JPH0816063B2 - Tumor cell growth inhibitor - Google Patents

Description

BACKGROUND OF THE INVENTION Field of the Invention The complexity of hematopoietic cells and thereby the differentiation and proliferation thereof is:
It is becoming increasingly clear as the list of isolated factors that coordinate these events grows continuously.
In most cases, these factors are present in very small amounts along with many other proteins that have a wide range of functions. Factors that have been isolated and shown to have activity include γ-
Includes polypeptides and proteins such as interferon, platelet growth factor, colony stimulating factor, interleukin-2, erythropoietin, and numerous other lymphokines. There is substantial interest in the isolation, purification and characterization of these blood components. Because they may be used in the treatment and description of diseases such as cancer.

There are many pitfalls in the isolation of naturally occurring factors. Separation systems must be developed that separate the factor of interest from other factors that may be present and have similar properties. Second, some means must be provided for measuring the various fractions that characterize the substance of interest in a specific or substantially specific manner relative to other traits present. If the polypeptide of interest has an extremely high activity, the difficulty of isolating the desired product is greatly enhanced. Thirdly, one must obtain a method that does not adversely affect the product of interest, especially one that avoids all denaturation. In addition, there are often other substances in the composition that act on and alter the substance of interest, resulting in
The finally obtained substance having some desired activity is not a naturally occurring substance. Finally, after isolation of the desired product in sufficiently pure form, for example amino acid sequencing,
Attempts must be made to physically characterize the polypeptide by glycosylation number, disulfide bridges, and the like. Furthermore, the substance must be characterized for its physiological properties.

Finally, even after isolation of the purified compound, characterization of the physiological activity of the compound will often prove elusive. In many situations activity is 1
Or to determine the physiological activity of a compound and to demonstrate other parameters that affect that activity, as it may depend on the presence of multiple other compounds, narrow concentration ranges, particular host cells, or the like. In addition, substantial intuitive effort and an extended experimental period are required.

Description of Prior Art Holley et al., Proc. Natl. Acad. Sci. USA (1980) 77 : 5989-
5992 describes an epithelial cell growth inhibitor. Nelson
-Hamilton and Holley, supra, (1983) 80 : 5636-5640.
Describe the effects of growth inhibitors and epidermal growth factor on the incorporation of radiolabeled methionine into proteins secreted by African green monkey cells (BSC-1). Morgan et al., Thromb. Haemost. (1980) 42 : 1652-60, which gives the amino acid sequence of human platelet factor 4. Da
Wes et al., Thromb. Res. (1983) 29 : 569-81, and Scherntha.
ner et al., Acta Med. Austraca (suppl.) (1976) 6 : 375-
9 reports a polyclonal antibody against platelet factor 4. Lawler, Thromb.Res. (1981) 21 : 121−
7 compares the sequences and structures of β-thromboglobulin and platelet factor 4.

SUMMARY OF THE INVENTION At least one of the following properties is capable of inhibiting the growth of tumors without inhibiting the growth of normal cells; pp6
At least a polypeptide capable of stimulating 0src autophosphorylation; capable of inducing secretion of a 52 kD protein from tumor cells; and isolated from mammalian platelets and exhibiting at least one of the above properties A polypeptide composition characterized by having an amino acid sequence substantially equivalent to a portion; and uses thereof. The polypeptide is provided in a substantially pure form.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a chart comparing the effect of Oncostatin-A on tumor growth in athymic mice.

Detailed Description of Specific Embodiments A polypeptide that inhibits mammalian neoplastic cell growth,
Compositions are provided that comprise other than derivatives, fragments, or the like. The polypeptide of the present invention relates to the natural polypeptide in the ethanolic HCl fraction obtained by extraction of platelets, which is referred to in the literature as platelet factor 4 and hereafter oncostatin-4 (Oncostatin-
A). This polypeptide has a mild temperature (0-25
℃), low pH, generally about pH 3 or less, usually pH 2 is stable. This polypeptide has a range of approximately 5,000 to 8,000,
More precisely, it has a molecular weight in the range of about 6,000 to 7,500, more specifically about 7,000. This polypeptide is obtained from platelets of higher animals, especially primates, and more particularly humans.

The polypeptide compound used has about 15-80 amino acids and the native polypeptide and its mimetic analogs have about 60-75 amino acids.
Amino acids, more usually about 65-72 amino acids, while fragments generally range from about 15-60 amino acids, more usually 15-72 amino acids.
It will be in the range of 35 amino acids. About 68-72 amino acids,
More specifically, polypeptides having 69, 70 or 71 amino acids are of particular interest. These polypeptides can bind to other compounds such as antigens, receptors, labels and the like.

The polypeptide is at least one biologically active,
For example, having an immunologically or epitopically active sequence, and having more than one biologically active sequence, where such a sequence competes with the natural product for biological properties. Could be

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

A composition having about 60 or more amino acids will have multiple charge domains separated by 25 or more amino acids, usually 40 or more amino acids, and less than about 70 and generally less than about 65 amino acids. Let's do it. The amino acid linking sequences that separate the charge domains have an excess of cationic amino acids over the anionic amino acids and generally range from about 1.5-
3, usually has a ratio of 2-1 and the compound has a pK of about 6.5-
It will be in the range of 8, especially about 7.4.

There are two disulfide bridges in the linking sequence,
These bridging disulfides are separated by about 20-45, usually 22-40 amino acids, preferably about 25 and 39 amino acids. The cysteines proximal to the N-terminus are located about 8-16 amino acids from the N-terminal end, and the cysteines proximal to the C-terminus are about 12-45 amino acids from the C-terminus, usually 16-40 amino acids. It is in.

The composition of interest is generally the pentapeptide EAE-
ED, more commonly the decapeptide E-A-E-E-
D-G-D-L-Q-C, often pentadecapeptide E-A-E-E-D-G-D-L-Q-C-L-C-V
-K-T, and more often the following formula: EA-EE
-D-C-D-L-Q-C-L-C-V-K-T-T-
It will have a sequence proximal to the N-terminus with the formula S-Q-V-R-P-R-H-. Here, the letters have the following meanings according to the conventional method.

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 composition of interest is generally at the C-terminus. Proximal to the sequence of formula K-K-I-I-K-K, and more generally of formula K-
K-I-I-K-K-L-L, and preferably P-L
-Y-K-K-I-I-K-K-L-L-E-S sequence.

