M.B. Usman1,*, S. Bhardwaj2, S. Roychoudhury3, D. Kumar4, A. Alexiou5,6, P. Kumar7, R.K. Ambasta7, P. Prasher8, S. Shukla9, V. Upadhye10, F.A. Khan11, R. Awasthi12, M.D. Shastri13, S.K. Singh14, G. Gupta15, D.K. Chellappan16, K. Dua9,17, S.K. Jha18, J. Ruokolainen19, K.K. Kesari19,20, S. Ojha21, N.K. Jha18
1. Department of Life Sciences, School of Basic Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India; 2. Department of Biotechnology, HIMT, CCS University, Greater Noida, UP, India; 3. Department of Life Science and Bioinformatics, Assam University, Silchar, India; 4. Amity Institute of Molecular Medicine and Stem Cell Research (AIMMSCR), Amity University Uttar Pradesh, Sec 125, Noida, India; 5. Novel Global Community Educational Foundation, Hebersham, 2770 NSW, Australia; 6. AFNP Med Austria, Wien, Austria; 7. Molecular Neuroscience and Functional Genomics Laboratory, Department of Biotechnology, Delhi Technological University (Formerly DCE), Delhi, India; 8. Department of Chemistry, University of Petroleum & Energy Studies, Energy Acres, Dehradun, India; 9. Discipline of Pharmacy, Graduate School of Health, University of Technology Sydney, Ultimo NSW 2007, Australia; 10. Centre of Research for Development (CRD4), Parul Institute of Applied Sciences, Parul University, Vadodara-391760, Gujrat, India; 11. Department of Stem Cell Biology, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia; 12. Amity Institute of Pharmacy, Amity University Uttar Pradesh, Noida, India; 13. School of Pharmacy and Pharmacology, University of Tasmania, Hobart, Australia; 14. School of Pharmaceutical Sciences, Lovely Professional University, Phagwara 144411, Punjab, India; 15. School of Pharmacy, Suresh Gyan Vihar University, Jagatpura, Mahal Road, Jaipur, India; 16. Department of Life Sciences, School of Pharmacy, International Medical University, Bukit Jalil, Kuala Lumpur, Malaysia; 17. Faculty of Health, Australian Research Centre in Complementary and Integrative Medicine, University of Technology Sydney, Ultimo, 2007 New South Wales, Australia; 18. Department of Biotechnology, School of Engineering & Technology, Sharda University, Greater Noida, Uttar Pradesh, India; 19. Department of Applied Physics, School of Science, Aalto University, Espoo, Finland; 20. Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Espoo, Finland; 21. Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain 17666, United Arab Emirates; * These authors contributed equally to this work
Corresponding Author: Dr. Niraj Kumar Jha, Assistant Professor, Department of Biotechnology, School of Engineering & Technology (SET), Sharda University, Knowledge Park III, Greater Noida, Uttar Pradesh-201310, India, Email: email@example.com; firstname.lastname@example.org, Tel: +91-7488019194, ORCID: https://orcid.org/0000-0001-9486-4069; Dr. Shreesh Ojha, Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, UAE University, PO Box – 17666, Al Ain, UAE, E-mail: email@example.com, Tel: +971-3-7137524, ORCID: https://orcid.org/0000-0001-7801-2966
J Prev Alz Dis 2021;4(8):534-551
Published online September 15, 2021, http://dx.doi.org/10.14283/jpad.2021.52
Alzheimer’s disease (AD) is a global health concern owing to its complexity, which often poses a great challenge to the development of therapeutic approaches. No single theory has yet accounted for the various risk factors leading to the pathological and clinical manifestations of dementia-type AD. Therefore, treatment options targeting various molecules involved in the pathogenesis of the disease have been unsuccessful. However, the exploration of various immunotherapeutic avenues revitalizes hope after decades of disappointment. The hallmark of a good immunotherapeutic candidate is not only to remove amyloid plaques but also to slow cognitive decline. In line with this, both active and passive immunotherapy have shown success and limitations. Recent approval of aducanumab for the treatment of AD demonstrates how close passive immunotherapy is to being successful. However, several major bottlenecks still need to be resolved. This review outlines recent successes and challenges in the pursuit of an AD vaccine.
Key words: Alzheimer’s disease, amyloid plaque, passive immunotherapy, active immunotherapy, monoclonal antibody.
Alzheimer’s disease (AD) is a brain disorder characterized by progressive, chronic neurodegenerative symptoms, such as memory loss, cognitive disabilities, and dementia. The global prevalence rates of dementia among people over 85 years and people over 60 years are 20% and 6%, respectively (1). AD is the most common form of dementia among aging individuals in North America and western Europe. It can lead to a decrease in cognitive function, judgment, decision-making, and language abilities among people over 65 years of age (2, 3). Gradual neurodegeneration in the cortex and hippocampus explains the continued loss of memory and dementia observed in patients with AD. This degenerative process can last for up to 25 years after the initial symptoms appear (4).
AD is a significant global health issue associated with a significant economic burden. The global AD prevalence is 24 million, with the United States (US) alone having nearly 5.5 million cases, including 200,000 cases of early-onset AD (2). According to the World Health Organization, an estimated 81.1 million people will have AD by 2040. Unfortunately, the number of people with the disease is projected to multiply in every 20 years (5, 6). AD is the fifth leading cause of death among elderly people globally and sixth in the US (7). While it ranks third in terms of total health care costs in the US after cancer and cardiovascular disease, it is projected to surpass the two diseases in terms of mortality rate and overall financial burden on US health care in the next two decades (8, 9). Approximately $172 billion is spent annually on AD-related healthcare costs (10).
AD, like other neurodegenerative disorders, is a proteinopathy, in that it arises due to protein misfolding or failure of certain peptides to adopt their usual functional and conformational state. Misfolding results in protein accumulation (and/or fibril formation), gain of toxic function, or loss of function. The major causes of protein misfolding include genetic mutations, exposure to external or internal toxins, impairments in the posttranslational modification machinery, and oxidative damage. AD is characterized by the pathological accumulation of two forms of proteinaceous inclusions: the extracellular amyloid beta (Aβ) plaques that develop during the initial disease phase and intracellular neurofibrillary tangles that manifest at later stages (11, 12).
Although we now have a deeper understanding of the pathological features of AD, several questions related to its complex pathways remain unanswered. At present, no single theory has accounted for the various risk factors leading to the pathological and clinical manifestations of dementia-type AD (3, 13). Treatment options targeting various molecules that play essential roles in the development of the disease have been unsuccessful due to multiple drawbacks (14, 15). However, these failures have led to a better understanding of the disease and a shift in focus toward preventive approaches that can avert or delay disease onset (5, 7, 16). One of the major therapeutic avenues being explored currently is immunotherapy, which involves manipulation of the immune system by suppressing, inducing, or enhancing its activity in vivo. Immunotherapy or vaccination against AD-specific peptides inspired considerable optimism in preventing or treating AD through an adaptive immune response (7, 16). Vaccines or immunotherapies for AD utilize the power of the immune system to attack the body’s own proteins or molecules that seem to be dangerous. Despite the practical challenges and decade of disappointments, hopes for Alzheimer’s vaccine are increasing again. Moreover, the enticing allure of being able to curtail the disease through vaccination makes the idea very appealing and worth striving for (16, 17). This review explores the recent successes and challenges in the pursuit of developing an AD vaccine.
