Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to novel factors which are polypeptides having the ability to inhibit the activation or reverse pre-existent activation of phagocytic cells (macrophages, and neutrophils) , to methods for preparing these factors, and to methods and compositions for the treatment of disorders using these factors.
• Phagocytic cells are activated by various environmental stimuli and cytokines to kill tumor cells and microbial pathogens by releasing reactive oxygen intermediates (e.g. hydrogen peroxide) during a respiratory burst, or by releasing reactive nitrogen intermediates (e.g., nitric oxide) .
• Cytokines that enhance the cytotoxic and antimicrobial function of macrophages and neutrophils have been extensively studied. More recently, cytokines have been identified that block the induction of macrophage activation. These include transforming growth factor (TGF)- ⁇ , (Tsunawaki et al., Nature 334- 260 (1988)); interleukin (IL)-4 (Abramson et al. , Clin. Res . 37, 825A (abstract) 1989) and calcitonin gene- related peptide (CGRP) (Nong et al. , _2. Immunol. 143- 45 (1989) ) .
• TGF transforming growth factor
• TCM tumor cell conditioned medium
• Phagocytic cell activation and the release of toxic products during respiratory burst can be deleterious to normal cells and has been implicated in inflammatory responses (Gallin et al., Inflammation-
• compositions that suppress phagocyte activation provides a means for reducing inflammatory conditions.
• European Patent Application 269408 hereby incorporated by reference, describes the use of TGF- ⁇ for the treatment of inflammatory disorders, but not as it pertains to the suppression of respiratory burst.
• An object of this invention is to provide novel factors that inhibit the activation or reverse pre-existent activation of macrophages and neutrophils .
• a further object of the invention is the use of such novel factors for the treatment of disorders characterized by inflammation.
• the subject invention comprises novel polypeptides herein referred to as respiratory burst suppression factor, or RBSF having the ability to inhibit activation of phagocytes.
• RBSF respiratory burst suppression factor
• These polypeptides have the property of inhibiting phagocyte respiratory burst, thereby inhibiting the release of toxic oxygen and nitrogen intermediates from phagocytes.
• These toxic intermediates provide a defense against diseases and infections by killing tumor cells and microbial pathogens.
• prolonged release of oxygen and nitrogen intermediates can be deleterious to normal cells. Therefore, RBSF can be used to treat disorders resulting from prolonged phagocyte activation.
• the subject invention provides a method for the production of RBSF comprising the steps of culturing a cell line which produces RBSF and isolating RBSF from the cell-conditioned culture medium.
• Any cell line producing RBSF can be used.
• the cell line is P815, a mouse mastocytoma cell line, or K562 a human cell line derived from a patient having chronic myelogenous leukemia.
• the subject invention also provides a method of purification of a polypeptide having the properties of RBSF wherein the polypeptide is distinct from TGF- ⁇ , IL-
• the method of purification of RBSF comprises the steps of gel filtration, ion exchange chromatography, and one or more steps of reverse phase high pressure liquid chromatography.
• the subject invention further relates to pharmaceutically acceptable compositions of RBSF and to the methods of treatment involving the administration of a therapeutically effective dose of RBSF.
• RBSF is effective as an anti-inflammatory agent, a wound healing factor and an immunosuppressant.
• Figure 1 shows the fractionation of RBSF by
• Figure 2 shows elution of RBSF from a Biogel P60 column.
• Figure 3 shows elution of RBSF from a Mono Q anion exchange column.
• Figure 4 shows the fractionation of RBSF by diphenyl reverse phase high pressure liquid chromatography (RPHPLC) .
• Figure 5 shows C4 reverse phase HPLC of RBSF using the bioactive peak from diphenyl C4 RPHPLC.
• Figure 6 shows a second C4 RPHPLC fractionation of RBSF using the bioactive peak from the first C4 column.
• Figure 7 shows inhibition by RBSF and TGF- ⁇ 's of macrophage release of hydrogen peroxide.
