Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs

We have used phage display to isolate a range of human domain antibodies (dAbs) that bind to mouse, rat and/or human serum albumin (SA) and can be expressed at very high levels in bacterial, yeast or mammalian cell culture. In contrast to non-SA-binding dAbs, which have terminal half-lives of less than 45 min, the half-lives of these 12 kDa 'AlbudAbs' can match the half-life of SA itself. To demonstrate the use of AlbudAbs for extending the half-lives of therapeutic drugs, we created a fusion of the interleukin-1 receptor antagonist (IL-1ra) with an AlbudAb. Soluble IL-1ra is potent inhibitor of IL-1 signalling that is approved for the treatment of rheumatoid arthritis but has a relatively short in vivo half-life. Here we show that although the AlbudAb/IL-1ra fusion has a similar in vitro potency, its in vivo efficacy can be dramatically improved due to its extended serum half-life. AlbudAbs could potentially be used to generate a range of long half-life versions of many different drugs in order to improve their dosing regimen and/or clinical effect.     

Spectral tuning of photoproteins by partnering site-directed mutagenesis strategies with the incorporation of chromophore analogs

Aequorin and obelin are photoproteins whose calcium controlled bioluminescent light emission is used for labeling in assays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. Both of these photoproteins emit blue light from a 2-hydroperoxycoelenterazine chromophore, which is non-covalently bound in the hydrophobic core of the proteins. In an effort to produce aequorin and obelin variants with improved analytical properties, such as alternative emission colors and altered decay kinetics, seven mutants of aequorin and obelin were prepared and combined with 10 different coelenterazine analogs. These semi-synthetic photoprotein mutants exhibited shifts in bioluminescent properties when compared with wild-type proteins. The bioluminescent parameters determined for these semi-synthetic photoprotein mutants included specific activity, emission spectra and decay half-life time. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants that had significantly altered bioluminescent properties. The largest emission maxima shift obtained was 44 nm, and the largest decay half-life difference was 23.91 s.     

The pharmacokinetics of an albumin-binding Fab (AB.Fab) can be modulated as a function of affinity for albumin

An AB.Fab (albumin-binding Fab) consists of a Fab and a phage-derived albumin-binding peptide. This molecule is capable of binding both antigen and albumin simultaneously. Using a Fab derived from Herceptin we generated a panel of AB.Fab variants with wide-ranging affinities for albumin. An assay that measured AB.Fab binding to albumin in solution was developed to most accurately reflect the binding affinity for albumin in vivo. Affinity varied depending upon the species of albumin tested. For rat and rabbit albumin, affinities ranged from 0.04 to 2.5 microM. Reduced affinity for albumin correlated with a reduced half-life and higher clearance rates in both species; the beta half-life ranged 6-fold while clearance ranged over 50-fold in rats and 20-fold in rabbits. To estimate the pharmacokinetic properties of an AB.Fab in humans, AB.Fab variants with similar affinities for rat and rabbit albumin were selected. Using their pharmacokinetic parameters and the principles of allometric scaling for albumin, we estimate an approximate beta half-life for an AB.Fab with 0.5 microM affinity for albumin of up to 4 days in humans with a clearance of 76 ml/h. These variants demonstrate the ability to modulate the clearance of a Fab fragment in vivo and help to establish guidelines for pharmacokinetic engineering of molecules through albumin binding.     

High solubility of random-sequence proteins consisting of five kinds of primitive amino acids

Searching for functional proteins among random-sequence librariesis a major challenge of protein engineering; the difficultiesinclude the poor solubility of many random-sequence proteins.A library in which most of the polypeptides are soluble andstable would therefore be of great benefit. Although modernproteins consist of 20 amino acids, it has been suggested thatearly proteins evolved from a reduced alphabet. Here, we haveconstructed a library of random-sequence proteins consistingof only five amino acids, Ala, Gly, Val, Asp and Glu, whichare believed to have been the most abundant in the prebioticenvironment. Expression and characterization of arbitrarilychosen proteins in the library indicated that five-alphabetrandom-sequence proteins have higher solubility than do 20-alphabetrandom-sequence proteins with a similar level of hydrophobicity.The results support the reduced-alphabet hypothesis of the primordialgenetic code and should also be helpful in constructing optimizedprotein libraries for evolutionary protein engineering.     

Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life

Chemical conjugation of small recombinant proteins with polyethylene glycol (PEG) is an established strategy to extend their typically short circulation times to a therapeutically useful range. We have investigated the production of a genetic fusion with a glycine-rich homo-amino-acid polymer (HAP) as an alternative way to attach a solvated random chain with large hydrodynamic volume. The anti-HER2 Fab fragment 4D5 was used as a model system and fused with either 100 or 200 residue polymers of the repetitive sequence (Gly(4)Ser)(n) to its light chain. Both fusion proteins were successfully produced in the periplasm of Escherichia coli and obtained as homogeneous preparations after two-step affinity chromatography via the His(6) tag fused to the heavy chain and the Strep-tag II fused to the extended light chain. Both modified Fab fragments showed binding activity towards the HER2 antigen indistinguishable from the conventional recombinant Fab fragment. When compared with the unfused Fab fragment, a significantly increased hydrodynamic volume, by ca. 120%, was observed during gel filtration for the 200 residue HAP fusion protein and, to a lesser extent, in the case of the 100 residue HAP. Difference CD measurements revealed a characteristic random coil spectrum for the 100 and 200 residue HAP fusion moieties. Finally, pharmacokinetic experiments were carried out in mice after radioiodination of the recombinant Fab fragments. Although the 100 residue HAP fusion showed a behavior very similar to the unfused Fab fragment, with a terminal plasma half-life of ca. 2 h, the 200 residue HAPylated Fab fragment gave rise to a significantly prolonged half-life of ca. 6 h. While this moderate effect may so far be most beneficial for specialized medical applications, such as in vivo imaging, the genetic engineering of optimized HAP sequences should yield pharmacokinetic properties similar to PEGylation, yet without necessitating in vitro modification steps.     

Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation

We have developed an efficient optimization technique, 'biased mutation-assembling', for improving protein properties such as thermostability. In this strategy, a mutant library is constructed using the overlap extension polymerase chain reaction technique with DNA fragments from wild-type and phenotypically advantageous mutant genes, in which the number of mutations assembled in the wild-type gene is stochastically controlled by the mixing ratio of the mutant DNA fragments to wild-type fragments. A high mixing ratio results in a mutant composition biased to favor multiple-point mutants. We applied this strategy to improve the thermostability of prolyl endopeptidase from Flavobacterium meningosepticum as a case study and found that the proportion of thermostable mutants in a library increased as the mixing ratio was increased. If the proportion of thermostable mutants increases, the screening effort needed to find them should be reduced. Indeed, we isolated a mutant with a 1200-fold longer activity half-life at 60 degrees C than that of wild-type prolyl endopeptidase after screening only 2000 mutants from a library prepared with a high mixing ratio. Our results indicate that an aggressive accumulation of advantageous mutations leads to an increase in the quality of the mutant library and a reduction in the screening effort required to find superior mutants.     

Humanization of the anti-CEA T84.66 antibody based on crystal structure data

Chimeric T84.66 (cT84.66) is a monoclonal antibody (mAb) of high specificity and affinity for the tumor-associated carcinoembryonic antigen (CEA). Radiolabeled cT84.66 has demonstrated utility in the clinic as a reagent for the radioimmunoscintigraphy and radioimmunotherapy of CEA-positive colorectal and breast malignancies. To extend the therapeutic efficacy of T84.66, humanization by complementary determining region (CDR) grafting was employed. CDR grafting is a well-established technique, though often a series of framework back-mutations is required to restore high affinity. Recently, the crystal structure of the T84.66 diabody (scFv dimer) derived from the murine T84.66 mAb was determined, facilitating the humanization process by the availability of crystal structure data for both the graft donor and graft acceptor. A search of the Protein Data Bank revealed close structural similarity (r.m.s.d. of 1.07 A) between the Fv of T84.66 and the Fv of 4D5v8, a humanized anti-p185HER2 antibody marketed as Herceptin (Trastuzumab). This resulted in two humanized versions of the T84.66 M5A and M5B mAbs that differed only in the number of murine residues present in the C-terminal half of CDR-H2. Biochemical analysis and animal biodistribution studies were conducted to evaluate the humanized mAbs. The M5A, M5B and cT84.66 mAbs showed sub-nanomolar affinity for CEA and as radiolabeled mAbs exhibited specific tumor localization in tumor bearing mice. The T84.66 M5A mAb was selected for clinical development due to a slightly higher tumor uptake and a larger content of human residues, and was renamed hT84.66. A limited-scale production and animal imaging study have demonstrated hT84.66's ability to support clinical trials. Planned clinical trials will determine the effective utilization of this structure-based approach in the development of a promising new therapeutic.     

