M~i:Mi❖Mt!i!f@t!SM
`Prevention of Experimental Choroidal
`Neovascularization With Intravitreal Anti-Vascular
`Endothelial Growth Factor Antibody Fragment
`
`Magdalena G. Krzystolik, MD; Mehran A. Afshari, MD; Anthony P. Adamis, MD;
`Jacques Gaudreault, PhD; Evangelos 5. Gragoudas, MD; Norman A. Michaud, MS;
`Wenjun Li, MS; Edward Connolly, BS; Charles A. O'Neill, PhD;Joan W. Miller, MD
`
`Oltlectlve: To evaluate the safety and efficacy of intra(cid:173)
`vitreal injections of an antigen-binding fragment of a re(cid:173)
`combinant humanized monoclonal antibody directed to(cid:173)
`ward vascular endothelial growth factor (rhuFab VEGF)
`in a monkey model of choroidal neovascularization
`(CNV).
`
`Methods: In phase 1 of the study, each animal re(cid:173)
`ceived intravitreal injections, 500 µg per eye, of rhuFab
`VEGF in one eye (prevention eye) , while the contralat(cid:173)
`eral eye received rhuFab VEGF vehicle (control eye) at
`2-week intervals. On day 21 , laser photocoagulation was
`performed to induce CNV. In phase 2, the vehicle(cid:173)
`treated eye was crossed over and both eyes received 500
`µg of rhuFab VEGF beginning 21 days following laser(cid:173)
`induced injury at days 42 and 56. The eyes were moni(cid:173)
`tored by ophthalmic examinations, color photographs,
`and fluorescein angiography.
`
`Results: rhuFab VEGF did not cause any ocular hem(cid:173)
`orrhages. All eyes treated with rhuFab VEGF developed
`
`acute anterior chamber inflammation within 24 hours of
`the first injection that resolved within 1 week, and this
`inflammation was less severe with subsequent injec(cid:173)
`tions. The incidence of CNV, defined angiographically,
`was significantly lower in the prevention eyes than the
`control eyes (P<.001). Subsequent treatments were as(cid:173)
`sociated with less leakage in eyes with established CNV
`that were crossed over from the control eyes to the treat(cid:173)
`ment eyes (P=.001).
`
`Conclusions: lntravitreal rhuFab VEGF injections pre(cid:173)
`vented formation of clinically significant CNV in cyno(cid:173)
`molgus monkeys and decreased leakage of already formed
`CNV with no significant toxic effects.
`
`eHnlcal Relevance: This study provides the nonclini(cid:173)
`cal proof of principle for ongoing clinical studies of in(cid:173)
`travitreally injected rhuFab VEGF in patients with neo(cid:173)
`vascular age-related macular degeneration.
`
`A rch Ophthalmol. 2002;120:338-346
`
`C HOROIDAL neovasculariza(cid:173)
`
`tion ( CNV) is the major
`cause of severe visual loss
`in patients with age-rela(cid:173)
`ted macular degenera(cid:173)
`tion (AMD). New blood vessels grow from
`the choroid and penetrate through the
`Bruch membrane into the subretinal pig(cid:173)
`ment epithelial and subretinal space. These
`vessels can leak and bleed, leading to exu(cid:173)
`dative retinal detachment and hemor(cid:173)
`rhage. Eventually, this process progresses
`to a fibrovascular scar with destruction of
`photoreceptors and vision loss.
`Until recently, the only beneficial treat(cid:173)
`ment demonstrated in clinical trials was la(cid:173)
`ser photocoagulation to obliterate the newly
`formed blood vessels. 1 However, this treat(cid:173)
`ment causes full-thickness retinal damage
`and, in the case of subfoveal lesions, leads
`to immediate loss of central vision. Most
`CNV lesions manifest in a subfoveal loca(cid:173)
`tion and are lesions that are frequently large,
`
`ill defined, or occult and do not qualify for
`laser photocoagulation.
`In an attempt to provide more selec(cid:173)
`tive and effective treatment for CNV in
`neovascular AMD , several innovative
`therapies have been studied, including
`photodynamic therapy (PDT) and agents
`that target angiogenesis. The goal of these
`treatments is to effectively treat CNV with(cid:173)
`out damaging adjacent retinal tissue or by
`inhibiting growth factor-mediated neo(cid:173)
`vascularization. In PDT, a photosensitiz(cid:173)
`ing drug is injected intravenously and ac(cid:173)
`tivated by a nonthermal laser light in
`affected vessels in the retina. Using the ap(cid:173)
`propriate drug dose, light dose, and tim(cid:173)
`ing of irradiation, relatively selective oc(cid:173)
`clusion of CNV vessels can be achieved
`with minimal effect on the retina. Several
`photosensitizing drugs have been stud(cid:173)
`ied, including verteporfin,2
`•3 lutetium
`texaphyrin,4 and tin ethyl etiopurpurin
`(SnET2/Purlytin).5 Verteporfin PDT (QLT
`
`From Retina Consultants,
`Providence, RI (Dr Krzystolik);
`Angiogenesis Laboratory and
`Laser Laboratory, Retina
`Research Institute, Department
`of Ophthalmology, Harvard
`Medical School, Massachusetts
`Eye and Ear Infirmary, Boston
`(Drs Afshari, Adamis,
`Gragoudas, and Miller and
`Messrs Michaud, Li, and
`Connolly); and
`Pharmacological Sciences,
`Genentech, Inc, South San
`Francisco, Calif
`(Drs Gaudreault and O'Neill).
