Abstract
It has been a year since we lost Dale Schenk on September 30, 2016. Dale’s visionary work resulted in the remarkable discovery in 1999 that an experimental amyloid-β (Aβ) vaccine reduced the neurodegeneration in a transgenic model of Alzheimer’s disease (AD). Following Dale’s seminal work, several active and passive immunotherapies have since been developed and tested in the clinic for AD, Parkinson’s disease (PD), and other neurodegenerative disorders. Here we provide a brief overview of the current state of development of immunotherapy for AD, PD, and other neurodegenerative disorders in the context of this anniversary. The next steps in the development of immunotherapies will require combinatorial approaches mixing antibodies against various targets (e.g., Aβ, α-syn, Tau, and TDP43) with small molecules that block toxicity, aggregation, inflammation, and promote cell survival.
INTRODUCTION: DALE SCHENK A LIFE IN SCIENCE FIGHTING FOR THE CURE
It has been a year since we lost Dale Schenk on September 30, 2016 after a two-year battle with cancer. Dale belonged to that generation of scientists that heralded a new era on research and therapeutics for neurodegenerative disorders. But Dale was also a musician, loyal colleague, and a beloved friend and family man who, with an amazing sense of humor and enthusiasm, brought out the best in people. He was always kind, positive, and generous as a person and as a scientist.
He received his Bachelor’s degree in Biology in 1979 and his Doctorate degree in Pharmacology and Physiology from the University of California, San Diego (UCSD) in 1984. He first accepted a scientist position at Scios Inc. in California and in 1987 joined Athena Neurosciences in San Francisco, where he remained as Chief Scientific Officer when the company was acquired by Elan in 1996. At a time when there were only sporadic and limited interactions between industry and academic scientists, Athena Neurosciences, under Dale’s leadership, became the leader in the field at developing collaborations that led to remarkable discoveries in the fields of Alzheimer’s disease (AD) pathogenesis [1, 2], transgenic animal modeling [3–6], and therapeutics for AD [7, 8] targeting amyloid-β protein precursor (AβPP) processing and amyloid-β (Aβ).
At this time, Dale’s visionary and innovative work resulted in the remarkable discovery in 1999 that an experimental Aβ vaccine reduced the accumulation of amyloid, neurodegeneration, and behavioral deficits in the PDAPP model of AD [1]. This work led to the testing of the first vaccine for AD targeting Aβ (AN1792) in humans. Although the Phase II clinical trial with this Aβ vaccine was stopped in 2002 due to the development of encephalitis in a handful of the patients [9, 10], Dale’s discovery propelled the field toward the new era of immunotherapy for neurodegenerative disorders for many years to come [11, 12]. Following Dale’s seminal work, several active and passive immunotherapies have since been developed and tested in the clinic for AD, Parkinson’s disease (PD), and other neurodegenerative disorders [13, 14]. Among many other honors, Dale was awarded in 2001 the American Academy of Neurology’s Potamkin Prize for his ground-breaking research in AD immunotherapeutics. Although Dale is best known for his work on immunotherapy, he was also working with his team at Elan (Fig. 1) at developing new therapeutics modulating β- and gamma-secretases [2, 15–19] as well as other pathways relevant to AD and PD. Then, in 2009 Dale became the head of Elan’s newly formed company-Neotope Biosciences where he continued in conjunction with his colleagues and friends, Dora Games (Fig. 1) and Pete Seubert, and in collaboration with our group at UCSD the work on immunotherapy with a renewed interest in PD and other degenerative disorders of the aging population. Afterwards in 2012, Dale co-founded Prothena, a spin-off of Neotope Biosciences, where he continued the experimental and clinical development of the passive immunization for PD targeting α-synuclein (α-syn) (PRX002), as well as immunotherapy programs for systemic amyloid disorders such as AL amyloidosis (NEOD001) and ATTR amyloidosis (PRX004) and inflammatory disorders.

