2. 1262 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
Platform technologies for next generation protein therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
Next generation antibodies and Fc fusion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
Engineered protein scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267
Introduction constraints limit examples here to a sampling of technologies of
broad applicability — so called platform technologies. The inter-
Since the early 1980s proteins have emerged as a major new class ested reader is referred to many excellent articles on protein
of pharmaceuticals with ~200 marketed products that are mainly therapeutics including those in this special issue and others cited
therapeutics with a small number of diagnostics and vaccines [1]. herein.
Protein therapeutics can be classified based upon their pharma-
cologic activity as drugs that: i) replace a protein that is deficient
or abnormal, ii) augment an existing pathway, iii) provide a novel Brief history of protein therapeutics
function or activity, iv) interfere with a molecule or organism, or
iv) deliver a payload such as a radionuclide, cytotoxic drug, or Protein therapeutics enabled by protein engineering
protein effector [2]. Alternatively, protein therapeutics can be
grouped into molecular types that include: antibody-based drugs, Protein therapeutics and its enabling sister discipline, protein
anticoagulants, blood factors, bone morphogenetic proteins, engineering, have emerged since the early 1980s. Protein engi-
engineered protein scaffolds, enzymes, Fc fusion proteins, growth neering by rational design or molecular evolution allows the
factors, hormones, interferons, interleukins, and thrombolytics systemic dissection of protein structure–function relationships and
(Fig. 1) [1,3]. Antibody-based drugs are the largest and fastest the generation of novel proteins with modified activities or entirely
growing class of protein therapeutics with 24 marketed antibody new properties. Indeed, protein engineering has revolutionized
drugs in the USA [4] and over 240 more in clinical development [5]. protein therapeutics by providing the tools to customize existing
This introductory article provides a brief history of protein proteins or to create novel proteins for specific clinical applications.
therapeutics, offers a rationale for developing better protein drugs, A brief history of protein therapeutics is provided here from a
and identifies some major future opportunities as well as emerging protein engineering perspective. The focus is on technologies with
technologies that may meet them. The 30-year history of protein the greatest impact on current protein therapeutics — engineered
drugs is illustrated here with examples that pioneered their class or versions of natural proteins, Fc fusion proteins, conjugation to the
have high clinical or commercial significance. The rationale for polymer, polyethylene glycol (PEG), and antibody therapeutics as
creating better protein drugs, including antibodies [6], comes from the quintessential protein therapeutic platform.
the convergence of clinical, scientific, technological and commer-
cial drivers that collectively have identified major unmet needs and Insulin — the first recombinant human protein therapeutic
provide strategies to address them. Major future opportunities for
protein therapeutics, are improved efficacy, greater safety, reduced The first human protein therapeutic derived from recombinant
immunogenicity or improved delivery. A plethora of emerging DNA technology was human insulin (Humulin®) created at
technologies for the generation and optimization of protein ther- Genentech [7], developed by Eli Lilly, and approved by the US
apeutics are anticipated to help address these opportunities. Space Food and Drug Administration (FDA) in 1982. Recombinant insulin
Recombinant Antibodies Fc fusion
proteins proteins
Fab fragment IgG
Anticoagulants
Blood factors Receptor
Bone morphogenetic Ligand
proteins Peptide
Enzymes
Growth factors
Hormones Fc
Interferons
Interleukins
Thrombolytics
Optional Conjugate PEG Conjugate PEG Bispecific
modifications Engineer protein Conjugate drug
Engineer glycosylation 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 1263
was used to replace its natural counterpart that can be deficient in Beyond half-life extension, Fc fusion can provide several
diabetes mellitus. Other recombinant human proteins were soon additional benefits such as facilitating expression and secretion
developed as therapeutics to replace the natural proteins deficient of recombinant protein, enabling facile purification by protein A
in some patients (e.g., growth hormone) or augment existing chromatography, improving solubility and stability, and enhancing
pathways (e.g., interferon-α, tissue plasminogen activator, and potency by increasing valency. Optional additional properties
erythropoietin) [2]. conferred by Fc fusion include binding to Fcγ receptors and/or
complement to support secondary immune functions [13,14]. The
Beginning of engineered proteins as therapeutics first demonstration of the Fc fusion protein concept was CD4-Fc
[16]. Five Fc fusion protein therapeutics were approved by 2010,
Beyond recombinant versions of natural proteins, the next major the prototypical and most successful drug of this class being
step in the evolution of protein therapeutics was the redesign of etanercept (Enbrel®), a TNF receptor 2-Fc fusion protein [1].
