Managed Care-Work in Progress or Stalled Experiment?

The symptomatic drugs currently on the market for Alzheimer's disease (AD) have no effect on disease progression, and this creates a large unmet medical need. The type of drug that has developed most rapidly in the last decade is immunotherapy: vaccines and, especially, passive vaccination with monoclonal antibodies. Antibodies are attractive drugs as they can be made highly specific for their target and often with few side effects. Data from recent clinical AD trials indicate that a treatment effect by immunotherapy is possible, providing hope for a new generation of drugs. The first anti-amyloid-beta (anti-A beta) vaccine developed by Elan, AN1792, was halted in phase 2 because of aseptic meningoencephalitis. However, in a follow-up study, patients with antibody response to the vaccine demonstrated reduced cognitive decline, supporting the hypothesis that A beta immunotherapy may have clinically relevant effects. Bapineuzumab (Elan/Pfizer Inc./Johnson & Johnson), a monoclonal antibody targeting fibrillar A beta, was stopped because the desired clinical effect was not seen. Solanezumab (Eli Lilly and Company) was developed to target soluble, monomeric A beta. In two phase 3 studies, Solanezumab did not meet primary endpoints. When data from the two studies were pooled, a positive pattern emerged, revealing a significant slowing of cognitive decline in the subgroup of mild AD. The Arctic mutation has been shown to specifically increase the formation of soluble A beta protofibrils, an A beta species shown to be toxic to neurons and likely to be present in all cases of AD. A monoclonal antibody, mAb158, was developed to target A beta protofibrils with high selectivity. It has at least a 1,000-fold higher selectivity for protofibrils as compared with monomers of A beta, thus targeting the toxic species of the peptide. A humanized version of mAb158, BAN2401, has now entered a clinical phase 2b trial in a collaboration between BioArctic Neuroscience and Eisai without the safety concerns seen in previous phase 1 and 2a trials. Experiences from the field indicate the importance of initiating treatment early in the course of the disease and of enriching the trial population by improving the diagnostic accuracy. BAN2401 is a promising candidate for A beta immunotherapy in early AD. Other encouraging efforts in immunotherapy as well as in the small-molecule field offer hope for new innovative therapies for AD in the future

Киберпространство и виртуальная реальность: попытка историко-философского анализа

Материалы IV Республик. науч. конф. студентов, магистрантов и аспирантов, Гомель, 12 мая 2011 г

The Uppsala APP deletion causes early onset autosomal dominant Alzheimer's disease by altering APP processing and increasing amyloid β fibril formation

Point mutations in the amyloid precursor protein gene (APP) cause familial Alzheimer's disease (AD) by increasing generation or altering conformation of amyloid beta (A beta). Here, we describe the Uppsala APP mutation (Delta 690-695), the first reported deletion causing autosomal dominant AD. Affected individuals have an age at symptom onset in their early forties and suffer from a rapidly progressing disease course. Symptoms and biomarkers are typical of AD, with the exception of normal cerebrospinal fluid (CSF) A beta 42 and only slightly pathological amyloid-positron emission tomography signals. Mass spectrometry and Western blot analyses of patient CSF and media from experimental cell cultures indicate that the Uppsala APP mutation alters APP processing by increasing beta-secretase cleavage and affecting alpha-secretase cleavage. Furthermore, in vitro aggregation studies and analyses of patient brain tissue samples indicate that the longer form of mutated A beta, A beta Upp1-42(Delta 19-24), accelerates the formation of fibrils with unique polymorphs and their deposition into amyloid plaques in the affected brain

Sensitive detection of Aβ protofibrils by proximity ligation - relevance for Alzheimer's disease

<p>Abstract</p> <p>Background</p> <p>Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of Aβ protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity.</p> <p>Results</p> <p>For specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation.</p> <p>Conclusions</p> <p>The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.</p

