William J. Meilandt

A Therapeutic Antibody Targeting BACE1 Inhibits Amyloid-β Production in Vivo

A human antibody inhibits BACE1 activity and Aβ peptide production in cultured neurons and in the central nervous system of mouse and monkey. A Trojan Horse Antibody Scales a Mighty Fortress As impenetrable as the walls of ancient Troy, the tight endothelial cell layer of the blood-brain barrier (BBB) allows only a few select molecules to enter the brain. Unfortunately, this highly effective fortress blocks passage of therapeutic antibodies, limiting their usefulness for treating diseases of the brain and central nervous system. Enter Ryan Watts and his team at Genentech with their ambitious dual goal of making a therapeutic antibody against a popular Alzheimer’s disease drug target, the enzyme β-secretase (BACE1), and developing a strategy to boost the amount of this antibody that enters the brain (Atwal et al. and Yu et al.). BACE1 processes the amyloid precursor protein into amyloid-β (Aβ) peptides including those molecular species that aggregate to form the amyloid plaques found in the brains of Alzheimer’s disease patients. By blocking the activity of BACE1, BACE1 inhibitors should reduce production of the aggregation-prone Aβ peptides, thus decreasing amyloid plaque formation and slowing Alzheimer’s disease progression. Although small-molecule inhibitors of BACE1 have been developed and can readily cross the BBB because of their small size, they do not show sufficient specificity and hence may have toxic side effects. Watts envisaged that a better approach to blocking BACE1 activity might be passive immunization with a highly specific anti-BACE1 antibody. So his team engineered an anti-BACE1 antibody that bound to BACE1 with exquisite specificity and blocked its activity (Atwal et al.). The investigators then showed that this antibody could reduce production of aggregation-prone Aβ peptides in cultured primary neurons. Next, Watts and his colleagues injected the antibody into mice and monkeys and demonstrated a sustained decrease in the concentrations of Aβ peptide in the circulation of these animals and to a lesser extent in the brain. The researchers knew that they must find a way to increase the amount of antibody getting into the brain to reduce Aβ peptide concentrations in the brain sufficiently to obtain a therapeutic effect. So Watts teamed up with fellow Genentechie, Mark Dennis, and they devised an ingenious solution (Yu et al.). The Genentech researchers knew that high-affinity antibodies against the transferrin receptor might be able to cross the BBB using a natural process called receptor-mediated transcytosis. However, when they tested their antibody, they found that although it readily bound to the BBB, it could not detach from the transferrin receptor and hence was not released into the brain. So, they made a series of lower-affinity mouse anti-transferrin receptor antibodies and found variants that could cross the BBB by receptor-mediated transcytosis and were released into the mouse brain once they got across the endothelial cell layer. Next, they designed a bispecific mouse antibody with one arm comprising a low-affinity anti-transferrin receptor antibody and the other arm comprising the high-affinity anti-BACE1 antibody that had shown therapeutic promise in their earlier studies. They demonstrated that their bispecific antibody was able to cross the BBB and reach therapeutic concentrations in the mouse brain. They then showed that this bispecific antibody was substantially more effective at reducing Aβ peptide concentrations in the mouse brain compared to the monospecific anti-BACE1 antibody. This elegant pair of papers not only demonstrates the therapeutic potential of an anti-BACE1 antibody for treating Alzheimer’s disease but also provides a strategy worthy of the ancient Greeks that could be applied to other therapeutic antibodies that require safe passage into the human brain. Reducing production of amyloid-β (Aβ) peptide by direct inhibition of the enzymes that process amyloid precursor protein (APP) is a central therapeutic strategy for treating Alzheimer’s disease. However, small-molecule inhibitors of the β-secretase (BACE1) and γ-secretase APP processing enzymes have shown a lack of target selectivity and poor penetrance of the blood-brain barrier (BBB). Here, we have developed a high-affinity, phage-derived human antibody that targets BACE1 (anti-BACE1) and is anti-amyloidogenic. Anti-BACE1 reduces endogenous BACE1 activity and Aβ production in human cell lines expressing APP and in cultured primary neurons. Anti-BACE1 is highly selective and does not inhibit the related enzymes BACE2 or cathepsin D. Competitive binding assays and x-ray crystallography indicate that anti-BACE1 binds noncompetitively to an exosite on BACE1 and not to the catalytic site. Systemic dosing of mice and nonhuman primates with anti-BACE1 resulted in sustained reductions in peripheral Aβ peptide concentrations. Anti-BACE1 also reduces central nervous system Aβ concentrations in mouse and monkey, consistent with a measurable uptake of antibody across the BBB. Thus, BACE1 can be targeted in a highly selective manner through passive immunization with anti-BACE1, providing a potential approach for treating Alzheimer’s disease. Nevertheless, therapeutic success with anti-BACE1 will depend on improving antibody uptake into the brain.

