FI91484C - Process for Preparing a New Cell Growth Regulating Factor Oncostatin M - Google Patents

Description

91484

A method for producing a new cell growth enhancer, oncostyl M

Leukocytes, both lymphocytes and monocytes, have been implicated in tumor growth inhibition in a few tumor models. The increased incidence of malignant tumors in people with impaired intuition supports the claim that white blood cells are involved in tumor growth. Protein factors already produced and characterized by these white blood cells that inhibit tumor growth and immune functions include tumor interferons α- and tumor necrosis factor, lymphotoxin, interleukin 2, and other lymphokines. Because each of the factors isolated from women has a different spectrum of action and may interact differently with mulden factors, there is still a strong interest in isolating and characterizing all the factors that leukocytes produce when regulating cell growth and immune function. These compounds may be found individually or in combination in the treatment or diagnosis of cancer, as promoters of injury healing, or as immunomodulators in the treatment of immunocompromised, autoimmune, transplant, and the like patients.

Many difficulties can be encountered in the detection, isolation, purification or characterization of naturally occurring factors. Methods must be developed to separate and purify the factor of interest from other factors present in the impure starting material without denaturing the activity of the desired factor; it is necessary to develop biological assays that allow the identification of fractions during differences in which a particular factor is concentrated; the new factor must be distinguished from already known factors or other unknown factors which may be lasna and which may affect, either negatively or positively, the activity of the desired factor; and the purified factor must be concentrated in a sufficient manner to allow identification and characterization of the factor. Therefore, as the number of isolated factors increases, it becomes more difficult for Kay to identify each new factor, Silia its role and function may be obscured by numerous other factors in the plasma.

Bealin et al. [Cancer Biochem. Biophys. 3 (1979) 93-96], peptides are present in human urine that inhibit growth and DNA synthesis in strongly altered normal cells. Holley et al. [Proc. Natl. Acad. Sci. 77 (1980) 5,989-5992] describe the purification of epithelial cell growth inhibitors. Letanskyn [Biosci. Rep. 2 (1982) 39-45], peptides purified from bovine placenta inhibit tumor growth and the incorporation of thymidine-15 nl into DNA in a larger normal adult marrow cell. Chen [Trends Biochem. Scl. 7 (1982) 364-365] describes the isolation of a heart suppressive peptide from vesicular fluid. Redding and Schally [Proc. Natl. Acad. Sci. 79 (1982) 7014-7018] describe the isolation of one or more peptides having antimitogenic activity against several normal and tumor cell lines from porcine hypothalamus. Sone et al. [Gann. 75 (1984) 920-928] describe the production of one or more factors produced by human macrophages that inhibit the growth of certain tumor cells in vitro. Ransom et al. [Cancer Res.

45 (1985) 851-862] describe the isolation of a factor called leukoregulin, which inhibits the replication of certain tumor cell lines and appears to be a different substance, lym-photoxin, interferon and interleukin 1 and 2. Most of the 30 female factors have not been fully characterized, nor their primary structure. unknown.

Aggarwal et al. [J. Biol. Chem. 259 (1984) 686-691] have purified and characterized human lymphotoxin (LT) produced by a lymphoblastoid cell line and subsequently sequenced it [Aggarwal et al., J. Biol. Chem. 260 3 91484 (1985) 2 334]. Gamma-Interferon (/ -IF), which is produced by lymphoid cells and has an immune and antitumor effect, has been produced through cloning and 11-mentoring (Gray et al., Nature 295 (1982) 503-5508). Tumor necrosis (TNF), which inhibits the growth of juvenile tumors and is produced by macrophages and certain leukemic cells, has been characterized, and TNF cDNA has been cloned and expressed in E. coli [Pennica et al., Nature 312 (1984) 724].

The invention relates to a method for producing a new peptide factor available from leukocytes. T3113 agents have utility in the accumulation of cell growth, such as inhibition of tumor cell growth and stimulation of normal fibroblast growth, and may elicit immune functions. The amino acid sequence of tek3πι3η factor is clearly different from that of other compounds reported to have similar properties.

Figure 1 shows the amino acid sequences of a fragment of oncostatin M; Figure 2 is a series of micrographs of cells treated with oncostatin M with (AC) A375 melanoma cells treated with 0.5 and 100 GIA units, respectively, and (DF) W138 fibroblasts treated with 0, 5 and 100 GIA units, respectively. , 5 and 100 GIA units, respectively; and Figure 3 is a photograph of an SDS-PAGE analysis of oncostatin M.

More particularly, the invention relates to a method for producing oncostatin M substantially free of cell debris and other leukocyte proteins, which has the ability to inhibit tumor cell proliferation and stimulate the proliferation of normal human fibroblasts, and which does not inhibit human proliferative cell proliferative cells. formation of granulocytic / myelocytic bone marrow colon cells with a molecular weight of 17-35 19 kD as determined by gel exclusion chromatography and about 4,91484 28 kD as determined by SDS-PAGE, and which is subjected to treatment with 1 N acetic acid and 1 N ammonium chloride and 1 N ammonium. for one hour. The methods of the invention are characterized in that a) leukocytes, such as histocytic lymphoma cells, are isolated from a normal human peripheral blood lymphocyte, from a mammalian cell culture to a mammal; b) activating the leukocytes with an inducing agent such as ingenol, phorbol tal mitogen; and c) isolating oncostatin M from the chromatographically activated leukocytes in pure form from the rest of the cellular material.

Leukocytes from which oncostatin M can be obtained include, for example, stimulated U937 cells or stimulated normal human arterial blood lymphocytes (PBL), into whose media the factor is secreted.

Polypeptide fragments that are polypeptides of at least 8 amino acids that are biologically active at least in the sense that they cross-react immunologically with naturally occurring oncostatin M are disclosed in FI patent application 911 853. Immunologically cross-reactive means that the polypeptide the agent cross-reacts with the original whole oncostatin M with at least 25 -costatin M in the denatured state. These polypeptides are therefore useful for inducing antibodies to oncostatin M, which antibodies can be used to determine the concentration of oncostatin M in a body fluid, to bind oncostatin M, and thus to inhibit its activity, and to promote oncostatin M. n, for example in an affinity column. Some polypeptides may retain the cell growth-promoting effect of myOs native whole oncostatin M, although this activity may be elicited, usually reduced compared to the original whole oncostatin M.