proteins to increase their clinical potential. Injected insulin fails to Etanercept was the top-selling protein therapeutic in 2009 with
adequately mimic endogenous insulin in two major ways: rapid $6.6 billion in world-wide sales in rheumatoid arthritis and other
onset of action after meals and prolonged maintenance of low level autoimmune diseases [1]. Romiplostim (Nplate®) is the first
insulin between meals [8]. Insulin has been engineered, commonly peptide-Fc fusion (“peptibody”) approved as a human therapeutic.
by one to three amino acid replacements, to create analogs that are Romiplostim is a thrombopoietin (TPO) receptor agonist approved
either rapid-acting or long-acting and better mimic different as a treatment for thrombocytopenia [17].
properties of endogenous insulin [8]. Multiple insulin analogs have
been approved for the treatment of diabetes mellitus including the Protein conjugation to polyethylene glycol
rapid-acting analogs, insulin lispro (Humalog®), insulin aspart
(NovoRapid® and Novolog®) and insulin glulisine (Apidra®) and Protein conjugation to chemicals such as radionuclide chelators,
the long-acting analogs, insulin glargine (Optisulin® and Lantus®) cytotoxic drugs and PEG can endow proteins with brand new
and insulin detemir (Levemir®) [1,8]. Insulin analogs offer mul- capabilities to increase their clinical potential.
tiple benefits over first generation recombinant insulin drugs that The most clinically and commercially successful protein
include improved physiologic profile, greater convenience for conjugates thus far have been with PEG. Protein conjugation to
patients, reduced risk of hypoglycemia, and less weight gain in PEG – “PEGylation” – increases their hydrodynamic volume,
some cases [9]. The commercial importance of engineered insulins prevents rapid renal clearance, and thereby increases the serum
is illustrated by Lantus®, which was one of the top ten selling half-life, as extensively reviewed [15,18,19]. At least 8 PEGylated
biopharmaceuticals in 2009 [1]. proteins are approved as human therapeutics [18]. Indeed, long-
Many protein therapeutics are produced in mammalian cells acting second generation interferon-α molecules that are PEGy-
and have one or more N- and/or O-linked glycans [10]. Engineering lated have largely superseded their first generation non-PEGylated
a protein with additional glycosylation sites can increase the in counterparts [20]. Benefits of PEGylation are that it is possible to
vivo serum half-life and thereby target exposure [11]. An early tune the serum-half of proteins by varying the number of attached
example of such “glycoengineering” of proteins for half-life PEG molecules, their size and extent of branching [21]. PEGylation
extension is provided by darbepoetin-α (Aranesp®), administered can also reduce the immunogenicity of proteins [18,19], although
to anemic patients to stimulate the production of red blood cells in PEGylated proteins can still sometimes be immunogenic [22]. A
the bone marrow. The first generation drug, epoetin-α, has 3 N- draw back to PEGylation is that it can impair protein function, a
linked glycosylation sites and was engineered with 2 additional limitation that is offset by the greatly increased half-life, and
glycosylation sites to create darbepoetin-α, a commercially potentially ameliorated by site-specific PEGylation and/or tailoring
successful longer-acting second generation drug [12]. Protein the PEG [18,19]. PEG is a non-natural and apparently non-
glycosylation can sometimes reduce bioactivity, albeit offset by biodegradable polymer, a molecular attribute that has unclear
longer half-life. Darbepoetin-α has higher specific activity in clinical significance. PEGylated proteins can induce renal tubular
patients than epoetin-α, reflecting that the 3-fold increase in vacuolation in animals, albeit with unaltered clinical pathology
serum half-life that increases drug exposure more than offsets a 5- [23] and unknown impact on long term safety.
fold reduction in receptor binding affinity [12].
