CA1340296C - Cell growth regulatory factor - Google Patents

Abstract

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

Description

13 1029~

NOVEL CELL GROWT~ REGULATORY ~ACTOR

BACKGROUND OF THE INVENTION
Field of the Invention Leukocytes, ~oth lymphocytes and monocytes, have been implicated in the inhibition of tumor growth in several animal tumor models. The increased inci-dence of malignancies in immunocompromised humans sup-ports the contention that the white blood cells play a role in the regulation of neoplastic growth. Protein factors produced by these white cells that inhibit tumor growth or modulate immune functions which have already been isolated and characterized include the interferons, ~- and Y-, tumor necrosis factor, lympho-toxin, interleukin-2, and other lymphokines. Since each of the factors which have been isolated have a different spectrum of activities and may interact dif-ferently in conjunction with other factors, there re-mains a continuing and strong interest in the isolation of and characterization of all of the factors which white cells ?roduce in the modulation of cell growth or immune functions. These compounds individually or to-gether may find use in the treatment or diagnosis of cancer, as promoters of wound healing or as 3~ immunomodulators for the treatment of patients with immunodeficiencies, autoimmunity, organ transplants, and the like.
There are several difficulties that may be encountered in the discovery, isolation, purification, or characterization of naturally occurring factors.
Methodologies must be developed for separating and purifying a factor of interest from other factors in ~'!10296 the crude starting material without denaturing the ac-tivity of the desired factor; bioassays must be devel-oped which allow for identifying the fractions during separations which concentrate a particular f~ctor; a novel factor must be distinguished from factors which are already known or other unknown factors which may be present and may affect, either negatively or positive-ly, the activity of the factor being pursued; the puri-fied factor must be characterized; and the purified factor must be concentrated in sufficient amount to permit identification and characterization of the factor. Therefore, with the increasing number of fac-tors which have been isolated, each new factor becomes more difficult to identify, since its role and function 15 may be obscured by the numerous other factors which are present.

Description of the Prior Art Beal et al., Cancer Biochem. BiophyS. (1979) 3:93-96 report the presence of peptides in human urine which inhibit srowth and DNA synthesis more in trans-formed cells than in normal cells. Holley et al., Proc. Natl. Acad. Sci. (1980) 77:5989-5992 describe the purification of epithelial cell growth inhibitors.
Letansky, Biosci. Rep. (1982) 2:39-45 report that pep-tides purified from bovine placenta inhibit tumor growth and thymidine incorporation in DNA to a greater extent in neoplasms than in normal cells. Chen, Trends Biochem. Sci. (1982) 7:364-365 reports the isolating of 3~ a peptide from ascites fluid with a cancer suppressing property. Redding and Schally, Proc. Natl. Acad. Sci.
(1982) 79:7014-7018 report isolation of purified pep-tide(s) from porcine hypothalmi which exhibit antimito-genic activity against several normal and tumor cell lines. Sone _ al., Gann (1984) 75: 920-328 report the production of a factor(s) produced by human macropha3es that inhibits the growth of certain tumor cells in ..

134~)296 vitro. Ransom et al., Cancer Res. (1985) 45:851-862, report the isolation of a factor called leukoregulin that inhibits replication of certain tumor cell lines and appears distinct from lymphytoxin, interferon and 5 interleukin 1 and 2. Most of these factors have not been fully characterized, nor are their primary struc-tures known.
Aggarwal et al., J. Biol. Chem. (1984) 259:
686-691 purified and characterized human lymphotoxin 0 (LT) produced by a lymphoblastoid cell line and subse-quently sequenced LT (Aggarwal et al., J. Biol. Chem.
(1985) 260:2334). Gamma interferon (Y-IF) which is produced by lymphoid cells and has immunomodulatory and tumor inhibitory activity has been cloned and ex-5 pressed. (Gray et al., Nature (1982) 2g5:503:508.) Tumor necrosis factor (TNF), which inhibits growth of some tumors and is produced by macrophages and certain leukemia cell lines has been characterized and the TNF
cDNA was cloned and expressed in E. coli (Pennica et 20 al., Nature ( 1984) 312 :724) .

SUMMARY OF THE INVENTION
A novel peptide factor and biologically active 25 fragments thereof are provided, which factor is available from leukocytes. The factor finds use in modulating cell growth, such as inhibiting tumor cell growth and stimulating growth of normal fibrobla~ts and may modulate immune functions. The factor has an 3~ amino acid sequence distinctively different from the sequences of other compounds which have been reported to have analogous properties.

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~'10296 BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 represents the amino acid sequence of fragments of Oncostatin M;
Figure 2 is a series of photomicrographs of cells treated with Oncostatin M wherein (A-C) are A375 melanoma cells treated with 0, 5 and 100 GIA units, respectively, and (D-F) are WI38 fibroblasts treated with 0, 5 and 100 GIA units, respectively; and Figure 3 is a photograph of an SDS-PAGE
analysis of Oncostatin M.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS
A novel polypeptide, polypeptide compositions, polypeptide fragments and mutations, their preparation and use are provided, with the compositions demonstrat-ing activity in the modulation of cell growth, particu-larly inhibiting tumor cell growth and stimulating growth of normal fibroblasts. A subject polypeptide, referred to as Oncostatin M is available from leuko-cytes; e.g., from conditioned media of stimulated U937cells or conditioned media of stimulated normal human peripheral blood lymphocytes (PBL). Fragments and mutants of the polypeptide having the biological activity of the intact Oncostatin M such as cell growth modulation activity or immunologic activity are also provided.
The polypeptide fragments of this invention are novel polypeptides of at least 8 amino acids that are biologically active, at least as to being immuno-logically cross-reactive with naturally occurring Oncostatin M. By immunologically cross-reactive it is meant that an antibody induced by a novel polypeptide of this invention will cross-react with intact Onco-statin M at least when Oncostatin M is in a denatured 35 state. Those polypeptides are tnerefore useful to induce antibodies to Oncostatin M which can be used to determine the concentration of Oncostatin M in a bodily ~'10296 fluid, to bind to Oncostatin M and thus modulate its activity, and to purify Oncostatin M, as by use in an affinity column. A portion of the polypeptides may also retain the cell growth modulatory activity of intact Oncostatin M, although that activity may be modulated, usually reduced in comparison to intact Oncostatin M.
Figure 1 represents the amino acid sequence of poly(amino acid)s cross-reactive with Oncostatin M with the first sequence representing the N-terminus of Oncostatin M.
Poly(amino acid)s of the present invention contain an amino acid sequence having at least 8 con-secutive amino acids that correspond to an amino acid sequence depicted in Figure 1 and differing from that sequence by no more than 3, usually no more than 1 amino acid. That difference can be either the inser-tion of an amino acid, the deletion of an amino acid or the substitution of one amino acid for another, partic-ularly a conservative substitution. Usually thepoly(amino acid)s will contain at least 10, more usually at least 12, consecutive amino acids that correspond to sequences depicted in the figure and differ by no more than one amino acid.
For purposes of the subject invention, the various amino acids will be divided into a number of subclasses. The following table indicates the sub-classes:

