Quan Dong Nguyen
`Eduardo Buchele Rodrigues
`Michel Eid Farah
`AARVoemmaMe\yNCCay
`
`nvaahel
`
`SPECeet
`
`Page 01 of 19
`
`SAUNDERS
`
`ELSEVIER
`
`Exhibit 2084
`
`€ e
`
`Mylan v. Regeneron, IPR2021-00880
`U.S. Pat. 9,669,069, Exhibit 2084
`
`Exhibit 2084
`Page 01 of 19
`
`

`

`
`
`Commissioning Editor: Russell Gabbedy
`Development Editor: Alexandra Mortimer
`Editorial Assistant: Poppy Garraway
`Project Manager: Beula Christopher
`Design: Charles Gray
`Mlustration Manager: Gillian Richards
`Illustrator: Cactus
`Marketing Managers (UK/USA): Richard Jones / Helena Mutak
`
`Exhibit 2084
`
`Page 02 of 19
`
`Exhibit 2084
`Page 02 of 19
`
`

`

`Pharmacotherapy
`
`Rvoahel
`
`!
`:
`
`Quan Dong Nguyen mp, MSc
`Associate Professor of Ophthalmology
`Diseases of the Retina and Vitreous, and Uveitis
`WilmerEyeInstitute
`Johns Hopkins University School of Medicine
`Baltimore, MD, USA
`
`Eduardo Buchele Rodrigues mp
`Department of Ophthalmology
`Vision Institute
`Federal University of Sao Paulo
`Sao Paulo, Brazil
`
`Michel Eid Farah mp
`Professor of Ophthalmology and Vice-chairman
`Retina and Vitreous Department
`Vision Institute
`Federal University of Sao Paulo
`Sao Paulo, Brazil
`
`William F. Mieler mp
`Professor and Vice-Chairman
`Department of Ophthalmology & Visual Sciences
`University ofIllinois at Chicago
`Chicago, IL, USA
`
`
`
` | ESTES
`
`Exhibit 2084
`
`Page 03 of 19
`
`Exhibit 2084
`Page 03 of 19
`
`

`

`SAUNDERS
`ELSEVIER
`
`SAUNDERSis an imprint of ElsevierInc.
`
`© 2010,Elsevier Inc. All rights reserved.
`
`First published 2010
`
`Chapter 35 in this publication is in the public domain and may be used and reprinted without special
`permission: citation of the source, however, is appreciated.
`
`No part of this publication may be reproduced or transmitted in any form or by any means,electronic or
`mechanical,
`including photocopying, recording, or any information storage and retrieval system, without
`permission in writing from the publisher. Details on how to seek permission, further information about
`the Publisher’s permissions policies and our arrangements with organizations such as the Copyright
`Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/
`permissions.
`
`British Library Cataloguing in Publication Data
`Retinal pharmacotherapy.
`1. Retina-Diseases—Chemotherapy.
`|. Rodrigues, Eduardo.
`617.7’35061 -dce22
`
`ISBN-13: 9781437706031
`
` Exhibit 2084
`
`Library of Congress Cataloging in Publication Data
`A catalog record for this book is available from the Library of Congress
`
`Notices
`Knowledge and bestpracticein this field are constantly changing. As new research and experience broaden
`our understanding, changesin research methods, professional practices, or medical treatment may become
`necessary. Practitioners and researchers mustalwaysrely on their own experience and knowledgein evaluat-
`ing and using any information, methods, compounds, or experiments describedherein. In using suchinfor-
`mation or methods they should be mindful of their own safety and the safety of others, including parties for
`whom they have a professional responsibility.
`With respect to any drug or pharmaceutical productsidentified, readers are advised to check the most
`current information provided(i) on procedures featured or(ii) by the manufacturer of each product to be
`administered, to verify the recommended doseor formula, the method and duration of administration, and
`contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of
`their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient,
`and to take all appropriate safety precautions.
`To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any
`liability for any injury and/or damage to personsor property as a matter of products liability, negligence or
`otherwise, or from any use or operation of any methods, products,instructions, or ideas contained in the
`material herein.
`
`The Publisher
`
`your source for books,
`OAC journals and multimedia
`in the health sciences
`www.elsevierhealth.com
`
`
`
`Working together to grow
`libraries in developing countries
`
`www.elsevier.com | www.bookaid.org | www.sabre.org
`BOOK AID
`International
`ELSEVIER
`Sabre Foundation
`
`
`
`The
`publisher's
`policy is to use
`paper manufactured
`from sustainable forests
`
`Printed in China
`Last digit is the printnumber:9 8
`
`7
`
`6
`
`5
`
`4
`
`3
`
`2
`
`1
`
`Page 04 of 19
`
`Exhibit 2084
`Page 04 of 19
`
`