The rise of antibody therapeutics
Fc fusion proteins
Antibody-based drugs are the largest and fastest growing class of
Another significant advance in protein therapeutics came with the protein therapeutics with 24 marketed antibody therapeutics in
advent of fusion proteins by joining the genes encoding 2 or more the USA (28 FDA-approved and 4 later withdrawn from the
different proteins. Fusion proteins combine properties of their market) [4] and at least 240 more in clinical development [5].
component parts. The most clinically and commercially successful Antibody-based drugs contributed $38 billion of $99 billion in
fusion protein therapeutics to date contain the Fc region of world-wide sales for protein biopharmaceuticals in 2009 [1].
immunoglobulins [1]. Such Fc fusions can endow peptides or Moreover, 5 of the 10 top-selling protein therapeutics in 2009
proteins with the IgG-like property of long serum half-life (days to were antibodies, namely, infliximab (Remicade®), bevacizumab
weeks), by virtue of binding to the salvage receptor, FcRn [13,14]. By (Avastin®), rituximab (Rituxan® and MabThera®), adalimumab
contrast, proteins of ~70 kDa and smaller are typically eliminated (Humira®) and trastuzumab (Herceptin®) [1].
rapidly from circulation by renal filtration and have half-lives of a The rapid growth of antibody therapeutics since the mid-1990s
few minutes to a few hours, that can in many cases render them was made possible by the advent of antibody chimerization
marginal or unsuitable for therapeutic applications [15]. and humanization [24] technologies that largely overcame the
4. 1264 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
limitations of rodent monoclonal antibodies as drugs, as well as other drug formats is that the success rate has been somewhat
the emergence of facile routes to human antibodies using higher — 17% for humanized antibodies from the first clinical trial
transgenic mice [25,26] or phage display [25,27] technologies. to regulatory approval [35].
Other advances that have contributed to the success of antibodies Proteins have several significant limitations as therapeutics. For
as therapeutics include the industrialization of antibody produc- example, protein therapeutics are very expensive, reflecting high
tion technologies [28] that can provide sufficient quantities of production costs, that may limit patient access and also clinical
antibodies to allow higher doses to be used in patients. The de- applications [28]. This high cost issue is exacerbated for protein
velopment of methods to express and secrete functional antibody therapeutics where multi-gram doses are needed for a treatment
fragments from E. coli [29,30] has significantly stimulated the field course, as is the case for some antibodies. Intracellular delivery of
of antibody engineering and enabled subsequent technologies proteins is possible in the laboratory setting, e.g., by protein
such as phage display. The next major advance in the expression of transduction [36]. However, targeted pharmaceutic delivery of
antibody fragments in E. coli was high titer (gram per liter) proteins to reach intracellular targets is in its infancy, so protein
production in a fermentor [31], that facilitated the development of therapeutics are currently limited to cell surface or extracellular
antibody fragment drugs such as the anti-VEGF Fab, ranibizumab targets.