Conservative substitution of amino acids
It should be understood that s) can be performed. Conservative changes include D and E; F and Y; K and R; G and A; N and G; V,
Substitutions involving I and R, etc. are included. In some cases, non-conservative changes, eg substitution of K or R for N or Q, may be preferred. This substitution is of particular interest when a 2-basic amino acid protease cleavage site, eg KR, is present, in which case the substitution protects this site against protease cleavage.

In addition, insertions or deletions may be included, in which case insertions or removals will normally involve 1-2 amino acids, especially 1 amino acid.

The novel polypeptides of interest most often have the following structural formula: Ac _R -M _R -Ba _r , where Ac _R (acidic region) is the N-terminal region, and 10-2
0 amino acids, 4-5 of which are acidic amino acids, at least 2 of the first 3 amino acids are acidic amino acids, 2 acidic amino acids are arranged in tandem, and The other two acidic amino acids are separated by a neutral aliphatic amino acid; two cys residues are separated by a neutral aliphatic amino acid;
X-cys is D or E-X-D depending on 2 to 6 amino acids
Or _R is an intermediate region, a short linking group of 2 to 30 carbon atoms or having about 25 to 40 amino acids; at least 10,
It generally has two cysteines separated from the cysteine of Ac _R by at least 20 amino acids, which of these cysteines form a disulfide bridge with one of the cysteines in Ac _R ; Basic amino acids and 2
-5, usually 3-4 acidic amino acids; Ba _R (basic region) is the C-terminal region, 12-30
C-terminal amino acids; having 2 amino acids; having 2 pairs of basic amino acids, each succeeded by 2 to 3 neutral aliphatic amino acids, polar or non-polar, usually non-polar; To 10 to 15 amino acids with one proline.

Preferably, Ac _R has the following formula: Where (H) intends hydrogen at the N-terminus; aa _1,3 is a bond or an aliphatic amino acid having 2 to 6 hydrogen atoms, usually an acidic amino acid or 2 to 3 carbons. Can be a non-polar amino acid having atoms; aa ² is a bond, or an aliphatic amino acid having 2 to 6 carbon atoms, a normally basic amino acid, a polar amino acid having 3 to 5 carbon atoms, or a carbon atom number. 2-3 non-polar amino acids; X ¹ is a bond, or an amino acid sequence consisting of 1 to 2 amino acids having 2 to 6 carbon atoms, usually 4 to 6 carbon atoms, which is an aliphatic Aa ⁶ is a nonpolar or polar amino acid; aa ⁶ is a nonpolar 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 2 carbon atoms ~ 6, usually 5 to 6 carbon atoms aliphatic amino acids, Be a conventional non-polar; aa ⁹ 2-6 carbon atoms, usually at 4-6 aliphatic amino acid carbon atoms, especially a polar carboxamide substituted be provided or basified; X ² is a bond, Or an amino acid sequence consisting of aliphatic amino acids having 2 to 6 carbon atoms, particularly 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 An amino acid, which may be non-polar or polar; aa ¹³ is an aliphatic amino acid having 2 to 6, usually 5 to 6 carbon atoms and is especially non-polar; X ³ Is a bond, hydroxy, an alkoxy group having 1 to 3 carbon atoms, amino, or 1 to 6, usually an amino acid sequence of 1 to 3 amino acids, usually neutral aliphatic, nonpolar or polar, It is particularly polar, the first three amino acids are generally positive, and In X ³ may be a terminal of the molecule, or M _R, it can be linked to Ba _R or antigen.

 The preferred amino numbers for the above symbols are:

aa ¹ -bond, D, E, G, A aa ² -bond, G, A, K, R, S, TX ¹ -bond, (S or T) _a- (V, L or I) _a a is 0 Or 1 aa ⁶ -S, T, G, A, D, E aa ^8- V, L, I aa ⁹ -N, Q, K, RX ^2- (G or A) _a (D or E) _a- ( V, L or I) _a aa ^11- V, L, I, M, S, T aa ^12- V, L, IX 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 to 3.

Preferably Ba _R has the following formula: ^Wherein aa ^44,56 is an aliphatic acidic amino acid having 4 to 5 carbon atoms or an amide thereof; aa ^{41,42,51,53,63,64,67} has 2 to 4 carbon atoms. 6 and, more specifically, aliphatic amino acids having 5 to 6 carbon atoms which are particularly non-polar; aa ⁴⁵ is aliphatic amino acids having 2 to 6 carbon atoms, especially 4 to 6 carbon atoms. Aa ⁴⁷ is an aliphatic amino acid having 3 to 5 carbon atoms, particularly a polar amino acid having a hydroxy or amide substituent,
Or an acidic amino acid; aa ⁵⁵ is a neutral aliphatic amino acid having 2 to 6 carbon atoms, particularly a polar amino acid having 4 to 6 carbon atoms and having a non-polar or carboxamide function and having 4 to 5 carbon atoms. 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 2 to 6 carbon atoms, usually carbon atoms Aa ⁶⁰ is an aliphatic or aromatic amino acid, usually a non-polar or basic amino acid having a number of 4 to 6; aa ⁶⁰ is an aliphatic or aromatic amino acid, especially an amino acid having a carbon number of 4 to 6 if aliphatic Aa ^64a is a bond or an aliphatic polar amino acid having 4 to 5 carbon atoms, especially 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 hydroxy, -C 1 3 alkoxy,
Amino, or an amino acid sequence of 1 to 4, usually 1 to 2 amino acids, which amino acids are especially aliphatic amino acids, more particularly polar and acidic amino acids,
The sequence has 0 to 1 non-polar amino acids having 2 to 3 carbon atoms, 0 to 3 acidic amino acids and 0 to 3 hydroxy-substituted aliphatic amino acids, where X ⁴ is a molecule. It is located at the end of or is linked to an antigen or immunoglobulin.

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

aa ^40,56 -D, E, N, Q aa ^{41,42,51,53,63,64,67} -V, L or I aa ⁴⁷ -N, Q, S, T, D, E aa ⁵⁵ -N , O, P, L, I, V; especially P or L aa ^45,59 -V, L, I, K, R aa ⁶⁰ -V, L, I, F, H, Y aa ^64a -bond, N or ^{Q aa 68 -G, A, P} , L, I, VX 4 - (G, A, D or _{E) a - (S, T} , D, A, G or E) a _- (D
Or _E) c - (T or S) _a c is 0-2.

