Researchers have devised a new way to sneak therapeutic cargo across the blood-brain barrier. Into the constant region of a human IgG1 antibody, they slid a domain that latches onto receptors that carry transferrin into the brain. On the other end of the IgG, the researchers attached therapeutic cargo, which the “transport vehicle” ferried across and released throughout the brain in both mice and monkeys. The strategy was reported in two papers published May 27 in Science Translational Medicine.
- Antibody Fc region engineered to bind the transferrin receptor.
- This “transport vehicle” carried BACE1 antibody, knocked down Aβ in mice and monkeys.
- Delivering an enzyme, it corrected a lysosomal storage disease in mice.
In one, Adam Silverman, Joy Zuchero, and colleagues at Denali in South San Francisco detailed the vehicle’s development, and showed that brain Aβ plummeted when animals were injected with the engineered IgG1 carrying two antibody Fab fragments that bind BACE1. In the second, Mihalis Kariolis, Anastasia Henry, and colleagues described how they used the vehicle to deliver iduronate 2-sulfatase into the brain. This enzyme is lacking in people with Hunter syndrome, a devastating lysosome-storage disease. In a mouse model of Hunter syndrome, the ETV, aka enzyme transport vehicle, restored normal lysosomal function while stemming neuroinflammation and axonal damage. In all, the findings suggest that the vehicle is capable of transporting myriad therapeutic cargo into the brain, including antibodies, enzymes, and potentially oligonucleotides.
Torben Moos of Aalborg University in Denmark called the approach highly promising. “I’m very optimistic that this approach will create major changes in our ability to deliver therapies into the brain,” he told Alzforum.
Berislav Zlokovic of the University of Southern California, Los Angeles, called the transport vehicle platform an important advance in the field of transferrin receptor (TfR) based approaches to cross the blood-brain barrier (BBB). However, he asked how a compromised BBB in neurological diseases, including Alzheimer’s, may affect the function of the TfR system.
The BBB is a formidable roadblock for therapeutics, especially for large ones such as antibodies or enzymes. To outsmart the barriers, scientists have attempted to co-opt receptor-mediated transcytosis pathways that transport essential cargo into the brain. The TfR is the most researched of these.
An early attempt tethered therapeutics to TfR antibodies (Pardridge, 2002). Alas, high-affinity versions of these antibodies tended to get snagged in the cerebrovasculature, stifling cargo delivery into the brain. Scientists at Genentech tried to get around this with bispecific antibodies, where one arm loosely bound TfR and the other latched onto a therapeutic target. This improved delivery (May 2011 news).
Roche (which acquired Genentech) took a modular approach with its Brain Shuttle (see video). It relies on a separate TfR-specific Fab fragment that can be attached to the constant region of a therapeutic antibody, or theoretically to other types of molecules (Jan 2014 news). Roche started a Phase 1 trial evaluating a shuttle version of the Aβ-immunotherapy gantenerumab in November 2019. Yet other configurations, such as a therapeutic antibody with TfR Fabs attached to its armpits, are also in the works (Hultqvist et al., 2017; Jan 2018 news).
Enter Denali’s BBB transport vehicle. As co-first authors Kariolis and Robert Wells described, it can carry all manner of cargo. Rather than attach an additional TfR-specific antibody fragment to the immunoglobulin Fc region, or waste a precious antibody arm targeting TfR, the researchers incorporated a slimmed-down, novel TfR-binding domain into the constant region of a human IgG1. They identified a nine-amino-acid stretch of the Fc stub that appeared to tolerate meddling, and randomized its amino acid sequence. Screening the mutant libraries in yeast, they pulled out a handful of constant region clones bestowed with a penchant for binding TfR. They purposefully selected one that cross-reacted with TfR from cynomolgus monkeys, allowing future studies in nonhuman primates.
Mix and Match. The BBB transport vehicle (left) is an antibody Fc region harboring a domain that binds to the transferrin receptor. The TV can be affixed with antibody Fab regions specific for one or two epitopes (middle), or with other types of molecules, e.g., enzymes (right). [Courtesy of Kariolis et al., Science Translational Medicine, 2020.]
After testing transport vehicles in cell culture studies, the researchers stuck them into chimeric, human TfR knock-in mice. In those, a portion of human TfR that the transport vehicle recognizes is knocked into one copy of the mouse TfR gene. The scientists injected an antibody transport vehicle (ATV) carrying two Fab arms specific for BACE1 intravenously into these knock-in mice. This did not douse normal expression of TfR in the brain, even after chronic dosing. Compared with mice injected with regular BACE1 antibodies, those treated with ATV-BACE1 had 40 times more anti-BACE1 in their brains 24 hours later. Depending on the timepoint, ATV-BACE1 had a 10- to 100-fold greater brain-to-plasma ratio than did typical anti-BACE1 antibodies. ATV-BACE1 engaged its target, triggering double the drop in soluble brain Aβ40 as that seen in anti-BACE1 antibodies.
