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Roche – Nature Biotechnology Symposium 2014:
Unlocking cell transport barriers to
deliver large therapeutics
3–5 September 2014, Roche Forum, Buonas, Switzerland
A
plethora of large molecules currently in the
clinic—monoclonal antibodies and antibody fragments, antibody-drug conjugates and
cytokine fusion proteins, are designed to target cells and tissues of interest. However, none
can precisely bring their therapeutic payloads
to targets within—rather than on—those cells.
“Our engineering capabilities seem to exceed
our knowledge on the biology that drives the
uptake and release of molecules in the cell,”
says Sylke Poehling, Acting Head of Large
Molecule Research and Head of Strategy and
Portfolio at Roche Pharma Research and Early
Development (pRED) in Basel, Switzerland.
“We have a good idea of how our [therapeutic] molecules get to the cell,” she adds, “but
we then have problems understanding how we
can then get the cargo into the cytosome”—
the cell’s internal machinery. “That’s basically
where I’d say we are still stuck to a large extent.”
Andrew Marshall, Chief Editor of Nature
Biotechnology in New York City, agrees. “Drug
delivery is one of the most important, fundamental problems we face,” he says. “Every
time a new therapeutic modality comes along
there’s this question: how do we get this new
therapeutic into the cell? This problem has
been around for decades, and we really haven’t
figured it out.”
To make headway on this problem, Roche
and Nature Biotechnology teamed up to bring
together some of the leading researchers in the
fields of drug delivery and cell biology. “This is
the right moment to have a meeting about the
intersection of these two communities,” notes
Marshall. “We need new thinking about this.
We need new ideas.”
At the 2014 Roche–Nature Biotechnology
Symposium, held over three days in early
September at the Roche Forum Buonas facilities on the shores of Lake Zug in Switzerland,
leading experts discussed how best to facilitate
Roche - Nature Biotechnology
the distribution and intracellular targeting of
macromolecule therapeutics.
Taking it all in
The meeting commenced with a keynote lecture outlining the current understanding of
how large molecules enter the cell — endocytosis 101. After going over the basics, Tomas
Kirchhausen, a symposium co-organizer and
cell biologist at Harvard Medical School in
Boston, Massachusetts, discussed a new technology that he describes as “transformative” for
teasing apart the molecular events by which
small transport organelles known as vesicles
traffic their cargo.
Kirchhausen recently acquired a Bessel
beam plane illumination microscope, also
known as a lattice light sheet microscope.
This imaging device—invented by biophysicist 2014 Nobel prize winner Eric Betzig at the
Howard Hughes Medical Institute Janelia Farm
Research Campus in Ashburn, Virginia—takes
advantage of very thin beams of light to noninvasively acquire hundreds of three-dimensional pictures per second from single living
cells. As an example, Kirchhausen played videos showing how the technique could track the
dynamics of vesicle formation via the recruitment of clathrin coat molecules at the surface
of the cell membrane.
According to Kirchhausen, the same method
could also be used to monitor the movement
of drug molecules. “Just let your imagination
now go: you can visualize any biological process,” he says.
One such process that could be better understood with this approach is how drugs escape
the endosome, a compartment within the cell
that holds molecules before they are typically
destroyed by the cell’s enzymatic machinery. “Getting out of the endosome is still the
big issue,” says James Wells, a cellular and
molecular pharmacologist at the University of
California, San Francisco, who was also a coorganizer of the meeting. “But how do we do it
without inducing toxicity?”
Following Nature’s lead
Several researchers are looking to pathogens
for inspiration. In his talk, Stephen Harrison, a
structural biologist at Harvard Medical School,
discussed how non-enveloped viruses such as
rotavirus manage to translocate their particles
across the cell membrane, through endosomes
and into the cytoplasm, using single virion live
cell imaging. He discussed how cell membrane
curvature and virion conformation changes
during the cell uptake process. The hope is that
by capitalizing on the ways by which bacteria
and viruses naturally evade the host’s intracellular defenses, drug developers should be able
to get therapies where they’re needed to have a
therapeutic effect. “That’s really the way to go,”
says Poehling, “because I truly believe that if
nature hasn’t invented something that we can
build on then it’s probably too tough to tackle.”
