Location of secretory component on the Fc edge of dimeric IgA1 reveals insight into the role of secretory IgA1 in mucosal immunity.

Secretory immunoglobulin A (SIgA) is the most prevalent antibody in the human body and a first line of defense in mucosal immunity. We located secretory component (SC) relative to dimeric IgA1 (dIgA1) within the SIgA1 structure using the constrained modeling of solution scattering and analytical ultracentrifugation data. The extended solution structure of dIgA1 is largely preserved within SIgA1. From conformational searches of SC locations, the best-fit SC models within SIgA1 show that SC is extended along the outermost convex edge of the Fc dimer in dIgA1. The topology of our SIgA1 structure reveals that it is able to bind to one FcalphaRI receptor molecule. SC binding to the Fc dimer confers protection to SIgA1 by the masking of proteolytically susceptible surface sites from bacterial proteases in the harsh environment of the mucosa. The models support a "zipper-like" unfolding of SC upon dIgA1 in the formation and transportation of SIgA1 into the mucosa.

An immunoglobulin E-reactive chimeric human immunoglobulin G1 anti-idiotype inhibits basophil degranulation through cross-linking of FcepsilonRI with FcgammaRIIb.

BACKGROUND: IgE binds to mast cells and basophils via its high-affinity receptor, FcepsilonRI, and cross-linking of FcepsilonRI-bound IgE molecules by allergen leads to the release of allergic mediators characteristic of type I hypersensitivity reactions. Previous work has shown that cross-linking of FcepsilonRI with FcgammaRIIb, an ITIM-containing IgG receptor, leads to inhibition of basophil triggering. 2G10, a chimeric human IgG1 anti-idiotype, has broad reactivity with human IgE and as such has the potential to bind simultaneously to FcepsilonRI-bound IgE, via its Fab regions, and the negative regulatory receptor, FcgammaRIIb, via its Fc region. OBJECTIVE: To assess the ability of human 2G10 to inhibit anti-IgE and allergen-driven basophil degranulation through cross-linking of FcepsilonRI-bound IgE with FcgammaRIIb. METHODS: 2G10 was assessed for its ability to bind to FcgammaRIIb on transfected cells and on purified basophils. In the basophil degranulation assay, basophils were purified from peripheral blood of atopic individuals and activated with either anti-IgE or the house dust mite allergen Der p 1, in the presence or absence of human 2G10. Basophil activation was quantified by analysis of CD63 and CD203c expression on the cell surface, and IL-4 expression intracellularly, using flow cytometery. RESULTS: Human 2G10 was able to bind to FcgammaRIIb on transfected cells and on purified basophils, and induce a dose-dependent inhibition of both anti-IgE and Der p 1-driven degranulation of basophils. CONCLUSION: The inhibition of basophil degranulation by the human IgG1 anti-idiotype 2G10 highlights the therapeutic potential of IgE-reactive IgG antibodies in restoring basophil integrity through recruitment of the inhibitory receptor FcgammaRIIb.

The production and characterisation of a chimaeric human IgE antibody, recognising the major mite allergen Der p 1, and its chimaeric human IgG1 anti-idiotype.

BACKGROUND: Two mouse monoclonal antibodies have been described, namely: mAb 2C7 (IgG2bkappa), which is directed against the major house dust mite allergen Der p 1, and mAb 2G10 (IgG1kappa), which is an anti-idiotypic antibody raised against mAb 2C7. Given its broad IgE specificity, anti-idiotype mAb 2G10 could potentially have immunomodulatory applications. For example, a chimaeric human IgG version of mAb 2G10 could prove to be a useful molecule for binding to mast cell and basophil FcepsilonRI bound IgE, and in doing so co-ligating FcepsilonRI with FcgammaRIIB, which has been reported to have downregulatory effects. AIMS: To produce a chimaeric human IgE version of mAb 2C7 (mAb 2C7huE) and a chimaeric human IgG1 version of its anti-idiotype mAb 2G10 (mAb 2G10huG1). METHODS: The Vkappa and VH regions of mAb 2C7 and its anti-idiotype mAb 2G10 were engineered into human constant regions of the IgE and IgG1 isotypes, respectively. RESULTS: The production of chimaeric mAb 2C7huE and its anti-idiotype mAb 2G10huG1 confirmed that the respective mouse antibody V regions were successfully engineered into human constant regions and still retained the specificity of the original murine V regions. CONCLUSION: The newly constructed chimaeric antibodies will be useful to investigate the downregulation of IgE mediated hypersensitivity by the crosslinking of FcepsilonRI with FcgammaRIIB.