3~ aliphatic neutral non-polar G A P V L I
polar S T C M N Q
acidic D E
basic K R
aromatic F H Y W

By conservative substitution, it is meant that amino acids from the same subclass (i.e., either neutral aliphatic, acidic aliphatic, basic aliphatic or aromatic), more particularly the same polarity, will be 5 substituted for each other. Desirably, amino acids of two to four carbon atoms or five to six carbon atoms will define monomer groupings in the aliphatic subclass.
The poly(amino acid)s will not exceed about 10 1000 amino acids in length. Usually they will have fewer than one hundred amino acids, more usually fewer than fifty amino acids. Thus, the poly(amino acid)s can be readily synthesized. Usually when poly(amino acid)s exceed 100 amino acids in length, those 15 poly(amino acid)s may be polymers of fragments of Oncostatin M having fewer than 100 amino acids each, or fusion proteins where the fragment is fused to an antigen, enzyme, enzyme fragments, etc. Particularly, the higher molecular weight poly(amino acid)s can be at 20 least one polypeptide fragment of fewer than about 100 amino acids joined covalently to a large immunogenic polypeptide carrier to provide for immunogenicity.
Exemplary of such protein carriers are bovine serum albumin, keyhole limpet hemocyanin (KLH) and the like.
25 Those conjugated polypeptides will be useful for inducing antibodies in an appropriate host organism.
U937 cells are a cell line derived from a histiocytic lymphoma cell line (Sundstrom and Nilsson, Int. J. Cancer (19760 17:565-577) that can be induced 30 to differentiate into cells having characteristics of macrophages following treatment with a variety of agents (Harris et al., Cancer Res. (1985) 45:9-13).
For production of Oncostatin M, the U937 cells may be srown in a conventional nutrient medium with serum and 35 treated with an appropriate inducer. Conveniently, phorbols or ingenols may be employed, particularly 12-O-tetradecanoylphorbol-13-acetate (TPA). Usually, from ~402~6 about 5-20ng/ml of the inducer may be employed. The initial number of cells is ~rom about 105 _1o6 cells/ml.
After allowing cells to be treated with the inducer for a sufficient time, generally three to six days, the supernatant is removed, the cells washed with serum-free nutrient medium, attached cells wasned again with serum-free medium and the cells allowed to incu-bate for at least 12 hours, usually not more than about 48 hours, in serum-free nutrient medium, e.g. RPMI-1640 medium. Supernatants are then collected and cells are removed by centrifugation. Cell-free supernatants were tested for cell growth inhibitory activity (GIA) as described in the experimental plan. The supernatant contains about 50 to 500 units of GIA/ml (see Experi-mental for definition of GIA units.) Oncostatin M can also be obtained from mitogen-stimulated normal human peripheral blood lym-phocytes (PBL's). PBL's can be isolated from leuko-fractions by diluting the fractions and centrifugingthem over Ficoll gradients. Cells collected from the gradient interface are washed and shock-lysed to remove red blood cells. Remaining cells are collected from that solution by centrifugation, resuspended in buffer containing serum and thrombin, agitated and the plate-let aggregate allowed to settle for a short period of time. The suspended cells are transferred, recovered by centrifugation, resuspended in serum and transferred to a column containing nylon wool. The column is incu-3~ bated to allow the attachments of monocytes and B-lymphocytes and then washed. Most peripheral blood T
lymphocytes do not adhere and are eluted from the column. Those cells were cultured at 37~ in culture medium, e.g. RPMI-1640 medium, and treated with an appropriate inducer, e.g. phytohemagglutinin (about 1 to 5 mg/l), for about 100 hours and then supernatants were collected. The supernatants were centrifuged to ~ 3 ~ 0 2 9 b remove cells and concentrated as by ultraiiltration or dialysis.
After isolating cell-free supernatant from either U937 cells or normal P8L's, the conditioned medium is concentrated, conveniently using a hollow fiber system or an ultrafiltration membrane, followed by dilution with acetic acid (to a concentration of 0.1N acetic acid) followed by concentrating about ten-fold and the dilution and concentration repeated. The concentrate may be lyophilized and used directly or the lyophilized product can be used for further purification.
The subject Oncostatin M can be purified by a procedure of gel permeation chromatography using aqueous 40d acetonitrile-0.1% trifluoroacetic acid as an isocratic mobile phase on a Bio-sil TS'~250 column, monitoring activity for each of the fractions. Purifi-cation provides for a composition having at least about 0.5-5x104 GIA units/ml in the active fractions.
The partially purified product from the gel permeation chromatography may be further purified by employing reverse phase high-pressure liquid chromatog-raphy employing a linear gradient, where the primary solvent is 0.1% trifluoroacetic acid in water and the secondary solvent is acetonitrile containing 0.1% tri-fluoroacetic acid. The schedule can be varied, ~en-erally the chromatographing requiring about 3-4 hours, with the major portion of the time, greater than about 50p of the time and not more than about 80p of the 3~ time, in the range of 30-45% of the secondary solvent.
Under these conditions the active fractions elute at about 41-42p acetonitrile.
The pooled active fractions may be further purified by repeating the reverse phase HPLC, employing a more rapid change in the gradient conditions and a slower flow.rate. Under these conditions, the activity emerges at about 40.5-41.5p acetonitrile.

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~t 10296 The reverse phase HPLC may then be repeated, changing the solvent system, where the secondary sol-vent is n-propanol-0.1% trifluoroacetic acid. A linear gradient is employed, where the gradient is changed slowly in the range of 23-35% n-propanol. The major activity is observed in the range of 25.5-27.5% pro-panol, to sive a substantially homogeneous product, having a specific activity of greater than 10 GIA
units/ng protein. Usually, the product is purified to provide a specific activity of at least about 100 GIA
units/ng protein, more usually 150 GIA units/ng.
The subject compounds are characterized by having a molecular weight of about 17 to 13 kiloDaltons (kD), particularly about 18kD, as determined by size exclusion chromatography The subject compounds are further characterized as having an apparent molecular weight of approximately 28kD as determined by poly-acrylamide gel electrophoresis under reducing or non-reducing conditions.
The amino acid sequence of fragments of puri-fied Oncostatin M was analyzed. Oncostatin has sub-stantially the amino acid sequences represented in Figure 1. Referring to Figure 1, the first sequence illustrates the N-terminus of Oncostatin M, while the remaining sequences illustrate internal fragments of the polypeptide.
Active preparations of isolated Oncostatin M
contained a mixture of high mannose and complex N-linked oligosaccharide. However, non-glycosylated preparations of Oncostatin M retained cell growth modulatory activity.
Oncostatin M is further characterized by its activity toward certain cell strains. The subject polypeptide lacks cytotoxic activity against WI26 and WI38 human fibroblasts, and mouse L929 cells which are sensitive to tumor necrosis factor, and a Y-interferon-sensitive human tumor cell line. It also is found not _ _. . ....

