PROTEIN COMPOUND CAPABLE OF INHIBITING TUMORAL GROWTH

Abstract: This invention relates to a substantially proteinaceous composition which is capable of inhibiting selectively the division of estrogen-sensitive tumoral cells and/or of exerting a cytotoxic activity on such cells. This invention also relates to a cell line isolated from a human liposarcoma, capable of dividing in a culture, which produces said composition, as well as to its culture medium has been conditioned, wherein said composition is present. This invention also relates to the various uses of said products in diagnostic and clinical applications.

Description: PROTEIN COMPOUND CAPABLE OF INHIBITING TUMORAL GROWTH This invention relates to a substantially proteic com¬ position which is capable of inhibiting selectively the div- ision of tu oral cells sensitive to estrogens and/or of exerting a cytotoxic activity on said cells. This invention also relates to a cell line isolated from a human Hposarcoma capable of dividing in a culture, which line produces said- composition, as well as to its culture conditioned medium in which- said composition is present. This invention is also concerned with the employments of said products in diagnostic and in clinical applications. A large number of, cell lines capable of growing n vitro and originally isolated from human- tumors are already known from the prior art. Such lines are particularly useful for th study of their biochemical and physiological properties, as well as for the production of specific factors " . In spite of the large number of attempts made up to the present time, no cell lines are known from the Drior art whic have a differentiated phenotype that can be associated to tha of the adipose cells or adipocytes, and no lines are known which are isolated from tumors belonging to the class of lipo sarco as. Such cells would be particularly useful as they derive from the stromal tissue, a tissue which at the present time has been very little characterized and for which a con¬ trolling role has been suggested as regards some of the func¬ tions performed by the adjacent tissues. Indeed, in vivo, in the mammary gland in the presence of the hormone testostero¬ ne, adipose cells induce the regression of epithelial tissue [Kratochwill , . Development and loss of androgen responsive¬ ness in the embryonic rudiment of the mouse mammary gland. Dev. Biol. 61: 358-365, 1977] so exerting paracrine action which possibly is mediated by one or more compounds released by said adipose cells [Kratochwill, K. 1987, in "Cellular and Molecular Biology of Mammary Cancer" (D. Medina, . Kidwell, G. Heppner, e E. Anderson, eds.), pp. 1-8 Plenum, New York]. The investigations mentioned above put particularly into evidence the need for isolating and growing in vitro a cell line capable of performing the functions observed in vivo. The Author of this invention has isolated and character- ized for the first time a cell line which is capable of div¬ iding in a culture, with a phenotype associable with that of adipose cells. Surprisingly, the cell line produces in the culture medium, denominated as conditioned medium, at least one compound that is capable of inhibiting selectively the division of epithelial tumoral cells deriving from epithelial tumors which are sensitive to estrogens. By the expression "phenotype associable to the phenotype of adipose cells" it is meant the manifestation of at least one differentiated character which is proper of adipose cells like the synthesis of lipids. By the expression "epithelial tumoral cells" it is meant tumoral cells derived from epithelial tumors, said cells being isolated or still present in the tumoral mass. By the expression "estrogen-sensitive epithelial tumors" it is meant - tumors comprising - cells with at least one receptor for a hormone belonging to the class of estrogens. By the expression "conditioned medium" it is meant the culture medium in the presence of which the cells are incubated under temperature, humidity and pH con¬ ditions suitable for growing the same, for at least 6 hours. By the "caDability for inhibiting cell division" it is meant the capability of a compound for inhibiting at least 50 % of the cell growth in a culture, after at least 3 days of incu- bation in the culture soil of said cells. Accordingly, there is evidently the need for identifyin and characterizing the comDound/s capable of inhibiting sel¬ ectively the division of tumoral cells which are derived fro estrogen-sensitive epithelial tumors. In a paper published recently [Mendelsohn M.E. et al., Proc. Natl. Acad. Sci. USA, 88, 11212-11216, 1991] it is dis closed that the "heat shock" p27 human protein (HSp27), al¬ ready identified and sequenced by Hickey E. et al., Nucl. Acids Res., 14, 10 4127-4145 (1986), has the amino acid se¬ quence of 199 aa. identical with that of a protein which is correlated to the estrogen-receptor and is called p29 [Coffe A.I. et al., Cancer Research 45, 3686-3693, 1990]. More recently [Carper S.W. et al., Nucl. Acids Res. 18, 6457, 1990], a cDNA clone obtained from the A549 cell line of human lung carcinoma has been sequenced. The nucleotide se¬ quence of such clone codes for a protein of 205 amino acids in which the first 193 amino acids coincide with those of human HSp27 already known. In correspondence with the codon that codes for the amino acid residue in the position 194, the insertion of two cytosine residues has been detected, which determines a shift of the reading frame to the followi stop codon in the positions 616-618 of the nucleotide se¬ quence. The insertion thus causes a modification of the C- terminal sequence of the HSp27 protein that diverges for the last 5 amino acids (from the 195 position to the 199 positio and shows an extension of 6 more amino acids (from the pos¬ ition 200 to the position 205). No inhibition activity of cell division of tumoral cell has been put into evidence in the prior art for any one of the proteins described. Among the compounds produced and secreted from the LSA line the Author of this invention has identified and charac¬ terized a compound, whose composition is substantially proteiji aceous in nature, capable of inhibiting selectively the div¬ ision of estrogen-sensitive tumoral epithelial cells and of exerting a cytotoxic activity on such cells. The Author has biochemically characterized said compound, which has been called plLSA, and has determined its amino acid sequence, which has turned out to be made up of 205 amino acids. The Author has found surprisingly that said amino acid sequence is identical with the sequence of the human HSp27 protein already determined by Carper S.W. (ibid.) except for a single amino acid. The difference observed is in the amino acid 194 (arginine instead of proline) and is determined by a sub¬ stitution of cytosine in the position 581 of the sequence that codes for the protein HSp27 with a guaπine. Accordingly, the Author has identified a new compound having an antineoplastic biological activity which is specific for estrogen-sensitive epithelial tumoral cells. The Author has identified the nucleotide sequence that codes for the protein pILSA and has determined its amino acid sequence. Accordingly, it is an object of this invention a compound whose composition is substantially proteinaceous in nature, said compound being capable of inhibiting selectively the cellular division of estrogen-sensitive epithelial tumoral cells and/or of exerting a cytotoxic activity on said cells; said compound having a molecular weight in the range from 27 to 30 kDa; said compound also comprising an amino acid se¬ quence from the amino acid 1 to the amino acid 193 which is substantially homologous to the corresponding sequence of human protein HSp27 (heat shock p27); or a fragment of said compound which is biologically active. By the expression "substantially homologous amino acid sequences" it is meant in the scope of this invention to designate sequences with homologies in the range from 50 % to 100 % which do not give rise to a loss of biological activity of the protein. By "biological activity" it is meant the capability of inhibiting selectively the division of estrogen sensitive epithelial tumoral cells (cytostatic activity) and/ or of exerting a cytotoxic activity on said cells. According to a preferred embodiment of this invention, said compound whose composition is substantially proteinaceou in nature comprises at its carboxyl end an amino acid sequenc which is substantially homologous to the sequence: GluAlaAlaLysSerAspGluThrAlaAlaLys-NH Preferably said substantially proteinaceous compound comprises the following amino acid sequence: MetThrGluArgArgValProPheSerLeuLeuArgGlyProSer 15 TrpAspProPheArgAspTrpTyrProHisSerArgLeuPheAsp 30 GlnAlaPheGlyLeuProArgLeuProGluGluTrpSerGlnTrp 45 LeuGlyGlySerSerTrpProGlyTyrValArgProLeuProPro 60 AlaAlalleGluSerProAlaValAlaAlaProAlaTyrSerArg 75 AlaLeuSerArgGlnLeuSerSerGlyValSerGluIleArgHis 90 T rAlaAspArgTrpArgValSerLeuAspValAsnHisPheAla 105 ProAspGluLeuT rValLysThrLysAspGlyValValGluIle 120 T rGlyLysHisGluGluArgGlnAspGluHisGlyTyrlleSer 135 ArgCysPheThrArgLysTyrT rLeuProProGlyValAspPro 15O ThrGlnValSerSerSerLeuSerProGluGlyThrLeuThrVal 165 GluAlaProMetProLysLevαAlaThrGlnSerAsnGluIleThr 180 IleProValThrPheGluSerArgAlaGlnLeuGlyGlyArgGlu 195 AlaAlaLysSerAspGluT rAlaAlaLys 205. According to a preferred embodiment of this invention, said substantially proteinaceous compound is produced by the LSA cells (DSM ACC. N. 2029) preferably is secreted by said cells in the culture medium. It is a further object of this invention a composition for pharmacological uses, preferably for antineoplastic ap- plications, and even more preferably for the treatment of estrogen-sensitive epithelial tumors, that comprises the sub¬ stantially proteinaceous compound of this invention or salts thereof which are physiologically acceptable. It is a further object of this invention the employment of said substantially proteinaceous compound according to