JPS61501708A - Tumor cell growth inhibitor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Platelet-related growth regulators The complexity of hematopoietic cells and their differentiation and proliferation is limited by the simple mechanisms that regulate these events. As the list of separated factors increases continuously, it becomes increasingly clear that Ru. In most cases, these factors are linked to many other proteins with a wide range of functions. It also exists in very small amounts. Factors that have been isolated and proven to be active The offspring are r-interferon, platelet-derived growth factor, colony stimulating factor, and interferon. - polypeptides and proteins such as leukin-2, erythropoietin, and Includes numerous other lymphokines. Isolation, purification and characterization of these blood components There is a substantial interest in This is because they are used to treat diseases such as cancer and This is because it may be used for explanation.

Many pitfalls exist in the isolation of naturally occurring factors. exist and similar Separation systems must be developed to separate the factor of interest from other factors that may have specific properties. It's not a big deal. Second, the substance of interest is uniquely or practically Several means are given for measuring the various components, which are characterized in a qualitatively specific manner. There must be. If the polypeptide of interest has extremely high activity, the target The difficulty of isolating the product is greatly increased. Third, unfavorable shadows on the product of interest We must find a way to avoid harm, especially one that avoids all degeneration. difference Additionally, there are often other substances in the composition that act on and change the substance of interest. The substance that exists and has some desired activity as a result is natural. It is not a substance that exists naturally. Finally, after isolating the desired product in sufficiently pure form, , such as amino acid sequencing, glycosylation numbers, disulfide bridges, and the like. Depending on the matter, one must attempt to physically characterize the polypeptide. No.

Furthermore, the substance must be characterized with respect to its physiological properties.

Finally, even after isolation of purified compounds, characterization of the physiological activity of said compounds Injuries may often prove elusive. In many situations, active may be due to the presence of one or more other compounds, a narrow concentration range, a particular host cell, or find out the physiological activity of a compound and determine its activity. It takes substantial intuitive effort and effort to prove other A' parameters that affect An extended experimental period is required.

Description of prior art It is listed. Nelson-Hamilton and Halley, supra.

(1983) 80:5636-5640. 1) Increase in the introduction of radiolabeled methionine into proteins secreted by The effects of growth inhibitors and epidermal growth factors are described.

Morgan et al. Thromb, Hiernost, (1980) 42 :1652-60 gives the amino acid sequence of human platelet factor 4. D awes et al., Thromb, Res, (1983) 29: Koita Fa reported a polyclonal antibody against vector 4. Lawler, Th romb, Res, (1981) 2 G121-7 is β-throndeglob The sequences and structures of phosphorus and platelet factor 4 are compared.

Summary of the invention At least one of the following properties, i.e. does not inhibit normal cell growth but tumor growth can inhibit pp608re autophosphorylation can induce the secretion of a 52 kD protein from tumor cells. and can be isolated from mammalian platelets and has at least one of the above properties. has an amino acid sequence substantially equivalent to at least a portion of a polypeptide that exhibits Polly! characterized by having; Zotide composition and its uses provided. The polypeptide is provided in substantially pure form.

Figure 1 shows the effect on tumor growth in athymic mice. It is a chart comparing the effects of costatin-A.

Polypeptides, derivatives, fragments, or the like that inhibit mammalian neoplastic cell growth Compositions are provided comprising the like. The polypeptide of this invention is effective for platelets. Regarding the natural poly(peptide) in the ethanolic HC1 fraction obtained by extraction, this It is called platelet factor 4 in the literature, and hereafter oncostatin-A. It is called (0ncostatin-A). This polypeptide is found at moderate temperatures (0-25°C), low-, generally below about pH 3, usually stable at pH 2 . This polypeptide ranges from about 5,000 to s, ooo, more precisely about 6,000 s, ooo. 000 to 7,500, more specifically about 7.000 to 7,500. It has a molecular weight of OOO . This polyglotide is found in platelets in higher animals, especially primates, and more specifically in humans. obtained from.

The polypeptide compounds used have approximately 15-80 amino acids and are naturally occurring poly-2f Tido and its mimetic analogs contain about 60-75 amino acids, more commonly about 65-72 amino acids. amino acids, while fragments generally have about 15 to 60 amino acids, more commonly 15 ~35 amino acids. Approximately 68-72 amino acids, further specified Of particular interest are the puri 27″ tides with 69.70 or 71 amino acids. These polypeptides can bind to other compounds, such as antigens, receptors, labels, etc. can be done.

The polypeptide contains at least one biologically active, e.g. have active sequences as Dogue, and have more than one biologically active sequence. and in which case such sequences cannot compete with natural products for biological properties. You will be able to do that.

The composition of interest is an acidic (anionic) N-terminus and a basic (cationic) C-terminus. This region has a charged region of 6 to 15 amino acids, usually 6 to 12 amino acids. contains an amino acid sequence of 6 to 8 amino acids, and the number of amino acids is 50 or more, usually 60 or more. The number of amino acids is ionic amino acids, and usually less than 90% of the number is ionic amino acids. It is an acid. Ionic amino acids are basic, K and R: acidic, D and E.

Compositions having about 60 or more amino acids include 25 or more amino acids, usually 40 or more amino acids. the above amino acids, and less than about 70 amino acids, and generally less than about 65 amino acids It will have multiple charged domains separated by. separate charged domains Amino acid concatenation sequences contain excess cationic amino acids over anionic amino acids. and generally have a ratio of about 1.5 to 3, usually 2 to 1, and the pK of the compound is about 6.5. ~8, particularly around 7.4.

There are two disulfide bridges in the linking sequence, and these bridge disulfides are about 20-45, usually 22-40 amino acids apart, preferably about 25 and They are separated by 39 amino acids. Multiple cysteines proximal to the N-terminus are N- Approximately 8 to 16 amino acids from the terminal bed, multiple cysteines proximal to the C-terminus - About 12-45 amino acids from the end, usually 16-40 amino acids.

Compositions of interest generally include the interpeptide F)-A-F)-1)(), and more generally and decapeptide E-A-E-ED-G-D-L-Q-C.

Often the interdecapeptide E-A-E-ED-G-D-L-Q-C-I, - C-V- to -T, and more often the formula: E-A-E-E-D-C-D -L-Q-C-L-C-V-ni-T-T-8-Q-V-R-P-R-H-<7) It will have an N-terminally proximal sequence having the formula:

Here, the characters have the following meanings according to common law.

A-alanine L-leucine C-cystine P-zoroline D-7s Al ragic acid Q-glutamine E-glutamic acid R-arginine G-Glycine S-Seri/ ■-Histizone-Threonine Sorbaline Compositions of interest generally have the formula -I -I- in the vicinity of the C-terminus. more generally in the formula -I-I- to-L-L, and preferably would have the sequence P-L-Y- to-I-I- to-L-L-E-8. cormorant.

Conservative substitution of amino acids It should be understood that ns) can be performed. D and E for conservative changes Substitutions involving LF and YAK and R; G and A: N and Q: v, Eng and R1, etc. is included. - In some cases non-conservative changes, such as K or R and N or Q would be preferred. This substitution results in 2-basic amino acid protease cleavage. Of particular interest is the presence of -R at the site, e.g., in which case the substitution This site is protected against aase cleavage.

Additionally, insertions or removals can be included, where insertions or removals typically include 1 to Two amino acids, especially one amino acid, will be involved.

