key: cord-0790049-xo34jk9u
authors: Plotkin, Steven S.; Cashman, Neil R.
title: Passive immunotherapies targeting Aβ and tau in Alzheimer's disease
date: 2020-07-16
journal: Neurobiol Dis
DOI: 10.1016/j.nbd.2020.105010
sha: 01d985de3be48744c128a1710dfdafdad99af84d
doc_id: 790049
cord_uid: xo34jk9u

Amyloid-β (Aβ) and tau proteins currently represent the two most promising targets to treat Alzheimer's disease. The most extensively developed method to treat the pathologic forms of these proteins is through the administration of exogenous antibodies, or passive immunotherapy. In this review, we discuss the molecular-level strategies that researchers are using to design an effective therapeutic antibody, given the challenges in treating this disease. These challenges include selectively targeting a protein that has misfolded or is pathological rather than the more abundant, healthy protein, designing strategic constructs for immunizing an animal to raise an antibody that has the appropriate conformational selectivity to achieve this end, and clearing the pathological protein species before prion-like cell-to-cell spread of misfolded protein has irreparably damaged neurons, without invoking damaging inflammatory responses in the brain that naturally arise when the innate immune system is clearing foreign agents. The various solutions to these problems in current clinical trials will be discussed.

There are currently about 132 therapeutic agents in 156 clinical trials for Alzheimer's disease (AD) (Cummings et al. (2019)). Among these are about 29 disease-modifying monoclonal antibody therapies involved in 24 clinical trials (Cummings et al. (2018 (Cummings et al. ( , 2019 ), nearly all of and the underlying biochemical mechanisms that motivate researchers to hypothesize that these therapies will be effective in treating AD.

In describing the common mechanisms that underly the effectiveness of potential antibody therapeutics, we found ourselves emphasizing general themes of antibody development that various different therapeutic strategies may have in common. As well, A  and tau have been shown to have intimately connected pathology, and therapeutic strategies targeting A  exclusively have had a long history fraught with ambiguous results and minimal therapeutic benefit. For these reasons it became almost inevitable to include both A  and tau therapies in the same review.

As some examples of biochemical similarity, both A  and tau have both been shown to have distinct, pathological species with conformations different from the healthy proteins, both are subject to isoform imbalance as a cause or symptom of pathology, both undergo post-translational modifications specific to pathological behavior that have been targeted by several candidate therapeutics, and both have been shown to form oligomers that propagate from cell-to-cell in prion-like fashion, which constitute therapeutic targets of specific interest. Wisniewski and Goñi (2015) ; Hung and Fu (2017) ; Dolan and Zago effect is hypothesized to be due to increased dissociation rates of the complex, resulting in reduced processivity, and thus the release of longer, incompletely processed A  peptides.

Apolipoprotein E (APOE) is a protein involved in the metabolism of fats in the body and is the principal cholesterol carrier in the brain (Puglielli et al. (2003) ). APOE exists in three main polymorphisms among the human population differing by two amino acid identities: namely the  2 (C112, C158),  3 (C112, R158), and  4 (R112, R158) isoforms. The  3 isoform is the most common (78% worldwide allele frequency). Individuals carrying APOE  2/  2 or  2/ 3 (8.4% of the population) are at decreased risk of AD (Liu et al. (2013) ). In vitro and in vivo evidence in APP transgenic mice has shown that APOE- 3, but not APOE- 4, attenuates A  protofibril-induced aggregation, by forming stabilizing complexes with A  (Hori et al. (2015) ).

As well, the APOE- 4 isoform is not as effective as the others at clearing A  (Jiang et al. (2008) ; Castellano et al. (2011)), and carriers of two copies of the  4 allele have on average 20 the risk of developing AD (Hauser and Ryan (2013) ). The  4 variant of APOE is currently the most significant known genetic risk factor for late-onset sporadic AD (Sadigh-Eteghad et al.

(2012); Roda et al. (2019) ).

Normally functioning TREM2, which encodes triggering receptor expressed on myeloid cells 2, facilitates microglia activation and clustering around amyloid and neurofibrillary tangles, increasing amyloid uptake, phagocytic activity, and plaque compaction in early stages of AD (D'Andrea et al. (2004) ; Hickman et al. (2018) ). These processes are impaired in AD-associated variants of TREM2, resulting in filamentous plaques associated with increased dystrophic neurites and a possible increase of tau pathology. (Jay et al. (2017) ; Ulrich et al. (2017) ; Gratuze et al. (2018) ; Zheng et al. (2018) ). Some variants of the TREM2 gene have been found to cause increased susceptibility to late onset AD with an odds ratio similar to that of ApoE- 4 (Guerreiro et al. (2012) ; Jonsson et al. (2013) ). The TREM2 mutant with the strongest AD association, R47H, has 3-4 the AD risk as wild-type, and shows significantly reduced A  -induced microglial responses in transgenic mouse models. Since TREM2 is exclusively expressed on immune cells, the above findings provide a direct link between dysregulation of the innate immune system as an active driver contributing to AD pathogenesis.

In summary, abundant evidence points to the progressive accumulation of A  in the brain, along with its impaired clearance and induced neuroinflammation, as very early features of mild-to-moderate AD (NCT01850238), it was found to be well-tolerated: The vaccine elicited no aberrant immune response or microhemorrhages compared to what was observed with AN1792 (Novak et al. (2017 (Novak et al. ( , 2019 ). Minor injection site reactions were the most common adverse event, observed in 53% participants. In a follow-up study 72 weeks after conclusion (NCT02031198)

involving 26 of the same participants, no aberrant immune responses were reported, except for microhemorrhages in one patient. Interestingly, cognitive decline as measured by baseline ADAS-cog11 value was shown to be significantly reduced in treated patients compared with placebo control (Novak et al. (2018b) ). These results have prompted AADvac-1 to move into a phase II clinical trial (NCT02579252) whose preliminary results have very recently been reported (Axon Neuroscience (2019)). The vaccine was again deemed safe and tolerable. Roughly 98% of patients generated antibodies against tau. Neurofilament Light Chain (NfL) biomarkers indicated significant slowing of neurodegenerative progression. AD-specific CSF pathological tau biomarkers, including phospho-tau181 and phospho-tau217, also appeared to show moderate to large reductions. Among the younger participants in the trial, there appeared to be positive signals for cognitive endpoints according to CDR-SB, MMSE, and ADCS-MCI-ADL tests, however the strength and significance has not yet been reported.

ACI-35 utilizes a liposomal-anchored 16-amino acid tetra-palmitoylated phospho-tau peptide, 393 VYKSPVVSGDTSPRHL 408 , with S396 and S404 phosphorylated, as they can be in pathological tau. The liposome sizes are such that they can accommodate  16 copies of the peptide. Presenting tau on liposomes alters the epitope conformation: Circular dichroism (CD)

shows a significant amount of  -sheet structure on the liposome surface similar to that of aggregated tau (Theunis et al. (2013) ).

In tau-transgenic mouse models, ACI-35 decreased both soluble and insoluble tau, increased retention of body weight, slightly extended lifespan, and improved the clinical phenotype of motor deficiency ; Theunis et al. (2013) ). The vaccine also did not induce marked CNS inflammation in spite of presenting multiple (16) copies of the epitope In guiding species selectivity and thus the efficacy of an antibody, the immunization strategy is central in determining which sub-population of the conformational ensemble of an epitope that an antibody will bind to. Guiding the epitope towards a desired conformational sub-population is often referred to as -epitope scaffolding‖ (see e.g. Skerra (2000) ; Ofek et al. (2010) ; Correia et al.

(2010); Azoitei et al. (2012)). A rational approach to immunization can save much effort by avoiding the subsequent high-throughput screens that are necessary when immunizing irrationally with generic polymorphic forms of a protein.

The protein drug targets A  and tau are both largely intrinsically disordered peptides when isolated as monomers in solution, and as such they are conformationally labile. Such polymorphic targets are inherently difficult to target for small-molecules, which are best-adapted to fit into well-structured binding pockets (Scott et al. (2016) ). On the other hand, antibodies are well suited to bind to disordered peptide segments. The selective binding of antibodies to regions of proteins that become disordered during the course of disease has been exploited to generate misfolding-specific antibodies for several proteins wherein misfolding is correlated with neurodegeneration (Paramithiotis et al. (2003) ; Glabe (2004) ; Rakhit et al. (2007) ; Broering et al. (2013) ; Ayers et al. (2014)).

In choosing the appropriate protein sequence and conformation for active immunization, ideally one would employ a reliable method for epitope prediction, and then use an immunogen appropriately displaying that epitope as it presents in toxic species. This is an enormous challenge at present: Our understanding of what proteinic features can be ubiquitously targeted on toxic species that are involved in the spread of AD is limited at present. Soluble oligomeric species that are thought to be central to the prion-like propagation of AD are conformationally plastic-they do not have a well-defined structure that would lend themselves to structural determination and subsequently epitope identification.

As described further in the examples below, epitopes are either left unidentified when immunizations with pathogenic species such as fibrils or oligomers are used, or a disease-specific J o u r n a l P r e -p r o o f Journal Pre-proof isoform is used in the immunogen (e.g. A 42

 rather than A 40  ), or a known post-translational modification observed in pathogenic species is incorporated onto a peptide fragment presented on the immunogen (e.g. phosphorylation of a serine residue, or pyroglutamate cyclization of a glutamic acid).

One method of epitope prediction used for the design of antibodies targeting tau and A  involves computation prediction of regions likely to be selectively exposed on the surface of soluble oligomers. This molecular dynamics approach applies the concept of a misfolding-specific epitope, useful in the context of other neurodegenerative diseases (Paramithiotis et al. (2003) ; Rakhit et al. (2007) ; Peng et al. (2018) ), to the problem of finding epitopes for A  and tau. In brief, a fibril structural model is weakened and disrupted by applying a global force along a collective coordinate. The weakest parts of the fibril complex are the first to become disordered, and constitute -stressed protofibril‖-specific epitopes (Plotkin (2017)).

We first describe an experiment providing strong evidence that Alzheimer's disease is a -conformational‖ disease of A  , which emphasizes the importance of conformation in the active immunization stage of antibody development. Peptides of A 1 15   or A 1 16   may be tethered to a liposome surface by conjugating two palmitoylated lysine residues at either end of the peptide, so that, for example the sequence of A 1 16   is Kpal Kpal DAE HQKK pal K pal (Nicolau et al. (2002) ). Additionally, the termini of the peptide may be PEGylated to provide an additional 77 units of spacing between the peptides and the liposome. Muhs et al. (2007) found, by CD and NMR measurements, that PEGylated, liposome-anchored A  preferred a random coil conformation, while non-PEGylated, liposome anchored A  preferred a  -sheet conformation, apparently due to enhanced proximity of the peptide to the liposome surface.

that target pathogenic, memory-reducing, species of A  . These liposomal compounds have been developed as an active vaccine (ACI-24) and are currently in phase 1/2 clinical trials (NCT02738450) in adults with Down syndrome. The pre-humanized murine antibody (mMABT) to crenezumab was generated by liposome-anchored A  inoculation, using an epitope subsuming residues 13-24.

Because of the above-mentioned disorder present in A  and tau, these proteins can present themselves to an antibody in multiple different conformations. It is often desirable for an antibody to be conformationally selective to a specific species (Westwood and Lawson (2015) ). For example, soluble A  is present in normal patient brains at a concentration of about a picomolar, while in AD brains it is present at concentrations 0.1 nM (Lue et al. (1999) ). Oligomer concentrations are less well-known but are thought to be about a 1000-fold lower in concentration (Yang et al. (2017) ). An antibody that is not conformationally selective for oligomer would suffer from target distraction by binding to the much more abundant monomer species, and thus lack sufficient target engagement.

