NO174556B - Procedure for Preparation of a Cell Growth Regulating Polyp pti - Google Patents

Description

The present invention relates to a method for producing a cell growth-regulating polypeptide.

Leukocytes, i.e. lymphocytes and monocytes, have been shown in several animal tumor models to be involved in inhibiting tumor growth. The increased incidence of malignant tumors in immunocompromised people supports the claim that white blood cells play a role in regulating neoplastic growth. Protein factors which are produced by these white blood cells and which inhibit tumor growth or modulate immune functions have already been isolated and characterized and include the α and β interferons, tumor necrosis factors, lymphotoxin, interleukin-2 and other lymphokines. As each of these isolated factors exhibits a different spectrum of activity and can interact in different ways in connection with other factors, there is a continued and strong interest in isolating and characterizing all the factors that the white blood cells produce when modulating cell growth or immune functions . These compounds can alone or together be used in the treatment or diagnosis of cancer, as activators for wound healing or as immunomodulators for the treatment of patients with immune deficiencies, autoimmunity, organ transplants, etc.

The discovery, isolation, purification or characterization of naturally occurring factors can present several difficulties. Methods must be developed for the separation and purification of a factor of interest from other factors in the impure starting material without denaturing the desired factor's activity, methods for biological analysis that allow identification of the fractions during separations whereby a particular factor is concentrated, methods for distinguishing a previously unknown factor from factors that are already known, or other unknown factors that may be present and which can either negatively or positively affect the activity of the sought-after factor, methods for characterizing the purified factor, and methods for concentration of the purified factor in a sufficient amount so that identification and characterization of the factor is permitted. As a result of the increasing number of factors that have been isolated, each new factor therefore becomes more difficult to identify as its role and function may be hidden by the numerous other factors present.

Beal et al., Cancer Biochem. Biophys. (1979) 3_:93~96' describes the presence of peptides in human urine, which peptides inhibit growth and DNA synthesis in transformed cells more than in normal cells. Holley et al., Proe. Nati. Acad. Pollock.

(1980) T7_ : 5989-5992, describes the purification of epithelial cell growth inhibitors. Letansky, Biosci. Rep. (1982) 2^ 39-45, describes that peptides purified from bovine placenta inhibit tumor growth and thymidine incorporation into DNA to a greater extent in neoplasm than in normal cells. Chen, Trends Biochem. Pollock. (1982) 7_ : 364-365, describes the isolation of a peptide from ascites fluid with cancer suppressive properties. Redding and Schally, Proe. Nati. Acad. Pollock. (1982) 79: 7014-7018, describes the isolation of one or more purified peptides from porcine hypothalamus, which peptides exhibit antimitogen activity against several normal cell lines and tumor cell lines. Sone et al., Gann (1984) 7_5_: 920-928, describe the production of one or more factors produced by human macrophages, which factors inhibit the growth of certain tumor cells in vitro. Ransom et al., Cancer Res. (1985) 4^:851-862, describes the isolation of a factor called leukoregulin, which factor inhibits the replication of certain tumor cell lines and appears to be distinct from lymphotoxin, interferon and interleukin 1 and 2. Most of these factors have not been fully characterized , and its primary structure is also not known.

Aggarwal et al., J. Biol. Chem. (1984) 259:686-691, have purified and characterized human lymphotoxin (LT) produced from a lymphoblastoid cell line, and subsequently sequenced LT (Aggarwal et al., J. Biol. Chemn. (1985) 260:2334) . Gamma interferon (γ-IF), which is produced by lymphoid cells and exhibits immunomodulatory and tumor inhibitory activity, has been cloned and produced by expression (Gray et al., Nature (1982) 295:503:508). Tumor necrosis factor (TNF) which inhibits the growth of certain tumors and is produced by macrophages and certain leukemic cell lines,

has been characterized, and TNF-cDNA has been cloned and pro-

silenced by expression in E. coli (Pennica et al., Nature (1984) 312:724).

The present invention relates to a method for producing a cell growth-regulating polypeptide which is capable of inhibiting the proliferation of neoplastic cells and stimulating the proliferation of normal, human fibroblast cells, which does not inhibit human, proliferative and cytotoxic T-cell reactions, does not inhibit granulocytic/myelocytic bone marrow cell colony formation, has a molecular weight in the range from 17 to 19 kD as determined by gel exclusion chromatography and 28 kD as determined by SDS-PAGE, and is relatively insensitive to moderately acidic and basic conditions and stable at a temperature of 56°C, and comprises at least one amino acid sequence that differs by at most three amino acids, and preferably by no more than one amino acid, from one of the following sequences:

which method is characterized by isolating leukocytes from a mammal, which leukocytes are selected from the group consisting of histocytic lymphoma cells and normal human peripheral blood lymphocytes, activating the leukocytes with an inducing agent selected from the group consisting of ingenols and phorbols, when the leukocytes are selected from histocytic lymphoma cells, or mitogens, when the leukocytes are selected from among normal human peripheral blood lymphocytes, and from the activated leukocytes free of cellular material, isolates the cell growth regulatory polypeptide in a purity that provides a specific activity of at least 10 GIA units/ng protein and preferably at least 100 GIA units/ng

protein.

