Download Protein Engineering for Cardiovascular Therapeutics

Review
This is the first Review in a new thematic series on Repairing the Heart: Thinking Outside the Stem Cell Box, which
includes the following articles:
Patching the Heart: Cardiac Repair From Within and Outside
Protein Engineering for Cardiovascular Therapeutics: Untapped Potential for Cardiac Repair
Cardiac Tissue Engineering—State of the Art
3D Engineering in the Heart Chamber
Jeffrey Robbins, Editor
Protein Engineering for Cardiovascular Therapeutics
Untapped Potential for Cardiac Repair
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Steven M. Jay, Richard T. Lee
Abstract: A number of new and innovative approaches for repairing damaged myocardium are currently undergoing
investigation, with several encouraging results. In addition to the progression of stem cell–based approaches
and gene therapy/silencing methods, evidence continues to emerge that protein therapeutics may be used to
directly promote cardiac repair and even regeneration. However, proteins are often limited in their therapeutic
potential by short local half-lives and insufficient bioavailability and bioactivity, and many academic laboratories
studying cardiovascular diseases are more comfortable with molecular and cellular biology than with protein
biochemistry. Protein engineering has been used broadly to overcome weaknesses traditionally associated with
protein therapeutics and has the potential to specifically enhance the efficacy of molecules for cardiac repair.
However, protein engineering as a strategy has not yet been used in the development of cardiovascular therapeutics
to the degree that it has been used in other fields. In this review, we discuss the role of engineered proteins in
cardiovascular therapies to date. Further, we address the promise of applying emerging protein engineering
technologies to cardiovascular medicine and the barriers that must be overcome to enable the ultimate success of
this approach. (Circ Res. 2013;113:933-943.)
Key Words: heart diseases
■
heart failure
A
n explosion of interest in new therapies for heart repair has occurred recently. Most notably, clinical trials
of cell-based cardiovascular therapy have garnered much attention, and preliminary results are encouraging.1,2 However,
the mechanism of action of cell-based therapies remains unclear, and many details with regard to characterization, quality
control, and delivery of cells remain to be worked out before
widespread therapeutic application. Nonetheless, the reported
efficacy of cardiac cell–based therapies in human trials has
generated interest in additional therapeutic development pathways. The identification of the release of paracrine-acting
proteins as one mechanism by which cell-based therapies in
the heart may improve cardiac function3 has spurred renewed
■
peptides
■
proteins
■
receptors
interest in protein-based therapeutic approaches for cardiac
repair.
Although protein therapeutics are increasingly becoming mainstream in a number of fields, including cancer and
inflammatory diseases, (9 of the 20 top-selling drugs in
2012 were proteins4) proteins have relatively low penetration in the cardiovascular market. This is not to suggest a
lack of progress in the cardiovascular therapeutic development, because small-­molecule drugs such as statins5,6 and
anticoagulants7 have been huge successes. Rather, as overall
new drug approvals shift predominantly to proteins, protein
therapeutic development represents an area of opportunity
for cardiovascular medicine. Protein therapies allow for
Original received March 26, 2013; revision received May 6, 2013; accepted May 6, 2013. In July 2013, the average time from submission to first decision
for all original research papers submitted to Circulation Research was 13.24 days.
From the Department of Medicine, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA (S.M.J., R.T.L.);
Harvard Stem Cell Institute, Cambridge, MA (S.M.J., R.T.L.); and Brigham Regenerative Medicine Center, Cambridge, MA (S.M.J., R.T.L.).
Correspondence to Richard T. Lee, MD, Brigham Regenerative Medicine Center, Partners Research Facility, 65 Landsdowne St, Room 280, Cambridge,
MA 02139. E-mail [email protected]
© 2013 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.113.300215
933
934 Circulation Research September 13, 2013
Nonstandard Abbreviations and Acronyms
BNP
EPO
EPS
G-CSF
IGF
IL-1
MI
ncAA
NRG
SDF-1
TNF
B-type natriuretic peptide
erythropoietin
engineered protein scaffold
granulocyte colony–stimulating factor
insulin-like growth factor
interleukin 1
myocardial infarction
noncanonical amino acid
neuregulin
stromal cell–derived factor-1
tumor necrosis factor
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more targeted interventions, with well-defined regulatory
pathways, improved scalability, and potentially reduced
overall cost, compared with cell-based approaches.
