The American Journal of Pathology, Vol. 181, No. 2, August 2012
`Copyright © 2012 American Society for Investigative Pathology.
`Published by Elsevier Inc. All rights reserved.
`http://dx.doi.org/10.1016/j.ajpath.2012.06.006
`
`ASIP Centennial Commentary
`
`A Brief History of Anti-VEGF for the Treatment of
`Ocular Angiogenesis
`
`Leo A. Kim and Patricia A. D’Amore
`From the Schepens Eye Research Institute, Massachusetts Eye and
`Ear Infirmary, Department of Ophthalmology, Harvard Medical
`School, Boston, Massachusetts
`
`In 1994, The American Journal of Pathology pub-
`lished a key article reporting that hypoxic retina pro-
`duces vascular endothelial growth factor (VEGF), sug-
`gesting a role for VEGF in ocular neovascularization.
`Subsequent developments in anti-VEGF treatment for
`neovascular eye disease have improved visual out-
`comes and changed the standard of care in retinal
`medicine and ophthalmology.
`(Am J Pathol 2012, 181:
`376–379; http://dx.doi.org/10.1016/j.ajpath.2012.06.006)
`
`This story starts in the early 1970s with the proposal by Judah
`Folkman1 that tumor growth and progression is dependent on
`the ability of the tumor to recruit and support formation of a
`vasculature. This concept prompted a significant effort to pu-
`rify a tumor-derived angiogenic factor, which led to the identi-
`fication and purification of acidic and basic fibroblast growth
`factors (FGF-1 and FGF-2, respectively). However, the very
`wide distribution of the two growth factors, the fact that both
`molecules lack a conventional signal sequence, and the sub-
`sequent finding of very modest phenotypes in mice lacking
`either FGF-1 or FGF-2 tempered enthusiasm regarding their
`possible role in tumor angiogenesis.
`The publication in 1989 of two back-to-back articles in Sci-
`ence2,3 began a new phase in this chronicle, one that culmi-
`nated in the relatively recent development of antiangiogenic
`therapies. One article reported the isolation of an endothelial
`mitogen from pituitary follicular cells, which the authors termed
`vascular endothelial cell growth factor (VEGF).2 The other ar-
`ticle described a tumor-derived factor, termed vascular per-
`meability factor (VPF), that was purified on the basis of its ability
`to induced vascular permeability.3 Subsequent cloning and
`sequencing of the genes encoding these factors led to the
`realization that the two factors are identical. (Under current
`nomenclature, the recommended name is vascular endothe-
`lial growth factor, with vascular permeability factor as an alter-
`native.) To date, antiangiogenesis has had the most dramatic
`effect in the treatment of neovascular diseases of the eye,
`which is addressed here in this commentary.
`
`376
`
`VEGF and Neovascular Eye Disease
`
`It had long been postulated that areas of ischemic retina,
`which characterize a number of ocular pathologies (most no-
`tably diabetic retinopathy and retinopathy of prematurity)
`would produce an agent, as yet unknown, that stimulates the
`growth of new blood vessels. In 1956 George Wise wrote,
`“Pure retinal neovascularization is directly related to a tissue
`state of relative retinal anoxia. Under such circumstances, an
`unknown factor x develops in this tissue and stimulates new
`vessel formation, primarily from the capillaries and veins.”4
`Early efforts to identify this factor x led to the isolation of acidic
`and basic fibroblast growth factors from retina.5 At about the
`same time, however, two studies using the rapidly growing
`and highly vascularized glioblastoma tumor model demon-
`strated that the expression of VEGF is associated with new
`vessel growth and is driven by hypoxia.6,7 These findings,
`together with the fact that VEGF not only acts as an angiogenic
`factor but is also able to induce permeability, made VEGF
`particularly attractive as a candidate for the long-sought-after
`factor x.
`A key demonstration that hypoxic retina produces VEGF
`was published in The American Journal of Pathology in 1994.8 In
`that study, the retinas of nonhuman primates were rendered
`ischemic by laser photocoagulation of the veins. This resulted
`in neovascularization of the iris (reminiscent of the rubeosis
`iridis sometimes associated with proliferative diabetic retinop-
`athy), suggesting the presence of a diffusible molecule. Levels
`of VEGF mRNA and protein were shown to be elevated in a
`manner that was spatially and temporally consistent with a role
`for VEGF in the growth of new vessels. In that same year, there
`was a report of elevated levels of VEGF in ocular fluids from
`patients with active neovascular ocular disease but not in oc-
`ular fluids from patients with no vessel growth.9 Together,
`these articles provided intriguing circumstantial evidence of a
`role for VEGF in ocular neovascularization.
