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Laboratory science - Extended reports
Influence of format on in vitro penetration of antibody fragments through porcine cornea
  1. H M Brereton1,
  2. S D Taylor2,
  3. A Farrall1,
  4. D Hocking2,
  5. M A Thiel3,
  6. M Tea1,
  7. D J Coster1,
  8. K A Williams1
  1. 1Department of Ophthalmology, Flinders University, Adelaide, Australia
  2. 2CSL Ltd, Parkville, Victoria, Australia
  3. 3Department of Ophthalmology, University of Zurich, Zurich, Switzerland
  1. Correspondence to: Dr Keryn Williams Department of Ophthalmology, Flinders Medical Centre, Bedford Park, SA 5042, Australia; keryn.williamsflinders.edu.au

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.

  • Ig, immunoglobulins
  • IMAC, immobilised metal affinity chromatography
  • MFI, mean fluorescence intensity
  • scFv, single chain variable domain antibody fragments
  • antibody fragments
  • cornea
  • perfusion chamber
  • drug delivery
  • pig model
  • Ig, immunoglobulins
  • IMAC, immobilised metal affinity chromatography
  • MFI, mean fluorescence intensity
  • scFv, single chain variable domain antibody fragments
  • antibody fragments
  • cornea
  • perfusion chamber
  • drug delivery
  • pig model

https://doi.org/10.1136/bjo.2005.066225

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    Footnotes

    • This work was supported by the National Health and Medical Research Council of Australia, the Ophthalmic Research Institute of Australia and the Flinders Medical Centre Foundation.

    • Competing interests: HMB, AF, MT, DJC and KAW have jointly received funds and reagents for research from CSL Ltd: SDT and DH are employees of CSL Ltd.

    • Ethical approvals: Experimentation on abattoir eyes was carried out with approval of Flinders University Animal Welfare Committee and conformed to the ARVO statement for the use of animals in ophthalmic and vision research. Experiments involving recombinant DNA were carried out with the approval of the Flinders University/Flinders Medical Centre Biosafety Committee.