Influence of format on in vitro penetration of antibody fragments through porcine cornea

Abstract

Aim: Antibody fragments, appropriately formulated, can penetrate through the ocular surface and thus have potential as therapeutic agents. The aim was to investigate the influence of protein fragment format on the kinetics and extent of ocular penetration in vitro.

Methods: Immunoglobulin single chain variable domain fragments of a murine monoclonal antibody with specificity for rat CD4 were engineered with a 20 or 11 amino acid linker by assembly polymerase chain reaction, expressed in Escherichia coli and purified by chromatography. Fab fragments of the parental antibody were prepared by papain digestion. Antibody fragments were formulated with a penetration and a viscosity enhancer and were applied to the surface of perfused pig corneas for up to 10 hours in vitro. Penetration was quantified by flow cytometry on rat thymocytes.

Results: 20-mer antibody fragments formed natural monomers and dimers following purification that could be separately isolated, while 11-mer fragments were dimeric. All formats of fragment (20-mer monomers and dimers, 11-mer dimers, Fab) showed penetration through the pig cornea after 6 hours of intermittent topical administration.

Conclusion: Antibody fragments of different shapes and sizes can penetrate the cornea after topical administration, thereby increasing the potential of this class of proteins for topical ophthalmic use.

Figure 1

Schematic representation of monomeric (scFv, Fab) and dimeric (scFv Dimer) antibody fragments in comparison with whole IgG molecule.

Figure 2

Anion exchange elution profile of 20-mer scFv. Stepwise elution separated monomeric scFv, dimeric scFv and contaminants. The 50 mM NaCl step eluted three peaks containing monomeric scFv. The 70 mM NaCl step eluted dimeric scFv and 1 M NaCl eluted scFv complexed with contaminant protein.

Figure 3

Size exclusion chromatography on a calibrated Superdex 75 10/30 column. Overlay of representative chromatograms of purified monomeric and dimeric 20-mer scFv. Monomer eluted at 25 minutes and dimer at 22 minutes. The elution times are consistent with calculated molecular masses of 27 kDa and 56 kDa respectively.

Figure 4

Representative pachymetry on four pig corneas perfused for 10 hours. Each cornea was treated topically with a different formulated antibody fragment. Corneal thickness increased gradually by approximately 30% over the 10 hour observation period.

Figure 5

Penetration of different antibody fragment formats across the perfused pig cornea. (A) Mean fluorescence intensity (as measured by flow cytometry on rat thymocytes) of perfusate sampled from representative corneas treated topically with scFv 20-mer monomer and dimer, scFv 11-mer dimer, and Fab monomer. (B) Titration series of purified protein for each antibody fragment format. Dilutions of purified fragment were tested for reactivity against rat thymocytes by flow cytometry.