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    Introduction to current and future protein therapeutics  a protein engineering perspective Introduction to current and future protein therapeutics a protein engineering perspective Document Transcript

    • 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/yexcrReviewIntroduction to current and future protein therapeutics: Aprotein engineering perspectivePaul J. Carter⁎Department of Antibody Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, USAA R T I C L E I N F O R M A T I O N A B S T R A C TArticle Chronology: Protein therapeutics and its enabling sister discipline, protein engineering, have emerged since theReceived 18 January 2011 early 1980s. The first protein therapeutics were recombinant versions of natural proteins. ProteinsRevised version received purposefully modified to increase their clinical potential soon followed with enhancements20 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 proteinAvailable 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 ofKeywords: clinical, scientific, technological and commercial drivers that have identified unmet needs andProtein therapeutics provided strategies to address them. Future protein drugs seem likely to be more extensivelyAntibody therapeutics engineered to improve their performance, e.g., antibodies and Fc fusion proteins with enhancedFc fusion proteins effector functions or extended half-life. Two old concepts for improving antibodies, namelyEngineered 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262 Brief history of protein therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262 Protein therapeutics enabled by protein engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262 Insulin — the first recombinant human protein therapeutic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262 Beginning of engineered proteins as therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263 Fc fusion proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263 Protein conjugation to polyethylene glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263 The rise of antibody therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263 Rationale for next generation protein therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264 Strengths and limitations of antibody and other protein therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264 Clinical rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264 Scientific rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264 ⁎ Fax: +1 650 467 8318. E-mail address: pjc@gene.com. Abbreviations: FDA, Food and Drug Administration; PEG, polyethylene glycol; TPO, thrombopoietin0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.yexcr.2011.02.013
    • 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267Introduction 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 proteinof pharmaceuticals with ~200 marketed products that are mainly therapeutics including those in this special issue and others citedtherapeutics 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 deficientor abnormal, ii) augment an existing pathway, iii) provide a novel Brief history of protein therapeuticsfunction or activity, iv) interfere with a molecule or organism, oriv) deliver a payload such as a radionuclide, cytotoxic drug, or Protein therapeutics enabled by protein engineeringprotein effector [2]. Alternatively, protein therapeutics can begrouped into molecular types that include: antibody-based drugs, Protein therapeutics and its enabling sister discipline, proteinanticoagulants, 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 thefactors, 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 entirelygrowing class of protein therapeutics with 24 marketed antibody new properties. Indeed, protein engineering has revolutionizeddrugs 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 aand identifies some major future opportunities as well as emerging protein engineering perspective. The focus is on technologies withtechnologies that may meet them. The 30-year history of protein the greatest impact on current protein therapeutics — engineereddrugs is illustrated here with examples that pioneered their class or versions of natural proteins, Fc fusion proteins, conjugation to thehave high clinical or commercial significance. The rationale for polymer, polyethylene glycol (PEG), and antibody therapeutics ascreating 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 therapeuticprovide strategies to address them. Major future opportunities forprotein therapeutics, are improved efficacy, greater safety, reduced The first human protein therapeutic derived from recombinantimmunogenicity or improved delivery. A plethora of emerging DNA technology was human insulin (Humulin®) created attechnologies for the generation and optimization of protein ther- Genentech [7], developed by Eli Lilly, and approved by the USapeutics 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.