`This work was supported in
`part by Genentech, Inc.
`
`( REPRINTED) ARC H OPHTHALMOL/VOL 120, MAR 2002
`338
`
`WWW.ARCHOPHTHALMO L.COM
`
`©2002 American Medical Association. All rights reserved.
`Downloaded From: https://j amanetwork.com/ by Andrew Calman on 10/18/2020
`
`Novartis Exhibit 228 1.001
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`MATERIALS AND METHODS
`
`FREQUENCY AND DOSING
`
`ANIMALS
`
`Ten cynomolgus monkeys (Macaca fascicularis), obtained
`from Covance Biomedical Products Inc, Alice, Tex, were
`used in accordance with the guidelines of Association
`for Research in Vision and Ophthalmology on the use of
`animals in research and according to the guidelines of
`the Animal Care Committee at the Massachusetts Eye
`and Ear Infirmary.
`Monkeys were anesthetized for all procedures with in(cid:173)
`tramuscular injections of ketamine hydrochloride, 20 mg/kg;
`acepromazine maleate, 0.125 mg/kg; and atropine sulfate,
`0.125 mg/kg. Supplemental anesthesia of 5 to 6 mg/kg of
`ketamine hydrochloride was administered as needed. In ad(cid:173)
`dition, 0.5% proparacaine hydrochloride was used for topi(cid:173)
`cal anesthesia. Supplemental anesthesia, with intravenous
`pentobarbital sodium solution (5 mg/kg), was adminis(cid:173)
`tered before enucleation. Animals were euthanized with
`an intravenous pentobarbital sodium veterinary euthana(cid:173)
`sia solution Q.A. Webster Inc, Sterling, Mass) adminis(cid:173)
`tered intravenously.
`
`ANTIANGIOGENIC DRUG INJECTIONS
`
`rhuFab VEGF was provided by Genentech Inc, South San
`Francisco, Calif. rhuFab VEGF was preserved in a lyophi(cid:173)
`lized powder form in a sterile vial and stored at 2°C to 8°C.
`The composition of the reconstituted rhuFab VEGF was 25
`mg/ml of rhuFab VEGF in lOmM histidine, 2.5% treha(cid:173)
`lose, and 0.01% polysorbate 20 (pH 5.5). The lyophilized
`powder was reconstituted in the vial before each use with
`sterile water for injection and physiologic buffer to yield a
`concentration of 10 µg/µL, which was confirmed by UV ab(cid:173)
`sorption. The control eye was injected with a vehicle con(cid:173)
`sisting of all components except the rhuFab VEGF protein.
`
`METHOD OF ADMINISTRATION
`
`Intravitreal injections of 50 µL per eye with either rhuFab
`VEGF or vehicle were performed on both eyes through the
`pars plana using a 30-gauge needle and tuberculin syringe
`after instilling topical anesthesia and 5% povidone iodine
`solution. Before each dose administration, the vial stop(cid:173)
`per was wiped with 70% alcohol and allowed to air dry.
`The drug was withdrawn through a 5-µm filter, and a new
`(sharp) 30-gauge needle was used for intraocular injec(cid:173)
`tion. After the injection, bacitracin ophthalmic ointment
`was instilled in the fomices. The injection sites were var(cid:173)
`ied to avoid trauma to the sclera. A 2-week interval was
`chosen based on previous toxicology studies. 15
`
`In phase 1, the right or left eye of each animal was ran(cid:173)
`domly assigned to receive intravitreal injections of rhuFab
`VEGF ata dose of 500 µg (50 µL per eye), and this eye was
`termed the prevention eye. The dose was based on previ(cid:173)
`ous toxicology studies.15 The fellow eye was assigned to in(cid:173)
`travitreal injections of rhuFab vehicle and was termed the
`control eye. All eyes received 2 intravitreal injections be(cid:173)
`fore laser treatment with either rhuFab VEGF or vehicle
`alone on days O and 14. On day 21, all eyes underwent ar(cid:173)
`gon green laser photocoagulation to induce CNV lesions.
`On day 28, 1 week after laser, the prevention eye received
`another injection of drug and the control eye received ve(cid:173)
`hicle. Phase 2 of the study began on day 4 2 or 3 weeks af(cid:173)
`ter laser induction, when CNV would be expected to have
`developed. Following fluorescein angiography on day 42 ,
`both eyes of each animal received intravitreal injections of
`rhuFab VEGF at a dose of 500 µg (50 µL per eye), and this
`was repeated on day 56 (Table 1 ) .
