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`NEENTENNIAL
`
`The Americanfournal ofPathology, Vol. 181, No. 2, August 2012
`Copyright © 2012 American Society for Investigative Pathology.
`Published by Elsem'er Inc. All rtghts reserved.
`http://dx.doi.owla1016/]'.a]path.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 anal
`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.(apt/1111201206006)
`
`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 v ry mod st ph notyp s in mic
`lacking
`either FGF—i or FGF—2 tempered enthusiasm regarding their
`possible role in tumor angiogenesis.
`The publication in 1989 of two back—to—back articles in Sci—
`
`encez'3 began a new phase in this chron'cle, one that culmi—
`
`nat d in th
`r
`lativ ly r c nt d v lopm nt of antiangiogenic
`th rapi s. On
`articl
`r port d th
`isolat'on of an endothelial
`mitogen from pituitary follicular cells, whicr the authors termed
`vascular endothelial cell growth factor (VEGF)? The other ar—
`ticle described a tumor—derived factor, termed vascular per—
`meability factor (VPF), that was purified on he 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 ident'cal. (Under current
`nom nclatur ,th r comm nd d nam is vascular endothe—
`lial growth factor, with vascular permeabil'ty 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 commen ary.
`
`
`
`
`
`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
`Jnknown 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?”7 These findings,
`
`
`together with the fact thatVEGF not only acts as an angiogenic
`factor but is also able to induce permeability, made V:G-
`particularly attractive as a candidate for the long—sought—after
`factor X.
`
`
`
`
`
`
`
`Eviderce in support of a direct role for VEGF in new vessel
`
`growth ir the eye came from studies using anti—VEGF anti—
`
`
`Supported by K127EY16335 (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.
`Email: patricia.damore@schepens.harvard.edu.
`
`Regeneron Exhibit 1054.001
`
`
`
`
`
`A key demonstration that hypoxic ret'na produces V:G-
`was published in The American Journal of Pathology in 1991.8 In
`that study, the retinas of nonhuman prirrates were rendered
`ischemic by laser photocoagulation of the veins. This resulted
`in neovascularization of the iris (reminiscent of the rubeosis
`iridis sometimes associated with prolifera ive diabetic retinop—
`athy , suggesting the presence of a diffusible molecule. Levels
`of VEGF mRNA and protein were showr to be elevated in a
`mar ner that was spatially and temporally consistent with a role
`for VEGF in the growth of new vessels. In hat same year, there
`
`was a report of elevated levels of VEGF in ocular fluids from
`patients with active neovascular ocular d'sease 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.
`
`
`

`

`377
`ASIP Centennial Commentary
`AjP August 2012, Vol, 18], No, 2
`
`
`
` st'mulated the
`
`sera,1O soluble V
`EGF aptamers,12 and
`EGF receptor, 1‘ anti—V
`
`
`Evidence that VEGF is not
`VEGFR1—neutralizing antisera.13
`only necessary b
`.lt sufficient was provid
`tion that injection ofV
`EGF into the eye 0
`
`growtr
`and permeability
`retina, and also induced neovascular g
`
`
`
`ed by the demonstra—
`a nonhuman primate
`of new vessels on the
`aucoma.14
`
`Anti— VEGF
`
`Therapy
`
`Neovascular Age—Related Macu/ar Degeneration
`
`EGF—neutralizing strat—
`The first treatment developed Jsing a V
`
`n), a humanized anti—VEGF an—
`egy was bevacizumab (Avasti
`EGF isoforms. In 1997, Genen—
`tibody designed to block all V
`tech (South San Francisco, CA)
`initia
`ed phase 1
`trials of
`bevacizumab for the treatmen
`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,
`'ncreased median survival times.17
`fluorouracil, and leucovorin
`s led to approval by the US. Food
`Taken together, these resul
`
`and
`DA) on February 26, 2004, of bev—
`Drug Administration (F
`acizumab for the treatment of colon cancer in combination with
`
`
`
`
`
`
`
`
` <J><U
`
`ranibizumab (Lucentis), was created by alteration of the com—
`plementary domain region of bevacizumab, followed by affinity
`selection by phage display.21 Subsequent prase 3 clinical
`studies determined ranibizumab to be an effective treatment
`
`for NVAlVD, with a significant improvement in v'sion. Contrary
`to the or'ginal understanding, full—length anti—VEGF antibody
`does, in act, diffuse well in diseased retinas. F'rst, the earlier
`studies examining antibody diffusibility were no ,
`in fact, con—
`
`ducted with anti—VEGF antibodies, but rather w' h humanized
`
`rhu Ab -lER2 antibody, wh'ch may bind specifically in the
`retina.22 Second, the fact tha the diseased retira is not intact
`likely faci itates diffusion of the antibodies.