Furthermore, proteins have much larger surface areas than
the small molecules of typical orally bioavailable drugs;
thus, many of the molecular targets that are probably
not druggable with small molecules can be targeted with
­proteins. A classic example of this concept is that no clinically successful small molecule activates the insulin receptor, and therefore patients with type I diabetes mellitus must
inject insulin.
With regard to proteins for cardiovascular therapy, much
effort has been put into enhancing cardiac microvasculature formation. More recently, the potential of proteins to
induce cardiomyocyte proliferation has been identified,8
increasing optimism that protein-based approaches can be
effective for cardiac regeneration. However, protein therapeutics often have limitations, including poor bioavailability, undesirable pharmacokinetics and biodistribution
profiles, and off-target effects.9–12 One approach to overcome these deficiencies is protein engineering, a strategy
that may not only render a therapeutic concept feasible but
also generate the intellectual property necessary for development in patients.
The term protein engineering is a general one that covers
many techniques for modifying proteins, including the use of
molecular display technologies to enable targeted delivery,13,14
structural modification of proteins to impart enhanced properties such as resistance to enzymatic degradation via rational
design15 or directed evolution,16 fusion of proteins to polymers
or other protein domains to promote immune evasion,17,18 and
incorporation of desired properties or functional groups via
noncanonical amino acids (ncAAs).19,20 Despite this promise
and widespread application in fields such as oncology, engineered protein therapeutics have to date failed to become a
major part of the toolkit of cardiologists. In this article, we
review the use of protein therapeutic approaches in the heart
to date, with emphasis on therapies that have reached the
clinical trial stage. We then highlight emerging and promising techniques in protein engineering and discuss how, when
combined with new discoveries in molecular cardiology, these
approaches might lead to a new generation of therapeutics for
repairing the heart.
Protein Engineering for Cardiovascular
Therapeutics to Date
Although none has yet achieved widespread clinical use, a
number of engineered proteins have been applied to cardiovascular diseases in clinical trials. Many other native protein
therapeutic approaches have also been applied to cardiac repair, and several of these may benefit from protein engineering. Some of the earliest and most high-profile examples of
protein engineering technology for cardiovascular therapy
have been clinical failures. However, other examples have
proven more successful, and ongoing clinical trials offer exciting possibilities.
Tumor Necrosis Factor Antagonists
One of the most prominent, and ultimately unsuccessful, examples of translation of protein engineering technology for
cardiovascular therapy involved the tumor necrosis factor
(TNF) antagonist etanercept (Enbrel). Developed based on research by Beutler et al,21 etanercept is a dimeric fusion protein
comprising the Fc region of human immunoglobulin G1 and
the soluble human TNF receptor 2 domain that is approved for
treatment of various forms of arthritis. After the description of
overexpression of TNF in heart failure,22 it was hypothesized
that TNF inhibition could be an effective treatment modality. However, despite initial promising results in carefully
performed studies, both preclinically23,24 and in patients,25,26
2 large-scale clinical trials revealed that etanercept did not
show clinical benefit in chronic heart failure patients and it
might have, in fact, increased the risk of chronic heart failure–
associated morbidity and mortality.27 Moreover, the chimeric
monoclonal antibody infliximab (Remicade), a TNF inhibitor
that works through a mechanism different from that of etanercept,28,29 also did not improve the clinical outcome of patients
with heart failure , with high doses showing profound adverse
effects.30 It should be noted that both etanercept and infliximab are efficacious against various forms of arthritis, and
infliximab is highly effective against Crohn’s disease. Thus,
protein engineering–based antagonism of TNF has proven to
be a case of a successful product development pathway that
has been unsuccessfully applied to cardiovascular therapy despite compelling preclinical investigation.
Natriuretic Peptides
The natriuretic peptides offer an example of a promising application of protein engineering to cardiovascular therapy.