Neprilysin Overexpression Inhibits Plaque Formation But Fails to Reduce Pathogenic Aβ Oligomers and Associated Cognitive Deficits in Human Amyloid Precursor Protein Transgenic Mice

The accumulation of amyloid-β (Aβ) peptides in the brain of patients with Alzheimers disease (AD) may arise from an imbalance between Aβ production and clearance. Overexpression of the Aβ-degrading enzyme neprilysin in brains of human amyloid precursor protein (hAPP) transgenic mice decreases overall Aβ levels and amyloid plaque burdens. Because AD-related synaptic and cognitive deficits appear to be more closely related to Aβ oligomers than to plaques, it is important to determine whether increased neprilysin activity also diminishes the levels of pathogenic Aβ oligomers and related neuronal deficits in vivo. To address this question, we crossed hAPP transgenic mice with neprilysin transgenic mice and analyzed their offspring. Neprilysin overexpression reduced soluble Aβ levels by 50% and effectively prevented early Aβ deposition in the neocortex and hippocampus. However, it did not reduce levels of Aβ trimers and Aβ*56 or improve deficits in spatial learning and memory. The differential effect of neprilysin on plaques and oligomers suggests that neprilysin-dependent degradation of Aβ affects plaques more than oligomers and that these structures may form through distinct assembly mechanisms. Neprilysins inability to prevent learning and memory deficits in hAPP mice may be related to its inability to reduce pathogenic Aβ oligomers. Reduction of Aβ oligomers will likely be required for anti-Aβ treatments to improve cognitive functions.

Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates

Bispecific antibodies engineered to both bind to the primate transferrin receptor and inhibit β-secretase are taken up by the nonhuman primate brain and reduce brain β-amyloid. A Two-Pronged Approach for Central Nervous System Therapeutics The brain has been considered off-limits to antibody therapies because of the blood-brain barrier (BBB), which protects the brain from circulating toxins while selectively transporting essential molecules into the brain. Efforts to use natural transport mechanisms to deliver antibody therapies into the brain have been successful in rodents. Whether a similar approach can be used in primates, including humans, remains unknown. Using bispecific antibodies with one arm binding to the transferrin receptor and the other to an Alzheimer’s disease drug target, we show that therapeutic antibodies can effectively and safely cross the BBB and enter the primate brain, thus paving the way for antibody therapeutics to treat central nervous system diseases in humans. Using therapeutic antibodies that need to cross the blood-brain barrier (BBB) to treat neurological disease is a difficult challenge. We have shown that bispecific antibodies with optimized binding to the transferrin receptor (TfR) that target β-secretase (BACE1) can cross the BBB and reduce brain amyloid-β (Aβ) in mice. Can TfR enhance antibody uptake in the primate brain? We describe two humanized TfR/BACE1 bispecific antibody variants. Using a human TfR knock-in mouse, we observed that anti-TfR/BACE1 antibodies could cross the BBB and reduce brain Aβ in a TfR affinity–dependent fashion. Intravenous dosing of monkeys with anti-TfR/BACE1 antibodies also reduced Aβ both in cerebral spinal fluid and in brain tissue, and the degree of reduction correlated with the brain concentration of anti-TfR/BACE1 antibody. These results demonstrate that the TfR bispecific antibody platform can robustly and safely deliver therapeutic antibody across the BBB in the primate brain.

Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier.

The safety of therapeutic bispecific antibodies that use TfR for delivery to the brain can be improved by reducing affinity for TfR and eliminating antibody effector function. Averting Roadblocks En Route to the Brain The blood-brain barrier represents a formidable blockade preventing therapeutic antibody delivery into the brain. Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting therapeutic antibody uptake into the brain. Although TfR can act as a molecular lift to promote brain uptake, little is known about the safety ramifications of this approach. Building on a pair of studies published in Science Translational Medicine, Couch and colleagues now report that when mice were dosed with therapeutic TfR antibodies, the animals showed acute clinical reactions and a reduction in immature red blood cells, known as reticulocytes. TfR bispecific antibodies engineered to lack Fc interactions with immune cells eliminated adverse acute clinical reactions and reduced reticulocyte loss; the extent of reticulocyte loss was also influenced by binding to TfR and interaction with the complement cascade. Because reticulocytes express high levels of TfR, other cell types that express high levels of TfR were also investigated. The authors observed, for example, that the blood-brain barrier remained completely intact after TfR antibodies were administered to mice, despite the high expression of TfR in brain endothelial cells. Finally, multiple doses of TfR/BACE1 bispecific antibodies reduced amyloid-β, a toxic protein implicated in Alzheimer’s disease, with minimal sustained toxicity. Investigation of monkey and human TfR levels in circulating reticulocytes suggested that loss of these cells may be less likely to occur in primates than in mice. The translational implications of these discoveries suggest that the blood-brain barrier is not the only obstacle to surmount on the way to the brain, at least when using TfR as a molecular lift. Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier.