• Figure 8 shows inhibition by RBSF and TGF- ⁇ of macrophage release of nitrites induced by gamma- interferon and the combination of gamma-interferon and tumor necrosis factor-alpha.
• Figure 9 shows stimulation of thymidine uptake by normal rat kidney fibroblasts in the presence of RBSF and TGF- ⁇ l.
• Figure 10 shows inhibition of thymidine uptake by mink lung epithelial cells in the presence of RBSF and TGF- ⁇ l.
• Figure 11 shows inhibition of thymidine uptake by concanavalin A-stimulated mouse splenocytes in the presence of RBSF and TGF- ⁇ l.
• Figure 12 shows the inability of RBSF to compete with TGF- ⁇ l for binding to fibroblasts and epithelial cells .
• Figure 13 shows the suppression by human RBSF of H2O2 release by activated neutrophils.
• Figure 14 shows a Biogel P60 fractionation of human RBSF.
• Figure 15 shows an SDS-PAGE analysis of Biogel P60 fractions.
• Figure 16 shows SP-5P chromatography of human RBSF using active Biogel P60 fractions.
• Figure 17 shows an SDS-PAGE analysis of SP-5P fractions .
• Respiratory burst suppression is defined herein as the suppression of release of reactive oxygen or nitrogen intermediates from activated phagocytes, for example, macrophages, and neutrophils.
• activated phagocytes for example, macrophages, and neutrophils.
• TCM tumor cell conditioned medium
• the present invention provides for factors that are free from other components in TCM and have the ability to inhibit release of reactive oxygen intermediates by macrophages which have been activated by a variety of soluble and microbial triggering agents (see Example 2) .
• polypeptides of the present invention inhibit the release of reactive nitrogen intermediates triggered by treatment of macrophages with ⁇ -interferon or the combination of ⁇ -interferon and TNF- ⁇ (see Example 3) . Prolonged release of reactive nitrogen or oxygen intermediates is deleterious to normal cells and is suppressed by treatment with an effective amount of RBSF.
• RBSF The biological activity of RBSF is not confined to the inhibition of macrophage activity. As described in Example 4, it has now been found that purified RBSF can affect the proliferation of nonmyeloid cell lines .
• RBSF stimulates the proliferation of kidney fibroblasts, and inhibits the proliferation of lung epithelial cells and T-lymphocytes.
• the stimulatory action of RBSF on fibroblasts is useful in accelerating wound healing while the inhibitory action on
• T lymphocytes is useful in suppressing immune responses.
• a method for the production of RBSF comprises the steps of culturing a cell line which produces RBSF and isolating RBSF from the cell-conditioned medium.
• the cell line is for example P815, a mouse mastocytoma cell line, or K562, a human cell line derived from a patient having a chronic myelogenous leukemia.
• a method for purifying RBSF from culture medium comprises the steps of: subjecting RBSF- containing material to gel filtration chromatography (e.g., Sephacryl S400 or Biogel P60), subjecting RBSF- containing material to ion exchange chromatography
• RBSF prepared by this method was purified over 6, 000-fold starting from TCM (see Table 1) .
• Purified RBSF is defined as a single, major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of about 13,000 daltons .
• SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
• purified RBSF is defined as a single peak of activity on reverse phase high pressure liquid chromatography (see Fig. 6A) , and as a single, major band on SDS-PAGE of about 13,000 daltons (see Fig. 6C) .
• RBSF is a novel factor distinct from other factors such as TGF- ⁇ i that are known to suppress the respiratory burst of activated phagocytes.
• RBSF suppresses respiratory burst of phagocytic cells in the presence of phagocyte activating factor gamma interferon, whereas TGF- ⁇ does not suppress respiratory burst when gam interferon is present.
• antibodies specifically binding TGF- ⁇ i fail to neutralize the ability of purified RBSF to suppress release of oxygen intermediates by macrophages.
• purified RBSF fails to compete with TGF- ⁇ i for binding to kidney fibroblasts and lung epithelial cells, which are cell lines which express TGF- ⁇ receptors .