Atrial natriuretic peptide and B-type natriuretic peptide
(BNP), also known as brain natriuretic peptide and basic natriuretic peptide, have similar activities and are well-established as useful biomarkers in the guidance of cardiovascular
therapy.31,32 In addition, exogenous administration of BNP
has been shown to improve multiple parameters in patients
with systolic heart failure.33 However, BNP as a therapeutic
(nesritide) may be limited because of its hypotensive effects.34
C-type natriuretic protein causes a lower hypotensive response
compared with atrial natriuretic peptide and BNP because of
its lack of activity on arteries attributable to differential receptor expression.35 Additionally, a related peptide isolated
from snake venom, Dendroaspis natriuretic peptide, acts
similarly to atrial natriuretic peptide and BNP but is highly
Jay and Lee Protein Engineering for Cardiac Repair 935
potent and resistant to enzymatic degradation.36 To leverage
the advantageous properties of C-type natriuretic protein
and Dendroaspis natriuretic peptide, Burnett et al37 synthesized a chimeric natriuretic peptide that comprises domains
of both (CD-NP). Initial results from clinical trials for this
engineered protein as a heart failure therapy were promising,38
and a follow-up trial may shed more light on the potential of
CD-NP for clinical use.39
Insulin-Like Growth Factor-1
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Insulin-like growth factor-1 (IGF-1) is a protein with a molecular structure similar that of insulin and has been shown
to provide protection from the progression of heart failure in
mice.40 In humans, low serum levels of IGF-1 are associated
with an increased risk of ischemic heart disease.41 However,
the undesirable side effects of IGF-1 systemic delivery
are well-noted and include increased risk of diabetic retinopathy and cancer.42–44 Thus, as of early 2013, there were
only 2 active clinical trials examining IGF-1 (Mecasermin)
as a cardiovascular therapy.45,46 As an attempt to overcome
these effects by promoting local delivery, Tokunou et al47
engineered an IGF-1 fusion with the heparin-binding domain of heparin-binding epidermal growth factor to make
heparin-binding IGF, which proved effective in stimulating
biosynthesis of chondrocytes. Additionally, Hubbell et al48
engineered a variant of IGF-1 with increased immobilization capacity within fibrin that improved smooth muscle cell
proliferation, introducing the possibility of a cofactorial local delivery approach. Notably, an IGF-1 modified to enable
interaction with self-assembling peptides for cardiac delivery has demonstrated efficacy in improved cardiac function
after myocardial infarction (MI).49,50 Thus, although not yet
applied in clinical trials, engineered variants of IGF-1 may
yield therapeutics for cardiovascular therapy.
Stromal Cell–Derived Factor-1α
One protein undergoing active clinical investigation for cardiac regeneration—although not currently as a protein therapy—is stromal cell–derived factor-1α (SDF-1). SDF-1 is
a chemokine that plays important roles in angiogenesis and
leukocyte trafficking.51 The discovery that SDF-1 induces
stem cell homing to the heart after injury52,53 spurred interest in its therapeutic application. However, SDF-1 is proteolytically cleaved by both matrix metalloproteinase-254,55 and
dipeptidyl peptidase IV56; thus, the likelihood of retained
bioactivity in the myocardium after injury—a highly inflammatory environment—is low. For this and other reasons, the
only active clinical trial of SDF-1 for cardiac therapy uses
plasmid delivery,57,58 which offers the potential for prolonged
SDF-1 expression but is also limited by issues of safety and
unpredictability common to gene therapy approaches. Protein
engineering applied to SDF-1 offers an alternative; we developed a protease-resistant form of SDF-1 by mutating a single
amino acid within the matrix metalloproteinase-2 cleavage
site.15 This protease-resistant SDF-1 successfully induced endothelial progenitor cell recruitment to the heart after MI that
resulted in improved cardiac function15 and led to increased
angiogenesis and improved ventricular function after onset of
myocardial ischemia.59 Protein engineering efforts to improve
delivery and tissue retention of SDF-1 have also been reported,60 as has the creation of a polypeptide analog of SDF-1 that
induced improved recovery after MI compared with the native
protein.61 Future synergy of these and other protein engineering strategies may allow for a therapeutic approach that overcomes the limitations of the native SDF-1 protein.
