OPEN Citation: Signal Transduction and Targeted Therapy (2016) 1, 15001; doi:10.1038/sigtrans.2015.

1
www.nature.com/sigtrans

ARTICLE
Human IgG1 antibodies suppress angiogenesis in
a target-independent manner
Sasha Bogdanovich1,21, Younghee Kim1,21, Takeshi Mizutani1,2,21, Reo Yasuma1,3,21, Laura Tudisco4,21, Valeria Cicatiello4,5,21,
Ana Bastos-Carvalho1, Nagaraj Kerur1, Yoshio Hirano1, Judit Z Baffi1, Valeria Tarallo1,4, Shengjian Li1, Tetsuhiro Yasuma1,
Parthasarathy Arpitha1, Benjamin J Fowler1, Charles B Wright1, Ivana Apicella4, Adelaide Greco6,7, Arturo Brunetti6,7, Menotti Ruvo8,
Annamaria Sandomenico8, Miho Nozaki2, Ryo Ijima3, Hiroki Kaneko3, Yuichiro Ogura2, Hiroko Terasaki3, Balamurali K Ambati9,10,
Jeanette HW Leusen11, Wallace Y Langdon12, Michael R Clark13, Kathryn L Armour13, Pierre Bruhns14,15, J Sjef Verbeek16,
Bradley D Gelfand1,17,18, Sandro De Falco4,19,21 and Jayakrishna Ambati1,20,21

Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world’s population. The most widely used anti-
angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does
not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse
models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration.
Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab,
omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited
angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc
cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab’s
Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased
developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a
potentially concerning off-target effect of hIgG1 therapies.

Signal Transduction and Targeted Therapy (2016) 1, 15001; doi:10.1038/sigtrans.2015.1; published online 28 January 2016

INTRODUCTION murine models of neovascularization.8–14 Yet nearly all these

Dozens of monoclonal antibodies are approved by the United reports have compared bevacizumab with saline or no treatment
States Food and Drug Administration, European Medicines controls rather than to a biologically appropriate human IgG1
Agency, and other regulatory agencies for treating numerous control. We suspected, therefore, that the angioinhibitory effect of
diseases including age-related macular degeneration (AMD), bevacizumab in murine models was misattributed to blockade of
asthma, autoimmune disorders and multiple cancers. These drugs Vegfa, and was instead due to an intrinsic property of the IgG1
are used in millions of people worldwide with global sales molecule independent of its antigenic specificity, namely a target-
independent effect.
exceeding $50 billion.1 In addition, there are hundreds of ongoing
In this study, we found that bevacizumab, and numerous other
clinical trials evaluating various other monoclonal antibodies.1 therapeutic human IgG1 antibodies, as well as mouse IgG2a,
Bevacizumab (Avastin), a humanized monoclonal IgG1 that suppressed angiogenesis in mice via FcγRI, the high-affinity IgG
targets VEGFA,2 inhibits blood vessel growth and has been
receptor.15–17 These effects were observed both with local and
approved for treating multiple cancers,3 and is widely used to systemic administration of these antibody preparations at doses
treat neovascular AMD.4 Bevacizumab is exquisitely specific for similar to or identical to those used in humans for various
human VEGFA, having no measurable binding affinity for, or ability diseases.
to functionally inhibit, murine Vegfa.5–7 Surprisingly, numerous A prospective randomized clinical trial reported in patients with
reports claim an anti-angiogenic effect of bevacizumab in various corneal angiogenesis that bevacizumab, a full-length antibody

1
Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, KY, USA; 2Department of Ophthalmology and Visual Science, Nagoya City University
Graduate School of Medical Sciences, Nagoya, Japan; 3Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 4Angiogenesis Lab,
Institute of Genetics and Biophysics—CNR, Naples, Italy; 5Bio-Ker, MultiMedica Group, Naples, Italy; 6Department of Advanced Biomedical Sciences, University of Naples ‘Federico
II’, Naples, Italy; 7CEINGE—Biotecnologie Avanzate, s.c.a.r.l., Naples, Italy; 8Istituto di Biostrutture e Bioimmagini, CNR, Naples, Italy; 9Department of Ophthalmology and Visual
Sciences, Moran Eye Center, University of Utah School of Medicine, Salt Lake City, UT, USA; 10Department of Ophthalmology, Veterans Affairs Salt Lake City Healthcare System,
Salt Lake City, UT, USA; 11Immunotherapy Laboratory, Laboratory for Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; 12School of
Pathology and Laboratory Medicine, University of Western Australia, Crawley, WA, Australia; 13Division of Immunology, Department of Pathology, University of Cambridge,
Cambridge, UK; 14Department of Immunology, Unit of Antibodies in Therapy and Pathology, Institut Pasteur, Paris, France; 15Institut National de la Santé et de la Recherche
Médicale (INSERM) U1222, Paris, France; 16Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands; 17Department of Biomedical Engineering,
University of Kentucky, Lexington, KY, USA; 18Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, USA; 19IRCCS
MultiMedica, Milano, Italy and 20Department of Physiology, University of Kentucky, Lexington, KY, USA.
Correspondence: S De Falco (sandro.defalco@igb.cnr.it) or J Ambati (jamba2@email.uky.edu)
21
These authors contributed equally to this work.
Received 28 August 2015; revised 25 November 2015; accepted 25 November 2015
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that neutralizes human VEGFA activity and is able to bind FcγRs, is clinical observation. In contrast, clinical trials in patients with
superior to ranibizumab, a humanized IgG1 Fab fragment that choroidal angiogenesis found no significant difference in the
blocks human VEGFA but cannot bind FcγRs, in inhibiting effects of bevacizumab versus ranibizumab, each tested at a single
angiogenesis.18 Our findings provide a molecular basis for this dose, on angiogenic lesion size.4,19 Our findings suggest that the

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dose of bevacizumab required to achieve FcγRI-mediated anti- Hind limb ischemia angiogenesis
angiogenic activity is roughly eight times higher than the dose Unilateral proximal femoral artery ligation was performed as previously
used in these trials, which is sufficient only to neutralize human described,27 and on day 7 after surgery, both anterior and posterior
VEGFA, thereby providing a molecular rationale for testing such muscles from ischemic and non-ischemic hind limbs were collected and
higher doses. processed for immunohistochemical analysis for vessel quantification.
Angiogenic diseases collectively affect half-a-billion people;20 Color laser Doppler analysis was also performed using a dedicated Laser
Doppler Perfusion Imaging System (LDPI, PeriScan PIM II System,
together, our data provide evidence that human IgG1 antibodies,
Perimed AB, Järfälla, Sweden).
as a class, form an important group of angioinhibitors, potentially
fill the need for developing inexpensive generic human IgG1
drugs,21 and raise awareness for monitoring possible unintended Statistical analyses
effects on blood vessels by these widely used therapeutics. We Choroidal angiogenesis volumes per laser lesion were compared by
hierarchical logistic regression using repeated measures analysis as
also found increased pathological and developmental angiogenic previously described.28 For other comparisons, we used the Mann–
responses in mice lacking FcγRI, suggesting that endogenous Igs Whitney U-test with Bonferroni correction for statistical comparison of
also have a role in vascular patterning. multiple variables. Results are expressed as mean ± s.e.m. Type-I error not
exceeding 0.05 was deemed significant.