A new and interesting poIJ <zotide, in most cases, has the following structural formula: AcR (acidic region) is the N-terminal region and has 10-20 amino acids. However, 4 to 5 of these are acidic amino acids (6=), and at least the first 3 amino acids are acidic. Also, two acidic amino acids are acidic, and the two acidic amino acids are arranged in tandem. , and the other two acidic amino acids are separated by a neutral aliphatic amino acid. There are two e711 residues separated by one neutral aliphatic amino acid; this ay s-X-Cys separated from D or g-X-D or E by 2 to 6 amino acids characterized by being; MR is in the intermediate range, with 1 short chain of 2 to 30 carbon atoms or about 25 to 40 carbon atoms. having at least 10, generally at least 20 amino acids; It has two cysteines separated from that of Acg, and these cysteines That of Tin forms a disulfide bridge with one of the cysteines in Acn. ; has 5 to 7 basic amino acids and 2 to 5, usually 3 to 4 acidic amino acids; ; Ban (basic region) is the C-terminal region and has 12 to 30 amino acids. It has two pairs of basic amino acids, each of which has two to three neutral aliphatic amino acids. polar or non-polar, usually non-polar; the C-terminal amino acid It is characterized by having one proline in every 10 to 15 amino acids.

Preferably, A is the following formula: %formula% (H) is intended to be hydrogen at the N-terminus; aa'.3 is a bond or has 2 carbon atoms ~6 aliphatic amino acids, usually acidic amino acids or having 2 to 3 carbon atoms aa2 can be a bond or a nonpolar amino acid having 2 to 6 carbon atoms; Aliphatic amino acids, usually basic amino acids, polar amino acids having 3 to 5 carbon atoms, or is a nonpolar amino acid having 2 to 3 carbon atoms; Xl is a bond or an amino acid having 2 to 6 carbon atoms, usually 4 to 6 carbon atoms; An amino acid sequence consisting of two aliphatic nonpolar or polar amino acids. ; aa' is a nonpolar or polar group having 2 to 6 carbon atoms, usually 2 to 4 carbon atoms. is an aliphatic amino acid or an acidic amino acid; aa8 is an aliphatic amino acid having 2 to 6 carbon atoms, usually 5 to 6 carbon atoms; and is usually non-polar; aa9 has 2 to 6 carbon atoms, usually 4 to 6 carbon atoms. aliphatic amino acids, especially polar cardexamide-substituted or basic X2 is a bond or an aliphatic amino acid having 2 to 6 carbon atoms, especially a neutral amino acid. An amino acid sequence consisting of 1 to 2 amino acids; aall is an aliphatic amino acid having 2 to 6 carbon atoms, usually 3 to 6 carbon atoms. As such, it can be non-polar or polar; lLa15 is an aliphatic amino acid of 2 to 6, usually 5 to 6 amino acids, with special is nonpolar; X3 is a bond, hydroxy, alkoxy group having 1 to 3 carbon atoms; , amino, or an amino acid sequence of 1 to 6, usually 1 to 3 amino acids, usually middle aliphatic, either nonpolar or polar, especially polar, and the first three amino acids is generally neutral, where X3 can be the terminal end of the molecule, or MR,B Can be linked to aR or antigen.

Preferred amino acids for the above symbols are as follows.

aa - bond, D, E, G, A aa2-bond-G + AtK-R-s eTXl-bond, (s or T )lL-(V, L or Engineering) aa is 0 or 1 aa6-S * T e G t A, D, Ban8-V, L, I aa9”’ N t Q t K t RX2-(G or A) a-(D or E) &-(V, L or I) aaa, 7'-'V, L, I*M, S, Taa12-V , L, l X3-bond-(S or T)b-(G, A, N or Q)lL-(V, I, or L) , -(K, R, N, Q, H, F or Y).

b is an integer from 0 to 3.

Preferably B a R is the following formula: %formula% aa40.56 is an aliphatic acidic amino acid having 4 to 5 carbon atoms or its amide. the law of nature; lLa41, 42, 51, 55, 65, 64.67 has 2 to 6 carbon atoms, and It is an aliphatic amino acid having 5 to 6 carbon atoms and is particularly nonpolar; aa45 is an aliphatic amino acid having 2 to 6 carbon atoms, especially a non-alphatic amino acid having 4 to 6 carbon atoms. Dust with polar amino acids or basic amino acids; aa is an aliphatic amino acid having 3 to 5 carbon atoms, especially hydroxy or amide A polar amino acid having a substituent or an acidic amino acid; , a55 is a neutral aliphatic amino acid having 2 to 6 carbon atoms, especially 4 to 6 carbon atoms. polar amino acids of 4 to 5 carbon atoms with a nonpolar or carmexamide functionality ; aa57 is an aliphatic amino acid having 2 to 6 carbon atoms, especially a non-alphatic amino acid having 2 to 3 carbon atoms. is a polar amino acid or a basic amino acid; a, 59 is an aliphatic amino acid having 2 to 6 carbon atoms, usually 4 to 6 carbon atoms; an ordinary non-polar or basic amino acid; aIL60 is an aliphatic or aromatic amino acid, especially an aliphatic amino acid having 4 to 6 carbon atoms. aa64'L is a bond or an aliphatic pole of 4 to 5 carbon atoms; amino acids, especially amino acids substituted with xamide; , a6B is an aliphatic amino acid having 2 to 6 carbon atoms, especially a nonpolar amino acid; X4 is hydroxy, alkoxy having 1 to 3 carbon atoms, amine, or , usually an amino acid sequence of 1 to 2 amino acids, which are particularly aliphatic amino acids. amino acids, more specifically polar and acidic amino acids, said series being 2-3 0 to 1 nonpolar amino acids with carbon atoms, 0 to 3 acidic amino acids and 0 ~3 hydroxy-substituted aliphatic amino acids, where X4 is located at the end of the molecule or to an antigen or immunoglobulin.

It is of particular interest if the symbol has the following definition:

aa40”’-D e E * N s Q, La41,42,51,53, 65, 64 + 67 - v, L again, Iaa, -N, Q, S, T, D, E jLa -N e O # P * L # I e V; especially P or L a, 64a-bond, N or Q aa　-GtA+P+L+ItV X'-(G, A, D or ") a-(s # T # D, A, G or E) 8 -(D or E)c-(T or 5)3 C is O~2° (If two amino acids are shown at the same position, which amino acid is shown at that position) may exist. ] Preferably, Mr has the following formula: %formula% comprising a sequence of at least 15 amino acids contained in here, a, 23 is an aromatic amino acid or an aliphatic polar amino acid having 3 to 5 carbon atoms, especially is an amide-substituted amino acid; , a24 is an aliphatic nonpolar amino acid having 2 to 6 carbon atoms, usually 5 or 6 carbon atoms. 1L1L is an aliphatic polar amino acid having 3 to 5 carbon atoms, especially an amino acid. or hydroxy-substituted amino acids; aa27.29.30 has 5 carbon atoms ~6 aliphatic non-polar amino acids; aa is an aliphatic amino acid having 2 to 6 carbon atoms; Can be either a non-polar amino acid or a basic amino acid; aa52 is a carbon Aliphatic amino acids having 2 to 6 atoms, nonpolar amino acids having 2 to 3 carbon atoms is either an acid or a basic amino acid; ,,54 is an aliphatic amino acid having 2 to 6 carbon atoms, usually 3 to 5 carbon atoms. is non-polar or polar, especially hydroxy-substituted; a& is carbon An aliphatic amino acid having 2 to 6 prime atoms, usually 3 to 5 carbon atoms, which can be nonpolar or polar, especially proline or cardexamide substituted: ,,5B is an aliphatic polar amino acid having 3 to 5 carbon atoms, usually amide or Hydroxy-substituted; ,,59 is an aliphatic nonpolar amino acid having 2 to 6 carbon atoms: &&40 aliphatic acidic amino acids having 4 to 5 carbon atoms or amides thereof; X5 is a bond, hydroxy, alkoxy having 1 to 3 carbon atoms, where X5 is located at the end of the sequence and is linked to B a R or to the antigen.