Granulocyte Colony–Stimulating Factor
Granulocyte colony–stimulating factor (G-CSF) is a glycoprotein that selectively induces a reduction of SDF-1 and an
increase in the SDF-1 receptor CXCR4 (C-X-C motif chemokine receptor) in the bone marrow.62 A majority of clinical trials involving G-CSF in the heart have focused on its use as an
adjunct to cell therapy,63–65 but others have assessed its potential as a primary therapy66–69 as G-CSF has been shown to prevent deleterious remodeling after MI.70 A potential next step
in these active lines of therapeutic investigation could involve
the use of fusion proteins incorporating G-CSF moieties, because improved functionality relating to delivery71 and stability72 of G-CSF has already been reported.
Interleukin Receptor Antagonists
Interleukins (ILs) are proteins that induce pleiotropic effects
in a wide variety of cell types.73 In the heart, IL-1 is a potent mediator of inflammation during ischemia/­reperfusion
injury74 and elevated plasma levels of IL-6 have been linked
with increased risk of future MI75; thus, their inhibition is
of therapeutic interest. IL-1 has a naturally occurring competitive inhibitor for its receptor (IL-1 receptor), the IL1–related protein IL-1 receptor antagonist (IL-1 receptor a),
also known as anakinra.76 Both forced overexpression and
injections of anakinra have been shown to reduce apoptosis
after heart injury in rodents,74,76 and this drug has been applied in clinical trials with promising results.77 In addition,
IL-6 inhibition has been pursued via antagonism of its receptor (IL-6 receptor or CD126) by an engineered, humanized
monoclonal antibody, tocilizumab. Encouragingly, a human
case report of tocilizumab administration resulting in successful improvement of cardiac dysfunction associated with
multicentric Castleman disease has been published,78 and a
clinical trial investigating the efficacy of tocilizumab in MI
is ongoing.79 Thus, IL receptor antagonists may represent a
promising path forward for protein engineering applied to
cardiovascular therapy.
Erythropoietin
A glycoprotein that regulates red blood cell production,
erythropoietin (EPO), has well-documented cardioprotective
properties and works via multiple mechanisms.80,81 Preclinical
studies in both rat82 and rabbit83 acute MI models showed that
EPO administration improves cardiac contractility and hemodynamic parameters, prompting human clinical trials. Initial
findings indicated that EPO, although failing to improve left
ventricular ejection fraction, may reduce heart failure in acute
MI patients.84 However, a more recent trial has cast doubt on
the potential for EPO as a therapy for heart failure, indicating
that it may lead to increased infarct size.85 Because of these
results, focus has shifted toward examining the efficacy of low
doses of EPO,86 an approach that might be aided by applying
936 Circulation Research September 13, 2013
protein engineering techniques to improve potency and stability of EPO, as has already been demonstrated.87
Neuregulin-1β
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Neuregulin (NRG) is an essential regulator of cardiovascular
development and plays an important role in cardiovascular
disease.88 The cardioprotective potential of NRG has been
well-established in preclinical models,89,90 and enthusiasm
for future therapeutic application of NRG in heart failure
increased on the report of its potential ability to induce
cardiomyocyte proliferation.8 Clinical trials with the epidermal growth factor–like domain of NRG in heart failure
have demonstrated safety and efficacy,91,92 and another NRG
molecule, glial growth factor-2, is currently in clinical trials for heart failure93 and has shown efficacy in a preclinical
model.94 The nature of the interactions between the cognate
receptor of NRG in the heart, ErbB4, and its preferred signal induction partner, ErbB2, may provide an opportunity
for exploitation via protein engineering. Our group hypothesized that ErbB receptor interactions, which are prerequisite
to signaling, could be biased away from the most commonly
induced partnerships by receptor–ligand affinity interactions imposed by a bivalent ErbB ligand.95 Bivalent NRG
(NN) was shown to induce differential signaling compared
with NRG95 and was shown to have superior cardioprotective efficacy compared with the epidermal growth factor-like
domain of NRG in a mouse model of doxorubicin-induced
cardiomyopathy.96 Further exploration of this strategy may
reveal a benefit of protein engineering for NRG in cardiac
repair and regeneration.
