Humanisation and characterisation of PR1A3, a monoclonal antibody specificfor cell-bound carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is highly expressed by most tumours of gastr ointestinal origin, but its use as a target for tumour therapy is complicat ed by the high levels of soluble CEA that are found circulating in the bloo d of cancer patients. A monoclonal antibody PR1A3 has been prepared, which binds preferentially to cell-surface rather than soluble CEA, this cell sel ectivity should make PR1A3 an ideal candidate for antibody-targeted tumour therapy. PR1A3 has been humanised and shown to retain its cell-surface spec ificity and affinity. Stable expression of the humanised antibody from chin ese hamster ovary (CHO) cells has been achieved after transfection and ampl ification. 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 develope d 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 t he Fc portion of Ige and three domains of biliary glycoprotein. Stable expr ession of this hybrid protein has been achieved from CHO cells. In ELISA bo th 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) an d the other at lower affinity (60 nM). Similar affinities were found for bo th murine and humanised antibodies. The data presented make it unlikely tha t 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 su pport 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.