunitinib did not induce kidney damage in this model.
doi:10.1371/journal.pone.0012031.g005
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PLoS ONE | www.plosone.org 11 August 2010 | Volume 5 | Issue 8 | e12031
shown by endomucin staining (***p,0.001). C, Immunocompetent
mice heterozygous for a spontaneous model of pancreatic
cancer received extended 8-week therapy with saline, 25 mg/kg/
week mouse chimeric r84 (mcr84), or 50 mg/kg/week sunitinib.
There was a trend towards a decrease in final pancreas weight at
time of sacrifice in mcr84-treated animals as compared to control,
although this decrease failed to reach statistical significance.
Found at: doi:10.1371/journal.pone.0012031.s004 (3.99 MB TIF)
Figure S3 Immunohistochemical analysis of r84 efficacy and
toxicity profile following long-term therapy. NOD/SCID mice
bearing subcutaneous PANC-1 tumors received long-term 12-
week therapy with 50 mg/kg/week r84 or a control IgG. A, Longterm
r84 therapy in TB or NTB animals did not change
pancreatic islet vessel density (endomucin, green) or pericyte
distribution (NG2, red) as compared to age-matched Nai¨ve
animals (**p,0.01). Blood chemistry analysis of serum samples
collected from mice at sacrifice revealed no change in glucose
levels between groups (B). TB or NTB animals receiving long-term
antibody therapy with r84 or a control IgG and Nai¨ve animals
showed no difference in insulin staining intensities (green) within
pancreatic islets (C).
Found at: doi:10.1371/journal.pone.0012031.s005 (7.39 MB TIF)
Acknowledgments
We thank the past and present members of the Brekken Laboratory and
Drs. Philip Thorpe, John Minna, Joan Schiller and Jason Fleming for
advice, support, and reagents. We also gratefully acknowledge the Mouse
Metabolic Phenotyping Core (5PL1DK081182, PI: D. Russell) and the
O’Brien Kidney Research Center (P30DK079328, PI: P. Igarashi) at UT
Southwestern for assistance in evaluating blood chemistries, urine analysis,
blood pressure, and histopathology. We are indebted to our colleagues at
Peregrine Pharmaceuticals and Affitech AS for provision of r84 and for
assistance in its characterization.
Author Contributions
Conceived and designed the experiments: LAS RAB. Performed the
experiments: LAS JGC CLR JET. Analyzed the data: LAS CLR JR RAB.
Contributed reagents/materials/analysis tools: LAS MNA AK KS. Wrote
the paper: LAS RAB.



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