Alzheimer's Disease Immunotherapy Research Articles

Epitope structure and binding affinity of single chain llama anti‐β‐amyloid antibodies revealed by proteolytic excision affinity‐mass spectrometry

ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors.

An Effector-Reduced Anti- -Amyloid (A ) Antibody with Unique A Binding Properties Promotes Neuroprotection and Glial Engulfment of A

Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

Harnessing adaptive immunity for AD

Immunotherapy has potential as a disease-modifying approach to the treatment of Alzheimer's disease. In this study, Santuccione et al. found that among individuals with Alzheimer's disease, those who exhibit slower cognitive decline have higher blood levels of antibodies specific for the neuronal cytoskeletal protein ankyrin G (ANKG; also known as ANK3). ArcAβ mice (a transgenic mouse model of Alzheimer's disease) that were vaccinated with ANKG had fewer and smaller amyloid plaques than unvaccinated animals, as well as less spine loss. Thus, taking advantage of the adaptive immune response to ANKG could be a potential approach for Alzheimer's disease immunotherapy.

Immunotherapy for Alzheimer disease—the challenge of adverse effects

Amyloid-β (Aβ) plays a crucial part in the pathogenesis of Alzheimer disease (AD), making this peptide an attractive therapeutic target. However, clearance of brain Aβ in clinical trials of Aβ-specific antibodies did not improve cognition in patients with AD, leading to reassessment of the current therapeutic strategies. Moreover, current immunotherapies are associated with autoimmunity-related adverse effects, and mobilization of neurotoxic insoluble Aβ-oligomers. Despite the fact that antibodies to the N-terminal domain of Aβ can promote Aβ production, immunotherapies in ongoing clinical trials predominantly target this peptide region. Here, we address the challenges of adverse effects of immunotherapy for AD. We discuss available evidence regarding the mechanisms of both endogenous and exogenous Aβ-specific antibodies, with a view to developing optimal immunotherapy based on peripheral Aβ clearance, targeting of the toxic domain of Aβ, and improvement of antibody specificity. Such strategies should help to make immunotherapy a safe and efficacious disease-modifying treatment option for AD.

P1‐242: Reactivity of human natural antibodies to various Aβ peptides

Accumulation of the Aβ peptide plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Many treatments for AD, delay the accumulation of Aβ, but evidence for reduced pre-existing plaques is unknown. Immunotherapy for AD, using human natural Aβ antibodies are currently in development. Anti-Aβ antibodies occur naturally in human blood and CSF in free or complex form with Aβ. Antibody reactivity was known prominent against oligomeric assemblies of Aβ and pyroglutamte or oxidized residues. But their diversity, abundance, and function remain largely unknown and large-scale analysis of Aβ antibody changes has not been described. Here, we demonstrated changes of natural Aβ antibody reactivity in human serum sample with peptide microarrays between healthy control (n = 100) and mild cognitive impairment (MCI, n = 100) and AD patients (n = 100), against various Aβ peptide. Some of Aβ peptides show increasing reactivity in AD patients serum compared with healthy control. Interestingly, N-terminal truncated pyroglutamte Aβ peptide, Aβ 22(pE)-42 immunoreactivity significantly (p < 0.001) increased in MCI and AD patients serum compared healthy control. Aβ 22(pE)-42 reduced cell viability in human neuroblastoma cell and aggregated in vitro. After active immunization with mice, antibody titration to Aβ 22(pE)-42 were increased but T-cell activation were not changed. These results suggest that Aβ22(pE)-42 may be helpful in better immunotherapy target of AD.

Longitudinal analysis of novel Alzheimer's disease proteomic cerebrospinal fluid biomarkers during intravenous immunoglobulin therapy

Intravenous immunoglobulin (IVIg) therapy has shown promising results in treating Alzheimer's disease (AD). In this study, a Random Forest (RF) classification model was used to identify possible effects of IVIg on a group of eight subjects who underwent immunotherapy. Cerebrospinal fluid (CSF) samples from eight AD subjects who underwent IVIg therapy were collected before therapy, after 6 months of therapy, and after a 3-month drug washout period. Samples were analyzed using 2DE and further studied using a RF classification model to identify effects of IVIg on a panel of 23 putative diagnostic AD biomarkers previously identified. Six of the eight subjects showed improvements with respect to the 23 AD diagnostic biomarkers after 6 months of therapy compared to the samples taken at the outset of the trial. All subjects reverted back to baseline during drug washout. These results are also consistent with clinical observations. The observed improvements in subjects during 6 months of IVIg therapy and the reversion back to baseline during drug washout provides preliminary evidence regarding the potential use of IVIg as an AD immunotherapy.

