Serum IgG-induced microglial activation enhances neuronal cytolysis via the NO/sGC/PKG pathway in children with opsoclonus-myoclonus syndrome and neuroblastoma.

BACKGROUND: Opsoclonus-myoclonus syndrome (OMS) is a rare neurological disease. Some children with OMS also have neuroblastoma (NB). We and others have previously documented that serum IgG from children with OMS and NB induces neuronal cytolysis and activates several signaling pathways. However, the mechanisms underlying OMS remain unclear. Here, we investigated whether nitric oxide (NO) from activated microglias and its cascade contribute to neuronal cytolysis in pediatric OMS. METHODS: The activation of cultured cerebral cortical and cerebellar microglias incubated with sera or IgG isolated from sera of children with OMS and NB was measured by the expression of the activation marker, cytokines, and NO. Neuronal cytolysis was determined after exposing to IgG-treated microglia-conditioned media. Using inhibitors and activators, the effects of NO synthesis and its intracellular cascade, namely soluble guanylyl cyclase (sGC) and protein kinase G (PKG), on neuronal cytolysis were evaluated. RESULTS: Incubation with sera or IgG from children with OMS and NB increased the activation of cerebral cortical and cerebellar microglias, but not the activation of astrocytes or the cytolysis of glial cells. Moreover, the cytolysis of neurons was elevated by conditioned media from microglias incubated with IgG from children with OMS and NB. Furthermore, the expression of NO, sGC, and PKG was increased. Neuronal cytolysis was relieved by the inhibitors of NO signaling, while neuronal cytolysis was exacerbated by the activators of NO signaling but not proinflammatory cytokines. The cytolysis of neurons was suppressed by pretreatment with the microglial inhibitor minocycline, a clinically tested drug. Finally, increased microglial activation did not depend on the Fab fragment of serum IgG. CONCLUSIONS: Serum IgG from children with OMS and NB potentiates microglial activation, which induces neuronal cytolysis through the NO/sGC/PKG pathway, suggesting an applicability of microglial inhibitor as a therapeutic candidate.

Overcoming Challenges of Implementing Closed System Transfer Device Clinical In-Use Compatibility Testing for Drug Development of Antibody Drug Conjugates.

Closed system transfer devices (CSTD) are a supplemental engineering control designed to reduce occupational exposure of hazardous drugs and are currently implemented in accordance with evolving regulations. Owing to the novelty and complexity of these devices and their importance in clinical in-use testing, here we evaluated FDA-approved CSTD, assessing product quality through stability indicating assays to determine any drug product incompatibilities. Six devices were used in a simulated compounding and administration of a late-phase IgG1 antibody-drug conjugate (ADC) and the resulting samples were analyzed for visible and subvisible particle counts by light obscuration and micro-flow imaging, physical stability by size exclusion chromatography, and biological activities by relative potency. Potential challenges included improper fit of CSTD components, loss of product to void volume, and material incompatibility. Results showed compatibility of the ADC with the 6 CSTD evaluated. One CSTD introduced subvisible particles into the ADC during compounding that were identified through morphological assessment as silicone oil. This study highlights the importance of clinical in use testing with new devices and proposes strategies to mitigate the risk of drug product incompatibility with CSTD.

Toxicological and pharmacological assessment of AGEN1884, a novel human IgG1 anti-CTLA-4 antibody.

CTLA-4 and CD28 exemplify a co-inhibitory and co-stimulatory signaling axis that dynamically sculpts the interaction of antigen-specific T cells with antigen-presenting cells. Anti-CTLA-4 antibodies enhance tumor-specific immunity through a variety of mechanisms including: blockade of CD80 or CD86 binding to CTLA-4, repressing regulatory T cell function and selective elimination of intratumoral regulatory T cells via an Fcγ receptor-dependent mechanism. AGEN1884 is a novel IgG1 antibody targeting CTLA-4. It potently enhanced antigen-specific T cell responsiveness that could be potentiated in combination with other immunomodulatory antibodies. AGEN1884 was well-tolerated in non-human primates and enhanced vaccine-mediated antigen-specific immunity. AGEN1884 combined effectively with PD-1 blockade to elicit a T cell proliferative response in the periphery. Interestingly, an IgG2 variant of AGEN1884 revealed distinct functional differences that may have implications for optimal dosing regimens in patients. Taken together, the pharmacological properties of AGEN1884 support its clinical investigation as a single therapeutic and combination agent.

