Cross-species Vascular Endothelial Growth Factor (VEGF)-blocking
Antibodies Completely Inhibit the Growth of Human Tumor Xenografts
and Measure the Contribution of Stromal VEGF*S
Received for publication, July 27, 2005, and in revised form,
November 1, 2005 Published, JBC Papers in Press, November 7, 2005,
DOI 10.1074/jbc.M508199200
Wei-Ching Liang‡1, Xiumin Wu§1, Franklin V. Peale¶, Chingwei V.
Lee‡, Y. Gloria Meng, Johnny Gutierrez, Ling Fu¶, Ajay K. Malik§2,
Hans-Peter Gerber§, Napoleone Ferrara§3, and Germaine Fuh‡4
From the Departments of ‡Protein Engineering, §Molecular Oncology,
¶Pathology, and Assay and Automation Technology, Genentech Inc.,
South San Francisco, California 94080
To fully assess the role of VEGF-A in tumor angiogenesis, anti-
bodies that can block all sources of vascular endothelial growth
fac- tor (VEGF) are desired. Selectively targeting tumor-derived
VEGF overlooks the contribution of host stromal VEGF. Other
strategies, such as targeting VEGF receptors directly or using
receptor decoys, result in inhibiting not only VEGF-A but also VEGF
homologues (e.g. placental growth factor, VEGF-B, and VEGF-C),
which may play a role in angiogenesis. Here we report the
identification of novel anti-VEGF antibodies, B20 and G6, from
synthetic antibody phage libraries, which block both human and
murine VEGF action in vitro. Their affinity-improved variants
completely inhibit three human tumor xenografts in mice of skeletal
muscle, colorectal, and pancreatic origins (A673, HM-7, and HPAC).
Avastin, which only inhibits the tumor-derived human VEGF, is 90%
effective at inhibiting HM-7 and A673 growth but is <50%
effective at inhibit- ingHPACgrowth. Indeed,HPAC tumors containmore
host stroma invasion and stroma-derived VEGF than other tumors.
Thus, the functional contribution of stromal VEGF varies greatly
among tumors, and systemic blockade of both tumor and
stroma-derived VEGF is sufficient for inhibiting the growth of
tumor xenografts.
Targeting angiogenesis, the process of new blood vessel formation,
has been validated as an effective approach for cancer therapy by
the recent Food and Drug Administration approval of bevacizumab
(AvastinTM antibody), a blocking antibody against vascular
endothelial growth factor A (VEGF-A or VEGF),5 for the treatment of
metastatic colorectal cancer (1, 2). Although many pro- or
anti-angiogenic factors have been implicated in tumor angiogenesis
(3, 4), the efficacy of a specific blocking antibody against VEGF
in suppressing cancer progres-
sion emphasizes the key role of VEGF. However, as VEGF is a member
of a family that also includes VEGF-B, VEGF-C, VEGF-D, and
placental growth factor (PlGF) with overlapping activities in three
tyrosine kinase receptors, VEGFR1 (Flt-1), VEGFR2 (KDR, or Flk-1 in
mouse), and VEGFR3 (5, 6), the questionswhetherVEGF-Ahas a
nonredundant role in tumor angiogenesis and whether blocking VEGF-A
is sufficient to inhibit tumor growth are fundamental. Systemic
blockade using specific blocking antibodies against tumor-
derived VEGF (7) or genetic deletion of VEGF in tumor cells (8–10)
severely suppressed tumor growth in mouse models. However, residual
growth or resistance of human tumor xenografts in nude mice treated
with antibodies against human VEGF has been reported (7, 11). One
possibility is that residual tumor angiogenesis and growth are
supported bymurine VEGF, which is produced by host stromal cells
recruited into the tumor (11). Significant VEGF expression has been
demonstrated by fibroblast and immune cells that surround and
invade the tumor mass (12–16), although the significance and extent
of the contribution of such stroma-derived VEGF to tumor growth
remain unclear. Other possible explanations for incomplete tumor
growth inhibition following treatment with anti-VEGF antibodies
include the following: insufficient binding affinity or tumor
penetration; VEGF-related molecules (e.g. PlGF and VEGF-C) that may
act independently or in concert with VEGF-A in promoting
angiogenesis (17); VEGF-independent angio- genesis pathways; and
“vascular mimicry” by tumor cells (18). To address these issues,
some of us (11) and others (19) have used the soluble VEGF receptor
extracellular domain (ECD) fragment, such as VEGFR1 Ig domain 1–3
Fc fusion (VEGFR1D1–3-Fc) or VEGF trap (VEGFR1D2- VEGFR2D3-Fc), and
showed more complete inhibition of human xenografts relative to
antibodies against tumor-derivedVEGF. It was reasoned that these
receptor decoys can block both human and murine VEGF (11) or that
the receptor fragments have higher affinity than the available
anti-h-VEGF antibody (19). However, these receptor fragmentsmay
also block other VEGF familymembers, i.e.VEGF-B and PlGF. Although
gene deletion of VEGF-B (20) or PlGF (21) was well tolerated in
embryonic and postnatal developments, suggesting that these factors
are not essential for developmental angiogenesis, recent studies
have shown that both VEGF-B and PlGF can contribute to path-
ological angiogenesis (21). Therefore, the specific contribution of
VEGF-A cannot be defined by using these receptor decoys. As for
affin- ity, direct comparison of anti-VEGF antibody with receptor
decoy was not meaningful because of their different specificities.
Other inhibitors such as inhibitors against VEGFR2 also cannot
reveal the specific role of VEGF-A because VEGF-C and VEGF-D can
also activate VEGFR2. To clarify these issues, antibodies that can
directly and specifically
* 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 Fig. 1 and Table.
