Hindawi Publishing Corporation
`Journal of Ophthalmology
`Volume 2012, Article ID 483034, 13 pages
`doi:10.1155/2012/483034
`
`Review Article
`Development of Anti-VEGF Therapies for Intraocular Use:
`A Guide for Clinicians
`
`Pearse A. Keane1 and Srinivas R. Sadda2
`
`1 NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of
`Ophthalmology, London EC1V 2PD, UK
`2 Doheny Eye Institute and Department of Ophthalmology, Keck School of Medicine, University of Southern California,
`Los Angeles, CA 90033, USA
`
`Correspondence should be addressed to Srinivas R. Sadda, ssadda@doheny.org
`
`Received 8 September 2011; Accepted 1 November 2011
`
`Academic Editor: Toshiaki Kubota
`
`Copyright © 2012 P. A. Keane and S. R. Sadda. This is an open access article distributed under the Creative Commons Attribution
`License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
`cited.
`
`Angiogenesis is the process by which new blood vessels form from existing vessel networks. In the past three decades, significant
`progress has been made in our understanding of angiogenesis; progress driven in large part by the increasing realization that
`blood vessel growth can promote or facilitate disease. By the early 1990s, it had become clear that the recently discovered “vascular
`endothelial growth factor” (VEGF) was a powerful mediator of angiogenesis. As a result, several groups targeted this molecule
`as a potential mediator of retinal ischemia-induced neovascularization in disorders such as diabetic retinopathy and retinal vein
`occlusion. Around this time, it also became clear that increased intraocular VEGF production was not limited to ischemic retinal
`diseases but was also a feature of choroidal vascular diseases such as neovascular age-related macular degeneration (AMD). Thus,
`a new therapeutic era emerged, utilizing VEGF blockade for the management of chorioretinal diseases characterized by vascular
`hyperpermeability and/or neovascularization. In this review, we provide a guide for clinicians on the development of anti-VEGF
`therapies for intraocular use.
`
`1. Introduction
`
`In 1948, Isaac Michaelson proposed that a diffusible factor
`(named afterward “factor X”) could be responsible, not
`only for the development of the normal retinal vasculature
`but also for pathological neovascularization in prolifera-
`tive diabetic retinopathy and other ocular disorders [1].
`By the early 1990s, it had become clear that the recently
`discovered “vascular endothelial growth factor” (VEGF)
`possessed many of the requisite characteristics of a “factor
`X” [2]. As a result, several groups targeted this molecule
`as a potential mediator of retinal ischemia-induced neovas-
`cularization in disorders such as diabetic retinopathy and
`retinal vein occlusion (RVO) [3, 4]. Around this time, it also
`became clear that increased intraocular VEGF production
`was not limited to ischemic retinal diseases but was also a
`feature of choroidal vascular diseases such as neovascular
`age-related macular degeneration (AMD) [5, 6]. Thus, a
`new therapeutic era emerged, utilizing VEGF blockade for
`
`the management of chorioretinal diseases characterized by
`vascular hyperpermeability and/or neovascularization.
`In this review, we begin by providing an overview of
`angiogenesis, the manner in which VEGF was discovered to
`be central to this process, and then a summary of VEGF
`biology. In this manner, we aim to provide the clinician with
`an understanding of the clinical scenarios in which VEGF
`blockade is likely to be successful and of patient benefit. We
`continue by describing the development of four key anti-
`VEGF therapies (pegaptanib, bevacizumab, ranibizumab,
`and aflibercept) and the results of their application in a
`selection of pioneering clinical trials. By describing the main
`features of their development in a manner accessible to clin-
`icians, we aim to highlight those molecular characteristics,
`of each agent, with implications for clinical outcomes and
`patient safety. We conclude the review by describing likely
`future directions in the application of anti-VEGF therapy in
`chorioretinal disease.
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`2. Angiogenesis
`
`2.1. Overview. Angiogenesis is the process by which new
`blood vessels form from existing vessel networks (by com-
`parison, vasculogenesis is a form of de novo blood vessel
`formation that is typically seen in the embryo) [7–9].
