FormalPara Key Summary Points

Therapeutic agents primarily targeting inhibition of vascular endothelial growth factor (VEGF)-A have been a major advance for treatment in oncology and retinal vascular diseases; however, there remains a need for improved therapies addressing additional mediators of the underlying pathophysiology because many patients do not respond optimally to current treatments.

Other ligand angiogenic mediators, VEGF-C and VEGF-D, are important in the pathogenesis of cancer and retinal vascular diseases; and evidence shows they are upregulated after VEGF-A suppression.

Broader inhibitory targeting of other members of the VEGF family of ligand–receptor signaling pathways, including ligands VEGF-C and/or VEGF-D, along with VEGF-A, as well as associated vascular endothelial growth factor receptors (VEGFR)-1, -2, and -3, may improve treatment outcomes.

The current review provides a summary of novel promising investigational therapies including those targeting VEGF-C and VEGF-D ligand–receptor signaling pathways currently in clinical development for retinal diseases.

Introduction

The vascular endothelial growth factor (VEGF) signaling family includes a group of secreted ligand glycoproteins and their receptors that together play an important role in the regulation of vasculogenesis, angiogenesis, and vascular homeostasis in a wide variety of tissues [1, 2]. The VEGF family consists of the protein ligands, VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PIGF), and virally encoded non-mammalian VEGF-E. With the exception of the last member, the first five genes of the VEGF family exist in mammalian genomes, including those of humans, and have a wide range of tissue distribution and functions [2]. The VEGF signaling family also includes three vascular endothelial growth factor receptors (VEGFR), VEGFR-1, VEGFR-2, and VEGFR-3, which are expressed on vascular endothelium. Aside from their essential role in vascular homeostasis, members of the VEGF ligand–receptor signaling family also play pathogenic roles in cancer by supporting tumor growth and metastasis [3], and also in retinal vascular diseases [4], primarily through effects on angiogenesis and vascular permeability [1]. Specific ocular disease examples include macular neovascularization, also known as choroidal neovascularization (CNV), in the retina, and anterior segment neoangiogenesis in proliferative diabetic retinopathy (PDR) and retinal vein occlusion [4,5,6,7].

Most research has focused on the ligand VEGF-A, for which signaling appears to contribute to tumor aggressiveness by mediating cancer cell migration and invasion [8], activating survival pathways, and promoting resistance to chemotherapy [9]. Additionally, VEGF-A is a major contributor to the pathogenesis of several angiogenesis-driven eye diseases, including the neovascular form of age-related macular degeneration (nAMD) as well as diabetic macular edema (DME) [10]. VEGF-A also regulates vascular permeability [4], and contributes to inflammation by inducing vascular cell adhesion molecule 1 (VCAM-1) expression, which enhances leukocyte recruitment and endothelial cell adhesion [11].

Since the identification of VEGF-A nearly 45 years ago, the targeting of VEGF-A, VEGFRs, or their signaling pathways has been studied in the context of manipulating pathological angiogenesis in a variety of disease indications, leading to the approval of several therapeutic inhibitors [12]. Although the mechanistic focus of these inhibitors has largely centered around VEGF-A, some therapeutic options use unique, dual or multitarget inhibition. The continued development of therapies that target these pathways has revolutionized treatment in retinal vascular diseases. However, unmet needs for improved outcomes in the treatment of these conditions remain because many patients do not respond optimally or may lose initial vision responses over time, despite requiring frequent injections. Specifically, retinal treatments with better efficacy and longer durability are still needed to help improve disease management. In this review, we will focus on the biology of the VEGF-C and VEGF-D ligands and their signaling receptors, as well as their role in retinal vascular diseases. This article is based on previously conducted studies and does not contain any new studies with human participants or animals.

VEGF Family of Ligands and Receptors: Understanding the Biology and Role in Disease

VEGF-A binds to both VEGFR-1 and VEGFR-2, whereas VEGF-B binds only to VEGFR-1 [2]. Unprocessed (i.e., full length) VEGF-C binds and activates VEGFR-3, whereas mature forms (i.e., proteolytically processed) of VEGF-C and VEGF-D can bind and activate both VEGFR-2 and VEGFR-3, which induces growth of both blood vessels and lymphatics [13, 14]. Most research around the VEGF family has been focused on VEGF-A because it was the first ligand member to be identified as a key regulator of angiogenesis both during homeostasis and in disease [1].

VEGF-A is considered a major regulator of angiogenesis and also plays a role in regulating vascular permeability by binding to VEGFR-2 to stimulate endothelial cell proliferation [12, 15]. The importance of VEGF-A signaling in solid tumors is supported by the association between elevated intratumoral VEGF-A expression and poorer prognosis or more aggressive disease [16,17,18,19,20,21]. Indeed, VEGF family members have been evaluated as potential diagnostic and prognostic biomarkers for several solid cancers [22, 23]. Various VEGF-A blocking therapies are currently approved and used to treat patients with cancer.

