CA2117073A1 - Mutated growth factor receptor as a drug and its use for the treatment of cancer - Google Patents
Mutated growth factor receptor as a drug and its use for the treatment of cancerInfo
- Publication number
- CA2117073A1 CA2117073A1 CA 2117073 CA2117073A CA2117073A1 CA 2117073 A1 CA2117073 A1 CA 2117073A1 CA 2117073 CA2117073 CA 2117073 CA 2117073 A CA2117073 A CA 2117073A CA 2117073 A1 CA2117073 A1 CA 2117073A1
- Authority
- CA
- Canada
- Prior art keywords
- receptor
- pharmaceutical composition
- composition according
- egf
- wild
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Abstract
Abstract The present invention relates to mutated growth factor receptors which are suited as a drug. The mutated growth factor receptors as especially advantageous for the treatment of cancer diseases, in particular of those types of cancer, in which the overactivity of growth factor receptors plays a role in the development of cancer and other diseases based on the overactivity of the receptors.
Mutants of the EGF receptor are disclosed as an especially effective drug for the treatment of cancer, in which the tyrosine kinase activity of the wild-type receptor was eliminated by a point mutation or deletion in the tyrosine kinase domain.
Mutants of the EGF receptor are disclosed as an especially effective drug for the treatment of cancer, in which the tyrosine kinase activity of the wild-type receptor was eliminated by a point mutation or deletion in the tyrosine kinase domain.
Description
02/~ ' ~4 17: 45 ~+4a 8~ 220287 PAe Gruenecker lal 004 2 1 1 7 0 ~ 3 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 -:
02/ ~ ~4 17:4~ ~+4~ 8~ 220~87 P~e Gruenecker ~005 ,~ Helg~ Schrapp ~ .. ; . j Veroniha~tr. 15 6ia2~ Munch~n ~ : .:
~ T~l. 08~143~6757 ' ~' ' ~' 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~
.. .,, . . . . . . ~ ..... . . .
: : : : : :: : : : :
02J,0~'~4 17:4~ ~+49 8~ 220287 PA~ Gruenecker l~100 . ,: .: ~ . :
2117073 ~
"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. ;
, , . ., .~"
,,~,,~ ~....
,~..: ~'', . . : ~- ~ .
. ~,~ ... . . .
02/~'~4 17:47 ~ 49 8~ 220_87 P~e Gruenecker lalO07 . .
2~7073 ~
"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. ~ ;
. .
' -: .
~ ~.. .. , . " ,.. ~ . . . : . . .
02/~ 4 17: 47 ~+4~ 8~ 220287 PAe Gruenecker 1~l008 ~ ~ ~
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. . ~
. - . ~ .
02~ 4 17:48 2S+4~ 8~ 220287 P~e Gruenecker lalO0~ .
2117073~ ~
.
.: ~`.,.`., "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 '.''''!''.;
. . .
02/~i' ~4 . 17: 4~ lB+4~ 8~ 220287 PAe Gruene~ker 12 010 ~:
;- 2~17073 ~ ;
~ a ~ ,;~................... ........ ,~
?
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~
.~
02/~, ' ~4 17: 4~ ~+4~ 8a 220287 PAe Gruene~ker 1~l 011 2 ~ 1 7 0 7 3 /~\
., ;~.;
. ~ ".. . .
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;
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 -:
02/ ~ ~4 17:4~ ~+4~ 8~ 220~87 P~e Gruenecker ~005 ,~ Helg~ Schrapp ~ .. ; . j Veroniha~tr. 15 6ia2~ Munch~n ~ : .:
~ T~l. 08~143~6757 ' ~' ' ~' 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~
.. .,, . . . . . . ~ ..... . . .
: : : : : :: : : : :
02J,0~'~4 17:4~ ~+49 8~ 220287 PA~ Gruenecker l~100 . ,: .: ~ . :
2117073 ~
"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. ;
, , . ., .~"
,,~,,~ ~....
,~..: ~'', . . : ~- ~ .
