KR910000735B1 - Platelet related growth regulator - Google Patents

Abstract

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Description

[Name of invention]

Platelet related proliferation control substances

[Brief Description of Drawings]

1 is a chart comparing the effects of oncostatin-A on the proliferation of human cancer cells in athymic mice.

Detailed description of the invention

[Field of Invention]

The complexity of hematopoietic cells and their differentiation and proliferation is becoming increasingly evident as the list of isolated factors regulating these actions continues to increase. In most cases these factors are present in extremely small amounts with many other proteins having a wide range of functions. Isolated particles that have been demonstrated to have activity include γ-interferon, platelet derived growth factors, colony stimulating factors, polypeptides and proteins such as interleukin-2, erythropoietin, and many other lymphokines. Isolation, purification and characterization of these blood components are of practical advantage because they are likely to be used for the treatment and explanation of diseases such as cancer.

There are many difficulties in the isolation of the factors present in nature.

First, a separation system must be developed that separates the target factor from other factors with similar characteristics.

Second, some means must be provided for measuring the various fractions that characterize the target substance specifically or substantially specific against other coexisting features. The higher the activity of the polypeptide of interest, the more difficult it is to isolate the desired product.

Third, a method must be obtained which does not have an undesirable effect on the desired product, in particular, a method that does not cause all denaturation. In the composition, there are sometimes other substances which act on and change the target substance, and the resultant substance having any target activity finally obtained is not a naturally occurring substance, but finally after the target product is isolated in a sufficiently pure form. For example, the polypeptide must be physically specified by amino acid sequence determination, glycosylated water, disulfide bridge, and the like. The substance must also be specified for its physiological properties.

Even after isolation of the last purified compound, it is sometimes difficult to characterize the physiological activity of the compound. In many situations, activity depends on the presence of one or more other compounds, narrow concentration ranges, specific host cells, etc., so that substantially intuitive efforts and efforts to identify the physiological activity of a compound and to demonstrate other parameters affecting its activity may occur. An extended experimental period is required.

[Description of the Prior Art]

Holley et al., Proc. Natl, Acad, Sci, USA (1980) 77: 5989-5992 published purification of inhibitors of epithelial cell proliferation.

Nelson-Hamiton & Holley et al. (1983) 80: 5636-5640 introduced a radiolabeled methionine into a protein secreted by African Green Monkey Cells (BSD-1) and inhibited proliferation. The effects of the substance and epithelial growth factor were reported. Morgan et al. Thromb. Haemost (1980) 42: 1652-60 published the amino acid sequence of human platelet factor-4. Dawes et al. Thromb. Res. (1983) 29; Schernthane et al. 569-81. Acta Med. Austriaca (Suppl.) (1979) 6: 375-9 reported polyclonal antibodies against platelet factor 4. Lawler et al., Thomb. Res (1981) 21: 121-7 compared the arrangement and structure of β-thromboglobulin and platelet factor 4.

Overview of the Invention

According to the present invention it is possible to induce the secretion of 52KD protein from tumor cells, which can stimulate pp60src autophosphorylation, which does not inhibit the proliferation of normal cells but does not inhibit the proliferation of normal cells. there is; And polypeptides characterized by having an amino acid sequence substantially isolated from at least a portion of the polypeptide that can be isolated from mammalian platelets and exhibit at least one of the above properties. This polypeptide is provided in a substantially pure form.

Detailed Description of Specific Embodiments

The present invention provides compositions comprising polypeptides, derivatives, fragments, or the like that inhibit mammalian neoplastic proliferation. The polypeptide of the present invention relates to a natural polypeptide in the methanolic HCl fraction obtained by extraction of platelets, which is described in the literature as platelet factor-4, hereinafter referred to as oncostatin-A. The polypeptide is stable at mild temperatures (0-25 ° C.), low pH, generally below pH 3, usually at pH 2. The molecular weight of this polypeptide is in the range of about 5,000-8,000, more precisely in the range of about 6,000 to 7,500 and more precisely about 7,000. This polypeptide is obtained from platelets of higher animals, particularly primates and more particularly human body.

Polypeptide compounds used have about 15 to 80 amino acids and natural polypeptides and mimic analogs thereof have about 60 to 75 amino acids, more specifically about 65 to 72 amino acids, while fragments are generally about 15 to 80 amino acids. It has 60 amino acids, more typically 15 to 35 amino acids. Of particular importance are polypeptides having about 68 to 75 amino acids, more specifically 69, 70 or 71 amino acids. These polypeptides may be linked to other compounds, such as antigens, receptors, labels, and the like.

A polypeptide has an array that is active at least one biologically active, eg immunologically or epitope, and has one or more biologically active sequences, in which case such arrangement is a natural product for biological properties. Can compete with

The target has an acidic (anionic) N-terminus and a basic (cationic) C-terminus with a charged region of 6-15 amino acids, usually 6-12 amino acids, which region comprises an amino acid sequence of 6-8 amino acids and is at least 50% The number, usually 60% or more, is the ionic amino acid, and usually 90% or less is the ionic amino acid. Ionic amino acids are basic K and R and acidic D and E.

Those having at least about 60% amino acids have domains separated by at least 25 amino acids, usually at least 40 amino acids or less than 70 and generally less than 65 amino acids. The amino acid linkage sequence separating the charged domains generally has an excess of cationic amino acids less than anionic amino acids at a ratio of about 1.5 to 3, usually 2 to 1, and the pK of the compound is in the range of about 6.5 to 8, especially about 7.4.

Two disulfide bridges are present in the linkage arrangement, and these crosslinked disulfides are sequestered at about 20-45, typically 22-40 amino acids, preferably about 25-39 amino acids. The plurality of cysteines adjacent to the N-terminus are at about 8-16 amino acids from the N-terminus and the plurality of cysteines adjacent to the C-terminus are at about 12-45 amino acids, usually 16-40 amino acids, from the C-terminus.

The object generally has a pentapeptide E-A-E-E-D more generally the decapeptide E-A-E-E-D-G-D-L-Q-C sometimes pentadecape peptide E-A-E-E-D-G-D-D-L-Q-C-L-C-V-K-T and sometimes the following formula: E-A-E-L- Letters in the formula have the following meanings in accordance with conventional practice. A-alanine, L-leucine, C-cysteine, P-proline, D-aspartic acid, Q-glutamine, E-glutamic acid, R-arginine, G-glycine, S-serine, H-histidine, T-threonine, V -Valine.

The object generally has the arrangement of the formula K-K-I-I-K-K at a position close to the C-terminus and more generally the arrangement of K-K-I-I-K-K-L-L preferably P-L-Y-K-K-I-I-K-K-I-I-E-S.

It will be appreciated that conservative substitutions of amino acids can be made.

Conservative changes include substitutions involving D and E: F and Y: K and R: G and A: N and Q: V, I and R. In some cases, non-conservative changes such as substitution of K or R with N or Q are preferred. This substitution is particularly useful where there is a two-basic amino acid protease cleavage site, for example K-R, in which case the protection protects this site from protease cleavage.

It is also possible to include insertions or removals, in which case usually insertions or removals will involve one or two amino acids, in particular one amino acid.

