anti-Aβ Antibodies Research Articles

Anti-A\u03b2 single-chain variable fragment antibodies restore memory acquisition in a Drosophila model of Alzheimer\u2019s disease

Alzheimer’s disease (AD) is a prevalent neurodegenerative disorder triggered by the accumulation of soluble assemblies of the amyloid-β42 (Aβ42) peptide. Despite remarkable advances in understanding the pathogenesis of AD, the development of palliative therapies is still lacking. Engineered anti-Aβ42 antibodies are a promising strategy to stall the progression of the disease. Single-chain variable fragment (scFv) antibodies increase brain penetration and offer flexible options for delivery while maintaining the epitope targeting of full antibodies. Here, we examined the ability of two anti-Aβ scFv antibodies targeting the N-terminal (scFv9) and C-terminal (scFv42.2) regions of Aβ42 to suppress the progressive memory decline induced by extracellular deposition of Aβ42 in Drosophila. Using olfactory classical conditioning, we observe that both scFv antibodies significantly improve memory performance in flies expressing Aβ42 in the mushroom body neurons, which are intimately involved in the coding and storage of olfactory memories. The scFvs effectively restore memory at all ages, from one-day post-eclosion to thirty-day-old flies, proving their ability to prevent the toxicity of different pathogenic assemblies. These data support the application of this paradigm of Aβ42-induced memory loss in Drosophila to investigate the protective activity of Aβ42–binding agents in an AD-relevant functional assay.

Single-step immunoassays and microfluidic droplet operation: Towards a versatile approach for detection of amyloid-β peptide-based biomarkers of Alzheimer’s disease

In this study, single-step magnetic-beads based immunoassays was developed and for the first time adapted to microfluidic droplet operation for sequential determination of well-established amyloid-β peptide biomarkers (i.e. monomeric Aβ 1–42 and Aβ 1–40) for molecular diagnosis of Alzheimer’s disease (AD). With the developed sandwich assay protocol, the capture antibodies (i.e. monoclonal anti-Aβ antibodies that are specific for N-terminus of Aβ 1–42 and Aβ 1–40) grafted onto magnetic beads and the detection antibodies (i.e. beta amyloid 1–16 monoclonal antibody labeled with horseradish peroxidase) can simultaneously bind to monomeric β peptides in a single step. Aβ 1–42 and Aβ 1–40 in cerebrospinal fluid (CSF) samples were successfully detected using the developed batchwise immunoassay approach. With the aim at expanding the spectrum of traced Aβ peptides beyond Aβ 1–42 and Aβ 1–40 for more precise diagnostics of AD, the developed sandwich assay was for the first time coupled as a downstream module to peptide fractionation and collection using capillary isoelectric focusing. Demonstration of this methodological combination was carried out with Aβ 1–40, Aβ 1–40 and Aβ 5–40. The immunoassay in batchwise format was downscaled into a purpose-made microfluidic droplet platform allowing significant sample volume reduction and higher throughput. Using a series of 4 programmable magnetic tweezers to manipulate a train of nano-scale confined droplets containing magnetic beads, sample, washing and detection solutions, a sequence of 8 analyses could be realized within 45min. This droplet based immunoassay was realized in a dedicated platform integrating an in-house-made light emitting diode (LED)-based fluorescent detector, replacing conventional microscopic setup so as to significantly reduce the construction cost and simplify the detection protocol. Using this microfluidic configuration coupled with LED-based detection, information on both Aβ 1–42 and Aβ 1–40 concentrations can be collected in a single sequence with less than 1μL of sample.

Chronic Verubecestat Treatment Suppresses Amyloid Accumulation in Advanced Aged Tg2576-AβPPswe Mice Without Inducing Microhemorrhage.

