Differential effects of FcRn antagonists on the subcellular trafficking of FcRn and albumin.

The homeostasis of IgG is maintained by the neonatal Fc receptor, FcRn. Consequently, antagonism of FcRn to reduce endogenous IgG levels is an emerging strategy for treating antibody-mediated autoimmune disorders using either FcRn-specific antibodies or an engineered Fc fragment. For certain FcRn-specific antibodies, this approach has resulted in reductions in the levels of serum albumin, the other major ligand transported by FcRn. Cellular and molecular analyses of a panel of FcRn antagonists have been carried out to elucidate the mechanisms leading to their differential effects on albumin homeostasis. These analyses have identified 2 processes underlying decreases in albumin levels during FcRn blockade: increased degradation of FcRn and competition between antagonist and albumin for FcRn binding. These findings have potential implications for the design of drugs to modulate FcRn function.

Effect of efgartigimod on muscle group subdomains in participants with generalized myasthenia gravis: post hoc analyses of the phase 3 pivotal ADAPT study.

BACKGROUND AND PURPOSE: Generalized myasthenia gravis (gMG) is a rare, chronic, neuromuscular autoimmune disease mediated by pathogenic immunoglobulin G (IgG) autoantibodies. Patients with gMG experience debilitating muscle weakness, resulting in impaired mobility, speech, swallowing, vision and respiratory function. Efgartigimod is a human IgG1 antibody Fc fragment engineered for increased binding affinity to neonatal Fc receptor. The neonatal Fc receptor blockade by efgartigimod competitively inhibits endogenous IgG binding, leading to decreased IgG recycling and increased degradation resulting in lower IgG concentration. METHODS: The safety and efficacy of efgartigimod were evaluated in the ADAPT study. Key efficacy outcome measures included Myasthenia Gravis Activities of Daily Living (MG-ADL) and Quantitative Myasthenia Gravis (QMG) scores. Efgartigimod demonstrated significant improvement in both the MG-ADL and QMG scores. This post hoc analysis aimed to determine whether all subdomains of MG-ADL and QMG improved with efgartigimod treatment. Individual items of MG-ADL and QMG were grouped into four subdomains: bulbar, ocular, limb/gross motor and respiratory. Change from baseline over 10 weeks in each subdomain was calculated for each group. RESULTS: Greater improvements from baseline were seen across MG-ADL subdomains in participants treated with efgartigimod compared with placebo. These improvements were typically observed 1 to 2 weeks after the first infusion and correlated with reductions in IgG. Similar results were observed across most QMG subdomains. CONCLUSIONS: These post hoc analyses of MG-ADL and QMG subdomain data from ADAPT suggest that efgartigimod is beneficial in improving muscle function and strength across all muscle groups, leading to the observed efficacy in participants with gMG.

The Fab region of IgG impairs the internalization pathway of FcRn upon Fc engagement.

Binding to the neonatal Fc receptor (FcRn) extends serum half-life of IgG, and antagonizing this interaction is a promising therapeutic approach in IgG-mediated autoimmune diseases. Fc-MST-HN, designed for enhanced FcRn binding capacity, has not been evaluated in the context of a full-length antibody, and the structural properties of the attached Fab regions might affect the FcRn-mediated intracellular trafficking pathway. Here we present a comprehensive comparative analysis of the IgG salvage pathway between two full-size IgG1 variants, containing wild type and MST-HN Fc fragments, and their Fc-only counterparts. We find no evidence of Fab-regions affecting FcRn binding in cell-free assays, however, cellular assays show impaired binding of full-size IgG to FcRn, which translates into improved intracellular FcRn occupancy and intracellular accumulation of Fc-MST-HN compared to full size IgG1-MST-HN. The crystal structure of Fc-MST-HN in complex with FcRn provides a plausible explanation why the Fab disrupts the interaction only in the context of membrane-associated FcRn. Importantly, we find that Fc-MST-HN outperforms full-size IgG1-MST-HN in reducing IgG levels in cynomolgus monkeys. Collectively, our findings identify the cellular membrane context as a critical factor in FcRn biology and therapeutic targeting.

Novel treatment strategies for acetylcholine receptor antibody-positive myasthenia gravis and related disorders.