1 Both authors contributed equally to this work. 2 Present address:
Hollis-Eden Pharmaceutical Inc., San Diego, CA 92121. 3 To whom
correspondence may be addressed: Dept. of Molecular Oncology,
Genentech
Inc., 1 DNA Way, South San Francisco, CA 94080. E-mail:
[email protected]. 4 To whom correspondence may be addressed: Dept. of
Protein Engineering, Genentech
Inc., 1 DNA Way, South San Francisco, CA 94080. E-mail:
[email protected]. 5 The abbreviations used are: VEGF, vascular
endothelial growth factor; h-VEGF, human
VEGF; m-VEGF, murine VEGF; VEGFR, VEGF receptor; PlGF, placental
growth factor; Fab, antigen binding fragment; ELISA, enzyme-linked
immunosorbent assay; ECD, extracellular domain; BSA, bovine serum
albumin; HRP, horseradish peroxidase; PBS, phosphate-buffered
saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonic acid; HUVEC, human umbilical vein endothelial
cells; CDR, comple- mentarity determining region.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 2, pp. 951–961,
January 13, 2006 © 2006 by The American Society for Biochemistry
and Molecular Biology, Inc. Printed in the U.S.A.
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block VEGF of both tumor and stromal sources would be the tool to
examine whether blocking VEGF alone is sufficient for complete
inhi- bition of tumor growth or whether the binding affinity of the
antibody affects the potency and efficacy. Furthermore, a
comparison between cross-reactive antibodies and antibodies that
block only tumor-derived VEGF may reveal the level of dependence on
stromal VEGF for tumor growth. VEGF-A is a member of the
homodimeric cysteine knot protein fam-
ily growth factor (6). Two tyrosine kinase receptors mediate the
signal activity of VEGF, VEGFR1 (Flt-1) andVEGFR2 (KDR), with the
latter as the main receptor mediating most of the endothelial cell
action as clearly demonstrated by VEGF variant and homologues that
only can bind VEGFR2 (22, 23). The role of VEGFR1 is less clear,
but it has high affinity for VEGF-A, VEGF-B, and PlGF, with the
latter two being VEGFR1-specific ligands. Structural and functional
studies of VEGF receptor binding domain in complex with Avastin Fab
(Fab-12) (24, 25) and affinity-improved Avastin, Y0317 (26), and
the primary binding domain of VEGFR1, the second Ig domain
(VEGFR1D2) (27), revealed that the VEGF epitope for Avastin and for
VEGFR1 or VEGFR2 have sufficient overlap for mutual exclusion.
Human and murine VEGFs are 85% identical in sequence. More than 10
hybridoma antibodies against human VEGF were generated, but none
can bind murine VEGF, which is expected asmice remove self-reactive
antibodies. The high homology of murine VEGF with rat or hamster
VEGF also made it difficult to generate the desired monoclonal
antibodies in these animals. To identify antibodies that
cross-react with murine and human
VEGFs, we employed phage-displayed synthetic antibody libraries,
which were generated by randomizing the complementarity determin-
ing regions (CDRs) of heavy chain in the humanized anti-ErbB2 anti-
body 4D5 (h4D5-8) template in amanner thatmimicked the diversity of
natural human antibodies (28). Humanized 4D5-8 contains the com-
monly used framework (VH-3/Vk-1); thus antibodies isolated from the
libraries would mimic natural human antibodies. Another main advan-
tage of antibody generation with phage libraries is its in vitro
selection process that should be indifferent to the cross-species
conservation (29, 30). In this study, we report the identification
of cross-human and murine VEGF binding clones, G6 and B20. We
examined the affinity- improved G6 and B20 variants for their
binding affinities, specificities, and VEGF blocking activities in
comparison to the hybridoma-derived anti-h-VEGF antibody, A4.6.1
(or the humanized version, Avastin), and we identified variants
suitable for in vivo studies, G6-31 and B20-4.1. In inhibiting
h-VEGF action in vitro, B20-4.1 was equipotent to Avastin, andG6-31
was equipotent to Y0317. The cross-species anti-VEGF anti- bodies
examined the specific role of VEGF in the tumor progression using
mouse models.
MATERIALS AND METHODS
Binding Specificity of G6 and B20—Competitive ELISAs were used to
determine the relative binding affinity of phage clones displaying
anti- VEGF Fab to h-VEGF andm-VEGF as described (28). Briefly, Fab
phage were incubated with serial dilutions of h-VEGF orm-VEGF in
PBS with 0.5% BSA and 0.05% Tween 20 (PBT) for 2 h, and then
unbound Fab phage were captured with VEGF-coated 96-well Maxisorp
ELISA plate (Nunc, Roskilde, Denmark) and detected by anti-M13
phage antibody conjugated with horseradish peroxidase (HRP)
(Amersham Bio- sciences) followed by substrate. EC50 values, the
concentrations of VEGF incubated with Fab phage in solution that
were effective in bind- ing 50% of Fab phage to block their binding
to immobilized VEGF were reported. Fab protein was prepared from
Escherichia coli harboring the plasmid of the G6 Fab expression
construct under the promoter of
alkaline phosphatase and secretion leader sequence stII andwas
purified with the protein G affinity column as described (28). IgGs
were gener- ated with mammalian cell transfection and purified with
protein A affinity column. For examining binding specificities,
VEGF and various homologues were first coated on an ELISA plate at
2 g/ml in PBS and blocked with 0.5% BSA and 0.05% Tween 20. Serial
dilutions of Fabs or IgGs were then incubated in VEGF
variant-coated wells for 1 h and measured with anti-human antibody
HRP diluted in PBT buffer. The VEGF homologues, mouse and human
PlGF-2, humanVEGF-C,mouse VEGF-D, human and mouse VEGF-B, were
purchased from R & D Systems (Minneapolis, MN). Solution
binding assays were also carried out in case some protein became
unfunctional upon coating on the plate. Fabs or IgG (0.5 nM) were
incubated with VEGF homologues at different concentrations for 1–2
h, and the unbound antibodies were captured with m-VEGF-coated
ELISA wells and detected as above. The results were consistent with
the direct binding ELISA format.
