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Strategies for extended serum half-life of protein therapeutics
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Strategies for extended serum half-life of protein therapeutics Roland E Kontermann With a growing number of protein therapeutics being developed, many of them exhibiting a short plasma half-life, half-life extension strategies find increasing attention by the biotech and pharmaceutical industry. Extension of the half-life can help to reduce the number of applications and to lower doses, thus are beneficial for therapeutic but also economic reasons. Here, a comprehensive overview of currently developed half-life extension strategies is provided including those aiming at increasing the hydrodynamic volume of a protein drug but also those implementing recycling processes mediated by the neonatal Fc receptor. Address Institut fu¨r Zellbiologie und Immunologie, Universita¨t Stuttgart, Allmandring 31, 70569 Stuttgart, Germany Corresponding author: Kontermann, Roland E ([email protected])
Current Opinion in Biotechnology 2011, 22:868–876 This review comes from a themed issue on Pharmaceutical biotechnology Edited by Luis Angel Ferna´ndez and Serge Muyldermans Available online 20th August 2011 0958-1669/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2011.06.012
Introduction In 2010, approximately 200 biologics were approved for therapeutic applications and more than 600 are currently in clinical development [1]. Besides monoclonal antibodies and vaccines, which account for more than 60% of these products, hormones, growth factors, cytokines, coagulation factors, enzymes, fusion proteins and other proteins are developed and used as therapeutics. Many of these protein drugs, with the exception of whole antibodies and Fc-fusion proteins, possess a molecular mass below 50 kDa and a rather short terminal half-life in the range of minutes to hours. However, most of the therapeutic applications of proteins benefit from maintaining a therapeutic effective concentration over a prolonged period of time. Often, this requires infusions or frequent administrations, or the drug is applied loco-regional or subcutaneously utilizing a slow adsorption into the blood stream. These limitations of small size protein drugs has led to the development and implementation of half-life extension strategies to prolong circulation of these recombinant antibodies in the blood and thus to improve Current Opinion in Biotechnology 2011, 22:868–876
administration and pharmacokinetic as well as pharmacodynamic properties.
Half-life extension strategies Several mechanisms are involved in clearance of protein drugs from circulation including peripheral bloodmediated elimination by proteolysis, renal and hepatic elimination, and elimination by receptor-mediated endocytosis [2]. Molecules possessing a small size, that is, a low molecular mass, are rapidly cleared by renal filtration and degradation, with a threshold in the range of 40–50 kDa. Responsible for renal clearance is the glomerular filtration barrier (GBM) formed by the fenestrated endothelium, the glomerular basement membrane and the slit diaphragm located between the podocyte foot processes [3]. While the fenestrae between the glomerular endothelial cells are rather large (50–100 nm) allowing free diffusion of molecules, the slit diaphragm represents the ultimate macromolecular barrier, forming an isoporous, zipper-like filter structure with numerous small, 4–5 nm diameter pores and a lower number of 8–10 nm diameter pores [4,5]. Furthermore, proteoglycans of the endothelial cells and the GBM form an anionic barrier, which partially prevents the traversal of negatively charged plasma macromolecules [3]. Consequently, the size of a protein therapeutic, that is, its hydrodynamic radius, but also its physicochemical properties represent starting points in order to improve half-life (Figure 1). Some plasma proteins such as serum albumin and IgG molecules possess an extraordinary long half-life in the range of 2–4 weeks, which clearly discriminates these molecules from all the other plasma proteins [6]. Responsible is a recycling through the neonatal Fc receptor (FcRn, Brambell receptor) [7]. Albumin and IgGs taken up by cells, for example, endothelial cells, through macropinocytosis will bind to the FcRn in a pH-dependent manner in the acidic environment of the early endosome. This binding diverges albumin and IgG from degradation in the lysosomal compartment and redirects them to the plasma membrane, where they are released back into the blood plasma due to the neutral pH. This offers additional opportunities to extend or modulate the half-life of proteins, for example, through fusion to albumin or the Fc-region of IgG [6] (Figure 1). Finally, protein drugs that bind to a cellular surface receptor will be internalized by receptor-mediated endocytosis and subjected to lysosomal degradation if the protein drug stays bound to the receptor [2,8]. Hence, engineering of the interaction of the therapeutic protein with its receptor(s) at acidic pH can therefore also prolong www.sciencedirect.com
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Figure 1
FcRn FcRn-mediated recycling
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Half-life extension strategies including firstly, those that increase the hydrodynamic volume, secondly, those that in addition utilize FcRn-mediated recycling, and thirdly, those that aim at modulating stability of protein-receptor complexes in the sorting endosome.
half-life of the protein by allowing recycling of the unbound molecules into the blood stream as shown for engineered G-CSF and an anti-IL6 receptor antibody [9,10] (Figure 1). In summary, strategies to extend the half-life of a therapeutic molecule can be grouped into firstly, those that increase the hydrodynamic volume, secondly, those that in addition utilize FcRn-mediated recycling, and thirdly, those that aim at modulating stability of protein-receptor complexes in the sorting endosome (Figure 1).
Increasing the hydrodynamic volume The hydrodynamic volume of a protein can be increased by attaching highly flexible, hydrophilic molecules such as polyethylene glycol and carbohydrates. PEGylation, that is, the chemical coupling of polyethylene glycol (PEG), was already developed more than 2 decades ago for half-life extension purposes and meanwhile nine PEGylated protein drugs are approved, including enzymes, interferon-a2b (INF-a2b), granulocyte-colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), and a Fab fragment (Table 1) [11]. In the approved drugs, one or several PEG chains of 5–40 kDa are conjugated. Different coupling methods have been established including random and site-directed approaches, for www.sciencedirect.com
example, to free thiol groups. The PEGylation strategy, that is, the site of PEGylation as well as the number and size of attached PEG chains, has to be carefully chosen in order to avoid a reduction or abrogation of the activity of the therapeutic protein [12]. Ideally, a single PEG chain is conjugated in a site-directed manner, for example, through the use of existing or genetically introduced cysteine residues. This is exemplified by certolizumab pegol (Cimzia), a bacterially produced anti-tumor necrosis factor (TNF) Fab’ fragment with a 40 kDa PEG chain attached to the free cysteine at the C-terminus of the heavy chain Fd chain, that is, opposite the antigen-binding site [13]. Meanwhile, PEGylation has been applied to a large number of other proteins of therapeutic interest [14]. Recently, novel coupling methods for a site-directed conjugation of PEG have been described, including coupling to histidine tags or an existing disulfide bond, with the intension to produce homogenous PEGylated products with increased purity [15]. Disulfide bridge-based PEGylation strategies are developed by PolyTherics and were already utilized to generate PEGylated IFNa, leptin and an anti-CD4 Fab fragment [15]. However, also with this technology a reduction of bioactivity was observed after PEGylation. In general, PEGylation of proteins is considered to be safe and well tolerated [11], although in animals the occurrence of renal tubular Current Opinion in Biotechnology 2011, 22:868–876
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Table 1 Half-life extended protein therapeutics (approved or in clinical trials) Drug
Modification/protein PEGylation Adenosine deaminase L-Asparaginase
Interferon a-2b Interferon a-2b G-CSF Human growth hormone (hGH) Erythropoietin Anti-TNF Fab0 Uricase EPO mimetic peptide Phenylalanine ammonia lyase IL-29 Coagulation factor IX Arginase Hyaluronidase N-glycosylation and polysialylation Erythropoietin Erythropoietin Insulin Albumin fusion Interferon a-2b Coagulation factor IX HER2 + HER3 specific single-chain Fv Albumin binding through a conjugated fatty acid chain Insulin Glucagon-like peptide-1 Fc-fusion proteins TNF receptor 2
LFA-3 CTLA-4 IL-1R TPO-mimetic peptide VEGF receptor BR3 Coagulation factor IX Coagulation factor VIII
Pegademase bovine (Adagen)
Indication
Status Approved 1990
Pegaspargase (Oncaspar) Peginterferon alfa-2b (Peg-Intron) Peginterferon alfa-2b (Pegasys) Pegfilgrastim (Neulasta) Pegvisomant (Somavert)
Severe combined immunodeficiency disease (SCID) Acute lymphoblastic leukemia Hepatitis C Hepatitis C Chemotherapy-induced neutropenia Acromegaly
mPEG-epoetin beta (Mircera) Certolizumab pegol (Cimzia) Pegloticase (Krystexxa) Peginesatide (Hematide) rAvPAL-PEG
Anemia Crohn’s disease Chronic gout Anemia Phenylketonuria
Approved 2007 Approved 2008 Approved 2010 Phase II Phase II
PEG-rIL-29 PEG-rFIX PEG-rArgI PEGPH20
Hepatitis C Hemophilia B Liver cancer Advanced solid tumors
Phase Phase Phase Phase
Darbepoetin alfa (Aranesp) PSA-EPO (ErepoXen) PSA-insulin (SuliXen)
Anemia Anemia Diabetes mellitus
Approved 2008 Phase II Phase I
Albinterferon alfa-2b (Joulferon, Zalbin) rIX-FP MM-111 (scFv-HSA-scFv)
Hepatitis C Hemophila B Cancer
Phase III Phase I Phase I/II
Insulin detemir (Levemir) Liraglutide (Victoza)
Diabetes mellitus Diabetes mellitus type 2
Approved 2003 Approved 2009
Etanercept (Enbrel)
Rheumatoid arthritis, ankylosing spondylitis, polyarticular juvenile idiopathic arthritis, psoriatic arthritis, plaque psoriasis Severe chronic plaque psoriasis Rheumatoid arthritis, juvenile idiopathic arthritis Cryopyrin-associated periodic syndromes Chronic idiopathic thrombocytopenic purpura Macular degeneration Rheumatoid arthritis Hemophilia B Hemophilia A
Approved 1998
Alefacept (Amevive) Abatacept (Orenica) Rilonacept (Arcalyst) Romiplostim (Nplate) Aflibercept (VEGF trap) Briobacept (BR3-Fc) rFIXFc rFVIII-Fc
vacuolization has been observed due to accumulation of the non-degradable PEG chains in the kidney [16].