Dale with Elan and later on Prothena colleagues Kelly Wood-Johnson and Dora Games (Photo courtesy of Dr. Lisa McConlogue, friend and colleague of Dale).
Fifteen years after Dale’s discoveries harnessing the immune system to target Aβ for the treatment of AD, there are currently several active and passive immunization programs under development targeting α-syn for PD and multiple system atrophy (MSA); tau for AD and frontotemporal dementia (FTD); TDP-43 for FTD; Htt for Huntington’s disease; and PrPsc for Creutzfeldt-Jakob disease among others. In the next sections, we will provide a brief overview of the current state of development of immunotherapy for AD and Lewy body disease (LBD).
NEW HOPE FOR FINDING TREATMENTS FOR NEURODEGENERATIVE DISORDERS
AD [20], LBD [21], and FTD [22] are incurable neurodegenerative disorders of the aging population characterized by the progressive accumulation of misfolded protein aggregates (e.g., Aβ, α-syn, tau, TDP-43) that initially trigger synaptic damage and network dysfunction, and that eventually lead to loss of selected neuronal populations [23, 24]. Clinically they are characterized by cognitive impairment and memory loss (progressing to dementia), behavioral alterations and motoric dysfunction [20], and LBD [21]. Prior to Dale’s work on immunotherapy in the late 1990s, there were very few or no disease modifying experimental therapies considered or under clinical testing for neurodegenerative disorders; now there are dozens of clinical trials testing immunotherapy targeting Aβ, α-syn, and tau.
Dementing neurodegenerative disorders are the cause of suffering for patients and caregivers; AD is currently considered a major medical problem affecting over 5 million in the US, and close to 35 million people worldwide with numbers projected to rise to 115 million by 2050 [25, 26]. AD is the 6th cause of death in the US, and AD and related dementing disorders (ADRD) that include LBD, FTD, and vascular dementia, are the only top causes of death for which no prevention or cure is currently available [27]. For this reason, the US as well as other countries have developed National plans to address this public health emergency [28, 29]. In the US, on January 4, 2011, President Barack Obama signed into law the National Alzheimer’s Project Act (NAPA) (Public Law 111-375). The Act defines “Alzheimer’s” as AD and ADRD and calls as a major goal to accelerate the development of treatments that would prevent, halt, or reverse the course of AD/ADRD by the year 2025. The law also requests for: 1) the creation of National Plan that is updated yearly; 2) coordination of AD research and services across all federal agencies; 3) improved early diagnosis and coordination of care and treatment of AD; and 4) coordination with international bodies to fight AD globally (https://aspe.hhs.gov/national-alzheimers-project-act), among other activities. Moreover, the US Congress appropriated an additional $350 million dollars for Fiscal Year (FY) 2016 and $400 million dollars for FY2017 to the National Institute on Aging (NIA) to accelerate research on AD/ADRD. The NIA and its sister Institutes and Centers at the National Institutes of Health use the milestones and recommendations from the AD (2012 and 2015) and ADRD (2013 and 2016) Summits held at the NIH in Bethesda, MD, to prioritize areas of research for support.
While in AD, Aβ and tau accumulate in various cortical regions [30], in LBD, which is an heterogeneous group of disorders that includes PD, PD dementia (PDD), and dementia with Lewy bodies (DLB) [31–33], the synaptic protein α-syn accumulates in cortical and subcortical nuclei [34, 35]. LBD in combination with other disorders with α-syn accumulation, such as MSA, are jointly referred to as synucleinopathies [36–38]. In FTD, aggregates of either tau, TDP-43, or Fused in sarcoma (FUS) are found [39, 40]. Moreover, in FTD cases with the GGGGCC expansion mutation located in the intron 1 of the C9ORF72 gene, there is accumulation of TDP43 and repeat-associated non-ATG (RAN) translation proteins [41]) in the affected brain regions. Moreover, α-syn can accumulate in selected brain regions in AD [42] and that TDP-43 aggregates are found in the limbic system in AD and DLB [43]. These findings reinforce the idea that abnormal protein accumulation is key in most neurodegenerative disorders.
