Antibodies and superantibodies in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps is associated with local immunoglobulin hyperproduction and the presence of IgE antibodies against Staphylococcus aureus enterotoxins (SAEs). Aspirin-exacerbated respiratory disease is a severe form of chronic rhinosinusitis with nasal polyps in which nearly all patients express anti-SAEs. OBJECTIVES: We aimed to understand antibodies reactive to SAEs and determine whether they recognize SAEs through their complementarity-determining regions (CDRs) or framework regions. METHODS: Labeled staphylococcal enterotoxin (SE) A, SED, and SEE were used to isolate single SAE-specific B cells from the nasal polyps of 3 patients with aspirin-exacerbated respiratory disease by using fluorescence-activated cell sorting. Recombinant antibodies with "matched" heavy and light chains were cloned as IgG1, and those of high affinity for specific SAEs, assayed by means of ELISA and surface plasmon resonance, were recloned as IgE and antigen-binding fragments. IgE activities were tested in basophil degranulation assays. RESULTS: Thirty-seven SAE-specific, IgG- or IgA-expressing B cells were isolated and yielded 6 anti-SAE clones, 2 each for SEA, SED, and SEE. Competition binding assays revealed that the anti-SEE antibodies recognize nonoverlapping epitopes in SEE. Unexpectedly, each anti-SEE mediated SEE-induced basophil degranulation, and IgG1 or antigen-binding fragments of each anti-SEE enhanced degranulation by the other anti-SEE. CONCLUSIONS: SEEs can activate basophils by simultaneously binding as antigens in the conventional manner to CDRs and as superantigens to framework regions of anti-SEE IgE in anti-SEE IgE-FcεRI complexes. Anti-SEE IgG1s can enhance the activity of anti-SEE IgEs as conventional antibodies through CDRs or simultaneously as conventional antibodies and as "superantibodies" through CDRs and framework regions to SEEs in SEE-anti-SEE IgE-FcεRI complexes.

Bispecific Antibody Binding To RANKL and Osteonectin with Enhanced Localization to the Bone.

Therapeutics reducing bone turnover, such as denosumab (Dmab), an anti-RANKL antibody, can provide treatments for patients with bone destruction. However, some patients with osteoporosis or localized primary bone tumors and many patients with various types of bone-metastatic cancer display unsatisfactory responses to Dmab. For achieving greater efficiency of RANKL neutralization in the bone microenvironment by enhancing the distribution of Dmab to the bone, we reengineered Dmab by fusing with single-chain variable fragments of an antibody specific for osteonectin (On), which is abundantly expressed in osseous tissues. The bispecific antibody, Dmab-FvOn, showed a similar activity as Dmab in inhibiting RANKL as examined in an osteoclast differentiation assay. When administered to mice, Dmab-FvOn was found to localize in increased proportions at the endosteum of the bone where osteonectin is abundant. Our study suggests that by linking anti-RANKL with an osteonectin-targeting moiety, a greater proportion of the therapeutic effector can be distributed in the bone. Future studies are needed to investigate whether the bispecific antibody can achieve higher therapeutic efficacy and lower toxicity.

Skewed Exposure to Environmental Antigens Complements Hygiene Hypothesis in Explaining the Rise of Allergy.

The Hygiene Hypothesis has been recognized as an important cornerstone to explain the sudden increase in the prevalence of asthma and allergic diseases in modernized culture. The recent epidemic of allergic diseases is in contrast with the gradual implementation of Homo sapiens sapiens to the present-day forms of civilization. This civilization forms a gradual process with cumulative effects on the human immune system, which co-developed with parasitic and commensal Helminths. The clinical manifestation of this epidemic, however, became only visible in the second half of the twentieth century. In order to explain these clinical effects in terms of the underlying IgE-mediated reactions to innocuous environmental antigens, the low biodiversity of antigens in the domestic environment plays a pivotal role. The skewing of antigen exposure as a cumulative effect of reducing biodiversity in the immediate human environment as well as in changing food habits, provides a sufficient and parsimonious explanation for the rise in allergic diseases in a highly developed and helminth-free modernized culture. Socio-economic tendencies that incline towards a further reduction of environmental biodiversity may provide serious concern for future health. This article explains that the "Hygiene Hypothesis", the "Old Friends Hypothesis", and the "Skewed Antigen Exposure Hypothesis" are required to more fully explain the rise of allergy in modern societies.

Treating IgE-mediated diseases via targeting IgE-expressing B cells using an anti-CεmX antibody.