MATERIALS AND METHODS

Animals RESULTS
All animal experiments were in accordance with the guidelines of the Bevacizumab and human IgG1 antibodies inhibit angiogenesis
relevant institutional authorities. Male mice, aged 4–8 weeks, were in mice
randomized 1:1 to treatment with active drug versus inactive drug or Bevacizumab has no detectable binding to mouse Vegfa
control treatments. by surface plasmon resonance and does not block mouse
Vegfa-induced retinal capillary endothelial cell proliferation.5–7
Corneal angiogenesis To further verify that bevacizumab does not functionally neutralize
Nylon sutures (Mani, Utsunomiya, Japan) were placed into the corneal mouse Vegfa, we tested its ability to inhibit the activation of the
stroma of mice, and on day 10 after injury, we calculated the mean Vegfr2 receptor tyrosine kinase in mouse Py4 hemangioma
percentage CD31+Lyve1− blood vessel areas for corneal flat mounts with endothelial cells. As expected, bevacizumab inhibited Vegfr2
ImageJ (US National Institutes of Health, Bethesda, MD, USA) as previously phosphorylation induced by human VEGFA but not by mouse
reported.22,23 Vegfa (Figure 1a).
We tested the effects of bevacizumab in a mouse model of
Choroidal angiogenesis suture injury-induced corneal angiogenesis, which is pathophy-
Laser photocoagulation (OcuLight GL, IRIDEX, Mountain View, CA, USA) siologically relevant to the human condition and is driven in large
was performed on both eyes of mice to induce neovascularization, and on part by Vegfa. We found that administration of bevacizumab into
day 7 after injury, choroidal angiogenesis volumes were measured by the cornea by intrastromal injection (4 μl of the commercial
scanning laser confocal microscopy (TCS SP5, Leica, Wetzlar, Germany) as 25 mg/ml preparation—a dose similar to that used in humans,
previously reported with 0.7% FITC-conjugated Isolectin B4 (Vector when corrected for relative size) inhibited corneal angiogenesis by
Laboratories, Burlingame, CA, USA).24,25 For intravitreous administration 46% compared with phosphate-buffered saline (PBS) administra-
in choroidal angiogenesis experiments, drugs was administered into tion in wild-type mice (Figure 1b). This angioinhibition occurred in
the vitreous humor of mice using a 33-gauge double-calibre needle a dose-dependent fashion (Supplementary Figure 1). We also
(Ito Corporation, Tokyo, Japan) as previously described.26 tested whether ranibizumab, a humanized monoclonal IgG1 Fab

Figure 1. Bevacizumab inhibited mouse angiogenesis via Fc region. (a) Western blot shows that bevacizumab inhibited Vegfr2
phosphorylation (pVegfr2) in Py4 mouse hemangioma endothelial cells when treated with human VEGFA, but not when treated with
mouse Vegfa, after 10 min. Image representative of three experiments. (b) Bevacizumab and human IgG1, but not ranibizumab, decreased
corneal angiogenesis in wild-type mice. Area of angiogenesis was measured 10 days after suture injury and normalized to PBS group. n = 10–38.
Representative photos of wild-type mouse eyes (upper row) and corneal flat mounts (lower row) showing reduced growth of blood vessels
(CD31+, red) in eyes treated with bevacizumab or human IgG1, but not in eyes treated with ranibizumab. Scale bars, 100 μm. (c) Bevacizumab
and human IgG1, but not ranibizumab, suppressed choroidal angiogenesis in wild-type mice 7 days after laser injury compared with PBS
(experiment performed in JA laboratory). Images depict representative choroidal angiogenesis lesions (endothelial cells stained in green) in
each treatment group. n = 12–20. (d, e) Treatment of ischemic hind limb with bevacizumab or human IgG1 in wild-type mice suppressed
muscle revascularization and decreased blood vessel perfusion, as seen in representative laser Doppler perfusion images (top), and
measured blood flow in the ischemic limbs (bottom), normalized to the contralateral non-ischemic limbs, 7 days after surgery. n = 6. I/NI,
ischemic/non-ischemic. Bevacizumab and human IgG1, but not ranibizumab, treatment of ischemic limbs reduced muscle angiogenesis
(CD31+, brown) as seen in representative images of muscle CD31 immunolocalization (e), and quantification of muscle CD31
immunolocalization (bottom), normalized to the contralateral non-ischemic limbs. (f) The Fc fragments, not the Fab fragment, of
bevacizumab suppressed corneal angiogenesis in wild-type mice. Area of angiogenesis was measured 10 days after suture injury and
normalized to PBS group. n = 10–38. (g) Co-administration of a peptide that prevents IgG-Fc binding to FcγR, but not a control peptide,
blocked inhibition of choroidal angiogenesis by bevacizumab in wild-type mice. (h) Co-administration of an IgG-Fc inhibitory peptide, but not
a control peptide, blocked inhibition of muscle angiogenesis (CD31+, brown) by bevacizumab, as seen in representative images of muscle
CD31 immunolocalization (left), and quantification of muscle CD31 immunolocalization (right), normalized to the contralateral non-ischemic
limbs. Scale bar, 100 μm. n = 6. (i) Bevacizumab suppressed choroidal angiogenesis in wild-type mice to the same extent as SU1498, a small
molecule tyrosine kinase inhibitor of Vegfr2. Combined administration of bevacizumab and SU1498 suppressed choroidal angiogenesis to a
greater extent than either of the agents alone. n = 6. (j) Choroidal angiogenesis, augmented by administration of human VEGFA, was
suppressed to similar extents by ranibizumab, bevacizumab-Fab, bevacizumab-Fc and human IgG1; and, to a greater extent, by bevacizumab.
n = 6–8. (k) Bevacizumab suppressed choroidal angiogenesis to a greater extent than ranibizumab in the humanized VEGFA mouse, a
transgenic model that expresses a VEGFA protein that can be neutralized by both bevacizumab and ranibizumab. n = 6. Results are means ± s.e.m.
*P o0.05 compared with PBS (b–h, k) or with vehicle (i) or with PBS+human VEGFA (j).