The novel polypeptide of interest has in most cases the following structural formula.

In the above formula, AC _R (acidic region) is N-terminal region having 10-20 amino acids, of which 4-5 are acidic amino acids, at least two of the first three amino acids are acidic amino acids, and two acidic amino acids are randomly arranged. Another two acidic amino acids are sequestered by neutral aliphatic amino acids and two Cys residues are sequestered by one neutral aliphatic amino acid; The Cys-X-Cys is isolated from D or EXD or E by 2 to 6 amino acids. M _R is an intermediate region having a single linkage of 2 to 30 carbon atoms or about 25 to 40 amino acids. Having two cysteines isolated from the cysteines of AC _R by at least 10, generally at least 20 amino acids, which of these cysteines form a disulfide bridge with one of the cysteines in AC _R ; 5 to 7 basic amino acids and 2 to 5 usually 3 to 4 acidic amino acids; Ba _R (basic region) is a C-terminal region having 12 to 30 amino acids; It is characterized by having two pairs of basic amino acids, each connected by two to three trialiphatic amino acids, polar or nonpolar, usually nonpolar, with one proline from 10 to 15 amino acids from the C-terminal amino acid.

Preferably AC _R has the following general formula.

Wherein (H) represents hydrogen at the N-terminus and aa ^1.3 is a bond or a nonpolar amino acid having 2 to 6 aliphatic amino acids, usually acidic amino acids or 2 to 3 carbon atoms; aa ² is a bond or an aliphatic amino acid having 2 to 6 carbon atoms, usually a basic amino acid, a polar amino acid having 3 to 5 carbon atoms, or a nonpolar amino acid having 2 to 3 carbon atoms; X ¹ is a bond or an amino acid sequence consisting of 1 to 2 amino acids of 2 to 6 carbon atoms, usually 4 to 6 carbon atoms, which is an aliphatic nonpolar or polar amino acid; aa ⁶ is an aliphatic or polar aliphatic amino acid having 2 to ⁶ carbon atoms, usually 2 to 4 carbon atoms, or an acidic amino acid; aa ⁸ is a nonpolar aliphatic amino acid having 2 to 6 carbon atoms, usually 5 to 6 carbon atoms; aa ⁹ is an aliphatic amino acid which is particularly polar carboxamide substituted or basic having 2 to 6 carbon atoms, usually 4 to 6 carbon atoms; X ² is a bond or an amino acid sequence of 2 to 6 aliphatic amino acids, in particular 1 to 2 neutral amino acids; aa ¹¹ is an aliphatic amino acid having 2 to 6 carbon atoms, usually 3 to 6 carbon atoms, which is nonpolar or polar; aa ¹³ is an aliphatic amino acid having 2 to 6 carbon atoms, usually 5 to 6 carbon atoms, especially nonpolar; X ³ is a bond, hydroxy, an alkoxy group having 1 to 3 carbon atoms, amino, or 1 to 6, usually 1 to 3 amino acid sequences and is usually neutral aliphatic and nonpolar or polar but especially polar and the first 3 amino acids are Generally neutral and X ³ is at the end of the molecule or linked to M _R , Ba _R or antigen.

Preferred amino acids represented by the above symbols are as follows.

X ³ -bond- (S or T) _b- (G ,, A, N or Q) _a- (V, I or L) _a- (K, R, N, Q, H, F or Y) _a b Is an integer of 0-3.

Preferably Ba _R has the following general formula.

^Wherein aa ^40,56 is an aliphatic acidic amino acid having 4 to 5 carbon atoms or an amide thereof; aa ^{41,42,51,53,63,54,67} is an aliphatic amino acid having 2 to 6 carbon atoms more specifically 5 to 6 carbon atoms, in particular nonpolar; aa ⁴⁵ is an aliphatic amino acid having 2 to 6 carbon atoms, in particular a nonpolar or basic amino acid having 4 to 6 carbon atoms; aa ⁴⁷ is a polar amino acid or acidic amino acid having 3 to 5 aliphatic amino acids, especially hydroxy or amide substituents, and aa ⁵⁵ is a neutral or aliphatic amino acid having 2 to 6 carbon atoms, especially 4-6 carbon atoms A polar amino acid having 4 to 5 carbon atoms having a mid function; aa ⁵⁷ is an aliphatic amino acid having 2 to 6 carbon atoms, especially a nonpolar amino acid having 2 to 3 carbon atoms, or a basic amino acid; aa ⁵⁹ is an aliphatic amino acid having 2 to 6 carbon atoms, usually 4 to 6 carbon atoms, usually a nonpolar or basic amino acid; aa ⁶⁰ is an aliphatic or aromatic amino acid, especially an aliphatic amino acid having 4 to 6 carbon atoms; aa ^64a is a bond or an aliphatic polar amino acid having 4 to 5 carbon atoms, in particular a carboxamide substituted amino acid; aa ⁶⁸ is an aliphatic amino acid having 2 to 6 carbon atoms, especially a non-polar amino acid; X ⁴ is an amino acid sequence of hydroxy, alkoxy having 1 to 3 carbon atoms, amino, or 1 to 4 usually 1 to 2 amino acids; These amino acids are in particular aliphatic amino acids, more specifically polar and acidic amino acids and the arrangement is 0 to 1 nonpolar amino acids having 0 to 3 carbon atoms, 0 to 3 acidic amino acids and 0 to 3 hydroxy substituted aliphatic amino acids. , Wherein X ⁴ is located at the end of the molecule or is linked to an antigen or immunoglobulin.

This is especially important if the symbol has the following definition:

(When two amino acids are shown in the same site, what kind of amino acid may exist in the position). Preferably M _r comprises an arrangement of at least 15 amino acids included in the formula:

Wherein aa ²³ is an aromatic amino acid or an aliphatic polar amino acid having 3 to 5 carbon atoms, especially an amide substituted amino acid; aa ²⁴ is an aliphatic nonpolar amino acid having 2 to 6 carbon atoms and usually 5 to 6 carbon atoms; aa ²⁵ is an aliphatic polar amino acid having 3 to 5 carbon atoms, in particular an amide or hydroxy substituted amino acid; aa ^27,29,30 is an aliphatic nonpolar amino acid of 5 to 6 carbon atoms; aa ³¹ is an aliphatic amino acid having 2 to 6 carbon atoms, a nonpolar amino acid having 2 to 3 carbon atoms, or a basic amino acid; aa ³² is an aliphatic amino acid having 2 to 6 carbon atoms, a nonpolar amino acid having 2 to 3 carbon atoms, or a basic amino acid; aa ³⁴ is an aliphatic amino acid having 2 to 6 carbon atoms, usually 3 to 5 carbon atoms, which is nonpolar or polar, in particular hydroxy substituted; aa ³⁷ is an aliphatic amino acid having 2 to 6 carbon atoms, usually 3 to 5 carbon atoms, which is nonpolar or polar, especially substituted with fluorine or carboxamide; aa ³⁸ is an aliphatic polar amino acid having 3 to 5 carbon atoms and is usually amide or hydroxy substituted; aa ³⁹ is an aliphatic nonpolar amino acid having 2 to 6 carbon atoms; aa ⁴⁰ is an aliphatic acidic amino acid having 4 to 5 carbon atoms or an amide thereof; X ⁵ is a bond, hydroxy, alkoxy having 1 to 3 carbon atoms where X ⁵ is located at the end of the sequence or is connected to Ba _R or to the antigen.

