Immunoadhesins Fc Fusions

Different engineering strategies have been employed to harness the protective action of FcRn to prolong the half-life of injected therapeutics. One of these strategies is to genetically or chemically fuse the protein or peptide of interest (referred to as the ligand in this text) to either the whole IgG, the Fc portion of an IgG, or an albumin, all of which interact with FcRn. The premise is that instead of rapid clearance by the kidney, or catabolism, IgG-, Fc-, or albumin-fused ligand can be recycled and protected by FcRn. We will first focus our discussion on Fc fusions. Albumin fusions will be discussed later in this chapter.

CD4-Fc: a Prototype for Immunoadhesins

CD4 is a cell surface glycoprotein present on a subset of peripheral T cells that recognize antigens presented by class II MHC molecules (54). It consists of four immunoglobulin-like domains followed by a transmembrane segment and a C-terminal cytoplasmic tail (55-57). CD4 biology became a hot research topic in the 1980s as CD4 was shown to mediate high-affinity (KD*nM) interaction with human immunodeficiency virus (HIV) envelop glycoprotein gp120 (58). Soluble recombinant CD4 ectodomain (rCD4) was created and successfully shown to block HIV infectivity, syncytium formation, and cell killing by gp120 (58). It was later shown that truncated rCD4 containing the first two N-terminal domains (trCD4) was also as effective as the full-length rCD4 (59,60). However, one major initial shortcoming of developing rCD4 as a drug was its short serum half-life (t1/2 * 4 hours in rabbits and t1/2 * 10 hours in human patients) (61,62).

Although FcRn function was not clearly elucidated at that time, Capon et al. observed that the Fc portion of an IgG, but not the Fab, had a long serum half-life like the whole IgG (61). They then constructed the first ligand-IgG fusion (named immunoadhesins) by genetically fusing the rCD4 or trCD4 to the constant region (CH1-CH2-CH3) of the human IgG1 heavy chain (61). Both the immunoadhesins showed similar binding to gp120 as rCD4 but exhibited more than 25-fold improved plasma half-lives in rabbits compared with rCD4 (61). An improved version of CD4 immunoadhesin was later generated by genetically fusing trCD4 to the Fc (CH2-CH3; residues 216 to 441, Kabat numbering) of a human IgG1 (63,64). The half-life of this trCD4-Fc variant was approximately two days in patients, a significant improvement over the t1/2 of approximately 10 hours observed for rCD4 following the same dosing regiment (62,65,66). However, the ii/2 of this CD4 immunoadhesin was still not as long as the ii/2 of the anti-gp120 antibody 2G12, which was determined to be approximately 15 to 20 days in patients (67).

Fc Fusions in Research and in the Clinic

In general, proteins and peptides of interest can be constructed as N- or C-terminal fusions to an IgG Fc. Linkers with or without protease sites can be inserted between the ligand and Fc region to increase the flexibility of the two domains, and the presence of a protease site allows release of the ligand from the fusion protein. Numerous studies have shown that fusing an Fc to a ligand can improve the ligand's half-life by more than 100-fold. Fc fusions also provide other benefits such as ease of purification using protein A or G, stability improvement achieved by the fusion, recruitment of Fc effector functions, and ability for placental transport. Currently, there are four marketed Fc fusion therapeutics; they are all constructed as C-terminal fusions to the human IgG1 Fc (Table 1). Multiple other Fc fusions are under development for various disease indications (68).

Given the proven utility of Fc fusions as therapeutic agents and the relative ease of their construction, one may ask whether an Fc fusion, instead of an antibody, should be developed for a particular target. Unfortunately, there is no straightforward answer, but several factors should be considered in choosing either format. Parts of the following discussion are based on current anti-TNF therapies, where both Fc fusions and antibodies were developed to block TNF: Etanercept (TNF-R-p75-IgG1-Fc fusion, Amgen, Inc., California, U.S., and Wyeth, Pennsylvania, U.S.), Lenercept (TNF-R-p55-IgG1-Fc fusion, Roche, New Jersey, U.S.), Adalimumab (fully human anti-TNF IgG1, Abbott, Illinois, U.S.), and Infliximab (chimeric anti-TNF IgG1, Centocor, Malvern, PA, U.S.).

Mechanisms of Action

Fc fusion proteins are an important class of protein therapeutics that may have activities not readily achieved with MAbs. Antibodies are usually developed to elicit antagonistic activities by blocking the antigens' interactions with their endogenous binding partners. In some cases, agonistic antibodies can be generated, often functioning though cross-linking the target receptors (69-71). Therefore, if a certain agonistic activity is required for the therapeutic action such as activating a cytokine receptor to trigger downstream signaling cascade, developing an Fc fusion, with the endogenous ligand being part of the fusion, should be more straightforward. Conversely, both Fc fusions and antibodies can be readily developed for antagonistic activities, as in the case of blocking TNF.

Binding Properties of an Antibody Vs. an Fc Fusion

Binding specificity is of concern when comparing the development of an antibody versus that of an Fc fusion. Antibodies typically mediate highly specific interactions with their antigens, so cross-reactions with other non-target proteins are very rare. In contrast, Fc fusions are sometimes made up of endogenous ligands or receptors, which can have broad specificities in vivo. For example, Etanercept binds both TNF and lymphotoxin, whereas Adalimumab and Infliximab bind only TNF. There may be unexpected activities as well as tox-icities associated with the bindings of these secondary targets. Therefore, it is important to investigate if an endogenous ligand-Fc fusion has any undesirable off-target binding partner in vivo.

Despite binding the same target, an antibody and an Fc fusion can have different binding properties such as affinity, binding kinetics, binding epitopes, and stoichiometry, all of which can impact the pharmacokinetics and pharma-codynamics of a drug. For example, Etanercept has a faster association rate than Adalimumab and Infliximab in binding soluble TNF, hence, it is more effective at neutralizing low concentrations of TNF (72,73). Moreover, because of the difference in binding stoichiometry (TNF/Etanercept 1:1 and TNF/Infliximab 1:3),

TABLE 1 Currently Marketed Immunoadhesin (Fe Fusion) Drugs

Drug name

Product

Target

MW (kDa)

Brand name

Company

Indications

Half-life (d)

Reference3

Etanercept

TN F-R2-lgG1 Fc

TNF, LT

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