current and future protein therapeutics

1. E XP E RI ME N T AL C E L L R E S EA RC H 31 7 ( 20 1 1) 1 2 6 1– 1 26 9
available at www.sciencedirect.com
www.elsevier.com/locate/yexcr
Review
Introduction to current and future protein therapeutics: A
protein engineering perspective
Paul J. Carter⁎
Department of Antibody Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, USA
A R T I C L E I N F O R M A T I O N
A B S T R A C T
Article Chronology:
Protein therapeutics and its enabling sister discipline, protein engineering, have emerged since the
Received 18 January 2011
early 1980s. The first protein therapeutics were recombinant versions of natural proteins. Proteins
Revised version received
purposefully modified to increase their clinical potential soon followed with enhancements
20 February 2011
derived from protein or glycoengineering, Fc fusion or conjugation to polyethylene glycol.
Accepted 24 February 2011
Antibody-based drugs subsequently arose as the largest and fastest growing class of protein
Available online 1 March 2011
therapeutics. The rationale for developing better protein therapeutics with enhanced efficacy,
greater safety, reduced immunogenicity or improved delivery comes from the convergence of
Keywords:
clinical, scientific, technological and commercial drivers that have identified unmet needs and
Protein therapeutics
provided strategies to address them. Future protein drugs seem likely to be more extensively
Antibody therapeutics
engineered to improve their performance, e.g., antibodies and Fc fusion proteins with enhanced
Fc fusion proteins
effector functions or extended half-life. Two old concepts for improving antibodies, namely
Engineered protein scaffolds
antibody-drug conjugates and bispecific antibodies, have advanced to the cusp of clinical success.
As for newer protein therapeutic platform technologies, several engineered protein scaffolds are in
early clinical development and offer differences and some potential advantages over antibodies.
© 2011 Elsevier Inc. All rights reserved.
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Brief history of protein therapeutics . . . . . . . . . . . . . . . . . . .
Protein therapeutics enabled by protein engineering . . . . . . . . .
Insulin — the first recombinant human protein therapeutic . . . . .
Beginning of engineered proteins as therapeutics . . . . . . . . . . .
Fc fusion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . .
Protein conjugation to polyethylene glycol . . . . . . . . . . . . . .
The rise of antibody therapeutics . . . . . . . . . . . . . . . . . . .
Rationale for next generation protein therapeutics . . . . . . . . . . . .
Strengths and limitations of antibody and other protein therapeutics
Clinical rationale . . . . . . . . . . . . . . . . . . . . . . . . . . .
Scientific rationale . . . . . . . . . . . . . . . . . . . . . . . . . . .
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⁎ Fax: +1 650 467 8318.
E-mail address: pjc@gene.com.
Abbreviations: FDA, Food and Drug Administration; PEG, polyethylene glycol; TPO, thrombopoietin
0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexcr.2011.02.013
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E XP E RI ME N T AL C E L L R E SE A RC H 31 7 ( 20 1 1) 1 2 61 – 1 26 9
Commercial rationale . . . . . . . . . . . . . . . . . . . .
Platform technologies for next generation protein therapeutics
Next generation antibodies and Fc fusion proteins . . . . .
Engineered protein scaffolds . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction
Since the early 1980s proteins have emerged as a major new class
of pharmaceuticals with ~200 marketed products that are mainly
therapeutics with a small number of diagnostics and vaccines [1].
Protein therapeutics can be classified based upon their pharmacologic activity as drugs that: i) replace a protein that is deficient
or abnormal, ii) augment an existing pathway, iii) provide a novel
function or activity, iv) interfere with a molecule or organism, or
iv) deliver a payload such as a radionuclide, cytotoxic drug, or
protein effector [2]. Alternatively, protein therapeutics can be
grouped into molecular types that include: antibody-based drugs,
anticoagulants, blood factors, bone morphogenetic proteins,
engineered protein scaffolds, enzymes, Fc fusion proteins, growth
factors, hormones, interferons, interleukins, and thrombolytics
(Fig. 1) [1,3]. Antibody-based drugs are the largest and fastest
growing class of protein therapeutics with 24 marketed antibody
drugs in the USA [4] and over 240 more in clinical development [5].
