Fc Engineering to Modulate Pharmacokinetics

As FcRn recycles IgGs or Fc fusions through its interaction with the Fc region, engineering the FcRn:Fc interaction is one of the methods for modifying the PK and PD of an IgG or Fc fusion (15,92). A number of studies have demonstrated that improving the affinity of an Fc variant against FcRn at acidic pH can prolong its serum half-life. Ghetie et al. first showed that an engineered murine Fc variant (T252L/T254S/T254F), which had threefold increase in murine FcRn affinity at pH 6, exhibited approximately 30% to 60% extension in serum half-life in mice (93). Subsequent studies have identified and validated various favorable mutations on human IgG1 Fc residues, namely Thr250, Met252, Thr254, Ser256, Thr307, Glu380, Met428, and Asn434 (Fig. 1C) (36,94-96). These mutations can be combined synergistically to give Fc variants of different FcRn affinities. For example, one human IgG1 Fc variant carrying the double mutations T250Q/ M428L exhibited a half-life of approximately 35 days in Rhesus monkeys (Rhesus macaques), a significant improvement over its wild-type counterpart (*14 days). However, it was observed that affinity increase of an Fc variant at acidic pH was usually coupled with an undesirable affinity increase at neutral pH (94,97,98). High levels of binding at neutral pH hinder the release of an FcRn-bound IgG variant back into the circulation and increase the binding of circulating IgGs to the cell surface-expressing FcRn, effectively accelerating the clearance of IgG and canceling out the benefit of increased affinity at acidic pH (94). Therefore, FcRn-binding affinities at both acidic and physiological pH are important determinants to balance for the PK engineering of an IgG or Fc fusion.

One important aspect of engineering the half-lives of IgGs is the use of suitable preclinical animal models to evaluate the variant's half-life in vivo and predict the variant's PK parameters in humans. Earlier studies involved testing human IgG1 Fc variants in mouse model; however, because of the recently discovered differences in human IgG's affinity and specificity to murine and human FcRn (99), PK results of the variants in mice were not expected to truly reflect the variants' PK in humans (94,100). Currently, the closest model system to humans is nonhuman primates, as nonhuman primates have similar levels of endogenous IgG and human IgG1 binds nonhuman primate FcRn with similar affinity and specificity as human FcRn (96,100,101). A promising alternative model to nonhuman primates is the human FcRn transgenic mouse, which lacks endogenous murine FcRn but expresses human FcRn (95). These mice allow the in vivo study of human IgG Fc variants' interactions with human FcRn in a higher throughput and more cost-effective manner than nonhuman primates. However, further developments like control of FcRn expression levels and patterns and endogenous IgG levels are needed to render these transgenic mice more humanlike.

An IgG can also be engineered to have a shorter in vivo half-life by reducing its FcRn affinity. It was shown that Fc variants' half-lives reduced in correspondence with the variants' decreasing FcRn affinities (92). These shortlived IgG Fc variants can be useful in situations where shorter IgG exposure is desired (e.g., imaging and toxin-conjugated antibody therapy).

PK-modifying IgG Fc variants are still in the research and early development phases; none of the variants has been evaluated in humans. Fc engineering does appear to be a promising way to modulate the PK of an antibody or Fc fusion, but it remains to be seen how much PK improvement and immunoge-nicity these Fc variants will have in humans.

ENGINEERING BINDING TO CARRIER PROTEINS Properties of Serum Albumin

Serum protein binding is a general property frequently responsible for extending the pharmacokinetic properties of small-molecule drugs. This property has been exploited as a strategy to increase the serum half-life of protein therapeutics. This approach takes advantage of the long half-life inherent in many highly abundant and long-lived serum proteins to enhance the properties of many therapeutic molecules. One of the most commonly exploited proteins is serum albumin.