Verubecestat is a potent BACE1 enzyme inhibitor currently being investigated in Phase III trials for the treatment of mild-to-moderate and prodromal Alzheimer’s disease. Multiple anti-amyloid immunotherapies have been dose-limited by adverse amyloid related imaging abnormalities such as vasogenic edema (ARIA-E) and microhemorrhage (ARIA-H) observed in human trials and mice. Verubecestat was tested in a 12-week nonclinical study for the potential to exacerbate microhemorrhage (ARIA-H) profiles in 18-22-month-old post-plaque Tg2576-AβPPswe mice. Animals were treated with verubecestat or controls including the anti-Aβ antibody analog of bapineuzumab (3D6) as a positive control for ARIA induction. ARIA-H was measured using in-life longitudinal T2*-MRI and Prussian blue histochemistry at study end. Verubecestat reduced plasma and cerebrospinal fluid Aβ40 and Aβ42 by >90% and 62% to 68%, respectively. The ARIA-H profile of verubecestat-treated mice was not significantly different than controls. Anti-Aβ treatment significantly increased ARIA-H detected by Prussian blue staining; however, anti-Aβ antibody treatment did not impact plaque status. Verubecestat treatment significantly suppressed the accumulation of total levels of brain Aβ40 and Aβ42 and Thioflavin S positive plaque load. Stereological analysis of cortex and hippocampus plaque load similarly revealed significantly reduced area of Aβ immunoreactivity and reduced plaque number in verubecestat-treated animals compared to controls. The absence of elevated ARIA events in verubecestat-treated mice was associated with a significant reduction in the level of accumulated CNS amyloid pathology and brain Aβ peptides; effects consistent with the desired therapeutic mechanism of verubecestat in AD patients. These data will be compared with longitudinal MRI profiles from ongoing clinical trials.

Production of an anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect.

ScFv-h3D6 has been shown as an efficient therapy in the 3xTg-AD mouse model of Alzheimer’s Disease. Because one of the major bottlenecks for the therapeutic uses of proteins produced in Escherichia coli is their potential contamination with endotoxins, LPS were extensively removed by a rather low-efficient, expensive, and time-consuming purification step. In addition, disulfide scrambling is favored in the reducing bacterial cytoplasm albeit the use of reductase deficient strains. To overcome these hurdles, as well as to improve the yield, the yeast Pichia pastoris, an endotoxin-free host system for recombinant protein production, has been used to produce scFv-h3D6, both in flask and in a fed-batch bioreactor. Comparison of the thermal stability of the obtained protein with that from E. coli showed no differences. Opposite to the case of the protein obtained from E. coli, no disulfide scrambled conformations or LPS traces were detected in that produced in P. pastoris. Cytotoxicity assays in SH-SY5Y neuroblastoma cell-cultures demonstrated that proteins from both expression systems were similarly efficient in precluding Aβ-induced toxicity. Finally, the 3xTg-AD mouse model was used to test the therapeutic effect of both proteins. Quantification of Aβ levels from cortex and hippocampus protein extracts by ELISA, and Aβ-immunohistochemistry, showed that both proteins reduced Aβ burden. This work demonstrates that scFv-h3D6 obtained from P. pastoris shows the same benefits as those already known for that obtained from E. coli, with multiple advantages in terms of recombinant production and safety.

Cerebrospinal Fluid and Blood Biomarkers in Alzheimer's Disease

To cope with an aging society, development of disease-modifying drugs for Alzheimer's disease (AD) is essential. Currently, only symptomatic treatments that suppress clinical manifestations are available. Amyloid-β₄₂ (Aβ₄₂) is an AD-related pathogenic molecule that triggers development of AD pathology; thus, decreasing Aβ₄₂ in the brain is a promising candidate for AD therapy. Numerous pharmaceutical companies have developed therapeutic drugs against Aβ₄₂, such as β-secretase inhibitors, γ-secretase inhibitors, and anti-Aβ monoclonal antibodies, but in clinical trials for patients with mild to moderate AD, these drugs did not meet the expected endpoints. These results suggest that earlier administration of these drugs to individuals who have not yet developed cognitive decline, but have AD pathological changes in the brain or high risk of developing these changes, may be beneficial. To enable such early treatment, preclinical AD biomarkers are required. In this review, we comment on current AD biomarkers in cerebrospinal fluid and in blood. We also explain CSF/blood APL1β, which is a candidate surrogate marker for Aβ₄₂ in the brain.

