FI98373B - Novel diagnostically utilizable polypeptide, oncostatin M - Google Patents

Abstract

The invention relates to a novel, diagnostically utilizable polypeptide which is essentially free of cell debris and other leukocyte proteins and which has the ability to prevent the in-vitro proliferation of tumour cells and stimulate the proliferation of normal human fibroblasts and which does not inhibit human proliferative or cytotoxic T cell responses or the formation of granulocytic/myelocytic bone marrow colony cells, whose molecular weight has been determined by gel exclusion chromatography to be approximately in the range 17-19 kD and determined to be approx. 28 kD by SDS-PAGE, which is relatively insensitive to moderately acid or basic conditions and to relatively elevated temperatures, whose degree of purity is such that the specific activity is at least approx. 10 GIA units/ng of protein and which contains amino acid sequences which correspond to the sequences in Figure 1 in such a way that the given sequence and the sequence which corresponds to the polypeptide differ from each other with regard to at most three amino acids, or a poly(amino acid) fragment which is formed by at least eight amino acids and which cross- reacts immunologically with such a polypeptide. The invention also relates to antibodies which have been formed against this polypeptide and to a diagnostic process for detecting this polypeptide and to a kit which can be used in this process. <IMAGE>

Description

i 98373

A novel diagnostically useful polypeptide, oncostatin M

Divided separated from patent application 865156 5

Leukocytes, both lymphocytes and monocytes, have been implicated in the inhibition of tumor growth in a few wild-type tumors. The increased incidence of malignancies in immunocompromised people supports 10 the claim that white blood cells are involved in the regulation of tumor growth. Protein factors already produced and characterized by these leukocytes that inhibit tumor growth or regulate immune function include interferons α and γ, tumor necrosis factor, lyrafotoxin, interleukin 2, and other lymphokines. Because each of these isolated factors has a different whitening spectrum and may interact differently with other factors, there is still a strong interest in isolating and characterizing the lime factors that leukocytes produce by regulating cell growth and immune function.

Many difficulties can be encountered in finding, isolating, purifying, or characterizing naturally occurring factors. Methods must be developed to separate and purify the factor of interest from the muleta factors present in the impure starting material without denaturing the activity of the desired factor; biological assays must be developed 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 present and which may affect, either negatively or positively, the activity of the desired factor; and the purified factor must be concentrated to a sufficient extent to allow identification and characterization of the factor. Therefore, as the number of erls-5 factors increases, each new factor becomes more difficult to identify, as its role and function may be obscured by the numerous other factors present ....... .. ...... ........

Bealln et al. 'n [Cancer Blochem. Biophys. 3 (1979) 109-96], peptides are present in human urine that inhibit growth and DNA synthesis in more strongly altered than normal cells. Holley et al. [Proc.

Natl. Acad. Sci. 77 (1980) 5,989-5992] describe the purification of epithelial cell growth inhibitors. Letanskyn 15 [Biosci. Rep. 2 (1982) 39-45], peptides purified from bovine placenta inhibit tumor growth and incorporation of thymidine into DNA to a greater extent in tumors than in a normal cell. Chen [Trends Blochem. Sci. 7 (1982) 364-365] describes the isolation of a cancer-suppressing peptide from 20 aqueous humor. 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] ** 25 describe the production of one or more factors produced by human macrophages that inhibit the growth of certain plant 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 from lym-photoxyl, interferon and interleukin 1 and 2. Most * · of these factors are not known. perfectly characterized,. ... ....... ..... ·· '"· A · ·. And their primary structure is not known.

·· ·. ......

·· .....

»• ....

0 0 0 00 0 3 '.

98373

Aggarwal et al. [J. Biol. Chem. 259 (1984) 686-691] have purified and characterized human lymphotoxin (LT) produced by the lymphoblastold1 cell line and subsequently sequenced it [Aggarwal et al., J. Biol. Chem. 260.: .- .. 5 (1985) 2 334]. Interferon gamma (γ-IF), which is produced by lymphoid cells and has an immunosuppressive and antitumor effect, has been produced through cloning and expression [Gray et al., Nature 295 (1982) 503-508]. Tumor necrosis factor (TNF), which inhibits the growth of some tumors and is produced by macrophages and certain leukemic cell lines, has been characterized, and TNF cDNA has been cloned and expressed in E. coli [Pennlca et al., Nature 312 (1984) 724].

The invention relates to a novel peptide factor available from leukocytes, the oncostatin M polypeptide, which is characterized by being substantially free of cellular debris and other leukocyte proteins, has the ability to inhibit tumor cell proliferation and stimulate the proliferation of normal human fibroblasts, and the proliferation of normal human fibroblasts. T cell responses and no formation of granulocytic / myelocytic bone marrow colony cells, has a molecular weight of 17 to 19 kD as determined by gel exclusion chromatography and approximately 28 kD as determined by SDS-PAGE, withstands treatment with 1 N acetic acid-25 pol and 1 N ammonium hydride n for one hour and can be prepared by a method comprising a) isolating leukocytes, such as histiocytic lymphoma cells or normal human peripheral blood lymphocytes, from a mammalian cell culture or a mammal; B) activating the leukocytes with an inducing agent such as ingenol, phorbol or mitogen; and c) isolating oncostatin M from chromatographically activated leukocytes in pure form from other cellular material. The invention also relates to antibodies raised against oncostatin M polypeptide or fragments thereof, 98373 M polypeptide and an antibody as defined above.