Targeting the IgE pathway is a clinically validated strategy for treating IgE-mediated diseases. Omalizumab, an anti-IgE antibody, which binds to free IgE and prevents the binding of IgE to FcεRI on mast cells and basophils has been approved for severe persistent allergic asthma and chronic spontaneous (idiopathic) urticaria. The therapeutic efficacy of anti-IgE has also been reported in allergic rhinitis, allergic bronchopulmonary aspergillosis, latex allergy, atopic dermatitis, allergic urticaria, anaphylaxis, and others. Anti-CεmX, which binds to membrane-bound IgE (mIgE) on IgE-switched B cells, lyses mIgE-expressing B lymphoblasts and prevents the allergen-induced generation of IgE-producing plasma cells, offers an alternative mechanism of intervening with the IgE inflammatory pathway. Because anti-CεmX does not bind to free IgE, it can modulate the IgE pathway regardless of the serum IgE levels in treated patients. These unique pharmacologic mechanisms potentially enable anti-CεmX to provide different clinical utilities from anti-IgE and serve as a therapeutic and a prophylactic in some IgE-mediated diseases, which are not adequately treated with current medicine.

The potential pharmacologic mechanisms of omalizumab in patients with chronic spontaneous urticaria.

In patients given a diagnosis of chronic spontaneous urticaria (CSU), there are no obvious external triggers, and the factors that initiate the clinical symptoms of wheal, flare, and itch arise from within the patient. Most patients with CSU have an autoimmune cause: some patients produce IgE autoantibodies against autoantigens, such as thyroperoxidase or double-stranded DNA, whereas other patients make IgG autoantibodies against FcεRI, IgE, or both, which might chronically activate mast cells and basophils. In the remainder of patients with CSU, the nature of the abnormalities has not yet been identified. Accumulating evidence has shown that IgE, by binding to FcεRI on mast cells without FcεRI cross-linking, can promote the proliferation and survival of mast cells and thus maintain and expand the pool of mast cells. IgE and FcεRI engagement can also decrease the release threshold of mast cells and increase their sensitivity to various stimuli through either FcεRI or other receptors for the degranulation process. Furthermore, IgE-FcεRI engagement potentiates the ability of mast cells to store and synthesize de novo inflammatory mediators and cytokines. Administration of omalizumab, by virtue of its ability to deplete IgE, attenuates the multiple effects of IgE to maintain and enhance mast cell activities and hence reduces the ability of mast cells to manifest inflammatory mechanisms in patients with CSU.

Changing patterns of antigen exposure and their impact on the prevalence of allergy.

Industrial development has advanced at a varying pace in different parts of the world over the past 200 years. Inhabitants of the most industrially advanced regions have experienced major changes in patterns of antigen exposure to infectious agents and to environmental biologic substances. This article analyzes the major factors that affect the amounts and variety of antigens to which the immune system of a young child is exposed. Depending on individual living environments and lifestyles, the types of antigen exposure of young children are graded into five patterns: 'primitive', 'pre-modern', 'early modern', 'modern', and 'ultramodern'. These patterns represent increasing deviation from the pattern of human immune system exposure to antigens prior to the industrial revolution. This article further discusses how such changes in antigen exposure have affected the immunologic system, especially with regard to the development of total IgE and allergic response-relevant antigen-specific IgE, and how the patterns of antigen exposure are related to the propensity to develop allergy.

An Antibody-Drug Conjugate for Multiple Myeloma Prepared by Multi-Arm Linkers.

First-line treatment of multiple myeloma, a prevalent blood cancer lacking a cure, using anti-CD38 daratumumab antibody and lenalidomide is often inadequate due to relapse and severe side effects. To enhance drug safety and efficacy, an antibody-drug conjugate, TE-1146, comprising six lenalidomide drug molecules site-specifically conjugated to a reconfigured daratumumab to deliver cytotoxic lenalidomide to tumor cells is developed. TE-1146 is prepared using the HighDAR platform, which employs i) a maleimide-containing "multi-arm linker" to conjugate multiple drug molecules creating a drug bundle, and ii) a designed peptide with a Zn2+-binding cysteine at the C-termini of a reconfigured daratumumab for site-specific drug bundle conjugation. It is shown that TE-1146 remains intact and effectively enters CD38-expressing tumor cells, releasing lenalidomide, leading to enhanced cell-killing effects compared to lenalidomide/daratumumab alone or their combination. This reveals the remarkable potency of lenalidomide once internalized by myeloma cells. TE-1146 precisely delivers lenalidomide to target CD38-overexpressing tumor cells. In contrast, lenalidomide without daratumumab cannot easily enter cells, whereas daratumumab without lenalidomide relies on Fc-dependent effector functions to kill tumor cells.

