Download Next-generation protein drugs

Next-generation protein drugs
Ian M Tomlinson
Ankyrin repeats generate high-affinity protein binders with biophysical properties that may favor
therapeutic applications.
Ask any major pharmaceutical company what
constitutes an ideal drug and the answer
would probably include the words ‘specificity,
affinity, solubility, stability and safety’ along
with the phrases ‘cheap to manufacture, easy
to formulate, simple to deliver and the right
pharmacokinetic profile.’ Ironically, many
drugs on the market fail to deliver in one or
more of these areas because of their sub-optimal biophysical makeup. Even blockbuster
biologics, such as therapeutic antibodies1,
suffer from drawbacks, such as the requirement for an expensive mammalian cell production system and the need for intravenous,
intramuscular or subcutaneous injection
(with molecular weights of around 150,000,
they are too large to be administered by any
other route). Clearly, there is room for
improvement. In this issue, Binz et al.2
describe a natural scaffold, ankyrin repeat
protein, that has promising biophysical properties for therapeutic application. Ankyrin
repeats are one of several new types of scaffold being developed for a new generation of
protein therapies.
An ideal drug would have the following
qualities: it would have very high affinity and
exquisite specificity for its target; it could
be manufactured by the bucket-load in bacteria or yeast; it would be both incredibly soluble and remarkably stable; it could be
delivered to any part of the human body by
any route of administration; and, once there,
it would hang around long enough to have
the desired therapeutic effect. Achieving all
these goals has been particularly difficult for
protein drugs.
Currently, protein drugs come in all shapes
and sizes: some are recombinant human proteins (for instance, insulin, growth hormone
Ian M. Tomlinson is Chief Scientific Officer of
Domantis Limited, 315 Cambridge Science
Park, Cambridge CB4 0WG, UK.
e-mail: [email protected]
Figure 1 All in a bind. Binz et al. randomized 6
of the 33 amino acids (red side chains) in three
ankyrin repeats (dark blue) and, using ribosome
display, isolated a range of nanomolar binders to
mannose-binding protein. The co-crystal structure
confirms the predicted binding of the engineered
ankyrin repeat protein to the mannose-binding
protein target.
Ankyrin repeat protein
Ribosome display selection for
high-affinity binders to
maltose-binding protein
Maltosebinding
protein
Ankyrin
repeat
protein
Bob Crimi
© 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology
NEWS AND VIEWS
and erythropoietin), others are monoclonal
antibodies (for instance, Remicade (infliximab; Johnson & Johnson, Kenilworth, NJ,
USA), Rituxan (rituximab; Genentech; S. San
Francisco, CA, USA) and Erbitux (cetuximab;
ImClone, New York, NY, USA) and still others
are viral or bacterial proteins used as vaccines
to elicit a specific immune response. Nature
did not evolve proteins for manufacture ex
vivo. For this reason, many human proteins
produced in recombinant form are difficult to
manufacture and some cannot be expressed
at all in microbial cell culture. Furthermore,
the serum half-life and tissue distribution of
endogenously expressed proteins is carefully
controlled in vivo to optimize their biological
activity. Most human proteins are not
NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 5 MAY 2004
designed to be administered from outside the
body. Recombinant proteins therefore tend to
be rapidly cleared and thus require frequent
injection (thus, the growing interest in
extending the serum half-life by, for example,
polyethylene glycol conjugation).
Antibodies have proved useful as human
protein therapeutics because they exhibit a
favorable pharmacokinetic profile. After a
single injection, they can persist for a long
time in the bloodstream, maintaining their
biological activity for several weeks. However,
antibodies have also evolved to be secreted
from mammalian cells and, for a variety of
reasons, cannot be expressed in yeast or bacterial cell culture.
Given the limitations of current protein
therapies, scientists are starting to develop
more tailored approaches to drug design
whereby you first assemble a list of the various properties you want the drug to have and
then engineer a drug with precisely those
properties. Over the past three years, several
new biotech companies have been set up to
exploit the use of ‘well-behaved’ human proteins as scaffolds to create a range of designer
protein drugs that have improved therapeutic
properties (see Table 1). This approach proceeds through the following steps: first, chose
a human protein that is well expressed in bacteria and/or yeast and has good biophysical
properties (solubility, stability and others);
second, create a repertoire by introducing
diversity into the loop regions of the given
scaffold, preferably in a way that does not disrupt the overall structure of the protein; third,
521
NEWS AND VIEWS
Table 1 Selected companies using human proteins as scaffolds to create nextgeneration drugs
© 2004 Nature Publishing Group http://www.nature.com/naturebiotechnology
Company
Protein scaffold
BioRexis (King of Prussia, PA, USA)
Transferrin
Borean Pharma (Aarhus, Denmark)
C-type lectins
Compound Therapeuticsa (Waltham, MA, USA)
Trinectins
Domantis (Cambridge, UK)
Domain antibodies
Dyax (Cambridge, MA, USA)
Kunitz domains
Pieris ProteoLab (Freising-Weihenstephan, Germany)
Lipocalins
aOn
9 March 2004, Compound Therapeutics announced the acquisition of the intellectual property estate of Phylos
(Lexington, MA, USA).
use a genotype-phenotype display system
(such as phage3 or ribosome display4) to
select a range of binders to a given therapeutic
target; and fourth, use some form of screen to
identify those leads that have the desired biological activity. If all goes according to plan,
the outcome should be a protein that has all
the desirable biophysical properties of the
parental scaffold and the required potency for
the therapeutic target.
