Note: Descriptions are shown in the official language in which they were submitted.
~0 91/18921 ~ 3 PCT/I S9I/03~3
Descri~tlon
of Cells overexeressinq the Human Oncogene er~B-2
Technical _ield
The pr~sent invention relates to a growth factor which
interacts with the hu~an oncogene er~B-2, and which inhibits
the growth of cells overexpressing this oncogene.
Bac~qround Art
Carcino~enesis is b~lieved to be a multi-step process
of alteration of genes which are involved in the growth
control of cells. A variety of proto-oncog~nes and
oncogenes have been implicated in t~e actiYation of tumor
cells as regulating factors. For example, oncogenic protein
~inases are b21ieved to induce cellular trans~orma~ion
through either inappropriate or excessive prot~in
phosphcrylation, resulting in the uncontroll~d growth Or
malignant neoplasms. See HistopathQloqY, Wrba, F. et al,
: 15, 71-7~ (l9a9)~
: one group of proto-oncogenes encodes cellular growt~ factors or their receptors. ThQ c-erbB-1 gene encodes the
epidermal growth ~actor or their receptors. T~e c-sis gene
encodes the ~-chain o~ the platelet-derived growth factor.
The c-fm5 qene encodes a ralated or identical molecule for
th~ ~ecap~or o~ the granulocyte-ma~rophag~ colony
stimulatinq fac~or. A fourth member of this group of
proto-onco~enQs~ called neu was iden~i~ied in
ethylni~rosourea-induced rat neuroblasto~as. The human
counterpart or nau, called ~ER-2/neu or c-erbB-2, has been
~, ~, . . . . . .
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i~;. W~ 921 ~ PCT/~S9]/0~3
--2
sequenced and ~apped to th~ chromosomal locus 17q21. See
Cancer Research, Schneider, P.M. et al, 49, 4968-4971
(September 15, 1989).
~he HE~-2/NEU O~ c-erb~-2 oncogene belongs to the
erbB like oncoqene group, and is related to, but distinct
fro~ the epidermal growth factor receptor ~EGFR). The
oncogene has ~ en demonstrated to be implicated in a number
of human adenocarcinomas leading to elev~ted levels of
expression of the pl85 protein productO Eor example, the
oncogene has been found to be amplified in breast, ovarian,
gas~ric an~ even lung adenocarcinomas.
Furthermore, the amplification of the c-erbB-2 oncogene
has been fcund in many cases to be a significant, if not the
most significant, predictor of ~oth overall survival time
and time to relapse in patients sufferin~ ~rom such forms of
cancer.
Carcinoma of the breast and ovary account for
approximately one-third of all cancers occurring in women
~: and together are responsible for approximately on~-fourth of
-~ 20 cancer related deaths in ~emales. Signif~cantly, the
c-srbB-2 oncagene has been found to ~e ampli~ied in 25 to
30% of human p~imary breast cancers. See Sc~nce., Slamon,
D. e~ al, 244, 707-712 (May 12, 1989).
The c-er~B-2 oncogene is known to express a 18SKd
: ~S trans~embran2 glycoprotein (pl85~bB-2). ~he expressed
protein ha~ ~een suggested to be a growth ~actor receptor
~ due tD its homology with EGFR. However, known EGFR ligands,
:~ such as E~F or ~GF~, do not bind to p185~rb8-2. At present,
:;
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WO 91 /18921 ~.., ~ ~; .J ~ ! ~3 ~3 PCl /1_ S9 i /03443
no ligand is known which binds to this protein.
Thus, a need continues to exist .or a li~and for
(pl85er~8-2). Such a ligand might be used to counteract the
e~fects of c-erbB-2 onco~ene over expression in facilitatlng
5 carcinogenesis.
DiscLQsur~ o~ the In~ention
Accordingly, it is an object of the present invention
to provid~-a growth factor which interacts directly with the
erbB-2 oncoqene.
It is also an object of the present in~ention to
provide a m~thod for the isolation and purification of the
abovedescribed growth factor.
It is also an object of the present invention to
provide a method for inhibiti~g t~e growt~ of cells which
overexpress the human oncogene erbB-2.
Accordingly, the above objects and others ar~ provided
~y a 30kDa TGF~-like glycoprotein.
BrLef~ s~ on o~ the Drawinqs
Fig~re 1 illus~ra~es the isolation of the present 30 Xd
growth factor. Portion A illustra~es th2 us~ of low
a~inity heparin chromatography, while portion B illustrates
t~e use of reversed-phase chromatography.
Figure 2 illus~rates the detection o~ phosphorylated
.
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WO 91/18921 ~ PCT/~S91/03~a3
- 4 -
proteins in SK-~r-3 cells.
Figure 3 illustrates the detection of phosphorylated
proteins in MDA-453 cells.
Figure 4 illustrates the phosphorylation of
5 pl85er~8-2 protein in intact CH0/DHFR and CH0/erbB-2 cells.
Figure 5 illustrates a pl85e~bB-2 reoeptor competition
assay in SX~ 3 cells.
Figure 6 illus~rates the inhibition of pl85~rbB-2
crosslinking with 4D5 antibcsdy by gp30.
10 Best Mode f~?~carr~inc7 out the_~nvent~on
The human c-er~3B-2 oncogene encodes a ~8~ Kd
transmem}~ran~ glycoprotein havin~ prot~in lcinase activity.
Thi glycoprotQin, known as pl85~rbB-2, ~hows esctensive
s1:ructural similarity with the pl70 epiders~al growth factor
receptor (EGFR) and is therefore thought to be growth factor
recep~or. Howaver, neither EGF nor TGF~, the normal ligands
~or the EGF~, interact directly with p~85erba-2. In ~act, no
ligand ~or this glycoprotein has b~en described. It would be
ext~emely de3irable to find a ligand ~or ~his 185 Kd
glycoprotein inasmuch as er~B-2 oncogene is amplified in
msny adenocarcinomas and is over oxpressed in nearly 30% of
human ~roast cancer patients. Additionally, it is known
that pl85rb~-2 iS necassary for the ~aintenance o~ the
malignant p~enotype of cells transfor~ed ~y the-oncogene.
., . 25 In accor~ance with the present invention, it has 'Deen
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WO91/18921 ~ pCT/~S91/0~W3
--5
surprisingly discovered that a 30 Kd growth factor which is
secreted fxom ~he estrogen rec~ptor negative cell ~in~
~DA-231 is effective as a ligand for pl85erbB-2
glycoprotein. The 30 Kd glycoprotein of the present
invention also exhibits TGFc-liXe activity. For example,
the present 30 Kd glycoprotein binds to EGFR, is capable of
phosphorylating EGFR as well as inducing NRX colony
for~ation. This is quite surprising inas~uch as the present
30 Kd growth factor is distinct from the normal precursor
for TG~ or mature TGF~ as shown by peptide mapping of the
translated proteins.
Generally, the 30 Kd glycoprotein was
i~munoprecipitated by an anti-TGF~ polyclonal antibody and
exhibited TGF~ e biological activity as assayed by E~F
radioreceptor a5say and NRK and AlN4T cell colony formati~r.
:assays. The 30 Kd growth factor also stimulated
autophosphorylation o~ the EGF receptor more ef~iciently
t~an ~atu~e 6 Kd TFG~.
The 30 Xd glycoprotein was observed, unlike EGF and
TGF~, to bind to heparin-sepharosa, and was purified to
apparent homogeneity by heparin a~inity chromato~raphy and
subsequ~nt reversed pha~e chromatography.
~ unicamycin treatment in vivo or N-~lyconase
deqlycosylation in vitr~ revealed a precursor of 2~ Kd in
: 25 contrast to th~ 18 Kd precursor for ~ature TGF~.
:Furt~er~ore, in vitro translation of total ~RN~ from
MDA-M8-231 cells confirmed these observations. Biochemical
characterization of the 30Xd TGF~-}ike protein was obtained
by V8-prot~ase digestion of the de-glycosylated polypeptides
:~ ` - ' . '
- . -~091/18921 ~ r ~ CT/~S91~0~3
- ~6-
and translated products. P~ptide mappinq of the
Va~diges~ed, immunoprecipita~2d material suggests an ~ln.e
acid sequence distinct from TGF~. Hence, the 30Kd
polypeptide, whil~ related to ~he EGF/TGF~ family, is
encoded by a different gene and is not a post-~ranslation
modification of mature TGF~.