3 '~L 0 2 ~ b l o to inhibit proliferation of normal human T-lymphocytes or to inhibit granulocytic/myelocytic colony formation from bone marrow cells at concentrations up to 100 GIA
units/ml. Further, Oncostatin M stimulates prolifera-tion of normal human fibroblasts as exemplified by WI38and WI2~ cells and inhibits proliferation of tumor cells such as A375, H3T10, A549 and SK-MEL28 and may augment growth of colony forming cells from normal bone marrow. Oncostatin M did not suQpress human prolifera-0 tive or cytotoxic T cell responses in mixed leukocyteculture reactions (MLC) at concentrations of 500 GIA
units/ml.
The subject polypeptide is found to be stable to moderate acid and base and to heat treatment at 5~~C.
The amino acid sequence of the subject poly-peptide may be completely determined using commercially available sequencers. The polypeptide may then be syn-thesized in accordance with known techniques, employing automated synt'nesizers which are also commercially available.
Alternatively, the subject polypeptide may be produced by recombinant DNA techniques. From a partial amino acid sequence, probes can be deduced which can then be used for screening a human genomic library.
The library may be a cDNA library or a chromosomal library. Once the clone(s) have been identified as annealing to the probe, the fragments containing the gene of interest may be identified in a number of ways 3~ and manipulated in a number of ways. The fragment may be reduced in size by endonuclease restriction, with the resulting fragments cloned and probed for the presence of the desired gene. Cells which produce the desired peptide or produce enhanced amounts of the de-sired peptide may be employed for production of messen-ger RNA. From the messenger RNA, sinsle stranded cDNA
may be prepared. The cDNA may then be annealed to -1~40296 . .
total messenger RNA from a cell which produces little, if any, of the polypeptide. The unannealed cDNA may then be isolated and used to prepare ds cDNA, which may be screened with the probes.
Alternatively, DNA fragments may be inserted into Agt11, so that coding fragments may be downstream from and in frame with the ~-galactosidase gene. Anti-bodies may be prepared to the Oncostatin M polypeptide and used for screening the resulting fused proteins for cross-reactivity. In this manner, fragments coding for the subject polypeptide or fragment thereof may be identified and used for identifying the desired gene.
Once a complete gene has been identified, either as cDNA or chromosomal DNA, it may then be manipulated in a variety of ways to provide for expres-sion. Where the gene is to be expressed in a host which recognized the wild-type transcriptional and translational regulatory regions of Oncostatin M, the entire gene with its wild-type 5'- and 3'-regulatory regions may be introduced into an appropriate expres-sion vector. Various expression vectors exist employ-ing replication systems from mammalian viruses, such as Simian Virus 40, adenovirus, bovine papilloma virus, vaccinia virus, insect baculovirus, etc. These repli-cation systems have been developed to provide formarkers which allow for selection of transfectants, as well as providing for convenient restriction sites into which the gene may be inserted.
Where the gene is to be expressed in a host which does not recognize the naturally occurring wild-type transcriptional and translational regulatory regions, further manipulation will be required. Con-veniently, a variety of 3'-transcriptional regulatory regions are known and may be inserted downstream from the stop codons. The non-coding 5'- region upstream from the structural gene may be removed by endonuclease restriction, Bal31 resection, or the like. Alterna--~?, -1029~

tively, where a convenient restriction site is present near the 5'-terminus of the structural gene, tne struc-tural gene may be restricted and an adaptor employed for linking the structural gene to the promoter region, where the adaptor provides for the lost nucleotides of the structural gene. Various strategies may be em-ployed for providing for an expression cassette, which in the 5'-, 3'-direction of transcription has a tran-scriptional and translational initiation region, which may also include regulatory sequences allowing for the induction of regulation, the structural gene under the transcriptional and translational control of the initi-ation region, and a transcriptional and translational termination region.
Illustrative transcriptional initiation re-gions or promoters include, for bacteria, the ~-gal promoter, the TAC promoter, lambda left and right pro-moters, etc.; for yeast, glycolytic enzyme promoters, such as ADH-I and -II promoters, GPK promoter, and PGI
promoter, TRP promoter, etc.; for mammalian cells, SV40 early and late promoters, adenovirus major late promo-ters, etc. As already indicated, the expression cas-sette may be included within a replication system for episomal maintenance in an appropriate cellular host or may be provided without a replication system, where it may become integrated into the host genome. The DNA
may be introduced into the host in accordance with known techniques, such a~ transformation, using calcium phosphate-precipitated DNA, transfection by contacting the cells with the virus, microinjection of the DNA
into cells or the like.
Once the structural gene has been introduced into the appropriate host, the host may be grown and will express the structural gene. In some instances, it may be desirable to provide for a signal sequence (secretory leader) upstream from and in reading frame with the structural gene, which provides for secretion ~ . ~
029~

of the structural gene and cleavage of the secretory leader, so as to provide for the mature polypeptide in the supernatant. Where secretion is not provided for, then the host cells may be harvested, lysed in accor-dance with conventional conditions, and the desiredproduct isolated and purified in accordance with known techniques, such as chromatography, electrophoresis, solvent extraction, or the like.
The subject compounds can be used in a wide 0 variety of ways, both in vivo and in vitro. The sub-ject compounds can be used for making antibodies to the subject compounds, which may find use in vivo or in vitro. The antibodies can be prepared in conventional ways, either by using the subject polypeptide as an immunogen and injecting the polypeptide into a mamma-lian host, e.g. mouse, cow, goat, sheep, rabbit, etc., particularly with an adjuvant, e.g. complete Freunds adjuvant, aluminum hydroxide gel, or the like. The host may then be bled and-the blood employed for iso-lation of polyclonal antibodies, or in the case of themouse, the peripheral blood lmphocytes or splenic lym-phocytes (B-cells) employed for fusion with an appro-priate myeloma cell to immortalize the chromosomes for monoclonal expression of antibodies specific for the subject compounds.
Either polyclonal or monoclonal antibodies may be prepared, which may then be used for diagnosis or detection for the presence of the subject polypeptide in a sample, such as cells or a physiological fluid, 3~ e.g. blood. The antibodies may also be used in affini-ty chromatography for purifying the subject polypeptide and isolating it from natural or synthetic sources.
The antibodies may also find use in controlling the amount of the subject polypeptide associated with cells in culture or in vivo, whereby growth of the cells may be modified.

