Neovascular Age-Related Macular Degeneration: Therapeutic Management and New-Upcoming Approaches

Age-related macular degeneration (AMD) constitutes a prevalent, chronic, and progressive retinal degenerative disease of the macula that affects elderly people and cause central vision impairment. Despite therapeutic advances in the management of neovascular AMD, none of the currently used treatments cures the disease or reverses its course. Medical treatment of neovascular AMD experienced a significant advance due to the introduction of vascular endothelial growth factor inhibitors (anti-VEGF), which dramatically changed the prognosis of the disease. However, although anti-VEGF therapy has become the standard treatment for neovascular AMD, many patients do not respond adequately to this therapy or experience a slow loss of efficacy of anti-VEGF agents after repeated administration. Additionally, current treatment with intravitreal anti-VEGF agents is associated with a significant treatment burden for patients, caregivers, and physicians. New approaches have been proposed for treating neovascular AMD. Among them, designed ankyrin repeat proteins (DARPins) seem to be as effective as monthly ranibizumab, but with greater durability, which may enhance patient compliance with needed injections.


Introduction
Age-related macular degeneration (AMD) constitutes a prevalent, chronic, and progressive retinal degenerative disease of the macula that affects elderly people and causes central vision impairment as a result of damage to retina, retinal pigment epithelium (RPE), and choriocapillaris [1][2][3].

Pegaptanib
The first intravitreal anti-VEGF treatment developed specifically for NVAMD was pegaptanib sodium. Pegaptanib is a pegylated oligoribonucleotide (aptamer) that binds with high specificity and affinity to VEGF 165 , sequestering and therefore preventing VEGF 165 from activating its receptor [123].
Two concurrent, multicenter, prospective, randomized, double-blind, and controlled clinical trials were conducted on AMD patients, at 117 sites in the United States, Canada, Europe, Israel, Australia, and South America [92]. These studies compared three pegaptanib intravitreal injection dosages (0.3 mg, 1.0 mg, and 3.0 mg) given every six weeks over a 48 week period versus (vs) sham injections. Among the 1208 patients randomly assigned to treatment, 1186 patients received at least one study treatment and were included in efficacy analyses [92]. As compared to sham injection (55%), a greater proportion of eyes with visual acuity (VA) loss < 15 letters was demonstrated for the 0.3 mg (70%, p < 0.001); 1.0 mg (71%, p < 0.001); and 3.0 mg (65%, p = 0.03) pegaptanib injections. Additionally, as compared to the sham injection group, a significantly higher proportion of patients maintained or improved their VA in the 0.3 mg (p = 0.003), 1.0 mg (p < 0.001), and 3.0 mg (p = 0.02) pegaptanib study groups [92].
However, due to its poorer efficacy compared with other currently available anti-VEGF drugs, pegaptanib is no longer recommended for the treatment of exudative AMD.
Regarding safety, incidence of ocular adverse events in the pegaptanib groups was greater than in the control group (see Table 3). Moreover, subjects in the pegaptanib groups were more likely to have a serious systemic adverse event than participants in the control one. Ocular adverse events are shown in Table 3. Table 3. An overview of the ocular adverse events reported by age-related macular degeneration patients treated with pegaptanib. Adapted from Solomon et al. [124].

Bevacizumab
Bevacizumab, a humanized monoclonal antibody that inhibits VEGF-A, was originally developed as a medication for use in combination with existing metastatic colorectal cancer chemotherapy regimens [125,126].
Intravitreal injections of bevacizumab are used widely as an off-label treatment for NVAMD [127,128].
The ABC trial was a prospective, multicenter, double-masked, and randomized clinical trial (RCT) that analyzed the efficacy and safety of bevacizumab for treating NVAMD [96,97]. The results of this study showed that a significantly greater proportion of patients achieved best-corrected visual acuity (BCVA) improvement ≥15 letters from baseline in the bevacizumab group than in the control group (32% vs. 3%, respectively; p < 0.0001). Additionally, the mean BCVA improvement in the bevacizumab group was 7.0 letters, while in the standard of care group the mean BCVA decreased 9.4 letters (p < 0.001) [96]. At the week-54 examination, bevacizumab-treated patients were more likely to gain at least 6 letters or more of contrast sensitivity than the patients receiving standard care (PDT [photodynamic therapy], pegaptanib intravitreal injections, or sham treatment) (35.4% vs. 15.2%, respectively; p = 0.009) [97].