The brains of healthy (non-AD presenting) elderly patients may contain insoluble A  amyloid plaque at concentrations comparable to AD patients (Lue et al. (1999) ); Diffuse senile plaques in the cerebral cortex have been considered to be age-related and unassociated with dementia (see e.g. Tagliavini et al. (1988) ). Thus, antibodies that bind generically to plaque may again suffer from target distraction, as well as additional clinical risks, particularly for antibodies binding to vascular deposits. In these cases, monocytes and other lymphocytes are recruited to clear the amyloid, and binding of antibody complexes to Fc receptors on macrophage-like cells stimulates the expression of proteases, which in turn degrade the extracellular matrix at those locations. Blood-brain barriers at the vessel wall are thus weakened, insterstitial fluid can enter the brain, and microhemorrhaging can occur (Schilling et al. (2018) ). This leakiness of brain vasculature manifests itself through amyloid-related imaging abnormalities (ARIA), which as mentioned above is generally accompanied by microhemorrhaging (ARIA-H) and/or edema (ARIA-E) (see Table 1 for ARIA levels for various A  immunotherapies).

Evidence has long been accumulating that soluble A  and tau oligomers are key J o u r n a l P r e -p r o o f pathogenic species that propagate cellular pathology throughout the brain in Alzheimer's disease (Kane et al. (2000) ; Thal et al. (2002) ; Walsh et al. (2005) ; Alonso et al. (2006) ; Haass and Selkoe (2007) ; Goedert et al. (2010) ; Hefti et al. (2013) ; Bloom (2014); Goedert (2015) ; Cline et al.

(2018); McAlary et al. (2019a) ). In AD patients, the amount of soluble A  species correlates more closely with cognitive decline than does amyloid plaque burden (Lue et al. (1999); McLean et al. (1999) ; Wang et al. (1999) , see also the comments on tau biomarker abnormalities below). In classic prion disease, soluble oligomers containing roughly 20 PrP molecules are by significant margin the most infectious when inoculated intracerebrally (Silveira et al. (2005) ), consistent with the notion that high molecular weight species such as plaques have the potential to play a protective role (Treusch et al. (2009) ). Consistently with these ideas, in brains of autopsy cases with similar amyloid load, the ratio of the amount of soluble oligomers over immunohistochemically determined plaque area fully differentiated demented vs. non-demented cases (Esparza et al. (2013) ).

The selectivity or promiscuity of an antibody can be understood within the context of energy lanscape theory. For an antibody-peptide system, the energy landscape for binding determines the peptide conformation bound by the antibody. For high-affinity binding, the transfer free energy to the bound conformation must be significantly negative, and the overall global structure of the energy landscape will have the topography of a funnel (Tsai et al. (1999) ; Papoian and Wolynes (2003) ; Wang and Verkhivker (2003) , see Figure 1 ). There is typically a significant amount of entropy loss, which is compensated for by the (negative) enthalpy gain concomitant with binding (Lafont et al. (2007) ; Chodera and Mobley (2013); Mills and Plotkin (2015) ). The degree to which this cancellation occurs determines how wide or how 'bottlenecked' the funnel is (Plotkin and Onuchic (2002)). For a wide funnel ( Fig. 1 left) , dissimilar conformations from the minimum free energy bound conformation also have favorable binding energy: There is more energetic guidance of dissimilar structures towards the minimum energy conformation. Conformations different from bound conformation that would be observed e.g. in the crystal structure, it is not particularly energetically selective to a specific conformational species (monomer, oligomer, or fibril/plaque).

The binding free energies for alternate conformations of A  or tau are still significant. Figure 1 : Schematics of energy landscapes of the binding free energy of an epitope to an antibody, as a function of conformational dissimilarity to the bound state structure, which is assumed to be at the lowest point. A conformationally-labile antibody is more prone to induced fit with different alternative conformations of a substrate ligand, and will thus lack binding selectivity (left). A conformationally-selective antibody will be unforgiving to even small conformational differences, which will be costly in terms of binding free energy.

For a steep funnel topography akin to the hole on a golf-green ( Fig. 1 right) , even slightly dissimilar conformations from the minimum free energy bound conformation do not have favorable binding energy, and so are not bound with significant affinity. The binding scenario is more reminiscent of conformational-selection, wherein the antibody is selective to a small ensemble of conformations consistent with a specific target species.

Polymorphism is an inherent aspect of A  fibril structures (Fig. 2) , and ultimately it is a consequence of the absence of any evolutionary selection towards a global free energy minimum, which structured proteins generally possess (Plotkin et al. (1997) ; Plotkin and Onuchic (2002) ).

That is, the misfolding energy landscape of the fibril does not have the global topography of a funnel, with a single dominant free energy basin. In contrast to the properties inhereint in well-folded proteins, we would thus expect mutants or alternate isoforms of A  (or tau), or altered environmental conditions, to result in alteration of the fibril morphology, which is exactly while being ineffective in binding alternate strains. The polymorphism in oligomers is even more profound, rendering structural determination difficult or moot, and making oligomer-selective targets particularly elusive (Sengupta et al. (2016) ; Lee et al. (2017) ).  , or the mutant A 40  (E22  )) are indicated for each image, along with the PDB entry: 2M4J (Lu et al. (2013) ), 2LMN (Paravastu et al. (2008) ), 2MVX (Schütz et al. (2015) ), 2MXU (Xiao et al. (2015) ), 5OQV (Gremer et al. (2017) ), and 2NAO (Wälti et al. (2016) ). An example of ionic salt-bridges stabilizing the fibril structure is shown for structure 2M4J (D23-K28) in licorice.

Structures 2LMN and 2MXU are incompletely resolved: Residues 1-8 are disordered in 2LMN

and residues 1-10 are disordered in 2MXU; These residues are thus missing from the respective solid state NMR structural models. For these structures, the missing amino acids have been added and the structures have been equilibrated using all-atom equilibrium molecular dynamics.

Consistent with the solid-state NMR data, these peptide regions remain disordered when molecular dynamics is implemented for these structures. For other structures such as 2M4J and 2NAO, these N-terminal peptide regions remain structured and are largely  -sheet.

With the above caveats acknowledged, an oligomer-selective antibody that was administered at the appropriate time would have the potential to block and neutralize some or all toxic, propagating species of misfolded protein. Prion-like propagation for both A  and tau is supported by multiple lines of in vitro and in vivo evidence. Tau prion-like propagation is discussed further below in the context of tau therapies. Here we focus on the evidence for A  prion-like propagation.

It has been noted that A  peptide exhibits many of the hallmarks of classical prionogenesis, including the adoption of  -rich architectures that are often resistant to proteolytic or denaturing forces, amyloidogenic polymerization that may template the misfolding and aggregation of healthy protein and which results in both structurally and functionally variable -strains‖, and systematic spread along neural connective networks that facilitates intercellular self-propagation (Rasmussen et al. (2017) ; Condello and Stöehr (2018); Watts and Prusiner (2018) ). That said, there is no current evidence of host-to-host transmission and systemic uptake of J o u r n a l P r e -p r o o f toxic A  species in the same sense as for the canonical prion diseases (see however the comments below). We thus refer to the intercellular propagation of misfolded A  as -prion-like‖. Meyer-Luehmann et al. (2006) have observed that brain extract from either human AD patient or APP23 transgenic mice induced numerous A  deposits in APP23 murine hosts beginning 2 months after injection. The same was not observed for WT donors or WT hosts, implying that misfolded A  needed to be present in the donor, and a host A  reservoir that is induction-competent was required for deposition of endogenous A  . The amyloid-inducing activity of extracts was prevented by immunodepletion of A  , and attenuated by pre-mixing with A  -specific antibodies, indicating that A  itself is the key species inducing deposition.

Similarly, weekly interperitonial passive immunization following injection blocked amyloid deposition, reinforcing the potential efficacy of an A  passive immunotherapy. Interestingly, pretreatment with formic acid, which does not dissociate high molecular weight species, but which does dissociate oligomers, completely prevented amyloid deposition of endogenous data.

In the study of Meyer-Luehmann et al. (2006) , no amyloid deposition was observed for aged, synthetic A 40  /A 42  preparations, synthetic oligomers, or even synthetic A  mixed with brain extract from WT mice. This observation, coupled with the above-mentioned blocking activity of A  antibodies, suggests the presence of polymorphic conformations with significantly variable and strain-dependent seeding efficacy, reminiscent of prions. Similarly, intracerebral inoculation of hAPPwt mice-which do not develop amyloid aggregates during their lifespan-with AD patient brain extract also induced pathological A  deposition, after 10 months (Morales et al. (2012) ). Extending the prion analogy, peripheral inoculation intraperitoneally with A  -containing brain homogenates from APP23 and APP-PS1 transgenic mice into either APP23 or R1.40 transgenic mice aged 1-2 months showed induction of cerebral  -amyloidosis in a pattern consistent with the entry of A  -templating seeds at multiple locations in the brain (Eisele et al. (2014) ).

The inability of synthetic A  preparations to induce cerebral amyloid deposition in the study of Meyer-Luehmann et al. (2006) is a cause for justifiable concern. However, more recently, Stöhr et al. (2012) have found that  -amyloid deposition can be induced by synthetic A  Induced A  amyloid deposition has been observed in individuals treated during childhood with cadaveric pituitary-derived growth hormone (c-hGH), which resulted in iatrogenic CJD (Swerdlow et al. (2003) ; Brown et al. (2012) ), but with additional A  amyloid pathology (Jaunmuktane et al. (2015) ). The samples of human c-hGH that induced A  pathology were shown by antibody capture and detection to contain high levels of A 40  , A 42  , and tau protein (Purro et al. (2018) ). This association between peripheral administration and brain deposition of A  was subsequently supported in APP NL-F knock-in mice (Purro et al. (2018) ). These mice were intracerebrally inoculated with the same A  and tau-containing samples of c-hGH that were administered to a subset of the above iatrogenic CJD patients. Intraperitoneal injections, though potentially very interesting, appear not to have been performed. The inoculated mice subsequently developed seeded formation of A  plaques and cerebral A  -amyloid angiopathy (CAA).

Together, the above results provide strong support for prion-like propagation of A  within a single host, and in rare cases between hosts under unusual environmental exposure. Table 1 lists the A  therapeutics currently in clinical trials, along with their epitopes, immunization strategies, selectivity for monomer (M), oligomer (O), or fibril/plaque (P) species.

Also included are antibody backbone isotype, current clinical trials and sponsor, and results for completed trials. For antibodies currently or recently in clinical trials with known epitopes on A 

, figure (3) shows the locations of those epitopes on the primary sequence. We begin by discussing antibodies whose development was relatively early historically, and/or whose clinical trials have been discontinued, moving to antibodies that have been developed more recently. 

The murine version of this antibody (3D6) was generated by active immunization of mice with sequence 1 DAEFR 5 of A  conjugated to sheep anti-mouse immunoglobulin (Johnson-Wood et al. (1997) ). The co-crystal structure of bapineuzumab (4HIX.pdb, see Fig. 4 ) in complex with a fragment of A  (residues 1-6) indicates a bound structure with a helical conformation of the epitope (Miles et al. (2013) ). The side chains of the acidic residues D1 and E3 on A  , as well as the positive N-terminus and aromatic ring of F4, all point into the binding cleft.

