**3. Tau**

While most passive immunotherapy clinical trials in AD have been directed at Aβ, key discoveries regarding tau function and contribution to disease mechanisms have prompted significant efforts directed towards tau. Hyperphosphorylated and aggregated tau protein are the main component of neurofibrillary tangles (NFTs), which, together with Abeta plaques, are considered a primary hallmark in Alzheimer's disease. Because of its intracellular localization, tau deposits have historically been thought to be unavailable to immunotherapeutic treatments. However, results outlined in this section indicate the potential for targeting tau through a passive immunotherapeutic approach.

#### **3.1. Tau biology and pathophysiology**

Since the discovery that NFTs are composed of the microtubule-associated protein tau [43– 45], many efforts have been devoted to elucidating molecular mechanisms of tau pathophysiology. Tau is an intracellular microtubule binding protein, which is involved in the regulation of microtubule stability and dynamics. In the brain, tau exists principally as six different isoforms, which vary in the absence or presence of N-terminal acidic repeats and a microtubule repeat; these differences are due to the splicing in or out of exons 2, 3, and 10 [46]. In normal physiological situations, the specific ratio of tau isoforms is developmentally regulated, likely due to the changing needs of microtubule fluidity versus stability throughout development and maturity [47].

Tau is an intrinsically disordered, natively-unfolded protein [48] whose physiological function is tightly regulated by post-translational modifications—principally via phosphorylation, which regulates microtubule binding affinity [49, 50]. In the AD brain, tau aggregates to form hyperphosphorylated NFTs and inclusions, composed of paired-helical and straight filaments [51]. In contrast to the intrinsically disordered nature of monomeric tau in solution, these structures adopt an ordered structure composed of a β-sheet core comprised of central residues, surrounded by a disordered coat comprised of the C- and N-termini of the molecule [52]. In AD, the appearance of tau pathological features positively correlates with dementia and disease progression [53, 54], leading to the hypothesis that the formation of tau pathology is a primary causative agent in the development of AD.

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 transmission.

#### **3.2. Tau passive immunotherapy**

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].

through a passive immunotherapeutic approach.

is a primary causative agent in the development of AD.

**3.1. Tau biology and pathophysiology**

134 Alzheimer's Disease - The 21st Century Challenge

and maturity [47].

While most passive immunotherapy clinical trials in AD have been directed at Aβ, key discoveries regarding tau function and contribution to disease mechanisms have prompted significant efforts directed towards tau. Hyperphosphorylated and aggregated tau protein are the main component of neurofibrillary tangles (NFTs), which, together with Abeta plaques, are considered a primary hallmark in Alzheimer's disease. Because of its intracellular localization, tau deposits have historically been thought to be unavailable to immunotherapeutic treatments. However, results outlined in this section indicate the potential for targeting tau

Since the discovery that NFTs are composed of the microtubule-associated protein tau [43– 45], many efforts have been devoted to elucidating molecular mechanisms of tau pathophysiology. Tau is an intracellular microtubule binding protein, which is involved in the regulation of microtubule stability and dynamics. In the brain, tau exists principally as six different isoforms, which vary in the absence or presence of N-terminal acidic repeats and a microtubule repeat; these differences are due to the splicing in or out of exons 2, 3, and 10 [46]. In normal physiological situations, the specific ratio of tau isoforms is developmentally regulated, likely due to the changing needs of microtubule fluidity versus stability throughout development

Tau is an intrinsically disordered, natively-unfolded protein [48] whose physiological function is tightly regulated by post-translational modifications—principally via phosphorylation, which regulates microtubule binding affinity [49, 50]. In the AD brain, tau aggregates to form hyperphosphorylated NFTs and inclusions, composed of paired-helical and straight filaments [51]. In contrast to the intrinsically disordered nature of monomeric tau in solution, these structures adopt an ordered structure composed of a β-sheet core comprised of central residues, surrounded by a disordered coat comprised of the C- and N-termini of the molecule [52]. In AD, the appearance of tau pathological features positively correlates with dementia and disease progression [53, 54], leading to the hypothesis that the formation of tau pathology

**3. Tau**

An overview of preclinical and clinical tau antibody efforts described in the following text is listed in **Table 2**.