Vascular Endothelial Growth Factor and
Fibroblast Growth Factor
Because of their well-characterized mitogenic effects on endothelial cells in many contexts, vascular endothelial growth
factor97 and fibroblast growth factor,98 as well as their splice
isoforms and variants, have been examined extensively for
therapeutic vascularization in the heart during the past ≈25
years.99–101 However, negative outcomes of early clinical trials with both fibroblast growth factor102 and vascular endothelial growth factor103 have stalled progress of therapeutic
vascularization approaches toward clinical translation, with
common limitations such as short half-life and systemic side
effects cited as reasons for the failures. Preclinical protein engineering studies of improved stability of angiogenic growth
factors through a variety of techniques are plentiful,104–110 and
the potential for local delivery of vascular endothelial growth
factor via fusion with a collagen-binding domain has been reported.111 Yet, there are still no protein drugs for therapeutic
vascularization of the heart used in clinical practice. Strategies
that combine protein engineering with nanotechnology109 or
other methods to improve delivery49,112–115 may hold promise to
enable protein-based therapies for therapeutic vascularization
in the heart in the future.
Additional Therapeutic Avenues
Beyond what is mentioned, additional ongoing cardiovascular
therapeutic efforts may benefit from protein engineering. Like
NRG, periostin has also been reported to induce cardiomyocyte proliferation116 and thus could be of further therapeutic
interest. Growth differentiating factor 11 has recently been
identified as a mediator of the reversal of age-related cardiac
hypertrophy117 and could open a new area of exploration for
protein therapeutics for the heart and other organs. Glucagonlike peptide-1–based therapies have been effective preclinically,118 and the cyclic peptide approach currently used119 may
lend itself to refinement through protein engineering. Biased
G-protein–coupled receptor ligands hold intriguing promise120
and represent a template for future protein engineering strategies designed to promote selective receptor activation, such
as has already been performed for the ErbB receptor system.95
Adipokines are peptides or proteins secreted by adipocytes
that can have beneficial or detrimental effects on the cardiovascular system depending on when and for how long they are
exposed to it.121,122 Protein engineering techniques that impart
enhanced control over tissue localization and delivery of this
class of peptides/proteins may prove critical in enabling their
eventual therapeutic application. Furthermore, intravenous immunoglobulin administration may be effective against certain
forms of heart failure.123 Engineering of proteins has perhaps
been most extensively applied to immunoglobulin molecules,
and so any effects observed in ongoing clinical trials124 could
potentially be augmented through robust protein engineering
approaches. Antibody-based therapeutic approaches and other
protein therapies for immunomodulation continue to emerge
as potential treatments for atherosclerosis.125 More recently,
mimetic peptides and monoclonal antibodies have been developed to inhibit PCSK9 (proprotein convertase subtilisin/kexin
type 9) to lower cholesterol levels.126 All of the aforementioned
approaches have promise, and the further application of protein
engineering techniques to augment these therapeutic strategies
could potentially enhance their efficacy.
Therapeutic Frontiers in Protein Engineering
Despite the implementation of some protein therapeutics for
cardiovascular therapy, many limitations still remain. The
ability to target delivery of a protein, or any drug, directly
to the heart would minimize both the required dose and undesirable off-target effects; however, this ability remains
mostly beyond our current reach. In general, proteins have
often been considered poor drug candidates because of their
low oral bioavailability and lack of long-term stability and
other characteristics; this applies to proteins for cardiovascular therapy as well. Many drug discovery professionals, especially those with chemistry training, are taught a common
mantra: proteins are not drugs. The protein engineering revolution in biotechnology has helped to change this perception,
as protein drugs for numerous applications—most notably
in cancer therapy—are now on the market and in the clinic.
However, this revolution has, for the most part, occurred outside of the realm of cardiovascular medicine. The existing
cardiovascular therapeutics created via protein engineering
noted demonstrate only a fraction of the potential of the field.
Innovative and exciting protein engineering approaches are
currently being developed, with many already having been
applied to noncardiovascular therapeutics. Application of
these burgeoning technologies to cardiovascular medicine,
for example, to enable heart-specific targeted drug delivery, to enhance protein stability and circulation times, or to
Jay and Lee Protein Engineering for Cardiac Repair 937
promote direct stimulation of specific pathways to promote
cardiomyogenesis via a therapeutic protein, may enhance the
utility of protein-based cardiac therapy.
Molecular Display
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Molecular display encompasses techniques that present molecules—typically peptides or proteins—on the surface of a cell,
virus, or other host entity. This approach enables a linkage of
genotype and phenotype of the displayed molecule, as its coding information is hybridized with that of the carrier. Thus,
selective enrichment is possible through successive propagation steps under specified conditions—a process known as
panning—and large polypeptide libraries can be screened for
desired properties or interactions (Figure 1).
