NL8603209A - NEW CELL GROWTH REGULATING FACTOR. - Google Patents

Description

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New cell growth regulating factor.

The invention relates to a new poly (amino acid) and and a method of obtaining it with at least eight amino acids which is immunologically cross-reactive with a compound, which compound is a polypeptide obtainable from leukocytes capable of inhibit the proliferation of neoplastic cells, stimulate the expansion of normal human fibroblasts, do not inhibit human proliferative cytotoxic T cell responses, nor inhibit granulocytic / myelocytic bone marrow colony cell formation, methods of inhibiting proliferation of neoplastic cells and promoting wound genes -10 sing, for the polypeptide specific antibodies, a DNA sequence encoding the polypeptide as well as diagnostic agents.

Leukocytes, both lymphocytes and monocytes, are involved in the inhibition of tumor growth in various animal tumor models. The increased incidence of malignant tumors in people with impaired immunosystem 15 supports the claim that white blood cells play a role in the regulation of neoplastic growth. Protein factors, which are produced by these white cells and which inhibit tumor growth or modulate immune functions, have already been isolated and characterized, and include the interferons, α and γ, tumor necrosis factor, lymphotoxin, interleukin-2 and other lymphokienes.

Since each factor isolated has a different spectrum of activities and will interact differently with other factors, there remains an ongoing strong interest in the isolation and characterization of all factors producing white cells in the modulation of cell growth or immune functions. These compounds may find use individually or together in the treatment or diagnosis of cancer, as wound healing promoters or as immunomodulators for the treatment of patients with immunodeficiency, autoimmunity, organ transplants, and the like.

Several difficulties are encountered in discovering, isolating, purifying or characterizing naturally occurring factors.

Methodologies should be developed to separate and purify an important factor from other factors in the raw starting material without denaturing the activity of the desired factor;

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ft c ΰ i ö 9 T f -2- Do biological analyzes need to be developed that allow the fractions to be identified during the separations with which a particular factor is concentrated? a new factor must be distinguished from other factors already known or other unknown factors which may be present and which either negatively or positively affect the activity of the target factor; the purified factor must be characterized and the purified factor must be concentrated in sufficient quantity to allow factor identification and characterization.

10 With the increasing number of factors already isolated, it is becoming increasingly difficult to identify each factor, as its role and function may be overshadowed by the numerous other factors present.

Description of the Related Art Beal et al., Cancer Biochem. Biophys. (1979) 3 ^ 93-96 report the presence of peptides in human urine, which inhibits growth and DNA synthesis in transformed cells more than in normal cells.

Holley et., Proc. Natl. Acad. Sci. (1980) 2Z! 5989-5992 describe the purification of epithelial cell growth inhibitors.

Letansky, Bisoci. Rep. (1982) 2: 39-45 report that purified peptides from bovine placenta inhibit tumor growth and thymidine incorporation into DNA to a greater extent in neoplasms than in normal cells.

Chen, Trends Biochem. Sci. (1982) 25364-365 discloses the isolation of a peptide from ascites fluid with cancer-suppressing properties.

Rescue and Schally, Proc. Natl. Acad. Sci. (1982) 79: 7014-7018 report the isolation of purified peptide (s) from porcine hypothalamus showing antimitogenic activity against various normal and tumor cell lines.

Sone et al., Gann (1984) 75: 920-928 report the production of a factor (s) produced by human macrophages and inhibiting the growth of certain tumor cells in vitro.

Ransom et al., Cancer Res. (1985) 45: 851-862 report the isolation of a factor, called leukoregulin, which inhibits the replication of certain tumor cell lines and is distinguished from lymphytoxin, interferon and interleukins 1 and 2. Most of these factors are not sufficient. > Λ y π ^ v i. y? 5 * 3-characterized; neither are their primary structures known.

Aggarwal et al., J. Biol. Chem. (1984) 259: 686-691 purified and characterized human lymphotoxin (LT) produced by a lympho-blastoid cell line and then determined the LT sequence (Aggarwal et al.

5 J. Biol. Chem. (1985) 260: 2334. Gamma interferon (γ-IF) produced by lymphoid cells and having an immunomodulatory and tumor inhibitory activity has been cloned and expressed. (Gray et al., Nature (1982) 295: 503-508). Tumor necrosis factor (TNF), which inhibits the growth of certain tumors and is produced by macrophages and has characterized the certain leukemia cell lines, cloning the TNFcDNA and expressing it in E. Coli (Pennica et al., Nature (1984) 312: 724).

Summary of the invention

A new peptide factor and biologically active fragments thereof are provided, which factor is available from leukocytes.

The factor is useful for modulating cell growth, such as inhibiting tumor cell growth and stimulating the growth of normal fibroblasts, and modulating immune functions. The factor has an amino acid sequence, which is clearly different from the sequences of other compounds, which have been reported to have analogous properties.

Brief description of the drawings

Fig. 1 represents the amino acid sequence of fragments of Oncostatin M; Figure 2 is a series of micrographs of cells treated with Oncostatin M, wherein (A-C) A375 are melanoma cells treated with 0.5 and 100 GIA units, respectively, and (D-F) WI38 are fibroblasts treated with. 0.5 and 100 GIA units, while Figure 3 is a photograph of an SDS-PAGE analysis of Oncostatin M.

A novel polypeptide, polypeptide preparations, polypeptide fragments and mutations, their preparation and use are provided, wherein the compositions demonstrate activity in the modulation of cell growth, in particular inhibiting tumor cell growth and stimulating growth of normal fibroblasts. This polypeptide, referred to as Oncostatin M, is available from leukocytes; for example, from conditioned media of stimulated D937 cells or conditioned media of stimulated normal human peripheral blood lymphocytes (PBL).

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Fragments and mutants of the polypeptide with the biological activity of the intact Oncostatin M, such as cell growth modulation activity or immunological activity, are also provided.

The polypeptide fragments of this invention are novel poly-5 peptides with at least eight amino acids that are biologically active in that they are at least immunologically cross-active with the naturally occurring Oncostatin M. Immunological cross-reaction means an antibody. induced by a novel polypeptide of this invention will cross-react with intact Oncostatin M, at least when Oncostatin M is in a denatured state. These polypeptides are therefore suitable for inducing antibodies to Oncostatin M which are useful for determining the concentration of Oncostatin M in a body fluid, to bind Oncostatin M and thus modulate its activity and to purify Oncostatin M , such as by use in an affinity column.

Some of the polypeptides may also retain the cell growth modulating activity of intact Oncostatin M, although that activity can be modified, usually reduced compared to the intact Oncostatin M.