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


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

immunized mice were peripherally injected into naïve transgenic mice and localized to neurons in the brain displaying tau pathology, demonstrating their ability to cross the bloodbrain barrier (BBB) and localize to their target. In a separate study performed by the same lab, passive administration of the mAb PHF1, directed at the Ser396/404 phosphoepitope, also resulted in reductions in tau pathology in mice compared to isotype control [68]. The findings from this series of studies were proposed to be due to two potential mechanisms: (a) antibody-mediated clearance of extracellular tau deposits and (b) intracellular uptake of tau antibodies. The efficacy of passive immunotherapy using PHF1, as well as the conformational antibody MC1, were also confirmed in independent labs [69, 70], bolstering early evidence of this novel promising therapeutic avenue.

tau oligomers and histopathology, and rescued deficits in rotarod and spontaneous alternation tests. Examination of serum revealed oligomeric tau and antibody/antigen complexes,

Passive Immunotherapy in Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.76299 137

Informed by studies indicating the potential for the propagation of tau pathology across cell membranes [64], as well as the demonstration of trans-synaptic transmission *in vivo* [60, 78], an independent effort to discover tau antibodies that interrupted cell-to-cell transmission yielded phosphorylation-independent antibodies that blocked uptake of tau aggregates to cultured cells [79, 80]. When administered to tau transgenic mice centrally via an Alzet minipump, these antibodies slowed the advance of tau pathology, as measured by immunohistochemical and biochemical means [79]. One of the efficacious antibodies used in this report, HJ8.5, was used in a peripheral administration model to further explore its potential as a therapeutic agent [81]. HJ8.5 is a high affinity anti-N terminal mAb that recognizes residues 25–30, which are present on all splice isoforms of tau. In this study, P301S tau transgenic mice were dosed intraperitoneally over a 3-month period with 10 or 50 mg/kg of HJ8.5. The high dose cohort displayed decreases of insoluble tau, AT8 staining, and thioflavin S staining. In addition, this cohort exhibited improvements in sensorimotor function compared to isotype control and low-dose cohorts. The preclinical efficacy profile, as well as the concordance of *in vivo* data with mechanistic *in vitro* studies, propelled the humanized analogue of this antibody into the clinic (clinicaltrials.gov; Identifier: NCT03391765) [82]. Interestingly, a separate effort focused on discovering antibodies and epitopes important for uptake and transmission determined that while N-terminal antibodies could indeed block uptake of recombinant and AD patient-derived tau, there were other epitopes with potentially more potent function,

A key component of the amyloid cascade hypothesis is that Aβ aggregation induces, either indirectly or directly, fibrillization of tau as well as other disease processes (reviewed in [84]). The finding that extracellular secreted and truncated forms of tau (termed eTau) could regulate Aβ levels demonstrated a potential upstream role of tau in relation to Aβ, complementary to the amyloid cascade hypothesis. In this study, secreted eTau was isolated from iPSC neurons derived from patients with AD; treatment of neurons with eTau displayed increases in secreted Aβ, and these increases could be prevented via application of eTau-binding antibodies such as MC1 and IPN002, which recognizes residues 17–28. Aβ levels were not affected by PHF1 antibody, as the PHF1 epitope is not present in eTau. This finding was recapitulated in transgenic P301L-tau mice; peripheral treatment with IPN002 resulted in reductions in Aβ in the interstitial fluid and cortical tissue [85]. These findings were recently confirmed by a different group using mAbs that target very similar N-terminal tau epitopes; in these studies, behavioral improvements as well as decreases in Aβ were noted in mice transgenic for mutant forms of presenilin, APP, and tau [86, 87]. IPN002 has been developed into a clinical therapeutic and is undergoing clinical trials as BIIB-092/BMS986168 (clinicaltrials.gov; Identifier:

Though the success of preclinical studies with tau antibodies has provided sufficient rationale to begin exploration in the clinic, a greater understanding of the full range of factors involved in tau toxicity and the mechanisms of action of tau passive immunotherapy are needed. These

suggesting peripheral clearance as a mechanism of action [77].

notably antibodies binding C-terminal to the acidic inserts [83].

NCT03068468) [88].

An antibody targeting a different phosphoepitope, pSer409, also shows promise in preclinical models; however, conclusions regarding the mechanism of antibody function were considerably different than those proposed in the initial active and passive studies described in the prior paragraph. In this study, a highly selective mAb was able to bind tau phosphorylated at Ser409 and specifically bind AD brain tissue. The mAb was shown to neutralize oligomer-induced neurotoxicity; however, the neutralization activity of the antibody was reduced in mixed neuron-microglial cultures. Antibody engineered with reduced effector function (REF) maintained neutralization activity in mixed neuron-microglial cultures, while the wild-type anti-pSer409 antibody did not prevent neurotoxicity and in fact promoted the release of pro-inflammatory cytokines from microglia [71]. Both wild-type and REF variants of the antibody prevented the progression of tau pathology in the tau P301L mouse, leading the authors to conclude that phagocytic clearance of tau structures was not a contributing mechanism of action to efficacy in the transgenic mouse model. In addition, the lack of FcR message found in isolated neurons prompted the conclusion that receptor-mediated uptake did not occur. The antibody examined in this report has been developed into a therapeutic candidate, which is currently in clinical development (clinicaltrials.gov; Identifier: NCT03289143).