Aβ Conformation Dependent Antibodies and Alzheimer's Disease

Soluble intermediates of the amyloid-β (Aβ) aggregation process are suggested to play a central role in the pathogenesis of Alzheimer’s disease (AD) by causing synaptic dysfunction and neuronal loss. In this thesis, soluble Aβ aggregates have been studied with a particular focus on the Aβ protofibril, which has served as the antigen for developing conformation dependent monoclonal antibodies. Antibodies generated from mice immunized with Aβ protofibrils were characterized regarding Aβ binding properties and the amino acid sequences of their antigen binding sites. A conformation dependent IgG antibody, mAb158, was further characterized and found to bind to Aβ protofibrils with a 200-fold higher affinity than to monomeric Aβ without affinity for soluble amyloid-β precursor protein (AβPP) or other amyloidogenic proteins. A sandwich enzyme-linked immunosorbent assay (ELISA) based on mAb158 was used to measure soluble Aβ protofibrils in brain extracts from AβPP-transgenic mice. Low levels of protofibrils could also be detected in human AD brain. However, positive signals generated from measurements in AD and control CSF samples were attributed to interference from heterophilic antibodies (HA), generating false positive signals by cross-binding the assay antibodies; consequently, a study on HA interference in Aβ oligomer ELISAs was initiated. A large set of plasma and CSF samples from AD and non-AD subjects were analyzed with and without measures taken to block HA interference, revealing that virtually all signals above the assay limit of detection were false and generated by HA interference. Many types of soluble Aβ aggregates have been described and suggested to impair neuron and synapse function. To investigate the soluble Aβ pool, synthetic Aβ and brain extracts from AβPP-transgenic mice and AD patients were ultracentrifuged on a density gradient to separate Aβ by size under native conditions. Four distinct gradient fractions were defined based on the appearance of synthetic Aβ in atomic force microscopy (AFM) and immunoreactivity in our protofibril specific sandwich ELISA. Interestingly, most Aβ from AD patients and AβPP-transgenic mice separated in the same fraction as toxic synthetic protofibrils

Engineered antibodies: new possibilities for brain PET?

Almost 50 million people worldwide are affected by Alzheimer's disease (AD), the most common neurodegenerative disorder. Development of disease-modifying therapies would benefit from reliable, non-invasive positron emission tomography (PET) biomarkers for early diagnosis, monitoring of disease progression, and assessment of therapeutic effects. Traditionally, PET ligands have been based on small molecules that, with the right properties, can penetrate the blood-brain barrier (BBB) and visualize targets in the brain. Recently a new class of PET ligands based on antibodies have emerged, mainly in applications related to cancer. While antibodies have advantages such as high specificity and affinity, their passage across the BBB is limited. Thus, to be used as brain PET ligands, antibodies need to be modified for active transport into the brain. Here, we review the development of radioligands based on antibodies for visualization of intrabrain targets. We focus on antibodies modified into a bispecific format, with the capacity to undergo transferrin receptor 1 (TfR1)-mediated transcytosis to enter the brain and access pathological proteins, e.g. amyloid-beta. A number of such antibody ligands have been developed, displaying differences in brain uptake, pharmacokinetics, and ability to bind and visualize the target in the brain of transgenic mice. Potential pathological changes related to neurodegeneration, e.g. misfolded proteins and neuroinflammation, are suggested as future targets for this novel type of radioligand. Challenges are also discussed, such as the temporal match of radionuclide half-life with the ligand's pharmacokinetic profile and translation to human use. In conclusion, brain PET imaging using bispecific antibodies, modified for receptor-mediated transcytosis across the BBB, is a promising method for specifically visualizing molecules in the brain that are difficult to target with traditional small molecule ligands

Age, dose, and binding to TfR on blood cells influence brain delivery of a TfR-transported antibody

Background Transferrin receptor 1 (TfR1) mediated brain delivery of antibodies could become important for increasing the efficacy of emerging immunotherapies in Alzheimer's disease (AD). However, age, dose, binding to TfR1 on blood cells, and pathology could influence the TfR1-mediated transcytosis of TfR1-binders across the blood–brain barrier (BBB). The aim of the study was, therefore, to investigate the impact of these factors on the brain delivery of a bispecific TfR1-transported Aβ-antibody, mAb3D6-scFv8D3, in comparison with the conventional antibody mAb3D6. Methods Young (3–5 months) and aged (17–20 months) WT and tg-ArcSwe mice (AD model) were injected with 125I-labeled mAb3D6-scFv8D3 or mAb3D6. Three different doses were used in the study, 0.05 mg/kg (low dose), 1 mg/kg (high dose), and 10 mg/kg (therapeutic dose), with equimolar doses for mAb3D6. The dose-corrected antibody concentrations in whole blood, blood cells, plasma, spleen, and brain were evaluated at 2 h post-administration. Furthermore, isolated brains were studied by autoradiography, nuclear track emulsion, and capillary depletion to investigate the intrabrain distribution of the antibodies, while binding to blood cells was studied in vitro using blood isolated from young and aged mice. Results The aged WT and tg-ArcSwe mice showed significantly lower brain concentrations of TfR-binding [125I]mAb3D6-scFv8D3 and higher concentrations in the blood cell fraction compared to young mice. For [125I]mAb3D6, no significant differences in blood or brain delivery were observed between young and aged mice or between genotypes. A low dose of [125I]mAb3D6-scFv8D3 was associated with increased relative parenchymal delivery, as well as increased blood cell distribution. Brain concentrations and relative parenchymal distribution of [125I]mAb3D6-scFv8D6 did not differ between tg-ArcSwe and WT mice at this early time point but were considerably increased compared to those observed for [125I]mAb3D6. Conclusion Age-dependent differences in blood and brain concentrations were observed for the bispecific antibody mAb3D6-scFv8D3 but not for the conventional Aβ antibody mAb3D6, indicating an age-related effect on TfR1-mediated brain delivery. The lowest dose of [125I]mAb3D6-scFv8D3 was associated with higher relative BBB penetration but, at the same time, a higher distribution to blood cells. Overall, Aβ-pathology did not influence the early brain distribution of the bispecific antibody. In summary, age and bispecific antibody dose were important factors determining brain delivery, while genotype was not