Of course, it’s not always that straightforward. In many cases, the mutations introduced to enable binding to the target
compromise the biophysical properties
and/or the three-dimensional structure of the
parental scaffold. In some cases, it may not
even be possible to achieve the desired
potency using such a small binding footprint
to the given target. And of course each starting scaffold has its unique pros and cons—
intracellular or extracellular expression, different binding sites for purification, immunogenicity and so on. Some scaffolds may have
intrinsically long serum half-lives5, whereas
others may show unusual properties, such as
the ability to refold reversibly after heat
denaturation6.
In the present paper, Binz et al. focus on the
use of one particular scaffold, based on
ankyrin repeats, to generate binders with biophysical properties designed for therapeutic
application. Ankyrins are proteins, first isolated in mammalian erythrocytes, involved in
the targeting, mechanical stabilization and
orientation of membrane proteins to specialized compartments within the plasma membrane and endoplasmic reticulum. Natural
ankyrin repeat proteins consist of many 33amino-acid modules, each comprising a βturn and two anti-parallel α-helices7. They do
not contain any disulfide bonds and therefore
can be expressed at very high yields in the
bacterial cytoplasm. They also seem to be
both highly soluble and stable.
The approach used by Binz et al. randomizes 6 of the 33 amino acids in each of
522
two or three ankyrin repeats. The diversity
thereby generated (12 or 18 randomized
residues) is sufficient to isolate a range of
nanomolar binders to mannose-binding
protein using ribosome display, all of which
have the desirable biophysical properties
of the parental ankyrin scaffold. Importantly, the authors also showed, by co-crystallization, that the selected binders have the
same structural fold as the parental scaffold.
Although the authors have yet to demon-
strate in vivo efficacy with an engineered
ankyrin repeat protein, the libraries they have
created should be a valuable resource for
the isolation of therapeutically relevant leads
that are both well expressed and highly
stable.
Undoubtedly, there is a big drive for the
drugs of the future to be much easier to manufacture and administer to patients. They
must also be highly efficacious with few, if
any, side effects. By wiping the slate clean and
designing potent drugs based on human protein scaffolds with good biophysical properties, we may find that the ideal drug is closer
than ever before.
1. Reichert, J.M. Nat. Biotechnol. 19, 819–822 (2001).
2. Binz, H.K. et al. Nat. Biotechnol. 22, 575–582
(2004).
3. Scott, J.K. & Smith, G.P. Science 249, 386–390
(1990).
4. Mattheakis, L.C., Bhatt, R.R. & Dower W.J. Proc. Natl.
Acad. Sci. USA 91, 9022–9026 (1994).
5. Ali, S.A., Joao, H.C., Hammerschmid, F., Eder, J. &
Steinkasserer, A. J. Biol. Chem. 274, 24066–24073
(1999).
6. Jespers, L., Schon, O., James, L.C., Veprintsev, D. &
Winter, G. J. Mol. Biol. 337, 893–903 (2004).
7. Sedgwick, S.G. & Smerdon, S.J. Trends Biochem. Sci.
24, 311–316 (1999).
Overcoming the gridlock in
discovery research
Jonathan Margolis & Greg D Plowman
Chemical screening in a zebrafish mutant has turned up two compounds
that rescue a heart defect, but will this yield new drugs?
Why is it so hard to find new drugs? A significant amount of time goes into finding and
validating new drug targets for the development of small-molecule or biotherapeutic
leads. Why not create in vivo disease models
and directly screen for compounds that can
ameliorate the disease state? In this issue,
Peterson et al.1 describe an effort to do just
that, using a zebrafish mutant with an
anatomical defect that resembles a malformation in the human heart.
In theory, whole-organism screening
should circumvent the need to identify specific drug targets, allowing the entire genome
to be screened in a single, unbiased assay. This
Jonathan Margolis and Greg D. Plowman
are at Exelixis, 170 Harbor Way, P.O. Box 511,
South San Francisco, CA, USA.
e-mail: [email protected]
approach is equivalent to a classical genetic
mutant suppressor screen, in which one
searches for secondary mutations that revert
the abnormal phenotype to wild type.
Sensitized cell-based screens have previously
been used to identify chemical suppressors of
a disease process—for example, drug leads
that block the proliferation of carcinoma
cells2. Peterson et al. extend this strategy to a
vertebrate organism. They describe compounds that rescue the abnormal vascular
development of a zebrafish mutant and suggest that this could provide a rapid path to
drug leads for diseases whose underlying biology is not well understood.
The gene encoding the gridlock transcription factor is a classic developmental selector
controlling the choice of angioblasts between
venous and arterial fates in the developing
fish heart (Fig. 1). With a partial reduction of
gridlock activity, the bifurcation of the lateral
VOLUME 22 NUMBER 5 MAY 2004 NATURE BIOTECHNOLOGY