In mouse models with > 10 -fold expression of APP over endogenous levels (PDAPP mice), 3D6 was shown to opsonize amyloid plaques, i.e. bind, decorate, and facilitate their clearance, as well as improve synaptic function and cognitive performance in behavioral assays (Bard et al. (2000) ; Spires-Jones et al. (2009); Kerchner and Boxer (2010)).

Bapineuzumab was the first monoclonal antibody to enter human testing after termination of the AN1792 active vaccination trial. Patients in these trials did not demonstrate significant cognitive benefits (Salloway et al. (2009) ), and MRI scans revealed significant adverse issues, including ARIA-H and ARIA-E (van Dyck (2018)). Interestingly, a retrospective review of MRI scans from the phase 2 studies revealed that about 7% (15) of participants had developed ARIA-E during the trials, but remained undetected (10% had ARIA that was detected). 13 of these 15 participants continued to receive additional immunotherapy infusions while ARIA-E was present, and these patients did not develop additional associated symptoms (Sperling et al. (2012) ). The J o u r n a l P r e -p r o o f occurrence of ARIA was strongly related to the ApoE- 4 copy number and arose predominantly during the first three infusions. All phase 3 trials with bapineuzumab were terminated in 2012 when phase III trials NCT00575055 and NCT00574132 showed no clinical benefit (Salloway et al. (2014) ). This decision was not based on any new safety concerns.

Nevertheless, a variant of Babineuzumab with reduced Fc-receptor-mediated effector function, AAB-003, has been developed, and two phase I clinical trials (NCT01193608,NCT01369225) to assess safety and tolerability have been completed. 

The murine precursor to solanezumab, m266, was generated by immunization of mice using A  peptide amino acids (13-28), conjugated to anti-mouse CD3  antibody as an immunogen (Schlossmacher and Selkoe (1993) ). Solanezumab is the humanized monoclonal IgG1 antibody of m266, with epitope in the mid-region of A  , spanning residues 16-26 (PDB structure 4XXD.pdb, see Fig. 4 , Crespi et al. (2015)). The conformation of the epitope is partly extended and partly helical (from F20-S26). Residues pointing into the binding cleft of the antibody are K16, F19, F20, E22, and D23 (F19, F20 are shown in magenta in Fig. 4 ). Solanezumab exhibits strong binding to monomers of A 40  or A 42  , with affinity in the low pM range. It also exhibits cross-reactivity to other proteins from brain homogenates (Watt et al. (2014) ). However, solanezumab has been J o u r n a l P r e -p r o o f Journal Pre-proof thought to deplete brain A  stores by sequestering A  monomers in the blood and thus shifting the brain-blood equilibrium (the peripheral sink hypothesis, see e.g. DeMattos et al. (2001)). In

 and A 42  concentrations in plasma and CSF. Subsequent observations have cast doubt on the peripheral sink mechanism, since a decrease in A  efflux due to m266 was observed in those experiments (Yamada et al. (2009) ), suggesting that the beneficial effect of m266 is due to inhibition of A  forming oligomers and fibrils in the brain. Additionally, antibody binding to A  in plasma substantially increases the half-life of A  , from approximately 5 minutes for free peptide, to up to several days for bound A  (Golde et al. (2009)). While not ruling out the peripheral sink hypothesis, such stabilizing effects must be disentangled from the potential effects of enhanced efflux from the brain.

Results from two large phase 3 trials involving over 2000 patients and completed in 2012 revealed no significant difference in cognition and memory between the solanezumab-treated and the placebo group (Doody et al. (2014) , see Table 1 ). However, subsequent analysis of subgroups in these trials revealed a statistically significant slowing in decline for some cognitive measures (34% slowing vs. placebo for ADAS-Cog 14 and MMSE) and a significant slowing for some functional measures (18% slowing vs. placebo for ADCS-iADL), for the subgroup of mild AD (Siemers et al. (2016) ). This suggested positive therapeutic effects may be seen if administered at earlier stages of progression. Follow-up phase 3 clinical trials (Expedition 3, NCT01900665) in mild AD patients showed no significant benefits over placebo however, and were terminated.

Currently, solanezumab is administered every 4 weeks in the Asymptomatic Alzheimer's Disease trial (A4 trial, NCT02008357), which has enrolled cognitively normal people with amyloid accumulation, to test whether earlier administration may be effective as a preventative measure.

Based on modest but encouraging results from previous clinical trials, the dosage was quadrupled from 400 to 1,600 mg in June 2017.

epitope to residues 30-40 of A 40  (Porte et al. (2012)). The co-crystal structure (PDB 3U0T.pdb, Figure 4 ) shows an extended, linear conformation of the epitope residues 30-40, with the C-terminal more buried than the N-terminal portion (Porte et al. (2012) ). The C-terminal carboxylic acid on residue 40 is critical to ponezumab binding activity; The antibody does not bind

ELISA binding assays along with immunohistochemistry show that ponezumab is not species-selective, binding to monomers, oligomers, and fibrils of A 40  (Porte et al. (2012)). Like solanezumab, it is hypothesized to deplete brain A  stores by sequestering A  in the blood and thus shifting the brain-blood equilibrium (the peripheral sink hypothesis, see e.g. DeMattos et al.

( 2001)).

Ponezumab shows low to moderate ARIA-H and low ARIA-E risk (Landen et al. (2017b,a) ). Although ponezumab revealed a favorable safety profile, two subsequent phase 2 studies revealed no significant clinical benefit, and development of ponezumab for AD was discontinued.

In the development of the murine precursor to crenezumab (MABT5102A or mMABT), liposomes containing anchored peptides using A 1 16   were used to immunize mice (Pfeifer et al. (2008) ;

Adolfsson et al. (2012)). Liposome presentation may present the epitope in  -sheet like conformations. Unusually, there was a shift in the binding epitope position of mMABT, from the region presented in the immunization peptide, to residues 13-24 on A  (Pfeifer et al. (2008)). An

IgG4 backbone isotype was selected for low effector function; The mutation S228P also appears to be implemented, which stabilizes inter-heavy chain disulfide bridges preventing -half-molecule‖ exchange (Silva et al. (2015) ).

The A  epitope comprising residues 13-24 is fairly linearized in the co-crystal structure 5VZY.pdb (Ultsch et al. (2016) , see Fig. 4 ). The antibody has high affinity for higher molecular weight species such as fibrils, plaques, and oligomers, while having low affinity for monomers (Table 1 ).

In phase II trials, crenuzumab lowered oligomer levels in CSF for the majority of patients (89% receiving subcutaneous doses and 86% receiving intravenous doses) (Yang et al. (2019) ), J o u r n a l P r e -p r o o f but PET amyloid load was not lowered, and no significant treatment-related change in cognitive outcome was observed (Table 1) . Incidence of ARIA was low, which, along with the high ARIA incidence of other amyloid-clearing antibodies, suggests that activation of effector mechanisms may be a key event in the clearance of plaque amyloid. Phase 3 trials were halted in January 2019, as interim analyses indicated that the trial was unlikely to reach its primary endpoint of slowing cognitive decline according to the CDR-SB test.

Motivated by the need for preventative intervention to modify the future course of the disease, the Alzheimer Prevention Initiative (API) is currently studying the efficacy of crenezumab vs. placebo for 300 asymptomatic presenilin-1 E280A mutation carriers, who are autosomal-dominant for AD (Tariot et al. (2018) ). This study will inform on the efficacy of crenezumab to either delay the onset, slow the decline, or prevent cognitive impairment in individuals with preclinical autosomal-dominant AD.

Rather than using an active immunization step, gantenerumab is a fully human IgG1 antibody selected from synthetic human combinatorial antibody libraries (HuCALs, Knappik et al. (2000)) using phage display, followed by in vitro affinity maturation using CDR cassette exchanges (Steidl et al. (2008) ). In the context of antibodies targeting influenza hemagglutinin, phage display libraries from isolated B cells have been used to isolate rare lead antibodies that were not detected directly by next-generation sequencing (Rajan et al. (2018) ).

Peptide screening assays (Bohrmann et al. (2012)) indicate that gantenerumab is capable of binding two discontiguous regions of A  , with highest affinity at residues 2 -11 and 18 -27 .

Such a binding mode to separate epitopes may allow binding to N-terminal truncated A  species, and facilitate avidity-enhanced binding on the fibril surface (Bohrmann et al. (2012)), potentially involving both variable domain arms of the antibody. It also implies a flexible binding pocket on the antibody that is capable of binding several sequences. This has implications for both the selectivity of the antibody for distinct A  species (the antibody binds all species, Table 1) , and the potential for off-pathway reactions. The structure of the antibody-epitope (PDB 5CSZ.pdb)

shows amino acids 1-10 of A  are extended in a linearized conformation (see Figure 4 ). (2012)).

Thus, gantenerumab preferentially interacts with aggregated A  , and may facilitate degradation of opsonized amyloid plaques by recruiting microglia and activating phagocytosis (Bard et al. (2000) ). These early studies indicated that even modest levels of peripherally administered antibody were able to cross the blood-brain barrier and enter the CNS, bind to plaques, and induce clearance of amyloid. Treatments combining BACE inhibitor R7129 with gantenerumab have shown an additive effect between the two drugs in APP transgenic mouse models, in that the combination reduced A  levels and plaque burden more strongly than either treatment alone (Jacobsen et al. (2014) ). In phase I clinical trials, gantenerumab was found to reduce plaque burden in AD patients, prompting further trials, including two ongoing additional phase III trials for patients with prodromal (NCT01224106) and mild (NCT02051608) AD (Table   1 ). As well, gantenerumab and solanezumab have both been tested in patients carrying autosomal-dominant mutations for AD in the DIAN-TU clinical trial (NCT01760005), discussed further below.

A disease-modified form of A  peptide (A 3 42 p   ) may occur through protease-cleavage of the first two amino residues, followed by cyclization of the side chain of glutamic acid residue E3 to pyroglutamate. This cyclization occurs either spontaneously or by the enzyme glutaminyl cyclase.

A 3 42 p   plays an important role in early AD pathology by seeding toxic oligomeric species (Wirths et al. (2009) (2015)). About 25% of patients taking gantenerumab developed ARIA-E, though mostly asymptomatic.

The latest phase II trial (NCT03367403) consists of 3 arms: One with both donanemab and the BACE inhibitor LY3202626, one with donanemab and placebo, and one with two placebos.

The arm of this trial involving BACE inhibitor was discontinued in October 2018, however the other two arms remain ongoing.

Aducanumab is a fully human IgG1 monoclonal antibody derived from a blood lymphocyte library that was collected from elderly patients who showed either no signs of cognitive impairment or unusually slow cognitive decline. It thus relies on the assumption that these patients would generate antibodies protective against AD. B cells are isolated from peripheral blood lymphocyte preparations by anti-CD22-mediated sorting, and were cultured on gamma-irradiated human peripheral blood mononuclear cell feeder layers. Supernatants from these patients' B-cells were screened for binding to A  plaques in tissue sections, in vitro binding to A 40  and A 42  , and extended conformation (Arndt et al. (2018)). The alanine residue A2 in A  points away from the antibody, and so is not included in the putative epitope in Table 1 . The complex is stabilized by a cation-pi interaction (Dougherty (2013) ) between an arginine on the antibody and phenylalanine F4 on A  , which likely contributes to the high binding affinity. Otherwise, the binding pocket is relatively shallow compared to other N-terminal binding antibodies such as bapineuzumab and gantenerumab.

