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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 S. Gragoudas, MD;
Norman A. Michaud, MS;
Wenjun Li, MS;
Edward Connolly, BS;
Charles A. O'Neill, PhD;
Joan W. Miller, MD
Arch Ophthalmol. 2002;120:338-346.
ABSTRACT
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Objective To evaluate the safety and efficacy of intravitreal injections of an
antigen-binding fragment of a recombinant humanized monoclonal antibody directed
toward vascular endothelial growth factor (rhuFab VEGF) in a monkey model
of choroidal neovascularization (CNV).
Methods In phase 1 of the study, each animal received intravitreal injections,
500 µg per eye, of rhuFab VEGF in one eye (prevention eye), while the
contralateral 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-treated eye was crossed over and both eyes received 500 µg
of rhuFab VEGF beginning 21 days following laser-induced injury at days 42
and 56. The eyes were monitored by ophthalmic examinations, color photographs,
and fluorescein angiography.
Results rhuFab VEGF did not cause any ocular hemorrhages. 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 injections. The incidence of CNV, defined
angiographically, was significantly lower in the prevention eyes than the
control eyes (P<.001). Subsequent treatments were
associated with less leakage in eyes with established CNV that were crossed
over from the control eyes to the treatment eyes (P
= .001).
Conclusions Intravitreal rhuFab VEGF injections prevented formation of clinically
significant CNV in cynomolgus monkeys and decreased leakage of already formed
CNV with no significant toxic effects.
Clinical Relevance This study provides the nonclinical proof of principle for ongoing clinical
studies of intravitreally injected rhuFab VEGF in patients with neovascular
age-related macular degeneration.
INTRODUCTION
CHOROIDAL neovascularization (CNV) is the major cause of severe visual
loss in patients with age-related macular degeneration (AMD). New blood vessels
grow from the choroid and penetrate through the Bruch membrane into the subretinal
pigment epithelial and subretinal space. These vessels can leak and bleed,
leading to exudative retinal detachment and hemorrhage. Eventually, this process
progresses to a fibrovascular scar with destruction of photoreceptors and
vision loss.
Until recently, the only beneficial treatment demonstrated in clinical
trials was laser photocoagulation to obliterate the newly formed blood vessels.1 However, this treatment 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 location and are lesions
that are frequently large, ill defined, or occult and do not qualify for laser
photocoagulation.
In an attempt to provide more selective 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 without damaging adjacent retinal
tissue or by inhibiting growth factor–mediated neovascularization. In
PDT, a photosensitizing drug is injected intravenously and activated by a
nonthermal laser light in affected vessels in the retina. Using the appropriate
drug dose, light dose, and timing of irradiation, relatively selective occlusion
of CNV vessels can be achieved with minimal effect on the retina. Several
photosensitizing drugs have been studied, including verteporfin,2-3
lutetium texaphyrin,4 and tin ethyl etiopurpurin
(SnET2/Purlytin).5 Verteporfin PDT (QLT 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 number of required treatments are high
and not all subfoveal lesions benefit from treatment.
A different approach to the treatment of ocular neovascularization is
antiangiogenic therapy. Angiogenesis refers to a process of new blood vessel
formation that involves 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 endothelial cells.8 It
has been shown to be present in surgically excised human CNV9-10
and in the aqueous and vitreous humor of patients with retinal neovascular
disorders, such as diabetic retinopathy and retinal vein occlusion.11 Antibodies to VEGF have been shown to inhibit neovascularization
in an experimental model of retinal ischemia and iris neovascularization in
cynomolgus monkeys.12
We wished to study the effects of intravitreal injections of an antigen-binding
fragment of a recombinant humanized monoclonal antibody directed toward VEGF
(rhuFab VEGF) in a monkey model of laser-induced CNV.
rhuFab VEGF is the Fab portion (the antigen-binding portion) of anti-VEGF
monoclonal antibody. It is a recombinant antibody that consists of 2 parts:
a nonbinding human sequence, which makes it less antigenic in primates, and
a high-affinity binding epitope derived from the mouse, which serves to bind
the antigen.13 Its molecular weight of 48 000
makes it a much smaller molecule than the full-length monoclonal antibody
with a molecular weight of 148 000.13
Unlike the full-length antibody, rhuFab VEGF has been shown to penetrate the
internal limiting membrane and access the subretinal space in animal models
when injected intravitreously.13-14
Therefore, rhuFab VEGF potentially 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 angiographic leakage
(M.G.K., unpublished data, December 1999) and therefore designed a study using
the contralateral eyes as controls. Safety and efficacy were evaluated in
2 phases of the study. Phase 1, the prevention phase, called for the initiation
of rhuFab VEGF treatment before laser induction of the CNV and 1 week after
laser to inhibit the formation of CNV, which typically 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 control 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.