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fragment that binds human VEGFA but not mouse Vegfa6,29 and is IL2-secretory sequence (phIgG1-Fc) by injecting it into the
approved for the treatment of neovascular AMD, was anti- subretinal space of wild-type mice before laser injury. We found
angiogenic in this model. We confirmed that, like bevacizumab, that phIgG1-Fc reduced choroidal angiogenesis in wild-type mice
ranibizumab inhibited Vegfr2 phosphorylation induced by human compared with a control null plasmid (Supplementary Figure 6).
VEGFA but not by mouse Vegfa (Supplementary Figure 2). These data show that angiogenesis can be suppressed not only by
However, unlike bevacizumab, ranibizumab, at equimolar exogenous administration of human IgG1 antibodies but also by
amounts, did not inhibit corneal angiogenesis (Figure 1b). endogenous overexpression of their Fc region.
Interestingly, the control isotype human IgG1 for bevacizumab
also reduced corneal angiogenesis in wild-type mice in a dose- Bevacizumab and human IgG1 antibodies inhibit angiogenesis in
dependent fashion (Figure 1b and Supplementary Figure 3). humanized VEGF mice
We next tested a mouse model of laser injury-induced choroidal Ranibizumab, which targets human VEGFA and does not have an
angiogenesis, a widely used model of neovascular AMD that is Fc region, is effective in treating neovascular AMD in humans.31,32
driven in large part by Vegfa and has been predictive of the We sought to compare the relative anti-angiogenic efficacies of
success of anti-VEGFA therapies in humans. Independent testing bona fide VEGFA targeting and of FcγR-mediated signaling. First
by three different laboratories (JA, YO and HT) determined that we found that the extent of suppression of choroidal angiogenesis
intraocular administration of bevacizumab (1 μl of the commercial by bevacizumab (48%) in wild-type mice was similar to that
25 mg/ml preparation—a dose approximately eight times that achieved by SU1498 (49%), a small molecule tyrosine kinase
was used in humans, when corrected for relative size) by inhibitor of Vegfr2 (Figure 1i), and to that achieved by various
intravitreous injection inhibited choroidal angiogenesis by inhibitors of Vegfa reported in previous studies (~40–55%).28,33–36
40–45% in wild-type mice compared with PBS administration, We also found that bevacizumab potentiated the angioinhibitory
whereas an equimolar amount of ranibizumab did not do so effects of SU1498 (Figure 1i).
(Figure 1c and Supplementary Figure 4). Similar to the corneal Next we used a model in which laser injury-induced choroidal
model, the isotype human IgG1 and human IgG1-Fc also angiogenesis is augmented by prior intravitreous administration
suppressed choroidal angiogenesis in wild-type mice (Figure 1c of human VEGFA.37 In this model where angiogenesis is driven
and Supplementary Figure 4). both by human VEGFA and endogenous mouse pathways, we
We tested the effect of bevacizumab in a third model of found that ranibizumab and bevacizumab-Fab, which target
angiogenesis, induced by hind limb ischemia produced by femoral human VEGFA but do not contain an Fc region, suppressed
artery ligation, which is a model of peripheral arterial disease. angiogenesis to a similar extent as bevacizumab-Fc and human
Intramuscular injection of either bevacizumab or isotype control IgG1, which contain an Fc region but do not target human VEGFA
human IgG1 suppressed limb revascularization and diminished (Figure 1j). In addition, full-length bevacizumab suppressed
perfusion, as monitored by color laser Doppler imaging, compared angiogenesis to a greater extent than any of the pure anti-
with PBS injection (Figure 1d). There was a corresponding VEGFA or Fc-containing agents alone, further indicating that
reduction in angiogenesis by 47–59% in the bevacizumab- or modulating these two anti-angiogenic pathways simultaneously
human IgG1-treated limbs compared with the PBS-treated limbs, can be additive. Next, we studied ‘humanized VEGFA’ mice,6
whereas ranibizumab did not suppress angiogenesis (Figure 1e). wherein the mouse Vegfa gene was mutated such that its protein
These data support the concept that human IgG1 antibodies can product could be neutralized by bevacizumab and ranibizumab. In
suppress angiogenesis in multiple tissues. this model, we found that bevacizumab suppressed choroidal
angiogenesis to a significantly greater extent than ranibizumab
Fc portion of human IgG1 critical for angioinhibition (Figure 1k). Collectively these data demonstrate that the Fc region
Since bevacizumab and ranibizumab had nonsynonymous effects of bevacizumab can potentiate its anti-angiogenic effect in
on angiogenesis in these mouse models, we suspected that the systems where human VEGFA is present.
anti-angiogenic action of bevacizumab was due not to Vegfa
neutralization but rather to IgG1-Fc-mediated effects. We con- FcγRI necessary for human IgG1-induced angioinhibition
firmed that angioinhibition was due to the Fc, and not Fab, We performed additional experiments to investigate the nature of
portion of bevacizumab by administering separately its Fab and Fc the Fc-mediated anti-angiogenic effect of bevacizumab. It is
fragments as prepared from papain enzymatic digestion of the known that deglycosylation of human IgG1 dramatically reduces
antibody (Supplementary Figure 5). Bevacizumab-Fab, but not its binding to both human and mouse FcγRs.38–40 We found that
bevacizumab-Fc, inhibited VEGFA-induced Vegfr2 phosphoryla- deglycosylated bevacizumab, despite retaining its ability to inhibit
tion, consistent with the location of the VEGFA-binding residues human VEGFA-induced Vegfr2 phosphorylation (Supplementary
on the Fab fragment and indicating that the VEGFA neutralizing Figure 7), did not reduce choroidal angiogenesis in wild-type mice
properties of bevacizumab were not affected by enzymatic (Figure 2a). These data suggest that the anti-angiogenic effect of
digestion (Supplementary Figure 5). In contrast, bevacizumab-Fc, bevacizumab in mice is mediated by an endogenous FcγR that
but not bevacizumab-Fab, reduced corneal angiogenesis in binds human IgG1.40
wild-type mice (Figure 1f), indicating that bevacizumab’s angioin- We found that bevacizumab did not suppress choroidal
hibitory activity in mice is due to its Fc domain and not because of angiogenesis in Fcer1g− / − (a.k.a. FcRγ− / −) mice (Figure 2b), which
binding Vegfa. lack the common gamma chain of the activating FcγRs: FcγRI,
Human IgG1 and human IgG1-Fc also suppressed choroidal FcγRIII and FcγRIV. To determine which activating FcγR was
angiogenesis in wild-type mice (Figure 1c and Supplementary responsible, we tested mice lacking these receptors. First, we
Figure 4). A peptide that prevents the binding of IgG to FcγRs by tested the involvement of FcγRI (encoded by Fcgr1), and found
interacting with the Fc portion of IgG (IgG-Fc peptide inhibitor),30 that bevacizumab failed to inhibit corneal or choroidal angiogen-
but not a control peptide, eliminated the ability of bevacizumab to esis in Fcgr1− / − mice (Figure 2c). In contrast, bevacizumab
inhibit choroidal and hind limb angiogenesis in wild-type mice inhibited corneal and choroidal angiogenesis in mice lacking
(Figure 1g and h), confirming a role for FcγR in these models. Fcgr3 (Supplementary Figure 8), which encodes FcγRIII, and in
We sought to determine whether human IgG1s would suppress mice lacking Fcgr4 (Supplementary Figure 9), which encodes
angiogenesis not only when exogenously administered but also if FcγRIV. Supporting the latter result, bevacizumab did not inhibit
produced endogenously. Therefore, we performed in vivo trans- angiogenesis in Fcgr1−/−; Fcgr2b−/−; Fcgr3−/−; Fcer1a−/− and
fection of a plasmid encoding human IgG1-Fc coupled to an Fcer2a−/− mice, which express FcγRIV but not any of the other