(Lucentis®). The emergence of antibody as therapeutics likely also Another shortcoming of proteins therapeutics is that they are
reflects improved choices of antibody and target antigen for denatured and/or proteolyzed in the gut and are thus not orally
intervention in the pathobiology of disease. bioavailable. Proteins, including antibodies [37], show very poor
The majority of approved antibody drugs are unmodified ability to penetrate the specialized endothelial structure of the
(“naked”) IgG molecules. However, other antibody formats have neurovasculature known as the blood-brain barrier. The integrity
been approved for therapy including IgG “armed” with radio- of the blood-brain barrier can be partially compromised in some
nuclides or cytotoxic drugs (antibody-drug conjugates) for cancer pathological settings such as chronic neurodegenerative diseases
treatment, as well as Fab fragments and a PEGylated Fab′ fragment [38]. Nevertheless, proteins with current technologies are poorly
[1]. 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
Rationale for next generation protein therapeutics other proteins and this large size may limit their tissue penetration
and ability to localize to their targets. For example, antibody
A major quest with protein therapeutics, especially antibodies localization to tumors in patients is typically very inefficient:
[6,32], is the development of even better next generation drugs ≤0.01% of the injected dose per gram of tumor [39]. Comparison of
with enhanced efficacy, greater safety or improved delivery. mathematical modeling with experimental data, strongly suggests
Current antibody therapeutics reveal major strengths that provide that molecular size and binding affinity are major determinants of
a foundation upon which to build, as well as significant limitations tumor targeting [40]. Many other factors likely contribute to tumor
to overcome that offers new opportunities [6,33]. Some antibody penetration including antigen expression level and turnover rate
strengths and many of their limitations apply more broadly to [41].
protein therapeutics and are discussed below as a compass to guide
the development of next generation protein therapeutics. The Clinical rationale
impetus to develop better protein therapeutics is also discussed
here as a convergence of clinical, scientific, and commercial forces From a clinical standpoint, first generation protein drugs can
that identify unmeet needs (this section), in conjunction with provide substantial patient benefit. However, in many cases there
technological tools that many help address them (next section). is room for improvement in efficacy and/or safety. For example,
several anti-cancer antibodies provide an overall survival benefit,
Strengths and limitations of antibody and other protein including bevacizumab [42] and trastuzumab [43] in combination
therapeutics with chemotherapy in metastatic colorectal and breast cancers,
respectively. Nevertheless, there remains plenty of opportunity for
One of the greatest strength of antibodies as a therapeutic platform enhancing antibodies for cancer treatment, such as elevating the
is the potential ease of generating high affinity and specificity overall survival rate of patients, as well as many strategies being
human antibodies to virtually any target of therapeutic interest, explored to do so [44]. All therapeutic proteins are potentially
e.g., using transgenic mice [25,26] or phage display [25,27] immunogenic in patients and occasionally an anti-drug antibody
technologies. Moreover, antibodies are readily tailored for thera- response can lead to a major safety issue. For example, a truncated
peutic applications by modulating (enhancing or attenuating) and PEGylated form of TPO (MGDF-PEG) elicited an antibody
their existing properties or endowing them with new activities as response in some patients that neutralized endogenous TPO
discussed in a later section and reviewed more extensively leading to severe and persistent anemia, i.e., the opposite of the
elsewhere [33,34]. As for other protein therapeutics, antibodies desired effect of increasing platelet counts [22]. This issue was
are commonly well tolerated by patients, but not invariably so. overcome with the peptibody, Romiplostim (see Fc fusion proteins
Antibody drug development benefits from recombinant produc- section) [17].
tion methods that are well-established, broadly applicable and
commonly support expression at high titer (multi-gram per liter) Scientific rationale
[28]. Widely available knowledge and experience across all facets
of antibody drug development also facilitates the generation of Scientifically, our expanding knowledge suggests drug mechanisms
future antibody therapeutics. An advantage of antibodies over 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 1265
overcome. For example, anti-CD3 antibodies illustrate the benefits of generation antibody and Fc fusion protein therapeutics and the
understanding and overcoming mechanisms of drug toxicity and emerging technologies of engineered protein scaffolds.