Passive and receptor mediated brain delivery of an anti-GFAP nanobody

Purpose: Antibody-based constructs, engineered to enter the brain using transferrin receptor (TfR) mediated transcytosis, have been successfully used as PET radioligands for imaging of amyloid-beta (Aβ) in preclinical studies. However, these radioligands have been large and associated with long circulation times, i.e. non-optimal properties for neuroPET radioligands. The aim of this study was to investigate the in vivo brain delivery of the radiolabeled nanobody VHH-E9 that binds to glial fibrillary acidic protein (GFAP) expressed by reactive astrocytes, without and with fusion to a TfR binding moiety, as potential tools to detect neuroinflammation. Methods: Three protein constructs were recombinantly expressed: 1) The GFAP specific nanobody VHH-E9, 2) VHH-E9 fused to a single chain variable fragment of the TfR binding antibody 8D3 (scFv8D3) and 3) scFv8D3 alone. Brain delivery of the constructs was investigated at 2 h post injection. Binding to GFAP was studied with autoradiography while in vivo brain retention of [125I]VHH-E9 and [125I]VHH-E9-scFv8D3 was further investigated at 8 h, 24 h and 48 h in wild-type (WT), and at the same time points in transgenic mice (ArcSwe) that in addition to Aβ pathology also display neuroinflammation. Results: At 2 h after administration, [125I]VHH-E9-scFv8D3 and [125I]scFv8D3 displayed 3-fold higher brain concentrations than [125I]VHH-E9. In vitro autoradiography showed distinct binding of both [125I]VHH-E9-scFv8D3 and [125I]VHH-E9 to regions with abundant GFAP in ArcSwe mice. However, in vivo, there was no difference in brain concentrations between WT and ArcSwe at any of the studied time points. Conclusions: Fused to scFv8D3, VHH-E9 displayed increased brain delivery. When radiolabeled and applied on brain sections, the bispecific construct was able to discriminate between WT and ArcSwe mice, but in vivo brain uptake and retention over time did not differ between WT and ArcSwe mice

Cationization increases brain distribution of an amyloid-beta protofibril selective F(ab')2 fragment

Antibodies and fragments thereof are, because of high selectivity for their targets, considered as potential therapeutics and biomarkers for several neurological disorders. However, due to their large molecular size, antibodies/fragments do not easily penetrate into the brain. The aim of the present study was to improve the brain distribution via adsorptive-mediated transcytosis of an amyloid-beta (A beta) protofibril selective F(ab')2 fragment (F(ab')2-h158). F(ab')2-h158 was cationized to different extents and the specific and unspecific binding was studied in vitro. Next, cationized F(ab')2-h158 was labelled with iodine-125 and its brain distribution and pharmacokinetics was studied in mice. Cationization did not alter the in vitro affinity to A beta protofibrils, but increased the unspecific binding somewhat. Ex vivo experiments revealed a doubling of brain concentrations compared with unmodified F(ab')2-h158 and in vivo imaging with single photon emission computed tomography (SPECT) showed that the cationized F(ab')2-h158, but not the unmodified F(ab')2-h158 could be visualized in the brain. To conclude, cationization is a means to increase brain concentrations of therapeutic antibodies or fragments and may facilitate the use of antibodies/fragments as imaging biomarkers in the brain