SE505059C2 - Method for cell growth regulating factors and antibodies reactive with them - Google Patents

Abstract

Novel cell growth regulatory compositions are provided. The compositions are obtainable from leukocytes, e.g., human peripheral blood lymphocytes (PBLs) or human histiocytic lymphoma lines cultured with one or more inducers. The naturally occurring peptide can be obtained by careful purification of the conditioned medium of such cells and is shown to have selective inhibitory activity against certain cell lines and selective stimulatory activity against other cell lines. The polypeptide may be isolated from conditioned medium of cultured human PBLs or of histiocytic lymphoma cell lines, may be synthesized, or may be prepared by cloning of the gene in an appropriate host. Mutants and fragments are also provided.

Description

505 059 must be characterized; and the purified factor must be concentrated in sufficient quantity to allow identification and characterization of the factor. With an increasing number of factors that have been isolated, each new factor therefore becomes more difficult to identify because its role and function can be obscured by the many other factors that are present.

Description of the Prior Art Beal et al., Cancer Biochem. Biophy_. (1979) å: 93-96 reports the presence of peptides in human urine, which inhibit growth and DNA synthesis more in transformed cells than in normal cells. Holley 25 âi., Prcc. Natl. Acaa.Asc1. (19ao) 11 = s9s9- -5992 describes purification of epithelial cell growth inhibitors. Le- tansky, Biosci. Rope. (1982) 2: 39-45 report that peptides, which have been purified from bovine placenta, inhibit tumor growth and thymidine incorporation with DNA to a greater extent in neoplasms than in normal cells. Chen, Trends Biochem. Sci. (1982) 1: 364-365 report isolation of a peptide from ascites fluid with anti-cancer properties. Redding and Schally, Proc. Natl. Acad. Sci. (1982) Z2: 7014-7018 report isolation of purified peptides from porcine hypothalamus, which exhibit antitumogenic activity against several normal and tumor cell lines.

Sone gt al., Gagn (1984) Z§: 920-928 reports the formation of a factor, which is produced by human macrophages and which inhibits the growth of certain tumor cells in vitro. Ransom et al., Cancer Res. (1985) í§: 851-862 reports the isolation of a factor called leukoregulin, which inhibits the replication of certain tumor cell lines and appears to differ from lymphytoxin, interferon and interleukin 1 and 2. Most of these factors do not fully characterized and not or are their primary structures known.

Aggarwal gt al., J. Biol. Chem. (1984) 2â2: 686-691 have purified and characterized human lymphotoxin (LT), which is produced by a lymphoblastoid cell line, and subsequently sequenced LT (Aggarwal et al., J. Biol. Chem. (1985) 2Q: 2334). Gamma interferon (YïIF), which is produced by lymphoid cells and has -un 505 059 immunomodulatory and tumor inhibitory activity, has also been cloned and subjected to expression. (Gray gt al., Nature (1982) g2§: 503: 508). Tumor necrosis factor (TNF), which inhibits the growth of certain tumors and is produced by macrophages and certain leukemia cell lines, has been characterized and TNF cDNA has been cloned and subjected to expression in E. coli (Pennica gt al., Nature (1984) § l3: 724).

SUMMARY OF THE INVENTION A novel peptide factor and biologically active fragments thereof are provided, which factor is available from leukocytes. The factor finds use in modulating cell growth, such as inhibiting tumor cell growth and simulating the growth Å of normal fibroblasts and can modulate immune functions.

The factor has an amino acid sequence that is clearly different from; the sequences of other compounds that have been reported exhibit analogous properties.

BRIEF DESCRIPTION OF THE DRAWINGS Pig. 1 represents the amino acid sequence of fragments of Oncostatin M; Fig. 2 is a series of photomicrographs of cells treated with Oncostatin M, where (AC) are melanoma cells A375 treated with 0, 5 and 100 GIA units respectively and (DF) fibroblasts W138 are treated with 0, 5 and 100 GIA devices; and Fig. 3 is a photograph of an SDS-PAGE assay of Oncostatin M.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS A novel polypeptide, polypeptide preparations, polypeptide fragments and mutations, their preparation and use are provided, which formulations show activity in modulating cell growth, in particular inhibition of tumor cell growth and stimulation of normal fibroblasts. A polypeptide of the invention, termed Oncostatin M, is available from leukocytes; for example, from conditioned media of stimulated U937 cells or conditioned media of stimulated normal human peripheral blood lymphocytes (PBL). Fragments and mutants of the polypeptide having the biological activity of intact Oncostatin M, such as cell growth modulating activity or immunological activity, are also provided.

The polypeptide fragments of this invention are novel polypeptides of at least 8 amino acids which are biologically active, at least immunologically cross-reactive with naturally occurring Oncostatin M. By "immunologically cross-reactive" is meant an antibody induced by a novel polypeptide of this invention. cross-reacts with intact Oncostatin M, at least when Oncostatin M is present in a denatured state. These polypeptides are therefore useful for inducing antibodies to Oncostatin M, which can be used to determine the concentration of Oncostatin M in a body fluid, to bind Oncostatin M and thus modulate its activity, and to purify Oncostatin M, as by use in an affinity column. Some of the polypeptides may also retain the cell growth modulating activity of intact Oncostatin M, whereas this activity may be modulated but is usually reduced compared to intact Oncostatin M.

Fig. 1 represents the amino acid sequence of poly (amino acids) which are cross-reactive with Oncostatin M, the first sequence representing the N-terminus of Oncostatin M.

Poly (amino acids) of the present invention contain an amino acid sequence of at least 8 consecutive amino acids, which corresponds to an amino acid sequence shown in Fig. 1 and does not differ from this sequence of more than 3 amino acids, usually not more than 1 amino acid. This difference can be either the introduction of one amino acid, the absence of one amino acid or the replacement of one amino acid with another, especially a conservative substitution. Usually the poly (amino acids) contain at least and usually at least 12 consecutive amino acids, which correspond to the sequences shown in the figure and do not differ by more than 1 amino acid.