The CATT (Comparison of Age-Related Macular Degeneration Treatments Trials) study was a randomized, single-blind, and non-inferiority study (non-inferiority margin 5 ETDRS letters) that compare the efficacy and safety of bevacizumab and ranibizumab in patients with NVAMD [28,100,101].
In this study, bevacizumab was non-inferior to ranibizumab, both when the drugs were given monthly and when the drugs were given as needed ( Figure 2) [100]. After two years, among those subjects who maintained the same treatment regimen, mean gain in VA was similar for both drugs (p = 0.21) [101]. However, as needed treatment posology resulted in less gain in VA (difference: −2.4 letters; p = 0.046) [104]. Interestingly, the 5-year results of the CATT study showed a decreased in BCVA (mean change −3 letters from baseline and −11 letters from 2 years), although 50% of eyes had a VA 20/40 or better [28].
The IVAN (alternative treatments to Inhibit VEGF in Age-related choroidal Neovascularization) study was a multicenter, randomized, and non-inferiority study that compared the efficacy and safety of bevacizumab (1.25 mg) and ranibizumab (0.5 mg) in patients with NVAMD. Subjects were randomly assigned to intravitreal injections of ranibizumab or bevacizumab in continuous (every month) or discontinuous (as needed) regimens, with monthly review [102,103]. In this study, the comparison of drugs was inconclusive. Using a 3.5 letter non-inferiority margin, bevacizumab was neither inferior nor equivalent to ranibizumab. Additionally, continuous treatment was equivalent to the discontinuous one [102]. The two-year results of the IVAN study confirmed these findings [103].
The efficacy and safety of fixed regimens of ranibizumab were evaluated in various trials, including ANCHOR [90,93]; MARINA [89,91], PIER [94,95], and EXCITE [132]. ANCHOR was a prospective RCT that compared the efficacy and safety of ranibizumab (0.3 and 0.5 mg) vs. verteporfin therapy in patients with NVAMD [90,93]. Mean BCVA increased by 8.5 letters in the 0.3-mg group and 11.3 letters in the 0.5-mg group, as compared with a decrease of 9.5 letters in the verteporfin group (p < 0.001 for each comparison) ( Figure 3).
Additionally, the results after two years of follow-up confirmed a clinically meaningful benefit of intravitreal ranibizumab vs. verteporfin therapy (34% to 41% patients treated with ranibizumab had gained ≥15 letters vs. 6.3% of them in the PDT group) [93].
MARINA (Minimally classic/occult trial of the Anti-VEGF antibody Ranibizumab in the treatment of Neovascular AMD) was a multicenter, 2-year, double-blind, and sham-controlled RCT designed to evaluate the efficacy and safety of intravitreal ranibizumab (0.3 mg and 0.5 mg) in patients with NVAMD [89,91]. At month 12, a significantly greater proportion of patients had lost fewer than 15 letters in the 0.3 mg ranibizumab group (94.5% of patients) or in the 0.5 mg ranibizumab group (94.6% of patients) than in the sham control group (62.2% of patients) with p < 0.001 for both comparisons [89]. Mean increases in visual acuity at month 12 were 6.5 letters in the 0.3 mg group and 7.2 letters in the 0.5 mg group, as compared with a decrease of 10.4 letters in the control group (p < 0.001 for both comparisons) [89] (Figure 4).  PIER was a multicenter, randomized, double-masked, and sham injection-controlled trial that evaluated the efficacy and safety of ranibizumab (0.3 mg and 0.5 mg) in patients with NVAMD treated quarterly after three monthly loading doses [94,95]. At month 12, mean changes from baseline VA were −16.3, −1.6, and −0.2 letters for the sham, 0.3 mg, and 0.5 mg groups, respectively (p ≤ 0.0001, for each ranibizumab dose vs. sham) [94]. At month 24, visual acuity VA had decreased an average of 21.4, 2.2, and 2.3 letters from baseline in the sham, 0.3 mg, and 0.5 mg groups (p < 0.0001 for each ranibizumab group vs. sham) [95]. It was suggested that in patients with NVAMD, retreatment need with intravitreal ranibizumab had a high intra-individual predictability, but that most patients required more frequent dosing than quarterly following the initial loading doses [133,134].
The efficacy of an individually tailored 'observe-and-plan' treatment regimen with ranibizumab was assessed in patients with NVAMD [135]. The results of this study indicated that mean VA improved by 8.7, 9.7, and 9.2 letters at months 3, 12, and 24, respectively. The mean treatment interval (after the loading doses) was 2.0 months during year-1 and 2.2 months during year-2 [135].