(When two amino acids are shown at the same site, any amino acid may be present at that position.) Preferably, Mr has the following formula: It comprises a sequence of at least 15 amino acids contained in, where, aa ²³ is an aromatic amino acid, or having 3 to 5 carbon atoms aliphatic polar amino acid, particularly an amide substituted amino acid; aa ²⁴ is the number of carbon atoms 2 to 6, usually 5 or 6 carbon atoms
^{Aa 25} is an aliphatic polar amino acid having 3 to 5 carbon atoms, especially an amide or hydroxy substituted amino acid; aa ^27,29,30 has 5 to 6 carbon atoms. An aliphatic non-polar amino acid; aa ³¹ is an aliphatic amino acid having 2 to 6 carbon atoms and is either a non-polar 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 and is either a nonpolar amino acid having 2 to 3 carbon atoms or a basic mino acid; aa ³⁴ is 2 to 6 carbon atoms Aa ³⁷ is 2 to 6 carbon atoms, usually 3 to 5 carbon atoms, which is non-polar or polar, especially hydroxy-substituted; 5 aliphatic amino acids, which are non-polar or polar, in particular Lorin or are carboxamide substituted; aa ³⁸ is a 3-5 aliphatic polar amino acid carbon atoms are usually amide or hydroxy substituted; aa ³⁹ is an aliphatic non of 2-6 carbon atoms A polar amino acid; an aliphatic acidic amino acid having 4 to 5 carbon atoms of aa ⁴⁰ or an amide thereof; X ⁵ is engagement, hydroxy, alkoxy having 1 to 3 carbon atoms, wherein X ⁵ Is located at the end of the sequence and is the linkage to Ba _R or to the antigen.

 The following definitions for symbols are of particular interest.

^{aa 23 -F, H, Y,} N, Q aa 24,27,29,30 -V, L, I aa 25,38 -S, T, N, Q aa 31,32 -G, A, K, R aa ³⁴ −P, S, T aa ³⁷ −P, N, Q aa ³⁸ −S, T, N, Q aa ³⁹ −G, A, P, V, L, I aa ⁴⁰ −D, E, N, Q Amino acids are classified as follows.

Aliphatic Neutral Nonpolar G, A, P, V, L, I Polar S, T, M, C, N, Q Acid D, E Basic K, R Aromatic F, H, Y, W As is apparent from the above, various conservative substitutions can be made in the above sequence without significantly affecting the physiological activity. Furthermore, removal and insertion of 1-2 amino acids can be used. Usually, no more than 5, and usually no more than 3 changes (substitutions or insertions) will be made in the sequence.

Of particular interest for use in this invention are the following sequences: A compound having ## STR3 ## or an analogue thereof, in particular 4 cysteines at their corresponding positions, and 12 amino acids at or near the C-terminus, especially 10 amino acids proximal to the C-terminus, and More specifically, 8 proximal to the C-terminus
Analogues or fragments containing 4 amino acids, including 4 basic and 4 neutral aliphatic amino acids. Analogs of the above composition are typically greater than about 80%, more usually about 85%.
%, And preferably about 90% or more of the same amino acids in the sequence or parts of the sequence.

The native polypeptide composition used in this invention can be obtained in high purity, as established by a sensitive bioassay. The natural polypeptide composition is
Less than about 20% by weight, more usually less than about 10% by weight, and preferably less than about 5% by weight, will include polypeptides other than the major components present in the composition. This contaminant polypeptide is associated with platelets.

The polypeptide compositions of this invention have various physiological activities. The compositions of this invention are in vitro and in-
It can be used to inhibit tumor growth in vivo. This composition can also be used to stimulate the autophosphorylation of pp60src.
Thus, this composition can function as a substrate for the pp60src enzyme and be phosphorylated at the tyrosine position of the polypeptide (residue 60). In addition, tumor cells can be induced to release the 52 kD protein when treated with Oncostatin-A. In addition, fusion proteins containing oncostatin-A or analogs, fragments thereof, or sub-sequences (fragments) with competitive immunological properties can be used for the production of monoclonal antibodies, or It can function as a reagent in a diagnostic assay to detect the presence of statin-A or immunologically competitive compounds or cell surface receptors for oncostatin-A.

The compounds of this invention have high activity for tumor inhibition. The composition can be used in-vivo or in-vivo to slow the growth rate of neoplastic cells. The polypeptide composition provides about 20% or more tumor cell growth, particularly lung, at 1 ng level.
It can result in the inhibition of cancer and sarcoma of the breast, skin, etc. Preferably, the polypeptide composition will result in about 40% or more, and more preferably about 50% or more inhibition of tumor cell growth according to the colony inhibition test described in the experimental section.

The compositions of this invention can be used in vivo by administration to a host suspected of having neoplasia. The composition may be applied to neoplastic sites, such as melanoma, to slow the rate of growth. The method of application depends on the site of the tumor, the dosage form of this composition,
Depending on the dose level and the like, injection, introduction by catheter, direct application, etc. are included. The dose will vary depending on whether it is systemic or local, with a dose concentration generally of about 0.1.
μg to 1000 μg / kg, and the total dose for large mammals, including primates, is about 0.01-10 mg per therapeutic dose.

Oncostatin-A-like substances including oncostatin-A and its congeners (congeners refer to oncostatin-A
Of at least one physiological property and containing at least one amino acid sequence substantially the same as the amino acid sequence of oncostatin-A, wherein the congener is more or less than oncostatin-A. (With a number of amino acids) can be formulated in a physiologically acceptable carrier, such as phosphate buffered saline, distilled water, excipients or the like, or can be used as such. it can.

Oncostatin-A and its congeners can be used indirectly to detect the presence of neoplastic cells.
Cells are about 1-500 ng / ml activator, preferably about 50-350 ng / ml
The 52 kD protein (p52) is secreted by the neoplastic cells when subjected to the concentration of ml of activator. Thus, the presence of neoplastic cells can be detected by detecting the secretion of p52 into external media such as nutrient media, blood, urine, or other physiological fluids. Therefore oncostatin-
A can be used to monitor host status and the presence of neoplastic status. Oncostatin-A can be used to diagnose the presence of ulcers, levels of neoplasms, etc. in surveillance surgery. Oncostatin-A will be administered in-vidro or in-vivo (to the medium or host) in an amount sufficient to result in the induction of p52 secretion. The fluid associated with the system will then be monitored for the presence of p52 as an indicator of the presence of parental cells.