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-β (Aβ). 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 Aβ 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 Aβ1-40 and Aβ1-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.

Active DNA Aβ42 vaccination as immunotherapy for Alzheimer disease

As a neurodegenerative disorder, Alzheimer disease (AD) is the most common form of dementia found in the aging population. Immunotherapy with passive or active immunizations targeting amyloid beta (Aβ) build-up in the brain may provide a possible treatment option and may help prevent AD from progressing. A number of passive immunizations with anti-Aβ42 antibodies are in different phases of clinical trials. One active immunization approach, AN-1792, was stopped after the development of autoimmune encephalitis in 6% of patients and a second one, CAD106, in which a small Aβ epitope is used, is currently in safety and tolerability studies. Besides active immunizations with proteins or peptides, active immunizations using DNA which codes for the protein against which the immune response will be directed, so called genetic immunizations, provide additional safety as the immune response in DNA immunizations differs quantitatively and qualitatively from the response elicited by peptide immunizations. In this review, we summarize our data using DNA Aβ42 immunizations in mouse models and discuss the results together with the results presented by other groups working on a DNA vaccine as treatment option for AD.

アルツハイマー病治療に応用可能な抗体の開発

Based on the amyloid cascade hypothesis, immunotherapy targeting amyloid β (Aβ) for Alzheimer's disease (AD) has been developed. It was reported that active immunization using Aβ peptide attenuates amyloid deposits and memory impairment in AD model mice. However, active immunization of patients with AD (AN-1792) was halted due to adverse effects in which a subset of patients developed meningoencephalitis. In order to avoid autoimmune encephalitis, passive immunotherapy using humanized monoclonal antibodies with specificity to Aβ are in clinical trials. We also developed an anti-Aβ monoclonal antibody 3.4A10, which react with AD brain-specific Aβ oligomers. On the other hand, some studies showed that immunotherapy approach targeting tau could attenuate pathology in AD model mouse. Here we introduce a current trend of immunotherapy for AD.

Mucosal immunotherapy in an Alzheimer mouse model by recombinant Sendai virus vector carrying Aβ1–43/IL-10 cDNA

Based on the amyloid cascade hypothesis, many reports have indicated that immunotherapy is beneficial for Alzheimer's disease (AD). We developed a mucosal immunotherapy for AD by nasal administration of recombinant Sendai virus vector carrying Aβ1–43 and mouse IL-10 cDNA. Nasal but not intramuscular administration of the vaccine induced good antibody responses to Aβ. When APP transgenic mice (Tg2576) received this vaccine once nasally, the Aβ plaque burden was significantly decreased 8 weeks after without inducing inflammation in the brain. The amount of Aβ measured by ELISA was also reduced in both soluble and insoluble fractions of the brain homogenates, and notably the Aβ oligomer (12-mer) was also apparently decreased. Tg2576 mice showed significant improvement in cognitive functions examined at 3 months after vaccination. Thus, this is an alternative immunotherapy for AD, which has an advantage in non-invasive, safe and relatively long lasting features.

Short amyloid-beta immunogens show strong immunogenicity and avoid stimulating pro-inflammatory pathways in bone marrow-derived dendritic cells from C57BL/6J mice in vitro

Amyloid beta peptide 1–15 (Aβ1–15) and its derivatives have attracted the attention of the scientific community as candidate vaccines for Alzheimer's disease (AD) immunotherapy. Recent studies suggested that Aβ1–42 modulated the immune system by inducing pro-inflammatory dendritic cells (DCs) with reduced antigen-presenting function. However, it remains elusive how Aβ1–15 impacts DCs function. We therefore investigated the modulation by short Aβ peptides of DCs from C57Bl/6J mice. Two new immunogens, a tandem repeat of two-lysine-linked Aβ1–15 sequences with or without an addition of a RGD motif, were tested. Chemotaxis, endocytosis, antigen presenting function and producing cytokines were measured. Both peptides increased migration/endocytosis of immature DCs and MHC II molecule expression/alloreactive T cell activation in TNF-α-matured DCs. In addition, they exhibited decreased production of Th1/Th2 cytokines and pro-inflammatory cytokines. Overall, the two peptides demonstrated strong immunogenicity but did not stimulate pro-inflammatory pathways. These results support the use of short Aβ immunogens in AD immunotherapy.