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Figure 2. Bevacizumab inhibited mouse angiogenesis via FcγRI. (a) Deglycosylated bevacizumab did not suppress choroidal angiogenesis in
wild-type mice; however, choroidal angiogenesis was inhibited by bevacizumab subjected to mock treatment. The deglycosylation buffer had
no effect on choroidal angiogenesis. n = 8–14. (b) Bevacizumab did not suppress corneal or choroidal angiogenesis in Fcer1g−/− mice. n = 8–10.
(c) Bevacizumab did not inhibit corneal or choroidal angiogenesis in Fcgr1−/− mice. No significant difference between groups. n = 10–13.
(d) Denosumab did not suppress corneal or choroidal angiogenesis in wild-type mice. n = 6–8. No significant difference between groups.
(e) Bevacizumab inhibited corneal angiogenesis in FcγR humanized mice. n = 8. Results are means ± s.e.m. *P o0.05 compared with PBS.
(f) Bevacizumab, but not denosumab, inhibited choroidal angiogenesis in FcγR humanized mice. n = 6–8. (g) Co-administration of a 17+2-nt
cholesterol conjugated human FCGR1A siRNA, but not a 17+2-nt cholesterol-conjugated control Luc siRNA, blocked inhibition of choroidal
angiogenesis by bevacizumab in FcγR humanized mice. n = 8. (h) Co-administration of an IgG-Fc inhibitory peptide, but not a control peptide,
blocked inhibition of choroidal angiogenesis by bevacizumab in FcγR humanized mice. n = 8. Results are means ± s.e.m. *P o0.05 compared
with PBS (a, e–h).

IgG or IgE receptors41 (Supplementary Figure 10). Human IgG2 We also found that subretinal transfection of a plasmid
binds to mouse FcγRII and FcγRIII, but not to FcγRI.40 The human encoding a mutant form of human IgG1-Fc engineered with
IgG2 denosumab (Prolia: anti-RANKL) did not inhibit corneal or point mutations that eliminate binding to FcγRI or of a plasmid
choroidal angiogenesis in wild-type mice (Figure 2d), suggesting encoding human IgG2-Fc did not suppress choroidal angiogenesis
that binding to FcγRI is required for IgG-induced angioinhibition. in wild-type mice (Supplementary Figure 11), further supporting

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the concept that angioinhibition is a target-independent class interaction with human FcγRI is mandatory for the anti-angiogenic
effect of human or humanized IgG1 monoclonal antibodies that is effect of IgGs in our models. The angioinhibitory effect of
mediated via FcγRI. bevacizumab was blocked by both the IgG-Fc peptide inhibitor
Administration of bevacizumab by i.v. injection every other day and a cholesterol-conjugated28 FCGR1A siRNA (Figure 2g and h).
(to account for the 6.5-fold higher serum clearance rate in mice These data demonstrate that target-independent FcγRI-mediated
compared with humans,42 in whom it is administered weekly or angioinhibitory activity of humanized monoclonal IgG1 antibodies
every other week) also suppressed choroidal angiogenesis in is operative in a FcγR humanized system.
wild-type mice in a dose-dependent fashion, but did not do so in
Fcgr1− / − mice (Supplementary Figure 12). Collectively, these data Bevacizumab interacts with FcγRI and initiates signaling in vivo
indicate that bevacizumab reduces mouse angiogenesis via FcγRI
As we found that bevacizumab suppressed angiogenesis via FcγRI,
and not via Vegfa inhibition.
we tested whether bevacizumab binds FcγRI in vivo using two
complementary strategies. First, using a pull-down assay, we
Bevacizumab reduces angiogenesis in FcγR humanized mice found that biotinylated bevacizumab, but not denosumab, that
Although human IgG1 binds both mouse FcγRI and human Fcγ was injected into wild-type mouse corneas following suture injury
RI,40 the structural diversity and unique cellular expression co-precipitated with mouse FcγRI (Figure 3a). Next, we injected
patterns of mouse and human FcγRs are not synonymous.43 The unlabeled bevacizumab into the corneas of FcγR humanized mice
generation of an FcγR humanized mouse via transgenic expres- that were subjected to suture injury, and found that immuno-
sion of the entire human FcγR family, under the control of their precipitation of human FcγRI pulled down human IgG1
human regulatory elements, on a genetic background lacking all (Figure 3b). Collectively, these data demonstrate an in vivo
mouse FcγRs has enabled better prediction of the functional interaction between bevacizumab and both human and mouse
consequences of engaging human FcγRs by IgGs.44 In these FcγR FcγRI. In addition, bevacizumab injected into the corneas
humanized mice, we found that intracorneal bevacizumab of FcγR humanized mice following suture injury-induced FcγRI
reduced corneal angiogenesis just as in wild-type mice phosphorylation (Figure 3b).
(Figure 2e). Bevacizumab also reduced choroidal angiogenesis in Crosslinking of FcγRI by human IgG1 aggregates can activate
FcγR humanized mice whereas the human IgG2 denosumab did FcγRI. However, we found, using dynamic light scattering, no
not (Figure 2f). As human IgG2 can bind human FcγRII and human evidence of aggregation of bevacizumab at the administered dose
FcγRIII but not human FcγRI,45 this result supports the notion that (Supplementary Figure 13), as might be expected from a clinical

Figure 3. Bevacizumab interacted with, induced phosphorylation of, and upregulated abundance of FcγRI in vivo. (a) Wild-type mouse corneas
that had been administered biotinylated bevacizumab or biotinylated denosumab following suture injury were subjected to streptavidin pull-
down and immunoblotting for mouse FcγRI. Biotinylated bevacizumab, but not denosumab, interacted with mouse FcγRI in vivo. Anti-
streptavidin immunoblotting confirmed efficient pull-down of both biotinylated antibodies. (b) FcγR humanized mouse corneas that had
been administered bevacizumab or PBS following suture injury were subjected to immunoprecipitation of human FcγRI followed by
immunoblotting for human IgG1 or phosphotyrosine. Bevacizumab, but not PBS, interacted with and induced phosphorylation of human
FcγRI in vivo. Reprobing confirmed efficient immunoprecipitation of human FcγRI in both bevacizumab- and PBS-treated corneas. Each image
is representative of three experiments (a, b). (c) Bevacizumab, but not PBS, increased Fcgr1 mRNA abundance in RAW264.7 mouse
macrophages and in wild-type mouse corneas following suture injury, as monitored by real-time reverse transcription PCR, and FcγRI protein
abundance in RAW264.7 cells, as monitored by western blotting. Densitometry of FcγRI normalized to Vinculin shown. n = 4–6. Results are
means ± s.e.m. *P o0.05 compared with PBS.