Recombinant PEG mimetics During the past 2–3 years, alternative methods mimicking the properties of PEGylation have emerged. The idea is to substitute PEG by a hydrophilic and flexible polypeptide chain. The advantages of this approach are that attachment can be achieved by genetic engineering, that is, avoiding any chemical coupling and additional purification steps, and that the attached polypeptide chain is fully biodegradable and of a defined length and composition. Amunix has recently established the XTEN technology based on polypeptide chains of varying length Current Opinion in Biotechnology 2011, 22:868–876
Approved Approved Approved Approved Approved
1994 2000 2002 2002 2002
I I I I
Approved 2003 Approved 2005 Approved 2008 Approved 2008 Phase III Phase II Phase II/III Phase I
composed of alanine, glutamic acid, glycine, proline, serine, and threonine [17]. An increase in solubility and stability of the attached protein and improved manufacturing has been attributed to this technology. Furthermore, animal studies demonstrated that the XTEN sequences are safe and poorly immunogenic [18]. In a recent study, an XTEN sequence was fused to glucagon (Gcg), possessing a half-life of <10 min, for the treatment of nocturnal hypoglycemia [18]. The XTEN sequence was adjusted to maintain efficacious Gcg-XTEN serum levels for approximately 8–10 hours, that is, during the patients sleeping period but not affecting normal insulin usage during the day. A sequence of 288 amino acid residues was found to fulfill these criteria www.sciencedirect.com
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as shown in various animal models, demonstrating the potential of this approach in fine-tuning the pharmacokinetic properties to meet the medical needs [18]. In a similar approach, recombinant PEG mimetics based on the three amino acids proline, alanine and serine were established by the group of Arne Skerra (reviewed in [6]). This so-called PASylation technology is further developed by XL-protein. PAS sequences, which adopt a stable random conformation, are composed of 200 to 600 residues and can be fused N-terminally and/or C-terminally resulting in plasma half-life extension by a factor of 10–100. These PAS sequences do not contain charged residues, thus, do not alter the isoelectric point of the protein. They have also been shown to be non-toxic and to lack T-cell epitopes, that is, being non-immunogenic as shown by animal experiments.
Glycosylation and conjugation of carbohydrates Many plasma proteins are N-glycosylated and/or Oglycosylated and it has been shown that glycosylation can influence half-life [19]. The presence of terminal sialic acids has been identified as important property of N-glycans in order to avoid rapid clearance of proteins containing galactose-terminating glycans through asialoglycoprotein receptor-mediated endocytosis but also of glycoproteins possessing glycans terminating in mannose, N-acetylglucosamine or fucose through lectin-like receptors [19]. The half-life of various protein drugs has been extended by glycoengineering. A prominent example is darbepoetin alfa (Aranesp), a hyperglycosylated form of EPO, which possesses two additional Nglycosylation sites and a 3-fold prolonged half-life in humans [20]. A similar approach was recently applied to generate long-lasting IFN-a derivatives possessing four and five N-glycans resulting in a 25-fold increased elimination half-life [21]. An increased antitumor activity was observed for this hyperglycosylated IFNa derivative in animal experiments, despite a reduced in vitro bioactivity. We have recently applied N-glycosylation to a recombinant bispecific antibody, a single-chain diabody molecule (scDb) with a molecular mass of 55 kDa and a terminal half-life in mice of 5–6 hours [22]. ScDbs are composed of the variable light and heavy chain domains of two different antibodies connected by three flexible linkers [23]. Three, six, or nine N-glycosylation sites (Asn-X-Thr) were introduced into two of the linkers as well as a Cterminal extension of varying length resulting in N-glycosylated molecules with increased hydrodynamic radii. Half-life of the scDb was prolonged leading to an approximately 2–3-fold increase of the area under the curve (AUC), compared with an approximately 10-fold increase by PEGylation or fusion to albumin, demonstrating that www.sciencedirect.com
N-glycosylation results only in a moderate prolongation of half-life [22]. Another approach utilizes the introduction of O-glycosylation sites into protein therapeutics. Such O-glycosylation sites are, for example, present in the C-terminal peptide (CTP) of human chorionic gonadotropin b-subunit. It was shown that fusion of one or several copies of the CTP to EPO or hGH, respectively, resulted in Oglycosylated derivatives with improved in vivo potency and half-life [24,25]. An alternative to the generation of glycosylated proteins by posttranslational modification represents the chemical conjugation of carbohydrates. For example, hydroxyethyl starch (HES), a modified, branched amylopectin, for example isolated from waxy maize starch, was used for half-life extension. HES is an approved plasma volume expander with a proven safety record. Because of its close similarity to glycogen, HES is not immunogenic [26]. The size and structure of HES and thereby its stability can be adjusted by acidic hydrolysis and by chemical hydroxyethylation at positions 2, 3, and 6 of the glucose unit. The HESylation technology was pioneered by Fresenius Biotech to improve the pharmacokinetic and pharmacodynamic properties of therapeutic proteins. Examples include a HESylated derivative of EPO, which was produced by chemical coupling of a 60 kDa HES (reviewed in [6]). Similarly, polysialic acid (PSA) has been used as an alternative to PEG [27]. PSA is found on the surface of a variety of cells including mammalian cells, thus is a biocompatible and biodegradable natural polymer. Colominic acid, a linear polymer of a-(2,8)-linked N-acetylneuraminic acid, was used for polysialylation of various proteins including asparaginase, insulin and antibody fragments and was shown to be capable of prolonging their half-lives [28]. Site-specific polysialylation of an anti-carcinoembryonic antigen (CEA) scFv at a C-terminally exposed cysteine residue increased half-life approximately 5-fold and led to a 30-fold improved tumor uptake as compared with the unmodified scFv [28]. Lipoxen is developing polysialylation as PolyXen technology and has different polysialylated protein drugs, including a long-acting EPO (ErepoXen), insulin (SuliXen), IFN-a2b, and G-CSF, at various preclinical and clinical stages (Table 1) [29].
Fc engineering to prolong half-life of immunoglobulins Naturally, immunoglobulins of the IgG1, IgG2 and IgG4 subclass exhibit long half-lives in the range of 3–4 weeks mediated by recycling through FcRn, although half-life is influenced also by antigen-dependent elimination, for example, through receptor-mediated endocytosis and intracellular degradation [30,31]. The site of interaction Current Opinion in Biotechnology 2011, 22:868–876
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of IgG with FcRn has been localized in the Fc region covering parts of the CH2 and CH3 domain. Mutations were identified which increase the affinity of IgG to FcRn at acidic pH without increasing affinity at neutral pH. Incorporation of such alterations into two therapeutic antibodies (bevacizumab, cetuximab) resulted in an increased half-life and established a direct correlation between affinity and half-life [32]. By mutating methonine 428 to leucine and asparagine 434 to serine (Xtend derivative), leading to a 11-fold increased affinity for FcRn at pH 6 (from 2.46 mM to 0.2 mM), half-life of bevacizumab was prolonged from 9.7 to 31.1 days and that of cetuximab from 2.9 to 13.9 days in cynomolgus monkeys [32]. The prolonged half-life translated into improved antitumor activity in xenograft tumor studies employing transgenic human FcRn/Rag1/ mice. However, a recent study with a modified version of bevacizumab carrying the mutations T307Q/N434A, resulting also in a 10-fold increased affinity for FcRn at acidic pH, indicated that along with FcRn affinity, other factors, such as antibody stability, tumor pH, growth rate, FcRn expression pattern, and specific clearance mechanisms, for example, antigen-dependent clearance, can influence the pharmacokinetics of human and humanized IgGs [33].
Fc-fusion proteins An increasing number of Fc-fusion proteins are developed for therapeutic applications, primarily with the aim to utilize bivalency of the molecules to improve activity but also to improve half-life (Table 1) [34]. Because of the homodimeric nature of these fusion proteins, the molecular mass is strongly increased resulting in a prolonged half-life, which is also influenced by FcRn-mediated recycling. In a recent study, a correlation between affinity for FcRn and half-life was established for various therapeutic antibodies and Fc-fusion proteins but also confirmed that other factors are critical for determining serum half-life [35]. Interestingly, the analyzed Fc-fusion proteins (abatacept, alefacept, and etanercept) exhibited a 2–3-fold lower affinity for FcRn than various human, humanized and chimeric antibodies, which was also reflected by a shorter half-life. It was speculated that the fused receptor domains have an influence on the structural environment of the FcRn-binding site.
Albumin fusion proteins and conjugates Serum albumin is the most abundant protein in the blood plasma, exhibiting also a long half-life, which is 19 days in humans. Responsible for this long half-life is also a pHdependent recycling by the FcRn. The binding site of albumin on FcRn is not overlaying with the IgG binding site, thus the two molecules do not interfere with each other for binding to FcRn [36]. The covalent attachment of albumin to a therapeutic protein is one approach to extend its half-life (Table 1). During the past decade, a large number of different proteins have been genetically Current Opinion in Biotechnology 2011, 22:868–876
fused to human serum albumin (HSA) including interferon alpha, erythropoietin, coagulation factors IX and VIIa, hGH, TNF, and antibody fragments [37]. The most advanced product is albinterferon alfa-2b exhibiting a terminal half-life of approximately 6 days [38]. Results from a phase III clinical trial for the treatment of hepatitis C demonstrated that albinterferon alfa-2b dosed every 2 weeks is not inferior to PEGylated IFN-a, thus providing an alternative efficacious treatment option [39]. Advanced are also studies with an albumin-coagulation factor IX fusion protein [40,41]. Coagulation factor IX has a half-life of 18–34 hours requiring regular infusions every 2–3 days to maintain a factor IX clotting activity of 1%. Factor IX was fused to albumin via a cleavable peptide linker, which is cleaved in parallel to factor IX activation. Clotting activity was 10–30-fold greater than that of a fusion protein generated with a non-cleavable linker and half-life of the fusion protein was improved by a factor of 1.5 to 3.9 in mice, rats and rabbits. This should be useful for a treatment with fewer infusions and lower doses. A phase I trials has been initiated to analyze safety and pharmacokinetics in subjects with hemophilia B (NCT01233440). Proteins can be fused either to the N-terminus or Cterminus of albumin, or even to both ends as shown for single-chain Fv fragments (scFv) [42–44]. Using antibody fragments, this allows for the generation of molecules exhibiting one or more antigen-binding sites. The fusion of antibody fragments is of special interest for applications where Fc-mediated effector functions are not required or even detrimental. For example, Merrimack is developing a bispecific scFv-albumin fusion protein (MM-111) directed against HER2 and HER3. Treatment is based on using HER2 as a marker to specifically target cancer cells and neutralization of HER3-mediated signaling implicated in disease progression. Two clinical phase I and II trials are currently conducted investigating MM-111 alone or in combination with trastuzumab in HER2-positive solid tumors (NCT00911898, NCT 01097460). The FcRn-binding site resides in domain III of albumin and recently it was shown that fusion of this domain to a bivalent anti-CEA diabody leads also to a strongly prolonged half-life, indicating that even a small 22 kDa fragment of albumin is suitable for half-life extension strategies [45]. Covalent linkage of a protein or peptide drug to albumin can also be achieved by chemical coupling. Of special interest for conjugation is a free and accessible cysteine residue at position 34 of albumin allowing for a defined and site-directed conjugation [46]. This approach was utilized to prolong the half-life of a 34 amino acid peptide with anti-HIV activity [47]. This peptide was functionalized with 3-maleimidopropionic acid (MPA), which www.sciencedirect.com
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allows formation of a stable thioether bond between albumin and the peptide. In rhesus monkeys, the albumin-peptide fusion protein exhibited a terminal half-life of 102.4 h, which was 9.4-fold longer than the half-life of the free peptide. A potent neutralizing activity against a number of HIV-1 isolates was observed in vitro and in vivo.
