THE STRUCTURE OF BIOLOGICAL THERAPEUTICS

1

THE STRUCTURE OFBIOLOGICAL THERAPEUTICSSheryl Martin-Moe, Tim Osslund, Y. John Wang, TahirMahmood, Rohini Deshpande, and Susan Hershenson

1.1. INTRODUCTION

The first synthetic drug, acetylsalicylate, produced in 1895 and patented as aspirin[1] (Bayer in 1900), marks the beginning of the modern pharmaceutical industry.Throughout the early to mid-1900s there was significant emphasis on developmentof synthetic antibiotics for infectious diseases and small organic molecules, whichcontinues to this day as the mainstay of the traditional (small-molecule) pharmaceuti-cal industry. Advances in understanding the mechanism of reproductive functions andmetabolic diseases, such as diabetes and short stature, led to the discovery of polypep-tide hormones. The early polypeptide hormone drugs were purified from organs, suchas insulin from animal pancreas or growth hormone from cadaver pituitary. Thefirst animal-derived insulin preparation to become commercially available was Iletin,derived from bovine or porcine sources (Eli Lilly in 1923) [2]. Growth hormone,which had to be derived from a human source, was originally produced by specialorder in hospital laboratories and only commercialized much later in the United Statesin 1976 [3]. Although these products were breakthroughs in the treatment of diabetesand dwarfism, there were serious limitations with this type of production, includingavailability of organs and issues with transmission of infectious diseases [4].

Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals,edited by Jameel and HershensonCopyright © 2010 John Wiley & Sons, Inc.

COPYRIG

HTED M

ATERIAL

4 THE STRUCTURE OF BIOLOGICAL THERAPEUTICS

During World War II, Edwin Cohn developed the plasma fractionation processwhereby blood components such as human serum albumin were produced to sup-plement blood loss in wounded soldiers [5]. Polyclonal antibody therapeutics derivedfrom human plasma, such as immunoglobulins, were available as treatment as early asthe 1940s [6]. In 1968 the first of the blood enzymes, antihemophilic factor VIII wascommercialized by Baxter’s Hyland Division [7,8]. Although these products repre-sented life-saving breakthroughs for the treatment of patients with chronic conditionssuch as hemophilia, the production of plasma proteins also suffered from safety andproduction-scale limitations since they, too, were isolated from natural sources [9,10].

Advances in the synthesis of peptides in solution and by solid phase made itpossible to produce peptides on the industrial scale [11]. However, until the adventof recombinant DNA technology, purification from natural or semisynthetic sources,with the attendant limitations of scale and/or concerns about impurities and infectiousagents, remained the only means of producing the larger polypeptide therapeutics,such as insulin and human growth hormone.

The biotechnology industry began formally in 1976 with the founding of thefirst biotechnology company, Genentech. Recognizing the significance of being ableto manipulate genes using the newly discovered tools of restriction endonucleasesand DNA ligases, Stanley Cohen and Herbert Boyer (a co-founder of Genentech)outlined in a series of papers the foundation of modern biotechnology [12]. Recom-binant DNA production systems are integrally related to the structure and functionof the protein products. The initial phase of recombinant production started withexpression and purification in bacterial systems such as Escherichia coil . Althoughsuccessful for many products, there can be serious challenges with refolding someproteins purified from E. coli . In addition, if posttranslational modifications, such asglycosylation, are required for activity, then E. coli is not viable as a host since themachinery for this is not present. Subsequently, methods were developed for cloningand expression of DNA sequences in yeast where refolding was not an issue but post-translation modifications were limited, and in insect cell systems using Baculoviruswhere modifications were closer (but still not identical) to those produced by humancells but where scalability was an issue [13]. It was not until mammalian cell systemssuch as Chinese hamster ovary (CHO) cells were established that recombinant DNAtechnology could produce products closely resembling the full range of human pro-tein structures. Mammalian systems enabled production of larger and more complexprotein therapeutics because of the ability of these cell systems to fold proteins cor-rectly and add posttranslational modifications such as N -linked and O-linked glycansessential for the biological activity and stability of many proteins. In addition, effortswere initiated to improve pharmaceutical or pharmacokinetic properties of proteins.Approaches included modification of the sequence, addition of glycosylation sites,and fusions with domains such as the Fc portion of an antibody or chemical modifi-cations such as addition of poly(ethylene glycol) or lipid. Today there are well overa hundred protein therapeutics on the market in the United States alone, representinga wide variety of structural classes; and the natural human proteins and chemicallymodified derivatives continue to be a fruitful source of new therapeutic products [14].

NATIVE HUMAN PROTEINS AND ANALOGS 5

The first monoclonal antibody therapeutics to be introduced were murine anti-bodies, based on the work of Kohler and Milstein on the continuous expression ofmonoclonal antibodies in mouse hybridoma systems [15]. The first monoclonal anti-body product produced by this technology was muromonab-CD3 (Orthoclone OKT-3by Ortho in 1986), for reversal of kidney transplant rejection [16]. Over the followingdecade methods for increasing the human content of therapeutic antibodies throughproduction of human–murine chimeras, followed by humanized and fully human MAbconstructions were developed. Today fully human antibodies are now commerciallyproduced [17]. Currently monoclonal antibodies constitute the most rapidly growingclass of human therapeutics and the second largest class of drugs after vaccines. Thereare 26 approved monoclonal antibody-based biopharmaceutical products, mainly forthe treatment of cancer and autoimmune diseases, and there are well over a 100 drugcandidates currently under clinical development [18].

Antibodies have served as a natural biomolecular scaffold for various applica-tions. The variable region serves as the antigen-binding site and provides an effectivehumoral response against foreign substances and invading pathogens. A key advan-tage of antibody therapeutics is their high level of specificity for the relevant diseasetargets, which minimizes cross-reactivity and off-target toxicity and can thereby serveto reduce adverse effects compared to other therapeutic approaches. New technologiesfor generating humanized and fully human monoclonal antibody therapeutics are beingdeveloped with the aims of extending the range of targets, increasing the efficiency ofproduction. In addition, the field continues to experiment with modified forms of thebasic monoclonal antibody platform to extend the already impressive performance ofthis dominant class of biological therapeutics [19,20].

The following section introduces some of the major structural classes of recom-binant therapeutic proteins. Consideration has been limited to this class and doesnot include related topics such as synthetically derived peptides, protein vaccines, ordiagnostic reagents. Even for therapeutic proteins, the chapter is by no means com-prehensive, but is rather intended to provide illustrative examples of each major class.In addition some of the recent trends in the development of new classes of therapeuticproteins are briefly considered. Figure 1.1 illustrates a few examples of the structuralrange of current therapeutic protein structures, in comparison to small-molecule drugssuch as aspirin or an antibiotic.

1.2. NATIVE HUMAN PROTEINS AND ANALOGS

1.2.1. Polypeptide Hormones, the First Recombinant Therapeutics

The first wave of the biotechnology industry in the 1980s targeted replacement ofexisting, nonrecombinant therapeutic proteins with high-purity, fully human proteinsproduced using the new recombinant DNA technology. The first human protein to becloned and expressed was a polypeptide hormone, somatostatin, in 1977, based onthe work by Herbert Boyer et al. in laboratories at the University of California, SanFrancisco and the City of Hope [21]. Soon after, the human polypeptide hormonesinsulin [22] and growth hormone, or somatotrophin [23], were cloned and expressed


Aspirin

Penicillin

MAB

Erythropoietin

Insulin

Figure 1.1. Molecular representations comparing the structural complexity of types of

parenteral drugs: aspirin (180 Da), penicillin (334 Da), insulin (5808 Da), erythropoietin

(36,000 Da), and a monoclonal antibody (MAB) (150,000 Da).

by recombinant DNA methods at Genentech for commercial purposes. To understandthe pressing medical need for recombinant sources, consider the cases of insulin andgrowth hormone.

Prior to the production of recombinant insulin, insulin-dependent diabetics weretreated with insulin preparations from either bovine or porcine sources. Human insulinwas made from conversion of porcine insulin using a combination of enzymatic andchemical treatment of the porcine product [24]. The three-dimensional conformationis similar for insulin analoges from human, bovine, or porcine sources, in that theA chain forms two antiparallel α helices, and the B chain forms a single α helixfollowed by a turn and a β strand. The arrangement of the chains buries the disulfidebonds and the aliphatic side chains in the nonpolar core. Insulin has the tendencyto self-associate forming dimer, hexamer, and multimers but is complexed with zincto form the hexamer [25,26]. Although the basic structure of the human, porcine,and bovine insulins is similar, the preparations derived from animal tissues containedmany impurities (proinsulin, arginine insulin, and desamidoinsulin), some of whichelicited immune responses exacerbated by the dosing frequency. Additionally, seem-ingly minor sequence differences between human and bovine insulin, in particular, mayhave elicited antibody responses that reduced biological activity over time, resultingin insulin resistance or altered pharmacokinetic profiles [27].

