Hyaluronic acid conjugation significantly extends the vitreal half-life of antibody fragments

Abstract

Purpose : 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.

Methods : 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 (R_H) were assessed for in vivo pharmacokinetic performance relative to unconjugated rabFab after intravitreal injection in rabbits.

Results : 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 R_H-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.

Conclusions : 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.