CA2130089A1 - Protein compound, coding nucleotide sequences, producing cell line and uses thereof - Google Patents

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

wo 93/18147 Pcrtr~93/0002() 213008~ -PROTEIN COMPOUND CAPABLE ~F INHIBITING TUMORAL GROWTH

ThiS invention relatos to a substantially proteic com-positlon which is capable of inhibiting selectively the div-S lsion of tumoral cells sensitivo to estrogons and/or of exerting a cytotoxic activity on said cells. This invention also rel~tes to a cell line isolated from a human liposarcom2, capable of divlding in a culture, which line produces said composition, as well as to its culture conditioned medium in t which.said composition is present.
Thts lnvention ls also concerned with the emPloyments of s~ld products tn tl~gnosttc and in clinical ~ppltc~tions.
A large number of cell l~nes capable of growing ~n v~tro and ortgtnally isolated from hum~n tumors ~re already known from the prior art. Such lines are p~rticul~rly useful .for the study of their b~ochemical anJ physiological properties, ~s ~211 ~S for the production of specific factOrs.
In spite of the l~rge number of ~ttempts maJe up to the present time, no cell lines are known from the Dr~or art which - 2D have a dtffenenti~ted phenotype that can be assoctated to that of the adlpose c911s or adioocytes, snd no lines sre known which ~re isolated ~rom tumors belonging to the class of lipo-sa~com~sO Such cells would be p~rticularly useful as they derive from the~stromal tissue, a tissue which at the present , : ~
time has been very little characterized and for wh k h a con-trolling role has been suggested ~s regards some of the func-tions performed by the ad~acent tissues. Indeed, in vivo, tn the mammary gl~nd in the presence of the hormQne testostero-~ ne, ~di~ose cells induce the regression of epltheli~l tissue ; 30 tKratochwlll. K. Development ~nd loss of androgen responsive-ness tn the embryonic rudiment of the mouse mammary gland.
~ . .

~ s3 WO 93/18147 pCr/lT93/0002(J

2~3o~8~

Dev. Biol. 61: 358-365, 1977~ so exerting paracrine actlon which poss~bly is medlated by one or more compounds released by said ad~ose cells tKratochwill, K. 1987, in "Cellular and Molecular Biology of Mammary Cancer" (D. Medina, W. Ktdwell, G. Heppner, e E. ~nderson, eds.), pp. 1-8 Plenum, New York~.
The investigations mentioned above put partlcularly into evldence the need for lsolating and growlng in vitro a cell line capable of performlng the functlons observed in v~vo.
~ he Author of this invention has isolated and character-lzed for the flrst tlme a cell line which ls capable of div-iding in a culture, wlth a phenotype associable with that of adipose cells. Surprisingly, the cell line produces ln the culture medium, denominated as conditioned mRdium, at least - one compound that is capable of ~nh~blting selectively the 1~ division of epithelial tumoral cells deriv~ng f~om epithellal tumors which are sensitive to estrogens.
By the expression ~phenotype associable to the phenotype of ~dipose cells" it is meant the manifestation of at least - one differentiated character which i5 proper of adipose cells, like the synthesls of lipids. By the expression "eplthe;ial tumoral cells~ ~t ~s me~nt tumoral cel~s deriYed from epithelial tumors, said cells being isolated or still present in the tumoral mass. By the express~on "estrogen-sensitive eplthelial tumors" ~t is meant ~umors comprising cells with zt least one receptor for a hormone belonging to the C12SS of estrogens. By the expression "conditioned medium"
it is meant the culturemediumin the presence of which the cells are incubated under temperature, humidity and pH con-ditlons sultable for growing the same. for at least 6 hours.
By the llcaDabsllty for inhlblting cell divlsion" it is meant the capability of a co~pound for inhiblting at least 50 X of the cell growth in a culture, after zt least 3 days of incu-~ ~ .