. ~,~ ... . . .
02/~'~4 17:47 ~ 49 8~ 220_87 P~e Gruenecker lalO07 . .
2~7073 ~
"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. ~ ;
. .
' -: .
~ ~.. .. , . " ,.. ~ . . . : . . .
02/~ 4 17: 47 ~+4~ 8~ 220287 PAe Gruenecker 1~l008 ~ ~ ~
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. . ~
. - . ~ .
02~ 4 17:48 2S+4~ 8~ 220287 P~e Gruenecker lalO0~ .
2117073~ ~
.
.: ~`.,.`., "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 '.''''!''.;
. . .
02/~i' ~4 . 17: 4~ lB+4~ 8~ 220287 PAe Gruene~ker 12 010 ~:
;- 2~17073 ~ ;
~ a ~ ,;~................... ........ ,~
?
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~
.~
02/~, ' ~4 17: 4~ ~+4~ 8a 220287 PAe Gruene~ker 1~l 011 2 ~ 1 7 0 7 3 /~\
., ;~.;
. ~ ".. . .
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 .
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. ~ ~ :
........
02/~ 4 17:50 ~+~ 8P 220287 PAe Gruenecker Q1012 ,,,'::
2117073 ~
~) ~,, g 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$singly found that the fi~e-step ~ignal transmi~sion ch~in oxplain-d above can be blocked or inhlbited by a mutated growth fac~o~ reeeptor, lf the ;
mutated receptor~ are 9i~ultaneously expres3ed with the wild-type receptor~ from on- c~ hus, muta~ed signalling de~ective growth factor reoeptors are suited a~ drug6 wlth m~
WhiCh diseases c~n be treated which are connected with an ~ -~
increased transm~s~lon of growth signals to the in~erior of -~
tho col} by corresponding receptors.
..... - .
~. . ..
! ' ' ' ."' ' ' ,', ', 02/~'~4 17:51 ~+4~ 8~ 220~87 PAe Gruenecker lalO13 2 1 1 7 0 7 3 ~
.
A pr~-rred r~ceptor ~utant does no longer have the ;~
tyro-in~ kina~e actlvity of th~ w~ld-typ~ receptor. .
~hl~ ~utant ie no longer capabl~ o~ phosphoryla~ing tyrooin~ re~idues ~n the recoptor dimer or in ~he polyp-pttde ~b~trate5 after llgand b~nding. ~hus, th~
con~-rston o~ the extrac-llular grow~h signal to an intrac411~1ar rign~ blocked or partly inh~bited.
~ ~, A point ~utatlon in the wild-type rëceptor can already bo our~iciont ~o th~t the w~ld-type re¢eptor does no ~ ~;
longer ~nction prop~rly if it lost th~ tyro~in- kina~e activity due to p~int mutation- Such a po$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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~T~l. 089/4i38~757 1~ -1 1 ' : . . : . :`
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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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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, .'','.'. . '"'.,',, ,,.'.' . . . ... ..
.''" ' . .'.''''..`.'' 02/~'~4 17:52 ~+4~ 8~ 220287 PAe Gruenecker 1~lol~ ~
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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. :;
~ .. ~ .;. .
:," . . .'' '.' ' `:,,;',' .... , , '';,' -~' .. ~.' 02/ ~ '~ 17:53 ~P+4~ 80 220287 PAe Gruenecker ~ 017 2117073 ~o~
v~ kSacshtl~P5P ~S
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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. .~.
, '~,' '' ' ''''''"'''"'`'"
02~ 4 17:54 ~+4~ 8~ 220287 PAe Gruenecker E~1018 ~
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lS
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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2117~73 ~
' 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 ~ ..
.".," ' ;~," ''~."