Figure 1 shows the amino acid sequences of fragments 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) are W138 fibroblasts treated with 0, 5, and 100 GIA units, respectively; and Figure 3 is a photograph of an SDS-PAGE analysis of oncostatin M.

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

Polypeptide fragments are novel 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. By immunologically cross-reactive is meant that the antibody induced by the novel polypeptide of this invention cross-reacts with the original whole oncostatin M at least when the oncostatin M is in a denatured state. These polypeptides are therefore useful for inducing antibodies to oncostatin M so that the antibodies can be used to determine the concentration of oncostatin M in body fluid, to bind oncostatin Min and thus to regulate its activity, and to purify oncostatin M, for example, using an affinity column. In some polypeptides, the cell growth regulating effect of the original whole oncostatin M may also be retained, although this activity may be regulated, usually decreased compared to the original whole oncostatin M.

. 98373 5

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

Poly (amino acids) generally contain an amino acid sequence of at least 8 5 consecutive amino acids, which corresponds to and differs from the amino acid sequence shown in Figure 1 by up to 3, usually at most 1 amino acid. This difference may be an amino acid insertion, an amino acid deletion, or the substitution of one amino acid for another, in particular a conservative substitution. Poly (amino acids) usually contain at least 10, more usually at least 12, consecutive amino acids that correspond to and differ from the sequences shown in the figure by at most one amino acid.

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

20 non-polar G A P V L I

polar S T C M N Q

acid D D

basic K R

. aromatic F H Y W

[. 25.

| By conservative substitution is meant that amino acids of the same subgroup (i.e., either neutral aliphatic, acidic alphatic, basic, or aromatic), more specifically, polar-30 amino acids, are used to replace amino acids. It is desirable that amino acids having 2 to 4 carbon atoms or 5 to 6 carbon atoms determine the grouping of monomers in each aliphatic subgroup.

Poly (amino acids) is up to about 1000 to 35 amino acids in length. Usually the carbon has less than 100 amino acids, more usually less than 50 amino acids. Poly (amino- 98373 6 acids) is thus easy to synthesize. When the length of the poly (amino acids) exceeds 100 amino acids, they may be polymers or fusion proteins of on-costatin M fragments, each containing less than 100 amino acids, in which 5 fragments are fused to antigen, enzyme, enzyme fragments, etc. Large molecules (amino molecules). in particular, may consist of at least one polypeptide fragment of less than about 100 amino acids covalently linked to a large immunogenic polypeptide carrier to provide imiLunogenicity. Examples of such protein carriers include bovine serum albumin, volcano hemocyanin (KLH), and the like. These conjugated polypeptides are useful for inducing antibodies in a suitable host organism.

U937 cells are a cell line derived from a histiocytic lymphoma cell line [Sundström and Nilsson, Int. J. Cancer 17 (1976) 565-577], which can be induced to differentiate into cells with macrophage characteristics by treatment 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 treated with an appropriate inducer. It is convenient to use phorbol or Inge-25 emulsions, especially 12-O-tetradecanoyl phorbol-13-acetate (TPA). Usually about 5-20 ng / ml inducer can be used. The initial number of cells is about 105 -10 * cells / ml.

After treatment of the cells with the inducer for a sufficient time, usually 3 to 6 days, the supernatant is removed, the cells are washed in serum-free medium, the adherent cells are washed again in serum-free medium and the cells are incubated for at least 12 hours, usually up to about 48 hours, in serum-free medium, e.g. 1640 media. The supernatants are then collected, and the cells are removed by centrifugation. So- .98373 7 ......

the cell-free supernatant was examined for cell growth inhibitory activity (GIA) as described in the experimental section. The supernatant contains about 50 to 500 GIA units / ml (the definition of the G1 unit is given in the experimental 5 'section) ·. ··; ··; .- ·: · ........

Oncostatin-1 M can also be obtained from normal human peripheral blood lymphocytes (PBL) stimulated with mitogen. PBLs can be isolated from the leukofraction 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. The remaining cells are recovered from the solution by centrifugation, resuspended in buffer containing serum and thrombin, mixed, and allowed to settle for a short period of blood-15 gluten aggregate. The suspended cells are removed, recovered 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 peripheral blood T lymphocytes do not attach and are thus washed away. These cells were cultured at 37 ° C in medium, e.g. RPMI-1640, and treated with an appropriate inducer, e.g. phytohemagglutinin (about 1-25 5 mg / l) for about 100 hours, and then the supernatants were collected. Supernatants were centrifuged to remove cells and concentrated, for example, by ultrafiltration or dialysis.

Once the cell-free supernatant has been isolated from either U937-30 cells or normal PBLs, the treated medium is concentrated, conveniently using a hollow fiber system or ultrafiltration membrane, then diluted with acetic acid (0.1 N acetic acid) and concentrated to about 10-fold concentration. -35 nos and concentration. The concentrate can be lyophilized • 98373 8 and used directly, or the lyophilized product can be further purified.