Additional studies have been conducted to identify and target post-translationally modified forms of tau to explore effects of antibody treatment. One compelling approach targets a unique structural isoform of tau induced by phosphorylation of tau at T231. Phosphorylation of tau at T231 occurs during disease progression; the prolyl isomerase Pin1 normally binds and converts the pT231/Proline motif from a toxic *cis* form to a soluble nontoxic *trans* form [72]. A mAb targeting *cis* but not *trans* pT231-tau detects pathology during mild cognitive impairment (MCI) [73]. In addition to AD, this post-translational signature (as well as others) appears in the brains of traumatic brain injury (TBI) patients. When administered peripherally in a murine TBI model carried out in tau transgenic mice, the *cis-*pT231 tau antibody prevented the spread of tauopathy and cortical LTP deficits, and improved performance in the elevated plus maze, which was correlated to TBI-induced disinhibition behavior in patients [74]. Another effort targeting disease-specific forms of tau is centered around developing antibodies that bind soluble oligomeric tau—hypothesized to be the most toxic form of the molecule [75]—and have minimal binding to monomeric or mature NFTs [76]. Tau oligomer-specific monoclonal antibodies (TOMAs) were dosed via intracerebroventricular (i.c.v.) infusion to tau P301L mice. Strikingly, a single i.c.v. injection reduced tau oligomers and histopathology, and rescued deficits in rotarod and spontaneous alternation tests. Examination of serum revealed oligomeric tau and antibody/antigen complexes, suggesting peripheral clearance as a mechanism of action [77].

immunized mice were peripherally injected into naïve transgenic mice and localized to neurons in the brain displaying tau pathology, demonstrating their ability to cross the bloodbrain barrier (BBB) and localize to their target. In a separate study performed by the same lab, passive administration of the mAb PHF1, directed at the Ser396/404 phosphoepitope, also resulted in reductions in tau pathology in mice compared to isotype control [68]. The findings from this series of studies were proposed to be due to two potential mechanisms: (a) antibody-mediated clearance of extracellular tau deposits and (b) intracellular uptake of tau antibodies. The efficacy of passive immunotherapy using PHF1, as well as the conformational antibody MC1, were also confirmed in independent labs [69, 70], bolstering early evidence of

An antibody targeting a different phosphoepitope, pSer409, also shows promise in preclinical models; however, conclusions regarding the mechanism of antibody function were considerably different than those proposed in the initial active and passive studies described in the prior paragraph. In this study, a highly selective mAb was able to bind tau phosphorylated at Ser409 and specifically bind AD brain tissue. The mAb was shown to neutralize oligomer-induced neurotoxicity; however, the neutralization activity of the antibody was reduced in mixed neuron-microglial cultures. Antibody engineered with reduced effector function (REF) maintained neutralization activity in mixed neuron-microglial cultures, while the wild-type anti-pSer409 antibody did not prevent neurotoxicity and in fact promoted the release of pro-inflammatory cytokines from microglia [71]. Both wild-type and REF variants of the antibody prevented the progression of tau pathology in the tau P301L mouse, leading the authors to conclude that phagocytic clearance of tau structures was not a contributing mechanism of action to efficacy in the transgenic mouse model. In addition, the lack of FcR message found in isolated neurons prompted the conclusion that receptor-mediated uptake did not occur. The antibody examined in this report has been developed into a therapeutic candidate, which is currently in clinical development (clinicaltrials.gov; Identifier:

