Analysis of the structural correlates for antibody polyreactivity by multiple reassortments of chimeric human immunoglobulin heavy and light chain V segments.

Abstract

SummaryPolyreactive antibodies (Abs) constitute a major proportion of the early Ab repertoire and are an important component of the natural defense mechanisms against infections. They are primarily immunoglobulin M (IgM) and bind a variety of structurally dissimilar self and exogenous antigens (Ags) with moderate affinity. We analyzed the contribution of Ig polyvalency and of heavy (H) and light (L) chain variable (V) regions to polyreactivity in recombinatorial experiments involving the V.-diversity(D)-JH and VK-J~ gene segments of a human polyreactive IgM, monoclonal antibody 55 (mAb55), and those of a human monoreactive anti-insulin IgG, mAb13, in an in vitro Cyl and C~ human expression system. These mAbs are virtually identical in their V, and VK gene segment sequences. First, we expressed the V.-D-J. and V~-J~ genes of the IgM mAb55 as V segments of an IgG molecule. The bivalent recombinant IgG Ab bound multiple Ags with an efficiency only slightly lower than that of the original decavalent IgM mAb55, suggesting that class switch to IgG does not affect the Ig polyreactivity. Second, we coexpressed the mAb55-derived H or K chain with the mAb13-derived K or H chain, respectively. The hybrid IgG Ab bearing the mAb55-derived H chain V segment paired with the mAb13-derived ~ V segment, but not that bearing the mAb13-derived H chain V segment paired with the mAb55-derived K V segment, bound multiple Ags, suggesting that the Ig H chain plays a major role in the Ig polyreactivity. Third, we shuffled the framework 1 (FR1) -FtL3 and complementarity determining region 3 (CDR3) regions of the H and K chain V segments of the mAB55-derived IgG molecule with the corresponding regions of the monoreactive IgG mAb13. The mAb55-derived IgG molecule lost polyreactivity when the H chain CDR3, but not the FR1-FR3 region, was replaced by the corresponding region of mAb13, suggesting that within the H chain, the CDR3 provides the major structural correlate for multiple Ag-binding. This was formally proved by the multiple Ag-binding of the originally monoreactive mAb13-derived IgG molecule grafted with the mAb55-derived H chain CDR3. The polyreactivity of this chimeric IgG was maximized by grafting of the mAb55-derived K chain FK1-FIL3, but not that of the K chain CDK3. The mAb55-derived K chain FR1-FK3 (an unmutated V~ 325 segment), however, failed to yield a polyreactive Ab when grafted onto an IgG that was in all other parts identical with mAb13. Rather, this chimeric molecule showed full specificity for insulin. Thus, the polyreactivity of the human mAb55 can be fully preserved after Ig class switch, and depends primarily on the contribution of structures that are generated through somatic rearrangement events (H chain CDK3), and structures that represent the expression of an unmutated gene (VK 325 segment). Sera of healthy humans and animals contain natural antibodies (Abs) that react with a variety of self and exogenous Ags. Most natural mAbs generated from humans and mice are polyreactive, i.e., they bind multiple, even very...

Polyreactive Binding of Antibodies Generated by Polyclonal B Cell Activation. I. Polyreactivity Could be Caused by Differential Glycosylation of Immunoglobulins

“…It has also been proposed that the combining sites of polyreactive Abs are enriched in structural elements that make them more 'sticky'. There is also evidence suggesting that the CDR3 of the heavy chain could contribute to polyreactive binding [10,[33][34][35][36][37]. Our finding that differential glycosylation could play an important role provides an alternative explanation to polyreactivity which is not in contradiction with other hypotheses.…”

B Cells Expressing a Natural Polyreactive Autoantibody Have a Distinct Phenotype and Are Overrepresented in Immunoglobulin Heavy Chain Transgenic Mice

“…NAAs are usually encoded by germline Ig V region genes with little or no somatic mutation (2,9,14,15), but they use the same spectrum of V H and V L genes as those used by conventional Abs (9,16). Previously, we have shown that NAAs differ from Ag-induced Abs in the third hypervariable region of the H chain (9), a finding subsequently confirmed by others in human polyreactive Abs (17)(18)(19).…”

“…It has been shown that H chain plays a central role in polyreactive Ab binding (17,19). Therefore, it is possible that the ppc1-5H/ Abs might also be polyreactive.…”

A Comparative Analysis of the Immunological Evolution of Antibody 28B4

“…While it is clear that CDR H3 plays an important role in determining the specificity of an antibody, it has also been shown that CDR H3 plays a critical role in distinguishing between polyspecific and monospecific antigen-combining sites of the antibody (34). Both monospecific and polyspecific antigen-combining sites can be generated utilizing identical light and heavy chain variable-region sequences in conjunction with a discrete CDR H3.…”