Fig. 1 represents the amino acid sequence of poly (amino acids) which are cross-reactive with Oncostatin M, the first sequence representing the N-terminal portion of Oncostatin M '. Poly (amino acids) according to the invention contain an amino acid sequence with at least 8 consecutive amino acids corresponding to the amino acid sequence represented in Fig. 1 and which differ from that sequence by no more than 3, usually no more than 1 amino acid. That difference can be either the insertion of an amino acid, the deletion of one amino acid and the substitution of one amino acid by another, in particular a conservative substitution. Usually, the poly (amino acid) will contain at least 10, usually at least 12, contiguous amino acids, corresponding to sequences shown in the figures, and differing by no more than 1 amino acid. For the purposes of the present invention, the different amino acids will be classified into a number of subclasses. The following table indicates the subclasses: 8603203 i i -5- aliphatic neutral

non-polar G A P V L I

polar S T C M N Q

5 acid D E

basic K R

aromatic F Η Y W

Conservative substitutions mean that amino acids from the same subclass (ie, either neutral aliphatic, acid aliphatic, basic aliphatic or aromatic), more particularly are substituted for one another with the same polarity- Amino acids with 2-4 carbon atoms or 5-6 carbon atoms will preferably fix monomer groupings in the aliphatic subclass.

The poly (amino acids) are no longer than 1000 amino acids. Usually they have less than 100 amino acids, but usually less than 50.

Thus, the poly (amino acid) can be easily synthesized. Usually, the poly (amino acids) when they are more than 100 amino acids in length may be polymers of fragments of Oncostatin M each less than 100 amino acids, or fusion proteins, in which the fragment is fused to an anti-20 enzyme enzyme fragments. In particular, the higher molecular poly (amino acids) at least one polypeptide fragment of less than about 100 amino acids can be covalently joined together to form a large immunogenic polypeptide carrier, providing immunogenicity. Examples of such protein carriers are bovine serum albumin, keyhole limpet hemocyanin (KLH), and the like. Such conjugated polypeptides are useful for inducing antibodies in a suitable host organism.

0937 cells are a cell line derived from a histiocytic lymphoma cell line (Sundstrom and Nilsson, Int. J. Cancer (1976) 17: 565-577) that can be induced to differentiate into cells with macrophage properties after treatment with a variety of agents (Harris et al., Cancer Res. (1985) 45: 9-13). For the production of Oncostatin M, the 0937 cells can be cultured in a conventional nutrient medium with serum and treated with a suitable inducer.

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Mostly, formalgols or ingenoles can be used, especially 12-o-tetradecanoylforbol-13-acetate (ΤΡΑ). Usually about 5-20 ng / ml of the inducer can be used. The starting number of cells is 5-6 approximately 10 -10 cells / ml.

After allowing the cells to be treated with the inducer for a sufficient time, generally 3-6 days, the top layer is removed, the cells are washed with serum-free nutrient medium, adhered cells are washed again with serum-free medium and the cells are incubated for at least 12 hours, usually no more than 48 hours, in serum-free food medium, for example RPMI-1640 medium. The top layers are then collected and the cells removed by centrifugation. Cell-free supernatants were examined for cell growth inhibitory activity (GIA) as described in the experimental review. The top layer contains about 50-500 units of GIA / ml (see experimental for definition of GIA units).

Oncostatin M can also be obtained from mitogen-stimulated normal human peripheral blood lymphocytes (PBLs). PBLs can be isolated from leukofractions by diluting the fractions and centrifuging them on Ficoll gradients. Cells from the gradient interface 20 are collected, washed and shock-lysed to remove red blood cells. The remaining cells are collected from that solution by centrifugation, resuspended in buffer containing serum and thrombin, stirred and the platelet aggregate allowed to settle for a short period of time. The suspended cells are transported, recovered by centrifugation, resuspended in serum and transported to a column containing nylon wool. The column is incubated to allow confirmation of monocytes and B lymphocytes and then washed. Most peripheral blood T lymphocytes will not stick and are eluted from the column. Said cells were grown at 37 ° C in a culture medium, for example RPMI-1640 medium and treated with a suitable inducer, for example phytohemagglutinin (about 1-5 mg / l) for about 100 hours, after which the supernatants were collected. The top layers were centrifuged to remove cells and concentrated by ultrafiltration or dialysis. After isolation of the cell-free top layer from either U937 cells or normal PBLs, the conditioned medium is concentrated, suitably 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 about tenfold concentration, repeating the dilution and concentration. The concentrate can be lyophilized and used directly or the lyophilized product can be used for further purification. The present Oncostatin M can be purified by a gel permeation chromatography procedure using aqueous 40% acetonitrile-0.1% trifluoroacetic acid as an isocreatic mobile phase on a Bio-Sil TSK250 column, the activity for each of the political groups are monitored. The purification yields a 4 composition with at least about 0.5-5x10 GIA units / ml in the active fractions. The partially purified product from the gel permeation chromatography can be further purified using reverse phase high pressure liquid chromatography using a linear gradient, the primary solvent being 0.1% trifluoroacetic acid in water and the secondary solvent acetonitrile containing Is 0.1% trifluoroacetic acid. The schedule can be changed, usually requiring chromatography about 3-4 hours, and most of the time period, more than about 50% but no more than about 80% thereof, in the 30-45% range of the secondary solvent. Under these conditions, the active fractions are eluted at about 41-42% acetonitrile. The pooled active fractions can be further purified by repeating reverse phase HPLC using a faster change in gradient conditions and a slower flow rate. Under these conditions, the activity emerges at about 40.5-41.5% acetonitrile.

The reverse phase HPLC can then be repeated, modifying the solvent system, the secondary solvent being n-pro-panol-0.1% trifluoroacetic acid.

A linear gradient is applied, with the gradient slowly changing in the range of 23-35% n-propanol. The main activity is observed in the range of 25.5-27.5 propanol to yield a substantially homogeneous product with a specific activity greater than 10 GIA units / ng protein. Usually, the product is purified to yield a specific activity of at least about 100 GIA units / ng protein, usually 150 GIA units / ng.

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The present compounds are characterized by a molecular weight of about 17-19 kilograms of Daltons (kD), especially about 18 kD, as determined by size exclusion chromatography. The present compounds are further characterized by an apparent molecular weight of approximately 28 kD determined by polyacrylamide gel electrophoresis under reducing or non-reducing conditions.