Aβ is well known to be secreted and to accumulate extracellularly [44], while α-syn, tau, and TDP-43 accumulate primarily intracellularly [45]. However, recent evidence has shown that aggregates of these proteins can be secreted [46–48] and transmit from cell to cell both in neuronal and non-neuronal cells [48–51]. This is important because the extracellular protein aggregates can be targeted with antibodies (Fig. 2). Under normal conditions most of these proteins can be found as soluble monomers in the cytoplasm or as dimers or tetramers associated with the plasma membrane [52–54]. However, under pathological situations such as those characterizing AD, LBD, and FTD, various forms of these protein arrays including small oligomers, larger oligomers, protofibrils, and fibrils [55–59] can be detected both intracellularly and as well as in the extracellular environment where it can transmit from cell to cell [60–65], providing a strong rationale for the development of immunotherapeutic approaches for AD, LDB, and FTD (Fig. 2).

Mechanisms of action of immunotherapy. Antibodies against Aβ, tau, and α-syn work via multiple mechanisms including targeting aggregates in the membrane, reducing oligomerization and fibrillization, enhancing clearance via autophagy, ESCRT and microglia, preventing cell-to-cell propagation, reducing neuro-inflammation, and promoting neuronal survival.
Examples of immunotherapy under consideration for AD, LBD, and FTD includes, active, passive and T cell based approaches. Active immunization (or vaccination) seeks to promote the immune system (self) to generate antibodies against target proteins such as Aβ, α-syn, tau, and TDP-43, while in passive immunization pre-prepared antibodies against these proteins are administered (Fig. 2; Table 1). A different type of immunotherapy involves the activation of T cells that are able to modulate inflammatory responses; this approach has also been investigated for AD and PD [66, 67] (Fig. 2). While the upside is that antibodies can be designed to target specific proteins and conformations of Aβ, tau, α-syn, or TDP-43 and have multiple mechanisms of action [68–73], the downside is the potential for non-specific inflammatory or immune reactions and limited penetration into the CNS. Active and passive immunization strategies are being explored in several clinical trials for AD and synucleinopathies (Table 1). Cellular immunization with activation of modulation of cells are under consideration for AD [74] and PD [75] although they are at earlier stage of development compared to antibody therapy.
Examples of immunotherapy clinical trials for AD, tauopathies, and α-synucleinopathies
AD, Alzheimer’s disease; DS, Down syndrome; FTD, frontotemporal dementia; PD, Parkinson’s disease; MSA, multiple system atrophy.
IMMUNOTHERAPY FOR AD AND TAUOPATHIES
The main targets for immunotherapy in AD are Aβ and tau, although recent studies have shown that α-syn [76] and TDP-43 [43, 78] also participate in some aspects of AD and as such are potential targets as well. In addition to AD, tau also accumulates in other neurodegenerative disorders such as progressive supranuclear palsy, Pick’s disease, cortico-basal degeneration, and FTLD-14. The most developed clinical studies are those on Aβ immunotherapy, for which at least two active immunization and five passive immunization trials are currently undergoing. For tauopathies, two vaccines and three antibodies are currently in Phase I studies (Table 1). For α-syn, there are two active immunization and one passive immunization trials that have completed Phase I and are now moving toward Phase II; these clinical studies are centered at PD and MSA (Table 1). Moreover, there is one immunotherapy trial targeting the AD pathology in Down syndrome.