`
`INDUCTION OF EXPERIMENTAL CNV
`
`The CNV membranes were induced in the macula, an area
`between the temporal vascular arcades, of cynomolgus mon(cid:173)
`keys with argon green laser bums (Coherent Argon Dye
`Laser 920; Coherent Medical Laser, Palo Alto, Calif) using
`a slitlamp and piano fundus contact lens. Nine lesions were
`symmetrically placed in the macula of each eye by a masked
`surgeon (M.G.K. and M.A.A.). The laser variables in(cid:173)
`cluded a 50- to 100-µm spot size, 0.1-second duration, and
`power ranging from 350 to 700 mW. The power used was
`assessed by the ability to produce a blister and a small hem(cid:173)
`orrhage. If no hemorrhage was noted, an additional laser
`spot was placed adjacent to the first spot following the same
`laser procedure. Color photographs and fluorescein angi(cid:173)
`ography were used to detect and measure the extent and
`leakiness of the CNV.
`
`OCULAR EXAMINATIONS
`
`The eyes of the animals were checked for relative pupil(cid:173)
`lary afferent defect and then dilated with 2.5% phenyleph(cid:173)
`rine hydrochloride and 0.8% tropicamide. Both eyes were
`examined using slitlamp biomicroscopy and indirect oph(cid:173)
`thalmoscopy on days 0, 14, 28, 42, and 56 (before drug in(cid:173)
`jection); days 1, 15, 29, 43, and 57 (24 hours after injec(cid:173)
`tion) ; day 21 (before laser); days 35 and 49 (intermediate
`days); and day 63 (enucleation and death).
`
`Continued on next page
`
`Phototherapeutics, Inc, Vancouver, British Columbia),
`the only approved drug for PDT to date, has been shown
`to reduce the risk of moderate vision loss in patients with
`subfoveal CNV, particularly those with predominantly
`classic CNV6•7; however, the recurrence rate and the num(cid:173)
`ber of required treatments are high and not all subfoveal
`lesions benefit from treatment.
`A different approach to the treatment of ocular neo(cid:173)
`vascularization is antiangiogenic therapy. Angiogenesis
`refers to a process of new blood vessel formation that in(cid:173)
`volves a complex interaction of different factors that can
`
`be either stimulatory or inhibitory and includes growth
`factors, extracellular matrix elements, and intracellular
`or cellular adhesion molecules. Some of these factors have
`been shown to be associated with CNV. Antiangiogenic
`agents inhibit neovascularization either by promoting the
`action of endogenous inhibitors of angiogenesis or by
`blocking the effect of angiogenic stimulators.
`One of the potential targets for antiangiogenic therapy
`is vascular endothelial growth factor (VEGF), which is a
`secreted polypeptide with mitogenic effects on vascular en(cid:173)
`dothelial cells.8 It has been shown to be present in surgi-
`
`(REPRINTED) ARCH OPHTHALMOL/VOL 120, MAR 2002
`339
`
`WWW.ARCHOPHTHALMOL.COM
`
`©2002 American Medical Association. All rights reserved.
`Downloaded From: https://jamanetwork.com/ by Andrew Calman on 10/18/2020
`
`Novartis Exhibit 2281.002
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`COLOR PHOTOGRAPHY AND FLUORESCEIN
`ANGIOGRAPHY
`
`Fundus photography was performed on all animals on the
`same days as the ocular examination except for days 28 and
`56. Photographs were obtained with a fundus camera
`( Canon Fundus CF-60Z; Canon USA Inc, Lake Success, NY)
`and 35-mm film.
`The Imagenet Digital Angiography System (Topcon
`501 A and Imagenet system; Topcon America Corp, Para(cid:173)
`mus, NJ) was used for fluorescein angiography. Red-free
`photographs of both eyes were obtained followed by fluo(cid:173)
`rescein angiography using 0.1 mllkg of bodyweight of 10%
`sodium fluorescein (Akom Inc, Abita Springs, La) at a rate
`of 1 mUs. Following the fluorescein injection, a rapid se(cid:173)
`ries of images were obtained in the first minute of the pos(cid:173)
`terior pole of first the right eye and then the left eye. Ad(cid:173)
`ditional pairs of images were obtained at approximately 1
`to 2 and 5 minutes. Between 2 and 5 minutes, 2 images of
`the midperipheral fields (temporal and nasal) were taken
`of each eye. Fluorescein angiography was performed at base(cid:173)
`line (day 0) and days 7, 14, 29, 42, 49, 57, and 63.
`
`ANALYSIS OF OPHTHALMIC DATA
`
`Photographs and angiograms were evaluated for evidence of
`angiographic leakage, hemorrhages, or any other abnormali(cid:173)
`ties. The fundus hemorrhages were graded based on a grad(cid:173)
`ing system with retinal hemorrhages that involved less than
`3 disc areas defined as grade 1, hemorrhages between 3 and
`6 disc areas defined as grade 2, and hemorrhages of more than
`6 disc areas defined as grade 3. The association of hemor(cid:173)
`rhages with CNV membranes or laser induction site was also
`assessed. Clinically significant bleeding was defined as any
`fundus hemorrhage greater than or equal to a 6-disc area.