Additional studies have been conducted to identify and target post-translationally modified forms of tau to explore effects of antibody treatment. One compelling approach targets a unique structural isoform of tau induced by phosphorylation of tau at T231. Phosphorylation of tau at T231 occurs during disease progression; the prolyl isomerase Pin1 normally binds and converts the pT231/Proline motif from a toxic *cis* form to a soluble nontoxic *trans* form [72]. A mAb targeting *cis* but not *trans* pT231-tau detects pathology during mild cognitive impairment (MCI) [73]. In addition to AD, this post-translational signature (as well as others) appears in the brains of traumatic brain injury (TBI) patients. When administered peripherally in a murine TBI model carried out in tau transgenic mice, the *cis-*pT231 tau antibody prevented the spread of tauopathy and cortical LTP deficits, and improved performance in the elevated plus maze, which was correlated to TBI-induced disinhibition behavior in patients [74]. Another effort targeting disease-specific forms of tau is centered around developing antibodies that bind soluble oligomeric tau—hypothesized to be the most toxic form of the molecule [75]—and have minimal binding to monomeric or mature NFTs [76]. Tau oligomer-specific monoclonal antibodies (TOMAs) were dosed via intracerebroventricular (i.c.v.) infusion to tau P301L mice. Strikingly, a single i.c.v. injection reduced

this novel promising therapeutic avenue.

136 Alzheimer's Disease - The 21st Century Challenge

NCT03289143).

Informed by studies indicating the potential for the propagation of tau pathology across cell membranes [64], as well as the demonstration of trans-synaptic transmission *in vivo* [60, 78], an independent effort to discover tau antibodies that interrupted cell-to-cell transmission yielded phosphorylation-independent antibodies that blocked uptake of tau aggregates to cultured cells [79, 80]. When administered to tau transgenic mice centrally via an Alzet minipump, these antibodies slowed the advance of tau pathology, as measured by immunohistochemical and biochemical means [79]. One of the efficacious antibodies used in this report, HJ8.5, was used in a peripheral administration model to further explore its potential as a therapeutic agent [81]. HJ8.5 is a high affinity anti-N terminal mAb that recognizes residues 25–30, which are present on all splice isoforms of tau. In this study, P301S tau transgenic mice were dosed intraperitoneally over a 3-month period with 10 or 50 mg/kg of HJ8.5. The high dose cohort displayed decreases of insoluble tau, AT8 staining, and thioflavin S staining. In addition, this cohort exhibited improvements in sensorimotor function compared to isotype control and low-dose cohorts. The preclinical efficacy profile, as well as the concordance of *in vivo* data with mechanistic *in vitro* studies, propelled the humanized analogue of this antibody into the clinic (clinicaltrials.gov; Identifier: NCT03391765) [82]. Interestingly, a separate effort focused on discovering antibodies and epitopes important for uptake and transmission determined that while N-terminal antibodies could indeed block uptake of recombinant and AD patient-derived tau, there were other epitopes with potentially more potent function, notably antibodies binding C-terminal to the acidic inserts [83].

A key component of the amyloid cascade hypothesis is that Aβ aggregation induces, either indirectly or directly, fibrillization of tau as well as other disease processes (reviewed in [84]). The finding that extracellular secreted and truncated forms of tau (termed eTau) could regulate Aβ levels demonstrated a potential upstream role of tau in relation to Aβ, complementary to the amyloid cascade hypothesis. In this study, secreted eTau was isolated from iPSC neurons derived from patients with AD; treatment of neurons with eTau displayed increases in secreted Aβ, and these increases could be prevented via application of eTau-binding antibodies such as MC1 and IPN002, which recognizes residues 17–28. Aβ levels were not affected by PHF1 antibody, as the PHF1 epitope is not present in eTau. This finding was recapitulated in transgenic P301L-tau mice; peripheral treatment with IPN002 resulted in reductions in Aβ in the interstitial fluid and cortical tissue [85]. These findings were recently confirmed by a different group using mAbs that target very similar N-terminal tau epitopes; in these studies, behavioral improvements as well as decreases in Aβ were noted in mice transgenic for mutant forms of presenilin, APP, and tau [86, 87]. IPN002 has been developed into a clinical therapeutic and is undergoing clinical trials as BIIB-092/BMS986168 (clinicaltrials.gov; Identifier: NCT03068468) [88].

Though the success of preclinical studies with tau antibodies has provided sufficient rationale to begin exploration in the clinic, a greater understanding of the full range of factors involved in tau toxicity and the mechanisms of action of tau passive immunotherapy are needed. These mechanisms may be different than those proposed for Aβ immunotherapy. There remain conflicting details from the studies presented here, such as the relative contribution of microglialmediated phagocytosis, the relative importance of eTau-mediated Aβ production, the extent of trans-synaptic transmission in transgenic mice with widespread expression in the brain, and the optimal epitope to target. Gaining a clearer understanding of these factors continues are a current research focus.