This introductory article provides a brief history of protein
therapeutics, offers a rationale for developing better protein drugs,
and identifies some major future opportunities as well as emerging
technologies that may meet them. The 30-year history of protein
drugs is illustrated here with examples that pioneered their class or
have high clinical or commercial significance. The rationale for
creating better protein drugs, including antibodies [6], comes from
the convergence of clinical, scientific, technological and commercial drivers that collectively have identified major unmet needs and
provide strategies to address them. Major future opportunities for
protein therapeutics, are improved efficacy, greater safety, reduced
immunogenicity or improved delivery. A plethora of emerging
technologies for the generation and optimization of protein therapeutics are anticipated to help address these opportunities. Space
Recombinant
proteins
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Brief history of protein therapeutics
Protein therapeutics enabled by protein engineering
Protein therapeutics and its enabling sister discipline, protein
engineering, have emerged since the early 1980s. Protein engineering by rational design or molecular evolution allows the
systemic dissection of protein structure–function relationships and
the generation of novel proteins with modified activities or entirely
new properties. Indeed, protein engineering has revolutionized
protein therapeutics by providing the tools to customize existing
proteins or to create novel proteins for specific clinical applications.
A brief history of protein therapeutics is provided here from a
protein engineering perspective. The focus is on technologies with
the greatest impact on current protein therapeutics — engineered
versions of natural proteins, Fc fusion proteins, conjugation to the
polymer, polyethylene glycol (PEG), and antibody therapeutics as
the quintessential protein therapeutic platform.
Insulin — the first recombinant human protein therapeutic
The first human protein therapeutic derived from recombinant
DNA technology was human insulin (Humulin®) created at
Genentech [7], developed by Eli Lilly, and approved by the US
Food and Drug Administration (FDA) in 1982. Recombinant insulin
Antibodies
Fab fragment
Conjugate PEG
Engineer protein
Engineer glycosylation
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constraints limit examples here to a sampling of technologies of
broad applicability — so called platform technologies. The interested reader is referred to many excellent articles on protein
therapeutics including those in this special issue and others cited
herein.
Anticoagulants
Blood factors
Bone morphogenetic
proteins
Enzymes
Growth factors
Hormones
Interferons
Interleukins
Thrombolytics
Optional
modifications
.
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.
Fc fusion
proteins
IgG
Receptor
Ligand
Peptide
Fc
Conjugate PEG
Bispecific
Conjugate drug
Conjugate radionuclide
Fig. 1 – Overview of currently marketed protein therapeutics from a protein engineering perspective.

3. E XP E RI ME N T AL C E L L R E S EA RC H 31 7 ( 20 1 1) 1 2 6 1– 1 26 9
was used to replace its natural counterpart that can be deficient in
diabetes mellitus. Other recombinant human proteins were soon
developed as therapeutics to replace the natural proteins deficient
in some patients (e.g., growth hormone) or augment existing
pathways (e.g., interferon-α, tissue plasminogen activator, and
erythropoietin) [2].
Beginning of engineered proteins as therapeutics
Beyond recombinant versions of natural proteins, the next major
step in the evolution of protein therapeutics was the redesign of
proteins to increase their clinical potential. Injected insulin fails to
adequately mimic endogenous insulin in two major ways: rapid
onset of action after meals and prolonged maintenance of low level
insulin between meals [8]. Insulin has been engineered, commonly
by one to three amino acid replacements, to create analogs that are
either rapid-acting or long-acting and better mimic different
properties of endogenous insulin [8]. Multiple insulin analogs have
been approved for the treatment of diabetes mellitus including the
rapid-acting analogs, insulin lispro (Humalog®), insulin aspart
(NovoRapid® and Novolog®) and insulin glulisine (Apidra®) and
the long-acting analogs, insulin glargine (Optisulin® and Lantus®)
and insulin detemir (Levemir®) [1,8]. Insulin analogs offer multiple benefits over first generation recombinant insulin drugs that
include improved physiologic profile, greater convenience for
patients, reduced risk of hypoglycemia, and less weight gain in
some cases [9]. The commercial importance of engineered insulins
is illustrated by Lantus®, which was one of the top ten selling
biopharmaceuticals in 2009 [1].
