**2. Aβ**

## **2.1. Mechanisms of Aβ pathophysiology**

The primary component of senile plaques is Aβ, a small peptide derived from the amyloid precursor protein (APP). In AD, Aβ is formed via sequential cleavage of APP by β-secretase [4] and the presenilin-1 (PS1) subunit of γ-secretase [5], respectively. This results in peptides of varying length, ranging from 38 to 43 amino acids [6], of which Aβ1–42 is the most amyloidogenic [7]. A central tenet in the understanding of causative factors of AD is the amyloid cascade hypothesis [8], which holds that the pathological increase of amyloidogenic Aβ in AD is a central initiating event in disease, that precedes and initiates a cascade of events that lead to other pathologies such as the formation of neurofibrillary tangles, inflammation, oxidative stress, neuronal dysfunction, and cell death [9]. While the amyloid cascade hypothesis has been challenged since first proposed [10, 11], there is abundant evidence from *in vitro* and *in vivo* studies confirming the significant role Aβ plays in inducing neurotoxicity, synaptic dysregulation, and pathology.

Degeneration of cultured neurons by treatment with aggregated forms of Aβ has been observed in multiple laboratories, and appears to correlate with extent of aggregation [7, 12]. Strong evidence indicates that soluble aggregated forms of Aβ might exert direct toxicity to neurons [13–15] through a variety of mechanisms, including (but not limited to) disruption of plasma membranes [16], dysregulation of mitochondrial function and dynamics via direct interaction [17], and excitotoxicity [18]. Confirming the centrality of Aβ's role in neurotoxicity, myriad transgenic mouse models expressing mutant APP or APP/PS1 recapitulate many AD phenotypes, including plaque pathology, synaptic dysfunction, decreased cognition, neuroinflammation, and neuronal loss (reviewed in [19]).

One of the earliest mouse models of Aβ plaque deposition was the PDAPP mouse (Line109). These transgenic mice exhibit high human APP expression (>10-fold higher than endogenous levels), which is accompanied by extracellular Aβ plaque deposition, development of neuritic dystrophy, gliosis, and loss of synaptic and dendritic structures in the hippocampus [20]. The PDAPP mouse model was instrumental to demonstrate that therapies developed to clear Aβ deposits could potentially ameliorate functional deficits. Schenk and colleagues were the first to develop an active immunization approach using aggregated Aβ1–42 [21], which resulted in prevention of plaque formation in mice immunized before the development of pathology, and more importantly demonstrated that the induced polyclonal response can promote plaque clearance in aged PDAPP mice via phagocytosis by resident microglia. This breakthrough was later extended by administering the anti-N-terminal Aβ monoclonal antibody (mAb) 3D6 directly to PDAPP mice (passive immunotherapy); antibodies crossed the blood-brain barrier (BBB), localized to pathological features, and induced the opsonization and clearance of senile plaques in a microglia-dependent manner [22]. These preclinical findings validated Aβ-directed passive immunotherapy as a potential therapeutic strategy for AD.

#### **2.2. Aβ passive immunotherapy in the clinic**

approaches, such as acetylcholinesterase inhibitors and NMDA receptor (NMDAR) antagonists, which aim to enhance the function of unaffected neurocircuitry but do not target the underlying cause of the disease, thus there is a desperate need for approved disease modify-

Alzheimer's disease is characterized by the dual pathological hallmarks of extracellular senile plaques and neurofibrillary tangles, composed of the amyloid-β (Aβ) peptide and tau protein, respectively. In addition, the primary familial forms of the disease are caused by mutations that directly affect Aβ homeostasis [2]. Due to both the pathological and genetic link to disease initiation, Aβ has been a prominent target for the development of disease-modifying

One such therapeutic approach is anti-Aβ immunotherapy. Active immunotherapy approaches utilize either the ability of the immune system to raise polyclonal antibodies against a therapeutic composed of an Aβ sequence-derived antigen and adjuvant, while passive immunotherapy approaches treat a patient with monoclonal antibodies with known antigen binding capabilities. While a large amount of research and development has been carried out regarding active immunotherapy towards AD targets [3], this chapter will focus on passive immunotherapy in AD, with the goal of describing what has been learned from