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1~ 102~6 The subject compound may be used as a ligand for detecting the presence of receQtors for tne subject compound. In this way, cells may be distinguished in accordance with the presence of and the density of re-ceptors for the subject compound, monitoring the effectof various compounds on the presence of such receptors.
The subject compound may be used in in vitro cultures to inhibit the growth of cells or cell lines sensitive to the subject polypeptide as distinguished 0 from cells which are not sensitive. Thus, heterogene-ous cell mixtures or cell lines can be freed of unde-sirable cells, where the undesirable cells are sensi-tive to the subject polypeptide. The subject polypep-tide may be administered in vivo in the case of neo-plastic conditions, for example, by injection, intra-lesionally, peritoneally, subcutaneously, or the like.
The subject compound may be used in vitro to eliminate malignant cells from marrow for autolosous marrow transpla~ts or to inhibit proliferation or eliminate .
malignant cells in other tissue, e.g. blood, prior to reinfusion.
The subject polypeptide may also be used in a method for treating wounds, such as cutaneous wounds, corneal wounds, and various other epithelial and stromal disruptions, such as chronic ulcers, burns, surgical incisions, traumatic wounds, and injuries to the hollow, epithelial-lined organs, such as the esoph-agus, stomach, large and small bowels, mouth, genital, and urinary tract. The method relies on the topical 3~ application of a treatment composition including Onco-statin M in a physiologically-accepable carrier.
The composition of the present invention may be used for treating a wide variety of wounds including substantially all cutaneous wounds, corneal wounds, and injuries to the epithelial-lined hollow organs of the body. Wounds suitable for treatment include those re-sulting from trauma such as burns, abrasions, cuts, and 1~ 10~6 the li'~e as well as from sur3ical procedures such as surgical incisions and skin grafting. Other conditions suitable for treatment with the compositions of the present invention include chronic conditions, such as chronic ulcers, diabetic ulcers, and other non-healing (trophic) conditions.
Oncostatin M may be incorporated in physio-103ically-acceptable carriers for application to the affected area. The nature of t'ne carriers may vary 0 widely and will depend on the intended location of application. For application to the skin, a cream or ointment base is usually preferred, suitable bases in-clude lanol'in, Silvadene (Marion) (particularly for the treatment of burns), Aquaphor ;Duke Laboratories, South Norwalk, Connecticut), and the like. If desired, it will be possible to incorporate Oncostatin M containing compositions in bandages and ot'ner wound dressings to provide for continuous exposure of the wound to the ~ peptide. Aerosol applications may also find use.
The concentration of polypeptide in the treat-ment composition is not critical. The polypeptide will be present in an epithelial cell proliferation-inducing amount. The compositions will be applied topically to the affected area, typically as eye drops to the eye or as creams, ointments or lotions to the skin. In the case of eyes, frequent treatment is desirable, usually being applied at intervals of ~ hours or less. On the skin, it is desirable to continually maintain the treatment composition on the affected area during the 3~ healing, with applications of the treatment composition from two to four times a day or more f~equently.
The amount employed of the subject polypeptide will vary with the manner of administration, the em-ployment of other active compounds, and the like, gen-erally being in the range of about lyg to lOOyg. Thesubject polypeptide may be employed with a physiolog-ically acceptable carrier, such as saline, phosphate-* Trade Mark B

buffered saline, or the like. The amount of compoundemployed will be determined empirically, based on the response of cells in vitro and response of experimental animals to the subject polypeptides or formulations containing the subject polypeptides. The subject com-pounds find use by themselves or in combination with other growth factors or inhibitors or immunomodulators, such as TNF, IL-2, Y-interferon, monoclonal antibodies, etc. The amounts of these other compounds will gener-ally be in the range of 1~g to 100~g. Conjugates ofthe subject compounds to site directing moieties, e.g., antibodies may be prepared, where the antibodies may be specific for particular malignant cells or organs.
The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL
Materials and Methods Oncostatin.M Isolated from U937 Cells Production of a Tumor Cell Growth Inhibitor From a Histiocyctic Lymphoma Cell Line U937 cells, a histiocyctic lymphoma cell line (Sundstrom and Nilsson, Int. J. Cancer (1976) 17:565-577), were cultured in 850cm2 roller bottles (Corning C2540) at a concentration of 4x105 cells/ml in a total volume of 300ml RPMI 1640 medium supplemented with lod fetal calf serum (FCS), penicillin/streptomycin (PS), L-glutamine and 1Ong/ml 12-0-tetradecanoylphorbol 13-acetate (TPA). Four days later, the; supernatants con-3~ taining the FCS and TPA were removed, the rollerbottles were washed five times with serum-free RPMI-1640, and the cells which detached (1x105 cells/ml) were washed 3 times with serum-free medium and added back to the bottles, resulting in a final volume of 125ml serum-free RPMI-1640 medium per roller bottle.
One day later, the supernatants were collected, centri-fuged to remove the cells, filtered through 0.45 micron (~) Nalgene filter and concentrated using a hollow fiber system (Amicon cartridge HIP10-20)~to a volume of 150ml (initial volume 1500ml). Oncostatin M was also isolated from supernatants of serum-free TPA-treated U937 cells in 150 CM2 tissue cultur~ flasks. The supernatant was concentrated with an Amicon Diaflo membrane PM-10,lOkD cut-off and dialyzed. Following dialysis, the concentrate was diluted with acetic acid resulting in a final concentration of 0.1~ acetic acid 0 in 500ml and concentrated to 50ml using an Amicon PM 10 filter. The 50ml concentrate was diluted to 400ml with 0.1N acetic acid and concentrated to 40ml with the same filter. The concentrate was diluted with 1N acetic acid and the resulting precipitate was removed by centrifugation. The resulting concentrate was frozen and lyophilized. The lyophilized material was used for purification steps.

