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• W2168848424 abstract "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-NH2) 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 vivoclearance 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. 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-NH2) 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 vivoclearance 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. The effectiveness of recombinant protein pharmaceuticals depends heavily on the intrinsic pharmacokinetics of the natural protein. Because the kidney generally filters out molecules below 60 kDa, efforts to reduce clearance have focused on increasing molecular size through protein fusions, glycosylation, or the addition of polyethylene glycol polymers (i.e.PEG). 1The abbreviations used are: PEG, polyethylene glycol; Fv, the variable light and variable heavy domains of an IgG; scFv, a single-chain Fv; Fab, the antigen binding fragment consisting of the light chain and the variable and first constant domains of the heavy chain; Fab′2, two Fab fragments joined by disulfides at the hinge region; TF, the extracellular domain of human tissue factor (residues 1–219); D3H44, a humanized IgG directed against human TF; D3H44 Fab, the Fab portion of D3H44; D3H44-L, D3H44 Fab with SA06 fused to the carboxyl terminus of the light chain; D3H44-Ls, D3H44 Fab lacking the light-heavy chain disulfide with SA06 fused to the carboxyl terminal of the light chain; FX, coagulation Factor X; HRP, horseradish peroxidase; TCEP, tri(2-carboxyethyl)phosphine hydrochloride; PBS, phosphate-buffered saline; LC/MS/MS, liquid chromatography/mass spectrometry/mass spectrometry; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ELISA, enzyme-linked immunosorbent assay. 1The abbreviations used are: PEG, polyethylene glycol; Fv, the variable light and variable heavy domains of an IgG; scFv, a single-chain Fv; Fab, the antigen binding fragment consisting of the light chain and the variable and first constant domains of the heavy chain; Fab′2, two Fab fragments joined by disulfides at the hinge region; TF, the extracellular domain of human tissue factor (residues 1–219); D3H44, a humanized IgG directed against human TF; D3H44 Fab, the Fab portion of D3H44; D3H44-L, D3H44 Fab with SA06 fused to the carboxyl terminus of the light chain; D3H44-Ls, D3H44 Fab lacking the light-heavy chain disulfide with SA06 fused to the carboxyl terminal of the light chain; FX, coagulation Factor X; HRP, horseradish peroxidase; TCEP, tri(2-carboxyethyl)phosphine hydrochloride; PBS, phosphate-buffered saline; LC/MS/MS, liquid chromatography/mass spectrometry/mass spectrometry; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ELISA, enzyme-linked immunosorbent assay. For example, fusions to large long lived proteins such as albumin (1Syed S. Schuyler P. Kulczycky M. Sheffield W.P. Blood. 1997; 89: 3243-3252Crossref PubMed Google Scholar, 2Yeh P. Landais D. Lemaitre M. Maury I. Crenne J.-Y. Becquart J. Murry-Brelier A. Boucher F. Montay G. Fleer R. Hirel P.-H. Mayaux J.-F. Klatzmann D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1904-1908Crossref PubMed Scopus (115) Google Scholar) or the Fc portion of an IgG (3Ashkenazi A. Chamow S.M. Curr. Opin. Immunol. 1997; 9: 195-200Crossref PubMed Scopus (67) Google Scholar), the introduction of glycosylation sites (4Keyt B.A. Paoni N.F. Refino C.J. Berleau L. Nguyen H. Chow A. Lai J. Pena L. Pater C. Ogez J. Etcheverry T. Botstein D. Bennett W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3670-3674Crossref PubMed Scopus (301) Google Scholar), and conjugation with PEG (5Clark R. Olson K. Fuh G. Marian M. Mortensen D. Teshima G. Chang S. Chu H. Mukku V. Canova-Davis E. Somers T. Cronin M. Winkler M. Wells J.A. J. Biol. Chem. 1996; 271: 21969-21977Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 6Lee L.S. Conover C. Shi C. Whitlow M. Filpula D. Bioconjugate Chem. 1999; 10: 973-981Crossref PubMed Scopus (115) Google Scholar, 7Tanaka H. Satake-Ishikawa R. Ishikawa M. Matsuki S. Asano K. Cancer Res. 1991; 51: 3710-3714PubMed Google Scholar) have been used. Through these methods, thein vivo exposure of protein therapeutics has been extended. Small molecule drugs have long relied on their association with various plasma components to improve their pharmacokinetic properties in vivo; however, a drug associated with plasma protein is