Note: Descriptions are shown in the official language in which they were submitted.
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MUTAT~ GROWT~ FACTO~ R~CEPTOR AS A DRUC AN~ ITS US~ ~OR TH~
TREATMEN~ OF CANCE~
The present invention relates to mutated receptor tyro~ine kina~es tha .:
dcfectlve in their sigrlallin~ actlvity and ha~e therapeutic properties,
drues containing at lea~t one mutated recep~or and the use of the ~utated
receptor(s) ~or the ~eat~cnt of diseases a~ociated with an ~ncontroll~d
hyperf~nction of r~ce~or tyro~ine kinases, in F2Lrtic~lar canc~r.
~ell grow~h is a carefully controlled pr~ce~s de~ending
on the 5pcci~1 needs of an organis~. In a young organ-
ism, the cell division rate oll~weighs t}~ dying rate of ~:~
cells, which leads to an increaso in size of the
organi~. In a ~ully grown organlsm, ~he new growth of
cells and cell death are so balanced t~at a
" 3teady ~tate " is fo~med. Howe~er, in infrequent
oaso~, the control of cell di~lsion c~llapes, a~ ~he
cells ~egin tc grow and to di~ide ~hemsel~es, although -~.
~here i~ no special need ~or a higher number of cells
o thi~ typ~ in tne organJsm. This uncontrolled Ge
~rowth i~ the cause of cance~: Factors which can ca~se ~
~he uncon~rolled c-ll grow~h connect~d with me~astasi~ are ~' .
often of ~ chemical n~tur~, but can also be of a physical :;~
nature sUCh a~ radioactivo radiation.
,
At pros-nt, t~o alternati~es are substantially avail- ' ~4
able ~or th- treatment of cancer. Elther one s~cceeds
in co~plet-ly remo~ing the cancer cell~ from the .
diseas~d organi~m by ~urglcal ~ntervention or it i8 .
at~empt-d to rendor the tran~for~ed cells in tho
organism innocuou~, e.g. by the adDinistration of ~rUg5 ~::
or by phy~ical treatm~nt method~ such as r~d~otherap~. . :
::
~rugs are often used in che~oth~rapy, which interfer~
with ~he DNA metabolism ~n some way or other and damage -~
rapidly growing cell~, ~hich must furni~h a higherDNs
~etabolic ff~ciency, more strongly than cells which
do not d~vide or only d~vide slowly. A ~erlous
disad~antagQ o~ many che~otherapies, howeYer, 1~ the
-:
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low ~pecifity of the u~ed active sub~tances, the result ~ -
of th~s being that healthy cells are dlso damaged in
chemotherapy. Thi~ low speci~ity of the aativ~ sub~
~tan~e~ require~ ~urthermoro that their dosage must in ~ . --
~ach case ba mad~ so that a~ ~ew healthy cells a~
posslbl~ ~rQ damaged with the simultaneous ~illing o~ ~.
th~ canc~r cell~. This i~ often not po~ibl~, and th~ :
patient 3uS~rinq from cancer dies duo the increa~ingly -~
sprQading cancor C8~ hlch causo the ~ailur- of v~tal ;~
~unctions in th~ t~rminal stage.
~t i~ tho ob~ect o~ tho present lnv ntion to mako
available a ~urth~r active substanc~ with valuablQ : ~.
therapeutic proper~es and a drug con~aining th~
active eubstanc~, tha active substance or the drug `,~
being osp~ci~lly ad~rantageou6 in the treatment of
cancex d~sea~aa. .;~
,:"-,;,','
Thi~ object is attained accordlng to the invention by a mutated
receptor tyrosin2 kinase that is defective in its si~nalling
activity and by a dru~ containing at least one ~utated receptor
tyrosine kina~e. : . ,i"
me t,erns which are u~ed in the present text shall now be explained
in mo~e de~ail for a better under~tanding of the present invention~
.. .,, . . . . . . ~ ..... . . .
: : : : : :: : : : :
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"Rece~tor tyrosine kina~e~ mean6 any kind of receptor
exhi~lting tyrosin~ klnase activity. ~he term includes
growth factor ~eceptor~ exhibiting tyrosine ki~ase
activity, ~8 well ~s H~R2 or met ~eceptors. -~
"De~ecti~e in its ~ignalling activity" means that ~ mutated
receptor i9 no longer capable o~ conve~ting an ~ .
extracellular g~owth signal or another signal to an
int~acellular signal, so th~t said signal is ~artly
lnhibited or fully blockod. .~
,' ':, '~ "
~Growth ~actor" me~ns any mitogenic chemical, normally a ;~
~olypoptide which is æocreted from normal and/or :
transfo~med mammalian cells and ~l~ys an important role in -;~:~ the regulation of cell ~rowth, especially in tSe
6timu1ation of the p~oli~0ration of cells and the .
maiPtenanco o~ their vi~bility. The term ~g~owth factor"
includos, e.g., the epldermal growth factor (EGF), ~he
~latelet-derived growth ~actor (PDGF) and the nerve growth
~acto~ (NGF).
. . - . .
"Growth factor ~eceptor" means a polypeptide which spans ;~
the cell me~brane ~nd bind~ a g~owth or differentiation
~actor ~nd has itself a tyro~ine kinase ac~ivity in its
~ntracellular pdrt or is asso~iated with such a~ activity. ;
, , . ., .~"
,,~,,~ ~....
,~..: ~'',
. . : ~- ~ .
. ~,~ ... . . .
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"Mutated recepto~ tyrosine kinase" means a tyrosine kinase
receptor wh~ch contains a otructuxal ch~ngo in com~arison
with the wild-type receptor, so that the receptor no longer
posso6ses the tyro~ine kinase activity of the wild~type
~eceptor. .
~, ,
"Nutated gxowth ~actor receptorH mean~ a g~owth factor
receptor wh~ch contAin~ a structural change in comparison
with the wild-ty~e receptor, 90 that the ~eceptor no longo~
possesses the tyro~ne kinase 4ctivity of the w~ld~type
recep~or. - .
"Wild-t~p~ growth factor recepto~" or other "wild-typen - .;
receptor mean~ a natu~ally occurring ~rowth factor receptor .
or other receptor ~hich exhibits tyrosine kinase acti~ity . ~ .
and is thus capabls o~ transmitting sig~als.
"Extracellular dom~in" of the g~owth ~actor receptor or
other receptor meaDs the part of the xeceptor that normally
pro~ect~ from the coll into the ext~acellular enviro~ment.
The extracellular domain comprisos, fo~ instance, the
receptor part to which a growth Sactor or another ~ole~ule ;~
binds.