VEGF Receptor Blocking Assay—Initial blocking assays were per-
formed by phage ELISA. Murine VEGF-coated ELISA wells were first
incubated with increasing concentrations of receptor fragments for
30 min, and phage displaying Fabs were then added. Bound phage were
measured as described above. Purified Fabs and IgGs were then used
to confirm receptor blocking activities. VEGF receptor-immobilized
plateswere prepared by capturingVEGFR2ECD (Ig domain 1–7) as
Fc
fusion with goat anti-human IgG Fc (Jackson ImmunoResearch, West
Grove, PA) on the ELISA plate, whereas VEGFR1 ECD fragment (Ig
domains 1–5) (VEGFR1D1–5) was coated directly. Biotinylated
h-VEGF165 or m-VEGF164 (2 nM) was first incubated with 3-fold seri-
ally diluted anti-VEGF antibodies in PBT. After 1–2 h of incubation
at room temperature, the mixtures were transferred to a VEGF
receptor- immobilized plate and incubated for 10 min. The unblocked
VEGF-A was capturedwith VEGF receptor-coatedwells and detected by
strepta- vidin-HRP conjugates as described above.
Anti-VEGF Antibodies Binding Affinities—For binding affinity meas-
urements, surface plasmon resonance measurements with BIAcoreTM-
3000 (BIAcore, Inc., Piscataway, NJ) were employed. Carboxymethyl-
ated dextran biosensor chips (CM5, BIAcore, Inc.) were activated
with N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride
and N-hydroxysuccinimide according to the supplier’s instructions.
Human or murine VEGF was immobilized to achieve 60 response units.
2-Fold serial dilutions of Fab or IgG (0.78–500 nM) were injected
in PBS with 0.05% Tween 20 (PBST) at 37 or 25 °C (data not shown)
at a flow rate of 25 l/min. Association rates (kon) and
dissociation rates (koff) were calculated using one-to-one Langmuir
binding model (BIAcore evaluation software version 3.2). The
equilibrium dissociation constant (Kd) was derived as the ratio
koff/kon.
HUVEC Assay of VEGF Inhibition—Human umbilical vein endothe- lial
cells (HUVEC) (Clontech) were grown and assayed as described (31).
Approximately 4000 HUVECs were plated in each well of the 96-well
cell culture plate and incubated in Dulbecco’s modified Eagle’s/
F-12 medium (1:1) supplemented with 1.5% (v/v) fetal bovine serum
(assay medium) for 18 h. Fresh assay medium with fixed amounts of
VEGF (0.15 nM final concentration), which was first titrated as a
level of VEGF that can stimulate submaximal DNA synthesis, and
increasing concentrations of anti-VEGF antibodies were then added
to the cells. After incubation at 37 °C for 18 h, cells were pulsed
with 0.5 Ci/well of [3H]thymidine for 24 h and harvested for
counting with TopCount Microplate Scintillation counter.
Tumor Implantation and in Vivo Treatments—HM-7, A673, and HPAC
cells (American Type Culture Collection) were maintained in culture
with Dulbecco’s modified Eagle’s/F-12 medium, supplemented
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with 10% fetal bovine serum. Cells were grown at 37 °C in 5% CO2
until confluent and then harvested and resuspended in sterile
Matrigel at a concentration of 25 106 cells/ml. Xenografts were
established in 6–8- week-old female Beige Nude XID mice by dorsal
flank subcutaneous injection of 5 106 cells/mouse and allowed to
grow. When tumors reached a volume of 500 mm3 for HM-7 and 150–200
mm3 for A673 and HPAC (48 h), a cohort was randomly selected (n 10)
as day 0
controls. The remaining mice were divided into groups of 10, and
anti- bodies were administered intraperitoneally at the same dose
for each group. Tumor sizes and weights were measured as described
(11). All statistical analyses used Student’s t test.
Histology and Staining—Tumor were fixed in 10% neutral buffered
formalin for 12–16 h prior to paraffin embedding. Histologic
sections of 4–5 m thickness were stained with hematoxylin and eosin
as
FIGURE 1. Binding specificities and receptor blocking activities of
the phage-derived antibodies. A, sequences of four different phage
clones initially identified were compared with template h4D5.
Shaded residues were not randomized in the antibody libraries. The
relative affinities for h-VEGF and m-VEGF were measured with
competitive ELISA (EC50). The receptor blocking activities of the
four clones were screened by measuring phage binding to
m-VEGF-coated wells in the presence of either serial dilutions of
VEGFR2 ECD (VEGFR2D1–7) (B) or second Ig domain of VEGFR1 ECD
(VEGFR1D2) (C). D, binding specificities of G6 or B20-4 Fabs were
measured by binding to h-VEGF (VEGF-A)-, m-VEGF-, h-PlGF-, m-PlGF-,
m-VEGF-D-, or h-VEGF-B-coated wells, and bound Fab was detected. E,
receptor blocking of Fab was confirmed by the binding of
biotinylated m-VEGF to immobilized VEGFR2D1–7 in the presence of
increasing concentration of G6, B20-4, Fab-12 (Fab of Avastin), or
Y0317.
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described previously (32). Vascular density was assessed in MECA-32
(anti-PLVAP)-stained histological sections of tumors, essentially
as described previously (33). For each tumor, three representative
images, centered 500 m from the tumor margin, were analyzed.