the invention or of its salts which are physiologically accept¬ able for the preparation of pharmacological compounds for anti-neoplastic treatment, preferably for the treatment of estrogen-sensitive epithelial tumors. It is an object of this invention a nucleic acid com¬ prising a nucleotide sequence that codes for the compound whose coϊiipcsition is substantially proteinaceous according to this invention, preferably said nucleic acid comprising the following nucleotide sequence: ATGACCGAGCGCCGCGTCCCCTTCTCGCTCCTGCGGGGCCCCAGC 45 TGGGACCCCTTCCGCGACTGGTACCCGCATAGCCGCCTCTTCGAC 90 CAGGCCTTCGGGCTGCCCCGGCTGCCGGAGGAGTGGTCGCAGTGG 135 TTAGGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCC 180 GCCGCCATCGAGAGCCCCGCAGTGGCCGCGCCCGCCTACAGCCGC 225 GCGCTCAGCCGGCAACTCAGCAGCGGGGTCTCGGAGATCCGGCAC 270 ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCAACCACTTCGCC 315 CCGGACGAGCTGACGGTCAAGACCAAGGATGGCGTGGTGGAGATC 360 ACCGGTAAGCACGAGGAGCGGCAGGACGAGCATGGCTACATCTCC 405 CGGTGCTTCACGCGGAAATACACGCTGCCCCCCGGTGTGGACCCC 450 ACCCAAGTTTCCTCCTCCCTGTCCCCTGAGGGCACACTGACCGTG 495 GAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540 ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCCGAGAA 585 GCTGCAAAATCCGATGAGACTGCCGCCAAGTAA 618 A further object of this invention consists in plasmid or phage type vectors comprising the nucleotlαe sequences of this invention. It is a further object of this invention a mammalian cell line capable of dividing in vitro, and having a pheno- tyDe associable to that of adipose cells, that produces also in its conditioned medium at least one compound caoable of inhibiting selectively the division of tumoral cells, preferably epithelial tumoral cells, which even more preferably derive from estrogen-sensitive epithelial tumors. According to a preferred embodiment of this invention, said cell line is isolated from a Hposarcoma, preferably of human origin, and also more preferably such line is the LSA cell line deposited with the DSM with the accession number 2029. Again according to this invention, said cell line pro¬ duces the substantially proteinaceous compound having the se¬ quence disclosed above. It is a further object of the invention a medium con¬ ditioned by the growth of cell lines herein disclosed and claimed. Preferably said conditioned medium compri ses at least one compound capable of inhibiting selectively the division of tumoral cells, preferably epithelial tumoral cells, and even more preferably cells deriving from estrogen-sensitive epi- thelial tumors, more preferably said compound being the pro¬ tein compound disclosed according to this invention. According to a preferred embodiment of the present in¬ vention, said medium is conditioned by the growth of the LSA cell line (DSM ACC. N. 2029). It is another object of this invention the employment of said conditioned medium for producing pharmaceutical compo¬ sitions for treating tumors, preferably epithelial tumors, and even more preferably for the treatment of estrogen-sensitive epithelial tumors. It is a further object of this invention the emDloy ent of said conditioned medium for the purification, identification and characterization of at least one compound capable of inhibiting selectively the division of tumoral cells, prefe¬ rably of epithelial tumoral cells, and even more preferably of cells derived from estrogen-sensitive epithelial tumors. This invention will be now disclosed by means of some examples that explain and illustrate the same but with no limitative purposes, and with reference to the enclosed fig¬ ures wherein: Figure 1 represents a growth curve of the LSA line both in the presence and in the absence of serum; Figure 2a shows a cytofluori etric analysis of human normal thymocytes; Figure 2b shows a cytofluorimetric analysis of LSA cells: Figure 3 shows an LSA-CM stimulation histogram of the 3 incorporation of [H]-thymidine in various cell lines, where 1 is the control sample, 2 is the LSA-CM at 1/4.dilution, 3 is the LSA-CM at 1/2 dilution, 4 is the LSA-CM undiluted, 5 is a control sample, 6 is the LSA-CM undiluted, 7 is the con¬ trol sample, and 8 is the LSA-CM undiluted; Figure 4 shows growth curves of the MCF-7 cell line, both in the presence and in the absence of LSA-CM: Figure 5 represents growth curves of the ZR-75-1 cell line, both in the presence and in the absence of LSA-CM; Figure 6 represents growth curves of the MDAMB-231 cell line, both in presence and in the absence of LSA-CM: Figure 7 reDreseπts growth curves of the NMMG cell line in the absence and in the presence of LSA-CM; Figure 8 represents growth curves of human cells from ovaric carcinoma both in the absence and in the presence of LSA-CM: Figure 9 represents a histogram of human cell growth from ovaric carcinoma both in the absence and in the presence of LSA-CM and/or of purified pILSA. Example 1 Isolation of the LSA line A fragment of human Hposarcoma of mixed liDOblastic- fibroblastic type is drawn in a sterile way from the leg of an adult woman, 65 years of age, by