2-Methoxyestradiol protects against IgG immune complex-induced acute lung injury by blocking NF-κB and CCAAT/enhancer-binding protein ß activities.

Increasing evidences indicate that 2-Methoxyestradiol (2ME2) plays an essential role in protecting against inflammatory responses. However, its effect on IgG immune complex (IC)-induced acute lung injury (ALI) remains enigmatic. In the study, by using i.p. administration of 2ME2, we evaluated its influence on IgG IC-induced pulmonary injury in mice. We found that during IgG IC-induced ALI, mice treated by 2ME2 displayed a substantial decrease in vascular permeability and neutrophil influx (represented by myeloperoxidase activity) when compared with their counterparts receiving vehicle treatment. Furthermore, 2ME2 treatment significantly decreased pro-inflammatory mediator production and inflammatory cell, especially neutrophil accumulation in bronchoalveolar lavage fluids (BALFs) upon IgG IC stimulation. In vitro, IgG IC-triggered inflammatory mediator production was markedly down-regulated by 2ME2 in macrophages. Moreover, we verified that the activation of the transcription factors, NF-κB and CCAAT/enhancer-binding protein (C/EBP) ß, were inhibited by 2ME2 in IgG IC-challenged macrophages. We demonstrated that alleviation of NF-κB-dependent transcription might be associated with reduced phosphorylation of NF-κB p65, and reduction of C/EBP activation was directly linked to its expression. In addition, we discovered that IgG IC-stimulated phosphorylation of both p38 MAPK and ERK1/2 was alleviated by 2ME2. These data indicated a novel strategy for blockade of IgG IC-induced inflammatory activities.

Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody.

The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species. The heterogeneities observed in the RP-HPLC have been determined to arise from unpaired cysteines (Cys-22 and Cys-96) that are buried in the V(H) domain. The Fab containing free thiols (referred to as "free-thiol Fab") and the Fab containing the disulfide (referred to as "intact Fab") of mAb A were generated through limited Lys-C digestion and purified with an ion exchange chromatography method. The binding of free-thiol Fab and intact Fab to its antigen was measured in a cell-based binding assay and an enzyme linked immunosorbent assay. The unpaired cysteines in the Fab of mAb A were found to have no significant impact on the binding to its target. Consistent with these Fab binding data, the enriched intact mAb A containing free thiols was determined to be fully active in a potency assay. The data reported here demonstrate that the redox status of cysteines is potentially a major source of heterogeneity for an antibody.

TEM8/ANTXR1 blockade inhibits pathological angiogenesis and potentiates tumoricidal responses against multiple cancer types.

Current antiangiogenic agents used to treat cancer only partially inhibit neovascularization and cause normal tissue toxicities, fueling the need to identify therapeutic agents that are more selective for pathological angiogenesis. Tumor endothelial marker 8 (TEM8), also known as anthrax toxin receptor 1 (ANTXR1), is a highly conserved cell-surface protein overexpressed on tumor-infiltrating vasculature. Here we show that genetic disruption of Tem8 results in impaired growth of human tumor xenografts of diverse origin including melanoma, breast, colon, and lung cancer. Furthermore, antibodies developed against the TEM8 extracellular domain blocked anthrax intoxication, inhibited tumor-induced angiogenesis, displayed broad antitumor activity, and augmented the activity of clinically approved anticancer agents without added toxicity. Thus, TEM8 targeting may allow selective inhibition of pathological angiogenesis.

39-week toxicity and toxicokinetic study of ponezumab (PF-04360365) in cynomolgus monkeys with 12-week recovery period.

