Targeted killing of colorectal cancer cell lines by a humanised IgG1 monoclonal antibody that binds to membrane-bound carcinoembryonic antigen

Abstract

The distribution of carcinoembryonic antigen (CEA) in colorectal cancer (CRC) differs from that in normal colorectal tissue, being found on all borders of the cell membrane and hence enabling access to intravenous antibody, making CEA a good target for antibody-based therapy. The distinctive anti-CEA antibody, PR1A3, binds only membrane-bound CEA. Humanised PR1A3 (hPR1A3) was assessed both in vitro cytotoxicity and binding assays with colorectal cancer cell lines expressing varying levels of CEA. Human peripheral blood mononuclear cells (PBMCs) and purified natural killer (NK) cells were used as effectors. The in vitro assays demonstrated hPR1A3 CEA-specific binding and antibody-dependent and CEA-specific killing of human colorectal cancer cell lines by human PBMCs. The effect increased with increasing concentration of antibody and surface CEA, and was lost by using the parent murine IgG1 PR1A3. Killing was also blocked by antibody to the Fc-gIIIA receptor. Purified human NK cells were effective at much lower effector:target ratios than unfractionated PBMCs, indicating that NK cells were the main mediators of hPR1A3-based CEAspecific killing. The results support the development of hPR1A3 for therapy of colorectal cancer.

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

“…The SKCO-1 (from ATCC), LS174T (from BH Tom, NW University, Chicago), LoVo (from ATCC), HCT116 (from ATCC) and MKN45 (from Cell Services LIF, CRUK, London, UK) cell lines were cultured as described (Conaghan et al, 2008). For cytotoxicity and phagocytosis assays, cells were suspended in 2% RPMI 1640 medium with 1% glutamine and 10% FCS (RPMI complete medium).…”

“…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).…”

“…Another uses glycoform engineering, which has been shown to improve in vitro ADCC by 10 -100-fold (Umana et al, 1999;Niwa et al, 2004b;Schuster et al, 2005). In this study, we have used glycoengineering to enhance the immune effector function of an antibody that we have shown earlier to kill CRC-derived cell lines by ADCC (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).…”

“…The three classes of FcgRs are expressed on NK cells, known to be potent activators of ADCC (Schmitz et al, 2002;Lazar et al, 2006;Conaghan et al, 2008), and on monocytes and granulocytes, which have also been identified as having at least some potential for antibody-dependent killing of target cells, for example, by antibody-dependent cellular phagocytosis (ADCP) (Munn et al, 1991;Huls et al, 1999;Dhodapkar et al, 2005;Lazar et al, 2006;Karagiannis et al, 2007;McEarchern et al, 2007). We describe here a modified phagocytosis assay, based on that developed by Munn et al (1991), and its application to the in vitro testing of the ability of humanised and glycoengineered PR1A3 to kill CRC-derived cell lines.…”