Brain Shuttle Ferries Antibodies Across the Blood-Brain Barrier

Researchers have deployed a natural transport system in the brain to give large molecules a lift across the blood-brain barrier (BBB). The Brain Shuttle, a transferrin receptor (TfR)-based system developed by scientists at Hoffmann-La Roche, Basel, Switzerland, boosted the delivery of an Aβ-specific antibody into the brains of mice that had an experimental form of Alzheimer’s disease. Reported in the January 8 Neuron, the findings showed that the shuttle increased the amount of antibody bound to plaques by 55-fold, upping plaque clearance.

Caught in the act:

Fifteen minutes after injection into a mouse, the sFab Brain Shuttle (red) infiltrates vesicles in brain endothelial cells (nuclei, blue) that line a microvessel (luminal side, green). Click to watch movie. Video courtesy of Jens Niewoehner, Neuron January 8, 2014

"The investigators weren't the first to tinker with the TfR system to enhance drug delivery. They optimized the 'Trojan Horse effect,'" David Morgan at the University of South Florida, Tampa, told Alzforum.

Whether the shuttle will work in humans, or how it will measure up to other new ways of penetrating the blood-brain barrier, remains to be seen. However, this system has the potential to enhance the delivery of a variety of therapeutic compounds into the brain, Luca Santarelli of Hoffmann-La Roche told Alzforum. The Roche study was co-led by Per-Ola Freskgård of the company’s Neuroscience Discovery and Translation Area in Basel and Anirvan Ghosh of Roche’s Large Molecule Research unit in Penzberg, Germany.

The Brain Shuttle takes advantage of TfR-mediated transcytosis, a well-known path across the BBB. Endothelial cells that tightly line blood vessels in the brain express high levels of TfR. Once transferrin binds to it, TfR shuttles from the luminal, or blood-facing, side of the endothelial cells to the abluminal, or brain-facing, side via a series of endosomal vesicles. 

Schemes to hijack this system have long been in the works. Researchers have had some success ferrying proteins through the BBB by attaching them to antibodies that bind TfR (see Pardridge, 2012). However, TfR’s unwillingness to release its cargo from its clutches on the brain side of the cell has limited the success of this approach.

Previously, researchers led by Ryan Watts at Genentech, San Francisco, California, addressed this problem. They generated a bispecific antibody whose one arm bound TfR, while the second arm bound BACE1 (see May 2011 news story on Yu et al., 2011). By choosing a low-affinity antibody for the TfR arm, the researchers improved release of the complex into the mouse brain. 

Roche’s team wanted to design a modular system they could latch onto any therapeutic cargo. The investigators attached either one or two single-chain Fab antibody fragments specific for TfR onto the stub (or Fc fragment) of mAb31, an anti-Aβ antibody. The researchers hypothesized that attaching one Fab fragment to mAb31 would interfere less with TfR’s normal function. “We had the idea that it could be very important to conserve this natural shuttling inside the cell,” Santarelli told Alzforum.

A series of in-vitro experiments supported this hypothesis. The anti-Aβ antibody with the double Fab attachment (dFab) got waylaid inside a monolayer of endothelial cells, while a significant fraction of the single anti-TfR complex (sFab) passed right through. Immunocytochemistry revealed dFab comingling with lysosomal vesicles more often than sFab, offering a possible explanation for dFab’s holdup inside the cells.

The authors hypothesized that dFab triggers dimerization of the TfR, which routes the complex into a vesicular pathway destined for lysosomal degradation, rather than transcytosis across the BBB. 

“We know that endocytosis and transcytosis occur at the BBB, but the specific mechanisms that dictate whether a molecule is transcytosed or sent to a lysosome for degradation are unclear,” Robert Bell of Pfizer, who was not involved in the research, told Alzforum. “This paper should influence efforts to understand how molecules are transcytosed in a much more specific manner, and to learn how to exploit those pathways.”

The authors next injected the two Brain Shuttles—sFab and dFab—intravenously into PS2APP double transgenic mice. Immunostaining of brain slices taken 15 minutes post-injection showed both constructs associated with cells lining the vessels. But eight hours later, the paths of the two constructs had diverged: DFab still lingered in the endothelial cells, while sFab had diffused into the brain parenchyma, where it latched onto Aβ plaques (watch movie above). 