Gel Permeation Chromatography A 3io-sil TSK-250 column (600x21.5mm) (Bio-Rad) was attached to a high pressure liquid chromato-graphic system. The crude fraction (10mg/ml) was dis-solved in 40% acetonitrile in 0.1~ aqueous trifluoro-acetic acid (0.1p TFA). A 2ml aliquot of the mixture was injected and elution was performed isocratically with a mobile phase of 40p acetonitrile in 0.1~ TFA.
The flow rate was 2.5ml/min and chart speed was set at 0.25cm/min. 5ml fractions were collected. The chroma-tography was performed at room temperature. An aliquot 3~ from each fraction was evaporated and assayed in trip-licate for growth inhibitory activity (GIA) of A375 cells.
The active fractions (Fractions 21 and 22) from six runs were pooled. The pooled material had a total of approximately 4.8x105 GIA units. The factor was found to have an apparent molecular weight of 18kD
as determined by size exclusion chromatography (Bio-Sil TS'~-250 column).
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. Reverse Phase High Pressure Chromatography (~PLC) ~f TSK-250 Fractions Pooled TSK-250 fractions 21 and 22 described above were diluted two-fold with O.lq TF.4. This mix-ture was injected isocratically on a ~-Bondapak-C18 *
column (7.8x300mm) (referred to as C181) at room temperature. The flow rate was set at 2.0 ml/min and the chart speed was 0.25 cm/min. The linear gradient was used between primary solvent 0.1d TFA and the sec-ondary solvent acetonitrile-TFA 0.1~. Tne gradient conditions were 0-30p in 20min; then 30-45~ in 150min;
45_55d in 20min; and 55-lOOp in lOmin. All solvents were HPLC grade. 4ml fractions were collected and aliquots of each fraction were assayed for growth inhiDitory activity. Fractions 72-75 were found to contain the majority of activity. The a~tive fractions eluted between 41-52p of acetonitrile concentration.
Fractions 72-75 were pooled. 16ml of 0.1~ TFA
was zdded to the pooled fractions. The mixture was in-jected into a ~-Bondap2k-Ct8 column (3.9x300mm) (re-ferred to as C182) at room temperature. The flow rate was set a lml/min and chart speed was 0.25cm/min. The gradient conditions were 0-35~ in lOmin; 35_45d in lOOmin; and 45-100~ in lOmin. Fractions were collected and aliquots wer~ taken and assayed for GIA. Most of the activity emer8ed from the column getween 40.7 to ~1.3~ acetonitrile concentration (retention time 83-86min).
Active fractions were pooled 2nd diluted two-fold with O.ld TFA and injected isocratically on a ~-Bondapa~-C18~column (3.9x300mm) (referred to as C?83) at room temperature. The flow rate was 1ml/min and ch2rt speed was 0.25 cm/min. A linear gradient was used betweem ?rim2ry solvent 0.1~ TFA 2nd the secondary solvent n-propanol-TFA (0.1~). The gr2dient conditions were 0-23d in 20min and 23-35~o in 120min. Fr2ctions were collected 2nd aliquots of each fr2ction were * Trade Mark 4 ~-.. . . ~, . . . . .

, 9 assayed for GIA. Most of the activity appeared between 25-26.5% propanol concentration (retention time 59min).
This apparently homogeneous fraction contained approxi-mately 300ng protein and about 40,000 GIA units.

Cell Growth Modulatory Assay Using , 3H-Thymidine Incorporation into DNA (GIA) The assays were Derformed in 96 flat well plates (Costar 3596). Human melanoma cells (A375) were used as a sensitive indicator cell line. Cells (3x103) in 0.1ml Dulbecco's modified Eagles medium (DMEM) sup-plemented with 10p FCS and PS were placed in each well.
Three hours later, 0.1ml of test samples was added to each well. Plates were incubated at 37~C for 3 days.
Then 0.025ml (0.5~Ci) of a solution of 3H-thymidine (specific activity 27~Ci/~g) was added to each well for the final 6 hours of incubation. The cells were then transferred to glass filter strips by using a multiwell harvester (PHD Cell Harvester, Cambridge Technology, Inc.). The filters were transferred to scintillation vials to which were added 2ml of scintillation fluid (ScientiVerse II,* Fisher Scientific Co.) prior to counting in a scintillation counter for quantitating 3H-thymidine incorporation.

Soft Agar Colony Inhibition Assay (TGI) A 0.5ml base layer of 0.5p agar (Agar Noble;
Difco Laboratories, Detroit, Michigan) in DMEM contain-ing 10p fetal calf serum (FCS) was added to 24 well 3~ Costar tissue culture plates. 0.5ml 0.3p agar contain-ing the same medium-FCS mixture, 1-2.5x103 A375 cells and the factor to be tested at various concentrations were overlaid on the base layer of agar. The plates were incubated at 37~C in a humidified atmosphere of 5%
C~2 in air and refed after 7 days by addition of 0.5ml of 0.3% agar containing the same medium and concentra-tions of factor. Colonies were enumerated unfixed and * Trademark F

.. . . . . . ...

~.3~0296 unstained and the number of colonies 3reater than 6 cells were scored between days 7 and 14.

Results Sequences of Oncostatin M Isolated from U937 Cells The N-terminal sequence and internal fragments of Oncostatin M were determined by microsequence analysis of the reduced and S-pyridinethylated poly-peptide and of peptides obtained from enzymatic digests of reduced and S-pyridinethylated Oncostatin M with the endoproteinase Lys-C and Staphylococcus aureus V8 protease. The peptide fragments were purified by reverse phase HPLC, using volatile solvents. The peptides were subjected to automated repetitive Edman degradation in the Model 470A protein sequencer (Applied Biosystems, Inc.). The phenylthiohydantoin amino acids were analyzed by reverse phase HPLC
(Applied Biosystems, Inc.) with a PTH-C18 column (2.1x220mm, ABI), usins a sodium acetate buffer/t2tra-hydrofuran/acetonitrile gradient for elution.
The resultant amino acid sequences aresubstantially as illustrated in Figure 1.
A comparison of these sequences wit,h those stored in the curent protein data base (PIR Release g.o, May 1986), revealed no significant sequence homol-ogies with any other known sequence. In addition, there is no homology with tumor necrosis factor, lymph-otoxin, colony stimulating factor, interleukin 1 or 2 or ~-transforming growth factor.

Inhibition of Proliferation of Tumor Cells and Augmen-tation of Proliferation of Normal Human Fibroblasts Employing the soft agar colony inhibition assay described above, the following results were obtained:

*Trade-mark ~-' Table l 13 ~0296 Inhibition of A375 Melanoma Cell Colony Formation in Soft Agar by Purified Oncostatin X
Isolated from U937 Calls*
% Inhibition of GIA UNits/Well% of Colonies Colony Formation ';A375 cells were plated in soft agar, with or without factor in a final volume of 2ml, as described above. The factor used was from a Cl8 propanol column fraction with peak tumor growth inhibitory activity (GIA). Eleven days later the number of colonies were enumerated. A colony was defined as a cluster of at least six cells. One GIA unit was defined as the amount to cause 50% inhibition of H-thymidine incorporation into A375 cells in micro wells as detailed above.