Oncostatin-A-like substances are also by themselves
However, it may preferably be used in combination with other lymphokines such as interferons, more particularly γ-interferon, to stimulate the immune system. That is, oncostatin-A-like substances can be combined with other polypeptides and administered to immunosuppressed hosts to stimulate the immune system. γ-Interferon is known to induce _Ia expression in monocytes and macrophages, and other tissues such as endothelial and fibroblasts. Oncostatin-A-like substances induce _Ia expression and stimulate γ-interferon _Ia induction to potentiate a given dose effect of γ-interferon. The amount of oncostatin-A-like substance is generally used to provide a concentration in the medium in the range of about 1-200, preferably about 2-70 ng / ml. The amount of γ-interferon will be routine for its use, such that the lymphokines are generally in the range of about 0.5-200 ng / ml. About 1.5 times or more, usually about 2
A more than 2-fold increase in expression is achieved with an Oncostatin-A-like agent when used as such or in combination with other lymphokines. Administration can be performed as described above.

Oncostatin-A-like substances are also kinases, especially pp
It can be used in combination with 60src to alter the substrate isomerism of the enzyme. In particular, contacting the enzyme with a small amount of an Oncostatin-A-like substance, especially at a concentration of about 0.05-50 μg / ml, enhances the kinase activity, which results in the observed amino acid changes being phosphorylated. In particular, trypsin is phosphorylated, as well as serine. Thus, the combination with pp60src or similar kinases can be used to modify a polypeptide having tyrosine and serine amino acids by causing phosphorylation of both tyrosine and serine in an enhanced ratio.

The oncostatin-A-like substances according to the invention can also be used as haptens or antigens, for example as immunogenic synergists such as haptens linked to antiparticles or the like, for the production of monoclonal antibodies or polchlor serum. Can be done. These antibodies can be used extensively, especially for diagnostic purposes. The antibody can be used by itself, or as an Oncostatin-A-like substance, as a reagent for the detection of Oncostatin-A and oncostatin-A receptors, including antibodies to Oncostatin-A.

 Various recipes and techniques for determining the analyte of interest
Can be used. These techniques are enzymatic, radioactive
Nuclide, fluorescent agent, chemiluminescent agent, enzyme substrate, enzyme inhibitor, granule
Using a variety of labels, including offspring and the like
It These methods involve separation steps (heterogeneous) or
It can include no separation step (uniform). Sign is onco
For statin-A-like substances or oncostatin-A-like substances
Covalently bound to an antibody (anti-oncostatin-A)
Or anti-oncostatin-A, such as anti-oncos
It is bound to an antibody directed against the Fc of tatin-A. Overall resistance
Hara, or Fab, F (ab) ′_{2 '}, F_VOr to these
Fragments that include similar ones can be used
It Describes various diagnostic techniques that may be used in the present invention.
A number of the US patents listed have been issued. These patents
A few examples of U.S. Pat.Nos. 3,766,162, No. 3,791,932, N
o.3,817,837, No.3,996,345, and No.4,233,402.
It RIA, EIA, EMIT for specific types of measurements , ELIS
A, SLFIA, FIA are included, all of which are commercially
Has been applied, and the reagents therefor are other analytes
Is available for. Various reagents in the kit
Can be used, in which case the sensitivity of the measurement is optimized
Thus, the types of reagents and their relative amounts are selected.

Antibodies can be prepared in a conventional manner depending on the preparation of monoclonal antibodies or polyclonal sera.
In each case, the appropriate host will be injected with an immunogen bearing the epitope site (s) of interest, usually followed by one or more booster injections. For polyclonal antisera, the host is bled and the globulin fraction is isolated. This immunoglobulin fraction can be further purified by affinity chromatography. For monoclonal antibodies, the host will be immunized as described above, but in this case usually the spleen will be removed and fused with an appropriate fusion partner. After selection of hybridomas expressing the desired antibody, the hybridomas are subjected to limiting dilution and then selected and cloned and further characterized.

The invention provides that the antibody can be of any naturally occurring type, such as IgA, IgD, IgE, EgG and IgM, especially IgM, and IgG.
Can be of various subtypes of IgG1, 2, 3, or 4.

The resulting monoclonal antibody can be used as an immunogen for the production of anti-idiotype antibodies that will have conformational similarities to Oncostatin-A type substances. They can then be used as alternative reagents for Oncostatin-A type substances in various applications.

Oncostatin-A can be obtained by extraction of platelets with about 0.3 M ethanolic hydrochloric acid. Phenylmethylsulfonyl fluoride and aprotinin can also be introduced as inhibitors for degradation, the former at a level of about 1-10% by weight of the extract composition and the latter at about 0.1-1 TIU / mg (of the extract composition. TIU ... introduced at the level of the trypsin inhibition unit). After raising the pH to about 5 with aqueous ammonium hydroxide, a small amount of ammonium acetate is added and the solution is clarified by centrifugation or other convenient means.

Cold ethanol (95%) and ether are then used to precipitate the protein, thicken the precipitate, and about 3,000
0.1 ~ 0.5M using dialysis membrane with cutoff less than 0Mr
Dialyze against acetic acid. The residue is lyophilized, resuspended in 1M acetic acid, clarified and then ready for further purification by gel permeation chromatography using Biogel P-10. The purified product is eluted with approximately 1 M acetic acid and the specie fraction is monitored using an appropriate measurement technique, such as tumor growth inhibition.

Fractions with growth inhibitory activity were lyophilized, resuspended in dilute aqueous trifluoroacetic acid (pH 2-3), clarified, and then chromatographed on a high pressure liquid chromatograph, where silica packing Has a coating of long aliphatic chains of about 16 to 20 carbon atoms, for example 18 carbon atoms. The column was equilibrated with dilute trifluoroacetic acid (0.02-0.1%),
And the product is rare (0.01 ~ 0.1%, usually about 0.04 ~ 0.05
%) Elute with an acetonitrile gradient to 60% acetonitrile in trifluoroacetic acid. Relatively slow flow rates, generally about 0.5-1 ml / min at ambient temperature are used. Fractions can be measured by the bioassays described above or other bioassays. For further purification, the product obtained from the column can be purified using high pressure gel exclusion chromatography.