    • 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 1263was used to replace its natural counterpart that can be deficient in Beyond half-life extension, Fc fusion can provide severaldiabetes mellitus. Other recombinant human proteins were soon additional benefits such as facilitating expression and secretiondeveloped as therapeutics to replace the natural proteins deficient of recombinant protein, enabling facile purification by protein Ain some patients (e.g., growth hormone) or augment existing chromatography, improving solubility and stability, and enhancingpathways (e.g., interferon-α, tissue plasminogen activator, and potency by increasing valency. Optional additional propertieserythropoietin) [2]. conferred by Fc fusion include binding to Fcγ receptors and/or complement to support secondary immune functions [13,14]. TheBeginning 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 beingstep 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 withadequately mimic endogenous insulin in two major ways: rapid $6.6 billion in world-wide sales in rheumatoid arthritis and otheronset of action after meals and prolonged maintenance of low level autoimmune diseases [1]. Romiplostim (Nplate®) is the firstinsulin 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 approvedeither rapid-acting or long-acting and better mimic different as a treatment for thrombocytopenia [17].properties of endogenous insulin [8]. Multiple insulin analogs havebeen approved for the treatment of diabetes mellitus including the Protein conjugation to polyethylene glycolrapid-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 newand 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 proteininclude improved physiologic profile, greater convenience for conjugates thus far have been with PEG. Protein conjugation topatients, 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 serumis illustrated by Lantus®, which was one of the top ten selling half-life, as extensively reviewed [15,18,19]. At least 8 PEGylatedbiopharmaceuticals 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-PEGylateda protein with additional glycosylation sites can increase the in counterparts [20]. Benefits of PEGylation are that it is possible tovivo serum half-life and thereby target exposure [11]. An early tune the serum-half of proteins by varying the number of attachedexample of such “glycoengineering” of proteins for half-life PEG molecules, their size and extent of branching [21]. PEGylationextension is provided by darbepoetin-α (Aranesp®), administered can also reduce the immunogenicity of proteins [18,19], althoughto anemic patients to stimulate the production of red blood cells in PEGylated proteins can still sometimes be immunogenic [22]. Athe bone marrow. The first generation drug, epoetin-α, has 3 N- draw back to PEGylation is that it can impair protein function, alinked glycosylation sites and was engineered with 2 additional limitation that is offset by the greatly increased half-life, andglycosylation sites to create darbepoetin-α, a commercially potentially ameliorated by site-specific PEGylation and/or tailoringsuccessful 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 unclearlonger half-life. Darbepoetin-α has higher specific activity in clinical significance. PEGylated proteins can induce renal tubularpatients than epoetin-α, reflecting that the 3-fold increase in vacuolation in animals, albeit with unaltered clinical pathologyserum 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 therapeuticsFc fusion proteins Antibody-based drugs are the largest and fastest growing class ofAnother significant advance in protein therapeutics came with the protein therapeutics with 24 marketed antibody therapeutics inadvent of fusion proteins by joining the genes encoding 2 or more the USA (28 FDA-approved and 4 later withdrawn from thedifferent 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 infusion 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 2009proteins with the IgG-like property of long serum half-life (days to were antibodies, namely, infliximab (Remicade®), bevacizumabweeks), by virtue of binding to the salvage receptor, FcRn [13,14]. By (Avastin®), rituximab (Rituxan® and MabThera®), adalimumabcontrast, 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-1990sfew minutes to a few hours, that can in many cases render them was made possible by the advent of antibody chimerizationmarginal or unsuitable for therapeutic applications [15]. and humanization [24] technologies that largely overcame the
    • 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 9limitations of rodent monoclonal antibodies as drugs, as well as other drug formats is that the success rate has been somewhatthe emergence of facile routes to human antibodies using higher — 17% for humanized antibodies from the first clinical trialtransgenic 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. Foras therapeutics include the industrialization of antibody produc- example, protein therapeutics are very expensive, reflecting hightion technologies [28] that can provide sufficient quantities of production costs, that may limit patient access and also clinicalantibodies to allow higher doses to be used in patients. The de- applications [28]. This high cost issue is exacerbated for proteinvelopment of methods to express and secrete functional antibody therapeutics where multi-gram doses are needed for a treatmentfragments from E. coli [29,30] has significantly stimulated the field course, as is the case for some antibodies. Intracellular delivery ofof antibody engineering and enabled subsequent technologies proteins is possible in the laboratory setting, e.g., by proteinsuch as phage