DO ANTI-AMYLOID BETA PROTEIN ANTIBODY CROSS REACTIVITIES CONFOUND ALZHEIMER DISEASE RESEARCH?

Alzheimer disease (AD) research has focussed mainly on the amyloid beta protein (Aβ). However, many Aβ-and P3-type peptides derived from the amyloid precursor protein (APP) and peptides thought to derive from Aβ catabolism share sequence homology. Additionally, conformations can change dependent on aggregation state and solubility leading to significant uncertainty relating to interpretations of immunoreactivity with antibodies raised against Aβ. We review evidence relating to the reactivities of commonly used antibodies including 6F3D, 6E10 and 4G8 and evaluate their reactivity profiles with respect to AD diagnosis and research. Antibody cross-reactivities between Aβ-type, P3-type and Aβ-catabolic peptides confound interpretations of immunoreactivity. More than one antibody is required to adequately characterise Aβ. The relationships between anti-Aβ immunoreactivity, neuropathology and proposed APP cleavages are unclear. We find that the concept of Aβ lacks clarity as a specific entity. Anti-Aβ antibody cross-reactivities lead to significant uncertainty in our understanding of the APP proteolytic system and its role in AD with profound implications for current research and therapeutic strategies.

Ponezumab in mild-to-moderate Alzheimer's disease: Randomized phase II PET-PIB study

IntroductionThe safety, pharmacokinetics, and effect on peripheral and central amyloid β (Aβ) of multiple doses of ponezumab, an anti-Aβ monoclonal antibody, were characterized in subjects with mild-to-moderate Alzheimer's disease treated for 1 year. MethodsSubjects were aged ≥50 years with Mini–Mental State Examination scores 16 to 26. Cohort Q was randomized to ponezumab 10 mg/kg (n = 12) or placebo (n = 6) quarterly. Cohort M was randomized to a loading dose of ponezumab 10 mg/kg or placebo, followed by monthly ponezumab 7.5 mg/kg (n = 12) or placebo (n = 6), respectively. ResultsPonezumab was generally well tolerated. Plasma concentrations increased dose dependently, but cerebrospinal fluid (CSF) penetration was low. Plasma Aβ increased dose dependently with ponezumab, but CSF biomarkers, brain amyloid burden, cognition, and function were not affected. ConclusionsBoth ponezumab dosing schedules were generally safe and well tolerated but did not alter CSF biomarkers, brain amyloid burden, or clinical outcomes.

Ameliorating effect of anti-Alzheimer's drugs on the bidirectional association between type 2 diabetes mellitus and Alzheimer's disease.