`Evidence in support of a direct role for VEGF in new vessel
`growth in the eye came from studies using anti-VEGF anti-
`
`Supported by K12-EY16335 (L.A.K.) and EY05318 and EY015435
`(P.A.D.).
`Accepted for publication June 25, 2012.
`Address reprint requests to Patricia A. D’Amore, Ph.D., Schepens Eye
`Institute and Harvard Medical School, 20 Staniford St., Boston, MA 02114.
`E-mail: patricia.damore@schepens.harvard.edu.
`
`Exhibit 2056
`Page 01 of 04
`
`

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`sera,10 soluble VEGF receptor,11 anti-VEGF aptamers,12 and
`VEGFR1-neutralizing antisera.13 Evidence that VEGF is not
`only necessary but sufficient was provided by the demonstra-
`tion that injection of VEGF into the eye of a nonhuman primate
`stimulated the growth and permeability of new vessels on the
`retina, and also induced neovascular glaucoma.14
`
`Anti-VEGF Therapy
`
`Neovascular Age-Related Macular Degeneration
`
`The first treatment developed using a VEGF-neutralizing strat-
`egy was bevacizumab (Avastin), a humanized anti-VEGF an-
`tibody designed to block all VEGF isoforms. In 1997, Genen-
`tech (South San Francisco, CA) initiated phase 1 trials of
`bevacizumab for the treatment of cancer and established that
`it had minimal toxicity.15 A phase 2 trial comparing bevaci-
`zumab combined with fluorouracil and leucovorin, against a
`control arm of fluorouracil and leucovorin alone, revealed a
`longer median survival time in the combined bevacizumab
`regimen (21.5 months, compared with 13.8 months for the
`control).16 A phase 3 trial indicated that the addition of bevaci-
`zumab to control groups receiving a regimen of irinotecan,
`fluorouracil, and leucovorin increased median survival times.17
`Taken together, these results led to approval by the U.S. Food
`and Drug Administration (FDA) on February 26, 2004, of bev-
`acizumab for the treatment of colon cancer in combination with
`chemotherapy.
`Concomitant with the development of anti-VEGF therapies
`for cancer, VEGF was found to play a pivotal role in neovas-
`cular age-related macular degeneration (NVAMD). NVAMD, or
`wet AMD, is the leading cause of blindness in the elderly
`population. One of the first anti-VEGF therapies for NVAMD
`was pegaptanib (Macugen), an RNA aptamer that binds and
`neutralizes VEGF165 (and likely also VEGF188, although this
`has not been substantiated). This therapy, developed by
`Eyetech Pharmaceuticals (New York, NY), was shown in two
`large phase 2 and 3 trials to decrease the progressive loss of
`vision associated with NVAMD.18 Pegaptanib was approved
`by the FDA on December 17, 2004, for the treatment of
`NVAMD, making it the first antiangiogenic therapeutic ap-
`proved for ocular neovascularization.
`After approval of bevacizumab for cancer therapy and
`given the suspected role of VEGF in NVAMD, systemic intra-
`venous bevacizumab began to be administered to treat
`NVAMD, as an off-label use. A small open-label, single-center
`uncontrolled study showed significant improvement in visual
`acuity, retinal thickness on optical coherence tomography,
`and angiographic outcomes; after 12 weeks of therapy, the
`median and mean visual acuity improved by 8 and 12 letters,
`respectively.19 Soon after, ophthalmologists began injecting
`bevacizumab directly into the vitreous cavity as an off-label
`use in the treatment of NVAMD. Intravitreal injection of bevaci-
`zumab was found to be effective in the treatment of NVAMD,
`with minimal systemic adverse effects, which led to the first
`studies to demonstrate an improvement in visual function in
`patients with NVAMD.20
`It was initially expected that bevacizumab would not diffuse
`through the retina efficiently enough to reach the choroid,
`prompting Genentech to generate an alternative molecule. A
`truncated and modified variant of bevacizumab, known as
`
`377
`ASIP Centennial Commentary
`AJP August 2012, Vol. 181, No. 2
`
`ranibizumab (Lucentis), was created by alteration of the com-
`plementary domain region of bevacizumab, followed by affinity
`selection by phage display.21 Subsequent phase 3 clinical
`studies determined ranibizumab to be an effective treatment
`for NVAMD, with a significant improvement in vision. Contrary
`to the original understanding, full-length anti-VEGF antibody
`does, in fact, diffuse well in diseased retinas. First, the earlier
`studies examining antibody diffusibility were not, in fact, con-
`ducted with anti-VEGF antibodies, but rather with humanized
`rhuMAb HER2 antibody, which may bind specifically in the
`retina.22 Second, the fact that the diseased retina is not intact
`likely facilitates diffusion of the antibodies.