WO 93/18~47 32 l 3 0 0 8 9 PCltlT9310002~

bation in the culture soil of sa~d cells.
Accordingly, there is evidently the need for ~dentlfying2nd charact~rizlng the comDound/s capabl~ of inhibittng sel-ectively the divlsion of tumoral cells which are derived from estrogen-sensitive epithelial tumors.
~ n a paper published recently [Mendelsohn M.E. et al., Proc. Natl. Acad. Sci. USA, 88, ~1212-11216~ 1991] it is dis-closed that the "heat shock" p27 human protein (HSD27), al-ready identifted 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 tCoffer A.I. et al., Cancer Research 45, 3686-3693, 1990].
More recently tcarPer S.W. et al., Nucl. Acids Res. 18, 6457, 1990],a cDNA clone obtained from the A549 cell line of hum~n lung carcinoma has been sequenced. The nucleotide se-quence of such clone codes for a protein of 205 amino acids in whlch the first 193 amino acids coincide with those of hum~n HSp27 already known. In correspondenGe with the codon that codes for the amino ~cid resldue in the posltlon 194, the insertion of two cytosine residues has been detecte~, which determines a shift of the re~ding frame to the following stop codon in the positions 616-618 of the nucleotide se-quenc~. The insertion thus causes a modification of the C-terminal sequence of the HSp27 protein that div_rges for th~last 5 amino acids ~from the 195 position to the 1~9 position) and shows an extension of 6 more amino acids (from the pos-ition 200 to the position 205).
No inhibit~on activity of cell division of tumoral cells ~ 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
, ' wog3/1814/ ?.~o~9 - 4 - pCI'/lT93/001~2U

line the Author of this invention has identified and charac-terized a compound, whose composition is substantially protein aceous tn nature, capable of inhibiting selectively the dtv-tslon of estrogen-sensitive tumoral ep~thelial cells and of exerting a cytotoxic activity on such cells. Th~ Author has ~iochemically characterized said compcund, which has ~en called plLSA, and has determined its amino acid sequence, whlch has turned out to be made up of 205 amino acids. ~he Author has found surprisingly that said amino acid sequence is identical wlth the sequence of the human HSp27 protein already determined by Carper S.W. (ibid.) except for a slngle amino acid. The difference observed is in the amino acid 194 (arginino instead of proline) and is determined by a sub-stitution of cytosine in the positton 581 of the sequence that codes for the protein HSp27 wlth a guanine.
Accordlngly, the Author has'identified a new compound having an antineopl~stic blological ~ctivity which is specific for estrogen-sensitive epithelial tumoral cells.
The Author has identified the nucleotide sequence that codes for the protein p1LSA and has determined its amino acid sequence.
Accordingly, it is an object of this invention a compound whose compos~tion is substantially proteinaceous in nature, said compound being capable of inhibitlng selectively the cellular division of estr3gen-sensitive epithelial tumoral cells and/or of exerting a cytotoxic activity on said cells:
said compound having a malecular welght in the range from 27 to 30 kDa; satd compound also comprlsing an amino acid se-quenco from the amlno 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 sald ¦~ comDound which ts biologically acttve.
I -WO93~18~47 pCTr~3l00020 ... ~ 5 213008~
By th~ expression ~'substantially ~omologous amlno actd se~uences" lt is me~nt in the scope of thls invention to designate sequences with homologies in the range from S0 t to 100 ~ wh~ch do not give rtse to a loss of btological activity S of the protetn. By "biological activity" tt ls meant the cap~bllity of inhlbiting selectively the divts~on of estrogen-sensitive epithelial tumoral cells (cytost~tlc activity) and/
or of exerting ~ cytotoxic activlty on sald cells.
Accordtng to ~ preferred embodiment of this invention, said compound whose composltion is subst~ntially proteinaceous in nature comprises at lts carboxyl end ~n amino acid sequence whlch is substantially ~omologous to the sequence:
, GluAl~AlaLysSerAspGluThrAlaAlaLys-NH2 Preferably sald substant~ally protetnaceous compound comprlses the following ~min~ actd sequence:

MetThrGluArgArgValProPheSerLeuLeuArgGlyProSer 15 TrpAspProPheArgAspTrpTyrProHisSerArgLeuPheAsp 30 GlnAlaP~eGlyLeuProArgLeuProGluGluTrpSerGlnTrp 45 ~-~ LeuGlyGlySerSerTrpProGlyTyrValArgProLeuProPro 60 AlaAlaIleGluSerProAlaValAlaAlaProAlaTyrSerArg 75 AlaLeuSerArgGlnLeuSerSerGlyValSerGluIleArgHis 90 ThrAlaAspArgTrpArgValSerLeuAspValAsnHisPheAla 105 ProAspGluLeuThrYalLysThrLysAspGlyValValGluIle 120 ThrGlyLysHisGluGlu~rgGlnAspGluHisGlyTyrIleSer 135 ArgCysPheThrArgLysTyrThrLeuProProGlyValAspPro lS0 - 25 ThrGlnValSerSerSerLeuSerProGluGlyThrLeuThrVal 165 GluAlaProMetProLysLeuAlaThrGlnSerAsnGluIleThr 180 IleProValThrPheGluSerArgAlaGlnLeuGlyGlyArgGlu 195 AlaAlaLysSerAspGluThrAlaAlaLys 20S.