This is especially important if the symbol has the following definition:

Amino acids are classified as follows.

As is apparent from the above formula, various conservative substitutions can be made in the above arrangement without significantly affecting the physiological activity. In addition, one or two amino acids can be removed and inserted. Usually 5 or less, usually 3 or less changes (substitution, removal or insertion) can be made in the above arrangement.

Particularly important compounds for use in the present invention are those having the following configuration.

Or analogs thereof, in particular approximately 4 cysteines at the position at which they correspond and 12 amino acids at or near the C-terminus, in particular 10 amino acids at a position near the C-terminus, and more particularly close to the C-terminus 8 Analogs or fragments containing 4 amino acids (including 4 basic and 4 trialiphatic amino acids). Analogs of such compounds usually have at least about 80%, more usually at least about 85%, preferably at least about 90%, of the same amino acid in the sequence or portion of the sequence.

Natural polypeptides used in the present invention can be obtained in high purity by sensitive biological assays. Natural polypeptides comprise less than about 20%, more usually less than about 10%, preferably less than about 5%, by weight of the non-primary components, which contaminate polypeptide is combined with platelets.

Polypeptides of the invention have a variety of physiological activities. The object of the present invention can be used to inhibit the proliferation of tumors in vitro and in vivo. This object can also be used to stimulate the autophosphorylation of pp60src. This mucus can therefore function as a substrate for the pp60sec enzyme and can be phosphorylated at the tyrosine position (residue 60) of the polypeptide. Tumor cells can also be induced to release 52KD protein when treated with oncostatin-A. Fusion proteins containing oncostatin-A or analogs, fragments thereof, or subarrays (fragments) with competitive immunological properties can also be used for the preparation of monoclonal antibodies or oncostatin-A or immunologically competitive It can function as a reagent in diagnostic measurements to detect the presence of a compound or cell surface receptor for oncostatin-A.

Compounds of the present invention have a high activity in tumor inhibition. This target may be used to slow the rate of proliferation of neoplastic cells in vitro or in vivo. This polypeptide can inhibit about 20% of proliferation of cancer and endothelial tumor cells, such as lung, breast, skin, etc., at 1 ng level. Preferably the polypeptide inhibits tumor cell proliferation of at least about 40%, more preferably at least about 50%, according to colony inhibition assays described in the experimental section.

The object of the present invention can be used in vivo by administering to a host suspected of having neoplasia. This object may also be applied to neoplastic sites such as melanoma in order to slow the rate of proliferation. The method of application depends on the site of the tumor, the formulation of the target, the dosage level and the like and includes injection, introduction by catheter, direct application and the like. The dosage depends on whether it is systemic or local and the dosage concentration is generally about 0.1 μg to 1000 μg / kg and the systemic dose for large mammals including primates is about 0.01 to 10 ng per treatment.

An oncostatin-AFB substance comprising oncostatin-A and its homologs (the homolog has at least one amino acid having at least one physiological property of oncostatin-A and substantially the same as the amino acid sequence of oncostatin-A A compound comprising an array, wherein the homolog has more or fewer amino acids than oncostatin-A), such as a physiologically acceptable carrier, e.g., dichloromide solution, excipient or the like Or may be used alone.

Oncostatin-A and its analogs can be used indirectly to detect the presence of neoplastic cells. When the concentration of the activator in the cell is 1 to 500 ng / ml, preferably about 50 to 350 ng / ml, 52 KD protein (p52) is secreted from the neoplastic cells. Thus, the presence of neoplastic cells can be detected by detecting p2 secretion in external media such as nutrient media, blood, urine or other physiological liquids. Thus oncostatin-A can be used to detect the state of a host and the presence of a neoplastic state. On-Costatin-A can be used to diagnose the presence of tumors, neoplastic levels, etc. in surgical procedures. Sufficient amounts of oncostatin-A isoforms may also be administered in vivo or in vitro (culture medium or host) to induce p52 secretion.

The fluid in the system is then measured for the presence of p52 as an indicator of the presence of neoplastic cells.

The oncostatin-A isoform can be used alone or preferably to stimulate the immune system, in combination with other lymphokines such as interferon, more specifically γ-interferon. That is, the oncostatin-A homolog can be administered to an immunosuppressed host in combination with other polypeptides to stimulate the immune system. γ-interferon is known to induce the expression of Ia in monocytes and macrophages and other tissues such as endothelial and fibroblasts. On-Costatin-A isoforms induce Ia expression and stimulate γ-interpellon Ia induction to enhance the dose effect of γ-interfelon. The amount of oncostatin-A isoform is generally used at a concentration in the range of about 1 to 200, preferably about 2 to 70 ng / ml in the medium. The amount of γ-interferon used is in the range of about 0.5-200 ng / ml of general lymphokine. Oncostatin-A homolog alone or in combination with other lymphokines enhances the expression of Ia by at least about 1.5 fold, usually about 2 fold. The method of administration is as described above. Oncostatin-A isoforms can be used in combination with kinases, particularly pp690src, to alter the substrate specificity of the enzyme. In particular, by contacting this effect with a small amount of oncostatin-A isoform, especially at a concentration of about 0.05-50 μg / ml, kinase activity is enhanced and phosphorylation, including changes in amino acids, is observed, especially in addition to trypsin phosphorylation. Phosphorylated. This combination with pp60src or similar kinase increases the phosphorylation rate of both tyrosine and serine and can therefore be used to denature polypeptides having tyrosine and serineamino acids.

The oncostatin-A homologs of the invention also produce monoclonal antibodies or polyclonal sera as haptens or antigens, for example as haptens linked to immunogenic synergists such as antiparticles or the like. Can be used for These antibodies can be broadly used, especially for diagnostic purposes. This antibody can be used on its own or as an oncotin-A homolog, as a reagent for the detection of an oncostatin-A receptor and an oncotin-A receptor comprising an antibody against oncostatin-A.

, ELISA, SLFIA, FIA가 있고, 이들 모두는 상업적으로 사용되고 있고 또 그들을 위한 시약이 다른 분석대상을 위하여 입수가능하다. 키트로 여러가지 시약을 사용할 수 있고 이 경우에 측정감도를 최적화하도록 시약의 종류 및 그들의 상대량이 선택된다.Various prescriptions and techniques can be used to determine the target analyte. Their technique uses a variety of labels, including enzymes, radioactive, nuclides, fluorescents, co-luminescent agents, enzyme substrates, enzyme inhibitors, particles, and the like. Their methods can include a separation step (homogeneous) or no separation step (homogeneous). The label is covalently bound to an oncostatin-A homolog or an antibody against an oncostatin-A homolog (anti-oncostatin-A) or to an Fc of an anti-oncostatin-A such as anti-oncostatin-A. Conjugated to oriented antibodies. All antigens or fragments thereof including Fab, F (ab) ₂ ′, Fr and the like can be used. A number of US patents have been published describing various diagnostic techniques that can be used in the present invention. Some examples of these patents include US Pat. Nos. 3,766,162, 3,791,932, 3,817,837, 3,996,345, and 4,233,402 and publications. Certain types of measurements include RIA, EIA, EMIT

, ELISA, SLFIA, FIA, all of which are commercially available and reagents for them are available for other analytes. Various reagents can be used in the kit, in which case the types of reagents and their relative amounts are selected to optimize the measurement sensitivity.