As described in previous sections, in the late 1990s Dale pioneered using immunotherapy for neurodegenerative disorders and in particular AD. As a result of his work, Dale developed an active immunization approach by vaccinating APP transgenic (tg) mice with Aβ1 - 42 (AN-1792) and adjuvant [1]. Clinical trials in patients with mild to moderate AD with AN-1792 (full length Aβ1 - 42) highlighted the risk of autoimmune responses when using this type of therapeutic strategy, as 6% of the patients in that study suffered with meningoencephalitis associated with T cell infiltration [79]. However neuropathological studies showed a considerable removal of amyloid in various brain regions [7, 80]. Current vaccines under development for AD includes CAD106 [81], Vanutide cridificar [82], and AD02 (AFFITOPE®) [83]. Unfortunately, patients with mild to moderate AD treated with AD02, a synthetic peptide that mimics the N-terminus structure of the Aβ peptide, did not showed significant improvements. The CAD106 vaccine contains an Aβ1–6 fragment as the immunogenic sequence attached to a carrier formed from the coat protein of bacteriophage Qβ as an adjuvant [15]. Phase II trials with CAD106 showed a good antibody response in AD patients with no side effects [26]; however, the study did not show clinical efficacy in patients with mild to moderate AD. Other active immunization trials against Aβ (Table 1) includes Lu AF20513 which consists of three repetitions of a modified Aβ1 - 12 sequence in which the natural T helper cell epitopes are engineered to reduce the possibility of inducing harmful autoreactive T cell responses and to improve the ability to mount an effective immune response [84]. ACI-24 (Europe, Phase I/II) is a liposome vaccine designed to elicit an antibody response against aggregated Aβ peptides without concomitant pro-inflammatory T cell activation [85, 86]. ACI-24 also improved novel object recognition without triggering pro-inflammatory responses [86]. Finally, with support from NIA, Phase I vaccination trials with ACI-24 are currently been conducted in adult patients with Down syndrome which are known to develop AD. The study is assessing safety, tolerability, and immunogenicity in patients aged 35–55 years. This study was developed following experimental studies in a mouse trisomy model [87].
Because of the limited effects observed with active immunization against Aβ, other immunotherapy approaches have been developed including passive immunization with antibodies targeting specific epitopes or conformations of Aβ. Early studies by Solomon and her group showed that monoclonal antibodies against Aβ reduce fibrillation in vitro. Moreover, Dale as well as other groups developed and tested in vivo antibodies against the N-terminus of Aβ [5, 90]. In this respect, his group showed that the monoclonal antibody 3D6 (which binds to deposited amyloid plaques and promotes clearance of Aβ) displayed efficacy in PDAPP mice [3, 5]. The humanized monoclonal antibody counterpart bapineuzumab, directed against the Aβ N-terminus (Aβ1–5), showed some initial promise in Phase II trials; however, subsequent parallel-group Phase III trials did not confirm the efficacy of this antibody in ameliorating the cognitive impairment in patients with AD while at the same time displaying adverse effects such as vasogenic edema [15, 27]. Interestingly, PET studies showed a reduction in Aβ accumulation in the brains of patients treated with bapinezumab (N-terminus) [91–93].
Other passive immunization approaches using monoclonal antibodies against Aβ1 - 40 [94], Aβ1 - 42 [3], pyroglutamate Aβ [95], oligomers [96], or protofibrils [97–99] have also been developed and tested in vivo. Currently, clinical trials with the antibodies BAN2401 (protofibrils) [100], crenezumab (aggregated species) [101], gantenerumab (fibrils) [102–104], and solanezumab (soluble Aβ mid-domain) [105, 106] are undergoing or have been completed (Table 1). Among them, clinical trials with solanezumab—an antibody that binds soluble Aβ systemically and probably works via a “sink” mechanism has completed Phase III in patients with mild to moderate AD. Although the primary end points were not attained in these trials, post hoc analysis showed a potential beneficial effect in patients with mild AD; subsequent studies confirmed a modest effect in cognitive scores that was not significant [107, 108]. Finally, recent studies with aducanumab (BIIB037), a monoclonal antibody binding to aggregated Aβ and derived from healthy aged donors, showed a dose-dependent (1, 3, and 10 mg/kg) reduction in amyloid deposition in six cortical regions of the brain. In a Phase