Recombinant human insulin was licensed to Eli Lilly by Genentech for develop-ment, and was the first recombinant human protein therapeutic to be approved by theUS Food and Drug Administration (FDA) in 1982. The original Genentech productionprocess involved insertion of the nucleotide sequence coding for the insulin A and B


chains into two different E. coli cells that were cultured separately at large scale. Afterpurification, the A and B chains were incubated together under appropriate conditionsto promote interchain disulfide bonds. Eli Lilly improved on this method by insertionof a proinsulin nucleotide sequence into E. coli , allowing a single fermentation andpurification process. After purification there is proteolytic excision of the C peptideto produce human insulin [24,28]. Today there are a number of insulin and insulinanalog products on the market produced by a variety of host cells and processes; forexample, in addition to a number of E. coli -derived products, at least one recombinanthuman insulin is produced in yeast (Novolin by Novo Nordisk, approved in 2005).

Insulin variants have also been designed with specific pharmacokinetic proper-ties. In 1987 the first group to design fast-acting insulin was at Novo Nordisk [29].The FDA eventually approved three analogs of insulin for fast onset: insulin lispro(Humalog by Lilly, in 1996), insulin aspart (NovoLog by Novo Nordisk in 2000), andinsulin glulisine (Apidra by Aventis in 2000). They differ from insulin in Lys-Pro atB28 and B29, Asp at B28, and Lys and Glu at B3 and B29, respectively [16]. Thesemodifications generally disrupt the hexamer association, shifting the equilibrium infavor of the monomer that is absorbed more rapidly [26].

For long-acting insulin, FDA granted approval of insulin glargin (Lantus by Aven-tis in 2000) and insulin detemir (Levemir by Novo Nordick in 2005) [16]. Insulinglargin contains an extra Gly on the A chain and two Arg residues on the B chain.Insulin detemir is novel as B30 is omitted and a fatty acid (myristate) is attached atB29 Thr [30,31] (see Table 1.1 for current insulin products).

In the case of growth hormone, patients (mainly children) had been treatedwith drug derived from the pituitary gland of human cadavers. These patients oftenexperienced loss of response to the therapy, which was also linked to induction ofneutralizing antibodies attributed to the quality of the preparations [32]. These obser-vations created a drive to introduce high-quality, native human protein therapeuticsthat would avoid or minimize these adverse reactions and exposure to infectiousagents [23]. Genentech’s somatrem (Protropin, met-hGH) was approved for treatinggrowth hormone deficiency in children in 1985. The approval process was expeditedby the FDA after a number of deaths caused by virus contamination in pituitarysomatropin [33]. The plasmid used at that time was designed for E. coli cytoplasmicexpression, resulting in a protein, somatrem, with an extra methionine at the N ter-minus of human growth hormone. By designing a plasmid with a signal sequence,it was shown that E. coli could remove the signal sequence during the secretionprocess to produce growth hormone without N -terminal methionine. On the basis ofthis sequence difference, Lilly’s recombinant somatropin, Humatrope, also receivedorphan drug exclusivity and received approval in 1987 [16]. By the end of 2008, therewere several recombinant somatropins on the market (Table 1.1).

Some of the smaller peptide hormones may be produced by either recombinantDNA technology or chemical synthesis. Nesiritide (Natrecor, commonly known as“brain” or B-type natriuretic peptide) has the same 32 amino acid sequence as theendogenous peptide, which is produced by the ventricular myocardium, a single chainwith a disulfide bond between cysteines at 10 and 26. In the late 1990s, Scios made

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12


nesiritide by total peptide synthesis for clinical development, then for cost reasonsswitched to manufacture the peptide using an E. coli expression system. The recom-binant version was approved by the FDA in 2001 for congestive heart failure [16].Together with glucagon (29 amino acids) and PTH (1–34), they are the smallest pro-teins produced by recombinant DNA technology. Salmon calcitonin (32 amino acids)was made either by total chemical synthesis in the Novartis product, or by the rDNAprocess in Unigene’s product. This trend suggests that the cost of recombinant DNAproduction is very competitive, with only small peptides consisting of up to 10 aminoacids produced exclusively by total chemical synthesis. Today there are a wide varietyof peptide hormone products on the market for treatment of diverse conditions (seeTable 1.1 for approved products in the United States).

1.2.2. Cytokines

Cytokines are proteins secreted from a variety of cells, including inflammatory leuko-cytes and some nonleukocyte tissues. Unlike hormones, which are produced in distinctglands and secreted into the bloodstream in order to act over long distances, cytokinesare produced and act locally where they regulate immune functions, inflammation,and hematopoiesis. They are generally produced in low concentrations in the humanbody and bind very tightly to their cognate receptors, often in the picomolar range(as compared to hormones that bind in the nanomolar range). The cytokine ligandoften causes dimerization of the receptor as either a homo- or heterodimer. The bind-ing of the cytokine to its receptor typically causes a phosphorylation event from anintracellular kinase that is either part of the receptor or is closely associated with theintracellular domain of the receptor, triggering a cascade of intracellular reactions [34].

1.2.2.1. Four-Helix Bundle Cytokines. This family of proteins shares a four-helix bundle motif in which the first two helices are oriented in the opposite directionrelative to the second set of helices. This “up–up–down–down” motif requires twolong loops between the first and second helix, a small loop between the second andthird helix, and another long loop between the third and forth helix [35]. This unusualconnectivity between the loops was not apparent when the first cytokine structure ofinterleukin-2 (IL-2) was published [36] but was later corrected [37]. IL-2 (aldesleukinor Proleukin by Chiron in 1992) is used for treatment of kidney (renal) carcinoma [16].

The four-helix bundle cytokines can be subdivided into two subclasses (Fig. 1.2)based on secondary and tertiary structure, those with long α helices and short crossingangles between the helices, such as granulocyte colony-stimulating factor (GCSF); andthose in which the helices are significantly shorter and the crossing angles betweenhelices are relative large, for example, granulocyte macrophage colony-stimulating fac-tor (GMCSF). Often the cytokines with the shorter helices have a small β strand con-necting the long loops between helices A and B and C and D. Both classes have yieldedtherapeutics, including methionyl GCSF or filgrastim (Neupogen by Amgen, in 1991)to treat chemotherapy-induced neutropenia and GMCSF or sargramostim (Leukineoriginally by Immunex, now Berlex in 1991) used postchemotherapy or bone marrowtransplantation to stimulate neutrophil recovery and mobilization of hematopoietic


(a) (b)

Figure 1.2. Ribbon structures of (a) GCSF and (b) GMCSF, illustrating the two major subclasses

of four-helix bundle cytokines. Note the shorter helices and longer crossing angles of GCSF.

See the insert for color representation of this figure.

progenitor cells into the bloodstream [16]. Endogenous human GM-CSF is a glyco-protein consisting of 127 amino acids. Sargramostim differs from that by a substitutionof the amino acid leucine for arginine at position 23 to stabilize the protein within theexpression system (yeast), and contains both N -linked and O-linked oligosaccharidescomposed of mannose and N -acetylglucosamine [38]. A modified version of GCSFhas been developed in which a single polyethylene glycol (PEG) is conjugated at theamino terminus, distal from the helical bundle and the lower half of the A helix, wheremuch of the receptor binding occurs [39]. Pegylation enhances the circulation time,allowing less frequent dosing than unmodified GCSF [40]. Pegfilgrastim has beencommercialized (Neulasta by Amgen in 2002) for treatment of patients undergoingchemotherapy to decrease the incidence of infection by febrile neutropenia [16].

Many members of the cytokine family of proteins are glycosylated in their nativestates, often including both N - and O-linked glycans. During the initial phase of thebiotechnology industry, therapeutic proteins were produced in bacteria, which are inca-pable of performing posttranslational glycosylation. This approach was successful fora number of cytokines, including GCSF, growth hormone, and interferons. However,bacterial production was not successful in all cases. For example, erythropoietin pro-duced in bacteria lacks N -linked carbohydrates and, as a result, was quickly eliminatedfrom circulation. Therefore, it was necessary to develop more complex manufactur-ing processes such as expression in CHO cells; CHO cell expression systems wereused for commercial production of erythropoietins such as epoietin alpha (Epogenby Amgen in 1989 and Procrit manufactured by Amgen for Johnson & Johnson) foranemia associated with kidney failure, generic Zidovudine-treated HIV, and cancerpatients on chemotherapy [16].