The concept of molecular display arose after George Smith’s
seminal report of phage display, the display of a foreign protein
on the surface of filamentous phage.13 Since then, a number of
platform approaches for molecular display have been developed
to enable high throughput screening of protein interactions.
Technologies such as cell-surface display, ribosomal display,
mRNA display, and others have allowed for directed evolution
of proteins to refine activity and stability. Phage display is the
most commonly used and is popular in the biotechnology industry, having been used in the development of a number of
protein drugs,127 especially antibodies.128 Beyond directed evolution, phage display has been used for panning of biological
and tissue samples—known as biopanning—to select for peptide ligands that enable tissue-specific homing129 and enhanced
tissue retention130 of drugs or drug carriers.
In addition to phage display, other display technologies
have emerged and may have relevance for the development
of cardiovascular therapies in the near future. Introduced by
Wittrup et al,14 yeast surface display enables, among other
things, high-throughput quantitative library screening via
fluorescent-activated cell sorting and has been used to evolve
extremely high-affinity antibodies131 and peptides132 that have
facilitated molecular targeting, for example, to promote highresolution vascular imaging.133 Cell-free protein evolution
methods such as ribosome display134 or mRNA display135 are
not encumbered by transformation or expression limitations
and also allow rapid evolution of high-affinity binding proteins. Overall, molecular display technologies are generally
mature methods for selection and refinement of protein characteristics, which have significant potential to promote cardiovascular protein therapies.
Engineered Protein Scaffolds
Antibodies have been the most successful protein therapeutics
to date. However, antibodies have weaknesses as therapeutics,
including limited tissue penetration because of their large size.
To address this and other issues, a number of engineered protein scaffolds (EPS) have been developed. For protein engineering, EPS comprise a minimal polypeptide framework that
can be based on either an immunoglobulin or a nonimmunoglobulin molecule.136 The framework is typically monomeric
without disulfide bonds or glycosylation sites and is usually
highly stable and readily soluble.137 EPS are also typically
able to be easily expressed in a host organism and have surface-accessible residues that allow incorporation of sequence
diversity and subsequent selection via molecular display.
A number of EPS have moved beyond the initial development stage toward clinical use.137 Among the most
Figure 1. Molecular display. A protein or peptide with specific properties can be derived via molecular display after multiple rounds of
selection. Polypeptide libraries can be made or purchased and either inserted into molecular hosts such as phage or yeast or appended
to carrier molecules such as mRNA or ribosomes. The schematic is representative of phage or cell-surface display, where proteins are
displayed on the surfaces of organisms that are subsequently exposed to various substrates, which serve as selection criteria. In the
example, protein-mediated binding of the organism with a specific substrate followed by washing away of nonspecific binders and elution
enable amplification of those organisms displaying proteins with the desired properties. Repetitive screening with additional or more
stringent selection criteria enables ultimate isolation of an optimized protein sequence based on phenotype, which can then be linked
with genotype after extraction of genetic material from the organism. Thus, molecular display facilitates discovery of information needed
to design a protein with specific properties evolved to match nearly any chosen selection criteria.
938 Circulation Research September 13, 2013
well-developed are the following: Adnectins,138 derived from
type III fibronectin domains; Anticalins,139 derived from lipocalin; Kunitz domains,140 derived from Kunitz-type protease
inhibitors; DARPins,141 derived from ankyrin repeat proteins;
and avimers,142 derived from the A-domain of low-density
lipoprotein receptors. Ecallantide (Kalbitor), a rationally designed Kunitz domain, was approved in 2009 for clinical use
to treat hereditary angioedema143 and could potentially be used
in cardiothoracic surgery as a replacement for aprotinin. Many
other EPS are being tested for myriad applications,137 and as
molecular targets for cardiac repair and regeneration continue
to be defined, EPS have the potential to play an important role
in future development of cardiovascular therapeutics.