The efficacy of a treat-and-extend (TREX) management strategy of intravitreal ranibizumab was assessed in treatment naïve NVAMD patients [116]. There were no significant differences in BCVA improvement between the monthly regimen (+9.2 letters) and the TREX strategy (+10.5 letters), p = 0.60. Nevertheless, the number of injections administered through the study follow-up was significantly lower in the TREX (10.1) than in the monthly (13.0) regime (p < 0.0001) [116].
In a separate study evaluating the efficacy and safety of ranibizumab administered in a TREX or in a monthly regimen, the TREX regimen was noninferior (p < 0.001) to the monthly regimen, with a mean BCVA improvement of 6.2 letters and 8.1 letters in the TREX and monthly groups, respectively [117].
The Canadian Treat-and-Extend Analysis Trial with ranibizumab (CANTREAT) was a 2-year, randomized, multicenter, and open-label study conducted on treatment-naive NVAMD patients [118,119]. At month 12, mean BCVA improvements of 8.4 ± 11.9 letters and 6.0 ± 11.9 letters were recorded in the TREX and monthly regimens, respectively [118]. Furthermore, at 24 months, mean BCVA improvement was not worse in the TREX treatment group (6.8 ± 14.1 letters) than in the monthly treatment group (6.0 ± 12.6 letters) [119].
Safety profile showed that incidence of adverse events, at both one-and two-year follow ups, were small (Table 5). With respect to ocular adverse events, eyes treated with ranibizumab more often developed cataracts compared with eyes in the control groups at both the one year (RR 1.48; 95% CI 0.83 to 2.66) and two year follow ups (RR 1.25; 95% CI 0.94 to 1.66) (See Table 5). Two eyes during the first year of ranibizumab injections and six more during the second year developed endophthalmitis, compared to no cases in the control eyes (Table 5). An overview of ocular adverse events is summarized in Tables 4 and 5.   [136][137][138].
VIEW 1 and VIEW 2 were parallel RCTs that included 2419 NVAMD patients in which monthly and every-2-month dosing of intravitreal aflibercept injection were compared with monthly ranibizumab injection. Subjects were randomly assigned to ranibizumab (0.5 mg) every 4 weeks (RQ4) or to 1 of 3 aflibercept doses: 0.5 mg every 4 weeks (0.5Q4), 2 mg every 4 weeks (2Q4), or 2 mg every 8 weeks after 3 monthly loading doses (2Q8) [120]. At week 52, the results of this study suggested that intravitreal aflibercept dosed monthly or every two months was not inferior to ranibizumab dosed monthly. The integrated analysis showed a mean BCVA improvement of +9.3, 8.7, 8.4, and 8.3 letters in the 2Q4, RQ4, 2Q8, and 0.5Q4 treatment groups, respectively [120]. At week 96 all the treatment regimens were equivalent. Mean BCVA gains ranged from 8.3 to 9.3 letters at week 52 and from 6.6 to 7.9 letters at week 96. The number of injections was mandated per protocol during the first 52 weeks of the study. From week 52 through 96, patients were treated PRN although injections were mandated at least every 12 weeks for both anti-VEGF agents (capped PRN posology). In a post hoc analysis, the number of injections from week 52 to week 96 was lower in the 2q4 and 2q8 groups vs. the Rq4 group: mean of aflibercept minus Rq4, −0.64 (95% CI, −0.89 to −0.40) for 2q4 and −0.55 (95% CI, −0.79 to −0.30) for 2q8 (p < 0.0001 for both) [139].
The comparative effectiveness of intravitreal ranibizumab and aflibercept was assessed in a 12-month RCT [140]. Although at month 12 there was a difference in mean BCVA improvement in favor of ranibizumab (mean letter score difference, 2.3; 95% confidence interval: −0.1 to 4.7), this difference was not statistically significant (p = 0.06) [140].

Brolucizumab
Brolucizumab (Beovu ® , Novartis, Basel, Switzerland) is a low molecular weight, single-chain antibody fragment that targets all forms of VEGF-A with high affinity. It was developed for the treatment of NVAMD, and it is in pivotal studies for diabetic macular edema (DME) and macular edema secondary to retinal vein occlusion [141]. The efficacy and safety of brolucizumab was compared to that of aflibercept in a prospective, randomized, double-masked, and multicenter phase 2 study [142]. At week 40, there were no significant differences in mean BCVA change from baseline. As compared to aflibercept, a greater proportion of brolucizumab-treated eyes had resolved intraretinal and subretinal fluid [142].
