Immunotherapy for choroidal neovascularization in a laser-induced mouse model simulating exudative (wet) macular degeneration

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness after age 55 in the industrialized world. Severe loss of central vision frequently occurs with the exudative (wet) form of AMD, as a result of the formation of a pathological choroidal neovasculature (CNV) that damages the macular region of the retina. We tested the effect of an immunotherapy procedure, which had been shown to destroy the pathological neovasculature in solid tumors, on the formation of laser-induced CNV in a mouse model simulating exudative AMD in humans. The procedure involves administering an Icon molecule that binds with high affinity and specificity to tissue factor (TF), resulting in the activation of a potent cytolytic immune response against cells expressing TF. The Icon binds selectively to TF on the vascular endothelium of a CNV in the mouse and pig models and also on the CNV of patients with exudative AMD. Here we show that the Icon dramatically reduces the frequency of CNV formation in the mouse model. After laser treatment to induce CNV formation, the mice were injected either with an adenoviral vector encoding the Icon, resulting in synthesis of the Icon by vector-infected mouse cells, or with the Icon protein. The route of injection was i.v. or intraocular. The efficacy of the Icon in preventing formation of laser-induced CNV depends on binding selectively to the CNV. Because the Icon binds selectively to the CNV in exudative AMD as well as to laser-induced CNV, the Icon might also be efficacious for treating patients with exudative AMD.

Figure 1

Immunohistology of an excised human subfoveal CNV complex showing staining of the Icon. (A) Excised tissue reveals a core of RPE cells surrounded by a well-developed capillary network (black arrows). Note that capillaries surrounded by RPE cells within the core of the lesion show signs of vascular collapse and atrophy (white arrowhead) and are eventually replaced by fibrous tissue (white arrows) (×500). (B) New vessels at the periphery of the CNV (white arrows) are stained intensely with the Icon (×850). (C) Binding of the Icon (green stain) to the vascular endothelium of the capillary marked with black arrowheads in A (×1,500). (D) Confocal micrograph of the same capillary showing speckled staining of the Icon on the endothelium (×4,500). An intense signal was detected in the perinuclear endothelial cell nucleus (white arrowhead) (27).

Figure 2

Confocal micrograph showing immunohistologic staining of the Icon in the CNV of the mouse. (A) Laser-induced CNV stained for TF. Vascular channels are stained positively with the Icon (arrows). Autofluorescence of photoreceptors is not seen (arrow heads) due to loss of outer nuclear layer during laser photocoagulation (×100). (B) Schematic diagram of laser-induced CNV complex showing RPE, choroid, and sclera. (C) Magnified view of CNV stained for TF (×600). Endothelium (arrows) lining vascular channels stain for TF. Cell nuclei are indicated by n.

Figure 3

Confocal micrograph showing immunohistologic staining of the Icon in the CNV of the pig. Choroidal flat mounts were prepared 1 week after diode laser photocoagulation in the pig, incubated with Icon protein, and stained with a FITC-labeled antibody against the Fc domain of the Icon. The endothelial cells of the CNV membrane (arrowhead) are stained green (speckled pattern), whereas the normal retinal vascular endothelium is not stained (arrow). The normal choroidal endothelium also did not stain (not shown) (×2,000).

Figure 4

Confocal micrograph of laser-induced CNV in the mouse. (A) Mice were injected i.v. with the control adenovirus on day 1 followed on day 7 by perfusion with FITC-Dextran. The eyes were excised and choroidal flat mounts were stained with a mAb against elastin and then with a CY3-conjugated second antibody. The prominent neovascular growth stained green whereas the underlying elastin in the Bruch's membrane stained red within a laser spot. The RPE (dark brown) was disrupted in the bed of the laser spot (×2,500). (B) Mice received an i.v. injection of the adenoviral vector encoding the Icon on day 1, and choroidal flat mounts were prepared on day 7 as described above. The inhibition of new vessel growth is indicated by the absence of vessels with a green stain (×2,500).