Mild to severe forms of nervous system damage were exhibited by approximately 60-70% of diabetics. It is important to understand the association between type 2 diabetes mellitus and Alzheimer's disease. The aim of the present work is to understand the bidirectional association between type 2 diabetes and Alzheimer's disease pathogenesis, that was monitored by glycaemic status, lipid profile, amyloid beta 40 and 42 (Aβ40 and Aβ42), C-reactive protein, total creatine kinase, total lactate dehydrogenase, D-dimer and magnesium measurements, to assess the association between theses biochemical markers and each other, to estimate the possibility of utilizing the amyloid beta as biochemical marker of T2D in Alzheimer's patients, and to evaluate the effect of piracetam and memantine drugs on diabetes mellitus. This study involved 120 subjects divided into 20 healthy control (group I), 20 diabetic patients (group II), 20 Alzheimer's patients (group III), 20 diabetic Alzheimer's patients with symptomatic treatment (group IV), 20 diabetic Alzheimer's patients treated with memantine (group V), and 20 diabetic Alzheimer's patients treated with piracetam (group VI). The demographic characteristics, diabetic index, and lipid profile were monitored. Plasma amyloid beta 40 and amyloid beta 42, C-reactive protein, total creatine kinase, total lactate dehydrogenase, D-dimer, and magnesium were assayed. The levels of amyloid beta 40 and amyloid beta 42 were significantly elevated in diabetic Alzheimer's patients with symptomatic treatment (group IV) compared to group II (by 50.5 and 7.5 fold, respectively) and group III (by 25.4 and 2.8 fold, respectively). In groups II, III, IV, V and VI, significant and positive associations were monitored between insulin and amyloid beta 40, amyloid beta 42, C-reactive protein, total creatine kinase, and D-dimer. Diabetic markers were significantly decreased in diabetic Alzheimer's patients treated with anti-Alzheimer's drugs (especially piracetam) compared to group IV. This study reveals the role of amyloid beta 40, amyloid beta 42, insulin, HbA1c, lipid profile disturbance, C-reactive protein, D-dimer, and magnesium in the bidirectional correlation between T2D and pathogenesis of Alzheimer's disease, that is powered by their correlations, and therefore the possibility of utilizing Aβ as a biochemical marker of T2D in Alzheimer's patients is recommended. Impact statement Several aspects associated with T2D that contribute to AD and vice versa were investigated in this study. Additionally, this work reveals the role of Aβ40, Aβ42, insulin, HbA1c, lipid profile disturbance, CRP, D-dimer, and magnesium in the bidirectional association between T2D and the pathogenesis of AD, that is powered by their correlations, and therefore the possibility of utilizing Aβ as a biochemical marker of T2D in Alzheimer's patients is recommended. Furthermore, the ameloriating effect of anti-Alzheimer's drugs on diabetes mellitus confirms this association. Hereafter, a new approach for treating insulin resistance and diabetes may be developed by new therapeutic potentials such as neutralization of Aβ by anti-Aβ antibodies.

Intradermal active full-length DNA Aβ42 immunization via electroporation leads to high anti-Aβ antibody levels in wild-type mice with variability in T cell responses depending on pulse parameters applied

Abstract Aβ immunotherapy has high potential as possible prevention treatment for Alzheimer’s disease, and anti-Aβ antibodies will help in the removal of excess amyloid from brain. We have previously shown that active DNA Aβ1-42 immunization via gene gun delivery led to a non-inflammatory immune response with efficient antibody levels which resulted in decreased Aβ levels in brains of the immunized AD mouse model mice. To optimize the DNA delivery and make DNA vaccination more applicable for clinical use, we have investigated here the use of intradermal electroporation using a variety of different voltages and electropulse parameters in wild-type mice. In the direct comparison to DNA delivery via gene gun, higher levels of anti-Aβ42 antibodies were detected already after three DNA immunizations. The antibody isotype profile following DNA electroporation was mixed with IgG1 and IgG2a antibodies. With a fine tuning of the electropulse protocol the T cell responses could be adjusted to similar findings we published earlier for DNA Aβ1-42 immunization via gene gun delivery with low levels of inflammatory cytokines (IFNγ, IL-17) in the in-vitro Aβ42 peptide re-stimulation assays. We found that short pulse durations (5 ms) were better than long pulse durations (10 ms) in regard to obtaining a Th2 type of immune response, and that a higher rate of pulses (16–20 pulses) increased the antibody response in our sets of experiments. Full-length DNA Aβ1-42 immunization delivered via electroporation with a fine tuning of the electropulse conditions has potential to be used in the clinical setting.

Evaluation of a DNA Aβ42 Vaccine in Aged NZW Rabbits: Antibody Kinetics and Immune Profile after Intradermal Immunization with Full-Length DNA Aβ42 Trimer.