Table 2 Effects of Various Treatments of Supernatants of TPA-Induced U937 Cells on Tumor Growth Inhibitory Activity '~
Final Dilution of Supernatant 1:4 1:8 1:16 Media Control 39,780 Untreated Super 7,206-';*13,896 16,000 IN Acetic Acid 6,670 17,07318,783 INH40H 6,956 15,01613,923 -'-U937 cells were treated with TPA (lOng/ml) for 3 days and then the cells were washed with media and incubated for 24 hr in serum-free media before collecting the supernatants. The supernatants were treated with lH acetic acid or lN ammonium hydroxide (NH40H). They were then displayed against medium and tested for their ability to inhibit H-thymidine incorporation into A375 cells. A375 cells were labeled with H-thymidine (3H-TdR) for the last 6 hr of a 3-day incubation.

~ ;-';Data shown are H-TdR incorporation counts per minute.

*Trade-mark - 21 -.. . ,, . .. _ . . . . . .

.~3 102~6 The above results demonstrate that Onco-statin M in the dialyzed supernatant is substantially resistant to inactivation by one normal acetic acid and one normal ammonium hydroxide. Thus, the subject com-pounds are relatively insensitive to both moderatelystrong acid and moderately strong base. The subject compound is stable to heat treatment at 56~C. for 1 hour but not to 90~C. for 30 minutes.
The subject compound was also tested for heat stability and was found to retain its activity after exposure at 56~C for lhr, but to lose substantially all its activity after exposure to 95~C for 30min.
In the next study, the ability of the subject polypeptides to inhibit tumor cell re~lication of a variety of neoplastic cells was investigated. The fol-lowing table indicates the results.

Table 3. Inhibition of Replication of Tumor Cells by Purified Oncostatin M from U937 Cells Units GIA to Cause 30% Inhibition Tumor Cells 3H-TdR Incorporation A549 (lung cancer) 21 HTB10 (neuroblastoma) 81 A375 (melanoma) 0.3 Tumor cells were plated in microwells 3hr prior to the addi-tion of various dilutions of Oncostatin M, purified by re-verse phase HPLC as detailed above. For the final 6hr of a 3-day incubation, cells in 0.2ml medium were labeled with 3H-thymidine (3H-TdR) (0.5~Ci/well). One unit tumor growth inhibitory activity (GIA) is defined in the legend to Table 1 as that amount which causes 50~ inhibition of 3H-TdR
incorporation into A375 melanoma cells. One unit was determined to be approximately 10pg of purified protein, therefore the concentration (ng/ml) to cause 30% inhibition 3H-TdR into A549, HTB10 and A375 cells was approximately 1.4, 4.0, and .015ng/ml, respectively. 3H-TdR incorporation into WI26 normal human fibroblasts was not suppressed by U937 factor in any experiment.

102g6 The above results demonstrate that Onco-statin M is selective in its ability to inhibit repli-cation, having widely varying effects depending upon the nature of the cell. The subject compound is effec-tive against melanoma cells, such as A375 melanomacells, squamous lung cancer cells, such as A549, and neuroblastoma cells, such as HTB10.

Tumor cells were seeded at 3x103 cells/well 10 and normal fibroblasts at 1.5x103 cells/well in 96 well pIates for 3 hours. Various concentrations of purified Oncostatin M, obtained from the fraction from the C183 column with peak antiproliferative activity against A375 cells were added and three days later 3H-thymidine incorporation into cells was measured in triplicate wells at each concentration. The results are shown in Table 4.

3o ...

~34~296 Table 4. Inhibition of Proliferation of Tumor Cells and Aug-mentation of Proliferation of Normal Fibroblasts by Oncostatin M
GIA
units/well % inhibition % stimulation Exp. 1 16 ~3 25 Exp. 2 27 NT 28 46 Exp. 3 75 ~9 30 Exp. 4 20 ~7 44 Results shown are ~ inhibition or % stimulation of 3H-thy-midine incorporated into tumor cells (A375, HTB10, A549, and SK-MEL28) and normal fibroblasts (WI26 and WI38~, respec- -tively. One GIA unit is defined in the legend to Table 1 as the amount of Oncostatin M that causes 50% inhibition of 3H-thymidine incorporation into A375 celIs.

In addition to the observed differential ef-fect on 3H-thymidine incorporation into tumor cells and normal human fibroblasts, there is also an observed differential effect of morphology and cell number fol-lowing 3 days of treatment of the two cell types with Oncostatin M as shown in Figure 2.
The Oncostatin M used was from the HPLC-C183 3o column fraction with peak activity for inhibiting pro-liferation of A375 cells. Figure 2 is a series of photomicrographs of A375 melanoma cells that were un-treated (A), treated with 5 growth inhibitory activity (GIA) units of Oncostatin M (B), or 100 units (C).
Photomicrographs of WI38 fibroblasts that were un-treated (D), treated with 5 units GIA (E), or 100 units .. ~

..... .

~ ~402~6 (F). The cells were stained with crystal violet in 0.5~ methanol. Magnification = 63X.

NaDodSO~/PAGE of Oncostatin M
Purified Oncostatin M, subjected to NaDodS04 performed under reducing conditions, was found to have an apparent molecular weight of approximately 28,kD as shown in Figure 3. The following proteins were used as standards (lane A): ovalbumin, Mr = 43 kD chymotryp-sinogen a, Mr = 25.7 kD; lactoglobulin ~, Mr = 18.4 kD;
lysozyme, Mr - 14.2 kD; bovine trypsin inhibitor = 6.2 kD; insulin A and B chain, Mr = 2.3 kD and 3.4 kD re-spectively. Oncostatin M was applied to lane a.
Oncostatin M, subjected to PAGE under non-reducing conditions, also has an apparent molecular weight of 28 kD and protein electroeluted from the band was found to inhibit proliferation of A375 cells.