At a concentration of 35 to 50 mg/mL (*600 pM), albumin is the most highly abundant protein in serum, comprising roughly 60% of the total serum protein (102). Albumin is broadly distributed throughout the body, serving as an important vascular and extravascular carrier of a wide variety of endogenous molecules such as bilirubin, thyroxine, fatty acids, and metals. Albumin has also been recognized as a major carrier of several small-molecule therapeutics such as ibuprofen, warfarin, and diazepam. Albumin plays an important role in maintaining the solubility of these compounds and protecting them from rapid

Cys34

Propos ec site of FcRn interactio

Domain II!

Cys34

Propos ec site of FcRn interactio

Domain II!

FIGURE 3 Structure of albumin. A ribbon diagram is shown on the basis of the X-ray crystal structure (Protein Data Bank accession number 2BXH), rendered using PyMol. The proposed site of FcRn interaction is indicated at left, and the region of small-molecule binding sites I and II are circled. Source: From Refs. 103 and 34.

FIGURE 3 Structure of albumin. A ribbon diagram is shown on the basis of the X-ray crystal structure (Protein Data Bank accession number 2BXH), rendered using PyMol. The proposed site of FcRn interaction is indicated at left, and the region of small-molecule binding sites I and II are circled. Source: From Refs. 103 and 34.

renal clearance. Many functional and structural studies of albumin have revealed multiple binding sites that enable albumin to accommodate the binding of this diverse set of ligands. Binding specificity and potential drug-drug interactions resulting from multiple highly adaptable binding sites on albumin have been investigated in a comprehensive study by Ghuman et al. (103).

Albumin is a 66-kDa globular protein consisting of three homologous domains oriented in a heart shape (Fig. 3). Although numerous drug interaction sites are distributed throughout the protein, there are two major drug-binding sites, sites 1 and 2, located in domains II and III, respectively, in addition to the metal-binding site near Cys34 in domain I. Albumin contains a total of 17 disulfides and one free thiol at Cys34 (102). Importantly, the very high serum concentration of albumin makes Cys34 the most abundant free thiol in serum.

In human, albumin has a half-life of 19 days, similar to that of IgG1. This long serum persistence can be partially explained by albumin's size that helps it to avoid renal clearance. On the other hand, like IgG, the fractional catabolic rate of albumin is directly related to its serum concentration. For example, in anal-buminemic patients, the half-life of albumin is as long as 115 days (104). This observation led Schultze and Heremans to propose, in a similar fashion to Brambell's hypothesis concerning the protection of IgG by FcRn, the existence of a set of albumin receptors that protect albumin from degradation (105). Only recently and quite accidentally, it was discovered that both albumin and IgG are protected in a noncompetitive manner by the same receptor, FcRn (30,31). FcRn is proposed to bind both albumin and IgG at low pH (* pH 6) during cellular pinocytosis, protecting them from lysosomal degradation. Upon recycling of the endocytic vessel back to the cell surface, both IgG and albumin are released back into serum at neutral pH. Molecules associated with albumin or IgG that do not interfere with FcRn binding and are able to maintain their association during this pH change would be expected to survive this pinocytosis process as well.

The abundance, long half-life in serum, and broad biodistribution of albumin make it an important target for enhancing the pharmacokinetic and pharmacodynamic properties of many therapeutic small molecules and protein therapeutics. General features and examples of the many approaches to capture the properties of albumin are described below and are outlined in Table 2. Although this review focuses on proteins and peptides, some of these approaches originate from attempts to extend the pharmacokinetic properties of small-molecule drugs; these pioneering examples have been included where appropriate.