Ponezumab (PF-04360365) is a novel humanized IgG2Δa monoclonal antibody that binds to amyloid-ß (Aß). It is designed to have reduced immune effector function compared to other passive immunotherapies for Alzheimer's disease (AD). Toxicity was evaluated in cynomolgus monkeys treated intravenously with vehicle or 10, 30, or 100 mg/kg of ponezumab every 10th day for up to 39 weeks, and after a 12-week recovery phase. The Aß peptide sequence of monkeys is identical to that of humans. No substantial difference in test article exposure between sexes was observed, and mean plasma Cmax and AUC0-n were approximately dose-proportional. Ponezumab was detectable approximately 9 weeks after cessation of dosing. All animals, except two males given 10 mg/kg, maintained exposure to test article. One of these males tested positive for anti-ponezumab antibodies. Ponezumab was detected in the cerebrospinal fluid (CSF) of animals given active treatment. The estimated CSF/plasma ponezumab concentration ratio was <0.008 after multiple doses. At the end of the dosing and recovery phases, plasma Aß1-40 and Aß1-x were increased in treated animals versus controls. No test article-related effects were seen after ophthalmogical, cardiovascular, physical examinations, and clinical and anatomic pathology evaluations. Plasma concentrations of ponezumab on day 261 at the no observed adverse effect level of 100 mg/kg were 22.4 and 5.3 times greater on a Cmax and AUC basis, respectively, than human exposures at the highest dose (10 mg/kg) in a single-dose Phase I trial. These data suggest an acceptable safety profile for ponezumab as an immunotherapy for AD.

The use of surrogate antibodies to evaluate the developmental and reproductive toxicity potential of an anti-TNFalpha PEGylated Fab' monoclonal antibody.

Certolizumab pegol (CZP) is a PEGylated Fab' fragment of a humanized monoclonal immunoglobulin G (IgG)1 antibody that binds to human tumor necrosis factor alpha (TNFα) with high affinity. As for many monoclonal antibodies (mAbs), nonclinical safety assessment of CZP has been constrained because of its limited species cross-reactivity and recognition of only nonhuman primate and human TNFα, which presents particular challenges for assessing reproductive and developmental safety. To comprehensively assess the potential liability of TNFα suppression on reproductive and developmental processes, a PEGylated Fab' anti-rat TNFα antibody surrogate (cTN3 PF) has been developed and evaluated for reproductive toxicity. Conventional rat fertility and early embryonic development, embryo-fetal toxicity and pre- and postnatal development studies have been shown to be free of maternal, reproductive, or development toxicity effects, following sustained TNFα inhibition with cTN3 PF. Importantly, these studies have also shown that in marked contrast to a whole IgG anti-TNFα antibody, the PEGylated Fab' antibody cTN3 PF homologous to certolizumab pegol demonstrated negligible fetal exposure following maternal administration during the period of organogenesis. In addition to minimal placental transmission, transfer to milk was lower and fetal absorption negligible compared with the whole IgG antibody cTN3 γ1, resulting in little or no detectable antibody in the plasma of lactating pups.

Disease exacerbation by etanercept in a mouse model of alphaviral arthritis and myositis.

OBJECTIVE: Mosquito-borne alphaviruses such as chikungunya virus, o'nyong-nyong virus, and Ross River virus (RRV) cause sporadic, sometimes large, outbreaks of rheumatic disease worldwide. This study was designed to test the effect of treating RRV-induced arthritis using the anti-tumor necrosis factor (anti-TNF) drug etanercept in a mouse model of rheumatic disease. METHODS: Mice were infected with RRV and treated with etanercept. Weight gain was measured, tissue viral titers were determined, and histologic changes in muscle and joint tissues were assessed. RESULTS: RRV-infected mice treated with etanercept showed decreased weight gain, higher viral titers in muscle, joints, and blood, and more tissue damage and inflammatory cell recruitment than RRV-infected mice without treatment. CONCLUSION: Anti-TNF therapy is unlikely to be useful in treating alphaviral arthritides. During alphaviral epidemics, careful monitoring of patients being treated with anti-TNF agents may be warranted.

Universal vaccine based on ectodomain of matrix protein 2 of influenza A: Fc receptors and alveolar macrophages mediate protection.

The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ(-/-), FcγRI(-/-), FcγRIII(-/-), and (FcγRI, FcγRIII)(-/-) mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII(-/-) mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)(-/-) mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG.