Second-generation products have been developed that are indicated for dosingintervals longer than that for recombinant erythropoietin. In one example, two


additional N -glycosylation sites were added to the molecule, resulting in a novelerythropoiesis-stimulating protein, darbepoietin alfa (Aranesp by Amgen in 2001),used for the treatment of chemotherapy-induced anemia [41]. In a second example,pegylation was employed to modify epoietin beta [42] (Mircera by Roche, in 2007),recently approved in the European Union (EU) for the treatment of anemia associatedwith chronic renal failure [16]. Both approaches have led to regulatory approval fortreatment of anemia with less frequent dosing.

The interferons provide additional examples of helical cytokine structures.Although the structure of interferon alpha has proved to be elusive, a homologymodel suggests that it falls in the typical four-helix bundle cytokine structure [43].IFN alpha-2 was the first cytokine to be approved by the FDA in two versions (Aand B), concurrently in June 1986. IFN alfa-2B (Intron A from Schering-Plough in1986 for the treatment of hairy cell leukemia, followed by numerous indications,including genital warts, AIDS-related Kaposi’s sarcoma, hepatitis B and C, malignantmelanoma, and follicular lymphoma) [16] and IFN alfa-2A (Roferon by Hoffman LaRoche in 1986 for hairy cell leukemia, and later hepatitis C and chronic mylegenousleukemia) [16]. PEGINTRON, PEGylated Intron A, was introduced subsequently(Schering-Plough in 2001) [16]. It is chemically conjugated using 12-kD PEG withthe greatest proportion (≈48%) derivatized to the His34 residue, and retains about28% in vitro antiviral activity relative to Intron A. In clinical studies, PEGINTRONdemonstrated a serum half-life of 48–72 h consistent with once-weekly dosing [44].Roferon has also been PEGylated using branched 40-kD PEG at lysine residues(PEGasys by Hoffmann-La Roche, in 2002). Plasma half-life was increased from3–8 h to 65 hours, and Tmax was prolonged from 10 to 80 h. Although it retains only7% of the in vitro antiviral activity, it is efficacious by weekly dosing [45].

The structure of interferon beta has been determined to be a helical bundle topol-ogy similar to other cytokines, but with five alpha helices in its bundle [46] instead offour. Interferon beta-1b, which is bacterially produced and therefore nonglycosylated,was approved for treatment of patients with multiple sclerosis (Betaseron by Chiron in1993). Cysteine 17 is mutated to serine to avoid disulfide scrambling [16]. Interferonbeta-1a (Avonex by Biogen, in 1996) is a 166–amino acid glycoprotein made in CHOcells, where it is also glycosylated. The impact of the glycosylation can be seen in therecommended doses and administration schedule for the two products, which are quitedifferent in that Betaseron is administered at 0.25 mg subcutaneously every other day,whereas Avonex is given 0.03 mg intramuscularly once a week [16].

Some of the more diverse structures in the cytokine family include interferongamma (Actimmune by Intermune in 1999), used for chronic granulomatous dis-ease and delaying progression of severe malignant osteoperosis, and stem cell factor(Ancestim by Amgen, currently licensed to Biovitrum AB) [16]. The core structureof IFN gamma contains six alpha helices that self-associate to form a homodimer inits active state [47]. The native factor contains a transmembrane domain in additionto the four-helix bundle core motif that accounts for the biological activity. It alsoself associates to form a noncovalent dimer with a head-to-head orientation betweenthe 2 four-helix bundles. Ancestim (Stemgen by Amgen, available in New Zealand),consists of the extracellular domain only [48].


Figure 1.3. Superimposition of the ribbon structures of FGF-1 (green), FGF-2 (blue), and

FGF-7/KGF (red). The β trefold is a common structural motif for the FGF family of proteins. See

the insert for color representation of this figure.

1.2.2.2. Fibroblast Growth Factor (FGF) Family of Cytokines. In contrastto the four-helix bundle cytokines, this family of proteins is structurally composed of β-trefoil architecture. Twelve antiparallel β strands are folded into three β–β–β (trefoil)units (Fig. 1.3). Twenty-three members of this family have been identified on the basisof sequence homology and structure. Although there is only 40% sequence homol-ogy between FGF1 and FGF7/KGF (keratinocyte growth factor), their conformationalstructures are almost identical [49]. The most significant difference between the twostructures is the receptor-binding domain, which is slightly larger in FGF7. Althoughboth FGF1 and FGF2 advanced to clinical trials, the only member of the FGF familyto be approved in the United States is KGF (Palifermin by Amgen in 2004, currentlylicensed to Biovitrum AB), used to alleviate mucositis, a common complication ofhigh-dose chemotherapy and radiation treatments associated with bone marrow trans-plant [16]. [FGF2 (Fiblast by Kaken Seiyaku) was approved in Japan in 2001 for use inwound healing and is currently (as of 2009) in clinical trials in the United States [50].]Anakinra, an interleukin-1 receptor antagonist (IL-1RA), adopts a similar β-trefoil fold[51], although not related to the FGF family of proteins in sequence, and has beenapproved for treatment of rheumatoid arthritis (Kineret by Amgen in 2001) [16].

1.2.2.3. Cystine Knot Cytokines. Resolution of the structures of theneurotrophic factors, nerve growth factor (NGF), brain-derived neurotrophic factor(BDNF), and neurotrophin 3 (NT3), revealed a novel protein topology in which twomolecules form a homodimeric, biologically active protein [52]. One unusual aspectof this characteristic fold is the position of six cysteines, which was eventually termedthe “cystine knot” motif. In this motif two disulfide bonds form an amino acid ringin the peptide backbone, which is penetrated by a third disulfide bond (Fig. 1.4). Thecystine knot motif is also shared by human transforming growth factor-β2 (TGFβ),vascular endothelial growth factor (VEGF), and a variety of sequence-unrelatedbut structurally similar proteins [53]. Although many of the original therapeutic


Figure 1.4. Ribbon structure of BDNF, representing the structure of the family of proteins

termed cystine knot cytokines. See the insert for color representation of this figure.

candidates in this structural class have not been commercialized, a few have made itto the market. Platelet-derived growth factor (Regranex or becaplermin by OMJ in1997) in carboxymethyl cellulose topical gel was approved for treatment of diabeticfoot ulcers [16]. A second use of PDGF is in a calcium phosphate matrix used forthe treatment of periodontal bone defects, (GEM 21S by Biomimetics in 2005). Othercases of growth factors incorporated into a matrix are bone morphogenic protein-7(BMP-7) (OP-1 Putty by Stryker Biotech in 2001) as osteoinductive and conductivebone graft material, and BMP in an absorbable collagen sponge (dibotermin-α asINFUSE® bone graft device by Wyeth in 2004), which is indicated for spinal fusionprocedures in skeletally mature patients with degenerative disk disease (all arementioned in Ref. 54).

1.2.3. Enzymes and Blood Factors

This is a highly varied class of proteins, comprising a number of diverse structures. Aswith insulin and human growth hormone, a number of products in this category hadprerecombinant antecedents. These include serum-derived versions of albumin, bloodfactors such as factor VIII, RhoGam [Rh(rhesus) factor], Gamimune, and immuneglobulins. The procurement of a safe blood supply has been difficult until relativelyrecently, and transmission of hepatitis and HIV viruses has occurred, resulting in infec-tion of patients using blood-derived products. In the past, over 60% of hemophilicpatients were likely to be accidentally infected [55]. Today the production of these


factors from human plasma is significantly safer with the addition of advanced meth-ods for blood screening for infectious agents and addition of viral inactivation stepsto the production process [6]. Although many blood-derived products now exist asrecombinants, even today not all members of the very diverse and complex categoryare available as recombinant products.