means of surgical tech¬ niques . The fragment is disgregated mechanically by a chisel so as to obtain 1 mm fragments and then this material is treated with a CTC solution equivalent to 20 U/ml of collagenase (CLSP, Worthington, . U.K. .), 0,75 mg/ml of trypsin (1/ 300, Inc. Pharmaceuticals), 2 % chicken serum, heat-inacti¬ vated and dialyzed (GIBC0) in Hank soil without Ca ++ and Mg ++ ions (GIBC0) and Ham F12 soil with 5 % bovine serum (GIBC0) for 4 hours at 37°C and under continuous stirring, in order to obtain a susDension of single cells. The cell suspension is diluted with the Ham F12 (GIBC0) culture medium supplemented with 5 % bovine serum (GIBC0), peni cillin (31 ug/ml), streptomycin (50 /jg/ml) and fungizone (2.5 μg/ml). The cells are plated at 200,000 cells/dish, 100 mm diameter (Costar). A clone of cells which is capable of dividing in a cul¬ ture and having the characteristics which are peculiar of the adipose cells has been obtained from ten dishes, said clone being called LSA and deposited with the DSM, accession num- ber ACC 2029. A growth curve of the LSA line is shown in Figure 1, in which the growth in the presence as well as in the absence of serum is evident. The LSA line shows a plating efficiency of 90 % and a duDlication time of 50-52 hours in the presence of serum and of 102 hours in the absence of serum. The medium is changed every 72 hours. Example 2 Characterization of the LSA line The DNA content of LSA cells has been analyzed by means of a cytofluorometer (Partec Pas II flow-cytometer) . The cell are trypsinized then fixed with 70 % ethanol and dyed with a solution containing 5 /jg/ml of ethidiu bromide, 12.5 g/nU of mitramycin and 1.5 mg MgCl_ in 0.1 M Tris buffer, pH 7.5. The cell percentage in the various steps of the itotic cycle is obtained as disclosed by Flintoff, W.E., Davidson, S.V., and Siminowitch, L. "Isolation and partial characterization of Methotrexate-Resistent phenotype from Chinese hamster ovar cells" Somatic Cell Genet. 2:245-261, 1976; and Ambesi-Impiom bato, F.S., Parks, L.A.M. and Coon H.G. Culture of hormone- dependent functional epithelial ceils from rat thyroid. Proc. Natl. Acad. Sci. USA 77: 3455-3459, 1980. As shown in Figure 2b, the values are compared to those obtained with normal human thymocytes as ploidy standard. The value of 1 has been assigned to the G1-G0 peak of thymocytes. Under the same con¬ ditions the G1-G0 peak of the LSA cells has a value of 1.79, which is indicative of a DNA content which is almost tetra- ploid. Also the DNA content/cell is almost twice as much as that obtained with thymocytes as shown in Figure 2a. The distribution of cells in the various steps of the cell cycle of an LSA cell population in the phase of logar¬ ithmic growth is shown in the following Table 1. Table 1 Phase Cell percentage G1/G0 59 S 27 G2/M 14 The presence of cells in the phase S confirms that the population is in the active growth phase. The counting of the chromosomes has put into evidence a number of chromosomes/cell between 80 and 90. The LSA cells as observed under an optical microscope look like elongated bodies with abudant cytoplasm. Under the electronic microscope, the cell nucleus contains many nu- cleoles and some organelles are present, localized in the perinuclear region. The cells are homogeneous to each other, the mitochondria are relatively abudant and the cytoplasmic vesicles are constant both in number and sizes. The most evi¬ dent feature is the presence of vacuolar areas inside the cytoplasm, said areas being electron-reflecting and typical of zones containing high amounts of lipids, which is removed as a result of subjecting the cells to a treatment for their preparation for observation under the electron microscope. Moreover, a well developed Golgi apparatus is also present. In order to estimate the content of lipids, the LSA cells are plated on culture slides of the Lab-Teck (NUNC) and are grown by semi-confluence. The cells are washed and fixed for 6 minutes with 4 % paraformaldehyde and 15 % picric acid saturated in PBS (phosphate saline buffer). The cells are washed twice with distilled water, then they are dyed with an Oil Red 0 solution for 5 minutes, then they are washed twice with PBS and dyed with 1 % toluidine blue for 10 seconds. If cells have been grown in the presence of 5 % bovine serum, lipids are disDersed throughout the cytoplasm, with round vesicles. The build up of lipids Is not present in cell grown in a medium without serum, but it is stimulated by in¬ cubating cells with methylxanthine/desamethasone for 48 hours and with insulin for a further period of 48 hours. Example 3 Effects of conditioned medium by LSA (LSA-CM) cells on growth of different cell lines in vitro LSA cells have been grown up to 80 % confluence on 100 mm plates (Costar) on F12 medium with 5% bovine serum. The cell monolayers are washed with PBS and incubated with F12 medium without serum. After 24 hours, the medium is replaced with fresh medium the conditioned medium is