The amino acid sequence of fragments of purified Oncostatin M was analyzed. Oncostatin has substantially the amino acid sequence as depicted in Fig. 1. In Fig. 1, the first sequence illustrates the N-terminal 10 part of Oncostatin M, while the remaining sequences are internal fragments of the. illustrate polypeptide. The active preparations of isolated Oncostatin M contain a mixture of high mannose and complex N-linked oligosaccharide. However, non-glycosylated preparations of Oncostatin M retained their cell growth modulating activity. Oncostatin M is further characterized by its activity against certain cell strains. The present polypeptide lacks cytotoxic activity towards WI26 and WI38 human fibroblasts and mouse L929 cells, which are sensitive to tumor necrosis factor and a γ-interferon-sensitive human tumor cell line.

It has also been found not to inhibit the extension of normal human T lymphocytes or to inhibit granulocytic / myelocytic colony formation from bone marrow cells at concentrations up to 100 GIA units / ml. Furthermore, Oncostatin M stimulates the expansion of normal human fibroblasts, as shown, for example, by WI38 and WI26 cells 25 and inhibits the proliferation of tumor cells, such as A375, HBT 10, A549 and SK-MEL28, and may contribute to the growth of colony-forming cells from normal bone marrow. Oncostatin M does not suppress human proliferative or cytotoxic T cell responses in mixed leukocyte culture (MLC) responses at concentrations of 500 GIA units / ml. It is found that the present polypeptide is stable to moderate acids and bases and to a heat treatment at 56 ° C. The amino acid sequence of the subject polypeptide can be fully determined with commercially available sequencing agents. The polypeptide can then be synthesized according to known techniques using automatic synthesizers available commercially. Optionally, the subject polypeptide can be produced by recombinant DNA techniques. Can be from a partial amino acid sequence

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"" Means "are deduced, which are useful for sorting a human genome library. The library can be a cDNA library or a chromosomal library. Once the clones have been identified as linked to the probe, the fragments containing the gene of interest can be identified in a number of ways and manipulated in a number of ways. The fragment can be resized by endonuclease restriction, cloning the resulting fragments and probing for the presence of the desired gene. Cells producing the desired peptide or increased amounts of the desired peptide are useful for the production of messenger RNA. Single stranded cDNA can be prepared from the shopping RNA. The cDNA can then be linked to the total message RNA from a cell, which produces little or no polypeptide. The unlinked cDNA can then be isolated and used to prepare cDNA, which can be sorted with the probes. Optionally, DNA fragments can be introduced into Agt11 so that coding fragments lie downstream of and within the 8-galactosidase gene. Antibodies to the Qncostatin M polypeptide can be prepared and used to screen the resulting fused proteins for cross-reactivity.

In this manner, fragments encoding the present polypeptide or a fragment thereof can be identified and used to identify the desired gene. Once a complete gene has been identified, either as a cDNA or simply as normal DNA, it can then be manipulated in various ways to express it. When the gene is to be expressed in a host that recognizes the wild-type transcription and translation regulatory region of Encostatin M, the entire gene with its wild-type 5 'and 31 regulatory regions may be in an appropriate expression. 30 sievector are entered. Several expression vectors exist employing mammalian virus replication systems, such as Simian virus 40, Adenovirus, bovine papillomavirus, vaccinia virus, insect baculovirus, etc. These replication systems have been developed to provide for markers enabling the selection of transfectants. in appropriate restriction sites into which the gene can be introduced.

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If the gene is to be expressed in a host that does not recognize naturally occurring wild-type transcription and translation regulatory regions, further manipulation will be required. It is appropriate that a number of 3 'transcription regulatory regions 5 are known which can be introduced downstream of the stop codons. The non-coding 5 'region upstream of the structural gene can be removed by endonuclease restriction, Ba 131 resection, and the like. Optionally, when a suitable restriction site is present near the 5 'end portion of the structural gene, the structural gene 10 can be cut and an adapter used to couple the structural gene to the promoter region, the adapter providing the lost nucleotides of the structural gene. Several strategies have been employed to provide an expression cassette which, in the 5 ', 3' direction of the transcription, has a transcription and translation initiation region, which may also include regulatory sequences with which the regulation can be turned on. structural gene under the transcription and translation control of the leader region and has a transcription and translation termination region.

Illustrative transcription initiation regions or promoters for bacteria include the β-GAL promoter, the TAC promoter, the LAMBDA left and right promoters, etc .; for yeast, glycolytic enzyme promoters, such as ADH-I eh-II promoters, GPK promoter and GPI promoter, TRP promoter, etc .; for mammalian SV40 early and late promoters, adenovirus major late promoters, etc. As already indicated, the expression cassette may be incorporated within a replication system into a plasmid in a suitable cellular host or may be provided without a replication system, when it is integrated into the host genome. The DNA can be introduced into the host according to known techniques, such as transformation, using calcium phosphate precipitated DNA, transfection by contacting the cells with the virus, microinjection of the DNA into the cells, etc. Once the structural gene has been introduced into the appropriate host, the host can be propagated and express the structural gene. In some instances, it may be desirable to provide a signal sequence 35 (secretory signal generator) upstream of and in the structure gene reading frame, which provides for the secretion of the structure gene and cleavage of the secretory signal generator in order to deliver finished polypeptide in the top layer. When secretion is not provided, the host cells can be harvested, lysed and the desired product isolated and purified by known techniques such as chromatography, electrophoresis, solvent extraction, and the like, under conventional conditions.

The present compounds can be used in many ways both in vivo and in vitro. The subject compounds are useful for making antibodies to the subject compounds and may find use in vivo or in vitro. The antibodies can be prepared in a conventional manner, either by using the subject polypeptide as an immunogen and injecting the polypeptide into a mammalian host, e.g., mice, cows, goats, sheep, rabbits, etc. especially with an adju -15 vans, for example, the complete Freunds adjuvant, aluminum hydroxide gel, and the like. The host can then be drained and the blood used for the isolation of polyclonal antibodies or, in the case of mice, the peripheral blood lymphocytes or spleen lymphocytes (B cells) used for fusion with an appropriate myeloma cell to express the chromosomes for monoclonal expression. present compound to immortalize specific antibodies.

Either polyclonal or monoclonal antibodies can be prepared, which can then be used for diagnosis or detection for the presence of the subject polypeptide in a sample, such as cells or a physiological fluid, eg blood. The antibodies are also useful in affinity chromatography for purifying the present polypeptide and isolating it from natural or synthetic sources. The antibodies can also be used to adjust the amount of the subject polypeptide associated with cells in culture or in vivo, allowing the growth of the cells to be modified.

The present compound is useful as a ligand for detecting the presence of receptors for the present compound. In this way, cells can be distinguished according to the presence and density of the receptors for the present compound, whereby the effect of different compounds on the presence of such receptors can be monitored.

