Y. Joy Yu Zuchero

Background: Although the first generation of immunotherapies for Alzheimer’s disease (AD) are now clinically approved, amyloid‐related imaging abnormalities (ARIA) remain a major safety problem for this class of drugs. Method: Here, we report an antibody transport vehicle (ATV) targeting the transferrin receptor (TfR) for brain delivery of amyloid beta (Aβ) antibodies that significantly reduced ARIA‐like lesions and improved plaque target engagement in a mouse model of amyloid deposition. Result: Asymmetrical Fc mutations (ATVcisLALA) allowed the molecule to selectively retain effector function only when bound to Aβ while mitigating TfR‐related hematology liabilities. Mice treated with ATVcisLALA:Aβ exhibited broad brain parenchymal antibody distribution; in contrast, anti‐Aβ IgG was highly localized to arterial perivascular spaces where vascular Aβ accumulates and likely plays a role in induction of ARIA. Importantly, ATVcisLALA: Aβ almost completely eliminated ARIA‐like lesions and vascular inflammation associated with anti‐Aβ treatment. Conclusion: Taken together, ATVcisLALA has the potential to significantly improve both safety and efficacy of Aβ immunotherapy through enhanced biodistribution mediated by transport across the blood‐brain barrier.

Transferrin receptor (TfR)-targeting of biologics has emerged as a promising strategy to improve drug delivery across the blood-brain barrier (BBB). However, most preclinical studies evaluating TfR-enabled drugs have been conducted in young adult animals. It remains unclear whether age and aging-related diseases impact TfR protein levels and/or BBB transport capacity. Here, we utilized a previously described TfR-targeting antibody transport vehicle (ATV ^TfR ) to investigate how healthy aging and disease progression in the 5xFAD mouse model of Alzheimer’s disease (AD) impact TfR protein and TfR-mediated brain delivery. ATV ^TfR transport capacity remained stable across 3- to 24-month-old healthy mice and 5xFAD progression did not impair ATV ^TfR brain transport up to 10.5 months, despite significant amyloid burden. Interestingly, neonates exhibited significantly elevated levels of vascular TfR protein and ATV ^TfR brain exposure compared to adult mice. Furthermore, vascular TfR in AD patient brains was similar to age-matched controls, suggesting conserved TfR transport is also likely in human AD. Overall, our data demonstrates broad functional utility for TfR-based brain delivery in both healthy aging and in an AD mouse model. Additionally, elevated TfR-mediated brain delivery during early mouse development highlights the potential of added efficacy in utilizing such platforms in disease treatment of infants and children.

Amyloid-related imaging abnormalities (ARIA), side effects of anti-amyloid drugs seen in magnetic resonance imaging of the brain, are a major safety concern in patients with Alzheimer’s disease. We developed an antibody transport vehicle (ATV) targeting transferrin receptor (TfR) for brain delivery of anti-amyloid-β protein (anti-Aβ) using asymmetrical Fc mutations (ATV ^cisLALA ) that mitigates TfR-related liabilities and retains effector function when bound to Aβ. Administration of ATV ^cisLALA :Aβ in mice exhibited broad brain distribution and enhanced parenchymal plaque target engagement. This biodistribution reduced ARIA-like lesions and vascular inflammation. Taken together, ATV ^cisLALA has the potential to improve the next generation of Aβ immunotherapy through enhanced biodistribution mediated by transport across the blood-brain barrier.

Targeting proteins highly expressed at the blood-brain barrier, including transferrin receptor (TfR) and CD98hc, is a transformative approach enabling more effective brain delivery of biotherapeutics for treatment of neurological diseases. TfR-mediated delivery promotes rapid, high brain uptake, while CD98hc-mediated delivery is slower with more prolonged exposure. Here, we engineer a huIgG Fc domain to bind both TfR and CD98hc to create a dual transport vehicle (TV) platform that drives distinct brain delivery properties. Dual TVs achieve significantly higher brain concentrations than TVs targeting either TfR or CD98hc alone. Modulation of TfR and CD98hc affinities shifts dual TV brain exposure kinetics and biodistribution. Stronger TfR affinity drives faster brain uptake and clearance, while stronger CD98hc affinity yields higher, more sustained concentrations, likely due to CD98hc affinity-dependent reduction in TfR-mediated neuronal internalization. This dual targeting strategy leverages the complementary properties of TfR and CD98hc-mediated brain exposure to increase optionality for brain delivery of biotherapeutics.

Blood brain barrier-crossing molecules targeting transferrin receptor (TfR) and CD98 heavy chain (CD98hc) are widely reported to promote enhanced brain delivery of therapeutics. Here, we provide a comprehensive and unbiased biodistribution characterization of TfR and CD98hc antibody transport vehicles (ATV^TfR and ATV^CD98hc) compared to control IgG. Mouse whole-body tissue clearing reveals distinct organ localization for each molecule. In the brain, ATV^TfR and ATV^CD98hc achieve enhanced exposure and parenchymal distribution even when brain exposures are matched between ATV and control IgG in bulk tissue. Using a combination of cell sorting and single-cell RNAseq, we reveal that control IgG is nearly absent from parenchymal cells and is distributed primarily to brain perivascular and leptomeningeal cells. In contrast, ATV^TfR and ATV^CD98hc exhibit broad and unique parenchymal cell-type distribution. Finally, we profile in detail brain region-specific biodistribution of ATV^TfR in cynomolgus monkey brain and spinal cord. Taken together, this in-depth multiscale characterization will guide platform selection for therapeutic targets of interest.

Transferrin receptor (TfR)-mediated transcytosis across the blood-brain barrier (BBB) is a promising strategy to improve delivery of biologics to the central nervous system (CNS). However, it remains unclear whether age and aging-related diseases impact TfR expression and/or BBB transport capacity. Here, we used the TfR-targeted antibody transport vehicle (ATV ^TfR ) to enhance CNS delivery in healthy mice and in the 5xFAD mouse model of Alzheimer’s disease (AD). Healthy neonates exhibited the highest vascular TfR expression and ATV ^TfR brain exposure, whereas BBB transport capacity remained stable across adulthood. Additionally, neither TfR expression nor ATV ^TfR brain uptake changed significantly in 5xFAD mice. Further, vascular TfR expression in AD patient brains was similar to age-matched controls, suggesting that TfR transport may be conserved for AD in humans. The elevated TfR-mediated brain delivery observed in early mouse development suggests the potential of added efficacy in utilizing TfR platforms for the treatment of early childhood diseases. Preservation of ATV ^TfR transport in adult mice across healthy aging and in an AD model supports continued application of TfR platforms in age-related diseases.

Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfR ^mu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfR ^mu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1 , across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfR ^mu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.