`Angiogenesis begins with vasodilatation and increases in vas-
`cular permeability, followed by activation and proliferation
`of vascular endothelial cells; these changes are accompa-
`nied by degradation of the surrounding extracellular matrix
`(ECM), facilitating endothelial cell migration. The migrat-
`ing endothelial cells assemble, form cords, and ultimately
`acquire lumens; further differentiation to accommodate local
`requirements then occurs and a network of similarly dif-
`ferentiated periendothelial cells and matrix develops. After
`further remodeling a complex vascular network is ultimately
`formed.
`
`2.2. Role of Angiogenesis in Disease. In the past three decades,
`significant progress has been made in our understanding of
`angiogenesis: progress driven in large part by the increasing
`realization that blood vessel growth can promote or facilitate
`disease [10]. This major conceptual advance first occurred
`in the 1930s and 1940s, when it was hypothesized that
`induction of blood vessel growth through release of vasopro-
`liferative factors would confer a growth advantage on tumor
`cells [11]. Subsequently, in the 1970s, Folkman hypothesized
`that blockade of angiogenesis could be a strategy to treat
`cancer and other disorders [12]. However, adoption of such a
`strategy first required the identification and characterization
`of the mediators of angiogenesis—a major technological
`challenge at that point.
`
`2.3. Putative Regulators of Angiogenesis. In the subsequent
`years, advances in molecular biology led to the identifica-
`tion of many putative regulators of angiogenesis, with well-
`known examples including basic fibroblast growth factor
`transforming growth factor (TGF)-β, and the
`(bFGF),
`angiopoietins [7]. In the 1980s, bFGF was thought to be the
`major angiogenic factor in the pituitary and other organs.
`However, this model was called into question when, in
`1986, it became clear that bFGF lacks a peptide sequence
`necessary for secretion and is thus confined intracellularly
`(angiogenesis is a process that requires diffusion in an
`extracellular environment) [13].
`
`2.4. Discovery of VEGF. In the mid-1980s, Ferrara and Hen-
`zel cultured a population of nonhormone secreting follicular
`cells—with unusual characteristics—from bovine pituitary
`glands (follicular cells have cytoplasmic projections that
`establish intimate connections with perivascular spaces and
`were thought to have a role in regulating growth and main-
`tenance of pituitary vasculature) [14]. Ferrara discovered
`that culture medium conditioned by these cells strongly
`promoted endothelial cell growth. He hypothesized that this
`mitogenic activity may be the result of a secreted protein;
`the subsequent isolation and sequencing of this protein led
`to discovery of the most important mediator of angiogenesis
`currently known—VEGF [15].
`
`Journal of Ophthalmology
`
`2.5. Vascular Permeability Factor. Independently, in the early
`1980s, Senger et al. had reported the identification of a per-
`meability-enhancing protein (in the supernatant of a guinea
`pig tumor cell line), which they named “vascular permeabil-
`ity factor” (VPF) [16]. In 1989, at the same time Ferrara
`and coworkers were reported their discovery of VEGF. Keck
`et al. reported the isolation and sequencing of VPF [17].
`Surprisingly, their findings indicated that VEGF and VPF
`were, in fact, the same molecule.
`
`2.6. Clinical Role for VEGF Blockade. Although multiple
`growth factors other than VEGF have been implicated in
`the angiogenic process (e.g., bFGF), VEGF appears critical
`for a number of reasons: its production is driven by hyp-
`oxia; it is highly selective for endothelial cells, it possesses
`diffusion characteristics that allow it to reach its target,
`and it affects multiple aspects of the angiogenic process
`[18, 19]. VEGF also causes vascular dilatation and promotes
`vasopermeability, both of which facilitate a rich environment
`for the growth of new vessels. Thus, despite the complexity
`of the angiogenic process, and the potential redundancy of
`the growth factors involved, VEGF blockade was quickly
`recognized as a promising approach for the restriction of
`blood vessel formation in a variety of pathologic scenarios
`[8].
