Neutralizing IgG4 antibodies are a biomarker of sustained efficacy after peanut oral immunotherapy.

BACKGROUND: Clinical efficacy of oral immunotherapy (OIT) has been associated with the induction of blocking antibodies, particularly those capable of disrupting IgE-allergen interactions. Previously, we identified mAbs to Ara h 2 and structurally characterized their epitopes. OBJECTIVE: We investigated longitudinal changes during OIT in antibody binding to conformational epitopes and correlated the results with isotype and clinical efficacy. METHODS: We developed an indirect inhibitory ELISA using mAbs to block conformational epitopes on immobilized Ara h 2 from binding to serum immunoglobulins from peanut-allergic patients undergoing OIT. We tested the functional blocking ability of mAbs using passive cutaneous anaphylaxis in mice with humanized FcεRI receptors. RESULTS: Diverse serum IgE recognition of Ara h 2 conformational epitopes are similar before and after OIT. Optimal inhibition of serum IgE occurs with the combination of 2 neutralizing mAbs (nAbs) recognizing epitopes 1.2 and 3, compared to 2 nonneutralizing mAbs (non-nAbs). After OIT, IgG4 nAbs, but not IgG1 or IgG2 nAbs, increased in sustained compared to transient outcomes. Induction of IgG4 nAbs occurs after OIT only in those with sustained efficacy. Murine passive cutaneous anaphylaxis after sensitization with pooled human sera is significantly inhibited by nAbs compared to non-nAbs. CONCLUSIONS: Serum IgE conformational epitope diversity remains unchanged during OIT. However, IgG4 nAbs capable of uniquely disrupting IgE-allergen interactions to prevent effector cell activation are selectively induced in OIT-treated individuals with sustained clinical efficacy. Therefore, the induction of neutralizing IgG4 antibodies to Ara h 2 are clinically relevant biomarkers of durable efficacy in OIT.

An engineered immunomodulatory IgG1 Fc suppresses autoimmune inflammation through pathways shared with i.v. immunoglobulin.

Immunoglobulin G (IgG) antibodies in the form of high-dose intravenous immunoglobulin (IVIG) exert immunomodulatory activity and are used in this capacity to treat inflammatory and autoimmune diseases. Reductionist approaches have revealed that terminal sialylation of the single asparagine-linked (N-linked) glycan at position 297 of the IgG1 Fc bestows antiinflammatory activity, which can be recapitulated by introduction of an F241A point mutation in the IgG1 Fc (FcF241A). Here, we examined the antiinflammatory activity of CHO-K1 cell-produced FcF241A in vivo in models of autoimmune inflammation and found it to be independent of sialylation. Intriguingly, sialylation markedly improved the half-life and bioavailability of FcF241A via impaired interaction with the asialoglycoprotein receptor ASGPR. Further, FcF241A suppressed inflammation through the same molecular pathways as IVIG and sialylated IgG1 Fc and required the C-type lectin SIGN-R1 in vivo. This contrasted with FcAbdeg (efgartigimod), an engineered IgG1 Fc with enhanced neonatal Fc receptor (FcRn) binding, which reduced total serum IgG concentrations, independent of SIGN-R1. When coadministered, FcF241A and FcAbdeg exhibited combinatorial antiinflammatory activity. Together, these results demonstrated that the antiinflammatory activity of FcF241A requires SIGN-R1, similarly to that of high-dose IVIG and sialylated IgG1, and can be used in combination with other antiinflammatory therapeutics that rely on divergent pathways, including FcAbdeg.

Improved low molecular weight Myc-Max inhibitors.

Compounds that selectively prevent or disrupt the association between the c-Myc oncoprotein and its obligate heterodimeric partner Max (Myc-Max compounds) have been identified previously by high-throughput screening of chemical libraries. Although these agents specifically inhibit the growth of c-Myc-expressing cells, their clinical applicability is limited by their low potency. We describe here several chemical modifications of one of these original compounds, 10058-F4, which result in significant improvements in efficacy. Compared with the parent structure, these analogues show enhanced growth inhibition of c-Myc-expressing cells in a manner that generally correlates with their ability to disrupt c-Myc-Max association and DNA binding. Furthermore, we show by use of a sensitive fluorescence polarization assay that both 10058-F4 and its active analogues bind specifically to monomeric c-Myc. These studies show that improved Myc-Max compounds can be generated by a directed approach involving deliberate modification of an index compound. They further show that the compounds specifically target c-Myc, which exists in a dynamic and relatively unstructured state with only partial and transient alpha-helical content.

Elucidation of stannin function using microarray analysis: implications for cell cycle control.

Stannin (Snn) is a highly conserved, vertebrate protein whose cellular function is unclear. We have recently demonstrated in human umbilical vein endothelial cells (HUVECs) that Snn gene expression is significantly induced by tumor necrosis factor-alpha (TNF-alpha) in a protein kinase C-epsilon (PKC-epsilon)-dependent manner. In HUVEC, TNF-alpha stimulation of HUVECs results in altered gene expression, and a slowing or halting of cell growth. An initial set of experiments established that Snn knockdown via siRNA, prior to TNF-alpha treatment, resulted in a significant inhibition of HUVEC growth compared to TNF-alpha treatment alone. In order to assess how Snn may be involved in TNF-alpha signaling in HUVEC growth arrest, we performed microarray analysis of TNF-alpha-stimulated HUVECs with and without Snn knockdown via siRNA. The primary comparison made was between TNF-alpha-stimulated HUVECs and TNF-alpha-exposed HUVECs that had Snn knocked down via Snn-specific siRNAs. Ninety-six genes were differentially expressed between these two conditions. Of particular interest was the significant upregulation of several genes associated with control of cell growth and/or the cell cycle, including interleukin-4, p29, WT1/PRKC, HRas-like suppressor, and MDM4. These genes act upon cyclin D1 and/or p53, both of which are key regulators of the G1 phase of the cell cycle. Functional studies further supported the role of Snn in cell growth, as cell cycle analysis using flow cytometry shows a significant increase of G1 cell cycle arrest in HUVECs with Snn knockdown in response to TNF-alpha treatment. Together these studies suggest a functional role of Snn in regulation of TNF-alpha-induced signaling associated with HUVEC growth arrest.

