Phosphate ester serum albumin affinity tags greatly improve peptide half-life in vivo

A series of phosphate ester based small molecules designed to bind tightly to serum albumin were applied to the amino terminus of an anticoagulant peptide in an effort to increase its protein binding in vivo. The tagged peptides exhibited high affinity for both rabbit and human serum albumin when passed through liquid chromatographic columns with serum albumins incorporated into the stationary phase. The peptides were then administered intravenously to rabbits and found to have a greater than 50-fold increase in plasma half life. The highest affinity peptides showed a reduction in bioactivity consistent with their sequestration away from their protein target in the presence of 0.1% rabbit serum albumin.     

Albumin binding as a general strategy for improving the pharmacokinetics of proteins

Plasma protein binding can be an effective means of improving the pharmacokinetic properties of otherwise short lived molecules. Using peptide phage display, we identified a series of peptides having the core sequence DICLPRWGCLW that specifically bind serum albumin from multiple species with high affinity. These peptides bind to albumin with 1:1 stoichiometry at a site distinct from known small molecule binding sites. Using surface plasmon resonance, the dissociation equilibrium constant of peptide SA21 (Ac-RLIEDICLPRWGCLWEDD-NH(2)) was determined to be 266 +/- 8, 320 +/- 22, and 467 +/- 47 nm for rat, rabbit, and human albumin, respectively. SA21 has an unusually long half-life of 2.3 h when injected by intravenous bolus into rabbits. A related sequence, fused to the anti-tissue factor Fab of D3H44 (Presta, L., Sims, P., Meng, Y. G., Moran, P., Bullens, S., Bunting, S., Schoenfeld, J., Lowe, D., Lai, J., Rancatore, P., Iverson, M., Lim, A., Chisholm, V., Kelley, R. F., Riederer, M., and Kirchhofer, D. (2001) Thromb. Haemost. 85, 379-389), enabled the Fab to bind albumin with similar affinity to that of SA21 while retaining the ability of the Fab to bind tissue factor. This interaction with albumin resulted in reduced in vivo clearance of 25- and 58-fold in mice and rabbits, respectively, when compared with the wild-type D3H44 Fab. The half-life was extended 37-fold to 32.4 h in rabbits and 26-fold to 10.4 h in mice, achieving 25-43% of the albumin half-life in these animals. These half-lives exceed those of a Fab'(2) and are comparable with those seen for polyethylene glycol-conjugated Fab molecules, immunoadhesins, and albumin fusions, suggesting a novel and generic method for improving the pharmacokinetic properties of rapidly cleared proteins.     

Selection of affinity ligands for protein purification from proteolytic digests

A simple method for the selection of affinity ligands from proteolytic digests by affinity chromatography is proposed. A small proportion of the peptides in the trypsin digest of bovine serum albumin (BSA) or the pepsin digest of cytochrome are retarded on insulin-immobilised or HSA (human serum albumin)-immobilised affinity columns, respectively. The peptides in these selected fractions can be immobilised onto solid phases and used in affinity chromatography procedures for the purification of insulin or HSA. © Rapid Science Ltd. 1998     

Development of mammalian serum albumin affinity purification media by peptide phage display

Several phage isolates that bind specifically to human serum albumin (HSA) were isolated from disulfide-constrained cyclic peptide phage-display libraries. The majority of corresponding synthetic peptides bind with micromolar affinity to HSA in low salt at pH 6.2, as determined by fluorescence anisotropy. One of the highest affinity peptides, DX-236, also bound well to several mammalian serum albumins (SA). Immobilized DX-236 quantitatively captures HSA from human serum; mild conditions (100 mM Tris, pH 9.1) allow release of HSA. The DX-236 affinity column bound HSA from human serum with a greater specificity than does Cibacron Blue agarose beads. In addition to its likely utility in HSA and other mammalian SA purifications, this peptide media may be useful in the proteomics and medical research markets for selective removal of mammalian albumin from serum prior to mass spectrometric and other analyses.     