collected every 6-24 hours for the successive 30 days. The cells go on growing and they show structurally and functionally viable under phase-con¬ trast microscope and after dying with Trypan Blue. The medium conditioned by LSA cells is capable of stimulat¬ ing the growth of normal epithelial cells like CH0 (CCL-61), FRTL-5 (CRL-1468) and NIH-3T3 (ECACC-87100803) grown for 24 hours in the absence of serum, as put into evidence by an 3 H-thymidine incorporation test shown in Figure 3. Contrarily to the preceding results , the medium conditioned by LSA cells shows an inhibition effect of growth and a cytolytic activity when it is analyzed with in vitro tumoral cells containing receptors for estrogens. The cells employed are: MCF-7 cells obtained from a human mammary carcinoma (Land, H. et al., Science 222:778 (1980), ECACC-86012803) that possess receptors for estrogens (Soule, H.D. et al., Natl. Cancer Inst. 51, 1409-1413 (1973)), androgens (Horowitz, K.B. et al., Steroids 26, 785-795 (1975)), insulin and triodothy- ronine (MacGrath, C . Cancer Res., 43: 1355-1360 (1983); the ZR-75-1 (CRL-1504) cells obtained from a human mam¬ mary carcinoma, said cells being positive for estrogen recep- tors; the MDAMB-231 (ATCC-HTB26) cells obtained froma a human mammary carcinoma, but that are negative for estrogen recep- tors ; the NMMG (Burker, R.E. et al., Cancer Res., 38, 3769- 3773 (1978), CRL-1636) cells, obtained from a mouse mammary epithelium, said cells being slightly positive for estrogen receptors; a human cell line of ovaric carcinoma obtained from a woman suffering from peritoneal effusion due to ovaric carci¬ noma metastasis, said line being positive for estrogen recep¬ tor. The growth curves of the cell lines mentioned herein have been obtained by plating 100,000 cells/dish of 100 mm diameter in 10 ml of DMEM (GIBC0) medium with 10 % bovine fetal serum (FCS, GIBC0). 2 ml of LSA-CM have been added to a set of dishes.. Figure 4 shows growth curves of the MCF-7 line in which the inhibition effect of LSA-CM is evident. In particular, after a 4 day treatment, the removal of LSA-CM cannot re¬ establish the proliferative capability of MCF-7 cells because of a cytolytic effect. Figure 5 shows growth curves of the 7R-75-1 line in the presence and in the absence of LSA-CM. The presence of LSA-CM gives rise to a strong inhibition of growth without any cyto¬ lytic effect. Figure 6 and 7 show the growth curves of MDAMB-231 cells and of normal epithelial NMMG cells, in which the absence of growth inhibition due to LSA-CM is evident. Such cells do not show estrogen receptors. Figure 8 shows that the LSA-CM conditioned medium has a cytotoxic effect on the cells of human ovaric carcinoma, even after just 24 hours of incubation. 3 The LSA-CM effect on the incorporation of H-thymidine in different cell lines is shown in the following Table 2. Table 2 3 Effect of LSA-CM on H-thymidine incorporation in different cell lines Cell lines No effect Stimulation Inhibition MCF7 + ZR-75-1 + NMMG + FRTL-5 + CHO + NIH-3T3 + Example 4 Effect of LSA-CM on animals 20 Balb/c fc3H mice (Charles River) which are affected by the Bittner virus are selected for the presence of an evident tumoral mass, an estrogen-dependent mammary carcinoma, which is induced by the virus itself. After a peritumoral injection practised every day of 200 JU1 of LSA-CM for 15 days, the inhibition of growth and the necrosis of the tumoral mass are observed in 80 % of the mice treated. Example 5 Isolation of the pILSA protein from LSA-CM The LSA-CM medium is concentrated 100 x, then it is sub- jected to dialysis against a isotonic phosphate buffer at pH 7.4 and then to gel filtration on P6 resin (Phar¬ macia). The fractions eluted are tested for their capability of inhibiting selectively the growth of MCF-7 cells. One only fraction shows said biological activity. A sample of said fraction is separated by electrophoresis on a polyacrylamide gel in non-denaturating conditions, identifying a protein of about 29 kDa which is called pILSA. Said protein is electro- eluted from the gel and employed for the production of poly- clonal antibodies as disclosed in the following and for the sequence analysis. The protein sequence is shown in the following Table 3. As shown in Figure 9, the purified pILSA protein has an evident cytotoxic effect on the growth of human ovaric tumora cells. Table 3 Amino acid sequence of the p1LSA protein and corresponding coding sequence ATGACCGAGCGCCGCGTCCCCTTCTCGCTCCTGCGGGGCCCCAGC 45 MetThrGluArgArgValProP eSerLeuLeuArgGlyProSer 15 TGGGACCCCTTCCGCGACTGGTACCCGCATAGCCGCCTCTTCGAC 90 TrpAspProPheArgAspTrpTyrProHisSerArgLeuP eAsp 30 CAGGCCTTCGGGCTGCCCCGGCTGCCGGAGGAGTGGTCGCAGTGG 135 GlnAlaP eGlyLeuProArgLeuProGluGluTrpSerGlnTrp 45 TTAGGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCC 180 LeuGlyGlySerSerTrpProGlyTyrValArgProLeuProPro 60 GCCGCCATCGAGAGCCCCGCAGTGGCCGCGCCCGCCTACAGCCGC 225 AlaAlalleGluSerProAlaValAlaAlaProAlaTyrSerArg 75 GCGCTCAGCCGGCAACTCAGCAGCGGGGTCTCGGAGATCCGGCAC 