Non-covalent interaction with serum albumin Serum albumin can also be engaged in half-life extension through modules with the capacity to non-covalently interact with albumin. In these approaches, an albumin-binding moiety is either conjugated or genetically fused to the therapeutic protein. A wide range of different moieties have been employed including molecules with intrinsic affinity for albumin but also other molecules such as peptides, antibody fragments, alternative scaffolds, and small chemicals generated and selected to exhibit albumin binding activity (for review see [6]). Albumin acts in the body, amongst other functions, as transport protein for a variety of substances including fatty acids, which bind with low to medium affinity to albumin. The chemical conjugation of a fatty acid chain to prolong the half-life of a therapeutic protein has been utilized for insulin. Insulin detemir (Levemir), which was approved in 2003, lacks ThrB30 and is generated by conjugation of a myristoyl fatty acid chain to LysB29 at the C-terminus of the insulin B-chain [48]. Similar to albumin binding through a conjugated fatty acid, a small organic molecule, 6-(4-(4-iodophenyl)butanamido)hexanoate, which was selected from a DNAencoded chemical library for albumin-binding activity, was employed as portable albumin binder for half-life extension purposes [49]. This compound has been chemically coupled to other small molecules but also to recombinant proteins. Thus, the maleimide-functionalized compound (Albu-tag) was chemically conjugated to an scFv fragment containing an additional C-terminal cysteine [50]. Half-life of this scFv directed against an alternatively spliced EDB domain of fibronectin, an antigen overexpressed by the tumor vasculature, was strongly prolonged from 20–30 min for the unmodified scFv to approximately 40 hours in mice, which translated into an approximately 10-fold increased tumor accumulation in tumor-bearing mice [50]. Proteins with albumin-binding activity are known from certain bacteria. For example, streptococcal protein G contains several small albumin-binding domains (ABD) composed of roughly 50 amino acid residues (6 kDa). Fusion of an ABD to a protein results in a strongly extended half-life as shown for various proteins, for example, antibody fragments and affibody molecules [51,52]. Using bispecific single-chain diabody molecule (scDb), we have shown that the half-life of an scDb-ABD www.sciencedirect.com
fusion protein is similar to that of an scDb-HSA fusion protein and a PEGylated scDb derivative with a 40 kDa branched PEG chain conjugated [22,51]. Further testing of ABD variants with increased or decreased affinity for albumin showed that even a low affinity (around 0.6 mM) is sufficient to strongly extend half-life and that increased affinity only marginally improves half-life of this antibody molecule [53]. Importantly, the prolonged half-life led to a strongly increased accumulation of the anti-CEA x antiCD3 scDb in CEA-positive tumors in animal studies [54]. Compared with a PEGylated scDb derivative an increased accumulation was observed in the antigenpositive tumors indicating a facilitated tissue penetration of the scDb-ABD fusion protein. That recycling by the FcRn is involved in the half-life was confirmed in a comparative study with FcRn wild-type and FcRn heavy chain knockout mice demonstrating also that the bound ABD does not interfere with FcRn binding of albumin [54]. The issue of being immunogenic was recently addressed by generating a deimmunized derivative of the ABD (ABD094), which is used by Affibody to develop their Albumod technology (see article by Fredrik Frejd in this issue). Furthermore, albumin-binding Nanobodies, singledomain antibodies, and alternative scaffolds are employed for half-life extension purposes. Nanobodies, which are based on variable domains (VHH) derived from camelid heavy chain antibodies, were used for prolonging the half-life of other therapeutically useful Nanobodies. For example, a Nanobody specific for mouse albumin (MSA21) was fused to two antagonistic anti-EGFR Nanobodies generating a bispecific, trivalent molecule with a molecular mass of approximately 50 kDa. Binding to albumin increased the half-life from 1 to 44 hours and efficiently delayed outgrowth of EGFR-positive tumors in animal models [55]. Tumor uptake of this trivalent Nanobody construct was similar to anti-EGFR monoclonal antibody cetuximab but the albumin-binding Nanobody construct showed faster and deeper tumor penetration [56]. In a similar approach, Domantis, a subsidiary of GSK, has developed the Albudab technology based on human domain antibodies (dAb). Various albumin-binding VL and VH dAbs were selected from phage libraries and analyzed for their half-life extension potential [57]. Fusion of one of these dAbs (dAbm16), exhibiting an affinity of 70 nM for mouse serum albumin (MSA), to IL-1 receptor antagonist (IL-1ra) prolonged half-life of IL-1ra in mice from 2 min to 4.3 hours, which was also longer than the half-life of an IL-1ra fused to an irrelevant dAb (24 min) [57]. In a collagen-induced arthritis model in DBA/1 mice, mean arthritis scores were significantly lower after treatment with the IL-1radAbm16 fusion protein compared with treatment with IL-1ra. In a further study, another albumin-binding dAb (DOM7 h-14) was fused to IFN-a2b [58]. After fusion to DOM7, in vitro potency of IFN-a2b was reduced Current Opinion in Biotechnology 2011, 22:868–876
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approximately 8-fold in the absence of HSA and 55-fold in the presence of HSA. Also, the affinity of DOM7 h-14 for HSA and MSA was reduced 7.7-fold and 22.3-fold, respectively. Nevertheless, half-life in mice was increased from 1.2 hours for IFN-a2b to 22.6 hours. Using a xenograft tumor model, an improved in vivo efficacy was observed for the IFN-a2b-DOM7 h-14 fusion protein compared with IFN-a2b but also an IFN-a2b-HSA fusion protein [58].
Conclusion Many new strategies to extend the half-life of therapeutic proteins and other molecules have emerged in the recent years. Increasing the hydrodynamic radius of a protein through PEGylation was established more than 20 years ago. New approaches such as genetic fusion of hydrophilic and flexible polypeptide chains are intended to mimic the biochemical properties of PEG, while incorporating further advantages, for example, biodegradability, lack of immunogenicity and facilitated production. Furthermore, a better understanding of the molecular mechanisms underlying the long half-lives of serum albumin and immunoglobulins has led to novel strategies based on fusion or non-covalent binding to albumin, fusion to the Fc region of IgG or improving the interaction of immunoglobulins with the FcRn. Most of these novel half-life extension strategies are still at the preclinical stage. However, it can be expected that in the near future more and more of these technologies will be evaluated in clinical trials and become established and accepted ways to prolong the half-life and thus to improve the pharmacokinetic and pharmacodynamic properties of therapeutic proteins.
Acknowledgment This work was supported by a grant from the Deutsche Forschungsgemeinschaft (KO1461/4).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Walsh G: Biopharmaceutical benchmarks 2010. Nat Biotechnol 2010, 28:917-924.
2.
Tang L, Persky AM, Hochhaus G, Meibohm B: Pharmacokinetic aspects of biotechnology products. J Pharmaceut Sci 2004, 93:2184-2204.
3.
Tryggvason K, Wartiovaara K: How does the kidney filter plasma? Physiology 2005, 20:96-101.
4.
Haraldsson B, So¨rensson J: Why do we not all have proteinuria? An update of our current understanding of the glomerular barrier. New Physiol Sci 2004, 19:7-10.
5.
Wartiovaara J, O¨fverstedt LG, Khoshnoodi J, Zhang J, Ma¨kela¨ E, Sandin S, Ruotsalainen V, Cheng RH, Jalanko H, Skoglund U, Tryggvason K: Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography. J Clin Invest 2004, 114:1475-1483.
6.
Kontermann RE: Strategies to extend plasma half-lives of recombinant antibodies. BioDrugs 2009, 23:93-109.
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This review provides a comprehensive summary of various half-life extension strategies. 7.
Roopenian DC, Akilesh S: FcRn the neonatal Fc receptor comes of age. Nat Rev Immunol 2007, 7:715-725.
8.
Lao BJ, Kamei DT: Improving therapeutic properties of protein drugs through alteration of intracellular trafficking pathways. Biotechnol Prog 2008, 24:2-7.
9.
Sarkar CS, Lowenhaupt K, Horan T, Boone TC, Tidor B, Lauffenburger DA: Rational cytokine design for increased lifetime and enhanced potentcy using pH-activated ‘‘histidine switching’’. Nat Biotechnol 2002, 20:908-913.
10. Igawa T, Ishii S, Tachibana T, Maeda A, Higuchi, Shimaoka S, Moriyama C, Watanabe T, Takubo R, Doi Y et al.: Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization. Nat Biotechnol 2010, 28:1203-1208. 11. Veronese FM, Mero A: The impact of PEGylation on biological therapies. BioDrugs 2008, 22:315-329. 12. Fishburn CS: The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics. J Pharm Sci 2008, 97:4167-4183. 13. Melmed GY, Targan SR, Yasothan U, Hanicq D, Kirkpatrick P: Certolizumab pegol. Nat Rec Drug Disov 2008, 7:641-642. 14. Jevsevar S, Junstelj M, Porekar VG: PEGylation of therapeutic proteins. Biotechnol J 2010, 5:113-128. 15. Brocchini S, Godwin A, Balan S, Choi J, Zloh M, Shaunak S: Disulfide bridge based PEGylation of proteins. Adv Drug Deliv Rev 2008, 60:3-12. 16. Gaberc-Porekar V, Zore I, Podobnik B, Menart V: Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr Opin Drug Discov Dev 2008, 11:242-250. 17. Schellenberger V, Wang CW, Geething NC, Spink BJ, Campbell A, To W, Scholle MD, Yin Y, Yao Y, Bogin O et al.: A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol 2009, 27:1186-1190. This work provides first data that fusion of recombinant PEG mimetics result in a drastic prolongation of serum half-life and that half-life can be adjusted by the length of the fused polypeptide chain. 18. Geething NC, To W, Spink BJ, Scholle MD, Wang CW, Yin Y, Yao Y, Schellenberger V, Cleland JL, Stemmer WPC, Silverman J: Gcg-XTEN: an improved glucagon capable of preventing hypoglycemia without increasing baseline blood glucose. PLoS ONE 2010, 5:e10175. This work describes the application of recombinant PEG mimetics to generate a long-acting glucagon and demonstrates that half-life can be fine-tuned and adjusted to the medical needs. 19. Sola´ RJ, Griebenow K: Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs 2010, 24:9-21. 20. Kiss Z, Elliott S, Jedynasty K, Tesar V, Szegedi J: Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact. Eur J Clin Pharmacol 2010, 66:331-340. 21. Ceaglio N, Etcheverrigaray M, Conradt HS, Grammel N, Kratje R, Oggero M: Highly glycosylated human alpha interferon: an insight into a new therapeutic candidate. J Biotechnol 2010, 146:74-83. 22. Stork R, Zettlitz KA, Mu¨ller D, Rether M, Hanisch FG, Kontermann RE: N-glycosylation as novel strategy to improve pharmacokinetic properties of bispecific single-chain diabodies. J Biol Chem 2008, 283:7804-7812. 23. Mu¨ller D, Kontermann RE: Bispecific antibodies for cancer immunotherapy: current perspectives. BioDrugs 2010, 24:89-98. 24. Fares F, Ganem S, Hajouj T, Agai E: Development of a longacting erythropoietin by fusing the carboxy-terminal peptide of human chorionic gonadotropin b-subunit to the coding sequence of human erythropoietin. Endrocrinology 2007, 148:5081-5087. www.sciencedirect.com
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25. Fares F, Guy R, Bar-Ilan A, Felikman Y, Fima E: Designing a long acting human growth hormone (hGH) by fusing the carboxylterminal peptide of human chorionic gonadotropin b-subunit to the coding sequence of hGH. Endocrinology 2010, 151:4410-4417. This study provides data from in vitro and in vivo studies showing that also O-glycosylation can lead to a prolonged half-life and improved efficacy. 26. Agreda-Va´squez GP, Espinosa-Poblano I, Sa´nchez-Guerrero SA, Crespo-Solı´s E, Cabrera-Va´squez S, Lo´pez-Salmora´n J, Barajas J, Pen˜aloza-Ramı´rez P, Tirado-Ca´rdenas N, Vela´zquez A: Starch and albumin mixture as replacement fluid in therapeutic plasma exchange is safe and effective. J Clin Apher 2008, 23:163-167. 27. Gregoriadis G, Jain S, Papaioannou I, Laing P: Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids. Int J Pharm 2005, 300:125-130.