Although the role of VEGF-A in disease is the most studied, VEGF-C and VEGF-D also play important roles in the pathogenesis of cancer and other diseases. VEGF-C binding to VEGFR-2 has garnered attention in oncology because of its angiogenic and vascular permeability activity. In addition, it has been reported that VEGF-C maintains VEGFR-2 activation in patient-derived glioblastoma cell lines treated with the anti-VEGF-A agent bevacizumab, potentially explaining its transient treatment response in patients with glioblastoma [24]. Further supporting this concept are reports that VEGF-C and VEGF-D are upregulated in response to VEGF-A inhibition with aflibercept or bevacizumab, or in conditions of experimentally induced underexpression of VEGF-A, which may in part contribute to the clinical sub-responsiveness reported in many patients treated with anti-VEGF-A monotherapy in both oncology [25,26,27,28,29] and ocular diseases [30]. In several types of cancers, VEGF-C is thought to be most important for lymphangiogenesis; a positive association between VEGF-C and lymph node metastasis has been reported, which has led to substantial interest in this relationship [31]. Further studies have shown that lymph node metastasis and other clinicopathological factors are dependent on the increased expression of both VEGF-C and VEGF-A but not on increased VEGF-C expression in the absence of increased VEGF-A expression, indicating a synergistic relationship between the two molecules. Similar to VEGF-C, VEGF-D is involved in inducing tumor angiogenesis and lymphangiogenesis [32]. It is also associated with metastatic spread, potential resistance to anti-angiogenic drugs, poor outcomes, and may be involved in modulating the immune response to cancer [32].

The VEGF family is also involved in the pathogenesis of neovascularization in nAMD, PDR, DME, retinal vein occlusion, and retinopathy of prematurity via their effects on angiogenesis and vascular permeability [12, 33, 34]. VEGF-A, -C, and -D, along with several other angiogenesis-promoting growth factors, are expressed in the retinal pigment epithelium (RPE) of surgical specimens of patients with nAMD; the lack of lymph vessels in ocular tissue suggests that VEGF-C and VEGF-D play a role in modifying ocular angiogenesis by stimulating this process [35]. An in vitro study using human RPE cells and human choroidal fibroblast cells implicated involvement of inflammatory mediators in the pathogenesis of nAMD by showing that age-related macular degeneration (AMD)-associated inflammatory cytokines induced substantial increases in the expression and secretion of VEGF-A and VEGF-C [36]. Additionally, both VEGF-C and VEGF-D have been detected in human vitreous; expression levels are positively associated with age and are affected by disease states, including AMD [37]. In patients with nAMD, VEGF-C and its cognate receptors (VEGFR-2 and VEGFR-3) are expressed in CNV membranes, plasma levels of both VEGF-C and VEGF-D are elevated, and the level of VEGF-D is elevated in aqueous humor [37, 38]. These findings further support the participation of VEGF-C and -D in pathogenic angiogenic processes in the absence of lymphangiogenesis. VEGF-C signaling via VEGFR-2 plays a critical role in control of vascular permeability and increased migration of capillary endothelial cells; this biological role of controlling vascular permeability is redundant with VEGF-A [39]. VEGF-C, like VEGF-A, can induce the formation of endothelial cell fenestrations, which leads to increased vascular permeability and is associated with vascular leakage and edema formation [40]. VEGF-C-induced vascular permeability is mediated through the binding and activation of VEGFR-2 [39].

Findings from several studies provide evidence of the involvement of VEGF-C/VEGFR-2 in PDR/DME. The human retina has a greater level of VEGFR-2 expression in patients with diabetes than in patients without diabetes, and VEGFR-2 is concentrated in microvascular endothelial cells, including those in the macula region [41, 42]. Furthermore, individuals with diabetes have greater retinal VEGF-C expression [43]. VEGF-C potentiates the angiogenic actions of VEGF-A by binding to VEGFR-2 and inhibiting tumor necrosis factor-α and hyperglycemia-induced apoptosis of microvascular endothelial cells [43]. These findings indicate that the interaction between VEGF-C and VEGFR-2 may play a functional role in the pathogenesis of both diseases.

VEGF-D signaling via either VEGFR-2 or VEGFR-3 plays a role in remodeling blood vessels and lymphatics [44]. VEGFR-3 is primarily considered critical for lymphangiogenesis, although it is also able to promote conversion of endothelial cells from a tip cell phenotype to a stalk cell phenotype at blood vessel sprout fusion points [45], and VEGFR-3 inhibition causes suppressed blood vessel sprouting, vascular density, vessel branching, and endothelial cell proliferation [46]. Although VEGFR-3 is universally expressed by the lymphatic epithelium, it can also be expressed by vascular endothelial cells during embryonic development and during active vessel remodeling in pathogenic processes [47]. VEGFR-3 has emerged as a useful marker of the proliferative vascular endothelial phenotype [47].

Both VEGF-C and VEGF-D are able to activate the proliferation of endothelial cells in vitro, and VEGF-D has been shown to promote angiogenesis in a corneal angiogenesis model [48, 49]. Interestingly, hypoxia-induced retinal expression of VEGF-C can induce pathological retinal neovascularization to an extent similar to that of VEGF-A [50]. In a study of the effects of adenoviral delivery of VEGF family ligands into skeletal muscle, VEGF-D was found to have a stronger angiogenic and lymphangiogenic effect than VEGF-A, VEGF-B, or VEGF-C [51].