MATERIALS AND METHODS
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 intramuscular 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 addition, 0.5% proparacaine
hydrochloride was used for topical anesthesia. Supplemental anesthesia, with
intravenous pentobarbital sodium solution (5 mg/kg), was administered before
enucleation. Animals were euthanized with an intravenous pentobarbital sodium
veterinary euthanasia solution (J.A. Webster Inc, Sterling, Mass) administered
intravenously.
ANTIANGIOGENIC DRUG INJECTIONS
rhuFab VEGF was provided by Genentech Inc, South San Francisco, Calif.
rhuFab VEGF was preserved in a lyophilized 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 10mM histidine, 2.5% trehalose, 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 absorption.
The control eye was injected with a vehicle consisting 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 stopper 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
injection. After the injection, bacitracin ophthalmic ointment was instilled
in the fornices. The injection sites were varied to avoid trauma to the sclera.
A 2-week interval was chosen based on previous toxicology studies.15
FREQUENCY AND DOSING
In phase 1, the right or left eye of each animal was randomly assigned
to receive intravitreal injections of rhuFab VEGF at a dose of 500 µg
(50 µL per eye), and this eye was termed the prevention eye. The dose
was based on previous toxicology studies.15
The fellow eye was assigned to intravitreal injections of rhuFab vehicle and
was termed the control eye. All eyes received 2 intravitreal injections before
laser treatment with either rhuFab VEGF or vehicle alone on days 0 and 14.
On day 21, all eyes underwent argon 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 vehicle. Phase 2 of the study
began on day 42 or 3 weeks after 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).
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Table 1. Experimental Design*
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INDUCTION OF EXPERIMENTAL CNV
The CNV membranes were induced in the macula, an area between the temporal
vascular arcades, of cynomolgus monkeys with argon green laser burns (Coherent
Argon Dye Laser 920; Coherent Medical Laser, Palo Alto, Calif) using a slitlamp
and plano 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
included 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 hemorrhage. 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 angiography were used to detect
and measure the extent and leakiness of the CNV.
OCULAR EXAMINATIONS
The eyes of the animals were checked for relative pupillary afferent
defect and then dilated with 2.5% phenylephrine hydrochloride and 0.8% tropicamide.
Both eyes were examined using slitlamp biomicroscopy and indirect ophthalmoscopy
on days 0, 14, 28, 42, and 56 (before drug injection); days 1, 15, 29, 43,
and 57 (24 hours after injection); day 21 (before laser); days 35 and 49 (intermediate
days); and day 63 (enucleation and death).
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, Paramus, NJ) was used for fluorescein angiography. Red-free
photographs of both eyes were obtained followed by fluorescein angiography
using 0.1 mL/kg of body weight of 10% sodium fluorescein (Akorn Inc, Abita
Springs, La) at a rate of 1 mL/s. Following the fluorescein injection, a rapid
series of images were obtained in the first minute of the posterior pole of
first the right eye and then the left eye. Additional 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 baseline (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 abnormalities. The fundus hemorrhages were
graded based on a grading 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 hemorrhages 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 biomicroscopy. 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 Ophthalmology (Table 2).
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Table 2. Anterior Chamber Inflammation Grading System*
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The CNV lesions were graded by reviewing fluorescein angiograms performed
on days 35, 42, 49, 56, and 63 by 2 masked and experienced examiners (E.S.G.
and J.W.M.) who graded by consensus opinion. The CNV lesions were graded according
to the following scheme, using standardized angiographs for comparison. Grade
1 lesions had no hyperfluorescence. Grade 2 lesions exhibited hyperfluorescence
without leakage. Grade 3 lesions showed hyperfluorescence in the early or
midtransit images and late leakage. 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-Aggregated Panel
Data with Generalized Estimating Equations and the incidence rate ratio (IRR).