The major peak of oncostatin-A activity separated by Novapak C ₁₈ reverse phase liquid chromatography is lyophilized and resuspended in 100 μl of 40% acetonitrile containing 0.1% trifluoroacetic acid. Sample is hydroxylated polyether gel column (Violad
TSK-250) and eluted with mobile phase containing 40% acetonitrile in 0.1% trifluoroacetic acid. Aliquots of each fraction are lyophilized and tested for oncostatin-A activity. Tumor cell inhibitory activity co-eluted with the major peptide peak (R _f -0.9), which also corresponds in molecular weight to that of the 6,000 Mr insulin marker used to guide this chromatographic system.

The product obtained from the column can be electrophoresed using SDS-PAGE. The band at a molecular weight of approximately 6,000-8,000 is isolated. This band is shown to have strong growth inhibitory activity on mammalian neoplastic cells.

Instead of isolating Oncostatin-A from natural sources or synthesizing the polypeptide or analog thereof on a solid support, Oncostatin-A can be prepared by hybrid DNA techniques. The gene producing Oncostatin-A can be obtained from the host cell genome using a probe prepared based on the amino acid sequence. A genomic library is probed with this probe (which should be reasonably redundant), hybridizing fragments are isolated, and the fragments are reduced in size and characterized by restriction mapping and sequencing.

Alternatively, one can similarly search the DNA for a genomic library and use these once the complete or partial structural gene has been isolated, since the latter will replace the missing codon. It is used by using the adapter of.

Conveniently, synthetic genes can be synthesized. Substantial flexibility is achieved by using synthetic genes,
It is possible to use preferred codons for the host and introduce unique or rare restriction sites. The restriction site is a deletion,
A certain degree of flexibility is added when transforming various parts of the gene by introducing transitions, transformations, insertions, etc. A strategy is devised to use single-stranded overlapping fragments that can be mixed into the hybridization ligation medium without interfering with heteroduplex formation. The resulting two genes can then be cloned and purified. Exemplary sequences are given in the experimental part.

Preferably, the ends of the gene are different to ensure the proper orientation for insertion. This gene can be inserted into an appropriate expression vector for expression. A large number of vectors can be used for expression in prokaryotes and eukaryotes, such as fungi, eg yeast, mammalian cells, eg masu cells, primate cells and the like. Multiple systems are plasmids,
It can be derived from a virus or a chromosome. Typical multiple systems are ColEl, Lambda, RSF1010, 2 μm plasmid,
SV40, adenovirus, bovine papilloma virus, etc. are included.

In addition to at least one multiple system, where episomal maintenance is desired (multiple systems are not required if integration is required), there will usually be a marker which allows for selection of the host containing the gene of interest. Let's do it. This marker will usually confer biocidal resistance, eg, antibiotics or heavy metals, or complement, prototrophy to an auxotrophic host.

Structural genes will usually be located between the transcriptional and translational initiation and termination control regions recognized by the expression host, by insertion into a polylinker (a sequence with multiple restriction sites). Transcription is constitutive or inducible, with proper selection of the transcription initiation region. Multiple promoter regions,
For example, E. coli trp and lac promoters, lambda P _L and P _R promoters, yeast glycolytic enzyme promoters, SV40 and adenovirus early and late promoters, and the like have been isolated and useful. Is shown.

In addition, a fusion gene can be prepared by providing the structural gene with a 5'-sequence encoding a secretory leader and processing signals. Representative secretory leaders described include the secretory leader of penicillinase, α-factor, immunoglobulins, T-cell receptors, outer membrane proteins, and the like. By fusion with an appropriate reading frame,
Mature oncostatin-A or the like can be secreted into the medium.

The construct containing the structural gene and the flanking regions that bring about the regulation of expression is transferred to an expression host by a convenient means, for example, transformation using calcium phosphate-precipitated DNA, transfection, transfection, conjugation, microinjection, etc. Can be introduced. The host can then be grown to high density in a suitable nutrient medium. Where the promoter is inducible, permissive conditions such as temperature changes, depletion or excess of metabolites or nutrients, or the like can be used.

If the product is retained in the host, cells are harvested, lysed and the product isolated and purified by extraction, precipitation, chromatography, electrophoresis etc. If the product is secreted, the nutrient medium can be thickened and the product isolated by conventional methods, eg affinity chromatography.

The structural gene sequences can be used for expression as well as probes for hybridization and detection of duplexing sequences. For example, the presence and amount of mRNA in the host cell can be detected.

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

Experimental abbreviations: DMEM ... Durbeco's modified Eagle medium; PBS ... Phosphate buffered salt solution; P / S ... Penicillin / streptomycin (0.57 mg / ml each); FCS ... Fetal bovine serum; SDS-PAGE ... Sodium dodecyl sulfate-poly Acrylamide gel electrophoresis.

Purification of Oncostatin-A Acid-ethanol extraction from human-platelets Two volumes of fresh or frozen and thawed room temperature thawed platelets (50 g): 375 ml Ethanol (95%), 7.5 ml
Concentrated HCl, 33mg Phenylmethylsulfonyl fluoride and 1m
1 aprotinin (23 TIU / ml; from bovine lung ... Sigma Chemical Co. A6012) was resuspended. The mixture was stirred overnight at 4 ° C and 30 at 8k rpm in a Beckman type 19 rotor.
Centrifuge for minutes and remove the supernatant. Of this supernatant
The pH was adjusted to 4.0 with concentrated ammonium hydroxide and the pH was adjusted to 5.2 with 1:10 diluted concentrated ammonium hydroxide.
I raised it to. After adding 1 ml of 2M ammonium acetate (pH5.2) per 0.1 supernatant, this solution was centrifuged for 30 minutes at 8k rpm in a type 19 rotor. The supernatant was removed, 2 volumes of cold 95% ethanol were added, followed by 4 volumes of cold diethyl ether, and the mixture was left at 0 ° C overnight. The precipitate was collected by centrifugation at 8k rpm for 30 minutes in a type 19 rotor, and the pellet approximately 10
Suspended in ~ 20 ml 1 M acetic acid. Dialyze the acetic acid dispersion against Spectrapol dialysis membrane (# 3) tubing (cutoff 3,500Mr) (Amersham Scientific Products) against 0.2M acetic acid with 2 changes of 5 each. did. The extract was dialyzed, resuspended in 7.5 ml 1 M acetic acid and then centrifuged at 30 k rpm.