`Ocular inflammation was assessed by slitlamp biomi(cid:173)
`croscopy. Anterior chamber and vitreal cells were counted
`with a 2-mm slitlight at a high magnification and graded
`using the schema of the American Academy of Ophthal(cid:173)
`mology (Table 2) .
`The CNV lesions were graded by reviewing fluores(cid:173)
`cein angiograms performed on days 35, 42, 49, 56, and 63
`by 2 masked and experienced examiners (E.S.G. and].W.M.)
`who graded by consensus opinion. The CNV lesions were
`graded according to the following scheme, using standard(cid:173)
`ized angiographs for comparison. Grade 1 lesions had no
`hyperfluorescence. Grade 2 lesions exhibited hyperfluo(cid:173)
`rescence without leakage. Grade 3 lesions showed hyper(cid:173)
`fluorescence in the early or midtransit images and late leak(cid:173)
`age. Grade 4 lesions showed bright hyperfluorescence in
`the transit and late leakage beyond the treated areas. Grade
`4 lesions were defined as clinically significant.
`
`Statistical analysis was performed using the Population(cid:173)
`Aggregated Panel Data with Generalized Estimating Equa(cid:173)
`tions and the incidence rate ratio (IRR). The incidence rate
`was defined as the number of grade 4 lesions that oc(cid:173)
`curred during a given interval divided by the total number
`oflesions induced. In phase 1, the IRR referred to the ratio
`of incidence rate of grade 4 lesions in the prevention eyes
`to the incidence rate in control eyes. An IRR of 1 would
`signify no difference between incidence rates . A number
`much smaller than 1 would indicate a reduction in the in(cid:173)
`cidence of grade 4 lesions in the prevention group vs con(cid:173)
`trol group. In phase 2, we compared the incidence of grade
`4 lesions in the control eyes vs the treatment eyes. This
`means that the incidence of grade 4 lesions was compared
`over time in the set of eyes that were first assigned to the
`control group but on days 42 and 56 were treated with
`rhuFab VEGF and became treatment eyes.
`
`SERUM PHARMACOKINETICS
`AND ANTIBODY ANALYSIS
`
`Blood (approximately 2 ml) was collected from a lower(cid:173)
`extremity vein before rhuFab VEGF injection and approxi(cid:173)
`mately 24 hours and 7 days after the injections. All samples
`were maintained at room temperature and allowed to clot,
`then chilled until centrifuged within 1 hour of blood col(cid:173)
`lection. Serum was transferred to 1.5-mL conical tubes and
`stored at -60°C to -80°C.
`Pharmacokinetics analysis of rhuFab VEGF was
`performed using the rhuFab VEGF antigen enzyme-linked
`immunosorbent assay method. Antibody analysis was
`performed using the anti-rhuFab VEGF antibody enzyme(cid:173)
`linked immunosorbent assay method.
`
`HISTOPATHOLOGIC ANALYSIS
`
`The globes were carefully removed from each animal, dis(cid:173)
`sected clean of orbital tissue, rinsed in isotonic sodium chlo(cid:173)
`ride solution, and placed in modified Karnovsky fixative
`consisting of 2% glutaraldehyde and 2.5% formaldehyde
`in 0.lM cacodylate buffer 7.4 on ice. Within 10 minutes,
`the globes were opened and the anterior segment re(cid:173)
`moved and the posterior pole placed in fixative overnight
`and then changed to buffer (0.lM cacodylate) until pro(cid:173)
`cessed for light microscopy.
`Each eye was prepared for light microscopy by section(cid:173)
`ing into blocks, which contained lesions of interest. Tissues
`were postfixed in 2% osmium tetroxide in 0.lM cacodylate
`buffer for 2 hours at room temperature then dehydrated in a
`series of ethanols, infiltrated with propylene oxide and Epon,
`and embedded in Epon. Blocks were cut for 1-µm sections
`and stained with 0.5% toluidine blue in borate buffer.
`
`cally excised human CN\19.1° and in the aqueous and vit(cid:173)
`reous humor of patients with retinal neovascular disorders,
`such as diabetic retinopathy and retinal vein occlusion.11
`Antibodies to VEGF have been shown to inhibit neovas(cid:173)
`cularization in an experimental model of retinal ischemia
`and iris neovascularization in cynomolgus monkeys. 12
`We wished to study the effects of intravitreal injec(cid:173)
`tions of an antigen-binding fragment of a recombinant
`humanized monoclonal antibody directed toward VEGF
`(rhuFab VEGF) in a monkey model oflaser-induced CNV.