5,91484

Figure 1 shows the amino acid sequences of poly (amino acids) cross-reacting with oncostatin;; the first sequence s1 represents the N-terminus of oncostatin M.

For purposes of this invention, various amino acids are divided into a number of subgroups. The subgroups are indicated in the following table: aliphatic neutrals

non-polar G A P V L I

10 polar S T C M N Q

acid D D

female K R

aromatic F Η Y W

15 U937 cells are a cell line derived from a histiocytic lymphoma cell line [Sundstrttm and Nilsson, Int. J. Cancer 17 (1976) 565-577], which can be induced to differentiate into cells with macrophage characteristics, treated with various agents [Harris et al., Cancer Res. 45 (1985) 9-13]. To produce oncostatin M, U937 cells can be grown in a conventional serum-containing medium and the cassette with an appropriate inducer. It is contemplated to use phorbols or in-genols, especially 12-O-tetradecanoyl phorbol 13-acetate (TPA). Usually about 5-20 ng / ml of inducer can be used. The initial number of cells is about 105 to 106 cells / ml.

After treatment of the cells with the inducer for a sufficient time, usually 3-6 days, the supernatant is removed, the cells are washed with serum-free medium, the adherent cells are washed again with serum-free medium and the cells are incubated for at least 12 hours, usually up to about 48 hours, in serum-free medium. In the 1640 platform. The supernatants are then collected and the cells are removed by centrifugation. Cell-free supernatants from 6,914,84 cells were examined for cell growth as described in the experimental section of es-tSvS activity (GIA). The supernatant contains about 50 to 500 GIA units / ml (the definition of GIA unit is shown in Experimental Section 5).

Oncostatin M can also be obtained from mitogen-stimulated normal human human thyroid lymphocytes (PBL). PBLs can be isolated from leukofractions by diluting the fractions and centrifuging them on Ficoll gradients. Cells recovered from the gradient interface are washed and shock disrupted to remove red blood cells. Cells divided by the well are recovered from the solution by centrifugation, resuspended in serum and thrombin-containing buffer, mixed, and the platelet aggregate allowed to settle for a short time. The suspended cells are removed, harvested by centrifugation, resuspended in serum and transferred to a column containing nylon wool. The column is incubated to allow attachment of monocytes and B lymphocytes and then washed. Most Såreisvere T-lymphocytes do not attach and are thus washed away. These cells were cultured at 37 ° C in medium, for example RPMI 1640, and treated with an appropriate inducer, for example phytohemagglutinin (about 25 1-5 mg / l) for about 100 hours, and then the supernatants were collected. The supernatants were centrifuged to remove cells and concentrated, for example, by ultrafiltration or dialysis.

After isolation of the cell-free supernatant from either U937-30 cells or normal PBLs, the treated medium is concentrated, covering the hollow fiber system or the ultrafiltration membrane, after dilution with acetic acid (to an acetic acid concentration of about 10 N) to a concentration of about 10 N, concentration and repeat dilution and concentration. The concentrate can be lyophilized 7,91484 and used directly, or the lyophilized product can be further purified.

Oncostatin M can be purified by gel permeation chromatography using an aqueous solution containing 40% acetonitrile and 0.1% trifluoroacetic acid as the isocratic mobile phase on a Bio-sil TSK250 column and monitoring the activity of each fraction. Purification gives a composition containing at least about 0.5 to 5 x 10 4 GIA units / ml in the active fractions.

The partially purified product from gel permeation chromatography can be further purified using RP-HPLC (lid phase high pressure liquid chromatography) using a linear gradient of 0.1% trifluoroacetic acid in water as the primary solvent and 0.1% acetic acid in secondary water as the secondary solvent. trifluoroacetic acid. The program can vary; in general, chromatography takes about 3-4 hours, most of the time, more than 50% and up to about 80% of the time, the proportion of 20 secondary solvents is 30-45%; the active fractions elute under female conditions with an acetonitrile content of about 41-42%.

The combined active fractions can be further purified by repeating RP-HPLC, changing the gradient conditions more rapidly and using a lower flow rate. Under these conditions, the activity elutes at an acetonitrile content of about 40.5 to 41.5%.

RP-HPLC can then be repeated by changing the solvent system to n-propanol containing 0.1% trifluoroacetic acid as a secondary solvent. A linear gradient is used, with the n-propanol content slowly changing from 23% to 35%. Most of the activity elutes at a propanol content of 25.5 to 27.5% to give a substantially homogeneous product with a specific activity of more than 10 GIA units / ng protein. The product 8 91484 is usually purified on Silia in such a way that the specific activity is at least about 100 GIA units / ng protein, more usually 150 GIA units / ng.

The compounds obtained according to the invention are characterized in that they have a molecular weight of 17 to 19 kD (kilodaltons), in particular about 18 kD, as determined by molecular size separation chromatography. The compounds are further characterized in that they have a native molecular weight of about 28 kD as determined by polyacrylic-10-lamide gel electrophoresis under reducing or non-reducing conditions.

The amino acid sequences of the purified oncostatin M fragments were analyzed. The amino acid sequences for oncostatin are approximately as shown in Figure 1. In Figure 1, the first sequence illuminates the N-terminus of oncostatin M, while the other sequences illuminate internal fragments of the polypeptide. In a more detailed analysis, the amino acid sequences obtained by sequencing the cDNA clones, which were published in Finland on 15 April 1993, differ somewhat from the sequences in Figure 1, which is not at all toothy. The sequences obtained by different methods of silia often differ somewhat. . The amino acid sequences obtained by sequencing the cDNA clones are shown below, using three-letter abbreviations for the amino acids. As in Figure 1, the first sequence illuminates the N-terminus of oncostatin M, while the other sequences illuminate internal fragments of the polypeptide.