Many protein therapeutics are produced in mammalian cells
and have one or more N- and/or O-linked glycans [10]. Engineering
a protein with additional glycosylation sites can increase the in
vivo serum half-life and thereby target exposure [11]. An early
example of such “glycoengineering” of proteins for half-life
extension is provided by darbepoetin-α (Aranesp®), administered
to anemic patients to stimulate the production of red blood cells in
the bone marrow. The first generation drug, epoetin-α, has 3 Nlinked glycosylation sites and was engineered with 2 additional
glycosylation sites to create darbepoetin-α, a commercially
successful longer-acting second generation drug [12]. Protein
glycosylation can sometimes reduce bioactivity, albeit offset by
longer half-life. Darbepoetin-α has higher specific activity in
patients than epoetin-α, reflecting that the 3-fold increase in
serum half-life that increases drug exposure more than offsets a 5fold reduction in receptor binding affinity [12].
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Beyond half-life extension, Fc fusion can provide several
additional benefits such as facilitating expression and secretion
of recombinant protein, enabling facile purification by protein A
chromatography, improving solubility and stability, and enhancing
potency by increasing valency. Optional additional properties
conferred by Fc fusion include binding to Fcγ receptors and/or
complement to support secondary immune functions [13,14]. The
first demonstration of the Fc fusion protein concept was CD4-Fc
[16]. Five Fc fusion protein therapeutics were approved by 2010,
the prototypical and most successful drug of this class being
etanercept (Enbrel®), a TNF receptor 2-Fc fusion protein [1].
Etanercept was the top-selling protein therapeutic in 2009 with
$6.6 billion in world-wide sales in rheumatoid arthritis and other
autoimmune diseases [1]. Romiplostim (Nplate®) is the first
peptide-Fc fusion (“peptibody”) approved as a human therapeutic.
Romiplostim is a thrombopoietin (TPO) receptor agonist approved
as a treatment for thrombocytopenia [17].
Protein conjugation to polyethylene glycol
Protein conjugation to chemicals such as radionuclide chelators,
cytotoxic drugs and PEG can endow proteins with brand new
capabilities to increase their clinical potential.
The most clinically and commercially successful protein
conjugates thus far have been with PEG. Protein conjugation to
PEG – “PEGylation” – increases their hydrodynamic volume,
prevents rapid renal clearance, and thereby increases the serum
half-life, as extensively reviewed [15,18,19]. At least 8 PEGylated
proteins are approved as human therapeutics [18]. Indeed, longacting second generation interferon-α molecules that are PEGylated have largely superseded their first generation non-PEGylated
counterparts [20]. Benefits of PEGylation are that it is possible to
tune the serum-half of proteins by varying the number of attached
PEG molecules, their size and extent of branching [21]. PEGylation
can also reduce the immunogenicity of proteins [18,19], although
PEGylated proteins can still sometimes be immunogenic [22]. A
draw back to PEGylation is that it can impair protein function, a
limitation that is offset by the greatly increased half-life, and
potentially ameliorated by site-specific PEGylation and/or tailoring
the PEG [18,19]. PEG is a non-natural and apparently nonbiodegradable polymer, a molecular attribute that has unclear
clinical significance. PEGylated proteins can induce renal tubular
vacuolation in animals, albeit with unaltered clinical pathology
[23] and unknown impact on long term safety.
The rise of antibody therapeutics
Fc fusion proteins
Another significant advance in protein therapeutics came with the
advent of fusion proteins by joining the genes encoding 2 or more
different proteins. Fusion proteins combine properties of their
component parts. The most clinically and commercially successful
fusion protein therapeutics to date contain the Fc region of
immunoglobulins [1]. Such Fc fusions can endow peptides or
proteins with the IgG-like property of long serum half-life (days to
weeks), by virtue of binding to the salvage receptor, FcRn [13,14]. By
contrast, proteins of ~70 kDa and smaller are typically eliminated
rapidly from circulation by renal filtration and have half-lives of a
few minutes to a few hours, that can in many cases render them
marginal or unsuitable for therapeutic applications [15].