The presence of autoantibodies directed against the muscle nicotinic acetylcholine receptor (AChR) is the most common cause of myasthenia gravis (MG). These antibodies damage the postsynaptic membrane of the neuromuscular junction and cause muscle weakness by depleting AChRs and thus impairing synaptic transmission. As one of the best-characterized antibody-mediated autoimmune diseases, AChR-MG has often served as a reference model for other autoimmune disorders. Classical pharmacological treatments, including broad-spectrum immunosuppressive drugs, are effective in many patients. However, complete remission cannot be achieved in all patients, and 10% of patients do not respond to currently used therapies. This may be attributed to production of autoantibodies by long-lived plasma cells which are resistant to conventional immunosuppressive drugs. Hence, novel therapies specifically targeting plasma cells might be a suitable therapeutic approach for selected patients. Additionally, in order to reduce side effects of broad-spectrum immunosuppression, targeted immunotherapies and symptomatic treatments will be required. This review presents established therapies as well as novel therapeutic approaches for MG and related conditions, with a focus on AChR-MG.

IgG regulation through FcRn blocking: A novel mechanism for the treatment of myasthenia gravis.

The neonatal Fc receptor (FcRn) is an MHC class I-like molecule that is widely distributed in mammalian organs, tissues, and cells. FcRn is critical to maintaining immunoglobulin G (IgG) and albumin levels through rescuing these molecules from lysosomal degradation. IgG autoantibodies are associated with many autoimmune diseases, including myasthenia gravis (MG), a rare neuromuscular autoimmune disease that causes debilitating and, in its generalized form (gMG), potentially life-threatening muscle weakness. IgG autoantibodies are directly pathogenic in MG and target neuromuscular junction proteins, causing neuromuscular transmission failure. Treatment approaches that reduce autoantibody levels, such as therapeutic plasma exchange and intravenous immunoglobulin, have been shown to be effective for gMG patients but are not indicated as ongoing maintenance therapies and can be associated with burdensome side effects. Agents that block FcRn-mediated recycling of IgG represent a rational and promising approach for the treatment of gMG. Blocking FcRn allows targeted reduction of all IgG subtypes without decreasing concentrations of other Ig isotypes; therefore, FcRn blocking could be a safe and effective treatment strategy for a broad population of gMG patients. Several FcRn-blocking antibodies and one antibody Fc fragment have been developed and are currently in various stages of clinical development. This article describes the mechanism of FcRn blockade as a novel approach for IgG-mediated disease therapy and reviews promising clinical data using such FcRn blockers for the treatment of gMG.

Safety, efficacy, and tolerability of efgartigimod in patients with generalised myasthenia gravis (ADAPT): a multicentre, randomised, placebo-controlled, phase 3 trial.