The oncostatin M of the invention 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 Blo-sil TSK250 column and monitoring the activity of each fraction. Purification yields 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 the gel permeation chromatograph can be further purified using RP-HPLC (reversed phase high performance liquid chromatography) using a linear gradient of 0.1% trifluoroacetic acid in water as the primary solvent and 0.1% acetonitrile as a secondary solvent. trifluoroacetic acid. The program can vary; in general, the chromatography takes about 3 to 4 hours, most of the time, more than 50% and up to about 80% of the time, the proportion of the secondary solvent is 30 to 45%; the active fractions elute under these conditions with acetonitrile *. with a concentration of about 41-42%.

m *. The combined active fractions can be purified / further repeated by RP-HPLC, changing the gradient more rapidly from the in 25 condition and using a lower flow rate. Female-. 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 so that the secondary solvent is n-propanol containing 0.1% trifluoroacetic acid. A linear gradient with n-propanol concentration is used. changes slowly 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. Product • ...

· 98373 9 is usually purified to give a specific activity of at least about 100 GIA units / ng protein, more usually 150 GIA units / ng.

The amino acid-5 subsequences of the purified oncostatin M fragments were analyzed. The amino acid sequences of oncostatin are approximately as shown in Figure 1. In Figure 1, the first sequence shows the N-terminus of oncostatin M, while the other sequences show internal fragments of polypeptide-dln. In a subsequent mass analysis of cDNA clones, the amino acid sequences obtained by the sequence node, which became public in Finland on 15 April 1993, differ somewhat from the sequences in Figure 1, which is by no means surprising, as the sequences obtained by different methods often differ somewhat. The amino acid sequences obtained from the cDNA clones by the sequence node 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.

1 5 10. Ala-Ala-Ile-Gly-Ser-Cys-Ser-Lys-Glu-Tyr-Arg-Val-Leu-Leu- X 15 20 25 »· .........

25 Gly-Gln-Leu-Gln-Lys-Gln-Thr-Asp-Leu-Met-Gln-Asp-Thr-Ser-! 30 35 * ·: ......

.. Arg-Leu-Leu-Asp-Pro-Tyr-Ile,; : 1 5 10

Gln-Arg-Leu-Pro-Lys-Ala-Gln-Asp-Leu-Glu-Arg-Ser-Gly-Leu-30 15 20 • · Asn-Ile-Glu-Asp-Leu-Glu-Lys and: 1. .... 5 10 l Leu-Arg-Glu-His-Cys-Arg-Glu-Arg-Pro-Gly-Ala-Phe-Pro-Ser- 15 20 35 Glu Glu-Thr-Leu-Arg-Gly • s ......

98373 10 wherein Ala is alanine, Cys is cystellin, Asp is Asparagic acid, Glu is glutamic acid, Gly is glycine, Hls is histidine, Ile is isoleucine, Lys is lysine, Leu is leucine, Met is metlonine, Asn is asparagine, 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 contained a mixture of high mannose 10 and a complex N-linked oligosaccharide. However, Glykosy created non-nuclear oncostatin M preparations were also effective in regulating cell growth.

Oncostatin M is, as previously mentioned, further characterized by activity against certain cell lines. The polypeptide of the invention lacks cytotoxic activity against W126 and Wlde human fibroblasts, tumor nectrosis factor-sensitive mouse L929 cells, and the γ-interferon-sensitive human tumor cell line. It has also been found not to inhibit the proliferation of normal 20 human T lymphocytes or the formation of granulocyte / myelocyte colonies from bone marrow cells at concentrations up to 100 GXA units / ml. Oncostatin M '. in addition to stimulating normal human fibroblasts, of which ·. examples are WI38 and WI26 cells, proliferation and inhibition of tumor cells such as A375, HBT10, A549 and SK. Proliferation of MEL28 cells and may promote the growth of colony-forming cells from the normal 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.

• Amino acid sequence of the polypeptide of the invention: can be determined in full using commercially available 9 ..... .....

j sequencers. The polypeptide can then be synthesized by known methods, using commercially available * 35 automated synthesizers as well.

m 98373 11

Alternatively, the polypeptide of the invention could be prepared by recombinant DNA technology. Probes can be deduced from the partial amino acid sequence, which can then be used to screen a human genomic library.

5 The library can be a cDNA or chromosome library. Once one or more clones mating with a probe have been identified, fragments containing the gene of interest can be identified in a variety of ways and processed 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. Single-stranded cDNA can be prepared from mRNA.

This cDNA can then be paired with total mRNA from a cell that produces little, if any, polypeptide. The unpaired cDNA can then be isolated and used to prepare a double-stranded cDNA that can be probed.

Alternatively, DNA fragments can be inserted

To Agt11 so that the coding fragments can follow and be in frame with the β-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, fragments encoding a polypeptide of the invention or a fragment thereof can be identified and used to identify the desired gene.

