A bioengineered anti-VEGF protein with high affinity and high concentration for intravitreal treatment of wet age-related macular degeneration.

Intravitreal (IVT) injection of anti-vascular endothelial growth factor (anti-VEGF) has greatly improved the treatment of many retinal disorders, including wet age-related macular degeneration (wAMD), which is the third leading cause of blindness. However, frequent injections can be difficult for patients and may lead to various risks such as elevated intraocular pressure, infection, and retinal detachment. To address this issue, researchers have found that IVT injection of anti-VEGF proteins at their maximally viable concentration and dose can be an effective strategy. However, the intrinsic protein structure can limit the maximum concentration due to stability and solution viscosity. To overcome this challenge, we developed a novel anti-VEGF protein called nanoFc by fusing anti-VEGF nanobodies with a crystallizable fragment (Fc). NanoFc has demonstrated high binding affinity to VEGF165 through multivalency and potent bioactivity in various bioassays. Furthermore, nanoFc maintains satisfactory chemical and physical stability at 4°C over 1 month and is easily injectable at concentrations up to 200 mg/mL due to its unique architecture that yields a smaller shape factor. The design of nanoFc offers a bioengineering strategy to ensure both strong anti-VEGF binding affinity and high protein concentration, with the goal of reducing the frequency of IV injections.

Quantifying Protein Shape to Elucidate Its Influence on Solution Viscosity in High-Concentration Electrolyte Solutions.

Therapeutic proteins with a high concentration and low viscosity are highly desirable for subcutaneous and certain local injections. The shape of a protein is known to influence solution viscosity; however, the precise quantification of protein shape and its relative impact compared to other factors like charge-charge interactions remains unclear. In this study, we utilized seven model proteins of varying shapes and experimentally determined their shape factors (v) based on Einstein's viscosity theory, which correlate strongly with the ratios of the proteins' surface area to the 2/3 power of their respective volumes, based on protein crystal structures resolved experimentally or predicted by AlphaFold. This finding confirms the feasibility of computationally estimating protein shape factors from amino acid sequences alone. Furthermore, our results demonstrated that, in high-concentration electrolyte solutions, a more spherical protein shape increases the protein's critical concentration (C*), the transition concentration beyond which protein viscosity increases exponentially relative to concentration increases. In summary, our work elucidates protein shape as a key determinant of solution viscosity through quantitative analysis and comparison with other contributing factors. This provides insights into molecular engineering strategies to optimize the molecular design of therapeutic proteins, thus optimizing their viscosity.

Polymorphic nanobody crystals as long-acting intravitreal therapy for wet age-related macular degeneration.

Wet age-related macular degeneration (wet AMD) is the most common cause of blindness, and chronic intravitreal injection of anti-vascular endothelial growth factor (VEGF) proteins has been the dominant therapeutic approach. Less intravitreal injection and a prolonged inter-injection interval are the main drivers behind new wet AMD drug innovations. By rationally engineering the surface residues of a model anti-VEGF nanobody, we obtained a series of anti-VEGF nanobodies with identical protein structures and VEGF binding affinities, while drastically different crystallization propensities and crystal lattice structures. Among these nanobody crystals, the P212121 lattice appeared to be denser and released protein slower than the P1 lattice, while nanobody crystals embedding zinc coordination further slowed the protein release rate. The polymorphic protein crystals could be a potentially breakthrough strategy for chronic intravitreal administration of anti-VEGF proteins.

Spray-Dried Monoclonal Antibody Suspension for High-Concentration and Low-Viscosity Subcutaneous Injection.

Administration of highly concentrated monoclonal antibodies (mAbs) through injection is often not possible as the viscosity can be readily above 50 mPa·s when the concentration exceeds 150 mg/mL. Besides, highly concentrated mAb solutions always exhibit increased aggregation propensity and lower stability, which raise the difficulty for the successful development of highly concentrated mAb formulations. We hereby explored the possibility of suspension as another formulation form for high-concentration proteins to reduce viscosity and maintain stability. Specifically, we demonstrated that spray drying can serve as a process to prepare particles for suspension. Particles prepared from formulations with different mAb/trehalose mass ratios displayed good physical stability and antibody binding affinity, as indicated by circular dichroism, fluorescence spectroscopy, and surface plasmon resonance (SPR)-based bioassay analyses. During spray drying, a surface tension-dominated enrichment of mAb on the particle surface was observed, but this did not show a significant negative impact on mAb stability. Spray-dried particles were subsequently suspended into benzyl benzoate, and the resulting suspension showed good stability and a lower viscosity when compared to its counterpart solution. Furthermore, mAbs recovered from the suspension maintained their conformational structure. Our study demonstrated that the suspension displayed low viscosity and good physical stability, so it may offer novel opportunities for the preparation of highly concentrated protein formulations.

Hypoxia Alters Epigenetic and N-Glycosylation Profiles of Ovarian and Breast Cancer Cell Lines in-vitro.

Background: Glycosylation is one of the most fundamental post-translational modifications. Importantly, glycosylation is altered in many cancers. These alterations have been proven to impact on tumor progression and to promote tumor cell survival. From the literature, it is known that there is a clear link between chemoresistance and hypoxia, hypoxia and epigenetics and more recently glycosylation and epigenetics. Methods and Results: Our objective was to investigate these differential parameters, in an in vitro model of ovarian and breast cancer. Ovarian (A2780, A2780cis, PEO1, PEO4) and triple negative breast cancer (TNBC) (MDA-MB-231 and MDA-MB-436) cells were exposed to differential hypoxic conditions (0.5-2% O2) and compared to normoxia (21% O2). Results demonstrated that in hypoxic conditions some significant changes in glycosylation on the secreted N-glycans from the ovarian and breast cancer cell lines were observed. These included, alterations in oligomannosylated, bisected glycans, glycans with polylactosamine extensions, in branching, galactosylation and sialylation in all cell lines except for PEO1. In general, hypoxia exposed ovarian and TNBC cells also displayed increased epithelial to mesenchymal transition (EMT) and migration, with a greater effect seen in the 0.5% hypoxia exposed samples compared to 1 and 2% hypoxia (p ≤ 0.05). SiRNA transient knock down of GATA2/3 transcription factors resulted in a decrease in the expression of glycosyltransferases ST3GAL4 and MGAT5, which are responsible for sialylation and branching, respectively. Conclusions: These glycan changes are known to be integral to cancer cell survival and metastases, suggesting a possible mechanism of action, linking GATA2 and 3, and invasiveness of both ovarian and TNBC cells in vitro.