,, ~
"T~ansmembrane regioD" of the growth factor receptor or
other ~eceptor means the hydrophobio part of the receptor
that is normally localized ~n the c~ll membr~ne of ~he cell ,~
which expresse~ the rec~ptor. ~ ;
. .
' -: .
~ ~.. .. , . " ,.. ~ . . . : . . .
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21~7073 ~ ~
"~yrosine kinase d~main" o~ "cytopla~matlc domain" of ~he
growth ~actor receptor o~ othe~ receptor means the part of
the receptor tha~ is no~lly positioned within the cell .
and e~fects the t~nsphospho~ylatlon of tyrosine residues.
"An e~fective amount" means an amount of the composition of ~ :
the invention which can achieve the desired therapeutic
e~r~ct . ',. . . ;~
. . . , ~ ,... .
, .. . ...
"Platelet-derived ~actor ~PDGF)" means a mltogenic
polypeptid~ which is contained in blood platelets and
stimulates m~sonchy~e-derived cells and stim~lates the
~tophos~horylating p~otein tyrosine ~lnase activity wh~n . .
~t binds to tho PDGF wild-type receptor.
, i ., .. ,~:
"Epidermal ~rowth ~actor (EGF)~ means a mitogenic
yolypeptide which ~ormally produces a ~itogenic response in ;~
~ibroblasts and which stimulates the autophosyhorylating .~ ,
protein tyrosin~ kin~se acti~ity o~ the ~GF wild-type : ;
receptor. .;~
UHyperplasia-based diseaseU me~ns a disea6e of a tissue o~
o~an, in~luding, for instanco, skin epidermis, the
in~estinal epithelium, hopa~ic cells, ~ibroblasts, marrow
colls, other bone cells, cartil~ge, and unstri~t~d mu6cles,
the di~ease being ch~racterized by an inc~ease in the .- ~ .
number of cells of tho tissue or organ, such as psoriasis . :.
and ~ndometric hyperpla ia. . ~
. - . ~ .
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.: ~`.,.`.,
"H~2" means ~ rec~ptor tyrosine Xinase which exhibits
sequence homolo~y to the epider~al growth factor receptor. ~ ;
"Lipoeomes" mean particlos in a~ aqueous medium which are
foxmed by lipid bilayers that enclose an aqueous
env~roment.
. :: .: - . . .
"overactivity" means an ~xcessiv~ u~controlled acti~ation
signal transmiseion path imparted by growth ~acto~
~eceptors, which results in an ex~essive cell dlvision
activity and oth~r consequences such as those occuxring in
some cancer cells in com~riso~ wlth the normal cells of a ~ v'.'r;;~imilar coll typ~
"Recombinant vectors" mean vectors which were genetically
ch~nged using reco,~binant DNA technology to incorporate
nucleic acid frag~ents that code fo~ normal or mutat~d
receptor ty~oslne kinases. The recombin~nt vectors can
in~ect target cells and induce the target cells to expres6
normal or mutated ~eceptors.
"Reco~binant retroviral vectors" mean recombinant vectors
w~ich are ret~ovirusos.
In acco~dance ~ith the ~resent invention, it was possible
to show that mutated receptor tyrosine kinases have '.''''!''.;
. . .
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valuable therapeutic ~rope~ties whioh can be e~ploited for
tr~ating disea~es associated with a hyperfunction of ;~receptor tyrosine kinases, the m~tate~ rec~ptors ~eing in
p~ic~lar suited ~or treating cancer di~as~
Growth ~actor rec~ptors play a decisivq role ln the .
dev~lopment and multiplication of human cancer cells. `;
In healthy cells, growth ~actor receptors are involved
in the control o~ c~ll growth. The actual ~ignal ror
cQll dlvlsion i~ the growth fa¢tor which is ~ormed a~ a
~un~tion o~ tA~ need~ Or thQ organi~m. The receptor .
as~umes the ~unction o~ ~ignal transmi~ion, i.~. it i~
involved in the conver~i~n of the Qxtracellular growth
~ignal to c~ll divi~$on actlvity in the interior o~ the
coll. I~ many growth factor receptor~, their ability to .~
trans~er phosphate re~idues to tyro~ine residue~ in - , .
protein~ after b;ndlng the growth factor to the extxa~
aell~l~r domain rop~es~nts A deaisi~e roll. Th~se . .. ::-
r~ceptor~ aro also de~ignatQd a~ r~ceptor tyro~in~
kinases. an overviow of r-ceptor tyrosine kinasQ~
found in Yarden, Y. ~nd Ullrlah A., Rev. Bioche~. 1988,
57, 443-7~- The dimerization of these growth ~actor ",;".,,,,,'",'~t,'.,,,~',,
xe~eptor~ a~tQr binding o~ the growth ~act~rs is
another i~portant phas~ Or the process o~ signal
tran~miscion. The conver~ion o~ an ex~raaellular
signal to an in~race~lular ~ignal by mean~ of growth
factor reo-ptors with tyrosine kinasQ activi~y eAn ~e
divlaed into the following ~iv~ ~topi~
.~
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1) The bindin~ o~ th- growth ~actor (also designated as
lig~nd~ to th~ extracellular domain of the receptor ~ ~;
lnduces a change in confor~a~ion~ the 6~e Cau~es
~) dlm~rization o~ rec-ptors which changed conforma~
tion; with
: ~ ,~', .':'
3) simultaneouJ induction o~ ~n allooteric change in ~:~
tho cytoplas~atic do~ain, by means o~ which, on the
other hand, th- k~n~se activlty is induced;
4) transphosphoryl~tion o~ tyrosin~ re~idues in th~ :
receptor dimer, whi~h, in ~urn, produces and stabilizes , ;~
an activated r-ceptor con~ormation; and .
5) pho4phory1ation o~ polypeptide substrates and
intera~tion with cellular factors.
:~ ' .; ,~ ! ~
Unaontrolled ov~ractivity of t~is signal ~ransmission
chain due to receptor overexpr44sion or ~utation can lead : :
to an exce~sive division activity of the corresponding cell, :.
and in th~ extreme ~ase, to a transformed canc~r cell.
An o~erViQW of grow~h factor r~ceptors and their funotion .
in signal transmis5ion from ~he extr~cellular to th~
intracellular envi~onmen~ and the pwsible influence o~
abn~ally expre6sed rec~ptor~ on the de~elopment of
cancs~ are indicated in Ullrich, A. and Schle~singer, J.
(199~) c~ll 61, 20~ - 212. ~ ~ :
........
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The ~pide~mal growth facto~ ~eceptor (EGF-R) iS a 1?0 kD
glycoprotein with ty~osine kinase act~vity ~Ullrich 0t al.