The protein stannin binds 14-3-3zeta and modulates mitogen-activated protein kinase signaling.

The molecular mechanisms underlying the selective toxicity of trimethyltin (TMT) remain unclear. Stannin (Snn), a protein preferentially expressed in TMT-sensitive cells, provides a direct link to the molecular basis for TMT toxicity. Recent evidence demonstrated that Snn peptides bind and de-alkylate TMT to dimethyltin (DMT); Snn may mediate both TMT and DMT toxicity. In this study, we demonstrate that Snn co-immunoprecipitates with a scaffolding protein 14-3-3, specifically with 14-3-3zeta isotype. Consistent with this, a detailed amino acid sequence analysis shows that Snn contains a putative 14-3-3 protein-binding site located within its hydrophilic loop. In addition, we present the evidence that Snn overexpression results in reduced extracellular regulated kinase activation and increased p38 activation. In contrast, the activity of c-Jun N-terminal kinase did not change following Snn overexpression. This is the first evidence that demonstrates a direct interaction between Snn and MAPK signaling molecules. Together, these findings indicate a role of Snn in modulation of MAPK signaling pathways through its interactions with 14-3-3zeta.

Protein kinase C epsilon regulates tumor necrosis factor-alpha-induced stannin gene expression.

Stannin (Snn) is a highly conserved vertebrate protein that has been closely linked to trimethyltin (TMT) toxicity. We have previously demonstrated that Snn is required for TMT-induced cell death. Others have shown that TMT exposure results in tumor necrosis factor-alpha (TNFalpha) production and that TNFalpha treatment induces Snn gene expression in human umbilical vein endothelial cells (HUVECs). In this study, we investigated a signaling mechanism by which Snn gene expression is regulated by TMT and demonstrated that TNFalpha stimulates Snn gene expression in a protein kinase C epsilon-dependent manner in HUVECs in response to TMT exposure. Supporting this, we show that TMT-induced toxicity is significantly blocked by pretreatment with an anti-TNFalpha antibody in HUVECs. Using a quantitative real-time polymerase chain reaction assay, we also show that the level of Snn gene expression is significantly increased in HUVECs in response to either TMT or TNFalpha treatment. This TNFalpha-induced Snn gene expression is blocked when HUVECs were pretreated with bisindolylmaleimide I, an inhibitor of protein kinase C (PKC). In contrast, when HUVECs were treated with phorbol 12-myristate 13-acetate, a PKC activator, we observed a significant increase in Snn gene expression. Using isotype-specific siRNA against PKC, we further show that knockdown of PKC epsilon, but not PKC delta or PKC zeta, significantly blocked TNFalpha-induced Snn gene expression. Together, these results indicate that TNFalpha-induced, PKC epsilon-dependent Snn expression may be a critical factor in TMT-induced cytotoxicity.

Stannin, a protein that localizes to the mitochondria and sensitizes NIH-3T3 cells to trimethyltin and dimethyltin toxicity.

Stannin (Snn) is a highly conserved, 88-amino acid protein that may mediate the selective toxicity of organotins. Snn is localized in tissues with known sensitivity to trimethyltin (TMT), including the central nervous system, immune system, spleen, kidney and lung. Cells in culture that do not express Snn show considerable resistance to TMT toxicity. In vitro, Snn peptide can bind TMT in a 1:1 ratio and can de-alkylate TMT to dimethyltin (DMT). We now show that transfection with Snn sensitized TMT-resistant NIH-3T3 mouse fibroblasts to both TMT and DMT cytotoxicity. Triple label confocal microscopy of Snn-transfected cells and Percoll gradient purification of mitochondria showed Snn localized to the mitochondria and other membrane structures. The mitochondrial localization of Snn, coupled with its ability to bind and dealkylate organotin compounds, indicates a possible mechanism by which selective alkyltin toxicity might be mediated.

The methyl-CpG binding protein MBD1 interacts with the p150 subunit of chromatin assembly factor 1.

DNA promoter hypermethylation has been shown to be a functional mechanism of transcriptional repression. This epigenetic gene silencing is thought to involve the recruitment of chromatin-remodeling factors, such as histone deacetylases, to methylated DNA via a family of proteins called methyl-CpG binding proteins (MBD1 to -4). MBD1, a member of this family, exhibits transcription-repressive activity, but to this point no interacting protein partners have been identified. In this study, we demonstrate that MBD1 partners with the p150 subunit of chromatin assembly factor 1 (CAF-1), forming a multiprotein complex that also contains HP1alpha. The MBD1-CAF-1 p150 interaction requires the methyl-CpG binding domain of MBD1, and the association occurs in the C terminus of CAF-1 p150. The two proteins colocalize to regions of dense heterochromatin in mouse cells, and overexpression of the C terminus of CAF-1 p150 prevents the targeting of MBD1 in these cells without disrupting global heterochromatin structure. This interaction suggests a role for MBD1 and CAF-1 p150 in methylation-mediated transcriptional repression and the inheritance of epigenetically determined chromatin states.