Strategies to Reduce Clearance Through Association with Albumin

Conjugation to Albumin

Early work by Poznansky and colleagues demonstrated many of the benefits of associating proteins with albumin. While searching for an approach to enhance enzyme therapy, glutaraldehyde was used as a cross-linking agent to form soluble conjugates of enzymes such as superoxide dismutase (SOD) with an excess of albumin (106). While unconjugated SOD was rapidly cleared with a half-life of six minutes, the SOD-albumin conjugates, fractionated by molecular weight and injected into rats, were found to clear through the liver with half-lives of up to 15 hours. Conjugates of increasing molecular weight were cleared progressively faster, suggesting an optimum conjugation ratio. Interestingly, conjugation to albumin also reduced the immunogenicity and antigenicity of SOD in rabbits. Similar findings of reduced immunogenicity were found in an earlier study linking uricase to albumin (107). These early studies demonstrated the potential of albumin conjugates as a means to prevent rapid clearance and reduce immunogenicity of heterologous proteins (108).

While encouraging in principal, these studies suffered from the heterogeneous preparations that were generated. Glutaraldehyde conjugation to albumin was random, and stoichiometry was difficult to control. Further, bio-activity of the conjugated protein was often compromised. For example, in a study conjugating porcine growth hormone (GH) to albumin, the average complex consisted of two molecules of albumin and six molecules of GH and although the conjugate increased the half-life of GH from five minutes to two to three hours, it did not stimulate growth in hypophysectomized rats (109).

An effective use of this approach has been to deliver cytostatic agents like methotrexate (MTX) to tumors. In a cancer therapy setting, albumin can provide additional benefits besides enhanced pharmacokinetics. Albumin accumulates in and is a major energy and nitrogen source for tumors, making it an ideal delivery vehicle for anticancer agents (110). In addition, association with albumin can alter the metabolism of associated agents, as clearance is shifted from the kidney to the liver. As a result of longer serum persistence, the slower metabolic release of free cytotoxic compounds can also lead to a reduction in toxicity (111). Finally, because of its smaller size, albumin is thought to diffuse through tissues ten times faster than IgG. In a study examining the loading rate of MTX on albumin, high MTX to albumin ratios were associated with more rapid clearance and decreased tumor uptake in rats, while a 1:1 ratio

TABLE 2 Characteristics of Albumin-Based Strategies

Characteristics

Effectiveness

Features

Covalent attachment to albumin

Random conjugation in Heterogeneous product; aggregates; poor reproducibility; very complex manufacturing process anticipated Defined product; requires albumin for in vitro conjugation; relatively complex manufacturing Simple defined product; conjugation to endogenous albumin becomes dominant species in vivo; relatively simple to manufacture Simple defined product that can be made recombinantly; manufacture requires expression in yeast or mammalian cell culture

Noncovalent association to albumin serum albumin ligands Design derived from known albumin ligands; typically low affinity for albumin vitro

Specific conjugation via C34 in vitro

Specific conjugation via C34 in vivo

Albumin fusions

Bacterial domains

Engineered Binding Domains

Bacterially derived surface antigens; modest to very high affinity for albumin; affinity relatively easy to modify Derived from engineered peptides or immunglobulin domains; modest to very high affinity for albumin; affinity relatively easy to modify

Can achieve modest PK improvement, up to the full half-life of albumin

Modest half-life extension; affinity for albumin affects half-life and can vary across species

Can achieve modest PK improvement, up to the full half-life of albumin; affinity for albumin affects half-life and can vary across species

Early demonstration of the use and benefits of albumin for prolonging half-life; difficult to control or optimize linkage Optimization required to identify linkage that maintains functionality of therapeutic molecule; possible to engineer time- or condition-dependent reversibility into linkage

Application limited to proteins and peptides; linkage limited to N- or C-terminal fusions that often impair functionality of therapeutic molecule; similar to Fc fusions but lacks Fey receptor functions

Requires numerous analogues to identify a variant with appropriate albumin-binding affinity while maintaining functionality of therapeutic molecule FDA-approved example (Levemir) Several examples have been shown to enhance immunogenicity to fused molecule; may be ideal for vaccine development Optimization required to identify linkage that maintains functionality of therapeutic molecule; possible to engineer time- or condition-dependent reversibility into linkage

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