~lg84), N~ture ~09, 418 - 425). Th~ molecular proce~ses in
the binding oi' it~ ligand and the s~imulation of its kinase
activi~y are described in detail in ~llrich et al., Cell
(19gO) 61, 203 - 212. Although EGF noDally causes a
mltog~nic re~ponse in ~ibro~last6, an ove~activity of the ~ ::
~ignal txansmission process by the EGF receptor on account
of overexpres~ed receptors leads to a ligand-dependent
transfo~mation o~ NI~ 3T3 mouse cells (Riedel et al. -~
~1988), Proc. Natl, Acad. 8ci., ~SA ~5: 1477 - 1481 and Di : ~.
F~o~e et ~ 12987), Cell 51, 1063 - 1070). Inten6ive .
cl~nic~l studios su~oxt the function of this recep~or ln
the de~elopment o~ speci~ic carcinomas, such as ,.
m~stocarcinoma, o~a~ian carGinoma and pulmonary carcinoma
(81amon et ~1. (15~7), Science 235, 177 - 182 and (198~
Scie~c~ 244, 707 - 712 and ~e~n et al. (1990) Cancer Res.
50, 5184 - 5191).
It w~s surpr$singly found that the fi~e-step ~ignal
transmi~sion ch~in oxplain-d above can be blocked or
inhlbited by a mutated growth fac~o~ reeeptor, lf the ;
mutated receptor~ are 9i~ultaneously expres3ed with the
wild-type receptor~ from on- c~ hus, muta~ed signalling
de~ective growth factor reoeptors are suited a~ drug6 wlth m~
WhiCh diseases c~n be treated which are connected with an ~ -~
increased transm~s~lon of growth signals to the in~erior of -~
tho col} by corresponding receptors.
..... - .
~. . ..
! ' ' ' ."' ' ' ,', ',
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A pr~-rred r~ceptor ~utant does no longer have the ;~
tyro-in~ kina~e actlvity of th~ w~ld-typ~ receptor. .
~hl~ ~utant ie no longer capabl~ o~ phosphoryla~ing
tyrooin~ re~idues ~n the recoptor dimer or in ~he
polyp-pttde ~b~trate5 after llgand b~nding. ~hus, th~
con~-rston o~ the extrac-llular grow~h signal to an
intrac411~1ar rign~ blocked or partly inh~bited.
~ ~,
A point ~utatlon in the wild-type rëceptor can already
bo our~iciont ~o th~t the w~ld-type re¢eptor does no ~ ~;
longer ~nction prop~rly if it lost th~ tyro~in- kina~e
activity due to p~int mutation- Such a po$nt mutant (e.g.
~ 21A) i3 espeoially pre~rred.
A ~tatQd receptor i3 furthernore preferred, which
carri~ a del~tion in the tyso~ine kinas- domain, which
leads to a 10~5 of tyrosine kinas- activity.
It i- prer rred t~t th~ receptor mu~at~d by a deletion
ln the cytoplasmatic do~eain H ~ ~ s, however, still ;~
~h- tranemembr~n- r-gion ~ utated rec~ptors with
xisting transmembrane region lead to a mor.e effective
inhibiton of growth slgn~l trans~ ion and thu~
exhiblt a b-tter therapeutic errect than receptors
wlthout transmembrane region 5uch as ~utants which only
conslst of tho extracellular do~ains (~.g. ~G. 1, ~E~CD-565).
- .. :.
' ' ' ' ~ . ~
:" ~ . '.,' '
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Mutants o~ receptor tyroisine kina~es such as o~ ~GF, PD~F, ',.'',~
ICF-I, M~T receptors, EaF rec~ptor-related receptors su~h
as H~R2, neu, C-erbB2 receptors or NGF receptors ar~
especially s~ited as a dr~g. The ME~ protein, for instance,
18 described in detail in Gio~dano et al. ~198~), Nolec.
cell. Biol. ~, 3510 - 3517 and in Giordano et al. (19~9) .
Natur~, 339. .
A mutated receptor of the pitermal growth fac~or (EGF~
~ v ry par~icularly w lted. ;. ; z.
. ' ~,,: '!' ,,
In a part~cul~rly.pre~orr~d nutant o~ tho EGF receptor
thQre i~ ~ point ~utation at the a~ino acid position ;~
721 o~ tho wild-typ~ recQptor sequenc~. In a pre~erred
mu~ant the ly~ reisidu at po~ition 72~ is replac-d
by an a~anine r-9~du- in the mutant. ~is mutant is
d-pos$ted undor DS~ 6678 wl~h th- German Collection of
Mi~roorgan~m- and Cell Cultur~ Cm~H under the ~ud~
p~t Treaty. In a furth~r pr~f-rr-d ~GF recejp~or ~utant `
thc S33 c-t-rm$nal amino acid~ o~ th- wlld-type recQpt~
or ar- deletod. Thls ~tant is depo~it-d under DSM
6679.
The receptor mutants can be produce~ according to
customary gen~tic ngln--rinq proce~seJ such a~ des- `.
cri~d in Sam~rook, J, et al (1~89) Molecular cloning,
Cold Spring ~arbor ~aboratory Pr~s~ ~tarting from the ~ i.
wild-type rec~pto~
' '~' " '"' ''''
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. . . -
., . ,',,.
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The drug according to the invention contain~ at least
one o~ t~e mutat~d gro~th factors described above and
tho customary ad~uvant and carrier substances.
., ., ~
A drug i3 o~peoi~lly preferred which contains the ,~
~eceptor ~utant~s) pac~ed in llposomes~ In order to
brtng thQ lipo~om~s col~ct~v-ly to the target ti~sue it : .:
i~ ad~an~ag-ou- i~ th- l~po~ome~ contain antibod~-s ~n
their membrane, the antibodi-~ recognizing ~peclfi~
epltopes or th~ targat cell~ and be~n~ bonded ~elect~
iv~ly to the~. Thu9, the receptor mutants are tar~eted
to ~he target tissue and can evolve their desired effect
ther~. ~h- admin~tration of ac~i~s s~bstance~ pac~ed in
liposom~s is a common form of ad~inistration nowaday~
A drug is furthermore pr~a~-d which contains t~
rQa-ptor~s) ln t~e for~ of on- or s-v~ral r~co~binant
ret~o~iral voctors. ~he r~¢ombinant vector~ contain
nucl~c acld ~ragmQnts coding for the receptor~8
Af!ter A ~in~str~tlon of th~ drug to tho patient, tl~
r~troviruse- ~n~ect the targ-t cell and le~ to ~hQ i.;::
expre~ion o~ t~ receptor ~utan~Qtherein. `;~: .. `
._. , . , . . ~ . . ~ . ..