The primary component of senile plaques is Aβ, a small peptide derived from the amyloid precursor protein (APP). In AD, Aβ is formed via sequential cleavage of APP by β-secretase [4] and the presenilin-1 (PS1) subunit of γ-secretase [5], respectively. This results in peptides of varying length, ranging from 38 to 43 amino acids [6], of which Aβ1–42 is the most amyloidogenic [7]. A central tenet in the understanding of causative factors of AD is the amyloid cascade hypothesis [8], which holds that the pathological increase of amyloidogenic Aβ in AD is a central initiating event in disease, that precedes and initiates a cascade of events that lead to other pathologies such as the formation of neurofibrillary tangles, inflammation, oxidative stress, neuronal dysfunction, and cell death [9]. While the amyloid cascade hypothesis has been challenged since first proposed [10, 11], there is abundant evidence from *in vitro* and *in vivo* studies confirming the significant role Aβ plays in inducing neurotoxicity, synaptic

Degeneration of cultured neurons by treatment with aggregated forms of Aβ has been observed in multiple laboratories, and appears to correlate with extent of aggregation [7, 12]. Strong evidence indicates that soluble aggregated forms of Aβ might exert direct toxicity to neurons [13–15] through a variety of mechanisms, including (but not limited to) disruption of plasma membranes [16], dysregulation of mitochondrial function and dynamics via direct interaction [17], and excitotoxicity [18]. Confirming the centrality of Aβ's role in neurotoxicity,

past clinical studies, and what lessons may be applied to future efforts.

**2.1. Mechanisms of Aβ pathophysiology**

dysregulation, and pathology.

ing therapies.

130 Alzheimer's Disease - The 21st Century Challenge

therapeutics.

**2. Aβ**

The first Aβ immunotherapy clinical trial utilized active vaccination with Aβ1–42 (AN1792) and was halted during Phase IIa due to the appearance of meningoencephalitis, likely due to the infiltration of T-cells in the brain as a result of the presence of T-cell epitope(s) in the antigen, which contained the full-length Aβ1–42 peptide [23]. However, long-term follow-up indicated that patients that developed an immune response displayed modest but significant sparing of function, as assessed by the Disability Assessment for Dementia (DAD) and the Dependence scale [24]; in addition, autopsy of a patient immunized with AN1792 without meningoencephalitis displayed an absence of plaque pathology at autopsy and the presence of Aβ-reactive microglia, indicating that AN1792 was successful at engaging phagocytes to remove plaques [25].

Concerns for safety in active Aβ vaccination trials shifted most development efforts to passive immunotherapy, which carries less risk of an inflammatory response to drug. An overview of clinical Aβ antibody efforts described in the following text is listed in **Table 1**.

#### *2.2.1. First-generation Aβ passive immunotherapies*

Bapineuzumab, directed at the N-terminus of Aβ, was the first monoclonal antibody therapy developed to target Aβ in AD. It was first tested in a phase I study in AD patients with single ascending doses ranging from 0.5 to 5 mg/kg administered every 13 weeks to evaluate safety, tolerability, and pharmacokinetics (PK) [26]. A significant safety finding of this study was the presence of vasogenic edema (VE) in the highest-dose cohort: 3/10 patients


Association-led workgroup composed of industry and academic experts to advise the FDA on potential routes to monitor VE and microhemorrhages. The term amyloid-related imaging abnormalities (ARIA) was adopted to address the spectrum of MR imaging abnormalities observed with anti-amyloid therapies, spanning from sulcal effusion and vasogenic edema seen on FLAIR MRIs to hypointensities (hemosiderin deposits) on T2\* MRI. The ARIA terminology was further subdivided to ARIA-E (sulcal effusion and edema) and ARIA-H (hemosiderin deposits) [31]. Recommendations from the workgroup included (a) standardization of technical and monitoring practices for MRI, (b) exclusion from trials of patients with preexisting ARIA-H, and (c) monitoring of symptoms potentially associated with ARIA. The adoption of these standards, and the understanding that ARIA is largely a short-lived treatment related effect inherent to many anti-amyloid therapies, opened the possibility of testing higher and more frequent drug administration regimens with appro-

Passive Immunotherapy in Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.76299 133

In parallel with bapineuzumab, two additional anti-Aβ passive immunotherapies underwent contemporaneous clinical trials: Ponezumab, directed at the C-terminus of Aβ, underwent Phase I and IIa trials, but was discontinued after Phase IIa [32]. Solanezumab, directed at an internal epitope of Aβ and hypothesized to function by binding soluble species in the CNS and periphery, failed a phase III trial in mild AD patients [33], and a trial conducted in prodromal patients was discontinued. However, it is currently being tested in geneticallydefined Alzheimer's disease populations, with results expected in 2021 (clinicaltrials.gov;