resistance. In 1986 the anti-CD3 antibody, muromonab-CD3
(OKT3®), became the first antibody approved by the FDA for Next generation antibodies and Fc fusion proteins
human therapy — for the treatment of acute allograft rejection in
renal transplant patients. In 1993 muromonab-CD3 was approved Many promising strategies are emerging to enhance the existing
for the treatment of acute rejection of heart and liver transplants in properties of antibodies or to endow them with new activities, as
patients resistant to steroid treatment. Unfortunately, muromonab- reviewed in detail elsewhere [6,32,33]. Antibodies (IgG) have
CD3 activates T cells resulting in transient cytokine release and acute multiple binding partners including antigen, Fcγ receptors,
and severe flu-like symptoms [45]. Another major limitation of complement and FcRn. Each of these interactions can be tailored
muromonab-CD3 is that its therapeutic benefit is reduced by in ways that may improve the clinical properties of antibodies
neutralizing antibodies that develop in ~50% of patients [45]. [32,34,49]. Platform technologies that seem likely to impact the
Muromonab-CD3 has since been voluntarily withdrawn from the next generation of antibody therapeutics include old concepts that
US market due to decreased sales. These major limitations of are finally coming to fruition, such as antibody-drug conjugates
muromonab-CD3 have been greatly reduced, if not overcome, in and bispecific antibodies. Technologies for improving Fc-mediated
some second generation anti-CD3 antibodies that have advanced to functions, such as half-life extension and effector function en-
late stage clinical development [6]. For example, the anti-CD3 hancement, seem likely to benefit the next generation of antibody
antibodies, teplizumab and otelixizumab, combine Fc engineering to therapeutics. Additionally, these technologies appear directly
attenuate Fcγ receptor binding with chimerization or humanization applicable to the optimization of Fc fusion proteins.
to reduce immunogenicity [34]. The 50-year old concept of bispecific antibodies [50] came of
Drug development, including for protein therapeutics, seems age in 2009 with the first regulatory approval of a bispecific
likely to gain from advances in the understanding of targets and antibody — catumaxomab (Removab®, anti-EpCAM x anti-CD3) in
their role in the pathobiology of disease. For example, there is a Europe. Catumaxomab is approved for the treatment of malignant
growing appreciation that targets are not isolated gene products ascites in patients with EpCAM-positive carcinomas [1,51].
but integral components of networks of interconnected signaling Recruitment of T cells via the anti-CD3 arm of catumaxomab and
pathways [46,47]. other immune effector cells via its Fc region may contribute to
antitumor activity. A tandem bispecific scFv format known as a
Commercial rationale “BiTE”, that binds CD3 and a tumor-associated antigen, is a
particularly effective way to retarget T cells to kill tumor cells.
Clinical and commercial success with protein therapeutics has Blinatumomab, a BiTE specific for CD19 and CD3, gave several
bred intense competition between different organizations striving complete responses in a phase I clinical trial in non-Hodgkin's
to develop protein therapeutics to the same antigen and/or lymphoma [52] and is currently in multiple phase II clinical trials.
overlapping therapeutic indications. Indeed, this is already Another major area of current interest with bispecific antibodies is
reflected in marketed drugs as illustrated by 5 different protein in the simultaneous blockade of 2 disease mediators, which may
therapeutics that block TNF including 3 different IgG (infliximab, lead to greater therapeutic benefit than targeting individual
adalimumab and golimumab), a PEGylated Fab′ fragment (certo- disease mediators [53]. Until recently, a major bottleneck in the
lizumab pegol) and a receptor-Fc fusion (etanercept) [6]. Such development of bispecific antibodies as therapeutics was the
competition is increasingly commonplace as patents dominating challenge of producing these complex molecules in sufficient
individual targets expire. The generation of high affinity and quantity and quality for clinical applications. This issue has been
potency IgG is often possible, arguably commoditized. This largely overcome by the advent of many alternative formats and
provides a strong commercial incentive to develop superior- production methods for bispecific antibodies [6].