II trial in 166 patients with early AD, aducanumab showed a dose dependent improvement in cognitive performance [109]. The reasons for the limited effects of passive immunization against Aβ in patients with AD are still under investigation; however, there are a number of possible pitfalls that have been proposed including: poor penetration of the antibodies, questions about antibody specificity, selecting the wrong target, insufficient Aβ reduction, or the immunotherapy should have been started earlier before substantial amyloid deposition in the brains of patients with AD occurred (too late). In fact, most of the studies described above were undertaken in patients with mild to moderate AD at a time when considerable amounts of amyloid have deposited in the brain. This emphasizes the need for clinical trials targeting earlier stages of the disease, that is primary or secondary prevention trials. In this regard and with NIA support, both solanezumab and gantenerumab are currently being tested for prevention of AD in individuals carrying autosome-dominant AD-causing mutations (adult children of parents with the familial form of the disease) as part of the Dominantly Inherited Alzheimer’s Network (DIAN) study which builds upon the results of earlier studies, suggesting effective antibody treatment of early-stage AD [15, 28]. Moreover and with NIA support, crenezumab has been investigated in a prevention trial as part of the Alzheimer Prevention Initiative (API) in individuals in the Medellin, Colombia area carrying the Presenilin 1 mutation (PS1) for which Aβ accumulation can be detected by biomarkers at around 25 years of age [15, 111]. Finally, another prevention trial is the so-called A4 study that is being conducted in older individuals who have evidence of amyloid plaque build-up in their brains and who may be at risk for memory loss and cognitive decline due to AD. The A4 study is testing the effects of solanezumab in older individuals, who do not yet show symptoms of AD clinically, with the aim of slowing memory and cognitive decline. The A4 study will also test whether solanezumab treatment can delay the progression of amyloid deposition in the brain by imaging and other biomarkers [112].
In AD and other neurodegenerative disorders of the aging population, tau accumulates in the brain [113–118]. Since cognitive alterations correlate with tau pathology [119, 120], removing tau has become a priority in AD and FTD [121, 122]. Both vaccination with tau antigens and immunization with monoclonal antibodies against tau have reduced accumulation and diminished behavioral deficits in tg mouse models of tauopathy [122–129]. Vaccination utilizing phosphorylated tau epitopes [123, 129] has displayed encouraging results in animal models, and two tau vaccines are currently in Phase I trial for AD, AADvac-1, and ACI-35 (Table 1). AADvac-1 consists of a synthetic peptide derived from amino acids 294 to 305 of the tau sequence, although the precise molecular nature of the antigen has not been disclosed. ACI-35 is a liposome-based vaccine that elicits an immune response against pathological conformers of phosphorylated tau without mounting autoimmune B cell or T cell responses against physiological tau conformations. The resulting antibodies bound pathological tau in the brain [130]. Passive immunization with anti-phospho-tau antibodies reduce tau pathology and functional deficits [124, 131–133], and antibodies targeting tau oligomers have also showed promise in tg models [123, 134], including a concomitant upstream reduction in Aβ pathology [135]. In this sense, there are 3 anti-tau antibodies currently being studied in Phase I trials (Table 1). BMS-986168 targets extracellular, N-terminally fragmented forms of tau (eTau). Finally, the recombinant humanized anti-tau antibody C2N-8E12 has recently begun a Phase I clinical study in patients with progressive supranuclear palsy.
Together, these studies suggest that passive immunization against Aβ and tau might hold promise for disease modification at earlier stages of AD, however more research is needed.
IMMUNOTHERAPY FOR SYNUCLEINOPATHIES
α-Syn is a small 140 amino acid long synaptic protein involved in synaptic transmission and vesicle release [136] that was initially identified in AD brains associated with plaque formation and neurodegeneration [137, 138] and in Lewy bodies (LBs) in PD [139]. Abnormal aggregation and accumulation of α-syn in synapses, axons, and in the neuronal cytoplasm as LBs are observed in PD and other synucleinopathies [55, 140], and therefore inhibiting α-syn aggregation would be a key mechanism for preventing its toxicity.