According to the present invention, a hitherto unknown polypeptide (or also referred to as peptide factor) is provided, as well as biologically active fragments thereof, which factor can be obtained from leukocytes. The factor finds application in the modulation of cell growth, such as inhibition of tumor cell growth and stimulation of the growth of normal fibroblasts, and can modulate immune functions. The factor has an amino acid sequence that clearly differs from the sequences of other compounds that have been reported to exhibit analogous properties.

The present invention is described in more detail below with reference to the drawing, in which

Figure 1 sets forth the amino acid sequence of fragments of Oncostatin M,

Figure 2 shows a series of photomicrographs of cells treated with Oncostatin M, where photomicrographs (A-C) represent A375 melanoma cells treated with, respectively. 0, 5 and 100 GIA units, and photograph (D-F) depicts W138 fibroblast cells treated with resp. 0, 5 and 100 GIA units, and

Figure 3 shows a photograph of an SDS-PAGE analysis of Oncostatin M.

A polypeptide produced according to the invention is referred to as Oncostatin M and can be obtained from leukocytes, e.g. from conditioned media of stimulated U937 cells or conditioned media of stimulated normal human peripheral blood lymphocytes (PBL). According to the invention, fragments and mutants of the polypeptide are also provided, which fragments and mutants exhibit the intact Oncostatin M's biological activity, such as cell growth modulating activity or immunological activity.

The polypeptide fragments produced according to the invention are hitherto unknown polypeptides with at least 8 amino acids, which polypeptides are biologically active, at least with regard to immunological cross-reactivity with naturally occurring Oncostatin M. Immunological cross-reactivity is understood to mean that an antibody that is elicited by a hitherto unknown polypeptide according to the invention will cross-react with intact Oncostatin M, at least when Oncostatin M is in denatured form. Such polypeptides are therefore useful for eliciting antibodies against Oncostatin M, which antibodies can be used for determining the Oncostatin M concentration in a body fluid, for binding to Oncostatin M and thus modulating its activity, and for purifying Oncostatin M, e.g. . when used in conjunction with an affinity column. Some of the polypeptides may also retain Oncostatin M's cell growth modulating activity, although the activity may be modulated, usually reduced in comparison to intact Oncostatin M.

Figure 1 shows the amino acid sequence of poly(amino acids) that cross-reacts with Oncostatin M, the first sequence constituting the N-terminal part of Oncostatin M.

Poly (amino acids) produced according to the invention contains an amino acid sequence with at least 8 consecutive amino acids which corresponds to an amino acid sequence depicted in Figure 1, and which at most differs from this sequence with respect to three, and usually no more than one amino acid. This difference can either consist of the insertion of an amino acid, the omission of an amino acid or the substitution of one amino acid with another, in particular a conservative substitution. Generally, poly(amino acids) will contain at least 10, and more usually at least 12, consecutive amino acids which correspond to sequences depicted in the figure, and which differ from them with respect to one amino acid at the most.

The different amino acids will be divided into a number of subclasses. The table below indicates these subclasses:

By conservative substitution is meant that amino acids from the same subclass (ie either neutral aliphatic, acidic aliphatic, basic aliphatic or aromatic), in particular with the same polarity, replace each other. Preferably, the monomer groupings in the aliphatic subclass will consist of amino acids containing 2-4 carbon atoms or 5-6 carbon atoms.

Specified poly(amino acids) will not have a length exceeding approx. 1,000 amino acids. Usually they will contain less than 100 amino acids, and usually less than 50 amino acids. Thus, specified poly(amino acids) can be easily synthesized. Poly(amino acids) whose length exceeds 100 amino acids will usually be polymers of fragments of Oncostatin M, which fragments each contain less than 100 amino acids, or fused proteins where the fragment is fused with an antigen, enzyme, enzyme fragment, etc. I in particular, higher molecular weight poly(amino acids) may exist

of at least one polypeptide fragment with less than approx. 100 amino acids covalently linked to a large immunogenic polypeptide carrier to provide immunogenicity. Examples of such protein carriers are bovine serum albumin, so-called keyhole limpet hemocyanin (KLH) etc. Such conjugated polypeptides will be useful in eliciting antibodies in a suitable host organism.

U937 cells are a cell line derived from a histiocytic lymphoma cell line (Sundstrøm and Nilsson,

Int. J. Cancer (1976) _17:565-577) which can be induced to differentiate into cells with macrophage characteristics after treatment with various agents (Harris et al., Cancer Res.

(1985) 4_5:9-13). For the production of Oncostatin M, the U937 cells can be cultured in a conventional nutrient medium with serum and treated with an appropriate inducer. Phorbols or ingenols, in particular 12-0-tetradecanoylphorbol-13-acetate (TPA), can be suitably used. Generally, it can be used from approx. 5-20 ng/ml of the inducer. The initial number of cells is from approx. 10 5 to 10 6 cells/ml. After treating the cells with the inducer for a sufficient period of time, usually from 1 to 6 days, the supernatant is removed, the cells are washed with serum-free nutrient medium, attached cells are washed again with serum-free medium, and the cells are allowed to incubate for at least 12 hours, usually no more than about 48 hours, in serum-free nutrient medium, e.g. RPMI-1640 medium. The supernatants are then collected, and the cells are removed by centrifugation. Cell-free supernatants are tested for cell growth inhibitory activity (GIA) as described in the experimental section. The supernatant contains approx. 50-500 units GIA per ml (with regard to the definition of GIA units, reference is made to the experimental part below). Oncostatin M can also be obtained from mitogen-stimulated normal human peripheral blood lymphocytes (PBL). PBL can be isolated from leukofractions by diluting the fractions and centrifuging them over "Ficoll" gradients. Cells collected from the gradient boundary layers are washed and shock-lysed to remove red blood cells. The remaining cells are collected from this solution by centrifugation, resuspended in buffered serum and thrombin, shaken, and the platelet aggregate is allowed to settle for a short time. The suspended cells are transferred, recovered by centrifugation, resuspended in serum and transferred to a column containing nylon wool. The column is incubated to allow monocytes and B lymphocytes to bind, then washed. Most peripheral blood T lymphocytes do not bind and are eluted from the column. These cells are cultured at 37° in culture medium, e.g. RPMI-1640 medium, and treated with a suitable inductor, e.g. phytohemagglutinin (approx. 1-5 mg/l) for approx. 100 hours, whereupon the supernatants are collected. The supernatants are centrifuged to remove cells and concentrated e.g. by ultrafiltration or dialysis.