performing antibody drugs to differentiate products from those of Antibody-drug conjugates, a concept that dates back to the
competitors. The success of innovator protein therapeutics has 1970s, use antibodies to deliver a cytotoxic payload to kill a target
brought a rise in biosimilar efforts (i.e., products that are deemed such as tumor cells [54,55]. One antibody-drug conjugate,
highly similar to innovator protein therapeutics according to drug gemtuzumab ozogamicin (Mylotarg®), was approved by the FDA
regulatory authorities). Indeed, several biosimilar approval appli- for the treatment of acute myeloid leukemia. Gemtuzumab
cations have been filed in the European Union with 13 biosimilars ozogamicin was withdrawn from the market in 2010 due to safety
being approved by European drug regulators by early 2009 [48]. concerns and insufficient patient benefit in post approval clinical
For creators of innovator drugs, the impetus to develop improved trials. Much progress has been made over the decades in many
next generation protein therapeutics that out perform the different facets of antibody-drug conjugates, including better
previous generation drugs has increased substantially [48]. 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,
Platform technologies for next generation [56]) in Hodgkin's disease and trastuzumab emtansine (T-DM1,
protein therapeutics [57]) in metastatic breast cancer over-expressing HER2/neu
following demonstration of robust antitumor activity and accept-
Many protein engineering tools have been developed that allow able safety profiles in earlier clinical trials.
one to optimize favorable properties of proteins, attenuate Engineering proteins to increase their serum half-life has the
undesired attributes and create proteins with entirely novel potential benefits of increased exposure leading to greater target
activities. Space constraints limit the discussion here to next localization and greater efficacy, lower or less frequent dosing and
6. 1266 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
lower cost. In the case of antibodies, the serum half-life in mice can ribosome display. Increased binding affinity – affinity maturation –
be extended by at least a few fold by engineering the Fc region for is often needed and readily accomplished by further library
higher affinity binding to the salvage receptor (FcRn), whilst building and selection.
preserving the pH dependence of binding [58]. Half-life extension Over 50 different engineered protein scaffolds have been
of Fc-engineered antibodies in non-human primates was subse- described with a few advancing into clinical development [64–
quently demonstrated by several different groups [59]. Antibody 66] including: Adnectins® from type III fibronectin domains,
half-life extension can result in increased target exposure and Affibodies® from the synthetic Z domain of staphylococcal protein
improved preclinical in vivo efficacy as recently shown for anti- A, Anticalins® from lipocalins, Avimers® from LDLR-A modules,
EGFR and anti-VEGF antibodies [60]. Engineering antibodies for DARPins® from ankyrin repeat proteins, and engineered Kunitz
pH-dependent interaction with their target antigen has also been domains. Engineered protein scaffolds also include single-domain
shown to extend antibody half-life in vivo [61]. The first half-life antibodies from human (dAbs®) and camelids (Nanobodies®).
extended antibody to enter clinical trials was the humanized anti- One engineered protein scaffold protein has been approved for
RSV antibody, MEDI-557, although pharmacokinetic data have yet therapy, namely, ecallantide (Kalbitor®), a potent and selective
to be reported. inhibitor of plasma kallikrein based upon a Kunitz domain [67].
