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We describe a method for the rational design of more effective therapeutic proteins using amino acid substitutions that reduce receptor binding affinity in intracellular endosomal compartments, thereby leading to increased recycling in the ligand-sorting process and consequently resulting in longer half-life in extracellular medium. We demonstrate this approach for granulocyte colony-stimulating factor by using computationally predicted histidine substitutions that switch protonation states between cell-surface and endosomal pH. Molecular modeling of binding electrostatics indicates two different single-histidine mutants that fulfill our design requirements; experimental assays demonstrate that each mutant indeed exhibits an order-of-magnitude increase in medium half-life along with enhanced potency due to increased endocytic recycling.

Chemical modification and mutagenesis of methionines in recombinant human granulocyte colony-stimulating factor (G-CSF) were investigated. Selective oxidation of G-CSF by H2O2 and t-butyl hydroperoxide leads to generation of different oxidized forms. Four modified forms were isolated and shown to contain 1 to 4 oxidized methionyl residues. All methionines in G-CSF are reactive, with reaction kinetics following the order of Met1>Met138>Met127>>>Met122. H2O2 oxidation of Met122 is relatively slow and is biphasic with a faster second reaction phase being affected by the oxidation of Met127. All oxidized forms retain gross G-CSF conformation similar to that of the native molecule and are able to bind the soluble G-CSF receptor. However, G-CSF form oxidized at both Met127 and Met122 is unstable and exhibits decreased ability to dimerize the receptor after exposure to acid or elevated temperature. All modified forms, except Met1-oxidized G-CSF, also show significantly lower biological activity. Our data suggest that Met138 is solvent accessible and its surrounding microenvironment may be critical for G-CSF function, whereas Met127 is less accessible to solvent and Met122 is near the hydrophobic core. Oxidation at both Met127 and Met122 results in alterations of G-CSF structure that affect the apparent molecular size, polarity, and stability and lead to the loss of G-CSF biological function. G-CSF variants with Leu replacement at Met127 or at Met138 are not completely resistant to oxidation-induced inactivation, while the variant with Leu replacement at both sites is more stable and can retain in vitro biological activity following oxidation., (Copyright 1999 Academic Press.)

A severe persistent neutropenia developed in a rabbit that was injected intradermally with 120, 60, 60, and 120 micrograms of recombinant canine granulocyte colony-stimulating factor (cG-CSF) on days 1, 22, 31, and 44, respectively. The neutropenia was present from day 44 to day 205. The nadir of the neutropenia (60 cells/microliters) occurred in conjunction with peak antibody titer (640,000) to cG-CSF on day 58. The immune antiserum from this rabbit reacted positively for cG-CSF on Western blot analysis. The immune antiserum also neutralized the activity of cG-CSF. On day 160, examination of the bone marrow showed marked granulocytic hypoplasia and mild erythroid hyperplasia. On day 205, the rabbit was still neutropenic (430 cells/microliters), even though the last injection of cG-CSF was given 161 previously. Necropsy on day 205 showed that there was still mild granulocytic hypoplasia with mild erythroid hyperplasia. Because of the lack of any inflammatory foci found at necropsy and the granulocytic hypoplasia, it was thought that the neutropenia was most likely due to decreased production and was not a consumptive process. It is hypothesized that the antibody that was produced to cG-CSF neutralized the effect of endogenous rabbit granulocyte colony-stimulating factor and prevented the normal proliferation and maturation of the rabbit neutrophils.