Intraocular inflammation was identified in 50 (4.6%) of the brolucizumab-treated patients. Of those, 36 subjects (3.3%) had concomitant retinal vasculitis. Of the 36 subjects with intraocular inflammation and vasculitis, 23 subjects (2.1%) had concomitant vascular occlusion. The absolute risk of developing intraocular inflammation and losing 15 or more letters was 0.7% (8/1088). Eight (22%) of 36 patients with vasculitis lost ≥3 lines of vision, and five of 36 (14%) lost ≥6 lines vision over 2 years. Among the 23 patients with occlusive retinal vasculitis, 7 (30%) lost ≥3 lines of vision, and 5 (22%) lost ≥6 lines [122]. The American Society of Retina Specialists (ASRS) conducted a post-approval analysis of brolucizumab-associated intraocular inflammation cases with short-term follow-up of 26 eyes from 25 patients with retinal vasculitis (of which 85% were designated as occlusive). In this consecutive series, ≥3-line vision loss and ≥6-line vision loss was seen in 46% and 35% of eyes, respectively [143]. Despite the risk of vision loss associated with retinal vasculitis following brolucizumab injection, the overall rate of vision loss in the study population was not different when comparing the brolucizumab to aflibercept arms in HAWK and HARRIER.
The most common ocular adverse events were conjunctival hemorrhage (brolucizumab 3 and 6 mg; HAWK) and reduced visual acuity (aflibercept; HAWK, both treatments; HARRIER) [122]. Adverse events of interest included uveitis and iritis (2.2% for each) with brolucizumab 6 mg vs. 0.3% and 0%, respectively, with aflibercept in HAWK; corresponding rates in HARRIER were <1% in both arms [122]. The incidence of serious ocular adverse events was low in both trials; no event occurred in >1% of eyes [127]. Table 7 summarizes the main ocular adverse events reported by the HAWK and HARRIER in Study Eye.

Real-World Outcomes
Despite the fact that RCTs represent the best method for establishing the efficacy and safety for different interventions for a disease process, they are not free of weaknesses that limit their external validity [144]. The limitations of RCTs that underlie differences in outcomes observed within the trial vs. those in clinical practice include limited sample size, restricted enrollment criteria (e.g., inclusion of patients with few comorbidities), rigorously enforced follow-up and treatment schedules, assessment of limited outcomes, limited follow-up period, and ready access to the medication [145]. Real-world evidence allows us to assess whether results from RCTs can be applied to the general population. For example, the 1-year effect of intravitreal bevacizumab, ranibizumab, and aflibercept on VA was compared in a real-world setting in NVAMD patients included in the IRIS (Intelligent Research in Sight) Registry. The mean number of injections was higher in the ranibizumab (6.4 ± 2.4) and aflibercept groups (6.2 ± 2.4) compared to bevacizumab group (5.9 ± 2.4; p < 0.0001). Once adjusted by different covariates, no differences were observed in VA outcomes, suggesting clearly that all 3 drugs improve VA similarly over 1 year of monotherapy [146]. However, as with RCTs, real-life studies have inherent limitations and cofounders that should be taken into consideration when interpreting their results such as the lack of standardized assessment of visual acuity.
SEVEN-UP was a multicenter and noninterventional cohort study that evaluated the long-term outcomes of ranibizumab in patients enrolled in the ANCHOR, MARINA, and HORIZON trials [147]. According to the results of this study, approximately one third of patients undergoing ranibizumab therapy in the ANCHOR or MARINA study achieved good VA outcomes after 7 years. However, as compared to baseline, vision in one third declined by 15 letters or more [147]. It should be mentioned that many patients were withdrawn of the study, and only a few completed the follow-up period. Interestingly, NVAMD fellow eyes remained at risk for further vision decline in later years under management with low-frequency anti-VEGF therapy [148].
AURA was a retrospective, observational, and multicenter study that assessed management of patients with NVAMD receiving anti-VEGF treatment in clinical practice (149). This study found that the number of intravitreal anti-VEGF injections was lower in clinical practice than in RCTs, despite the observed initial improvement in VA. However, such improvement was not maintained over time [149]. Similarly, the Pan-American Collaborative Retina Study Group reported a retrospective case series that evaluated the long-term anatomical and functional outcomes of bevacizumab in patients with CNV secondary to AMD. This study found that the early VA gains achieved with intravitreal bevacizumab were not maintained after 5 years of follow-up [150]. Ozkaya et al. [151] found that approximately half of ranibizumab NVAMD treated patients remained stable after 5-years of follow-up.
