In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody |
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Authors: | Rob C Roovers Edith van der Linden Adriaan P de Bruïne Jan-Willem Arends Hennie R Hoogenboom |
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Institution: | (1) Department of Pathology, University of Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands e-mail: HHO@LPAT.AZM.NL Tel.: +31-43-3874630; Fax: +31-43-3876613, NL;(2) University Hospital Maastricht, Maastricht, The Netherlands, NL |
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Abstract: | Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for
therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety
of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies
to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody
fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1)
cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2)
specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity
and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody
and had an off-rate of 5 × 10−2 s−1. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity,
the genes of scFv VEL-2 were re-formatted by fusion to a human (γ1) hinge region and CH3 domain. This “minibody” was expressed
in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These
results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules
with promising characteristics for in vivo use in tumour targeting.
Received: 13 July 2000 / Accepted: 12 October 2000 |
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