Another major area of interest for improving antibody per- Many potential advantages of engineered protein scaffolds over
formance is effector function enhancement, as reviewed exten- antibodies have been proposed [64–66], albeit with limited
sively elsewhere [34,62] and discussed briefly below. Antibody supporting evidence to date in the context of developing protein
effector functions include ADCC (antibody-dependent cellular therapeutics. For example, the smaller size and higher stability of
cytotoxicity), ADCP (antibody-dependent cellular phagocytosis) some engineered protein scaffolds may allow for alternative routes
and complement-dependent cytotoxicity (CDC). Effector functions of administration such as pulmonary delivery. The smaller size of
may be advantageous where destruction of the target cells is engineered protein scaffolds over antibodies favors more efficient
desired, as is often the case for oncology targets. tissue penetration, but this can be more than offset by much faster
Indeed many antibodies approved for oncologic indications clearance. Engineered protein scaffolds may be more readily
(e.g., rituximab, trastuzumab, alemtuzumab, cetuximab and expressed in microbial hosts reflecting their high stability and
ofatumumab) support one or many effector functions that may solubility, small size, simple structure (single polypeptide), and
contribute to their clinical antitumor activity, with the strongest that they commonly lack disulfide bonds and/or glycosylation
evidence being for rituximab [63]. sites. The small and modular architecture of engineered protein
For antibody and Fc fusion protein therapeutics potential scaffolds seems particularly well suited to the construction of
benefits from enhancing effector functions include greater efficacy, multivalent and/or multispecific protein therapeutics (Fig. 2).
lower or less frequent dosing and decreased cost-of-goods. As for Engineered proteins are commonly associated with independent
risks, these include more frequent or severe adverse events. ADCC, intellectual property that is outside the scope of antibody patents.
ADCP and CDC have been augmented by Fc protein engineering as Several of these proposed advantages of engineered protein
extensively reviewed elsewhere [62]. ADCC has also been scaffolds have been achieved with antibodies by engineering for
augmented to a similar level by modifying the glycan attached to improved stability or expression, or by exploiting the modular
the Fc, e.g., afucosylation achieved through cell line engineering architecture of antibodies to create different formats including
[62]. At least 8 effector function enhanced antibodies have ones that are smaller and simpler than IgG [6,32,33].
advanced into early clinical development [34]. Engineered protein scaffolds lack Fc-mediated properties of
antibodies such as long serum half-life and effector functions. This
Engineered protein scaffolds does not appear to be a major limitation, as many different
strategies have been developed to extend the half-life of proteins
The success of antibodies as protein therapeutics has inspired the as comprehensively reviewed [15]. For example, single-domain
development of so-called “engineered protein scaffolds” as alterna- antibodies binding to the long-circulating protein, serum albumin,
tive platform technologies for the development of novel next have been identified and fused to small proteins, including other
generation protein therapeutics [64–66]. Engineered protein scaf- single-domain antibodies to extend their half-life [68,69]. As for
folds provide binding sites that can be tailored to specifically effector functions, these are desired in some clinical settings but
recognize desired target molecules, in an analogous way that not others. Fc fusion offers a simple way to endow engineered
antibodies can be developed to recognize their target antigens. protein scaffolds with effector functions if needed. Engineered
Here the basic features of engineered protein scaffolds are described, protein scaffolds, like antibodies, offer significant opportunity for
together with their strengths, limitations and some possible future sequence diversity but individual protein scaffolds appear to
directions. provide less opportunity for structural diversity than do anti-
Protein scaffolds chosen for engineering have known 3- bodies. The functional impact of lower structural diversity on the
dimensional structures that together represent diverse protein ease of being able to generate binding proteins to specific sites on
folds [64–66]. Protein scaffolds are commonly selected with the targets of interests remains to be determined.
following attributes: small, monomeric, high solubility and All protein therapeutics, including engineered protein scaffolds,
stability, and that lack disulfide bonds and glycosylation sites. are potentially immunogenic in patients and may elicit an anti-
Protein scaffolds are typically readily expressed in microbial hosts. drug antibody response that may neutralize or otherwise interfere
Sequence diversity is introduced into the protein scaffold, e.g., by with the drug's activity. This issue can be more significant for
randomizing surface accessible residues, to create a so-called chronic therapy involving repeated drug administration. Clinical
library. Binders to a target of therapeutic interest are then iden- trials are ultimately needed to understand the extent to which
tified by selection using technologies such as phage, yeast or individual engineered protein scaffold drugs are immunogenic in
7. 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 1267
Engineered scaffold Representative Target Target Long
proteins assemblies valencies specificities 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 [6] A.C. Chan, P.J. Carter, Therapeutic antibodies for autoimmunity
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insulin, Proc. Natl Acad. Sci. USA 76 (1979) 106–110.