A pathological hallmark of Alzheimer’s disease (AD) are amyloid plaques in the brain consisting of aggregated amyloid-β 42 peptide (Aβ42) derived from cellular amyloid-β protein precursor (AβPP). Based on successful experiments in mouse AD models, active immunization with Aβ42 peptide and passive immunizations with anti-Aβ42 antibodies were started in clinical trials. Active Aβ42 peptide immunization in humans had led to an inflammatory autoimmune response, and the trial was stopped. Passive immunizations had shown some effects in slowing AD pathology. Active DNA Aβ42 immunizations administered with the gene gun into the skin elicits a different immune response and is non-inflammatory. While in rodents, good responses had been found for this type of immunization, positive results in larger mammals are missing. We present here results from sixteen New Zealand White Rabbits, which underwent intradermal DNA Aβ42 immunization via gene gun. The humoral immune response was analyzed from blood throughout the study, and cellular immune responses were determined from spleens at the end of the study. A good anti-Aβ antibody response was found in the rabbit model. The T cell response after re-stimulation in cell culture showed no IFNγ producing cells when ELISPOT assays were analyzed from PBMC, but low numbers of IFNγ and IL-17 producing cells were found in ELISPOTS from spleens (both 5 immunizations). Brains from immunized rabbits showed no signs of encephalitis. Based on these results, DNA Aβ42 immunization is highly likely to be safe and effective to test in a possible clinical AD prevention trial in patients.

Concentrations of antibodies against β-amyloid 40/42 monomer and oligomers in Chinese intravenous immunoglobulins

Intravenous immunoglobulin (IVIg) preparations are being investigated as a potential agent for treatment or prevention of Alzheimer’s disease (AD). Antibodies towards soluble β-amyloid (Aβ) contained in IVIg were considered to be the major component contributing to the beneficial effect of the preparations in pilot studies. This study compared the antibody concentrations against Aβ in Octagam® IVIg (Octapharma) and 9 IVIg preparations from different Chinese manufacturers by ELISA, using Aβ40 monomer, Aβ40 soluble oligomers, Aβ42 monomer and Aβ42 soluble oligomers as the antigens. The results showed that each preparation contained different antibody levels against the four Aβ forms. The median values of the four antibody concentrations in Chinese IVIg preparations were 16.53, 8.47, 24.36 and 33.25μg/mL, which were remarkably higher than that in Octagam® IVIg (1.66, 2.07, 4.61 and 4.64μg/mL). Moreover, the anti-Aβ42 oligomer antibody levels in almost all IVIg preparations were higher than the anti-Aβ42 monomer antibody, and the concentrations of anti-Aβ42 antibodies in most of the IVIg preparations were significantly higher than that of anti-Aβ40 antibodies. These findings will contribute to an increased understanding of the uniqueness of Chinese IVIg preparations, and could provide support for a trial of a Chinese IVIg product in AD patients.

Prophylactic active immunization with a novel epitope vaccine improves cognitive ability by decreasing amyloid plaques and neuroinflammation in APP/PS1 transgenic mice

Both amyloid-β peptide (Aβ) deposition and neuroinflammation are considered to be early events that play pivotal roles in Alzheimer’s disease (AD) pathogenesis and its associated cognitive impairment. Prophylactic anti-Aβ active immunotherapy is a promising therapeutic strategy for AD, if the Aβ-specific autoimmune responses to self T cell epitopes of Aβ can be avoided. This can be achieved by the use of antigen, which contains the B cell epitope of Aβ and excludes the Aβ-specific T cell epitope. In this study, we developed a novel peptide epitope vaccine, Aβ3-10-KLH, by coupling the B cell epitope Aβ3-10 to keyhole limpet hemocyanin (KLH) as the carrier protein, and subcutaneously injected it into 2.5-month-old APP/PS1 transgenic mice. Aβ3-10-KLH immunization induced high levels of anti-Aβ antibodies and significantly improved cognitive ability in APP/PS1 transgenic mice. Immunohistochemistry and immunofluorescence revealed that Aβ3-10-KLH immunization significantly reduced cerebral amyloid plaque formation and alleviated gliosis. The results indicate that Aβ3-10-KLH immunization successfully rescued cognitive impairment in APP/PS1 transgenic mice via decreasing cerebral Aβ deposition and neuroinflammation. Aβ3-10-KLH may potentially be safe and effective for prevention and treatment of AD.