To measure the difference in plaque targeting among the two brain shuttle constructs and the native mAb31, first author Jens Niewoehner and colleagues used confocal microscopy and imaging software to analyze brain slices eight hours after injecting mice with equal amounts of each antibody. Plaques bound 55-fold more sFab than dFab or mAb31. To test long-term effects, the researchers injected PS2APP mice weekly with two doses of sFab (0.53 mg/kg or 2.67 mg/kg) or mAb31 (0.4 mg/kg or 2mg/kg). After three months, mice treated with the higher dose of sFab had approximately two-thirds as many plaques in the cortex as untreated animals. The native mAb31 had no discernable effect on plaque number at either dose. SFab also reduced plaques in the hippocampus, although slightly less so. Previously, researchers reported that much higher doses (20mg/kg) of mAb31 are needed to have any effect on plaques (see Bohrmann et al., 2012). 

Whether Genentech’s bispecific antibody or Roche’s sFab Brain Shuttle is the best approach to smuggling drugs across the BBB still isn’t clear. Although Roche owns Genentech, the two approaches were developed independently, according to Santarelli. They aim at different targets in the brain (BACE1 and Aβ, respectively), yet both showed a similar fold change in brain penetrance. Some scientists voiced caution about the Brain Shuttle’s reported 55-fold increase in plaque binding. This number relied on quantification by immunofluorescence, a technique Ben Barres at Stanford University in Stanford, California, called semi-quantitative.

Roy Weller of the University of Southampton in England expressed some concern that accelerating the disposal of Aβ plaques in humans could damage blood-vessel walls, a problem that plagued early clinical trials for lone anti-Aβ antibodies (see Jul 2011 conference news). “But if you treat patients earlier, before the whole system becomes clogged up, then this Brain Shuttle could be an advantage,” Weller told Alzforum. 

The Brain Shuttle’s unique potential may lie in its ability to transport a variety of molecules—not just antibodies—across the BBB, Santarelli said. In fact, although the researchers tried out the shuttle on an anti-Aβ antibody, Santarelli said the company has no plan to pursue a Brain Shuttle version of its anti-Aβ antibody gantenerumab, which is currently in prevention (see Oct 2012 news story) and treatment (see Alzforum’s Therapeutics database) trials for AD. Amyloid plaques are an abundant target and bind irreversibly to the antibody, so the small amount of gantenerumab that enters the brain is already sufficient to get the job done, Santarelli said (see Oct 2011 news story). 

The shuttle might be necessary for gene suppression strategies, enzyme replacement approaches, or for targets that are harder to reach, such as tau, said Santarelli.

Roche has teamed up with Isis Pharmaceuticals of Carlsbad, California, to attach the Brain Shuttle to antisense oligonucleotides that target the huntingtin gene, a key player in Huntington’s disease. Another Roche partnership with South San Francisco-based Prothena aims to fit the shuttle to an anti-α-synuclein antibody to treat Parkinson’s disease.—Jessica Shugart

Comments

  1. Niewoehner and colleagues set out to further improve approaches around targeting the transferrin receptor (TfR) for brain uptake of antibodies. This study is similar to those published by others, however, it further advances the possibilities by engineering a Fab to a traditional antibody, as opposed to using a bivalent anti-TfR format, or the more recently described bispecific monovalent anti-TfR format. The group shows that monovalent binding to TfR, similar to reduced affinity for the receptor, improves uptake and biodistribution of the antibody/anti-TfR complex. As stated in the preview by Bell and Ehlers, the study does not carefully evaluate pharmacokinetics, safety, or the relative contribution of valency and affinity for TfR. The claim of 55-fold increased target engagement at eight hours is based on quantification of immunofluorescence, which is semi-quantitative at best. Indeed, careful evaluation of the supplemental data (Figure S4) shows ~10-fold increased uptake when measuring drug levels in the brain at eight hours, which is reduced to 1.9-fold in wild-type mice at 24 hours. These fold differences are similar to what has been published previously. It is also interesting to note the quick clearance and relatively low total concentrations in the brain achieved at these doses (~2 nM max concentration). Overall this study provides an interesting next step in the effort to develop brain-penetrating molecules for CNS diseases. The modulatory nature of the approach opens opportunities that will need further investigation.

