Pegylated CB2782: a Complement Factor C3-Inactivating Protease and Potential Long-Acting Treatment for Dry AMD

Abstract

Purpose : Complement factor C3 (C3) blockade received clinical validation for the treatment of geographic atrophy with APL-2 treatment; however, the drug required monthly intravitreal (IVT) injections for maximal efficacy. Previously, an unmodified CB-2782 completely eliminated vitreal C3 from monkey eye for at least a week after an intravitreal injection but the 2-day vitreal half-life in monkeys was modeled as not adequate for quarterly administration in humans. Pegylation is an established method to extend the vitreal half-life of macromolecules. The biophysical and biochemical properties of a site-specific pegylated analogue of 2782 that has potential for an extended vitreal residence time are described herein.

Methods : CB-2782, is engineered from a human serine protease (matriptase) with a single unpaired cysteine and produced in E. coli. Conjugation of a CB-2782 with 40 kDa linear PEG was accomplished by maleimide reaction with the unpaired cysteine and confirmed by mass spectrometry. Enzymatic activity and specificity of CB-2782 were evaluated in hydrolysis assays and inhibition of complement activation was evaluated in standard sheep red blood cell hemolysis assays. Molecular weight (MW) and purity were established by SDS-PAGE and size-exclusion chromatography (SEC).

Results : Pegylated CB-2782 was a single band on an SDS-PAGE gel with a molecular weight of 100 kDa, close to the calculated MW of 68 kDa. SEC indicated that the preparation contained less than 5% contamination with aggregated forms or unpegylated material and migrated with a MW of approximately 500 kDa due to the PEG-induced large hydrodynamic radius measured by this nondenaturing sizing method. The pegylated enzyme had indistinguishable enzymatic activity from the unmodified CB-2782 on a C3-mimicing peptide substrate, specifically hydrolyzing C3 at a single site into inactive components, and blocked ex vivo C3-dependent complement-mediated sheep red blood cell hemolysis.

Conclusions : Pegylated CB-2782 was a highly pure product with indistinguishable enzymatic activity inactivating C3 as the unmodified enzyme. The denatured and native molecular weight increases resulting from the pegylation were in line with pegylation of other small proteins and is likely to produce an extended half-life in the vitreous. Rabbit ocular pharmacokinetics will be presented at the time of the conference.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.