Development of a novel, nonimmunogenic, soluble human TNF receptor type I (sTNFR-I) construct in the baboon.

Pharmacokinetics and immunogenicity of six different recombinant human soluble p55 tumor necrosis factor (TNF) receptor I (sTNFR-I) constructs were evaluated in juvenile baboons. The constructs included either an sTNFR-I IgG1 immunoadhesin (p55 sTNFR-I Fc) or five different sTNFR-I constructs covalently linked to polyethylene glycol. The constructs were administered intravenously three times, and pharmacokinetics and immunogenicity were examined over 63 days. All of the constructs were immunogenic, with the exception of a 2.6-domain monomeric sTNFR-I. To evaluate whether the nonimmunogenic 2.6-domain monomeric construct could protect baboons against TNF-alpha-induced mortality, baboons were pretreated with 1, 5, or 10 mg/kg body wt and were compared with baboons receiving either placebo or 1 mg/kg body wt of the dimeric 4.0-domain sTNFR-I construct (n = 3 each) before lethal Escherichia coli bacteremia. The monomeric construct protected baboons and neutralized TNF bioactivity, although greater quantities were required compared with the dimeric 4.0-domain sTNFR-I construct. We conclude that E. coli-recombinant-derived human sTNFR-I constructs can be generated with minimal immunogenicity on repeated administration and still protect against the consequences of exaggerated TNF-alpha production.

Use of ion-exchange chromatography and hydrophobic interaction chromatography in the preparation and recovery of polyethylene glycol-linked proteins.

Cation- and anion-exchange chromatography can be used to purify a polyethylene glycol-linked protein dimer (PEG dimer) made with M, 20 000 PEG bis-vinylsulfone, even when there are no net charge differences between the components that are being separated. The retention time on ion-exchange generally is inversely proportional to the PEG:protein ratio (on a mass basis). One of the biggest challenges in developing the process for making this PEG dimer was the quality of the PEG linker. Reversed-phase HPLC can be used to determine both size heterogeneity and the degree of end-group activation of Mr 20 000 PEG bis-vinylsulfone. In addition, we have found that hydrophobic interaction chromatography can be used make more size homogeneous preparations of Mr 20000 PEG bis-vinylsulfone, which significantly increased the recovery of the PEG dimer.

Phase I/II trial of recombinant methionyl human tumor necrosis factor binding protein PEGylated dimer in patients with active refractory rheumatoid arthritis.

OBJECTIVE: To evaluate the safety, immunogenicity, pharmacokinetics, and efficacy of intravenous administration of tumor necrosis factor binding protein (TNFbp) dimer in patients with rheumatoid arthritis (RA). METHODS: This phase I/II study was a multicenter, randomized, double blind, placebo controlled, ascending dose study evaluating TNFbp dimer administered by i.v. infusion. Thirty-three patients with RA divided into 3 cohorts received TNFbp dimer (30, 100, 300 microg/kg) or placebo during a 5 min infusion at baseline and at 3 and 6 weeks; patients were followed at routine intervals after each infusion through 77 days postinfusion. Pharmacokinetics were analyzed using a log-linear regimen and comparisons were made between half-life after first, 2nd, and 3rd doses. Plasma TNFbp dimer concentrations and serum antibody levels were used in the measurement of pharmacokinetics. RESULTS: Administration of 30 microg/kg of TNFbp dimer was generally well tolerated; the maximum tolerated dose was 100 microg/kg. No serious adverse events were reported. A significant antibody response affected the half-life and clearance of TNFbp dimer at each dose group. Anti-TNFbp antibodies were noncytotoxic and nonagonistic. Clinical evaluations provided evidence of in vivo activity of TNFbp dimer in these patients. CONCLUSION: TNFbp dimer administered to patients with long standing RA resulted in significant antibody production to the study drug. This effect reduced the half-life and clearance of the TNFbp. This TNFbp will not be a viable option for treating patients with RA secondary to immunogenicity.

Receptor binding and Nb2 cell mitogenic activities of glycosylated vs. unglycosylated porcine prolactin.