display. The next major advance in the expression of transduction [36]. However, targeted pharmaceutic delivery ofantibody fragments in E. coli was high titer (gram per liter) proteins to reach intracellular targets is in its infancy, so proteinproduction in a fermentor [31], that facilitated the development of therapeutics are currently limited to cell surface or extracellularantibody 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 arereflects improved choices of antibody and target antigen for denatured and/or proteolyzed in the gut and are thus not orallyintervention 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 integritybeen approved for therapy including IgG “armed” with radio- of the blood-brain barrier can be partially compromised in somenuclides or cytotoxic drugs (antibody-drug conjugates) for cancer pathological settings such as chronic neurodegenerative diseasestreatment, 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 toRationale 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, antibodyA 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 ofwith enhanced efficacy, greater safety or improved delivery. mathematical modeling with experimental data, strongly suggestsCurrent antibody therapeutics reveal major strengths that provide that molecular size and binding affinity are major determinants ofa foundation upon which to build, as well as significant limitations tumor targeting [40]. Many other factors likely contribute to tumorto overcome that offers new opportunities [6,33]. Some antibody penetration including antigen expression level and turnover ratestrengths and many of their limitations apply more broadly to [41].protein therapeutics and are discussed below as a compass to guidethe development of next generation protein therapeutics. The Clinical rationaleimpetus to develop better protein therapeutics is also discussedhere as a convergence of clinical, scientific, and commercial forces From a clinical standpoint, first generation protein drugs canthat identify unmeet needs (this section), in conjunction with provide substantial patient benefit. However, in many cases theretechnological 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 combinationtherapeutics with chemotherapy in metastatic colorectal and breast cancers, respectively. Nevertheless, there remains plenty of opportunity forOne of the greatest strength of antibodies as a therapeutic platform enhancing antibodies for cancer treatment, such as elevating theis the potential ease of generating high affinity and specificity overall survival rate of patients, as well as many strategies beinghuman antibodies to virtually any target of therapeutic interest, explored to do so [44]. All therapeutic proteins are potentiallye.g., using transgenic mice [25,26] or phage display [25,27] immunogenic in patients and occasionally an anti-drug antibodytechnologies. Moreover, antibodies are readily tailored for thera- response can lead to a major safety issue. For example, a truncatedpeutic applications by modulating (enhancing or attenuating) and PEGylated form of TPO (MGDF-PEG) elicited an antibodytheir existing properties or endowing them with new activities as response in some patients that neutralized endogenous TPOdiscussed in a later section and reviewed more extensively leading to severe and persistent anemia, i.e., the opposite of theelsewhere [33,34]. As for other protein therapeutics, antibodies desired effect of increasing platelet counts [22]. This issue wasare commonly well tolerated by patients, but not invariably so. overcome with the peptibody, Romiplostim (see Fc fusion proteinsAntibody drug development benefits from recombinant produc- section) [17].tion methods that are well-established, broadly applicable andcommonly support expression at high titer (multi-gram per liter) Scientific rationale[28]. Widely available knowledge and experience across all facetsof antibody drug development also facilitates the generation of Scientifically, our expanding knowledge suggests drug mechanismsfuture antibody therapeutics. An advantage of antibodies over of action to enhance, and mechanisms of resistance or toxicity to
    • 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 1265overcome. For example, anti-CD3 antibodies illustrate the benefits of generation antibody and Fc fusion protein therapeutics and theunderstanding 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 proteinshuman therapy — for the treatment of acute allograft rejection inrenal transplant patients. In 1993 muromonab-CD3 was approved Many promising strategies are emerging to enhance the existingfor the treatment of acute rejection of heart and liver transplants in properties of antibodies or to endow them with new activities, aspatients resistant to steroid treatment. Unfortunately, muromonab- reviewed in detail elsewhere [6,32,33]. Antibodies (IgG) haveCD3 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 tailoredmuromonab-CD3 is that its therapeutic benefit is reduced by in ways that may improve the clinical properties of antibodiesneutralizing antibodies that develop in ~50% of patients [45]. [32,34,49]. Platform technologies that seem likely to impact theMuromonab-CD3 has since been voluntarily withdrawn from the next generation of antibody therapeutics include old concepts thatUS market due to decreased sales. These major limitations of are finally coming to fruition, such as antibody-drug conjugatesmuromonab-CD3 have been greatly reduced, if not overcome, in and bispecific antibodies. Technologies for improving Fc-mediatedsome 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 antibodyantibodies, teplizumab and otelixizumab, combine Fc engineering to