**4. Tie-2/angiopoietin pathway**

Recently, there has been great interest in drug development within the Tie-2 transmembrane tyrosine kinase receptor pathway in exudative diseases such as DME and nAMD. This receptor is found on endothelial cells and is responsive to the opposing angiopoietins, Ang-1 and Ang-2 (see **Figure 2**) [29]. These growth factors are integral players in vessel homeostasis, permeability, and angiogenesis. Ang-1 activates the Tie-2 receptor and leads to vascular stability [30]. Ang-2 acts as a competitive antagonist to Ang-1, turning off the Tie-2 receptor, leading to abnormal vascular growth, leakage, and increased inflammatory signals within endothelial cells [31, 32]. Tumor necrosis factor α (TNFα), a central inflammation mediator, induces Ang-2 release from endothelial cells to enhance its stimulation of inflammation and vascular leakage [33]. Additionally, the enzyme vascular endothelial protein tyrosine phosphatase (VE-PTP) inactivates the Tie-2 receptor and therefore interferes with the vascular stabilizing effects of Ang-1 [34].

A number of preclinical studies have supported the importance of the Tie-2/ Ang-2 pathways in DR and DME pathophysiology. Studies in the developing retina and in ischemic retinal mouse models have shown increased expression of both Ang-2 and VEGF which correlated to increased neovascularization [35, 36]. A study in double transgenic mice expressing both Ang-1 and VEGF showed that increased expression of Ang-1 led to decreased neovascularization and suppression of VEGF [37]. Additionally, high levels of Ang-2 and VEGF have been found in samples from diabetic patients following vitrectomy [38, 39]. During times of stress such as, hyperglycemia, ischemia, and oxidative stress, Ang-2 levels increase and result in aberrant vascular leakage and growth. Therapeutic interventions for exudative diseases may focus on inhibiting Ang-2 or VE-PTP therefore preventing their counterregulatory effects on the Tie-2 receptor.

#### **4.1 Faricimab**

Faricimab, previously RG7716 (Roche, Basel, Switzerland and Genentech, South San Francisco, CA, USA) is a bispecific antibody that binds to and inhibits both VEGF and Ang-2. Phase 1 and 2 trials showed promising results, supporting advancement to phase 3 trials [40, 41]. Results from four phase 3 trials from both patients with DR/DME and nAMD were released in February, 2021 from Roche [42]. All four studies demonstrated non-inferior results when administered in longlasting dosing intervals compared to aflibercept.

#### **Figure 2.**

*Molecular targets and approaches to re-establish homeostasis in Ang–Tie-2 and VEGF–VEGFR pathways (reproduced with permission from [9]).*

The YOSEMITE and RHINE studies are two identical phase 3 clinical studies that compared faricimab to aflibercept in patients with DME. The YOSEMITE and RHINE trials enrolled 940 and 951 patients respectively. These studies compared faricimab 6.0 mg given at individualized intervals (one, two, three or four months based on DR activity), faricimab 6.0 mg given at two-month intervals, and aflibercept 2.0 mg given at two-month intervals. Sham injections were given when patients within a treatment group were not scheduled to receive treatment to maintain blinding. The primary outcomes were average change in best corrected visual acuity (BCVA) at one year. Secondary outcomes were percent of individualized interval arm receiving doses at one, two, three, and four months at 52 weeks, percent of participants with a two-step or more improvement in diabetic retinopathy severity score (DRSS) from baseline, percent of participants with gain and percent without loss of 15 letters or more in BCVA, and change in central subfield thickness [43, 44].

YOSEMITE showed an average improvement of +11.6 ETDRS letters in the individualized interval faricimab arm, +10.7 ETDRS letters in the two-month interval faricimab arm, and +10.9 ETDRS letters in the aflibercept arm. Similarly, RHINE showed an average improvement of +10.8 ETDRS letters in the individualized interval faricimab arm, +11.8 ETDRS letters in the two-month interval faricimab arm, and +10.3 ETDRS letters in the aflibercept arm. Within the individualized interval faricimab arm, there were 151/286 (52.8%) participants in YOSEMITE that were dosed with a four-month interval at one year and 60/286 (21%) that were dosed with a three-month interval. Similarly, in RHINE, 157/308 (51%) participants were dosed with a four-month interval at one year and 62/308 (20.1%) achieved a three-month dosing interval. When compared to the aflibercept two-month dosing interval arm, both studies showed that the participants that achieved up to four-month dosing intervals of faricimab had larger reductions in central subfield thickness (CST) [42]. Roche has announced plan to submit a new drug application to the U.S. Food and Drug Administration and the European Medicines Agency for the treatment of DME and nAMD.

