Maya H. Maloney, MD and Sophie J. Bakri, MD
As vascular endothelial growth factor (VEGF) was identified as a regulator of tumor angiogenesis, it was also identified as a mediator of angiogenesis in the eye. VEGF was suggested to play a role in neovascularization secondary to ischemic retinal disorders, such as active proliferative diabetic retinopathy, ischemic central retinal vein occlusion, and retinopathy of prematurity.1,2 Later, the importance of VEGF in choroidal neovascularization, and therefore age-related macular degeneration (AMD), was also recognized.3,4 During this time, multiple animal models helped shed light on ocular angiogenesis. Occlusion of retinal veins by laser in a cynomolgus monkey stimulated rubeosis iridis and showed increases in aqueous VEGF levels proportional to the severity of iris neovascularization.5 Subsequent studies demonstrated that intravitreal injection of VEGF was sufficient to produce iris neovascularization and injection of anti-VEGF antibodies prevented such neovascularization.6,7 Mouse models of ischemia-induced proliferative retinopathy showed similar results of suppressed neovascularization by inhibition of VEGF with VEGF receptor chimeric proteins and antisense oligodeoxynucleotides.8,9 As we learned that the inhibition of VEGF action could have therapeutic applications, further strategies were developed and honed to inhibit VEGF production, neutralize free VEGF, block VEGF receptors, and interfere with the endothelial cell response to VEGF.10
Pegaptanib
Pharmacology
Pegaptanib (Macugen) is an RNA aptamer that binds the heparin-binding site of the VEGF165 isoform, thereby preventing it from binding to VEGF receptors. Aptamers (from the Latin aptus, to fit, and the Greek meros, part or region) are RNA or DNA oligonucleotide ligands that can bind extracellular target molecules with high affinity and specificity. Unlike monoclonal antibodies, they are easily produced and non-immunogenic. Aptamers are selected during systematic evolution of ligands by exponential enrichment (Figure 2-1). In this process, up to 1015 random oligomer sequences are flanked by binding sites for reverse transcriptase and polymerase chain reaction primers, promoter sequences for T7 RNA polymerase, and restriction endonuclease sites. These 20 to 40 nucleotide-long regions that bind targets are selected, amplified, and reselected. In the case of anti-VEGF aptamer development, iterations of the systematic evolution of ligands by exponential enrichment process identified ligands that were subsequently modified for improved stability and nuclease resistance.11 The VEGF antagonist that was ultimately chosen for development, and became known as pegaptanib is shown in Figure 2-2. It significantly reduced VEGF-induced vascular permeability in adult guinea pigs compared to its counterparts.12 Pegaptanib was developed specifically to antagonize the pathologic 165 isoform of VEGF because of concern that inhibition of all VEGF isoforms would interfere with physiologic angiogenesis in addition to antagonizing pathologic neovascularization. It became the first aptamer successfully developed as a therapeutic for human use when the United States Food and Drug Administration (FDA) approved it for the treatment of neovascular AMD in 2004.11
Figure 2-1. A diagram of systematic evolution of ligands by exponential enrichment (SELEX). A library of random oligonucleotide sequences are flanked by fixed regions, labeled A and B, that allow for enzyme binding and other chemical reactions. Sequences are selected during repeated rounds of partitioning and amplification. (Adapted from Eaton BE, Gold L, Hicke BJ, et al. Post-SELEX combinatorial optimization of aptamers. Bioorg Med Chem. 1997;5(6):1087-1096. Used with permission of Mayo Foundation for Medical Education and Research. All rights reserved.)
Prior to clinical trials, studies were conducted to better characterize the aptamer. Pegaptanib was stable after 18 hours of incubation at ambient temperature. The half-life was 9.3 hours in monkeys after intravenous administration. In another study using a monkey model, biologically active pegaptanib remained in the vitreous for at least 28 days following a single 0.5 mg intravitreal injection.11 Other preclinical studies confirmed the anti-angiogenic effects of pegaptanib with its inhibition of corneal neovascularization in rats and of retinal neovascularization in the rat model of retinopathy of prematurity.13 Pegaptanib is cleared by the kidneys11 and has been shown not to elicit an immunogenic response.14 Previous studies supported that it was the 165 isoform of VEGF, and not other isoforms like VEGF121, that was responsible for pathological ocular neovascularization, and further research suggested that pegaptanib inhibited VEGF165 by binding a residue in the heparin-binding domain of VEGF165 that is not found in VEGF121.11 With these results, this targeted anti-VEGF agent entered preliminary clinical trials.
Figure 2-2. Structure of pegaptanib. Sequence of modified nucleotides in pegaptanib. Uridine-14 (pink) interacts with the heparin-binding domain of VEGF165. There is a 40 kDa polyethylene glycol moiety at the 5’ end and a 3’-terminal deoxythymidine at the 3’ end. (Adapted from Ng EW, Shima DT, Calias P, Cunningham ET Jr, Guyer DR, Adamis AP. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov. 2006;5(2):123-132. Used with permission of Mayo Foundation for Medical Education and Research. All rights reserved.)