The following larger definition of the symbol is particularly interesting.

aa-F, H, Y, N, QalL24, 27129jO-y, I, , 1, La25.58-3, T, Ne Qaa39-G g A r P r” a L e Iaa-D, E, N, Q Amino acids are classified as follows.

Non-polar G, A, P, V, L, I Basic K, R As is clear from the above formula, the above without any significant effect on physiological activity. Various conservative substitutions can be made in the sequence of. Additionally, 1-2 amino acids Removal and insertion of can be used. Usually 5 or less changes, usually 3 or less changes ( Substitutions, deletions or insertions) may be made in the above sequences.

Of particular interest for use in this invention are the following sequences: Glu Ala Glu Glu Asp Gly Asp Lau Gin Cys LeuCys Val Lys Thr Thr Ser Gin V al Arg Pro ArgHis IIs Thr Ser Leu Gl u Val Ile Lys Ala GlyPro His Cys Pro Thr Ala Gin Leu Ile Ala ThrLeu Lys Asp Gly Arg Lys Ile Cys Leu Asp LeuG in Ala Pro Leu Tyr Lye Lye Ile Ile L ye LysLauLeuGluSer or analogues thereof, especially those having four syllables in approximately their respective positions. stin, and 12 amino acids at or near the C-terminus, particularly near the C-terminus. and more specifically 8 amino acids proximal to the C-terminus. (including 4 basic and 4 neutral aliphatic amino acids) It's a piece. Analogs of the above compositions are typically about 80% or higher, more usually about 85%. or more, and preferably about 90% or more, of the above sequence or part of the above sequence. Contains amino acids.

The natural polypeptide compositions used in this invention can be used in sensitive bioassays. can be obtained in high purity, as established by. Detide composition on natural poly is less than about 20% by weight, more usually less than about 10% by weight, and preferably about Contains less than 5 parts by weight of polybezotide other than the main component present in the composition. cormorant. This contaminating polybezotide is associated with platelets.

The polypeptide compositions of this invention have a variety of physiological activities. Composition of this invention The product is used to inhibit tumor growth in vitro and in vitro. be able to. The composition also facilitates autophosphorylation of pp6osrc. It can be used to stimulate. Therefore, this composition has a pp60 sr can function as a substrate for the c enzyme, and the tyrosine of the l IJ peptide can be phosphorylated at position (residue 60). Furthermore, oncostatin-A When treated, tumor cells can be induced to release the 52kD protein.

Additionally, oncostatin-A or analogs, fragments thereof, or competitive immunological properties. A fusion protein containing a sub-sequence (fragment) with the quality of a monoclonal antibody is Oncostatin-A or immunologically competitive Detecting the presence of a competitive compound or cell surface receptor for oncostatin-A It can serve as a reagent in diagnostic measurements.

The compounds of this invention have high activity for tumor inhibition. This composition is in- Vitro or Impi! used to slow the growth rate of neoplastic cells in can be used. This poly-2ftide composition has an In,F level of about Inhibits tumor cell proliferation of 20 or more cancers, especially lung, breast, skin, etc. cancers and sarcomas. can bring. Preferably, the polypezotide composition is as described in the experimental section. According to the colony inhibition test listed above, about 40% or more, and more preferably will result in inhibition of tumor cell growth of about 50% or more.

The composition of this invention is suspected of having neoplasia. By being administered to the host, it is possible to reduce the rate of tumor reproduction at the neoplastic site, e.g. can be applied to melanoma. The method of application depends on the site of the tumor, the dosage form of this composition, the administration C, injection, catheterization, direct application, etc. depending on the amount level, etc.

The dosage will vary depending on whether it is systemic or local, and the dosage concentration will generally be around 0. ．． 1 μF - 1000 μb to total The dosage is about 0.01-10rn9 per therapeutic dose.

Oncostatin-A-like substances, including oncostatin-A and its congeners substance has at least one physiological property of oncostatin-A and at least one amino acid sequence substantially the same as the amino acid sequence of ncostatin-A oncostatin-A, where analogs are compounds containing more or less oncostatin-A. (with no number of amino acids) in a physiologically acceptable carrier, e.g. phosphate buffer. can be formulated in salt solution, distilled water, excipients or the like; Or it can be used as is.

Oncostatin-A and its congeners are used indirectly to detect the presence of neoplastic cells. can be used for. Cells are approximately 1-500n! i’/mA! activator, good Preferably, when subjected to a concentration of active agent of about 50-350 ng/ml, the new A 52 kD protein (,52) is secreted by biological cells. Therefore, external media , detecting the secretion of p52 into, e.g., nutrient media, blood, urine, or other physiological fluids. By doing so, the presence of neoplastic cells can be detected. Follow Oncosta Chin-A can be used to monitor the condition of the host and the presence of neoplastic conditions. O Ncostatin-A is used to diagnose the presence or absence of tumor and the level of neoplasm during surveillance surgery. can be used to Oncostatin-A leads to induction of secretion of 52 in vitro or in vitro (in a culture medium or host) in a sufficient amount to ) will be administered. The fluid associated with the system is then an indicator of the presence of parental biological cells. will be monitored for the presence of 52.

Oncostatin-A-like substances can also be used by themselves, but preferably in combination with other phosphorus Hokines, such as interferon, more specifically gamma-interferon Can be used in combination to stimulate the immune system. i.e. oncostatin -A-like substance is combined with other +]'lj pezotide and to stimulate the immune system can be administered to immunosuppressed hosts. γ-interferon stimulates monocytes and macrophages. expression of phage, as well as in other tissues such as endothelial and fibroblast cells. is known to induce Oncostatin-A-like substances induce I expression and to stimulate γ-interferon I induction after a given administration of γ-interferon. Enhances the quantity effect. The amount of oncostatin-A-like substance in the vehicle is generally about 1 to 20 0, preferably in the range of about 2 to 70 n,9/d. It will be done. The amount of γ-interferon depends on its use, as the lymphokine is generally It will be in the range of 0.5 to 200 n,li'/ml as usual.

Expression was enhanced by about 1.5 times or more, usually about 2 times or more, by oncostatin-A-like substances. and when used by itself or in combination with other lymphokines. , achieved. Administration may be carried out as described above. Oncostatin-A-like substances also combine with kinases, particularly f1p60src. can be used to alter the substrate specificity of the enzyme. In particular, the enzyme Small amounts of Oncostatin-A-like substances, especially at concentrations of about 0.05 to 50 μI/1 rLl. Kinase activity is enhanced by contacting the phosphate at Includes the observed amino acid changes that are phosphorylated, especially tridicine. In addition, serine is also phosphorylated.

Thus, combinations with ppsosrc or similar kinases tyrosine and phosphorus, resulting in phosphorylation of both at an enhanced ratio It can be used to modify polypeptides with serine amino acids.

Oncostatin-A-like substances of this invention can also be used as hasitenes or antigens, e.g. a hapten linked to an immunogenic synergist, such as an antiparticle or the like; can be used for the production of monoclonal antibodies or polyclonal serum. . These antibodies can be used in a wide range of ways, especially for diagnostic purposes. The antibody is Oncostatin A and oncostatin-A receptor, including antibodies against oncostatin-A and oncostatin-A receptors. It can be used as a reagent for detection.