`
`rhuFab VEGF is the Fab portion (the antigen(cid:173)
`binding portion) of anti-VEGF monoclonal antibody. It is
`a recombinant antibody that consists of 2 parts: a nonbind(cid:173)
`ing human sequence, which makes it less antigenic in pri(cid:173)
`mates, and a high-affinity binding epitope derived from the
`mouse, which serves to bind the antigen.13 Its molecular
`weight of 48000 makes it a much smaller molecule than
`the full-length monoclonal antibody with a molecular weight
`of 148000.13 Unlike the full-length antibody, rhuFab VEGF
`has been shown to penetrate the internal limiting mem-
`
`(REPRINTED) ARCH OPHTHALMOL/VOL 120, MAR 2002
`340
`
`WWW.ARCHOPHTHALMOL.COM
`
`©2002 American Medical Association. All rights reserved.
`Downloaded From: https://jamanetwork.com/ by Andrew Calman on 10/18/2020
`
`Novartis Exhibit 2281.003
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`Table 1. Experimental Design*
`
`Day
`0
`7
`21
`28
`42
`56
`63
`
`Control/Treatment Eye
`PrevenUon Eye
`rhuFab VEGF (500 µg per eye) Vehicle (50 µL per eye)
`rhuFab VEGF
`Vehicle
`Laser
`Laser
`rhuFab VEGF
`Vehicle
`rhuFab VEGF (500 µg per eye)
`rhuFab VEGF
`rhuFab VEGF
`rhuFab VEGF
`Enucleation
`Enuclealion
`
`*rhuFab VEGF indicates recombinant humanized monoclonal antibody
`fragment directed toward vascular endothelial growth factor.
`
`Table 2. Anterior Chamber Inflammation Grading System*
`
`Grade
`0
`1+
`2+
`3+
`4+
`
`Definition
`No inflammatory cells
`Up to 1 O inflammatory cells
`10-20 cells
`20-30 cells
`Too numerous to count
`
`*American Academy of Ophthalmology. Basic Clinical Science Course.
`American Academy of Ophthalmology; 1995;9:86.
`
`brane and access the subretinal space in animal models when
`injected intravitreously.13·H Therefore, rhuFab VEGF po(cid:173)
`tentially offers better retinal and choroidal distribution and
`a better therapy than its full-length antibody counterpart.
`The purpose of this study was to assess the safety
`and efficacy of intravitreal injections of rhuFab VEGF in
`the laser-injury CNV model. This model uses argon green
`laser to induce CNV in the monkey macula and has been
`used in the past to study PDT. We have previously shown
`that there is a good correlation between fellow eyes in
`the number of CNV lesions with significant angio(cid:173)
`graphic leakage (M.G.K., unpublished data, December
`1999) and therefore designed a study using the contra(cid:173)
`lateral eyes as controls. Safety and efficacy were evalu(cid:173)
`ated in 2 phases of the study. Phase 1, the prevention
`phase, called for the initiation of rhuFab VEGF treat(cid:173)
`ment before laser induction of the CNV and 1 week af(cid:173)
`ter laser to inhibit the formation of CNV, which typi(cid:173)
`cally appears by 2 to 3 weeks after laser injury. Phase 2,
`the treatment phase, began on day 42 or 3 weeks after
`laser when CNV lesions would be expected in the con(cid:173)
`trol eyes from phase 1. Therefore, in phase 2 the effect
`of rhuFab VEGF treatment on attenuating the extent and
`leakiness of existing CNV lesions was assessed.
`
`RESULTS
`
`SAFETY OF INTRA VITREAL
`rhuFab VEGF INJECTIONS
`
`Clinical examination and review of fundus photographs did
`not show any hemorrhages before the laser photocoagu(cid:173)
`lation, with the exception of one eye that developed a mild
`vitreous hemorrhage after the first intravitreal injection
`through the pars plana. This eye was hypotonus before in(cid:173)
`travitreal injection due to a paracentesis for aqueous hu-
`
`Table 3. Total Number of Fundus Hemorrhages
`for All Animals According to Grade
`
`Grade 1
`(< 3 Disc Areas)
`0
`5
`6
`7
`2
`0
`
`Grade 2
`(3·6 Disc Areas)
`0
`1
`1
`0
`0
`0
`
`Grade 3
`(> 6 Disc Areas)
`1
`0
`0
`0
`0
`0
`
`Prelaser days
`Day 21 (laser)
`Day28
`Day35
`Day42
`Days 56 and 63
`
`Table 4. Anterior Chamber (AC) Cellular Inflammatory
`Response After Recombinant Humanized Monoclonal
`Antibody Directed Toward Vascular Endothelial Growth
`Factor (rhufab VEGF) and Vehlcle lntravitreal Injections
`
`Prevention Eye
`
`Control/Treatment Eye
`
`Injection
`rhuFab VEGF
`
`rhuFab VEGF
`
`rhuFab VEGF
`
`rhuFab VEGF
`
`rhuFab VEGF
`
`AC Cells
`
`1 to 4+
`0 to trace
`
`t to 2+
`o to trace
`
`0 to 3+
`
`Oto 2+
`0
`
`Oto 3+
`0
`
`Injection
`Vehicle
`
`Vehicle
`
`Vehicle
`
`rhuFab VEGF
`
`rhuFab VEGF
`
`AC Cells
`
`o to 1+
`0
`
`0
`0
`
`Oto trace
`
`3+ to 4+
`0
`
`Oto 3+
`0
`
`Day
`0
`1
`7
`14
`15
`21
`28
`29
`42
`43
`49
`56
`57
`63
`
`mor collection. The hemorrhage resolved within the next
`2 weeks and did not recur with the future injections. Within
`1 week of the laser, retinal hemorrhages were seen asso(cid:173)
`ciated with laser injury sites as expected both in the rhuFab
`VEGF- and vehicle-injected eyes (Table 3 ). On day 28
`(1 week after laser), there were 6 grade 1 hemorrhages ob(cid:173)
`served in all animals. There was only 1 grade 2 hemor(cid:173)
`rhage noted at this time, and by day 35 (1 week later) , this
`hemorrhage was less than 3 disc areas and became grade
`1. All of these hemorrhages resolved within the next 4
`weeks. No retinal or choroidal hemorrhages were noted as(cid:173)
`sociated with intravitreal rhuFab VEGF or vehicle injec(cid:173)
`tions in either prevention or treatment eyes.