Antibodies can be prepared by conventional methods by the preparation of monoclonal antibodies or polyclonal serum.

In each case, an immunogen having one or more epitope sites is injected into a suitable host. Usually, one or more additional immunological scans are performed.

To obtain polyclonal antiserum, the blood of the host is bled and the groverin fraction is isolated.

This globulin fraction can be further purified by affinity chromatography.

To obtain monoclonal antibodies, the spleen of the host is removed and in this case fused with an appropriate fusion partner.

After selection of the hybridomas expressing the desired antibody, the hybridomas are marginally diluted and then further selected and selected for further purification.

Antibodies of the invention can be of any type present naturally, for example IgA, IgD, IgE, IgG and IgM, in particular IgM and various subtypes of IgG, IgG 1, 2, 3 or 4.

The resulting monoclonal antibody has structural similarity with the oncostatin-A type of substance and can be used as an immunogen for the preparation of a flaviodiode antibody. They can then be used as alternative reagents for oncostatin-A type substances in various applications.

On-Costatin-A can be obtained by extraction of platelets with about 0.3 M ethanol hydrochloric acid. Phenylmethylsulfonyl fluoride and aprotinin can also be introduced as inhibitors to degradation, the former at about 1-10% by weight of the extract composition and the latter at about 0.1-1TIU / mg (TIU ... ..... is introduced at the level of tripsin inhibitory unit).

The pH is raised to about 5 with aqueous ammonium hydroxide and then a small amount of ammonium acetate is added to clarify by centrifugation or other convenient means.

The protein is then precipitated using successively cold ethanol (95%) and ether and the precipitate is collected and dialyzed with 0.1-0.5 M acetic acid using a dialysis membrane having a cutoff of less than about 3,000 Mr. The residue is lyophilized and resuspended and quenched in 1M acetic acid and then prepared for further purification by gel permeation chromatography using Biogel P-10.

The product is eluted with about 1 M acetic acid and several fractions are also measured for suitable measuring techniques, such as tumor growth inhibitory activity.

Fractions having anti-proliferative activity are freeze-dried, resuspended and quenched in Trifluuric acetic acid recovery solution (pH 2 to 3), followed by chromatography on high pressure liquid chromatography.

In this case, the silica filler uses a film having a long aliphatic chain of about 16 to 20 carbon atoms, for example 18 carbon atoms. The column is equilibrated with dilute trifluoroacetic acid (0.02 to 0.1%) and the product is eluted with 60% acetonitrile dissolved in dilute (0.01 to 0.1%, usually about 0.04 to 0.05%) trifluoroacetic acid.

It is carried out at a relatively gentle flow rate, generally about 0.5-1 ml / min at ambient temperature.

Fractions are analyzed by the above biological analysis or other biological analysis.

For further purification, the product obtained in the column can be purified using high pressure gel exclusion chromatography.

The main peak of oncostatin-A activity isolated by Novapak C ₁₈ reverse phase liquid chromatography is lyophilized and resuspended in 100 μl of 40% acetonitrile containing 0.1% trifluoroacetic acid.

Samples are injected into a hydroxylated polyether gel column (Bio Rad TSK-250) and eluted by a mobile phase containing 40% acetonitrile dissolved in 0.1% trifluoro acetic acid.

Aliquots of each fraction were lyophilized and tested for oncostatin-A activity and co-eluted with tumor cell inhibitory main peptide peak (Rf-0.9), which was also used to detect the 6000Mr insulin used to measure this chromatographic system. It corresponds to the molecular weight of the marker.

The product obtained in the column is electrophoresed using SDS-PAGE to isolate bands at molecular weights of about 6,000-8,000.

This band was shown to have a strong proliferative inhibitory activity on mammalian neoplastic cells.

Instead of the isolation of oncostatin-A in its natural source or the synthesis of this polypeptide or its analogues on a solid support, oncotin-A may be prepared by hybrid DNA techniques. The structural gene of oncostatin-A can be obtained from the host cell genome using probes purified based on amino acid sequence.

Using this probe (with a reasonable margin), genome libraries are examined to isolate hybridized fragments, reduce the size of these fragments, and specify by restriction map preparation and alignment.

As with other methods, DNAs can be examined for genomic libraries to isolate complete or partial structural genes, which can be used and the latter can be used as adapters to compensate for lost codons.

Conveniently, synthetic genes can be synthesized.

Substantial flexibility is achieved by using synthetic genes and single or few restriction sites can be introduced using codons desired for the host. Restriction sites provide some degree of flexibility in modifying many parts of the gene by introducing loss, transition, transversal, and insertion.

It is also possible to use short chain overlap fragments which can be mixed in the hybridization linking medium without disturbing heteroduplex formation. The resulting double stranded gene can then be purified by cronization. Exemplary arrangements are shown in Experimental Examples.

Preferably the ends of the genes are different to ensure proper orientation upon insertion. This gene can be inserted into an appropriate expression vector for expression.

Many vectors are available for expression in prokaryotes and eukaryotes such as fungi such as yeasts, mammalian cells such as mouse cells, primate cells and the like. Replication systems can be derived from plasmid viruses or chromosomes. Representative replication systems include ColE1, lambda, RSF 1010, 2 μm prasmid, SV40, adenovirus, bovine papilloma virus, and the like.

Where episomal maintenance is desired (a replication system is not required when assembly is required), markers are usually present that allow selection of a host containing the gene of interest in addition to at least one replication system.

These markers typically provide biocidal resistance and provide for example antibiotics or heavy metals or protonutrients to complement, nutritionally demanding hosts. One or more markers may be present, but generally no more than three.

Structural genes are typically located between transcriptional and translational initiation and end control regions recognized by the expression host by insertion into a polylinker (an array with multiple restriction sites).

By appropriate selection of the transcription initiation region, transcription is either constitutive or inductive.

Many promoter regions have been isolated and demonstrated usefulness, such as the E, coli, trp and lac promoters, lambda P _L and lambda P _R promoters, yeast glycolytic promoters, SV40 and early kimchi late promoters of adenoviruses. .

In addition, a fusion gene can be prepared by providing a structural gene with a 5′-array encoding an isolation leader and a processing signal.

Representative separation leaders published include penicillase secretion leaders, α-factor, immunity, globulin, and T-cell receptor outer membrane proteins.

Mature oncostatin-A or the like can be secreted in culture by fusion to the appropriate reading frame.

Constructs containing the structural genes and flanking regions that provide control of expression can be introduced into the expression host by convenient means, for example, by transfection, transfection, track reduction junction, micro injection using calcium phosphate precipitated DNAFMF. Can be.