Having obtained t~e present 30 ~d glycoprotein, in
accordanca with another aspect of the present in~ention, the
same i~ used to inhibit the growth of cells which
overexpress ~e c-~rbB-2 oncoyene.
In accordance with the present invention, the pr~sent
30 Kd glycoprotein may be used, by itself, or in conjunction
with other medicinal substances to inhibit the growth of any
cells which overexpress the c~erb~-2 oncogene.
Generally, the present 30 Kd glycoprotein may be used
advantageausly to inhibit the growth of adanocarcinoma
cells, preferably those of breast, ovarian, gastric and lung
tissue which overexpress the erbB-2 oncogene and EGFR.
.,
In order to furt~er exemplify t~e present invention,
reference will now bc made to certain examples which are
: provided solely for purposes of illustration and are not
intended ~o be limitati~e.
~ER~A~S AN~ ME~DS FOR OBT~INI~G TRE 30 Kd GLYCO~ROTEIU
l ~L~ -
Z5 Cell~ from th~ following sources were used: MDA-M3 231
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WO91/18921 ~:,r ~ !~3
and NRX clone 49F fibroblasts were obtalned from the
American type Culture Collection (Rockville, MD). HsS78T
cells, A43l cells, and H8 cells, a TGF~trans~ected MCF-7
br~ast cancer cell line, were available upon request from a
variety of sources. Carcinogen~immortalized normal mammary
epithelial cell subline 184AlN4 and its SV40-transfected
derivative l84AlN4T, were also available on request.
Rat-FeSrV transfected cells w~re also provided upon reques~.
All cell lines were propagated in improved modi~ied Eagle ~ s
10 medium (IMEM, Gibco, Grand Island NY) supplemented with 10%
fetal bovine serum tFss~ Gibco~.
Condi~i~ed Media Pre~aration~ Collection and Concentration
Conditioned media collections were carried using a
well-known procedure. The media were concan~rated lO0-fold
in an Amicon ultr~-filtration c811 (Y~5 membrane) (Amicon,
Danvers, MA) . Once clarif ied and conc~ntrated, the media
were stored at -20C while consecuti~e collections were ~ade
during the following days. The concen~rated media were
dialyzed using Spec~raphore 3 tubing (Spec~ral Medical
Industries,
: Los An~eles, CA) against lO0 volumes of O.l M acetic acid
over a two day period at 40C. The material that
precipitated during dialysis was removed by centrifugation
at 4000 rpm ~or 30 min. at 4C; protease inhibitors were
2~ a~dQd. The clariSied sample was then lyophilized.
~ç~aboLîc ~abel}L~a-~ad I~muno~recipitation
Cells were grown ~o 80% confluen~e in IMEM. C~ll
:~ monolayers were washed three times with PBS and incubated
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.. WO91/18921 PCT/~91/0~W3
--8--
for two hours in serum-free IMEM which lacked methionine and
cysteine and was supple~ented with glu~a~ine (2.9 g/l)
(Biofluids, Roc~ville, MD). This medium was then re~oved
and replaced with serum-free IMEM witAout met~ionine and
cysteine containing 2.5 mCi/ml ~35S~ cyst~ine and methionine
: (~mersham, ~rli~gton H~iqhts, IL, 1175 Ci/mmole). A total
of 2.5 ml of this m~dium was used for a 5 cm dish. The
medium was harvested from the culture after 16 hrs at 37OC
and clarified by centrifugation. Cells were washed once
with PBS, har~ested by scraping, and lysed in 1 ~l of RIPA
buffer (300 mM ~aCl, 100 ~M Tris-HCl, containing 2% Triton
xlOO, 2~ Nadeoxycholat~, Q.2% SDS, 0.4S BSA and 2 mM PMSF).
Following an incu~ation o~ 30 minutes on ice, the lysate was
clarified by centrifugation (30 mi~utes a~ 4000 rp~ and
: 15 used i~mediately or was stored at -70C. ~35S]- labelled
proteins released into the conditioned media by the
dlfferent cell line~ wer~ immunoprQcipitated with lO g
(specific or non specific) antibody partially puri~ied by
45% ammonium sulfat~ p~ecipitation. A~ter solubilization
the immunoprecipitates were analyzed by 15% SDS-PAGE and
subsequent fluorography. ~res~ained molecular weight
markers (Biorad, Richmond, CA) ware run in parallel lanes.
unic~y~LT~çatment
Tunicamycin ~Sigma, St. Louis, MO) was dissol~ed in 50
mM sodiu~ carbo~ate (pH 10.0) and filter-sterilized with a
0.~2 m ~ilter. Confluen~ monolay~rs of MDA-M3-231, MCF-7
and ~s578~ cells were grown in IME~ in the p~esence of 20
g/ml tunicamycin ~unless otherwise specified) for 4 hours
prior to ~etabolic labelling. Metabolic labelling was ~hen
performed as described above with continued tunicamycin
.
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WO 91/18921 ~ PCr/l ~i91 03~3
_g_
treatment.
Elastase Treatment
The samples containing TGF~-like activity were
incubated with 20 g porcine pancreatic elastase (Si~ma)
dissolved in 50 mM ~l~cylglycine, pH 7.9, for 1 hour at
22C. The samples were then subjected to
immunoprecipitation and SDS-PAGE analy~is.
PolYclonal Antibodies; Antiserum against human TGF2 was
obtained by immu~ization of a rabbit on day O with 400 g of
reco~binant TGF~ synthesized in E. coli, provided by
; Genentech Corp. The immunogen was first conjugated to
keyhole limpet hemocyanin ~XLH) and wa~ emulsifled in
complete Freund's adjuvant and was injected intradermally at
multlple sites. Additional injeotions were given as
follows: day 60, 175 g TGF~ and day5 90, 150~ 180, and 210,
100 g TGF~. The booster injections were giYen
subc~taneously at multiple sites in incomplete Freund's
adjuvant. The rabbit serum was assayed for antibody titer
by ELISA at 10 to 14 days following each i~jection. The
antiser~ collected at day 180, designated R399, was used
~or immunoprecipitation and radioimmunoassay.
Monocl~nal Antibodies; A monoclonal antibody against
recombinant TGF~ w~s kind~y provided by Genetech Corp.
ZS Measurement of Anti TGFU An~ibod~ ~R1199)_~evels~by_~3~L~B
~icro-Elisa plates (Dynatech-Immunolon II, Dynatech
- WO91/læ92l ~ `' PCT/~S91/03~3
--~o--
Laboratories, I~c. Chantilly VA.) were coated for 16 hours
at 4C with 500 -.g~l of recombinant TGF~ in 50 mM sodium -
carbonate buffer (pH 9.6). Th~ samples to be assayed
(antibody) were serially diluted 1:1,000-1:64,000 with O.lS
M NaCl, 0.05 M Tris-HCl (pH 7.4), 2 ~M EDT~, 5 mg/ml bovine
serum albumin, 0 . 05% 5~een 20 (TBS-BSA-'rween) and were
incubated in the wells for 2 hours at 37C. 'rhe plates were
washed fi~a ti~es with PBS-Tween and then incubated for 1 hr
at 37ac with horseradish peroxidase-conju~ated goat
anti-rabbit i~munoglobulin in ~8S-8SA-Tween. The plates
were then washed five ~i~es with P~S-Tween and incubated for
4 hrs at 22C with 100 l per WPll 0~ O- 1 mg/ml
o-phenylenediamine, 0O012% H202 in o.l M Phosphate-citrate
buf ~er ( p~ 5.0). ~h~ reaction was stopped by the addition
of so l/well of 2.5 N H2SO4 and th~ absorbance was measl~red
at 492 nm using a U~ 700 Microplate Reader (Dynatech Lab.,
Inc. Chantilly, VA).