Pathophysiology and Molecular Concept of AD
AD was first described in 1907 by the German physician, Alois Alzheimer (18). This discovery was ensued by many studies that led to various discoveries and hypothesis on the disease. While the exact cause of AD remains unknown, the most widely acknowledged hypothesis involves abnormal processing of the β-amyloid precursor protein (AβPP), resulting in the overproduction of or reduced clearance of amyloid β-protein (Aβ) in the cortex (19). AβPP, the progenitor molecule of Aβ, is a membrane-bound protein that plays crucial roles in the regulation of neuronal survival, synaptic stabilization and plasticity, cell adhesion, and neuritic outgrowth formation (20, 21). Under normal circumstances, α-secretase cleaves the large AβPP molecule at the middle of the Aβ sequence. In AD, the β-secretase-mediated endoproteolytic cleavage of AβPP generates the primary N-terminal cut, while γ-secretase generates pathogenic Aβ fragments. The length of the deadly Aβ peptide fragments can be determined from the exact site of γ-secretase cleavage. Although β- and γ-secretases are active throughout a person’s lifetime, their undesirable effects on Aβ production are observed in individuals aged ≥60 years (22, 23). The two major forms of Aβ peptides are the 40-residue (Aβ1-40) and 42-residue (Aβ1-42) moieties, which are considered more pathogenic due to their higher aggregation tendency, longer length, and higher quantity in amyloid plaques incases of sporadic and early-onset AD (4, 24). Aβ peptides, like other proteins, have N and C terminals; the N-terminal constitutes the hydrophilic domain with 1-28 residues that are mostly charged, while the C-terminal domain is completely hydrophobic with 29–40 or 29–42 residues. Aβ42, when produced, assumes a beta-pleated structure that clumps to form fibrils that are insoluble in the extracellular space. Over time, amyloid plaques are formed by the deposition of complement protein, microglia, and reactive astrocytes (11, 25). Amyloid plaque formation results in a cascade of neuropathogenic events characterized by neurotoxicity, local inflammation, neuronal apoptosis, complement activation, and disruption of calcium homeostasis, ultimately leading to cognitive decline and AD manifestations. Aβ impairs neuronal function even before its deposition in amyloid plaques. Similarly, Aβ oligomers induce hyperphosphorylation of microtubule-associated protein tau (cytoskeletal protein), leading to the formation of insoluble intracellular neurofibrillary tangles and consequent tauopathy that affects neuronal function (26-28). Hence, the two abnormal protein deposits, amyloid plaques and neurofibrillary tangles, result in the pathophysiological, clinical, and microscopic manifestations of AD (Figure 1).
Figure 1. Neurobiology of Alzheimer’s disease
Although the amyloid hypothesis suggests that Aβ deposition and plaque formation are the first steps in the pathogenesis of AD, the relationship between amyloid burden and cognitive symptoms remains unclear. Similarly, the order and timing of amyloidosis and other processes of AD that result in the clinical onset of dementia are not well understood (12). Moreover, the failure of different therapeutic approaches in preventing Aβ aggregation or production raises questions about the hypothesis. So far, there has not been a single successful treatment based on the amyloid hypothesis (3). Recent studies point to the protective and anti-microbial roles of Aβ peptides along with increased formation of tau-positive tangles in AD cell lines, rodent models, and nematodes. In some cases, Aβ is produced in response to bacterial and neurotoxic fungal infection, indicating its neuroprotective role (29). Hence, overproduction of Aβ may be due to downstream immune dysregulation and not the disease process itself.
AD can be divided into two types based on symptom onset: (i) late-onset or sporadic AD is the most common type of AD, in which a majority of patients are diagnosed after 65years of age, and its incidence increases with age (30, 31) and (ii) early-onset AD accounts for 1–2% of AD cases and is characterized by symptom presentation before the age of 65 years (32, 33). Early-onset AD is also referred to as autosomal-dominant AD as it results from mutations in the following genes: amyloid precursor protein (APP) (chromosome 21), presenilin 1/PSEN1(chromosome 14), and presenilin 2/PSEN2 (chromosome 1). Mutations in these genes can lead to abnormal Aβ processing, its excessive accumulation, and consequently, AD with complete penetrance (12). The age of clinical onset of autosomal-dominant AD is influenced by genetic background and is similar among different generations in a family (34, 35). While dominantly inherited mutations have no significant role in sporadic AD, polymorphisms in the apolipoprotein E gene (ε4 allele) increase the risk of developing AD, particularly in females (36, 37). Increasing evidence shows that sporadic and autosomal-dominant AD share pathophysiological features (12, 32).
Modified vaccine formulations use Aβ-specific sequences and epitope-based DNA, while emerging vaccine candidates target other proteins and molecules involved in AD etiology.
Vaccines and Immunotherapies for AD
Most immunotherapies and vaccines directly or indirectly target Aβ42 peptides to elicit an appropriate immune response (anti-Aβ antibodies) that will not only clear the Aβ deposits, but also help in improving cognitive and functional abilities (38). Immunotherapy related to AD may be divided into two forms: injection of Aβ42-containing antigens is termed active immunotherapy (vaccination), whereas passive immunotherapy involves administering preformed antibodies against the Aβ42 peptide (such as monoclonal antibodies) (24). Thus, an immunotherapeutic approach involves active injection of Aβ-based immunogens or passive infusion of Aβ-specific antibodies (Figure 2).
Figure 2. Classification of Alzheimer’s disease immunotherapy and hypothetical mechanisms of anti-Aβ antibody action
In active immunotherapy, patients are injected with a purified form of an antigen, usually coupled with a different protein carrier or adjuvant that helps in the optimization of the immune response. Active AD vaccines are aimed at eliciting an appropriate immune response that clears accumulated proteins. While active immunotherapy has the potential to generate long-term polyclonal antibodies through short-term administration of vaccines at a limited cost, it may cause inconsistent immune responses and long-lasting adverse reactions, especially in older people with low immune competence (25, 39). Most active vaccine trials involve the administration of Aβ42 antigenic peptides. However, more recent studies make use of small Aβ peptides, their DNA sequences, or prime-boost approaches to elicit the anti-Aβ antibody production. This is usually achieved through B-cell activation while avoiding T-cell activation, which may cause autoimmunity (24, 39). As the presence of Aβ plaques is common across different forms of AD, the Aβ peptide is a notable target across immunotherapeutic approaches.
Mechanism of Anti-Aβ Antibodies
Anti-Aβ antibodies are versatile in nature owing to the intrinsic diversity of the human immune system. This versatility is necessary because of the uncertainty about the role of Aβ in physiological conditions and lack of knowledge of the pathogenic forms of Aβ (40). The mechanism by which anti-Aβ antibodies are transported into the central nervous system (CNS) is not well understood. However, it is thought to involve the lymphatic system, passive diffusion through perivascular spaces, and leaky areas in the CNS within the blood-brain barrier (BBB). Consequently, only a small fraction of antibodies in the peripheral circulation is detectable in the CNS (25).