Once the entire gene has been identified, either as cDNA or chromosomal DNA, it can be processed in different ways to effect expression. Once the gene is to be expressed in a host that has recognized wild-type oncostatin M replication and read regulatory regions, the entire gene with its wild-type 5 'and 3' regulatory regions can be introduced into an appropriate expression vector. There are a variety of expression vectors that use replication systems for mammalian viruses such as Simianvirus 40, adenovirus, bovine papillomavirus, vaccinia virus, insect baculovirus, etc.

5 These replication systems have been developed to provide markers that allow selection of transfectants as well as the provision of convenient restriction sites to which the gene can be inserted.

When a gene is to be expressed in a host that does not recognize naturally occurring wild-type copies and reads regulatory regions, further processing is required. Various convenient 3 'end copy control regions are known that can be placed after the stop codons. The 5 'unencoded region can be removed in front of the structural gene by endonuclease restriction, Bal31 resection, etc. When there is a convenient restriction site near the 5' end of the structural gene, the structural gene can alternatively be cleaved and an adapter used to link the structural gene to the promoter region. lost nucleotides. Various strategies can be used to provide an expression vector with a 5 'to 3' copy and read start region in the copy direction, which may also include regulatory sequences that allow for regulatory induction, a structural gene under the control of the initiation region, and copy and read stop area.

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 prorotor, etc. for yeast; Early and late promoter of SV40, late major promoter of adenovirus, etc. for n cells. As already mentioned, the expression unit may be included in a replication system to maintain the eplsome in a suitable cell host, or it may be without a replication system, allowing it to integrate into the host genome. DNA can be introduced into a host by known methods, such as transformation using calcium phosphate-doped DNA, transfection in which cells are contacted with a virus, microinjection of DNA into cells, and the like.

manner .; - - ......

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 a signal sequence (secretion precursor) in front of and in the same reading frame as the sequence that causes the secretion of the structural gene and the secretion of the secretion precursor so as to effect secretion of the mature polypeptide into the supernatant. If no secretion is caused, the host cells can be harvested, lysed in a conventional manner, and purified by conventional methods such as chromatography, electrophoresis, solvent extraction, and the like.

The polypeptide of the invention can be used in a wide variety of ways in vitro. The polypep of the invention. tide can be used to make its antibodies that-. are also the subject of the present invention. Antibodies can be prepared by conventional methods, e.g. using a polypeptide of the invention as an immunogen and injecting it into a mammalian host, for example, mouse, cow, goat, sheep, rabbit, etc., especially with an excipient, for example, a complete Freund's adjuvant, aluminum hydroxide gel or the like. From the host: blood can then be taken and used to isolate polyclonal antibodies or, in the case of mice, »| fusing peripheral blood lymphocytes or splenic lymphocytes (B cells) with a suitable myeloma cell to render chromosomes immortalized to produce monoclonal antibodies specific for the polypeptide of the invention - 98373 14.

Either polyclonal or raono-clonal antibodies can be prepared, which can then be used to diagnose in vitro or to detect the polypeptide in question in a sample, such as cells, or in a physiological fluid, such as blood. Antibodies can also be used in affinity chromatography to purify the present polypeptide and isolate it from a natural or synthetic source. Antibodies can also be used to regulate the amount of cellular polypeptide in question in culture.

The polypeptide of the invention can be used as an 11-gandant to detect the presence of receptors for this compound. In this way, receptors can be distinguished in terms of the presence and density of receptors in a polypeptide by monitoring the effect of various compounds on the presence of such receptors.

The polypeptide of the invention can be used in in vitro cultures to inhibit the growth of cells or cell lines sensitive to this polypeptide that react differently from insensitive cells. In this way, undesired cells can be removed from heterogeneous cell mixtures or cell lines when the undesired cells are sensitive to the polypeptide of the invention.

e #. The following examples are provided to illustrate the invention and are not intended to limit it.

Experimental part

Materials and Methods 30 Oncostatlin M isolated from U937-8 cells: tumor cells • ..... · ...........

; production of a growth inhibitor from a histiolytic lymphoma cell line U937 cells, a histiolytic lymphoma cell line [Sundström and Nilsson, Int. J. Cancer 17 (1976) 565 - 577) • ....., v, .. .... ......

.: 35 were cultured in 850 cm 2 spinner flasks (Corning C2540) at a distance of 4 x 10 5 cells / ml in a total of 300 ml of RPMI 1640 medium supplemented with 10% fetal bovine serum (FCS), penicillin / streptomycin (PS), L-glutamyl and 12-O-tetradecanoyl phorbol 13-acetate 5 (TPA, 10 ng / ml). After four days, the supernatants containing FCS and TPA were removed, the spinner flasks were washed five times with serum-free RPMI 1640, and the detached cells (1 x 10 * cells / ml) were washed three times with serum-free medium and added back to the flasks, Jolio created the final volume was 125 ml of serum-free RPMI 1640 medium per spinner flask. After 24 hours, the supernatant was collected, centrifuged to remove cells, filtered through a 0.45 Nalgene filter, and concentrated using a hollow fiber system 15 (Amicon cartridge HIP10-20) to a final volume of 150 ml (initial volume 1500 ml). Oncostatin M was also isolated from the supernatants of U937 cells treated with serum-free TPA in 150 cm 2 tissue culture flasks. The supernatant was concentrated on Amicon Diaflo-cal-20 vola PM-10 (cut-off 10 kD) and dialyzed. After dialysis, the concentrate was diluted with acetic acid to a concentration of 0.1 N acetic acid 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 while filtering. The concentrate was diluted with 1 N acetic acid, and the resulting precipitate was removed by centrifugation. The resulting concentrate was frozen and freeze-dried. The lyophilized material was used for the purification step.