therapeutics. Additionally, these technologies appear directlyattenuate 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 bispecificlikely to gain from advances in the understanding of targets and antibody — catumaxomab (Removab®, anti-EpCAM x anti-CD3) intheir role in the pathobiology of disease. For example, there is a Europe. Catumaxomab is approved for the treatment of malignantgrowing 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 andpathways [46,47]. other immune effector cells via its Fc region may contribute to antitumor activity. A tandem bispecific scFv format known as aCommercial 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 severalbred intense competition between different organizations striving complete responses in a phase I clinical trial in non-Hodgkinsto 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 isreflected in marketed drugs as illustrated by 5 different protein in the simultaneous blockade of 2 disease mediators, which maytherapeutics that block TNF including 3 different IgG (infliximab, lead to greater therapeutic benefit than targeting individualadalimumab and golimumab), a PEGylated Fab′ fragment (certo- disease mediators [53]. Until recently, a major bottleneck in thelizumab pegol) and a receptor-Fc fusion (etanercept) [6]. Such development of bispecific antibodies as therapeutics was thecompetition is increasingly commonplace as patents dominating challenge of producing these complex molecules in sufficientindividual targets expire. The generation of high affinity and quantity and quality for clinical applications. This issue has beenpotency IgG is often possible, arguably commoditized. This largely overcome by the advent of many alternative formats andprovides 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 thecompetitors. The success of innovator protein therapeutics has 1970s, use antibodies to deliver a cytotoxic payload to kill a targetbrought 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 FDAregulatory authorities). Indeed, several biosimilar approval appli- for the treatment of acute myeloid leukemia. Gemtuzumabcations have been filed in the European Union with 13 biosimilars ozogamicin was withdrawn from the market in 2010 due to safetybeing approved by European drug regulators by early 2009 [48]. concerns and insufficient patient benefit in post approval clinicalFor creators of innovator drugs, the impetus to develop improved trials. Much progress has been made over the decades in manynext generation protein therapeutics that out perform the different facets of antibody-drug conjugates, including betterprevious 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 Hodgkins 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 theundesired attributes and create proteins with entirely novel potential benefits of increased exposure leading to greater targetactivities. Space constraints limit the discussion here to next localization and greater efficacy, lower or less frequent dosing and
    • 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 9lower 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 libraryhigher 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 beenof 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 proteinimproved 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 KunitzpH-dependent interaction with their target antigen has also been domains. Engineered protein scaffolds also include single-domainshown 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 forRSV antibody, MEDI-557, although pharmacokinetic data have yet therapy, namely, ecallantide (Kalbitor®), a potent and selectiveto 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 overformance is effector function enhancement, as reviewed exten- antibodies have been proposed [64–66], albeit with limitedsively elsewhere [34,62] and discussed briefly below. Antibody supporting evidence to date in the context of developing proteineffector functions include ADCC (antibody-dependent cellular therapeutics. For example, the smaller size and higher stability ofcytotoxicity), ADCP (antibody-dependent cellular phagocytosis) some engineered protein scaffolds may allow for alternative routesand complement-dependent cytotoxicity (CDC). Effector functions of administration such as pulmonary delivery. The smaller size ofmay be advantageous where destruction of the target cells is engineered protein scaffolds over antibodies favors more efficientdesired, 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 andofatumumab) support one or many effector functions that may solubility, small size, simple structure (single polypeptide), andcontribute to their clinical antitumor activity, with the strongest that they commonly lack disulfide bonds and/or glycosylationevidence 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 ofbenefits 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 independentrisks, 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 proteinextensively reviewed elsewhere [62]. ADCC has also been scaffolds have been achieved with antibodies by engineering foraugmented to a similar level by modifying the glycan attached to improved stability or expression, or by exploiting the modularthe 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. ThisEngineered protein scaffolds does not appear to be a major limitation, as many different strategies have been developed to extend the half-life of proteinsThe success of antibodies as protein therapeutics has inspired the as comprehensively reviewed [15]. For example, single-domaindevelopment 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 othergeneration protein therapeutics [64–66]. Engineered protein scaf- single-domain antibodies to extend their half-life [68,69]. As forfolds provide binding sites that can be tailored to specifically effector functions, these are desired in some clinical settings butrecognize desired target molecules, in an analogous way that not others. Fc fusion offers a simple way to endow engineeredantibodies can be developed to recognize their target antigens. protein scaffolds with effector functions if needed. EngineeredHere the basic features of engineered protein scaffolds are described, protein scaffolds, like antibodies, offer significant opportunity fortogether with their strengths, limitations and some possible future sequence diversity but individual protein scaffolds appear todirections. 