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 V(H) and V(L) 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 1.52x10-8 M, and IgG1κ 2.09x10-7 M). To substantiate these results, the V(H) and V(L) 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 (7.7x10-9 M), whereas the IgG1κ construction displayed a significantly lower affinity (3.28x10-7 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.