Accordlng to a preferred embodiment of this invention, sa~d substanttally protetnaceous comDound is produced by the LSA

., , ~ . .
, ~

W093/18147 2 ~3 00~9 - 6 - PCTr~3/~2U

cells tDsM ACC. N. 2029) preferably is secret~d by said cells ln th~ culture mediu~.
It is a furthDr object of this invention a compos~tion for pharmacological uses, pref2rably for antineoplast k ap-S plicat~ons, and ev~n more preferably for the treatment ofestrogen-s~nsitive epitheltal tumors, th~t comprlses the sub-st~ntlally protein~ceous compound of this invent~on or salts thereof which ~re physiologically ~cceptable.
It is a f.urther object of this invention the employment of s~ld subst~ntl~lly protelnaceous compound according to the ~nventlon or of lts s~lts whlch are phys~ologically accept-~ble for the prep~r~tion of pharm~cological compounds for anti-neopl~st~c tre~tment, preferably for thè treatment of estrogen-sensit~ve epithel~al tumors.
It is ~n object of thls invent~on ~ nucleic ~cid com-pr~s~ng ~ nucleotide sequence that codes for the compound whose co~slt~on ~s subst~nt~lly protelnaceous accordlng to this inventlon. prefer~bly said nucleic acld ::~ comprlsing the following nucleot~de sequenc~:
,~ ~

. TTAGGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCC 180 GCGCTC~GCCGGCAACTCAGCAGCGGGGTCTCGGAGATCCGGCAC 270 . 25 ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCAACCACTTCGCC 315 ~ 30 GAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540 :~: ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCCGAGAA 585 ,,. - , ,~'','~'' ' , ~
" ~' ",, w093/1814~ - 7 - PCTr~3/0002~

A further object of this invention consists in plasmld or phage type vectors comprising the nucleot~ sequences of th~s invention.
It is a further object of this inventlon a mammalian cell line capable of dividing in vitro, and hav~ng a phono-tyDe associablc to that of ~di~os~ colls, that produces also in lts conditioned medium at least one compound caoablo of inhibiting selectively the division of tumorai cells, preferably epitholial tumoral cells, which even more preferably dcrive from estrogen-sensitive epithelial tumors.
Accordtng to a preferred embodimont of this invention, said cell line is isolzted from a liposarcoma, preferably of human origin, ~nd also more preferably such line ~s the LSA
cell line deposited with the DSM with the accession number t5 2029. Agair according to this inv~ntion, said cell line pro-duces the substantially proteinaceous compound having the se-quence disclosed above.
It is a further object of the invention a ~.edium con-ditioned by the growth of c~ll llnes herein ~lsclosed and claimed.
Prefer~bly sa~d conditioned medium comprises at le~st one compound cap2ble of inhibiting selectively the dtvision of ~umoral cells, preferably eDithelial tumoral cells, and even more preferably cells deriving from estrogen-sensitive epi-thelial tumors, mcre preferably sald compound being the pro-tein compound disclosed according to this invention.
Accord~ng to a preferred embodiment of the present in-Yention, said medium isconditioned by the growth of the ~S~
cell line (DSM ACC. N. 2029).
It is another object of this invention the employment of said conditioned nedlum for producing pharmaceutical compo-sitions for treating tumors. Dreferably epithelial tumors~ and .~"~
;~

w093/18147 00~ - 8- PCrtll~3~10020 even more preferably for the treatment of estrogen-sensit~ve epithelial tumors.
It is a further object of this învention the emDloymont of said conditioned medium for t~ purification, ident~f~cation S and characterization of at l~ast ono compound capable of inhibiting selectively the division of tumoral cells, prefe-rably of epithellal tumoral cells, and even more preferably of cells derived from estrogen-sensitive eplthelial tumors.
This lnvention will be now disclosed by means of some examples that ~xplain and illustrate the same but with no lim~t~tive purposes, and wlth reference to the enclosed flg-ures wherein:
Figure 1 represents a growth curve of the LSA l~ne both in the presence ~nd in the ~bsence of serum:
~5 Figure 2a shows a cytofluorimetric analysis of human norm~l th~mocytes;
Figure 2b shows a cytofluor~metrlc an~!ysis of LSA cells:
Figure 3 shows an LSA-CM stimulation histogram of the incorporation Of tH]-thymidine in various cell lines, where 1 is the control sample, 2 is the LSA-CM ~t 1/4 dilution, 3 is the LSA-CM at 1/2 dilution. 4 is the LSA-CM undilute~, 5 is a control sample, 6 is the LSA-CM undiluted, 7 is the con-trol sample, ~nd 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:
Flgure 5 represents growth curves of the ZR-75-1 cell llne, 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 ~Sence of ~SA-CM:
~;~ 30 Figure 7 reDresents growth curves of the NMMG cell line ln the ~bsence and in t~e presence of LSA-CM;
figure 8 represents growth curves of human col l S from ~, .