ELISAs for VEGF Protein in Tumor Extract and Anti-VEGF in Mice
Serum—For VEGF in tumors, 400–800 g of excised tumor was
homogenized in RIPAbuffer (100l/g) containing 20mMTris, pH7.5, 150
mMNaCl, 2 mM EDTA, 1% deoxycholic acid, 1% Triton X-100, and 0.25%
SDS, and supernatants were collected. Human VEGF ELISA of high
sensitivity was performed as described (34). Briefly, sandwich
ELISA used monoclonal antibody 3.5F8 (Genentech) to h-VEGF as coat,
and biotinylated A4.6.1 as detecting antibody. VEGF165 standards
(1–128 pg/ml in 2-fold serial dilution) and samples were measured
side by side. This ELISA does not detect m-VEGF. To measure m-VEGF,
sandwiched ELISA used goat anti-m-VEGF antibody (R&D Systems)
as coat and the same antibody in the biotinylated form for
detection; m-VEGF standards (3.9–500 pg/ml in 2-fold serial
dilutions) and tumor extract in sample buffer were measured side by
side. The cross-reactiv- ity of h-VEGF occurred when concentration
was above 2000 ng/ml at 0.4%. For serumanti-VEGF (uncomplexed),
ELISAplateswere coated overnight with 0.5 g/ml VEGF165 in 50 mM
sodium carbonate, pH 9.6, blocked, washed, and then incubated with
2-fold serial dilutions of serum samples or anti-VEGF standards
(0.20–25 ng/ml for Y0317 and Avastin, 0.39–50 ng/ml for B20-4.1,
and 0.1–12.8 ng/ml for G6-31) in 0.05% BSA, 0.2% bovine -globulin,
0.25% CHAPS, 5 mM EDTA, 0.35 M
NaCl, 0.05% Tween 20 in PBS, pH 7.4 (samples buffer), for 2 h.
Bound antibodies were detected by appropriate anti-IgG-HRP
conjugates, e.g. anti-human Fc-HRP (Jackson ImmunoResearch) for
Avastin, Y0317, and G6-31 as human IgG1, and anti-mouse IgG2a-HRP
(Pharmingen) for B20-4.1, which had mouse IgG2a constant domains.
Total antibod- ies (complexed and uncomplexed) were measured with
ELISA plates coatedwith appropriate anti-IgG antibodies as above.
Bound antibodies were detected with the same anti-IgG antibodies
conjugated with HRP. By using the same assay for B20-4.1, we
observed that serum from con- trol nudemicewith no injected
antibodies contained less than 0.2g/ml mouse IgG2a.
RESULTS
G6 and B20 Bind Human and Murine VEGF—G6 and B20 were among several
clones isolated from a synthetic antibody phage library with
humanized anti-ErbB2 antibody, h4D5, as the template by
selec-
tion against m-VEGF. The binding specificity and receptor blocking
activity of these phage clones displaying the antigen-binding
fragment (Fab) were first examined (Fig. 1A). G6, the clone with
the best apparent affinity form-VEGF (EC50 0.6 nM), also showed
near equivalent bind- ing for h-VEGF at 1.4 nM (EC50). B20,
however, binds m-VEGF and h-VEGF with relative affinity of 44 and
300 nM, respectively. To assess which antibody clone can blockVEGF
binding to its receptors, VEGFR2 ECD with seven Ig-like domains
(VEGFR2D1–7) (Fig. 1B) or the second Ig domain of VEGFR1 ECD
(VEGFR1D2) (Fig. 1C) was used to block the interaction of
Fab-displaying phage with m-VEGF. Strong blocking was observed with
G6 and B20 by both receptor fragments, suggesting that their
epitopes on VEGF overlapped with the epitopes for receptor frag-
ments. As B20 hadweak and different affinity for h-VEGF andm-VEGF,
it went through a first step of affinity improvement by selection
in a phage-displayed library with light chain CDR residues
randomized. Variant B20-4 exhibited equal apparent affinities for
both h-VEGF and m-VEGF (EC50 15 nM) (see supplemental Fig.
1).
Next, G6 and B20-4 Fab were generated to confirm the binding spec-
ificities. G6 and B20-4 as Fabs indeed demonstrated near equal
affinity for human andmurine VEGF (see Table 1 forKd values), and
no detect- able binding to other VEGF homologues, h- and m-PlGF,
h-VEGF-B (and m-VEGF-B, not shown), h-VEGF-D (Fig. 1D), and VEGF-C
(data not shown). G6 also bound rat and rabbit VEGF with similar
affinity as for m-VEGF (data not shown). The receptor blocking
activity of G6 or B20-4 Fab was confirmed by blocking both h- and
m-VEGF binding to VEGFR2 (1–7 Ig domain ECD) (Fig. 1E) or VEGFR1
(1–5 Ig domain ECD) (m-VEGFblocking forVEGFR2binding shown, and the
rest is not shown). Avastin Fab (Fab-12), in comparison, can only
block h-VEGF but notm-VEGF binding to the receptor as expected
(Fig. 1E). AnAvas- tin variant with improved affinity for h-VEGF,
Y0317, showed slight m-VEGF-blocking activity at high
concentrations as it acquired a weak binding affinity for m-VEGF
(26) (Kd 333 nM, see below).
The Affinity-improved G6 and B20 Variants—To improve the affinity
of G6, light chain CDR residues were mutated as described (28).
Many G6 variants were isolated that differed fromG6 by four or five
residues in the CDR-L3, e.g. G6-8, G6-23, and G6-31. B20-4 was
further improved by mutations in heavy chain CDRs to B20-4.1 (see
supplemental Fig. 1). Binding affinities (Kd) of the improved
variants as Fab or IgG protein were measured at 37 °C with surface
plasmon resonance (BIAcoreTM) on immobilized human or murine VEGF
in
TABLE 1 The binding affinities and kinetic parameters of anti-VEGF
antibodies Affinities of Fab or IgG are measured on BIAcore using
sensor chip immobilized with human or murine VEGF at 37 °C (see
“Materials andMethods”). Each measurement represents an average of
three independent assays that vary by 20–50%. NB denotes no binding
detected; ND denotes calculation not done.