• the molecular weight of RBSF distinguishes it from two other factors known to suppress macrophage activity, IL-4, which is about 20,000 daltons, and CGRP, which is about 4,500 daltons.
• the factors of the present invention also encompass variations in the primary structure (i.e., amino acid sequence) of RBSF. Such variants include precursor forms of RBSF having additional amino acids at the amino terminus which are processed during secretion from host cells and allelic variants.
• the invention also provides chemically modified forms of RBSF which exhibit increased solubility, stability and/or circulating half-life compared to unmodified RBSF. Such modifications include, for example, covalent linkage to polyethylene glycol (see U.S. Patent No. 4179337 hereby incorporated by reference) .
• the factors of the invention may be covalently attached to a radioactive (e.g., 1-125) or nonradioactive (e.g., fluorescent dye reagent) reporter group to provide reagents useful in the detection or quantification of RBSF in solid tissues and fluid samples .
• a radioactive e.g., 1-125
• nonradioactive e.g., fluorescent dye reagent
• compositions comprising therapeutically effective amounts of RBSF together with suitable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
• Therapeutically effective amounts of RBSF e.g., those that reduce the level of respiratory burst activity
• a pharmaceutical composition can be determined by the ordinary artisan, taking into account such variables as the half-life of RBSF preparations, route of administration and the clinical condition being treated.
• the RBSF compositions are administered by continous infusion, sustained release formulation, aerosol spray, or injection.
• the invention provides for the use of RBSF alone or in combination with other therapy in the treatment of disorders characterized by inflammation resulting from excessive phagocyte activation.
• disorders include, but are not limited to, the following: rheumatoid arthritis and other arthritic diseases including crystal induced inflammation (Gallin et al., Inflammation, Basic Principles and Clinical Correlates, Raven Press, New York, 1988, pp. 751-783); asthma (Calhoun et al., Am. Rev. Respir. Dis . 135, 224A (abstract) 1987; Serhan et al., PNAS 81, 5335-5339 (1984)); emphysema (Mittman et al.
• the invention provides for the use of RBSF alone, or in combination with other therapy, as an antagonist to factors which, when administered, may promote deleterious effects associated with macrophage activation.
• RBSF may be used to counteract the effects of macrophage activation resulting from treatments with ⁇ —interferon, the combination of ⁇ -interferon and TNF, M-CSF, G-CSF or GM-CSF.
• the invention encompasses the use of RBSF alone or in combination with other therapy for the treatment of wounds.
• Administration of RBSF at a wound accelerates tissue regeneration by stimulating the proliferation of fibroblasts and by suppressing the release of toxic molecules by macrophages at the wound site.
• Activated macrophages were washed from the peritoneal cavities of female CD1 mice (Charles River Laboratories, Wilmington, MA) with Eagle's minimal essential medium, ⁇ —variant ( ⁇ MEM) (KC Biologicals,
• TCM Tumor cell-conditioned medium
• P815 mouse mastocytoma cells (American Type Culture Collection, Rockville, MD) were cultured at an initial density of 1.5 x 10 ⁇ /ml in ⁇ -MEM. After 48 h, the medium was centrifuged (5225 x g, 30 min, 4°C) and the supernatant sterilized by filtration (0.45 ⁇ , Millex-HA; Millipore, Bedford, MA) and stored at 4°C.
• TCM was concentrated by ultrafiltration on either YM-300, YM-100, YM-10, or YM-5 membranes (Amicon, Lexington, MA) under nitrogen at 4°C with slow stirring. Filtrate and retentate were both reserved.
• TCM was extracted with acid ethanol according to method of Roberts et al., Proc. Nat. Acad. Sci. USA 77, 3494 (1980)) with minor modifications.
• precipitated proteins were collected by centrifugation (5225 x g, 20 min, 4°C) and the supernatant discarded. The precipitate was dissolved in 1 M acetic acid and dialysed against the same overnight at 4°C. Insoluble material was discarded and the supernatant lyophilized (Savant Instrument Co., Farmingdale, NY) and stored at -20°C.