`
`3. VEGF Biology
`
`3.1. Gene Family. VEGF-A, first discovered in 1989 (see
`above), is the prototype member of a gene family (i.e.,
`a group of genes with shared sequences and with similar
`biochemical functions) that also includes placental growth
`factor (PLGF), VEGF-B, VEGF-C, VEGF-D, and VEGF-E
`(prior to the discovery of other family members, VEGF-A
`was known simply as VEGF; the terms are used interchange-
`ably in this review) [10, 18]. Of note, VEGF-C and VEGF-
`D are involved in the regulation of lymphatic angiogenesis
`[20], demonstrating the unique role of this gene family in
`controlling multiple structural components of the vascular
`system.
`
`3.2. Regulation of VEGF Gene Expression. Oxygen tension
`has a key role in regulating the production of VEGF. VEGF
`mRNA expression is induced by exposure to low oxygen
`tension under a variety of pathophysiological circumstances,
`and it is now well established that a transcription factor,
`hypoxia-inducible factor-1 (HIF-1), is a key mediator of this
`response [21, 22]. Recent studies have also shown that Von
`Hippel Lindau (VHL) protein, a product of the VHL tumor
`suppressor gene, provides negative regulation of VEGF and
`other hypoxia-inducible genes (inactivation of this gene leads
`to development of capillary hemangioblastomas in the retina
`and cerebellum, and in many cases, renal cell carcinomas)
`[23].
`Several major growth factors, such as epidermal growth
`factor, also upregulate VEGF mRNA expression, suggesting
`that paracrine or autocrine release of such factors works
`in concert with local hypoxia to increase production of
`VEGF [24, 25]. In addition, inflammatory cytokines, such as
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`Journal of Ophthalmology
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`3
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`interleukin-1α and interleukin-6, induce expression of VEGF
`in several cell types (an observation in agreement with the
`hypothesis that VEGF plays a role in the angiogenesis and
`hyperpermeability seen in some inflammatory disorders)
`[25].
`
`3.3. VEGF Isoforms. The human VEGFA gene is organized as
`eight exons separated by seven introns (i.e., eight expressed
`regions that are joined together in the final mature RNA)
`[26]. Alternative splicing of the VEGFA gene results in
`the generation of four major isoforms (VEGF121, VEGF165,
`VEGF189, and VEGF206), having, respectively 121, 165, 189,
`and 206 amino acids. VEGF165 is the predominant isoform
`[27].
`Native VEGF is a heparin-binding glycoprotein (heparin
`is commonly used during protein purification due to its
`structural similarity to RNA and DNA), with a protein
`molecular weight of 45 kDa, the properties of which corre-
`spond closely to those of VEGF165 [27]. Loss of the heparin-
`binding domain of VEGF results in a significant loss in
`its mitogenic activity [28]. VEGF121, while freely diffusible
`in the ECM, is acidic and does not bind heparin [27].
`Conversely, VEGF189 and VEGF206, while being highly basic
`and capable of binding heparin with high affinity, are almost
`completely sequestered in the ECM. Thus, VEGF165, with
`intermediary properties, possesses the optimal characteris-
`tics of bioavailability and biological potency [27].
`
`3.4. VEGF Receptors. VEGF binds to two, related, receptor
`tyrosine kinases: VEGF Receptor 1 (VEGFR1) and VEGF
`Receptor 2 (VEGFR2) [27]. Both VEGFR1 and VEGFR2
`have seven immunoglobulin-like domains in the ECM, a
`single transmembrane region, and a tyrosine kinase sequence
`interrupted by a kinase-insert domain. In 1992, VEGFR1 was
`the first VEGF receptor discovered and was found to bind
`VEGF with high affinity [29]. However, despite its lower
`binding affinity for VEGF relative to VEGFR1, there is now
`agreement that VEGFR2 is the major mediator of the mito-
`genic, angiogenic, and permeability-enhancing effects of
`VEGF (the precise function of VEGFR1 is still under debate
`but may provide a “decoy effect” on VEGF signaling) [27]. In
`addition, VEGF interacts with a family of nonsignaling core-
`ceptors, the neuropilins—neuropilin-1 (NRP-1) appears to
`present VEGF165 to VEGFR2 in a configuration that increases
`the effectiveness of VEGFR2-mediated signal transduction
`[18, 30].