`

`CHAPTER
`
` LEO
`
`
`
`
`Ocular angiogenesis: vascular
`endothelial growth factor
`and other factors
`
`Anthony P. Adamis, MD
`
`
`
`INTRODUCTION
`
`Theoriginal visionary proposal by Dr. Judah Folkman!that antiangio-
`genic therapy couldoffer an approachto the treatment of manycancers
`ultimately led to a major research effort into the mechanisms which
`control both physiological and pathological angiogenesis. His work
`also contemplated the use of antiangiogenic drugs in ophthalmology.
`A principal focus of this research effort has been the identification of
`Specific molecules involved in the promotion andinhibition of angio-
`enesis, an effort that has already led to the developmentof targeted
`therapies against vascular endothelial growth factor (VEGF). In addi-
`tion, manyother factors have been identified that act as promoters or
`inhibitors of angiogenesis (Table 4.1). This chapter will focus on those
`molecules whose roles have been best validated to date, and which
`possess particular relevance to ocular neovascularization.
`
`
`
`PROMOTERSOF ANGIOGENESIS
`
`VASCULAR ENDOTHELIAL
`GROWTH FACTOR
`
`VEGFin physiologic and pathologic
`angiogenesis
`VEGF(also known as VEGF-A)is a 45-kDa homodimeric glycoprotein
`belonging to a family that also includes VEGF-B through VEGF-E,
`platelet-derived growth factor (PDGF), and placental growth factor?
`Initially isolated as a vascular permeability factor, VEGF was
`subsequently cloned and found to be a potent proangiogenic factor,
`acting as a master regulator of angiogenesis (reviewed by Ferrara and
`Davis-Smyth’ and Ferrara’). VEGFhas subsequently been foundto act
`in a wide variety of other physiological contexts,’ some of which,
`such as neuroprotection, are completely independentof its role in
`angiogenesis.
`Alternative splicing of the human VEGFgeneyields six principal
`isoforms of 121, 145, 165, 183, 189, and 206 amino acids.> The corre-
`sponding rodent isoforms are one aminoacid shorter.? Many studies
`have focused on characterizing the functions of VEGF,2, VEGFy¢5, and
`VEGFigo. VEGF,.;is freely diffusible, while VEGF) and larger isoforms
`are found sequestered in the extracellular matrix; VEGFy5 exists in both
`diffusible and matrix-bound forms.’ VEGFacts as a ligand for VEGF
`teceptor 1 (VEGFR1) and VEGFR2; these receptor tyrosine kinases in
`turn activate downstream signaling cascades.
`VEGFacts in many capacities in angiogenesis, including as an endo-
`thelial cell mitogen’ and survivalfactor,” and as a chemoattractantfor
`bone marrow-derived endothelial progenitorcells.* In addition, VEGF
`induces the upregulation of extracellular matrix-degrading enzymes,
`such as matrix metalloproteinases (MMPs)’ and plasminogen activa-
`tor,'” as well as nitric oxide," a downstream mediator of VEGFsignal-
`ing.'’ Moreover, VEGFhas twoadditional properties whichareofdirect
`relevance for the pathophysiology of ocular neovascular diseases.First,
`
`Exhibit 2084
`
`Page 05 of 19
`
`it is the most potent known inducerof vascular permeability," an action
`related to the edema which often accompanies ocular neovasculariza-
`tion. Secondly, the retinal expression of VEGF, which is produced by
`a wide variety ofretinal cell types,'*"* is upregulated by hypoxia,'*"”
`a responsethatis believed to be important in maintaining the health