The 5-year outcomes of the CATT study suggested that VA improvements achieved during the first 2 years were not maintained at 5 years. Nevertheless, it should be mentioned that at the 5-year visit, 50% of eyes had VA of 20/40 or better, confirming the positive long-term effect of anti-VEGF in NVAMD patients [28].
According to the results of two meta-analyses, intravitreal ranibizumab for NVAMD prevents severe visual loss in real-world practice. However, patient outcomes are considerably worse than those reported in RCTs for both fixed and PRN regimens [27,153] (Figure 5). Among other factors, this difference in outcomes can be explained by the lower number of injections in real-world practice [152]. in real-world studies and randomized controlled trials after 12 months of ranibizumab treatment in patients with neovascular age related macular degeneration. Real world studies results have been adapted from Chong [27] and Kim et al. [153]. RLS: Real-life studies; gm: grand means (mean of the means of the several studies); wm: weighted means (mean weighted against number of eyes per study).

Non-Responders
Although anti-VEGF therapy has become the standard treatment for NVAMD [131], many patients do not respond adequately to this therapy or experience a slow loss of efficacy of anti-VEGF agents after repeated administration over time [154,155]. Resistance can occur at any time during the course of therapy [154,155]. Patients who do not respond adequately to anti-VEGF therapy can be classified into two groups, namely initial-non-responders (do not respond to the drug at the loading dose) and tachyphylactic patients (patients respond to the loading dose, but the response decreases gradually after repeated administrations) [155]. A framework of the potential causes of resistance to anti-VEGF therapy and their possible therapeutic approaches is summarized in Figure 6. A retrospective study reported that 22 eyes (10.1%) were identified as initial non-responders to ranibizumab [156]. Another retrospective study found that 14 (22.2%) eyes (8 eyes receiving aflibercept and 6 ones receiving ranibizumab) were classified as non-responders, while 8 (12.7%) eyes were identified as tachyphylactic (7 eyes treated with aflibercept and one eye with ranibizumab) [157]. The results of a post-hoc subanalysis of the CATT study, which involved "pseudo-switching", highlighted the importance of having a control group to evaluate the effect of switching therapy, an apparent improvement following a switch may be nothing more than a manifestation of regression to the mean (i.e., if a random variable is extreme on its first or first few measurements, it is likely to become closer to the mean or average on subsequent measurements) [158].
A post hoc subanalysis of the HARBOR trial, in which patients meeting specific futility criteria were maintained on the original therapy and followed, also demonstrated that apparent improvements in vision and optical coherence tomography (OCT) results attributed to switching may be nothing more than a manifestation of regression to the mean [159]. In fact, visual function and OCT improvements observed in these two "pseudo switching" trials were almost identical to that observed after actual switching studies [160]. Generally, apparent treatment futility can be a manifestation of inadequate treatment frequency as well as biological resistance based on the pharmacology of the applied treatment.

Cost
Monthly intravitreal injections are associated with a significant treatment burden for patients, caregivers, and physicians, often making such a regimen unachievable in clinical practice. According to the annual National Report of the Use of Drugs in Italy, a total of 137,387 patients with NVAMD received anti-VEGF therapy during the year 2018 [161]. Among them, 55.3% received treatment with ranibizumab; 25.3% were treated with aflibercept; 18.8% were treated with bevacizumab; and 0.5% received treatment with pegaptanib. The mean (standard deviation) number of injections during the first 12 months was 3.4 (3); 3.8 (84); 3.4 (3); and 2.6 (2), respectively [161]. As mentioned above, in real clinical practice patients usually received fewer anti-VEGF injections and achieved worse visual outcomes compared with patients receiving frequent therapy according to a fixed regimen in RCTs [27,153]. In fact, BCVA improvement correlated with treatment intensity at 1 year [162].
Additionally, differences in the overall treatment approach and how ophthalmologists use these anti-VEGF therapies have been identified. These differences may critically influence treatment effectiveness in real clinical practice [163]. According to the results of a meta-analysis, intravitreal aflibercept was associated with a higher overall treatment cost than ranibizumab (18,187 € vs. 17,168 €, respectively), although with an incremental gain in effectiveness (4.92 vs. 4.88 quality-adjusted life years, respectively) [163].
Despite the fact that bevacizumab is "off label" and not considered the standard of care for NVAMD in Europe (nor it is approved for treating NVAMD by regulatory agencies such as the US Food and Drug Administration [FDA] and the European Medicines Agency [EMA]), it has been identified as the most cost-effective treatment [164]. Table 8 shows a cost analysis comparison between bevacizumab, ranibizumab, and aflibercept.