Overall, there has been more research on the biological function of VEGF-C than of VEGF-D in ocular angiogenesis. Given that individual inhibitors of VEGF-C or VEGF-D have not yet been investigated, it is difficult to quantify and decouple the relative effects of each individual ligand on retinopathy. Broader therapeutic targeting for inhibition of VEGF-C/-D and VEGF-A results in blocking of all ligands that signal through VEGFR-2 and VEGFR-3. Additionally, preclinical in vivo disease models in which this dual-blocking approach was evaluated have demonstrated a reduction of vascular leakage and leukocyte adhesion in diabetic retinal edema, as well as blood vessel growth or leakage in models of angiogenesis [52, 53]. Considering the role of angiogenesis and vascular permeability in the pathogenesis of retinopathy, these two processes are important targets for ophthalmic therapies, and evidence presented in this review suggests that targeting VEGF-C and VEGF-D can inhibit them.

Targeting Ligands VEGF-C, VEGF-D and Receptors VEGFR-2 and VEGFR-3 in Disease

Lessons from Oncology

Therapeutic targeting to inhibit VEGF-A is widely used in oncology and, although treatment responses are often achieved, many patients may experience relapse or lack an adequate or complete therapeutic response [1]. The reasons for this are complex but, in part, may be attributable to the multifactorial pathophysiology of the disease process and compensatory mechanisms in other VEGF ligand and VEGFR signaling. Studies have shown that there is upregulation of VEGF-C and VEGF-D and increased tumor blood flow after VEGF-A inhibition [25,26,27,28,29,30]. Thus, therapeutic targeting of the VEGF-C/-D ligands or their receptors (VEGFR-2/R-3) is attractive for novel drug development. Several tumor models have demonstrated a benefit for VEGF-C/-D blockade, including models of inflammatory skin carcinogenesis, mammary cancer, and bladder cancer [54,55,56]. One study showed that an anti-VEGF-C antibody inhibited tumor growth in an animal model of experimental clear-cell renal cell carcinoma, and the therapeutic activity of anti-VEGF-C was enhanced when combined with bevacizumab [57]. A phase 1 study of LY3022856 (Eli Lilly), an anti-VEGFR-3 monoclonal antibody, demonstrated good tolerability, but had minimal anti-tumor activity in colorectal cancer [58]. Another molecule, VGX-100 (Vegenics Pty Ltd), a fully human monoclonal antibody that binds and inhibits VEGF-C to prevent its interaction with the VEGFR-2 and VEGFR-3 receptors, suppressed corneal angiogenic responses and prevented the trafficking and maturation of antigen-presenting cells in mouse models of corneal transplant [59]. In a phase 1 study, VGX-100 was generally well tolerated at intravenous doses of up 20 mg/kg weekly, as monotherapy and in combination with bevacizumab in patients with advanced solid tumors [60].

Ramucirumab (Eli Lilly), a monoclonal antibody against the receptor VEGFR-2 that effectively neutralizes signal activation by all three extracellular ligands VEGF-A/-C/-D, has been approved for treating gastric cancer, gastro-esophageal junction adenocarcinoma, metastatic non-small cell lung cancer, metastatic colorectal cancer, and hepatocellular carcinoma [61, 62]. The efficacy of ramucirumab in clinical practice is similar to that observed in clinical trials [63]. In metastatic colorectal cancer, ramucirumab is indicated for disease progression after prior therapy with bevacizumab plus chemotherapy [61], indicating that a more comprehensive blockade of VEGF signaling (VEGF-A/-C/-D vs. VEGF-A alone) may be more beneficial for treatment. The trap molecule ziv-aflibercept, a recombinant fusion protein that contains the VEGF binding portions of VEGFR-1 and VEGFR-2, sequestering multiple extracellular ligands VEGF-A, VEGF-B, and PIGF, is approved for treating metastatic colorectal cancer [64]. It is recommended in combination with chemotherapy (5-fluorouracil, leucovorin, irinotecan) and has been established as a safe and effective treatment regimen for metastatic colorectal cancer as second-line chemotherapy [65].

Several inhibitors of protein tyrosine kinases are also approved for treating solid cancers (hepatocellular carcinoma, renal cell carcinoma, lung cancer, and so on); these include sunitinib, sorafenib, axitinib, and pazopanib, which non-selectively target multiple downstream intracellular signaling pathways of angiogenesis driven by varying inhibitory activity against receptor types including VEGFR-1, VEGFR-2, VEGFR-3, platelet-derived growth factor receptor (PDGFR)α/β, and/or C-KIT [66,67,68]. However, these small-molecule anti-angiogenic tyrosine kinase inhibitors (TKIs) inhibit a broad spectrum of serine-threonine and tyrosine kinases, which often means they are associated with toxicity profiles that can prevent long-term clinical use in many patients [62].

Many of the anti-angiogenesis/VEGF-targeting therapies that initially were developed for treating solid tumors, such as bevacizumab, aflibercept, and TKIs, have since been utilized clinically to treat retinal diseases.