37 °C kon (104) M1 s1 koff (104) s1 Kd (koff/kon) nM
h-VEGF m-VEGF h-VEGF m-VEGF h-VEGF m-VEGF Fab G6 24 28 48 19 20 6.8
G6-8 79 65 27 12 3.4 1.8 G6-31 145 126 13 8 0.9 0.6 G6-23 337 231
25 10 0.7 0.4 B20-4.1 16 12 6.0 14 3.8 12 Fab-12 7.5 NB 6.4 NB 8.5
NB Y0317 7.4 ND 0.11 ND 0.15 ND
IgG G6 18 12 11 6.4 6.2 5.2 G6-8 61 41 6.5 3.9 1.1 0.9 G6-31 133 58
2.4 1.6 0.2 0.3 G6-23 241 135 5.5 3.0 0.2 0.2 B20.4.1 7.5 11 1.2
2.2 1.6 2.0 A4.6.1 8.1 NB 1.4 NB 1.7 NB Avastin 9.2 NB 2.0 NB 2.2
NB Y0317 8.1 1.2 0.02 40 0.02 333
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comparison to A4.6.1, Avastin (Fab-12 as Fab), and the affinity-im-
proved version Y0317 (Table 1). The avidity advantage exhibited by
the IgG reduced the apparent off-rates, but the overall ranking of
these antibodies held. The improved G6 and B20 variants have sim-
ilar affinities for human and murine VEGF. Compared with
hybri-
doma-derived Avastin (or Fab-12 as Fab), B20-4.1 binds h-VEGF with
similar affinity (4–8 nM as Fab and 2 nM as IgG) and kinetic
profile of on-rate, kon, and off-rate, koff. Avastin was used
inter- changeably with A4.6.1 in the study as they have equivalent
activities in vitro and in vivo (35). Among the high affinity
variants, G6-23 and
FIGURE 2. Characterization of affinity-improved G6 and B20-4
variants. A, binding of G6-31 and B20-4.1 as IgG (10 g/ml) to ELISA
wells coated with VEGF homologues is shown. The binding of
biotinylated m-PlGF to VEGFR1 (B) or biotinylated h-VEGF (C) and
m-VEGF (D) to VEGFR2 was inhibited with the anti-VEGF antibodies or
receptor ECD with various potencies. To test the in vitro activity
of the anti-VEGF antibodies, HUVECs were incubated with 0.15 nM
h-VEGF (E) or m-VEGF (F) with increasing concentrations of IgG
variants for 18 h and pulsed with [3H]thymidine for 24 h. The
incorporated [3H]thymidine (in counts/min) as percentages of cells
treated with VEGF alone was plotted. Triplicate data were used, and
error bars represent means S.E.
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G6-31 bind 5–10-fold weaker than Y0317 (20 pM), but G6 variants
bind with nearly 20-fold faster kinetics (on-rate and off-rate)
than Y0317. As for binding specificity, G6-31 shows no detectable
binding for
any of the VEGF homologues (PlGF, VEGF-B, -C, and -D) as its parent
(Fig. 2A). However, B20-4.1 as IgG, but not as Fab, shows some weak
interaction with m-PlGF; the avidity advantage of IgG apparently
enhanced the weak interaction. To assess whether B20- 4.1 binding
to m-PlGF is biologically significant, we examined how well B20-4.1
inhibits m-PlGF from binding m-VEGFR1 ECD, and we saw weak
inhibition at 10 M (estimated EC50 30–100 M) (Fig. 2B). VEGFR1, as
control, shows physiologically relevant binding for the m-PlGF
(EC50 1 nM). G6-31 and B20-4.1 block both h-VEGF
and m-VEGF from binding VEGFR2 and VEGFR1 with high potency (EC50
subnanomolar) (Fig. 2, C and D, and data not shown). The weak
interaction of B20-4.1 and m-PlGF should not play a significant
role for its effect in vivo. We next determined the activity of G6
and B20 variants in inhibiting the VEGF-stimulated growth of HUVEC
(Fig. 2, E and F). Both G6 and B20 affinity-improved variants were
shown to inhibit the activity of both human and murine VEGF,
whereas Y0317 or Avastin, as expected, blocked human VEGF selec-
tively. B20-4.1 exhibited similar potency as Avastin in inhibiting
h-VEGF, whereas G6-31 and G6-23 were 20-fold more potent than
B20-4.1 in inhibiting human and murine VEGF and 20-fold more potent
than Avastin in inhibiting h-VEGF, consistent with their binding
affinities. G6-31 and G6-23 were equipotent to Y0317; the
FIGURE 3. In vivo potency and efficacy of anti-VEGF antibodies in
inhibiting HM-7 (human colorectal) xenografts. A, dose-responsive
inhibition of tumor growth by A4.6.1, Y0317, B20-4.1, and G6-31 (5,
0.5, and 0.1 mg/kg, twice weekly) in HM-7 bearing mouse was shown
by the tumor size (mm3) as a function of time (days). Error bars
represent the S.E. (n 10). B, tumor weight reduction by different
doses of antibodies was measured at day 23. Statistically
significant reduction by each treatment versus control antibody (5
mg/kg) was denoted with * (p 0.05, Student’s t test). In comparing
the potency of G6-31 and B20-4.1, a statistically significant (p
0.05) advantage of the high affinity G6-31 was seen only when dosed
at 0.1 mg/kg. C, tumor sections were stained for vessel-specific
marker as described under “Materials and Methods” to determine the
vessel density (vessel perimeter (m)] per tumor area (m2), m1).
G6-31 and A4.6.1 treatment significantly reduced the tumor vessel
density compared with control antibody (p 0.001).
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assay was unable to differentiate antibodies with affinities higher
than the concentration of VEGF used in the assay (0.15 nM).
In Vivo Activity of B20-4.1 and G6-31 Compared with A4.6.1 and
Y0317 in Inhibiting Human Tumor Growth in Nude Mice—To investi-
gate whether a more complete blockade of VEGF will achieve greater
tumor suppression, B20-4.1,G6-31, or isotype control IgGwere
injected into nudemice after the human colorectal cancer cell
lineHM-7 formed a sizable tumor under the mouse skin (500 mm3, 2
days). Treatments that could not block VEGF originating from host,
A4.6.1 and Y0317, were included in the study for comparison.