• Lyophilized RBSF extracted by method (3) was dissolved in 1 M acetic acid.
• the precipitate was removed by centrifugation and the supernatant further clarified by filtration (0.45 ⁇ ) and applied on a 1.5x100 cm column of Biogel P60 (Biorad Laboratories, Richmond, CA) equilibrated with 1 M acetic acid at room temperature.
• Six-ml fractions were eluted in 1 M acetic acid at a flow rate of 20ml/h.
• Most of the RBSF activity eluted in a broad peak between ubiquitin (8.7 kilodalton marker) and chymotrypsingen (25 kilodalton marker) as shown in Fig. 2A.
• SDS-PAGE under reducing conditions Fig. 2B
• the bioactive fraction was further purified by two sequential fractionations on a RP C4 column (4.6 x 50 mm, 6.5 ⁇ pore) (Synchrom, Lafayette, IN) equilibrated with 0.1% TFA (phase A) .
• Phase B was 100% acetonitrile in 0.1% TFA.
• Elution was monitored at 214 and 280 nm and 1 ml fractions were collected.
• RBSF activity eluted in a single peak at 50 ⁇ 2% acetonitrile (Fig. 5) .
• RBSF activity in fractions 49 and 50 corresponded to polypeptides in the 13 kilodaltons range (Fig. 5, inset) .
• a second C4 fractionation the single peak of RBSF activity (Fig. 6A) corresponded to the sole absorbance peak (Fig. 6B) .
• Silver-stained SDS-PAGE revealed one band at about 13 kilodaltons (Fig. 6C) .
• purified RBSF from this and two other preparations contained variable amounts of a 66 kilodalton protein that has been identified as albumin by amino acid sequencing.
• the transfer buffer contained 0.1% SDS in 40 mM glycine, 25 mM Tris, 20% methanol pH 8.3.
• Immune complexes were visualised after reaction with indolyl phosphate and nitroblue tetrazolium (United States Biochemical Co., Cleveland, OH) in veronal acetate buffer pH 9.6.
• Protein was determined by the method of Lowry et al. (J.Biol. Chem. 193, 265 (1959)) with bovine serum albumin as the standard. The concentration of protein in RBSF fractions of highest purity was determined by amino acid analysis after hydrolysis in 6 N HC1.
• TCM was prepared without serum (to avoid an exogenous source of TGF- ⁇ ) and was acidified, neutralized and dialyzed as described (Tsunawaki et al. , supra 1989) to activate latent TGF- ⁇ -related molecules, then used as the retentate after filtration on a YM-5 membrane (nominal molecular wieght cut-off, 5 kilodaltons)
• RBSF was purified from serum-containing TCM by a 7-step procedure: acid-ethanol extraction, ether precipitation, size exclusion chromatography, anion exchange chromatography, and 3 steps of reverse phase chromatography.
• RBSF was purified only through step 5. Purification was monitored by the macrophage H2O2 release assay described below, in which 1 unit of RBSF is defined as the volume required to inhibit by 50% the phorbol ester-induced release of H2O2 by in vivo-activated macrophages after a 24-h incubation in a final volume of 0.135-0.150 ml. This corresponds to 6.7-7.4 U/ml.
• the fluorescent microtest plate assay for caseinate-activated mouse peritoneal macrophage H2O2 release has been detailed (de La Harpe et al. , supra 1985) .
• Test cytokines were added to peritoneal cell cultures in ⁇ -MEM with 10% horse serum for 24 h. The adherent cell monolayers were then washed and placed in Krebs '-Ringer's phosphate buffered saline containing glucose, sodium azide to inhibit catalase, the fluorescent indicator scopoletin, horseradish peroxidase to catalyze oxidation of scopoletin by H2O2, and phorbol myristate acetate to trigger the respiratory burst .
• Loss of fluorescence of scopoletin was recorded over 1-3 h, following which the amount of cell protein adherent in each well at the start of the assay was measured as described, without removing the assay medium. Results are expressed as nmol H202 per mg adherent cell protein per unit time.
