The migration and invasion of cells are necessary for many normal and pathological processes, including tissue remodeling, embryo implantation, angiogenesis, and tumor cell invasion and metastasis (1-4). Recent reports suggest that these processes require an active cell-surface proteolytic cascade (5, 6). Important components of this cascade are the plasminogen activator/plasmin system, as well as the matrix metalloproteinases (6). The requirement for both protease expression and a cell-surface protease binding protein has been demonstrated most clearly in the case of urokinase plasminogen activator (uPA) and the uPA receptor (uPAR) but has also been recently described for type IV collagenase (7,8). It has been shown in human colon and breast carcinomas that urokinase is expressed in stromal, fibroblast-like cells and uPAR is expressed on tumor epithelial cells or macrophages, respectively (9, 10). This suggests that a paracrine relationship between uPA and its receptor occurs in these pathological conditions. The in vitro observation that human tumor cell invasion is proportional to receptor-bound urokinase, not total urokinase synthesis, further supports the hypothesis that cell-surface protease localization is a key for invasion (11,12). Other results show that plasminogen activation is more efficient when both uPA and plasminogen are bound on a cell surface, and that cell-surface plasmin is resistant to inhibition by a2-antiplasmin (7 (23). We report here the identification and characterization of peptide antagonists with nanomolar affinity for the human uPAR by using a 15-mer peptide library. This extension of bacteriophage peptide display to cell-surfaceexpressed proteins expands the utility of the method to a wide variety of biologically interesting targets. MATERIALS AND METHODSReagets and Stains. Bacteriophage library construction and bacteriophage growth and isolation were performed as described by Devlin et al. (15). The Escherichia coli strains H249, a recA, sup0, F' derivative of MM294, and JM103 [F' traD36 proAB+ lacJq lacZAM15 A(pro-lac) supE hsdR endAI sbcBl5 thi-1 strAAi] were used for these experiments. Recombinant DNA manipulations were according to Sambrook et aL (24); electrocompetent E. coil HB101 (Stratagene) were used for subcloning unless otherwise noted. Restriction enzymes were from New England Biolabs; high molecularweight human uPA, plasminogen, and the anti-uPAR monoclonal antibody 3936 were from American Diagnostica (Greenwich, CT). Streptavidin was from Molecular Probes or Sigma, and bovine serum albumin (BSA) was from Sigma. Immulon-2 96-well plates were fom Dynatech. The plasmin substrate S-2251 was from Kabi Pharmacia Diagnostics (Piscataway, NJ). Linear synthetic peptides were prepared on an Applied Biosystems model 430A peptide synthesizer using 9-fluorenylmethoxycarbonyl-based chemistry and were purified by reversed-phase HPLC after trifluoroacetic acid cleavage. Alternatively, peptides were obtained from Chiron Mimotopes (Melbourne, Australia). The cyclic uPA peptide encom...

We have modified recombinant interleukin-2 (rIL-2) to facilitate site-directed covalent attachment of monomethoxy polyethylene glycol (PEG). The site chosen for modification and subsequent covalent attachment with PEG (PEGylation) was the single glycosylation position found in the native interleukin-2 (IL-2). The mutant protein was expressed in E. coli, purified, and PEGylated with a PEG-maleimide reagent to obtain PEG-cys3-rIL-2. The PEG-cys3-rIL-2 had full bioactivity relative to the unmodified molecule and had an increase in hydrodynamic size sufficient to increase its systemic exposure by approximately 4 fold. This method has general applicability for modifying any therapeutic protein at a specific site and thereby alter its potency. In particular, it can be used to attach PEG to prokaryotically expressed recombinant proteins at their glycosylation sites.