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02i~ 4 17:55 ~-4~ 8~ 220287 PAe Gruenecker 121020 ~
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~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 2117073 ~
Helg~ Sohra~p V~lonika~tr. 15 ~ : ~ :
~; ~1827 MGnoh~n -~
5 Tel. 089~4398 : ~ , .:.:,. -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 ~\ ~.
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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 ~
~ Hd~ll S~h~pp g~ ~lerr~nika~U 15 ~1827 M~nchr~n T~l. 0991439 6757 ~ .' ~'~ ~,,';',, :. .' .: .: '.: ' '.~ ~ .
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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 y~ 27 IlJlunchn ~ :: S :. :.::
~el. 0~14390757 ~ -~
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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 :,:
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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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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. ~ ~ :
........
02/~ 4 17:50 ~+~ 8P 220287 PAe Gruenecker Q1012 ,,,'::
2117073 ~
~) ~,, g 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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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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2117~73 ~
' 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 ~
'PP'~ ~
,,",~
~ ~ .
~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 2117073 ~
Helg~ Sohra~p V~lonika~tr. 15 ~ : ~ :
~; ~1827 MGnoh~n -~
5 Tel. 089~4398 : ~ , .:.:,. -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~
. . . .
, ~ ....~ ,. .~ .
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 ~
~ Hd~ll S~h~pp g~ ~lerr~nika~U 15 ~1827 M~nchr~n T~l. 0991439 6757 ~ .' ~'~ ~,,';',, :. .' .: .: '.: ' '.~ ~ .
. ' ', ...
''~', '. ''.; ~ ,'' "
~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 y~ 27 IlJlunchn ~ :: S :. :.::
~el. 0~14390757 ~ -~
Zl ~
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 2 1 1 7 ~ 7 3 ~ ;~
H~19 fic~u~pp ~,, :~
4~27 M~nch~
T~ 996~57 ~OB914996~5~,~
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. ,~
''' ~' ",.:
~, ~'',"`~
' ;. ~ '.' '' ,; ~, ~ , t !
~, .~.: . . .. .
.. . . .
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 ln~ectiOn ~~~~~~~~~~~~~~~-~~~~~~ ~~ ~.i`~.
+10 ng/ml EGF ~ 42 TGFa ________________________________ ., ,~.
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~
," ", ~
. ~' ';-,...
' '"~':'"~ ,''"
," ~;'' '~;,, .
,~, . , . , ~ ,; : . - ,, ,.. , ~ ., . . , . - . ~ , ~ - -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~
_________________________________~___.. _________________ :.;~ ~
NTK-HERg721A 0 0 ; '`
NTX-HERCD-533 o 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.
, -, ' " '~','. '.
~ ~ "'.''...
......... ',''''`';'''''.,'',''', Table 1: Colo~
~umber of colonies ln~ectiOn ~~~~~~~~~~~~~~~-~~~~~~ ~~ ~.i`~.
+10 ng/ml EGF ~ 42 TGFa ________________________________ ., ,~.
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~
," ", ~
. ~' ';-,...
' '"~':'"~ ,''"
," ~;'' '~;,, .
,~, . , . , ~ ,; : . - ,, ,.. , ~ ., . . , . - . ~ , ~ - -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~
_________________________________~___.. _________________ :.;~ ~
NTK-HERg721A 0 0 ; '`
NTX-HERCD-533 o 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.
, -, ' " '~','. '.
Claims (28)
1. A pharmaceutical composition containing vectors carrying nucleic acid fragments coding for a mutated receptor tyrosine kinase that is defective in its signalling activity.
2. A pharmaceutical composition according to claim 1, characterized in that the vectors are recombinant retroviral vectors.
3. A pharmaceutical composition according to claim 1, characterized in that the receptor no longer has the tyrosine kinase activity of the corresponding wild-type receptor.
4. A pharmaceutical composition according to claim 3, characterized in that the receptor carries a deletion in its tyrosine kinase domain.
5. A pharmaceutical composition according to claim 3, characterized in that the receptor carries a point mutation in its tyrosine kinase domain.
6. A pharmaceutical composition according to claim 3, characterized in that the receptor includes an extracellular domain and a transmembrane region.