Whereas the first generation of Aβ therapeutic mAbs differed in binding to distinct antibody domains (N-, mid-, and C-terminus), the second generation are intended to primarily bind specific conformations and aggregation states. Gantenerumab, currently in two phase III trials for mild and prodromal AD, binds a discontinuous epitope consisting of the N-terminus and an internal epitope, implying a unique conformational binding specificity (clinicaltrials.gov; Identifiers: NCT01224106, NCT02051608) [34]. Crenezumab, currently in phase II and phase III trials for autosomal dominant AD and prodromal-to-mild AD, respectively, is reported to selectively bind soluble and insoluble aggregates, but not monomers (clinicaltrials.gov; Identifiers: NCT01998841, NCT03114657) [35]. In contrast to other therapeutic mAbs, crenezumab is engineered on an IgG4 backbone to reduce effector function, and microglialmediated phagocytosis of Aβ deposits is not anticipated. BAN-2401, is in clinical development in a large phase II study in early AD patients; is proposed to selectively bind Aβ protofibrils

A promising antibody candidate from this group that is currently in the clinic is aducanumab. Aducanumab is a human mAb that selectively targets soluble aggregates and fibrils, and binds the N-terminus of Aβ. Preclinical studies demonstrate that the chimeric form of aducanumab peripherally administered to an APP transgenic mouse (a) crosses the BBB and binds to plaques (b) reduces calcium overload in neurons [37], and (c) reduces plaque burden in a dose-dependent manner [38]. An interim report from a double-blind, placebo controlled

priate patient safety monitoring.

Identifier: NCT02008357).

*2.2.2. Second-generation Aβ passive immunotherapies*

(clinicaltrials.gov; Identifier: NCT01767311) [36].

**Table 1.** Past and current Aβ antibody therapeutics.

displayed these abnormalities, two of whom were asymptomatic. Due to the observation of VE at 5 mg/kg a dose regimen ranging from 0.15 to 2 mg/kg, administered every 13 weeks for 18 months was selected for the multiple ascending dose phase II trial [27]. In the phase II trial, study completers that received all 6 planned infusions displayed significant improvements in DAD score and the Alzheimer's Disease Assessment Scale-Cognitive (ADAS-cog), though this effect was not observed in the intent-to-treat population. VE was observed in ~10% of bapineuzumab treated patients (half of whom were asymptomatic), in comparison to 0% of the placebo group; the appearance of VE was dose-dependent and appeared early during the course of treatment. Interestingly, the majority (10/12) of VE cases occurred in carriers of the *APOε4* allele, a risk factor for aggressive AD [28].

Two phase III trials for bapineuzumab were completed to evaluate efficacy in patients with mild to moderate AD who were either *APOε4* carriers or non-carriers in separate trials, with a lower dose regimen in the carrier trial [29]. These trials did not meet the co-primary cognitive and functional endpoints, though CSF phospho-tau, a proposed biomarker of neurodegeneration in AD, did decrease in both studies and positron emission tomography-Pittsburgh B (PET-PIB) imaging revealed less amyloid pathology in the *APOε4* carrier group treated with bapineuzumab compared to placebo. One important finding is that of the subgroup that underwent PET-PIB imaging, 6.5% of *APOε4* carriers and 36.1% of non-carriers did not have detectable PET-PIB signal at trial entry, raising concerns about misdiagnosis and improper subject selection in the trials. While these studies did not succeed in meeting primary endpoints, they did provide information to guide future trials, particularly in understanding MRI abnormalities, such as VE and microhemorrhages.

During the course of the phase III trials, the observation that VE and microhemorrhages correlated with anti-amyloid dose levels was more pronounced in *APOε4* carriers, and were normally transient and asymptomatic [30] led to the formation of an Alzheimer's Association-led workgroup composed of industry and academic experts to advise the FDA on potential routes to monitor VE and microhemorrhages. The term amyloid-related imaging abnormalities (ARIA) was adopted to address the spectrum of MR imaging abnormalities observed with anti-amyloid therapies, spanning from sulcal effusion and vasogenic edema seen on FLAIR MRIs to hypointensities (hemosiderin deposits) on T2\* MRI. The ARIA terminology was further subdivided to ARIA-E (sulcal effusion and edema) and ARIA-H (hemosiderin deposits) [31]. Recommendations from the workgroup included (a) standardization of technical and monitoring practices for MRI, (b) exclusion from trials of patients with preexisting ARIA-H, and (c) monitoring of symptoms potentially associated with ARIA. The adoption of these standards, and the understanding that ARIA is largely a short-lived treatment related effect inherent to many anti-amyloid therapies, opened the possibility of testing higher and more frequent drug administration regimens with appropriate patient safety monitoring.