Neovascular Age-Related Macular Degeneration

Development of nAMD, and the Role of the VEGF Family in Pathogenesis

There are two subtypes of AMD. Non-neovascular (dry) AMD, encompassing early, intermediate, and late atrophic AMD, accounts for approximately 90% of AMD cases, and neovascular (wet) AMD (nAMD) accounts for the remaining 10% [69]. The global prevalence of AMD among individuals aged 30–97 years is estimated to be 8.7%, and it is projected that 288 million people will be living with AMD in 2040 [70]. The wet (neovascular) form of AMD is a leading cause of irreversible blindness; it affects the macular region of the retina and thus can cause progressive loss of central vision [71].

With nAMD, immune cells are recruited to the macula, where they secrete proangiogenic cytokines (e.g., VEGF-A and VEGF-C) and proinflammatory cytokines (e.g., IL-31, LIF, and SDF1α) [38, 72]. VEGF-A, VEGF-C, and VEGF-D, which play important roles in CNV via their signaling through VEGFR-2 and VEGFR-3, are secreted at high levels by these inflammatory cells and also by several types of retinal cells, including RPE cells [40, 45, 72, 73]. These VEGF ligand molecules stimulate endothelial cell proliferation and migration, angiogenesis, and increased vascular permeability, ultimately resulting in leakage of blood vessel fluid that damages the photoreceptor layer and impairs vision [72].

Therapeutic Strategies Targeting VEGF-C and VEGF-D Signaling Pathways in nAMD

Biologic protein-based therapeutics that primarily target inhibition of VEGF-A represent the current standard of care for nAMD. Despite the great advances made, frequent intravitreal injections are required to maintain the effects of these treatments as a result of their limited durability, and the associated cost is relatively high [74]. After 12 months treatment of standard care VEGF-A inhibition, visual acuity (VA) of 20/40 is achieved in less than 50% of patients, and up to a quarter of patients experience further visual loss during treatment [75]. Some patients with nAMD who receive anti-VEGF-A treatment continue to have persistent fluid or recurrent exudation, and others become resistant to anti-VEGF-A therapy, resulting in a diminished clinical effect [76]. Similar to the findings in oncology, inhibition of VEGF-A leads to increased production of other VEGF family members that can participate in angiogenesis. Bevacizumab treatment in patients with CNV secondary to nAMD results in significantly decreased aqueous humor levels of VEGF-A, but this is accompanied by significant increases in other biomarkers of angiogenesis, including VEGF-C, which is elevated within 1 month of treatment initiation and VEGF-A suppression [30]. These collective findings demonstrate that, given the multifactorial pathophysiology of nAMD, there remains a need to investigate other therapies that target alternative mediators of the disease, which may lead to further improvements in outcomes for patients with nAMD.

The shortcomings of anti-VEGF-A therapy can potentially be improved or overcome through blockade of other ligand members of the VEGF family. Sozinibercept (OPT-302; Opthea Limited, Australia) is a fusion protein “trap” molecule, comprising a modified form of the first three extracellular ligand binding domains of human VEGFR-3, fused to human fragment crystallizable immunoglobulin G1 (IgG1 Fc); it binds to and neutralizes VEGF-C and VEGF-D by preventing ligand binding to the endogenous receptors (VEGFR-2 and VEGFR-3) [77]. Extracellular targeting of VEGF-C and VEGF-D using sozinibercept in combination with VEGF-A inhibition (ranibizumab or aflibercept) has shown promising results in nAMD and DME [77, 78].

Current Treatment Landscape for nAMD

Approved Treatments

The goal of nAMD treatment is to reverse or minimize visual loss and to stabilize or improve visual function [79]. Intravitreal injection of anti-VEGF-A agents is the foundation of nAMD treatment. These agents are considered the most effective method to manage nAMD and should be used as first-line treatment. Bispecific anti-VEGF-A plus anti-angiopoietin 2 therapy was recently introduced and the treatment duration performed favorably to anti-VEGF-A monotherapy in clinical trials [80, 81]. The real-world use and knowledge of bispecific therapy is still evolving [82, 83]. Although photodynamic therapy and laser photocoagulation surgery are approved treatments, they are rarely used in current clinical practice [79, 84].

