COMPOSES PROTEIQUES, SEQUENCES NUCLEOTIDIQUES CODANTES, LIGNEE CELLULAIRE PRODUCTRICE ET LEURS APPLICATIONS

PROTEIN COMPOUND, CODING NUCLEOTIDE SEQUENCES, PRODUCING CELL LINE AND USES THEREOF

Abrégé anglais

2130089 9318147 PCTABS00025
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.

Revendications

CLAIMS
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:
<IMG>
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:
<IMG>

24
<IMG>
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:
<IMG>

<IMG>
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.

26
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.

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~
-- 2
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~:
,~ ~
ATGACCGAGCGCCGCGTCCCCTTCTCGCTCCTGCGGGGCCCCAGC 4S
TGGGACCCCTTCCGCGACTGGTACCCGCATAGCCGCCTCTTCGAC 9O
CAGGCCTTCGGGCTGCCCCGGCTGCCGGAGGAGTGGTCGCAGTGG 135
. TTAGGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCC 180
GCCGCCATCGAGAGCCCCGCAGTGGCCGCGCCCGCCTACAGCCGC 225
GCGCTC~GCCGGCAACTCAGCAGCGGGGTCTCGGAGATCCGGCAC 270
. 25 ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCAACCACTTCGCC 315
CCGGACGAGCTGACGGTCAAGACCAAGGATGGCGTGGTGGAGATC 360
ACCGGTAAGCACGAGGAGCGGCAGGACGAGCATGGCTACATCTCC 405
CGGTGCTTCACGCGGAAATACACGCTGCCCCCCGGTGTGGACCCC 450
ACCCAAGTTTCCTCCTCCCTGTCCCCTGAGGGCACACTGACCGTG 495
~ 30 GAGGCCCCCATGCCCAAGCTAGCCACGCAGTCCAACGAGATCACC 540
:~: ATCCCAGTCACCTTCGAGTCGCGGGCCCAGCTTGGGGGCCGAGAA 585
GCTGCAAAATCCGATGAGACTGCCGCCAAGTAA 618
,,. - ,
,~'','~'' ' , ~
" ~'
",,

w093/1814~ - 7 - PCTr~3/0002~
2130089
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
G1/G0 59

WO 93/1814 / - 1 1 21 3 0 0 $ 9 P~tlT93/0002(~
S 27
G2/M 1 4
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 +
ZR-75-1
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
ATGACCGAGCGCCGCGTCCCCTTCTCGCTCCTGCGGGGCCCCAGC 45
MetThrGluArgArgValProPheSerLeuLeuArgGlyProSer 15
TGGGACCCCTTCCGCGACTGGTACCCGCATAGCCGCCTCTTCGAC 9O
TrpAspProPheArgAspTrpTyrProHisSerArgLeuPheAsp 30
CAGGCCTTCGGGCTGCCCCGGCTGCCGGAGGAGTGGTCGCAGTGG 135
GlnAlaPheGlyLeuProArgLeuProGluGluTrpSerGlnTrp 45
TTAGGCGGCAGCAGCTGGCCAGGCTACGTGCGCCCCCTGCCCCCC 180
LeuGlyGlySerSerTrpProGlyTyrValArgProLeuProPro 60
GCCGCCATCGAGAGCCCCGCAGTGGCCGCGCCCGCCTACAGCCGC 225
AlaAlaIleGluSer~roAlaValAlaAlaProAlaTyrSerArg 75
GCGCTCAGCCGGCAACT~AGGAGCGGGGTCTCGGAGATCCGGCAC 270
AlaLeuSerArgGlnLeuSerSerGlyValSerGluIleArgHis 90
ACTGCGGACCGCTGGCGCGTGTCCCTGGATGTCA~CCACTTCGCC 315
ThrAlaAspArgTrpArgValSerLeuAspValAsnHisPheAla 105
CCGGACGAGCTGACGGTCAAGACCAAGGATGGCGTGGTGGAGATC 360
ProAspGluLeuThrValLysThrLysAspGlyValValGluIle 120
ACCGGTAAGCACGAGGAGCGGCAGGACGAGCATGGCTACATCTCC 405
ThrGlyLysHisGluGluArgGlnAspGluHisGlyTyrIleSer 135
CGGTGCTTCACGCGGAAATACACGCTGCCCCCCGGTGTGGACCCC 450
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
GCTGCAAAATCCGATGAGACTGCCGCCAAGTAA 618
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~
- 19 -
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
- 20-
(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 4
Met Thr Glu Arg Arg Val Pro Phe Ser Leu Leu Arg Gly Pro Ser Trp
5 10 15
GAC CCC TTC CGC GAC TGG TAC CCG CAT AGC CGC CTC TTC GAC CAG GCC 96
Asp Pro Phe Arg Asp Trp Tyr Pro His Ser Arg Leu Phe Asp Gln Ala
20 25 30
TTC GGG CTG CCC CGG CTG CCG GAG GAG TGG TCG CAG TGG TTA GGC GGC 144
Phe Gly Leu Pro Arg Leu Pro Glu Glu Trp Ser Gln Trp Leu Gly Gly
35 40 45
AGC AGC TGG CCA GGC TAC GTG CGC CCC CTG CCC CCC GCC GCC ATC GAG 192
Ser Ser Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala I le Glu
50 55 60
AGC CCC GCA GTG GCC GCG CCC GCC TAC AGC CGC GCG CTC AGC CGG CAA 240
Ser Pro Ala Val Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gln
65 70 75 80
CTC AGC AGC GGG GTC TCG GAG ATC CGG CAC ACT GCG GAC CGC TGG CGC 288
Leu Ser Ser Gly Val Ser Glu Ile Arg His Thr Ala Asp Arg Trp Arg
85 90 95
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
100 105 110
ACC AAG GAT GGC GTG GTG GAG ATC ACC GGT AAG CAC GAG GAG CGG CAG 384
Thr Lys Asp Gly Val Val Glu Ile Thr Gly Lys His Glu Glu Arg Gln
115 120 125
GAC GAG CAT GGC TAC ATC TCC CGG TGC TTC ACG CGG AAA TAC ACG CTG 432
A~p Glu His Gly Tyr Ile 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 480
Pro Pro Gly Val Asp Pro Thr Gln Val Ser Ser Ser Leu Ser Pro G1U
145 150 155 ' 160
GGC ACA CTG ACC GTG GAG GCC CCC ATG CCC AAG CTA GCC ACG CAG TCC 523
Gly Thr Leu Thr Val Glu Ala Pro Met Pro Lys Leu Ala Thr Gln Ser
165 170 11S
AAC GAG ATC ACC ATC CCA GTC ACC TTC GAG TCG CGG GCC CAG CTT GGG 576
Asn Glu Ile Thr Ile Pro Val Thr Phe Glu Ser Arg Ala Gln Leu Gly
180 - 185 190
GGC CGA GAA GCT GCA AAA TCC GAT GAG ACT GCC GCC AAG TA 618
Gly Arg Glu Ala Ala Lys Ser Asp Glu Thr Ala Ala Lys
I95 200 205
:~
.,~,

WO93~18147 2 1 3 0 0 8 ~ pCTt~3/0002(~ ;
- 21-
(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
130 135 140
Pro Pro Gly Val Asp Pro Thr Gln 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 Gln Ser
165 170 175
Asn Glu Ile Thr Ile Pro Val Thr Phe Glu Ser Arg Ala Gln Leu Gly
180 185 190
~: Gly Arg Glu Ala Ala Lys Ser Asp Glu Thr Ala Ala Lys
195 200 205
:,