Enkephalin Elevations Contribute to Neuronal and Behavioral Impairments in a Transgenic Mouse Model of Alzheimer's Disease

The enkephalin signaling pathway regulates various neural functions and can be altered by neurodegenerative disorders. In Alzheimers disease (AD), elevated enkephalin levels may reflect compensatory processes or contribute to cognitive impairments. To differentiate between these possibilities, we studied transgenic mice that express human amyloid precursor protein (hAPP) and amyloid-β (Aβ) peptides in neurons and exhibit key aspects of AD. Met-enkephalin levels in neuronal projections from the entorhinal cortex and dentate gyrus (brain regions important for memory that are affected in early stages of AD) were increased in hAPP mice, as were preproenkephalin mRNA levels. Genetic manipulations that exacerbate or prevent excitotoxicity also exacerbated or prevented the enkephalin alterations. In human AD brains, enkephalin levels in the dentate gyrus were also increased. In hAPP mice, enkephalin elevations correlated with the extent of Aβ-dependent neuronal and behavioral alterations, and memory deficits were reduced by irreversible blockade of μ-opioid receptors with the antagonist β-funaltrexamine. We conclude that enkephalin elevations may contribute to cognitive impairments in hAPP mice and possibly in humans with AD. The therapeutic potential of reducing enkephalin production or signaling merits further exploration.

GluN2B Antagonism Affects Interneurons and Leads to Immediate and Persistent Changes in Synaptic Plasticity, Oscillations, and Behavior

Although antagonists to GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) have been widely considered to be neuroprotective under certain pathological conditions, their immediate and lasting impacts on synaptic, circuit, and cognitive functions are poorly understood. In hippocampal slices, we found that the GluN2B-selective antagonist Ro25-6981 (Ro25) reduced synaptic NMDAR responses and consequently neuronal output in a subpopulation of GABAergic interneurons, but not pyramidal neurons. Consistent with these effects, Ro25 reduced GABAergic responses in pyramidal neurons and hence could affect circuit functions by altering the excitation/inhibition balance in the brain. In slices from Ts65Dn mice, a Down syndrome model with excess inhibition and cognitive impairment, acutely applied Ro25-rescued long-term potentiation (LTP) and gamma oscillation deficits, whereas prolonged dosing induced persistent rescue of LTP. In contrast, Ro25 did not impact LTP in wild-type (wt) mice but reduced gamma oscillations both acutely and following prolonged treatment. Although acute Ro25 treatment impaired memory performance in wt mice, memory deficits in Ts65Dn mice were unchanged. Thus, GluN2B–NMDARs contribute to the excitation/inhibition balance via impacts on interneurons, and blocking GluN2B–NMDARs can alter functions that depend on this balance, including synaptic plasticity, gamma oscillations, and memory. That prolonged GluN2B antagonism leads to persistent changes in synaptic and circuit functions, and that the influence of GluN2B antagonism differs between wt and disease model mice, provide critical insight into the therapeutic potential and possible liabilities of GluN2B antagonists.

A Death Receptor 6-Amyloid Precursor Protein Pathway Regulates Synapse Density in the Mature CNS But Does Not Contribute to Alzheimer's Disease-Related Pathophysiology in Murine Models

Recent studies implicate death receptor 6 (DR6) in an amyloid precursor protein (APP)-dependent pathway regulating developmental axon pruning, and in a pruning pathway operating during plastic rearrangements in adult brain. DR6 has also been suggested to mediate toxicity in vitro of Aβ peptides derived from APP. Given the link between APP, Aβ, and Alzheimers disease (AD), these findings have raised the possibility that DR6 contributes to aspects of neurodegeneration in AD. To test this possibility, we have used mouse models to characterize potential function(s) of DR6 in the adult CNS and in AD-related pathophysiology. We show that DR6 is broadly expressed within the adult CNS and regulates the density of excitatory synaptic connections onto pyramidal neurons in a genetic pathway with APP. DR6 knock-out also gives rise to behavioral abnormalities, some of which are similar to those previously documented in APP knock-out animals. However, in two distinct APP transgenic models of AD, we did not observe any alteration in the formation of amyloid plaques, gliosis, synaptic loss, or cognitive behavioral deficits with genetic deletion of DR6, though we did observe a transient reduction in the degree of microglial activation in one model. Our results support the view that DR6 functions with APP to modulate synaptic density in the adult CNS, but do not provide evidence for a role of DR6 in the pathophysiology of AD.