Currently, there are six commercially available anti-VEGF-A agents to treat nAMD, including bevacizumab (Avastin, Genentech, USA) a humanized monoclonal IgG1 antibody, which is not approved for nAMD treatment but is frequently used off-label. At present, the other agents most common in clinical practice are ranibizumab (Lucentis, Genentech, USA), a humanized IgG1 antibody fragment against VEGF-A and aflibercept (Eylea, Regeneron, USA), a protein “trap” composed of the second and third binding domains of VEGFR-1 and VEGFR-2 respectively, fused with the Fc region of human IgG1. Aflibercept also binds and inhibits VEGF-B and PIGF. Pegaptanib (Macugen, OSI Pharmaceuticals, USA) is a pegylated synthetic RNA-based oligonucleotide (aptamer) that binds only to VEGF-A isoform 165 and was the first available anti-VEGF-A agent for nAMD treatment; however, unlike the other anti-VEGF-A drugs, it does not improve VA [79]. As such, it is rarely used in clinical practice and is no longer available commercially in the USA. Another molecule, conbercept (Lumitin; Chengdu Kang Hong Biotech), a protein comprising the extracellular domain of VEGFR-1 and binding domains 3 and 4 of VEGFR-2 fused to the Fc region of human IgG1, which binds VEGF-A/-B and PIGF, is commercially available for nAMD in China, but the two phase 3 trials in the USA were terminated without reporting the primary endpoint [85, 86]. The more recently approved molecules are brolucizumab (Beovu, Novartis, Switzerland), a humanized single-chain antibody fragment against VEGF-A, and faricimab (Vabysmo, Genentech, USA), a bispecific monoclonal antibody that inhibits VEGF-A and angiopoietin 2, allowing for the inhibition of two pathways involved in the pathology of nAMD [80]. Both brolucizumab and faricimab were formulated to be longer acting than the other anti-VEGF-A agents, which typically require frequent dosing every 4 or 8 weeks, with the hope of fewer injections being required over time (i.e., extended dosing intervals) [80, 87]. Also commercially available are biosimilars for ranibizumab including Razumab (Intas Pharmaceuticals Ltd, India), Cimerli (Coherus Biosciences, USA), Ranivisio (Midas Pharma GmbH, Germany), Ximluci (STADA Arzneimittel AG, Germany), and Byooviz (Samsung Bioepsis, South Korea) [88,89,90,91]. In addition, the first biosimilar for aflibercept, Yesafili (Biocon Biologics, Germany) was recently approved in Europe and others are in late-stage development [91, 92]. The biosimilars have been developed to coincide with the patent expirations of the originator molecules, and aim to offer a less expensive alternative, even though bevacizumab is still widely used off-label in many countries owing to its lower treatment cost.

Some research has been aimed at understanding the specific advantages of the available anti-VEGF-A therapies. A meta-analysis of randomized clinical trials of patients with nAMD demonstrated that bevacizumab was noninferior to ranibizumab in regard to VA change at 1 year of treatment; however, ranibizumab produced better anatomic results, and bevacizumab was associated with a higher incidence of serious systemic adverse events [93]. Some real-world comparisons of aflibercept and ranibizumab showed no statistically significant differences in VA improvement between these treatments after 1 or 2 years of therapy in patients with nAMD [94, 95]. However, the DRCR.net Protocol T study demonstrated that the average change in VA from baseline to 12 months was greater with aflibercept than with bevacizumab or ranibizumab in patients with DME whose mean baseline Snellen-equivalent VA was 20/50 or worse; safety profiles were comparable for these treatments [96, 97]. Aflibercept targets VEGF-A/-B and PIGF; however, in regard to the improved results in this subpopulation, the relative contribution of PIGF inhibition versus its substantially higher affinity for VEGF-A is unclear [98]. Thus, further prospective analyses are required to determine whether the additional targeting of PIGF can lead to better efficacy than can be achieved by targeting of VEGF-A alone. The superior VA results observed in patients with DME at 1 year for aflibercept versus ranibizumab were not maintained through 2 years; however, in eyes with baseline VA of 20/50 or worse, aflibercept was superior to bevacizumab [97, 99].

A major concern about the current standard of care anti-VEGF-A therapies is their high treatment burden. In an effort to decrease this, recent clinical research has focused on therapeutic approaches with extended dosing regimens, and these include brolucizumab, faricimab, high-dose 8 mg aflibercept (Regeneron, USA), the Port Delivery System (PDS; Susvimo, Genentech, USA) and emerging gene therapies. Brolucizumab dosed every 2 or 3 months was shown to be noninferior to two monthly aflibercept in the HAWK and HARRIER nAMD trials [100], and faricimab proved its potential to extend dosing intervals up to 16 weeks and was not clinically inferior to aflibercept 2 mg every 8 weeks in the TENAYA and LUCERNE nAMD trials [80]. The high-dose 8 mg formulation of aflibercept was recently approved for commercial use on the basis of the PULSAR and PHOTON phase 3 results studying nAMD and DME respectively, with every 12 or 16 week dosing showing non-inferiority at 48 weeks to the aflibercept 2 mg dose given every 8 weeks [101]. The PDS is a surgically placed permanent refillable ocular implant designed to continuously deliver a customized formulation of ranibizumab into the vitreous [102]. The phase 3 Archway study demonstrated that the PDS with ranibizumab 100 mg/ml with fixed 24-week refill-exchanges was non-inferior to that of monthly ranibizumab in patients with nAMD [102]. However, after approval, the device was recalled, and resolution of manufacturing issues are pending [103, 104]. Gene therapy offers an exciting potential to have a one-time administration typically utilising an adeno-associated virus (AAV) to deliver a long-term expression of an anti-VEGF-A protein to manage nAMD [105]. Gene therapies in clinical development include ixoberogene soroparvovec (Adverum Biotechnologies, USA) currently in phase 1/2 trials that utilizes a novel vector capsid, AAV2.7m8 to deliver a gene that codes for an aflibercept-like protein intravitreally, and ABBV-RGX-314 (Abbvie-Regenxbio, USA) an AAV8 vector currently in two phase 2/3 nAMD studies evaluating intraretinal delivery of genes that code for a ranibizumab-like antibody fragment against VEGF-A, and another phase 2  nAMD trial using suprachoroidal delivery of the same gene product [106,107,108]. Some earlier gene therapy candidates including rAAV.sFLT-1 (Avalanche, USA) and AAV2-sFLT01 (Sanofi Genzyme, USA) did not advance past early clinical evaluation and challenges still remain for those in development regarding the optimal target/mode of administration and potential safety concerns [105, 109].