`of both retinal neurons® and the choriocapillaris” while also creating
`a proangiogenic environment.
`Reflecting the original focus of Dr. Folkman’s proposal on the impor-
`tance of angiogenesis in cancer growth and metastasis,’ initial investiga-
`tions of the role of VEGF in pathological angiogenesis demonstrated
`that interference with VEGFsignaling inhibited tumor growth.!? Over
`the course of a decade, a role for VEGFin ocular neovascular disease
`also wasestablished based on three main lines of evidence: (1) correla-
`tions of VEGFelevation with the presence of ocular neovascular disease
`in the eyesofpatients; (2) preclinical studies demonstrating that experi-
`mental elevation of VEGFlevels in the eye led to neovascularization;
`and(3) the converse experiment, in which inhibition of VEGFsignaling
`decreased neovascularization.
`Correlations betweenelevations in ocular levels of VEGF and ocular
`neovascular disease have been reported and include conditions such
`as iris neovascularization, retinal vein occlusion, diabetic retinopathy
`(DR), diabetic macular edema (DME), neovascular glaucoma,andreti-
`nopathy of prematurity (reviewed byStarita etal."). Elevated expres-
`sion of VEGFalso has been detected in surgically removed maculae”
`and choroidal neovascularization (CNV) membranesof eyes with age-
`related macular degeneration (AMD).”
`A variety of approaches have been employed to demonstrate that
`elevated ocular levels of VEGFare sufficient to induce ocular neovas-
`cularization. These haveincluded direct intravitreal injection of VEGE”
`and retinal vein photocoagulation” in monkeys; in rodent models,
`studies have included intravitreal
`injection of VEGF-expressing
`vectors,“4 and the use of transgenic mice engineered to overexpress
`VEGFin the retinal pigment epithelium (RPE).”
`The experiments demonstrating that VEGFelevations are necessary
`for the developmentof ocular neovascularization have also employed
`various techniques. Agents used to block the actions of VEGF have
`included VEGFR fusion proteins,”anti-VEGFantibodies,” an anti-
`VEGF monoclonalantibody antigen-binding fragment (Figure 4.1);
`an aptamer directed against VEGFys," and VEGF,sb, a VEGE variant
`which binds VEGFR2 but cannotactivateit.” Agents used to block the
`ocular production of VEGF or VEGFR1atthetranscriptional or trans-
`lational level have included small interfering RNAs (siRNAs) specific
`for VEGF® or VEGFR1,™ and antisense oligonucleotides specific for
`VEGE®*Blocking the actions of VEGF in the eye by various means
`inhibited neovascularization of theiris,” cornea,” retina," and
`choroid.2731334
`Further detailed investigations into the mechanisms underlying
`VEGF’s importance have revealedthat the isoform VEGF«5 is especially
`pathogenic. In a murine model of ischemia-associated ocular neovas-
`cularization, retinal expression of VEGF,,; was foundto be dramatically
`elevated compared to other isoforms; moreover, intravitreal injection
`of a VEGFi¢5-specific RNA aptamer wasasefficient at inhibiting the
`pathological neovascularization as injection of a VEGFR-Fc fusion
`protein that inactivated all VEGF isoforms (Figure 4.2).”* In addition,
`VEGF;5 acts as an especially potent
`inflammatory cytokine, a
`property of direct relevance given the importance of inflammation in
`
`23
`
`Exhibit 2084
`Page 05 of 19
`
`