Antibody-based drugs are the largest and fastest growing class of
protein therapeutics with 24 marketed antibody therapeutics in
the USA (28 FDA-approved and 4 later withdrawn from the
market) [4] and at least 240 more in clinical development [5].
Antibody-based drugs contributed $38 billion of $99 billion in
world-wide sales for protein biopharmaceuticals in 2009 [1].
Moreover, 5 of the 10 top-selling protein therapeutics in 2009
were antibodies, namely, infliximab (Remicade®), bevacizumab
(Avastin®), rituximab (Rituxan® and MabThera®), adalimumab
(Humira®) and trastuzumab (Herceptin®) [1].
The rapid growth of antibody therapeutics since the mid-1990s
was made possible by the advent of antibody chimerization
and humanization [24] technologies that largely overcame the

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limitations of rodent monoclonal antibodies as drugs, as well as
the emergence of facile routes to human antibodies using
transgenic mice [25,26] or phage display [25,27] technologies.
Other advances that have contributed to the success of antibodies
as therapeutics include the industrialization of antibody production technologies [28] that can provide sufficient quantities of
antibodies to allow higher doses to be used in patients. The development of methods to express and secrete functional antibody
fragments from E. coli [29,30] has significantly stimulated the field
of antibody engineering and enabled subsequent technologies
such as phage display. The next major advance in the expression of
antibody fragments in E. coli was high titer (gram per liter)
production in a fermentor [31], that facilitated the development of
antibody fragment drugs such as the anti-VEGF Fab, ranibizumab
(Lucentis®). The emergence of antibody as therapeutics likely also
reflects improved choices of antibody and target antigen for
intervention in the pathobiology of disease.
The majority of approved antibody drugs are unmodified
(“naked”) IgG molecules. However, other antibody formats have
been approved for therapy including IgG “armed” with radionuclides or cytotoxic drugs (antibody-drug conjugates) for cancer
treatment, as well as Fab fragments and a PEGylated Fab′ fragment
[1].
Rationale for next generation protein therapeutics
A major quest with protein therapeutics, especially antibodies
[6,32], is the development of even better next generation drugs
with enhanced efficacy, greater safety or improved delivery.
Current antibody therapeutics reveal major strengths that provide
a foundation upon which to build, as well as significant limitations
to overcome that offers new opportunities [6,33]. Some antibody
strengths and many of their limitations apply more broadly to
protein therapeutics and are discussed below as a compass to guide
the development of next generation protein therapeutics. The
impetus to develop better protein therapeutics is also discussed
here as a convergence of clinical, scientific, and commercial forces
that identify unmeet needs (this section), in conjunction with
technological tools that many help address them (next section).
Strengths and limitations of antibody and other protein
therapeutics
One of the greatest strength of antibodies as a therapeutic platform
is the potential ease of generating high affinity and specificity
human antibodies to virtually any target of therapeutic interest,
e.g., using transgenic mice [25,26] or phage display [25,27]
technologies. Moreover, antibodies are readily tailored for therapeutic applications by modulating (enhancing or attenuating)
their existing properties or endowing them with new activities as
discussed in a later section and reviewed more extensively
elsewhere [33,34]. As for other protein therapeutics, antibodies
are commonly well tolerated by patients, but not invariably so.
Antibody drug development benefits from recombinant production methods that are well-established, broadly applicable and
commonly support expression at high titer (multi-gram per liter)
[28]. Widely available knowledge and experience across all facets
of antibody drug development also facilitates the generation of
future antibody therapeutics. An advantage of antibodies over
other drug formats is that the success rate has been somewhat
higher — 17% for humanized antibodies from the first clinical trial
to regulatory approval [35].
Proteins have several significant limitations as therapeutics. For
example, protein therapeutics are very expensive, reflecting high
production costs, that may limit patient access and also clinical
applications [28]. This high cost issue is exacerbated for protein
therapeutics where multi-gram doses are needed for a treatment
course, as is the case for some antibodies. Intracellular delivery of
proteins is possible in the laboratory setting, e.g., by protein
transduction [36]. However, targeted pharmaceutic delivery of
proteins to reach intracellular targets is in its infancy, so protein
therapeutics are currently limited to cell surface or extracellular
targets.