This work provides first clinical data showing that an albumin-interferona2b fusion protein has an activity similar to a PEGylated derivative. 40. Schulte S: Half-life extension through albumin fusion technologies. Thromb Res 2009, 124:S6-S8. This work provides data on the application of a coagulation factor IXalbumin fusion protein implementing a cleavable linker between albumin and factor IX for release of the coagulation factor upon activation. 41. Metzner HJ, Weimer T, Kronthaler U, Lang W, Schulte S: Genetic fusion to albumin improves the pharmacokinetic properties of factor IX. Thromb Haemost 2009, 102:634-644. 42. Mu¨ller D, Karle A, Meißburger B, Ho¨fig I, Stork R, Kontermann RE: Improved pharmacokinetics of recombinant bispecific antibody molecules by fusion to human serum albumin. J Biol Chem 2007, 282:12650-12660.
28. Constantinou A, Epenetos AA, Hreczuk-Hirst D, Jain S, Wright M, Chester KA, Deonarain MP: Site-specific polysialylation of an antitumor single-chain Fv fragment. Bioconjug Chem 2009, 20:924-931.
43. Yazaki PJ, Kassa T, Cheung CW, Crow DM, Sherman MA, Bading JR, Anderson ALJ, Colcher D, Raubitschek A: Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein. Nucl Med Biol 2008, 35:151-158.
29. Zhang R, Jain S, Rowland M, Hussain N, Agarwal M, Gregoriadis G: Development and testing of solid dose formulations containing polysialic acid insulin conjugate: next generation of long-acting insulin. J Diabetes Sci Technol 2010, 4:532-539.
44. Evans L, Hughes M, Waters J, Cameron J, Dodsworth N, Tooth D, Greenfield A, Sleep D: The production, characterisation and enhanced pharmacokinetics of scFv-albumin fusions expressed in Saccharomyces cerevisiae. Protein Expr Purif 2010, 73:113-124.
30. Tabrizi MA, Tseng CML, Roskos LK: Elimination mechanisms of therapeutic monoclonal antibodies. Drug Discov Today 2006, 11:81-88.
45. Kenanova VE, Olafsen T, Salazar FB, Williams LE, Knowles S, Wu AM: Tuning the serum persistence of human serum albumin domain III: diabody fusion proteins. Protein Eng Des Sel 2010, 23:789-798.
31. Keizer RJ, Huitema ADR, Schellens JHM, Beijnen JH: Clinical pharmacokinetics of therapeutic monoclonal antibodies. Clin Pharmacokinet 2010, 49:493-507.
46. Kratz F: Albumin as drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Res 2008, 132:171-183. 47. Xie D, Yao C, Wang L, Min W, Xu J, Xiao J, Huang M, Chen B, Liu B, Li X, Jiang H: An albumin-conjugated peptide exhibits potent anti-HIV activity and long in vivo half-life. Antimicrob Agents Chemother 2010, 54:191-196.
32. Zalevsky J, Chamberlain AK, Horton HM, Karki S, Leung IWL, Sproule TJ, Lazar GA, Roopenian DC, Desjarlais JR: Enhanced antibody half-life improves in vivo activity. Nat Biotechnol 2010, 28:157-159. This very detailed study shows that the half-life of monoclonal antibodies can be extended by introducing mutations into the Fc region that increase affinity for the FcRn at acidic pH without affecting release at neutral pH. Further, it demonstrates a correlation between affinity for FcRn and halflife.
48. Sørensen AR, Stidsen CE, Ribel U, Nishimiura E, Sturis J, Jonassen I, Bouman SD, Kurtzhals P, Brand CL: Insulin detemir is a fully efficacious, low affinity agonist at the insulin receptor. Diabetes Obes Metab 2010, 12:665-673.
33. Yeung YA, Wu X, Reyes AE, Vernes JM, Lein S, Lowe J, Maia M, Forrest WF, Meng YG, Damico LA et al.: A therapeutic anti-VEGF antibody with increased potency independent of pharmacokinetic half-life. Cancer Res 2010, 70:3269-3277.
49. Dumelin CE, Tru¨ssel S, Buller F, Trachsel E, Bootz F, Zhang Y, Mannocci L, Beck SC, Drumea-Mirancea M, Seeliger MW et al.: A portable albumin binder from a DNA-encoded chemical library. Angew Chem Int Ed 2008, 47:3196-3201.
34. Huang C: Receptor-Fc fusion therapeutics, traps, and MIMETIBODY technology. Curr Opin Biotechnol 2009, 20:692-699.
50. Tru¨ssel S, Dumelin C, Frey K, Villa A, Buller F, Neri D: New strategy for the extension of the serum half-life of antibody fragments. Bioconjug Chem 2009, 20:2286-2292. A small organic chemical is described which is capable of binding to albumin thus allowing its use as half-life extension module after conjugation to a therapeutic molecule as shown for a recombinant antibody molecule.
35. Suzuki T, Watabe AI, Tada M, Kobayashi T, Kanayasu-Toyoda T, Kawanishi T, Yamaguchi T: Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR. J Immunol 2010, 184:1968-1976. A detailed analysis of monoclonal antibodies and Fc-fusion proteins for FcRn established a correlation between affinity for FcRn and half-life and also showed that Fc-fusion proteins exhibit shorter half-lives than IgG molecules. 36. Chaudhury C, Brooks CL, Carter DC, Robinson JM, Anderson CL: Albumin binding to FcRn: distinct from the FcRn-Igg interaction. Biochemistry 2006, 45:4983-4990. 37. Chuang VTG, Kragh-Hansen U, Otagiri M: Pharmaceutical strategies utilizing recombinant human serum albumin. Pharm Res 2002, 19:569-577. 38. Rustgi VK: Albinterferon alf-2b, a novel fusion protein of human albumin and human interferon alf-2b, for chronic hepatitis C. Curr Med Res Opin 2009, 25:991-1002. 39. Nelson DR, Benhamou Y, Chuang WL, Lawitz EJ, Rodriguez Torres M, Flisiak R, Rasenack JW, Kryczka W, Lee CM, Bain VG et al.: Albinterferon alfa-2b was not inferior to PEGylated interferon-a in a randomized trial of patients with chronic hepatitis C virus genotype 2 or 3. Gastroenterology 2010, 139:1267-1276. www.sciencedirect.com
51. Stork R, Mu¨ller D, Kontermann RE: A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G. Protein Eng Des Sel 2007, 20:569-576. 52. Andersen JT, Pehrson R, Tolmachev V, Bekele MD, Abrahmsen L, Ekblad C: Extending half-life by indirect targeting of the neonatal Fc receptor (FcRn) using a minimal albumin binding domain (ABD). J Biol Chem 2010, 286:5234-5241. 53. Hopp J, Hornig N, Zettlitz KA, Schwarz A, Fuss N, Mu¨ller D, Kontermann RE: The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a singlechain diabody-ABD fusion protein. Protein Eng Des Sel 2010, 23:827-834. This study provides evidence that the affinity of an albumin-binding domain for albumin can influence half-life but that even a medium affinity is sufficient to strongly increase half-life as shown for a recombinant antibody molecule. 54. Stork R, Campigna E, Robert B, Mu¨ller D, Kontermann RE: Biodistribution of a bispecific single-chain diabody and its half-life extended derivatives. J Biol Chem 2009, 284:25612-25619. Current Opinion in Biotechnology 2011, 22:868–876
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This study compares the biodistribution of various half-life extended singlechain diabody fusion proteins and demonstrates that fusion of an albuminbinding domain results in superior tumor accumulation compared, for example, to PEGylation or N-glycosylation. It also shows that the long half-life of the ABD fusion protein is in part due to recycling by the FcRn. 55. Roovers RC, Laeremans T, Huang L, De Taeye S, Verkleij AE, Revets H, de Haard HJ, van Berg en Henegouwen PMP: Efficient inhibition of EGFR signalling and of tumour growth by antagonistic anti-EGFR nanobodies. Cancer Immunol Immunother 2007, 56:303-317. 56. Tijink BM, Laeremans T, Budde M, Stigter-van Walsum M, Dreier T, de Haard HJ, Leemans CR, van Dongen GA: Improved tumor targeting of anti-epidermal growth factor receptor nanobodies through albumin binding: taking advantage of
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modular nanobody technology. Mol Cancer Ther 2008, 7:2288-2297. 57. Holt LJ, Basran A, Jones K, Chorlton J, Jespers LS, Brewis ND, Tomlinson IM: Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs. Protein Eng Des Sel 2008, 21:283-288. 58. Walker A, Dunlevy G, Rycroft D, Topley P, Holt LJ, Herbert T, Davies M, Cook F, Holmes S, Jespers L, Herring C: Anti-serum albumin domain antibodies in the development of highly potent, efficacious and long-acting interferon. Protein Eng Des Sel 2010, 23:271-278. This study demonstrates that an albumin-binding single domain antibody is a useful tool to prolong the half-life and increase in vivo efficacy of protein drugs as shown for IFN-a.