Ultra-Early Phase pathologies of Alzheimer's disease and other neurodegenerative diseases.

The concept of neurodegenerative diseases and the therapeutics targeting these intractable diseases are changing rapidly. Protein aggregation as the top of pathological cascade is now challenged, and many alternative ideas are proposed. Early molecular pathologies before microscopic detection of diseases protein aggregates, which I propose to call “Ultra-Early Phase pathologies or phase 0 pathologies”, are the focus of research that might explain the failures of clinical trials with anti-Aβ antibodies against Alzheimer’s disease. In this review article, I summarize the critical issues that should be successfully and consistently answered by a new concept of neurodegeneration. For reevaluating old concepts and reconstructing a new concept of neurodegeneration that will replace the old ones, non-biased comprehensive approaches including proteome combined with systems biology analyses will be a powerful tool. I introduce our recent efforts in this orientation that have reached to the stage of non-clinical proof of concept applicable to clinical trials.

Clinical Development of Aducanumab, an Anti-Aβ Human Monoclonal Antibody Being Investigated for the Treatment of Early Alzheimer's Disease.

The amyloid hypothesis has been the dominant framework for Alzheimer's disease (AD) research, including the development of anti-AD therapies. However, none of the phase III clinical trials conducted to date that targeted amyloid β (Aβ) production, aggregation, or clearance demonstrated a statistically significant treatment effect in patients with AD. This includes the approach of using monoclonal antibodies that recognize various Aβ epitopes and display different binding selectivity. While some monoclonal antibodies have failed in phase III trials, several are still in development. Aducanumab (BIIB037) is a human antibody that selectively targets aggregated forms of Aβ, including soluble oligomers and insoluble fibrils. In PRIME (NCT01677572), an ongoing phase Ib trial (N=196 patients dosed), aducanumab was shown to reduce Aβ plaques and slow decline in clinical measures in patients with prodromal or mild AD, with acceptable safety and tolerability. The main safety finding was amyloid-related imaging abnormalities (ARIA), a side effect associated with removal of Aβ, which was dose-dependent and occurred more often in ApoE ε4 carriers than non-carriers. ENGAGE (NCT02477800) and EMERGE (NCT02484547), the ongoing phase III trials of aducanumab in early AD, have been designed based on the outcomes of PRIME and on lessons from past clinical trials in patients with AD. Those study design features include patient selection with confirmed Aβ pathology, ensuring sufficient target engagement, and conducting clinical trials in patients at earlier symptomatic stages of AD.

Comparison of Efficacy of Preventive and Therapeutic Vaccines Targeting the N Terminus of β-Amyloid in an Animal Model of Alzheimer’s Disease

Previously, we reported that Alzheimer's disease (AD) epitope vaccines (EVs) composed of N-terminal β-amyloid (Aβ42) Bcell epitope fused with universal foreign T helper (Th) epitope(s) were immunogenic, potent, and safe in different amyloid precursor protein (APP) transgenic mice with early AD-like pathology. However, developing an effective therapeutic vaccine is much more challenging, especially when a self-antigen such as Aβ42 is a target. Here, we directly compare the efficacy of anti-Aβ42 antibodies in Tg2576 mice with low or high levels of AD-like pathology at the start of immunizations: 6-6.5months for preventive vaccinations and 16-19months for therapeutic vaccinations. EV in a preventive setting induced high levels of anti-Aβ antibodies, significantly reducing pathologic forms of Aβ in the brains of Tg2576 mice. When used therapeutically for immunesenescent Tg2576 mice, EV induced low levels of antibodies not sufficient for clearing of AD-like pathology. Separately, we demonstrated that EV was also not effective in 11-11.5-month-old Tg2576 mice with moderate AD-like pathology. However, we augmented the titers of anti-Aβ antibodies in transgenic (Tg) mice of the same age possessing the pre-existing memory Th cells and detected a significant decrease in diffuse and core plaques in cortical regions compared to control animals along with improved novel object recognition performance.