Another shortcoming of proteins therapeutics is that they are
denatured and/or proteolyzed in the gut and are thus not orally
bioavailable. Proteins, including antibodies [37], show very poor
ability to penetrate the specialized endothelial structure of the
neurovasculature known as the blood-brain barrier. The integrity
of the blood-brain barrier can be partially compromised in some
pathological settings such as chronic neurodegenerative diseases
[38]. Nevertheless, proteins with current technologies are poorly
suited to the treatment of chronic neurodegenerative diseases or
tumors that originate or have metastasized to the brain.
Antibodies are relatively large (IgG ~150 kDa) compared to
other proteins and this large size may limit their tissue penetration
and ability to localize to their targets. For example, antibody
localization to tumors in patients is typically very inefficient:
≤0.01% of the injected dose per gram of tumor [39]. Comparison of
mathematical modeling with experimental data, strongly suggests
that molecular size and binding affinity are major determinants of
tumor targeting [40]. Many other factors likely contribute to tumor
penetration including antigen expression level and turnover rate
[41].
Clinical rationale
From a clinical standpoint, first generation protein drugs can
provide substantial patient benefit. However, in many cases there
is room for improvement in efficacy and/or safety. For example,
several anti-cancer antibodies provide an overall survival benefit,
including bevacizumab [42] and trastuzumab [43] in combination
with chemotherapy in metastatic colorectal and breast cancers,
respectively. Nevertheless, there remains plenty of opportunity for
enhancing antibodies for cancer treatment, such as elevating the
overall survival rate of patients, as well as many strategies being
explored to do so [44]. All therapeutic proteins are potentially
immunogenic in patients and occasionally an anti-drug antibody
response can lead to a major safety issue. For example, a truncated
and PEGylated form of TPO (MGDF-PEG) elicited an antibody
response in some patients that neutralized endogenous TPO
leading to severe and persistent anemia, i.e., the opposite of the
desired effect of increasing platelet counts [22]. This issue was
overcome with the peptibody, Romiplostim (see Fc fusion proteins
section) [17].
Scientific rationale
Scientifically, our expanding knowledge suggests drug mechanisms
of action to enhance, and mechanisms of resistance or toxicity to

5. E XP E RI ME N T AL C E L L R E S EA RC H 31 7 ( 20 1 1) 1 2 6 1– 1 26 9
overcome. For example, anti-CD3 antibodies illustrate the benefits of
understanding and overcoming mechanisms of drug toxicity and
resistance. In 1986 the anti-CD3 antibody, muromonab-CD3
(OKT3®), became the first antibody approved by the FDA for
human therapy — for the treatment of acute allograft rejection in
renal transplant patients. In 1993 muromonab-CD3 was approved
for the treatment of acute rejection of heart and liver transplants in
patients resistant to steroid treatment. Unfortunately, muromonabCD3 activates T cells resulting in transient cytokine release and acute
and severe flu-like symptoms [45]. Another major limitation of
muromonab-CD3 is that its therapeutic benefit is reduced by
neutralizing antibodies that develop in ~50% of patients [45].
Muromonab-CD3 has since been voluntarily withdrawn from the
US market due to decreased sales. These major limitations of
muromonab-CD3 have been greatly reduced, if not overcome, in
some second generation anti-CD3 antibodies that have advanced to
late stage clinical development [6]. For example, the anti-CD3
antibodies, teplizumab and otelixizumab, combine Fc engineering to
attenuate Fcγ receptor binding with chimerization or humanization
to reduce immunogenicity [34].
Drug development, including for protein therapeutics, seems
likely to gain from advances in the understanding of targets and
their role in the pathobiology of disease. For example, there is a
growing appreciation that targets are not isolated gene products
but integral components of networks of interconnected signaling
pathways [46,47].