`
`[33]. Coverage by pericytes is thought to be one of the key
`events, resulting in loss of VEGF dependence [34].
`VEGF has also been shown to act as a chemotactic agent
`for bone marrow-derived monocytes [35], a pro-inflam-
`matory cytokine through upregulation of intercellular adhe-
`sion molecule-1 (ICAM-1) with consequent leukocyte adhe-
`sion [36], and a promoter of blood vessel extravasation
`through the upregulation of matrix metalloproteinases and
`decreased release of metalloproteinase inhibitors [37].
`The effects of VEGF on the promotion of vascular leak-
`age, both in inflammation and in other pathologic circum-
`stances, are also well established (prior to its isolation and
`sequencing, VEGF was initially characterized as “vascular
`permeability factor” by Senger et al. (see above)) [16]. Con-
`sistent with this role, VEGF has been shown to promote
`dissolution of tight junctions between endothelial cells and to
`induce endothelial fenestration in a number of vascular beds
`[38]. VEGF also induces vasodilatation in a dose-dependent
`fashion as a result of release of endothelial cell-derived nitric
`oxide—systemic blockade of VEGF may thus result in a
`clinically significant adverse hypertensive effect [39].
`Taken together, blockade of the biologic effects of VEGF
`results in rapid vessel remodeling with regression of pericyte-
`poor capillaries, reductions in vascular lumen diameter, and
`reductions in vascular permeability [33, 34]. More recently,
`evidence has suggested that VEGF could have additional
`neuroprotective effects [40].
`
`3.6. Role of VEGF in Ocular Disease. In 1994, Aiello et al.
`found a striking correlation between intraocular VEGF con-
`centrations and active proliferative retinopathy in patients
`with diabetes and ischemic central retinal vein occlusion
`(CRVO) [3]. Around the same time, Adamis et al. reported
`increased concentrations of VEGF in the vitreous of patients
`with diabetic retinopathy [4]. In 1996, it also became clear
`that increased intraocular levels of VEGF were not limited to
`ischemic retinal disorders: in a pair of influential studies, the
`localization of VEGF to choroidal neovascular membranes
`in patients with neovascular AMD was reported [5, 6].
`Proof-of-concept studies then demonstrated that blockade
`of VEGF, in animal models, led to marked decreases in
`retinal and iris neovascularization [41, 42]. Furthermore,
`exogenous administration of VEGF was demonstrated to
`produce retinal ischemia and vascular hyperpermeability in
`primates [43].
`
`4. Pegaptanib
`
`3.5. Activities of VEGF. Vascular endothelial cells are the
`primary targets for VEGF biologic activity, with their mito-
`genic effects well documented, both in vitro and in vivo [27].
`In particular VEGF induces a potent angiogenic effect in a
`variety of animal models in vivo [15, 31].
`VEGF also acts as a survival factor for endothelial cells
`in a variety of circumstances. Inhibition of VEGF results in
`extensive apoptotic changes in the vasculature of neonatal,
`but not adult mice [32]; furthermore, a marked VEGF de-
`pendence has been demonstrated in the endothelial cells of
`newly formed but not of established vessels within tumors
`
`Pegaptanib sodium is an RNA aptamer that binds to the
`heparin-binding domain of VEGF and, thus, prevents the
`predominant VEGF165 isoform from binding to VEGF recep-
`tors [44]. Pegaptanib was licensed to EyeTech Pharmaceuti-
`cals (now OSI Pharmaceuticals) for late stage development
`and marketing in the United States as “Macugen” (outside
`the USA, pegaptanib is marketed by Pfizer Inc.).