The host is then grown at high density in nutrient medium. If the promoter is inducible a depletion or excess of, for example, temperature-changing metabolic products or nutrients, or such acceptance conditions are used.

If the product is retained in the host, the cells are harvested and the product is isolated and purified by lysis, extraction, precipitation, chromatography, electrophoresis and the like. When the product is secreted, the nutrient medium is collected and the product is isolated by conventional methods such as affinity chromatography.

Structural gene sequences can be used as probes for hybridization and detection of duplexed arrays in addition to those used for expression.

For example, the presence and amount of mRNA in the host cell can be detected.

The following examples are intended to illustrate the invention, but the invention is not limited thereto.

[Tablet of oncostatin-A]

Acid-Ethanol Extraction from Human Platelets

Platelets (wet weight 50g) thawed at room temperature after fresh or freezing: 2 doses: 375ml ethanol (95%), 7.5ml concentrated HCl, 33mg phenylmethylsulfonyl fluoride and 1ml aprotinin (23TIU / ml; bovine lung) Origin …… Sigma Chemicals A 6012).

The mixture is stirred overnight at 4 ° C. and centrifuged at 8 Krpm for 30 minutes in a Beckman type 19 rotor to separate the supernatant.

The pH of this supernatant was adjusted to 4.0 with concentrated ammonium hydroxide and then the pH was increased to 5.2 using concentrated ammonium hydroxide in a 1: 10 dilution. After adding 1 ml of 2M ammonium acetate (pH 5.2) per 0.1 liter of supernatant, the solution was centrifuged for 30 minutes at 8 Krpm in a Type 19 rotor.

The supernatant was collected and two volumes of cold 95% ethanol were added followed by four volumes of cold diethyl ether and the mixture was left at 0 ° C. overnight. The precipitate was collected by centrifugation at 8 Krp for 30 minutes in the Type 19 rotor, and the pellet was suspended in about 10-20 ml of 1 M acetic acid.

Acetic acid dispersion was sufficiently dialyzed twice with 5 L of 0.2 M acetate in a spectrum and a pore dialysis membrane (# 3) tube (cut-op 3.500Mr) (American Scientific Prodact). The extract was dialyzed, resuspended in 7.5 ml of 1 M acetic acid and then centrifuged at 30 Krpm.

Gel Permeation Chromatography

Biogel P-10 (200-400 mesh; Bio Rad Labs) was swollen overnight in 1M acetic acid, thoroughly degassed, and then injected into a 100 × 2.5 cm siliconized glass column and equilibrated with 1M acetic acid overnight.

Acid ethanol soluble peptide (50-70 mg of protein) was dissolved in 7.5 ml of 1 M acetic acid from 29 g of human platelets and added to the column.

To collect fractions (3.5ml) and freeze-dried again A549 human lung cancer cells were tested for 5- ¹²⁵ I- iodo-2'-deoxy-uridine inhibited introduced.

[Reverse High Pressure Liquid Chromatography]

The column containing the peak of tumor growth inhibitory activity (200 ng of protein) was lyophilized and resuspended in 0.05% (V / V) trifluoroacetic acid in the column.

The column was then eluted by linear gradient using acetonitrile 0.60% solution dissolved in 0.045% trifluoro acetic acid at 23 ° C at a flow rate of 0.8 ml / min.

A portion of each fraction was lyophilized and measured three times as above.

The fraction containing inhibitory activity was then dissolved in 40% acetonitrile containing 0.1% trifluoroacetic acid, added to a hydroxylated polyethergel column (Bio Rad TSK-250), and dissolved in 0.1% trifluoro chosane. Elution was carried out using 40% acetonitrile as mobile phase.

Fractions were collected, lyophilized, and growth inhibition activity was measured three times.

Insulin markers eluted and showed activity in fractions of 6-8 KD.

The highest activity fraction SDS-PAGE was then electrophoresed as follows.

The peptide corresponding to the main oncostatin-A activity in the reverse phase HPLC purification step was lyophilized and 12.5 mM Tris-CL pH 6.7, 4% SDS, 10% β-mercaptoethanol, 20% glycerin and 0.01% brominephenol blue Resuspend and boil in 0.03 ml of containing sample preparation buffer (2 min). This sample was poured onto layered gel in 5% polyacrylamide predominantly on 17-27% polyacrylamide gradient slab gel at pH 8.8 containing 0.1% SDS.

The gel was run at 10 milliamperes until the sample moved through the middle gel and 20 milliamperes until the dye front moved to the bottom of the gel. The gel was fixed and stained overnight in a solution of 0.2% comablue, 50% methanol and 9% acetic acid.

After staining, a hopper density band was used to determine the position of the coma positive band.

Markers include insulin (6,000Mr), trypsinogen (24,500Mr), RNAase (13,700Mr), and aprotinin (6,500Mr).

Zupeptides were run with 6,500Mr aprotinin standard under the above electrophoretic conditions.

The measurement method used was as follows. A549 cells (human lung cancer) developed in NUNC 96 well plates (Kamstruprej 90, DK-4,000 Roskilde, Denmark) on the morning of the second day.

These cells pass through at 30 or less.

45,000 cells / 50 μl / well 10% FCS, P / S, 9 × 10 ⁴ cells per ml of DMEM containing glutamine) were introduced into all wells except the surrounding wells).

50 μl of PBS was poured into the surrounding wells, and the whole plate was incubated at 37 ° C.

In the afternoon, the test samples were resuspended in DMEM containing 10% FCS, P / S, glutamine and run three times. 50 μl was supplied to each test well while 50 μl DMEM was supplied to the control wells and the plates were incubated at 37 ° C. for 3 days.

On day 4 50 ml of a solution of ¹²⁵ I-iodine-2 'deoxyuridine (4Ci-mg-0.5m Ci / ml) (1 μl / isotope / 10% FCS, DMEM ml with glutamine) were added and the plate was brought to 37 ° C. Incubated overnight.

On day 5, the medium was aspirated from the wells, washed once with PBS, 100 μl of methanol was added and left for 10 minutes before methanol was aspirated.

Then, 200 μl of 1 M sodium hydroxide was added and the plate was incubated at 37 ° C. for 30 minutes, and then 1 M sodium hydroxide was removed by a Titertek plug (Flow Labs).

This plug was then counted for radioactivity in a gamma counter.

In order to prove the effectiveness of the on-Costatin-A prepared as described above, the following test was conducted. This test is called Yeonhancheon Colony. Materials used were 75% autoclaved distilled water 75ml, 5% agar [3.75g Nobel agar (Diffco)] DMEM containing 10% FCS, 100U penicillin, 100U streptomycin, 200mM glutamine , And human melanoma cells (A 375).

The material to be tested is lyophilized in a 12 × 75 mm sterile test tube. Use DMEM to make a 1: 10 dilution of 5% agar and heat to 46 ° C in a water bath.

A substrate is formed by pipetting 1 ml of 0.5% agar solution into each well of a 6-well culture plate (35 × 14 mm).

This layer is left at room temperature until cured. SA6 cells are prepared by trypsinization and the cell number is counted.

Dilute the cells to a final concentration of 1 × 10 ⁴ cells / ml and add 0.35 ml of cells to each test sample tube.