Gel Permeation Chromatography Biogel P-10 (200-400 mesh; Bio Rad Labs) was swelled overnight in 1M acetic acid, thoroughly degassed, and then placed on a 100 x 2.5 cm siliconized glass column. Injection and equilibration with 1M acetic acid overnight.

29 g of the acid-ethanol solubilizing peptide from human platelets (50-70 mg protein) was dissolved in 7.5 ml of 1M acetic acid and applied to the above column. Collect the fractions (3.5 ml),
The lyophilized aliquots and tested for inhibition of introduction of ^5-125 I- iodo-2'-deoxyuridine to A549 hydrate lung cancer cells.

Reversed phase high pressure liquid chromatography The fraction containing the peak tumor growth inhibitory activity (200 ng protein) from the above column was lyophilized and 0.05% (v / v)
Resuspended in v) trifluoroacetic acid. The column was then eluted with a 0.60% linear gradient of acetonitrile in 0.045% trifluoroacetic acid at a flow rate of 0.8 ml / min at 23 ° C. Lyophilize an aliquot of each fraction,
And it measured 3 times as mentioned above.

Fractions containing inhibitory activity were then dissolved in 40% acetonitrile containing 0.1% trifluoroacetic acid and a hydroxylated polyether gel column (Bio-Rad.
TSK-250) and 4 in 0.1% trifluoroacetic acid
Eluted with a mobile phase of 0% acetonitrile. Fractions were collected, lyophilized and measured for growth inhibitory activity in triplicate. The activity elutes in the fraction in which the insulin marker elutes and corresponds to a molecular weight of 6-8 kD.

Next, the fraction with the highest activity was electrophoresed using SDS-PAGE as follows.

The peptide corresponding to the main oncostatin-A activity from the reverse phase HPLC purification step was lyophilized to 12.5 mM Tris-ClpH.
6.7 Resuspended in 0.03 ml of sample preparation buffer containing 4% SDS, 10% β-mercaptoethanol, 20% glycerin and 0.01% bromphenol blue and boiled (2 minutes). This sample contains 0.1% SDS
Loaded on a 5% polyacrylamide overlay gel cast on a 17-27% polyacrylamide gradient slab gel at pH 8.8. Gels were run at 10 milliamps until the sample migrated through the overlay gel and 20 milliamps until the dye front migrated to the bottom of the gel. Fix gel, and 0.2% Coomassie blue, 50
Stain overnight in a solution of% methanol and 9% acetic acid. After destaining, the position of Coomassie positive bands was determined using a Hoffer densitometer. Markers are insulin (6,000Mr), trypsinogen (24,50
0Mr), RNase (13,700Mr), and aprotinin (6,5Mr)
00Mr) was included. The major peptide ran with a 6,500 Mr aprotinin standard under these electrophoretic conditions.

The measurement method used was as follows. On the morning of the second day, Nunc 96 well plate (Kamstruprej 90.DK-4,00
0, Roskilde, Denmark) A 549 cells (human lung cancer) developed. These cells were passaged if less than 30.
45,000 cells / 50 μl in all wells except surrounding wells
/ Well (9% per DMEM ml containing 10% FCS, P / S, glutamine)
X 10 ⁴ cells) were introduced. 50 μl PBS in the surrounding wells
And the whole plate was incubated at 37 ° C. In the afternoon, the test sample was resuspended in DMEM containing 10% FCS, P / S, and glutamine to perform a triplicate test. 50 μl was given to each test well, while 50 μl DMEM was given to control wells and the plates were incubated at 37 ° C. for 3 days.
On day 4, ¹²⁵ I-iodo-2′-deoxyuridine (4
Ci / mg-0.5m Ci / ml) solution (1μl / isotope / 10%
FCS, P / S, glutamine in DMEM ml) was added and the plates were incubated overnight at 37 ° C. On the 5th day, aspirate the medium from the wells, wash once with PBS and
μl of methanol was added, the methanol was left for 10 minutes and then the methanol was aspirated. Next, add 200 μl of 1M sodium hydroxide to the wells and plate at 37 ° C for 30 min.
Incubate for 1 min and then remove 1M sodium hydroxide with a Titertek plug (Flow Labs). The plug was then counted for radioactivity in a gamma counter.

The following tests were performed to demonstrate the efficacy of Oncostatin A prepared as described above. This test is referred to as soft agar colony inhibition. The materials used are% agar [3.75g Nobel agar (Difco)], 125ml of Wheston
75 ml of distilled water autoclaved in a bottle, DMEM with 10% FCS, 100 U penicillin, 1000 streptomycin, 200 mM glutamine, and human melanoma cells (A375).

The material to be tested is lyophilized in a 12 x 75 mm sterile test tube. Make a 1:10 dilution of 5% agar with DMEM and heat to 46 ° C in a water bath. Substrates are prepared by pipetting 1 ml of 0.5% agar solution into each well of a 6-well culture plate (35 x 14 mm). This layer is left at room temperature until it cures. SA ₆ 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, spin the mixture slowly,
And the contents of the test sample tube (test sample, cells,
Agar) on the base layer, and about until the agar hardens.
Leave at room temperature for 20 minutes. The plates are then stored in a 37 ° C humidified chamber with 5% carbon dioxide.

The plates are inspected for chilling of colony growth up to 10 days after 3 days depending on the titer of the test material. The number of colonies is counted in 8 random low-power microscope fields. If the plate should be retained for more than 5 days,
Further drying of the test sample layer is prevented by overlaying a further layer of 1 ml of 0.3% agar solution on the test sample layer.

The above method was used with varying concentrations of purified Oncostatin-A. The table below shows the results, the amount of Oncostatin-A shown is the lyophilized amount introduced into the test tube. Results are reported as% maximum inhibition.

From the above results, it is clear 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 give a fine 50% inhibition. Therefore, this compound has widespread use in inhibiting cell growth, including neoplastic cell growth. For example, the compound also inhibits various cultured human tumor cells but not normal non-neoplastic human foreskin fibroblasts, as shown in the following table.