One route into the cell involves toxins
secreted by pathogenic bacteria. In the case of
the Shiga toxin—which is produced by Shigella
dysenteriae and Escherichia coli—this transport
system goes through the Golgi network and
endoplasmic reticulum to arrive at the cytoplasm. Ludger Johannes, a cell biologist at the
Curie Institute in Paris, France, highlighted
how his team has used a harmless subunit
of the Shiga toxin (STxB) to deliver antigens
into cells as a cancer immunotherapy strategy.
Again he discussed the dynamics of membrane
curvature, membrane ganglioside content and
the importance of shape of the toxin in uptake
and the subsequent process of endosome sorting through SNARE.
In a similar vein, Andreas Plückthun, a
protein engineer at the University of Zürich
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The organizers and faculty on the first day of the Roche – Nature Biotechnology Symposium 2014.
in Switzerland, has used toxin subunits from
anthrax and Pseudomonas bacteria to efficiently target an antibody-like scaffold format
termed DARPins (designed ankyrin repeat
proteins). “The combination of evolution and
design is really quite powerful,” Plückthun says
of this approach. Plückthun went on to show
how the use of a simple biotin ligase assay to
measure intracellular uptake of DARPin. The
talk highlighted the importance of developing
simple assays to measure intracellular delivery
of therapeutic payloads.
No brain, no gain
Endosome escape is one of the key challenges
in drug delivery. Another is breaching the socalled blood-brain barrier (BBB), which will
need to be overcome if companies wish to
deliver large molecules in diseases with neurological or psychiatric pathologies
The BBB is essential for protecting the
central nervous system (CNS) from injury
and neuronal damage. As molecular biologist Richard Daneman from the University
of California, San Diego, discussed, it does so
through the action of pericytes, contractile cells
that wrap around the endothelial cells in the
capillaries to restrict vascular permeability and
therefore the movement of molecules between
the bloodstream and the brain. “And we think
that they do this by inhibiting the expression
of genes that normally make the blood-brain
barrier leaky,” Daneman says.
Even so, the BBB seals itself off, drug companies would like to find some way to penetrate
the brain’s defenses. According to Anirvan
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Ghosh, Global Head of CNS Discovery at
Roche pRED, the BBB “has really hindered
the development of a large class of therapeutics
that could be of great benefit.”
A Roche-designed platform could help
give large molecules a lift across the BBB. The
technology works by engaging the transferrin
receptors that are involved in the transport
of molecules into the brain. By binding an
amyloid-β–targeted antibody to the transferrin receptor, scientists at Roche showed that
they could ferry the therapy into the brain
and reduce the formation of plaques in mouse
models of Alzheimer’s disease.
According to Per-Ola Freskgård, Roche’s
preclinical project leader for the Brain Shuttle,
the platform can be used with all manners of
biologic drugs. “You can link on this module
to any type of cargo — like an antibody, a peptide, an enzyme or an antisense oligonucleotide,” he says. To this end, Roche has teamed
up with other pharmaceutical companies to fit
the transferrin shuttle to antibodies capable of
treating Parkinson’s disease and to antisense
therapies for Huntington’s.
Matthew Wood, a neuroscientist at the
University of Oxford in the UK, presented two
other methods of trans-BBB drug delivery, at
least for nucleic acid–based therapies. In one,
his group engineered extracellular vesicles to
transport small RNAs to the brain. In the other,
his team tethered an exon-skipping oligonucleotide therapy with cell-penetrating peptides.
Wood had been designing the peptides for
optimal heart tissue delivery as a treatment for
Duchenne muscular dystrophy. But after see-
ing some infiltration into the brain, he hopes
to modify the peptides for targeted delivery of
therapeutics for neuromuscular diseases such
as spinal muscular atrophy. “We’ve really just
skimmed the surface of the peptide design
space,” Wood says, “and it seems highly likely
that we’ll be able to optimize and improve these
peptides so they penetrate much, much better
into the brain.”
Meanwhile, Richard Ransohoff, a neurologist
at the Cleveland Clinic in Ohio, is studying what
goes wrong with the BBB in a disease called neuromyolitis optica (NMO) in hopes both of finding new therapies for this inflammatory CNS
condition and to reveal biological processes that
could be exploited for smuggling drugs across
the BBB in healthy people. NMO is characterized by antibodies to the astrocyte water channel
aquaporin 4 (AQP4), but it has been a mystery
how the antibodies enter the brain. “If we can
puzzle out the target and the underlying signaling pathway that causes this mild leakiness
[of the BBB], then I think we’re going to have
some potentially very valuable tools for facilitating transport of therapeutic proteins or other
molecules into the CNS,” Ransohoff says. Using
an innovative in vitro BBB model, Ransohoff
has identified an Ig component in NMO sera
distinct from AQP4. He believes that the action
of this antibody on endothelial cells results in
changes in porosity of the BBB that enable AQP4
antibodies to enter the brain.
