10 research outputs found
Effect of mAb 2A4 on the resolution of human ALÎș amyloidomas in mice.
<p>The mAbs 2A4 or JH70 were administered subcutaneously into scid mice bearing human ALÎș amyloidomas (50 mg Hig extract), 7 days after injection. Mice were euthanized and the mass of the residual amyloidoma was measured (A) and the amyloidomas photographed (B). Amyloidomas were fixed and processed for paraffin sectioning. Panel C shows immunohistochemical staining for macrophages (anti-Iba-1) in representative sections of amyloidomas from mice treated either with mAbs 2A4 or JH70. Arrowheads designate the positive staining for macrophages; arrows are the band of polymorphonuclear cells, and; âAâ designates the major mass of amyloid material.</p
Shi biodistribution and amyloidâ¶organ ratio.
<p>Shi biodistribution and amyloidâ¶organ ratio.</p
EuLISA of 2A4 (blue) or control JH70 (red) mAb at 50 nM binding to human AL amyloid extracts.
<p>(A) Each amyloid extract derived from the liver (L) or spleen (S) was dried onto the well of a 96-well microplate normalized to the ThT signal equivalent to that of 10 ”g/mL Hig, L. The far right bars of panel A show binding to synthetic WIL fibrils normalized to the same ThT value. (B) Summary of 2A4 binding to ALÎș or ALλ amyloids, normalized to the binding to rV<sub>λ</sub>6WIL synthetic fibrils.</p
Binding of 2A4 to ALÎș Hig amyloid extract and rV<sub>λ</sub>6Wil fibrils in the presence of different Bence Jones proteins.
<p>(A) ALÎș amyloid extract, 10 ”g/mL Hig, L (blue) or 0.83 ”M rV<sub>λ</sub>6Wil fibrils (red) were dried onto the wells of a 96-well microplate and the binding of 2A4 evaluated in the presence of BJp pre-incubated with 2A4. Concentrations of 2A4 were 5 nM for wells containing rV<sub>λ</sub>6 Wil (red) and 200 nM for the Hig samples (blue). Competitor BJps, designated on the abscissa by 3 letter code were present at 41.5 ”M. The data are expressed as the % of the positive control, i.e., mAb 2A4 in the absence of any BJp. Competition with rV<sub>λ</sub>6 Wil fibrils (far right) served as a positive control. (B) EuLISA titration of 2A4 on Hig, L amyloid extract, 10 ”g/mL (closed) and rV<sub>λ</sub>6 Wil fibrils, 0.25 ”M (open). The EC<sub>50</sub> for Wil was âŒ3 nM and for Hig, âŒ40 nM.</p
Evaluation of size-fractionated Kir BJp preparation as competitors.
<p>(A) Absorbance profile of size fractionated soluble Kir BJp preparation using a Superdex 75 gel filtration column. (B) Fractions, 46, 52, 59 and 66, selected on the basis of the A280 chromatogram trace, were analyzed by SDS-PAGE, and then (C) tested for their ability to inhibit the reaction between 2A4 (200 nM) and Hig L amyloid extract (10 ”g/mL) in the standard ELISA. Each fraction was used at the same concentration based on A280 to inhibit binding as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052686#pone-0052686-g004" target="_blank">Fig. 4A</a>.</p
Binding of radioiodinated 2A4 or isotype matched control IgG MOPC 141 to human ALÎș and ALλ amyloidomas.
<p>Antibodies were radiolabeled with <sup>125</sup>I and injected iv in the lateral tail vein (âŒ150 ”Ci, 25 ”g) of mice bearing human AL amyloidomas (50 mg of human Îș1 AL [Hig] or λ2 AL [Shi]), at 7 days post-implantation. Mice were imaged post mortem at 48 h post injection using SPECT/CT and the site of the amyloidoma was determined from the CT imaging and is indicated by the arrows (left panels). Tissues including the amyloid mass were harvested, fixed and sectioned for analysis. The right hand panels show autoradiography of the radioiodine tracer (ARG), hematoxylin and eosin staining for tissue morphology (H&E) or staining with Congo red to show amyloid deposits (CR).</p
Hig biodistribution and amyloidâ¶organ ratio.
<p>Hig biodistribution and amyloidâ¶organ ratio.</p
Immunohistochemistry of 2A4 binding to human ALÎș and ALλ amyloid in tissue samples.