The incidence rate was defined as the number of grade 4 lesions that occurred
during a given interval divided by the total number of lesions 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 incidence of grade 4 lesions in the
prevention group vs control 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-extremity vein
before rhuFab VEGF injection and approximately 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 collection. 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-linked immunosorbent
assay method.
HISTOPATHOLOGIC ANALYSIS
The globes were carefully removed from each animal, dissected clean
of orbital tissue, rinsed in isotonic sodium chloride solution, and placed
in modified Karnovsky fixative consisting of 2% glutaraldehyde and 2.5% formaldehyde
in 0.1M cacodylate buffer 7.4 on ice. Within 10 minutes, the globes were opened
and the anterior segment removed and the posterior pole placed in fixative
overnight and then changed to buffer (0.1M cacodylate) until processed for
light microscopy.
Each eye was prepared for light microscopy by sectioning into blocks,
which contained lesions of interest. Tissues were postfixed in 2% osmium tetroxide
in 0.1M 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.
RESULTS
SAFETY OF INTRAVITREAL rhuFab VEGF INJECTIONS
Clinical examination and review of fundus photographs did not show any
hemorrhages before the laser photocoagulation, 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 intravitreal injection
due to a paracentesis for aqueous humor 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 associated 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 observed in all animals. There was
only 1 grade 2 hemorrhage 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 associated with intravitreal rhuFab VEGF or vehicle injections
in either prevention or treatment eyes.
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Table 3. Total Number of Fundus Hemorrhages for All Animals According
to Grade
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All eyes treated with rhuFab VEGF developed acute anterior chamber cells
within 24 hours of the first intravitreal 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). Subsequent 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 inflammation. 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 was less pronounced.
No other clinical or angiographic abnormalities were observed.
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Table 4. Anterior Chamber (AC) Cellular Inflammatory Response After
Recombinant Humanized Monoclonal Antibody Directed Toward Vascular Endothelial
Growth Factor (rhuFab VEGF) and Vehicle Intravitreal Injections
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EFFICACY OF INTRAVITREAL rhuFab VEGF INJECTIONS
Fluorescein angiograms of both eyes in each animal were evaluated according
to the grading system described in the "Materials and Methods" section for
phase 1 and phase 2. Examples of the photographic and angiographic appearance
of paired eyes in phase 1 and 2 are shown in Figure 1, Figure 2, and Figure 3.
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Figure 1. Posterior fundus color photographs
demonstrating choroidal neovascular lesions 2 weeks after laser induction
on day 35. A, Control eye (recombinant humanized monoclonal antibody directed
toward vascular endothelial growth factor [rhuFab VEGF] vehicle); B, prevention
eye (rhuFab VEGF).
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Figure 2. Fluorescein angiogram of the same
animal as in Figure 1 on day 35 demonstrating laser-induced lesions. A, Early
frame of the control eye (recombinant humanized monoclonal antibody directed
toward vascular endothelial growth factor [rhuFab VEGF] vehicle). B, Early
frame of the prevention eye (rhuFab VEGF). C, Late frame of the control eye
(rhuFab VEGF vehicle). D, Late frame of the prevention eye (rhuFab VEGF).
Late frames of the fluorescein angiogram demonstrate presence of grade 4 lesions
in the control eye but not in the prevention eye.
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Figure 3. Fluorescein angiogram of the same
animal as in Figure 7 at the time of enucleation (day 63). A, Early frame
of the control eye (recombinant humanized monoclonal antibody directed toward
vascular endothelial growth factor [rhuFab VEGF] vehicle crossed over to rhuFab
VEGF treatment). B, Early frame of the prevention eye (rhuFab VEGF). C, Late
frame of the control eye (rhuFab VEGF vehicle crossed over to rhuFab VEGF
treatment). D, Late frame of the prevention eye (rhuFab VEGF). Late frames
of the fluorescein angiogram demonstrate decreased leakage in both eyes.
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In phase 1, analysis of the CNV lesions at days 35 and 42 (2 and 3 weeks
after laser induction) showed a reduction in the likelihood of reaching grade
4 leakage (P<.001) in the rhuFab VEGF prevention
group compared with the vehicle control group (Figure 4). The IRR for the prevention group and the control group
was 0.041 (95% confidence interval, 0.009-0.176), indicating that intravitreal
rhuFab VEGF injections prevented formation of clinically significant CNV.