30 1 5 10

Ala-Ala-Ile-Gly-Ser-Cys-Ser-Lys-Glu-Tyr-Arg-Val-Leu-Leu-15 20 25

Gly-Gln-Leu-Gln-Lys-Gln-Thr-Asp-Leu-Met-Gln-Asp-Thr-Ser-30 35 35 Arg-Leu-Leu-Asp-Pro-Tyr-Ile, 9 91484 15 10

Gln-Arg-Leu-Pro-Lys-Ala-Gln-Asp-Leu-Glu-Arg-Ser-Gly-Leu- 5 15 20

Asn-Ile-Glu-Asp-Leu-Glu-Lys and 15 10

Leu-Arg-Glu-His-Cys-Arg-Glu-Arg-Pro-Gly-Ala-Phe-Pro-Ser- 10 15 20

Glu Glu-Thr-Leu-Arg-Gly where Ala is alanine, Cys is cysteine, Asp is Aspara-15 ginic acid, Glu is glutamic acid, Gly is glycine, His 10 is histidine, Ile is isoleucine, Lys is lysine, Leu is leucine, Met is methionine, Asn is asparagine. Pro is proline, Gln is glutamine, Arg is arginine, Ser is serine, Thr is threonine, Val is valine and Tyr is tyrosine.

Active preparations containing isolated oncostatin M would contain a mixture of high mannose and complex N-linked oligosaccharide. However, glycosylated non-oncostatin M preparations were also associated with cell growth.

As previously mentioned, oncostatin M is further characterized by activity against certain cell carriers. The polypeptide of the invention lacks cytotoxic activity against WI26 and Wl38 human fibro-30 blasts, murine L929 cells sensitive to tumor necrosis factors, and a human tumor cell line sensitive to interferon-β. MyOs has been shown not to inhibit the proliferation of normal human T lymphocytes or the formation of granulocyte / myelocyte clots from bone marrow cells.

35 concentrations up to 100 GIA units / ml. Oncostatin M

10,91484 further stimulates the proliferation of normal human fibroblasts, such as WI38 and W126 cells, and inhibits the proliferation of tumor cells, such as A375, HBT10, A549, and SK-MEL28 cells, and may promote the growth of colony-forming cells. in the bone marrow. Oncostatin M did not attenuate human proliferative or cytotoxic T cell responses in leukocyte mixed culture (MLC) reactions at concentrations of 500 GIA units / ml.

The amino-10 acid sequence of the polypeptide of the invention can be determined entirely using commercially available sequencers. The polypeptide can then be synthesized by known methods, as well as commercially available automated synthesizers.

Alternatively, the polypeptide of the invention could be prepared by recombinant DNA methods. The partial amino acid sequence can be probed into probes, which can then be used to screen a human genomic library. The library can be a cDNA or chromosome library. Once one or more clones mating with probe 20 have been identified, the binding fragments of the gene of interest can be identified in a variety of ways and kasi-Telia in a number of ways. The size of the fragment can be reduced by endonuclease digestion and the resulting fragments cloned and examined for the presence of the desired gene. Cells that produce the desired peptide or produce it in elevated amounts can be used to produce mRNA. A single-stranded cDNA can be prepared from the mRNA. This cDNA can then be paired with total mRNA from a cell that produces a waxy polypeptide, if at all. The unpaired cDNA can then be isolated and used to prepare a double-stranded cDNA that can be probed.

Alternatively, the DNA fragments may be inserted into 35 gtllre so that the coding fragments may follow and be in frame with the β-1191484 galactosidase gene. Antibodies to the oncostatin M polypeptide can be prepared and used to study the cross-reactivity of the resulting fused proteins. In this way, the polypeptide obtained according to the invention can be encoded by a fragment encoding a fragment thereof and the desired gene can be identified and used.

Once the entire gene1 has been identified, either as cDNAs or chromosomal DNAs, it can be processed in different ways to effect expression. Once the gene1 has been introduced into the 11-host host that has recognized wild-type oncostatin M replication and readable regions, the entire gene1 with its wild-type 5 'and 3' envelope regions can be inserted into a suitable expression vector. There are a variety of expression vectors that utilize the replication systems of mammalian viruses such as Simianvirus 40, adenovirus, bovine papillomavirus, vaccinia virus, hybrid baculovirus, and the like. These replication systems have been developed to provide markers that allow selection of transfectants as well as the provision of overlapping restriction sites into which gene1 can be inserted.

When the gene is present, it may be expressed in a host that does not recognize naturally occurring wild-type copies and reads the accompanying regions, further processing is required. Various overlapping 3 'transcription regions are known to be located at the stop codons. The unencoded 5 * end region can be removed in front of the structural gene by endonuclease restriction, Bal31 resection, 30, etc. When the 5 'end region of the structural gene has a covering restriction site, the structural gene can alternatively be cleaved and an adapter used to attach the structural gene to the promoter region. lost nucleotides of a structural gene. Various strategies can be used to provide an expression vector with a 5 'to 3' copy and read initiation region in the copy direction, which may include myOs insertion sequences that allow for the induction of saately, a structural gene that is read and transcribed. under the control of the initiation area, and 5 the end area of copying and reading.

Typical replication initiation regions or promoters include a β-gal promoter, a TAC promoter, a lambda phage left and right promoter, etc. for bacteria; promoters of glycolytic enzymes such as ADH-I and II promoters, GPK promoter, PGI promoter, TRP promoter, etc. for yeast; SV40 early and late promoter, adenovirus late parent promoter, etc. for mammalian cells. As already mentioned, the expression unit may be included in the replication system to maintain the episome in a suitable cellular host, or it may be without the replication system, allowing it to integrate into the host genome. The DNA can be introduced into the host by known methods by transformation using calcium phosphate-precipitated DNA, transfection, contacting the cells with the virus, microinjection of the DNA into the cells, and the like.

Once the structural gene has been introduced into a suitable host, the host can be cultured, and expression of the structural gene can be induced. In some cases, it may be desirable to place the signal sequence (secretion precursor) before and in the same reading frame as the sequence that causes the secretion of the structural gene and the secretion of the secretion precursor to effect secretion of the mature polypeptide into the supernatant. If no secretion is caused, the sphinct cells can be harvested, lysed in a conventional manner, and purified by conventional methods such as chromatography, electrophoresis, solvent extraction, and the like.