Despite the advances made with VEGF-A inhibitors for the management of nAMD, there still remain unmet needs to be addressed to further improve visual gains and durability of treatment responses for patients. The compensatory mechanisms of upregulation of other VEGF family ligands, such as VEGF-C/-D after VEGF-A suppression, beg the question of whether broader parallel inhibition of other members may improve efficacy.

Investigational Therapies in Clinical Development

Sozinibercept (OPT-302) “Trap” Inhibitor of VEGF-C and VEGF-D

As previously noted, sozinibercept (Opthea Limited, Australia) is a so-called trap biologic that targets both VEGF-C and VEGF-D. It is a soluble fusion protein that contains extracellular domains 1–3 of VEGFR-3 fused to the human IgG1 Fc domain [77]. The extracellular domains of VEGFR-3 act as a ligand trap, binding and sequestering VEGF-C and VEGF-D. Once bound to sozinibercept, VEGF-C and VEGF-D are no longer able to bind or activate their receptors (VEGFR-2 and VEGFR-3) [110]. This, in turn, prevents VEGF-C- and VEGF-D-mediated activation of VEGFR-2 and VEGFR-3 signaling that promote angiogenesis, further inflammatory response, and vascular leakage [72]. Additionally, broader blockade of the ligand signaling pathways is achieved when sozinibercept inhibition of VEGF-C/-D is used in combination with standard-of-care therapies primarily targeting VEGF-A, as illustrated in Fig. 1.

Fig. 1
figure 1

The vascular endothelial growth factor (VEGF) family of ligands includes mammalian VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PIGF), which each have selective interactions with three cell-surface receptors: VEGFR-1, VEGFR-2, and VEGFR-3. Currently approved standard-of-care treatments are shown in green boxes, and these biologics primarily target the inhibition of VEGF-A, acting through the VEGFR-2 receptor (aflibercept also targets VEGF-B and PIGF, whereas faricimab also targets angiopoietin 2 [Ang-2]). Emerging therapies are shown in blue boxes; these have additional effects for either inhibiting extracellular VEGF-C and/or VEGF-D ligand binding or disrupting intracellular receptor signal transduction pathways reducing VEGFR-2- and/or VEGFR-3-mediated angiogenesis and vascular permeability associated with the pathogenesis of retinal diseases including nAMD. (Refer to the text for more detail on the mechanism of action for each therapeutic agent.) Assistance with the preparation of this figure was provided by IsoForm

Full size image

In a laser-induced mouse model of CNV (a major cause of visual loss in patients with nAMD), the ability of anti-VEGF-C/-D monotherapy to reduce vascular leakage and CNV area was similar to that of aflibercept (which targets VEGF-A/-B and PIGF); and when used in combination, the efficacy was superior to that of either therapeutic approach alone [52].

The biodistribution of sozinibercept also warrants consideration. Free sozinibercept binds VEGF-C or VEGF-D to form a stable, inert complex; it is not expected to undergo degradation in vitreous humor given the low levels of metabolic enzymes present [111]. Intraocular pharmacokinetics for biologic drugs are largely determined by structural properties, particularly molecular weight and dose. Increased molecular weight is associated with longer intraocular half-life. For example, the intravitreal half-life of aflibercept (9–11 days) is longer than that of ranibizumab (7.2 days), which likely can be explained by the larger molecular weight of aflibercept (approx. 115 kDa vs. 48 kDa) [112, 113]. In rabbits, intravitreal administration of sozinibercept has shown a relatively prolonged intravitreal half-life, similar to that of aflibercept; this also may relate to the large molecular weight of sozinibercept (approx. 140 kDa), effectively slowing distribution from the ocular space [114]. After intravitreal injection of 125I-radiolabeled sozinibercept in rabbits, systemic exposure was minimal relative to vitreous exposure, which likely resulted from clearance via absorption into the choroid and aqueous humor outflow. Analysis of the distribution of 125I-sozinibercept in rabbits indicated that concentrations were highest in the retina/choroid and RPE 1 to 12 h after intravitreal administration, similar to observations for 125I-aflibercept and overall exposure was similar for both molecules in ocular tissues.

In regard to its pharmacokinetics in humans, data from the first-in-human phase 1 study demonstrated that sozinibercept concentrations after intravitreal injection are similar whether sozinibercept (2 mg dose) is injected alone or in combination with ranibizumab (0.5 mg) [110]. Systemic exposure for sozinibercept was low (half-life [t1/2] = 8 ± 2 days; mean maximum serum concentration [Cmax] = approx.  21 ng/mL) at  approximately 31 h after intravitreal administration, and no accumulation occurred after repeated multiple intravitreal dosing every 4 weeks [110].

