Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs.

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.

Novel approach to antiangiogenic factors in age-related macular degeneration therapy.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss among the population above 85 worldwide. There are two main types of AMD: neovascular and dry AMD. Neovascular AMD leads to macular changes resulting from abnormal choroidal neovascularization. Untreated neovascular AMD leads to scar formation and irreversible sight deterioration. Dry AMD in consequence leads to atrophic changes of the macula. The last decades brought a breakthrough in the therapy of neovascular age-related macular degeneration by introduction of, firstly, photodynamic therapy and, later, anti-VEGF agents administered intravitreally in order to stop neoangiogenesis. However, the treatment of dry AMD is still challenging. Among the directions in dry AMD treatment, the most promising are complement cascade inhibitors and complement cascade targeted gene therapy. In the article we outline the main directions in up-to-date experimental and practical approaches to wet and dry AMD therapy with the emphasis on antiangiogenic factors and gene therapy focused on the inhibition of pathological angiogenesis.

Emerging vascular endothelial growth factor antagonists to treat neovascular age-related macular degeneration.

INTRODUCTION: Evolving anti-vascular endothelial growth factor (VEGF) treatments for neovascular age-related macular degeneration (nAMD) include long acting agents, combination strategies involving new pathways, topical agents, sustained-release, and genetic therapy strategies. Areas covered: Brolucizumab and abicipar pegol have smaller molecular size, facilitating higher concentrations and potentially longer duration than current anti-VEGF agents. Agents being combined with anti-VEGFs include OPT-302 (to inhibit VEGF-C and VEGF-D); pegpleranib and rinucumab (to inhibit platelet derived growth factor, PDGF - but both failed to show consistently improved visual outcomes compared to anti-VEGF monotherapy); and RG7716, ARP-1536 and nesvacumab (to activate the Tie-2 tyrosine kinase receptor, which reduces permeability). X-82 is an oral anti-VEGF and anti-PDGF being tested in phase 2 studies. Topical anti-VEGF ± anti-PDGF drugs under study include pazopanib, PAN-90806, squalamine lactate, regorafinib, and LHA510. Sustained-release anti-VEGF delivery treatments, such as the ranibizumab Port Delivery System, GB-102, NT-503, hydrogel depot, Durasert, and ENV1305 aim to reduce the burden of frequent injections. Gene therapies with new viral vectors hold the potential to induce sustained expression of anti-angiogenic proteins via the retina's cellular apparatus, and include AVA-101/201, ADVM-202/302, AAV2-sFLT01, RGX314, and Retinostat. Expert opinion: There are many emerging anti-VEGF treatments that aim to improve visual outcomes and reduce the treatment burden of nAMD.

Vascular Endothelial Growth Factor Antagonists: Promising Players in the Treatment of Neovascular Age-Related Macular Degeneration.

Neovascular age-related macular degeneration (nAMD) treatment has been revolutionized by the introduction of vascular endothelial growth factor antagonists (anti-VEGF), but the need for frequent intravitreal injections poses a heavy burden to patients and physicians. Evolving anti-VEGF therapies include longer duration agents, approaches that target multiple pathways, topical anti-VEGF agents, sustained-release, and genetic therapies. Abicipar pegol, a designed ankyrin repeat protein (DARPin), demonstrated the ability to maintain stable visual acuity with 12-week dosing, but was not approved by the FDA due to higher than usual rates of intraocular inflammation. Conbercept, a recombinant anti-VEGF fusion protein, has been approved in China, and is in Phase 3 trials globally. KSI-301 is an anti-VEGF antibody biopolymer conjugate that allowed 66% of nAMD patients to maintain at least a 6-month treatment-free interval in Phase 1b studies. OPT-302, an inhibitor of VEGF-C/D, will be tested in phase 3 studies that compare anti-VEGF-A monotherapy against combination therapy with OPT-302. Faricimab is a bispecific anti-VEGF/Ang-2 antibody that upregulates the Tie-2 signaling pathway and promotes vascular stability; it is undergoing phase 3 trials with potential for 12- or 16-week dosing. PAN-90806 is a topical anti-VEGF agent that showed the ability to reduce injection frequency by 79% compared to ranibizumab monotherapy in a phase 1/2a trial. Sustained-release anti-VEGF therapies include the ranibizumab Port Delivery System (in phase 3 studies), GB-102 (Phase 2b), OTX-TKI (phase 1), and Durasert (preclinical). Suprachoroidal delivery of the tyrosine kinase inhibitor, axitinib, is in preclinical studies. Genetic therapies in phase 1 studies include RGX-314 and ADVM-022, which introduce a viral vector that modifies the retina's cellular apparatus to create an anti-VEGF biofactory, potentially serving as a one-time treatment. Further investigation is warranted for drugs and delivery systems that hope to advance visual outcomes and reduce treatment burden of nAMD.