8303209 -12-

The subject compound can be used in in vitro cultures to inhibit the growth of cells or cell lines that are sensitive to the subject polypeptide as distinct from cells that are not sensitive. Thus, heterogeneous cell mixtures or cell lines can be freed from unwanted cells when the unwanted cells are sensitive to the subject polypeptide. The subject polypeptide can be administered in vivo in the case of neoplastic conditions, for example, by injection, intralesional, peritoneal, subcutaneous, and the like.

The present compound can be used in vitro to eliminate malignant cells from marrow for autologous marrow transplants or to inhibit proliferation of malignant cells or remove them in other tissues, eg blood, before re-infusion.

The subject polypeptide is also useful in a method of treating wounds, such as skin wounds, corneal wounds, and various other epithelial and stromal disorders, such as chronic ulcers, burns, surgical incisions, traumatic wounds, and lesions of the epithelial sacs, such as the esophagus, stomach, large and small intestines, mouth, genital organs and urinary tract.

The method is based on the topical administration of a treatment composition comprising Oncostatin M in a physiologically acceptable carrier.

The composition of the present invention can be used to treat a wide variety of wounds including substantially all skin wounds, corneal wounds and lesions of the epititically coated hollow organs of the body. Wounds suitable for treatment include those caused by violent action, such as burns, abrasions, cuts, and the like, as well as from surgical procedures, such as surgical incisions and skin grafting.

Other conditions suitable for treatment with the compositions of the present invention include chronic conditions, such as chronic ulcers, diabetic ulcers and other non-healing (trophic) conditions. Oncostatin M can be incorporated into physiologically acceptable carriers for administration to the affected area. The nature of the carriers can vary widely and depends on the intended site of administration. For skin application, preference is usually given to a cream or ointment base, with suitable bases 8 8 0 3. 2 0 9 -¾ 4 -13-lanolin, Silvadeen (Marion) (especially for the treatment of burns) , Aquaphor (Duke Laboratories, South Norwalk, Connecticut), and the like. If desired, it is possible to incorporate Oncostatin M-containing compositions into bandages and other wound dressings to provide continuous wound contact with the peptide. Aerosol administrations can also be used.

The concentration of the polypeptide in the treatment composition is not critical. The polypeptide will be present in an epithelial cell extension promoting amount. The compositions will be applied externally to the affected area, typically as eye drops on the eye or as creams, ointments or lotions on the skin. In that case of eyes, repeated treatment is desirable, which is usually repeated every 4 hours or less. For the skin, it is desirable to maintain the treatment composition continuously in the affected area during healing, with the treatment composition being administered 2-4 times a day or more. The amount of the subject polypeptide used depends on the mode of administration, the use of other active compounds, and the like, and is generally in the range of about 1-100 micrograms.

The subject polypeptide can be used with a physiologically acceptable carrier, such as brine, phosphate buffered brine, and the like. The amount of compound used will be determined empirically based on the response of cells in vitro and the response of experimental animals to the subject polypeptides or preparations containing the subject polypeptides.

The present compounds are useful on their own or in combination with other growth factors or inhibitors or immunomodulators, such as. TNF, IL-2, γ-interferon, monoclonal antibodies, etc. The amounts of these other compounds will generally range from 1 microgram to 100 micrograms. Conjugates of the subject compounds with locating units, e.g. antibodies, can be prepared when the antibodies are specific for certain malignant cells or organs.

The following examples are presented for illustrative and not limiting purposes.

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Materials and methods;

Oncostatin M isolated from U937 cells

Production of a tumor cell growth inhibitor from a histiocytic lymphoma cell line 5 U937 cells, a histiocytic lymphoma cell line (Sundstrom and

Nilsson, Int. J. Cancer (1976) (1976) 17: 565-577) were grown in 2 850 cm roller bottles (Corning C2540) at a concentration of 4x10 cells / ml in a total volume of 300 ml RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), penicillin / streptomycin (PS), L-glutamine and 10 10 ng / ml 12-O-tetradecanoylforbol 13 acetate (TPA). Four days later, the top layers containing the FCS and TPA were removed, the roller bottles were washed 5x with serum-free RPMI 1640 and the cells that dislodged (1x105 cells / ml) were washed 3x with serum-free medium and returned to the bottles, leading to up to a final volume of 125 ml of serum-free RPMI 1640 medium / roll bottle. One day later the supernatants were collected, centrifuged to remove the cells, filtered through a 0.45 micrometer Nalgen filter and concentrated using a hollow fiber system (Amicon cartridge HIP 10-20) to a volume of 150 ml (initial volume 1500 ml ). Oncostatin M was also isolated from top layers 2 of serum-free TPA-treated U937 cells in 150 cm tissue culture flasks. The top layer was concentrated with an Amicon-Diaflo membrane PM-10, 10 kD cut-off and dialized. After the dialysis, the concentrate was diluted with acetic acid resulting in a final concentration of 0.1N acetic acid in 500 ml and concentrated to 50 ml with a 25 Amicon PM 10 filter. The 50 ml concentrate was diluted to 400 ml with 0.1N acetic acid and concentrated to 40 ml with the same filter. The concentrate was diluted with 1N acetic acid and the resulting precipitate was removed by centrifugation. The resulting concentrate was frozen and freeze-dried. The lyophilized material was used for purification steps,

Gel permeation chromatography

A Bio-Sil TSK-250 column (600x21.5 mm) (Bio-Rad) was attached to a high pressure liquid chromatography system. The crude fraction (10 mg / ml) was dissolved in 40% acetonitrile in 0.1% aqueous trifluoroacetic acid (0.1% TFA). 2 ml aliquot of the mixture was injected and the elution was performed isocratically with a mobile phase of 40% acetonitrile in 0.1% 86 0 3 2 0 9-15 TFA. The flow rate was 2.5 ml / minute and the recording rate was set at 0.25 cm / minute. 5 ml fractions were collected. The chromatography was performed at room temperature. An aliquot of each fraction was evaporated and analyzed in triplicate for growth inhibitory activity (G1A) of A375 cells. The active fractions (fractions 21 and 22) from six experiments were pooled. The pooled material contained approximately 4.8x10 GIA in total. units. The factor was found to have an apparent molecular weight of 18 kD, as determined by size exclusion chromatography (Bio-Sil-TSK-250 column).

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

The pooled TSK-250 fractions 21 and 22 as described above were diluted 2-fold with 0.1% TFA. This mixture was injected isocratically into a ^ i-Bondapak C18 column (7.8x300 mm) (designated 018 ^) at room temperature. The flow rate was set at 2.0 nu./minute and the recording rate was 0.25 cm / minute. A linear gradient between the primary solvent 0.1% TFA and the secondary solvent 0.1% TFA and the secondary solvent Acetonitrile-TFA 0.1% was used.