Pipette 0.750 ml of 0.5% agar solution into each of the 10 test sample tubes, slowly rotate the mixture and pour the contents of the test sample tubes (test samples, cells, agar) onto the substrate.

And leave at room temperature for about 20 minutes until agar passes. The plates are then stored in a humidification chamber at 37 ° C. containing 50% carbon dioxide.

Plates are measured for inhibition of colony proliferation depending on the titer of the sample until 10 days after 3 days. The number of colonies is counted in a random eight low magnification microscope field of view.

If the plate is to be retained for more than 5 days, dry 1 ml of 0.3% agar solution layer on the test sample layer to prevent drying of the test sample layer.

The above method was used while using oncostatin-A purified at various concentrations.

The table below shows the results and the amount of oncostatin-A shown here is the lyophilized amount introduced into the test tube. Results are expressed in% of maximum inhibition.

TABLE 1

The above results show that the polypeptide of the present invention is an effective inhibitor of cell proliferation. Based on the results observed with melanoma cells, about 1 ng is sufficient to provide about 50% inhibition. Thus, the compounds have a wide range of uses in the inhibition of cell proliferation, including neoplastic cell proliferation.

For example, this compound inhibits proliferation of various cultured human tumor cells as shown in the following table, but does not inhibit the proliferation of normal non-neoplastic human foreskin gland cells.

TABLE 2

* Maximum inhibition of ¹²⁵ I-deoxyuridine binding to A549 cells observed at saturation concentration (100 ng / well) of oncostatin-A using the described measurement conditions does not exceed 50% for untreated control cultures. .

[Effect of On-Costatin-A on Proliferation of Human Cancer Cells in Athymic Mice]

Young (8-10 week) athymic mice were injected subcutaneously with 5 × 10 ⁶ human lung cancer cells (A549) suspended in 100 μl of phosphate buffer solution (PBS). Seven days after the inoculation, a detectable tumor with a diameter of 2.5 to 3.0 mm developed. Tumor-infected mice were randomly selected from this group, and on day 7, subcutaneous injection of 50 μl of PBS containing 0.30 μg of oncotin-A purified uniformly from human platelets as described above was performed subcutaneously. PBS without oncostatin-A was injected into tumor-infected control mice, and had no effect on tumor proliferation.

Tumor-infected mice were then injected with the same amount of oncostatin-A dissolved in 50 μl of PBS on days 11 and 16 after injection of A549 cells. On-Costatin-A was injected directly into the tumor mass using 30 1/2 "g saliva.

Tumor size at the indicated date was monitored by measuring the horizontal and vertical dimensions of the tumor with a caliper: (a) Tumor size shown in ordinates represents the product of these two dimensions. The results are shown in FIG.

[Specific Release of 52,000Mr Polypeptides in Human Lung Cells Treated with On-Costatin-A]

Human lung cancer cells (A549) were treated with oncostatin-A (200 ng / ml culture) to determine the effect on the polypeptide released in the cell culture supernatant. Treated cultures and control cultures (without oncostatin-A treatment) were added with ³⁵ S-methionine (5μ Ci / ml SA-800Ci / m mol) at 0 [Oncostatin-A addition or medium (control) addition. After culturing, the culture supernatant after 12 hours was taken out, and was first quenched at a low speed (15 minutes at 1,500 × g) and then at a high speed (1 hour at 30,000 × g).

Trichloroacetic acid (TCA) was added to the cleared supernatant to precipitate the polypeptide, followed by electrophoresis of sodium dodecyl sulfate polyacrylamide gel on 12.5% acrylamide slab gel.

Radioautography of the gel showed that the supernatant obtained from A549 cells treated with oncostatin-A contained polypeptides labeled 52,000Mr ³⁵ S-methionine, whereas untreated cancer cells secreted trace amounts of protein. At least a 10-fold increase was seen after treatment A) No other qualitative or quantitative difference was seen between the treated culture and the control culture.

[Stimulation of pp60src Autophosphorylation by On-Costatin-A]

pp60src from Proc, Natl, Acad, Sci, USA (1978) 76: 6260-6264 by Erickson et al .; Cold, Spring Harbor Symp, Quant, Biol (1979) 44: 902-917 by Erickson et al. Were used to perform purification by mission no affinity chromatography. 5 μL of purified enzyme (about 0.47 pmoles) contains 100 ng of purified homogeneous oncostatin-A, 20 mM ATP, 5 mM MgCl ₂ , 10 mM Tris-Cl (pH 7.2) and the final reaction volume is 30 ㎕ Incubated for 30 minutes. Two times sample buffer was added to stop the reaction and analyzed by SDS polyacrylamide gel electrophoresis as described in Laemmeli, UK, Nature (1970) 227: 680-685. Slavic gel's autoradiography has clearly shown that it promotes 10 times the autophosphprission of pp60src.

The increase in phosphorylation in the SRC enzyme was not limited to the tyrosine residue but was also seen at the serine position.

[Effect of On-Costatin on Macrophage Ia Antigen Expression]

Wehi-3 is a mouse macrophage cell line that can be induced to express H2 class II antigen by gamma interfelon (γ-IFN). The characteristics of this derivation have been studied by several laboratories and have been found to be identical to normal macrophages induction.

These cells were grown at various concentrations of small plate oncostatin-A in the presence or absence of small amounts of γ-IFN. In either case with or without γ-IFN, oncostatin-A showed proliferation of class II antigens according to the oncostatin-A dose as measured by direct immunofluorescence on FACS. The degree of effect of oncostatin-A was approximately twice the concentration used (˜2-70 ng.ml). Preparation of Monoclonal and Polyclonal Antibodies Specific for On-Costatin-A

[Cross Junction of On-Costatin-A to Bacteria Lipopolysaccharide]

The method of cross-conjugating oncostatin-A to bacterial lipopolysaccharide is a modification of the method developed by Primi and Caszenave J. Immunol (1982) 129 (3): 1124-1129.

10 ng of oncostatin-A and 12.5 ng of cell lipopolysaccharide (LPS; Sigma # L-263) were diluted with distilled water to a volume of 500 μl. 50 µl of 2.5% glutaraldehyde dissolved in PBS was added and the mixture was incubated at room temperature for 30 minutes. The reaction was stopped by adding 50 µl of 2M glycine dissolved in PBS and incubating for 1 hour at room temperature. On-Costatin-A: LPS conjugates were diluted with 10 ml of mixed lymphocyte conditioned media and the filters were sterilized for use in in vitro immune sensitization.

[In Vitro Immunosensitization of Balb / C Splenocytes by Conjugates of On-Costatin with LPS]

In vitro immune sensitization of non-immune splenocytes using a variation of the method described in Reading Immunol Meth. (1982) 53: 261-269.

Mixed lymphocyte conditioned (MLS) media were prepared by incubating the same number of Balb / C and C57 black mouse thymic cells at 4 × 10 ⁶ cells / ml for 48 hours in DMEM containing 2% rabbit serum. This medium was collected and stored at -20 ° C. PBCs were collected by flushing sterile PBS into thioglycolate treated Balb / C mice.