270 AlaLeuSerArgGlnLeuSerSerGlyValSerGluIleArgHis 90 ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCAACCACTTCGCC 315 ThrAlaAspArgTrpArgValSerLeuAspValAsnHisPheAla 105 CCGGACGAGCTGACGGTCAAGACCAAGGATGGCGTGGTGGAGATC 360 ProAspGluLeuThrValLysThrLysAspGlyValValGluIle 120 ACCGGTAAGCACGAGGAGCGGCAGGACGAGCATGGCTACATCTCC 405 ThrGlyLysHisGluGluArgGlnAspGluHisGlyTyrlleSer 135 CGGTGCTTCACGCGGAAATACACGCTGCCCCCCGGTGTGGACCCC 450 ArgCysPheThrArgLysTyrThrLeuProProGlyValAspPro 150 ACCCAAGTTTCCTCCTCCCTGTCCCCTGAGGGCACACTGACCGTG 495 T rGlnValSerSer :ΣSerLeuSerProGluGlyThrLeuThrVal 165 GAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540 GluAlaProMetProLysLeuAlaT rGlnSerAsnGluIleThr 180 ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCCGAGAA 585 IleProValThrPheGluSerArgAlaGlnLeuGlyGlyArgGlu 195 GCTGCAAAATCCGATGAGACTGCCGCCAAGTAA 618 AlaAlaLysSerAspGluThrAlaAlaLys 205 From a comparison with the sequences deposited with data banks, the sequence turns out to be identical with that corresponding to the human HSp27 protein described by Carper S.W. mentioned above, except for just one amino acid. The difference consists in a substitution of the amino acid in the position 194, said amino acid turning out to be arginine instead of proline. Example 6 Production of anti-p1LSA polyclonal anti- bodies, and inhibition of the activity of the pILSA protein 100 ug of pILSA protein electroeluted from a 10 % poly- acrylamide gel is dissolved in 1 ml of PBS (phosphate saline buffer) and mixed with 1 ml of a complete Freund adjuvant. The mixture is injected through intramuscolar injection in a rabbit four times at 10 day intervals. 30 ml of immune serum are drawn from rabbits every other day for 20 days. The immu- noglobulin (Ig) fraction is purified by the Mab Trap G (Phar¬ macia) according to the procedure recommended by the producer, ImmunoDrecipitation and i muπoblotting tests show that immunoglobulins react in a specific way with the pILSA pro¬ tein from LSA-CM. A pre-incubation of 100 ( ul of LSA-CM with 10 ul of an Ig solution at 1 mg/ l for 30 minutes at 37°C stops the ac¬ tivity of inhibition of tumoral epithelial cell (MCF-7) div- 3 ision, restoring an H-thymidine incorporation and growth curves similar to the untreated control sample. The LSA-CM activity turns out to be unaltered after incubation with pre im une serum. Such experimental data show that the pILSA protein isolated and purified according to the procedures disclosed in Example 5 and having the sequence disclosed in Table 3 is actually the active principle responsible for the activity o inhibition of epithelial cell division disclosed herein. Example 7 Nucleotide sequence coding for the pILSA protein The nucleotide sequence coding for the pILSA protein is determined by purifying the RNA polyA from LSA cells follow¬ ing standard methods and employing the following primers for the PCR reaction: oligo 5' : CGGAATTCTGAGCAGACGTCCAGAG EcoRI oligo 3' : CGGΆΆTTCCGGGCTAAGGCTTTACTTG EcoRI The sequences are deduced respectively from the sequenc at the positions 5 1 and 3' not translated of the gene that codes for the HSp27. The amplification products are cloned directly in the EcoRI site of the vectors pGEM4z (Promega) and sequenced by means of the dideoxy method. The complete sequence of the coding portion is shown in the preceding Table 3. This invention has been disclosed with reference to som preferred embodiments of the same but it is to be understood that modifications and/or changes can be introduced by those who are skilled in the art without departing from the spirit and scope of the invention. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: 1st. Naz. per Studio e Cura dei Tumori Fond. G. Pascale (B) STREET: Via M. Semmola (C) CITY: Naples (E) COUNTRY: Italy (F) POSTAL CODE (ZIP): 80131 (ii) TITLE OF INVENTION: Protein compound, coding nucleotide sequences, producing cell line and uses thereof (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: IT RM92/A000161 (B) FILING DATE: 09-MAR-1992 (Vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: IT RM92/A000716 (B) FILING DATE: 30-SEP-1992 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 618 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (C) INDIVIDUAL ISOLATE: patient with liposarcoma (F) TISSUE TYPE: Adipose tissue (G) CELL TYPE: Adipocyte (H) CELL LINE: LSA cell line deposited at DSM N. ACC2029 (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA library from poly A+ RNA (B) CLONE: pILSA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..618 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG ACC GAG CGC CGC GTC CCC TTC TCG CTC CTG CGG GGC CCC AGC TGG Met Thr Glu Arg Arg Val Pro Phe Ser Leu Leu Arg Gly Pro Ser Trp 1 5 10 15 GAC CCC TTC CGC GAC TGG TAC CCG CAT AGC CGC CTC TTC GAC CAG GCC Asp Pro Phe Arg Asp Trp Tyr Pro His Ser Arg Leu Phe Asp Gin Ala 20 25 30 TTC GGG CTG CCC CGG CTG CCG GAG GAG TGG TCG CAG TGG TTA GGC GGC 1 Phe Gly Leu Pro Arg Leu Pro Glu Glu Trp Ser Gin Trp Leu Gly Gly 35 40 45 AGC AGC TGG