Attenuation of green fluorescent protein half-life in mammalian cells

The half-life of the green fluorescent protein (GFP) was determinedbiochemically in cultured mouse LA-9 cells. The wild-type proteinwas found to be stable with a half-life of ~26 h, but couldbe destabilized by the addition of putative proteolytic signalsequences derived from proteins with shorter half-lives. A C-terminalfusion of a PEST sequence from the mouse ornithine decarboxylasegene reduced the half-life to 9.8 h, resulting in a GFP variantsuitable for the study of dynamic cellular processes. In anN-terminal fusion containing the mouse cyclin B1 destructionbox, it was reduced to 5.8 h, with most degradation taking placeat metaphase. The combination of both sequences produced a similarGFP half-life of 5.5 h. Thus, the stability of this marker proteincan be controlled in predetermined ways by addition of the appropriateproteolytic signals.     

Leptin in Atherosclerosis: Focus on Macrophages,Endothelial and Smooth Muscle Cells

Increasing adipose tissue mass in obesity directly correlates with elevated circulating leptin levels. Leptin is an adipokine known to play a role in numerous biological processes including regulation of energy homeostasis, inflammation, vascular function and angiogenesis. While physiological concentrations of leptin may exhibit multiple beneficial effects, chronically elevated pathophysiological levels or hyperleptinemia, characteristic of obesity and diabetes, is a major risk factor for development of atherosclerosis. Hyperleptinemia results in a state of selective leptin resistance such that while beneficial metabolic effects of leptin are dampened, deleterious vascular effects of leptin are conserved attributing to vascular dysfunction. Leptin exerts potent proatherogenic effects on multiple vascular cell types including macrophages, endothelial cells and smooth muscle cells; these effects are mediated via an interaction of leptin with the long form of leptin receptor, abundantly expressed in atherosclerotic plaques. This review provides a summary of recent in vivo and in vitro studies that highlight a role of leptin in the pathogenesis of atherosclerotic complications associated with obesity and diabetes.     

A novel tri-functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single-chain diabody with an albumin-binding domain from streptococcal protein G

The therapeutic application of small recombinant antibody molecules is often limited by a short serum half-life. In order to improve the pharmacokinetic properties, we have investigated a strategy utilizing fusion with an albumin-binding domain (ABD) from streptococcal protein G. This strategy was applied to a bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. This novel tri-functional fusion protein (scDb-ABD) was expressed in mammalian cells and recognized both antigens as well as human and mouse serum albumin. scDb-ABD was capable to retarget T cells to CEA-expressing target cells in vitro and to activate the effector cells as measured by stimulation of IL-2 release. Although activity was reduced 3-fold compared with scDb and further reduced 4-fold in the presences of human serum albumin, this assay demonstrated that scDb-ABD is active when exposed to all three antigens. Compared with scDb, the circulation time of scDb-ABD in mice was prolonged 5- to 6-fold similar to a previously described scDb-HSA fusion protein. This strategy, which adds only a small protein domain (46 amino acids) and which utilizes high-affinity, non-covalent albumin interaction, should be broadly applicable to improve serum half-lives of small recombinant antibody molecules.     