`All eyes treated with rhuFab VEGF developed acute
`anterior chamber cells within 24 hours of the first intra(cid:173)
`vitreal injection. As given in Table 4 , prevention eyes
`developed 1 to 4+ cells on day 1 after the drug injection.
`Inflammation resolved within 1 week (day 7) . Subse(cid:173)
`quent injections produced less inflammation when eyes
`were examined 24 hours later (days 15, 29, 43, and 57).
`Eyes injected with vehicle showed minimal or no inflam(cid:173)
`mation. However, on day 42 (in phase 2 of the study),
`control eyes were crossed over to receive rhuFab VEGF,
`and these eyes developed 3+ to 4+ anterior chamber cells
`within 24 hours. Following the second administration
`of rhuFab VEGF to these eyes (at day 56), inflammation
`
`(REPRINTED) ARCH OPHTHALMOL/VO L 120 , MAR 2002
`341
`
`WWW.ARCHO PHTHALMOL.COM
`
`©2002 American Medical Association. All rights reserved.
`Downloaded From: https://jamanetwork.com/ by Andrew Calman on 10/18/2020
`
`Novartis Exhibit 2281.004
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`IDI\$ 2 mkS il~er asu I'1Clllctlon Oil~ 35. A, c«i. 41 eye (reeom'birlan1
`m un 1. Post.erl11t hPidUS ootor phOl;OOrapliS duml!Jnstr.ittnci Ch()1ol!ilal nevva,:st~r
`lllowtil iador ll'llllfa.iJ, VEGF) veflioM): II, 1111•1•t11nion IS')'fl (rtmFab \liEGf}.
`numl!11k!Dd m1111oclona1 iUlti!Kldy dlffJCtN ttJwaJ'd vastU
`01100\hali
`
`flg;un 2, 'Fluorcsceil! anulo11r.1m of Iha samunlm as in fllliml 1 on It 35 dnmo11slra~rio laset-lnducl!d IMloos" A, Ead)' trame irrlli10, lltlf11rll1 O'JfJ jf!oCOfl'.li)lnanl
`~r(l'Mf] l'ador llfllJfu, VEGF) velllcle), 8, eait, lrame of 11le Pffl'B111XH'1 ')'B (lli!!M VEGF), C,
`humanl\l~d JllOC10Cl01iil a111ilMKl)r dlreotecl wwa:rd V'il!iCU
`e11ilolhlll
`. ·e, fta;111t ol Ue coml I/I'll' (rlwfall VEGJ vellfdel, 0, latt! lra_me or Ill~ prel'~nOOll ")'B (lll!!f@• V-El.lF), Lab lratnes ol llie ll!Klresll\1'1 anglo11ram dem{ilfl$1Qt,
`p!ffl"°° 01 orMe 4 leSIOM In 1119 ~nl!NII eye l,ul no, In Ill~ Jlffll'Gnli0ll l!'j4!.
`
`was less pmoounced. o other cliniral or angiographic
`abnormalities wue observe
`
`FA
`Y F NTRA lTREAL
`rh1.tf'ab VEGF iNJ no
`
`Flm;m:scein an:giogra.ms of bolh eyes in each .anim.31 were
`evaluated a~oording to the gr-a.ding systmi. described in
`1h "Mruerialsand .. ff:thods~ section for phase l mid phase
`
`2. Examplr.s or die photographk and. angjogaphic ap
`prarance or paired e es in phase 1 and 2 ar; shown in
`N 9uM1 1 , 2 , aod 3 ..