NVAMD Management: Multi Target Approach
New approaches have been proposed for treating NVAMD. Indeed, VEGF is the main but not the only player in the NVAMD pathophysiology. However, over the past two years we have witnessed the failure of 3 phase II and III clinical trials that evaluated AMD treatments [165][166][167].
Pegpleranib (Fovista, Ophthotech, New York, NY, USA), a PDFG inhibitor, combined with bevacizumab, ranibizumab, or aflibercept did not result in benefit as measured by the mean change in BCVA at the 12-month time point [165]. The concomitant administration of squalamine (a supposed anti-VEGF booster and PDGF inhibitor) eye drops in combination with monthly intravitreal ranibizumab did not show significant advantages vs. intravitreal ranibizumab monotherapy [167]. Finally, nevascumab (Regeneron Inc., New York, NY, USA) is a fully human immunoglobulin G1 (IgG1) VelocImmune monoclonal antibody that selectively binds Ang2 with high affinity (K d = 24 pM), blocks Ang2 binding to the Tie2 receptor, and does not bind to Ang1 [168]. The aim of nesvacumab is to rebalance the angiogenic Ang1-Ang-2 imbalance occurring in case of pathologic angiogenesis. Unfortunately, the results of a phase 2 study that evaluated the effect of adding nevascumab to intravitreal injections of aflibercept did not provide additional benefit, which might suggest a peripheral role of Ang-Tie pathway in comparison to VEGF-VEGFR2 one [169].
Nevertheless, Ang-Tie pathway blockade has recently come under additional scrutiny. Faricimab (Genentech/Roche, South San Francisco, CA) is the first bispecific antibody targeting both Ang2 and VEGF-A [170,171]. This single molecule has dual inhibition of both Ang2 and VEGF targets in NVAMD. Rather than monthly dosing, faricimab dosing ranges from 12 weeks to 16 weeks [170,171].
The AVENUE [172] and STAIRWAY [173] trials found faricimab to be noninferior to ranibizumab for treatment of NVAMD.
AVENUE was a head-to-head trial of 273 patients comparing 0.5 mg ranibizumab every 4 weeks to 1.6 mg or 6 mg faricimab given every 4 or 8 weeks in the treatment of NVAMD [172]. Over a follow-up period of 36 weeks, changes in BCVA, CMT, and CNV were comparable between treatment arms [172].
STAIRWAY is a 52-week study that assessed two extended dosing regimens of faricimab 6.0 mg given every 16 weeks or every 12 weeks, compared to ranibizumab 0.5 mg every four weeks [173]. At week 24 (three months after the last of four loading doses), patients randomized to faricimab every 16 weeks switched to 12-week dosing if they were shown to have active disease, per pre-defined criteria. At week 24, 65% (n = 36/55) of eyes treated with faricimab had no active disease, highlighting the potential of 16-week dosing in nearly two-thirds of patients. BCVA was fully maintained through to week 52 with both 16-and 12-week dosing regimens. Patients treated with faricimab dosed every 16 weeks had a mean improvement of 11.4 letters from baseline, compared to 10.1 letters in patients treated with faricimab injected every 12 weeks, and 9.6 letters in patients treated with monthly 0.5 mg ranibizumab. The three treatment regimens were similar in both the proportion of patients gaining more than 15 letters and avoiding a loss of more than 15 letters. Comparable reductions in central retina thickness were also observed [173]. The rates of ocular and systemic adverse events observed with faricimab were similar to the rates observed with ranibizumab. No new safety signal was observed [173].
Phase 3 studies with faricimab are ongoing. OPT-302 (Opthea Ltd., Melbourne, Australia), a VEGF-C/D trap molecule, has been devised to be used in conjunction with existing standard-of-care anti-VEGF-A therapies [174]. The results of a phase 1/2a study that included 51 NVAMD patients showed that OPT-302 was noninferior as both monotherapy and in combination with ranibizumab [175].

Designed Ankyrin Repeat Protein in NVAMD Treatment
Repeat proteins are the second most abundant protein classes functioning in protein-protein binding after immunoglobulins [176,177]. Proteins composed of ankyrin repeats (ARs), a motif of 33 amino acid residues, are important in modulating diverse cellular pathways necessary for the evolution of a more complicated multicellular organism [177]. Designed ankyrin repeat protein (DARPin) molecules, which are composed of natural ARs, are small, single-domain proteins that can selectively bind to a target protein with high affinity and specificity [178,179].