CCA GGC TAC GTG CGC CCC CTG CCC CCC GCC GCC ATC GAG 1 Ser Ser Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala He Glu 50 55 60 AGC CCC GCA GTG GCC GCG CCC GCC TAC AGC CGC GCG CTC AGC CGG CAA 2 Ser Pro Ala Val Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gin 65 70 75 80 CTC AGC AGC GGG GTC TCG GAG ATC CGG CAC ACT GCG GAC CGC TGG CGC 2 Leu Ser Ser Gly Val Ser Glu He Arg His Thr Ala Asp Arg Trp Arg 85 90 95 GTG TCC CTG GAT GTC AAC CAC TTC GCC CCG GAC GAG CTG ACG GTC AAG 3 Val Ser Leu Asp Val Asn His Phe Ala Pro Asp Glu Leu Thr Val Lys 100 105 110 ACC AAG GAT GGC GTG GTG GAG ATC ACC GGT AAG CAC GAG GAG CGG CAG Thr Lys Asp Gly Val Val Glu He Thr Gly Lys His Glu Glu Arg Gin 115 120 125 GAC GAG CAT GGC TAC ATC TCC CGG TGC TTC ACG CGG AAA TAC ACG CTG Asp Glu His Gly Tyr He Ser Arg Cys Phe Thr Arg Lys Tyr Thr Leu 130 135 140 CCC CCC GGT GTG GAC CCC ACC CAA GTT TCC TCC TCC CTG TCC CCT GAG Pro Pro Gly Val Asp Pro Thr Gin Val Ser Ser Ser Leu Ser Pro Glu 145 150 155 160 GGC ACA CTG ACC GTG GAG GCC CCC ATG CCC AAG CTA GCC ACG CAG TCC Gly Thr Leu Thr Val Glu Ala Pro Met Pro Lys Leu Ala Thr Gin Ser 165 170 175 AAC GAG ATC ACC ATC CCA GTC ACC TTC GAG TCG CGG GCC CAG CTT GGG Asn Glu He Thr He Pro Val Thr Phe Glu Ser Arg Ala Gin Leu Gly 180 185 190 GGC CGA GAA GCT GCA AAA TCC GAT GAG ACT GCC GCC AAG TA .Gly Arg Glu Ala Ala Lys Ser Asp Glu Thr Ala Ala Lys 195 200 205 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 205 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Thr Glu Arg Arg Val Pro Phe Ser Leu Leu Arg Gly Pro Ser Trp 1 5 10 15 Asp Pro Phe Arg Asp Trp Tyr Pro His Ser Arg Leu Phe Asp Gin Ala 20 25 30 Phe Gly Leu Pro Arg Leu Pro Glu Glu Trp Ser Gin Trp Leu Gly Gly 35 40 45 Ser Ser Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala He Glu 50 55 60 . Ser Pro Ala Val Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gin 65 70 75 80 Leu Ser Ser Gly Val Ser Glu He Arg His Thr Ala Asp Arg Trp Arg 85 90 95 Val Ser Leu Asp Val Asn His Phe Ala Pro Asp Glu Leu Thr Val Lys 100 105 110 Thr Lys Asp Gly Val Val Glu He Thr Gly Lys His Glu Glu Arg Gin 115 120 125 Asp Glu His Gly Tyr He Ser Arg Cys Phe Thr Arg Lys Tyr Thr Leu 130 135 140 Pro Pro Gly Val Asp Pro Thr Gin Val Ser Ser Ser Leu Ser Pro Glu 145 150 155 160 Gly Thr Leu Thr Val Glu Ala Pro Met Pro Lys Leu Ala Thr Gin Ser 165 170 175 Asn Glu He Thr He Pro Val Thr Phe Glu Ser Arg Ala Gin Leu Gly 180 185 190 Gly Arg Glu Ala Ala Lys Ser Asp Glu Thr Ala Ala Lys 195 200 205 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13£ * ts) A. The indications made below relate lo the microorganism referred lo in the description on page " » ' . ° » , line ' » " , JU, B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet j { Na e of e p o it ar y i nst i tut i o n DS - Deutsche Sammlung von Mikroorganismen und Zellkultureπ GmbH - Address of depositary institution (including postal code e d country) Mascheroder eg 1 B D-3300 BRAUNSCHWEIG - GERMANY Date of deposit Accession Number 4th FEBRUARY, 1992 ACC2029 C. ADDITIONAL INDICATIONS (lcaveblank if no. applicable) This information is continued on an additional sheet \ { D. DES 1 GN ATED STATES FOR ΛVHI CH INDICATI ONS ARE MADE (if the indications ere no,' for ell designate*! States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will besubmi ll ed to ihelnternalional Bureau later (sp_cifytheg_ j ιcralnaiureofιhcin__catioι_. _-g., 'Accession Ku her of Deposit') For receiving Office use only For international Bureau use only J J This sheet was received with the international application j I This sheet was received by the International Bureau on: Au'horΪ7.ed offtcci Authorized officer

Claims: C LA I MS 1. A substantially proteinaceous compound, characterize in that it is caoable of inhibiting selectively the cellular division of estrogen-sensitive eDithalial tumoral cells, and or of exerting a cytotoxic activity on such cells; and chara terized in that it has a molecular weight in the range between 27 and 30 kDa: it comprises an amino acid sequence from the amino acid 1 to the amino acid 193 which is substan tially homologous to the corresponding sequence of human HSp (heat shock p27) protein; or a biologically active fragment of said compound. 2. A substantially proteinaceous compound according to claim 1, said comDound comprising at its carboxyl end an amino acid sequence which is homologous to the sequence: GluAlaAlaLysSerAsDGluThrAlaAlaLys-NH . 3. A substantially proteinaceous compound according to claim 2, such compound comprising an amino acid sequence whic is substantially homologous to the following amino acid se¬ quence: MetThrGluArgArgValProPheSerLeuLeuArgGlyProSer 15 TrpAspProPheArgAspTrpTyrProHisSerArgLeuPheAsp 30 GlnAlaPheGlyLeuProArgLeuProGluGluTrpSerGlnTrp 45 LeuGlyGlySerSerTrpProGlyTyrValArgProLeuProPro 60 AlaAlalleGluSerProAlaValAlaAlaProAlaTyrSerArg 75 AlaLeuSerArgGlnLeuSerSerGlyValSerGluIleArgHis 90 ThrAlaAspArgTrpArgValSerLeuAspValAsnHisPheAla 105 ProAspGluLeuThrValLysThrLysAspGlyValValGluIle 120 ThrGlyLysHisGluGluArgGlnAspGluHisGlyTyrlleSer 135 ArgCysPheThrArgLysTyrThrLeuProProGlyValAspPro 150 ThrGlnValSerSerSerLeuSerProGluGlyThrLeuThrVal 165 GlxiAlaPro etProLysLeuAlaThrGlnSerAsnGluIleTtir 180 HeProValThrPheGluSerArgAlaGlnLeuGlyGlyArgGlu 195 AlaAlaLysSerAspGluThrAlaAlaLys 205. 