The gradient conditions were 0-30% in 20 minutes; then 30-45% in 150 minutes; 45-55% in 20 minutes and 55-100% in 10 minutes. All solvents were HPLC grade. 4 ml fractions were collected and aliquots of each fraction were examined for growth inhibitory activity.

Fractions 72-75 were found to have the greatest activity. The active fractions were eluted between 41-52% acetonitrile concentration. Fractions 72-75 were pooled together. 16 ml of 0.1% TFA was added to the pooled fractions. The mixture was injected into a u-Bondapak-C18 2 column (3.9x300 mm) (referred to as CIS) at room temperature. The flow rate was set at 1 ml / minute and the card reading speed at 0.25 cm / minute. The gradient conditions were 0-35% in 10 minutes and 35-45% in 100 minutes and 45-100% in 10 minutes.

The fractions were collected and aliquots taken and examined for GIA. Most activity arose from the column between 40.7 - 41.3% acetonitrile concentration (retention time 83 - 86 minutes). Active fractions were pooled and diluted 2-fold with 0.1% TFA and injected isocratically into a 1-Bondapak-C18 column (3.9-200 mm) (designated C183) at room temperature. The flow rate was 1 ml / minute and the card reading speed 0.25 cm / minute. A linear gradient was applied between the primary solvent 0.1% TFA and the secondary solvent n-propa-. nol-TFA (0.1%). The gradient conditions were 0-23% in 20 minutes and 23-35% in 120 minutes. Fractions were collected and aliquots of each fraction were examined for GIA. Most activity appeared between 25-26.5% propanol concentration (retention time 59 minutes). This apparently homogeneous fraction contained 300 ng of protein and approximately 40,000 GIA units.

Cell growth modulating analysis using 3 H-thymidine incorporation into DNA (GIA). The analyzes were performed on 96-well flat plates (Costar 3596). Human melanoma cells (A375) were used as the sensitive indicator cell line. Cells (3x103) in 0.1 ml Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% FCS and PS were placed in each well. 3 hours later, 0.1 ml of the test samples were added to each cup. The plates were incubated at 37 ° C for 3 days. Then 0.025 ml (0.5 µCi) of a solution of 3 H-thymidine (specific activity 27 µC ± / q) was added to each well during the last 6 hours of incubation. The cells were then transferred to glass filter strips using a multi-cup winch (PHD cell Harvester, Cambridge Technology, Inc.). The filters were transferred to scintillation vials containing 2 ml of scintillation fluid (ScientiVerse II,

Fisher Scientific Co.) were added, before counting in a scintillation counter, to quantify the 3 H-thymidine incorporation.

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Agaron Oil Soft Inhibition Test (TGX)

A 0.5 ml base layer of 0.5% agar (Agar Noble; Difco

Laboratories, Detroit, Michigan) in DNEM containing 10% fetal calf serum (FCS) was added to 24-well Costar tissue culture plates.

3 0.5 ml 0.3% agar containing the same medium-FCS mixture, 1-2.5x10 A375 cells and the factor to be tested were coated at different concentrations on the base layer of agar,

The plates were incubated in air at 37 ° C in a humid atmosphere of 5% CO 2 with re-feeding after 7 days by adding 0.5 ml of 0.3% agar containing the same medium and concentrations of the factor. The colonies were numbered unfixed and unstained and the number of colonies larger than 6 cells were counted between days 7 and 14.

Results 15 Sequences of Oncostatin M isolated from U937 cells

The N-terminal sequence and internal fragments of OncostatinM were determined by micro-sequence analysis of the reduced and S-pyridine-ethylated polypeptide and of peptides obtained from enzymatic digestion products of reduced and S-20-pyridine-ethylated 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 sequence meter (Applied Biosysterns, Ine.). The phenylthiohydantholine amino acids were analyzed by reverse phase HPLC (Applied Biosystems, Inc.) with a PTH-C18 column (2.1x220 mm, ABI) using a sodium acetate buffer / tetrahydrofuran / acetonitrile gradient for elution.

The resulting amino acid sequences are substantially the same as those illustrated in Figure 1. A comparison of these sequences with those stored in the current protein databases (PIR Release 9.0, May 1986) revealed no significant sequence homologs with any other known sequence. Also, there is no homology with tumor necrosis factor, lymphotoxin, colony stimulating factor, interleukin 1 or 2 or β-trans-35 forming growth factor.

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Inhibition of tumor cell proliferation and proliferation or extension of normal human fibroblasts

Using the soft agar colony inhibition analysis as described above, the following results were obtained: Table A. Inhibition of A375 Melanoma Cell Colony Formation in Soft Agar by Purified Oncostatin M Isolated from U937 Cells * GIA Units / Cup% Colony Inhibition Colony Formation 250 4 96 83 6 94 t 10 27 11 89 45 32 69 0 106 * A375 cells were plated on soft agar plates, with or without factor in a final volume of 2 ml, as described above. The factor used was from a C18 propanol column fraction with peak tumor growth inhibitory activity (GIA). Eleven days later, the numbers of colonies were numbered. A colony was defined as one. group of at least 6 cells. A GIA unit was defined as the amount that caused a 50% inhibition of 3 H-thymidine-20 uptake into A375 cells in microcups as described in detail above.

In the following study, various treatments were used to determine the effect of the treatment, whether chemical or physical, on the activity of the subject polypeptide. The following table gives an indication of the results.

Table B. Effect of different treatments of top layers of TPA-induced U937 cells on tumor growth inhibitory activity *

Final dilution of top coat 30 1: 4 1: 8 1:16

Media Control - - 39,780

Untreated top 7,206 ** 13,896 16,000 low IN acetic acid 6,670 17,073 18,783 IN.OH 6,956 15,016 13,923 4 35 * U937 cells were treated with TPA (10 ng / ml) for 3 days after which the cells were washed with media and left in for 24 hours serum-free media were incubated before collection

SoO 3208 -19- of the top layers. The top layers were treated with 1N acetic acid or 1N ammonium hydroxide (NH 2 OH). They were then dialyzed against hst medium and tested for their ability to inhibit 3H-thymidine uptake in A375 cells. A375 cells were labeled with 3H-thymidine (3H-TdR) 5 for the last 6 hours of a 3 day incubation.

** Data shown is 3H-TdR recording counts per minute.

The above results demonstrate that Oncostatin M in the dialyzed top layer is substantially resistant to inactivation by one normal acetic acid and one normal ammonium hydroxide. Thus, the present compounds are relatively insensitive to both moderately strong acid and moderately strong base. The present compound is stable to heat treatment at 56 ° C for 1 hour but not at 90 ° C for 30 minutes.