Besides their high selectivity and affinity, DARPin molecules also display remarkable stability that confers some advantages over currently available antibodies or antibody fragments as potential therapeutics. In addition, DARPin molecules can be specifically designed to modulate local or systemic pharmacokinetics [179,180]. DARPins have different clinical applications, including biochemical research, diagnosis, and therapy [181]. As a diagnostic tool, a specific DARPin for human epidermal growth factor receptor 2 was developed, demonstrating similar sensitivity but significantly higher specificity than a FDA-approved antibody [182]. Additionally, DARPins have been involved in preclinical tumor targeting, tumor killing, and as a vehicle in tumor therapy [183][184][185].
DARPins can be used in their monovalent form or conjugated to other chemical moieties, e.g., polyethylene glycol, for half-life modulation. DARPins can also be engineered to produce bi-specific or tri-specific compounds that bind different epitopes of the same target protein or two different target proteins [179]. A notable example of a tri-specific DARPin is MP0250, a drug candidate that can bind VEGF-A and hepatocyte growth factor (HGF) as well as one molecule of MP0250 binding two molecules of human serum albumin (HSA), to extend half-life [186]. MP0250 has successfully completed preclinical development and is currently in phase 2 clinical development for relapsed refractory multiple myeloma [187]. The strategy of double VEGF/HGF inhibition was suggested by the observation that resistance to VEGF inhibition can be induced by the activation of HGF/cMet signaling pathway [186]. In the same line of reasoning, bi-specific or tri-specific DARPins might be conceived and projected in the field of AMD, targeting the simultaneous inhibition of VEGF and PDGF or any other of the factors involved in the pathogenesis of disease, as described above. At present, we have MP0112, a DARPin engineered to bind VEGF-A with a K d of 1-4 pmol/L [188,189]. Abicipar pegol (AGN-150998, MP0112, abicipar; Allergan plc/Molecular Partners) is a 14-kDa recombinant DARPin protein coupled to a 20-kDa polyethylene glycol (PEG) moiety to yield a 34-kDa molecule [190].
MP0112 was tested in a series of clinical trials for treatment of AMD [191] and DME [192]. The safety and effectivity of the DARPin MP0112 in treatment naïve NVAMD patients was primarily assessed in a phase 1/2, multicenter, open-label, and dose-escalation study [191]. Six cohorts of patients received a single MP0112 dose of 0.04 mg, 0.15 mg, 0.4 mg, 1.0 mg, 2.0 mg, or 3.6 mg, respectively. The BCVA remained stable and did not show any difference among the dosing cohorts during the study follow-up. CMT reduction was greater in those cohorts receiving 1.0 mg and 2.0 mg MP0112 (−95 µm and −111 µm, respectively), whereas patients who received 0.04 mg, 0.15 mg, and 0.4 mg of MP0112 had CMT reduction of −7 µm, −12 µm, and −62 µm, respectively. Treatment-related adverse events were reported in 13 (41%) patients, including one (3%) case of severe ocular inflammation with hypopyon in a patient who received 2.0 mg of MP0112 [191].
CEDAR and SEQUOIA were two randomized, multicenter, double-masked, parallel-group, active-controlled, 2-year, and phase 3 clinical trials designed to compare the efficacy and safety of abicipar 2 mg (administered at baseline, week 4, week 8, and every 8 weeks thereafter through week 96) (Abicipar Q8); abicipar 2 mg (administered at baseline, week 4, week 12, and every 12 weeks thereafter through week 96) (Abicipar Q12); and ranibizumab 0.5 mg (administered at baseline and every 4 weeks through week 96) (ranibizumab Q4). The primary end point was stable vision (≤15 ETDRS letter loss) at week 52 [195]. The number of patients with stable vision at week 52 was 496 (93.2%), 481 (91.3%), and 564 (95.8%) in the abicipar Q8, abicipar Q12, and ranibizumab Q4 study groups, respectively. Mean CMT was reduced significantly in all the treatment groups from baseline to week 52 (see Table 9) [195]. At year 2 BCVA improvements achieved during the first year were maintained [196]. Moreover, only four intravitreal injections of abicipar were required to maintain the outcomes, as compared to monthly intravitreal ranibizumab injections [196].  Unfortunately results of phase 3 studies showed that abicipar-treated patients had higher risk of developing intraocular inflammation (IOI) and endophthalmitis compared with ranibizumab-treated patients. The incidence of drug-related ocular adverse events (AEs) was higher in the abicipar Q8 (16.8%) and abicipar Q12 (20.4%) groups than in the ranibizumab Q4 group (4.5%) because of the occurrence of IOI. Intraocular inflammation AEs in the study eye were reported for 96 patients (15.4%) in the abicipar Q8 group, 96 patients (15.3%) in the abicipar Q12 group, and 2 patients (0.3%) in the ranibizumab Q4 group. Uveitis, vitritis, and retinal vasculitis were the main IOI AEs. The occurrence of these complications led to both a higher rate of study discontinuations and a higher rate of vision loss in the abicipar groups. The most commonly reported treatment related adverse events have been summarized in Table 10.