WO 93/1814, pCI'111`93/00020 2~3008~

ov~ric carcinoma both in the absence and in the presence of LSA-CM:
Figure 9 re~resents a histogram of human cell growth from ovaric carcinoma both ln the absence and in the presence of LSA-CM and/or of purified D1LsA.
xample 1 Isol~tion of the LSA line A fragment of hu~an liPos3rcs~a of mixed l~Doblastic-fibroblastic type is drawn in a sterile way from the leg of an adult wom~n, 65 years of age, by m~ans of surgical tech-niqu-s.
The fragment is disgregated mechanically ~y a chlsel so as to obtain 1 ~m fragments and then thls material is tr~ated with a CTC solution equivalent to 20 U/ml of collagenase (CLSP, Worthlngton, .U.K. .), 0,75 mg/ml of tryps~n (1/
300, lnc. Ph~rmaceuticais~, 2 X ehick~n serum. heat~inacti vated and dialyzed (6IBC0) in Hank soil wlthout Ca~+ ~nd Mg+~
ions (6IBC0) and H2m F12 soil with 5 X boviRe serum (GIBC0) for 4 hours at 37~C and under c~ntinuous stirrlng, ln order to obtaln ~ susDension of single cells.
T~e cell suspension is dilut~d with the Ham F12 (GI8~0) culture medium s~pple~n~d ~ith S ~ b~vine serum (~lBCO)i Deni-cillin (31 ~g/ml~, str~Dtomycin (S0 ~g/ml) and fung~zone (2.5 ~g/ml). the cells are plated at 200.00~ cellsldish, 100 m~ di~meter (Costar).
A clone of cells which is capable of dlviding in a cul-tura and having the characteristics wh~ch are pecullar of the adiposa cells has been obtained from ten dis~s, said clone being called LSA and deposited wittl the DSM, accession num-ber ACC 2029.
A growth curve of the LSA line i5 shown in Fiaure 1, in which the growth in the presenc- as well as in the 2bsence wos3/1814~ 2~3~ 1 o - PCTt~93/0002U

of serum is evident. The LSA l~ne shows a plating effic~ency of 90 X and a du~lication 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 Characterizztion of the LSA line The ~NA content of LSA cells has been analyzed by me~ns of a cytofluorometer (Partec Pas II flow-cytometer). The cells ~re trypsini~ed then fixed with 70 ~ ethanol and dyed with a solution containlng S ~g/ml of eth~dlum bromide, 12.5 ~g/ml of mltramycin and 1.5 mg MgCl2 in 0.~ M Tris ~uffer, pH 7.5.
The cell percentage in the v~rious steps of the mitotlc cycle is obtained as disclosed by Flintoff, W.E., DaYidson, S.Y., ~nd Siminowitch, L. "Isol~tion and partial characterization of Metho~rex~t~-Resistent ph~notype from chinese hamster ovary cells" Somatic Cell Genet. 2:245-261, ~976: and Ambesi-lmp~om-bato, f.S., P~rks, L.A.M. ~nd Coon H.G. Culture of hormone-dep~ndent funct~on~l epitheli~l cells from rat thyrold. Proc.
Natl. Acad~ Sci. USA 77: 3455~3459~ 1980. A~s shown in Figure 2b, the ~alues are compared to those obtained with normal human thymocytes as ploity standard . The value of 1 haS 'been assigned to the G1-G0 peak of thymocytes. Under the s~me con-dltions the 61-G0 peak of ~he ~SA cel ls has a value o~ 1.79, which is indlc~tive of a DNA content which is almost tetra-ploid. Also the DNA content/cell is almost twice as mu~h as th~t obtained with thymocytes as shown in Figure 2a.
The d~stribution 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.
~30 Table 1 Phase Cell percentage WO 93/1814 / - 1 1 21 3 0 0 $ 9 P~tlT93/0002(~