Association of fibronectin with carboxy-group-modified proteins in vitro

Treatment of human immunoglobulin G, albumin and fibronectin with water-soluble carbodi-imide at pH4.75 in the presence of glycine ethyl ester resulted in an avid binding of ¹²⁵I-labelled native fibrinectin to the modified proteins. Succinoylation, reduction and alkylation or heat-denaturation had no such effect. In affinity chromatography under physiological conditions, serum was depleted of fibronectin when run through columns of the carbodi-imide-treated proteins coupled to agarose. Fractions eluted from such columns with urea were enriched in fibronectin. The binding of radiolabelled fibronectin to the carbodi-imide-treated proteins was inhibited by unlabelled fibronectin in relatively low concentrations, but also by albumin in higher concentrations. Heat-denatured albumin inhibited at concentrations approx. 10–30 times lower than native albumin. The binding reaction had a pH optimum of 6–8. It was inhibited at high ionic strength and in the presence of urea. Anionic detergents inhibited at millimolar concentrations, but non-ionic detergents did not inhibit the binding reaction. The results were interpreted as showing that: (1) fibronectin is capable of binding to itself, to immunoglobulin G and to albumin after a reduction of the negative surface charge of these proteins, and may have a general ability to bind such modified proteins; (2) this binding can take place under physiological conditions; (3) carboxy-group-modified proteins selectively bind fibronectin from serum. This novel binding phenomenon could be important in terms of the opsonin function of circulatory fibronectin. We propose that fibronectin may recognize modified (denatured) proteins and mediate their uptake by the reticuloendothelial system.     

Binding of thyroid hormones and their analogues to thyroxine-binding globulin in human serum.

The present study was undertaken to study the binding of several thyroid hormones and structurally related compounds to human serum thyroxine-binding alpha-globulin (TBG). The source of TBG was normal human serum diluted 1:100 in 0.035 M barbital buffer, pH 7.4. In the binding assays, 125I-thyroxine, unlabeled thyroxine, and diluted serum were incubated for 20 h at 37 degrees in Plexiglas equilibrium dialysis units. Two orders of binding sites were discerned: a high affinity, low capacity binding site with an affinity constant of approximately 2.5 X 10(9) M-1, and a low affinity, very high capacity binding site with an affinity constant of less than 10(6) M-1. Studies with purified TBG, serum deficient in TBG, and purified human serum albumin indicated that the high affinity site represented binding to TBG and the low affinity site represented binging to albumin. The ability of several groups of thyroid hormone analogues to bind to TBG was then investigated. As a result of these studies, the following structural features of thyroid hormones were found to be important for optimal binding activity: (a) the L-alanine side chain conformation, (b) the presence of a 4'-hydroxyl group, (c) the presence of two substituents in the inner and outer rings (positions 3, 5, 3', and 5'), and (d) the presence of either bromines or iodines in the inner ring and iodines in the outer ring. Of lesser importance was the presence of an oxygen atom in the ether position.     

Mass spectrometric identification of serum peptides employing derivatized poly(glycidyl methacrylate/divinyl benzene) particles and mu-HPLC

Biomarkers play a key role in preclinical screening and diagnosis of a disease. Various support materials are utilized for this task, in combination with MALDI-TOF-MS. The way to effectively bind serum contents and their profiling is well-elaborated by the material-enhanced laser desorption ionization (MELDI) approach. In this particular work, focus is placed on the development of a strategy to identify low molecular weight serum peptides. Poly(GMA/DVB) is derivatized in a way to achieve an affinity termed as immobilized metal ion affinity chromatography (IMAC). Iminodiacetic acid (IDA) is used as a chelating ligand, whereas copper (Cu2+) acts as a metal ion for complexing peptides and proteins out of blood serum. Polymer binds the serum compounds over a broad mass range, which includes low mass peptides and high mass albumin (66 kDa). Bound contents are eluted from material by an acetonitrile/trifluoroacetic acid mixture, which proves the reversible nature of metal and amino acid linkage. Polystyrene/divinyl benzene (PS/DVB) monolithic capillary column is used for fractionation through RP-HPLC, prior to the target spotting. The tandem TOF fragment ion mass spectra of each fraction is acquired and used to search against the Swiss-Prot database, using the Mascot search engine for the identification of peptides.     