The compounds of the invention can be used in a wide variety of ways, both in vivo and in vitro. The compounds can be used to prepare their antibodies, which antibodies can be used in vitro or in vivo. Antibodies can be prepared by conventional methods, for example, using the polypeptide of the invention as an immunogen and injecting it into a mammalian strain, e.g., mouse, cow, goat, sheep, rabbit, etc., especially with an excipient, e.g., complete Freund's excipient, aluminum hydroxide gel, and the like. The host can then be bled and used to isolate poly-10 clonal antibodies, or, in the case of a mouse, to fuse arterial blood lymphocytes or spleen lymphocytes (B cells) with a suitable myeloma cell to immobilize chromosomes immortalized with the multicomponent compounds of the invention. -ai-15 to produce them.

Either polyclonal or monoclonal antibodies can be prepared, which can then be used for diagnosis or to detect the polypeptide in question from a sample, such as cells, or a physiological fluid, such as blood. The antibodies can be used in myds affinity chromatography to purify the polypeptide in question and isolate it from a natural or synthetic source. Antibodies can be used myds to obtain the amount of the polypeptide in question associated with the cells in culture or in vivo, through which cell growth can be induced.

The compound of the invention can be used as a ligand to detect the presence of receptors for this compound. In this way, the receptors can be distinguished in terms of the receptor depletion and density of this compound by monitoring the effect of different compounds on the receptor depletion.

The compound of the invention can be used in in vitro cultures to inhibit the growth of tail-sensitive cells or cell lines that react differently from insensitive cells. Heterogeneous cell mixtures or cell lines can thus be stained for undesired cells when the depolarizing cells are sensitive to the polypeptide. The polypeptide prepared according to the invention can be administered in vivo in the case of tumor conditions, for example, by injection, intratumorally, peritoneally, subcutaneously and the like. The compound can be used in vitro to remove malignant cells from a bone marrow used for autologous bone marrow transplants or to prevent the proliferation of malignant cells in another tissue, such as blood, or to remove them prior to re-infusion.

The polypeptide obtained according to the invention can also be used for the treatment of epithelium of injuries such as skin injuries, corneal injuries and various epithelial and basal tissue injuries of the earth, such as chronic ulcers, burns, surgical wounds, gastric lesions, accidental injuries and hollow, epithelium. for the treatment of small and large intestinal, oral, genital and urinary tract injuries. Patients are administered topically a Hoi composition comprising oncostatin M in a physiologically acceptable carrier.

The composition containing oncostatin M can be used to treat a wide variety of injuries, including virtually all skin injuries, including corneal injuries and epithelial-covered hollow organ injuries. Injuries suitable for treatment include injuries resulting from accidents, such as burns, abrasions, surgical wounds, etc., as well as injuries resulting from surgical procedures, such as surgeries and skin grafts. Other conditions suitable for treatment with oncostatin-containing compositions include chronic conditions, such as chronic ulcers, diabetic ulcers, and other incurable (trophic) conditions.

35 15 91484

Oncostatin M can be incorporated into physiologically acceptable carriers for application to the affected area. The nature of the carriers may vary widely and will depend on the particular site of administration. For subcutaneous administration, a cream or ointment base is usually preferred: suitable bases include lanolin,

Silvadene (Marion) (especially for burns), Aguaphoe (Duke Laboratories, South Norwalk, Connecticut), etc. Oncostatin M-containing compositions may, if desired, be incorporated into dressings and other wadding products to continuously treat the wound with peptide. MyOs aerosol applications can be used.

The concentration of polypeptide in the therapeutic composition is not critical. The polypeptide should be an inducer of proliferation of epithelial cells. The compositions are applied topically to the affected area, typically as eye drops to the eye or as creams, ointments or milks on the skin. In the case of the eyes, it is desirable to repeat the cases frequently, usually in 4-hour increments or more frequently. In the case of the skin, it is desirable to maintain the treatment composition in the affected area continuously during healing by applying the treatment composition 2-4 times a day or more often.

The amount of polypeptide prepared according to the invention to be used varies depending on the mode of administration, the use of other active compounds and the like, and is generally about 1 to 100 pg. The polypeptide may be used in conjunction with a physiologically acceptable carrier such as physiological saline, phosphate buffered saline, and the like. The use of a compound is determined experimentally based on the in vitro response of the cells and the response of the test antibodies to the polypeptides of the invention or formulations containing them. The compounds may be used alone or in combination with other growth factors, inhibitors or immunomodulators such as TNF: S3n, IL-2, interferon, monoclonal antibodies, etc. The matrices of other compounds are generally in the range of 1 to 100 pg. Conjugates of the compounds can be prepared with groups targeted at a particular site 5, for example, antibodies, whereby the antibodies may be specific for certain malignant cells or organs. The following examples are provided to illustrate the invention and are not intended to limit it.

10 Experimental part

Materials 1a Methods Oncostatin M isolated from U937 cells: production of a tumor cell growth inhibitor from a histiocytic lymphoma cell line 15 U937 cells, a histiocytic lymphoma cell line [Sundstrom and Nilsson, Int. J. Cancer 17 (1976) 565-577] were cultured in 850 cm 2 flasks (Corning C2540) at a concentration of 4 x 10 5 cells / ml in a total of 300 ml of RPMI 1640 medium supplemented with 10% fetal bovine serum. 20 mia (FCS), penicillin / streptomycinyl (PS), L-glutamine and 12-O-tetradecanoyl phorbol 13-acetate (TPA, 10 ng / ml). After four days, the supernatants containing FCS: S3 and TPA were removed, the flasks were washed five times with serum-free RPMI 1640: 113, and the detached cells (1 x 10 s cells / ml) were washed three times with serum-free medium and added back to the flasks. came 125 ml of serum-free RPMI 1640 medium per pyridrity flask. After 24 hours, the supernatants were collected, centrifuged to remove cells, filtered through a 0.45 μπκη Nalgene filter, and concentrated using a 3 μm hollow fiber system (Amicon cartridge HIP10-20) to a final volume of 150 ml (initial volume 1500 ml). Oncostatin M was also isolated from supernatants of U937 cells treated with serum-35 TPA in 150 cm2 tissue culture flasks. The supernatant was concentrated on an Amicon Diaflo membrane PM-10 (1SpSy limit 10 kD) and dialyzed. The concentrate of the dialysis fraction was diluted with acetic acid to give an acetic acid content of 0.1 N in a volume of 500 ml and concentrated to 50 ml using an Amicon PM 10 filter. This concentrate (50 mL) 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 1 N acetic acid, and the resulting precipitate was removed by centrifugation. The resulting concentrate was frozen and lyophilized. The lyophilized material was used in the purification steps.