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important class of therapeutics. Experience with protein thera- [10] R.J. Solá, K. Griebenow, Glycosylation of therapeutic proteins: an
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Commercial rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
Platform technologies for next generation protein therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
Next generation antibodies and Fc fusion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265
Engineered protein scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267
Introduction constraints limit examples here to a sampling of technologies of
broad applicability — so called platform technologies. The inter-
Since the early 1980s proteins have emerged as a major new class ested reader is referred to many excellent articles on protein
of pharmaceuticals with ~200 marketed products that are mainly therapeutics including those in this special issue and others cited
therapeutics with a small number of diagnostics and vaccines [1]. herein.
Protein therapeutics can be classified based upon their pharma-
cologic activity as drugs that: i) replace a protein that is deficient
or abnormal, ii) augment an existing pathway, iii) provide a novel Brief history of protein therapeutics
function or activity, iv) interfere with a molecule or organism, or
iv) deliver a payload such as a radionuclide, cytotoxic drug, or Protein therapeutics enabled by protein engineering
protein effector [2]. Alternatively, protein therapeutics can be
grouped into molecular types that include: antibody-based drugs, Protein therapeutics and its enabling sister discipline, protein
anticoagulants, blood factors, bone morphogenetic proteins, engineering, have emerged since the early 1980s. Protein engi-
engineered protein scaffolds, enzymes, Fc fusion proteins, growth neering by rational design or molecular evolution allows the
factors, hormones, interferons, interleukins, and thrombolytics systemic dissection of protein structure–function relationships and
(Fig. 1) [1,3]. Antibody-based drugs are the largest and fastest the generation of novel proteins with modified activities or entirely
growing class of protein therapeutics with 24 marketed antibody new properties. Indeed, protein engineering has revolutionized
drugs in the USA [4] and over 240 more in clinical development [5]. protein therapeutics by providing the tools to customize existing
This introductory article provides a brief history of protein proteins or to create novel proteins for specific clinical applications.
therapeutics, offers a rationale for developing better protein drugs, A brief history of protein therapeutics is provided here from a
and identifies some major future opportunities as well as emerging protein engineering perspective. The focus is on technologies with
technologies that may meet them. The 30-year history of protein the greatest impact on current protein therapeutics — engineered
drugs is illustrated here with examples that pioneered their class or versions of natural proteins, Fc fusion proteins, conjugation to the
have high clinical or commercial significance. The rationale for polymer, polyethylene glycol (PEG), and antibody therapeutics as
creating better protein drugs, including antibodies [6], comes from the quintessential protein therapeutic platform.
the convergence of clinical, scientific, technological and commer-
cial drivers that collectively have identified major unmet needs and Insulin — the first recombinant human protein therapeutic
provide strategies to address them. Major future opportunities for
protein therapeutics, are improved efficacy, greater safety, reduced The first human protein therapeutic derived from recombinant
immunogenicity or improved delivery. A plethora of emerging DNA technology was human insulin (Humulin®) created at
technologies for the generation and optimization of protein ther- Genentech [7], developed by Eli Lilly, and approved by the US
apeutics are anticipated to help address these opportunities. Space Food and Drug Administration (FDA) in 1982. Recombinant insulin
Recombinant Antibodies Fc fusion
proteins proteins
Fab fragment IgG
Anticoagulants
Blood factors Receptor
Bone morphogenetic Ligand
proteins Peptide
Enzymes
Growth factors
Hormones Fc
Interferons
Interleukins
Thrombolytics
Optional Conjugate PEG Conjugate PEG Bispecific
modifications Engineer protein Conjugate drug
Engineer glycosylation Conjugate radionuclide
Fig. 1 – Overview of currently marketed protein therapeutics from a protein engineering perspective.](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)