After isolation of the cell-free supernatant from

either U937 cells or normal PBL, the conditioned medium is suitably concentrated using a hollow fiber system or an ultrafiltration membrane, followed by dilution with acetic acid (to a concentration of 0.1 N acetic acid), then concentrated approx. 10 times, after which dilution and concentration are repeated. The concentrate can be lyophilized and used directly, or the lyophilized product can be used for further purification.

Indicated Oncostatin M can be purified by gel permeation chromatography using 40% acetonitrile-0.1% trifluoroacetic acid as the isocratic mobile phase on a "Bio-Sil TSK250" column, monitoring the activity of each fraction. The purification gives a composition with at least 0.5-5x10 4 GIA units/ml in the active fractions.

The partially purified product from the gel permeation chromatography can be further purified using reverse phase high pressure liquid chromatography using a linear gradient where the primary solvent is 0.1% trifluoroacetic acid in water, and the secondary solvent is acetonitrile containing 0.1% trifluoroacetic acid. The time course can be varied, as the chromatography usually requires approx. 3-4 hours, whereby most of the time, i.e. more than approx. 50% of the time, and no more than approx. 80% of the time is consumed in the gradient interval where the concentration of secondary solvent is 30-45%. Under these conditions, the active fractions elute at a concentration of approx. 41-42% acetonitrile.

The combined active fractions can be further purified by repeating the indicated reverse phase HPLC using a faster change in gradient conditions, and a slower flow rate. Under these conditions, the active fractions appear at a concentration of approx. 40.5-41.5% acetonitrile.

Specified reverse-phase HPLC can then be repeated with a change in the solvent system, n-propanol-0.1% trifluoroacetic acid being used as the secondary solvent.

A linear gradient is used where the gradient changes slowly in the interval from 23-35% n-propanol. The most significant activity is observed in the interval from 25.5-27.5% propanol, which gives an essentially homogeneous product with a specific activity exceeding 10 GIA units/ng protein. Generally, the product is purified to produce a specific activity of at least

100 GIA units/ng protein, and usually 150 GIA units/ng.

Indicated compounds are characterized by a molecular weight of approx. 17-19 kiloDaltons (kD), in particular 18 kD,

according to determination by size exclusion chromatography. Said compounds are further characterized by exhibiting

an apparent molecular weight of approx. 28 kD as determined by polyacrylamide gel electrophoresis under reducing or non-reducing conditions.

The amino acid sequence of fragments of purified Oncostatin M is analyzed. Oncostatin mainly exhibits the amino acid sequences indicated in Figure 1. Referring to Figure 1, the first sequence illustrates the N-terminal portion of Oncostatin M, while the remaining sequences illustrate internal fragments of the polypeptide.

Active compositions of isolated Oncostatin M contained a mixture with a high content of mannose and complex N-linked oligosaccharide. However, non-glycosylated compositions of Oncostatin M retained cell growth modulatory activity.

Oncostatin M is further characterized by its activity against certain cell lines. The indicated polypeptide lacks cytotoxic activity against WI26 and WI38 human fibroblast cells and tumor necrosis factor-sensitive L929 mouse cells, and a -jr interferon-sensitive human tumor cell line. It has also been shown that it does not inhibit proliferation of normal human T-lymphocytes or inhibit granulocytic/myelocytic colony formation from bone marrow cells at concentrations up to 100 GIA units/ml. Oncostatin M further stimulates the proliferation of normal human fibroblast cells as exemplified by WI38 and WI26 cells, and inhibits the proliferation of tumor cells such as A375, HBT10, A549 and SK-MEL28, and can increase the growth of colony-forming cells from normal bone marrow. Oncostatin M does not suppress human proliferative or cytotoxic T-cell responses in mixed leukocyte culture (MLC) reactions at concentrations of

500 GIA units/ml.

Said polypeptide has been shown to be stable against moderate acid and base exposure, and against heat treatment at 56°C.

The amino acid sequence of the subject polypeptide can be fully determined using commercially available sequence analysis apparatus.

The indicated compound can be used in in vitro cultures to inhibit the growth of cells or cell lines which are sensitive to the indicated polypeptide, as opposed to cells which are not sensitive. Heterogeneous cell mixtures or cell lines can thus be freed of unwanted cells when the unwanted cells are sensitive to the specified polypeptide. The indicated polypeptide can be administered in vivo in the case of neoplastic conditions, e.g. by intralesional, peritoneal, subcutaneous or similar injection. The indicated compound can be used in vitro for the elimination of malignant cells from the marrow of autologous marrow transplants, or for the inhibition of proliferation or elimination of malignant cells in other tissues, e.g. blood, before reinfusion.