The PEC cells were incubated with 10 ml of MLC medium containing 1 mouse splenocytes and 10 ng of oncostatin-A-LPS conjugate. Cells were incubated for 7 days.

[Production of Monoclonal Antibody]

Hybridomas that synthesize oncostatin-A specific monoclonal antibodies were prepared by collecting immune splenocytes and fusion with SP 2/0 myeloma cells in a 1: 1 ratio.

Hybridomas were tested for the production of oncostatin-A antibodies by Emzyme Linked Mission Norsay (ELSA). Positive hybridomas were cloned twice by limiting dilution. Clones were developed and tested for immunoclobulin classes and injected into Balb / C mice for ascites.

Forty positive hybridoma clones were first developed and retested for anti-oncostatin-A activity. The seven most reactive clones were used to produce ascites in Balb / C mice. The rest was developed and frozen. ELISA was used to test the specificity for a plurality of oncostatin-A, oncostatin-A peptide-KLH conjugates and BSA.

It was reacted with a 1: 3000 dilution of a plurality of oncostatin-A and oncostatin-A peptides. The reaction with the latter was more passive at this time. Immunoglobulins in Russo et al. Anal. Purification was carried out by the caprylic acid precipitation method described by Biochem (1983) 65: 269-271. Paragon analysis and Ouchterlony analysis of immunoglobulins indicated that all immunoglobulins were type IgM.

ELISA Analysis for On-Costatin-A

On-Costatin-A was diluted in 0.1M acetic acid and pipet injected into 96-well Dynatech flakes at 10 ng / well. The solution was dried overnight at room temperature. Plates were blocked by incubating the wells at 37 ° C. for 1 hour with 2.5% BSA, 2.5% bovine fetal serum (FCS) dissolved in PBS.

This plate was then washed twice with 2.5% FCS dissolved in PBS. The hybridomas or antiserum were then added in appropriate dilution. The plates were then incubated at 37 ° C. for 2 hours and washed three times with 2.5% FCS dissolved in PBS.

Vectorabix avidin-biotin-HRP ELISA reagent was used according to the manufacturer's instructions. Between each step the wells were washed with 2.5% FCS dissolved in PBS.

Positive wells were visualized by adding 0.4 ng / ml oxo-phenylene diamine dissolved in 0.1 M sodium citrate solution containing 4 μl of 30% hydrogen peroxide / 10 ml solution. The 30-bunan reaction was continued at room temperature. The reaction was stopped by the addition of 50 μl 1.4 NH ₂ SO ₄ / well.

[Preparation of polyclonal anti-oncostatin antiserum]

Balb / C mice were immunosensitized by nitrocellulose immobilized oncostatin-A. The purpose of this immunosensitization method is to prevent rapid release of the polypeptide by the host. In this way, immunosensitization can be performed by a very small amount of oncostatin-A.

A solution of oncostatin-A dissolved in 0.1 M acetic acid was attached to a small piece of nitrocellulose (Schielcher & Schuell 0.45 탆) and left to dry. For initial immunization nitrocellulose was placed in the abdominal cavity of three Balb / C mice (0.375 ml / mouse) (mouse injected with 0.1 ml of Freund's adjuvant intraperitoneally). Was further sensitized by nitrocellulose to which oncotin was immobilized twice at intervals of two weeks. For further immunization nitrocellulose was cut and subcutaneously injected (0.125 ml / mouse) with homogenization with 0.1 ml of water and 0.1 m of incomplete Freund's adjuvant.

Mouse serum was tested for specificity for oncostatin-A by the ELISA measurement and horseradish peroxidase conjugate protein A was used as the reagent of the second step. Serum was tested for specificity for oncostatin-A peptide-KLH conjugates and blocked plates.

The following arrangements were made in accordance with conventional methods.

This arrangement is designed for use in E. coli and a number of restriction enzyme recognition sites have been devised to facilitate modification of the code sequence. Short gene overlap arrays were prepared and combined in an annealed medium and combined with leading faces to produce a fusion protein from which oncostatin-A could be isolated to obtain a complete gene with the appropriate ends for insertion into the expression vector.

Short chain segments were 5'-phosphorylated with T ₄ polynucleotides and 200 P mol of each segment was reacted with 30 μl of reaction volume (30 mM ATP, 10 mM DTT, 10 mM MgCl ₂ , 1 μg / ml spermidine, 100 mM Tris HCl, pH 7.8 And T4 DNA ligase). dsDNA is digested with BssH II and BamH I and purified on 7% natural polyacrylamide gels.

Plasmid ptrPED 5-1 [HAllewell and Entage Gene (1980) 9: 27; Tacon et al., Mol. Gen. Genet (1980) 177: 427) is digested with endonuclease BssH II and BamH I and a fragment of substantially sufficient length with trpD gene is isolated by preparative gel electrophoresis.

The oncostatin-A gene is linked to the linearized ptrPED 5-1 prasmid to obtain the plasmid ptrP (Onc-A) and transformed E. coli HB101 cells using the ligation mixture.

Transformants are selected by ampicillin resistance and the prasmid is analyzed by restriction endonuclease digestion.

Transformants were grown in Luria broth at 37 ° C. at about 10 ⁸ cells / ml and 3-indol acetic acid (IAA) was added at about 1 mm and proliferation was continued for 1 hour. A portion (1 ml) is taken and centrifuged for a few seconds in an Eppendorf centrifuge tube and the pellet suspended in 500 μl of 7% formic acid containing 5 mg / ml cyanogen bromide. After 24 hours at room temperature a portion is diluted 10-fold in water and oncostatin-A of the diluted sample is measured.

Since oncostatin-A does not have internal methionine but has N-terminal methionine, oncostatin-A is produced in which the cleavage of the fusion protein has the same amino acid sequence as natural oncostatin-A.

The above results demonstrate that the compounds of the present invention have a wide range of uses. In particular, the compounds are useful for the diagnosis and treatment of neoplastic conditions. In treatment, the compounds can slow down tumor cell proliferation and can therefore be used in combination with other aspects of treatment for the destruction of tumor cells. For the treatment, the compound of the present invention induces the production of p52, and as a result, the presence of tumor cells can be known by increasing the p52 level when the compound is administered to a host.

This is very important in determining whether tumor cell removal was successful or metastasis occurred during the treatment of neoplasia.

The compounds of the present invention are also useful as reagents in diagnostic measurements to detect the presence of oncostatin-A or oncostatin-A receptors.

Although described by the detailed description and experimental examples of the present invention for clarity of understanding, it may be practiced with a slight modification within the scope of the appended claims.