Fibrin specific peptides derived by phage display: characterization of peptides and conjugates for imaging

Peptides that bind to fibrin but not to fibrinogen or serum albumin were selected from phage display libraries as targeting moieties for thrombus molecular imaging probes. Three classes of cyclic peptides (cyclized via disulfide bond between two Cys) were identified with consensus sequences XArXCPY(G/D)LCArIX (Ar = aromatic, Tn6), X(2)CXYYGTCLX (Tn7), and NHGCYNSYGVPYCDYS (Tn10). These peptides bound to fibrin at ～2 sites with K(d) = 4.1 μM, 4.0 μM, and 8.7 μM, respectively, whereas binding to fibrinogen was at least 100-fold weaker. The peptides also bind to the fibrin degradation product DD(E) with similar affinity to that measured for fibrin. The Tn7 and Tn10 peptides bind to the same site on fibrin, while the Tn6 peptides bind to a unique site. Alanine scanning identified the N- and C-terminal ends of the Tn6 and Tn7 peptides as most tolerant to modification. Peptide conjugates with either fluorescein or diethylenetriaminepentaaceto gadolinium(III) (GdDTPA) at the N-terminus were prepared for potential imaging applications, and these retained fibrin binding affinity and specificity in plasma. Relaxivity and binding studies on the GdDTPA derivatives revealed that an N-terminal glycyl linker had a modest effect on fibrin affinity but resulted in lower fibrin-bound relaxivity.     

Engineered human IgG antibodies with longer serum half-lives in primates

The neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies. A correlation has been established between the pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives in mice. In this study, molecular modeling was used to identify Fc positions near the FcRn binding site in a human IgG antibody that, when mutated, might alter the binding affinity of IgG to FcRn. Following mutagenesis, several IgG2 mutants with increased binding affinity to human FcRn at pH 6.0 were identified at Fc positions 250 and 428. These mutants do not bind to human FcRn at pH 7.5. A pharmacokinetics study of two mutant IgG2 antibodies with increased FcRn binding affinity indicated that they had serum half-lives in rhesus monkeys approximately 2-fold longer than the wild-type antibody.     

Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G

Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.     

Structural interrogation of phosphoproteome identified by mass spectrometry reveals allowed and disallowed regions of phosphoconformation

Background

Many biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics.

Results

The binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the μM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs.

Conclusions

The biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful consideration in antimicrobial peptide studies, as the systemic distribution can be significantly affected by HSA interactions.     

Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin—azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.     

Newly designed modifier prolongs the action of short-lived peptides and proteins by allowing their binding to serum albumin

We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO(3)S-(CH(2))(15)-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a K(a) value of 0.83 ± 0.08 × 10(6) M(-1), a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH(2))(15)-SO(3)(-) into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn(2+)-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH(2))(15)-SO(3)(-) to mice yielded prolonged and stable reduction in glucose level, 5-9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH(2))(15)-SO(3)(-)](3) to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.     

A Structure-Based Model for Predicting Serum Albumin Binding

One of the many factors involved in determining the distribution and metabolism of a compound is the strength of its binding to human serum albumin. While experimental and QSAR approaches for determining binding to albumin exist, various factors limit their ability to provide accurate binding affinity for novel compounds. Thus, to complement the existing tools, we have developed a structure-based model of serum albumin binding. Our approach for predicting binding incorporated the inherent flexibility and promiscuity known to exist for albumin. We found that a weighted combination of the predicted logP and docking score most accurately distinguished between binders and nonbinders. This model was successfully used to predict serum albumin binding in a large test set of therapeutics that had experimental binding data.     

Identification of β-Lactamase Inhibitory Peptide Using Yeast Two-Hybrid System

Random oligonucleotide fragments were designed and amplified by PCR and fused with the activating domain of pGAD424 to construct a random peptide library. The DNA fragment encoding beta-lactamase was fused with the binding domain of pGBT9(+2). Subsequently, using yeast two-hybrid system we found two positive clones encoding peptides P1 and P2 that have the ability to bind beta-lactamase in vivo. The genes encoding P1 and P2 were cloned into pGEX-4T-1. GST-peptide fusion proteins were expressed in Escherichia coli and isolated by glutathione-Sepharose 4B affinity chromatography. Finally, P1 and P2 were cleaved from the fusion protein with thrombin and purified by ultrafiltration. Inhibition assay of peptides with beta-lactamase in vitro indicated that only P1 has the ability to inhibit beta-lactamase.     