### **4.2 AXT107**

AXT107 (Asclepix Therapeutics, Baltimore, MD, USA) is a peptide that modifies Ang-2 to function more similarly to Ang-1. This peptide is derived from the non-collagenous domain of collagen IV and ultimately activates the Tie-2 receptor and stabilizes vascular permeability [9]. In studies using confluent monolayers of endothelial cells, AXT107 attached to Ang-2 binds to the Tie-2 receptor and disrupts α5β1 integrin, causing Tie-2 and α5 to move to cell junctions. Ultimately, Ang-2 modified by AXT107 serves as an agonist of the now junctional Tie-2 receptor and acts similarly to Ang-1 even in the presence of low concentrations of Ang-1 [45]. AXT107 also suppresses TNF-α induced vascular inflammation in endothelial cells, which may provide additional benefit in treating the chronic inflammatory component of DME and other retinal vascular diseases [33]. Additionally, in animal models, AXT107 increased breakdown of VEGFR2 which ultimately decreases the effects of VEGF [46]. Studies conducted in rabbit eyes injected AXT107 into the vitreous as a clear gel depot. This gel formulation slowly releases the AXT107 and could potentially decrease the need for many repeat injections. The rabbit model studies showed that AXT107 decreased leakage by 86% and 70% at one and two months, respectively. This was compared to aflibercept which reduced leakage by 69% at one month and did not reduce leakage at two months [47].

The CONGO study is a phase 1/2a clinical trial that will evaluate the safety and bioactivity of AXT107. This is a non-randomized open label study with 18 participants with DME. Three treatment arms of low (0.1 mg), medium (0.25 mg), and

high (0.5 mg) doses are included. The primary outcome is safety measured by incidence of adverse effects. Secondary outcomes are efficacy assessed by change in CST, change in BCVA, and percentage of participants improving by greater than 5, 10, 15 letters on the eye chart. This study is expected to be concluded by May 2022 [48].

#### **4.3 Nesvacumab**

Nesvacumab (Regeneron, Tarrytown, NY, USA) is a human immunoglobulin G1 (IgG1) monoclonal antibody that binds to and blocks Ang-2. Nesvacumab was coformulated with aflibercept and phase 1 trials showed promising results for nAMD and DME. The phase 2 ONYX (nAMD) and RUBY (DME) trials did not duplicate that success however; patients treated with nesvacumab-aflibercept combo did not show any significant benefit in BCVA or CST compared to aflibercept monotherapy. For the RUBY study, however, there was a significant difference in the proportion of patients with resolution of foveal edema and ≥2 step improvement in DRSS in the high dose co-formulation arm [49]. In 2017, Regeneron announced that Nesvacumab would not advance to phase 3 trials [50].

#### **4.4 AKB-9778**

AKB-9778 (Aerpio Pharmaceuticals, Cincinnati, OH, USA) is an antagonist of VE-PTP given via subcutaneous injection. The TIME-2a study was a phase 2 study that sought to determine the safety and efficacy of AKB-9778 in DME patients. There were three treatment arms within 144 participants: 15 mg AKB-9778 twice daily and monthly placebo intravitreal injections, 15 mg AKB-9778 twice daily and monthly 0.3 mg ranibizumab intravitreal injections, and placebo subcutaneous injection twice daily and monthly 0.3 mg ranibizumab injections. The primary outcome was mean change in CST at three months and secondary outcomes were BCVA, DRSS, and safety [51]. Mean change CST was significantly greater in participants receiving both AKB-9778 and ranibizumab (−164.4±24.2 μm) than in participants receiving ranibizumab alone (−110.4±17.2 μm). Regarding the secondary outcomes, the percentage of participants that gained ≥10 or ≥15 letters across the treatment arms were: 8.7% and 4.3% in the AKB-9778 alone group, 29.8% and 17.0% in the ranibizumab along group, and 35.4% and 20.8% in the combination group, respectively. The DRSS remained similar across the three groups and AKB-9778 was found to be safe [52].

The safety and efficacy of AKB-9778 in 167 patients with nonproliferative DR was studied in the Time-2b study. This study did not utilize anti-VEGF therapy in its treatment arms and instead used AKB-9778 alone. The treatment arms included: AKB-9778 15 mg once daily, AKB-9778 15 mg twice daily, and subcutaneous placebo injected twice daily [53]. The primary outcome of percentage of participants with improvement in their DRSS was not met at 48 weeks. There was a significant 20% improvement in urine albumin creatinine ratio in patients treated with AKB-9778 twice daily and there was also a significant reduction in intraocular pressure among the treatment groups versus placebo. Based on these results, Aerpio Pharmiceuticals is investigating the use of AKB-9778 in diabetic nephropathy and open angle glaucoma. AKB-9778 is also in a phase 2 study for use in hospitalized subjects with COVID-19 acute respiratory distress syndrome [54].

#### **4.5 ARP-1536**

ARP-1536 (Aerpio Pharmiceuticals, Cincinnati, OH, USA) has the same biologic activity as AKB-9778 however it is delivered by intravitreal injection rather than

subcutaneously. ARP-1536 is an antagonist to VE-PTP which activates the Tie-2 receptor and provides vascular stability. It is in pre-clinical studies in combination with anti-VEGF therapy for DME and wet AMD [54].