Directed evolution converts subtilisin E into a functional equivalent of thermitase

We used directed evolution to convert Bacillus subtilis subtilisinE into an enzyme functionally equivalent to its thermophilichomolog thermitase from Thermoactinomyces vulgaris. Five generationsof random mutagenesis, recombination and screening created subtilisinE 5-3H5, whose half-life at 83°C (3.5 min) and temperatureoptimum for activity (T_opt, 76°C) are identical with thoseof thermitase. The T_opt of the evolved enzyme is 17°C higherand its half-life at 65°C is >200 times that of wild-typesubtilisin E. In addition, 5-3H5 is more active towards thehydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-typeat all temperatures from 10 to 90°C. Thermitase differsfrom subtilisin E at 157 amino acid positions. However, onlyeight amino acid substitutions were sufficient to convert subtilisinE into an enzyme equally thermostable. The eight substitutions,which include known stabilizing mutations (N218S, N76D) andalso several not previously reported, are distributed over thesurface of the enzyme. Only two (N218S, N181D) are found inthermitase. Directed evolution provides a powerful tool to unveilmechanisms of thermal adaptation and is an effective and efficientapproach to increasing thermostability without compromisingenzyme activity.     

Leptin,Acting at Central Level,Increases FGF21 Expression in White Adipose Tissue via PPARβ/δ

The altered function of adipose tissue can result in obesity, insulin resistance, and its metabolic complications. Leptin, acting on the central nervous system, modifies the composition and function of adipose tissue. To date, the molecular changes that occur in epididymal white adipose tissue (eWAT) during chronic leptin treatment are not fully understood. Herein we aimed to address whether PPARβ/δ could mediate the metabolic actions induced by leptin in eWAT. To this end, male 3-month-old Wistar rats, infused intracerebroventricularly (icv) with leptin (0.2 μg/day) for 7 days, were daily co-treated intraperitoneally (ip) without or with the specific PPARβ/δ receptor antagonist GSK0660 (1 mg/kg/day). In parallel, we also administered GSK0660 to control rats fed ad libitum without leptin infusion. Leptin, acting at central level, prevented the starvation-induced increase in circulating levels of FGF21, while induced markedly the endogenous expression of FGF21 and browning markers of eWAT. Interestingly, GSK0660 abolished the anorectic effects induced by icv leptin leading to increased visceral fat mass and reduced browning capacity. In addition, the pharmacological inhibition of PPARβ/δ alters the immunomodulatory actions of central leptin on eWAT. In summary, our results demonstrate that PPARβ/δ is involved in the up-regulation of FGF21 expression induced by leptin in visceral adipose tissue.     

Conservation of mechanism, variation of rate: folding kinetics of three homologous four-helix bundle proteins

The amino acid sequence of a protein determines both its final folded structure and the folding mechanism by which this structure is attained. The differences in folding behaviour between homologous proteins provide direct insights into the factors that influence both thermodynamic and kinetic properties. Here, we present a comprehensive thermodynamic and kinetic analysis of three homologous homodimeric four-helix bundle proteins. Previous studies with one member of this family, Rop, revealed that both its folding and unfolding behaviour were interesting and unusual: Rop folds (k(0)(f) = 29 s(-1)) and unfolds (k(0)(u) = 6 x 10(-7) s(-1)) extremely slowly for a protein of its size that contains neither prolines nor disulphides in its folded structure. The homologues we discuss have significantly different stabilities and rates of folding and unfolding. However, the rate of protein folding directly correlates with stability for these homologous proteins: proteins with higher stability fold faster. Moreover, in spite of possessing differing thermodynamic and kinetic properties, the proteins all share a similar folding and unfolding mechanism. We discuss the properties of these naturally occurring Rop homologues in relation to previously characterized designed variants of Rop.     

Variants of human tissue-type plasminogen activator substituted at the protease cleavage site and glycosylation sites, and truncated at the N- and C-termini