Biogen initially reported in March 2019 that aducanumab did not meet its primary end points for slowing cognitive decline in phase III clinical trials (NCT02477800 (ENGAGE), NCT02484547 (EMERGE)), although the antibody was effective at clearing A  plaque from patients, likely through FcγR-mediated phagocytosis by microglia (Sevigny et al. (2016) ). The high affinity for abundant, insoluble A  along with significant effector function of the antibody gave rise to a 37% or 41% risk of ARIA-E,H in the two highest dosage groups (Sevigny et al. (2016) ). The trial recruited patients in the early symptomatic phase of AD, however it appears that this stage is already late in deriving clinical benefit by targeting A  , and tau pathology and neuroinflammation may be the predominant neurodegenerative drivers at this stage.

Biogen initially halted development of aducanumab in March 2019 after the preliminary data from the EMERGE and ENGAGE trials suggested it would not meet primary endpoints. The initial conclusion that there was a failure to show cognitive benefit indicated that removal of amyloid was ineffective-at least on the time scale of 2-3 years-for patients who have progressed to mild or moderate stages of the disease (Selkoe (2019)). It should be noted however that these results do not preclude drugs such as aducanumab as potentially effective in prodromal cases of AD. There was also the speculation that, while on average there appeared to be no significant cognitive benefit, some patients could have experienced favorable effects.

On October 22, 2019, Biogen announced that the interim futility analysis was incorrect, and that subsequent analysis of a larger data set instead showed EMERGE had in fact met its primary endpoint (data was presented at the 2019 CTAD conference (Haeberlein et al. (2019) )).

Specifically, patients on the highest dose (titrated to 10 mg/kg over 26 weeks) had a significant reduction in decline in cognition, according to CDR-SB test-the primary endpoint. As well, the high-dose group declined less on secondary cognitive endpoints such as the MMSE, ADAS-Cog, and ADCS-ADL-MCI tests. The lower dose group (titrated to either 3 mg/kg (ApoE- 4  ) or 6 mg/kg (ApoE- 4 )) appeared to show slowing of cognitive, but the changes did not reach J o u r n a l P r e -p r o o f statistical significance.

Oddly, the cognitive trajectories in the ENGAGE trial appear significantly different from those in the EMERGE trial, and the ENGAGE study arm did not meet its primary endpoint. This was explained through differences in the enrollment between the study arms during the dosing titration increase. Unlike the EMERGE data, the ENGAGE data also did not show dose response for phospho-tau and total tau biomarkers. That said, a subgroup analysis (post protocol version 4) of patients in both arms who had received 10 or more 10 mg/kg doses of aducanumab did show dose-dependent and statistically-significant reduction in CDR-SB-measured cognitive decline.

Based on this latest data and the revised analysis, eligible patients from phase III trial arms have been asked to return for continued dosing and testing, and Biogen has announced plans to apply in early 2020 for regulatory approval for aducanumab in the U.S.

SAR-228810 is a humanized IgG4 antibody based on murine antibody 13C3. 13C3 was itself raised by immunization using incubated synthetic A 42  , which forms multimers/oligomers of various size (Schupf et al. (2008) The precise epitope location has not been determined/disclosed, but is likely in the N-terminal region between residues 4-20 (Ravetch and Fukuyama (2009) (2018)). The antibody binds to protofibrillar and fibrillar aggregates with approximately 100-fold selectivity over A  monomer in ELISA assays.

SAR-228810 has two mutations on a human IgG4 backbone, one (S241P) that promotes inter-heavy chain disulfide bridges preventing -half-molecule‖ exchange (Angal et al. (1993) ), and another (L248E) that significantly reduces effector function (Reddy et al. (2000) ). The antibody has low binding affinity for activating Fc  Rs on human microglia, and shows no binding to complement C1q, which is a pro-inflammatory component of the innate immune system J o u r n a l P r e -p r o o f Journal Pre-proof (Pradier et al. (2013) 

Patients carrying the E22G (APP E693G) mutation of A  (the -Arctic‖ mutation) show particularly high levels of A  protofibrils (Nilsberth et al. (2001) ), abundant parenchymal plaques but without a dense amyloid core (Basun et al. (2008) ), and are autosomal-dominant for early-onset AD. (Weggen and Beher (2012)). Murine antibody mAb158 was generating by immunizing mice against E22G mutant A  protofibrils (Tucker et al. (2015) ). Soluble protofibrils are an abundant toxic species in AD brains (Sehlin et al. (2012) ). mAb158 binds to protofibrils with much higher affinity than monomers (Englund et al. (2007) ), and reduces protofibrils in the brain and CSF of transgenic mice expressing both the above Arctic mutation and the -Swedish‖ double mutation in APP (K670N/M671L; tgArc-Swe mice) (Tucker et al. (2015) ).

Studies in embryonic mouse-derived co-cultures of astrocytes, neurons, and oligodendrocytes

show that mAb158 can protect neurons from A 42  -induced death by preventing the accumulation of A  through astrocyte-uptake (Söllvander et al. (2018) ).

BAN-2401 is the humanized version of the mAb158. In phase 2b trials, the antibody reduced plaques by 93% in patients in the highest dosage arm (Swanson et al. (2018) ). This is consistent with immunohistochemical observations that the antibody binds to plaque as well as high molecular weight oligomer (ProMIS Neuroscience (2018)). As a likely consequence to plaque binding however, ARIA-E was observed in 14.6% of APOE  4 carriers in the largest, most-frequent dosage arm (10 mg/kg bi-weekly). In this dosage arm however, cognitive decline was slowed by 47% on the ADAS-Cog, and by 30% on the ADCOMS (Swanson et al. (2018) ). In a controversial decision arising from safety concerns related to ARIA, european regulators limited the number of APO  4 carriers in the highest most frequent dosage arm compared to the placebo arm and other dose groups, partway through the trial. The concern arising over this imbalance was then whether it contributed significantly to the appearance of a benefit in the high dosage arm, MEDI-1814 has an IgG1 backbone, but has a triple mutation in its Fc tail to reduce effector function. Consistently, initial phase 1 results (NCT02036645, clinicaltrials.gov]) report no serious adverse effects, and MRI scans showed no evidence of ARIA (Ostenfeld et al. (2017) ).

Participants in phase I clinical trials showed also no signs of either ARIA-H or ARIA-E.

KHK6640 is a humanized IgG4 antibody with mutations to limit effector function. CSF analysis 2019))). The rationale is based on the hypothesis that A  may be bound to albumin and the complex then circulates in plasma, so extracting this plasma could flush amyloid from the brain, similar to the peripheral sink hypothesis but without potential confounding effects of stabilized bound complexes in the blood. As well, albumin has been shown to have antioxidant, immune-modulatory, and anti-inflammatory properties (Gleeson and Dickson (2015) ; Bar-Or et al. (2006) ), which may diminish neuroinflammation.

The AMBAR study revealed some impressive data that at the very least warrants further studies. Perhaps consistently with the performance of IVIgs in previous clinical trials for AD, there was no significant effect on whether PE was accompanied with IVIGs; There was also no significant effect on whether PE also had low or high dose albumin replacement. Cognitive endpoints such as CDR-SB showed significant difference from placebo and even potential improvement among mild AD participants (Páez et al. (2019) ). Among moderate AD participants there was a statistically significant reduction in cognitive decline by CDR-SB. There were also significant differences in both groups according to psychometrics such as the Clinical Global 

Many bacteria form functional amyloid assemblies on their cell surface, which aid in biofilm formation and other community behaviors involving cell-cell interactions (Zhou et al. (2012) ).

These amyloids can enhance virulence, facilitate cell adherence and invasion, and aid the survival and spread of the pathogen (Gerven et al. (2018) ). M13 is a filamentous bacteriophage that recognizes amyloids on the bacterial cell surface through a two-domain fragment of the phage capsid protein g3p (gene 3 protein). NMR studies have shown that g3p can also recognize A  fibrils, predominantly through an epitope involving the middle and C-terminal residues of A  (Krishnan et al. (2014)).

NPT088 is an antibody made from a fusion of g3p with the Fc region of a human IgG1. The chimeric antibody targets many different amyloids, including amyloid beta, tau, alpha-synuclein, antibody light chain, and transthyretin (Messing (2016)). The recognition portion is thus referred to as a general amyloid interaction motif (GAIM). Based on the ability of the antibody to remove A  plaque, reduce phospho-tau pathology, and improve cognitive performance in mouse models (Levenson et al. (2016) ), NPT088 has moved into clinical trials (phase I, NCT03008161). A second candidate utilizing GAIM recognition (NPT189) is also currently in phase I clinical trials (NCT03610035). (2010)). Given the early dates of these initial findings, the prospects of the above antibodies entering clinical trials are uncertain, but would appear to be unlikely. . (2010a,b) ). The antibody shows preferential binding affinity for 3-24mers of A  , vs.

monomeric A  or A  plaque, and no visible binding to vascular amyloid. In fact, the binding epitope sequence of 3B3 was not able to be determined by linear epitope mapping in ELISA, as the antibody failed to bind any members of the overlapping peptide set, even at high concentrations.

However it could bind A  1-20 peptide, which was used as a positive control. Similarly, binding of 3B3 to ADDLs was not blocked by short linear peptides of  10 amino acids in A 42  , but interestingly binding was blocked by A  1-28, indicating an epitope based on a conformational structure also found in A  1-28 fragments (and possibly also A  1-20). 3B3 was observed to be effective in blocking the assembly of ADDLs, as observed through fluorescence quenching of flourescein-labelled oligomers by unlabelled monomers, and fluorescence polarization increase as oligomers assembled (Acton et al. (2010a,b) ). The murine precursor 3B3 was able to restore long-term potentiation in rat hippocampal slices (Cline et al. (2019)), and to reverse the dysregulation of cytosolic calcium concentration ).

PMN310 is a humanized IgG4 antibody that binds a conformational epitope consisting of 13 HHQK 16 , specifically when presented on low-molecular weight oligomers and protofibrils. The antibody shows no apparent binding to A  monomer, amyloid plaque, or vascular deposits (Gibbs et al. (2019) ). This is a potential advantage to avoid target distraction by more abundant monomers, and if clearing plaque does not correlate with cognitive benefit. The epitope was predicted based on computational modelling of A  oligomers, by using molecular dynamics to find the regions most-likely to be solvent-exposed in a protofibril ; Cashman and Plotkin (2016)). Immunization proceeded by conjugating cyclic peptides containing the epitope to KLH as an immunogen, wherein the cyclic peptide constructs were chosen based on oligomer-selective epitope scaffolding (Silverman et al. (2018) ). Immunohistochemical studies show that PMN310 exhibits essentially no binding to A  plaque in AD brain samples, supporting greater selectivity of PMN310 to A  oligomers and reduced risk of ARIA-related adverse effects 

Tau protein binds to and stabilizes microtubules, enabling transport of cellular cargo along neurons in the central nervous system (Drubin and Kirschner (1986) ). Through microtubule regulation, tau mediates neuronal signaling and synaptic plasticity (Arendt et al. (2016)). Tau There are more than 20 neurodegenerative diseases associated with the pathology of tau (Williams (2006) ). In AD, pathologic tau manifests as neurofibrillary tangles (NFTs) in the brain.

In healthy, terminally differentiated, post-mitotic cells such as neurons, cytosolic tau is phosphorylated at about 2 sites on average, while in NFTs, tau is hyperphosphorylated (2019)). Jackson et al. (2016) have shown that immunodepleting brain lysates from P301S transgenic mice with phospho-specific antibodies abolishes prion-like tau seeding activity in HEK cell cultures and transgenic P301S mice. Only oligomers containing more than about 10 tau molecules were seed-competant. Thus, tau hyperphosphorylation may inadvertently contribute to a conformational reorganization inducing large tau oligomers to become prion-like. The relative abundance in human brain of prion-like, phosphorylated tau, rather than the total amount of inert,

insoluble tau, appears to correlate with AD patient longevity (Aoyagi et al. (2019)).