Purified fractions of glycosylated (pGPrl) and unglycosylated (pUGPrl) porcine prolactin were prepared by affinity chromatography on Concanavalin A-Sepharose. The relative binding activities of these two forms of prolactin for receptors from porcine mammary, adrenal cortex and rabbit mammary, as well as their Nb2 cell mitogenic activity were determined. In both the porcine mammary and adrenal cortex receptor binding assays pGPrl had a 2-3 fold lower activity than pUGPrl. In the rabbit mammary binding assay pGPrl had a about a 5 fold lower activity than pUGPrl. Similarly, pGPrl had only about 20% of the activity of pUGPrl in the Nb2 cell proliferation assay.

Monoclonal antibodies specific for non-glycosylated porcine prolactin and for pituitary porcine prolactin.

Glycosylated and non-glycosylated forms of porcine pituitary prolactin were prepared using Concanavalin A Sepharose chromatography. Anti-prolactin monoclonal antibodies were screened for their ability to distinguish these two forms. One monoclonal antibody (17D9) exhibited high affinity binding for the non-glycosylated form of porcine prolactin, but little or no affinity for the glycosylated form. Using this antibody in conjunction with other monoclonals which equally recognize both forms, we developed immunoassays which can be used to determine the amount of the glycosylated vs. non-glycosylated prolactin in serum or other tissue samples.

Comparison of ornithine decarboxylase from rat liver, rat hepatoma and mouse kidney.

Comparisons were made of ornithine decarboxylase isolated from Morris hepatoma 7777, thioacetamide-treated rat liver and androgen-stimulated mouse kidney. The enzymes from each source were purified in parallel and their size, isoelectric point, interaction with a monoclonal antibody or a monospecific rabbit antiserum to ornithine decarboxylase, and rates of inactivation in vitro, were studied. Mouse kidney, which is a particularly rich source of ornithine decarboxylase after androgen induction, contained two distinct forms of the enzyme which differed slightly in isoelectric point, but not in Mr. Both forms had a rapid rate of turnover, and virtually all immunoreactive ornithine decarboxylase protein was lost within 4h after protein synthesis was inhibited. Only one form of ornithine decarboxylase was found in thioacetamide-treated rat liver and Morris hepatoma 7777. No differences between the rat liver and hepatoma ornithine decarboxylase protein were found, but the rat ornithine decarboxylase could be separated from the mouse kidney ornithine decarboxylase by two-dimensional gel electrophoresis. The rat protein was slightly smaller and had a slightly more acid isoelectric point. Studies of the inactivation of ornithine decarboxylase in vitro in a microsomal system [Zuretti & Gravela (1983) Biochim. Biophys. Acta 742, 269-277] showed that the enzymes from rat liver and hepatoma 7777 and mouse kidney were inactivated at the same rate. This inactivation was not due to degradation of the enzyme protein, but was probably related to the formation of inactive forms owing to the absence of thiol-reducing agents. Treatment with 1,3-diaminopropane, which is known to cause an increase in the rate of degradation of ornithine decarboxylase in vivo [Seely & Pegg (1983) Biochem. J. 216, 701-717] did not stimulate inactivation by microsomal extracts, indicating that this system does not correspond to the rate-limiting step of enzyme breakdown in vivo.

Investigation of structure and rate of synthesis of ornithine decarboxylase protein in mouse kidney.

An immunoblotting technique was used to study the forms of ornithine decarboxylase present in androgen-induced mouse kidney. Two forms were detected which differed slightly in isoelectric point but not in subunit molecular weight (approximately 55 000). Both forms were enzymatically active and could be labeled by reaction with radioactive alpha-(difluoromethyl)-ornithine, an enzyme-activated irreversible inhibitor. On storage of crude kidney homogenates or partially purified preparations of ornithine decarboxylase, the enzyme protein was degraded to a smaller size (Mr approximately 53 000) without substantial loss of enzyme activity. The synthesis and degradation of ornithine decarboxylase protein were studied by labeling the protein by intraperitoneal injection of [35S]methionine and immunoprecipitation using both monoclonal and polyclonal antibodies. The fraction of total protein synthesis represented by renal ornithine decarboxylase was increased at least 25-fold by testosterone treatment of female mice and was found to be about 1.1% in the fully induced androgen-treated female. Both forms of the enzyme were rapidly labeled in vivo, and the immunoprecipitable ornithine decarboxylase protein was almost completely lost after 4-h exposure to cycloheximide, confirming directly the very rapid turnover of this enzyme. Treatment with 1,3-diaminopropane which is known to cause a great reduction in ornithine decarboxylase activity did not greatly selectively inhibit the synthesis of the enzyme. However, 1,3-diaminopropane did produce an increase in the rate of degradation of ornithine decarboxylase and a general reduction in protein synthesis. These two factors, therefore, appear to be responsible for the loss of ornithine decarboxylase activity and protein in response to 1,3-diaminopropane.