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grade preparation. This suggests that monomeric bevacizumab interaction between these human antibodies and the mouse
can induce FcγRI-mediated signaling in vivo in the systems homologs of their human protein targets by testing them in mice
we studied. Indeed, monomeric IgG engagement of other deficient for the homologous genes. Such interactions were not
activating FcγRs has been shown to induce phosphorylation and responsible for the angiosuppression as we found that corneal
signaling.46,47 Nevertheless, we cannot exclude the possibility that angiogenesis was inhibited by adalimumab in Tnf− / − mice,
once the non-aggregated liquid formulation is administered into alemtuzumab in CD52− / − mice, ofatumumab in CD20− / − mice
the mouse, bevacizumab might undergo in vivo aggregation. and omalizumab in IgE-deficient mice (Figure 4d), just as in wild-
However, this seems unlikely given the lack of any known mouse type mice. Collectively, these data indicate that multiple
ligand for bevacizumab. Moreover, if such in vivo aggregation of therapeutic human IgG1 antibodies can suppress angiogenesis
bevacizumab occurred in the mouse, it would also be expected to via FcγRI and independent of their intended target.
occur in human eyes because of the similar dose injected and the Next we tested some of these antibodies in FcγR humanized
presence of a bona fide ligand—human VEGFA. mice. We found that intracorneal palivizumab reduced corneal
We next wondered how bevacizumab could bind FcγRI given angiogenesis (Figure 4e). In addition, alemtuzumab, but not
the high serum concentration of endogenous mouse IgG that alemtuzumab G1Δab, which does not bind FcγRI, suppressed
might be expected to compete for binding. Indeed, it has been choroidal angiogenesis in FcγR humanized mice (Supplementary
shown that FcγR can still bind IgG under serum conditions48,49 and Figure 16). These data demonstrate that target-independent
can execute numerous biological functions in vivo in response to angioinhibitory activity of humanized monoclonal IgG1 antibodies
exogenous mouse IgG2a and human IgG1 antibodies.50–58 This is operative in a FcγR humanized system.
ability of FcγRI to contribute to biological signaling has been
attributed to the short half-life of the interaction of FcγRI with its Mouse IgG2a and mouse IgG2c inhibit angiogenesis via FcγRI
ligand (turnover within minutes), de novo synthesis of free FcγRI, To determine whether antibodies potentially produced by
receptor reorganization or conformational changes on the mice against human IgGs might have a role in the angioinhibition
membrane, sampling IgG as a scavenger receptor, and ‘inside- we observed, we tested Rag2− / − mice, which lack B and T cells and
out’ stimulation by cytokines that rapidly increases the binding of are devoid of Igs. Bevacizumab inhibited corneal and choroidal
FcγRI to exogenous monomeric IgG.48,49,51,56,59,60 Indeed we found angiogenesis in Rag2− / − mice (Supplementary Figure 17), indicat-
that bevacizumab increased FcγRI levels in mouse macrophages ing that such an immune response potentially mounted against
and in wild-type mouse corneas following suture injury (Figure 3c). bevacizumab is not responsible for its angioinhibitory effect.
More importantly, we found that the concentrations of To exclude other potential cross-species biological effects, we
endogenous mouse IgG2c (an allelic variant of mouse IgG2a that tested mouse IgG2a, which like human IgG1 binds to FcγRI with
is expressed in C57BL/6 mice61,62) in the extravascular portion of high affinity.17,41,57,68,69 Intracorneal or subretinal transfection of a
the injured tissues are minute compared with circulating plasmid encoding mouse IgG2a-Fc coupled to an IL2-secretory
levels and far lower than the extravascular tissue concentrations sequence inhibited corneal or choroidal angiogenesis, respec-
of exogenously administered bevacizumab (Supplementary tively, in wild-type mice (Supplementary Figure 18). In contrast, a
Figure 14a and b). This paucity of extravascular mouse IgG plasmid encoding a mutant form of mouse IgG2a-Fc engineered
(Supplementary Figure 14c) and the excess of bevacizumab in the with point mutations that eliminate binding to FcγRI and coupled
injured tissues combined with increased FcγRI abundance can to the same IL2-secretory sequence, did not suppress angiogenesis
explain the ability of the exogenous human IgG1 to bind FcγRI (Supplementary Figure 18). Recombinant mouse IgG2a-Fc inhibited
in vivo on extravascular cells, e.g., macrophages, which express choroidal angiogenesis in wild-type mice in a dose-dependent
FcγRI63 and can modulate angiogenesis,64 and initiate signaling. fashion, whereas mouse IgG2b-Fc, which has high binding affinity for
FcγRIV but not for FcγRI,17,41,43,68,69 did not suppress angiogenesis
Numerous therapeutic human IgG1s inhibit angiogenesis via FcγRI (Supplementary Figure 19). In addition, neither recombinant mouse
Next we assessed the anti-angiogenic effects of several human or IgG2a-Fc nor a plasmid encoding mouse IgG2a-Fc reduced
humanized IgG1 monoclonal antibodies that are approved for angiogenesis in Fcgr1− / − mice (Supplementary Figure 20). Further,
treatment of various human diseases, and either do not bind the mouse IgG2c also suppressed choroidal angiogenesis in wild-type
mouse homologs of their intended human protein targets or have mice but not in Fcgr1− / − mice (Supplementary Figure 21).
no mammalian target: adalimumab (Humira: anti-TNFα), alemtu- Together these data further support the concept that suppression
zumab (Campath: anti-CD52), ofatumumab (Arzerra: anti-CD20), of angiogenesis via FcγRI is not limited to human IgG1 but also is a
omalizumab (Xolair: anti-IgE), palivizumab (Synagis: anti- property of mouse IgG2a and mouse IgG2c.
respiratory syncytial virus protein F), and tocilizumab (Actemra:
anti-IL-6R). All of these human IgG1 antibodies reduced Host Igs modulate angiogenesis
both corneal and choroidal angiogenesis in wild-type mice As we found that recombinant and endogenously over-expressed
(Figure 4a and b) in contrast to the human IgG2 denosumab mouse IgG2a and mouse IgG2c suppressed injury-induced
(Figure 2e). We tested two of these antibodies—omalizumab and angiogenesis, we explored whether native host Igs modulate
palivizumab—in Fcgr1− / − mice, and found that they did not vascularization. Indeed, we found that corneal and choroidal
suppress corneal or choroidal angiogenesis (Figure 4c). We also angiogenesis responses to suture or laser injury (without adminis-
found that a mutant version of alemtuzumab (G1Δab), which was tration of bevacizumab), respectively, were higher in Fcgr1− / − and
engineered with point mutations in the CH2 domain of its Fc Rag2− / − mice compared with littermate wild-type controls
region that eliminate binding to FcγRI and reduce binding to other (Figure 5a and b). Physiological vascularization of the retina during
FcγRs,65 yet retains binding to human CD52 (Supplementary development proceeds from the central optic nerve to the
Figure 15a and b), did not inhibit corneal or choroidal angiogen- periphery. This process is not complete in mice until several days
esis in wild-type mice (Supplementary Figure 15d and e). after birth. We found that at postnatal day 4, both the area of
Conversely, we found that another mutant version of alemtuzu- vascularized retina and density of retinal vessels were greater in
mab (D270A), which preserves binding to FcγRI but not to FcγRII Fcgr1− / − and Rag2− / − mice compared with littermate wild-type
and FcγRIII,66,67 suppressed choroidal angiogenesis in wild-type controls (Figure 5c). Taken together, these data suggest an anti-
mice but not in Fcgr1− / − mice (Supplementary Figure 15f). angiogenic role for endogenous Igs in vascular patterning both
We sought to exclude the possibility that the observed during development and response to injury that is mediated
angioinhibition could be due to unforeseen or illegitimate via FcγRI.