Immunotherapy with Aducanumab Restores Calcium Homeostasis in Tg2576 Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is currently without a cure. Aducanumab is an anti-amyloid-β antibody being developed for the treatment of AD. Interim analyses of a phase 1b clinical trial have suggested potential beneficial effects on amyloid pathology and cognitive status in patients treated with aducanumab (Sevigny et al., 2016). Here, we show that a murine analog of aducanumab clears amyloid plaques in an acute setting and restores calcium homeostasis disrupted in a mouse model of AD upon chronic treatment. Therefore, we demonstrate that aducanumab reverses a functional outcome measure reflective of neural network activity.

Active immunization with the peptide epitope vaccine Aβ3-10-KLH induces a Th2-polarized anti-Aβ antibody response and decreases amyloid plaques in APP/PS1 transgenic mice

Active amyloid-β (Aβ) immunotherapy is effective in preventing Aβ deposition, facilitating plaque clearance, and improving cognitive functions in mouse models of Alzheimer's disease (AD). Developing a safe and effective AD vaccine requires a delicate balance between inducing adequate humoral immune responses and avoiding T cell-mediated autoimmune responses. In this study, we designed 2 peptide epitope vaccines, Aβ3-10-KLH and 5Aβ3-10, prepared respectively by coupling Aβ3-10 to the immunogenic carrier protein keyhole limpet hemocyanin (KLH) or by joining 5 Aβ3-10 epitopes linearly in tandem. Young APP/PS1 mice were immunized subcutaneously with Aβ3-10-KLH or 5Aβ3-10 mixed with Freund's adjuvant, and the immunopotencies of these Aβ3-10 peptide vaccines were tested. Aβ3-10-KLH elicited a robust Th2-polarized anti-Aβ antibody response and inhibited Aβ deposition in APP/PS1 mice. However, 5Aβ3-10 did not induce an effective humoral immune response. These results indicated that Aβ3-10-KLH may be a safe and efficient vaccine for AD and that conjugating the antigen to a carrier protein may be more effective than linking multiple peptide antigens in tandem in applications for antibody production and vaccine preparation.

ImmunoPEGliposome-mediated reduction of blood and brain amyloid levels in a mouse model of Alzheimer's disease is restricted to aged animals.

The accumulation of extracellular amyloid-beta (Aβ) and intracellular neurofibrillary tangles (hyper-phosphorylated Tau) in the brain are two major neuropathological hallmarks of Alzheimer's disease (AD). Active and passive immunotherapy may limit cerebral Aβ deposition and/or accelerate its clearance. With the aid of a newly characterized monoclonal anti-Aβ antibody we constructed immunoPEGliposomes with high avidity for capturing Aβ in the periphery. The functionality of these vesicles in modulating Aβ uptake by both human brain capillary endothelial hCMEC/D3 cells (suppressing uptake) and THP-1 phagocytes (stimulating uptake) was confirmed invitro. The multivalent immunoliposomes dramatically reduced circulating and brain levels of Aβ1-40, and particularly Aβ1-42, in "aged" (16 month-old), but not "adult" (10 month-old) APP/PS1 transgenic mice on repeated intraperitoneal administration. Furthermore, the immunoPEGliposome-mediated reduction in amyloidosis correlated with lower levels of glial fibrillary acidic protein (GFAP) and reactive glia (GFAP-positive cells). This treatment also lowered the ratio of phosphorylated Tau to total Tau. The therapeutic efficacy of immunoliposome treatment was superior to free monoclonal antibody administration (at an equivalent antibody dose). The potential mechanisms and significance of age-dependent immunoliposome therapy in AD is discussed.