In the phase 1 first-in-human trial, the investigators evaluated sozinibercept (up to 2 mg) given by repeat intravitreal injections, once every 4 weeks, either as monotherapy or in combination with ranibizumab (0.5 mg) in 51 patients with nAMD [110]. In general, sozinibercept was safe and well tolerated, and improved visual and anatomic responses were observed with sozinibercept/ranibizumab combination therapy through week 12 [110]. The mean change in best-corrected VA (BCVA) from baseline to week 12 was greater in treatment-naïve patients (+ 10.8 letters; 95% confidence interval [CI], 4–17) than in those who previously received anti-VEGF-A monotherapy (+ 4.9 letters; 95% CI, 3–7) [110].

A subsequent phase 2b study of sozinibercept 0.5 mg, 2.0 mg, or sham, each combined with ranibizumab 0.5 mg, was conducted in treatment-naïve patients with nAMD [77]. Intravitreal injections were given at 4-week intervals, for a total of six treatments. The mean improvement in Early Treatment of Diabetic Retinopathy Study (ETDRS) BCVA from baseline to week 24 was statistically superior with 2 mg sozinibercept combination therapy (+ 14.2 letters) in comparison to ranibizumab sham control (+ 10.8 letters) (p = 0.01). Secondary outcomes (proportion of participants gaining or losing ≥ 15 ETDRS BCVA letters and reductions in intraretinal fluid and/or subretinal fluid on spectral domain optical coherence tomography for baseline vs. week 24) were supportive and also favored the sozinibercept 2 mg combination group. Notably, greater reductions in CNV and total lesion areas from baseline to week 24 were observed with sozinibercept combination therapy in the phase 2b study [115]. Previous findings have shown expression of VEGF-C/-D occurs in endothelial cells involved in CNV, suggesting a rationale contributing to the increased efficacy for combined sozinibercept/ranibizumab treatment compared to ranibizumab monotherapy [115]. The incidence of adverse events was similar for sozinibercept combination therapy and standard ranibizumab monotherapy. Based on the promising results of the phase 1 and phase 2b studies, two phase 3 clinical studies are being conducted concurrently to investigate sozinibercept in combination with standard-of-care therapy in treatment-naïve patients with nAMD: ShORe (sozinibercept plus ranibizumab; [NCT04757610) and COAST (sozinibercept plus aflibercept; NCT04757636]) [116, 117].

Tyrosine Kinase Inhibitors

In contrast to the biologic therapies that target extracellular ligands of the VEGF family preventing their binding to VEGF receptors, the TKIs are small molecules that act intracellularly to prevent downstream signaling cascade pathways of the VEGFRs and possibly those of other receptor types. Novel sustained-release polymer drug-delivery systems are being utilized to improve on the safety and pharmacokinetic challenges with small molecule TKIs, with the aim of showing durable treatment effects in nAMD owing to the potential for broad VEGFR inhibition [109, 118]. Those in clinical-stage development include vorolanib (EYP-1901; Eyepoint, USA) using Durasert delivery technology, axitinib (CLS-AX; Clearside, USA) being developed as an injectable suspension, and Axpaxli (Ocular Therapeutix, USA) which is an intravitreal hydrogel implant [119,120,121,122]. Each of these TKIs target the extracellular domains of VEGFR-1/-2/-3, putatively inhibiting all VEGF signaling (Fig. 1) as well as PDGFRα/β [119,120,121,122]. Of these TKIs, axitinib has the highest affinity for VEGFRs [119]. In early-stage clinical trials, these TKIs have shown biological activity with minimal safety concerns in patients with nAMD. Recent interim results from the phase 2 DAVIO 2 clinical trial in which 160 patients with previously treated nAMD demonstrated that the change in vision at 28 and 32 weeks after EYP-1901 (2 mg and 3 mg) of + 1.0 and + 0.9 letters versus the + 1.3 letter change for aflibercept (+ 1.3 letters). In addition, approximately 65% of patients receiving EYP-1901 did not require rescue treatment for up to 6 months with good tolerability [123]. CLS-AX is a suprachoroidal injection of axitinib and has been evaluated in the phase 1/2a OASIS trial in prior-treated patients with nAMD, where 57% and 67% receiving the 0.5 mg (n = 7) and 1 mg (n = 5) doses, respectively, were rescue therapy free at 6 months with no SAEs reported [124]. Intravitreally administered Axpaxli has completed a phase 1 nAMD study in which 23 patients at doses of 0.2–0.6 mg, with 61% and 15% across all-dose subjects not needing rescue therapy at 6 and 12 months, respectively, and the hydrogel polymer insert was well tolerated [125]. A follow-on phase 1b study in 21 previously treated patients with 0.6 mg Axpaxli showed 73% and 33% were rescue-free at 6 and 12 months with no SAEs [126]. A phase 3 trial in treatment-naïve patients aims to assess Axpaxli versus aflibercept after two loading doses of aflibercept with the primary endpoint of the proportion of patients with ≤ 15 letters loss from baseline at 36 weeks [127, 128].