`The effectiveness of ranibizumab was determined by two
`pivotal trials: the Minimally Classic/Occult Trial of the Anti-
`VEGF Antibody Ranibizumab in the Treatment of Neovascular
`Age-Related Macular Degeneration (MARINA) and the Anti-
`VEGF Antibody for the Treatment of Predominantly Classic
`Choroidal Neovascularization in Age-Related Macular Degen-
`eration (ANCHOR). MARINA and ANCHOR were the first
`phase 3 trials to show improvement in visual outcomes for all
`forms of choroidal neovascularization in NVAMD.23,24 Based
`on this evidence, ranibizumab was approved by the FDA on
`June 30, 2006, for the treatment of NVAMD.
`Recently, bevacizumab and ranibizumab were compared
`and found to have equivalent visual outcomes. The Compari-
`son of Age-Related Macular Degeneration Treatment Trials
`(CATT) revealed equivalent effects on visual acuity after 1 year
`of monthly administration of either bevacizumab or ranibi-
`zumab.25 Similarly, the two drugs were equivalent when given
`as needed. The results suggest that these two closely related
`molecules have equivalent clinical efficacy (as might be ex-
`pected, given their similar modes of action). The current stan-
`dard of care in the treatment of NVAMD is the use of anti-VEGF
`antibodies.
`Another anti-VEGF strategy, developed by Regeneron
`Pharmaceuticals (Tarrytown, NY), consists of a chimeric fusion
`protein comprising the second immunoglobulin domain of
`VEGF receptor 1, the third immunoglobulin domain of VEGF
`receptor 2, and the Fc portion of human IgG1.26 This so-called
`VEGF-trap (aflibercept) functions as a decoy receptor to se-
`quester VEGF, thereby blocking its biological effects. Afliber-
`cept was developed to improve the pharmacokinetics of VEGF
`binding. Aflibercept exhibits a binding affinity near 0.5 pmol/L,
`compared with 50 pmol/L for ranibizumab or bevacizumab,
`which represents a 100-fold increase in binding affinity. In
`addition, the intravitreal half-life of aflibercept is 4.8 days, com-
`pared with 3.2 days and 5.6 days for ranibizumab and bev-
`acizumab, respectively.27 The improved pharmacokinetics of
`aflibercept is thought to decrease the frequency of dosing in
`patients, with similar efficacy as anti-VEGF antibodies. Phase 3
`results from the VIEW trials (VEGF Trap: Investigation of Effi-
`cacy and Safety in Wet AMD) revealed that 2 mg of aflibercept
`dosed every 2 months was not inferior to ranibizumab dosed
`monthly. Based on these studies, aflibercept was approved by
`the FDA on November 18, 2011.
`
`Diabetic Retinopathy
`
`In addition to its role in NVAMD, VEGF plays a critical role in
`diabetic retinopathy and contributes to the development of
`diabetic macular edema (DME). DME is the leading cause of
`
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`Kim and D’Amore
`378
`AJP August 2012, Vol. 181, No. 2
`
`vision loss in the working-age population in developed coun-
`tries. Analogous to the use of anti-VEGF treatment in NVAMD,
`bevacizumab, ranibizumab, and aflibercept have all been
`shown to have some efficacy in the treatment of DME. How-
`ever, given the relatively rapid improvement of macular edema
`with anti-VEGF treatment, anti-VEGF therapy for DME is likely
`mediated by modulating VEGF-induced vascular permeabil-
`ity. To date, the FDA has not approved the use of any of the
`anti-VEGF agents for the treatment of DME. However, ranibi-
`zumab has been approved for the treatment of DME in Europe
`and Australia.
`The treatment of DME with bevacizumab has been evalu-
`ated in a variety of trials. One of the larger trials investigated
`intravitreal bevacizumab alone or in combination with intravit-
`real triamcinolone versus macular laser photocoagulation.28
`The results of the 2-year study showed superiority of visual
`improvement in the bevacizumab-alone group at the 6-month
`time point, and these findings were sustained over the 24-
`month study period. Intravitreal bevacizumab demonstrated
`only slight superiority in visual acuity over either intravitreal
`bevacizumab combined with intravitreal triamcinolone or mac-
`ular laser photocoagulation.