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.     

Fluorescence and analytical ultracentrifugation analyses of the interaction of the tyrosine kinase inhibitor, tyrphostin AG 1478-mesylate, with albumin

Quantifying the interaction of drugs with carrier proteins in plasma is of importance for understanding effective drug delivery to disease-affected tissues. In this study, we employed analytical ultracentrifugation and steady-state fluorescence spectroscopy to characterize the interaction of a potential new anticancer drug, AG 1478-mesylate, with plasma proteins in a suspension of normal serum albumin (NSA). We found that mesylate salt of AG 1478, an epidermal growth factor receptor kinase inhibitor, sediments in 0.1%(w/v) NSA as a complex with a sedimentation coefficient of 3.8 S. This is consistent with the size of human serum albumin. This interaction was quantitated by meniscus depletion sedimentation and fluorescence titration analyses. AG 1478-mesylate binds to albumin with an apparent single-site affinity (K(d)) of 120 microM. In this article, we show that the cyclodextrin carrier molecule, Captisol, increases the apparent affinity of the hydrophobic AG 1478-mesylate for albumin (K(d)=4-6 microM), and we propose that the AG 1478-mesylate-Captisol (1:1) complex binds to albumin with at least 10-fold higher affinity than does AG 1478-mesylate ligand alone. A fluorenylmethoxycarbonyl-sulfonic acid (FMS) derivative of the 6-aminoquinazoline analog of AG 1478, which was designed to have improved serum-binding properties, was shown by fluorescence analysis to bind with approximately 100-fold greater affinity than the parent compound. This has significant implications in the effective delivery of therapeutic agents in vivo.     

Purification and specificity of antibodies to inosine 5'-monophosphate

Antibodies to inosine 5'-monophosphate elicited in rabbits by immunization with a conjugate of IMP (oxidized with periodate) and bovine serum albumin have been purified by affinity chromatography. By the use of two affinity columns, Sepharose-IMP and Sepharose-oligo(I), the antibodies have been fractionated into three fractions. By gel diffusion, the three fractions were found to react with the conjugates of bovine serum albumin and IMP, GMP and AMP respectively. The association constants for the binding of the Fab fragments purified on the Sepharose-oligo(I) column and several haptens have been deduced from fluorescence experiments. It is shown that the base and the phosphate group play an important part in the binding of IMP to Fab fragments. No reaction has been found between the antibodies and poly(I).poly(C) by gel diffusion. However, the antibodies interact with poly(I).poly(C) since they decrease the thermal stability of poly(I).poly(C).     

Evaluation of quantitative performance of sequential immobilized metal affinity chromatographic enrichment for phosphopeptides

We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. Although our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semiquantitative method when used in large-scale phosphoproteomics studies in complex backgrounds.     

Reduction of the amyloidogenicity of a protein by specific binding of ligands to the native conformation

It is known that human muscle acylphosphatase (AcP) is able, under appropriate conditions in vitro, to aggregate and form amyloid fibrils of the type associated with human diseases. A number of compounds were tested for their ability to bind specifically to the native conformation of AcP under conditions favoring denaturation and subsequent aggregation and fibril formation. Compounds displaying different binding affinities for AcP were selected and their ability to inhibit protein fibrillization in vitro was evaluated. We found that compounds displaying a relatively high affinity for AcP are able to significantly delay protein fibrillization, mimicking the effect of stabilizing mutations; in addition, the effectiveness of such outcome correlates positively to both ligand concentration and affinity to the native state of AcP. By contrast, the inhibitory effect of ligands on AcP aggregation disappears in a mutant protein in which such binding affinity is lost. These results indicate that the stabilization of the native conformation of amyloidogenic proteins by specific ligand binding can be a strategy of general interest to inhibit amyloid formation in vivo.