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

In the following study, the ability of the subject polypeptides to inhibit tumor cell replication from a plurality of neoplastic cells was investigated. The following table gives an indication of the results.

Table C. Inhibition of tumor cell replication by purified OncostatinMuit U937 cells

Units GIA causing 30% inhibition 25 Tumor cells 3H-TdR uptake A549 (lung cancer) 21 HTB1Q (neuroblastoma) 81 A375 (melanoma) 0.3

Tumor cells were plated into microcups 3 hours before the addition of various dilutions of Oncostatin M, purified by reverse phase HELC as explained in detail above.

During the last 6 hours of a 3 day incubation, cells in 0.2 ml medium were labeled with 3β-thymidine (3H-TdR) (0.5 µCi / cup). One unit of tumor growth-inhibitory activity (GIA) is described in Note 8 A η τ 9 r »§.

V V V V> Μ -20- of Table A defined as that amount causing 50% inhibition of 3H-TdR uptake in A375 melanoma cells. It was determined that one unit was approximately 10 µg of purified protein, so therefore the concentration (ng / ml) to cause 30% inhibition of 3 H-TdR in A549, HTB10 and A375 cells was approx. 1.4, 4.0 and 0.015 ng / ml. 3H-TdR uptake in W126 normal human fibroblasts was not suppressed by the U937 factor in any experiment.

The above results demonstrate that Oncostatin M is selective in its ability to inhibit replication, and has widely varying effects depending on the nature of the cell. The present compound is effective against melanoma cells such as A375 melanoma cells, lung cancer cells such as A549 and neuroblastoma cells such as HTB10.

Tumor cells were seeded at 3 x 10 3 cells per cup and 3 normal fibroblasts at 1.5 x 10 cells per cup in 15 96-cup plates for 3 hours. Different concentrations of purified Oncostatin M obtained from the fraction of the C18 column with peak anti-proliferation activity against A375 cells were added and three days later the 3 H-thymidine incorporation in the cells in triplicate cups was measured at each concentration. The results are shown in Table D.

Table D. Inhibition of tumor cell proliferation and proliferation of normal fibroblasts by Oncostatin M

GIA

units / cup% Inhibition% Stimulation A3 75 WI38 25 Example 1 16 83 25 4 62 30 1 46 46 A375 HTB10 WI26

Example 2 21 NT 28 46 30 9 87 22 36 3 76 11 52 A375 A549

Example 3 75 89 30 25 85 22 35 8 71 16 A375 SK-MEL28

Example 4 20 87 44 5 75 25 1 59. 11 «’ λ fï ΌΛΟ

J J v i · V W

-21-

Results shown are% inhibition or% stimulation of 3H-thymidine incorporated into tumor cells (A375, HTB1Q, A549 and SK-MEL28) and normal fibroblasts (WI26 and WI38). A GIA unit is defined in the Notes to Table A as the amount of OncostatinM that causes 50% inhibition of 3 H-thymidine incorporation in A375 cells.

In addition to the observed differential effect on 3H-thymidine uptake in tumor cells and normal human fibroblasts, a differential effect of morphology and cell number was also observed after 3 days of treatment of the two cell types with OncostatinM, as shown in Figure 2.

The Oncostatin ™ used came from the HPLC-C183 column fraction with peak activity to inhibit proliferation of A375 cells.

Fig. 2 is a series of micrographs of A375 melanoma cells consisting of untreated (A), treated with 5 growth inhibitory activity (GIA) units, 15 Oncostatin Μ (B), or 100 units (C). Furthermore, micrographs of WI38 fibroblasts that were untreated (D) treated with 5 units of GIA (E), or 100 units (F). The cells were stained with crystal violet in 0.5% methanol. Magnification = 63x.

NaDodSO4 / PAGE of Oncostatin M

Purified Oncostatin M subjected to NaDOdSO4 conducted 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 chymotrypsinogen a, Mr = 25.7 kD; lactoglobulin 8, 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. OncostatinM was applied in strip B.

Oncostatin. M, subjected to PAGE under non-reducing conditions, also has an apparent molecular weight of 28 kD and protein electroluted from the band was shown to inhibit proliferation of A375 cells.

Antibody to a synthetic peptide of Oncostatin M 125 reacts in I-labeled Oncostatin. M in radioimmunoprecipitations a) Peptide synthesis: The peptide corresponds to residues 6-19 of the OncostatinM protein and was synthesized by solid phase 35 techniques using a Beckman automated instrument as described (Gentry et al., J. Biol. Chem. (1983). 258; 11219). The peptide was cleaved from the resin according to the "low-high" HF procedure (Tam et al ..

8 60 3 20 9.

5-22 Amer. Chem. Soc. (1983) 105: 6442-6445). Purification was accomplished by preparative HPLC.

b) Purification of Antibodies The peptide was coupled to bovine γ globulin as described (Gentry and Lawton, Virology (1986) 5 152: 421-431). New Zealand white rabbits were prepared and stimulated four times subcutaneously (5 times) as described (Gentry and Lawton, Virology (1986) 152: 421-431). The antisera used was obtained 2 weeks after the fifth stimulus.

c) Iodination of Oncostanine MD: A sample of partially purified Oncostatin M was radiolabelled with iodine-125 according to published procedures (Linsley et al., PNAS (1985) 82: 356-360).

A portion of the labeled preparation containing 100,000 cpm was mixed with 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 µg) and subjected to immunoprecipitation analysis as described (Linsley et al., Biochemistry (1986) 25: 2978-2986.

In particular, one tube containing 5 µl was made in advance

Incubated with 2 yag of the N-terminal peptide in 10 µl TNEN

(20 mM Tris pH 7.5, 5mM EDTA, 150 mM NaCl, 0.05% Nonidet P-40) containing 0.1% 125

BSA contained at 4 ° C for 30 minutes before the addition of I.

Oncostatin in 85λ TNEN containing 0.1% BSA and 40 mM dithiothreltol (DTT). Seven tubes containing 5 ul of antisera were incubated with 125 I Oncostatin Min 85 ul of THEN containing 0.1% BSA and 40 mM DTT for 30 min at 4 ° C before the addition of 50 filter of 10% formalin-fixed Staphylococcus aureus ( Pansorbine, Calbiochem).

After an additional incubation for 30 minutes at 4 ° C, the tubes were microcentrifuged, and the pellets were washed four times with 1 ml TNEN before subjecting to PAGE analysis. After SDS / PAGE analysis of the immunoprecipitates, a diffuse band of Mr = 32 kD was observed. Precipitation of this type was inhibited by including an excess of unlabelled peptide corresponding to the N-terminal 17 amino acids of Oncostatin M 35 indicating that the precipitation for this peptide was specific.