In 2019)). Alzheimer's disease appears to manifest as an A  -exacerbated tauopathy. Roberson et al. (2007) found that in transgenic mice overexpressing human A  , reducing endogenous tau levels could prevent AD-like behavioral decline. High A  level was unaffected.

Reducing tau could also protect neurons from excitotoxic dysfunction. Similar findings have been observed by administration of tau antibody 43D in triple transgenic (3×Tg) mice, where the antibody reduced total tau and hyperphosphorylated tau, decreased APP production and thus A  production, and increased microglial activation and complement C1q and C9 levels (Dai et al.

(2017)). These latter effects resulted in less A  plaque load. In another study using a phospho-specific C-terminal tau antibody, in addition to the expected decreases in soluble and insoluble tau, A  deposits were decreased by  84% (Rajamohamedsait et al. (2017) ). Taken together, the above evidence suggests that reducing tau levels via passive or active J o u r n a l P r e -p r o o f Journal Pre-proof immunotherapies could thus represent an effective strategy for treating AD and related tauopathies. A concern in pursing such an approach however is to avoid targeting functional tau.

An antibody selective to pathological tau is most desirable as a safe and effective therapeutic.

Current immunotherapies directed against tau are listed in Table 2 , along with their epitopes, immunization strategy, binding selectivity if known, antibody backbone isotype, and current and completed trial information. For antibodies currently in clinical trials directed against tau, figure (5) shows the locations of their corresponding epitopes on the tau primary sequence.

The order of antibodies below is loosely based on their current stage in clinical trials as well as their historical development. Specific epitope locations are listed in Table 2 .

As described above, extracellular tau is thought to mediate the onset, cell-to-cell propagation, and neurodegenerative progression of AD as well as the other tauopathies, including progressive supranuclear palsy (PSP), chronic traumatic encephalopathy (CTE), and some cases of frontotemporal lobar degeneration. BIIB092 was developed to target extracellular tau. Bright et al. (2015) reprogrammed skin cells from patients with sporadic or presenilin-1-mutant AD to induced pluripotent stem cells (iPSCs), which they then differentiated into cortical neurons. Compared to age-matched control neurons, the AD-derived cells secreted N-terminal fragments of tau (eTau) into the extracellular space. Electrophysiological analysis showed that the secreted eTau induced neuronal hyperactivity, which could itself increase A  production in a kind of toxic pas de deux (Ittner and Götz (2010) ; Dai et al. (2017)).

Bright et al.'s immunization strategy was to raise a mouse monoclonal antibody by standard immunization using in vitro aggregated full-length (2N4R) tau, rather than selectively J o u r n a l P r e -p r o o f Journal Pre-proof presenting a particular epitope. The purified antibodies were then screened and selected afterward for high binding affinity to both the secreted eTau and full-length tau, as well as their ability to ameliorate eTau-induced neuronal hyperactivity.

BIIB092, the humanized version of the above mouse monoclonal antibody (IPN002), is an IgG4 monoclonal that recognizes a linear, non-phosphorylated epitope in the N-terminal region of tau consisting of amino acid residues 15 AGTYGLGDRK 24 (Griswold-Prenner et al. (2014); Qureshi et al. (2018) ).

In phase 1 trials, BIIB092 was found to be safe and well-tolerated; there were no severe adverse effects in the low and moderate dose arms, and 8% (2/24) severe adverse effects in the highest dosage arm. These were not considered to be related to the drug, and they all eventually for AD and PSP (see Table 2 ). The PSP trial (PASSPORT, NCT03068468) was discontinued in December 2019, for failure to meet or either primary or secondary endpoints (AlzForum.org;

Gosuranemab, Biogen's Anti-Tau Immunotherapy...). The AD trial (TANGO trial, NCT03352557) is currently ongoing, and has a completion date of 2024.

The mouse monoclonal IgG2b antibody HJ8.5 has been raised by conventional immunization using recombinant full-length human tau. The state of tau (monomeric, aggregated) as well as the adjuvant immunogen has to our knowledge not been disclosed ). The antibody is selective to human tau and does not bind to mouse tau. The epitope has been published as either 25 DQGGYT 30 (Yanamandra et al. (2013) ) or 22 DRKDQGGYTMHQD 34 ). The discontiguous epitope is present in an aberrant conformation of tau that is present in a nonfilamentous, soluble form of tau indistinguishable from NFTs by ELISA and immunoblotting, and is also present in PHFs (Weaver et al. (2000) ). Reactivity to the antibody appears to correlate with the severity and progression of AD (Vitale et al. (2018) ). Passive immunization experiments 2017)), but which is also discontiguous like MC-1, and involves amino acids 7 EFE 9 and 312 PVDLSKVTSKC 322 (Alvarado et al. (2016)).

One possible issue in this regard is the potential loss of reactivity to N-terminally truncated species. LY3303560 is selective for tau aggregates over monomer (Alam et al. (2017) 

The development of semorinemab (RO7105705) seeks to minimize Fc  receptor activation, and seeks to maximize binding across different extracellular tau species. The argument for a pan-tau antibody is that nearly all antibody-accessible tau is extracellular, and that any extracellular tau could drive pathology and so is a viable target for elimination.

-Effectorless‖ antibodies may be engineered by making D265A and N297G (DANG) mutations in the Fc region of the antibody, which when combined, abolish binding to microglial Fc  receptors. (Couch et al. (2013) ). Preclinical studies have shown that effectorless antibodies J o u r n a l P r e -p r o o f protected neurons from toxicity better than the unmodified version did, and that effectorless antibodies can remove aggregates in mouse models as well as normal antibodies ). Similarly, RO7105705 is a humanized IgG4, which only weakly activates microglial Fc  receptors, minimizing inflammation. It should be noted that strict adherence to IgG4 antibodies to minimize effector response is not universally embraced. It is argued for example that the benign safety profile of the active immunotherapy AADvac-1, which induces predominantly an IgG1 antibody response, implies that at least pathological tau can be safely targeted with IgG1

antibodies (Novak et al. (2017 (Novak et al. ( , 2018a ).

Antibody generation for RO7105705 likely proceeded by vaccination of mice with recombinant, phosphorylated, and oligomerized human tau (Adolfsson et al. (2016)). Antibody selection then proceeded by assaying for binding to full-length tau, then to phosphorylated tau and oligomerized tau, with the aim to find antibodies that bound equally well to both tau and post-translationally modified tau. Binding to all 6 isoforms of human tau was also used as a selection criterion. To maintain pan-tau properties, epitopes that mapped to regions with a high density of phosphorylated residues (S,T,Y) were avoided. The epitope of RO7105705 is likely within residues 2-24 ( 2 AEPRQEFEVMEDHAGTYGLGDRK 24 , Adolfsson et al. (2016)). The antibody reacts with all 6 isoforms of human and primate tau, but not mouse tau (residues 19 GLGDRK 24 are absent in mouse tau), implying that the epitope resides in the C-terminal portion of the above sequence.

RO7105705 was found to protect neurons from tau-mediated toxicity in cell-based studies.

In transgenic mice expressing human mutant P301L tau, 13 weeks of treatment with either 3, 10, or 30 mg/kg of RO7105705 reduced pathological tau in the brain in a dose-dependent fashion; The antibody also raised tau levels in blood plasma, implying target engagement and stabilization in the periphery, akin to the peripheral sink mechanism proposed for A  antibodies such as solanezumab and ponezumab. Chronic dosing was safe in both mice and cynomolgus monkeys (Ayalon (2017)).

Phase 1 studies have shown that the antibody is safe and tolerable in healthy volunteers, even at extraordinarily high single doses of 16,800 mg (Kerchner et al. (2017)). Two current phase II trials, one for prodromal/probable AD participants (TAURIEL trial, NCT03289143), and one for moderate AD participants (NCT03828747) are ongoing.

Journal Pre-proof

Healthy human subjects who are at risk for AD, either because of advanced age or genetic

predisposition, yet who exhibit no AD symptoms or unusually slow progression, provide a valuable therapeutic resource for the isolation of antibodies to AD-related proteins. This strategy has been also exploited in the development of aducanumab, as mentioned above in Section 4.7.

Tau disease-selective monoclonal antibodies isolated from memory B cells in such healthy subjects with no signs of a neurodegenerative tauopathy are expected to have excellent safety profile and lack of immunogenicity, and to be already evolutionarily optimized and affinity matured by the human immune system. This therapeutic strategy has motivated the development BIIB076 is a fully human IgG1, which binds with subnanomolar affinity to both human and cynomolgus monkey recombinant tau. (Czerkowicz et al. (2017)). It is a -pan-tau‖ antibody, recognizing monomeric and fibrillar forms, as well as tau isolated from healthy human and Alzheimer's disease brains. No cell-based or mouse preclinical work with this antibody has been published. BIIB076 exhibited no adverse toxicology or pathology in cynomolgus monkeys, and CSF total and free tau levels were significantly reduced in the highest BIIB076 dose animals in this study (Czerkowicz et al. (2017)). These results established a positive safety profile for inclusion of BIIB076 into phase I trials. This trial (NCT03056729) recruited healthy and mild-AD volunteers to monitor adverse events and pharmacokinetics ( Table 2 ). The trial protocol was modified in June 2019, to drop the more advanced AD cohort and adopt adverse events as the sole primary outcome.

The trial has recently finished in March 2020.

Phosphorylation at S422 is a part of the maturation process of PHFs, and generally precedes proteolytic cleavage at least at some locations such as D421 (Guillozet-Bongaarts et al. (2006) ).

Journal Pre-proof pS422 is prominent in early stages of Alzheimer's disease and persists until late-stages, making it an attractive target. Active immunizations also support pS422 as a viable target. In a transgenic mouse model overexpressing a mutant, alternative isoform of tau (the 412 amino acid isoform missing N-terminal domain N2, with mutations G272V and P301S, under a neuron-specific promotor (Thy1.2)), active immunization with a peptide containing pS422 decreased insoluble tau in the brain, and this decrease correlated with significant memory improvement using the Y-maze spatial memory task (Troquier et al. (2011) ).

Polyclonal antibodies to epitopes containing phospho-serine 422, which resides near the C-terminus of tau, have been shown to be reactive to brain extracts from patients with AD, PSP, corticobasal degeneration (CBD), and other neurodegenerative diseases, while such polyclonal antibodies are unreactive to controls (Bussière et al. (1999) ). pS422 has thus been identified as a pathological epitope found in several diseases with neurofibrillary degeneration. Polyclonal pS422 antibodies recognized intra-neuronal NFTs in cells that had lost their integrity; extra-neuronal NFTs were also recognized. In contrast to some other phospho-selective antibodies with epitopes at sites pT153, pS262, and T231, staining of pre-tangles with pS422 was rare (Augustinack et al.

( 2002)).

RG7345 was developed from mice and/or rabbits immunized with phospho-peptide 416 SIDMVD(pS)PQLATLAD 430 coupled to KLH, where antibodies were subsequently screened for selective binding to the peptide with pS422 (Bohrmann et al. (2010)). The antibody was then recombinantly expressed with a murine IgG1 or a human IgG1 isotype. The isotype of the humanized antibody has not been disclosed to our knowledge, and patent protection specifies both IgG1 and IgG4 isotypes (Emrich et al. (2016) ). In triple transgenic mice expressing mutant APP, 

There is some evidence that antibodies binding to the mid-region of tau are more effective at blocking cell-to-cell propagation than those targeting the N-terminus, though this notion is still speculative, as some N-terminal binding antibodies such as BIIB092 and ABBV-8E12 were selected for blocking cell-to-cell transmission of pathology, and findings for these antibodies as well as RO7105705 indicate at least partial efficacy of N-terminal antibodies in blocking the spread of pathogenic tau. 2018)). One explanation is that these antibodies target the N-terminal region of tau as that tends to be where affinity is highest, however N-terminal tails are thought to be exposed outside the core of tau fibrils where they may be cleaved by proteases (Courade et al. (2018) ). Thus at least some proteopathic tau seeds may be missing N-termini.

