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Immunoadhesin (Fe fusion protein)

FIGURE 1 Three general strategies for enhancing the pharmacokinetic half-life of injected peptides and proteins: (A) use of an antibody (IgG) platform or Fc fusion (immunoadhesin), which can be further engineered for enhanced FcRn binding; (B) covalent or noncovalent association with serum albumin; and (C) chemical modification to increase hydrodynamic volume.

Key considerations in the application of protein engineering methods for therapeutic polypeptides are that the final molecule should have appropriate specificity, target-binding affinity, and half-life to achieve the intended therapeutic benefit while retaining manufacturing, formulation, and delivery feasibility. The modified or mutated protein must also evade the human immune system, particularly if it is to be used over an extended period of time. The consequences of an immunogenic response to a drug may include severe adverse events such as anaphylactic (hyperimmune) antibody responses, or simply faster pharmacokinetic clearance as a result of complexes formed with antidrug antibodies. We have also omitted discussion of approaches to reduce immunoge-nicity, which have been reviewed elsewhere for antibodies and other proteins (6).

MAbs represent a particularly attractive class of injected protein therapeutics, in part because of the highly developed technologies available for the discovery, humanization, and affinity maturation of highly specific, targeted agents that in full-length immunoglobulin G (IgG) form typically have long pharmacokinetic half-lives on the order of three weeks in humans, as reviewed in the previous chapter of this volume. However, even these relatively long-lived proteins may benefit from protein engineering efforts. Here we review recent studies that have shown significant increases in circulating half-life in nonhuman primates for antibodies that have been mutated at a few sites in the Fc region of the molecule. These mutations serve to enhance binding to the neonatal Fc receptor (FcRn), an abundant cell surface receptor that is found throughout the reticuloendothelial system and that serves to scavenge IgG from the circulation, thus protecting it from metabolic degradation.

Albumin

Fusion/Conjugation

Albumin Formulation

Glycosylation or PEGylation

Serum albumin, another long-lived serum protein, also benefits from interaction with FcRn, persisting in the circulation with a half-life of about three weeks. This observation has given rise to a variety of strategies to use albumin as a "carrier protein" for peptides and small proteins that can be fused to—or otherwise engineered to associate with—serum albumin.

Finally, the pharmacokinetic half-life of small proteins and peptides can often be greatly increased by simply increasing the hydrodynamic radius through chemical modification, for example, by conjugation to polyethylene glycol (PEG) or similar polymers. An established strategy has been to modify the protein with one or more PEG groups, with the goal of achieving a larger effective molecular weight (EMW), and this strategy has proved successful in several marketed therapeutics, including pegvisomant (Somavert®), which was engineered for higher binding affinity to its target [human growth hormone (hGH)] receptor, for antagonist activity, and for longer half-life through PEGy-lation (5). Yet even larger, particulate formulations are also being investigated using conjugates that form micelles or nanoparticles (7). These approaches create additional opportunities for delivering otherwise short-lived drugs in Trojan horse fashion, either specifically or nonspecifically, to target tissues.

It is worth noting at this point that not all therapeutic polypeptides will benefit from engineering efforts to maximize half-life. This is illustrated by insulin and other family members, which can have unexpected or undesired consequences if dosed on a schedule incompatible with their metabolic role (8). Among antibodies, bevacizumab (Avastin®) anti-VEGF benefits from long halflife allowing for dosing by intravenous infusion every three or four weeks (9). However, a different anti-VEGF, ranibizumab (Lucentis®), a Fab fragment engineered for high antigen-binding affinity and tissue penetration to the retina, is delivered intravitreally (10). In this case, prolonged residence in the eye may be beneficial, whereas increased systemic half-life may raise safety issues.

The central role of FcRn in mediating the half-life of antibodies and the fact that FcRn effects are mediated through interaction with a specific region of the antibody—the Fc—translates to a simple approach to improve the half-life of many small proteins, including receptor extracellular domains, namely, by fusing the protein to the Fc region of an IgG. Such an approach has additional advantages of (i) providing a bivalent molecule, which may benefit in potency from avidity effects, (ii) minimizing immunogenicity risks because both protein (e.g., an extracellular domain (ECD)) and antibody Fc may be of human origin (though the junction site is typically a nonnative sequence), and (iii) facilitating manufacturing and clinical assay development activities that are analogous to those for therapeutic antibodies. Etanercept (Enbrel®) is a marketed anti-TNF-a (tumor necrosis factor a) agent in this category and is discussed below in comparison with anti-TNF antibodies. Both types of molecules may benefit from Fc engineering that enhances binding interactions with FcRn.

ENGINEERING BINDING TO FcRn

The Neonatal Fc Receptor: Function and Expression

Typical serum proteins have half-lives of less than one week, for example, fibrinogen (1-3 days), IgD (2-5 days), IgM (4-6 days), IgA (3-7 days), and haptoglobin (*5 days) (11-14). However, serum IgG and albumin have half-lives of approximately three weeks. The prolonged half-lives of IgGs and albumins are mainly due to the protective action of the FcRn (15,16). FcRn is also known as Fc receptor Brambell (17) (FcRB) and Fc receptor protection (FcRp), named after Professor Brambell who first described it and its protective function, respectively. In the late 1950s, a saturable receptor system was proposed by Professor Brambell for mediating the transport of IgG from mothers to infants through the yolk sacs and intestines (17,18). Observing the similarity between passive transmission and catabolism of IgG, he later postulated that a similar or identical receptor system was responsible for the protection of IgG from catabolism (19). It was not until thirty years later that this IgG receptor, FcRn, was cloned (18) and confirmed to carry out both the important functions of transcytosis (18) and homeostasis of IgG (20-23). FcRn of multiple mammalian species have been cloned (National Center for Biotechnology Information), and functional expression of FcRn has been reported in mammals like rat, mouse, rabbit, sheep, bovine, nonhuman primate, and human (17,24-26).