`Tre ef'ectiveness o ranib'zumab was determined by two
`'votal
`trials:
`the lVlinimally Classic/Occult Tria of the Anti—
`:G- An ibody Ranibizumab in the Treatment 0 Neovascular
`ge—Rela ed lVlacular Degereration (MARINA) and the Anti—
`:G- Artibody for the Treatment of Predomirantly Classic
`
`Choroida Neovascularizatior in Age—Related lVlacular Degen—
`eration (ANCHOR).
`ARINA and ANC-lOR were tre first
`phase 3 rials to show 'mprovement 'n visJal ou comes for all
`
`forms of choroidal neovascu arization in VAl\/D.23’24 Based
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
` Dist
`
`chemotherapy
`EGF therapies
`Concomitant with the development of anti—V
`
`for cancer, VEGF was found to play a pivotal role in neovas—
`
`cular age—related macular degeneratior (NVAlVl D). NVAlVD, or
`
`wet AM),
`is the leading cause of bl'ndness in the eldery
`population. One of the first anti—VEGF therapies for NVAlVD
`was pegaptanib (lVlacugen), an R A aptamer hat binds ard
`
`neutralizes VEGF165 (and likely aso VEGF188, although tr's
`has not been substantiated
`. Tris therapy, developed by
`Eyetech Pharmaceuticals (New York, NY), was shown in two
`arge phase 2 and 3 trials to decrease
`vision associated with NVAlVl
`3.18 Pegaptanib was approved
`by the FDA on December 17, 2004,
`for the treatment of
`3, making it the first antiangiogenic therapeutic ap—
`proved for ocular neovascularization.
`After approval of bevaciZJmab for carcer therapy and
`given he suspected role of VEGF in NVAlVD, systemic intra—
`venous bevacizumab begar to be adm'nistered to treat
`NVAlVD, as ar
`off—label use. A small open—label, single—center
`uncon rolled s
`udy showed significant improvement in visual
`
`thickness on optical coherence tomography,
`ret'nal
`acuity,
`and angiographic outcom s; aft
`r 12 w ks of th rapy, th
`median ard rr
`ean visual acu'
`ty improved by 8 and 12 letters,
`Soon after, ophthalmologists began injecting
`directly into the vitreous cavity as an off—label
`ment of NVAlVl
`
`
` he progressive loss of
`
`
`
`
`
`
`
`
`
`
`
` D. lntravitreal injection of bevaci—
`
`
`zumab was fOJnd to be effective in the treatment of NVAlVD,
`with minirral s
`ystemic adverse effects, which led to the first
`studies to
`demonstrate an improvement in visual function in
`
`VAl\/D.20
`patients with
`It was iritially expected that bevacizumab would not diffuse
`
`through tre retina efficiently enough to reach the choroid,
`prompting G n nt ch tog n rat an alt rnativ mol cul
`.A
`truncated and modified variant of bevacizumab, known as
`
` VA
`
`
`
`respective y. ‘9
`bevacizumab
`use in the rea
`
`
`
`
`
`on this evidence, ranibizumab was approved by the FDA on
`June 30, 2006, for the reatment of VAlVD.
`Recently, bevacizumab and ranibizumab were corrpared
`and found to have equivalent visual outcomes. The Compari—
`
`son of Age—Related
`acular Degereration Treatment Trials
`(CATT) revealed equivalent effects or visual acuity after 1 year
`of monthly administra ion of either bevacizumab or ranibi—
`zumab.25 Similarly, the two drugs were equivalent when given
`as needed. The results suggest that hese two closely related
`molecules have equivalent clinical e'
`ficacy (as m'ght be ex—
`r similar modes of action). The CJrrent stan—
`pected, given the'
`dard of care in the
`he use 0 anti—V
`treatment of NVA
` EGF
`antibodies.