`The efficacy and safety of intravitreal ranibizumab was eval-
`uated in the Ranibizumab Injection in Subjects With Clinically
`Significant Macular Edema With Center Involvement Second-
`ary to Diabetes Mellitus (RISE and RIDE) trials. These parallel
`studies evaluated monthly intravitreal ranibizumab injections at
`0.5 or 0.3 mg versus sham injections in the treatment of DME,
`with macular laser photocoagulation available according to
`protocol guidelines. These studies revealed significant im-
`provement in visual acuity (in approximately 63% of patients),
`decreased macular edema, decreased worsening of retinop-
`athy, and increased likelihood of improvement with laser ther-
`apy in the ranibizumab-treated groups. A significant proportion
`of patients exhibited persistently poor vision despite resolution
`of macular edema, suggesting that anti-VEGF therapy does
`not restore damaged retinal tissue. Ocular safety was similar to
`that in previous ranibizumab studies.29
`A phase 2 study of aflibercept for the treatment of DME
`assessed different doses of aflibercept versus macular laser
`photocoagulation. In general, aflibercept therapy was well tol-
`erated in the eye and resulted in statistically significant visual
`gains and reduction in macular thickness. One-third of the
`aflibercept patients gained 15 or more letters from baseline,
`compared with only 21% in the laser-treated patients. Mean
`reductions in macular thickness ranged from 127.3 to 194.5
`␮m, compared with only 67.9 ␮m in the laser-treated group.
`
`ranibizumab group also had a statistically significant decrease
`in retinal thickness, compared with the control group.30
`Central retinal vein occlusions are also amenable to ranibi-
`zumab therapy. The Ranibizumab for the Treatment of Macu-
`lar Edema after Central Retinal Vein Occlusion Study (CRUISE)
`trial yielded similar results. The proportion of patients who
`gained 15 or more letters in visual acuity at 6 months was
`46.2% (0.3 mg) and 47.7% (0.5 mg) in the ranibizumab groups
`and 16.9% in the sham injection group. Similar to findings from
`other trials, central foveal thickness as determined by optical
`coherence tomography was significantly reduced in the ranibi-
`zumab groups.
`
`Summary
`
`Other significant findings relevant to understanding the role of
`VEGF in the eye have appeared in the pages of The American
`Journal of Pathology. Insight into the mechanisms of VEGF
`up-regulation came from studies demonstrating that the inhi-
`bition of NAD(P)H oxidase could block ischemia-induced
`VEGF up-regulation.31 Consistent with a known role for VEGF
`in vascular development, retinal pigment epithelial cell-derived
`VEGF has been shown to play a critical role for in the formation
`of the choriocapillaris,32 whereas overexpression of VEGF
`leads to choroidal neovascularization.33 The fact that virtually
`every adult tissue expresses VEGF in a cell type-specific fash-
`ion points to a postdevelopmental role for VEGF.34,35 Consis-
`tent with this idea, evidence indicates VEGF is a survival factor
`and neuroprotectant for retinal neurons,36–38 observations that
`have led some to raise concerns regarding chronic VEGF
`neutralization in patients. One of the efforts to improve the
`efficacy of anti-VEGF therapy involves the simultaneous block-
`ade of PDGF-B signaling.39,40
`The progress in scientific development and in treatment of
`diseases caused by pathological ocular angiogenesis high-
`lights the importance of basic research dedicated to improving
`patient care. The use of anti-VEGF therapies has introduced a
`paradigm shift in the treatment of a wide array of ocular dis-
`eases, including NVAMD, diabetic retinopathy, and retinal vein
`occlusions. Before the development of anti-VEGF therapies,
`these conditions were most often treated with a combination of
`ablative and nonspecific laser treatment or were simply given
`careful observation and monitoring, with a universal decline in
`vision. The current use of anti-VEGF treatment has resulted in
`improvement of visual outcomes and has changed the stan-
`dard of care in retinal medicine and ophthalmology.
`
`Retinal Vein Occlusions
`
`References
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`VEGF neutralization has also been found to be effective in the
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`zumab at doses of 0.3 and 0.5 mg resulted in a higher pro-
`portion of subjects who gained 15 or more letters at the
`6-month time point. Specifically, 55.2% (0.3 mg) and 61.1%
`(0.5 mg) of patients in the ranibizumab groups and 28.8% in
`the sham group gained 15 or more letters at 6 months. The
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`
`Exhibit 2056
`Page 04 of 04
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