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Figure 4. Human IgG1s inhibited mouse angiogenesis via FcγRI. Treatment with the human IgG1 antibodies adalimumab, alemtuzumab,
ofatumumab, omalizumab, palivizumab or tocilizumab reduced (a) corneal and (b) choroidal angiogenesis in wild-type mice. n = 8–19.
(c) Palivizumab and Omalizumab did not inhibit corneal or choroidal angiogenesis in Fcgr1−/− mice. n = 6–8. No significant difference between
groups. (d) Adalimumab, a human anti-TNFα monoclonal antibody, inhibited corneal angiogenesis in Tnf−/− mice. n = 9. Alemtuzumab, a
humanized anti-CD52 monoclonal antibody, inhibited corneal angiogenesis in CD52−/− mice. n = 8. Ofatumumab, a human anti-CD20
monoclonal antibody, inhibited corneal angiogenesis in CD20−/− mice. n = 8. Omalizumab, a humanized anti-IgE monoclonal antibody,
inhibited corneal angiogenesis in IgE-deficient mice. n = 10. (e) Palivizumab inhibited choroidal angiogenesis in FcγR humanized mice. Results
are means ± s.e.m. *P o0.05 compared with PBS (a, b, d, e).

Human IgG1 reduces angiogenesis via bone marrow-derived cells These results suggest that FcγRI in bone marrow-derived cells is
expressing FcγRI critical for bevacizumab-induced angioinhibition.
To determine whether bone marrow-derived or resident cell Among the various types of bone marrow-derived cells,
expression of FcγRI was the critical effector in IgG1 mAb-mediated macrophages are best known to have a critical role in
angioinhibition, we created bone marrow chimeric mice. angiogenesis.70 Both bevacizumab and human IgG1 inhibited
Bevacizumab suppressed corneal and choroidal angiogenesis in mouse Vegfa-induced migration of wild-type mouse bone
Fcgr1− / − mice receiving wild-type bone marrow but did not do so marrow-derived macrophages (BMDMs) but not of Fcgr1− / −
in wild-type mice receiving Fcgr1− / − bone marrow (Figure 6a and b). BMDMs (Figure 6c). Corroborating these data, we found that

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Figure 5. Endogenous Igs suppressed mouse angiogenesis. Corneal angiogenesis area (a) and choroidal angiogenesis volume (b) are greater
in Fcgr1−/− and Rag2−/− mice compared with wild-type mice. n = 8–20. (c) The vascular density and total area of vascularized retina at postnatal
day 4 is greater in Fcgr1−/− and Rag2−/− mice compared with wild-type mice. n = 8. Results are means ± s.e.m. *P o0.05 compared to wild-type
mice (a–c). Vascular density in the retina is normalized to wild-type mice. Representative flat mounts of corneal (a, red), choroidal
(b, green) and retinal (c, red), vessels are shown.

bevacizumab reduced the infiltration of F4/80+ macrophages into bevacizumab-Fab, following suture injury (Figure 6f). Neither
the sutured cornea, laser-injured choroid, and ischemic hind limb bevacizumab nor human IgG1 inhibited mouse Vegfa-induced
of wild-type mice (Supplementary Figure 22). These findings migration of c-Cbl− / − BMDMs (Figure 6c). Further, we found that
are in concert with the abundant expression of FcγRI by bevacizumab did not inhibit corneal or choroidal angiogenesis in
macrophages.63,71 c-Cbl− / − mice (Figure 6g), indicating that c-Cbl activation is
We next assessed whether bevacizumab induces intracellular essential for this process.
FcγR-mediated signaling events. First we tested the FcRγ-chain One of the principal signaling pathways employed by mouse
signaling-deficient NOTAM mice, which exhibit normal cell surface Vegfa to induce macrophage migration is activation of Vegfr1
Fc receptor expression and normal IgG binding, but have non- receptor tyrosine kinase and downstream activation of PI3K and
signaling Fc receptors because their associated γ-chains have PLCγ1.75–78 Via its E3 ubiquitin ligase activity, c-Cbl is capable of
been mutated in their immunoreceptor tyrosine-based activation inducing degradation of numerous tyrosine kinases including
motif, which is responsible for signal transduction.72 We found Vegfr1.79 Indeed, we found that bevacizumab treatment of mouse
that bevacizumab did not suppress choroidal angiogenesis in FcR macrophages induced degradation of Vegfr1 in wild-type but not
NOTAM mice (Figure 6d), suggesting that this angioinhibition is FcR NOTAM BMDMs (Figure 6h). Bevacizumab also reduced mouse
dependent on FcγR-mediated signaling. Bevacizumab induced Vegfa-induced phosphorylation of PI3K and PLCγ1 in mouse
phosphorylation of FcγRI in the mouse cornea (Figure 3b); macrophages (Figure 6i). Consistent with these findings, neither
therefore, we examined the potential involvement of c-Cbl, a bevacizumab nor human IgG1 inhibited mouse Vegfa-induced
major regulator of tyrosine kinase signaling that is downstream of migration of BMDMs isolated from c-Cbl (C379A) mutant mice
FcγRI-initiated signaling.73,74 We found that bevacizumab induced (Figure 6c), which lack a functional RING finger domain necessary
phosphorylation of c-Cbl in wild-type but not FcR NOTAM mouse for the E3 ubiquitin ligase activity of c-Cbl.80 Also consistent
BMDMs (Figure 6e). This was also corroborated in vivo: increased with these findings, and the lack of angioinhibition observed in
phosphorylation of c-Cbl was observed in the corneas of wild-type c-Cbl− / − mice, was the finding that bevacizumab did not
mice treated with bevacizumab and bevacizumab-Fc, but not reduce corneal angiogenesis in c-Cbl (C379A) mutant mice