Ra~ munoassaY (RIA)
TGF~RIA; The presence of peptides i~unclogically
related to TGF~ was determined using a RIA kit with a
polyclonal anti-rat ~GF~ and rat t2sI]TGFQ (aiotope~ Inc.,
Seattle, WA). This antibody does not cross-react with human
EGF. Aliquots o~ conditioned media were reduced wit~ 40 mm
dithiothreitol and denaturQd by immersion for 1 minute in a
~5 boiling water bath. Assays were done in duplicate according
to the manu~oturer's protocol and each collection of
conditioned media was assayed at least twic~.
- Solid P~ase RI~; 96 well microtiter plates were coated
with anti-TGF~ antibody (R399 or monoclonal antibody) for 2
.
. :
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-~W~91/18921 ~ PCT/~S91/0~3
--11~
hours at 3~C. The wells were ~hen filled with lO0 l of the
column fraction to be assayed for TGF~ activi~y. A standard
curve was constructed using 0.075 to 15 ng unlabelled TGF~.
After the 2 hours incubation 5x104 cpm of [l25I] TFG~ or
2x105 cpm o4 metabolically labelled antigen was added per
well. The plates were incubated further for 16 hours at
4C. The wells were then washed and counted using a ga~ma
counter (Model B5002, Packard Instru~ants Co~, Sterling,
VA). The E~F RIA was per~ormed with an anti-EGF antibody
(Oncogene Science clone 144-8, ~anhasset, NY). A standard
cur~e was constructed using human EGF (HEGF, receptor grade,
Gollaborative Research, Waltham, MA).
T~e puri~ied 30 kDa ~GF~-like protein was subjec~d to
diqestion with N-glyconase. Sa~ples equivalent to iO0 ng
were incubated with 50 1 ot 0.2 M sodium phosphate (pH 8.6~,
1.25% NP40 and 2-6 g N-Glycanase (Genzym2 Corp., Boston, MA)
were subsequently added to each sample and incubated at 37C
for 16 hours. 50 l of 3-fold concentrated loading buffer
was added ~e~ore e~ectrophoretic analysis, per~ormed as
outlined above. The gel was silver stained.
EGF ~adiorece~ptor Assay
A431 membranes wer~ prepared according to t~e method of
Xlmball and Warner~ A431 cells were disrupted under
nitrogen and the nuclei and organelles pelle~ed by low speed
cen~ri~Ugation. The ~embranes were then pelleted by
cent~ifugation at 35,000 rpm for l hour and re~uspended in -
20 mM HEPES b~ffer, p~ 7.4. Membranes (2.5 gt~l~ were
-~.
:
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W091~18921 ~ 3 PCT/~S91/0~3
-12-
plated into ~6 well plates and allowed to dry overnight at
3,~C before use. Standard binding competition studies we~e
performed using tl25I]EGF (ICN, Costa M~sa, California,
specific activity- lOO ci/g, about 50,000 CPM/well). A
standard curve was constructed with 0.075-lOng of unlabelled
hEGF (rece~tor grade, Collaborative Res~arch). Tha
di~ferent fractions to be analyzed were lyophili ed and
reconstituted in PBs (0.5 ~1/500 ml conditioned media).
After incubation of the labelled EGF and 10 1 of the ~a~ples
for 2 hours at 37~ in binding bu~fer (IMEM containing 50 mM
HEPES a~d 0.1% BSA pH 7.7), the w~lls were washed, cut from
the plate and counted. EGF comp~Eing activity, was computed
using a Hewlett Pac~ard RIA Progr~m.
Anchoraae-indePendent Growth Assay
lS Soft a~ar cloning assay~ were carried out using a l ~i
botto~ layer of IMEM contalning 0.6% Bacto agar (Di~co,
Detroit, MI), lOS FBS, and 2 mM glu~amine in 35~m tissue
~ishes (Costar, Ca~ridge, MA). A O.8 ~l top layer of IMEM
containing the test samples, 0.3~% agar, 10% FBS, and 3 X
104 NRX cell~ was added after solidi~ication of the bottom
layer. Each sample was plated in triplicate. All samples
were s~erilized by filtration using a 0.22 m Millex CU
millipora ~ilter before platiny. Platas were incubated in a
humidi~i~d, 5~ C02 atmosphere at 37C and were counted aPter
25 12 days incubat~on with a Bausch and Lo~b S~em Cell Colony
Count~r (Artex Sy~tem~ Corp, Farmingdale, NY).
Ancho~aae-deDen~ent Growth Assay
CQ11~ w2re grown in IMEM containing 5% FCS. Upon
.
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~091/1~9~ PCTt~S91/0~3
-13-
confluence cells were detached using trypsin-versene
(Bi~fl~ids, Rockville MD) and ~a~sed at 1:20 to 1:50
dilutions. Cells were seeded in l2~well plates at
- 4,000-lO,000 cells/well, depending on the cell type
(MDA-MB-231~8,000 cells/well in serum free IMEM). After 24
hours the media was changed and the cells were treated with
: EGF, TGF~ or ~G~a-like protein weEe harvested at l, 2 and 4
days using t~ypsin-versen~. The cells were coun~ed using a
coulter counter.
Heparin Affinity Chromato~raphy
Media conditioned by MDA-M~-231 cells were clarified by
centrifugation for 20 minutes at 2,000 rp~ at 4C. The
supernatant was collected and stored at -70C. After
allowin~ the heparin-sepharose (Pharmacia, Piscataway, NJ)
to expand in PBS, 2 ml of gel was loaded on an Econo column
~Biorad, ~ichmond; CA) and washad with abou~ lOO bead
volumes of PBS. Conditioned media were run through the
beads by gravity (flow rat~ 20 to 50 ~l/hr). The gel was
: then wa~hed with 5 volumes of PBS and ~luted stepwise with
an increasing qradient o~ NaCl in lO mM Tris-~Cl, p~ 7.0
(elution ~uffer). Gradient steps of 0.4 M, l.l M, 2.0 M and
3.0 M NaCl were used in the elution bu~er until the 280nm
; abqorption during each step returned to baselin~ (usually 3
to 5 column bed volumes). The eluate was d~salted on G-25
columns (Pharmaci , Piscataway, NJ) and filter-s~eri~ized
: hePore use in the dif~erent ~ioa~says. Poaled fraction~
containin~ active materials were also de~alted on PDlO
columns tPharmacia, Piscataway, NJ) before running t~rough
HPLC and FP~C.
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WO 91/1~921 ~ `S'r~ 3 PCr/~S91/03443
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Lyophilized conditioned medium was dissolved in 1 M
acetic acid to a final concentration of a}~out 25 mg/3IIl total
protein. Insoluble ~naterial was removed by centrifugation
at 10, 000 rpm for 15 minutes. The sample was then loaded
o;~to a Sephadex G-100 cs:~lumn (XX 16, Pharmac:ia, Piscataway,
NJ~, was e~uilibrated and wa~ subjected to elution with 1 M
acetic acid at 4 C with an upward ~low of 30 ml/hr . 100 ng
of protein was processed from 4 ml of 100-fold concentrated
medium. F~actions containing 3 ml of elu~te were
lyophilized and resuspended in 300 1 PBS for assay and
served as a source for further purification.
Reversed-phase Hiqh Pressure Liquid Chromatoqraphy (~PLC~
~teeD Ace~Q~i~ril~_Ç~adient; Step acetonitrile gradiant
and all ot~er HPLC step~ were carried out at roo~
temperat~re after equilibration o~ the C3-Reversed phase
column with o.oS% TFA (Trifluoroacetic acid) in water
~H~LC-grad~). The sampleq were loaded and fractions eluted
with a linear gradient (0-45% acetonitrile in 0.05~ TFA) at
a ~low rate of 1 ml/min over a 30 minute period, Absorbance
was monitored at 280nm. One ml ~ractions were collected and
lyophilized bQfore analysis for EGF receptor-competing
activity.
~hallQw Ac~toni~ile ~radlen~ pool of active
~ractions ~o~ th~ pr~vious ~P~C step was rechomatographed
over th~ saMe colu~n. Elution was performed with a 0-13~
ace~onitril~ gradient in 0.05S TF~ over a 5 minute period
followed by a linear 18-45~ acetonitrile gradient in 0.05%
, . , . ~ , .
