Kwame Hoyte

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

The prevalence of dementia in the Western world in people over the age of 60 has been estimated to be greater than 5%, about two-thirds of which are due to Alzheimer’s disease. The age-specific prevalence of Alzheimer’s disease nearly doubles every 5 years after age 65, leading to a prevalence of greater than 25% in those over the age of 90 (ref. 3). Here, to search for low-frequency variants in the amyloid-β precursor protein (APP) gene with a significant effect on the risk of Alzheimer’s disease, we studied coding variants in APP in a set of whole-genome sequence data from 1,795 Icelanders. We found a coding mutation (A673T) in the APP gene that protects against Alzheimer’s disease and cognitive decline in the elderly without Alzheimer’s disease. This substitution is adjacent to the aspartyl protease β-site in APP, and results in an approximately 40% reduction in the formation of amyloidogenic peptides in vitro. The strong protective effect of the A673T substitution against Alzheimer’s disease provides proof of principle for the hypothesis that reducing the β-cleavage of APP may protect against the disease. Furthermore, as the A673T allele also protects against cognitive decline in the elderly without Alzheimer’s disease, the two may be mediated through the same or similar mechanisms.

Boosting Brain Uptake of a Therapeutic Antibody by Reducing Its Affinity for a Transcytosis Target

Brain uptake of a therapeutic bispecific antibody by receptor-mediated transcytosis is enhanced by reducing the antibody’s affinity for the transferrin receptor. 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. Monoclonal antibodies have therapeutic potential for treating diseases of the central nervous system, but their accumulation in the brain is limited by the blood-brain barrier (BBB). Here, we show that reducing the affinity of an antibody for the transferrin receptor (TfR) enhances receptor-mediated transcytosis of the anti-TfR antibody across the BBB into the mouse brain where it reaches therapeutically relevant concentrations. Anti-TfR antibodies that bind with high affinity to TfR remain associated with the BBB, whereas lower-affinity anti-TfR antibody variants are released from the BBB into the brain and show a broad distribution 24 hours after dosing. We designed a bispecific antibody that binds with low affinity to TfR and with high affinity to the enzyme β-secretase (BACE1), which processes amyloid precursor protein into amyloid-β (Aβ) peptides including those associated with Alzheimer’s disease. Compared to monospecific anti-BACE1 antibody, the bispecific antibody accumulated in the mouse brain and led to a greater reduction in brain Aβ after a single systemic dose. TfR-facilitated transcytosis of this bispecific antibody across the BBB may enhance its potency as an anti-BACE1 therapy for treating Alzheimer’s disease.

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.

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.

Lack of Widespread BBB Disruption in Alzheimer's Disease Models: Focus on Therapeutic Antibodies

The blood-brain barrier (BBB) limits brain uptake of therapeutic antibodies. It is believed that the BBB is disrupted in Alzheimers disease (AD), potentially increasing drug permeability de facto. Here we compared active versus passive brain uptake of systemically dosed antibodies (anti-transferrin receptor [TfR] bispecific versus control antibody) in mouse models of AD. We first confirmed BBB disruption in a mouse model of multiple sclerosis as a positive control. Importantly, we found that BBB permeability was vastly spared in mouse models of AD, including PS2-APP, Tau transgenics, and APOE4 knockin mice. Brain levels of TfR in mouse models or in human cases of AD resembled controls, suggesting target engagement of TfR bispecific is not limited. Furthermore, infarcts from human AD brain showed similar occurrences compared to age-matched controls. These results question the widely held view that the BBB is largely disrupted in AD, raising concern about assumptions of drug permeability in disease.

Subcutaneous bioavailability of therapeutic antibodies as a function of FcRn binding affinity in mice

The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid β monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased binding affinity at pH 6.0 to mFcRn and affinity comparable to the WT antibody at pH 7.4, had the highest bioavailability (94.7%). A semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A.H435A, is consistent with the hypothesis that the decreased bioavailability of variant I253A.H435A was due to loss of the FcRn-mediated protection from catabolism at the absorption site. Together, these data demonstrate that FcRn plays an important role in SC bioavailability of therapeutic IgG antibodies.

Prospective Design of Anti-Transferrin Receptor Bispecific Antibodies for Optimal Delivery into the Human Brain

Anti‐transferrin receptor (TfR)‐based bispecific antibodies have shown promise for boosting antibody uptake in the brain. Nevertheless, there are limited data on the molecular properties, including affinity required for successful development of TfR‐based therapeutics. A complex nonmonotonic relationship exists between affinity of the anti‐TfR arm and brain uptake at therapeutically relevant doses. However, the quantitative nature of this relationship and its translatability to humans is heretofore unexplored. Therefore, we developed a mechanistic pharmacokinetic‐pharmacodynamic (PK‐PD) model for bispecific anti‐TfR/BACE1 antibodies that accounts for antibody‐TfR interactions at the blood‐brain barrier (BBB) as well as the pharmacodynamic (PD) effect of anti‐BACE1 arm. The calibrated model correctly predicted the optimal anti‐TfR affinity required to maximize brain exposure of therapeutic antibodies in the cynomolgus monkey and was scaled to predict the optimal affinity of anti‐TfR bispecifics in humans. Thus, this model provides a framework for testing critical translational predictions for anti‐TfR bispecific antibodies, including choice of candidate molecule for clinical development.

Modest reductions in BACE1 activity significantly reduce beta-amyloid plaque load and neuroinflammation in a mouse model of Alzheimer's disease

Background:Amyloidosis in Alzheimer’s disease (AD) starts with gradual aggregation of amyloid b (A beta) species into neurotoxic soluble dimers and oligomers, and insoluble fibrils and plaques. The aggregation process could be divided into two categories: reversible aggregation due to physical hydrophobic stacking, and irreversible aggregation due to covalent crosslinking. Many compounds have been shown to attenuate the aggregation process or to accelerate the aggregation into fibrils. However, few compounds have been able to inhibit covalent crosslinking of A beta.Methods: Fluorescent spectra and H NMRwere used to demonstrate the specificity of curcumin analogues towards specific region of Abeta. SDS-Page gel and western blots were used to evaluate the anti-crosslinking effects of the designed compounds. Results: Both fluorescence data and 1H NMR spectra indicated that one of the derivatives, CRANAD-3, could specifically interact with the core fragment of A b. In addition, our data suggested that CRANAD-3 certainly interactedwith H13 (histidine13), whose imidazolemoiety is an essential binding site for copper, which plays a critical role in covalent crosslinking of A b, and has been considered as an important triggering factor for AD. Furthermore, we designed and synthesized curcumin derivative CRANAD-17, in which imidazole rings were incorporated to compete with Histidine13 of A b for copper binding. SDS-PAGE and western blot results showed that CRANAD-17 was capable of inhibiting A b 42 crosslinking induced by copper. Conclusions: Our results indicate the possibility of using this compound for AD therapy. Since CRANAD-17 is also a fluorescent probe and could be easily adapted into a PET probe, it could potentially be used as a theranostic probe.