<p>2A4 mAb was used to immunostain AL amyloid (arrows) in formalin-fixed paraffin-embedded tissues derived from the liver, kidney or pancreas. Top panels show staining with mAb 2A4 alone; middle panels show results with mAb 2A4 adsorbed with immunogen peptide -GHEDT- (imm) and bottom panels mAb 2A4 adsorbed with control peptide -GHETMADQE- (ctrl).</p
2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis<sup>â </sup>
<p>Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agencyâapproved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid <i>in vitro</i>. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both Îș and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin Tâpositive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits <i>in vitro</i>. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis.</p
ELN484228 provides protection in cellular models of αSyn-mediated dysfunction.
<p><b>A.</b> ELN484228 alleviates αSyn-mediated impairment of vesicular dynamics. H4 neuroglioma cells over-expressing αSyn from a tetracycline inducible promoter were cultured for 24 hours in the absence or presence of 1 ”g/ml tetracycline to induce αSyn and ELN484228 or control compound ELN484217 (compound number 38 in table S4 in Supporting Information text <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087133#pone.0087133.s001" target="_blank">file S1</a>). Open bars: without compound, black bars: with indicated amount of compound. Cells were assayed for phagocytic activity as a measure of αSyn-mediated impairment of vesicular function. 4 ÎŒ beads were added for 90 minutes and a phagocytic index was calculated by microscopic visualization. Each sample was run in triplicate and experiments were run three independent times. The phagocytic indices for each individual experiment were averaged and statistical analyses run on the final averages from the three experiments. T-test analysis of the combined averages of the three experiments revealed a significant difference in phagocytosis between Tet-induced samples with and without ELN484228 (nâ=â3+/â s.e.m *pâ€0.001 versus no compound Tet-induced sample). <b>B.</b> Microglia isolated from postnatal day 1 to 3 pups from hSNCA<sup>E46K</sup> transgenic (αSyn ) or non-transgenic littermates were incubated for 24 hours with 100 ”M ELN484217 or ELN484228 followed by addition of 10 ”m beads for 90 minutes. A phagocytic index was calculated by microscopic visualization (nâ=â3+/â s.e.m *pâ€0.001). <b>C.</b> ELN484228 alleviates loss of dopaminergic neurons and neurite retraction induced by the A53T mutant of αSyn. Primary rat embryonic midbrain cultures were non-transduced (âcontrolâ) or transduced with adenovirus encoding A53T αSyn, in the absence or presence of 10 ”M ELN484228. The cells were then stained immunocytochemically for MAP2 and TH. Preferential dopaminergic cell death was assessed by evaluating the percentage of MAP2-positive cells that also stained positive for TH. The lengths of neurites staining positive for both MAP2 and TH were measured using the NIS-Elements software. Data are plotted as the mean ± s.e.m. nâ=â3 for neuron viability analysis; nâ=â160â206 for neurite length analysis. *p-valueâ€0.05, ***p-valueâ€0.001 versus aSyn A53T virus in the absence of compound; one-way ANOVA with Newman-Keuls post-test. <b>D.</b> ELN484228 reduces translocation of αSyn to the phagocytic cup<b>.</b> To assess αSyn translocation, H4 cells were treated with 100 ”M ELN484228 and 1 ”g/ml tetracycline for 24 hours; cells were then stimulated with 4 ÎŒ beads for 90 minutes. Samples were fixed and stained with 5C12 antibody to detect αSyn (red). Cells were counterstained with 488-phalloidin (green) and Hoechts (blue). A dotted circle indicates the position of the bead. <b>E.</b> ELN484228 reduces translocation of αSyn to synapses. Rat hippocampal neurons (âŒ21DIV) grown in serum-free conditions were treated for 24 hours with 1 ”M ELN484228 or 0.01% DMSO vehicle. On the left side is a representative confocal microscopic image showing localization of αSyn (red) detected with 5C12 antibody, and localization of the presynaptic marker synaptophysin (green). Scale bar is 5 ”m. Images were subjected to quantitative analysis and synaptic αSyn levels were determined as the amount of signal that colocalizes with the synaptic synaptophysin marker. Automated measurements were performed in Metamorph imaging analysis software to determine synaptic αSyn and synaptophysin levels by integrated intensity or pixel area, respectively. Values represent mean +/â SEM, nâ=â1000 terminals (αSyn) or 18 optical fields (synaptophysin) per condition, and derived from 2â3 independent cultures. Quantitation demonstrates that ELN484228 reduces the synaptic levels of αSyn in rat hippocampal neurons.</p