~n ~specially pr~f~rr~d drug contains the retroYiral .
vec~ors p~TK-HER-R7ZLA and/or pNTR-HERCD-533 codlng for
EG~ recepto~ mutants,; which are deposited with DSM ~. -
(German Collectlon of ~ioroor~ ms), M~scheroden We~ ~B~ D-3300 : ~'
Braun~chwelg, under DSM 6678 and DSM ~679, respectively,
.'','.'. . '"'.,',, ,,.'.'
. . . ... ..
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The mut~ted r~cep~ors ca~ be incorporated into a drug in
tho conventional manne~, as is, ~or instance, described in : .
Remington's PharmHc~utical Scienc~6 (osol~ A. editor) Nack
P~lishing Co~pany, East~n, P~ (1980) and consecuti~e
vol~mos.
Th- ~utated receptor~ de~cri~e~ abo~e and/o~ the drug~
co~taining them ar~ e~pea~ally ~ui~ed for th~ trea~cent
o canc~r. ~uch type~ of cana~r can b~ trsa~ed ; ~;~
npec~ally w~ll, wh~ch are a result of an overact~lty .
o~ growth fac~o~ receptor~. ~hQSe type8 of canc-r
include ln par~icular ma~toCarc-nowa~ ovarian ~cino~a and puloonary
~ arcinon~a., The role o~ ~ur~ace receptors in
cancer di-eas~ de cribed in detail in Slamon, D.~
et al (1987), sci-nc~, 235, 177 - 182 and ~1989)
SclencQ, 244, 707 - 112 and XQrn, ~.A. et al ~1990),
Canc~r Re~ ., SO, ~184 - 51~
The pharmaceutical com~osition ~a~ contain salts, buffers,
additives and oth~r substances that are desirable for ~--
improving the efficiency o$ the mutated receptors. :;
~ .. ~ .;. .
:," . . .'' '.' ' `:,,;','
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compositions for ~ parenteral admini~tration comprise
5terile aqueous ~r non-aqueous solutions, suspension~ ~nd
emul~ions. Aqueou~ susp~ns~on6 ~or injec~ion may contain
substances increasinq the vloco8ity of the suspension and
com~rise, ~or in~t~nce, sodium car~oxymethylcellulo~e,
Borbit and/or d~xtran. The ~uspension msy optionally
contaln 6tabilizer~. ~xamplos of no~-aqueous solvents are
propyl~ne glycol, polyethylene glycol, vegetable oils, such ;~ -,
48 olive oil, and in~ctable o~ganic esters such as ethyl
olo~tQ.
Carrier ~u~stances o~ occlusiv~ plasters may be used or
inc~e4sing skin pe~m~ability as well as the dermal
adsorption of the drug. ;.
~i~uid fo~ms o$ do6age may typically include a liposome
~olution containing the liquid form of do~age. Suitable , ......... ,-
forms for susyendlng liposome6 include emul~ions, ;~
su~pensions, solutions, 6yrup aDd elixirs with ine~t
dilutants which ars no~mally used in this field, e.g., : . :
pu~i~ied Wate~. .. ;
Apar~ from th- inert dilutants, these compositions may al60 `~
include additiv~s, wettin~ aqents, em~lsi~ying or : -
6usFending agents o~ aromat~cs. Exampl~s of other materials
suited for use in the ~resent pharmaceutical Composition
are indicat2d in R~mington' Ph~rmaceutical Sciences (Osol,
A., edi~or), Mack Publi6hi~g Co., Easton, PA ~19801 and ..
conse~utive volume6. .~.
, '~,'
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The treatment o an in~i~idual having a tu~ox includes the ~ -
admlni~tration of an e~fective *~ount of the mutated
recepto~ or recombinant vectors, which produce the ~utated
receptor6, i~ a single do~e, several do~es or in the ~orm
o an in~usion on ~ patlQnt or an animal.
In accordance with tho pre6~nt inv~ntion an "eff~c~ive
amount" o~ a pharmuce~tical compo~ition i5 an amount which
ls su~icient to achi~ve the desired ~iological e~ect. A~
4 rule, the dosage which ~ needod ~or ~oviding an
e~rective a~ount o~ the compo8ition and which can be SQt by
an export will depend on ~acto~s, such as the receptor to
be specirically us~d, the presence a~d kind of ot9her
thera~eutical ~e~ts, the age of the p~t$~nt o~ ani~al and . .
tho condition, SQX an~ clinical state thereo~, including
the extent of the disease and o~her ~arisbles.
The pt~rred dose of the phar~aceutical Compo8ition of the
invention in a human being is >109 plaque-formi~g u~it6 .
~pfu) per petson and depend~ on the type o$ cancer and the
extent of the rece~tor hyperfun~tion.
The pre~erred method o~ a~nistering the phar~aceutical .
composition Or the invent~on i8 a parenteral method. ~he
mo~t preferred way is a~ intravenous, intraperitoneal or .
topic~l one or directly into the ~rain, the spinal cord
liquid or the tumor itself. .
"~
-.. -; ... ...
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. .. : . .. . -
02~ 4 17:5~ ~+4~ 8~ 220287 PAe Gruenecker Qlol~
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'
Without ~eing commltte~ to a specific theory, it is assumed
that th~ d~scribed rec~ptor mutants evolve their effect in
~he ~arge~ Cell9 ~ that the mutants are incorporated into
~he membra~ of th~ target cells in addition to the ~ild~
type xeceytor, ~nd th~ receptor muta~t the~ lmpairs ~he
func~ion o t~e wi}d-typ~ recepto~ by forming siqnalling
incompetent dimers con~isting of one wild-type ~ oncogenic
roce~tor and one ~ignalling d~f~ctive muta~ed receptor.
Ths present inYe~tion i8 described in more detail by means
o~ m~tant~ of the RGF receptor as a model. . .~,
~t ~a~ surpri~ingly found that the expre~sion of EGF : .
receptor mutants ~hich do no longer have any tyrosine ;~
kinase activity ca~ reverse the transfomred phenotype in . ;
transformed c~ncer cells exp~essing the EGF wild-type ~.: . :
rece~tor. . !.
Mat~rial and M~thod~
,..,,., ~" , .,, :.,
Production Qf r~co~binan~ retrovirus
Th~ retroviral exp~ession vectors pN2, pNTR2 and ,,',
p~TR-RERc are d~scribed in detail in Keller, c. et ~1, ,. ;;.~
(~985), Nature, 318, 149 - 154; Stewart, C.L. et al, .;.
(~987~ EMBO J.t 6, 383-388; von R~den, ~. and wagner, ~ '~
E.F. ~1988), EM90 J., 7, 2749-2756. pN~K-~ER-K 721A was .
produced by clonlng a sgl II fragment o~ CMVHER-K721A
in pNTR~ c. pNTK-HE~CD-533 wa~ produced by making a ~ ..