usually unavailable for binding to the target although its half-life is extended. Since only the unbound fraction of the small molecule is generally functionally active, a fine balance must be maintained between the concentration of free drug required for efficacy and the frequency at which it must be administered (8). Albumin (molecular mass ∼67 kDa) is the most abundant protein in plasma, present at 50 mg/ml (600 μm), and has a half-life of 19 days in humans (9Peters Jr., T. Adv. Protein Chem. 1985; 37: 161-245Crossref PubMed Scopus (2515) Google Scholar, 10Peters Jr., T. All about Albumin. Academic Press, Inc., San Diego, CA1996Google Scholar). Albumin serves to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in plasma. There are several major small molecule binding sites in albumin that have been described. Warfarin is known to bind at site I, benzodiazepines and indoles at site II, and cardenolides and biliary acids at site III. In addition, there is an important metal ion binding site. Noncovalent association with albumin has been shown to extend the half-life of short lived proteins. A recombinant fusion of the albumin binding domain from streptococcal protein G to human complement receptor type 1 increased its half-life 3-fold to 5 h in rats (11Makrides S.C. Nygren P.-A. Andrews B. Ford P.J. Evans K.S. Hayman E.G. Adari H. Levin J. Uhlen M. Toth C.A. J. Pharmacol. Exp. Ther. 1996; 277: 534-542PubMed Google Scholar). In addition, fusion to this domain has served to enhance the immunological response directed to peptide antigens (12Sjolander A. Nygren P.-A. Stahl S. Berzins K. Uhlen M. Perlmann P. Andersson R. J. Immunol. Methods. 1997; 201: 115-123Crossref PubMed Scopus (54) Google Scholar). In another example, when insulin was acylated with fatty acids to promote association with albumin (13Kurtzhals P. Havelund S. Jonassen I. Kiehr B. Larsen U.D. Ribel U. Markussen J. Biochem. J. 1995; 312: 725-731Crossref PubMed Scopus (304) Google Scholar, 14Markussen J. Havelund S. Kurtzhals P. Andersen A.S. Halstrom J. Hasselager E. Larsen U.D. Ribel U. Schaffer L. Vad K. Jonassen I. Diabetologia. 1996; 39: 281-288Crossref PubMed Scopus (244) Google Scholar), a protracted effect was observed when injected subcutaneously in rabbits or pigs. Together, these studies demonstrate a linkage between albumin binding and prolonged action. In this report, peptide phage display was used to develop peptides that selectively bind albumin with high affinity. These peptides bind to albumin from multiple species at a novel site distinct from the known classical binding sites. To test whether association of a short lived protein with albumin could improve its pharmacokinetic properties, one albumin binding peptide was added to a Fab through the use of a simple recombinant fusion that rendered it capable of binding albumin without affecting antigen binding. We demonstrate this approach as a viable route to increasing the half-life of potentially important protein pharmaceuticals. Eighteen phage libraries expressing random peptide sequences fused to the major coat protein, P8 (15Lowman H.B. Chen Y.M. Skelton N.J. Mortensen D.L. Tomlinson E.E. Sadick M.D. Robinson I.C.A.F. Clark R.G. Biochemistry. 1998; 37: 8870-8878Crossref PubMed Scopus (92) Google Scholar), were pooled into four groups: pool A contained CX 2GPX 4C,X 4CX 2GPX 4CX 4, and X iCX jCX k, where j = 8–10; pool B containedX 20 andX iCX jCX k, where j = 4–7; pool C containedX 8 andX 2CX jCX 2, where j = 4–6; pool D containedX 2CX jCX 2where j = 7–10. X represents any of the 20 naturally occurring l-amino acids, and in pools A and B,i + j + k = 18 and ‖i − k‖ < 2. Each library has in excess of 1010 clones. The phage library pools were suspended in binding buffer (PBS, 1% ovalbumin, 0.05% Tween 20) and sorted against rabbit, rat, or human albumin (Sigma) immobilized directly on Maxisorp plates (Nunc, Roskilde, Denmark) at 10 μg/ml in PBS overnight at 4 °C. Plates were blocked for 1 h at 25 °C using PBS, containing 1% ovalbumin except for round 4, where Tris-buffered saline-casein blocker (Pierce) was used. Phage were allowed to bind for 2 h. Unbound phage were removed by repetitive washing with PBS, 0.05% Tween 20, and bound phage were eluted with 500 mm KCl, 10 mmHCl, pH 2. Eluted phage were propagated in XL1-Blue cells with VCSM13 helper phage (Stratagene, La Jolla, CA). Enrichment was monitored by titering the number of phage