There was no laterality effect (P = .48) between
the eyes.
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Figure 4. Phase 1. Total number of grade
4 choroidal neovascular (CNV) lesions in the prevention group (recombinant
humanized monoclonal antibody directed toward vascular endothelial growth
factor [rhuFab VEGF]) (empty bar) vs control (rhuFab VEGF vehicle) (shaded
bar) 2 weeks after laser induction (day 35) and 3 weeks after the laser induction
(day 42).
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In phase 2, all eyes in the control group were injected with 500 µg
of rhuFab VEGF on days 42 and 56, and these eyes became the crossover or treatment
group. The number of grade 4 lesions in the control/treatment group is presented
in Figure 5. Using the population-aggregated
panel data model, the chance of being classified as grade 4 on days 49, 56,
and 63 (treatment eyes) compared with days 35 and 42 (control eyes) was assessed.
The rate of grade 4 lesion occurrence was reduced in the treatment group and
was statistically significant (P = .001; IRR = 0.074;
95% confidence interval, 0.032-0.174), indicating decreased leakage of already
formed CNV membranes with rhuFab VEGF treatment. There was no laterality effect
(P = .15) between eyes.
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Figure 5. Phase 2. Total number of grade
4 lesions in the control (recombinant humanized monoclonal antibody directed
toward vascular endothelial growth factor [rhuFab VEGF] vehicle)/treatment
group (rhuFab VEGF) over time. The control eye was injected with rhuFab VEGF
vehicle until day 42. After fluorescein angiogram was performed on day 42,
the eyes in this group were injected with rhuFab VEGF. The bar graph demonstrates
the number of grade 4 lesions over time before and after rhuFab VEGF injection.
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PHARMACOKINETICS AND ANTIBODY ANALYSIS
Anti–rhuFab VEGF assay performed on blood serum showed that 1
of 10 animals developed antibodies to rhuFab VEGF. The antibodies were first
detected on day 42 and persisted until the day of death. The rhuFab VEGF antigen
assay showed that the average detectable drug level in the vitreous was 32
ng/mL after the first injection and increased with subsequent treatments as
shown in Figure 6. These levels
continued to increase when both eyes were injected with rhuFab VEGF on days
42 and 56. Levels decreased within 7 days of treatment but increased further
with subsequent injections, indicating accumulation.
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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, decrease within
a week. With the subsequent injections, the serum drug levels tend to increase
further.
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HISTOPATHOLOGIC ANALYSIS
Histopathologic evaluation was performed on representative lesions,
and the data are summarized in Table 5.
The first lesion presented in Table 5
is from an eye in the prevention group and was graded as grade 2 on the fluorescein
angiogram 2 weeks after the laser induction (day 35) and at the time of death
on day 63. It consisted of a depression in the retina with fibroblasts and
lacked the normal outer nuclear layer. There were some macrophages present
in the choroid but no capillaries (Figure
7A). This lesion looked similar to lesion 2 (Table 5), which 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 developed a grade 4 lesion with profound
angiographic leakage 3 weeks after laser induction but then decreased leakage
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-laden macrophages, with
the occasional fibroblast, but the lesion was small and covered by retinal
pigment epithelium (Figure 7B).
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Table 5. Summary of Histopathologic Data on Selected Choroidal Neovascularization
Laser-Induced Lesions
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Figure 7. Photomicrographs of a 1-µm
section of a lesion from the eye in Figure 3. A, A lesion assessed as grade
2 on the fluorescein angiogram (prevention, rhuFab VEGF treatment). There
are a few macrophages and proliferating retinal pigment epithelial cells forming
a barrier at the level of the Bruch membrane (arrowhead) to which remnants
of the outer nuclear layer are opposed. There are no capillaries. B, A lesion
assessed as grade 4 at 2 weeks after laser induction (control recombinant
humanized monoclonal antibody directed toward vascular endothelial growth
factor [rhuFab VEGF] vehicle) that was then treated with rhuFab VEGF and evaluated
as grade 2 at the time of death (treatment). There are capillaries (arrow)
and macrophages covered by proliferating retinal pigment epithelial cells.