Recombinant antihemophilic factor (recombinant factor VIII) is, at 2332 aminoacids and 280 kDa, the largest commercial protein made by recombinant technology. Inits native form it is a glycoprotein with a domain structure of A1–A2–B–A3–C1–C2,in which the heavy chain is composed of A1–A2–B domains and the light chaincomprises the remaining domains [56]. There are 25 Asn glycosylation sites [57], sixtyrosine sulfation sites [58], eight disulfide bonds, and three free cysteines [59], andthe presence of a metal ion [60]. Recombinant factor VIII (rFVIII) was first cloned byGenentech, licensed to Bayer, and approved by the FDA (Kogenate in 1992). SeveralrFVIII products are currently on the market, including Kogenate FS (Bayer); ReFacto,an engineered version of factor VIII with a deleted B domain, resulting in a moleculethat is 110 kDa, smaller than the full-length protein (Genetics Institute/Wyeth). Xyntha,an albumin-free formulation of ReFacto, and Advate (Baxter) are serum-free factorVIII products [16].

In 1987 Genentech received approval for Alteplase, a recombinant tissue plas-minogen activator (r-tPA) to dissolve blood clots in patients with acute myocardialinfarction. tPA is a multidomain serine protease that converts plasminogen to plasmin,a required step in blood coagulation [61,62]. Alteplase is a single-polypeptide chaincomposed of 527 amino acids held together by 17 disulfide bridges with four poten-tial sites for N -linked glycosylation. By homology with other proteins, alteplase isdivided into five domains. A domain homologous to fibronectin type I finger extendsfrom residues 1 to 43; residues 44–91 are homologous to human epidermal growthfactor; the kringle 1 (92–173) and kringle 2 (180–261) regions are homologous tothe kringle regions found in plaminogen and prothrombin; the C -terminal region ofalteplase, comprising residues 276–527, is homologous to the trypsin family of ser-ine proteases and contains a catalytic active site formed by His [322], Asp [371],and Ser [478] [63]. Retavase (Reteplase, by Boehringer Mannheim in 1996, also fordissolving blood clots) is a nonglycosylated deletion mutein of tissue plasminogenactivator (tPA), containing the kringle 2 and the protease domains of human tPA.Retavase contains 355 of the 527 amino acids of native tPA (amino acids 1–3 and176–527) which allows production in E. coli . It has a longer half-life than does itsparent molecule, tPA, which is attributed in part to lack of glycosylation and resul-tant inability to be cleared by the mannose receptor pathway [16,64]. Tenecteplase(TNKase by Genentech in 2000) is another r-tPA variant. TNKase is approved fortreatment of acute myocardial infarction. It contains a number of amino acid changes(Asn for Thr [102], Glu for Asn [116], and tetraalanine for amino acids 296–299)compared to alteplase, which results in enhanced fibrin selectivity [16].

Following the observation that it was effective in reducing the viscosity of humansputum, DNase was developed as a therapeutic to reduce the concentration of freeDNA in the lungs of patients with cystic fibrosis. The original product was isolatedfrom bovine sources; however, this was replaced by the recombinant form dornase alfa,


(Pulmozyme by Genentech in 1993). Dornase alfa is a single-polypeptide chain of 260amino acids with two N -linked glycosylation sites. It is classified as an αβ proteinwith 2 six-stranded β-pleated sheets packed against each other into a hydrophobiccore [16]. The α helices and extensive loop regions flank the two antiparallel β sheets.The conformation and stability are both highly dependent on the binding of calcium.Pulmozyme is delivered by inhalation using a jet nebulizer [65].

Lysosome storage disorders are genetic diseases caused by a deficiency in enzymeactivity, and patients have benefited from enzyme replacement therapy. The first prod-ucts were modified placental proteins that have been largely replaced by recombinantproteins. Gaucher’s disease is an example of an autosomal recessive lipid storagedisorder caused by the deficiency of the lysosomal enzyme β-glucocerebrosidase. Themannose terminated recombinant product is imiglucerase (Cerezyme, by Genzymein 1994) and is produced by recombinant DNA technology using CHO cells. Puri-fied imiglucerase is a monomeric glycoprotein of 497 amino acids, containing fourN -linked glycosylation sites and has a molecular weight of 60,430 Da. Imiglucerasediffers from placental glucocerebrosidase by one amino acid at position 495, where his-tidine is substituted for arginine. Somewhat different from the carbohydrate structuresof native placental glucocerebrosidase, the oligosaccharide chains have been modifiedto terminate in mannose sugars. These mannose-terminated oligosaccharide chains arespecifically recognized by endocytic carbohydrate receptors on macrophages, the cellsthat accumulate lipid in Gaucher’s disease. This recognition facilitates the enzyme toenter these cells more efficiently to cleave the intracellular lipid that has accumulatedin pathological amounts [16]. This example illustrates molecular modifications thatmay be introduced to influence biodistribution, thereby enhancing activity [66].

The foregoing examples serve to illustrate a fraction of the highly diverse cat-egory of enzymes and blood factors that are currently produced using recombinantDNA technology, including factors that were originally commercialized from humanplasma sources and later converted to recombinant DNA production and others whoseproduction was initially enabled by the advent of recombinant DNA technology. Thelatest breakthrough in this field occurred in February 2009, when the FDA approvedrecombinant human antithrombin expressed in genetically engineered goats (ATrynby GTC Biotherapeutics) for the prevention of perioperative and peripartum throm-boembolic events, in hereditary antithrombin deficient patients [16]. (For a completelisting of enzymes and blood factors approved in the United States, see Table 1.1.)At the same time, there are a number of blood enzymes and factors that continue tobe isolated from human serum, presumably due to the complexity of producing theseproteins using recombinant technology combined with substantial improvements inthe safety of the blood supply [67], such as albumin (by Octapharma in 2006) used torestore blood volume, alpha-1 proteinase inhibitor (Aralast by Baxter in 2002, Zemairaby Aventis Behring in 2003) for genetic emphysema, antihemophilic factor/von Wille-brand factor complex (Alphanate, by Grifols Biologics in 2007 and Humate-P by CSLBehring in 2007), for hemophilia, C1 esterase inhibitor (Cinryze by Lev Pharmaceuti-cals in 2008) for use in hereditary angioedema attacks, immune globulin (by numerous


companies) for immune deficient diseases, thrombin (Evithrom by Omrix Biopharma-ceuticals in 2007) for an aid in hemostasis, and protein C (Ceprotin by Baxter in2007) for venous thrombosis and purpura fulminans [16].

1.2.4. Fusion Proteins

In the early 1980s the preferred approach for most biotechnology companies was tomake recombinant proteins that were structurally as close as possible to the naturallyoccurring human form, with the expectation that this would minimize antigenicity.Almost from the beginning, however, the need to overcome stability, pharmacokinetic,or other limitations of the native proteins led to the introduction of various strategiesto engineer protein therapeutics to improve their pharmaceutical properties (see earlierdiscussion). Fusion proteins represent a further evolution in the technology to improvepharmacokinetics. One of the initial purposes for developing fusion protein productswas to join two different activities in a single molecule in the hope that the combinedfunctionality would have a synergistic effect. A more widespread application hasbeen to fuse an active binding protein or domain with the Fc domain of an IgG(see Table 1.1, p. 12). The binding domain confers the desired activity, while theFc domain serves to increase overall molecular size and prolong circulation time.The complete molecule can be expressed recombinantly, which eliminates the needfor a chemical modification step such as pegylation. A well-studied fusion protein isetanercept (Enbrel by Immunex/Amgen in 1998 for moderate to severe rheumatoidarthritis, 2002 for psoriatic arthritis, 2003 for ankylosing spondylitis, and 2004 formoderate to severe plaque psoriasis). The fusion protein is bivalent, having two TNFα

receptors fused to the Fc region of an IgG1 antibody (see Fig. 1.5). It functions bybinding to tumor necrosis factor alpha (TNFα) to interrupt the immunological cascadethat triggers rheumatoid arthritis in many patients [16,68].

Peptibodies may be considered a class of Fc fusion proteins in which a bio-logically active, nonnative peptide is fused with the Fc portion of an antibody (seenext section for discussion of Fc properties). The fusion can take place at either theamino or carboxyl terminus or even, in some cases, within the Fc domain. Pepti-bodies can be expressed and refolded from bacteria, whereas the more complex Fcfusion proteins generally require mammalian cell expression systems. The first pep-tibody to be approved by was romiplostim (Nplate by Amgen in 2008) [16,69,70].The molecule was designed to target chronic immune thrombocytopenic purpura andhas been demonstrated to be an effective thrombopoiesis-stimulating protein. It iscomposed of a disulfide-bonded human IgG1 heavy-chain and κ light-chain constantregions with two peptide sequences fused at residue 228 of the heavy chain with apolyglycine linker between the peptide and FC fragment.