The specified polypeptide is used in a

method for the treatment of wounds, such as cutaneous wounds, corneal wounds and various other epithelial and stromal wounds, such as chronic wounds, burns, surgical interventions, traumatic wounds and injuries to the hollow epithelial-covered organs such as the esophagus, stomach, large and small intestine , mouth, genitals and urinary tract. The method depends on the topical application of a treatment preparation containing Oncostatin M in a physiologically acceptable carrier.

The preparation can be used to treat various types of wounds, including mainly all cutaneous wounds, corneal wounds and damage to the epithelial-lined hollow organs in the body. Wounds suitable for treatment include those resulting from trauma, such as burns, scrapes, incisions, etc., as well as from surgical procedures such as surgical incisions and skin grafting. Other conditions which are suitable for treatment with the compositions according to the invention include chronic conditions such as chronic ulcer, diabetic ulcer and other non-legendary (trophic) conditions.

Oncostatin M can be incorporated into physiologically acceptable carriers for application to an affected area. The nature of the carrier may vary and will depend on the intended application area. For application to the skin, a cream or ointment base is normally preferred, such a suitable base including lanolin, "Silvaden"

(Marion) (especially for treating burns), Aquaphor®

(Duke Laboratories, South Norwalk, Connecticut), et al. If desired, it will be possible to incorporate Oncostatin M-containing compositions into bandages and other wound dressings to provide continuous exposure of the wound to the peptide. Aerosol administration can also be used.

The concentration of the polypeptide in the treatment preparation is not critical. The polypeptide will be present in an epithelial cell proliferation-inducing amount. The preparations are applied topically to the affected area, typically as eye drops to the eye or as creams, ointments or lotions on the skin. When treating the eyes, a frequent treatment is desirable, usually applications at intervals of 4 hours or less. On the skin, it is desirable to continuously maintain the treatment preparation on the affected area during treatment, with applications of the treatment preparation from 2 to 4 times a day or more often.

The amount of the specified polypeptide used will vary depending on the method of administration, the use of other active compounds and the like, and will generally lie in the interval from approx. 1 ug to 100 ug. The indicated polypeptide can be used with a physiologically acceptable carrier such as saline, phosphate-buffered saline or the like. The amount of compound used is determined empirically on the basis of the reaction of cells in vitro, and the reaction of experimental animals to the indicated polypeptides or compositions containing the indicated polypeptides.

The specified compounds are used alone or in combination with other growth factors or inhibitors or immunomodulators such as TNF, IL-2, r-interferon, monoclonal antibodies etc. The amount of these other compounds will generally lie in the range from 1 µg to 100 ug. Conjugates of the indicated compounds with site-directing residues, e.g. antibodies, can be produced in cases where the antibodies are specific to particularly malignant cells or organs.

The invention is explained in more detail below.

Materials and methods

Oncostatin M isolated from U937 cells

Production of a tumor cell growth inhibitor from a histiocytic lymphoma cell line

U937 cells which are a histiocytic lymphoma cell line (Sundstrøm and Nilsson, Int. J. Cancer (1976) 17; 565-577) were grown in 850 cm 2 roller flasks (Corning C2540) at a concentration of 4 x 10 5 cells/ml , in a total volume of 300 ml RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), penicillin/streptomycin (PS), L-glutamine and 10 ng/ml 12-0-tetradecanoylphorbol-13-acetate (TPA). 4 days later, the supernatants containing FCS and TPA were removed, the roller flasks were washed 5 times with serum-free RPMI-1640, and detached cells (1 x 10<5> cells/ml) were washed 3 times with serum-free medium and returned to the flasks , which resulted in a final volume of 125 ml of serum-free RPMI-1640 medium per roller flask. One day later, the supernatants were collected, centrifuged to remove the cells, filtered through a 0.45 micrometer (u) Nalgene<®> filter, and concentrated using a hollow fiber system ("Amicon Cartridge HIP10-20") to a volume of 150 ml (starting volume 1500 ml). Oncostatin M was also isolated from supernatants of serum-free TPA-treated U937 cells in 150 cm 2 tissue culture flasks. The supernatant was concentrated with an "Amicon Diaflo PM-10" membrane exhibiting a cutoff value of 10 kD, and dialyzed. After dialysis, the concentrate was diluted with acetic acid, resulting in a final concentration of 0.1 N acetic acid in 500 ml, and was concentrated to 50 ml using an "Amicon PM-10" filter. This 50 ml concentrate was diluted to

400 ml with 0.1 N acetic acid and was concentrated to 40 ml with the same filter. The concentrate was diluted with 1 N acetic acid, and the resulting precipitate was removed by centrifugation. The resulting concentrate was frozen and lyophilized. The lyophilized material was used for purification steps.