Geellpermeaatiokromatoarafia

A Bio-sil TSK-250 column (600 x 21.5 mm) (BioRad) 15 was connected to a high pressure liquid chromatographic system.

The crude fraction (10 mg / ml) was dissolved in an aqueous solution containing 40% acetonitrile and 0.1% trifluoroacetic acid (0.1% TFA). A 2 ml portion of the mixture was injected and eluted isocratically using 20 solutions of 40% acetonitrile and 0.1% TFA in water as the mobile phase. The flow rate was 2.5 ml / min and the plotter paper flow rate was 0.25 cm / min. 5 ml fractions were collected. Chromatography was performed in a room lamp room. A sample from each fraction was evaporated and tested for growth inhibitory activity (GIA) of A375 cells in triplicate.

The active fractions (21 and 22) from the six runs were pooled. The combined material contained a total of approximately 4.8 x 10 5 GIA units. The apparent molecular weight of the factor was found to be 18 kD as determined by size exclusion chromatography (Bio-sil TSK-250 column).

RP-HPLC (lid phase high pressure chromatography) of TSK-250 fractions

The combined TSK-250 fractions 21 and 22 described above were diluted to twice the volume with 0.1% 18,91484 TFA. The TSma mixture was injected isocratically onto a μ-Bonda-pak-Cl8 column (7.8 x 300 nun) (designated C181) at room temperature. The flow rate was adjusted to 2.0 ml / min and the funnel speed was 0.25 cm / min.

A linear gradient from primary solvent (0.1% TFA) to secondary solvent (acetonitrile, 0.1% TFA) was used. Gradient conditions were 0 -> 30% at 10 min, then 30 -> 45% at 150 min; 45 -> 55% at 20 min, and 55 -> 100% at 10 min. All solvents were HPLC grade. 4 ml fractions were collected, and a sample taken from each fraction was tested for growth inhibitory activity. Fractions 72-75 were found to contain the majority of the activity. The active fractions eluted at an acetonitrile content of 41-52%. Fractions 72-15 were pooled. To the combined fractions was added 16 ml of 0.1% TFA. The mixture was injected onto a p-Bondapak C18 column (3.9 x 300 nun) (referred to as C182) in a room lamp room. The flow rate was set to 1 ml / min and the plotter speed was 0.25 cm / min. Gradient-20 conditions were 0 -> 35% in 10 min; 35 -> 45% in 100 min; and 45 -> 100% in 10 min. Fractions were collected, sampled, and GIA examined. Most of the activity eluted from the column at an acetonitrile content of 40.7-41.3% (retention time 83-25 86 min).

The active fractions were pooled, diluted to twice the volume with 0.1% TFA and injected isocratically onto a μ-Bondapak-Cie column (3.9 x 300 mm) (designated C183) at room temperature. The flow rate was 1 ml / min, and the plotter paper rate was 0.25 cm / min. A linear gradient in primary solvent (0.1% TFA) to secondary solvent (n-propanol, 0.1% TFA) was used. Gradient conditions were 0 -> 23% at 20 min and 23 -> 35% at 120 min. 35 Fractions were collected and GIA was examined for 19,91484 samples taken from each fraction. Most of the activity was eliminated at a propanol content of 25-26.5% (retentloalkala 59 min). This apparently homogeneous fraction contained about 300 ng of protein and about 40,000 GIA units.

5 Maarltvs. In this study, cell growth was induced by the incorporation of 3 H-tvmidine into DNA (GIA).

The experiments were performed in 96-well, flat-bottomed well plates (Costar 3596). Human melanoma cells (A375) were used as a sensitive indicator cell line. Cells (3 x 103) in 1.1 ml of Dulbecco's modified Eagles' medium (DMEM) supplemented with 10% FCS and PS were placed in each well. After three hours, 0.1 ml of test sample was added to each well. The plates were incubated at 37 ° C for 3 days. 0.025 ml (0.5 pCi) of 3 H-thymidine solution (specific activity 27 pCi / ug) was then added to each of the 15 wells and incubated for a further 6 hours. The cells were then transferred to glass filter strips using a multi-candy pipette (PHD Cell Harvester, Cambridge Techology, Inc). The filters were transferred to scintillation vials containing 2 ml of scintillation fluid (ScientiVerse II, Fisher Scientific Co.) and then scintillated to quantify 3H-thymidine incorporation.

A 1-ml bottom layer containing 0.5% agar (Agar Noble; Difco Laboratories, Detroit, Michigan) in DMEM containing 10% fetal bovine serum (FCS). was added to 24-well Costar tissue culture plates.

0.5 ml of the same medium-FCS mixture containing 0.3% agar, 1 to 2.5 x 103 A375 cells and test factor at different concentrations was applied to the agar base layer.

The plates were incubated at 37 ° C in a humidified atmosphere containing 5% CO 2 in air, and after 35 days, 0.5 ml of 20,91484 0.3% agar in the same medium was added. was the same. Pe colonies were counted unattached and unshielded, and colonies containing more than 6 cells on days 7 and 14 were taken into account in the calculation.

5 Results Sequences of Oncostatin M Isolated from U937 Cells The N-terminal sequence and internal fragments of oncostatin M were determined by microsequence analysis of the reduced and S-pyridine ethylated polypeptide and peptides obtained by digestion and peptide digestion. Enzymatically with endoproteinase Lys-C and Staphylococcus aureus V8 proteinase. The peptide fragment was purified by RP-HPLC using volatile solvents. Peptides were subjected to automatic repeat Edman digestion in a Model 470A protein sequencer (Applied Biosystems, Inc.). Phenyl lithiohydantoin amino acids were analyzed by RP-HPLC (Applied Biosystems, Inc.) using a PTH-C18 column (2.1 x 220 mm, ABI) and eluted using a sodium acetate-20 drop buffer-tetrahydrofuran-acetonitrile gradient.

