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

Abstract

BACKGROUND: The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. METHODS: The antibody was modified using EBNA cells cotransfected with b-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. RESULTS: The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. CONCLUSION: The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

Direct and immune mediated antibody targeting of ERBB receptors in a colorectal cancer cell-line panel

“…In keeping with our work on anti-CEA antibodies and with other studies, we have shown that immune mediated cetuximab killing of ERBB1 + CRC cell lines that express moderate to high levels of ERBB1 receptor occurs both by ADCC and ADCP (14,15).…”

“…Previous evidence shows that the main mediators of ADCC in humans are natural killer (NK) cells (14,15). We have therefore checked that the cetuximab-mediated ADCC against the cell lines is effective at a NK cell: target cell ratio of 10:1 and is blocked by antibody to the FcγRIII receptor, as would be expected from our own and others previous results with cell line in vitro studies (Fig.…”

Glycovariant anti-CD37 monospecific protein therapeutic exhibits enhanced effector cell-mediated cytotoxicity against chronic and acute B cell malignancies

“…Previous studies have shown that changing the glycosylation pattern of the Fc portion of antibodies can influence their ability to elicit effector function by enhancing their binding affinity to FcγRIIIa on human immune cells. 14,27 We saw similar enhanced affinity to FcγRIIIa with SMIP-016 GV (Fig. 1C).…”

Glycoengineering of Therapeutic Antibodies Enhances Monocyte/Macrophage-Mediated Phagocytosis and Cytotoxicity

“…NK cells are considered as one of the players because they rapidly elicit potent ADCC in vitro in comparison with freshly isolated monocytes or granulocytes (6,17,22,23) and infiltrate tumors in trastuzumab-treated patients (24). On the other hand, several preclinical studies in murine models convincingly demonstrated that macrophage subsets are primarily responsible for eliminating circulating and tissue B cells upon anti-CD20 mAb therapy and essentially excluded a role for NK cell-, T cell-or perforin-dependent mechanisms in vivo in those particular models (25)(26)(27)(28)(29).…”