For the purposes of the present invention, the various amino acids are divided into a number of subclasses. The following table indicates the subclasses. aliphatic neutral non-polar GAPVLI polar STCMNQ Acid DE basic KR aromatic FHY wi By "conservative substitution" is meant that amino acids from the same subclass (ie either neutral aliphatic, acidic aliphatic, basic aliphatic or aromatic), more specifically matched with the same polarity, exchanged with each other.Additionally, amino acids having 2 to 4 carbon atoms or 5 to 6 E carbon atoms define monomer groups in the aliphatic subclass.

Poly (amino acids) do not have a length exceeding approx. 1000 amino acids. They usually contain less than 100 amino acids and usually less than 50 amino acids. Thus, the poly (amino acids) γ can be easily synthesized. When the poly (amino acids) have a length exceeding 100 amino acids, these poly (amino acids) can usually be polymers or fragments of Oncostatin M with less than 100 amino acids each or fusion proteins, where the fragment is linked to an antigen, enzyme, enzyme fragment etc.

In particular, the high molecular weight poly (amino acids) may consist of at least one polypeptide fragment with less than about 100 amino acids, which are covalently linked to a large immunogenic polypeptide carrier to provide immunogenicity. Examples of such protein carriers are bovine serum albumin, "keyhole limpet hemocyanin (KLH)" and the like. These conjugated polypeptides are useful in inducing antibodies in a suitable host organism.

U937 cells are a cell line derived from a histiocytic lymphoma cell line (Sundstrom and Nilsson, Int. J. Cancer (1976) IZ: 565-577), which can be induced to differentiate into cells with the properties of macrophages after treatment with a variety of agents ( Harris gt al., Cancer Res. (1985) g§: 9-13).

To produce Oncostatin M, the U937 cells can be grown in a conventional serum nutrient medium and treated with a suitable inducer. Suitably phorbol or ingenols may be used, especially 12-O-tetradecanoylphorbol-13-acetate (TPA). Usually approx. 5-20 ng / ml of the inducer is used. The original number of cells is approx. 105-106 cells / ml.

After the cells have been treated with the inducing agent for a sufficiently long time, generally 3 to 6 days, the supernatant is removed, the cells are washed with a serum-free nutrient medium, adherent cells are washed again with serum-free medium and the cells are incubated for at least 12 hours. , usually not more than approx. 48 hours, in serum-free nutrient medium, for example the medium RPMI-1640. Excess fluids are then collected and the cells are removed by centrifugation. Cell-free overlying fluids have been tested for cell growth inhibitory activity (GIA), as described in the experimental section. The supernatant contains approx. 50- -500 units GIA / ml (see experimental section on the definition of GIA units).

Oncostatin M can also be obtained from mitogen-stimulated, normal human peripheral blood lymphocytes (PBL). PBL can be isolated from leuko fractions by diluting the fractions and centrifuging them over Ficoll gradients. Cells, which are recovered from the gradient interface, are washed and shock lysed to remove red blood cells. Remaining cells are recovered from said solution by centrifugation, resuspended in buffer containing serum and thrombin, stirred and the platelet aggregate allowed to settle for a short period of 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 adhesion of monocytes and B lymphocytes and then washed. Most peripheral blood T lymphocytes do not attach and elute from the column. These cells have been cultured at 37 ° C in culture medium, for example the medium RPMI-1640 and treated with a suitable inducing agent, for example phytohemagglutinin (approx. 1-5 mg / liter) for approx. 100 hours and then the supernatants have been recovered. The supernatants were centrifuged to remove cells and concentrated by ultrafiltration or dialysis.

After isolating cell-free supernatant from either U937 cells or normal PBL cells, the conditioned medium is concentrated, preferably using a semi-fiber system or an ultrafiltration membrane, followed by dilution with acetic acid (to a concentration of 0.1 N acetic acid) and then through an approx. 10-fold concentration, after which the dilution and concentration are repeated. The concentrate can be lyophilized and used directly or the lyophilized product can be used for further purification.

The present Oncostatin M can be purified by a gel filtration chromatography procedure using an aqueous solution of 40% acetonitrile - 0.1% trifluoroacetic acid as the isocratic mobile phase on a column of Biosil TSK250 while monitoring the activity of each of the fractions. .

Purification gives a preparation with an activity of at least approx. 0.5 - x 104 GIA units / ml in the active fractions.

The partially purified product from the gel filtration chromatography can be further purified using reverse phase high performance liquid chromatography using a linear gradient, the primary solvent being 0.1% trifluoroacetic acid in water and the secondary solvent being acetonitrile. containing 0.1% trifluoroacetic acid. The program can vary and in general the chromatography requires approx. 3 - 4 hours with most of the time, more than approx. 50% of the time and no more than approx. 80% of the time, within the range of 30 - 45% of the secondary solvent. Under these conditions, the active fractions are eluted with approx. 41 - 42% acetonitrile.

The pooled active fractions can be further purified by repeating reverse phase HPLC using a faster change of gradient conditions and a lower flow rate. Under these conditions, the activity is eluted at approx. 40.5 - 41.5% acetonitrile.

Reverse phase HPLC can then be repeated while changing the solvent system, the secondary solvent being | n-propanol-0.1% trifluoroacetic acid. A linear gradient is used, whereby the gradient changes slowly within the interval | 23 - 35% n-propanol. The main activity is observed in the range 25.5 - 27.5% propanol, giving a substantially homogeneous product with a specific activity of more than 10 GIA-3 units / ng protein. Usually the product is purified to give a specific activity of at least approx. 100 GIA units / ng protein, in particular 150 GIA units / ng.

