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.

Pharmacokinetic Simulations of Intravitreal Biologicals: Aspects of Drug Delivery to the Posterior and Anterior Segments

“…This is understandable, because diffusion in the vitreous and clearance to the anterior chamber are inversely related to the cubic root of the hydrodynamic radius ( R h 1/3 ) [8]. More significant delays may be reached by modulating the drug interactions with the vitreous components, such as hyaluronic acid [29]. The vitreous may become more heterogenous and its viscosity may decrease in elderly patients.…”

Toward Drug-Like Multispecific Antibodies by Design

“…A majority of preclinical and clinical ocular therapies utilize antibody fragments rather than full-length antibody formats, as their smaller size enables the fragments to cross the cornea in topical applications, which is the preferred route of administration with minimal systemic side effects [ 149 ]. Additionally, due to the small acceptable volumes required for both topical and intravitreal administration of antibody-based therapies for ocular targets, such as age-related macular degeneration, the final drug products need to be formulated at high protein concentrations [ 150 ]. While antibody fragments are generally less soluble than full-length antibodies, Fab fragments have the potential to be very soluble.…”

“…As such, CDR engineering is a major focus in the development of antibodies with favorable solubility in concentrated formulations. Researchers increased the solubility of anti-Factor D by light chain CDR1 engineering, resulting in a variant that can be formulated at high concentrations (>250 mg/mL) and low viscosities (<17 cP) for intravitreal injection [ 150 ]. Importantly, the mutations that improved solubility and stability also retained antigen-binding capacity and favorable PK properties.…”

Identification and characterization of an octameric PEG-protein conjugate system for intravitreal long-acting delivery to the back of the eye

“…Here, PEG scaffolds were conjugated to an anti-factor D Fab therapeutic (AFD.v14), previously engineered for physical and chemical stability in ocular compartments [22]. AFD is a Fab of a humanized immunoglobulin G1 (IgG1) monoclonal antibody directed against complement factor D (CFD), a highly specific chymotrypsin-like serine protease that is a rate-limiting enzyme in the activation of the alternative complement pathway (ACP) [23].…”