Three hypotheses have been formulated to outline the mechanisms by which anti-Aβ antibodies achieve plaque clearance and reduce AD symptoms (Figure 2). First, anti-Aβ antibodies bind directly to the peptides in the senile plaques, protofibrils, fibrils, or oligomers to destabilize their aggregates and eventually disrupt them (direct action hypothesis). Second, specific antibodies would bind to Aβ plaques and trigger phagocytosis mediated by microglial cells and Fc receptors (41). Third, specific antibodies do not cross the BBB, but bind to and remove the Aβ molecules circulating in the plasma. This generates a concentration gradient that leads to the efflux of Aβ molecules from the brain to the plasma (peripheral sink hypothesis) (14). Most AD vaccine studies prioritize the reduction in senile plaques in the brain by active immunization, which can stimulate the production of anti-Aβ antibodies (38, 42, 43).
Anti-Aβ antibodies are also involved in several other mechanisms that contribute to Aβ reduction or clearance. For instance, antibodies can interact with and alter the transport system of Aβ that includes the receptor for advanced glycation end products (RAGE), the influx channel for Aβ in the CNS, and efflux via the low-density lipoprotein receptor. Theoretically, antibodies that block RAGE could enhance reduction in Aβ levels in the cerebrospinal fluid (CSF) by hindering their transport from the blood (44, 45). While some antibodies may interfere with the interaction between Aβ and other molecules, thereby reducing toxicity, others could act as signals that induce or reduce inflammation by binding to receptors on immune effectors. Further, when antibodies enter the synaptic cleft between neurons or are internalized by neurons, they can alter the cell-to-cell transmission of Aβ and its aggregates (46).
First- and Next-Generation Active Vaccines
Active vaccines aim to stimulate the patient’s immune system to prevent or reduce amyloidosis and restore cognitive and functional abilities. It is commonly believed that immunotherapy must start when the two common features amyloid plaques and neurofibrillary tangles are not obvious. Efforts to develop active AD vaccines have been punctuated by drawbacks, which have led to the evolution of vaccine generations (Table 1).
Table 1. Summary of active vaccines of AD in clinical trial stage
First-generation active vaccines
AD immunotherapy research began with a major breakthrough published by Schenk et al., who demonstrated that active immunization with Aβ42 and an immune-stimulating adjuvant improved cognition in transgenic mice (47). They also showed prevention of or reduction in β-amyloid plaque formation in transgenic mice overexpressing human APP. This discovery led to the rapid development of a first-generation active vaccine called AN-1792.
AN-1792, the first anti-Aβ immunotherapy candidate, consists of aggregated human Aβ42 coupled to a saponin-based adjuvant (QS-21). It elicits an immunological response against the host Aβ42, which can improve cognition and reduce plaque burden (48). The phase 1 trial showed evidence of the tolerability and safety of the vaccine. Moreover, anti-Aβ42 antibodies developed by the recipient patients could recognize the β-amyloid plaque in the extracellular space and the β-amyloid within the blood vessels of the brain. The antibodies were also selective and did not cross-react with native full-length APP or other physiological components (43). Amyloid clearance is facilitated by the solubilization of Aβ42, leading to its exit from the brain through the perivascular pathway. Vaccination also resulted in reduced hippocampal tau pathology mediated by a decrease in tau phosphorylation and inhibition of inflammatory processes that result in neurodegeneration (49-51). Approximately 20% of the vaccinated patients developed antibody titers above the present therapeutic cut-off level (52, 53). However, despite the desirable outcomes, AN-1792 clinical trials were halted in phase 2, owing to adverse inflammatory reactions resulting in subacute meningoencephalitis in nearly 6% of the patients and one death. Subsequent follow-up studies attributed these consequences to the activation of proinflammatory T helper (Th)-1 cell-mediated responses that result in autoimmunity (25, 54). Inflammatory infiltrates in the CNS of the deceased patient were mainly CD8+ cells; to a lesser extent, CD4+, CD3+, and CD5+ cells; and rarely CD7+ cells. In contrast, the patient tested negative for T cytotoxic markers such as CD16 and CD57, turbidimetric immunoassay, granzymes, and B lymphocytes (54). The Aβ42 epitopes are located in the carboxyl-terminal and central region of the Aβ peptide (55). These findings were supported by studies conducted to develop next-generation vaccines containing only B-cell epitopes (primarily located in the N-terminal region of the Aβ peptide). As vaccines that induce only humoral or Th2-mediated responses aim to avoid the undesirable inflammatory effects of Th1 stimulation (Table 1), next-generation vaccines usually contain B-cell epitopes as antigenic determinants coupled to an appropriate adjuvant (56-59).
Next-generation active vaccines
Next-generation active vaccines target the N-terminal regions of Aβ peptides (B-cell epitope) to stimulate humoral immune responses.
ACC-001: ACC-001 (VanutideCridificar) contains1-7 amino acid-long N-terminal Aβ peptide fragments connected to a carrier protein (CRM197) via a surface-active saponin adjuvant (QS-21). The CRM197 carrier protein is a nontoxic Diphtheria toxin mutant (60, 61). ACC-001 elicits an Aβ-specific B-cell response without the adverse T-cell response recorded following AN-1792 administration (62). A phase 1, single ascending dose trial of ACC-001 showed safety and tolerability, which paved the way for phase 2, multiple ascending dose studies (61) conducted in Europe (ClinicalTrials.gov Identifier: NCT00479557), US (ClinicalTrials.gov Identifier: NCT00498602), and Japan. These trials involved administration of different doses of the vaccine (3, 10, and 30μg) with or without the adjuvant. The patients who received doses of ACC-001+QS-21 adjuvant showed sustained anti-Aβ IgG titers and consistently higher peaks. While no case of meningoencephalitis was reported, few patients showed side effects such as insignificant microhemorrhage, treatment-related vasogenic edema, local injection reaction, and headache (61,62). Phase 2a extension studies carried out in these countries showed that long-term exposure to ACC-001+ QS-21 was well-tolerated and gave the highest anti-Aβ IgG titer compared to other regimens (63). However, the phase 2 trial of this vaccine was aborted in 2014 owing to adverse effects linked to autoimmune responses, lack of efficacy, and case of treatment-related angina pectoris recorded in a patient who received ACC-001 (30μg) + QS-21 (62, 64).
AD01, AD02, AD03: While AD01 and AD02 contain Aβ1-6 (B-cell epitope) peptides that mimic the N-terminal region of Aβ42 coupled with an Alum adjuvant, AD03 consists of N-terminal-truncated and pyroglutamated Aβ conjugated with an Aluma djuvant (58). Phase 1 trials of AD01/ AD02 have been announced to be completed by AFFiRiS (Wien, Austria). So far, the trial has demonstrated safety of AD02 and its ability to stabilize cognitive parameters based on a potential correlation between cognitive function and post-vaccination antibody levels; however, these data have not yet been published (58). Phase 2 trials of AffitopeAD02 have been performed in patients with early-onset AD; however, these trials were terminated due limited efficacy and adverse side effects (64). AFFiRiS also conducted a phase 1 trial usingAD03 (58). However, the follow-up study was aborted due to organizational reasons (65, 66).