`

`
`
`
`
`$10]08-]JBUIOPUP1O}Oe4YIMOJD[eljeyJOPUFJejnoseA‘siseueHo!BuyJIN°yHALdWHO
`
`
`
`
`
`
`
`
`
`
`
`Table 4.1 Proangiogenic and antiangiogenic factors
`
`Vasostatin (calreticulin fragment)
`
`
`
`Proangiogenic
`factors
`
`Angiogenin
`Angiopoietin- 1
`Complementfactors C3
`and C5
`
`Cryptic collagen IV
`fragment
`Developmentally
`regulated endothelial
`locus 1 (Del-1)
`Fibroblast growth factors:
`acidic (aFGF) and basic
`(bFGF)
`Follistatin
`
`Granulocyte colony-
`stimulating factor
`(G-CSF)
`Hepatocyte growth factor
`(HGF)/scatter factor (SF)
`Interleukin-8 (IL-8)
`a5 integrins
`Leptin
`Midkine
`
`Pigment epithelium-
`derived growth factor
`Placental growth factor
`Platelet-derived
`endothelial cell growth
`factor (PDECGF)
`Platelet-derived growth
`factor-BB (PDGF-BB)
`Pleiotrophin (PTN)
`Progranulin
`Proliferin
`
`Transforming growth
`factor-a (TGF-a)
`Transforming growth
`factor-B (TGF-B)
`Tumor necrosis factor-a.
`(TNF-a)
`Vascular endothelial
`growth factor (VEGF)
`
`Antiangiogenic factors
`
`Angioarrestin
`Angiostatin (plasminogen
`fragment)
`Antiangiogenic antithrombin Ill
`Cartilage-derived inhibitor (CDI)
`CD59 complement fragment
`Endostatin (collagen XVIII
`fragment)
`Fibronectin fragment
`Growth-related oncogene (Gro-B)
`Heparinases
`Heparin hexasaccharide
`fragment
`Human chorionic gonadotropin
`(hCG)
`Interferon o/B/y
`Interferon-inducible protein
`(IP-10)
`Interleukin-12
`
`Kringle 5 (plasminogen fragment)
`Metalloproteinase inhibitors
`(TIMPs)
`2-Methoxyestradiol
`Pigment epitheliurn-derived
`growth factor
`Placental ribonuclease inhibitor
`
`Plasminogenactivator inhibitor
`Platelet factor-4 (PF4)
`Prolactin 16-kDa fragment
`Proliferin-related protein (PRP)
`Retinoids
`Soluble VEGFR-1
`
`Tryptophanyl-tRNA synthase
`fragment
`VEGF0
`Tetrahydrocortisol-S
`Thrombospondin-1 (TSP-1)
`Transforming growth factor-B
`(TGF-B)
`Vasculostatin
`
`prematurity (Figure 4.4)* and in laser-induced CNV.* VEGF,; was
`more potent at chemotaxis of monocyte/macrophages than VEGF,).”
`Since macrophages produce VEGE,” their infiltration serves as an
`amplification mechanism in further promoting angiogenesis.
`
`Investigational approaches to VEGF inhibition
`in ocular neovascularization
`
`The extensive research effort into elucidating VEGF’s role in ocular
`neovascularization has provided a sound foundation for the develop-
`mentof anti-VEGFtherapies. Three agents, pegaptanib,” ranibizumab,"
`and bevacizumab,” are already in widespread use, and are discussed
`in dedicated chapters of this text. A brief account of other approaches
`currently under evaluation in clinicaltrials follows.
`
`RNAinterference
`
`RNAinterference abrogates gene expression througha cellular defense
`mechanism mediated by double-stranded RNA sequencesofatleast
`21 nucleotides long, resulting in targeted destruction of specific mRNA
`species.” RNA interference has been used to target VEGF mRNAin
`animal models, leading to suppression of corneal neovascularization™
`as well as CNV induced either by laser® or by overexpression of VEGF
`from a transgene."* Sirna-027, an agent targeting the expression of
`VEGEFR1,also has been shown to suppress both retinal and CNV in
`murine models.™
`Currently there are two siRNA agents undergoing evaluation in