Several difficulties with systemic and topical delivery of TKIs in development highlight the challenges associated with those routes of administration for small molecules. When administered systemically, TKIs have been associated with unfavorable side effects, including hematological, cardiovascular, gastrointestinal, and endocrine events; and skin toxicity [129]. For example, X-82 (an oral pan-VEGFR and PDGFR inhibitor) caused dose-dependent transaminase elevations in several participants of a phase 1 trial in nAMD [130]; moreover, 29% of patients did not complete the 24-week endpoint, and 17% withdrew as a result of adverse events. Another challenge with small-molecule TKIs is their wide pharmacokinetic variability [131]. Research on other TKIs that were in clinical development for topical administration (eye drops) was halted because of lack of clinical efficacy, possibly relating to poor bioavailability of the small molecules at the back of the eye; these TKIs include pazopanib, regorafenib, and squalamine [132].

4D-150 Dual-Transgene Vector

Another drug in early clinical development is 4D-150 (4D Molecular Therapeutics, USA), which utilizes an intravitreal vector (R100) and a transgene payload expressing aflibercept and a inhibitory RNA to inhibit the expression of VEGF-C [133]. Together, this payload targets the inhibition of VEGF-A, -B, and -C, as well as PIGF (Fig. 1). Altogether, 88 patients with nAMD that received 4D-150 in either a phase 1 PRISM dose exploration (N = 15), dose expansion (N = 41), or a phase 2 population extension (N = 32) had no clinically significant treatment-emergent inflammation reported [133]. Recent data from the randomized PRISM dose expansion demonstrated favorable tolerability and clinical activity for 4D-150 in patients treated previously for nAMD [134]. Enrolled patients were assigned randomly (2:2:1) to receive a single intravitreal high dose (3E10 vg/eye) or low dose (1E10 vg/eye) of 4D-150 or a regimen of intravitreally administered aflibercept (2 mg) every 8 weeks (control). By 24 weeks of follow-up, 63% and 50% of patients did not require supplemental aflibercept injection in the high- and low-dose 4D-150 arms, respectively. The change from baseline in BCVA to week 24 was − 1.8 and + 1.8 letters for the high- and low-dose arms, respectively, and central subfield thickness was − 8.3 and + 29.9 µm, respectively [135]. There was no significant intraocular inflammation reported and 97% of patients completed 20 weeks of prophylactic topical corticosteroid taper on schedule. In light of these findings, 4D-150 was granted the “Regenerative Medicine Advanced Therapy” designation from the US Food and Drug Administration for intravitreal treatment of nAMD [134]. Future plans include the first phase 3 trial, which would be a BCVA noninferiority comparison study of the 3E10-vg/eye (high) dose of 4D-150 versus a regimen of aflibercept (2 mg every 8 weeks).

AXT107 Peptide

Another promising agent in early clinical development is the integrin-regulating peptide AXT107 (gersizangitide: AsclepiX Therapeutics, USA), which utilizes a microparticulate suspension suitable for intraocular injection. AXT107 inhibits VEGFR-2 (Fig. 1) and activates TIE-2 [74], mediated by an interaction of the molecule with integrin αvβ3 and integrin α5β1, to suppress vascular leakage. The intraocular injection of AXT107 microparticulate suspension will be explored in the DISCOVER trial, an open-label, single-injection, dose-escalating, 40-week, phase 1/2a clinical study of patients with nAMD, designed to evaluate the safety, tolerability, bioactivity, and duration of action for three dose levels of AXT107.

IBI333 (VEGF-A/VEGF-C Bispecific Fusion Protein)

IBI333 is an investigational anti-VEGF-A/and anti-VEGF-C bispecific fusion protein being developed by Innovent (China and USA) [136]. It can simultaneously bind and neutralize the activities of VEGF-A and VEGF-C, thus inhibiting the potential compensatory mechanism of VEGF-C upregulation after VEGF-A neutralization, to further inhibit VEGF signaling compared with VEGF-A blockade alone (Fig. 1). A phase 1 study is underway in China to evaluate the safety and tolerability of intravitreal injection of IBI333 in patients with nAMD.

Conclusion and Future Perspectives

A growing body of research is providing compelling evidence that, in retinal diseases such as nAMD, the pathophysiology is broader than simply dysregulation or overproduction of VEGF-A. Despite the tremendous progress in standard-of-care intravitreal anti-VEGF-A agents for nAMD, regular injections are often required to maintain treatment responses, while visual gains can be limited in some patients, as evidenced by clinical and real-world studies. More recent treatments based primarily on VEGF-A inhibition have shown noninferiority with extended dosing intervals to earlier agents, offering reduced treatment burden. However, the visual results of the newer agents have not been superior to those of the established anti-VEGF-A inhibitors, which have been the gold standard for nAMD. Patients and clinicians strongly desire improved visual outcomes from the next generation of nAMD treatments. Combination inhibition of extracellular ligands VEGF-A, VEGF-C, and VEGF-D, and/or intracellular blockade of VEGFR signaling, which has been successfully applied in oncology, also may hold promise for addressing some current challenges of nAMD management, which could lead to better outcomes for patients.