The present compounds are characterized by a molecular weight of approx. 17 - 19 kilodaltons (kD), in particular approx. 18 kD, as can be determined by size exclusion chromatography. The present compounds are further characterized by having 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 has been analyzed. Oncostatin essentially has the amino acid sequences shown in Fig. 1. Referring to Fig. 1, the first sequence illustrates the N-terminus of Oncostatin M, while the other sequences illustrate internal fragments of the polypeptide.

Active preparations of isolated Oncostatin M contain a mixture of high mannose and complex N-linked oligosaccharide. However, non-glycosylated preparations of Oncostatin M retain cell growth modulating activity.

Oncostatin M is further characterized by its activity against certain cell lines. The present polypeptide is in the absence of cytotoxic activity against human fibroblasts W126 and W138 and mouse cells L929, which are sensitive to tumor necrosis factor, and an β-interferon-sensitive human tumor cell line. The polypeptide has also been shown not to inhibit the proliferation of normal human T lymphocytes or to inhibit granulocytic / myelocytic colony formation from bone marrow cells at concentrations up to 100 GIA units / ml. Furthermore, Oncostatin M stimulates the proliferation of normal human fibroblasts as exemplified by WI38 and WI26 cells and inhibits the profiling of such tumor cells as A375, HBT10, A549 and SK-MEL28 and may enhance the growth of colony forming cells from normal bone marrow. Oncostatin M suppresses non-human proliferative or cytotoxic T cell responses in mixed leukocyte culture reactions (MLC) at concentrations of 500 GIA units / ml.

The present polypeptide has been found to be stable to medium strength acids and bases and to heat treatment at 56 ° C.

The amino acid sequence of the present polypeptide has been completely determined using commercially available sequencing apparatus. The polypeptide can then be synthesized according to the prior art using automated synthesizers, which are also commercially available.

Alternatively, the present polypeptide can be prepared by recombinant DNA techniques. Samples can be derived from a partial amino acid sequence, which can then be used to screen a human genomic library. The library can be a cDNA library or a chromosomal library. Once the clone or clones have been identified as bound to the sample, the fragments containing the gene of interest can be identified in a number of different ways and manipulated in a number of different ways. The fragment can be reduced in size by endonuclease restriction, with the resulting fragments being cloned and tested for the presence of the desired gene.

Cells that produce the desired peptide or produce increased amounts of the desired peptide can be used to produce messenger RNA. Single-stranded cDNA can be prepared from messenger RNA. The cDNA can then be bound to total messenger RNA from a cell, which produces little if any amount of the polypeptide. Unbound cDNA can then be isolated and used to prepare ds cDNA, which can be screened with the samples.

Alternatively, DNA fragments can be inserted into μg11, so that coding fragments can be downstream of and in frame with the β-galactosidase gene. Antibodies can be prepared against the Oncostatin M polypeptide and used to screen the resulting pooled proteins for cross-reactivity. In this way, fragments encoding the present polypeptide or fragments thereof can be identified and used to identify the desired gene.

Once a complete gene has been identified, either as cDNA or chromosomal DNA, it can then be engineered in a number of different ways to provide expression. When the gene is to be expressed in a host organism which recognizes the transcriptional and translational regulatory wild-type regions of Oncostatin M, the whole gene with its 5 'and 3' -regulating wild-type regions can be inserted into a suitable expression vector. Various expression vectors exist utilizing replication systems from mammalian viruses such as Simian Virus 40, adenovirus, bovine papillomavirus, vaccine virus, insect baculovirus, etc. These replication systems have been developed to provide ._.___ 1__! 11 505 059 hold markers which allow selection of transfectants as well as suitable restriction sites where the gene can be introduced.

When the gene is to be expressed in a host organism which does not recognize the naturally occurring transcriptional and translational regulatory wild-type regions, further manipulation is required. Conveniently, a variety of 3 'transcriptional regulatory regions are known and can be introduced downstream of the stop codons. The 5 'non-coding region upstream of the structural gene can be removed by endonuclease restriction, §al31 resection or the like. Alternatively, when a suitable restriction site is present near the 5 'end of the structural gene, the structural gene may be restricted and an adapter may be used to link it - \ ~.-.._ ..,. the structural gene to the promoter region, the adapter providing the lost nucleotides of the structural gene. Different strategies can be used to provide an expression cassette which, in 5 ', 3' control of the transcription, has a transcriptional and translational initiation region, which may also include regulatory sequences, such as -v- fl- wwwmuv- p-.ar- ..... takes into account the induction of regulation, the structural gene under the transcriptional and translational control of the initiation region and a transcriptional and ... -.- vv ---. .__ translational termination region.

Examples of transcriptional initiation regions or promoters include, for bacteria, the α-kgal promoter, the TAC promoter, the Å left and right promoters, etc .; for yeast glycolytic enzyme promoters, such as ADH-I and -II promoters, s GPK promoter and PGI promoter, TRP promoter, etc .; for mammalian cells, early and late SV40 promoters, later adenovirus promoters, etc. As already indicated, the expression cassette may be incorporated into an episomal maintenance replication system in a suitable cellular host organism or may be provided without a replication system, as can be integrated with the host genome. DNA can be introduced into the host organism according to known techniques, such as transformation, using calcium phosphate-precipitated DNA, transfection by contacting the cells with the virus, microinjection of the DNA into the cells or the like.

Once the structural gene has been introduced into the appropriate host organism, it may grow and will express the structural gene. In some cases, it may be desirable to provide a signal sequence (secretory leader) upstream of and within the reading frame of the structural gene, which signal sequence provides for secretion of the structural gene and cleavage of the secretory leader to provide the mature polypeptide in the the supernatant. If secretion is not provided, the host cells can be harvested, lysed according to conventional techniques, and the desired product isolated and purified according to known techniques, such as chromatography, electrophoresis, solvent extraction or the like.

The present compounds can be used in a wide variety of ways, both in vitro and in vitro. The present compounds can be used for the production of antibodies to the present compounds, which may find use in or in use.