Effect of Oncostatin-A on Human Cancer Cell Proliferation in Athymic Mice Young (8-10 week old) athymic mice were challenged with 5 × 10 5 suspended in 100 μl of phosphate buffered saline (PBS). ^Six human lung cancer cells (A549) were injected subcutaneously in the groin. Seven days after inoculation, a palpable tumor with a diameter of 2.5-3.0 mm developed.
Tumor-bearing mice were randomly selected from this population and on day 7 50 μl P containing 0.30 μg of Oncostatin-A uniformly purified from human platelets as described above.
BS was injected subcutaneously above the tumor site. Oncostatin-
Injection of A-free PBS into tumor-bearing control mice had no effect on tumor growth. Tumor-bearing mice were then injected with 50 μl of 11 and 16 days after injection of A549 cells.
The same amount of Oncostatin-A was injected in PBS. These doses of Oncostatin-A were administered using a 301/2 "g needle
Injection directly into the tumor mass. By measuring the tumor in both horizontal and vertical dimensions with calipers,
Tumor size was monitored on the indicated days; (a) Tumor size shown on the ordinate represents the product of these two dimensions. The results are shown in Fig. 1.

Specific release of 52,000 Mr polypeptide by human lung cancer cells treated with oncostatin-A Human lung cancer cells (A549) were transformed with oncostatin-A (200 ng /
ml culture) and the effect on the polypeptide released into the cell culture supernatant was determined. Treated and control cultures (without oncostatin-A) were treated with 0
When [Oncostatin-A or medium alone (control) was added], ³⁵ S-methionine (5 μCi / ml SA-800Ci / mmo
pulsed according to l). The culture supernatant after 12 hours was removed and clarified first at low speed (1,500 xg for 15 minutes) and then at high speed (30,000 xg for 1 hour). Peptides were precipitated from the clarified supernatant with trichloroacetic acid (TCA) and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 12.5% acrylamide slab gel.

Radioautography of the gel shows Oncostatin-A
It was shown that ³⁵ S-methionine-labeled polypeptide was present in the supernatant from A549 cells treated with S. cerevisiae, while untreated cancer cells secreted a minimal amount of this protein (oncostatin-A treated. Later, at least a 10-fold increase was seen). No other qualitative or quantitative differences were observed between treated and control cultures.

Stimulation of pp60src autophosphorylation by oncostatin-A pp60src was isolated from Erickson et al., Proc. Natl. Acad. Sci. USA (1
979) 76 : 6260-6264; Erickson et al., Cold Spring Harbor.
Purified by immunoaffinity chromatography as described in Symp. Quant. Biol. (1979) 44 : 902-917. 5 μl of purified enzyme (approximately 0.47 p mo
le) 100 ng of purified homogeneous oncostatin-A
And a final reaction volume of 30 μl containing 20 mM ATP, 5 mM MgCl, 10 mM Tris-Cl (pH 7.2) was incubated at 30 ° C. for 30 minutes. The reaction was stopped by adding 2 volumes of sample buffer, and Laemmeli, UK, Nature (197
0) 227 : 680-685 and analyzed by SDS polyacrylamide gel electrophoresis. Slab gel autoradiography clearly showed a 10-fold stimulation of pp60src autophosphorylation. sr
In c enzyme, increased phosphorylation was not limited to tyrosine residues but was also found at serine positions.

Effect of oncostatin on macrophage Ia antigen expression Wehi-3 is a gamma-interferon (γ-IFN)
Is a mouse macrophage cell line that can be induced to express H2 class II antigens by. The characteristics of this induction have been studied by several laboratories and have been shown to be an exact reproduction of normal macrophage induction.

These cells were grown with several concentrations of platelet oncostatin-A in the presence or absence of small amounts of γ-IFN. Oncostatin-A, in the presence or absence of γ-IFN, showed a dose-dependent enhancement of class II antigens as measured by direct immunofluorescence on FACS. The extent of the effect of Oncostatin-A was approximately 2-fold at the concentrations used (~ 2-70 ng / ml).

Production of Monoclonal and Polyclonal Antibodies Suitable for Oncostatin-A Crosslinking of Oncostatin-A to Bacterial Lipopolysaccharide A method for crosslinking Oncostatin-A to bacterial lipopolysaccharide is described in Primi and Cazenave, J. .Immunol.
(1982) 129 (3): a variant of the method developed by 1124-1129.

10 ng of Oncostatin-A and 12.5 ng of bacterial lipopolysaccharide (LPS; Sigma # L-263) were distilled 5
Diluted to a volume of 00 μl. 50 μl of 2.5% glutaraldehyde in PBS was added and the mixture was incubated for 30 minutes at room temperature. Add 50 μl of 2M Glycine in PBS,
Then, the reaction was stopped by incubating at room temperature for 1 hour. Oncostatin-A: LPS conjugate was diluted with 10 ml of mixed lymphocyte conditioned medium and the filter was sterilized for use in in vitro immunization.

In-vitro immunization of Balb / C splenocytes with a conjugate of oncostatin and LPS Non-immune splenocytes using a modification of the method described by Reading, Immunol. Meth. (1982) 53 : 261-269. Were immunized in vitro.

Equal numbers of Balb / c and C57 black mouse thymocytes in DMEM containing 2% rabbit serum at 4 x 10 ⁶ cells / ml.
Mixed lymphocyte conditioning (MLC) media was prepared by culturing for hours. Collect this medium, and −20
Stored at ° C.

Balb / c mice treated with thioglycollate in sterile PBS
Peritoneal exudate cells (PEC) were collected by flushing with. PEC cells were placed in culture with 1 mouse equivalent of splenocytes and 10 ml of MLC medium containing 10 ng of Oncostatin-A-LPS conjugate. The cells were cultured for 7 days.

Production of Monoclonal Antibodies Oncostatin-A by collecting immune splenocytes and fusing with SP2 / 0 myeloma cells in a 1: 1 ratio.
A hybridoma was prepared that synthesizes a specific monoclonal antibody. Enzyme linked immunoassay (ELIS
Hybridomas were tested for production of oncostatin-A antibody by A). Positive hybridomas were cloned twice by limiting dilution. Clones were expanded, tested for immunoglobulin class, and injected into Balb / c mice for ascites production.

Forty positive hybridoma clones were first expanded and retested for anti-oncostatin-A activity.
The seven most reactive clones were used to produce ascites in Balb / c mice. The rest unfolded and frozen. Ascites was tested in an ELISA for specificity for oncostatin-A, oncostatin-A peptide-KLH conjugate and BSA.