A unique fusion protein is denileukin diftitox (ONTAK by Seragen in 1999),approved for the treatment of patients with persistent or recurrent cutaneous T-celllymphoma whose malignant cells express the CD25 component of the IL-2 receptor.Denileukin diftitox is a genetically engineered cytotoxic fusion protein consisting ofthe amino acid sequences for the enzymatically active portion of diphtheria toxin fusedto the sequence of human interleukin-2, resulting in a molecule that is cytotoxic forcells bearing the target IL-2 receptor [16].

ANTIBODIES 21

TNF Receptor Domain

TNF Receptor Domain

Figure 1.5. Structure of entanercept, a fusion protein with two tumor necrosis factor

receptors fused to the Fc of an IgG1.

1.3. ANTIBODIES

Antibodies or immunoglobulin proteins are produced in response to non-self antigensand are a key mechanism for defence against pathogenic organisms and toxins. Thedistinguishing characteristics of antibodies are the enormous diversity and specificityof antigens that can be recognized. The antibody protein consists of two light (∼25-kDa) and two heavy (55–65-kDa) chains that adopt β-pleated sheet structuresarranged in a symmetric fashion producing an overall Y-shaped quaternary structure.There are five classes of antibodies, IgG, IgM, IgD, IgA, and IgE, which are definedby their particular heavy chains. The main serum antibody, IgG, is the antibodyclass used almost exclusively in therapeutic antibodies (Fig. 1.6). The two heavychains are joined at the hinge region by disulfide bonds, and the two light chainsare each linked to a heavy chain by a disulfide bond such that the intact moleculeis about 160 kDa. Each chain has a variable (V) domain at the amino terminuscontaining about 110 residues that is involved in antigen binding and a constant(C) domain at the carboxyl terminus that determines the subclass or isotype. Somemouse and human antibodies are glycosylated at a conserved site in the CH2 domain(Asn297) with an N -linked fucosyl biantennary complex that can be sialylated. O-Linked oligosaccharides have been reported mainly in the hinge region and possiblythe Fc region [71]. Glycans can affect binding to antigens and are important forinteractions with other components of the immune system, including complement andFcγ receptors on cytotoxic T lymphocytes.

There are two major variations within the light chain that are designated kappaand lambda . Most antibodies developed for therapeutic use have the kappa version


Figure 1.6. Examples of therapeutic antibody constructs. Cartoon structural representations

of an intact human IgG1 monoclonal antibody [72] and F(ab′)2 and Fab fragments derived

from it, an scFv [72], and an scFv dimer diabody [74]. The heavy chains are shown in magenta

and green and light chains, in yellow and wheat. The carbohydrate component of the whole

IgG1 molecule is shown as a stick representation and the cystiene residues linking the heavy

and light chains are shown in red. The images were produced from PDB files using Pymol

(DeLano Scientific, LLC). See the insert for color representation of this figure.

of the light chain; this bias is also found in the structural database, where there arealmost 20 times as many kappa structures than lambda. Differences in amino acidsequence between the two light chains lead to specific variations in domain packing,thermodynamics, and structure [78]. The insertion of a single amino acid at the elbowbetween the variable domain and the constant domain within the light chain of thelambda variation may introduce more flexibility for the overall domain allowing theelbow angle to be significantly larger than the kappa version of the light chain [76].

There are four subclasses of IgG in humans: IgG1, IgG2, IgG3, and IgG4. From asequence and structural perspective, the major difference between the IgG subclassesresides in the length and flexibility of the hinge region. This region links domainsof β-pleated sheets and can be cleaved by proteases to produce large fragments thatdelineate specific functions. The portion of the constant region between the C terminusand the hinge is the Fc (crystallizable) fragment, and the remaining Fab fragmentcontains the antibody-combining (i.e., antigen-binding) site. Most of the approvedtherapeutic human(ized) monoclonal Abs are the IgG1 kappa subclass [71]. The IgG1

ANTIBODIES 23

subtype has a hinge region 15 amino acids in length, with two interchain disulfidebridges. This configuration allows the Fab region to rotate freely on the tetheringregion of the first disulfide bond. In contrast, the IgG2 hinge region is 12 aminoacids in length, with a polyproline helix and four disulfide bonds. This more rigidconfiguration restricts the flexibility of the Fab [80]. Subclass IgG3 has an extendedhinge region of 62 amino acids with up to 11 disulfide bonds. Although this results ina larger molecule [78], it is more susceptible to proteases, resulting in a shorter half-life [79–81]. The hinge region of the IgG4 is three amino acids shorter than the IgG1hinge and contains a proline substitution for serine, which allows for an intrachaindisulfide bond. The intrachain disulfide bond changes the position of the Fab relativeto the Fc allowing direct contact of the CH1 domain with the CH2 domain, resultingin a more compact structure relative to the other IgG molecules. In addition, althoughIgG4 antibodies are expressed as bivalent molecules similar to the other IgGs, IgG4antibodies have a propensity to swap light and heavy chains [82], creating bispecificstructures and half molecules [83].

The human subclasses of IgGs also differ in effector functions, mediated by theirFc domains. The Fc domain recruits cytotoxic effector functions through comple-ment, which is referred to as complement-dependent cytotoxicity (CDC) and/or throughinteractions with Fc-gamma receptors (on gamma globulins) which is referred to asantibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) [84]. Complement-dependent cytotoxicity results frominteraction of cell-bound antibodies with proteins of the complement cascade systemstarting with binding of complement protein C1q to the Fc domain. This causes aconformational change in C1q that initiates an enzymatic cascade of complement pro-teins that spreads rapidly and ends in formation of the membrane attack complex thatcauses lysis of the target [85]. ADCC and ADCP are governed by engagement of theFc region with a family of Fcγ receptors (FcγRs) [86]. In humans, this protein familycomprises FcγRI (CD64); FcγRII (CD32), including isoforms FcγRIIa, FcγRIIb, andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa and FcγRIIIb. FcγRs areexpressed on a variety of immune cells, and formation of the Fc/FcγR complex recruitsthese cells to sites of bound antigen, typically resulting in signaling and subsequentimmune responses such as release of inflammation mediators, B-cell activation, endo-cytosis, phagocytosis, and cytotoxic attack of the target cell. All FcγRs bind IgG Fc,yet with differing high (FcγRI) and low (FcγRII and FcγRIII) affinities [87]. Further-more, whereas FcγRI, FcγRIIa/c, and FcγRIIIa are activating receptors characterizedby an intracellular immunoreceptor tyrosine-based activation motif (ITAM), FcγRIIbhas an inhibition motif (ITIM) and is therefore inhibitory. Engineered Fc variantshave been created with improved FcγR affinity and specificity that provide significantenhancements in cytotoxicity [88,89].

Broadly, IgG1 and IgG3 are more competent at inducing cytotoxicity than areIgG2 and IgG4 [18,90]. If the mechanism of drug action requires a cytotoxic event,then IgG1 may be the most desirable subclass with the best half-life. If effector func-tion is not needed or is considered undesirable then IgG2 or IgG4 may be preferred.For example, denosumab, recently filed for postmenopausal osteoporosis and patients


undergoing hormone ablation for prostate or breast cancer, is an IgG2, which mayprovide a safety advantage (Amgen, filed in 2008) [91–93].

The first monoclonal antibodies were murine antibodies obtained by hybridomatechnology. The half-life of murine MAbs is typically relatively short, likely becauseof the inability of the MAbs to interact with the human neonatal Fc receptor [90].This property of murine MAbs is used to advantage for diagnostics or therapeutics,where it is desirable to clear the MAb quickly after the procedure. For example,two radioisotope-conjugated anti-CD20 MAb preparations, ibritumomab tiuxetan-111Inor- 90Y, (Zevalin by IDEC in 2002) and tositumomab- 131I, (Bexxar by SmithKlineBeecham in 2003), both for treatment of non-Hodgkin’s lymphoma, utilize murineMAbs that are cleared relatively rapidly [16]. There are relatively few commercialmurine MAb therapeutics, most likely owing to the potential for human anti-mouseimmunogenic responses [94].

Several years after the introduction of murine MAbs, a new class of therapeu-tic MAbs consisting of mouse/human chimeric antibodies received FDA approval.Chimeric MAbs generally combine the variable regions of a mouse antibody withthe effector or constant regions of a human antibody, resulting in antibodies that areapproximately 65% human in sequence. Chimeric antibodies generally exhibit reducedimmunogenicity and increased plasma half-life compared with those of murine Mabs[90]. Examples of chimeric MAb products include rituximab that targets CD20 (Rit-uxan by IDEC and Genentech in 1997) for the treatment of patients with B-cellnon-Hodgkin’s lymphoma, and infliximab, which targets tumor necrosis factor alpha(TNFα) (Remicade by Centocor in 1998 for Crohn’s disease and in 1999 for rheuma-toid arthritis) [16].