Antibodies were adminis- tered intraperitoneally at doses of 0.1,
0.5, or 5 mg/kg (2.5, 12.5, and 125 g/mouse) twice weekly for 3
weeks (n 10); tumor volume wasmeas- ured twice weekly (Fig. 3A),
and tumor weight was determined at the
end of the experiment (Fig. 3B). All four antibodies inhibited
tumors significantly relative to the control group in a
dose-dependent manner. G6-31was able to inhibit significantly tumor
growth (70%) even at the very low dose (0.1mg/kg). Higher doses
resulted in complete inhibition. Furthermore, G6-31-treated tumor
shows significant reduction of ves- sel density like A4.6.1,
suggesting inhibition of angiogenesis as the mechanism of action
(Fig. 3C). To examinewhether a correlation exists between binding
affinity and
potency of tumor inhibition, the low affinity antibody B20-4.1 was
com- pared with the high affinity G6-31 antibody. The two
antibodies have identical cross-species reactivity. We observed
that both antibodies achieved nearly complete tumor inhibition at
the high dose (5 mg/kg) (Fig. 3). However, at lower doses (0.5 and
0.1 mg/kg), B20-4.1 was less
FIGURE 4. Stroma-derived VEGF has different levels of contributions
to the growth of xenografts in mice. A, HPAC and A673 tumor sizes
in response to treatment with different antibodies at saturating
doses (5 mg/kg) (n 10) compared with antibody control. B, efficacy
of inhibiting the growth of HPAC, A673, and HM-7 (5 mg/kg) was
determined by tumor weights measured at the end of the experiments.
p 0.05 (see boldface) indicates statistically significant
difference in the denoted pair.
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effective than G6-31 (p 0.065 and 0.007, respectively) (Fig. 3B),
sug- gesting that higher affinity may result in greater in vivo
potency. In apparent conflict with this conclusion, A4.6.1 and
Y0317 were not sig- nificantly different in potency despite100-fold
different binding affin- ities for h-VEGF andmore than a 20-fold
advantage for Y0317 in inhib- iting h-VEGF-stimulated endothelial
cell growth in vitro (Fig. 2). At all doses, tumor shrinkage in the
Y0317-treated groupwas not significantly better than the
A4.6.1-treated group (p 0.05). The only group that showed a slight
advantage of Y0317 was the high dose group (5 mg/kg),
but the difference was not statistically significant (p 0.059)
(Fig. 3B). One possible explanation is that stromal VEGF (m-VEGF)
was up-reg- ulated andmasked the effect of blocking tumor-derived
VEGF. The fact that Y0317 does have some weak binding affinity for
m-VEGF whereas A4.6.1 (orAvastin) has no detectable binding
tom-VEGF confounds the interpretation of the observation. Another
possible explanation is that the twice-weekly dosing regimen and/or
the metabolic turnover rate of VEGF did not permit the off-rate
differences of these two antibodies to impact the potency. G6-31
and B20-4.1, in contrast, varied mainly in their on-rate.
Stromal VEGFContributionMeasurements—To examine the level of
stromal VEGF contribution to tumor growth, we compared the efficacy
of antibodies that blocked both stromal and tumor-derived VEGF,
G6-31 and B20-4.1, and antibodies that only blocked tumor derived
VEGF, Avastin. We added a slow-growing pancreatic tumor xenograft
HPAC, which contains extensive stromal cell infiltration,6 and
another fast growing human rhabdomyosarcoma A673 xenograft (Fig.
4A). HPAC and A673 tumor inhibition was compared with HM-7 results
at a saturating dose of these antibodies (5mg/kg/week, n 10). For
HM-7 inhibition, the cross-species binding antibodies (G6-31 and
B20-4.1) were more efficacious than the non-cross-species
antibodies (A4.6.1), but the inhibition by the latter was
substantial (90%), indicating some but minimal contribution of
stromal VEGF (Fig. 4B). For A673, the
6 F. V. Peale, and N. Ferrara, unpublished observations.
TABLE 2 Circulating anti-VEGF antibody measurements Serum samples
were collected 7 days after threeweekly injections (day 28) (n 5)
in the HPAC group. Uncomplexed IgG and total IgG (complexed plus
uncomplexed) were measured (mean S.E.) as described under
“Materials and Methods.” The differences between the uncomplexed
and total IgG levels of all four antibodies are statistically
insignificant (p 0.05). The circulating levels (total IgG) of
Avastin are significantly higher than all three other antibodies (p
0.05), yet the levels of two anti-human/mouse VEGF antibodies,
B20-4.1 and G6-31, are statistically indistin- guishable (p 0.
34).
Antibodies Uncomplexed Total g/ml
Avastin 105 1.2 104 11 Y0317 69 3.8 77 4.5 B20-4.1 8.3 3.0 13 4.7
G6-31 5.9 6.3 7.6 6.6
FIGURE 5. Examination of tumor sections. A, hematoxylin and
eosin-stained 5-m sections were scanned at low magnification (bar 5
mm). Images of two independent tumors representa- tive of each
treatment group are shown: control (a and b), Avastin (c and d) (or
A4.6.1 for HM-7 group), or G6-31 antibodies (e and f). Viable
tissue appears dark blue (hematoxylin-stained nuclei); necrotic
tissue stains pink (eosin-stained protein debris, absence of intact
nuclei). Typical viable and necrotic areas are marked v and n,
respectively. B, medium magnification images (bar 500 m) of
pancreatic HPAC (a, e, and i) and colonic HM-7 (b, f, and j)
xenografts treated with control antibody (a and b), anti-human
VEGF, Avastin (A4.6.1 for HM-7) (e and f), or anti-mouse/human
VEGF, G6-31 (i and j). All images are centered 1 mm from the tumor/
host margin. High magnification images (bar 50 m) of HPAC (c, g,
and k) and HM-7 (d, h, and l) tumor xenografts treated with control
antibody (c and d), anti-human VEGF, Avastin (g and h), or anti-
mouse/human VEGF, G6-31 (k and l). Compared with HM-7, HPAC
xenografts are relatively stroma- rich (compare c and d). Control
antibody-treated HPAC tumors have extensive stromal cells infiltra-
tion (arrowheads, c), with indistinct small-caliber vessels
surrounded by connective tissue cells and matrix. Treatment of
these tumors with anti-hu- man (g) or anti-mouse/human VEGF (k)
results in little change in tumor-stroma proportion. In con- trast,
control HM-7 tumors (d) have relatively sparse stroma, and large
caliber thin walled ves- sels (arrow). Treatment with anti-human
VEGF (h) reduces vessel diameter and increases the relative amount
of stromal matrix and cells around remaining vessels. Similar but
more dramatic changes occur when HM-7 tumors are treated with
anti-mouse/human VEGF, G6-31 (l). Images c, d, g, h, and k are
centered 1 mm from the tumor/ host margin (left in the images);
image l is centered 200 m from the tumor/host margin (in this sam-
ple, only necrotic tumor is present at 1 mm from the host
margin).