Antibody to a Synthetic Peptide of Oncostatin M Reacts in 125I-labeled Oncostatin M in Radioimmune Precipitations a) Peptide Synthesis: The peptide corresponds to residues 6-19 of the Oncostatin M protein and was syn-thesized by solid phase techniques on a Beckman auto-mated instrument as described (Gentry et al., J. Biol.
Chem. (1983) 258:11219). The peptide was cleaved fromthe resin using the "low-high" HF procedure (Tam et al., J. Amer. Chem. Soc. (1983) 105:6442-6445).
Purification was accomplished by preparative HPLC.
b) Production of Antibodies: The peptide was coupled to bovine Y-globulin as described (Gentry and Lawton, Virology (1986) 152:421-431). New Zealand white rabbits were primed and boosted (5 times) at 4 sites subcutaneously as described (Gentry and Lawton, Virology (1986) 152:421-431). Antisera used were ob-tained 2 weeks after the fifth boost.
-~' 3~0~96 c) Iodination of Oncostatin M: A sample of par-tially purified Oncostatin M was radiolabeled with iodine-125 using published procedures (Linsley et al., PNAS (1985) 82:356-350). An aliquot of the labeled preparation containing 100,000 cpm was mixed ~ith rabbit antiserum directed against the N-terminal 17 amino acids of Oncostatin M (final dilution of 1:20), in the absence or presence of the N-terminal peptide (the N-terminal 17 amino acids of Oncostatin M) (2 ~g) and subjected to immune precipitation analysis as described (Linsley et al., Biochemistry (1986) 25:2978-2986.
Specifically, one tube containing 5 ~l was preincubated with 2 ~g of the N-terminal peptide in 10 Ml TNEN (20mM Tris pH 7.5, 5mM EDTA, 150mM NaCl, 0.05p Nonidet P-40~ containing 0.1~ BSA for 30 minutes at 4~C
prior to tne addition of I125 Oncostatin M in 851 TNEN
con~aining 0.1~ BSA and 40 mM dithiothreitol (DTT).
Seven tubes containing 5 ~l antisera were incubated with I125 Oncostatin M in 85 ~l TNEN containing 0.1%
BSA and 40 mM DTT for 30 minutes at 4~C prior to the addition of 50 yl of 10p formalin-fixed Staphylococcus aureus (Pansorbin,* Calbiochem).
Following an additional incubation for 30 min.
at 4~C, the tubes ~ere microfuged and the pellets were washed 4 times with 1 ml TNEN prior to subjecting them to PAGE analysis. A diffuse band of Mr ~ 32 kD was ob-served after SDS/PAGE analysis of the immune precipi-tates. Precipitation of this species was inhibited by the inclusion of an excess of unlabeled peptide corres-ponding to the N-terminal 17 amino acids of Onco-statin M, indicating that the precipitation was spe-cific for this peptide.
The carbohydrate composition of Oncostatin M
was examined by testing for glycosidase sensitivity.
Immune precipitates prepared as in c above were treated with buffer, endoglycosidase H, or neuraminidase, as * Trademark F

.. ....

0~96 described by Linsley et al. (1986). Treatment with endoglycosidase H, an enzyme with specificity for N-linked high mannose oligosaccharides resulted in the appearance of a lower molecular weight s~ecies of ~r =
24 kD. Only a portion of the radiolabeled material ~as sensitive to this enzyme, indicating that not all mole-cules contained high mannose oligosaccharides. Treat-ment with neuraminidase resulted in the appearance of a sin~le band of Mr = 27 kD, indicating that the hetero-geneity in size of untreated 125I-labeled Oncostatin ~
was due to molecular heterogeneity in sialyation of the glycoprotein core. The results indicated that active preparations of Oncostatin M contained a mixture of high mannose and complex N-linked oligosaccharide side chains.

Oncostatin M Isolated from Normal Human Peripneral Blood Lymphocytes (P~L) Production of a Tumor Cell Growth Inhibitor from PBL's Leucofractions containing P3L's, obtained from the Blood Bank, were diluted 1:1 with phosphate buf-fered saline, pH 7.4 (PBS). Thirty five ml of diluted blood were underlayed with 10 ml of a solution con-sisting of 9~ Ficoll containing 20d by volume of 50p sodium diatrizoate (final specific gravity of 1.080).
The gradients were centrifuged at room temperature for 20 minutes at 850 x g. Cells were collected from the gradient interface and washed with PBS. Red blood cells were shoc'~-lysed for 3-4 minutes with 10-20 ml of a solution containing 0.8p ammonium chloride and 0.1p Na4-EDTA.
Cells were collected from red blood cell lysing solution by centrifugation at 600 x g for 10 ~inutes 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 U/ml. The cell suspension was agitated for 5 minutes at 37~C and the B~ * Trade Mark , 10296 platelet aggregate allowed to settle for 5 minutes.
The suspended cells were transferred to new tubes, re-covered by centrifugation, resuspended in 1 ml fetal bovine serum, and tr~nsferred into a column containin3 0.5 g of brushed, prewetted nylon wool, type 200 (Fenwal).
The nylon wool column was incubated at 37~C
for 60 minutes to allow attachment of monocytes and B
lymphocytes. The column was th-en washed with 3 x vol-umes of RPMI 1640 medium (37~C) containing 5~ fetalbovine serum and tne eluted nonadherent cells (~BL) collected.
PBL (2 x lo6 cells/ml) were cultured at 37~C, 5% C02-95P air for 96 hours in RPMI-1640 medium (10.4g/l) containin3 FeS04 7H20 (1 mg/l), ZnS04 7H20 (2 mg/l) Na2SeO3-5H20 (0.017 mg/l), 1-aminoethanol (1 mg/l) human transferrin (5 mg/l), bovine serum albumin-linoleic acid conjugate (Sigma) (200 mg/l), L-glutamins (300 mg/l), penicillin/streptomycin (100,000 units/l) and phytohemagglutinin-P (Wellcome) (2 mg/l). The supernatants were collected, centrifuged to remove the cells, concentrated by ultrafiltr~tion (Amicon Diaflo membrane YM-10, 10 Kd cut-off), and dialyzed against 0.1 M acetic acid (Spectrapore 3*dialy~is tubing). The clarified retentate was lyophilized.

Gel Permeation Chromatosraphy The crude fraction was reconstituted in 20 ml of 1 M acetic acid (50 mg/ml) and applied to a BioGel*
3~ P-100 column (2.6 x 88 cm) equilibrated with 1 M acetic acid at a flow rate of 0.5 ml/min. Twelve ml fractlons were collected. An aliquot from each fraction was evaporated and assayed in triplicate for growth inhibi-tory activity (GIA) of A375 cells. The active frac-tions were pooled, lyophilized, and rechromatographed on a Bio-Sil TSK-250 column (600 x 21.5 mm), as described.
.
* Trademark . . , - .. , . ~ ...... . .

Iq29~

Reverse Phase High Pressure Chromatography of TS~-250 Fractions:
The final purification of pooled TSK-250 fractions was achieved by reverse phase HPLC essen-tially as described. PBL-derived tumor cell inhibitor eluted from ~Bondapak C18 support at 40-41% ace-tonitrile and at 26.5% n-propanol concentrations, respectively.

Cell Growth Modulatory Assay Using 125I-Iodo-deoxyuridine Incorporation into DNA (GIA):
These assays were performed in flat-bottomed 96-well tissue culture trays (Costar 3596). Human melanoma cells, A375 (4x103), in 50 ~l of test sample was added to each well and incubated for 3 days at 37~C. Cells were labeled for 24 hours with 125I-IdU
(0.05 ~Ci/well) and incubated an additional 24 hours.
Cells were washed three times, ha~vested with a multiple sample harvester, and radioactivity was counted in a gamma counter.