The resulting amino acid sequences are approximately as shown in Figure 1.

When comparing these sequences to an up-to-date protein database (PIR Release 9.0, May 1986), no significant sequence homology was observed with any of the known sequences. There is no homology with tumor necrosis factor, lymphotoxin, colony stimulating factor, interleukin 1 or 2, or β-transforming growth factor.

30 Inhibition of tumor cell proliferation and stimulation of normal human fibroblast proliferation

The following results were obtained using the previously described soft-backed inhibition test: t 21 91484

Table 1 Inhibition of A375 melanoma cell colony formation by purified oncostatin M isolated from U937 cells * 5 GIA units / Colony inhibition of colony formation (% 1_ 250 4 96 83 6 94 27 11 89 10 45 32 69 0 * Cells were inoculated as described above into soft agar with the manufacturer at a final air volume of 2 ml, the factor used was the fraction from the C18 propanol column with the highest tumor growth inhibitory activity (GIA), and the number of colonies was counted after 11 days. a cluster containing at least 6 cells One GIA unit6 was defined as the amount that causes the incorporation of 3 H-thymidine into A375 cells in the microwell by 50% inhibition in the experiment described above.

In the following experiment, the effect of various treatments, either chemical or physical, on the polypeptide in question was determined. The results are given in the following table.

Table 2 Effect of different treatments of TPA-induced U937 cell supernatants on tumor growth inhibitory activity *

Final dilution ratio of supernatant 1: 4 1: 8 1:16

Chassis, reference sample - - - 39,780 35 UntreatedOn supern. 7,206 ** 13,896 16,000 1 N acetic acid 6,670 17,073 18,783; · 1N NH 4 OH 6,956 15,016 13,923 22,91484 * U937 cells were treated with TPA (10 ng / ml) for 3 days, then the cells were washed with medium, incubated for 24 hours in serum-free medium and supernatant recovered. The supernatants were treated with 1 N acetic acid and 1 N ammonium hydroxide (NH 4 OH). They were dialyzed against medium and examined for their ability to inhibit the incorporation of 3 H-thymidine into A375 cells. A375 cells were labeled with 3 H-thymidine (3 H-TdR) for the last 6 hours of a 3 day incubation time.

** The results show the incorporation of 3H-TdR in pulses per 10 minutes.

The above results show that oncostatin M in the dialyzed supernatant is hardly inactivated by 1 N acetic acid or 1 N ammonium hydroxide. The compounds 15 prepared according to the invention are thus relatively insensitive to both a moderately strong acid and a moderately strong base. The compound undergoes a 1 hour lamp treatment at 56 ° C, but not a 30 minute treatment at 90 ° C.

The lamp stability of the myOs compound was examined and it was assumed that the activity of the compound was maintained when kept at 56 ° C for 1 hour, but disappeared approximately completely when kept at 90 ° C for 30 minutes.

The following experiment examined the ability of the polypeptides of the invention to inhibit the replication of various tumor cell lines. The results are shown in the following table.

Table 3

Inhibition of tumor cell replication GIA unit causing 30% inhibition by purified oncostatin M obtained from U937 cells Omar Tumor cells Uptake of 3H-TdR into cells A549 (lung heart) 21 HTB10 (neuroblastoma) 81 35 A375 (melano) 3 23 91484

Tumor cells were seeded in microwells 3 hours before RP-HPLC after addition of purified oncostatin M as described above diluted in various ratios. For the last 6 hours of incubation for 3 days, 5 cells in 0.2 ml of medium were labeled with 3 H-thymidine (3 H-TdR) (0.5 pCi / well). One unit of tumor growth inhibitory activity (GIA) was defined in the description of Table 1 in a field that reduces the incorporation of 3 H-TdR into A375 melanone cells by 50%. One one-10 kOn was determined to be about 10 pg of purified protein; thus, the concentration (ng / ml) that causes 30% inhibition of 3 H-TdR incorporation into A549, HTB10, and A375 cells was about 1.4, 4.0, and 0.015 ng / ml, respectively.

Factor U937 did not reduce the incorporation of 3H-TdR into human normal WI16 fibroblasts in any of the experiments.

The above results show that the ability of oncostatin M to inhibit replication is selective; the effect varies greatly depending on the nature of the cell. The compound is effective against melanoma cells such as A375 melanoma cells, lung lymphocytes such as A549 cells, and neuroblastoma cells such as HTB10 cells.

Tumor cells were seeded at a density of 3 x 10 3 cells / well and normal fibroblasts at a density of 1.5 x 10 3 cells / well in 96-well plates for 3 hours. Purified oncostatin M obtained from the fraction of the C183 column with the highest antiproliferative activity against A375 cells was added at various concentrations, and the incorporation of 3 H-thymidine at each concentration was measured from three parallel stalks. The Tu-30 results are shown in Table 4.

24 91484

Table 4

Inhibition of tumor cell proliferation and progression of normal fibroblast proliferation by oncostatin M 5 GIA units /% inhibition stimulation (%) swennvs A375 WI38

Experiment 1 16 83 25 10 4 62 30 1 46 46 A375 HTB10 WI26

Experiment 2 27 NT 28 46 9 87 22 36 15 3 76 11 52 A375 A549

Experiment 3 75 89 30 25 85 22 8 71 16 20 A375 SK-MEL28

Experiment 4 20 87 44 5 75 25 1 59 11 25

The results show inhibition (%) and stimulation (%) of 3 H-thymidine incorporation into tumor cells (A375, HTB10, A549 and SK-MEL28) and normal fibroblasts (W126 and W138), respectively. One GIA unit is defined in the description of Table 1 as the oncostatin M, which causes 50% inhibition of 3 H-thymidine incorporation into A375 cells.

In addition to the fact that the effect on the incorporation of 3 H-thymidine into tumor cells is different from that on normal human fibroblasts, a different effect of myOs on morphology and cell number is observed after treatment of these two cell types for 3 days with oncostatin M, as shown in Figure 2.