Absence of inactivation or phosphorylation of ornithine decarboxylase by nuclear protein kinase NII and of immunological cross-reactivity between RNA polymerase I and ornithine decarboxylase.

Incubation with protein kinase NII did not result in phosphorylation or inactivation of mouse kidney ornithine decarboxylase. Partially purified ornithine decarboxylase preparations contained a protein kinase activity and stimulated the activity of RNA polymerase I. However, these properties were due to contaminating protein(s) since further purification reduced the kinase activity and removal of the ornithine decarboxylase with a specific antiserum did not abolish the ability to stimulate RNA polymerase I. Antibodies to RNA polymerase I did not interact with ornithine decarboxylase and antibodies to ornithine decarboxylase did not interact with RNA polymerase I. These results indicate that: a) mammalian ornithine decarboxylase activity is not regulated by phosphorylation by protein kinase NII or the contaminating kinase, and b) the ability of impure preparations of ornithine decarboxylase to stimulate RNA polymerase I is due to a contaminating unrelated protein.

Autoradiographic localization of ornithine decarboxylase in mouse kidney by use of radiolabeled alpha-difluoromethylornithine.

Ornithine decarboxylase, a key enzyme in polyamine biosynthesis and cell growth, has been localized in mouse kidney by autoradiography after administration of radiolabeled alpha-difluoromethylornithine. This drug is an enzyme-activated irreversible inhibitor of ornithine decarboxylase and forms a covalent bond with the enzyme. It was found that ornithine decarboxylase is present in all cell types studied but that the highest content occurs in the proximal convoluted tubules followed by the distal convoluted tubules and the collecting tubules. The majority of the enzyme is located in the cytoplasm but about 10-15% is present in the nuclei (often associated with nucleolus-like components) of the cells of the proximal and distal convoluted tubules. The labeled ornithine decarboxylase was lost rapidly from both nucleus and cytoplasm of all the cell types examined, and labeling by radioactive alpha-difluoro-methylornithine was greatly reduced if the mice were pretreated for 5 h with cycloheximide to block protein synthesis. These results indicate that ornithine decarboxylase turns over rapidly in all of the cells.

Studies of mammalian ornithine decarboxylase using a monoclonal antibody.

A monoclonal antibody of the immunoglobulin M class was produced against mouse kidney ornithine decarboxylase. Screening for the antibody was carried out using alpha-difluoromethyl[5-3H]ornithine-labelled ornithine decarboxylase. The antibody reacted with this antigen and with native ornithine decarboxylase. The antibody attached to Sepharose could be used to form an immunoaffinity column that retained mammalian ornithine decarboxylase. The active enzyme could then be eluted in a highly purified form by 1.0M-sodium thiocyanate. The monoclonal antibody could also be used to precipitate labelled ornithine decarboxylase from homogenates of kidneys from androgen-treated mice given [35S]methionine. Only one band, corresponding to Mr of about 55000, was observed. The extensive labelling of this band is consistent with the rapid turnover of ornithine decarboxylase protein, since this enzyme represents only about 1 part in 10000 of the cytosolic protein.

Effect of 1,3-diaminopropane on ornithine decarboxylase enzyme protein in thioacetamide-treated rat liver.

