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Aglycosylated IgG Variants Expressed in Bacteria That Selectively Bind FcγRI Potentiate Tumor Cell Killing by Monocyte-Dendritic Cells

Sang Taek Jung, Sai T. Reddy, Tae Hyun Kang, M. Jack Borrok, Inger Sandlie, Philip W. Tucker, George Georgiou and Frances H. Arnold
Proceedings of the National Academy of Sciences of the United States of America
Vol. 107, No. 2 (Jan. 12, 2010), pp. 604-609
Stable URL: http://www.jstor.org/stable/40535817
Page Count: 6
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Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Aglycosylated IgG Variants Expressed in Bacteria That Selectively Bind FcγRI Potentiate Tumor Cell Killing by Monocyte-Dendritic Cells
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Abstract

The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Feγ receptors on effector cells and the clearance of target cells via antibody dependent cell-mediated cytotoxicity (ADCC). Escherichia coli expressed, aglycosylated Fc domains bind effector FcγRs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to FcγRI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative FcγRI binding epitope within the CH3 domain. These mutations did not adversely affect its pH-dependent interaction with FcRn in vitro nor its serum persistence in vivo. Remarkably, the anti-Her2 IgG trastuzumab containing the E382V, M428I substitutions and expressed in E. coli exhibited highly selective binding to FcγRI but not to the other activating receptors (FcγRlla, FcγRllla) nor to the inhibitory receptor, FcγRllb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293Tcells bound to all Fcγ receptors in a manner similar to that of clinical grade trastuzumab. E. coli-purified trastuzumab (E382V, M428I), but not glycosylated trastuzumab (E382V, M428I) or clinical grade trastuzumab, was capable of potentiating the killing of Her2 overexpressing tumor cells with dendritic cells (DCs) as effectors. These results indicate that aglycosylated IgGs can be engineered to display unique FcγR selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.

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