The oncostatin M used was obtained from the fraction of the HPLC-C183 column 40 which had the highest anti-proliferative activity of A375 cells. Figure 2 is a series of photomicrographs of A375 melanoma cells treated with 25,91484 untreated Gm (A) treated with 5 GIA-one kGlia (growth inhibitory activity units) of oncostatin M (B) and treated with 100 unitsGG (B). C), as well as micrographs of W138 fibroblasts that were 5 untreated (D), treated with 5 GIA units (E) or 100 units (F). The cells were covered with a 0.5% methanol solution of crystal violet. Magnification is 63x.

NaDodSO 4 / PAGE of oncostatin

The purified oncostatin 1 M subjected to 10 NaDodSO 4 -PAGE under reducing conditions was found to have a net molecular weight of about 28 kD, as shown in Figure 3. The following proteins were used as standards (lane A): ovalbumin, molecular weight 43 kD; chymotrypsino gene α, molecular weight 25.7 kD; lactoglobulin β, Mole-15 wt weight 18.4 kD; lysozyme, molecular weight 14.2 kD, bovine trypsin inhibitor, molecular weight 6.2 kD; insulin A and B chains, molecular weight 2.3 kD and 3.4 kD, respectively. Oncostatin M was placed in lane B.

Oncostatin M, which was PAGEed under unreduced conditions under 20 conditions, had a apparent molecular weight of 28 kD myGs, and a protein electroeluted from the band was found to inhibit A375 cell proliferation.

The antibody of the synthetic oncostatin M peptide reacts with 125 I-labeled oncostatin Mrn in radioimmune-25 precipitations a) Peptide synthesis: The peptide corresponds to groups 6-19 of the oncostatin M protein and was synthesized by the solid phase method as described by the automated Beckman apparatus. Gentry et al., J. Biol.

30 Chem. 258 (1983) 11,219]. The peptide was cleaved from the resin for use in the "low-high" HF procedure [Tam et al., J. Amer. Chem. Soc. 105 (1983) 6,442-6,445].

b) Antibody production: The peptide was coupled to bovine gamma globulin as described in the literature 35 [Gentry and Lawton, Virology 152 (1985) 421-431]. White rabbits were administered the peptide as a basal dose and as a booster dose at 4 sites subcutaneously as described in the literature [Gentry and Lawton, Virology 152 (1986) 421-431]. Antisera were taken 5 2 weeks after the fifth booster dose.

c) Logging of oncostatin M: A sample of partially purified oncostatin M was radiolabeled using iodine-125 according to published procedures [Linsley et al., PNAS 82 (1985) 356-360]. A portion of the labeled preparation containing 10,000 pulses / min was mixed with rabbit antiserum raised against the 17 N-terminal amino acids of oncostatin M (final dilution ratio 1:20), the N-terminal peptide (oncostatin M: n 17 N-terminal amino acids, 2 pg) in the absence or absence, and immunoprecipitation analysis was performed as described in the literature [Linsley et al. Biochemistry 25 (1986) 2,978-2,986].

More specifically, one tube containing 5 μΐ of sample was preincubated with 2 pg of N-terminal 20 peptide in 10 ml of TNEN solution (20 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0 mM .05% Nonidet P-40) containing 0.1% BSA for 30 min at 4 ° C, to which 125 I-oncostatin M in 85 μl of TNEN solution containing 0.1% BSA was added. BSA and 40 mmol / l dithiothreitol (DTT). Seven 25 tubes containing 5 μΐ of antiserum were incubated with 125 I-oncostatin M in 85 μl of TNEN solution containing 0.1% BSA and 40 mmol / l DDT for 30 min 4 At 50 ° C, to which was added 50 μyl of Staphylococcus aureus (Pansorbin, 30 Calbiochem) fixed with 10% formalin.

After an additional 30 min incubation at 4 ° C, the tubes were centrifuged in a microcentrifuge, and the pellets were washed four times with 1 ml of TNEN solution, which was then subjected to PAGE analysis. A diffuse band corresponding to a molecular weight of 32 kD was detected in 27,91484 fractions of SDS / PAGE analysis of immunoprecipitates. Precipitation of this band was prevented by an excess of unlabeled peptide corresponding to the 17 N-terminal amino acids of oncostatin M, indicating that the precipitation was specific for this peptide.

The carbohydrate composition of oncostatin M was tested by testing for glycosidase sensitivity. Immunoprecipitates prepared as described in c) above were treated with buffer, endoglycosidase H, or neutramidase as described by Linsley et al. (1986). Treatment with endoglycosidase H, an enzyme specific for N-linked, mannose-rich oligosaccharides, resulted in the appearance of a smaller molecule (molecular weight 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 neuramidase resulted in the appearance of a single band (molecular weight 27 kD), indicating that the size heterogeneity of untreated 125 I-labeled oncostatin M was due to heterogeneity in sialylation of the Gly-20 coprotein core. The results showed that the active oncostatin M preparations contained a mixture of run-asmanmanned and complex N-linked oligosaccharide side chains.

Oncostatin M isolated from normal human blood vessels (PBL); production of a tumor cell growth inhibitor from PBL

PBL-containing leukofractions from a blood bank were diluted 1: 1 with phosphate-buffered saline, pH 7.4, (PBS), 10 ml of a solution containing 9% Ficoll and 20% v / v 50% sodium disitroate (final specific gravity). 1.080), 35 ml of diluted blood was applied. The gradients were centrifuged at 850 x g for 20 min at room temperature. Cells were harvested from the gradient interface and washed with 35 PBS. The red blood cells were lysed by treatment with 28,91484 for 3-4 min in 10-20 ml of a solution containing 0.8% ammonium chloride and 0.1% Na4-EDTA.

Cells were harvested from the erythrocyte lysis solution by centrifugation at 600 x g for 10 min and resuspended in 10 ml of RPMI 1640 medium (GIBCO) containing 5% bovine sicide serum. Thrombin was added to a final concentration of 0.5 units / ml. The cell suspension was stirred for 5 min at 37 ° C and the platelet aggregate was allowed to settle for 5 min. The suspended cells were transferred to new tubes, harvested by centrifugation, resuspended in 1 ml of bovine serum 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 min to allow monocytes and B lymphocytes to attach. The column was then rinsed with three volumes of RPMI 1640 medium (37 ° C) containing 5% bovine sicide serum and the eluted non-adherent cells (PBLs) were collected.