Site-specific Conjugation of 6 DOTA Chelators to a CA19-9-targeting scFv-Fc Antibody for Imaging and Therapy.

Antibodies conjugated with diagnostic/therapeutic radionuclides are attractive options for inoperable cancers lacking accurate imaging methods and effective therapeutics, such as pancreatic cancer. Hence, we have produced an antibody radionuclide conjugate termed TE-1132 comprising a α-CA19-9 scFv-Fc that is site-specifically conjugated at each C-terminus to 3 DOTA chelators via a cysteine-containing peptide linker. The smaller scFv-Fc size facilitates diffusivity within solid tumors, whereas the chelator-to-antibody ratio of six enabled 177Lu-radiolabeled TE-1132 to exhibit high radioactivity up to 520 MBq/nmol. In mice bearing BxPC3 tumors, immuno-SPECT/CT imaging of [111In]In-TE-1132 and the biodistribution of [177Lu]Lu-TE-1132 showed selective tumor accumulation. Single and multiple doses of [177Lu]Lu-TE-1132 effectively inhibited the BxPC3 tumor growth and prolonged the survival of mice with no irreversible body weight loss or hematopoietic damage. The adequate pharmacokinetic parameters, prominent tumor accumulation, and efficacy with good safety in mice encourage the further investigation of theranostic TE-1132 for treating pancreatic cancer.

The IgE gene in primates exhibits extraordinary evolutionary diversity.

Membrane-bound IgE (mIgE) on B lymphocytes is essential for IgE production. Earlier studies showed that the ε chain of mIgE (mε) on human B cells has a "long" isoform, with an extra "CεmX" domain of 52 amino acid (aa) residues between the CH4 domain and the membrane-anchor segment, as compared to the conventional "short" isoform. Because CεmX provides an antigenic site for targeting IgE-expressing B cells to down-regulate IgE production in patients with allergy, analysis of CεmX in various animals is of great interest. Hence, we analyzed the ε Ig gene, in particular, its membrane exon regions encoding the membrane anchor peptide segment and CεmX domain, of 26 species of the order Primates and 12 species of seven non-Primate orders using data obtained experimentally or retrieved from GenBank. Our analyses reveal the unexpected finding that the genes of three extant tarsier species do not contain the membrane exons for mIgE. Another striking finding is that early evolved Strepsirhini primates such as lemurs and lorises do not have gene segments for the long isoform, whereas New World monkeys such as marmosets and squirrel monkeys allow the transcription of only the long isoform. In Old World monkeys and apes, including humans, the ε gene allows the transcription of both isoforms. This work thus reveals the dramatic differences in the gene segment encoding the mε C terminal region among the four major primate lineages: the Strepsirhini primates, the tarsiers, New World monkeys, and Old World monkeys and apes/humans.

Unique epitopes on C epsilon mX in IgE-B cell receptors are potentially applicable for targeting IgE-committed B cells.

Membrane-bound IgE (mIgE) is part of the IgE-BCR and is essential for generating isotype-specific IgE responses. On mIgE(+) B cells, the membrane-bound epsilon-chain (mepsilon) exists predominantly in the long isoform, mepsilon(L), containing an extra 52 aa CepsilonmX domain between CH4 and the C-terminal membrane-anchoring segment; the short isoform of mepsilon, mepsilon(S), exists in minor proportions. CepsilonmX thus provides an attractive site for immunologic targeting of mIgE(+) B cells. In this study, we show that nine newly prepared CepsilonmX-specific mAbs, as well as the previously reported a20, bound to mIgE.Fc(L)-expressing CHO cells, while only 4B12 and 26H2 bound to mIgE.Fc(L)-expressing B cell line Ramos cells. The mAb 4B12 bound to the N-terminal part, 26H2 the middle part, and all others the C-terminal part of CepsilonmX. Expression of Igalpha and Igbeta on the mIgE.Fc(L)-CHO cells reduces the binding of a20 to CepsilonmX as compared with that of 4B12 and 26H2. The chimeric mAbs c4B12 and c26H2, when cross-linked by secondary antibodies, lysed mIgE.Fc(L)-Ramos cells by apoptosis through a BCR-dependent caspase pathway. Using PBMCs as the source of effector cells, c4B12 and c26H2 demonstrated Ab-dependent cellular cytotoxicity toward mIgE.Fc(L)-Ramos cells in a dose-dependent fashion. In cultures of PBMCs from atopic dermatitis patients, c4B12 and c26H2 inhibited the synthesis of IgE driven by anti-CD40 and IL-4. These results suggest that 4B12 and 26H2 and an immunogen using the peptide segments recognized by these mAbs are potentially useful for targeting mIgE(+) B cells to control IgE production.