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Figure 6. Bevacizumab inhibited angiogenesis via macrophage FcγRI and c-Cbl. Bevacizumab suppressed corneal (a) and choroidal
(b) angiogenesis in Fcgr1−/− mice transplanted with wild-type mouse bone marrow, but not in wild-type mice receiving Fcgr1−/− bone marrow.
n = 11–16. (c) Bevacizumab and human IgG1 inhibited mouse Vegfa-induced migration, over 12 h, of bone marrow-derived macrophages
isolated from wild-type mice but not from Fcgr1−/−, c-Cbl−/− or c-Cbl (C379A) mice, which lack E3 ubiquitin ligase activity. n = 3. Results are
means±s.e.m. *P o0.05 compared with PBS (a–c). (d) Bevacizumab did not inhibit choroidal angiogenesis in NOTAM mice. n = 10. (e) Western
blot shows induction of c-Cbl phosphorylation in wild-type mouse BMDMs treated with bevacizumab for 15 min. Protein loading was assessed
by α-Tubulin abundance. (f) Western blot shows in vivo induction of c-Cbl phosphorylation in wild-type mouse corneas following suture injury
that were treated with bevacizumab or its Fc fragment, but not by its Fab fragment. (g) Bevacizumab did not inhibit corneal or choroidal
angiogenesis in c-Cbl−/− mice. n = 11–30. NS, no significant difference between groups. (h) Western blots show time-dependent Vegfr1
degradation in wild-type but not NOTAM mouse BMDMs treated with bevacizumab. Protein loading was assessed by HSP70 abundance.
(i) Western blots show that RAW264.7 mouse macrophages pre-treated with bevacizumab, but not PBS, 2 h before stimulation with mouse
Vegfa, exhibited reduced phosphorylation of PI3K and PLCγ1 at 10 min after Vegfa exposure. Protein loading was assessed by β-actin
abundance. (j) Western blots show induction of c-Cbl phosphorylation in human peripheral blood mononuclear cells (PBMC) or in THP-1
human monocytic cells, treated with bevacizumab, but not PBS, for 15 min. Treatment with bevacizumab, but not PBS, reduced VEGFR1
abundance in human PBMCs and THP-1 cells. Protein loading was assessed by Vinculin or α-Tubulin abundance. Images representative of
three experiments (e, f, h–j).