Geellpermeaatiokromatografia

A Bio-sil TSK-250 column (600 x 21.5 mm) (BloRad) was connected to a euro liquid chromatography system. The crude fraction (10 mg / ml) was dissolved in an aqueous solution containing 40% acetonitrile and 0.1% trifluoroacetic acid 98373 16 (0.1% TFA). A 2 ml portion of the mixture was injected and eluted isocratically using a solution of 40% acetonitrile and 0.1% TFA in water as the mobile phase. The flow rate was 2.5 ml / min and the flow rate of the plotter paper 5 was 0.25 cm / min. 5 "fractions were collected. Chromatography was performed at room temperature. 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 10s GIA units. The apparent molecular weight of the factor was found to be 18 kD as determined by size exclusion chromatography (Bio-s11 TSK-250 column).

RP-HPLC (reverse phase high performance chromatography) of 15 TSK-250 fractions

The combined TSK-250 fractions 21 and 22 described above were diluted to twice the volume with 0.1% TFA. This mixture was injected isocratically onto a μ-Bonda-20 pak-C18 column (7.8 x 300 mm) (referred to as C181) at room temperature. The flow rate was adjusted to 2.0 ml / min and the plotter 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% for 10 min, then 30-45% for 150 min; 45 - * 55% 20 min: get, and 55 - * 100% 10 min: get. The lime solvents were HPLC grade. 4 ml fractions were collected, and growth inhibitory activity was tested on a sample taken from each fraction. Fractions 72-75 were found to contain most of the activity. The active fractions elute with an acetonitrile flux of 41-52%.

Fractions 72-75 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 mm) (referred to as C183) at room temperature. The flow rate f was adjusted to 1 ol / min and the plotter speed was 0.25 cm / min. Gradient conditions were 0 to ♦ 35% in 10 min; 35 - * 45% in 100 million; and 45 - »100% 5 in 10 min. Fractions were collected, sampled, and examined from samples 6IA. Most of the activity eluted from the column at an acetonitrile content of 40.7-41.3% (retention time 83-86 min).

The active fractions were pooled, diluted to 10-fold volume with 0.1% TFA and injected isocratically onto a p-Bondapak-C18 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 was used in 15 primary solvent (0.1% TFA) to secondary solvent (n-propanol, 0.1% TFA). Gradient conditions were 0 -> 23% at 20 min and 23 - * 120 min. Fractions were collected and examined for GIA from a sample taken from each fraction. Most of the activity eluted at a propanol content of 25-26.5% (retention time 59 min). This apparently homogeneous fraction contained about: 300 ng of protein and about 40,000 GIA units.

Assay to investigate the regulation of cell growth using the incorporation of 3H-tvraidilnln OMA than (GIA) 25 Experiments were performed in plates with 96 flat-bottomed wells (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 3 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 gravity 27 pCi / μg) was then added to each well and incubated for a further 6 hours. Cells 35 were then transferred to glass filter strips using a multiwell collector (PHD Cell Harvester, Cambridge Techology, 98373 18

Inc). The filters were transferred to scintillation counting flasks to which 2 ml of scintillation counting solution (SclentiVerse il, Fisher Scientific Co.) was added, and then scintillation counting was performed to determine the incorporation of 3 H-thymidine.

On soft agar, a 0.5 ml basal layer of anti-colony inhibition assay (TGI) containing 0.5% agar (Agar Noble; Difco Laboratories, Detroit, Michigan) in 10 DMEM containing 10% fetal bovine serum (FCS) was added 24- for Costar tissue culture plates. 0.5 ml of the same medium-FCS mixture containing 0.3% agar, 1-2.5 x 10 * A375 cells and test factor at different concentrations were applied to the agar base layer.

The plates were incubated at 37 ° C in a humidified atmosphere containing 5% CO in air, and after 7 days an additional 0.5 ml of 0.3% agar in the same medium at the same concentration of factor was added. Colonies were counted as unfixed and unstained 20 and colonies containing more than 6 cells between days 7 and 14 were counted.

s, 5 Results Sequences of oncostatin1 M isolated from U937 cells The N-terminal sequence and fragments of oncostatin1 M were determined by making a microsequence; analysis of reduced and S-pyridine ethylated polypeptides and peptides obtained by digestion of reduced and S-pyridine ethylated oncostatin 1 M enzymatically with endoproteinase Lys-C and Staphylococcus aureus V8 protinase. The peptide fragment was purified by RP-HPLC J using volatile solvents. Peptides were subjected to automatic • repeated Edman digestion in a Model 470A protein sequencer (Applied Biosystems, Inc.). Phenylthiohydanoylamino acids were analyzed by RP-HPLC (Applied Bio- * 35 systems, Inc.) using a PTH-C18 column (2.1 x 98373 19 220 mm, ABI) and eluted using sodium acetate buffer-tetrahydrofuran acetone triiligradienttia.