Pathogenic autoantibodies to IFN-γ act through the impedance of receptor assembly and Fc-mediated response.

Anti-interferon (IFN)-γ autoantibodies (AIGAs) are a pathogenic factor in late-onset immunodeficiency with disseminated mycobacterial and other opportunistic infections. AIGAs block IFN-γ function, but their effects on IFN-γ signaling are unknown. Using a single-cell capture method, we isolated 19 IFN-γ-reactive monoclonal antibodies (mAbs) from patients with AIGAs. All displayed high-affinity (KD < 10-9 M) binding to IFN-γ, but only eight neutralized IFN-γ-STAT1 signaling and HLA-DR expression. Signal blockade and binding affinity were correlated and attributed to somatic hypermutations. Cross-competition assays identified three nonoverlapping binding sites (I-III) for AIGAs on IFN-γ. We found that site I mAb neutralized IFN-γ by blocking its binding to IFN-γR1. Site II and III mAbs bound the receptor-bound IFN-γ on the cell surface, abolishing IFN-γR1-IFN-γR2 heterodimerization and preventing downstream signaling. Site III mAbs mediated antibody-dependent cellular cytotoxicity, probably through antibody-IFN-γ complexes on cells. Pathogenic AIGAs underlie mycobacterial infections by the dual blockade of IFN-γ signaling and by eliminating IFN-γ-responsive cells.

Genetic variations in the C epsilon mX domain of human membrane-bound IgE.

The epsilon chain of membrane-bound IgE (mIgE) is expressed predominantly as a "long" isoform, containing an extra segment of 52 amino acid (a.a.) residues, referred to as C epsilon mX, between the CH4 domain and the C-terminal membrane-anchoring transmembrane peptide. C epsilon mX results from an alternative splicing of the epsilon RNA transcript at 156-bp upstream of the splicing acceptor site used by the "short" isoform. Here, based on an analysis of the C epsilon mX genomic DNA sequences of 320 subjects residing in Taiwan, we report that single-nucleotide polymorphisms have been found at two positions, namely, G/T at #46 and A/G at #93 (along the 156 bp of C epsilon mX), with the former creating an amino acid change from Val to Leu at #16 (along the 52 a.a. of C epsilon mX) and the latter resulting in no change (Gly). Among the 640 C epsilon mX sequences identified, the previously known 46G93A allelic form appeared 293 times, the newly discovered 46T93A allelic form (GeneBank accession no. GU208817) 26 times, and the 46G93G allelic form (GU208818) 321 times. No 46T93G allelic form was found. Serum IgE measurements showed that the polymorphisms did not correlate with the levels of serum IgE. The anti-C epsilon mX monoclonal antibody, 4B12, could bind equally well to mIgE.Fc(L)(16V) and mIgE.Fc(L)(16L). While genetic variation of C epsilon mX of broader populations should also be investigated, these newly discovered genetic variants of C epsilon mX in the Taiwanese population do not seem to affect the feasibility of using an anti-C epsilon mX mAb, such as 4B12, to target mIgE-expressing B cells.

Cumulative environmental changes, skewed antigen exposure, and the increase of allergy.

The human immune system evolved over many hundreds of million of years in the ancestors of vertebrates and mammals to defend them against infectious and parasitic organisms in their natural habitats. By the time the Primates and Rodentia orders diverged about 88 million years ago, the human immune system was largely configured. From about 125,000 years ago, marked by the use of fire, Homo sapiens began to make substantial changes in their living environment and lifestyle. Here, we examine those changes in two phases, before and after the Industrial Revolution, and analyze their impact on the exposure of our immune system to infectious organisms and to harmless environmental antigens. Our analyses show that the cumulative changes in environment and lifestyle in many regions of the world have drastically altered the pattern by which humans are exposed to infectious organisms and harmless environmental antigens and that these changes have profoundly impacted the function of the immune system and enhanced the development of allergy. Our analyses expand the hygiene hypothesis by taking into consideration the immunological impact of a broader range of antigen exposure changes than simply decreased microbial infection during childhood. We subsequently examine the proposed mechanisms of TH1 to TH2 shift and Treg downregulation with regard to the hygiene hypothesis and present an immunological basis for the increased activity of the IgE-mediated pathway in allergic patients. In our "skewed antigen exposure" theory, we propose that, for many individuals living in modern societies: (i) reduced exposure to a large variety of infectious organisms and environmental antigens and (ii) increased exposure to a small variety of environmental antigens, resulting from the cumulative changes in individuals' living environment and lifestyle, together alter the balance of the immune system, and increase production of IgE and the sensitization of mast cells toward a limited variety environmental antigens unique to affected individuals, resulting in an overall increase in allergy.