A radioimmunoassay for ornithine decarboxylase was used to study the regulation of this enzyme in rat liver. The antiserum used reacts with ornithine decarboxylase from mouse, human or rat cells. Rat liver ornithine decarboxylase enzyme activity and enzyme protein (as determined by radioimmunoassay) were measured in thioacetamide-treated rats at various times after administration of 1,3-diaminopropane. Enzyme activity declined rapidly after 1,3-diaminopropane treatment as did the amount of enzyme protein, although the disappearance of enzyme activity slightly preceded the loss of immunoreactive protein. The loss of enzyme protein after cycloheximide treatment also occurred rapidly, but was significantly slower than that seen with 1,3-diaminopropane. When 1,3-diaminopropane and cycloheximide were injected simultaneously, the rate of disappearance of enzyme activity and enzyme protein was the same as that seen with cycloheximide alone. These results show that the rapid loss in enzyme activity after 1,3-diaminopropane treatment is primarily due to a loss in enzyme protein and that protein synthesis is needed in order for 1,3-diaminopropane to exert its full effect. A macromolecular inhibitor of ornithine decarboxylase that has been termed antizyme is induced in response to 1,3-diaminopropane, but our results indicate that the loss of enzyme activity is not due to the accumulation of inactive ornithine decarboxylase-antizyme complexes. It is possible that the antizyme enhances the degradation of the enzyme protein. Control experiments demonstrated that the antiserum used would have detected any inactive antizyme-ornithine decarboxylase complexes present in liver since addition of antizyme to ornithine decarboxylase in vitro did not affect the amount of ornithine decarboxylase detected in our radioimmunoassay. Anti-(ornithine decarboxylase) antibodies may be useful in the purification of antizyme since the antizyme-ornithine decarboxylase complex can be immunoprecipitated, and antizyme released from the precipitate with 0.3 M-NaCl.

Mechanism of stimulation of ornithine decarboxylase activity in transformed mouse fibroblasts.

Stimulation of confluent serum-starved SV-3T3 cells by serum produces a transient enhancement of ornithine decarboxylase activity which fluctuates at least 20-fold over a 12-h period. The mechanism by which this fluctuation is produced was investigated. Two techniques for assaying the amount of enzyme protein in these cells were utilized: radioimmunoassay and titration with alpha-(difluoromethyl) [5-3H]ornithine. The radioimmunoassay was carried out by using a specific antiserum prepared in rabbits against homogeneous mouse kidney ornithine decarboxylase and by using alpha-(difluoromethyl) [5-3H]ornithine-labeled kidney ornithine decarboxylase as the tracer ligand. An exact correlation between the amount of enzyme protein and the amount of enzyme activity was seen during the rise and fall of ornithine decarboxylase activity after serum stimulation. Similarly, the amount of protein which was labeled covalently by reaction with alpha-(difluoromethyl) [5-3H]ornithine (a specific enzyme-activated irreversible inhibitor) correlated with the enzyme activity. Investigation of the protein labeled in this way by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that no change in the size of the protein which had a molecular weight of 53 000 occurred during this time period. These results indicate the alteration in ornithine decarboxylase activity can be accounted for entirely by changes in the amount of enzyme protein rather than by posttranslational modifications, activators, or inhibitors.

Changes in mouse kidney ornithine decarboxylase activity are brought about by changes in the amount of enzyme protein as measured by radioimmunoassay.

Antibodies were produced in rabbits to homogeneous mouse kidney ornithine decarboxylase and used to determine the amount of this protein present in kidney extracts by a competitive radioimmunoassay procedure. The labeled ligand for this assay was prepared by reacting renal ornithine decarboxylase with [5-3H] alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor. The sensitivity of the assay was such that 1 ng of protein could be quantitated and the binding to ornithine decarboxylase of a macromolecular inhibitor (antizyme) or alpha-difluoromethylornithine did not affect the reaction. It was found that treatment of female mice with testosterone produced a 400-fold increase in ornithine decarboxylase protein in the kidney within 4-5 days. Exposure to cycloheximide or to 1,3-diaminopropane led to a rapid disappearance of the protein which paralleled the loss of enzyme activity. There was no sign of any immunoreactive but enzymatically inactive form of mouse kidney ornithine decarboxylase under any of the conditions investigated. The results indicate that fluctuations of the enzyme activity in this organ are mediated via changes in the amount of enzyme protein rather than by post-translational modifications or interaction with inhibitors or activators.

Polyamine biosynthesis and interconversion in rodent tissues.