The resulting amino acid sequences are approximately as shown in Figure 1. When comparing these sequences to the up-to-date protein database (PIR Release 9.0, May 1986), no significant sequence homologs were observed with any known sequence. Homo-logla is also absent with tumor necrosis factor, lymphotoxin-10, colony stimulating factor, interleukin 1 and 2, and 8-transforming growth factor.

Inhibition of Tumor Cell Proliferation, 1a Normal Stimulation of Human Fibroblast Proliferation Using the soft tag colony hlbi-15 test described above, the following results were obtained:

Table 1 Inhibition of A375 melanoma cell colony formation by purified oncostatil M isolated from U937 cells1 20 GIA units / Inhibition of colony formation well_number_% (250) 96: 96. 83 6 94! 27 11 89 1 25 45 32 69 0 106 - A375 cells were not detached as described above in soft agar with or without factor with a final volume of 2 ml. The factor used was the fraction from the C18 propanol column with the highest tumor growth inhibitory activity (GIA). The number of colonies was counted after 11 days. A cluster of at least 6 cells was defined as a colony. One GIA unit was defined as the amount that causes the incorporation of 3 H-thymidine 20 - ......

98373 to A375 cells in its microwell in the 50% inhibition assay 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 U937 cell supernatants on TPA-induced cells on tumor growth inhibitory activity *

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

Substrate, reference sample - - - 39,780 Untreated supern. 7,206 ** 13,896 16,000 15 1 N acetic acid 6,670 17,073 18,783 1 N NH 4 OH 6,956 15,016 13,923 * 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 supernatants were collected.

Supernatants were treated with 1 N acetic acid or 1 N amino acid. nlumhydroxyl (NH 4 OH). They were dialyzed against medium. and examined their ability to inhibit the incorporation of 3 H-thymidine into •. * A375 cells. A375 cells were labeled with 3 H-thymidine \ 25 (3H-TdR) for the last 6 hours of the 3 day incubation period.

* '** The results show the incorporation of 3H-TdR in pulses per minute.

: The above results show that oncostatlin M in the dlylated supernatant is hardly / inactivated by IN acetic acid or 1 N ammonium hydroxide. The compounds of the invention are thus. relatively insensitive to both moderately strong acid l and moderately strong base. The compound of the invention undergoes a heat treatment at 56 ° C for 1 hour, but not a treatment at 90 ° C for 35 minutes.

· »21 98372

The thermal stability of the compound of the invention was also examined and it was found that the activity of the compound was maintained when kept at 56 ° C for 1 hour, but disappeared almost completely when kept at 5 ........ 90 ° C for 30 minutes. '"ί -

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

10 Table 3

Inhibition of tumor cell replication Number of GIA units causing 30% inhibition by purified oncostatin M from U937 cells Tumor cells Uptake of 3H-TdR into cells 15 A549 (lung cancer) 21 HTB10 (neuroblastoma) 81 A375 (melanoma)

Tumor cells were seeded in microwells for 3 hours before the addition of oncostatin M purified by RP-HPLC as described above diluted in various ratios. For the last 6 hours of incubation for 3 days, cells in 0.2 ml of medium were labeled with 3H- »thymidine (3H-TdR) (0.5 pCi / well). One unit of tumor growth inhibitory activity (GIA) was defined in the description of Table 1 as the amount that reduces the incorporation of 3 H-TdR into A375 melanoma cells by 50%. One unit 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.

: U937 factor did not reduce 3H-TdR incorporation in Human 9 .....

• normal WI16 fibroblasts in any experiment.

The above results indicate that the ability of oncostatin M to inhibit replication is selective; : 98373 22 The effect varies greatly depending on the nature of the cell. The compound of the invention is effective against melanoma cells such as A375 melanoma cells, lung cancer cells such as A549 cells, and neuroblastoma cells such as HTBIO 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 1: 103 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 uptake of 3 H-thymidine at each concentration was measured from three parallel wells. The results are shown in Table 4.

15 Table 4

Inhibition of tumor cell proliferation and progression of normal fibroblast proliferation by oncostatin M GIA-20 units / inhibition (%) stimulation (%) well A375 WI38

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

Experiment 2 27 NT 28 46 9 87 22 36 3 76 11 52 30 A375 A549

Experiment 3 75 89 30 25 85 22 8 71 16 A375 SK-MEL28 35 Experiment 4 20 87 44 5 75 25 1 59 11 40 The results show the incorporation of 3 H-thymidine into tumor cells (A375, HTB10, A549 and SK-MEL28) and normal fibroblasts ( W126 and W138) inhibition (%) and 98373 23 stimulation (%), respectively. One GIA unit is defined in the description of Table 1 as the amount of oncostatin M that causes 50% inhibition of 3 H-thymidine incorporation into A375 cells.

5 In addition to the fact that the effect on the incorporation of 3H-thymidine into tumor cells is different from that in normal human fibroblasts, a different effect is also observed .......................... ....... · .. ·. · ·. ..

morphology and cell number after treatment of these two cell types with oncostatin M for 3 days, as shown in Figure 10 2.