Commercial rationale
Clinical and commercial success with protein therapeutics has
bred intense competition between different organizations striving
to develop protein therapeutics to the same antigen and/or
overlapping therapeutic indications. Indeed, this is already
reflected in marketed drugs as illustrated by 5 different protein
therapeutics that block TNF including 3 different IgG (infliximab,
adalimumab and golimumab), a PEGylated Fab′ fragment (certolizumab pegol) and a receptor-Fc fusion (etanercept) [6]. Such
competition is increasingly commonplace as patents dominating
individual targets expire. The generation of high affinity and
potency IgG is often possible, arguably commoditized. This
provides a strong commercial incentive to develop superiorperforming antibody drugs to differentiate products from those of
competitors. The success of innovator protein therapeutics has
brought a rise in biosimilar efforts (i.e., products that are deemed
highly similar to innovator protein therapeutics according to drug
regulatory authorities). Indeed, several biosimilar approval applications have been filed in the European Union with 13 biosimilars
being approved by European drug regulators by early 2009 [48].
For creators of innovator drugs, the impetus to develop improved
next generation protein therapeutics that out perform the
previous generation drugs has increased substantially [48].
Platform technologies for next generation
protein therapeutics
Many protein engineering tools have been developed that allow
one to optimize favorable properties of proteins, attenuate
undesired attributes and create proteins with entirely novel
activities. Space constraints limit the discussion here to next
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generation antibody and Fc fusion protein therapeutics and the
emerging technologies of engineered protein scaffolds.
Next generation antibodies and Fc fusion proteins
Many promising strategies are emerging to enhance the existing
properties of antibodies or to endow them with new activities, as
reviewed in detail elsewhere [6,32,33]. Antibodies (IgG) have
multiple binding partners including antigen, Fcγ receptors,
complement and FcRn. Each of these interactions can be tailored
in ways that may improve the clinical properties of antibodies
[32,34,49]. Platform technologies that seem likely to impact the
next generation of antibody therapeutics include old concepts that
are finally coming to fruition, such as antibody-drug conjugates
and bispecific antibodies. Technologies for improving Fc-mediated
functions, such as half-life extension and effector function enhancement, seem likely to benefit the next generation of antibody
therapeutics. Additionally, these technologies appear directly
applicable to the optimization of Fc fusion proteins.
The 50-year old concept of bispecific antibodies [50] came of
age in 2009 with the first regulatory approval of a bispecific
antibody — catumaxomab (Removab®, anti-EpCAM x anti-CD3) in
Europe. Catumaxomab is approved for the treatment of malignant
ascites in patients with EpCAM-positive carcinomas [1,51].
Recruitment of T cells via the anti-CD3 arm of catumaxomab and
other immune effector cells via its Fc region may contribute to
antitumor activity. A tandem bispecific scFv format known as a
“BiTE”, that binds CD3 and a tumor-associated antigen, is a
particularly effective way to retarget T cells to kill tumor cells.
Blinatumomab, a BiTE specific for CD19 and CD3, gave several
complete responses in a phase I clinical trial in non-Hodgkin's
lymphoma [52] and is currently in multiple phase II clinical trials.
Another major area of current interest with bispecific antibodies is
in the simultaneous blockade of 2 disease mediators, which may
lead to greater therapeutic benefit than targeting individual
disease mediators [53]. Until recently, a major bottleneck in the
development of bispecific antibodies as therapeutics was the
challenge of producing these complex molecules in sufficient
quantity and quality for clinical applications. This issue has been
largely overcome by the advent of many alternative formats and
production methods for bispecific antibodies [6].
Antibody-drug conjugates, a concept that dates back to the
1970s, use antibodies to deliver a cytotoxic payload to kill a target
such as tumor cells [54,55]. One antibody-drug conjugate,
gemtuzumab ozogamicin (Mylotarg®), was approved by the FDA
for the treatment of acute myeloid leukemia. Gemtuzumab
ozogamicin was withdrawn from the market in 2010 due to safety
concerns and insufficient patient benefit in post approval clinical
trials. Much progress has been made over the decades in many
different facets of antibody-drug conjugates, including better
choices of target antigen and antibody, and the development of
more potent drugs and linkers of greater stability [54,55]. Pivotal
clinical trials are in progress for brentuximab vedotin (SGN-35,
[56]) in Hodgkin's disease and trastuzumab emtansine (T-DM1,
[57]) in metastatic breast cancer over-expressing HER2/neu
following demonstration of robust antitumor activity and acceptable safety profiles in earlier clinical trials.
