Kunitz type domains

Proteases have important biological functions, they have been identified as key regulators of cellular processes such as ovulation, fertilization, wound healing, angiogenesis, apoptosis, peptide hormone release, coagulation and complement activation (Nixon and Wood, 2006). Because unregulated proteolysis in any of these processes will have undesirable effects, effective control of protease activity is critical. Indeed, unregulated activity has been implicated in the pathogenesis of many diseases (e.g. cancer, inflammatory diseases, chronic obstructive pulmonary disease (COPD), chronic pancreatitis, muscular dystrophy and Alzheimer’s disease (Naruse et al., 1999; Egeblad and Werb, 2002; Nunan and Small, 2002; Shapiro, 2002; Laval and Bushby, 2004; Parks et al., 2004), making proteases important target proteins. It is notable, that there are only a few marketed drugs (such as HIV protease inhibitors, angiotensin-converting enzyme inhibitors, the proteasome inhibitor bortezomib and the renin inhibitor aliskiren), since a key challenge in the discovery of new inhibitors is the ability to identify drugs which are both potent and specific. Structural similarities within the active site of most proteolytic enzyme families often result in a simultaneous inhibition of several family members, which can lead to an unacceptable toxicity profile. One solution to this problem represents the use of endogenous, engineered protease inhibitors that make more contacts with the target protease and therefore allow for tighter control over specificity. One type of endogenous inhibitors include the kunitz domain inhibitors, e.g. bovine pancreatic trypsin inhibitor (BPTI) (Ascenzi et al., 2003).

In 1992, Ladner and co-workers produced a small library of mutants of kunitz domain BPTI, displayed on phage and screened for the ability to bind to elastase (Roberts et al., 1992), a serine protease that is believed to play a causative role in a variety of lung diseases, including COPD and cystic fibrosis (Hautamaki et al., 1997). Isolates that were able to bind to elastase were expressed, purified and tested for inhibition of elastase. The P-loop (see Figure 13.1A), the region that was varied in the library, of the best inhibitor (K_i ∼1 pM) was transferred to a human kunitz domain without subsequent loss of potency (Ley et al., 1997). The resulting engineered molecule, depelestat (Dyax/Debiopharm SA), inhibited human neutrophile elastase (HNE) activity in the sputum of cystic fibrosis patients, was demonstrated to be safe in an aerolized format in monkeys and no adverse effects were reported in Phase I clinical trials (Delacourt et al., 2002; Grimbert et al., 2003; Nixon and Wood, 2006). Pharmacodynamic reports of a Phase IIa clinical trial have been shown to inhibit completely sputum HNE in 52.6% of the patients and to decrease interleukin-8 levels, a biomarker of inflammation, in the sputum of treated patients (Saudubray et al., 2003).

In 1996, another human kunitz domain, the lipoprotein-associated coagulation inhibitor (LACI-D1), was used as a scaffold for the successful isolation of a very specific and potent inhibitor of human kallikrein (Markland et al., 1996). Kallikrein is believed to be an important mediator of hereditary angioedema (HAE), a rare disorder with attacks of oedema in the hands, face, feet, abdomen and/or throat. This condition is caused by a genetic deficiency of C1 esterase inhibitor (C1-Inh), which is an endogenous inhibitor of plasma kallikrein. In Europe, HAE is treated with plasma-derived C1-Inh which attenuates attacks and may prove life-saving, but C1-Inh is expensive and requires the use of pooled blood product (Nixon and Wood, 2006). After an iterative selection and screening approach chosen by the authors (Markland et al., 1996), the plasma kallikrein inhibitor DX-88 (Dyax Corp/Genzyme Corp) was isolated and proved to be a potent inhibitor (K_i = 40 pM) with high selectivity (Table 1 in Williams and Baird, 2003). The use of DX-88 in a C1-Inh deficient mouse model demonstrated that DX-88 is effective in preventing changes in vascular permeability (Han et al., 2002). In December 2009, DX-88 (ecallantide) was approved by FDA for the treatment of HAE.