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Strategies for extended serum half-life of protein therapeutics Roland E Kontermann With a growing number of protein therapeutics being developed, many of them exhibiting a short plasma half-life, half-life extension strategies find increasing attention by the biotech and pharmaceutical industry. Extension of the half-life can help to reduce the number of applications and to lower doses, thus are beneficial for therapeutic but also economic reasons. Here, a comprehensive overview of currently developed half-life extension strategies is provided including those aiming at increasing the hydrodynamic volume of a protein drug but also those implementing recycling processes mediated by the neonatal Fc receptor. Address Institut fu¨r Zellbiologie und Immunologie, Universita¨t Stuttgart, Allmandring 31, 70569 Stuttgart, Germany Corresponding author: Kontermann, Roland E ([email protected])
Current Opinion in Biotechnology 2011, 22:868–876 This review comes from a themed issue on Pharmaceutical biotechnology Edited by Luis Angel Ferna´ndez and Serge Muyldermans Available online 20th August 2011 0958-1669/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2011.06.012
Introduction In 2010, approximately 200 biologics were approved for therapeutic applications and more than 600 are currently in clinical development [1]. Besides monoclonal antibodies and vaccines, which account for more than 60% of these products, hormones, growth factors, cytokines, coagulation factors, enzymes, fusion proteins and other proteins are developed and used as therapeutics. Many of these protein drugs, with the exception of whole antibodies and Fc-fusion proteins, possess a molecular mass below 50 kDa and a rather short terminal half-life in the range of minutes to hours. However, most of the therapeutic applications of proteins benefit from maintaining a therapeutic effective concentration over a prolonged period of time. Often, this requires infusions or frequent administrations, or the drug is applied loco-regional or subcutaneously utilizing a slow adsorption into the blood stream. These limitations of small size protein drugs has led to the development and implementation of half-life extension strategies to prolong circulation of these recombinant antibodies in the blood and thus to improve Current Opinion in Biotechnology 2011, 22:868–876
administration and pharmacokinetic as well as pharmacodynamic properties.
Half-life extension strategies Several mechanisms are involved in clearance of protein drugs from circulation including peripheral bloodmediated elimination by proteolysis, renal and hepatic elimination, and elimination by receptor-mediated endocytosis [2]. Molecules possessing a small size, that is, a low molecular mass, are rapidly cleared by renal filtration and degradation, with a threshold in the range of 40–50 kDa. Responsible for renal clearance is the glomerular filtration barrier (GBM) formed by the fenestrated endothelium, the glomerular basement membrane and the slit diaphragm located between the podocyte foot processes [3]. While the fenestrae between the glomerular endothelial cells are rather large (50–100 nm) allowing free diffusion of molecules, the slit diaphragm represents the ultimate macromolecular barrier, forming an isoporous, zipper-like filter structure with numerous small, 4–5 nm diameter pores and a lower number of 8–10 nm diameter pores [4,5]. Furthermore, proteoglycans of the endothelial cells and the GBM form an anionic barrier, which partially prevents the traversal of negatively charged plasma macromolecules [3]. Consequently, the size of a protein therapeutic, that is, its hydrodynamic radius, but also its physicochemical properties represent starting points in order to improve half-life (Figure 1). Some plasma proteins such as serum albumin and IgG molecules possess an extraordinary long half-life in the range of 2–4 weeks, which clearly discriminates these molecules from all the other plasma proteins [6]. Responsible is a recycling through the neonatal Fc receptor (FcRn, Brambell receptor) [7]. Albumin and IgGs taken up by cells, for example, endothelial cells, through macropinocytosis will bind to the FcRn in a pH-dependent manner in the acidic environment of the early endosome. This binding diverges albumin and IgG from degradation in the lysosomal compartment and redirects them to the plasma membrane, where they are released back into the blood plasma due to the neutral pH. This offers additional opportunities to extend or modulate the half-life of proteins, for example, through fusion to albumin or the Fc-region of IgG [6] (Figure 1). Finally, protein drugs that bind to a cellular surface receptor will be internalized by receptor-mediated endocytosis and subjected to lysosomal degradation if the protein drug stays bound to the receptor [2,8]. Hence, engineering of the interaction of the therapeutic protein with its receptor(s) at acidic pH can therefore also prolong www.sciencedirect.com
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Figure 1
FcRn FcRn-mediated recycling
mutagenesis
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carbohydrates conjugation modification
increased hydrodynamic radius
recombinant polymer mimetics fusion
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conjugation fusion binding
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Half-life extension strategies including firstly, those that increase the hydrodynamic volume, secondly, those that in addition utilize FcRn-mediated recycling, and thirdly, those that aim at modulating stability of protein-receptor complexes in the sorting endosome.
half-life of the protein by allowing recycling of the unbound molecules into the blood stream as shown for engineered G-CSF and an anti-IL6 receptor antibody [9,10] (Figure 1). In summary, strategies to extend the half-life of a therapeutic molecule can be grouped into firstly, those that increase the hydrodynamic volume, secondly, those that in addition utilize FcRn-mediated recycling, and thirdly, those that aim at modulating stability of protein-receptor complexes in the sorting endosome (Figure 1).
Increasing the hydrodynamic volume The hydrodynamic volume of a protein can be increased by attaching highly flexible, hydrophilic molecules such as polyethylene glycol and carbohydrates. PEGylation, that is, the chemical coupling of polyethylene glycol (PEG), was already developed more than 2 decades ago for half-life extension purposes and meanwhile nine PEGylated protein drugs are approved, including enzymes, interferon-a2b (INF-a2b), granulocyte-colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), and a Fab fragment (Table 1) [11]. In the approved drugs, one or several PEG chains of 5–40 kDa are conjugated. Different coupling methods have been established including random and site-directed approaches, for www.sciencedirect.com
example, to free thiol groups. The PEGylation strategy, that is, the site of PEGylation as well as the number and size of attached PEG chains, has to be carefully chosen in order to avoid a reduction or abrogation of the activity of the therapeutic protein [12]. Ideally, a single PEG chain is conjugated in a site-directed manner, for example, through the use of existing or genetically introduced cysteine residues. This is exemplified by certolizumab pegol (Cimzia), a bacterially produced anti-tumor necrosis factor (TNF) Fab’ fragment with a 40 kDa PEG chain attached to the free cysteine at the C-terminus of the heavy chain Fd chain, that is, opposite the antigen-binding site [13]. Meanwhile, PEGylation has been applied to a large number of other proteins of therapeutic interest [14]. Recently, novel coupling methods for a site-directed conjugation of PEG have been described, including coupling to histidine tags or an existing disulfide bond, with the intension to produce homogenous PEGylated products with increased purity [15]. Disulfide bridge-based PEGylation strategies are developed by PolyTherics and were already utilized to generate PEGylated IFNa, leptin and an anti-CD4 Fab fragment [15]. However, also with this technology a reduction of bioactivity was observed after PEGylation. In general, PEGylation of proteins is considered to be safe and well tolerated [11], although in animals the occurrence of renal tubular Current Opinion in Biotechnology 2011, 22:868–876
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Table 1 Half-life extended protein therapeutics (approved or in clinical trials) Drug
Modification/protein PEGylation Adenosine deaminase L-Asparaginase
Interferon a-2b Interferon a-2b G-CSF Human growth hormone (hGH) Erythropoietin Anti-TNF Fab0 Uricase EPO mimetic peptide Phenylalanine ammonia lyase IL-29 Coagulation factor IX Arginase Hyaluronidase N-glycosylation and polysialylation Erythropoietin Erythropoietin Insulin Albumin fusion Interferon a-2b Coagulation factor IX HER2 + HER3 specific single-chain Fv Albumin binding through a conjugated fatty acid chain Insulin Glucagon-like peptide-1 Fc-fusion proteins TNF receptor 2
LFA-3 CTLA-4 IL-1R TPO-mimetic peptide VEGF receptor BR3 Coagulation factor IX Coagulation factor VIII
Pegademase bovine (Adagen)
Indication
Status Approved 1990
Pegaspargase (Oncaspar) Peginterferon alfa-2b (Peg-Intron) Peginterferon alfa-2b (Pegasys) Pegfilgrastim (Neulasta) Pegvisomant (Somavert)
Severe combined immunodeficiency disease (SCID) Acute lymphoblastic leukemia Hepatitis C Hepatitis C Chemotherapy-induced neutropenia Acromegaly
mPEG-epoetin beta (Mircera) Certolizumab pegol (Cimzia) Pegloticase (Krystexxa) Peginesatide (Hematide) rAvPAL-PEG
Anemia Crohn’s disease Chronic gout Anemia Phenylketonuria
Approved 2007 Approved 2008 Approved 2010 Phase II Phase II
PEG-rIL-29 PEG-rFIX PEG-rArgI PEGPH20
Hepatitis C Hemophilia B Liver cancer Advanced solid tumors
Phase Phase Phase Phase
Darbepoetin alfa (Aranesp) PSA-EPO (ErepoXen) PSA-insulin (SuliXen)
Anemia Anemia Diabetes mellitus
Approved 2008 Phase II Phase I
Albinterferon alfa-2b (Joulferon, Zalbin) rIX-FP MM-111 (scFv-HSA-scFv)
Hepatitis C Hemophila B Cancer
Phase III Phase I Phase I/II
Insulin detemir (Levemir) Liraglutide (Victoza)
Diabetes mellitus Diabetes mellitus type 2
Approved 2003 Approved 2009
Etanercept (Enbrel)
Rheumatoid arthritis, ankylosing spondylitis, polyarticular juvenile idiopathic arthritis, psoriatic arthritis, plaque psoriasis Severe chronic plaque psoriasis Rheumatoid arthritis, juvenile idiopathic arthritis Cryopyrin-associated periodic syndromes Chronic idiopathic thrombocytopenic purpura Macular degeneration Rheumatoid arthritis Hemophilia B Hemophilia A
Approved 1998
Alefacept (Amevive) Abatacept (Orenica) Rilonacept (Arcalyst) Romiplostim (Nplate) Aflibercept (VEGF trap) Briobacept (BR3-Fc) rFIXFc rFVIII-Fc
vacuolization has been observed due to accumulation of the non-degradable PEG chains in the kidney [16].