Engineering proteins to increase their serum half-life has the
potential benefits of increased exposure leading to greater target
localization and greater efficacy, lower or less frequent dosing and

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lower cost. In the case of antibodies, the serum half-life in mice can
be extended by at least a few fold by engineering the Fc region for
higher affinity binding to the salvage receptor (FcRn), whilst
preserving the pH dependence of binding [58]. Half-life extension
of Fc-engineered antibodies in non-human primates was subsequently demonstrated by several different groups [59]. Antibody
half-life extension can result in increased target exposure and
improved preclinical in vivo efficacy as recently shown for antiEGFR and anti-VEGF antibodies [60]. Engineering antibodies for
pH-dependent interaction with their target antigen has also been
shown to extend antibody half-life in vivo [61]. The first half-life
extended antibody to enter clinical trials was the humanized antiRSV antibody, MEDI-557, although pharmacokinetic data have yet
to be reported.
Another major area of interest for improving antibody performance is effector function enhancement, as reviewed extensively elsewhere [34,62] and discussed briefly below. Antibody
effector functions include ADCC (antibody-dependent cellular
cytotoxicity), ADCP (antibody-dependent cellular phagocytosis)
and complement-dependent cytotoxicity (CDC). Effector functions
may be advantageous where destruction of the target cells is
desired, as is often the case for oncology targets.
Indeed many antibodies approved for oncologic indications
(e.g., rituximab, trastuzumab, alemtuzumab, cetuximab and
ofatumumab) support one or many effector functions that may
contribute to their clinical antitumor activity, with the strongest
evidence being for rituximab [63].
For antibody and Fc fusion protein therapeutics potential
benefits from enhancing effector functions include greater efficacy,
lower or less frequent dosing and decreased cost-of-goods. As for
risks, these include more frequent or severe adverse events. ADCC,
ADCP and CDC have been augmented by Fc protein engineering as
extensively reviewed elsewhere [62]. ADCC has also been
augmented to a similar level by modifying the glycan attached to
the Fc, e.g., afucosylation achieved through cell line engineering
[62]. At least 8 effector function enhanced antibodies have
advanced into early clinical development [34].
Engineered protein scaffolds
The success of antibodies as protein therapeutics has inspired the
development of so-called “engineered protein scaffolds” as alternative platform technologies for the development of novel next
generation protein therapeutics [64–66]. Engineered protein scaffolds provide binding sites that can be tailored to specifically
recognize desired target molecules, in an analogous way that
antibodies can be developed to recognize their target antigens.
Here the basic features of engineered protein scaffolds are described,
together with their strengths, limitations and some possible future
directions.
Protein scaffolds chosen for engineering have known 3dimensional structures that together represent diverse protein
folds [64–66]. Protein scaffolds are commonly selected with the
following attributes: small, monomeric, high solubility and
stability, and that lack disulfide bonds and glycosylation sites.
Protein scaffolds are typically readily expressed in microbial hosts.
Sequence diversity is introduced into the protein scaffold, e.g., by
randomizing surface accessible residues, to create a so-called
library. Binders to a target of therapeutic interest are then identified by selection using technologies such as phage, yeast or
ribosome display. Increased binding affinity – affinity maturation –
is often needed and readily accomplished by further library
building and selection.
Over 50 different engineered protein scaffolds have been
described with a few advancing into clinical development [64–
66] including: Adnectins® from type III fibronectin domains,
Affibodies® from the synthetic Z domain of staphylococcal protein
A, Anticalins® from lipocalins, Avimers® from LDLR-A modules,
DARPins® from ankyrin repeat proteins, and engineered Kunitz
domains. Engineered protein scaffolds also include single-domain
antibodies from human (dAbs®) and camelids (Nanobodies®).
One engineered protein scaffold protein has been approved for
therapy, namely, ecallantide (Kalbitor®), a potent and selective
inhibitor of plasma kallikrein based upon a Kunitz domain [67].