• the suppression of macrophage H2O2 release by RBSF is shown in Fig 7B.
• the suppression of H2O2 release by added TGF- ⁇ , TGF- ⁇ 2 or TGF- ⁇ 3 is shown in Fig. 7A for comparison.
• RBSF was purified from P815 mastocytoma cell conditioned medium by the procedures described in Example 1. Its specific activity was 5.7 x 10 4 units/mg protein.
• a unit of RBSF is defined as that amount of RBSF in a final culture volume of 0.15 ml which causes 50% suppression of macrophage H2O2 releasing capacity after a 24-h incubation.
• One unit of RBSF per well is equivalent to 6.7 units/ml.
• the potency of RBSF in the assay for suppression of macrophage H2O2 releasing capacity increases by a factor of -10 when the incubation with macrophages is extended to 48 h.
• Murine interferon- ⁇ (5.2 x 10 7 units/mg protein) and human TNF- ⁇ (4 x 10 7 units/mg protein) were from Genentech, Inc. (South San Francisco, CA) .
• Human TGF- ⁇ l 50 ⁇ g/ml
• human TGF- ⁇ 2 (20 ⁇ g/ml)
• human TGF- ⁇ 3 (30 ⁇ g/ml)
• Murine IL-4 (10 ⁇ units/mg protein) was from Immunex, Seattle, WA.
• LPS endotoxin
• LPS prepared by phenol extraction from Escherichia coli 0111:B or Salmonella minnesota (wild type) was from List Biologicals Laboratories, Campbell, CA.
• Adherent monolayers were obtained by plating 1 x 10 ⁇ cells/well in 96-well plates (Costar Data Packaging, Cambridge, MA) in complete medium, consisting of ⁇ MEM with 8% heat-inactivated fetal calf serum (Hyclone Laboratories, Logan, UT) , 50 U/ml of penicillin, and 50 ⁇ g/ml of streptomycin.
• the LPS level in complete medium was ⁇ 20 pg/ml.
• the nonadherent cells were removed by aspiration and freshly prepared complete medium with cytokines was added. Assays for 0 ⁇ and 0j3 ⁇ .
• nitrite in the medium was measured by an automated colorimetric assay based on the Griess reaction (Green et al., Proc. Natl. Acad. Sci. USA 23., 7764 (1981)) . Briefly, samples were reacted with 1% sulfanilamide, 0.1% naphthylethylene-diamine dihydrochloride, and 2.5% H3PO4 at room temperature for 10 min, and 02 ⁇ concentration was determined by absorbance at 550 nm in comparison with sodium nitrite standards . Where indicated, nitrate in the sample was reduced to nitrite as described (Green et al. , supra, 1981) .
• RBSF and TGF- ⁇ , TGF- ⁇ 2, TGF- ⁇ 3, is shown in Fig. 8A.
• Suppression of ⁇ -interferon plus TNF-CX induced nitrite release is shown in Fig. 8B.
• Cytokines were prepared as described in Example 2.
• the mitogenic assay on NRK49-F cells was essentially as described (Assoian et al. , J_. Biol. Chem. 258, 7155 (1983)) . 4 x 10 3 cells in ⁇ -MEM with 10% bovine calf serum (BCS;Hy-Clone) were incubated overnight in 6-mm-diam flat-bottom wells in 96-well plates (Costar Data Packaging, Cambridge, MA) . The next day, the cells were washed twice with ⁇ -MEM and incubated for 2 d in ⁇ -MEM with 0.2% BCS at 37°C in 5%
• the growth-arrested cells were then incubated with cytokines for 24 h. Four h before the end of the incubation, 1 ⁇ Ci thymidine-methyl— ⁇ H (2Ci/mmol, Dupont-New England Nuclear, Wilmington, DE) was added (final volume, 200 ⁇ l) (the same amount of the same 3H-thymidine preparation was used for the assays described below) .
• the cells were washed with ⁇ -MEM and the DNA was precipitated with cold 10% TCA, washed with 90% ethanol and solubilized in 0.5 N sodium hydroxide.