ACI-24: ACI-24 is based on tetra-palmitoylated amyloid 1–15 peptide in β conformation coupled with liposomes containing monophosphorylated lipid A as an adjuvant. ACI-24 aims to induce antibodies specific to the beta-sheet conformation, thereby targeting Aβ1-15 (67). It is similar to the liposomal vaccine against Aβ1-15, which showed the ability to restore memory defects and reduced plaques in mice (67, 68). Having achieved the desired outcomes in the preclinical trial, a combined phase1/2a clinical trial was initiated (67, 69). The trial compared vaccine doses of 10,100, 300, and 1000 µg/ml to placebo; the dose was administered subcutaneously for the first year, followed by an additional 1 or 2 years. The primary outcomes included tolerability, safety, and serum titers of anti-Aβ42 IgG antibodies. The secondary outcomes included biomarker measures such as T-cell activation measures; magnetic resonance imaging (MRI)-based volumetry; and tau, phospho-tau, and Aβ levels in the CSF. ACI-24 was the first anti-Aβ vaccine to be examined for the treatment of AD patients with Down’s syndrome. The study involved subcutaneous injection of ACI-24 in 24 patients (age: 35–55 years). The study ended in June 2020 and reported positive outcomes and no serious adverse effects. The AC Immune registered additional phase 2 trial in the same syndrome by May 2020, it was set to commence in October 2020 and designed to enroll 72 patients aged 40–50 years who had only brain amyloid deposition without dementia. The primary outcome measures include safety parameters and incidence of adverse events such as suicidal ideation, heart rate, and changes in blood pressure studied for up to 2 years. The secondary outcome measures include changes in cognitive and behavioral measures, levels of amyloid and tau in the blood, neurodegeneration, blood Aβ antibody titers, and levels of amyloid and tau in the brain as determined by positron emission tomography (PET). The trial is projected to end in October 2024 (70).
CAD-106 (Novartis): Novartis’s CAD-106 is composed of multiple copies of B-cell epitope (Aβ1-6) fragments as the immunogenic sequence, attached to a carrier with 180 copies of bacteriophage QB protein coat as an adjuvant (57, 69). The formulation stimulates Aβ-specific antibodies unique to the N-terminus, while avoiding T-cell autoimmune responses (57). As the vaccine could reduce Aβ plaques in APP transgenic mice in a preclinical trial, a phase 1 trial was conducted among patients with mild AD. The trial showed reasonable antibody response and evidence of safety, with no meningoencephalitis, autoimmunity, or other adverse reactions (71). Although phase 2 trials showed adequate antibody production in 75% of the patients without the adverse effects observed in the AN-1792 trials, there was no significant difference between the control and treated groups (71). Phase 2a randomized control trials and two open extension studies showed effective antibody response in approximately 64% of the treated patients. There were sustained anti-Aβ IgG titers in extension versus core studies. Although there was no evidence of Aβ-specific T-cell response or vasogenic edema, a few patients showed intracerebral hemorrhage and imaging abnormalities corresponding to amyloid-related microhemorrhage (57). The phase 2/3 clinical trial (GENERATION 1) sponsored by Novartis Pharmaceuticals was initiated in 2015. It aimed to investigate whether CAD-106 and CNP520, an inhibitor of aspartyl protease beta-secretase or beta-site APP cleaving enzyme, can stall the onset and progression of clinical symptoms in cognitively unimpaired individuals with two APOE4 genes. The clinical trial consists of 1340 enrolled patients and is set to end in 2024. While half of the participants will receive CAD-106 injections four times a year, the other half will receive 50 mg CNP520 once daily; the outcomes in both groups will be compared to that of an age-matched placebo group (72). An additional phase 2/3 prevention study (GENERATION 2) was initiated in August 2017, which enrolled 2000 heterozygous carriers with evidence of brain amyloid protein (age 65–70 years) or homozygous ApoE4 carriers. Patients were randomized to one of three groups: while groups 1 and 2 are given one capsule of CNP520 (group 1: 15 mg; group 2: 50 mg) daily for 60–84 months, group 3 is given one capsule of placebo daily. The GENERATION 1 and 2 trials of CNP520 were both prematurely terminated by the sponsors in July 2019, owing to worsening of cognitive abilities in the treatment groups (73). A phase 3 trial is expected to show whether CAD-106 is more effective than placebo in delaying AD symptoms among individuals with genetic susceptibility to AD. Therefore, CAD-106 remains the only vaccine to advance to phase 3 trials and was selected for an AD prevention initiative (API) in theAPOEε4 homozygote study (39).
Lu AF20513: Lu AF20513 consists of three B-cell epitopes (Aβ1-12) attached to two Th epitopes obtained from tetanus toxoid P2 and P30 (74). The formulation is designed to activate memory Th cells present in majority of the population immunized with the conventional tetanus vaccine, thereby enhancing response against Aβ1-12 in elderly people. The phase 1 study aimed to determine the tolerability and safety of multiple immunizations of the drug. The trial enrolled 24 patients with a recent MRI consistent with an AD diagnosis and Aβ antibodies in the CSF. Multiple shots of either low-, medium-, or high-dose Lu AF20513 were administered to the participants. Although the study aimed to evaluate the safety, tolerability, and antibody titers for around 2 years, the study was terminated on account of new efficacy data from another study (59).
UB-311: UB-311 contains synthetic Aβ1-14 (B-cell epitope) coupled with CpG/Alum as an adjuvant (58). A novel form of the vaccine contains two synthetic Aβ targeting peptides, each of which is conjugated with different Th epitopes and designed in a Th2-based delivery system (56). A successful phase 1 trial led to the advancement to phase 2. The recruitment for this trial is now complete, and the outcomes show early evidence of safety and immunogenicity (59).
V-950: V-950 is a multivalent vaccine containing Aβ1-15 coupled with Alum/ISCOMATRIX as an adjuvant. Although a phase 1 study was initiated to determine its safety, tolerability, and immunogenicity, the study was suspended for unknown reasons (69).
Anti-tau Vaccines: Given the failure of vaccine candidates that target Aβ to provide the desired results in clinical trials, recent efforts seek to include tau protein as another target antigen in preventing or controlling AD.
ACI-35: ACI-35 is a liposomal vaccine based on a synthetic human tau protein sequence phosphorylated at S396 and S404 (75); phase 1 trials to study ACI-35 are ongoing (64, 76).
AADvac1: AADvac1 contains synthetic peptides that mimic the naturally occurring truncated and misfolded tau protein, conjugated with keyhole limpet hemocyanin and aluminum hydroxide as adjuvants (77). AADvac1 is formulated to elicit antibodies against the pathological tau protein, prevent the aggregation or progression of the tau protein aggregates, and thereby hinder the spread of the pathology and the disease. A phase 1 trial was conducted in patients with mild-to-moderate AD. A 24-month, randomized, placebo-controlled, parallel group, double-blind, multi-center, phase 2 study aimed at assessing the safety and efficacy of AADvac1 in patients with mild AD (ADAMANT) is ongoing. Patients with pathological tau protein and/or hippocampal atrophy and CSF amyloid were enrolled in the phase 2 trial, in which they would be given 11 vaccinations within a period of 11 months. Although the study was set to conclude in summer 2019 (77), the results are yet to be published.