Abicipar pegol: an investigational anti-VEGF agent for the treatment of wet age-related macular degeneration.

INTRODUCTION: Several approaches have been investigated for the management of wet age-related macular degeneration (w-AMD); however, the first-line treatment option for w-AMD currently constitutes anti-VEGF agents. Abicipar pegol is a designed ankyrin repeat protein (DARPin), a novel, promising anti-VEGF agent for the treatment of w-AMD and is reviewed in this article. AREAS COVERED: We discuss the pharmacokinetic, pharmacodynamic, clinical, and tolerability profile revealed by phase II REACH, CYPRESS, and BAMBOO and phase III CEDAR and SEQUOIA Trials. These two latter phase III trials revealed the non-inferiority of abicipar pegol administered with a bimonthly and quarterly regimen when compared with monthly ranibizumab. EXPERT OPINION: Abicipar pegol has been proven to be an emerging, promising anti-VEGF agent in the management of w-AMD. The possibility of adopting a quarterly regimen would allow a decrease in treatment burden and improve patient compliance; however, further larger-scale studies should better characterize abicipar pegol clinical efficacy over longer follow-up periods.

New Anti-VEGF Drugs in Ophthalmology.

This review focuses on 5 new anti-VEGF drugs in the advanced stage of clinical development (i.e., phase 3): conbercept, brolucizumab, port delivery system with ranibizumab, abicipar pegol and faricimab. Results of clinical trials and the advantages of each drug compared to the available molecules are discussed in detail.

Abicipar pegol for neovascular age-related macular degeneration.

INTRODUCTION: The development of intravitreal anti-vascular endothelial growth factor (VEGF) therapy has revolutionized management of neovascular age-related macular degeneration (nAMD) and serves as the standard of care for treating this chronic, progressive disease. One shortcoming is the need for frequent intravitreal injections to maintain visual gains, which has led to pursuit of long-acting agents to reduce treatment burden. AREAS COVERED: A literature search was conducted using the keywords 'abicipar pegol' and 'DARPin' on PubMed. EXPERT OPINION: DARPin (Designed Ankyrin Repeat Proteins) molecules such as abicipar pegol offer potential therapeutic advantages over antibodies or antibody fragments, including high affinity, stability, and high molar concentration. The phase III SEQUOIA and CEDAR clinical trials suggest that abicipar allows >90% of patients to maintain stable vision with 12-week dosing intervals, comparable to results achieved with monthly ranibizumab injections. Relative to other anti-VEGF agents, intraocular inflammation has been noted in a concerning percentage of patients, which is hypothesized to be related to the manufacturing process rather than the drug itself. Modifications to reduce pro-inflammatory components resulted in reduced inflammation (8.9%) in the MAPLE study. If this high inflammation rate can be further reduced, abicipar has the potential to decrease treatment burden for nAMD patients.

Innovative therapies for neovascular age-related macular degeneration.