7. A pharmaceutical composition according to claim 3, characterized in that the receptor includes an extracellular domain.
8. A pharmaceutical composition according to claim 6, characterized in that the extracellular domain and the transmembrane region derive from the wild type.
9. A pharmaceutical composition according to claim 7, characterized in that the extracellular domain derives from the wild type.
10. A pharmaceutical composition according to any one of claims 1 to 9, characterized in that the mutated receptor is a growth factor receptor.
11. A pharmaceutical composition according to claim 10, characterized in that the receptor is a mutated receptor for the epidermal growth factor (EGF).
12. A pharmaceutical composition according to claim 10, characterized in that the receptor is a mutated receptor for the platelet-derived growth factor (PDGF).
13. A pharmaceutical composition according to any one of claims 1 to 9, characterized in that the receptor is a mutated HER2 receptor.
14. A pharmaceutical composition according to any one of claims 1 to 9, characterized in that the receptor is a met receptor.
15. A pharmaceutical composition according to claim 5, characterized in that the point mutation is at amino acid position 721 of theEGFwild-type receptor sequence.
16. A pharmaceutical composition according to claim 15, characterized in that the point mutant carries an alanine residue at amino acid position 721 and is deposited with the German Collection of Microorganisms under DSM 6678.
17. A pharmaceutical composition according to claim 4, characterized in that the 533 C-terminal amino acids of the F wild-type receptor are deleted.
18. A pharmaceutical composition according to claim 4, characterized in that the 566 C-terminal amino acids of the EGF wild-type receptor are deleted and deposited with the German Collection of Microorganisms under DSM 6680.
19. A pharmaceutical composition according to claim 2, characterized in that the retroviral vector is selected from pNTR-HER-X721A and pNTK-HERCD-533, deposited with the German Collection of Microorganisms under DSM 6678 and DSM
6679.
6679.
20. A pharmaceutical composition including a mutated EGF
receptor which carries a point mutation at amino acid position 721 of the EGF wild-type receptor sequence.
receptor which carries a point mutation at amino acid position 721 of the EGF wild-type receptor sequence.
21. A pharmaceutical composition according to claim 20, characterized in that the point mutant carries an alanine residue at amino acid position 721 and is deposited with the German Collection of Microorganisms under DSM 6678.
22. A pharmaceutical composition including a mutated EGF
receptor of which the 533 C-terminal amino acids of the EGF
wild-type receptor are deleted.
f
receptor of which the 533 C-terminal amino acids of the EGF
wild-type receptor are deleted.
f
23. A pharmaceutical composition including a mutated receptor of which the 566 C-terminal amino acids of the EGF
wild-type receptor are deleted and which is deposited with the German collection of Microorganisms under DSM 6680.
wild-type receptor are deleted and which is deposited with the German collection of Microorganisms under DSM 6680.
24. A method of treating man or animal affected by cancer, including the administration of an effective amount of the pharmaceutical composition according to any one of claims 1 to 23.
25. A method according to claim 24, characterized in that the cancer is the result of a hyperfunction of receptor tyrosine kinases.
26. A method according to claim 25, characterized in that the cancer is selected from mastocarcinoma, ovarian carcinoma and pulmonary carcinoma.
27. A method of treating human or animal diseases based on hyperplasma, which are characterized by the hyperfunction of receptor tyrosire kinases, including the administration of an effective amount of the pharmaceutical composition according to any one of claims 1 to 23.