Given the failures and practical uncertainties associated with several peptide vaccines in clinical trials, new formulations that do not require adjuvant-like peptides such as DNA vaccines, epitope/protein-based vaccines, and the prime-boost approach have been developed (Table 2).
Table 2. Summary of some active vaccines at preclinical stage
DNA Vaccines (genetic vaccines): These are considered as third-generation vaccines; they are constructed by inserting a gene of interest or target gene (Aβ) into an expression vector. The construct is then introduced into a host, which expresses the protein of interest that elicits an immune response in the recipient host (78). DNA vaccines have been found to elicit both humoral and cellular immune responses characterized by Th2 cell stimulation and IgG1 antibody generation in animals (79). The vaccine formulations employ the concept of fusion with immune-modulatory sequences, such as the pan-human leucocyte antigen DR-binding peptide (PADRE) sequence, a non-self Th-cell epitope being used together with other modulators or by itself (7, 80, 81). The vaccine formulation demonstrated evidence of induction of an Aβ-specific immune response without the undesired cytotoxic response.
Some epitope vaccines are obtained from the fusion of Aβ with immunomodulatory sequences such as PADRE, which are either attached to adjuvants or incorporated into chimeric vaccines, such as virus-like particles. The formulation shows good immunogenicity, induction of humoral immune response, and Th2 modulation (58, 82, 83). Vaccines based on recombinant viruses encode an Aβ-specific epitope. However, they are costly and may have adverse effects due to the generation of antibodies with altered epitope specificities (84).
The prime-boost approach seeks to enhance the immune response by administering priming doses (like synthetic peptides) followed by booster doses (like DNA vaccines). This delivery approach facilitates the expansion and selection of B cells with a high degree of affinity for the target gene. Further, the initial boost stimulates T-cell generation, while the second boost activates regulatory T cells that help in the Aβ-specific T-cell-mediated prevention of autoimmune reactions (85, 86).
Passive immunotherapy involves the administration of preformed antibodies to stimulate the immune system. These antibodies are either derived from humanized murine monoclonal antibodies (mAbs) or naturally occurring polyclonal antibodies obtained from various young healthy donors (intravenous immunoglobulin [IVIG]). Humanized mAbs are derived from non-human sources and have their protein sequences modified to increase similarity with naturally produced human antibodies, whereas fully human mAbs are obtained using phage display or transgenic mice to avoid the side effects of human antibodies (93). Unlike active immunotherapy, passive immunotherapy ensures consistent antibody titer volumes (through infusion of known amount of antibody) and rapid antibody clearance. Drawbacks of the therapy include repeated infusion of antibodies, high cost of production, BBB penetration, proper selection of antigen targets, and generation of an immune response to the injected antibodies (67,94). The antibodies injected into human subjects have different modes of action based on their antigenic targets (Table 3).
Table 3. Summary of monoclonal antibodies (mAbs) with their targets and current statuses
Mechanisms of Action of Anti-Aβ Monoclonal Antibodies
Monoclonal antibodies (mAbs) originate from a single clone of a unique parent cell and bind to a single epitope given their monovalent affinity. For the treatment of AD, various mAbs have been designed to target various epitopes of Aβ species (95) and are administered either subcutaneously or through intravenous infusions.
Monoclonal antibody action begins with binding to a specific antigenic epitope, which triggers an effector function mediated by the Fc portion of the mAb (96). While one hypothesis suggests that mAb binding to amyloid initiates a cascade of processes resulting in complement activation and macrophage-mediated phagocytosis, another suggests that the peripheral sink leads to the efflux of Aβ from the CNS (see mechanism of anti-Aβ antibody and Figure 2 and 3). However, the first hypothesis is based on the assumption that mAbs enter the CNS in sufficient amounts and enhance the phagocytic action of resident microglia or infiltrating monocytes (97). This hypothesis is not widely acknowledged because only 0.1% of the mAbs cross the BBB; the failures of these agents can be linked to poor CNS penetration (67). A novel approach targets receptors on the BBB to induce active transport of the antibodies into the CNS or deliver the gene encoding the antibodies (98).
A recent approach for mAbs is targeting pyroglutamate-3 Aβ, which may be considered as a seed of Aβ aggregation owing to its neurotoxicity and resistance to degradation (93). A preclinical study showed that passive immunization with mAbs reduces plaque deposits while minimizing vaccination side effects (99,100). Another approach involves targeting the N-terminus of Aβ, which could be the most effective way of removing aggregated Aβ (98).
Figure 3. Alzheimer’s disease immunotherapy or anti-Aβ vaccines associated adverse effects
Monoclonal Antibodies in Clinical Trials
Bapineuzumab was the first mAb developed for passive immunotherapy in AD; it entered testing after the failure of the AN-1792 trial. It is a humanized mAb (IgG1) targeting the Aβ N-terminus (Aβ1-5), which binds to and clears fibrillar Aβ42 as well as amyloid plaques. A 12-month, phase 1, single ascending dose trial of 0.5, 1.5, or 5 mg/kg of bapineuzumab showed safety and tolerability in patients with mild-to-moderate AD (101). The phase 2 study involved intravenous administration of either 0.15, 0.5, 0.1, or 2 mg/kg of bapineuzumab in 124 patients with the same form of AD; vasogenic edema was recorded, especially among APOEε4 carriers (102). APOEε4 is one of the alleles of polymorphic apolipoprotein E involved in cholesterol metabolism. It is associated with an increased risk of late-onset AD and Aβ production (103). Given differences in the incidence of vasogenic edema between APOEε4 carriers and non-carriers, phase 3 trials included separate protocols for the two. The mAb was intravenously administered to 2452 patients with mild-to-moderate symptoms in two 18-month phase 3 trials. The results of the two large, multi-center, randomized, double-blind, placebo-controlled, parallel group phase 3 studies did not match the expected outcomes, which were largely negative. Although there was a small reduction in the CSF tau level, there were no significant differences between the bapineuzumab-treated groups and placebo-treated control group (104). The adverse effects included significant vasogenic edema and intracerebral microhemorrhages, referred to as amyloid-related imaging abnormalities with parenchymal edema (ARIA-E) and hemorrhage (ARIA-H), respectively. These conditions could be detected by MRI even when lower doses were administered to APOEε4 carriers (105). Other adverse effects include neuropsychiatric and gastrointestinal symptoms, headache, and confusion. The bapineuzumab trial was terminated because of these side effects and lack of clinical efficacy (94, 106).