Various formulations and techniques can be used to determine the analytes of interest. child These techniques involve enzymes, radionuclides, fluorescent agents, chemiluminescent agents, enzyme substrates, and enzyme inhibitors. , particles, and the like. These methods take minutes Can include a separation step (heterogeneous) or not include a separation step (homogeneous). Label is an oncostatin-A-like substance or an antibody against an oncostatin-A-like substance (anti- covalently linked to oncostaf7-A) or anti-oncostatin-A1 example For example, it is conjugated to an antibody directed against the Fc of anti-oncostatin-A. whole antigen, or Fab　F(ab)'2', Fv, or similar. You can use fragments of it. Various diagnostics that can be used in this invention A number of US patents have been issued describing cutting techniques. A few examples of these patents U.S. Patent No. 3,766,162, No. 3,791,932, No. 3,817,83 7, A3,996,345, and A4,233,402. specific type Measurement methods include RIA, EIA, EMIT■, ELISA, 5LFIA, F IA is included, all of which are commercially applicable, and the Reagents are available for other analytes.

Various reagents can be used in the kit, in which case the sensitivity of the measurement can be optimized. The types of reagents and their relative amounts are selected so as to

Antibodies can be prepared in a conventional manner depending on the preparation of monoclonal antibodies or polyclonal sera. It can be prepared using In each case, a suitable host has one or more An immunogen containing an epitope site is injected, usually followed by one or more booster injections. A booster injection will be given. For polyclonal antisera, the host has blood drawn. and the globulin fraction is isolated. This immunoglobulin fraction has affinity Further purification can be achieved by chromatography.

For monoclonal antibodies, the host is immunized as described above, but In this case, the spleen is usually removed and fused with an appropriate fusion agent. It will be done. After selection of hybridomas expressing the desired antibody, this Ibridomas are subjected to critical dilution, then selected and cloned, and then further characterized.

The antibodies of this invention may be of any naturally occurring type, such as IgA, IgD, gE, EgG and IgM, especially IgM, and the various subtypes of IgG, such as It can be G1.2.3 or 4.

The resulting monoclonal antibodies conformate to oncostatin-A type substances. Immunogens and immunogens for the production of anti-ideotizo antibodies that will have similar properties. It can be used as These are then used as oncostatin-A drugs in a variety of applications. It can be used as an alternative reagent for other types of substances.

Oncostatin-A is obtained by extraction of platelets with approximately 0.3M ethanolic hydrochloric acid. can be obtained. Phenylmethylsulfonyl fluoride as an inhibitor against degradation. Ruolide and aprotinin can also be introduced, the former being about 1-1% of the extract composition. At a level of 0% by weight, and the latter approximately 0.1 to ITIU/r of the extract composition It is introduced at the level of n9 (TIU... trypsin inhibitory unit). water After raising the pH to about 5 using ammonium hydroxide, a small amount of ammonium acetate is added and the solution is clarified by centrifugation or other convenient means.

The proteins were then precipitated using cold ethanol (95%) and ether. , collect the precipitate, and use a dialysis membrane with a cutoff of about 3,000 Mr or less. Dialyze against 0.1-0.5M acetic acid using The residue was lyophilized and dissolved in 1M acetic acid. resuspended, clarified, and then gel permeabilized using Bior/I/P-10. Prepare for further purification by chromatography. The product is diluted with approximately IM acetic acid. and separate the various fractions using appropriate measurement techniques, e.g. tumor growth inhibition. Monitor.

Both components having growth inhibitory activity were lyophilized and added to a dilute trifluoroacetic acid aqueous solution (pH 2~ 3), resuspend, clarify and then chromatograph on a high pressure liquid chromatograph. rough treatment, in which case the silica filling has about 16 to 20 carbon atoms, e.g. It has a coating of long aliphatic chains of carbon atoms. The column was filled with dilute trifluoroacetic acid (0,0 2-0.1%] and the product is diluted (0.01-0.1%, usually approx. 0.04-0.05 thiacetonitrile up to 60 thiacetonitrile in trifluoroacetic acid Elute with a nitrile gradient. Relatively slow flow rate, typically at ambient temperature Approximately 0.5 to 1 m 11 minutes is used in this case. Both parts are the bioassay described above or It can be measured by other bioassays. Column for further purification The product obtained from can be purified using high-pressure exclusion chromatography. can.

By Novapak CI B reverse phase liquid chromatography The separated main peak of oncostatin-A activity was lyophilized and added to 0.1% Resuspend in 100μ of 40 thiacetonitrile containing lifluoroacetic acid. sa sample in a hydroxylated polyether column (BioRad TSK-250). and a solution containing 40 thiacetonitrile in 0.1 trifluoroacetic acid. Elute with mobile phase. Both aliquots of each were lyophilized and Oncosta Test for Chin-A activity. Tumor cell inhibitory activity is the main pezotide peak (R4- 0,9), which also provides guidance for this chromatographic system. The molecular weight of the 6,000 Mr incilin marker used for We will respond accordingly.

The product obtained from the column can be electrophoresed using SDS-PAGE. Wear. A band at a molecular weight of approximately 6,000-s, ooo is isolated. this van is shown to have strong growth inhibitory activity against mammalian neoplastic cells.

Isolation of Oncostatin-A from natural sources or the polypeptide on a solid state Alternatively, oncostatin-A can be synthesized using hybrid DNA technology. It can be prepared from The structural gene of oncostatin-A has the amino acid sequence can be obtained from host cells using probes prepared based on . child probe the r-Tsum library using probes (which would be suitably redundant) and Isolate the fragments that hybridize and reduce the size of the fragments and restrict the Characterize by diagramming and sequencing.

Another method is to examine the DNA for the r-Tsum library in the same way, and These can be used once a complete or partial structural gene is isolated; used by using an adapter to replace the deleted codon. .

Conveniently, synthetic genes can be synthesized.

Substantial flexibility is achieved through the use of synthetic genes, allowing host-preferred code unique or rare restriction sites can be introduced. restriction site Gene species can be modified by introducing deletions, transitions, transpersions, insertions, etc. Adds a degree of flexibility when deforming different parts. heteroduplex Can be mixed into the hybridization coupling medium without interfering with the formation We devised a strategy using single-stranded overrunning fragments. Next, the two that occurred The chain gene can be cloned and purified. Example sequences in experimental section show.

Preferably, the ends of the genes are different to ensure proper orientation upon insertion. child The gene can be inserted into a suitable expression vector for expression.

Prokaryotes and eukaryotes, such as fungi, such as yeast, mammalian cells, such as mice. A large number of vectors are available for expression in cells, primate cells, etc. multiple Production systems can be derived from cilasmids, viruses or chromosomes. Representative reproduction The system is Co1E1, Lambda, R8F 1010.2pmf Lasmi I', SV40. Includes adenovirus, bovine papilloma virus, etc.

If episomal maintenance is desired (if integration is required, a replication system is not required). In addition to at least one replication system, selection of a host containing the gene of interest is necessary for There will usually be an enabling marker. This marker usually indicates biocide resistance. providing, e.g. antibiotics or heavy metals, or complementing, prototrophy to the auxotrophic host will bring sex.

Structural genes are usually inserted into polylinkers (sequences with many restriction sites). However, if the transcription and translation initiation and termination control regions are recognized by the expression host, There will be. By appropriate selection of the transcription initiation region, transcription can be constitutive or inducible. It is. Multiple bromotor regions, such as E. coli (E. colt) trp and lac promoter, lambda PL and PR promoter, yeast glycolytic enzyme promoter promoter, SV40 and adenovirus early and late promoters; Others similar to these have been isolated and shown to be useful.

