Convergent recognition of the IgE binding site on the high-affinity IgE receptor.

Two structurally distinct classes of peptides were recently identified by phage display that bind the high-affinity IgE receptor, FcepsilonRI, and block IgE binding and subsequent receptor activation. Both classes adopt highly stable structures in solution, one forming a beta hairpin, with the other forming a helical "zeta" structure. Despite these differences, the two classes bind competitively to the same site on the receptor. Structural analyses of both peptide-receptor complexes by NMR spectroscopy and/or X-ray crystallography reveal that the unrelated peptide scaffolds have nevertheless converged to present a similar three-dimensional surface to interact with FcepsilonRI and that their modes of interaction share a key feature of the IgE-FcepsilonRI complex, the proline/tryptophan sandwich.

Evaluation of IgE Antibodies to Omalizumab (Xolair®) and Their Potential Correlation to Anaphylaxis.

Omalizumab (Xolair®) is a recombinant humanized monoclonal antibody that selectively binds to human immunoglobulin E (IgE). Omalizumab is used to treat IgE-mediated diseases such as chronic idiopathic urticaria (CIU) and moderate to severe allergic asthma. In pre-marketing clinical trials in patients with asthma, anaphylaxis was reported in 3 of 3,507 (0.1%) patients. In post-marketing spontaneous reports, the frequency of anaphylaxis attributed to omalizumab use was estimated to be at least 0.2% of patients based on an estimated exposure of about 57,300 patients from June 2003 through December 2006. To better understand the risk of anaphylaxis in patients with allergic asthma receiving omalizumab, a post-marketing pharmacosurveillance study was initiated in 2009. As part of this study, an assay was developed to detect antibodies of IgE isotype to omalizumab. Serum samples from patients in the study were evaluated using this assay. Our results indicated that there was no observable correlation between either anaphylaxis or skin test reactivity and the presence of antibodies of IgE isotype to omalizumab. Here, we discuss the development of this assay as well as the results of the immunogenicity assessment.

Rapid identification of small binding motifs with high-throughput phage display: discovery of peptidic antagonists of IGF-1 function.

A panel of 22 naïve peptide libraries was constructed in a polyvalent phage display format and sorted against insulin-like growth factor-1 (IGF-1). The libraries were pooled to achieve a total diversity of 4.4 x 10(11). After three rounds of selection, the majority of the phage clones bound specifically to IGF-1, with a disulfide-constrained CX(9)C scaffold dominating the selection. Four monovalently displayed sub-libraries were designed on the basis of these conserved motifs. Sub-library maturation in a monovalent format yielded an antagonistic peptide that inhibited the interactions between IGF-1 and two cell-surface receptors and those between IGF-1 and two soluble IGF binding proteins with micromolar potency. NMR analysis revealed that the peptide is highly structured in the absence of IGF-1, and peptides that preorganize the binding elements were selected during the sorting.

Monoclonal antibodies to the V2 domain of MN-rgp120: fine mapping of epitopes and inhibition of α4ß7 binding.

BACKGROUND: Recombinant gp120 (MN-rgp120) was a major component of the AIDSVAX B/E vaccine used in the RV144 trial. This was the first clinical trial to show that vaccination could prevent HIV infection in humans. A recent RV144 correlates of protection study found that protection correlated with the presence of antibodies to the V2 domain. It has been proposed that antibodies to the α4ß7 binding site in the V2 domain might prevent HIV-1 infection by blocking the ability of virions to recognize α4ß7 on activated T-cells. In this study we investigated the specificity of monoclonal antibodies (MAbs) to the V2 domain of MN-rgp120 and examined the possibility that these antibodies could inhibit the binding of MN-rgp120 to the α4ß7 integrin. METHODOLOGY/PRINCIPAL FINDINGS: Nine MAbs to the V2 domain were isolated from mice immunized with recombinant envelope proteins. The ability of these MAbs to inhibit HIV infection, block the binding of gp120 to CD4, and block the binding of MN-rgp120 to the α4ß7 integrin was measured. Mutational analysis showed that eight of the MAbs recognized two immunodominant clusters of amino acids (166-168 and 178-183) located at either end of the C strand within the four-strand anti-parallel sheet structure comprising the V1/V2 domain. CONCLUSIONS/SIGNIFICANCE: These studies showed that the antigenic structure of the V2 domain is exceedingly complex and that MAbs isolated from mice immunized with MN-rgp120 exhibited a high level of strain specificity compared to MAbs to the V2 domain isolated from HIV-infected humans. We found that immunization with MN-rgp120 readily elicits antibodies to the V2 domain and some of these were able to block the binding of MN-rgp120 to the α4ß7 integrin.

Isolation and characterization of the B-cell marker CD20.

The integral membrane protein CD20 has been identified as an important therapeutic target in the treatment of non-Hodgkin's lymphoma (NHL). CD20 binding of many antibodies including the therapeutic antibody, rituximab, has been shown to be critically dependent upon the conformation of a loop structure between the third and fourth helical transmembrane regions. In this work, human and murine CD20 proteins expressed in Escherichia coli are shown to be localized with the cell membrane and are purified in nondenaturing detergent solutions. The purified human and murine CD20 proteins have a substantial helical structure as measured by circular dichroism spectroscopy. Only small changes in the secondary structure are observed following the reduction of CD20, with the addition of SDS, or after heating. The rituximab antibody is shown to bind to purified human CD20 with nanomolar affinity. Rituximab binding is abolished by reduction and alkylation of CD20, with data consistent with the proposed antibody epitope being within the disulfide-bonded loop formed between cysteine residues 167 and 183. Disulfide-bond-dependent antibody binding is partially recovered following reoxidation of reduced CD20. Antibody binding is unaffected by mutations of cysteines proposed to be in the intracellular domain of CD20. The affinities of intact rituximab and its Fab fragment to the isolated and purified CD20 are similar to the observed affinity of rituximab Fab for CD20 on the surface of B cells. However, the intact rituximab antibody shows much higher affinity for CD20 on B cells. This suggests that B cells display CD20 in such a way that allows for marked avidity effects to be observed, perhaps through cross-linking of CD20 monomers into lipid rafts, which limits receptor diffusion in the membrane. Such cross-linking may play a role in partitioning CD20 into lipid rafts and in enhancing antibody-dependent B-cell depletion activities of rituximab and other therapeutic anti-CD20 antibodies.

Stable "zeta" peptides that act as potent antagonists of the high-affinity IgE receptor.

Recently we described a family of peptides, unrelated in sequence to IgE, that form stable beta-hairpins in solution and inhibit IgE activity in the microM range [Nakamura, G. R., Starovasnik, M. A., Reynolds, M. E. & Lowman, H. B. (2001) Biochemistry 40, 9828-9835]. Using an expanded set of peptide-phage libraries, we found a simpler motif, X(2)CPX(2)CYX, for binding to the high-affinity IgE receptor. In solution, one of these peptides spontaneously formed a covalent antiparallel dimer. We subsequently linked these monomers in a single-chain construct on phage and optimized receptor binding. Ultimately, peptides with 30 nM affinity were produced. NMR studies showed that the peptide adopts a stable fold consisting of two "zeta" (zeta)-shaped moieties. Structure-activity analyses reveal a single binding site created by the zeta-dimer, with two tyrosine residues important for structural stability and two proline residues important for Fc epsilon RI binding. The peptides inhibit histamine release from cultured cells and are extremely stable in biological fluids. The zeta peptides appear to act as competitive IgE inhibitors and suggest possibilities for design of novel IgE antagonists.