.".," ' ;~," ''~."
''.', ~' '' . ~',':
~. :~ '- -, '
. :, ~ . . . : , - . , . : .
02i~ 4 17:55 ~-4~ 8~ 220287 PAe Gruenecker 121020 ~
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17
,,",~
~ ~ .
~laI site at both sideo of the 2 kb large fragment
XbaI/XhoI of pLSXN~ 8~ de~cr~b-d in Livneh, E. et
J. ~ol. Chem., 260, 12490-12~97 by mean~ of customary
cloning processe~ a~ describQd in Sambrook, J. et al
(1989), Molecular Cloning~ Cold Spring Harbol~ ~a~ora-
~ory Pre~s, and ~ubseguently th- 2 kb ~1~1 fragment was
ligated with Cla~-cleav~d pNTX~. ThQ NTR-HERC~-566
con~truct was produced by cloning a ClaI ~ragment o~
CVNHERX~D into thQ ~laI site o~ pNTX2. The construct
wa~ d~po~ited under DSM 6680. Ecotrophi¢ recom~inant
retroviru~e~ were produced from the helper-virus-fre~
pr~duc~r line GP+E-86~ de~cribed in Markowitz, D.
(lg88) J. Virol., 62, llZ0-1124. Stable GPIB-86 pro~
ducer lines werQ produced by meahs of a modified
in~ect~on in~truction as d~sc~ibed in Miller, ~.D. and
Butti~ore, C. (1986) Mol. Cell. Biol., 6, 289~-2902. An
amphotrophic virus with low titer was produced by th~
tran~ient transf~ction o~ retroviral expre~sion pla~
mids into the help~r-virus-~ree packaging cell line
PA317, described in Niller, A.D. et al ~1985) Mol.
Cell. Biol., 5, 431-437, and was used to infect second-
ary packaging cells GP+E-86, ~ollowed by ~ selection o~
clones Or the producer line GPIE-86 in G418 (1 mg/ml).
Th~ vlrus titer w~s de~er~in-d by infecting NIH 3T3
cclls with dilution seri-s of retrovirus which centain-
ed cellfree GPIE-86 ~upernatants, and determination o~
the number o~ the ~418 rooi~tant colonies. A retroviru~
( ~2TGFa) which contains thQ g~ne for ~he tumou~ growth
factor ~GF~) i8 de~cribed in Blasband, A.J. (199O),
oncogene~ 5, 1213-la21.
02/0jl~'~4 17:5~ ~+48 8~ 220287 P.4e Gruenecker ~021
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Gene ~rans~er by means of r~troviru~e6 .
Su~confluqnt NIH 3T3 oells ~105 cells/6 cm plate) were
lncubated with sup~rnatants of GP~E-8~ cells which , .-
r~loa~e high tlteirs of ~T~-HERc ~rus (5x105 G418R .;~
~olony-~ormlng unit~ per ml) for 4 to 12 hours in the
preOEence o~ 4 ~g/ml Polybrene (Aldrich) and subse~ue~t- .
ly in a supernatant of GP+E-Q6 cells which release high : .
tlter~ o~ ~ither N2, NTK-HERK721A, ~TK-HERCD-533 or .
NTK-HERCD-5~6 viruse~. The expre~sion level of th~ . : '.s
r~eptor~ was increa~ed by 6everal in~ection cy~les a~
de~c~lbed ln Bordign~n, C. ~t al (l989), ~roc. Natl, .
Acad. Sci, USA 86, 6748 - 6752. In the described
experi~ents, the in~ction was once carried out with l . : i
~1 of a dilut~d supernatant (1.25 x lO5 colon~-forming
units) or 1 to 4 times with the sam~ ~olume of undilut- .
ed supernatant ~ x 105 colony-forming units) or . :- ~
CP~E-86 cells which release high titers of eithQr N2, ~"~;,','',,~:.";~!~"'.'':.
NTK-HERK72~A, ~K-HE~CD-S33 or NTK-HERCD-566 ~iruses.
Receptor phosphorylation in intact cells ~ ~;
The cells infected as indicated abovR were ~ultivated : ~.
ln lOcm - plates up to ~ 90S ~onfluenoe, wa~hed and . ~.. ;
cultlvated for 16 hours ln ~ethionine-free DME~ ~Gibco)
supplemented with 1% FCS containing 50~Ci/ml35~-meth~
lonin~ (A~ers~a~ he c~lls wer~ stimulated with 20 .
ng/ml ~GF (Amgen CorF.) for lO minute~ and lysed ~n 0.5
ml lyee burfe~ (50 mM Hepes pH 7.2, 150 mN NaCl, 1.5 mM
MgCl2, 1 mM EG~A, 10% gly¢~rol, l~ ~riton X-lO0, 1 mM
P~SF, lO m~ml Aprotinin , lO0 ~M sodium ortho~anadate)
at 4~. The lys~tes w~r~ centrifuged in ~n Eppendorf ;
02/~'~4 17:5~ ~+~ 8~ 220287 PAe Gruenecker 1~l022
` 2~17073 ~\ ~.
-;-. ~.`.;
1 9 .. i~
. . . .
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c~ntrifuge at abo~t 12,000 g at 4C for 10 min~tes. The .
~uperna~ant~ were ~han incubated with an nxcess of
monoclonal antibody 10~.1, de~crlbed in Hon~igger, A.~
~llrich A. and Schleç~inger, J. (1989) Proc. Natl.
Acad. Sci, USA, 86, pages 925 - 929, and protein ~ .
A-~epha~os~ at 4~C for 4 hour~ no- precipi- ., . ,j 2
tates wero washed twice with NNTC (20 mM H-pe~ pH 7.3, .
150 mM NaCl, 0~1~ Trito~ X-100 and 10~ glycerol). ~he
pellet wa~ r~suspend-d in ~ sample bu~fer, boiled ~or 5
minut-~ and analysed by m~ano of SDS-PAGE (7.5%). $he ;.. . ~;~
prot~in~ w~r~ elRctrophore~lcally transferred to
~ltroc-llulo~o and 8ubs~qu~ntly incubat~d with a ^~.
monoclonal mou~e antibody again~t phosphotyrosine ~5 ,.
E2), de~cribed in ~endly, B.M. ~t al (19gO) Canc-r .. .
Research, 50, 1~0-1558. For d-tection, th~ nitrocel~
lulo~o ~ilter was incuba~ed with a peroxidas--coupl~d
goat anti-mous- antibody, ~o~lowed by an EC~ substratQ ;~
re~ction ~A~er~h~m~. A~ter de~e~tl~n o~ the ECL
substrate roaction w~th a Kodak X-Omat film, t~Q nitro- :s
cellulo~e $ilter~ w re wash~d with PBS containing 0.2~
$ween 20. Subs~quently th¢ 35S-methionine-marked pro-
t-ins wore det-cted by me~ns of autoradiography. Ihe - :~
den~ity of the bands was ascertained by means of .. ~ ,.
dRn~tome~ry.
