Fig. 2. Induction of IgG4 Fab arm exchange in vitro. (A) Incubation of IgG4-Betv1/IgG4-Feld1 mixtures (circles) with human erythrocytes (closed symbols) at 37°C resulted in generation of bispecific antibodies; no bispecificity was generated in IgG1 mixtures (squares). As controls, an excess of irrelevant IgG4 was added (triangles) or antibody mixtures were incubated without erythrocytes (open symbols). Error bars represent range of duplicate measurements. (B) IgG4-Betv1/IgG4-Feld1 mixtures (black bars) were incubated for 24 hours at 37°C in the presence of different reducing agents, and the generation of bispecific IgG was determined. IgG1 antibody mixtures were used as controls (gray bars). Error bars represent SEM calculated from three measurements. (C) IgG4-CD20/IgG4-EGFr mixtures (squares) were incubated at 37°C with (closed symbols) or without (open symbols) 0.5 mM GSH. The formation of bispecific antibodies was measured in ELISA. OD450, optical density (absorbance) at 450 nm. Mixtures of IgG1 antibodies were used as controls (circles). Data are representative of three experiments. (D) IgG1 and IgG4 mutants were prepared for the Betv1 and Feld1 antibodies. P228S represents a point mutation that introduces the IgG4 core hinge sequence into IgG1. CH3(
1) and CH3(4) represent domain swap mutations in which the original third constant domain is replaced with the IgG1 or IgG4 domain, respectively. Fab arm exchange between corresponding mutants is shown. (E) IgG4-EGFR/IgG4-CD20 mixtures were incubated for 24 hours in the absence (E) or presence (F) of 0.5 mM GSH, after which the antibodies were deglycosylated with peptide N-glycosidase F, and the molecular masses of the resulting antibodies were determined by ESI-TOF mass spectrometry. Deconvoluted ESI-TOF spectra are shown. Data are representative of two experiments.
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