`
`4.1. Chemistry. Aptamers (from the Latin aptus, to fit, and
`the Greek meros, part or region) are oligonucleotides that
`bind to specific target molecules and that are usually created
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`Journal of Ophthalmology
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`by selection from a large random sequence pool [45]. In
`this manner, aptamers are commonly used for basic research
`and clinical purposes as macromolecular drugs. Aptamers
`constitute one of four classes of oligonucleotide reagents,
`the others being antisense oligonucleotides, ribozymes, and
`small interfering RNAs (siRNAs) [44]. However, in contrast
`with these other entities, aptamers can act on extracellular
`targets and, therefore, are not required to cross cell mem-
`branes to exert their therapeutic effects.
`The selection of aptamers has become relatively straight-
`forward with the advent of “systematic evolution of ligands
`by exponential enrichment” (SELEX) [46]; in this process,
`aptamers are engineered to bind to various target molecules
`through repeated rounds of in vitro selection. Aptamers
`offer molecular recognition properties that rival that of
`antibodies, but with a number of advantages: (1) they can be
`engineered completely in vitro, (2) they are readily produced
`by chemical synthesis, (3) they possess desirable storage
`properties, and (4) they elicit little or no immunogenicity
`[44, 45]. Pegaptanib has the distinction of being the first
`aptamer therapeutic approved for use in humans [44].
`Having chosen VEGF165 as the target for selection of
`a prospective anti-VEGF aptamer, three separate iterations
`of the SELEX methodology were carried out by scientists
`at NeXstar Pharmaceuticals [44]. By 1998, three, stable,
`high-affinity anti-VEGF165 aptamers had been characterized,
`one of which was selected for development as pegaptanib
`(initially designated NX1838, and then, EYE001) (all three
`aptamers demonstrated little or no binding to VEGF121)
`[47].
`
`4.2. Preclinical Studies. The fact that pegaptanib offers se-
`lective inhibition of a single isoform offers the theoretical
`advantage that “normal” vessels may be maintained by
`VEGF121 and other isoforms, while pathologic neovascular-
`ization may be suppressed [18, 44, 48]. Indeed, prior to
`clinical trials in humans, basic research demonstrated that
`administration of EYE001 (pegaptanib) could lead to both
`reduced vascular permeability and inhibition of both corneal
`and retinal neovascularization [49]. It has subsequently been
`shown, however, that various proteases activated during
`angiogenesis may cleave VEGF165 (and longer isoforms) to
`generate nonheparin binding fragments—such fragments
`may be sufficient to drive angiogenesis while evading pegap-
`tanib blockade [50, 51].
`
`4.3. Pharmacokinetics and Metabolism. Nonmodified aptam-
`ers are rapidly cleared from the body, with a half-life of mi-
`nutes to hours, as a result of nuclease degradation and renal
`clearance (a result of the inherently low molecular weight
`of aptamers). Therefore, modification of aptamers, such as
`(cid:2)
`-fluorine-substituted pyrimidines, and polyethylene glycol
`2
`(PEG) linkage, can be used to increase their stability and
`terminal half-life (both approaches are used in the case of
`pegaptanib) [44]. Using these approaches pegaptanib has
`been found to be stable in human plasma, at ambient tem-
`peratures, for more than 18 hours [52].
`Pegaptanib pharmacokinetics have been evaluated fol-
`lowing intravitreal injection in monkeys and rabbits [49,
`
`52, 53]. In both animal models, pegaptanib was detected in
`the vitreous at biologically active levels for at least 28 days
`following a single 0.5 mg intravitreal injection. In rabbits,
`after a single dose of pegaptanib, the initial vitreous humor
`levels were approximately 350 μg/mL and decreased by an
`apparent first-order elimination process to approximately
`1.7 μg/mL by day 28. By comparison, the plasma concentra-
`tions of pegaptanib were significantly lower, ranging from
`0.092 μg/mL to 0.005 μg/mL (day 1 to day 21). Plasma levels
`also declined by an apparent first-order elimination. In a
`human pharmacokinetic study, pegaptanib was not found
`to accumulate in the plasma after multiple doses (i.e.,
`systemic exposures were similar at different time-points);
`furthermore, no antipegaptanib antibodies (IgG or IgM)
`were detected [54].