BACKGROUND: There is an unmet need for treatment options for generalised myasthenia gravis that are effective, targeted, well tolerated, and can be used in a broad population of patients. We aimed to assess the safety and efficacy of efgartigimod (ARGX-113), a human IgG1 antibody Fc fragment engineered to reduce pathogenic IgG autoantibody levels, in patients with generalised myasthenia gravis. METHODS: ADAPT was a randomised, double-blind, placebo-controlled, phase 3 trial done at 56 neuromuscular academic and community centres in 15 countries in North America, Europe, and Japan. Patients aged at least 18 years with generalised myasthenia gravis were eligible to participate in the study, regardless of anti-acetylcholine receptor antibody status, if they had a Myasthenia Gravis Activities of Daily Living (MG-ADL) score of at least 5 (>50% non-ocular), and were on a stable dose of at least one treatment for generalised myasthenia gravis. Patients were randomly assigned by interactive response technology (1:1) to efgartigimod (10 mg/kg) or matching placebo, administered as four infusions per cycle (one infusion per week), repeated as needed depending on clinical response no sooner than 8 weeks after initiation of the previous cycle. Patients, investigators, and clinical site staff were all masked to treatment allocation. The primary endpoint was proportion of acetylcholine receptor antibody-positive patients who were MG-ADL responders (≥2-point MG-ADL improvement sustained for ≥4 weeks) in the first treatment cycle. The primary analysis was done in the modified intention-to-treat population of all acetylcholine receptor antibody-positive patients who had a valid baseline MG-ADL assessment and at least one post-baseline MG-ADL assessment. The safety analysis included all randomly assigned patients who received at least one dose or part dose of efgartigimod or placebo. This trial is registered at ClinicalTrials.gov (NCT03669588); an open-label extension is ongoing (ADAPT+, NCT03770403). FINDINGS: Between Sept 5, 2018, and Nov 26, 2019, 167 patients (84 in the efgartigimod group and 83 in the placebo group) were enrolled, randomly assigned, and treated. 129 (77%) were acetylcholine receptor antibody-positive. Of these patients, more of those in the efgartigimod group were MG-ADL responders (44 [68%] of 65) in cycle 1 than in the placebo group (19 [30%] of 64), with an odds ratio of 4·95 (95% CI 2·21-11·53, p<0·0001). 65 (77%) of 84 patients in the efgartigimod group and 70 (84%) of 83 in the placebo group had treatment-emergent adverse events, with the most frequent being headache (efgartigimod 24 [29%] vs placebo 23 [28%]) and nasopharyngitis (efgartigimod ten [12%] vs placebo 15 [18%]). Four (5%) efgartigimod-treated patients and seven (8%) patients in the placebo group had a serious adverse event. Three patients in each treatment group (4%) discontinued treatment during the study. There were no deaths. INTERPRETATION: Efgartigimod was well tolerated and efficacious in patients with generalised myasthenia gravis. The individualised dosing based on clinical response was a unique feature of ADAPT, and translation to clinical practice with longer term safety and efficacy data will be further informed by the ongoing open-label extension. FUNDING: argenx.

Phase 2 study of efgartigimod, a novel FcRn antagonist, in adult patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder, characterized by a low platelet count (<100 × 109 /L) in the absence of other causes associated with thrombocytopenia. In most patients, IgG autoantibodies directed against platelet receptors can be detected. They accelerate platelet clearance and destruction, inhibit platelet production, and impair platelet function, resulting in increased risk of bleeding and impaired quality of life. Efgartigimod is a human IgG1 antibody Fc-fragment, a natural ligand of the neonatal Fc receptor (FcRn), engineered for increased affinity to FcRn, while preserving its characteristic pH-dependent binding. Efgartigimod blocks FcRn, preventing IgG recycling, and causing targeted IgG degradation. In this Phase 2 study, 38 patients were randomized 1:1:1 to receive four weekly intravenous infusions of either placebo (N = 12) or efgartigimod at a dose of 5 mg/kg (N = 13) or 10 mg/kg (N = 13). This short treatment cycle of efgartigimod in patients with ITP, predominantly refractory to previous lines of therapy, was shown to be well tolerated, and demonstrated a favorable safety profile consistent with Phase 1 data. Efgartigimod induced a rapid reduction of total IgG levels (up to 63.7% mean change from baseline), which was associated with clinically relevant increases in platelet counts (46% patients on efgartigimod vs 25% on placebo achieved a platelet count of ≥50 × 109 /L on at least two occasions, and 38% vs 0% achieved ≥50 × 109 /L for at least 10 cumulative days), and a reduced proportion of patients with bleeding. Taken together, these data warrant further evaluation of FcRn antagonism as a novel therapeutic approach in ITP.

Randomized phase 2 study of FcRn antagonist efgartigimod in generalized myasthenia gravis.