Notably, while most of the ATV-BACE1 was spotted in the cerebrovasculature four hours after dosing, by 24 hours it had penetrated the brain parenchyma of the cortex, hippocampus, and cerebellum. Super-resolution confocal imaging of brain sections revealed that neurons had readily internalized ATV-BACE1. By contrast, anti-BACE1 antibodies made a sparse showing in both the vasculature and the parenchyma.
Finally, the researchers tested ATV-BACE1 in cynomolgus monkeys, which express TfR in their brain vasculature. They dosed the monkeys with ATV-BACE1, anti-BACE1, or a control IgG, then tracked various APP products. In animals treated once with ATV-BACE1, CSF concentrations of Aβ40 plummeted by 70 percent, while the ratio of APPβ/APPα, an indicator of BACE1 cleavage activity, dropped by 75 percent two days after dosing. By 14 days, levels had returned to baseline. CSF Aβ40 or APPβ/APPα did not drop in monkeys treated with anti-BACE1 or control IgG. In another experiment, the researchers found that two days after dosing, levels of ATV-BACE1 were 26- to 35-fold higher across multiple brain regions than levels of anti-BACE1 antibodies, suggesting that the transport vehicle significantly boosted delivery into the brain.
The BACE1-ATV studies were designed to test delivery and target engagement in the brain, Steve Krognes of Denali told Alzforum. In part due to disappointing results of BACE1 inhibitor trials, the company will not pursue BACE1 as a target in AD clinical trials, he said.
Instead, Denali set its sights on the microglial receptor TREM2, for which activating antibodies have been developed and fused to the transport vehicle (May 2019 conference news; Mar 2020 news). For amyotrophic lateral sclerosis and frontotemporal dementia, Denali is testing out a TV loaded with progranulin, a lysosomal protein that is dysfunctional in some people with those neurodegenerative diseases.
However, the cargo nearest to clinical development is one to treat Hunter syndrome. This childhood lysosomal-storage disease wreaks havoc throughout the body. In the second STM paper, first authors Julie Ullman and Annie Arguello reported that an ETV loaded with iduronate 2-sulfatase (IDS) crossed into the mouse brain and restored lysosomal function there.
IDS deficiency leads to a buildup of the enzyme’s substrates, namely glycosaminoglycans (GAGs), in the lysosome. This in turn hobbles lysosomal function, leading to an accumulation of myriad other lysosomal substrates that normally would be destroyed. The disease affects multiple organs, and in about two-thirds of patients, the brain. Intravenous injection of recombinant IDS is the standard of care; however, the recombinant enzyme bounces off the BBB and thus does nothing to stem neurological symptoms, which can include cognitive impairment.
To learn if a transport vehicle could deliver the enzyme to the brain, the scientists injected their ETV-IDS intravenously into human TfR knock-in mice that had their IDS knocked out. Just like recombinant IDS, ETV-IDS reduced GAGs in the spleen and liver. However, only ETV-IDS entered the brain and disposed of GAGs there, as well. This dramatically reduced the build-up of other lysosomal lipids and proteins, and dampened neuroinflammation, a hallmark of Hunter disease. The transported enzyme also lowered the concentration of neurofilament light (NfL) in the mouse CSF, suggesting that the treatment staunched neurodegeneration.
Lipid Clean-Up. These heat maps show how IDS knockout mice (left) have a build-up of two types of lysosomal lipid in the brain (top and bottom). Treatment with ETV-IDS (right) reduced accumulation, while vehicle (left) or IDS alone (middle) did not. [Courtesy of Ullman et al., Science Translational Medicine, 2020.]
“This is a cool tool that could be potentially applied to treat many lysosome-storage diseases as well as adult-onset neurodegenerative diseases,” commented Fenghua Hu of Cornell University in Ithaca, New York.
Paul Saftig of the University of Kiel in Germany agreed. “This is certainly a major step forward in the development of an effective therapy of Hunter syndrome (MPSII) and maybe other lysosomal-storage disorders,” he wrote. That said, Saftig offered a word of caution. Because the ETV-IDS fusion protein is unknown to the body and requires chronic dosing, it could rouse problematic immune reactions, including generation of antibodies that could erode the treatment’s efficacy (see comment below).
Denali will begin a Phase 1/2 clinical trial testing an ETV-IDS in children with Hunter syndrome. Beyond enzymes and therapeutic antibodies, the scientists are also working on attaching anti-sense oligonucleotides to the transport vehicles. “The strongest feature of this platform is its modularity,” Kariolis said.—Jessica Shugart
- Smuggling Antibodies to BACE Across the Blood-Brain Barrier
- Brain Shuttle Ferries Antibodies Across the Blood-Brain Barrier
- Antibody Shuttle Rouses Anti-Aβ Response in Brain without Waking the Periphery
- Antibodies Against Microglial Receptors TREM2 and CD33 Head to Trials
- Paper Alert: Mouse TREM2 Antibody Boosts Microglial Plaque Clean-Up
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