Additionally, the 5'-sequences encoding the secretory leader and processing signals were constructed. A fusion gene can be prepared by adding a fusion gene to a gene. Are listed Typical secretory leaders include the inicilinase secretory leader, α-factor, Immunoglobulins, T-cell receptors, outer membrane proteins, and similar proteins is included. By merging into the appropriate reading frame, mature oncosta Tin-A or analogs may be secreted into the medium.

Constructs containing structural genes and flanking regions that provide control of expression are transformation using convenient means, e.g. calcium phosphate precipitated DNA. transfection, transduction 1, conjugation, microinduction It can be introduced into an expression host by injection or the like. Next, set the host to a suitable It can be grown to high density in nutrient media. the promoter is inducible permissive conditions, e.g. temperature changes, depletion or excess of metabolic products or nutrients. , or something similar to these can be used.

If the product is retained in the host, cells are harvested, lysed, and extracted, precipitated, The product is isolated and purified by chromatography, electrophoresis, etc.

If the product is secreted, collect the nutrient medium and use conventional methods, e.g. The product can be isolated by chromatography.

Structural gene sequences are used for expression as well as for duplexing sequences. It can be used as a probe for imridation and detection.

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

The following examples are given by way of illustration and not limitation.

experiment Abbreviations: DMEM...Dulpeco's improved Eagle medium; PBS...phosphoric acid Buffered salt solution; P/S... Yesillin/Streptomycin (0.57 μm each) /mA; FC8...-Fetal bovine serum; SDS-PAGE...Dodecyl sulfur Sodium acid-polyacrylamide electrophoresis.

Purification of oncostatin-A Double volumes of fresh or frozen platelets (50 g wet weight) thawed at room temperature: 375 mJ ethanol/' (95%), 7.5 ml concentrated HCt, 33rn9 phenyl Methylsulfonyl fluoride and 1-aprotinin (23 TIU/d; bovine Lung-derived: resuspended in Sigma Chemical Co., Ltd. A6012). Bring this mixture to 4℃ Stir overnight at The mixture was centrifuged for 0 minutes, and the supernatant was removed.

The - of this supernatant was adjusted to 4.0 with concentrated ammonium hydroxide, and the voice was adjusted to 1. = 5.2 using a 10 dilution of concentrated ammonium hydroxide. 0. IJ's Kamikiyoto After adding 2M ammonium acetate (pH 5,2) from the first sample, this solution was added to the third sample. Centrifugation was performed for 30 minutes at 8 krpm in a 90-meter. Remove supernatant and double volume of cold 95% ethanol, then 4 times the volume of cold diethyl ether, and then The mixture was left at 0°C overnight. 8k rpm in Thailand 7'190-tar Collect the precipitate by centrifugation for 30 minutes at Suspended in mJ of 1M acetic acid. The acetic acid dispersion was mixed with Spectrapol (Spectra). -por) Dialysis membrane (3) tube (cutoff 3,500 Mr) (flax - Jam Scientific Products), with 0.51 exchanged twice each. Extensive dialysis was performed against 2M acetic acid. The extract was dialyzed and diluted with 7.5 ml of 1M acetic acid. and then centrifuged at 30 krpm.

Glue permeation chromatography Bioglu P-10 (200-400 meth; Bio-Rad Lapus) was added to 1M acetic acid. Allow to swell overnight in silico, thoroughly degasse, and then add 100×2.5cn1 silico. and equilibrated with 1M acetic acid overnight. .

Acid-ethanol solubilized isotide from 29 g human platelets (50-70 m9 protein white matter) was dissolved in 7.5 grams of 1M acetic acid and applied to the column described above. Fraction ( 3, sy) were collected and aliquots were lyophilized and A349 human lung cancer cells were collected. Regarding inhibition of the introduction of 5 + 1251-iobi 2'-deoxyuridine into cells. It was tested.

Reversed phase high pressure liquid chromatography Both fractions containing the peak of tumor growth inhibitory activity from the above column (protein 200n, ! 7) was lyophilized and dissolved in 0.05% (v/v) trifluoroacetic acid. Resuspend. The column was then run at a flow rate of 0.8 m//min at 23°C. 0.60% linear gradient of acetonitrile in 0.45% trifluoroacetic acid. It was more eluted. An aliquot of each fraction was lyophilized and three times as above. It was measured.

Next, the fraction containing inhibitory activity (0.1%) containing IJ fluoroacetic acid 40 dissolved in thiacetonitrile and hydroxylated polyether Glucara (Bio-Rad TSK-250) and 0.1% trifluoroacetic acid. It was eluted with a mobile phase of 40% acetonitrile in medium. Both parts were collected, freeze-dried, and The growth inhibitory activity was measured three times. Activity is eluted by insulin marker It elutes in both minutes and corresponds to molecules of 6-8 kD.

Next, the fraction with the highest activity was extracted using SDS-PA (J) as follows. Electrophoresis was performed.

(reversed phase) peptide corresponding to the main oncostatin-A activity from the IPLC purification step. Lyophilize and add 12.5mM Tria-CtpH6,7,4%SDS, ICI β-mercaptoethanol, 20% glycerin and 0.01% bromophenol Resuspend in 0.03-ml of sample preparation buffer containing Blue and bring to a boil. (2 minutes 5% polyacrylic poured onto a 7% polyacrylamide gradient slab. loaded onto a Ruamide overlay. 10 until the sample moves through the layered glue. mA and 20 mA until the dye front moves to the bottom of the glue. Glue migration was performed in pairs. Fix the glue and 0.2 thikuma sea pull -, stained overnight in a solution of 50 timethanol and 9% acetic acid. After destaining, hora Location of Coomassie positive bands using Hoffar densitometer It was determined. Markers are incilin (6, OOOMr), trypsinogen ( 24,500 Mr), RNase (13,700 Mr), and Abrochi = 7 (6 , soogr). The main peptide is 6,50% under these electrophoretic conditions. 0 Mr aprotinin standard.

The measurement method used was as follows. On the morning of the second day, Nunc96 well pre -) (Kamstruprej 90°DK -4,000, Roski l A349 cells (human lung cancer) were generated in the chin mark). These cells are If less than 30, it was passaged. 45.0 in all wells except surrounding wells 00 cells 150 μl/well (10% FC3, P/S, D 9 x 10' cells) were introduced. 50 μl of PBS in surrounding wells and incubated the entire plate at 37°C. Afternoon, 10 Resuspend the test sample in DMEM containing ChiFC8, P/S, and glutamine. A series of tests was conducted. Give 50 μl to each test well, while control wells receive 50 μl. Plates were incubated at 37° C. for 3 days with DMEM. 4 days In the eyes, 125I-E-)'-2'-y'Oxiu IJ Schiff (4Ci 7m 9-0, 5 mCi/lrt) solution (1 μl/isotope/10% F DMEMtd containing C8, P/S, glutamine) 50 d, and the plate. Incubated overnight at 37°C. On day 5, the medium was aspirated from the wells and PB Wash once with S, add 100 μl of methanol, and leave the methanol for 10 minutes. First, methanol was sucked out. Next, add 20 mL of 1M sodium hydroxide to the wells. 0 μIt was added, the plate was incubated for 30 minutes at 37°C, and then Titertek plug (Flow Lapse) 1M sodium hydroxide was taken out. Next, connect this plug to the gamma counter were counted for radioactivity.

In order to prove the effectiveness of Oncostatin A prepared as above, a first trial was carried out. Test was carried out. This test is called soft agar colony inhibition. The materials used are 5chi. Agar (3,751! Nobel Agar (Difco)), 125-Wh　e　at　t 75 m of distilled water autoclaved in a bottle, including 10 FC8'Jk D-M, 100U penicillin, 1000 streptomycin, 200mM gluta min, and human melanoma cells (A375).