Characterisation of a mouse monoclonal anti-idiotype reactive with a V region sequence commonly used by human immunoglobulins.

BACKGROUND: A mouse monoclonal antibody (2C7/IgG2b kappa) has been described recently, which is directed against the major house dust mite allergen Der p 1, and whose epitope specificity is representative of a major component of the human IgE anti-Der p 1 response. AIMS: To characterise an anti-idiotypic antibody (2G10/IgG1 kappa) raised against monoclonal antibody 2C7 as surrogate human IgE anti-Der p 1. METHODS: The specificity of the anti-idiotype antibody 2G10 was determined by competitive inhibition experiments using human and mouse immunoglobulins of known VH gene families. The epitope recognised by monoclonal antibody 2G10 was located on the molecular model of the Fv (fragment variable) region of monoclonal antibody 2C7. RESULTS: The data suggest that monoclonal antibody 2G10 is directed against a crossreactive idiotype on human IgE that is shared by polyclonal IgG. Competitive inhibition studies against human immunoglobulins, representative of VH2, VH3, and VH4 gene families, showed that monoclonal antibody 2G10 is mostly likely to be directed against sequences encoded by either VH3 or VH4 genes. The fact that monoclonal antibody 2G10 binds to the humanized (complementarity determining region (CDR) grafted) CAMPATH-1H antibody, but not to the original rat CAMPATH-1 YTH34.5.6 antibody, indicates that it is directed against a framework region rather than the CDRs. Analysis of amino acids in the VH region for charge, hydrophobicity, and accessibility suggests that reactivity with monoclonal antibody 2G10 is defined by a hexapeptide spanning residues 74-79 within framework region 3. CONCLUSION: The anti-idiotype monoclonal antibody 2G10 could potentially be used as a probe for determining the contribution of the VH3 and VH4 gene segments to antigenic specificity.

Prediction of the interacting surfaces in a trimolecular complex formed between the major dust mite allergen Der p 1, a mouse monoclonal anti-Der p 1 antibody, and its anti-idiotype.

BACKGROUND: Two mouse monoclonal antibodies (mAbs) have been described recently; namely, mAb 2C7 (IgG2b kappa), which is directed against the major house dust mite allergen Der p 1, and mAb 2G10 (IgG1 kappa), which is an anti-idiotypic antibody raised against mAb 2C7. The anti-idiotype mAb 2G10 does not block the binding of mAb 2C7 to Der p 1, which means that mAb 2C7 can simultaneously bind to Der p 1 and to mAb 2G10, thereby generating a trimolecular complex consisting of antigen-idiotype-anti-idiotype. AIMS: To sequence and model the V region of the anti-idiotypic antibody mAb 2G10 to enable the prediction of the interacting surfaces in the trimolecular complex consisting of Der p 1-mAb 2C7-mAb 2G10. METHODS: DNA sequencing of mAb 2G10 was carried out and the Swiss Model and Swiss PDB-Viewer programs were used to build a three dimensional model of the trimolecular complex. RESULTS: Complementarity of shape and charge was revealed when comparing the protrusion of the previously determined Der p 1 epitope (Leu147-Gln160) with the cavity formed by the complementarity determining regions (CDRs) of mAb 2C7. Such complementarity was also observed between the mAb 2C7 epitope predicted to be recognised by mAb 2G10 (residues Lys19 from framework region 1 (FRW1) and Ser74-Gln81 from FRW3) and residues from the CDRs of mAb 2G10 (a negatively charged patch flanked by the residues Asp55H/Glu58H and Glu27L/Glu27cL). As expected, the location of the mAb 2C7 epitope recognised by mAb 2G10 does not appear to interfere with the binding of Der p 1 to mAb 2C7. CONCLUSION: Although the results obtained represent only an approximation, they nevertheless provide a rare insight into how an antigen (Der p 1) might bind to its antibody (mAb 2C7) while in complex with an anti-idiotype (mAb 2G10).