Solanezumab (Eli Lilly) is another humanized IgG1mAb that binds to monomeric, soluble, and toxic Aβ species at the mid-region of the peptide (Aβ16-26) (107). After its apparent success in improving cognitive deficits in transgenic mice, a phase 1 trial of solanezumab at doses of 0.5, 1.5, 4.0, or 10.0 mg/kg in 19 patients with mild-to-moderate AD and healthy volunteers showed good tolerability without any MRI evidence of microhemorrhage, vasogenic edema, or inflammation (108, 109). No adverse events were observed in a multiple-dose study involving 33 patients with mild-to-moderate AD taking 400 mg/month intravenous solanezumab. However, pharmacodynamic biomarker studies showed changes in plasma and CSF levels of Aβ40 and Aβ42. The phase 2 study involved administering 100–1600 mg/month of solanezumab to patients with mild-to-moderate AD. The drug showed a good safety profile and adequate tolerability even at high doses; although the dose-dependent increases in Aβ (Aβ40 and Aβ42) levels in the plasma and CSF indicate mobilization of Aβ from the senile plaques in the brain, there was no change in cognitive function (110). Double-blind, placebo-controlled phase 3 studies (EXPEDITION-1 and EXPEDITION-2) were conducted with over 2000 patients with mild-to-moderate AD who were administered a drug dose of 400 mg/month. Subgroup analysis in the EXPEDITION-1 trial revealed a 34% reduction in cognitive decline in patients with mild AD. The incidences of ARIA-E and ARIA-H in the treatment and placebo groups across the two studies were not significantly different (107). Consequently, Lilly launched the phase 3 (EXPEDITION-3) trial on 2100 patients with brain amyloid burden and mild AD. Although the secondary outcome of this trial slightly favored the drug, solanezumab had no effect on Aβ and tau PET biomarkers; therefore, its development was discontinued (111). Nonetheless, given the drug’s good safety profile and encouraging performance in mild AD cases, it was considered as a candidate in two secondary prevention studies. One prevention study was conducted by the Dominantly Inherited Alzheimer’s Network Trials Unit (DIAN-TU) in 2012. The study was targeted at 210 asymptomatic and very mildly symptomatic carriers of APP, PSEN1, and PSEN2 mutations. The study began as a two-year, phase 2 biomarker study and later proceeded to phase 3 registration with endpoint measurement of cognition after 4 years of treatment. The dose was 400 mg/month initially, which was increased to 1600 mg/month halfway through the trial. However, as the trial did not meet its primary endpoint and there was no reasonable treatment-related change on the DIAN multivariate cognitive endpoint, it was considered to have failed (112).
The other prevention study was initiated by the Alzheimer’s Disease Cooperative as a three-year trial in February 2014. The study recruited 1150 very mildly symptomatic or asymptomatic patients (age: 65 years or more) and investigated biomarker-based evidence of brain amyloid deposition. Solanezumab or placebo was administered intravenously once every four weeks and the drug dose was increased from 400 to 1600 mg/month in June 2017. This trial is expected to continue until mid-2020 (113). Therefore, solanezumab is being evaluated for treatment in patients with mild AD and for prevention in cognitively normal individuals at risk of AD (NCT02008357) and those with familial AD mutations (NCT01760005) (114, 115).
Gantenerumab (Hoffman-La, Roche/Ganentech) is the first fully human anti-Aβ mAb(IgG1) that binds specifically to the fibrillar form of Aβ (116). Gantenerumab is a conformational protein that binds to epitopes expressed on Aβ fibrils at the N-terminal (3-12) and central (18-27) amino acids of Aβ. Therefore, the antibody shows a higher affinity for Aβ oligomers and fibrils than for Aβ monomers (116). Gantenerumab significantly reduced Aβ plaques in transgenic mice by mobilizing microglia and hindering the formation of new plaques without altering plasma Aβ levels (116).
Four phase 1 trials in 308 patients conducted internationally showed safety, tolerability, and reduction in brain amyloid plaques in a dosage-dependent manner; however, ARIA remains a major concern. A phase 2 trial was started by Roche in 2010, consisting of 360 participants receiving subcutaneous gantenerumab injections (105 or 225 mg). The study was later expanded into a multinational, 159-center, phase 2/3 registration trial called SCarlet RoAD and recruited 799 participants. Double-blind, placebo-controlled, phase 2/3 studies conducted on Aβ-PET-positive patients with prodromal AD were terminated due to lack of efficacy and incidence of ARIA that increased in a dose-dependent and APOEε4 genotype-dependent manner; this was subsequently converted to an open extension study (117). Participants of SCarlet RoAD who became part of the open-label extension study were administered up to 1200 mg subcutaneous gantenerumab, and slow titration resulted in less ARIA-E (118). Although the open-label trial was set to continue until July 2020, it was later reported to have failed futility analysis (119). GRADUATE-1 and GRADUATE-2 are two new phase 3, double-blind, placebo-controlled studies initiated in 2018, each with the goal of recruiting 760 patients with Aβ pathology and prodromal-to-mild AD; the target enrollment was later raised to 1016 in 2020 (14). The participants will be administered up to 1020 mg subcutaneous gantenerumab or placebo for 2 years. The trial might be completed in 2023. Gantenerumab together with solanezumab are being tested by DIAN-TU for prevention of AD in a phase 2/3 trial for 210 individuals at risk of AD due to autosomal-dominant APP, PSEN1, and PSEN2 mutations (114, 115). The researchers increased the dosage of the two drugs and began a two-year, phase 2 biomarker study. The trial failed to meet its primary endpoint as gantenerumab did not provide reasonable treatment-related changes on the DIAN multivariate cognitive endpoint (114).
Crenezumab (Genetech/Hoffman-La Roche) was obtained from a mouse antibody and modified to a novel human IgG4mAb that binds to pentameric oligomeric forms of Aβ oligomers, plaques, and fibrils. It also promotes disaggregation while hindering aggregation (120). Phase 1 studies in patients with mild-to-moderate AD reported no case of ARIA-E in either single dose or multiple ascending doses (121). A phase 2, double-blind, placebo-controlled study of patients with mild-to-moderate AD did not report sufficient efficacy (122). A smaller phase 2 imaging study (BLAZE) also failed to show cognitive or clinical benefits of the drug, while a double-blind, placebo-controlled phase 1b trial reported adverse effects like ARIA-H. Currently, a phase 3, double-blind, placebo-controlled study (NCT02670083) is being conducted on Aβ-PET-positive patients with prodromal-to-mild AD to evaluate higher doses of crenezumab (39). As part of the API, crenezumab has also been tested in secondary prevention trials in cognitively normal PSEN1 mutation carriers from the world’s largest early-onset AD kindred in Columbia (NCT01998841) (123).
Ponezumab (Pfizer Inc.) is a humanized IgG2mAb designed to recognize the C-terminus of Aβ40 (Aβ30-40) (124). It elicits lower immune effector functions than IgG1. Although phase 1 trials showed a good safety profile without evidence of ARIA, CSF antibody levels were poor. The development of ponezumab was halted when two consecutive phase 2 studies revealed no clinical efficacy (125).