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:
W091/18921 ~3'~ PCT/~S91/0~3
-15-
TFA over a 30 minute period. The flow rate was 1.0 ml/min
and 1 ml fractions were collected. H~man TGF~-likP factor
was eluted at a '0-32% acetonitrile concentration as a
single peak detectable by RRA.
Electrophoretic Elution of Radiolabelled Protein from Gels
After fluorography of an SDS-PAGE, bands of interest
were excised and the protein eluted by electrophoresis into
a di~lysis tubing over 16 hrs at 120 volts. The contents of
the dialysis bag were cooled at 4 C and then precipitated by
lo the addition o~ trichloroac~tic acid to a final
concentration of 20~. The precipitates were pelleted by
centrifugation, washed twice with ethyl ether, and
resuspended in loading buffer.
Di~estion P~ocedure for Pu~lfied E~uted.Proteins
Electroeluted proteins were dissolved at approximately
0.~ mg/ml in loading buffer which contained 0.125 M Tris-HCl
(pH 6.8), 0.5~ SDS, 10% Glycerol and 0.001~ Bromophenol
Blue. The samples were then heated at 100C for 5 minutes.
Proteolytic digestion were carried out at 37C for 30
minUtes by the addition of StaDhylococcus ~ure~s Protease V8
~Sigma, St. Louis, M0) to a final concentration of 25 g/ml
according to methods. P-mercaptoethanol and SDS were
subsequently added to final concentrations of 20~ and 2%,
respectivQly. Proteolysis was stopped by boiling for 2 min.
Th~ sample~ were then injec~ed on a C18 Reversed Phase HPLC
column.
~- Ph~sphoryl~h~n_~ 5he EGF ~eceDtor
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W091/1X921 ~ PCT/~S9l/~3
-16-
Subconfluent A431 c~lls were cultured i~ IMEM or 10-12
hours. The cells ~r~ trcated with 10-30 nM TGFQ, EGF or
TFG~-like growth factor for 30 minutes at 37C. Cells were
lyse~ in 20 mM Trls-HCl (pH 7.4), 150 ~M NaCl, 1% NP40, 1
S EDTA, 2 mM PMSF, 42 mM leupeptin and immunoprecipitated as
described a~ove using monoclonal antibody 225 directed
against the E~;F receptor Oncogene Science, Manhasset, NY)~
The i~munoprecipitates were washed three ti~nes with RIPA
bu~fer and resuspended in 40 1 TNE ~O.OlM Tris-HCl, pH 7.5,
0.15 M NaCl, 1 mM EDTA). Five Ci o~ [~-32P]ATP was added to
the immunoprecipitates and the total ATP concentration was
adjusted to lS mM (final) in a volu~e of 60 1. The reaction
mixture was incubated for 5 minutes on ice before addition
of 20 l of 3x sample bu~fer. The samples were boiled for 5
minutes and analyzed by denaturing 7 . 5~ SDS-PAGE.
RNA Extraction
Total cellular RNA was extracted from ce~ls by
` homogeni2ing in quanidine isothiocyanate followed ~y
; c~ntrifugation over a cesium chloride cushion. Poly (A)+
~RNA was eluted in 10 ~M ~ris after passing total cellular
RNA over an oligo (DT) cellulose column (Pharmacia,
~iscataway, NJ) equilibrated with 10 mM TrisoO.5 M NaCl pH
8Ø A~ter precipitation in ethanol ~66~ vol/vol~ and 0.1 M
ac~tic acid, both total and poly(A)~ selected RNA were
resusp~nded in lO m Tris-l m~ EDTA buffer and separated on
1% agaros~, 6% formaldehyde gels. Electrophoresis was
carried out at 20 volt~ over ~4-16 hours in: 5 mm NaAs 1 mM
EDTA, 20 mM 3-~N-morpholino~ propane sulfonic aeid pH 7.0
~` ~MOPS-Sigma). The gels were stained with ethidium bro~ide
2.Og/ml to allow inspection of th~ quality and quantity o~
:; .
::'
- ~ , -. . :, .
.. - ,
WO91/189~ PCT/~S91/0~3
-17-
RNA (). In vitro translation assays were perfor~ed using
Weat Germ kit accor~lng to tha manufacturer's instructions
(Promega).
Identification of a TGF~-like Poly~eptide in MDA MB 231
Human 3reast Cancer Cells
To determine whether the 30 Kd TGF~-like pro~ein was
recognized by antibodies developed against mature 6 kDa
TFG~, MDA-MB-231 cells were metabolically labelled With
~35S] methlonine and [35S~ cysteine. M~tabolically labelled
condikioned ~edia from MDA-MB-231, TGF~-transfected MCF-7
(H8), and HS578T cells were te~ted by solid phase RIA for
i~unoreactivity with a polyclonal antibody (R399) and a
monoclonal antibody raised against reco~binant 6 XDa TGF~.
Metabolically labelled TGF~-like mat~rial fro~ MDGA-mb-~31
cells reacted only with the polyclonal antibody. In
contrast, the two anti~odies cro~s-reacted with
meta~olically labelled material derived from H8 cells and no
immune-reaction was noted with prei~mune serum (normal
rabbit seru~ NRS) or metabolically labelled conditioned
media ~rom H~5~Bt breast carcinosarco~a cells (Fig. l),
which do not produce TGF~ M~N~. Thus only the monoclonal
an~i~ody is able to distinguish bstween the di~ferent
species of TGF~. Specificity of the assay was demonstrated
~ing a co~petition RIA with unla~elled recombinant TGF~.
Labelled material from MDG-MG-231, H8, ~nd ~at-FeSrY
c~lls was i~munoprecipitated with the an~i-TGF3 polyclonal
an~body. D~ection of.an immunoreactive specie~ of
app~oximately 30 kDa size veri~ied the secretion of a high
molecular weight TGF~-like polypeptide in M~A-`~B-231 cells.
,
W091/1892~ ,3~ PCT/~91/0
-18-
~8 cells, which overexpress classical TGF~, yielded a 6 kDa
product. The expected 18 kDa precursor of the classical 6
kDa TGF~ was precipitated fro~ Rat-FeSrV, which are ~nown to
secrete the "normal" precursor. The intensi~y of the bands
diminished when the immunoprecipitation was performed in the
presencP of excess unlabelled TGF~. No specific bands were
immunoprecipitated by preimmune rabbit serum.
The apparent heterogeneity in size o~ the larger TFG~
spç~ies and the potential ~or N-linked glycosylation of the
TFG~ precursor at ~sn 25 led to the consid~ration of whether
the high molecular weight TGF~-like polypeptide secreted
from MDA-NB-231 cell~ was a glycosylated ~orm of TGF~. When
MDA-MB-231 cells were incubated with tunicamycin, an
i~i~itor o~ co-translatio~al N-linked glyco~ylation, and
the media was immunoprecipitated with the anti-TGFc
polyclonal antibody a species of 22 kDa substituted tha~
: , previously observed of 30 kDa. Additional cleavage of the
22 kDa polypeptide with elastase yi~lded an apparent ll kDa
product different ~rom the matu~a 6 kDa TGFa that was
ob~erved in Rat-FeSrV labelled ~edia. The ll kDa product
h~d a higher immunoreactivity with the R399 antibody than
the 30 kDa and the 22 kDa polypeptides. Shor~er exposure of
th~ gel showed clearly a p~ecipitated ~and ~ear the ll kDa
molecular weight. Tunicamycin treatment did not
significantly affect the l2vels of se~reted TGF~ ac~ivity as
determined by both RIA ~nd EGF receptor binding assays.
When the purified 30 ~Da polypeptide was treated with
Nglycona~e a 22 kDa product was detected ~y silver staining.
WO91/18921 ~ `3 ~ PCT/~S91/0~3
The absence of cleavage of the purified 30 kDa polypeptide
2f~er O-glyconase treatment suggests that no O-glycosylatle~
occurs in this system.