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inhibition by the cross-species antibodies was not significantly
better than Avastin, thus contribution by stromal VEGF was nearly
nonexist- ent. In contrast, Avastin inhibited less than 50% of the
HPAC growth, whereas both B20-4.1 and G6-31 completely inhibited
the tumor growth, indicating significantly more stromal VEGF
contribution in the growth of HPAC than HM-7 and A673 (Fig. 4, A
and B). Y0317 inhibi- tion of HPAC and HM-7 was also less effective
than the cross-species reactive antibodies, but it surpassed
Avastin with statistical significance in HPAC but not HM-7, which
could be due to the fact that Y0317 also weakly binds m-VEGF. The
differences in efficacy between Avastin, which clearly cannot bind
m-VEGF, and the cross-species antibodies verified that host-derived
VEGF does contribute to the tumor growth and, for the first time,
demonstrated clearly that there were significant differences in the
level of stromal VEGF contribution in the growth of different
tumors. Tumor sections were next examined for the effect of the
antibodies
on these tumors with different contributions of stromal VEGF (Fig.
5). Macroscopically, the section of the HM-7 and HPAC tumors
treated with Avastin IgG (or the equivalent A4.6.1), G6-31
(representative of B20-4.1 group), or control antibodies showed
that Avastin indeed was not effective in decreasing the tumor size
and increasing the area of necrosis of HPAC, whereas G6-31
treatment resulted in extensive necrosis in both HPAC and HM-7
tumor (Fig. 5A). Microscopically, HM-7 and HPAC exhibited quite
different characteristics (Fig. 5B). HM-7 is a solid mass of tumor
cells interspersed with large thin-walled vessels, which appeared
to be lined only with endothelial cells; treat- ment with A4.6.1
resulted in collapse of these large vessels and appear- ances of
small vessels next to clusters of host-derived stromal matrix and
various host cell types. Treatment with G6-31 resulted in a largely
necrotic mass thinly margined by the remaining HM-7 tumor cells,
presumably surviving by accessing nutrients diffusing from the sur-
rounding tissues. Tumor sections of A673 were highly similar to
HM-7, as studied previously (11). HPAC tumors, however, appeared
densely interlaced with host-derived cells and matrix, and small
size vessels were visible. Avastin treatment did not result in
marked histological differences; G6-31 treatment, however, reduced
the tumor to a mostly necrotic mass, but the surviving tumor cells
on the margin still main- tained a high proportion of host-derived
cells, similar to the control tumors. Recruitment of host cells was
indeed much more a property of HPAC than HM-7.
VEGF Generated by Tumor or Stromal Cells and Antibody Levels in
Serum—We next determined the level of tumor versus host stroma-
derived VEGF present in the tumor mass to see whether it was
consist- ent with tumor inhibition by antibodies of different
specificities. In the tumor extract of HM-7 (control group, n 5),
tumor-derived h-VEGF was dominating (1802 35 pg/mg protein, mean
S.E.), although stroma-derived m-VEGF was detected (35 9 pg/mg). In
A673, h-VEGF was present at a moderate level (163 39 pg/mg), and
m-VEGF was minimal (15 4 pg/mg). In contrast, HPAC tumors con-
tained nearly equivalent amounts of h-VEGF (116 30 pg/mg) and
m-VEGF (147 60 pg/mg), consistent with the finding that blocking
h-VEGF alone was very efficient at inhibiting the growth of HM-7
and A673 but not sufficient for HPAC inhibition (see supplemental
Table for summary). Circulating concentrations of injected
antibodies in the tumor-bear-
ing mice were measured to correlate the observed effects to serum
con- centrations. Serum samples on day 7 after the 3rd week of
treatment of the HPAC group were assayed with ELISA for uncomplexed
antibody (VEGF-coated wells for capture and anti-Fc-HRP conjugate
for detec- tion) or total antibody (anti-Fc-coated wells for
capture and anti-Fc-
HRP for detection) (Table 2), which were representative of the
serum IgG levels of animals with the other tumors. The levels of
Y0317, G6-31, and B20-4.1 were lower than Avastin (p 0.05). The
reason for this is unclear, but it is possible that the
cross-reactive antibodies, including Y0317, either cleared faster
or were bound by host VEGF in tissues because the free and total
IgG levels in circulation were statistically indistinguishable.
Studies are ongoing to understand the pharmacoki- netics and tissue
distribution of these antibodies in animals. For the current goal
of examining the efficacy of cross-blocking antibodies, the
equivalent circulating concentration of B20-4.1 and G6-31
antibodies supports the interpretation of the role of affinity in
potency and efficacy using these two antibodies, and the lower
circulating levels ofG6-31 and B20-4.1 when compared with Avastin
and Y0317 further emphasize the result that cross-reactive
antibodies had greater efficacy than Avastin and Y0317 in two of
three tumor models.