`
`4.4. Selected Clinical Studies: Neovascular AMD. In 2004, fol-
`lowing publication of results from two, concurrent, phase
`III clinical trials (the VEGF Inhibition Study in Ocular Neo-
`vascularization, or VISION, trials), pegaptanib was licensed
`for use in the USA by the Food and Drug Administration
`(FDA) [55]. The VISION trials—two large-scale, multicen-
`ter, randomized, controlled, clinical trials—demonstrated
`that intravitreal administration of 0.3 mg of pegaptanib at
`six weekly intervals, for a period of 48 weeks (a total of nine
`treatments), was effective in reducing moderate vision loss
`in patients with neovascular AMD (higher doses were not
`shown to provide clinical benefit). In these studies, 70% of
`pegaptanib-treated patients avoided further moderate visual
`loss (defined in most AMD studies as a loss of fewer than 15
`letters of visual acuity) compared with 55% of sham-treated
`patients. However, treated eyes still lost, on average, 1.5
`lines of visual acuity over the course of a year of treatment.
`There was no evidence of either systemic toxicity or an
`increased risk of potential VEGF inhibition-related adverse
`events (a safety profile confirmed following three years of
`treatment/follow-up) [56].
`
`4.5. Selected Clinical Studies: Diabetic Macular Edema. In
`2011, the results of a phase II/III-randomized controlled
`trial, of intravitreal pegaptanib for the treatment of diabetic
`macular edema (DME), were published [57]. In this study,
`subjects with DME received injections of 0.3 mg of intravit-
`real pegaptanib, or sham injections, every six weeks for a year,
`and then according to prespecified criteria in a second year.
`In all, 36.8% of patients receiving pegaptanib, versus 19.7%
`of those in the sham group, experienced an improvement
`in visual acuity greater than 10 letters when compared to
`baseline. After two years, pegaptanib-treated patients gained,
`on average, 6.1 letters of visual acuity (versus 1.3 letters
`for controls). Pegaptanib-treated patients also received fewer
`focal/grid laser treatments (subjects were eligible for this
`beginning at week 18).
`
`5. Bevacizumab
`
`Bevacizumab (Avastin, Genentech, South San Francisco, CA)
`is a full-length monoclonal antibody, first derived from a
`murine source and prepared for intravenous administration,
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`5
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`which binds to and inhibits all isoforms of VEGF [18, 58].
`Bevacizumab was originally developed and approved for the
`treatment of metastatic colorectal cancer but may also be
`of benefit in the treatment of nonsmall cell lung cancer,
`metastatic breast cancer, and glioblastoma multiforme [59].
`Use of bevacizumab in these contexts has been associated
`with increased incidences of hypertension, bleeding, and
`thromboembolic events [59]. However the doses employed
`for intraocular use are many times lower than those used
`systemically, and the efficacy and safety of bevacizumab,
`for the treatment of neovascular AMD, has recently been
`demonstrated in phase III clinical trials [60, 61].
`
`5.1. Chemistry. Bevacizumab was originally developed from
`a mouse antihuman VEGF antibody (A.4.6.1), generated
`from mice immunized with the VEGF165 isoform [58].
`A.4.6.1 recognizes all isoforms of VEGF and, in 1992, was
`shown to inhibit growth of human tumor cell
`lines in
`vivo [62]. Subsequently, in 1996, intraocular administration
`of A.4.6.1 was found to inhibit
`iris neovascularization
`occurring secondary to retinal ischaemia in a primate model
`[42]. In 1997, bevacizumab was developed by humaniza-
`tion of A.4.6.1 [63]. In this process, six complementarity-
`determining regions (CDRs) (i.e., regions that determine
`antibody-binding) were transferred from A.4.6.1 to a human
`antibody framework previously used for humanizations.
`However, this transfer reduced VEGF binding over 1000
`fold—to reduce this effect, eight framework residues were
`changed from human to murine.