FcRn, which is structurally homologous to major histocompatibility complex (MHC) class I molecules, is a heterodimer consisting of a transmembrane a-chain and p2-microglobulin (b2m) (27,28). However, unlike MHC molecules, FcRn is incapable of binding peptides because the counterpart of the MHC peptide-binding groove in FcRn is occupied by its own residues (27,28). FcRn can simultaneously bind both IgG and albumin, but the binding stoichiometries are different, with an FcRn/IgG ratio of 2:1 and a FcRn/albumin ratio of 1:1 (29-32). Although a crystal complex structure of human Fc-FcRn is still not available, the major contact residues in the human complex can be deduced from the crystal structure of a rat Fc:FcRn complex (Fig. 2A) (29,33), and site-directed mutagenesis studies (35-38). FcRn binds the Fc portion of IgG at the CH2-CH3 interface (28,29,36). Major contact residues of the human FcRn are Glu115, Asp130, Trp131, Glu133, and Leu135 on the a-chain and Ile1 on the p2m (Fig. 2B). On the Fc side, residues Ile253, Ser254, His310, His435, and Tyr436 are important for the interaction (Fig. 2C), as alanine substitution at these positions results in a greater than 10-fold reduction in binding to FcRn (36). Meanwhile, the contact residues for FcRn and albumin are not as clearly defined as those for the FcRn and IgG interaction. FcRn was found to bind albumin around His166 (Fig. 2B), opposite from the Fc binding region (39); this may explain why FcRn can simultaneously bind both IgGs and albumins. It is not clear whether p2m is involved in binding to the albumin, but p2m is required for the expression of FcRn (40).

FcRn protects IgGs and albumins from catabolism in a pH-dependent manner. IgGs and albumins bind FcRn with high affinity at acidic pH; as the pH is raised to neutral, the binding affinity drops considerably. The pH-dependent interaction is mainly attributed to the titration of histidine residues (41). Specifically, pinocytosed IgGs or albumin are captured by FcRn in the acidic endosome (42), recycled back to the cell surface (43) and then released back into the circulation at physiological pH of 7.4 (44). IgGs or albumins that are not bound by FcRn are targeted to the lysosome and degraded (42). Previous studies in knockout mice illustrated that the serum half-lives of IgGs and albumin in FcRn- or p2m-deficient mice were greatly reduced (16,20-22,30). It was also observed in familial hypercatabolic hypoproteinemia patients that their low levels of serum IgG and albumin were caused by the reduction of FcRn expression, resulting from p2m deficiency (23). Functional FcRn expression has been reported in a variety of tissues and cells such as vascular endothelium (45), monocytes (46), macrophages (46), dendritic cells (46), intestinal epithelium (47),

FIGURE 2 The crystal structures of a rat Fc:FcRn complex (A), human FcRn (B), and human IgG1 Fc (C). (A) A rat Fc:FcRn complex structure (PDB accession number 1I1A) shows that CH2-CH3 interface of the IgG Fc region (right side ribbons) interacts with both the a-chain and the p2-microglobulin of FcRn left side ribbons. Carbohydrates are shown as spheres. (B) Critical binding residues on human FcRn (PDB 1EXU) for IgG and albumin interactions are shown as labeled spheres. Human IgGs and albumin appear to bind at the opposite sides of a human FcRn. The a-chain and p2-microglobulin of human FcRn are shown as ribbons. (C) Residues where alanine substitutions result in greater than 10-fold reduction in FcRn affinity are shown as Ile253, Ser254, His310, His435, Tyr436 spheres on a human Fc structure (PDB 1DN2). These critical residues are located at the CH2-CH3 interface of a human IgG1 Fc. Mutations of the Fc residues shown as Thr250, Met252, Thr256, Thr307, Glu380, Met428, Asn434 spheres have been shown to improve human FcRn affinity. These residues are surrounding the critical binding residues. Molecular models in this figure were created using PyMol. Abbreviation: PDB, Protein Data Bank. Source: From Ref. 34.

FIGURE 2 The crystal structures of a rat Fc:FcRn complex (A), human FcRn (B), and human IgG1 Fc (C). (A) A rat Fc:FcRn complex structure (PDB accession number 1I1A) shows that CH2-CH3 interface of the IgG Fc region (right side ribbons) interacts with both the a-chain and the p2-microglobulin of FcRn left side ribbons. Carbohydrates are shown as spheres. (B) Critical binding residues on human FcRn (PDB 1EXU) for IgG and albumin interactions are shown as labeled spheres. Human IgGs and albumin appear to bind at the opposite sides of a human FcRn. The a-chain and p2-microglobulin of human FcRn are shown as ribbons. (C) Residues where alanine substitutions result in greater than 10-fold reduction in FcRn affinity are shown as Ile253, Ser254, His310, His435, Tyr436 spheres on a human Fc structure (PDB 1DN2). These critical residues are located at the CH2-CH3 interface of a human IgG1 Fc. Mutations of the Fc residues shown as Thr250, Met252, Thr256, Thr307, Glu380, Met428, Asn434 spheres have been shown to improve human FcRn affinity. These residues are surrounding the critical binding residues. Molecular models in this figure were created using PyMol. Abbreviation: PDB, Protein Data Bank. Source: From Ref. 34.

brain and choroids plexus endothelium (48,49), podocytes (50), placental endothelium (51,52), and lung epithelium (53). FcRn has been shown in these studies to either recycle or transport IgG across the cellular barriers. Overall, FcRn has been shown to extend the half-lives of IgGs and albumins in circulation.

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