that bound to an albumin coated well compared with a well coated with ovalbumin or casein. Phage clones (∼1011phage) were added to Maxisorp plates coated with mouse, rat, rabbit, bovine, rhesus, or human albumin (Sigma) as described above. The microtiter plate was washed with PBS, 0.05% Tween 20, and bound phage were detected following incubation with HRP/anti-M13 conjugate (Amersham Biosciences) in PBS, 0.05% Tween 20. The amount of HRP bound was measured using ABTS/H2O2 substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and monitoring the absorbance at 405 nm. A soft randomized library was designed using an oligonucleotide coding for clone RB but synthesized with a 70:10:10:10 mixture of bases as described (16Dennis M.S. Eigenbrot C. Skelton N.J. Ultsch M.H. Santell L. Dwyer M.A. O'Connell M.P. Lazarus R.A. Nature. 2000; 404: 465-470Crossref PubMed Scopus (203) Google Scholar). A fully randomized library was designed holding highly selected residues (underlined) constant:X 5D X CLP X WGCLW X 4; randomized positions (X) were coded by NNS. Both the soft randomized and fully randomized libraries were sorted against rat, rabbit, and human serum albumin as above. Peptides were synthesized by either manual or automated (Milligen 9050) Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based solid phase synthesis on a 0.25-mmol scale using a PEG-polystyrene resin as described (17Dennis M.S. Roberge M. Quan C. Lazarus R.A. Biochemistry. 2001; 40: 9513-9521Crossref PubMed Scopus (41) Google Scholar). The carboxyl terminal lysine of peptide SA08 was derivatized withN-hydroxysuccinimide-LC-biotin as recommended by the manufacturer (Pierce) and purified by reversed phase high pressure liquid chromatography, yielding SA08b (Ac-QGLIGDICLPRWGCLWGDSVKb-NH2, where Kb refers to lysine-biotin). Rat, rabbit, or mouse albumin was immobilized directly on Maxisorp plates and blocked as above. Samples, serially diluted in binding buffer were added to the plate, followed immediately by the addition of 10 nm SA08b for 1 h at 25 °C. SA08b has an EC50 of 2 and 4 nm for rat and rabbit albumin, respectively. The microtiter plate was washed with PBS, 0.05% Tween 20, and bound SA08b was detected with streptavidin/HRP (Roche Molecular Biochemicals). The amount of HRP bound was measured using ABTS/H2O2 substrate as above. The binding affinities between SA peptides and albumin were obtained using a BIAcore 3000 (BIAcore Inc., Piscataway, NJ). Human, rabbit, and rat albumin were captured on a CM5 chip using amine coupling at ∼5000 resonance units. SA peptides at 0, 0.625, 1.25, 2.5, 5, and 10 μm were injected at a flow rate of 20 μl/min for 30 s. The bound peptides were allowed to dissociate for 5 min before matrix regeneration using 10 mm glycine, pH 3. The signal from an injection passing over an uncoupled cell was subtracted from that of an immobilized cell to generate sensorgrams of the amount of peptide bound as a function of time. The running buffer, PBS containing 0.05% Tween 20, was used for all sample dilutions. BIAcore kinetic evaluation software (version 3.1) was used to determineK D from the association and dissociation rates using a one-to-one binding model. Three male New Zealand White rabbits were administered an intravenous bolus dose of 2 mg/kg of SA21 in PBS. Eighteen blood samples were collected at serial time points just prior to dosing and from 1 min to 21 days postdosing. Samples were collected in tubes containing sodium citrate as an anticoagulant and then centrifuged, and the plasma portion was frozen at −70 °C until analysis using an electrospray ionization, LC/MS/MS method. The mass spectrometer used was an API 4000 (Applied Biosystems/MDS Sciex, Foster City, CA). The autosampler was a CTCPAL System (Leap Technologies, Chapel Hill, NC) equipped with a cooling stack. The high pressure liquid chromatography system consisted of the Shimadzu SCI-10A system controller with two Shimadzu LC-10A pumps. A prefilter was placed in front of the analytical column (C18, 2.1 × 50 mm; Phenomenex Synergi 4μ MAX-RP 80A). The column flow was set at 500 μl/min. Solvent systems A (100% H2O) and B (100% acetonitrile) both contained 1% formic acid. A fast gradient (0.0–0.4 min, 90% A; 0.4–0.8 min, from 90% A to 10% A; 0.8–1.8 min, 10% A; 1.8–2.0 min, from 10% A to 90% A; 2.0–3.0 min, 90% A) was used for all analysis. The injection volume was 10 μl. SA 21 was initially characterized