In P301L human tau transgenic mice given hippocampal injections with AD brain homogenate or PHF from AD brains, intraperitoneal administration with high doses (30 mg/kg) of antibody D were able to block the progression of tau seeding pathology to distal brain regions (Albert et al. (2019)). This result was recapitulated for a tau fragment injectant containing only the 4 microtubule binding repeats with mutation P301L, which antibody D does not bind given its J o u r n a l P r e -p r o o f Journal Pre-proof epitope location. This latter result explicitly demonstrates blockage of seeded endogenous tau.

UCB0107 is the humanized version of antibody D. Its specific isotype is likely an IgG4 (AlzForum.org; Can Clinical Trials...; Knight et al. (2017) ). The developers are fairly agnostic as to whether at least some effector function is desirable or if it is to be avoided. The epitope of UCB0107 has been mapped to residues 234 SPSSAKSRLQTA 246 , which is at the end of tau's second proline-rich region and just before its first microtubule-binding domain. The (undeposited) co-crystal structure contains a bound tau peptide with residues 234-244, at least part of which is in a helical structure (Knight et al. (2017)).

Two phase I clinical trials for UCB0107 (NCT03464227 and NCT03605082) were completed in December 2018 and March 2019 respectively, but results have not yet been reported.

A phase I trial for PSP initiated in December 2019 (NCT04185415) has been halted as a precautionary measure due to the COVID-19 pandemic.

Functional tau that is bound to axonal microtubules is hypo-phosphorylated, while aggregated tau in AD is hyper-phosphorylated. This post-translational process along the pathological maturation pathway can provide unique epitopes that are distinct from the physiologically active pool of tau.

JNJ-63733657 is a monoclonal antibody likely of IgG1 isotype, with selective affinity for paired helical filament (PHF) tau and tau phosphorylated at select sites described below.

The murine precursor to JNJ-63733657, PT3, was derived from immunization of a Balb/c mouse with enriched PHF-tau (ePHF-tau) from AD brain. The antibody was tested for target selectivity to phospho-tau versus non-phospho-tau by ELISA, western blot, and immunohistochemistry (IHC). The humanized antibody B296 has the same CDR sequences as PT3, and was humanized by pairing variable regions of PT3 with a human IgG1/  constant region ); This antibody was affinity matured to generate JNJ-63733657.

B296 has a strong affinity of 27 pM to PHF tau. B352, an IgG4 variant of B296 with the same variable regions, had an affinity of 43 pM to PHF-tau. Both of these antibodies did not bind unphosphorylated tau.

The antibody binds to a phosphorylated epitope in the proline rich domain P2 of tau protein between residues G204-K225. For high-affinity binding by the murine precursor PT3 ( < 25 D K nM to the phospho-peptide), either T212 or T217 must be phosphorylated. If both T212 and T217

J o u r n a l P r e -p r o o f ). The epitope of PT3 is distinct from other reported epitopes of phospho-dependent anti-tau antibodies, such as AT8, AT180, and anti-tau pS422.

The co-crystal structure of the antibody-epitope has been obtained but not yet deposited on the PDB. Structurally, the bound epitope is extended and linearized when bound to the antibody in the co-crystal structure.

Similarly to the assay described above in Section 5.7, a cell-based assay may be used to measure the inhibition of tau propagation by anti-tau antibodies, when the cells are transfected with a co-incubated mixture of AD brain homogenate and anti-tau antibody. Vandermeeren et al. An in vivo P301L tau transgenic mouse model was tested by Mercken et al. (2018) , wherein intraperitoneal injection of PT3 (or controls) was followed by seeding induction by intracranial injection of AD-brain-derived PHF-tau. In this model, peripheral administration of PT3 was able to significantly reduce the seeded propagation of tau aggregation in mouse brains.

A recent phase I clinical trial in healthy Japanese participants aged 55-75 finished in July 2019 (NCT03689153). JNJ-63733657 was found to be generally safe and well-tolerated. CSF levels were  0.2% of serum levels, and a dose-dependent reduction in phospho-S217 tau was J o u r n a l P r e -p r o o f Journal Pre-proof observed in the CSF following antibody administration (Galpern et al. (2019) ). A 2nd phase I trial testing safety and pharmacokinetics in both healthy participants or participants with prodromal or mild Alzheimer's disease has recently completed in December 2019. Results have not yet been reported.

As mentioned above in the context of A  -targeting therapies (Section 4.13), NPT088 is a generic amyloid-binding antibody that is reactive to both A  and tau. In transgenic mouse models overexpressing mutant APP that elevates levels of A  (Tg2576 mice expressing K670N/M671L APP) Y-maze performance was significantly increased and novel object recognition was significantly improved after weekly intraperitoneal dosing with NPT088 for 10 weeks or 14 weeks respectively (Levenson et al. (2016) ). Staining of brain sections with a PHF-tau-specific monoclonal antibody (AT8, see below) shows a reduction in PHF in these systemically treated mice compared to controls (Levenson et al. (2016) ). Since NPT088 is specifically reactive to amyloid fibrils (Table 1) , these results suggest that binding to monomeric tau may be unnecessary for efficacy of an immunotherapeutic, and that targeting oligomers and/or fibril species may be sufficient. This notion bears some analogy with the problem of avoiding target distraction due to abundant monomer concentration for A  therapeutics. Another potential advantage of a therapeutic such as NPT088 with reactivity to both A  and tau is that if a combination therapy is required, NPT088 alone may still be effective clinically.

BIIB080 is an antisense oligonucleotide (ASO) that silences the translation of tau mRNA as described below, so it can be thought of as a passive but not a protein immunotherapeutic. This antisense oligonucleotide targets the beginning of exon 5 (at the 5' end). In a study investigating 31 candidate morpholinos, the morpholino targeting this specific site (extended by 3 bases to bracket the intron-exon boundary of the splice acceptor site) was one of the most effective in significantly reducing total MAPT transcript levels (Sud et al. (2014) ). Exon skipping of exon 5 (as well as 1 and 7) results in changing the open reading frame of the mRNA, leading to a premature stop codon likely resulting in nonsense-mediated decay.

In preclinical studies, the ASOs of BIIB080 had a phosphorothioate backbone to improve nuclease resistance and promote cellular uptake. In human P301S tau-transgenic mice, reduction J o u r n a l P r e -p r o o f Journal Pre-proof of tau expression by BIIB080 ASOs resulted in fewer tau inclusions, reversal of preexisting phosphorylated tau and Thioflavin S pathology, reduced rates of neuronal death, and extension of mouse survival time (DeVos et al. (2017) ). Although the effects of tau knock-down by an anti-sense oligonucleotide may be different in humans than in a mouse model that by design is overexpressing mutant tau, these encouraging initial results have led to an active clinical trial (NCT03186989) for BIIB080.

DC8E8 will bind to any one of 4 separate epitopes on tau having similar sequence motif (Kontsekova et al. (2014) structures of the DC8E8 Fab in the complex with a 14-mer tau peptide have been determined; The antigen-binding region from 5MO3.pdb is shown in Fig. 6 , which contains the epitope K 298 HVPGGGS 305 . The epitope is mainly linear, with a  -turn in the glycine region. The backbone contacts in the glycine region are apparently important for antibody affinity: The above 6-residue epitope motifs compete with tau 151 391  for binding to antibody DC8E8, but removal of the C-terminal glycine from the epitope eliminates the ability of the corresponding peptide to compete (Kontsekova et al. (2014) ).

Binding of the antibody to this epitope on tau interfered with pathological tau-tau interactions in an in vitro assay, reducing the amount of oligomeric tau by 84% (Kontsekova et al.

(2014)). In vivo, DC8E8 significantly reduced the amount of insoluble oligomerised tau and the number of early and mature neurofibrillary tangles in transgenic mouse brains. The humanized version of the murine antibody DC8E8 is AX004. AX004 has been shown to block cell-to-cell propagation of tau by preventing neuronal internalization of extracellular tau, as mediated between the microtubule binding domain containing DC8E8 epitopes, and Heparan Sulfate Proteoglycans (HSPGs) on the neuron surface (Weisová et al. (2019) ). As mentioned above in Section 2.1.2, the epitope when used as an active vaccine in humans is AADvac-1.

The murine monoclonal IgG1 antibody AT8 (Mercken et al. (1992) ; Goedert et al. (1995) ) is a widely used anti-tau antibody to probe phosphorylated PHFs and assess tau phosphorylation at J o u r n a l P r e -p r o o f Journal Pre-proof the amino acids Ser 202, Thr 205 and Ser 208. AT8 recognizes an epitope doubly phosphorylated at serine 202 and threonine 205. AT8 is about 10% cross-reactive to the doubly phosphorylated epitopes S199/S202 and T205/S208. The antibody has no cross-reactivity with unphoshorylated tau. The epitope is 200 PG(pS)PG(pT)PG 207 (Porzig et al. (2007) ). The co-crystal structure of the antigen-binding fragment of AT8 bound to a triply phosphorylated tau peptide 194 Ac-RSGYSSPG(pS)PG(pT)PG(pS)RSR-OH 211 (residues 202-209 are resolved in the crystal structure) is shown in Fig. 6 (Malia et al. (2016) ). Currently AT8 is a useful research antibody; It is unclear if AT8 or a humanized/modified variant will be clinically-relevant.

A tau oligomer-selective monoclonal antibody (TOMA) was developed by Kayed and colleagues (Castillo-Carranza et al. (2014) ), by immunizing BALB/c mice with recombinant (-synthetic‖) tau oligomers. TOMA is a murine IgG1 that selectively recognizes tau oligomers over either monomeric tau or tau NFTs. Its epitope has not been determined/disclosed. In JNPL3 mice, which expresses the mutant human P301L tau, a single intracerebroventricular injection of TOMA cleared tau oligomers but did not significantly affect levels of tau monomers or NFTs. The injection also reversed both rotorod locomotor and Y-maze memory deficits-improvements that 

It is probably too perfunctory to say that current AD therapeutics have had no effect on disease progression. More precisely, AD therapeutics in current clinical trials have generally failed to meet their desired endpoints for the slowing of cognitive decline. It must be noted however that when subgroups of patients from a full cohort have been subsequently analyzed for some therapeutics, cognitive benefits have been observed.

A recurring theme in Alzheimer's therapies is the importance of early or even preventative treatment (Dubois et al. (2016) ; Strobel (2010)). Treating other chronic conditions such as J o u r n a l P r e -p r o o f Journal Pre-proof atherosclerosis (with statins) or hypertension (with lifestyle modification or anti-hypertensives) as early as possible has generally been advised. There are currently 9 prevention-based clinical trials ongoing since 2018 (Cummings et al. (2018)). The occurrence of molecular pathology that appears to undergo prion-like propagation long before any behavioral symptoms manifest may make presymptomatic treatment mandatory for Alzheimer's disease. It also may imply that perhaps some of the halted or discontinued therapeutics described above may be effective when used in a preemptive manner.