`clinical trials for treatment of neovascular AMD.Bevasiranib (Ophi
`Health), directed against VEGF, has successfully completed a phase II
`trial and is currently recruiting patients for the phase III COBALTtrial
`in which it will be combined with ranibizumab.” In addition, a phase
`I trial of the anti-VEGFR1 agent AGN211745 (Allergan; previously
`Sirna-027) has been completed,” and enrollment for a phaseII trial is
`ongoing.** Recent evidence suggests that antiangiogenic siRNAs work
`nonspecifically and through a nonclassical siRNA mechanism in sup-
`pressing CNV.”
`
`Soluble VEGFRfusion protein: VEGF-Trap —
`Work demonstrating the potential of soluble VEGFR fusion proteins to
`suppress retinal neovascularization” provided a basis for the develop-
`ment of VEGF-Trap, a fusion protein combining components of both
`VEGFR1 and VEGFR2.” VEGF-Trap, which was engineered with a
`view to optimizing pharmacokinetic properties as well as efficacy,
`binds to all isoforms of VEGF as well as placental growth factor.”
`Intravitreal injection of VEGF-Trap inhibited laser-induced CNV in
`mice, as well as preventing VEGF-induced blood-tetinal barrier break-
`down.”It is now being evaluated in a phase III study.”
`
`Anecortave acetate
`
`Anecortaveacetate is a memberof a group of corticosteroids,first iso-
`lated in Dr. Folkman’slaboratory,” that have angiostatic properties but
`lack conventional anti-inflammatory activity.” In a rat retinopathy of
`prematurity model, anecortave significantly reduced pathologic retinal
`neovascularization without affecting normalretinal angiogenesis.” In
`other studies with this model, anecortave was foundto reduceretinal
`expression of VEGE®andofinsulin growth-factor-1 andits receptor.”
`Anecortave also inhibited VEGFR2 expression in a murine model of
`retinoblastoma.” These findings suggest that the angiostatic effects of
`anecortave mayatleast in part be mediated through VEGFsignaling
`pathways.”
`Anecortave acetate has shown some promise as a treatment for
`neovascular AMD, administered as a juxtascleral depoteither alone®
`or in combination with photodynamic therapy.” Although anecortave
`acetate did not meetits efficacy endpointin a phase II noninferiority
`trial comparing it
`to photodynamic therapy with verteporfin,® it
`remains understudy as a prophylactic treatment to slow the progres-
`sion of neovascular AMD.”
`
`Adapted from: Angiogenesis Foundation. Understanding angiogenesis.
`List of known angiogenic growth factors. Available online at: http://www.
`angio.org/understanding/content_understanding. html.
`
`pathological neovascularization. Laser injury has been shown to up-
`regulate retinal expression of intercellular cell adhesion molecule-1
`(ICAM1), thereby promoting leukocyte adhesion to the vascular endo-
`thelium through CD18, the leukocyte ligand for ICAM1.* Genetic
`ablation of either molecule significantly reduced the formation of
`laser-induced CNV (Figure 4.3). In this context, it is noteworthy that
`VEGF,,; was found to be significantly more potent at upregulating
`ICAM1expression on endothelial cells than VEGF;>,.” In addition,
`depletion of macrophages has been found to inhibit the development
`of pathological neovascularization in a rat model of retinopathy of
`
`24
`
`Exhibit 2084
`
`Page 06of 19
`
`Exhibit 2084
`Page 06 of 19
`
`