The antibodies can be prepared in a conventional manner, either by using the present polypeptide as an immunogen and injecting the polypeptide into a host mammal, for example mouse, cow, goat, sheep, rabbit, etc., in particular with an adjuvant, for example complete Freund's adjuvant, aluminum hydroxide gel or similar. The host animal can then be bled and used to isolate polyclonal antibodies or, in the case of mice, peripheral blood lymphocytes or spleen lymphocytes (B cells) can be used for fusion with a suitable myeloma cell to immortalize the chromosomes for monoclonal expression of antibodies specific for the present compounds.

Either polyclonal or monoclonal antibodies can be prepared, which can then be used to diagnose or detect the presence of the present polypeptide in a sample, such as 505 cells or a physiological fluid, such as blood. The antibodies can also be used in affinity chromatography to purify the present polypeptide and isolate it from natural or synthetic sources. The antibodies may also find use in controlling the amount of the present polypeptide associated with cells in culture or inoculation, whereby the growth of the cells can be modified.

The present compound can be used as a ligand to detect the presence of receptors for the present compound. In this way, cells can be distinguished according to the presence and density of receptors for the present compound, monitoring the effect of different compounds on the presence of such receptors.

The present compound can be used in in vitro cultures to inhibit the growth of cells or cell lines that are sensitive to the present polypeptide, as opposed to cells that are not sensitive. Thus, heterogeneous cell mixtures or cell lines can be freed from unwanted cells, as the unwanted cells are sensitive to the present polypeptide.

The present polypeptide may be administered by administration in neoplastic conditions, for example by injection intralesionally, peritoneally, subcutaneously or the like. The present compound can be used to eliminate malignant cells from marrow for autologous marrow transplants or to inhibit proliferation or eliminate malignant cells in other tissues, for example blood, before reinfusion.

The present polypeptide can also be used in a method of treating wounds, such as cutaneous wounds, corneal wounds and various other epithelial and stromal disorders, such as chronic ulcers, burns, surgical incisions, traumatic wounds and injuries to the epithelial-lined cavities. , such as the esophagus, stomach, large and small intestine, oral cavity, genital area and urinary tract. The method is based on topical application of a treatment formulation comprising Oncostatin M in a physiologically acceptable carrier. 505 059 14.

The formulation of the present invention can be used to treat a variety of wounds including substantially all cutaneous wounds, corneal wounds and damage to the epithelial-lined puncture organs in the body. Wounds suitable for treatment include those derived from trauma, such as burns, abrasions, cuts and the like, as well as from surgical procedures such as surgical incisions and skin grafting. Other conditions suitable for treatment with the formulations of the present invention include chronic conditions such as chronic ulcers, diabetic ulcers and other non-healing (trophic) conditions.

Oncostatin M can be incorporated into physiologically acceptable carriers for application to the exposed area. The nature of the carriers can vary widely and depends on the intended application site. For application to the skin, a cream or ointment base is usually preferred; Suitable bases include lanolin, Silvadene (Marion) (especially for the treatment of burns), Aquaphor (Duke Laboratories, South Norwalk, Connecticut) and the like. If desired, it is possible to incorporate Oncostatin M-containing formulations with bandages and other wound dressings to provide continuous exposure of the wound to the peptide. Aerosol applications can also be used.

The concentration of the polypeptide in the treatment preparation is not critical. The polypeptide is present in an epithelial cell proliferation-inducing amount. The preparations are applied topically to the affected area, typically as eye drops in the eye or as creams, ointments or lotions on the skin. In the case of the eyes, it is desirable to have an often repeated treatment, which is usually repeated at intervals of 4 hours or less.

On the skin, it is desirable to continuously keep the treatment preparation on the affected area during healing with applications of the treatment compound from 2 to 4 times daily or more often. The amount used of the present polypeptide varies with the mode of administration, the use of other active compounds and the like, and is generally in the range of from about 1 Pg to 100 pg. The present polypeptide can be used with a physiologically acceptable carrier such as saline, phosphate buffered saline or the like. The amount of the compound used is determined empirically, based on the response of cells in Xitrg and the response of experimental animals to the present polypeptides or preparations containing the present polypeptides. The present compounds can be used as such or in combination with other growth factors or inhibitors or immunomodulators such as TMF, IL-2, γ-interferon, monoclonal antibodies, etc. The amount of these other compounds is generally in the range of 1 pg to 100 pg. ..._- w Conjugates of the present compounds to target cell-directed elements, for example antibodies, can be prepared when the antibodies are specific for particular malignant cells or organs.

The invention is further illustrated by the following working examples.

EXPERIMENTAL REPORT Materials and methods Oncostatin M is isolated from U937 cells, Preparation of a tumor cell growth inhibitor from a histiocytic lymphoma cell line U937 cells, a histiocytic lymphoma cell line (Sundstrom and Nilsson, Int. J. Cancer (1976) 1Z: 565-577) 850 cm roller bottles (Corning C2540) in a concentration of 4 x 105 cells / ml in a total volume of 300 ml medium RPMI 1640 supplemented with 10% fetal calf serum (FCS), penicillin / streptomycin (PS), L-glutamine and 10 ng / ml 12-O-tetradecanoylphorbol-13-2-acetate (TPA). Four days later, the supernatants containing FCS and TPA were removed, the roller bottles were washed sus us 16 five times with serum-free RPMI-640 and the cells released (1 x 105 cells / ml) were washed three times with serum-free medium and returned to bottles, resulting in a final volume of 125 ml of serum-free medium RPMI-1640 per roller bottle. One day later, the supernatants were collected, centrifuged to remove the cells, filtered through a 0.45 micron (p) Nalgene filter and concentrated using a half-fiber system (Amicon cartridge HIP10-20) to a volume of 150 ml ( initial 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 membrane PM-10, 10kD limitation, and dialyzed. After dialysis, the concentrate was diluted with acetic acid, resulting in a final concentration of 0.1N acetic acid in 500 ml, and concentrated to 50 ml using an Amicon PM 10 filter. The 50 ml concentrate was diluted to 400 ml with 0.1 N acetic acid and concentrated to 40 ml with the same filter. The concentrate was diluted with 1N acetic acid and the resulting precipitate was removed by centrifugation. The resulting concentrate was frozen and lyophilized. The lyophilized material was used for the purification steps.