'
~ . .',, ;''
,
02/~ '~4 17:57 ~-4~ 8~ 220287 PAe Gruenecker 1~l023
2117073 ~
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~ .' ~'~ ~,,';',,
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~3~]-thymidine incorporation - ; -.
.~: .. . . .. . . .. .
Subaon~luent NI~ c~lls 3T3 (105 cells/6cm- plate) were . . '.
coln~ected with NTK-HERc as descri~d, followed by 4 .~
lnfection cy¢l~s ~th ith~r N2, ~TK-HERK721A, NTX- :; '''"r
HERCD-533 or NIX~H.ERCD-566. Thc cells were distributed
to 12-well ~ostar plates. After th- rea~h~ng o~ oon- : ~:
Yluona~, the c~ll monolayers were starved in 0.5 ml ;.'
DNE~, O.S~ F~S for 24 hours, and, 18 hours a~ter EGF
addi~ion, the c~lls were labeled with 0.~ ~Ci Methyl~
[3H~-thymidine (A~rsha~) ~or 4 hours. The cell~ were ~,5~ ,'. j ~,~,~.'. .,~
~ashed twic~ with PBS and subsequently precipitated on ~;
ice with 10~ TCA for 1 hour. ~he precipitate was ~ashed .. ;~
with 10% TCA and dlssol~d again in 200 ~1 0.2 N'`~'~''''''"'~h''~'~'""'
NaO~/~.2% SDS. Th~ lysates we~e n-utralized, and the 'i~
incorporated radio~cti~ity was quantitativ~ly determln~
~d by ~cintillatlon countilng. ..
Tr~ne~o~ation test~
In order to exa~ine the abillty of N~ 3T3 cells for
colony ~ormation in ~o~t agar, subconfluent NIH 3T3
cell~ (105 cells/6cm-p~ate) ~ere in~ected with .
NIX-HERC, followed by 4 cycle~ of infection with either
~, NTX-HERK721A, ~X-HERCD-533 or NTK-H~R~D-S66~ In
th~ cases in which an autocrin~ s~mulation was to b~ :
caused, the cells were inf~ct~d with ~2TGFa Yirus ~s x
104 C418R of colony-forming units per ml). ~IH 3T3
`. ''~'.'
02~ 4 17: 57 ~+~ 8~ 220287 PAe Gruenecker 1~ 024
2~17073 ~h
~ Vr1ron~ r
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cells (105) were plated on 6cm-plates in the presence
or absence o~ lO ng/ml ~GF ~n a tdp~layer of 3 ~l M~q .
containlng 10% FCS and 0.2~ ~.gar (G~b~o).
Th~ bottom lay~r contained MEM, lO~ FCS and 0.4~ agar. ''~"''~.~'' '~"!.""''~
Visible coloniee were counted after 4 weeks. "~
For ~oc~ forma~ion tests ~ubcon~luent NIH 3~3 cell~ ` :
~lO5 cell~ /6 cm-pL~t~) were coinfQc~ed with NTK--.~ER~ (1x104
G418R colony-forming units per ml), followed by 4
cyclefi of infection with eithQr N2, ~TR-HERK721A,
NTK-HERcD-533 or NTX-HERCD-566 viru~es- In some ex-
periments the cell~ were supe~infected wi~h 2~F~
virus (l x 103 G418R colony-forming units per ml). ~l,
Inf~cted cell~ were cultivated on 6 cm-pl~te~ with D~E~ ;.
con~aining 4% F~S in the presenc~ or a~senc~ of lO
ng/ml ~. Tho medium wa~ replaced e~ery 3 day6. ~h~
plate~ were 3~aine~ with crys'al violet, an~ the roc~
were counted ~n day 1.8. . x
Legends ~or the ~igs.
Fig. l: Schematic representation of ~he human EGF
~ild-typ~ receptor and muta~ed EGF receptors. The -
position o~ domains rlch in cysteine (cys), o$ the
tyrosine kina~e (TK) and the ~rans~em~rane (~M) domains ~ .:
is indi~ated. Th~ mu~ant HEK~72lA ca~riQs a point : .
mutation at position 721 (an exch~nge Q~ lysine to
alanine), while ~ERCD-533 and HERCD-566 carry C-t~rm- :
inal deletion6 o~ 533 and ~65 amino acids, re~pectively.
The mutants are de~cribed ir. detail in Livneh, E., ~t al
(1986) J. Biol. ~h~., 260, 12490-12497 and Honegger
A .M . et al ~1987 ) Cell, 51, 1 99-20g .
Fig. 2 ~A): Tyr~sin~ phosphoryla~ion of t~e wild-type
and mutat~d EGF receptors. Cells which either
:,:
.., .. ~ --
'::' .'' , ' ' ~ ' ' -
02/~' ~4 17: 58 ~+4~ 8~ 220287 PAe GruenecXer E~ 025
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express the wild-type receptor alone or coex~ress the .
wild-type receptor and mutated receptora were la~ei~d ~ :
wi~h ~35S]-methionine overnight and ~ubsequently .~
incubated ~n the presenoe or abssn~e of 2 ng~ml ~GF for ~; :
10 minute~. Th~ cell~ were di6solved and precipated
with anti-EGF receptor antibody tmAb 108), separated by .
SDS-PAGE and analysed immunolog~cally with
anti-ph~sphotyrosine ~ntibodies (5E2), followed by an :. .~.. ~`.
ECL ~ubstrate react~on. . '~
~B): Expression of the EGF receptor on NIH 3T3 -"''`''''-;"~'~';~",';'!''!
cell~. Cell~ whlch éxpres~ oith~r ~he wild-type ~ . ';;.~ .
roc~ptor alone or coexpre3s th~i wil~-t~pe receptor an~ mutated ;
receptor3 were la~eled with t355]-methionine overnight . .
and sube~guently lncubated in th~ presence or ab~ence
of 20 ng/ml EGF for 10 m~n~te~. ~he cells were dis~olv~
ed and precipitat~d with anti-EGF r~ceptor antibody ..
~mAb 108), separated by meanis of SDS-PAGE and i~muno- .~
logi~ally de~Qcted with an anti-phosphotyrosine anti- ~ ...
body t5E2), ~ollowed by an EC~ ubatrat~ reaction. The
EC~ ~ubstrate was washed with PBS containing 0.2% Twe~n
20, and the t3~S~-methionine-marked proteins were
detscted by ~eans o~ autoradiography.
