title: A standardized conversion of IgG antibody to bispecific form with inversely altered affinities for Fcγ-receptors II and III.
abstract: This work aims to enhance killing of antibody-coated human tumor cells by altering the antibody's affinity for two effector-cell Fcγ-receptors (FcγR). Affinity for the activating FcγRIII is raised, affinity for the inhibitory FcγRIIB is reduced, with the ratio between the two association constants increasing >1000-fold. We use as a standard tool the Fab'γ from a monoclonal antibody specific for human FcγRIII. This Fab'γ module is bonded to an IgG antibody by a tandem thioether link running between cysteine residues in the hinge vicinity of each protein, thus forming a bispecific FabIgG construct. Simultaneously, effector function of the IgG module is adjusted by leaving its hinge open and adding negative charges. FabIgG constructs derived from the chimeric IgG1 antibody rituximab show the following properties. (1) The titer for antibody-dependent cellular cytotoxicity is enhanced by 12-100-fold, reflecting the affinity of the Fab'γ module for effector-cell FcγRIII. (2) Two functions of the construct's Fcγ, activation of complement and prolonged metabolic survival, are moderately reduced. (3) In contrast, affinities of the Fcγ for all FcγR are severely reduced, with two anticipated consequences. First, attacks by macrophages on antibody-coated cells are favored by reduced engagement of the inhibitory FcγRIIB. Second, reduced engagement of activating FcγR by the Fcγ lowers the probability of untoward crosslinkings of FcγR, which have been shown to provoke toxicity. If the Fab'γ module possesses human constant regions, the linkage strategy requires prior genetic deletion of at least one cysteine residue. With both Fab'γ and IgG modules available, FabIgG can be prepared in 35 h.
authors.affiliation: Tenovus Research Laboratory, Cancer Sciences Division, Southampton University School of Medicine, Southampton SO16 6YD, UK.
authors.forename: Weng S
authors.initials: WS
authors.lastname: Leong
authors.forename: Karen-Anne
authors.initials: KA
authors.lastname: Thomas
authors.forename: Claude H
authors.initials: CH
authors.lastname: Chan
authors.forename: George T
authors.initials: GT
authors.lastname: Stevenson
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D017452:Receptors, IgG / Q000276:immunology*
chemical_list: D018033:Antibodies, Bispecific; D007074:Immunoglobulin G; D017452:Receptors, IgG