Purification of the TGF~-like PolypeDtide
TG~-like, material was isolated from s~rum free
conditi~ned ~dia of MDA MB 231 cells. L~vels of T~Fu-like
polypeptide were quantified by three inclependent assays:
capacity to indu~a anchorage-indapendent growth of NRK
fibroblasts in soft agar, ability to compete with (l2sI]EGF
for EGF receptor binding on A431 human carcino~a cell
membranes and cross-reactivity with polyclonal antibodies
raised against mature TGF~. EGF receptor binding activity
and TGF~ i~unoreactivity were detected ~siny a R~A kit
provided by Biotops. To determine th~ approximate molecular
weight o~ the MDA-MB-231 deri~ed TGFQ-l~X~ polypeptide, ~ ~i
o~ 100-~old concentrated, dialyzed conditioned mediu~ was
chromatographed by g~l filtration usin~ Sephadex G-lO0.
Elution was performed with l.0 M acetic acid and ~ractions
were characterized ~or protein content. TGF~-like
activi~i~s were elut~d fram the column in a sin~le broad
peak. Maximal activity was observed at an apparent
molecular weight of 30 kDa and was separat~d from the bulk
of contaminating proteins prasant in the bad volume. All
t~e ~ractlons demonstrating TGF~ immunoreactivity also
:~ 25 contained EGF receptor binding activity. The ralative
a~ounts o~ rQceptor binding a~tivity and immunoreactivity
present in these fractions, however appe~red to di~er.
Further analysis of the TGF~-~ike polypeptide from
MD~-MB-231 cells was carried out using heparin-sepharose
affini~y chromatography. Heparin-sepharos~ afPinity
,, . -
. .
.~. . .
'
: .
~091/18921 ~ PCT/~S91/0~3
-20-
chromatography was perf ormed on uAconcentrated conditioned
medla from MDA-MB-231 cells. In all experiments, less t~an
20~ of the TGF~ activity loaded onto the column was
recovered in the unabsorbed fractions. A sharp pea~ of EGF
receptor binding activity was eluted by heparin-sephar~s~
chromatography at a concentration of 0.4-0.5 ~ NaCl. This
activity represented one major 30 k~a molecular weight
protein, which retained 70%-80~ of the load activity.
The TGF~-like polypeptidP was further purif~ed by
reversed phase chromatography ~HPLC) in two steps. A pool
of fractions containing EGF receptor-competing activity from
heparin-sepharose chromatography Was reconstituted in 0.05%
TFA in water and then chromatographed on a ~ondapak C3
column. A steep acetonitrile gradient (0-100%) was used in
thiS step. TGFQ-like polypeptide elutes as a sharp peak
in 30~ acetonitrile and is separated from the bulk ~f the
contaminating proteins. The capacity of the individual
fractions to compet~ for EGF receptor binding and to
stimulate the growth ~f NRK cells in ~oft a~ar was
determined. A pool of the active fractions ~indicated with
an horizontal bar) was rechromatographed on the same column.
~rac~ions w~re eluted with a 0-20% acetonitrile gradient in
O . 05% TFA for 5 minutes followed by a linear 20-40~
acetonitrile gradient. The TGF~-like polypeptid~ activity
Was eluted at 25-30% acetonitrile and sffectively separa~ed
~rom oth~r contaminant proteins.
In order to achieve a complete separa~ion of TGF~-like
polypeptide from those i~pUritie detected by sLlver
staining (data no~ shown) we used size exclusion
chromatography under acidic conditions. The ~ctive
:, ~ - , . . .
: ' . - '
. W09~ll892l PCT/~Sgl/0~3
-21-
fractions for EGF receptor-competing activity were pooled
and analyzed hy S~-PAGE. One single polypeptlde band was
observed after silver staining.
A summary of the steps leading to the isolation and
purifica~ion of T~F~olike polypeptide is presented in Table
1. A 27% recovery of activity and approximate 5400 fold
purirication was achieved.
.
,~
'
:
.. ~ ' '
': ,
wo 91/18921 PCr/~S91/i)3~3
--22--
_ ~ _~
O ~ ~ ~ ~ ~
_~ C ~ _~ C
c~ o e ~ _~
_ ~ C~
, _ " :
. _ __, 1. C
~ ~ IJ ~ E ~ :~ c 3
e u c o-0 ~ :r e :
e c~ ~ ul 3 ~ c
e ~ E . O c G
u ~ J C O ~ O, ~ ~ c~ ~ 0
0 ~ u ~
~. ~ c~ ~e~ ~c, c ~ O
.~ O O U C~O ~ ,~, _ o O U -- -- ~ U
U~ O--I U _ ~ L C
O 3 ~ E ~
E~ ~ o C C ~ O ~ ~
' U 3 C _ ~ ~ ~ C U C C
L 1: S' C U~ _ O ~ U 3 ~
O '3 --~ ~- I ~ ~ 1~.C~l O .CI --I
_ ~ _
L~ ~ ~
.. ~ O ~ 0 Ul L~
~.1 ~ " O X O
~U ~ _ I ~ E~ 0 0 v~
Q ~ ~ 5 ~ ~ I I I t~ y
~q C~ U~
,
~2r~
WO91/18921 PCT/~'S91/0~3
-23-
Bio1Oaical Characterization of the TGF~liXe ~aterial
The EGF receptor binding aotivity of the 30 ~Da TGF~-
like prot~in was compared with that of EGF in a
radioreceptor assay. Both growth factors competed With
[l25I]EGF or receptor sites on A431 membranes. The
specific EGF-competing activity of the purified TGF~olike
polypeptida was found to ~e 1-1.5 x lO6 units/~g; l.lng o~
TGF~-like polypeptide was r~quired to inhibit EGF binding by
50%. TGF~-like polypeptide was as e~fective as EGF in EGF
receptor bindin~. Furthermore, the purified 30 kDa
TGF~-like polypeptide stimulated the growth of serum NRX
fibrobl~sts ~nd induced colsny formation of t~ese cells in
so~ agar. The bioactivity of the purified T~F~-like
polypeptide was also tested by anchorage-depend~nt growth
assays of the carcinogen- immortalized huma~ ~ammary
epithelial cell~ 184AlN4 and anchorage-independent growth
assays of 184AlN4-derived cells partially transfor~d by
SV~O T antig~n, 184AlN4T. Dose respo~se curve~ of TGF~-like
polypeptide on these cells were similar to those observed
with EGF and TGFU. The biological activity o~ the purified
30 kDa TGF~-like factor wa3 ~urther assessed ~y examinin~
its ability to induce autophosphorylation o~ the EGF
recep~or. A431 cells, which oversxpress th~ EGF receptor,
w~re incubatQd with Yarious concentrations ~f EGF, TCFU or
25 TGF~-like growth factor~ Each of thQ three peptides
s$milarly st$mulated phosphorylat$on of th~ EGF receptor.
Pep~ide Mappin~
In order to determine the degree of homology between
the no~el 30 kDa TGF~-like growth factor and ~aturs TGF~,
., ~ .
' ' ' .
WO 91/18921 PCT/~S91/03~3
--24--
peptide lsLapping was performed using th~ method of Clevand.
Immur.opr~c~ L _tation of metabolically labelled conditi~ned
media from MDA MB-231, H8, and ~at-FeSrV cells was carried
out with the R3g9 anti-TCF~ polyclonal antibody.
Precipitate~ were analyzed on SDS-PAGE and the specific
bands were electroeluted (~0 Kd from ~DA-MB-231 cells, 6 kDa
from H8 cells, and 18 kDa from l:he Rat-F~SrV cells)~ These
proteins were subjected to enzynatic treatment with
N-glyoona~a and elastac~. The precipitated bands sizes are
summarized in Table 2. The products were then su~jected to
a peptide~~igestion using 25 g/ml V8-protease. After
complete digestion, the samples were analyzed by Cl8-
reversed phase chromatography. Three major peptide peaXs
eluted at different acetonitrile concentrations by reversed
phase chromatography. However, the concentrations at which
thos2 peptides isolated from MDA-MB-231 cells eluted (16%,
: 18.7~, and 21.~%) were di~ferent fro~ the peptides isolated
from H8 and FeSrV cells (24~, 29~, and 32.6%). T~e peptide
elution pattern of the TGF~ (6 Kd) d~rived from H8 cells and
Rat-FeSrV cells was essential identical. Th~ same results
were obtained with 40 g of V8 protease, indicating that
concentration oi~ the enzyme wa~ not responsible for t~se
differential peptide cleava~e. Moreover, in ~itro
translation o~ mRNA d2ri~ed from MDA-MB-231 ~ells and X8
c~lls wa~ dona, the resulting polypeptide had the same
pep~ mappi~g profile than th~ purified 30 kDa fac~or
arter tr~tment with N-glyconase and elastase. These
results p~ovide evidence that a precursor different than the
l'normal" TGF~ precursor is translated from the mRNA of
MDA-MB-231 cells. Moreover, the above results indicate that
: th~ MDA-MB-231 derived TGF~-like polypeptide shares Yery
raw, i~ any, common peptide sequences ~ith mature ~GF~.