TREM2 deficiency reduces the efficacy of immunotherapeutic amyloid clearance.

Immunotherapeutic approaches are currently the most advanced treatments for Alzheimer's disease (AD). Antibodies against amyloid β‐peptide (Aβ) bind to amyloid plaques and induce their clearance by microglia via Fc receptor‐mediated phagocytosis. Dysfunctions of microglia may play a pivotal role in AD pathogenesis and could result in reduced efficacy of antibody‐mediated Aβ clearance. Recently, heterozygous mutations in the triggering receptor expressed on myeloid cells 2 (TREM2), a microglial gene involved in phagocytosis, were genetically linked to late onset AD. Loss of TREM2 reduces the ability of microglia to engulf Aβ. We have now investigated whether loss of TREM2 affects the efficacy of immunotherapeutic approaches. We show that anti‐Aβ antibodies stimulate Aβ uptake and amyloid plaque clearance in a dose‐dependent manner in the presence or absence of TREM2. However, TREM2‐deficient N9 microglial cell lines, macrophages as well as primary microglia showed significantly reduced uptake of antibody‐bound Aβ and as a consequence reduced clearance of amyloid plaques. Titration experiments revealed that reduced efficacy of amyloid plaque clearance by Trem2 knockout cells can be compensated by elevating the concentration of therapeutic antibodies.

DNA AB42 VACCINATION LEADS TO HIGH ANTIBODY RESPONSES IN RABBITS AND RHESUS MONKEYS

After successful experiments in mouse AD models, active immunization with Aβ42 peptide and passive immunizations with anti-Aβ42 antibodies were started in clinical trials. In humans, active Aβ42 peptide immunization resulted in menigo-encephalitis and inflammation, and the trial was stopped. Passive immunizations had shown some slowing of AD pathology. Active DNA Aβ42 immunizations into the skin elicits a non-inflammatory immune response in mouse models. While in rodents good immune responses had been found for this type of immunization, positive results in larger mammals are missing. We present here results from NZW rabbits and Rhesus macaques after DNA Aβ42 immunization via gene gun delivery into the skin. Sixteen NZW rabbits and six Rhesus macaques were immunized five times with two different doses of a DNA Aβ42 trimer vaccine: Half of the animals in each species received 8 μg DNA per immunization time point (low dose); the other half received 16 μg DNA per immunization (high dose). Antibody responses were analyzed from blood throughout the study. In rabbits, cellular immune responses were determined from spleens after five vaccinations. In monkeys, cellular immune responses were determined with ELISPOT assays from PBMC following the third vaccination, and will be further followed. High antibody responses were found in both species. In the rabbit, 277.1 ± 118.4 μg/ml plasma were measured after 5 immunizations with no significant differences between the dose groups. Antibody levels declined slightly to 246.6 ± 109.5 μg/ml after a resting period of two months. Low levels of IFNγ and IL-17 secreting splenocytes were found in the ELISPOT assays from rabbit splenocytes. Rhesus monkeys reached mean antibody levels of 114.2 ± 41.67 μg/ml plasma after five immunizations. The isotype profile was high on IgG4 antibodies, indicative of a Th2 immune response. After three immunizations, no IL-17 or IFNγ producing cells were found in ELISPOT assays from PBMCs. DNA Aβ42 immunization leads to high antibody titers in large mammals, and is likely to produce high antibody levels and a safe (Th2 biased) immune response in humans as well. supported by NIA/NIH P30AG12300-21, Zale Foundation, Rudman Foundation, AWARE, Presbyterian Village North, Freiberger, and Denker Family Funds