Abstract
Human IgG4 antibody shows therapeutically useful properties compared with the IgG1, IgG2, and IgG3 subclasses. Thus IgG4 does not activate complement and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we studied the solution structure of wild-type IgG4(Ser(222)) and a hinge mutant IgG4(Pro(222)) in different buffers and temperatures where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients s20,w(0) of 6.6-6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the x-ray radius of gyration Rg was unchanged with concentration in 50-250 mm NaCl buffers, whereas the neutron Rg values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves (P(r)) revealed two peaks, M1 and M2, that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures in addition to IgG4 dimer formation at high concentration in heavy water. Constrained x-ray and neutron scattering modeling revealed asymmetric solution structures for IgG4(Ser(222)) with extended hinge structures. The IgG4(Pro(222)) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement and clarify aspects of its stability and function for therapeutic applications.
Highlights
The human IgG4 antibody subclass does not activate complement and forms half-antibodies
Human IgG4 antibody shows therapeutically useful properties compared with the IgG1, IgG2, and IgG3 subclasses
Using high throughput scattering methods, we studied the solution structure of wild-type IgG4(Ser222) and a hinge mutant IgG4(Pro222) in different buffers and temperatures where the proline substitution suppresses the formation of half-antibody
Summary
The human IgG4 antibody subclass does not activate complement and forms half-antibodies. Conformational modeling on the two core hinge peptides predicted that the wild-type IgG4(Ser222) hinge is more flexible than the mutant IgG4(Pro222) hinge, perhaps explaining the formation of stable intrachain disulfide bonds in the mutant [12] The equilibrium between these disulfide bridges leads to heterogeneous IgG4 populations, each with two different heavy chains [13]. IgG4(Ser222) had been studied by ultracentrifugation and x-ray scattering to show that IgG4 undergoes a concentration-dependent Fab-arm rearrangement [20] This concentration dependence is explored here using more detailed solution structural data for both IgG4(Ser222) and IgG4(Pro222). We show that IgG4(Ser222) and IgG4(Pro222) are principally monomeric, but IgG4(Ser222) shows self-associative behavior in heavy water Both forms display concentration-dependent structural changes corresponding to Fab-arm rearrangements at low concentrations. The implications of this result for the reduced immune reactivity of IgG4 are discussed
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