Recombinant PEG mimetics During the past 2–3 years, alternative methods mimicking the properties of PEGylation have emerged. The idea is to substitute PEG by a hydrophilic and flexible polypeptide chain. The advantages of this approach are that attachment can be achieved by genetic engineering, that is, avoiding any chemical coupling and additional purification steps, and that the attached polypeptide chain is fully biodegradable and of a defined length and composition. Amunix has recently established the XTEN technology based on polypeptide chains of varying length Current Opinion in Biotechnology 2011, 22:868–876
Approved Approved Approved Approved Approved
1994 2000 2002 2002 2002
I I I I
Approved 2003 Approved 2005 Approved 2008 Approved 2008 Phase III Phase II Phase II/III Phase I
composed of alanine, glutamic acid, glycine, proline, serine, and threonine [17]. An increase in solubility and stability of the attached protein and improved manufacturing has been attributed to this technology. Furthermore, animal studies demonstrated that the XTEN sequences are safe and poorly immunogenic [18]. In a recent study, an XTEN sequence was fused to glucagon (Gcg), possessing a half-life of <10 min, for the treatment of nocturnal hypoglycemia [18]. The XTEN sequence was adjusted to maintain efficacious Gcg-XTEN serum levels for approximately 8–10 hours, that is, during the patients sleeping period but not affecting normal insulin usage during the day. A sequence of 288 amino acid residues was found to fulfill these criteria www.sciencedirect.com
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as shown in various animal models, demonstrating the potential of this approach in fine-tuning the pharmacokinetic properties to meet the medical needs [18]. In a similar approach, recombinant PEG mimetics based on the three amino acids proline, alanine and serine were established by the group of Arne Skerra (reviewed in [6]). This so-called PASylation technology is further developed by XL-protein. PAS sequences, which adopt a stable random conformation, are composed of 200 to 600 residues and can be fused N-terminally and/or C-terminally resulting in plasma half-life extension by a factor of 10–100. These PAS sequences do not contain charged residues, thus, do not alter the isoelectric point of the protein. They have also been shown to be non-toxic and to lack T-cell epitopes, that is, being non-immunogenic as shown by animal experiments.
Glycosylation and conjugation of carbohydrates Many plasma proteins are N-glycosylated and/or Oglycosylated and it has been shown that glycosylation can influence half-life [19]. The presence of terminal sialic acids has been identified as important property of N-glycans in order to avoid rapid clearance of proteins containing galactose-terminating glycans through asialoglycoprotein receptor-mediated endocytosis but also of glycoproteins possessing glycans terminating in mannose, N-acetylglucosamine or fucose through lectin-like receptors [19]. The half-life of various protein drugs has been extended by glycoengineering. A prominent example is darbepoetin alfa (Aranesp), a hyperglycosylated form of EPO, which possesses two additional Nglycosylation sites and a 3-fold prolonged half-life in humans [20]. A similar approach was recently applied to generate long-lasting IFN-a derivatives possessing four and five N-glycans resulting in a 25-fold increased elimination half-life [21]. An increased antitumor activity was observed for this hyperglycosylated IFNa derivative in animal experiments, despite a reduced in vitro bioactivity. We have recently applied N-glycosylation to a recombinant bispecific antibody, a single-chain diabody molecule (scDb) with a molecular mass of 55 kDa and a terminal half-life in mice of 5–6 hours [22]. ScDbs are composed of the variable light and heavy chain domains of two different antibodies connected by three flexible linkers [23]. Three, six, or nine N-glycosylation sites (Asn-X-Thr) were introduced into two of the linkers as well as a Cterminal extension of varying length resulting in N-glycosylated molecules with increased hydrodynamic radii. Half-life of the scDb was prolonged leading to an approximately 2–3-fold increase of the area under the curve (AUC), compared with an approximately 10-fold increase by PEGylation or fusion to albumin, demonstrating that www.sciencedirect.com
N-glycosylation results only in a moderate prolongation of half-life [22]. Another approach utilizes the introduction of O-glycosylation sites into protein therapeutics. Such O-glycosylation sites are, for example, present in the C-terminal peptide (CTP) of human chorionic gonadotropin b-subunit. It was shown that fusion of one or several copies of the CTP to EPO or hGH, respectively, resulted in Oglycosylated derivatives with improved in vivo potency and half-life [24,25]. An alternative to the generation of glycosylated proteins by posttranslational modification represents the chemical conjugation of carbohydrates. For example, hydroxyethyl starch (HES), a modified, branched amylopectin, for example isolated from waxy maize starch, was used for half-life extension. HES is an approved plasma volume expander with a proven safety record. Because of its close similarity to glycogen, HES is not immunogenic [26]. The size and structure of HES and thereby its stability can be adjusted by acidic hydrolysis and by chemical hydroxyethylation at positions 2, 3, and 6 of the glucose unit. The HESylation technology was pioneered by Fresenius Biotech to improve the pharmacokinetic and pharmacodynamic properties of therapeutic proteins. Examples include a HESylated derivative of EPO, which was produced by chemical coupling of a 60 kDa HES (reviewed in [6]). Similarly, polysialic acid (PSA) has been used as an alternative to PEG [27]. PSA is found on the surface of a variety of cells including mammalian cells, thus is a biocompatible and biodegradable natural polymer. Colominic acid, a linear polymer of a-(2,8)-linked N-acetylneuraminic acid, was used for polysialylation of various proteins including asparaginase, insulin and antibody fragments and was shown to be capable of prolonging their half-lives [28]. Site-specific polysialylation of an anti-carcinoembryonic antigen (CEA) scFv at a C-terminally exposed cysteine residue increased half-life approximately 5-fold and led to a 30-fold improved tumor uptake as compared with the unmodified scFv [28]. Lipoxen is developing polysialylation as PolyXen technology and has different polysialylated protein drugs, including a long-acting EPO (ErepoXen), insulin (SuliXen), IFN-a2b, and G-CSF, at various preclinical and clinical stages (Table 1) [29].
Fc engineering to prolong half-life of immunoglobulins Naturally, immunoglobulins of the IgG1, IgG2 and IgG4 subclass exhibit long half-lives in the range of 3–4 weeks mediated by recycling through FcRn, although half-life is influenced also by antigen-dependent elimination, for example, through receptor-mediated endocytosis and intracellular degradation [30,31]. The site of interaction Current Opinion in Biotechnology 2011, 22:868–876
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of IgG with FcRn has been localized in the Fc region covering parts of the CH2 and CH3 domain. Mutations were identified which increase the affinity of IgG to FcRn at acidic pH without increasing affinity at neutral pH. Incorporation of such alterations into two therapeutic antibodies (bevacizumab, cetuximab) resulted in an increased half-life and established a direct correlation between affinity and half-life [32]. By mutating methonine 428 to leucine and asparagine 434 to serine (Xtend derivative), leading to a 11-fold increased affinity for FcRn at pH 6 (from 2.46 mM to 0.2 mM), half-life of bevacizumab was prolonged from 9.7 to 31.1 days and that of cetuximab from 2.9 to 13.9 days in cynomolgus monkeys [32]. The prolonged half-life translated into improved antitumor activity in xenograft tumor studies employing transgenic human FcRn/Rag1/ mice. However, a recent study with a modified version of bevacizumab carrying the mutations T307Q/N434A, resulting also in a 10-fold increased affinity for FcRn at acidic pH, indicated that along with FcRn affinity, other factors, such as antibody stability, tumor pH, growth rate, FcRn expression pattern, and specific clearance mechanisms, for example, antigen-dependent clearance, can influence the pharmacokinetics of human and humanized IgGs [33].
Fc-fusion proteins An increasing number of Fc-fusion proteins are developed for therapeutic applications, primarily with the aim to utilize bivalency of the molecules to improve activity but also to improve half-life (Table 1) [34]. Because of the homodimeric nature of these fusion proteins, the molecular mass is strongly increased resulting in a prolonged half-life, which is also influenced by FcRn-mediated recycling. In a recent study, a correlation between affinity for FcRn and half-life was established for various therapeutic antibodies and Fc-fusion proteins but also confirmed that other factors are critical for determining serum half-life [35]. Interestingly, the analyzed Fc-fusion proteins (abatacept, alefacept, and etanercept) exhibited a 2–3-fold lower affinity for FcRn than various human, humanized and chimeric antibodies, which was also reflected by a shorter half-life. It was speculated that the fused receptor domains have an influence on the structural environment of the FcRn-binding site.
Albumin fusion proteins and conjugates Serum albumin is the most abundant protein in the blood plasma, exhibiting also a long half-life, which is 19 days in humans. Responsible for this long half-life is also a pHdependent recycling by the FcRn. The binding site of albumin on FcRn is not overlaying with the IgG binding site, thus the two molecules do not interfere with each other for binding to FcRn [36]. The covalent attachment of albumin to a therapeutic protein is one approach to extend its half-life (Table 1). During the past decade, a large number of different proteins have been genetically Current Opinion in Biotechnology 2011, 22:868–876
fused to human serum albumin (HSA) including interferon alpha, erythropoietin, coagulation factors IX and VIIa, hGH, TNF, and antibody fragments [37]. The most advanced product is albinterferon alfa-2b exhibiting a terminal half-life of approximately 6 days [38]. Results from a phase III clinical trial for the treatment of hepatitis C demonstrated that albinterferon alfa-2b dosed every 2 weeks is not inferior to PEGylated IFN-a, thus providing an alternative efficacious treatment option [39]. Advanced are also studies with an albumin-coagulation factor IX fusion protein [40,41]. Coagulation factor IX has a half-life of 18–34 hours requiring regular infusions every 2–3 days to maintain a factor IX clotting activity of 1%. Factor IX was fused to albumin via a cleavable peptide linker, which is cleaved in parallel to factor IX activation. Clotting activity was 10–30-fold greater than that of a fusion protein generated with a non-cleavable linker and half-life of the fusion protein was improved by a factor of 1.5 to 3.9 in mice, rats and rabbits. This should be useful for a treatment with fewer infusions and lower doses. A phase I trials has been initiated to analyze safety and pharmacokinetics in subjects with hemophilia B (NCT01233440). Proteins can be fused either to the N-terminus or Cterminus of albumin, or even to both ends as shown for single-chain Fv fragments (scFv) [42–44]. Using antibody fragments, this allows for the generation of molecules exhibiting one or more antigen-binding sites. The fusion of antibody fragments is of special interest for applications where Fc-mediated effector functions are not required or even detrimental. For example, Merrimack is developing a bispecific scFv-albumin fusion protein (MM-111) directed against HER2 and HER3. Treatment is based on using HER2 as a marker to specifically target cancer cells and neutralization of HER3-mediated signaling implicated in disease progression. Two clinical phase I and II trials are currently conducted investigating MM-111 alone or in combination with trastuzumab in HER2-positive solid tumors (NCT00911898, NCT 01097460). The FcRn-binding site resides in domain III of albumin and recently it was shown that fusion of this domain to a bivalent anti-CEA diabody leads also to a strongly prolonged half-life, indicating that even a small 22 kDa fragment of albumin is suitable for half-life extension strategies [45]. Covalent linkage of a protein or peptide drug to albumin can also be achieved by chemical coupling. Of special interest for conjugation is a free and accessible cysteine residue at position 34 of albumin allowing for a defined and site-directed conjugation [46]. This approach was utilized to prolong the half-life of a 34 amino acid peptide with anti-HIV activity [47]. This peptide was functionalized with 3-maleimidopropionic acid (MPA), which www.sciencedirect.com
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allows formation of a stable thioether bond between albumin and the peptide. In rhesus monkeys, the albumin-peptide fusion protein exhibited a terminal half-life of 102.4 h, which was 9.4-fold longer than the half-life of the free peptide. A potent neutralizing activity against a number of HIV-1 isolates was observed in vitro and in vivo.