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 thedimensional structures that together represent diverse protein ease of being able to generate binding proteins to specific sites onfolds [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 interfereSequence diversity is introduced into the protein scaffold, e.g., by with the drugs activity. This issue can be more significant forrandomizing surface accessible residues, to create a so-called chronic therapy involving repeated drug administration. Clinicallibrary. Binders to a target of therapeutic interest are then iden- trials are ultimately needed to understand the extent to whichtified by selection using technologies such as phage, yeast or individual engineered protein scaffold drugs are immunogenic in
    • 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 YesFig. 2 – Modular assembly of engineered protein scaffolds into multivalent and multispecific fusion proteins. Engineered scaffoldproteins binding to different targets or different epitopes on the same target are identified by selection from libraries. The modularnature 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 supporttherapeutic applications. Many different half-life extension strategies for small proteins have been developed [15], including forengineered protein scaffolds (shown), e.g., binding to serum albumin. Linkers between modules may be included but are omittedfor 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 autoimmunityproteins are often selected for engineered protein scaffolds, which and inflammation, Nat. Rev. Immunol. 10 (2010) 301–316. [7] D.V. Goeddel, D.G. Kleid, F. Bolivar, H.L. Heyneker, D.G. Yansura, R.may help reduce the risk of immunogenicity in patients for ther- Crea, T. Hirose, A. Kraszewski, K. Itakura, A.D. Riggs, Expression inapeutic applications. Escherichia coli of chemically synthesized genes for human insulin, Proc. Natl Acad. Sci. USA 76 (1979) 106–110. [8] R. Vigneri, S. Squatrito, L. Sciacca, Insulin and its analogs: actions via insulin and IGF receptors, Acta Diabetol. 47 (2010) 271–278.Conclusions [9] J.S. Freeman, Insulin analog therapy: improving the match with physiologic insulin secretion, J. Am. Osteopath. Assoc. 109 (2009)Proteins are now well established as a clinically and commercially 26–36.important class of therapeutics. Experience with protein thera- [10] R.J. Solá, K. Griebenow, Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy, BioDrugs 24 (2010) 9–21.peutics has provided an understanding of their strengths and [11] S. Elliott, T. Lorenzini, S. Asher, K. Aoki, D. Brankow, L. Buck, L.limitations and ways in which they might be improved. Clinical Busse, D. Chang, J. Fuller, J. Grant, N. Hernday, M. Hokum, S. Hu, A.and commercial success with protein therapeutics provides strong Knudten, N. Levin, R. Komorowski, F. Martin, R. Navarro, T.motivation to develop better protein drugs, whereas new tools, Osslund, G. Rogers, N. Rogers, G. Trail, J. Egrie, Enhancement ofespecially platform technologies provide the likely means to do so. therapeutic protein in vivo activities through glycoengineering,The next generation of protein therapeutics will likely include Nat. Biotechnol. 21 (2003) 414–421.some of the many enhanced antibodies in early clinical develop- [12] Z. Kiss, S. Elliott, K. Jedynasty, V. Tesar, J. Szegedi, Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs),ment, optimized Fc fusion proteins, as well as new formats such as including the hyperglycosylated ESA, darbepoetin alfa: an updateengineered protein scaffolds. of the rationale and clinical impact, Eur. J. Clin. Pharmacol. 66 (2010) 331–340. [13] C. Huang, Receptor-Fc fusion therapeutics, traps, andREFERENCES MIMETIBODY technology, Curr. Opin. Biotechnol. 20 (2009) 692–699. [14] J.A. Jazayeri, G.J. Carroll, Fc-based cytokines: prospects for [1] G. Walsh, Biopharmaceutical benchmarks 2010, Nat. Biotechnol. engineering superior therapeutics, BioDrugs 22 (2008) 11–26. 28 (2010) 917–924. [15] R.E. Kontermann, Strategies to extend plasma half-lives of [2] B. Leader, Q.J. Baca, D.E. Golan, Protein therapeutics: a summary recombinant antibodies, BioDrugs 23 (2009) 93–109. and pharmacological classification, Nat. Rev. Drug Discov. 7 [16] D.J. Capon, S.M. Chamow, J. Mordenti, S.A. Marsters, T. Gregory, H. (2008) 21–39. Mitsuya, R.A. Byrn, C. Lucas, F.M. Wurm, J.E. Groopman, S. Broder, [3] N.C. Nicolaides, P.M. Sass, L. Grasso, Advances in targeted D.H. Smith, Designing CD4 immunoadhesins for AIDS therapy, therapeutic agents, Expert Opin. Drug Discov. 5 (2010) Nature 337 (1989) 525–531. 1123–1140. [17] D.B. Cines, U. Yasothan, P. Kirkpatrick, Romiplostim, Nat. Rev. [4] J.M. Reichert, Metrics for antibody therapeutics development, Drug Discov. 7 (2008) 887–888. mAbs 2 (2010) 695–700. [18] S. Jevševar, M. Kunstelj, V.G. Porekar, PEGylation of therapeutic [5] J.M. Reichert, Antibodies to watch in 2010, mAbs 2 (2010) 28–45. proteins, Biotechnol. J. 5 (2010) 113–128.
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