The material to be tested is lyophilized in a 12 x 75 m sterile test tube. D Prepare a 1:10 dilution of 5% agar using MEM and heat to 46°C in a water bath. do. 0.5 in each well of 6-'7Ay culture 7'1z-) (35X14m) Prepare the substratum by injecting 1-% agar solution. This layer, it Leave at room temperature until hardened. By treatment with Trizocine, SA, 5 fine particles Prepare cells and count cell numbers. Cell t-IX 10' cell/- final Dilute to concentration and add 0.35 mJ of cells to each test sample tube.

Add 0.750 ml of 0.5% agar solution to each of the 10 test sample tubes. inject, slowly rotate the mixture, and remove the contents of the test sample tube (test (sample, cells, agar) onto the substratum and leave for approximately 20 minutes until the agar hardens. Leave at room temperature for a while. Next, play) a humidified chamber at 37 °C with 1-15% carbon dioxide. Store inside.

3 calibers for up to 10 days, depending on the titer of the test material, for cold inhibition of colony growth. Check the route. The number of colonies was counted in eight random low-power microscopic fields. Ru. If the plate is to be kept for more than 5 days, an additional 1-0. Dry the test sample layer by overlaying a layer of 3T agar solution on top of the test sample layer. Prevents dryness.

Using the method described above, using various concentrations of purified oncostatin-A. Ta. The table below shows the results and the amounts of Oncostatin A shown are Chill with the lyophilized amount introduced into the tube.

Results are reported as maximum inhibition.

Table I Oncostatin-A maximum inhibition The above results demonstrate that the polypeptide of the present invention is an effective inhibitor of cell proliferation. is clear. Based on these results observed using melanoma cells, a small 50-chi. Approximately 1 nI is sufficient to cause inhibition.

Therefore, this compound has a wide range of uses in inhibiting cell proliferation, including neoplastic cell proliferation. Has a range of uses.

For example, this compound has also been tested in various cultured human tumors, as shown in the following table. tumor cells, but not normal, non-neoplastic human foreskin fibroblasts.

Table ■ Effect of oncostatin-A on DNA synthesis Cell line 12! iI-deoki Maximum % inhibition of ciuridine uptake (*) transformed Human lung cancer (A549) 100 Human lung adenocarcinoma (H125) 41 Human melanoma (A375) 67 Human breast cancer (MCF-7) 37 not transformed Human foreskin fibroblasts (HuFp6) 0 (*) Using the measurement conditions described, A34 observed at saturating concentration of ncostatin-A (100 nJ/pressure) Maximal inhibition of I-deoxyuridine uptake into 9 cells relative to untreated control cultures does not exceed 50%.

Young (8-10 weeks old) athymic mice were given 100 μl of phosphate buffered saline solution (P 5 x 106 human lung cancer cells (A349) suspended in BS) were placed in the inguinal region. It was injected subcutaneously. 7 days after inoculation, diameter 2.5-3. Palpable tumor of Otra There has occurred. Tumor-bearing mice were randomly selected from this population and on day 7 , Oncostatin-Ao, 30 homogeneously purified from human platelets as described above. 50 μl of PBS containing μI was injected subcutaneously over the tumor site. oncosta Injection of PBS without Chin-A into tumor-bearing control mice had no effect on tumor growth. I didn't give it. Tumor-bearing mice were then given 11 days post-injection of A349 cells and On day 166, the same volume of Oncostatin-A't-injected in 50 μl of PBS. O These administrations of ncostatin-A were administered into the tumor mass using a 30 1/2” F needle. Injected directly. Calculate the tumor in both horizontal and vertical dimensions. Tumor size was monitored on the indicated days by measuring; (a) ordinate; The tumor size shown in represents the product of these two dimensions. The results are shown in Figure 1.

Human lung cancer cells (A349) were treated with oncostatin-A (200 ng/d culture). to determine the effect on polypeptides treated and released into the cell culture supernatant. The treated cultures and control cultures (without oncostatin-A) were incubated at 0:00 [o. 358-methionine (5 μCi / ml S, A, -800Ct/mmol) j)) 4 / l/ I passed. After 12 hours, the culture supernatant was removed and first incubated at low speed (1, soo xg for 15 minutes) and then at high speed (30,000X, 9 for 1 hour). I made it. Precipitation of iptide from the clarified supernatant with trichloroacetic acid (TCA) then sodium dodecyl sulfate on a 12.5% acrylamide slab. Subjected to polyacrylamide gel electrophoresis Radioautography of glu A349 treated with oncostatin-A The presence of S-methionine-labeled polypeptide in the supernatant from cells On the other hand, untreated cancer cells secreted minimal amounts of this protein (oncostatin). At least a 10-fold increase was seen after treatment with N-A). Treated cultures and controls No other qualitative or quantitative differences were observed between the cultures.

pp60src t+, Er1ckson et al., Proc, Natl, Aca d.

Sci, USA (1979) 76:6260-6264; and purified by immunoaffinity chromatography. Purification of 5μ Enzyme (approximately 0.47 pmole) f 100 n,! The refined uniformity of i’ Oncostatin-A, 20mM ATP, 5mM MgC2,1 In a final reaction volume of 30 μl containing 0 mM Tri 5-CL (pH 7,2) Incubate for 30 minutes at 30°C. Add twice the volume of sample buffer. The reaction is stopped by and Laemmeli, U, to, Nat ure (1970) 227:680-685. It was analyzed by DS polyacrylamide gel electrophoresis. Slabdal autoradiation ophography shows a 10-fold increase in the autophosphorylation of pp60src. It clearly showed irritation. In the 8re enzyme, increased phosphorylation It was not limited to syn residues, but was also found at serine positions.

Effect of oncostatin on macrophage Ia antigen expression Wehi-3 is activated by gamma interferon (γ-IFN). A murine macrophage cell line that can be induced to express S. antigen. child The characteristics of induction of macrophages have been studied by several laboratories, and normal macrophage induction It has been shown that this is an accurate reproduction of the

These cells were incubated with platelet oncostatin in the presence or absence of γ-IFH in a small dish. Several concentrations of In-A were used for growth. In the presence or absence of γ-IFH In both cases, oncostatin-A was detected by direct immunofluorescence on FAC8. When measured, a dose-dependent enhancement of class ■ antigens was observed. Oncostatin-A The magnitude of the effect is approximately 2-fold at the concentrations used (~2-70iIi/mj) Met.

Monoclonal and polyclonal antibodies specific for oncostatin-A manufacturing A method for cross-linking oncostatin-At bacterial lipopolysaccharide is r1m1 and Cazenave.

J. Immunol, (1982) 129 (3): 1124-11 This is a modification of the method developed by No. 29.

10 n# of Oncostatin-A and 12.5 n# of bacterial lipopolysaccharide (LPS: Sigma + I, -263) - Diluted with distilled water to a capacity of 500μ I interpreted it. Add 50 μl of 2.5 μl of glutaraldehyde in PBS and add this mixture. The compounds were incubated for 30 minutes at room temperature.

Add 50 μl of 2-glyglycine in PBS and incubate for 1 hour at room temperature. In this way, the reaction was stopped. Oncostatin-p: Lps conjugate 10 Dilute the td mixed phospho-AI spheres in conditioned medium and filter. Sterilized for use in in vitro immunization.

Non-immune tile cells were immunized in vitro using a modification of the method described by 269. Made.