The carbohydrate composition of Oncostatin M was tested by testing for glycosidic sensitivity. Immunoprecipitates, prepared 8603209-23- as in c above were treated with buffer, endoglycosidase H or neuraminidase, as described by Linsley et al »(1986). Treatment with endoglycosidase H, an enzyme with specificity for N-linked high mannose oligosaccharides, resulted in the appearance of a low molecular compound type of Mr = 24 kD. Only part of the radiolabelled material was sensitive to this enzyme, indicating that not all molecules contain high mannose oligosaccharides. Neuraminidase treatment resulted in the appearance of a single band of Mr = 27 kD, indicating that the heterogeneity in the size of untreated I-labeled Oncostatin M was due to molecular heterogeneity in the sialyation of the glycoprotein nucleus. The results indicated that active preparations of Oncostatin M contained a mixture of high mannose and complex N-linked oligosaccharide side chains.

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

Production of a tumor cell growth inhibitor from PBLs

Leukofractions containing PBLs obtained from the blood bank were diluted 1: 1 with phosphate buffered brine, pH 7.4 (PBS). 35 ml of diluted blood was layered 10 ml of a solution consisting of 9% Ficoll containing 20% by volume of 50% sodium diatrizoate (final specific gravity of 1,080). The gradients were centrifuged at 850 x g for 20 minutes at room temperature.

Cells were collected from the gradient interface and washed with PBS.

Red blood cells were lysed by shock for 10 minutes with 10-20 ml of a solution containing 0.8% ammonium chloride and 0.1%

After -EDTA contained.

4

Cells were collected from the red blood cell lysing solution by centrifugation at 600 x g for 10 minutes and resuspended in 10 ml RPMI 1640 medium (G1BC0) containing 5% fetal bovine serum. Thrombin was added to a final concentration of 0.5 µ / ml. The cell suspension was stirred at 37 ° C for 5 minutes and the platelet aggregates were allowed to settle for 5 minutes. The suspended cells were transferred to new tubes, recovered by centrifugation, resuspended in ml of fetal bovine serum and transferred to a column containing 0.5 g of brushed, pre-wetted nylon wool, type 200 (Fenwal).

R «n" o o 9 -24-

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

g PBL '(2 x 10 cells / ml) were cultured in RPMI-1640 medium (10.4 g / l) at 37 ° C, 5% CO2 ~ 95% air for 96 hours, which medium was FeS04.7H20 (1 mg / 1), ZnS04.7H20 (2 mg / 1), Na2Se03.5H20 (0.017 mg / 1), 1-aminoethanol (1 mg / 1), human transferrin (5 mg / 1), bovine serum 10 albumin-linoleic acid conjugate (Sigma ) (200 mg / 1), L-glutamine (300 mg / 1), penicillin / streptomycin (100,000 units / 1) and phytohemagglutinin-P (Wellcome) (2 mg / 1). The supernatants were collected, centrifuged to remove the cells, concentrated by ultrafiltration (Amicon Diaflo membrane YM-10, 10 Kd cut-off point), and dialyzed against 0.1 M acetic acid (Spectrapore 3 dialysis tubes). The clarified retentate was freeze-dried.

Gel permeation chromatography

The crude fraction was prepared in 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.

12 ml fractions were collected. An aliquot from each fraction was evaporated and analyzed in triplicate for growth inhibitory activity (GIA) of A375 cells. The active fractions were pooled, lyophilized and re-chromatographed on a Bio-Sil TSK-250 column (600 x 25 21.5 mm) as described.

Reversed phase high pressure chromatography of TSK-250 fractions

Final purification of pooled TSK-250 fractions was accomplished by reverse phase HPLC essentially as described. PBL-derived tumor cell inhibitor was eluted from Bondapak C 18 carrier 30 at 40-41% acetonitrile and at 26.5% n-propanol concentrations.

125

Cell growth modulating analysis using I-iodo-deoxyuridine incorporation into DNA (GIA):

These analyzes were performed in flat bottom culture trays containing 96 cups (Costar 3596). Human melanoma cells, 35 A375 (4x103) in 50 filter of the test sample were added to each well and incubated at 37 ° C for 3 days. The cells were

ö λ A 7 O A

y Ό J i u b 125 / c -25- labeled with I-IdU (0.05 uCi / cup) for 24 hours and incubated an additional 24 hours. The cells were washed three times, recovered with a multiple sample recovery device and the radioactivity counted in a gamma counter.

5 Results:

A PBL-derived tumor cell inhibitor preparation was subjected to automated repetitive Edman degradation. The amino terminal amino acid sequence is as follows: 15 10 15

10 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 yet been identified.

A comparison of this sequence with that of the CJ937 factor clearly demonstrates the identity with the N-terminal group of PBL-derived factor.

15 1 5 10 15

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

In the following study, the ability of PBL derived Oncostatin M to effect replication of a plurality of cells was investigated. Mouse L929 cells were found to be insensitive to PBL-derived Oncostatin using up to 1000 GIA units / ml. Human fibroblasts, WI25 were stimulated by treatment with 1000 GXA units / ml. Normal human T lymphocyte extension at 72 hours after mitogenesis was not affected by up to 500 GIA units / ml.

It is clear from the above results that new polypeptide and polypeptide fragments are provided which are useful for modulating cellular growth. The compound appears to have a varying activity depending on the nature of the cell line involved, so that it can be used alone or associated with other compounds to modulate cellular growth. The subject polypeptides thus add an additional polypeptide that can be used with mixtures of cells, both in vivo and in vitro, to selectively reduce or enhance cellular proliferation or extension of a particular type of cell.

8303209 -26-

In particular, the factor can be used to treat cells for autologous bone marrow transplants. By applying the factor, the growth of tumor cells in the marrow is inhibited and colony cell formation can be stimulated. Oncostatin M can also be used to stimulate the growth of epithelial cells thereby promoting wound healing. Also, the intact polypeptide or fragments thereof can be used as immunogens to induce antibody formation. The induced antibodies find use for the titration of Oncostatin M contained in a body fluid or for modulating the activity of the factor by binding to it. Furthermore, these antibodies together with purified Oncostatin M or fragments thereof serve as a component of diagnostic kits in conjunction with other reagents, in particular antibodies to detect and quantify Oncostatin M.

