Aglycosylated IgG variants expressed in bacteria that selectively bind FcgammaRI potentiate tumor cell killing by monocyte-dendritic cells.
The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Fcgamma 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 FcgammaRs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to FcgammaRI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative FcgammaRI 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 FcgammaRI but not to the other activating receptors (FcgammaRIIa, FcgammaRIIIa) nor to the inhibitory receptor, FcgammaRIIb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293T cells bound to all Fcgamma 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 FcgammaR selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.
Jung
Sang Taek
ST
Department of Chemical Engineering, University of Texas, Austin, TX 78712, USA.
Reddy
Sai T
ST
Kang
Tae Hyun
TH
Borrok
M Jack
MJ
Sandlie
Inger
I
Tucker
Philip W
PW
Georgiou
George
G
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D061067:Antibodies, Monoclonal, Humanized; D000920:Antibody-Dependent Cell Cytotoxicity; D003713:Dendritic Cells / Q000276:immunology*; D004926:Escherichia coli / Q000276:immunology; D005434:Flow Cytometry; D005818:Genetic Engineering; D014644:Genetic Variation; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics*; D008958:Models, Molecular; D009000:Monocytes / Q000276:immunology*; D011487:Protein Conformation; D017452:Receptors, IgG / Q000737:chemistry / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D000068878:Trastuzumab
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; C489017:FCGR1A protein, human; D007074:Immunoglobulin G; D017452:Receptors, IgG; D011994:Recombinant Proteins; D000068878:Trastuzumab