Neuropathology and Amyloid-β Spectrum in a Bapineuzumab Immunotherapy Recipient

The field of Alzheimer's disease (AD) research eagerly awaits the results of a large number of Phase III clinical trials that are underway to investigate the effectiveness of anti-amyloid-β (Aβ) immunotherapy for AD. In this case report, we review the pertinent clinical history, examine the neuropathology, and characterize the Aβ profile of an AD patient who received bapineuzumab immunotherapy. The patient received four bapineuzumab infusions over a 39 week period. During the course of this treatment, there was no remarkable change in cognitive impairment as determined by MMSE scores. Forty-eight days after the fourth bapineuzumab infusion was given, MRI revealed that the patient had developed lacunar infarcts and possible vasogenic edema, probably related to immunotherapy, but a subsequent MRI scan 38 days later demonstrated resolution of vasogenic edema. The patient expired due to acute congestive heart failure complicated by progressive AD and cerebrovascular accident 378 days after the first bapineuzumab infusion and 107 days after the end of therapy. Neuropathological and biochemical analysis did not produce evidence of lasting plaque regression or clearance of Aβ due to immunotherapy. The Aβ species profile of this case was compared with non-immunized AD cases and non-demented controls and found to be similar to non-immunized AD cases. SELDI-TOF mass spectrometric analysis revealed the presence of full-length Aβ₁₋₄₂ and truncated Aβ peptides demonstrating species with and without bapineuzumab specific epitopes. These results suggest that, in this particular case, bapineuzumab immunotherapy neither resulted in detectable clearance of amyloid plaques nor prevented further cognitive impairment.

Administration of Amyloid-β42 Oligomer-Specific Monoclonal Antibody Improved Memory Performance in SAMP8 Mice

Amyloid-β peptide (Aβ) is recognized by many as the leading cause of Alzheimer's disease (AD), and Aβ oligomers play a major role in the early-onset form of AD. Recently, the application of passive immunization targeting Aβ has been investigated as a potential method of AD immunotherapy. We used a strain of monoclonal antibody against Aβ42 oligomers, designated A8, as an Aβ inhibitor to suppress Aβ aggregation and Aβ-derived cell toxicity in vitro, and as a passive immunotherapy approach to treat SAMP8 (senescence accelerated mouse sub-line P8) mice, an animal model of AD, in vivo. First, our results showed that pre-incubation of A8 with Aβ oligomers inhibited both the maturation of Aβ fiber and Aβ oligomer toxicity on SH-SY5Y cells. Second, learning and memory was improved through intraperitoneal administration of A8 in SAMP8 mice. Third, Aβ pathology was ameliorated with decreased Aβ oligomers and phospho-tau levels in SAMP8 mice. Our data suggest that our monoclonal antibody A8 may be a candidate as a potential immunotherapeutic agent in AD.

Recent advances in the development of immunotherapies for tauopathies

The use of immunotherapy for Alzheimer's disease (AD) has traditionally focused on the amyloid-β (Aβ) peptide and has shown great potential in both animal and human studies. However, an emerging body of work has begun to concentrate on tau and to develop immunization protocols designed to decrease tau pathology in AD and other tauopathies. This commentary will discuss the use of immunotherapy for AD, focusing on tau immunotherapy in the context of recent reports on the use of tau phospho-peptides in transgenic models of tau pathology.

Reduction of amyloid-beta formation towards Alzheimer's disease immunotherapy

Reduction of amyloid-beta formation towards Alzheimer's disease immunotherapy

Amyloid-&amp;#946; Immunotherapy for Alzheimers Disease

Alzheimer's disease (AD) is a progressive, degenerative disorder of the brain and the most common form of dementia among the elderly. As the population grows and lifespan is extended, the number of AD patients will continue to rise. Current clinical therapies for AD provide partial symptomatic benefits for some patients; however, none of them modify disease progression. Amyloid-beta (Abeta) peptide, the major component of senile plaques in AD patients, is considered to play a crucial role in the pathogenesis of AD thereby leading to Abeta as a target for treatment. Abeta immunotherapy has been shown to induce a marked reduction in amyloid burden and an improvement in cognitive function in animal models. Although preclinical studies were successful, the initial human clinical trial of an active Abeta vaccine was halted due to the development of meningoencephalitis in approximately 6% of the vaccinated AD patients. Some encouraging outcomes, including signs of cognitive stabilization and apparent plaque clearance, were obtained in subset of patients who generated antibody titers. These promising preliminary data support further efforts to refine Abeta immunotherapy to produce highly effective and safer active and passive vaccines for AD. Furthermore, some new human clinical trials for both active and passive Abeta immunotherapy are underway. In this review, we will provide an update of Abeta immunotherapy in animal models and in human beings, as well as discuss the possible mechanisms underlying Abeta immunotherapy for AD.