OBJECTIVE: To investigate safety and explore efficacy of efgartigimod (ARGX-113), an anti-neonatal Fc receptor immunoglobulin G1 Fc fragment, in patients with generalized myasthenia gravis (gMG) with a history of anti-acetylcholine receptor (AChR) autoantibodies, who were on stable standard-of-care myasthenia gravis (MG) treatment. METHODS: A phase 2, exploratory, randomized, double-blind, placebo-controlled, 15-center study is described. Eligible patients were randomly assigned (1:1) to receive 4 doses over a 3-week period of either 10 mg/kg IV efgartigimod or matched placebo combined with their standard-of-care therapy. Primary endpoints were safety and tolerability. Secondary endpoints included efficacy (change from baseline to week 11 of Myasthenia Gravis Activities of Daily Living, Quantitative Myasthenia Gravis, and Myasthenia Gravis Composite disease severity scores, and of the revised 15-item Myasthenia Gravis Quality of Life scale), pharmacokinetics, pharmacodynamics, and immunogenicity. RESULTS: Of the 35 screened patients, 24 were enrolled and randomized: 12 received efgartigimod and 12 placebo. Efgartigimod was well-tolerated in all patients, with no serious or severe adverse events reported, no relevant changes in vital signs or ECG findings observed, and no difference in adverse events between efgartigimod and placebo treatment. All patients treated with efgartigimod showed a rapid decrease in total immunoglobulin G (IgG) and anti-AChR autoantibody levels, and assessment using all 4 efficacy scales consistently demonstrated that 75% showed a rapid and long-lasting disease improvement. CONCLUSIONS: Efgartigimod was safe and well-tolerated. The correlation between reduction of levels of pathogenic IgG autoantibodies and disease improvement suggests that reducing pathogenic autoantibodies with efgartigimod may offer an innovative approach to treat MG. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that efgartigimod is safe and well-tolerated in patients with gMG.

Efgartigimod improves muscle weakness in a mouse model for muscle-specific kinase myasthenia gravis.

Myasthenia gravis is hallmarked by fatigable muscle weakness resulting from neuromuscular synapse dysfunction caused by IgG autoantibodies. The variant with muscle-specific kinase (MuSK) autoantibodies is characterized by prominent cranial and bulbar weakness and a high frequency of respiratory crises. The majority of MuSK MG patients requires long-term immunosuppressive treatment, but the result of these treatments is considered less satisfactory than in MG with acetylcholine receptor antibodies. Emergency treatments are more frequently needed, and many patients develop permanent facial weakness and nasal speech. Therefore, new treatment options would be welcome. The neonatal Fc receptor protects IgG from lysosomal breakdown, thus prolonging IgG serum half-life. Neonatal Fc receptor antagonism lowers serum IgG levels and thus may act therapeutically in autoantibody-mediated disorders. In MuSK MG, IgG4 anti-MuSK titres closely correlate with disease severity. We therefore tested efgartigimod (ARGX-113), a new neonatal Fc receptor blocker, in a mouse model for MuSK myasthenia gravis. This model involves 11 daily injections of purified IgG4 from MuSK myasthenia gravis patients, resulting in overt myasthenic muscle weakness and, consequently, body weight loss. Daily treatment with 0.5 mg efgartigimod, starting at the fifth passive transfer day, reduced the human IgG4 titres about 8-fold, despite continued daily injection. In muscle strength and fatigability tests, efgartigimod-treated myasthenic mice outperformed control myasthenic mice. Electromyography in calf muscles at endpoint demonstrated less myasthenic decrement of compound muscle action potentials in efgartigimod-treated mice. These substantial in vivo improvements of efgartigimod-treated MuSK MG mice following a limited drug exposure period were paralleled by a tendency of recovery at neuromuscular synaptic level (in various muscles), as demonstrated by ex vivo functional studies. These synaptic improvements may well become more explicit upon longer drug exposure. In conclusion, our study shows that efgartigimod has clear therapeutic potential in MuSK myasthenia gravis and forms an exciting candidate drug for many autoantibody-mediated neurological and other disorders.

Neonatal Fc receptor antagonist efgartigimod safely and sustainably reduces IgGs in humans.