Anti-IgE antibodies for the treatment of IgE-mediated allergic diseases.

The pharmacological purposes of the anti-IgE therapy are to neutralize IgE and to inhibit its production to attenuate type I hypersensitivity reactions. The therapy is based on humanized IgG1 antibodies that bind to free IgE and to membrane-bound IgE on B cells, but not to IgE bound by the high-affinity IgE.Fc receptors on basophils and mast cells or by the low-affinity IgE.Fc receptors on B cells. After nearly 20 years since inception, therapeutic anti-IgE antibodies (anti-IgE) have been studied in about 30 Phase II and III clinical trials in many allergy indications, and a lead antibody, omalizumab, has been approved for treating patients (12 years and older) with moderate-to-severe allergic asthma. Anti-IgE has confirmed the roles of IgE in the pathogenesis of asthma and helped define the concept "allergic asthma" in clinical practice. It has been shown to be safe and efficacious in treating pediatric allergic asthma and treating allergic rhinitis and is being investigated for treating peanut allergy, atopic dermatitis, latex allergy, and others. It has potential for use to combine with specific and rush immunotherapy for increased safety and efficacy. Anti-IgE thus appears to provide a prophylactic and therapeutic option for moderate to severe cases of many allergic diseases and conditions in which IgE plays a significant role. This chapter reviews the evolution of the anti-IgE concept and the clinical studies of anti-IgE on various disease indications, and presents a comprehensive analysis on the multiple intricate immunoregulatory pharmacological effects of anti-IgE. Finally, it reviews other approaches that target IgE or IgE-expressing B cells.

Alleles and isoforms of human membrane-bound IgA1.

In humans, IgA exists as two subclasses, IgA1 and IgA2, which contain distinct alpha1 and alpha2 heavy chains, respectively. Both subclasses also have membrane-bound forms (mIgA1 and mIgA2) containing the corresponding malpha1 and malpha2 heavy chains, which differ from alpha1 and alpha2 by an additional "membrane-anchor" peptide segment extending from the CH3 domain of alpha1 and alpha2. The membrane-anchor segment has three parts: an extracellular, a transmembrane, and an intracellular segment. The heavy chain malpha1 exists in short and long isoforms, referred to as malpha1S and malpha1L, with the latter containing extra 6 amino acid residues, GSCSVA, at the N-terminus of the extracellular segment (residues 453-458). By studying the genomic and mRNA sequences of malpha1 and malpha2 from 30 individuals residing in Taiwan, we have found that, in addition to the known malpha1 allele, referred to as malpha1(456S), malpha1 also has a previously unknown allele, referred to as malpha1(456C) (GenBank accession no. EU431191). This newly identified allele is present in the donor population at a similar proportion to malpha1(456S), and appears to exist only as the long isoform, i.e. malpha1L, rather than the short isoform, malpha1S. Furthermore, we confirmed that malpha2 exists only as the short isoform. Future studies will examine whether these mIgA1 variations affect the regulation of IgA synthesis and whether mIgA1 can provide an antigenic site for the immunological targeting of IgA-expressing B cells.

Structural basis for selective inhibition of immunoglobulin E-receptor interactions by an anti-IgE antibody.

Immunoglobulin E (IgE) antibodies play a central role in the allergic response: interaction with FcεRI on mast cells and basophils leads to immediate hypersensitivity reactions upon allergen challenge, while interaction with CD23/FcεRII, expressed on a variety of cells, regulates IgE synthesis among other activities. The receptor-binding IgE-Fc region has recently been found to display remarkable flexibility, from acutely bent to extended conformations, with allosteric communication between the distant FcεRI and CD23 binding sites. We report the structure of an anti-IgE antibody Fab (8D6) bound to IgE-Fc through a mixed protein-carbohydrate epitope, revealing further flexibility and a novel extended conformation with potential relevance to that of membrane-bound IgE in the B cell receptor for antigen. Unlike the earlier, clinically approved anti-IgE antibody omalizumab, 8D6 inhibits binding to FcεRI but not CD23; the structure reveals how this discrimination is achieved through both orthosteric and allosteric mechanisms, supporting therapeutic strategies that retain the benefits of CD23 binding.

