title: Effective phagocytosis of low Her2 tumor cell lines with engineered, aglycosylated IgG displaying high FcγRIIa affinity and selectivity.
abstract: Glycans anchored to residue N297 of the antibody IgG Fc domain are critical in mediating binding toward FcγRs to direct both adaptive and innate immune responses. However, using a full length bacterial IgG display system, we have isolated aglycosylated Fc domains with mutations that confer up to a 160-fold increase in the affinity toward the low affinity FcγRIIa-R131 allele as well as high selectivity against binding to the remarkably homologous human inhibitory receptor, FcγRIIb. The mutant Fc domain (AglycoT-Fc1004) contained a total of 5 amino acid substitutions that conferred an activating to inhibitory ratio of 25 (A/I ratio; FcyRIIa-R131:FcγRIIb). Incorporation of this engineered Fc into trastuzumab, an anti-Her2 antibody, resulted in a 75% increase in tumor cell phagocytosis by macrophages compared to that of the parental glycosylated trastuzumab with both medium and low Her2-expressing cancer cells. A mathematical model has been developed to help explain how receptor affinity and the A/I ratio relate to improved antibody dependent cell-mediated phagocytosis. Our model provides guidelines for the future engineering of Fc domains with enhanced effector function.
authors.affiliation: Department of Chemical Engineering, University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: Sang Taek
authors.initials: ST
authors.lastname: Jung
authors.forename: William
authors.initials: W
authors.lastname: Kelton
authors.forename: Tae Hyun
authors.initials: TH
authors.lastname: Kang
authors.forename: Daphne T W
authors.initials: DT
authors.lastname: Ng
authors.forename: Jan Terje
authors.initials: JT
authors.lastname: Andersen
authors.forename: Inger
authors.initials: I
authors.lastname: Sandlie
authors.forename: Casim A
authors.initials: CA
authors.lastname: Sarkar
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D045744:Cell Line, Tumor; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology*; D010587:Phagocytosis; D015202:Protein Engineering; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D017452:Receptors, IgG / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D007074:Immunoglobulin G; D018719:Erb-b2 Receptor Tyrosine Kinases; D017452:Receptors, IgG; C080568:Fc gamma receptor IIA; C508053:ERBB2 protein, human