`

` |
`
`
`
`
`
`BuljyoYyUlS99USIDSJISege|NOILOAS
`
`| Prevention
`
`| Control
`
`No.ofgrade4CNVlesions
`
`35
`
`42
`
`Days
`
`Control/Treatment
`
`30
`
`No.ofgrade4CNVlesions
`
`Days
`
`Es
`
`x
`Figure 4.1 Inactivation ofall vascular endothelial growth factor (VEGF) isoforms potently inhibits laser-induced choroidal neovascularization
`(CNV) in the nonhuman primate. (A) Cynomolgus monkeys(n = 10) received 500 pig of recombinant humanized monoclonal anti-VEGF
`
`antibody (rhuFab VEGF) in one eye andvehicle in the other, every 2 weeks. On day 21, CNV wasinducedby laser wounding. The bar
`“graph showsthe total number of grade 4 CNV lesions in the eyes receiving rhuFab VEGF(gold bar) compared to thosein control eyes that
`‘Teceived vehicle (blue bar); assessments were made 2 weeksafter laser induction (day 35), and 3 weeksafter the laser induction (day 42)
`Adapted from Krzystolik MG, Afshari MA, Adamis AP,et al. Prevention of experimental choroidal neovascularization with intravitreal anti-
`vascular endothelial growth factor antibody fragment. Arch Ophthalmol 2002;120:338-346.
`1.0-
`
`
`
`
`
`(mm?) on oSie)
`
`|Control 4
`|
`[BB VEGF46q-selective blockade
`a8
`_ |) Nonselective VEGF blockade
`
`baadfo)
`
`Area
`
`0.0-
`
`especially critical for the recruitment of mural cells (pericytes and
`smooth-muscle cells) to the developing vasculature.” Genetic ablation
`of PDGF-Bleads to perinatal death from hemorrhages and vascular
`system abnormalities” while ablation of the PDGFR-generesults in a
`similar phenotype.” Proliferation of mural cells was significantly
`reduced in mice lacking either PDGF-B or PDGER-B.* Also, administra-
`tion of an aptamerspecific for PDGF-Bledfirst to pericyte loss and then
`to regression of tumorvessels in a murine tumor model. These find-
`ings indicate that PDGF-B produced by endothelial cells is essential for
`the proliferation, migration, and recruitmentof muralcells to the devel-
`oping capillaries (Figure 4.5)."°
`Studies of ocular neovascularization in mice have provided further
`evidence in support of this model. Inhibition of PDGF-B signaling,
`whether by genetic ablation in endothelial cells or PDGFR kinase
`inhibitors,” led to deficient pericyte recruitment in models ofretinal”
`and corneal” neovascularization.
`Studies using three different models of ocular neovascularization, in
`which PDGF-B and VEGFsignaling were blocked by administration of
`an antibody to PDGFR-f or pegaptanib,respectively, have furtherdelin-
`eated the respective roles of these molecules.” Physiological retinal
`angiogenesis wasinhibited on postnatal day 3 by blocking PDGF-B, but
`notby blocking VEGF,«:; however, combined blockadeprovided greater
`inhibition. Conversely, VEGFblockadealoneinhibited the development
`of laser-induced CNV, whereas blocking PDGF-B signaling was ineffec-
`tive on its own,again,greater inhibition occurredifboth pathways were
`blocked. Finally, in a corneal model of neovascularization, PDGF-B
`blockade between days 10 and 20 postinjury led to detachmentof mural
`cells from corneal neovessels; in contrast, VEGF blockade reduced neo-
`vascularization when applied immediately after wounding, butit did
`not induce regression ofvessels after they were established. However,
`vessel regression was enhancedif both inhibitors were given (Figure
`4.6).These experiments suggest that a combination strategy targeting
`both VEGF and PDGF-B may be more effective, both in treating estab-
`lished neovascularization and in preventing new vessel growth.
`
`FIBROBLAST GROWTH FACTOR2 (FGF2)
`
`FGF2(also knownasbasic FGF)is a heparin-binding growth factorthat
`occurs in several isoforms. FGF2 signals throughfour receptor tyrosine
`kinases (FGFreceptor 1 through FGFreceptor 4) andacts ina variety
`of developmentalprocesses, including angiogenesis.”
`
`25
`
`
`
`
`The PDGF family consists offour related dimeric polypeptides (PDGF-A
`thtough PDGF-D)" that are structurally related to VEGF? In general
`
`they occur as homodimers, although the PDGF-AB heterodimer has
`also been identified." PDGFs are ligands for two receptor tyrosine
`
`Kinases, PDGFR-o. and PDGER-B, of which PDGER-B is principally
`Tesponsible for signal transduction on cells associated with the vascular
`system,
`including endothelial cells, pericytes, and smooth-muscle
`
`cells. Similarly, PDGFalso has a widespread distribution among these
`“samecell types.” In addition to its central role in vascular system
`_development, PDGF signaling is important for processes such as
`_Woundhealing and central nervous system development.”
`
`__
`Studies have revealed a central role for the PDGF-B homodimerin
`vascular development, as it was found to stimulate the proliferation,”
`
`and inducecapillary tube formation™ of endothelial cells. PDGF-B is
`
`e Figure 4.2 Vascular endothelial growth factor (VEGF64/165) is
`€specially potent in promoting pathological neovascularization. In a
`_fat modelof ischemia-induced retinal neovascularization, intravitreal
`injection of an aptamer specific for VEGF164165 Was as effective in
`"inhibiting pathological neovascularization as a VEGFR1-Fe fusion
`_ protein which binds all VEGF isoforms. Adapted from Ishida S, Usui
`T, Yamashiro K,et al. VEGF164-mediated inflammation is required
`
`for pathological, but not physiological, ischemia-induced retinal
`
`“neovascularization. J Exp Med 2003;198:483-489.
`
`LATELET-DERIVED GROWTH FACTOR
`
`
`Exhibit 2084
`
`Page 07 of 19
`
`Exhibit 2084
`Page 07 of 19
`
`