Gel filtration chromatography A Bio-strainer TSK-250 column (600x21.5 mm) (Bio-Rad) was connected to a high pressure liquid chromatography system. The crude fraction (10 mg / ml) was dissolved in 40% acetonitrile in a 0.1% aqueous solution of trifluoroacetic acid (0.1% TFA). A 2 ml aliquot of the mixture was injected and elution was performed isocratically with a mobile phase of 40% acetonitrile in 0.1% TFA. The flow rate was 2.5 ml / minute and the paper speed 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 three times for growth inhibitory activity (GIA) in A375 cells.

The active fractions (fractions 21 and 22) from six runs were pooled. The pooled material had an activity of a total of approx. 4.8 x 105 apparent molecular weight of 18 kD when determined by size GIA units. The factor was found to have an exclusion chromatography (Bio-Sil TSK-250 column).

High pressure reverse phase 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 isocratically injected onto a p-Pondapak-C18 column (7.8 x 300 mm) (designated C181) at room temperature. The flow rate was set at 2.0 ml / minute and the paper rate at 0.25 cm / minute. The linear gradient was used between the primary solvent 0.1% TFA and the secondary solvent acetonitrile-TFA 0.1%. The gradient conditions were 0 - 30% for 20 minutes; thereafter - 45% for 150 minutes; 45 - 55% for 20 minutes and 55 - 100% for 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 majority of the activity. The active fractions were eluted between 41 - 52% of the acetonitrile concentration.

Fractions 72 - 75 were pooled. 16 ml of 0.1% TFA was added to the pooled fractions. The mixture was injected onto a p-Pondapak-C18 column (3.9 x 300 mm) (designated C182) at room temperature. The flow rate was set at 1 ml / minute and the paper rate was 0.25 cm / minute. The gradient conditions were 0- -35% over 10 minutes; 35 - 45% for 100 minutes; and 45 - 100% for 10 minutes. Fractions were collected and aliquots were taken and analyzed for GIA. Most of the activity originated from the column between 40.7 and 41.3% acetonitrile concentration (retention time 83 - 86 minutes).

Active fractions were pooled and diluted twice with 0.1% TFA and injected isocratically onto a P-Bondapak C18 column 505 059 18 (3.9 x 300 mm) (designated C183) at room temperature. The flow rate was 1 ml / minute and the paper rate was 0.25 cm / minute. A linear gradient was used between the primary solvent 0.1% TFA and the secondary solvent n-propanol-TFA (0.1%). The gradient conditions were 0 - 23% for minutes and 23 - 35% for 120 minutes. Fractions were collected and aliquots of each fraction were analyzed for GIA. Most of the activity occurred between 25 and 26.5% propanol concentration (retention time 59 minutes).

This apparently homogeneous fraction contained approximately 300 ng of protein and approx. 40,000 GIA devices.

Cell growth modulating assay by incorporating 3 H-thymidine into DNA (GIA) The assays were performed in 96-well plate plates (Costar 3596). Human melanoma cells (A375) were used as a sensitive indicator cell line. Cells (3x10 3) in 0.1 ml of Dulbecco's modified Eagles medium (DMEM), supplemented with 10% FCS and PS, were placed in each compartment. Three hours later, 0.1 ml of sample was added to each compartment. The plates were incubated at 37 ° C for 3 days. Then, 0.025 ml (0.5PCi) of a solution of 3 H-thymidine (specific activity 27pCi / pg) was added to each well during the last six hours of the incubation. The cells were then transferred to glass filter strips using a multi-compartment 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 quantify the 3 H-thymidine addition.

Soft agar colony inhibition assay (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-compartment tissue culture plates made by Costar. 0.5 ml of 0.3% agar containing the same mixture of medium and FCS, 1 - 2.5 x 103 A375 cells and the factor to be tested in different concentrations were layered on the base layer of agar. The plates were incubated at 37 ° C in a humidified atmosphere of 5% CO 2 in air and after 7 days again charged with 0.5 ml of 0.3% agar containing the same medium and concentrations of factor. Colonies were counted unfixed and unstained and the number of colonies larger than 6 cells was noted between days 7 and 14.

Results Sequences of Oncostatin M are isolated from U937 cells The N-terminal sequence and internal fragments of Oncostatin M were determined by microsequencing of the reduced and S-pyridine-ethylated polypeptide and of peptides obtained by enzymatic digestion of reduced and S-pyridinylated Oncostatin M with the endoproteinase Lys-C and Staphylococcus aurggs V8 protease. The peptide fragments were purified by reverse phase HPLC using volatile solvents. The peptides were subjected to automated repetitive Ednan degradation in a Model 47OA protein sequencer (Applied Biosystems, Inc.). The phenylthiohydantoin amino acids were analyzed by reverse phase HPLC (Applied Biosystems, Inc.) with a PTH-C18 column (2.1 x 220 mm, ABI) using a sodium acetate buffer / tetrahydrofuran / acetonitrile gradient for elution.

The resulting amino acid sequences are essentially those illustrated in Figure 1.