`Another
`
`anti—V
`
`
`
`
`
`
`
`
`
`
`
`Gl’OlT
`Regen
`EGF strategy, developed by
`sists of a chimeric fusion
`Pharmaceuticals (
`Tarrytown, NY), cor
`
`domain of
`g the second immunoglobul'n
`protein comprisir
`V:G
`- receptor 1, the third immunoglobulin doma'n of VEGF
`
`receptor 2, and the Fc portion of human lgG1.26 "h's so—called
`V:G
`-—trap (aflibercept) functions as a decoy receptor to se—
`
`q .iester V
`EGF, thereby blocking its biological ef'ects. Afl'ber—
`was developed to improve the pharmacokinet'cs ofVEGF
`ng. Aflibercept exhibits a bind'
`ng affinity near 0.5 prrol/L,
`pared with 50 pmol/L for ranibizumab or bevacizumab,
`h represents a 100—fold increase in binding affinity.
`ln
`the intravi real half—life of a
`libercept is 4.8 days, com—
`for ranibizumab and bev—
`
`cep
`
`b'nd'
`
`whic
`add'
`
` ion,
` mon
`
`
`
`
`
`
`
`pared with 3.2 days and 5.6 days
`aciz.imab, respect
`'vely.27 The improved pharmacokinetics of
`aflibercept is thought to decrease
`he frequency of dosing in
`EGF antibodies. Phase 3
`
`patients, with similar efficacy as anti—V
`TGSU
`
` Effi—
`ts from the VIEW trials (VEGF
`Trap: Investigation of
`D) revea
`cacy and Safety in Wet AlVl
`ed that 2 mg of aflibercept
`dosed every 2 months was not inferior to ranibizumab dosed
` Based on these st
`.idies, af
`hly.
`ibercept was approved by
`
`DA on November 18, 2011.
`
`the F
`
`Diabetic Retinopathy
`
`
`In addition to its role in NVAlVD, V
`EGF plays a critical role in
`
`diabetic retinopathy and cortrithes to the development of
`
`
`
`
`
`diabetic macular edema (DlVl-). D
`- is the leading cause of
`
`Regeneron Exhibit 1054.002
`
`

`

`vision loss in the work'ng—age population in developed coun—
`
`
`tries. Analogous to the use of arti—VEGF treatment 'n NVAIVID,
`bevacizurrab,
`ranibiz.imab, ard aflibercept have all been
`
`
`shown to have some efficacy ir the treatment 0 DIVI E. How—
`ever, giver the relative y rapid irrprovement of maCJlar edema
`
`with anti—VEGF treatment, anti—VEGF trerapy for D E is likely
`mediated by modulat'rg VEGF—induced vascular permeabil—
`
`
`ity. To date, the FDA has rot approved the use 0 any of the
`
`anti—VEGF agents for re treatrrent of DIVIE. However, ranibi—
`zumab has been approved for the treatment of DIVI- in -urope
`and Australia.
`
`
`rials. One of the larger trials investigated
`ated in a variety of
`intravitreal bevacizumab alone or in combination with 'ntravit—
`
`
`
`
`
`
`
`
`
`The treatment of D E with bevaciZJmab has beer evalu—
`
`
`
`
`
`
`
`real triamcinolone VGTSJS macular laser photocoagulation.28
`The results of the 2—year study showed superiority 0 visual
`improvement in the bevacizumab—alone group at the 6—month
`time point, and these indings were sustained over he 24—
`month study period.
`lrtravitreal bevacizumab derrorstrated
`only sight superiority in VISJaI acuity over either 'ntravitreal
`bevac'zumab combined witr intravitreal triamcinolore or mac—
`
`ular laser photocoagulation.
`The efficacy and safety of 'ntravitreal ranibizumab was eval—
`uated 'n the Ranibizumab Ir'ection ir Subjects Witr Clinicaly
`Significant IVIacular Edema With Cen er lnvolvemen Second—
`ary to Diabetes IVIeI itus (RISE and RIDE) trials. These paral el
`studies evaluated rronthly intravitreal ranibizumab in'ections at
`
`0 5 or 0 3 mg versus sham injections r the treatmert of DIVIE,
`with rracular laser photocoagulatior available accordirg 0
`protocol guidelines. These studies revea ed sign' ican im—
`provement in visual acuity (in approx'rrately 63% 0 patients),
`decreased macular d ma, d or as d wors ning ofr t'nop—
`athy, and increased likelihood of improverrent with aser ther—
`apy in the ranibizumab—treated groups. A significant proportion
`of patients exhibited persistently poor vision despite reso ution
`of macular edema, suggesting that anti—VEGF therapy does
`not restore damaged retinal tissue. Ocular safety was sim'lar to
`that in previous ranibizumab studies.29
`
`A phase 2 study of aflibercept for the treatment of DIVI
`ass ss d diff
`r nt dos s of aflib rc ptv rsus macular ase
`photocoagulation. In general, aflibercept therapy was well to—
`erated in the eye and resulted in statistically significant visual
`gains and reduction in macular thickness. One—third o the
`aflibercept patients gained 15 or more letters from baseline,
`compared with only 21% in the laser—treated patients.