DISCUSSION
Both genetic and pharmacological approaches have been employed to
inhibit VEGF and to demonstrate the key role of VEGF in tumor
angiogenesis (36, 37). As discussed in Introduction, due to target
or species-specificity, previously developed pharmacological
inhibitors do not adequately allow assessment of the contribution
of VEGF-A to tumor angiogenesis (7, 11, 19). We have developed two
phage library- derived antibodies, G6-31 and B20-4.1, which block
both human and mouse VEGF-A. These VEGF blockers achieved complete
inhibition of the growth of implanted human colorectal cancer
(HM-7), rhadomyo- sarcoma (A673), and pancreatic cancer (HPAC)
cells inmice. The com- pleteness of the tumor inhibition was
remarkable as demonstrated by the extent of tumor reduction and
necrosis. As the antibody itself did not kill tumor cells or
inhibit endothelial cell growth stimulated with growth factors
other than VEGF (e.g. basic fibroblast growth factor) (data not
shown), the inhibitionwas a consequence of specific inhibition of
VEGF. This model established that blocking VEGF-A is sufficient to
fully inhibit tumor angiogenesis and tumor growth. This conclusion
is consistent with recent studies of an endogenously induced
pancreatic -cell tumor model where specifically knocking out VEGF
expression locally severely inhibited tumorigenesis (10). Our
observations demon- strated that blocking all sources of VEGF at an
early stage of tumorigen- esis was sufficient to inhibit tumor
progression and thus validated their application for assessing the
role of VEGF at different stages of tumor- igenesis in a variety of
tumor models in mouse. Mouse models represent powerful, often
indispensable, means to
understand the biology of many diseases. The contribution of host
stro- ma-derived VEGF in the growth of different tumors, however,
is a con- founding factor in the interpretation of the effects of
various pharma- cological blockers or genetic modulation of tumor
VEGF expression. Furthermore, it has been a controversial issue as
to the functional sig- nificance of stromal VEGF. For example,
according to a recent study, stromal VEGF does not contribute
significantly to the growth of Ras- transformed adult fibroblasts
(38). In contrast, in a study that used a VEGF-null Ras-transformed
fibrosarcoma of mouse embryonic origin, the growth of the implanted
tumor inmouse was substantially inhibited by a blocking antibody
(G6-23) against VEGF (murine VEGF) (39), indi- cating the
significant functional importance of stromal VEGF. The dif- ferent
conclusions of the two studies could be due not only to the dif-
ferent potencies and efficacies of the blocking agents used but
also may be related to differences in the levels of stromal VEGF
involvement in the two tumors. The availability of cross-species
blocking antibodies enables measurements of functional contribution
of stromal VEGF
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directly, which is clearly demonstrated to vary significantly among
human tumor types in this study. A conclusion of the present study
is that increases in affinity of VEGF
antibodies above1 nM (Kd at 37 °C)) did not result inmarked
increases in efficacy in these models. However, G6-31 inhibited
tumor growth better than B20-4.1 at low dose (0.1 mg/kg). In in
vitro systems such as VEGF-stimulated HUVEC proliferation assays,
the potency correlated well with affinity of anti-VEGF antibodies.
However, when VEGF was added to the cells at concentrations above
1–2 nM, such affinity advan- tage of antibodies was reduced (data
not shown). VEGF synthesis is finely regulated such that
inactivating even a single copy of the VEGF gene results in
embryonic lethality (40, 41). A partial blockade of VEGF may be
sufficient to block in vivo tumor angiogenesis. The effective
concentration of available VEGF in vivo and themetabolic turnover
rate of VEGF in tumor angiogenesis that could determine the impact
of affinity of blocking antibodies are not known because the
circulatory VEGF only represented a portion of VEGF activities in
vivo. VEGF tran- scriptional activation in tumors has been shown to
last an extended period of time (13), and the release of VEGF from
a matrix-bound state to activate angiogenesis is highly regulated
(42). It is likely that a low level of VEGF is generated,
continuously or in pulses, throughout the process of tumorigenesis,
which could vary significantly from tumor to tumor. As the stromal
VEGF contributes to tumor angiogenesis, the environment of tumor
growth could also influence the dynamics of VEGF recruitment to
fuel tumor angiogenesis. The impact of the affin- ity of
VEGF-blocking antibodies thus could vary significantly among
different tumors and tumor models. The particularly high VEGF
levels in HM-7 extracts might have reduced the impact of the
affinity of the blocking antibody. It would be important to
continue to assess the impact of binding affinity in relation to
the potency and efficacy in different tumor models and stages, e.g.
orthotopically implanted tumor, metastasized tumor, and
endogenously induced tumor models. The cross-species blocking
anti-VEGF antibodies are suitable to test these hypotheses. Ever
since the initial proposal that blocking angiogenesis may
affect
tumorigenesis (43), decades of research have shown its validity
and, at the same time, recognized the complexity of tumor
angiogenesis (44). Now that specific VEGF blockade has been shown
to significantly sup- press tumor progression in patients (2), it
is important to be able to establish preclinical models that more
closely reflect the complexity of tumor progression. The
cross-reactive antibodies described in the pres- ent study provide
tools to further understand this approach of therapy. For example,
tumor types and progression stages that indeed escape VEGF blockade
may now be identified and analyzed so that factors and mechanisms
that contribute to tumor escape can be studied; combina- tion
treatment can be explored. Furthermore, the specific role of VEGF
in other pathological and physiological processes can be dissected
using these antibodies in mouse models.
Acknowledgments—We thank Kenneth Hillan, Lisa Damico, Henry Lowman,
and Abraham de Vos for discussions. We thank Michael J. Elliot,
Phil Hass, Barbara Moffat, and Richard Vandlen for purifying the
antibodies and m-VEGF. We also thank Christian Wiesmann and David
Wood for graphics.
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Nonredundant Role of VEGF-A in Xenograft Growth
JANUARY 13, 2006 • VOLUME 281 • NUMBER 2 JOURNAL OF BIOLOGICAL
CHEMISTRY 961
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Contribution of Stromal VEGF Completely Inhibit the Growth of Human
Tumor Xenografts and Measure the
Cross-species Vascular Endothelial Growth Factor (VEGF)-blocking
Antibodies
doi: 10.1074/jbc.M508199200 originally published online November 7,
2005 2006, 281:951-961.J. Biol. Chem.
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