Humanisation and characterisation of PR1A3, a monoclonal antibody specific for cell-bound carcinoembryonic antigen |
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| Authors: | Lorna M Stewart Susan Young Graham Watson Stephen J Mather Paul A Bates Heather A Band Robert W Wilkinson Elizabeth L Ross David Snary |
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| Institution: | (1) Applied Development Laboratory, Imperial Cancer Research Fund, Dominion House, St. Bartholomew's Hospital, 59 Bartholomew Close, London EC1A 7BE, UK e-mail: d.snary@icrf.icnet.uk Tel.: +44-171-726-4784, Fax: 0171 796 3907, GB;(2) Nuclear Medicine Group, Imperial Cancer Research Fund, St. Bartholomew's Hospital, London, UK, GB;(3) Biomolecular Modelling Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, UK, GB |
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| Abstract: | Carcinoembryonic antigen (CEA) is highly expressed by most tumours of gastrointestinal origin, but its use as a target for
tumour therapy is complicated by the high levels of soluble CEA that are found circulating in the blood of cancer patients.
A monoclonal antibody PR1A3 has been prepared, which binds preferentially to cell-surface rather than soluble CEA, this cell
selectivity should make PR1A3 an ideal candidate for antibody-targeted tumour therapy. PR1A3 has been humanised and shown
to retain its cell-surface specificity and affinity. Stable expression of the humanised antibody from chinese hamster ovary
(CHO) cells has been achieved after transfection and amplification. Since PR1A3 binds preferentially to cell-associated CEA,
a cell-free enzyme-linked immunosorbent assay (ELISA) has been developed to allow characterisation and routine assay of the
antibody. This assay was developed using a recombinant chimeric protein constructed by cloning the domain of CEA that is bound
by PR1A3 (the B3 domain) into a hybrid gene containing the Fc portion of IgG and three domains of biliary glycoprotein. Stable
expression of this hybrid protein has been achieved from CHO cells. In ELISA both humanised and murine PR1A3 bound strongly
to this antigen but only at a minimal level to soluble CEA. Two binding sites for the antibody were found on the gastric carcinoma
cell line MKN45, one of higher affinity (1 nM) and the other at lower affinity (60 nM). Similar affinities were found for
both murine and humanised antibodies. The data presented make it unlikely that the differential binding to cell-surface as
distinct from soluble CEA can be accounted for by low affinity of PR1A3 for CEA, and provides further support for the hypothesis
that some conformational change takes place on CEA release from cells and that it is this change that blocks PR1A3 binding
to its epitope.
Received: 5 October 1998 / Accepted: 19 November 1998 |
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| Keywords: | Carcinoembryonic antigen Monoclonal antibody Humanisation Reombinant antigen |
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