BACKGROUND: Intravenous Ig (IVIg), plasma exchange, and immunoadsorption are frequently used in the management of severe autoimmune diseases mediated by pathogenic IgG autoantibodies. These approaches modulating IgG levels can, however, be associated with some severe adverse reactions and a substantial burden to patients. Targeting the neonatal Fc receptor (FcRn) presents an innovative and potentially more effective, safer, and more convenient alternative for clearing pathogenic IgGs. METHODS: A randomized, double-blind, placebo-controlled first-in-human study was conducted in 62 healthy volunteers to explore single and multiple ascending intravenous doses of the FcRn antagonist efgartigimod. The study objectives were to assess safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity. The findings of this study were compared with the pharmacodynamics profile elicited by efgartigimod in cynomolgus monkeys. RESULTS: Efgartigimod treatment resulted in a rapid and specific clearance of serum IgG levels in both cynomolgus monkeys and healthy volunteers. In humans, single administration of efgartigimod reduced IgG levels up to 50%, while multiple dosing further lowered IgGs on average by 75% of baseline levels. Approximately 8 weeks following the last administration, IgG levels returned to baseline. Efgartigimod did not alter the homeostasis of albumin or Igs other than IgG, and no serious adverse events related to efgartigimod infusion were observed. CONCLUSION: Antagonizing FcRn using efgartigimod is safe and results in a specific, profound, and sustained reduction of serum IgG levels. These results warrant further evaluation of this therapeutic approach in IgG-driven autoimmune diseases. TRIAL REGISTRATION: Clinicaltrials.gov NCT03457649. FUNDING: argenx BVBA.

ARGX-110, a highly potent antibody targeting CD70, eliminates tumors via both enhanced ADCC and immune checkpoint blockade.

Overexpression of CD70 has been documented in a variety of solid and hematological tumors, where it is thought to play a role in tumor proliferation and evasion of immune surveillance. Here, we describe ARGX-110, a defucosylated IgG1 monoclonal antibody (mAb) that selectively targets and neutralizes CD70, the ligand of CD27.   ARGX-110 was generated by immunization of outbred llamas. The antibody was germlined to 95% human identity, and its anti-tumor efficacy was tested in several in vitro assays. ARGX-110 binds CD70 with picomolar affinity. In depletion studies, ARGX-110 lyses tumor cells with greater efficacy than its fucosylated version. In addition, ARGX-110 demonstrates strong complement-dependent cytotoxicity and antibody-dependent cellular phagocytosis activity. ARGX-110 inhibits signaling of CD27, which results in blocking of the activation and proliferation of Tregs. In a Raji xenograft model, administration of the fucosylated version of ARGX-110 resulted in a prolonged survival at doses of 0.1 mg/kg and above. The pharmacokinetics of ARGX-110 was tested in cynomolgus monkeys; the calculated half-life is 12 days. In conclusion, ARGX-110 is a potent blocking mAb with a dual mode of action against both CD70-bearing tumor cells and CD70-dependent Tregs. This antibody is now in a Phase 1 study in patients with advanced malignancies expressing CD70 (NCT01813539).

Identification of interaction sites for dimerization and adapter recruitment in Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 4.

Toll-like receptor signaling requires interactions of the Toll/IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4-TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization.

Caspase-1 targets the TLR adaptor Mal at a crucial TIR-domain interaction site.

Toll-like receptors (TLRs) are crucial components of innate immunity, ensuring efficient responses against invading pathogens. After ligand binding, TLR signaling is initiated by recruitment of adaptor molecules, a step mediated by homotypic Toll-IL-1 receptor (TIR) domain interactions. Four TIR-containing TLR adaptor molecules are described, all of which are susceptible to modification and strict regulation. For example, caspase-1 is reported to cleave the TLR adaptor Mal at position D198, an event that is indispensible for Mal function. In this report, we use the mammalian two-hybrid technique MAPPIT to study the implications of Mal cleavage. We show that a Mal mutant, which mimics caspase-1 cleavage and a caspase-1-uncleavable MalD198A mutant, are abrogated in their bridging function and lose the ability to activate NF-kappaB. A MalD198E mutant is still fully functional, suggesting that caspase-1 cleavage of Mal is not necessary for Mal-mediated signaling. D198 of Mal is conserved in MyD88 and TLR4 TIR domains and the negatively charged amino acid at this position is crucial for the interactions and function of Mal, MyD88 and TLR4 TIR. Our data suggest an inhibitory, rather than an activating role for caspase-1 in Mal regulation, and show that the caspase-1 cleavage site in Mal is part of a TIR-domain interaction site.

MAPPIT (mammalian protein-protein interaction trap) analysis of early steps in toll-like receptor signalling.