Oh-eda et al. have shown instability of granulocyte-colony stimulating factor (G-CSF) upon storage above pH 7.0 [J. Biol. Chem. (1990) 265, 11,432-11,435]. To clarify the mechanism of this instability, the accessibility of a free cysteinyl residue at position 17 for disulfide exchange reaction was examined using a sulfhydryl reagent. The results show that the cysteine is partially solvent-exposed in both glycosylated and nonglycosylated forms, suggesting that the exposure of the cysteine plays a critical role in the instability of the protein. This is supported by the facts that at low pH where the cysteine is protonated, both proteins have much greater stability and that a Cys17-->Ser analog is extremely stable at neutral pH and 37 degrees C. It was observed that the rate of sulfhydryl titration is slower for the glycosylated form than for the nonglycosylated form, suggesting that the cysteine residue is less solvent-exposed for the former protein or that the pKa is somewhat more basic. In either case, the carbohydrate appears to affect the reactivity of the sulfhydryl group through steric hindrance or alteration in local conformation. Both the glycosylated and nonglycosylated proteins showed essentially identical conformation as determined by circular dichroism, fluorescence, and infrared spectroscopy. Unfolding of these two proteins, induced either by guanidine hydrochloride or by pH, showed an identical course, indicating comparable conformational stability. Contribution of conformational changes to the observed instability at higher pH is unlikely, since little difference in fluorescence spectrum occurs between pH 6.0 and 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant human granulocyte colony stimulating factor (G-CSF) produced in Chinese hamster ovary cells is glycosylated. The carbohydrate compositional analysis indicated that G-CSF molecule contains sialic acid, galactose and galactosamine. By isolation and characterization of the purified glycopeptides obtained from cleavages by Staphylococcal aureus V-8 protease and cyanogen bromide, the O-linked glycosylation site was confirmed to be a Thr residue at position 133. Neuraminidase and O-glycanase digestion followed by sodium dodecyl sulfate polyacrylamide and isoelectric focusing gel electrophoreses distinguished two possible carbohydrate structures attached at Thr-133: structure A, NeuNAc-Gal-beta(1,3)-GalNAc-O-Thr; and structure B, NeuNAc-Gal-beta(1,3)-[NeuNAc]-GalNAc-O-Thr. Different glycoforms, undigested or after glycosidase digestion, can also be separated by ion-exchange or reversed-phase high-performance liquid chromatography. The approach described in this report provides a simple and valuable procedure to characterize glycoprotein structures containing simple carbohydrate moieties.

Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered subcutaneously at a dosage of 5 micrograms/kg/day to five healthy, young adult cats for 42 days. Mean neutrophil counts +/- standard deviation increased significantly (P < 0.001) from 10,966/microL +/- 2324 to 30,688/microL +/- 5296 within 24 hours after administration of the first dosage of rcG-CSF. Mean neutrophil counts reached 52,978/microL +/- 11,207 on day 6, representing a second significant increase (P < 0.01) over the previous 5 days. Mean neutrophil counts continued to increase, reaching 66,994/microL +/- 12,419 on day 14, then remaining within a range of 66,994 to 87,839/microL throughout the remainder of the study. The maximum mean neutrophil count was 87,839/microL +/- 8,695 on day 42. Neutrophil counts remained high until the administration of recombinant canine granulocyte colony-stimulating factor was discontinued 42 days after initiation of therapy. Once the rcG-CSF administration was discontinued, neutrophil counts returned to pretreatment values within 5 days. There were no significant changes in numbers of any of the other cell lines. There was no clinically significant toxicosis associated with the administration of rcG-CSF.

The effects of recombinant canine granulocyte colony-stimulating factor (rcG-CSF) on leukocyte counts and neutrophil function in clinically normal cats and in cats heterozygotic and homozygotic for Chediak-Higashi syndrome (CHS) were examined. CHS is a genetic disease characterized by neutropenic episodes and defects in a variety of phagocyte functions. Short-term administration of rcG-CSF at 10 micrograms/kg body weight resulted in a five- to tenfold increase in circulating granulocytes by day 10 of administration and normalizes CHS neutrophil counts by day 3. The drug was specific for neutrophils as determined by differential cell counts. Neutrophil chemotaxis under agarose and phagocytosis of Escherichia coli were characterized following administration of rcG-CSF in vivo. Granulocytes elicited by rcG-CSF show enhanced chemotactic abilities toward activated serum, increased spontaneous migration, and an enhanced ability to ingest opsonized E. coli. At a concentration of 1 nM rcG-CSF in vitro, chemotaxis and spontaneous migration were increased, with no effect on phagocytosis. CHS neutrophil function was improved by administration of rcG-CSF in all parameters studied, although the defect in chemotaxis was present throughout the treatment period. We conclude from this study that neutrophils elicited by rcG-CSF are functionally enhanced and that rcG-CSF may be a viable therapy for CHS and other related disorders.

Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered to clinically normal dogs, cyclic-hematopoietic dogs, and dogs undergoing autologous bone marrow transplantation, to determine whether rcG-CSF could be used to stimulate WBC production and function in normal and neutropenic dogs. To the normal dogs, rcG-CSF was administered by SC injection at rates of 1 microgram/kg of body weight, q 12 h; 2 micrograms/kg, q 12 h; or 5 micrograms/kg, q 12 h. A significant dose-dependent increase in the WBC count resulted from the stimulation of bone marrow progenitor cells. The increased WBC count was characterized by mature neutrophilia and monocytosis. Neutrophil myeloperoxidase and phagocytic activity were normal in rcG-CSF-treated normal dogs, demonstrating the production of normal functional neutrophils in response to rcG-CSF treatment. Recombinant canine G-CSF prevented neutropenia and associated clinical signs but did not completely eliminate the cycling of neutrophils in cyclic-hematopoietic dogs when it was administered at rates of 1 microgram/kg, q 12 h, and 2.5 micrograms/kg, q 12 h. The time to bone marrow reconstitution was not decreased in dogs treated with rcG-CSF at a rate of 2.5 micrograms/kg, q 12 h, for 13 days following autologous bone marrow transplantation. On the basis of our findings, we suggest that treatment with rcG-CSF is an effective way to stimulate myelopoiesis in dogs, but that the dose of rcG-CSF required to stimulate WBC production will vary depending on the cause of neutropenia. Recombinant canine G-CSF should be useful in stimulating production and maintaining function of WBC for treatment of clinical diseases seen commonly in veterinary practice.

The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.

Routine quantitation of recombinant human granulocyte colony-stimulating factor charge isoforms in the purified protein product requires development of a reliable analytical method. In this report, isoelectric focusing gel electrophoresis, peptide mapping, and cation-exchange high-performance liquid chromatography are compared and evaluated in the analysis of charge isomers that may be present in the recombinant factor. Due to a lack of sensitivity and reliability, isoelectric focusing gel electrophoresis and peptide mapping are not recommended. However, peptide mapping can distinguish aberrant peptides with differences in charges and provide separation for subsequent structural characterization. By this approach, an N-terminally blocked formylmethionyl species was identified to be the minor charge isoform in the purified preparations of recombinant human granulocyte colony-stimulating factor. In contrast to electrophoresis and peptide mapping, a strong cationic-exchange chromatographic procedure was found to be the most selective, sensitive, and reproducible analytical method. The sensitivity and reliability of the method were evaluated and validated using the formylmethionyl isoform and several deamidated analogs (Gln----Glu) made by site-directed mutagenesis. Recombinant human granulocyte colony-stimulating factor preparations contain a very low to undetectable level of the formylmethionine isoform and have no detectable deamidated isoforms.

Ten dogs were given mitoxantrone at a dose of 5 mg/m2 body surface area intravenously. Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered subcutaneously daily for 20 days after an infusion of mitoxantrone in five of these dogs to determine the effect of the hematopoietic growth factor on the duration and severity of myelosuppression. The median neutrophil counts dropped below normal (less than 3,000/uL) for 2 days in the dogs that received rcG-CSF, and for 5 days in the dogs that received only mitoxantrone. Four of five dogs not treated with rcG-CSF and none of those receiving rcG-CSF developed serious neutropenia (less than 1,500/uL). The neutrophil counts were significantly (P less than 0.05) higher in the rcG-CSF treated dogs at all time points except before the administration of the colony-stimulating factor, and the sixth day after the mitoxantrone was administered. These findings demonstrate that rcG-CSF is capable of reducing the duration and severity of mitoxantrone-induced myelosuppression.

Immunomodulatory effects of recombinant bovine granulocyte colony-stimulating factor were evaluated in periparturient dairy cows. Eleven of 21 cows were experimentally infected with Staphylococcus aureus in one mammary quarter prior to the study. Cows were assigned to four groups in a randomized complete block design to evaluate the effects of recombinant bovine granulocyte colony-stimulating factor (5 micrograms/kg of body weight or placebo injected subcutaneously once daily beginning 14 d prepartum through 10 d postpartum) on infected and uninfected cows during the periparturient period. Blood lymphocytes were isolated and evaluated from 5 wk before expected parturition through 7 wk postpartum. Lymphocyte function was evaluated using a blastogenesis assay, a mitochondrial methylthiazoltetrazolium cleavage activity assay, and an in vitro assay of IgM production. Serum concentrations of IgM, IgG1, conglutinin, and hemolytic complement were also determined. Injections of cows with recombinant bovine granulocyte colony-stimulating factor resulted in enhanced lymphocyte blastogenesis and mitochondrial methylthiazoltetrazolium cleavage activity in unstimulated cultures, higher serum IgM, and increased in vitro IgM production by B lymphocytes. These data provide support for the use of recombinant bovine granulocyte colony-stimulating factor to alleviate immunosuppression in periparturient cows.