,
- .
9~ ~;J"~ P~T/~S91/0~3
-25-
In order ~o characterize the cellular effects of the
present 30 Xd glycoprotein, various experimen~s were
conducted. The following Examples are provided solely for
the purpose of illustra~ing the present inventi~n and are
not intended to be limitative.
Exam~le 1
In order to characterize t~e cellular ef~ects o~ the
present 30 Xd glycoprote~n ligand, its induc~ion of tyrosine
phosphorylation was assessed in t~e hu~an breast cancer
lines MDA-468 and SK-Br~3. Notably, MDA-46 8 cells ~ave
amplification ~nd over expression of th~ EGFR gsne and do
not expresi erbB-2 xeceptor-like protein. SK-Br-3 cells have
a~plification and ove~ expression of the erhB-2 gene as well
as ~elatively elevated levels o~ EGFR. T~e 30 Xd liqand,
15 TGF~ and EGF were found to induce tyrosine phosphorylation
in both cell lines an EGFR blocking antibody abolished the
phosphorylation induced by the three growth ~actors in
~DA-468 cells. This antibody did not, however, completely
block the phoiphorylation induced by the present 30 Kd
ligand and SK-Br-3 cells. ~owever, it did block the
phosphorylation induced by ~F~.
From th~ abov~ result, it appears t~at tyrosine
phosphorylation of a protein is di~ferent from EGFR occurs
in sX-ar-3 cells treated with 30 Xd fac~or. No
phosphorylation was obserYed in untreated SK-Br-3 cell~, and
cells ~reated with the anti-EGF~ antibody alone.
: ~ .
.~ Exam~
.
:: :
S~?D ~ 3 ~
W091/189~1 PCT/~S91/0~3
-26-
In human mammary carcinoma cell line MDA-453, which
over expresses er~B-2, but which has undetecta~le levels of
the EGF receptor protein or mRNA, the 30 Kd ligand -~as
observed to induce a sisnificant increase in ~yrosine
S phosphorylation in a dose dependent manner at concentrations
ranging fro~ 1.25 mg/ml to SO mg/~l. By contrast, EGF and
TG~U were unable to induce tyrosine phosphorylation in the
185 kDa range, at a concentration of 25 mg/ml. No
phosphorylation was observed in untrea~ed cells. Hence,
o from the above, a direct interac~ion between the 30 Rd
ligand and the 185 K~ glycopro~ein appears to occur.
Exam~le 3
In order to determine t~e effects of the present 30 Kd
ligand on the proliferation in colony ~ormation of breast
carcinoma cell lines, the followinq experiment wa~
conducted.
CQ11S were treated with the present 30 Kd growth
~actor, EGF, TGF~ and anti-QrbB-2 antibody in order to
inhibit t~a proliferation o~ S~-Br-3 c~lls.
It was observed that the ~nti-erbB-2 antibody inhibited
the proliferation of the SK-Br-3 and MDA~453 calls by 60-70%
bu~ did not inhibit the proliferation of MDA-468 cells.
Surprisingly, by exposing SK-Br-3, MDA-453 and ~DA-468 cells
to the 30 Kd ligand protein of the present invention, a
60 70% inhibition of cell gro~th was observed ~or all cell
lines.
Inhibition o~ growth by the 30 Kd ligand pro~ein was
' ~ ~
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J r \ (_~ ~
WO91/18921 PCT/~S91/0~3
-27-
reversed by an EGFR blocking antibody in ~DA-468 cells, but
not in SX-~r-3 or ~A-4~3 cells. This is an indica~ion that
the effects of Ihe 30 Xd protein on SK-Br~3 and MDA-453
cells are not mediated through EGFR.
By contrast, the present 30 ~d glycoprotein exhibited
no effect on MCF-7 cells, which have nor~al levels of EGFR
and ~rbB-2. Additionally, EGF and TGF~ inhibi~ed the
anchorag dependant growth of MDA-468 cells and SX-Br-3
cells, but not that of MDA-453 or MCF 7 oells. EGF-induced
anchorage dependant growth inhibition of SK~Br-3 and MDA-468
cells was reversed by an anti-EGFR blocking anti~ody. In
the presence of the 30 Kd glycoprotein, the growth
inhibition of SR-Br-3, ~DA-453 and MDA-468 cell~ was neaxly
complete.
The growth inh~bitory property of the pres~nt 3 0 Kd
i ligand appears to be similar to that described ~or EGF on
; size whichever express EGFR such as A431 cells and MDA-46
. cells.
; Exampl9_4
Further, t~e growth of CHO/erb~-2 trans~ected cells was
inhibi~ed by 70-80~ after treatment with th~ present 30 Kd
. glycopro~Qin. No e~ct was obser~ed on thQ CH0/~HFR
control trans~ectants and the parenteral CHO line. TGF~ at
the qame molar concentration did not exhibit any effect on
th~ proliferat$on of any of the three lines. Tyrosin~
; phosphorylat~on and cel.l proliferation of thQ CHOJDHFR cells
and the parenteral C~0 cell line is not ~ ected after
tr~at~ente by t~e present 30 Xd ligand or TGF~.
-
~ ,
. . .
... .
,
s ~ 3~3
WO91/18~2~ PCT/~S91/0~w3
-2~-
~xam~le 5: Cell Growth Inhibitlon by gp30
.Sk-Br-3, MDA 453, MDA 468 and MCF-7 cells were plated
in 24 well plates in I~EM (Bio~luids) supplemented with 5%
FCS. Parental CHO cells, and CHO cells trans~ected With the
5 DHFR gene or the erbB-2 gene were plated in ~4 well plates
(Costar) in ~-~EM (Biofluids) supplemented by 10% dialyzed
FCS, 0.75 mg/ml G418 and Methotrexate (M~X) 50 n~ for the
CH0 parental and CH0-DHFR CELLS for 259 nM ~or the
C~0-erbB-2. After 24 hours media was removed and replaced
with control serum free media (SFM) containing fibronectin,
transferrin, hepes, glutamine, trace elements, and BSA, or
SFM with the addition of 2.0 ng/ml gp30, lO ng/ml
recombinant TGF~ (Genetech), or with 2.5 MgJ~l 4DS spacific
anti-pl85~rb8 2 monoclonal antibody. Cells were grown in
lS 90% confluence of control and counted. Each Group was
assayed in triplicate. Resul~s are shown as growth relative
to con'crol. The experiments were performed thre~ times and
the results were reproducible. The results are shown ~elow
in Table 2.
,~ ~
. _ _ _ _ _
_ _ SK-Br-3 _MDA153 CHO/crbB 2 C~O~HFR MDA~ M~
t ~30 3l 2~ 20 ~ la l~
4DS~nd~ 32 34 22 98 l~ ~
MF~ 73 9l 89 9S 7~ IOS
Con~l~ ~y __ _87 9l 87 ~ _9~ _ ~
In order to further d~scribe the various aspects of t~e
present invention, reference will now be made to the ~igures
of th~ present specification.
~,; F~qure 1: lsolatlon of qp30
. :
, . .
- . . , .~.......... . .
~ . ~ ~ , . . .
. ,
W091/18921 ~r~ PCT/~'S91/0~3
-29-
Part A illustrates the use of low affinity heparin
chroma~ography. In particular, affinity chromatography of
conditioned media ~rom MDA~231 cells was performed on a
heparin sepharose column. Fractions were analy~ed for EGF
receptor binding ac ivity of A431 cell membranes. Ali~uots
from the input media and ~rom the fractions containing
activity were analyzed by a 15% SDS~PAGE, followed by silver
staining. Lane 1 shows unconc~ntrated conditioned media.