The next step in increasing the human component of MAb therapeutics was to gen-erate humanized MAbs. These antibodies generally combine the hypervariable regionsof a mouse (or rat) antibody within the framework of a human antibody molecule.Most of the MAbs on the market today are in this class. Examples include daclizumab,which targets CD25 (Zenapax by Hoffman-La Roche in 1997) [16] for prophylaxisof acute organ rejection in patients receiving renal transplants; anti-HER2/neu anti-body, trastuzumab (Herceptin by Genentech in 1998), approved for treatment ofmetastatic breast cancer in HER2-positive patients [16]; and bevacizumab, which tar-gets VEGF (Avastin by Genentech, in 2004 for treatment of colorectal cancer, in 2006for advanced lung cancer, and in 2008 for metastatic breast cancer) [16].

Fully human MAbs can now be produced by replacing murine immunoglobulingenes with human immunoglobulin genes, leading to the production of fully humanmonoclonal antibodies [95]. Examples of fully human MAb products include adali-mumab (Humira, by Abbott in 2002), a fully human anti-TNF MAb for treatment ofrheumatoid arthritis and subsequently for psoriastic arthritis in 2005, Crohn’s diseasein 2007, and plaque psoriasis in 2008; and panitumumab (Vectibix by Amgen in 2006)for treatment of colorectal cancer [16].

As with other classes of protein therapeutics, immunogenicity continues to be asafety consideration for antibodies, although the advent of humanized and fully humanMAb constructs has led to a reduction in the general level of risk and facilitated theirapplication to an increasingly wide range of therapeutic indications [90,96]. Today,

ANTIBODIES 25

monoclonal antibodies constitute by far the largest and fastest growing category ofbiological therapeutics, with 26 currently approved in the United States alone (seeTable 1.2) and hundreds in various stages of clinical and preclinical development[18]. The majority of the current MAb products are approved for cancer therapy[18]; however, as experience with this class of therapeutics increases, the range ofindications is expanding rapidly.

1.3.1. Antibody Conjugates

Attempts to produce antibody conjugates carrying a toxin can be traced to the 1970s[97]. Early attempts included conjugation of diphtheria toxin (a protein) to an anti-body, followed a few years later by conjugates of daunomycin and adriamycin, smallmolecules that were covalently bound to antibodies [98]. Interest was driven by thepotential advantages of conjugates, including reduced toxicity due to better localiza-tion of the toxin as well as, in some cases, greater efficacy based on a combination ofeffects from both the targeting moiety and the toxin. Early drug candidates failed for anumber of reasons including insufficient information regarding tumor antigens, murineantibody prompted immune responses, short biological half-life of murine antibodies,and issues with linker chemistries. For conjugates containing protein toxins, inefficientinternalization of the toxin posed another hurdle [99].

Despite the considerable challenges and early low success rate, interest in thisformat has persisted, based on its obvious potential advantages, and several antibodyconjugates have been approved by the FDA. These include conjugates with isotopessuch as Bexxar and Zevalin [100], and the only toxin conjugate, gemtuzumab ozogam-icin (Mylotarg by Wyeth in 2000), approved for the treatment of elderly patientssuffering from CD33-positive acute myeloid leukemia [16,100]. A humanized IgG4kappa MAb was selected for this application because the effector functions are notneeded, and the IgG4 subclass reportedly has reduced nonspecific binding to nor-mal tissues [101]. The anti-CD33 antibody is conjugated with a cytotoxic antitumorantibiotic, ozogamycin (N -acetyl-γ-calicheamicin). The drug load is about four tosix molecules of ozogamycin per antibody molecule, with approximately 50% of theantibody conjugated and the remaining 50% unconjugated. The linker contains twocleavable bonds (disulfide and hydrazone) [102].

Today most conjugates are based on humanized or human MAbs, and newlinker chemistries have also been developed. A comprehensive review article onantibody–drug conjugates has recently been published [103]. Advances in theunderstanding of cancer biology have led to selection of improved targeting agentsand a wider range of toxins; and a number of antibody conjugates are currently invarious stages of clinical testing and preclinical development [99].

1.3.2. Fab

For some applications, the Fc-mediated effector functions and prolonged serumcirculation times are not required or may even be detrimental to the desired activ-ity of the therapeutic. Eliminating the Fc domain to create F(ab′)2 results in a molecule

TAB

LE1.

2.Re

com

bina

ntAn

tibod

yPa

rent

eral

Prod

ucts

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oved

Non

prop

riet

ary

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deIg

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alf-

Lif

e,In

dica

tions

,A

dmin

istr

atio

nM

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rer

and

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eaN

ame

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days

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te,b

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eA

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val

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r

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ine

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omab

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ersa

lof

kidn

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lant

reje

ctio

n,IV

bolu

s5

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day

for≤

2w

eeks

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hoB

iote

ch;

1986

(US)

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tum

omab

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uxet

an(90

Y)

Zev

alin

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Non

-Hod

gkin

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mph

oma,

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2m

gID

EC

(US)

2002

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ngA

G(E

U)

2004

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tum

omab

(IgG

,Ig

G-13

1I)

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xar®

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7cN

on-H

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mer

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)

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uxim

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mat

oid

arth

riti

s,1

g,fo

llow

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ks

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ente

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C;

1997

(US)

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xim

abR

emic

ade®

19.

5cA

nti-

TN

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rC

rohn

’sdi

seas

e,rh

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dar

thri

tis,

psor

iasi

s,IV

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5m

g/kg

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toco

r;19

98(U

S),

1999

(EU

)

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ilix

imab

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ulec

t®1

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xis

ofor

gan

tran

spla

nt,

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lus,

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rto

tran

spla

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ion,

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ter

4da

ys

Nov

artis

;19

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S,E

U)

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uxim

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rbit

ux®

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8cE

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ptor

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ress

ing

colo

rect

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ncer

;IV

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S)

26

Hum

aniz

ed(.

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mab

)

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lizum

abZ

enap

ax®

120

cA

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eytr

ansp

lant

reje

ctio

n,IV

1m

g/kg

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he/P

rote

inD

esig

nL

ab;

1997

(EU

),19

99(U

S)Pa

liviz

umab

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gis®

119

–27

cPr

ophy

laxi

sof

pedi

atri

cre

spir

ator

ysy

ncyt

ial

viru

s,IM

15m

g/kg

Med

Imm

une;

1998

(US)

Abb

ott

1999

(EU

)T

rast

uzum

abH

erce

ptin

®1

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–10

cH

ER

2ov

erex

pres

sed

met

asta

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east

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er,

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mg/

kg,

foll

owed

by2

mg/

kgev

ery

wee

kfo

r52

wee

ks

Gen

ente

ch19

98(U

S);

Hof

fman

n-L

aR

oche

2000

(EU

)A

lem

tuzu

mab

Cam

path

®1

12c

Chr

onic

lym

phoc

ytic

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emia

,es

cala

tion

from

IV3

to30

mg/

day,

3ti

mes

per

wee

k

Ber

lex/

ILE

X/

Mil

leni

um20

01(U

S)M

ille

nium

/IL

EX

2001

(EU

)G

emtu

zum

aboz

ogam

icin

Myl

otar

g®4

1.9

–2.

5cA

cute

mye

loid

leuk

emia

,IV

9m

g/m

2,

seco

nddo

sein

14da

ysW

yeth

;20

00(U

S)

Om

aliz

umab

Xol

air®

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cA

nti-

IgE

for

asth

ma,

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0–

375

mg

ever

y2

or4

wee

ksG

enen

tech

/N

ovar

tis/

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x;20

03(U

S)E

fali

zum

abR

apti

va®

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ose-

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nden

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oria

sis,

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ngle

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kgfo

llow

edby

wee

kly

1m

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ente

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2003

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2004

(US)

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(Ant

egre

n®)

411

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tiple

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rosi

san

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rohn

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e,IV

300

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ever

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wee

ksB

ioge

n/E

lan;

2004

(US)

(con

tinu

ed)

27

TAB

LE1.

2.Re

com

bina

ntAn

tibod

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rent

eral

Prod

ucts

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d)

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deIg

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alf-

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dica

tions

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and

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Type

days

Rou

te,b

and

Dos

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then

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Ale

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S)

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(...