The presence of cells in the ph2se S confirms that the population is in the active growth ph2se.
The c~unting of the chromosomes has put into evidence a number of chromosomes/cell between 80 and 90.
The LSA cells as observ~d under an optical microscope look like elongated bodles with abudant cytoplasm. Under the electronic mlcroscope, the cell nucleus contains many nu-cleoles and some organelles ~re present, locallzed in the p~rinuclear region. The cells are homogeneous to each other, the mitochondria ~re relatively abudant and the cytoplasmic vesicies are const~nt bo-th-in number and sizes. The most ev~-dent feature is the presence of vacuoltr areas inside the cytoplasm, said ~reas being el~ctron-reflecting and typical of zones containing high amounts~of lipids. which is removed as a result of subjecttng the cells to a treatment for the1r prepar~tion for observation under the electron microscope.
Moreover, ~ well developed Golgl apparatus ~s also present.
ln order to estimate the content of liplds, the LSA cells are plated on culture slides of the Lab-Teck (NUNC) ~nd are grown by semi-confluence. The cells are washed snd fixed for 6 mlnutes with 4 ~ paraformaldehyde and 15 X Picric acid s~tur~ted in P8S (phosphate saline buffer). The cells are washed twice with dlstilled water, then they are dyed ~ith an Oil Red 0 solution for S minutes, then they are washed twic~
with PBS and dyed with 1 X toluidine blue for 10 seconds.
- If cells have been grown in the presence of 5 X bovine serum, lipids are disDersed throughout the cytoplasm, with round ves~cles. The build up of lipids is not present in cells grown in a nedium without serum, but it is stimul~ted by in-cubating cells with methylxanthine/desamethasone for 48 hours ' ~, wo 93/~814~ pCrlrr93/0002(~
~,~3~ 1 2 -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 SX bovine serum. The cell monolayers are washed wlth P8S and incubated with f12 medium without serum. After 24 hours, the medium is replaced with ~resh medium the conditioned medium is collected every 6-24 hours for the successive 30 days. The cells go on growing and they show structur~lly and functionallyviable under phase-con-trast microscope and after dying with Trypan Blue.
The medium conditionod by ~SA cells is capable of stimulat-ing the growth of normal epithellal cells like CH0 (CCL-61), fRTL-5 (CRL-1468) and NlH-3T3 (ECACC-87100803) grown for 24 hours ~n the absence of serum, as put into evldence by an 3H-thymidine lncorporation test shown in Figure 3. Contrarily to the preceding results,t~ medium conditionod by LSA cells shows ~n inhibit$on effect of growth ~d ~ cytolyt~c activ1ty when it is analyzed wlth in vitro tumor21 cells contai~ing "
receptors for estrogens. T~e cells emDloyed are:
~CF-7 cells obtained from a human mammary carcinoma (L~nd. ~. et al., Sc~ence 222:778 (1980), ECACC-86012803) that possess receptors for estrogens (Soule, H~D. et al., Natl.
2~ Cancer Inst. 51, 1409-iS'3 (1973)), androgens (H~rowitz, K.B.
et al., Steroids 26, 785-795 (197~ insulin ~nd triodothy-ronine (MacGrath, C.M. Cancer ~es., 43: 1355-1360 (1983);
the ZR-75-1 (CRL-1504) cells obtained from a human mam-mary carc~noma, said cells being posltive for estrogen recep-tors;
-~ the MDAMB-231 (ATCC-HTB26) cells obtained froma a human mammary carcinoma. but t~at are negative for estrogen recep-, ~ .

wos3/18147 - 13 - PCT~3/0~2~
213()o8~
tors;
the NMMG (Burker, R.E. et al., C2ncer Res., 38, 3769-3773 (1978), CRL-1636) cells, obtained from a mouse mammary epithelium, said cells being slightly positive for estrogen receptors:
~ human cell line of ov~ric carcinoma obtained from a woman suffering from perltoneal effusion due to ovaric carci-noma metast~sis, sald line being positive for estrogen recep-tor.
The growth curves of the cell lines mentioned herein h~ve been obttined by plating 100,000 cells/dish of 100 mm diameter ln 10 ml of DMEM (GIBCO)medium with 10 X bovtne fetal serum (FCSi GI8C0). 2 ml of LSA-C~ have been added to a set of dishes flgure 4 shows growth curves of th~ MCf-7 line in which the inhibltion effect of LSA-CM is evident. In partlcular, 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 ~nd in the absence of LSA-CM. The presence of ~SA-CM
gives rise to a strong inhibltion of growth without any cy~o-lytic effect.
Figure 6 ~nd 7 show the growth curves of MDAMB-231 cells and of normal epithelial NMM6 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 incubatlon.
The LSA-CM effect on the incorporation of H-thymidine in dlfferent cell lines is shown in the following Table 2.