With the first approved drug kunitz type domains represent the most advanced scaffold in this field. The strategy of taking a human protease inhibitor as a scaffold and improving it by protein engineering works well for certain therapeutic applications. At the same time, kunitz type domains have the limitation of binding only to proteases, and thus, at present, they do not represent a universal source for the generation of binding proteins to a variety of targets.

Tenth type III domain of fibronectin (Adnectins)

The tenth type III domain of human fibronectin (¹⁰Fn3) is a 94 aminoacid residue structural protein with an immunoglobulin-like fold that is used as an antibody-mimic scaffold. High thermal stability (T_M = 90°C) and solubility (>15 mg/mL), high expression yields in Escherichia coli and the lack of cysteines in its structure are attractive properties that make the ¹⁰Fn3 a good scaffold candidate (Xu et al., 2002). Three of the solvent exposed loops in ¹⁰Fn3, named BC, DE and FG are structurally analogous to the V_H complementary-determining regions (CDR) (Figure 13.1B).

The first report on the use of the ¹⁰Fn3 domain as a protein with artificial binding sites was published in 1998 (Koide and Koide, 1998). In that study, a library of 10⁸ distinct ¹⁰Fn3 mutants was made by deleting three residues in the FG loop and by randomizing five residues in the BC loop and four residues in the FG loop. The library, displayed on phage, was used in affinity selection experiments with immobilised ubiquitin as model target protein. After five rounds of panning, clones were randomly picked for sequencing. A clone designated as Ubi4-Fn3 dominated the population of selected protein mutants and was subjected to further analysis. Ubi4-Fn3 was demonstrated to bind in phage enzyme-linked immunosorbent assay (ELISA) experiments. However, when expressed as a single domain protein in E. coli, Ubi4-Fn3 exhibited low solubility at neutral pH, unspecific binding to the dextran matrices of the size-exclusion chromatography column and the dextran coated biosensor chips used for the subsequent characterization of Ubi-Fn3. More importantly, the affinity was relatively low; the IC₅₀ determined by competition ELISA was 5 µM.

In order to select ¹⁰Fn3 domains binding to a target of interest with improved properties than Ubi4-Fn3, an alternative library design in combination with a fully in vitro selection system (mRNA display (Wilson et al., 2001)) was used for the isolation of ¹⁰Fn3 variants binding to tumour necrosis factor-α (TNF-α) (Xu et al., 2002). Clones from the ninth and tenth round of selection were cloned in E. coli, sequenced and expressed. Affinity constants were determined by incubating the in vitro translated ³⁵S-methionine labelled proteins with biotinylated TNF-α at different concentrations. The solution containing the ¹⁰Fn3 mutants bound to TNF-α were aspirated by vacuum onto a membrane coated with streptavidin, therefore capturing protein complexes on the membrane. Binding was analysed by measuring the radioactivity on the membrane, the K_D for selected mutants were found in the range of 1-24 nM. Further affinity maturation procedures revealed K_D values around 100 pM, the best being 20 pM, and analytical gel filtration showed that the apparent molecular weight of purified, soluble wild-type (wt) and mutant domains was consistent with the variants being monomeric. The reported affinities, especially for TNF-α, are very high. However, these values should be evaluated critically because first, the capture step of the biotinylated TNF-α-Fn3 protein complexes was performed in a solid phase format, and second, TNF-α is homotrimeric protein, so avidity effects may contribute to the high apparent affinity observed.