Gel permeation chromatography

A "Bio-Sil TSK-250" column (600 x 21.5 mm)

(Bio-Rad) was connected to a high-pressure liquid chromatographic system. The crude fraction (10 mg/ml) was dissolved in 40% acetonitrile in 0.1% aqueous trifluoroacetic acid (0.1% TFA). A 20 ml aliquot of the mixture was injected and the elution was carried out isocratically with a mobile phase consisting of 40% acetonitrile in 0.1% TFA. The flow rate was 2.5 ml/minute and the advance speed of the chart paper was set at 0.25 cm/minute. 5 ml fractions were collected. The chromatography was performed at room temperature. An aliquot from each fraction was evaporated and assayed in triplicate for growth inhibitory activity (GIA) against A375 cells.

The active fractions (fractions 21 and 22) from six runs were united. The combined material showed an activity of approx. 4.8 x 10 5GIA units. The factor was found to have an apparent molecular weight of 18 kD as determined by size exclusion chromatography ("Bio-Sil TSK-250" column).

Reverse-phase high-pressure liquid chromatography (HPLC) of TSK-250 fractions

The pooled TSK-250 fractions 21 and 22 described above were diluted twice with 0.1% TFA. This mixture was injected isocratically onto a "u-Bondapak-C18" column (7.8 x 300 mm) (designated as C18<1>) at room temperature. The flow rate was set at 2.5 ml/minute and the chart speed of the printer was set at 0.25 cm/minute. A linear gradient was used between primary solvent-0.1% TFA and the secondary solvent acetonitrile-0.1% TFA. The gradient conditions were 0-30% over 20 minutes, then 30-45% over 150 minutes, 45-55% over 20 minutes and 55-100% over 10 minutes. All solvents were of HPLC grade. 4 ml fractions were collected and aliquots of each fraction were analyzed for growth inhibitory activity. Fractions 72-75 were found to contain the predominant activity. The active fractions were eluted at acetonitrile concentrations of between 41 and 52%.

Fraction 72-75 were united. 16 ml of 0.1% TFA was added to the combined fractions. The mixture was injected into a "u-Bondapak-C18" column (3.9 x 300 mm) (designated as C18 2 ) at room temperature. The flow rate was set at 1 mL/minute, and the plotting speed of the printer was set at 0.25 cm/minute. The gradient conditions were 0-35% over 10 minutes, 35-45% over 100 minutes and 45-100% over 10 minutes. The fractions were collected, and aliquots were taken and analyzed for GIA. Most of the activity left the column at an acetonitrile concentration of between 40.7 and 41.3% (retention time 83-86 minutes).

The active fractions were combined and diluted twice with 0.1% TFA and injected isocratically onto a "u-Bondapak-C18" column (3.9 x 300 mm) (designated as C18^) at room temperature. The flow rate was 1 ml/minute,

and the chart speed of the printer was 0.25 cm/minute. A linear gradient was used between primary solvent-0.1% TFA and the secondary solvent n-propanol-TFA (0.1%). The gradient conditions were 0-23% over 20 minutes and 25-35% over 120 minutes. The fractions were collected, and aliquots of each fraction were examined for GIA. Most of the activity appeared at a propanol concentration of between 25 and 26.5% (retention time 59 minutes). This apparently homogeneous fraction contained approx. 300 ng of protein and exhibited an activity of 40,000 GIA units.

Cell growth modulatory analysis using 3 H-thymide incorporation into DNA (GIA)

The analyzes were carried out using plates provided with 96 flat wells (Costar 3596). Human melanoma cells (A375) were used as a sensitive indicator cell line. Cells (3 x 10<3>) in 0.1 ml Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and PS were plated in each well. 3 hours later, 0.1 ml of sample was added to each well. The plates were incubated at 37°C for 3 days. Then 0.025 ml (0.5 uCi) of a solution of <3>H-thymidine (specific activity 27 uCi/ug) was added to each well for the last 6 hours of incubation. The cells were then transferred to glass filter strips using an ultrawell harvester (PHD Cell Harvester, Cambridge Technology, Inc.). The filters were transferred to scintillation vials to which 2 ml of scintillation fluid (ScientiVerse II, Fisher Scientific Co.) was added before counting in a scintillation counter to determine <3>H-thymidine incorporation.

Colony inhibition test on soft agar (TGI)

A 0.5 ml base layer of 0.5% agar ("Agar Noble", Difco Laboratories, Detroit, Michigan) in DMEM containing 10% fetal calf serum (FCS) was added to 24-well Costar tissue culture plates. 0.5 ml of 0.3% agar containing the same mixture of medium and FCS, 1-2.5x10<3> A375 cells and different concentrations of the factor to be investigated was placed on top of the agar base layer. The plates were incubated at 37°C in a humidified atmosphere of 5% CO2 in air and after 10 days 0.5 ml of 0.3% agar containing the same medium and factor concentrations were again added. The colonies were counted, detached and destained, and the number of colonies containing more than 6 cells was assessed between days 7 and 14.

Results

Sequences of Oncostatin M isolated from U937 cells

The N-terminal sequence and internal fragments of Oncostatin M were determined by microsequencing analysis of the reduced and S-pyridine-ethylated polypeptide, and of peptides obtained by enzymatic digestion of reduced and S-pyridine-ethylated Oncostatin M with endoproteinase Lys-C and Staphylococcus aureus V8 protease. The peptide fragments were purified by reverse phase HPLC using volatile solvents. The peptides were subjected to automated repetitive Edman digestion in a "Model 470A" protein sequencer (Applied Biosystems, Inc.). The phenylthiohydantoin amino acids were analyzed by reverse phase HPLC (Applied Biosystems, Inc.) with a "PTH-C18" column (2.1x220 mm, ABI) using a sodium acetate buffer/tetrahydrofuran/acetonitrile gradient for the elution.