`

`CD18-/-
`
`Wildtype C57BL/6J
`ICAM-1 -/-
`Percent Week1
`
`P=0.02
`
`P=0.015
`
`P=0.042
`
` 9= 4+mJa
`
`ae@
`OQO
`
`c= 52o©@
`
`Q
`=,
`QO
`De
`
`9<»Q9
`
`°
`cS
`
`=m
`
`M2
`
`Q 2aDa ) >T
`
`Figure 4.3 Evidenceofthe role of inflammation in the model
`oflaser-induced choroidal neovascularization (CNV). (A) Genetic
`ablation of either CD18 or intercellular cell adhesion molecule-1
`(ICAM1) led to marked diminution of the size of laser-induced CNV.
`Two weeks following laser injury, stacked confocal images were
`takenof fluorescein Griffonia simplicifolia lectin |-labeled tissue within
`the laser scars. CNV membranes were significantly reduced in both
`mutant strains, compared to wild-type mice. Scale bar, 100 um.
`(B) Loss of either CD18 or ICAM1 resulted in fewer lesions of
`pathological significance. Fluorescein angiography performedat
`1, 2, and 4 weeksafter laser photocoagulation demonstrated that
`ablation of either CD18 (blue bar) or ICAM1 (purple bar) resulted in
`Significantly fewer grade 2B lesions (those showing pathologically
`significant leakage) than were seen in wild-type mice (gold
`lbet)
`bar) (mean + SEM: n =5forall groups). Adapted from Sakurai E
`=°x
`Week2
`Week 4
`Taguchi H, AnandA,etal. Targeted disruption of the CD18 or
`(B)
`ICAM-1 geneinhibits choroidal neovascularization. Invest Ophthalmol!
`»=2)
`Vis Sci 2003;44:2743-2749,
`
`The role of FGF2 in ocular neovascular disease is not well defined.
`Elevated expression of FGF2 has been detected in CNV membranes
`from patients with AMD” and in epiretinal membranes from patients
`with proliferative DR.” However, exogenous administration of FGE2
`produced only subretinal neovascularization that did not penetrate
`Bruch’s membrane in an experimental model of CNV. Other studies
`foundthat transgenic mice with elevated retinal FGF2 expression devel-
`oped CNV following low-intensity laser (sufficient to disrupt photo-
`receptors but not Bruch’s membrane) while wild-type mice did not.”
`Taken together with studies demonstrating that genetic ablation of the
`FGF2 genedid notinhibit the formation oflaser-induced CNV,”these
`findings suggest that FGF2isin itself not sufficient to provoke CNV in
`the absence of an additional stimulus and that FGF2 mayalso not be
`required to induce CNV.
`
`TUMOR NECROSIS FACTOR-o (TNF-a)
`TNF-a is the prototypic member ofa superfamily of cytokines that
`mediate a variety of biological functions, signaling through a corre-
`spondingly large family of receptors.” Several studies have examined
`the role of TNF-c. as a mediatorof angiogenesis, but a unified picture
`is not yet apparent.
`TNF-o. has been found to stimulate angiogenesis in the corneas of
`rats® and rabbits.” It is notclear if these representdirect or indirect
`effects since TNF-a has been demonstrated to induce expression of
`VEGF” and VEGFR2"potently in cultured endothelial cells. TNEF-o.
`also upregulates the synthesis of other factors associated with angio-
`genesis, including angiopoietin 1 and angiopoietin 2as well as MMP2
`and MMP9.*
`
`a Sa
`
`e
`@Q=
`
`ob!
`
`Qoa
`
`26
`
`Several studies have assessed therole of TNF-o. signaling in angio-
`genesis. In ischemic-induced neovascularization in the limbs of mice,
`TNF-a wasessentialfor the mobilization andsurvival ofbone marrow-
`derived endothelial progenitorcells, induction of VEGF expression and
`collateral vessel development.® In another report, administration of
`infliximab (a monoclonal antibody to TNF-a)* or etanercept(a soluble
`TNF receptor fusion protein) both inhibited the size of laser-induced
`CNV in mice. Gene knockout studies, however, have been inconsis-
`tent; somestudies found a dependenceofretinal neovascularization on
`TNF-a function® whereas others did not.
`In clinical studies, elevated levels of TNF-o. have been found in
`fibrovascular membranes of patients with proliferative DR®’ and
`in surgically excised CNV membranes. Intriguingly,
`intravenous
`administration of infliximab for treatment of rheumatoid arthritis
`caused regression of CNV in patients with AMD”; moreover, intrave-
`nous infliximab also led to reductions in macular edemain patients
`with DME.” It is notclear if these effects of TNF-a are independent of
`its upregulation of VEGF:if separate pathwaysare involved, TNF-a
`inhibition aloneorin combination with VEGFinhibition could provide
`an additional therapeutic option.
`
`EPHS AND EPHRINS
`ee
`Ephs comprisea large family of receptortyrosine kinases that are acti-
`vated upon binding with their cognate membrane-bound ligands, the
`ephrins.””” EphrinAsare attached to the cell membrane by a glyco-
`sylphosphatidy] anchor while the ephrinBs have transmembrane and
`cytoplasmic signaling domains (Figure 4.7).The Ephs alsofall into two
`
`|
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Serre
`Page 08 of 19
`
`Exhibit 2084
`Page 08 of 19
`
`