via direct infusion (1 μm in 20% acetonitrile) into the API 4000. The transition of triply charged ion 758 to fragment 948 was optimized for analysis in plasma matrix. Standard curves were prepared in citrated rabbit plasma in 96-well plates by adding 10 μl of diluted SA21 into 190 μl of plasma over a range of 2500 to 4.9 nm. Tri(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma-Aldrich) was used as a reducing agent to enhance peptide recovery and was added to all samples at a final concentration of 2 mm for 20 min at 37 °C. Plasma proteins were then precipitated by the addition of 160 μl of 80% acetonitrile to 40 μl of plasma for 10 min and removed by centrifugation for 10 min at 10 °C. The supernatant was transferred to another 96-well plate, and the plate was sealed with silicone sealing mat (AxyGen, Inc., Union City, CA). Samples were placed in the autosampler at 5 °C to be analyzed by LC/MS/MS. Samples with a high concentration of SA21 (PK samples from 1 min to 7 h) were diluted 10-fold with blank rabbit plasma prior to TCEP addition. Pharmacokinetic parameters were fitted to a one-compartment elimination model using WinNonlin software, version 3.1 (Pharsight Corp., Mountain View, CA) to obtain the clearance (CL), volume of distribution (V 1), elimination half-life ( t12), and drug exposure (AUC). D3H44 Fab was produced as described (18Presta 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. Kirchhofer D. Thromb. Haemost. 2001; 85: 379-389Crossref PubMed Scopus (55) Google Scholar). D3H44-L was constructed by inserting DNA encoding a linker sequence (GGGS) followed by SA06 (QRLMEDICLPRWGCLWEDDF) onto the carboxyl-terminal end of the light chain of D3H44 using Kunkel mutagenesis (19Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar). D3H44-Ls was constructed in the same manner; however, additional mutations were added to remove the disulfide between the light and heavy chains of the Fab. Each chain was terminated one residue prior to the heavy-light interchain disulfide followed by the addition of the linker sequence and peptide SA06 to the light chain. The resulting plasmids, pD3H44-L and pD3H44-Ls, were confirmed by DNA sequencing. Expression of D3H44-L and D3H44-Ls was carried out as described (18Presta 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. Kirchhofer D. Thromb. Haemost. 2001; 85: 379-389Crossref PubMed Scopus (55) Google Scholar). Cells were harvested; frozen; suspended in 1 mm EDTA, 10 mm Tris, pH 8, 0.5 mm phenylmethylsulfonyl fluoride; and disrupted using a tissue homogenizer. D3H44-L and D3H44-Ls were rapidly purified using a Hi-Trap tissue factor (TF) affinity column followed by a Hi-Trap rabbit albumin affinity column (Amersham Biosciences), each generated as recommended by the manufacturer. Both columns were washed with PBS and eluted with 50 mm HCl. Eluted fractions were immediately neutralized using 1 m Tris, pH 8. D3H44-L and D3H44-Ls were further purified using Sephacryl S-200 gel filtration (Amersham Biosciences) in PBS followed by an extraction with Triton X-114 to remove traces of endotoxin (20Aida Y. Pabst M.J. J. Immunol. Methods. 1990; 132: 191-195Crossref PubMed Scopus (479) Google Scholar). D3H44-L and D3H44-Ls were judged greater than 99% pure by SDS-PAGE. The FX activation assay and the prothrombin time assay were performed as described previously (18Presta 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. Kirchhofer D. Thromb. Haemost. 2001; 85: 379-389Crossref PubMed Scopus (55) Google Scholar). A soluble mutant of TF (E219C) (21Kelley R.F. Methods. 1994; 6: 111-120Crossref Scopus (24) Google Scholar) was specifically biotinylated using a 4-fold molar excess of biotin BMCC (Pierce) in 200 mm Tris, pH 7.5, 20% Me2SO. The reaction was desalted using an NAP5 (AmershamBiosciences) column and concentrated via Centricon YM10 (Millipore, Bedford, MA) to 260 μm. Biotinylated TF binds to D3H44 Fab immobilized directly on a Maxisorp plate (10 μg/ml in PBS, overnight at 4 °C and blocked for 1 h at 25 °C using casein blocker (Pierce)) with an EC50 of 11 nm. For the albumin/TF sandwich assay, rabbit albumin was immobilized as described above. Dilutions of D3H44 Fab, D3H44-L, or D3H44-Ls were added in binding buffer for 1 h. The plate was washed with PBS, 0.05% Tween 20, and 50 nm biotinylated TF in binding buffer was added for 1 h. The microtiter plate was washed with PBS, 0.05% Tween 20, and streptavidin/HRP