The resulting amino acid sequences are essentially as depicted in Figure 1.

A comparison of these sequences with sequences stored in the current protein database (PIR Release 9.0, May 1986) revealed no significant sequence homologies with any other known sequence. In addition, there is no homology with tumor necrosis factor, lymphotoxin colony-stimulating factor, interleukin 1 or 2 or (3-transforming growth factor.

Inhibition of proliferation of tumor cells and increase of proliferation of normal human fibroblast cells

Using the colony inhibition test described above on soft agar, the following results were obtained:

In connection with the next investigation, different treatments were used to determine the effect of the treatment, which was either of a chemical or physical nature, on the activity of the indicated polypeptide. The results are indicated in the following table.

It appears from the above results that Oncostatin M in the dialyzed supernatant is mainly resistant to inactivation with 1N acetic acid and 1N ammonium hydroxide. The specified compounds are thus relatively insensitive to both moderately strong acids and moderately strong bases. The indicated compound is stable to heat treatment at 56°C for 1 hour, but not at 90°C for 30 minutes.

The title compound was also tested for heat stability and was found to retain its activity after a heat treatment at 56°C for 1 hour, but to lose essentially all activity at 95°C for 30 minutes.

Furthermore, the ability of the indicated polypeptides to inhibit tumor cell replication in various neoplastic cells was investigated. The results appear from the following table.

Tumor cells were plated in microwells 3 hours prior to the addition of various dilutions of Oncostatin M that had been purified by reverse phase HPLC as indicated above. In the last 6 hours of an incubation over 3 days, the cells were labeled in 0.2 ml of medium with 3 H-thymidine ( 3 H-TdR) (0.5 uCi/well). One unit of tumor growth inhibitory activity (GIA) is defined in the footnote to Table 1 as the amount that causes 50% inhibition of <3>H-TdR incorporation in A3 75 melanoma cells. It was demonstrated that a unit responded to approx. 10 pg of purified protein, and therefore the concentration (ng/ml) which caused 30% inhibition of incorporation of <3>H-TdR in A549, HTB10 and A-375 cells, respectively, was about. 1.4, 4.0 and 0.015 ng/ml. <3>H-TdR incorporation into WI26 normal human fibroblast cells was not suppressed by the U937 factor in any experiment.

It appears from the above results that Oncostatin M's ability to inhibit replication is selective, and that Oncostatin M has highly varying effects depending on the type of cell. The specified compound is effective against melanoma cells such as A375 melanoma cells, squamous cell carcinoma cells such as A549 and neuroblastoma cells such as HTB10.

Tumor cells were seeded for 3 hours on plates containing

96 wells, using 3 x 10 3 cells/well, and for normal fibroblast cells, 1.5 x 10 3 cells/well. Different concentrations of purified Oncostatin obtained from the C18 3-column fraction with maximal antiproliferative activity against A375 cells were added, and 3 days later <3>H-thymidine incorporation into the cells was measured in triplicate experiments for each concentration. The results appear from table 4.

The indicated results are the percentage inhibition

or percentage stimulation of H-thymidine mcorporation in resp. tumor cells (A375, HTB10, A549 and SK-MEL28) and normal fibroblast cells (WI26 and WI38). One GIA unit is defined in the footnote to Table 1 as the amount of Oncostatin M that causes a 50% inhibition of H-thymidine incorporation in A375 cells.

In addition to the observed differential effect on <3>H-thymidine incorporation in tumor cells and in normal human fibroblast cells, as shown in Figure 2, a differential effect on morphology and cell number was also observed after treatment for 3 days of the two cell types with Oncostatin M.

The Oncostatin M used was derived from the HPLC-C18 column fraction with maximal activity in inhibiting the proliferation of A375 cells. Figure 2 shows a series of photomicrographs of A375 melanoma cells that were untreated (A), treated with 5 GIA units (growth inhibitory activity units)

Oncostatin M (B) or 100 units (C). Also shown are photomicrographs of WI38 fibroblast cells that were untreated (D), treated with 5 GIA units (E) or 100 units (F). The cells were stained with crystal violet in 0.5% methanol. The magnification is 63 times.

NaDodSO^/ PAGE of Oncostatin M

—Purified Oncostatin M was subjected to NaDodSO -examination under reducing conditions and was found to have an apparent molecular weight of approx. 2 8 kD, as can be seen from figure 3. The following proteins were used as standards (lane A): ovalbumin, Mr = 43 kD, chymotrypsinogen a, Mr = 25.7 kD, lacto-globulin |3, Mr = 18.4 kD , lysozyme, Mr = 14.2 kD, bovine trypsin inhibitor, Mr = 6.2 kD and insulin A-chain and B-chain, resp.

Mr = 2.3 kD and 3.4 kD. Oncostatin M was placed in slot B.

Oncostatin M which was subjected to PAGE examination

under non-reducing conditions, also exhibits an apparent molecular weight of 28 kD, and protein electroeluted from the band was shown to inhibit proliferation of A375 cells.

Antibody to a synthetic peptide of Oncostatin M reacts

125 . ■

with I-labeled Oncostatin M in radioimmunological precipitations

a) Peptide synthesis

The peptide corresponds to residues 6-19 in the Oncostatin M protein

and was synthesized by solid phase technique on an automated Beckman instrument as described in Gentry et al., J. Biol. Chem.