In parallel with bapineuzumab, two additional anti-Aβ passive immunotherapies underwent contemporaneous clinical trials: Ponezumab, directed at the C-terminus of Aβ, underwent Phase I and IIa trials, but was discontinued after Phase IIa [32]. Solanezumab, directed at an internal epitope of Aβ and hypothesized to function by binding soluble species in the CNS and periphery, failed a phase III trial in mild AD patients [33], and a trial conducted in prodromal patients was discontinued. However, it is currently being tested in geneticallydefined Alzheimer's disease populations, with results expected in 2021 (clinicaltrials.gov; Identifier: NCT02008357).

#### *2.2.2. Second-generation Aβ passive immunotherapies*

displayed these abnormalities, two of whom were asymptomatic. Due to the observation of VE at 5 mg/kg a dose regimen ranging from 0.15 to 2 mg/kg, administered every 13 weeks for 18 months was selected for the multiple ascending dose phase II trial [27]. In the phase II trial, study completers that received all 6 planned infusions displayed significant improvements in DAD score and the Alzheimer's Disease Assessment Scale-Cognitive (ADAS-cog), though this effect was not observed in the intent-to-treat population. VE was observed in ~10% of bapineuzumab treated patients (half of whom were asymptomatic), in comparison to 0% of the placebo group; the appearance of VE was dose-dependent and appeared early during the course of treatment. Interestingly, the majority (10/12) of VE cases occurred in carriers of the

**phase**

PhIII [41]

**References**

**Name Epitope Most recent clinical** 

Bapineuzumab 1–6 PhIII (terminated) [22] Solanezumab 16–26 PhIII [33, 41] Ponezumab 35–40 PhIIa (terminated) [32, 42]

(aggregate-selective)

Gantenerumab 3–11, 18–27 PhIII [34] BAN-2401 Protofibrils PhII [36] Aducanumab N-terminus PhIII [37, 38]

*First-generation Aβ passive immunotherapeutics*

132 Alzheimer's Disease - The 21st Century Challenge

*Second-generation Aβ passive immunotherapeutics* Crenezumab 16–26

**Table 1.** Past and current Aβ antibody therapeutics.

Two phase III trials for bapineuzumab were completed to evaluate efficacy in patients with mild to moderate AD who were either *APOε4* carriers or non-carriers in separate trials, with a lower dose regimen in the carrier trial [29]. These trials did not meet the co-primary cognitive and functional endpoints, though CSF phospho-tau, a proposed biomarker of neurodegeneration in AD, did decrease in both studies and positron emission tomography-Pittsburgh B (PET-PIB) imaging revealed less amyloid pathology in the *APOε4* carrier group treated with bapineuzumab compared to placebo. One important finding is that of the subgroup that underwent PET-PIB imaging, 6.5% of *APOε4* carriers and 36.1% of non-carriers did not have detectable PET-PIB signal at trial entry, raising concerns about misdiagnosis and improper subject selection in the trials. While these studies did not succeed in meeting primary endpoints, they did provide information to guide future trials, particularly in understanding MRI

During the course of the phase III trials, the observation that VE and microhemorrhages correlated with anti-amyloid dose levels was more pronounced in *APOε4* carriers, and were normally transient and asymptomatic [30] led to the formation of an Alzheimer's

*APOε4* allele, a risk factor for aggressive AD [28].

abnormalities, such as VE and microhemorrhages.