Many potential advantages of engineered protein scaffolds over
antibodies have been proposed [64–66], albeit with limited
supporting evidence to date in the context of developing protein
therapeutics. For example, the smaller size and higher stability of
some engineered protein scaffolds may allow for alternative routes
of administration such as pulmonary delivery. The smaller size of
engineered protein scaffolds over antibodies favors more efficient
tissue penetration, but this can be more than offset by much faster
clearance. Engineered protein scaffolds may be more readily
expressed in microbial hosts reflecting their high stability and
solubility, small size, simple structure (single polypeptide), and
that they commonly lack disulfide bonds and/or glycosylation
sites. The small and modular architecture of engineered protein
scaffolds seems particularly well suited to the construction of
multivalent and/or multispecific protein therapeutics (Fig. 2).
Engineered proteins are commonly associated with independent
intellectual property that is outside the scope of antibody patents.
Several of these proposed advantages of engineered protein
scaffolds have been achieved with antibodies by engineering for
improved stability or expression, or by exploiting the modular
architecture of antibodies to create different formats including
ones that are smaller and simpler than IgG [6,32,33].
Engineered protein scaffolds lack Fc-mediated properties of
antibodies such as long serum half-life and effector functions. This
does not appear to be a major limitation, as many different
strategies have been developed to extend the half-life of proteins
as comprehensively reviewed [15]. For example, single-domain
antibodies binding to the long-circulating protein, serum albumin,
have been identified and fused to small proteins, including other
single-domain antibodies to extend their half-life [68,69]. As for
effector functions, these are desired in some clinical settings but
not others. Fc fusion offers a simple way to endow engineered
protein scaffolds with effector functions if needed. Engineered
protein scaffolds, like antibodies, offer significant opportunity for
sequence diversity but individual protein scaffolds appear to
provide less opportunity for structural diversity than do antibodies. The functional impact of lower structural diversity on the
ease of being able to generate binding proteins to specific sites on
targets of interests remains to be determined.
All protein therapeutics, including engineered protein scaffolds,
are potentially immunogenic in patients and may elicit an antidrug antibody response that may neutralize or otherwise interfere
with the drug's activity. This issue can be more significant for
chronic therapy involving repeated drug administration. Clinical
trials are ultimately needed to understand the extent to which
individual engineered protein scaffold drugs are immunogenic in

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E XP E RI ME N T AL C E L L R E S EA RC H 31 7 ( 20 1 1) 1 2 6 1– 1 26 9
Engineered scaffold
proteins
Representative
assemblies
Target
valencies
Target
specificities
Long
half-life
Target A binder
1
1
No
Target B binder
1
1
Yes
Target C binder
1, 1
2
Yes
Half-life extender
2, 1
2
Yes
1, 2
2
Yes
2, 2
2
Yes
1,1,1
3
Yes
Fig. 2 – Modular assembly of engineered protein scaffolds into multivalent and multispecific fusion proteins. Engineered scaffold
proteins binding to different targets or different epitopes on the same target are identified by selection from libraries. The modular
nature of engineered protein scaffolds allows them to be readily assembled into multimers with specific valency for each target
[64–66]. However, steric considerations may prevent all modules from being able to bind their respective targets simultaneously.
Serum half-life extension is commonly required for engineered scaffold proteins to obtain sufficient exposure to support
therapeutic applications. Many different half-life extension strategies for small proteins have been developed [15], including for
engineered protein scaffolds (shown), e.g., binding to serum albumin. Linkers between modules may be included but are omitted
for simplicity in this figure.
patients and whether or not this limits their clinical utility. Human
proteins are often selected for engineered protein scaffolds, which
may help reduce the risk of immunogenicity in patients for therapeutic applications.
Conclusions
Proteins are now well established as a clinically and commercially
important class of therapeutics. Experience with protein therapeutics has provided an understanding of their strengths and
limitations and ways in which they might be improved. Clinical
and commercial success with protein therapeutics provides strong
motivation to develop better protein drugs, whereas new tools,
especially platform technologies provide the likely means to do so.
The next generation of protein therapeutics will likely include
some of the many enhanced antibodies in early clinical development, optimized Fc fusion proteins, as well as new formats such as
engineered protein scaffolds.
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