Fig. 3: EGF-simulated [3~-thy~idine in~orporation.
Cells which eithQr bxpres.sed the wild-type ~eceptor
alone (d~sh~d line) or coexpres~ed th~ wild-type recepLor and
muta~ed receptor~ a~ in A: wild-type EGF recep~or +
K721A, B: w~ld-~ypo EGF receptor ICD-533, C: wild-type -::
EGF receptor t CD-566 werQ cultivated in 12- well C05tar
plates up to conflu~nco and starved for 2 days in DME~ :
contain1ng 0.~% PCS. 10~ FCS or dif~erent EGF concen- . :.
trations wer~ added, and, 18 hours a~ter the EGF addi-
tion, [3H~-thymidlne (0.5 ~ ell ) was added ~or 4 .
hours and it$ in~orporat~on in DNA was determined. The ~
mitogenic response was re~orded in order to show the
relation ~etween dose and response. The values were
corrected b~ the ~asal thymidin~ incorpora~ion and the
maximally observed responee to EGF was defined a~ 100~.
The ~illed triangles indicat~ the semi-maximal ~hymid-
ine incorporation.
Results:
Cells whi~h oxpre~ the wild-type receptor ~lone or
together with the mutated receptor6 were lab~led with
[3~s~-methionino~ incuba~ed in the presence or absence
o~ ECF for 1~ minute~, lysed and
im~uno_ pre-
aipated with a mouse antibody against human EGF recept-
or ~mAb 108). ~he ~amples were s~parated with SDS-PAGE,
t~ansrerrd to nitrooellulose rilters, and the tyrosine
pho~phorylation wa~ de~ected by me~n~ of the phospho-
tyro~ine-np~clric mouse ant~body 5E2 (F~.g. 2A~. The
amount Or th~ receptor present in the ~.no-
precipitate was detected by means of autoradiography o~
the sam~ nitrocellulo~ filter (Flg. Z~).
As shown in Fig. 2A, lane~ b and c, the ~GF addition
to intact NI~ 3T3 ~lls, whi~h are infected with the
viru~ containing t~e wild-type EGF ~eceptor, indu~es a
strong tyrosine phosp~oryla~lon o~ the 170 kD EGF
recep~or band. Due to phosphorylation, the elec~rophore~ic
mobility o~ the ~G~ recep'cor decrea~es in the SDS-PAGE as
compared with the unphosE~horyla~ed EGF receptor as can
be seen ~rom Fig. 2B, LanRs b and c. ~he level o~ the
EGF-stimulated phosphoryla~ion o~ the wild-type rocept-
or wa~ not reduced ~y tho ~o-expression of ~he soluble
oxtracellular domain Or the EGF receptor a~ en~oded by
the ~TX-HERCD~S66 virus genome even if tbQ extracel-
lular domain wag expres~ed in a 4-fold excess to the
02~ 4 17:5~ ~+~ 8~ 220~87 PAe Gruenecker 1~027 : : .
wild-type receptor (Fig. 2A, lane~ d to fS Fig. 2
L~s d to f),
,` . ;.. .: ~ . . .~.
On t~ contrary, in an analogous experiment in which
virus which expressæs the me~brane- anchored EGF recept~
or dRletion ~utant HE~CD-533 (Fig. 1) was used, a
~trong dose-dependent inhibition of the EGF-induced
phosphorylation o~ th~ wild-type ECF receptor was
observed ~F~g.2A, lan~s g to i), althou~h tte level of
th~ 170 ~ EGF receptor protein remalned constant (Fig
2B, lane~ g to i). The intensity of the tyrosine-
phosphorylat~d bands decreased from 100~ to 71% or 30~
re~pectiYely. Und-r th~ conditions the EGF receptor
had the ~ame ~lectrophoretic properties as a non-phos~
phoryla~d receptor (Fig. 2B, trace i), which is in
con~ormity ~ith i~s 3tate of tyrosine phosp;~oryla
tion, detected by m~b 5E2 (Fig. Z~, L~
;. ; , ,:
If ~he wild-type receptor wa~ co-expressed with the
kina6e-negative mutant HERX721A, an increasQd tyrosine
phosphorylation of the lZ0 kD band was detected (Fig.
2A, laneg k to m). The intensity o~ the signal of the
tyro~ine pho~phorylation of the re~eptor increased from
251~ to 337~ or 450%, respecti~ely, according to the
densitometric analy~is of the autoradiograFhY . Since
~he wild-typ~ receptor and the k~nase-negative mutant
axe of the sa~e size, th~ increased 17~ kD signal in
Fig. 2B, lanes k to ~, represents the ~um ~ the
phosphorylation of both receptors sincQ the mu~atQd
receptor can be ~ranspho~ylated by the wild-type
rec~ptor.
Inhibition of thQ EGF-inducedcell di~ision rate.
,,' ,
02~ 4 18:00 ~+4~ 8~ 220~87 PAe Gruenecker Q1028
~ ' ' .' ,
2117 0 7 3 $~ IIG~ ~5 ~ ~
EGF stimulates cell division in ~IH 3T3 fibroblasts
expressing the EGF receptor as described in Riedel, H.
et al (ls88) Proc. N~tl. A~ad. Sci. USA, 85, 1477
1481 and Prywes, ~. et al ~1986) EM30 J., 5, 2179-21~0.
~he influeno~ of ~utated receptor~ on the cell division
controlled by the ~GF wild-type re~ptor was deter~ined
by mean~ of the induction of the DNA synthe~is.
~he DNA synthQsi~ ~a~ determined in cells infected wi~h
the ~K-HERc virus and th~ N2 virus control as
~3H~-thymidin~ incorporation, and the synthesis was
maximally stimul~ted at 2 ng/ml EGF, with a semi-
maximal sti~ulation (~D50) at 0.66 ng/ml (Fig. 3). In
similar fashion as ~n earlier results a8 described ~n
Honegger, A.M. et al (1988) EMBO J., ?~ 3045 - 30S2,
and RiQdel, H., Qt al tl988) Proc. Natl. Aaad. SC~
USA, 85, 1477-1481 higher EGF concentrations led to
lower levels o~ [3H]-thymidine incorporation as shown
in Fig. 3. The co-expression of the EGF receptor with
HER~D-533 ~nd ~ERK721A led to a marked shifting of the
dose-dependen. curve towards higher EGF concentrations
a~ter four cy~le~ of infec~ion with the correspond$ng
viruse~ (Figs. 3A and ~). mis indicates t~at the cell~
have become less ~en~itive to the growth factor as
compared with HERc/~2 cell~. Both the deletion mutant
HERCD-533 and the point mutant HERK721A (~ig. 1) ~howed
si~ilar ~ffec~s on the cell division signal imparted by
the EGF wild-type rceptor ~nd caused a ten-~old in-
creas~ of ED50 to 6.6 ng~l EGF. As opposed to this,
the euperinfeotion with NTK-HERCD-566 virus did not
hav~ any significant ~ffect on the DNA synthesis
sti~ulated by the wild-type receptor by EGF ~Fig. 3C~
Antl-oncogenic aativ~ty of the EGF reGeptor ~utants
-
:,~ :. ,. : .