(1983) 2_58:11219. The peptide was cleaved from the resin using the "low-high" HF method (Tam et al., J. Amer. Chem. Soc. (1983) 105:6442-6445). The purification was carried out by means of preparative HPLC.

b) Production of antibodies

The peptide was coupled with bovine 7-globulin as described

in Gentry and Lawton, Virology (1986) 152:421-431. White New Zealand rabbits were injected and received booster injections

(5 times) subcutaneously in 4 sites as described by Gentry and Lawton, Virology (1986) 152:421-431. The antiserum used was obtained 2 weeks after the fifth booster injection.

c) Iodination of Oncostatin M

A sample of partially purified Oncostatin M was radiolabelled

with iodine 125 using those of Linsley et al., PNAS

(1985) 82^:356-360 described methods. An aliquot of the labeled composition containing 100,000 cpm was mixed with rabbit antiserum directed against the N-terminal 17 amino acids of Oncostatin M (final dilution 1:20) in the absence or presence of the N-terminal peptide (the N-terminal 17 amino acids of Oncostatin M) (2 vig) and subjected to immunoprecipitation analysis as described by Lindley et al., Biochemistry (1986) 25:2978-2986.

Specifically, a tube containing 5 µl was preincubated with 2 µg of the N-terminal peptide in 10 ml TNEN (20 mM Tris pH 7.5,

5 mM EDTA, 150 mM NaCl, 0.05% Nonidet<®> P-40) containing 0.1% BSA for 30 min at 4°C, before addition of I Oncostatin M

in 85 µl TNEN containing 0.1% BAS and 40 mM dithiothreitol (DTT). 7 tubes containing 5 µl antiserum were incubated with I<125>Oncostatin M in 85 µl TNEN containing 0.1% BSA and 40 mM

DTT for 30 min at 4°C before adding 50 µl 10% formalin-fixed Staphylococcus aureus (Pansorbin, Calbiochem).

After incubation for an additional 30 min at 4°C, the tubes were microcentrifuged, and the precipitates were washed 4 times with 1 ml of TNEN before being subjected to PAGE analysis. A diffuse band with Mr = 32 kD was observed after SDS/PAGE analysis of the immunoprecipitates. Precipitation of this species was inhibited by including an excess of unlabeled peptide corresponding to the N-terminal 17 amino acids of Oncostatin M, suggesting that the precipitation was specific for this peptide.

The carbohydrate composition of Oncostatin M was investigated by glycosidase sensitivity tests. Immunoprecipitates prepared as described under c) above were treated with buffer, endoglycosidase H or neuraminidase as described by Linsley et al. (1986). Treatment with endoglycosidase H, which is an enzyme with specificity for N-linked high mannose oligosaccharides, resulted in the appearance of a lower molecular weight compound, Mr = 24 kD. Only a portion of the radiolabeled material was sensitive to this enzyme, indicating that not all molecules contained high mannose oligosaccharides. Treatment with neuraminidase resulted in the appearance of a single band with Mr = 27 kD, indicating that the size heterogeneity of untreated 125 I-labeled Oncostatin M was due to molecular heterogeneity in the sialylation of the glycoprotein core. The results indicated that active compositions of Oncostatin M contained a mixture of high-mannose and complex N-linked oligosaccharide side chains.

Oncostatin M isolated from normal human peripheral blood lymphocytes (PBL)

Production of a tumor cell growth inhibitor from PBL

Leukofractions containing PBL obtained from the blood bank were diluted 1:1 with phosphate-buffered saline, pH 7.4 (PBS).

10 ml of a solution consisting of 9% "Ficoll" containing

20% by volume of 50% sodium diatrizoate (final specific gravity of 1.080) was placed under 35 ml of diluted blood. The gradients were centrifuged at room temperature for 20 min at 850 x G.

The cells were collected from the gradient interface and were washed with PBS. Red blood cells were shock-lysed for 3-4 minutes with 10-20 ml of a solution containing 0.8% ammonium chloride and 0.0% Na 2 -EDTA.

Cells were collected from the red blood cell lysis solution by centrifugation at 600 x G for 10 minutes, and these were resuspended in 10 ml of RPMI-1640 medium (Gibco) containing 5% fetal bovine serum. Thrombin was added to give a final concentration of 0.5 U/ml. The cell suspension was shaken for 5 minutes at 37°C, and the platelet aggregate was allowed to settle for 5 minutes. The suspended cells were transferred to new tubes, recovered by centrifugation, resuspended in 1 ml of fetal bovine serum and transferred to a column containing 0.5 g of brushed, pre-moistened nylon wool, type 200 (Fenwal).

The nylon wool column was incubated at 37°C for 60 minutes to allow attachment of monocytes and B lymphocytes. The column was then washed with 3 vol. RPMI-1640 medium (37°C) containing 5% fetal bovine serum, and the eluted non-adherent cells (PBL) were collected.