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dar

thri

tis,

SC40

mg

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y1

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Cro

hn’s

dise

ase

160

mg

follo

wed

by80

mg

ever

y2

wee

ks

Cam

brid

geA

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ody/

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ott;

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(US)

,20

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U)

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tum

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tibi

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met

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a,IV

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g/kg

ever

y14

days

Am

gen/

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enix

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S)

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imum

abSi

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ni™

114

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i-T

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for

rheu

mat

oid

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tive

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car

thri

tis

and

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vean

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sing

spon

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once

am

onth

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toco

r;20

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S)

Fab

and

Its

Con

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tes

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ixim

ab(c

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29c

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ent

bloo

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mg/

kgIV

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sfo

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edby

IVin

fusi

onof

0.12

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for

12h

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toco

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illy

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S)

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ibiz

umab

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entis

®1

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-rel

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ular

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nera

tion

,0.

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gin

trav

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lon

cea

mon

thG

enen

tech

;20

06(U

S)

Cer

toli

zum

abpe

gol

Cim

zia®

114

Cro

hn’s

dise

ase,

2×SC

200

mg

each

and

onda

ys14

and

28C

ellt

ech/

UC

B;

2008

(US)

aN

onpr

opri

etar

yna

me:

U.S

.ad

opte

dna

me.

bT

heIV

rout

eis

indi

cate

dfo

rin

trav

enou

sin

fusi

onun

less

othe

rwis

est

ated

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omTa

ble

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inR

efer

ence

[130

];al

lot

hers

from

Ref

eren

ce[1

6].

28

ANTIBODIES 29

that retains the bivalent character of the antibody structure in a smaller construct,whereas Fab’s are monovalent (Fig. 1.6). Both constructs can enable bacterial pro-duction and may enhance tissue penetration. Ranibizumab (Lucentis, by Genentech in2006), approved for treatment of wet acute macular degeneration, is a recombinanthumanized IgG1 kappa isotype Fab that binds to and inhibits the biological activityof human VEGF-A [16]. Designed for intravitreal injection, the Fc domain functionswere not needed; and elimination of complement-mediated or cell-dependent cytotox-ity, as well as rapid systemic clearance were additional advantages [104]. In addition,point mutations were introduced into the CDR region of the parent anti-VEGF Fabto enhance binding potency [105]. Pharmacokinetic studies of primates showed that,after a single intravitreal administration, biologically effective retinal concentrations ofranibizumab were maintained for one month, while serum levels were 1000–2000-foldlower than the ocular levels [106].

Certolizumab pegol (Cimzia, by UCB in 2008) is a PEGylated version of ananti-TNFα monoclonal antibody fragment approved for treatment of Crohn’s disease.The humanized antibody fragment is a F(ab′)2, (i.e., two Fab fragments linked by adisulfide bond), manufactured in E. coli , with a light chain of 214 amino acids and aheavy chain of 229 amino acids. In order to improve half-life without losing bindingavidity, two PEG moieties (20 kDa each) are conjugated to one cysteine residue in thehinge region, distant from the antigen-binding region. This enhances the eliminationhalf-life up to 311 hours. Given that the construct does not contain an Fc portion, it isincapable of inducing in vitro complement activation, ADCC or apoptosis [16,107].

1.3.3. Newer Antibody-Derived Structures

With increasing application in research, biotechnology, and medical therapy, it hasbecome obvious that antibodies, with all their merits, also suffer inherent disadvan-tages under some circumstances. Immunoglobulin (Ig), molecules are rather large,thus limiting tissue penetration. For solid tumors, there are the additional problemsimposed by penetration through the vasculature and dispersion against an interstitialpressure. In addition, IgGs are composed of two different polypeptides, the light andheavy chains, which requires complicated cloning steps for recombinant expression;they are also glycosylated, which requires eukaryotic cell culture, time-consumingoptimization, and limited production capacities. The complex architecture of theirantigen-binding site formed by six hypervariable loops, three from each chain, canbe difficult to manipulate simultaneously if synthetic libraries are to be generated.The constant Fc region mediates immunological effector functions that are crucial foronly a few biopharmaceutical applications and may lead to undesired interactions;in some cases the long serum half-life of a full-length MAb can be a disadvantage.Consequently, there is an emerging need for antibody alternatives with improved fea-tures. Beyond Fabs, even more radical approaches are being investigated, includingdissecting the molecule into constituent domains and recombining them to generatemonovalent, bivalent, or even multivalent fragments [19]. Some of these products arenow in preclinical and clinical trials [108].

Single-chain variable fragments (scFv’s) are an increasingly popular format inwhich the VH and VL domains are joined with a flexible polypeptide linker to prevent


dissociation (Fig. 1.6). As with Fab’s, scFv fragments, comprising both VH and VL

domains, may retain the specific, monovalent, antigen-binding affinity of the parentIgG, while showing improved tissue penetration and rapid pharmacokinetics [108].Despite early excitement concerning the functional activity of single V domains, thesefragments were poorly soluble and prone to aggregation. More recent reports oncamelid antibodies have revived interest in this class since, unlike mouse VH domains,camelid VhH domains are in general soluble and can be produced as stable in vitrotargeting reagents for diagnostic platforms and nanosensors [109]. For therapeuticapplications, humanization may be required to reduce immunogenicity.

It may be desirable for some applications to design multivalent antibody frag-ments with greater functional avidity and slower dissociation rates for cell surface ormultimeric antigens. In addition to increased avidity, multivalent engagement of cellsurface receptors may result in increased activation of transmembrane signaling, whichmay improve the therapeutic efficiency of some antibodies [110,111]. Fab and scFvfragments have been engineered into dimeric, trimeric, or tetrameric conjugates usingeither chemical or genetic crosslinks [19]. One strategy to genetically encode multi-meric scFv’s has been the reduction of scFv linker length (to five or fewer residues),which directs self-assembly into these multivalent antibody forms [112]. The diabod-ies may prove to have stability advantages in vivo, although additional optimizationof the linker sequences may be required to control protease degradation. Alternativelydisulfide linkages can stabilize bivalent diabodies (Fig. 1.6). More recently, stable Fvtetramers have also been generated by noncovalent association [113].

For solid tumors, a number of approaches are being explored to increase vascularpenetration and improve tissue distribution. One such approach is targeting fragmentsto surface molecules on endothelial caveolae, specialized plasmalemmal invaginationsthat can transcytose across the normally restrictive endothelial cell barriers [114].This may allow greater penetration into and accumulation throughout solid tumorsto enable more effective delivery of imaging agents or therapeutic payloads [108].Another emerging format is produced by heterodimeric association of two domain-swapped scFv’s of different specificity. These molecules are designed to bind twodifferent epitopes on the same target, thereby increasing avidity and specificity, or tobind two different antigens for different applications such as recruitment of cytotoxicT and natural-killer cells. One class of bispecific scFvs and diabodies are designedto bind CD3 T-cell coreceptor and thereby recruit cytotoxic T cells to the tumor site.Diabody–Fc fusions can target two cell surface receptors such as IGFR and EGFRsimultaneously, to elicit rapid IGFR internalization and recruit effective antibody-dependent cellular cytoxicity. Another application of multiple specificity constructs ispretargeting (e.g., multistep delivery of an antibody–strepavidin conjugate to a recep-tor followed by biotin labeled with a radioisotope) [108]. Additionally, scFv–cytokinefusion proteins are another format being explored as cancer therapeutics. In intialevaluations, poor protein production, adverse immune reactions, and vascular leaksyndrome limited their preclinical evaluation. However, some scFv–cytokines havebegun to show promise, such as scFv–IL12 fusion proteins that were found to besuperior to IL-12 alone in a lung metastasis model [115].

THERAPEUTIC PROTEIN ALTERNATIVES TO ANTIBODY-DERIVED SCAFFOLDS 31

1.4. THERAPEUTIC PROTEIN ALTERNATIVES TO ANTIBODY-DERIVEDSCAFFOLDS

As crystallography and genome sequencing converge, it has become apparent thatbiological recognition is dependent on properties of shape and charge, rather thansimply the primary sequences involved in protein binding [116]. This observation hastriggered efforts to create alternative protein scaffolds with potential for high-affinitybinding of a broad range of specific targets. Similar to the novel antibody-based con-structs, it is hoped that these alternative formats may confer additional advantages orspecific capabilities, such as bacterial production, enhanced stability, increased tissuepenetration, multivalent structures, alternate routes of delivery, and/or other features.By exploring more diverse sequence and structural space, it may be possible to extendthe performance range of protein therapeutics even further. The alternative scaffoldscan be derived from natural proteins found in eukaryotic or bacterial organisms. Corestructures may be modified by incorporating sequences for target binding and/or otherfunctions, or novel binding surfaces may be derived from these sources. A few ofthese emerging scaffolds, intended to illustrate some of the basic approaches in thisarea, are described below.