BAN2401 (Biogen/Eisai) is a humanized IgG1mAb that selectively binds, clears, or neutralizes the large soluble Aβ protofibrils. A multi-center phase 1 trial comprised a randomized, double-blind, placebo-controlled study to assess the safety, tolerability, pharmacokinetics, immunogenicity, and pharmacodynamic response to repeated intravenous infusions of BAN2401 (up to 10 mg/kg every 2 weeks for 4 months) in 80 subjects with mild AD and mild cognitive impairment due to AD. While the tolerability of BAN2401 at all the tested doses was good, dosage-dependent increases in ARIA-H and ARIA-E were observed in the treatment and placebo groups. Although the serum elimination half-life was short (7 days) and there was no clear effect on CSF biomarkers, the antibodies entered the CSF and showed dose-dependent exposure (126). A phase 1/2a study of the drug showed adequate tolerability with no cases of ARIA-E (127). Consequently, an 18-month phase 2b trial recruited 856 participants with prodromal-to-mild AD to evaluate the safety, tolerability, and efficacy of BAN2401 at five different intravenous dosages. The study revealed a 47% reduction in cognitive decline and a 93% reduction in brain amyloid with the highest antibody dose (10 mg/kg) administered twice monthly. MRI reports in the highest dose group revealed ARIA in only 10% of the participants and in less than 15% of those with ApoE4 (128). Eisai began a phase 3 trial known as Clarity AD in March 2019, and enrolled 1566 patients with early symptomatic AD across 250 sites in the world. Participants will receive 10 mg/kg drug or placebo every 2 weeks for a period of 18 months, followed by a two-year open-label extension. Changes in Clinical Dementia Rating Scale Sum of Boxes (CDR-SB) at 18 months and the brain amyloid subscale constitute the primary and secondary outcomes, respectively. The trial will continue till 2024. The Alzheimer’s Clinical Trial Consortium began a large BAN2401 phase 3 study, co-funded by Eisai and National Institute of Health (NIH) (AHEAD 3-45). The trial was expected to start in July 2020 and recruit 1400 people who would be divided into two sub-studies. A3 will consist of 400 participants with sub-threshold amyloid levels, and BAN2401 (5 mg/kg titrated to 10 mg/kg) or placebo will be administered every month for 216 weeks; changes in brain amyloid PET at week 216 will constitute the primary outcome. A45 will comprise 1000 participants with amyloid-positive PET scans; BAN2401 (titrated to 10 mg/kg) will be administered at two-week intervals for 96 weeks, followed by a dose of 10 mg/kg every 4 weeks for 216 weeks. A change from baseline in the Preclinical Alzheimer Cognitive Composite 5 score at week 216 constitutes the primary outcome, while changes in brain amyloid PET and cognitive function constitute the secondary outcomes (128).
Aduhelm (Neurimmune/Biogen) is another fully human IgG1mAb that selectively binds to soluble Aβ aggregates and insoluble fibrils (129).The drug was developed by screening libraries of B-memory cells from healthy elderly individuals for reactivity against aggregated Aβ. The analog of aducanumab has been shown to cross the BBB in transgenic mice; dose-dependent reductions in soluble and insoluble Aβ have also been observed in mice (129). A 12-month phase 1b trial conducted on patients with Aβ-PET-positive prodromal-to-mild AD showed evidence of a dose- and time-dependent reduction in brain fibrillar Aβ. However, the ARIA-E incidence among APOEε4 carriers was high (129). Two identical 18-month phase 3 studies were launched based on the success of the phase 1b trial. These trials sought to evaluate the efficacy of monthly doses of aducanumab in improving cognitive and functional abilities. Although only the data related to doses of 1, 3, and 10 mg/kg were reported, the drug appeared to reduce decline in a dose-dependent manner. Exploratory analyses showed that instances of ARIA-E increased with ApoE4 carriage and dosage (55% inApoE4 homozygotes at 10 mg/kg); these instances occurred in the initial phase of the trial and were later resolved (130).
The development study on patients with mild-to-moderate AD began in Japan in May 2015, with a phase 1 trial of increasing doses up to 6 mg/kg. Later, a phase 3 trial with two efficacy trials was initiated:221AD301ENGAGE and 221AD302EMERGE. 221AD301 ENGAGE enrolled 1350 patients with mild AD or mild cognitive impairment due to AD, as determined by a positive amyloid PET scan. The study, set to continue until 2022, aimed at comparing placebo with monthly infusions of one of the three doses of aducanumab over a period of 18 months. 221AD302 EMERGE, identical to ENGAGE, was conducted at 131 sites in North America with 1350 additional patients. In 2016, Biogen published and presented PRIME data, indicating that a dose titration schedule mitigated ARIA-E and announced its usage in phase 3 (131). However, in March 2019, Biogen and Eisai announced a plan for termination of all aducanumab trials based on an interim analysis that suggested that ENGAGE and EMERGE would miss their primary endpoints; the drug was subsequently removed from the pipeline (132). Interestingly, in October 2019, Biogen faulted the futility analysis and subsequent analysis showed that EMERGE achieved its primary endpoint. Although ENGAGE did not meet the primary endpoint, some exploratory analysis suggested a slow decline in the subgroup that received 10 or more doses of 10 mg/kg.
Following some interactive sessions with the Food and Drug Administration (FDA), Biogen announced plans to apply for regulatory approval of aducanumab in the US and to re-engage eligible patients from the EMERGE, ENGAGE, and PRIME trials with renewed dosing and observations (133).
In January 2020, Biogen launched a phase 3b open-label study called EMBARK, targeting 2400 previous aducanumab trial participants who will receive monthly injections of 10 mg/kg for 2 years. EMBARK has the same endpoints for efficacy as EMERGE and ENGAGE, while biomarker endpoints consist of tauPET, amyloidPET, volumetric MRI, and CSF in a subset of participants. The study is expected to end in 2023.
Biogen submitted the license application in July 2020, demanding priority review (134), and later applied for approval in Japan and the European Union. In November 2020, the FDA advisory committee cited weaknesses in efficacy and voted against approval, while recommending a confirmatory trial. In April 2021, the committee renewed its argument against approval with complaints from public citizens (135). Ultimately, the FDA approved aducanumab in June 2021 under its accelerated approval pathway that requires reasonable likelihood of a meaningful clinical benefit, substantial evidence of effect on an intermediate marker, and phase 4 evidence for such a benefit to be gathered in a subsequent trial after the marketing license has been granted (136).
Intravenous Immunoglobulin (IVIG)
IVIG is closely related to passive immunotherapy. It involves the intravenous administration of naturally occurring polyclonal antibodies obtained from the plasma of thousands of healthy young donors. IVIG has already been used as replacement therapy in various clinical conditions, such as certain forms of cancers, immunodeficiency syndromes, and hematological and autoimmune disorders. IVIG primarily contains IgG antibodies, only about 0.5% of which bind to Aβ. The use of IVIG as a potential treatment for AD began in 2002 when human pooled antibodies were shown to have strong affinity for Aβ fibrils and neurotoxic oligomers, while weakly interacting with its monomeric form (16, 137). Moreover, IVIG has some immunomodulatory effects pertinent to the treatment of AD. Early trials of IVIG revealed some benefits in reducing cognitive decline, paving the way for further studies. A phase 2 open-label IVIG trial revealed symptomatic benefits, and a futility study of Gammagard IVIG (Baxter) conducted on patients with mild-to-moderate AD showed positive cognitive scores (25). Baxter and the US funded the phase 3 trial of Gammagard IVIG to determine its efficacy and safety among patients with mild-to-moderate AD. Baxter announced that the primary endpoint for this study was not achieved; the trial has now been discontinued (138).