Purkinje cell death after uptake of anti-Yo antibodies in cerebellar slice cultures.

Paraneoplastic cerebellar degeneration accompanying gynecological and breast cancers is characteristically accompanied by a serum and cerebrospinal fluid (CSF) antibody response, termed "anti-Yo," which reacts with cytoplasmic proteins of cerebellar Purkinje cells. Because these antibodies interact with cytoplasmic rather than cell surface membrane proteins, their role in causing Purkinje cell death has been questioned. To address this issue, we studied the interaction of anti-Yo antibodies with Purkinje cells in slice (organotypic) cultures of rat cerebellum. We incubated cultures with immunoglobulin G (IgG)-containing anti-Yo antibodies using titers of anti-Yo antibody equivalent to those found in CSF of affected patients. Cultures were then studied in real time and after fixation for potential uptake of antibody and induction of cell death. Anti-Yo antibodies delivered in serum, CSF, or purified IgG were taken up by viable Purkinje cells, accumulated intracellularly, and were associated with cell death. Normal IgG was also taken up by Purkinje cells but did not accumulate and did not affect cell viability. These findings indicate that autoantibodies directed against intracellular Purkinje cell proteins can be taken up to cause cell death and suggest that anti-Yo antibody may be directly involved in the pathogenesis of paraneoplastic cerebellar degeneration.

Patient autoantibodies deplete postsynaptic muscle-specific kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice.

The postsynaptic muscle-specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody-positive myasthenia gravis patients (MuSK IgG; 45 mg day(1)i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51-73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR beta-subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK-rapsyn system of AChR clustering.

A chromatographic approach for elevating the antibody-dependent cellular cytotoxicity of antibody composites.

Recent studies have shown that antibodies with low fucose content in their oligosaccharides exhibit highly potent antibody-dependent cellular cytotoxicity (ADCC). However, composites of therapeutic antibodies produced by conventional production systems using cell lines such as Chinese hamster ovary (CHO) and SP2/0 do not necessarily contain sufficient amounts of non-fucosylated antibody species. In this study, we combined two lectin-affinity chromatography techniques, Concanavalin A and Lens culinaris agglutinin, to enrich the non-fucosylated species from therapeutic material using the anti-Her2/neu model antibody. Oligosaccharide analysis by matrix-assisted laser desorption/ionization-time of flight MS following peptide-N-glycosidase F digestion suggested that non-fucosylated antibody could be enriched in the purified fraction with efficient removal of high-mannose species. The ADCC activity of the purified fraction was about 100-fold higher than that of the initial material. The chromatographic strategy presented here can be a useful tool to elevate ADCC activity of antibody materials without concentrating high-mannose oligosaccharides.

Synaptophysin is an autoantigen in paraneoplastic neuropathy.

Paraneoplastic neurological syndromes (PNS) are often associated with antineuronal autoantibodies and many of them could be identified in the recent years. However, there are still new antineuronal binding patterns with yet unidentified autoantigens. We here describe a new autoantibody associated with paraneoplastic sensorimotor and autonomic neuropathy in a patient with small cell lung cancer. In indirect immunofluorescence test, the patient's serum colocalised with the synaptic protein synaptophysin in the cerebellum and myenteric plexus of the gut. Immunoblotting showed a 38 kDa reactivity, which is also the molecular weight of synaptophysin. Therefore a Western Blot with recombinant synaptophysin has been used and revealed reactivity of the serum against synaptophysin. In patients with non-paraneoplastic neuropathies or healthy controls, anti-synaptophysin autoantibodies were not detectable. In 20 SCLC patients without neurological syndromes, two patients had low-titer anti-synaptophysin autoantibodies. The patient's serum and IgG fraction showed cytotoxicity to primary cultured myenteric plexus neurons. We conclude that synaptophysin is an autoantigen in paraneoplastic neurological syndromes.

Targeting the follicular lymphoma microenvironment through blockade of TNFalpha with etanercept.