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(Supplementary Figure 23). We also found that bevacizumab demonstrate in a companion manuscript.88 Additional anti-
induced phosphorylation of c-CBL and degradation of VEGFR1 in angiogenic efforts might be directed toward developing peptides
primary human peripheral blood monocytes as well as in THP-1 or small molecules that induce signaling via FcγRI or c-Cbl.
human monocytes (Figure 6j). The dose of bevacizumab (100 μg) we injected into the mouse
cornea, whose volume is ~ 2 μl, is similar in concentration to the
Human IgG1 does not inhibit angiogenesis via ADCC, ADCP dose of bevacizumab (2.5–5 mg) that has been administered into
or CDC human corneas, whose volume is ~ 70 μl. Our findings suggest
that in human corneas, bevacizumab would, at this dose, exert
Antibody-dependent cell-mediated cytotoxicity (ADCC) and
anti-angiogenic activity both via VEGFA inhibition and via FcγRI-
antibody-dependent cellular phagocytosis (ADCP) are two-step
mediated pathways, and that it might be expected to suppress
processes initiated by full-length IgGs that couple Fab binding
angiogenesis to a greater extent than ranibizumab, which
to a target cell antigen with Fc binding to an activating FcγR on possesses only the anti-VEGFA activity. Indeed, a recent prospec-
an effector cell.71 These effector functions, as well as tive randomized study reported that in humans with corneal
complement-dependent cytotoxicity (CDC) have a major role in angiogenesis, bevacizumab was superior to ranibizumab.18
the mode of action of several monoclonal antibodies employed In contrast, no significant difference was found between
in cancer therapy.81–83 Our findings that numerous human bevacizumab and ranibizumab in human eyes with choroidal
IgG1 antibodies, each with different Fab targeting domains angiogenesis due to neovascular AMD.4,19 We suggest that the
(and none of which target mouse antigens), similarly suppressed reason for this lack of difference is that the amount of
angiogenesis argue against ADCC and ADCP as the mediators of bevacizumab that is currently administered in these patients,
this class effect. Moreover, bevacizumab-Fc and human IgG1-Fc, while sufficient to neutralize VEGFA, is insufficient to induce FcγRI-
each devoid of Fab domains, also suppressed angiogenesis mediated signaling. The dose of intravitreously administered
like full-length antibodies. We have already shown that bevacizumab required to suppress choroidal angiogenesis via
bevacizumab inhibited corneal and choroidal angiogenesis in FcγRI in mice (25 μg) translates, based on relative vitreous humor
mice lacking FcγRIII, a receptor on NK cells that mediates volumes, to ~ 10 mg in the human eye, which is eightfold the
ADCC,84 and in mice lacking FcγRIV, which also has an important currently administered clinical dose. These values are compatible
role in ADCC85 (Supplementary Figures 8 and 9). In addition, with
bevacizumab inhibited corneal and choroidal angiogenesis in the relative lower affinity of human IgG1-Fc for human FcγRI
Il2rg−/− mice, which are deficient in NK cells (Supplementary (KD = 15–40 nmol/l)45,56,89 compared with that of bevacizumab for
Figure 24). These data support the thesis that this effector human VEGFA (KD = 0.5–2.2 nmol/l).3,5 Our findings predict that a
function is not involved in the angioinhibitory effect of eightfold higher dose bevacizumab would achieve both VEGFA
bevacizumab in mice. inhibition and FcγRI-mediated angioinhibition, and provide a
The inability of denosumab to suppress corneal or choroidal rationale for testing such higher doses of bevacizumab or
angiogenesis suggests that ADCC and CDC, which can be induced combining human IgG1-Fc to anti-human VEGFA drugs in patients
by both human IgG1 and human IgG2,86 are not responsible for with neovascular AMD to potentiate therapeutic angioinhibition.
angioinhibition induced by human IgG1s. We also found that a It is reasonable to query whether it would be possible to inject
mutant version of alemtuzumab (G1Δa), which was engineered 10 mg of bevacizumab into the human eye. The viability of
with point mutations in the CH2 domain of its Fc region that injecting 10 mg of a biological drug has been demonstrated in a
eliminate its CDC activity, yet retains binding to human CD52 and Phase 2 trial of lampalizumab, a Fab fragment. At present in the
to FcγRI (Supplementary Figure 15a–c), inhibited corneal clinic, 1.25 mg of bevacizumab is injected in a 50-μl volume. Retina
and choroidal angiogenesis in wild-type mice (Supplementary specialists routinely inject 100 μl of corticosteroids or 200 μl of
Figure 15d and e). In addition, bevacizumab suppressed choroidal antibiotics into the vitreous humor of humans for various
angiogenesis in C1qa− / − mice (Supplementary Figure 25), which disorders. With such higher delivery volumes, 10 mg of bevacizu-
are deficient in complement C1QA, confirming that the angioin- mab can be administered by increasing the concentration of the
hibitory activity of human IgG1 antibodies does not require CDC. formulation from the current 25 mg/ml to 50–100 mg/ml, a value
Subretinal transfection of a plasmid encoding a mutant form of similar to that of therapeutic human IVIg preparations in current
human IgG1-Fc engineered with the K322A or D270A point use. Alternatively, bevacizumab-Fc or human IgG1-Fc could be
mutations, which eliminates binding to C1q and induction of CDC administered, at correspondingly lower doses, to induce FcγRI-
while preserving binding to FcγRI,66,67,87 also reduced choroidal dependent angioinhibition.
angiogenesis in wild-type mice (Supplementary Figure 26). It would be interesting to explore to what extent the
Collectively, these data support the concept that angioinhibition therapeutic effects of IgG1 antibodies used in the treatment of
is a target-independent class effect of human or humanized IgG1 AMD, arthritis, asthma and solid tumors—disorders in which
monoclonal antibodies that is mediated via FcγRI, and not ADCC, angiogenesis plays a critical role20—might be mediated by FcγRI.
ADCP or CDC. Our data also suggest that it might be prudent to monitor
potential effects of human IgG1 antibodies on the vasculature in
other diseases, as we demonstrate is the case in IVIg-treated
DISCUSSION patients in a companion manuscript.88 Indeed, the minimal
We have shown that human or humanized IgG1 antibodies are, as angioinhibitory dose of bevacizumab in mice, 15 mg/kg, is used
a class, angioinhibitory in multiple mouse models of ocular and in humans with many forms of cancer, suggesting that at this dose
muscle angiogenesis via Fc-dependent signaling. Our findings in people, the drug might have dual anti-angiogenic activity: via
introduce angiosuppression to the list of important biological VEGFA inhibition and FcγRI-dependent pathways. Although most
functions that are triggered by FcγRI in vivo.57 Exploiting this human IgG1 antibodies are administered systemically at doses of
intrinsic property of human IgG1s could offer new therapeutic 5–10 mg/kg, several are administered at 15 mg/kg and some as
opportunities to treat diseases driven by angiogenesis that high as 30 mg/kg. Whether these antibodies might modulate
collectively affect nearly 10% of the world’s population.20 For other cellular processes, apart from angioinhibition, via FcγRI/c-Cbl
example, several human IgG1 drugs or human IgG1-Fc fusion signaling also merits future study. Such effects, if they occur, could
proteins approved for other indications could be repurposed as be mitigated by the use of miniaturized configurations such as Fab
angiogenesis inhibitors. IVIg or non-targeted, ‘generic’ human IgG- or single chain variable fragments, fully deglycosylated antibodies,
Fc might represent even more inexpensive alternatives, as we or Fc region engineering. Prolonged and frequent therapeutic IgG

© 2016 West China Hospital, Sichuan University Signal Transduction and Targeted Therapy (2016) 15001
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injections could potentially interfere with natural activation of DISCLAIMER
FcγRs by endogenous IgGs. Therefore, targeted local therapy on The content is solely the responsibility of the authors and does not necessarily
an intermittent basis might be preferable for the treatment of represent the official views of the NIH.
chronic diseases.
Our bone marrow chimera experiments point to FcγRI on
circulating myeloid cells as being critical for bevacizumab-induced AUTHOR CONTRIBUTIONS
angioinhibition. In human AMD as well as murine laser-induced SB, YK, TM, RY, LT, VC, AB-C, NK, YH, JZB, VT, SL, TY, PA, BJF, IA, AG, AB, MR, AS,
angiogenesis, macrophages are highly spatially and temporally MN, RI, HK, YO, HT, BKA, KLA, BDG and SDF, performed experiments. JSV, PB,
coincident with areas of choroidal neoangiogenesis.64,90 Indeed, of KLA, MRC, WYL and JHWL provided animals, tissues or reagents. JA conceived
the various circulating myeloid cells in mice, only macrophages and directed the project, and, with assistance from BDG, BJF and BKA wrote the
express FcγRI.91 Furthermore, we documented a reduction in paper. SDF directed the execution of the hind limb ischemia experiments. All
macrophage infiltration following bevacizumab treatment that authors had the opportunity to discuss the results and comment on the
corresponds to angioinhibition. Nevertheless, in addition to manuscript.
disrupting Vegfr1 levels and signaling in macrophages, FcγRI-
mediated events might also affect other myeloid cells, endothelial
cells, or their bone marrow-derived precursors, and could COMPETING INTERESTS
transduce complex crosstalk among these cell types to modulate JA is a co-founder of iVeena Holdings, iVeena Pharmaceuticals, iVeena Delivery
angiogenesis. Signaling pathways downstream of c-Cbl activation, Systems and Inflammasome Therapeutics, and has received honoraria from
as well as other yet to be determined molecular signals triggered Allergan and research funding from Olix Pharmaceuticals unrelated to this
via FcγRI, could be additionally responsible for human IgG1- work. JA and SDF are named as inventors on patent applications filed by the
induced angioinhibition. University of Kentucky relating to the technology described in this work. MRC is
Our data suggest that endogenous Igs could have a homeo- listed as an inventor on patents covering alemtuzumab and MRC and KLA are
static role in modulating physiological or pathological angiogen- listed as inventors on patents covering the Fc mutated forms of alemtuzumab.
esis. Future studies could explore the extent to which Igs regulate
developmental vasculature. Polymorphisms in various FCGR genes
have been associated with clinical responses to certain mono- REFERENCES
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