`IVIean
`reductions in macular thickness ranged from 127.3 to 194.5
`[.Lm, compared with only 67.9 pm in the laser—treated group.
`
`
`
`
`
`w
`
`
`
`Retinal Vein Occlusions
`
`References
`
`
`
` VEGF neutralization has also been found to be effective in the
`reatment of macular edema associated witr vein occlusions.
`Retinal vein occlusion is the second most common retinal
`
`vascular disease after diabetic retinopathy. Results from the
`Ranibizumab for the Treatment of IVIacular Edema following
`
`3ranch Retinal Vein Occlusion (BRAVO) trial ound that ranibi—
`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
`
`
`
`Kim and D’Amore
`378
`AjP August 2012, Vol, 18], No, 2
`
`
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`
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`
`
`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 IVIacu—
`
`Iar Edema after Central Retinal Vein Occlusion Study (CRUISE)
`
`tria yielded similar results. The proportion of patients who
`gained 15 or more Ieters 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
`oth rtrials, c ntral fov al thickn ss as d t rmin d by optical
`coherence tomography was significantly reduced in the ranibi—
`zumab groups.
`
`Summary
`
`
`
`
`
`
` <e'fdl
`
`
`
`
`
`Other significant findings relevant to understanding the role of
`
`VEGFin th
`y hav app ar din th pag s of The American
`ourna/ of Pathology.
`Insight into the mechanisms of V:G-
`CL
`p—regulation came from studies demonstrating that the inh'—
`
`
`tion of NAD(P)H oxidase could block ischemia—induced
`- up—regulation.31 Consistent with a known role for VEG-
`vascular development, retinal pigment epithelial cell—derived
`
`:G- has been shown to play a critical role for in the formation
`0 tre choriocapillaris,32 whereas overexpression of VEG-
`leads to choroidal neovascularization.33 The fact that virtJally
`
`every adult tissue expresses VEGF in a cell type—specific fasr—
`ion points to a postdevelopmental role for VEGIES‘I’35 Consis—
`
`
`tent with this idea, evidence indicates VEGF 's a survival factor
`
`and neuropro ectant for retinal neurons?”8 observations that
`
`hav
`I d sorr
`to rais
`conc rns r garding chronic VEGF
`neutralization 'n patients. One of the efforts to improve the
`
`efficacy of ant—VEGF therapy involves the simultaneous block—
`
`ade of PDGF—3 signaling.39'4O
`Th progr ss in sci ntific d v lopm nt and in treatment of
`diseases caused by pathological ocular angiogenesis high—
`lights the importance of basic research dedicated to improving
`
`patient care. "he use of anti—VEGF therapies has introduced a
`
`paradigm shit in the treatment of a wide array of ocular dis—
`
`eases, including NVAIVI D, diabetic retinopathy, and retinal vein
`
`occlusions. Before the development of anti—VEGF therapies,
`
`these condit'ons were most often treated with a combination of
`ablative and nonspecific laser treatment or were simply given
`careful observation and monitor'ng, 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.
`
`therapeutic implications. N Engl
`
`1. Folkman J: Tumor angiogenesis:
`J Med 1971, 28511821186
`2. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N: Vase
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`Science, 1989, 246:130641309
`3. Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Peder J, Connolly
`DT: Vascular permeability factor, an endothelial cell mitogen related
`to PDGF. Science, 1989, 246:130941312
`4. Wise GN: Retinal neovascularization. Trans Am Ophthalmol Soc
`1956, 54:7294826
`5. D’Amore PA, Klagsbrun M: Endothelial cell mitogens derived from
`retina and hypothalamus: biochemical and biological similarities.
`J Cell Biol 1984, 99154541549
`
`Regeneron Exhibit 1054.003
`
`

`

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`ASIP Centennial Commentary
`AjP August 2012, Vol, 18], No, 2
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