Mutations were directed to specific regions of the human tissue-typeplasminogen activator (t-PA) gene in an effort to better definestructure–function relationships of the enzyme. Threetypes of modifications were effected by in vitro mutagenesis:elimination of glycosylation sites; substitutions of amino acidsat the cleavage site for conversion of single-chain t-PA totwo-chain t-PA; and truncations of the N- and C-termini. Thirteenvariants were purified from permanent CHO cell lines and analyzedfor specific activity, fibrin stimulation, fibrin binding, inhibitionby plasminogen activator inhibitor-2 (PAI-2) and half-life.The results of these analyses are: (i) variants with carbohydrate–depletedkringle domains possessed higher specific activities than wild-typet-PA; (ii) a cleavage site variant substituted at Arg275 withGly had greatly reduced specific activity; (iii) two variantssubstituted at Lys277 exhibited altered interactions with PAI-2;(iv) the variant with a truncated C-terminus had reduced activityin the absence of fibrin; and (v) no variants had significantlyaltered half–lives. In order to test the effects of combiningmutations, four additional variants were produced. Each combinationvariant retained at least one of the altered properties observedin the original variants, and in three of the variants the diverseproperties were additive.     

Site-directed mutagenesis of Escherichia coli translation initiation factor IF1. Identification of the amino acids involved in its ribosomal binding and recycling

Starting from a synthetic modular gene (infA^*) encoding Escherichiacoli translation initiation factor IF1, we have constructedmutants in which amino acids are deleted from the carboxyl terminusor in which His29 or His34 are replaced by Tyr or Asp residues.The mutant proteins were overproduced, purified and tested invitro for their properties in several partial reactions of thetranslation initiation pathway and for their capacity to stimulateMS2 RNA-dependent protein synthesis. The results allow for theconclusion that: (i) Arg69 is part of the 30S ribosomal subunitbinding site of IF1 and its deletion results in the substantialloss of all IF1 functions; (ii) neither one of its two histidinesis essential for the binding of IF1 to the 30S ribosomal subunit,for the stimulation of fMet-tRNA binding to 30S or 70S ribosomalparticles or for MS2 RNA-dependent protein synthesis; but (iii)His29 is involved in the 50S subunit-induced ejection of IF1from the 30S ribosomal subunit.     

Efficient production of recombinant human factor VIII by co-expression of the heavy and light chains

We have developed a high-level expression system for human bloodcoagulation factor VID (FVUI) consisting of a 90 kDa heavy (H-)chainand an 80 kDa light (L-)chain. Two expression plasmids wereprepared, one expressing the H-chain and the other expressingthe L-chain. These recombinant plasmids were designed to produceeach chain linked to short additional ammo acid residues derivedfrom the FVm precursor sequence. Furthermore, Kozak's translationinitiation consensus sequence was introduced into the startcodon for the H-chain. These modifications have dramaticallyincreased the levels of expression of these chains. Chinesehamster ovary (CHO) cells co-transfected with these two recombinantplasmids were subjected to gene amplification and cloning. Thefinal cell line, designated CTC-CF8, secretes 15 IU/day/106cells of active FVm which is indistinguishable from plasma-derivedFVm in its structure and biochemical properties. This systemis suitable for large-scale production of pathogen-free recombinanthuman FVm which can be used for the treatment of haemophiliaA patients.     

Improvement of Fc-erythropoietin structure and pharmacokinetics by modification at a disulfide bond

Erythropoietin (Epo) is a cytokine that controls the production of red blood cells (RBCs). Epo acts continuously on RBC precursors to prevent apoptosis, so it is important to maintain high levels of Epo activity when treating anemic patients. We describe here modified human Epo [Epo(NDS)] with mutations His32Gly, Cys33Pro, Trp88Cys and Pro90Ala that result in the rearrangement of the disulfide bonding pattern from Cys29-Cys33 to Cys29-Cys88 and that, in the context of an Fc-Epo(NDS) fusion protein, lead to significantly improved properties. Fc-Epo was secreted from NS/0 myeloma cells as about 35% high molecular weight aggregates, was unstable upon removal of N-linked oligosaccharides and showed poor pharmacokinetics and little efficacy in mice. In contrast, a corresponding Fc-Epo(NDS) was secreted almost exclusively as a unit dimer, was relatively stable to removal of N-linked oligosaccharides, had much improved pharmacokinetic properties and had a significantly improved effect on RBC production. These results indicate that rearrangement of the disulfide bonding pattern in a therapeutic protein can have a significant effect on pharmacokinetics and, potentially, the dosing schedule of a protein drug.     

Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor

Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 25-50 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents for radionuclide based imaging applications in various carcinomas is discussed.