was added. After a final wash, bound HRP was measured as above. Groups of three New Zealand White rabbits were given an intravenous bolus of 400–525 μg/kg D3H44 variants (D3H44 Fab, D3H44-L, D3H44-Ls) into the marginal ear vein. Plasma samples were obtained from an arterial catheter placed in the contralateral ear over a 21-day period for analysis by TF ELISA (see below). Individual plasma concentration versus time curves were fitted to a two-compartment elimination model using WinNonlin version 3.0 (Pharsight, Inc., Mountain View, CA). The pharmacokinetic parameters of clearance, V 1, steady state volume (V ss), t12, AUC, and AUC corrected for actual dose administered (AUC/dose) were averaged for each treatment group. Differences between groups were determined by analysis of variance, with significance at p < 0.05. Groups of nine BALB/c mice received a 5.0 mg/kg intravenous bolus of D3H44 Fab or D3H44-L into the tail vein. Plasma samples were obtained by eye bleed from three mice per time point over 2–9 days and assayed for concentration of D3H44 using the TF ELISA. The average plasma concentration was obtained for each time point and fitted to a two-compartment elimination model using WinNonlin version 3.0. As the analysis of the mouse experiment produces one average concentrationversus time profile for each variant, PK parameters are presented as a single estimate for the group of nine mice. The concentration of D3H44 Fab, D3H44-L, and D3H44-Ls in rabbit plasma was determined using a TF ELISA. Maxisorp plates were coated overnight at 4 °C with 1 μg/ml TF (Genentech, Inc., South San Francisco, CA) in 50 mmsodium carbonate buffer, pH 9.6. Plates were blocked with 0.5% ovalbumin in PBS, pH 7.4. Diluted antibody standards (0.23–50 ng/ml) and samples (minimum dilution 1:100) in PBS containing 0.5% ovalbumin, 0.05% polysorbate 20, 0.35 n NaCl, 5 mm EDTA, 0.25% CHAPS, 0.2% bovine γ-globulins (Sigma), and 1% plasma were added to the plates for 2 h. Antibody bound to the plates was detected with HRP-conjugated goat anti-human Fab′2 antibody (Jackson ImmunoResearch, West Grove, PA). Bound HRP was measured using the substrate 3,3′,5,5′-tetramethyl benzidine/H2O2 (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and the change in absorbance was monitored at 450 nm. Data falling in the linear range of the standard curve was used to calculate D3H44 concentrations in the samples. D3H44 Fab and D3H44-L in mouse plasma were assayed in the same enzyme-linked immunosorbent assay except that samples were diluted in buffer without 1% plasma and the standard curve range was 0.31–40 ng/ml. Naive peptide phage library pools A through D were selected against rat, rabbit, and human albumin. Each pool, with the exception of pool D when human albumin was the target, showed enrichment for each species of albumin. The sequences from the enriched pools revealed in each case that a single clone had taken over the pool. The inferred peptide sequences from these clones are shown in Table I.Table ISequences of phage clones selected from polyvalent naive libraries for binding rat, rabbit, or human albuminThe locations of fixed cysteines from the library design are shaded. Sequence identity among clones derived from different phage libraries is boxed. A qualitative assessment of the ability of phage bearing the indicated peptide sequence to bind human (HSA), rabbit (BuSA), or rat (RSA) albumin is indicated. Open table in a new tab The locations of fixed cysteines from the library design are shaded. Sequence identity among clones derived from different phage libraries is boxed. A qualitative assessment of the ability of phage bearing the indicated peptide sequence to bind human (HSA), rabbit (BuSA), or rat (RSA) albumin is indicated. Interestingly, albumin has greater than 70% amino acid sequence identity between these species, yet unique peptide sequences originating from within a given phage library pool were identified for each species. The sequence similarity observed between clones HB and HC, RA and RD, and RB and RC, despite their origins from independent phage library pools, suggests the importance of the homologous residues in binding to the respective species of albumin. Individual phage clones were examined using a phage binding assay, a quick qualitative screen to assess species selectivity. Whereas phage clones generally bound only to the albumin for which they were selected, clones