28. Use of claim 27, characterized in that the disease is psoriasis.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19914129533 DE4129533A1 (en) | 1991-09-05 | 1991-09-05 | MUTTED GROWTH FACTOR RECEPTOR AS A MEDICINAL PRODUCT AND ITS USE FOR TREATING CANCER |
| DEP4129533.1 | 1991-09-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2117073A1 true CA2117073A1 (en) | 1993-03-18 |
Family
ID=6439926
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2117073 Abandoned CA2117073A1 (en) | 1991-09-05 | 1992-09-07 | Mutated growth factor receptor as a drug and its use for the treatment of cancer |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP0667899A1 (en) |
| JP (1) | JPH07502884A (en) |
| CN (1) | CN1071586A (en) |
| AU (1) | AU669857B2 (en) |
| CA (1) | CA2117073A1 (en) |
| DE (1) | DE4129533A1 (en) |
| FI (1) | FI941053A (en) |
| MX (1) | MX9205084A (en) |
| NO (1) | NO940778L (en) |
| NZ (1) | NZ244239A (en) |
| PT (1) | PT100844A (en) |
| WO (1) | WO1993005148A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AUPN274295A0 (en) * | 1995-05-02 | 1995-05-25 | Garvan Institute Of Medical Research | GDU, a novel signalling protein |
| US6465623B2 (en) | 1995-05-02 | 2002-10-15 | Garvan Institute Of Medical Research | GDU, a novel signalling protein |
| US6790614B1 (en) | 1999-11-19 | 2004-09-14 | Novartis Ag | Selectable cell surface marker genes |
| CN104480200B (en) | 2004-03-31 | 2017-12-29 | 综合医院公司 | Determine method of the cancer to EGF-R ELISA magnetic target therapy reactivity |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0325224B1 (en) * | 1988-01-22 | 1996-07-31 | ZymoGenetics, Inc. | Methods of producing secreted receptor analogs |
| WO1990008160A1 (en) * | 1989-01-20 | 1990-07-26 | Imperial Cancer Research Technology Ltd. | Growth factor receptor-like peptides without tyrosine-kinase activity |
| GB9001466D0 (en) * | 1990-01-23 | 1990-03-21 | Erba Carlo Spa | Extracellular form of the human fibroblast growth factor receptor |
| CA2055441C (en) * | 1989-05-19 | 2003-01-07 | Robert M. Hudziak | Her2 extracellular domain |
| JP2975679B2 (en) * | 1989-09-08 | 1999-11-10 | ザ・ジョーンズ・ホプキンス・ユニバーシティ | Structural change of EGF receptor gene in human glioma |
-
1991
- 1991-09-05 DE DE19914129533 patent/DE4129533A1/en not_active Ceased
-
1992
- 1992-09-04 PT PT10084492A patent/PT100844A/en not_active Application Discontinuation
- 1992-09-04 MX MX9205084A patent/MX9205084A/en unknown
- 1992-09-05 CN CN 92111396 patent/CN1071586A/en active Pending
- 1992-09-07 WO PCT/EP1992/002058 patent/WO1993005148A1/en not_active Application Discontinuation
- 1992-09-07 EP EP92918949A patent/EP0667899A1/en not_active Withdrawn
- 1992-09-07 NZ NZ24423992A patent/NZ244239A/en unknown
- 1992-09-07 CA CA 2117073 patent/CA2117073A1/en not_active Abandoned
- 1992-09-07 AU AU25185/92A patent/AU669857B2/en not_active Ceased
- 1992-09-07 JP JP5504969A patent/JPH07502884A/en active Pending
-
1994
- 1994-03-04 FI FI941053A patent/FI941053A/en not_active Application Discontinuation
- 1994-03-04 NO NO940778A patent/NO940778L/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| DE4129533A1 (en) | 1993-03-11 |
| AU2518592A (en) | 1993-04-05 |
| CN1071586A (en) | 1993-05-05 |
| MX9205084A (en) | 1993-05-01 |
| NO940778D0 (en) | 1994-03-04 |
| FI941053A (en) | 1994-04-08 |
| EP0667899A1 (en) | 1995-08-23 |
| FI941053A0 (en) | 1994-03-04 |
| NZ244239A (en) | 1995-07-26 |
| JPH07502884A (en) | 1995-03-30 |
| NO940778L (en) | 1994-05-04 |
| WO1993005148A1 (en) | 1993-03-18 |
| AU669857B2 (en) | 1996-06-27 |
| PT100844A (en) | 1994-05-31 |
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