An epitope on carcinoembryonic antigen defined by the clinically relevant antibody PR1A3.

Abstract

The monoclonal antibody PR1A3 has been used successfully for in vivo imaging of colorectal cancers, and several properties associated with this antibody, including minimal reactions of the antibody with circulating antigen in patients' sera, differentiate it from anti-carcinoembryonic antigen (CEA) antibodies used in similar studies. However, the antigen bound by PR1A3 was identified as CEA by analysis of somatic cell hybrids and by antigen expression from yeast artificial chromosomes, cosmids, and cDNA clones. The molecular weight, presence of a glycosyl-phosphatidylinositol anchor, elevation of surface expression by y-interferon, and N-terminal amino acid sequence all confirmed the antigen identification as CEA. A series of biiary glycoprotein-CEA hybrid proteins was produced which demonstrated that the epitope bound by the antibody was at the site of membrane attachment and involved parts of the glycosyl-phosphatidylinositol anchor and the B3 domain of CEA to form a conformational epitope. Access to this epitope, although possible when the antigen was on the cell surface, appeared to be blocked when CEA was released from the cell. The nature and location of the epitope on CEA are proposed to be responsible for the unique properties of the antibody.

Enhancement of colorectal tumor targeting using a novel biparatopic monoclonal antibody against carcinoembryonic antigen in experimental radioimmunoguided surgery

“…12 Otherwise, PR1A3 binds to the epitope on the CEA-specific domains of A3 with exclusive affinity to the membrane-bound CEA, whereas T84.66 binds to that of B3 with its affinity to soluble CEA. 20,21 Binding to 2 adjacent epitopes may increase affinity and avidity by divalent binding to a single CEA molecule as well as divalent to quadrivalent binding to the 2 adjacent molecules of both membrane-bound or soluble CEA in our study. We used BMH (bismaleimidohexane) rather than p-PDM (N,NЈ-p-phenylene dimaleimide) as a linker because the former is able to promote greater flexibility of 2 FabЈ fragments with its longer thioether linkage.…”

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).…”

“…This antibody has been shown to bind specifically to CEA in the B3-GPI domains. This epitope is at the point CEA anchors into the membrane, and this presumably explains why PR1A3 binds to membrane-bound CEA but not soluble CEA (Durbin et al, 1994;Conaghan et al, 2008).…”

“…Earlier work from our laboratory has shown that the antibody PR1A3, which is specific for membrane-bound carcinoembryonic antigen (CEA, formally designated CEACAM5), elicits ADCC against CEA-positive CRC cell lines even in the presence of super-physiological levels of free CEA (Durbin and Bodmer, 1987;Durbin et al, 1994;Conaghan et al, 2008). CEA is present on a high proportion of colorectal cancers, in which it is accessible to intravenously administered antibody, whereas in normal colorectal epithelium, due to the apical localisation of the antigen, it is not (Granowska, 1993).…”

Biological therapy: approaches in colorectal cancer

“…Recently, the novel anti-CEA monoclonal antibody PR1A3 has been successfully used in radioimmunoscintigraphy for the detection of CT-negative and CEA-negative recurrent colorectal cancer as well as to investigate the cell-based epitope domain of CEA (Granowska et al, 1989(Granowska et al, , 1993Durbin et al, 1994). This antibody was originally produced in mice against components of normal human colonic epithelium and has been demonstrated to be highly sensitive by immunohistochemistry for nearly all human colorectal carcinomas, regardless of differentiation.…”