Aglycosylated IgG variants expressed in bacteria that selectively bind Fc gamma RI potentiate tumor cell killing by monocyte-dendritic cells

作者：Jung Sang Taek; Reddy Sai T; Kang Tae Hyun; Borrok M Jack; Sandlie Inger; Tucker Philip W; Georgiou George^*

来源：Proceedings of the National Academy of Sciences, 2010, 107(2): 604-609.

DOI：10.1073/pnas.0908590107

• antibody engineering
• bacterial display
• bacterial expression
• directed evolution
• effector function

The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Fc gamma 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 Fc gamma Rs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to Fc gamma RI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative Fc gamma RI 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 Fc gamma RI but not to the other activating receptors (Fc gamma RIIa, Fc gamma RIIIa) nor to the inhibitory receptor, Fc gamma RIIb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293T cells bound to all Fc. 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 Fc gamma R selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.

• 出版日期2010-1-12