WO 93~18147 ~ 4 pCl'llT93/0002() ~,~3~0~

Table 2 Effect of LSA-CM on 3H-thymidine incorporation in different cell lines Cell linesNo effect Stimulation Inhibition S
MCF7 +

NMMG +
fRTL-5 +
CHO
NIH-3T3 +
Example 4 Eff~ct of LSA-CM on animals 20 Balb/c fc3H mice (Charles River) which are affected tS by the Blttner virus are selected for the presence of an evident tumoral mass, an estrogen-dependent mammary carcinoma, whlch is tnduce-d by the virus ltself.
After a peritumoral injection practised eYery day of 200 ~ul of LSA-CM for 15 days, the lnhibition of ~growth and the necrosis of the tumoral mass ~re observed ~n 80 ~ of the mice treated. -Example 5 Isolation of the p1LSA proteln from LSA-CM
The LSA-CM medium is concentrated 100 x, then it is sub-jected to dialysls ~gainst a isotonic phosphatebufferat pH 7.4 and then to gel filtration on P6 resin (Phar-macia). The fractions eiuted are tested for their capability of inhibiting s~lectively the grow~h of MCF-7 cells. One only fraction shows sald biological activity. A sample of said fraction ls separ~ted by electrop~oresls on a polyacrylamide ~-~ gel in non-denaturating condi~lons, identlfylng a protein of about 29 kDa which is called p1LSA. Said protein ls electro-,, .

,~

wos3/18l47 - 15 ~1 3 0 ~ ~ ~ pcT/n~

eluted from the gel and employed for ~h e production of po 1 y - `
clonal 2ntlbodi~s as dlsclosed in the followbg and for the sequence analysis.
The protein sequence is shown in the follow~ng Tabl~ 3.
As shown in Figure 9, the purifi~d p1LSA prote~n has an evldent cytotoxic effect on the growth of human ovaric tumoral cells.
- .. Table 3 Amino acid sequence of th ptLSA proteln and correspondlng coding sequence MetThrGluArgArgValProPheSerLeuLeuArgGlyProSer 15 TrpAspProPheArgAspTrpTyrProHisSerArgLeuPheAsp 30 GlnAlaPheGlyLeuProArgLeuProGluGluTrpSerGlnTrp 45 LeuGlyGlySerSerTrpProGlyTyrValArgProLeuProPro 60 AlaAlaIleGluSer~roAlaValAlaAlaProAlaTyrSerArg 75 GCGCTCAGCCGGCAACT~AGGAGCGGGGTCTCGGAGATCCGGCAC 270 AlaLeuSerArgGlnLeuSerSerGlyValSerGluIleArgHis 90 ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCA~CCACTTCGCC 315 ThrAlaAspArgTrpArgValSerLeuAspValAsnHisPheAla 105 ProAspGluLeuThrValLysThrLysAspGlyValValGluIle 120 ThrGlyLysHisGluGluArgGlnAspGluHisGlyTyrIleSer 135 ArgCysPheThrArgLysTyrThrLeuProProGlyValAspPro 150 ':

,~

WO 93/t8147 pCrtlT93/0002U
300~ t 6 ACCCAAGmCCTCCTCCCTGTCCCCTGAGGGCACACTGACCGTG 4 9 5 ThrGlnValSerSerSerLeuSerProGluGlyThrLeuThrVal 165 &AGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540 GluAlaProMetPro~ysLeuAlaThrGlnSerAsnGluIleThr 180 ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCC~AGAA 585 IleProValThrPheGluSerArgAlaGlnLeuGlyGlyArgGlu 195 AlaRlaLysSerAspGluThrAlaAlaLys 205 from a compartson with the saquences dopostted with data banks, the sequence turns out to be identical with that corresponding to the human HSp27 protein describ2d by Carper S.W. mentioned aboYe, except for just one amino acid. The dlfference consists in a substitution of the amino acid in the position 194, said amino ~cid turning out to be arginine instead of proline.
Example 6 Productlon of anti-p1LSA polyclonal ~nti-bodies, and inhibition of the ~ctivity of the p1LSA protein ' 100 ,ug of p1LSA protein electroeluted from a 10 X poly-acrylamide g~l is dissolYed in 1 ml of PBS (phosphate saline buffer) and mtxed with 1 ml of a complete Freund adjuvant.
The mixture is injected through intramuscolar injectio~ in a rabbit four times at 10 day interYals. 30 ml of immune serum are drawn from rabbits every other day for 20 days. The immu-noglobulin (lg) fraction is purified by the Mab Trap G (Phar-macia) ~ccording to the procedure recommended by the producer.
- 30 lmmunoDreclpltation ~nd immunoblotting tests show that : tmmunoglobulins re~ct in a sDecific way wlth the p1LSA pro-tetn from LSA-CM.
, :