classical complement cascade, C1q, is enriched in the developing mouse CNS and localizes to synapses; genetic ablation of this protein results in misregulated innervation due to increased presence of synapses [96]. While C1q is normally downregulated after development, it is elevated in normal aging [97] and disease, including AD [98]. In a transgenic APP mouse, C1q localizes to synapses, and is required for pathological synapse loss. Treatment of C1q knockout mice with oligomeric Aβ displayed no synaptic loss, indicating that C1q is a required mediator of Aβ-induced toxicity. Interestingly, an anti-C1q antibody rescued Aβ-induced synaptotoxicity *in vivo*, and LTP impairment *in situ*, when compared to isotype control [99]. These data hinted at the promise of C1q immunotherapy to provide protective benefits by neutralizing a key mediator of Aβ-induced microglial overactivation, which results in synaptic loss. The anti-C1q antibody used in this study has been developed into a human therapeu-

Passive Immunotherapy in Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.76299 139

tic, and is beginning clinical trials (clinicaltrials.gov; Identifier: NCT03010046) [100].

The mounting evidence of involvement of the adaptive immune system in restraining the advance of AD pathology has opened the possibility of directing passive immunotherapies to the periphery, which considerably eases the challenge of achieving sufficient drug exposure in the CNS to affect pathology. Microglia resident in the brain are known to be recruited to sites of injury such as senile plaques, but the finding that peripherally-derived bone marrow stem cells are able to enter the CNS, and differentiate into microglia [101, 102], was the first direct evidence that repopulation and recruitment of microglia from the periphery was an active process. This finding was extended to AD mouse models with the finding that peripherally-recruited microglia are mobilized by Aβ, recruited to the site of senile plaques, and are able to clear plaques via phagocytosis [103]. The protective role of these immune cells in the presence of AD-like pathology was confirmed with the observation that (a) knocking out the chemokine receptor CCR2 in an APP-transgenic mouse resulted in decreased recruitment of monocytes to Aβ plaques [104], and (b) the specific ablation of bone-marrow derived cells via diphtheria-toxin receptor expression resulted in increased Aβ plaques [105]. Furthermore, increasing trafficking of macrophages by inhibiting the normally immunosuppressive regulatory T-cells through pharmacologic or genetic methods results in reduced Aβ pathology [106].

Elucidation of the biology of inhibitory signaling pathways and proteins such as Programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), known as immune checkpoints, led to the development of antibody therapeutics for use in cancer (reviewed in [107]). These therapies function by neutralizing immune checkpoints and activating T-cells, which prompts antitumor activity. The characterization of checkpoint signaling pathways, along with the findings that peripheral immune cells modulate AD-like pathology in a regulatory T-cell (Treg)-dependent manner, has prompted examination of the PD1/PD-L1 axis in AD. In a recent study, AD transgenic mice were treated with an anti-PD1 antibody to blockade the PD1/PD-L1 axis. Remarkably, checkpoint blockade in this model resulted in substantial rescue of performance in a behavioral assay of memory and cognition after a single dose, and mice exhibited decreases in Aβ pathology with only two dose administrations [108]. The effect on pathology was observed even in mice with profound amyloid burden. While the findings of a profound effect on functional measures after such a short dose regimen are very exciting, they should be taken with a note of caution. A follow-up study, carried out by three pharmaceutical companies using three transgenic models and numerous

Clinical trials with Aβ immunotherapies have demonstrated the importance of proper clinical diagnosis, patient selection, sensitive cognition tests, and effective biomarkers to monitor efficacy and disease progression. Though some general commonalities may exist in the clinical design of Aβ and tau passive immunotherapy trials, there are substantial differences in the targets and any potential clinical development approaches. In contrast to Aβ, there are a number of non-AD tauopathies such as progressive supranuclear palsy (PSP) [89] and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) [90] that may provide alternative clinical development pathways to test novel tau-directed therapeutic approaches. In contrast to AD, these diseases present pathological signatures composed almost uniformly of tau and neurofibrillary tangles; in addition, FTDP-17 is an autosomal dominant disorder, genetically validating the causative role of tau. Diagnosis of these and other tauopathies have historically been made solely based on clinicopathology; due to the difficulty of diagnosis from to the overlap of symptomologies with other neurodegenerative disorders, as well as the lack of clear biomarkers, diagnosis is only confirmed at autopsy [91]. Modern tau PET imaging agents are currently under clinical investigation [92]; while early generations of tau PET tracers displayed nonspecificity and suboptimal binding and PK characteristics, the newest class of tracers display improved specificity, PK properties, and may allow for improved diagnosis in tauopathies as well as an ability to monitor tau pathology in AD clinical trials [93].