Loss of dual leucine zipper kinase signaling is protective in animal models of neurodegenerative disease

Blocking dual leucine zipper kinase slows disease progression in animal models of ALS and Alzheimer’s disease. A new therapeutic target zips into view The genetics, pathology, and clinical manifestations of chronic neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), are heterogeneous, which has made the development and testing of candidate therapeutics difficult. Here, Le Pichon et al. identify dual leucine zipper kinase (DLK) as a common regulator of neuronal degeneration in mouse models of ALS and Alzheimer’s disease and in human patient postmortem brain tissue. Deletion of DLK or treatment with a DLK inhibitor resulted in neuronal protection and slowing of disease progression after diverse insults in several mouse models of neurodegenerative disease. This suggests that DLK may have broad applicability as a therapeutic target for the treatment of a number of neurodegenerative diseases. Hallmarks of chronic neurodegenerative disease include progressive synaptic loss and neuronal cell death, yet the cellular pathways that underlie these processes remain largely undefined. We provide evidence that dual leucine zipper kinase (DLK) is an essential regulator of the progressive neurodegeneration that occurs in amyotrophic lateral sclerosis and Alzheimer’s disease. We demonstrate that DLK/c-Jun N-terminal kinase signaling was increased in mouse models and human patients with these disorders and that genetic deletion of DLK protected against axon degeneration, neuronal loss, and functional decline in vivo. Furthermore, pharmacological inhibition of DLK activity was sufficient to attenuate the neuronal stress response and to provide functional benefit even in the presence of ongoing disease. These findings demonstrate that pathological activation of DLK is a conserved mechanism that regulates neurodegeneration and suggest that DLK inhibition may be a potential approach to treat multiple neurodegenerative diseases.

Chronic GluN2B Antagonism Disrupts Behavior in Wild-Type Mice Without Protecting Against Synapse Loss or Memory Impairment in Alzheimer's Disease Mouse Models

Extensive evidence implicates GluN2B-containing NMDA receptors (GluN2B-NMDARs) in excitotoxic-insult-induced neurodegeneration and amyloid β (Aβ)-induced synaptic dysfunction. Therefore, inhibiting GluN2B-NMDARs would appear to be a potential therapeutic strategy to provide neuroprotection and improve cognitive function in Alzheimers disease (AD). However, there are no reports of long-term in vivo treatment of AD mouse models with GluN2B antagonists. We used piperidine18 (Pip18), a potent and selective GluN2B-NMDAR antagonist with favorable pharmacokinetic properties, for long-term dosing in AD mouse models. Reduced freezing behavior in Tg2576 mice during fear conditioning was partially reversed after subchronic (17 d) Pip18 treatment. However, analysis of freezing behavior in different contexts indicated that this increased freezing likely involves elevated anxiety or excessive memory generalization in both nontransgenic (NTG) and Tg2576 mice. In PS2APP mice chronically fed with medicated food containing Pip18 for 4 months, spatial learning and memory deficits were not rescued, plaque-associated spine loss was not affected, and synaptic function was not altered. At the same time, altered open field activity consistent with increased anxiety and degraded performance in an active avoidance task were observed in NTG after chronic treatment. These results indicate that long-term treatment with a GluN2B-NMDAR antagonist does not provide a disease-modifying benefit and could cause cognitive liabilities rather than symptomatic benefit in AD mouse models. Therefore, these results challenge the expectation of the therapeutic potential for GluN2B-NMDAR antagonists in AD.

Selective Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12) with Activity in a Model of Alzheimer’s Disease

Significant data exists to suggest that dual leucine zipper kinase (DLK, MAP3K12) is a conserved regulator of neuronal degeneration following neuronal injury and in chronic neurodegenerative disease. Consequently, there is considerable interest in the identification of DLK inhibitors with a profile compatible with development for these indications. Herein, we use structure-based drug design combined with a focus on CNS drug-like properties to generate compounds with superior kinase selectivity and metabolic stability as compared to previously disclosed DLK inhibitors. These compounds, exemplified by inhibitor 14, retain excellent CNS penetration and are well tolerated following multiple days of dosing at concentrations that exceed those required for DLK inhibition in the brain.