Grifols conducted a pilot study that involved plasma removal and replacement with Albutein in seven patients with mild-to-moderate AD (7). This procedure was performed twice weekly with a follow-up period of 6 months. Grifols concluded that this is a feasible approach for AD treatment. In 2017, Grifols conducted a phase 2 trial using the same approach and measured similar parameters as the pilot, involving 20 sham-treated and 19 actively treated patients with mild-to-moderate AD. A sawtooth pattern for plasma Aβ40/Aβ42 was seen in the treatment group, while both groups showed similar incidence of adverse events. Grifols’ recent Alzheimer Management by Albumin Replacement (AMBAR) study was a multi-center, randomized, double-blind, placebo-controlled study involving 496 patients with mild-to-moderate AD treated for 14 months. This approach is under phase 3 trial in Europe, while a phase 2 trial in the US is investigating the effect of plasmapheresis with albumin replacement and IVIG. The treatment groups were divided into a sham-treated control group and three treatment groups: plasmapheresis with albumin replacement, plasmapheresis with low dose albumin and IVIG, and plasmapheresis with high-dose albumin and IVIG (139). Changes in the Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) and Alzheimer’s Disease Cooperative Study-Activities of Daily Living (ADCS-ADL) scores between baseline and the endpoint constitute the primary outcome measures. The secondary measures include changes in functional, cognitive, and behavioral tests; measures of disease progression; changes in CSF total tau, p-tau, Aβ40, and Aβ42 levels; changes in plasma Aβ40 and Aβ42 levels; and changes in brain structure and brain glucose metabolism. Subjects in the treatment groups showed 50–75% less worsening of ADAS-Cog scores and 42–70% less worsening of ADCS-ADL scores than control subjects. In addition, pooled data from treated subjects showed that the average decline in ADAS-Cog and ADCS-ADL scores in the treatment group were 66% and 52% lower, respectively, then in the control group. Although some patients with mild AD showed slower disease progression, sham-treated patients with mild AD unexpectedly showed a similar pattern. Grifols reported significant differences in memory, processing speed, quality of life, and language between the control and high albumin/high IVIG treatment groups. Moreover, actively treated patients with moderate AD demonstrated better memory and quality of life than their sham-treated counterparts. Similarly, actively treated patients with mild AD showed better outcomes in language and processing speed tasks than their control counterparts. However, some instances of mild adverse events were noted during high-volume plasma exchange. While the outcomes of AMBAR are promising, some important gaps need to be addressed: mechanism(s) leading to reduction in disease progression; effectiveness of the approach in mild AD as in moderate AD; necessity of including IVIG in the protocol; and how ApoE genotype, age, and sex influence the treatment response (140).
Adverse effects of anti-Aβ vaccines
Importantly, both passive and active Aβ immunization elicit CNS inflammation, and can also induce cerebral microhaemorrhage and vasogenic oedema in the already inflamed milieu (141). With the administration of vaccine/antibodies against Aβ, many factors have led to compromised efficacy of immunotherapy in clinical trials. These adverse effects include brain cerebral amyloid angiopathy (CAA), microhemorrhage and meningoencephalitis, which have led to the suspension of clinical trials (Figure 3). Furthermore, patients with AD have well-established neurotic plaques, which are obstacles for a successful vaccine-mediated immune response. Aβ peptide production leads to the activation of the innate immune response marked by activated microglia and elevated levels of complement protein, together they are known to release chemokines and proinflammatory cytokines. Moreover, endogenous sugars can modify Aβ fibrils to advanced glycation end products (AGEs), resulting in proinflammatory signal transduction pathways pertaining to the overproduction of reactive oxygen species and upregulation of AGE receptors. These pathological events constitute a secondary inflammatory response to the early aggregation of Aβ peptides. When the vaccine is administered, the Aβ–antibody complex activates the complement system and microglia, eliciting inflammation in the CNS. Furthermore, activation of T-lymphocytes triggers an adaptive immune response. T-lymphocytes insinuate the brain parenchyma and damage the neural tissue, which is the primary cause of aseptic meningoencephalitis reported in many immunotherapy clinical trials. Moreover, mobilization of Aβ plaques may be an additional concern. As Aβ species cross the BBB, there is a potential risk of neurotoxicity from the brain to the periphery (141). Aβ monomers readily aggregate into oligomers and then into fibrils with β-pleated sheet structures. Aβ oligomers are reported to be more neurotoxic than other Aβ species. Aβ toxicity can be reduced by targeting Aβ oligomers in the early stages rather than plaques. Additionally, current clinical trials based on Aβ-based immunotherapies target Aβ aggregates and do not affect the amount of soluble Aβ. An AN1972 active immunization study reported that increased concentrations of detergent-soluble and water-soluble forms of Aβ in the brain are linked to reduced Aβ plaque load. A series of events such as this aids the formation of Aβ oligomers, which may cause damage to neurons during Aβ clearance (141). This effect of immunotherapy is a significant safety concern and must be investigated.
Conclusion and Future Perspectives
After a decade of disappointment in AD prevention through vaccination against Aβ, some vaccine candidates have entered phase 3 clinical trials, while other approaches are in preclinical trials. One of the challenges related to vaccination is its timing. It is now clear that vaccination must start early because plaque removal at later stages does not curtail the progression of the disease, possibly due to progressive tau aggregation. Thus, amyloid removal at pre-symptomatic stages can avert the clinical onset of AD. However, determining the appropriate time for commencement of vaccination is quite challenging. Another point of concern in vaccination is anti-Aβ specificity and antibody titer volumes. Therefore, future studies should evaluate the appropriate timing for vaccination, pathogenic Aβ specificity, and optimization of the titer for antibody response.
Currently, passive immunotherapy appears more promising than active vaccination. The recent approval of aducanumab by the FDA, albeit with some controversies, demonstrates the potential of passive immunotherapy. One of the advantages of passive immunotherapy is that mAbs are amenable to dose and specificity modulation. However, the challenges of short-term antibody effects, low improvement in cognition, and instances of ARIA constitute bottlenecks that need to be addressed.
Given the complex pathophysiology of AD, it is necessary to re-strategize future research in both active and passive immunotherapy. Combination therapy may help in targeting tau protein and Aβ protein, while specific formulations may be beneficial in individuals with specific APOE genotypes, immune phenotypes, and/or Aβ strains. Thus, considering inter-individual differences could improve the prospects of immunotherapeutic prevention of AD.
Author’s contributions: NKJ and SO conceptualized the study and hypotheses. MBU, SB, SR, DK, AA and SKS performed literature search. NKJ draw the schemes and drafted the artwork. GG, DKC, KD, JR and KKK drafted the tables. NKJ, and other authors contributed significantly in editing the manuscript. PK, RKA, PP, SS, VU, FAK, RA, SKJ and MDS significantly contributed during revision. All authors read, edited and approved the manuscript.
Acknowledgements: The authors would like to express their gratitude to the unknown referees for carefully reading the paper and giving valuable suggestions.
Conflict of Interest: The authors declare that they have no conflict of interest.
Consent for publication: All authors have read the final version of the manuscript and have given their consent for publication.
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