Noncanonical Amino Acids
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The concept of incorporating ncAAs (also referred to as unnatural amino acids, non-natural amino acids, and artificial
amino acids) site-specifically into proteins offers stunning
potential: an avenue to attach any chemical functional group
of interest to a protein, allowing for coupling with other proteins or molecules such as fluorophores or small-molecule
drugs using straightforward, efficient, and tunable chemical
reactions. Although several variations exist, the general approach involves aminoacylation of a tRNA that has an incorporated suppressor anticodon with an ncAA. This chemical
modification facilitates site-specific ncAA incorporation during mRNA translation at a nonsense codon site, which can be
inserted (site-specifically) in a gene of interest using standard
gene synthesis or molecular biology techniques (Figure 2).144
This development of ncAA technology was pioneered by
Schultz et al,19 and this group and many others have contributed to important advances that have made it robust for
imparting desired functionality to proteins for therapeutic
applications. For example, ncAA incorporation enables sitespecific PEGylation of therapeutic proteins145 to enhance evasion of the immune system and to promote longer circulation
times. This approach also allows for insertion of molecular
staples—hydrocarbon linkers that can stabilize protein secondary structure in part by physically preventing entropic
structural relaxation—into proteins to improve stability.146
This strategy could be extended via expressed protein ligation techniques147 to fuse other engineered proteins for similar
purposes.148 Using a related process developed by Tirrell et
al,149 residue-specific ncAA incorporation of interferon β-1b
was PEGylated for improved pharmacological properties.150
The ncAA site–specific incorporation has further been used to
generate bispecific antibodies151 and thus could be adapted to
the creation of bivalent NRG molecules95 that might stimulate
cardiomyogenesis. As ncAA technology continues to evolve,
its potential for application toward cardiovascular therapy,
which is already promising, should only be enhanced.
Enabling Methods for Drug Delivery
Protein engineering techniques have begun to be used for the
development of proteins and peptides that enable more effective and efficient small-molecule, protein, and nucleic acid
delivery across biological barriers targeted to specific tissues.
This is a critical consideration because effective cardiovascular protein therapies will almost certainly require targeted or
localized delivery to the heart. Some of these strategies have
already been mentioned or implied, such as the functionalization of a therapeutic protein or a microcarrier or nanocarrier
with a targeting sequence derived by molecular display or from
an EPS. This general concept has also been applied to enable
Figure 2. Incorporation of noncanonical amino acids (ncAAs) into proteins. The ncAA incorporation into proteins has been shown via
multiple methods. The schematic contrasts canonical amino acid (AA) incorporation into proteins with site-specific ncAA incorporation.
Canonical AA incorporation proceeds after aminoacylation catalyzed by an endogenous aminoacyl-tRNA synthetase, which charges
tRNA with a cognate amino acid. The ncAA aminoacylation is possible via incorporation of a heterologous orthogonal (ie, not crossreactive with host cell machinery) tRNA:synthetase pair into the cell responsible for protein production. Because of this incorporation, an
orthogonal ncAA can be inserted site-specifically in response to a specific codon (typically a stop codon). A given ncAA typically has a
structure similar to that of a canonical AA except that a desired atypical chemical functional group is included such that it is accessible to
participate in a reaction. Thus, once translated, a protein incorporating an ncAA is readily modifiable with molecules such as polyethylene
glycol (PEG), which improves circulation and limits immediate renal clearance, or with a molecule such as a fluorescent probe that
facilitates imaging, as well as many other potential possibilities.
Jay and Lee Protein Engineering for Cardiac Repair 939
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gene delivery with enhanced tissue specificity.152 In addition,
Dowdy et al153 developed the concept of protein transduction
domain–containing fusion proteins for protein delivery. In this
method, the protein of interest is fused with a protein transduction domain, also referred to as a cell-penetrating peptide,
such as that derived from the human immunodeficiency virus
transactivator of transcription protein.153 This allows, among
other things, proteins to cross biological barriers that would
normally restrict them because of size and charge considerations (Figure 3).154 Protein transduction domains have also
been used in gene delivery.155 A conceptually similar fusion
protein approach whereby the protein of interest is fused with
an antibody or peptide that is transported across a biological
barrier, such as the blood–brain barrier, by receptor-mediated
transport has also been explored156 and is sometimes referred to
as the molecular Trojan horse approach.157 Some of these techniques have already been applied to facilitate targeted delivery
to the myocardium,158 and continued expansion of these and
other efforts159 will likely prove critical to the ultimate success
of protein-based cardiovascular therapies.