The first α-syn immunotherapeutic vaccine was a collaborative effort between our group at UCSD and Dale’s group at Elan, for these studies we used full length human α-syn protein and Freund adjuvant [141]. In these studies, α-syn aggregates and neurodegeneration were decreased in the α-syn tg mouse model of LBD, and antibodies isolated from the immunized mice promoted the degradation of α-syn aggregates [141]. Other active immunization approaches include using AFFITOPEs®, which mimic abnormal conformations of α-syn, have been studied in animal models of PD and MSA [72, 73]. Vaccination with the AFFITOPEs® AFF1 reduced neurodegeneration and improved motor and memory deficits in two α-syn tg models [73] and in a tg mouse model of MSA [72] and led to Phase I clinical trials with the AFFITOPEs® PD01A and PD03A for PD and MSA.
Passive immunization approaches are also being pursued using antibodies against α-syn. Antibodies that recognize a C-terminus epitope of α-syn cleared intracellular aggregates, inhibited α-syn propagation and prevented C-terminus cleavage of the protein that may lead to increased aggregation [12, 143]. Antibodies against the N-terminus were also effective at clearing α-syn aggregates, reducing their propagation and diminishing motor dysfunctions [144, 145]. Together, these reports support the value of immunotherapy with antibodies directed against α-syn for PD, and in this sense the C-terminus antibody PRX002 is currently the focus of a Phase I clinical trial (Table 1).
Since passive immunization uses immunoglobulins, which are voluminous proteins that do not easily cross the BBB and recognize a limited variety of epitopes, recent strategies have focused on using single chain antibodies (single chain variable fragment, scFv). scFvs retain antigen-binding properties and can be easily screened using phage display methodology. Using this approach, scFvs have been identified [146–148] that detect individual conformational species of α-syn and could be potentially used to discriminate among “strains” [149] to treat different synucleinopathies or for diagnostic purposes. scFvs have been further modified with the LDL domain of apolipoprotein B to increase BBB penetrability and facilitate the clearance of α-syn in a tg model of DLB. The brain-targeted fusion antibody easily crossed the BBB and was internalized by neurons using the ESCRT pathway for enhanced degradation of α-syn aggregates [146], thus attenuating neuronal degeneration in vivo. The use of gene therapy with intracellular scFv (intrabodies) is also being explored for the detection and clearance of intracellular α-syn aggregates [150–152].
Therefore, antibodies against α-syn ameliorate α-syn pathology by multiple mechanisms (Fig. 2) including reducing oligomerization and fibrillization [73, 154]. Furthermore, antibodies may also facilitate clearance of extracellular α-syn and prevent cell-to-cell propagation [49, 144], inflammation and promote cell survival. Importantly, both aggregation and cell-to-cell propagation are intimately related to α-syn toxicity and inflammation PD pathology, suggesting that these processes are promising therapeutic targets for immunotherapy. Likewise, cellular immunotherapy strategies are been developed for PD utilizing compounds such as copaxone that might block migration of cytotoxic T cells [75], although they are at earlier stage of development compared to antibody therapy. Future studies are investigating combination therapies with antibodies and T-cell mediated immunity.
CONCLUSIONS
In summary, immunotherapy strategies have been developed for AD, tauopathies, and α-synucleinopathies that are now at various stages of clinical development. The most advanced trials are those targeting Aβ; it is expected that by 2019 preliminary results might be available for the secondary prevention trials. Antibodies against Aβ, tau, and α-syn work via multiple mechanisms (Fig. 2) including reducing oligomerization and fibrillization, enhancing clearance via autophagy, ESCRT and microglia, preventing cell-to-cell propagation, reducing neuro-inflammation, and promoting neuronal survival. Next phase in the development of immunotherapies will require combinatorial approaches mixing for example antibodies against various targets (e.g., Aβ, α-syn, Tau) with small molecules that block toxicity, aggregation, and inflammation and promote cell survival [14, 155].
DISCLOSURE STATEMENT
Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/17-1071).