Ascites reacted with both oncostatin-A and, to a lesser extent, oncostatin-A peptide at dilutions of 1-3000. Immunoglobulin, Russo et al., A
N. Biochem. (1983) 65: 269-271 and purified by the caprylic acid precipitation method. Paragon and Octerlonie analysis of immunoglobulins showed that all immunoglobulins were type IgM.

ELISA measurement for oncostatin-A Oncostatin-A was diluted in 0.1M acetic acid and
The ng / well was pipetted into a 96-well Dynatech Immunolon plate. The solution was dried at room temperature overnight. PBS
Wells with 2.5% BSA, 2.5% fetal calf serum (FCS) in
Plates were blocked by incubating at 37 ° C for 1 hour. The plate was then washed twice with 2.5% FCS in PBS. Hybridoma medium, immunoglobulins or antisera were then added to the appropriate dilutions. The plate is then incubated at 37 ° C for 2 hours and 2.5% in PBS.
Washed 3 times with% FCS. Vector Lazus avidin-biotin-HRP ELISA reagent was used according to the manufacturer's instructions. Between each step the wells were washed with 2.5% FCS in PBS. Contains 4 μl of 30% hydrogen peroxide / 10 ml solution
Positive wells were visualized with 0.4 mg / ml ortho-phenylenediamine in 0.1 M sodium citrate solution. At room temperature
The reaction was allowed to continue for 30 minutes. The reaction was stopped by adding 50 μl of 1.4 NH ₂ / SO ₄ / well.

Production of polyclonal anti-oncostatin antiserum Balb / c mice were immunized with nitrocellulose-immobilized oncostatin-A. The purpose of this immunization procedure is to avoid rapid clearance of the polypeptide by the host. Thus, immunization can be performed with a very small amount of oncostatin-A.

A solution of Oncostatin-A in 0.1M acetic acid was applied to a small piece of nitrocellulose (Schleicher & Schuell, 0.45 μm) and left to dry. For the first immunization, pieces of nitrocellulose were placed in the abdominal cavity of three Balb / c mice (0.375 ng / mouse). (The mice were further injected intraperitoneally with 0.1 ml of complete Freund's adjuvant. The mice were boosted twice with oncostatin-immobilized nitrocellulose at intervals of 2 weeks. Cut, 0.1 ml water and 0.1 ml
Was homogenized with Incomplete Freund's Adjuvant and injected subcutaneously (0.125 ng / mouse). Mouse sera were tested for specificity for oncostatin-A by the ELISA assay described above, using horseradish peroxidase-conjugated protein A as the second step reagent. Sera were tested against oncostatin-A peptide-KLH conjugate and blocked plates to show specificity.

 The following sequences were prepared according to a conventional method.

This sequence was designed for use in E. coli and numerous restriction enzyme recognition sites devised to facilitate modification of the coding sequence. Single stranded overlapping sequences were prepared, brought together in an annealing medium and ligated,
The complete gene was obtained with suitable ends for insertion into an expression vector with a combined reading phase to prepare a fusion protein from which Oncostatin-A could be isolated. 5'-phosphorylated by a single chain segment and T4 polynucleotide ligase, and each segment 200p mole
To a reaction volume of 30 μl (30 mM ATP, 10 mM DTT, 10 mM MgCl ₂ , 1
μg / ml spermidine, 100 mM Tris-HCl, pH 7.8, and T 4
Anneal by combining in DNA ligase). ds DNA was digested with Bss H II and Bam HI, and 7
% Purify on native polyacrylamide gel.

Plasmid ptrpED5-1 [Hallewell and Entage, Gene
(1980) 9:. 27; Tacon , etc., Mol.Gen.Genet (1980) 177: 42
7] is digested with endonucleases Bss H II and Bam HI, and a substantially full-length fragment lacking the trpD gene and having a truncated trpE gene is isolated by preparative gel electrophoresis.

PtrpEP5-linearized oncostatin-A gene
One plasmid is ligated to obtain plasmid ptrp (Onc-A), and the ligation mixture is used to transform E. coli HB101 cells. Transformants with ampicillin resistance are selected and plasmids are analyzed by restriction endonuclease digestion. Transformants are grown in Luria broth at 37 ° C. to approximately 10 ⁸ cells / ml and 3-indoleacetic acid (IAA) is added to approximately 1 mM and growth is continued for approximately 1 hour. An aliquot (1 ml) is centrifuged for a few seconds in an Eppendorf centrifuge tube and the pellet is suspended in 500 μl of 7% formic acid containing 5 mg / ml cyanogen bromide. After 24 hours at room temperature, aliquots are diluted 10-fold in water and diluted samples are measured for oncostatin-A. Since oncostatin-A has an N-terminal methionine without an intermediate methionine, cleavage of the fusion protein results in oncostatin-A having the same amino acid sequence as native oncostatin-A.

From the above results, it is clear that the compounds of this invention have a wide range of uses. In particular, this compound can be used in the diagnosis and treatment of neoplastic conditions. In medicine, this compound can result in slowing of tumor cell growth and therefore can be used in combination with other modes of treatment for the destruction of tumor cells. For diagnostic purposes, the compounds of this invention induce p52 production,
As a result, p5 is enhanced when this compound is administered to the host.
Two levels are found to be indicative of the presence of tumor cells. This can be very important for determining whether tumor cells have been successfully removed or metastases have occurred during the treatment of neoplastic conditions. The compounds of this invention may also be used as a test in diagnostic measures for the presence of oncostatin-A or oncostatin-A receptors.

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. Let's do it.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical indication location C07K 7/02 8318-4H 14/47 8318-4H 16/18 8318-4H C12N 15/02 15/09 ZNA C12P 21/08 9358-4B G01N 33/53 D 33/577 B (C12P 21/08 C12R 1:91) 9281-4B C12N 15/00 C

Claims (3)

[Claims] 1. The following amino acid sequence (I): (Wherein X is Asp or Asn), or at least the following amino acid sequence (II) in the amino acid sequence (I): Lys Lys Ile
Ile Lys Lys Leu Leu A tumor cell growth inhibitor comprising a peptide fragment containing Leu as an active ingredient. 2. The active ingredient comprises the following amino acid sequence (II
I): The tumor cell growth inhibitor according to claim 1, which is a peptide fragment comprising a sequence of 15 amino acids in (wherein X is Asp or Asn). 3. The active ingredient is the amino acid sequence (I).
The tumor cell growth inhibitor according to claim 1, which is a peptide fragment comprising a sequence of 60 amino acids.