Lane 2 r~p~sents the actiYe fraction.
Part B illustrates the use of reversed-phase
chromatography. Notably, the EGF/TFGA active fractions
obtained after heparin-sepharose chromatography were
chromatographed twice on a ~Bondapak C3 column in O.O~S TFA.
Samples were eluted with a steep gradient of ac~tonitrile.
Fractions ~hat showed EGF r~ceptor binding activity were
~hen rechromatographed and e}uted wi~b a shallow
acetonitrile gradient. EGF comp~eting activity waC
constantly eluted at a 25 30T acetonitrile gradient. The
resulting fraction was analyzed on a 15% SDS-PAGE followed
by silver staining. Sizes are shown in kilodaltons.
Fiqure 2: ~etection of Phos~ho~Ylated Proteins Ln-~K-ar-3
Cells
SK-~3r-3 cells were grown in 90~ con~luence in 24-2311
platss (Costar). Cells were treated at 30C with I~E~
~lanes 1 and 2), I~EM containing 25 nb/ml recombinant TGF~
(Genetech, CA) (lanes 3 and 4), and I~EM contain~ng 5 ng/ml
of gp30 (laneB S and 6~, all o~ these in tAe pres nce (lanes
1, 4, 5) and khe absenc~ ~lanes 2, 3, 6) Of an anti~EGF
r~ceptor blocking an~ibody (G~netech, CA). After 20 ~inutes
the ~edi~ was removed and cell5 were lysed in 100 ~1 of
:
, .
~r ~ $~
~091~18921 ~ pcT/~ssl/o~3
~30
sample buffer containing 1% SDS, O.l~ ~mercaptoethanol,
0015 M Tr~s~ h 6.8), lO~ ~lycerol, 0.02 ~
bromophenol blue, lMm EDTA, 2 Mm Pmsf and 42 Mm leupeptin.
After 5 minutes at 95C, 50 ~ of protein were loaded in a
7.5% SDS-PAGE. Proteins were then transferred to
nitr~cellulose membrane for immunoblotting (Hoefer
Scientific Instruments, California) by electrophoresis in a
~odified method of Towbin et al, using a electrophoretic
transfer unit (Hoefer, TE 22). Electrophoretic trans~er was
carried out at room tempsrature for one hour at 125 Ma in a
buffer containing 25 Mm glycine, 129 Mm Tris (Ph 8.3) and
20% methanol. Followlng transfer, the filter was blocked
with 5~ BSA in Tris-Buffered Saline containing 0.5% Tween
20. An antiphosphotryosine antibody (Amersham) was reactPd
with ~he immobilized proteins in 5% BSA ~Sigma RIA Grade).
Immunecomplexes were detected by a ~oat anti-mou~e antibody
conjugated to al~aline phosphata~e. Blots were then
incubated with a color dev~lopment substrate solution
containing N~T and BCIP (Promega).
Figure 3: Detection ~f PhQs~horYlated Proteins i n MDA-453
Cells
~`
MDA-45e cell~ were grown to 90~ confluence in 24-2311
plates (Costar) and treated at 37C with IME~ ~lane l), IMEM
containing 25 ng/ml of recombinant TGF~ (Genetech, CA) (lane
25 lO)~ or I~EM containing 1.25-40 ng/ml of gp30 ~lanes 2-9).
After 20 ~inute~ media was remo~ed` and c~115 were lysed in
100 ~1 of sample buffer as described in Figure 2. After 5
minutes at s50C, 50 ~g of protein was loaded in a 7.5~
SDS~AGE. Proteins were then transferred to nitrocellulose
~embrane for immunoblotting with an antiphosphotryosine
: .
. .
, ~ ~, i, . .. .,.-
:.,-
.
WO 91/1~92I PCT/~S91/03~3
antlbody (Amersham) as described in Figure 2.
Fiqure 40 P~o sphorYlation o~ P185 Protein in IntactCHo/DHFR, and C~O/erb~-2 Cells
Cells were grown to 90% confluence in 24-2311 plates
(Costar) in A~EM (Biofluids~ supplemented with lOS dialyzed
FCS, 0.75 mg/ml G418, and Methotrexate (~rX) at
concentra~ions o~ 50nM (CHO parental and C~O-DHFR) OR 250 Nm
(CHO-er~B-2~. CXO-DHFR (Fig. 4A) and CHO-erbB-2 ~Fig. 4B)
cells, were treated at 37C with control media supplemented
with 20 M~ Hepes (Ph 7.4) ~A and B lanes 1 and 4), With lO
ng/ml of recombinant TGFa (Genetech, CA) (A and B lanes 2
and 5), and control media supplemented ~.0 ng/ml of gp30 ~A
and B lanes 3 and 6). After 20 minutes, media was removed
and cells were lysed in 100 gl of sampla buffer (as
d~cr~bed in Figure 2). An anti-phosphotyrosine antioody (A
and a lane~ 1 to 3) [Amersham) and an anti-erbB-2 antibody
(A and B lanes 4 to 6) (NEN), were reacted with tha
` im~obil~zed proteins in 5~ BSA (Sigma RIA Grade~. -
Im~unocomplexes were detected as ~e~cribed for Fiqure 2.
Cell~
SK-Br-3 calls were plated in 24 wç~l plates in IMEM
(E~iofluid~) supplemented with 5% FC5. Ai~er a wash with
binding bu~er tD~/Fl2 pH 7 . 4, containing 1 mg/ml ssA, lO
- 25 M~ hepes and 20 ~ glutamine) cells were incubat~d for ~0
minutes at 37C with binding buffer. The EGF~R were
satu~a~ed with 30 nM EGF for 2 hours at 4 ~c. plas binding
*l studi~s were t~en perfo~ed for 3 hours at 40c w1th 1 nM
.
.
~'091/18~21 ~ ~ PCT/~S91/~W3
-32-
iodinated 4D5 in the presence of various c~ncentrations of
unlabeled gp30 ~or 4D5. After the incubation, cells w~_~
~ashed 3 times with binding buffer and then solubilized with
1% SDS. No specific binding was det~rmined with excess (100
: S nM) of unlabeled antibody. Each ~roup was assayed in
triplicate. The experiments were p~rformed f ive times and
th~ resul s were reproducible.
Fiqure 6: Inhibition_o~ Pl85-cross-linkinq with 4D5
The binding assays were performed as described in
Figure 5. Binding was performed with iodinated 4D5 ~1 n~)
alone (lane 1), in the presence o~ 100 nFM unlabeled 4D5
(lane 2) and in t~e presence of 2nM gp30 (lane 3). 100 nM
EG.F were used as a control (lane 4). Cell~ were then
i~ treated with a cros5-linking agent EGS for 45 minutes at
4C, then quenched ~y adding 0.1 ml of 20 Mm NH4Cl. The
solubilized c811s wer~ immunoprecipitated with a polyclonal
antibody to the C-ter~inal domain o~ erbB 22 ~Genetech, CA).
The precipitate~ were analyzed on a 5% SDS-PAGE.
The 30 Xd gly~oprotein cf th~ present inven~ion may
thus be used advantageously to in~ibit the growth o~ ~arious
: types of ad~noca~cinoma cells which overexpres~ the erb3-2
oncogen~ and EGFR. Preferably, the present 30 Kd
glycoprotein i~ used in inhibit the growth of ~denocarcinoma
call~ of breast, ovarian, gastric and lun~ tissue which
. overexpress ~he erbB-2 oncogene and EGFR.
In ucing the present 30 Xd ~lycoprotein to inhibit the
growth o~ the above malignant cells in a ma~mal, pre~erably
:
.
.- -
,
- - -
:
r ~s~
~091/18921 PCT/~91~0~3
-33-
a human, relatlvely low concentrations of th~ glycoprotein
ma-~ ~G ~s~d. For example, an aqueous solutio~ having a
concentration of about 1-50 ng/ml may be convenie~tly
ad~inistered to a patie~t such that a total of from about -
l-lO,000 ng of glycoprotein are administered per day. It is
preferred, howevar, if about l-l,000 ng are administered per
day.
