title: A variant human IgG1-Fc mediates improved ADCC.
abstract: Ribosome display was applied to the Fc region of human immunoglobulin G (IgG1) to select for improved binding to human FcγRIIIa, the receptor expressed on human natural killer cells that mediates antibody-dependent cellular cytotoxicity (ADCC). A library of human Fcγ1 variants was generated using error-prone polymerase chain reaction, and subjected to multiple rounds of ribosome display selection against progressively decreasing concentrations of soluble human FcγRIIIa, to enrich for improved binders. Radioimmunoassay and alphascreen analyses of the aglycosylated IgG-Fc output revealed variants with improved binding to FcγRIIIa relative to wild-type IgG-Fc. Subsequent expression in human (HEK-EBNA) cells generated glycosylated IgGs with modified activity in ADCC assays. One particular variant, 125_B01 triggered enhanced ADCC (EC(50) up to four-fold reduced with increased maximal lysis) relative to wild-type antibody, having more equal levels of ADCC for each allotype (V158/F158) of FcγRIIIa. Deconvolution of individual replacements within the variant showed that improved function arose from the Phe243Leu replacement within the CH2 domain, rather than the CH3 domain replacements Thr393Ala or His433Pro. Surprisingly, the oligosaccharide profiles of 125_B01 indicated more oligosaccharide chains lacking fucose, or with bisecting N-acetylglucosamine relative to wild-type IgG1, which correlates with improved function and the replacement Phe243Leu that is a carbohydrate contact residue within the C(H)2 domain.
authors: MedImmune, Granta Park, Cambridge, UK. stewartr@medimmune.com
authors: Ross
authors: R
authors: Stewart
authors: George
authors: G
authors: Thom
authors: Michaela
authors: M
authors: Levens
authors: Gülin
authors: G
authors: Güler-Gane
authors: Robert
authors: R
authors: Holgate
authors: Pauline M
authors: PM
authors: Rudd
authors: Carl
authors: C
authors: Webster
authors: Lutz
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authors: Jermutus
authors: John
authors: J
authors: Lund