`- Ln phase l , a.nalys of the C V ~
`lo - ac days
`__ nd 4 2 (2 and 3 wee after I ·er lnduclion) h ved
`reduclion in the likelihood of reachim grade' 4 leakage
`(P<.001) in the rbu ab VEG prevention group com•
`pared with I.he vehicle control gc-oup (Pl1v.,. 4 ). be
`lR.R for the pr-EYenlion group :md the control gxou p was
`
`CREl'RL'(l'ED) ARO! OJ"l-11l1A11- LIVOL lln. MAil. 20111
`141
`
`WWW,ARCHOrl:fTIIALMOL.OOM
`
`lkn\•.oludNI Frum: hl~ps::Jflam ■ nm r'.Ju11ml by Andn:w Oalm■n on llll8/2020
`
`Novartis Exhibit 2281.005
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`fl g;ure a. ;fJu!lfflce111 .inukl11ram o1 lhe wme anlm as Ill 1F1111ne 7111 lbe ~me 111 enucleatioo (d'a', 63), A, Eatt, ffil/Tl111 i,f tti~ llOOlr01 ~ lreoomll 111111 hulllilllze!I
`Jell U!\'1J!I ~!liar e'!ld~e _ IIIVl'#fll fattQr [rl\11~!1 VEGFJ ~ 0!'0$$Gd ovu 1(1 iflYFi!l! v.EGF 1reit11lOm). 8. Ealt; rr.Jlle Of 1li~
`mo,u~cl1J1~l ~~bo(!y
`PIMl11i!ln ~ lll1UFab Vl:GF}. C. latll liam!i or Ill~ CCilllf01 Jjll (lllUFa.11 Vt(lf \'O~ic[e CFl!ISffll 0/ltf tll rllufab Val'F lraalm.,1). D. Lalo lrama or ltle ~ntklfl 11)'9
`? frames oJ 11111 ll111Jiftsen1 11111io11rarn demons1Rte decreaed le
`In bolfl rres.
`(rllll
`VEGf),
`
`lC
`
`11g~[
`
`JO
`
`C\!nllOll'TttlUIJtnl
`I
`
`g
`3211
`
`it .. ....
`
`~ 10
`!
`
`0
`
`iii$
`
`0
`
`3S
`
`a
`
`li!i
`
`1,31
`
`numtJeJ or arade 4 e11or0illal naovasmw jC-W\Q
`fla:un. •·· Pl:llsa • T
`leslDM In 1118 prBW11ll0n ~ roop (reco111b'lnan1 llum;ml-lad rno111ocional
`r,ec ed f(IINird 1/\llSClulir endolll
`I growtll rai:oor [rl!ufill \IECifJ)
`il!Ulbod1
`(empty barJ Vii COfltrol fllulfatJ VlGh>ielliel~HsNIJell barJ 2 wuell$ ill$r
`lase indtJclioo (da')l 3S) ilfld a wookul'I
`Ille laser lnllucllon (day 42).
`
`0.041 (95% OOHfidence interval, 0·.009-0.176), imlical•
`iog that intravitreal rhuFab _ EG mjeetioos pre"e:med
`ornmtion oklinically significant C
`. Th - was no lat-
`-ra11 ,.cl)'. Cl (P- .4-S) b l\ -en th ·e}
`In ph _ e 2, all c
`in the oonlrol gr up wcr
`inJc l with 500 µg o[ rhu 11b VEG on da , 42 and
`56, and th~ eyes became the crossov :r or Lrealme:nt
`group. The number oJ gradf 4 lesiom: in the cont:Jlolltreat(cid:173)
`men t g.roup is presen1e:d in F11u•• 9 . lJ.sing the
`
`Ffa11115. l'NSll 2. f!!lal 1n~11I l\!d$418mMinlheC011il'IJl(ffflOfflbi1U11C
`hu1111111zed1 m1111odof1111 ~rrtloody mirected wward ,ra,sQJiar emilioilleilal growlll
`VEGf) owr m The
`lictor lrllufBII IIEGfJ ,·.e~~t o•oup tmu
`ce,-rci ey$ '.\'la$ ~Jecttll -,,iitJ; rtlufall \!OOf vthi;le umll' Gal" .f2.. Mer II orvscelO
`~rannor.1s polforme'II en dll'! 42, Ill 8'j'fl& in tllls ·~rilllp wtmt lojtll;l?d
`111 vll!de 4 leslo.ns ow,
`rtrufa!J, '111:Clif, The baJ ~rapn dem::inilr[tes ttle· numb
`lectiOffl.
`befor11iPClatlllrrlMIMVE<if
`
`populmion~egllled pam::I darn mode]. Lhe chance or
`b ing classill d gntde 4 on days 49, 56, nd 63 {tt
`l(cid:173)
`m nl cy ) compared with da
`3 and 42 (c nu-ol
`eyes} was II essed. The nil of radc 4
`too oc.cu·r•
`~nee was !'!;OOc:-eci in Lbe treattnenl group and was sm(cid:173)
`dsticalJy signilk:anl (P ... 001~ lRR .. 0.074; 95% confi(cid:173)
`dence interval. 0.032-0.174) , indicating decreased
`
`CREl'RL'(l'ED) ARO! OJ"l-lTI-IAU LIVOL lln. MAil. 20111
`kl
`
`WWW,ARCHOrl:fTIIALMOL.OOM
`
`lkn\•.oludNI Frum: hl~ ps::Jflam ■ nm r'.Ju11ml by Andn:w Oalm■n on llll8/2020
`
`Novartis Exhibit 2281.006
`Regeneron v. Novartis, IPR2021-00816
`
`

`

`Table 5. Summary of Histopathologic Data on Selected Choroidal Neovascularization Laser-Induced Lesions
`
`Lesion No.