.. . . . .
02/,~ ' ~4 18: 01 ~-4~ 8~ 220_87 PAe Gruenecker 1~l o2
It i~ known that the overexpression of the EGF receptor
causes an EGF^dependen~ cell transformation of NIH 3T3
cell~ as d~cribed in ~i Fiore, P.P. et al (1987),
Cell, 51, 10~3-1070, Velu, T.J. et al ~1987), SCiencQ,
237, 1408-1410 and Riedel, H. et al (1988) Proc. Natl.
Acad. sci., tJSA 85, 1477-1481. In order to examine
wh~ther th~ t~ansforming potential of o~erexpresoea EGF
receptor c~n ~ inhibited ~y EGF receptor mutants, the
EGF reoeptor was co-expxessed with recep~or mutants,
and subsequently their ability of producing c~loni~s in
soft agar or foci in a monolayer cell cul~ure was
examined. Thq ~timulation of ovorexpressed EGF recept~r
was elther achleved by the ~ddition of EGF to the
medium or by inf~ction with a virus ( ~2~GF~), which
carri~ a TGF-~ DNA in order to produce an autocrin~
activation sy3tem (tablo 1: average values from ~our
expariments ar~ shown).
After inf~ction with the ~TK-HERc virus and th- N2
control, ~IH iT3 cell~ ~ormed abou~ 250 colonie~ in
~oft agar in the presence of 10 ng/ml EGF. By means of
Coin~QCt~on with ~aTGF~ virus the formation of 148
ooloni-s under otherwise identical conditions ~table 1)
was achievQd. HOWQVQr, if th~ cells in~cted ~ith the
EGF receptor W-rO ~uperinfected either with NTR-HER~
K721A or NTX-~ERCD-533 ~iruses, the colony-forming
c~pacity ~as suppres~ed almo~t co~pletely. The co-ex-
pression o~ the ~CF receptor with th~ extracellular
domain HERCD-566 r~duced the colony-forming ability ~y
about 50~ with stimulat$on with lO ng/ml EGF in tho
agar layer and by about 33% with s~imulation by tbe
au~ocrin~ TCF aftQr infection with th~ 4~TCF~ virus.
In ~imilar fashion, th- focus-for~ing potential o~ the
NTK-HERc virus was determ~n~d in ~1~ 3T3 monolayer
02/p~ 4 18:01 ~P+4~ 80 220287 PAe Gruenecker ~ 030
~ `~ 2 1 1 7 0 7 3 f ~
T~l. 08a/4~3P6757
2. ~ ~1 ~ ,~
c~ltures, either in the presence of lo ng/ml EGF, which
led to 9~0 foci per loG viru~e~ or after coinfection
with ~2TGF~ virus, which led ~o 48~ ~oci per 1o6 :
NTK-HERC viruces. Superinf~ction with ~TRK-HERR721A or
NTX-~ERCD-533 vi~u~e~ suppre~-d the number of the ~oci
by 100% or g~, respec~i~ely, i~ the -c~imulation with
EGF was e~f~c~ed and by 7~% or 71~, respectively, in ~:~
the case o~ ~ti~ulation with the 4~TGF~ viru~ ~table ~ ~:
2). :-
~ell~ wh~ch co-expressed the EGF wild-typ- receptor and - .
HERCD-566 showed thQ ~ame number of foci as cells .
expres~ng the EGF receptor and in~ect~d wlth the
~ontrol virus N2. This rQsult was ob~erved both in the
sti~ulation with EGF and with the 4~TGF~ virus.
The af~rem~ntioned state~-nt~ reveal ~hat the EGF
recept~r mutant~ have both a marked antiprolifera~ve
and an antl-cnco~enic potential and ar- thus excellent- :
ly suited ~or the treat~nt o~ cancer. ,~
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02/9~i~4 18:02 ~+4~ 8~ 220287 PAe Gruenecker 1~031 . :~ ~
: . . . .; .~ . .:
:: 2 1 1 7 0 7 3 ~, .. ~
/~ Helga Sohr~g 00\
a Vrilronlka~r. 1 ~ :.-:: ,. --: :. .
6~827 M6nchen
Tol, 0091439~75T
~ ~ "'.''...
......... ',''''`';'''''.,'',''',
Table 1: Colo~
~umber of colonies
/106 CFU
ln~ectiOn ~~~~~~~~~~~~~~~-~~~~~~ ~~ ~.i`~.
+10 ng/ml EGF ~ 42 TGFa
________________________________ ., ,~.
N2
Nl~C-HER}t? 2 lA o
Nq~X-HERCl)-533 o o .
~TX-HERCD-566 0 o
NTX-~ERc/N2 246 148
NTK-HERc/NTK-HERK721A ~ 2 .
N~K-HERe/N'rK-HERCD-53 3 6 4 :~
N~K-HERC/Nl~-~Il~CD-566 128 lOO
___------------ ,,.
Tho colonies were counted aft~r 4 weeks. The valuQs -
repreoont ave~age value~ fro~ four indepondent expori-
mon~s. CFU mean~ colony-forming unit~
," ", ~
. ~' ';-,...
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," ~;'' '~;,, .
,~, . , . , ~ ,; : . - ,,
,.. , ~ ., . . , . - . ~ , ~ - -
02/~ '~4 18:02~4~ 8~ 220_87 P~e Gruenecker ~ 032 : -
- 2117Q73 ~ ~
~9 (~3 ~
~ . .
Num~er of foci/106 CFa
'.~ "`'''. :.''
cell line ----~
~10 ng/ml EGF + ~2 TGF~
_________________________________~___.. _________________ :.;~ ~
N2
NTK-HERg721A 0 0 ; '`
NTX-HERCD-533 o 0
NTK-HERCD-566 0 0
NTR-HERc/N2 gao 480 ,. .~ .'
NTK-HERclNTR-~K~721A 40 18 .~
NTX-HERc/NTK-~ERCD-533 90 14 .,,
NTg-HE~RC/NTR-HERCD-566 910 !500
_______________________________________________________ :.. ~,
The ~oc~ wero count~d after 14 to 16 d~y~. The valu4s ,~:
represent average value~ f~om four independent exper~
nt~. CFU me~n~ colony-~orming units.
, -,
' " '~','. '.