On our initial discovery that prion protein (PrP)-derived peptides were capable of capturing the pathogenic prion protein (PrP Sc ), we have been interested in how these peptides interact with PrP Sc . After screening peptides from the entire human PrP sequence, we found two peptides (PrP 19 -30 and PrP100-111) capable of binding full-length PrP Sc in plasma, a medium containing a complex mixture of other proteins including a vast excess of the normal prion protein (PrP C ). The limit of detection for captured PrP Sc was calculated to be 8 amol from a Ϸ10 5 -fold dilution of 10% (wt/vol) human variant Creutzfeldt-Jakob disease brain homogenate, with >3,800-fold binding specificity to PrP Sc over PrP C . Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the peptides and PrP Sc . Neither hydrophobic nor polar interactions appear to correlate with binding activity. The peptide-PrP Sc interaction was not sequence-specific, but amino acid composition affected binding. Binding occurs through a conformational domain that is only present in PrP Sc , is species-independent, and is not affected by proteinase K digestion. These and other findings suggest a mechanism by which cationic domains of PrP C may play a role in the recruitment of PrP C to PrP Sc .plasma ͉ Creutzfeldt-Jakob disease ͉ detection ͉ cationic interaction ͉ diagnostic

Cardiac troponins (cTns) are released and cleared slowly after myocardial injury. Cardiac myosin–binding protein C (cMyC) is a similar cardiac-restricted protein that has more rapid release and clearance kinetics. Direct comparisons are hampered by the lack of an assay for cMyC that matches the sensitivity of the contemporary assays for cTnI and cTnT. Using a novel pair of monoclonal antibodies, we generated a sensitive assay for MyC on the Erenna platform (Singulex) and compared serum concentrations with those of cTnI (Abbott) and cTnT (Roche) in stable ambulatory cardiac patients without evidence of acute cardiac injury or significant coronary artery stenoses. The assay for cMyC had a lower limit of detection of 0.4 ng/L, a lower limit of quantification (LLoQ) of 1.2 ng/L (LLoQ at 20% coefficient of variation [CV]) and reasonable recovery (107.1 ± 3.7%; mean ± standard deviation), dilutional linearity (101.0 ± 7.7%), and intraseries precision (CV, 11 ± 3%) and interseries precision (CV, 13 ± 3%). In 360 stable patients, cMyC was quantifiable in 359 patients and compared with cTnT and cTnI measured using contemporary high-sensitivity assays. cMyC concentration (median, 12.2 ng/L; interquartile range [IQR], 7.9–21.2 ng/L) was linearly correlated with those for cTnT (median, <3.0 ng/L; IQR, <3.0–4.9 ng/L; R = 0.56, P < 0.01) and cTnI (median, 2.10 ng/L; IQR, 1.3–4.2 ng/L; R = 0.77, P < 0.01) and showed similar dependencies on age, renal function, and left ventricular function. We have developed a high-sensitivity assay for cMyC. Concentrations of cMyC in clinically stable patients are highly correlated with those of cTnT and cTnI. This high correlation may enable ratiometric comparisons between biomarkers to distinguish clinical instability.

Background: There is a paucity of information regarding cardiac troponin (cTn) concentrations in peripheral blood of nonhuman primates (NHP). Even less is known regarding cTn concentrations in monkeys that are restrained for oral or intravenous (iv) dosing. Objectives: The objectives of these studies were to (1) determine cardiac troponin I (cTnI) concentration in resting Cynomolgus monkeys and investigate biologic variability in cTnI concentration over time, (2) determine cTnI changes in restrained monkeys given sham oral dosing, and (3) determine cTnI changes in restrained NHP given a sham intravenous dosing. Methods: The Research Use Only Erenna cTnI ultrasensitive immunoassay based on single molecule counting technology was used to determine serum cTnI concentration in longitudinal studies of male Cynomolgus monkeys at rest, and after sham oral and intravenous dosing. Animals were catheterized prestudy, and blood samples were collected by an automated sampling device to limit disturbance of the animals during studies. Results: In resting monkeys cTnI concentrations were relatively low and constant and ranged from 0.2 to 9.6 pg/mL (mean = 2.5 pg/mL), with minimal variability during a 24-hour period. Animals given sham oral dosing also had low cTnI concentration with little variability similar to the resting values. Several animals restrained for intravenous dosing had a small transient increase in cTnI concentration (~5-25 pg/mL) that resolved quickly within one to 3 hours postinjection. Conclusions: Results of this longitudinal study provide information that may be important in differentiating effects of animal handling from those associated with compound-related effects in preclinical toxicology studies of drugs in development.