Rapid Cerebral Amyloid Binding by Aβ Antibodies Infused into β-Amyloid Precursor Protein Transgenic Mice

Passive immunization for the treatment of Alzheimer's disease (AD) was rapidly translated into clinical trials. However, basic mechanisms of AD immunotherapy remain only partially understood. We analyzed the dynamic changes of amyloid-β (Aβ) levels in plasma, brain, and cerebrospinal fluid (CSF) as well as cerebral amyloid binding by Aβ antibody after a single β1-antibody infusion into APP(Swedish) and APP(wildtype) transgenic mice at preplaque and plaque-bearing age. Following intravenous Aβ antibody treatment, plasma Aβ increased rapidly, reaching significantly higher levels in preplaque compared with plaque-bearing mice, whereas cerebral and CSF Aβ remained unchanged. Strikingly, Aβ antibodies exhibited strong cerebral amyloid plaque binding rapidly after intravenous administration in a subset of animals with more severe vascular amyloid. Rapid plasma Aβ increase after Aβ antibody infusion results primarily from stabilization of Aβ. Nevertheless, the smaller plasma Aβ increase in plaque-bearing mice might be of diagnostic use. Importantly, intravenously administered antibodies can rapidly bind to cerebral plaques, potentially facilitated by vascular-amyloid-mediated damage of the blood-brain barrier.

Changing the Kinetics of Amyloid Beta Plaques Formation: Implications for Alzheimer's Disease Immunotherapy

Clear evidence exists linking the presence of neuritic amyloid beta (As) peptides plaques with the brain's tissue deterioration and cognitive impairment in patients with Alzheimer's disease (AD). Removing these plaques results a logic approach to treat patients with AD. Understanding the plaque formation mechanisms is key to developing strategies to remove them. In previous studies our group has investigated the plaque formation process using attenuated total reflection Fourier infrared (ATR-FTIR) spectroscopy and atomic force microscopy (AFM), observing how different As peptide aggregates formed and under what kinetic conditions they assembling into mature fibrils. We have been able to determine the changes in the secondary structure of the peptide molecules during this process. In this work, we combined the same analytical techniques to investigate the use of different anti-As monoclonal antibodies to analyze the process of destabilization and prevention of AD plaque formation. We compared the changes of kinetic rates of fibrillization when the peptides were incubated with different antibodies from the early stage of aggregation, at pH 7.4 and 37°C. The molar ratio of antibodies to peptide used was 1:1000. We found that some antibodies considerable decrease the formation of parallel beta sheets structures increasing the formation of alpha helix structures or unordered, driving the peptide away from normal fibrillization and rather producing amorphous aggregates. The results of the peptide's morphological evolution and secondary structure changes revealed that some antibodies have the potential of preventing formation of parallel beta sheets structures, which may be an indication of As fibrillization preclusion. This work provides insights to understand the mechanistic effects in which antibodies alter the secondary structure of amyloid peptides modifying their fibrillization, critical for As plaque clearance in the brain of patients with AD.

Recombinant GST-I-Aβ28-induced efficient serum antibody against Aβ42

Six recombinant proteins GST-Aβ28/Aβ35/Aβ42 and GST-I-Aβ28/Aβ35/Aβ42 [I was the abbreviation for an immunostimulatory sequence that consisted of pan HLA DR binding epitope (PADRE) and Tetanus toxin epitope (TT)] were used as antigens after expressed and purified to immunize mice. The strongest antibody response against Aβ42 (titer 1:3200) was achieved by GST-I-Aβ28 or GST-Aβ42 immunization. However, IgG1 and IgG2b were the predominant serum antibody isotype responses by GST-I-Aβ28 immunization, whereas did IgG2a by GST-Aβ42 immunization. Thus, it indicated that GST-I-Aβ28 immunization in a mouse mainly evoked a stronger Th-2-type response; whereas, GST-Aβ42 immunization mainly elicited a Th-1-type response. Moreover, GST-I-Aβ28-induced serum antibodies had higher specificity to Aβ42 monomers and oligomers than to protofibrils and mature fibrils and exhibited the highest efficacy to block Aβ42 aggregation or fibrillogenesis and to disassemble Aβ42 aggregates in vitro. GST-I-Aβ28-induced serum antibodies also showed the most protective and restorative effects on target cells in vitro by inhibiting or neutralizing Aβ42-induced cytotoxicity. All of the above results indicated that Aβ28 could be speculated to substitute for Aβ42 and would become a better antigenic peptide for Alzheimer's disease immunotherapy in the presence of additional Th-cell epitopes such as the immunostimulatory sequence (I) applied in this study.

Abeta-oligomer selective antibody A-887755 exhibits a favorable profile for Alzheimer's disease immunotherapy compared to Abeta-peptide unselective antibodies

Abeta-oligomer selective antibody A-887755 exhibits a favorable profile for Alzheimer's disease immunotherapy compared to Abeta-peptide unselective antibodies