Can isotype switch modulate antigen-binding affinity and influence clonal selection?

Abstract

Four different monoclonal Ig (MIg) (IgA1κ, IgG1κ, IgG2κ and IgG4κ) displaying anti‐tubulin activity were detected in the serum from a lymphoma patient. The complete sequence of three of these MIg showed identical VH and VL domains and the presence of mutations compatible with an antigen‐driven process. Surprisingly, despite complete homology in their variable domains, IgA1κ, IgG1κ, or their Fab fragments bound to a common motif recognized in β tubulin, with significant differences in affinity (IgA1κ 1.52×10–8 M, and IgG1κ 2.09×10–7 M). To substantiate these results, the VH and VL domains from IgA1κ were cloned and introduced into expression vectors containing the constant κ exon and either the μ or the γ1 constant exon, and complete recombinant IgMκ and IgG1κ were obtained. Like the IgA1κ, the IgMκ construction bound to the tubulin epitope with consistent affinity (7.7×10–9 M), whereas the IgG1κ construction displayed a significantly lower affinity (3.28×10–7 M). These results provide definitive evidence that isotype can influence binding affinity to antigen and suggest that malignant transformation occurred at the germinal center once the mutational process was achieved and the switch process was still active.

A Systematic Comparison of Free and Bound Antibodies Reveals Binding-Related Conformational Changes

“…Thus, a conformational change in this loop may affect the relative orientation of the CH1 domain versus the Fc, thereby influencing effector binding to the Fc. There are now many examples of Abs with identical variable domains but different isotypes that bind the same Ag with a different affinity or specificity (13,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Some of these studies suggested that the CH1 domain accounts for the observed changes in binding, because it was identified as the only region with sequence diversity between the tested Abs and because a similar effect was observed using only the Fab instead of the entire Ab scaffold (13,16,19).…”

“…However, recent reports provide some evidence for binding-related allosteric effects in Abs. It was demonstrated that Ag binding, as well as the structure of the variable domains, may be influenced by changes in the constant domains (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). For example, Pritsch et al (13) demonstrated that two human mAbs sharing identical variable domains, but expressing different isotypes, bind tubulin with significantly different affinities.…”

Immunoglobulin isotype influences affinity and specificity

“…Several years later, IgG1 and IgA mAbs, as well as their Fabs, all with identical V regions binding the same epitope, were found to manifest differences in affinity (8). These observations failed to change the existing dogma, possibly because the field was reluctant to abandon a cherished paradigm without additional data.…”

“…In addition, two other groups, including the report by Tudor et al (5), described additional examples in which Abs expressing identical V region sequences manifested altered specificity and/or affinity (13). Given that five independent groups have now reported that C region can affect V region affinity and/or specificity (5,7,8,10,13)…”

Variable Region Identical Immunoglobulins Differing in Isotype Express Different Paratopes

“…This tidy view of separate structural and functional domains comprising an immunoglobulin G molecule has unraveled in recent years with various observations that C regions can affect the interaction of certain V regions with their Ag (2-7). At least six independent groups have reported findings that isotype switching is associated with altered specificity despite conservation of V region sequences (7)(8)(9)(10)(11)(12)(13). However, the molecular mechanisms for these phenomena are not understood.…”