Introduction: Investigational anti-VEGF treatments for neovascular age-related macular degeneration (nAMD) aim to improve visual outcomes and reduce treatment burden; these include long-acting agents, combination strategies, topical agents, sustained-release, and genetic therapies. Areas covered: The authors provide a comprehensive review of investigational therapies for nAMD, focusing on therapies currently in clinical trial. Expert opinion: Long-acting anti-VEGF agents have demonstrated promising results in phase 3 studies, and include Brolucizumab, a single-chain antibody fragment, and Abicipar, a designed ankyrin repeat protein (DARPin). Other unique anti-VEGF agents in current trials include Conbercept - a fusion protein of the VEGF receptor domains, KSI-301 - an anti-VEGF antibody biopolymer conjugate, and OPT-302 - an inhibitor of VEGF-C/D. Strategies to activate the Tie-2 receptor, some in combination with VEGF inhibition, are of interest, with recent trials of Faricimab, ARP-1536, and nesvacumab. Topical anti-VEGF ± anti-PDGF agents, such as pazopanib, squalamine lactate, regorafenib, and LHA510 have shown limited efficacy and/or have not been advanced, although PAN-90806 continues to advance with promising initial results. Sustained-release anti-VEGF treatments, to address treatment burden, include the ranibizumab Port Delivery System, GB-102, NT-503, hydrogel depot, Durasert, and ENV1305. Similarly, genetic therapies, including RGX-314 and ADVM-022, aim to provide sustained anti-VEGF expression from the retina.

Double-Masked, Randomized, Phase 2 Evaluation of Abicipar Pegol (an Anti-VEGF DARPin Therapeutic) in Neovascular Age-Related Macular Degeneration.

Purpose: To evaluate safety and efficacy of the vascular endothelial growth factor binding protein abicipar pegol (abicipar) versus ranibizumab for neovascular age-related macular degeneration. Methods: Phase 2, multicenter, randomized, double-masked comparison (REACH study, stage 3). Patients (n = 64) received intravitreal injections of abicipar 1 mg or 2 mg at baseline, week 4, and week 8 (3 injections) or ranibizumab 0.5 mg at baseline and monthly (5 injections). Results: In the abicipar 1 mg (n = 25), abicipar 2 mg (n = 23), and ranibizumab (n = 16) arms, respectively, least-squares mean best-corrected visual acuity (BCVA) change from baseline was +6.2, +8.3, and +5.6 letters at week 16 (primary endpoint) and +8.2, +10.0, and +5.3 letters at week 20. Least-squares mean central retinal thickness (CRT) reduction from baseline was 134, 113, and 131 µm at week 16 and 116, 103, and 138 µm at week 20. Intraocular inflammation adverse events (AEs), reported in 5/48 (10.4%) abicipar-treated patients, resolved without sustained vision loss or other sequelae. Conclusions: Abicipar demonstrated durability of effect: BCVA and CRT improvements were similar between abicipar and ranibizumab at weeks 16 and 20 (8 and 12 weeks after the last abicipar injection and 4 weeks after the last ranibizumab injection). No serious AEs were reported.

Designed Ankyrin Repeat Proteins: A Look at their Evolving Use in Medicine with a Focus on the Treatment of Chorioretinal Vascular Disorders.

BACKGROUND: Antibodies constitute an important drug development platform for drugs to treat several ophthalmic, oncologic, and immunologic conditions, but due to limitations inherent in antibody production and structure, a wide range of other protein binding scaffolds are being investigated. Designed ankyrin repeat proteins (DARPins) are simple to produce and offer a range of advantages over antibodies because of their stability, high binding affinity, and rigid structure. OBJECTIVE: DARPins are being developed for a wide variety of medical applications, and the most studied molecule, abicipar pegol, is used to treat chorioretinal vascular diseases. This mini-review will discuss the current state of DARPin technology and will summarize drug development with a focus on abicipar. METHODS: PubMed searches with keywords "DARPin" and "designed ankyrin repeat proteins" were performed and the reference lists of identified articles were examined for additional research material. Studies using DARPin molecules were identified at Clinicaltrials. gov. Non-peer reviewed data were found through Google searches of pertinent websites. RESULTS: Abicipar prevents angiogenesis by binding all isoforms of vascular endothelial growth factor (VEGF)-A with single-digit picomolar affinity. Abicipar has a long intraocular half-life in rabbits and has produced promising results in pre-clinical studies. Pivotal phase III registration trials for the treatment of neovascular age-related macular degeneration are ongoing and a phase II/III trial for the treatment of diabetic macular edema has been announced. CONCLUSION: Abicipar pegol has the potential to effectively treat chorioretinal vascular conditions with an extended duration of action beyond those of currently used anti-VEGF drugs.