`(Animal No.)
`1 (4511)
`2(4511)
`3(4511)
`
`Fluoresceln
`Anglogram Grade
`
`Eye Group
`Prevention
`ControVtreated
`ControVtreated
`
`Day 35 Day63
`2
`2
`2
`2
`4
`2
`
`Width, Height,
`pm
`pm
`300
`0
`500
`0
`560
`80
`
`No. of
`Capillaries
`0
`0
`4
`
`No. of
`Aclnl
`0
`0
`0
`
`No. of
`Macrophages
`12
`22
`27
`
`Retinal Pigment
`Epithelial Coverage
`Unable to assess
`Unable to assess
`Yes
`
`Subretinal
`Fluid
`No
`No
`No
`
`800
`
`0
`
`400
`..,
`j 200
`t 100
`.,,
`50
`i
`2l
`<:
`0 u
`j
`"' ~
`'€
`
`0 ......
`
`.....
`
`0
`
`14
`
`15
`
`1 • rhufab VEGF Conctntrations in the Animal I
`With Antibodies Against r!lufab VEGF
`• rhufab VEGF Concentrations
`
`0
`
`'!'
`:
`
`0
`0
`
`0
`
`0
`
`0
`
`L
`
`0
`
`0
`0
`git
`
`0
`0
`
`!
`
`•
`
`0
`
`0
`
`""
`"'
`
`•
`
`0
`
`g>
`• 0
`"'
`.,
`
`0
`
`0
`
`..
`
`0
`
`.._
`28
`
`•
`"""'
`42
`
`29
`Day
`
`....
`
`56
`
`43
`
`omo
`
`57
`
`63
`
`was also assessed as grade 2 on the angiogram, but came
`from an eye in the control/treatment group. Lesion 3 is
`from a control eye in the prevention phase that devel(cid:173)
`oped a grade 4 lesion with profound angiographic leak(cid:173)
`age 3 weeks after laser induction but then decreased leak(cid:173)
`age to grade 2 after treatment with rhufab VEGF. At the
`end of the study, this treated lesion was smaller than the
`typical untreated CNV lesions studied previously in the
`same model (M.G.K., unpublished data, December 1999).
`Within the lesion there were few capillaries and pigment(cid:173)
`laden macrophages, with the occasional fibroblast, but
`the lesion was small and covered by retinal pigment epi(cid:173)
`thelium (Figure 7B).
`
`COMMENT
`
`Intravitreal injections of 500 µg of rhufab VEGF admin(cid:173)
`istered every 2 weeks in a laser-induced CNV model in
`10 cynomolgus monkeys showed no significant toxic ef(cid:173)
`fects and prevented formation of clinically significant
`CNV. The results also suggested that rhuFab VEGF may
`have a beneficial effect in treating established CNV as seen
`in neovascular AMD.
`In our study, transient anterior chamber inflamma(cid:173)
`tion that resolved without any sequelae and retinal hem(cid:173)
`orrhages associated with laser induction were observed
`as expected and reabsorbed over several weeks. A pre(cid:173)
`vious study of rhuFab VEGF injection in normal cyno(cid:173)
`molgus monkey eyes showed a similar safety profile re(cid:173)
`garding ocular inflammation.15 In that study, intravitreal
`administration of rhuFab VEGF every 2 weeks into eyes
`of otherwise untreated monkeys showed transient in(cid:173)
`flammation at doses up to 2000 µg per eye, doses that
`were higher than the levels used in our study. Perivas(cid:173)
`cular lesions that had been reported at the high doses in
`some animals were not observed in this study. This could
`also be related to the longer dosing interval of 13 weeks
`as opposed to 9 weeks in our experiment. Additionally,
`O'Neill et al15 showed that intravitreal administration of
`rhuFab VEGF had no effect on electroretinographic vari(cid:173)
`ables, including visual evoked potential.
`The phase 1 part of the study indicated that
`rhuFab VEGF prevented the formation of angiographi(cid:173)
`cally leaking CNV. Three of 10 animals did not develop
`grade 4 lesions in either eye. However, all the other 7
`animals showed significantly fewer grade 4 lesions in
`the eyes receiving rhuFab VEGF then the eyes receiving
`vehicle.
`The phase 2 part of the study suggests a treatment
`benefit for established CNV lesions. Three weeks after
`laser induction, eyes previously in the control group were
`
`Figure 6. Recombinant humanized monoclonal antibody directed toward
`vascular endothelial growth factor (rhufab VEGF] serum levels detected over
`time. The box plot illustrates that the rhufab VEGF levels increase 1 day after
`injection but, generally, decrea