Poster X17 - Dendrimer-enabled Intracellular Delivery of Aflibercept Fusion Protein: Advancing Nano therapies for Age-related Macular Degeneration and beyond

Introduction: Age-related macular degeneration (AMD) is a leading cause of blindness in adults over the age of 60 in developed countries worldwide. Currently, an estimated 1.5 million people have progressive exudative (wet) AMD, and it is projected that the incidence of wet AMD will increase to 2.5 million by 2025. Choroidal neovascularization (CNV) is a major pathological feature of neovascular AMD and primarily affects the macula, impacting central vision while leaving peripheral vision unaffected. Vascular endothelial growth factor (VEGF) has emerged as a key treatment target, and the fusion protein aflibercept (Eylea), which binds to extracellular VEGF, is used in the current standard of care for wet AMD. However, the periodic intravitreal injection of aflibercept still does not work in a subset of patients. Targeted therapies that can address multiple pathways, including inflammation, intracellular VEGF production, and neovascularization, and that deliver drugs specifically to affected cells in the choroid and retina, will be highly important. We explore dendrimer-conjugated antibodies and fusion proteins (e.g., afibercept®) for extracellular and intracellular attenuation of disease targets.

Materials and

Methods: We have developed a hydroxyl-terminated poly(amidoamine), PAMAM dendrimer-conjugated aflibercept (D-aflibercept) targeting choroidal/retinal microglia and macrophages. The synthesis of D-aflibercept was carried out using generation 6 hydroxyl PAMAM dendrimer (D6-OH), and conjugation was carried out using a trans-cyclooctene-tetrazine (TCO-Tz) strain-promoted click chemistry strategy under catalyst-free, mild conditions. The PEGylated trans-cyclooctene (PEG4-TCO) labeled hydroxyl PAMAM dendrimer and PEGylated tetrazine(PEG2-Tz) labeled aflibercept were reacted under physiological conditions to produce dendrimer-aflibercept (D-aflibercept).

Results, Conclusions, and Discussions: Results
The novel dendrimer conjugate, D-aflibercept, without the assistance of a targeting ligand, specifically targeted reactive macrophages and retinal pigment epithelial (RPE) cells in the disease-affected area, with minimal uptake in healthy tissue/cells. Also, intracellular dendrimer-aflibercept led to significant CNV suppression over 21 days in a mouse laser-induced CNV model, performing better than intravitreal aflibercept (positive control) and vehicle controls. Intravenous dendrimer-aflibercept conjugates are retained in the CNV lesions for at least 28 days in this model. At longer times, dendrimer-conjugation led to improved attenuation of intracellular VEGF, compared to free aflibercept, leading to improved CNV results. Delivering aflibercept into the key pathological cells via dendrimer significantly improves efficacy compared to free aflibercept.

Conclusions
The delivery of aflibercept to intracellular targets in key pathological cells via dendrimer significantly improves efficacy compared to free aflibercept. Our study also shows that D-aflibercept constructs suppress both extracellular and intracellular VEGF produced by macrophages and RPE, while aflibercept only removes extracellular VEGF.

Discussion
The study lays the groundwork for the development of dendrimer-enabled intracellular delivery of antibodies and fusion proteins, as demonstrated through conjugation of aflibercept for the treatment of AMD in animal models. Maintaining the extracellular properties of these moieties, while providing for the additional benefits from intracellular delivery of these large moieties has significant implications. Targeting intracellular VEGF with dendrimer-conjugated aflibercept is therefore a promising strategy for controlling CNV and preventing wet AMD-induced blindness.

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