As a result of the improvements in the manufacturing process, the incidence of intraocular inflammation (IOI) was 8.9 percent in the MAPLE study, which was lower than the rate observed in prior Phase 3 studies [197]. Most IOI events were assessed as mild to moderate in severity. The incidence of severe IOI was 1.6 percent with one reported case of iritis and one reported case of uveitis. There were no reported cases of endophthalmitis or retinal vasculitis in this study.
This IOI rate was lower than that observed in the abicipar arms in the SEQUOIA and CEDAR studies but higher than the rate observed in the ranibizumab arms in those studies [195].
In June 2020 the FDA has issued a Complete Response Letter to the Biologics License Application (BLA) for Abicipar pegol.
The letter from the FDA indicates that the rate of intraocular inflammation observed following administration of abicipar pegol 2mg/0.05 mL results in an unfavorable benefit-risk ratio in the treatment of NVAMD.   In July 2020 the company decided to withdraw application filings with both the EMA and the Japanese Regulatory Agency (PMDA) for abicipar and it is committed to working with the regulatory agencies to determine the appropriate next steps and discuss requirements for potential resubmissions.
Based on the latest news, the need for the new treatment options for NVAMD remains unmet.

Conclusions
AMD is a prevalent condition that constitutes one of the leading causes of irreversible visual impairment in industrialized countries. The overall prevalence is approximately 8.7%, although it varies significantly among different populations. From a clinical perspective, late-stage AMD can be classified atrophic and neovascular AMD. Although nonexudative AMD represents the most prevalent form, NVAMD is nonetheless a major cause of severe vision loss.
The exact pathogenesis of AMD has not been fully elucidated although the activation of a cascade of proinflammatory and proangiogenic responses, originating from damage to the choriocapillaris, the RPE and the outer retina may play an important role. The most studied factor related to neovascularization in AMD is VEGF-A. According to currently available scientific evidence, there are no clinically important differences in efficacy among the three anti-VEGF agents with which we have the greatest experience, i.e., aflibercept, bevacizumab, and ranibizumab. Moreover, the results achieved in clinical practice seem to be worse than those reported in clinical trials.
The next generation of VEGF inhibitors for ocular applications must offer significant advantages over the currently licensed products. Ideally, the administration should be simplified, and the frequency of intraocular injections should be reduced. DARPins directed against VEGF-A can specifically and potently inhibit angiogenesis and vascular leakage in vitro and in vivo. These treatments have been shown to be as effective as ranibizumab, but with greater durability, which may enhance patient compliance with needed injections and reduce the risk of serious adverse events such endophthalmitis, all of which may lead to better results in clinical practice (i.e., real world results) than have been achieved with aflibercept, bevacizumab, and ranibizumab. The differences in the structure of the paratope of DARPin broaden the spectrum of target molecules, while the ease of creating hybrid fusion proteins provides exceptional opportunities for creating bispecific and multivalent constructs. Faricimab, a bispecific antibody directed against VEFG-A and Ang2, also may offer greater durability than the currently used anti-VEGF agents.
Although there have been new and promising developments in AMD treatment, unmet needs continue. They include the high number of intravitreal injections and indefinite evaluations; the duration of the treatment effects, progression of atrophy or subretinal fibrosis, and scar formation. Additionally, there is a need to identify alternative treatments that target other biological pathways, which might help to halt progression and restore vision in those affected by NVAMD.
Author Contributions: All authors met the ICMJE authorship criteria. All authors contributed to the drafting and critical revision of the manuscript, commented on previous versions of the manuscript, and read and approved the final manuscript prior to submission. All authors have read and agreed to the published version of the manuscript.
Funding: Logistic for writing services has been provided by Allergan, an AbbVie company. Allergan did not participate in either data analysis or redaction of the manuscript.

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