Whereas the first generation of Aβ therapeutic mAbs differed in binding to distinct antibody domains (N-, mid-, and C-terminus), the second generation are intended to primarily bind specific conformations and aggregation states. Gantenerumab, currently in two phase III trials for mild and prodromal AD, binds a discontinuous epitope consisting of the N-terminus and an internal epitope, implying a unique conformational binding specificity (clinicaltrials.gov; Identifiers: NCT01224106, NCT02051608) [34]. Crenezumab, currently in phase II and phase III trials for autosomal dominant AD and prodromal-to-mild AD, respectively, is reported to selectively bind soluble and insoluble aggregates, but not monomers (clinicaltrials.gov; Identifiers: NCT01998841, NCT03114657) [35]. In contrast to other therapeutic mAbs, crenezumab is engineered on an IgG4 backbone to reduce effector function, and microglialmediated phagocytosis of Aβ deposits is not anticipated. BAN-2401, is in clinical development in a large phase II study in early AD patients; is proposed to selectively bind Aβ protofibrils (clinicaltrials.gov; Identifier: NCT01767311) [36].

A promising antibody candidate from this group that is currently in the clinic is aducanumab. Aducanumab is a human mAb that selectively targets soluble aggregates and fibrils, and binds the N-terminus of Aβ. Preclinical studies demonstrate that the chimeric form of aducanumab peripherally administered to an APP transgenic mouse (a) crosses the BBB and binds to plaques (b) reduces calcium overload in neurons [37], and (c) reduces plaque burden in a dose-dependent manner [38]. An interim report from a double-blind, placebo controlled phase Ib study revealed a dose-dependent decrease of amyloid PET signal that corresponded with significant slowing of cognitive decline at 52 weeks at the highest dose level, 10 mg/kg [38]. While ARIA was reported at a similar frequency compared with previous trials, adherence to guidelines formalized by the Alzheimer's Association ARIA working group [31] allowed for higher and more frequent dosing, potentially contributing to the positive results seen in these early studies. Aducanumab is currently in phase III trials in prodromal early AD patients, with endpoints and patient populations informed by the successful phase Ib study [39]. Interestingly, enrollment for these phase III clinical trials was recently increased by approximately 15% due to patient variability in the primary functional endpoint [40].

While the stereotypic appearance and progression of tau pathology down the perforant pathway—the neurocircuit from the entorhinal cortex to the hippocampus—has been described [55, 56], the molecular mechanisms underpinning this observation had remained elusive. Neurons in the performant path have long been known to be selectively vulnerable to insult such as hyperactivity [57] and expression of AD-related presenilin mutations [58], but the discovery that, when injected into the brain parenchyma, tau from a mutant mouse could simulate the formation of tau aggregates in a previously healthy animal [59] allowed the possibility that this progression may be mediated by aggregated and misfolded forms of the protein. This was strikingly confirmed in mice with tau expression restricted to the entorhinal cortex: in these mice, tau pathology propagated from the region of expression to distant efferent neurons [60, 61], demonstrating that direct cell-cell contact was not required for propagation, and that the pathological signal could be spread trans-synaptically. The demonstration that tau itself was present in interstitial fluid [62], could be secreted from neurons [63], and passed between cells [64] and neurons [65] provided evidence that tau species themselves could be directly transmitted between neurons *in vivo*, providing a potential mechanistic basis for the propagation of tau pathology. Although tau and Aβ are likely associated with different pathophysiological processes in Alzheimer's disease, the presence of pathogenic extracellular tau species could theoretically also be targeted by immunotherapeutic approaches, in this case by a different mechanism of action: interception/sequestration and prevention of cell-to-cell

Passive Immunotherapy in Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.76299 135

An overview of preclinical and clinical tau antibody efforts described in the following text is

Pioneering tau immunotherapy studies demonstrated that immunization with phospho-tau peptides (phosphorylated at Ser396/404) in two different tau transgenic lines raised anti-tau antibodies, which immunohistochemically stained the brains of P301L-tau transgenic mice. In addition, active immunization resulted in reductions in tau pathology. The mice also displayed improved performance in motor tasks [66, 67]. Purified anti-tau antibodies from

**Name Epitope Most recent development phase References** MC1 7–9, 313–322 Preclinical [69, 70] BIIB092/BMS986168 17–28 PhII (recruiting) [85, 88] ABBV-8E12 25–30 PhI open label extension [81, 82] *Cis* mAb *Cis*-pT231 Preclinical [74] RO7105705 pSer409 PhII (recruiting) [71] PHF1 pSer396/404 Preclinical [67, 69, 70] TOMA Tau oligomer Preclinical [76]

**Table 2.** Tau clinical and preclinical antibodies discussed in this chapter.

transmission.

listed in **Table 2**.

**3.2. Tau passive immunotherapy**
