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


CONCLUSIONS
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.
Brereton
H M
HM
Department of Ophthalmology, Flinders University, Adelaide, Australia.
Taylor
S D
SD
Farrall
A
A
Hocking
D
D
Thiel
M A
MA
Tea
M
M
Coster
D J
DJ
Williams
K A
KA
D000287:Administration, Topical; D000818:Animals; D015704:CD4 Antigens / Q000276:immunology*; D003315:Cornea / Q000378:metabolism*; D019281:Dimerization; D019573:Epithelium, Corneal / Q000378:metabolism; D004926:Escherichia coli / Q000276:immunology; D005434:Flow Cytometry; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000378:metabolism*; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D010316:Particle Size; D051381:Rats; D013552:Swine; D046509:Tissue Culture Techniques
D015704:CD4 Antigens; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region