The mammalian protein-protein interaction trap (MAPPIT) is a two-hybrid technique founded on type I cytokine signal transduction. Thereby, bait and prey proteins are linked to signalling deficient cytokine receptor chimeras. Interaction of bait and prey and ligand stimulation restores functional JAK (Janus kinase)-STAT (signal transducers and activators of transcription) signalling, which ultimately leads to the transcription of a reporter or marker gene under the control of the STAT3-responsive rPAP1 promoter. In the subsequent protocol, we describe the use of MAPPIT to study early events in Toll-like receptor (TLR) signalling. We here demonstrate a "signalling interaction cascade" from TLR4 to IRAK-1.

MAPPIT analysis of early Toll-like receptor signalling events.

Toll-like receptor (TLR) signalling is initiated by the recruitment of one or more adaptor proteins to the activated receptor complex. At present, four of these proteins are identified, namely MyD88, Mal, Trif and Tram and their selective usage by different TLRs in part accounts for TLR-specific transcriptional responses. Recent findings described unique biochemical properties for each of these TIR-domain containing adaptors and revealed that these adapters are subjected to post-translational modification. We used mammalian protein-protein interaction trap (MAPPIT), a two-hybrid technique that functions in a mammalian cell context, to study the molecular interactions downstream of TLR activation. We demonstrate pathway walking from TLR4 to IRAK-1 and identified Mal as a bridging adaptor, linking MyD88 to the activated TLR4.

MAPPIT analysis of TLR adaptor complexes.

Toll-like receptors (TLRs) are crucial components of the innate immune system, coupling pathogen recognition to a cellular response. We used the MAPPIT mammalian two-hybrid technique to investigate protein-protein interactions in the early steps in TLR signalling. A partial TLR-adaptor interaction map was constructed confirming several known but also documenting novel interactions. We show that the TLR adaptor Mal is critical for linking Myeloid Differentiation primary response protein 88 (MyD88) to TLR2 and TLR4. Analysis of the contributions of the different sub-domains of MyD88-adaptor-like protein (Mal) and MyD88 in adaptor homo- and hetero-dimerisation provides an initial mechanistic insight in this bridging function of Mal.

The C-terminus of CIS defines its interaction pattern.

Proteins of the SOCS (suppressors of cytokine signalling) family are characterized by a conserved modular structure with pre-SH2 (Src homology 2), SH2 and SOCS-box domains. Several members, including CIS (cytokine-inducible SH2 protein), SOCS1 and SOCS3, are induced rapidly upon cytokine receptor activation and function in a negative-feedback loop, attenuating signalling at the receptor level. We used a recently developed mammalian two-hybrid system [MAPPIT (mammalian protein-protein interaction trap)] to analyse SOCS protein-interaction patterns in intact cells, allowing direct comparison with biological function. We find that, besides the SH2 domain, the C-terminal part of the CIS SOCS-box is required for functional interaction with the cytokine receptor motifs examined, but not with the N-terminal death domain of the TLR (Toll-like receptor) adaptor MyD88. Mutagenesis revealed that one single tyrosine residue at position 253 is a critical binding determinant. In contrast, substrate binding by the highly related SOCS2 protein, and also by SOCS1 and SOCS3, does not require their SOCS-box.

Immunostimulatory potential of hepatitis B nucleocapsid preparations: lipopolysaccharide contamination should not be overlooked.

The nucleocapsid of hepatitis B virus (HBV) allows insertions of heterologous peptides and even complete proteins. Because of its outstanding capacity to induce B-cell, T-helper and cytotoxic T-cell responses, this structure is considered to be an important instrument for future vaccine development. Most of the evidence for the unique immunogenic qualities of nucleocapsids has been generated in mice, which are not natural hosts of HBV. Moreover, most nucleocapsid preparations used in these studies were produced in a recombinant manner in Escherichia coli. Such preparations have been shown to contain lipopolysaccharide (LPS). Not unexpectedly, it is shown here that contaminating LPS, rather than the nucleocapsid structure itself, is responsible for the activation of human antigen-presenting cells. Careful examination of the literature dealing with the immunogenicity of HBV nucleocapsids suggests that the possible presence of LPS has been largely ignored or underestimated in several studies. This raises doubts on some of the underlying mechanisms that have been proposed to explain the unique immunogenicity of the HBV nucleocapsid.

Mapping of the leptin binding sites and design of a leptin antagonist.

The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.