4. A substantially proteinaceous compound according to any one of the preceding claims, which is produced by LSA cells (DSM ACC. N. 2029), which compound is preferably se¬ creted by said calls in the culture medium. 5. A composition for pharmacological use, preferably f c.use as anti-neoplastic, comprising the substantially protein- aceous compound according to any one of the preceding claims, or physiologically acceptable salts thereof. 6. A composition according to claim 5 to be employed for treatment of estrogen-sensitive epithelial tumors. 7. Use of the substantially proteinaceous compound according to any one of the preceding claims or of its salts physiologically acceptable, for the preparation of pharmaco¬ logical comoositions for antineoplastic treatment, preferably of estrogen-sensitive epithelial tumors. 8. A nucleic acid comorising a nucleotide sequence coding for said substantially proteinaceous compound according to any one of the preceding claims. 9. A nucleic acid according to claim 8 comprising the following nucleotide sequence: ATGACCGAGCGCCGCGTCCCCTTCTCGCTCCTGCGGGGCCCCAGC 45 TGGGACCCCTTCCGCGACTGGTACCCGCATAGCCGCCTCTTCGAC 9O CAGGCCTTCGGGCTGCCCCGGCTGCCGGAGGAGTGGTCGCAGTGG 135 TTAGGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCC 180 GCCGCCATCGAGAGCCCCGCAGTGGCCGCGCCCGCCTACAGCCGC 225 GCGCTCAGCCGGCAACTCAGCAGCGGGGTCTCGGAGATCCGGCAC 270 ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCAACCACTTCGCC 315 CCGGACGAGCTGACGGTCAAGACCAAGGATGGCGTGGTGGAGATC 360 ACCGGTAAGCACGAGGAGCGGCAGGACGAGCATGGCTACATCTCC 405 CGGTGCTTCACGCGGAAATACACGCTGCCCCCCGGTGTGGACCCC 450 ACCCAAGTTTCCTCCTCCCTGTCCCCTGAGGGCACACTGACCGTG 495 GAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540 ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCCGAGAA 585 GCTGCAAAATCCGATGAGACTGCCGCCAAGTAA 618 10. Plasmid or phage vectors comprising the nucleotide sequences according to any one of the preceding claims 8 or 11. A mammalian cell line caDable of dividing in vitro, having a phenotype associable to that of adipose cells, whic produces in its culture medium,or conditioned medium, at least one compound according to any one of the preceding claims 1-4 which is capable of inhibiting selectively the division of tumoral cells. 12. A mammalian cell line according to claim 11 wherein said tumoral cells comorise epthelial tumoral cells. 13. A mammalian cell line according to claim 12 wherein said epithelial tumoral cells derive from estrogen-sensitive epithelial tumors. 14. A mammalias cell line according to any one of the Drecεding claims 11-13 and capable of dividing in vitro both in the presence and in the absence of serum in the culture medium. 15. A mammalian cell line according to any one of. the preceding claims 11-14 wherein said cell line is isolated from a Hposarcoma. 15. A mammalian cell line according to claim 15 wherein said cell line is isolated from a human Hposarcoma. 17. A mammalian cell line according to claim 15, wherei said cell line is the LSA line (DSM ACC. N. 2029). 18. A mediumthat has been conditioned by the growth of a cell line according to any one of the preceding claims 11-17 19. A medium that has been conditioned according to claim 18, said medium comprising at least one compound capable of in¬ hibiting selectively the division of tumoral cells. 20. A medium that has been conditioned according to claim 19, wherein said tumoral cells are eoithelial tumoral cells. 21. A medium that has been conditioned according to claim 20 wherein said eoithelial tumoral cells derive from estrogen- sensitive epithelial tumors. 22. A medium that has been conditioned according to claim 21, wherein said cell line is the LSA line (DSM ACC.N. 2029). 23. Use of the conditioned medium according to any one of the preceding claims 18-22 for the production of pharmaceutical compositions for treatment of tumors. 24. Use of the conditioned medium according to claim 23 wherein said tumors are epithelial tumors. 25. Use of the conditioned medium according to claim 24 wherein said eoithelial tumors are estrogen-sensi¬ tive. 25. Use . of the conditioned medium according to any one of the preceding claims 18-22 for the purification, identi¬ fication and characterization of at least one compound which is capable of inhibiting selectively the division of tumoral cells. 27. Use of the conditioned medium according to claim 26, wherein said tumoral cells are epithelial cells. 28. Use of the conditioned medium according to claim 27 wherein said cells are derived from estrogen-sensitive epithelial tumors.

Inventors: MANCINI ALDO, MANCINI, ALDO;
Applicants: IST NAZ STUD CURA DEI TUMORI, ISTITUTO NAZIONALE PER LO STUDIO E LA CURA DEI TUM
Application year: 1993
Main classification: C12N15
IPC codes: A61K38, C07K14, C12N15