In line with the above notions of preemptive therapy, solanezumab and gantenerumab have both been tested in a clinical trial (the DIAN-TU trial, NCT01760005) involving subjects with known autosomal-dominant Alzheimer's disease mutations, and who are within -15 to + 10 years of the predicted or actual age of cognitive symptom onset ; clinicaltrials.gov). Initial topline results on this trial reported in February 2020 indicated that, disappointingly, the trial had missed a cognitive endpoint consisting of a composite of four cognitive tests developed by the Dominantly Inherited Alzheimer Network. Site investigators agreed that the drugs were likely markedly underdosed, both in dosage amount and duration.

Dosage was increased mid-study, but many participants received relatively short durations of the higher dose (roughly 25% of the total duration). Results reported at the April 2020 AAT-AD/PD meeting clarified that the patient cohort was also highly heterogeneous-participants who were symptomatic had descended into moderate dementia before they could be titrated up to a high dose, whereas asymptomatic participants remained stable througout the trial regardless of drug or placebo arm.

In fact, the degree of symptoms appeared to strongly determine how effective the treatment was: Participants who were presymptomatic at baseline improved significantly on logical memory tests and digit symbol substitution tests, and remained stable on MMSE, CDR-SB and functional assessment score (FAS) tests; Participants who were symptomatic did not improve on the logical memory test, and declined on all other tests. These results, though still not conclusive, appear to support the earliest possible intervention.

Additionally, gantenerumab, but not solanezumab, appeared to improve biomarkers. In addition to removing brain amyloid plaques and improving (increasing) the CSF A 42  /A 40  ratio, gantenerumab lowered levels of CSF total tau and phospho-tau181 by 1/ 3  2018)). Combination therapies may be particularly effective in later stages of AD when multiple biochemical pathways have been altered. Even early stages of AD pathology likely involve multiple proteins and druggable pathways, including both A  and tau. Significant mixed pathology is present in up to 70% of dementia cases, further supporting the idea that combination therapies may be most effective and perhaps even necessary. As mentioned in the above section on fibril and oligomer polymorphism, the presence of multiple conformational strains of misfolded protein will limit the efficacy of a single antibody to block propagation and spread of all distinct conformational strains. Concerning challenges for such a combinatoric approach are the timelines required for regulatory approval for separate individual therapeutics, and the disconcerting possibility that if combination therapy is required for benefit, individual drugs will fail to meet primary end points, and support for further development will be difficult to obtain.

By achieving a more thorough understanding of the underlying biochemical mechanisms of Alzheimer's disease, we have been able to develop precision therapeutics that target key processes in the molecular pathology, including the prion-like propagative aspects that are central to the spread of the disease. With each trial, we have gained new insights and learned often painful lessons about the insidious nature of this disease. It is up to us to continue to innovate novel solutions to target and stop the spread of AD pathology, and discover the truly effective therapies that natural biology and rational design will someday provide.

Commercial human albumin preparations for clinical use are immunosuppressive in vitro

Peripherally administered antibodies against amyloid  -peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease

Clinical and Neuropathological Features of the Arctic APP Gene Mutation Causing Early-Onset Alzheimer Disease

The DIAN-TU next generation Alzheimer's prevention trial: Adaptive design and disease progression model

The genetic epidemiology of neurodegenerative disease

Neocortical Neurofibrillary Tangles Correlate With Dementia Severity in Alzheimer's

Human secreted tau increases amyloid-beta production

Identification of human monoclonal antibodies specific for human SOD1 recognizing distinct epitopes and forms of SOD1

Iatrogenic Creutzfeldt-Jakob disease, final assessment

Phosphorylated serine422 on tau proteins is a pathological epitope found in several diseases with neurofibrillary degeneration

Differential in vitro and in vivo binding profiles of BIIB037 and other anti-abeta clinical antibody candidates

Mutations in presenilin 2 and its implications in Alzheimer's disease and other dementia-associated disorders

Alzheimer's Disease. IntechOpen, Rijeka. chapter 8

Spreading of amyloid- peptides via neuritic cell-to-cell transfer is dependent on insufficient cellular clearance

Phase 3 trials of solanezumab for mild-to-moderate alzheimer's disease

The cation- interaction

Tau protein function in living cells

Preclinical Alzheimer's disease: Definition, natural history, and diagnostic criteria

Tau Prion-Like Propagation: State of the Art and Current Challenges

Neuron-to-neuron wild-type tau protein transfer through a trans-synaptic mechanism: relevance to sporadic tauopathies

Anti-amyloid- monoclonal antibodies for alzheimer's disease: Pitfalls and promise

Multiple factors J o u r n a l P r e -p r o o f Journal Pre-proof contribute to the peripheral induction of cerebral  -amyloidosis

Humanized anti-tau(pS422) antibodies and methods of use

Sensitive ELISA detection of amyloid- protofibrils in biological samples

Amyloid-beta oligomerization in Alzheimer dementia versus high-pathology controls

Tau oligomers impair artificial membrane integrity and cellular viability

Widespread tau seeding activity at early Braak stages

P1-052: A single ascending dose study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of the anti-phospho-tau antibody JNJ-63733657 in healthy subjects

The role of functional amyloids in bacterial virulence

Clinical effects of A  immunization (AN1792) in patients with AD in an interrupted trial

Conformation-dependent antibodies target diseases of protein misfolding

Albumin gains immune boosting credibility

Alzheimer's and Parkinson's diseases: The prion concept in relation to assembled A  , tau, and  -synuclein

The propagation of prion-like protein inclusions in neurodegenerative diseases

Monoclonal antibody at8 recognises tau protein phosphorylated at both serine 202 and threonine 205

Quantitative and mechanistic studies of A  immunotherapy

New insights into the role of TREM2 in Alzheimer's disease

Fibril structure of amyloid- (1-42) by cryo-electron microscopy

An overview of FDA-approved vaccines & their innovators

Methods of treating a tauopathy. US Patent

Antibodies to amyloid beta

Abnormal phosphorylation of the microtubule-associated protein tau (tau) in alzheimer cytoskeletal pathology

TREM2 variants in Alzheimer's disease

Pseudophosphorylation of tau at serine 422 inhibits caspase cleavage: in vitro evidence and implications for tangle formation in vivo

Unique pathological tau conformers from Alzheimer's brains transmit tau pathology in nontransgenic mice

Roles of tau protein in health and disease

Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid  -peptide

EMERGE and ENGAGE Topline Results: Two Phase 3 Studies to Evaluate Aducanumab in Patients With Early Alzheimer's Disease

Conformational selection or induced fit: A flux description of reaction mechanism

Tau phosphorylation: the therapeutic challenge for neurodegenerative disease

Association of amyloid and tau with cognition in preclinical Alzheimer disease: A longitudinal study

A hundred years of Alzheimer's disease research

The amyloid hypothesis of Alzheimer's disease: Progress and problems on the road to therapeutics

Alzheimer's Disease: The Amyloid Cascade Hypothesis

Impact of apolipoprotein E on Alzheimer's disease

The case for soluble A  oligomers as a drug target in Alzheimer's disease

Microglia in neurodegeneration

Prion-like properties of tau protein: The importance of extracellular tau as a therapeutic target

Proteopathic tau seeding predicts tauopathy in vivo

Role of Apolipoprotein E in  -amyloidogenesis: Isoform-specific effects on protofibril to fibril conversion of A  in vitro and brain A  deposition in vivo

Tau immunotherapies for Alzheimer's disease

Nanoparticle-assisted combination therapies for effective cancer treatment

Hyperphosphorylation determines both the spread and the morphology of tau pathology

Drug candidates in clinical trials for Alzheimer's disease

Subgroups of alzheimer's disease based on cerebrospinal fluid molecular markers

Recent developments with tau-based drug discovery

Amyloid- and tau -a toxic pas de deux in Alzheimer's disease

Tracking pathophysiological processes in Alzheimer's disease: an updated hypothetical model of dynamic biomarkers

Short fibrils constitute the major species of seed-competent tau in the brains of mice transgenic for human P301S tau

Combined treatment with a BACE inhibitor and anti-A  antibody gantenerumab enhances amyloid reduction in APP london mice

A walk through tau therapeutic strategies

Evidence for human transmission of amyloid- pathology and cerebral amyloid angiopathy

TREM2 in neurodegenerative diseases

ApoE promotes the proteolytic degradation of A 

Alz-50 and MC-1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau

Amyloid precursor protein processing and A 42  deposition in a transgenic mouse model of Alzheimer disease

A mutation in APP protects against Alzheimer's disease and age-related cognitive decline

Variant of TREM2 associated with the risk of Alzheimer's disease

Pathogenic protein seeding in Alzheimer disease and other neurodegenerative disorders

Evidence for seeding of  -amyloid by intracerebral infusion of Alzheimer brain extracts in  -amyloid precursor protein-transgenic mice

Selective targeting of amyloid-beta oligomer species by PMN310, a monoclonal antibody rationally designed for greater therapeutic

Alzheimer's association international conference (AAIC)

Antibodies to watch in 2019

The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics

The role of antibodies and their receptors in protection against ordered protein assembly in neurodegeneration

Alzheimer's disease: Safety and efficacy

Alzheimer brain-derived tau oligomers propagate pathology from endogenous tau

Antibody-mediated targeting of tau in vivo does not require effector function and microglial engagement

Towards an understanding of amyloid- oligomers: characterization, toxicity mechanisms, and inhibitors

NPT088 reduces both amyloid- and tau pathologies in transgenic mice

Propagation of tau pathology: hypotheses, discoveries, and yet unresolved questions from experimental and human brain studies

Antibody-based drugs and approaches against amyloid- species for Alzheimer's disease immunotherapy

Trans-synaptic spread of tau pathology in vivo

Intravenous immunoglobulin and Alzheimer's disease: what now?

Molecular structure of  -amyloid fibrils in Alzheimer's disease brain tissue

Soluble amyloid  peptide concentration as a predictor of synaptic change in Alzheimer's disease

Epitope mapping and structural basis for the recognition of phosphorylated tau by the anti-tau antibody AT8

Molecular mechanisms of Alzheimer disease protection by the A673T allele of amyloid precursor protein

Biochemistry and cell biology of tau protein in neurofibrillary degeneration. Cold Spring Harbor perspectives in medicine

Immunotherapy with anti-A  monoclonal antibodies in Alzheimer's disease: A critical review on the molecules in the pipelines with regulatory considerations

Emerging developments in targeting proteotoxicity in neurodegenerative diseases

Prion-like propagation of protein J o u r n a l P r e -p r o o f Journal Pre-proof misfolding and aggregation in amyotrophic lateral sclerosis

Soluble pool of A  amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease

An overview on the clinical development of tau-based therapeutics

Anti-PHF-tau antibodies and uses thereof. US Patent

Monoclonal antibodies with selective specificity for Alzheimer tau are directed against phosphatase-sensitive epitopes

Phage M13 for the treatment of Alzheimer and Parkinson disease

Exogenous induction of cerebral  -amyloidogenesis is governed by agent and host

Combination versus sequential single-agent therapy in metastatic breast cancer

Bapineuzumab captures the N-terminus of the Alzheimer's disease amyloid-beta peptide in a helical conformation

Protein transfer free energy obeys entropy-enthalpy compensation

De novo induction of amyloid- deposition in vivo

Passive anti-amyloid immunotherapy in J

Alzheimer's disease: What are the most promising targets?

What is the evidence that tau pathology spreads through prion-like propagation?

Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice

Modeling the association between 43 different clinical and pathological variables and the severity of cognitive impairment in a large autopsy cohort of elderly persons

Correlation of alzheimer disease neuropathologic changes with cognitive status: A review of the literature

A liposome-based therapeutic vaccine against  -amyloid plaques on the pancreas of transgenic NORBA mice

The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced A  protofibril formation

Human anti-tau antibodies

Ten years of tau-targeted immunotherapy: The path walked and the roads ahead

b. FUNDAMANT: an interventional 72-week phase 1 follow-up study of AADvac1, an active immunotherapy against tau protein pathology in Alzheimer's disease

Safety and immunogenicity of the tau vaccine AADvac1 in patients with Alzheimer's disease: a randomised, double-blind, placebo-controlled, phase 1 trial

AADvac1, an active immunotherapy for alzheimer's disease and non alzheimer tauopathies: An overview of preclinical and clinical development

Elicitation of structure-specific antibodies by epitope scaffolds

Prion-like seeding and nucleation of intracellular amyloid-

Evaluation of safety, tolerability, pharmacokinetics and pharmacodynamics of MEDI1814, A beta-amyloid 42 (A  42)-specific antibody, in patients with mild-moderate Alzheimer's disease

AMBAR (Alzheimer's management by albumin replacement) phase 2B/3 trial: Complete clinical, biomarker and neuroimaging results. The Journal of Prevention of Alzheimer

Immunotherapy for alzheimer's disease: from anti- -amyloid to tau-based immunization strategies

Amyloid- immunotherapy for alzheimer disease: Is it now a long shot

The physics and bioinformatics of binding and folding-an energy landscape perspective

A prion protein epitope selective for the pathologically misfolded conformation

Molecular structural basis for polymorphism in Alzheimer's  -amyloid fibrils

Tau immunotherapy for Alzheimer's disease

Prediction of misfolding-specific epitopes in SOD1 using collective coordinates

Humanized antibody. US Patent

Efficacy of ACI-35, a lipsomal anti-phospho tau vaccine in two different mouse models of

Alzheimer's & Dementia

Systems and methods for predicting misfolded protein epitopes by collective coordinate biasing

Understanding protein folding with energy landscape theory i: Basic concepts

Statistical mechanics of a correlated energy landscape model for protein folding funnels

Structural basis of C-terminal  -amyloid peptide binding by the antibody ponezumab for the treatment of Alzheimer's disease

Epitope mapping of mabs at8 and tau5 directed against hyperphosphorylated regions of the human tau protein

SAR228810: an antibody for protofibrillar amyloid  -peptide designed to reduce the risk of amyloid-related imaging abnormalities (ARIA)

comparison to other amyloid beta-directed antibodies in clinical development

Alzheimer's disease: the cholesterol connection

Antibody-based therapy in Alzheimer's disease

Transmission of amyloid- protein pathology from cadaveric pituitary growth hormone

Structural variation in amyloid- fibrils from Alzheimer's disease clinical subtypes

A randomized, single ascending dose study of intravenous BIIB092 in healthy participants

Alzheimer's & Dementia

Prophylactic active tau immunization leads to sustained reduction in both tau and amyloid- pathologies in 3xTg mice

Recombinant human B cell repertoires enable screening for rare, specific, and natively paired antibodies

An immunological epitope selective for pathological monomer-misfolded SOD1 in ALS

A  seeds and prions: How close the fit?

Antibodies specific for the protofibril form of beta-amyloid protein. US Patent

Elimination of fc receptor-dependent effector functions of a modified (IgG4) monoclonal antibody to human CD4

A phase 3 trial of iv immunoglobulin for alzheimer disease

Edward Jenner and the history of smallpox and vaccination

Reducing endogenous tau ameliorates amyloid  -induced deficits in an Alzheimer's disease mouse model

The role of apolipoprotein e isoforms in alzheimer's disease

Association of apolipoprotein E epsilon 4 allele with sporadic late onset Alzheimer's disease. A meta-analysis

Two phase 3 trials of bapineuzumab in mild-to-moderate alzheimer's disease

A phase 2 multiple ascending dose trial of bapineuzumab in mild to moderate Alzheimer disease

Distinct tau prion strains propagate in cells and mice and define different tauopathies

Combination therapy and the evolution of resistance: The theoretical merits of synergism and antagonism in cancer

Safety, tolerability and efficacy of the glutaminyl cyclase inhibitor PQ912 in Alzheimer's disease: results of a randomized

Alzheimer's Research & Therapy 10

Secreted amyloid  -protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease

Passive A  immunotherapy: Current achievements and future perspectives

Methods of screening for compounds which inhibit soluble  -amyloid peptide production

Peripheral A  subspecies as risk biomarkers of Alzheimer's disease

Atomic-resolution three-dimensional structure of amyloid  fibrils bearing the Osaka mutation

Small molecules, big targets: drug discovery faces the protein-protein interaction challenge

Large aggregates are the major soluble A  species in AD brain fractionated with density gradient ultracentrifugation

Preventing Alzheimer's disease

Alzheimer disease and Aducanumab: Adjusting our approach

The role of amyloid- oligomers in toxicity, propagation, and immunotherapy

Neuropathological alterations in Alzheimer disease

The antibody aducanumab reduces A  plaques in Alzheimer's disease

Hopes and hindrances‖

A single dose study of a novel humanized anti-amyloid beta (A  ) aggregate specific antibody KHK6640 in Japanese patients with Alzheimer's disease. The Journal of Prevention of Alzheimer

Phase 3 solanezumab trials: Secondary outcomes in mild Alzheimer's disease patients

Tau immunotherapies for Alzheimer's disease and related tauopathies: Progress and potential pitfalls

The s228p mutation prevents in vivo and in vitro igg4 fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation

The most infectious prion protein particles

A rational structured epitope defines a distinct subclass of toxic amyloid-beta oligomers

Engineered protein scaffolds for molecular recognition

The A  protofibril selective antibody mAb158 prevents accumulation of A  in astrocytes and rescues neurons from A  -induced cell death

Amyloid-related imaging abnormalities in patients with Alzheimer's disease treated with bapineuzumab: a retrospective analysis

Passive immunotherapy rapidly increases structural plasticity in a mouse model of Alzheimer disease

In vitro affinity maturation of human gm-csf antibodies by targeted cdr-diversification

Purified and synthetic Alzheimer's amyloid beta (A  ) prions

Alzheimer's prevention initiative

Antisense-mediated exon skipping decreases tau protein expression: A potential therapy for tauopathies

Treatment of early AD subjects with BAN2401, an anti-A  protofibril monoclonal antibody, signficantly clears amyloid plaque and reduces clinical decline

Creutzfeldt-Jakob disease in United Kingdom patients treated with human pituitary growth hormone

Measurement of anti-beta amyloid antibodies in human blood

Alzheimer's-causing mutations shift A  length by destabilizing  -secretase-A  n interactions

Preamyloid deposits in the cerebral cortex of patients with Alzheimer's disease and nondemented individuals

The Alzheimer's prevention initiative autosomal-dominant Alzheimer's disease trial: A study of crenezumab versus placebo in preclinical PSEN1 E280A mutation carriers to evaluate efficacy and safety in of autosomal-dominant Alzheimer's disease, including a placebo-treated noncarrier cohort

Quantification of plasma phosphorylated tau to use as a biomarker for brain alzheimer pathology: pilot case-control studies including patients with Alzheimer's disease and down syndrome

Oligomer formation of tau protein hyperphosphorylated in cells

Phases of A  -deposition in the human brain and its relevance for the development of AD

Efficacy and safety of a liposome-based vaccine against protein tau

Observations on the brains of demented old people

Amyloid deposits: Protection against toxic protein species?

Targeting phospho-ser422 by active tau immunotherapy in THY-tau22, a suitable therapeutic approach

Folding funnels, binding funnels, and protein function

The murine version of BAN2401 (mAb158)

Elucidating the role of TREM2 in Alzheimer's disease

Structure of crenezumab complex with a  shows loss of  -hairpin

The genetic landscape of Alzheimer disease: clinical implications and perspectives

Anti-tau monoclonal antibodies derived from soluble and filamentous tau show diverse functional properties in vitro and in vivo

Advancing alzheimer's disease treatment: Lessons from ctad 2018

Long-term follow-up of patients immunized with AN1792: Reduced functional decline in antibody responders

Safety and pharmacokinetics of anti-protofibrillar mAb SAR228810 in first-in-man study after single and multiple ascending IV and SC dosing in patients with mild to moderate Alzheimer's disease

Amyloid  deposition, neurodegeneration, and cognitive decline in sporadic

Alzheimer's disease: a prospective cohort study

Mitotic mechanisms in alzheimer's disease?

Anti-tau conformational scFv MC1 antibody efficiently reduces pathological tau species in adult JNPL3 mice

The role of cell-derived oligomers of A  in Alzheimer's disease and avenues for therapeutic intervention

Atomic-resolution structure of a disease-relevant A  amyloid fibril

The levels of soluble versus insoluble brain A  distinguish Alzheimer's disease from normal and pathologic aging

Energy landscape theory, funnels, specificity, and optimal criterion of biomolecular binding

An acute functional screen identifies an effective antibody targeting amyloid- oligomers based on calcium imaging

Do current therapeutic anti-a  antibodies for alzheimer's disease engage the target?

Serial propagation of distinct strains of A  prions from Alzheimer's disease patients

 -Amyloid Prions and the Pathobiology of Alzheimer's Disease. Cold Spring Harbor Perspectives in Medicine

Conformational change as one of the earliest alterations of tau in alzheimer's disease

Molecular consequences of amyloid precursor protein and presenilin mutations causing autosomal-dominant Alzheimer's disease. Alzheimer's Research & Therapy 4

Human anti-tau antibodies

Therapeutic antibody targeting microtubule-binding domain prevents neuronal internalization of extracellular tau via masking neuron surface proteoglycans

Preclinical and clinical development of ABBV-8E12, a humanized anti-tau antibody, for treatment of Alzheimer's disease and other tauopathies

Opportunities for conformation-selective antibodies in amyloid-related diseases

Tauopathies: classification and clinical update on neurodegenerative diseases associated with microtubule-associated protein tau

Active immunotherapy options for Alzheimer's disease. Alzheimer's Research & Therapy 6

Intraneuronal pyroglutamate-A  3-42 triggers neurodegeneration and lethal neurological deficits in a transgenic mouse model

Identification of low molecular weight pyroglutamate A  oligomers in Alzheimer disease: A novel tool for therapy and diagnosis

Immunotherapeutic approaches for Alzheimer's disease

A neuronal antigen in the brains of alzheimer patients

Development of novel combination therapies

A  (1-42) fibril structure illuminates self-recognition and replication of amyloid in Alzheimer's disease

A  immunotherapy: Intracerebral sequestration of a  by an anti-a  monoclonal antibody 266 with high affinity to soluble a 

Anti-tau antibody reduces insoluble tau and decreases brain atrophy

Anti-tau antibodies that block tau aggregate seeding in vitro markedly decrease pathology and improve cognition in vivo

Assay of plasma phosphorylated tau protein (threonine 181) and total tau protein in early-stage Alzheimer's disease

Target engagement in an alzheimer trial: Crenezumab lowers amyloid  oligomers in cerebrospinal fluid

Large soluble oligomers of amyloid  -protein from Alzheimer brain are far less neuroactive than the smaller oligomers to which they dissociate

Trem2 in alzheimer's disease: Microglial survival and energy metabolism

From induced fit to conformational selection: A continuum of binding mechanism controlled by the timescale of conformational transitions

Bacterial amyloids

We acknowledge support from the Canadian Institutes of Health Research (S.S.P. and N.R.C.), the Alberta Prion Research Institute (S.S.P. and N.R.C.), Compute Canada (S.S.P.) and Brain Canada (N.R.C.).