Equal numbers of Ba1b/a and C57 rabbits in DMEM containing 2% rabbit serum. By culturing bear mouse thymocytes at 4 x 10' cells/- for 48 hours, the cells were mixed. A lean lymphocyte conditioned (MLC) medium was prepared. Collect this media and Stored at -20°C.

Flush thioglycolate-treated Balb/c mice with sterile PBS Peritoneal exudate cells (PEC) were collected by this method. PEC cells into a tile equivalent to one mouse cells, and MLC medium containing 10 nJ of oncostatin-A-LPS conjugate. 10- in culture. Cells were cultured for t-7 days.

Immune tile cells were collected and fused with 5P210 myeloma cells at a 1:1 ratio. A method for synthesizing dionchostatin-A-specific monoclonal antibodies by Breedomas were prepared. By endeme-linked immunoassay (ELISA) Hybridomas were tested for production of dioncostatin-A antibodies. limit dilution Two positive hybridomas were cloned by the method. Expand clones and immunize Balb/c mice tested for globulin class and for ascites production. was injected into.

Forty positive hybridoma clones were first expanded and anti-oncostatin -Retested for A activity. Ba1b using the 7 most reactive clones /c produced ascites in mice. The residue was expanded and frozen. Ascites 1 ELIs Oncostatin-A, oncostatin-A peptide-KLH conjugate and and specificity for BSA.

Ascites is treated with oncostatin-A and, to a lesser extent, at dilutions of 1 to 3000. It reacted with both oncostatin-A”2 peptide and immunoglobulin. Purification was carried out by the caprylic acid precipitation method described by 71. of immunoglobulin Parabon analysis and Auchtellrony analysis showed that all immunoglobulins were type I. gM.

Oncostatin-A'i diluted in 0.1 M acetic acid and 10n&/wa Pipette into a t-96 well dynamometer long plate. chamber the solution It was dried overnight at a warm temperature. 2.5 ml BSA, 2.5 ml fetal serum (Fe2) in PBS ) for 1 hour at 37°C. blocked.

The plate was then washed twice with 2.5% FC8 in PBS. Next is Hypride Media, immunoglobulin or antiserum were added at appropriate dilutions. Then this program The rates were incubated for 2 hours at 37°C and 2.54 Fe2 in PBS. Washed three times with Vectorlasus avinone-biotin according to the manufacturer's instructions. -HRP ELISA reagent was used. 2. In PBS during each step. Washed with 5% FC3. Contains 4μl of 30% hydrogen peroxide/10-solution 0.4 mg/-ortho-phenylenediamine in 1M sodium citrate solution Positive wells were visualized. The reaction was allowed to continue for 130 minutes at room temperature. 50 The reaction was stopped by adding pl of 1.4 NH2So4/well.

Production of polyclonal anti-oncostatin antiserum Ba1b/c mouse wokotrose The cells were immunized with ululose-immobilized oncostatin-A. Purpose of this immunization method is to avoid rapid elimination of the polypeptide by the host. In this way, very Immunization can be performed with small amounts of oncostatin-A.

A solution of Oncostatin A in 011M acetic acid was prepared using nitrocellulose (Schleicher & Schuell, 0.45 μm) and left to dry. the first For immunization, nitrocellulose strips were prepared from the abdomen of 3 (7) Ba1b/c mice. (0.375 nJi'/mouse). (An additional 0.1- Complete Freund's adjuvant was injected intraperitoneally. Mouse t-2 times at 2 week intervals , and a booster immunization with oncostatin-immobilized nitrocellulose. Additional exemption For epidemics, nitrocellulose is cut and mixed with 0.1 - of water and 0.1 - of incomplete Homogenized with Freund's adjuvant and injected subcutaneously (0.12 5nJ/mouse). Mouse serum was tested for oncostatin by the above ELISA measurement. horseradish as a second step reagent. G-oxidase-conjugated protein Af was used. Serum was omitted to demonstrate specificity. For ncostatin-A peptide-KLH conjugate and blocked plates. Tested.

The following sequences were prepared according to conventional methods.

■TACCITα included qτ−απaλQsu GπΣN℃α℃αz　m　GAA　c ar　GAA　GAA　Glsmmc’ic　-39island πCGrA　AA A　ACT　ACT　TCT　(&　(’rA　AGOαr　Qlr　CAC- 781eu ays val lya thr thr ser gin va l　arg　pro　arg　hisXhoI　Nael 'r71GTGr　AGT　G/Gσ[OCAT　TAG　TrrαDαnαE G’lu a GGCg ACA TCA C’fCG71GGrA AE AAA 1ffl G$ CCCCACTGCCCG -120flu thr aer leu glu val ilu lys ala gly prc his ays pr.

ACTGCTcIGC’1tffA! GCCACrmAAAAAAACTCXll rAAG -159thr ala gin leu flu ala thr leu lys aan gly arg lysBgl II Xbal Pstl TIG A C & CID C7’GαπαD (XG TACAAAAAATCflu cys leu aap leu gin ala pro leu tyr lya lys fluATCAAMACICCIGGAATCT TAA G-22s This arrangement is double-needled for use in E, Cori, and corded. A number of restriction enzyme recognition sites have been devised to facilitate sequence modification. single chain over -wrap sequences are prepared, brought together in an annealing medium, and ligated and turned on. A reading phase was used to prepare a fusion protein from which costatin-A could be isolated. Complete genes with suitable ends for insertion into expression vectors I got it. Single chain segment) '5'-phosphate by tT4 polynucleotide ligase and a reaction volume of 30 μm (3 0mM ATP, 10mM DTT, 10mM MgC12, 1μ9 7ml spermidine, 100mM Tris-HO2, pH 7,8, and T 4.Purify together on Midoglu in DNA ligase).

Plasmid ptrpED51 (Hallewell and Entage.

- Digested with enzyme Bss HIf and Bam HI to delete the trpD gene. A fragment of substantially sufficient length containing the trpE gene cleaved by Isolate by electrophoresis.

Linking the oncostatin-A gene to the linearized ptrpEP5-1 plasmid to obtain the plasmid ptrp(Onc-A)tl- and the ligation mixture was used to create E , transform KoIJ HBIOI cells. Ampicillin-resistant transfectant are selected and the plasmids are analyzed by restriction endonuclease digestion. shape Transformants were grown in Luria broth at 37°C to approximately 108 cells/- and separated. 3-indoleacetic acid (IAA) was added to approximately 1 mM, and growth was allowed to occur for approximately 1 hour. Continues for a while. Centrifuge for a few seconds in a T-Ebbendorf centrifuge tube. Separate and pellet t-5m9/,7 containing nt of cyanodanprohuman Suspend in 500 μj of thiformic acid.

After 24 hours at room temperature, the aliquot was diluted 10 times in water and The sample is measured for Oncostatin-A. Oncostatin-A is medium Because it has an N-terminal methionine and no intervening methionine, cleavage of the fusion protein is naturally difficult. Naturally, oncostatin-A has the same amino acid sequence as oncostatin-A. vinegar.

From the above results, it is clear that the compounds of this invention have a wide range of applications. . In particular, this compound can be used in the diagnosis and treatment of neoplastic conditions. . In medicine, this compound can slow down tumor cell growth; Can be used in combination with other aspects of treatment for the destruction of tumor cells Wear. For clinical purposes, the compounds of this invention induce the production of p52 and thus The enhanced 952 level when the compound is administered to the host is an indicator of the presence of tumor cells. It is found that This means that tumor cell removal during treatment of neoplastic conditions is It can be very important to determine whether success or metastasis has occurred. This issue The light compound is also. Presence of oncostatin-A or oncostatin-A receptor It can be used as a test in diagnostic measurements for the presence of cancer.

Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, the appended Certain changes and modifications may be made within the scope of the appended claims. It is clear that it can be done.

international search report lal--rllll-mna+＾＠＠１噸ealla++１ζLPGT/USf +51004110

Claims (13)

[Claims] 1. tumor cells by bringing a tumor cell growth-inhibiting amount of the composition into contact with a large number of cells. A composition for use in inhibiting cell proliferation, the composition comprising: Masu】 (In the array, (H) is intended to be hydrogen indicating the N-terminus; aa1,3 is a bond or the number of carbon atoms 2 to 6 aliphatic nonpolar or acidic amino acids; aa2 is a bond or carbon a neutral or basic aliphatic amino acid having 2 to 6 prime atoms; X1 is a bond or an amino acid consisting of 1 to 2 aliphatic amino acids having 2 to 1 carbon atoms; aa6 is a neutral or acidic amino acid having 2 to 6 carbon atoms; ; aa8, 13, 24, 27, 29, 30, 41, 42, 51, 53, 63, 64 , 67 is a nonpolar aliphatic amino acid having 5 to 6 carbon atoms; aa9 is a carbon an aliphatic polar or basic amino acid having 4 to 6 atoms; X2 is a bond or an amino acid sequence of 1 to 2 aliphatic amino acids; aa11 is a neutral aliphatic amino acid having 2 to 6 carbon atoms; X3 is a bond or an amino acid sequence of 1 to 6 aliphatic amino acids; aa23 is a neutral aliphatic polar amino acid having 3 to 5 carbon atoms, or an aromatic amino acid is an acid; aa25,38 is a neutral polar aliphatic amino acid having 3 to 5 carbon atoms; aa31,32,57 are basic or nonpolar amino acids having 2 to 6 carbon atoms; the law of nature; aa34 is proline or a neutral polar aliphatic amino acid having 3 to 5 carbon atoms. the law of nature; aa37 is a neutral aliphatic amino acid having 3 to 5 carbon atoms; aa39,68 is an aliphatic nonpolar amino acid having 2 to 6 carbon atoms; aa40,56 is an aliphatic acidic amino acid having 4 to 5 carbon atoms or an amide thereof; can be; aa45,59 is a basic or nonpolar aliphatic amino acid having 5 to 6 carbon atoms. the law of nature; aa47 is an aliphatic polar or acidic amino acid having 3 to 5 carbon atoms; aa55 is a neutral aliphatic amino acid having 4 to 6 carbon atoms; aa60 is an aromatic or neutral non-polar aliphatic amino acid; aa64a is a bond or a polar aliphatic amino acid having 4 to 6 carbon atoms; X4 is hydroxy, alkoxy having 1 to 3 carbon atoms, or 1 to 4 amino acids substantially identical to the sequence of at least 15 amino acids of ], which is the amino acid sequence of A composition having an array. 2. Array in which the above fragment extends to the next part: [There is an array] The composition according to claim 1, comprising at least the 15 amino acid sequence of. 3. The partially extended array is: [There is an array] (In the sequence, aa70 is S, T, A or G.) Claim 2 Composition of. 4. Said partially extended sequence has at its N-terminus the following sequence: [There is an array] (In the array, aa24, 27, 29, 30 are V, L or I; aa31 is G, A, K or is R; aa34 is P, S or T; and aa39 is G, A or P) The composition according to claim 3, comprising: 5. The following growing array: [There is an array] (In the array, aa* is V, L or I; aa3,6 is G, A, D or E; aa18 is G, A, N or Q; aa20 is K, R, N or Q; aa31,57 is G, A, K or R; aa34 is P, S or T; aa39 is G, A or P; aa45 is V, L, I, K or R; aa47 is N, Q, S or T; aa55 is V, L, I, N or Q; aa70 is G, A, S or T, ) has a sequence substantially identical to the sequence of at least 60 amino acids of The composition according to item 1. 6. The following amino acid sequence: [There is an array] The composition according to claim 1, comprising: 7. Presence of tumor cells in a large number of cells by contacting a large number of cells with a composition and the induced secretion of p52 protein. A composition of the following sequence: [There is an array] (In the array, (H) is intended to be hydrogen indicating the N-terminus; aa1,3 is a bond or the number of carbon atoms 2 to 6 aliphatic nonpolar or acidic amino acids; aa2 is a bond or carbon a neutral or basic aliphatic amino acid having 2 to 6 prime atoms; X1 is a bond or an amino acid consisting of 1 to 2 aliphatic amino acids having 2 to 1 carbon atoms; aa6 is a neutral or acidic amino acid having 2 to 6 carbon atoms; ; aa8, 13, 24, 27, 29, 30, 41, 42, 51, 53, 63, 64 , 67 is a nonpolar aliphatic amino acid having 5 to 6 carbon atoms; aa9 is a carbon an aliphatic polar or basic amino acid having 4 to 6 atoms; X2 is a bond or an amino acid sequence of 1 to 2 aliphatic amino acids; aa11 is a neutral aliphatic amino acid having 2 to 6 carbon atoms; X3 is a bond or an amino acid sequence of 1 to 6 aliphatic amino acids; aa23 is a neutral aliphatic polar amino acid having 3 to 5 carbon atoms, or an aromatic amino acid is an acid; aa25,38 is a neutral polar aliphatic amino acid having 3 to 5 carbon atoms, aa31,32,57 are basic or nonpolar amino acids having 2 to 6 carbon atoms; the law of nature; aa34 is proline or a neutral polar aliphatic amino acid having 3 to 5 carbon atoms. the law of nature; aa37 is a neutral aliphatic amino acid having 3 to 5 carbon atoms; aa39,68 is an aliphatic nonpolar amino acid having 2 to 6 carbon atoms; aa40,56 is an aliphatic acidic amino acid having 4 to 5 carbon atoms or an amide thereof; can be; aa45,59 is a basic or nonpolar aliphatic amino acid having 5 to 6 carbon atoms. the law of nature; aa47 is an aliphatic polar or acidic amino acid having 3 to 5 carbon atoms; aa55 is a neutral aliphatic amino acid having 4 to 6 carbon atoms; aa60 is an aromatic or neutral non-polar aliphatic amino acid; aa64a is a bond or a polar aliphatic amino acid having 4 to 6 carbon atoms; X4 is hydroxy, alkoxy having 1 to 3 carbon atoms, or 1 to 4 amino acids A composition having the amino acid sequence of ]. 8. The following growing array: [There is an array] (In the array, aa* is V, L or I; aa3,6 is G, A, D or E; aa18 is G, A, N or Q; aa20 is K, R, N or Q; aa31,57 is G, A, K or R; aa34 is P, S or T; aa39 is G, A or P; aa45 is V, L, I, K or R; aa47 is N, Q, S or T; aa55 is V, L, I, N or Q; aa70 is G, A, S or T, ) has a sequence substantially identical to the sequence of at least 60 amino acids of The composition according to item 7. 9. A monoclonal antibody specific for the composition of claim 6. 10. The labeled monoclonal antibody according to claim 9. 11. The following code sequence: [There is an array] DNA sequence containing. 12. A method for expressing oncostatin-A or a fragment thereof, comprising: The DNA sequence according to claim 11 or a fragment thereof can be used as a transcription promoter that functions in a host. Propagating cells containing under the control sequences and translational control sequences; thus producing oncostatin-A or a fragment thereof; A method comprising: 13. The following amino acid sequence: [There is an array] (In the array, aa* is V, L or I; aa45 is V, L, I, K or R; aa47 is N, Q, S or T; aa55 is V, L, I, N or Q; aa70 is G, A, S or T, ) or a fragment thereof of at least 15 amino acids.