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

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.

Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis

“…This ADCC activity is still dependent on CD16 (FcgRIIIa) on the NK cells, as shown by Fab 2 anti-CD16 blocking and enhanced ADCC of ghPR1A3 over uhPR1A3 in the presence of enriched NK cells. The improved ADCC activity of the glycoengineered antibody is achieved without affecting PR1A3's binding activity, leaving it membrane specific, as described earlier (Durbin et al, 1994;Stewart et al, 1999). This is an important property of PR1A3, given the finding that soluble CEA can accumulate in lymph nodes and lead to false positive detection of cancers in lymph nodes when using other anti-CEA antibodies for immunoscintigraphy (Granowska et al, 1989).…”

“…The original murine IgG1k monoclonal antibody to CEA (Richman and Bodmer, 1987) was humanized by Stewart et al (1999). Murine (mPR1A3, IgG1) and unmodified humanized (uhPR1A3, IgG1) antibodies were acquired from the Biotherapeutics Development Unit, Clare Hall, CRUK, London, UK.…”

Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA) Gold 4 epitope and designed for radioimmunotherapy (RIT) of colorectal cancers

“…Different chimeric or humanized anti-CEA MAbs have been described and evaluated in experimental and clinical studies [8,14,15,19,20]. In the present study using the XenoMouse ® technology, we describe the generation and the characterization of two fully human anti-CEA antibodies, one IgG2κ and one IgM, designed for RIT of colorectal cancers.…”

Reply: In vitro and in vivo anticancer efficacy of unconjugated humanised anti-CEA monoclonal antibodies

“…This can be explained by the specific binding of PR1A3 to membrane-bound CEA. Previous work has identified the B3-GPI anchor of CEA as being the epitope of PR1A3 (Durbin et al, 1994;Stewart et al, 1999). The authors feel that this information can be used to further engineer PR1A3 for maximal clinical effectiveness in humans.…”