Structural and Physical Basis for Anti-IgE Therapy.

Omalizumab, an anti-IgE antibody, used to treat severe allergic asthma and chronic idiopathic urticaria, binds to IgE in blood or membrane-bound on B lymphocytes but not to IgE bound to its high (FcεRI) or low (CD23) affinity receptor. Mutagenesis studies indicate overlapping FcεRI and omalizumab-binding sites in the Cε3 domain, but crystallographic studies show FcεRI and CD23-binding sites that are far apart, so how can omalizumab block IgE from binding both receptors? We report a 2.42-Šomalizumab-Fab structure, a docked IgE-Fc/omalizumab-Fab structure consistent with available experimental data, and the free energy contributions of IgE residues to binding omalizumab, CD23, and FcεRI. These results provide a structural and physical basis as to why omalizumab cannot bind receptor-bound IgE and why omalizumab-bound IgE cannot bind to CD23/FcεRI. They reveal the key IgE residues and their roles in binding omalizumab, CD23, and FcεRI.

Down regulation of B cells by immunization with a fusion protein of a self CD20 peptide and a foreign IgG.Fc fragment.

In vivo studies of mice were performed to investigate whether auto-reactive antibodies specific for self CD20 antigen on B cells could be induced by immunizing with a CD20 peptide linked to a foreign, human IgG.Fc fragment through a T cell immunologically inert linker peptide and how such an auto-reactivity, if generated, would affect the levels of B cells. The dimeric Fc fusion protein containing the extracellular 44-amino acid portion of CD20, and the CH2-CH3 domains of human gamma 1 immunoglobulin were prepared. After several subcutaneous immunizations with this CD20-Fc protein, mice produced anti-CD20 antibodies that can bind to native CD20 on normal B cells and B-lymphoma cells. In mice immunized with the CD20-Fc protein, the fraction of B cells in total peripheral blood lymphocytes decreased to about 40%, significantly lower than that of mice immunized with human IgG. In addition, antibody response towards an irrelevant bystander antigen, chicken ovalbumin, was weakened compared with that of mice immunized with human IgG. These results show that auto-reactive antibodies specific for CD20 can be induced by immunizing with an autologous CD20 peptide fused with a foreign IgG.Fc and that the auto-antibodies can partially reduce the levels of B cells and their response to other antigens.

Improving pharmacokinetic properties of adrenocorticotropin by site-specific lipid modification.

Although many peptides are potentially good therapeutic agents for treating various diseases, only a few have been developed for limited applications. A major shortcoming is that peptides have generally very short serum half lives. In the present study, we use adrenocorticotropin (ACTH) as a model and explore the potential of combining site-specific amino acid substitution and lipid modification to increase the circulating half-lives of peptides. Phe39 of ACTH was substituted by Cys, which has a free sulfhydryl group that can react specifically with iodoacetamide derivatives of lipophilic groups. The biological activities of lipophilized ACTH(F39C)s were higher than native ACTH. Lipophilized ACTH(F39C)s bound more tightly to human serum albumin and cell membranes in vitro and had longer serum half-lives in vivo than native ACTH. These results indicate that the pharmacokinetic properties of peptides can be improved by site-specific substitution with cysteine residues and subsequent conjugation with lipophilic moieties.

Two potential therapeutic antibodies bind to a peptide segment of membrane-bound IgE in different conformations.

IgE mediates hypersensitivity reactions responsible for most allergic diseases, which affect 20-40% of the population in developed countries. A 52-residue domain of membrane-bound IgE (mIgE) called CεmX is currently a target for developing therapeutic antibodies; however, its structure is unknown. Here we show that two antibodies with therapeutic potential in IgE-mediated allergic diseases, which can cause cytolytic effects on mIgE-expressing B lymphocytes and downregulate IgE production, target different conformations of an intrinsically disordered region (IDR) in the extracellular CεmX domain. We provide an important example of antibodies targeting an extracellular IDR of a receptor on the surface of intended target cells. We also provide fundamental structural characteristics unique to human mIgE, which may stimulate further studies to investigate whether other monoclonal antibodies (mAbs) targeting intrinsically disordered peptide segments or vaccine-like products targeting IDRs of a membrane protein can be developed.