• Mink epithelial cells (American Type Culture Collection CCL64) were used as described (Tucker et al. , Science 236, 705 (1984)) .
• 3xl0 4 cells were cultured in 200 ⁇ l of a-MEM with 1% BCS overnight at 37°C in 5% C02 • The cells were washed, then incubated with cytokines in a total volume of 150 ⁇ l for 24 h.
• 3 H-Thymidine was added during the last 4-8 h of incubation and DNA prepared for liquid scintillation counting as above.
• mice (Charles River Laboratories, Wilmington, MA) were sacrificed by cervical dislocation and the spleens removed aseptically.
• Single- cell suspensions were prepared as described (Kehrl et al., J. Exp. Med. 163, 1037 (1986) and the cells suspended in a ⁇ -MEM with 10% BCS.
• 5x10 ⁇ cells were cultured in 6-mm-diam flat-bottom wells in 96-well plates with 4 ⁇ g of concanavalin A (Sigma Chemical Co., St. Louis, MO) in a total volume of 250 ⁇ l for 24 h, followed by one wash with 0.3 M methyl-a-D-mannoside (Sigma) in a-MEM.
• Cytokines were added in ⁇ -MEM with 1% BCS and the plates incubated for 72 h at 37°C in 5% C02.
• 3 H-Thymidine was added for the last 24 h, after which the samples were processed with a semi-automated cell harvester (Cambridge, Technology Inc., Watertown, MA) for liquid scintillation counting. Inhibition of 3 H-thymidine uptake into mouse spleen cells stimulated with the selective T-lymphocyte mitogen concanavalin A by RBSF and TGF- ⁇ , is shown in Fig. 11B and 11A, respectively.
• Example 1 Preparation of cytokines and antibodies and assays for macrophage H2O2 release are described in Example 1.
• RBSF purified as described in Example 1 was incubated with turkey anti-TGF- ⁇ antibody which specifically binds TGF- ⁇ i.
• Table 2 shows that the antibody completely neutralized the activity of TGF- ⁇ i, on activated macrophages, but had no effect on the activity of RBSF on activated macrophages .
• Anti-TGF- ⁇ antisera were raised in turkeys and rabbits as described (Danielpour et al., J_. Cell Ph ⁇ siol. 138, 79 (1989)) .
• Transiently acidified TCM, transiently acidified control medium, and TGF- ⁇ l or TGF- ⁇ 2 diluted in control medium were incubated with rabbit pre-immune or immune sera (final dilution 1:250) for 30 min at 37°C, followed by the addition of 25 ⁇ l of a 50% suspension of Protein-A agarose (Pharmacia Fine Chemicals, Piscataway, NJ) . The mixture was further incubated for 30 min with intermittent shaking, then centrifuged.
• test samples were incubated with turkey antisera or nonimmune sera (1:125) for 30 min at 37°C and added directly to macrophage cultures.
• Table 3 demonstrates that antibodies to TGF- ⁇ i, and/or TGF- ⁇ 2 have no effect on the activity of RBSF on activated macrophages.
• RBSF in this experiment is assayed in TCM and is not purified.
• TCM and control medium were acidified, neutralized, and dialyzed.
• tAntisera and control sera were used for immunodepletion at 1:250 (rabbit) or for neutralization at 1:125 (turkey) .
• ⁇ Activity in untreated control medium was 481 ⁇ 14.
• Results are means ⁇ SEM from 3 experiments, each in triplicate .
• TGF- ⁇ i Five ug of purified recombinant TGF- ⁇ i in protein-free vehicle was iodinated with carrier free 125I (Amersham, Arlington Heights, IL) using chloramine- T (Sigma) as described (Frolik et al. , 1984; Massague, 1987) to a specific activity of 133 ⁇ Ci/ ⁇ g, based on 60% recovery. Nonspecific binding was measured in the presence of 100- to 200-fold excess of nonradiolabelled TGF- ⁇ i. Binding to NRK-49F and mink lung epithelial cells was carried out in 16-mm-diam wells in 24-well plates .
