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Susan Crowell, Amin Famili, C. Andrew Boswell, Joyce Chan, Julia Gray, Kelly Loyet, Laetitia Comps-Agrar, Amrita kamath, Karthikan Rajagopal; Hyaluronic acid conjugation significantly extends the vitreal half-life of antibody fragments. Invest. Ophthalmol. Vis. Sci. 2018;59(9):235. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Treatment of ocular diseases associated with neovascularization requires frequent intravitreal injection of anti-vascular endothelial growth factor (aVEGF) therapies. Reducing the required frequency of aVEGF injections—and associated clinical visits—may improve patient adherence to the prescribed treatment regimen and improve outcomes. Frequent re-dosing of biologic aVEGF therapies is dictated by their clearance from the vitreous humor; strategies to modify vitreal clearance rates may extend dosing intervals. In this work, we explore conjugation of fragment antibodies (Fab) to the biopolymer hyaluronic acid (HA) as a half-life modifying strategy and assess the impact on Fab biophysical properties and vitreal pharmacokinetics.
Two model Fab molecules—one species-matched rabbit Fab (rabFab) used for rabbit PK experiments and one human aVEGF Fab (Fab1) used for biophysical characterization—were expressed with C-terminal cysteine residues and conjugated to maleimide-modified linear HA of varying molecular weights. HA-Fab1 conjugates were characterized for solution physical properties, melting behavior, and higher-order structure. HA-rabFab conjugates of three distinct molecular weights and hydrodynamic radii (RH) were assessed for in vivo pharmacokinetic performance relative to unconjugated rabFab after intravitreal injection in rabbits.
Covalent conjugation of Fab1 to HA did not measurably alter the biophysical properties of the protein: melting temperature (Fab1: 90.9°C; HA-Fab1: 90.4°C), and secondary and tertiary structure measured by circular dichroism were unchanged. Conjugation to HA significantly slowed the in vivo clearance of rabFab from the rabbit vitreous in a RH-dependent manner. Compared to free rabFab (observed vitreal half-life of 2.8 days), HA-rabFab conjugates cleared with observed half-lives of 7.6, 10.2 and 18.3 days for 40 kDa, 200 kDa and 600 kDa HA conjugates, respectively.
Conjugation of therapeutic Fab molecules to HA resulted in significant half-life extension while maintaining required Fab biophysical properties. This strategy may enable long-acting delivery of proteins targeting posterior ocular diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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