Therapeutic Efficacy
In phase Ia and II trials, pegaptanib was administered to small groups for evaluation of safety and efficacy. In the phase Ia trial, patients with poor vision from neovascular AMD received intravitreal injections of varying doses of pegaptanib to establish a safe dosage range. No serious side effects were noted, and vision stabilized or improved in 80% of patients at 3 months.13 The phase II trial involving 21 patients compared the use of pegaptanib alone to pegaptanib treatment with concomitant photodynamic therapy. Eight patients completed the 3-month course of monthly intravitreal injection of pegaptanib alone; 7 had stabilization or improvement of their vision.15 This encouraging preliminary data led to larger-scale studies.
Pegaptanib was subsequently tested in pivotal clinical trials for the treatment of neovascular AMD and diabetic macular edema (DME). In the phase III VEGF Inhibition Study in Ocular Neovascularization (VISION) trials, intravitreal injection of pegaptanib was compared to sham injection at 1 and 2 years. Results demonstrated that pegaptanib was both safe and effective, reducing vision loss in patients when compared to sham injection. Another phase II trial was also conducted to investigate the efficacy of pegaptanib in the treatment of DME and similarly showed improved visual acuity outcomes in treated patients.11 These trials supported the successful development of a therapeutic aptamer targeted at VEGF and pathologic ocular neovascularization, a novel treatment not dependent on the classification of neovascularization by fluorescein angiogram like the previously available therapies.
Bevacizumab
Pharmacology
Bevacizumab (Avastin) was developed to inhibit VEGF for the treatment of metastatic colorectal cancer and FDA approved for this use in 2004. The original mouse anti-human VEGF antibody, A.4.6.1, was produced by mice immunized with VEGF165 but recognized all VEGF-A isoforms. This full-length monoclonal antibody was later humanized by transferring the VEGF-binding regions of A.4.6.1 to a human antibody framework (Figure 2-3); the final product contains 93% human amino acid sequence. Large quantities are produced in Chinese hamster ovary cells using expression plasmids.16
Initial preclinical work with bevacizumab was focused on tumor anti-angiogenesis. Further research regarding intravitreal injection and ocular use was performed only after the medication was already in active use off-label for ocular indications. As with pegaptanib, safety studies were performed in cynomolgus monkeys as VEGF isoforms were found to be identical between the Macaca fascicularis and humans.17 In rabbits receiving the 1.25 mg dose that is commonly administered today, the half-life of intravitreal bevacizumab was 4.32 days, compared to 2.88 days for 0.5 mg ranibizumab.18,19 Interestingly, bevacizumab can be found in the vitreous of the contralateral eye after intravitreal injection18,19 and has been shown to penetrate throughout the retina on confocal immunohistochemistry.20
Therapeutic Efficacy
Phase I trials started in 1997 for systemic administration and showed that the addition of bevacizumab to standard chemotherapy regimens was safe. Multiple phase II trials were then conducted to study the role of bevacizumab in the treatment of different cancers, including colorectal cancer, renal cell cancer, and non-small cell lung cancer, among others. These oncology trials suggested that adverse events such as thrombosis, bleeding, and hypertension may be systemic adverse events related to bevacizumab administration.17 Results from the Systemic Avastin for Neovascular AMD trial supported the hypothesis that systemic bevacizumab improved vision in affected patients.21,22 Later, larger-scale phase III studies to assess the efficacy of intravitreal bevacizumab in the treatment of neovascular AMD were initiated, such as the Avastin for Treatment of Choroidal Neovascularization (ABC) trial and the Comparison of Age-Related Macular Degeneration (CATT) trial. Internationally, there have now been multiple comparative treatment trials, including the Inhibition of VEGF in Age-Related Choroidal Neovascularisation (IVAN), the Multicentre Anti-VEGF Trial in Austria (MANTA), the Avastin Versus Lucentis for Neovascular AMD (GEFAL), and others.
Figure 2-3. Structures and roles of bevacizumab, ranibizumab, and aflibercept. Bevacizumab and ranibizumab were both developed from a mouse immunoglobulin G. Bevacizumab is a humanized full-length monoclonal antibody and ranibizumab is a humanized Fab fragment. Aflibercept is a fusion protein comprised of ligand-binding domains 2 and 3 of VEGFR1 and VEGFR2, respectively, attached to the Fc region of a human immunoglobulin G. Fab = fragment antigen binding; Fc = fragment crystallizable; VEGFR1 = vascular endothelial growth factor receptor 1; VEGFR2 = vascular endothelial growth factor receptor 2. (Adapted from Au A, Singh RP. A multimodal approach to diabetic macular edema. J Diabetes Complications. 2016;30(3):545-553. Used with permission of Mayo Foundation for Medical Education and Research. All rights reserved.)
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