Many of the emerging scaffolds attempt to exploit common families of human pro-teins involved in molecular recognition functions. In one example protein scaffoldshave been engineered based on the fibronectin type III domain (FN3). Fibronectinis a large glycoprotein that is found in the extracellular matrix of tissues, and isinvolved in cell–substrate and cell–cell interactions. The FN3 domain is found in∼2% of all human proteins, including cell surface receptors, carbohydrate-bindingdomains, and cell adhesion proteins, with a configuration that has been character-ized as a three-dimensional β-sandwich structure, similar to the antibody VH domainwith fewer β strands [117]. The molecular recognition function that is characteristicof these proteins resides in a binding site consisting of surface loops; and it is thisfeature that may be manipulated by introducing novel sequences or engineering theinterfaces to modulate target specificity and binding affinity [68,118]. Another verycommon protein family that is being explored as a framework for novel therapeuticsis the ankyrin repeat proteins (ARPs), which are among the most naturally abun-dant binding proteins encoded by the human genome [119] These proteins providethe backbone for DARPins (designed ankyrin repeat proteins), in which structuralmotifs are assembled onto a fairly rigid protein framework consisting of α helixesand β sheets [76,120–122]. Along with several other classes of emerging scaffolds(e.g. tetranectins and avimers, see below), DARPins can be engineered to producemultivalent molecules.

A number of other families of human proteins are being explored as potentialscaffolds for therapeutic proteins. Lipocalins are composed of a central cylindrical β

sheet (β barrel) with eight antiparallel strands carrying four loops that are variablein their number of amino acid residues, sequence, and conformation. In their naturalform, lipocalins typically bind to low-molecular-weight targets, such as vitamins [123].The β barrel provides a rigid backbone onto which specific binding sequences can begrafted to yield constructs, termed anticalins , which can be engineered for specificity


and binding affinity to particular targets [124,125]. Affilin scaffolds are based on therigid β-sheet folds of human ubiquitin and γ-crystallin. Randomized residues of eightamino acids are isolated from phage libraries against specific protein and peptidetargets, to create a binding site that is grafted onto the γ-crystallin backbone [126].The “knottin family” of proteins, known as microbodies , consist of an antiparallel β

sheet and cystine knot motif with three disulfide bonds. These constructs are usuallycomposed of 30 amino acids, with an introduced peptide ligand sequence for targetpresentation. Because of their relatively small size and stability, the cystine knotmicroproteins have been investigated for oral delivery [127,128].

Other constructs have been designed to provide modular structures consisting ofseveral units for increased avidity or carrying multiple functions. Avimers are com-posed of binding domains based on the A domain of the human low-density lipoproteinreceptor. Peptide sequences against specific targets are obtained via phage display, then“stitched” onto the ∼35–amino acid A-domain backbone to form an a multiepitopebinding scaffold with high avidity to its target [129]. Another construction is basedon human tetranectin, which is found in tissues and plasma, and has a natural abilityto bind to a range of proteins at the ligand-binding C-type lectin domain. Althoughinitially monomeric, the protein trimerizes through coiled-coil helical trimerization atthe N -terminal domain to present a multivalent interface. As a result of the multivalentbinding domains, the modified tetranectins are able to bind to their targets with highavidity [130].

In efforts to exploit an even broader sequence and structural space, nonhumanproteins have gained some consideration as sources of human therapeutics. Phylom-ers, small, nonimmunoglobulin protein fragments encoded by bacterial DNA, are oneexample. Since bacteria contain the greatest known source of sequence divergence,highly diverse libraries of polypeptides can be generated through varying combina-tions of gene sequences. Sequence mutagenesis allows the selection of moleculeswith affinity on par with antibodies and other native proteins, in molecules that rangebetween 15 and 80 amino acid residues [131].

These approaches highlight some of the efforts to produce novel classes of ther-apeutic proteins with the potential to address multiple targets, similar to antibodies,while also providing diverse characteristics that may enable new applications or conferother benefits. In addition to very challenging performance requirements, a commonconcern underlying all alternative protein scaffolds is the risk of immunogenicity.Nonetheless, if successful, these approaches have the potential to extend the rangeof protein therapeutics even beyond the very considerable achievements of today’snative proteins, monoclonal antibodies, and antibody-related products in the therapeu-tic biologics space.

1.5. CONCLUSION

Proteins represent the most diverse range of functions in living organisms, with enor-mous potential to generate highly potent and specific therapeutics to treat a wide rangeof disease states. The advent of recombinant DNA technology combined with mon-oclonal antibody technology has greatly expanded the range of protein therapeutics,

CONCLUSION 33

from a very limited number of factors that could be isolated from natural animalor human sources, to include close to the full range of human protein structuresas potential therapeutics. Further these technologies have enabled production of thishighly diverse range of protein structures and functions in formats that are generallyacceptable in terms of their immunological and safety properties, allowing applicationof biological therapeutics for treatment of a widening range of indications and diseasestates. At the same time, the field continues to experiment with a variety of techniquesto modify or engineer protein structures to expand further the range of activities andpharmaceutical properties of this highly diverse class. Although the requirement forinjection still poses a barrier in some settings, adoption of more convenient injectiondevices, particularly for chronic home administration, has increased acceptance ofbiologics for a wider range of therapeutic applications. As a result, there are well overa hundred approved protein therapeutic products on the market today for treatmentof a broad range of diseases and indications, and hundreds more in various stages ofclinical and preclinical development, representing an impressive potential to alleviatecurrently unmet medical needs and benefit patients.

Nevertheless, there are still significant constraints and barriers to realizing the fullpotential of this therapeutic class. Many of these hurdles are related to the inherentsize, flexibility, and structural complexity of this class of molecules; ironically, theseare the same properties that also enable the broad range of activities and functionsas well as the often exquisite specificity of action. Characterization of these complexand labile molecules remains a challenge, and the field continues to seek additionaltechniques to probe physical structure, association states, and degradation pathways.The requirement for injection still constitutes a hurdle in some disease and geographicsettings, and more complex delivery devices pose their own challenges in terms offormulation and stability for many products. Further, administration by routes otherthan intravenous (IV) is limited in the amounts that can be delivered via a singleinjection; and the drive to higher doses introduces additional formulation and pro-cessing challenges, including solubility, viscosity, and aggregation [132]. While greatstrides have been made in the elimination of impurities and infectious agents duringmanufacturing, and the general safety of protein therapeutics has advanced tremen-dously, safety remains a significant consideration. Immunogenic reactions in particularcan impose constraints on molecular design as well as strict standards for stability inproduction, storage, and handling. To maintain the integrity of these labile molecules,most protein therapeutics must be shipped and stored under refrigerated conditionsfor the majority of their shelf lives, posing additional challenges in manufacturingand distribution, as well as restricting the use of these products in areas where thecold chain is less developed or unavailable. Later chapters in this volume outline thegreat strides that have been made in the field in terms of the formulation of proteintherapeutics, as well as the processes for manufacturing the final product, and describesome of the ongoing research to address these and other challenges.

The ultimate hurdles for protein therapeutics may lie in the realm of biology moreso than chemistry. Because of their great specificity, the most problematic toxicitiesfor biologics are often related to their mechanism of action. Incorporation of targeting


strategies in the design and delivery of biological therapeutics may assist in over-coming some of these impediments. The limited capacity for tissue penetration andmembrane transport further restricts the potential of this class of therapeutics. Theseconstraints, which are again related to their size and structural complexity, signifi-cantly limit the targets that can be addressed with this class, despite their great rangeof activities and functions. For example, the inability of most proteins to penetrate theblood–brain barrier restricts their application in many neurological disorders, as wellas other protected compartments. Moreover, many of the most desirable targets fortherapeutic intervention are intracellular and hence beyond the range of the vast major-ity of current protein therapeutics. Hopefully the effort and ingenuity dedicated to thedesign new classes of protein therapeutics will eventually result in breakthroughs thatwill extend the application of these molecules, with their inherent advantages in termsof function, potency, and specificity, even to these most challenging frontiers.

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THE STRUCTURE OF BIOLOGICAL THERAPEUTICS