~ .
~ ' wos3/ls147 17 21~ 0089 A pre-incub~tion of 100 ,ul of LSA-CM w~th 10 ~l of an 19 solutlon at 1 mg/ml for 30 mtnutes at 37C stops the ac-tivity of tnhtbition of tumoral ePtthelial cell (MCF-7) div-ision, restorlng an H-thymidlne lncorporatlon and growth curves similar to the untreated control sample. The LSA-CM
activlty turns out to be unaltered after incubation Wtth pre-tmmune serum.
Such experimental data show that the p1LSA protein isolated and purtfied according to the procedures disclosed ln Example 5 and havtng the sequence disclosed in T~ble 3 is actually the act1ve prtnctple responslble for the activity of inhtbitton of epithellal cell divlsion disclosed herein.
Example 7 Nucleotide sequence codtng for the p1LSA
tS prote~n The nucleotlde sequence coding for the p1LSA protein ls determlned by purlfylng the RNA polyA~ from LSA cells follow-lng stand~rd methods ~nd employing the followtng primers for the PCR reaction:

oligo 5': CG9~$~TGAGCAGACGTCCAGAG

:: EcoRI

oligo 3': CGG~CCGGGCTAAGGC~TTACTTG

EcoRI

The sequences ~re deduced respectively from the sequences at the positions 5' and 3' not translated of the gene that codes for the HSp27.
I The ampltflcation products are cloned directly tn the EcoRI site of the vectors pGEM4z (Promega) and sequenced by means of the dideoxy method. The complete sequence of the ¦-~ codlng portion ls shown in the preceding Table 3.
li~ This invention has been dtsclosed with reference to some 1~',' :
1,~
l,i,, ,,, I`~
i-, WO 93/18147 1 8 - - PCrl~T93/0002() ~,~3~0~9 preferred embodiments of the same but lt is to be understoo~
that modiflcatlons and/or changes can be introduced by those who are skilled in the art without departing from the spirit and scope of the invention.

, , ~ ~

W093/1814/ pCT/~3/0002U
213008~

SEQUENCE LISTING

(1) GENERAL INFORMATION:
( i ) APPLICANT:
(A) NAME: Ist. 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 ~equences, producing cell line and uses thereof (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM:
(A) MEDIUN 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 NUNBER: IT RM92/A000161 (B) FILING DATE: O9-NAR-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) C~ELL LINE: LSA cell line deposited at DSM N. ACC2029 (vii) IMMEDIATE SOURCE:
(A) LIBRARY: cDNA library from poly A+ RNA
~ ~ (B) CLONE: plLSA
,~

,~

WO93/18147 ~ pCT/n~3/0002U

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..618 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Met Thr Glu Arg Arg Val Pro Phe Ser Leu Leu Arg Gly Pro Ser Trp Asp Pro Phe Arg Asp Trp Tyr Pro His Ser Arg Leu Phe Asp Gln Ala Phe Gly Leu Pro Arg Leu Pro Glu Glu Trp Ser Gln Trp Leu Gly Gly Ser Ser Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala I le Glu Ser Pro Ala Val Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gln Leu Ser Ser Gly Val Ser Glu Ile Arg His Thr Ala Asp Arg Trp Arg GTG TCC CTG GAT GTC AAC CAC TTC GCC CCG GAC GAG CTG ~CG GTC AAG 336 Val Ser Leu Asp Val Asn His Phe Ala Pro Asp Glu Leu Thr Val Lys Thr Lys Asp Gly Val Val Glu Ile Thr Gly Lys His Glu Glu Arg Gln A~p Glu His Gly Tyr Ile Ser Arg Cys Phe Thr Arg Lys Tyr Thr Leu 130 135 . 140 Pro Pro Gly Val Asp Pro Thr Gln Val Ser Ser Ser Leu Ser Pro G1U
145 150 155 ' 160 Gly Thr Leu Thr Val Glu Ala Pro Met Pro Lys Leu Ala Thr Gln Ser Asn Glu Ile Thr Ile Pro Val Thr Phe Glu Ser Arg Ala Gln Leu Gly Gly Arg Glu Ala Ala Lys Ser Asp Glu Thr Ala Ala Lys :~
.,~, WO93~18147 2 1 3 0 0 8 ~ pCTt~3/0002(~ ;