A comparison of these sequences with those stored in the present protein database (PIR version 9.0, May 1986) did not reveal any significant sequence homologies with any other known sequence. Furthermore, there is no homology with tumor necrosis factor, lymphotoxin, colony stimulating factor, interleucine 1 or 2 or ß-transforming growth factor. 29 'sus 059 20 Inhibition of tumor cell proliferation and enhancement of normal human fibroblast proliferation Using lymphoma colony inhibition assay described above, the following results were obtained: Inhibition of colony formation of melanoma cells 3375 Table 1 in soft agar by purified Onoostatin M cells isolated from GIA 379 units / Number% inhibition of compartment colonies colony formation 250 4 96 83 1 6 94 27 11 89 45 32 '69 0 106 - * A375 cells were plated in soft agar} with or without factor, in a final volume of 2 ml as described above . The factor used was from a C18 propanol column fraction with tumor growth inhibitory peak activity (GIA). 11 days later, the number of colonies was counted. A colony was defined as a cluster of at least 6 cells. A GIA unit was defined as the amount that causes § 50 inhibition of the incorporation of 3H-thymidine into A375- or microwells as described in detail above.

In the next study, different treatments were used to determine the effect of the treatment, which was either chemical or physical on the activity of the present polypeptide.

The following table shows the results.

Effect of Different Treatments of Supernatants of TPA-Induced U937 Cells on Tumor Growth Inhibitory Activity * Final Dilution of Supernatant 1: 4 1: 8 1:16 Table 2 157 21 5Û5'Û59 Medium Control - - - 39,730 Untreated. supernatant 7206 13896 16000 1N acetic acid 6670 17073 18783 1N NH 4 OH 6956 15016 ~ 13923 ø * U937 cells were treated with TFA (10ng / ml) for 3 days and then the cells were washed with media and incubated for 24 hours. in serum-free media before recovering the supernatant. The supernatants were treated with 1N acetic acid or 1N ammonium hydroxide (NH 4 QH). They were then dialyzed against the medium and tested for their ability to inhibit the incorporation of 3 H-thymidine into A375 cells. VA375 cells were labeled with 38-thymidine (3 H-Tdk) for the last six hours of a three-day incubation.

** Displayed data are BH-TdR incorporation pulses per minute.

The above results show that Oncostatin M in the dialyzed supernatant is mainly resistant to inactivation of abnormal acetic acid and normal ammonium hydroxide. The present compounds are thus relatively insensitive to both medium acid and medium has. The present compound is stable to heat treatment at 56 ° C for 1 hour but not at 90 ° C for 30 minutes.

The present compound was also tested for thermal stability and was found to retain its activity after exposure for 1 hour at 56 ° C but was found to lose substantially all activity after exposure for 30 minutes to 95 ° C.

In the next study, the ability of the present polypeptides to inhibit tumor cell replication of a variety of neoplastic cells was determined. The following table lists the results. 505 059 22 Table 3 Inhibition of the replication of purified Oncostatin M tumor cells from U937 cells GIA units causing 30% inhibition Tumor cells 3H TdR incorporation A549 (lung cancer) 21 HTB1O (neuroblastoma) 81 A375 (melanoma) 0.3 * Tumor cells was plated in micro-compartments 3 hours before the addition of various dilutions of Oncostatin M, which had been purified by reverse phase HPLC as described in detail above. Before the last six hours of a three-day incubation, the cells were labeled in 0.2 ml of medium with 3n-thymidine (3n-TaR) (0.5 Pci / feek). One unit of tumor growth inhibitory activity (GIA) is defined in the commentary to Table 1 as the amount that causes 50% inhibition of 3 H-TdR incorporation into A375 melanoma cells. One unit was determined to correspond to approx. 10pg purified protein, so the concentration (ng / ml) that gives rise to 30% inhibition of '3n-mn in A549, Hfrmo and A375 cells was ce. 1.4, 4.0 respectively 0.015 ng / ml. The incorporation of 3 H-TdR into normal human fibroblasts WI26 was not suppressed by the U937 factor in any experiment.

The above results show that Oncostatin M is selective in its ability to inhibit replication, with widely differing effects depending on the nature of the cell. The present compound is effective against melanoma cells, such as melanoma cells A375, squamous cell carcinoma cells, such as A549, and neurobal cells such as HTB10. 3 Tumor cells were inoculated with 3 x 10 6 cells / compartment and normal fibrous cells / compartment in plates fitted with 1.5 x 10 96-well roblasts for 3 hours. Various concentrations of purified Oncostatin M, which had been obtained from the fraction of the C183 colon with antiproliferative peak activity against A375 cells, were added and three days later the incorporation of 3 H-thymidine into the cells was measured in triplicate compartments for each concentrate. - tion. The results are shown in Table 4.

/ Table 4 Inhibition of tumor cell proliferation and enhancement of normal fibroblasts proliferation by Oncostatin M Experiment GIA units / compartments% inhibition% stimulation A375 WI38 1 16 83 25 4 '62 30 46 46 A375 HTB10 WI26 Experiment 2 27 NT 28 46 9 87 22 36 3 76 11 52 A375 A549 Experiments 3 75 89 30 85 22 8 71 16 A375 SK-MEL28 Experiments 4 20 87 44 75 25 59 11 The results shown are percentage inhibition or percentage stimulation of 3H-thymidine incorporated with tumor cells (A375 , HTB10, A549 and SK-MEL28) and normal fibroblasts (WI26 and WI38), respectively. A GIA unit is defined in the footnote to Table 1 505 059 24 as the amount of Oncostatin M that gives rise to 50% inhibition of the incorporation of 3 H-thymidine into A375 cells.

In addition to the observed different effect on the incorporation of 3 H-thymidine into tumor cells and normal human fibroblasts, there is also an established differential effect in terms of morphology and cell number after 3 days of treatment of the two cell types with Oncostatin M as shown in Figure 2.

The Oncostatin M used was derived from the HPLC-C183 column fraction with peak activity in inhibiting the proliferation of A375 cells. Fig. 2 is a series of photomicrographs of A375 melanoma cells, which were untreated (A), treated with 5 growth inhibitory activity units (GIA) of Oncostatin M (B) or 100 units (C). The photomicrographs of WI38 fibroblasts refer to untreated (D), treated with 5 units of GIA (E) or 100 units (F). The cells were stained with crystal violet in 0.5% methanol. Magnification = 63 X.