PBL (2 x 10<6> cells/ml) were cultured at 37°C and using 5% CO 2~95% air for 96 hours in RPMI-1640 medium (10.4 g/l) containing FeSO 4 .7H 2 O (1 mg/l), ZnSO4.7H2O

(2 mg/l), Na2Se03.5H20 (0.017 mg/l), 1-aminoethanol (1 mg/l), human transferrin (5 mg/l), bovine serum albumin-linoleic acid conjugate (Sigma) (200 mg/l), L -glutamine (300 mg/l), penicillin/streptomycin (100,000 units/l) and phytohemagglutinin-P' (Wellcome)

(2 mg/l). The supernatants were collected, centrifuged to remove the cells, concentrated by ultrafiltration ("Amicon Diaflo YM-10" membrane with 10 kD cutoff) and dialyzed against 0.1 M acetic acid ("Spectrapore 3" dialysis tubing system). The clarified retentate was lyophilized.

Gel permeation chromatography

The crude fraction was reconstituted in 20 ml of 1 M acetic acid (50 mg/ml) and loaded onto a "BioGel P-100" column (2.6 x 88 cm) equilibrated with 1 M acetic acid, at a flow rate of 0.5 ml/minute. 12 ml fractions were collected. An aliquot of each fraction was evaporated and a triplicate assay was performed for growth inhibitory activity (GIA) on A375 cells. The active fractions were combined, lyophilized and rechromatographed on a "Bio-Sil TSK-250" column (600 x 21.5 mm) as described above.

Reverse-phase high-pressure chromatography of TSK-250 fractions

The final purification of the pooled TSK-250 fractions was accomplished essentially as described by reverse phase HPLC. PBL tumor cell inhibitor was eluted from the "u-Bondapak-C18" column at an acetonitrile concentration of 40-41% and an n-propanol concentration of 36.5%.

125 Cell growth modulating test using I-iodo-deoxyuridine incorporation into DNA (GIA)

These tests were performed in flat-bottomed tissue culture trays (Costar 3596) with 96 wells. Human melanoma cells, A375 (4 x 10 3 ) in 50 µl of the test sample were added to each well and incubated for 3 days at 37°C. The cells were labeled for 24 hours with 125

I-IdU (0.05 uCi/well) and was further incubated for 24 hours. The cells were washed 3 times, were harvested with a multiple harvester, and the radioactivity was determined using a gamma counter.

Results

A PBL-derived tumor cell inhibitor composition was subjected to automated repeated Edman digestion. The N-terminal amino acid sequence is the following:

X denotes an amino acid that has not been identified.

A comparison of this sequence with the U937 factor sequence clearly indicates identity with the N-terminal part of the PBL-derived factor.

The ability of PBL-derived Oncostatin M to affect the replication of a number of different cells was then investigated. L929 mouse cells were found to be insensitive to PBL-derived Oncostatin M using up to 1,000 GIA units/ml. Human fibroblast cells, WI26, were growth-stimulated by treatment with 1,000 GIA units/ml. Normal human T-lymphocyte formation 72 hours after mitogenesis was not affected by up to 500 GIA units/ml.

It is clear from the results stated above that a hitherto unknown polypeptide and unknown polypeptide fragments have been provided which can be used to modulate cellular growth. The compound has been shown to exhibit different activity depending on the type of cell line involved so that it can be used alone or together with other compounds to modulate cellular growth. The specified peptides can therefore be used with mixtures of cells both in vivo and in vitro to selectively reduce or increase cellular proliferation of a particular type of cells.

In particular, the factor can be used to treat cells with a view to autologous bone marrow transplantation. Application of the factor inhibits the growth of tumor cells in the marrow and can stimulate colony cell formation. Oncostatin M can also be used to stimulate the growth of epithelial cells and thus promote wound healing. In addition, the intact polypeptide or fragments thereof can be used as immunogens for inducing antibody formation. The formed antibodies can be used to determine the strength of Oncostatin M which is present in a body fluid, or to modulate the activity of the factor by binding to it. Furthermore, these antibodies together with purified Oncostatin M or fragments thereof can be used as components

in a diagnostic kit together with other reagents, in particular antibodies, for the detection and quantification of Oncostatin M.

Claims (4)

1. Process for the production of a cell growth-regulating polypeptide which is capable of inhibiting the proliferation of neoplastic cells and stimulating the proliferation of normal human fibroblast cells, which does not inhibit human proliferative and cytotoxic T-cell reactions, does not inhibit granulocytic/myelocytic bone marrow cell colony formation, has a molecular weight in the range from 17 to 19 kD as determined by gel exclusion chromatography and 28 kD as determined by SDS-PAGE, and is relatively insensitive to moderately acidic and basic conditions and stable at a temperature of 56°C, and comprises at least one amino acid sequence which differs by at most three amino acids, and preferably by no more than one amino acid from one of the following sequences: characterized by isolating leukocytes from a mammal, which leukocytes are selected from the group consisting of histocytic lymphoma cells and normal human peripheral blood lymphocytes, activating the leukocytes with an inducing agent selected from the group consisting of ingenols and phorbols, when the leukocytes are selected from histocytic lymphoma cells, or mitogens, when the leukocytes are selected from normal human peripheral blood lymphocytes, and from the activated leukocytes free of cellular material, isolates the cell growth regulatory polypeptide in a purity that provides a specific activity of at least 10 GIA units/ng protein and preferably at least 100 GIA purity units/ng protein. 2. Method according to claim 1, characterized by selecting U937 cells as histocytic lymphoma cells. 3. Method according to claims 1-2, characterized in that 12-0-tetra-decanoylphorbol-13-acetate is selected as phorbol. 4. Method according to claim 1, characterized by choosing phytohemagglutinin-P as a mitogen.