`Bevacizumab is produced in Chinese hamster ovary cells
`using expression plasmids (plasmids are DNA molecules sep-
`arate from chromosomal DNA that can be used to manu-
`facture large quantities of proteins) [58]. Bevacizumab is a
`149 kDa full-length antibody, composed of two light chains
`and two heavy chains, and with a 93% human amino acid
`sequence.
`
`5.2. Preclinical Studies. The effects of bevacizumab have been
`examined in a number of in vitro and in vivo studies [58];
`as bevacizumab was not developed with the intention of
`intraocular administration, many of these studies were per-
`formed only after its widespread adoption in this manner for
`clinical practice. In both murine and porcine models, beva-
`cizumab has been demonstrated to reduce VEGF-induced
`permeability and proliferation of choroidal endothelial cells
`and to inhibit VEGF-induced migration of human umbilical
`vein endothelial cells [64–66]. In addition, bevacizumab has
`been demonstrated as nontoxic, or not to alter the viability
`of, neurosensory retinal cells, retinal ganglion cells, and
`human retinal pigment epithelium (RPE) cells [58]. Concern
`has also been raised about the Fc component present in
`full-length antibodies such as bevacizumab—Fc domains
`are known to initiate complement activation and immune
`cell destruction [18]. Recent studies have demonstrated that
`choroidal neovascular membranes from patients with neo-
`vascular AMD treated with bevacizumab are characterized by
`significantly higher inflammatory activity [67]. Preliminary
`results have also demonstrated that bevacizumab Fc domains
`are capable of binding effectively to human RPE and human
`
`umbilical vascular endothelial cell (HUVEC) membranes via
`Fc receptors, activating the complement cascade and leading
`to cell death [58].
`
`5.3. Pharmacokinetics and Metabolism. Bevacizumab was
`developed for intravenous administration in diseases such as
`colorectal cancer [59]. As a result, compounding into smaller
`doses is required for intraocular administration. Studies have
`demonstrated differences in bevacizumab concentration and
`the presence of particulate contaminants following this pro-
`cess, emphasizing the need for implementation of optimal
`protocols when compounding pharmacies prepare this drug
`for intravitreal use [58, 68].
`The pharmacokinetics of bevacizumab, following intrav-
`itreal administration, have not been well characterized.
`Knowledge of the vitreous half-life is an important consid-
`eration when optimizing retreatment frequencies, whereas
`serum concentrations are an important factor with respect
`to systemic adverse effects (e.g., stroke). In rabbits receiving
`1.25 mg of bevacizumab, the vitreous half-life was 4.32 days
`(versus 2.88 days for ranibizumab), and the maximum serum
`concentrations were reached after eight days [69, 70]. Small
`amounts of bevacizumab were also detected in the vitreous of
`the fellow, uninjected eye. In a more recent study performed
`in humans, an aqueous half-life of 9.82 days was found after
`intravitreal injection of 1.5 mg of bevacizumab [71].
`The retinal penetration of bevacizumab has also been
`studied in animal models (experience with retinal pene-
`tration of other, full-length antibodies suggested that their
`large size would act as a limiting factor). In rabbits, Shahar
`et al. demonstrated, using confocal immunohistochemistry,
`that full thickness retinal penetration occurred 24 hours
`after intravitreal injection; this study also demonstrated the
`essential absence of bevacizumab from the retina by four
`weeks post injection [72].
`
`5.4. Selected Clinical Studies: Neovascular AMD. In 2010, the
`results of the ABC (Avastin (Bevacizumab) for treatment
`of Choroidal Neovascularization) trial provided the first
`evidence from a phase III-randomized controlled study
`for the efficacy of intravitreal bevacizumab in neovascular
`AMD [60, 73]. In this single year trial, 32% of patients
`treated with bevacizumab gained 15 or more letters from
`baseline visual acuity (initial three month loading phase, and
`then retreatment as required). In addition, 91% of patients
`receiving bevacizumab lost fewer than 15 letters of visual
`acuity from baseline, and mean visual acuity increased by 7.0
`letters over the study period.