• Confluent monolayers were incubated in binding buffer ( ⁇ -MEM with 0.1% bovine serum albumin in 25 mM Hepes, pH 7.5) for 2 h at 37°C and washed twice with binding buffer before addition of cytokines as potential competitors of binding of 100 pM 125 ⁇ TGF- ⁇ l in a final volume of 375 ⁇ l. After 1 h at 37°, the plates were placed on ice. The monolayers were washed 4 times with cold binding buffer and solubilized in 1% Triton X-100, 10% glycerol and 25 mM Hepes, pH 7.5 for ⁇ -counting.
• binding buffer ⁇ -MEM with 0.1% bovine serum albumin in 25 mM Hepes, pH 7.5
• TCM from human cell lines was prepared by culturing cells in RPMI-1640 medium (Gibco, Grand Island, NY) supplemented with L-glutamine to 2 mM and horse serum to 10%. After incubation at 37°C for four to five days, cells were pelleted by low speed centrifugation. TCM from each cell line was assayed for respiratory burst suppression and for the presence of LPS as described in Example 1. Of the four human cell lines tested, only TCM from K562 gave at least
• TCM Medium conditioned by P815 mouse mastocytoma cells and K562 human cells were prepared as described in Example 6. Release of H2O2 bovine neutrophils which were untreated or treated with ⁇ -interferon was assayed as a function of increasing amounts of TCM as described in Example 1. Tne suppression of H2O2 release from bovine neutrophils by human, but not mouse, TCM is shown in Figure 13. F250 refers to TCM from P815 cells.
• K562 a human cell line from a patient having chronic myelogenous leukemia, was cultured at an intial density of 1 x 10 5 /ml in RPMI-1640 medium (Gibco, Grand Island, NY) supplemented with L-glutamine to 2 mM and horse serum to 1%. Cells were incubated at 37°C for four to five days prior to harvesting.
• Active fractions 80-96 were predominately polypeptides having a weight of less than about 25 kilodaltons.
• 1.75 g. of protein from acetic acid/ethanol extraction (about 20 to 50% of the total amount extracted) was loaded at one time onto a 13.6 liters bed volume Biogel P60 column.
• Four or five loadings were required to process the entire acetic acid/ethanol extract.
• the column was equilibrated in 1 M acetic acid and proteins were eluted at a flow rate of about 15 ml./min. 50 ml fractions were collected and 1 ml of each fraction was assayed for absorbance at 280 nm and RBSF activity.
• Active fractions were pooled (1.8 liter per run) and concentrated 50 to 100-fold by lyophilization or by filtration using a YM-5 membrane filter (Amicon, Danvers, MA) after adding 0.01% PEG-600 to prevent binding of RBSF to the filter.
• YM-5 membrane filter Amicon, Danvers, MA
• Pool-E is predominately a single polypeptide having a molecular weight of about 13 kilodaltons. Based on the above observation, active pooled and concentrated fractions from the 13.6 liter. Biogel P60 chromatography were applied directly to an SP-5PW column under the conditions described above. A linear NaCl gradient from 0 to 0.4 M NaCl eluted the bulk of the 280 absorbing material that had bound to the column. Fractions collected during gradient elution are assayed for RBSF activity.
• the material from pool-E (about 3 ⁇ g) is concentrated and applied to a diphenyl reverse phase column equilibrated in 0.1% TFA and eluted in an increasing gradient of acetonitrile in 0.1% TFA as described in Example 1. Active fractions eluting from a dephenyl reverse phase column are then purified on a reverse phase C4 column equilibrated in 0.1% TFA (phase A) . Phase B was 100% acetonitrile in 0.1% TFA. In both steps, fractions are assayed for UV absorbance and RBSF activity.

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  • 1991-04-03 NZ NZ237664A patent/NZ237664A/xx unknown
  • 1991-11-29 FI FI915643A patent/FI915643A0/fi not_active Application Discontinuation
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