The oncostatin M used was obtained from the fraction of the HPLC-C183 column in which the proliferative inhibitory activity of A375 cells was highest. Figure 2 is a series of microscopic photographs of A375 melanoma cells that were untreated (A) treated with 5 GIA units (units of growth inhibitory activity) oncostatin M (B) and treated with 100 units (C) , as well as micrographs of W138 fibroblasts that were untreated (D) treated with 5 GIA units (E) or 20,100 units (F). Cells were stained with 0.5% crystal violet methanol solution. Magnification is 63x.

1 Oncostatin M? N NaDodSO./PAGE

: Apparent molecular weight of purified oncostatin M subjected to NaDodSO 4 under reducing conditions | 25 was found to be about 28 kD, as shown in Figure 3.

The following proteins were used as standards (lane A): ovalbumin, Mp - 43 kD; chymotrypsinogen α, Mp - 25.7 kD; lactoglubulin 8, Mp - 18.4 kD; lysozyme, Mp - 14.2 kD, bovine trypsin inhibitor Mp - 6.2 kD; the A and B chains of the insu-30 line, Mp »2.3 kD and 3.4 kD, respectively. oncostatin M was placed in lane B.

Oncostatin M, which was PAGEed under non-reducing conditions, also had an apparent molecular weight of 28 kD, and a protein electroeluted from the zone was found to inhibit A375 cell proliferation.

98373 24

The antibody of the synthetic oncostatin M peptide reacts with 1Z5I-labeled oncostatin M in radioimmunoassays a) Peptide synthesis: The peptide corresponds to groups 6 to 19 of the oncostatin „M- ________ 5 protein and was synthesized by the solid phase method by automated Beckman as described in [Gentry et al., J · Biol.

Chem. 258 (1983) 11219] · The peptide was cleaved from the resin using the "low-high" HF procedure [Tain et al., J.

10 Amer. Chem. Soc. 105 (1983) 6442-6445].

b) Antibody production: The peptide was coupled to bovine gamma globulin as described in the literature [Gentry and Lawton, Virology 152 (1985) 421-431]. White New Zealand rabbits were administered the peptide at a base dose and five booster doses at 4 sites subcutaneously as described in the literature [Gentry and Lawton, Virology 152 (1986) 421-431]. Antisera were taken 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 et al., PNAS 82 (1985) 356-360]. A dose of the labeled preparation containing 100,000 pulses / min was mixed with rabbit antiserum raised against the 17 N-ter - ..: 25 terminal amino acids of oncostatin M (final dilution ratio 1:20), the N-terminal peptide ( 17 n - terminal amino acid of oncostatin M, 2 pg) in the presence or absence, and immunoprecipitation analysis was performed as described in the literature [Linsley et al. Biochemistry 25 (1986) 30 2978-2986].

: More specifically, one tube containing 5 μΐ sample, preincubated with 2 μg of N-terminal peptide in 10 ml of TNEN solution (20 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.05% Nonidet P- 40) containing 0.1% BSA for 30 min at 4 ° C, after which 98373 of 125 I-oncostatin M in 85 μ 85 of a THEN solution containing 0.1% BSA and 40 mmol / l dithiothreitol (DTT). Seven tubes containing 5 μΐ of antiserum were incubated with 125 I-oncostatin M in 85 μl of TNEN solution-5 containing 0.1% BSA and 40 mmol / l DDT for 30 min at 4 ° C. followed by the addition of 50 μΐ of 10% formalin-fixed Staphylococcus aureus (Pansorbin, Calbio-chem).

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, followed by PAGE analysis. A diffusion band corresponding to Mp 32 kD was observed after SDS / PAGE analysis of immunoprecipitates. Precipitation of this band was prevented by an excess of unlabeled peptide-15 corresponding to the 17 N-terminal amino acids of oncostatin M, indicating that precipitation was specific for this peptide.

The carbohydrate composition of oncostatin Mm was examined by testing for glycosidase sensitivity. Immunoprecipitates prepared as described in c) 20 above were treated with buffer, endoglycosidase H or neutramidase *. * Linsley et al. (1986). Treatment with endo- 9 • glycosidase H: 11a, an enzyme specific for N-linked, mannose V-rich oligosaccharides, resulted in the appearance of a more small molecule band (Mp 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 (Mp -30 27 kD), indicating that the size heterogeneity of untreated 125 I-labeled oncostatin M was due to heterogeneity in sialylation of the glycoprotein core. The results showed that the active oncostatin M preparations contained a mixture of high mannose and complex N-linked oligosaccharide side chains.

98373 26

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

Production of inhibitors of tumor cell growth from PBLs

PBL-containing leukofractions obtained from a blood bank were diluted 1: 1 with phosphate-buffered saline, 10 ml of solution / containing 9% Ficoll and 20% by volume of 50% sodium diatrizoate (final specific gravity 1.080). Gradient te and centrifuged at room temperature for 20 imlh at 850 x g. Cells 10 were harvested from the gradient interface and washed with PBS. The red blood cells were lysed by treating for 10 min with 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% fetal bovine 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 fetal bovine serum, and transferred to a column containing 0.5 g of brushed, pre-moistened nylon wool (type 200, Fenwal).