    View all comments by Ben Barres

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References

News Citations

  1. Smuggling Antibodies to BACE Across the Blood-Brain Barrier
  2. Paris: Renamed ARIA, Vasogenic Edema Common to Anti-Amyloid Therapy
  3. DIAN Trial Picks Gantenerumab, Solanezumab, Maybe BACE Inhibitor
  4. Aβ Antibody Gantenerumab Clears Plaques

Research Models Citations

  1. PS2APP

Therapeutics Citations

  1. Gantenerumab

Paper Citations

  1. Pardridge WM. Drug transport across the blood-brain barrier. J Cereb Blood Flow Metab. 2012 Nov;32(11):1959-72. PubMed.
  2. Yu YJ, Zhang Y, Kenrick M, Hoyte K, Luk W, Lu Y, Atwal J, Elliott JM, Prabhu S, Watts RJ, Dennis MS. Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target. Sci Transl Med. 2011 May 25;3(84):84ra44. PubMed.
  3. Bohrmann B, Baumann K, Benz J, Gerber F, Huber W, Knoflach F, Messer J, Oroszlan K, Rauchenberger R, Richter WF, Rothe C, Urban M, Bardroff M, Winter M, Nordstedt C, Loetscher H. Gantenerumab: A Novel Human Anti-Aβ Antibody Demonstrates Sustained Cerebral Amyloid-β Binding and Elicits Cell-Mediated Removal of Human Amyloid-β. J Alzheimers Dis. 2012;28(1):49-69. PubMed.

Other Citations

  1. [[{"fid":"2671","view_mode":"default","type":"media","attributes":{"height":169,"width":240,"style":"line-height: 1.538em; width: 240px; height: 169px; float: left; margin-left: 3px; margin-right: 3px;","class":"media-element file-default"}}]]

Further Reading

Papers

  1. Bohrmann B, Baumann K, Benz J, Gerber F, Huber W, Knoflach F, Messer J, Oroszlan K, Rauchenberger R, Richter WF, Rothe C, Urban M, Bardroff M, Winter M, Nordstedt C, Loetscher H. Gantenerumab: A Novel Human Anti-Aβ Antibody Demonstrates Sustained Cerebral Amyloid-β Binding and Elicits Cell-Mediated Removal of Human Amyloid-β. J Alzheimers Dis. 2012;28(1):49-69. PubMed.
  2. Pardridge WM. Drug transport across the blood-brain barrier. J Cereb Blood Flow Metab. 2012 Nov;32(11):1959-72. PubMed.
  3. Couch JA, Yu YJ, Zhang Y, Tarrant JM, Fuji RN, Meilandt WJ, Solanoy H, Tong RK, Hoyte K, Luk W, Lu Y, Gadkar K, Prabhu S, Ordonia BA, Nguyen Q, Lin Y, Lin Z, Balazs M, Scearce-Levie K, Ernst JA, Dennis MS, Watts RJ. Addressing Safety Liabilities of TfR Bispecific Antibodies That Cross the Blood-Brain Barrier. Sci Transl Med. 2013 May 1;5(183):183ra57. PubMed.
  4. Yu YJ, Zhang Y, Kenrick M, Hoyte K, Luk W, Lu Y, Atwal J, Elliott JM, Prabhu S, Watts RJ, Dennis MS. Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target. Sci Transl Med. 2011 May 25;3(84):84ra44. PubMed.
  5. Ostrowitzki S, Deptula D, Thurfjell L, Barkhof F, Bohrmann B, Brooks DJ, Klunk WE, Ashford E, Yoo K, Xu ZX, Loetscher H, Santarelli L. Mechanism of amyloid removal in patients with Alzheimer disease treated with gantenerumab. Arch Neurol. 2012 Feb;69(2):198-207. Epub 2011 Oct 10 PubMed.
  6. Bell RD, Ehlers MD. Breaching the blood-brain barrier for drug delivery. Neuron. 2014 Jan 8;81(1):1-3. PubMed.

Primary Papers

  1. Niewoehner J, Bohrmann B, Collin L, Urich E, Sade H, Maier P, Rueger P, Stracke JO, Lau W, Tissot AC, Loetscher H, Ghosh A, Freskgård PO. Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle. Neuron. 2014 Jan 8;81(1):49-60. PubMed.

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