The Bruch membrane is indicated by arrowhead. The subretinal space is an artifact
of processing (toluidine blue stain, original magnification x20).
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COMMENT
Intravitreal injections of 500 µg of rhuFab VEGF administered
every 2 weeks in a laser-induced CNV model in 10 cynomolgus monkeys showed
no significant toxic effects 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 inflammation that resolved
without any sequelae and retinal hemorrhages associated with laser induction
were observed as expected and reabsorbed over several weeks. A previous study
of rhuFab VEGF injection in normal cynomolgus monkey eyes showed a similar
safety profile regarding ocular inflammation.15
In that study, intravitreal administration of rhuFab VEGF every 2 weeks into
eyes of otherwise untreated monkeys showed transient inflammation at doses
up to 2000 µg per eye, doses that were higher than the levels used in
our study. Perivascular 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 variables,
including visual evoked potential.
The phase 1 part of the study indicated that rhuFab VEGF prevented the
formation of angiographically 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 crossed over to receive rhuFab VEGF, with both eyes receiving active
rhuFab VEGF treatment. The number of grade 4 lesions that were counted after
the rhuFab VEGF injection on day 42 was significantly lower than the number
of grade 4 lesions before rhuFab VEGF injection. This suggests a significant
treatment effect with rhuFab VEGF. However, previous studies have shown that
the natural history of these lesions is to angiographic regression with loss
of leakage as early as 2 or 3 weeks after the laser induction, with a mean
regression period of 13 weeks.16 Although our
data suggest that rhuFab VEGF successfully attenuated the appearance of leaking
CNV lesions, spontaneous regression of these lesions may have played a role.
Levels of rhuFab VEGF in the blood are highest on the first day following
rhuFab VEGF injection, but then they decrease rapidly by the seventh postdose
day. At day 43, the increase in serum rhuFab VEGF may be attributed to switching
the previously control eye to active treatment. Only 1 of the 10 animals developed
antibodies to the drug in the serum. In a previous toxicology study, 15 of
28 animals developed antibodies toward rhuFab VEGF; however, these animals
were treated for a longer period (13 weeks) and were treated at doses up to
and including 2000 µg per eye at 2-week intervals.15
It is not unexpected to elicit an antibody response following the administration
of a heterologous protein. Additionally, since antibodies bind the rhuFab
VEGF, it is not surprising that the animal with rhuFab VEGF antibodies exhibited
elevated serum levels of the rhuFab VEGF more than other monkeys due to reduced
clearance of the complex. It should be noted that these antibodies were nonneutralizing
and as expected were generated toward the humanized backbone of the rhuFab
VEGF and not the binding epitope.
In this study, all of the representative histopathologically examined
lesions were relatively smaller than lesions studied previously in this CNV
model (M.G.K., unpublished data, December 1999). This finding suggests that
rhuFab VEGF inhibited growth of CNV lesions and also led to regression of
established CNV lesions. However, this finding is confounded by the relatively
long period of follow-up (6 weeks after laser induction) coupled with natural
history of laser-induced CNV lesion regression in this model.
This study showed that intravitreal injections of rhuFab VEGF in the
cynomolgus monkeys were safe and prevented formation of clinically significant
CNV. Also, rhuFab VEGF may have a beneficial effect in treating established
CNV. In view of these positive results in the animal model, rhuFab VEGF may
be a promising agent in treating human CNV lesions associated with neovascular
AMD. Clinical trials of intravitreal injections with rhuFab VEGF in patients
with neovascular AMD are currently ongoing.
AUTHOR INFORMATION
Submitted for publication July 10, 2000; final revision received October
3, 2001; accepted November 16, 2001.
Magdalena G. Krzystolik, MD, and Mehran A. Afshari, MD, contributed
equal amounts of work and are both considered first authors.
This study was presented in part at the annual meeting of the Association
for Research in Vision and Ophthalmology; Fort Lauderdale, Fla; May 3, 2000.
We thank Robin Holzer, BS (Massachusetts Eye and Ear Infirmary), for
her technical support and Mark L. Hartmangruber, BS (Genentech, Inc), for
his work on rhuFab VEGF assays.
Corresponding author and reprints: Joan W. Miller, MD, Retina Service,
Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114 (e-mail: jwmiller{at}meei.harvard.edu).
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.
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