Enabling Technologies for Cell-Based Therapy
In addition to providing an alternative for cell-based cardiac
therapy, protein engineering can be used to augment this therapeutic approach. Cardiac cell transplantation has been limited by poor localization of injected cells,160 an ≈90% death
rate for cells within 1 week of the injection or implantation,161
and, in the case of stem cells, uncontrolled cell proliferation or
differentiation after transplantation.162 Some of these limitations have been overcome in the most recent clinical trials1,2;
however, the efficiency and efficacy of cell-based cardiac
therapy can still be improved. Our group has used a therapeutic approach utilizing molecularly designed self-assembling peptide scaffolds combined with engineered proteins
to create a microenvironment for improved engraftment and
regenerative potential of transplanted cells for myocardial regeneration.15,49,113 In this case, self-assembly occurs through
electrostatic interactions between heterospecific complementary amino acid sequences; a peptide scaffold is engineered
to express one sequence, and the therapeutic protein entity
is engineered to express the complement. This approach allows for nearly any chosen growth factor(s) to be controllably displayed or delivered along with cells, which may bind
or otherwise interact with the peptide scaffold. In this way,
differentiation and engraftment of transplanted cells can be
improved. Moreover, the starting materials are easy to synthesize and purify on a large scale. This and other methods for
enabling cell-based therapies for the heart provide yet another
therapeutic frontier for cardiovascular protein engineering.
Conclusion
Protein engineering is a powerful approach with untapped potential for cardiovascular therapeutic development. As previously stated, approximately half of new drugs are proteins,4 and
many of these therapeutics were created via protein engineering. The application of this technology to therapeutic development should only increase in the future; yet, no engineered
protein drugs are currently being routinely applied for treatment of cardiovascular diseases, perhaps because of deficiencies in our current understanding of the molecular mechanisms
of cardiac repair and regeneration, which is still evolving (the
authors direct the reader to several extensive recent reviews on
the topic).163–166 From the standpoint of therapeutic development,
2 phenomena that contribute to myocardial repair and regeneration have been the focus of much of the effort: vascularization
and cardiomyogenesis. Although the fundamental processes that
constitute vascularization—angiogenesis, arteriogenesis, and
vasculogenesis—are relatively well-defined, the mechanistic
Figure 3. Protein engineering
methods to enable drug
delivery. A number of
techniques within the realm
of protein engineering could
be used to enhance delivery
of small molecules, proteins,
and nucleic acids to the heart.
Examples shown include the
targeting of a nanocarrier
to a cell type of interest (eg,
cardiomyocytes, endothelial
cells) by a peptide molecule
derived via molecular display
and the enhancement of
cellular internalization of a
therapeutic entity via fusion
with a protein transduction
domain (PTD). siRNA indicates
small interfering RNA.
(Illustration credit: Ben Smith).
940 Circulation Research September 13, 2013
underpinnings of cardiomyogenesis remain the subject of controversy. Hopefully, advances in the molecular cardiology field and
resolution of fundamental controversies within it will facilitate a
leap forward for cardiovascular medicine through increased use
of engineered protein therapeutics.
Acknowledgments
We apologize to those whose work could not be cited due to space
limitations.
Sources of Funding
Funding was provided by National Institutes of Health grants K99
HL112905 (Dr Jay), AG040019 (Dr Lee), and AG032977 (Dr Lee),
and by the Harvard Stem Cell Institute (Dr Lee).
Disclosures
Downloaded from http://circres.ahajournals.org/ by guest on June 12, 2017
Brigham and Women’s Hospital has filed for patents pertaining to the
described bivalent ligand technology, listing Dr Jay and Dr. Lee as inventors. Dr Lee is a cofounder and co-owner of Provasculon, Inc, is a
paid consultant for the company, and serves on the company’s Board
of Directors. Provasculon has interests in stromal cell–derived factor1α therapy, an area related to the topics discussed in this review. Dr
Lee’s interests were reviewed by Brigham and Women’s Hospital and
Partners HealthCare in accordance with their institutional policies.
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Protein Engineering for Cardiovascular Therapeutics: Untapped Potential for Cardiac
Repair
Steven M. Jay and Richard T. Lee
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Circ Res. 2013;113:933-943
doi: 10.1161/CIRCRESAHA.113.300215
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