(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 Asp Pro Phe Arg Asp Trp Tyr Pro His Ser Arg Leu Phe Asp Gln Ala Phe Gly Leu Pro Arg Leu Pro Glu Glu Trp Ser Gln Trp Leu Gly Gly Ser Ser Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala Ile Glu .
Ser Pro Ala Val Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gln Leu Ser Ser Gly Val Ser Glu Ile Arg His Thr Ala Asp Arg Trp Arg 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 Ile Thr Gly Lys His Glu Glu Arg Gln ~ 115 120 125 : Asp Glu His Gly Tyr Ile Ser Arg Cys Phe Thr Arg Lys Tyr Thr Leu Pro Pro Gly Val Asp Pro Thr Gln Val Ser Ser Ser Leu Ser Pro Glu Gly Thr Leu Thr Val Glu Ala Pro Met Pro Lys Leu Ala Thr Gln Ser Asn Glu Ile Thr Ile Pro Val Thr Phe Glu Ser Arg Ala Gln Leu Gly ~: Gly Arg Glu Ala Ala Lys Ser Asp Glu Thr Ala Ala Lys :,

Claims (28)

1. A proteinaceous compound suitable for inhibiting selectively the cellular division of estrogen-sensitive epithelial tumoral cells and/or of exerting a cytotoxic activity on such cells; having an amino acid sequence from the amino acid 1 to the amino acid 194 which is homologous to the corresponding sequence of human HSp27 (heat shock p27) protein, characterized in that:
- it has a molecular weight in the range between 27 and 30 kDa by size fractionation on a polyacrylamide gel in non-denaturing conditions, - its carboxy end has an amino acid sequence from aminoacid 195 to aminoacid 205 which is homologous to the sequence:
or a fragment of said compound, suitable for inhibiting selectively the cellular division of estrogen-sensitive epithelial tumoral cells and/or of exerting a cytotoxic activity on such cells.

2. A proteinaceous compound according to claim 1, such compound having the following amino acid sequence:

3. A proteinaceous compound according to any one of the preceding claims which is produced by LSA
cells (DSM ACC. N.2029), which compound is preferably secreted by said cells in the culture medium.

4. A composition suitable for pharmacological anti-neoplastic therapy, comprising the proteinaceous compound according to any one of the preceding claims, or physiologically acceptable salts thereof.

5. A composition according to claim 5 suitable for treatment of estrogen-sensitive epithelial tumors.

6. Use of the proteinaceous compound according to any one of the preceding claims or of its physiologically acceptable salts, for the preparation of pharmacological compositions for antineoplastic treatment.

7. Use of the proteinaceous compound according to claim 6 wherein said pharmacological compositions are for antineoplastic treatment of estrogen-sensitive epithelial tumors.

8. A nucleic acid having a nucleotide sequence coding for said proteinaceous compound according to any one of preceding claims 1-3.

9. A nucleic acid according to claim 8 having the following nucleotide sequence:

10. Plasmid or phage vectors comprising the nucleotide sequences according to any one of the preceding claims 8 or 9.

11. A mammalian cell line capable of dividing in vitro, expressing at least one differentiated character proper of adipose cells which produces in its culture medium, or conditioned medium, at least one compound according to any of the preceding claims 1-3 which is capable of inhibiting selectively the division of tumoral cells.

12. A mammalian cell line according to claim 11 wherein said tumoral cells comprise epithelial tumoral ceils.

13. A mammalian cell line according to claim 12 wherein said epithelial tumoral cells derive from estrogen-sensitive epithelial tumors.

14. A mammalian cell line according to any one of the preceding 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 obtained from a liposarcoma.

16. A mammalian cell line according to claim 15 wherein said cell line is isolated from a human liposarcoma.

17. A mammalian cell line according to claim 18, wherein said cell line is the LSA line (DSM ACC. N.
2029).

18, A medium that 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 inhibiting selectively the division of tumoral cells.

20. A medium that has been conditioned according to claim 19, wherein said tumoral cells are epithelial tumoral cells.

21. A medium that has been conditioned according to claim 22 wherein said epithelial 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 epithelial tumors are estrogen-sensitive.

26. Use of the conditioned medium according to anyone of the preceding claims 18-22 for the purification, identification 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.