Results:
One preparation of PBL-derived tumor cell inhibitor was subjected to automated repetitive Edman degradation. The aminoterminal amino acid sequence is as follows:

A-A-I-G-X-X-X-K-E-Y-X-V-L-X-X-Q-L-Q-K
X represents an amino acid that has not been 3~ identified.
A comparison of this sequence with that of U937 factor clearly indicates indentity with the 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-Q-K

.

~ 3~0296 3o In the next study, the ability of PBL derived Oncostatin M to effect replication of a variety of cells was investigated. It was found that mouse L929 cells were insenstive to PBL-derived Oncostatin M using up to 1000 GIA units/ml. Human fibroblasts, WI26, were stimulated to grow by treatment with 1000 GIA
units/ml. Normal human T-lymphocyte proliferation at 72 hours post mitogenesis was not affected by up to 500 GIA units/ml.
It is evident from the above results, that a novel polypeptide and polypeptide fragments are provided, which can be used for modulating cellular growth. The compound is found to have varying activity depending upon the nature of the cell line involved, so that it may be used by itself or in conjunction with other compounds in modulating cellular srowth. The subject polypeptides therefore add an additional poly-peptide which may be used with mixtures of cells, both in vivo and in-vitro, to selectively reduce or enhance cellular proliferation of a particular type of cell.
In particular, the factor can be used to treat cells for autologous bone marrow transplants. Use of the factor inhibits the growth of tumor cells in the marrow and may stimulate colony cell formation.
Oncostatin M may also be used to stimulate growth of epithelial cells thereby promoting wound healing. In addition, the intact polypeptide or fragments thereof can be used as immunogens to induce antibody forma-tion. The induced antibodies find use to titer 3~ Oncostatin M present in a bodily fluid or to modulate the activity of the factor by binding to it. Further, those antibodies together with purified Oncostatin M or fragments thereof serve as a component of diagnostic kits in conjunction with other reagents, particularly antibodies to detect and quantitate Oncostatin M.

~ 3402~6 Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modificatlons may be practiced within the scope of the appended claims.

3o

Claims (14)

1. A polypeptide compound obtained from leukocytes, comprising a component, said component being a polypeptide cell growth regulatory factor, being characterized as capable of inhibiting proliferation of neoplastic cells, stimulating proliferation of normal human fibroblasts, not inhibiting proliferative and cytotoxic human T cell responses and not inhibiting granulocytic/myelocytic bone marrow colony cell formation, having a molecular weight of about 17 to 19 kD as determined by gel exclusion chromatography and about 28 kD as determined by SDS-PAGE, and being relatively insensitive to moderate acid and base and moderately elevated temperatures; and being at a purity providing a specific activity of at least about 10 GIA units/ng protein; and the component comprising amino acid sequences corresponding to one of the sequences depicted below:

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

T-D-L-M-Q-D-T-S-T-L-L-T-P-Y-I

Q-R-L-P-K-A-Q-D-L-E-R-S-G-L-N-I-E-D-L-E-K

L-R-E-H-C-R-E-R-P-G-A-F-P-S-E-Q-Q-L-I-G
with no more than three amino acid differences between the depicted sequence and said corresponding sequence, wherein the differences comprise conservative substitutions, insertions and deletions but not comprising any of the depicted sequences which are the same as a part of a sequence of Oncostatin M.

2. The polypeptide compound of claim 1, wherein said leukocytes are mitogen-activated normal human peripheral blood lymphocytes.

3. The polypeptide compound of claim 2, wherein said mitogen is phytohemagglutinin.

4. The polypeptide compound of claim 1, wherein said leukocytes are phorbol diester-induced human histiocytic lymphoma cells.

5. The polypeptide compound of claim 1, substantially free of cellular components.

6. The polypeptide compound of claim 1, wherein the specific activity of said component is at least about 100 GIA
units/ng protein.

7. A polypeptide cell growth regulatory factor substantially free of cellular debris and other leukocytic proteins, said polypeptide cell growth regulatory factor being capable of inhibiting proliferation of neoplastic cells, stimulating proliferation of normal human fibroblasts, not inhibiting proliferative and cytotoxic human T cell responses and not inhibiting granulocytic/myelocytic bone marrow colony cell formation, having a molecular weight of about 17 to 19 kD
as determined by gel exclusion chromatography and about 28 kD
as determined by SDS-PAGE, and being relatively insensitive to moderate acid and base and moderately elevated temperatures and comprising amino acid sequences corresponding to one of the sequences depicted below:

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

T-D-L-M-Q-D-T-S-T-L-L-T-P-Y-I

Q-R-L-P-K-A-Q-D-L-E-R-S-G-L-N-I-E-D-L-E-K

L-R-E-H-C-R-E-R-P-G-A-F-P-S-E-Q-Q-L-I-G

with no more than three amino acid differences between the depicted sequence and said corresponding sequence, wherein the differences comprise conservative substitutions, insertions and deletions but not comprising any of the depicted sequences which are the same as a part of a sequence of Oncostatin M.

8. A peptide containing an amino acid sequence having at least ten consecutive amino acids that correspond to an amino acid sequence depicted below:

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

T-D-L-M-Q-D-T-S-T-L-L-T-P-Y-I

Q-R-L-P-K-A-Q-D-L-E-R-S-G-L-N-I-E-D-L-E-K

L-R-E-H-C-R-E-R-P-G-A-F-P-S-E-Q-Q-L-I-G

with no more than three amino acid differences between the depicted sequence and said corresponding sequence, wherein the differences comprise conservative substitutions, insertions and deletions but not comprising any of the depicted sequences which are the same as a part of a sequence of Oncostatin M.

9. A method for inhibiting the proliferation of neoplastic cells in vitro, which comprises contacting said cells with a proliferation inhibiting amount of a peptide compound according to claim 8.

10. Polyclonal antibodies specific for a polypeptide according to claim 1 including wherein said amino acid sequences of claim 1 comprise any of the depicted sequences which are the same as a part of a sequence of Oncostatin M.

11. A method for detecting the presence of a polypeptide according to claim 1, which comprises:
combining a sample suspected of containing said polypeptide with a polyclonal antibody according to claim 10;
and determining the amount of immune complex formation with said polyclonal antibody.

12. A method for detecting the presence of a peptide according to claim 8, which comprises:
combining a sample suspected of containing said peptide with a polyclonal antibody according to claim 10; and determining the amount of immune complex formation with said polyclonal antibody.

13. A diagnostic kit comprising a polyclonal antibody of claim 10 and at least one peptide of claim 8 or polypeptide of claim 1, including wherein said amino acid sequences of claims 8 and 1 comprise any of the depicted sequences which are the same as a part of Oncostatin M, wherein the specific activity of said polypeptide is at least about 100 GIA units/ng protein.

14. A diagnostic kit comprising a polyclonal antibody of claim 10 or a polypeptide obtained from a composition of claim 1.