Follicular dendritic cells (FDCs) support the survival of follicular lymphoma (FL). Tumor necrosis factor alpha (TNFalpha) is overexpressed by FL cells and is critical in the development and maintenance of FDCs. We hypothesised that TNFalpha might be an ideal therapeutic target. We treated seven patients with relapsed/refractory FL with 8 weeks of etanercept, 25 mg SC on day 1 and 4 of each week. Patients without progression received 16 additional weeks of etanercept. All patients completed at least 8 weeks of etanercept and two patients completed 24 weeks. At the 8 week evaluation five patients had SD. Of the five with SD, two progressed at 9 and 12 weeks on therapy and the remaining three progressed between 12 and 24 weeks after initiating therapy. Minimal toxicity was observed. FDG-PET imaging demonstrated decreases in standardized uptake value (SUV) following treatment with etanercept in five patients. Further studies in FL targeting the microenvironment in conjunction with standard cytotoxic therapy are warranted.

Anti-MuSK patient antibodies disrupt the mouse neuromuscular junction.

OBJECTIVE: A subset of myasthenia gravis patients that are seronegative for anti-acetylcholine receptor (anti-AChR) antibodies are instead seropositive for antibodies against the muscle-specific kinase (anti-MuSK-positive). Here, we test whether transfer of IgG from anti-MuSK-positive patients to mice confers impairment of the neuromuscular junction and muscle weakness. METHODS: IgG from anti-MuSK-positive myasthenia gravis patients or control IgG (seronegative for AChR and MuSK) was injected intraperitoneally (45 mg daily for 14 days) into 6-week-old female FVB/NJ and C57BL/6J mice. Changes at neuromuscular junctions in the tibialis anterior and diaphragm muscles were assessed by confocal fluorescent imaging of AChRs stained with fluorescent-alpha-bungarotoxin. Loss of function was assessed by electromyography. RESULTS: In experimental mice injected with anti-MuSK-positive patient IgG, postsynaptic AChR staining was reduced to as little as 22% of that seen in control mice. Experimental mice showed reduced apposition of the nerve terminal (labeled with antibodies against synaptophysin and neurofilament) and the postsynaptic AChR cluster (labeled with fluorescent-alpha-bungarotoxin). Mice injected with IgG from two of three anti-MuSK-positive patients lost weight and developed muscle weakness associated with a decremental electromyographic trace on repetitive nerve stimulation. INTERPRETATION: IgG from anti-MuSK-positive patients can cause myasthenia gravis when injected into mice. This may be explained by a progressive reduction in the density of postsynaptic AChR combined with changes in the nerve terminal and its relation to the postsynaptic structure.

Neutralization of Yersinia pestis-mediated macrophage cytotoxicity by anti-LcrV antibodies and its correlation with protective immunity in a mouse model of bubonic plague.

Plague is a life-threatening disease caused by Yersinia pestis, for which effective-licensed vaccines and reliable predictors of in vivo immunity are lacking. V antigen (LcrV) is a major Y. pestis virulence factor that mediates translocation of the cytotoxic Yersinia protein effectors (Yops). It is a well-established protective antigen and a part of currently tested plague subunit vaccines. We have developed a highly sensitive in vitro macrophage cytotoxicity neutralization assay which is mediated by anti-LcrV antibodies; and studied the potential use of these neutralizing antibodies as an in vitro correlate of plague immunity in mice. The assay is based on a Y. pestis strain with enhanced cytotoxicity to macrophages in which endogenous yopJ was replaced by the more effectively translocated yopP of Y. enterocolitica O:8. Mice passively immunized with rabbit anti-LcrV IgG or actively immunized with recombinant LcrV were protected against lethal doses of a virulent Y. pestis strain, in a mouse model of bubonic plague. This protection significantly correlated with the in vitro neutralizing activity of the antisera but not with their corresponding ELISA titers. In actively immunized mice, a cutoff value for serum neutralizing activity, above which survival was assured with high degree of confidence, could be established for different vaccination regimes. The impact of overall findings on the potential use of serum neutralizing activity as a correlate of protective immunity is discussed.

Intravitreal toxicity of high-dose etanercept.