The nylon wool column was incubated at 37 ° C for 60 min to allow monocytes and B lymphocytes to adhere. The column was then rinsed with three volumes of RPMI 1640 medium (37 ° C) containing 5% fetal bovine serum and the eluted non-adherent cells (PBLs) were collected.

PBLs (2 x 10 * 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 · 7H 3 O (1 mg / l), ZnSO 4 * 7H 2 O (2 mg / l), Na 2 SeO 3 · 5H 2 O (0.017 mg / l), 35 1-aminoethanol (1 mg / l), human transferrin (5 mg / l), bovine serum albumin linoleic acid conjugate 27 yvt (Sigma) (200 ®g / l), L-glutamine (300 mg / l), penicillin / streptomycin (100,000 units / l) and phytohemagglutinin-Keep (2 mg / l) / 1). Supernatants were collected, centrifuged to remove cells, concentrated by ult-5 fat filtration (Amicon Diaflo membrane YM-10, permeation limit 10 kD) and dialyzed against 0.1 M acetic acid (Spectrapore 3 dialysis tubing). The clarified retentate was lyophilized.

Gel Permeation Chromatography 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 / min. 20 ml fractions were collected. A sample from each fraction was evaporated and tested for A375 cell growth inhibitory activity (GIA) in triplicate. The active fractions were combined, lyophilized and rechromatographed on a Bio-sil TSK-250 column (600 x 21.5 mm) as described above.

RP-HPLC of 20 TSK-250 fractions

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 the regulation of cell growth using “incorporation of 5I-iodideoxyuridine into DNA (GIA) These experiments were performed in flat-bottomed 96-well tissue culture plates (Costar 3596). Human melanoma cells (A375, 4 x 103) in 50 of the test sample were added to each well and incubated for 3 days at 37 ° C. Cells were labeled with 129Z-ZdU (0.05 yCi / well) for 24 hours, and incubated for an additional 24 hours. The cells were washed three times, collected on a multi-sample ice, and the radioactivity was determined in a gamma counter.

28 98375

Score:

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

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

X represents an unidentified amino acid.

Comparison of this sequence with the sequence of U937 factor clearly shows identity with the 10 N-terminus of PBL-derived factor.

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-O-K

The following study investigated the ability of PBL-derived oncostatin M to affect the replication of various cells. Mouse L929 cells were found not to be sensitive to PBL-derived oncostatin M when used at a maximum concentration of 1000 GIA units / ml.

Growth of human WI26-ibroblasts is stimulated by treatment with 1000 GIA units / ml. Up to 500 GIA units / ml did not affect normal human T-lymphocyte proliferation 72 hours after mitogenesis.

From the above results, it is apparent that a novel polypeptide having the ability to regulate cell growth has been provided. The compound has activity varying according to the nature of the cell line in question, so that it can be used alone or in combination with other compounds to regulate cell growth in vitro. The polypeptides of the invention thus represent a novel polypeptide that can be used in cell mixtures in vitro to selectively reduce or promote a particular cell type proliferator. In addition, the entire polypeptide or fragments thereof can be used as immunogens to induce antibody production. Induced antibodies can be used to determine oncostatin in body fluids 98373 29 ...........

M or to regulate factor activity through binding in vitro. In addition, this antibody, together with purified oncostatin M or fragments thereof, act as Korn-5 components in diagnostic kits, together with other reagents, in particular antibodies for the detection and quantification of oncostatin M.

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

Claims (7)

98373 30. : Oncostatin M polypeptide, characterized in that it is substantially free of cellular debris and other leukocyte proteins, has the ability to inhibit tumor cell proliferation and stimulate the proliferation of normal human fibroblasts, does not inhibit human proliferative or cytotoxic T cell responses or formation of cellular bone marrow colony cells, has a molecular weight of 17 to 19 kD as determined by gel exclusion chromatography and about 28 kD as determined by SDS-PAGE, it is resistant to treatment with 1N acetic acid and 1 N ammonium hydroxide and 56 ° C for one hour and it can be prepared by a method comprising a) isolating leukocytes, such as histiocytic lymphoma cells or normal human peripheral blood lymphocytes, from or in a mammalian cell culture; b) activating the leukocytes with an inducing agent such as ingenol, phorbol or mitogen; and c) isolating oncostatin M from the chromatographically activated leukocytes in pure form from the rest of the cellular material. Polypeptide according to claim 1, characterized in that the polypeptide is of such a purity that its specific activity is at least 100 GIA units / ng protein. Polypeptide according to Claim 1, characterized in that the leukocytes are human histiolytic lymphoma cells induced by phorbol ester. Polypeptide according to Claim 1, characterized in that the leukocytes are mitogen-activated normal human peripheral blood lymphocytes. 31 98375 Antibodies, characterized in that they are specific for a polypeptide or a poly (amino acid) fragment according to claim 1. A method for detecting the presence of a poly-5 peptide according to claim 1, characterized by combining a sample suspected of containing said polypeptide with an antibody according to claim 5 and quantifying the formation of a complex with the antibody. Diagnostic kit, characterized in that it comprises an antibody according to claim 5 and a polypeptide according to claim 1. 98373 32