Non-covalent interaction with serum albumin Serum albumin can also be engaged in half-life extension through modules with the capacity to non-covalently interact with albumin. In these approaches, an albumin-binding moiety is either conjugated or genetically fused to the therapeutic protein. A wide range of different moieties have been employed including molecules with intrinsic affinity for albumin but also other molecules such as peptides, antibody fragments, alternative scaffolds, and small chemicals generated and selected to exhibit albumin binding activity (for review see [6]). Albumin acts in the body, amongst other functions, as transport protein for a variety of substances including fatty acids, which bind with low to medium affinity to albumin. The chemical conjugation of a fatty acid chain to prolong the half-life of a therapeutic protein has been utilized for insulin. Insulin detemir (Levemir), which was approved in 2003, lacks ThrB30 and is generated by conjugation of a myristoyl fatty acid chain to LysB29 at the C-terminus of the insulin B-chain [48]. Similar to albumin binding through a conjugated fatty acid, a small organic molecule, 6-(4-(4-iodophenyl)butanamido)hexanoate, which was selected from a DNAencoded chemical library for albumin-binding activity, was employed as portable albumin binder for half-life extension purposes [49]. This compound has been chemically coupled to other small molecules but also to recombinant proteins. Thus, the maleimide-functionalized compound (Albu-tag) was chemically conjugated to an scFv fragment containing an additional C-terminal cysteine [50]. Half-life of this scFv directed against an alternatively spliced EDB domain of fibronectin, an antigen overexpressed by the tumor vasculature, was strongly prolonged from 20–30 min for the unmodified scFv to approximately 40 hours in mice, which translated into an approximately 10-fold increased tumor accumulation in tumor-bearing mice [50]. Proteins with albumin-binding activity are known from certain bacteria. For example, streptococcal protein G contains several small albumin-binding domains (ABD) composed of roughly 50 amino acid residues (6 kDa). Fusion of an ABD to a protein results in a strongly extended half-life as shown for various proteins, for example, antibody fragments and affibody molecules [51,52]. Using bispecific single-chain diabody molecule (scDb), we have shown that the half-life of an scDb-ABD www.sciencedirect.com
fusion protein is similar to that of an scDb-HSA fusion protein and a PEGylated scDb derivative with a 40 kDa branched PEG chain conjugated [22,51]. Further testing of ABD variants with increased or decreased affinity for albumin showed that even a low affinity (around 0.6 mM) is sufficient to strongly extend half-life and that increased affinity only marginally improves half-life of this antibody molecule [53]. Importantly, the prolonged half-life led to a strongly increased accumulation of the anti-CEA x antiCD3 scDb in CEA-positive tumors in animal studies [54]. Compared with a PEGylated scDb derivative an increased accumulation was observed in the antigenpositive tumors indicating a facilitated tissue penetration of the scDb-ABD fusion protein. That recycling by the FcRn is involved in the half-life was confirmed in a comparative study with FcRn wild-type and FcRn heavy chain knockout mice demonstrating also that the bound ABD does not interfere with FcRn binding of albumin [54]. The issue of being immunogenic was recently addressed by generating a deimmunized derivative of the ABD (ABD094), which is used by Affibody to develop their Albumod technology (see article by Fredrik Frejd in this issue). Furthermore, albumin-binding Nanobodies, singledomain antibodies, and alternative scaffolds are employed for half-life extension purposes. Nanobodies, which are based on variable domains (VHH) derived from camelid heavy chain antibodies, were used for prolonging the half-life of other therapeutically useful Nanobodies. For example, a Nanobody specific for mouse albumin (MSA21) was fused to two antagonistic anti-EGFR Nanobodies generating a bispecific, trivalent molecule with a molecular mass of approximately 50 kDa. Binding to albumin increased the half-life from 1 to 44 hours and efficiently delayed outgrowth of EGFR-positive tumors in animal models [55]. Tumor uptake of this trivalent Nanobody construct was similar to anti-EGFR monoclonal antibody cetuximab but the albumin-binding Nanobody construct showed faster and deeper tumor penetration [56]. In a similar approach, Domantis, a subsidiary of GSK, has developed the Albudab technology based on human domain antibodies (dAb). Various albumin-binding VL and VH dAbs were selected from phage libraries and analyzed for their half-life extension potential [57]. Fusion of one of these dAbs (dAbm16), exhibiting an affinity of 70 nM for mouse serum albumin (MSA), to IL-1 receptor antagonist (IL-1ra) prolonged half-life of IL-1ra in mice from 2 min to 4.3 hours, which was also longer than the half-life of an IL-1ra fused to an irrelevant dAb (24 min) [57]. In a collagen-induced arthritis model in DBA/1 mice, mean arthritis scores were significantly lower after treatment with the IL-1radAbm16 fusion protein compared with treatment with IL-1ra. In a further study, another albumin-binding dAb (DOM7 h-14) was fused to IFN-a2b [58]. After fusion to DOM7, in vitro potency of IFN-a2b was reduced Current Opinion in Biotechnology 2011, 22:868–876
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approximately 8-fold in the absence of HSA and 55-fold in the presence of HSA. Also, the affinity of DOM7 h-14 for HSA and MSA was reduced 7.7-fold and 22.3-fold, respectively. Nevertheless, half-life in mice was increased from 1.2 hours for IFN-a2b to 22.6 hours. Using a xenograft tumor model, an improved in vivo efficacy was observed for the IFN-a2b-DOM7 h-14 fusion protein compared with IFN-a2b but also an IFN-a2b-HSA fusion protein [58].
Conclusion Many new strategies to extend the half-life of therapeutic proteins and other molecules have emerged in the recent years. Increasing the hydrodynamic radius of a protein through PEGylation was established more than 20 years ago. New approaches such as genetic fusion of hydrophilic and flexible polypeptide chains are intended to mimic the biochemical properties of PEG, while incorporating further advantages, for example, biodegradability, lack of immunogenicity and facilitated production. Furthermore, a better understanding of the molecular mechanisms underlying the long half-lives of serum albumin and immunoglobulins has led to novel strategies based on fusion or non-covalent binding to albumin, fusion to the Fc region of IgG or improving the interaction of immunoglobulins with the FcRn. Most of these novel half-life extension strategies are still at the preclinical stage. However, it can be expected that in the near future more and more of these technologies will be evaluated in clinical trials and become established and accepted ways to prolong the half-life and thus to improve the pharmacokinetic and pharmacodynamic properties of therapeutic proteins.
Acknowledgment This work was supported by a grant from the Deutsche Forschungsgemeinschaft (KO1461/4).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Walsh G: Biopharmaceutical benchmarks 2010. Nat Biotechnol 2010, 28:917-924.
2.
Tang L, Persky AM, Hochhaus G, Meibohm B: Pharmacokinetic aspects of biotechnology products. J Pharmaceut Sci 2004, 93:2184-2204.
3.
Tryggvason K, Wartiovaara K: How does the kidney filter plasma? Physiology 2005, 20:96-101.
4.
Haraldsson B, So¨rensson J: Why do we not all have proteinuria? An update of our current understanding of the glomerular barrier. New Physiol Sci 2004, 19:7-10.
5.
Wartiovaara J, O¨fverstedt LG, Khoshnoodi J, Zhang J, Ma¨kela¨ E, Sandin S, Ruotsalainen V, Cheng RH, Jalanko H, Skoglund U, Tryggvason K: Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography. J Clin Invest 2004, 114:1475-1483.
6.
Kontermann RE: Strategies to extend plasma half-lives of recombinant antibodies. BioDrugs 2009, 23:93-109.
Current Opinion in Biotechnology 2011, 22:868–876
This review provides a comprehensive summary of various half-life extension strategies. 7.
Roopenian DC, Akilesh S: FcRn the neonatal Fc receptor comes of age. Nat Rev Immunol 2007, 7:715-725.
8.
Lao BJ, Kamei DT: Improving therapeutic properties of protein drugs through alteration of intracellular trafficking pathways. Biotechnol Prog 2008, 24:2-7.
9.
Sarkar CS, Lowenhaupt K, Horan T, Boone TC, Tidor B, Lauffenburger DA: Rational cytokine design for increased lifetime and enhanced potentcy using pH-activated ‘‘histidine switching’’. Nat Biotechnol 2002, 20:908-913.
10. Igawa T, Ishii S, Tachibana T, Maeda A, Higuchi, Shimaoka S, Moriyama C, Watanabe T, Takubo R, Doi Y et al.: Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization. Nat Biotechnol 2010, 28:1203-1208. 11. Veronese FM, Mero A: The impact of PEGylation on biological therapies. BioDrugs 2008, 22:315-329. 12. Fishburn CS: The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics. J Pharm Sci 2008, 97:4167-4183. 13. Melmed GY, Targan SR, Yasothan U, Hanicq D, Kirkpatrick P: Certolizumab pegol. Nat Rec Drug Disov 2008, 7:641-642. 14. Jevsevar S, Junstelj M, Porekar VG: PEGylation of therapeutic proteins. Biotechnol J 2010, 5:113-128. 15. Brocchini S, Godwin A, Balan S, Choi J, Zloh M, Shaunak S: Disulfide bridge based PEGylation of proteins. Adv Drug Deliv Rev 2008, 60:3-12. 16. Gaberc-Porekar V, Zore I, Podobnik B, Menart V: Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr Opin Drug Discov Dev 2008, 11:242-250. 17. Schellenberger V, Wang CW, Geething NC, Spink BJ, Campbell A, To W, Scholle MD, Yin Y, Yao Y, Bogin O et al.: A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol 2009, 27:1186-1190. This work provides first data that fusion of recombinant PEG mimetics result in a drastic prolongation of serum half-life and that half-life can be adjusted by the length of the fused polypeptide chain. 18. Geething NC, To W, Spink BJ, Scholle MD, Wang CW, Yin Y, Yao Y, Schellenberger V, Cleland JL, Stemmer WPC, Silverman J: Gcg-XTEN: an improved glucagon capable of preventing hypoglycemia without increasing baseline blood glucose. PLoS ONE 2010, 5:e10175. This work describes the application of recombinant PEG mimetics to generate a long-acting glucagon and demonstrates that half-life can be fine-tuned and adjusted to the medical needs. 19. Sola´ RJ, Griebenow K: Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs 2010, 24:9-21. 20. Kiss Z, Elliott S, Jedynasty K, Tesar V, Szegedi J: Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact. Eur J Clin Pharmacol 2010, 66:331-340. 21. Ceaglio N, Etcheverrigaray M, Conradt HS, Grammel N, Kratje R, Oggero M: Highly glycosylated human alpha interferon: an insight into a new therapeutic candidate. J Biotechnol 2010, 146:74-83. 22. Stork R, Zettlitz KA, Mu¨ller D, Rether M, Hanisch FG, Kontermann RE: N-glycosylation as novel strategy to improve pharmacokinetic properties of bispecific single-chain diabodies. J Biol Chem 2008, 283:7804-7812. 23. Mu¨ller D, Kontermann RE: Bispecific antibodies for cancer immunotherapy: current perspectives. BioDrugs 2010, 24:89-98. 24. Fares F, Ganem S, Hajouj T, Agai E: Development of a longacting erythropoietin by fusing the carboxy-terminal peptide of human chorionic gonadotropin b-subunit to the coding sequence of human erythropoietin. Endrocrinology 2007, 148:5081-5087. www.sciencedirect.com
Strategies for extended serum Kontermann 875
25. Fares F, Guy R, Bar-Ilan A, Felikman Y, Fima E: Designing a long acting human growth hormone (hGH) by fusing the carboxylterminal peptide of human chorionic gonadotropin b-subunit to the coding sequence of hGH. Endocrinology 2010, 151:4410-4417. This study provides data from in vitro and in vivo studies showing that also O-glycosylation can lead to a prolonged half-life and improved efficacy. 26. Agreda-Va´squez GP, Espinosa-Poblano I, Sa´nchez-Guerrero SA, Crespo-Solı´s E, Cabrera-Va´squez S, Lo´pez-Salmora´n J, Barajas J, Pen˜aloza-Ramı´rez P, Tirado-Ca´rdenas N, Vela´zquez A: Starch and albumin mixture as replacement fluid in therapeutic plasma exchange is safe and effective. J Clin Apher 2008, 23:163-167. 27. Gregoriadis G, Jain S, Papaioannou I, Laing P: Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids. Int J Pharm 2005, 300:125-130.