PURPOSE: The aim of this study was to evaluate the retinal toxicity of high-dose intravitreal etanercept, a U.S. Food and Drug Administration-approved anti-inflammatory drug, in the rabbit model. METHODS: Twenty (20) New Zealand albino rabbits were divided into 5 groups (n=4); eyes in each group were intravitreally injected with one of the following doses of etanercept: 125 microg, 250 microg, 500 microg, 1 mg, or 2.5 mg. One (1) eye in each animal was used for the study dose; the fellow eye was injected with buffered sterile saline as a control. All animals were examined using indirect ophthalmoscopy and slit-lamp biomicroscopy before and after intravitreal injection and at days 1, 7, and 14. Electroretinography (ERG) was performed on all animals before intravitreal injection and 14 days after injection. The animals were euthanized on day 14. Histological preparations of the enucleated eyes were examined with light microscopy for retinal toxicity. RESULTS: Clinical examination, histological evaluation, and ERG results of all 5 groups demonstrated no signs of retinal toxicity. CONCLUSIONS: Intravitreal doses as high as 2.5 mg of etanercept did not cause retinal toxicity. Intravitreal doses of up to 2.5 mg of etanercept may provide a more potent, prolonged effect than the lower doses previously recommended.

A peptide that mimics the Vth region of beta-2-glycoprotein I reverses antiphospholipid-mediated thrombosis in mice.

Antiphospholipid (aPL) antibodies bind to beta2glycoprotein I (beta2GPI) and cause endothelial cell (EC) activation and thrombosis in mice. beta2GPI binds to EC through its Vth domain and induces their activation. TIFI is a 20 amino acid synthetic peptide that shares similarity with the Vth domain of beta2GPI. Our objectives were to examine the ability of TIFI to affect aPL-mediated thrombosis in mice and the interactions of TIFI, beta2GPI with phospholipid surfaces and target cells. CD1 mice were injected with IgG from a patient with antiphospholipid syndrome (IgG-APS) or with control IgG-NHS and with either TIFI or with control peptide (VITT). Size of induced thrombi was determined. Inhibition and competition studies were done using aPL antibodies, cardiolipin (CL) liposomes in the presence of varying amounts of TIFI and beta2GPI. Binding of fluorescinated beta2GPI to human ECs and to murine macrophages in the presence or absence of TIFI, was also examined. TIFI significantly decreased thrombus size in mice injected with IgG-APS. TIFI reverted the beta2GPI-dependent binding of aPL antibodies to CL liposomes in a dose-dependent fashion. This effect was abrogated by addition of beta2GPI, suggesting that TIFI displaces the binding of beta2GPI to phospholipids. TIFI inhibited the binding of fluorescinated beta2GPI to human EC and to murine macrophages. The data indicate that TIFI abrogates thrombogenic properties of aPL in mice by competing with beta2GPI and preventing its binding to target cells. This may be important in designing new modalities for the treatment of thrombosis in APS.

Targeting melanoma cells with human high molecular weight-melanoma associated antigen-specific antibodies elicited by a peptide mimotope: functional effects.

Human high molecular weight-melanoma associated Ag (HMW-MAA) mimics have been shown to elicit HMW-MAA-specific humoral immune responses that appear to be clinically beneficial. This finding has stimulated interest in characterizing the mechanism(s) underlying the ability of the elicited Abs to exert an anti-tumor effect. To address this question, in the present study, we have generated HMW-MAA-specific Abs by sequentially immunizing rabbits with the peptide P763.74, which mimics the HMW-MAA determinant recognized by mAb 763.74, and with HMW-MAA(+) melanoma cells. HMW-MAA-specific Abs isolated from immunized rabbits mediated cell-dependent cytotoxicity but did not mediate complement-dependent cytotoxicity of HMW-MAA(+) melanoma cells. These Abs also effectively inhibited spreading, migration and Matrigel invasion of HMW-MAA(+) melanoma cells. Besides contributing to our understanding of the role of HMW-MAA in the biology of melanoma cells, these results suggest that both immunological and nonimmunological mechanisms underlie the beneficial clinical effects associated with the induction of HMW-MAA-specific Abs in melanoma patients immunized with a HMW-MAA mimic.