Claims (13)

A platelet-related proliferation control agent that inhibits tumor cell proliferation by contacting a plurality of cells with a tumor cell proliferation inhibitory substance having an arrangement substantially identical to at least 15 amino acid fragments having

[In array (H) is hydrogen representing the N-terminus; aa ^1,3 is a bond or an aliphatic nonpolar or acidic amino acid having 2 to 6 carbon atoms; aa ² is a bond or a neutral or basic aliphatic amino acid of 2 to 6 carbon atoms; X ¹ is a bond or an amino acid sequence consisting of ¹ to 2 aliphatic amino acids having 2 to ¹ carbon atoms; aa ⁶ is a neutral or acidic amino acid having 2 to ⁶ carbon atoms; aa ^{8,13,24,27,29,30,41,42,51,53,63,64,67} are nonpolar aliphatic amino acids having 5 to 6 carbon atoms; aa ⁹ is an aliphatic polar or basic amino acid having 4 to 6 carbon atoms; X ² is a binding or aliphatic amino acid of 1-2 amino acids; aa ¹¹ is a neutral aliphatic amino acid having 2 to 6 carbon atoms; X ³ is a binding or aliphatic amino acid sequence of 1 to 6 amino acids; aa ²³ is a neutral aliphatic polar amino acid having 3 to 5 carbon atoms, or an aromatic amino acid; aa ^25,38 is a neutral polar aliphatic amino acid having 3 to 5 carbon atoms; aa ^31,32,57 is a basic or nonpolar amino acid having 2 to 6 carbon atoms; aa ³⁴ is proline or a neutral polar aliphatic amino acid having 3 to 5 carbon atoms; aa ³⁷ is a neutral aliphatic amino acid having 3 to 5 carbon atoms; aa ^39,68 is an aliphatic nonpolar amino acid having 2 to 6 carbon atoms; aa ^40,56 is an aliphatic acidic amino acid having 4 to 5 carbon atoms or an amide thereof; aa ^45,59 is a basic or nonpolar aliphatic amino acid having 5 to 6 carbon atoms; aa ⁴⁷ is an aliphatic polar or acidic amino acid having 3 to 5 carbon atoms; aa ⁵⁵ is a neutral aliphatic amino acid having 4 to 6 carbon atoms; aa ⁶⁰ is an aromatic or neutral nonpolar aliphatic amino acid; aa ^64a is a bond or a polar aliphatic amino acid having 4 to 6 carbon atoms; X ⁴ is hydroxy, alkoxy having 1 to 3 carbon atoms or amino acid sequence of 1 to 4 amino acids] The platelet-related proliferation controlling substance of claim 1, wherein the fragment has a 15 amino acid sequence comprising at least the following covalently extended sequence.

3. The platelet-related proliferation controlling substance according to claim 2, which is the next arrangement of the partially elongated arrangement.

[Aa ⁷⁰ in the array is S, T, A or G] 4. The platelet related proliferation controlling substance according to claim 3, wherein said partially elongated arrangement has the following arrangement at the n-terminus.

[ ^{Aa 24,27,29,30} in the array is V, L or ^Idl and aa ³¹ is G, A, K or R; aa ²⁴ is P, S or T; aa ³⁹ is G, A or P] The platelet-related proliferation controlling substance according to claim 1, wherein the platelet-related proliferation controlling substance has a configuration substantially identical to a 60-amino acid sequence of the following extended sequence.

[In array aa ^* is L or I; aa ^3,5 is G, A, D or E; aa ¹⁸ is G, A, N or Q; aa ³⁰ is K, R, N or Q; aa ^31,57 is G, A, K or R; aa ³⁴ is P, S or T; aa ³⁹ is G, A or P; aa ⁴⁵ is V, L, I, K or R; aa ⁴⁷ is N, Q, S or T; aa ⁵⁵ is V, L, I, N or Q; aa ⁷⁰ is G, A, S or T] The platelet-related proliferation control material according to claim 1, wherein the platelet-related proliferation control material has the following amino acid sequence.

Tumor cell detection material that can be used to detect the presence of tumor cells in cells by contacting a plurality of cells with the following arrangements to detect induced secreted p52 protein:

[In array (H) is hydrogen representing the N-terminus; aa ^1,3 is a bond or an aliphatic nonpolar or acidic amino acid having 2 to 6 carbon atoms; aa ² is a bond or a neutral or basic aliphatic amino acid of 2 to 6 carbon atoms; X ¹ is a bond or an amino acid sequence consisting of ¹ to 2 aliphatic amino acids having 2 to ¹ carbon atoms; aa ⁶ is a neutral or acidic amino acid having 2 to ⁶ carbon atoms; aa ^{8,13,24,27,29,30,41,42,51,53,63,64,67} are nonpolar aliphatic amino acids having 5 to 6 carbon atoms; aa ⁹ is an aliphatic polar or basic amino acid having 4 to 6 carbon atoms; X ² is a binding or aliphatic amino acid of 1-2 amino acids; aa ¹¹ is a neutral aliphatic amino acid having 2 to 6 carbon atoms; X ³ is a binding or aliphatic amino acid sequence of 1 to 6 amino acids; aa ²³ is a neutral aliphatic polar amino acid having 3 to 5 carbon atoms, or an aromatic amino acid; aa ^25,38 is a neutral polar aliphatic amino acid having 3 to 5 carbon atoms; aa ^31,32,57 is a basic or nonpolar amino acid having 2 to 6 carbon atoms; aa ³⁴ is proline or a neutral polar aliphatic amino acid having 3 to 5 carbon atoms; aa ³⁷ is a neutral aliphatic amino acid having 3 to 5 carbon atoms; aa ^39,68 is an aliphatic nonpolar amino acid having 2 to 6 carbon atoms; aa ^40,56 is an aliphatic acidic amino acid having 4 to 5 carbon atoms or an amide thereof; aa ^45,59 is a basic or nonpolar aliphatic amino acid having 5 to 6 carbon atoms; aa ⁴⁷ is an aliphatic polar or acidic amino acid having 3 to 5 carbon atoms; aa ⁵⁵ is a neutral aliphatic amino acid having 4 to 6 carbon atoms; aa ⁶⁰ is an aromatic or neutral nonpolar aliphatic amino acid; aa ^64a is a bond or a polar aliphatic amino acid having 4 to 6 carbon atoms; X ⁴ is hydroxy, alkoxy having 1 to 3 carbon atoms or amino acid sequence of 1 to 4 amino acids] 8. The tumor cell detection substance according to claim 7, wherein the tumor cell detection substance has a configuration substantially identical to at least a 60 amino acid sequence having the following extended configuration.

[In array aa ^* is V, L or I; aa ^3,6 is G, A, D or E; aa ¹⁸ is G, A, N or Q; aa ²⁰ is K, R, N or Q; aa ^31,57 is G, A, K or R; aa ³⁴ is P, S or T; aa ³⁹ is G, A or P; aa ⁴⁵ is V, L, I, K or R; aa ⁴⁷ is N, Q, S or T; aa ⁵⁵ is V, L, I, N or Q; aa ⁷⁰ is G, A, S or T] A monoclonal antibody specific for the platelet-related proliferation control agent according to claim 6. The labeled monoclonal antibody of claim 9. DNA in the following code sequence.

A method of expressing oncostatin-A or a fragment thereof which proliferates a cell under a transcriptional and translational control function that functions in a host cell containing the DNA of claim 11 or a fragment thereof. A polypeptide of the following amino acid sequence or a fragment thereof of at least 15 amino acids.

[Aa ^* in the array is V, I or Idl; aa ⁴⁵ is V, L, I, K or R; aa ⁴⁷ is N, Q, S or T; aa ⁵⁵ is V, L, I, N or Q; aa ⁷⁰ is G, A, S or T]

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