Download Alphabodies – working inside the cell

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Commentary: Mark Vaeck
Alphabodies – working inside the cell
A large proportion of all known human protein targets cannot
be addressed by either small chemical drugs or biologics.
Small chemicals typically interact with hydrophobic pockets,
which limit their target space to about 10% of all human
proteins; similarly, biologics, including antibodies, lack the
ability to penetrate through cell membranes, and therefore
can only address another 10%, that exist as extracellular
proteins. It is therefore estimated that the vast majority of
all potential protein targets, more than 80%, are currently
considered ‘undruggable’ by the two main classes of
therapeutic drugs.1 A large number of these undruggable
targets belong to the interesting class of intracellular
protein-to-protein interactions. Intracellular protein-toprotein interactions regulate a wide variety of essential
cellular processes, many of which are known to be involved in
important disease processes, such as those causing cancer and
central nervous system and metabolic diseases. The potential
to modulate protein interactions inside the cell across multiple
targets and disease categories represents a significant medical
and commercial opportunity.
This opportunity was seized by the founding scientists of
our company in 2007 when they synthesised an artificial
protein which we now call the ‘alphabody’. The alphabody
is a small single-chain protein, 70-100 amino acids long,
with a molecular weight in the range of 8-12,000 dalton,
and comprises three alpha-helices organised in a stable and
compact ‘coiled coil’ structure. The target-binding moiety of an
alphabody is created by engineering patches of varying amino
acid residues on the surface of an alphabody structure.
Although alpha-helix coils or bundles do exist in nature,
the typical three alpha-helix coiled coil of an alphabody is
not derived from any naturally occurring protein. Instead it
is an artificial protein scaffold, entirely developed in silico,
via computer design, by the protein engineering scientists at
Complix. Despite being artificial, alphabodies have shown in
animal studies to be non-toxic and non-immunogenic.
Given these characteristics, we believe the molecule is
suitable to act as a drug scaffold against a broad range of
disease targets including cytokines, cell receptors, viral entry
regulators and most importantly, intracellular protein-toprotein interactions. Alphabodies share features of other
small therapeutic protein scaffolds such as nanobodies
including stability in serum and an ability to penetrate tissue.
Significantly, alphabodies can be engineered to penetrate
intracellular space, while retaining stability. Recently our
research team generated data showing that alphabodies
specific for the intracellular cancer target MCL-1 can interact
with this target and cause biological effects on the cancer cells
in vitro as well as in vivo. We call this subset of alphabody the
‘cell penetrating alphabody’.
The discovery process at Complix involves a combination of
in silico design with library screening, allowing us to obtain
leads in less than three months. The rigid alpha-helical coil
structure of alphabodies allows them to be created with
precision and a high probability of success in silico. Moreover
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the in silico design procedure does not require isolation and
purification of the target molecule, which is an advantage in
case targets are unstable in a test tube, as many membrane
receptors and intracellular proteins can be.
To transform an alphabody into a cell penetrating
alphabody, specific sequence patterns are introduced to the
alphabody. Typical sequence patterns comprise a mixture
of positively charged amino acids and hydrophobic residues.
The cell penetrating alphabody residues are integrated at
spatially fixed positions giving optimal contact with certain
membrane components in order to induce effective membrane
transduction. The cell penetrating alphabody design can
be combined with target binding capacity, thanks to the
versatility of the alphabody scaffold.
Cellular uptake studies, conducted with cell penetrating
alphabodies on a variety of cancer cell types, have revealed
that uptake can be rapid and concentration dependent,
allowing them to reach therapeutically relevant intracellular
concentrations within a few hours of incubation. The proteins
mostly enter directly into the cytosolic space of the cells,
and do not end up in the endosomal pathway, in most cases
an important prerequisite for achieving effective target
interaction. Importantly, alphabodies that are not designed
as cell penetrating alphabodies do not have membrane
penetrating capacity and therefore remain outside cells,
as expected. Such unmodified alphabodies are suitable for
addressing extracellular targets such as our programme
targeting Interleukin-23.
For our initial therapeutic programme for cell penetrating
alphabodies, we have selected the intracellular target protein
MCL-1. MCL-1 belongs to the family of BCL-2 proteins, which
regulate cellular apoptosis, determining whether a cell dies
or survives. In cancer cells, MCL-1 is often overexpressed and
prevents these cells from entering the apoptosis phase. We
have now designed and produced proteins that antagonise
MCL-1, and consequently induce cell death in the cancer
cells. MCL-1 is a potential drug target and is suspected to be
implicated in several types of haematological cancers. More
recently we have generated a panel of anti-MCL-1 alphabodies
that display various cross-reactivities against other different
members of the BCL-2 family.
Today Complix has several discovery programmes including
an agreement with the International AIDS Vaccine Initiative
to apply the technology to understand the structure of the
HIV protein complex. In short, we are looking for innovative
solutions to some of the world’s most intractable diseases.
Reference:
1.Verdine, 2010, The Harvey Lectures, Series 102: 1-16.
This commentary was written by Mark Vaeck, a
co-founder and currently chief executive officer, of
Complix NV, which is located in Hasselt, Belgium.
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