NaDodSO4 / PAGE of Oncostatin M Purified Oncostatin M, which was exposed to NaDodSO4 under reducing conditions, was found to have an apparent molecular weight of approx. 28 kD as shown in Fig. 3. The following proteins were used as standard (lane A): ovalbumin, Mr = 43 kD; chymotrypsinogen gf, Mr = 25.7 kD; lactoglobulin ß, Mr = 18.4 kD; lysozyme, Mr -14.2 kD; bovintrypsin inhibitor = 6.2 kD; insulin A and B chains, Mr = 2.3 kD and 3.4 kD, respectively. Oncostatin M was applied in lane B. Oncostatin M, which was subjected to PAGE under non-reducing conditions, also had an apparent molecular weight of 28 kD and protein electroeluted from the band was found to inhibit the proliferation of A375 cells. 505 059 Antibody to a synthetic peptide of Oncostatin M reacts 125I with -labeled Oncostatin M in radioimmunoprecipitate a) Peptide synthesis: The peptide corresponded to residues 6 - 9 of. The oncostatin M protein and was synthesized by solid state techniques in an automated Beckman apparatus as described by Gentry gt eel., J. Biol. Chem. (1983) 2â§: 11219. The peptide was cleaved from the resin using the HF "low-high" method (Tam et al., J. Amer. Chem. Soc. (1983) 105: 6442-6445). Purification was performed by preparative HPLC. b) Production of antibodies: The peptide was coupled to bovine-γ 'globulin as described by Gentry and Lawton, Virologx (1986) §§: 421-431. White New Zealand rabbits were primed and given booster doses (5 times) at 4 sites subcutaneously as described by Gentry and Lawton, Virology (1986) 152: 421-431.

The use of antisera was obtained 2 weeks after the fifth booster dose. c) Iodination of Oncostatin M A sample of partially purified Oncostatin M was radiolabeled with iodine-125 using published procedures (Linsley gt al., §§ê§ (1985) §š: 356-360). An aliquot of the labeled preparation containing 100,000 pulses per minute was mixed with rabbit antiserum directed to the 17 N-terminal amino acids of Oncostatin M (final dilution 1:20) in the absence or presence of the N-terminal peptide (the 17 The N-terminal amino acids of Oncostatin M) (2 pg) and subjected to immunoprecipitation analysis as described by Linsley et al., Gig; chemistry (1986) 25: 2978-2986.

More specifically, a tube containing 5 μl was incubated with 2 pg of the N-terminal peptide in 10 ml of TNEN (20 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.05% Nonidet P-40) containing 0.1% BSA for 30 minutes at 4 ° C before the addition of I125- -Oncostatin M in 85A TNEN containing 0.1% BSA and 40mM diethiothreitol (DTT). Seven tubes containing 5 μl of antisera were incubated with I125-Oncostatin in 85 μl of TNEN containing 0.1% BSA and 40 mM DTT for 30 minutes at 4 ° C before the addition of 50 μl of 10% formalin-fixed Staphylococcus aureus (Pan-sorbin, Calbiochem). .

After a further incubation for 30 minutes at 4 ° C, the tubes were microfuged and the pellets were washed four 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. The precipitation of this substance was inhibited by the inclusion of an excess of unlabeled peptide corresponding to the 17 N-terminal amino acids of Oncostatin M, which showed that the precipitation was specific for this peptide.

The carbohydrate composition of Oncostatin M was tested by testing the glycosidase sensitivity. Immunoprecipitates, which had been prepared according to c) above, were treated with buffer, endoglycosidase H or neuraminidase, as described by Linsley et al. (1986). Treatment with endoglycosidase H, an enzyme with specificity for N-linked high mann oligosaccharides, resulted in the appearance of a low molecular weight substance with Mr = = 24 kD. Only a portion of the radiolabeled material was sensitive to this enzyme, indicating that not all molecules contained high mannosol oligosaccharides. Treatment with neuraminidase resulted in the appearance of a single band with Mr = 27 kD, which showed that the heterogeneity of 1251-labeled Oncostatin M was that the size of untreated attributed to a molecular heterogeneity in sialylation of the glycoprotein nucleus. The results showed that active preparations 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) Preparation of a tumor cell growth inhibitor from PBL Leukofractions containing PBL, obtained from the blood bank, ___., 2, sos 059 were diluted 1: 1 with phosphate buffered saline, pH 7.4 (PBS). 35 ml of the diluted blood was underlayered with 10 ml of a solution consisting of 9% Ficoll containing 20% by volume of 50% sodium diatrizoate (final specific gravity 1,080). The gradients were centrifuged at room temperature for 20 minutes at 850 x g.

Cells were recovered from the gradient interface and washed with PBS. Red blood cells were shock lysed for 3-4 minutes with -20 ml of a solution containing 0.8% ammonium chloride and 0.1% Na4-EDTA.

Cells were recovered from the lysed red blood cell solution by centrifugation at 600 x g for 10 minutes and resuspended in 10 ml of RPMI-1640 medium (GIBCO) containing 5% bovine fetal serum. Thrombin was added to a final concentration of 0.5 U / ml. The cell suspension was stirred for 5 minutes at 37 ° C and the platelet aggregates were allowed to settle for 5 minutes. The suspended cells were transferred to new tubes, recovered by centrifugation, resuspended in 1 ml of bovine fetal 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 for _ _ ................._......_............. ....__..........._ @. e ... to allow adhesion of monocytes and B lymphocytes. The column was then washed with three volumes of RPMI 1640 medium (37 ° C) containing 5% bovine fetal serum and the eluted non-adherent cells (PBL) were recovered.