`In 2011, the results of the CATT (Comparison of Age-
`Related Macular Degeneration Treatments Trials) study pro-
`vided further evidence, from larger phase III trial, for the
`efficacy of bevacizumab in neovascular AMD [61]. In this
`trial, 31.3% of patients treated with bevacizumab on a fixed,
`monthly regimen gained 15 or more letters from baseline
`visual acuity (28.0% for patients treated with bevacizum-
`ab as required). In addition, 94.0% of patients receiving
`bevacizumab on a fixed, monthly regimen lost fewer than
`15 letters of visual acuity from baseline (91.5% in the
`bevacizumab as required group). Finally, mean visual acuity
`
`Novartis Exhibit 2025.005
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`6
`
`Journal of Ophthalmology
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`increased by 8.0 letters over the study period in those re-
`ceiving bevacizumab monthly (5.9 letters in bevacizumab as
`required group). Of note, statistical comparisons between
`bevacizumab given as needed, and given on a fixed, monthly
`regimen, were inconclusive.
`
`6. Ranibizumab
`
`Ranibizumab (formerly known as rhuFAb V2) is an antibody
`fragment that binds and inhibits all
`isoforms of VEGF
`[18]. Specific development of ranibizumab for intraocular
`use was driven, in part, by preliminary studies suggesting
`that full-length monoclonal antibodies would not distribute
`across all retinal
`layers [74]. Furthermore, the relatively
`long systemic half-life of full-length antibodies (versus an-
`tibody fragments) raised concerns about systemic toxicity in
`patients requiring long-term anti-VEGF blockade [18].
`
`6.1. Chemistry. Bevacizumab is constructed from A.4.6.1
`using one of 12 possible Fab (fragment antigen binding)
`variants: “Fab 12” [58]. Ranibizumab is constructed using
`a different Fab variant from A.4.6.1: “Fab MB1.6”, in an
`effort to obtain higher binding affinities for VEGF [75].
`Ranibizumab is produced as a 48 kDa antibody fragment,
`in E. coli, using expression plasmids [58]. It is a chimeric
`molecule, consisting of an antigen-binding murine compo-
`nent, and a nonbinding human component that serves to
`make it less antigenic (in Greek mythology, the chimera was a
`monster with a lion’s head, a goat’s body, and a serpent’s tail).
`On a molar basis, ranibizumab is between five- and 20-times
`more potent than bevacizumab at binding of VEGF [75].
`
`6.2. Preclinical Studies. Preclinical studies have demonstrat-
`ed the safety, tolerability, and efficacy of ranibizumab in an-
`imal models. In particular, intravitreal administration of
`ranibizumab reduced vascular leakage in a monkey model
`of choroidal neovascularization (CNV), while pretreatment
`with ranibizumab prevented laser-induced development of
`CNV in this model [76].
`
`6.3. Pharmacokinetics and Metabolism. The pharmacokinet-
`ics of ranibizumab, after intravitreal administration, have
`been studied both in animal models and in human trials [69,
`77, 78]. Ranibizumab is thought to exit the vitreous cavity
`posterior via retinal penetration and choroidal vascular
`drainage or anteriorly via the aqueous drainage route. In
`animal studies, ranibizumab is cleared from the vitreous
`with a half-life of approximately three days [69]. Therefore,
`ranibizumab is thought to maintain biologically active retinal
`concentrations for approximately one month. After reaching
`a maximum at approximately one day, the serum concen-
`tration of ranibizumab declines in parallel with this. In
`human studies, following monthly intravitreal ranibizumab
`administration, maximum serum concentrations were dose
`dependent but low (0.3 ng/mL to 2.36 ng/mL—levels more
`than 1000 fold lower than in the vitreous and thought to be
`below the concentrations necessary for reduction in biolog-
`ical activity of VEGF by 50%) (http://www.gene.com/). In a
`recent study by Bakri et al., no ranibizumab was detected in
`
`the serum, or the fellow uninjected eye, of rabbits injected
`with 0.5 mg of intravitreal ranibizumab; by comparison,
`small amounts of bevacizumab were detected, both in