A human anti-insulin IgG autoantibody apparently arises through clonal selection from an insulin-specific "germ-line" natural antibody template: Analysis by V gene segment reassortment and site-directed mutagenesis

We analyzed the structural correlates underlying the insulin-dependent selection of the specific anti-insulin IgG1 κ mAb13-producing cell clone, derived from a patient with insulin-dependent diabetes mellitus treated with recombinant human insulin. First, we cloned the germ-line genes that putatively gave rise to the expressed V_H and Vκ segments and used them to generate the full (unmutated) "germ-line revertant" of the "wild-type" (somatically mutated) mAb13, using recombinant PCR methods and an in vitro human Cγ1 and Cκ expression system. The full "germ-line revertant" bound insulin specifically and in a dose-saturable fashion, but with a relative avidity (Av_rel) more than three-fold lower than that of its wild-type counterpart (Av_rel, 1.69 × 10^-8vs4.91 × 10^-9 g/μl). Second, we established, by reassorting wild-type and germ-line revertant forms of the mAb13 V_H and Vκ segments, that the increased Av_rel for insulin of mAb13 when compared with its full "germ-line revertant" counterpart was entirely dependent on the mutations in the V_H not those in the Vκ chain. Third, we determined, by site-directed mutagenesis experiments, that of the three mutations in the mAb13 V_H segment (Ser→Gly, Ser→Thr, and Ser→Arg at positions 31, 56, and 58, respectively), only Arg58 was crucial in increasing the mAb13 Av_rel (from 1.44 × 10^-8 to 5.14 × 10^-9 g/μl) and affinity (K_d, from 189 to 59 nM) for insulin. The affinity enhancement mediated by the VH segment Arg58 residue reflected about a threefold decrease in dissociation rate constant (K_off, from 4.92 × 10^-3 to 1.54 × 10^-3 s^-1) but not an increase in association rate constant (K_on, from 2.60 × 10⁴ to 2.61 × 10⁴ M^-1 s^-1), and it contrasted with the complete loss of insulin binding resulting from the substitution of the V_H segment Asn52 by Lys. The present findings suggest that human insulin, a self Ag, has the potential to recruit a natural autoantibody-producing cell precursor expressing a specific surface receptor for Ag in unmutated configuration, and drive it through affinity maturation. They also show that binding of insulin by such a receptor can be enhanced or completely abrogated by a single amino acid change.