PBL (2 x 106 cells / ml) is dissolved at 37 ° C, so co2-9s air for 96 hours in RPMI 1640 medium (10.4 g / liter) containing FeSO4-7H2O (1 mg / l) , ZnSO4-7H2O (2 mg / l) Na2SeO3-5H2O (0.017 mg / l), 1-aminoethanol- (1 mg / l), human transferrin (5 mg / l), bovine serum albumin linolenic 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 membrane YM-10, 10 Kd restriction) and dialyzed against 0.1 M acetic acid (dialysis tube Spectropore 3) . The managed retentate was lyophilized.

Gel filtration chromatography The crude fraction was reconstituted in 20 ml of 1 M acetic acid (50 mg / ml) and applied to a BioGel-P-100 column (2.6 x 88 cm), which had equilibrated with 1 M acetic acid at a flow rate of 0.5 ml / min. Fractions of 12 ml were collected. An aliquot from each fraction was evaporated and analyzed threefold for growth inhibitory activity (GIA) on A375 cells. The active fractions were pooled, lyophilized and chromatographed on a Bio-Sil TSK-250 column (600 x 21.5 mm) as described.

Reverse phase high pressure chromatography of TSK-250 fractions: The final purification of pooled TSK-250 fractions is accomplished by reverse phase HPLC essentially as described above.

Tumor cell inhibitors derived from PBL were eluted from Pßondapak C18 carriers at concentrations of 40-41% acetonitrile and at 26.5% n-propanol, respectively.

Cell growth modulation assay with the incorporation of 125 deoxyuridine into DNA (GIA): I-iodine- These assays were performed in 96-well flat-bottomed tissue culture dishes (Costar 3596). Human melanoma cells, A375 (4 x 103), in 50 ml test samples were added to each compartment and incubated for 3 days at 37 ° C. The cells were labeled for 24 hours with 125 I -IdU (0.05 pCi / compartment) and incubated for an additional 24 hours. The cells were washed three times, harvested with a multiple sample harvester and the radioactivity was measured in a gamma counter. 505 059 29 Results A preparation of tumor cell inhibitor derived from PBL was subjected to automated repeated Edman degradation. The amino-terminal amino acid sequence was as follows: A-A-1-G-x-x-x-x-E-x-x-v-L-x-x-Q-L-Q-K X represents an amino acid that has not been identified.

A comparison of this sequence with the sequence of U937 factor shows clear identity with the N-terminus of factor derived from PBL. 1 5 10 15 PBL factor A-A-I-o-x-x-x-K-s-x-x-v-L-x-x-Q-L-Q-K u937- factor A-A-I-G-s-c-s-K-E-1-R-v-L-L-G-Q-L-Q-x In the next study, the ability of PBL to examine It was found that L929 mouse cells were insensitive to Oncostatin M derived from PBL when using up to 1000 GIA units / ml. Human fibroblasts, WI26, were stimulated to grow by treatment with 1000 GIA units / ml. Proliferation of normal human T lymphocytes 72 hours after mitogenesis was not achieved at up to 500 GIA units / ml.

The results above show that a new polypeptide and polypeptide fragments are provided, which can be used to modulate cell growth. The compound has been shown to exhibit varying activity depending on the nature of the cell line involved, so that it may be used as such or in conjunction with other compounds for modulating cell growth. The present polypeptides therefore involve the addition of an additional polypeptide, which can be used with mixtures of cells, both in vivo and in vivo, to selectively reduce or enhance the cell proliferation of a particular type of cell.

In particular, the factor can be used to treat cells for autologous bone marrow transplants. The use of the factor inhibits the growth of tumor cells in the marrow and can stimulate colon cell formation. Oncostatin M can also be used to stimulate the growth of epithelial cells and thus promote wound healing. Furthermore, the intact polypeptide or fragments thereof can be used as immunogens to induce antibody formation. The induced antibodies can be used to determine the level of Oncostatin M present in a body fluid or to modulate the activity of the factor by binding thereto. Furthermore, these antibodies together with purified Oncostatin M or fragments thereof serve as a component in diagnostic test kits together with other reagents, especially antibodies for detecting and quantifying Oncostatin M.

Claims (5)

10 15 31 505 059 Patent claims A method for producing a cell growth regulating polypeptide capable of inhibiting the proliferation of neoplastic cells and stimulating the proliferation of normal human fibroplastes and which does not inhibit proliferative and cytotoxic human T cell responses and does not inhibit granulocytic / myelocytic bone marrow colonic cell formation. from 17 to 19 kD, determined by gel chromatography, and 28 kD, determined by SDS-PAGE, and is relatively insensitive to medium acids and bases and is stable at a temperature of 56 ° C and includes at least one amino acid sequence that differs by no more than three amino acids and preferably not more than one amino acid from any of the following sequences: 1 s 1o 1s 2o zs ao ss AA-1-cscsKEY - RvLLeQLQKQTnLMQnTsTLL-TPv-1 1 5 10 15 20 QRLPKAQDLERSGLNIEDLE LREHCRERPGAFPSEQQLI-VG characterized by isolating leukocytes from a mammals, which leukocytes are selected from the group comprising histiocytic lymphoma cells and normal human peripheral blood lymphocytes, activate said lymphocytes with an inducing agent selected from the group consisting of ingenols and phorbol when said lymphocytes are selected from histiocytic lymphocyte cells are selected from normal human peripheral blood lymphocytes and from said activated leukocytes isolate, free from cellular material, said cell growth regulating polypeptide in a purity which provides a specific activity of at least about 10 GlA units / ng protein and preferably at least 100 GIA units / ng protein. The method according to claim 1, characterized in that U937 cells are selected as histiocytic lymphoma cells. Process according to Claim 1 or 2, characterized in that O-tetradecanoylphorbol-13-acetate is chosen as phorbol. The method according to claim 1, characterized in that phytohemagglutinin-P is selected as mitogen. Antibodies, characterized in that they are immunologically reactive with the polypeptide produced according to claim 1.