Penetration of engineered antibody fragments into the eye

Abstract

SUMMARY Antibodies are powerful immunotherapeutic agents but their use for treating ocular disorders is limited by their poor penetration into the eye. We hypothesized that antibody fragments of relatively small size might penetrate the cornea more readily. Monovalent single chain variable region (scFv) antibody fragments and divalent miniantibodies were engineered from existing monoclonal antibodies, expressed in a bacterial expression system, and purified by metal ion affinity chromatography. Corneoscleral preparations from normal pig and cat eyes were mounted in a corneal perfusion chamber. Intact antibodies and antibody fragments were applied topically to the anterior corneal surface over 12‐h periods, and samples were collected from the artificial anterior chamber. Similar experiments were performed with whole enucleated pig and human eyes. Penetration of antibodies and fragments was quantified by high‐sensitivity flow cytometry on appropriate target cells. Both monovalent scFv and divalent miniantibody fragments (but not whole immunoglobulin molecules) passed through de‐epithelialized and intact corneas after topical administration, and could be detected by antigen binding. Addition of 0·5% sodium caprate facilitated penetration through intact corneas. Topically‐applied scFv was found to penetrate into the anterior chamber fluid of rabbit eyes in vivo. The engineered fragments were stable and resistant to ocular proteases. Monovalent and divalent antibody constructs of molecular weight 28 kD and 67 kD, respectively, can penetrate through intact corneas into the anterior chamber, with retention of appropriate antigen‐binding activity. Such constructs may form novel therapeutic agents for topical ophthalmic use.

Topically applied antibody fragments penetrate into the back of the rabbit eye

“…Delivery into the posterior segment via systemic absorption (either from nasolacrimal drainage or ingestion as a result of grooming) and subsequent diffusion through the paucivascular retinal circulation of the rabbit may have contributed to the outcomes observed in the third experiment, but not to the outcomes of the first and second experiments, given that serum levels of scFv in the latter were low. As anticipated from the work of others 3 and from our own previous findings, 4 whole immunoglobulin was unable to penetrate through the ocular surface into the eye over the time frame of the experiments described.…”

“…4 The scFv was expressed in Escherichia coli and purified on an immobilised metal affinity column followed by passage over Q-Sepharose resin at CSL Ltd (Parkville, VIC, Australia). Three separate batches of scFv were fermented and purified to 91-94% scFv protein.…”

“…4 Assays were performed in duplicate or triplicate. Geometric mean fluorescence intensity (MFI) was used as a relative quantitative measure for scFv or intact IgG concentration, by reference to a dilution series of purified protein of known concentration.…”

“…3 We have recently shown that some monovalent and divalent antibody fragments can penetrate across the ocular surface into the anterior chamber of the eyes of pig, cat, rabbit, and human when administered in eye drop form. 4 We thus decided to investigate the penetration of topically applied antibody fragments into the posterior segment of the eye.…”

Antibody-based immunosuppressive agents for corneal transplantation

“…4 ScFv fragments have approximately half the molecular size of regular antigen-binding fragments (Fab), which enable the former to penetrate into the anterior chamber and the vitreous when administered topically. [5][6][7] In contrast to IgG antibodies, which have two antigen-binding sites per molecule, Fab and scFv antibody fragments have a single-binding site, permitting monovalent binding only. Divalent binding allows formation of a tighter bond and cross-linking of non-soluble antigens, such as receptors on cell surfaces.…”