Deliver the goods
The BBB is not the only barrier to drug delivery
in our bodies — and many engineering soluRoche - Nature Biotechnology
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tions will be needed to traverse them. Take the
mucosal surface, for example. This epithelial
layer separates the host tissues from the environment. Wayne Lencer, a cell and molecular biologist at Boston Children’s Hospital in
Massachusetts, used GM1 ganglioside as a
vehicle for trans-mucosal delivery of bioactive peptides. As a proof of principle, his team
fused GM1 to a hormone drug called glucagon
like peptide (GLP-1) with the hope of creating an orally delivered therapy for diabetes. At
the meeting, Lencer won first prize in a poster
competition for this work.
Rakesh Jain, a chemical engineer turned
cancer biologist at the Massachusetts General
Hospital in Boston, argued at the meeting
that drug penetration in solid tumors is frequently impaired by vascular compression due
to extravascular solid stress. He eloquently
related how the efficacy of many cancer
therapies is thwarted by this stress, which is
generated as dividing cells push and pull on
their surroundings. Jain suggested that treatments, such as hyaluronidase, might be of
use in increasing delivery of cancer therapies
to solid tumors. “The problem is that a halfchoked tumor is worse than what you started
with,” Jain says.
Targeting anti-cancer drugs so they penetrate effectively into the tumor microenvironment was a major focus for many presenters at
the symposium. Mauro Ferrari, a bioengineer
at the Houston Methodist Research Institute
in Texas, discussed the physical barriers to
effective delivery of a nanoparticle vector in
different cancerous tissues and lamented the
lack of an appreciation for the physics of the
process by many practitioners. His group is
developing vectors to sequentially address
different parts of the delivery process to
the cell cytoplasm, an approach he terms
multistage nanovectors. In another talk,
Ronald Raines, a chemical biologist at the
University of Wisconsin–Madison, discussed
work involving an engineered ribonuclease
enzyme that due to its highly cationic nature
can enter cells. By engineering the protein to
evade endogenous inhibitor in tumor cells,
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Poster sessions were a source for vigorous discussions among the attendees
Raines hopes to create a new type of anticancer therapy.
Some experimental therapeutic modalities
beyond traditional antibodies and proteins
also need specialized solutions to treat human
disease. Small gene-silencing RNA molecules,
for instance, are highly charged molecules that
usually must be formulated in lipid nanoparticles to enable efficient delivery in cells. Steven
Dowdy, a molecular biologist at the University
of California, San Diego, underwent “six tortuous years of chemistry” to synthesize short
interfering ribonucleic neutral (siRNN) molecules, which are uncharged, easier to deliver
and result in robust therapeutic responses in
animal models. “These siRNNs have all these
drug-like properties,” Dowdy says. “I think this
has legs. I think it’s scalable. And I think we’ve
fleshed out most of the chemistry.”
Such strategies will be needed for most
types of large molecule drugs. Fortunately,
however, even inefficient delivery endosome
escape can have large clinical consequences for
small RNAs. Judy Lieberman, an immunolo-
gist at Boston Children’s Hospital, presented
evidence showing that only around 1% of these
therapeutic RNA molecules get released into
the cytoplasm of liver cells. Still, that’s enough
to achieve dramatic gene knockdown. “You
don’t need very many molecules of small RNAs
to get a very powerful effect,” Lieberman says.
Known unknowns, unknown unknowns
All these findings discussed at the September
meeting often raised more questions than
offered answers. But at least they’re starting to
fill in some of the knowledge gaps in the field
of large molecule drug delivery. “There’s a little
clink of light of how these different mechanisms might be working,” Marshall says.
Poehling, reflecting on the meeting, adds:
“We appreciate how much we still don’t know
about basic biology and basic mechanisms, but
I’ve seen a lot of really good dialogue going on
at this intersection of molecular biology and
engineering and cell biology. People really
started to identify how we could forge collaborations to help us advance the field.”
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