~ he present invention thus relates to the use of the
present 30 Xd TGF~-like glycoprotein in direct interactions
With EGFR and pl85erb8-2o Hence, in another aspect, the
present invention provides conjugates of the ~0 Kd
glycoprotein ligand with either EGFR or pl85~8-2. In still
another aspect, the present invention provides diagnostic
and therapeutic methods using t~ese conjugates. Further,
lS the present invent~on provides a diagnostic test ~it using
the present con~ugates.
In another aspect, the present invention relates to the
preparation of monoclonal antibodies of gp3O, and the use of
these monoclonal antibodies to detect the presence of gp30
in patient sera.
As gp30 is known to be produced by MDA-MB-231 breast
cancer cells, and is also lik~ly to be produced by other
adenocarcino~a cancer cells, the presen~ invention also
provides a method for detecting gp30 in patient sera.
Generally, in accordance with the present invention,
t~e mer~ d~tectio~ of eit~er pl8s or gp30 is a ~asis for
concluding that the detected pro~ein is being overexpressed.
This conclusion, in turn, leads to a poor patient prognosis
necessitating the use of more aggressive treatment of the
tumor.
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W091/t~921 P~T/~S91/0~W3
. -34-
In more detail, the present invention specifically
first contemplateS the use of con~sate, ~' the 30 Kd
glycoprotein and EGFR, and of the 30 Kd glycoprotein and
pl85e~b8-2 in detecting the presence of adenocarcinoma cells
which overexpress either EGFR or erbB-2 oncogene.
Preferably, the adenocarcinoma cells dete ted are of breast,
ovarian, gastric and lung tissue.
Generally, the pr~sent conjugates may be used
advantageously in a biochemical detPction method in which
the 30 Kd glycoprotein ligand is bound to a surface and put
into contact with aqueous solution containing a tumor
portion containing cells whi~h are suspected o~
overexpressing either E~FR or erbB-2 oncogene. This is
conveniently done as either EGFR or pl85 may be found on the
cell surfaces. If such calls are present, eit~er t~e EGFR
or pl85erb~-2 will become bound to the ligand. Iherea~ter,
the aqueous solution is separated ~ro~ t~e bound antiligand
material, a~d the antiligand material may ~e conveniently
detected with a known detection mean5 associatad therewith.
For example, an ampli~ied enzymelinked immunoa~say may be
used. ~he sur~ace to which the li~and i8 bound is treated
with one or more agents ~or limiting the amoun~ of
non-speci~ic binding. Such agents reduce the "noise"
arising due to non-specific binding when int~rpreting the
as ay.
In acco~dance with the abov~ procedure, a diagnostic
t2st kit may be constructed in a variety of ways.
.
For example, a test kit may be constructed to contain a
: vessel con~aining a test liquid having a surface to which
gp30 llgand is bound. This is preferably a multi-well test
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3~-
plate. Also contained is at least one other vessel
containing reagent sclut~ he agQnt for limitlng
non-specific binding may be incorporatPd wil:hin a solution
of the kit or may have been used to treat the surface of the
first vessel before it is supplied.
Then, a portion of the tumor or a tumor sample may be
worked up into an aqueous solution and put into contact wit~
: the bound gp30.
In order to conveniently detect the overexpression of
EGFR or erbB-2 oncogene in a human patient it is
advantageous to use the well-known sandwich assay tech~ique.
For example, one assay method and test kit which may be
used in accordance with the present invention are described
in U.S. Patent 4,668,639 which is incorporated herein in the
entirety.
Henc~, th~ presant invention contemplates and is
specificalay ~i~ected to any diagnos~ic o~ therapeutic
.. method ~ar the-~detect~on of adenocarcinoma cells whic~
`!
overexpre~S EGFR or erbB-2 oncogene, whic~ method uses the
formation o~ a conjugate between the 30 Xd glycoprotein of
the present inYention and either EGFR or pl85~r~-2.
AQ noted above, the present invention also provides an
assay and a test kit for the detection of gp30 using
monoclonal antibodies to gp30.
.~
It is noted that although either polyclonal or
monoclonal antibodies can be used for this purpose, it is
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W091/18921 ~ PCT/~S91/03
-36-
preferred that monoclonal antibodies be used.
In such an assay, the monoclonal antibodies to gp~0 are
preferably bound to the microtiter or ~ulti-well plate and
exposed to patient sera suspected of containing gp30.
Upon de~ecting the presence of gp30 by a conventional
de~ecting means, a conclusion o~ poor prognosis would be
mad~ necessitating the use o~ more aggressive treatment for
the tumor. IDportantly, however, the presence oS the 30 Rd
glycoprotein (gp30) in patient sera can ~e detected
utilizing either monoclonal or polyclonal antibodies in
virtually any type of immunoassay. This includes both
single-site or twosite or "sandwich" assays of the
non-competitive types, as well as in traditional competiti~e
binding assays.
With the above assay, a test kit is also provided.
Generally, the kit contains a first container containing an
antibody having specificity for gp 30 and a seoond ~ontainer
containinq a s~cond antibody having specificity for gp30 and
: being labelled with a reporter molecule capabl~ giving a
detec~ahle si~nal. The first antibody i9 immobilized on a
solid surface.
; The above assay and test kit for the detection of gp30
may b~, re3pectively, conducted and constructed by analogy
in accordance with U.S. Patent 4,92l,790, which is
in~orporated herein in the entirety.
The ~0 Kd glycoprotein of the present invention is
wellcharacterized by:
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.W09~/18921 P~Ti~S9t/0~3
-37-
1) being a heparin-bInding protein;
2 ) being capable of binding to EGF reoeptor;
3) exhibiting cross-reactivity to antibodies to TGF~;
4~ being capable of cleavage by elastase; and
5) being capable of stimulating transforming activity in
normal rat kidney (NRK) cells.
T~ polyclonal or monoclonal antibo~ies produced
against gp30 may be produced in accordance with well-known
techniques. For example, see current P~otocols in ~olecular
10 3ioloa~, edited by F.M. Ausubel et al (Wiley 1987), in
particular Chapter 11 on Immunology. Also, the i~munoassays
used in the assays and diagnostic test.kit~ of the pr-sent
invention are well ~nown to the artisan as evidenced by the
above treatise, and by thQ methods disclosed in U.S. Patent
15 4,g21790 which patent has been specifically incorpurated
herein in the entirety.
., .
As descri~ed above, the diagnostic aspects of the
pressnt invention relate to the use of methods and test Xits
for the detection of either pl85, EGFR or gp30. T~e
. 20 de~ection of any one of these proteins may form the basis
:~ ~or a poor prognosis necessitating the use of aggressive
treatment of one or more adenocarcinomas.
~e present invention also relates to gp30, itself, and
.~ conjugates of gp30-EGF~ and/or gp30-pl85e~b3~2.
.~ .
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~ WO91/1~92~ s~ ~ ~ PCT/~591/0~3
-38-
The therapeutic aspects of the present inv~ntion relate
~O ~.le ~ls2 of gp30 to inhibit the growth of adenocarcinom~
cells which overexpress EGFR and/or erbB-2 oncogene.
Generally, the amount of gp30 to be administered as a
th~rapeutic agent will bê determinêd on a case by case basis
by the attending physician. As a guideline, the extent of
the adenocarcinoma, body weight and age o~ th~ patient are
considered while up to about 10,000 ng per day may be used,
generally not ~ore than 1,000 ng per day of gp30 is
administered~ It is preferred, however, if ~rom about 5-500
ng per day are used. Notably, however, the above amounts
may vary on a case-by-case basis.
While the present 30 Xd glyccoprotein may be
administered by itself, as a therapeutic agent, it ~ay be
administered in comb~nation wit~ one or more other
therapeuti~ aqents. For example, the 30 Kd qlycoprotein ~ay
administered with any chemotherapeutic substance, growth
inhibitor or immune~timulating substance. The present
invention specifioally contemplates such combinations.
.
: 20 Havinq now descri~ed the invention, it will now be
apparent to one of ordinary skill in the art that many
ch~nge and mod~fications can be made to the above
em~odiments without departing ~rom t~e scope and spirit of
the present invention.
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