HB and HC, selected for binding to human albumin, also bound to rat albumin, and clone RB, selected for binding to rat albumin, bound albumin from all three species (Table I). None of the phage clones bound to structurally unrelated ovalbumin, indicating that the interaction with albumin was specific. Because of its broad recognition of rat, rabbit, and human albumin, clone RB was chosen for sequence maturation on phage using “soft randomization.” The potential diversity of a soft randomized library is the same as the starting naive libraries; however, a soft randomized library maintains a bias toward a particular sequence, in this case DNA coding for the peptide sequence from clone RB. Sequences after four rounds of selection against rat, rabbit, or human albumin are shown in Table II along with results from the phage binding assay. All clones were specific for the albumin to which they were selected based upon their lack of binding to immobilized ovalbumin and casein; however, several clones also bound to albumin from other species including bovine, rhesus, and mouse albumin (TableII).Table IISequences of phage clones selected for binding rat, rabbit, or human albumin following soft randomization of clone RBAmino acids positions identical in clone RB, the starting sequence used for soft randomization are shaded. A qualitive assessment of the ability of phage bearing the indicated peptide sequence to bind human (HSA), rabbit (BuSA), rat (RSA), bovine (BSA), rhesus (RhSA), and mouse (MSA) albumin is indicated (nd indicates not determined). Open table in a new tab Amino acids positions identical in clone RB, the starting sequence used for soft randomization are shaded. A qualitive assessment of the ability of phage bearing the indicated peptide sequence to bind human (HSA), rabbit (BuSA), rat (RSA), bovine (BSA), rhesus (RhSA), and mouse (MSA) albumin is indicated (nd indicates not determined). Since at any given position, the amino acid present in the parent sequence was designed to appear approximately half the time in these libraries, only fully conserved positions are likely to indicate important structural or contact elements that support albumin binding. A final library that kept these highly selected residues (underlined) constant,X 5D X CLP X WGCLW X 4, while allowing all 20 amino acids at the 11 remaining positions allowed a more extensive search of pertinent sequence space. The sequence preferences at each randomized position resulting from selection against rabbit albumin are shown in Fig.1. A similar profile was observed from sequences selected for binding rat and human albumin (not shown). For each species of albumin, there was a strong preference for Ile at position 7 and Arg at position 11, thus generating a core consensus of DICLPRWGCLW. Additionally, there was a general preference for negatively charged residues (Asp or Glu) at positions flanking this core, particularly on the carboxyl terminus. Several peptides patterned after the sequences selected for albumin binding were synthesized. Their binding to human, rabbit, and rat albumin was assessed by Biacore, and a peptide competition assay was used to assess their affinity for rabbit and mouse albumin (Tables III andIV). The IC50 values obtained for binding to rabbit albumin compared favorably withK d values determined by Biacore (Table III). In comparison with rabbit and rat albumin, the peptides bind more weakly to human and more tightly to mouse albumin; however, the rank affinity of a given peptide is generally maintained from species to species. Peptide SA15, representing the consensus for binding rabbit albumin (Fig. 1), had the lowest IC50 value in the peptide binding assay and highest affinity by surface plasmon resonance for rabbit albumin (Table III). A linear peptide, identical to SA06 but with both Cys residues changed to Ala, had an IC50 greater than 50 μm, demonstrating the importance of the disulfide. In addition, the affinity of the peptides for rabbit albumin diminished with reduction in the length of the peptides (T" @default.
• W2168848424 created "2016-06-24" @default.
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• W2168848424 date "2002-09-01" @default.
• W2168848424 modified "2023-10-05" @default.
• W2168848424 title "Albumin Binding as a General Strategy for Improving the Pharmacokinetics of Proteins" @default.
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