15 8 60 3209

Claims (23)

A novel poly (amino acid) with at least eight amino acids which is immunologically cross-reactive with a compound, which compound is a polypeptide obtainable from leukocytes capable of inhibiting proliferation of neoplastic cells, stimulating 5 of extension of normal human fibroblasts, failure to inhibit human extension and cytotoxic T cell responses and failure to inhibit granulocytic / myelocytic bone marrow colony cell formation, with a molecular weight in the range of about 17 to 19 kD as determined by gel exclusion chromatography and about 28 kD as determined by SDS-PAGE, which is relatively insensitive to moderate acids and bases and moderately elevated temperatures. Poly (amino acid) according to claim 1, characterized in that it contains an amino acid sequence with at least ten consecutive amino acids corresponding to an amino acid sequence as indicated in Fig. 1 and that of said sequence with no more than three amino acids differs. Poly (amino acid) according to claim 2, characterized in that it contains twelve consecutive amino acids corresponding to an amino acid sequence represented in Fig. 1 and which differs from said sequence 20 by no more than one amino acid, which difference is either a deletion or is a conservative substitution. 4. Novel polypeptide preparation obtainable from leukocytes, containing a component capable of preventing the proliferation of neoplastic cells, stimulating the expansion of normal human fibroblasts, not inhibiting human expanding and cytotoxic T cell responses, and not to inhibit granulocytic / myelocytic bone marrow colony cell formation, with a molecular weight in the range of about 17 to 19 kD as determined by gel exclusion chromatography and about 28 kD as determined by SDS-PAGE, which is relatively insensitive to moderate acid and moderate base and moderately elevated temperatures ; and with a purity that provides a specific activity of at least about 10 GIA units / ng protein. S o 0 3 2 0 9: r * -28- Polypeptide preparation according to claim 4, characterized in that said leukocytes are mitogen-activated normal human peripheral blood lymphocytes, Polypeptide preparation according to claim 4, characterized in that said leukocytes are pholester diester-induced human histiocytic lymphoma cells. Polypeptide preparation according to claim 4, characterized in that it is substantially free of cellular components. Polypeptide obtained from a composition according to claim 4, characterized in that the specific activity of said polypeptide is at least about 100 GIA units / ng protein. 9. Polypeptide substantially free of cellular debris and other leukocytic proteins capable of inhibiting proliferation of neoplastic cells, stimulating the expansion of human fibroblasts, not inhibiting human proliferating and cytotoxic T cell responses and not inhibit granulocytic / myelocytic bone marrow colony cell formation, with a molecular weight in the range of about 17 to 19 kD as determined by gel exclusion chromatography and about 28 kD as determined by SDS-PAGE, and which is relatively insensitive to moderate acids and bases and moderately elevated temperatures and amino acid sequences corresponding to the sequences indicated in Fig. 1, wherein there are no more than three amino acid differences between the proposed sequence and said corresponding sequence. Polypeptide according to claim 9, characterized in that said corresponding sequence differs by no more than one amino acid from said indicated sequence. 11. A method of inhibiting the proliferation of neoplastic cells, comprising contacting said cells with an anti-proliferation amount of a composition according to claim 8. A method of enhancing wound healing comprising contacting a wound with a fibroblast extension-stimulating amount of a polypeptide according to claim 8. Antibodies specific for a polypeptide preparation according to claim 1 or 4. Monoclonal antibodies according to claim 13. 8S032O9 -29-m 15. Method for detecting the presence of a polypeptide preparation. according to claim 4, characterized in that it comprises: combining a sample suspected of containing said composition with an antibody according to claim 13, and determining the amount of complex formation with said antibody. DNA sequence encoding a polypeptide according to claim 8. A method of producing a polypeptide according to claim 8, comprising: growing a cell culture containing cells with an expression construct consisting of a DNA sequence encoding a polypeptide according to claim 4 under the regulatory control of transcription and translation regulatory signals that are functional in said cells, thereby expressing said polypeptide; and isolating said polypeptide substantially free of cellular material. 18. Diagnostic kit comprising an antibody according to claim 13 and at least one of a poly (amino acid) according to claim 1 or a polypeptide according to claim 8. P. & & ono V Ki &lt; United Patent Application No. 8603209 &quot; Sogravenhage (holuand) beh. when writing dd. May 14. 19 87 Ln / 657 - Improvement (s) of erratum (a) in the description associated with patent application No. 8603209 proposed by applicant (star) under date May 14, 1987 cc oc cc cc tc «<r cc cc cc cc« cc cc The following claims 19-23 are added. · -, 19. A method of producing a novel poly (amino acid) having at least eight amino acids which is immunologically cross-reactive with a compound, said compound being a polypeptide obtainable from leukocytes capable of inhibiting the proliferation of neoplastic cells , capable of stimulating the extension of normal human fibroblasts, does not inhibit human extension and cytotoxic T cell responses and does not inhibit granulocyte / myelocytic bone marrow colony cell formation, with a molecular weight in the range of about 17-19 kD as determined by gel exclusion chromatography and about 28 kD as determined by SDS-PAGE, and which is relatively insensitive to moderate acids and bases and moderately elevated temperatures, cultivating a cell culture containing cells with an expression construct containing a DNA sequence encoding said poly (amino acid) under the regulatory control of transcription and translation control signals mentioned in said the cells are functional, thereby expressing the poly (amino acid) and isolating the poly (amino acid) essentially free of cellular material. Sa Λ V O Λ · Λ o w j l "j y ...... 545 33- * Μ The method of claim 19, wherein the poly (amino acid) contains an amino acid sequence having at least ten contiguous amino acids corresponding to one shown in FIG. 1 amino acid sequence and differ from said sequence in no more than three amino acids. The method of claim 19, wherein the poly (amino acid) contains twelve contiguous amino acids corresponding to one shown in FIG. 1 amino acid sequence and differ from that sequence in no more than one amino acid, which difference is a deletion or a subsequent substitution. 22. A method of producing a polypeptide, which is essentially free of cellular debris and other leukocytic proteins, which polypeptide is capable of inhibiting the proliferation of neoplastic cells, capable of stimulating the extension of normal human fibroblasts, human does not inhibit proliferative and cytotoxic T cell responses and does not inhibit granulocytic / myelocytic bone marrow colony cell formation, with a molecular weight in the range of about 17-19 kD as determined by gel exclusion chromatography and determined by SDS-PAGE, relative is insensitive to moderate acids and bases and moderately elevated temperatures, and includes amino acid sequences corresponding to those shown in FIG. 1 sequences with no more than 3 amino acid differences between the sequence shown and said corresponding r sequence, culturing a cell culture containing cells with 8603209 '-32: an expression construct comprising a DNA sequence encoding said polypeptide under the regulatory control of transcription and translation control signals functional in said cells, wherein said polypeptide is expressed, and the polypeptide is isolated essentially from cellular material. The method of claim 22, wherein said corresponding sequence differs in no more than one amino acid from said sequence shown. 0 γΛ f \ W \ J ** '