This work provides first clinical data showing that an albumin-interferona2b fusion protein has an activity similar to a PEGylated derivative. 40. Schulte S: Half-life extension through albumin fusion technologies. Thromb Res 2009, 124:S6-S8. This work provides data on the application of a coagulation factor IXalbumin fusion protein implementing a cleavable linker between albumin and factor IX for release of the coagulation factor upon activation. 41. Metzner HJ, Weimer T, Kronthaler U, Lang W, Schulte S: Genetic fusion to albumin improves the pharmacokinetic properties of factor IX. Thromb Haemost 2009, 102:634-644. 42. Mu¨ller D, Karle A, Meißburger B, Ho¨fig I, Stork R, Kontermann RE: Improved pharmacokinetics of recombinant bispecific antibody molecules by fusion to human serum albumin. J Biol Chem 2007, 282:12650-12660.
28. Constantinou A, Epenetos AA, Hreczuk-Hirst D, Jain S, Wright M, Chester KA, Deonarain MP: Site-specific polysialylation of an antitumor single-chain Fv fragment. Bioconjug Chem 2009, 20:924-931.
43. Yazaki PJ, Kassa T, Cheung CW, Crow DM, Sherman MA, Bading JR, Anderson ALJ, Colcher D, Raubitschek A: Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein. Nucl Med Biol 2008, 35:151-158.
29. Zhang R, Jain S, Rowland M, Hussain N, Agarwal M, Gregoriadis G: Development and testing of solid dose formulations containing polysialic acid insulin conjugate: next generation of long-acting insulin. J Diabetes Sci Technol 2010, 4:532-539.
44. Evans L, Hughes M, Waters J, Cameron J, Dodsworth N, Tooth D, Greenfield A, Sleep D: The production, characterisation and enhanced pharmacokinetics of scFv-albumin fusions expressed in Saccharomyces cerevisiae. Protein Expr Purif 2010, 73:113-124.
30. Tabrizi MA, Tseng CML, Roskos LK: Elimination mechanisms of therapeutic monoclonal antibodies. Drug Discov Today 2006, 11:81-88.
45. Kenanova VE, Olafsen T, Salazar FB, Williams LE, Knowles S, Wu AM: Tuning the serum persistence of human serum albumin domain III: diabody fusion proteins. Protein Eng Des Sel 2010, 23:789-798.
31. Keizer RJ, Huitema ADR, Schellens JHM, Beijnen JH: Clinical pharmacokinetics of therapeutic monoclonal antibodies. Clin Pharmacokinet 2010, 49:493-507.
46. Kratz F: Albumin as drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Res 2008, 132:171-183. 47. Xie D, Yao C, Wang L, Min W, Xu J, Xiao J, Huang M, Chen B, Liu B, Li X, Jiang H: An albumin-conjugated peptide exhibits potent anti-HIV activity and long in vivo half-life. Antimicrob Agents Chemother 2010, 54:191-196.
32. Zalevsky J, Chamberlain AK, Horton HM, Karki S, Leung IWL, Sproule TJ, Lazar GA, Roopenian DC, Desjarlais JR: Enhanced antibody half-life improves in vivo activity. Nat Biotechnol 2010, 28:157-159. This very detailed study shows that the half-life of monoclonal antibodies can be extended by introducing mutations into the Fc region that increase affinity for the FcRn at acidic pH without affecting release at neutral pH. Further, it demonstrates a correlation between affinity for FcRn and halflife.
48. Sørensen AR, Stidsen CE, Ribel U, Nishimiura E, Sturis J, Jonassen I, Bouman SD, Kurtzhals P, Brand CL: Insulin detemir is a fully efficacious, low affinity agonist at the insulin receptor. Diabetes Obes Metab 2010, 12:665-673.
33. Yeung YA, Wu X, Reyes AE, Vernes JM, Lein S, Lowe J, Maia M, Forrest WF, Meng YG, Damico LA et al.: A therapeutic anti-VEGF antibody with increased potency independent of pharmacokinetic half-life. Cancer Res 2010, 70:3269-3277.
49. Dumelin CE, Tru¨ssel S, Buller F, Trachsel E, Bootz F, Zhang Y, Mannocci L, Beck SC, Drumea-Mirancea M, Seeliger MW et al.: A portable albumin binder from a DNA-encoded chemical library. Angew Chem Int Ed 2008, 47:3196-3201.
34. Huang C: Receptor-Fc fusion therapeutics, traps, and MIMETIBODY technology. Curr Opin Biotechnol 2009, 20:692-699.
50. Tru¨ssel S, Dumelin C, Frey K, Villa A, Buller F, Neri D: New strategy for the extension of the serum half-life of antibody fragments. Bioconjug Chem 2009, 20:2286-2292. A small organic chemical is described which is capable of binding to albumin thus allowing its use as half-life extension module after conjugation to a therapeutic molecule as shown for a recombinant antibody molecule.
35. Suzuki T, Watabe AI, Tada M, Kobayashi T, Kanayasu-Toyoda T, Kawanishi T, Yamaguchi T: Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR. J Immunol 2010, 184:1968-1976. A detailed analysis of monoclonal antibodies and Fc-fusion proteins for FcRn established a correlation between affinity for FcRn and half-life and also showed that Fc-fusion proteins exhibit shorter half-lives than IgG molecules. 36. Chaudhury C, Brooks CL, Carter DC, Robinson JM, Anderson CL: Albumin binding to FcRn: distinct from the FcRn-Igg interaction. Biochemistry 2006, 45:4983-4990. 37. Chuang VTG, Kragh-Hansen U, Otagiri M: Pharmaceutical strategies utilizing recombinant human serum albumin. Pharm Res 2002, 19:569-577. 38. Rustgi VK: Albinterferon alf-2b, a novel fusion protein of human albumin and human interferon alf-2b, for chronic hepatitis C. Curr Med Res Opin 2009, 25:991-1002. 39. Nelson DR, Benhamou Y, Chuang WL, Lawitz EJ, Rodriguez Torres M, Flisiak R, Rasenack JW, Kryczka W, Lee CM, Bain VG et al.: Albinterferon alfa-2b was not inferior to PEGylated interferon-a in a randomized trial of patients with chronic hepatitis C virus genotype 2 or 3. Gastroenterology 2010, 139:1267-1276. www.sciencedirect.com
51. Stork R, Mu¨ller D, Kontermann RE: A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G. Protein Eng Des Sel 2007, 20:569-576. 52. Andersen JT, Pehrson R, Tolmachev V, Bekele MD, Abrahmsen L, Ekblad C: Extending half-life by indirect targeting of the neonatal Fc receptor (FcRn) using a minimal albumin binding domain (ABD). J Biol Chem 2010, 286:5234-5241. 53. Hopp J, Hornig N, Zettlitz KA, Schwarz A, Fuss N, Mu¨ller D, Kontermann RE: The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a singlechain diabody-ABD fusion protein. Protein Eng Des Sel 2010, 23:827-834. This study provides evidence that the affinity of an albumin-binding domain for albumin can influence half-life but that even a medium affinity is sufficient to strongly increase half-life as shown for a recombinant antibody molecule. 54. Stork R, Campigna E, Robert B, Mu¨ller D, Kontermann RE: Biodistribution of a bispecific single-chain diabody and its half-life extended derivatives. J Biol Chem 2009, 284:25612-25619. Current Opinion in Biotechnology 2011, 22:868–876
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This study compares the biodistribution of various half-life extended singlechain diabody fusion proteins and demonstrates that fusion of an albuminbinding domain results in superior tumor accumulation compared, for example, to PEGylation or N-glycosylation. It also shows that the long half-life of the ABD fusion protein is in part due to recycling by the FcRn. 55. Roovers RC, Laeremans T, Huang L, De Taeye S, Verkleij AE, Revets H, de Haard HJ, van Berg en Henegouwen PMP: Efficient inhibition of EGFR signalling and of tumour growth by antagonistic anti-EGFR nanobodies. Cancer Immunol Immunother 2007, 56:303-317. 56. Tijink BM, Laeremans T, Budde M, Stigter-van Walsum M, Dreier T, de Haard HJ, Leemans CR, van Dongen GA: Improved tumor targeting of anti-epidermal growth factor receptor nanobodies through albumin binding: taking advantage of
Current Opinion in Biotechnology 2011, 22:868–876
modular nanobody technology. Mol Cancer Ther 2008, 7:2288-2297. 57. Holt LJ, Basran A, Jones K, Chorlton J, Jespers LS, Brewis ND, Tomlinson IM: Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs. Protein Eng Des Sel 2008, 21:283-288. 58. Walker A, Dunlevy G, Rycroft D, Topley P, Holt LJ, Herbert T, Davies M, Cook F, Holmes S, Jespers L, Herring C: Anti-serum albumin domain antibodies in the development of highly potent, efficacious and long-acting interferon. Protein Eng Des Sel 2010, 23:271-278. This study demonstrates that an albumin-binding single domain antibody is a useful tool to prolong the half-life and increase in vivo efficacy of protein drugs as shown for IFN-a.
www.sciencedirect.com