Polyamine levels in rodent tissues are regulated by the activities of three enzymes: ornithine decarboxylase, S-adenosylmethionine decarboxylase, and spermidine/spermine N1-acetyltransferase. These enzymes are present in the cell in very small amounts, have very short half-lives, and are highly inducible. Ornithine decarboxylase was purified to homogeneity (about 10,000-fold) from androgen-treated mouse kidneys, which have enzyme levels several hundred times higher than those in other fully induced mammalian tissues. This decarboxylase could be specifically labeled either in vitro or in vivo by reaction with radioactive alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor. Such covalent binding of alpha-difluoromethylornithine was used to titrate the number of molecules of the enzyme and to estimate its purity. It was also used for autoradiographic localization of the enzyme within tissues and to follow the degradation of the protein in vivo. S-Adenosylmethionine decarboxylase has been purified from rat liver and psoas muscle, and significant differences between the enzyme forms present in these tissues were observed. The rate-limiting enzyme in the interconversion of the polyamines, spermidine/spermine N1-acetyltransferase was purified more than 100,000-fold from carbon tetrachloride-induced rat liver. This acetylase acts on both spermine and spermidine to form N1-acetyl derivatives, which are then oxidized by polyamine oxidase forming spermidine and putrescine, respectively.

Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled alpha-difluoromethylornithine.

The binding of alpha-difluoromethylornithine, an irreversible inhibitor, to ornithine decarboxylase was used to investigate the amount of enzyme present in rat liver under various conditions and in mouse kidney after treatment with androgens. Maximal binding of the drug occurred on incubation of the tissue extract for 60min with 3mum-difluoromethyl[5-(14)C]ornithine in the presence of pyridoxal phosphate. Under these conditions, only one protein became labelled, and this corresponded to ornithine decarboxylase, having M(r) about 100000 and subunit M(r) about 55000. Treatment of rats with thioacetamide or carbon tetrachloride or by partial hepatectomy produced substantial increases in ornithine decarboxylase activity and parallel increases in the amount of enzyme protein as determined by the extent of binding of difluoromethyl[5-(14)C]ornithine. Similarly, treatment with cycloheximide or 1,3-diaminopropane greatly decreased both the enzyme activity and the amount of difluoromethyl-[5-(14)C]ornithine bound to protein. In all cases, the ratio of drug bound to activity was 26fmol/unit, where 1 unit corresponds to 1nmol of substrate decarboxylated in 30min. These results indicate that even after maximal induction of the enzyme in rat liver there is only about 1ng of enzyme present per mg of protein. When mice were treated with androgens there was a substantial increase in renal ornithine decarboxylase activity, the magnitude of which depended on the strain. There was an excellent correspondence between the amount of activity present and the capacity to bind labelled alpha-difluoromethylornithine in the mouse kidney extracts, but in this case the ratio of drug bound to activity was 14fmol/unit, suggesting that the mouse enzyme has a higher catalytic-centre activity. After androgen induction, the mouse kidney extracts contain about 170ng of enzyme/mg of protein. These results indicate that titration with alpha-difluoromethylornithine provides a valuable method by which to quantify the amount of active ornithine decarboxylase present in mammalian tissues, and that the androgen-treated mouse kidney is a much better source for purification of the enzyme than is rat liver.

Effect of androgens on turnover of ornithine decarboxylase in mouse kidney. Studies using labeling of the enzyme by reaction with [14C] alpha-difluoromethylornithine.

Treatment of male mice with excess androgens increased the activity of renal ornithine decarboxylase 60-fold in the BALB/c strain and 4-fold in the CD-1 strain. Part of the increase in the activity of ornithine decarboxylase was due to a decreased rate of degradation of the enzyme since activity declined more slowly (t1/2 80 min) in androgen-treated BALB/c mice than in controls (t1/2 20 min) when protein synthesis was inhibited by cycloheximide. When ornithine decarboxylase protein was labeled in vivo by injection of [5-14C]difluoromethylornithine, the rate of disappearance of the labeled protein was exactly the same as the rate of loss of ornithine decarboxylase activity when protein synthesis was inhibited by cycloheximide, confirming that ornithine decarboxylase protein does turn over rapidly in vivo. The half-life of another rapidly turning over enzyme important in polyamine metabolism, S-adenosylmethionine decarboxylase, was also increased in the mouse kidney by androgen treatment. These results indicate that steroid hormones can affect the level of certain proteins by changing the rate of degradation and that the labeling of ornithine decarboxylase by reaction with radioactive alpha-difluoromethylornithine in vivo provides a useful method for studying the degradation of this protein.