The objective of this study was to determine how recombinant human granulocyte colony-stimulating factor (rhG-CSF) affects hematopoiesis in normal cats. Recombinant human G-CSF was given at 3.0, 5.0, and 10.0 micrograms/kg to two cats each s.c. twice daily for 21 days. This resulted in significant (p less than 0.01) elevations of peripheral blood neutrophils from 3.0- to 9.2-fold above pretreatment levels and significantly (p less than 0.02) above levels of nontreated control cats (n = 4). A statistically significant dose-related response was not seen at these dosages in any parameter evaluated. The period of maximum neutrophilia occurred between days 10 and 14 of rhG-CSF treatment, with maximum neutrophil counts ranging from 20,370 cells/microliters to 61,400 cells/microliters (normal is less than 12,500). Lymphocytosis (greater than 7000 lymphocytes/microliters) and monocytosis (greater than 850 monocytes/microliters) were observed in 50% of the cats receiving rhG-CSF during the period of maximal neutrophil stimulation. Monocyte counts in treated cats were significantly (p less than 0.01) elevated over those of treatment controls on days 12-17. Lymphocyte numbers in rhG-CSF-treated cats were significantly elevated (p less than 0.05) over pretreatment controls on days 12 and 14 of rhG-CSF treatment. No significant changes were observed in reticulocyte counts, platelet counts, or hematocrit levels. By day 19, neutrophil levels had dropped significantly (p less than 0.01) from the maximum neutrophil levels, with one cat attaining a normal blood neutrophil count by day 21 of rhG-CSF treatment. Marrow aspirates revealed an overall increase in marrow cellularity through day 14 of treatment in rhG-CSF-treated cats, with increased myeloid:erythroid ratios (two- to ninefold) over those of nontreated controls. The erythroid and lymphoid component of the marrow decreased from day 0 to day 14, whereas the early myeloid progenitors (myeloblasts, progranulocytes, and myelocytes) increased significantly (p less than 0.05). No significant differences in the percentage of later myeloid forms in the marrow were observed over the treatment period. In vitro colony-forming assays of marrow obtained from treated cats revealed increases in granulocyte-macrophage colony-forming units (CFU-GM) through day 14, with subsequent decreases by day 21 of rhG-CSF treatment. Recombinant human G-CSF was also effective at in vitro stimulation of feline marrow cells from untreated cats in a dilution study, with maximal CFU-GM formation at 0.1 microgram rhG-CSF/ml assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration of recombinant bovine granulocyte colony stimulatory factor to periparturient dairy cows was evaluated as a method to prevent periparturient immunosuppression. Eleven of 21 cows were experimentally infected with Staphylococcus aureus in one mammary quarter prior to the study. Cows were randomly assigned to four groups in a 2 x 2 factorial design to evaluate the effects of placebo or recombinant bovine granulocyte colony stimulatory factor administration on chronic, subclinically infected and uninfected cows during the periparturient period. Blood neutrophils were isolated and evaluated for phagocytic activities 5 wk before expected parturition through 7 wk postpartum. Administration of recombinant bovine granulocyte colony stimulatory factor (5 micrograms/kg body weight or placebo subcutaneously beginning 14 d prepartum through 10 d post-partum) resulted in a prepartum and postpartum leukocytosis of 35,600/microliters and 53,500/microliters, respectively. This was attributed to a mature neutrophilia of 24,010/microliters during prepartum and 38,080/microliters during postpartum treatment periods (pretreatment baseline = 2330/microliters). Mononuclear cell counts averaged 7610/microliters during prepartum and 9830/microliters during postpartum treatment periods (baseline = 3450/microliters). Neutrophil random and directed migration were reduced during recombinant bovine granulocyte colony stimulatory factor treatment compared with placebo or baseline levels. Ingestion of bacteria and cytotoxicity by neutrophils was increased during recombinant bovine granulocyte colony stimulatory factor therapy compared with placebo or baseline levels. Shedding of S. aureus in lacteal secretions was unaffected by recombinant bovine granulocyte colony stimulatory factor treatment. In summary, administration of recombinant bovine granulocyte colony stimulatory factor increased the number and functional activity of neutrophils and prevented some aspects of periparturient immunosuppression in dairy cows.