`

`(a o
`
`an
`E
`g°°
`oc
`
`304
`mo
`a
`
`0.2
`
`0
`
`7 (clodronate)
`
`u
`
`P>0.5
`
`(D) 30
`
`=
`E 20
`8
`os
`
`z
`= 10
`a
`
`Clodronate-
`aes liposome
`
`:
`
`PBS-
`liposome
`.
`
`Clodronate-
`;
`
`
`
`
`
` BulyeyUlSBOUSIDSJIsege|NOILOAS
`liposome (kK)
`
`Normoxia hypoxia
`
`
`164 —>
`120 —>
`
` 188 —>|
`
`
`Figure 4.4 Monocytes contribute to pathological retinal neovascularization. In a retinopathy of prematurity model, postnatal day zero (PO)
`rats were maintained for 10 days in 80% oxygen, interrupted daily by 30 minutes in room air, followed by a progressive return to 80%
`Oxygen. This treatment led to an avascular retina. On P10, corresponding to study day 0 (D0), retinal revascularization was induced by
`maintaining the rats in room air for an additional 7 days (D7). (A-C) At D7, pathological neovascularization (PaNV; arrowsin A and B) was
`Significantly inhibited by treatment with clodronate liposomes compared to controlliposomes (n = 8 for both treatments; means + standard
`deviation). (D) Physiological neovascular area (PhRV) wasnotsignificantly affected by treatment with clodronateliposomes(P > 0.05).
`(E-J) Influx of monocytes was observed just before and during pathological neovascularization. (H-J) Monocytes were labeled with a
`fluorescein conjugated antibody to CD13 (E and H), while rhodamine-conjugated Concanavalin A was used to label the retinal vasculature
`and adherent leukocytes (F and |). As shown by superposition of these figures (panels G and J), the concanavalin A and CD13 staining
`co-localized, indicating that the adherent leukocytes were monocytes. (K) In cultured peripheral blood monocytes obtained from
`retinopathologic rats at D7, exposure to hypoxia (1% oxygen) led to marked increase in expression of vascular endothelial growth factor
`mRNA compared to exposure to normoxia (21% oxygen). PBS, phosphate-buffered saline. Scale bars: (A and B) 0.5 mm and (E-J) 50 um.
`Reproduced from Ishida S, Usui T, Yamashiro K,et al. VEGF164-mediatedinflammation is required for pathological, but not physiological,
`ischemia-inducedretinal neovascularization. J Exp Med 2003;198:483-489.
`
`Wild type
`
`Figure 4.5 Platelet-derived growth factor (PDGF)-B regulates
`the developmentof blood vessel walls. During blood vessel
`development, the nascent endothelial tube (yellow) is surrounded
`by undifferentiated mesenchymalcells (gray) which are induced to
`
`differentiateintovascularsmooth-musclecells(VSMC), andtoform
`
`a surrounding sheath (red). During further development of the
`vascular network, with concomitant growth and sprouting of blood
`vessels, PDGF-B derived from the endothelium further promotes
`VSMCproliferation and migration. These proliferative and migratory
`responses are reducedin mice in which PDGF-B or PDGFR-B have
`been genetically ablated, leading to defective coating of capillaries
`by pericytes, as well as to VSMC hypoplasiain larger vessels.
`Reproduced from Hellstrom M, Kalen M, Lindahl P, et al. Role of
`PDGF-B and PDGFR-betain recruitment of vascular smooth muscle
`Cells and pericytes during embryonic blood vessel formation in the
`mouse. Development 1999;126:3047-3055.
`
`Exhibit 2084
`
`Page 09 of 19
`
`PDGF-Bdriven
`VSMCproliferation
`and migration
`
`vSMC
`induction
`
`
`o~«@eG“”
`PDGF-B
`and migration
`
`Reduced vSMC
`proliferation
`
`PDGFR-B
`knock-out
`
`27
`
`Exhibit 2084
`Page 09 of 19
`
`

`

`
`
`
`
`
`
`$10}0e4JA8UJOPUeJOJOB4YIMOIHJeljayyOpuUyZJejnose/\ssiseueBolbuyJeINdOefHALdVHO
`
`
`
`
`
`
`
`
`
`
`
`PBS
`control
`
`Anti-VEGF
`aptamer
`
`Ant-PDGFR-B
`antibody
`
`Ant-VEGF aptamer +
`anti-PDGFR-6 antibody
`
`Figure 4.6 The role of platelet-derived growth factor (PDGF)-B on blood vessel growth and muralcell coverage in a corneal
`neovascularization model. (A) Endothelial cells were labeled by staining with lectin (green) and mural cells were stained with an antibody
`against smooth-muscle actin (red). Starting at 10 days following cornealinjury, mice received daily intraperitoneal injections of an anti-
`PDGFR-B antibody or phosphate-buffered saline (PBS), and were sacrificed at 20 days postinjury. Treatment with the anti-PDGF-B antibody
`led to reduced mural cell coverage compared to controls (arrow). Scale bar = 20 um. (B) Following induction of cornealinjury, mice received
`daily intraperitonealinjections of one of the following: PBS, a polyethylene-glycolated anti-vascular endothelial growth factor (VEGF) aptamer,
`an anti-PDGFR-B antibody, or both the anti-VEGF aptamer and the anti-PDGFR-B antibody. Neovasculature (green) was stained by
`fluorescein isothiocyanate-concanavalin A. Neovascularization wassignificantly reduced by the anti-VEGF aptamer compared with either
`PBSorthe anti-PEGFR-B antibody (P < 0.01); inhibition of both VEGF and PDGF-Bsignaling led to a furthersignificant reduction (P < 0.05),
`comparedto inhibition of VEGF signaling alone. Scale bar = 100 um. Adapted from Jo N, Mailhos C, Ju M, etal. Inhibition of platelet-
`derived growth factor B signaling enhancestheefficacy of anti-vascular endothelial growth factor therapy in multiple models of ocular
`neovascularization. Am J Pathol 2006; 168:2036-2053.
`
`broad groups, EphA and EphB, with the EphAs binding primarily,
`although notexclusively, to members of ephrinA subclass, while EphBs
`similarly tend to bind preferentially to ephrinB ligands.
`Owing tothe association of ephrins to cell