Other ¹⁰Fn3 variants were shown to recognize ανβ3-integrin expressed on cell surface and inhibiting ανβ- dependent cell adhesion (Richards et al., 2003) and to bind to vascular endothelial growth factor receptor 2 (VEGF-R2) inhibiting the activation by VEGF-A, -C and –D (Parker et al., 2005; Getmanova et al., 2006). Indeed, the test case of ¹⁰Fn3 for the in vivo behaviour in human patients is the VEGF-R2 binding CT-322 (K_D = 11 nM), currently in clinical development by Bristol Myers Squibb in cancer patients (Choy et al., 2002; Molckovsky and Siu, 2008). The CT-322 ¹⁰Fn3 domain is coupled via a C-terminal cysteine to a 40-kDa polyethylene glycol (PEG) to increase the size of the molecule above the threshold for kidney-mediated clearance. In a Phase I study in patients with solid tumours, CT-322 administered intravenously at 1 mg/kg showed a terminal half-life of 68.7 hours, based on the first dose of a weekly dosing regimen (Molckovsky and Siu, 2008). The half-life in humans is substantially shorter than that of IgG (7–26 days), as expected for a molecule lacking FcRn binding, and also shorter than the 14-day half-life reported for Cimzia, a pegylated Fab that binds to TNF-α (Choy et al., 2002). Still, the pharmacokinetics of CT-322 supported weekly intravenous dosing, and a biomarker for VEGF-R2 blockade showed sustained elevation over several repeated weekly doses. Immunogenicity was also assessed in this Phase I trial showing that 31 out of 39 patients developed antibodies to CT-322 (Beyond Antibodies Conference, 2009, San Diego) but these did not lead to a clinical significant immunogenicity (Bloom and Calabro, 2009). As for other biologics, immunogenicity should be monitored carefully for this scaffold, especially for the generation of cross-reactive antibodies binding not only to the injected mutant, but also to the endogenous fibronectin domain. In the near future we will learn more about this scaffold, since patients are currently being enrolled in a Phase II trial in glioblastoma multiforme, in which CT-322 will be tested alone or in combination with irinotecan (Campto, Pfizer), a cytostatic agent inhibiting DNA-topoisomerase I. The technology of using the ¹⁰Fn3 domain was commercialized by Adnexus, which was acquired by Bristol Myers Squibb for $430 million in 2007, proving the interest and significant potential of antibody alternatives in the field of pharmaceutical biotechnology.

A-domains (Avimers)

A family of A-domains (Huang et al., 1999; North and Blacklow, 1999) has been described to be suitable as a scaffold for the generation of new binding proteins (Silverman et al., 2005). A-domains occur as strings of multiple domains in several cell-surface receptors (Figure 13.1C). Domains of this family bind over 100 different known targets, including small molecules, proteins and viruses (Krieger and Herz, 1994; Gliemann, 1998). Such a target is typically contacted by multiple A-domains with each domain binding independently to a unique epitope, thereby binding with high avidity. Each of the 217 human A-domains comprises approximately 35 amino acids and the domains are separated by linkers that average five amino acids in length. Native A-domains fold quickly and efficiently to a uniform, stable structure mediated by calcium binding and disulfide formation. A conserved scaffold motif of only 12 amino acids is required for this common structure (Koduri and Blacklow, 2001). Stemmer and co-workers have designed a phage display library of A-domains, consisting of a conserved consensus sequence in the scaffold motif and variable amino acids at different positions (Silverman et al., 2005). Like other directed evolution technologies, the process the authors developed to isolate binding proteins, included multiple recursive cycles, each consisting of library generation and screening in functional assays. However, rather than mutagenizing the protein between cycles (e.g. for affinity maturation purposes), they added a new domain library adjacent to the domain(s) selected in previous rounds.

The result of this process is a single protein chain containing multiple domains, each of which having a separate binding function. The A-domains are therefore called ‘avimers’, from avidity multimers. A heterotetramer consisting of three IL-6 binding A-domains and an IgG binding domain (named C326) showed a remarkable affinity (picomolar range) to its target and exhibited sub-picomolar IC₅₀s in cell-proliferation assays (Silverman et al., 2005). Moreover, C326 completely abrogated acute-phase protein induction by human IL-6 in mice in a dose-dependent manner, suggesting that C326 inhibited IL-6 functions in vivo. The technology of using the modular platform of A-domains was commercialized by Avidia Inc. that was acquired by Amgen for $290 million in 2006. A placebo-controlled Phase I study of C326 was registered in the United States in September 2006, with patients suffering from Crohn’s disease. Since then, to our knowledge, new results have not been published in the public domain. Recent data (Beyond Antibodies Conference, 2009, San Diego) indicate that Amgen is further engineering avimers for the neutralization of IL-6 mediated effects.