20 PBLs (2 x 10 6 cells / ml) were cultured at 37 ° C in an atmosphere containing 5% CO 2 and 95% air for 96 hours in RPMI 1640 medium (10.4 g / l) containing FeSO 4. .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), 25 bovine animals 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). 1). Supernatants were collected, centrifuged to remove cells, concentrated by ult-30 fat filtration (Amicon Diaflo membrane YM-10, permeation limit 10 kD) and dialyzed against 0.1 M acetic acid (Spectrapo-re 3 dialysis tubing). The clarified retentate was lyophilized.

29 91484

Geellpermeaatiokromatoarafia

The crude fraction was mixed with 20 ml of 1 M acetic acid (50 mg / ml) and applied to a BioGel P-100 column (2.6 x 88 cm) equilibrated with 1 M acetic acid at a flow rate of 0.5 ml / ml. min. 20 ml fractions were collected. The nSyte from each fraction was evaporated and assayed for A375 cell growth inhibitory activity (GIA) in triplicate. The active fractions were pooled, lyophilized and reprocessed Kro-10 on a Bio-sil TSK-250 column (600 x 21.5 mm) as described above.

RP-HPLC of TSK-250 fractions

The final purification of the combined TSK-250 fractions was performed by RP-HPLC approximately as described above. Tumor cell inhibitor from PBL eluted from the Bonda-pak C18 column at an acetonitrile concentration of 40-41% and an n-propanol concentration of 26.5%.

Investigation of cell growth uptake by incorporation of 125 I-1oddeoxyuridine into DNA (GIA) 20 Ndma experiments were performed in flat-bottomed 96-well tissue culture plates (Costar 3596). Human melanoma cells (A375, 4 χ 103) in 50 μl of test sample were added to each well and incubated for 3 days at 37 ° C. Cells were labeled with 125 I-IdU (0.05 pCi / well) for 24 hours, and incubated for another 24 hours. The cells were washed three times, swirled with a polycandel pipette, and the radioactivity was determined in a gamma counter.

Score:

One PBL-derived tumor cell inhibitor selection was subjected to automatic repeated Edman lysis. The amino - terminal amino acid sequence is as follows: 1 5 10 15

A-A-I-G-X-X-X-K-E-Y-X-Y-L-X-X-Q-L-Q-K

wherein X represents an unidentified amino acid.

35 91484 30

Comparison of this sequence with the sequence of U937 factor clearly shows identity with the N-terminus of PBL-based factor.

5 PBL factor A-A-I-G-X-X-X-K-E-Y-X-V-L-X-X-Q-L-Q-K

U937 factor A-A-I-G-S-C-S-K-E-Y-R-V-L-L-G-Q-L-Q-K

The following study examined the ability of PBL-derived oncostatin M to affect the replication of various cells. It was found that hliren L929 cells were susceptible to PBL-proliferative oncostatin M when used at a maximum concentration of 1,000 GIA units / ml. The growth of human WI26 fibroblasts is stimulated by Treatment at 1,000 GIA units / ml. Up to 500 GIA units / ml did not affect the proliferation of normal human T lymphocytes 72 hours after mitogenesis.

From the above results, it is apparent that a novel polypeptide and polypeptide fragments have been provided that can be used to accompany cell growth. The compound has activity that varies according to the nature of the cell line in question, so that it can be used alone or in combination with other compounds to inhibit cell growth. Polypeptides prepared in accordance with the invention thus represent a novel polypeptide that can be used in cell mixtures both in vivo and in vitro to selectively reduce or promote the proliferation of a particular cell type. The factor can be used in particular for the processing of cells for autologous bone marrow transplants. The use of this factor inhibits the growth of tumor cells in the bone marrow and may stimulate the formation of colony cells. Oncostatin M can be used to stimulate the growth of myds epithelial cells and thus promote injury healing. In addition, the entire polypeptide or fragments thereof can be used as immunogens to induce antibody production. Induced antibodies can be used in the determination of oncostatin M present in body fluids to induce factor activity through binding. In addition, this antibody, together with purified oncostatin M or fragments thereof, act as components in diagnostic kits, together with antibodies for the detection and quantification of mulden reagents, in particular oncostatin M.

Although the invention has been described in some detail in an illustrative and exemplary manner in order to facilitate its understanding, it will be apparent that certain changes and modifications may be made therein without departing from the scope of the appended claims.

Claims (10)

A process for the preparation of oncostatin M that is substantially free of cellular debris and other leukocyte proteins and which is capable of preventing proliferation of tumor cells and stimulating proliferation of normal human fibroblasts and which does not inhibit human proliferative or cytotoxic T cell response granulocytic / myelocytic bone marrow colonies, and spring molecular weight Or 17 - 19 kD analyzed by gel exclusion chromatography and about 28 kD analyzed by SDS-PAGE and enduring treatment with 1 N acetic acid and 1 N ammonium hydroxide and a temperature of 56 ° C for one hour , it can be characterized that a) leukocytes, such as histiocytic lymphoma cells or normal human peripheral blood lymphocytes, are isolated from a mammalian cell culture or from a mammal; b) the leukocytes are activated with an inducing amne such as ingenol, phorbol or a mitogen; C) Oncostatin M is chromatographically separated from the activated leukocytes purely f from other cellular material. The method of claim 1, wherein the leukocytes are histiocytic lymphoma cells. 3. A method according to claim 2, characterized in that the histiocytic lymphoma cells are U937 cells. 4. A method according to claim 2 or 3, characterized in that the inducing agent is ingenol or premol. 5. A process according to claim 4, characterized in that the compound is 12-O-tetradecanoyl-compound-13-acetate. 6. A process according to claim 1, characterized in that the leukocytes are normal lymphocytes in human peripheral blood. 35 91484 7. A process according to claim 6, characterized in that the inducing agent is a moderate. 8. A process according to claim 7, characterized in that the mitogen is phytohemagglutinin P. Process according to any one of claims 1-8, characterized in that the purity of the on-cost statin M is such that the specific activity is at least about 10 GIA units / ng of protein. 10. A process according to any one of claims 1-8, characterized in that the purity of the on-cost statin M is such that the specific activity is at least about 100 GIA units / ng of protein.