Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye

Abstract

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.

Keywords: age-related macular degeneration; deamidation; high concentration formulation; isomerization; long-acting delivery; protein engineering; solubility; stability.

Figure 1.

Key contacts observed in structure of AFD:CFD complex (4D9R). AFD and CFD are shown as green and aqua ribbons, respectively. Residues in contact with (A) CFD-Lys223 or (B) CFD-Arg172 are shown in space-filling and numbered. Figure prepared using Pymol (Schrödinger).

Figure 2.

Solubility of AFD molecules in 20 mM His-HCl, pH 6.0. Protein concentration in supernatant following centrifugation was determined by UV absorbance measurements. Photograph shows precipitate obtained upon centrifugation of the dialyzed sample.

Figure 3.

Self-association and molecular charge of AFD. (A) One potential cluster from molecular docking simulations of AFD self-association. (B) Electrostatic surface modeled for AFD (left) and AFD.v8 (right). Surfaces calculated and displayed using Pymol (Schrödinger) and AFD coordinates from AFD:fD complex structure (pdb code: 4D9R). AFD.v8 substitutions were modeled onto 4D9R structure.

Figure 4.

Fraction of antibody Fab active for antigen binding as determined using SPR. Protein solutions (100 mg/mL in PBS) were incubated at 37°C for various times.

Figure 5.

Concentration-time profile observed for ranibizumab and AFD.v8 following intravitreal injection (1.0 mg/eye) in rabbits. Concentrations in vitreous humor and retina were determined by ELISA. Pharmacokinetic parameters derived from a noncompartmental analysis are shown in the inset.

Figure 6.

Simulated human vitreous concentration-time profiles for 10- and 25-mg doses. Simulations are based on the previously published population pharmacokinetic model for lampalizumab. The 10-mg and 25-mg dose lines represent predictions for a typical patient.